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Identification of effectors with avirulence functions in the pathogenic barley smut fungus, using marker-based… Ali, Shawkat 2011

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IDENTIFICATION OF EFFECTORS WITH AVIRULENCE FUNCTIONS IN THE PATHOGENIC BARLEY SMUT FUNGUS, USING MARKERBASED APPROACHES AND COMPARISON AMONG GENOMES OF RELATED SPECIES  by  Shawkat Ali M. Phil., Quaid-I-Azam University, Islamabad, Pakistan, 2004  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in The Faculty of Graduate Studies  (Botany)  THE UNIVERSITY OF BRITISH COLUMBIA  (Vancouver)  August 2011  © Shawkat Ali, 2011  ABSTRACT  In plant pathology, the molecular genetic analysis of the interaction between pathogen and host yields knowledge applicable to combat crop disease. During infection, pathogens secrete effector proteins to reprogram the host for its benefit. In special cases, recognition of certain effectors by resistance genes, essential components of the host surveillance system, induces resistance to infection. No effectors with such avirulence function have been described for basidiomycete fungi infecting cereals. Ustilago hordei is a biotrophic basidiomycete fungus that infects barley. One of its effectors functions as an avirulence protein, UhAVR1, rendering it avirulent on barley cultivar Hannchen, having corresponding resistance gene Ruh1. I have located UhAvr1 within the genome using a deletion approach and confirmed its resistance-triggering function. I provide evidence that transposable element (TE) activity in the UhAvr1 promoter region and translocation of the coding region are likely responsible for enabling virulence on Hannchen. This region of the genome harbours a cluster of predicted secreted proteins and is syntenic to a cluster in closely-related corn pathogens, U. maydis and Sporisorium reilianum. In U. maydis, deletion of this region results in dramatic reduction in virulence on corn. This region is under selection pressure in both U. maydis and U. hordei likely to avoid recognition by the host. Evolution of the region in U. maydis seems to involve gene duplication and diversification, while in U. hordei this region is saturated with TEs and repeats which can play a role in genome rearrangements. Computational analysis of the U. hordei genome sequence identified 372 candidate secreted effector proteins (CSEPs), many of which are expected to contribute to virulence and some to trigger resistance in analogy to UhAVR1. Most CSEPs are Ustilago-specific proteins of unknown function and without similarities to sequences in public databases. Evidence for accelerated evolution was observed when comparing CSEPs among smut species. More than half of these CSEPs have four or more cysteine residues in characteristic patterns, possibly involved in disulphide bridge formation and protein folding. The study of effectors with avirulence function can reveal resistance genes which can be used for crop breeding programs to obtain disease-resistant cultivars.  ii  PREFACE  Below is a list of manuscripts that have been published or are in preparation for publication and that comprise this thesis. The contributions made by the candidate are mentioned.  Chapter 1: A shortened version of the chapter will be submitted as review for publication. Anticipated author: Ali, S. and Bakkeren, G.  The candidate wrote the chapter. Dr. Bakkeren supervised the manuscript preparation and provided editorial support.  Chapter 2: A version of the chapter entitled “The avirulence gene UhAvr1 clusters with predicted secreted proteins in the U. hordei genome and is inactivated by transposon activity in virulent strains”, is in preparation for publication. Anticipated co-author list: Ali, S., Linning, R., Laurie, J. and Bakkeren, G.  The candidate designed and performed the experiments, as well as wrote the manuscript. Mr. Linning assisted with the preparation of Figures 2.12 and 2.16 and provided bioinformatic support. Dr. Bakkeren supervised the work and manuscript preparation and provided editorial support.  Chapter 3: A version of this chapter will be included in a joint manuscript entitled “Genomewide analysis of U. hordei candidate secreted effectors proteins; comparison with U. maydis.” which is in preparation for publication. Anticipated co-author list: Laurie, J., Ali, S., Linning, R., Bakkeren G., Schirawski, J., Kahmann, R. and others.  The candidate designed and performed the experiments, as well as wrote the chapter. Mr. Linning provided bioinformatic support and Dr. Bakkeren supervised the work and manuscript preparation and provided editorial support.  iii  Chapter 4: This work was published in its entirety in Current Genetics, 57: 63-73, Ali, S. and Bakkeren, G. 2011. ”Introduction of large DNA inserts into the barley pathogenic fungus, Ustilago hordei, via recombined binary BAC vectors and Agrobacterium-mediated transformation.” .  The candidate designed and performed the experiments, as well as wrote the manuscript. Dr. Bakkeren supervised the work and manuscript preparation and provided editorial support.  Chapter 5: “Towards the cloning of UhAVR6.” The candidate designed and performed the experiments, as well as wrote the chapter. Dr. Bakkeren supervised the work and manuscript preparation and provided editorial support.  Chapter 6: “General discussion.” The candidate wrote the chapter. Dr. Bakkeren provided editorial support.  iv  TABLE OF CONTENTS  ABSTRACT ...................................................................................................................... ii PREFACE ....................................................................................................................... iii TABLE OF CONTENTS ................................................................................................... v LIST OF TABLES............................................................................................................ ix LIST OF FIGURES .......................................................................................................... x LIST OF ABBREVIATIONS ............................................................................................ xi ACKNOWLEDGEMENTS ............................................................................................. xiv 1. Introduction ................................................................................................................1 1.1. Plant-microbe interactions ........................................................................... 1 1.1.1. Strategies used by pathogens to infect host plants ..............................2 1.1.2. Resistance in plants to pathogens ........................................................3 1.1.3. Pathogen associated molecular patterns ..............................................5 1.1.4. Types of resistance in plants ................................................................6 1.2. Effectors in plant-microbe interactions ......................................................... 8 1.2.1. Role of effectors in infection .................................................................9 1.2.1a. Breaking the physical barrier ..................................................... 10 1.2.1b. Disarming plant defense enyzymes ........................................... 11 1.2.1c. Suppression of receptor activation ............................................. 12 1.2.1d. Suppression of R gene-triggered resistance .............................. 13 1.2.1e. Down-regulation of defense signaling ........................................ 14 1.2.1f. Alteration of the plant defense transcriptome.............................. 15 1.2.1g. Destruction of antimicrobial compounds .................................... 15 1.2.1h. Killing of host cells ..................................................................... 17 1.2.1i. Suppression of host defense by symbionts ................................. 18 1.3. Gene-for-gene or R and Avr interaction ..................................................... 19 1.3.1 Fungal Avr genes ................................................................................ 21 1.3.1a. Cladosporium fulvum ................................................................. 21 1.3.1b. Rynchosporium secalis .............................................................. 23 1.3.1c. Blumeria graminis ...................................................................... 23 1.3.1d. Melampsora lini .......................................................................... 24 1.3.1e. Magnaporthe oryzae .................................................................. 26 1.3.1f. Leptosphaeria maculans ............................................................. 27 1.3.2. Oomycete Avr genes .......................................................................... 29 1.3.2a. Hyaloperonospora arabidopsidis ............................................... 29 1.3.2b. Phytophthora sojae .................................................................... 30 1.3.2c. Phytophthora infestans .............................................................. 31 1.4. Resistance proteins (R) in plants ............................................................... 31 1.5. Recognition of Avr proteins by R proteins .................................................. 32 1.6. Marker-based approaches for cloning of Avr genes................................... 34 1.7. Comparative genomics (secretomics) ........................................................ 36 1.8. Smut fungi.................................................................................................. 37 v  1.9. Proposed research project ......................................................................... 38 1.10. Research objectives................................................................................. 41 2. The avirulence gene UhAvr1 clusters with predicted secreted proteins in the U. hordei genome and is inactivated by transposon activity in virulent strains ...................................................................................................... 50 2.1. Introduction.............................................................................................. 50 2.2. Material and methods ............................................................................. 52 2.2.1. Barley cultivars and U. hordei strains used in this study..................... 52 2.2.2. Growth conditions of U. hordei and barley and U. hordei transformation .......................................................................................... 52 2.2.3. Mapping of the UhAvr1 ....................................................................... 53 2.2.4. Sequencing of ORFs in the UhAvr1 locus from the virulent parent and field isolates............................................................................53 2.2.5. Sequencing of BAC clones from the avirulent and virulent parents by 454 method ............................................................................. 54 2.2.6. Deletion of the UhAvr1-containing region ........................................... 54 2.2.7. Analysis of deletion mutants ............................................................... 55 2.2.8. Plasmids to complement U. hordei deletion mutants .......................... 55 2.2.9. Western blot analysis .........................................................................57 2.2.10. Mating test ........................................................................................ 57 2.2.11. Pathogenicity assays ........................................................................57 2.2.12. Nucleic acid manipulation ................................................................. 58 2.2.13. qRT-PCR analysis ............................................................................58 2.3. Results ....................................................................................................... 59 2.3.1. Sequencing of ORFs from the virulent parent and field isolates ..................................................................................................... 59 2.3.2. Delimiting of the UhAvr1-containing region by deletion analysis .................................................................................................... 60 2.3.3. Deletion of fragment C19A2 in both mating partners does not impair virulence towards Odessa........................................................ 61 2.3.4. Complementation of C19A2 deletion mutants .................................... 62 2.3.5. Fragments C19A2-C and C19A2-D contain UhAvr1........................... 63 2.3.6. Overlapping regions of the fragments C19A2-C and C19A2D contain UhAvr1 ..................................................................................... 64 2.3.7. Sequence comparison between the Avr1 and avr1 loci in the parental strains ......................................................................................... 65 2.3.8. Variable sequences at the UhAvr1 locus point to TE activity .............. 66 2.3.9. Lack of complementation of the C19A2 deletion mutant with U. maydis homologs of Uh10022 ............................................................. 67 2.3.10. Synteny between U. hordei and U. maydis at the UhAvr1 locus ......................................................................................................... 67 2.4. Discussion ............................................................................................ 68 3. Genome-wide analysis of Ustilago hordei candidate secreted effectors proteins; comparison with U. maydis ......................................... 107  vi  3.1. Introduction .............................................................................................. 107 3.2. Materials and methods............................................................................. 109 3.2.1. Genomic resources .......................................................................... 109 3.2.2. Prediction of secreted proteins ......................................................... 109 3.2.3. Genome comparison ........................................................................ 110 3.2.4. Phylogenetic analysis of U. hordei candidate secreted effectors proteins...................................................................................110 3.3. Results ..................................................................................................... 111 3.3.1. Candidate predicted secreted proteins of U. hordei.......................... 111 3.3.2. Phylogeny of CSEPs and their paralogs ........................................... 111 3.3.3. Cysteine- rich secreted protein ......................................................... 112 3.3.4. Comparison of U. hordei and U. maydis secretomes ....................... 112 3.3.5. A subset of predicted secreted proteins and their paralogs reside in clusters ...................................................................................113 3.3.6. Comparison between the U. hordei and U. maydis secreted protein clusters ...................................................................................... 113 3.4. Discussion ............................................................................................... 114 4. Introduction of large DNA inserts into the barley pathogenic fungus, Ustilago hordei, via recombined binary BAC vectors and Agrobacterium-mediated transformation...................................................... 126 4.1. Introduction .............................................................................................. 126 4.2. Materials and methods............................................................................. 128 4.2.1. Strains and plasmid ..........................................................................128 4.2.2. Recombineering ...............................................................................128 4.2.3. Fungal transformation....................................................................... 129 4.2.4. Analysis of transformants ................................................................. 130 4.3.1. Recombineering ...............................................................................132 4.3.2. Fungal transformation....................................................................... 133 4.3.3. Molecular analysis of fungal transformants ...................................... 134 4.4. Discussion ............................................................................................... 135 5. Towards the cloning of UhAVR6 ......................................................................... 144 5.1. Introduction .............................................................................................. 144 5.2. Material and methods .............................................................................. 145 5.2.1. DNA manipulation.............................................................................145 5.2.2. SSR analysis .................................................................................... 145 5.2.3. RAPD analysis.................................................................................. 146 5.2.4. AFLP analysis................................................................................... 146 5.3. Results ..................................................................................................... 146 5.3.1. Construction of populations segregating for UhAvr6 and uhavr6 .................................................................................................. 146 5.3.2. Pools for bulked segregant analysis (BSA) ...................................... 147 5.3.3. SSR primer screening ...................................................................... 148 5.3.4. AFLP primer screening ..................................................................... 149 5.3.5. RAPD primer screening .................................................................... 149  vii  5.4. Discussion ............................................................................................... 150 6. General Discussion and Future Perspectives ................................................... 159 6.1. General discussion ................................................................................... 159 6.2. Future perspectives ................................................................................. 165 6.2.1. Localization of UhAVR1 .................................................................... 165 6.2.2. Novel host protein interactions with UhAVR1 ................................... 166 6.2.3. Function of UhAVR1 ......................................................................... 167 6.2.4. Cloning of other U. hordei virulence and avirulence genes .............. 168 6.3. Conclusion ............................................................................................... 169 References ................................................................................................................. 170  viii  LIST OF TABLES Table 1.1 Effector proteins of filamentous plant pathogens ................................................ 44 Table 2.1 Strains used in this work ......................................................................................... 93 Table 2.2 Primers used in this work ....................................................................................... 96 Table 2.3 U. hordei genes located on BAC3-A2 (117 kb) and their homologs in U. maydis ................................................................................................................................ 103 Table 3.1 U. hordei candidate secreted effectors proteins with characteristic patterns of occurring cysteine residues (C) and spacing (number of X amino acid residues) 122 Table 4.1 Recombineering and transformation efficiencies .............................................. 143 Table 5.1 SSR and AFLP primers for U. hordei used in this study. ................................. 154 Table 5.2 Composition of pools used for bulked segregant analysis. ............................. 158 Table 5.3 Results of different marker analyses on the pools and progeny..................... 158  ix  LIST OF FIGURES  Figure 1.1 Infection process of U. hordei on barley.. .......................................................... 42 Figure 2.1 Plasmid map of pUBlexInt:GateWayHA. ............................................................ 76 Figure 2.2 The UhAr1 locus.. .................................................................................................. 77 Figure 2.3 Deletion analysis of cluster C19A. ...................................................................... 78 Figure 2.4 Pathogenicity test of the deletion mutants. ........................................................ 79 Figure 2.5 Analysis of virulence towards barley cultivars of a cross of strains both deleted for the C19A2 fragment....................................................................................... 80 Figure 2.6 Position of BAC1-6 subclone at the UhAvr1 locus and pathogenicity tests. 81 Figure 2.7 Complementation analysis of the C19A2 deletion mutant transformed with Uh10021, Uh10022 and Uh10024 and their virulence toward barley. ....................... 82 Figure 2.8 Deletion analysis of fragment C19A2. ................................................................ 83 Figure 2.9 Pathogenicity test of the mutants deleted for sub-fragments of C19A2. ....... 85 Figure 2.10 Deletion analysis of Uh10022 and pathogenicity test. ................................... 86 Figure 2.11 Comparison of the UhAvr1 locus between the avirulent and virulent parents. ................................................................................................................................ 87 Figure 2.12 Sequence comparison of the U. hordei UhAvr1 locus between the virulent parent Uh362 (upper) and the avirulent parent Uh364 (lower). .................................. 88 Figure 2.13 Sequence comparison of the intergenic region between Uh10021 and Uh10022 at the UhAvr1 locus. ......................................................................................... 89 Figure 2.14 Comparison of UhAVR1 to a U. hordei paralog and U. maydis homologs. 90 Figure 2.15 Pathogenicity tests of the C19A2 deletion mutant complemented with U. maydis homologs. .............................................................................................................. 91 Figure 2.16 Comparison of the U. hordei UhAvr1 locus to the syntenic region in U. maydis harbouring cluster 19A. ....................................................................................... 92 Figure 3.1 Overview of the molecular relatedness between U. hordei CSEPs and NPCSEPs showing a large diversity and small families. ........................................... 120 Figure 3.2 Diversity among U. hordei and U. maydis proteins ........................................ 121 Figure 4.1 Schematic representation of the BAC to BIBAC conversion method. ......... 139 Figure 4.2 Verification of conversion of BAC clones to BIBAC vectors. ........................ 140 Figure 4.3 PCR analysis of genomic DNA of six independent BIBAC_2-1 and two independent BIBAC_1-6 U. hordei transformants. ..................................................... 141 Figure 4.4 DNA blot analysis of the genomic DNA of independent U. hordei BIBAC transformants. ................................................................................................................... 142 Figure 5.1 PCR amplification with SSR and AFLP primers showing polymorphisms in parents, pools and progeny. ........................................................................................... 153  x  LIST OF ABBREVIATIONS  aa ABA AD AFLP AMT AS Avr BAC BD Bgh BIBAC BL BR BSA CAT CC-NBS-LRR cDNA CF Cm CM CSEPs CSP DAMPs DCM DCM-S ECL ECP Ef-Tu ETI Fol GFP GUS HESP HR HTS Hyg B IM JA Kb Km LB LPS  Amino acid Abscisic acid Activation domain Amplified fragment length polymorphisms Agrobacterium mediated transformation Acetosyringone Avirulence Bacteria artificial chromosome Binding domain Blumeria graminis f. sp. hordei Binary BAC Left border Right border Bulked segregant analysis Chloramphenicol acetyl transferase Coiled-coil nucleotide-binding site, leucine-rich repeat Complementary DNA Cladosporium fulvum Chloramphenicol Complete medium Candidate predicted secreted proteins Cold shock protein Danger associated molecular patterns Double complete medium Double complete medium-sorbitol Enhanced chemiluminescence system Extracellular cysteine-rich proteins Elongation factor Tu Effector-triggered immunity Fusarium oxysporum f. sp. lycopersi Green fluorescent protein Bacterial uidA gene (beta-glucuronidase) Haustoria-expressed secreted proteins Hypersensitive response Host targeting signal Hygromycin B Induction medium Jasmonic acid Kilo base Kanamycin Luria-Bertani Lipopolysaccharides  xi  LTR MAMP MAT-1 MAT-2 mg Mig Ml ml-1 MPa Nd ng NJ NLP NLS NO NPSPCs NRPS o C ORF PAL PAMP PCD PCR PDA PEG % PKS PR PRR Psi Pst PTI qRT-PCR R RAPD RFLP RIP RLK ROS Ruh1 RXLR SA SCAR SDS SDS-PAGE SIMAP  Long terminal repeat Microbe associated molecular Mating type 1 Mating type 2 Millegram Maize induced gene Mildew Per milleliter Mega pascal Niederzenze Nanogram Neighbor-joining NEP1-like proteins Nuclear localization signals Nitric oxide Non-predicted secreted paralogs of CSEPs Nonribosomal peptide synthetase Degree Celsius Open reading frame Phenylalanine ammonia lyase Pathogens associated molecular pattern Programmed cell death Polymerase chain reaction Potato dextrose agar Polyethylene glycol Percent Polyketide synthase Pathogenesis-related PAMP recognition receptors Pounds per square inch Psedomonas syringae pv tomato PAMP triggered immunity Quantitative reverse transcriptase polymerase chain reaction Resistance Randomly amplified polymorphic DNA Restriction fragment length polymorphisms Repeat-induced point Receptor-like kinases Reactive oxygen species Resistance to Ustilago hordei Avr1 Arginine any amino acid leucine arginine Salicylic acid Sequence-characterized amplified region Sodium dodecyl sulphate Sodium dodecyl sulphate polyacrylamide gel electrophoresis Similarity matrix of proteins  xii  SNP SSR SP SSPs SUMO TBE Tc T-DNA TE Ti TILLING TIR TMD TTSS UAS UhAvr1 UPA v/v) w/v w/w WsB Xcc YXC Zeo µg µl  Single nucleotide polymorphism Simple sequence repeat Signal peptide Small secreted proteins Small ubiqiuitin-like modifier Tris-borate-EDTA Tetracycline Transfer DNA Transposable element Tumor inducing Targeting induced local lesions IN genomes Toll or interleukin 1 receptor Transmembrane domain Type three secretion systems Upstream activating sequences Ustilago hordei Avr1 Upregulated by AVRBS3 Volume/volume Weight/volume Weight/weight Wassilewskkija Xanthomonas campestris pv campestris Tyrosine any amino acid cysteine Zeocin Microgram Microliter  xiii  ACKNOWLEDGEMENTS  First and foremost I wish to express my gratitude and appreciation to my supervisor, Dr. Guus Bakkeren, for his thorough guidance, continuous support, encouragement, and constructive criticism during the course of my Ph.D. research pursuits as well as during the write up of my thesis. Many thanks to Guus for his extreme patience, even in the darkest days of UhAvr6 cloning. I wish to thank my co-supervisor Dr. Xin Li and research committee members; Dr. Steven Lund, and Dr. Colette Breuil, who carefully assessed my thesis and returned me thoughtful feedback and valuable suggestions. I also wish to express my appreciation to Dr. Jim Kronstad, Xin Li and Guus Bakkeren for reference letters which were crucial during my search for a post doctorate position. Special thanks go to Rob Linning for his excellent technical assistance and bioinformatics support, lab organization and availability assistance. I am also thankful to all the present and past members of Bakkeren lab, including Dr. Xiao Song, Dr. John Laurie, Dr. José Antonio Cervantes-Chávez, and Dr. David Joly for their useful discussion and help. Great thanks go to Pacific Agriculture and Agri-Food Canada Summerland for allowing me to use their facilities. Special thanks to Dr. Kenna MacKenzie, Research Manager and Widdis Laura Human Resources Coordinator. I am also thankful for all the staff at Pacific Agriculture and Agri-Food Canada Summerland for their help during my research. Many thanks to Dr. Paul Wiersma, Joan Chisholm, Jane Theilmann, Melanie Walker, Ron Reade, Virginia Dickison, Les Willis, Colleen Harlton and Michael Weis for their help. I am also thankful to Dr. Helene Sanfaçon and Guus Bakkeren for their hospitality during my first visit to Okanagan in 2005. Also I would like to thank the staff members of the department of Botany especially Mrs.Veronica Oxtoby (Graduate Secretary), and Mrs. Judy Heys for their assistance. I also wish to thanks our collaborators at the Max Planck Institute for Terrestrial Microbiology in Marburg Germany for their contributions to the Ustilago hordei genome project and for their willingness to share unpublished information. I would like to thank all my friends at the station for their help and fun times together playing volleyball, skiing, hiking, camping and fishing- Erkan Karacabey, Tanja Durbic, Bahram Soltani, Nadia Sokal, Choothaweep Palakawong, Ting Wei, Yingchao Nie, Minggang Fang, Yasantha Athukorala, Carl Pronky, Raquel K. Cruz Bravo, Haydé Azeneth Vergara Castañeda, Coralie Blanchard, Zhou Wei, Guangzhi Zhang, Dongbao Fu, Eunice Randall, Paul Randall, Liz Hui, Julie Boulé, Yukihiro Tamaki, Juan Jovel, Basudev Ghoshal, Kankana Ghoshal, and Ikeda Laurie. Special thanks go to my friend Dr. Sajid Ali Barlas and his family for his help in everything. I am especially indebted to my family for their strong support during my lengthy graduate student career. I wish to express my heartfelt appreciation and gratitude to my father, mother, brothers, and sisters and to all my teachers for their continuous love, support, and encouragement. Words alone are inadequate to describe what they have given me. I dedicate this thesis to my parents. I am particularly indebted to my wife Lubna whose continued support and encouragement over the last three years enabled me to overcome the much travail during my studies. xiv  CHAPTER 1 Introduction 1.1. Plant-microbe interactions Plant diseases cause large crop losses worldwide. Approximately 14.1% of total crop yields are destroyed by diseases caused by both biotic and abiotic sources (Agrios 2005). Plants are attacked by many pathogenic organisms including fungi, oomycetes, bacteria, viruses, aphids and nematodes. Fungi cause the most serious damage among all plant pathogens, with an estimated annual loss of 200 billion $US (Horbach, et al. 2011), and account for 12 of the 19 most serious pathogens in the US (Madden and Wheelis 2003). There are more than 100,000 species of fungi, and more than 10% of them are plant pathogens, while only 50 fungal species are human pathogens and a similar number are animal pathogens (Agrios 2005). In North America, more than 8,000 fungal species are plant pathogens that cause more than 100,000 diseases (Horbach, et al. 2011). By definition, plant pathogens are organisms that have the ability to cause disease on plants; however, it is widely accepted that plants are resistant to most pathogens, which makes plant disease the exception rather than the rule. Such pathogens are called non-adapted or nonhost pathogens and the plants are called nonhost plants. When a pathogen can infect one or more members of a plant species, the pathogens are called adapted or host pathogens and the plant is called a host for that particular pathogen. Phytopathogens can be divided into three classes: necrotrophs, biotrophs, and hemibiotrophs, based on their lifestyles and mode of interaction with their hosts. Necrotrophs kill cells of their host plants often through the production of toxins at the first attempt of colonization and subsequently feed on dead cells (Horbach, et al. 2011). Besides toxins, necrotophs also elicit cell death by secreting reactive oxygen species (ROS) and large amounts of cell wall degrading enzymes that also causes significant tissue damage (Rohe, et al. 1995, Tudzynski and Kokkelink 2009, Walton 1994). A shortened version of the chapter will be submitted as a review for publication. Anticipated author: Ali, S. and Bakkeren, G.  1  Some of the host plants have developed abilities to recognize the secreted toxins at early infection stages of necrotrophs; thus, these weapons become liabilities for the pathogens (Rohe, et al. 1995). Biotrophs do not produce toxic secondary metabolite or toxic proteins since they need living host cells to supply the nutrients for long periods of time to complete their life cycle (Dangl and Jones 2001). The obligate biotroph has evolved a mechanism to suppress the host defense response or to avoid recognition during infection of living tissues (Greenberg 1997); however, some host plants can recognize pathogen-specific molecules which then trigger rapid and localized cell death that can stop pathogen growth to the neighboring cells, a the so-called, hypersensitive response (HR). Hemibiotrophs combine these two lifestyles by acting as biotrophs in the beginning of the infection to avoid detection and multiply in association with living cells, and then as necrotrophs, by killing their host cells toward the end prior to dispersal (Horbach, et al. 2011).  1.1.1. Strategies used by pathogens to infect host plants For a pathogen to infect a plant, it first needs to recognize its plant host. Pathogens use different strategies to enter their host plants. For example, viruses use other organisms as vectors to enter plant cells and proliferate intracellularly, while pathogenic bacteria enter plants through natural openings normally used for gas and water (stomata and hydathods, respectively) or through wounds; they proliferate in the apoplast. Fungi and oomycetes enter either through natural openings or directly through the plant epidermal cells by mechanical and chemical means, or expand their hyphae on the top of, within, or between the plant cells (Jones and Dangl 2006). The pathogens need to adhere to the plant before penetrating the plant cuticle. Fungal hyphae and spores use mucilaginous and adhesive substances at their tips and the intermolecular forces between plant and pathogen are responsible for the close contact. Most rust fungi enter plants through stomata by developing appressoria over the stomata to penetrate into the cavity below, while ascomycetes such as Magnaporthea grisea and the powdery mildews, such as Blumeria graminis f. sp. hordei (Bgh) penetrate the cuticle of plants directly through appressorium. For direct cuticle penetration, M. grisea uses turgor pressure in its melanized appressorium (which is about 8 MPa) for penetration while B. graminis f. sp. hordei penetrates cell walls by the combined activity of cellulases and a turgor pressure of about 2-4 MPa (Pryce-Jones, et al. 1999, Talbot 2003). Other pathogens secrete cell wall degrading enzymes, such as cutinases, cellulases, 2  pectinases, and wax degrading enzymes for penetration of their host. After gaining entrance to the host plants, pathogens require additional “weapons” to neutralize the defense reaction of the host and gain access to nutrients.  1.1.2. Resistance in plants to pathogens Resistance to most non-adapted pathogenic microbes is achieved by preformed physical barriers, such as waxy cuticular surface layers of the leaves that prevent pathogens from entering into the plant. Even after successful penetration of the cuticle, the pathogen needs to overcome the biochemical barriers that include low pH of the apoplast, broad spectrum antimicrobial compounds, and “defense” enzymes that degrade microbial cell walls (Felle 1998, Dangl and Jones 2001, Huckelhoven 2007). In addition, plants cells are surrounded by thick and stable cell walls that most microbes are not equipped to penetrate. The cell wall components such as actin microfilaments play an important role in defense against fungal penetration and whose disruption results in loss of nonhost resistance against several nonhost fungi (Kobayashi, et al. 1997a, Kobayashi, et al. 1997b, Mysore and Ryu 2004). Plants also produce a number of peptides, proteins, and non-proteinaceous secondary antimicrobial metabolites which may determine the host range of some pathogens (Broekaert, et al. 1995, Morrissey and Osbourn 1999). Other plants like tomato and sugar beet produce fungitoxic substances on the surface of leaves to inhibit the germination of spores of Botrytis and Cercospora, respectively (Agrios 2005). Similarly, some red scale varieties of onion contain the phenolic compounds, protocatecuic acid and catechol, which inhibit the germination of conidia causing them to burst, thereby protecting the onion plant from infection (Agrios 2005). Phytoanticipins (the preformed antimicrobial compounds) like tannins and fatty acid-like compounds such as dienes, present in high concentration in cells of young fruits, leaves, or seeds are responsible for resistance to pathogens such as Botrytis (Agrios 2005). Saponins, another group of phytoanticipins, such as tomatine in tomato and avenacin in oat, provide resistance to Septoria and Gaeumannomyces graminis var. avena, respectively, when the pathogens lack the corresponding detoxifying enzymes, such as saponinase (Osbourn 1996). Protease inhibitors are another class of antimicrobial compound which were originally isolated for their anti-feedant activities against insects. Reports have shown that proteinase inhibitors such as cysteine protease inhibitor produced by pearl millet have anti-fungal activities (Joshi, et al. 1999). Tomato and potato produce several cysteine proteases 3  and secrete them to the apoplast to control pathogenic bacteria, fungi, and oomycetes (Axtell and Staskawicz 2003, Lucas 1998). Besides preformed defense systems, plants also have inducible defense systems to produce antimicrobial compounds such as the phytoalexins. Pea produces pisatin when attacked by Nectria spp. and rice produces sakuranetin against M. grisea (Agrios 2005). Pathogenesisrelated (PR) proteins are defense proteins that are induced in response to pathogen attack. The accumulation of these proteins is usually associated with the acquisition of systemic resistance in plants against a wide range of pathogens (van Kan, et al. 1992). The PR proteins produced in plants are chitinases, phenylalanine ammonia lyase (PAL), β -1, 3 glucanase, PR-1, PR-4 to PR14, and peroxidase (Agrios 2005). In tobacco, five groups of PR proteins have been identified, each group consisting of an acidic, extracellular and basic, intracellular protein (van Loon, et al. 1987). PAL is one of the key enzymes involved in the synthesis of aromatic compounds, like phytoalexins which are involved in stress and disease resistance (Dixon and Harrison 1990). Chitinase and β-1,3 glucanase are capable of hydrolyzing the chitin and β-1,3 glucans, which are two major polysaccharides of the cell walls of many pathogenic fungi (Kauffmann, et al. 1987, Legrand, et al. 1987). In addition to induced chemical defenses, plants also use induced physical defenses against pathogens, for example, by forming cell wall depositions (papilla) directly under the penetration sites of the pathogens; this can stop up to 90% of the penetration attempts in the B. graminis f. sp. hordei /Arabidopsis nonhost interaction (Collins, et al. 2003). Some physiological responses seen upon incompatible interactions are the increase in 2+  Ca levels in the cells surrounding the recognition site, which is the first measurable defense response and is required for the HR (Grant and Loake 2000). The oxidative burst, the production of reactive oxygen species (ROS), is another early defense response. The accumulation of ROS is involved in cell wall cross-linking, up-regulation of defense gene expression, and induction of the HR (Torres and Dangl 2005, Torres, et al. 2006). Salicylic acid (SA), activates plant defense responses against invading pathogens (Durner, et al. 1997). SA is required for systemic defense in both host and nonhost resistance and is rapidly induced during the HR. Nitric oxide (NO) also acts as a signalling molecule and promotes the HR. The early physiological and biochemical events and signalling requirements in defense responses are almost similar in host, nonhost, and virulent pathogen interactions. The difference in the strength and timing of activation of these  4  defence pathways makes a resistant interaction different from a compatible interaction (da Cunha, et al. 2006).  1.1.3. Pathogen associated molecular patterns In addition to preformed and inducible physical and biochemical barriers, plants also have surveillance systems that evolved to recognize various pathogen surface-exposed and cytoplasmic molecules known as pathogen (microbe) associated molecular patterns: PAMPs or MAMPs (Shiu and Bleecker 2003). MAMPs are highly conserved molecules of microbes and are perceived by host receptors called PAMP recognition receptors (PRRs) at an early stage of infection. Recognition results in induction of PAMP triggered immunity (PTI). Examples of surface-exposed PAMPs that have been shown to be capable of triggering PTI are flagellin (Felix, et al. 1999), lipopolysaccharides (LPS) (Erbs and Newman 2003, Meyer, et al. 2001), lipooligosaccharide from gram-negative bacteria, chitin from cell walls of higher fungi (Bartnicki-Garcia 1968, Ren and West 1992), invertase from Saccharomyces cerevisiae (Basse, et al. 1992), and 1,3-1,6-hepta-β-glucoside from the cell walls of Phytophthora sojae (Sharp, et al. 1984a, Sharp, et al. 1984b). Examples of cytoplasmic MAMPs that induce host defense are cold shock protein (CSP) and elongation factor Tu (Ef-Tu; (Felix and Boller 2003, Kunze, et al. 2004). One efficient strategy for pathogens to avoid PAMP recognition and triggered host defense would be changing PAMP sequences that are recognized by the PRRs, but mutating PAMPs is not very easy due to their often essential roles for pathogen survival (Gohre and Robatzek 2008). One of the best characterized PAMPs is flagellin from gram-negative bacteria that is present in both animal and plant pathogenic bacteria. In Pseudomonas syringae, flg22, a highly-conserved peptide from the N-terminal part of flagellin, is a strong elicitor of defense in tomato and Arabidopsis (Felix, et al. 1999, Gomez-Gomez and Boller 2000); however, there are some examples such as in Agrobacterium tumefaciens and Ralstonia solanacearum where these bacteria have changed their flagellins in order to avoid recognition by corresponding PRRs (Felix, et al. 1999, Pfund, et al. 2004). Sun and co-workers also reported that isolates of Xanthomonas campestris pv campestris (Xcc) which causes black rot disease on crucifers, also show a high degree of variation in their flagellin sequences and that some flagellin variants had less elicitor activity than others without affecting the motility of bacteria (Sun, et al. 2006). 5  Lipopolysaccharides (LPS) from the cell wall of gram-negative bacteria are recognized by host plants as PAMPs and elicit the defense response (Dow, et al. 2000, Erbs and Newman 2003, Meyer, et al. 2001). Pretreating tobacco plant with LPS prevented the HR and accelerated the production of SA and subsequent resistance to both host and non-host pathogens (Graham, et al. 1977, Newman, et al. 1997, Newman, et al. 2002); however, symbiotic microorganisms need to avoid this recognition in order to overcome the plant defense for colonization of their host. The LPS from Sinorhizobium meliloti is actually able to suppress the elicitor-triggered defense reaction instead of inducing it by blocking ROS production in cell suspension cultures from its symbiotic host Medicago truncatula (Albus, et al. 2001) while it induces ROS production in cell suspension cultures from nonhost tobacco; this shows that this suppression is a very specific effect of this LPS for launching a symbiotic relationship (Albus, et al. 2001). Tellstrom et al. (2007) showed that S. meliloti LPS can suppress the expression of PR genes triggered by yeast invertase by comparing PR gene expression in two treatments, one with LPS and invertase, and one with invertase alone. The variation in LPS is proposed to be responsible for this difference between suppression and triggering of PTI leading to symbiosis and pathogenicity, respectively (Gohre and Robatzek 2008).  1.1.4. Types of resistance in plants Resistance in plants is of three different types; Race-specific, race nonspecific and nonhost resistance. Race-specific resistance is qualitative, usually controlled by dominant avirulence or Avr genes in the pathogen and controlled by one or a few dominant resistance or R genes in a specific plant genotype or cultivar of an otherwise susceptible host species. This resistance is of the “gene-for-gene interaction” type and will be discussed in section 1.3 in detail. Race nonspecific resistance is controlled by many genes and is known as general, quantitative, or partial resistance and is generally durable. This type of resistance depends on pre-existing and induced structural and biochemical defenses provided by dozens or perhaps hundreds of defenseassociated genes and the possible ability of pathogenicity-related (PR) proteins to induce pathogens to release molecules that elicit host defenses. Nonhost resistance means the resistance of an entire plant species against a specific pathogen and is the most common and durable form of disease resistance exhibited by plants (Heath 2000). Nonhost resistance is the result of both preformed and inducible defense mechanisms (Heath 2000, Mysore and Ryu 2004), seems to be 6  under complex genetic control, and can involve multiple defense factors that individually may segregate within host species without compromising overall resistance (Heath 2000). Both nonhost and host resistance sometimes show common components, including deposition of physical barriers, production of reactive oxygen species (ROS), accumulation of antimicrobial compounds, and the HR (Able 2003, Huckelhoven 2007, Zhao, et al. 2005). It is still not clear whether the signal transduction pathways are similar in race specific and nonhost resistance (Mysore and Ryu 2004). Several signaling components such as ethylene and SA which are important in host resistance to activate the plant defense system also play an important role in nonhost resistance. Transgenic tobacco plants expressing the Arabidopsis etr11 gene responsible for loss of ethylene perception, become susceptible to various nonhost pathogens (Knoester, et al. 1998). Similarly, sid2 mutant Arabidopsis plants, which are defective in salicylic acid synthesis, and Arabidopsis plants expressing NahG which encodes salicylate dehdroxylase that degrade SA, become susceptible to the cow pea rust fungus Uromyces vigna which is a nonhost pathogen for Arabidopsis (Mellersh and Heath 2001). Some nonhost resistance genes, such as NHO1 and EDS1 in Arabidopsis, are involved in nonhost resistance and disruption of theses genes results in the loss of nonhost resistance against nonhost pathogens (Kang, et al. 2003, Parker, et al. 1996), while mutations in Pen1, Pen2 and Pen3 disrupt the ability of plants to arrest penetration of both host and nonhost pathogens (Collins, et al. 2003, Stein, et al. 2006, Thordal-Christensen 2003). The nonhost resistance in plants against bacteria, oomycetes, and fungi is of two types; type I and type II (Mysore and Ryu 2004). Type I nonhost resistance is a more basal defense, does not produce an HR or any visible symptoms, and usually arrests the penetration and multiplication of the pathogen in the plant cells (Mysore and Ryu 2004). Type II nonhost resistance is the most common type of nonhost resistance and produces a visible HR at infection sites. Type II nonhost resistance looks phenotypically more similar to a “gene-for-gene based” incompatible interaction which is associated with a HR as a result of recognition of pathogen elicitor molecules. It is hypothesized that type II nonhost resistance may be due to the production of two or more avirulence proteins in the pathogen species which are recognized by all genotypes of a particular plant species and will, therefore, remain a nonhost pathogen (Collins, et al. 2003). For example, a functional type three secretion system (TTSS) is required for bacteria to deliver effectors (which often are avirulence gene products; see next section) into plants and  7  cause the HR on nonhost plants (Alfano and Collmer 1996). Similarly, INF1, an avirulence factor secreted by Phytophthora infestans, is required for producing a HR in nonhost plants such as Nicotiana benthamiana (Kamoun, et al. 1998). The type of nonhost resistance produced in nonhost plants depends not only on the pathogen species but also on the plant species. There are several examples in which the same pathogen species produces a type I nonhost resistance on one nonhost plant species and a type II nonhost resistance on another nonhost plant species (reviwed by Klement, et al. 1999, Mysore and Ryu 2004). Similarly, the same plant species can produce a Type I nonhost resistance to one nonhost pathogen and a Type II nonhost resistance to another nonhost pathogen (Lu, et al. 2001, Thomma, et al. 1999).  1.2. Effectors in plant-microbe interactions Successful pathogens secrete a wide range of so-called “effector” molecules widely believed to function in suppressing host defense responses at multiple levels and/or in evading basal immunity that otherwise would be sufficient to stop infection. Effectors are small molecules and proteins produced by pathogens that can modify host-cells structures or functions, either by contributing to diseases progression (virulence factors and toxins) or induce defense responses (avirulence factors and elicitors) or both (Hogenhout, et al. 2009, Huitema, et al. 2004, Kamoun 2006). Effectors are also thought to have roles in establishing feeding interactions and/or nutrient leakage from the host to the benefit of the pathogens. Phytopathogen effectors are the products of pathogen genes that function inside the plant cells or at the interface of pathogen and host plants (Kamoun 2006, Kamoun 2007). Individual pathogens genomes encode dozens of secreted effectors that are targeted to the host plant apoplast or cytoplasm (Cunnac, et al. 2009, Jiang, et al. 2008, Kamoun 2006, Lindeberg, et al. 2009a, Lindeberg, et al. 2009b, Tyler, et al. 2006). Effectors from several groups of cellular phytopathogens such as bacteria, oomycetes, fungi and nematodes can enter plant cells (Chisholm, et al. 2006, Huang, et al. 2003, Kamoun 2007). Bacteria use the Type II, Type III and Type IV secretion systems to deliver many effector proteins to the host plant (Cunnac, et al. 2009, Lindeberg, et al. 2006). Oomycetes and fungi likely secrete even more effectors than bacteria; many genomes from phytopathogenic oomycetes and fungi have been sequenced and hundreds of potential effectors have been identified using bioinformatics (Haas, et al. 2009, Jiang, et al. 2008, Kamper, et al. 2006, Tyler,  8  et al. 2006, Mueller, et al. 2008, Schirawski, et al. 2010). Predicted oomycete effectors, in addition to having an N-terminal signal peptide (SP), carry a host targeting signal (HTS) next to the SP that contains a conserved RXLR and a DEER motif that can target them to the host cell in the absence of the pathogen (Dou, et al. 2008b, Haas, et al. 2009, Jiang, et al. 2008, Kale, et al. 2010, Rehmany, et al. 2005, Tyler, et al. 2006, Whisson, et al. 2007). The predicted sets of effectors from fungi have an N-terminal SP and, in some cases, an RXLR-like motif (Dean, et al. 2005, Kamper, et al. 2006, Kale, et al. 2010, Godfrey, et al. 2010, Schirawski, et al. 2010). Several examples exist where predicted effectors with a SP are shown to have been delivered to the host cytoplasm (Catanzariti, et al. 2006, Gan, et al. 2010a, Kemen, et al. 2005, Khang, et al. 2010). Recently, Kale et al. (2010) showed that at least three effectors; AVRL567 from Melampsora lini, AVRLm6 from Leptosporia maculans, and AVR2 from Fusarium oxysporum f. sp. lycopersi (Fol) contain a RXLR-like motif and can enter plant cells without the presence of the pathogen. They also showed that the conserved RXLR motif from oomycetes and the RXLRlike motif from other fungi bind specifically to phospholipids, in particular phospahatidylinositol-3-phosphate (PI3P) on the surface of the plasmamembrane and enter the cell through lipid raft-mediated endocytosis (Kale, et al. 2010). Godfrey et al. (2010) recently showed that small secreted protein from haustoria-forming fungal pathogens have Y/F/WXC motif in addition to N-terminal secretion signal. There may be a motif equivalent to RXLR in the effectors from other fungal pathogens that could target them to the host cytoplasm.  1.2.1. Role of effectors in infection Secreted effectors help the phytopathogens to colonize the host plant at multiple levels. They may facilitate entry into the host, acquisition of nutrients from the host, stop recognition of PAMPs, prevent phytotoxin production, inactivate the plant defense enzymes, or target the PRRs to interfere with the downstream signaling and defense gene expression. Since bacteria have smaller genomes and are normally easier to manipulate, many more plant-pathogenic bacteria have been studied in great detail, as compared to fungal pathogens however, the number of fungal genomes being generated or already available is growing, which is quickly stimulating studies on fungal and oomycete effectors.  9  1.2.1a. Breaking the physical barrier After landing on the surface of the host plant, the pathogen either enters the host plant through natural openings (stomata and hydathode) or penetrates the plant surface tissue directly in order to overcome the physical barrier. Bacteria proliferate on the surface of the host plants and use natural openings or wounds to enter the apoplast for colonization. The opening and closing of stomata is highly controlled in plants, by a complex hormone system. Stomata are open during photosynthesis in most plants to allow proper exchange of gasses and phytopathogenic bacteria profit from this opportunity to move towards stomata by sensing compounds released during photosynthesis. The PAMP recognition receptor (PRR) of the guard cells can sense PAMPs such as bacterial flagellin or lipopolysaccharides or lipooligosaccharides from bacteria, which induce the closure of stomata systemically (Gohre and Robatzek 2008). Phytopathogenic bacteria such as P. syringae produce coronatine, a phytotoxin that mimics jasmonic acid (JA) to interfere with SA and abscisic acid (ABA) signaling for reopening stomata so that pathogenic bacteria can gain access to the host apoplast (Melotto, et al. 2006). It is not yet known whether MAMP associated defense pathways also close stomata to eukaryotic pathogens and whether these pathogens use effectors in a similar way to overcome this hurdle. After getting into the plant apoplast, the next physical barrier to the phytopathogen is the plant cell wall which prevents them from obtaining nutrients. To promote nutrient leakage from the cytosol into the apoplast, bacteria use a Type II secretion system to secret lytic enzymes that degrade the cell wall locally. In the generated open channel, a component of the Type III secretion system, a nano-scale injection structure, is then formed which penetrates the cell wall and cell membrane (Gohre and Robatzek 2008). At the same time, these bacteria secrete effector molecules to suppress the host defense that is activated by danger associated molecular patterns (DAMPs) from the degrading cell wall molecules (Jha, et al. 2007). Powdery mildew fungi, such as B. graminis f. sp. hordei penetrate the cuticle of plant cells directly through a special penetration structure, called the appressorium. It has been shown that B. graminis f. sp. hordei secreted effectors AVRa10 and AVRk1 increase the penetration efficiency on susceptible barley cultivars but the exact mechanism is not yet known (Ridout, et al. 2006).  10  1.2.1b. Disarming plant defense enyzymes Plants produce antimicrobial enzymes like proteases, hydrolases, glucanases, and chitinases that can degrade the cell wall of invading pathogenic fungi in the apoplast without detrimental effects to the plant (Lucas 1998). This has a dual role, the degradation of cell walls can attenuate fungal growth on the one hand, while on the other hand, the molecules released from degraded cell walls serve as elicitors for inducing plant defense. Pathogens use effector molecules either to stop the delivery of these antimicrobial enzymes and compounds by preventing their secretion or by inhibiting their activity after they are secreted (Bent and Mackey 2007). The apoplastic fungus Cladosporium fulvum secretes AVR2, a cysteine protease inhibitor, during infection that binds directly to RCR3, a tomato cysteine protease, to protect the fungus from the deleterious effect of the enzyme. AVR2 also promotes virulence for other fungal pathogens that cause disease in tomato, such as Verticillium dahliae and Botrytis cinerea, when expressed heterologously in Arabidopsis (van Esse, et al. 2008). Similarly, effector AVR4, a chitin binding lectin from C. fulvum, binds to chitin of fungal cell walls to protect it from chitinases of the host plant, tomato (van den Burg, et al. 2006). AVR4 can also protect chitin against plant chitinases in the cell wall of other fungi, such as Trichoderma viride and Fusarium solani f. sp. phaseoli (van den Burg, et al. 2006). In this way, AVR4 not only protects the fungi from hydrolysis by plant chitinases but also keeps chitin fragments from eliciting PTI (Libault, et al. 2007). ECP6 from C. fulvum has a Lys-M domain that binds to carbohydrates including chitin and protects the pathogen from plant chitinases or may be involved in scavenging of chitin fragments that are released during cell wall degradation by plant chitinases, thus, preventing them from inducing PTI (Bolton, et al. 2008). The oomycete pathogen, P. infestans, is known to secrete a suite of Cysteine and Kazal family protease inhibitors (Tian and Kamoun 2005, Tian, et al. 2007, van Esse, et al. 2008). The tomato papain-like protease, PIPI, which is induced by SA, is blocked by the EPIC2B inhibitor of P. infestans (Tian and Kamoun 2005, Tian, et al. 2007, van Esse, et al. 2008). PIP1 is related to RCR3, and a new report shows that AVR2 from C. fulvum can inhibit PIP1 and two other cystein protease, aleurain and TDI65, in plants (Rooney, et al. 2005, Shabab, et al. 2008, van Esse, et al. 2008). Also EPIC1 and EPIC2B from P. infestans can bind and inhibit RCR3, similar to AVR2, but unlike AVR2, these EPICs do not elicit an HR on Cf-2/Rcr3pimp tomato plants suggesting that P. infestans evolved stealthy effectors that can inhibit tomato proteases without  11  activating defense responses (Song, et al. 2009). These findings show that effectors from different pathogens can target the same apoplastic enzymes to increase pathogen fitness in the host (Shabab, et al. 2008). Other effectors, like EPI1 and EPI10 from P. infestans, target the subtilisin-like serine protease of tomato P69B, a PR protein (Tian, et al. 2004, Tian and Kamoun 2005, Tian, et al. 2007). AVRP123, a secreted protein from M. lini, the flax rust fungus, also shows similarity to Kazal serine protease inhibitors (Catanzariti, et al. 2006). The soybean pathogen, Phytophthora sojae, secretes glucanase inhibitor proteins, GIP1 and GIP2, that target the endo-β-1,3-glucanase-A of the host plant to protect the pathogen during infection and also to prevent PTI induced by oligoglucoside (Rose, et al. 2002). Bacteria also secrete plant cell walldegrading enzymes locally in order to construct the Type III secretion system and use effectors such as HOPP1 to suppress defense induced by DAMPs in the N. benthamiana apoplast (Gust, et al. 2007, Oh, et al. 2007). HOPP1 either sequesters or processes the fragments that are produced during cell wall degradation so that they cannot function as PAMPs (Gohre and Robatzek 2008). These examples of disarming the apoplastic enzymes from plants by different classes of pathogen (fungi, oomycets and bacteria) show the importance of effectors in preventing PTI and promoting pathogen fitness in the host. 1.2.1c. Suppression of receptor activation The recognition of PAMPs by PRRs of plants leads to the activation of defense responses against the pathogen by triggering a cascade of defense signaling. It was proposed in the beginning that resistance induced by PRRs is only basic and not as strong as that induced by resistance genes, but it is clear now, at least in the case of FLS2 (the flagellin receptor), that the contribution of PRRs towards overall resistance is huge (de Torres, et al. 2006, Hann and Rathjen 2007, He, et al. 2006). PRRs make significant contributions to resistance in nonhost pathogens. Bacterial strains mutated in the Type III secretion system show that suppression of PTI by effectors is important for full virulence in these pathogens (Hauck, et al. 2003, Kim, et al. 2005). Several effectors, particularly the Type III secretion system effectors of phytopathogenic bacteria, target these PRRs directly to jam all the downstream resistance responses (Blocker, et al. 2008, Jamir, et al. 2004, Jones and Dangl 2006). AVRPTO and AVRPTOB are two unrelated Type III effectors of P. syringae that inhibit FLS2 recognition, upstream of MAPKKK signaling in the plant (He, et al. 2006). Several PTI responses such as the production of ROS, MAPK cascade  12  induction, elicitation of PR genes, and callose accumulation are prevented by the action of AVRPTO when it is localized in the membrane (He, et al. 2006). The AVRPTO and AVRPTOB target the resistance gene product, PTO, in tomato, a protein which is guarded by resistance gene product PRF and induces an HR (Kim, et al. 2002). The Arabidopsis genome did not contain an ortholog of PTO but the kinase domains of FLS2, BAK1, and EFR show significant homology to PTO (Chinchilla, et al. 2007, Kim, et al. 2002). It has been shown by structural modeling that the kinase domain of PTO is similar to FLS2 and EFR (Xiang, et al. 2008). AVRPTO interacts with FLS2 and EFR both in vitro and in protoplast, which shows that it targets multiple PRRs and most likely the same, may be true for AVRPTOB (Xiang, et al. 2008). The C-terminus of AVRPTOB carries a functionally active E3 ligase activity that ubiquitinates FEN kinase, a member of the PTO family, for degradation; that activity can then prevent an HR in tomato plants lacking PTO (Janjusevic, et al. 2006, Rosebrock, et al. 2007). AVRPTO and AVRPTOB can also suppress nonhost HR in N. benthamiana induced by FLG22 or INF1 of P. infestans (Hann and Rathjen 2007). DSPA/E that is related to the AVRE family of the apple fire blight pathogen, Erwinia amylovora, interacts directly with four apple proteins that are putative receptor-like kinases (RLKs; (Meng, et al. 2006). Several oomycete RXLR effectors can suppress host cell immunity in a similar manner, as do the bacterial Type III secretion system effectors. AVR3A from P. infestans can block an HR induced by INF1 (Bos, et al. 2009). Similarly, AVR1B from P. sojae also suppresses programmed cell death triggered by the mouse protein, BAX, in plants and yeast (Dou, et al. 2008a). Several alleles of ATR1 and ATR13 from Hyaloperonospora arabidopsidis increased the virulence of P. syringe DC3000 on susceptible Arabidopsis thaliana when these effectors were delivered by the P. syringae Type III secretion system (Sohn, et al. 2007). ATR13 targets PTI by suppressing callose accumulation and ROS production in susceptible A. thaliana (Sohn, et al. 2007). 1.2.1d. Suppression of R gene-triggered resistance Phytopathogenic fungi like Fusarium oxysporum f.sp. lycopersici can avoid host defense by evolving effectors that can suppress R gene-triggered resistance (Houterman, et al. 2008). AVR3 and AVR2 from F. oxysporum f.sp. lycopersici are required for full virulence on tomato plants but they are also recognized by tomato lines that have resistance genes I-3 or I-2, respectively, that trigger an HR and cause arrest of the pathogen (Huang and Lindhout 1997, Rep, et al. 2005,  13  Rep, et al. 2004). In contrast, AVR1 is a small cysteine-rich secreted protein from F. oxysporum f.sp. lycopersici that is recognized by resistance gene I or I-1 but is not required for virulence. Tellingly, AVR3 is present in all F. oxysporum f.sp. lycopersici strains analysed, while Avr1 is present only in F. oxysporum f.sp. lycopersici strains that are virulent on I-3 lines. Houterman et al. (2008) showed that AVR1 actually suppresses the resistance triggered by I-2 and I-3, as the transformation of Avr1 to F. oxysporum f.sp. lycopersici strains that were avirulent on I-2 or I-3 became virulent on these lines. It is proposed that F. oxysporum f.sp. lycopersici l strains acquired Avr1 as a mechanism of partial functional redundancy, so that they can avoid the consequences of losing Avr3 and probably Avr2 that are required for full virulence (Stergiopoulos and de Wit 2009). In the flax rust fungus, M. lini, the interaction of AvrL567 in strain CH5-89 with the flax cultivar Barnes that contains the L7 gene is inhibited by an inhibitor gene and thus results in a lower virulence reaction (Lawrence, et al. 2010). 1.2.1e. Down-regulation of defense signaling As already mentioned, the perception of a PAMP by a PRR triggers a cascade of defense signaling including MAPK cascades. In Arabidopsis, the recognition of FLG22 by FLS2 induces downstream signaling through a MAPK cascade (Asai, et al. 2002, Gomez-Gomez and Boller 2000). Bacterial Type III secretion system effectors use different mechanisms to dephosphorylate the MAP kinase signaling components in order to suppress the defense response (Gohre and Robatzek 2008). HOPAI1 encodes an enzyme, phosphothreonine lyase, that removes a phosphate group from phosphothreonine residues of MAPKs to stop phosphorylation, which in turn blocks FLG22 signaling at early stages (Zhang, et al. 2007). Protein pull-down assay showed that HOPAI1 directly interacts with both MAP3 and MAP6 kinases (Zhang, et al. 2007). The inactivation of MAPK by HOPAI1 also stops downstream defense responses, such as the production of ROS and the expression of PR genes (Zhang, et al. 2007). HOPAO1, another Type III secretion system effector, has a protein tyrosine phosphatase motif and posseses this enzymatic activity in vitro (Bretz, et al. 2003, Espinosa, et al. 2003). The transient expression of HOPAO1 in N. tabacum suppresses an HR induced by the constitutively active MAPKK, NtMEK2, in N. tabacum (Espinosa, et al. 2003). The heterologous expression of HOPAO1 in Arabidopsis also suppresses PAMP-induces ROS production, callose deposition, and thereby enhances virulence and multiplication of P. syringae pv tomato (Pst) DC3000 hrpA mutants  14  (Underwood, et al. 2007). In that study, transgenic Arabidopsis plants expressing a catalytically inactive derivative did not show these phenotypes, which argues that the phosphatase activity is required for HOPAO1 function (Underwood, et al. 2007). 1.2.1f. Alteration of the plant defense transcriptome The perception of PAMPs such as flagellin changes the expression of at least 1,000 genes in Arabidopsis (Zipfel, et al. 2004). As discussed, bacterial Type III secretion system effectors obstruct the defense signaling on the one hand, while on the other hand, they target the plant transcriptome directly. This can be achieved by different mechanisms. Several effectors change RNA stability. For example, HOPU1, a mono-ADP-ribosyltransferase, acts on glycine-rich RNA-binding proteins such as AtGRP7 and AtGRP8 which are RNA chaperones (Fu, et al. 2007); thus, HOPUI changes the plant transcriptome by reducing transcripts and the expression of defense response genes. Arabidopsis grp7 mutants promote growth of P. syringae pv tomato DC3000 compared to the wild type (Fu, et al. 2007). Besides HOPUI, HOPO1-1 and HOPO1-2 also encode ADP-RTs. Some pathogen effectors act as transcription factors and can induce the expression of host genes for their own benefits. For example, the AVRBS3 family of effectors from X. campestris pv vesicatoria has plant nuclear localization signals and binds to an “upa-box” (upregulated by AVRBS3) that is found in the promoter of Upa20, a master regulator of cell size inducing hypertrophy, and several other host genes, ensuring proper nutrient supply for pathogen multiplication (Gurlebeck, et al. 2006, Kay, et al. 2007, Szurek, et al. 2002); however, in resistant plant cultivars, the promoter of Bs3 also carries an “upa-box” and, thus, binding of the ABRBS3 effecor induced transcription of Bs3 and cell death (Kay, et al. 2007). This shows that under selection pressure, plants can evolve to recognize effectors and use them for their own defense. 1.2.1g. Destruction of antimicrobial compounds Microbial effectors also attack and degrade antimicrobial compounds and proteins that play roles in PAMP perception, defense signaling, or any other defense reaction. This degradation is achieved either by protease action on the plant substrate or by exploiting the plant protein degradation machinery (Zeng, et al. 2006). SUMO (small ubiquitin-like modifier) modification  15  or sumoylation controls several processes in plants like pathogen infection, abiotic stress, hormone signaling, and flowering time (Hanania, et al. 1999, Kurepa, et al. 2003, Lois, et al. 2003, Murtas, et al. 2003). It was recently discovered that several bacterial Type III secretion system effectors such as YOPJ, XOPD, YOPT, AVRXV4, AVRPPHB and AVRRPT2 possesses cysteine protease functions, suggesting that the degradation of host proteins is an important approach to subvert plant defenses (Hotson and Mudgett 2004). YOPJ and XOPD effectors have deSUMOylating activities and XOPD accumulates in the subnuclear foci, revealing its function in deSUMOylation of transcription factors (Hotson, et al. 2003, Hotson and Mudgett 2004). The YOPT protease has a cytotoxic effect that destroys the actin cytoskeleton during the entry into the cell, resulting in severe cell damage (Shao and Dixon 2003). AVRPPHB effector cleaves itself before entering into host plant by the TTSS and is fatty acylated to target PBS1 cleavage (Shao, et al. 2002). In resistant plants, the cleavage and PBS1 kinase activities are required for RPS5 resistance (Shao, et al. 2002). It has also been suggested that AVRRPT2 functions as a cysteine protease in planta (Hotson and Mudgett 2004). In Arabidopsis plants, AVRRPT2 degrades RIN4 which in turn activates RPS2 to develop resistance to the pathogen (Mudgett and Staskawicz 1999). Besides AVRRPT2, two other P. syringae effectors, AVRRPM1 and AVRB, independently target RIN4 and induce its phosphorylation, which in turn induces RPM1 resistance (Mackey, et al. 2002). As AVRRPT2 degrades RIN4, AVRRPM1 and AVRB cannot activate RPM1 in the presence of AVRRPT2 (Axtell and Staskawicz 2003, Mackey, et al. 2003). It is proposed that RIN4 might be acting as an adapter for holding multiple PRR signaling pathways under negative control (Gohre and Robatzek 2008). TTSS effectors also exploit the proteasome degradation machinery of host plants for degrading host defense related proteins (Angot, et al. 2007). AVRPTOB is a modular protein that contains an N-terminal domain that induces the HR by interacting with PTO/PRF in a gene-forgene manner (Abramovitch, et al. 2003, Jamir, et al. 2004). The C-terminal domain of AVRPTOB is capable of suppressing the HR triggered by AVRPTO/PTO recognition in N. benthamiana and the HR induced by other bacterial Type III secretion system effectors, fungal specific HR inducing proteins, and even pre-apoptotic mouse BAX protein (Abramovitch, et al. 2003, Jamir, et al. 2004). C-terminal domain of AVRPTOB carries an E3-ligase activity and is capable of auto-ubiquitination and possibly of ubiquitination of plant substrates (Abramovitch, et al. 2006, Abramovitch, et al. 2003, Janjusevic, et al. 2006). MAMPs from several bacterial and  16  fungal phytopathogens induce cell wall-based responses. Papilla formation, which is a localized cell wall thickening mainly by addition of callose near pathogen penetration sites, is induced by a variety of phytopathogens (reviewed by Bent and Mackey 2007). During papilla production, cellular vesicles deliver cell wall reinforcement and antimicrobial compound to the site of pathogen penetration (Robatzek, et al. 2006). Several effectors can act on proteins involved in this transportation and redirect their cargo to suppress MAMP-induced callose deposition. HOPM1, a Type III secretion system effector from P. syringae, is required for virulence and able to suppress host cell wall-associated defense (DebRoy, et al. 2004). HOPM1 manipulates the plant ubiquitination system to alter vesicle trafficking. It interacts specifically with AtMIN7, one of the Arabidopsis ARF-GEFs (adenosine diphosphate ribosylation factor guanine nucleotide exchange factor) that is involved in vesicle trafficking (Nomura, et al. 2006). The interaction of HOPM1 with AtMIN7 mediates proteasome-based degradation of AtMIN7 and, thus, callose deposition is prevented (Nomura, et al. 2006). HOPM1 by itself did not have any classical E3ubiquitin ligase features, leading to the hypothesis that HOPM1 may act as an adapter protein mediating the detection of AtMIN7 by the plant ubiquitin/26S proteasome system (Angot, et al. 2007). Similarly, AVRPTO also suppresses cell wall-based defenses independent of SA signaling (Hauck, et al. 2003). It is possible that AVRPTO interferes with vesicle trafficking in a similar manner as HOPM1, as a yeast two-hybrid assay showed that it interacts with two RabGTPases (Hauck, et al. 2003). 1.2.1h. Killing of host cells Necrotrophic phytopathogens produce several phytotoxins in addition to cell wall hydrolyzing enzymes in order to kill the host tissue for colonization during infection. Some necrotrophic fungi produce proteinaceous effectors, also called host selective toxins (HST), that are required for infection on susceptible host plants that have the corresponding dominant receptor gene (Wolpert, et al. 2002). This represents a situation opposite of the classical gene-for-gene interaction in which a dominant gene is required for disease resistance rather than pathogenicity (Wolpert, et al. 2002). Two wheat necrotrophs, Stagonospora nodorum and Pyrenopora triticirepentis, produce several host-specific peptide effectors, such as PTRTOXA, SNTOX1, SNTOX2, and SNTOX4, that are recognized by their corresponding dominant susceptibility genes in wheat (TsN1, Snn1, Snn2 and Snn4) to causes disease (Abeysekara, et al. 2009, Friesen,  17  et al. 2007, Liu, et al. 2006, Liu, et al. 2004, de Wit, et al. 2009, Manning, et al. 2009). PTRTOXA, the best-studied effector from P. tritici-repentis has an N-terminal secretion signal, followed by an RGD domain for host targeting and a C-terminal domain with effector function (Manning, et al. 2007, Sarma, et al. 2005). After entering the host cell, PTRTOXA was reported to enter the chloroplast and interfere with TOXABP1 function of the chloroplast, thereby affecting photosystem I and II function in a light-dependent manner (Manning, et al. 2007). PTRTOXA is an ortholog to SNTOX1 from S. nodurum and both effectors are recognized by the same wheat susceptibility gene, Tsn1 (Liu, et al. 2006). Some phytopathogenic bacteria, oomycetes, and fungi produce NEP1-like proteins (NLPs) that are toxic to only dicotyledonous plants, possibly by the different molecular compositions of dicot and monocot cell membranes (Gijzen and Nurnberger 2006, Ottmann, et al. 2009, Pemberton and Salmond 2004). The common heptapeptide motif, GHRHDWE, and two conserved cysteine residues make it structurally similar to actinoporins, cytolytic toxins from marine organisms (Gijzen and Nurnberger 2006, Ottmann, et al. 2009). Hemibiotrophs, such as P. infestans and P. sojae, produce NLPs such as NPP1 and PiNPP1, in the late necrotrophic phase which could contribute to disease development with their cytolytic activities (Kanneganti, et al. 2006, Qutob, et al. 2002). 1.2.1i. Suppression of host defense by symbionts Symbiotic microorganisms also secrete effectors into host plants to suppress host defense responses, a condition essential for their lifestyle. Many rhizobial strains use the Type III secretion system to deliver effectors into host cells in a similar way that pathogenic bacteria suppress host defense responses (Deakin and Broughton 2009). The relationship between symbiotic bacteria and their host plants is very specific, which is determined by molecular signals recognized by the two organisms. For example, each rhizobial strain can establish symbiosis with only limited species of host plants (Perret, et al. 2000, Yang, et al. 2010). The host range is defined by bacterial recognition of flavonoid compounds secreted by the host, which induce the NOD factors and the perception of these NOD factors by the host plants receptors (Geurts, et al. 1997, Limpens, et al. 2003, Radutoiu, et al. 2003, Radutoiu, et al. 2007). The transformation of NFR1 and NFR5 Nod-factor receptors from Lotus japanicus into M. truncatula make it a host for Mesorhizobium loti that usually has a symbiotic relationship only  18  with L. japanicus (Radutoiu, et al. 2003, Radutoiu, et al. 2007). LPSs from phytopathogenic bacteria are recognized by plants as elicitors of PTI, while symbiotic bacteria such as rhizobia use these LPSs to suppress host defenses and assist in making infection threads and inducing nodule growth (D'Haeze and Holsters 2004, Jones, et al. 2008). It was reported a few decades ago that dominant R genes present in plants regulate symbiotic relationships with particular rhizobial strains which is similar to a gene-for-gene interaction in plant-pathogen interaction (Caldwell 1966, Devine and Kuykendall 1996). Two R genes, Rj2 and Rfg1 have been cloned from soybean, which restrict symbiotic relationships between Bradyrhizbium japanicum and Sinorhizobium fredii in a gene-for-gene manner (Yang, et al. 2010). In the fungal kingdom, the genome of Laccaria bicolor, an ectomycorrhizal fungus, revealed more than 3,000 predicted secreted proteins of which 10% are small secreted proteins (SSPs) of the effector-type (Martin, et al. 2008, Martin and Selosse 2008). Some of these SSPs are homologous to rust fungus “haustoria-expressed secreted proteins” (HESPs) and are differentially expressed during infection, suggesting a possible role during infection and evading the host defense (de Wit, et al. 2009).  1.3. Gene-for-gene or R and Avr interaction Adapted pathogens secrete effector molecules into the host plant to surmount physical barriers, neutralize preformed antimicrobial compounds, and overcome PTI. As a result, during coevolution of plant host and adapted pathogen, plants have developed very sophisticated recognition systems, usually encoded by R genes to recognize these effectors or their action and trigger defense responses; this induced resistance has recently been called effector-triggered immunity (ETI) and leads to a rapid and enhanced defense response in the host plant often including HR (Shirasu and Schulze-Lefert 2000). These effectors are called avirulence (Avr) factors since they activate the plant defense system and make the pathogen unable to cause disease when the plant has the corresponding R gene (Jones and Dangl 2006). This genetically superimposed R and Avr interaction is called “gene-for-gene” resistance, or host/cultivar-level resistance, as particular cultivars of the host with a certain R gene product recognize an Avr gene product from a particular race of the pathogen. When a pathogen cannot cause disease in plants, the interaction is incompatible, the pathogen is avirulent, and the plant is resistant. The  19  hypothesis of “gene-for-gene” resistance, first proposed by Flor (1942), states that for every dominant Avr gene in the pathogen, there is a corresponding dominant R gene in the resistant host. The (genetic) interaction between these gene products leads to activation of defense responses in the host and suppresses pathogen growth (Flor 1942). Some Avr genes encode effectors molecules that suppress host defense and, thus, act as virulence factors when the plant does not have the corresponding resistance gene. ETI represents the qualitative, secondary layer of resistance and leads to an evolutionary arms race between the pathogen and the plant in which the pathogen either mutates or loses the effectors to avoid recognition by the host or develops new effectors to suppress ETI, while the plant develops new R genes to recognize the mutants or new effectors (de Wit 2007, Bent and Mackey 2007, Jones and Dangl 2006). The cloning and characterization of various Avr and R genes in the past three decades has increased our knowledge of the biochemical and molecular basis of the gene-for-gene interaction (Hammond-Kosack and Parker 2003). The first Avr gene was cloned from a bacterium in 1984 (Staskawicz, et al. 1984), which was followed by the cloning and characterization of more than 40 bacterial Avr genes in the following decades (Mudgett 2005, Van't Slot and Knogge 2002). The cloning and characterization of Avr genes from fungi and oomycetes lagged behind because of these organisms’ larger genome sizes and sometimes inefficient transformation systems. The first Avr gene from a fungus was isolated from C. fulvum in 1991 (van Kan, et al. 1991) and the first oomycete Avr gene was cloned from P. sojae and P. infestans in 2004 (Shan, et al. 2004). Fungal Avr genes are mainly isolated from ascomycetes, such as C. fulvum, Rynchosporium secalis, B. graminis, Magnaporthe oryzae, F. oxysporum, and L. maculans, but a handful have been reported from a basidiomycete, the flax rust fungus, M. lini (Catanzariti, et al. 2006). Several more oomycete Avr genes have since been isolated from H. arabidopsidis, P. sojae, and P. infestans (Table 1.1). The sequencing of the complete genomes of many bacterial, fungal, and oomycete plant pathogens has revealed a myriad of effectors, many of which are potential avirulence genes that can trigger ETI. Avr genes isolated and characterized to date differ among one another both in sequence and function, and those from different plant pathogens do not seem to share many common features (Agrios 2005). More than 40 bacterial Avr genes have been cloned and sequenced primarily from Pseudomonas and Xanthomonas species and most of them share little or no homology to each other with few exceptions, such as in the avrbBs3 and avrRxv/yopJ families  20  (Deslandes, et al. 2003, Lahaye and Bonas 2001). The AVRBS3-like proteins from all Xanthomonas share 90-97% sequence identity in a central region of nearly identical 34-amino acid repeats (Lahaye and Bonas 2001). Besides the repeat domain, all members of the AVRBS3 family contain nuclear localization signals (NLS) and an acidic transcriptional activation domain. Bioinformatic analyses showed that all family members of AVRRXV/YOPJ have common invariant residues in a putative protease catalytic site (Orth, et al. 2000). All of the fungal and oomycete Avr genes characterized to date encode small proteins (28-311 amino acids), except the ACE1 of M. grisea which has 4,034 amino acids, and all have a secretion signal/protein transport motif at the N-termini (Ellis, et al. 2006); however, it has been shows that AVRa10 and AVRk1 from B. graminis f. sp. hordei do not have secretion signals like other fungal AVR proteins (Ridout, et al. 2006). In spite of the absence of secretory signals, these AVR proteins are recognized by intracellular barley resistance proteins when expressed transiently in planta. It has been suggested that AVRa10 and AVRk1 may be secreted from the fungus by nonendomembranous pathways (Ridout, et al. 2006).  1.3.1 Fungal Avr genes Several Avr genes have been cloned and characterized from extracellular fungi such as C. fulvum, F. oxysporum f. sp. lycopersici, R. secalis, M. oryzae and L. maculans and from obligate biotrophic fungal pathogens that produce haustorial feeding structures within cells, such as B. graminis and M. lini (Table 1.1). 1.3.1a. Cladosporium fulvum C. fulvum is an apoplastic fungal pathogen of the ascomycete subgroup that reproduces asexually and causes leaf mold of tomato (de Wit, et al. 1997, Joosten and de Wit 1999, Thomma, et al. 2005). Four avirulence genes have been cloned: Avr2, Avr4, Avr4E and Avr9, which all encode small secreted cystein-rich effector proteins that are recognized in tomato by CF2, CF4, CF4E and CF9 resistance proteins, respectively, that mediate the HR in tomato (de Wit, et al. 1997, Joosten and de Wit 1999, Thomma, et al. 2005). The virulence function of AVR2 and AVR4 has been discussed previously in the effectors section. AVR4E is a secreted cysteine-rich, 101 amino acid (aa) protein that induces HR in tomato lines that contain the Cf-4E gene; it does not have a known virulence function (Westerink, et al. 2004). Several field isolates that overcome CF-4E21  mediated resistance reveal point mutations in AVR4E or a complete loss of the Avr4E gene, which shows that it probably does not affect fitness of the pathogen (Stergiopoulos, et al. 2007). Avr9 encodes a 28 aa mature protein with six cycteine residues after it is processed by plant and fungal proteases at its C- and N-termini (Ackerveken, et al. 1993, van Kan, et al. 1991). Structurally, AVR9 is similar to carboxypeptidase inhibitor but no definitive function has been identified so far (Van den Ackerveken, et al. 1993, van den Hooven, et al. 2001, van Kan, et al. 1991). All natural strains of C. fulvum that overcome CF9 resistance lack Avr9, suggesting that it is not required for full virulence (Stergiopoulos, et al. 2007). C. fulvum deleted for Avr9 is fully virulent on tomato plants, but the heterologous expression of Avr9 in tomato plants makes it more susceptible to C. fulvum strains lacking Avr9, indicating some (redundant) virulence function (de Wit, et al. 2009, Marmeisse, et al. 1993). Besides Avr effectors, four extracellular cysteine-rich proteins (ECP), such as ECP1, ECP2, ECP4 and ECP5, have been cloned and characterized from C. fulvum that induce the HR in tomato lines containing the corresponding Cf-Ecp resistance genes (de Kock, et al. 2005, Lauge, et al. 2000). Bolton et al. (2008) reported the cloning of Ecp6 and Ecp7 from C. fulvum but the corresponding tomato lines that recognize these genes have not yet been identified. ECPs are present in all strains of C. fulvum and are secreted during infection. They contain an even number of cysteine residues that are most likely involved in disulphide bridge formation to protect them from apoplastic proteases (Luderer, et al. 2002a). Three of the ECPS, ECP1, ECP2 and ECP6, have virulence functions on the host plants, based on data showing that deletion or suppression of expression of these genes reduced virulence of C. fulvum on host plants (Bolton, et al. 2008, Lauge, et al. 1997). Orthologs for AVR4 and ECP6 have been identified in several fungal species because of the presence of CMB14 and LysM domains in these proteins (Bolton, et al. 2008). The ortholog of AVR4 and ECP2 have been identified in Mycosphaerella fijiensis that causes black Sigatoka disease of banana (Stergiopoulos et al. 2010). The M. fijiensis ortholog of AVR4 induces HR in tomato lines containing the corresponding Cf4 gene and binds to chitin of fungal cell walls to protect against cell wall degradation, similar to C. fulvum AVR4 (Stergiopoulos, et al. 2010). Similarly, ECP2 of M. fijiensis is a functional ortholog of C. fulvum CF-ECP2 and is recognize by CF2 of tomato to induce a HR, while in the absence of CF2, it promotes virulence on tomato plants (Stergiopoulos, et al. 2010).  22  1.3.1b. Rynchosporium secalis The imperfect fungus, R. secalis, causes leaf scald disease on barley by secreting low molecularweight toxic proteins. Three of these effectors, designated as NIP1, NIP2 and NIP3, have been cloned (Hahn, et al. 1993, Rohe, et al. 1995, Steiner-Lange, et al. 2003) and encode small secreted toxic proteins in a genotype non-specific manner on barley and related cereal plant species. Mature NIP1 is a 60 aa protein with ten cysteine residues that are involved in intramolecular disulphide bond formation. NIP1 triggers specific defense responses without a HR on barley cultivars that have the corresponding resistance gene, Rrs1 (Lehnackers and Knogge 1990). The injection of NIP1 into leaves of barley and other cereal plant species causes scald-like lesion formation (van 't Slot, et al. 2007, Wevelsiep, et al. 1991). A nip1 disruption mutant of R. secalis is slightly less virulent than the wild type on susceptible plants, demonstrating its role in virulence (Knogge and Marie 1997). R. secalis virulent strains overcome Rrs1 resistance of barley either by a point mutation in the ORF that results in a single aa substitution or by jettison of the Nip1 gene (Rohe, et al. 1995). It has been shown that NIP1 interacts with a single plasma membrane receptor (different from RRS1) that is involved both in necrosis and defense induction (van 't Slot, et al. 2007). A field population study of this pathogen showed a positive diversifying selection on the Nip1 locus, as three out of the 14 isoforms gained virulence on Rrs1 barley lines and a high deletion frequency was observed in the Nip1 locus compared to Nip2 and Nip3 (de Wit, et al. 2009). The deletion frequency of Nip1 was higher than the occurrence of the point mutation that gains virulence indicating a reduced fitness penalty for the loss of the Nip1 gene. Nip2 encodes a 109 aa protein with a predicted secretion signal of 16 aa and a mature protein with six cysteine residues, while Nip3 encodes a 115 aa protein with a predicted secretion signal of 17 aa and a mature protein with eight cysteine residues (de Wit, et al. 2009). 1.3.1c. Blumeria graminis Powdery mildews are biotrophic ascomycete fungi that cause diseases on various mono- and dicotyledonous plant species, including food crops, feed crops and ornamental plants (Bushnell 2002). They are obligate biotrophs that need a living host for growth and reproduction and produce intracellular feeding structures, the haustoria, in the epidermis of their host plants  23  (Glawe 2008, Yarwood 1957). B. graminis f. sp. hordei causes powdery mildew on barley and is the most thoroughly studied powdery mildew fungus. It interacts with its host in a “gene-forgene” manner (Both, et al. 2005, Zhang, et al. 2005). The gene-for-gene interaction revealed that there are more than 85 dominant or semi-dominant mildew (Ml) resistant genes that recognize different races of B. graminis f. sp. hordei, including 28 highly similar genes at the Mla locus on barley chromosome 5 (Jensen, et al. 1980). Seven Mla genes at this locus have been cloned and they all encode closely related intracellular coiled-coil nucleotide-binding site, leucine-rich repeat (CC-NBS-LRR) type R proteins (Halterman, et al. 2001, Shen, et al. 2003) that recognize different B. graminis f. sp. hordei avirulence proteins (Halterman, et al. 2001, Halterman and Wise 2004). Two effectors proteins, AVRa10 and AVRk1, have been isolated from B. graminis f. sp. hordei; they are virulence factors in that they promote infection on susceptible barley cultivars (Ridout, et al. 2006); however, these effectors are recognized by the barley resistance genes, Mla10 and Mlk1, respectively, where they induce a HR (Ridout, et al. 2006). Both Avr genes belong to multi-gene families that have more than 30 paralogs and they also have orthologs in other forma speciales that are pathogenic on other grasses (Ridout, et al. 2006). The AVRa10 and AVRk1 predicted proteins lack any N-terminal secretion signal or host targeting sequence, but it has been shown by fluorescence microscopy that the MLA10 protein is present intracellularly, both in the cytoplasm and the nucleus of invaded barley cells. This means that AVRa10 is taken up by the cell by an as yet unknown mechanism (Bieri, et al. 2004, Shen, et al. 2007). Besides AVRa10 and Avrk1, two other Avr genes, Avra22 and Avra12, have been mapped recently (Skamnioti, et al. 2008). 1.3.1d. Melampsora lini M. lini is an obligate biotrophic fungus that belongs to the basidiomycetes and causes flax rust disease not only in flax but also in other species of the genus Linum (Stergiopoulos and de Wit 2009). A number of flax R proteins have been analyzed; these are highly polymorphic cytoplasmic TIR-NBS-LRR proteins that recognize effector proteins that are delivered into flax cells during colonization. This interaction triggers HR and arrests growth of the fungi in a genefor-gene manner (Lawrence, et al. 2007). The genetic analysis of M. lini with its host plant flax revealed at least 30 Avr genes and corresponding R genes (Ellis, et al. 1997). Several Avr genes have been cloned from M. lini, mainly from four different loci: AvrL567, AvrM, AvrP123 and  24  AvrP4. These genes encode haustoria expressed secreted proteins (HESPs), suggesting that they have virulence functions, but elicit defence responses in hosts that have the corresponding R genes (Catanzariti, et al. 2006). The AvrL567 locus has a cluster of three polymorphic Avr genes: AVRL567A, AVRL567B and AVRL567C. All three encode 127 aa mature proteins after cleavage of a 23 aa signal peptide and are recognized directly by the L5, L6 and L7 proteins inside the cell (Dodds, et al. 2004). The mature AVR proteins are highly polymorphic with at least one or more aa substitutions in the exposed surface of the proteins, suggesting functional interactions (Ellis, et al. 2007c, Wang, et al. 2007). Some of the isolates harbouring these AVR proteins became virulent on plants and overcame matching resistance genes, indicating that genes in the AvrL567 locus are under positive diversifying selection (Dodds, et al. 2006). The analysis of six flax rust isolates revealed twelve members of the AvrL567 gene family, including the three previously isolated AvrL567A-C genes and seven of these were avirulent while five were virulent and could no longer be recognized by L5, L6, or L7 (Dodds, et al. 2006). The AvrM gene family is a small family that consists of five avirulence paralogs, AvrMA to AvrME, and one virulent one, avrM, that is not recognized by any known flax R protein (Catanzariti, et al. 2006). These AVRM proteins do not have any known homologs and are highly variable both in sequence and size due to deletions or insertions of DNA, or to “pre-mature” termination of the protein because of the location of stop codons (Catanzariti, et al. 2006). AVRP123 is a small cycteine-rich protein that contains the characteristic CX7CX6YX3CX2-3C signature of the kazal family of serine protease inhibitors, suggesting its role as an inhibitor of host proteases. This is similar to the function of AVR2 from C. fulvum that inhibits the RCR3 cysteine protease in the tomato apoplast (Catanzariti, et al. 2006). AVRP4 also encodes a small cyteine-rich protein of 67 aa after cleavage of a 28 aa signal peptide. The 28 aa C-terminal part of AVRP4 has six cysteine residues with the spacing consensus of a typical “cysteine knotted” peptide, similar to the C. fulvum AVR9 protein (van den Hooven, et al. 2001). AvrP4 is expressed only in planta while AvrM is expressed both in planta and in vitro (Stergiopoulos and de Wit 2009). Agroinfiltration of AvrP4 and AvrM in flax plants with matching resistance genes results in a HR which indicates that these are functional in host cells and are, therefore, likely translocated into the host during infections; this is in agreement with the predicted cytoplasmic location of P and M resistance proteins in flax (Anderson, et al. 1997).  25  1.3.1e. Magnaporthe oryzae M. oryzae (formerly known as M. grisea) is a filamentous ascomycete fungus that causes rice blast disease, destructive to rice production worldwide, but can also cause disease in many other members of gramineous plants (Couch and Kohn 2002, Kato, et al. 2000). More than forty resistance genes have been identified in rice against the blast fungus and several of them have been extensively used in resistant rice lines in the past few decades (Bryan, et al. 2000, Chen, et al. 2006). These resistant rice lines in the fields are overcome quickly by the emergence of new races of the pathogen through various mechanisms such as deletion of the Avr genes from the genome (Yoshida, et al. 2009) or changes in gene expression (Kang, et al. 2001, Fudal, et al. 2005) or point mutations in the Avr genes (Orbach, et al. 2000) resulting in escaping recognition by R genes; (Kolmer 1989, Leach, et al. 2001, McDonald and Linde 2002). Eight cultivar- and species-specific Avr genes have been cloned and characterized from M. oryzae: Avr-Pita, Avr1CO39, Ace1, Pwl1, Pwl2, AvrPiz-t, Avr-Pia, Avr-Pii and Avr-pik/km/kp (Bohnert, et al. 2004, Collemare, et al. 2008, Farman and Leong 1998, Orbach, et al. 2000, Valent, et al. 1991, Li, et al. 2009, Miki, et al. 2009, Yoshida, et al. 2009). The AVR-PITA effector shows similarity to fungal metalloproteases of the deuterolysin family and is not required for full virulence on rice plants (Jia, et al. 2000, Orbach, et al. 2000). Avr-Pita encodes a 223 aa protein that is predicted to be secreted and processed into a 176 aa active form (AVR-PITA 176) that interacts with rice PITA resistance protein of the NBS-LRR class (Jia, et al. 2000). Only the processed form can trigger PITA-mediated defense responses by directly expressing it in rice cells (Jia, et al. 2000). Yeast two-hybrid assays and in vitro binding analyses showed a direct interaction between AVR-PITA 176 and the LRR of the PITA resistance gene protein (Jia, et al. 2000). In certain strains of M. oryzae, Avr-Pita undergoes spontaneous mutations in the laboratory and also under field conditions, such as deletion, point mutation, and the insertion of transposons, all resulting in overcoming Pi-ta resistance in rice cultivars (Kang, et al. 2001, Orbach, et al. 2000, Khang, et al. 2008, Zhou, et al. 2007). Avr-Pita is located close to the telomere of chromosome 3 in the genome of M. oryzae and this may be responsible for the genetic instability of this gene. Avr-Pita was renamed as Avr-Pita1 after identification of Avr-Pita2 and Avr-Pita3 (Khang, et al. 2008). Avr-Pita2 is recognized by the Pita gene from rice and elicits the defence response while AvrPita3 is not recognized by PITA (Khang, et al. 2008).  26  Avr1-Co39 was isolated from M. oryzae isolate 4091-5-8 pathogenic on weeping lovegrass and specifies avirulence on rice cultivar CO39 that contains the resistant gene, PiCO39(t) in a gene for gene manner (Valent, et al. 1991, Chauhan, et al. 2002). The virulent isolates of M. oryzae on rice cultivar CO-39 lack Avr1-CO39 in most of the cases (Farman, et al. 2002). It has been shown that M. oryza Avr1-CO39 is a species-specific rather than a cultivarspecific type of Avr gene (Zheng, et al. 2011). Another Avr gene, Ace1, encodes a 4035 aa polyketide synthase (PKS) fused to a nonribosomal peptide synthetase (NRPS); these are two different classes of enzymes that are probably involved in the production of a secondary metabolite that triggers Pi33-mediated resistance in rice cultivars (Bohnert, et al. 2004). M. grisea genome analysis revealed that Ace1 is present in a cluster of 15 genes of which 14 encode enzymes such as a second PKS-NRPS (Syn2), two enoyl reductases (Rap1 and Rap2) and a putative Zn(II)(2)Cys(6) transcription factor (BC2) which probably all play a role in secondary metabolism (Collemare, et al. 2008). Ace1 and all other genes in the cluster are specifically expressed during penetration into the host plant, defining an infection-specific gene cluster, which suggests that Ace1 might have a role in virulence; however, an Ace1 disruption mutant did not show any reduction in virulence (Bohnert, et al. 2004, Fudal, et al. 2005). The Pwl genes isolated from a M. oryzae stop this pathogen from causing disease on weeping lovegrass and finger millet in a species-specific manner, but they still can infect rice (Kang, et al. 1995, Sweigard, et al. 1995). PWL effectors are small glycine-rich secreted proteins that are evolving fast and belong to a gene family designated as PWLI-PWL4. Virulent strains of Pwl2 on weeping lovegrass appear due to spontaneous mutations, predominantly by genetic rearrangement and large deletions (Kang, et al. 1995, Sweigard, et al. 1995). In the three homologs of Pwl2, identified by homology searches, only Pwl1 is the functional homolog while Pwl3 and Pwl4 are not functional; however, Pwl4 is functional only when expressed under the control of the Pwl1 or Pwl2 promoter, while Pwl3 is not functional in that case (Kang, et al. 1995). Recently, three new Avr genes, Avr-Pia, Avr-Pii and Avr-Pik/Km/kp, have been isolated from M. oryzae by association genetics (Yoshida, et al. 2009). 1.3.1f. Leptosphaeria maculans L. maculans is an ascomycete fungus that causes blackleg (phoma stem canker) disease on oilseed rape (Brassica napus). Genetic analysis of the interaction has revealed at least nine  27  avirulence genes designated as AvrLm1-AvrLm9 that are recognized by corresponding resistance genes Rlm1-Rlm9 of the host (Balesdent, et al. 2002, Fitt, et al. 2006, Rouxel and Balesdent 2005, Yu, et al. 2005). Seven of these nine genes are present in two unlinked clusters (AvrLm1-26 and AvrLm 3-4-7-9) while the remaining two are individual genes (Balesdent, et al. 2002). AvrLm1, AvrLm6 and AvrLm4-7 have been cloned by a map-based strategy and all encode small putative secreted proteins and have no similarity to sequences in public databases (Fudal, et al. 2007, Gout, et al. 2006, Parlange, et al. 2009). AvrLm1 has been cloned from this fungus and is clustered with two other Avr genes in the genome; AvrLm6 has also been cloned from this cluster (Balesdent, et al. 2002, Gout, et al. 2006). Both AvrLm1 and AvrLm6 are located in a gene-poor, AT-rich, non-coding heterochromatin-like region as solo genes in stretches of 269 and 131 kb, respectively, which contain a number of degenerated nested copies of long terminal repeat (LTR) retrotransposon (Fudal, et al. 2007, Gout, et al. 2006). Also, both AvrLm1 and AvrLm6 are single copy genes that encode small proteins of 205 and 144 aa, respectively, with an N-terminal secretion signal but no other conserved motif or any similarity to each other (Fudal, et al. 2007, Gout, et al. 2006). The expression of both genes is strongly induced during early leaf infection but also expressed in the media at relatively low levels (Fudal, et al. 2007, Gout, et al. 2006). AVRLM1 has only one cysteine residues and is likely taken up by the host cell (Gout, et al. 2006), while AVRLM6 contains six cysteine residues that make disulphide bridges that could provide stability in the apoplastic environment (Fudal, et al. 2007). The L. maculans virulent strains on Rlm1 oilseed rape cultivars lack the AvrLm1 gene, like in other fungi. It has been shown that gain of virulence on Rlm1 cultivars has been attained in 98% of the field isolates in France by deletion of an entire 260 kb locus (Gout, et al. 2006). Repeat-induced point (RIP) mutation and deletion were also responsible for gain of virulence on Rlm1 cultivars (Fudal, et al. 2009). The AvrLm4-7 gene was cloned via a map-based approach from a 238 kb genetic locus and is similar to the AvrLm1 and AvrLm6 loci by the presence of multiple LTR retrotransposon and a single gene in a gene-poor, AT-rich, 60 kb isochor (Parlange, et al. 2009). In this 238 kb region, a total of 40 predicted ORFs were identified of which 35 were in a “GC-equilibrated” region while only 5 were located in the AT-rich region (Parlange, et al. 2009). AvrLm4-7 was the only gene in the 60-kb AT-rich region that was recognized by two resistance genes, Rlm4 and Rlm7 (Parlange, et al. 2009). AvrLm4-7 encodes a cysteine-rich small secreted protein of 143 aa with a predicted N-terminal secretion signal of 21 aa (Parlange, et al. 2009). Like other effectors  28  from this fungus, the expression of AvrLm4-7 is induced in planta but is also expressed at low levels in media (Parlange, et al. 2009). The partial or complete loss of AvrLm4-7 in field isolates is responsible for gain of virulence on both Rlm4 and Rlm7 cultivars, while a point mutation that changes a glycine to an arginine residue, can overcome recognition by Rlm4 (Parlange, et al. 2009).  1.3.2. Oomycete Avr genes Oomycetes are filamentous fungus-like organisms that belong to the kingdom Stramenopila and are evolutionary related to algae. They are eukaryotic microorganisms and include some wellknown pathogens of both cultivated and forest plants such as P. infestans, H. arabidopsidis, P. sojae and P. ramorum. Several effector proteins have been isolated and characterized from oomycetes (Table 1.1). 1.3.2a. Hyaloperonospora arabidopsidis H. arabidopsidis, formerly known as H. parasitica, is an oomycete pathogen that causes downy mildew disease on the model plant A. thaliana. From this pathogen, two avirulence genes have been cloned and characterized, Atr1NdWsB and Atr13Nd; the products are recognized by RPP1NdWsB and RPP13Nd from Arabidopsis (Allen, et al. 2004, Rehmany, et al. 2005). ATR1, recognized by the products of two Rpp1 genes from two Arabidopsis ecotypes, Niederzenze (Rpp1Nd) and Wassilewskkija (Rpp1WsB), was identified by map-based cloning. RPP1Nd recognizes the product of a single allele of Atr1, while RPPIWsB recognizes the products of four different alleles and provides resistance to a wide range of isolates (Rehmany, et al. 2005). Atr1NdWsB encodes a 311 aa protein with a predicted secretion signal and a conserved RXLR motif that is present in most oomycete effectors (Rehmany, et al. 2005). Transient expression of Atr1NdWsB in Arabidopsis leaves by particle bombardment triggered cell death (Kamoun 2006). ATR1NdWsB is a highly polymorphic protein with six different alleles in eight isolates that differ in about one third of all residues (Kamoun 2006). Intense diversifying selection and recombination played an important role in the evolution of this locus (Kamoun 2006). Atr13 encodes a 187 aa protein with no similar sequences found in public databases. It triggers a HR when transiently expressed by particle bombardment in Arabidopsis plants (Allen, et al. 2004). In addition to the N-terminal signal peptide and an RXLR motif, it has a heptad leucine/isolecine 29  repeat motif that is required for RPP13 recognition, followed by an imperfect direct repeat of 4 x 11 aa which lies between aa residues 93 and 136. ATR13 was apparently under intense diversifying selection and shows a high level of aa polymorphism, similar to it corresponding resistance protein, RPP13, suggesting a co-evolutionary arms race at these loci (Allen, et al. 2004). Both ATR1 and ATR13 suppress basal defense responses of host plants when delivered by the P. syringae DC3000 Type III secretion system, revealing their effector virulence function (Sohn, et al. 2007). 1.3.2b. Phytophthora sojae P. sojae causes root and stem rot of soybean, resulting in huge damage to soybean production in North America (Shan, et al. 2004). Four avirulence genes designated as Avr1b-1, Avr1a, Avr3a and Avr3c have been cloned from this pathogen; they are recognized by soybean resistance genes Rps1b, Rps1a, Rps3a and Rps3c (Dong, et al. 2009, Qutob, et al. 2009, Shan, et al. 2004). Avr1b1 was cloned by map-based cloning and is predicted to encode a secreted protein of 138 aa with a RXLR motif and required another gene, Avr1-b2, at the locus for accumulation of Avr1b-1 mRNA (Shan, et al. 2004). Virulent isolates of P. sojae, such as P6497 and P9073, harbour a complete Avr1b-1 gene but cannot accumulate Avr1b-1 mRNA like avirulent strains (Shan, et al. 2004). In addition to recognition by RPS1B, AVR1B-1 is also recognized by Rpsk1 plants which can trigger limited cell death (Kamoun 2006). As effectors from the Rpsk1 gene clusters are cytoplasmic, it is assumed that AVR1-B is recognized inside the host cytoplasm (Bhattacharyya, et al. 1997, Shan, et al. 2004). P. sojae Avr1a, Avr3a and Avr3c were cloned by a combinatorial approach of genetic mapping, transcript profiling, and functional analysis (Qutob, et al. 2009). All P. sojae strains virulent and avirulent on Rps1 plants contain the Avr1a gene that is present in four nearly identical copies of 5.2 kb but in some virulent strains, Avr1a is transcriptionally silenced (Qutob, et al. 2009). In other virulent strains, two fragments containing Avr1a are deleted (Qutob, et al. 2009). Avr3a also occurs in four duplicated copies of about 10.8 kb in addition to four other predicted ORFs located on the DNA fragment (Qutob, et al. 2009). Transcriptional silencing of these four copies of Avr3a is responsible for avoiding recognition by Rps3 plants in some P. sojae virulent strains, while in other virulent strains three of the segments are deleted and the fourth one is transcriptionslly silenced (Qutob, et al. 2009). The Avr3c gene is present on a 33.7  30  kb fragment in addition to eight other predicted ORFs and three identical copies of this fragment are present in the P. sojae genome (Dong, et al. 2009). Avr3c virulent strains avoid recognition by RPS3A through specific mutations in the effector domain and subsequent sequence exchange between two copies of AVR3C (Dong, et al. 2009). 1.3.2c. Phytophthora infestans P. infestans causes late blight disease in potato and tomato and was responsible for the ‘Irish Famine’ in 1840. Association genetics was used for the cloning of Avr3a, which encodes a cytoplasmic RXLR effector (Armstrong, et al. 2005). It is a secreted protein of 147 aa with two polymorphic residues in the mature protein (Armstrong, et al. 2005). AVR3AK80/I103 (AVR3A KI) is avirulent on potato R3a-expressing plants while the virulent allele has two aa substitutions AVR3AE80/M103 (AVR3AEM) (Armstrong, et al. 2005). AVR3AKI functions as an elicitor of defense in R3a plants, while in the absence of R3a it strongly suppresses P. infestans INF1 elicitin induced cell death in plants, thereby illustrating a major virulence function (Armstrong, et al. 2005, Bos, et al. 2009). The virulent allele, Avr3a EM, cannot induce an R3a-mediated HR, but can weakly suppress INF1-induced cell death (Bos, et al. 2009, Bos, et al. 2006). These two different activities have been separated by making a series of mutants in important aa residues (Bos, et al. 2009). Deletions or mutations in the C-terminal residue, tyrosine 147, can maintain the R3A-mediated HR activity while it causes the loss of the ability to suppress INF1-induced cell death (Bos, et al. 2009, Bos, et al. 2006)). Also, AVR3AKI interacts and stabilizes the host ubiquitin E3-ligase CMPG in host plants which is required for INF1-induced cell death (Bos, et al. 2010). Silencing of Avr3a reduces pathogenicity which means it is required for full virulence. Transient expression of Avr3a in plants without a signal peptide induces R3a-mediated HR and, therefore, this effector is considered to be recognized in the plant cytoplasm (Armstrong, et al. 2005). Avr3a of P. infestans is located in a syntenic region to H. arabidopsidis ATR1NdWsB, suggesting that this locus is ancient in these oomycetes (Armstrong, et al. 2005).  1.4. Resistance proteins (R) in plants AVR proteins are delivered either to the apoplast, the cytoplasm, or to the nucleus of plant cells depending on their target. This is consistent with the location of matching R proteins. For  31  example, most of the bacterial effector proteins are injected directly into the cytoplasm by the Type III secretion system and the corresponding R proteins to most of these effector proteins are also intracellular (Staskawicz, et al. 2001). Some nematodes, oomycetes, and fungi also translocate their AVR proteins to the cytoplasm (Bryan, et al. 2000, Dodds, et al. 2004, Dodds, et al. 2006, Jia, et al. 2000, Orbach, et al. 2000). On the other hand, the AVR2, AVR4, AVR4E and AVR9 proteins of C. fulvum, are secreted into the apoplast (Joosten and de Wit 1999, Lauge, et al. 1998) which is consistent with the membrane localization of their matching CF proteins (Piedras, et al. 2000). The AVRBS3-like protein has a nuclear localization signal (NLS), which would suggest that its corresponding R protein is located in the nucleus (Lahaye and Bonas 2001). Several functional R genes encoding resistance against bacterial, fungal, viral, oomycete, nematode, and insect pathogens have been isolated from various model and crop plants. This wide range of R proteins can be divided into five or six classes on the basis of domain structure (Dangl and Jones 2001, Martin, et al. 2003, Nimchuk, et al. 2003). Class I has only one member, PTO from tomato, which has a myristylation site at the N-terminal end and a serine/threonine kinase catalytic activity domain. The majority of the R proteins are Nucleotide Binding SiteLeucine Rich Repeat (NBS-LRR) proteins, which make up classes II and III of R proteins. The only difference between these two classes is located in the N-terminal region of their members. Class II members have a coiled-coil domain and class III members have a Toll or Interleukin 1 Receptor (TIR) domain at the N-terminal end. Proteins in all these three classes are intracellular, as they do not have any predicted transmembrane domains. R proteins in classes IV and V are similar to each other in the sense that both have extracellular LRRs and a transmembrane domain, but class IV members have a short cytoplasmic tail with unknown function. Class V members have a cytoplamic serine/threonine kinase domain like PTO. Resistance proteins such as HM1, RPW8, MLO, and several others do not easily fit into any of the above mentioned classes and are put into class VI.  1.5. Recognition of Avr proteins by R proteins The recognition of invading pathogens is a critical step in the activation of the host defense response by R proteins. Plant resistance proteins use either a direct or an indirect mode of  32  recognition of specific elicitors from pathogens. In the direct recognition systems, the AVR protein acts as a ligand and the R protein as a receptor. For example, based on yeast two-hybrid screens, the rice Pita R gene product binds the cognate AVR-PITA from M. grisea (Jia, et al. 2000). Similarly, RRS1-R from Arabidopsis binds POPP2 from R. solanacearum (Deslandes, et al. 2003). Direct recognition has been shown to operate in recognition of AVRL567 from M. lini by flax L5, L6 and L7 (Dodds, et al. 2006) Dodds PN 2006). In indirect recognition systems, the R protein recognizes the pathogen effectors through detection of changes in their host protein target (Van der Biezen and Jones 1998). This suggested model is called the “guard model” in which the R protein guards the effector target, also called guardee, and can then detect changes in the guardee. This seems to be a more common recognition system than the receptor-ligand system (Jones and Dangl 2006). Several studies of bacterial Type III secretion system effector proteins and their corresponding R proteins support the guard model. The RPM1 protein in Arabidopsis recognizes effector proteins AVRRPM1 and AVRB from P. syringae indirectly by changes in the host protein RIN4 (Mackey, et al. 2002). Both of these Type III effectors induce phosphorylation of RIN4, which serves as a signal for activation of RPMI. Changes in RIN4 are also perceived by another protein, RPS2, when the pathogen delivers AVRRPT2, a cysteine protease, inside the cell which cleaves RIN4 (Day, et al. 2005, Mackey, et al. 2003). RIN4 interacts with another protein, NDR1, which is required for the activation of RPM1 and RPS2 (Day, et al. 2006). AVRRPT2 causes degradation of RIN4 at two different sites through its cysteine protease activity. The down-regulation of RIN4 activates RPS2 (Chisholm, et al. 2005, Kim, et al. 2005). RIN4 is not the only target of these effector proteins in the host, as AVRRPT2 can degrade several other Arabidopsis proteins that contain its corresponding cleavage site (Chisholm, et al. 2005). It means that the contribution of an effector to virulence might involve several host targets and the generation of several modified host molecules, but the perturbation of only one is sufficient for activation of the R gene (Belkhadir, et al. 2004, Day, et al. 2005). In rpm1 rps2 double mutants, AVRRPT2 and AVRRPM1 manipulate RIN4 for suppressing PAMP-triggered immunity (Dodds, et al. 2006). RPS5 from Arabidopsis monitors changes in PBS1, which is targeted by AVRPPHB of P. syringae (Shao, et al. 2002). CF-2 in tomato monitors changes in RCR3 brought about by the activity of C. fulvum AVR2 (Chang, et al. 2000). Two effectors, EPIC1 and EPIC2B from P. infestans, bind to RCR3 that plays a role in defense against P. infestans (Dangl and Jones 2001). Similarly, tomato PRF monitors changes in  33  PTO caused by AVRPTO and AVRPTOB from P. syringae (Jones and Dangl 2006, Van der Biezen and Jones 1998). Recently, the decoy model has been proposed for the indirect recognition of AVR proteins by R proteins, based on a lack of evidence that guardees increase host susceptibility in the absence of their matching R proteins (van der Hoorn and Kamoun 2008). In the decoy model, the target of the effector is required for the function of the R protein but it does not have any direct function in host susceptibility or resistance (van der Hoorn and Kamoun 2008). Whether R and AVR proteins interact directly or indirectly, the ultimate result is an incompatible interaction between plant and pathogen, usually associated with HR. The HR in plants directed against microbial pathogens is a type of rapid and localized programmed cell death (PCD) similar to that in mammalian tissues and differs from developmental PCD only in its consistent association with the induction of local and systemic defence responses. The number of dead cells varies from one to dozens and depends on both genotypes of pathogen and plants. This HR in plants is usually visible macroscopically, but in some incompatible plant-pathogen interactions, such as the Ustilago hordei-barley interaction, it can be seen only microscopically (Hu, et al. 2002); however, there are also a few other reported cases of gene-for-gene resistance that do not produce any visible HR, such as the Arabidopsis dndI (defense no death) mutant, which produces resistance in a gene-for-gene manner without any visible HR against avirulent bacteria (Yu, et al. 1998). Similarly, the Rx gene of potato recognizes the potato virus X gene in a gene-for-gene manner but does not produce any microscopic or macroscopic HR lesions on potato plants or transgenic tobacco plants (Kang, et al. 1995).  1.6. Marker-based approaches for cloning of Avr genes Most known pathogen Avr genes are highly diverged in DNA sequence and many do not have annotated homologs in public databases, although similar sequences can often be found in the genomes of related species. Identifying functional Avr genes has been a challenge (Gan, et al. 2010b, Van't Slot and Knogge 2002). In the case of bacterial phytopathogens, several Avr genes have been isolated by classical genetic techniques commonly used for bacterial gene isolation, such as transformation of a genomic library from an avirulent strain into a virulent strain and subsequently testing for a HR response on host plants to select for Avr-containing clones  34  (Collmer 1998, Van den Ackerveken and Bonas 1997). This method is not easily applicable for isolating Avr genes from fungi and oomycetes due to large genome sizes and inefficient transformation methods in many cases (Lauge, et al. 1998). Reverse genetics and map-based cloning are the two main strategies that have been successfully used for isolating fungal and oomycetes Avr genes. A reverse genetics approach is based on the isolation and purification of proteins encoded by Avr genes that elicit the defense response in specific resistant cultivars or nonhost plants. Using this approach, several fungal Avr genes have been isolated, such as Nip1 from the barley leaf scald pathogen, R. secalis (Rohe, et al. 1995), Avr9 (van Kan, et al. 1991), Avr4 (Joosten, et al. 1994), Ecp1, Ecp2 (Van den Ackerveken, et al. 1993), Ecp3 and Ecp4 (Lauge, et al. 2000) from C. fulvum, AvrM, AvrP4, AvrP123 and AvrL567 from M. lini (Catanzariti, et al. 2006, Dodds, et al. 2006), and Avr2 (Houterman, et al. 2007), Avr3 (Rep, et al. 2004) and Avr1 (Houterman, et al. 2008) from F. oxysporum. Similarly, a few oomycete Avr genes have been isolated by this approach such as Inf1 from P. infestans (Kamoun, et al. 1998) and Gip1 and Gip2 from P. sojae (Rose, et al. 2002). For intracellular pathogens such as M. oryzae, a reverse genetics approach was not very successful for isolating Avr genes. M. oryzae Avr genes such as Pwl2 (Sweigard, et al. 1995), AvrPita (Farman and Leong 1998), AvrCo39 (Miki, et al. 2009) and Avr-Pia (Miki, et al. 2009, Chen, et al. 2007) were isolated by positional cloning strategy. In addition, a few Avr genes, AvrPi15 (Ma, et al. 2006) and Pre1 (Miki, et al. 2009), have been mapped on a short genetic interval by linkage mapping in M. oryzae. Similarly, AvrLm6 (Fudal, et al. 2007), AvrLm4-7 (Parlange, et al. 2009) and AvrLm1 (Gout, et al. 2006) from L. maculans, Avra10 and Avrk1 from B. graminis f. sp. hordei (Ridout, et al. 2006) were isolated by map-based cloning and Avra22 and Avra12 from B. graminis f. sp. hordei were mapped on short genetic intervals (Skamnioti, et al. 2008). Several oomycete Avr genes have been isolated by map-based cloning, such as Avr1b1 and Avr1b-2 (Shan, et al. 2004), Avr1a (Qutob, et al. 2009) and Avr3c (Dong, et al. 2009) from P. sojae, and Atr1 (Rehmany, et al. 2005) and Atr13 (Allen, et al. 2004) from H. arabidopsidis. Relevant to this thesis, UhAvr1 was mapped to an 85 kb genetic interval in the barley covered smut fungus, U. hordei, by a marker-based approach (Linning, et al. 2004). Several other techniques have been employed, including using bioinformatics and association genetics. AvrPia, Avr-Pii and Avr-Pik/km/kp were isolated from M. oryzae (Yoshida, et al. 2009) and Avr3a from P. infestans (Armstrong, et al. 2005). Avr3a was identified in P. sojae by correlating gene  35  transcript profiling data with phenotypically pooled progeny using microarrays (Qutob, et al. 2009). Stergiopoulos et al. (2010) isolated orthologs of Avr4 and Ecp2 from M. fijiensis, the causal agent of black sigatoka disease of banana, by means of a bioinformatic homology search approach.  1.7. Comparative genomics (secretomics) The continuous arms race between pathogens and their host plants has intensively affected the co-evolution of pathogen and plant genomes. Whole genome sequencing and analyses show that R genes are the most polymorphic genes as compared to the rest of the genome (Clark, et al. 2007). Similarly, effector genes from pathogens are also evolving at a fast pace and in few cases, these effectors are present on unstable parts of the genome such as at telomeres, or reside on small, dispensible chromosomes (Gout, et al. 2006, Orbach, et al. 2000). Comparative genomics is a powerful tool and can be very useful for the identification of new virulence and avirulence effectors from phytopathogens by comparing the genome sequences of closely related pathogens. Several clusters of effectors have been identified by comparing the genomes of two related basidiomycete smut fungi, U. maydis and Sporisorium reilianum, both infecting corn (Schirawski et al. 2010). In M. oryzae, 316 new candidate effectors have been identified by genome comparison of isolate 70-15 and a field isolate, Ina 168 (Yoshida, et al. 2009), and three of them have proved to be Avr genes. The effectors from oomycetes pathogens have a hosttargeting RXLR-dEER motif, in addition to a secretion signal at the N-terminal end (Tyler 2009). The genome sequences of P. sojae, P. ramorum, P. infestans, and H. arabidosidis have a large number of RXLR-dEER effectors with 40-50 % identity across species (Jiang, et al. 2008). The majority of effectors from different cellular pathogens are predicted to be secreted or have been proven to be secreted by different mechanisms (Chisholm, et al. 2006, Huang, et al. 2003, Kamoun 2007). The effector proteins of certain oomycetes and fungi can be largely predicted computationally (Ellis, et al. 2007b, Kamoun 2007). Because many fungal and oomycete genomes have been sequenced or are close to completion, an opportunity exists to predict a complete suite of secreted proteins. The processing of these sequences by tools of comparative genomics (Tyler, et al. 2006) and in silico prediction of secreted proteins (Torto, et al. 2003) has  36  identified a substantial number of candidate effector genes that could be involved in pathogenesis.  1.8. Smut fungi The work presented in this thesis involves the experimental organism, the barley covered smut fungus, U. hordei. Smut fungi belong to Order Ustilaginales of the basidiomycetes and occur throughout the world. There are approximately 1400 species of smut fungi in over 70 genera (Agrios 2005, Fisher and Holton 1957). These are facultative obligate biotrophs that cause diseases in 4,000 species of angiosperms belonging to approximately 75 different families. The genus, Ustilago, mainly infects cereal crops and grasses belonging to the Gramineae family, which are used as food and feed. Most smut fungi infect the ovaries of grains and grasses and destroy the fruits (kernels) of grains, completely; however, some smuts can infect and produce spores on vegetative and floral parts, such as the corn smut, U. maydis (Alexopoulos, et al. 1996, Fisher and Holton 1957). Usually, smut fungi do not kill their hosts but in some cases, the diseased plant may be severely stunted. Most of the smut diseases can be controlled by treatment of seeds with fungicides or the use of resistant varieties. The fungal pathogen, U. hordei, causes covered smut on barley and oats and not only decreases the yield of the crops, but also greatly reduces the quality of the remaining yield due to the presence of black smut spores on the surface of healthy kernels. U. hordei is a representative of smut fungi that infect small grains. This fungus infects the seedling and grows as dikaryotic hyphae within the developing plant without any visible symptoms until flowering. During flowering, the fungal cells proliferate, making thick-walled teliospores during which karyogamy takes place (Hu, et al. 2002). Teliospores disseminate from the diseased head and contaminate healthy barley seeds of nearby plants and usually overwinter under the seed hull. When the conditions are favorable for germination, both seed and teliospores germinate. Upon germination, teliospores undergo a process of meiosis giving rise to haploid basidiospores that segregate 1:1 for mating types MAT-1 and MAT-2 (Bakkeren and Kronstad 1994). The basidiospores multiply by budding and are amenable to different types of molecular genetic techniques. The opposite mating types, MAT-1 and MAT-2, can recognize each other by a pheromone/receptor system (Bakkeren and Kronstad 1996), forming dikaryotic hyphae by  37  fusion. These resulting dikaryotic hyphae can penetrate emerging seedlings by direct penetration (Hu, et al. 2002), thereby completing the life cycle (Figure 1). The smuts are important pathogens that cause disease world-wide, resulting in crop losses in many countries, including Canada (Menzies, et al. 1996, Thomas 1989). The barley/U. hordei pathosystem is an excellent model system for small grain-infecting basidiomycetes due to the presence of race cultivar-, ‘gene-for-gene’-based resistance and the availability of resources such as a genetic transformation system, gene deletion techniques, genetic crosses, many field isolates and races, and several differential barley cultivars. Also, the complete genome sequence of U. hordei became available during the course of my thesis work (collaborative effort with a group from the Max Planck Institute for Terrestrial Microbiology in Marburg, Germany: Drs. J. Schirawski and R. Kahmann), and complete genome sequence data and many ESTs are available from the closely related fungi, U. maydis and S. reilianum (Kamper, et al. 2006, Schirawski, et al. 2010). Six Avr genes have been described in U. hordei which in different combinations constitute fourteen different reported races (Tapke 1945). Six corresponding resistance genes have been identified in barley (Thomas 1976). At the onset of my thesis work, a population of 54 progeny, resulting from a cross between two parents possessing dominant and recessive avirulence genes and segregating genetically for three Avr genes, UhAvr1, UhAvr2 and UhAvr6, were available in our laboratory (Linning, et al. 2004). Two dominant genes, UhAvr1 and UhAvr6, act consistently in a stable genetic manner while UhAvr2 expression is influenced by environmental conditions (Linning, et al. 2004). The UhAvr1 gene has been identified on a genomic region of 85 kb (Laurie and Bakkeren, unpublished). In addition, BAC libraries from both the virulent and avirulent parents of the mapping population and a physical BAC map for this pathogen were available in our laboratory (Bakkeren, et al. 2006).  1.9. Proposed research project The isolation and characterization of Avr genes from pathogens and R genes from their hosts is an important focus of research in molecular plant pathology to understand the biochemical and molecular basis of effector-triggered immunity in plants (Hammond-Kosack and Parker 2003). This will help us to understand the compatibility and disease potential, suppression of host defenses, functions of effectors as elicitors, the host targets of these effectors, virulence of the  38  pathogen, and defense initiation. The cloning and characterization of Avr genes is made feasible because they are single genes and are usually dominant, thus making them easy to follow genetically. Currently, our molecular genetic knowledge of fungal Avr genes is based on only seven of the above-mentioned fungal species. Most of these Avr gene sequences are much diverged from one another and also do not have many similar sequences in public databases (Gout, et al. 2006). M. oryzae, an ascomycete, is the only fungal pathogen infecting cereal crops from which Avr genes have been characterized (Chen, et al. 2007, Farman and Leong 1998, Miki, et al. 2009, Sweigard, et al. 1995). M. lini, the flax rust fungus, is the only basidiomycete from which Avr genes have been characterized (Catanzariti, et al. 2006, Dodds, et al. 2004, Dodds, et al. 2006). No Avr genes have been isolated from basidiomycetes infecting monocots thus far. To fill this gap, we need to build a model system to study the molecular interaction of R and Avr genes in basidiomycetes and monocots to which all cereal crops belong. U. maydis, the corn smut, is a widely-studied pathogen with a sequenced genome but no Avr genes have been described (Kamper, et al. 2006). U. hordei and barley offer an excellent model system in this regard to understand the Avr-R gene interaction between basidiomycetes and monocots. UhAvr1 has been isolated on a 85-kb region by a marker based approach (Linning, et al. 2004). The corresponding resistance gene UhR1 has been mapped to the short arm of chromosome 1 in barley by our collaborators at the University of Saskatchewan (Grewal, et al. 2008). Barley cultivar Morex that contains the Ruh1 gene, is available on a BAC library (Dr. A. Kleinhofs, Washington State University; (Brueggeman, et al. 2002) and a barley cDNA library in a yeast two-hybrid specific vector is available (Dr. R. Hueckelhoven, University of Giessen, Germany), allowing for follow-up research involving R gene and effector target studies. Studying the interaction between UhAvr1 and Ruh1 will help to understand the mechanism of pathogenesis and elicitation of the defense response. Identifying UhAvr1, the analysis of the effector locus might shed some light on mechanisms involved in its evolution since, as I will show, these Ustilago effector loci are somewhat conserved between U. hordei and U. maydis. My research project was mainly focused on identifying and characterizing the UhAvr1 gene on the 85-kb region of U. hordei genomic DNA. I also used comparative genomics to understand the evolution of this locus between two related smut fungi. Pathogen effectors are rapidly evolving and in many cases, pathogen avirulence genes are present in regions displaying high genome flexibility, such as telomeric (Orbach, et al. 2000), or heterochromatic locations (Fudal, et al. 2007, Parlange, et al. 2009), or  39  they are surrounded by transposable elements (Fudal, et al. 2007, Gout, et al. 2006, Khang, et al. 2008, Zhou, et al. 2007). I was therefore interested in finding out in what sort of molecular environment the UhAvr1 gene is located and what the molecular basis was of the change from avirulent U. hordei strains to overcome UhR1 resistance in the field. I was also interested in other small secreted proteins (SSPs) of U. hordei that may have virulence or avirulence functions. For this purpose, I searched for U. hordei-specific SPs by comparing all predicted SPs from the U. hordei genome to the predicted suites of SPs from U. maydis (Kamper, et al. 2006, Schirawski, et al. 2010) using bioinformatic approaches. In the population of U. hordei, an additional Avr gene, UhAvr6, was segregating; this gave me the opportunity to work towards the cloning of this gene as well. For the cloning of UhAvr6, I used SSR, RAPD and AFLP techniques to find molecular markers linked to this gene. A collaborator tested U. hordei strains on new barley lines and found potential new Avr genes and corresponding R genes. In order to initiate cloning of this gene(s) as well, I developed a U. hordei population segregating for these new Avr genes. The genetic transformation system for U. hordei is not very efficient and does not easily allow for the transfer of large genomic fragments. The current method of protoplast transformation is dependent on the use of lytic enzymes which need to be optimized for each batch of enzyme. This makes this method inconsistent. Since many effectors reside in paralogous clusters on large genomic fragments, the transfer of such regions into recipient U. hordei strains would possibly allow for functional complementation analyses. Agrobacterium mediated transformation (AMT) has been used efficiently for several filamentous fungi and worked better for several fungi that were difficult to transform by traditional transformation methods (Chen, et al. 2000, Degefu and Hanif 2003, Meyer, et al. 2003, Mikosch, et al. 2001, Michielse, et al. 2005b). In addition to its efficiency, AMT results in transformation of single copy integration of the T-DNA at random sites in the genome making it suitable for insertion mutagenesis (Combier, et al. 2003, Mullins, et al. 2001, Takahara, et al. 2004). I therefore developed an AMT-based transformation protocol for U. hordei. To subsequently be able to transfer to U. hordei large genomic fragments without the need for cumbersome cloning with restriction and ligation enzymes, I adapted a technique to engineer Agrobacterium binary vectors by in vitro recombineering so these vectors can contain BAC clones (BIBACs).  40  The knowledge obtained from my dissertation research will not only be helpful in understanding molecular interactions between plant and pathogen but will also be helpful in designing novel ways to increase crop resistance.  1.10. Research objectives 1. Identification and characterization of the Ustilago hordei avirulence gene 1 within the UhAvr1 locus, and possibly UhAvr6, to extend our knowledge of effector-triggered immunity in plants to now include an interaction between a basidiomycete pathogen and a monocot host, and to further our knowledge of fungal effectors in disease establishment and defense induction 2. To study how U. hordei overcomes Ruh1-triggered resistance in barley 3. To understand evolutionary pressures acting on the UhAvr1 locus in light of its similarity to a locus in the closely-related corn smut fungus, U. maydis 4. To gain insight into the potential repertoire of small secreted proteins (effectors) of U. hordei likely involved in virulence and avirulence towards the host plant 5. To study the function of clusters of predicted secreted proteins by developing a reliable technique for introducing large genomic DNA fragments into U. hordei  41  Haploid basidiospore MAT-1s MAT-2  Diploid teliospores  a  Meiosis  Mating and infection  Sporulation  Infected inflorescence  Germinating seedling  c  b t  d  t b 5 µm  25 µ m  e  f  5 µm  5 µm  g  20 µ m  42  Figure 1.1 Infection process of U. hordei on barley. a. schematic representation of the U. hordei life cycle. Dispersed teliospores become lodged under seed hulls and germinate together with the seed; mating needs to precede infection which can only occur on the young coleoptile. Photographic insert depicts a light microscopic picture of an immature inflorescence at 5 weeks after infection showing blue-colored fungi expressing the β-glucuronidase gene after treatment with glucuronide. b. light microscopic picture of germinated teliospores (t) on the surface of a barley coleoptile 17 hrs after inoculation having produced a basidium (b) from which haploid basidiospores (arrows) are emerging (cotton blue-staining). c. scanning electron micrograph of two mated cells of opposite mating type fused through conjugation hyphae to produce the dikaryotic infection filament on a barley coleoptile. d. SEM of a dikaryotic infection hypha entering the barley coleoptile wall by direct penetration. e. SEM showing a penetration site from which the hypha has been removed. f. light microscopic picture of an immature inflorescence showing extensive, early teliospores formation (arrows). g. emerged head where all kernels have been replaced by black teliospores, next to a healthy head (see Hu et al. 2002, for details).ced by black teliospores, next to a healthy head (see Hu et al. 2002, for details).  43  Table 1.1 Effector proteins of filamentous plant pathogens Protein  Organism  Length aa residues (mature)  No: of Cysteines  Signal Peptide in aa  Biological activity/homology  Protein localization  Role in virulence/pathogenicity  Corresponding R-gene  Avr2  C. fulvum  78 (58)  8  20  Avr4  C. fulvum  135 (86)  8  18  induces HR in the presence of Tomato Rcr3, Protease inhibitor, induces HR, Chitinbinding, orthologs in some other fungi  Avr4E  C. fulvum  121 (101)  6  10  Avr9  C. fulvum  63 (28)  6  23  Ecp1  C. fulvum  96 (65)  8  23  Ecp2  C. fulvum  165 (101)  4  Apoplast  Inhibits Rcr3 and other proteases  Cf-2  (Stergiopoulos, et al. 2010)  Apoplast; Fungal cell wall chitin  Protects against chitinases  Cf-4  induces HR,  Apoplast  Unknown  Hcr9-4E  induces HR, Carboxypeptidase inhibitor induces HR, Tumornecrosis factor receptor  Apoplast  Unknown  Cf-9  Apoplast  Cf-Ecp1  induces HR,  Apoplast  18  induces HR,  Apoplast  Disruption leads to reduced virulence Disruption leads to reduced virulence Unknown  Cf-Ecp4  6  17  Induce necrosis,  Apoplast  Unknown  Cf-Ecp5  8  23  LysM-domains; chitinbinding, ortholog found in different pathogen and non-pathogenic sp.  Apoplast  Knock-down leads to reduced virulence  Unknown  (Joosten, et al. 1994, Stergiopoulos, et al. 2010) (van den Burg, et al. 2006) (Westerink, et al. 2004) (Van den Ackerveken, et al. 1993) (Van den Ackerveken, et al. 1993) (Van den Ackerveken, et al. 1993) (Bolton, et al. 2008) (Bolton, et al. 2008) (de Jonge and Thomma 2009)  Ecp4  C. fulvum  119 (101)  6  Ecp5  C. fulvum  115 (98)  Ecp6  C. fulvum  222 (199)  22  Cf-Ecp2  References  44  Protein  Organism  Length aa residues (mature)  No: of Cysteines  Signal Peptide in aa  Ecp7  C. fulvum  - (100)  6  -  Nip1  R. secalis  82 (60)  10  22  Nip2  R. secalis  109 (?)  7 (6)  16  Nip3  R. secalis  115 (?)  9 (8)  17  Avra10  B. graminis  286  4  -  Avrk1  B. graminis  177  3  -  AvrL5 67 (A, B and C)  M. lini  150 (127)  1  23  Biological activity/homology  Protein localization  Role in virulence/pathogenicity  Corresponding R-gene  References  Unknown  Apoplast  Unknown  Unknown  Non-specific toxin/induces necrosis and plasma-membrane H+ ATPase Non-specific toxin/induces necrosis in several plants species  Probably in apoplast  Not required for virulence  Rrs-1  Probably in apoplast  Not required for full virulence  Unknown  Non-specific toxin/induces necrosis in several plants species,  Probably in apoplast  Not required for full virulence  Unknown  More than 30 paralogues in Bgh and other f. sp. No Nterminal SP, induces HR, More than 30 paralogues in Bgh and other f. sp. No Nterminal SP, induces HR, Unknown induces HR, Functional RXLR like motif  Probably in cytoplasm  Unknown  Mla10)  Probably in cytoplasm  Unknown  Mlk1)  (Ridout, et al. 2006)  Cytoplasm  Unknown  L5, L6 and L7  (Dodds, et al. 2004) (Kale, et al. 2010) (Rafiqi, et al. 2010)  (Bolton, et al. 2008) (Rohe, et al. 1995)  (Rohe, et al. 1995) (Stergiopoulos and de Wit 2009) (Rohe, et al. 1995) (Stergiopoulos and de Wit 2009) (Ridout, et al. 2006)  45  Protein  Organism  Length aa residues (mature)  No: of Cysteines  Signal Peptide in aa  Biological activity/homology  Protein localization  Role in virulence/pathogenicity  Corresponding R-gene  AvrM  M. lini  314  1  28  Unknown, induces HR, RXLR like motif  Cytoplasm  Unknown  M  AvrP1 23 AvrP4  M. lini  117 (94)  11  23  95 (67)  7  28  P, P1, P2 and/or P3) P4  AvrPita  M. oryzae  224 (176)  8  16  Probably in cytoplasm Probably in cytoplasm Cytoplasm  Unknown  M. lini  induces HR,, Kazal Ser protease inhibitor induces HR, Cystine knotted peptide Homology to Metalloproteases, RXLR like motif  Not required for virulence on rice  Pi-ta  AvrPita2  M. oryzae  224 (?)  8  16  Homology to Metalloproteases  Probably in apoplast  Pi-ta  AvrPita3  M. oryzae  226 (?)  8  16  Homology to Metalloproteases  Probably in cytoplasm  Probably not required for virulence on rice Probably not required for virulence on rice  Unknown  (Khang, et al. 2008)  Pwl1  M. oryzae  147 (124)  2  23  Glycine-rich hydrophilic protein  Biotrophic interfacial complex  Unknown  Unknown  (Kang, et al. 1995) (Khang, et al. 2010)  Pwl2  M. oryzae  145 (126)  2  21  Glycine-rich hydrophilic protein  cytoplasm  Unknown  Unknown  Pwl3  M. oryzae  137 (116)  0  21  Glycine-rich hydrophilic protein  Probably in apoplast  Non-functional  Unknown  (Sweigard, et al. 1995) (Khang, et al. 2010) (Kang, et al. 1995)  Unknown  References  (Catanzariti, et al. 2006) (Kale, et al. 2010) (Rafiqi, et al. 2010) (Catanzariti, et al. 2006)) (Catanzariti, et al. 2006) Orbach, MJ 2000, (Kale, et al. 2010), (Khang, et al. 2010) (Khang, et al. 2008)  46  Protein  Organism  Length aa residues (mature)  No: of Cysteines  Signal Peptide in aa  Biological activity/homology  Protein localization  Role in virulence/pathogenicity  Corresponding R-gene  Pwl4  M. oryzae M. oryzae  138 (117)  0  21  Unknown  43  -  Probably in apoplast Not secreted  Non-functional  4035  Unknown  Pi33  M. oryzae  Not cloned yet  -  -  Glycine-rich hydrophilic protein Hybrid polyketide synthase/nonribosomal peptide synthetase Unknown  Unknown  Unknown  Pi-CO39(t)  Probably in cytoplasm Probably in cytoplasm Probably in cytoplasm  Unknown  Ace1  Avr1CO39  References  (Kang, et al. 1995) (Bohnert, et al. 2004) (Farman, et al. 2002)  AvrPia Avr-Pii  2  induces HR  (Yoshida, et al. 2009) (Yoshida, et al. 2009) (Yoshida, et al. 2009)  3  induces HR  AvrPik/km /kp AvrLm 1  3  induces HR  L. maculans  205 (183)  1  22  induces HR,  Probably in cytoplasm  Required for full virulence  Rlm1  (Gout, et al. 2006)  AvrLm 6  L. maculans  144 (124)  6  20  Probably in apoplast  Unknown  Rlm6  AvrLm 4-7  L. maculans  143 (122)  8  21  Unknown, Functional RXLR like motif Unknown  Probably in apoplast  Required for full virulence  Avr3 (Six1)  F. oxysporu m f. sp. hordei  284 (189)  6 or 8  21  Unknown  Xylem  Required for full virulence  Rlm4 and/or Rlm7 I-3  (Fudal, et al. 2007) (Kale, et al. 2010) (Parlange, et al. 2009)  Unknown Unknown  (Rep, et al. 2004) (Stergiopoulos and de Wit 2009)  47  Protein  Organism  Length aa residues (mature)  No: of Cysteines  Signal Peptide in aa  Avr4 (Six2)  F. oxysporu m f. sp. hordei  232 (172)  8  20  Avr2 (Six3)  F. oxysporu m f. sp. hordei  163 (144)  3 (2)  Avr1 (Six4)  F. oxysporu m f. sp. hordei  242 (184)  6  ATR1Nd  H. arabidop sidis H. arabidop sidis P. sojae  WsB  ATR13  Biological activity/homology  Protein localization  Role in virulence/pathogenicity  Corresponding R-gene  Unknown  Xylem  Probably not required for virulence  Unknown  (Houterman, et al. 2007) (Stergiopoulos and de Wit 2009)  19  induces HR, Unknown, Functional RXLR like motif  Xylem  Required for full virulence  I-2  17  Unknown  Xylem  Suppression of I2 and I-3 resistance  I or I-1  311 (296) -  15  induces HR, RXLR domain  cytoplasm  Suppress host defense  187 (168) -  19  induces HR, RXLR domain  cytoplasm  Suppress host defense  RPP1WsB and RPP1Nd RPP13  138 (117)  21  Probably cytoplasm  Unknown  Rps1b and RpsK1  Unknown  Rps1a  (Houterman, et al. 2007) (Kale, et al. 2010, Stergiopoulos and de Wit 2009) (Houterman, et al. 2007) (Stergiopoulos and de Wit 2009) (Rehmany, et al. 2005, (Sohn, et al. 2007) (Allen, et al. 2004) (Sohn, et al. 2007) (Shan, et al. 2004) (Dou, et al. 2008a) (Qutob, et al. 2009) (Qutob, et al. 2009) (Dong, et al. 2009)  Avr1a  P. sojae  induces HR, suppress BAX induced cell death, RXLR domain RXLR domain  Avr3a  P. sojae  RXLR domain  Unknown  Rps3a  Avr3c  P. sojae  induces HR, RXLR domain  Unknown  Rps3c  Avr1b1  220  References  48  Protein  Organism  Length aa residues (mature)  Avr3a  P. infestans  147  MfAvr 4  M. fijiensis  121 (100)  MfEcp 2  M. fijiensis  161(142)  No: of Cysteines  Signal Peptide in aa  Biological activity/homology  Protein localization  Role in virulence/pathogenicity  Corresponding R-gene  21  10  21  4  19  References  induces HR, suppress INF1 induced HR RXLR domain, interact with CMPG1 ubiquitin E3 ligase  cytoplasm  Required for full virulence  R3a  Chitin-binding peritrophin-A, induces HR or necrosis in Cf4 toamto, induces HR or necrosis in CfEcp2 toamto, Unknown  Probably in apoplast  Protect fungi against chitinases  (Stergiopoulos, et al. 2010)  Probably in apoplast  Promote virulence by interacting with host cell target causing necrosis  (Stergiopoulos, et al. 2010)  (Armstrong, et al. 2005) (Bos, et al. 2006) (Bos, et al. 2009) (Bos, et al. 2010)  49  CHAPTER 2 The avirulence gene UhAvr1 clusters with predicted secreted proteins in the U. hordei genome and is inactivated by transposon activity in virulent strains1 2.1. Introduction Plants use a variety of defense mechanisms to avoid pathogen invasion and subsequent disease. These defense mechanisms include physical barriers, preformed antimicrobial compounds, and activation of induced defenses. As discussed in Chapter 1, triggers for such defenses can be highly conserved molecules from pathogens (Pathogens or Microbe Associated Molecular Pattern or PAMPs or MAMPs) resulting in PAMP-triggered immunity (PTI). On the other hand, pathogens secrete effector molecules into the host plant to avoid or suppress PTI and help in nutrient acquisition for pathogen growth and development (Kamoun 2007, van der Does and Rep 2007). Plants use a highly sophisticated system encoded by resistance (R) genes to recognize these effectors and trigger a variety of defense mechanisms, including a localized cell death called the hypersensitive response (HR) to arrest pathogen development (Keen 1990, Van der Biezen and Jones 1998). The pathogen molecules that are recognized by R genes are called avirulence (Avr) genes, since they render the pathogen unable to cause disease on that particular host. This type of resistance in the host and avirulence in the pathogen is called a gene-for-gene interaction (Flor 1942). Pathogen effectors are rapidly evolving and, in many cases, avirulence genes are present in regions displaying high genome flexibility, such as telomeres (Orbach, et al. 2000), or heterochromatic locations (Fudal, et al. 2007, Parlange, et al. 2009), or they are surrounded by transposable elements (Fudal, et al. 2007, Gout, et al. 2006, Khang, et al. 2008, Zhou, et al. 2007). 1  A version of the chapter entitled “The avirulence gene UhAvr1 clusters with predicted secreted proteins in the U. hordei genome  and is inactivated by transposon activity in virulent strains”, is in preparation for publication. Anticipated co-author list: Ali, S., Linning, R., Laurie, J. and Bakkeren, G.  50  Pathogen Avr proteins are recognized by plant R proteins either directly through the receptorligand model or indirectly by changes in the host protein (Keen 1990, Van der Biezen and Jones 1998). Direct interaction results in diversifying selection of both R and Avr genes and an arms race between the pathogen and the host plant (Stahl and Bishop 2000). In indirect recognition systems, the R protein recognizes the pathogen’s effectors through detection of changes in the host proteins that the effectors target, also called the guardees (Van der Biezen and Jones 1998). This model is called the “guard model” and seems to be a more common system than the receptor-ligand model. The smuts are important pathogens that cause disease world-wide (Menzies, et al. 1996, Thomas 1989). In our laboratory, we are working on two closely related smuts, U. hordei and U. maydis, that infect barley and corn, respectively. U. maydis is a widely-studied pathogen and the complete genome for this pathogen has been sequenced, although no Avr genes have been described (Kamper, et al. 2006). The barley/U. hordei pathosystem is an excellent model system for small grain-infecting basidiomycetes due to the presence of “gene-for-gene”-based resistance. Six Avr genes have been described in U. hordei which in different combinations constitute 14 different reported races (Tapke 1945); six corresponding resistance genes have been identified in barley (Thomas 1976). The UhAvr1 gene has been identified in a genomic region of 85 kb (Linning, et al. 2004). To our knowledge, no Avr gene from basidiomycetes infecting monocots has been isolated so far. In this study, I present the identification of the UhAvr1 gene from U. hordei by targeted deletions of clusters of predicted secreted proteins, located between genetic markers that were previously identified (Linning, et al. 2004). I also used a complementation-based approach to restore the avirulence function in virulent deletion mutants. The region containing the UhAvr1 gene is syntenic to cluster 19A of U. maydis which contains small secreted proteins that have virulence functions (Kamper, et al. 2006). The data show that UhAvr1 is located in a transposon- and repeat-rich region and that (retro) transposon activity is responsible for breaking the avirulence towards Hannchen (Ruh1). In the virulent parent, it appears that this gene has been separated from its promoter and a region of more than 14 kb has translocated to another part of the genome.  51  2.2. Material and methods 2.2.1. Barley cultivars and U. hordei strains used in this study Four barley cultivars, namely universal susceptible Odessa (ruh1, ruh2, ruh6), and differentials Hannchen (Ruh1), Excelsior (Ruh2), and Plush (Ruh6) were used for pathogenicity assays of U. hordei in this study. Table 2.1 lists all wild-type and deletion mutant U. hordei strains used and generated in this study.  2.2.2. Growth conditions of U. hordei and barley and U. hordei transformation All haploid strains of U. hordei (Table 2.1) were grown in liquid complete medium (CM; Holliday et al. 1961) always supplemented with Ampicillin 100 µg ml-1 (Fisher Scientific), while Carboxin 2.5 µg ml-1 (Sigma-Aldrich), Hygromycin B 100 µg ml-1 (Calbiochem, La Jolla CA, USA) or Zeocin 40 µg ml-1 (Invitrogen, Valencia, CA, USA), were added when appropriate. Strains were grown for two to three days at 22 oC and then preserved in cryovials at -80 oC after adding 9% v/v filter-sterilized DMSO. When needed, strains were recovered on solid potato dextrose agar (PDA; potato starch 0.4%, dextrose 2%, agar 1.5% ), CM or YEPS (1% yeast extract, 2% peptone, 2% sucrose, 2% agar for solid media) media incubated at 22 oC for 3 days. For genetic transformation of U. hordei, protoplasts were prepared according to a modified protocol (Tsukuda, et al. 1988) but using 384 mg ml-1 Vinoflow FCE (Gusmer Enterprises) as enzymes for dissolving the fungal cell wall (Szewczyk, et al. 2006). The prepared protoplasts were used either fresh or stored at -80 oC like haploid sporidia. The protoplasts were transformed with 5 µg DNA mixed with 1 µl of 15 mg ml-1 heparin (Sigma H-3125) in STC (10 mM TrisHCL pH. 7.5, 100 mM CaCl2, 1M sorbitol) and selected on double-complete medium plate (DCM) supplemented with 1 M sorbitol and appropriate antibiotic. After 5-7 days incubation at 22 oC, colonies from DCM-S were transferred to CM medium and incubated for two days at 22 o  C before transferring to liquid CM medium for further analysis. Barley seeds were planted in general potting mix (Pro-Mix BX) in pots of 3 X 3 inches  that were placed in trays to a density of 3 seeds per pot and 18 pots per tray; both pots and trays had small holes for water drainage. Trays were placed in controlled-environment chambers  52  (Conviron, Winnipeg Manitoba Canada) or in the green house with an 18 hour light-6 hour dark cycle and held at 22°C.  2.2.3. Mapping of the UhAvr1 The UhAvr1 gene was mapped (Linning, et al. 2004) using a marker-based approach in a population of U. hordei haploid strains, Uh4857-4 (alias Uh364), Uh 4857-5 (alias Uh365), Uh4854-10 (alias Uh362), Uh4854-4 (alias Uh359) and their progeny. Briefly, a population was created by crossing Uh364 and Uh362 haploid strains on universal susceptible barley cultivar Odessa and fifty-four random progeny were selected, half had mating type MAT-1 and half MAT2. To identify virulence genotype, all the progeny were subsequently backcrossed to virulent parents (Uh362 or Uh359) depending on mating type, and tested for pathogenicity on differential cultivar Hannchen (Ruh1) and on Odessa (ruh1) as an inoculation control. This created a mapping population for measuring recombination frequencies. Bulked-segregant analysis was used for mapping Uhavr1 in pools of eight progeny segregating for UhAvr1 or uhavr1. BAC clone 3-A2 was isolated from the BAC library of the avirulent parent (Uh364) that contains the entire 85 kb UhAvr1 locus. Subsequently, two overlapping BAC clones 1-E2 and 2-G7 were isolated from the virulent parent that span the Uhavr1 locus.  2.2.4. Sequencing of ORFs in the UhAvr1 locus from the virulent parent and field isolates All open reading frames (ORFs) that coded for proteins predicted to be secreted and located in the UhAvr1 locus between the genetic markers, were amplified by PCR (using a mixture of Pfu and Taq polymerase) from strain Uh362 (avr1) virulent on barley cultivar Hannchen, and field isolates. Primers for these ORFs were designed 100 bp upstream and 100 bp downstream of the ORFs (Table 2.2) using the Primer3 software. The PCR products were run on 1% agarose gels and purified using a QIAquick Gel extraction kit (QIAGEN) following the manufacturer’s instruction. Sequencing of the purified products was carried out either at PARC (Summerland, BC) using the Big Dye terminator mix from Applied Biosystems and an ABI310 Genetic Analyzer (Foster City, CA, USA), or at the UBC-Okanagan campus in Kelowna, BC. The sequence data were compared using the sequence analysis and alignment software of the VectorNTI software package (Invitrogen). When appropriate, data were compared to the genome 53  sequence from U. hordei strain 364 (MAT-1) at MIPS (http://mips.helmholtzmuenchen.de/genre/proj/MUHDB/, to be made public in 2011) and from U. maydis strain 521 (a1 b1) at MIPS (http://mips.helmholtz-muenchen.de/genre/proj/ustilago/).  2.2.5. Sequencing of BAC clones from the avirulent and virulent parents by 454 method The UhAvr1 region of the avirulent parent (strain Uh364, represented by BAC clone 3-A2) had been sequenced using the GPS-Mutagenesis System (New England Biolabs) by J. Laurie (UBC Thesis, 2008; and unpublished). These sequences and those for this region obtained from the BAC end-sequencing of the source BAC genomic library (Genome Sciences Center, Vancouver, BC; unpublished data) were assembled using the PCAP.REP software suite (Huang, et al. 2006). Some manual sequencing was done to confirm regions and fill gaps. Genes were predicted using VectorNTI (Invitrogen) and FGENESH (Salamov and Solovyev 2000). Two BAC clones (1-E2 and 2-G7) were also obtained for the Uhavr1 region from the virulent parent (strain Uh362) via hybridization, by J. Laurie (UBC Thesis, 2008; and unpublished). These two, as well as the BAC3-A2 clone from the avirulent parent were sequenced using the 454 technology at the Plant Biotechnology Institute (Saskatoon, Sask.) and the resulting reads assembled using the Newbler program (Roche Applied Science). Alignment of the BAC sequences from the virulent parent along the avirulent backbone was facilitated by a custom Perl script provided by Matthew Links (Agriculture Canada, Saskatoon, Sask.). This was again followed by confirmatory manual sequencing and PCR reactions.  2.2.6. Deletion of the UhAvr1-containing region To make deletion mutants of the UhAvr1 gene or clusters of genes in the UhAvr1 locus, several plasmids were constructed using the DelsGate method (Garcia-Pedrajas, et al. 2010). Briefly, primers were designed separately for each construct to amplify 1.5-2 kb, 5/- and 3/- sequences flanking the target region (Table 2.2) by PCR using the Uh364 genomic DNA as a template. Primers 5L and 5R were then used for the amplification of the 5/ -flanking fragment which contains an I-SceI recognition sequence tail upstream and an attB1 sequence tail downstream. Primers 3L and 3R were used to amplify the 3/-flanking fragment which then contained the attB2 sequence upstream and the I-SceI sequence tail downstream. The two PCR-amplified fragments 54  were then gel-purified using the QIAquick Gel extraction kit and subsequently recombined into the pDonorCbx vector (NCBI accession number EU360889; Garcia-Pedrajas, et al. 2010) using the Gateway BP Clonase II enzyme Mix (Invitrogen). Two PCR reactions were performed for the verification of the deletion construct using 5/- gene-specific primer 5R in combination with the SceIF primer (Table 2.2), and 3/- gene-specific primer 3L in combination with primer SceIR primer (Table 2.2). SceIF and SceIR primers were designed for the I-SceI enzyme recognition site in the forward and reverse orientation, respectively. All deletion constructs used in this study were verified by sequencing. The deletion constructs were then linearized with I-SceI enzyme (New England, Biolabs), ethanol precipitated, and subsequently resuspended in STC (section 2.2.2) and used directly for transformation of U. hordei strains as described, above. Putative transformants (resistant to carboxin) on DCM-S media were transferred to CM media plates supplemented with carboxin for further analysis.  2.2.7. Analysis of deletion mutants Carboxin-resistant mutants were analyzed for proper gene deletion by two PCR reactions amplifying the 5/- flanking part that was used for making the deletion construct with primer DonF sits on the construct and another primer 150-200 bp upstream of 5/-flanking gene in the genome (Table 2.2). The second PCR reaction was carried out to verify the 3/-flanking part using primer DonR from the construct and another primer 150-200 bp downstream of the 3/ flanking part in the genome. U. hordei deletion mutants that were positive for both PCR reactions were further verified by Southern bloting analysis to confirm proper homologous deletion of the gene. Southern blot analysis was carried out in the same way as described in Chapter 4 of this thesis. Briefly, genomic DNA from either PCR-positive deletion mutants or wild type strains was digested with two different restriction enzymes. A probe for either the 5/- or 3/- flanks was amplified using PCR in a way that would yield different size bands in the wild-type strain and deletion mutants.  2.2.8. Plasmids to complement U. hordei deletion mutants ORFs of Uh10021, Uh10022, and Uh10024 of U. hordei and ORFs Um05295 and Um05296 of U. maydis, either with or without the sequence coding for the signal peptide (SP), but without  55  their stop codon, were each amplified by PCR with a CACC tetranucleotide sequence at the 5/ end to allow for directional cloning into Gateway entry vector pENTRTM /D-TOPOTM (Invitrogen). Kanamycin-resistant colonies were verified by PCR and inserts were sequenced using primers M13F and M13R. Plasmid DNA was purified from E. coli using the plasmid mini extraction kit (QIAGEN) following the manufacturer’s instruction. The genes were subsequently transferred to the expression vector, pUBleX1Int:GateWayHA (Fig. 2.1), using the LR recombineering reaction (Invitrogen). For the construction of pUBleX1Int:GateWayHA, a synthetic linker containing a HA epitope tag was used (bold, translates into YPYDVPDYA) and a stop codon (red), flanked by BamHI/BglII and KpnI cohesive ends (underlined) and an NruI restriction enzyme site (italic) for blunt-end cloning of GateWayTM recombineering cassette, reading frame B (to allow in-frame fusions with the HA epitope tag). Primers that were annealed to obtain this linker were: Bam-HAtag-Kpn_fw GATCCTCGCGATATCCGTACGACGTACCAGACTACGCATGAGGTAC and Bam-HAtag-Kpn_rev CTCATGCGTAGTCTGGTACGTCGTACGGATATCGCGAG. The annealed product was ligated to a shuttle vector, cut with BamHI and KpnI. The GateWayTM cassette, reading frame B (Invitrogen), was inserted into the unique Nru1 site. Subsequently, the GateWay-HAtag-STOP cassette was amplified by PCR with primer Gateway-5'_BglII (GGAAGATCTCGATCAACAAGTTTGTAC) which adds a BglII site (underlined) and primer MCG161_nos_fw (agaccggcaacaggattcaatc) which sits upstream of another flanking BglII site in the shuttle vector. Because there is a third BglII site in the GateWayTM cassette, reading frame B fragment, the PCR products was digested partially with BglII to yield a 1,860 bp fragment which was inserted into the unique BglII site of integrative, Ustilago-specific expression vector pUBleX1Int (Hu, et al. 2007). To complement U. hordei deletion mutants with a whole-gene construct, Uh10022 with its native promoter and terminator sequences was amplified by PCR using primers 1616 and 1617, each containing a NotI restriction enzyme site. The PCR product was digested with NotI and cloned in the NotI site of plasmid pHyg101 (Mayorga and Gold 1998) using T4 DNA ligase (Invitrogen). The construct was verified by PCR and linearized by SspI to allow stable integration in the genome of U. hordei following the transformation method described, above.  56  2.2.9. Western blot analysis Total protein was isolated from frozen ground cells, as described (Laurie, et al. 2008). Protein samples were boiled for five minutes and spun briefly for 30 sec before being applied on a 12.5% SDS-PAGE for separation using a Bio-Rad Mini-Protean III. Protein was transferred from the gel to Eeqi-Blot PVDF Western blotting membrane (Bio-Rad) using a Bio-Rad liquid transfer apparatus following the manufacturer’s recommended protocols. Western blot hybridizations were carried out according to a standard protocol (Harlow and Lane, 1988). Membranes were probed with 200 ng ml-1 rat anti-HA high affinity monoclonal antibody. For detection of primary bound antibody, membranes were incubated with peroxidase-conjugated AffiniPure Goat AntiRat-Ig (H+L) secondary antibody according to supplier’s instruction. For visualization of bound antibody, the Enhanced Chemiluminescence system (ECL) plus Western Blotting Detection Reagents (Amersham Biosciences/GE Healthcare) were used.  2.2.10. Mating test U. hordei mating tests were carried out for all deletion mutants before pathogenicity tests were performed on barley cultivars. Strains of interest were grown in CM liquid medium with appropriate antibiotics for 36-48 hours in a shaking incubator at 22 oC to reach an OD600 of 0.60.8. Two haploid strains of opposite mating type were then mixed 1:1 (v/v) and 30-50 µl were spotted on CM media plates supplemented with 1% (w/v) activated charcoal and incubated at 22 o  C for 36-48 hours. A positive reaction in a colony, indicating mating, was visible as a white  “fuzzy” phenotype.  2.2.11. Pathogenicity assays Pathogenicity assays were performed as follows: Haploid U. hordei strains of opposite matingtype backgrounds (such as deletion mutant strains in the Uh364 (MAT-1, Avr1) to the Uh362 (MAT-2, avr1) parent) were grown separately in CM media with appropriate antibiotics in a shaking incubator for 36-48 hours to reach an OD600 of 1-1.5. The two cultures of opposite mating types were mixed 1:1 v/v before inoculation of the barley seeds. The deletion mutants were also mixed with Uh365 (MAT-2, Avr1) wild-type strains to complement the deletion mutant. Wild-type combination Uh364 x Uh362 was used as a control. Seeds of barley cultivar  57  Hannchen (Ruh1) and Odessa (ruh1) that were previously surface sterilized and dried were dipped in the mixed cultures and a vacuum of 20 psi was applied for 20 minutes. Subsequently, the seeds were placed in sterilized plates on tissues to drain the excess culture at room temperatures for 4-6 hours. The seeds were then sown in potting mix (Pro-Mix BX) as described, above. Disease rating was scored 2 months after heading of the plants by counting infected plants in all inoculated plants. All pathogenicity tests were repeated at least three times.  2.2.12. Nucleic acid manipulation For deletion mutant analysis, total genomic DNA was isolated according to a modified protocol for miniprep of Ustilago genomic DNA (Elder, et al. 1983). For cloning and DNA blot analysis, total genomic DNA was isolated using the DNeasy Plant Maxi kit (QIAGEN Mississauga, Ontario, Canada) following the manufacturer’s instructions (for more detail, see Material & Methods section 4.2.4 of genomic DNA isoalation in Chapter 4). Routine Polymerase Chain Reaction (PCR) was conducted using recombinant Taq polymerase (Invitrogen) or when required, Pfu polymerase (Fermentas Life Science). For cloning and labeling, PCR products were either gel-purified using a QIAquick Gel extraction kit (QIAGEN) or they were directly purified using the QIAquick PCR purification Kit (QIAGEN) according to manufacturer’s instruction.  2.2.13. qRT-PCR analysis Mated U. hordei wild-type strains Uh362 and Uh364 were inoculated on three day-old seedlings of barley cultivars Odessa and Hannchen. Inoculum was prepared by collecting cells from two day-old cell cultures by centrifugation, mixing of opposite mating types and then painting the cell paste onto coleoptiles with cotton swabs. Barley seeds were also inoculated in the same way as described, above, in section 2.2.11, and then germinated in the dark on moist filter paper in Petri plates. Samples were collected from inoculated plants at 24, 48, 72, 96, and 120 hours post inoculation and used for RNA isolation. RNA was also isolated from infected immature barley heads dissected from approximately 5 week-old plants and from mature heads filled with teliospores. As a control, RNA was isolated from U. hordei cells mated on charcoal plates. 1 µg of total RNA that was isolated and purified using a QIAquick RNeasy extraction kit (QIAGEN)  58  according to the manufacturer’s instructions, was treated with DNaseI, amplification grade (Invitrogen). First-strand cDNA was synthesized using a Dynamo SYBR Green 2-step qRT-PCR kit (FINNZYMES) following the manufacturer’s instruction. These samples were then diluted ten times and real-time qPCR was performed with specific primers on an Mx3000P qPCR instrument (Stratagene, La Jolla, CA, USA). Amplification cycles were as follows: 15 min at 95 o  C, followed by 40 cycles of 30 s at 94 oC, 30 s at 63 oC, 30 s at 72 oC; fluorescence data were  collected at 63 oC at each cycle. The reliability of the product was verified by acquisition of a dissociation curve at the end of each run. The sequence of the primers for the genes of interest and the housekeeping control gene are shown in Table 2.2.  2.3. Results 2.3.1. Sequencing of ORFs from the virulent parent and field isolates Lining, et al. (2004) identified three genetic markers linked to UhAvr1 in a mapping population of 54 progeny segregating for this gene (Fig 2.2). The AFLP marker was converted to a probe for screening a cosmid library. Part of a positive cosmid clone was then used as a probe to identify from a BAC library from avirulent parent, Uh364 (Avr1), five overlapping BAC clones that spanned the whole locus (Lining R 2004). A single BAC clone, BAC3A-2 was selected because it contained the entire UhAvr1 locus. This clone was sequenced by GPS transposon insertion resulting in a sequence of 117 kb with two small gaps (J. Laurie, UBC Thesis, 2008; and unpublished). Genes were predicted using VectorNTI (Invitrogen) and FGENESH (Salamov and Solovyev 2000) and in this region 47 ORFs were identified (Table 2.3). The sequence analysis of this BAC clone revealed that UhAvr1 locus is syntenic to a region in U. maydis on contig 1.191 spanning a complete cluster, called 19A, the largest cluster in U. maydis harbouring 26 small secreted proteins (Kamper, et al. 2006). To identify the UhAvr1 gene by sequence comparison, I hypothesized that it could encode a secreted protein. Predicted secretion signals in eight ORFs were identified by SignalP 3.0 and TargetP 1.1 in the region identified by genetic marker analysis; three fall outside this region (Fig 2.2, RAPD and RFLP; Lining et al., 2004). A change from avirulence to virulence (or vice versa) could potentially be caused by mutations in the candidate gene; therefore, I wanted to compare candidate genes encoding putative secreted proteins between the avirulent 59  and virulent parent, and among a collection of eight field isolates collected from different parts of the world whose virulence was known (Table 2.1; two avirulent and six virulent on barley cultivar Hannchen, Ruh1). These predicted ORFs with secretion signals were sequenced from virulent strain Uh362 (avr1) and the eight field isolates. ORFs were amplified by PCR from genomic DNA with primers designed 100 bp upstream and 100 bp downstream of each ORF (Table 2.2). The PCR products were sequenced directly using the primers listed in table 2.2. Sequence analysis revealed point mutations in four ORFs, Uh10021, Uh08127, Uh08128 and Uh08132, between the parental strains. In Uh10021, two point mutations were identified, one at 21 nt upstream of the ATG start codon and the other at 165 bp downstream of the ATG. The latter mutation, G-to-A, changes a valine to an isoleucine in the virulent parent. Uh08127 had one point mutation in the ORF, 657 bp downstream of the start codon, an A-to-G that translates into a single amino acid difference between the parental strains, a methionine into an isoleucine in the virulent parent. One point mutation was identified in Uh08128, 264 bp downstream of the ATG, a T-to-C translating a serine into a proline in the virulent parental strain. In gene Uh08132, a single point mutation in the stop codon shifts the frame, resulting in a longer protein sequence in the virulent parent. Among the field isolates, only one, Uh813 collected from Iran, revealed a point mutation in the ORFs of Uh08127, Uh08128, Uh08132, Uh08139 and two bp changes in Uh10022, that each translated into single amino acid changes. Unfortunately, all these above mentioned point mutations did not correlate with the genotypes Avr1 or avr1 in the field isolate collection and were, therefore, not followed further for avirulence gene analysis.  2.3.2. Delimiting of the UhAvr1-containing region by deletion analysis To identify the UhAvr1 gene in the 85 kb region between the markers, it was divided into four overlapping fragments (Fig 2.3), ranging from 15 to 38.5 kb based on the number of ORFs in the region coding for predicted secreted proteins. The DelsGate method (Garcia-Pedrajas, et al. 2010) was used to prepare the deletion mutants of these fragments (see Material and Methods). The deletion constructs were then transferred to Uh364 (Avr1) protoplasts to make the Avr1 deletion mutants. Sixty to as many as 300 carboxin-resistant colonies sometimes needed to be screened to get at least four PCR positive transformants for each construct. These putative deletion mutants were further verified by Southern blot analyses (Fig. 2.3) to confirm expected homologous deletion mutants. The efficiency of homologous recombination was different for 60  different constructs and seemed dependent on the size of the deletion fragment; the efficiency was higher for small fragments. No phenotypic differences or abnormal growth were observed for any of the tested haploid basidiospore mutants and, also, proper mating with compatible haploid basidiospores was observed. At least one deletion mutant for each fragment was inoculated on barley cultivars Hannchen and Odessa after mixing with compatible virulent parent Uh362. The deletion mutants that were used for pathogenicity tests are listed in Table 2.1. Inoculation of deletion mutants and the wild-type U. hordei strains on barley cultivars Odessa (ruh1) and Hannchen (Ruh1) clearly indicated that the fragment called C19A2 contained the avirulence gene Avr1 (Fig 2.4). The wild-type strain Uh364 and the mutants deleted for three fragments, C19A3, C19A4 and C19A5, when mated with virulent partner Uh362 of the opposite matting type, caused disease on Odessa but not on Hannchen, indicating that these deletion mutants were avirulent on barley cultivar Hannchen and, therefore, still contain a functional Avr1. The C19A2 deletion mutants however, caused disease on both barley cultivars when mated with virulent parent Uh362, proving that the UhAvr1 gene is present on this fragment. This deletion mutant strain was named Uh1041 (Uh364-∆19A2 Table 2.1) and will be referred to as such for the remainder of the thesis. Strain Uh1041 also causes a higher rate of disease towards Odessa than the wild-type strains (Fig 2.4). To independently test whether the disease on barley cultivar Hannchen was the result of UhAvr1 deletion, I also inoculated this barley cultivar with a cross of Uh1041 with the avirulent strain Uh365 (Avr1), which resulted in disease on Odessa but not on Hannchen. I also used another control in which Uh1041 (∆Avr1, Avr6, Avr2) was inoculated on two other barley differential cultivars, Plush and Excelsior, that have the resistance gene, Ruh6 or Ruh2, respectively, after crossing with Uh362 (avr6, avr2). Uh1041 did not produce disease on any of these cultivars, which verifies that the virulence of this mutant on Hannchen is due to deletion of the UhAvr1 gene in this region.  2.3.3. Deletion of fragment C19A2 in both mating partners does not impair virulence towards Odessa In other pathosystems, the deletion of avirulence genes/effectors was shown to affect virulence. To determine whether genes on fragment C19A2, which includes 5 SSPs, have any virulence functions in U. hordei, I needed to construct a deletion mutant in the mating partner as well. A mutant deleted for the C19A2 fragment was obtained in the other mating type partner by  61  crossing Uh1041 (C19A2 deletion mutant, MAT-I) with the virulent parent, Uh362 (MAT-2), on barley cultivar Hannchen. Basidiospores were collected from teliospores of infected heads and selected for carboxin resistance. Carboxin-resistant basidiospores of mating type 2 (MAT-2) were obtained by performing mating tests with Uh364 (MAT-1) and Uh359 (MAT-I) on DCM plates supplemented with active charcoal. Deletion mutants of mating type 2 were further verified by Southern blot analysis (Fig. 2.5). Seeds of barley cultivar Odessa and Hannchen were then inoculated after mixing equal amounts of Uh1041 culture with C19A2-deletion mutants of mating type 2, and disease was scored after heading of the barley plants. Three individual C19A2 deletion mutants, Uh1116, Uh1117 and Uh1118, of mating type 2 were tested in this study. Virulence of the cross lacking any C19A2 components towards Odessa was similar to the wild-type cross (Fig. 2.5), as measured by counting percent diseased plants out of total inoculated plants. The percentage disease on Hannchen seemed also not affected compared to the single deletion mutant, indicating that the genes located on fragment C19A2 did not contribute significantly to virulence on barley.  2.3.4. Complementation of C19A2 deletion mutants A 38.5 kb fragment, C19A2, contains the functional UhAvr1 gene; this fragment encodes five predicted secreted proteins, three of which are located between the genetic markers delineating the locus. I focused on these three genes as potential candidates for UhAvr1. A BAC subclone was identified in this region that contains two of the predicted secreted protein-coding ORFs, Uh10021 and Uh10022 (Fig 2.6). BAC 1-6 was previously cloned in pUSBAC5 (BAC vector derivative of pEcBAC1; (Frijters, et al. 1997). and converted for use in Ustilago species by introducing a specific hygromycin B resistance cassette (Linning, et al. 2004). Two C19A2 deletion mutants complemented with BAC1-6 (Uh1205 and Uh1207; Table 2.1) were inoculated on barley cultivars Odessa and Hannchen after mixing with the compatible virulent strain. No abnormal growth or defect in mating behavior was observed in these haploid complemented strains. The complemented strains caused the same level of disease on Odessa as the wild-type combination and the deletion mutants, while on Hannchen the level of disease was severely reduced (Fig 2.6). On Odessa, the disease level varied from 25-40% while on Hannchen the disease was only 2-2.5%, which strongly suggests that BAC 1-6 contains the functional UhAvr1 gene. 62  To find the UhAvr1 gene, the deletion mutant strain Uh1041 was individually complemented with each of the three genes, Uh10021, Uh10022 and Uh10024. For complementation with the individual genes, these ORFs were cloned both with signal peptide (predicted by SignalP 3.0) and without signal peptide in integrative plasmid pUblexInt:GateWayHA under control of the HSP70 constitutive promoter. Deletion mutant strains complemented with these different genes were analyzed by Western blot analysis to confirm the expression of the transgenes. As shown in Fig 2.7, all genes were expressed at a high level in all of the complemented lines tested. Two complemented strains for each individual gene were selected for pathogenicity tests on the barley cultivars, Odessa and Hannchen. The pathogenicity data did not confirm that these individual genes are able to complement the avirulence function in the 38.5 kb deletion mutant strain, Uh1041 (Fig. 2.7).  2.3.5. Fragments C19A2-C and C19A2-D contain UhAvr1 In order to determine the location of the UhAvr1 gene on the 19A2 fragment of 38.5 kb, this region was divided into five sub-fragments, C19A2A-C19A2E (Fig 2.7), for making further deletion mutants. To generate the sub-deletion mutant constructs, the primers were designed in such a way that the three predicted secreted protein encoding ORFs would be deleted in two different deletion constructs (Fig 2.8). The 1.5 to 2 kb flanking regions of the targeted fragments were amplified by PCR using the primer combinations listed in Table 2.2 and subsequently cloned in pDONR-Cbx by BP clonase II. The sub-deletion constructs were then linearized with I-SceI enzyme for integration into the genome of Uh364. Sixty-four PCR-positive deletion mutants were obtained for the five deletion constructs, which were further verified by Southern blot analysis (Fig 2.8). Nine deletion mutants were selected, two for each deletion mutant, except for C19A2-B for which only one expected deletion mutant was obtained, and were tested for virulence towards Hannchen. The virulence of each of these deletion mutants after mating with the virulent partner Uh362 (avr1) on Hannchen was assessed the same way as described, above. Four deletion mutants, two for C19A2-C and two for C19A2-D, were virulent towards both barley cultivars Odessa and Hannchen (Fig 2.9). The virulence of these mutants towards Hannchen is because of disruption of the UhAvr1 gene. The deletion mutants for the other three fragments, two for each of C19A2-A and C19A2-E, and one for C19A2-B, produced disease on Odessa, the universal susceptible host but not on Hannchen (Ruh1). These findings indicate that 63  each deletion mutant had the intact UhAvr1 gene and, therefore, could not cause disease on Hannchen that recognizes UhAvr1.  2.3.6. Overlapping regions of the fragments C19A2-C and C19A2-D contain UhAvr1 As deletion mutants of both fragments, C19A2-C and C19A2-D, were virulent towards Hannchen, the overlapping region in these fragments was hypothesized to contain the functional UhAvr1 gene. I found Uh10022 as the only ORF in this region using VectorNTI (Invitrogen) and FGENESH (Salamov and Solovyev 2000). Uh10022 encodes a predicted secreted protein (as identified by SignalP 3.0, TargetP 1.1 and ProtComP 9 prediction) and was a strong candidate for the UhAvr1 gene. Another deletion mutant was produced in which the 3′-end (319 bp) of the Uh10022 ORF was deleted by making a construct (Fig. 2.10) in which 2 kb flanking each side of this fragment was amplified by PCR using primer combinations listed in Table 2.2. The two amplified DNA fragments were cloned into pDONR-cbx as described, above, and linearized for integration into genomic DNA of the avirulent strain Uh364. A total of 110 carboxin-resistant colonies were screened by PCR for the proper deletion mutant and ten colonies were identified to be positive for both flanks. The Southern blot analysis of the eight transformants showed that five of them were proper homologous deletion mutants. Two of these transformants were selected and inoculated on the barley cultivars, Hannchen and Odessa. Both deletion mutants were virulent toward Hannchen and produced 30-40 % disease, which confirmed that Uh10022 is the UhAvr1 gene (Fig 2.10). Quantitative RT-PCR was used to analyse the expression of Uh10022 at various life cycle stages. RNA was isolated from plants infected with virulent and avirulent strains from mature or immature infected heads, or from in vitro-grown or mated basidiospores (see Material and Methods); however, the expression of this gene was not detected during infection under the conditions tested (data not shown). The expression of the housekeeping control gene was detected in the RNA samples collected from cells mated on charcoal plates, in the mated cell mix inoculated on the coleoptiles, and in both immature and mature infected heads; however, in all these conditions, expression of Uh10022 was not detected. The expression of the housekeeping gene or Uh10022 in RNA samples collected from inoculated seeds was not detected, likely because of limited fungal biomass (data not shown).  64  2.3.7. Sequence comparison between the Avr1 and avr1 loci in the parental strains After genetically confirming that Uh10022 is the UhAvr1 gene, this gene was sequenced from BAC clone 1E-2 isolated from virulent parent Uh362 (avr1) using several primers combinations as listed in the Table 2.2. The homolog of Uh10022 was previously obtained by PCR from genomic DNA of the virulent parent, Uh362, and sequenced; however, further analysis revealed that this gene was not present on BAC clone 1-E2, nor on BAC2-G7, although these two BAC clones are overlapping and span the whole region as shown in Figure 2.11. The sequence obtained from BAC1-E2 (Material and Methods) matched the sequence of the avirulent parent up to 134 bp upstream of the Uh10022 start codon. After this point, which I called the break point, the sequence was no longer syntenous with the avirulent parent (Fig. 2.12). WUBLAST analysis of the 400 bp sequence after the break point indicated matches to two retrotransposon genes (Uh14086, Uh14170) in the U. hordei genome. PCR analysis of Uh10022 and several other downstream genes on the BAC3-A2 clone revealed that almost 14 kb (containing nine predicted ORFs) was not present at this locus on BAC clones 2-G7 and 1-E2 from the virulent parent. PCR analysis confirmed the presence of all other ORFs flanking this 14 kb gap on these two BAC clones (Fig. 2.12). This was further verified by sequencing the ends of these two BAC clones and re-confirmed that they are overlapping and reside in this region. PCR was used on genomic DNA of the virulent parent, Uh362, for amplification across the break point using several primers combinations (Table 2.2), but no combination resulted in a PCR product. This suggested that these two regions are not physically close in the genome; however, as shown in section 2.3.1, I can amplify by PCR and sequence the ORFs of Uh10021 and Uh10022 from genomic DNA of the virulent parent but cannot amplify them as one physical fragment by PCR from the virulent parent, although both ORFs and the intergenic region span less than 2 kb in the avirulent parent. To measure the insertion, a PCR product of 3 kb was amplified from genomic DNA from the virulent parent using primer 1513 (600 bp downstream of the break point in the forward direction) and primer 1741 (within gene, Uh10026, in the reverse orientation; Table 2.2, Fig. 2.12). The combined data suggest that in the virulent parent, this 14 kb-region at this locus has translocated to another part of the genome and has been replaced by an insertion of 3.6 kb, harbouring transposable element (TE) sequences. It is likely that this event was caused by TE activity (Fig. 2.11). This translocation separated the Uh10022 ORF from its promoter and likely  65  caused changes in the expression of the Uh10022 (UhAvr1) gene and, hence, a conversion to a virulence genotype.  2.3.8. Variable sequences at the UhAvr1 locus point to TE activity The Uhavr1 locus was sequenced from several virulent field isolates (Uh805, Uh815, Uh820, Uh822, Uh811 and Uh818) collected from different parts of the world (Table 2.1). PCR amplification across the break point failed in all virulent field isolates when several primer combinations matching different positions for the avirulent parent Uh364 were used (Table 2.2). To sequence the region spanning the break point, PCR products were generated using the primer combinations used for the sequencing of the region from the virulent parent BAC clone 1-E2. The sequence analysis of the PCR-amplified fragments confirmed the insertion of TE sequences (as identified in virulent parent Uh362) in all virulent field isolates sequenced in this study. This suggests that Uh10021 and Uh10022 are not physically connected to each other in the genome of all of these field isolates and is similar to the virulent parental strain Uh362; TE sequence insertions are present at this locus. Several mutations were revealed among the transposable element sequences in the different virulent strains. One predominant mutation found in four virulent strains (Uh362, Uh805, Uh815, and Uh820) is a ten bp-insertion of a repeat (GAGAGAGAGC) that is absent from three other virulent strains (Uh811, Uh818, and Uh822). The UhAvr1 locus was also sequenced from three avirulent field isolates (Uh813, Uh1273, and Uh1283) that were collected from different parts of the world (Table 2.2). In avirulent field isolate Uh813, a two bp mutation in the ORF of Uh10022, 506 bp downstream of the ATG, was identified that translates into a single amino acid change (isoleucine to argenine) in the C-terminal end of this protein. Since this strain is still avirulent on Hannchen, this change does not seem to affect its avirulence function. The locus from these field isolates was similar to the avirulent parent, Uh364. Uh10021 and Uh10022 are close to each other in the genome and could be amplified by PCR. Primers 13897F and 10022R (Table 2.2) were used for amplification of Uh10021 and Uh10022 on one PCR product but revealed that these products were roughly 340 bp larger in these strains than in the parental strain Uh364. Interestingly, in all the three avirulent field isolates tested here, the 340 bp addition was caused by an insertion in the intergenic region between Uh10021 and Uh10022 (Fig. 2.13). WUBLAST analysis of the 340 bp-insertion in these three avirulent strains indicated matches to TE sequences in U. hordei. The 66  340 bp-insertion was flanked by six bp repeats (TGGGTT), possibly a footprint of TE activity (Fig. 2.13). This insertion was not found in the virulent parent Uh362 or in the six virulent field isolates tested here. Overall, these sequence analyses and the presence of TE-related sequences suggests TE activity at this locus.  2.3.9. Lack of complementation of the C19A2 deletion mutant with U. maydis homologs of Uh10022 A search using WUBLAST of the Uh10022 protein sequence identified one paralog in the U. hordei genome (Uh10021) and three orthologs in the U. maydis genome (Um05295, Um05296/Um12302, and Um05294). Phylogenetic analysis of these protein sequences, including two more related U. maydis proteins, Um05297 and Um05298, revealed that Uh10022 is a homolog to Um05295 and Um05296 (Fig. 2.14). To determine whether the two U. maydis genes are functional homologs of Uh10022, both genes were cloned in the pUblexInt:GateWayHA expression vector expressed from the constitutive Hsp70 promoter, using primers listed in Table 2.2. Upon transformation, the expression of these genes in the complemented deletion mutant strains was similar to those complemented with Uh10022 based on protein blot data (not shown). Four complemented U. hordei C19A2 deletion mutants, two for each of Um05295 and Um05296, were selected for pathogenicity assays on barley cultivars Odessa and Hannchen. These complemented deletion mutant strains were crossed with virulent strain Uh362 before inoculation on barley seeds. All complemented strains were virulent on Hannchen and produced levels of disease on this cultivar similar to Uh1041 (Fig. 2.15).  2.3.10. Synteny between U. hordei and U. maydis at the UhAvr1 locus As mentioned above, the UhAvr1 locus is syntenic to the region harbouring U. maydis cluster C19A, which contains twenty six predicted secreted protein encoding genes. In U. maydis, deletion of this cluster resulted in reduced disease on corn seedlings (Kamper, et al. 2006). SIMAP analysis and two-directional BLASTP searches were used to find orthologs of all proteins at this locus in the U. maydis genome. Orthologous proteins were found for several proteins at this locus (Table 2.3). The synteny is highly conserved on both flanks of cluster C19A (Fig 2.16); however, the sequences of the predicted secreted protein encoding ORFs are much diverged and rearrangements, including changes of gene orientation and several translocations of 67  genes within the cluster, are apparent. Genes encoding DigA protein on one side of the region are co-linear between the two species revealing proteins of very similar lengths. On the other side of the locus, two genes encoding a tubulin beta chain protein and another protein related to a VPS10 domain-containing receptor, SorCS1 precursor, are conserved, revealing a similar transcriptional orientation. In U. maydis, adjacent to the digA gene, an oligosaccharyltransferase gene is directly flanking the cluster of genes for secreted proteins, while in U. hordei this gene is transcriptionally inverted and located on the other side of the fragment that is shown by the red colored two-sided arrows (Fig. 2.16). This region is 38 kb larger than in U. maydis, seemingly the result of insertions of transposons and repetitive DNA (Fig. 2.16). In U. maydis, there are five families of SSP genes that are tandemly arranged in clusters of several paralogs. In U. hordei, most of these families are represented by a single gene; in two cases, the family is represented by two paralogs. UhAvr1 shares little homology with two U. maydis genes however they are in strikingly syntenic locations. In contrast, in U. hordei, there are several transposons and repeats located at this locus interspersed with the SSP coding-genes. As shown by the twosided arrows in Figure 2.16, two fragments of U. maydis containing several ORFs appear in opposite orientation in U. hordei. Also, both of these fragments in U. hordei are much larger than in U. maydis, possibly caused by insertion/activities of transposons, since three of these genes are related to TEs.  2.4. Discussion The isolation and characterization of avirulence genes from different plant pathogens including fungi has been a long-term goal in plant pathology. Avirulence genes play key roles in determining genetic compatibility with plants and can induce resistance in host plants having corresponding resistance genes, the so-called effector-triggered immunity. I have shown in this study that Uh10022 is the UhAvr1 gene responsible for the induction of the defense response in Hannchen in a gene-for-gene manner. Avirulence towards Ruh1 is broken down by TE activity and translocation of the coding region of UhAvr1, removing its promoter region and likely disrupting expression. In this study, I aimed to identify the UhAvr1 gene in U. hordei that was previously mapped to an 85 kb genetic interval through a genetic marker-based approach (Linning, et al.  68  2004). Eleven ORFs were identified at this locus that encodes predicted secreted proteins. They were annotated as ‘hypothetical’ with no known matches in public databases. To identify UhAvr1 in the 85 kb region, I first used sequence comparisons of the predicted secreted protein coding ORFs between the two parental strains of the population and several field isolates collected from different parts of the world. The objective was to look for mutations linking genotypes UhAvr1 or Uhavr1 to phenotypes among the virulent and avirulent parental and field strains; however, the sequence comparisons of these ORFs did not provide conclusive data for the identification of a gene candidate for UhAvr1, despite several point mutations revealed in several ORFs between the virulent and avirulent parents (i.e., Uh10021, Uh08127, Uh08128 and Uh08132). It is possible that some of the point mutations that I identified in several of the small secreted proteins could be related to some other avirulence or virulence genes in this pathogen not recognized by Ruh1 in Hannchen. Nevertheless, the data presented demonstrate that the change from avirulence (UhAvr1) to virulence (uhavr1) is not due to the mutations in the ORFs or the presence or absence of ORFs in these two strains. The alternative hypothesis was that the avirulence gene may not be expressed in virulent strains because of a mutation(s) in the promoter region, possibly a promoter disruption by a transposable element insertion, as has been shown for several other avirulence genes from fungi (Kang, et al. 2001). A targeted deletion and complementation-based approach was used to identify the UhAvr1 gene and demonstrate that this gene is required for Ruh1-based resistance in barley cultivars towards U. hordei. A deletion mutant was produced by the DelsGate method (GarciaPedrajas, et al. 2010) that showed virulence towards barley cultivar Hannchen (Ruh1) while still avirulent to other cultivars such as Plush (Ruh6) and Excelsior (Ruh2). The deletion mutant Uh1041 in which a 38.5 kb region (C19A2) was deleted, could be complemented with 11 kb genomic fragment containing the predicted secreted proteins Uh10021 and Uh10022; however, this restoration of avirulence towards Hannchen was not complete and a very low level of disease remained that varied from 2-2.5% compared to the deletion mutant which produced 40% disease. I speculate that the reason for this low level of disease could be the result of reduced expression of transgenes not at the same level as in the wild type avirulent strain. Inadequate expression could result from two reasons; either the fragment, including the genes contained within it, being integrated into a part of the genome that expressed at a low level or the fragment was not complete and transgenes did not contain sufficient promoter sequences. I did not verify  69  the expression of the genes. Similar results have been shown for Fusarium oxysporum f. sp lycopersici mutant strains complemented with the Six1 (Avr1) avirulence gene that did not restore complete avirulence towards tomato lines that contained the resistance gene I-3 (Rep, et al. 2004). To identify the gene that is specifically responsible for the avirulence phenotype in Hannchen, the C19A2 fragment was further divided into five deletion fragments. This result in the identification of a region overlapping in fragments C19A2-C and C19A2-D, pointing to Uh10022 as UhAvr1. This was further confirmed by deleting 340 bp from the 3/-end of Uh10022; however, complementation of the C19A2 deletion mutant with Uh10022, both with and without the N-terminal signal peptide, was not successful in restoring the avirulence function towards Hannchen. The complementing gene in these integrative constructs was expressed from the constitutive Hsp70 promoter, causing over-expression, which may have interfered with proper processing and translocation into plant cells. Alternatively, it is possible that the transgenes were hampered in their activity because of position effects and may be functional only at their native avirulence gene’s locus. Another scenario is that UhAVR1 needs another gene close by for its proper function, possibly required either for mRNA stability or proper folding of protein after translation. In the C19A2 deletion mutant, 38.5 kb is deleted that contains several genes in addition to Uh10022. In the oomycete Phytophthora sojae at least one avirulence gene Avr1b-1 needs another gene Avr1b-2 for mRNA accumulation and the avirulence function towards soybean (Shan, et al. 2004). The two orthologs of Uh10022 from U. maydis also did not complement the avirulence function towards Hannchen. However, my experiments are not conclusive as to whether they are properly transferred and active in the host and are thus true functional homologs of Uh10022, similar to the lack of complementation as disussed for Uh10022. One characteristic feature of Avr genes is that their expression is induced inside the plant during infection (Dodds, et al. 2004, Lauge and de Wit 1998, Rep, et al. 2004). I did not detect the expression of Uh10022 in any of the conditions tested. As discussed earlier, after penetration, U. hordei proliferates heavily only in the developing head and seeds in inflorescences, where the fungus induces the disease symptoms. The RNA samples collected from germinating, inoculated seeds did not have detectable levels of expression even of the housekeeping gene, suggesting very low levels of fungal biomass at this stage. In all other samples, i.e. immature and mature  70  infected heads and mated cells from charcoal plates, high levels of expression were detected for the housekeeping gene but not of Uh10022. Based on these findings, I speculate that the expression of Uh10022 is highly regulated and might be expressed only during the early stage of infection and possibly at a very low level. Some fungal Avr genes are expressed in specific infection structures such as appressoria or haustoria (Bohnert, et al. 2004, Dodds, et al. 2004, Catanzariti, et al. 2006). On the other hand, some fungal Avr genes are expressed during the whole infection process uniformly and are not organ-specific (Lauge and de Wit 1998, Luderer, et al. 2002b, Rohe, et al. 1995). Two Avr genes from L. maculans, AvrLm1 and AvrLm6, have been shown to be expressed constitutively at low levels (Fudal, et al. 2007). UhAvr1 is predicted to encode a small secreted protein of 171 aa after cleavage of a signal peptide with no cysteine residues in the mature protein. Such low content of cysteine residues has also been found for other fungal genes cloned by map-based strategies. Cysteinepoor effectors are common in biotrophic pathogenic fungi such as the basidiomycetes and oomycetes that form close associations with the host plants through haustoria (Allen, et al. 2004, Armstrong, et al. 2005, Ellis, et al. 2006, Rehmany, et al. 2005, Shan, et al. 2004). These secreted effectors are suggested to be translocated to the cytoplasm and, thus, spend only a short time in the extracellular space where they could be degraded by proteases. (Birch, et al. 2006, Dodds, et al. 2004, Dou, et al. 2008b, Haas, et al. 2009, Jiang, et al. 2008, Kale, et al. 2010, Khang, et al. 2010, Tyler, et al. 2006, Whisson, et al. 2007). Although U. hordei does not make haustoria within barley plant cells, it forms close interactions with interfaces surrounding mycelial tubes sometimes transversing plant cells within the host plant (Hu, et al. 2003). Similarly, putative secreted protein, such as AVRLm1 from L. maculans, has only one cysteine and does not form any disulphide bond; this fungus also does not form haustoria in its host (Gout, et al. 2006). In contrast, several fungal pathogens that colonize the plant apoplast encode avirulence proteins that are cysteine-rich and often have an even number of cysteine residues providing the means to form disulphide bridges (Rep, et al. 2004, Thomma, et al. 2005, van den Burg, et al. 2003, van den Hooven, et al. 2001, Stergiopoulos, et al 2010). Several bacterial avirulence proteins have been shown to enter host cells and interact with a virulence target inside the cell, thus, playing a role in suppression of the host defense when the plant does not have the corresponding resistance protein (Chapter 1). The fact that no difference in disease is seen on Odessa when inoculated with U. hordei having avirulence gene UhAvr1 or  71  the recessive allele uhavr1, suggests UhAVR1 is not significantly involved in virulence toward barley; however, this assessment was not done using isogenic strains. The C19A2 deletion mutant was created in the Uh364 (MAT-1 Avr1) parental strain and since this mutant Uh1041 became virulent towards Hannchen, it was deduced that it no longer contained a functional UhAVR1; therefore, a progeny of the other mating-type (MAT-2) was selected in which the C19A2 fragment was also deleted by crossing Uh1041 to parental line Uh362 (MAT-2 avr1). Inoculation of the new cross on barley cultivars Odessa and Hannchen revealed no significant difference in virulence towards these cultivars. This suggests that UhAvr1 does not contribute significantly to virulence toward these barley cultivars. This may be explained by different scenarios. The first explanation may be that measuring infected plants out of the total number of inoculated plants is not a very sensitive assay for quantifying the virulence of pathogens towards their host. It is possible that they cause subtle variations that might become detectable in a population study. Second, these genes may have only additive effects on virulence. In U. maydis, some genes in cluster 19A also have additive effects on virulence towards maize seedlings (Brefort 2008). Therefore, this experiment needs to be reassessed in light of the later finding that in the virulent strain Uh362, genes 17 to 25 are translocated to another genomic location. Since it is not know yet whether this translocated fragment is on the same chromosome, when selecting for the C19A2 fragment deletion in the progeny with mating type MAT-2 (using carboxin resistance), it cannot be sure these genes are no longer present in this progeny. I cannot deduce that genes 17 to 25 are not contributing to virulence; however, since gene number 17 (UhAvr1) no longer has a promoter and is not causing an avirulent phenotype in the virulent parent, it is likely not expressed. On these grounds, I conclude that UhAVR1 does not contribute significantly to virulence. We are currently verifying the location of the translocated fragment in virulent strains, but the experiment should be repeated in isogenic strains by crossing the generated UhAvr1 3′-deletion mutant with avirulent parental strain Uh365 (MAT-2 Avr1) and selecting MAT-2 progeny on carboxin. There are only a few examples of avirulence proteins from eukaryotic pathogens that have shown a clear role in virulence. Examples include AVR-a10 and AVR-k1 from Blumeria graminis that enhance the penetration of the fungus in plant epidermal cells, and AVR2 and AVR4 from C. fulvum that inhibit an apoplastic protease and bind to fungal chitin, respectively (Dixon, et al. 1996, Joosten, et al. 1994, Ridout, et al. 2006).  72  Similarly, AVR3a from Phytophthora infestans can suppress necrotic responses in Nicotiana benthamiana induced by INF1 elicitor (Bos, et al. 2006). UhAvr1 encodes an extremely monomorphic protein. This gene was sequenced from nine field isolates (six virulent and three avirulent strains) collected from different parts of the world, in addition to the parental strains. Surprisingly, only two point mutations were identified in only one avirulent strain Uh813 that translated into a single amino acid substitution. When plants R proteins recognize a particular effector from pathogens, natural selection pushes the pathogens to escape this recognition either by acquisition of additional effectors to suppress ETI, or by jettison or diversification of the recognized gene, or by disruption of gene expression. The direct interaction of an avirulence protein with a resistance protein (according to the ‘receptor-ligand’ model) results in diversifying selection that generates highly divergent alleles as a result of gene duplication and subsequent point mutation in order to avoid recognition by host R proteins (Dodds, et al. 2006, Ellis, et al. 2007a, Wang, et al. 2007). Twelve different alleles have been identified for AvrL567 from six different rust strains from geographically separated locations showing 20% amino acid difference (Dodds, et al. 2006). The plant resistance locus (L) from flax that recognizes this gene has also undergone diversifying selection and thirteen different alleles have been identified to recognize the diverged avirulence alleles. This is consistent with an evolutionary arms race between the pathogen and the host (Dodds, et al. 2006, Ellis, et al. 1999, Stahl and Bishop 2000). Similarly, six diverged alleles of ATR1-1NdWsB have been cloned from eight strains of the oomycete pathogen, Hayaloperonospora arabidopsidis (Rehmany, et al. 2005). Diversifying selection also acted on ATR13 from H. arabidopsidis and its corresponding locus, RPP13, in Arabidopsis (Deslandes, et al. 2003, Jia, et al. 2000). The indirect recognition of AVR and R, the “guard model”, results in purifying selection as the guard recognizes modifications of the AVR protein on the guardee and imposes selection pressure against its function (Rohmer, et al. 2004). The indirect recognition favours gene inactivation or deletion (Bent and Mackey 2007). As not many polymorphisms have been detected at the UhAvr1 locus but have shown inactivation of the gene through the activity of transposable elements in the promoter region, I speculate that the interaction between UhAvr1 and Ruh1 is indirect. This needs to be confirmed experimentally, for example by using the yeast two-hybrid assay or protein pull-down after cloning of the barley Ruh1 gene. I am not sure whether the single amino acid substitution in UhAVR1 (isoleucine to arginine) in strain Uh813 is  73  still recognized by the RUH1 protein or whether there is another allele of Ruh1 in Hannchen that recognizes the mutated avirulence protein. A double-recognition strategy for at least one avirulence protein from L. maculans, AVRLm4-7, has been described previously in which a single amino acid substitution changes recognition by the corresponding resistance R protein (Parlange, et al. 2009). Transposable elements can alter gene expression by insertion into a promoter element or by disrupting the protein by insertion into the ORF of the gene (Daboussi and Capy 2003, Ganko, et al. 2003, Hua-Van, et al. 2002, Kang, et al. 2001). Repetitive elements also play an important role in genome rearrangements and can result in deletion, inversion, duplication, and translocation, based on the orientation and location of the repeat on the chromosome (Daboussi and Capy 2003, Hua-Van, et al. 2000, Kim, et al. 1998, Nitta, et al. 1997, Khang, et al. 2008). The UhAvr1 gene is located in a region of the genome that is rich in transposons and repeats and is considered an unstable part of the genome. Based on sequence analysis of the virulent and avirulent parents and the field isolates, it is likely that the activity of a TE in the promoter element of UhAvr1 and translocation resulted in inactivation of this gene and thus the breaking of avirulence on Hannchen. Moreover, PCR and sequence analysis of BAC clones from the virulent and avirulent parents revealed a translocation of a fourteen kb-fragment including Uhavr1 in the virulent parent to another location in the genome. Several avirulence genes from bacteria, fungi, and oomycetes have been found close to transposable elements and repeats and seem to have been inactivated by these elements (Orbach, et al. 2000, Rep, et al. 2004, Houterman, et al. 2008, Kim, et al. 1998, Rehmany, et al. 2003). Transposon-mediated mutations have been documented for several M. oryzae avirulence genes such as AvrPita, AvrCo39 and Ace1 (Bohnert, et al. 2004, Farman, et al. 2002, Farman and Leong 1998, Kang, et al. 2001, Orbach, et al. 2000, Zhou, et al. 2007). In C. fulvum, the insertion of a transposon resulted in truncation and inactivation of the AVR2 (Luderer, et al. 2002b). In the genome of B. graminis f.sp hordei, the Avr-a22 and Avr-a12 loci are close to repetitive DNA and Avr-k1 paralogs are located near a retrotransposon (Ridout, et al. 2006, Skamnioti, et al. 2007). The insertion of a Pot3 transposon in the promoter region 302 bp upstream the ATG in AvrPita1, resulted in breaking its avirulence activity (Kang, et al. 2001). In P. sojae, an insertion of 3 kb upstream of Avr1b-1 was suggested to be responsible for the loss of transcription in some avr1b-1 isolates (Shan, et al. 2004).  74  Analysis of the 120 kb region spanning the UhAvr1 gene in U. hordei revealed conserved synteny with the U. maydis cluster 19A. This U. maydis cluster is the biggest cluster of predicted secreted protein encoding genes whose transcription is induced after infection of corn inside tumor tissue; deletion of this cluster severely reduced disease on corn seedlings (Kamper 2006). The coding regions on both sides of the UhAvr1 locus are similar to the coding regions flanking cluster 19A in U. maydis. It appears that both species shared some of these ancestral genes but because of their obligate biotrophic interaction with different hosts, these effectors evolved differently in the two organisms. In U. maydis there are several alleles of the same secreted protein coding genes and diversifying selection likely acted on these effectors to avoid host recognition, while in U. hordei the mechanism to avoid host recognition might have involved the activity of transposons and recombination through repetitive elements. Interestingly, in U. maydis-corn pathosystem, no effetor-R gene interaction involving avirulence and resistance genes has been genetically identified although the diversified multi-member families of effectors could indicate there might have been such an interaction in the past.  75  TrpC terminator  Um Hsp70 prom  phleo res ORF GateWay cassette, reading frame B Um GAP promoter LacZ pUBleXInt:GateWayHA 8171 bp  ori  HA epitope tag STOP codon Amp-res  Um Hsp70 terminator  Figure 2.1 Plasmid map of pUBlexInt:GateWayHA. For the construction of pUBleX1Int:GateWayHA, a synthetic linker containing a HA epitope tag and a stop codon flanked by BamHI/BglII and KpnI cohesive ends and an Nru1 restriction enzyme site for bluntend cloning of GateWayTM recombineering cassette was used. The annealed product was ligated to a shuttle vector, cut with BamHI and Kpn1. The GateWayTM cassette, reading frame B (Invitrogen) was inserted into the unique Nru1 site. Subsequently, the GateWay-HAtag-STOP cassette was amplified by PCR to clone in the shuttle vector and transferred to Ustilago-specific expression vector pUBleX1Int.  76  BAC2-G7 (362)  Vir  BAC1-E2 (362) Avir  C19A  BAC3-A2 (364) 117Kb  A  RAPD 1 rec  AFLP 1 rec  RFLP 3 rec  18 17  10 11 1 2  3 4 5  6  7  8  9  15 13 14 12  27 21  16  19 20 22  28 23 24 25  26  29  30 31  32  33  34  35  36  39 37 38 40 41  42  43  44 45  46  47  B Uh BAC3A2-Avr1 region 0001  UhAvr1  Figure 2.2 The UhAr1 locus. (A) Three markers spanning the UhAvr1 locus are shown by black vertical lines. BAC3-A2 from the avirulent parent (Uh364) contains the entire locus and is shown by a blue bar. The two overlapping BAC clones, 1E-2 and 2-G7 from the virulent parent Uh362, are show as pink bars at the top of BAC-3A2. (B) The entire 117 kb region from BAC3-A2 was sequenced using a transposon insertion technique. The arrows represent all the ORFs with their direction of transcription. Asterisks represent the predicted secreted proteins encoding genes at the locus.  77  18 17  10  1 2  3 4 5  6  7  8  13 14 12  9  27  15  11  21 16  1920 22  28 23 24 25  26  29  30 31  32  33  34  35  39 37 38 40 41  36  42  43  44 45  46  47  Uh BAC3A2-Avr1 region 0001  UhAvr1  A  C19A2: 38.5 kb  C19A3: 14.9 kb C19A5: 18.4 kb  ∆ C19A2  C19A2:: XhoI  C19A4:26.8 kb  XhoI  8kb  XhoI  SalI  5.4kb  C19A2  Cbx - DG  4.6kb  1  2  3  4  SalI  2.1kb  SalI  Cbx - DG  5  1  6  2  3  4  5 6  Wt ∆ C19A2  C19A3 SalI  Wt ∆ C 19Α5 3A  :: ∆ C19A3  SalI  SalI  4.6kb  C19A3  5.8kb  AvaI  AvaI  AvaI  3.5kb  Cbx - DG  2  ∆ C19A4  C19A4:: SalI  AvaI  7.2kb  C19A4  Probe 3F 1  D  Probe 3F  1A  C  SalI  C19A5  Probe 3F  B  ∆ C19A5  C19A5::  XhoI  Cbx - DG  Probe 3F 3  4  1  ∆ C19A3 Wt  2  3  4  5  ∆ C19A4 Wt  E  Figure 2.3 Deletion analysis of cluster C19A (A) The four overlapping bars (C19A2, C19A4, C19A3 and C19A5) under the blue bar (genomic region) represent the fragments that were deleted in the four independent deletion mutants, respectively, with their sizes in kb. (B) Southern blot analysis of C19A2 transformants: genomic DNA digested with XhoI. One of the transformants, number 5 in the gel shows a band of the expected size of 5.4 kb for deletion mutant and was used for pathogenicity analysis; transformants 1-4 reveal both wild-type bands and the expected fragment of deletion mutant. (C) Southern blot analysis of C19A3 transformants: genomic DNA digested with SalI. All transformants reveal a band of expected size of 5.8 kb of the deletion mutant. (D) Southern blot analysis of C19A5 transformants: genomic DNA digested with SalI. The analysis of these transformants revealed bands for both wild type and mutant of the expected size of 2.1 kb of deletion mutant. Two of these transformants were used for pathogenicity analysis. (E) Southern blot analysis of C19A4 transformants: genomic DNA was digested with AvaI. One of the transformants, lane 3, revealed a band of the expected size of 7.2 kb and was used for pathogenicity analysis; the other transformants contained one or more additional bands. In all these Southern blot analyses, the probe used was for the 3’ flank of the deletion mutant. The last sample in each gel is wild-type U. hordei. The little cartoon on the top of each gel is a schematic representation of the wild-type region and deletion mutant, respectively.  78  70  Infected Plants (%)  60 50 40 30 20  364x362  Wild type  1041x362  ∆C19A2  1046x362  1048x362  ∆C19A3  1051x362  ∆C19A4  Hannchen  Odessa  Hannchen  Odessa  Hannchen  Odessa  Hannchen  Odessa  Odessa  Hannchen  Odessa  0  Hannchen  10  1053x362  ∆C19A5  Figure 2.4 Pathogenicity test of the deletion mutants. Fragment C19A2 (deletion mutant strain Uh1041) contains the functional UhAvr1, as the deletion mutant of this fragment was virulent towards Hannchen, shown by the red bar. All other deletion mutants and wild type are virulent towards Odessa but avirulent towards Hannchen which shows that they have a functional UhAvr1. Details of all mutants and wild-type strains used in this experiment are given in Table (2.1). Bars on X-axis show the deletion mutants for different fragments mated with virulent strains and inoculated on barley cultivars Odessa and Hannchen. The Y-axis shows the percent of infected plants out of the total number of inoculated plants. The data shown here is an average of three independent experiments with standard deviation shown as error bars.  79  C19A2:: ∆C19A2 XhoI  XhoI  XhoI  8kb  5.4kb  C19A2  Cbx-DG  Probe 3F  Wt ∆C19A2 A 70  Infected Plants (%)  60 50 40 30 20 10 0 Odessa  Hannchen  364x362  Wild Type  Odessa  Hannchen  1041xU1116  Odessa  Hannchen  1041xU1118  B  C19A2 deletion mutant in both mating partner  Figure 2.5 Analysis of virulence towards barley cultivars of a cross of strains both deleted for the C19A2 fragment. (A) DNA blot analysis of C19A2 deletion mutants having mating type 2. DNA was digested with XhoI and probed with the 3’-flank that was used for construction of the deletion construct. Two of the deletion mutants that show a band of the expected size of 5.4 kb were used for the pathogenecity tests. (B) Pathogenicity tests of crosses between mating partners both deleted for fragment C19A2. Virulence towards both barley cultivars seemed not significantly affected. Vertical bars on the X-axis show the pathogenicity of the crosses inoculated on barley cultivars Odessa (in blue) and Hannchen (in red). The Y-axis shows the percent of infected plants out of total inoculated plants.  80  18 17  10  15  11 1 2  3 4 5  6  7  8  9  13 14 12  27 21  16  1920 22  28 23 24 25  26  30  29  31  32  33  34  35  36  39 37 38 40 41  42  43  44 45  46  47  Uh BAC3A2-Avr1 region 0001  C19A2: 38.5 kb 10  10022  11  A  08127 5 608128  7  8  9  12  13  18  15  10021  14  19  21  22 23  10024  BAC 1-6 70  B Infected Plants (%)  60 50 40 30 20 10  364-362  364-362  Wild type  1205x362  1205x362  1207x362  1207x362  1041x362  BAC1-6 complemented ∆C19A2  Hannchen  Odessa  Hannchen  Odessa  Hannchen  Odessa  Hannchen  Odessa  0  1041x362  ∆C19A2  Figure 2.6 Position of BAC1-6 subclone at the UhAvr1 locus and pathogenicity tests. (A) The 38 kb-fragment C19A2 is enlarged to show the different ORFs and predicted secreted proteins in red with arrows indicating the direction of transcription. The position of BAC1-6 is shown in by a purple bar. The figure is drawn to scale. (B) Pathogenicity test of the deletion mutant strain (Uh1041) complemented with BAC1-6 (11 kb) fragment that contains two predicted secreted proteins Uh10021 and Uh10022 as shown in A. The complemented strains cause very low disease on Hannchen while they are fully virulent towards Odessa. The deletion mutants are fully virulent towards Hannchen and the wildtype strains are virulent towards Odessa and avirulent towards Hannchen which show that they have UhAvr1. The details of all mutant and wild strains used in this experiment is given in the table 2.1. Bars on X-axis show different strains inoculated on barley cultivars Odessa and Hannchen. The Y-axis shows the percent of infected plants out of total inoculated plants. The data shown here is an average of three independent experiments with standard deviation as the error bar. The blue bars represent disease on Odessa and red bars represent disease on Hannchen.  81  1  2 3  4  5 6  7  8  9 10 11 12 13  14 15 16 17 18 31  31 31  21  21  14..5  21  10021  10021− 10021−SP  10022− 10022−SP  10022  Uh1041  10024  10024-SP Uh1041  A 40 35  Infected plants (%)  30 25 20 15 10 5  Uh1041  10021  10024  Odessa  Hanchen  Odessa  Hanchen  Odessa  10022− 10022−SP  Hanchen  Odessa  Hanchen  Odessa  9 10 11 12 13 14 15 16 17 18 19 20 21 22  10022  Hanchen  8  Odessa  7  10021− 10021−SP  Hanchen  Odessa  Hanchen  6  Odessa  5  Hanchen  4  Odessa  3  Hanchen  2  Odessa  1  Hanchen  Odessa  Hanchen  0  10024-SP  B  Figure 2.7 Complementation analysis of the C19A2 deletion mutant transformed with Uh10021, Uh10022 and Uh10024 and their virulence toward barley. (A) Western blot analysis of the deletion mutant Uh1041 (control; lanes 13 and 18) and Uh1041 complemented with the full length Uh10021 ORF (10021; lanes 1 and 2), Uh10022 ORF (10022; lanes 6 and 7), or Uh10024 ORF (10024; lanes 14 and 15), or with the ORFs without the signal peptide 10021-SP (lanes 3-5), 10022-SP (lanes 8-12), or 10024-SP (lanes 16 and 17) as indicated. Different lanes in the gel numbered 1-12 and 14-17, represent Uh1041 (C19A2 deletion mutant) each complemented with the respective genes shown at the bottom of the gel. On the left side of each gel the relevant protein marker is indicated in kDa. The arrows indicate the expected protein size as several non-specific bands were observed in both wild type and complemented strains. (B) Results of the pathogenicity tests with the deletion mutant strain Uh1041 and complemented with individual genes (Uh10021, Uh10022 and Uh10024) both with full length and without signal peptide under Hsp70 promoter, as indicated in the figure. The blue bars represent the percentage of infected plants of Odessa and red bars represent disease on Hannchen, given as percentage of diseased plants among the total inoculated number of plants (Yaxis). 82  10  10022  11  08127 5 608128  7  8  9  12  13  18  15  10021  14  19  21  22 23  C19A2  10024  C19A2-D: 4.3 kb  C19A2-A: 21.4kb  C19A2-B: 9.5 kb C19A2-C: 2 kb C19A2-E: 5.8 kb A  C19A2-B :: ∆ C19A2-B  C19A2-A :: ∆C19A2-A BgllI  BgllI  2.1kb C19A2  BgllI  -A  BgllI  2.7kb Cbx  C19A2  Probe 5F  ∆ C19A2-A  BgllI  2.6kb  - DG  BgllI  BgllI  3.2kb  -B  Cbx  - DG  Probe 5F  -1  2  3  4  ∆ C19A2-B 1  5  2  3  4  5  6  7  8 Wt  Wt  B BglII  BglII  BglII  1.8kb C19A2  1  2.7kb  C19A2  HindIII  Cbx  3  4  5  ∆ C19A2 6  7  PstI  - DG  1  2  -D 3  5  PstI  1.4kb  -E  Cbx  PstI  - DG  Probe 3F  ∆ C19A2-E 4  PstI  7.8kb C19A2  Probe 3F  ∆ C19A2  -C  Wt  D  HindIII  6.2kb  Cbx  - DG  8  C19A2-E :: ∆ C19A2-E  HindIII  -D  3.6kb  -C  2  HindIII  BglII  C  C19A2-D :: ∆C19A2-D  C19A2-C :: ∆ C19A2-C  6  7  1  8  2  3  4  5  6  7  8  9  Wt Wt  E  F  Figure 2.8 Deletion analysis of fragment C19A2. (A) Cartoon representing the fragment C19A2 (black bar) which covers the genomic region with the genes indicated (top line), and five overlapping fragments (C19A2-A, C19A2-B, C19A2-C, C19A2-D and C19A2-E, colored bars) with their sizes in kb that were deleted in five independent mutants, respectively. Wt, wild-type fragment expected (lane 6) (B) Southern blot analysis of transformants generated with deletion construct C19A2-A; total genomic DNA was digested with BglII. Three of the deletion mutants, lanes 1, 3 and 4, show a band of the expected size of 2.7 kb. One shows an additional band representing a random insertion in the genomic DNA (lane 2). Two of the mutants were used for pathogenicity analysis. (C) Southern blot analysis of transformants generated with deletion construct C19A2-B; gDNA digested with BglII. Two of the deletion mutants show a band of the expected size of 3.2 kb. (D) Southern blot analysis of transformants generated with deletion construct C19A2-C; gDNA digested with BglII. All deletion mutants analysed contain the expected size band of 3.6 kb. Two of these transformants were used for pathogenicity analysis. (E) Southern blot analysis of transformants generated with deletion construct C19A2-D; gDNA digested with HindIII. Several mutants analysed show the band of expected size of 6.2 kb while 83  some additionally contain the wild-type band. (F) Southern blot analysis of transformants generated with deletion construct C19A2-E; gDNA digested with PstII. Several mutants analysed show the band of expected size of 6.2 kb while some additionally contain the wild-type band. The little cartoon on the top of each gel represents a schematic representation of the wild-type region and deletion mutant, respectively. In the Southern blot analysis of the transformants generated with constructs C91A2-A and C19A2-B, the probe used was a fragment of the 5’-flank of the deletion constructs, while for C19A2-C, C19A2-D and C19A2-E, probes were taken from the 3’-flanks (indicated by little black bars under the cartoons.  84  80  infected Plants (%)  70 60 50 40 30 20  7  8  9 10 11 12 13 14 15 16 17 18 19 20  ∆C19A2-D  Hannchen  6  Odessa  Odessa  Hannchen  Hannchen  5  Odessa  Odessa  Hannchen  Hannchen  4  Odessa  Odessa  3  Wild Type ∆C19A2-A ∆C19A2B ∆C19A2-C  Hannchen  Hannchen  2  Odessa  Odessa  Hannchen  Hannchen  1  Odessa  Odessa  0  Hannchen  10  ∆C19A2-E  Figure 2.9 Pathogenicity test of the mutants deleted for sub-fragments of C19A2. All mutants were crossed with virulent parent Uh362. Analysis shows that the mutants deleted for fragments C19A2-C and C19A2-D were virulent towards Hannchen, shown by red bars in the figure, indicating that the functional UhAvr1 gene is located on these fragments. All other deletion mutants and the wild type were virulent towards Odessa and avirulent towards Hannchen which showed that they had an intact UhAvr1 gene. Details of all mutants and wild strains used in this experiment are given in the Table 2.1. Horizontal bars on the X-axis show the mutants deleted for the different fragments. The Y-axis shows the percent of infected plants out of the total inoculated plants. The data shown here is an average of three independent experiments with standard deviation as the error bar. 85  10022  10021  10022-3F  10022-5F  A  Cbx− −∆10022 60  10022:: ∆10022 BgllI BgllI  1.8kb  3.6kb  10022  Cbx-DG  BgllI  Infected plants (%)  BgllI  50  Probe 3F  Cbx-∆ ∆10022 1  40  30  20  10  2  3  4  5  6  0 Odessa  Hannchen 364-362  Wild Type  Odessa  Hannchen  1289x362  Odessa  Hannchen  1297x362  ∆Cbx-10022  Wt C B  Figure 2.10 Deletion analysis of Uh10022 and pathogenicity test. (A) Schematic representation of the deletion mutant; the pink bar represents the 3’-part of Uh10022 that is deleted in deletion mutant. The green bars represent the flanks used in the deletion construct. (B) Southern blot analysis of several transformants; total gDNA was digested with BglII. Two of the deletion mutant strains showing a band of expected size of 3.6 kb, were used in pathogenicity tests. (C) All strains were mated with virulent strain Uh362 and inoculated on barley cultivars Odessa and Hannchen as indicated. Pathogenicity tests of the deletion mutants confirmed that Uh10022 is the functional UhAvr1 gene: the mutants deleted for the 3’-end of the gene were virulent towards Hannchen, as shown by red bars in the figure. Horizontal black bars on X-axis indicate the deletion mutant and wild type. The Y-axis shows the percent of infected plants out of the total inoculated plants. The data shown here is an average of three independent experiments with standard deviation as the error bar.  86  BAC3-3A2 (364) 18 17  10 11 1  2  3 4  5  6  7  8  12 13  9  15 14 16  21 19 20 22  23 24 25  26  27 28 30 29 31  32  33  34  35  36  39 37 38 40 41  42  43  44 45  46  47  Uh BAC3A2-Avr1 region 0001  Probable transposase  14 kb Related to Gag-pol poly  BAC3-A2  22 16  17 18  16  19  3.6 kb  20  21  23  24  25  26  26  Related to retransposon  BAC1-E2 (362)  BAC2-G7 (362)  Figure 2.11 Comparison of the UhAvr1 locus between the avirulent and virulent parents. The blue bar represents the BAC clone 3-A2 from the avirulent parent Uh364 with the genes identified underneath. The 14 kb-region containing Uh10022 (UhAvr1) and 8 other ORFs, indicated by a yellow line, is enlarged below. The pink lines represent the two overlapping BAC clones from the virulent parent (Uh362). The 14 kb-region in the virulent parent is replaced by an insertion of 3.6 kb, part of which matches transposable element (TE)-related sequences shown by the dotted line in the figure. The retrotransposon was found 134 bp upstream of the Uh10022 ATG start site.  87  BAC (Uh362)  13 14 1516  TE  30 2829 31 26 27  32 33 34 35  Legend: Length of matches in bp  22  BAC (Uh364)  13 14 1516 17 1819 20 21  23 24  25 26  30 29 31  27  32  33 34 35  28  Figure 2.12 Sequence comparison of the U. hordei UhAvr1 locus between the virulent parent Uh362 (upper) and the avirulent parent Uh364 (lower). Both loci are drawn to scale and PatternHunter output (Ma et al., 2002) was used to provide an overview of the regions being compared between the two strains. In the virulent strain, the Avr1 gene Uh10022 (number 17 in red in the lower panel) and seven downstream genes are absent as a deletion of a 14 kb-stretch which is present at another location in the Uh362 genome. In the virulent parent we find at this location a 3.6 kb stretch with matches to transposable element sequences as shows at the top panel (TE). Two black arrows at the upper line indicate the position of primers 1513 and 1741 in the forward and reverse orientation respectively, used for PCR to investigate the size of the insert at the Uhavr1 locus in the virulent parent.  88  340 bp Ins. related to TE  Promoter  10021  10022  TGGGTT U1273 (UhAvr1) ICARDA Azerbaijan U1283 (UhAv1) Turkey U364 (UhAvr1) U813 (UhAvr1) Iran Consensus  690 700 710 720 730 740 750 760 770 780 790 805 (679) 679 (660) GTGAGTGGGTTAATTGAAGATAACAATTAATATACGGCGTTATCGGGGTCACGATCAGACTCCCTTTGCCACAGCCGTATGTTCTATGTAATGCTTAGTTTTCTTGGTGTGAGACTAAATGACAAAC (641) GTGAGTGGGTTAATTGAAGATAACAATTAATATACGGCGTTATCGGGGTCACGATCAGACTCCCTTTGCCACAGCCGTATGTTCTATGTAATGCTTAGTTTTCTTGGTGTGAGACTAAATGACAAAC (675) GTGAGTGGGTT-------------------------------------------------------------------------------------------------------------------(642) GTGAGTGGGTTAATTGAAGATAACAATTAATATACGGCGTTATCGGGGTCACGATCAGACTCCCTTTGCCACAGCCGTATGTTCTATGTAATGCTTAGTTTTCTTGGTGTGAGACTAAATGACAAAC (679) GTGAGTGGGTTAATTGAAGATAACAATTAATATACGGCGTTATCGGGGTCACGATCAGACTCCCTTTGCCACAGCCGTATGTTCTATGTAATGCTTAGTTTTCTTGGTGTGAGACTAAATGACAAAC  U1273 (UhAvr1) ICARDA Azerbaijan U1283 (UhAv1) T urkey U364 (UhAvr1) U813 (UhAvr1) Iran Consensus  U1273 (UhAvr1) ICARDA Azerbaijan U1283 (UhAv1) T urkey U364 (UhAvr1) U813 (UhAvr1) Iran Consensus  820 830 840 850 860 870 880 890 900 910 920 932 (806) 806 (787) ACATATATATACTCCATTCACTGCTCTGATCCCATCCGAGGCAGGGTGTGTTCCTTCATTTGTGCCCCTGTGTTGCTATGATACCGGGTGCCACTGTGCTGTCACCATGACAACAATTGCGTACCTC (768) ACATATATATACTCCATTCACTGCTCTGATCCCATCCGAGGCAGGGTGTGTTCCTTCATTTGTGCCCCTGTGTTGCTATGATACCGGGTGCCACTGTGCTGTCACCATGACAACAATTGCGTACCTC (686) ------------------------------------------------------------------------------------------------------------------------------(769) ACATATATATACTCCATTCACTGCTCTGATCCCATCCGAGGCAGGGTGTGTTCCTTCATTTGTGCCCCTGTGTTGCTATGATACCGGGTGCCACTGTGCTGTCACCATGACAACAATTGCGTACCTC (806) ACATATATATACTCCATTCACTGCTCTGATCCCATCCGAGGCAGGGTGTGTTCCTTCATTTGTGCCCCTGTGTTGCTATGATACCGGGTGCCACTGTGCTGTCACCATGACAACAATTGCGTACCTC  940 950 960 970 980 990 1000 1010 1020 1030 1040 1050 1060 (934) 934 (915) TTGTGCTCCTCGCTTATGTGAGCCTCCCGTATCTCGATGTCTCGGCCGTAACACGTGCACCTTGCCTGAGATGCCGATGCATCTTCAACTGGGTTGCTCAGACTGAACACCGGTGCACTGCATTTGA (896) TTGTGCTCCTCGCTTATGTGAGCCTCCCGTATCTCGATGTCTCGGCCGTAACACGTGCACCTTGCCTGAGATGCCGATGCATCTTCAACTGGGTTGCTCAGACTGAACACCGGTGCACTGCATTTGA (686) -----------------------------------------------------------------------------------------------GCTCAGACTGAACACCGGTGCACTGCATTTGA (897) TTGTGCTCCTCGCTTATGTGAGCCTCCCGTATCTCGATGTCTCGGCCGTAACACGTGCACCTTGCCTGAGATGCCGATGCATCTTCAACTGGGTTGCTCAGACTGAACACCGGTGCACTGCATTTGA (934) TTGTGCTCCTCGCTTATGTGAGCCTCCCGTATCTCGATGTCTCGGCCGTAACACGTGCACCTTGCCTGAGATGCCGATGCATCTTCAACTGGGTTGCTCAGACTGAACACCGGTGCACTGCATTTGA  Figure 2.13 Sequence comparison of the intergenic region between Uh10021 and Uh10022 at the UhAvr1 locus. Several different avirulent field isolates were compared to the avirulent parent Uh364 (see Table 2.1). Note the insertion of 340 bp, matching transposable element sequences, upstream of the Uh10022 promoter in three avirulent field isolates (highlighted in blue). The insert is flanked by 6 bp direct repeats (TGGGTT, boxed) and this sequence matches sequences in the avirulent parental strain, possibly representing a “footprint” indicating (past) TE activity. The sequences were aligned using AlignX (Vector NTI, Invitrogen).  89  A  Uh_ 1 0 0 2 1 MIP S Um_ 0 5 2 9 4 Uh_ 1 0 0 2 2 P ro t e in MIP S Um1 2 3 0 2 o r 0 5 2 9 6 Um_ 0 5 2 9 5 Um_ 1 0 5 5 3 o r 5 2 9 7 Um_ 1 0 5 5 4 o r 0 5 2 9 8 Co nse nsus  U h _ 1 0 0 2 1 M IP S (0 . 3 7 3 5 ) U m_ 0 5 2 9 4 (0 . 3 8 9 6 ) U h _ 1 0 0 2 2 P ro t e in M IP S (0 . 4 0 4 1 ) U m1 2 3 0 2 o r 0 5 2 9 6 (0 . 3 4 2 1 ) U m_ 0 5 2 9 5 (0 . 3 4 2 7 ) U m_ 1 0 5 5 3 o r 5 2 9 7 (0 . 4 3 2 7 ) U m_ 1 0 5 5 4 o r 0 5 2 9 8 (0 . 3 9 9 0 ) 10 20 30 40 50 60 70 80 90 100 110 120 136 (1 ) 1 (1 ) ----------------------MLTQPANVILFMVAFLFSTTALPGRSYK----PSR----FRPYNEPFVVHSVS-KFQ---DEYADRLVLVLQAYFHQQYAKIKLLDRDPISLQDLKQDLRGDRNPKRFIHLGQV (1 ) -------------------MSFLSKILWITVLVMAAVLPTTLAARSARVLPIDPEPQSPPPESRVNMPVLVTRWDGLTD---DPMQLYLRYHLRHTYGMDAAHLEPAHYDPVTLEELEQQIRESGNQKRFIQLGQT (1 ) ----------------------MRSFSLFLVLCIWVIGVIAPGDKASSSAAPAQQHQP-----SFKLEIAENPN-------VDPFLEKISKLGNSHDLYP--HVALMRTTLYGKDKLTTNLGAYPDFRRFIYLGNS (1 ) --------------------------MKILSLLLFALSATLSVIATRYT----NVFN------LYNSETPHESP-----------AARLPDHLNNEWWLH-VQSQSYPPNAMDHDTLRRDLSSDINHRRFLYLGHT (1 ) -----------------------MRSPALLLLLLFAIEVIATGGSGEPSGAQGPVSSAPVNGDPYDGEYAIDMRPSSLR---APFSARLENHIGDVWQVPNVHVEPVELQSFSPTDLRANFNYDRNLRRHLYLYNT (1 ) --------------------MLKVQPRPLLVLASFVILLLLCLSCQAVKPSVERDSSRALAFDPRHVSFTLSLDR------TPERVERLLTQLRTTFRFHDAEVGHLPARTMVKVRLAESFFSSPTKPRFLNLGFI (1 ) MQQVAEKKLPILENDSKDFTDGAVYDPPAIATRLDDMSINVKSASATSLRFLLLSAYLDVEEPRPSFPMALRFDRDQEDRWTNIYTIRLDLHLYQSVGLRNYHVSELTSITLNLRELEYQLRYNRSLRQFLHLGPA (1 ) V ILVL LFAI V A A S Y L F RL HL F L HV L M DL NL N RRFLYLGNT  B 140  150  160  170  180  190  200  210  220  230  242  VVPHRANMVVATYLNRPPN------SDGSRKFVLLSILRPQS-SDAPHVFIHGYADVAGLEDIEDQLRNTVNSAS--DDPQFGHVLSIEGVFETLSRM--------TAPGRSAMRVAFALQNTYQ------GHGQKYFALLHVGMERR-AISPYIFSLGFVKASGVYGFEDAIPVPADLRNARVVTLHGAPLTANEMLRAALFL--------SP-GVPEMYFAVPLHLNPHG-----VDRNLAWSLIYAHSDQP----KTLVHHGFVSASGGHLVLDKVKKTNYPSSR--SFEIGDVLTLREILDIELPALRFAG---TAWGRPDMVLAVPLQNGAN------ADRTHTWAILSVHKSENPKRPPYFFVHNYVKVSDGRATLARLAQAYGPQN--GVLEHGQALTLEEVFDELKMLQPADWPH-TYSNFPDMVFATPMHREPR------PQGNHLWALFSAHQPPFQGDHPLIRVHGFVTVSNGGAVVQRLSELPGPQNR-AAVEKGHVLNIQELFQQLGRLRWPHWDRNW IRPSAQSDVRGFAIALPMTTLQPVYHPNVRGFALFSVYPDRAN---PVGTDVNKSGGRLGWSTVHFCPETLCLWKR--FIERSYTGTIDDIVEGCDLEA-------AMGELPGIAAGFPIAVDWG------EESHYLFALLSVHPPNR--DIPSITLHGFAKVRAVDETPIEEMLAGVPRQPDDQVGPGDVLSIRGIFRQVLEHHPV-----T P MV A PL D FALLSVH P I VHGFV VSGG L L P N VE G VLTI EIFD L L  Figure 2.14 Comparison of UhAVR1 to a U. hordei paralog and U. maydis homologs. (A) A phylogeny was based on alignment of U. hordei UhAVR1 paralog, Uh10021, and U. maydis homologs, Um05294, Um05295, Um05296, Um05297 and Um05298 using the alignment from B. (B) Alignment of the protein sequences using AlignX (VectorNTI, Invitrogen).  90  45 40 Infected Plants (%)  35 30 25 20 15 10 5  Odessa  Hannchen  Odessa  Hannchen  Odessa  Hannchen  Odessa  Hannchen  Odessa  Hannchen  0  1  2  3  4  5  6  7  8  9  10  ∆ C19A2  Uh1041-Um05295  Uh1041-Um05296  Figure 2.15 Pathogenicity tests of the C19A2 deletion mutant complemented with U. maydis homologs. Complemented strains and the original C19A2 deletion strain (horizontal black bars), after crossing to the virulent parent Uh362, were equally virulent towards Hannchen. Crosses were inoculated on barley cultivars Odessa (blue bars) and Hannchen (red bars). The Y-axis shows the percent of infected plants out of total inoculated plants. Note: As complementation of this deletion mutant with Uh10022 expressed in the same construct was not successful in restoring avirulence towards Hannchen, it is uncertain whether these U. maydis genes, although expressed in U. hordei, were properly directing protein transfer and therefore active in the host. Therefore the result of this experiment is not conclusive at this level.  91  U. hordei  3 4 5  9  6  2  39  22  7 1  12 1011 1314151617 181920 21  23 24  30 29 31  25 26  8  27  32  33 3 4  36 35  45  3738 40  41  42  43  44  46  47  28  Legend: Length of matches in bp  5 1 2  3  4  10 11 67 8 9 12  13 14 15 16 17 18 19 20 21  23 2224  25  26  28 30  32  33  3435  36  37  38  U. maydis  27 29 31  Figure 2.16 Comparison of the U. hordei UhAvr1 locus to the syntenic region in U. maydis harbouring cluster 19A. The genomic U. hordei region, based on the sequenced BAC clone 3-A2, is at the top with arrows indicating the position and direction of transcription of the genes. Green rectangles and hatched boxes represent regions with LTRs and repeats, respectively. The red and green color twosided-arrows represent the two fragments that are inverted in U. hordei. Blue vertical lines represent small repeats. The U. maydis sequence (bottom) was obtained from MIPS and vector NTI (Invitrogen) was used to annotate the sequences. Homologous U. hordei and U. maydis coding sequences are represented by similar colours. Gene numbering in U. hordei is as in Table 3.1; gene number 17 is Uh10022/Avr1 in the top panel. Both loci are drawn to scale and PatternHunter output (Ma et al., 2002) was used to provide an overview of the regions being compared between the two species and to illustrate the gross rearrangements present in the two genomes.  92  Table 2.1 Strains used in this work Strain ID  Relevant Genotype  Source or Origin  Uh364  MAT-1 Avr1,Avr2,Avr6  Uh365  MAT-2 Avr1,Avr2,Avr6  Uh359  MAT-1 avr1,avr2,avr6  Uh362  MAT-2 avr1,avr2,avr  Um324  a2b2  Uh1041  Uh364-∆19A2 (167) cbxR  wild type; (Lining et al. 2004) wild type; (Lining et al. 2004) wild type; (Lining et al. 2004) wild type; (Lining et al. 2004) wild type; Um521 in (Kronstad and Leong 1989) S. Ali, PhD thesis, 2011  Uh1116  Uh364- ∆19A2 (31) cbxR  S. Ali, PhD thesis, 2011  Uh1117  Uh364-∆19A2 (33) cbxR  S. Ali, PhD thesis, 2011  Uh1118  Uh364-∆19A2 (35) cbxR  S. Ali, PhD thesis, 2011  Uh1046  Uh 364-∆19A3 (10) cbxR  S. Ali, PhD thesis, 2011  Uh1051  Uh364-∆19A4 (105) cbxR  S. Ali, PhD thesis, 2011  Uh1053  Uh364-∆19A5 (2) cbxR  S. Ali, PhD thesis, 2011  Uh1166  Uh364-∆19A2-A (76) cbxR  S. Ali, PhD thesis, 2011  Uh1173  Uh364-∆19A2-A (316) cbxR  S. Ali, PhD thesis, 2011  Uh1131  Uh364-∆19A2-B (52) cbxR  S. Ali, PhD thesis, 2011  Uh1137  Uh364-∆19A2-C (19) cbxR  S. Ali, PhD thesis, 2011  Uh1142  Uh364-∆19A2-C (59) cbxR  S. Ali, PhD thesis, 2011  Uh1149  Uh364-∆19A2-D (1) cbxR  S. Ali, PhD thesis, 2011  Uh1155  Uh364-∆19A2-D (82) cbxR  S. Ali, PhD thesis, 2011  Uh1189  Uh364-∆19A2-E (64) cbxR  S. Ali, PhD thesis, 2011  Uh1197  Uh364-∆19A2-E (109) cbxR  S. Ali, PhD thesis, 2011  93  Strain ID  Relevant Genotype  Source or Origin  Uh1289  Uh364-∆10022 (37) cbxR  S. Ali, PhD thesis, 2011  Uh1297  Uh364-∆10022 (106) cbxR  S. Ali, PhD thesis, 2011  Uh1205  Uh1041 plus BAC 1-6int (2) hygR Uh1041 plus BAC 1-6int (10) hygR Uh1041 plus Uh10024int (6) ZeoR Uh1041 plus Uh10024int (7) ZeoR Uh1041 plus Uh10024-SPint (1) ZeoR Uh1041 plus Uh10024-SPint (2) ZeoR Uh1041 plus Uh10021int (3) ZeoR Uh1041 plus Uh10021int (4) ZeoR Uh1041 plus Uh10021-SPint (2) ZeoR Uh1041 plus Uh10021-SPint (3) ZeoR Uh1041 plus Uh10022int (1) ZeoR Uh1041 plus Uh10022int (4) ZeoR Uh1041 plus Uh10022-SPint (4) ZeoR Uh1041 plus Uh10022-SPint (6) ZeoR Uh1041 plus Um25295int (2) ZeoR Uh1041 plus Um25295int (4) ZeoR Uh1041 plus Um25296int (14) ZeoR Uh1041 plus Um25296int (4) ZeoR Uh1041 plus Um10022 int (19) HygR  S. Ali, PhD thesis, 2011  Uh1207 Uh1212 Uh1213 Uh1268 Uh1269 Uh1250 Uh1251 Uh1253 Uh1254 Uh1255 Uh1256 Uh1257 Uh1258 Uh1298 Uh1299 Uh1301 Uh1302 Uh1311  S. Ali, PhD thesis, 2011 S. Ali, PhD thesis, 2011 S. Ali, PhD thesis, 2011 S. Ali, PhD thesis, 2011 S. Ali, PhD thesis, 2011 S. Ali, PhD thesis, 2011 S. Ali, PhD thesis, 2011 S. Ali, PhD thesis, 2011 S. Ali, PhD thesis, 2011 S. Ali, PhD thesis, 2011 S. Ali, PhD thesis, 2011 S. Ali, PhD thesis, 2011 S. Ali, PhD thesis, 2011 S. Ali, PhD thesis, 2011 S. Ali, PhD thesis, 2011 S. Ali, PhD thesis, 2011 S. Ali, PhD thesis, 2011 S. Ali, PhD thesis, 2011  94  Strain ID Uh1319  Relevant Genotype  Source or Origin int  S. Ali, PhD thesis, 2011  int  S. Ali, PhD thesis, 2011  Uh805  Uh1041 plus Um10022 HygR Uh1041 plus Um10022 HygR MAT-1 avr1  Uh815  MAT-2 avr1  Canary Island  Uh820  MAT-2 avr1  Tunisia  Uh795  MAT-1  Avr1  unknown  Uh813  MAT-1  Avr1  Iran  Uh822  MAT-1  avr1  Canada  Uh811  MAT-1  avr1  Ethiopia  Uh818  MAT-1  avr1  Spain  Uh1273  MAT (unknown) Avr1  ICARDA Azerbaijan  Uh1283  MAT (unknown) Avr1  Turkey  Uh1320  Kenya  Strain ID refers to Bakkeren Lab inventory, Uh for U. hordei and Um for U. maydis. All mutants were generated in the Uh364 background as indicated. R, resistant to the indicated antibiotic: Hyg, hygromycin B; Zeo, zeocin; cbx, carboxin. int, integrative complementing plasmid. The number in parentheses represents the specific transformant chosen for analysis. ∆:deletion mutant, indicating specific gene or region.  95  Table 2.2 Primers used in this work # 1244  Name of the Sequence primer UH_13897_Fw CACCATGCTTACTCAACCGGCCAAC  Cloning of Uh10021  728  Uh05294plus_f  GGAACTGTGCTCTGGTAGTGG  Sequencing of Uh10021  1246  UH_13897_Rev  CATTCGTGACAACGTCTCAAAAAC  1245  UH_13897SP_FW UH_10022_Fw  CACCATGGCACTACCCGGTCGCAGCTAC  Cloning and sequencing of Uh10021 Cloning of 10021  CACCATGCCTGGCGACAAAGCTTCTTC  1249  UH_10022SP_Fw UH_10022_Rev  TCCGGCAAATCGGAGCGCAG  736  Uh05311aPlus_f  GCTTTTCATCAGAGCCATACCT  Cloning and sequencing of Uh10022 Sequencing of Uh08130  737  Uh05311aPlus_r  TGGCTTGTTTACAGAGTGCAA  Sequencing of Uh08130  1253  UH_08132_Fw  CACCATGGCCACAACATCACTCTTAC  Sequencing of Uh08132  1281  Uh08132-plus-L  CGCTATGGAAGCACTTCATTTT  Sequencing of Uh08132  661  Uh5311R2  GCATTCGGCCTGATACCAAC  Sequencing of Uh08132  1256  UH_08128_Fw  CACCATGCTTTTCTTTATTCTCGCC  Sequencing of Uh08128  1258  UH_08128_Rev  AGAAAAGTGCGGCAGTGATGC  Sequencing of Uh08128  1247 1248  CACCATGCGATCGTTTTCCCTTTTCC  Purpose  Cloning and sequencing of Uh10022 Cloning of Uh10022  96  #  Purpose  730  Name of the Sequence primer Uh05306plus_f ATAGCCCTCCTTCATGGTGT  731  Uh05306plus_r  TCGCAGTGCCTTTCATATTG  Sequencing of Uh08128  1272  Uh08127-plusL  GCAGAGCATGACGTGAAACTAC  Sequencing of Uh08127  1273  Uh08127-plusR  GTTCAAGGCCCTATATCCCTCT  Sequencing of Uh08127  1261  UH_08127_Rev  TCCGTGGCCTCTAACAGCAAG  Sequencing of Uh08127  1265  UH_08139_Fw  CACCATGTTCCGAATCGGCTTTGTC  Sequencing of Uh08139  1267  UH_08139_Rev  TCCAGGCAATCTGATCAGGC  Sequencing of Uh08139  727  Uh05318homo_r  GCGCACTAGTTCAATTCAAAGCTGGAGGTATG  Sequencing of Uh10033  966  5319 plus_r  TCTCCCTTCCTCGTCAACCTG  Sequencing of Uh10033  1282  Uh10033-plus-L  GGTCAAGTCGACCTCCAACAG  Sequencing of Uh13916  1283  Uh10033-plus-R  GTCCCTTCCGTCACTTCCAT  Sequencing of Uh13916  1277  Uh10024-plusL  TGAGATCTGTCATAGAGCTGTTTC  Sequencing of Uh10024  1278  Uh10024-plusR  GCATCTTCGGATGTCAGGTAGT  Sequencing of Uh10024  1424  UH_10024_Fw  CACCATGTTGCCTGCAACACTGCCTTC  Cloning of Uh10024  1425  UH_10024_rev  CAGCATCGCCAGCGATGCTGCTC  Cloning of Uh10024  Sequencing of Uh08128  97  #  Cloning of Uh10024  1152  Name of the Sequence primer UH_10024CACCATGCCTGGATGCTCAAGAAGCAGGAG SP_Fw UH_08134_Fw CACCATGAAGGTACATCTGTCTAC  1154  UH_08134_Rev  GCCTCGAATGGTCACAGG  Sequencing of Uh08134  1284  C19-A1-5L1Sce-1F C19-A1-5Rflank–attB1 C19-A1-ko-5 FlankL C19-A2-3LattB2 C19-A2-3R1Sce-1R C19-A2-ko-3 FlankR C19-A3-5L-1sce1F C19-A3-5R– attB1 C19-A3-ko-5 FlankL C19-A4-5L1Sce-1F C19-A4-5RattB1 C19-A4-ko-5 FlankL  AAAATAGGGATAACAGGGTAATCGACAGATCTCGAGGAAACC  5F of Deletion construct C19A2 5F Deletion construct C19A2 Confirmation of deletion mutant C19A2 3F of Deletion construct C19A2 3F of Deletion construct C19A2 Confirmation of deletion mutant C19A2 5F Deletion construct C19A3 5F Deletion construct C19A3 Confirmation of deletion mutant C19A2-A 3F of Deletion construct C19A3 3F of Deletion construct C19A3 Confirmation of deletion mutant C19A5  1510  1285 1286 1289 1290 1291 1292 1293 1294 1295 1296 1297  GGGGACAAGTTTGTACAAAAAAGCAGGCTATTGAATTGTTTGCCACACCTG TCACTTCAGGAGGTGATCAAGA GGGGACCACTTTGTACAAGAAAGCTGGGTAGGAGAGAAGAAGCAGAGCT AAAAATTACCCTGTTATCCCTATTGTGCTTCACTGCACCTTC TCCCTGTCGGTGTCTTCTTACT AAAATAGGGATAACAGGGTAATCCTGTCGATTGCTAGGAAGG GGGGACAAGTTTGTACAAAAAAGCAGGCTATTGAGGGTCAATCGGAGAGAT TTGTTGTCTTGGTTTCCTGTGT AAAATAGGGATAACAGGGTAATAAGCCCTGCTTCTTCTCTCC GGGGACAAGTTTGTACAAAAAAGCAGGCTATGAGTGGATCCCCATTGTCAT AGCTTGCAGTCTGTTCATCATC  Purpose  Sequencing of Uh08134  98  # 1298 1299 1300 1301 1302 1303 1428 1429 1430 1431 1432 1433 1434 1435 1436  Name of the Sequence primer C19-A4-3L attB2 GGGGACCACTTTGTACAAGAAAGCTGGGTACGTACAGGACCGTGAGGACT C19-A4-3R1Sce-1-R C19-A5-5L1Sce1-F C19-A5-5RattB1 C19-A5-3L-attB2  AAAAATTACCCTGTTATCCCTAGTGGATCAGCTGTTCACTCG  C19-A5-3R1Sce-1R C19A2-A-3FattB2-L C19A2-A-3F1sce1R-R C19A2-b-5F1sce1F-L C19A2-b-5FattB1-R C19A2-b-3FattB2-L C19A2-b-3F1SceR-R C19A2-c-5F1sce1F-L C19A2-c-5FattB1-R C19A2-c-3FattB2-L  AAAAATTACCCTGTTATCCCTAGGACTGGACTTTAGGGCACA  AAAATAGGGATAACAGGGTAATCCCCCTATCTGGCTCTCTTC GGGGACAAGTTTGTACAAAAAAGCAGGCTATGAACGTGTGGTATGCTGAGG GGGGACCACTTTGTACAAGAAAGCTGGGTAGATCGTGGCTCCAAGACAGT  GGGGACCACTTTGTACAAGAAAGCTGGGTAGCATTGTGCTCAAGCTGTGT AAAAATTACCCTGTTATCCCTAACTGCTGGGCAAGAATGACT AAAATAGGGATAACAGGGTAATCTCAAACCCAATCTGCAGTG GGGGACAAGTTTGTACAAAAAAGCAGGCTATAGGTTAGCGGTCCAGATCAA GGGGACCACTTTGTACAAGAAAGCTGGGTACTAGGACGAAACAGCCAAGC AAAAATTACCCTGTTATCCCTAACTCCAATCACGGGAATCAC AAAATAGGGATAACAGGGTAATTGGGTAGAGGTTTGGTGAGG GGGGACAAGTTTGTACAAAAAAGCAGGCTATAAGAATCCTGCCTTGCTTCA GGGGACCACTTTGTACAAGAAAGCTGGGTACCTTAGCCTAGTCCCGCTCT  Purpose 5F Deletion C19A4 5F Deletion C19A4 5F Deletion C19A5 5F Deletion C19A5 3F Deletion C19A5 3F Deletion C19A5 3F Deletion C19A-A 3F Deletion C19A-A 5F Deletion C19A-B 5F Deletion C19A-B 3F Deletion C19A-B 3F Deletion C19A-B 5F Deletion C19A-C 5F Deletion C19A-C 3F Deletion C19A-C  construct construct construct construct construct construct construct construct construct construct construct construct construct construct construct  99  #  1506  Name of the primer C19A2-c-3F1SceR-R C19A2-d-5F1sce1F-L C19A2-d-5FattB1-R C19A2-d-3FattB2-L C19A2-d-3F1SceR-R C19A2-e-3FattB2-L C19A2-e-3F1SceR-R C19A2-A-KO-3F  TTACAATTGCAGGCAACCAG  1507  C19A2-B-KO-5F  GCATATGGCTTCTTGCCATT  1508  C19A2-D-KO-3F  TGTCATACAGCCCCAGATCA  1551  C19A2-E-ko-3F  TGATGCTCATGCTGATTTCA  1614  Uh10022-5FattBI Uh10022F-for RNAi LP-10022 with Pro-NotI RP-10022 with Pro-NotI  GGGGACAAGTTTGTACAAAAAAGCAGGCTATAGGTTAGTGGTCAGTTTATC  1437 1438 1439 1440 1441 1446 1447  1615 1616 1617  Sequence  Purpose  AAAAATTACCCTGTTATCCCTAGAGAAGAAGCAGGGCTTTCA  3F Deletion construct C19A-C 5F Deletion construct C19A-D 5F Deletion construct C19A-D 3F Deletion construct C19A-D 3F Deletion construct C19A-D 3F Deletion construct C19A-E 3F Deletion construct C19A-E Confirmation of deletion mutant C19A2-A Confirmation of deletion mutant C19A2-B Confirmation of deletion mutant C19A2-D Confirmation of deletion mutant C19A2-D 5F Deletion construct Uh10022  AAAATAGGGATAACAGGGTAATTTCATCTTCGCCCATTCTTC GGGGACAAGTTTGTACAAAAAAGCAGGCTATTTGAAGCTCCTCGTCAGACA GGGGACCACTTTGTACAAGAAAGCTGGGTACATCATCATAGGCTGAGTGGA AAAAATTACCCTGTTATCCCTAGGCAAGCTTTGACTTGGAAT GGGGACCACTTTGTACAAGAAAGCTGGGTAGAGACGATCGTGCGTATGTG AAAAATTACCCTGTTATCCCTATTCACTGCGATCTGCCATAG  CACCGTGCACCATGGATTCGTCT ggtagcggccgcAACGTTTGTTCAGCCCTGTT ggatgcggccgcTCCGGCAAATCGGAGCGCAG  Cloning of Uh10022 in pHYG101 Cloning of Uh10022 in pHYG101  100  # 1632  Name of primer Um5295F  the Sequence  1633  Um5295R  CCAGTTGCGATCCCAATGGG  1634  Um5296F  CACCATGGGCAAAGCAACAGAGAT  1635  Um5296R  ATGAGGCCAGTCGGCTGGCT  1513  VirC17R1  CTGCAGGTCGACTCTAGAGG  1741  10026-60K-REV  GGGAAGACCAACAACCGACA  1685  10021F1(QPCR)  CGATGTAGCGGGTCTCGAAG  Cloning Um05295 in pUBleX1Int:GateWayH Cloning of Um05295 in pUBleX1Int:GateWayHA Cloning of Um05296 in pUBleX1Int:GateWayHA Cloning of Um05296 in pUBleX1Int:GateWayHA For PCR of transposable element For PCR of transposable element For QPCR analysis  1686  10021R1(QPCR)  CCCCTTCGATCGAGAGAACA  For QPCR analysis  1687  10021F2(QPCR)  ATCTTCTGATGCCCCACACG  For QPCR analysis  1688  10021R2(QPCR)  CGAGGCAGAGTTCACGGTGT  For QPCR analysis  1689  10022 qPCR-L 1  GGTGGACACCTGGTCCTAGA  For QPCR analysis  1690  10022 qPCR-R 1  CTGAGGGTCAGAACGTCTCC  For QPCR analysis  1690  10022 qPCR-R 1  CTGAGGGTCAGAACGTCTCC  For QPCR analysis  1692  10022 qPCR-R 2  GCAGTTCAATATCAAGTATCTCTCTG  For QPCR analysis  1595  Uh-0772 eIFB2  AAATGGTGTCCGCTCATCTC  For QPCR analysis  CACCATGAGATCTCCCGCTCTTC  Purpose  101  #  Purpose  1596  Name of the Sequence primer Uh-0772 eIFB2 CAACCCACGATGTTCTCCTC  1604  Uh-08813 Actin  TCGATCCTTCGTCTCGATCT  For QPCR analysis  1605  Uh-08813 Actin  CAGAGCCGAAGACTGGGTAG  For QPCR analysis  1668  10026F  TGTCGGTTGTTGGTCTTCCC  For PCR amplification  1669  10027R  TGATCAACCACATGGGTGCT  For PCR amplification  1670  10028F  CCAGTAGCCTGGAAGTCAGC  For PCR amplification  1671  10028R  TAGACTTTCGTGCGTTGTGC  For PCR amplification  1672  13901F  GAATTTCCGAGTCGATCCAA  For PCR amplification  1673  10030R  GCAAGAGGGAGCAACAAGTC  For PCR amplification  For QPCR analysis  F, forward. R, reverse. 3F and 5F indicates that primers were used for the amplification of 3’- and 5’-ends of deleted regions. The ISceI recognition sequence is in bold type and underlined, while only bold type represents the attB1 and attB2 sequences on the primers used for the deletion constructs. The tetranucleotide CACC in bold type indicates the sequence used for directional cloning in the pENTR/DTM Gateway plasmid (Invitrogen). Lower case in bold type on primers 1616 and 1617 represents the restriction recognition sequence for NotI enzymes. #; Bakkeren Lab inventory primer number.  102  Table 2.3 U. hordei genes located on BAC 3-A2 (117 kb) and their homologs in U. maydis U. hordei MIPS ID 2 1 UH_08121  U. maydis homolog 3 um05292  ExpValue  2 UH_13886  um10151  3 UH_13887  No hit in Um  4 UH_13888  Number 1  Function8  0  Percent Identity 5 82.8  Percent Similarity6 94.4  score/selfscore ratio query7 0.85  2.09E-38  42.9  63.9  0.15  hypothetical protein  um04317  0.04  36  52  0.13  hypothetical protein  5 UH_08127  um04656.2  0.02  26.4  45.5  0.06  hypothetical protein  6 UH_08128  um05306  1.54E-44  34.2  62.6  0.28  7 UH_13890  um00543  0.01  36.4  56.8  0.14  conserved hypothetical Ustilaginaceae-specific protein hypothetical protein  8 UH_10014  um02565  0  62.4  88.6  0.54  conserved hypothetical protein  9 UH_10015  um12288  0  33.5  56.3  0.29  conserved hypothetical protein  10 UH_10016  um02285  4.60E-05  20.3  54.2  0.05  hypothetical protein  11 UH_13893  um11726  0.01  25  52  0.1  hypothetical protein  12 UH_10017  um02745  2.39E-16  30.6  57.5  0.23  conserved hypothetical protein  13 UH_10018  um00776  6.96E-18  46.1  68.5  0.18  14 UH_10019  um06249  8.76E-05  25.3  53.2  0.11  conserved hypothetical Ustilaginaceae-specific protein hypothetical protein  15 UH_10020  um02747  4.95E-12  25.8  60.3  0.14  conserved hypothetical protein  4  related to DigA protein  103  U. hordei MIPS ID 2 16 UH_10021  U. maydis homolog 3 um05294  ExpValue  17 Uh_10022  um12302  18 Uh_13899  No hit in Um  19 UH_10023  Number 1  Function8  2.28E-15  Percent Identity 5 27.8  Percent Similarity6 59.3  score/selfscore ratio query7 0.17  1.87E-16  30.5  65.2  0.18  um04172.2  0.00066  18.7  56  0.05  probable transposase  20 UH_10024  um03280  0.00027  29.4  51  0.09  hypothetical protein  21 Uh_13901  No hit in Um  22 UH_10025  um06075  0.00808  27  57  0.05  probable transposase  23 UH_08123  um05293  0  91.6  97.2  0.9  probable oligosaccharyltransferase  24 Uh_13903  No hit in Um  25 Uh_13904  No hit in Um  26 UH_10026  um04367  0  58.3  75.4  0.12  related to Gag-pol polyprotein  27 UH_10027  um10618  1.10E-11  32.9  71.4  0.12  hypothetical protein  28 UH_10028  um02565  0  63  87.3  0.49  conserved hypothetical protein  29 Uh_13907  No hit in Um  30 Uh_10029  No hit in Um  31 UH_10030  um05274  0.02  31.2  64.1  0.1  hypothetical protein  4  conserved hypothetical Ustilaginaceae-specific protein conserved hypothetical Ustilaginaceae-specific protein  104  U. hordei MIPS ID 2 32 UH_10031  U. maydis homolog 3 um02565  ExpValue  33 UH_08130  Number 1  Function8  0  Percent Identity 5 46.1  Percent Similarity6 63  score/selfscore ratio query7 0.12  um05311  5.89E-21  36.8  67.2  0.21  34 UH_08132  um05311  1.97E-21  33.1  64.5  0.24  35 UH_08134  um05312  2.27E-43  45.8  74.8  0.38  36 UH_10032  um04367  0  57.9  75.4  0.08  related to retrotransposon protein conserved hypothetical Ustilaginaceae-specific protein conserved hypothetical Ustilaginaceae-specific protein conserved hypothetical Ustilaginaceae-specific protein related to pol protein  37 UH_13915  No hit in Um  38 UH_13916  um05318  2.01E-08  58.3  83.3  0.28  hypothetical protein  39 UH_10033  um05319  4.24E-13  36.7  65.1  0.28  40 UH_08135  um10558  4.92E-300  93.2  96.8  0.93  conserved hypothetical Ustilaginaceae-specific protein probable tubulin beta chain  41 UH_08136  um02237  0  26.7  53.9  0.13  conserved hypothetical protein  42 UH_08137  um10560  0  49.1  73.3  0.4  conserved hypothetical protein  43 UH_08138  um10561  0  81.6  93.4  0.84  44 UH_08139  um03753  8.65E-19  23.4  55.7  0.13  related to VPS10 domaincontaining receptor SorCS1 precursor conserved hypothetical Ustilaginaceae-specific protein  45 UH_13921  No hit in Um  46 UH_13922  um05325  0  64.9  78.4  0.66  4  conserved hypothetical protein  105  U. hordei MIPS ID 2 47 UH_13925  Number 1  1 2  U. maydis homolog 3 um05326  ExpValue 4  0  Percent Identity 5 54.3  Percent Similarity6 72  score/selfscore ratio query7 0.5  Function8 conserved hypothetical protein  Number corresponds to predicted genes in the figure. MIPS Ustilago hordei Database gene ID; The protein sequences of U. hordei strain 364 (MAT-1) were obtained from our  collaborators and will be publicly available at http://mips.helmholtzmuenchen.de/genre/proj/MUHDB/ after the publication of the analysis. 3  MIPS Ustilago maydis Database gene ID  4  SIMAP results of the best hit; SIMAP is a program that measures protein similarity based on identities of amino acids in homologous  fragments multiplied by the length of the homologous region and divided by the protein length (Rattei, et al. 2010) 8  Function of U. hordei gene (query).  106  CHAPTER 3 Genome-wide analysis of Ustilago hordei candidate secreted 1 effectors proteins; comparison with U. maydis 3.1. Introduction Pathogens secrete numerous effectors that play important roles in mediating infection of their hosts. Individual pathogen genomes encode dozens of secreted effectors that, in case of plant pathogens, are directed to the host apoplast, cytoplasm or nucleus depending on their target (Cunnac, et al. 2009, Jiang, et al. 2008, Kamoun 2006, Lindeberg, et al. 2009a, Lindeberg, et al. 2009b, Tyler, et al. 2006). As effector proteins interact with proteins in host cells and tissues, they are the direct targets of evolutionary forces and are expected to rapidly evolve. Most of the virulence and avirulence effectors from eukaryotic phytopathogens described to date are small secreted proteins (SSPs) (Rep 2005). It has been shown that these effector-encoding genes have orthologs in related species but due to an accelerated rate of evolution, they have diverged sequences compared to the rest of the genome (Liu, et al. 2011, Schirawski, et al. 2010). In Phytophthora sojae and Phytophthora ramorum, the RXLR effector-encoding genes have evolved at a faster pace compared to the rest of the genome (Jiang, et al. 2006). A three-genome comparison of P. sojae, P. ramorum, and Phytophthora infestans showed that effector families are evolving expeditiously in each species, apparently due to evolutionary pressure from their host plant (Tyler 2009). Only 25% of the effectors from these pathogens have orthologs in other species and less that 25% of these occur in a syntenic region, although the overall synteny is very high in these oomycete genomes (Tyler 2009, Tyler, et al. 2006). Raffaele et al. (2010) compared four genomes of closely related Phytophthora species that cause disease on different host plant species, in which they found that most of the pathogen genes and genome regions are highly conserved but the RXLR effectors are highly diverged. 1  A version of this chapter will be included in a joint manuscript and submitted for publication: Laurie, J., Ali, S Linning, R., Bakkeren, G., Schirawski, J, Kahmann, R. et al.  107  Also, most of these genes are located in transposon-rich, gene-poor regions, which suggests a rapid evolution of effector loci after a change in the host (Raffaele, et al. 2010). The secreted effectors of Blumeria graminis f. sp. hordei (Bgh) which contain the conserved motif, YXC, are also very species specific and only 4% have orthologs in two other powdery mildew fungi that infect dicotyledonous plants (Spanu, et al. 2010). U. hordei and U. maydis are two closely related basidiomycete phytopathogens, causing disease on two related cereal crops, barley and corn respectively, and having a similar life cycle leading to the infectious form (Hu, et al. 2002, Kamper, et al. 2006). As biotrophs, both U. hordei and U. maydis make a close association with the host and, analogous to other plant pathogens, likely secrete effectors into the host plant. The secretion of an effector in the host plant has been shown for at least one U. maydis effector, PEP1 (Basse, et al. 2000, Doehlemann, et al. 2009). In U. maydis, 554 protein-encoding genes have been predicted to be secreted and the expression of several of them has been shown to be induced during colonization of the host plant (Mueller, et al. 2008). More than 26% of these genes are clustered in seventeen clusters (Kamper, et al. 2006, Schirawski, et al. 2010). The increasing number of complete genome sequences of related fungal and oomycete pathogens provides an opportunity to predict a complete set of secreted effector proteins for comparison. The genome of U. hordei has been sequenced by our laboratory in collaboration with J. Schirawski, Georg-August-Universität Göttingen, Germany, and R. Kahmann, Max Planck Institute for Terrestrial Microbiology, Marburg, Germany. The draft genome sequence of U. hordei strain 364 is approximately 26 Mb and encodes 7,113 predicted proteins (Laurie, J., Ali, S Linning, R., Bakkeren, G., Schirawski, J, Kahmann, R. et al., in preparation). The predicted sets of effectors from fungi have an N-terminal SP and in some cases a non-conserved RXLR-like motif that could be involved in targeting these effectors to host cells (Dean, et al. 2005, Godfrey, et al. 2010, Kale, et al. 2010, Kamper, et al. 2006, Schirawski, et al. 2010). This N-terminal SP can be predicted by a number of computational tools to arrive at a comprehensive suite of potentially secreted proteins, tentatively called the “secretome” of a particular organism (Lee, et al. 2006a, Lee, et al. 2006b). In this chapter, I describe computational methods to arrive at a set of predicted secreted proteins from U. hordei to identify candidate secreted effector proteins (CSEPs) for screening for virulence or avirulence functions. In addition, I present a comparative analysis with the predicted set of U. maydis secreted proteins.  108  3.2. Materials and methods 3.2.1. Genomic resources The genome and protein sequences of U. hordei strain 364 (MAT-1) were obtained from our collaborators and will be publicly available at http://mips.helmholtzmuenchen.de/genre/proj/MUHDB/ after the publication of the genome analysis. Sequences for U. maydis strain 521 (a1b1) were obtained from http://mips.helmholtzmuenchen.de/genre/proj/ustilago/.  3.2.2. Prediction of secreted proteins A combination of several computational tools and software programs, SignalP 3.0, TMHMM 2.0, Target P 1.1 (http://www.cbs.dtu.dk/services/SignalP/ TMHMM/TargetP) and ProtComp 9.0 (http://linux1.softberry.com/berry.phtml) were used (Emanuelsson, et al. 2007) in a bioinformatic pipeline for the prediction of secreted proteins in U. hordei. All 7,113 predicted proteins were first analyzed by the SignalP 3.0 program to predict a SP based on SignalP-HMM results. This resulted in 1,142 SP-predicted proteins, which were subsequently run through TMHMM 2.0 to identify transmembrane proteins. Proteins with 1 transmembrane domain (TMD) were kept in the data set if the TMD was located close to the predicted signal peptide, since this could indicate a function related to translocation and not necessarily predict membrane retention (Mueller, et al. 2008). All other proteins with predicted transmembrane domains were removed. The resulting 931 proteins were subsequently screened by TargetP 1.1 to identify and remove proteins that were predicted to be mitochondrial; resulting in a set of 540 predicted secreted proteins. These 540 protein sequences were then analyzed with ProtComP 9.0 which compared them to proteins in the LocDB and PotLocDB databases, which hold proteins with data on known or reliably predicted localizations. Proteins with a predicted extra-cellular localization or with no similarity in the database were kept in the data set, resulting in 515 predicted secreted proteins.  109  3.2.3. Genome comparison Predicted proteins from U. hordei strain 364 and U. maydis strain 521 were compared using Similarity Matrix of Proteins (SIMAP) at the Munich Institute for Protein Sequences (MIPS). SIMAP is a program that measures protein similarity based on identities of amino acids in homologous fragments multiplied by the length of the homologous region and divided by the protein length (Rattei, et al. 2010). After comparing all U. hordei proteins to all U. maydis proteins, they were divided into three groups on the basis of amino acid identities (SIMAP values; as described in Schirawski et al, 2010). Proteins with an amino acid identity of less than 20% were considered species-specific, proteins with values between 20 and 57% were grouped as moderately conserved, and above 57%, proteins were judged to be highly conserved between the two species. In addition, within the set of secreted proteins of U. hordei, I investigated manually whether the corresponding genes were arranged in clusters on the genome and then looked for possible synteny in U. maydis.  3.2.4. Phylogenetic analysis of U. hordei candidate secreted effectors proteins All U. hordei proteins were run against one another at the MIPS using SIMAP to obtain a list of paralogs. Among the U. hordei CSEPs, proteins were considered true paralogs at a SIMAP value of 20% or greater. The phylogram of U. hordei CSEPs and their paralogs was constructed by using the multiple sequence alignment function in MEGA software, version 5 (Tamura, et al. 2011). A total of 495 proteins (372 CSEPs and 123 Non-Predicted Secreted Paralogs of CSEPs or NPSPCs) were used for sequence alignment. The resulting alignment was then used as input in MEGA version 5 to generate the neighbor-joining (NJ) tree using the Poisson correction method and number of amino acid substitution per site as the unit. The tree was subsequently depicted as a circular linearized tree. The main objective of the phylogram is to provide an overview of the effector diversity.  110  3.3. Results 3.3.1. Candidate predicted secreted proteins of U. hordei U. hordei strain 364 has 515 predicted secreted proteins according the computational screening described in the Materials and Methods section, out of a total of 7,113 annotated ORFs. Previously, predicted secreted proteins in U. maydis had been further analyzed on the basis of similarity to known proteins with annotated functions such as enzymatic functions as to arrive at a set of CSEPs (Mueller, et al. 2008). In analogy, I subdivided the set of 515 predicted secreted proteins into two classes using annotations from MUHMB (a combination of computational analysis and manual curation). 157 of the proteins had a specific annotated function and the majority of them encoded secreted enzymes involved in cell wall modifications of plants, degradation of other plant components, modification of fungal cell walls and modification of metabolites. Fourteen of these annotated proteins are related to Mig (Maize induced gene) proteins that are induced during biotrophic growth in U. maydis and are considered effectors (Basse, et al. 2000). The remaining 358 predicted secreted U. hordei proteins are hypothetical, conserved hypothetical, or conserved hypothetical Ustilaginaceae-specific proteins that did not have any annotated function and could be potential effector proteins. After adding the 14 Mig proteins to the hypothetical predicted secreted proteins, the total number of CSEPs in U. hordei strain 364 is 372.  3.3.2. Phylogeny of CSEPs and their paralogs I also searched the paralogs of the 372 U. hordei CSEPs and found 1,446 unique paralog proteins. At a SIMAP value of ≥20% identity, this number was reduced to 135 non-predicted secreted unique proteins. Twelve of these had annotated functions and were therefore removed from the candidate list, leaving 123 of these paralogs as hypothetical proteins. Adding these resulted in a total set of 495 proteins, 481 of which were accepted in the alignment algorithm (Material and Methods); the remaining proteins were discarded from the analysis because the pairwise distance could not be estimated. Interestingly, the 123 paralogs pulled into the set did not have any signal peptides and were therefore not included in the initial set based on SignalP and TargetP searches; however, by definition, they are related to the predicted effectors. An  111  example is the four additional Mig1-related proteins that have a similar cysteine pattern (Table 3.1). Such non-predicted secreted paralogs of CSEPs (NPSPCs) expand phylogenetic groups resulting in greater sequence diversity. Overall, many NPSPCs grouped with various CSEP protein families (Fig 3.1).  3.3.3. Cysteine- rich secreted protein A number of pathogen effector molecules, among which are proven avirulence proteins, have specific folding requirements: secondary and tertiary structures that often require the presence of disulphide bonds which help in protein stability and function (Doehlemann, et al. 2009, Joosten, et al. 1994, Müller, et al. 2008, Rooney, et al. 2005, Stergiopoulos, et al. 2010, Teertstra, et al. 2006, Tian, et al. 2008, Tian, et al. 2004, van den Burg, et al. 2006). Disulphide bonds or ‘bridges’ formed between cysteine residues. Several examples exist of avirulence proteins having mutations in a virulence allele where the cysteine residue has been replaced resulting in a changed conformation and, consequently, a loss of function (Joosten, et al. 1994, Doehlemann et al 2009). The 372 U. hordei CSEPs and their 123 paralogs with SIMAP value of ≥20% were, therefore, analyzed for their cysteine residue content. This analysis revealed different numbers of cysteine residues, varying from zero to as many as 28. Of the 372 CSEPs, 190 had four or more cysteine residues potentially involved in disulphide bridge formation, while this number was 90 among the 123 paralogs. Among these cysteine-rich effectors, I identified 71 proteins which could be tentatively placed in 20 classes based on their characteristic spacing of the cysteine residues (Table 3.1).  3.3.4. Comparison of U. hordei and U. maydis secretomes Based on the similarity criteria outlined in the Materials and Methods section, a comparison to the U. maydis protein complement categorized 16.9% (1,203) of the total proteins as U. hordeispecific (having amino acid identities, i.e., SIMAP values, of less than 20%); out of these, 11.6 % (140 proteins) are predicted to be secreted. In contrast, 83.1% of U. hordei proteins (5,910) have homologs in U. maydis (having amino acid identities, i.e., SIMAP values, of 20-100%), whereas only 6% of these are predicted to be secreted. Among the homologous proteins, 15% (1,067) fall in the moderately conserved group with 10.9% predicted to be secreted, while 68%  112  (4,837) are in the highly-conserved group in which only 5.5% are predicted to be secreted. As a general observation, more predicted secreted proteins are found in the species-specific and lessconserved “homologous” proteins (Fig 3.2). 1,203 proteins are present only in U. hordei, while 631 exist only in U. maydis. Among predicted secreted proteins, 355 U. hordei proteins, out of the 495, have orthologs to, and are more or less conserved with, U. maydis proteins, while 140 proteins seem U. hordei-specific.  3.3.5. A subset of predicted secreted proteins and their paralogs reside in clusters Many of the predicted secreted effector proteins in U. maydis (Kamper, et al. 2006) and Sporisorium reilianum (Schirawski, et al. 2010) are arranged in clusters. I analyzed the U. hordei genome for the localization and arrangement of the 372 CSEPs and 123 NPSPCs and found at least 62 clusters containing a subset of these predicted effector proteins and their paralogs. Clusters were defined as regions of the genome that contained at least two consecutive CSEPs and NPSPCs or were interrupted by only a few non-related proteins. The following findings substantiated the cluster definition: the 62 U. hordei clusters contained 389 genes and more than 51% of these genes encoded CSEPs or NPSPCs, while overall only 7% of the total U. hordei genes encoded CSEPs and NPSPCs. These clusters contained from two to as many as 32 CSEPs and NPSPCs. There are 199 CSEPs and NPSPCs in the 62 identified clusters accounting for approximately 40% of the total CSEPs and NPSPCs in U. hordei.  3.3.6. Comparison between the U. hordei and U. maydis secreted protein clusters Among the 62 identified U. hordei clusters, 9 had predicted secreted proteins that had highlyconserved homologs in U. maydis (SIMAP value higher than 57%), whereas 39 had predicted secreted proteins that were “least-conserved” or species-specific (SIMAP value between 20% and 57%, or 20% and below, respectively);14 clusters harboured both of those last two classes. In U. maydis, seventeen of the clusters, accounting for more than 26% of the predicted secreted proteins in that species, had been deleted individually. Deletion of either of six clusters reduced disease, while for two clusters, disease ratings on corn seedlings increased; it was concluded that many of the effectors residing in these clusters had a function in virulence (Kamper, et al. 2006, Schirawski, et al. 2010). Ten of these seventeen investigated U. maydis clusters were conserved 113  in U. hordei although two of those ten U. maydis clusters (e.g. 3A and 9A; Kamper, et al. 2006) were not identified in U. hordei genome analysis using the criteria for effectors in this study because they had annotated functions; these were discarded from further analysis. Among the remaining eight syntenic clusters, four contained U. hordei CSEPs and NPSPCs that are least conserved in an otherwise highly-conserved, syntenic region and the deletion of the homologous U. maydis clusters either increased or decreased pathogenicity. The remaining four syntenic clusters contained conserved homologs for almost all CSEPs in both U. hordei and U. maydis and the deletion of these clusters in U. maydis did not have any effect on pathogencity (Kamper, et al. 2006, Schirawski, et al. 2010). There seems to be a strong correlation between the degree of conservation among homologous effectors between the fungal species and the effect they have on virulence. The less-conserved, more-diverged effectors seem to have a more-defined role in virulence.  3.4. Discussion The sequencing of plant pathogen genomes provides an opportunity for pathologists to identify all predicted secreted proteins of pathogens and develop hypotheses regarding their potential interaction(s) with host plants. Here, I used a bioinformatic approach to identify a set of predicted secreted proteins (the secretome) from the recently sequenced genome of the barley smut pathogenic fungus, U. hordei (Laurie, J., Ali, S Linning, R., Bakkeren, G., Schirawski, J, Kahmann, R. et al in preparation). Among these, I expect to find many candidates effector genes with virulence or avirulence functions. I also determined whether these effectors are clustered and compared the secretomes of U. hordei and the closely-related corn smut fungi, U. maydis and S. reilianum. In U. maydis, there are 386 predicted secreted proteins that lack any annotated enzymatic function (Mueller, et al. 2008) and this is similar to the number of effectors I identified in U. hordei. It has been shown that effector PEP1 from U. maydis, which is required for penetration of the host plant, is conserved in U. hordei and is a functional ortholog since it can complement a Pep1-deficient U. maydis mutant (Doehlemann, et al. 2009). These two fungi are closely related; 84% of the U. hordei genes have orthologs in U. maydis and 90% of U. maydis genes have orthologs in U. hordei; however, there is a difference in conservation among secreted and nonsecreted proteins between the two species. The majority of the predicted secreted U. hordei  114  effectors are either species-specific or fall into the `least conserved` category of genes in U. maydis (Fig 3.2). Only 31% of the predicted U. hordei effectors are highly conserved in U. maydis, which is very low compared to 68% of all proteins which are highly conserved between U. hordei and U. maydis. This suggests that the effectors are evolving faster than the rest of the genome, most likely because of the selection pressure they face from interactions with their respective host plants. This trend of higher evolution among effectors has been shown for several other plant pathogens. In three closely related oomycetes, P. sojae, P. ramorum, and P. infestans, which show very high overall synteny in their genomes, only 25% of the effectors have orthologs in other species and more than 75% of these are not in syntenic regions (Tyler 2009, Tyler, et al. 2006). Similarly, only 4% of the secreted effectors of B. graminis f. sp. hordei that contain the conserved motif, YXC, have orthologs in two other powdery mildew fungi infecting dicotyledonous plants (Spanu, et al. 2010). The different degree of conservation among the CSEPs in U. hordei and in U. maydis may be related to their function. The effectors that are highly conserved between the two species may have some basic role in causing infection on plants. On the other hand, the species-specific effectors probably evolved more recently because of the arms race between the pathogen and its specific host plant. The number of predicted secreted effectors in smut fungi are relatively small compared to other fungal pathogens such as the poplar leaf rust Melampsora larici-populina and the wheat steam rust Puccinia graminis f.sp tritici and ascomycetes such as M. oryzae (Duplessis, et al. 2011, Yoshida, et al. 2009). In M. larici-populina, a set of 1,184 predicted SSPs has been identified, 74% of which are speciesspecific while 85% of 1,103 predicted SSPs are species-specific in P graminis f.sp tritici (Duplessis, et al. 2011). In M. oryzae, 1,206 putative secreted proteins have been identified (Yoshida, et al. 2009). In obligate biotroph B. graminis f. sp. hordei, 248 predicted secreted proteins have been identified that did not have any homologs outside the group of mildew fungi analyzed (Spanu, et al. 2010). However, one should keep in mind that different groups may be using slightly different approaches for identification of predicted secreted proteins. 105 CSEPs (28%) have at least one paralog in the U. hordei genome not predicted to be secreted, i.e., have no SP identified. Moreover, some of the CSEPs have paralogs that group together in gene families with non-predicted secreted proteins. These paralogs may have either lost their signal peptide during recombination and duplication, or they may still be secreted through a non-classical secretion pathway. Gene families generally arise by duplication of some  115  part of the chromosome and one copy can keep the original function while others can undergo mutations and can get a diverged function. Several extracellular effectors from Phytophthora species are encoded by multigene families, and some of these families have orthologs in other plant pathogens, like bacteria, while some are Phytophthora-specific (Dong, et al. 2009, Liu, et al. 2005, Qutob, et al. 2009, Torto, et al. 2003). More than 50% (190) of the U. hordei CSEPs, have four or more cysteine residues. In several fungal pathogens that colonize the apoplastic space, secreted cysteine-rich effectors have been found; disulphide bridge formation serves as a mechanism to protect them from apoplastic proteases (Kamoun 2006b, Rep 2005, Tian, et al. 2004, Tian and Kamoun 2005, Tian, et al. 2007, van Esse, et al. 2007, van Esse, et al. 2008). This number of cysteine-rich effector proteins is higher for intracellular biotropic pathogens that make a close association with host cells, but the disulphide bond may be required for structure and function rather than protection from apoplastic enzymes in biotrophs (Catanzariti, et al. 2007). In biotrophic fungus, M. lacariapopulina that parasitizes poplar, a large number of potential effectors have been identified to be cysteine-rich (Duplessis, et al. 2011). In U. hordei, several of the cysteine-rich proteins were grouped into twenty classes of effector proteins based on characteristic cysteine content and spacing (Table 3.1). The length of the proteins and the number of cysteine residues is not the same among these classes. Class I contains Mig1-related and Mig-like proteins that have been shown to be induced in U. maydis during maize infection (Basse, et al. 2000, Basse, et al. 2002). Although a Mig1 deletion mutant did not affect pathogenicity of U. maydis on corn (Basse, et al. 2000), the deletion of the whole cluster of Mig1 paralogs increased virulence on corn (Schirawski, et al. 2010). It was suggested that this Mig1 cluster might contain an avirulence effector because of its conserved similarity to some other fungal avirulence proteins. In U. maydis there are four Mig1 effectors, while in S. reilianum there are ten (Schirawski, et al. 2010). This group with characteristic cysteine spacing is represented by 18 effectors in U. hordei; 13 of these are predicted to be secreted while five are not because no SPs can be identified. The SP-encoding sequences may have been lost during duplication by recombination in these genes. Interestingly, in U. maydis and S. reilianum, these Mig1 genes are clustered together, while in U. hordei, there are two clusters each with two genes and the rest are dispersed and reside at 14 different loci. Class II cysteine-rich effectors contain Mig2-like proteins with similar characteristic cysteine spacing, while one member, UH_14902, has one cysteine residue  116  less and is 60 amino acid shorter than the other two members of the class. The remaining 18 classes all contain hypothetical secreted proteins with no known functions. In several fungal and oomycete pathogens, it has been shown that these cysteine-rich apoplastic effectors are inhibitors of host hydrolytic enzymes, protecting the pathogen from their devastating effect, or they are protectors that bind to fungal chitin and provide a shield (Rooney, et al. 2005, Tian, et al. 2004, Tian, et al. 2007, Shabab, et al. 2008, van den Burg, et al. 2006, van Esse, et al. 2008). To assess whether the predicted secreted proteins were encoded by genes residing in clusters, CSEP genes were located on the U. hordei genome and the flanking regions were searched manually. The majority of the genes in these clusters encoded hypothetical, conserved hypothetical or Ustilaginaceae-specific conserved hypothetical proteins. They were very similar to gene clusters in U. maydis and S. reilianum (Kamper, et al. 2006, Schirawski, et al. 2010). Schirawski et al (2010) performed a comparative genomic study of U. maydis and S. reilianum and identified 43 diverged gene clusters in which 94% of the genes encoded hypothetical proteins and 61% predicted secreted proteins. In Chapter 2, I discussed one such related cluster and the involvement of at least one member, UhAvr1, in gene-for-gene interactions in U. hordei. In this manner, the U. hordei clusters are different from the U. maydis and S. reilianum clusters of secreted proteins, as no such avirulence function has been identified in these two fungi with corn so far. This type of clusters in these Ustilaginomycete fungi is unique among fungi; no such arrangement has been identified in other completed fungal genomes such as M. oryzae (Dean, et al. 2005) and B. graminis f. sp. hordei (Spanu, et al. 2010). Gene clusters in other fungi are usually involved in sexual reproduction, biosynthesis, or degradation of secondary metabolites (Gardiner, et al. 2004, Kupfahl, et al. 2006). In several ascomycetes such as Fusarium solani, F. oxysporum, Alternaria alternata and Cochliobolus spp., genes for putative virulence functions are located on the same chromosome close to one another and sometimes interspersed with repeats, transposable elements, or other genes (van der Does and Rep 2007). In Leptosphaeria maculans, avirulence genes are clustered in a large region full of retrotransposons and their remnants, interrupted by housekeeping genes (Fudal, et al. 2007, Gout, et al. 2006). It is not understood how genes involved in virulence or avirulence evolve to cluster in fungal pathogens. Van der Does and Rep (2007) proposed two possible mechanisms for the evolution of virulence gene clusters in fugal pathogens. According to the first hypothesis, virulence genes may appear  117  at random positions in the genome and then cluster together as a result of random gene shuffling and a selection force driving clustering as a strong selective advantage. Clustering may be necessary for co-regulation of the genes, especially if several virulence genes are required for infection of a particular plant host. The clustering of the virulence genes or at least the colocation on the same chromosome will be necessary for genes that are horizontally transferred from a pathogen to a non-pathogen or a different lineage of fungi. In F. oxysporum f. sp. lycopersici, there are at least six avirulence genes on lineage-specific chromosome 14 that are considered to have been gained through horizontal gene transfer (Ma, et al. 2010). Similarly, it has been shown for M. oryzae that at least 316 candidate effectors are present on a 1.68 Mbregion in strain Ina168 but absent in the sequenced isolate, 70-15 (Yoshida, et al. 2009). The second hypothesis is that some parts of the genome are more receptive to the emergence of new genes or virulence gene insertion. It may be the reason that several effectors encoding genes are located close to repetitive elements and transposable elements that may be involved in their accumulation (Fudal, et al. 2005, Fudal, et al. 2007, Gout, et al. 2006, Kang, et al. 2001, Khang, et al. 2008, Zhou, et al. 2007). Transposons are also involved in chromosome rearrangements and might assist the insertion of new genes or chimeric gene synthesis as a result of recombination (Thon, et al. 2006, van der Does and Rep 2007, Wostemeyer and Kreibich 2002). I described here a genome-wide, computational prediction and analysis of CSEPs in U. hordei and a comparison of that set with a similar set in the closely related corn smut fungi, U. maydis and S. reilianum. Having identified these CSEPs, it will first be necessary to show that these effectors are actually secreted, which may be possible using the Yeast Secretion Trap system (Lee, et al. 2006). Transcriptome profiling using U. hordei microarrays or RNA sequencing could be used to look for effectors that are up-regulated during biotrophic growth to reduce the number of CSEPs further for experimental approaches. The functional analysis of these effectors could involve transient expression in different barley cultivars using Agroinfiltration in order to identify effectors that have an avirulence function. Interaction of such an avirulence effector and a matching resistance gene could result in a HR response; however, current investigated incompatible interactions between U. hordei and barley normally do not result in a macroscopically visible HR response (Chapter 2). An alternative approach could be co-infiltration of effector genes together with marker genes such as GUS or GFP; an avirulence function will result in cell death and consequently in a decrease in marker gene expression  118  (Dong, et al. 2009, Rehmany, et al. 2005). To look for any virulence function of these CSEPs, such as suppression of host defense responses, Agroinfiltration of these effectors followed by inoculation of a secondary barley pathogen such as B. graminis f. sp. hordei will be helpful. One bigger challenge will be to demonstrate the site of action of these effectors, i.e. to determine whether they function in the apoplast to disarm a plant defense enzyme or can enter the host cytoplasm or even the nucleus and have a function there to suppress host defense and establish compatibility. Chimeric constructs with fluorescent proteins and confocal microscopy could possibly be used for such an approach.  119  Figure 3.1 Overview of the molecular relatedness between U. hordei CSEPs and NPCSEPs showing a large diversity and small families. Circular neighbor-joining phylogenetic tree of 481 amino acid sequences (372 CSEPs and 123 non-predicted secreted paralogs, the latter marked by diamond shapes). Fourteen sequences whose pair-wise distance could not estimated, were discarded from the analysis. The tree is drawn to scale with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method and are in the units of the number of amino acid substitutions per site.  120  25  Total Proteins (%) CSEPs & NPCSEPs (%)  Percentage  20  15  10  5  0 20-30  30-40  40-50  50-60  60-70  70-80  80-90  90-10  SIMAP identity (% age)  Figure 3.2 Diversity among U. hordei and U. maydis proteins. Depicted is the distribution of amino acid identities in 10% increments (X-axis) among all U. hordei and U. maydis proteins (blue bars) as a percent of the total complement of 7,113 U. hordei proteins (Y-axis). The red bars represent the distribution of amino acid identities compared to U. maydis homologs of the predicted U. hordei CSEPs and NPCSEPs as a percentage of the 495 predicted SSPs.  121  Table 3.1 U. hordei candidate secreted effectors proteins with characteristic patterns of occurring cysteine residues (C) and spacing (number of X amino acid residues) Class Protein ID I UH_06051 UH_06234 UH_04736 UH_08826 UH_06702 UH_05040* UH_12818* UH_13038* UH_16113* UH_08403* UH_15214 UH_04923 UH_08252 UH_04676 UH_04675 UH_04922 UH_04990 UH_06803  Location of cysteine residue and spacing C-X14-C-X9-C-X13-C-X53-C-X39-C C-X14-C-X9-C-X13-C-X53-C-X39-C C-X14-C-X9-C-X13-C-X52-C-X39-C C-X14-C-X9-C-X13-C-X53-C-X39-C C-X14-C-X9-C-X13-C-X53-C-X39-C C-X3-C-X12-C-X52-C-X39-C C-X39-C C-X39-C C-X39-C C-X2-C-X9-C-X31-C-X2 -C-X9 -C-X14-C-X2 -C-X9 -C-X14-C-X2-C-X9-C C-X62-C-X15-C-X6 -C-X68-C-X42-C-X21 C-X11-C-X31-C-X15-C-X9 -C-X67-C-X37-C C-X40-C-X15-C-X9 -C-X50-C-X8 -C-X39-C C-X16-C-X26-C-X15-C-X9 -C-X66C-X37-C C-X11-C-X4- C-X16-C-X14-C-X66C-X39-C C-X16-C-X27-C-X15-C-X9- C-X42-C-X9 -C-X37-C-X37-C C-X17-C-X9 -C-X65-C-X41-C C-X17-C-X9 -C-X69-C-X41-C  II  UH_14902 UH_08758 UH_12644  C-X34-C-X9-C-X21-C-X2-C-X12-C C-X45-C-X35-C-X9-C-X21-C-X2-C-X12-C C-X47-C-X34-C-X9-C-X21-C-X2-C-X12-C  III  UH_05003 UH_01593 UH_07232 UH_01836  C-X22-C-X20-C-X2-C C-X22-C-X20-C-X2-C C-X22-C-X21-C-X2-C C-X23-C-X22-C-X2-C-X123-C-X27-C  122  Class Protein ID IV UH_02419 UH_03994  Location of cysteine residue and spacing C-X15-C-X29-C-X5-C-X15-C-X28-C-X8-C-X77-C-X2-C-X40-C C-X15-C-X30-C-X6-C-X15-C-X30-C-X8-C-X45-C-X2-C-X24-C  V  UH_08738 UH_05606  C-X21-C-X88-C-X3-C-X12-C-X8-C-X15-C-X59-C C-X21-C-X88-C-X3-C-X12-C-X8-C-X15-C-X59-C  VI  UH_03442 UH_03402  C-X50-C-X195-C-X54-C C-X12-C-X163-C-X14-C-X166-C-X54-C  VII  UH_00093 UH_00478  C-X10-C-X12-C-X80-C-X42-C-X16-C-X19-C-X13-C-X40-C-X14-C C-X10-C-X11-C-X20-C-X92-C-X3 -C-X8 -C-X8 -C-X15-C-X34-C  VIII  UH_14977 UH_16458 UH_01705  C-X6 -C-X6-C-X6 -C-X28-C-X9 -C-X5-C-X10-C C-X6 -C-X6-C-X6 -C-X28-C-X9 -C-X5-C-X10-C C-X14-C-X6-C-X44-C-X19-C-X37-C  IX  UH_14136 UH_03910 UH_04742  C-X4C-X59-C-X8-C-X29-C-X3-C-X12-C-X17-C-X3-C-X4-C-X2-C C-X4-C-X2-C-X55-C-X8-C-X29-C-X3-C C-X4C-X54-C-X8-C  X  UH_02707 UH_04984  C-X50-C-X6-C-X52-C-X4-C-X29-C C-X42-C-X6-C-X39-C-X4-C-X10-C-X40-C  XI  UH_08916 UH_08915 UH_07874 UH_12082 UH_14135  C-X17-CC-X17-C-X17-CC C-X24 -C-X17-CC-X17-C-X17-CC C-X1-C-X23-CC-X17-C C-X1-C-X11- C-X9- C -X47-C-X7 -C C-X6-C-X11- C-X11-C -X44-C  XII  UH_06080  CC-X59-C-X17-C-X10-C  123  Class Protein ID UH_04531  Location of cysteine residue and spacing C-X5- C-X17-C-X10-C-X13-C-X12-C-X1-C-X6-C  XIII  UH_06316 UH_14081 UH_02007 UH_00491 UH_05093  CC-X11C-X10-C-X3 -C C-X3 -CC C-X4-C-X3-C-X10-C-X3 -C-X4 -C-X3 -C C-X3 -C-X12-C-X62-C-X21-C-X10-C-X4-C C-X4-C-X4-C-X4 -C-X4 -C-X56-C-X18-C-X24-C C-X4-C-X5-C-X6-C-X1-C-X7 -C-X33-C-X5 -C  XIV  UH_04400 UH_04949  C-X6-C-X3-C-X4-C-X22-C-X5 -C-X9 -C-X18-C-X40-C-X3-C C-X6-C-X7-C-X1-C-X53-C-X50-C-X63-C  XV  UH_14114 UH_14108  C-X3-C-X15-C-X108-C-X33-C-X157-C-X16 -C-X9 -C-X6-C C-X3-C-X15-C-X108-C-X33-C-X52 -C-X104-C-X31-C  XVI  UH_01209 UH_01211  C-X6-C-X20-C-X29-C-X6-C-X3-C-X21-C-X5-C-X9-C C-X6-C-X20-C-X30-C-X6-C  XVII  UH_01635 UH_04865  C-X6-C-X4-C-X10 -C-X8 -C-X1 -C-X18-C-X6 -C-X3 -C-X12 -C-X3 -C-X7-C C-X6-C-X4-C-X6 -C-X27 -C-X9 -C-X5 -C-X12 -C-X75-C-X6 -C-X5 -C-X6-C-X27C-X9-C-X5-C-X8-C-X186-C C-X5-C-X7-C-X109-C-X78 -C-X75-C-X39-C-X158-C-X9 -C-X180-C-X29-C C-X5-C-X7-C-X27 -C-X124-C-X5 -C-X22-C-X26 -C-X7 -C-X12 –C C-X5-C-X4-C-X4 -C-X12 -C-X5 -C-X5 -C-X4 -C-X9 -C-X5 -C-X4 -C-X4-C-X11C-X5-C-X5-C-X4-C-X9-C -X5-C-X4-C-X4-C-X11-C-X5C-X4-CX4-C-X9-C-X5-C-X4-C-X5-C C-X6-C-X5-C-X5 -C  UH_00039 UH_08589 UH_03101  UH_14724 XVIII UH_01807 UH_01944  C-X8-C-X15-C-X169-C-X4 -C-X71-C-X79-C C-X8-C-X15-C-X60 -C-X19-C  124  Class Protein ID  Location of cysteine residue and spacing  XIX  UH_00393 UH_01539  C-X27-C-X20-C-X6-C C-X2- C-X29-C-X6-C  XX  UH_01947 UH_01952  C-X11-C-X62-C-X19-C C-X13-C-X57-C-X18-C  * Proteins representing the non-predicted secreted protein paralogs of CSEPs.  125  CHAPTER 4 Introduction of large DNA inserts into the barley pathogenic fungus, Ustilago hordei, via recombined binary BAC vectors and Agrobacterium-mediated transformation1 4.1. Introduction An understanding of cell development and pathogenicity in U. hordei will require an efficient genetic transformation procedure for gene complementation, gene replacement and other genome manipulations. It is also desirable to transfer large DNA fragments containing complete genes with regulatory sequences necessary for gene function, or complete clusters of genes, to assess the location of specific functions/genes on genome-size fragments as represented by Bacterial Artificial Chromosome (BAC) inserts (Shizuya, et al. 1992). For example, U. hordei harbours gene clusters coding for related predicted secreted proteins that could be effectors during the interaction with its host, similar to the clusters described in U. maydis (Kamper, et al. 2006). There is no efficient and reproducible transformation system for U. hordei. Current methods use partial protoplasts and the addition of 1% polyethylene glycol (PEG) followed by electroporation (Bakkeren, unpublished). The generation of partial protoplasts involves the use of lytic enzymes, which have to be calibrated for each enzyme batch rendering this method not very reproducible. Agrobacterium tumefaciens is a well known plant pathogen causing crown gall disease on plants by transferring a part of its tumor inducing (Ti) plasmid DNA to plant cells and integrating this stably into the host genome. Any DNA between specific 25 base-pair imperfect repeats, termed left and right border sequences, can be transferred. Such constructs can be located on smaller replicating plasmids, so-called binary vectors (Hoekema, et al. 1983, Lee and Gelvin 2008). Commonplace in plant biotechnology as the agent of choice for genetic transformation, the Agrobacterium-mediated transformation (AMT) system 1  A version of this chapter has been published: Ali, S. and Bakkeren, G. 2011 Introduction of large DNA inserts into the barley pathogenic fungus, Ustilago hordei, via recombined binary BAC vectors and Agrobacterium-mediated transformation. Current Genetics 57: 63-73.  126  has been exploited extensively for fungal transformation as well (Amey, et al. 2002, de Groot, et al. 1998, Michielse, et al. 2005a, Michielse, et al. 2005b, Sugui, et al. 2005, Tucker and Orbach 2007). Compared to conventional transformation methods, AMT is very efficient for many filamentous fungi and has worked well for the development of transformation systems for fungi which are refractory to transformation using conventional methods, such as Agaricus bisporus, Aspergillus giganteus and Helminthosporium turcicum (Chen, et al. 2000, Degefu and Hanif 2003, Meyer, et al. 2003, Mikosch, et al. 2001, Michielse, et al. 2005b). Among basidiomycetes, AMT is very efficient in Cryptococcus neoformans and Cryptococcus gattii (McClelland, et al. 2005) and U. maydis (Ji, et al. 2010) and was recently also used successfully for the genetic transformation of the flax rust, Melampsora lini (Lawrence, et al. 2010). AMT is not only more efficient compared to conventional methods but often results in single copy integration events at random sites in the fungal genome which is desirable for creating insertion mutations (Combier, et al. 2003, Mullins, et al. 2001, Takahara, et al. 2004). An additional advantage of AMT, compared to classical mutagenesis is the ease with which fungal sequences flanking the T-DNA insertion site can be recovered and identified (Bundock and Hooykaas 1996, Bundock, et al. 2002, de Groot, et al. 1998, Leclerque, et al. 2004). For example, sporulation deficient mutants, pathogenicity deficient mutants, antibiotic deficient mutant and mutants altered in pigmentation have been obtained in several fungi (Blaise, et al. 2007, Li, et al. 2005, Rogers, et al. 2004). Conventional cloning methods for the generation of transformation constructs using restriction enzymes are often inefficient and time consuming, especially for BAC clones because of their large sizes and few convenient restriction sites (Nagano, et al. 2007). Recombineering as an alternative method uses the DNA double-strand repair machinery and bacteriophage lambda RED recombination proteins (Lee, et al. 2001) and does not require restriction sites for conventional cloning or specific recombination sites such as the ones required for ‘GatewayTM’ cloning. Recombineering is therefore useful for cloning large DNA fragments and facilitates the cloning of whole or specific regions of BAC or PAC clones (Lee, et al. 2001, Raymond, et al. 2002). Methods to transfer large genomic fragments in BAC clones to plants using AMT have been established many years ago when Agrobacterium-specific binary BAC vectors, so-called BIBAC vectors, were developed (Hamilton, et al. 1996). Combining above-mentioned techniques, Takken et al. (2004) developed a strategy to convert BAC vectors into fungal-  127  specific BIBAC vectors suitable for Agrobacterium-mediated transfer into fungal strains. This was efficiently achieved by a one-step procedure making use of in vivo recombineering (Lee, et al. 2001) of a linear ‘fungal- and binary-specific’ fragment into BAC clones. In the present study I investigated the feasibility of using this system in U. hordei and testing whether large DNA fragments could be delivered stably into its genome via AMT. I converted two BAC clones containing genomic inserts into BIBACs and showed that A. tumefaciens can deliver these genomic fragments stably into the genome of U. hordei.  4.2. Materials and methods 4.2.1. Strains and plasmid E. coli strain SW102, a recombineering strain derived from strain DY380 (Warming, et al. 2005), was obtained from Dr. N. Copeland (National Cancer Institute, Frederick, MD). Supervirulent Agrobacterium strain COR309 is a recA-deficient C58 nopaline strain UIA143 harbouring disarmed pTiB6 derivative plasmid pMOG101 (Hamilton, et al. 1996) and a special vir helper plasmid pCH32 which provides extra copies of the virA and virG two-component signaling genes. Agrobacterium strain COR308 is similar to COR309 except that it has disarmed pTi derivative plasmid pMP90 instead of pMOG101; they were obtained from Cornell University (http://www.biotech.cornell.edu/BIBAC/BIBACHomePage.html). Ustilago hordei haploid strain Uh4857-4 (alias Uh364, MAT-1) has been described (Linning et al. 2004) and U. maydis haploid strain 324 (a2b2) is identical to Um521 (Kronstad and Leong 1989). The REC plasmid pFT41 was obtained from Dr. F. Takken (Swammerdam Institute for Life Sciences, University of Amsterdam, The Netherlands; Takken, et al. 2004). pUSBAC5 is a BAC vector derivative of pEcBAC1 (Frijters, et al. 1997) converted for use in Ustilago species by introducing a specific hygromycin B resistance cassette (Linning et al. 2004).  4.2.2. Recombineering Target constructs pUSBAC5, pUSBAC5_2-1 and pUsBAC5_1-6 were transformed into recombineering E. coli strain SW102, selected on Luria-Bertani (LB) plates supplemented with chloramphenicol (Cm) 20 µg ml-1 at 30 oC to prevent premature induction of the phage  128  recombineering genes (RED gene). Details of the protocol can be found in Lee et al. (2001). The recombining REC part was amplified from pFT41 by Polymerase Chain Reaction (PCR) with primers cat-f2 (CCGTTGATATATCCCAATGGC) and catR (ACAAACGGCATGATGAACCT) using TaKaRa LA TaqTm polymerase (TAKARA Bio INC) and the following program on a MyCycler (BioRad): an initial denaturation step of 5 min at 95 o  C followed by 35 cycles of 30 sec at 95 oC, 40 sec at 58 oC and 10 min at 68 oC, with a final  extension at 68 oC for 15 min. The PCR product was digested with DpnI to remove the template and then purified on an agarose gel using the QIAquick Gel extraction kit (QIAGEN) according to manufacturer’s instruction. The SW102 cells harbouring the target BAC vectors were grown in LB medium supplemented with 20 µg Cm ml-1 at 30 oC to an optical density at 600 nm (OD600) of 0.6-0.9, and incubated for 15 min at 42 oC to induce the phage recombination RED genes whose expression is under control of a temperature-sensitive λ-repressor. Cells were cooled immediately by chilling on ice for 20 min, then centrifuged at 4000 rpm for 10 min and washed three times with sterile ice-cold water. Cells were resuspended in an appropriate volume of ice cold sterile water. Cells were either used fresh for electroporation or mixed with 30% sterile glycerol and stored at -80 oC for future use. The electroporation of 40 µl cells in a 0.2 cm gap electroporation cuvette was carried out in a Gene Pulser (BioRad) with 120 ng of REC DNA, using a pulse of 2.5 kV (at 25 µF and 200 Ω). Immediately after electroporation, 0.5 ml of SOC medium (2 % Bacto-tryptone, 0.5% , Bacto-yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4, 20 mM glucose, pH 7.0) was added and cells were incubated at 32 oC for 1 h with gentle shaking (120 rpm) to initiate recombineering, recover and express antibiotic resistance. Cells were subsequently plated on LB medium containing 50 µg/ ml-1 kanamycin (Km) and incubated overnight at 30 oC. Transformants were tested for sensitivity to chloramphenicol; correct recombination of the REC vector into the BAC vector disrupts the CAT gene. Further verification of generated BIBAC vectors was carried out by restriction enzyme digests and PCR.  4.2.3. Fungal transformation BIBAC constructs were introduced by standard electroporation into A. tumefaciens strain COR309 or COR308, and a fresh colony was grown overnight at 28 oC in LC medium (0.8% NaCl w/v, 1% Bacto-tryptone w/v, 0.5% Bacto-yeast extract w/v) supplemented with 5µg ml-1 129  tetracycline (Tc) to select for the helper plasmid pCH32 and 50 µg Km ml-1 to maintain the BIBAC construct. Five ml of BIBAC-containing A. tumefaciens culture was spun down for 10 minutes at 4000 rpm and the pellet resuspended in 5 ml induction medium (IM, minimal medium as in Takken et al. (2004), supplemented with 40 mM MES, 0.5% glycerol, 0.2% glucose). After centrifugation as above, the pellet was resuspended to an OD600 of 0.4 in 5 ml IM containing the appropriate antibiotics and supplemented with 200 µM acetosyringone (AS, PhytoTechnology Laboratories, Shawnee Mission, KS). Cells were incubated at 28 oC for 6-8 hours to reach OD600 of 0.5. Control cells were treated identically in the same medium but without AS. U. hordei strain Uh364 and U. maydis strain 324, grown in 5 ml complete medium (CM; Holliday. 1974) for 2 days, were used to re-inoculate 20 ml fresh CM to an OD600 of 0.15 and subsequently grown to an OD600 of 0.5. U. hordei was always incubated at 22 oC and U. maydis at 28 oC unless mentioned otherwise. Both U. hordei and U. maydis cell cultures were diluted 10-fold in IM and mixed with an equal volume of AS-induced A. tumefaciens culture and 200 µl of the mixture was plated onto ME-25 filters (Schleicher and Schuell, 0.45 µm pore size, 47 mm diameter) which were placed on co-cultivation medium plates (IM but with 0.1% glucose and 200 µM AS added); negative controls contained no AS. The membranes were air-dried briefly for 10-60 minutes and incubated at 20-24 oC for 2-5 days on co-cultivation media. To select for transformants, membranes were transferred to CM plates containing Cefotaxime 200 µg ml-1 to kill off A. tumefaciens and 300 µg ml-1 hygromycin B (hyg B, Calbiochem EMD Biosciences, Inc. La Jolla, CA) to select for fungal transformants. After 4 days on selection media, individual transformants were transferred to CM medium supplemented with 300 µg ml-1 hyg B.  4.2.4. Analysis of transformants Genomic DNA isolation and PCR amplification – Individual putative U. hordei and U. maydis transformants were grown in 5 ml CM medium supplemented with 300 µg hyg B ml-1 for two days. Subsequently, 1 ml of this culture was inoculated in 100 ml fresh CM medium with the same antibiotic. Cultures were spun down and pellets frozen rapidly in liquid nitrogen for direct use or storage at -80 oC. Frozen pellets were ground to a fine powder in liquid nitrogen and used for DNA extraction using the DNeasy Plant Maxi kit (QIAGEN) following the manufacturer’s instructions. PCR was used to confirm the presence of T-DNA by amplifying an internal 1023 bp 130  fragment of the hygromycin B phosphotransferase open reading frame using primers hyg B-F (GTACCATGGAAAAGCCTGAACTCACCGCGACG) and hyg B-R (GCATCTAGACTCTATTCCTTTGCCCTCGGAC). The cycling conditions were as follows: an initial denaturation step of 5 min at 95 oC followed by 35 cycles of 30 sec at 95 oC, 30 sec at 60 o  C and 1 min at 72 oC, and a final extension at 72 oC for 10 min. To verify the presence of an  intact T-DNA insertion, another PCR reaction was performed using primers near the left border: cat-f2 (see above) and LB-r2 (CACAGCGACTTATTCACACGA). An intact left T-DNA border would result in the amplification of a 302 bp fragment. The cycling conditions were the same as above except for an annealing temperature of 64 oC and extension time of 30 sec.  DNA gel blot hybridization – For DNA blot analysis, 8 µg of genomic DNA was digested with AvaI or BglII run out slowly on a 0.8 % (w/v) agarose gel in 1X Tris-borate-EDTA (TBE) buffer (89 mM Tris base, 89 mM boric acid, 2 mM EDTA pH 8.0), and transferred overnight to a Hybond-N+ membrane as recommended by the supplier (Amersham Biosciences/GE healthcare). Two hybridization probes were used: for the left border of the T-DNA, a fragment of 302 bp was amplified from BIBAC_2-1 by PCR using Taq polymerase and primers cat-f2 and LB-r2, and for right border of the T-DNA, a fragment of 564 bp was generated by PCR using primers cat-r2 (ACAAACGGCATGATGAACCT) and Rb-r2 (CACAGCGACTTATTCACACGA). These fragments were gel-purified using a QIAquick Gel extraction kit (QIAGEN) following the manufacturer’s instruction. Probe labeling was carried out with αP32-dCTP, using the Rediprime II Random Prime Labeling system (Amersham Biosciences/GE healthcare), and hybridized to the membrane using ULTRA-hyb buffer (Ambion) at 42 oC according to manufacturer’s instructions. The blots were washed twice for 5 minutes with 2X SSC (0.3 M sodium chloride, 0.03 M sodium citrate, pH 7.0), 0.1% w/v sodium dodecyl sulphate (SDS) followed by two washes each of 15 min in 0.1 X SSC, 0.1% SDS. All washes were carried out at 42 oC and the blots were exposed to Kodak Biobax film (Kodak Canada, Toronto, ON, Canada).  131  4.3. Results 4.3.1. Recombineering The recombineering technology is based on the RED homologous recombination system and uses functions that are provided by a defective λ prophage that is present on chromosomal DNA of E. coli strain SW102. These λ prophage gene products supply the functions that protect and integrate the linear introduced REC DNA into BAC vectors (Warming, et al. 2005). To convert BAC vectors into BIBACs, Agrobacterium-specific functions such as a bacterial selectable marker (kanamycin resistance), T-DNA specific border sequences BL and BR and a broad-host range origin of replication need to be introduced on the linear, recombining part of the REC vector. Flanking this transforming fragment are 40 bp ends providing the homologous termini for integration into the CAT resistance gene. Such REC vector was developed by Takken et al. (2004) for use in ascomycete fungi by introducing a fungal-specific selectable resistance cassette to allow AMT of Fusarium and Aspergillus species. I used the pFT41 backbone to convert a previously developed Ustilago-specific BAC vector, pUsBAC5, already containing an Ustilagospecific hygromycin B cassette under the control of the HSP70 promoter and terminator signals (Wang, et al. 1988). Integration of the REC fragment from pFT41 would create a binary construct that has the complete pUsBAC5 construct including the hygromycin B cassette and any genomic insert residing on pUsBAC5, in between the T-DNA borders BR and BL (Fig. 4.1). Two different target constructs were made in Ustilago-specific BAC vector, pUsBAC5, by inserting a 11 kb Sac1 and a 9.3 kb Xba1 fragment from the U. hordei avirulence gene 1 (UhAvr1)-containing genomic region (Linning et al. 2004). The 11 kb SacI-fragment was cloned in the SacI site of pUsBAC5, creating pUsBAC5_1-6, and the 9.3 kb Xba1 fragment was inserted in the HindIII site by partially filling in 2 base pairs in each of the 5-overhanging tails generated by HindIII and XbaI with the Klenow fragment of DNA polymerase 1 to create only 2 bp-sticky overhangs (Korch 1987) ligation generated construct pUSBAC5_2-1. pUsBAC5 was used as an “empty vector” control. The introduction via electroporation of 120 ng of linear, PCRamplified REC DNA into E. coli strain SW102 previously transformed with the BAC target constructs and heat-induced to activate the λ RED genes, usually resulted in 50-100 kanamycinresistant colonies. No colonies were obtained from cells that were not heat-induced. Kanamycin resistance colonies were tested for chloramphenicol-sensitivity to select for proper recombination  132  in the CAT gene of the BAC vector. In general, 25 to 30% of kanamycin-resistant colonies became chloramphenicol-sensitive in RED induced cells (Table 4.1). Figure 4.2 shows an EcoRI restriction enzyme pattern of BIBAC plasmids purified from colonies that were chloramphenicol-sensitive and kanamycin-resistant. The restriction analysis of these recombinants showed that the REC vector had integrated at the proper position without causing any rearrangements of the BAC clones. These data demonstrate that recombineering works well and allows for the conversion of U. hordei BAC library clones into BIBAC vectors.  4.3.2. Fungal transformation Constructs BIBAC_2-1, BIBAC_1-6 and BIBAC_5 (“empty vector” control), were introduced into A. tumefaciens strain COR309 and COR308 via electroporation. These Agrobacterium strains are recA- and contain extra copies of virA and virG on a helper plasmid. Frary and Hamilton (2001) have shown that extra copies of these AS inducer-sensing and signaling components are essential for successful transformation of plants with large pieces of DNA such as contained on BIBAC vectors. Co-cultivation of Agrobacterium cells harbouring BIBAC_5, BIBAC_2-1 and BIBAC_1-6 with U. hordei strain Uh364 in the presence of AS led to the formation of hygromycin B-resistant colonies, while no colonies were obtained on co-cultivation medium without AS. I also included the related corn smut pathogen U. maydis strain 324 (a2b2) for comparison. The AMT transfer efficiency of these BIBAC constructs into U. hordei is lower than that for U. maydis (Table 4.1). Interestingly, for U. maydis, some hygromycin B-resistant colonies were obtained in the absence of AS in co-cultivation medium, although AS was used in the induction medium. For U. hordei, hygromycin B-resistant colonies were obtained only when AS was used in both induction and co-cultivation media. I tested whether U. hordei cells without cell walls (protoplasts) would be more sensitive to the Agrobacterium T-DNA transfer machinery. AMT transformation efficiencies were found to be essential the same as for sporidia (Table 4.1). The transformation frequency of BIBAC_5, the “empty vector” control which still harboured a T-DNA insert of approximately 10 kb, was on average three times higher in U. hordei than that of BIBAC_2-1 and BIBAC_1-6 (Table 4.1). Various parameters have been reported to affect AMT efficiencies in other fungi, such as drying of co-cultivation medium plates with Agrobacterium and fungal mixtures for various times (Almeida, et al. 2007), the length of cocultivation period (Rho, et al. 2001), the use of different ratios of Agrobacterium to 133  fungal cells (Michielse et al. 2005b), and various cocultivation temperatures (Michielse et al. 2005b). However, due to low overall transformation efficiencies, no significant differences could be measured.  4.3.3. Molecular analysis of fungal transformants To test the mitotic stability of the transgenes, eight randomly selected, hygromycin B-resistant transformants (selected on CM medium supplemented with 300 µg hygromycin B ml-1) were transferred to selection-free PDA plates for 5 successive cycles (4 days of growth at 22 oC per cycle). Cells from non-selective PDA plates were then transferred to 100 ml CM liquid medium supplemented with 300 µg hygromycin B ml-1 for total genomic DNA isolation. To confirm the presence of intact T-DNA, two PCR analyses were performed: one to test for the presence of the internal hygromycin B phosphotransferase gene and one for the left T-DNA border. Using the primers hyg B-F and hyg B-R, a PCR product of expected size (1020 bp) was amplified from DNA of all eight putative transformants (Fig 4.3A) which correlated with the observed growth in selective hygromycin B medium. Agrobacterium generates T-DNA directionally from an initial nick at the right border which is then linked to the virD2 protein, ending at the left border. Integrated T-DNA therefore frequently has variable left border truncations in contrast to the right border junction which is often more precise (Tinland 1996; Bundock and Hooykaas 1996; Zhong et al. 2007). The second PCR was performed to verify the presence of intact left border sequences by using primers LB-r2 and cat-f2, which is expected to amplify 302 bp immediately adjacent to the left border of the T-DNA. Seven out of the eight transformants amplified a PCR product of the expected size (Fig 4.3B). The genomic DNA of the eight selected, PCR-positive, stable U. hordei transformants was analyzed on DNA blots to determine the extent of random T-DNA integration events and assess copy number of the insertions. I analyzed both ends of the T-DNA insertion by hybridizing two separate blots with a left T-DNA border- or a right border-specific probe. Transformant C which was negative for the left border in the PCR analysis, was positive upon hybridization (Fig. 4.4B). Single fragments were revealed for all transformants for both right border and left border junctions, indicating that the T-DNA had inserted as a single copy in each strain, since the selected probes did not span the chosen restriction enzyme sites. At least seven of the transformants revealed junction fragments of different sizes indicating random insertion 134  events at different locations in the genome. One transformant, Uh364 BIBAC_1-6 transformant Z, did not show a positive hybridization signal with the right border-specific probe (Fig. 4.4C); in this case it is likely that the right border end of the T-DNA became truncated upon the integration event thereby deleting the probe binding site. Truncation of T-DNA upon integration is not uncommon but is normally more prominent at left border junctions.  4.4. Discussion The main objective of this work was to establish an Agrobacterium-mediated transformation (AMT) protocol for transferring large fragments of genomic DNA, contained on BAC library clones, into the barley smut fungus, Ustilago hordei. To this end I evaluated the use of an in vivo recombineering method for converting BAC library clones into binary BAC (BIBAC) vectors and to subsequently develop an AMT protocol for this fungus. The recombineering method is based on a modification of cloned DNA in E. coli via a λ RED mediated homologous recombination and avoids the cumbersome restriction and ligation reactions usually carried out to modify DNA (Warming, et al. 2005). The recombination of the REC vector into a BAC clone is based on the expression of RED genes from a stably integrated defective λ prophage under the control of a temperature sensitive repressor, cI857. The REC vector provides all the required functions for the construction of a binary vector (Takken, et al. 2004). Recombineering uses stretches of homologous DNA which in this method is provided by the bacterial chloramphenicol resistance gene. Proper insertion of the REC vector into BAC clones results in the loss of chloramphenicol resistance while resistance to kanamycin, present on the REC vector, is gained. This provides an easy tool to select for likely proper recombinants which can then be verified by restriction enzyme analysis. I obtained an efficiency of proper recombination of 25-34% based on gain of kanamycin and loss of chloramphenicol resistance. This is comparable to the 40% reported for the conversion of Fusarium oxysporum and Aspergillus awamori BAC clones into BIBACs by recombineering (Takken, et al. 2004). The remaining colonies, found to be both kanamycin and chloramphenicol resistant, may be the result of integration of the transforming REC fragment into BAC vector locations other than the CAT gene, including homologous stretches in the BAC genomic insert, or may be due to dimerization of the generated BIBAC with the original BAC clone and thus contain both selectable markers (Takken, et al. 2004). Yu  135  et al. (2000) reported that such dimerization can be suppressed by transformation of both REC and BAC plasmids at the same time. Compared to plant transformation, AMT of fungi is relatively new (Bundock, et al. 1995, de Groot, et al. 1998) but has been very successful for a number of species and the number of fungi that can be transferred by Agrobacterium is still increasing. However, optimization of the transformation protocol is required for each species (Amey, et al. 2002, Mata, et al. 2007, Michielse, et al. 2005a, Michielse, et al. 2005b). To our knowledge, this is the first report on AMT of the barley pathogen U. hordei. U. hordei strains Uh364 and U. maydis strain 324 were transformed with either BIBAC_2-1, BIBAC_1-6 or “empty-vector” control BIBAC_5, using two Agrobacterium strains, COR308 and COR309. Overall, the transformation efficiency of U. hordei was low compared to U. maydis using the same protocol. In a recent study, Ji et al. (2010) reported on the development of an efficient AMT method for U. maydis, employing a series of optimization steps. In my study, no difference in transformation efficiencies was observed when using the two Agrobacterium strains COR309 or COR308, indicating that the origin of the virulence functions on the Ti plasmid do not influence efficiency. The transformation efficiency of BIBAC_5 was at least three times higher than that of both BIBAC_2-1 and BIBAC_1-6; it was slightly higher for BIBAC_2-1 compared to BIBAC_1-6. This suggests that the size of the insert (T-DNA) in the vector could influence the transformation efficiency. Alternatively, the inserts in both BIBAC_21 and BIBAC_1-6 clones originate from a region in the U. hordei genome where many repeats and transposable elements have been found and this could also be the reason for the low transformation efficiencies. Such elements might inhibit integration or, additionally, it may be that such repeats present on these T-DNAs and homologies in the genomic DNA in U. hordei make integration in the genome less efficient as to avoid duplication (Yu et al. 2000). Takken et al. (2004) also observed a low transformation efficiency and truncation of integrated T-DNA when using BAC clones containing genomic DNA inserts with large stretches of homologous DNA, compared with the empty vector without any homologous region. I evaluated the effect of acetosyringone (AS), a plant phenolic compound that is produced in wound sites of plants. It is an inducer of the vir genes in A. tumefaciens (Gelvin 2003) and serves as inducing agent for in vitro transformation. I obtained U. hordei hygromycin B resistant colonies only when AS was used both in the induction and co-cultivation media, which suggest  136  that AS is essential for AMT of U. hordei, a requirement found for the majority of fungal species (Amey, et al. 2002, Michielse, et al. 2005a, Michielse, et al. 2005b, Duarte, et al. 2007, Marchand, et al. 2007, Zhang, et al. 2008). The presence of AS in induction medium before cocultivation is not necessary for Agrobacterium growth but it has been reported to improve transformation efficiencies in A. carbonarius, F. oxysporum and M. grisea (Morioka, et al. 2006, Mullins, et al. 2001). Indeed, in parallel experiments of AMT transformation of U. maydis 324, I obtained a few hygromycin B resistant colonies in the absence of AS in the co-cultivation media, although AS was included in the induction medium. The mitotic stability of the transformants was verified by growth on non-selective media plates for five successive transfers and subsequent comparative growth on selective versus nonselective medium plates. Subsequent PCR amplification of the hyg B phosphotransferase gene and the left T-DNA border and analysis of the genomic DNA by DNA blot hybridization was consistent with stable T-DNA integration into chromosomal DNA previously reported for TDNA transfer to filamentous fungi (Gelvin 2003, Ji et al. 2010, Gelvin. 2003, Covert, et al. 2001). Previous reports showed that several parameters affect the T-DNA copy number in fungal genome (Michielse et al. 2005b). For example, the addition of AS in IM and the length of cocultivation time seem to affect the number of T-DNA integrations per transformant (Combier, et al. 2003, Rho, et al. 2001). The addition of AS in IM as carried out in our experiments, reduced the occurrence of multiple integrations in the ectomycorhizal fungus, Hebeloma cylindrosporum (Combier, et al. 2003), while in M. grisea the addition of AS in IM increased multiple integrations (Rho, et al. 2001). DNA blot analysis revealed at least seven fragments of different sizes among the eight transformants analyzed, suggesting a random mode of integration. It has been suggested that the mode of T-DNA integration either by homologous or non-homologous recombination depends on the organism (Bundock, et al. 1995, Covert, et al. 2001, van Attikum, et al. 2001). Cloning and sequencing of the junctions between the integrated T-DNA and the genomic insertion sites would be necessary to verify true random integration and to assess the precise mode of integration. The T-DNA transfer process in Agrobacterium starts at the right border after nicking and the attachment of a VirD2 protein on the 5′-end of the nascent single strand. The production of the T-DNA molecule proceeds until the left border sequence is reached and another nick is  137  introduced, generating an unprotected end (Tzfira and Citovsky 2006). After transfer and during integration, deletion of T-DNA nucleotides occurs at the junctions of the T-DNA repeats, most frequently at the unprotected left border end in plants, yeast and other filamentous fungi (Bundock and Hooykaas 1996, de Groot, et al. 1998, Tinland 1996, Zhong, et al. 2007). Recipient insertion sites in the genomic DNA also often suffer deletions. PCR analysis revealed that the left border end was missing from only one of the transformants but the DNA blot analysis showed that this must be a minor truncation of T-DNA at this left border (including a primer binding site) because the left border end probe still hybridized. In conclusion, the strategy of using recombineering to convert BAC library clones into BIBAC constructs and to use A. tumefaciens for the transfer of these BIBAC constructs to U. hordei is feasible. We have several BAC libraries containing U. hordei genomic inserts of various sizes (Bakkeren et al. 2006). One is derived from a strain which has several avirulence genes and the complete genome of which has been sequenced (Laurie, J., Ali, S Linning, R., Bakkeren, G., Schirawski, J, Kahmann, R. et al in preparation). The method described in this paper will facilitate the functional analyses of individual genes and whole gene clusters by complementation studies.  138  Figure 4.1 Schematic representation of the BAC to BIBAC conversion method. Conversion of Bacterial Artificial Chromosome vector, pUSBAC5, harbouring genomic inserts, into a binary BAC (BIBAC) vector using a linear 7,087 bp, PCR-amplified DNA fragment (REC-vector) from pFT41 (top line; Takken et al. 2004). The REC-vector recombines into the chloramphenicol resistance gene (CAT) present on pUSBAC5, using homologous regions present on both left (cat-f2) and right end (cat-r2) of the REC-vector (dashed arrows). Recombinants are selected for kanamycin resistance (Kan) present on the REC-vector and screened for loss of chloramphenicol resistance indicating proper integration. The location of the U. hordei genomic fragments in the respective BAC clones, 1-6 in the SacI site or 2-1 in the HindIII site, are indicated (solid arrows). The resulting BIBAC constructs are then transformed into a suitable Agrobacterium strain for subsequent transformation into U. hordei. Any DNA present between the right border (BR) and left border (BL) sequence elements is considered T-DNA and is transferred by Agrobacterium to the host. U. hordei transformants were selected on hygromycin B; a Ustilago-specific hyg B phosphotransferase-cassette present on the T-DNA resulted from recombination of the RECvector with pUSBAC5.  139  Figure 4.2 Verification of conversion of BAC clones to BIBAC vectors. Ethidium bromidestained 1.2% agarose gel showing EcoRI-digested pUSBAC5 (lane 4), pUSBAC5_2-1 (lane 5), seven independent BIBAC_2-1 clones (lanes 6-12), pUSBAC5_1-6 (Lane 13), and two BIBAC_1-6 clones (lanes 14 and 15). The molecular markers are: 5 kb ladder (lane 1), λHindIII fragments (lane 2) and 1kb ladder (lane 3) with sizes indicated in kb on the left.  140  Figure 4.3 PCR analysis of genomic DNA of six independent BIBAC_2-1 and two independent BIBAC_1-6 U. hordei transformants. (A) Ethidium bromide-stained 1% agarose gel showing the amplification of a 1020 bp fragment constituting the hygromycin B phosphotransferase open reading frame, using primers hyg B-F and hyg B-R. (B) Ethidium bromide-stained 1% agarose gel showing PCR amplification products of 302 bp using primers LB-r2 and cat-f2, representing the T-DNA left border end. Lane 1: BIBAC_1-6 vector (positive control), lane 2: BIBAC_2-1 vector (positive control), lane 3: Uh364 untransformed (negative control), lane 4: Uh365 untransformed (negative control). Lanes 5-10: independent Uh364 BIBAC_2-1 transformants (named A, B, C, D, E and F, respectively), lanes 11 and 12: independent Uh364 BIBAC_1-6 transformants (named Y and Z, respectively). M: 1 kb plus molecular weight DNA ladder. Size bars at the left side of the gels are in kbp.  141  Figure 4.4 DNA blot analysis of the genomic DNA of independent U. hordei BIBAC transformants. (A) Genomic DNA was digested with AvaI, which cuts 761 bp proximal to the left border of the T-DNA. The membrane was probed with a 302 bp-fragment located within the left border of the T-DNA and the most-proximal AvaI site (black line in the cartoon above the blot; the location of the genomic inserts, BAC_2-1 or BAC_1-6, is indicated). (B) Genomic DNA was digested with BglII, which cuts 1831 bp proximal to the right border of the T-DNA. The membrane was probed with a 564 bp-fragment located within the right border of the T-DNA and the most-proximal BglII site (black line in the cartoon above the blot). Lane 1: Uh364 untransformed (negative control), lanes 2-7: Uh364 BIBAC_2-1 transformants A, B, C, D, E and F, respectively, lanes 8 and 9: Uh364 BIBAC_1-6 transformants Y and Z. Size bars at the left side of the blots are in kbp.  142  Table 4.1 Recombineering and transformation efficiencies BAC  RE  Inoculation  Cocultivation  U. hordei  U. hordei  U. maydis  (%)  medium 1  medium  TF  TF  TF  (transformants/ 4  2-1  1-6  pUSBAC5  30  25  34  (transformants/ 4  (transformants/  1.2x10 sporidia  1.2x10 protoplasts  1.2x104 sporidia  +AS  -AS  0  0  0.4  +AS  +AS  1  1  32  +AS  -AS  0  0  0.5  +AS  +AS  0.8  0.9  27  +AS  +AS  3  NT  40  Recombination efficiencies of conversion of BAC clones into binary BAC constructs in E. coli (RE), and transformation efficiencies (TF) of U. hordei sporidia, protoplasts and of U. maydis sporidia using AMT. 1 AS, medium with (+) and without (-) acetosyringone added. NT, not tested.  143  CHAPTER 5 Towards the cloning of UhAVR6 5.1. Introduction Avirulence (Avr) genes from different pathogens do not share sequence significantly with each others. Many do not have annotated homologs in public databases, although similar sequences can often be found in genomic sequences of related species. Consequently, identification of Avr genes is a constant challenge (Gan, et al. 2010b, Van't Slot and Knogge 2002). Several Avr genes have been isolated by classical genetics techniques from pathogens with small genomes such as bacteria. Such methodologies included the transformation of a genomic library from an avirulent to a virulent strain, followed by subsequent testing for an HR response on host plants (Collmer 1998, Van den Ackerveken and Bonas 1997). Due to large genome sizes and inefficient transformation methods in fungi and other eukaryotic pathogens, this method cannot be used efficiently (Lauge, et al. 1998). The most common methods for the cloning fungal avirulence genes are reverse genetic techniques and map-based cloning. Several types of molecular markers have been developed for determining DNA sequence variation within and among species. These include amplified fragment length polymorphisms (AFLP), restriction fragment length polymorphisms (RFLP), simple sequence repeats (SSR), randomly amplified polymorphic DNA (RAPD) and single nucleotide polymorphism (SNP) markers. These methods are based on the detection of polymorphisms through the analysis of total genomic DNA among various isolates and or progeny of crosses. Each of these techniques has advantages and limitations, and the choice of fingerprinting technique depends on its application. In this study to clone the U. hordei UhAvr6 gene, I used a PCR-based marker approach. The marker-based approach has been successful in the cloning of several fungal genes, including U. hordei Avr1 (Linning et al 2004). I used 115 SSR primer pairs designed from the U. maydis genome sequence and 55 SSR primers pairs from U. hordei BAC clone end-sequences to find a marker linked to UhAvr6. Additionally, I used sets of AFLP and RAPD primers in an alternate approach. All primers were tested on the genomic DNA of the avirulent parent (Avr6) and virulent parent (avr6), as well as 144  on four pools of combined progeny, two for each of genotype Avr6 and avr6, respectively. I also constructed a new population segregating for this locus from two U. hordei strains collected from geographically distant areas. Several of these primer pairs amplified polymorphic bands, in the parents and pools but unfortunately no linked markers were confirmed after testing on individual progeny.  5.2. Material and methods 5.2.1. DNA manipulation DNA preparations were carried out as described in Chapter 2 section 4.2.4. Bulked pools were made by mixing equal amounts of genomic DNA from four progeny giving a final concentration of 10 ng µl-l.  5.2.2. SSR analysis 115 SSR primer pairs (5 per chromosome) representing microsatellite markers of U. maydis, a closely related corn smut, were obtained from our collaborators (Munkacsi and May University of Minnesota). For the sequences of the primers see (Munkacsi, et al. 2008). 55 SSR primer pairs were designed for U. hordei based on sequences obtained from end-sequences of 2300 BAC clones from the avirulent parent Uh364 (Avr6) and are listed in Table 5.1. Primers were designed to all microsatellite repeat sequences identified and those pairs giving a 100-400 bp PCR product were retained. More than half of these primers were selected for trinucleotide repeats while the rest represent di-, tetra- and penta-nucleotide repeats. PCR reactions were carried out in 25 µl volumes containing 2 mM MgCl2, 100 µM of each of the four dNTPs, 0.5 unit of recombinant Taq polymerase (Invitrogen), 25 ng of genomic DNA as template in 1X PCR reaction buffer (Invitrogen) with 0.4 µΜ οf each forward and reverse primer. The cycling conditions were as follows: an initial denaturation step of 5 min at 95 oC followed by 35 cycles of 30 sec at 95 oC, 30 sec at 55 oC and 1 min at 72 oC, and a final extension at 72 oC for 10 min. Reaction products were run on 2-4% agarose gels (MetaPhor) depending the size of the product, for 6-8 hours at 110-160 W. The running buffer consisted of 1 X TBE (45 mM Tris-Borate, 1mM EDTA, pH 8.0).  145  5.2.3. RAPD analysis The set of RAPD primers was designed and synthesized at the Nucleic Acid and Protein Synthesis unit at UBC Vancouver. For RAPD analysis (Williams, et al. 1990), PCR reactions were carried out as described for SSR analysis (section 5.2.2), but using instead a single primer at a concentration of 0.6 µΜ. The reaction was carried out with an initial denaturation step of 5 min at 95 oC followed by 40 cycles of 12 sec at 95 oC, 60 sec at 36 oC, 60-sec ramp to 72 oC and 1 min at 72 oC, and a final extension at 72 oC for 10 min. The low annealing temperature was used for shorter primers (series 1-800) while a higher annealing temperature of 42 oC was used for longer primers (series 801-890). PCR products were run on 1.5% agrose gels in 1 X TBE.  5.2.4. AFLP analysis AFLP analysis was carried out as described Linning, et al. (2004) and Vos, et al. (1995). In short, genomic DNA was digested by a combination of a “six-cutter” restriction enzyme, BamH1, and a “four-cutter” restriction enzyme, Mse1, followed by ligation with the corresponding adapters for these enzymes. The digested DNA was pre-amplified with three different primer combinations with one specific nucleotide (i) BamPc-MsePt, (ii) BamPc-MsePc and (iii) BamPtMsePc. Twenty different primer combinations were subsequently used for each of these preamplified fragments having two specifying nucleotides at each primer end. The “six cutter” primers were labeled with [γ 33P] dATP (6000 Ci/ mMol, Perkin Elmer, LAS Canada Inc. Wood Bridge, Ontario, Canada) using the standard T4 polynucleotide kinase labeling procedure (Vos et al 1995). 4 µl of AFLP products were mixed 1:1 (v/v) with formamide and dye, heated at 95 oC for 5 min and then separated on a 4.5% polyacrylamide gel [the ratio of acrylamide and bisacrylamide was 20:1 (w/w)], 7.5 M urea, 1X TBE at constant power of 110 W at 50 V/cm. Gels were dried and exposed to Kodak X-OMAT AR X-ray film.  5.3. Results 5.3.1. Construction of populations segregating for UhAvr6 and uhavr6 Two populations were used in this study. One had been previously constructed to analyze the segregation of three avirulence genes (UhAvr1, UhAvr2 and UhAvr6; Linning, et al. 2004). The 146  other one was constructed in this study, because it was revealed during the course of my screening experiments that the existing population was insufficiently genetic diverse with respect to finding polymorphisms as they related to the UhAvr6 and uhavr6 alleles. This new population was generated by crossing two haploid strains: Uh365 (isolated from southern Manitoba, Canada) and Uh813 (isolated from Iran) on universal susceptible barley cultivar Odessa. Prior to the final selection of the parents, I screened six geographically diverse U. hordei strains using several RAPD and SSR primers to select ones that had increased genetic diversity. Teliospores were collected from infected plants; individual basidiospores were isolated and tested in mating tests against haploid basidiospores of known mating type in order to determine their mating type. Fifty-two progeny; 26 for mating type 1 and 26 for mating type 2, were selected for evaluation of their genotypes on barley cultivars Odessa (ruh6) and Plush (Ruh6) by backcrossing with the virulent partner of opposite mating type. Progeny producing infection of more than 10% on Odessa with no disease on Plush were considered to have the UhAvr6 genotype. U. hordei strains having the UhAvr6 allele do not produce disease on Plush, while the virulent strain (uhavr6) can produces up to 100% infection in control experiments (Linning, et al. 2004). Progeny producing more than 10% disease on both Odessa and Plush were categorized as virulent, having the uhavr6 genotype.  5.3.2. Pools for bulked segregant analysis (BSA) To identify markers linked to the UhAvr6 or uhavr6 alleles, a bulked segregant analysis or BSA (Michelmore, et al. 1991) approach was used. Four pools were made, two for each UhAvr6 and uhavr6 and each consisting of four progeny (Table 5.2). Small pools containing four progeny each were made in this study, in the hope that this would reveal weak linkage to UhAvr6 which would indicate a possible location on the available U. hordei genome sequence. Only those progeny revealing a clear virulence or avirulence phenotype were selected for the pools, which is critical for the identification of linkage to the avirulence gene. Pools were made in such a way that the bulked progeny were uniform for mating type as well as the other avirulence genes, if known, such as UhAvr1 and UhAvr2. In this manner, the pools differed only for the desired gene.  147  5.3.3. SSR primer screening 115 SSR primers pairs from U. maydis were used in PCR reactions on two parents and four pools with bulked genomic DNA from UhAvr6 and uhavr6 to screen for differences in amplification profiles. 56 (50%) of these primers produced fragments in U. hordei, showing that the two organisms are at least moderately related at the DNA level. These primers amplified from one to as many as five fragments with an average of three fragments per primer. Some of these primers also produced more than one fragment in U. maydis (Munkacsi, et al. 2008, Munkacsi AB University of Minnesota Thesis, 2005). The PCR fragments amplified by these primers were of different intensities; some were very strong while others were hardly visible in ethidium bromide-stained gels. The weak bands were more polymorphic, but not reproducible in different PCR reactions and could have resulted from mismatches at primer binding sites. Primers not producing products were re-tested to confirm that a lack of amplification was not a PCR reaction artifact. Consistent failure of primers may be attributed to the absence of a complementary binding site or prohibitive length of intervening sequences between primer pairs. Of the 56 successful primer pairs, ten gave a polymorphic band in both the parents and the pools (Table 5.3). In the next step, I screened all the available progeny of these populations for polymorphisms, but was unable to find any linked to UhAvr6 or uhavr6. An example of a polymorphic banding pattern is shown in Figure 5.1. In addition to the U. maydis SSR primers, I also used 55 SSR primer pairs designed from BAC end-sequences of U. hordei for PCR analysis of bulked genomic DNAs to investigate the difference in amplification profiles. All but one of the tested primer pairs (Table 5.3) produced fragments of expected sizes in the PCR reactions. Unlike the SSR primers from U. maydis, these primers produced a single product except for a few pairs which yielded two. Most of the PCR products were of uniform intensity in both the parents and pools. Only three of the primer pairs amplified polymorphic fragments in the parents and pools but none were found linked to UhAvr6 or uhavr6 after screening the individual progeny (Table 5.3, Fig. 5.1).  148  5.3.4. AFLP primer screening The screening of SSR primers from U. maydis and U. hordei was unsuccessful in finding markers linked to UhAvr6 or uhavr6. I decided to attempt the AFLP marker-based approach for the cloning of the UhAvr6 gene in the population segregating for these avirulence genes. To find a marker linked to UhAvr6 or uhavr6, I used 60 different primer combinations after digesting the bulked DNA with different restriction enzymes (see Materials and Methods). All the AFLP primers gave an average of 20 fragments per primer pair tested. Two of the tested pairs revealed a polymorphic fragment between the parents and pools (Fig. 5.1, Table 5.3). These primers were further analyzed on individual progeny for linkage to UhAvr6 or uhavr6 but none was seen (Table 5.3).  5.3.5. RAPD primer screening The UhAvr6 and uhavr6 parents and progeny pools were screened for polymorphisms with more than 200 RAPD primers. 150 of these primers yielded several fragments ranging from 1 to 13 with an average of six fragments per primer. To detect polymorphisms, I looked carefully to find differences in the sizes of fragments and/or the presence/absence of fragments in corresponding genotype pools. These amplified products varied in length from 150 to 3500 bp. The intensities of the bands were also not uniforme; some bands were very bright while others were very faint in the ethidium bromide-stained gels. The faint bands were also not reproducible while the stronger bands were more consistent across different PCR reactions. Fifty primers did not amplify any RAPD fragments during either the initial or confirmatory PCR reactions. I postulate that this could be due to the absence of complementary binding sites for these primers. Alternately, the primer binding sites could have been too widely separated and could not amplify the DNA in between by PCR under the conditions tested. In the initial analysis, several primers produced polymorphic fragments but most of them were faint and were not reproducible under different PCR reactions. Only two of the tested primers, 714 and 719, produced a polymorphic band in the pools, but after testing on individual progeny only 714 gave a polymorphic band that appeared to be weakly linked. The band that was present in the UhAvr6 parent was excised from the gel, cloned and sequenced in order to design sequence-characterized amplified region (SCAR) primers for more robust visualization of the polymorphism; however, analysis with the SCAR  149  primers on individual progeny could not confirm the linkage, so this experimental approach was not pursued further.  5.4. Discussion The existing population segregating for the dominant avirulence gene, UhAvr6 did not yield a linked marker after testing many AFLP, RAPD and SSR markers. It became apparent that this population was not sufficiently genetically diverse as the two parents used to generate the population were back-crossed several times. In this study, a new population of U. hordei was generated which was segregating for the avirulence gene UhAvr6. In this new population, the UhAvr6 and uhavr6 alleles segregated independently from the mating-type locus, MAT, as has been shown in the previous populations (Linning, et al. 2004). Pathogenecity tests of backcrosses on Plush confirmed that the avirulence gene UhAvr6 segregated as a dominant gene in the population. BSA was used in this study to find markers linked to the UhAvr6 gene in both populations segregating for this gene. The main principle behind BSA is the randomization of the genetic background of unlinked loci and the saturation of the region of interest (Michelmore, et al. 1991). The use of two pools for the same trait as used in this study, has been suggested because of the poor reproducibility of RAPD markers in some cases and to ensure that polymorphisms are not artifactual PCR variations (Ellsworth, et al. 1993, Penner, et al. 1993). Small pools of four progeny were made in this study because we hypothesized that by using smaller pool sizes, a weaker linkage could be identified, since a previous study found no markers linked to UhAvr6 in pools of eight progeny (Linning, et al. 2004). However, it is acknowledged that in small pools the frequency of false positives is increased relative to that found for larger pools (Michelmore, et al. 1991). This may be the reason that we identified several polymorphic bands in the pools and progeny, but could not confirm them as linked to UhAvr6 or uhavr6 after testing them on individual progeny. U. hordei SSR primers yielded fewer polymorphic fragments when compared to the U. maydis SSR primers. The reason for this may be that U. hordei SSR primers amplified comparatively fewer loci. These 54 primer pairs amplified a total of 65 loci while the 56 primer pairs from U. maydis amplified a total of 170 loci, almost three times as much. Generally, the  150  AFLP markers appeared to be more polymorphic when compared to the products obtained with the SSR primers. This could be because AFLP markers amplify many loci per reaction and can therefore quickly scan the whole genome, while SSR primers amplify fewer loci, necessitating a more comprehensive commitment to screen a genome. On the other hand, SSRs detect variations within repetitive DNA regions which are highly variable compared to the rest of the genome because of the slippage that can occur during recombination. This is more frequent than other types of mutations such as point mutations or insertion/deletions (Palombi and Damiano 2002). From this study it is clear that the different virulent and avirulent strains of U. hordei are rather similar to one another at the UhAvr6 locus and that a very small variation could be responsible for the avirulence/virulence allelic differences. In several pathogens, it has been shown that single base pair mutations are responsible for changing avirulence to a virulence phenotype (Joosten, et al. 1994, Parlange, et al. 2009, Schurch, et al. 2004, van Esse, et al. 2007, Westerink, et al. 2004). The sequence analysis of several predicted secreted proteins from different field isolates actually confirms this high degree of sequence homology between geographically diverse strains (Chapter 2). The molecular marker-based approaches that were used for the cloning of UhAvr6 genes in this study, may not be very efficient for revealing point mutations. A possible alternative approach may be using molecular biological techniques that are efficient for revealing point mutations. One such technique is the Targeting Induced Local Lesions IN Genomes (TILLING) to reveal point mutations (Comai, et al. 2004, Till, et al. 2003a, Till, et al. 2003b). An alternative approach could be to sequence the genomes of different strains by next generation sequencing technologies which has become relatively cheap, and then search for single nucleotide polymorphisms (SNPs) and link them in the population.  151  A A A A V A v A  M  M  V A  V  A  V  A  V  A V  A  V  A V  A  V  A  V  A  V  A  V  A  V A  M  200  200 100  100  M  V A  V  A  V  A  V  A  V  A  V  A  V  A V  A Ap Vp Ap Vp AP VP 364 362 /M  A 200  100  200  M  V  A  V  A  V  A  M  V  A  V  A  V  A  V  V  A  A  V  V  A  A  V  V  A  A  V  A  V  A  V  A  Vp  Ap  Vp  Ap  364  V  A  V  A  V  A  V  A  V  A  V  362  A  M  M  100  A  A  A  A  V  A  M  200  362  364  Ap  Ap  Vp  Vp  M  100  B  C  600  152  Figure 5.1 PCR amplification with SSR and AFLP primers showing polymorphisms in parents, pools and progeny. (A) Ethidium bromide-stained agarose gel revealing polymorphisms generated by PCR amplification with an SSR primer pair from the U. maydis genome sequence as an example, on pooled progeny having dominant allele UhAvr6 (AP), pooled progeny having recessive allele uhavr6 (Vp), individual progeny having dominant allele UhAvr6 (A), individual progeny having recessive allele uhavr6 (V),, Parents were Uh362 (uhavr6) and Uh364 (UhAvr6). M; Marker (Low DNA mass ladder). (B) Ethidium-bromide-stained agrose gel revealing polymorphisms generated by PCR amplification with an SSR primer pair from the U. hordei BAC end sequences. (C) Section of an autoradiograph showing polymorphic AFLP markers in four BSA pools and parents. Size bars at the side of the blots are in bp.  153  Table 5.1 SSR and AFLP primers for U. hordei used in this study. #  Primer Name  Clone Name  829 830 831 832 833 834 835 836 837 838 839 840 841 842 843 844 845 846 847 848 849 850 851 852 853 854 855 856 857 858  Uh SSR IL Uh SSR IR Uh SSR 2L Uh SSR 2R Uh SSR 3L Uh SSR 3R Uh SSR 4L Uh SSR 4R Uh SSR 5L Uh SSR 5R Uh SSR 6L Uh SSR 6R Uh SSR 7L Uh SSR 7R Uh SSR 8L Uh SSR 8R Uh SSR 9L Uh SSR 9R Uh SSR 10L Uh SSR 10R Uh SSR 11L Uh SSR 11R Uh SSR 12L Uh SSR 12R Uh SSR 13L Uh SSR 13R Uh SSR 14L Uh SSR 14R Uh SSR 15L Uh SSR 15R  H001D03_CR  96 Trinucleotide  H001G04_C7  1 Trinucleotide  H001J18_C7  Contig No  Repeat type  101 Trinucleotide  H001M23_CR  37 Trinucleotide  H002B09_C7  100 Trinucleotide  H002J09_CR H003M04_CR  6 pentanucleotide not known  Trinucleotide  H004F11_C7  3 Dinucleotide  H004F24_CR  62 Dinucleotide  H004G03_CR  5 tetranucleotide  H004L07_C7  65 Dinucleotide  H006M20_C7  1 Trinucleotide  H006N02_CR  9 pentanucleotide  H006P13_C7  10 Trinucleotide  H001A10_CR  57 Trinucleotide  Primer sequence TCCTTTCAGAGCTTGCTAAC ACAGTACCACAGGTATTCGG ACTAGCATTCGCAATCTCAT AGAAGAACGTGGCTATTGAG AGCTTTCAGAGCAGAGACAG AGTATCCTCAACTGACAGCG TTTCCTATCGTCAACATCCT ATCTTGCTTGAACAATGGAC GATTGGAGCAGTAGACAAGC TGTCTTTGCCTCAACTACCT TTGGTAGTTCGGAGTAAGGA TCACGTGCTGTACCTAGTTG AGCTTTCAGAGCAGAGACAG AGTATCCTCAACTGACAGCG GAATGAGGTCAAGAGTCAGC CTCTTGGTGTCTTCTTGGAG cGACTGTGGTTGTGTATCTG TCGTTGTTAGGTGGAGAGAT CTTGGCAAGGCTAATACCTA ACTACCtttgGATTGCAAGA GGTCACTCGAGTAAGTCTGC GACTGTCCTCGTCAACTTGT GCTGCTAGTCTTCCACACTT ATATGGTTCCATCGTTTCAG GTTGAACGACCTCTCGTAAG CCATCTTTGTCAGTCTGGAT GTAACCAACTTTGTCGGAAG AAAGAATCATATCTCGCCAA ACATTTGGAGTCTGATGAGG GATGTCGGAAGAGTTGTAGC  Product Size (bp) 224 241 228 389 349 264 228 137 214 373 244 353 336 246 238  154  #  Primer Name  Clone Name  Contig No  Repeat type  859 860 861 862 863 864 865 866 867 868 869 870 871 872 873 874 875 876 877 878 879 880 881 882 883 884 885 886 887 888 889  Uh SSR 16L Uh SSR 16R Uh SSR 17L Uh SSR 17R Uh SSR 18L Uh SSR 18R Uh SSR 19L Uh SSR 19R Uh SSR 20L Uh SSR 20R Uh SSR 21L Uh SSR 21R Uh SSR 22L Uh SSR 22R Uh SSR 23L Uh SSR 23R Uh SSR 24L Uh SSR 24R Uh SSR 25L Uh SSR 25R Uh SSR 26L Uh SSR 26R Uh SSR 27L Uh SSR 27R Uh SSR 28L Uh SSR 28R Uh SSR 29L Uh SSR 29R Uh SSR 30L Uh SSR 30R Uh SSR 31L  H001A13_CR  44 tetranucleotide  H001A14_C7  20 Dinucleotide  H001A21_C7  2 Trinucleotide  H001B06_CR  23 Trinucleotide  H001C15_CR  95 Trinucleotide  H001D12_C7  3 Dinucleotide  H001F03_CR  266 pentanucleotide  H001G10_CR  5 pentanucleotide  H001I05_CR  4 Dinucleotide  H001L06_CR  101 Dinucleotide  H001M01_C7  76 Trinucleotide  H002C04_C7  8 Trinucleotide  H002H17_CR  47 tetranucleotide  H002K02_C7  100 Trinucleotide  H002K10_C7  12 Dinucleotide  H002N01_CR  4 Trinucleotide  Primer sequence GTTAGACCTGCAATTGCTTT CTTTGATCTGATGGGTGTCT GAGCGTAAGCAAGACCTAAA GAAGCAAGGAGTTGAAGATG ATTGCTCCAGTAGGTCcc CGTTCAGCTGTAATACCTCC TCACATCAACACTGACATCC AGACAGCTTTCTCTAGGGCT AGAGCATCGTCAGATAGCAT CAACGATGACAGTACAGAGC GTTCAAATTCGAGTCTCTGC AAGAACGGCAAAGTCATAAA AAGATCAATGTACCACAGCC CCAATCAATATGTATGTgcg TTACTGAGAGGCTCATTCGT CAGGACCTTTCTGTATCTGC TGGATAAGGATCCACTTGAC CCCTGTACAGTaCTCGATGAA CTGCAGCACAGTAGTCGTAA GAAGCGATACTTCTTGGCTA GCTAGAATTTAGGTTGGTGC CGAGCATGGTCTATTAGCTC GACCTCTGTGGACACTCTGT ACATTCACCAGCCTTATCAC CTCGAAGAGGATGTAGTTGG AAGGATTCAGGAAAGGAGAC AGGTTATGATGACACCAAGG AAGCATCAAGTCAACACACA TGAAACTCTCCTCTTCCAAA CAGAGGACAACAAAGAGGAG GAATGGAGTGGTGTTGCTAT  Product Size (bp) 304 304 253 125 398 133 111 134 129 125 108 135 119 113 106 121  155  #  Primer Name  890 891 892 893 894 895 896 897 898 899 900 901 902 903 904 905 906 907 908 909 910 911 912 913 914 915 916 917 918 919 920  Uh SSR 31R Uh SSR 32L Uh SSR 32R Uh SSR 33L Uh SSR 33R Uh SSR 34L Uh SSR 34R Uh SSR 35L Uh SSR 35R Uh SSR 36L Uh SSR 36R Uh SSR 37L Uh SSR 37R Uh SSR 38L Uh SSR 38R Uh SSR 39L Uh SSR 39R Uh SSR 40L Uh SSR 40R Uh SSR 41L Uh SSR 41R Uh SSR 42L Uh SSR 42R Uh SSR 43L Uh SSR 43R Uh SSR 44L Uh SSR 44R Uh SSR 45L Uh SSR 45R Uh SSR 46L Uh SSR 46R  Clone Name  Contig No  Repeat type  H002N17_C7  55 tetranucleotide  H002O08_CR  86 pentanucleotide  H003H10_C7  115 Trinucleotide  H004A16_CR  20 pentanucleotide  H004A22_CR  9 Trinucleotide  H004C11_CR  125 Trinucleotide  H004D14_C7  57 Dinucleotide  H004H18_C7  12 Dinucleotide  H004I22_C7  10 Trinucleotide  H004K03_C7  44 Trinucleotide  H005A12_C7  2 Trinucleotide  H005A14_C7  86 Trinucleotide  H005B12_CR  76 tetranucleotide  H005C16_C7  55 Trinucleotide  H005D07_C7  96 pentanucleotide  Primer sequence CTGTGTAATTGATATTGACATTGA AGACGATGCTTTAGGGAt GCGTCAACGAGAACGCAT TCACTGCTGTTGTTGTCATT GCACAccTACAAAGAGAAGG GTTGGTGGGACTAGTTGAGA GAACGAGGATGATGAACTGT TATGGATAAGGAGCAAAGGA CTTGCTAACCTatCAGACGC TCACCATGTTTGGTTCTT CTTACCTCCAGTCGTACCAA TCACATCAACACTGACATCC AGACAGCTTTCTCTAGGGCT ACTTTACACGACCACGACTT ACGTGATTACCATTCTCGAC TGAAACTCTCCTCTTCCAAA CAGAGGACAACAAAGAGGAG TGAACGAAAGAGTGTGAGTG GTCTTTCCTCCTCTCTCCAT AACCTCAATCTCAACCACAG AGTAACGGCTGCTGATATTT ATTGTGTTGCAGTGGTGTTA TGACTGATGACAAGACGGTA ATGTTGCCCAGATACAGAAG TTTCCTTCTCCTCAACTGAA AAGGATTCAGGAAAGGAGAC CTCGAAGAGGATGTAGTTGG ACTTTGCCTTTATCACTGGA AAGGCACAACAACAGCTC CTATTGATGAAGAAGCCCAG TCACTGAGATGTGAGGTTGA  Product Size (bp) 104 144 142 130 151 125 135 106 118 100 189 199 119 137 146  156  #  Primer Name  Clone Name  Contig No  Repeat type  921 922 923 575 576 577 578 579 580 581 582 583 584 575 829 830 831 832 833 834 835 836 837 838 839  Uh SSR 47L Uh SSR 47R Uh SSR 48L Uh SSR 48R Uh SSR 49L Uh SSR 49R Uh SSR 50L Uh SSR 50R Uh SSR 51L Uh SSR 51R Uh SSR 52L Uh SSR 52R Uh SSR 53L Uh SSR 53R Uh SSR 54L Uh SSR 54R Uh SSR 55L Uh SSR 55R MsePt BamPc BamPt BamPca BamPcc BamPta Adapter MseI MseAI Adapter MseI MseA2 Adapter BamHI/Bgl II BamAI Adapter BamHI/Bgl II BamA2 MsePt  H005I08_CR  47 Trinucleotide  H005I09_C7  55 tetranucleotide  H005K09_CR  86 Trinucleotide  H006K19_CR  6 Dinucleotide  H006L11_C7  65 Trinucleotide  H001E02_CR  43 Trinucleotide  H002P11_C7  43 Dinucleotide  H002H22_CR  118 Trinucleotide  H003H10_C7  115 Dinucleotide  Primer sequence ATGGTCGTCACGAGAATAAC TCGTCGTAGAGACCAATACC GATGCTTTAGGGATGCag GCGTCAACGAGAACGCAT TCACTGCTGTTGTTGTCATT GCACACCTACAAAGAGAAGG CTCAAGGACGAAGTAACCAG TTCGGATCACGTAACCTAAC CCAAAGGAACTGTCACTGAT CTGTGcctcTTGAgctGT CATTCTGCGTATTGTTGATG CATGTCTCCTTCTCTCTTCG AAGTGTTGTCCCGATAACTG ATGGTCATTAAGTGGAATGC AGGAGAAAGAGCATGATGAA CAGAAATGACTTTGCATTGA GACAGTTAGTGTGTCAGCGA same primer like UhSSR34 R GATGAGTCCTGAGTAAt GGACTGCGTACGATCCc GGACTGCGTACGATCCt GGACTGCGTACGATCCca GGACTGCGTACGATCCcc GGACTGCGTACGATCCta GACGATGAGTCCTGAG TACTCAGGACTCAT CTCGTGGACTGCGTAC  Product Size (bp) 193 100 144 133 112 182 180 205 313  GATCGTACGCAGTCCAC GATGAGTCCTGAGTAAt  #Primer number refer to Bakkeren Lab primer inventory 157  Table 5.2 Composition of pools used for bulked segregant analysis. Pools from new population Pools or parent  Strain number with mating type I  Strain number with the mating type II  V6-1  852 (V6), 863 (V6)  848 (V6), 855 (V6)  V6-2  895 (V6), 912 (V6)  873 (V6), 898 (V6)  v6-3  878 (v6), 902 (v6)  861 (v6), 891 (v6)  v6-4  917 (v6), 924 (v6)  893 (v6), 897 (v6)  Parent  365 (V6)  Parent  813 (v6)  Pools from old population V6-1  420 (V6), 398 (V6)  392 (V6), 407 (V6)  V6-2  411(V6), 427(V6)  385 (V6), 382 (V6)  v6-1  391 (v6), 408 (v6)  386 (v6), 380 (v6)  v6-2  381 (v6), 409 (v6)  414 (v6), 405 (v6)  Parent  364 (V6)  Parent 1  362 (v6)  Genotype: V6 = UhAvr6 dominant allele, v6 = uhavr6 recessive allele  Table 5.3 Results of different marker analyses on the pools and progeny. Approach  No. of primer pairs tested  Primers that work in U. hordei 56 (50 %)  Polymorphic in pools and parents 10  Linked to UhAvr6 or uhavr6 0  SSRs (U. maydis) SSRs (U. hordei) AFLP  115 55  55  3  0  60  60  2  0  RAPD  > 200  150  10  0  158  CHAPTER 6 General discussion and future perspectives 6.1. General discussion The main theme of my dissertation research was to study the avirulence gene UhAvr1 and other effectors from U. hordei. The goal of my research was to extend our knowledge of effectortriggered immunity in plants to include an interaction between a basidiomycete pathogen and a monocot host, and to further our knowledge of fungal effectors in disease establishment and defense induction. To this end, research was conducted towards five objectives (Chapter 1). The first objective was the identification and characterization of the Ustilago hordei avirulence gene 1 within the UhAvr1 locus, and possibly UhAvr6, to improve our knowledge of effector-triggered immunity in plants. I have identified Uh10022 as the UhAvr1 gene (Chapter 2) which was previously mapped to an 85 kb genetic segment through a genetic marker-based approach (Linning, et al. 2004). UhAVR1 is a relatively small, predicted secreted protein with no homolog with known function in the public databases. In this regard, UhAvr1 is similar to most of the identified fungal avirulence proteins, which are also usually small proteins, possesses an N-terminal secretion signal peptide and lack homology to sequences available in public databases (Dean, et al. 2005, Fudal, et al. 2007, Gout, et al. 2006, Rep, et al. 2005). In the analysis of this gene, I showed by both deletion of the C-terminal end of UhAVR1 and complementation approach, that this gene is responsible for inducing resistance in the barley cultivar Hannchen having resistance gene Ruh1. The expression of the UhAvr1 gene was not detected in any conditions tested in this study. The reason for this result may be that the expression of this gene is highly regulated and might be expressed at a very low level only during the early stage of infection. But obtaining enough fungal material from an early infected plant is a challenge. The deletion of this gene from an avirulent strain did not affect the disease on a barley cultivar not having the cognate Ruh1 gene this suggest that this gene might be dispensable for infection or that some other genes had similar function. Another explanation is that this gene might cause only subtle variations in virulence that could only be detected in a population, or that the gene might have additive effects 159  on virulence. The virulence was measured by the number of diseased plants out of a total of inoculated plants; this is not a sensitive assay. I did not succeed in the isolation of the UhAvr6 gene using DNA marker-based approaches (Chapter 5). However, this study made it clear that the different virulent and avirulent strains of U. hordei that were used in this study for finding markers linked to the UhAvr6 gene, were similar to each other at the DNA level at this locus. It is possible that small variations could be responsible for the avirulence/virulence allelic differences. A single base pair mutation, as shown for several other Avr genes from different pathogens, might be responsible for changing the avirulence to virulence phenotype at this locus (Joosten, et al. 1994, Parlange, et al. 2009, Schurch, et al. 2004, van Esse, et al. 2007, Westerink, et al. 2004). The molecular marker-based approaches used in this study were not very efficient for revealing point mutation. The second objective dealt with how U. hordei overcomes Ruh1-triggered resistance in barley. To overcome ETI due to the recognition of avirulence proteins by plant R proteins, natural selection pushes the pathogens to escape recognition by the host (Dawkins and Krebs 1979). ETI can be overcome by several mechanisms to avoid recognition at the gene level by point mutations, frameshifts, recombination, deletions, gene duplications, gene disruption by transposable element (TEs) and acquiring novel genes. Deletion of effectors is feasible if their functions are dispensable or when genes with redundant functions are available in the genome. The loss of indispensable genes can also be countered by acquiring novel genes with a redundant function through horizontal gene transfer. Insertions of TEs into a promoter element or into the ORF of the gene can change gene expression, or the expressed protein itself, respectively (Daboussi and Capy 2003, Ganko, et al. 2003, Hua-Van, et al. 2002, Kang, et al. 2001). The influence of repetitive elements can result in deletions, inversions, duplications and translocations based on the relative orientation and location of the repeat with respect to the target gene (Daboussi and Capy 2003, Hua-Van, et al. 2000, Khang, et al. 2008, Kim, et al. 1998, Nitta, et al. 1997). I have shown in Chapter 2 that the UhAvr1 gene is embedded in transposons and repeats and that the activity of TE is responsible for the inactivation of the gene. In contrast to gene deletion, the mutation in the avirulence gene caused by TE activity resulted in a nonfunctional allele, that could be beneficial for U. hordei as it is preserved in the genome and can be available for reversion once the selection pressure from the host is over (Stergiopoulos, et al. 2007). Several bacterial, fungal and oomycete avirulence genes have been shown to be  160  inactivated by TE activity (Orbach, et al. 2000, Rep, et al. 2004, Houterman, et al. 2008, Kim, et al. 1998, Rehmany, et al. 2003, Bohnert, et al. 2004, Farman, et al. 2002, Farman and Leong 1998, Kang, et al. 2001, Luderer, et al. 2002b). After entry to the host cell or apoplast, the interaction between the AVR and R proteins can be either direct or indirect. It has been shown that effector proteins that bind directly to R proteins contain two distinct effectors and avirulence domains enabling them to change their binding domains without changing their effector function (Wang, et al. 2007). Such proteins are subject to diversifying selection which generates highly divergent alleles by gene duplication followed by point mutation as shown for the AvrL567 locus in Melmpsora lini (Dodds, et al. 2006, Ellis, et al. 2007a, Wang, et al. 2007), ATR1NdWsB and ATR13 in Hayaloperonospora arabidopsidis strains (Deslandes, et al. 2003, Jia, et al. 2000, Rehmany, et al. 2005) and Avr1-b1 in Phytophthora sojae (Dou, et al. 2008a). In contrast, indirect recognition of effectors by R proteins results in selection on AVR effector functions and therefore purifying selection (Bent and Mackey 2007). Under this regime, these effectors will favour mutations that render these proteins either non-functional or non-expressed or removal from the organism (Armstrong, et al. 2005, Rooney, et al. 2005). As revealed by the monomorphic nature of UhAVR1 among several field isolates and its inactivation by TE activity in virulent isolates (Chapter 2), I speculate that the interaction between UhAVR1 and RUh1 is indirect. However, this needs to be verified experimentally. The isolation of the Ruh1 gene from barley could shed light on whether the interaction is indirect or direct. The third objective was focused on obtaining an understanding of the evolutionary pressures acting on the UhAvr1 locus. The region containing this locus is syntenic to U. maydis cluster 19, harbouring many predicted effectors (Kamper, et al. 2006) and a similar region in Sporisorium reilianum (Schirawski, et al. 2010). The synteny is highly conserved between the three organisms over the genes flanking the cluster on each side. Most of the predicted secreted proteins in the U. maydis cluster are represented by at least one homolog in U. hordei. However, in contrast to U. hordei, the gene families are much more conserved between the two corn pathogens. It can be assumed that this cluster is generally important for all three organisms. U. hordei contains several unique genes at this locus, and several genes are transcriptionally inverted compared to the two corn pathogens. In this regard, the region is much more diverged in U. hordei compared to the other two organisms possibly because the host is different; U. maydis  161  and S. reilianum infect corn while U. hordei infects barley. The region is enlarged in S. reilianum and U. hordei in comparison to U. maydis; both U. hordei and S. reilianum have more genes in this region and the intergenic region is enlarged compared to U. maydis (Schirawski, et al. 2010). In U. hordei the region is saturated with repeats and transposable elements which may be involved in the re-arrangement of this region and breaking of avirulence on barley cultivars containing the Ruh1 gene. The co-localization of repeat and TE elements with Avr genes is a common feature in several pathogens and appears to supply a common mechanism used by pathogens to overcome host ETI (Orbach, et al. 2000, Rep, et al. 2004, Houterman, et al. 2008, Kang, et al. 2001, Kim, et al. 1998, Luderer, et al. 2002b, Rehmany, et al. 2003). Given the overall conservation in three organisms, it is likely that these genes in a cluster-like setting were obtained from a common ancestor before speciation but that each pathogen evolved independently in response to their specific biotropic life styles and hosts. In U. maydis and S. reilianum, there are several alleles of the same secreted proteins at this locus which indicate that diversifying selection might have acted upon this locus as a result of direct interaction between the effector and corresponding resistance gene. In these species, diversification might have acted on these genes to overcome host recognition. This is a bit surprising as no effector-R gene interaction has been shown to exist for U. maydis and S. reilianum with corn. I speculate from the gene duplications found at this locus in the two corn-infecting smut that there may have been effector-R gene interactions sometime in the past. It will be interesting to test a series of U. maydis and S. reilianum isolates on corn and other related wild species of corn and grasses to find out if some of these effectors are avirulence factors. The fourth objective was geared towards gaining insight to the potential repertoire of small secreted proteins (effectors) of U. hordei likely involved in virulence and avirulence towards the barley host. My hypothesis was that secreted proteins that have functions in host cells, i.e effectors, would be direct targets of evolution. In biotrophic pathogens such as U. hordei, these effectors likely reprogram the host cells for their own benefit. My approach was to mine the genome of U. hordei to obtain its secretome and to compare it with other publicly available genomes of basidiomycetes and other fungal pathogens (chapter 3). The CSEPs (candidate secreted effector proteins) that were identified in U. hordei in this study, were either hypothetical or did not have a known function. More than 50% of the CSEPs have four or more cysteine residues and several have a cysteine-content and spacing  162  characteristic for secreted apoplastic avirulence proteins. The percentage of CSEPs that have orthologs in related smut was very low as compared to non-secreted proteins this suggest that these proteins evolved at a faster pace compared to the rest of the proteome. In oomycete pathogens such Phytophthora sojae, P. ramorum and P. infestans, only one quarter of the effectors have orthologs in other species (Tyler, et al. 2006). The secreted effectors of Blumeria graminis f. sp. hordei also have very low numbers of orthologs in two other powdery mildew fungi infecting dicotyledonous plants (Spanu, et al. 2010). The high rate of sequence diversification, gene duplication and genome rearrangement in the effector-encoding genes may be the result of the ongoing molecular arms race between the pathogen and the host. This may also be responsible for the lack of orthologs in other pathogens. Effector-encoding genes are also usually located in highly flexible regions of the genome such as telomeres or embedded in transposable elements and thus can easily mutate and overcome the R-mediated resistance. In U. maydis, 17 clusters of predicted secreted protein-coding genes were identified which were distributed randomly over the genome and comprised 26% of the complement of predicted secreted proteins (Kamper, et al. 2006). Genes in a variety of these clusters were induced during infection and deletion of eight of them affected pathogenicity on corn seedlings (Kamper, et al. 2006, Schirawski, et al. 2010). The induction of some cluster genes was also tissue specific and the effects of deleted clusters were variable depending on the host tissue infected (Skibbe, et al. 2010). In a comparative genomic study between U. maydis and S. reilianum, 43 clusters in which genes diverged between these species, were identified and 61% of the genes encoded predicted secreted proteins (Schirawski, et al. 2010). Analyzing the predicted secreted proteins of U. hordei, 62 clusters were identified (Chapter 3). The majority of the genes in these clusters encoded hypothetical, conserved hypothetical or Ustilaginaceaespecific conserved hypothetical proteins and some were very similar to gene clusters in U. maydis and S. reilianum (Kamper, et al. 2006, Schirawski, et al. 2010). In most other completed fungal genomes, no cluster arrangements of CSEPs were identified (Dean, et al. 2005, Spanu, et al. 2010). Gene clusters in other fungi are usually involved in sexual reproduction, or biosynthesis or degradation of secondary metabolites (Gardiner, et al. 2004, Kupfahl, et al. 2006). In several ascomycetes such as Fusarium solani, F. oxysporum, Alternaria alternata and Cochliobolus spp., genes with putative virulence functions are co-located on the same  163  chromosome, sometimes they interspersed with repeats, transposable elements, or other genes (van der Does and Rep 2007). It is not understood how genes involved in virulence or avirulence evolved in clusters in fungal pathogens. Gene clusters in fungi with unknown functions are extremely unusual. Clustering may be necessary for co-regulation of the genes, especially if several virulence genes are required for infection of a particular plant host (van der Does and Rep 2007). It might be true for at least some of the clusters in U. maydis as genes in these clusters co-induced in the host (Kamper, et al. 2006, Schirawski, et al. 2010). The alternative hypothesis, suggested by van der Does and Rep (2007) is that virulence genes may appear at random positions in the genome and then cluster together as a result of random gene shuffling due to a strong selective advantage. For example, gene duplications that have been observed in several clusters in U. maydis, U. hordei and S. reilianum might be the result of direct interactions with R genes of the host as a mechanism to avoid host recognition. In objective five, I developed an efficient Agrobacterium-mediated transformation (AMT) system for U. hordei for transferring large fragments of genomic DNA (chapter 4). Prior to this work, the transformation was carried out by electroporation of partial protoplasts, a procedure which is poorly reproducible and inefficient. This AMT method will facilitate greater understanding of the pathogenicity in U. hordei by allowing gene complementation especially through the transfer of large DNA fragments containing genes with regulatory sequences necessary for gene function, or complete gene clusters, to assess the location of specific functions/genes on such genome-size fragments as represented by Bacterial Artificial Chromosome (BAC) inserts. As shown, U. hordei harbours several gene clusters coding for predicted secreted proteins that could be effectors acting during host interactions, similar to those clusters described in U. maydis (Kämper et al. 2006, Chapter 3). In conclusion the work described in this thesis chronicles the isolation of the UhAvr1 gene and the characterization of the locus through comparison to the syntenic locus in U. maydis. New tools for functional genetic analysis have also been developed. Computational mining of the sequenced genome has yielded an inventory of a complement of potential secreted effector proteins. However, my work has generated new, exciting questions that should be addressed in future work. Below, I suggest some follow-up experiments that could make significant contributions to the field.  164  6.2. Future perspectives 6.2.1. Localization of UhAVR1 Fluorescent protein tagging is a powerful tool in molecular and cell biology used to monitor subcellular activities such as protein-protein interaction, protein localization, gene expression, protein movement, cell division and vesicle and organelle trafficking in living cells (Leffel, et al. 1997, Takemoto, et al. 2003). Chimeric constructs of the green fluorescent protein (GFP) or mCherry and the UhAVR1 protein will be helpful in investigating its localization during the U. hordei infection process (Khang, et al. 2010, Kale, et al. 2010, Kemen, et al. 2005). Effector proteins from fungal pathogens act either in the apoplast or after cell entry in the cytoplasm or nucleus (Bryan, et al. 2000, Dodds, et al. 2004, Dodds, et al. 2006, Jia, et al. 2000, Orbach, et al. 2000, Staskawicz, et al. 2001). This can be done by fusing UhAvr1 to fluorescent protein sequences at either the N-terminal or C-terminal extension and then transferring the constructs into the Uh10022 deletion strain (Uh364-∆10022). These transformant strains should then first be tested on barley cultivar Hannchen for their ability to complement the mutation and show that the chimer still retained its avirulence function. They can then be inoculated on resistant (Hannchen) and susceptible (Odessa) barley cultivars and monitored throughout the infection cycle of U. hordei at the tissue and cellular levels by using fluorescent and confocal microscopy. This will enable visualization of the expression and location of the UhAVR1 protein during both compatible and incompatible interactions. In addition to fluorescent protein tagging, immunochemistry can be used to obtain a more precise localization of UhAVR1. Immunochemistry utilizes antibodies to visualize antigens in sections of tissue using light or electron microscopy (Walker, et al. 2001). For visualization, specific antibodies must be produced and incubated with pre-prepared embedded sections of cells and tissues. This immunochemistry analysis will also be helpful in investigating whether the UhAvr1 gene is constitutively expressed or expressed under specific conditions. This analysis should also allow to determine the extent to which UhAVR1 is secreted into plant tissue during infection. If these experiments prove that UhAVR1 is indeed secreted into the host plant, it will then be highly interesting to unravel the mechanism by which host barriers are crossed.  165  6.2.2. Novel host protein interactions with UhAVR1 The interaction between UhAVR1 and the host target(s) will provide more insight into the infection process of barley by U. hordei. The yeast two-hybrid assay system could be used to find fungal and barley proteins interacting with UhAVR1. It is an easy, comparatively quick and flexible technique used for protein-protein interaction studies. This technique is based on the transcriptional activation of GAL4, a modular protein that requires the interaction of two domains with different specific functions (a DNA-binding domain, BD and an activation domain, AD). The UhAvr1 gene can be fused with the Gal4 BD and transfected into a receptor yeast strain harbouring the upstream activating sequences (UAS) and reporter gene which results in a hybrid bait protein. A barley cDNA library in hunter specific yeast two hybrid vectors with GAL4 AD is available from our collaborator Dr. R. Hückelhoven (J-L. University, Giessen, Germany) and will be transfected into the yeast strain containing the UhAvr1-GAL4 construct. Colonies expressing the reporter gene will be those where UhAVR1 interacts physically with the cognate protein coded by cDNA clones from barley. It might be possible to find the corresponding Ruh1 gene in barley if its product interacts with UhAVR1 directly. If the interaction is not direct, the UhAVR1 trap will identify other host targets. The Ruh1 gene has been genetically identified to a 300 kb region on the short arm of chromosome 1. A more efficient approach might be to identify cDNA clones located in that region such as R-gene like genes, and apply them to the UhAVR1 trap assay. Many other avirulence proteins that are secreted into the host and which interact directly with R proteins of the host or other host factors have been identified using this system. It is possible that no interaction is found between UhAVR1 and barley proteins coded by the cDNA library. A complementary approach would be to transfer a cDNA library from U. hordei into a yeast strain transformed with the UhAvr1-baitconstruct. In this way, interactions between UhAVR1 and possibly other proteins of U. hordei could be found. Positive cDNA clones of barley and U. hordei can be sequenced to identify the genes. Further molecular characterization of the interacting proteins can be validated in planta by bimolecular fluorescence complementation studies.  166  6.2.3. Function of UhAVR1 I have shown that UhAVR1 did not exhibit virulence functions towards barley cultivars Odessa and Hannchen by inoculating them with U. hordei strains where both mating partners carried the deletion for C19A2 (chapter 2). However, this experiment should be repeated based on our later finding that in the virulent strain Uh362, this region is translocated to another part of the genome. Truely isogenic strains should be created by crossing the generated UhAvr1 3/-deletion mutant in the Uh364 (MAT-1) background with the avirulent parental strain Uh365 (MAT-2 Avr1) and selecting MAT-2 progeny on carboxin; which should have then the UhAvr1 3/-deletion as well. Work in Bakkeren laboratory is also ongoing to find the location of the translocated fragment in the virulent parent. More work is required to establish the molecular function of UhAVR1. Future efforts should focus on gaining insight into the intrinsic function of UhAVR1 in pathogenicity. This can be done in several ways. One approach would be to inoculate barley cultivars Odessa (ruh1) and Hannchen (Ruh1) with both the UhAvr1 deletion mutants (Uh364 ∆UhAvr1) and U. hordei wild type (Uh364 UhAvr1) strains followed by inoculation with a secondary barley pathogen such as Blumeria graminis f. sp hordei (Bgh). B. graminis f. sp. hordei is a barley pathogen that penetrates epidermal cells and whose subsequent infection process can be easily monitored. If UhAVR1 suppresses the host defense response, the secondary pathogen would produce increased severity of symptoms on Odessa when pre-inoculated with the wild type strain as opposed to the deletion mutant strain. On the other hand, on Hannchen, UhAVR1 should elicit a host defense and as a result the B. graminis f. sp. hordei should produce decreased symptoms on barley that is pre-inoculated with the wild type strains compared to mutant strains. An alternative to this approach would be the co-bombardment or agro-infiltration of resistant and susceptible barley coleoptiles with a fluorescent marker such as GFP as a reporter gene plus UhAVR1, followed by assays and testing for reduced reporter gene expression as a result of cell death or HR (Dong, et al. 2009, Rehmany, et al. 2005). Another approach would be to clone UhAvr1 and a fluorescent protein-coding gene such as luciferase in different cereal-expressing plasmids and transform barley protoplasts with the two plasmid mixed in equal ratio and monitor cell viability. This approach has been used successfully to identify virulence functions for three avirulence genes from Magnaporthe oryzae by transferring them into rice protoplasts (Yoshida, et al. 2009). Finally, additional work is ongoing in the Bakkeren laboratory, using a cereal-adapted  167  Pseudomonas species for type III secretion-mediated translocation of the UhAVR1 protein into barley leaves and monitoring for suppression or induction of the defense response in susceptible and resistant barley cultivars, respectively.  6.2.4. Cloning of other U. hordei virulence and avirulence genes For the cloning of UhAvr6 and other avirulence genes (UhAvr2 and UhAvrx) which segregate in the two populations in Bakkeren laboratory, a more targeted approach should be used. The majority of known avirulence factors from fungal and oomycete pathogens are secreted into the host (Ellis, et al. 2007b, Kamoun 2007). To identify genes showing polymorphisms correlated with avirulence functions, primers should be designed 1 kb upstream and downstream of all genes coding for the predicted secreted effector proteins in U. hordei (Chapter 3). PCR amplification will reveal presence/absence and/or difference in size of the amplified fragments. The use of these primers to screen the parents and bulked segregant pools could clearly indicate any marker linked to these avirulence genes. Any linked marker could be further verified on individual progeny of the mapping population. The same PCR products could be used for Targeting Induced Local Lesions IN Genomes (TILLING) studies to look for point mutations (Comai, et al. 2004, Rakshit, et al. 2007). An alternative would be the deletion of all the clusters of predicted secreted protein-coding genes that have been identified in this study (Chapter 3). These deletion mutants could be subsequently inoculated on the set of differential barley cultivars available. UhAvr1 is located in a cluster that has the least-conserved genes between U. hordei and U. maydis. All clusters representing such diverged genes should be targeted first. Another strategy would be to employ an association mapping approach (Yoshida, et al. 2009). This would entail sequencing of the genomes of different strains, especially from the other parents of the populations, by next-generation sequencing technologies. Variation in predicted secreted effector proteins/genes, such as organization, deletions and single nucleotide polymorphisms (SNPs), can be revealed and correlated to virulence phenotypes (Yoshida, et al. 2009). To reveal candidate secreted effector proteins (CSEPs, Chapter 3) with virulence or avirulence functions, it should first be confirmed whether these candidates are secreted. This can be done using the Yeast Secretion Trap system (Lee, et al. 2006). To assess whether effectors are induced during biotrophic growth, which has been shown to occur for most of effectors, U. 168  hordei microarrays or RNA sequencing could be employed. Functional analysis of these effectors could then be undertaken by transient expression in different barley cultivars using coinfiltration of effector genes together with marker genes such as GUS or GFP as discussed, above (Rehmany, et al. 2005). Virulence functions of these CSEPs, such as the suppression of host defense responses in susceptible plants or the elicitation of the host defense in resistant plants, can be carried out using secondary pathogens as described above. For protein localization, fluorescent protein tagging or immuno-chemistry could be used, as previously described.  6.3. Conclusion The identification of UhAvr1 has broadened our knowledge of how pathogens overcome resistance in host plants. In particular, UhAvr1 is located in a repeat-and transposon-rich genomic environment prone to variation and under selection pressure. This region is syntenic to a similar region in closely-related basidiomycete smut fungi. Much more work is required to fully understand the intrinsic virulence function of this effector in host infections. To my knowledge, this is the first Avr gene isolated from a basidiomycete pathogen infecting cereal crops and as such can serve as a stepping stone to further our knowledge of plant-microbe interactions. The isolation of the corresponding R gene from barley using the cloned UhAVR1 effector will provide a potential new source for crop resistance. In my dissertation, I also exhaustively identified other candidate effector genes in this fungus which can be targeted in future studies. The virulence or avirulence functions of these effectors can be investigated by several approaches as suggested in the future perspectives section. This could lead to the isolation of other corresponding R genes, allowing for R gene pyramiding and increasing general crop resistance. 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