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Charaterization of RNA silencing and avirulence in two related smut fungi Laurie, John Drummond 2008

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CHARATERIZATION OF RNA SILENCING AND AVIRULENCE IN TWO RELATED SMUT FUNGI  by John Drummond Laurie  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) December 2008  ©John Drummond Laurie, 2008  ABSTRACT The basidiomycete cereal pathogens Ustilago hordei and U. maydis are closely related and possess genomes with a high degree of homology and synteny. I report on the disparity of the RNAi phenomenon between U. hordei and U. maydis. Using an RNAi expression vector I targeted both a GUS transgene and an endogenous mating-type gene and confirmed the presence of double-stranded (ds)RNA in transgenic cells of both species. However, downregulation of the GUS gene and production of siRNAs were seen only in U. hordei. The biological effect was a reduction in GUS protein and activity, and reduced mating only in U. hordei. In support of this experimental evidence, homologs to Dicer and Argonaute were found in the U. hordei genome but not in the published U. maydis genome. Interestingly, preliminary U. hordei sequences reveal conservation and synteny in U. maydis in the regions spanning these loci, with the only noticeable difference being the lack of Dicer and Argonaute genes in U. maydis. U. maydis also appears to differ from U. hordei with respect to genes presumed to be involved in transcriptional gene silencing and also has far fewer transposons in its genome. Efforts to clone the avirulent gene UhAvr1 led to a locus containing a large number of small proteins predicted to be secreted. This locus appears to be heterochromatic and is orthologous to the largest cluster of secreted proteins in U. maydis. Other laboratories have reported that deletion of this cluster in U. maydis results in a dramatic reduction in virulence. Genetic evidence for an avirulence gene at this locus in U. hordei suggests that the locus may also be important for U. hordei. Differences between these two smut fungi at this locus and at others identified in this study point to key differences in gene regulation and genome evolution.  ii  TABLE OF CONTENTS Abstract............................................................................................................................... ii Table of Contents............................................................................................................... iii List of Tables .......................................................................................................................v List of Figures.....................................................................................................................vi List of Abbreviations ........................................................................................................ vii Acknowledgements.............................................................................................................ix Co-Authorship Statement ....................................................................................................x 1. Introduction..................................................................................................................1 1.1. General plant-pathogen interactions......................................................................1 1.2. Plant-microbe gene-for-gene interactions .............................................................2 1.3. Acquired resistance in plants.................................................................................5 1.4. Introduction to Smut fungi ....................................................................................6 1.4.1. Ustilago maydis as a model for the Smut fungi...........................................8 1.4.2. Ustilago hordei as a model for studies into avirulence .............................10 1.5. RNA silencing .....................................................................................................11 1.6. RNA silencing and plant defence ........................................................................12 1.7. RNA silencing defends the fungal genome .........................................................14 1.8. Proposed research................................................................................................14 1.9. Research objectives .............................................................................................16 1.10 References............................................................................................................20 2. Hallmarks of RNA silencing are found in the smut fungus Ustilago hordei but not in its close relative Ustilago maydis..................................................32 2.1. Introduction ..........................................................................................................32 2.2. Materials and methods..........................................................................................34 2.2.1. Vector construction....................................................................................34 2.2.2. Strains and genetic transformation ............................................................35 2.2.3. GUS fluorometic MUG assay....................................................................36 2.2.4. RNA extraction and detection ...................................................................37 2.2.5. Quantitative-RT-PCR ................................................................................38 2.2.6. Protein extraction and detection ................................................................39 2.3. Results ..................................................................................................................39 2.3.1. Ustilago RNAi vector construction and expression...................................39 2.3.2. RNAi of an endogenous mating-type gene................................................41 2.4. Discussion.............................................................................................................42 2.5. References ............................................................................................................52 3. Identification of RNA silencing genes in Ustilago hordei provides clues to the uniqueness of Ustilago maydis ........................................................................60 3.1. Introduction ..........................................................................................................60 3.2. Materials and methods..........................................................................................67 3.2.1. Amino acid and DNA sequences...............................................................67 3.3. Results ..................................................................................................................68 3.3.1. Phylogenetic relationship...........................................................................68 3.3.2. Ustilago hordei genome sequencing .........................................................68 iii  3.3.3. Blast search for RNAi components in the U. hordei genome ...................68 3.3.4. Synteny between the U. hordei RNAi component loci and the U. maydis genome.........................................................................70 3.3.5. Search for genes lost from U. maydis........................................................70 3.3.6. Intron comparison......................................................................................72 3.4. Discussion.............................................................................................................73 3.5. References ............................................................................................................94 4. The Ustilago hordei UhAvr1 locus is syntenic to cluster 19A of Ustilago maydis and appears to be heterochromatic ...........................................................102 4.1. Introduction ........................................................................................................102 4.2. Materials and methods........................................................................................106 4.2.1. Plant varieties and fungal strains used for mapping UhAvr1 ..................106 4.2.2. Chlorazol Black E staining of U. hordei hyphae.....................................107 4.2.3. Comparison of U. hordei sequences from the UhAvr1 locus to the U. maydis genome .........................................................................107 4.2.4. BAC 3-A2 sequencing and analysis ........................................................107 4.2.5. Virulent parent BAC library screening....................................................108 4.2.6. Gene ID and comparisons........................................................................109 4.2.7. Knockout of gene UH_ 08134 (#8), ortholog of Um05312.....................109 4.2.8. Pathogenicity tests ...................................................................................110 4.3. Results ................................................................................................................111 4.4. Discussion...........................................................................................................116 4.5. References ..........................................................................................................134 5. General discussion ...................................................................................................142 5.1. RNA silencing experiments................................................................................142 5.2. Characterization of the UhAvr1 locus ................................................................144 5.3. Future experiments .............................................................................................147 5.3.1. Recombination.........................................................................................147 5.3.2. Lack of RNAi: convergent transcripts, histones......................................149 5.3.3. U. hordei virulence and avirulence genes................................................149 5.3.4. U. maydis as a model for the smuts .........................................................150 5.4. Concluding remarks............................................................................................152 5.5. References ..........................................................................................................153  iv  LIST OF TABLES Table 2.1 Primers for vector construction and qPCR ........................................................51 Table 3.1 Location of RNA silencing genes in U. hordei and their top Blast hits ............86 Table 3.2 Number of RNAi genes in selected fungal species ...........................................87 Table 3.3 Syntenic loci horboring RNA silencing and chromodomain genes ..................88 Table 3.4 Comparison of predicted proteins of U. hordei to U. maydis............................90 Table 3.5 Genes found in U. hordei but not in U. maydis.................................................91 Table 3.6 Comparison of intron number............................................................................93 Table 4.1 Predicted genes at the UhAvr1 locus ...............................................................130 Table 4.2 Primers used to sequence genes for putative secreted proteins .......................131 Table 4.3 Primers for deletion, expression and RNAi cloning........................................132 Table 4.4 Pathology test results for the null mutant of candidate UhAvr1 gene, UH_08134 (gene #8) .......................................................................................133  v  LIST OF FIGURES Figure 1.1 Potential outcomes of plant-microbe interactions............................................17 Figure 1.2 Symptoms of Covered Smut of barley and Corn Smut....................................18 Figure 1.3 Life cycle of Ustilago hordei, Covered Smut of barley...................................19 Figure 2.1 Construction of RNAi vectors..........................................................................47 Figure 2.2 Expression of dsRNA and production of siRNA .............................................48 Figure 2.3 RNA interference by dsRNA ...........................................................................49 Figure 2.4 RNA interference with the endogenous bW gene transcripts disrupts mating in U. hordei but not in U. maydis ..............................................................50 Figure 3.1 Phylogenetic relationship of selected fungal species based on β-tubulin protein sequences ...................................................................................79 Figure 3.2 Predicted U. hordei Dicer protein ....................................................................80 Figure 3.3 Predicted U. hordei Argonaute protein ............................................................81 Figure 3.4 Predicted U. hordei RdRP proteins ..................................................................82 Figure 3.5 Predicted U. hordei Chromodomain proteins ..................................................84 Figure 3.6 Comparison of orthologous histone proteins showing differences between U. hordei and U. maydis..........................................................................85 Figure 4.1 The UhAvr1 locus...........................................................................................121 Figure 4.2 Using the GPS-Mutagenesis System to sequence BAC 3-A2........................122 Figure 4.3 Chlorazol Black E staining of three week old barley seedlings.....................123 Figure 4.4 Characterization of Gene X (UH_13922) ......................................................124 Figure 4.5 BAC 3-A2 sequencing results ........................................................................125 Figure 4.6 Comparison of duplicated genes at the UhAvr1 locus to homologous genes in U. maydis...............................................................................................126 Figure 4.7 Search for BAC clones from the virulent parent library corresponding to the UhAvr1 locus .............................................................................................127 Figure 4.8 Comparison of parental alleles for secreted proteins at the UhAvr1 locus to cluster 19A in U. maydis........................................................................128 Figure 4.9 Candidate UhAvr1 (UH_08134, gene #8) knock-out and analysis ................129  vi  LIST OF ABBREVIATIONS a AFLP AGO AN AT Avr BAC bE bW CA CC CenH3 Chp Clr4 CN DCL / DCR dsRNA DSRM ET GUS H3K9 H3K9me HDAC HR ISR JA KO LB MAMP MAT MG MGb miRNA mRNA MSUD MUG NC nt PAMP PAZ PC PCR piRNA Piwi PR PTGS R  Mating-type locus that encodes pheromones and pheromone receptors Amplified fragment length polymorphism Argonaute Aspergillus nidulans Arabidopsis thaliana Avirulence gene Bacterial artificial chromosome Mating-type locus that encodes homeodomain protein subunit Mating-type locus that encodes homeodomain protein subunit Candida albicans Coprinus cinereus Centromeric histone H3 variant Chromodomain proteins Chromodomain methyl transferase Cryptococcus neoformans Dicer Double-stranded RNA dsRNA-binding domain Ethylene Bacterial uidA gene (beta-glucuronidase) Histone H3 lysine 9 Methylation on histone H3 lysine 9 Histone deacetylase Hypersensitive response Induced Systemic Resistance Jasmonic acid Gene knock-out Laccaria bicolor Microbe-associated molecular pattern Mating type Magnaporthe oryzae (grisea) Malassezia globosa Micro RNA Messenger RNA Meiotic silencing of unpaired DNA 4-methylumbelliferyl B-D-Glucuronide Neurospora crassa Nucleotide Pathogen-associated molecular pattern Domain found in Argonaute and Piwi related proteins Phanerochaete chrysosporium Polymerase chain reaction Piwi-interacting RNAs P-element induced wimpy testis in Drosophila Pathogenesis-related gene Post-transcriptional gene silencing Resistance gene vii  RdRP/Rdp RFLP RISC RITS RNAi ROS RT-PCR Ruh1 SA SAR SC SET siRNA SP Swi6 tasiRNA TGS UH UhAvr1 UM  RNA-dependent RNA polymerase Restriction fragment length polymorphism RNA-Induced Silencing Complex RNA-induced transcriptional silencing complex RNA interference Reactive oxygen species PCR from reverse transcribed RNA Barley resistance gene 1 that recognizes UhAvr1 Salicylic acid Systemic Acquired Resistance Saccharomyces cerevisiae Domain found in lysine methyltransferase enzymes Small interfering RNA Schizosaccharomyces pombe Chromodomain protein Transacting siRNA Transcriptional gene silencing Ustilago hordei Ustilago hordei avirulence gene 1 recognized by Ruh1 Ustilago maydis  viii  ACKNOWLEDGEMENTS I would like to express profound gratitude to my advisor, Dr. Guus Bakkeren, for his invaluable support, encouragement, and supervision throughout my doctoral studies. His guidance and willingness to allow me to explore new avenues enabled me to find success in my work. I am also thankful to my committee members for valuable suggestions and guidance throughout this study. I am grateful for the cooperation and assistance of students and staff at Agriculture and Agri-Food Canada in Summerland. Their kindness and friendship helped my studies progress smoothly and made my time in the Okanagan memorable and enjoyable. I also wish to thank collaborators at the Max Planck Institute for Terrestrial Microbiology in Marburg for their contributions to the Ustilago hordei genome project and for their willingness to share information. I am especially indebted to my parents for their patience and support and to my brothers and sisters for their eagerness to contribute to my studies. Special thanks go to Lorraine Bennest for editorial suggestions and for employment when times were tough. Most of all, I am particularly indebted to my wife and best friend Ekida, whose love and support enabled me to endure the ups and downs of graduate studies. I am forever grateful for her sacrifices and encouragement.  ix  CO-AUTHORSHIP STATEMENT Below is a list of papers that have been published or are in preparation for publication. The contributions made by the candidate are mentioned. Chapter 2: Laurie, J., Linning, R. and Bakkeren, G. 2008. Hallmarks of RNA silencing are found in the smut fungus Ustilago hordei but not in its close relative Ustilago maydis. Current Genetics 53: 49-58. The candidate conceived and designed experiments, as well as wrote the manuscript. Linning, R. and Bakkeren, G. provided editorial support. Chapter 3: Laurie, J., Linning, R., Bakkeren, G., Schirawski, J, Kahmann, R. et al. A version of the chapter entitled “Identification of RNA silencing genes in Ustilago hordei provides clues to the uniqueness of Ustilago maydis” is in preparation for publication. The candidate conceived and designed experiments, as well as wrote the manuscript. Bakkeren, G. provided editorial support. Linning, R. provided bioinformatics support. Chapter 4: Laurie, J., Ali, S., Linning, R. and Bakkeren, G. A version of the chapter entitled “The Ustilago hordei UhAvr1 locus is syntenic to cluster 19A of Ustilago maydis and appears to be heterochromatic” is in preparation for publication with the alternative title “A cluster of predicted secreted proteins from the basidiomycete barley smut fungus, Ustilago hordei, contains avirulence gene Uhv1”. The candidate performed the experiments following genetic mapping and wrote the manuscript. Bakkeren, G. provided editorial support. Linning, R. provided bioinformatics support.  x  CHAPTER 1:  Introduction  1.1 General plant-pathogen interactions It is generally accepted that plants are either hosts or non-hosts to particular pathogenic microorganisms, or in other words, either susceptible or resistant. Non-host resistance may result from a plant’s innate immunity or by an induced response that prevents a microbe from causing disease on any member of that plant species (Hammond-Kosack and Parker 2003, Nurnberger and Brunner 2002, Thordal-Christensen 2003). These innate defenses include structural (cuticle and cell wall), enzymatic and chemical (tannins and phenolics) barriers that provide immunity to a broad range of potential pathogens (Heath 2002, Wittstock and Gershenzon 2002). They provide immunity by either directly arresting microbe advance or by revealing microbe presence so that more specific localized defenses can be mobilized. By using mutagenesis in the laboratory, these preformed barriers can be weakened resulting in mutant plants lines becoming hosts to microbes that previously could not infect that species (Papadopoulou, et al. 1999). In nature, plants have a surveillance system evolved to recognize various molecules essential to the function of potential pathogens (Shiu and Bleecker 2001). This type of defense recognizes such molecules as flagellin, a structural component of the tail from certain bacteria and chitin from fungal cell walls, and collectively such molecules from diverse microorganisms are referred to as pathogen or microbe-associated molecular pattern (PAMPs or MAMPs) (Nurnberger and Brunner 2002, Zipfel and Felix 2005). Interestingly, the nature of PAMP recognition is highly conserved and similarities exist between the plant and animal kingdoms. It is likely that, in addition to other innate defense mechanisms, recognition of PAMPs contributes significantly to non-host resistance (Hayashi, et al. 2001). 1  For a microbe to become a pathogen, it must be able to overcome a plant’s defenses. During this process the potential pathogen needs to secrete molecules to assist penetration of mechanical barriers, to suppress plant defenses, and to acquire nutrients for survival. These socalled “effectors” are therefore virulence factors. Consequently, the microbe provides a variety of molecules that can potentially be recognized by the plant and be used to mount a very restrictive defense response. A subset of such molecules are recognized by specific plant resistance (R) genes (Martin, et al. 2003) and have been termed avirulence factors, the products of so-called avirulence (Avr) genes, since they render the pathogen isolate avirulent on that particular plant cultivar (Van't Slot and Knogge 2002). It is thought that these genes are maintained in the pathogen population because they offer some benefit to the pathogen when interacting with hosts lacking the matching R-genes (White, et al. 2000). When a race of a pathogen containing a certain Avr-gene is recognized by a host cultivar possessing the corresponding R-gene, defenses are activated and the plant is resistant to that race of the pathogen (Hammond-Kosack and Parker 2003, Nurnberger and Scheel 2001, Rathjen and Moffett 2003). This type of recognition and defense is known as the “gene-forgene” interaction (Flor 1971). When both the Avr and R genes are present, an interaction is said to be “incompatible” since the pathogen is arrested and unable to cause disease. When either of the matching genes is absent, however, the interaction is said to be “compatible” and disease is the outcome (Fig. 1.1).  1.2 Plant-microbe gene-for-gene interactions Gene-for-gene resistance is often characterized by rapid production of reactive oxygen species (ROS) (Bolwell, et al. 2002) and a rapid, localized cell death initiated at the site of the infection called the hypersensitive response (HR) (Beers and McDowell 2001, Flor 1971, Heath 2000). As well, localized increases in salicylic acid (SA) (Delaney, et al. 1994), phytoalexins, 2  cell wall components and the induction of pathogenesis-related (PR) genes occur (Muthukrishnan, et al. 2001). Interestingly, susceptible plants react to pathogen advance with many of the same responses as resistant plants. They produce ROS and express many of the same defense genes, but unlike resistant plants these responses are delayed or subsequently suppressed in susceptible plants, allowing the invading pathogen to spread within the plant. Currently, it is thought that many of the same responses occur in resistant and susceptible plants, but that it is the timing or quantitative nature of the response that results in an appropriate defense enabling resistance (Tao, et al. 2003). Gene-for-gene resistance is achieved through highly specific recognition of pathogen effector proteins by specialized plant resistance genes. Plant R-proteins were initially thought to be receptors that interact directly with pathogen Avr-proteins acting as ligands (de Wit 1997). However, as more and more Avr-proteins and matching R-proteins were identified, R-proteins often appeared to indirectly recognize their matching Avr-protein. One model suggests that an R-protein may “guard” another protein or complex of proteins that is somehow affected by the matching Avr-protein (Van der Biezen and Jones 1998). Support for this “guard” hypothesis has been demonstrated in the RPS2/AvrRpt2 and RPS5/AvrPphB combinations between Arabidopsis and Pseudomonas syringae (Swiderski and Innes 2001). A more recent interpretation of data from many diverse systems has pointed out that interactions between Avr proteins and proteins guarded by R-proteins has no impact on virulence during compatible interactions (Van der Hoorn and Kamoun 2008). This observation has lead to the formation of the decoy model, where guarded proteins have the sole function of detecting pathogen effector molecules. Other recent studies involving the oomycete, Phytophthora infestans, and the flax rust, Melampsora lini, have provided more examples for direct Avr/R-protein interactions (Catanzariti, et al. 2007, Dodds, et al. 2006). Both direct and indirect interactions, therefore, appear to be universal strategies used by plants to detect microbe invaders. 3  The large number of diverse Avr-genes characterized to date, necessitate a correspondingly comprehensive complement of host defense genes. Using bioinformatics it was estimated that nearly 1% of Arabidopsis genes resemble R-genes (Meyers, et al. 1999). Utilization of a “guard” gene strategy would extend the limited potential for defense afforded by R-genes. Therefore, it makes sense that other proteins are involved in the recognition of diverse pathogen effectors. Since R-genes represent the last line of defence, guarding essential cellular machinery or processes targeted by microbial effectors is an effective strategy whereby plant R-genes could potentially detect diverse pathogens. Since direct interaction with effectors would result in diversifying selection of both R-genes and Avr-genes (Dodds, et al. 2006), an indirect surveillance strategy would complement the use of direct detection methods. So, in addition to the R-genes being acted upon by diversifying selection, other R-genes working through the guard model would be under balancing selection (Hoorn, et al. 2002). Having Rgenes of both forms would provide an evolutionary advantage to plants. Most R-proteins have been placed into five classes based on recognizable structural motifs (Martin, et al. 2003). Classes 1, 2 and 3 lack transmembrane domains and therefore likely reside within the cytoplasm. This implies that the corresponding pathogen effector proteins must enter the plant cell or cause an effect that is recognized within the cell. Class 1 is characterized by a serine/threonine kinase domain and an N-terminal myristylation motif, while classes 2 and 3 have leucine rich repeats (LRR), nucleotide binding sites (NBS) and either Nterminal coiled coils (CC) (class 2), or N-terminal Toll and Interleukin1 receptor-like (TIR) regions (class 3). Classes 4 and 5 proteins have an extracellular LRR, a transmembrane domain and either a cytoplasmic region of unknown function (class 4) or a cytoplasmic serine/threonine kinase region (class 5).  4  1.3 Acquired resistance in plants One interesting result of an initial plant/pathogen incompatible interaction is the induction of systemic defenses arming a plant for subsequent attacks. Investigations into the nature of this response have identified SA, ethylene (ET) and jasmonic acid (JA) as signaling intermediates (Dong 1998, Pieterse and van Loon 1999). From these, two alternative pathways for resistance have been identified (Hammond-Kosack and Parker 2003). One pathway depends on SA and protects plants from biotrophic pathogens through the use of the HR (Glazebrook 2005). The other depends on JA and ethylene and protects plants from necrotrophs and herbivorous insects where an HR response would be advantageous for the pest. Biotrophic pathogens that induce SA signalling, during incompatible interactions, have been shown to activate Systemic Acquired Resistance (SAR) while JA and ET induction by incompatible necrotrophs activates Induced Systemic Resistance (ISR) (Hammond-Kosack and Parker 2003). Although these pathways act independently, they can also be additive in their effects (Clarke, et al. 2000, van Wees, et al. 2000). Additionally, careful crosstalk must occur between these pathways as a plant attempts to recognize the kind of pathogen and mount the appropriate response. Systemic acquired resistance (SAR) is a vital plant response that primes a plant for subsequent pathogen invasion (Dong 2001, Ryals, et al. 1996). During an incompatible plantpathogen interaction a localized HR is accompanied by an increase in SA, both locally and later systemically. The nascent effect of SA was mimicked by exogenous application of SA which resulted in the induction of SAR and the expression of several PR genes in the absence of pathogens (Ye, et al. 1989). Further support for the role of SA in SAR came from transgenic Arabidopsis plants expressing a bacterial salicylate hydroxylase gene (nahG) known to degrade SA (Gaffney, et al. 1993). These plants were unable to accumulate SA and similarly failed to develop SAR. The strongest support, however, came through grafting experiments where SA 5  was demonstrated to be necessary but not the mobile signal for SAR (Vernooij, et al. 1994). To dissect the pathway leading from SA accumulation to acquisition of SAR, genetic screening identified a single gene responsible for SA induction of SAR (Dong 2001). This gene has been named NPR1 and exists as a homo-oligomer in the cytosol (Mou, et al. 2003). Deletion mutants of NPR1 fail to develop SAR and are hypersensitive to pathogen attack. Interestingly, however, over-expression of NPR1 does not lead to constitutive SAR, but instead results in a stronger SAR response when plants are challenged by pathogens, suggesting that NPR1 is somehow activated by SA accumulation (Fan, et al. 2002). In support of this, changes in the cell redox state upon pathogen challenge, results in cleavage of disulfide bridges releasing the monomeric form of NPR1 (Mou, et al. 2003). Monomeric NPR1 then enters the nucleus where it interacts with transcription factors inducing expression of a variety of genes including numerous PR genes.  1.4 Introduction to Smut fungi Smuts are obligate biotrophic plant pathogenic fungi responsible for significant agricultural losses worldwide. They belong to the order Ustilaginales of the basidiomycetes and comprise an estimated 1200 species, causing disease in over 4000 plant species worldwide (Alexopoulos, et al. 1996). The genus Ustilago infects members of the Gramineae, which include cereal crops and forage grasses, but individual species have a narrow host range (Agrios 1997). Typically, infected plants appear normal until flowering at which point the fungus produces dark spores from which the smuts took their name (Fig. 1.2). For the barley smut Ustilago hordei, infection takes place at the embryo or seedling stage where the fungus penetrates a young plant and resides near the shoot apical meristem (Hu, et al. 2002). Infective mycelium is dikaryotic, that is, the fungus maintains two haploid nuclei per cell (a common phenomenon among basidiomycetes). Infected plants show no signs of disease until flowering 6  when the fungus undergoes massive sporulation and fills floral tissues with smut sori. For many smut species, nuclear fusion precedes teliospore maturation. Diploid teliospores are shed from the sori of the diseased heads and attach to uninfected seeds. The teliospores persist under the seed hull until conditions are suitable for germination at which point they germinate together with the seed. Meiosis follows, yielding four haploid basidiospores that segregate 1:1 for the mating types MAT-1 and MAT-2 (Bakkeren and Kronstad 1994). These cells are yeastlike, multiply by budding and are amenable to molecular genetic techniques. When opposite mating types are present they recognize each other via a pheromone/receptor system (Bakkeren and Kronstad 1996). Following recognition, conjugation tubes carry out fusion between the spores resulting in a dikaryotic infection hypha, which penetrates the germinated seed thereby completing the cycle (Fig. 1.3). Diseased flowers are common in many smuts, but variation exists in their mode of infection. Ustilago maydis, for example, can infect corn throughout vegetative growth as well as its floral tissue, and has the added ability to sporulate independent of flowering (Alexopoulos, et al. 1996). As a whole, the smuts are important pathogens for study due to their interesting lifecycles and their impact on our precious cereal crops. Much of the general knowledge about smuts has been gained through earlier studies that classified the different smuts by virtue of the hosts they infect. In these studies, lifecycles were described and resistant hosts and virulent strains were identified. More recently, use of molecular biology and the model smut U. maydis has increased our knowledge about smut biology (Bolker 2001, Martinez-Espinoza, et al. 2002). A complete genome sequence together with powerful experimental tools has placed U. maydis at the forefront of many fields, including fungal mating, cell signalling, DNA recombination, and plant-fungal interactions (Kamper, et al. 2006). As new sequencing technologies bring down the cost and speed of DNA sequencing, however, comparative studies of related smuts is becoming possible. Such studies  7  will allow insights into the evolution of host specificity, evolution of virulence factors and overall genome organization.  1.4.1 Ustilago maydis as a model for the Smut fungi U. maydis has emerged as a model organism for the study of a variety of processes relating to eukaryotes in general and to more specialized fields (Basse and Steinberg 2004, Kahmann and Kamper 2004, Martinez-Espinoza, et al. 2002). An important contribution from earlier studies was the work of Robin Holliday, and others, on DNA recombination and repair. This work led to formation of the Holliday model to describe cross-over events during DNA recombination which is still taught in classrooms around the world. Since the model was initially proposed, refinements have been made and many important genes have been identified (Holloman, et al. 2008). An interesting feature of U. maydis, and perhaps all smuts, is that mating is required for pathogenicity. Consequently, extensive work has led to detailed descriptions of the events surrounding the switch from haploid cell budding on the plant surface to the dikaryotic filaments or hyphae responsible for infection and disease symptoms. In U. maydis, mating is governed by a tetrapolar mating system consisting of genetically unlinked a and b mating-type loci. Mating is initiated by the biallelic a mating-type locus that encodes pheromones and pheromone receptors (Banuett 1995) and involves fusion of conjugation tubes emanating from compatible haploid basidiospores. The multiallelic b mating-type locus encodes two homeodomain protein subunits (bE and bW) that form a heterodimeric transcription factor required for activation of genes responsible for dikaryotic filament formation post-conjugation. As many as 23 b specificities have been reported from natural populations (Silva 1972, Zambino, et al. 1997). With the realization that b genes form transcription factors, much work has been done to identify the DNA motif recognized by the transcription factors, as well as the genes that are controlled by these factors (Brachmann, et al. 8  2001). As well, mutational analysis identified a protein, Rum1, similar to a human retinoblastoma binding protein, that is responsible for normal silencing of b-dependent genes and suppression of filament formation (Quadbeck-Seeger, et al. 2000). Analysis of signalling events during pheromone response and transition to filamentous growth has identified MAPkinase and cAMP dependent pathways (Garcia-Pedrajas, et al. 2008, Gold, et al. 1994). Work on the cell cycle has demonstrated arrest in G2 upon mating, marking the end of budding (Garcia-Muse, et al. 2003, Perez-Martin, et al. 2006). Details of the infection process and completion of the U. maydis lifecycle on maize plants have also been studied. At the microscopic level, infection has been monitored from initial penetration, through tumour formation to differentiation of teliospores. Studies have looked at differential gene expression between normal-healthy and infected maize tissues showing initial recognition and triggering of defence responses (Basse 2005). As disease progresses, however, defense responses are attenuated and cell death is suppressed (Doehlemann, et al. 2008). Another study demonstrated prevention of C4 photosynthesis and maintenance of sink metabolism in infected leaf tissues (Horst, et al. 2008). With completion of the U. maydis genome sequence assembly and annotation, details are starting to fill in on previous studies and new studies have been initiated (Kamper, et al. 2006). One topic of considerable interest is the characterization of U. maydis secreted proteins (Mueller, et al. 2008). From the genome, 426 proteins were predicted to be secreted, with 18.6 % of these residing within 12 clusters. Microarray expression analysis showed that many of these genes are up-regulated during plant infection (Kamper, et al. 2006). Additionally, four of the clusters resulted in either reduced or complete loss of pathogenicity when deleted. This suggests that these genes encode important virulence factors. Interestingly, one of the clusters resulted in increased virulence when deleted, suggesting a role for proteins in the cluster to  9  control fungal aggressiveness. Current efforts are aimed at identifying the function of secreted protein in U. maydis (Mueller, et al. 2008).  1.4.2 Ustilago hordei as a model for studies into avirulence The barley/U. hordei interaction is an excellent candidate to be developed as a model system for the study of cereals and their fungal pathogens. Since isolation of the first R-gene was reported in 1992 (Johal and Briggs 1992), exhaustive research is continuing to add new Rgenes to the inventory. From these, however, only Mla, mlo and Rpg1 have been cloned in barley (Brueggeman, et al. 2002, Buschges, et al. 1997, Halterman, et al. 2001, Zhou, et al. 2001). Additionally, only a limited number of Avr-genes have been cloned from fungal pathogens infecting monocots, but none of these have come from basidiomycetes (Van't Slot and Knogge 2002). This lack of information on interactions between monocots and their pathogens supports the need for more study. To fill this void, important model systems need to be developed, as in the dicots. A major advantage for using the barley/U. hordei system is the existence of gene-for-gene resistance. Previous studies have mapped not only Avr genes, but also virulence factors (Caten, et al. 1984, Pope and Wehrhahn 1991, Thomas and Huang 1985). Additionally, the cloned barley R-genes will also enable comparative studies. Another advantage is that U. hordei is closely related to U. maydis, and is amenable to the same laboratory tools. It, however, has a number of distinguishing features that sets it apart from U. maydis. In contrast to U. maydis, U. hordei has a bipolar mating system where the a and b loci are genetically linked (Bakkeren, et al. 2006). Such a system would favour inbreeding since fewer partners would be available after meiosis. Additionally, sporulation is also associated with flowering like for most other smuts. Recently, the U. hordei genome was sequenced providing further support for the use of this smut as a model for small grain cereal smut diseases. 10  1.5 RNA silencing RNA silencing or RNA interference (RNAi) can affect gene expression at both the transcriptional and post-transcriptional levels and possibly evolved as a mechanism for controlling invasive nucleic acids (Grewal and Elgin 2007, Mello and Conte 2004, Plasterk 2002). Some biological functions of the RNA silencing machinery include degradation of RNA from invading viruses and bacteria as well as degradation of RNA emanating from mobile genetic elements. RNA silencing therefore plays an important role in maintaining genome stability. Many organisms have also evolved to use the RNA silencing machinery to control gene expression by encoding double-stranded (ds) RNA from endogenous loci. This form of control is often used for key steps in development and for response to stress (Axtell, et al. 2006, Bartel 2004). RNA silencing is an evolutionary conserved mechanism for gene regulation and genome defence. Initial studies leading to its discovery involved transgenic plants engineered to express multiple copies of homologous sequences (Matzke, et al. 1989, Napoli, et al. 1990, van der Krol, et al. 1990). In all cases, gene expression was essentially silenced. Subsequent work lead to the discovery of dsRNA as the instigator of RNA silencing (Fire, et al. 1998, Kennerdell and Carthew 1998, Ngo, et al. 1998, Waterhouse, et al. 1998). Shortly thereafter, small RNA was found to be associated with the silencing phenomenon (Hamilton and Baulcombe 1999). In the last decade, significant progress has been made at identifying the different RNA silencing pathways and the genes involved. From this work, it was determined that dsRNA emanating from foreign nucleic acids or from endogenous sources is processed by a dicer enzyme into small interfering (si) RNA or micro (mi) RNA. Dicers are RNase III endonucleases whose primary role is to seek out dsRNA and to chop it up into small ca. 20 nucleotide (nt) fragments (Bernstein, et al. 2001). These small RNAs are then loaded into Ago proteins of the Argonaute-Piwi family that associate with other proteins to form a multiprotein 11  complex termed RISC (RNA-Induced Silencing Complex) (Liu, et al. 2004, Rand, et al. 2004). The mRNA population is then scanned by RISC for regions complementary to siRNA, resulting in mRNA cleavage or translational repression depending upon the organism and pathway. Ago proteins are the catalytic engines of both the RISC involved in posttranscriptional gene silencing (PTGS) where mRNA is degraded, and the RNA-induced transcriptional silencing complex (RITS) where methylation of DNA and associated histones accomplish transcriptional gene silencing (TGS). In many organisms, TGS is achieved using RNA silencing through siRNA loading of Ago proteins that target homologous DNA for methylation or direct histone H3 methylation. In Arabidopsis, the DCL3 Dicer cleaves dsRNA into 24 nt siRNA that are loaded into Ago4. Ago4 then directs methyl transferases to methylate DNA homologous to the 24 nt siRNA. In Schizosaccharomyces pombe, Dcr1 (Dicer), Ago1 and Rdp1 (RNA-dependent RNA polymerase) are required for TGS within centromeric DNA repeats (Volpe, et al. 2002). In animals, X-chromosome inactivation may require Dicer and the RNAi machinery to accomplish silencing by directing histone H3 lysine 27 trimethylation (H3-3meK27) (Ogawa, et al. 2008). Taken together, these examples illustrate the use of the RNAi machinery to both target and stop the transcription of repetitive DNA in addition to homology-dependent degradation of repetitive DNA transcripts.  1.6 RNA silencing and plant defence RNA silencing has relevance to plant-microbe interactions and is studied by numerous laboratories working on very diverse systems. Work done on plant-viral interactions has made significant contributions to this field. In particular, the discovery of viral-encoded silencing suppressors has helped dissect RNA silencing pathways. This was possible since viral suppressors were identified that block various different steps in RNA silencing pathways, 12  thereby counteracting the defense response of the host plant. In addition to early work on viruses, recent findings now support a role for RNA silencing in combating bacterial infection. One of the first studies showed that siRNAs are found corresponding to transferred T-DNA during Agrobacterium infection of plants (Dunoyer, et al. 2006). Successful infection depended on the anti-silencing state of plant tumours where siRNA production, but not miRNA, was essentially abolished. In addition to targeting expression of DNA from Agrobacterium, endogenous small RNA defence pathways are now coming to light that play more general roles in plant defence. Recognition of a bacterial PAMP activates the miRNA, miR393, in Arabidopsis contributing to resistance to P. syringae (Navarro, et al. 2006). Auxin receptors are targeted by miR393 allowing a plant to control the auxin response during infection. A recent genetic screen looked for secreted bacterial effectors that interfere with the miRNA pathway (Navarro, et al. 2008). This study implicated miRNAs as key components of plant basal defence and identified several effectors that interfere with various stages of miRNA pathways. Further support for RNA silencing in plant defence responses comes from a mutant of the Arabidopsis Ago4 (Agorio and Vera 2007). Ago4 is involved in small RNA-directed DNA methylation (Zilberman, et al. 2003). A mutation in this gene resulted in enhanced disease susceptibility to virulent, avirulent and non-host pathogens suggesting that epigenetic changes directed by Ago4 are important for proper plant resistance responses. An interesting finding is that upon pathogen challenge, plants produce numerous small RNA molecules, some of which are homologous to R-gene loci. In addition to these events, somatic recombination is enhanced at R-gene loci during pathogen challenge. Examples from viruses and bacteria are helping to dissect the intricacies of defence pathways involving small RNAs. Although such studies using fungi are currently lacking, fungal effectors likely target many of the same defence pathways.  13  1.7 RNA silencing defends the fungal genome Due to their ease of study, fungi have played important roles in defining RNA silencing pathways. Shortly after co-suppression was discovered in plants and RNAi in nematodes, quelling was termed for essentially the same process in the ascomycete fungus, Neurospora crassa (Romano and Macino 1992). Like in other organisms, fungi also use RNA silencing to fight viral infections and to control transposable elements (Murata, et al. 2007, Segers, et al. 2007). An additional pathway has also been found in N. crassa where the expression of unpaired DNA is silenced during meiosis and requires the RNA silencing machinery (Alexander, et al. 2008, Shiu, et al. 2001). It is therefore becoming clear that RNA silencing maintains careful watch over invading nucleic acids from both exogenous and endogenous sources throughout mitotic and meiotic phases of the cell cycle in fungi as it does in plants and animals.  1.8 Proposed research Cloning of a host R-gene and its matching U. hordei Avr-gene would allow better understanding of the compatible and incompatible interactions between plants and fungi. Of the three monocot R-genes cloned, only one matching Avr-gene has also been cloned (Orbach, et al. 2000). The AVR-Pita protein from the ascomycete rice blast fungus Magnaporthe grisea was recently demonstrated to interact directly with its matching Pi-ta protein from rice (Jia, et al. 2000). This is a good step towards understanding plant/fungal molecular interactions, but more gene pair interactions need to be characterized, especially between cereal plants and basidiomycete fungi. The UhAvr1/Ruh1 combination between U. hordei and barley is being developed as such a model. UhAvr1 has recently been located through positional cloning (Linning, et al. 2004), while the Ruh1 gene has been mapped to the short arm of chromosome 1 (7H) between markers iPgd1A and BCD129 by collaborators at the University of Saskatchewan 14  (Grewal, et al. 2008). In addition, preliminary data indicate that Ruh1 is located in the genome of barley cv. Morex for which a BAC library is available through a collaboration with Washington State University (Brueggeman, et al. 2002). My project has involved searching for the UhAvr1 gene in U. hordei as well as characterization of its locus. I am particularly interested in small secreted proteins that likely act as virulence factors and might be responsible for the avirulence observed. Since other fungal avirulent genes have been found to reside in heterochromatic loci, I am also interested in looking for signs of heterochromatin at the UhAvr1 locus. Finally, through comparisons with U. maydis I hope to reveal interesting aspects of fungal evolution with regard to virulence and avirulence. Additionally, I will study RNA silencing pathways in U. hordei and U. maydis. Since RNA silencing is important for genome defence and endogenous gene regulation in diverse eukaryotes, study of these mechanisms might provide insight into the biology of smut fungi. An earlier report indicated that RNA silencing pathways might not be functional in U. maydis (Keon, et al. 1999). My initial experiments showed that RNA silencing was indeed functional in U. hordei. Although these two species are closely related and appear very similar, this difference in a fundamental mechanism of genome maintenance might provide clues to other differences between these species. RNA silencing is known to play key roles in PTGS, controlling transposons, TGS and establishment of heterochromatin, centromere function and chromosome separation and suppression of recombination. Could variation in any of these processes contribute to differences observed between U. hordei and U. maydis? Organization of mating-type genes, mode of plant infection, gene-for-gene resistance causing avirulence and occurrence of DNA repetitive elements are notably different between these two species. It is likely that after divergence from a common ancestor, differences in RNA silencing capabilities contributed to some of these differences. My work attempts to address some of these issues by first demonstrating experimentally that RNA silencing pathways are present in U. hordei, but 15  absent from U. maydis (Chapter 2). I then go on to identify key RNA silencing genes from the U. hordei genome and through comparative analyses show synteny with U. maydis at these loci (Chapter 3). In my final discussion (Chapter 5), I relate this work to information gained from studying the UhAvr1 locus (Chapter 4). My work reveals many important features of U. hordei and U. maydis and provides a foundation for important future studies.  1.9 Research objectives  Objective 1. Cloning UhAvr1 and characterization of its locus  Objective 2. Identifying genes involved in RNA silencing in U. hordei  Objective 3. Comparative genomics between U. hordei and U. maydis with an emphasis on differences in evolution of RNA silencing capabilities and evolution of virulence  16  Microbe  “NON-HOST”  PATHOGEN  RECOGNITION  HOST  (SPECIES LEVEL) basic compatibility  GENETICALLY SUPERIMPOSED (SUB-SPECIES LEVEL)  RACE (Avr)  CULTIVAR (R)  Figure 1.1 Potential outcomes of plant-microbe interactions. Non-host defences prevent most microbes from causing disease (top). Compatibility is seen between certain species and disease is the outcome (middle). Within species, gene-for-gene interactions involving avirulence (Avr) and resistance (R) genes lead to plant resistance (bottom).  17  A  B  FreeFoto.com  Photo by C. Dubeau  C  Wikipedia  Figure 1.2 Symptoms of Covered Smut of barley and Corn Smut. (A) Healthy barley plants growing in a field. Note the extension of the floral spikes and fullness of the seeds. (B) The typical symptom of Covered Smut of barley caused by Ustilago hordei is the black deformed floral spike. The spike is often stunted with a twisted stem, and although most of the smut spores are within the spike some smut can be seen on flag leaves of certain sensitive barley cultivars. (C) Corn Smut caused by U. maydis is similar to U. hordei both genetically and microscopically. It has the added advantage of infecting any above ground maize tissue and is capable of inducing large tumors on leaves and cobs.  18  III Haploid Basidiospores  Teliospores Meiosis  Spores  MAT-1 MAT-2 IV  II  In plant hyphae  I  Figure 1.3 Life cycle of Ustilago hordei, Covered Smut of barley. Plant infection begins when fungal dikaryotic hyphae penetrate germinating seeds (I). Fungal hyphae then grow toward the apical meristem(s) where they reside until flowering. As the floral meristem begins to develop, the fungus simultaneously proliferates eventually replacing most of the floral tissues with fungal biomass. During the final stages of proliferation black teliospores are formed giving the spikes a smutty appearance (II). When barley seeds are harvested, teliospores, the survival structures, are shed and attach to healthy seeds. Seed and teliospore overwinter together and germinate in the spring when conditions are favorable. 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Current opinion in plant biology 8: 353-360  31  CHAPTER 2:  Hallmarks of RNA silencing are found in the smut fungus Ustilago hordei but not in its close relative Ustilago maydis 1  2.1 Introduction RNA silencing or interference (RNAi) has had a significant impact on many different fields and has challenged the central dogma of molecular biology. Early studies in plants demonstrated that transgenes could silence, in trans, homologous endogenous genes (Napoli et al. 1990; van der Krol et al. 1990). Soon after, a similar phenomenon was seen in Neurospora crassa and was termed ‘quelling’ (Romano and Macino 1992). However, not until 1998 was double-stranded (ds)RNA shown to be responsible for initiating the silencing process (Fire et al. 1998). Since that time, a role for RNA as cell regulator has come to light and intense investigation has identified many of the components and pathways involved. From these studies, RNAi-like phenomena have been implicated in transcriptional, post-transcriptional as well as meiotic gene silencing (Mette et al. 2000; Sijen et al. 2001; Shiu et al. 2006). RNA has been demonstrated to guide DNA methylation, histone modification, and induce the silencing of transgenes, viruses, and transposons (Volpe et al. 2002; Hall et al. 2003; Sijen and Plasterk 2003). Additionally, specialized RNAi pathways utilizing micro (mi)RNA have evolved in plants and animals that play important roles in their development (Bartel 2004). Although specialized RNAi pathways have been reported, a common mechanism has emerged. In general, dsRNA, sometimes formed by RNA-dependent RNA polymerases (RdRPs) (Cogoni and Macino 1999; Dalmay et al. 2000; Smardon et al. 2000), is 1  A version of this chapter has been published: Laurie, J., Linning, R. and Bakkeren, G. 2008. Hallmarks of RNA silencing are found in the smut fungus Ustilago hordei but not in its close relative Ustilago maydis. Current Genetics 53: 49-58.  32  cut into small 21-26 nt fragments by an RNaseIII endonuclease called Dicer (Bernstein et al. 2001). Dicer fragments or short interfering (si)RNAs subsequently become associated with a multiprotein complex termed RISC (RNA-Induced Silencing Complex) containing a protein from the Argonaute-Piwi family that plays a central role in RISC activity (Liu et al. 2004; Rand et al. 2004). The mRNA population is then scanned by RISC for regions complementary to siRNA resulting in mRNA cleavage or translational repression depending upon the organism and pathway. Repression of translation is often associated with animal miRNA and is thought to occur due to a lack of complementarity in the central part of the miRNA (Doench et al. 2003; Zeng et al. 2003). Under translational repression, messages are targeted to subcellular compartments called P-bodies where they are degraded or stored (Liu et al. 2005; Pillai et al. 2005). Under stressful conditions, messages have been observed to leave P-bodies and resume translation (Bhattacharyya et al. 2006). Messenger RNA targeted for cleavage, however, have complementarity with the central region of the siRNA and are thought to be cleaved immediately. The ability of RNAi to work in trans has been exploited as a tool for manipulating gene expression (e.g.; Boutros et al., 2004; Nakayashiki et al., 2005; Travella et al., 2006). RNAi has been especially valuable for organisms in which transformation and subsequent generation of gene deletions by homologous recombination is difficult. An added advantage is that silencing of a particular gene creates a series of mutants exhibiting a range of phenotypes because interference or knock-down is rarely complete. This allows functions of essential genes to be discovered that otherwise would be lethal when deleted. RNAi mutagenesis has proven to be useful in various basidiomycete fungi (Liu et al. 2002; Namekawa et al. 2005) and should also be a valuable tool in the smut fungi. Smuts belong to the order Ustilaginales and are pathogenic basidiomycete fungi responsible for significant agricultural losses worldwide. The genus Ustilago infects members 33  of the Poaceae, which include cereal crops and forage grasses. Infection typically involves mating of haploid yeast-like cells on the host plant surface resulting in a dikaryotic infection hypha that penetrates the surface and grows between plant cells in a biotrophic manner. Although smuts execute a variety of infection strategies, diseased floral tissue is a common feature. Some smuts, such as Ustilago maydis, can induce large tumors on floral and vegetative tissues of their host plant (corn, Zea mays) which in later stages fill with masses of dark teliospores. U. maydis is perhaps the best-known smut and has become a model for this group of fungi (Martinez-Espinoza et al. 2002; Kahmann and Kamper 2004). As a result of the extensive studies with U. maydis, a vast number of molecular tools are available including the genome sequence (Kamper et al. 2006). I am interested in its close relative, U. hordei, because this pathogen has a genetically defined gene-for-gene resistance relationship with barley (Hu et al. 2002; Hu et al. 2003; Linning et al. 2004) which is not observed between U. maydis and corn. In order to exploit the advantages of RNAi, I developed an RNAi vector suitable for expression of dsRNA in U. hordei and U. maydis. I demonstrate the feasibility of RNAi mutagenesis in U. hordei and the lack of the RNAi phenomenon in U. maydis by targeting both a transgene and an endogenous mating-type gene.  2.2 Materials and Methods 2.2.1 Vector construction With the goal of using PCR products for cloning into both fungal and plant expression vectors, I decided to mimic the pMCG161 plant RNAi vector from ChromDB (www.chromdb.org; NCBI accession no. AY572837). To start, a 262 bp intron from the U. hordei bW2 gene (NCBI Accession no. Z18531) was placed between the Hsp70 promoter and terminator in the Ustilago vector, pUBleX1 (Hu et al. 2007); this episomal vector provides resistance to bleomycin/phleomycin/zeomycin-related compounds. For Ustilago species we 34  routinely use selection on 50 μg/ml zeomycin (Zeocin, Invitrogen) in Complete Medium (CM) (Holliday 1974). The intron was flanked by restriction sites Asc1 and Avr2 upstream, and Sgf1 and Spe1 downstream, yielding pUBleX1-RNAi (Fig. 2.1a and Table 2.1, primers 1 + 2). PCR of target gene fragments using primers containing Asc1 and Spe1 sites in one primer and Avr2 and Sgf1 sites in the other primer, resulted in products that could be ligated into either Ustilago-specific pUBleX1-RNAi or pMCG161 in tandem inverted orientation flanking the intron. A 563 bp region towards the 3'-end of the GUS gene (nucleotides 1214 to 1777) was cloned into pUBleX1-RNAi to form the pUBleX1-iGUS plasmid (Fig. 2.1b and Table 2.1, primers 3 + 4). A 131 bp conserved region of the bW gene was amplified from the U. maydis bW2 gene (NCBI accession no. M84182; nucleotides 675 to 805; Table 2.1, primers 5 + 6) and the U. hordei bW1 gene (NCBI accession no. Z18532; nucleotides 488 to 618; Table 2.1, primers 7 + 8) and cloned into pUBleX1-RNAi to form the plasmids pibWUh and pibWUm (Fig. 2.1c).  2.2.2 Strains and genetic transformation U. hordei haploid strains Uh4857-4 (alias Uh364, MAT-1) and Uh 4857-5 (alias Uh365, MAT-2) (Linning et al. 2004) and U. maydis haploid strains 521 (a1b1) and 518 (a2b2) (Kronstad and Leong 1989) were used in this study. Stable GUS-expressing, transgenic lines were generated from U. hordei strain Uh4857-5 (MAT-2) and U. maydis strain 518 (a2b2) using a fungal expression vector containing a hygromycin resistant gene (Hu et al. 2002), and the bacterial uidA gene (beta-glucuronidase or GUS). These GUS expressing lines were subsequently used for transformation with the pUBleX1-iGUS vector (Fig. 2.1b). Strains were cultured by shaking at 200 rpm in liquid CM at 22°C (U. hordei) or 28°C (U. maydis). For analysis of RNAi on the endogenous bW mating-type genes, plasmid pibWUh was transformed into U. hordei strain Uh4857-4 and plasmid pibWUm was transformed into U. maydis strain 35  518. RNAi lines generated from strain Uh4857-4 were tested for mating against U. hordei lines Uh4854-10 (MAT-2) and Uh4857-4 (MAT-1), while lines generated from U. maydis strain 518 were tested against strains 521 (a1b1) and 518 (a2b2). Mating was done by mixing 5 µl of each culture on 1.5 % agar plates containing Double Complete Medium (DCM) supplemented with 1.0 % (w/v) activated charcoal (Holliday 1974). Biolistic transformation was done as described for Cryptococcus neoformans (Davidson et al. 2000). In brief, U. hordei haploid cells were grown overnight in 50 ml of CM to a cell density of OD600=1. Cells were then pelleted and resuspended in 10 ml of CM. 200 µl was then spread on a 1.0 % agar plate containing DCM medium and 1M sorbitol. Plates were dried for at least 4 hrs with opened lids in a laminar air-flow hood before particle bombardment. U. maydis was transformed using polyethylene glycol (PEG) after enzymatic removal of cell walls as previously described (Brachmann et al. 2004).  2.2.3 GUS fluorometic MUG assay Basidiospore cultures were grown to an approximate density of OD600=1. One ml was removed from each culture for the GUS histochemical assay (Jefferson et al. 1987, data not shown) while the remainder was used for RNA and protein extraction. For quantitative GUS assays, basidiospores were pelleted by centrifugation, frozen in liquid nitrogen, and ground to a fine powder. Proteins were extracted by mixing 100 mg of ground basidiospores with 1 ml of GUS extraction buffer (50 mM sodium phosphate buffer at pH 7.0, 10 mM EDTA, 10 mM βmercaptoethanol, 0.1 % Triton X-100, and 0.1 % sodium lauryl sarcosine). After thorough mixing, protein extracts were centrifuged twice for 10 min at 14, 000 rpm and 4°C to remove cell debris. Protein concentrations were determined using a Bradford protein assay kit (BioRad) by mixing 20 µl of basidiospore extract with 180 µl of diluted reaction mix before spectrophotometry. Samples were read along with BSA protein standards. Care was taken to 36  ensure that extracts were within the linear range of the assay. GUS activity was measured by incubating equal protein concentrations in GUS extraction buffer containing 0.8 M MUG (4methylumbelliferyl B-D-Glucuronide, Sigma) at 37°C. After equal incubation times each sample was stopped by addition of two volumes of stop solution (0.2 M sodium carbonate). Fluorescence was measured in triplicate from each biological replicate along with MU (4methylumbelliferone, Sigma) as the standard using a SpectraMAX GeminiEM microtiter-plate reader (Molecular Devices) with emission, excitation and cut-off filters set at 460 nm, 365 nm and 420 nm respectively. For each sample, nmol MU per mg of protein per minute of incubation time was calculated and compared to the positive control GUS cell line.  2.2.4 RNA extraction and detection Basidiospore cultures were pelleted by centrifugation before rapid freezing in liquid nitrogen. Frozen pellets were then ground to a fine powder and stored at -80°C until extraction of either RNA or protein. RNA was extracted using a modified RNeasy protocol (Qiagen). Briefly, RNA was extracted from ground basidiospores using RLT buffer containing 1 % βmercaptoethanol. After passing through a QIAshredder, one volume of 80 % ethanol was added to the supernatant prior to loading an RNeasy mini column. After a short spin, long RNA (>200 nt) remained bound to the RNeasy column while short RNA (<200 nt) passed through. Columns containing longer RNA were washed and eluted as per the RNeasy protocol. To obtain shorter RNA, 1.4 volumes of 100 % ethanol were added to the supernatant before loading to a second RNeasy mini column and processing as per the RNeasy protocol. Longer RNA (5 µg) was separated on a MOPS/formaldehyde gel (2 % formaldehyde) before capillary blotting to Hybond-N+ as recommended by the supplier (Amersham Biosciences/GE Healthcare). For detection of dsRNA, 5 µg of RNA was treated with RNase A/T1 (1:100 dilution of RNase A/T1 in RNase Digestion Buffer; mirVana miRNA Detection Kit, Ambion) 37  for 60 min at 37°C, precipitated with ethanol and subjected to electrophoresis in a MOPS/formaldehyde gel before blotting to Hybond-N+. A GUS PCR probe (Table 1, primers 3 + 4) was labelled with [32P]dCTP (PerkinElmer) using the Rediprime II Random Prime Labelling System (Amersham Biosciences/GE Healthcare) and hybridized to membranes using ULTRAhyb buffer (Ambion) at 42°C. After washing twice for 5 min in 2X SSC (0.3 M sodium chloride, 0.03 M sodium citrate, pH 7.0), 0.1 % SDS (sodium dodecyl sulphate) followed by twice for 15 min in 0.1X SSC, 0.1 % SDS (all at 42°C), membranes were scanned using the Cyclone Plus Storage Phosphor System (PerkinElmer). Short RNA (10 µg) was separated in a 15 % denaturing PAGE gel (8 M urea/1X TBE buffer: 89 mM Tris-Borate, 2 mM EDTA, pH 8.0) and electro-blotted onto Hybond-N+. Probe generation, hybridization and detection were the same as for longer RNA, but washing was less stringent and included a single 5 min wash with 2X SSC at 37°C followed by two 15 min washes with 2X SSC, 0.1% SDS at 42°C.  2.2.5 Quantitative-RT-PCR Two µg of RNA, extracted using the RNeasy kit and quantified using a Nanodrop ND1000 spectrophotometer, was treated with DNaseI (Invitrogen) prior to cDNA synthesis using SuperScript II reverse transcriptase and random primers (Invitrogen). cDNA was diluted (8X) before quantification by Q-PCR using SYBR Green and an Mx4000 cycler (Stratagene). Equal amounts of cDNA generated from quantified RNA were amplified using AmpliTaq Gold (Perkin Elmer). For each cell line tested, data represent the average of three separate RNA preparations from biological replicates. For measurement of GUS mRNA, samples were normalized to a housekeeping gene (Succinate dehydrogenase, NCBI Accession no. XM_751898, for U. maydis and a putative vacuolar protein sorting-associated protein, NCBI Accession no. AM118080, for U. hordei) and compared to control cell lines. Amplification 38  involved 10 min at 95°C followed by 40 cycles of 30 sec at 95°C, 60 sec at 63°C, and 60 sec at 72°C. Fluorescent data was collected at the end of the 63°C annealing step in every cycle. Melting curves were done at the end of each run to verify the reliability of the results.  2.2.6 Protein extraction and detection Proteins were extracted from ground frozen cells with two volumes of protein extraction buffer (10 mM KCl, 5 mM MgCl2·6H2O, 400 mM sucrose, 100 mM Tris-HCl, pH 8.1, 10 % glycerine, 0.007 % β-mercaptoethanol) and centrifuged at 14,000 rpm for 5 min at 4°C. 100 µl of each of the supernatants were then mixed with one volume of 2X protein loading buffer (100 mM Tris-HCl, pH 6.8, 4 % sodium dodecyl sulphate, 20 % glycerine, 0.2 % bromo-phenol-blue, 200 mM dithiothreitol) and boiled for 5 min before SDS-PAGE (8 % acrylamide, 1.5 M TrisHCl, pH 8.8). A test gel was stained with coomassie blue so that each sample could be visually quantified. For subsequent gels, equal amounts of protein extracts were loaded for each sample. Proteins were transferred onto Sequi-Blot PVDF membrane (Bio-Rad) and probed with a rabbit polyclonal antibody against GUS (Molecular Probes A-5790) followed by a goat anti-rabbit immunoglobulin G conjugated to horseradish peroxidase (Bio/Can). Immunoreactions were detected using the ECL Western blotting detection system (Amersham Biosciences/GE Healthcare).  2.3 Results 2.3.1 Ustilago RNAi vector construction and expression An RNAi expression vector was developed as an initial step in studying the efficacy of using RNAi as a tool for reducing gene expression in Ustilago species. The pUBleX1 expression vector (Hu et al. 2007) was chosen based on its utility for obtaining high expression levels for inserted genes and it was engineered to contain an intron with flanking restriction 39  sites between the constitutive Ustilago-specific Hsp70 promoter and terminator elements (Fig. 2.1a). To test the RNAi vector, GUS expressing lines were first obtained for both U. hordei and U. maydis (see Materials and Methods). Next, a 563 bp region near the 3'-end of the GUS gene was amplified by PCR and digested with Asc1 and Avr2 for sense cloning and Sgf1 and Spe1 for anti-sense cloning into the RNAi vector (Fig. 2.1b). The episomal iGUS vector, pUBleX1-iGUS, was then introduced into haploid, GUS-expressing cell lines from both species and transformants were maintained by zeomycin selection. For confirmation that the pUBleX1-iGUS vector was functioning properly, the first step was to look for dsRNA corresponding to the cloned GUS fragments, since RNA expressed from this vector is designed to fold back upon itself. To do this, RNA was extracted from multiple RNAi lines and from GUS expressing control lines from both species. Next, an RNase protection assay (RNase A/T1-treated RNA) was conducted to eliminate all single stranded RNA. The RNase A/T1treated RNA was then transferred to nylon membrane by Northern blotting and probed with a PCR product corresponding to the GUS fragment in the pUBleX1-iGUS vector. A single band of the expected 563 nucleotides was clearly seen in all RNAi lines carrying the pUBleX1-iGUS vector demonstrating the presence of GUS dsRNA and thus proper expression of the RNAi construct and subsequent folding of the transcript (Fig. 2.2). As well, no GUS mRNA or dsRNA were detected in the GUS expressing control lines. Furthermore, in blots without digestion no bands could be detected at 563 nt and the dsRNA ran at roughly 1000 nt, and in the digested samples no ethidium bromide-stained rRNA could be seen in the gel indicating successful RNase A/T1 digestion (data not shown). For starting concentration comparison, however, undigested RNA was separated by electrophoresis and stained with ethidium bromide for visualization of rRNA (Fig. 2.2). Since a distinguishing feature of RNAi is the generation of siRNA from dsRNA the next step was to isolate and separate small RNAs for analysis. Northern blot analysis of PAGE-separated RNA probed with the same GUS fragment used to 40  detect dsRNA, confirmed the presence of siRNA in U. hordei (Fig. 2.2). On the contrary, after repeated efforts, no such GUS siRNA could be detected in U. maydis (Fig. 2.2). RNAi is achieved through the RISC-guided cleavage of mRNA complementary to siRNA generated by Dicer (Liu et al. 2004; Meister and Tuschl 2004). To determine the influence of GUS dsRNA/siRNA on GUS mRNA levels, quantitative-RT-PCR analysis was performed on cDNA from control and RNAi lines. Normalized data revealed a significant drop in GUS mRNA levels in U. hordei but not U. maydis (Fig. 2.3a) indicating that siRNA in U. hordei effectively silenced the GUS transgene. The effects of RNAi could also be seen at the protein level as GUS enzymatic activity was significantly reduced in U. hordei RNAi lines as measured in a MUG fluorometric assay (Fig. 2.3b). In this assay, a decrease in GUS activity was measured as a marked reduction in enzymatic cleavage of MUG substrate in normalized protein extracts. The results of the MUG assay were supported by western blot analysis of GUS protein (Fig. 2.3c). A GUS polyclonal antibody confirmed that GUS protein levels were concomitantly reduced in equivalent protein extracts from U. hordei RNAi lines (Fig. 2.3c), as indicated by a strong band of approximately 74 kDa only in the positive control GUS sample. Neither reduction in GUS protein activity or level could be detected in U. maydis lines expressing the pUBleX1-iGUS construct (Fig. 2.3b and c).  2.3.2 RNAi of an endogenous mating-type gene Since RNAi proved to be successful in U. hordei for silencing the GUS transgene, our next goal was to measure its ability to silence an endogenous gene. Given that mating of compatible haploid strains can easily be visualized on charcoal-containing medium in the form of white mating hyphae, a domain of the bW mating-type genes, conserved among different alleles (Fig. 2.1c), was targeted in both U. hordei and U. maydis. Primers flanking a 131 bp region were designed for both species and PCR products were ligated into the episomal RNAi 41  vector, pUBleX1-RNAi (Fig. 2.1c and Table 2.1, primers 5 to 8). Transformants were obtained for both U. hordei Uh4857-4 (MAT-1) and U. maydis 518 (a2b2) and tested for their mating ability. For this assay, haploid cultures were placed as drops on the charcoal-containing medium (Fig. 2.4). To the left was spotted on the agar surface a drop of the haploid line to be tested. An equivalent drop was co-spotted with haploid culture of either mating type in the center row and to the right, respectively. Successful mating resulted in white fuzzy colonies, whereas haploid colonies containing the same mating type remained smooth and grey. Three independent transformants showed a considerable reduction in the production of mating hyphae for U. hordei (Fig. 42.a). Because the transgenic line was MAT-1, normal mating would have resulted when mixed with a MAT-2 strain, as seen for the control (Fig. 2.4a, arrow). Reduced mating in the U. hordei RNAi lines indicated successful functioning of the pibWUh construct. Mating in four U. maydis transformants seemed to be unaffected since mating of all lines was indistinguishable from the control (Fig. 2.4b).  2.4 Discussion In this paper Idescribed the construction of a functional RNAi vector for Ustilago hordei and demonstrated the feasibility of using RNAi to knock-down gene expression in this smut fungus. For my experimental design I chose to separate sense and anti-sense PCR fragments by an intron since previous studies established an increase in RNAi efficiency with the use of an intron (Smith et al. 2000; Wesley et al. 2001). Also for ease of transformation, I elected to express the hairpin constructs from a strong promoter on an episomal vector known to yield high expression levels (Hu et al. 2007). As anticipated, expression of dsRNA induced the RNAi pathway in U. hordei. This was evident by the down-regulation of a GUS transgene and the endogenous bW mating-type genes. Occurrence of GUS-specific siRNAs provided experimental evidence for the presence of a Dicer gene in U. hordei. Additionally, down42  regulation of transcripts homologous to the introduced dsRNA points to a functional RISC. Based on data from other basidiomycetes (Nakayashiki et al. 2006), this complex is likely to contain a protein in the Argonaute-Piwi family. RNAi lines showed variation in the degree of silencing achieved, similar to reports on RNAi in other species (Tanguay et al. 2006; Walti et al. 2006). This was especially evident in the GUS histochemical assay where several silenced lines displayed a range of faint blue color indicating incomplete knock-down of the GUS transgene (data not shown). The quantitative MUG and RT-PCR results supported the histochemical assay and showed a reduction in steady-state mRNA levels of as much as 75% and a protein activity reduction to less than 1%. Such levels are consistent with those reported in other species (Liu et al. 2002). Incomplete knock-down of genes highlights the value of this technique as a tool for studying essential genes which might otherwise be lethal if deleted. When the endogenous U. hordei bW1 mating-type gene was targeted by RNAi, a clear phenotype was seen upon mating in the form of a significant reduction in white mating hyphae. Even though knock-down was probably incomplete, a distinct phenotype was nevertheless seen. In addition, since each mating type possesses a bE and a bW allele which, upon mating, interact and produce an active bE1/bW2 and a bE2/bW1 heterodimer complex (Gillissen et al. 1992; Bakkeren and Kronstad 1993), it follows that the pibWUh construct must have affected both bW alleles in the dikaryon. Despite the 9.2 % divergence between these sequences (12 out of 131 nucleotides, Fig. 2.1c), the RNAi construct seemed able to target both bW alleles. These results support the use of RNAi as a tool for targeting several members of a closely related gene family, and demonstrate the feasibility of using RNAi to study gene function in U. hordei. Previous studies have indicated that the RNAi phenomenon may be lacking in U. maydis. First, an earlier study showed that antisense transcripts fail to down-regulate the pyr3 gene (Keon et al. 1999). More recently, components from the RNAi pathway could not be 43  identified in the U. maydis genome (Kamper et al. 2006; Nakayashiki et al. 2006). However, since bioinformatic searches have failed in the past to identify RNA components for organisms known to posses RNAi, more direct experimental proof was needed. Our study is the first to demonstrate the presence of dsRNA upon introduction of the RNAi construct in U. maydis and to show the failure to detect corresponding siRNA. When the GUS gene was targeted by our RNAi vector, no negative effect was observed on GUS mRNA level, protein level, or gene function. Similarly, dsRNA targeted to the endogenous bW mating-type genes had no apparent negative effect on bW2 and bW1 functions, as normal mating ensued. These results support the previous pyr3 report, as well as the bioinformatic studies, and suggest that no functional homolog to Dicer exists in U. maydis. The results presented here are significant because U. hordei and U. maydis are phylogenetically closely related (Stoll et al. 2005). Comparison of DNA sequences between several large regions of the U. hordei genome and the corresponding regions of the U. maydis genome show conservation of synteny and a high level of sequence similarity (Bakkeren et al. 2006 and unpublished). Furthermore, these two species have very similar life cycles and can be artificially forced to mate (Bakkeren and Kronstad 1996). Since a common ancestor to all eukaryotes is considered to have possessed the basic machinery for RNAi (Cerutti and CasasMollano 2006), one must ask why or how U. maydis lost its RNAi capability. Furthermore, what are the consequences of such a loss? One immediate concern is vulnerability to foreign or mobile genetic elements. First, how does U. maydis protect itself against attacks from viruses? RNAi is the prime safeguard used by plants against viruses, as evident in the fact that plant viruses contain suppressors of silencing to aid in their attack (Li and Ding 2001; Qu and Morris 2005). Furthermore, RNAi was recently shown to protect the chestnut blight fungus, Cryphonectria parasitica, from viral attack (Segers, et al. 2007). Since viruses are known to infect U. maydis (Martinez-Espinoza et 44  al. 2002), it remains to be determined what type of defence U. maydis employs and if such viruses have lost their suppressors of silencing. A second concern is the control of mobile genetic elements. Numerous studies in plants, animals, and fungi demonstrate the importance of RNAi-like mechanisms to control transposable elements (Nolan et al. 2005; Tran et al. 2005; Brennecke et al. 2007; Houwing et al. 2007). Surprisingly, the U. maydis genome is relatively devoid of transposable elements; in particular, no class II (DNA) elements or otherwise active endogenous elements can be found (Ladendorf et al. 2003; Kamper et al. 2006). On the other hand, U. hordei appears to contain a large number of transposable elements in its genome. Numerous transposable elements are evident throughout the genome as determined from sequencing 527 kb in the MAT-1 region (Bakkeren et al. 2006) and 100 kb in a region containing an avirulence gene, Avr1 (Linning et al. 2004), together with BAC end sequencing and genome hybridizations (Bakkeren, unpublished). This observation suggests that U. maydis has a different evolutionary history with transposable elements and may deploy a different but very effective strategy to control mobile genetic elements. Another intriguing question is the possible consequence of RNAi loss on transcription regulation. It is tantalizing to think that transcription from both strands of a gene could occur coincidentally in a single cell without the expected degradation triggered by dsRNA. Recent reports on a number of organisms indicate that overlapping transcripts are more common than previously thought (Williams et al. 2005; Steigele et al. 2007). Such strategies may reflect more efficient utilization of genomic information and means of transcriptional control (Hongay et al. 2006). In support of these studies, U. maydis EST and SAGE libraries possess evidence of overlapping transcripts (Ho et al. 2007; J. Kronstad and G. Hu, personal communication). Recent data for a number of diverse organisms point to life cycle stage-specific RNAi pathways (Nolan et al. 2005; Brennecke et al. 2007; Carmell et al. 2007; Gunawardane et al. 2007; Houwing et al. 2007). A recent report on N. crassa shows that the presence of dsRNA 45  induces expression of RNAi components as well as numerous other genes including homologs of antiviral and interferon-stimulated genes, thus demonstrating an active response in vegetative cells (Choudhary et al. 2007). Additionally, N. crassa is known to silence unpaired DNA during meiosis, a process known as Meiotic Silencing of Unpaired DNA (MSUD) (Shiu et al. 2001). Similarly, recent reports on mice and Drosophila suggest an ancient mechanism exists to silence unwanted DNA during meiosis (Brennecke et al. 2007; Carmell et al. 2007). Since we have shown that U. maydis does not possess a functional RNAi machinery, it remains to be seen how U. maydis deals with mobile genetic elements in vegetative cells and what U. maydis does with unpaired DNA during meiosis. Furthermore, it would be interesting to see which genes, if any, are up-regulated in the dsRNA iGUS lines in both U. hordei and U. maydis. The loss of RNAi is not unique to U. maydis. Using bioinformatics on completed genomes, RNAi components could not be detected in the fungi Saccharomyces cerevisiae and Candida lusitaniae (Nakayashiki et al. 2006), in the parasites Trypanosoma cruzi, Leishmania major, Plasmodium falciparum and in the red alga Cyanidioschyzon merolae (Cerutti and Casas-Mollano 2006). Intriguingly, the majority of these organisms, like U. maydis, are obligate parasites with relatively compact genomes and are devoid of the DNA class of transposable elements. This study has shown that RNAi is a useful tool for the down-regulation of genes in U. hordei, and has raised a number of questions concerning an apparent lack of this phenomenon in U. maydis. Our laboratory is collaborating on a project to sequence the U. hordei genome. We anticipate identification of homologs to Dicer, Argonaute-Piwi, RdRP and perhaps MSUD genes from the genome sequence. Since a high degree of synteny has been observed between U. hordei and U. maydis, it will be interesting to compare U. maydis syntenic regions to U. hordei loci containing RNAi components. Such a comparison may provide clues to the evolutionary history of RNAi in U. maydis. 46  A  Bgl2, Asc1, Avr2  phleor  Sgf1, Spe1, Bgl2  Intron Hsp70 term.  Hsp70 prom.  pUBleX1-RNAi  B  Asc1 Avr2  Sgf1 Spe1 GUS  GUS  pUBleX1-iGUS  C  UhbW1 GTCGAGGGCACGACTCTGACGCAGTCCGAATCCTCGAACAAGCCTTCCAG UhbW2 GCCGCGGGCACGACTCTGACGCAGTCCGCGTCCTCGAACAAGCCTTCCAA UmbW2 GTCGTGGACACGACTCCGAAGCAGTCCGAATCCTGGAGCAAGCCTTCAAA UhbW1 CACACACCCAACATCACGCAAGCCGAGAAATATCGATTAGCTGAAGTCAC UhbW2 CACACACCGAACATCACGCAAGCCGAAAAGTATCGACTGGCTGAAGTCAC UmbW2 CATTCACCAAACATCACCCCAGCCGAGAAGTTCCGACTTTCAGAGGTCAC UhbW1 CGGACTCAAGCCAAAGCAAGTGACCATCTGG UhbW2 CGGACTCAAGCCAAAGCAGGTTACCATCTGG UmbW2 TGGACTCAAACCAAAGCAAGTCACTATCTGG  pibWUm  ibW2  ibW2  pibWUh  ibW1  ibW1  Figure 2.1 Construction of RNAi vectors. (A) pUBleX1-RNAi plasmid. A 262 bp intron from the U. hordei bW2 gene (Accession Z18531) is flanked by cloning sites between the constitutive Ustilago Hsp70 promoter and terminator. (B) The pUBle-iGUS plasmid. A 563 bp GUS PCR product was ligated in inverted repeats to the pUBleX1-RNAi vector. (C) The pibWUm and pibWUh RNAi vectors. Homologous regions were obtained from respective bW alleles using PCR and ligated into pUBleX1-RNAi.  47  A  B +  1  2  3  +  1  2  3 1 kb  dsRNA 0.5 kb 35 nt siRNA 15 nt rRNA  Figure 2.2 Expression of dsRNA and production of siRNA. Transformants (1, 2, and 3) containing pUBleX1-iGUS were generated from stable GUS expressing (+) U. hordei (A) and U. maydis (B) lines. GUS dsRNA was detected after an RNase protection assay using RNase A/T1 in RNA from only the transformants (upper panel); this single product represents the double stranded hair-pin structure which when denatured migrates at the predicted length of 563 nucleotides when denatured. siRNA in the 25 nt-range was detected only in U. hordei iGUS lines (middle panel). Equivalent amounts of RNA were used to start each experiment as shown by EtBr staining of rRNA (lower panel). The scale indicated on right is based on singlestrand RNA markers (upper panel) and DNA oligonucleotides (middle panel). All Northern blots were probed with radiolabelled DNA corresponding to the original GUS fragment in the pUBleX1-iGUS construct: kb, kilobases; nt, nucleotides.  48  A Relative GUS m RNA level  2  U. hordei 1.5  1  0.5  0  B  3.5  R elativ e G U S ac tivity  U. maydis  +  1  2  3  U. hordei  +  1  2  3  2  3  U. maydis  3 2.5 2 1.5 1 0.5 0  +  1  2  3  +  1  C  Figure 2.3 RNA interference by dsRNA. (A) Presence of GUS dsRNA in transformants 1, 2 and 3 decreased the level of GUS mRNA in U. hordei, but not in U. maydis as measured by QPCR. (B) MUG assay showing that GUS protein activity was significantly reduced in U. hordei iGUS lines but not in U. maydis iGUS lines. (C) Immuno blot showing that the GUS protein level was correspondingly reduced in U. hordei iGUS lines, but not U. maydis iGUS lines (upper panel). Coomassie staining shows equal loading of protein (lower panel). (+), stable GUS-expressing control lines.  49  A  B MAT-2  MAT-1  a1b1  a2b2  1 1 2 2  3  3  4 MAT-1 X MAT-2  a1b1 X a2b2  Figure 2.4 RNA interference with the endogenous bW gene transcripts disrupts mating in U. hordei but not in U. maydis. Haploid transformants containing pibWUm or pibWUh were generated and tested for mating ability. (A) U. hordei ibWUh lines (1, 2, and 3, MAT-1) were paired against tester lines of mating type MAT-1 and MAT-2. (B) U. maydis ibWUm lines (1, 2, 3 and 4, a2b2) were mated with tester lines 521 (a1b1) and 518 (a2b2). Successful mating produces dikaryotic aerial hyphae which appear as a white “fuzzy” colony phenotype on activated-charcoal plates: white boxes, cultures of compatible mating types; arrows, compatible mating controls. Shown are typical examples of test results which were repeated at least three times.  50  Table 2.1 Primers for vector construction and qPCR Primer  Sequence (5' to 3')  Description  1  ttAGATCTGGCGCGCCCCTAGGgtcagtaaag ctttgtcctgtacgcgactc ttAGATCTACTAGTGCGATCGCctgtagaaag gggatcagaacaagaaggg ttACTAGTGGCGCGCCtggtgatgtggagtattgcc ttGCGATCGCCCTAGGagttcatgccagtccagcgt ttACTAGTGGCGCGCCgtcgtggacacgactccga agcagt ttGCGATCGCCCTAGGccagatagtgacttgctttg gtttga ttACTAGTGGCGCGCCgtcgagggcacgactctga cgcagt ttGCGATCGCCCTAGGccagatggtcacttgctttg gcttga gactttgcaagtggtgaatccgca ttcagcgtaagggtaatgcgaggt ggtgtcgacatgacagtctcgttgcg ctcttcgaagtgttctgggagcagat gtcgctattcaacgtcagcaacggtcttcg gagcgaaaaggtgttctcgagcttctgtc  Cloning; U. hordei bW2 intron with Bgl2, Asc1 and Avr2 sites.  2 3 4 5 6 7 8 9 10 11 12 13 14  Cloning; U. hordei bW2 intron with Bgl2, Spe1 and Sgf1 sites. Cloning; 3' end of GUS with Spe1 and Asc1 sites. nt 1214* Cloning; 3' end of GUS with Sgf1 and Avr2 sites. nt 1777 Cloning; U. maydis bW2 with Spe1 and Asc1 sites. Cloning; U. maydis bW2 with Sgf1 and Avr2 sites. Cloning; U. hordei bW1 with Spe1 and Asc1 sites. Cloning; U. hordei bW1 with Sgf1 and Avr2 sites. qPCR; for 5' end of GUS. nt 696 qPCR; for 5' end of GUS. nt 1005 qPCR; for U. hordei control gene. nt 1415 qPCR; for U. hordei control gene. nt 1806 qPCR; for U. maydis control gene. nt 3 qPCR; for U. maydis control gene. nt 747  Restriction sites are in uppercase. * Gene location of 5′-most nucleotide (nt); see “Materials and methods” for GenBank accession numbers  51  2.5 References Bakkeren G, Jiang G, Warren RL, Butterfield Y, Shin H, Chiu R, Linning R, Schein J, Lee N, Hu G, Kupfer DM, Tang Y, Roe BA, Jones S, Marra M, Kronstad JW (2006) Mating factor linkage and genome evolution in basidiomycetous pathogens of cereals. 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Plant J 27:581-590 Williams BA, Slamovits CH, Patron NJ, Fast NM, Keeling PJ (2005) A high frequency of overlapping gene expression in compacted eukaryotic genomes. Proc Natl Acad Sci USA 102:10936-10941 Zeng Y, Yi R, Cullen BR (2003) MicroRNAs and small interfering RNAs can inhibit mRNA expression by similar mechanisms. Proc Natl Acad Sci USA 100:9779-9784  59  CHAPTER 3:  Identification of RNA silencing genes in Ustilago hordei provides clues to the uniqueness of Ustilago maydis 1  3.1 Introduction RNA silencing can affect gene expression at both the transcriptional and posttranscriptional levels and possibly evolved as a mechanism for controlling invasive nucleic acids (Plasterk 2002). Through genetic and biochemical analyses in diverse eukaryotes, genes encoding important proteins for different RNA silencing pathways have now been identified. Among the first to be discovered were the RNase III-like enzymes called Dicer, the Argonaute (Ago) proteins and the RNA-dependent RNA polymerases (RdRP) (Mello and Conte 2004, Shabalina and Koonin 2008). The underlying mechanism of RNA silencing involves doublestranded (ds) RNA from endogenous or exogenous sources being cut by Dicer into short interfering (si) RNA or micro (mi) RNA with lengths ranging from ~21-26 nucleotides (nt) (Bernstein, et al. 2001). The small RNA fragments are then loaded into Ago proteins where most often it is the antisense strand that is retained and necessary for target recognition. Ago proteins are the catalytic engines of both the RNA-induced silencing complex (RISC) involved in post-transcriptional gene silencing (PTGS) where mRNA is degraded, and the RNA-induced transcriptional silencing complex (RITS) where methylation of DNA and associated histones accomplishes transcriptional gene silencing (TGS)(Hutvagner and Simard 2008, Tolia and Joshua-Tor 2007). RdRPs contribute to certain pathways by forming dsRNA that initiates RNA silencing. As well, RdRP can form secondary dsRNA after initial cleavage of mRNA by 1  A version of this chapter will be included in a joint manuscript and submitted for publication: Laurie, J., Linning, R., Bakkeren, G., Schirawski, J, Kahmann, R. et al. Upon publication, the U. hordei genome sequence, assembly and gene ID numbers mentioned in this chapter, will be released in the public domain.  60  a miRNA-loaded Ago protein, thus amplifying the silencing response and allowing a second phase of siRNA to be produced (Axtell, et al. 2006, Faehnle and Joshua-Tor 2007). Some biological functions of the RNA silencing machinery include degradation of RNA from invading viruses and bacteria as well as degradation of RNA emanating from mobile genetic elements. RNA silencing therefore plays an important role in maintaining genome stability. Many organisms have also evolved to use the RNAi machinery to control gene expression by encoding dsRNA (miRNA precursors) from endogenous loci (Bartel 2004, Jones-Rhoades, et al. 2006). This form of control is often used for key steps in development and for response to stress. Since related RNA silencing proteins have been implicated in both PTGS and TGS, it has been suggested that an evolutionary arms race has taken place between RNA silencing genes and invasive genetic elements (Aravin, et al. 2007, Hartig, et al. 2007, Marques and Carthew 2007). Whether RNA silencing simultaneously evolved to function in both PTGS and TGS pathways, or one preceded the other, is not clear. However, examples of both mechanisms are present in diverse eukaryotes and RNA silencing is considered to have been present in the last common ancestor to all eukaryotes (Cerutti and Casas-Mollano 2006, Shabalina and Koonin 2008). In plants, distinct Dicers cut dsRNA for either the TGS or PTGS pathways (Margis, et al. 2006). The siRNAs are then preferentially loaded into different Ago proteins to direct each pathway (Mi, et al. 2008, Montgomery, et al. 2008). In the fission yeast, Schizosaccharomyces pombe, single Dicer and Ago proteins are responsible for both PTGS and TGS (Martienssen, et al. 2005). In animals a single Dicer is responsible for generating miRNA and siRNA (Tijsterman and Plasterk 2004). Phased siRNA have recently been identified in plants and nematodes that, like miRNA, are important for development and stress tolerance (Axtell, et al. 2006, Sijen, et al. 2001). This PTGS pathway in the model plant Arabidopsis thaliana requires 61  the RdRP, RDP6, to produce dsRNA from non-coding mRNA after cleavage by a miRNA. The dsRNA is then chopped by the DCL4 Dicer enzyme and the siRNA loaded into Ago proteins that target endogenous genes. Loci producing these non-coding transcripts produce siRNA referred to as transacting siRNA or tasiRNA (Axtell, et al. 2006). Another PTGS pathway first discovered in the germ-line of animals involves loci rich in DNA having homology to mobile genetic elements that produce small RNA (piRNA) (Aravin, et al. 2007). These piRNA are loaded into the Piwi class of Ago proteins that effectively silence mobile genetic elements throughout the genome. To date no Dicer has been implicated in this process and the mechanism for generating piRNA is unclear. Recently, a similar mechanism has also been observed to occur in animal somatic cells (Czech, et al. 2008). In many organisms, TGS is achieved using RNAi through siRNA loading of Ago proteins that target homologous DNA for methylation or direct histone H3 methylation (Bender 2004, Matzke and Birchler 2005). In Arabidopsis the DCL3 Dicer cleaves dsRNA into 24 nt siRNA that are loaded into Ago4. Ago4 then directs methyl transferases to methylate DNA homologous to the 24 nt siRNA. This pathway can be stimulated artificially by expressing dsRNA homologous to gene promoters and occurs naturally during imprinting and paramutation by transcriptional silencing of genes located near repetitive DNA (Chandler and Alleman 2008, Kinoshita, et al. 2004). In S. pombe, Dcr1 (Dicer), Ago1 and Rdp1 (RdRP) are required for TGS by histone H3 methylation within centromeric DNA repeats (Volpe, et al. 2002). In animals, X-chromosome inactivation may require Dicer and the RNAi machinery to accomplish silencing by directing histone H3 lysine 27 trimethylation (H3-3meK27) (Ogawa, et al. 2008). Taken together, these examples illustrate the use of RNAi machinery to both target and stop the transcription of repetitive DNA in addition to homology-dependent degradation of repetitive DNA transcripts. Use of RNAi machinery for genome control at both transcriptional and post-transcriptional levels is supported by many examples and likely 62  evolved later as a general method of endogenous gene regulation (miRNA and tasiRNA) in multicellular organisms (Shabalina and Koonin 2008). Dicers are large multi-domain proteins that cleave dsRNA into small fragments of defined length (Bernstein, et al. 2001). These proteins are often characterized by their Nterminal helicase domains and the two tandem copies of the catalytic RNase III domain toward their C-terminus. As well, Dicers may include a domain of unknown function (DUF283), a PAZ domain, and a dsRNA-binding domain (DSRM). From the crystal structure of the Dicer of Giardia intestinalis it was proposed that the two RNase III domains form an internal dimer responsible for dsRNA cleavage (Macrae, et al. 2006). Furthermore, it was recently shown that the PAZ domain together with the RNase III domains act to measure the length of RNA for cleavage (MacRae, et al. 2007). The positional relationship of the PAZ domain with respect to the RNase III domains is an important factor in defining cleavage product length. Dicers therefore have evolved as specialized molecular rulers. Since a Dicer protein is considered to have been present in the last common ancestor to eukaryotes, over time, Dicers have undergone diversification and multiplication in various taxa (Margis, et al. 2006, Shabalina and Koonin 2008). For example, Arabidopsis has four Dicer genes while humans have only one. In Arabidopsis, the Dicer enzyme DCL1 processes dsRNA in the miRNA pathway into 21-nt, while the Dicer enzyme DCL3 processes dsRNA from genomic repeats into 24-nt siRNA designated for the TGS pathway. Characterization of the Dicer pathways in Arabidopsis was initially impaired by the ability of certain Dicers to act redundantly, and as a consequence double and triple mutants were needed to assign specificities to the different Dicers (Blevins, et al. 2006, Deleris, et al. 2006, Gasciolli, et al. 2005). In fungi, the ascomycete Neurospora crassa has two Dicers that act redundantly in Quelling (PTGS) (Catalanotto, et al. 2004). For meiotic silencing of unpaired DNA (MSUD), however, only the N. crassa DCL-1 Dicer is required, and deletion of the gene resulted in sterility (Alexander, et al. 2008). As previously 63  mentioned, S. pombe has a single Dicer enzyme required for both PTGS and TGS. Since Dicer null mutants often have serious negative phenotypes affecting development, it is becoming clear that most organisms use Dicer enzymes for endogenous small RNA pathways that impinge on basic processes. Examples now include Dicer generated miRNA and siRNA that control endogenous gene expression. So, in addition to protecting genomes against mobile and invading nucleic acids, Dicers have become an integral component of gene regulation of an organism. The Argonaute/PIWI family of proteins (Ago) determines which direction or pathway a small RNA will enter (Hutvagner and Simard 2008, Tolia and Joshua-Tor 2007). Like Dicer, the Ago proteins are considered to have been present in the last common ancestor to eukaryotes (Cerutti and Casas-Mollano 2006, Shabalina and Koonin 2008). Unlike Dicer, however, the Ago proteins have multiplied and diverged with most eukaryotes having multiple members. The nematode Caenorhabditis elegans for example has 27 Ago related genes. The many Ago related genes identified from diverse taxa can be divided into three groups (Vaucheret 2008). Group 1 binds miRNA and siRNA, while group 2 is represented by the Piwi proteins that bind piRNA from mobile elements in the germ-line of animals. Group 3 is somewhat diverged from the other two groups and binds secondary siRNA in C. elegans. Ago proteins are relatively large (90-100 kDa) and contain conserved domains toward their C-terminus but lack conservation in their variable N-terminus. The conserved domains consist of MID, PAZ and PIWI domains (Hutvagner and Simard 2008, Tolia and Joshua-Tor 2007). The MID domain binds 5′ phosphates of the small RNAs, while the PAZ domain interacts with the 3′ end. The Piwi domain adopts a folded structure like the bacterial RNase H enzyme and possesses endonuclease activity. Various studies have demonstrated “slicer” activity for a few Ago proteins in plants, fungi and animals. However, many Ago proteins do not appear to slice and have been shown to act by repressing translation. In animals, Ago proteins that bind miRNA 64  most often repress translation of homologous mRNA, and target the mRNA to P-bodies which are small RNA processing centers (Liu, et al. 2005, Pillai, et al. 2007). Initially it was thought that mRNA cleavage or translational repression was due to the degree of complementarity between the small RNA and the mRNA, since animal miRNA possessed mismatches with their target sites in mRNA and repressed translation, while plant miRNA often showed nearly complete homology to their targets and most often cleaved their targets. As more small RNA and their targets are studied, however, some exceptions to the rule are being noticed. Some recent work also suggests that the mode of action of Ago proteins may depend on interacting proteins and subcellular localization (Brodersen, et al. 2008). Since Ago proteins have multiplied and diverged in many organisms, further research is needed to define each and every role of a particular Ago protein. However, since many fungi, such as S. pombe, have only one Ago protein, they make perfect model organisms to study Ago function. Numerous studies have linked RNA silencing components to DNA repeat-induced gene silencing and heterochromatin formation (Grishok, et al. 2005, Henderson and Jacobsen 2007, Pal-Bhadra, et al. 2004). Currently, the nature of heterochromatin is under intense investigation. Since it was first described using cytological observation, heterochromatin was thought to be an inactive structural component of chromatin. Today, however, the nature of heterochromatin is much more defined, with many of its components and properties identified (Ebert, et al. 2006, Grewal and Jia 2007). In brief, 147 bp of DNA makes one complete wrap around histone proteins (H3, H4, H2A and H2B) to form the nucleosome. Interaction between DNA and histones is controlled by histone N-terminal tail modifications, including among others, acetylation, methylation, phosphorylation, and ubiquitination (Jenuwein and Allis 2001, Kouzarides 2007). Heterochromatin differs from euchromatin in that it often contains repetitive sequences, is mainly devoid of protein coding genes, does not participate in recombination during meiosis, and replicates late in S-phase of the cell cycle (Grewal and 65  Moazed 2003). Large heterochromatic regions occur at centromeres, telomeres and matingtype regions. It was initially thought that heterochromatic DNA was wrapped so tightly around histones that transcription was prevented. Several studies, however, demonstrated the need for low level transcription in order for proper formation of heterochromatin (Grewal and Elgin 2007, Motamedi, et al. 2004, Volpe, et al. 2002). This paradox was resolved recently by two groups working with fission yeast that demonstrated that during S-phase of the cell cycle both strands of DNA are transcribed and that the nascent sense strand is targeted by the RITS complex containing an siRNA-loaded Ago protein (Chen, et al. 2008, Kloc, et al. 2008). Such targeting also recruits Rik1 that loads Clr4, a chromodomain methyl transferase that performs methylation on histone H3 lysine 9 (H3K9me). Methylated histones at H3K9 then provide binding sites for the chromodomain proteins Chp2 and Swi6 which are required for heterochromatin formation and silencing throughout the rest of the cell cycle. This model nicely supports the observation that transcription is required for heterochromatin formation and predicts how heterochromatin is passed from parent to daughter cells during mitosis and how epigenetic information is inherited over generations. Deletion of the core RNA silencing genes (Dcr1, Ago1 and Rdp1) abolishes H3K9me and association of Swi6 resulting in the loss of silencing at centromeric repeats leading to chromosome segregation and cohesion defects (Hall, et al. 2003, Provost, et al. 2002, Volpe, et al. 2003). It is therefore apparent from the work on S. pombe that RNAi-directed heterochromatin formation is important for not only silencing of repetitive DNA, but also for proper packaging of DNA required for cell cycle progression and overall cell viability. I recently reported on the disparity of RNAi between the two smut fungi Ustilago hordei and Ustilago maydis (Laurie, et al. 2008). Using an RNAi expression vector, I targeted a reporter gene and an endogenous gene and was able to detect dsRNA in both species. However, knock-down of the reporter gene and siRNA production was seen only in U. hordei. 66  Through a collaborative project, a rough draft of the U. hordei genome is now available to our laboratory and from it I have found genes for Dicer, Ago and RdRP as well as other genes presumed to be involved in TGS. Here I report these findings and present a comparative genomics study showing loss of RNA silencing genes from the U. maydis genome that are important for both PTGS and TGS and point out features of the U. maydis genome that likely correlate with this loss. Additionally, since issues relating to the use of RNAi for genome protection and stability can easily be studied using smut fungi, I provide a rationale for the use of smut fungi to study genome evolution.  3.2 Materials and Methods 3.2.1 Amino acid and DNA sequences Amino acid sequences for the RNaseIII domain of Dicer, the PIWI domain of Ago, and the RdRP domain of RdRP proteins were described previously for other fungi (Nakayashiki, et al. 2006). Using these sequences Blast searches (tBlastx) were performed on the initial draft of the U. hordei genome compiled by the Munich Information Center for Protein Sequences (MIPS). Retrieved sequences were used to search NCBI for hits supporting their identity. Genes were predicted using FGENESH and domains predicted using the CDART program at NCBI. Phylogenies were done using Mega version 4 (Neighbor Joining with 1000 bootstrap replicates) with sequences described previously (Nakayashiki, et al. 2006), but with the addition of Laccaria bicolor dicers LB 1 (scaffold_4, 1619948-1616065) and LB 2 (scaffold_6, 1342360-1348160) from the DOE Joint Genome Institute (http://genome.jgipsf.org/Lacbi1/Lacbi1.home.html). U. maydis genome sequences were obtained from the databases at MIPS (http://mips.gsf.de/genre/proj/ustilago) and the Broad Institute of MIT and Harvard (http://www.broad.mit.edu/annotation/genome/ustilago_maydis).  67  3.3 Results 3.3.1 Phylogenetic relationship U. hordei and U. maydis are close relatives that have diverged relatively recently (Fig. 3.1). Separation has been estimated to have occurred between 21 and 27 million years ago (Bakkeren and Kronstad 2007). A phylogeny based on tubulin was constructed for selected fungi and shows the relationship between U. hordei and U. maydis amongst other basidiomycete fungi. When compared to the publicly available genomes, U. hordei and U. maydis are the most closely related fungi having differences in RNAi capability. Budding yeast and Candida species do not have RNAi, but to date, no close relatives possessing RNAi have been found.  3.3.2 Ustilago hordei genome sequencing A U. hordei BAC library of 2304 clones was constructed and fingerprinted as described previously (Bakkeren, et al. 2006). BAC end sequencing was performed yielding approximately 3 Mbp of paired BAC end reads, accounting for roughly 10-15 % of the U. hordei genome. This sequence was contributed to a joint effort to sequence the U. hordei genome. The other part of the collaboration involved high-throughput pyrosequencing (454 Life Sciences) of U. hordei haploid strain Uh4857-4 (alias Uh364, MAT-1)(Linning, et al. 2004) by our collaborators Drs. R. Kahmann and J. Schirawski at the Max Planck Institute for Terrestrial Microbiology (Marburg, Germany) and genome assembly was done by MIPS.  3.3.3 Blast search for RNAi components in the U. hordei genome Using the amino acid sequences for the RNaseIII domain of Dicer, the PIWI domain of Ago, and the RdRP domain of RdRP genes, Blast searches (tBlastx) were performed on the 68  initial draft of the U. hordei genome, revealing homologs for all three RNAi components. From these searches, one Dicer, one Ago and two RdRP genes were identified (Table 3.1). This finding is consistent with numbers found for other fungi, but represents a reduced number compared to other basidiomycetes (Table 3.2). The U. hordei gene for Dicer, UhDcl1, is predicted (CDART prediction software at NCBI) to contain two exons producing a protein of 1754 aa and contains predicted DEXDc and Helicc helicases, and two Riboc RNase III domains as found in Dicers from other organisms (Fig. 3.2a). Prediction software was unable to detect a dsRNA binding domain or a PAZ domain found in many other Dicers. In this regard, UhDcl1 has a domain architecture similar to Dcr1 from S. pombe. A Blast search of NCBI nr found UhDcl1 to be most similar to other fungal Dicers (Table 3.1). Phylogenetic analysis of RNase III domains from selected basidiomycetes shows that the UhDcl1 protein is most similar to the C. neoformans Dicers (Fig. 3.2b). It also appears that there are two families of Dicers and that one of the families has further multiplied in certain fungi. Lacaria bicolor has a single representative in each family. The C. neoformans Dicers likely evolved recently after divergence from the common ancestor to U. hordei. The U. hordei gene for Argonaute, UhAgo1, is predicted (CDART prediction software at NCBI) to contain a single exon producing a protein of 973 aa with predicted DUF, Paz_agolike and Piwi_ago-like domains (Fig. 3.3a). Of all the RNAi components identified, the Ago proteins are the most expanded. Blastp search of the NCBI database found UhAgo1 to be similar to other fungal Ago proteins, the greatest match being an Ago from C. neoformans (Table 3.1). A phylogenetic analysis of the Piwi domain of Ago proteins from selected basidiomycetes again shows UhAgo1 to be most similar to C. neoformans Ago proteins (Fig. 3.3b). Similar to the Dicers, C. neoformans Ago proteins likely evolved from a single gene  69  after divergence from U. hordei. Also, the occurrence of groups of related Ago genes from the same species indicates that gene multiplication is common for Ago genes within fungi. The two U. hordei RdRP genes both contain a single predicted (CDART prediction software at NCBI) RdRP domain (Fig. 3.4a). UhRdRP1 and UhRdRP2 are similar to other fungal RdRP genes (Table 3.1). From a phylogeny using selected basidiomycetes, the two U. hordei RdRP genes are more similar to each other than to related RdRP genes from other basidiomycetes and likely arose from a recent duplication event occurring after the progenitor to U. hordei diverged from the other species (Fig. 3.4b).  3.3.4 Synteny between the U. hordei RNAi component loci and the U. maydis genome Neighboring genes flanking the U. hordei RNAi components were used to Blast search the U. maydis genome. Orthologous proteins were found for all genes and their location and order was recorded. Interestingly, loci for all four RNAi components showed conservation of synteny and were mostly co-linear in U. maydis (Figs. 3.2c, 3.3c, and 3.4c). Homologous genes were more or less in the same order, but the RNAi components were completely lacking in U. maydis, with no apparent remnants or obvious footprints left behind. Slight differences at the RNAi loci included differential intron number and gene rearrangements resulting in genes from another location falling in the locus for one of the RdRP genes (Fig. 3.4c). An interesting observation is that the two genes flanking Dicer seem more diverged than their immediate neighboring genes (Table 3.3). Syntenic loci are listed in Table 3.3 including gene identification, E-value and annotation.  3.3.5 Search for genes lost from U. maydis Predicted proteins were compared using BlastP. Of the roughly 6800 proteins, 2725 have an E-value of zero (Table 3.4). Proteins having an E-value of less than e-20 represented 70  87 % of the proteins. This shows that these two fungi have a high degree of similarity and supports phylogenetic studies. To investigate the difference in gene complement between U. hordei and U. maydis, all U. hordei predicted proteins were compared to U. maydis proteins; proteins having an E-value smallar than e-5 were selected for analysis. This search revealed 693 putative genes without close-matching genes in U. maydis. These proteins were then used to search NCBI nr for hits against other organisms. This search revealed many genes with strong matches to fungi and other organisms. A summary of these results is presented in Table 3.5. In addition to confirming RNAi genes involved in PTGS (UhAgo1, UhRdRP1, and UhRdRP2), genes resembling ones demonstrated to be involved in TGS in other organisms were also revealed (Table 3.5). Interestingly, UhDcl1 was not identified in this screen, since it hits the U. maydis gene Um00911 with an E-value of e-6. Since Um00911 is orthologous to UH_01371 from U. hordei and lacks recognizable domains for Dicer, the Blast hit of e-6 most likely represents a false hit. With this in mind, the true number of genes present in U. hordei, but lacking from U. maydis is likely somewhat larger than 693 genes. Also, at the time of this analysis, only a rough assembly of the U. hordei genome was available. Of the genes that were identified in this screen, most notably were the chromodomain containing proteins potentially orthologous to S. pombe Chp2 and Swi6. Additionally, a DNA methyl transferase and other genes containing domains with roles in gene regulation were found. Like the other RNAi components analyzed, the chromodomain genes were at loci showing synteny with the genome of U. maydis (Fig. 3.5 and Table 3.3). Blast searches were also performed to look for other genes known to be involved in TGS including histone deacetylases (HDAC) and methyltransferases. This search identified a unique HDAC found only in U. hordei (Table 3.5). The gene matches other U. maydis HDAC genes at E-values lower than e-5, but all of which have orthologous genes elsewhere in the U. hordei genome. By comparing the locus of this gene to the U. maydis genome, synteny is conserved and the gene is lacking from U. maydis as 71  was seen for the other RNAi genes (data not shown). So, similar to UhDcl1, the HDAC gene UH_01301 has homology to other genes, but is lacking from the U. maydis genome. This HDAC gene also has no clear ortholog in other fungi, so probably represents a Ustilago specific HDAC. Since evidence for differential histone modification was revealed in genes recovered in these searches, a comparison of histones was done for both species (Fig. 3.6). Protein alignments showed that H2A, H3-1, H3-2, H4 and H2B.2 are identical (data not shown). However, histones cenH3, H1 and H2A F/Z show clear differences (Fig. 3.6). Since RNAi is known to play a role in proper centromere function in S. pombe, it is interesting that the Nterminal regions are variable between the cenH3 histones of the two smut fungi. Most notable are the change and loss of residues representing possible phosphorylation sites and the loss of a potential methylation site.  3.3.6 Intron comparison Organisms that have been sequenced and do not have RNAi genes generally show a reduction in repetitive DNA and number of introns (Cerutti and Casas-Mollano 2006). U. maydis was reported to be relatively devoid of repetitive DNA and to have a low frequency of introns (Kamper, et al. 2006). To see if loss of RNAi correlated with a reduced number of introns, the number of introns was compared between orthologous genes in U. maydis and U. hordei (Table 3.6). U. maydis has 3098 introns in 1990 predicted genes and 4795 genes (70.7% of total) without introns. U. hordei has 4068 predicted introns in 2357 predicted genes and 4482 (65.6% of total) genes without introns. By comparison there are 970 more introns in U. hordei. Since roughly 70% of the genes in both organisms do not have introns, it appears that a low number of introns is independent of the presence or absence of RNAi.  72  3.4 Discussion Studying mechanisms and pathways of RNA silencing is providing enormous insight into the function of eukaryotic cells. As important as the molecular and genetic studies, however, is the study of evolutionary history and diversity of RNA silencing components. My work shows that two related fungi have very different evolutionary histories with respect to RNA silencing genes. U. hordei has retained the common RNA silencing genes Dicer, Ago and RdRPs as well as genes similar to ones required for TGS in S. pombe (Buhler and Moazed 2007). These genes produce proteins with the common domains found in their well characterized relatives. The UhDcl1 protein has N-terminal helicase domains and C-terminal RNase III domains, but similar to S. pombe’s Dcr1, apparently lacks dsRNA-binding and PAZ domains. This observation may be the result of software prediction failure due to sequence divergence or to actual lack of the domains. Lack of the PAZ domain seems odd since the PAZ and RNase III domains have been shown to coordinate the measuring of dsRNA length for cleavage (Gan, et al. 2006, Zhang, et al. 2004). Alternatively, every step in RNA silencing pathways is likely governed by multiprotein complexes. Dicers that apparently lack PAZ domains could be assisted by other proteins at the cleavage step (MacRae, et al. 2007). Since only one Dicer gene was identified in U. hordei, it is likely that this gene participates in multiple pathways. I have already shown that dsRNA is cleaved into siRNA (Chapter 2) and therefore assume that this cleavage was due to the now identified UhDcl1 gene. The Dcr1 gene of S. pombe is responsible for both PTGS and TGS and has similar recognizable domains as UhDcl1. It would be interesting to determine if UhDcl1 also participates in TGS, especially at centromeres. In S. pombe, transcription of repetitive DNA at pericentromeric regions during Sphase of the cell cycle leads to heterochromatic silencing in an RNAi-dependent manner (Chen, et al. 2008, Kloc, et al. 2008). Chromodomain proteins Chp2 and Swi6 associate with this heterochromatin as well as the histone H3 variant cenH3. Interestingly, the centromers of U. 73  maydis are quite unique in that they are relatively short and possess very little repetitive DNA (Kamper, et al. 2006). The U. hordei and U. maydis H3 histone proteins are identical, but the cenH3 proteins have differences in their N-terminal region. These differences include variable phosphorylation and methylation sites. It is tantalizing to think that coincident with the loss of RNAi components that repetitive DNA was removed from pericentromeric regions resulting in differential evolution of cenH3 N-termini. It could also be that while U. maydis began to rely on an alternative means of regulating mobile DNA elements, it also evolved a means to form heterochromatin independent of RNAi. This mechanism may be reflected in the divergent evolution of the N-terminal regions of its cenH3 protein and not the H3 histones. It would be of interest, therefore, to see if CenH3 localizes to centromeres in U. maydis and in U. hordei, as it does in fission yeast (Mellone, et al. 2003, Takahashi, et al. 2000). Such a study would show that the unique centromeres of U. maydis either evolved a method of heterochromatin formation by different recruitment methods or use of different structural components. Since RNAi is involved in recruitment of machinery to loci destined for heterochromatin, and U. maydis has lost RNAi, it can be assumed that U. maydis uses a different method for recruitment of heterochromatin forming machinery. It is possible that the initiating signal may also be dsRNA. In budding yeast, Saccharomyces cerevisiae, that also does not have RNAi, readthrough transcription at neighboring convergent genes results in dsRNA formation that is normally prevented by the exosome (Camblong, et al. 2007). However, as cells age and exosomes begin to fail, cells detect dsRNA and recruit histone deacetylases. Histone deacetylation then recruits transcription repressors. If U. maydis uses this form of silencing, one might expect to see controlled expression of convergent transcripts at loci programmed to form heterochromatin. There is one recognizable dsRNA binding protein in the U. maydis genome but it resembles the mitochondrial precursor of the 60S ribosomal protein L3 from  74  budding yeast (Becht, et al. 2005). It therefore remains to be determined if dsRNA results in localized histone deacetylation as in budding yeast. U. hordei possesses one Argonaute gene, UhAgo1. The presence of its function in RNAi was shown experimentally (Chapter 2). An UhAgo1 null mutant is required to see if this gene is primarily responsible for the silencing observed. It should also be determined if UhAgo1 acts through mRNA cleavage, translational repression or both. As well, it remains to be determined if this gene plays a role in controlling endogenous gene expression and TGS. This could be alluded to by the phenotype of the null UhAgo1. If it plays a role in TGS at centromeric repeats like its S. pombe ortholog, one might expect to see chromosomal segregation defects in the null mutant (Hall, et al. 2003). It should also be determined if the UhAgo1 protein forms a RITS complex and if the two chromodomain proteins identified in this study are actively recruited by the RITS. Since the S. pombe RITS directs methylation of histone H3 one should determine the methylation status of H3 proteins, especially the CenH3 proteins in U. hordei and U. maydis. A bioinformatics search for U. hordei and U. maydis orthologs to the S. pombe histone methyl transferase Clr4 responsible for H3K9 methylation at centromeric repeats was inconclusive. Proteins containing SET domains were found, but none contained an N-terminal chromodomain like Clr4 (Horita, et al. 2001, Nakayama, et al. 2001). By experimentally checking the methylation status of the CenH3 proteins one could conclude if this form of regulation is different between these two smut fungi. As well, the different predicted phosphorylation sites should also be experimentally validated. U. hordei possesses two RdRP genes. Since RdRP genes are known to form dsRNA important for quelling and meiotic silencing in N. crassa, it remains to be determined if the two U. hordei genes play similar roles (Fulci and Macino 2007, Shiu, et al. 2001). Since my experimental study used a fold-back method of forming dsRNA, a RdRP was not required for the RNA silencing I observed. To see if either of the U. hordei RdRP genes is involved in 75  RNA silencing, one needs to either express a gene at a high level or place multiple copies of a gene in U. hordei (Catalanotto, et al. 2006). If silencing is observed, then an RdRP is likely involved. Null mutants of the two RdRP genes will show if either is responsible for RNA silencing individually or redundantly. The fact that there are two RdRP genes in U. hordei suggests that there may be two separate RNA silencing pathways involving dsRNA. From the phylogeny based on the RdRP domain, the two UhRdRP genes likely evolved relatively recently from a common ancestral gene after divergence from the other fungi used in the phylogeny. Perhaps one of the UhRdRP genes is involved in the PTGS pathway analogous to quelling and the other in TGS or meiotic silencing as in S. pombe and N. crassa. The use of mutants should help define these roles. The fact that no RdRP gene can be found in U. maydis raises many questions. Firstly, how does U. maydis regulate expression of highly expressed genes or genes that multiply yielding many copies in the genome? It has been reported that transgenes often produce truncated transcripts in U. maydis (Zarnack, et al. 2006). This may be due to the ability of U. maydis to regulate transcription based on G+C content. Without a high G+C content, expression of foreign genes will result in truncated transcripts and poor expression. Perhaps lack of RNA silencing allows U. maydis to multiply certain genes and still maintain expression. U. maydis possesses clusters of duplicated genes. It is possible that these evolved after the loss of RNAi since homologs are present in U. hordei, but not in multiples of more than two genes (see Chapter 4). As well, U. maydis also has very little repetitive DNA and therefore has evolved to rely on an alternative means to cleanse its genome. This mechanism may act during meiosis when homologous chromosomes pair, but does not involve an RdRP like meiotic silencing of unpaired DNA as in N. crassa (Shiu, et al. 2001). Instead it likely involves some form of homologous recombination leading to DNA removal possibly through active DNA breaks and homologous end joining (Holloman, et al. 2008). In other species, Ago mutants show a higher level of homologous recombination (Tsukioka, et al. 2006). 76  The loss of RNAi might help explain the high frequency of homologous recombination often observed in U. maydis, e.g. during creation of targeted gene deletions using homologous recombination. Experimental evidence is needed to track unpaired DNA through meiosis. It is possible that U. maydis uses some form of DNA elimination that may rely on RNA, but unlike in the ciliated protozoa (Nowacki, et al. 2008), does not involve RNAi components. An interesting find in this study is the complete elimination of RNA silencing genes from U. maydis. Conservation of synteny with U. hordei allowed the identification of loci in U. maydis that formerly housed the RNA silencing genes. I initially expected to see some form of remnant like a truncated, pseudogene and was surprised by the complete elimination of the RNAi genes. This observation suggests that U. maydis not only possesses a means to actively eliminate repetitive DNA, but also to cleanly remove unwanted or unnecessary genes. By performing a Blast comparison of proteins from the two smuts, 693 proteins were identified in U. hordei that either do not exist in U. maydis or have diverged beyond recognition. Comparative genomics looking for conservation of synteny will resolve the issue of which genes were eliminated from U. maydis. This list has many genes with homologs in other fungi and possessing conserved domains that can be used to suggest function. Interestingly, some of the genes resemble genes involved in gene regulation and TGS. The entire list requires further study and should form a basis for defining genetic and biochemical differences between these two smuts. A number of other species from diverse taxa have also lost RNAi (Cerutti and CasasMollano 2006, Nakayashiki, et al. 2006, Shabalina and Koonin 2008). Some general features of these organisms are that they are more or less unicellular and possess compact genomes. As well, most have very few introns in their genes. U. maydis fits all three of these criteria. The U. maydis genome has 970 fewer introns than U. hordei. These introns are distributed in roughly 35 % of the U. hordei genes. An interesting experiment would be to take a U. maydis gene that 77  has multiple introns and replace it with the same gene without introns and to similarly take a U. maydis gene without introns and replace it with the same gene engineered to contain an intron. Would regulation of either of these genes be affected? There are many questions regarding the consequences of losing RNAi which can be addressed by studying these two smut fungi. As previously mentioned, many other species have lost RNAi, but none have a close relative that has retained RNAi. This makes these two species unique and a powerful resource for studying the role of RNAi in both PTGS and TGS. The genomic differences identified in this report along with the list of different genes will provide the ground work for important studies that should allude to the roles and requirements of RNA silencing in eukaryotic evolution.  78  100 43  UM UH  UM05828.1 TBB2 UM05828.1 UH  CN  CN  60  PC CC 100 62 LB LB NC NC MG MG AN1 AN PC  CC1  87 100  SP  SP  35  SC CA SC  92 98  CA  AT  AT6  0.02  Figure 3.1 Phylogenetic relationship of selected fungal species based on β-tubulin protein sequences. The phylogenetic tree was drawn from aligned protein sequences using Mega version 4. The Neighbor Joining method was used to construct the tree and values represent 1000 bootstrap replicates. Ascomycete fungi are in blue while basidiomycetes are represented in red. Arabidopsis β-tubulin is in green and was used to root the tree. AT, Arabidopsis thaliana; AN, Aspergillus nidulans; MG, Magnaporthe oryzae (grisea); NC, Neurospora crassa; CA, Candida albicans; SC, Saccharomyces cerevisiae; SP, Schizosaccharomyces pombe; UH, Ustilago hordei; UM, Ustilago maydis; CN, Cryptococcus neoformans; CC, Coprinus cinereus; LB, Laccaria bicolor; PC, Phanerochaete chrysosporium.  79  A DEXDc  RIBOc HELICc  RIBOc  UH_08937 (UhDcl1) 1754 a.a. B  CN 2  100 97  CN 1 UH 1  33  PC 1 26  1  CC 1  75 100  LB 1 PC 2  100  CC 2  2  PC 3 CC 3  100 57  LB 2  At-DCL3  0.2  C  UhDcl1  1  2  3  4  5  6  Figure 3.2 Predicted U. hordei Dicer protein. (A) Domain features of the U. hordei Dicer protein as predicted by the CDART program at NCBI. (B) Phylogenetic relationship of UhDcl1 (UH 1) to other basidiomycete Dicer proteins using the conserved RNase III domain. Abbreviations as in Fig. 3.1. (C) Conservation of synteny between the UhDcl1 locus and a region of the U. maydis genome. UhDcl1 is presented in green, while the U. hordei flanking genes are colored blue. U. maydis genes are presented in red. Introns are shown as grey boxes within genes.  80  A  DUF  Piwi_ago-like PAZ_ago.  UH_06256 (UhAgo1) 973 a.a. PC 2  42 100  B  PC 3  98  PC 4  100  CC 5  1  PC 7 71  100  CC 4 UH 1 CN 2  54 100  CN 1 PC 1  83 45  CC 7  99  CC 6  2  CC 3 PC 5  99  99  PC 6 100  CC 1 100  0.1  CC 2  At Ago5  C  UhAgo1  1  2  3  4  5  6  Figure 3.3 Predicted U. hordei Argonaute protein. (A) Domain features of the UhAgo1 protein as predicted by the CDART program at NCBI. (B) Phylogenetic relationship of the UhAgo1 protein (UH 1) to other basidiomycete Ago proteins using the conserved Piwi domain. Abbreviations as in Fig. 3.1. (C) Conservation of synteny between the UhAgo1 locus and a region of the U. maydis genome. UhAgo1 is presented in brown, while the U. hordei flanking genes are coloured blue. U. maydis genes are presented in red. Introns are shown as grey boxes within genes.  81  Figure 3.4 Predicted U. hordei RdRP proteins. (A) Domain features of the two U. hordei RdRP proteins as predicted by the CDART program at NCBI. (B) Phylogenetic relationship of the U. hordei RdRP proteins (UH 1 and UH 2) to other basidiomycete RdRP proteins using the conserved RdRP domain. Abbreviations as in Fig. 3.1. (C) Conservation of synteny between the two RdRP gene loci and corresponding regions of the U. maydis genome. RdRP genes are presented in green, while the U. hordei flanking genes are coloured blue. U. maydis genes are presented in red. Introns are shown as grey boxes within genes.  A RdRP superfamily  I) UH_08874 (UhRdRP1) 1462 a.a. RdRP superfamily  II) UH_01631 (UhRdRP2) 1557 a.a.  B  CC 2  100 57  CC 1  89  PC 4  78  PC 6 PC 7  95  CC 7 95  PC 2  100 63  CC 3 UH 1  89 100  UH 2 CN 1  100  PC 8 PC 9  100  CC 6 PC 1  97  63  PC 3 PC 5  100  CC 4  99 95  CC 5  At 6  0.1  82  C  UhRdRP1  1  2  3  4  5  6  UhRdRP2  * 1  2  3  4  5  6  83  A UhChp1  1  2  3  4  5  6  5  6  B UhChp2  1  2  3  4  7  Figure 3.5 Predicted U. hordei Chromodomain proteins. (A) UhChp1, UH_05111, contains three exons and has flanking genes syntenic to U. maydis. (B) UhChp2, UH_07750, has one exon and is also in a region syntenic to U. maydis. Chromodomain genes are presented in green while the U. hordei flanking genes are coloured blue. U. maydis genes are presented in red. Introns are shown as grey boxes within genes.  84  A UH_08061 um10546  UH_08061 um10546  UH_08061 um10546  UH_08061 um10546  1 P P 50 (1) MASRPRPSLADSSYPES-TYAETYDEGSYASSSEGGGSAMLSRRAPSIQS (1) MASRARPSLADTSYPESTTYAETYEEASYASSSEGGGSAMLSRRAPSILS P M 100 (50) SLSRRRFEEYSVTEPSLSHIGQRKRFKPGTVALREIRKYQKSTDLLLRKL (51) GISRRRFEEYSMPEPSLSNVGQKKRFKPGTVALREIRKYQKSTDLLLRKL αN 101 150 (100) PFARLVREIANDFVTSYEHGEYGSGLRWQSSAILALQEATEAFLVHLFED (101) PFARLVREIANDFVTSYEHGEYGSGLRWQSSAILALQEATEAFLVHLFED α1 α2 151 185 (150) ANLCAIHGKRVTLMKKDFQLARRLRGRWDGLGGY(151) ANLCAIHGKRVTLMKKDFQLARRLRGRWDGLGGYα3  B UH_05357 um10447 UH_05357 um10447 UH_05357 um10447 UH_05357 um10447  1 50 (1) MSAAAAKPVAKKAASKKAAGTSVSYEAMIKEAILAHPADARAGIGRATIK (1) -MSAAAKPAAKKAATKKAAGSSVSYEAMIKEAILAHPAEARAGIGRATIK 51 100 (51) KYIQSHHPETAKGSEASFNTRVNQAITRGAEKKTFLLPKGPSGKVKLAPK (50) KYIQSHHPETAKGSEATFNTRVNQAITRGAQKKTFVLPKGPSGKVKLAPK 101 150 (101) AKVEKK---------APAAKKPAAKKVAAKKPAAS------KTAAKKPTA (100) AKVEKKPATTPITTTAAVAKKPAAKKVAAKKPAAAKKPAAKKTAAKKPAA 151 184 (136) KKVASSTATKAKKPAAKKAAKPAAKKTAAKKA-(150) KKVASSTTTKAKKPAAKKVAKPAAKKAAPAKKA-  C UH_00733 um00469 UH_00733 um00469 UH_00733 um00469 UH_00733 um00469  1 50 (1) MSDPRAKGGKG--GNAKVEPKKSVTQSSRAGLQSPHRLAEDGRQAPLIDI (1) MSDPRAKGGKGGAANAKNEPKKQTTQSARAGLQ----------------51 100 (49) VMKHLKPAVARPGPSMDASSREDAASNFPVGRIHRHLKNRTQNHVRIGAK (34) ---------------------------FPVGRIHRHLKSRTQNHVRIGAK 101 150 (99) AAVYTSAILEYLTAEVLELAGNASKDLRVKRITPRHLQLAIRGDEELDSL (57) AAVYTSAILEYLTAEVLELAGNASKDMRLKRITPRHLQLAIRGDEELDSM 151 182 (149) VRATIAGGGVLPHIHKTLIKAPSKKKAAGDA(107) VRATIAGGGVLPHIHKTLIKAPSKKKALE---  Figure 3.6 Comparison of orthologous histone proteins showing differences between U. hordei and U. maydis. (A) Alignment of putative CenH3 proteins from U. hordei and U. maydis using AlignX. Conserved helix domains are represented by red amino acid with grey background and are labelled αN, α1, α2 and α3. The variable N-termini are predicted to have differential phosphorylation (P) and methylation (M) sites. (B) Alignment of putative histone H1 proteins. (C) Alignment of putative histone H2A F/Z proteins.  85  Table 3.1 Location of RNA silencing genes in U. hordei and their top Blast hits Gene ID UhDcl1  UhAgo1  UhRdRP1  UhRdRP2  UhChp1 (Swi6/ Chp2)  MIPS ID & Coordinates UH_08937 - related to cell cycle control protein dicer Contig: uh_contig_2.00072 / Exons: 112426108212,108018107552,107458106876 (C) UH_06256 - conserved hypothetical protein Contig: uh_contig_2.00050 / Exons: 212545-209624 (C) UH_08874 hypothetical protein Contig: uh_contig_2.00071 / Exons: 133588-137976 (W) UH_01631 - related to RNA-directed RNA polymerase Contig: uh_contig_2.00012 / Exons: 277223-281896 (W) UH_05116 hypothetical protein Contig: uh_contig_2.00040 / Exons: 204862204686,204557204156,204044203262 (C)  tBlastn NCBI  Score (Bits) 245  E Value 1e-61  ref|NM_001051683.1| Oryza sativa (japonica cultivar-group) Os…  228  3e-56  gb|AY352639.1| Brugia malayi dicer-like mRNA, complete sequence  219  1e-53  ref|XM_567302.1| Cryptococcus neoformans var. neoformans JEC2…  470  1e-129  ref|XM_567314.1| Cryptococcus neoformans var. neoformans JEC2…  437  1e-119  dbj|AK250484.1| Hordeum vulgare subsp. vulgare cDNA clone: FL... ref|XM_001828822.1| Coprinopsis cinerea okayama7#130 hypothet...  347  1e-92  386  5e-104  ref|XM_381758.1| Gibberella zeae PH-1 hypothetical protein pa…  355  7e-95  ref|XM_001217665.1| Aspergillus terreus NIH2624 predicted pro… ref|XM_001828822.1| Coprinopsis cinerea okayama7#130 hypothet…  354  2e-94  349  7e-93  ref|XM_001826673.1| Aspergillus oryzae RIB40 hypothetical pro...  339  7e-90  ref|XM_655229.1| Aspergillus nidulans FGSC A4 hypothetical pr... dbj|AK107686.1| Oryza sativa Japonica Group cDNA clone:002-13…  322  8e-85  272  2e-70  ref|NM_001017150.2| Xenopus tropicalis chromobox homolog 1 (D..  54.3  1e-04  gb|M14131.1|DROC1A9 Drosophila melanogaster nonhistone chromosomal pro…  53.1  3e-04  ref|NM_001127228.1| Homo sapiens chromobox homolog 1 (HP1 bet…  52.8  3e-04  ref|XM_001798780.1| Phaeosphaeria nodorum SN15 hypothetical ...  86  Table 3.1 continued Gene ID  MIPS ID & Coordinates UH_07750 - conserved hypothetical protein Contig: uh_contig_2.00061 / Exons: 140340-141446 (W)  UhChp2 (Swi6/ Chp2)  tBlastn NCBI  Score (Bits) 75.9  E Value 2e-11  ref|XM_001831666.1| Coprinopsis cinerea okayama7#130 hypothet…  70.9  8e-10  ref|NM_001023829.1| Schizosaccharomyces pombe chromodomain pr…  64.3  7e-08  ref|XM_001883761.1| Laccaria bicolor S238N-H82 hypothetical p…  Table 3.2 Number of RNAi genes in selected fungal species Ascomycetes  Basidiomycetes  NC  CA  SC  SP  PC  CC  LB  CN  MGb  UH  UM  Ago  2  1  0  1  7  8  6*  2  0  1  0  Dicer  2  0  0  1  3  3  2  2  0  1  0  RdRP  3  0  0  1  9  7  6*  1  0  2  0  *Database: Laccaria bicolor Best Models (proteins) JGI Abbreviations as in Fig. 3.1 except MGb, Malassezia globosa Table adapted from Nakayashiki et al. 2006  87  Table 3.3 Syntenic loci horboring RNA silencing and chromodomain genes Locus Dicer  Ago  RdRP1  Flanking genes 1  U. hordei U. maydis gene ID gene ID UH_08933 um06394  E-value  annotation  2.8e-108  2 3 UhDcl1 4  UH_08935 UH_08936 UH_08937 UH_08940  um06395 um06396 No hit um06397  2.0e-162 3.5e-57 --9.6e-67  5  UH_08941 um06398  6.0e-200  6  UH_08942 um06399  1.6e-135  1  UH_06253 um04175  E=0  2 3  UH_06254 um04176 UH_06255 um04177  3.2e-277 E=0  UhAgo1 4  UH_06256 No hit UH_06258 um10893  --8.0e-150  5  UH_06259 um04179  3.2e-98  6 1  UH_06260 um04180 UH_01626 um01076  3.9e-134 3.8e-159  2 3  UH_01628 um01077 UH_01630 Um11277  1.9e-164 2.0e-89  UhRdRP1 4  UH_01631 No hit UH_01632 um01079  --E=0  5  UH_01633 um01080  3.6e-241  6  UH_01634 um01081  3.7e-129  Related to 50S ribosomal protein L19 Related to NOT3 Putative protein Dicer Conserved hypothetical protein Conserved hypothetical protein Conserved hypothetical protein Probable GCV2 – glycine decarboxylase P subunit Probable FRS1 Conserved hypothetical protein Ago Conserved hypothetical protein Probable multicatalytic endopeptidase complex chain PRE1 Related to MTW1 Conserved hypothetical protein Putative protein Conserved hypothetical protein RdRP Conserved hypothetical protein GatA 4-aminobutyrate aminotransferase Related to AOS1 – Smt3p activating protein  88  Table 3.3 continued Locus RdRP2  Chp1  Chp2  Flanking genes 1  U. hordei U. maydis gene ID gene ID UH_08870 um06256  E-value  annotation  2.3e-285  2  UH_08871 um06257  E=0  3  UH_08873 um06258  E=0  UhRdRP2 * 4*  UH_08874 No hit UH_08875 No hit UH_05573 um06259  ----1.2e-144  5*  UH_05572 um06260  9.5e-289  6  UH_08876 um06261  8.2e-212  1 2  UH_05111 um03282 UH_05113 um03283  2.4e-126 6.3e-63  3  UH_05114 um03284  8.4e-52  UhChp1 4  UH_05116 No hit UH_05117 um03285  --8.2e-226  5  UH_05119 um03286  E=0  6  UH_05120 um10830  2.8e-225  1  UH_07745 um04852  3.3e-176  2 3  UH_07746 um10850 UH_07747 um10851  5.3e-39 1.4e-96  UhChp2 4 5 6  UH_07750 UH_07751 No hit UH_07752  --E=0 --5.1e-61  7  UH_07754 um04856  Conserved hypothetical protein Conserved hypothetical protein Conserved hypothetical protein RdRP Hypothetical protein Probable U-snRNPassociated cyclophilin Conserved hypothetical protein Conserved hypothetical protein Related to MAK32 protein Conserved hypothetical protein Conserved hypothetical protein Chromodomain protein Conserved hypothetical protein Related to MYO1 – myosin-1 isoform Conserved hypothetical protein Conserved hypothetical protein Putative protein Conserved hypothetical protein Chromodomain protein Related to MET30 Putative protein Probable 60S ribosomal protein L26 Conserved hypothetical protein  No hit um10852 um10853 um04855  2.8e-122  89  Table 3.4 Comparison of predicted proteins of U. hordei to U. maydis  Proteins 2725 1562 1635 214 693 BlastP results  E-value 0 -100 to -180 -20 to -99 -6 to -19 No hit  90  Table 3.5 Genes found in U. hordei but not in U. maydis  U. hordei Gene ID UH_08509  Blastp hit  E-value  annotation  Laccaria bicolor gb|EDR14876.1| Aspergillus niger ref|XP_001401620.1| Malassezia globosa ref|XP_001730359.1| Cryptococcus neoformans ref|XP_777758.1| Cryptococcus neoformans ref|XP_776330.1| Candida albicans ref|XP_714037.1| Malassezia globosa ref|XP_001732161.1| Gibberella zeae ref|XP_386545.1| Phaeosphaeria nodorum gb|EAT80904.1| Saccharomyces cerevisiae ref|NP_009310.1|  3e-51  C5-DNA-methyltransferase  5e-28  4e-42  SAM-dependent methyltransferase WD40, WD40 domain protein YDG/SRA domain protein  9e-30  DUF567 domain protein  4e-07  3e-68  putative nuclear envelope fusion chaperone component Transcription elongation factor Elf1 like Jacalin-like lectin domain  2e-27  ankyrin-like protein  2e-103  UH_05250*  Saccharomyces cerevisiae ref|NP_009308.2|  1e-60  UH_01301  Ustilago maydis um10914(orthologous to UH_07428, 6.5e-267)  1e-44  Reverse transcriptase required for splicing of the COX1 pre-mRNA Endonuclease I-SceIII, encoded by a mobile group I intron within the mitochondrial COX1 gene related to HOS3 Trichostatin A-insensitive homodimeric histone deacetylase (HDAC)  UH_05253 UH_06113 UH_08116 UH_00036 UH_05697 UH_01464 UH_02609 UH_03224 UH_10010*  3e-71  9e-27  91  Table 3.5 continued U. hordei Gene ID Enzymes UH_03771  Blastp hit  E-value  annotation  Cryptococcus neoformans ref|XP_568133.1| Aspergillus nidulans gb|EAA63948.1| Laccaria bicolour gb|EDR10808.1| Aspergillus clavatus ref|XP_001276385.1|  0.0  CAT1 catalase  3e-23  Putative tannase precursor  4e-20  UH_01768  Laccaria bicolour gb|EDR11338.1|  9e-27  UH_04077  Coprinopsis cinerea gb|EAU92986.1| Phanerochaete chrysosporium gb|AAG24510.1| Aspergillus niger gb|ABB59678.1| Phaeosphaeria nodorum gb|EAT92053.1| Cryptococcus neoformans ref|XP_570407.1|  2e-67 6e-10  polysaccharide lyase family 14 protein aldo/keto reductase / aflatoxin B1-aldehyde reductase NAD-dependent epimerase/dehydratase family galactan 1,3-betagalactosidase alpha-galactosidase  2e-63  extracellular invertase  2e-94  Putative arabinanase  1e-26  Putative N,Ndimethylglycine oxidase  UH_00862 UH_00435 UH_01106  UH_06273 UH_07406 UH_07793 UH_09006  2e-33  92  Table 3.6 Comparison of intron number Genome comparison U. hordei 6829 Genes 4068 Introns 2357 (34.5%) Genes with introns 4472 Genes without introns  U. maydis 6785 3098 1990 (29.3%) 4795  93  3.5 References Alexander WG, Raju NB, Xiao H, Hammond TM, Perdue TD, Metzenberg RL, Pukkila PJ, Shiu PK (2008) DCL-1 colocalizes with other components of the MSUD machinery and is required for silencing. 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Pathogens then attempt to manipulate their hosts by secreting effecter molecules that block defenses and aid in the acquisition of nutrients necessary for growth and reproduction (Grant, et al. 2006, Kamoun 2007, van der Does and Rep 2007). A plant’s defense system involves multiple layers (Jones and Dangl 2006). Innate or preformed barriers include physical obstacles such as the cuticle and cell wall, as well as chemical deterrents. By artificially disrupting these layers in the laboratory, penetration of otherwise non-host microbes is possible (Lipka, et al. 2005, Stein, et al. 2006). If a potential microbial pathogen is able to pass or withstand these barriers it must then hide itself from the plant’s basal surveillance system. This system is capable of recognizing microbe-associated-molecular-patterns (MAMPs) or what are more frequently referred to as pathogen-associated-molecular-patterns (PAMPs), and recognition is achieved through receptor-like kinases that recognize conserved often indispensable proteins, or motifs, of the invading microbe (Nurnberger and Brunner 2002, Zipfel and Felix 2005). Examples from bacteria include the flg22-epitope of flagellin that is recognized by FLS2 in Arabidopsis, and EF-Tu that is recognized by ERF receptor-like kinases only in the brassicaceae family 1  A version of this chapter will be submitted for publication: Laurie, J., Ali, S., Linning, R. and Bakkeren, G. A cluster of predicted secreted proteins from the basidiomycete barley smut fungus, Ustilago hordei, contains avirulence gene Uhv1.  102  (Kunze, et al. 2004, Nurnberger, et al. 2004). Arabidopsis has >600 receptor-like kinases that play numerous important functions in plant life (Zipfel, et al. 2004). Although many of these genes have roles in processes other than defense, some could be responsible for recognizing other PAMPs, like chitin oligosaccharides from fungi, which are also potent elicitors of plant defense (Akimoto-Tomiyama, et al. 2003, Kaku, et al. 2006, Shabalina and Koonin 2008). Once past a plant’s basal defense system, a microbe is then capable of growth and multiplication that ultimately leads to disease, unless the microbe is detected by a plant’s resistance genes or R-genes (Martin, et al. 2003). R-genes represent a plant’s last line of defense. Pathogen effectors recognized by R-genes activate a strong and effective defense response similar to recognition of PAMPs (Jones and Dangl 2006). This includes production of reactive oxygen species, the induction of many pathogen-related (PR) defense genes and ultimately activation of the hypersensitive response (HR) which is a form of localized programmed cell death (Greenberg and Yao 2004, Mur, et al. 2008). Resistance at this level occurs through specific R-genes recognizing specific pathogen effectors which have been termed avirulence or Avr genes because they trigger resistance and render the pathogen avirulent. This is represented genetically by the classical gene-for-gene relationship (Flor 1971). R-genes have been divided into several classes including kinases and receptor kinases, however, most of the R-genes identified fall into the nucleotide binding site leucine-rich repeat (NB-LRR) class (Martin, et al. 2003). Arabidopsis has roughly 150 genes of this class. Recognition of microbial effectors by R-genes occurs in different cellular locations depending on the location of the R-gene. Some R-genes are membrane spanning and recognize effectors in the apoplasm or at the cell surface, while others are intracellular and therefore recognize effectors that make it into plant cells. Recognition of effectors by R-genes places strong selective pressure on effectors for mutations that avoid R-gene detection yet maintain or enhance virulence (Allen, et al. 2004). Similarly, evolution of plant R-genes is essential for 103  plant species survival. An arms race therefore exists between a host plant and its microbial pathogen (Dawkins and Krebs 1979). Plant pathogens use effectors to increase virulence (Sacristan and Garcia 2008). Although the mode of action of these effectors remains largely unknown, significant progress has been made in the last decade. Most of our current knowledge comes from bacterial effectors which are secreted through the type-three secretion system (TTSS) (Alfano and Collmer 2004, Grant, et al. 2006). About 15 years ago it was observed that TTSS-deficient bacteria activate plant basal defenses, while TTSS-competent wild-type bacteria prevent such activation (Brown, et al. 1995, Jakobek, et al. 1993). From these studies it was proposed that plant pathogenic bacteria secrete effector proteins capable of suppressing plant basal defenses. This idea was later confirmed when the AvrPto effector from Pseudomonas syringae pv. tomato was shown to suppress basal defenses acting at the plant cell wall (Hauck, et al. 2003). Since that time extensive efforts have helped define a number of TTSS effectors including recent examples of effectors that block host proteasome function and interfere with endogenous small RNA pathways (Groll, et al. 2008, Navarro, et al. 2008). In addition to the bacterial studies, light is being shed on fungal and oomycete effectors as well (Tyler 2008). In the oomycete Phytophthora infestans that causes late blight on potato, many effectors contain an RXLR motif resembling a motif used by malaria parasites to translocate proteins into their host cells (Rehmany, et al. 2005). Studies show that, as in the malaria parasites, the oomycete effectors similarly rely on their RXLR domains to enter host plant cells (Bhattacharjee, et al. 2006). The ToxA host-selective toxin from the ascomycete fungus Pyrenophora tritici-repentis, the causal agent of tan spot of wheat, possesses an RGDcontaining loop that is responsible for internalization of the ToxA protein into wheat mesophyll cells (Manning, et al. 2008). Additionally, effectors from the flax rust fungus, Melampsora lini, have been shown to act within the cytoplasm of their host cells and interact directly with R104  genes predicted to be cytoplasmic (Dodds, et al. 2004). Through direct interaction, diversifying selection has resulted in highly variable flax rust Avr567 genes that have different recognition specificities by corresponding L5, L6 and L7 R-genes (Dodds, et al. 2006). Prior to the recent findings from fungal and oomycete effectors, only a few examples demonstrated direct interaction of pathogen effector molecules with their corresponding Rgenes, fitting a receptor-ligand model. These include the AvrPto of P. syringae interacting with Pto in tomato, AvrPita of the rice blast fungus interacting with Pita in rice and the bacterial wilt PopP2 interacting with RRS1-R in Arabidopsis (Deslandes, et al. 2003, Jia, et al. 2000, Pedley and Martin 2003). Other effectors could not be shown to interact directly, but instead involved a third partner. To help explain these observations the “guard” hypothesis was proposed (Innes 2004, Van der Biezen and Jones 1998). Classical examples supporting the guard hypothesis include Arabidopsis RPS2 detecting AvrRpt2 degradation of the Arabidopsis protein RIN4, and Arabidopsis RPS5 detecting AvrPphB cleavage of the Arabidopsis protein PBS1. With intense investigation of Avr and R-genes in many different systems, examples of both direct and indirect interactions are coming to light, including examples from fungi (Ellis, et al. 2007). Our laboratory studies smut fungi which are obligate biotrophic plant pathogens that form a separate order, the Ustilaginales, within the basidiomycetes. Ustilago hordei and Ustilago maydis are closely related members of this group that infect barley and maize, respectively. Both fungi are dimorphic, having a haploid yeast-like cell stage and a dikaryotic filamentous form that is responsible for plant infection. U. maydis has been used in molecular biology as a model for several decades and was instrumental for studies on recombination and mating within fungi (Martinez-Espinoza, et al. 2002). Both U. hordei and U. maydis are capable of causing severe damage to their respective crop hosts and therefore are of interest to plant pathologists and breeders. Plant cultivar and fungal isolate screening has lead to identification of resistant cultivars of barley that recognize isolates of U. hordei on a gene-for105  gene basis (Tapke 1937). In contrast, no gene-for-gene resistance has been observed between U. maydis and maize in spite of the enormous diversity of maize (Buckler, et al. 2006). Since these two fungi are so closely related and can be manipulated using the same laboratory techniques, they provide a unique opportunity for making genomic and pathogenicity comparisons. With this in mind and with powerful tools and resources including a genomic DNA sequence for both organisms, I report cloning efforts that locate the UhAvr1 gene from U. hordei to a cluster of secreted proteins syntenic to cluster 19A, the largest cluster of secreted proteins in U. maydis (Kamper, et al. 2006).  4.2 Materials and Methods 4.2.1 Plant varieties and fungal strains used for mapping UhAvr1 Barley cultivars Hannchen (Ruh1), Plush (Ruh6) and Odessa (universally susceptible) were used for pathogenicity tests. U. hordei haploid strains Uh4857-4 (alias Uh364, MAT-1 V1 V6), Uh 4857-5 (alias Uh365, MAT-2 V1 V6), Uh4854-10 (alias Uh362, MAT-2 v1 v6), Uh4854-4 (alias Uh359, MAT-1 v1 v6) and their progeny from a mapping population were described previously (Linning, et al. 2004). In brief, Uh364 was crossed with Uh362 and progeny were collected to form the mapping population. Fifty-four random progeny were backcrossed to virulent strains (Uh362 or Uh359) and tested on differential barley cultivars recognizing the different Avr genes; this created the mapping population for measuring recombination frequencies. Bulked-segregant analysis was performed on pools of eight progeny segregating for virulence and avirulence. For UhAvr1, analysis using AFLP and RAPD methods identified three linked markers that spanned five tiled BAC clones and covered a distance of roughly 170 kb. BAC subcloning and RFLP analysis delimited the window spanning UhAvr1 to 80 kb (Fig. 4.1).  106  4.2.2 Chlorazol Black E staining of U. hordei hyphae Avirulent strains of U. hordei containing UhAvr1 are blocked at initial penetration on barley cultivars containing Ruh1 (Hu, et al. 2003). Infection with virulent strains on susceptible cultivars of barley is difficult to detect before flowering. In an attempt to score disease early during the infection cycle, I chose to stain hyphae within plant tissues using Chlorazol Black E (Brundrett, et al. 1984). Stem segments (2-3 cm in length) containing the shoot apical meristem, were collected from barley seedlings three weeks after exposure to compatible or incompatible U. hordei strains. Stem segments were stored for four days in 70% ethanol before autoclaving at 120°C for twenty minutes in potassium hydroxide (5% w/v). After autoclaving, the stems were rinsed in distilled water and then stained for 16 hours at 60°C in staining mix containing Chlorazol Black E (0.1%), lactic acid (85%) and glycerol (1:1:1). Following staining the stems were rinsed several times in 50% glycerol before being sliced longitudinally and observed using a stereomicroscope.  4.2.3 Comparison of U. hordei sequences from the UhAvr1 locus to the U. maydis genome DNA sequences generated from BACs and subclones were compared to DNA sequences from both the MIPS Ustilago maydis database (MUMDB; http://mips.gsf.de/genre/proj/ustilago/) and the Ustilago maydis Sequencing Project, Broad Institute of MIT and Harvard (http://www.broad.mit.edu).  4.2.4 BAC 3-A2 sequencing and analysis BAC 3-A2 from a library of the Uh364 parent was sequenced using the GPSMutagenesis System (New England Biolabs) with a few modifications (Fig. 4.2). In the donor vector, the kanamycin resistance cassette within the transprimer was replaced with a phleomycin resistance cassette driven by both the Em7 bacterial promoter and the U. maydis 107  glyceraldehydes-3-phosphate dehydrogenase (GAP) promoter and terminator (Kinal, et al. 1991) so that gene constructs could be used to generate knockouts within U. hordei through homologous recombination. Some optimization was necessary to establish the correct amount of BAC and donor vector required for efficient in vitro transprimer transfer and E. coli (Electromax DH10B/r, Invitrogen) transformation. Random bacterial colonies were picked and analyzed to determine if BACs contained single transprimers inserted randomly into BACs in a 1:1 ratio. After this was confirmed, individual colonies were picked to start cultures in six 96well plates. Cultures were grown in LB freezing medium prior to freezing and shipping to a sequencing service provider (Genome Sciences Centre, Vancouver). Sequencing was done using primers N and S as described in the GPS-Mutagenesis System protocol, yielding paired sequence reads from the ends outwards of the randomly inserted transprimers. All DNA sequences generated were entered in the PCAP.REP genome assembly program (Huang, et al. 2006). To place certain sequences and to verify their location, physical mapping was performed by using the unique Not1 restriction enzyme sites in the transprimer and BAC insert and measuring generated fragment sizes on CHEF gels (data not shown). DNA contigs from the PCAP.REP were further analyzed by FGENESH (Salamov and Solovyev 2000) and VectorNTI (Invitrogen) to predict genes. Predicted genes were searched for secretion signals using the SignalP 3.0 Server (http://www.cbs.dtu.dk/services/SignalP/).  4.2.5 Virulent parent BAC library screening One end of BAC 3-A2 sits in a gene related to DigA and is orthologous to Um05292. The other end sits in a gene related to VPS10 and is orthologous to Um05324 (Fig. 4.1b). Primers (CTCGAACCCGGCAACAGA-forward and CCAGCGATGAGTGCATC-reverse) were designed from the end sequence of BAC 3-A2 sitting in the DigA-like gene. The PCR product (206 bp using BAC 3-A2 as a template) was radiolabeled with [32P]dCTP 108  (PerkinElmer) using the Rediprime II Random Prime Labelling System (Amersham Biosciences/GE Healthcare) and used to probe a BAC library from the virulent parent Uh362 spotted on Hybond-N+ nylon membrane (Amersham Biosciences/GE Healthcare). Similarly, a probe was made from the end sequence of subclone 2-1 that sits in a gene homologous to Um05311 (primers; AGTGGCTTGATGAAACATAG-forward and GCATTCGGCCTGATACCAAC-reverse, 358 bp product) and used to search the same BAC library. Hybridized membranes were exposed to screens and positive colonies were detected using the Cyclone Plus Storage Phosphor System (PerkinElmer).  4.2.6 Gene ID and comparisons Primers were designed for all genes called by FGENESH and VectorNTI in the BAC 3A2 sequence and were predicted to be secreted. All corresponding genes were amplified by PCR from the BACs recovered from the virulent parent that corresponded to the UhAvr1 locus (Table 4.1). Primers were placed 50-100 bp upstream of predicted start codons and 50-100 bp downstream of stop codons to ensure sequencing of entire open reading frames (ORFs, Table 4.2). Sequencing was done at PARC (Summerland, BC, Canada) and resulting alleles were compared using alignment software in the VectorNTI software package (Invitrogen).  4.2.7 Knockout of gene UH_ 08134 (#8), ortholog of Um05312 A double-jointed PCR method (Yu, et al. 2004) was selected to make a replacement construct capable of removing UH_08134 from the avirulent U. hordei strain Uh364. A PCR product was generated from the promoter region of UH_08134 with one primer having overlapping sequence to a hygromycin resistance cassette (Table 4.3, primers 1 and 2). Another PCR product was generated from the sequence downstream of the stop codon of UH_08134 with one primer overlapping the other end of the hygromycin resistance cassette 109  (Table 4.3, primers 3 and 4). PCR-amplified hygromycin resistance cassette (Table 4.3, primers 5 and 6) was mixed first with the upstream flank and then with the downstream flank and all three fragments were fused through successive rounds of PCR. Nested primers were then needed to amplify the final construct to generate enough DNA for fungal transformation (Table 4.3, primers 7 and 8). The resulting linear construct was confirmed using restriction digest analysis and used to transform Uh364 as described previously (Bakkeren and Kronstad 1993), but with 384 mg/ml Vinoflow FCE (Gusmer Enterprises) used as enzymes to digest the fungal cell wall (Szewczyk, et al. 2006). DNA was extracted from 36 transformants having resistance to hygromycin and analyzed to find a homologous knockout of UH_08134 by PCR screening. One candidate was identified, Uh364Δ8-27, and its DNA was then analyzed by Southern Blotting. In brief, Hind III and Bam H1 digests were done each separately on 5 μg of DNA from Uh364Δ8-27 and four non-homologous transformants. DNA was separated by agarose-gel electrophoresis and capillary-blotted onto Hybond N+ nylon membrane (GE Healthcare). A probe was made using PCR (ACTCCTGTGACCATTCGAGG-forward and GTGCATGGTGTTGGAGGTC-reverse) for a 649 bp region just downstream of the gene replacement and used to probe the blotted DNA. For the wild type locus a restriction fragment size of 2212 bp was expected, while elimination of this fragment and appearance of a 4112 bp fragment presented a homologous knockout. Radioactivity was detected as for the BAC library screening.  4.2.8 Pathogenicity tests The knockout line Uh364Δ8-27 (MAT-1 ΔUH_08134 putative ΔV1) was mated with a virulent line Uh362 (MAT-2 v1) by mixing haploid cultures just prior to inoculation of barley seeds. Additionally, the knockout line was mated with an avirulent line Uh365 (MAT-2 V1) to complement the deleted UH_08134 gene. Both crosses were used to inoculate barley cultivars 110  Hannchen (Ruh1) and Odessa (ruh1) by vacuum infiltrating seeds for 15 minutes with mixed haploid cultures using standard laboratory vacuum. Seeds were then planted in general potting mix (Pro-Mix BX) in flats to a density of roughly 50 seeds per flat and having small holes for water drainage. At least two flats were planted for each treatment. For controls, each haploid strain tested was used to inoculate Odessa by inoculating with only the haploid cultures. This was done to ensure that pure cultures were used and to check for seed contamination. Flats were placed in controlled-environment chambers (Conviron) with a 16 hour photoperiod and held at 22°C. Plants were scored at flowering for smut symptoms.  4.3 Results Chlorazol Black E staining of hyphae within plant tissues was done with the goal of scoring disease early during the infection cycle. Staining worked well to help visualize U. hordei hyphae in barley tissues (Fig. 4.3). Even though the method was very harsh, enough cell wall material remained to distinguish plant tissues. U. hordei hyphae were seen as a network of mycelia beneath the shoot apical meristem in stems only from compatible interactions. This technique is somewhat labor intensive, but could still prove useful for rapid analysis of mutant U. hordei strains. A summary of the results delimiting the UhAvr1 locus is presented in Figure 4.1a. Gene X (#17) possesses large variable repeats that were used to identify three recombinants in the mapping population (Fig. 4.4 and Table 4.3, primers 13 and 14). This gene is located near the one end of subclone 5-12 and was initially considered a candidate for UhAvr1 because of the high degree of variability between alleles. However, when the variable repeat region was checked among progeny of the mapping population recombinants were found. Moreover, an RNAi knock-down mutant had an interesting phenotype in which haploid spores failed to separate after budding, but pathogenicity was unaffected (on ruh1) and virulence (on Ruh1) 111  was intact (data not shown). The other marker (AFLP) defining the UhAvr1 locus maps to subclones 1-1 and 1-6 and identified one recombinant. Between these markers are three RFLP markers having no recombinants in our population of 54 progeny, thus defining the UhAvr1 locus. Sequencing done at PARC previously produced large stretches of DNA sequence data in the vicinity of gene X and at the ends of several subclones. This sequence was used to search for homologous sequences in the U. maydis genome. Interestingly, all the genes identified at the UhAvr1 locus were syntenic to a region in U. maydis on contig 1.191 (the Ustilago maydis Sequencing Project, Broad Institute of MIT and Harvard) as depicted in Figure 1b. Furthermore, the locus seemed to span cluster 19A, the largest cluster of genes encoding small secreted proteins in U. maydis. This information prompted us to obtain DNA sequence for the entire region spanning UhAvr1 in U. hordei. Since BAC 3-A2 from the Uh364 library spanned the entire syntenic region, it was chosen for sequencing (Fig. 4.1b). PCAP.REP analysis of paired sequence reads from BAC 3-A2 resulted in formation of 17 contigs ranging in size from 749 to 42,679 bp. From these, three contigs were found that in tandem spanned the defined UhAvr1 locus (Fig 4.5a). Two short gaps remained that were either not sequenced or not bridged by the analysis software due to repetitive sequences. An interesting feature of the UhAvr1 locus is the abundance of repetitive DNA, seemingly similar to those observed in the mating-type region (Bakkeren, et al. 2006). Large stretches of repetitive DNA surrounded most of the genes within the cluster at the UhAvr1 locus, making primer design and placement problematic. PCR across the gaps from flanking regions or from flanking genes, however, indicated that the gaps were no more than 610 bp each (Fig. 4.5b). For gap one, the forward primer was 1478 bp from the end of Contig 0.1rc while the reverse primer was 1156 bp from the end of Contig 3.1, combining for a total of 2634 bp. PCR using these primers yielded a band of approximately 3000 bp and indicated that the remaining gap size was roughly 366 bp. Primers for gap two were both roughly 45 bp from the ends of 112  contigs 2.1 and 3.1. A PCR product of approximately 700 bp minus the 90 bp of flanking sequences indicated that gap two was roughly 610 bp. Subsequent to BAC 3-A2 sequencing, a joint project involving our laboratory together with the Max Planck Institute for Terrestrial Microbiology (Marburg, Germany) and the Munich Information Center for Protein Sequences (MIPS) combined resources to sequence the genome of strain Uh364. Recently, the MIPS U. hordei database was updated with large contigs formed from high-throughput pyrosequencing (454 Life Sciences) together with BAC end sequences from the Uh364 BAC library. Contig 5.00017 spans the entire UhAvr1 locus but includes several presumably small gaps (data not publicly available yet) and confirms our previous results. Using the VectorNTI ORF finding feature together with FGENESH, numerous putative genes were identified from our contigs (Table 4.1 and Fig 4.5a). Intriguingly, each group of tandem related genes from the 19A cluster in U. maydis were represented by single genes in U. hordei with the exception of two groups. The group of Um05309, Um05310 and Um05311 is represented by two genes in U. hordei (UH_08130 and UH_08132). These two genes, however, are both more related to Um05311, than to Um05309 or Um05310 (Fig. 4.6). As well, the group of Um05294, Um05295, Um05296, Um05297 and Um05298 is represented by two genes in U. hordei (UH_13897 and UH_10022). Synteny with U. maydis contig 1.191 is for the most part maintained across the entire region with the exception of gene shuffling at the one end of the cluster, placing the gene orthologous to Um05293 (UH_08123) within the cluster of secreted proteins in the U. hordei genome. In an attempt to find the UhAvr1 gene through simple ORF sequence comparisons, we first needed to find a BAC representing the corresponding region from the BAC library of the virulent parent Uh362. A probe for the DigA-like gene at the one end of BAC 3-A2 was used to identify BAC 1-E2 (Fig. 4.7). Unfortunately, after careful analysis using PCR and BAC end sequencing, this BAC was found to only partially span the UhAvr1 locus. Another screen was 113  therefore undertaken using the end of subclone 2-1 as a probe. This screen identified BAC 2G7, but like 1-E2 it also did not span the locus. Fortunately, the two BACs overlapped and together spanned the entire UhAvr1 locus (Fig. 4.7). Primers were designed for all predicted ORFs from BAC 3-A2 between the markers and that contained a secretion signal as predicted by SignalP (Table 4.2). The ORFs, together with 50-100 bp of flanking sequences, were amplified by PCR from BACs 1-E2 and 2-G7 and sequenced. Comparisons of the resulting sequences to the ORFs from BAC 3-A2 and cluster 19A of U. maydis are shown in Figure 4.8. Genes #4a (UH_13897) and #8 (UH_08134) both possessed differences between alleles from the two parents. UH_13897 contained single nucleotide differences at a base 21 nt upstream of the start codon and a base at position 165 downstream from the start within the ORF. This translates to a one amino acid difference between parental alleles (valine or isoleucine). According to the mapping data, however, this gene is outside the genetic interval and therefore was not studied further. UH_08134, on the other hand, was the only gene predicted to produce a secreted protein between our markers showing a difference (base 233 in the ORF) between parental alleles (Figs. 4.5a and 4.8). To determine if UH_08134 was the UhAvr1 gene, a knockout of the gene was performed (Fig. 4.9). The double-jointed PCR method worked well and was relatively easy to perform (Yu, et al. 2004). The critical step in the protocol seemed to be the use of nested primers at the final step. Introduction of the linear knockout construct into Uh362 was straight forward. After analysis of 36 transformants, one true homologous knockout was recovered and confirmed by Southern blotting. This homologous recombination frequency is consistent with attempts to knockout other genes in U. hordei. Since colony #27 represented the knock-out of gene #8, the cell line was named Uh364Δ8-27. Transgenic line Uh364Δ8-27 grew without any observable phenotypic abnormalities. No abnormal growth was seen in haploid basidiospore cultures and mating with compatible haploid strains did not seem to be affected (data not 114  shown). Mating of the Uh364Δ8-27 line with Uh365 (V1) and Uh362 (v1) strains and testing for virulence on barley cultivar Hannchen and Odessa clearly indicated that deletion of UH_08134 did not eliminate the avirulent phenotype of strain Uh364 and therefore is not the UhAvr1 gene (Table 4.4 and Fig. 4.9). The only observable effect of knocking-out this gene was that smut appeared later on plants containing the Uh364Δ8-27 X Uh362 cross on Odessa and that smut was seen in heads of shorter tillers (data not shown). The overall infection, however, was comparable to the other cross, indicating that initial infection may be equivalent, but that growth within the plant may have been negatively affected alluding to a possible function in virulence. In addition to deleting the candidate UhAvr1 gene, a transient expression assay was also developed for detecting avirulence functions of candidate genes. Infiltration of leaves with an Agrobacterium strain able to express a GUS marker from the strong plant-specific 35S promoter contained on its T-DNA, will produce blue-staining leaf cells. Co-infiltration with another strain expressing an ORF from an avirulence gene would elicit programmed cell death and thereby reduce the number of blue-staining cells. The ORF alleles of the UH_08134 gene from both parents of the mapping population were cloned between the Asc1 and Spe1 sites of the pMCG161 binary vector (www.chromdb.org; NCBI accession no. AY572837; Table 4.3, primers 9 and 10). The plasmids were then introduced into Agrobacterium strain LBA4404 (Invitrogen) and forced into barley cv Hannchen (Ruh1) leaves together with an LBA4404 strain expressing GUS from the same binary plasmid. Faint blue color was observed in all tissues including the negative controls (data not shown). Some darker regions were seen in tissues exposed to the strain carrying the GUS plasmid, but no differences were observed when co-infiltrated with strains containing either of the UH_08134 alleles. Caution must be taken in using this approach since faint blue cells are common when assaying for GUS activity in grass species. An alternative might be to use one of the newer fluorescent proteins instead of GUS, 115  or to directly stain for HR following leaf infiltration with Agrobacterium containing candidate genes.  4.4 Discussion Fungi are masters of secretion and for free-living forms this may not be much of a problem, but for parasitic forms much care must be taken to avoid detection by their hosts (van der Does and Rep 2007). For smut fungi like U. hordei and U. maydis, detection could very easily be lethal for the fungus. For U. hordei, several avirulent isolates have been identified through crossing on differential varieties of barley (Tapke 1937, Thomas 1976). Genetic mapping of UhAvr1 demonstrated that a single locus is responsible for fungal isolate recognition and plant resistance (Linning, et al. 2004). In this report, I have delimited the UhAvr1 locus to a region spanning several secreted proteins and containing large amounts of repetitive DNA including retrotransposons. This locus is syntenic to cluster 19A, the largest group of secreted proteins in U. maydis (Kamper, et al. 2006). In comparing the loci between the two smuts one can see that either the secreted proteins have multiplied in U. maydis or have been reduced in U. hordei (Fig. 4.8). Since U. maydis has many clusters of duplicated genes predicted to be secreted and since the U. maydis genome is reduced both in its number of introns and amount of repetitive DNA, it is probable that the genes in the 19A cluster multiplied after the divergence of the two species from a common ancestor. Multiplication of the secreted proteins within cluster 19A in U. maydis indicates that diversifying selection may have acted upon this locus helping the fungus avoid detection (Allen, et al. 2004), and indicating the importance of these genes to U. maydis. Deletion of these genes by the fungus may not have been an option, since forced deletion of the 19A cluster in U. maydis resulted in markedly reduced pathogenicity (Kamper, et al. 2006). Evidence for diversifying selection at this locus, points to direct recognition of these secreted effectors by plant R-genes (Allen, et al. 116  2004). Alternatively, indirect recognition through the “guard model” would likely have resulted in purifying selection, which is not the case (Rohmer, et al. 2004). Genes at this locus are therefore essential virulence factors. This locus is similarly important for U. hordei since it contains a factor (UhAvr1) that, once recognized, leads to plant resistance and ultimately fungal death. If UhAvr1 was not essential, one might expect it to have been eliminated from the U. hordei genome due to negative selective pressure. Having an essential role in pathogenicity would have prevented the gene from being lost to avoid detection (Skamnioti and Ridout 2005, Thrall and Burdon 2003). In a diverse population of plant hosts or on barley cultivars lacking the matching R-gene (Ruh1), the UhAvr1 gene would have been maintained in the smut population. Since the secreted proteins at this locus are not duplicated and diverged to the same extent as in U. maydis, more work is needed to analyze alleles from other strains to confirm if diversifying selection worked at the UhAvr1 locus as it appears to have on the 19A cluster in U. maydis. What then are these genes doing? Blast search failed to find related genes with known function. As well, no recognizable domains could be found in these proteins. Furthermore, none to very few cysteine residues are present within the secreted proteins in this cluster. Presence of numerous cysteine residues can result in disulfide bond formation leading to three dimensional structures resilient to protease activity (van den Hooven, et al. 2001, van den Burg, et al. 2003). Effectors from biotrophic fungi that form intimate feeding structures like haustoria usually contain very few cysteines (Ellis, et al. 2006). Such secreted effectors would have a short extracellular half-life or would be translocated into plant cells to avoid proteases in the apoplasm. Although no haustorial structures are formed by these two smuts, intimate contact with their hosts has been observed (Hu, et al. 2003, Snetselaar and Mims 1994). These secreted proteins therefore represent a novel cluster of fungal effectors important for virulence. The fact that one of these genes is the most likely candidate to be recognized by barley and that deletion 117  of the whole cluster has serious detrimental effects for U. maydis, makes study of these genes important for plant pathologists and breeders. Deletion of the entire cluster is required to determine the importance of this cluster for virulence in U. hordei. As well, each gene should be deleted individually and in combinations. Some of the genes in the 19A cluster appear to have an additive effect on virulence (Thomas Brefort at the 4th International Ustilago Meeting, Marburg, Germany 2008). Since the proteins are only predicted to be secreted this also needs to be confirmed experimentally. The yeast secretion-trap system may prove useful in this regard (Lee, et al. 2006). As well, it should be determined if the proteins function in the apoplasm at the plant-fungal interface or somehow make it into the plant cells to exert their effects. An interesting feature of this locus in U. hordei is that considerable amounts of repetitive DNA flank many of the genes. Repetitive DNA is known to correlate with heterochromatic regions of the genome (Buhler and Moazed 2007). Heterochromatin is known to be repressed in meiotic recombination, and may help explain the low homologous recombination frequency observed while generating the knockout line Uh364Δ8-27. Interestingly, for other organisms, species specific genes and genes that are developmentally regulated can be found in heterochromatin (Fitzpatrick, et al. 2005, Yasuhara, et al. 2005). Heterochromatin-like regions may therefore be important for evolution of certain virulence factors helping fungal pathogens avoid R-gene detection. Some Avr genes from the ascomycete, Leptosphaeria maculans, can be found in heterochromatin-like DNA that has very few genes, is rich in repetitive DNA and has a higher A+T content than gene rich areas (Fudal, et al. 2007). Similarly, the AVR-Pita gene is located in heterochromatic DNA near a telomere of Magnaporthe grisea (Orbach, et al. 2000). The largest contig at the UhAvr1 locus has a G+C content of 49 % similar to the 527 kb MAT-1 locus known to harbor over 50 % repetitive and transposon-related DNA (Bakkeren, et al. 2006). Three random gene rich areas of U. 118  hordei had an average G+C content of 54 %. Although more data is needed, this slight reduction is consistent with the idea that the UhAvr1 locus is heterochromatic. UhAvr1 was not found by comparing ORF DNA sequences between secreted proteins within the defined UhAvr1 locus. This can be explained by a number of different scenarios. Perhaps the difference(s) between alleles is outside the ORF and was not revealed by the sequence data we generated. It is also possible that cryptic ORFs have not been identified by the available software used in this study. Alternatively, the avirulent phenotype may be due to a nearby transposon that affects gene expression level as observed for other phenotypes in plants, animals and fungi (Kang, et al. 2001, Kinoshita, et al. 2007). UH_08130 is less than 600 bp from the start of a retrotransposon gene (UH_10031). Bidirectional transcription from the promoter of the retrotransposon may influence the expression of UH_08130. Since UH_08130 is the only duplicated gene in this cluster it may have been made transcriptionally inactive after duplication as a means to avoid R-gene detection. Insertion of the nearby retrotransposon may have reactivated the gene causing recognition of that strain. Sequencing of the other parental strain to look for presence of the retrotransposon together with comparing gene expression is needed to test this hypothesis. Another possibility is that UhAvr1 is not a small secreted protein. In the vicinity of the UhAvr1 locus, candidate genes would include tubulin (UH_08135), an ion channel gene (UH_08136), a MADS-box transcription factor (UH_08137), a vacuolar protein sorting gene (VPS10, UH_08138) and transposon-related genes. Lastly, it is possible that one of the progeny of the mapping population has been scored erroneously as being avirulent or virulent, obscuring recombination frequencies, or that markers have been placed at wrong locations on the newly sequenced BAC clone. Indeed, there are five small secreted proteins outside the current genetic interval separated by just one recombinant (UH_10024, UH_10022, UH_13897, UH_08128 and UH_08127). UH_13897 has been sequenced and shows differences in the ORF and promoter between the parental alleles 119  and would be an immediate candidate under this scenario. Current efforts in the laboratory are focused on eliminating each scenario. As well, a strategy whereby larger sections of the UhAvr1 locus will be deleted should help define the exact location of UhAvr1. This can be done by independently deleting 3 or 4 different sections of the locus. In summary, UhAvr1 was mapped to a region of the U. hordei genome syntenic to cluster 19A in U. maydis containing a large number of secreted proteins. The number of secreted proteins was reduced in U. hordei and at least one member of each class defined in U. maydis was represented in U. hordei. An interesting feature of the locus in U. hordei is that a significant amount of repetitive DNA is present giving the appearance of heterochromatin. In support of this, meiotic recombination is suppressed within the cluster and knock-out (homologous recombination) of a cluster gene was difficult. Given the importance of this cluster to U. maydis and U. hordei, identification of UhAvr1 will provide insights into the evolution of these two pathogens and lead to a better understanding of gene-for-gene resistance between U. hordei and barley, as well as provide clues as to why gene-for-gene resistance is not seen between U. maydis and maize.  120  A  AFLP  Gene X  BAC 1-J3 Y  VPS 4-1 (19kb)  FMO  X 5-12 (26 kb)  2-1 (9kb) 1-1 (9.5kb) 1-6 (9kb) BAC 3-A2 BAC 1-P19  B Secreted proteins  U. maydis contig 1.191  Y (  U hordei Avr1 locus  VPS X  FMO  )  BAC 3-A2  Figure 4.1 The UhAvr1 locus. (A) Markers spanning the UhAvr1 locus identified overlapping BAC clones (black bars). BAC 1-J3 was subcloned (green bars; length of insert in Kb) and used for RFLP analysis. Sequencing from subclones (red bars) identified a number of genes (arrows). (B) Comparison of the DNA sequences from the UhAvr1 locus to U. maydis resulted in identification of a syntenic region on U. maydis contig 1.191 (synteny represented by dotted lines). The mapping interval (pink bars in A, parentheses in B) seemed to span a large portion of a cluster of U. maydis secreted proteins (cluster 19A, pink oval, Kamper, et al. 2006). Based on sequences from flanking genes it was apparent that U. hordei BAC 3-A2 completely spanned U. maydis cluster 19A. 121  A  Transprimer Phleomycin-R (M)  pDonor Amp-R  PI-SceI ori  Gene 1  Gene 2  Target DNA (BAC 3-A2)  B  N  ∆ Gene 1  S M  Gene 2 N  ∆ Gene 2  Gene 1  M N  Intergenic  Gene 1  S  S M  Gene 2  Figure 4.2 Using the GPS-Mutagenesis System to sequence BAC 3-A2. (A) The donor vector was engineered to express a phleomycin resistance gene (M) from bacterial and Ustilago promoters within a transprimer (transposon). An in vitro transposition reaction moves the transprimer containing the phleomycin resistance marker into random locations of the target DNA (BAC 3-A2). A PI-SceI digest then destroys the donor vector. (B) BAC 3-A2 clones are then sequenced bi-directionally off both ends of the transprimer using primers N and S resulting in paired sequence reads from random location across the BAC.  122  A  L  B SAM  L  SAM  Hyphae  Figure 4.3 Chlorazol Black E staining of three week old barley seedlings. (A) Longitudinal median section of barley cv Hannchen (Ruh1) inoculated with a mixed culture of U. hordei strains Uh364 (UhAvr1) and Uh365 (UhAvr1). (B) Longitudinal median section of barley cv Odessa (ruh1) inoculated with a mixed culture of U. hordei strains Uh364 (UhAvr1) and Uh365 (UhAvr1). Note the mass of hyphae growing within the barley tissues below the SAM. See text for details. SAM, shoot apical meristem; L, leaves.  123  A  RNAi Start  0  1st Coiled-Coil  702-839  2nd Coiled-Coil  1987-2083  Repeats  2329-2842  Stop  3350  B  wt  RNAi  Figure 4.4 Characterization of Gene X (UH_13922). Gene X was sequenced from parents of the mapping population and from selected strains from around the world. (A) The gene is large (1099 aa) with two coiled-coil domains and a domain containing large repeats resembling collagen. From the alleles identified, variation is seen in the first coiled-coil domain and in the large repeats. Knock-down of this gene was conducted using an RNAi approach targeting a region of the gene between the coiled-coil domains. (B) Phenotype of the RNAi mutant. Haploid basidiospores were grown in liquid culture. Wild type cells are shown on the left while RNAi mutant cells are shown on the right. RNAi mutant cells show difficulties separating after budding.  124  A U. maydis contig 1.191  Secreted proteins  Y 1 2  3 4a 4b 5 6  ( BAC 3-A2  VPS X  7a 7b 8  9,10,11,12,13,14, 15 16 17  U. hordei Avr1 locus  Contig 0.1 rc  Contig 3.1 Gap 1  FMO )  Contig 2.1  Gap 2  B 12000 Across Gap 1  3000 2000 1650 1000 850  Across Gap 2  650 500 400 300  Figure 4.5 BAC 3-A2 sequencing results. (A) Three large contigs span the markers (parentheses) and together with several contigs of various sizes represent most of the BAC 3A2 sequence (green bars). Due to repetitive DNA, several contigs could not be bridged by the PCAP.REP software. Several genes were identified and are numbered consecutively along the red bar representing the UhAvr1 locus and flanking regions. VPS, vacuolar protein sorting gene; FMO, FAD-containing monooxygenase gene. Synteny is represented by dotted lines that transverses the U. hordei DNA and U. maydis contig 1.191. Cluster 19A is indicated by a large oval. Two small gaps within the UhAvr1 locus are marked by arrows. (B) PCR across the gaps indicates the amount of DNA missing from our analysis. For gap 1, primers were used from genes near the end of each contig to bridge the gap using PCR (UH_08130 from contig 0.1 rc to UH_08132 from contig 3.1). Flanking DNA sequences (2660 bp) subtracted from the roughly 3000 bp PCR product estimates the gap to be around 340 bp. For gap 2, primers were designed from the ends of contigs 3.1 and 2.1. The resulting PCR band of approximately 700 bp minus the 90 bp of flanking sequence allows for an estimate of around 610 bp for gap 2. 125  A UH 08132 UH 08130 75  UM05311 UM05309 100  UM05310  0.1  B UM05309 UM05310 UM05311 UH_08130 UH_08132  (1) (1) (1) (1) (1)  UM05309 UM05310 UM05311 UH_08130 UH_08132  (50) (50) (37) (45) (46)  UM05309 UM05310 UM05311 UH_08130 UH_08132  (99) (99) (86) (95) (96)  UM05309 UM05310 UM05311 UH_08130 UH_08132  (147) (147) (135) (143) (145)  UM05309 UM05310 UM05311 UH_08130 UH_08132  (196) (196) (185) (193) (174)  1 50 -MLVFP -MLVF PLVQNALVLFALVLGCSAPRTSKSRSTRFRGAKATIRKGQITDQV -MLVSPLFRLTLLLIALVLECSAPKADKSKSARSRGTKSTIGKGLVTDQV MQLCVSNKLRVSLIWACCLALMGLGAPVSVEF--------------SSSL -MLLTSKVGSLLSIFSFFLITSEIVEGQGYTT--AGTSAARSQ---SSAL -MATTSLLRRCAPYLLSSLLLVLQILATAATMTPSGIPE---K-LFPASL 51 100 QTFITRLSEQELFALNQVKYWIDTQAHPEYQRRLLN-NLFQGGTNRSPTL ASFLARLSGQKLLSPNQVKYWMDTQALPNHQGRLLN-NLFQSGTNRSPRL AVFVDRL-EDASVPVLPRQYHSTWDFSSNKVHHWLTSFLFKDGKNFNPKL EPFLDQLLKDQNLDSRSSIYAITELTQQSSKHHLLTQYIYRNGQDFNPQI DPFVNELSLRGFLYTSRIELAFDLTNMQFRSHHALTDHFFKNGQDWNPRV 101 150 LSLGTNDAG--DHMAVSILKPDIETAALLAGPERRRPRGWSTRKGLMLMH LSLGTNDAG--DHMAVSILKPDIETAAMLAGSEYARPKGWNTRNGVVLML FYLGRDPLKLDTHLAVAHWYPNTFLLNRVRPIG-AQTVNLEYRHGLLAMK VYLGTDPKDPSAHLAVKPFAPDLDLADDILRNG--KFHRWKNREGLLAMR VYLGEHPALPSTHMAASVFHPNIQLTNHLVPKS-GLMKHRNGKNGVLAIN 151 200 LTSNEPPSVVGWIFMKNHRSANALTTSSEMVSLDNFEWLHGLSGIVREVLRSNERPNVIGWLLMKNHNSAMGLKTSSEMVSLDNFEWLHGLSGIVREVLASHEEPEFVGILWIKDKRNAKKARAADRAKRSGKHREERRESQGRSKSD LRSREEPEFAGIIWLSKRQMTKGIKWEAVQTFDQLQSDLSLGVIERVLHH LRANEEPEFVGVLWLKSHRAVNVLERSGM--------------------201 236 ----------------------------------------------------------------------PPWDYLIEVRCSLDPLSITSLSTILVYIHRITCLLR LPVRRK-----------------------------------------------------------------  Figure 4.6 Comparison of duplicated genes at the UhAvr1 locus to homologous genes in U. maydis. (A) A phylogeny was drawn based on the alignment of homologous genes using the Neighbor joining method and 1000 bootstrap replicates (Mega version 4). (B) Alignment of the homologous genes using AlignX (VectorNTI, Invitrogen).  126  A  BAC 3-A2 Sub-clone 2-1  1  2  B BAC 1-E2  BAC 2-G7  1 2  C BAC 3-A2 Sub-clone 2-1  BAC 1-E2  BAC 2-G7  Figure 4.7 Search for BAC clones from the virulent parent library corresponding to the UhAvr1 locus. (A) location of probes used to search the BAC library. Probe 1 was made using BAC 3-A2 end sequence data. Probe 2 was made using subclone 2-1 end sequence data. (B) Identification of positive BAC clones BACs 1-E2 and 2-G7, within the BAC library laid out on nylon filters. (C) Relationship of BACs 1-E2 and 2-G7 to BAC 2-A2.  127  A 6  4a  4b  3  19A  7a 7b  8  9  10 11  B catacgT(A)gcctcgacaacctcataaatatgcttactcaaccggccaacgtcatccttttcatggtcgcct tcctcttttcaacgactgcactacccggtcgcagctacaaaccttcccgatttcggccctataacgagccgttc gtggtccattccgtctccaaattccaagacgagtatgctgatcgtctgG(A)tcctcgtcctccaagcctactt ccatcagcaatatgcaaaaatcaagctgcttgaccgagatccgatcagcctacaagatttgaaacaggac cttcgaggcgatcgtaacccaaaacgcttcatccacttaggccaagtggtcccacacagagctaatatggt ggttgcaacgtatctgaaccgtccacccaactcggacggttctcggaaattcgtactcttgtcaattcttcgac ctcaatcttctgatgccccacacgtcttcatacacggttatgccgatgtagcgggtctcgaagacattgaaga tcaacttcggaacaccgtgaactctgcctcggatgatccgcagttcggacatgttctctcgatcgaaggggtt tttgagacgttgtcacgaatgtaa  C atgaaggtacatctgtctaccgcttcaatcttcttcttttcagccatttgctgtcgaacggcacacccacagaat ccgttgccacaccaaggtatcttctacccaggcacagacctcgaatttcacatggccgagacgagtaaag ctagcagtgccgcaggcatcgccgaacacgtcttcagtcttccctcgagtcatctctcccctatctacggcta cgaggaaggccagcG(A)tcaagcagtgatcaatcatatcgaaaatcctgcttctcgttacgtcgtgctgat cgaggaaggccagcG(A)tcaagcagtgatcaatcatatcgaaaatcctgcttctcgttacgtcgtgctgat aaagtacagaagaggctggacctttgtcatttctccatgggtcgtgcggcgtgggcctgatgcacctacaca gaaaggcgtactgtttctgcaggcttatccaggcggggtactgtctcctattggatacgctgaggtgaagga gggtattccggaaggacatagtcgagacttctgggatactctcaaccgtgcagccagaacgacaaggga agagctattgaggaggtttgagattgataggatcgtcactcctgtgaccattcgaggctag  Figure 4.8 Comparison of parental alleles for secreted proteins at the UhAvr1 locus to cluster 19A in U. maydis. (A) The number of small secreted proteins is reduced in U. hordei as depicted by aligning homologous genes to cluster 19A (Kamper, et al. 2006). Only genes numbered 4a and 8 show differences when comparing the ORFs of the two parents. Of these two genes, only number 8 lies within the genetic markers defining UhAvr1 (compare Fig 4.5A). DNA sequence for gene #4a (B) and gene #8 (C) is shown indicating start and stop codons as well as the differences between parental alleles (grey shaded boxes). The first uppercase letter is the nucleotide of the avirulent parental allele while next to it in parentheses is the nucleotide of the virulent parental allele. Nucleotides in lowercase are identical between parental alleles. 128  A BamH1  BamH1 Hygromycin-R  4112 bp  UH_08134  Probe  2212 bp  BamH1  BamH1  B KO 1 2 3 4  4112 bp 2212 bp C Pathology Test  Infected Plants (%)  60.00 50.00 40.00 30.00 20.00 10.00 0.00 1  2  3  4  5  6  7  8  Treatment Number  Figure 4.9 Candidate UhAvr1 (UH_08134, gene #8) knock-out and analysis. (A) Doublejointed PCR was used to build a linear deletion construct consisting of a Ustilago-specific hygromycin resistance cassette flanked by roughly 1000 bp of DNA both upstream and downstream of UH_08134. The construct was used to replace UH_08134 through homologous recombination. A probe recognizing the downstream flank (blue bar) was used to verify the knock-out. (B) Southern blot analysis of Bam H1 digested genomic DNA was probed with the downstream probe. The knock-out (KO) shows the expected 4112 bp band, while nonhomologous transformants possess the wild type 2212 bp band and bands representing random insertions. (C) Pathogenicity tests show that deletion of UH_08134 does not prevent the avirulent phenotype of strain Uh364. The arrow shows the knock-out line mated with a virulent strain and inoculated onto barley cultivar Hannchen. See Table 4.4 for treatments . Treatments 1 to 4 are various compatible interactions while 6 to 8 are incompatible. Treatment 5 is the null UH_08134 stain mated with Uh362 (avr1) and tested on barley cultivar Hannchen (Ruh1); normally an incompatible interaction due to UhAvr1. 129  Table 4.1 Predicted genes at the UhAvr1 locus Number1 MIPS ID. 2  U. maydis homolog3  E - value4  U. hordei Function 5 Coordinates  1 2 3  UH_08121 UH_08127 UH_08128  UM05292 No hit UM05306  0.0 --1.87066E-30  ? ? ?  4a  UH_13897  UM05294  7.63671E-11  4b  UH_10022  UM05296  1.70128E-10  Contig 0.1rc 29747-30316 Contig 0.1rc 28425-28994  5 6  UH_10024 UH_08123  No hit UM05293  0.0  7a  UH_08130  UM05311  1.71205E-16  7b  UH_08132  UM05311  2.29326E-16  8  UH_08134  UM05312  6.38569E-31  9 10  Transposase UH_13916  UM05316? UM05318  --4.15971E-7  11  UH_10033  UM05319  3.73423E-8  12  UH_08135  UM05320  0.0  13  UH_08136  UM05321  0.0  14  UH_08137  UM05323  0.0  15  UH_08138  UM05324  0.0  16  UH_08139  UM03752  1.34596E-14  17  UH_13922  UM05325  0.0  Contig 0.1rc 19449-21854 Contig 0.1rc 885-1478 Contig 3.1 10242-10760 Contig 3.1 7899-8456 --Contig 2.1 29717-29956 Contig 2.1 28923-29237 Contig 2.1 26578-28071 Contig 2.1 23694-26240 Contig 2.1 18893-20818 Contig 2.1 13397-17949 Contig 2.1 11617-12246 Contig 2.1 4929-8040  DigA Hypothetical Conserved hypothetical Hypothetical Hypothetical Hypothetical Oligosaccharyltransferase Conserved hypothetical Conserved hypothetical Conserved hypothetical --Hypothetical Conserved hypothetical Tubulin Beta chain Conserved hypothetical Conserved hypothetical VPS10 Conserved hypothetical Hypothetical  1  Numbers correspond to predicted genes  2  MIPS Ustilago hordei Database gene ID (http://mips.gsf.de/genre/proj/MUHDB/); upon publication of a joint manuscript describing the U. hordei genome sequence, assembly and called gene ID numbers, this information will be released in the public domain.  3  Ustilago maydis database at the Broad Institute (http://www.broad.mit.edu/annotation/genome/ustilago_maydis/Home.html)  4  BlastP results  5  Position of gene on contigs generated from sequencing BAC 3-A2 130  Table 4.2 Primers used to sequence genes for putative secreted proteins Number1 MIPS ID. 2  Primer sequence  4a 4a 4a 4a 7a 7a 7a 7a 7b 7b 7b 7b 8 8 8 8 10 10 10 10 11 11 11 11  5′-GGAACTGTGCTCTGGTAGTGG-3′ 5′-TGCTTTGAGTCGGGCATTAT-3′ 5′-TGAAGGCGCGCCATGCTTACTCAACCGGCCAAC-3′ 5′-GCGCACTAGTTTACATTCGTGACAACGTCTC-3′ 5′-GCTTTTCATCAGAGCCATACCT-3′ 5′-TGGCTTGTTTACAGAGTGCAA-3′ 5′-TGAAGGCGCGCCATGCTTCTGACCTCGAAAGTAG-3′ 5′-GCGCACTAGTTCACTTTCTCCTCACGGGTAG-3′ 5′-GGAACCGGTTGACGATGTAT-3′ 5′-GGCTGGAATCGGTATGATGT-3′ 5′-TGAAGGCGCGCCATGGCCACAACATCACTCTTAC-3′ 5′-GCGCACTAGTTCACATTCCACTGCGCTCCAG-3′ 5′-ACAAACTAGCCAACTTCAAG-3′ 5′-ACGCCGAGATCGAGAGCGTG-3′ 5′-TGAAGGCGCGCCATGAGGGGCATGAGGGCCCTC-3′ 5′-GCGCACTAGTCTAGCCTCGAATGGTCACAGGAG-3′ 5′-CTGTGATAAGGGGTAACGAC-3′ 5′-TTGTGCAACTTCTTCGGTAC-3′ 5′-TGAAGGCGCGCCATGACCCGTCTCAAGCAAAAC-3′ 5′-GCGCACTAGTTCACCCAACACTTCGGCTCGTAG-3′ 5′-CCGTGTCAATCGTGTTGCTC-3′ 5′-TCTCCCTTCCTCGTCAACCTG-3′ 5′-TGAAGGCGCGCCATGCGCTTCGCATCCCTCGT-3′ 5′-GCGCACTAGTTCAGAGTTCGTACAGAGAGA-3′  UH_13897 UH_13897 UH_13897 UH_13897 UH_08130 UH_08130 UH_08130 UH_08130 UH_08132 UH_08132 UH_08132 UH_08132 UH_08134 UH_08134 UH_08134 UH_08134 UH_13916 UH_13916 UH_13916 UH_13916 UH_10033 UH_10033 UH_10033 UH_10033  Usage3 P,S P,S S S P,S P,S S S P,S P,S S S P,S P,S S S P,S P,S S S P,S P,S S S  1  The numbers correspond to genes in Table 4.1  2  MIPS Ustilago hordei Database gene ID; upon publication of a joint manuscript describing the U. hordei genome sequence, assembly and called gene ID numbers, this information will be released in the public domain.  3  P, primer used to amplify gene; S, primer used for sequencing  131  Table 4.3 Primers for deletion, expression and RNAi cloning #  Primer name  Purpose Deletion construct Deletion construct Deletion construct Deletion construct Deletion construct  1  5′-flank, f  2  5′-flank, r  3  3′-flank, f  4  3′-flank, r  5  Hygromycin cassette, f  6  Hygromycin cassette, r  7  Nested, f  8  Nested, r  9  UH_08134, f  10  UH_08134, r  11  Gene X, f  RNAi  12  Gene X, r  RNAi  13  Gene X repeats, f  Mapping  14  Gene X repeats, r  Mapping  Deletion construct Deletion construct Deletion construct ORF cloning, expression ORF cloning, expression  Sequence 5′-GGTACGCCGATCACTTCAAC-3′ 5′-ATAATCCTTAAAAACTCCATTTCCA CCCCTGTAGACAGATGTACCTTCAT-3′ 5′-ACTTTATTGTCATAGTTTAGATCTAT TTTGACTCCTGTGACCATTCGAGG-3′ 5′-CCGATACCGAGACAATAGCTG-3′ 5′-CAAAATAGATCTAAACTATGACAAT AAAGTTCATGTTTGACAGCTTATCATC G-3′ 5′-AGGGGTGGAAATGGAGTTTTTAAGG ATTATGAACGTGGTAACTACCAGCGAG TTC-3′ 5′-TGAAGGCGCGCCATGTCAGAGGAA GCGAG-3′ 5′-GTGCATGGTGTTGGAGGTC-3′ 5′-TGAAGGCGCGCCATGAAGGTACATC TGTCTAC-3′ 5′-GCGCACTAGTCTAGCCTCGAATGGT CACAGGAG-3′ 5′-TTACTAGTGGCGCGCCCTCCAAGAG CCTGGTTTCATC-3′ 5′-TTGCGATCGCCCTAGGGAAGAAATG GACGTCGCGCATG-3′ 5′-GTGGGCCATCTAGGCCCACGCCAAC AAGGACTGGTA-3′ 5′-TCGAATAAGCACTGGAAG-3′  132  Table 4.4 Pathology test results for the null mutant of candidate UhAvr1 gene, UH_08134 (gene #8)  Treatment 1  U. hordei strains 2  Barley cultivar 3  Infected plants (%)  Standard deviation  Number of flats planted 5  1  Δ-8 X 365  Odessa  49.14  5.48  3  2  Δ-8 X 362  Odessa  38.86  6.78  3  3  364 X 365  Odessa  39.44  0.79  2  4  362 X 359  Hannchen  23.67  3.02  2  5  Δ-8 X 362  Hannchen  0.00  0.00  3  6  Δ-8 X 362  Plush  0.83 4  1.18  2  7  362  Odessa  3.23 4  -  1  8  Δ-8  Hannchen  0.00  -  1  1  Treatments 1 to 4 represent various compatible interactions while 6 to 8 are non-pathogenic controls; treatment 5 is the null UH_08134 stain mated with 362 (Uhavr1) and tested on barley cultivar Hannchen (Ruh1), normally an incompatible interaction when UhAvr1 is present  2  Δ-8, strain 364 (MAT-1, ΔUhAvr1, UhAvr6) = null mutant of UH_08134 (UhAvr1 candidate); strain 365 (MAT-2, UhAvr1, UhAvr6); strain 362 (MAT-2, Uhavr1); strain 359 (MAT-1, Uhavr1)  3  Odessa (ruh1); Hannchen (Ruh1); Plush (ruh1, Ruh6)  4  a low level of seed contamination was found (possibly loose smut, U. nuda)  5  Average of 46 plants per flat  133  4.5 References Akimoto-Tomiyama C, Sakata K, Yazaki J, Nakamura K, Fujii F, Shimbo K, Yamamoto K, Sasaki T, Kishimoto N, Kikuchi S, Shibuya N, Minami E (2003) Rice gene expression in response to N-acetylchitooligosaccharide elicitor: comprehensive analysis by DNA microarray with randomly selected ESTs. 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My results confirmed the lack of RNAi genes in U. maydis and showed experimentally the presence of functional Dicer and Argonaute (Ago) genes in U. hordei. Given the close phylogenetic relatedness of these two smut fungi, my results prompted a search for the genes in U. hordei. As part of a collaborative effort, the genome of strain Uh364 was sequenced. This strain was selected mainly because it was used as a parent for mapping the UhAvr1 gene and contains at least two other Avr-genes (Linning, et al. 2004); in addition, from this strain, a BAC library of 2,300 clones was ordered using HindIII restriction fragment profiles (fingerprint map) and 527 kb spanning the mating-type region (MAT-1) was sequenced and analyzed (Bakkeren, et al. 2006). Upon receipt of the initial draft of the sequence, I continued my work on RNA silencing and identified genes for Dicer, Ago and RdRP. As well, the initial contigs were large enough to identify flanking genes for all of the RNAi genes. Comparison of these genes to the U. maydis genome revealed conservation of synteny. Complete lack of RNAi genes at these loci in U. maydis indicated that an active removal process had occurred after divergence of these two species from a common ancestor. With the demonstrated role for RNAi genes in TGS in diverse eukaryotes (Kloc and Martienssen 2008), a search for homologs in U. hordei and U. maydis was conducted. Additionally, a search for genes present in U. hordei genome, but lacking from the U. maydis 142  genome, was performed using BlastP analysis of predicted proteins from both species. These searches resulted in the identification of numerous enzymes, two chromodomain (Chp) genes and one histone deacetlyase (HDAC) gene seemingly present in U. hordei, but absent from U. maydis. Work done in S. pombe, demonstrated that RNAi recruits histone methyl transferases to loci expressing repetitive DNA (Zhang, et al. 2008). Chromodomain proteins then associate with methylated histones, helping to establish heterochromatin and ensure TGS. These studies were done looking at pericentromeric regions containing repetitive DNA. A similar study should be done to look at the localization of the two chromodomain proteins that I identified to see if they are also associated with repetitive DNA sequences and heterochromatin. Regions to compare would include the MAT locus and the UhAvr1 locus because of their known repetitive DNA content, against gene-rich loci. The observation that these two chromodomain proteins are not found in U. maydis might suggest that U. maydis uses different means for heterochromatin formation. It is possible that when U. maydis began to rely on a different mechanism for controlling repetitive elements through a process of active DNA elimination, that the two chromodomain proteins became redundant and were therefore deleted. The presence of a unique HDAC in U. hordei supports this notion of differences between these two organisms in methods of TGS. Histone deacetylation is the main driving force for TGS in many organisms. Following deacetylation, methylation of histones H3 on lysine 9 or 27, results in strong transcriptional repression (Brosch, et al. 2008, Chen and Tian 2007, Isaac, et al. 2007, Loyola and Almouzni 2007). Evidence for differences in gene regulation in relation to histones, prompted me to compare histones between these two smuts. Differences were found in three histones, but of interest is the cenH3 histone known to associate with centromeres in other species (Baker and Rogers 2006). These orthologous genes possess multiple differences in their N-termini but remain conserved throughout their C-termini. Since chromatin formation is regulated by 143  several known N-terminal modifications in histone H3, including acetyation, methylation and phosphorylation, the variability seen in the cenH3 genes of these two smuts strongly suggests possible differences in gene regulation. Variability in cenH3 genes also suggests differences in centromere formation and regulation. Deletion of RNAi components in S. pombe results in chromosomal segregation defects (Provost, et al. 2002, Volpe, et al. 2003). Since RNAi silences repetitive DNA near centromeres by active heterochromatin formation, it appears that repetitive elements could be essential components of centromeres. Large stretches of repetitive DNA have been identified at the U. hordei MAT-1 and UhAvr1 loci, and might be present at centromeres as well. Presumably, the RNAi machinery acts to silence repetitive elements and initiates heterochromatin formation at the centromeres of U. hordei as in S. pombe. Intriguingly, U. maydis also has repetitive elements near its centromeres, even though the genome is relatively devoid of repetitive elements (Kamper, et al. 2006). This implies that repetitive elements may also play a role at centromeres independent from RNAi. Analysis of the repetitive elements at the U. maydis centromeres suggests that they may be nonfunctional retroelements. The fact that they appear only at centromeres suggests that they are not capable of jumping or are actively removed after jumping. Since U. maydis seems so efficient at eliminating unnecessary DNA, it is likely that U. maydis retained these repetitive element remnants for a specific purpose. Alternatively, due to the importance of centromeres, U. maydis might not have been able to completely eliminate the repetitive elements, possibly due to a competing mechanism involved in centromere maintenance.  5.2 Characterization of the UhAvr1 locus Plant pathogenic microbes possess specialized tools that allow them to navigate through preformed and induced basal defenses of their host, as well as to avoid or suppress R-gene resistance. Current interests are focused on the proteins that microbes secrete into their hosts. 144  For bacteria, many virulence factors are delivered directly into plant cells through the type three secretion system. For fungi and oomycetes, proteins are secreted through membrane trafficking networks that require proteins to have N-terminal secretion signals (Ellis, et al. 2006). Once secreted into the apoplast, protein effectors then perform a function, or have additional means of entering the plant cell cytoplasm, like the RXLR domain of some oomycete effectors (Bhattacharjee, et al. 2006). In either the apoplast or within the plant cell, an effector recognized by an R-gene, either directly or indirectly, most often leads to microbe failure and disease prevention. Gene-for-gene interactions between avirulent effectors and Rgenes are therefore of considerable interest to plant breeders and molecular biologists. Mapping of the UhAvr1 gene has lead to some interesting findings. The most important find is that the UhAvr1 locus is syntenic to cluster 19A, the largest cluster of secreted proteins in U. maydis (Kamper, et al. 2006). This cluster is significant for both organisms for a number of reasons. First, when the cluster is deleted in U. maydis, pathogenicity is severely affected, demonstrating presence of these effectors to be essential for disease (Kamper, et al. 2006). Since synteny is conserved flanking the cluster and most of the U. maydis secreted proteins in the cluster have at least one homolog in U. hordei, it can be assumed that the cluster is similarly important for U. hordei. Secondly, U. hordei possesses an avirulence gene at this locus. Strong selective pressure would be against U. hordei strains carrying this gene when landing on cultivars of barley containing the matching Ruh1 gene. The presence of the UhAvr1 gene at this cluster poses many questions, especially about U. maydis. In comparison, this locus in U. maydis has many more secreted proteins that are arranged in groups of tandem duplicated genes (Kamper, et al. 2006). Gene duplication indicates that diversifying selection may have acted upon these genes. For this to have happened, the effectors must have been directly recognized by maize. This is extremely odd, since no gene-for-gene resistance relationship has been observed between U. maydis and maize. To stress this point, it should be noted that maize is 145  the most diverse crop plant, and therefore should have numerous R-genes within even the cultivated varieties capable of detecting at least one strain of U. maydis. Either people have not looked carefully enough or something special about U. maydis allows it to avoid gene-for-gene resistance. Since I have already shown that U. maydis is very special in its lack of RNA silencing, I favor the latter scenario. Furthermore, using microscopic and gene expression analyses, localized defense responses including HR were seen early during leaf infection in some penetration sites (Doehlemann, et al. 2008). U. maydis, therefore, activates either basal or R-gene receptors at some initial penetration sites, but makes appropriate changes to prevent such responses as it searches for alternative penetration sites. Signaling downstream of R-gene activation is highly conserved in plants, as demonstrated by effective signaling when R-genes are expressed in unrelated species and challenged with the matching Avr-gene (HammondKosack, et al. 1998, Piedras, et al. 1998, Rentel, et al. 2008). Perhaps U. maydis has evolved a method to block this conserved signaling, downstream of R-gene recognition, allowing it to live on maize unimpeded by R-genes. This is very unlikely, because if this was possible, bacteria would most likely have exploited such mechanisms before fungi. U. maydis, therefore, probably has a way to fine-tune gene expression whereby avirulence is tuned-down in the presence of an R-gene. This could be reflected in the multiplication of secreted effectors in the19A cluster. The observation that certain hyphae activate HR in maize epidermal cells, while other hyphae do not might suggests that U. maydis is able to fine-tune gene expression to avoid R-gene detection at certain penetration sites. An experiment that might help explain some of these observations would be to ectopically express a known Avr-gene in U. maydis and infect maize containing the matching R-gene. If U. maydis grows, then blockage of R-gene signaling is likely the reason for the overall success of U. maydis. If U. maydis does not grow, then tuning of gene expression is likely the reason for success. Each individual effecter in the 19A cluster should be transiently expressed using a leaf assay on several maize varieties or 146  ectopically expressed in U. maydis to test for possible HR reactions on maize. If in isolation, the effectors can be recognized, then fine-tuning is likely the means by which U. maydis avoids R-gene recognition and defense. The final interesting feature of these two loci is that in U. hordei the locus appears to be heterochromatic. This is evident by the suppressed recombination and presence of repetitive elements, as well as by a slight reduction in G+C content. Species-specific and developmentally important genes are often found associated with heterochromatin (Yasuhara, et al. 2005). If this holds true for U. hordei, then the effectors at the UhAvr1 locus may also be tightly regulated and important for development. Suppressed recombination may allow for mutations to create new alleles enabling strains to quickly evolve and escape R-gene recognition. It would be interesting to know the recombination frequency of genes in the 19A cluster of U. maydis. Perhaps differences at these orthologous loci are mainly due to differences in recombination. That is, U. hordei maintains few genes that evolve through repressed recombination, while U. maydis uses gene duplication to evolve different alleles and some form of gene regulation involving fine-tuning to escape R-gene recognition.  5.3 Future experiments My work has lead to the discovery of numerous important differences between two closely related plant pathogenic fungi. From this work, many follow-up studies are possible. A list of experiments that I think will make significant contributions is presented below.  5.3.1 Recombination If recombination is the driving force for differences between these two smuts, then a direct comparison is essential. Recombination frequency is high in U. maydis as demonstrated by efficiency of creating homologous gene deletions (Garcia-Pedrajas, et al. 2008) and by the 147  recovery of U. maydis after DNA damage caused by ionizing radiation (Holloman, et al. 2007). Side-by-side comparisons would involve independent homologous deletion in both species of multiple orthologous genes in both gene rich areas and in heterochromatic regions. This should resolve the possibility that previously observed differences in recombination frequency are due to the heterochromatic nature of the U. hordei MAT and UhAvr1 loci. If homologous recombination frequencies are equivalent in syntenic gene rich regions, but different at the MAT-1 and UhAvr1 loci, then heterochromatin may be the reason for previously observed differences. If homologous recombination frequency is different at all genes tested, then the two smuts are mechanistically different with respect to homologous recombination capability. Additionally, a side-by-side experiment that measures the lethal dosage of ionizing radiation may indirectly point to differences in homologous recombination capabilities. This would involve measuring the number of DNA breaks and the ability to recover. Since U. maydis is known to be highly resistant to ionizing radiation as a result of its high recombination frequency (Holloman, et al. 2008), U. hordei also needs to be tested for its resistance to ionizing radiation. Although some preliminary work has been done in this regard, a quantitative comparison in now warranted (Hood 1968). On the same theme, transgenes or introduced transposable elements should be followed through multiple sexual cycles to see if there exists a difference in fate of unpaired DNA through meiosis. With the observations that U. maydis has very few repetitive elements and has completely eliminated various enzymes and genes involved in RNA silencing, it is possible that unnecessary DNA is eliminated at some point in the cell cycle. This experiment needs to be conducted and homologous recombination should be monitored as a possible mechanism.  148  5.3.2 Lack of RNAi: convergent transcripts, histones Since RNAi was found in U. hordei, gene deletion mutants need to be generated for the Dicer, Ago and the two RdRP genes. These mutants should indicate whether RNAi is essential for normal U. hordei growth. As well, they may reveal multiple RNAi pathways, especially with respect to specific functions of the two RdRP genes. This work could be done in the stable transgenic GUS line (Chapter 2). Deletion of RNAi genes and introduction of the pUBleX1-iGUS plasmid in this line will show the role that each deletion mutant plays in the various steps of PTGS. RNAi mutants should be monitored for effects on mating, meiosis and chromosome separation. An experiment must also be done to test if RNAi is involved in TGS in U. hordei. This might be accomplished by introducing a marker gene with an inducible promoter immediately downstream of the ORF and facing in the opposite direction. Induction of this promoter would create dsRNA at this gene. Expression after induction would need to be measured, as well as the methylation status of DNA and associated histones. Gene deletions for Dicer, Ago, the two RdRP and the two Chp genes should help determine if any are involved in this form of TGS.  5.3.3 U. hordei virulence and avirulence genes As already mentioned, strategies for identifying UhAvr1 include making sub-deletions of the UhAvr1 locus to pinpoint its precise location, as well as obtaining more DNA sequences for genes from BAC clones of the virulent parent. Since UhAvr1 is located at a cluster of secreted proteins in a region that appears to be heterochromatic, all such regions need to be identified in U. hordei now that the genome has been sequenced. This would require looking for genes embedded in loci rich with repetitive elements. Markers would need to be created for all such loci and used to check the mapping population (Uh364 X Uh362) for linkage to avirulence. Besides UhAvr1, at least two other Avr-genes are present in this mapping population. Similarly, 149  now that the genome has been sequenced, virulence factors that were previously identified through genetic crosses should now be found (Caten, et al. 1984, Pope and Wehrhahn 1991, Thomas and Huang 1985). This would involve linking the old mapping data to the genome sequence and identifying loci possessing the virulence factors. Fine mapping might lead to a small number of genes that could be knocked-down using RNAi to see virulence reduced. This study would be important for understanding virulence and possibly host specificity.  5.3.4 U. maydis as a model for the smuts. Although U. maydis and its pathogenicity on maize have been studied extensively, there appears to be a significant oversight, and as a result important information is missing that has relevance to other smut fungi. As far as I can see, all recent plant infection studies have looked at infection and lifecycle completion in maize seedlings. This is because seedlings are readily infected, take up very little space and data can be collected relatively quickly. The issue I have with this approach is that most related smuts cause disease on flowers. For U. maydis to be used appropriately as a model for other smuts, there needs to be a comprehensive study looking at infection of maize floral tissue, especially cobs. Such a study would have to look at effects on maize both at the cellular and molecular levels. It has been reported that U. maydis infection with the goal of producing edible “cuitlacoche” is most successful when plants are emasculated and unpollinated cobs are infected (Hermilo Leal Lara at the 4th International Ustilago Meeting, Marburg, Germany 2008). This is interesting since ovules develop to a certain stage and then remain quiescent, until pollination at which point seed development commences. From the appearance of U. maydis-infected maize cobs it is clear that individual kernels or ovules grow to an enormous size prior to being overtaken by the fungus. A detailed histological study is needed to see which cells are responsible for this growth. During normal seed development a fertilized embryo sac is surrounded by maternal nucellus and integuments. 150  Double fertilization of the egg and central cell within the embryo sac leads to formation of the embryo and endosperm, respectively. In the mature maize seed, the endosperm and embryo make up most of the biomass, while the nucellus is absorbed and the integuments form the seed coat. Prior to fertilization, the central cell and the egg cell are in cell cycle arrest (Huh, et al. 2007). It is essential to know if U. maydis targets one or both of these cells. Studies of mutants in Arabidopsis show that genes related to polycomb group proteins repress genes involved in cell cycle proliferation in central cells prior to fertilization (Huh, et al. 2008). Mutation of the polycomb genes results in central cell proliferation without fertilization. If U. maydis is able to break cell cycle arrest within maize ovules, then the mechanism and effectors need to be determined. Such information would not only be of interest to plant pathologists, but also to seed biologist. In addition to the cellular studies, gene expression data also need to be collected from both U. maydis and maize cobs at various stages of infection. This can be compared to expression data recently collected from infected leaves. During leaf infection, U. maydis converts leaves into carbon sinks and prevents them from entering C4 photosynthesis (Horst, et al. 2008). Since cobs are already carbon sinks and do not partake in photosynthesis, very different gene expression could occur in both organisms during this route of infection. Information gained from studying cob infections should prove more relevant to the biology of related smuts. Another screen that might prove useful is to search for effectors that allow U. maydis to infect leaves. Since U. maydis is related to flower-infecting smuts, it is likely that infection and lifecycle completion on leaves is an acquired trait. Mutagenesis of a solo-pathogenic line (eg. Strain SG200; Kamper, et al. 2006) and screening for mutants that are unable to infect leaves, but retain the ability to develop disease on cobs might reveal some interesting genes.  151  5.4 Concluding remarks The smut fungi are in a good position to remain as model organisms for the study of plant-microbe interactions well into the future. Genetic manipulation is routine for both U. maydis and U. hordei due to the abundant tools and resources available. With the soon to be released genome sequence for U. hordei and the related smut Sporisorium reilianum, even more comparative studies will be possible. Furthermore, the maize genome is near completion and will facilitate rapid advancement of knowledge through detailed studies of interactions between maize and U. maydis. Current work in several laboratories to characterize the many U. maydis secreted proteins will likely make important contributions. These studies in parallel with studies on U. hordei and barley should help address important questions regarding virulence and avirulence. Furthermore, the close relatedness of U. hordei to U. maydis puts these species at the forefront of studies on RNA silencing and genome evolution. It is my hope that the work I presented here helps to answer important questions and stimulate further study.  152  5.5 References Baker RE, Rogers K (2006) Phylogenetic analysis of fungal centromere H3 proteins. Genetics 174: 1481-1492 Bakkeren G, Jiang G, Warren RL, Butterfield Y, Shin H, Chiu R, Linning R, Schein J, Lee N, Hu G, Kupfer DM, Tang Y, Roe BA, Jones S, Marra M, Kronstad JW (2006) Mating factor linkage and genome evolution in basidiomycetous pathogens of cereals. Fungal Genet Biol 43: 655-666 Bhattacharjee S, Hiller NL, Liolios K, Win J, Kanneganti TD, Young C, Kamoun S, Haldar K (2006) The malarial host-targeting signal is conserved in the Irish potato famine pathogen. 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