INVESTIGATING THE MOLECULAR MECHANISMS OF FUSARIUM HEAD BLIGHT RESISTANCE IN WHEAT by Nora Afsaneh Foroud B.Sc., The University of Lethbridge, 2002 M.Sc., The University of Lethbridge, 2005 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in The Faculty of Graduate Studies (Plant Science) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) March 2011 © Nora Afsaneh Foroud, 2011 ii Abstract Fusarium Head Blight is a disease of cereal crops caused by a group of trichothecene- producing Fusarium species. Two major forms of resistance to Fusarium Head Blight are Type I resistance (resistance to initial infection) and Type II resistance (resistance to disease spread). Using functional genomics approaches, the effect of FHB-elicitors on the defense response of three wheat genotypes that share the susceptible cv. 'Superb' pedigree were evaluated. Distinct differences were observed between the resistant genotypes and 'Superb', as well as between the Type I and Type II genotypes. The data presented in this thesis suggests that different molecular mechanisms exist not only between susceptibility and resistance responses, but also between different forms of genetic resistance. It is proposed here that Type I resistance involves a combination of structural features that slow fungal penetration and the activation of a systemic response in uninfected tissues adjacent to the site of infection to prevent and minimize secondary infection; whereas, Type II resistance is more likely a form of local resistance. Based on the results from the functional genomics study, follow up experiments where wheat heads were primed with FHB elictiors and subsequently inoculated with a virulent F. graminearum strain and evaluated for changes in disease outcomes, support the hypothesis that Type I resistance involves the activation of a systemic response. Furthermore, an analysis of the role of plant hormone signalling using a combination of genetic and biochemical analysis suggest that jasmonic acid signalling is involved in Fusarium Head Blight resistance. iii Preface This thesis and all of its contents, including figures and diagrams, is original work, prepared by Nora A. Foroud, using Microsoft Office programs and ChemSketch (http://www.acdlabs.com/resources/freeware/chemsketch/). A version of Chapter 1 has been published as a review (Foroud NA., Eudes F. 2009. Trichothecenes in cereal grains. Int. J. Mol. Sci. 10:147-173). The experiments presented in Chapter 2 were designed by Dr. François Eudes and Nora A. Foroud, with feedback provided by Dr. Brian Ellis. The remaining experiments (Chapters 3-4), were designed by Nora A. Foroud, with feedback provided by Drs. François Eudes and Brian Ellis. The project was funded by Agriculture and Agri-Food Canada (AAFC)-Lethbridge Research Centre, and the Alberta Agriculture Research Initiative. All experimental work was conducted by Nora A. Foroud, with support provided as defined below: 1. Hybridization of fragmented and labeled cRNA to Affymetrix microarray chips (Chapter 2) was conducted by Brenda Oosterveen from Dr. Mark Jordan’s lab (AAFC-Winnipeg). 2. Dr. Thérèse Ouellet (AAFC-Ottawa) provided an improved annotation of the Affymetrix Wheat Gene Chip (Chapter 2). 3. Jenna Friedt (Co-op, University of Lethbridge), helped in the disease evaluation of FHB-infected heads for the hormone silencing experiment (Chapter 3). 4. Additional support is described within the thesis, including: a. protein sequencing (Chapter 2), hormone and DON quantification (Chapter 3) conducted at various service labs; b. transgenic plant material provided from other research teams within and outside of AAFC (Chapters 3 and 4). iv Table of Contents Abstract.............................................................................................................................. ii Preface............................................................................................................................... iii Table of Contents ............................................................................................................. iv List of Tables ..................................................................................................................... x List of Figures................................................................................................................... xi List of Schemes............................................................................................................... xiii List of Abbreviations ..................................................................................................... xiv Acknowledgements ...................................................................................................... xviii Dedication ....................................................................................................................... xix 1. Introduction................................................................................................................... 1 1.1 History and Background ......................................................................................... 2 1.1.1 Fusarium Head Blight: a brief history ........................................................... 2 1.1.2 The impact of Fusarium Head Blight............................................................. 3 1.1.3 Fusarium Head Blight management strategies .............................................. 5 1.2 Fusarium Species.................................................................................................... 7 1.2.1 Diseases caused by Fusarium species............................................................ 7 1.2.2 Fusarium morphology and classification....................................................... 9 1.3 Trichothecenes ...................................................................................................... 11 1.3.1 Trichothecene structure and toxicity............................................................ 11 1.3.2 Trichothecenes as aggressiveness factors in Fusarium Head Blight ........... 13 1.4 Fusarium Head Blight Resistance......................................................................... 19 v 1.4.1 Mechanisms of Fusarium Head Blight resistance........................................ 19 1.4.2 Sources of Fusarium Head Blight resistance ............................................... 23 1.4.3 Breeding for Fusarium Head Blight resistance............................................ 25 1.4.4 Engineering for Fusarium Head Blight resistance ....................................... 27 1.5 Overview............................................................................................................... 31 2. Differential Gene Expression and Protein Accumulation Patterns in Resistant and Susceptible Fusarium Head Blight Interactions in Wheat.......................................... 33 2.1 Introduction........................................................................................................... 33 2.2 Materials and Methods.......................................................................................... 36 2.2.1 Plant material ............................................................................................... 36 2.2.2 Inocula.......................................................................................................... 36 2.2.3 Inoculations and tissue collection ................................................................ 37 2.2.4 Microarray.................................................................................................... 39 2.2.5 Real-time RT-PCR....................................................................................... 40 2.2.6 Protein sample preparations ......................................................................... 41 2.2.7 2D-electrophoresis ....................................................................................... 42 2.2.8 2D-electrophoresis optimization for resolution in the basic range .............. 44 2.3 Results................................................................................................................... 46 2.3.1 Real-time RT- PCR validation of microarray results .................................. 76 2.3.2 Constitutive differences in gene expression ................................................ 76 2.3.3 Challenge-induced differences in global gene expression........................... 76 2.3.4 Challenge-induced differential gene expression related to defence response pathways ...................................................................................................... 77 vi 2.3.5 2D-electrophoresis: optimization and resolution ......................................... 80 2.3.6 Genotype differences in protein accumulation ............................................ 81 2.3.7 Challenge-induced differential protein accumulation.................................. 83 2.4 Discussion ............................................................................................................. 84 2.4.1 Constitutive differences in gene expression ................................................ 84 2.4.2 Challenge-induced differences in gene expression and protein accumulation patterns ......................................................................................................... 85 2.4.3 Deoxynivalenol-induced up-regulation of genes encoding ribosomal proteins in Fusarium Head Blight-resistant wheat..................................................... 87 2.4.4 Early expression of pathogenesis-related proteins in resistant wheat.......... 88 2.4.5 Up-regulation of phenylpropanoid metabolism in Type I resistant DH1 .... 90 2.4.6 Jasmonic acid signalling and Type II resistance.......................................... 92 2.5 Conclusions........................................................................................................... 93 3. The Role of Plant Hormone Signalling in Mediating Fusarium Head Blight Resistance in Wheat........................................................................................................ 95 3.1 Introduction........................................................................................................... 95 3.2 Materials and Methods........................................................................................ 110 3.2.1 Plant material ............................................................................................. 110 3.2.2 Screening for hormone silencing ............................................................... 111 3.2.3 Inoculations................................................................................................ 112 3.2.4 Fusarium Head Blight disease assays ........................................................ 113 188.8.131.52 Priming experiment ............................................................................ 113 184.108.40.206 Point-inoculation controls for priming experiment............................ 113 vii 220.127.116.11 Hormone silencing experiment .......................................................... 114 3.2.5 Deoxynivalenol quantification from priming experiment ......................... 114 3.2.6 Hormone profiling of primed wheat spikes ............................................... 115 3.3 Results................................................................................................................. 117 3.3.1 The effect of point-inoculation on disease outcomes ................................ 117 3.3.2 Priming experiment disease evaluation ..................................................... 118 3.3.3 Priming has no effect on SA or JA accumulation within point-inoculated wheat spikes ............................................................................................... 120 3.3.4 DON accumulation in mature kernels from primed and spray-inoculated wheat spikes is primarily correlated with disease severity ........................ 121 3.3.5 Screening for hormone silencing ............................................................... 123 18.104.22.168 Screening for NAHG over-expression and SA degradation .............. 123 22.214.171.124 Screening for silencing of JA biosynthesis ........................................ 124 126.96.36.199 Screening for silencing of ET signalling............................................ 125 3.3.6 Hormone silencing disease evaluation....................................................... 132 3.4 Discussion ........................................................................................................... 135 3.4.1 Priming induces changes in FHB-resistance and -susceptibility of wheat 135 3.4.2 The role of hormone signalling in FHB-resistance.................................... 137 3.5 Conclusions......................................................................................................... 140 4. Investigating a Putative Role for TaLTP3, a Wheat Lipid Transfer Protein, in Fusarium Head Blight Resistance ............................................................................... 142 4.1 Introduction......................................................................................................... 142 4.2 Materials and Methods........................................................................................ 144 viii 4.2.1 Plant material ............................................................................................. 144 4.2.2 TaLTP3 and TaPIP1 gene expression analysis.......................................... 145 4.2.3 Microscopy ................................................................................................ 146 4.2.4 Recombinant expression and purification of TaLTP3 ............................... 147 188.8.131.52 Recombinant protein expression ........................................................ 147 184.108.40.206 Recombinant protein purification....................................................... 148 220.127.116.11 Trx tag removal trial from recombinant TaLTP3 fusion protein ....... 149 4.2.5 LTP binding and antifungal assays ............................................................ 151 4.2.6 Transgenic modification of TaLTP3 expression in DH1 and ‘Superb’ ..... 153 4.3 Results................................................................................................................. 154 4.3.1 Tissue-specific expression of TaLTP3 and TaPIP1 in DH1...................... 154 4.3.2 Cuticle thickness ........................................................................................ 154 4.3.3 Purification and binding activity assay for recombinant LTP3trx............. 156 4.3.4 rLTP3trx-F. graminearum inhibition assays ............................................. 163 4.3.5 Transgenic modification of TaLTP3 expression in DH1 and ‘Superb’ ..... 164 4.4 Discussion ........................................................................................................... 164 4.4.1 Validation of TaLTP3 and TaPIP1 transcript accumulation and evaluation of organ-specific expression in DH1.............................................................. 164 4.4.2 Measurement of cuticle thickness in DH1 compared with ‘Superb’ ......... 166 4.4.3 TaLTP3 does not exhibit antifungal activity towards F. graminearum..... 167 4.5 Conclusions......................................................................................................... 167 5 Conclusions................................................................................................................. 170 Bibliography .................................................................................................................. 178 ix Appendix: Supplementary Tables and Figures.......................................................... 212 x List of Tables Table 1.1 Genes involved in trichothecene production. ................................................... 15 Table 1.2 FHB resistance mechanisms in cereals. ............................................................ 20 Table 2.1 Primer sequences for qPCR validation. ............................................................ 40 Table 2.2 Constitutive difference between transcriptomes of FHB-resistant genotypes and susceptible ‘Superb’.......................................................................................................... 47 Table 2.3 Genotype differences in challenge-induced protein accumulation................... 52 Table 2.4 Challenge-dependent changes in defence-related genes in FHB-susceptible ‘Superb’ and resistant genotypes. ..................................................................................... 56 Table 2.5 Challenge-induced differential protein accumulation....................................... 68 Table 2.6 qPCR validation of 2FD in microarray............................................................. 75 Table 3.1 Transgenic crosses for hormone silencing...................................................... 111 Table 3.2 Constitutive levels of SA and JA in untreated wheat heads. .......................... 122 Table 3.3 Time-course analysis of SA and JA accumulation in wheat spikes primed with FHB elicitors. .................................................................................................................. 122 Table 3.4 Effect of priming in DON accumulation in wheat kernels. ............................ 123 Table 3.5 Screen of transgenic crosses by hormone quantification and qPCR. ............. 126 Table 4.1 Tracking TaLTP3 and TaPIP1 expression within the wheat spikelet............. 156 Table A1. Challenge-dependent differentially regulated genes in FHB-susceptible ‘Superb’ amd resistant genotypes. .................................................................................. 212 Table A2. Effect of hormone silencing on disease severity at different days after inoculation....................................................................................................................... 286 xi List of Figures Figure 1.1 Macroconidia of Fusarium graminearum. ........................................................ 9 Figure 1.2 Type A and B trichothecene structures. .......................................................... 12 Figure 2.1 Inoculation of wheat spikes and harvesting of spikelets for RNA and protein extractions. ........................................................................................................................ 38 Figure 2.2 Number of treatment-dependent differentially regulated transcripts. ............. 78 Figure 2.3 2D-electrophoresis optimization. .................................................................... 82 Figure 3.1 Structure of chemical priming agents.............................................................. 98 Figure 3.2 Phytohoromone biosynthesis and signalling pathways ................................. 103 Figure 3.3 Disease progression in three wheat genotypes: ‘Superb’, DH1, and DH2.... 120 Figure 3.4 Effect of hormone silencing on FHB-disease progression in wheat ............. 134 Figure 4.1 Components of the wheat floret for organ dissection.................................... 145 Figure 4.2 Wheat lipid transfer protein 3 sequence and construct designs..................... 150 Figure 4.3 TEM of GC from ‘Superb’ and DH1. ........................................................... 157 Figure 4.4 Recombinant LTP3trx digest trial ................................................................. 159 Figure 4.5 Purification of recombinant TaLTP3trx and Trx........................................... 161 Figure 4.6 TaLTP3 binding and inhibition assays .......................................................... 163 Figure 5.1 Model for molecular mechanisms of FHB defence response........................ 177 Figure A1. NahG expression cassette ............................................................................. 291 Figure A2. AOS sequence and RNAi silencing construct. ............................................. 292 Figure A3. EIN2 sequence and RNAi silencing construct ............................................. 295 Figure A4. Elongation factor 1-alpha (EF1α) housekeeping gene sequence.................. 296 xii Figure A5. TaLTP3 sequence alignment and primer and probe design ......................... 300 Figure A6. TaPIP1 sequence alignment and primer and probe design........................... 312 xiii List of Schemes Scheme 1.1 Trichothecene biosynthesis pathways ........................................................... 16 xiv List of Abbreviations 15-ADON 15-O-acetyl deoxynivalenol 3-ADON 3-O-acetyl deoxynivalenol 4,15-DAS 4,15-diacetoxyscirpenol 4-VP 4-vinylpyridine ACC 1-aminocyclopropane-1-carboxylic acid ACO 1-aminocyclopropane-1-carboxylic acid oxidase ACS 1-aminocyclopropane-1-carboxylic acid synthase AOC allene oxide cyclase AOS allene oxide synthase ATA alimentary toxic aleukia BA2H benzoic acid 2-hydrolase BSMV barley stripe mosaic virus BTH benzothiadiazole COI1 coronatine-insensitive 1 CTR1 constitutive triple response 1 DON 4-deoxynivalenol DTT dithiothreitol EC expression construct EDS5 enhanced disease susceptibility 5 EIN ethylene insenstive EIP ethylene-inducible protein xv EK enterokinase ET ethylene ETR ethylene receptor ETRF ethylene response factor FD fold-difference FDK Fusarium Damaged Kernels FEB Fusarium Ear Blight FgTri5- Fusarium graminearum GZ3639 trichothecene non-producing mutant FgTri5+ Fusarium graminearum GZ3639 wild-type strain FHB Fusarium Head Blight GST glutathione-S-transferase HC hairpin construct HST host-selective toxin IAA iodoacetamide ICS isochorismate synthase IEF isoelectric focusing JA jasmonic acid JIP jasmonic acid-inducible protein LBB lipid transfer protein binding buffer LOX lipoxygenase LTP lipid transfer protein MeJA methyl jasmonate MFS major facilitator superfamily xvi MS mass spectrometry NBB native binding buffer NEB native elution buffer NIV nivalenol NPR1 non-pathogenesis related 1 OPR 12-oxophytodienoic acid reductase PAL phenylalanine ammonia lyase PIP aquaporin plasma membrane intrinsic protein PK protein kinase POX peroxidase PR pathogenesis-related QTL quantitative trait locus rEK recombinant enterokinase RG rosetta gami 2 cells RISC RNA-induced silencing complex rLTP3trx recombinant wheat lipid transfer protein-thioredoxin fusion protein RNAi RNA interference ROS reactive oxygen species rTrx recombinant thioredoxin SA salicylic acid SAM S-adenosylmethionine SEC size exclusion chromatography SID1 SA induction-deficient 1 xvii ssRNA single stranded RNA TaLTP wheat lipid transfer protein TBP tributylphosphine TCEP tris(2)carboxyethylphosphine TEM transmission electron microscopy TF transcription factor TOF time-of-flight TRI5 trichodiene synthase Trx thioredoxin VIGS virus-induced gene silencing ZON zearalenone xviii Acknowledgements My supervisors, Dr. François Eudes and Dr. Brian Ellis, thank you for your confidence, your support and your guidance throughout my thesis program. I could not have asked for two better mentors. My supervisory committee, Dr. Leonard Foster and Dr. Steven Lund, thank for your advice and feedback, and for keeping me on track. My friends, colleagues, and mentors at the Agriculture and Agri-Food Canada, Denise Nilsson, Eric Amundsen, Dr. Ana Badea, Dr. André Laroche, Dr. Denis Gaudet, Dr. Thérèse Ouellet, Michele Frick, Carolyn Penniket, Byron Puchalski, Dr. Fran Legget, Jenna Friedt, Thérèse Despins, Dr. Rob Graf, Dr. Harpinder Randhawa, Dr. Gopal Subramaniam, thank you for your friendship, your feedback and support. My fellow graduate students and post-docs at the University of British Columbia, Hardy Hall, QingNing Zeng, Mathias Schuetz, Apruv Bhargava, Jia Cheng, Adrienne Nye, thank you for your friendship and for helping see things from another perspective. Thank you to the greenhouse staff, at the Lethbridge Research Centre, for your support. Thank you to my family: my parents Zahra and Nader Foroud, my sister and her husband, Afra Foroud and Brad Gom, my sister Rebecca Foroud, and my husband Paul Hazendonk, for your never ending support, love and friendship. xix Dedication To my mother and father, Zahra Foroud and Dr. Nader Foroud, for your support and guidance. ع To my husband Dr. Paul Hazendonk, for your patience and love. 1 1. Introduction Fusarium Head Blight (FHB) is a destructive disease of grain crops, with worldwide economic and health impacts. Wheat is one of the most widely FHB-affected crops and suffers the largest damage. The disease is caused by a range of trichothecene-producing Fusarium species, F. graminearum (teleomorph: Gibberella zeae) being the most economically relevant (Parry et al., 1995; Tóth et al., 2005). The fungus infects the cereal inflorescence during anthesis and grain development. Once established within the floret, fungal hyphae can spread from spikelet to spikelet through the vascular bundles of the rachis (Parry et al., 1995). Fusarium species produce trichothecene mycotoxins that accumulate in the kernels of infected spikelets, reducing both food quality and grain germination. Trichothecenes are phytotoxic and are associated with aggressiveness of F. graminearum disease spread in Triticeae (Proctor et al., 1995a; Eudes et al., 2001; Bai et al., 2002; Langevin et al., 2004; Jansen et al., 2005; Maier et al., 2006), but are not necessary for establishing initial infection (Bai et al., 2002; Jansen et al., 2005). The most effective means to prevent damage caused by this disease is to cultivate crops with high levels of genetic resistance to FHB (Eudes et al., 2004; Foroud and Eudes, 2009). Two major forms of FHB resistance have been characterized in wheat (Schroeder and Christensen, 1963). Type I resistance is defined as resistance to initial infection, and Type II resistance is resistance to disease spread within an infected spike. An improved understanding of the molecular mechanisms associated with resistance has the potential to provide essential new tools for the development of improved FHB- resistant germplasm. 2 1.1 History and Background 1.1.1 Fusarium Head Blight: a brief history The first documented FHB-outbreak occurred in England in 1884, where the disease was named “wheat scab” (Stack, 2003). Outbreaks have since been reported in the Americas (Moschini and Fortugno, 1996; Gilbert and Tekauz, 2000; McMullen, 2003), Eurasia (Bhat et al., 1989; Parry et al., 1995; McCormick, 2003), Australia (Burgess et al., 1987), and South Africa (Scott et al., 1988; Kriel and Pretorius, 2008). The most notorious epidemic in North America spanned the 1990s, where in the United States alone, estimated economic losses approached 3 billion USD (McMullen et al., 1997). Rice-wheat rotations are routine in many Asian countries, and while FHB is more endemic in the latter, rice crops still serve as a host for inoculum buildup that could lead to an outbreak. FHB of barley and wheat is a pervasive problem in China. From 1951 to 1990, wheat farmers were burdened with seven severe epidemics (exceeding 40% yield losses) and 14 moderate epidemics (10-20% yield losses). A reduced frequency of severe epidemics has subsequently been observed with the introduction of moderately resistant cultivars (Hongxiang et al., 2008). In South Africa, a double cropping system (with maize as a summer crop and wheat as a winter crop) in combination with conservation tillage has led to a growing FHB problem, especially in regions where irrigation is required (Kriel and Pretorius, 2008). Maize crop debris is one of the worst culprits for harbouring Fusarium ascospores. Increased maize production in the United States driven by the biofuel initiatives (Cassman and Liska, 2007) may introduce an increased threat of FHB in North America—a threat that may become an issue of food security. Maize is a major 3 staple food and the increase in maize production for non-food products is already removing valuable farmland from food production (Cassman and Liska, 2007), thus reducing the volume of wheat and barley, among other crops, available for food uses. If the soil-borne inocula were to increase as a result of expanded maize cultivation, this would threaten the already dwindling food supply of wheat and other grains. This concern also emphasizes the need to improve FHB-management strategies and the importance of producing highly resistant cultivars. 1.1.2 The impact of Fusarium Head Blight FHB-related yield losses are directly related to effects of trichothecenes, which accumulate in the grain of wheat spikes that become infected during or post-anthesis (Del Ponte et al., 2007), although significant yield losses are more relevant in early infections (during anthesis to the early stages of kernel development) (Bushnell et al., 2003; Steffenson, 2003). The kernels that do develop from FHB-infected spikes are usually contaminated with trichothecenes, 4-deoxynivalenol (DON) being the most prevalant. Trichothecene-contaminated grain is readily distinguished from its shriveled and discolored appearance. Several studies have shown that the percentage of Fusarium Damaged Kernels (FDK) serves as a reliable estimate of DON content (Miedaner et al., 2001; Mesterházy, 2002). This allows for rapid visual screening of FDK in order to prevent contaminated grain entering the food chain in unsafe quantities. Nevertheless, FDK screening for toxin contamination should be used with caution since reduced physical damage is observed if infection occurs past the soft dough stages of kernel development (Pestka and Smolinski, 2005; Del Ponte et al., 2007). FDK can therefore be 4 used as a preliminary screen when sorting and grading grain, but quantitative trichothecene testing is advisable before the grain enters the food chain. Maximum FDK limits in place for different Canadian wheat classes and varieties are enforced by the Canadian Grain Commission at licensed grain elevators. DON-testing is carried out on end-products by the Canadian Food Inspection Agency and Health Canada to ensure the maximum allowable levels of this trichothecene in food-stuffs are not exceeded. Currently, the maximum limit of DON is set at 2 ppm for Canadian soft wheat (1 ppm for use in baby food), and the establishment of maximum limits of DON in hard wheat is currently being evaluated (Tom Nowicki and Randall Clear, Canadian Grain Commission, Winnipeg, Manitoba, personal communications). The established DON limits are also being reviewed, and more rigid standards may be imposed in order to harmonize the Canadian standards with those of the European Union. DON tolerance in China, Hungary, Russia, Switzerland, and the United States is 1 ppm; and in Austria, Germany, and the Netherlands is 0.5 ppm (Pestka and Smolinski, 2005). Standards are also in place for grain that is used in animal feed, since trichothecenes cause similar ailments in farm animals as in humans (Rotter et al., 1996). Ingestion of contaminated grain can cause severe intestinal irritation in mammals, resulting in feed refusal in livestock (Eriksen and Pettersson, 2004), and can lead to a potentially fatal condition in humans and other mammals known as alimentary toxic aleukia (ATA). In addition to gastrointestinal irritation, symptoms of ATA can include ataxia, dyspenia, and subcutaneous hemorrhaging (Lutsky et al., 1978). The first recorded Fusarium-related ATA outbreak occurred in Siberia in 1913, but the human impacts of ATA may go as far back as the 5th century B.C.; it has been suggested that the plague of 5 Athens in 430-426 B.C. was an outbreak of ATA (Schoental, 1994). Symptoms of a reported disease epidemic in New Hampshire in the 1730s are also reminiscent of ATA (reviewed in Stack, 2003). The most devastating outbreak on record occurred in Russia between 1942 and 1948, where at least 100,000 people died. In this case, a post-world war II famine in Russia led people to consume moldy over-wintered grain which had become contaminated with the T-2 trichothecene toxin producers F. sporotrichioides or F. poae (Mayer, 1953; Yagen and Joffe, 1976). However, despite these massive food- borne illness outbreaks, it was not until 1950 that the connection between Fusarium toxins and ATA was established. While some Fusarium species may proliferate on grain during storage, or over-wintered in the field, the main source of trichothecenes in the food chain is from FHB-infection of growing grain. The most effective means to minimize mycotoxin contamination in food-stuff is therefore to find ways to prevent or minimize the impact of this disease on wheat crops in the field. 1.1.3 Fusarium Head Blight management strategies FHB-management strategies are essential for reducing the economic damage and potential health hazards associated with this disease. Three components have been identified as being necessary for an FHB outbreak: (1) the source of inoculum, (2) the presence of susceptible hosts, and (3) favorable environmental conditions. Strategies have been developed to target each of these components. The first component, inoculum source, is usually present in the form of ascospores in the soil. It has been demonstrated that crop-rotation, tillage, chemical/ biological control, and the use of FHB-resistant cultivars can all contribute to reducing the presence of Fusarium/Gibberella- 6 contaminated crops and/or crop debris and thereby reducing inoculum buildup in the soil (Miller et al., 1998; Dill-Macky and Jones, 2000; Jones, 2000; Gilbert and Fernando, 2004; Horsley et al., 2006; Khan et al., 2006; Paul et al., 2007). The second essential component for an outbreak is a suitable host on which spores can germinate and establish disease. Unfortunately, the number of registered cultivars possessing good resistance is limited, and the cultivation of resistant cultivars is the only way, short of growing only non-host crops, to deny a suitable host for the disease. Moreover, farmers preferentially select cultivars with good agronomic characteristics, and these, regrettably, tend to correlate with poor resistance (see section 1.4). The third component necessary for disease establishment, namely favorable environmental conditions, is difficult to affect. FHB thrives in wet, humid conditions with an optimum temperature of 25oC during the anthesis and grain filling stages of crop development (Parry et al., 1995). While the weather cannot be controlled, disease forecasting models can be used to devise an effective spraying schedule for chemical control, and for more organized post-harvest management of potentially diseased kernels (Moschini and Fortugno, 1996; De Wolf et al., 2003; Xu, 2003; Prandini et al., 2008). Application of more than one management strategy (McMullen et al., 2008) has been shown to be the most effective route to reducing FHB severity and trichothecene accumulation in grains—although, the development of highly resistant cultivars with good agronomic qualities would have the largest impact. The use of resistant cultivars can manage each of the three components essential for disease. First, it can reduce the amount of inoculum buildup in host-crop debris (and subsequently in the soil). Second, the suitability of host crops can be reduced by using highly-resistant cultivars. Finally, 7 while a strong genotype-environment interaction has been observed in FHB disease outcomes (Miedaner et al., 2001), studies have shown that the effect of the environment can be indirectly managed by using highly-resistant genotypes. In contrast, while genotypes with moderate or intermediate resistance can reduce the impact of FHB, this resistance is not stable under high disease pressure. In a two-year FHB field trial of winter wheat, rye and triticale genotypes, grown in three regions of South-West Germany, Miedaner et al. (2001) observed strong interactions between genotype and disease outcomes, and between genotype and trichothecene accumulation. They also observed a significant impact of the environment on these interactions, but the effect of the environment was reduced in more resistant genotypes. Similar interactions have been observed between wheat genotypes and F. graminearum strains (Foroud et al., in preparation), where resistance to both disease spread and the development of FDK is stable in highly resistant lines, but variable in intermediate/moderate sources of resistance. These studies emphasize the importance of developing highly resistant cultivars. Furthermore, the level of resistance “is more important in governing DON accumulation in a given cultivar than is the aggressiveness of an isolate (Mesterházy, 2002)”. 1.2 Fusarium Species 1.2.1 Diseases caused by Fusarium species The genus Fusarium consists of a wide range of soil-borne saprophytic and pathogenic fungi. Some species of Fusarium, including F. moniliforme, oxysporum, solani, and 8 verticillioides, are known to infect immune-compromised humans (Guarro and Gené, 1995), but plant species are the main target of Fusarium pathogenicity. Fusarium fungi are known to be the causative agents in a variety of plant diseases in addition to FHB; it is said that most species of plants are susceptible to at least one disease caused by this genus of fungi (Leslie and Summerell, 2006). A broad range of host plants are susceptible to vascular wilt disease caused by Fusarium species, in which the fungus colonizes the xylem via the roots and the growing mycelium eventually causes vessel obstruction, blocking transport of water to the aerial parts of the plant (Pietro et al., 2003). Over 100 F. oxysporum formae speciales have been identified for various crops species, including F. oxysporum f. sp. cucumerinum, lycopersici, and pisi, which infect cucumber, tomato, and pea crops, respectively (Pietro et al., 2003). F. oxysporum f. sp. cubense was responsible for the vascular wilt epidemic of bananas, known as panama disease, in the 1950s which forced growers to switch cultivars from Gros Michael to a more resistant cultivar, Cavendis, although the threat of this disease is still pervasive (Ploetz, 1994). F. oxysporum, among other species, is are also responsible for crown rot disease in a variety of crops, including tomatoes (Pharand et al., 2002), asparagus (Hartung and Stephens, 1983), and alfalfa (Couture et al., 2002). Wheat and barley are susceptible to crown rot, caused by species similar to those responsible for FHB, including F. graminearum, pseudograminearum, and culmorum (Backhouse et al., 2004; Smiley et al., 2005; Mudge et al., 2007). Interestingly, DON accumulation in wheat spikes, similar to that observed by FHB, can be observed in wheat inoculated with the crown rot pathogens, F. graminearum or pseudograminearum (Mudge et al., 2007). 9 A. B. Figure 1.1 Macroconidia of Fusarium graminearum. A. The F. graminearum strain GZ3639 produce macroconidia with a straight ventral (lower) and slightly arched dorsal (upper) side, with well defined septa. The apical cell (left) is tapered and the basal cell (right) is foot shaped. B. Macroconidia develop off the hyphae of growing mycelium, and are eventually released by pinching off at the basal cell (arrow). Photographs were taken under light microscopy with 100 x magnification. 1.2.2 Fusarium morphology and classification Fusarium species are ascomycetes with teleomorphs in Gibberella, Albonectria, and Haematonectria, but in some species, such as F. culmorum and sporotrichioides, no teleomorph has yet been identified. While it is standard practice to use the teleomorphic name when referring to the holomorph, in the case of Fusarium the anamorphic name is more commonly used (Leslie and Summerell, 2006). Fusarium species produce crescent- shaped macroconidia spores and some also produce microconidia. The size and number of septa in the macroconidia are often used to differentiate between species (Leslie and 10 Summerell, 2006). F. graminearum produce elongated 5-6 septate pale orange macroconidia with a tapered apical and a foot-shaped basal cell (Figure 1.1). The ascospores from the sexual stage, G. zeae, are slightly curved, fusoid-shaped spores with 1-3 septa, which are released from a dark purple-black perithecium. F. culmorum, which does not have a known sexual stage, produces short orange macroconidia that are wide relative to their length, with 3-4 septa, and a rounded apical and notched basal cell. F. sporotrichioides produces macroconidia with variable morphological features, generally with 3-5 septa, and also produces microconidia that are 1-septate and ellipsoid in shape (Leslie and Summerell, 2006). The F. graminearum species complex (Fg complex), the main group responsible for FHB in North America, is composed of more than 10 phylogenetically distinct species that produce primarily DON and DON-derivatives (O'Donnell et al., 2000; Starkey et al., 2007; Ward et al., 2008). Over the past decade, the nature of the pathogen has been evolving, and a shift in the trichothecene genotype and/or chemotypes of the Fg complex has been observed (Ward et al., 2008). Historically, the 15-O-acetyl DON (15-ADON) chemotype had been the most prevalent chemotype in North America. However, in recent years, 3-O-acetyl DON (3-ADON) chemotypes, and strains with increased 15- ADON production, have been identified on this continent. The high-producing 15- ADON population is composed of strains that are genetically similar to, and are believed to have parts of their genomes derived from the 3-ADON population (Gale et al., 2005; Ward et al., 2008). Additionally, an increased presence of NIV chemotypes has also been observed in North America (Starkey et al., 2007). The 3-ADON chemotypes appear to be more virulent than the 15-ADON chemotypes (Ward et al., 2008), although there is no 11 evidence linking the trichothecene-chemotype itself to this observed phenotypic difference. It has been proposed that additional factors of aggressiveness in the 3-ADON genotypes may be responsible, at least in part, for the difference in aggressiveness between these populations, since differences in aggressiveness were observed in a study conducted with strains that are genetically coded for 3-ADON production, but where 3- ADON production has not been observed in planta (Foroud et al., in preparation). 1.3 Trichothecenes 1.3.1 Trichothecene structure and toxicity Trichothecenes are toxic sesquiterpenoid compounds composed of a central core of fused cyclohexene/tetrahydropyran rings, with a cyclopentyl moiety fused to the tetrahydropyran ring through C-2 and C-5 (Figure 1.2). Furthermore, C-12 comprises part of an epoxide functionality which has been deemed crucial for toxicity (Desjardins et al., 1993). There are five positions at which functionality varies, most commonly featuring hydroxyl or acetyl groups. Four types of trichothecenes have been identified from trichothecene-producing fungi: types A, B, C and D. The major type A trichothecenes in Fusarium species include T-2 toxin and HT-2 toxin, both of which possess an isovalerate ester at C-8 (Mirocha et al., 2003). F. sporotrichioides and F. poae are some of the major type A trichothecene producers (Liddell, 2003). Type A trichothecenes are highly toxic; T-2 toxin has been reported to be roughly ten times more toxic in mammals than DON (Ueno, 1983). Type A toxins are thought to have been responsible for the previously described ATA outbreak of post-world war Russia. On the other hand, DON is the most 12 prevalent toxin associated with FHB, and belongs to the more phytotoxic (Eudes et al., 2001) Type B trichothecenes which feature a ketone at C-8 (Mirocha et al., 2003). F. culmorum and F. graminearum, which are the major disease agents in FHB of wheat, produce mainly DON, NIV, and their derivatives (Liddell, 2003). Type C and D trichothecenes, which are characterized by a second epoxide (C-7,8 or C-9,10) or an ester-linked macrocycle (C-4,16), respectively, are not associated with FHB (Sudakin, 2003). Other mycotoxins, such as zearalenone (ZON), fumonisins, moniliformin and butenolide are also produced by Fusarium species (Mirocha et al., 2003; Desjardins et al., 2007b; Harris et al., 2007), but trichothecenes are the toxins of main interest in this disease. The trichothecene biosynthesis pathway is summarized in Scheme 1, and genes associated with trichothecene production are presented in Table 1.1. Figure 1.2 Type A and B trichothecene structures. Type A trichothecenes include T-2 toxin, HT-2 toxin, 4,15-diacetoxyscirpenol (4,15-DAS). Type B trichothecenes include nivalenol (NIV), 4-deoxynivalenol (DON), 3-O-acetyl DON (3-ADON), and 15-O-acetyl DON (15-ADON). OAc = acetyl function; OIsoval = isovalerate function. Toxin R1 R2 R3 R4 R5 DON –OH –H –OH –OH =O 3-ADON –OAc –H –OH –OH =O 15-ADON –OH –H –OAc –OH =O NIV –OH –OH –OH –OH =O T-2 toxin –OH –OAc –OAc –H –OIsoval HT-2 toxin –OH –OH –OAc –H –OIsoval 11 6 10 7 9 8 2 12 O 5 15 14 3 4 16 R1 R3 R5 R4 13 O R2 13 Trichothecene exposure can lead to growth retardation in eukaryotes, reproductive dysfunction in mammals and inhibition of seedling growth/regeneration in plants (reviewed in Rocha et al., 2005). The toxicity of trichothecenes is attributed to their ability to inhibit the peptidyl transferase activity of 60S ribosomes (McLaughlin et al., 1977). Additional impacts of trichothecene toxicity (reviewed in Ueno, 1977; Ueno, 1985; Rocha et al., 2005) include disruption of nucleic acid synthesis, mitochondrial function, membrane integrity, and cell division. Trichothecenes have also been shown to induce apoptosis in animal cells (Shifrin and Anderson, 1999), and may induce programmed cell death in plants (reviewed in Rocha et al., 2005). 1.3.2 Trichothecenes as aggressiveness factors in Fusarium Head Blight Different levels of aggressiveness and pathogenicity have been observed in different isolates of a given Fusarium species. These differences can be attributed only in part to fitness, which suggests that aggressiveness factors may contribute to disease outcomes. Toxins have long been implicated as aggressiveness factors in various pathogen systems. Host-selective toxins (HSTs) are pathogen-produced toxins shown to be necessary for disease to occur in some plant-pathogen interactions. HSTs are toxic only toward the target host (reviewed in Wolpert et al., 2002). Examples of HSTs include AK-toxin of Alternaria alternata, victorin of Cochliobolus victoriae, and T-toxin of C. heterostrophus, the causal agents in black spot of Japanese pear (Tanaka et al., 1999), Victoria blight of oats (Navarre and Wolpert, 1999), and corn leaf blight (Baker et al., 2006), respectively. Other examples of toxins with less host specificity, but still involved plant pathogen aggressiveness or pathogenicity, include botrydial and its derivatives from 14 Botrytis cinerea, which causes grey mould in over 200 plant species (Choquer et al., 2007; Williamson et al., 2007), and zinniol, which is produced by Alternaria species responsible for a variety of plant diseases (Cotty and Misaghi, 1984; Robeson and Strobel, 1984; Thuleau et al., 1988; Moreno-Escobar et al., 2005). Over the past few decades, evidence that trichothecenes act as non-host-selective toxins involved in aggressiveness of Fusarium-related diseases has been accumulating. Observations by Beremand et al. (1991) suggested a link between trichothecene production, fertility, and pathogenicity of F. sambucinum isolates. Correlations between fungal biomass (measured by ergosterol quantification) and DON accumulation in cereal grains have been observed (Perkowski et al., 1996; Miedaner et al., 2000; Miedaner et al., 2001). A link between DON accumulation and disease outcomes has also been observed (Hart et al., 1984; Snijders and Perkowski, 1990). The trichothecene chemotype of Fusarium isolates (Foroud et al., in preparation), and the cumulative impact of multiple trichothecenes produced either by a single isolate (Mesterhazy et al., 1999) or by a composite of isolates (Arseniuk et al., 1993), may increase disease severity. In addition, some FHB-resistant plant genotypes have been shown to detoxify DON, primarily by glycosylation (reviewed in Boutigny et al., 2008). Together, these data suggest that the ability of Fusarium species to cause disease is linked to trichothecene accumulation in the host, and that reduced aggressiveness may be linked to either reduced toxin production by the pathogen, or removal/degradation of the toxin by the host. 15 Table 1.1 Genes involved in trichothecene production. For more details on trichothecene gene cluster see (Hohn et al., 1993; Alexander et al., 1997; Kimura et al., 2003; Brown et al., 2004; Kimura et al., 2007). Gene Cluster Description References Enzymes: for pathway reactions see also Scheme 1 Tri1 Tri1- Tri16 C-7 monooxygenase (F. graminearum); C-8 monooxygenase (F. graminearum, F. sporotrichioides) (Beremand, 1987; Brown et al., 2003; Meek et al., 2003; McCormick et al., 2006) Tri3 Core Tri 15-O-acetyltransferase (McCormick et al., 1996) Tri4 Core Tri monooxygenase (Hohn and Vanmiddlesworth, 1986; Tokai et al., 2007) Tri5 Core Tri sesquiterpene cyclase (Hohn and Beremand, 1989) Tri7 Core Tri 4-O-acetyltransferase; functional F. graminearum TRI7 required for NIV- chemotype; functional F. sporotrichioides TRI7 required for T-2 toxin production (Brown et al., 2001; Lee et al., 2002) Tri8 Core Tri C-3 deacetylase; functional F. sporotrichioides TRI8 required for T-2 toxin production (Brown et al., 2001; McCormick and Alexander, 2002) Tri9 Core Tri (Brown et al., 2001) Tri11 Core Tri C-15 monooxygenase (McCormick et al., 1999) Tri13 Core Tri monooxygenase; functional F. graminearum TRI13 required for NIV-chemotype (Lee et al., 2002; Kim et al., 2003) Tri14 Core Tri Tri16 Tri1- (Peplow et al., 2003) Tri101 None 15-O-acetyltransferase (McCormick et al., 1990; Kimura et al., 1998) Transcription Factors Tri6 Core Tri zinc-finger; binding motif (YNAGGCC) in most promoter regions within Tri5 cluster (Proctor et al., 1995b; Hohn et al., 1999; Brown et al., Tri10 Core Tri (Tag et al., 2001) Other Tri12 Core Tri MFS transporter involved in trichothecene efflux (Alexander et al., 1999a; Tag et al., 2001) 16 Proctor et al. (1995a) developed a trichothecene non-producing strain of F. graminearum (FgTri5-; wild-type FgTri5+) by disruption of the locus encoding trichodiene synthase (Tri5), whose gene product catalyzes the first committed step in trichothecene biosynthesis (Scheme 1.1) (Hohn and Vanmiddlesworth, 1986; Desjardins et al., 1993; Proctor et al., 1995a). Other Tri5 mutants have also been produced in other strains. In order to clarify the role of trichothecenes in FHB-aggressiveness, these mutants have been compared with the wild type strains, and the results are consistent with a role for trichothecenes in disease spread in Triticeae (Proctor et al., 1995a; Bai et al., 2001; Eudes et al., 2001; Langevin et al., 2004; Maier et al., 2006) and in maize (Harris et al., 1999; Maier et al., 2006). The current view is that trichothecenes are not necessary for initial infection (Bai et al., 2002), or infection through the wheat fruit coat, but that they are required for entry into the rachis and subsequently for disease spread (Jansen et al., 2005). In the absence of trichothecene production, F. graminearum has been shown to be contained in point-inoculated spikelets by cell wall thickening at the rachis node (Jansen et al., 2005). Scheme 1.1 Trichothecene biosynthesis pathways (pages 17-18). Steps in the pathway are catalyzed by Tri-gene products (see Table 1 for more details), and have been identified in either F. graminearum (Fg), F. sporotrichioides (Fs), or both. OAc = acetyl function; IsovalO = isovalerate function. Diagram is a compilation adapted from (Hohn et al., 1993; Brown et al., 2001; Mesterházy, 2002; McCormick, 2003). 17 farnesyl-pyrophosphate trichodiene 2-hydroxytrichodiene 12,13-epoxy-9,10- trichoene-2-ol 6 11 10 7 9 8 5 12 4 2 3 13 OH OH O TRI4 TRI5 4,15-diacetoxyscirpenol O OH AcO OAc O O OAc AcO OAc OH O 3-acetyl T-2 toxin T-2 toxin 3-acetoxyineosolaniol 3-acetyl HT-2 toxinHT-2 toxin O OH AcO OH IsovalO O O OH AcO OAc IsovalO O O OAc AcO OAc IsovalO O O OAc AcO OH IsovalO O O OH AcO O O OH 15-acetyldeoxynivalenol OPP FsTRI8? TRI4 TRI4 FsTRI1 O OH AcO O O OH OAc 4,15-diacetylnivalenol FgTRI8 O OH OH O O OH OAc 4-acetylnivalenol O OAc AcO O O OH OH 3,15-diacetylnivalenol O OAc AcO O O OH OAc 3,4,15-triacetylnivalenol FgTRI7 FgTRI8 FgTRI13 ∆FsTRI8 Scheme 1.1 (page 1 of 2) 18 isotrichodiol isotrichotriol trichotriol 2O 3 OH O OH OH O OH OH OH O 2 3 OH OH OH O isotrichodermol 3,4,15-triacetoxyscirpenol O OAc AcO OAc O 3,15-diacetyl T-2 tetraol O OAc O isotrichodermin O OAc OH O 15-deacetylcalonectrin O OH OH O 3,15-dideacetylcalonectrin O OAc AcO O calonectrin O OH OH O OH O deoxynivalenol TRI101 3,15-diacetoxyscirpenol O OH OH O OH OH O nivalenol O OAc AcO O OH OH O OAc AcO OH O O OAc AcO OH OH O 7,8-dihydroxycalonectrin O OAc AcO O O OH 3,15-diacetyldeoxynivalenol TRI4 TRI4 TRI11 TRI3 FgTRI3 TRI13 FsTRI3FsTRI1 FgTRI8 FgTRI13 ∆FsTRI8 ∆FsTRI7 Scheme 1.1 (page 2 of 2) 19 1.4 Fusarium Head Blight Resistance 1.4.1 Mechanisms of Fusarium Head Blight resistance FHB resistance is both dominant and quantitative. A gene-for-gene resistance interaction has not been identified in FHB-resistance, nor has immunity to the disease been observed. Stability of resistance is dependent on environmental factors at the time of infection and/or aggressiveness factors associated with the invading Fusarium strain, although resistance has been shown to be stable in genotypes with very high levels of resistance (Mesterházy, 1995; Miedaner et al., 2001; Foroud et al., in preparation). Several different forms of resistance have been identified (Table 1.2). These mechanisms of resistance can interact with each other to improve the overall resistance. Type I (resistance to initial infection) and Type II (resistance to disease spread) resistances, first described in wheat by Schroeder and Christensen (1963), are the best documented forms of resistance, since they are the most readily assayed. Due to the physiological differences between maize and other cereals, resistance to Fusarium Ear Blight (FEB) of maize is defined separately from that of FHB. Type I resistance in FHB is phenomenologically comparable to silk resistance in maize, where the fungus cannot penetrate the silk channel to infect the kernels (Reid et al., 1992). Type II resistance in FHB is comparable to kernel-resistance in FEB, where the fungus cannot penetrate the rachis, or ‘cob’, and hence does not spread from kernel to kernel (Chungu et al., 1996). 20 Table 1.2 FHB resistance mechanisms in cereals. Resistance Description Reference Resistance in Small Grain Cereals (as defined in Mesterházy, 2003) Type I Resistance to initial infection (Schroeder and Christensen, 1963) Type II Resistance to disease spread (Schroeder and Christensen, 1963) Type III Resistance to kernel infection (Mesterházy, 1995) Type IV Tolerance against FHB and trichothecenes (Mesterházy, 1995) Type V Resistance to trichothecene accumulation (Miller et al., 1985) Class 1 By chemical modification of trichothecenes (Boutigny et al., 2008) Class 2 By inhibition of trichothecene synthesis (Boutigny et al., 2008) Resistance in Maize Silk Resistance Resistance to silk penetration (Reid et al., 1992) Kernel Resistance Resistance to kernel disease spread (Chungu et al., 1996) Type I resistance can be measured as the percentage of spikelets exhibiting symptoms after exposure to the pathogen. Plants are typically sprayed during anthesis with macroconidial (or ascosporic) suspensions and high humidity is maintained (by bagging infected heads or by mist-irrigation) for a few days post-inoculation. Alternatively, the grain spawn method, where infected wheat or corn is dispersed in the field, can be used to better mimic natural conditions of infection (Rudd et al., 2001; Dill-Macky, 2003). 21 Resistance is measured 7 to 21 days after anthesis, and is typically reported as a ‘disease index’, and scored for average ‘incidence’ (percentage of diseased spikes) and ‘severity’ (percentage of infected spikelets on diseased spikes). Acquiring accurate assessments of Type I resistance is challenged by several factors: (a) the amount of inoculum that actually reaches the spikelets is unquantified, resulting in variability in the exposure of different spikes or plants within and between experiments; (b) environmental conditions are difficult to control, especially in field experiments; and (c) ‘disease index’ is not a measure of Type I resistance alone, but rather a combination of resistance to initial infection, disease spread and tolerance (Rudd et al., 2001; Eudes et al., 2004). Some researchers equate ‘incidence’ with Type I resistance and ‘severity’ with Type II resistance, while others treat ‘disease index’ as an estimate of both Type I and II resistances; thus, it has been proposed that the definition of Type I resistance is in need of re-evaluation (Mesterházy et al., 2008). Evaluation of Type II resistance is more straightforward (Rudd et al., 2001). A quantifiable amount of inoculum is injected into individual spikelets at anthesis, and high humidity is maintained for several days. Resistance is subsequently measured as the number of infected spikelets below the inoculation point (note that the disease typically spreads down the spike through the rachis). Delayed hyphal colonization of the vascular bundles in the rachis is observed in Type II resistant genotypes (Kang and Buchenauer, 2000a). As previously mentioned, trichothecenes are necessary for disease spread (Proctor et al., 1995a; Eudes et al., 2001; Bai et al., 2002; Langevin et al., 2004), but they do not appear to play a role in establishing initial infection by spray or point inoculation (Bai et al., 2002) or for infection of the fruit coat (Jansen et al., 2005). Using GFP 22 expression under control of the TRI5 promoter as a reporter for trichothecene gene expression, Ilgen et al. (2009) recently demonstrated that the trichothecene biosynthetic pathway is induced following colonization of the developing kernel, and later at the rachis node . Together, these data suggest that trichothecenes, which are necessary for disease spread, do not accumulate until after initial infection has been established. If trichothecenes are not necessary for establishing initial infection, and since evaluation methods for resistance to initial infection are confounded by resistance to disease spread, perhaps a more accurate estimate of resistance to “initial infection” would be obtained by using spray inoculation with FgTri5-, or even by using grain spawn infected with FgTri5- . This would eliminate the interaction between Type I and II resistance in the evaluation of resistance to initial infection. Such a strategy would also eliminate the interaction of Type I resistance with other forms of resistance that are directly related to trichothecenes (namely Type IV and V resistance). Type III, IV, and V resistance cannot be directly quantified. This is, in part, because the nature of these forms of resistance is often intermingled with each other and/or with Type I and II resistance. Type III resistance can be assessed by FDK evaluation. Type IV resistance is defined as tolerance to FHB, meaning that yield and quality of the grain product is maintained in spite of disease presence. Type IV resistance may also be defined as DON tolerance, in which case it could be evaluated by comparing FDK values to DON content; if FDK is low but DON content is high, tolerance to DON and FHB would then be observed. Type V resistance (resistance to toxin accumulation) can be estimated by DON quantification of FHB-infected plants or by an in vitro tissue assay (Miller et al., 1985; Wang and Miller, 1988). Type V resistance can be subdivided into 23 two classes as defined by Boutigny et al. (2008): Type V-1 resistance refers to the host’s ability to chemically modify trichothecenes, resulting in toxin degradation or detoxification; Type V-2 resistance refers to the host’s ability to inhibit trichothecene biosynthesis in the invading fungus. 1.4.2 Sources of Fusarium Head Blight resistance The development of FHB-resistant cultivars has proven to be a difficult task. While cereal breeders worldwide have invested considerable effort in the development of FHB- resistant germplasm (Miedaner, 1997; Gilbert and Tekauz, 2000; Tekauz et al., 2000; Bai et al., 2003; Mesterházy, 2003; Tekauz et al., 2004), relatively few resistant cultivars have been generated by conventional breeding methods. Moreover, most of the work has focused on wheat and barley breeding, since they are the most heavily FHB-affected crops (especially wheat). Barley has an inherent Type II resistance, but unconventional disease spread can be observed externally from spikelet to spikelet without penetration of the rachis (Langevin et al., 2004). Six-row barley, which is more susceptible than two- row barley and is preferred for malting, is nearly as susceptible as wheat (Tekauz et al., 2004). Resistance in two-row barley is attributed to a quantitative trait locus (QTL) that is associated with the Vrs1 locus, which controls spike type. It is not clear whether resistance is functionally linked to Vrs1, or if there is a pleiotropic effect at play (de la Pena et al., 1999; Zhu et al., 1999; Ma et al., 2000). Durum wheat (Triticum turgidum subsp. durum) is one of eight subspecies of tetraploid (AABB) wheat (Oliver et al., 2008), and is far more FHB-susceptible than hexaploid (AABBDD) bread wheat (Gilbert and Tekauz, 2000). Rye is generally more resistant than wheat and barley (Miedaner, 24 1997; Langevin et al., 2004). Oats are also considered to be more FHB-resistant than wheat and barley (Langevin et al., 2004), but this pattern may be deceptive since FHB symptoms are not as readily discerned in oats compared with other crops (Tekauz et al., 2008). Furthermore, DON and T-2 toxin accumulation in oats is more severe than in wheat (Tekauz et al., 2004; Bjørnstad and Skinnes, 2008). Over 100 FHB resistance-related QTLs have been reported from FHB-resistant wheat sources, 22 of which have been detected in multiple mapping populations (summarized in Bürstmayr et al., 2009). The best characterized and most widely used source of resistance in hexaploid wheat is the Chinese cultivar, ‘Sumai3’ (Bürstmayr et al., 2009). ‘Sumai3’- derived resistance is attributed to the Fhb1 locus on chromosome 3BS, the major QTL conferring Type II resistance. Positional cloning of Fhb1 is underway, and gene identification may become available within the next few years (Liu et al., 2008). Additional QTLs from ‘Sumai3’ include Fhb2 (6BS) and Qfhs.ifa-5A (5A); the latter is associated with Type I resistance and has been found in different germplasm sources from around the world (Bürstmayr et al., 2009). Another popular resistance source comes from the Brazilian cultivar, ‘Frontana’, which possesses moderate FHB resistance, and whose resistance-associated QTLs have been mapped to positions 3A, 5A, 2B, 3AL, and 7AS (Steiner et al., 2004; Han et al., 2005; Mardi et al., 2006; Bürstmayr et al., 2009). An interesting set of QTLs for FHB resistance may be associated with Rht, plant height regulators. Some studies have shown that plant height is correlated with FHB-resistance (Couture, 1982; Steiner et al., 2004; Klahr et al., 2007), although rare exceptions may be found (A. Comeau, personal communications). Rht-D1 co-localizes with a FHB resistance QTL on 4DS, found in the European winter wheat cultivar, ‘Arina’ (Draeger et 25 al., 2007). Rht-B1 is on the same chromosome (4B) as an FHB-resistant QTL, and Rht8 is located close to a QTL on 2D. Rht-B1 and Rht-D1 are de-repressors of gibberellins signalling, and Rht8 conditions gibberellin-responsiveness, while the so-called semi- dwarfing alleles, Rht-B1b and Rht-D1b, confer gibberellin-insensitivity (Zhang et al., 2006b). Rht-D1b, but not Rht-B1b, also confers FHB-susceptibility (Draeger et al., 2007; Nicholson et al., 2008). It has not yet been determined whether there is a pleiotropic effect that would explain this interaction (Draeger et al., 2007). 1.4.3 Breeding for Fusarium Head Blight resistance Some of the challenges breeders are confronted with include: (1) poor agronomic traits associated with highly-resistant germplasm sources, which are often derived from exotic sources, (2) the polygenic nature of resistance, and (3) variability in disease rating accuracy, as previously described. Unfortunately, breeders have been limited in their choices of resistance sources, and therefore some of these problems perpetuate themselves. For example, while gene-for-gene resistance brings with it its own set of difficulties (such as the development of an arms-race between host and pathogen; Dodds et al., 2006), the lack of vertical resistance in FHB-host interactions precludes the prospect of immunity. As a consequence, the few available sources of strong resistance are recycled through various breeding programs, a phenomenon that may ultimately lead to the arms race scenario observed in gene-for-gene resistance. This limitation brings us back to the original problem: FHB-resistance is quantitative, and thus, the use of only a limited number of resistant sources in the development of resistant germplasm is not very effective. Furthermore, most of these resistance sources have poor agronomic traits. 26 ‘Sumai3’ is the prime example of a stable resistant source that is used in breeding programs worldwide. The stability and level of resistance is higher in this germplasm than in any other known source of resistance. Unfortunately, ‘Sumai3’ germplasm brings with it several agronomic deficiencies that seem to co-segregate with the 3BS QTL, including susceptibility to kernel shattering and reduced yields (Zhang and Mergoum, 2007). Over the past few decades, several tactics have emerged as enhancements, or as an alternative to, traditional breeding methods. Traditional breeding is a long and tedious process, requiring many generations of screening, which can be costly and time consuming. Breeders often screen thousands of lines, narrowing them down every year, for up to ten years, before they are ready to apply for the registration of one or two cultivars. A promising alternative approach to developing FHB-resistant cultivars involves an in vitro selection process, as proposed by Bruins et al., (1993). It was suggested that single-cell microspores might be well suited for in vitro selection for resistance, since a large proportion of the plant genome is expressed in both the sporophyte and the gametophyte. This approach can be used to efficiently screen a large population of cells for trichothecene or Fusarium resistances. Fadel and Wenzel (1993) were the first to report a selection scheme using mixed culture filtrates of 99 F. graminearum isolates co-cultured with 10-day old anther cultures, but they had little success in selecting for resistance among plants regenerated from the anthers. Following a similar strategy, Eudes et al. (2008) applied crude trichothecenes to anther cultures and reported successful selection for both reduced mycotoxin accumulation and FHB resistance in the resulting regenerated double haploid plants. 27 1.4.4 Engineering for Fusarium Head Blight resistance Genetic engineering can be effective both as tool for studying the mechanisms of FHB-resistance, and towards the development of FHB-resistant crops. Some of the major challenges in any genetic manipulation of wheat are directly related to the complexity of the wheat genome. Common bread wheat, Triticum aestivum, is a hexaploid (AABBDD) with seven homologous chromosome pairs derived from each of three ancestors, T. uratu (AA), Aegilops speltoides (SS, closely related to the B genome), and A. tauschii (DD), all of which co-evolved from a common ancestor. T. uratu and A. speltoides first converged to form the tetraploid (AABB) durum wheat (T. turgidum), which later (over 8000 years ago) converged with A. tauschii to produce T. aestivum (reviewed in Huang et al., 2002; Dubcovsky and Dvorak, 2007). The genetic redundancy encoded within the wheat genome, in combination with the large genome size (16,000 Mb; Gill et al., 2004), makes for a system that is difficult to manipulate using either conventional genetic approaches or transgenic technology, since transgenes are easily lost to segregation after a few generations. The in vitro selection process described by Eudes et al. (2008) for the development of FHB-resistant plants can be used in combination with cell penetrating peptides to introduce foreign genes into the microspore genome, providing a promising tool for the production of double haploid transgenic wheat lines. This methodology should, in theory, overcome the problems of transgene segregation. Furthermore, this approach can also be used for gene silencing by employing RNA-interference (RNAi). RNAi technology takes advantage of the plant’s endogenous machinery responsible for degradation of double- stranded RNA (dsRNA), and subsequent degradation of any single stranded RNA 28 (ssRNA) with the same sequence. The RNAi gene silencing machinery has been identified in many organisms, including nematodes, plants, and mammals. Foreign dsRNA species, often introduced by viruses, are targeted by Dicer and Dicer-like proteins (Hammond et al., 2000; Liu et al., 2009), nucleases that cleave dsRNA into 21- to 26-mer products known as short interfering RNA (siRNA) (Elbashir et al., 2001), which are then incorporated into an RNA-induced silencing complex (RISC) (Paroo et al., 2007). The RISC subsequently targets single-stranded RNA (ssRNA) homologous to the siRNA, and degrades the ssRNA. Similarly, micro-RNAs (miRNAs), endogenous regulatory non- coding RNAs 20- to 24-mer in length, are also targeted by RISCs (Zhang et al., 2006a). Thus, RNAi-mediated gene silencing can be conferred by transgenically expressing a sequence homologous to the gene of interest, followed by a short spacer sequence and the antisense sequence (reviewed in Mansoor et al., 2006). The transcribed RNA product will form a hairpin loop, where the reverse antisense sequence folds onto the sense strand, forming dsRNA, which would then be targeted by Dicer and RISC. The RISC would subsequently degrade any messenger RNAs (mRNAs) with homologous sequences, thus preventing translation of the gene of interest. Similarly, virus-induced gene silencing (VIGS) (van Kammen, 1997; Ruiz et al., 1998; Baulcombe, 1999), provides a transient gene silencing approach based on RNAi. The VIGS technology has been applied in a variety of species, where a sequence from the target gene for silencing is encoded in a vector within an RNA virus, which is then used to systemically infect the plant. Once infected, the RNA expressed by the virus is targeted by RISC for degradation. Tobacco mosaic virus, potato virus X, and barley stripe mosaic virus (BSMV) are examples of some viruses that have been used in the VIGS system. 29 Using BSMV, this technology was recently adapted for use in wheat, and has been successfully implemented in identifying disease resistance genes (Scofield et al., 2005). Both gene silencing and over-expression of genes of interest have been successfully implemented in the study of gene function associated with FHB-resistance in wheat (Anand et al., 2003; Makandar et al., 2006; Chen et al., 2009). Genetic engineering towards FHB-resistance could be an effective approach to reducing mycotoxin accumulation in cereals, bypassing the problems of both limited sources of resistance and the co-segregation of resistance with poor agronomic traits. Transgenic crops could directly reduce trichothecene accumulation when the modifications include expression of genes or pathways within the host that inhibit trichothecene synthesis, detoxify trichothecenes, or remove them from the cell by efflux. Examples of enzymes that may be involved in detoxification include epoxidases, acetyltransferases, and glycosyltransferases (Boutigny et al., 2008). While trichothecene de-epoxidation has not been observed in planta (Boutigny et al., 2008), the epoxide group has been shown to be essential for trichothecene toxicity (Ehrlich and Daigle, 1987). Trichothecene 3-O- acetyltransferase activity of TRI101, which leads to DON-acetylation, has been shown to reduce toxicity of trichothecenes in F. graminearum (Kimura et al., 1998). It has also been demonstrated that a C-3 acetylation reduces the phytoxicity of various trichothecenes in plants (Alexander et al., 1999b; Muhitch et al., 2000; Desjardins et al., 2007a; Ohsato et al., 2007). However, this may be species-dependent since DON and its C-3 acetylated counterpart, 3-ADON have been shown to be equally phytotoxic in wheat coleoptile growth inhibition assays (Eudes et al., 2000). Since TRI101 activity reduces trichothecene toxicity in Fusarium by C-3 acetylation, and since C-3 acetylation has been 30 shown to reduce phytotoxicity of trichothecenes in some plant species, it was thought that similar outcomes may be observed in plants. In fact, transgenic expression of this gene has shown improved FHB- and trichothecene-resistance in plants (Okubara et al., 2002; Ohsato et al., 2007; Alexander, 2008). Another Fusarium gene, Tri12, which is a Major Facilitator Superfamily (MFS) transporter responsible for trichothecene efflux from F. sporotrichioides (Alexander et al., 1999a), might also have the potential to reduce trichothecene accumulation in plants if it were ectopically expressed in transgenic wheat. Wheat UDP-glucosyltransferase has been shown to detoxify DON by condensation of a glucose molecule with the hydroxyl group at C-3 (Poppenberger et al., 2003), and glycosylated-DON derivatives have been observed in Fusarium-infected cereals (Berthiller et al., 2005; Dall’Asta et al., 2005; Lemmens et al., 2005). It has been hypothesized that the 3BS QTL of ‘Sumai3’ is in fact a UDP-glucosyltransferase (Lemmens et al., 2005). However, fine-mapping of the 3BS QTL by James Anderson’s research group in Minnesota has narrowed it down to a few candidate genes, and none of these encode UDP-glucosyltransferase. Nevertheless, this does not eliminate the possibility that the 3BS QTL is involved in the regulation of UDP-glucosyltransferase expression (Liu et al., 2008). Interestingly, differential accumulation of UDP- glucosyltransferase transcripts and protein has been observed in maize upon exposure to Fusarium (Harris et al., personal communications). Transgenic expression of UDP- glucosyltransferase might lead to improved FHB-resistance and reduced toxin accumulation in cereals and maize crops, although this approach may be unsuitable for food crop production since glycosylated-ZON has been shown to be converted back to ZON in the intestinal tract of swine (Gareis et al., 1990). 31 1.5 Overview Both Type I and Type II resistance to FHB are multigenic, and because of their complex nature it has been difficult to define the genetic basis in either case. The physiological mechanism of Type II resistance is more clearly defined, in part due to the relative simplicity of evaluation of this resistance compared with that of Type I. Furthermore, high levels of Type II resistance have thus far been found only in ‘Sumai 3’ and derived genotypes with the 3BS QTL. In contrast, many QTL have been identified for Type I resistance, but the different QTLs for Type I resistance tend to be associated with different sources of resistance; thus, no one QTL has repeatedly been correlated with Type I resistance. It is proposed here that the molecular mechanisms of Type I resistance are variable and genotype-dependent, whereas Type II resistance likely involves a more specific set of molecular responses to the pathogen. Furthermore, since trichothecenes are involved in FHB-disease spread, I hypothesized that the molecular mechanism of Type II resistance involves a specific response to the effect of trichothecenes, and that this response could be resolved from the more complex responses to live pathogen challenge by an appropriately designed evaluation of elicited changes in gene/protein expression. Since previous functional genomics studies to date have not been able to clearly identify a specific pathway for Type II resistance, I designed and carried out a series of studies using molecular plant pathology approaches in three related wheat genotypes (‘Superb’, DH1, and DH2) in an effort to isolate the differences in Type I and Type II resistance. ‘Superb’ is a susceptible Canadian cultivar; DH1 (‘CIMMYT 11’/’Superb’*2; Type I resistant) and DH2 (‘CM82036’/’Superb’*2; Type II resistant) are resistant double 32 haploid lines generated by in vitro selection of microspore-derived embryos using a trichothecene toxin screen (0.23 mg L-1 deoxynivalenol, 0.23 mg L-1 15-O-acety-4- deoxynivalenol, 0.47 mg L-1 nivalenol, and 0.7mg L-1 T2 toxin) as described by (Eudes et al., 2008). In order to compare the interaction between trichothecenes and disease outcomes in a Type II resistant genotype with their interaction in genotypes that do not have the 3BS QTL for FHB-resistance, the effects of three elicitors (FgTri5+, FgTri5-, and the trichothecene DON) were compared. In Chapter 2, experiments in functional genomics were performed to provide preliminary insights into the molecular pathways that are differentially regulated in resistant and susceptible wheat genotypes in their response to the same three elicitors. Chapters 3 and 4 present results from experiments designed to follow up novel results obtained from functional genomics studies. A summary of my findings, their implications and proposed future research directions are presented in Chapter 5. 33 2. Differential Gene Expression and Protein Accumulation Patterns in Resistant and Susceptible Fusarium Head Blight Interactions in Wheat 2.1 Introduction Functional genomics studies of the FHB-host interaction have been reported throughout the past decade (Pritsch et al., 2000; Pritsch et al., 2001; Zhou et al., 2005; Boddu et al., 2006; Zhou et al., 2006; Bernardo et al., 2007; Boddu et al., 2007; Golkari et al., 2007; Li and Yen, 2008; Golkari et al., 2009; Steiner et al., 2009). Pritsch et al. (2000) tracked the F. graminearum-induced transcript accumulation of six genes encoding pathogenesis-related (PR) proteins: PR-1, PR-2 (β-1,3-glucanase), PR-3 (chitinase), PR-4, PR-5 (thaumatin-like protein), and peroxidase (POX), and observed increased accumulation for all six. Among these, PR-4 and -5 accumulated earlier and in higher quantities in Type II resistant ‘Sumai 3’ than in the susceptible cv. ‘Wheaton’. In a second study, they found that PR-1, -2 and -5 accumulated in the uninvaded spikelets of wheat heads (Pritsch et al., 2001). F. graminearum-induced up-regulation of PR-2, -4 and -5 has also been observed in ‘Sumai 3’, but not in two ‘Sumai 3’ near-isogenic lines that are susceptible to FHB (Golkari et al., 2009). In another resistant cultivar, ‘Ning 7840’, F. graminearum infection induced up-regulation of PR-1 and a chitinase precursor, relative to the response of the same genes in the susceptible cv. ‘Clark’ (Bernardo et al., 2007). Protein accumulation studies in barley have shown PR-3 and PR-5 up-regulation in resistant lines compared with susceptible ones (Geddes et al., 2008). 34 Fewer studies have used global protein profiling to explore the differential proteomics of FHB and wheat. Zhou et al. (2005) observed up-regulation of antioxidant and PR proteins in F. graminearum-infected spikelets of ‘Ning7840’. In the other protein accumulation studies in wheat, F. graminearum has been shown to induce downregulation of metabolism-related proteins and upregulation of defence-related proteins; these responses were observed as early as 6 hai in a resistant wheat cv. ‘Wangshuibai’ (Wang et al., 2005), and at 3 dai in an independent study in the susceptible wheat cv. ‘Crystal’ (Zhou et al., 2006). Consistent with functional genomics analyses, over-expression of various PR or defence-related proteins has been shown to improve cereal resistance to FHB, as well as Arabidopsis resistance to F. oxysporum (Epple et al., 1997; Anand et al., 2003; Makandar et al., 2006). Regulation of PR protein expression is often mediated by host signalling metabolites, including salicylic acid (SA), jasmonic acid (JA), and ethylene (ET) (Glazebrook, 2005). The related signalling pathways are well characterized in dicots, but increasing evidence indicates that they may differ in monocots (Huckelhoven et al., 1999; Molina et al., 1999; Lu et al., 2006). JA/ET signalling is typically seen as the preferred pathway mediating a resistance response to necrotrophic pathogens in both monocots and dicots, whereas SA signalling tends to be associated with resistance to biotrophic pathogens (Glazebrook, 2005). In the case of FHB, it remains unclear which pathways are involved in either Type I or Type II resistances responses. Despite advances in our knowledge of the genes and pathways associated with FHB- resistance, the molecular processes that actually confer a resistance or susceptible response remain unclear. The absence of gene-for-gene resistance increases the 35 complexity of the disease response, and makes it difficult to identify discrete mechanisms underlying resistance or susceptibility. In order to better understand the cellular processes controlling disease outcomes and to create opportunities for developing novel forms of resistance, we need to better define the molecular pathways in FHB-challenged wheat tissues that result in resistance. Since changes in gene expression are expected to reflect the suite of host cell responses during FHB infection, I conducted a time-course study of the differential transcriptome of three wheat genotypes in their response to isolated components of FHB. The three wheat genotypes, ‘Superb’, DH1 and DH2 (as described in Chapter 1), were challenged with four inocula (water, FgTri5+, FgTri5- and DON). Transcriptome and proteome analysis was performed on uninoculated spikelets harvested from point-inoculated wheat spikes, a sampling strategy that would be expected to capture the changes resulting from a systemic, rather than local, host tissue response. This experimental design allowed for comparison of the molecular biology of susceptible vs resistant host responses, as well as a comparison of Type I vs Type II resistant host responses, while at the same time allowing an exploration of how different components of FHB disease affect the disease outcomes. Furthermore, by isolating the effects of aggressiveness factors and the effects of trichothecens in these three wheat genotypes, this design allowed me to investigate the hypothesis presented in Chapter 1, that the molecular mechanisms of Type II resistance involves a specific response to the effect of trichothecenes. 36 2.2 Materials and Methods 2.2.1 Plant material The three wheat genotypes defined in Chapter 1, namely ‘Superb’, DH1 and DH2, were used in this study. Seeds were sown in 4 L pots (1 plant per pot) containing Cornell Mix (Boodley and Sheldrake, 1977) and grown in a greenhouse (21/18oC, 16 h photoperiod). Plants were watered as needed and fertilized biweekly with 20-20-20 (N-P- K). At the 5-7 leaf stage they were treated with Tilt™ (2.5 mL L-1 propiconazole, Syngenta Crop Protection Canada, Guelph, ON) and Intercept™ (0.004 g L-1of soil, Imidacloprid, Bayer Crop Science Canada, Toronto, ON) as preventative measures against powdery mildew and aphids. 2.2.2 Inocula All four inocula defined in Chapter 1 were used in this study: water, FgTri5+, FgTri5-, and DON. For FgTri5+ or FgTri5- inocula, four mycelium plugs (1 cm2 each) from cultures grown on potato dextrose agar plates were used to inoculate 500 mL CMC broth (1.5% carboxymethylcellulose (SIGMA C1011), 0.1% NH4NO3, 0.1% KH2PO4, 0.05% MgSO4·7H2O, 0.1% yeast extract; Cappellini and Peterson, 1965). The culture was incubated at room temperature with gentle agitation (150 rpm) for two weeks and then filtered through cheese cloth. The filtrate was diluted with sterile ddH2O to a working concentration of 40,000 macroconidia mL-1. For DON-containing inocula, DON (D0156, Sigma-Aldrich, Oakville, ON, Canada) was dissolved in 40% ethanol to a concentration of 1 mg mL-1, and diluted to a working concentration of 2 1 mg mL-1 in ddH2O. 37 2.2.3 Inoculations and tissue collection Plants were moved to a mist-irrigated greenhouse (25oC, 95% humidity, 16 h photoperiod) during anthesis. Two spikelets (one on each row) near the center of the spike were point-inoculated with 10 µL inoculum. In order to reduce the variation in transcript profiles due to the circadian cycle, all inoculations took place between 0700 and 1000. Up to six spikelets above and below each inoculation point (for a total of up to 12 spikelets; see Figure 2.1) were collected at 3, 8 and 24 hours after inoculation (hai) for RNA extractions and at 3 days after inoculation (dai) for protein extraction. Zero hour uninoculated controls were also collected for RNA extractions (up to 12 spikelets above/below the centre were harvested between the hours 0700 and 1000). Three experimental repetitions were performed, each with three biological replicates, where experimental repetitions were differentiated by seeding dates. Each biological replicate consisted of one spike from one plant for a given condition. The three biological replicates (spikelets from three spikes) were pooled together for RNA or protein extractions. Thus, for each experimental repetition, a total of 39 RNA extractions were performed, as well as 15 protein extractions. 38 A. B. Figure 2.1 Inoculation of wheat spikes and harvesting of spikelets for RNA and protein extractions. A. The two central spikelets (4 florets; shaded in figure) of the wheat head were each point-inoculated with 10 µL inoculum, and incubated in a mist- irrigated greenhouse. Spikelets above and below the inoculation point were collected at 3, 8 and 24 hai, and the remainder of the head was discarded For 0 hai controls, plants were not inoculated, but spikelets were collected above and below the two central spikelets at anthesis. Total RNA or protein was extracted from the collected spikelets. B. Analysis of differential transcriptomics or proteomics included four treatment comparisons: I. effect F. graminearum wild-type (FgTri5+); II. effect of trichothecene non-producing F. graminearum (FgTri5-); III. effect of DON; and IV. effect of aggressiveness factors (FgTri5+ vs FgTri5-). FgTri5+ FgTri5- H2O I II III DON IV Affymetrix RNA spikelets 2D-electrophoresis Protein discard 39 2.2.4 Microarray For each of the three experimental repetitions, harvested spikelets were ground under liquid nitrogen, and total RNA was extracted from 0.1 g of the resultant powder using QIAGENs RNeasy® Plant Mini Kit with an on-column DNase digestion. RNA quality was verified on a 1% agarose gel and 5 µg total RNA was used for biotin labelling. The two-cycle cDNA synthesis, cRNA labeling and hybridization to the Affymetrix GeneChip® Wheat Genome Array were done according to manufacturers instructions (Affymetrix). A total of 117 chips were used, corresponding to the 39 RNA extractions in each of three experimental repetitions. The Affymetrix cel files resulting from scanning will be deposited in the Gene Expression Omnibus (NCBI) for public accessibility. Cel files were subjected to GCRMA (Wu et al., 2004b) and MAS5 (Hubbell et al., 2002) algorithms for background subtraction, data normalization, and probe summarization, using the ArrayAssist® (Stratagene) software. Default settings were used in statistical analysis to calculate fold difference (FD) and p-value. Treatment comparisons were made within each plant line, at each harvest time (3, 8 and 24 hai), with a 2.0 FD cut-off (p < 0.05). Treatment comparisons are presented in Figure 2.1 and include: (I) effect of wild- type F. graminearum (FgTri5+); (II) effect of the trichothecene non-producing F. graminearum (FgTri5-); and (III) effect of DON; (IV) effect of FgTri5+ compared directly with FgTri5-. Comparisons were also made between plant lines for the 0 hai harvest with a 5.0 FD cut-off (p < 0.05). Probe sets were initially annotated using an automated procedure by BLASTint against the SwissProt non-redundant protein database and the plant and fungal sections of the non-redundant nucleotide database (NCBI). Probe sets of potential interest were then manually curated to identify those with BLAST 40 match(es) that strongly supported annotation of the corresponding wheat gene as an homolog of a biochemically characterized gene in wheat or another plant species (Ouellet et al., unpublished). Most of the gene annotations are putative gene descriptions; thus, the gene descriptions in this text are mainly putative descriptions. Table 2.1 Primer sequences for qPCR validation. Sequences were selected to have overlapping regions corresponding to the probes used in the Affymetrix GeneChip®. Probe set Forward Primer Reverse Primer AFFX-Ta_Sucsyn_3_at 5’-AGTGTCGGCTGCGTTATGAT 5’-CACAAATGTGCCGAGACAAC TaAffx.106139.1.S1_at 5’-GCTGCTGAGCAAAAACAAATC 5’- AATCAAATCTTGGAGGGGATG TaAffx.108735.1.S1_at 5’-CTACGACTGTGGCTGGCGGC 5’-TGCAACCGCCACAACACCTTCA TaAffx.111195.1.S1_at 5’-GAGTTTAACGCGGTTTCCAC 5’-ATAGCTGGGTGCTCACGATT Ta.526.1.S1_x_at 5’-GGTGTTCATCCGCACCAT 5’-TGCCCGAGGTAAATCTCATC Ta1207.1.S1_x_at 5’-CGACCCACATGCACTCGGGA 5’-GGGCAGGAGCCCGTGAGGTA Ta.1967.1.S1_x_at 5’-CCCAACAGCATCTCCATTTA 5’- TGTCCCATTCCAATTATACCC Ta.16723.2.S1_x_at 5’-GTGCTCGCGCAGATGTGCCT 5’-AGCATGGCTCTGCCCCAGGA Ta.24501.1.S1_at 5’-GCTATCCTGCTCCCTCTCC 5’-TCGTAGAAGTCCTGGGTGCT 2.2.5 Real-time RT-PCR cDNA synthesis was performed on 2 µg total RNA using Invitrogen’s SuperScriptTM III Reverse Transcriptase according to manufacturer’s instructions, and the product was diluted 20-fold in nuclease-free water. Primers were designed to amplify gene products from eight unique probe sets (Table 2.1). The gene product corresponding to the housekeeping probe set AFFX-Ta_Sucsyn_5_at was selected as a housekeeping gene for real-time RT-PCR (qPCR), as this gene showed little variation accross gene chips for the various conditions and genotypes used for the microarray experiment, with an average signal for log-transformed data of 7.2 + 0.8. The qPCR reaction mix was set up using 41 Qiagen’s QuantiTectTM SYBR® Green Master Mix with 2 µL of cDNA, and 0.5 µM of both forward and reverse primers, in a final reaction volume of 15 µL. Samples were run on an Applied Biosystems’ 7900HT Fast Real-Time PCR System, with a 15 min hot- start at 95oC, followed by 40 cycles through 95oC (30 s) and 60oC (60 s). A melting curve was included in order to confirm amplification of a single gene product. Fold-differences and standard error were calculated from CT-values using Qiagen’s REST 2006 software package. 2.2.6 Protein sample preparations For total protein extractions, harvested spikelets were ground under liquid nitrogen and the powder suspended 10 mL Precipitation Buffer (90% acetone, 10% trichloroacetic acid, 0.07% β-mercaptoethanol) for an overnight protein precipitation at -20oC. Following an initial centrifugation, the pellet was washed and spun three times using cold 10 mL Wash Buffer (acetone with 0.07% β-mercaptoethanol). All centrifugation steps were performed at 12,000 x g for 20 min at 4oC. After the third wash step, the pellet was air dried in the fume hood on ice, for 20 min. The protein was solubilized for 20 min at room temperature in 3 mL Rehydration Buffer 3-10 (7 M Urea, 2 M thiourea, 4% CHAPS, 0.5% ZOOM® Carrier Ampholytes pH 3-10, 10 mM tris(2)carboxyethylphosphine (TCEP)), and after a centrifugation step the supernatant was transferred to a new tube. In order to concentrate the samples, the protein was precipitated from the supernatant for 2 h at -20oC in 30 mL Precipitation Buffer, after which the precipitate was spun down. The resulting pellet was washed and spun three times in 5 mL pre-chilled Wash Buffer, air dried at room temperature for up to 10 min, 42 and then resuspended in 600 µL Rehydration Buffer 3-10. Samples were cleaned using ReadyPrepTM 2-D Cleanup Kit (BioRad), and resuspended in 600 µL Rehydration Buffer 3-10. Purified protein was quantified by Bradford Assay (BioRad) and 2 mg of total protein in 600 µL Rehydration Buffer 3-10 with 400 mM 4-vinylpyridine (4-VP) was vortexed at approximately 1000 rpm for 60 min at room temperature (reduction- protection technique adapted from Bai et al. (2005)). Pyridilation was terminated by the addition of 400 mM dithiothreitol (DTT) and incubation at room temperature for 15 min. Samples were diluted to a final volume of 3.25 mL in Rehydration Buffer 3-10 for 2D- electrophoresis. 2.2.7 2D-electrophoresis Samples were pre-fractionated according to isoelectric point using a ZOOM® isoelectric focusing (IEF) Fractionator with ZOOM® discs pH 3, 7, and 10, according to manufacturer’s instructions (Invitrogen). The resulting pH 3-7 and 7-10 fractions were quantified by Bradford Assay (BioRad) and loaded onto a 17 cm ReadyStripTM immobilized pH gradient (IPG) Strips (BioRad) which was allowed to rehydrate for 16 h at 20oC. For pH 3-7 fractions, a total of 100 µg protein in 300 µL Rehydration Buffer 4-7 (7 M Urea, 2 M thiourea, 4% CHAPS, 10mM TCEP, 0.5% ZOOM® Carrier Ampholytes pH 4-7) was loaded onto pH 4-7 IPG strips. For pH 7-10 fractions, a total of 150 µg protein in 300 µL Rehydration Buffer 7-10 (7 M Urea, 2 M thiourea, 4% CHAPS, 10mM TCEP, 0.5% Bio-Lyte® 7/10) was loaded onto pH 7-10 IPG strips. Isoelectric focusing was performed in a Protean IEF Cell (BioRad) with 5 µA maximum per gel for pH 4-7 strips (ramp to 250V for 30 min, slow ramp to 10,000 V for 2 h, hold at 10,000 V for 40 43 kVh) and for pH 7-10 strips (ramp to 150 V for 2 h, 300 V for 4 h, 1500 V for 1 h, 5000 V for 5 h, 7000 V for 6 h, 10,000 V for 3 h). Strips were equilibrated twice for 10 min in Equilibration Buffer (6 M Urea, 15% (w/v) glycerol, 1% SDS, 0.1 M Tris-HCl, pH 8.8), with 10 mM TCEP included in the second equilibration. Strips were rinsed in Running Buffer (192 mM glycine, 0.1% SDS, 25 mM Tris-HCl, pH 8.3) and loaded onto 12% acrylamide gels for second dimension electrophoresis (15 mA per gel for 30 min, 30 mA per gel for 4.5 h). For each pH range a total of 45 gels were run, corresponding to the 45 protein extractions. 2D-gels were stained with Sypro-Ruby (BioRad) and the resulting images were captured using a Typhoon 9400 scanner (600 V, normal sensitivity; 610 BP30/532 nm; GE Healthcare). 2D-gel patterns were analyzed using Phoretix 2D Expression v2005 (Nonlinear Dynamics). For each treatment and plant line, an average gel was created from three experimental replicates (see Inoculations and Tissue Isolation). A normalized spot volume for each spot was calculated as a percentage of the total spot volume. Warping, spot matching between average gels and comparison gels, and mean spot volumes were calculated by the software. Spot matches were then manually confirmed. For comparison of average gels, differentially regulated spots were defined as having greater than a 2FD (p < 0.05). The same four treatment comparisons were performed within each plant line as described for the microarray analysis. Comparisons were also made between plant lines for each of the four treatments. All differentially regulated spots were selected for protein sequencing. Selected spots were excised from the gels and sent for sequencing at the Proteomics/Mass Spectrometry Core Facility at the Institute for Biological Sciences, 44 National Research Council of Canada. Spots were trypsin (Promega)-digested overnight at 37oC at a ratio of 30:1 (protein:enzyme, v/v) in 50 mM ammonium bicarbonate. The resulting peptide solutions were injected onto a C18 trap column (Symmetry C18, 5 µm, 180 µm x 20 mm) on a nano-Acquity UPLC (Waters, Milford, MA). The C18 trap was brought in line with a C18 capillary column (100 µm x 100 mm nano-Acquity UPLC column). A 14-minute gradient from 1% acetonitrile/0.2% formic acid (99% 0.2% formic acid in water) to 40% acetonitrile/0.2% formic acid (60% 0.2% formic acid in water) was used from chromatographic separation. The nano-Acquity UPLC effluent was connected to a nano-electrospray source on a Q-TOF-Ultima hybrid quadrupole time-of-flight (TOF) mass spectrometer (Waters, Milford, MA). The ions were analyzed by TOF-MS (survey scan) and when a 2+ or a 3+ ion is detected, an MSMS scan on this ion triggered. 2.2.8 2D-electrophoresis optimization for resolution in the basic range Several reduction/protection techniques were used for optimization of resolution in the basic pH range of the IEF gradient. These include: (1) ReadyPrepTM Reduction- Alkylation (BioRad) using tributylphosphine (TBP) and iodoacetamide (IAA); (2) reduction with TBP and pyridylation with 4-VP, as described in Bai et al. (2005); and (3) reduction with TCEP and pyridylation with 4-VP, adapted from Bai et al. (2005). Total protein was extracted as described in section 2.2.6, but it was solubilized in 1 mL Rehydration Buffer 3-10 with 50 mM DTT and without TCEP. The total protein was then cleaned using the ReadyPrepTM 2-D Cleanup Kit (BioRad) as described in section 2.2.6, except the samples were resuspended in 600 µL Rehydration Buffer 3-10 without TCEP 45 or other reducing agents. All reduction-protection protocols were conducted at room temperature. In the first approach, 250 µL total protein in Rehydration Buffer 3-10 without TCEP was combined and vortexed at approximately 1000 rpm with 7.5 µL alkylating buffer and TBP to a final concentration of 5 mM. After a 30 min incubation, 15 mM fresh iodoacetamide (IAA) was added and the mixture vortexed. Alkylation was terminated after 60 min by adjusting the TBP concentration to 10 mM, followed by 15 min incubation. In the second and third reduction-protection approaches, samples were first reduced with TBP and TCEP, respectively, and then pyridylated with 4-VP to protect the sulphur groups. These reactions were performed as described in section 2.2.6, except, in the second approach, the Rehydration Buffer 3-10 without TCEP was combined with 5 mM TBP and 40 mM Tris-HCl pH 8.8 and pyridilation was performed with 20 mM 4-VP and terminated with 20 mM DTT. Following each of the three reduction protection steps used, samples were cleaned again using ReadyPrepTM 2-D Cleanup Kit (BioRad), as described in section 2.2.6, and then quantified by the Bradford Assay (BioRad). The 17 cm ReadyStripTM IPG pH 7-10 Strips (BioRad) were rehydrated for 11 h at 20oC in 330 µL Rehydration Buffer 7-10 (7 M Urea, 2 M thiourea, 4% CHAPS, 0.5% Bio-Lyte® 7/10). The reduced/protected total protein samples (450 µg protein in 130 µL) were then loaded into a cup placed on the anode side of the strips. IEF was performed in a Protean IEF Cell (BioRad) with 5 µA maximum current per gel (ramp to 250V for 30 min, linear ramp to 10,000 V for 1 h, hold at 10,000 V for 30 kVh). Strips were equilibrated twice for 10 min in Equilibration Buffer first with 2% DTT and then with 46 2.5% IAA. The second dimension separation was performed as described in section 2.2.7, and resolution of protein gels were compared visually. 2.3 Results Affymetrix GeneChip® microarray and 2D-electrophoresis analyses were used to investigate induced transcriptional and proteomic differences, respectively, in the wheat response upon exposure to different treatments (defined in Figure 2.1). Genotype-specific differences in constitutive gene expression between ‘Superb’, DH1, and DH2 were examined in the zero-hour uninoculated control samples. Genes showing at least five-fold differences (FD) in constitutive expression (p < 0.05) are listed in Table 2.2 and discussed below. Constitutive differences were not evaluated in the proteome, but genotype differences were observed within each of the four treatments, and those with > 2-FD (p < 0.05) are listed in Table 2.3. In challenge experiments, analysis was conducted on the uninoculated spikelets from inoculated heads collected at 3, 8 and 24 hai for microarray analysis and at 3 dai for protein profiling. Four treatment comparisons (Figure 2.1) were made within each genotype: (I) FgTri5+ vs. water (effect of FgTri5+); (II) FgTri5- vs. water (effect of FgTri5-); (III) DON vs. water (effect of DON); and (IV) FgTri5+ vs FgTri5-. Genes showing challenge-dependent expression changes of at least 2-FD (p < 0.05) are presented in Table A1, and a subset of those (presented in Table 2.4) are discussed in the current report. Proteins showing challenge-dependent changes of at least 2-FD (p < 0.05) are presented in Table 2.5. Technical validation of the 2-FD cut-off values observed in the microarray experiments was conducted by qPCR (Table 2.6). 47 Table 2.2 Constitutive difference between transcriptomes of FHB-resistant genotypes and susceptible ‘Superb’. Positive and negative values (FD > 5.0, p < 0.05) indicate higher transcript accumulation in the resistant line and ‘Superb’, respectively. DH1 vs ‘Superb’ DH2 vs ‘Superb’ Probe Set ID Gene Description FD p-val FD p-val Defense-Related Protein Ta.352.1.S1_at Dehydration-responsive protein RD22 [Oryza sativa (japonica cultivar- group)] +73.7 0.001 TaAffx.36658.1.S1_at Disease resistance protein Hcr2-5D [O. sativa (japonica cultivar- group)] +5.9 0.006 Ta.21646.1.S1_at Lipid transfer protein [O. sativa (japonica cultivar-group)] -10.8 0.026 Ta.21646.1.S1_x_at Lipid transfer protein [O. sativa (japonica cultivar-group)] -5.0 0.035 Ta.1282.4.S1_at Lipid transfer protein 3 (LTP3) [Triticum aestivum] +38.8 0.000 Ta.28695.6.S1_at Metallothionein (LOC542898) [T. aestivum] -28.4 0.006 Transport Protein Ta.2895.1.S1_at Aquaporin PIP1 (Pip1) +120.5 0.000 Ta.2895.1.S1_x_at Aquaporin PIP1 (Pip1) +143.2 0.000 TaAffx.113846.1.S1_s_at Aquaporin PIP1 (Pip1) [T. aestivum] +259.1 0.000 Ta.9751.1.A1_at ABC transporter [O. sativa] -13.2 0.000 48 DH1 vs ‘Superb’ DH2 vs ‘Superb’ Probe Set ID Gene Description FD p-val FD p-val Structural Protein Ta.7378.6.S1_at Alpha-tubulin (LOC543387) +8.1 0.001 TaAffx.14498.1.S1_at Alpha/beta-gliadin precursor (LOC543192) +56.5 0.000 Cell Signalling TaAffx.27775.1.S1_at Receptor-like protein kinase 4 [O. sativa (japonica cultivar-group)] +5.1 0.017 Ta.20570.1.A1_at 1-aminocyclopropane-1-carboxylate oxidase [Sorghum bicolor] -16.7 0.002 Gene Expression Ta.9409.1.S1_at Transcriptional coactivator p15 (PC4) family protein-like [O. sativa (japonica cultivar-group)] +11.7 0.042 Hydrolysis TaAffx.97452.1.A1_at Lipase [O. sativa (japonica cultivar-group)] -11.0 0.003 TaAffx.97535.1.S1_at Lipase [O. sativa (japonica cultivar-group)] -8.4 0.008 Oxidases Ta.25832.1.A1_at Cytochrome P450 [Triticum aestivum] -14.6 0.000 Ta.5022.1.A1_at NADH dehydrogenase (ubiquinone) +5.7 0.020 49 DH1 vs ‘Superb’ DH2 vs ‘Superb’ Probe Set ID Gene Description FD p-val FD p-val Metabolism Ta.15908.1.S1_at Argininosuccinate lyase -6.7 0.034 Ta.27757.1.S1_at Phosphoglycerate mutase family [O. sativa (japonica cultivar-group)] -8.4 0.018 Other Ta.26907.1.S1_at RNase S-like protein precursor [Hordeum vulgare] +6.3 0.046 Protein with Unknown Function Ta.22794.1.S1_x_at LOC543011 +8.5 0.011 Ta.22794.1.S1_at LOC543011 +9.0 0.005 Ta.24733.1.S1_at Hypothetical protein [O. sativa (japonica cultivar-group)] -6.8 0.001 Ta.12402.1.S1_at Hypothetical protein [O. sativa (japonica cultivar-group)] -6.2 0.000 Ta.913.1.S1_x_at O. sativa (japonica cultivar-group) cDNA clone:001-103-C02 -12.6 0.002 Ta.913.1.S1_at O. sativa (japonica cultivar-group) cDNA clone:001-103-C02 -12.4 0.001 TaAffx.8202.1.S1_at Unknown -9.6 0.004 TaAffx.43135.1.A1_x_at Unknown +5.7 0.014 TaAffx.16415.2.S1_at Unknown -6.0 0.001 TaAffx.100436.1.S1_at Unknown -9.8 0.004 50 DH1 vs ‘Superb’ DH2 vs ‘Superb’ Probe Set ID Gene Description FD p-val FD p-val Ta.28862.1.S1_at Unknown +5.2 0.031 Ta.24761.1.S1_at Unknown -16.8 0.000 TaAffx.92842.1.S1_at Unknown +13.3 0.007 TaAffx.78888.1.S1_x_at Unknown -5.3 0.011 TaAffx.78888.1.S1_at Unknown -5.7 0.011 TaAffx.7810.2.S1_at Unknown +5.8 0.001 +5.9 0.000 TaAffx.50853.1.S1_at Unknown -6.8 0.049 TaAffx.31525.1.S1_at Unknown -5.6 0.003 Ta.8888.1.A1_at Unknown +6.7 0.002 Ta.7304.1.A1_at Unknown +6.4 0.001 Ta.3780.2.S1_a_at Unknown -8.8 0.001 Ta.3764.1.S1_at Unknown -6.5 0.001 Ta.30583.1.S1_at Unknown -9.9 0.035 Ta.30560.1.S1_at Unknown +27.9 0.008 Ta.2963.1.S1_at Unknown +11.3 0.000 Ta.29371.1.S1_at Unknown +5.8 0.000 51 DH1 vs ‘Superb’ DH2 vs ‘Superb’ Probe Set ID Gene Description FD p-val FD p-val Ta.28366.1.S1_a_at Unknown -5.5 0.003 Ta.28236.1.S1_x_at Unknown -6.3 0.017 Ta.28079.1.S1_x_at Unknown +5.4 0.023 Ta.25945.1.S1_at Unknown +11.5 0.002 Ta.24736.1.S1_at Unknown +8.3 0.016 Ta.14115.2.S1_at Unknown +5.4 0.006 Ta.13689.1.S1_at Unknown +6.5 0.000 Ta.1038.1.S1_a_at Unknown +18.4 0.000 TaAffx.144000.1.S1_s_at -5.2 0.031 52 Table 2.3 Genotype differences in challenge-induced protein accumulation. Positive and negative FD values indicate higher and lower protein accumulation, respectively, observed in spots from 2D-electrophoresis in the pH 4-7 (A) and 7-10 (B) ranges. Accessions and protein descriptions were determined by MASCOT searches. Protein predictions with scores above 100 and a minimum of two queries matched are considered acceptable. The organism indicates which species to which the protein prediction was based upon. FD p- value Spot No. Accession/ Gene ID Protein Description Pathway Score Queries Coverage Organism DH1 Tri5+ vs water +2.09 0.002 386A AY646352.1 spermidine synthase polyamine synthesis 194 3 9% Cucumis sativus -2.30 0.039 458A NM_001059187.1 ATP synthase subunit alpha, mitochondrial ATP synthesis 105 5 4% Oryza sativa +2.37 0.050 167B gi|54304033 catalase oxidative stress 223 8 13% Secale cereale +4.45 0.006 198A - - - - - - - DON vs water -2.53 0.006 193B - - - - - - - 53 FD p- value Spot No. Accession/ Gene ID Protein Description Pathway Score Queries Coverage Organism +3.11 0.009 44B gi|132270 Rubber elongation factor protein lipid metabolism 52 1 7% Hevea brasiliensis Tri5+ vs Tri5- -2.46 0.018 380A P18492 Glutamate-1-semialdehyde 2,1-aminomutase (GSA), chloroplastic - 184 4 11% Hordeum vulgare -2.09 0.001 123A - - - - - - - +2.12 0.026 177B - - - - - - - +2.37 0.045 198A - - - - - - - DH2 Tri5- vs water -3.62 0.015 240A - - - - - - - +2.01 0.033 74A AJ313311.1 phosphoglucomutase glycogenesis 274 9 11% Triticum aestivum +2.02 0.042 256B - - - - - - - +3.30 0.022 123B - - - - - - - +3.32 0.006 198A - - - - - - - DON vs water -3.79 0.004 240A - - - - - - - -2.98 0.007 128A - - - - - - - -2.55 0.046 309A NC_002762.1 ATP synthase CF1 alpha subunit ATP synthesis 673 19 32% Triticum aestivum 54 FD p- value Spot No. Accession/ Gene ID Protein Description Pathway Score Queries Coverage Organism -2.49 0.034 205B gi|226316441 fructose-bisphosphate aldolase glycolysis 307 11 23% Triticum aestivum -2.08 0.034 95B gi|120680 Glyceraldehyde-3- phosphate dehydrogenase, cytosolic glycolysis 567 19 41% Hordeum vulgare -2.01 0.045 187B gi|148508784 glyceraldehyde-3- phosphate dehydrogenase glycolysis 246 9 18% Triticum aestivum +2.53 0.013 226A gi|115450493 glyceraldehyde 3- phosphate dehydrogenase; Os03g0129300 glycolysis 248 8 15% Oryza sativa +4.03 0.037 123B - - - - - - - Tri5+ vs Tri5- -3.88 0.048 319A - - - - - - - -3.44 0.001 299A gi|56606827 calreticulin-like protein signal transduction 57 2 4% Triticum aestivum -2.12 0.033 447A gi|14017579 ATP synthase CF1 beta subunit ATP synthesis 535 12 26% Triticum aestivum +2.55 0.047 321A gi|66276267 ATP synthase beta subunit ATP synthesis 106 2 2% Coriaria ruscifolia +3.16 0.028 225A - - - - - - - ‘Superb’ 55 FD p- value Spot No. Accession/ Gene ID Protein Description Pathway Score Queries Coverage Organism Tri5+ vs water -3.49 0.010 565A - - - - - - - -2.08 0.000 190B gi|225465670 PREDICTED: hypothetical protein Kalvin cycle 58 2 6% Vitis vinifera +2.22 0.044 298A DQ435668.1 alpha tubulin-5D cytoskeleton 160 5 11% Triticum aestivum +5.06 0.038 279B - - - - - - - Tri5- vs water -3.78 0.013 558A DQ435659.1 alpha tubulin-2A cytoskeleton 356 11 13% Triticum aestivum -2.17 0.030 9A gi|15808779 ascorbate peroxidase oxidative stress 177 4 22% Hordeum vulgare +2.07 0.022 444A AY236152.1 cytosolic malate dehydrogenase Kalvin cycle 247 7 18% Triticum aestivum +2.24 0.007 538A - - - - - - - +2.26 0.013 279B - - - - - - - DON vs water -4.65 0.024 564A - - - - - - - -2.71 0.034 470A gi|18181983 myo-inositol-1-phosphate synthase inositol phosphate metabolism 216 6 10% Avena sativa Tri5+ vs Tri5- -2.09 0.033 9A gi|15808779 ascorbate peroxidase oxidative stress 177 4 22% Hordeum vulgare 56 Table 2.4 Challenge-dependent changes in defence-related genes in FHB-susceptible ‘Superb’ and resistant genotypes. Positive and negative FD (FD > 2.0, p < 0.05) values indicate higher and lower transcript accumulation, respectively, in FgTri5+ (FgTri5+ vs water), FgTri5- (FgTri5- vs water), DON (DON vs water), and in FgTri5+ vs FgTri5-. Probe Set ID Predicted Gene Function Plant Line Treatment Comparison hai FD p-value Kinases TaAffx.31923.1.S1_at Serine/threonine protein kinase [Oryza sativa (japonica cultivar-group)] Superb Tri5+ vs water 3 2.035 0.028 TaAffx.12878.1.A1_at Wall-associated kinase 3 [Triticum aestivum] Superb Tri5+ vs water 8 2.077 0.006 TaAffx.113624.2.S1_at Serine/threonine-protein kinase BRI1-like 3 precursor (Brassinosteroid insensitive1-like protein 3) Superb Tri5+ vs water 8 2.656 0.005 TaAffx.113624.2.S1_at Serine/threonine-protein kinase BRI1-like 3 precursor (Brassinosteroid insensitive 1-like protein 3) Superb Tri5- vs water 8 2.345 0.006 Ta.18587.1.S1_x_at Systemin receptor SR160 precursor (Brassinosteroid LRR receptor kinase) [Oryza sativa (japonica cultivar- group)] Superb Tri5- vs water 24 -2.296 0.000 Ta.3748.1.A1_at Hexokinase1 Superb Tri5- vs water 24 -2.201 0.015 Ta.3748.1.A1_at Hexokinase1 Superb DON vs water 24 -2.233 0.012 Ta.4972.1.A1_at Aspartate kinase-homoserine dehydrogenase [Oryza sativa (japonica cultivar-group)] (contig annotation) Superb DON vs water 24 -2.096 0.046 Ta.10354.2.S1_x_at Pyrophosphate--fructose 6-phosphate 1- phosphotransferase alpha subunit (PFP) (6- phosphofructokinase, pyrophosphate dependent) Superb DON vs water 24 -2.053 0.026 57 Probe Set ID Predicted Gene Function Plant Line Treatment Comparison hai FD p-value TaAffx.110222.1.S1_x_at Leucine-rich repeat-containing extracellular glycoprotein [Sorghum bicolor] / somatic embryogenesis receptor kinase SERK [Medicago truncatula] Superb DON vs water 24 2.217 0.004 Ta.20980.2.S1_at Serine/threonine kinase receptor precursor (S-receptor kinase) (SRK) Superb Tri5+ vs Tri5- 24 2.202 0.012 TaAffx.64399.1.S1_at Receptor-like kinase RHG1 [Glycine max] DH1 Tri5+ vs water 3 -2.750 0.003 Ta.19909.1.A1_at MAP kinase activating protein C22orf5 DH1 Tri5+ vs water 3 -2.482 0.026 TaAffx.84282.1.S1_at CDPK-related protein kinase (PK421) DH1 Tri5+ vs water 3 -2.401 0.000 Ta.1357.+2.A1_at Protein kinase [Oryza sativa (japonica cultivar-group)] DH1 Tri5+ vs water 8 2.002 0.031 Ta.18665.1.S1_at Protein kinase G11A [Oryza sativa (japonica cultivar- group)] DH1 Tri5+ vs water 8 2.027 0.010 TaAffx.18131.1.S1_at Putative phytosulfokine receptor precursor (Phytosulfokine LRR receptor kinase) DH1 Tri5+ vs water 8 2.073 0.048 Ta.5204.1.S1_at ATP binding / kinase/ protein serine/threonine kinase [Arabidopsis thaliana] DH1 Tri5+ vs water 8 2.188 0.022 Ta.3631.2.S1_at 3,4-dihydroxy-2-butanone kinase DH1 Tri5+ vs water 8 2.203 0.035 TaAffx.37109.1.S1_at Protein kinase [Arabidopsis thaliana] DH1 Tri5+ vs water 8 2.382 0.003 Ta.5481.1.S1_at Glucosidase II beta subunit precursor (Protein kinase C substrate, 60.1 kDa protein, heavy chain) (PKCSH) (80K-H protein) (Vacuolar system) DH1 Tri5+ vs water 8 2.498 0.004 Ta.6683.1.A1_x_at Mitogen-activated protein kinase kinase kinase 12 (Leucine-zipper protein kinase) (ZPK) (Dual leucine zipper bearing kinase) (DLK) DH1 Tri5+ vs water 8 2.705 0.040 TaAffx.12878.1.A1_at Wall-associated kinase 3 [Triticum aestivum] DH1 Tri5+ vs water 24 -2.062 0.015 58 Probe Set ID Predicted Gene Function Plant Line Treatment Comparison hai FD p-value Ta.6954.3.S1_x_at Choline kinase [Oryza sativa (japonica cultivar-group)] DH1 Tri5- vs water 3 -2.404 0.037 Ta.3322.3.S1_x_at Ankyrin protein kinase-like [Poa pratensis] DH1 Tri5- vs water 3 -2.190 0.023 TaAffx.38017.1.A1_at Receptor-type protein kinase LRK1 [Oryza sativa (japonica cultivar-group)] DH1 Tri5- vs water 3 -2.003 0.011 TaAffx.54339.1.S1_at Mitogen-activated protein kinase homolog MMK2 DH1 Tri5- vs water 3 2.087 0.008 TaAffx.37109.1.S1_at Receptor protein kinase [Arabidopsis thaliana] DH1 Tri5- vs water 8 2.021 0.007 TaAffx.101059.2.S1_at 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase, chloroplast precursor (CMK) DH1 Tri5- vs water 8 2.238 0.010 TaAffx.52905.1.S1_x_at Phosphoribulokinase DH1 Tri5- vs water 8 2.251 0.002 TaAffx.12878.1.A1_at Wall-associated kinase 3 [Triticum aestivum] DH1 Tri5- vs water 8 2.398 0.000 TaAffx.56854.1.S1_at Serine/threonine kinase receptor precursor-like protein [Oryza sativa (japonica cultivar-group)] DH1 Tri5- vs water 24 2.029 0.005 Ta.3931.1.S1_at 30 kDa pollen allergen (GIX1) [Hordeum vulgare] DH1 Tri5- vs water 24 2.082 0.020 Ta.8249.3.S1_at Similar to calmodulin-domain protein kinase [Oryza sativa (japonica cultivar-group)] DH1 Tri5- vs water 24 2.294 0.025 Ta.29379.1.A1_at Kinase interacting protein 1 -like [Oryza sativa (japonica cultivar-group)] DH1 DON vs water 8 2.017 0.001 TaAffx.488+2.1.S1_at Receptor-type protein kinase LRK1 [Oryza sativa (japonica cultivar-group)] DH1 DON vs water 8 2.028 0.015 TaAffx.30003.1.S1_x_at Aegilops tauschii protein kinase 1 mRNA, complete cds DH1 DON vs water 8 2.076 0.003 TaAffx.50893.1.S1_s_at Serine/threonine protein kinase [Triticum aestivum] DH1 DON vs water 8 2.269 0.008 Ta.5481.1.S1_at Glucosidase II beta subunit precursor (Protein kinase C substrate, 60.1 kDa protein, heavy chain) (PKCSH) (80K-H protein) (Vacuolar system DH1 DON vs water 8 2.429 0.027 59 Probe Set ID Predicted Gene Function Plant Line Treatment Comparison hai FD p-value TaAffx.129414.2.S1_at Receptor-like protein kinase precursor DH1 DON vs water 24 -2.429 0.032 Ta.13013.2.S1_x_at Pathogenesis-related protein 10b [Sorghum bicolor] DH1 DON vs water 24 -2.416 0.004 Ta.22638.1.A1_at YRK1 DH1 DON vs water 24 -2.326 0.030 Ta.27812.1.A1_at Receptor-protein kinase [Oryza sativa (japonica cultivar-group)] DH1 DON vs water 24 -2.074 0.008 TaAffx.10874.1.S1_at Receptor-protein kinase [Oryza sativa (japonica cultivar-group)] DH1 DON vs water 24 -2.035 0.010 TaAffx.104820.1.S1_at Receptor kinase [Oryza sativa (japonica cultivar- group)] DH1 DON vs water 24 -2.013 0.044 TaAffx.54339.1.S1_at Mitogen-activated protein kinase homolog MMK2 DH1 Tri5+ vs Tri5- 3 -2.316 0.014 TaAffx.5699.1.S1_at Receptor protein kinase CRINKLY4 precursor DH1 Tri5+ vs Tri5- 3 2.005 0.030 TaAffx.101059.2.S1_at 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase, chloroplast precursor (CMK) DH1 Tri5+ vs Tri5- 8 -2.096 0.023 TaAffx.59615.1.S1_at Receptor-like protein kinase [Oryza sativa (japonica cultivar-group)] DH1 Tri5+ vs Tri5- 24 -2.136 0.021 TaAffx.4882.1.S1_at Receptor-type protein kinase LRK1 [Oryza sativa (japonica cultivar-group)] DH1 Tri5+ vs Tri5- 24 -2.055 0.026 Ta.4696.1.S1_at Receptor protein kinase-like protein [Oryza sativa (japonica cultivar-group)] DH2 Tri5+ vs water 8 2.646 0.029 Ta.1684.3.S1_at Nucleoside diphosphate kinase III, chloroplast precursor (NDK III) DH2 Tri5- vs water 3 -2.055 0.042 Ta.19909.1.A1_at MAP kinase activating protein C22orf5 DH2 Tri5- vs water 8 2.098 0.009 TaAffx.114172.1.S1_s_at Serine/threonine-specific protein kinase [Oryza sativa (japonica cultivar-group)] DH2 Tri5- vs water 24 -2.238 0.010 TaAffx.5982.1.S1_at MAPKK kinase [Hordeum vulgare subsp. vulgare] DH2 DON vs water 3 -2.091 0.044 60 Probe Set ID Predicted Gene Function Plant Line Treatment Comparison hai FD p-value Ta.1684.3.S1_at Nucleoside diphosphate kinase III, chloroplast precursor (NDK III) DH2 Tri5+ vs Tri5- 3 2.731 0.009 Ta.16965.2.A1_at UNR-interacting protein (serine-threonine kinase receptor-associated protein) DH2 Tri5+ vs Tri5- 8 -2.068 0.044 Ribosomal Proteins TaAffx.108878.1.S1_x_at 60S ribosomal protein L5 [Neurospora crassa] Superb Tri5+ vs water 24 -2.086 0.019 TaAffx.6593.2.S1_at Chloroplast 50S ribosomal protein L33 Superb Tri5+ vs water 24 -2.070 0.010 TaAffx.35350.1.S1_at Ribosomal protein S4 [Panax ginseng] Superb Tri5- vs water 3 2.246 0.001 TaAffx.128418.38.S1_at 18S ribosomal RNA [Glycine max] Superb DON vs water 3 -2.039 0.000 TaAffx.107003.1.S1_at 50S ribosomal protein L4, chloroplast (CL4) [Arabidopsis thaliana] Superb DON vs water 3 -2.018 0.016 TaAffx.128418.24.S1_at 28S ribosomal RNA [Triticum aestivum] Superb DON vs water 8 -2.637 0.030 Ta.12751.3.A1_at Ribosomal protein L13a Superb Tri5+ vs Tri5- 8 2.274 0.012 Ta.10990.1.A1_at 40S ribosomal protein S19 DH1 Tri5+ vs water 8 2.022 0.019 TaAffx.107897.1.S1_at Chloroplast 30S ribosomal protein S18 DH1 Tri5+ vs water 8 2.180 0.039 TaAffx.129824.5.S1_x_at ribosomal protein S11 [Triticum aestivum] DH1 Tri5+ vs water 8 2.393 0.041 TaAffx.113782.1.S1_at 60S ribosomal protein L11-1 (L16A) DH1 Tri5+ vs water 24 -2.034 0.022 TaAffx.128418.24.S1_at 28S ribosomal RNA [Triticum aestivum] DH1 Tri5- vs water 8 2.359 0.032 Ta.9507.2.S1_x_at Ribosomal protein L6 DH1 DON vs water 8 2.678 0.026 Ta.9507.2.S1_at Ribosomal protein L6 DH1 DON vs water 8 2.722 0.037 TaAffx.111195.1.S1_at 5S ribosomal RNA [Triticum aestivum] DH1 DON vs water 8 3.004 0.012 61 Probe Set ID Predicted Gene Function Plant Line Treatment Comparison hai FD p-value Ta.30687.2.S1_x_at Ribosomal protein S29 DH2 Tri5+ vs water 3 -3.951 0.013 Ta.13787.1.S1_x_at Plastid-specific 30S ribosomal protein 1, chloroplast precursor (CS-S5) (CS5) (S22) (ribosomal protein 1) (PSRP-1) DH2 Tri5+ vs water 24 -2.313 0.032 TaAffx.111195.1.S1_at 5S ribosomal RNA [Triticum aestivum] DH2 Tri5- vs water 3 -2.237 0.049 TaAffx.56089.1.S1_at Ubiquitin/ribosomal fusion protein DH2 Tri5- vs water 3 2.018 0.022 Ta.30687.2.S1_x_at Ribosomal protein S29 DH2 Tri5- vs water 8 2.992 0.021 TaAffx.129824.5.S1_x_at ribosomal protein S11 [Triticum aestivum] DH2 Tri5- vs water 24 -3.485 0.049 TaAffx.129824.9.S1_x_at 30S ribosomal protein S11 DH2 Tri5- vs water 24 -3.466 0.045 Ta.30687.2.S1_x_at Ribosomal protein S29 DH2 Tri5- vs water 24 -2.624 0.008 TaAffx.128896.21.S1_x_at Chloroplast 30S ribosomal protein S8 DH2 Tri5- vs water 24 -2.372 0.020 TaAffx.129824.1.S1_s_at Chloroplast 30S ribosomal protein S11 DH2 Tri5- vs water 24 -2.128 0.044 TaAffx.111195.1.S1_at 5S ribosomal RNA [Triticum aestivum] DH2 DON vs water 3 -3.190 0.022 Ta.9507.+2.S1_x_at Ribosomal protein L6 DH2 DON vs water 8 2.028 0.026 Ta.9507.2.S1_at Ribosomal protein L6 DH2 DON vs water 8 2.083 0.027 Ta.30687.2.S1_x_at Ribosomal protein S29 DH2 DON vs water 8 2.211 0.037 Ta.30687.2.S1_x_at Ribosomal protein S29 DH2 DON vs water 24 -4.267 0.033 Ta.30687.2.S1_x_at Ribosomal protein S29 DH2 Tri5+ vs Tri5- 3 -3.020 0.031 Ta.30687.2.S1_x_at Ribosomal protein S29 DH2 Tri5+ vs Tri5- 24 2.411 0.011 62 Probe Set ID Predicted Gene Function Plant Line Treatment Comparison hai FD p-value Phenylpropanoid pathway Ta.7022.1.S1_x_at Phenylalanine ammonia-lyase Superb DON vs water 3 -2.258 0.016 Ta.9220.3.S1_at Phenylalanine ammonia-lyase DH1 DON vs water 8 2.012 0.013 Ta.3609.1.S1_a_at 4-coumarate-CoA ligase [Arabidopsis thaliana] DH1 DON vs water 8 2.096 0.001 Ta.10418.2.S1_x_at Chalcone synthase [Oryza sativa (japonica cultivar- group)] DH1 DON vs water 8 2.242 0.033 TaAffx.84154.1.S1_at Phenylalanine ammonia-lyase DH1 DON vs water 8 2.272 0.020 Ta.9220.3.S1_x_at Phenylalanine ammonia-lyase DH1 Tri5+ vs water 8 2.129 0.003 Ta.8086.1.A1_at Hydroxycinnamoyl transferase [Oryza sativa (japonica cultivar-group)] DH1 Tri5- vs water 8 2.291 0.036 Ta.9220.3.S1_x_at Phenylalanine ammonia-lyase DH1 Tri5- vs water 8 2.660 0.003 Ta.9172.3.S1_at Naringenin-chalcone synthase DH1 Tri5- vs water 24 2.466 0.050 TaAffx.111664.1.S1_at Cinnamyl alcohol dehydrogenase [Oryza sativa (japonica cultivar-group)] DH2 Tri5- vs water 3 2.046 0.006 Pathogenesis-related (PR) Ta.2256+2.1.S1_at Glucan endo-1,3-beta-D-glucosidase Superb Tri5+ vs water 24 2.072 0.045 Ta.82.1.S1_at Peroxidase Superb Tri5+ vs water 24 2.659 0.027 Ta.21120.1.S1_at Glucan endo-1,3-beta-D-glucosidase Superb Tri5+ vs water 24 3.080 0.017 Ta.14461.3.S1_x_at Nectarin 1 precursor (Superoxide dismutase [Mn]) Superb DON vs water 8 -2.564 0.024 Ta.29496.2.S1_at Peroxidase 12 precursor (Atperox P12) (PRXR6) (ATP4a) Superb DON vs water 8 -2.200 0.037 63 Probe Set ID Predicted Gene Function Plant Line Treatment Comparison hai FD p-value TaAffx.32266.1.A1_at Phospholipid hydroperoxide glutathione peroxidase (PHGPx) (Salt-associated protein) Superb DON vs water 24 -2.664 0.047 Ta.24715.1.S1_at Peroxidase Superb DON vs water 24 2.019 0.037 Ta.8304.1.S1_x_at Pathogenesis-related PR1a [Triticum monococcum] Superb DON vs water 24 2.084 0.010 TaAffx.116570.1.S1_at Pathogenesis-related protein 4 Superb DON vs water 24 2.207 0.050 Ta.28354.3.S1_x_at Glutathione transferase Superb DON vs water 24 2.293 0.005 Ta.21307.1.S1_x_at peroxidase [Oryza sativa (japonica cultivar-group)] Superb DON vs water 24 2.311 0.016 Ta.24501.1.S1_at thaumatin-like protein Superb DON vs water 24 2.537 0.030 Ta.22562.1.S1_at Glucan endo-1,3-beta-D-glucosidase Superb DON vs water 24 2.541 0.002 Ta.27762.1.S1_x_at Thaumatin-like protein Superb DON vs water 24 2.764 0.029 Ta.21120.1.S1_at Glucan endo-1,3-beta-D-glucosidase Superb DON vs water 24 3.205 0.001 Ta.82.1.S1_at Peroxidase Superb DON vs water 24 4.072 0.007 Ta.14946.1.S1_at ATHCHIB (basic chitinase) [Arabidopsis thaliana] Superb DON vs water 24 5.850 0.010 TaAffx.78864.1.S1_at Glutathione S-transferase [Oryza sativa (japonica cultivar-group)] Superb Tri5+ vs Tri5- 8 -2.304 0.011 Ta.21386.1.S1_at Nonspecific lipid-transfer protein 2 (LTP 2) Superb Tri5+ vs Tri5- 8 2.066 0.048 Ta.5235.1.S1_x_at Peroxidase precursor Superb Tri5+ vs Tri5- 24 2.055 0.003 TaAffx.86266.1.S1_at Beta-glucosidase homologue precursor DH1 Tri5+ vs water 8 2.088 0.038 Ta.14580.1.S1_at Peroxidase precursor DH1 Tri5+ vs water 8 2.166 0.012 64 Probe Set ID Predicted Gene Function Plant Line Treatment Comparison hai FD p-value Ta.30755.1.S1_at Nonspecific lipid-transfer protein 2G (LTP2G) (Lipid transfer protein 2 isoform 1) (LTP2-1) (7 kDa lipid transfer protein 1) DH1 Tri5+ vs water 8 2.420 0.044 Ta.14281.1.S1_at Defensin DH1 Tri5+ vs water 8 2.422 0.014 Ta.5481.1.S1_at Glucosidase II beta subunit precursor (Protein kinase C substrate, 60.1 kDa protein, heavy chain) (PKCSH) (80K-H protein) (Vacuolar system) DH1 Tri5+ vs water 8 2.498 0.004 Ta.13754.1.S1_s_at Lipid transfer protein-related [Arabidopsis thaliana] DH1 Tri5+ vs water 8 2.589 0.043 Ta.4601.2.S1_at Beta-glucosidase DH1 Tri5+ vs water 8 2.593 0.033 Ta.8258.1.S1_x_at Type 2 non-specific lipid transfer protein precursor [Triticum aestivum] DH1 Tri5+ vs water 24 -2.528 0.040 Ta.8258.2.S1_at Type 2 non-specific lipid transfer protein [Triticum aestivum] DH1 Tri5+ vs water 24 -2.463 0.042 Ta.11124.1.A1_at Glucan endo-1,3-beta-D-glucosidase [Hordeum vulgare subsp. vulgare] (contig annotation) DH1 Tri5- vs water 3 -2.312 0.034 Ta.23376.2.S1_s_at Peroxidase 47 precursor (Atperox P47) (ATP32) DH1 Tri5- vs water 3 -2.079 0.018 Ta.4328.1.S1_at Pathogenesis-related protein 10b [Sorghum bicolor] DH1 Tri5- vs water 8 2.299 0.015 Ta.4876.1.A1_x_at Peroxidase DH1 Tri5- vs water 8 2.342 0.045 Ta.761.1.S1_at Glutathione S-transferase [Oryza sativa (japonica cultivar-group)] (contig annotation) DH1 Tri5- vs water 8 2.358 0.038 Ta.26983.1.A1_at Chitinase DH1 Tri5- vs water 8 2.364 0.037 Ta.14281.1.S1_at Defensin DH1 DON vs water 8 2.059 0.047 TaAffx.32251.1.S1_at Nonspecific lipid transfer protein 4.3 precursor (LTP 4.3) DH1 DON vs water 8 2.064 0.022 65 Probe Set ID Predicted Gene Function Plant Line Treatment Comparison hai FD p-value Ta.5481.1.S1_at Glucosidase II beta subunit precursor (Protein kinase C substrate, 60.1 kDa protein, heavy chain) (PKCSH) (80K-H protein) (Vacuolar system) DH1 DON vs water 8 2.429 0.027 Ta.26048.1.S1_x_at Glucan endo-1,3-beta-D-glucosidase DH1 DON vs water 24 -2.516 0.013 Ta.303.3.S1_x_at Glutathione transferase DH1 DON vs water 24 -2.482 0.043 Ta.28.1.S1_at Glucan endo-1,3-beta-D-glucosidase DH1 DON vs water 24 -2.463 0.012 Ta.13013.2.S1_x_at Pathogenesis-related protein 10b [Sorghum bicolor] DH1 DON vs water 24 -2.416 0.004 Ta.28354.3.S1_x_at Glutathione transferase DH1 DON vs water 24 -2.291 0.048 Ta.30739.1.A1_at Pathogenesis-related 1a [Triticum monococcum] DH1 Tri5+ vs Tri5- 8 -2.049 0.038 Ta.8828.3.S1_a_at Peroxidase [Ananas comosus] DH2 Tri5- vs water 3 2.427 0.038 TaAffx.113452.1.S1_at Chitinase [Triticum aestivum] DH2 Tri5- vs water 24 -2.198 0.025 TaAffx.113452.1.S1_at Chitinase [Triticum aestivum] DH2 Tri5- vs water 24 -2.198 0.025 Ta.4876.1.A1_x_at Peroxidase DH2 Tri5- vs water 24 -2.182 0.011 Ta.18560.1.S1_s_at Class III peroxidase [Oryza sativa (japonica cultivar- group)] DH2 DON vs water 8 2.051 0.011 TaAffx.108531.2.S1_at Peroxidase DH2 DON vs water 8 2.266 0.001 Ta.30944.1.S1_s_at Glutathione transferase F2 DH2 DON vs water 8 2.699 0.035 Ta.18647.1.S1_s_at Nonspecific lipid-transfer protein AKCS9 precursor (LTP) DH2 DON vs water 24 -2.062 0.025 Ta.14850.1.S1_at Glutathione S-transferase [Oryza sativa (japonica cultivar-group)] DH2 Tri5+ vs Tri5- 3 -2.139 0.042 Ta.25024.1.S1_x_at Peroxidase [Arabidopsis thaliana] DH2 Tri5+ vs Tri5- 8 -2.044 0.029 66 Probe Set ID Predicted Gene Function Plant Line Treatment Comparison hai FD p-value Ta.27389.1.S1_at Gamma-purothionin - poulard wheat DH2 Tri5+ vs Tri5- 24 2.093 0.003 Ta.30501.1.S1_at Chitinase DH2 Tri5+ vs Tri5- 24 2.420 0.031 Ta.5385.1.S1_at Peroxidase DH2 Tri5+ vs Tri5- 24 2.716 0.021 SA signalling Ta.8304.1.S1_x_at Pathogenesis-related PR1a [Triticum monococcum] Superb DON vs water 24 2.1 0.010 Ta.3322.3.S1_x_at Ankyrin protein kinase-like [Poa pratensis] DH1 Tri5- vs water 3 -2.2 0.023 JA signalling Ta.526.1.S1_x_at Lipoxygenase Superb DON vs water 3 -2.8 0.010 TaAffx.58772.1.S1_at 12-oxophytodienoate reductase 3 (12- oxophytodienoate-10,11-reductase 3) (OPDA- reductase 3) (LeOPR3) Superb Tri5- vs water 8 2.2 0.001 TaAffx.128684.1.S1_x_at 2-oxo-phytodienoic acid reductase [Zea mays] DH1 DON vs water 24 -2.1 0.022 Ta.12757.1.A1_at Lipoxygenase-like protein (lox gene) [Hordeum vulgare subsp. vulgare] DH1 DON vs water 24 -2.4 0.015 Ta.1207.1.S1_at Oxo-phytodienoic acid reductase [Oryza sativa (japonica cultivar-group)] DH1 DON vs water 24 -2.1 0.015 Ta.30827.1.A1_x_at 32.6 kDa jasmonate-induced protein [H. vulgare] DH1 Tri5- vs water 24 2.2 0.044 Ta.1967.1.S1_x_at Lipoxygenase DH2 Tri5- vs water 3 2.9 0.049 Ta.12757.1.A1_at Lipoxygenase-like protein (lox gene) [H. Vulgare subsp. vulgare] DH2 Tri5- vs water 24 -2.2 0.017 Ta.526.1.S1_x_at Lipoxygenase (contig annotation) DH2 Tri5+ vs Tri5- 3 -2.0 0.038 67 Probe Set ID Predicted Gene Function Plant Line Treatment Comparison hai FD p-value Ta.1207.1.S1_at Oxo-phytodienoic acid reductase [O. sativa (japonica cultivar-group)] DH2 Tri5+ vs Tri5- 3 2.2 0.043 TaAffx.134501.1.A1_at Lipoxygenase DH2 Tri5+ vs Tri5- 24 2.2 0.048 Ta.1207.1.S1_x_at Oxo-phytodienoic acid reductase [O. sativa (japonica cultivar-group)] DH2 Tri5+ vs water 3 2.2 0.032 Ta.1207.1.S1_at Oxo-phytodienoic acid reductase [O. sativa (japonica cultivar-group)] DH2 Tri5+ vs water 3 3.2 0.004 ET signalling Ta.4470.1.S1_at Ethylene-binding protein-like / AP2 domain-containing transcription factor-like [O. sativa (japonica cultivar- group)] Superb Tri5- vs water 3 2.3 0.049 Ta.6397.1.A1_at Adenosylmethionine decarboxylase Superb DON vs water 24 -2.2 0.002 TaAffx.93223.1.A1_at 1-aminocyclopropane-1-carboxylate oxidase (ACC oxidase) (Ethylene-forming enzyme) (EFE) Superb DON vs water 8 -2.1 0.025 TaAffx.128576.1.S1_at ethylene-forming enzyme [O. sativa (japonica cultivar- group)] DH1 Tri5+ vs water 8 2.0 0.019 TaAffx.57475.1.S1_x_at Methionine adenosyltransferase DH2 Tri5- vs water 3 2.1 0.047 68 Table 2.5 Challenge-induced differential protein accumulation. Positive and negative FD values indicate higher and lower protein accumulation, respectively, observed in spots from 2D-electrophoresis in the pH 4-7 (A) and 7-10 (B) ranges. Accessions and protein descriptions were determined by MASCOT searches. Protein predictions with scores above 100 and a minimum of two queries matched are considered acceptable. The organism indicates which species to which the protein prediction was based upon. FD p- value Spot No. Accession/ Gene ID Protein Description Pathway Score Queries Coverage Organism DH1 vs ‘Superb’ Water -2.39 0.024 141A gi|167096 ribulose 1,5-bisphosphate carboxylase activase isoform 1 photosynthesis 375 15 Hordeum vulgare -2.37 0.012 163B gi|13873336 dihydrolipoamide dehydrogenase precursor Kalvin cycle 162 6 10 Bruguiera gymnorhiza -2.32 0.035 17A gi|114145394 glycine-rich RNA-binding protein - 389 10 48 - 2.01 0.004 16B BAF18429 nucleoside diphosphate kinase; Os05g0595400 - 202 8 20 Oryza sativa Tri5+ -2.55 0.045 17A gi|114145394 glycine-rich RNA-binding protein - 389 10 48 - -2.39 0.005 260A P12782 Phosphoglycerate kinase, chloroplastic glycolysis 367 10 17 Triticum aestivum -2.30 0.017 174A - - - - - - - -2.05 0.014 586A - - - - - - - 2.08 0.048 387A BT039007.1 unknown 73 3 6 Zea mays 2.15 0.005 296A - - - - - - - 69 FD p- value Spot No. Accession/ Gene ID Protein Description Pathway Score Queries Coverage Organism Tri5+ 2.31 0.046 21B - - - - - - - 2.99 0.012 380A P18492 Glutamate-1-semialdehyde 2,1-aminomutase (GSA), chloroplastic chlorophyl synthesis 184 4 11 Hordeum vulgare Tri5- -3.71 0.004 75A AB059557.2 myo-inositol-1-phosphate synthase 501 19 - - -2.81 0.000 369A gi|4239821 germin-like protein 1 oxidative stress 186 6 20 Triticum aestivum 2.01 0.021 85B gi|195622500 serine hydroxymethyltransferase amino acid synthesis 143 8 9 Zea mays 2.01 0.000 8A gi|310561 ascorbate peroxidase oxidative stress 121 5 5 Glycine max 2.10 0.041 299A gi|56606827 calreticulin-like protein signal transduction 57 2 4 Triticum aestivum 2.24 0.044 86B 2.34 0.023 29CO gi|83267753 FT-like protein flowering 125 6 - Hordeum vulgare 2.93 0.050 357A - - - - - - - 2.97 0.002 387A BT039007.1 unknown - 73 3 6 Zea mays 3.99 0.008 198A - - - - - - - DON 2.04 0.023 134B gi|205830697 RecName: Full=Unknown protein 18 - 112 3 100 Pseudotsuga menziesii 2.08 0.014 110B gi|75138360 Phospho-2-dehydro-3- deoxyheptonate aldolase 2, chloroplastic; AltName: Full=Phospho-2-keto-3- shikimic acid pathway 79 1 2 Oryza sativa 70 FD p- value Spot No. Accession/ Gene ID Protein Description Pathway Score Queries Coverage Organism +2.12 0.042 208B - - - - - - - +2.13 0.010 158B gi|91694277 glucose-6-phosphate isomerase glycolysis 404 11 13 Triticum aestivum +2.36 0.014 120B gi|115450835 phosphoserine aminotransferase, chloroplast 03g0157900 amino acid synthesis 227 5 9 Oryza sativa +2.64 0.015 69B gi|118748148 UCW116, putative lipase lipid metabolism 90 4 7 Hordeum vulgare DH2 vs ‘Superb’ Water -2.06 0.047 183B - - - - - - - -2.05 0.016 538A - - - - - - - +2.11 0.036 75B - - - - - - - +2.38 0.005 53B gi|223974435 unknown - 82 2 6 Zea mays +2.40 0.019 71B gi|115488340 RNA binding protein, putative, expressed; Os12g0420200 - 320 10 19 Oryza sativa +2.75 0.027 447A gi|14017579 ATP synthase CF1 beta subunit ATP synthesis 535 12 26 Triticum aestivum Water +2.84 0.017 280B - - - - - - - Tri5+ -3.63 0.019 165B X61626.1 catalase oxidative stress 49 1 1 Oryza sativa -2.63 0.016 434A gi|115461739 legumin-like protein, putative, expressed; Os05g0116000 storage protein 199 7 11 Oryza sativa 71 FD p- value Spot No. Accession/ Gene ID Protein Description Pathway Score Queries Coverage Organism -2.29 0.004 321A gi|66276267 ATP synthase beta subunit ATP synthesis 106 2 2 Coriaria ruscifolia +2.14 0.012 116B gi|115488340 RNA binding protein, putative, expressed; Os12g0420200 - 338 15 18 Oryza sativa +2.23 0.007 93B - - - - - - - +2.27 0.034 447A gi|14017579 ATP synthase CF1 beta subunit ATP synthesis 535 12 26 Triticum aestivum +2.41 0.026 53B gi|223974435 unknown - 82 2 6 Zea mays +3.08 0.004 71B gi|115488340 RNA binding protein, putative, expressed; Os12g0420200 - 320 10 19 Oryza sativa +3.11 0.004 565A - - - - - - - Tri5- -2.73 0.021 25A AP003510.3 putative chaperonin 21 precursor protein folding 108 3 9 Oryza sativa -2.33 0.008 444A AY236152.1 cytosolic malate dehydrogenase Kalvin cycle 247 7 18 Triticum aestivum -2.20 0.014 591A - - - - - - - Tri5- -2.19 0.001 369A gi|4239821 germin-like protein 1 oxidative stress 186 6 20 Triticum aestivum +2.04 0.018 134B gi|205830697 Unknown - 112 3 100 Pseudotsuga menziesii +2.35 0.026 5A gi|81176509 atp1 ATP synthesis 151 3 6 Triticum aestivum +2.62 0.040 378A gi|115450493 glyceraldehyde 3-phosphate dehydrogenase; Os03g0129300 glycolysis 331 8 15 Oryza sativa +2.87 0.012 357A - - - - - - - +2.87 0.019 528A - - - - - - - 72 FD p- value Spot No. Accession/ Gene ID Protein Description Pathway Score Queries Coverage Organism +3.66 0.025 52A - - - - - - - +4.28 0.006 198A - - - - -- - - DON -3.72 0.032 130A gi|242089295 hypothetical protein SORBIDRAFT_09g001680 - 85 2 5 Sorghum bicolor +2.28 0.024 86A - - - - - - - +2.43 0.032 110B gi|75138360 Phospho-2-dehydro-3- deoxyheptonate aldolase 2, chloroplastic shikimic acid pathway 79 1 2 Oryza sativa +2.53 0.007 116B gi|115488340 RNA binding protein, putative, expressed; Os12g0420200 - 338 15 18 Oryza sativa +2.66 0.043 347A - - - - - - - +2.74 0.015 5A gi|81176509 atp1 ATP synthesis 151 3 6 Triticum aestivum +4.40 0.043 4B - - - - - - - DH2 vs DH1 water -3.27 0.013 226A gi|115450493 glyceraldehyde 3-phosphate dehydrogenase; Os03g0129300 glycolysis 248 8 15 Oryza sativa -2.77 0.034 458A NM_0010591 87.1 ATP synthase subunit alpha, mitochondrial ATP synthesis 105 5 4% Oryza sativa -2.45 0.034 170A gi|974605 single-stranded nucleic acid binding protein - 90 5 20 Triticum aestivum +2.53 0.015 71B gi|115488340 RNA binding protein, putative, expressed; Os12g0420200 - 320 10 19 Oryza sativa +2.63 0.045 280B - - - - - - - +3.36 0.004 163B gi|13873336 dihydrolipoamide dehydrogenase precursor Kalvin cycle 162 6 10 Bruguiera gymnorhiza 73 FD p- value Spot No. Accession/ Gene ID Protein Description Pathway Score Queries Coverage Organism Tri5+ -4.67 0.000 225A - - - - - - - -3.27 0.044 22B - - - - - - - -3.08 0.009 209A EQ973790.1 pyruvate dehydrogenase, putative Kalvin cycle 133 3 - Ricinus communis -2.84 0.026 380A P18492 Glutamate-1-semialdehyde 2,1-aminomutase, chloroplastic - 184 4 11 Hordeum vulgare Tri5+ -2.21 0.005 123A - - - - - - - -2.19 0.037 39A gi|20302473 ferredoxin-NADP(H) oxidoreductase photosynthesis 178 4 12 Triticum aestivum -2.03 0.009 204B - - - - - - - +2.03 0.007 30B gi|37788312 cyclophilin-like protein Signal transduction 97 4 12 Triticum aestivum +2.28 0.003 71B gi|115488340 RNA binding protein, putative, expressed; Os12g0420200 - 320 10 19 Oryza sativa +2.31 0.026 134B gi|205830697 Unknown - 112 3 100 Pseudotsuga menziesii +6.21 0.031 401A - - - - - - - Tri5- -3.34 0.017 299A gi|56606827 calreticulin-like protein signal transduction 57 2 4 Triticum aestivum -3.17 0.025 86B - - - - - - - -3.16 0.003 177B - - - - - - - -2.39 0.001 434A gi|115461739 legumin-like protein, putative, expressed; Os05g0116000 storage protein 199 7 11 Oryza sativa 74 FD p- value Spot No. Accession/ Gene ID Protein Description Pathway Score Queries Coverage Organism -2.26 0.029 39A gi|20302473 ferredoxin-NADP(H) oxidoreductase photosynthesis 178 4 12 Triticum aestivum +2.42 0.011 4B - - - - - - - +2.56 0.005 75A AB059557.2 myo-inositol-1-phosphate synthase 501 19 25 Avena sativa DON -3.28 0.007 174A - - - - - - - -2.86 0.016 205B gi|226316441 fructose-bisphosphate aldolase glycolysis 307 11 23 Triticum aestivum -2.39 0.041 319A - - - - - - - -2.16 0.032 85B gi|195622500 serine hydroxymethyltransferase amino acid synthesis 143 8 9 Zea mays 75 Table 2.6 qPCR validation of 2FD in microarray. Positive and negative FD values indicate up- and down-regulation in the given treatment comparison. microarray qPCR Probe Set ID Gene Description Plant line comparison hai FD p-val FD std error TaAffx.106139.1.S1_at unknown Superb FgTri5- vs water 24 -2.1 0.004 -2.4 ±0.16 Ta.526.1.S1_x_at Lipoxygenase DH2 FgTri5+ vs FgTri5- 3 -2.0 0.038 -3.1 ±0.92 TaAffx.108735.1.S1_at unknown DH1 FgTri5+ vs FgTri5- 8 +2.1 0.003 +2.2 ±0.27 Ta.1207.1.S1_at Oxo-phytodienoic acid reductase [O. sativa (japonica cultivar-group)] DH2 FgTri5+ vs FgTri5- 3 +2.2 0.043 +4.2 ±0.06 Ta.16723.2.S1_x_at unknown protein [O. sativa (japonica cultivar-group)] gi|48716457| DH1 DON vs water 8 +2.2 0.020 +2.4 ±1.54 Ta.24501.1.S1_at Thaumatin-like protein Superb DON vs water 24 +2.5 0.030 +2.2 ±0.68 Ta.1967.1.S1_x_at Lipoxygenase DH2 FgTri5- vs water 3 +2.9 0.049 +2.3 ±0.78 TaAffx.111195.1.S1_at 5S ribosomal RNA [T.aestivum] DH1 DON vs water 8 +3.0 0.012 +2.0 ±0.49 Ta.1207.1.S1_at Oxo-phytodienoic acid reductase [O.sativa (japonica cultivar-group)] DH2 FgTri5+ vs water 3 +3.2 0.004 +3.3 ±0.98 76 2.3.1 Real-time RT- PCR validation of microarray results Eight genes from nine comparisons were randomly selected from the > 2.0 FD microarray data set for confirmation by quantitative RT-PCR (qPCR). While the absolute expression FD values obtained by qPCR differed from those observed in the microarray system, all nine comparisons showed the same expression trend in the qPCR and microarray data sets, and displayed FD values >2.0 in both systems (Table 2.6). 2.3.2 Constitutive differences in gene expression Expression levels of 44 and 14 transcripts were constitutively different in spikelets of ‘Superb’ vs. DH1 (Type I resistant) and ‘Superb’ vs DH2 (Type II resistant), respectively (Table 2.2). The most dramatic constitutive difference observed was that of a wheat aquaporin PIP1 (identified by three unique probe sets), which was highly over-expressed (143 to 259 FD) in DH1 compared with ‘Superb’. Other transcripts with potential connections to plant defence that were over-expressed in DH1, compared with ‘Superb’, include a gene with similarity to a dehydration-responsive protein from rice (RD22; 74 FD) and a wheat LTP (TaLTP3; 39 FD). 2.3.3 Challenge-induced differences in global gene expression Differences > 2.0 FD in transcript accumulation were observed in all treatment comparisons and at all harvest times (Figure 2.2), with an average of 36 differentially regulated transcripts for any combination of genotype and treatment. The largest number of expression differences between treatments was observed in Type I resistant DH1 compared with treatment-induced differences observed in Type II resistant DH2 and susceptible cv. 77 ‘Superb’. Additionally, by 24 hai no up-regulation of transcripts was observed in DH2 in response to the three elicitors being evaluated (FgTri5+, FgTri5-, or DON). DON treatment led mainly to down-regulation of transcripts in all three wheat lines at 3 hai. In ‘Superb’, strong DON-induced down-regulation of gene expression was also observed at 8 hai, and by 24 hai, two-thirds of the differentially regulated genes were down-regulated. In the resistant genotypes, DON-induced down-regulation was also observed at 24 hai. However, at 8 hai, a total of 122 genes were up-regulated and none were down-regulated in DH1, while 40 genes were up-regulated and only 6 were down-regulated in DH2. 2.3.4 Challenge-induced differential gene expression related to defence response pathways The genes displaying differential expression following challenge were associated with several distinct pathways. Some of these genes are known to be involved directly in the plant defence response or potentially involved in regulating defence responses (Table 2.4). Differential expression of protein kinases was observed in all three wheat lines. In DH1, a total of 38 differences in kinase gene expression were observed across the various treatment conditions, and these differences occurred in response to all treatment comparisons (less so in the FgTri5+ vs. FgTri5- comparison). In contrast, only 8 and 11 kinase gene expression differences were observed across the various conditions in DH2 and ‘Superb’, respectively. 78 Figure 2.2 Number of treatment-dependent differentially regulated transcripts. For each VEN diagram, the top left circle indicates the number of up (+) or down (-) regulated genes in DH1 for a given treatment comparison at a given harvest time; similarly, for the top right circle representing DH2 and for the bottom one representing ‘Superb’. The overlapping regions between circles indicate the number of differentially regulated transcripts in common between plant lines. 79 Differential expression of genes encoding ribosomal components was also observed. At 8 hai in DH1, all three elicitors (FgTri5+, FgTri5-, and DON) induced up-regulation of ribosomal gene expression. In DH2, DON (8 hai) and FgTri5- (3 and 8 hai) also induced up-regulation of similar genes. Down-regulation of these genes was observed in susceptible ‘Superb’ in response both to DON (3 and 8 hai) and FgTri5+ (24 hai). Challenge-induced differences in expression of pathogenesis-related (PR) genes, primarily up-regulation, was observed in all three wheat genotypes, but occurred mainly in DH1 and ‘Superb’, and typically occurred earlier in the resistant genotypes (Table 2.4). Differential expression of phenylpropanoid pathway genes was primarily limited to DH1, where up-regulation of two genes encoding L-phenylalanine ammonia-lyase (PAL) was observed at 8 hai: one of these genes (probeset Ta.845145.1.S1_at) occurred in response to DON, and the other (probeset Ta.9220.S1_at) was induced by DON, FgTri5+ and FgTri5- (Table 2.4). In susceptible ‘Superb’, DON induced down-regulation of a different PAL (probeset Ta.7022.1.S1_x_at) at 3 hai. PAL is the entry point to phenylpropanoid metabolism and is involved in the formation of a variety phenolic metabolites, including lignins, flavonoids, and SA (Dixon et al., 2002). In DH1, up- regulation of genes involved in both lignin and flavonoid biosynthesis was observed in response to both FgTri5- and DON. Up-regulation of genes whose products have often been associated with SA signalling in Arabidopis, but not necessarily in wheat (Huckelhoven et al., 1999; Molina et al., 1999; Lu et al., 2006), were an ankyrin kinase-like protein that was down-regulated in DH1 (FgTri5- at 3 hai) and the SA-regulated PR-1, which was up-regulated in ‘Superb’ 24 hai after point inoculation with DON. 80 Jasmonic acid synthesis involves a well-characterized multi-step pathway that converts linolenic acid to the final hormone. Among the corresponding genes, up-regulation of oxo- phytodienoic acid reductase was observed in DH2 in response to FgTri5+ (3 hai) when compared to either FgTri5- or the water controls. Two genes encoding lipoxygenases, a class of enzymes thought to initiate JA biosynthesis, were also upregulated in response to the FgTri5+ treatment when compared with water (3 hai) or FgTri5- (24 hai) (Table 2.3). Up-regulation of JA biosynthesis or responsive genes was also observed in the ‘Superb’ and DH1 genotypes, but only in response to FgTri5- treatment. Point-inoculation with DON, on the other hand, induced down-regulation of JA signalling genes in both DH1 (24 hai) and ‘Superb’ (3 hai), but not in DH2 (Table 2.4). 2.3.5 2D-electrophoresis: optimization and resolution 2D-gels in the pH 4-7 range have successfully resolved wheat spikelet proteins in previous studies (Zhou et al., 2005; Zhou et al., 2006), but to my knowledge, the only reports on resolution of wheat proteins in the basic range were on pH 3-10 gels (Wang et al., 2005). In the current study, several techniques were tested for their ability to generate higher resolution of proteins in the basic range. First, the extracted proteins were reduced and protected prior to IEF. Three reduction/protection techniques were evaluated, using different reducing and protecting agents including (1) TBP and IAA; (2) TBP and 4-VP; and (3) TCEP and 4-VP. The reduction/protection step was followed by cup-loading onto the IPG strips, as recommended for basic range proteins (Biorad). Poor protein spot resolution was observed in the 2D-gels when using the TBP/IAA reagents. Overall 2D- gel resolution was improved in the other two approaches, which used 4-VP as a protecting 81 agent, but no resolved proteins were observed in the range of pH 8-9 (Figure 2.3A and B). In the next attempt to improve resolution, samples were reduced and protected using TCEP and 4-VP, and then pre-fractionated into proteins with isoelectric points in the ranges of pH 3-7 and pH 7-10 using a ZOOM® IEF Fractionator (Invitrogen). These pre-fractionated samples were then used to rehydrate IPG strips, in the pH 4-7 and 7-10 range, respectively, followed by IEF. The use of 4-VP as a protecting agent in combination with pre- fractionation according to pH resulted in improved resolution in the pH 7-10 gels (Figure 2.3D), although the resolution from pH 9-10 was inconsistent, and thus no analysis was performed in this range. This approach also provided good resolution in pH 4-7 range (Figure 2.3C). A total of 660 and 312 unique spots were resolved on pH 4-7 and 7-10 gels, respectively. For both sets of comparisons (genotype differences and challenge-induced differences), spots with more than a 2FD (p < 0.05) were sequenced. A total of 130 comparisons met these criteria (40 for the treatment comparisons, and 90 for the wheat genotype comparisons). Among the 130 differences, there were a total of 74 unique spots: 44 and 30 from the pH 4-7 and 7-10 ranges, respectively. Of the 74 unique spots, 45 had successful matches in the MASCOT searches with scores > 100. 2.3.6 Genotype differences in protein accumulation Constitutive differences in protein accumulation were not investigated in the spikelets of uninoculated spikes, as was done in the differential gene expression analysis, but genotype differences in protein accumulation were compared in spikelets of inoculated spikes (Table 82 2.3). Most of the genotype differences in the spikelets of water-challenged spikes were of metabolic proteins. Figure 2.3 2D-electrophoresis optimization. A. Reduction-protection trials with TBP/4- VP or B. TCEP/4-VP followed by cup loading onto pH 7-10 Biorad IPG strips (17 cm) and second dimension separation by SDS-PAGE. C. Reduction-protection with TCEP/4-VP, followed by pre-fractionation with ZOOM IEF fractionators into pH 3-7 and 7-10 fractions, which were then separated by IEF (passive rehydration) on pH 4-7 and D. 7-10 Biorad IPG strips (17 cm), respectively, and second dimension separation by SDS-PAGE. A. B. C. D. 83 The most interesting differences among the genotypes in response to challenge occurred in response to DON-challenge. This led to higher accumulation of a range of different proteins in the resistant genotypes, compared with ‘Superb’; only one protein (with unknown function) showed lower accumulation in the Type II resistant genotype, DH2. Among the four protein spots whose accumulation differed between the two resistant genotypes after DON-challenge, all showed lower expression in DH2. Most of the genotype differences in DON-challenged heads involved proteins predicted to be engaged in primary metabolism. 2.3.7 Challenge-induced differential protein accumulation Differential protein accumulation was observed within each of the three wheat genotypes challenged with the various elicitors as defined in Figure 2.1. As was observed in the gene expression differences of elicited wheat heads, most of the observed treatment- induced changes in protein accumulation within a given genotype were of proteins involved in primary metabolism (Table 2.5). Again, some differences in defence-related proteins were also observed, some examples include down-regulation in the susceptible cv. ‘Superb’ of an oxidative stress-related protein, ascorbate peroxidase (spot 9A), in response to FgTri5-, and also in FgTri5+ compared with FgTri5-. Another oxidative stress-related protein, catalase (spot 167B), was up-regulated in Type I resistant DH1 in response to FgTri5- . In the Type II resistant genotype, DH2, FgTri5- treatments resulted in up- regulation of four out of five differentially accumulated protein spots, one of which was identified as a metabolism-related protein (spot 74A). In contrast, challenge with DON in 84 DH2 led to down-regulation of six out of eight protein spots, mainly of proteins involved in metabolism. 2.4 Discussion 2.4.1 Constitutive differences in gene expression Among the observed genotype-dependent differences was the constitutively higher expression of a Type I lipid transfer protein (LTP), TaLTP3 (Jang et al., 2003; Jang et al., 2005), in DH1 compared with ‘Superb’. LTPs are small extracellular plant proteins with the capacity to transfer phospholipids between membranes in vitro (Kader et al., 1984; Yeats and Rose, 2008). Despite their conserved localization at the plant cell wall and their in vitro activity, the cellular/physiological function(s) of this multigene family remain elusive. Changes in LTP expression have been observed in response to both biotic stresses, including pests and fungal pathogens of wheat (Jang et al., 2003; Lu et al., 2005), and abiotic stress, such as cold-tolerance or wound-response (Gaudet et al., 2003; Yubero- Serrano et al., 2003; Wu et al., 2004a), Some LTPs have been demonstrated to possess antimicrobial activity (Castro and Fontes, 2005; Gonorazky et al., 2005; Sun et al., 2008) or to be involved in cell signalling (Buhot et al., 2001; Blein et al., 2002). To our knowledge, TaLTP3 has not been functionally characterized, but it is possible that it could be contributing to resistance to F. graminearum penetration either via direct antifungal activity or through a role in cuticle strengthening in DH1. The higher steady-state expression TaLTP3 in DH1 was accompanied by a higher steady-state expression of an aquaporin, TaPIP1. Aquaporins are involved in transport of 85 water and uncharged solutes across the plasma membrane (Tornroth-Horsefield et al., 2006), and some LTPs have been reported to take part in cuticular wax deposition at the plant cell wall (Cameron et al., 2006; Kim et al., 2008; Samuels et al., 2008; DeBono et al., 2009; Kunst and Samuels, 2009). It is therefore intriguing that over-expression of a barley aquaporin PIP in transgenic rice was earlier reported to lead to increased cuticle thickness (Hanba et al., 2004). The cuticle layer can be an effective first line of defence against plant diseases (Martin, 1964), and the primary mode of invasion by FHB-causing species is by direct penetration of the host epidermis within the floral structure, most likely by cutinase- or lipase-mediated cuticular degradation (Kang and Buchenauer, 2000b; Voigt et al., 2005; Cuomo et al., 2007). The simultaneously elevated expression of TaLTP3 and TaPIP1 could potentially be contributed to cuticular strengthening as a defence against F. graminearum penetration in DH1. Further functional characterization of both TaLTP3 and TaPIP1 will be needed to define the nature of their relationship, if any, to Type I resistance. 2.4.2 Challenge-induced differences in gene expression and protein accumulation patterns The largest number of expression differences occurred in Type I resistant DH1, in response to all three elicitors (FgTri5+, FgTri5-, or DON). The high degree of differential gene expression observed in this genotype included numerous protein kinases (PKs), which are often involved in early signalling events in response to various stimuli. Thus, the systemic (neighbouring but uninoculated) tissues in DH1 appear to be the most strongly affected by FHB elicitors compared with the other two lines. In contrast to the impressive response observed in DH1, the Type II resistant DH2 showed fewer differences in global 86 gene expression, and by 24 hai, none of the three elicitors induced up-regulation of transcripts in this genotype. These data suggest that by 24 hai, DH2 has either already completed the changes involved in setting in motion a resistance response, or that the plant is reallocating resources for a defence response at the site of infection. Interestingly, while the DON treatment led mainly to down-regulation of transcripts in all wheat lines at the 3 and 24 hai time points, in ‘Superb’, a strong down-regulation pattern in response to DON was also observed at 8 hai. In contrast to this, a total of 122 genes were up-regulated at 8 hai in DH1 in response to DON, and none were down-regulated, while in DH2, 40 genes were up-regulated, and only 6 were down-regulated. Since several proteins accumulated to higher levels in either resistant line compared with ‘Superb’ following challenge with DON (see section 2.4.2), this result could potentially be related to the stronger pattern of DON-induced down-regulation of a gene expression in ‘Superb’. The pattern of DON-induced down-regulated molecular events suggests that trichothecenes may delay the plant’s defence response pathway in susceptible genotypes, but is less effective in doing so in resistant lines. These results demonstrate that when wheat spikelets respond to challenge with either F. graminearum or DON, the challenge is perceived in distal tissues and induces systemic changes in gene expression. The resulting gene expression patterns may be important in determining whether or not effective resistance develops in the challenged plant. Despite the 75% shared parentage between the three wheat genotypes being tested, very few of the transcriptional changes induced in systemic tissues were common among these lines, suggesting that the gene expression differences may be arising directly from the genome 87 fraction derived from the original resistant parents in the pedigrees, or from the interactions between that fraction and the shared genomic complement. 2.4.3 Deoxynivalenol-induced up-regulation of genes encoding ribosomal proteins in Fusarium Head Blight-resistant wheat The trichothecenes produced during infection by F. graminearum are known to induce ribotoxic stress and programmed cell death in eukaryotic cells (Shifrin and Anderson, 1999) as a result of their direct inhibition of ribosomal peptidyl transferase activity (McLaughlin et al., 1977). Interestingly, in the current study, direct application of DON induced up-regulation of a range of genes encoding ribosomal components in both resistant wheat lines, but not in the susceptible genotype. In both DH1 and DH2, this up-regulation was observed at 8 hai. Ribosomal genes were also up-regulated in DH1 (8 hai) in response to both FgTri5+ and FgTri5- inoculation, even though the FgTri5- fungal strain does not produce trichothecenes. Fungal challenge is expected to invoke a more complex set of responses than challenge with a defined aggressiveness factor, and thus, in DH2, FgTri5- induced up-regulation of ribosomal genes at 3 and 8 hai, but many ribosomal genes were down-regulated again by 24 hai. In the susceptible ‘Superb’ genotype, down-regulation of ribosomal genes was observed in response to both DON and FgTri5+. Thus, when challenged with either DON or DON-producing fungi, the resistant wheat genotypes displayed increased expression of genes encoding components of the ribosomal machinery, whereas expression of these genes declined in susceptible wheat. It may be relevant that both of the resistant wheat lines evaluated here were originally recovered from an in vitro selection screen using a trichothecene toxin (Eudes et al., 2008). The resistant lines have, 88 therefore, already demonstrated both resistance to FHB and tolerance for the trichothecene virulence factor, which may result from induced over-production of ribosomes or DON- sensitive ribosome components. Furthermore, the up-regulation of ribosomal component synthesis may also provide yet another explanation for the observed increase in protein spot intensity in the resistant genotypes compared with the susceptible (see section 2.4.2). Despite the fact that ribosomes are known targets of DON-toxicity, this is the first report, to my knowledge, on differential expression of ribosomal proteins in the plant response to trichothecenes or trichothecene-producing fungi. On the other hand, this is not the first report of microbial elicited changes in ribosomal gene expression (Dai et al., 2004; Gabriëls et al., 2006; Hall et al., 2007), and it has been proposed that modification of ribosomal composition can have a direct impact on regulating the translation of specific gene products (Mauro and Edelman, 2002). Thus, in addition to the potential alleviation of trichothecene-induced ribotoxicity, trichothecene-induced changes in ribosomal gene expression may represent a plausible mechanism for regulating translation of specific defence responses. 2.4.4 Early expression of pathogenesis-related proteins in resistant wheat Toxic stress in plants during pathogenic invasion arises not only from the direct impact of toxins produced by the pathogen, but also from the plant’s production of reactive oxygen species (ROS) as part of its suite of defence response. In order to protect itself against ROS-induced cellular damage, plants also produce antioxidants, including the PR proteins glutathione-S-transferase (GSTs) and peroxidases (POXs). The elicitor-induced up- regulation of PR gene expression observed in DH2 consisted mainly of antioxidant gene 89 expression responses (Table 2.4). In response to challenge with DON, antioxidant genes were up-regulated at 8 hai in Type II resistant DH2, but later in ‘Superb’ (24 hai). In response to challenge with the fungus, POX up-regulation was observed earliest in DH2 at 3 hai (FgTri5-), then later (8 hai) in DH1 (FgTri5+ and FgTri5-), and finally only by 24 hai in ‘Superb’ (FgTri5+). In the proteomics analysis, no differential accumulation of PR proteins was observed, but some additional antioxidant-related proteins were observed. For instance, accumulation of a putative catalase (spot 167B) increased in DH1 when challenged with FgTri5-, and in ‘Superb’ a reduction in accumulation of a putative ascorbate peroxidase (spot 9A) was observed in response to FgTri5- (compared to water), and in FgTri5+ vs FgTri5-. Most of the differential PR protein expression, which consisted mainly of genes expressing antifungal proteins, was observed primarily in susceptible ‘Superb’ and Type I resistant DH1, occurring earlier in the resistant genotype. At 8 hai, challenge with the fungus (either FgTri5+ or FgTri5-) induced up-regulation of several antifungal genes in DH1, while fungus-induced up-regulation of PR genes in ‘Superb’ was delayed (24 hai), and involved fewer genes. DON itself also induced up-regulation of antifungal genes, but this was observed mainly in ‘Superb’ (24 hai). Thus, up-regulation of PR genes in DH1 not only occurred earlier than in the resistant genotypes, but was more pronounced in response to the fungus than to DON. In ‘Superb’, on the other hand, up-regulation occurred later and primarily in response to DON. Since DON is presumably a single effector, whereas challenge with a live fungus brings many potential effectors into play, this response difference is not surprising. 90 Early expression of PR genes was observed in both resistant genotypes compared with ‘Superb’, consistent with the model that an earlier plant defence response will often distinguish a susceptible from a resistant response, where the earlier response is observed in the resistant varieties. This has been previously reported for FHB, where early expression of PR proteins was observed in Fusarium-infected tissues of resistant cereal crops, when compared to the susceptible lines (Geddes et al., 2008). PR gene expression in the systemic tissues of resistant and susceptible wheat has also previously been observed in response to FHB (Pritsch et al., 2001). Thus, the observations presented here corroborate previous reports that early PR protein expression may contribute to FHB resistance. Accumulation of these gene products in the systemic tissue could presumably prevent secondary infection or reduce disease spread into these tissues. 2.4.5 Up-regulation of phenylpropanoid metabolism in Type I resistant DH1 With the exception of a single gene in DH2, up-regulation of genes encoding enzymes of the phenylpropanoid pathway was observed only in DH1. Phenylpropanoid compounds are secondary metabolites with diverse roles in the plant defence response. The first step in this pathway is the conversion of phenylalanine to cinnamate via the activity of PAL (Dixon et al., 2002). In susceptible ‘Superb’, DON induced down-regulation of a PAL gene at 3 hai. Up-regulation of a different PAL transcript was observed in DH1 at 8 hai in response to all three elicitors, and an additional isoform was also up-regulated in response to DON. It is not known how the different isoforms are functionally distinct. Perhaps the expression of the second PAL observed in response to DON occurs in different tissues within the spikelet of DH1. Alternatively, the dual expression in DH1 may be indicative of 91 DON-induced up-regulation of two different pathways since the product of PAL activity, namely cinnamate, is the precursor for multiple metabolic pathways, including flavonoid and lignin biosynthesis (Dixon et al., 2002). Up-regulation of genes from both of these branches of the phenylpropanoid pathway was observed in DH1 at 8 hai. Cell wall lignification or thickening of the lemma and of the rachis node has been implicated in FHB-resistance (Kang and Buchenauer, 2000a; Jansen et al., 2005). POX activity and hydrogen peroxide metabolism are involved in the process of cell wall lignification (Schopfer, 1996). In DH1, up-regulation of POX, which was limited to the fungal treatments (FgTri5+ and FgTri5-), was detected at the same time (8 hai) as the observed up-regulation of the lignin biosynthesis pathway in this genotype. Aquaporins, in addition to their ability to transport water across cellular membranes (del Martínez-Ballesta et al., 2006), can move small molecules, including H2O2, which is the substrate for POX (Bienert et al., 2006). The higher constitutive expression of aquaporin TaPIP1 in DH1 may facilitate accumulation of H2O2 at the cell wall for lignification. If this lignification were to occur in the glumes or lemma of uninfected spikelets, prevention of Fusarium penetration through the spike surface may be observed, thus increasing Type I resistance. On the other hand, cell wall lignification at the rachis node of an already infected spikelet would improve Type II resistance. It has already been demonstrated that the rachis node is a significant barrier for disease spread in FHB, and since it is a barrier that trichothecenes play a significant role in overcoming (Kang and Buchenauer, 2000a; Jansen et al., 2005; Voigt et al., 2005; Maier et al., 2006; Ilgen et al., 2009), it is perhaps surprising that up- regulation of the phenylpropanoid pathway was so limited in DH2 tissues. On the other hand, since the analysis was performed on the systemic, uninoculated tissues, rather than 92 on the directly inoculated tissues, the up-regulation of lignin biosynthesis genes in this Type II resistant genotype may be occurring in a much more restricted location, at or near the site of infection. 2.4.6 Jasmonic acid signalling and Type II resistance No significant differential expression patterns were observed for those genes believed to be involved in SA and ET signalling, but up-regulation of JA biosynthesis and signalling genes was observed in treatment/genotype combinations where a resistance to disease spread was anticipated: i.e. in response to FgTri5+ challenge in DH2 (‘Sumai 3’-derived resistance with the 3BS QTL), and in response to FgTri5- challenge in ‘Superb’ and DH1 (where point-inoculation with the trichothecene non-producing strain phenocopies classical Type II FHB-resistance). Interestingly, treatment with pure DON, an important aggressiveness factor in FHB disease spread within the host, resulted in down-regulation of JA signalling genes in both ‘Superb’ and DH1, suggesting that the JA defense response pathway may be interdicted by DON-producing fungi in a susceptible interaction. It is well established that trichothecene production is an important factor for overcoming the host barrier at the rachis node (Jansen et al., 2005; Voigt et al., 2005; Ilgen et al., 2009), and that trichothecenes are required for disease spread (Proctor et al., 1995a; Eudes et al., 2001; Bai et al., 2002; Langevin et al., 2004; Maier et al., 2006). JA signalling can be a positive regulator of genes involved in lignin biosynthesis (Xue et al., 2008) and signalling through this pathway could potentially help modify cell wall lignification at the rachis node of an infected spikelet. 93 2.5 Conclusions Both Type I and Type II resistance to FHB are multigenic and, because of their complex nature, it has been difficult to define the genetic basis in either case. Little new information was provided from the current differential proteomics study, which probably reflects the relative lack of sensitivity of classical 2D gel approaches in differential protein accumulations studies. In order to more deeply examine the proteome for changes in less abundant protein regulators, such as transcription factors and signal transduction components, more sensitive proteomics techniques, in combination with a more fully annotated wheat proteome database, will be necessary. On the other hand, gene expression analysis was sufficiently comprehensive to provide useful insights into the different mechanisms of resistance, even with an incomplete genome database as a reference. One of the earliest points at which resistance to pathogens can occur in plant tissues is by blocking initial pathogen invasion. In the case of FHB, this is thought to be the main mechanism underlying Type I resistance. Consistent with this model of slowing or preventing initial fungal penetration, constitutive gene expression differences observed between the Type I resistant DH1 and its susceptible parent cultivar, ‘Superb’, include higher expression of several genes that may participate in producing a form of constitutive defence against invading pathogens through accumulation of antifungal proteins or providing additional physical protection against the invading fungus by enhancing cuticle thickness and/or cell wall lignification. Several induced defence responses were observed in both of the resistant genotypes that were not observed in the susceptible genotype, including a more rapid elicitor-induced regulation of gene 94 expression. Compared with ‘Superb’, up-regulation of PR genes occurred earlier in DH1 and DH2, although more prominently in the former. While DON treatments induced down- regulation of global gene expression in all three wheat genotypes, some transcript up- regulation was observed at 8 hai, a response that was delayed in ‘Superb’ relative to the resistant genotypes. A number of DON-induced up-regulated genes were observed only in the resistant genotypes, including those encoding ribosomal components, which is the main target of DON toxicity. Despite these commonalities in the regulation of gene expression between the Type I and Type II resistant lines, in many cases more transcripts were up- regulated in DH1. In fact, by 24 hai, DH2 showed no transcript up-regulation in the systemic tissues of wheat heads challenged with any of the three elicitors evaluated. Since disease containment is the mechanism of resistance to FHB spread, it would be logical for Type II resistance to be dictated primarily by host responses at the site of infection rather than a systemic response. It is proposed here that Type I resistance in DH1 involves a combination of i) structural features that slow fungal penetration and ii) activation of a systemic response in uninfected tissues adjacent to the site of infection to prevent or minimize secondary infection. Type II resistance, on the other hand is more likely a form of local resistance that may be regulated by JA signalling. 95 3. The Role of Plant Hormone Signalling in Mediating Fusarium Head Blight Resistance in Wheat 3.1 Introduction The regulation of plant disease resistance responses by hormone signalling metabolites is a common process in many pathogen-host systems (Thomma et al., 1998; Ton et al., 2002; Park et al., 2007). For example, SA-signalling is necessary for a resistance response to Pseudomonas syringae in Arabidopsis (Cameron et al., 1999). In this case, the accumulation of SA leads to the activation of a hypersensitive response in which a localized burst of programmed cell death ultimately prevents this biotrophic pathogen from feeding on living plant material (Summermatter et al., 1995). A similar response to necrotrophs would not be as effective, since host cell death would be advantageous to these pathogens. JA and JA/ET signalling pathways are more commonly observed in a resistant response to nectrophic pathogens (Glazebrook, 2005). It is not clear which signalling pathways are involved in FHB resistance in cereals, in part because the available evidence points in disparate directions, as described in Chapter 2 (Yu and Muehlbauer, 2001; Makandar et al., 2006; Zhou et al., 2006; Bernardo et al., 2007; Geddes et al., 2008; Li and Yen, 2008; Chen et al., 2009; Thatcher et al., 2009; Makandar et al., 2010). These conflicting observations may be related to the observation that F. graminearum is believed to be a hemibiotroph that requires live tissues in the early stages of infection, but subsequently switches to a necrotrophic phase. This switch to necrotrophy presumably occurs around the time that the fungus commences trichothecene production. Activation of 96 the Tri5 gene promoter in F. graminearum, which regulates expression of the first enzyme in the trichothecene biosynthesis pathway (Scheme 1-1), has been observed when the invading mycelium reaches the rachis node of wheat spikes (Ilgen et al., 2009). Furthermore, immunocytological studies have demonstrated that DON accumulates ahead of the growing hyphae during F. culmorum invasion of wheat spikes, and that this accumulation is coincident with host cell death (Kang and Buchenauer, 1999). Thus, when Fusarium species reach the barrier between the infected spikelet and the rachis; namely, the rachis node, trichothecene production commences, leading to cell death. This not only provides nutrients for the growing fungus, but facilitates invasion of the rachis and subsequent disease spread. It is thus conceivable that the specific signalling pathways that mediate an FHB-resistance response would be dependent on the timing of this expression with respect to the phase of disease progression. Several techniques are available to study the roles of the hormone signalling molecules in FHB-resistance mechanisms, including (1) analysis of the differential expression of genes associated with these pathways in response to FHB-elicitors; (2) biochemical analysis of hormone accumulation in resistant and susceptible wheat genotypes in response to FHB-elicitors; and (3) modifying the signalling pathway by genetic approaches or chemical treatment, and observing the impact on FHB disease outcomes. The first of these approaches was pursued in Chapter 2. In the second approach (biochemical analysis), plant material can be harvested following treatment with pathogen elicitors and the endogenous concentrations of the metabolites of interest can be determined. Several methods are available for the quantitation of plant hormones; the most sensitive techniques utilize HPLC- or GC-MS (Engelberth et al., 2003; Pan et al., 2010). 97 In the third approach described for studying the role of hormone signalling pathways, modification of these pathways can be conducted by ‘priming’ plants through exogenous chemical treatments aimed at affecting the pathway of interest, an approach that has been shown to activate systemic defense responses and improve resistance to various pathogens (Conrath et al., 2006; Jung et al., 2009). Common priming agents (Figure 3.1) include: benzothiadiazole (BTH), a synthetic SA analogue that has been shown to entice a similar response in plant cells as those observed for SA application (Friedrich et al., 1996); methyl jasmonate (MeJA), a JA derivative that is produced by plants as part of the JA signalling pathways (Yamane et al., 1980; Creelman and Mullet, 1997); ethephon, a chemical precursor of ethylene (Maynard and Swan, 1963); silver ions, which inhibit ET perception by competitive inhibition of the copper binding site in the ET receptors (Beyer, 1976), but have also been shown to increase auxin efflux in plant cells (Strader et al., 2009). Priming with pathogens, or with elicitors derived from them, has also been shown to improve resistance, provided that the priming response successfully activates the appropriate set of pathways for an effective defense against the pathogen in question (Conrath et al., 2006; Jung et al., 2009). Modification of hormone signalling can also be conducted by manipulation of the genes involved in the biosynthesis of these molecules or of genes encoding key proteins in their downstream response. Both of these approaches, priming or genetic manipulation, are effective in altering the plant’s phenotype and if the targeted pathways are involved in mediating a resistant or susceptible outcome to specific plant diseases, changes in disease response can be observed. In Arabidopsis, loss-of-function mutants provide a popular resource for studying the role of a given gene in different processes, such as plant defense 98 (Delaney et al., 1995; Yu et al., 1998; Greenberg et al., 2000). In wheat, however, where gene redundancy and an unsequenced genome complicate matters, an alternative approach to silencing genes is needed. Fortunately, targeted silencing of specific genes or groups of genes by RNA interference (RNAi) can be used in this species (Hannon, 2002). Over- expression of the gene of interest in wild-type plants can also provide additional insights into its biological function. However, such targeted modifications of hormone signalling pathways requires knowledge of the identity of the genes involved in their biosynthesis and downstream responses. OH OH OCl P S N O O O A. B. C. Figure 3.1 Structure of chemical priming agents. A. benzothiadiazole (BTH), B. ethephon, and C. methyl jasmonate (MeJA). 99 SA biosynthesis can occur via the phenylpropanoid or shikimic acid pathways (Figure 3.2a). In the phenylpropanoid pathway, phenylalanine is first converted to trans-cinnamic acid by phenylalanine ammonia lyase (PAL). After beta-oxidation of the cinnamic acid to shorten its side-chain the resulting benzoic acid derivative is hydroxylated to yield SA (Lee et al., 1995). SA biosynthesis can also take place via the shikimic acid pathway, where chorismate is first converted to isochorismate by isochorismate synthase (ICS), and isochorismate it then converted to salicylate. This pathway was first identified in bacteria (Marshall and Ratledge, 1971, 1972; Serino et al., 1995), but it was more recently determined that ICS is also involved in SA biosynthesis in plants (Nugroho et al., 2001; Wildermuth et al., 2001), and represents the main pathway for SA biosynthesis in some species, including Arabidopsis (Abreu and Munné-Bosch, 2009). It is not known which pathway is dominant in wheat, although a wheat EST for ICS (accession EV254155) has been identified. Enhanced Disease Susceptibility 5 (EDS5), a multidrug and toxin extrusion protein localized at the plastid membrane in Arabidopsis, is believed to be involved in SA transport into the cytosol (Ishihara et al., 2008). Studies conducted with allelic mutants of the Eds5 gene (eds5 and salicylic acid induction-deficient 1, sid1) have demonstrated that EDS5 is necessary for induction of SA-mediated defense (Nawrath and Metraux, 1999; Nawrath et al., 2002). SA accumulation in the cytosol has been proposed to lead to reduction of the intermolecular disulfide bonds that keep NPR1 in an oligomeric state, releasing NPR1 monomers that can then enter the nucleus and interact with transcription factors and up-regulate PR1 gene expression (Zhang et al., 1999; Mou et al., 2003). 100 Figure 3.2A 101 Figure 3.2B 102 Figure 3.2C 103 Figure 3.2 Phytohoromone biosynthesis and signalling pathways (pages 100-102). A. Salicylic acid (SA) biosynthesis can occur via the phenylpropanoid or shikimic acid pathway. In the former, SA biosynthesis involves phenylalanine ammonia-lyase (PAL) and benzoic acid 2-hydroxylase (BA2H) activities. The latter pathway involves isochorismate synthase (ICS) activity, and Enhanced Disease Susceptibility 5 (EDS5) is believed to mediate transport of SA from the chloroplast into the cytosol. SA accumulation in the cytosol leads to de-oligomerization of Non-expressor of Pathogenesis-Related 1 (NPR1), and monomeric NPR1 enters the nucleus and interacts with the TGA transcription factor (TF), resulting in up-regulation of Pathogenesis-Related Protein 1 (PR1). Transgenic expression of salicylate hydroxylase (NahG) can lead to the degradation of SA into catechol. B. Jasmonic acid (JA) biosynthesis is initiated in the chloroplast by activities of lipoxygenase (LOX), allene oxide synthase (AOS) and allene oxide cyclase (AOC). The 12-oxophytodienoic acid product is then reduced in peroxisomes via the activity of 12- oxophytodienoic acid reductase (OPR), followed by three β-oxidation reactions. JA accumulation results in activation on COI1, which then forms an ubiquitination complex, and targets proteosomal degradation of inhibitors of TFs involved in the up-regulation of JA-inducible proteins (JIP). C. The ethylene (ET) biosynthesis pathway involves the activities of S-adenosylmethionine (SAM) synthase, to 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS), and ACC oxidase (ACO). In the absence of ET, the ET receptors (ETRs) constitutively activates Constitutive Triple Response 1 (CTR1), which in turn constitutively inhibits Ethylene Insensitive 2 (EIN2). Once ET binds the ETR, CTR1 is deactivated. EIN2 activity is de-repressed and interacts with TFs such as Ethylene Response Factors (ERFs), leading to up-regulation of ET-inducible proteins (EIP). 104 Many of the genes involved in SA signalling have been well characterized in Arabidopsis by studying mutants compromised in their SA signalling pathways (Cao et al., 1997; Li et al., 1999; Klessig et al., 2000; Loake and Grant, 2007). Evidence from gene expression studies and priming with BTH has suggested that downstream SA signalling in wheat differs substantially from that in Arabidopsis (Huckelhoven et al., 1999; Molina et al., 1999; Yu and Muehlbauer, 2001; Lu et al., 2006), although the overall pathway remains poorly characterized in monocots. Therefore, targeting genes involved in SA signalling for silencing of this pathway would be difficult in wheat. Furthermore, the precursors to SA accumulation are also precursors for other metabolic pathways. Thus, the most effective way of attenuating the SA response would be to express salicylate hydroxylase, an enzyme which catalyzes the hydroxylation and decarboxylation of salicylic acid to produce catechol. Salicylate hydroxylase was first purified in 1962 (Katagiri et al.) and the corresponding gene was cloned 20 years later from Pseudomonas putida (Yen and Gunsalus, 1982). While salicylate hydroxylase activity has been identified in some plant species (Ellis and Towers, 1969), transgenic expression of the bacterial gene ( NAHG), in plants has been shown to result in SA degradation, and this depletion compromises SA- mediated defense responses (Gaffney et al., 1993; Vernooij et al., 1994). However, over- expression of functional NAHG in some plant species may not have an impact on steady- state SA levels within the tissues of the transgenic plants, as was demonstrated in poplar, where endogenous free SA and catechol levels were unaffected in NAHG over-expressing transgenic lines, but the levels of glucosylated SA decreased while levels of glucosylated catechol increased (Morse et al., 2007). 105 Key points from the JA signalling pathways are summarized in Figure 3.2b. In addition to defense signalling, such as the plant response to wounding (Howe, 2004) and biotic/abiotic stress responses (Creelman and Mullet, 1995), JA is involved in several physiological processes including root growth inhibition, floral development, and senescence (reviewed in Wasternack, 2007). JA biosynthesis is initiated at the plastid membrane where phospholipases release linolenic acid from membrane phospholipids (Ishiguro et al., 2001; Pohnert, 2002). Lipoxygenase (LOX) (Vick and Zimmerman, 1983; Bell et al., 1995; Feussner et al., 1995) and allene oxide synthase (AOS) (Vick and Zimmerman, 1981; Laudert and Weiler, 1998) sequentially catalyze the next two reactions to produce allene oxide, a highly unstable molecule which can spontaneously cleave into α- and γ-ketols (Hamberg, 1987; Vick and Zimmerman, 1987). Cyclization of allene oxide at C-9,13, catalyzed by allene oxide cyclase (AOC), yields 12-oxo-phytodienoic acid (Hamberg, 1988), which undergoes a reduction of the 10,11-double bond by oxo- phytodienoic acid reductase (OPR) in the peroxisome (Schaller and Weiler, 1997; Schaller et al., 2000), followed by three β-oxidation steps to produce jasmonic acid. Several enzymes have been implicated in catalyzing the β-oxidation steps, including acyl-CoA oxidase (ACX1) (Li et al., 2005; Schilmiller et al., 2007), multifunctional protein (MFP) (reviewed in Wasternack, 2007), and L-3-ketoacyl-CoA thiolase (KAT) (reviewed in Wasternack, 2007). JA-regulated gene expression in plant defense and other pathways is mediated by coronatine insensitive 1 (COI1), an F-box protein involved in targeted protein ubiquitination for proteosome-mediated degradation (Xie et al., 1998; Devoto et al., 2002). This protein degradation is believed to de-repress JA responsive genes (Beckers and Spoel, 2006). Examples of stress-induced JA-responsive genes include systemin (Li et al., 2002) 106 and the PR-protein, defensin (Manners et al., 1998). JA is also a positive regulator of genes in the lignin (Xue et al., 2008) and ET biosynthesis pathways (Saniewski and Czapski, 1985; Creelman and Mullet, 1995). The ET signalling pathway is summarized in Figure 3.2c. Methionine, the biosynthetic precursor of ET (Lieberman and Mapson, 1964), is converted to S-adenosyl methionine (SAM) via the activity of SAM synthase (Adams and Yang, 1977; Wang et al., 2002). SAM, which is also a precursor in other metabolic pathways, including polyamine biosynthesis (Lu, 2000), is converted to 1-aminocyclopropane-1-carboxylic acid (ACC) (Adams and Yang, 1979) in the first committed step of ET biosynthesis (reviewed in Wang et al., 2002). This step is catalyzed by ACC synthase (ACS) in a reaction that also yields 5’-methylthioadenosine, which can be re-converted to methionine (Adams and Yang, 1977; Yu et al., 1979; Wang et al., 1982). The final step in ethylene biosynthesis is catalyzed by ACC oxidase (ACO; formerly known as ‘ethylene-forming enzyme’) (Kuai and Dilley, 1992). ET accumulation is perceived by ET-receptors at the PM membrane. Five ET receptors have been identified in Arabidopsis from two subfamilies, namely ET receptor 1 (ETR1) and ET response sensor 1 (ERS1) from subfamily I, and ETR2, ERS2, and ET insensitive 4 (EIN4) from subfamily II (Hua et al., 1998). Similarly, five receptors have also been identified in rice: ERS1and ERS2 from subfamily I, and ETR2-like (ERL1), ETR4 and ETR5 from subfamily II (reviewed in Wuriyanghan et al., 2009). A single ET receptor has been identified to date from wheat: the ETR1 homologue W-er1 (Ma and Wang, 2003). ET receptors are negative regulators of the ET signalling pathway, and unless bound by ET and copper as a co-factor, they constitutively activate the mitogen- activated protein kinase kinase kinase, constitutive triple response 1 (CTR1), which is also 107 a negative regulator of the ET response pathway (Kieber et al., 1993; Hua and Meyerowitz, 1998; Rodriguez et al., 1999). Once CTR1 is no longer activated, EIN2, a positive regulator of the ET response pathway, is activated, leading to up-regulation of EIN3 and ET response factor (ETRF) TFs (Alonso et al., 1999; Guo and Ecker, 2003). Similarly to JA, ET signalling is involved in the regulation of both physiological processes and plant defense. ET is well known for its role in senescence and fruit ripening (Gane, 1934; Grbić and Bleecker, 1995; Klee and Clark, 2010), and in some pathogen interactions this activity can result in increased disease susceptibility (reviewed in van Loon et al., 2006). In general, the role of ET in plant defense occurs in conjunction with JA signalling. Up-regulation of certain PR proteins requires concomitant JA and ET signalling (Xu et al., 1994), although antagonistic interactions have also been observed between these two molecules (Tuominen et al., 2004). Cross-talk also occurs between JA and SA signalling; in this case, an antagonistic interaction is more commonly observed (Glazebrook, 2005; Leon-Reyes et al., 2010a). This antagonistic interaction is mediated by the cytosolic monomer of NPR1, which is believed to interfere with COI1 activity (Spoel et al., 2003; Beckers and Spoel, 2006). Interestingly, all three hormone signalling systems may interact, since ET has recently been shown to block SA-mediated suppression of JA signalling (Leon-Reyes et al., 2009; Leon-Reyes et al., 2010b). Cross-talk between these three hormone signalling molecules may, in part, explain the contrasting results described in the literature describing the mechanism of FHB resistance. Makandar et al. (2010) recently proposed that the timing of SA/JA signalling may be crucial in differentiating an FHB-resistant outcome from a susceptible one. Silencing the SA signalling pathway in Arabidopsis (sid2, npr1, wrky18 mutant lines and a NAHG 108 overexpression line) resulted in increased susceptibility to F. graminearum; whereas, JA silencing (opr3, coil and jar1 mutant lines) led to increased resistance. Interestingly, the double mutant, jar1-npr1, was more susceptible than the npr1 mutant, suggesting that JA signalling is somehow involved in FHB-resistance in Arabidopsis. Furthermore, priming of Arabidopsis host tissues by exogenous application of MeJA led to improved resistance, but only when the priming occurred 12 h after F. graminearum infection. These data suggest that late JA signalling is involved in regulating resistance outcomes in Arabidopsis and since JA signalling is associated with resistance to necrotrophic pathogens (Glazebrook, 2005), this may be coincident with a switch in the pathogen’s life cycle from biotrophy to necrotrophy. Since the switch to necrotrophy in F. graminearum likely occurs when the pathogen is beginning to spread into the rachis, it may be relevant that JA-signalling has been implicated in Type II resistance in wheat (Thesis Chapter 2; Li and Yen, 2008). Furthermore, JA signalling is a positive regulator of lignin biosynthesis (Xue et al., 2008), and cell wall lignification at the rachis node appears to be important in preventing F. graminearum spread (Kang and Buchenauer, 2000b; Jansen et al., 2005). ET signalling has been implicated in susceptibility to F. graminearum in both wheat and Arabidopsis (Chen et al., 2009). Using RNAi to target the EIN2 transcripts, Travella et al. (2006) successfully silenced the ET signalling pathway in the FHB-susceptible wheat cv. ‘Bobwhite’. These lines were subsequently evaluated for changes in their response to FHB, and a notable decrease in susceptibility was observed (Chen et al., 2009). Furthermore, exogenous applications of ethephon or silver thiosulphate demonstrated that ET signalling enhances FHB-susceptibility in wheat and barley, an outcome that may be related to ET- mediated host senescence (Chen et al., 2009). Since these studies were conducted in FHB- 109 susceptible cultivars, and since cross-talk and timing of expression may have an impact on disease outcomes, as was observed in the case of JA-signalling in Arabidopsis (Makandar et al., 2010), there is still much to be learned about the roles of these signalling molecules in mediating FHB-resistance and -susceptibility. In the differential transcriptomics study presented in Chapter 2, elicitor-induced systemic changes in wheat spikes were evaluated, where isolated components of FHB pathosystem were used to induce these changes. The effect of elicitors (FgTri5+, FgTri5-, and DON) was evaluated in three wheat genotypes (DH1, DH2, and ‘Superb’). Based on the differential gene expression pattern observed in the systemic, or uninoculated, tissue of the Type I and Type II resistant genotypes, it was proposed that Type I resistance in DH1 can be systemically induced, while Type II resistance in DH2 is a local response that cannot be systemically induced. In order to test the hypothesis presented in Chapter 2, a priming experiment was conducted on the same three wheat lines using the same three elicitors. To further explore the putative roles of hormone signalling in FHB-resistance of wheat, this study was complemented by hormone profiling of primed spikes. Additionally, the impact of suppressing specific hormone signalling pathways in wheat lines with ‘Superb’, DH1, and DH2 backgrounds was assessed in terms of the resistance/susceptibility of these lines to FHB. Silencing was performed by crossing the three experimental lines with cultivars that have been genetically compromised in SA, JA or ET signalling. 110 3.2 Materials and Methods 3.2.1 Plant material The three wheat genotypes defined in Chapter 1, namely ‘Superb’, DH1, and DH2, were used for the priming experiment in the current chapter. For the hormone silencing experiment, a total of 18 wheat crosses were used (Table 3.1), where each of three female parents (‘Superb’, DH1, and DH2) were crossed with each of six male parents (‘Bobwhite’, ‘Bobwhite’-∆EIN2, ‘Blizzard’, ‘Blizzard’-NAHG, Fielder’, and Fielder’-∆AOS). All of the non-transgenic male parents are FHB-susceptible cultivars. NAHG, AOS, and EIN2 gene sequences and construct design for transgenics are presented in Supplementary Figures 1, 2, and 3, respectively. Both ‘Bobwhite’- ∆EIN2 (Travella et al., 2006) and ‘Fielder’-∆AOS are RNAi lines (Eudes et al., unpublished), the former was kindly donated by B. Keller (Institute of Plant Biology, University of Zurich), and the latter was designed by N. Foroud and produced by Eudes et al. (unpublished) using cell penetrating peptides to introduce the silencing construct into microspore-derived embryos. The ‘Blizzard’-NAHG line was produced by biolistic transformation of the NAHG sequence from P. putida (Eudes et al., unpublished), and the transgenic lines were screened by Gaudet et al. (unpublished). Both ‘Blizzard’-NAHG and Fielder’-∆AOS lines are double haploids, the latter generated using the corn pollination technique (as described by Kisana et al., 1993), and the former produced by colchicine treatments, although often double haploidy is achieved spontaneously in microspore-derived embryogenesis. Transgenic crosses were screened for pathway silencing as described below. All plants were grown as described in Chapter 2 (section 2.2.1). 111 Table 3.1 Transgenic crosses for hormone silencing. Columns are arranged according to female parents, and rows according to male parents. Parent ‘Superb’ DH1 DH2 ‘Blizzard’ S-SA 1-SA 2-SA ‘Blizzard’-NahG S-SAi 1-SAi 2-SAi ‘Bobwhite’ S-ET 1-ET 2-ET ‘Bobwhite’-∆ein2 S-ETi 1-ETi 2-ETi ‘Fielder’ S-JA 1-JA 2-JA ‘Fielder’-∆aos S-JAi 1-JAi 2-JAi 3.2.2 Screening for hormone silencing The transgenic crosses were screened at three weeks after seeding for silencing of the EIN2 or AOS genes, or for overexpression of NAHG. Total RNA was extracted from the described tissues (Qiagen RNeasy® Plant Mini Kit with an on-column DNase digestion). cDNA synthesis and quantitative real-time PCR (qPCR) was performed as described in 2.2.5. Primer sequences are presented in Supplementary Figures 1-4. Fold-differences (FD) and standard errors were calculated from CT-values using Qiagen’s REST 2006 software package, where CT-values were normalized against the housekeeping gene, elongation factor (EF1α; Figure A4). Furthermore, crosses with ‘Blizzard’-NAHG and Fielder’-∆AOS, were screened for reduced accumulation of SA and JA, respectively, at six weeks after seeding. A total of 500 mg of leaf tissue was collected from three crosses for each of the transgenic and parent crosses, at each seeding date (thus, a total of 216 samples were collected) and hormone extraction and quantification was performed as described in section 3.2.6. 112 An additional screening was performed for the ‘Blizzard’-NAHG lines, where three week old seedlings of ‘Blizzard’ or ‘Blizzard’-NAHG were sprayed with SA as described below, and the leaves collected at 12 and 24 h after spraying for SA quantification. A total of three experimental repetitions were performed, where 60 seedlings divided into three 5” x 5” pots consisted of a single repetition. A 300 ppm SA solution was sprayed in 5 and 10 passes using a spray cabinet (Model 822-1547; Research Instrument Manufacturing Co. Ltd., Guelph, Ont.) set at 45 psi (1 psi = 6.895 kPa), 3.1 km h-1, spray width of 57 cm, and nozzle output of 370 mL min-1 set at 35 cm above the canopy. 3.2.3 Inoculations The same four inocula described in Chapter 2 (water, FgTri5+, FgTri5-, and DON) were prepared as described in section 2.2.2. For all FHB-disease experiments, three experimental repetitions were performed, where each repetition represented a different seeding date. For each repetition, a minimum of three biological replicates were used, where one plant for a given condition and plant line represents one biological replicates, and multiple spikes were treated within each biological replicate. For point inoculations (or priming), 10 µL of inoculum (water, FgTri5+, FgTri5- or DON) was injected into a single spikelet during anthesis, between 0700 and 1000 in the morning. For spray inoculations, an FgTri5+ spore suspension was sprayed in four passes with a spray cabinet as described in section 3.2.2. Spray inoculated plants were returned to the mist-irrigated greenhouse. Following point or spray inoculations, plants were incubated in a mist-irrigated greenhouse, maintaining 95% humidity at 25oC. Five days after spray 113 inoculation plants were moved to a normal humidity greenhouse at 22oC/18oC (day/night), where they were rated for disease. 3.2.4 Fusarium Head Blight disease assays Three types of experiments were performed for disease assays: (1) point-inoculation controls for the priming experiment, (2) priming experiment, and (3) hormone silencing experiments. The three wheat genotypes, ‘Superb, DH1, and DH2, were used in the first two experiments, and the crosses from Table 3.1 were used in the latter experiment. 18.104.22.168 Priming experiment Plants were primed by point inoculation as described in section 3.2.3, and then spray inoculated at 8 h after point inoculation (hai). The 8 h priming period was selected based on microarray results (Chapter 2) where a large number of changes in elicited transcript accumulation was observed at 8 hai compared with 3 and 24 hai, particularly in DH1. Plants were rated for FHB severity (percent of diseased spikelets per spike) at 7, 9, 12, and 18 days after inoculation (dai). Statistical analysis was performed as described in section 22.214.171.124. Plants were allowed to mature, and the grain harvested for DON quantification. 126.96.36.199 Point-inoculation controls for priming experiment Wheat spikes were point inoculated as described in section 3.2.3. Since point inoculation was not followed spray inoculation in this experiment, incubation in the mist-irrigated greenhouse lasted for 5 days and 8 h after point inoculation. Disease 114 rating was performed as described in section 188.8.131.52. Wilting was not evaluated, instead the total number of discolour spikelets were considered to be infected. Statistical analysis was performed on the combined data generated from the priming experiment (sections 184.108.40.206) and its point-inoculation controls (this section). Statistical analysis for disease rating was performed with SAS least square mean (LSMean) routine and grouped by dai. Data were considered statistically different when p < 0.05. 220.127.116.11 Hormone silencing experiment Crosses were spray inoculated in the evenings between 1700 and 1900 as described in section 3.2.2; no point inoculations were performed on these lines. Plants were rated for severity (percent of diseased spikelets per spike) and for FHB-index (average of severity and incidence (percent of diseased spike per plant)) at 6, 9, 12, 15 and 18 dai. Statistical analysis for disease rating was performed with SAS LSMean and grouped by dai or by cross. Data were considered statistically different when p < 0.05. 3.2.5 Deoxynivalenol quantification from priming experiment Kernels were harvested from matured spikes following the priming experiment, finely ground, and trichothecenes extracted from 1 g of material. Biological replicates within each experimental repetition were pooled for trichothecene extractions. Thus for the three wheat lines, four treatments, three harvest times, and three experimental repetitions, a total of 108 extractions were performed. The amount of DON per gram of kernel was determined by ELISA at the Eastern Cereal and Oilseed Research Centre, Agriculture and Agri-Food 115 Canada, as previously described (Savard et al, 2000). Statistical analysis was performed with SAS LSMean routine; data were considered statistically different when p < 0.05. 3.2.6 Hormone profiling of primed wheat spikes For hormone profiling, plants were point inoculated, or ‘primed’, as described in section 3.2.3. Inoculation was not followed by spray inoculation in this case, but the entire spike of primed spikes were collected at 3, 8 and 24 hai, frozen in liquid nitrogen and stored at - 80oC for hormone extractions. Biological replicates within each experimental repetition were pooled for hormone extractions, where a total of 108 extractions were performed. Hormone extraction and quantification was performed at the National Research Council, Plant Biotechnology Institute of the National Research Council, Saskatoon, Canada (http://www.nrc-cnrc.gc.ca/eng/facilities/pbi/plant-hormone.html). The pooled spikes were ground to a fine powder in liquid nitrogen and 500 mg of the frozen powder was extracted in 3 mL of extraction solvent (methanol:water:glacial acetic acid (90:9:1, v/v/v)) to which 100 µL solution containing internal standards were added (acetonitrile:water, 50:50 v/v with 0.1% formic acid, containing 1 ng/µL of 3,4,5,6-d4-2-hydroxybenzoic acid and 0.5 ng/µL of 2,2-d2-jasmonic acid). The mixture was sonicated (5 min), incubated on an orbital shaker (4oC, 5 min), and then spun down (4.4 krpm, 10 min). The supernatant was collected, and the extraction procedure was repeated twice on the pellet first with 2 mL of extraction solvent, followed by 1 mL of methanol. The supernatants from all three extractions were pooled and evaporated under reduced pressure. To each dried sample kept on ice, 1 mL of aqueous 0.3 N NaOH was added, followed by extraction with 3 mL dichloromethane. The aqueous layer was collected, and the organic layer re-extracted with 116 2 mL aqueous 0.3 N NaOH. On ice, the combined aqueous layers were acidified with 1 mL 5% aqueous HCl, and then extracted with a 1 mL mixture of ethylacetate:cyclohexane (1:1, v/v). The organic phase was collected and the aqueous phase re-extracted with 0.5 mL ethylacetate:cyclohexane (1:1, v/v). The solvent was evaporated from combined organic fractions under nitrogen gas, and the samples reconstituted in 200 µL methanol:water (30:70 v/v) with 0.1 % formic acid and containing external standards (100 ng of 1,2,3,4,5,6-13C6-2-hydroxybenzoic acid and 50 ng of 12,12,12-d3-jasmonic acid). Extracted salicylic acid (SA) and jasmonic acid (JA), both free non-conjugated acids, were analyzed by UPLC/ESI-MS/MS, utilizing a Waters ACQUITY UPLC system equipped with a binary solvent delivery manager and sample manager coupled to a Waters Micromass Quattro Premier XE quadrupole tandem mass spectrometer via a Z-spray interface. The UPLC used an HSS C18 column (2.1 x 100 mm, 1.8 µm) and a gradient elution of mobile phase A (1% formic acid in HPLC-grade water) and mobile phase B (1% formic acid in HPLC-grade methanol). The analysis utilized the Multiple Reaction Monitoring (MRM) function of the MassLynx v4.1 (Waters Inc.) control software and Quanynx v4.1 software was used for SA and JA quantifications employing similarly to analytical procedures described by Ross et al. (2004). For treatment comparisons, the values for the water control were subtracted from the treatment values. All statistical analysis was performed with SAS LSMean routine; data were considered statistically different when p < 0.05. 117 3.3 Results 3.3.1 The effect of point-inoculation on disease outcomes Since the priming experiments combined point-inoculation with a subsequent spray- inoculation, an initial evaluation of the disease outcomes associated with point-inoculation by itself was performed as a control. These controls enabled us to determine the extent to which any symptoms directly induced by the initial point inoculation would impact the overall disease outcomes in the priming experiment. No disease symptoms were observed for the point-inoculation controls using either water or DON in ‘Superb’, DH1 or DH2 (data not shown). Disease symptoms were observed in inoculated spikelets of FgTri5- point-inoculation controls, at 7 dai, but little to no disease spread was observed in any of the three wheat lines evaluated (Figure 3.3a). The method of disease evaluation, a measurement of severity (percent infected spikelet per spike) at multiple dai, was selected since an evaluation of Type I and II resistance was of interest; however, since the average number of total spikelets per spike differed between plant lines (data not shown), the infection of a single spikelet resulted in a different percentage of diseased spikelets for each plant line. For ‘Superb’, this ranged from 7-10 % across the four rating dates; 2-5 % for DH1; and 6-9 % in DH2 (Figure 3.3a). These results differed (p < 0.05) from priming with FgTri5- followed by spray inoculation with FgTri5+ in ‘Superb’, where 20 % of the spike was diseased at 7 dai, and 66 % by 18 dai. In both resistant genotypes, disease was higher in the priming experiment compared with the FgTri5- point-inoculation controls, but there was only one case where this was significant: DH1 at 18 dai, where 5% (18 dai) and 30 % (18 dai) of the spike was diseased. For FgTri5+ point-inoculation controls, little to no 118 disease spread was observed in DH2 which has good Type II resistance. There was also no difference observed in this genotype with either the FgTri5+ priming experiment, or the FgTri5- point-inoculation controls. In the Type I resistant genotype, DH1, disease spread was observed from 7 to 18 dai (FgTri5+), but no differences were observed between the point-inoculation controls and priming experiment (p > 0.05). Disease spread was also observed in FgTri5+ point-inoculation controls in susceptible ‘Superb’, and differences were observed between the point inoculation control and the priming experiment at 7 dai. 3.3.2 Priming experiment disease evaluation Wheat spikes were primed by point inoculation with water, FgTri5-, or DON, and then spray inoculated 8 hours later with FgTri5+. The water control for the priming experiment resulted in 7 % disease in the ‘Superb’ wheat spike at 7 dai, which increased over the remaining evaluation days (Figure 3.3b). Priming with FgTri5- led to an increase in disease severity (p < 0.05) at 7 dai compared with the water primed spikes, but these two treatments did not differ (p > 0.05) by 9 and up to 18 dai. In contrast, priming with FgTri5- in DH1 led to a reduction in disease (p < 0.05) by 18 dai compared with water priming. Thus, FgTri5- priming increased susceptibility to early (or initial) infection in the susceptible cv. ‘Superb’, but increased resistance to disease in the later stages in Type I resistant DH1. In Type II resistant DH2, no significant differences were observed between the water-primed spikes and in the spikes primed with either FgTri5-. Furthermore, priming with DON did not have an impact on disease outcomes in any of the three genotypes when compared with water priming (p > 0.05) 119 A. B. Figure 3.3 Disease progression in priming experiment a ab b a a a a a a a a a a ab b a a b a a a a a a a a a a a a a ab b a ab b s e v e ri ty ‘Superb’ DH1 DH2 dai dai dai a a a a a a a a a a a a a a a a a ab bc c a ab c d a b bc d a a b c a a b c a ab bc d a a cd d a a cd d s e v e ri ty Disease progression in point inoculation controls vs priming experiment ‘Superb’ DH1 DH2 dai dai dai 120 Figure 3.3 Disease progression in three wheat genotypes: ‘Superb’, DH1, and DH2 (page 119). A. FgTri5+ (○) and FgTri5- (∆) point inoculation controls were compared with priming experiment (FgTri5+ priming (●) and FgTri5- priming (▲), followed by spray inoculation with FgTri5+ at 8 hai). B. Disease progression was also compared between wheat heads priming with water (□), FgTri5- (▲), or DON (♦), followed by spray inoculation with FgTri5+ at 8 hai. Significant differences (p < 0.05) are represented for each plant line at a given dai. 3.3.3 Priming has no effect on SA or JA accumulation within point-inoculated wheat spikes Constitutive levels of free SA and JA were quantified in uninoculated wheat spikes at anthesis, and results showed that the steady-state levels of JA in DH2 were roughly twice as high as those observed in the other two wheat lines (p < 0.05) (Table 3.2). The level of SA detected in uninoculated wheat spikes was 240 parts per billion (ppb) (data not shown), which is quite low compared with what has been observed in other species. Free SA accumulation has been reported in the range of 2000 to 9000 ppb from mustard and rice seedlings, respectively (Chen et al., 1997; Dat et al., 1998). On the other hand the low levels of JA accumulation, 45 ppb from uninoculated wheat spikes (data not shown), seems to be consistent with what has been observed in other species. Free JA accumulation has been reported to range from 5 to 20 ppb in tomato and rice (Schweizer et al., 1997; Thoma et al., 2003). 121 Free SA and JA were also measured in wheat spikes primed by point-inoculation with FgTri5+, FgTri5-, or DON, at 3, 8 and 24 hai. Spikes primed with water served as controls. Differences in SA accumulation, compared with the water-primed spikes, ranged between - 52 and +84 ppb, and no significant differences (p > 0.05) were observed over time or between treatments (Table 3.3). Likewise, JA accumulation in point-inoculated spikes did not differ significantly among treatments or over time, when compared with the water- primed spikes. 3.3.4 DON accumulation in mature kernels from primed and spray-inoculated wheat spikes is primarily correlated with disease severity DON accumulation was quantified from kernels of mature spikes that were primed with water, FgTri5+, FgTri5-, or DON at anthesis and subsequently spray inoculated with FgTri5+. DON accumulation ranged from 30 to 215 ppm in ‘Superb’, 12 to 31 ppm in DH1, and 6 to 20 ppm in DH2 (Table 3.4). Thus, the highest DON accumulation was observed in genotypes where disease was highest. In all cases, priming with FgTri5+ resulted in the highest accumulation. Despite some of the large differences observed between treatments or genotypes, no significant differences were observed among any of these comparisons. This lack of significance may be attributed to the variation introduced by the small sample size within experimental replicates. The total mass of kernels harvested per experimental replicate did not exceed 2-3 g, and while trichothecene extractions were conducted on only 1 g of ground kernel, a larger sample size from which to sample 1 g of material would have reduced the variation observed between experimental replicates. 122 Table 3.2 Constitutive levels of SA and JA in untreated wheat heads. Letter grouping indicates differences (p < 0.05) for LSMean of SA or JA accumulation in leaf tissues among the three wheat genotypes. Plant Line SA (ppb) JA (ppb) ‘Superb’ 129 a 25 a DH1 122 a 34 a DH2 154 a 61 b Table 3.3 Time-course analysis of SA and JA accumulation in wheat spikes primed with FHB elicitors. LSMean for SA and JA accumulation in wheat spikes primed by point inoculation with FgTri5+, FgTri5-, or DON, are presented, with values from water priming subtracted. Letter grouping indicates that no significant differences (p < 0.05) in SA or JA accumulation, where levels were compared across all conditions and wheat lines evaluated. Condition SA (ppb) JA (ppb) 3 hai 8 hai 24 hai 3 hai 8 hai 24 hai ‘Superb’ FgTri5+ vs water +20 a +4 a -13 a -6 a +12 a -8 a FgTri5- vs water -12 a +51 a -4 a -18 a +7 a -6 a DON vs water +56 a +26 a +31 a -6 a +7 a -5 a DH1 FgTri5+ vs water +33 a -11 a +69 a +8 a +7 a +12 a FgTri5- vs water +30 a -5 a +33 a +9 a +3 a +2 a DON vs water 0 a +54 a +84 a +1 a -2 a +3 a DH2 FgTri5+ vs water +39 a -26 a -37 a +13 a -11 a -7 a FgTri5- vs water -52 a -4 a -27 a +3 a -12 a -3 a DON vs water +10 a +15 a -26 a +6 a -12 a -1 a 123 Table 3.4 Effect of priming in DON accumulation in wheat kernels. DON accumulation (ppm) in mature kernels of three lines (rows) primed by point-inoculation with one of four inocula (columns) 8 h prior to spray inoculation with FgTri5+. Letter grouping (superscript) indicates that no significant differences (p < 0.05) in DON accumulation were observed among the conditions evaluated. Plant line/Condition water FgTri5+ FgTri5- DON ‘Superb’ 29.6 a 214.6 a 64.1 a 45.2 a DH1 11.5 a 30.8 a 12.4 a 23.9 a DH2 8.1 a 19.7 a 7.9 a 5.9 a 3.3.5 Screening for hormone silencing 18.104.22.168 Screening for NAHG over-expression and SA degradation The Blizzard-NAHG parent was screened for the effects of SA silencing by Gaudet et al. (unpublished): (1) F1 seeds were germinated on mannitol as a screen for SA- deficiency, since it has previously been demonstrated that osmotic stress using mannitol causes necrosis wild-type Arabidopsis seedlings, but yields healthy green tissue in NAHG over- expression lines (Borsani et al., 2001); (2) the presence of the gene was confirmed by PCR, although transcript accumulation was not observed by qPCR using the NahG1 primers (Figure A1); (3) disease assays showed that the transgenic lines were more susceptible to wheat leaf and stripe rusts and to snow mold, but had no effect on bunt resistance. Since ‘Blizzard’-NAHG was produced by biolistic transformation and the resulting transgenic lines were made into double haploids after transformation, all F1 124 crosses with this line should be positive for the NAHG gene. All crosses with ‘Blizzard’-NAHG were intended to be screened for NAHG transcript accumulation by qPCR, but no transcript accumulation was detected by qPCR using any of the four primer sets presented in Figure A1. A sample set of the crosses was randomly selected for SA quantification, but SA accumulation was highly variable and it was not possible to observe any differences in accumulation among the crosses with ‘Blizzard’ compared with ‘Blizzard’-NAHG (Table 3.5). Due to the inconclusive results from these screens, an additional test was conducted: both ‘Blizzard’ and ‘Blizzard’-NAHG were treated with SA, followed by SA quantification 12 and 24 h after treatment to determine whether SA degradation could be observed in the transgenic line. Final data are not yet available from this experiment. 22.214.171.124 Screening for silencing of JA biosynthesis Although the ‘Fielder’-∆AOS parent is a double haploid line, experimental evidence suggests that transgene incorporation into the microspore genome when using cell penetrating peptides may occur after the stage of chromosome doubling (Eudes et al.unpublished data). Thus, crosses of other wheat genotypes with ‘Fielder’-∆AOS may result in segregation of the transgene, and PCR-based genotyping of the progeny of the transgenic crosses was necessary prior to disease evaluation. Some variability in AOS transcript accumulation among wild-type crosses made it difficult to set a clear FD cut- off value when selecting for JA-silenced crosses (Table 3.5). JA quantification generally supported a CT-value cut-off at a difference of > 8.0 between the housekeeping gene and AOS for JA silencing. However, a large degree of variability in the JA accumulation 125 was also observed, and low levels of JA accumulation were observed in some of the wild-type crosses, especially in the second experimental repetition for 2-JA. Due to the low number of JA-silenced crosses available, the selected transgenic plants were ‘cloned’ at 4-5 weeks by dividing them into three or four separate plants, in an effort to effectively increase the biological replicates. 126.96.36.199 Screening for silencing of ET signalling ‘Bobwhite’-∆EIN2 lines are not double haploids, and since segregation of the transgene was expected, all of the ‘Bobwhite’-∆EIN2 crosses were genotyped by PCR for reduced expression of the EIN2 transcript. qPCR screens of crosses with ‘Bobwhite’ or ‘Bobwhite’-∆EIN2 was initially performed with the EIN2-RT primer set that was used in the screening when these transgenic lines were originally produced (Travella et al., 2006). Unexpectedly high CT-values (>30) were typically observed with the crosses, resulting in highly variable data (data not shown). When new primers sets were designed, both Ein2-1 and Ein2-2 had CT-values of approximately 24, and yielded reproducible results. Thus screening for EIN2 silencing in the current study was conducted with primer set Ein2-1. Unfortunately, only a few transgenic crosses were found to display reduced expression of EIN2, and this was using a low FD cut-off value (1.5 FD) in comparisons with the wild-type crosses (Table 3.5). 126 Table 3.5 Screen of transgenic crosses by hormone quantification and qPCR. Three biological replicates (BR) from each of the three experimental repetitions (ER) were randomly selected from each of the crosses with the wild-type male parents for controls in hormone quantification and/or qPCR analysis. All crosses with the transgenic parents were screened by qPCR. The FD was calculated between a given transgenic cross, CT- differences (CT-diff; CT-value of gene of interest with CT-value of the housekeeping gene) of a single BR from the transgenic cross was compared with the average CT-diff of BR within the same ER of the wild-type parent. qPCR with primer sets AOS1 and EIN1 for were used for JA/JAi and ET/ETi crosses, respectively, both yielded CT-values higher than the housekeeping gene (EF1α); thus, higher CT-diff indicates reduction in transcript accumulation of the gene of interest. qPCR data for transgenic crosses are presented only for the samples which were selected for FHB disease assays, with the exception of two replicates (red font, marked with an asterisk), which are presented here since SA and JA data were collected for these samples and provide some support for the selected CT-diff. ppb qPCR ER BR SA JA CT-diff FD stdev S-SA 1 1 50.6 16.8 NA NA NA 1 2 64.0 17.1 NA NA NA 1 3 69.2 9.3 NA NA NA 2 1 48.7 3.0 NA NA NA 2 2 40.6 2.5 NA NA NA 2 3 265.5 10.5 NA NA NA 3 1 157.0 3.6 NA NA NA 3 2 156.7 4.6 NA NA NA 3 3 186.1 4.1 NA NA NA S-SAi 1 1 49.9 4.7 NA NA NA 1 2 105.2 31.8 NA NA NA 127 ppb qPCR ER BR SA JA CT-diff FD stdev 1 3 108.2 24.6 NA NA NA 2 1 35.1 4.1 NA NA NA 2 2 127.8 4.0 NA NA NA 2 3 295.7 3.8 NA NA NA 3 1 40.6 4.3 NA NA NA 3 2 56.4 4.4 NA NA NA 3 3 120.6 3.2 NA NA NA 3 4 148.2 4.6 NA NA NA 1-SA 1 1 12.0 4.9 NA NA NA 1 2 19.0 12.5 NA NA NA 1 3 50.1 6.9 NA NA NA 2 1 26.2 4.7 NA NA NA 2 2 30.7 5.5 NA NA NA 2 3 34.6 4.5 NA NA NA 3 1 119.4 9.3 NA NA NA 3 2 141.8 3.1 NA NA NA 3 3 216.8 5.7 NA NA NA 1-SAi 1 1 12.5 8.9 NA NA NA 1 2 63.5 12.7 NA NA NA 1 3 203.0 13.7 NA NA NA 2 1 17.4 5.6 NA NA NA 2 2 55.5 7.6 NA NA NA 2 3 49.9 3.7 NA NA NA 3 1 147.8 5.6 NA NA NA 3 2 124.5 5.4 NA NA NA 3 3 270.1 9.1 NA NA NA 3 4 27.8 5.5 NA NA NA 2-SA 1 1 22.5 9.9 NA NA NA 1 2 34.1 12.3 NA NA NA 1 3 19.5 7.0 NA NA NA 2 1 73.3 4.7 NA NA NA 2 2 22.0 3.3 NA NA NA 2 3 37.4 3.3 NA NA NA 3 1 47.5 3.5 NA NA NA 128 ppb qPCR ER BR SA JA CT-diff FD stdev 3 2 87.5 21.5 NA NA NA 3 3 59.6 3.3 NA NA NA 2-SAi 1 1 73.2 6.9 NA NA NA 1 2 24.9 4.7 NA NA NA 2 1 15.3 3.6 NA NA NA 2 2 47.1 5.9 NA NA NA 2 3 61.3 4.8 NA NA NA 3 1 175.3 10.1 NA NA NA 3 2 62.9 4.1 NA NA NA 3 3 165.8 7.0 NA NA NA 'Blizzard' NA NA 26.8 7.3 NA NA NA NA NA 36.7 7.0 NA NA NA 'Blizzard'-NahG NA NA 17.0 7.7 NA NA NA NA NA 17.1 8.0 NA NA NA NA NA 42.8 7.0 NA NA NA S-Field 1 1 36.4 7.4 8.2 NA NA 1 2 45.9 13.1 3.4 NA NA 1 3 54.1 14.0 6.6 NA NA 2 1 130.3 6.8 5.1 NA NA 2 2 194.3 7.5 5.0 NA NA 2 3 820.7 10.1 5.3 NA NA 3 1 67.4 4.2 2.5 NA NA 3 2 176.3 6.5 4.7 NA NA 3 3 181.3 10.1 2.5 NA NA S-JAi 1 1 15.1 1.7 8.6 -14.9 ±0.08 1 2 30.3 1.5 8.3 -13.1 ±0.04 2 1 217.8 5.3 8.4 -12.5 ±0.02 2 2 96.3 3.4 9.0 -8.8 ±0.03 3 1 96.3 2.5 9.5 -72.9 ±0.00 3 2 54.3 3.3 9.3 -63.3 ±0.01 3 3* 138.4 37.0 2.2 +2.2 ±0.60 129 ppb qPCR ER BR SA JA CT-diff FD stdev 1-Field 1 1 19.0 2.8 6.6 NA NA 1 2 27.3 7.9 7.1 NA NA 1 3 50.2 8.0 5.4 NA NA 2 1 84.2 2.4 5.8 NA NA 2 2 179.2 2.4 3.6 NA NA 2 3 171.6 2.6 5.7 NA NA 2 4 233.0 2.7 3.6 NA NA 3 1 124.4 3.6 NA NA NA 3 2 242.2 7.1 2.9 NA NA 3 3 476.5 14.5 3.7 NA NA 1-JAi 1 1 58.6 2.1 8.7 -5.2 ±0.09 1 2 58.0 2.5 8.6 -4.9 ±0.09 1 3 22.1 2.0 8.2 -3.5 ±0.14 2 1 246.6 4.9 10.7 -43.9 ±0.01 2 2 NA NA 9.1 -19.3 ±0.05 3 1 63.3 2.3 8.1 -15.1 ±0.03 3 2 129.7 5.5 NA NA NA 3 3 38.9 2.6 NA NA NA 2-Field 1 1 13.0 6.6 6.1 NA NA 1 2 21.6 8.0 NA NA NA 1 3 16.0 9.9 4.4 NA NA 2 1 78.4 5.1 6.5 NA NA 2 2 221.6 9.9 5.6 NA NA 2 3 157.8 11.1 5.4 NA NA 3 1 159.6 5.0 NA NA NA 3 2 165.1 5.0 NA NA NA 3 3 159.8 6.7 NA NA NA 2-JAi 1 1* 162.1 27.7 7.3 -4.0 ±0.10 2 1 64.5 3.1 9.0 -9.3 ±0.04 3 1 70.5 2.2 9.4 -12.7 ±0.03 3 2 22.0 3.9 8.8 -6.8 ±0.04 3 3 113.6 2.9 NA NA NA 3 4 226.6 4.3 NA NA NA 130 ppb qPCR ER BR SA JA CT-diff FD stdev S-ET 1 1 NA NA 4.9 NA NA 1 2 NA NA 4.3 NA NA 1 3 NA NA 4.3 NA NA 2 1 NA NA 6.1 NA NA 2 2 NA NA 5.8 NA NA 2 3 NA NA 5.7 NA NA 3 1 NA NA 6.8 NA NA 3 2 NA NA 5.6 NA NA S-ETi 1 1 NA NA 5.8 -2.4 ±0.08 1 2 NA NA 5.2 -1.7 ±0.14 1 3 NA NA 5.2 -1.6 ±0.16 3 1 NA NA 6.8 -1.5 ±0.57 1-ET 1 1 NA NA 5.4 NA NA 1 2 NA NA 4.8 NA NA 1 3 NA NA 4.3 NA NA 2 1 NA NA 5.6 NA NA 2 2 NA NA 6.3 NA NA 3 1 NA NA 5.4 NA NA 3 2 NA NA 4.3 NA NA 1-ETi 1 1 NA NA 6.3 -2.6 ±0.10 1 2 NA NA 5.8 -2.0 ±0.13 1 3 NA NA 5.7 -1.7 ±0.11 1 4 NA NA 5.6 -1.7 ±0.16 1 5 NA NA 5.6 -1.7 ±0.12 1 6 NA NA 5.5 -1.5 ±0.14 2 1 NA NA 6.6 -1.5 ±0.16 3 1 NA NA 5.5 -1.6 ±0.23 3 2 NA NA 5.9 -2.1 ±0.15 3 3 NA NA 6.7 -3.8 ±0.08 3 4 NA NA 6.1 -2.5 ±0.11 2-ET 1 1 NA NA 5.3 NA NA 1 2 NA NA 4.8 NA NA 131 ppb qPCR ER BR SA JA CT-diff FD stdev 1 3 NA NA 4.6 NA NA 2 1 NA NA 6.3 NA NA 2 2 NA NA 5.5 NA NA 2 3 NA NA 5.4 NA NA 3 1 NA NA 6.9 NA NA 3 2 NA NA 6.7 NA NA 2-ETi 2 1 NA NA 6.5 -1.7 ±0.15 2 2 NA NA 6.4 -1.6 ±0.15 3 1 NA NA 6.7 -2.0 ±0.17 3 2 NA NA 6.7 -1.9 ±0.14 3 3 NA NA 6.5 -1.7 ±0.22 3 4 NA NA 6.5 -1.6 ±0.13 NA = not available 132 3.3.6 Hormone silencing disease evaluation Comparisons were made in disease severity and FHB-index among the different crosses at selected times post-challenge. The observed differences (p < 0.05) in disease severity were identical to the observed differences in FHB-index (data not shown) and, thus, only severity is considered in the current analysis. Significant differences in disease severity (percent of infected spikelets per spike) were observed between crosses at specific time points, and differences in disease progression (the change in disease severity over time) were also observed over time within a given cross. SA silencing in the DH2 background resulted in increased FHB susceptibility: 2-SA and 2-SAi showed an average difference of 17% in disease severity at a given dai, where disease was higher in the 2-SAi line (Figure 3.4). On the other hand, SA silencing had little or no impact on disease outcomes in the other two genetic backgrounds. JA silencing in both the DH1 and DH2 backgrounds resulted in an increase in disease severity, which was significant as early as 6 dai. Thus, resistance to initial infection increased when JA signalling had been compromised in the resistant backgrounds. On the other hand, disease progression was relatively consistent in all of the JAi crosses compared with the respective wild-type crosses. In other words, the change in disease severity over time occurred at similar rates between the wild-type and JA-silenced crops, suggesting that, in contrast to initial infection, disease spread was unaffected by JA silencing. 133 Figure 3.4 134 Figure 3.4 Effect of hormone silencing on FHB-disease progression in wheat (page 133). Multivariate analysis was used to determine whether there were differences in disease severity and in disease progression among crosses (see Table A2 for the complete analysis), and is presented above in groups, where the ‘Superb’ (S), DH1 (1), or DH2 (2) crossed with the ‘Blizzard’ (SA), ‘Fielder’ (JA), or ‘Bobwhite’ (ET), are compared directly with the respective hormone silenced cross (SAi, JAi or ETi). Differences marked with an asterisk indicate differences between the crosses at a given dai (p < 0.05). Grouping by letters indicate disease progression, or differences over time, within a given cross (p < 0.05), upper and lowercase letters are used to demonstrate disease progression within the wild-type and silenced crosses, respectively. ET silencing resulted in significant changes in resistance to initial infection (disease severity observed at 6 dai) in both the susceptible and DH2 backgrounds. However, due to the low number of replicates available in the ETi crosses the reproducibility of this data has not been tested. In the susceptible background, ET silencing (S-ETi) increased host susceptibility, but in the Type II resistant background, ET silencing (2-ETi) resulted in an increase in resistance compared with the respective wild-type crosses (S-ET and 2-ET). In both cases differences were observed in initial infection (observed at 6 dai). Some minor but statistically significant differences (p < 0.05) were observed among the three genetic backgrounds (‘Superb’, DH1, and DH2) crossed with the wild-type (non- transgenic) lines (Table A2). The most notable differences were observed in DH2, where 135 disease severity in 2-ET was generally higher than the other wild-type crosses in this genetic background (namely, 2-SA and 2-JA). 3.4 Discussion 3.4.1 Priming induces changes in FHB-resistance and -susceptibility of wheat Point-inoculation controls were used to determine whether the disease symptoms which develop directly from the point of inoculation would have an impact on disease progression following the subsequent spray inoculation for the priming experiment. Point inoculations with DON or FgTri5- did not adversely affect the interpretation of results in the priming experiment, since disease symptoms (if any) were confined to the inoculated floret. Priming with DON had no impact on subsequent infection by FgTri5+ in any of the three wheat lines evaluated. Priming with FgTri5-, on the other hand, resulted in increased susceptibility to initial infection by FgTri5+ in ‘Superb’, observed at 7 dai. In contrast, improved resistance to disease spread in DH1was observed when FgTri5- priming was used. A trend toward improved resistance was observed as early as 7 dai in this genotype, but the difference became significant by 18 dai. Thus, priming with FgTri5- increased resistance to subsequent infection by wild-type F. graminearum in the Type I resistant genotype. In contrast with the susceptible and Type I resistant genotypes, resistance in DH2 was unaffected by priming with FgTri5- inoculation. DH2, which has good Type II resistance, allowed little or no disease spread in any of the treatments. It has already been demonstrated that resistance to disease spread occurs at the rachis node of the infected 136 spikelet (Kang and Buchenauer, 2000a). Our results suggest that the strong local resistance mechanism exhibited in the ‘Sumai 3’-derived genotype precludes the induction of a systemic defense response in spikelets distal to the point of infection. It is interesting that initial infection (assessed at 7 dai) and the final disease outcome (assessed at 18 dai) did not appear to differ between water-primed spikes of DH1 and ‘Superb’. This was unexpected since DH1 was previously evaluated to have good Type I resistance, and Superb is a susceptible cultivar. If one considers the methodology used to define their resistance/susceptibility, in combination with the observations made in the current report, this discrepancy can be explained. Type I FHB resistance for both DH1 and ‘Superb’ were previously evaluated under field conditions (unpublished data), where the plants were sprayed with FgTri5+ macroconidia on three successive occasions (as described by Eudes et al. 2008). Together, these data imply that the first spray inoculation date during the evaluation of DH1 Type I resistance, which occurred when 50 % of the spikes had emerged from the boot, could have effectively primed the plants by inducing some level of systemic resistance to the subsequent inoculations, which occurred two and four days later. When DON profiling was performed on kernels from mature spikes at the end of the priming experiment, no significant differences in DON levels could be correlated with the different priming treatments, but much lower DON accumulation was detected in both the resistant lines, as might be predicted, based on their reduced levels of fungal colonization. 137 3.4.2 The role of hormone signalling in FHB-resistance In order to determine which signalling pathways might be induced in the non-inoculated tissues of primed wheat spikes, SA and JA accumulation patterns were examined in primed wheat spikes at 3, 8 and 24 hai, and tested for correlations with the observed disease resistance and susceptibility outcomes. Unfortunately, the low endogenous levels of SA and JA in this material, possibly combined with minor variations in developmental staging for the point-inoculated spikelets, made it impossible to obtain replicable differences in analyte data from the available tissue. Interestingly, while no major challenge-induced differences were observed in SA or JA accumulation patterns, the phytohormone analyses did establish that spikes from Type II resistant DH2 constitutively accumulate almost twice as much JA as do either ‘Superb’ or DH1. Higher steady-state JA levels may play a role in prompt activation of a local resistance mechanism in this genotype. As previously reported, priming with MeJA increased susceptibility to F. graminearum infection in Arabidopsis during the early stages of infection, but improved resistance could be observed if priming was performed at 12 hai (Makandar et al., 2010). Furthermore, (FgTri5-)-priming induced up-regulation of JA signalling-related transcripts at 3 hai in ‘Superb’ (Chapter 2) and also increased susceptibility to initial infection by FgTri5+ in the current study. In DH1, (FgTri5-)- priming induced a more delayed (24 hai) up-regulation of JA signalling transcripts (Chapter 2) and also led to increased resistance to FgTri5+ infection (observed by 18 dai in the current study), suggesting that priming had improved resistance to disease spread in this genotype by triggering JA-mediated responses at an effective time-point (24 hai). It is interesting that JA signalling is associated with resistance to necrotrophs (Glazebrook, 138 2005) and that late JA signalling is involved in resistance to F. graminearum in Arabidopsis, presumably when the fungus has entered a necrotrophic phase (Makandar et al., 2010). These observations, taken in combination with the results from differential transcriptomics studies presented in Chapter 2, suggest that appropriate timing of JA signalling may be an important feature of the mediation of Type II FHB-resistance mechanisms in wheat. Furthermore, trichothecene production, which is known to be necessary for disease spread, may inhibit the JA-mediated response in a susceptible interaction, since DON was shown to down-regulate genes involved in JA signalling in ‘Superb’ and DH1 (Chapter 2). However, FHB-disease assays in the JAi lines suggests that JA may play a more prominent role in Type I resistance (see below). The remaining discussion, based on the silencing of SA, JA, or ET and their impacts on disease outcomes, is based on the assumption that silencing was successful and that the data are reproducible, although, as previously discussed, more work is necessary to validate these assumptions. Putative SA silencing resulted in an increase in susceptibility, but only in the Type II resistant background. These conclusions would be consistent with results presented by Makander et al. (2010), where Arabidopsis plants with compromised SA accumulation or signalling pathways showed an increase in susceptibility to F. graminearum infection. If one considers the hypothesis that JA signalling increases susceptibility to initial infection, but improves resistance to disease spread, I could predict that SA signalling serves to suppress the potentially adverse effects of the constitutively higher JA accumulation on Type I resistance in DH2. However, the impact of JA silencing in both resistant backgrounds suggest that JA signalling is positive regulator of Type I resistance. Whether JA signalling impacts both Type I and II resistance or whether this is 139 specifically associated with a Type I resistance mechanism remains unclear. A more accurate assessment of Type II resistance, using point inoculation studies, would provide a clearer indication of the impact of JA silencing on this resistance mechanism. ET silencing also had an impact in disease severity in the Type II resistant background, since the 2-ETi genotype showed an overall improvement in resistance compared with 2- ET. Interestingly, the disease severity in 2-ET was generally higher than in the other wild- type crosses in this genetic background (namely, 2-SA and 2-JA). It was previously demonstrated that ET silencing in the male parent line used here, ‘Bobwhite’, reduced susceptibility to FHB (Chen et al., 2009). Thus, it was surprising that a positive interaction between ET silencing and FHB resistance in the current study was observed only in the Type II resistant background. The observed increase in disease severity in 2-ET compared with 2-SA and 2-JA, and subsequent reduction in severity in the ET-silenced genotype (2- ETi), suggests that the susceptibility in this cross arises directly from the interaction of the ‘Bobwhite’ and DH2 backgrounds and that this susceptibility is correlated with ET signalling. Since similar differences were not observed in the other two genetic backgrounds silenced in the ET signalling pathway, and in fact the opposite response was observed in the susceptible background in this study, it is likely that the specific mechanisms of resistance or susceptibility are highly genotype-dependent. In an analogous situation, affecting ET signalling by chemical treatments was shown to enhance susceptibility in susceptible wheat and barley cultivars, ‘Hobbit’ and ‘Golden Promise’, respectively (Chen et al., 2009), while similar experiments in another susceptible wheat line, landrace Y1193-6, had the opposite effect (Li and Yen, 2008). 140 3.5 Conclusions Results from both the priming and hormone silencing experiments emphasize the complex nature of genetic resistance or susceptibility to FHB, and suggest that the mechanisms of resistance and susceptibility are diverse. For instance, priming with FgTri5- elicited opposite responses in ‘Superb’ and DH1, where susceptibility increased in the latter, but resistance improved in the former. These differences may simply be a question of a delayed response in one genotype compared with the other, but could also mean that the different pathways were activated in each genotype. The incongruent effects of ET signalling in different host backgrounds support the hypothesis that different disease outcomes are mediated by different pathways. While ET signalling appears to be correlated with susceptibility in ‘Bobwhite’, it does not seem to explain the susceptibility in ‘Superb’. In fact, in the ‘Bobwhite’ by ‘Superb’ cross, ET silencing increased susceptibility, suggesting that ET signalling effectively reduces susceptibility in this background. These opposing outcomes are likely mediated by cross-talk with other pathways. Improved resistance was observed in the ‘Bobwhite’ and DH2 cross when the ET signalling pathway was silenced. In this DH2 background, both SA and JA signalling seem to be involved in mediating a resistance outcome, whereas these two pathways have little impact on FHB development in ‘Superb’. Furthermore, in DH1, silencing of ET signalling had no effect on disease, but JA signalling may be important in mediating resistance in this genotype. Perhaps ET signalling only increases susceptibility if it occurs in conjunction with an active role for JA and/or SA signalling. 141 In the Type I resistant DH1, priming-induced systemic resistance was observed, and results from the current study, in combination with those from Chapter 2, suggest that JA signalling may be involved both in the induced, as well as the innate resistance mechanisms of this genotype. By contrast, Type II resistance in DH2 is a local response, and in this case, the innate resistance may be mediated by both JA and SA signalling. Thus, these three wheat genotypes, which share a high degree of genetic identity in their genomes, not only appear to express different types or mechanisms of resistance/susceptibility, but also differ in their biochemical/molecular responses to FHB infection. One commonality observed in both resistant backgrounds, which nevertheless differ in their mechanism of resistance, is that JA signalling appears to mediate resistance, at least in part, in both genotypes. 142 4. Investigating a Putative Role for TaLTP3, a Wheat Lipid Transfer Protein, in Fusarium Head Blight Resistance 4.1 Introduction Lipid transfer proteins (LTPs), first identified in 1975 (Kader) from potato tubers, are small extracellular proteins that are highly conserved in structure and in vitro activity. Two major types of LTPs have been identified. Type 1 LTPs are 9 kDa proteins with four α- helices, four disulfide bridges and a hydrophobic cavity. Type 2 LTPs are similar in structure, except smaller (7 kDa) with only three α-helices. The hydrophobic cavity allows enhanced rates of phospholipid exchange between membranes, as observed in vitro (Kader et al., 1984; Yeats and Rose, 2008). However, the in vivo biological activity of LTPs is still poorly understood. Furthermore, while most LTPs that have been functionally characterized belong to the Type 1 group, it is not clear if and how these two groups may be functionally distinct. Some LTPs have been implicated in the plant stress response (Gaudet et al., 2003; Jung et al., 2003; Yubero-Serrano et al., 2003; Wu et al., 2004a; Lu et al., 2005), with putative roles in cell signalling (Buhot et al., 2001; Blein et al., 2002; Maldonado et al., 2002) and antimicrobial behaviour (Castro and Fontes, 2005; Gonorazky et al., 2005; Sun et al., 2008; Cruz et al., 2010). Sun et al. (2008) evaluated the in vitro antifungal activity of seven Type 1 and one Type 2 stress-inducible wheat LTPs, and found that a few of the Type 1 LTPs inhibited spore germination and/or hyphal growth of several fungal species, including F. graminearum, Verticillium dahliae, and Puccinia graminis. 143 LTPs have also been shown to partake in physiological and developmental processes (reviewed in Carvalho Ade and Gomes, 2007). LTP5 from Arabidopsis and an LTP from Lilium longiflorum (stigma/style cysteine-rich adhesion; SCA) have been shown to mediate fertilization during sexual reproduction (Chae et al., 2009). Several LTPs have been reported to be involved in cuticular wax deposition at the plant cell wall (Cameron et al., 2006; Kim et al., 2008; Samuels et al., 2008; DeBono et al., 2009; Kunst and Samuels, 2009). The main function of the plant cuticle is thought to be in the regulation of water loss (Goodwin and Jenks, 2007), but it also serves as the first line of defense against many invasive pathogens and pests (Martin, 1964; Mendgen et al., 1996). Constitutively higher expression of the TaLTP3 transcript was observed in the spikelets of untreated Type 1 FHB-resistant DH1 genotype compared with its susceptible parent, ‘Superb’ (Chapter 2). The TaLTP3 gene product has not been functionally characterized, but it is a stress inducible Type 1 LTP gene. First cloned from a differential cDNA library of a wheat-rye translocation line infested with Hessian fly (Mayetiola destructor) larvae (Jang et al., 2003), TaLTP3 was later found to be SA- (but not MeJA-) inducible and to be differentially expressed in the phloem and the epidermis (Jang et al., 2005). Constitutively higher transcript accumulation of a plasma membrane intrinsic protein, aquaporin TaPIP1, was also observed in the spikelets of untreated DH1 compared with ‘Superb’ (Chapter 2). Aquaporins PIPs are involved in water transport across the plasma membrane (Tornroth- Horsefield et al., 2006), and transgenic over-expression of a barley aquaporin PIP in rice has been reported to result in increased cuticle thickness (Hanba et al., 2004). Cuticle thickness or composition may play a role in preventing or minimizing FHB host-infection, since the primary mode of Fusarium invasion of plant cells is by direct penetration of the 144 host epidermis, and apparent cuticular degradation has been observed during F. culmorum invasion of wheat spikes (Kang and Buchenauer, 2000b). Cuticle degradation can occur by hydrolytic activity of cutinases or lipases, and expression of Fusarium genes encoding both enzyme classes has been implicated in FHB aggressiveness (Voigt et al., 2005; Cuomo et al., 2007). Furthermore, it has been observed that F. graminearum germinates and grows more easily on leaves of A. thaliana CER1, a cuticular wax-deficient mutant, than on wild- type plants (Ouellet et al., unpublished data). Thus, the simultaneously higher expression of TaLTP3 and TaPIP1 observed in spikelets of Type I FHB-resistant DH1, compared with its susceptible parent ‘Superb’, could potentially contribute to cuticular strengthening and ultimately minimize F. graminearum penetration in this genotype. I therefore conducted a series of experiments to determine whether the constitutively higher expression of TaLTP3 in DH1 compared with the FHB-susceptible cv. ‘Superb’ has an impact on FHB-resistance, and preliminary results are presented here. 4.2 Materials and Methods 4.2.1 Plant material Two wheat genotypes were used for analysis of cuticle thickness and for TaLTP3 and TaPIP1 expression analysis: ‘Superb’ and DH1. Plants were grown as described in Chapter 2 (section 2.2.1). 145 Figure 4.1 Components of the wheat floret for organ dissection. Total RNA extraction was performed on four organs, namely the glume (G), lemma (L) palea (P), and ovary (O). For microscopy, two regions of the G were isolated, the edge (GE; highlighted in blue) and the centre (GC; highlighted in red), one region of the L (highlighted in yellow) and one region of the P (highlighted in green). 4.2.2 TaLTP3 and TaPIP1 gene expression analysis. Spikelets were collected from ‘Superb’ and DH1 at anthesis, and frozen in liquid nitrogen. For DH1, spikelet organs (glumes (G), lemma (L), palea (P), and ovaries (O)) were also collected and frozen at anthesis (Figure 4.1). Total RNA was extracted from the 146 described tissues according to manufacturers’ instructions (Qiagen RNeasy® Plant Mini Kit with an on-column DNase digestion) and cDNA synthesis was conducted as described in 2.2.5. Primer and probe sequences are presented in Supplementary Figures 4, 5, and 6, for EF1α, TaLTP3, and TaPIP1, respectively. Fold-differences (FD) and standard errors were calculated from CT-values using Qiagen’s REST 2006 software package, where CT- values were normalized against the housekeeping gene (EF1α). 4.2.3 Microscopy The G, L, P, O and anthers (A) were collected from ‘Superb’ and DH1 at anthesis. For G, L and P, the tissue were cut into 2-5 x 2-5 mm pieces from the center and edge of the glume (GC and GE, respectively), and from the top center of the L or P (Figure 4.1). The tissue was fixed overnight in 2% gluteraldehyde and 0.1 M phosphate buffer, pH 7.2. All steps prior to embedding were performed at room temperature under gently agitation. The tissue was then washed three times in 0.1 M phosphate buffer, pH 7.2, for 10 min each, fixed for 4 h in 1% osmium tetraoxide in 0.1 M phosphate buffer, pH 7.2, followed by three 15 min washes in water. An overnight perfusion with 2% uranyl acetate in water was performed, followed by three 15 min washes in water. The tissue was then dehydrated in a series of increasing ethanol concentrations (30%, 50%, 70%, 70%, 95%, 95%, 100%, 100% and 100%) each for 15 min. Resin (25% (w/v) vinyl-4-cyclohexene dioxide, 15% (w/v) diglycidyl ether of polypropyleneglycol, 65% (w/v) nonenyl succinic anhydride, and 0.01% (w/v) 2-dimethylaminoethanol) infiltration was conducted in a series of ratios of absolute ethanol:resin, first for 4 h with 3:1 (v/v), then overnight at a ratio of 1:1 (v/v) and twice at 3:1 (v/v) for 12 h each. The tissue was then infiltrated with pure resin for three days, 147 replacing with fresh resin twice per day, and then embedded in resin at 70oC for 8 h. Thin (70 nm) and thick (1 µm) sections were prepared with a microtome for transmission electron microscopy (TEM) and confocal microscopy, respectively. Thin sections were stained with lead citrate and thick sections were differentially stained with methylene blue- azure A and basic fuchsin. 4.2.4 Recombinant expression and purification of TaLTP3 The sequence for TaLTP3, excluding the signal peptide, was synthesized by Genscript and cloned into a pET32a(+) expression vector (Novagen). with a 17 kD N-terminal tag (composed of an N-terminal thioredoxin (Trx), internal His-tag, a C-terminal S-tag followed by an enterokinase site). The fusion protein rLTP3trx has a predicted molecular weight of 26 kD. The predicted product of the rTrx expression, from the empty pET32a(+) vector, is 20 kD, where the extra 3 kD consists of ‘junk’ sequence from the multiple cloning site downstream of the enterokinase site. The purified plasmid (pET32a-TaLTP3) was provided by Genscript (Figure 4.2A). Both pET32a and pET32a-TaLTP3 were transformed into Novagen’s Rosetta-Gami 2 cells (RG) according to manufacturer’s instructions. 188.8.131.52 Recombinant protein expression Cells for each of pET32a(RG) for Trx-tag (rTrx) expression and pET32a-TaLTP3(RG) for the TaLTP3-trx fusion protein (rLTP3trx) expression were each cultured in 250 mL erlenmeyer flasks with 50 mL LB with 50 µg mL-1 carbenicillin. When an OD600 of 0.8-0.9 was reached, expression was induced with 1 mM IPTG. A total of eight-50 mL 148 reactions were combined, and the 400 mL of cells were collected and spun at 8000 x g for 5 min, resuspended in 40 mL Native Binding Buffer (NBB; 0.5 M NaCl; 20 mM sodium phosphate, pH 7.4), and spun for another 5 min. Cells were resuspended in 36 mL NBB and 4 mL 10x FastBreak (Promega) with 20 µg DNase I, 40 µg RNase A and 8 mg lysozyme. The cells were lysed for 60 min at room temperature under gently agitation, and the cell lysate was pushed through a 22 gauge needle syringe, spun at 8000 x g for 5 min, and supernatant was filtered through a 0.45 µm syringe filter. 184.108.40.206 Recombinant protein purification Optimization of His-tag purification was conducted on 1 mL of the cell lysate, which was loaded onto a 1 mL HisTrapTM HP connected to an ÄKTA purifier 10 FPLC at 4oC (GE Healthcare). The column was washed with 5 mL of NBB, followed by a 40 mL gradient elution with 0 to 500 mM imidazole in NBB (1 mL min-1). Following optimization, up to 50 mL was loaded onto a 1 mL HisTrapTM HP connected to the FPLC at 4oC, washed with 10 mL of NBB with 200 mM imidazole, eluted in 5 mL NBB with 300 mM imidazole. The eluate was diluted to 15 mL with NBB and then concentrated in an Amicon Ultra-15 with a 10 kD molecular weight cut-off by centrifugation at 20oC 3220 x g in a swinging bucket rotor for 30 min. The concentrated sample (approximately 200 µL) was diluted with 1 mL LTP Binding Buffer (LBB; 10 mM MOPS, 8 mM β-mercaptoethanol). The sample was then injected (0.9 mL) onto a HiLoad 16/60, Superdex 75 prep grade size exclusion column connected to the FPLC at 4oC for size exclusion chromatography (SEC) and eluted with LBB (0.3 mL min-1). rTrx and rLTP3trx were eluted at 207 and 359 min, respectively. Various fractions from the 149 different stages of rTrx and rLTP3trx purification were analyzed by SDS-PAGE and Western blotting as described in section 4.2.5. 220.127.116.11 Trx tag removal trial from recombinant TaLTP3 fusion protein rLTP3trx expression was conducted as described in section 18.104.22.168, and purified using ProBondTM Purification System (Invitrogen). A total of 50 mL cells were collected 3 h after induction, washed and resuspended in NBB (with 20 mM imidazole) as described in section 4.2.4, with a final cell lysate volume of 8 mL. The cell lysate was loaded onto a 2 mL ProBond nickel-chelating resin and incubated at room temperature with gentle agitation for 60 min. The unbound proteins were eluted by gravity, and the column was washed by gravity elution twice with 8 mL NBB with 20 mM imidazole, once with 8 mL NBB with 40 mM imidazole, then with 100 mM imidazole. The rLTP3trx was then either digested on-column with enterokinase, or eluted in 2 mL NBB with 250 mM imidazole and followed by post-purification enterokinase digest. For post-column digest, the purified rLTP3trx was digested in 50 µL reactions for 8 and 16 h with 1 U Tag-offTM High Activity rEK (rEK) and 1x rEK buffer (Novagen) at room temperature. For on-column digest, following the 100 mM imidazole wash, the resin was washed with 8 mL LBB and then suspended in 2 mL LBB with 10 µL rEK and 10 µL of 10x rEK buffer. The digest proceeded at room temperature under gentle agitation, and 20 µL aliquots were collected at 0.75, 2, 6, 17 and 24 h intervals. Digested fractions were analyzed by SDS-PAGE and some fractions were also evaluated by Western blotting, using the HRP substrate detection method as described in section 4.2.5. 150 A. B. Figure 4.2 Wheat lipid transfer protein 3 sequence and construct designs (page 148). A. The nucleotide (lowercase) and corresponding the protein sequence (uppercase) for TaLTP3 (accession AY22680). The highlighted amino acid sequence was used to develop a rabbit polyclonal antibody (Genscript). The signals peptide sequence is in blue font. B. For the TaLTP3 over-expression and silencing, an expression construct (EC) and a hairpin construct (HC) were designed, respectively. The TaLTP3 sequence for the EC consisted of the coding domain of TaLTP3 from ‘A’. The hairpin sequence for the HC was based on a 27 mer sequence (highlighted in grey in ‘A’). 151 22.214.171.124 Western blotting of recombinant proteins SDS-PAGE gels were stained in Coomasie R-250, or transferred to a 0.2 µm nitrocellulose membrane. Prior to transfer, both the gel and membrane were soaked in Transfer Buffer (NuPAGE® Transfer Buffer (Invitrogen) with 20% methanol) for 15 min, and the transfer was performed at 100 V for 60 min in Transfer Buffer. Immunodetection was conducted using a One-Hour WesternTM Advanced Kit, according to manufacturer’s instructions (GenScript). Each blot was incubated at room temperature for 40 min with 1 µg of the appropriate antibody in 10 mL of buffer. The blot was either developed directly on the membrane using the LumiSensorTM Super Chemiluminescent HRP Substrate (GenScript) or by exposing the membrane to a plastic film for 20 s and then developed according to manufacturers’ instructions (GenScript). Two different antibodies were used for detection: a His-tag rabbit polyclonal antibody (His-pAb) (GenScript) and a polyclonal antibody that was raised in rabbits (GenScript) against a peptide sequence from TaLTP3 (LTP-pAb) (LNGLARSSPDRKIAC; Figure 4.2A). 4.2.5 LTP binding and antifungal assays The purified rLTP3trx and rTrx were quantified by Bradford activity assay (BioRad), and diluted to a concentration of 10 µM each in LBB. An LTP binding assay was performed (adapted from Zachowski et al., 1998), where different concentrations (0 to 15 µM final concentrations) of the fatty acid analog D3821 (4,4-difluoro-5,7-dimethyl-4-bora- 3a,4a-diazas-indacene-3-hexadecanoic acid; Invitrogen) from a 100-µM stock solution in LBB with 1% ethanol to 100 µL protein in a 96-well plate. Control reactions were also run, 152 with 1 µM BSA and 10 µM lysozyme, both in LBB, and a blank reaction (LBB). The excitation wavelength employed was 490 nm and the emission spectrum was recorded at 520 nm on a BioTek Gen5. Antifungal assays were performed using a mycelium growth inhibition test (as described by Sun et al. (2008) and a spore germination inhibition test. For the mycelium growth inhibition test, 1 µL of macroconidia in water (300,000 macroconida mL-1) was used to inoculate the center of a 100-mm petri dish with 20 mL potato dextrose agar (PDA) with supplemented 50 µg mL-1 spectromycin sulfate. After two days of growth at room temperature, three 0.5 cm discs of Whatman Filter paper No.1 were placed 1 cm from the growing hyphae. A total of 100, 150, 200, 250 or 300 µL of 10 µM rTrx or rLTP3trx was pipetted onto the discs, 25 µL at a time and a 90 min period was provided for sample absorption on to the discs between each application. A control sample was pipetted onto the third disc (50 µg of the antimicrobial peptide MsrA2, which was previously reported to inhibit mycelium growth of certain F. graminearum strains (Badea et al., 2009). Mycelium growth was subsequently monitored at room temperature for 24 h. For spore germination inhibition tests, 100 µL of F. graminearum spores (300,000 macroconidia mL-1) were spread onto petri dishes of PDA with supplemented 50 µg mL-1 spectromycin sulfate. Three discs were immediately placed onto the media, and a total of 100, 150, 200, 250 or 300 µL of 10 µM rTrx or rLTP3trx was pipetted onto the discs as described above. The control reaction on the third disc consisted of 50 µg 10R, an antimicrobial peptide previously reported to inhibit spore germination of select F. graminearum strains (Badea et al., 2009). The petri dishes were incubated at room temperature and spore germination was monitored for 24 h. In order to determine whether 153 rLTP3trx activity remained consistent over the incubation period, protein samples were also incubated at room temperature for 24 h, after which a fatty acid binding assay was conducted as described in above. 4.2.6 Transgenic modification of TaLTP3 expression in DH1 and ‘Superb’ An expression cassette designed for RNAi-mediated silencing of TaLTP3 (hairpin construct; HC) and another for over-expression of TaLTP3 (expression construct; EC) were constructed as described in Figure 4.2. The TaLTP3 sequence for the EC consisted of the coding domain of TaLTP3. The hairpin sequence for the HC was based on a 27 mer sequence with high GC-content for the design of hairpin loop sequence using Integrated DNA Technologies SciTools RNAi Design (http://www.idtdna.com/Scitools/Applications/RNAi/RNAi.aspx?source=menu). Both the TaLTP3 and hairpin sequences were synthesized by synthesized by Geneart with appropriate adapter sequences for direct cloning with MultiSite Gateway® Pro 3.0 system. The EC and HC was constructed into the pDEST vector from which it excised and is composed of an actin promoter sequence, followed by the TaLTP3 sequence for over- expression or the hairpin sequence for TaLTP3 silencing, and a nos1 terminator sequence. The EC and HC were introduced into microspores of ‘Superb’ and DH1, respectively, using cell penetrating peptides by E. Amundsen. Details of the transformation procedure are proprietary information (F. Eudes). Plantlets were colchicine treated by M. Fast for double haploid production. The seed produced from the resulting plants can be seeded and the leaf tissue screened by qPCR for changes in TaLTP3 expression as described in section 154 4.2.1, followed by FHB-disease assays by spray inoculation as described in Chapter 3 (section 3.2.3). 4.3 Results 4.3.1 Tissue-specific expression of TaLTP3 and TaPIP1 in DH1 No differences in TaLTP3 transcript accumulation were observed between DH1 and ‘Superb’ (Table 4.1). TaPIP1 transcript accumulation was also examined, in order to determine whether the constitutively higher TaPIP1 and TaLTP3 expression that was observed in DH1 in the microarray experiment described in Chapter 2 occurred in the same organs. qPCR results demonstrated higher TaPIP1 transcript accumulation in DH1 vs ‘Superb’, and also showed higher and lower TaPIP1 accumulation in the palea (P) and ovaries (O), respectively, compared with accumulation in the spikelet of DH1. Similar expression patterns in the spikelets of DH1 were observed with the TaLTP3 primer/probe set. 4.3.2 Cuticle thickness Measurement of cuticle thickness by TEM proved to be a difficult task, yielding inconsistent results. Sections were prepared only for GE and GC, for preliminary investigation; however, as a result of the problems encountered, the remaining tissue has not been evaluated. Some of the challenges faced included: (1) difficulties infiltrating fixed tissue sufficiently for the preparation of thin sections; (2) irregularities in the cuticle thickness from spikelet to spikelet, and also within a given section; (3) difficulties 155 acquiring a consistent sectioning plane. The first major challenge described did not become apparent until thin sections were prepared, where the cells would ‘fall out’ (Figure 4.3A) of the section, although the cell wall remained attached to the resin. The most feasible explanation for this phenomenon is that the tissue was not completely infiltrated with the resin. In case this was a result of incomplete dehydration of the tissue prior to infiltration, fresh plant material was grown and new GE, GC, and L, tissue was fixed and embedded in resin. In this case, an initial infiltration at 3:1 (alcohol:resin) was conducted and the final infiltration in pure resin proceeded for three days, changing the resin twice per day, as described in section 4.2.2. This differed from the original protocol, where the first 3:1 infiltration was omitted and the final infiltration in pure resin proceeded for only 24 h. Unfortunately, the additional steps were unsuccessful in preventing the ‘falling out’ phenomenon, but it was possible to proceed with TEM, since the cell wall and cuticle layer remained embedded in the resin. As noted above, cuticle thickness was highly variable, both between spikelets and within a section. The spikelet to spikelet variation may be, at least in part, attributed to the inconsistency of the sectioning plane. An image of the cuticles from ‘Superb’ and DH1 is presented in Figure 4.3B and C, respectively. Thick sections for confocal microscopy differentially stained with a combination of methylene blue-azure A and basic fuchsin (stains cellulose walls and cuticle red and lignified walls green) are currently being prepared for further investigation. 156 Table 4.1 Tracking TaLTP3 and TaPIP1 expression within the wheat spikelet. qPCR was conducted on cDNA from RNA collected from spikelets of untreated wheat spikes from (A) microarray experiment, where plants were incubated in the mist-irrigated greenhouse for 24 h prior to sampling and from (B) spikelets and spikelet organs of untreated wheat spikes that were not exposed to high humidity. CT-values from amplification and hybridization with The TaLTP3 primer and probe set 2 (Figure A5) and TaPIP1 primer and probe set (Figure A6) were each normalized against the housekeeping gene, EF1α (Figure A4). Positive and negative FD values indicate up- and down- regulation in the given treatment comparison. TaLTP3 TaPIP1 Comparison RNA FD stdev FD stdev DH1 vs 'Superb' spikelet A +1.1 0.3 +1.7 0.3 DH1 vs 'Superb' spikelet B +1.3 0.3 +2.7 0.3 DH1 glume vs spikelet B +1.2 0.3 +1.2 0.2 DH1 lemma vs spikelet B +1.2 0.3 -1.2 0.2 DH1 ovary vs spikelet B -2.1 0.2 -5.0 0.3 DH1 palea vs spikelet B +2.6 0.3 +1.8 0.2 4.3.3 Purification and binding activity assay for recombinant LTP3trx Results from initial purification of rLTP3trx demonstrate that the Trx-tag cannot be removed from the fusion protein without simultaneously losing the LTP. The His-tagged rLTP3trx recombinant protein has a predicted molecular mass of 26 kD, and both on- column and post-purification digests yielded two products, one with an apparent molecular mass of 26 kD, and the other with a mass of 17 kD (Figure 4.4). Furthermore, both digest products were co-eluted from the on-column digest, indicating that both products have an 157 affinity for nickel ions, suggesting that both products still possess a His-tag. This would be consistent with an incomplete digest where the 26 kD product corresponds to the uncleaved fusion protein, and the 17 kD product corresponds to the Trx-tag with TaLTP3 removed. No product was observed with a mass of 9 kD, suggesting that the purified TaLTP3 was insoluble or unstable. Figure 4.3 TEM of GC from ‘Superb’ and DH1. A. The ‘falling out’ phenomenon can be observed in this section of ‘Superb’. The black arrow indicates a white space where the cell should be. The cell wall (CW) and cuticle (C) remained attached to the resin. Guard cells were observed in this image, as indicated by the white arrows. Magnification x 1500. B. and C. Examples of good sections of ‘Superb’ and DH1, respectively, where the cells remained attached to the resin, and the cuticle layer (CL), cuticle proper (CP), and epicuticular wax (EP) can be clearly distinguished. Magnification x 10,000. 158 rLTP3trx was therefore directly purified by FPLC, and a preliminary gradient elution with an increasing concentration of imidazole (0 to 500 mM) from a 1 mL HisTrap nickel column established that the fusion protein can be eluted off the column at 250 mM imidazole (data not shown). A stepwise elution was then performed to maximize both purity and yield, and Western blotting confirmed elution of rLTP3trx, as well as rTrx, at 300 mM imidazole. Since the desired recombinant proteins were co-eluting with degradation products (Figure 4.5), the eluted products were further fractionated by size exclusion chromatography (SEC) with a Superdex 75 column. rTrx was co-eluted with some lower-molecular weight products, but rLTP3trx was free of detectable contaminants following SEC. The product identity was confirmed by Western blotting with both anti-His and anti-TaLTP3 polyclonal antibodies. A lipid binding assay was performed in order to test rLTP3trx activity. The fatty acid analogue D3821 contains a fluorescent group that emits a strong fluorescence in non- aqueous environments, such as the fatty acid binding cavity of LTP molecules (Invitrogen). This approach has previously been utilized to demonstrate LTP binding activity by other authors (Zachowski et al., 1998; Sun et al., 2008). The increase in fluorescence with increasing concentrations of D3821 incubated with rLTP3trx, but not with Trx, demonstrates successful binding activity of rLTP3trx (Figure 4.6A). 159 Figure 4.4 Recombinant LTP3trx digest trial. A. rLTP3trx purified with a ProBond nickel-chelating resin (lane 1) was digested for 6 h (lane 2) or 18 h (lane 3). Gel was stained with Sypro Ruby. B. Whole cell lysate of pET32a-LTP3(RG) collected 3 h after IPTG induction (IL) (lane 1), IL was digested with rEK for 6 h (lane 2), and purified rLTP3trx digested with rEK for 6 h (lane 3), were resolved by SDS-PAGE, transferred to a 0.2 µm nitrocellulose membrane, and immunodetection with the LTP-pAB was conducted directly on the membrane. C. IL (lane 10), was loaded (8 mL) onto ProBond nickel- chelating 2 mL resin, washed three times with NBB, and once with LBB. The resin was then resuspended in 2 mL LBB with 20 U Tag-offTM High Activity rEK (rEK). Samples were collected from the suspension at 0.75 h (lane 1), 2 h (lane 2), 6 h (lane 3), 17 h (lane 4), 24 h (lane 5), and then the resin washed with NBB with 100 mM imidazole (lane 6), 250 mM imidazole (land 7), and 500 mM imidazole. Gel was stained with Coomasie Blue. 160 Figure 4.5 161 Figure 4.5 Purification of recombinant TaLTP3trx and Trx (page 160). A. rTrx purification by affinity chromatography using a HisTrapTM HP column (top chromatogram), and fraction 2 (F2) was further resolved by SEC (bottom chromatogram). rTrx was eluted in F4 by SEC (0.3 mL min-1) a retention time of 207. Uninduced (UT) and induced (IT) whole cell lysate of pET32a(RG), the flow through (FT) from affinity chromatography and selected fractions were resolved by SDS-PAGE (stained in Coomassie blue; top image) and Western blotting with a His-pAb developed on a plastic film (bottom image) was used to track purification. B. Similarly to rTrx purification, rLTP3trx was purified affinity chromatography (top chromatogram), and fraction 2 (F2) was resolved by SEC (bottom chromatogram). rLTP3trx was eluted in F6 by SEC (0.3 mL min-1) a retention time of 359. Uninduced (UL) and induced (IL) whole cell lysate of pET32a- TaLTP3(RG), the FT and selected fractions were resolved by SDS-PAGE (top image), and Western blotting was performed using His-pAb (middle image) and LTP-pAb (bottom image) developed on a plastic film. 162 A. B. Figure 4.6 163 Figure 4.6 TaLTP3 binding and inhibition assays (page 162). A. Binding assay with the fatty acid analogue D3821 (4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaz-indacene-3- hexadecaonoic acid) which emits fluorescence when captured in a non-aqueous environment. An increase in fluorescence was observed with increasing concentrations of D3821 applied to rLTP3trx and rLTP3trx which has been incubated at room temperature for 24 h (rLTP3trx(RT). The fluorescence for rLTP3trx and rLTP3trx(RT) for > 2 µM D3821 continued; however, the instrument was unable to provide a reading because the detector was ‘overflowed’. The limited fluorescence emitted from rTrx did not exceed fluorescence emitted by the blank reaction, LBB. B. F. graminearum mycelium (left) and spore germination (right) inhibition tests with 50 µg 10R (1), 50 µg MsrA2 (2), 100 µL of 10 µM rTrx (3), and 100 µL of 10 µM rLTP3trx (4). Similar results to those observed in 3 and 4, were also observed at all molar quantities of rTrx and rLTP3trx evaluated: 150 µL, 200 µL, 250 µL, and 300 µL of 10 µM protein (data not shown). Only MsrA2 showed antifungal activity towards F. graminearum, and both mycelium growth and spore germination were affected. Although the photograph was not able to clearly capture MsrA2 mycelium inhibition, the results were quite evident by visual inspection: the hypha extended over all the other discs, except for disc 2, where the mycelium clearly grew around the disc. 4.3.4 rLTP3trx-F. graminearum inhibition assays It was previously demonstrated that some wheat LTPs can inhibit mycelium growth of F. graminearum in in vitro assays (Sun et al., 2008). Using the same approach in the 164 current study, I found that rLTP3trx does not exhibit antifungal activity towards the growing mycelium or germinating spores of F. graminearum strain GZ3639 (Figure 4.6B). 4.3.5 Transgenic modification of TaLTP3 expression in DH1 and ‘Superb’ A total of seven and two plantlets were produced from the DH1-HC and ‘Superb’-EC transformation, respectively, but only two DH1-HC and one ‘Superb’-EC transformant survived colchicine treatment. Seed is not yet available. 4.4 Discussion 4.4.1 Validation of TaLTP3 and TaPIP1 transcript accumulation and evaluation of organ-specific expression in DH1 In the microarray experiment (Chapter 2) both TaLTP3 and TaPIP1 showed constitutively higher transcript accumulation in untreated DH1 spikelets compared with its susceptible parent ‘Superb’. qPCR was used here to confirm this expression pattern. qPCR was conducted both on RNA collected during the microarray experiment (spikelets of untreated wheat spikes incubated under high humidity in the mist-irrigated greenhouse) and on RNA collected from spikelets of untreated spikes that were not exposed to high humidity (Table 4.1). The constitutive higher TaPIP1 expression in DH1 compared with ‘Superb’ was substantially higher (259 FD) on the Affymetrix gene chip compared with qPCR results (Table 4.1). By contrast, the qPCR reaction conducted on cDNA synthesized from the same RNA collected for the microarray experiment, showed only a 1.7 FD higher transcript accumulation in DH1 spikelets compared with ‘Superb’. A slightly greater 165 difference was observed from RNA collected from spikelets grown under normal humidity, where transcript accumulation was 2.7 FD higher in DH1 compared with ‘Superb’. Thus, the constitutively higher TaPIP1 transcript accumulation in DH1 spikelets was validated by qPCR, but on a much lower scale than observed in the microarray experiment. Higher TaLTP3 transcript accumulation in DH1 vs ‘Superb’, on the other hand was not validated. LTPs are highly conserved sequences, and while steps were taken to maximize primer and probe specificity in the qPCR design (Figure A5), the probability of cross-reactivity remains high. By contrast, the Affymetrix gene chip contains multiple probes which would increase the specificity of a specific LTP over the gene family. No other LTPs genes were shown to be constitutively over-expressed in DH1 vs ‘Superb’ in the microarray experiment (Table 2.2). Furthermore, since the FD in the microarray experiment was so high for this gene (> 30 FD), and since it was not only observed in the uninoculated controls (as shown in Chapter 2) but also in elicitor treated-DH1 compared with elicitor treated-‘Superb’ (where differences ranged from 30 to 100 FD; data not shown), it is unlikely that the results of the microarray experiment were artifactual. An effort to determine in which organ(s) TaLTP3 expression occurred, and also to observe whether or not this coincided with TaPIP1 expression, transcript accumulation of both genes was tracked in specific organs, namely the glumes (G), lemma (L), palea (P), and ovaries (O). I postulated that by tracking organ-specific expression, some insight into the putative functions of these genes as they pertain to FHB-resistance may be revealed. For instance, if high levels of TaLTP3 occurs in any of the described tissues, and if TaLTP3 expresses antifungal activity, this could contribute to FHB-resistance; if expression occurs in outer tissues of the spikelet (G or L), then putative antifungal activity 166 could contribute more specifically to Type I resistance. If both TaLTP3 and TaPIP1 expression occurs in G, expression of these genes could result in enhanced cuticle deposition, since some LTPs and PIPs have been implicated in cuticle thickening or deposition (Hanba et al., 2004; Samuels et al., 2008). qPCR with the selected TaLTP3 and TaPIP1 primers and probes suggest that transcript accumulation were highest in the P and lowest in the O of DH1. Since the primer/probe specificity for TaLTP3 has not been confirmed, the biological significance of these results remains elusive. 4.4.2 Measurement of cuticle thickness in DH1 compared with ‘Superb’ To determine whether or not the constitutively higher transcript accumulation of TaLTP3 has an impact on cuticle thickness in DH1, I attempted to measure cuticle thickness by quantitative analysis of TEM sections. Unfortunately, technical challenges in the experimental design were encountered, and more work will be necessary to address this question. It may be possible to overcome some of these challenges by evaluating a large number of replicates. An analysis of cuticle thickness in ‘Superb’ and DH1 compared with the transgenic lines (‘Superb’-EC and DH1-HC) will also be necessary to address the question of whether TaLTP3 expression quantitatively influences cuticle deposition. It would also be worthwhile to evaluate cuticle composition in the different genotypes, since this may also affect resistance. The types of waxes and fatty acids present in the cuticle can affect a resistance or susceptible response to different plant pathogens (Bessire et al., 2007; Lee et al., 2009; Curvers et al., 2010). Leaf cuticle waxes have been known induce spore germination (Podila et al., 1993) and/or appresorium formation (Uchiyama et al., 1979; Uchiyama and Okuyama, 1990; Podila et al., 1993) of various pathogens, including 167 the rice blast fungus Pyricularide oryzae and the causative agent of anthracnose in avocado plants, Colletotrichum gloeosporioides. 4.4.3 TaLTP3 does not exhibit antifungal activity towards F. graminearum Antifungal properties of recombinant TaLTP3 (rLTP3trx) were evaluated by inhibition assays of F. graminearum spore germination and mycelial growth. Using a series of rLTP3trx concentrations, no adverse effects were observed in either type of assay, suggesting that this LTP does not exhibit antifungal properties. It should be noted that some LTPs have been shown to exhibit antimicrobial properties towards some genera of fungi, but not others (Sun et al., 2008). Furthermore, the antimicrobial properties of the peptides LR10 and MsrA2, which have been shown to inhibit mycelia growth and/or spore germination in a variety of Fusarium species, were found to be strain-specific; even within F. graminearum species, some strains were unaffected by these peptides (Badea et al., 2009). Since the antifungal assays in the current study were conducted using a single F. graminearum strain, namely GZ3639, the possibility that these results are exclusive to this particular strain cannot be eliminated until these studies have been repeated with different strains and/or species. 4.5 Conclusions The premise of these experiments was that TaLTP3 might play a role in the Type I resistance mechanism of DH1. However, the results have provided little support for such a role. It is therefore possible that TaLTP3 does not, in fact, play a role in FHB-resistance, 168 since the hypothesis presented in Chapter 2 was based on inferences from comparative transcriptomics data. Nevertheless, recent studies by T. Ouellet and colleagues (Agriculture and Agri-Food Canada, Ottawa) suggest that TaLTP3 gene expression is highly correlated with the 5A QTL associated with Type I resistance to FHB in wheat cultivars ‘Wuhan’ and ‘NuyBai’, and in a series of derived lines (personal communication). Interestingly, no QTLs for resistance have been identified specifically in DH1 (Eudes et al. unpublished data). It is nonetheless possible that TaLTP3 is involved in the mechanism of resistance in wheat genotypes with the 5A QTL and also in DH1, if the gene(s) of interest associated with the 5A QTL interact with TaLTP3 in mediating resistance. The ambiguous results of my experiments do not conclusively eliminate a role for TaLTP3 in FHB-resistance; additional studies will be required to address this question thoroughly. It will be necessary to validate the constitutively higher transcript accumulation of TaLTP3 in DH1 compared with ‘Superb’. This information could also provide some insight into the broader biological role of TaLTP3. F. graminearum spore germination and mycelium growth inhibition assays have suggested that TaLTP3 does not have any direct antifungal properties towards Fusarium, but it will be necessary to extend these results using additional F. graminearum strains and Fusarium species. In order to gain some insight into TaLTP3’s possible role in cuticle deposition, improved methods will have to be developed that allow cuticle thickness and composition to be assessed in a consistent fashion. Since the biological role of LTPs in plants is not well defined, it is still conceivable that this protein may participate in conferring FHB-resistance through a mechanism that we are not yet aware of. Analysis of the impact of transgenic modification of the expression of 169 this gene on disease outcomes should provide some insight into whether or not this protein can, in fact, impact resistance. Furthermore, these studies can be complemented with Fusarium leaf colonization assays to determine whether the change in TaLTP3 expression can impact germination on wheat leaves, and whether cuticle composition or thickness has been affected. 170 5 Conclusions While the molecular mechanisms of FHB-resistance are poorly understood, the physiological mechanisms are more clearly defined, particular in the case of Type II resistance. Studies using a Type II resistant background inoculated with trichothecene- producing Fusarium species (Kang and Buchenauer, 2000a), and studies using susceptible genotypes inoculated with trichothecene non-producing F. graminearum (Jansen et al., 2005), have demonstrated that resistance to disease spread is associated with cell wall thickening at the rachis node of the infected spikelet, thereby preventing the pathogen from invading the rachis. Less is known about the mechanisms of Type I resistance. In theory, Type I resistance could be conferred by the presence of structural features at the spikelet surface, such as thick cuticles or cell walls, that would block or slow penetration of the fungus. Alternatively, the host could reduce the build-up of fungal biomass available for host invasion by producing antimicrobial proteins that would inhibit spore germination or hyphal growth. It is interesting that Type II resistance, which has a well understood physiological mechanism of resistance, is tightly correlated with the presence of the 3BS QTL, whereas no single QTL has been consistently correlated with Type I resistance, whose physiological mechanism is less well defined. Based on the available information, I proposed at the outset of this study that the mechanisms of Type I resistance would be variable and genotype-dependent, and that Type II resistance involves a very specific molecular response to Fusarium species. Furthermore, since Type II resistance can be phenocopied in 171 otherwise susceptible wheat inoculated with trichothecene non-producing Fusarium strains, I hypothesized that the specific pathway(s) that are upregulated during a Type II resistance response interact in some way with trichothecenes to suppress their effectiveness. In Chapter 2, a functional genomics study using both differential transcriptomics and proteomics was used to evaluate this hypothesis. The systemic response in uninoculated wheat spikes primed with different elicitors of FHB (FgTri5+, FgTri5-, and DON) was compared in three wheat genotypes (susceptible Canadian cv. ‘Superb’, Type I resistant DH1, and Type II resistant DH2). The results led to the development of three new hypotheses: (1) Type II resistance is mediated by JA-signalling, a response that is inhibited by trichothecenes in genotypes that do not display the 3BS QTL for resistance to disease spread, (2) Type I resistance can be systemically induced in DH1, whereas Type II resistance is a local response conferred by the 3BS QTL, and cannot be systemically induced in DH2, and (3) Type I resistance in DH1 involves a combination of structural features and antifungal activities of proteins that slow fungal penetration. The first of these new hypotheses was an extension of the original hypothesis, where it was proposed that the specific pathway(s) that are up-regulated interact in some way with trichothecenes to minimize their impact. An interaction was observed between the Type II resistant response and trichothecenes, and involved the up-regulation of a specific pathway, namely JA-signalling. Differential transcriptomics data revealed up-regulation of JA biosynthesis/responsive genes in DH2 elicited by FgTri5+. While, DH1 and ‘Superb’ also showed up-regulation of similar genes, this occurred only under conditions where these two genotypes would phenocopy Type II resistance, namely in response to FgTri5-. 172 Furthermore, DON induced down-regulation of genes from this pathway in ‘Superb’ and DH1, but not in DH2. In order to test this hypothesis, the role of JA signalling was evaluated in Chapter 3. The hormone silencing studies suggest that JA-signalling is important in Type I resistance, but point inoculation studies will be necessary to determine whether or not it also plays a role in Type II resistance. Hormone quantification did, however, show that JA constitutively accumulates in higher quantities in the Type II resistant background DH2. It is not known how this signalling pathway would lead to a resistant outcome, but it is known that JA signalling can be a positive regulator of the lignin biosynthesis pathway (Xue et al., 2008). Furthermore, increasing evidence suggests that cell wall lignification at the rachis node is an important feature of Type II resistance. It is also likely that cell wall lignification at the spikelet surface would improve Type I resistance, since Fusarium infects by direct penetration of the epidermis. Interestingly, I observed that the lignin biosynthesis pathway was strongly up-regulated by FHB-elicitors in the systemic tissues of DH1 in the differential transcriptomic study (Chapter 2). Other hormone signalling pathways were also investigated in the hormone silencing experiment, and the FHB disease assay revealed a complex interaction between hormone signalling and resistance vs susceptible outcomes. The data suggest that SA and ET signalling have a positive and negative effect, respectively, on resistance to initial infection in Type II resistant DH2; whereas, no effect of either hormone pathways was observed on resistance in the DH1 background. In the susceptible genotype, neither JA nor SA signalling appeared to have an impact on resistance, but ET signalling was shown to reduce susceptibility to initial infection—the opposite of what was observed in DH2 in this 173 experiment and also in the susceptible wheat cv. Bobwhite in an experiment conducted by Chen and colleagues (2009). Perhaps, ET-signalling only leads to susceptibility when it occurs in conjunction with increased SA signalling. Future studies in silencing multiple pathways simultaneously in wheat, as was recently conducted in Arabidopsis (Makandar et al., 2010), should help to elucidate the role of cross-talk in these interactions. It was also interesting that silencing of the SA-signalling pathway was shown to have an adverse impact on resistance to initial infection in DH2, but not in Type I resistant DH1. The wild-type DH2, which has a good Type II resistance, also has a moderate Type I resistance. Together, these data suggest that not only are the mechanisms of Type I resistance genotype-dependent, as proposed in the initial hypothesis presented in Chapter 1, but so are the mechanisms of susceptibility. The priming experiment in Chapter 3 demonstrated that systemic resistance can be improved by priming with an FHB elicitor in DH1, and not in DH2. These results support the second hypothesis proposed in Chapter 2. Interestingly, while FgTri5- priming improved resistance in DH1, the opposite effect was observed in ‘Superb’. These results, together with the hormone silencing data, demonstrate the complexity of the FHB interaction in wheat, and provide additional support to the first part of the original hypothesis presented in Chapter 1; i.e. that the mechanisms of resistance are genotype- dependent. Future priming studies and hormone silencing experiments using a wider range of plants, with different backgrounds of Type I or Type II resistance, will be valuable in further testing this hypothesis. The third hypothesis presented in Chapter 2, states that Type I resistance in DH1 is conferred by combination structural features and antifungal activities of proteins that 174 reduces or inhibits fungal penetration. The functional genomics experiment revealed constitutively higher expression of two genes of interest, namely TaPIP1 and TaLTP3, in the spikelets of untreated spikes of DH1 compared with its susceptible parent ‘Superb’. Since fungal treatments (FgTri5+ and FgTri5-) induced up-regulation of lignin biosynthetic genes, and since aquaporins can transport both water and hydrogen peroxide, increased levels of TaPIP1 in DH1 may facilitate accumulation of H2O2 at the cell wall for lignification, thus providing additional physical protection against the invading fungus. TaLTP3 may be involved in either directly affecting the pathogen through its potential antifungal properties, or may contribute, together with TaPIP1, to enhance cuticle deposition, which would provide a protective barrier against pathogen invasion. In Chapter 4, the putative roles for TaLTP3 in FHB-resistance were investigated. Antifungal assays did not reveal any antimicrobial activities of TaLTP3 towards F. graminearum strain GZ3639, and the comparisons of cuticle thickness in the glumes of ‘Superb’ vs DH1 by TEM was inconclusive. More work will be necessary to determine the putative role of TaLTP3 and/or TaPIP1 in FHB-resistance. The most crucial experiment will be to assess the impact of modified expression of these genes on disease outcomes. If expression of these genes does in fact affect FHB-resistance, then it would be worthwhile to further pursue the studies of cuticle thickness or composition in DH1 compared with ‘Superb’. Additional studies to investigate putative FHB-elicited changes in cell wall lignification of DH1 would also provide insights into the mechanisms of Type I resistance in DH1. In summary, results from Chapters 2 and 3 support the original hypothesis that the mechanisms of resistance are genotype-dependent, and also suggest that the mechanisms of 175 susceptibility are also genotype-dependent. Furthermore, the JA signalling pathway was implicated in FHB-resistance. It is not yet clear whether this pathway is involved in both Type I and Type II resistance, and how SA and ET signalling interact with JA-signalling in conferring different types of resistance. The first hypothesis presented in Chapter 2, suggests that Type II resistance is conferred by JA-signalling, and that trichothecenes inhibit this response in other genotypes. If JA-signalling is also important in Type I resistance, as is suggested in Chapter 3, trichothecene-mediated suppression of JA- signalling would not have any impact on Type I resistance since trichothecene biosynthesis does not occur until after initial infection has been established. The second hypothesis presented in Chapter 2, states that Type I resistance can be systemically induced in DH1, but not in DH2, and results from the priming experiment Chapter 3 support this hypothesis. The next step will be to determine whether or not these outcomes can be observed in different Type I and Type II resistant backgrounds. More work will be necessary to evaluate the third hypothesis presented in Chapter 2, which states that Type I resistance in DH1 involves a combination of structural features and antifungal activities of proteins that slow fungal penetration. Based on all of my results, I have proposed a model for the molecular basis of FHB- resistance in wheat (Figure 5.1). In a susceptible interaction, the Fusarium spores germinate and the hyphae easily penetrate the surface of spikelet. In some relatively susceptible cultivars, such as ‘Superb’, this penetration can be suppressed by ET signalling; in other backgrounds, such as ‘Bobwhite’ or DH2, this ease of penetration is actually enhanced by ET signalling. These genotypic differences are likely mediated by cross-talk with other hormone signalling pathways. Upon spikelet invasion, DON synthesis begins, 176 and as this metabolite accumulates, it inhibits a JA signalling response that would normally up-regulate lignin production for cell wall strengthening at the rachis node. Suppression of the wall strengthening response ultimately results in hyphal penetration of the rachis. In a Type I resistant interaction, spore germination or mycelial growth may be inhibited by antimicrobial peptides or proteins (AMPs). Upon recognition of the presence of Fusarium, JA biosynthesis is induced, and this may lead to lignin biosynthesis and subsequent lignification of the glume or lemma, ultimately inhibiting fungal penetration. Penetration may be also be inhibited by the structure or composition of the cuticle. SA signalling may also improve Type I resistance in some genotypes, possibly by inducing a hypersensitive response which would prevent the fungus from establishing initial infection during the biotrophic phase of disease progression. 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Probe Set ID regulation hai FD p-val Gene Symbol Gene Title Superb Tri5+ vs water Ta.12319.1.A1_at down 3 2.3 0.018 ribosomal protein S5 (animal contaminant) Ta.1979.2.S1_x_at down 3 2.2 0.048 31 kDa ribonucleoprotein, chloroplast precursor Ta.5633.3.A1_at down 3 2.0 0.003 NITRATE REDUCTASE (NR) TaAffx.27931.1.S1_at down 3 2.1 0.009 synaptobrevin [Triticum monococcum] TaAffx.57167.1.S1_at down 3 2.0 0.036 similar to uroporphyrinogen decarboxylase [Oryza sativa (japonica cultivar-group)] Ta.497.2.S1_x_at down 8 2.6 0.008 PROTEIN TRANSPORT PROTEIN SEC23A (SEC23-RELATED PROTEIN A) TaAffx.52404.1.S1_at down 8 2.7 0.000 unknown Ta.14235.2.S1_x_at down 24 2.1 0.037 unknown Ta.16011.1.S1_at down 24 2.3 0.010 unknown Ta.2638.1.S1_at down 24 2.6 0.046 rab 15B rab protein Ta.27542.1.S1_at down 24 2.0 0.007 Ketol-acid reductoisomerase, chloroplast precursor (Acetohydroxy-acid reductoisomerase) (Alpha-keto-beta- hydroxylacil 213 Probe Set ID regulation hai FD p-val Gene Symbol Gene Title Ta.9085.3.S1_x_at down 24 2.2 0.012 unknown protein [Arabidopsis thaliana] gi|16649041|gb|AAL24372.1| unknown protein [Arabidopsis thaliana] gi|21618051|gb|AAM67101.1| unknown [Arabidopsis thaliana] TaAffx.108878.1.S1_x_at down 24 2.1 0.019 60S RIBOSOMAL PROTEIN L5 [Neurospora crassa] TaAffx.111598.2.S1_s_at down 24 2.3 0.035 unknown TaAffx.121246.1.S1_at down 24 2.1 0.003 unknown TaAffx.64328.1.S1_at down 24 2.0 0.004 LOC543447 GTP-binding protein TaAffx.6593.2.S1_at down 24 2.1 0.010 Chloroplast 50S ribosomal protein L33 TaAffx.88122.4.S1_at down 24 2.1 0.002 Cytochrome P450 90D2 (C6-oxidase) Ta.11565.1.A1_at up 3 2.6 0.016 putative Ca2+-dependent lipid-binding protein [Oryza sativa (japonica cultivar-group)] TaAffx.111915.1.S1_at up 3 2.2 0.040 unknown TaAffx.31923.1.S1_at up 3 2.0 0.028 serine/threonine protein kinase [Oryza sativa (japonica cultivar-group)] TaAffx.56177.1.S1_at up 3 2.2 0.038 hypothetical protein [Oryza sativa (japonica cultivar-group)] Ta.10769.1.A1_at up 8 2.4 0.000 unknown Ta.10859.1.A1_at up 8 2.1 0.048 Transcribed locus, moderately similar to XP_473516.1 OSJNBa0017B10.7 [Oryza sativa (japonica cultivar-group)] Ta.20029.1.S1_at up 8 2.2 0.002 unknown Ta.223.1.S1_at up 8 2.2 0.033 Beta-1,3-glucanase precursor (Glb3) Ta.23165.1.S1_at up 8 2.3 0.041 unknown Ta.26556.1.A1_at up 8 2.1 0.004 Zea mays unknown mRNA, partial sequence Ta.5445.2.S1_at up 8 2.0 0.029 PREDICTED P0654B04.18 gene product [Oryza sativa (japonica cultivar-group)] gi|50912235|ref|XP_467525.1| unknown protein [Oryza sativa (japonica cultivar-group)] Ta.6578.2.S1_x_at up 8 2.6 0.027 transaldolase ToTAL2 [Oryza sativa (japonica cultivar-group)] 214 Probe Set ID regulation hai FD p-val Gene Symbol Gene Title Ta.7702.2.S1_x_at up 8 2.1 0.039 Calcineurin B-like protein 3 (SOS3-like calcium binding protein 6) Ta.8582.2.S1_a_at up 8 2.0 0.001 unknown TaAffx.106775.1.S1_at up 8 2.1 0.017 unknown TaAffx.107918.1.S1_at up 8 2.0 0.015 CYTOCHROME P450 71A1 (CYPLXXIA1) (ARP-2) TaAffx.108743.1.S1_at up 8 2.1 0.007 similar to beta-1,3-glucanase 2a [Hordeum vulgare] TaAffx.109085.1.S1_at up 8 2.2 0.010 glucosyltransferase [Oryza sativa (japonica cultivar-group)] (contig annotation) TaAffx.113515.1.S1_at up 8 2.1 0.003 unknown TaAffx.113624.2.S1_at up 8 2.7 0.005 Serine/threonine-protein kinase BRI1-like 3 precursor (BRASSINOSTEROID INSENSITIVE 1-like protein 3) TaAffx.128541.59.A1_at up 8 2.9 0.041 transposon [Triticum aestivum] TaAffx.12878.1.A1_at up 8 2.1 0.006 wall-associated kinase 3 [Triticum aestivum] TaAffx.218.1.S1_at up 8 2.2 0.036 unknown TaAffx.36998.1.S1_at up 8 2.1 0.034 unknown TaAffx.56014.3.S1_at up 8 2.6 0.001 41 kD chloroplast nucleoid DNA binding protein (CND41) [Oryza sativa (japonica cultivar-group)] TaAffx.56793.1.S1_x_at up 8 2.8 0.020 diaphanous homologue-like [Oryza sativa (japonica cultivar-group)] (contig annotation) TaAffx.65440.1.S1_at up 8 2.7 0.026 peptide chain release factor subunit 1 (ERF1) [Oryza sativa (japonica cultivar-group)] TaAffx.83019.1.S1_at up 8 3.2 0.032 Inorganic diphosphatase TaAffx.96741.2.S1_at up 8 2.2 0.047 OSJNBa0039K24.21 [Oryza sativa (japonica cultivar-group)] gi|38345518|emb|CAE01802.2| OSJNBa0039K24.21 [Oryza sativa (japonica cultivar-group)] Ta.18832.2.S1_at up 24 2.1 0.032 unknown Ta.21120.1.S1_at up 24 3.1 0.017 Glucan endo-1,3-beta-D-glucosidase (contig annotation) 215 Probe Set ID regulation hai FD p-val Gene Symbol Gene Title Ta.21650.1.A1_at up 24 2.0 0.015 Transcribed locus, weakly similar to NP_921072.1 putative disease resistance gene [Oryza sativa (japonica cultivar-group)] Ta.22562.1.S1_at up 24 2.1 0.045 Glucan endo-1,3-beta-D-glucosidase Ta.22981.3.S1_a_at up 24 2.5 0.002 cytochrome P450 protein [Arabidopsis thaliana] Ta.5042.1.A1_at up 24 2.0 0.047 unknown Ta.82.1.S1_at up 24 2.7 0.027 LOC543287 peroxidase Ta.8545.2.S1_at up 24 2.3 0.024 unknown TaAffx.110215.1.S1_x_at up 24 2.7 0.013 unknown TaAffx.131249.1.S1_s_at up 24 2.4 0.036 beta-1,3-glucanase [Oryza sativa (japonica cultivar-group)] TaAffx.27956.1.S1_at up 24 2.2 0.010 unknown TaAffx.4083.5.A1_x_at up 24 3.1 0.000 similar to vacuolar sorting receptor protein homolog PV72 [Cucurbita cv. Kurokawa Amakuri] TaAffx.57405.1.S1_x_at up 24 2.2 0.017 similar to tonoplast membrane integral protein [Oryza sativa (japonica cultivar-group)] (contig annotation) TaAffx.59664.1.S1_at up 24 2.1 0.016 unknown TaAffx.6823.1.S1_at up 24 2.4 0.014 SH6.2 S-adenosyl-L-homocysteine hydrolase TaAffx.71003.1.S1_at up 24 2.1 0.028 unknown TaAffx.79727.1.S1_at up 24 2.2 0.049 C2 domain-containing protein [Hordeum vulgare subsp. vulgare] TaAffx.81742.1.S1_at up 24 2.7 0.027 unknown TaAffx.83360.1.S1_at up 24 2.0 0.005 Annexin A13 (Annexin XIII) TaAffx.9022.1.S1_at up 24 2.8 0.028 beta-1,3-glucanase precursor [Oryza sativa (japonica cultivar-group)] TaAffx.92097.1.S1_at up 24 2.2 0.037 unknown TaAffx.92097.1.S1_x_at up 24 2.3 0.019 unknown Tri5- vs water 216 Probe Set ID regulation hai FD p-val Gene Symbol Gene Title Ta.10781.1.A1_at down 3 3.5 0.010 60S acidic ribosomal protein P0 (DNA- (apurinic or apyrimidinic site) lyase) (animal contaminant) Ta.12470.1.A1_at down 3 2.0 0.030 unknown Ta.9346.3.S1_x_at down 3 2.2 0.049 ABSCISIC STRESS RIPENING PROTEIN 1 TaAffx.83824.1.S1_at down 3 2.7 0.047 dioscorin class A precursor [Oryza sativa (japonica cultivar-group)] Ta.497.2.S1_x_at down 8 2.4 0.004 PROTEIN TRANSPORT PROTEIN SEC23A (SEC23-RELATED PROTEIN A) TaAffx.52404.1.S1_at down 8 2.7 0.019 unknown Ta.10357.2.A1_s_at down 24 2.0 0.049 zinc finger protein [Oryza sativa] (contig annotation) Ta.10781.1.A1_at down 24 4.9 0.009 60S acidic ribosomal protein P0 (DNA- (apurinic or apyrimidinic site) lyase) (animal contaminant) Ta.11242.1.A1_at down 24 2.2 0.009 unknown Ta.11332.1.A1_at down 24 2.1 0.050 P0421H07.26 [Oryza sativa (japonica cultivar- group)] gi|20804607|dbj|BAB92298.1| P0421H07.26 [Oryza sativa (japonica cultivar- group)] gi|13872913|dbj|BAB44019.1| P0684B02.6 [Oryza sativa (japonica cultivar- grou Ta.11565.1.A1_at down 24 2.3 0.043 putative Ca2+-dependent lipid-binding protein [Oryza sativa (japonica cultivar-group)] Ta.12109.1.A1_at down 24 2.2 0.041 unknown Ta.12319.1.A1_at down 24 4.6 0.041 ribosomal protein S5 (animal contaminant) Ta.12402.1.S1_at down 24 2.5 0.039 hypothetical protein [Oryza sativa (japonica cultivar-group)] gi|48717095|dbj|BAD22868.1| hypothetical protein [Oryza sativa (japonica cultivar-group)] Ta.16011.1.S1_at down 24 2.7 0.002 unknown Ta.18587.1.S1_x_at down 24 2.3 0.000 systemin receptor SR160 precursor (Brassinosteroid LRR receptor kinase) [Oryza sativa (japonica cultivar-group)] 217 Probe Set ID regulation hai FD p-val Gene Symbol Gene Title Ta.21213.3.S1_x_at down 24 2.1 0.018 similar to myb family transcription factor [Arabidopsis thaliana] Ta.24110.1.A1_s_at down 24 2.5 0.028 putative proline-rich protein [Oryza sativa (japonica cultivar-group)] gi|14488319|gb|AAK63900.1| Putative proline- rich protein [Oryza sativa] Ta.3748.1.A1_at down 24 2.2 0.015 HEXOKINASE 1 Ta.426.1.A1_at down 24 2.5 0.022 linalool synthase [Oryza sativa (japonica cultivar-group)] / terpene synthase [Oryza sativa (japonica cultivar-group)] TaAffx.106139.1.S1_at down 24 2.1 0.004 unknown TaAffx.111283.1.S1_at down 24 2.0 0.040 unknown TaAffx.120138.1.A1_at down 24 2.3 0.025 Transcribed locus, weakly similar to NP_919846.1 putative proline-rich protein [Oryza sativa (japonica cultivar-group)] TaAffx.120404.1.S1_at down 24 2.1 0.006 putative permease 1 [Oryza sativa (japonica cultivar-group)] gi|38637273|dbj|BAD03537.1| putative permease 1 [Oryza sativa (japonica cultivar-group)] gi|38637220|dbj|BAD03486.1| putative permease 1 [Oryza sativ TaAffx.128835.3.S1_x_at down 24 2.1 0.014 unknown TaAffx.23052.1.S1_at down 24 2.1 0.004 unknown TaAffx.23875.1.S1_at down 24 2.4 0.011 unknown TaAffx.50737.1.S1_at down 24 2.0 0.047 Transcribed locus, weakly similar to NP_178396.1 protein binding / ubiquitin- protein ligase/ zinc ion binding [Arabidopsis thaliana] TaAffx.56516.1.S1_at down 24 2.5 0.049 F-box containing protein transport inhibitor response TIR1-like [Oryza sativa (japonica cultivar-group)] TaAffx.59491.1.S1_at down 24 2.2 0.005 T.aestivum atp-2 mRNA for ATP synthase beta subunit TaAffx.82948.1.S1_at down 24 2.5 0.021 unknown 218 Probe Set ID regulation hai FD p-val Gene Symbol Gene Title TaAffx.86251.1.S1_at down 24 2.0 0.018 Oryza sativa (japonica cultivar-group) cDNA clone:J023087L18, full insert sequence Ta.10200.1.A1_x_at up 3 2.1 0.027 Xylem serine proteinase 1 precursor (AtXSP1) (Cucumisin-like protein) Ta.1614.1.S1_at up 3 2.1 0.000 unknown Ta.22966.3.S1_at up 3 2.0 0.001 F1F0-ATPase inhibitor protein [Oryza sativa (japonica cultivar-group)] (contig annotation) Ta.4470.1.S1_at up 3 2.3 0.049 similar to ethylene-binding protein-like [Oryza sativa (japonica cultivar-group)] / AP2 domain- containing transcription factor-like [Oryza sativa (japonica cultivar-group)] Ta.6173.3.S1_at up 3 2.2 0.004 hypersensitive-induced reaction protein 4 [Hordeum vulgare subsp. vulgare] TaAffx.111915.1.S1_at up 3 2.3 0.012 unknown TaAffx.128541.69.S1_at up 3 7.4 0.046 TaAffx.25679.1.S1_at up 3 2.3 0.015 unknown TaAffx.35350.1.S1_at up 3 2.2 0.001 ribosomal protein S4 [Panax ginseng] TaAffx.56177.1.S1_at up 3 2.0 0.020 hypothetical protein [Oryza sativa (japonica cultivar-group)] TaAffx.6790.2.S1_at up 3 2.0 0.000 Triticum monococcum BAC clone 453N11, complete sequence TaAffx.77918.1.S1_at up 3 2.2 0.005 Hordeum vulgare Ty3/gypsy retrotransposon cereba gag-pol polyprotein gene, partial cds TaAffx.82114.1.S1_at up 3 2.0 0.001 unknown TaAffx.83405.1.S1_at up 3 2.1 0.001 unknown Ta.16143.1.A1_at up 8 2.1 0.009 Transcribed locus, weakly similar to NP_914180.1 P0475H04.16 [Oryza sativa (japonica cultivar-group)] Ta.20297.1.S1_at up 8 2.1 0.017 unknown Ta.22172.1.S1_x_at up 8 2.0 0.035 HGWP repeat containing protein-like [Oryza sativa (japonica cultivar-group)] 219 Probe Set ID regulation hai FD p-val Gene Symbol Gene Title Ta.28224.2.S1_x_at up 8 2.1 0.008 Transcribed locus, weakly similar to NP_918838.1 OSJNBb0093M23.11 [Oryza sativa (japonica cultivar-group)] Ta.6578.2.S1_x_at up 8 2.6 0.008 transaldolase ToTAL2 [Oryza sativa (japonica cultivar-group)] Ta.8071.1.A1_at up 8 2.0 0.011 Oryza sativa chromosome 3 BAC OSJNBa0052F07 genomic sequence, complete sequence TaAffx.105364.1.S1_at up 8 2.0 0.011 unknown TaAffx.111861.1.S1_x_at up 8 2.5 0.001 Ty3/gypsy-like retrotransposon [Triticum aestivum] TaAffx.113624.2.S1_at up 8 2.3 0.006 Serine/threonine-protein kinase BRI1-like 3 precursor (BRASSINOSTEROID INSENSITIVE 1-like protein 3) TaAffx.128541.59.A1_at up 8 4.9 0.025 transposon [Triticum aestivum] TaAffx.218.1.S1_at up 8 2.2 0.007 unknown TaAffx.25602.1.S1_s_at up 8 2.1 0.042 tandem repeat sequence specific for chromosome arm 4AS [Triticum monococcum] TaAffx.29302.1.S1_at up 8 2.0 0.013 retrotransposon protein, putative, Ty1-copia sub-class [Oryza sativa (japonica cultivar- group)] TaAffx.30272.3.A1_at up 8 2.0 0.040 unknown TaAffx.51102.1.S1_at up 8 2.0 0.002 unknown TaAffx.58772.1.S1_at up 8 2.2 0.001 12-oxophytodienoate reductase 3 (12- oxophytodienoate-10,11-reductase 3) (OPDA- reductase 3) (LeOPR3) TaAffx.59356.1.S1_at up 8 2.1 0.001 Aegilops tauschii TaAffx.65484.1.A1_at up 8 2.0 0.001 calcineurin B-like protein 8 (CBL8) [Oryza sativa (japonica cultivar-group)] (contig annotation) TaAffx.81381.1.S1_at up 8 2.0 0.047 unknown TaAffx.83019.1.S1_at up 8 3.2 0.027 Inorganic diphosphatase Ta.22981.3.S1_a_at up 24 2.4 0.006 cytochrome P450 protein [Arabidopsis thaliana] 220 Probe Set ID regulation hai FD p-val Gene Symbol Gene Title Ta.3420.2.S1_at up 24 2.2 0.006 similar to acyl-CoA oxidase ACX3 [Arabidopsis thaliana] TaAffx.12576.1.A1_at up 24 2.1 0.005 unknown TaAffx.4083.5.A1_x_at up 24 2.5 0.000 similar to vacuolar sorting receptor protein homolog PV72 [Cucurbita cv. Kurokawa Amakuri] TaAffx.57405.1.S1_x_at up 24 2.4 0.000 similar to tonoplast membrane integral protein [Oryza sativa (japonica cultivar-group)] (contig annotation) TaAffx.71003.1.S1_at up 24 2.3 0.007 unknown DON vs water Ta.10532.1.A1_s_at down 3 2.9 0.005 LOC778394 pore-forming toxin-like protein Hfr-2 Ta.12088.1.S1_at down 3 2.2 0.030 unknown Ta.12470.1.A1_at down 3 2.2 0.022 unknown Ta.13423.1.S1_at down 3 2.0 0.034 unknown Ta.14129.3.S1_x_at down 3 2.1 0.035 unknown Ta.19355.1.S1_at down 3 2.0 0.010 unknown Ta.20147.2.S1_at down 3 2.4 0.003 unknown Ta.20205.1.A1_s_at down 3 2.3 0.014 ver2 ver2 protein Ta.2278.2.S1_a_at down 3 2.0 0.022 LOC542963 Cyc07 Ta.27268.1.S1_at down 3 2.1 0.025 Clone wl1.pk0012.d7:fis, full insert mRNA sequence Ta.27725.1.S1_at down 3 2.0 0.024 wpi6 plasma membrane protein Ta.28171.1.S1_at down 3 2.3 0.045 Clone wdk2c.pk011.j13:fis, full insert mRNA sequence Ta.526.1.S1_x_at down 3 2.8 0.010 Lipoxygenase (contig annotation) Ta.7022.1.S1_at down 3 2.1 0.026 Phenylalanine ammonia-lyase Ta.7022.1.S1_x_at down 3 2.3 0.016 Phenylalanine ammonia-lyase Ta.9361.2.S1_x_at down 3 2.0 0.004 Transcribed locus, moderately similar to NP_914565.1 putative zinc finger transcription factor [Oryza sativa (japonica cultivar-group)] 221 Probe Set ID regulation hai FD p-val Gene Symbol Gene Title Ta.9590.1.S1_at down 3 2.4 0.037 unknown TaAffx.105521.1.S1_at down 3 2.4 0.020 pore-forming toxin-like protein Hfr-2 [Triticum aestivum] TaAffx.107003.1.S1_at down 3 2.0 0.016 similar to 50S ribosomal protein L4, chloroplast (CL4) [Arabidopsis thaliana] TaAffx.112290.2.S1_at down 3 2.4 0.024 putative acid phosphatase [Hordeum vulgare subsp. vulgare] gi|41529149|emb|CAB71336.2| putative acid phosphatase [Hordeum vulgare subsp. vulgare] TaAffx.128418.38.S1_at down 3 2.0 0.000 18S Soybean (Glycine max) 18S ribosomal RNA TaAffx.29617.1.S1_at down 3 2.0 0.014 Zea mays PCO130571 mRNA sequence TaAffx.52983.1.S1_at down 3 2.1 0.027 unknown TaAffx.55998.1.S1_at down 3 2.1 0.012 pentatricopeptide (PPR) repeat-containing protein-like [Oryza sativa (japonica cultivar- group)] TaAffx.61713.1.S1_at down 3 2.1 0.020 Hypothetical protein C13C5.04 in chromosome I TaAffx.6797.2.S1_at down 3 2.2 0.022 putative 41 kD chloroplast nucleoid DNA binding protein (CND41) [Oryza sativa (japonica cultivar-group)] gi|51964318|ref|XP_506944.1| PREDICTED OJ1008_D06.19 gene product [Oryza sativa (japonica TaAffx.86830.1.S1_at down 3 2.0 0.037 OSJNBb0049O23.17 [Oryza sativa (japonica cultivar-group)] gi|15528730|dbj|BAB64676.1| P0697C12.11 [Oryza sativa (japonica cultivar- group)] gi|20161129|dbj|BAB90058.1| OSJNBb0049O23.17 [Oryza sativa (japonica cu TaAffx.9179.1.S1_at down 3 2.1 0.029 unknown 222 Probe Set ID regulation hai FD p-val Gene Symbol Gene Title Ta.10234.2.S1_x_at down 8 2.1 0.009 Transcribed locus, strongly similar to XP_001076100.1 PREDICTED: similar to Actin, cytoplasmic 2 (Gamma-actin) [Rattus norvegicus] Ta.10915.2.S1_at down 8 2.1 0.028 unknown Ta.11540.1.A1_x_at down 8 2.1 0.030 similar to prenylated Rab acceptor protein 1 [Oryza sativa (indica cultivar-group)] Ta.12057.1.S1_at down 8 2.3 0.003 unknown Ta.14461.3.S1_x_at down 8 2.6 0.024 Nectarin 1 precursor (Superoxide dismutase [Mn]) Ta.15782.1.S1_at down 8 2.1 0.046 putative GAMYB-binding protein [Oryza sativa (japonica cultivar-group)] gi|55773651|dbj|BAD72190.1| putative GAMYB-binding protein [Oryza sativa (japonica Ta.18832.2.S1_a_at down 8 2.1 0.002 unknown Ta.18981.1.S1_at down 8 3.3 0.037 putative laccase [Oryza sativa (japonica cultivar-group)] gi|19571025|dbj|BAB86452.1| putative laccase [Oryza sativa (japonica cultivar-group)] Ta.2188.3.S1_at down 8 2.5 0.036 unknown Ta.26475.1.A1_at down 8 2.0 0.001 similar to rhomboid protein-related [Arabidopsis thaliana] (contig annotation) Ta.27021.1.A1_at down 8 2.2 0.017 P0034A04.28 [Oryza sativa (japonica cultivar- group)] gi|29837187|dbj|BAC75569.1| leaf senescence related protein-like [Oryza sativa (japonica cultivar-group)] Ta.28351.1.S1_at down 8 3.0 0.038 LOC542989 adenosine diphosphate glucose pyrophosphatase Ta.28351.1.S1_x_at down 8 3.5 0.022 LOC542989 adenosine diphosphate glucose pyrophosphatase Ta.28669.3.S1_at down 8 2.4 0.016 protein phosphatase 2C [Arabidopsis thaliana] 223 Probe Set ID regulation hai FD p-val Gene Symbol Gene Title Ta.2929.2.S1_at down 8 2.1 0.005 DEOXYURIDINE 5'-TRIPHOSPHATE NUCLEOTIDOHYDROLASE (DUTPASE) (DUTP PYROPHOSPHATASE) (P18) Ta.29496.2.S1_at down 8 2.2 0.037 Peroxidase 12 precursor (Atperox P12) (PRXR6) (ATP4a) Ta.29587.3.A1_at down 8 2.2 0.044 LOC543465 Ribulosebisphosphate carboxylase small subunit Ta.29895.2.A1_at down 8 2.1 0.036 Probable phosphomannomutase (PMM) Ta.3249.3.A1_at down 8 3.1 0.014 chlorophyll a/b binding protein [Oryza sativa (japonica cultivar-group)] Ta.3909.2.A1_at down 8 2.1 0.009 unknown protein [Oryza sativa (japonica cultivar-group)] Ta.4147.1.S1_at down 8 3.1 0.045 MtN19 [Oryza sativa (japonica cultivar-group)] (contig annotation) Ta.4245.2.S1_x_at down 8 2.2 0.004 terbinafine resistance locus protein [Oryza sativa (japonica cultivar-group)] TaAffx.104550.2.S1_at down 8 2.4 0.023 PREDICTED P0443H10.4 gene product [Oryza sativa (japonica cultivar-group)] gi|50936599|ref|XP_477827.1| putative CDPK substrate protein 1 [Oryza sativa (japonica TaAffx.105412.1.S1_at down 8 2.3 0.000 unknown protein [Oryza sativa (japonica cultivar-group)] TaAffx.107244.2.S1_at down 8 2.6 0.002 hydrolase, alpha/beta fold protein-like [Oryza sativa (japonica cultivar-group)] TaAffx.110801.1.S1_at down 8 2.5 0.003 unknown TaAffx.117382.1.S1_at down 8 2.2 0.002 Transcribed locus, weakly similar to NP_173021.1 AVP1; ATPase [Arabidopsis thaliana] TaAffx.117651.2.S1_at down 8 2.3 0.005 PREDICTED OJ1442_E05.19 gene product [Oryza sativa (japonica cultivar-group)] gi|50904779|ref|XP_463878.1| putative bHLH protein [Oryza sativa (japonica cultivar-group)] TaAffx.118866.1.S1_at down 8 2.2 0.034 unknown 224 Probe Set ID regulation hai FD p-val Gene Symbol Gene Title TaAffx.119097.1.S1_at down 8 2.2 0.008 Oryza sativa (japonica cultivar-group) chromosome 5 clone OSJNOa0048I04, complete sequence TaAffx.119225.1.S1_s_at down 8 2.4 0.024 Major Facilitator Superfamily, putative [Oryza sativa (japonica cultivar-group)] TaAffx.12424.2.S1_at down 8 2.2 0.035 mitochondrial transcription termination factor- like [Oryza sativa (japonica cultivar-group)] TaAffx.128418.24.S1_at down 8 2.6 0.030 28S ribosomal RNA [Triticum aestivum] TaAffx.128682.1.S1_at down 8 2.0 0.001 endonuclease [Hordeum vulgare subsp. vulgare] TaAffx.128862.4.S1_at down 8 2.1 0.012 similar to senescence-associated protein-like [Oryza sativa (japonica cultivar-group)] TaAffx.24121.1.S1_at down 8 2.3 0.013 unknown TaAffx.24125.1.S1_at down 8 2.0 0.012 unknown TaAffx.25572.1.S1_at down 8 2.2 0.000 calmodulin-binding protein -like [Oryza sativa (japonica cultivar-group)] TaAffx.27404.1.S1_at down 8 2.1 0.022 unknown TaAffx.28135.1.S1_at down 8 2.0 0.004 hydrolase-like [Oryza sativa (japonica cultivar- group)] (contig annotation) TaAffx.28815.1.S1_at down 8 2.1 0.049 unknown protein [Oryza sativa (japonica cultivar-group)] gi|33146668|dbj|BAC80014.1| unknown protein [Oryza sativa (japonica cultivar-group)] TaAffx.32506.1.S1_at down 8 2.3 0.025 unknown TaAffx.38460.1.S1_s_at down 8 2.6 0.026 similar to (contig annotation) lectin [Hordeum vulgare subsp. vulgare] TaAffx.42438.1.A1_at down 8 2.2 0.045 unknown TaAffx.52929.1.S1_at down 8 2.5 0.007 unknown TaAffx.56300.1.S1_at down 8 2.0 0.000 unknown TaAffx.62625.1.S1_at down 8 2.1 0.010 unknown TaAffx.66676.2.S1_at down 8 2.0 0.032 similar to reverse transcriptase [Oryza sativa (japonica cultivar-group)] 225 Probe Set ID regulation hai FD p-val Gene Symbol Gene Title TaAffx.71746.1.A1_at down 8 2.2 0.029 PHOTOSYSTEM II 10 KD POLYPEPTIDE PRECURSOR TaAffx.77811.1.S1_at down 8 2.1 0.001 unknown TaAffx.79035.1.S1_at down 8 2.0 0.002 Hydroquinone glucosyltransferase [Oryza sativa (japonica cultivar-group)] TaAffx.86355.1.S1_at down 8 2.0 0.036 Hypothetical 171.1 kDa protein in RPL6A- DAK1 intergenic region TaAffx.93223.1.A1_at down 8 2.1 0.025 1-aminocyclopropane-1-carboxylate oxidase (ACC oxidase) (Ethylene-forming enzyme) (EFE) Ta.10087.1.A1_at down 24 2.3 0.025 unknown Ta.10354.2.S1_x_at down 24 2.1 0.026 Pyrophosphate--fructose 6-phosphate 1- phosphotransferase alpha subunit (PFP) (6- phosphofructokinase, pyrophosphate dependent) Ta.10781.1.A1_at down 24 4.1 0.015 60S acidic ribosomal protein P0 (DNA- (apurinic or apyrimidinic site) lyase) (animal contaminant) Ta.11005.1.S1_at down 24 2.1 0.048 myosin 2 light chain [Lonomia obliqua] (animal contaminant) (contig annotation) Ta.11242.1.A1_at down 24 2.1 0.011 unknown Ta.11332.1.A1_at down 24 2.6 0.011 P0421H07.26 [Oryza sativa (japonica cultivar- group)] gi|20804607|dbj|BAB92298.1| P0421H07.26 [Oryza sativa (japonica cultivar- group)] gi|13872913|dbj|BAB44019.1| P0684B02.6 [Oryza sativa (japonica cultivar- grou Ta.1166.2.S1_a_at down 24 2.1 0.015 Fructose-bisphosphatase Ta.1197.1.S1_at down 24 2.2 0.046 LOC542862 alpha 1,4-glucan phosphorylase Ta.12319.1.A1_at down 24 4.7 0.041 ribosomal protein S5 (animal contaminant) Ta.14235.2.S1_x_at down 24 2.7 0.025 unknown Ta.1619.3.A1_x_at down 24 2.0 0.001 leucine-rich repeat-like protein [Oryza sativa (japonica cultivar-group)] 226 Probe Set ID regulation hai FD p-val Gene Symbol Gene Title Ta.16225.1.A1_at down 24 2.0 0.017 similar to C4-dicarboxylate transporter/malic acid transport family protein [Arabidopsis thaliana] (contig annotation) Ta.16547.1.S1_x_at down 24 2.0 0.040 unknown Ta.16611.1.S1_at down 24 2.3 0.033 unknown Ta.1876.1.S1_s_at down 24 2.1 0.036 cold shock protein [Erwinia carotovora subsp. atroseptica SCRI1043] Ta.19172.1.S1_at down 24 2.1 0.005 unknown Ta.20218.1.A1_x_at down 24 4.2 0.045 Triticum aestivum clone wrsu1.pk0006.a3:fis, full insert mRNA sequence Ta.20528.1.A1_at down 24 2.1 0.017 Dihydrolipoyl dehydrogenase Ta.21394.2.A1_x_at down 24 2.1 0.037 putative protodermal factor [Oryza sativa (japonica cultivar-group)] gi|49388954|dbj|BAD26174.1| putative protodermal factor [Oryza sativa (japonica Ta.24110.1.A1_s_at down 24 2.4 0.027 putative proline-rich protein [Oryza sativa (japonica cultivar-group)] gi|14488319|gb|AAK63900.1| Putative proline- rich protein [Oryza sativa] Ta.25836.1.S1_at down 24 2.1 0.034 drought-induced protein RDI [Oryza sativa Japonica Group] Ta.2638.1.S1_at down 24 2.4 0.045 rab 15B rab protein Ta.26970.1.A1_at down 24 2.0 0.049 Leucoanthocyanidin reductase (LAR) (BANYULS) (Anthocyanin spotted testa) (ast) Ta.27983.2.S1_a_at down 24 2.5 0.048 putative potyviral helper component protease- interacting protein 2 [Oryza sativa (japonica cultivar-group)] gi|46390299|dbj|BAD15748.1| putative potyviral helper component protease- interacting protein 2 Ta.28063.1.S1_x_at down 24 2.1 0.028 unknown Ta.28496.1.A1_x_at down 24 2.4 0.039 chlorophyll a/b binding protein Ta.30702.1.S1_x_at down 24 2.5 0.027 chlorophyll a/b-binding protein WCAB precursor [Triticum aestivum] Ta.3249.1.S1_at down 24 2.8 0.038 LOC542973 Thioredoxin M 227 Probe Set ID regulation hai FD p-val Gene Symbol Gene Title Ta.3249.2.S1_x_at down 24 2.0 0.014 LOC542973 Thioredoxin M Ta.3249.3.A1_at down 24 2.6 0.021 chlorophyll a/b binding protein [Oryza sativa (japonica cultivar-group)] Ta.3748.1.A1_at down 24 2.2 0.012 HEXOKINASE 1 Ta.3795.1.S1_x_at down 24 3.4 0.027 Wcab chlorophyll a/b-binding protein WCAB precursor Ta.4057.1.S1_at down 24 2.2 0.040 unknown Ta.4280.2.S1_at down 24 2.0 0.034 Oryza sativa (japonica cultivar-group) cDNA clone:J023012A09, full insert sequence Ta.4889.1.S1_at down 24 2.0 0.037 WPI2 PISTILLATA-like MADS box protein Ta.4940.1.A1_at down 24 2.5 0.000 unknown Ta.4972.1.A1_at down 24 2.1 0.046 similar to aspartate kinase-homoserine dehydrogenase [Oryza sativa (japonica cultivar-group)] (contig annotation) Ta.5004.1.S1_at down 24 2.9 0.036 Vignain precursor (Cysteine endopeptidase) Ta.6397.1.A1_at down 24 2.2 0.002 Adenosylmethionine decarboxylase Ta.7839.1.A1_at down 24 2.0 0.005 Probable xyloglucan endotransglucosylase/hydrolase protein 27 precursor (At-XTH27) (XTH-27) Ta.7907.1.A1_at down 24 2.2 0.026 putative transthyretin, having alternative splicing products [Oryza sativa (japonica cultivar-group)] Ta.9799.1.S1_at down 24 2.1 0.001 GAMMA-GLIADIN B PRECURSOR TaAffx.105964.1.S1_at down 24 2.2 0.005 unknown TaAffx.114127.1.S1_x_at down 24 3.1 0.040 CHLOROPHYLL A-B BINDING PROTEIN PRECURSOR (LHCII TYPE I CAB) (LHCP) TaAffx.12303.2.S1_at down 24 2.3 0.037 unknown TaAffx.12523.1.A1_at down 24 2.6 0.036 APETALA3-like MADS box protein [Triticum aestivum] TaAffx.23875.1.S1_at down 24 2.2 0.010 unknown TaAffx.29871.1.S1_at down 24 2.3 0.043 unknown 228 Probe Set ID regulation hai FD p-val Gene Symbol Gene Title TaAffx.32266.1.A1_at down 24 2.7 0.047 Probable phospholipid hydroperoxide glutathione peroxidase (PHGPx) (Salt- associated protein) TaAffx.37096.2.S1_at down 24 2.0 0.008 gene_id:MVI11.8~unknown protein [Arabidopsis thaliana] TaAffx.43723.1.A1_at down 24 2.4 0.017 Betaine-aldehyde dehydrogenase, chloroplast precursor (BADH) TaAffx.50369.1.S1_at down 24 2.2 0.000 unknown TaAffx.59876.1.S1_at down 24 3.1 0.041 unknown TaAffx.65101.2.S1_at down 24 2.1 0.005 OSJNBa0089N06.20 [Oryza sativa (japonica cultivar-group)] gi|39546250|emb|CAE04259.3| OSJNBa0089N06.20 [Oryza sativa (japonica cultivar-group)] TaAffx.79227.1.S1_at down 24 5.1 0.034 unknown TaAffx.80666.1.S1_x_at down 24 2.1 0.001 unknown TaAffx.80911.1.S1_at down 24 2.2 0.034 unknown TaAffx.8262.1.S1_x_at down 24 2.2 0.006 glutamyl-tRNA reductase [Hordeum vulgare] (poor sequence quality) Ta.14946.1.S1_at up 24 5.8 0.010 Transcribed locus, weakly similar to NP_566426.1 ATHCHIB (BASIC CHITINASE); chitinase [Arabidopsis thaliana] Ta.21120.1.S1_at up 24 3.2 0.001 Glucan endo-1,3-beta-D-glucosidase (contig annotation) Ta.21307.1.S1_x_at up 24 2.3 0.016 peroxidase [Oryza sativa (japonica cultivar- group)] Ta.21353.1.S1_a_at up 24 2.0 0.005 Transcribed locus, weakly similar to NP_179942.1 catalytic/ hydrolase, acting on ester bonds [Arabidopsis thaliana] Ta.21353.1.S1_at up 24 2.2 0.017 Transcribed locus, weakly similar to NP_179942.1 catalytic/ hydrolase, acting on ester bonds [Arabidopsis thaliana] 229 Probe Set ID regulation hai FD p-val Gene Symbol Gene Title Ta.21650.1.A1_at up 24 2.2 0.001 Transcribed locus, weakly similar to NP_921072.1 putative disease resistance gene [Oryza sativa (japonica cultivar-group)] Ta.22562.1.S1_at up 24 2.5 0.002 Glucan endo-1,3-beta-D-glucosidase Ta.22981.3.S1_a_at up 24 2.0 0.006 cytochrome P450 protein [Arabidopsis thaliana] Ta.24501.1.S1_at up 24 2.5 0.030 LOC543292 thaumatin-like protein Ta.24715.1.S1_at up 24 2.0 0.037 pox3 peroxidase Ta.27757.1.S1_at up 24 2.8 0.034 similar to (contig annotation) phosphoglycerate mutase family, putative [Oryza sativa (japonica cultivar-group)] Ta.27762.1.S1_x_at up 24 2.8 0.029 Ta-TLP thaumatin-like protein Ta.27882.1.S1_s_at up 24 2.2 0.049 unknown Ta.28354.3.S1_x_at up 24 2.3 0.005 gstu3 Glutathione transferase Ta.82.1.S1_at up 24 4.1 0.007 LOC543287 peroxidase Ta.8304.1.S1_x_at up 24 2.1 0.010 pathogenesis-related PR1a [Triticum monococcum] Ta.97.2.S1_x_at up 24 2.8 0.034 pathogen-induced protein WIR1A [Triticum aestivum] TaAffx.108743.2.S1_at up 24 2.1 0.005 beta-1,3-glucanase precursor [Triticum aestivum] TaAffx.110222.1.S1_x_at up 24 2.2 0.004 leucine-rich repeat-containing extracellular glycoprotein [Sorghum bicolor] / somatic embryogenesis receptor kinase SERK [Medicago truncatula] TaAffx.116570.1.S1_at up 24 2.2 0.050 PR4 Pathogenesis-related protein 4 TaAffx.131249.1.S1_at up 24 2.8 0.027 beta-1,3-glucanase [Oryza sativa (japonica cultivar-group)] TaAffx.131249.1.S1_s_at up 24 2.8 0.008 beta-1,3-glucanase [Oryza sativa (japonica cultivar-group)] TaAffx.28047.1.S1_at up 24 2.5 0.038 Sterol 14-demethylase / cytochrome P450 [Oryza sativa (japonica cultivar-group)] TaAffx.81099.1.S1_at up 24 4.2 0.041 Cycloartenol synthase 230 Probe Set ID regulation hai FD p-val Gene Symbol Gene Title Tri5+ vs Tri5- TaAffx.108604.1.S1_at down 3 2.3 0.037 unknown TaAffx.109291.1.S1_at down 3 2.6 0.006 Aegilops tauschii TaAffx.126278.1.S1_at down 3 2.2 0.031 unknown TaAffx.128510.10.S1_s_at down 3 2.0 0.043 OSJNBa0013K16.16 [Oryza sativa (japonica cultivar-group)] gi|38344284|emb|CAE03767.2| OSJNBa0013K16.16 [Oryza sativa (japonica cultivar-group)] TaAffx.95411.1.S1_at down 3 2.1 0.039 unknown Ta.14032.1.S1_at down 8 2.1 0.003 T-complex protein 1 subunit epsilon (TCP-1- epsilon) [Avena sativa] (contig annotation) TaAffx.5790.1.S1_at down 8 2.1 0.007 one helix protein [Deschampsia antarctica] (contig annotation) TaAffx.7349.1.S1_at down 8 2.1 0.005 immediate-early fungal elicitor protein CMPG1 [Oryza sativa (japonica cultivar-group)] TaAffx.78864.1.S1_at down 8 2.3 0.011 glutathione S-transferase [Oryza sativa (japonica cultivar-group)] TaAffx.82012.1.S1_at down 8 2.2 0.019 Genomic sequence for Oryza sativa, Nipponbare strain, clone OJ1113A07, from chromosome 3, complete sequence Ta.18870.1.S1_at down 24 2.3 0.014 Oryza sativa (japonica cultivar-group) cDNA clone:J023075G11, full insert sequence TaAffx.32362.1.S1_at down 24 2.2 0.019 similar to lipase acylhydrolase [Arabidopsis thaliana] TaAffx.81741.1.S1_at down 24 2.4 0.036 unknown Ta.3255.1.S1_at up 3 2.0 0.001 unknown TaAffx.54615.1.S1_at up 3 2.2 0.005 unknown Ta.12751.3.A1_at up 8 2.3 0.012 LOC606336 Ribosomal protein L13a Ta.16366.1.S1_at up 8 2.3 0.012 BTB and TAZ domain protein [Oryza sativa (japonica cultivar-group)] (contig annotation) Ta.21386.1.S1_at up 8 2.1 0.048 Probable nonspecific lipid-transfer protein 2 (LTP 2) 231 Probe Set ID regulation hai FD p-val Gene Symbol Gene Title TaAffx.128541.20.S1_at up 8 2.2 0.005 unknown TaAffx.129222.10.S1_at up 8 2.2 0.003 Triticum monococcum BAC clones 116F2 and 115G1 gene sequence TaAffx.53890.1.S1_at up 8 2.1 0.007 TaAffx.78352.1.S1_at up 8 2.0 0.000 OSJNBa0020P07.12 [Oryza sativa (japonica cultivar-group)] gi|38344869|emb|CAE01295.2| OSJNBa0020P07.12 [Oryza sativa (japonica cultivar-group)] TaAffx.80607.1.S1_at up 8 2.0 0.021 unknown Ta.10184.2.S1_at up 24 2.0 0.002 Pre-mRNA cleavage complex II protein Clp1 Ta.1562.3.S1_s_at up 24 2.9 0.016 2'-hydroxyisoflavone reductase (contig annotation) Ta.20980.2.S1_at up 24 2.2 0.012 PUTATIVE SERINE/THREONINE KINASE RECEPTOR PRECURSOR (S-RECEPTOR KINASE) (SRK) Ta.28879.3.S1_at up 24 2.1 0.044 protein phosphatase type 2C [Arabidopsis thaliana] (contig annotation) Ta.30400.1.A1_at up 24 2.2 0.027 bHLH protein-like [Oryza sativa (japonica cultivar-group)] gi|41052738|dbj|BAD07594.1| bHLH protein-like [Oryza sativa (japonica cultivar-group)] gi|41052625|dbj|BAD08134.1| bHLH protein-like [Oryza sativa (jap Ta.5235.1.S1_x_at up 24 2.1 0.003 prx Peroxidase precursor Ta.6987.2.S1_at up 24 2.2 0.025 putative AT hook-containing MAR binding protein [Oryza sativa (japonica cultivar-group)] gi|29824463|gb|AAP04178.1| putative AT hook-containing MAR binding protein [Oryza sativa Ta.8245.2.S1_at up 24 2.4 0.033 OSJNBa0060N03.12 [Oryza sativa (japonica cultivar-group)] gi|38567892|emb|CAE03647.2| OSJNBa0060N03.12 [Oryza sativa (japonica cultivar-group)] 232 Probe Set ID regulation hai FD p-val Gene Symbol Gene Title Ta.9983.1.S1_s_at up 24 2.1 0.031 DFR dihydroflavonol 4-reductase TaAffx.105995.1.S1_at up 24 2.0 0.014 TaAffx.110215.1.S1_x_at up 24 2.1 0.045 TaAffx.111546.1.S1_s_at up 24 2.4 0.028 unknown TaAffx.27956.1.S1_at up 24 2.4 0.004 TaAffx.50140.1.S1_at up 24 2.1 0.014 GRAS family transcription factor, putative [Oryza sativa (japonica cultivar-group)] (contig annotation) TaAffx.5139.1.S1_at up 24 2.1 0.027 unknown TaAffx.6827.1.S1_s_at up 24 2.1 0.026 TaAffx.79727.1.S1_at up 24 2.0 0.033 C2 domain-containing protein [Hordeum vulgare subsp. vulgare] TaAffx.81742.1.S1_at up 24 2.1 0.044 TaAffx.92097.1.S1_x_at up 24 2.1 0.049 unknown DH1 Tri5+ vs water Ta.11460.1.A1_at down 3 2.0 0.042 hypothetical protein LOC_Os11g14070 [Oryza sativa (japonica cultivar-group)] Ta.18959.1.S1_at down 3 2.2 0.009 DNA-binding protein-like [Oryza sativa (japonica cultivar-group)] gi|37806155|dbj|BAC99660.1| DNA-binding protein-like [Oryza sativa (japonica cultivar- group)] gi|29467557|dbj|BAC66727.1| DNA- binding protein-li Ta.19909.1.A1_at down 3 2.5 0.026 Putative MAP kinase activating protein C22orf5 Ta.2107.2.S1_at down 3 2.0 0.003 Aldehyde dehydrogenase family 7 member A1 (Antiquitin 1) (Matured fruit 60 kDa protein) (MF-60) Ta.27238.1.S1_at down 3 2.0 0.010 Clone wre1n.pk0020.d2:fis, full insert mRNA sequence Ta.5989.3.S1_x_at down 3 2.0 0.001 unknown 233 Probe Set ID regulation hai FD p-val Gene Symbol Gene Title TaAffx.119611.1.A1_at down 3 2.1 0.008 DEGREENING RELATED GENE DEE76 PROTEIN TaAffx.129134.2.S1_at down 3 2.3 0.046 a2b Glycosyltransferase TaAffx.20193.1.A1_at down 3 2.1 0.021 hypothetical protein [Arabidopsis thaliana] TaAffx.64399.1.S1_at down 3 2.8 0.003 similar to receptor-like kinase RHG1 [Glycine max] TaAffx.64857.1.S1_at down 3 2.2 0.022 unknown TaAffx.84282.1.S1_at down 3 2.4 0.000 CDPK-RELATED PROTEIN KINASE (PK421) Ta.10895.1.S1_at down 24 2.9 0.022 unknown Ta.12713.1.S1_at down 24 3.2 0.019 unknown Ta.12774.1.A1_at down 24 2.0 0.033 unknown Ta.14612.1.S1_at down 24 2.4 0.034 unknown Ta.22319.2.S1_a_at down 24 3.4 0.004 unknown Ta.28379.1.S1_x_at down 24 2.6 0.015 5a2 protein [Triticum aestivum] Ta.8258.1.S1_x_at down 24 2.5 0.040 type 2 non-specific lipid transfer protein precursor [Triticum aestivum] Ta.8258.2.S1_at down 24 2.5 0.042 type 2 non-specific lipid transfer protein [Triticum aestivum] TaAffx.113782.1.S1_at down 24 2.0 0.022 60S ribosomal protein L11-1 (L16A) TaAffx.12878.1.A1_at down 24 2.1 0.015 wall-associated kinase 3 [Triticum aestivum] TaAffx.65750.1.S1_at down 24 2.0 0.010 Transcribed locus, moderately similar to NP_908628.1 B1012D10.25 [Oryza sativa (japonica cultivar-group)] TaAffx.79682.1.S1_x_at down 24 2.3 0.011 HISTONE H2B.2 Ta.13682.1.A1_at up 3 2.1 0.010 progesterone 5-beta-reductase [Oryza sativa (japonica cultivar-group)] Ta.20940.2.S1_at up 3 2.9 0.001 Bowman-Birk type trypsin inhibitor (WTI) [Triticum aestivum] TaAffx.108604.1.S1_at up 3 2.1 0.036 unknown 234 Probe Set ID regulation hai FD p-val Gene Symbol Gene Title Ta.10185.2.S1_x_at up 8 2.5 0.037 P0018C10.44 [Oryza sativa (japonica cultivar- group)] gi|20161436|dbj|BAB90360.1| B1065E10.10 [Oryza sativa (japonica cultivar- group)] gi|21952826|dbj|BAC06242.1| P0018C10.44 [Oryza sativa (japonica cultivar- gro Ta.10465.1.S1_at up 8 2.0 0.049 unknown Ta.10574.1.S1_a_at up 8 3.1 0.008 Transcribed locus, moderately similar to XP_470664.1 Hypothetical protein [Oryza sativa (japonica cultivar-group)] Ta.10729.3.S1_x_at up 8 2.3 0.049 unknown Ta.10918.1.S1_at up 8 2.4 0.000 unknown Ta.10990.1.A1_at up 8 2.0 0.019 40S ribosomal protein S19 (contig annotation) Ta.10993.1.S1_a_at up 8 2.1 0.047 unknown Ta.11358.2.A1_x_at up 8 2.0 0.016 nuclear transport factor 2 (NTF2)-like protein [Oryza sativa (japonica cultivar-group)] Ta.11360.1.A1_at up 8 2.1 0.049 putative 2,4-dihydroxydec-2-ene-1,10-dioic acid aldolase [Oryza sativa (japonica cultivar- group)] Ta.11584.3.S1_x_at up 8 2.6 0.004 unknown Ta.12060.2.S1_at up 8 2.5 0.030 putative agenet domain-containing protein [Oryza sativa (japonica cultivar-group)] gi|51536260|dbj|BAD38428.1| putative agenet domain-containing protein [Oryza sativa (japonica Ta.12176.1.S1_at up 8 2.1 0.034 unknown Ta.1291.1.A1_x_at up 8 2.2 0.049 Transcribed locus, weakly similar to NP_191286.1 BG1 (BETA-1,3-GLUCANASE 1); hydrolase, hydrolyzing O-glycosyl compounds [Arabidopsis thaliana] Ta.13468.1.S1_x_at up 8 2.5 0.030 unknown Ta.1357.2.A1_at up 8 2.0 0.031 protein kinase [Oryza sativa (japonica cultivar- group)] 235 Probe Set ID regulation hai FD p-val Gene Symbol Gene Title Ta.13754.1.S1_s_at up 8 2.6 0.043 similar to lipid transfer protein-related [Arabidopsis thaliana] Ta.13824.1.S1_x_at up 8 3.9 0.042 unknown Ta.13829.1.S1_at up 8 2.8 0.047 H.vulgare mRNA (clone NUC1) Ta.13835.1.S1_at up 8 2.7 0.038 unknown Ta.13838.1.S1_at up 8 2.5 0.002 similar to leucine-rich repeat protein [Oryza sativa] (contig annotation) Ta.13926.1.S1_a_at up 8 2.0 0.030 hypothetical protein [Oryza sativa (japonica cultivar-group)] gi|24899450|gb|AAN65020.1| hypothetical protein [Oryza sativa (japonica cultivar-group)] Ta.14281.1.S1_at up 8 2.4 0.014 Tad1 defensin Ta.1435.1.S1_at up 8 2.2 0.047 Clone wle1n.pk0039.d2:fis, full insert mRNA sequence Ta.14580.1.S1_at up 8 2.2 0.012 PEROXIDASE PRECURSOR Ta.15095.1.S1_at up 8 2.4 0.028 unknown Ta.16011.1.S1_at up 8 2.1 0.044 unknown Ta.16120.1.A1_at up 8 2.1 0.009 hypothetical protein [Oryza sativa (japonica cultivar-group)] gi|13236665|gb|AAK16187.1| hypothetical protein [Oryza sativa (japonica cultivar-group)] Ta.16270.1.S1_at up 8 2.0 0.009 unknown Ta.16901.1.S1_at up 8 2.1 0.018 NOD26-like membrane integral protein ZmNIP2-1 [Zea mays] Ta.18152.1.S1_at up 8 2.2 0.028 unknown Ta.18223.2.S1_at up 8 2.2 0.028 Anter-specific proline-rich protein APG precursor Ta.18225.1.A1_at up 8 2.8 0.042 alpha-L-arabinofuranosidase/beta-D- xylosidase Ta.18438.1.S1_at up 8 2.0 0.009 putative ATP-dependent Clp protease proteolytic subunit [Oryza sativa (japonica cultivar-group)] 236 Probe Set ID regulation hai FD p-val Gene Symbol Gene Title Ta.18665.1.S1_at up 8 2.0 0.010 putative protein kinase G11A [Oryza sativa (japonica cultivar-group)] gi|55296796|dbj|BAD68122.1| putative protein kinase G11A [Oryza sativa (japonica Ta.19173.1.S1_at up 8 2.3 0.033 unknown Ta.20971.1.S1_at up 8 2.2 0.050 nodulin-like protein 5NG4 [Oryza sativa (japonica cultivar-group)] (contig annotation) Ta.21285.1.A1_at up 8 2.7 0.015 Transcribed locus, weakly similar to XP_483682.1 putative auxin induced protein [Oryza sativa (japonica cultivar-group)] Ta.21327.3.A1_x_at up 8 2.1 0.045 PROTEIN TRANSPORT PROTEIN SEC61 BETA SUBUNIT Ta.21803.1.S1_at up 8 2.0 0.013 Zea mays PCO074907 mRNA sequence Ta.21906.1.S1_at up 8 2.4 0.019 Clone wdk2c.pk007.a4:fis, full insert mRNA sequence Ta.22046.1.A1_at up 8 2.0 0.022 2-oxoglutarate dehydrogenase E1 component (Alpha-ketoglutarate dehydrogenase) Ta.22525.3.S1_x_at up 8 2.3 0.026 proteinase inhibitor-related protein [Triticum aestivum] (contig annotation) Ta.22628.1.S1_at up 8 2.2 0.043 TaHSP70d HSP70 Ta.22694.1.A1_at up 8 2.3 0.048 unknown Ta.22766.1.S1_a_at up 8 2.3 0.042 unknown Ta.22825.2.S1_at up 8 2.0 0.014 similar to embryogenesis transmembrane protein-like [Oryza sativa (japonica cultivar- group)] Ta.22954.1.S1_a_at up 8 2.2 0.048 Triticum aestivum cultivar Renan clone BAC 930H14, complete sequence Ta.23392.2.S1_x_at up 8 2.3 0.010 similar to membrane protein [Oryza sativa (japonica cultivar-group)] Ta.24041.2.A1_x_at up 8 2.3 0.018 unknown Ta.2490.3.S1_at up 8 2.0 0.031 METHIONINE AMINOPEPTIDASE 2 (METAP 2) (PEPTIDASE M 2) (INITIATION FACTOR 2 ASSOCIATED 67 KDA GLYCOPROTEIN) (P67) 237 Probe Set ID regulation hai FD p-val Gene Symbol Gene Title Ta.25282.1.S1_x_at up 8 2.0 0.034 putative beta-glucuronidase precursor [Oryza sativa (japonica cultivar-group)] Ta.25470.1.A1_at up 8 2.6 0.019 Homeobox-leucine zipper protein ATHB-4 (HD-ZIP protein ATHB-4) Ta.26020.1.A1_at up 8 2.1 0.037 unknown Ta.26106.1.A1_at up 8 2.3 0.022 OSJNBa0036M16.17 [Oryza sativa (japonica cultivar-group)] gi|28071338|dbj|BAC56026.1| hypothetical protein [Oryza sativa (japonica cultivar-group)] Ta.26606.1.A1_at up 8 2.1 0.022 Zea mays CL672_1 mRNA sequence Ta.26923.1.S1_at up 8 2.1 0.048 unknown protein [Oryza sativa (japonica cultivar-group)] Ta.27039.1.S1_at up 8 2.1 0.040 Clone wr1.pk182.b10:fis, full insert mRNA sequence Ta.27766.1.S1_at up 8 2.8 0.046 similar to endosperm specific protein [Zea mays] Ta.2798.1.S1_x_at up 8 2.2 0.001 LOC543497 Em protein (AA 1-93) Ta.28132.1.S1_x_at up 8 2.0 0.005 Histone H2A Ta.2889.2.S1_a_at up 8 2.3 0.026 beta-1,3-glucanase [Oryza sativa (japonica cultivar-group)] Ta.2954.1.A1_at up 8 2.1 0.040 beta-expansin/allergen protein [Arabidopsis thaliana] (contig annotation) Ta.3035.2.S1_at up 8 2.2 0.018 RNA-binding protein [Oryza sativa (japonica cultivar-group)] (contig annotation) Ta.30535.1.S1_at up 8 2.1 0.015 Zea mays clone EL01N0559A08.c mRNA sequence Ta.30755.1.S1_at up 8 2.4 0.044 Nonspecific lipid-transfer protein 2G (LTP2G) (Lipid transfer protein 2 isoform 1) (LTP2-1) (7 kDa lipid transfer protein 1) Ta.3278.1.A1_x_at up 8 2.6 0.034 calreticulin precursor [Oryza sativa (japonica cultivar-group)] (contig annotation) 238 Probe Set ID regulation hai FD p-val Gene Symbol Gene Title Ta.3368.2.A1_at up 8 2.4 0.006 Polyadenylate-binding protein 2 (Poly(A)- binding protein 2) (PolyA binding protein II) (PABII) (Polyadenylate-binding nuclear protein 1) Ta.3605.3.S1_x_at up 8 2.2 0.038 P0470A12.42 [Oryza sativa (japonica cultivar- group)] gi|20161390|dbj|BAB90314.1| P0470A12.42 [Oryza sativa (japonica cultivar- group)] gi|20804983|dbj|BAB92659.1| P0004D12.3 [Oryza sativa (japonica cultivar- grou Ta.3631.2.S1_at up 8 2.2 0.035 PUTATIVE 3,4-DIHYDROXY-2-BUTANONE KINASE Ta.3663.1.A1_a_at up 8 2.4 0.037 OSJNBb0002J11.20 [Oryza sativa (japonica cultivar-group)] gi|38567850|emb|CAE05693.2| OSJNBb0002J11.20 [Oryza sativa (japonica cultivar-group)] gi|32489520|emb|CAE04723.1| OSJNBa0043L24.11 [Oryza sativa (japoni Ta.3744.1.S1_at up 8 2.1 0.011 unknown Ta.3788.2.A1_x_at up 8 2.5 0.025 Transcribed locus, weakly similar to NP_172176.1 ATTIC110/TIC110 [Arabidopsis thaliana] Ta.4245.2.S1_x_at up 8 2.5 0.011 terbinafine resistance locus protein [Oryza sativa (japonica cultivar-group)] Ta.46.1.A1_at up 8 2.4 0.042 LOC543224 imidazoleglycerolphosphate dehydratase Ta.4601.2.S1_at up 8 2.6 0.033 TaGlu1a beta-glucosidase Ta.5204.1.S1_at up 8 2.2 0.022 Transcribed locus, weakly similar to NP_200638.1 ATP binding / kinase/ protein serine/threonine kinase [Arabidopsis thaliana] Ta.5269.1.A1_at up 8 2.2 0.007 LOC606351 Beta-expansin TaEXPB1 Ta.5481.1.S1_at up 8 2.5 0.004 Glucosidase II beta subunit precursor (Protein kinase C substrate, 60.1 kDa protein, heavy chain) (PKCSH) (80K-H protein) (Vacuolar system 239 Probe Set ID regulation hai FD p-val Gene Symbol Gene Title Ta.556.1.S1_x_at up 8 2.8 0.045 Transcribed locus, weakly similar to XP_467967.1 lipase class 3-like [Oryza sativa (japonica cultivar-group)] Ta.5601.1.A1_at up 8 2.1 0.045 unknown Ta.6134.1.A1_at up 8 2.1 0.044 P0682B08.20 [Oryza sativa (japonica cultivar- group)] gi|14495231|dbj|BAB60950.1| P0682B08.20 [Oryza sativa (japonica cultivar- group)] Ta.6334.1.S1_at up 8 2.0 0.021 Oryza sativa (japonica cultivar-group) cDNA clone:001-102-H11, full insert sequence Ta.6642.2.S1_at up 8 2.3 0.012 wali1 Metallothionein-like protein Ta.6683.1.A1_x_at up 8 2.7 0.040 Mitogen-activated protein kinase kinase kinase 12 (Leucine-zipper protein kinase) (ZPK) (Dual leucine zipper bearing kinase) (DLK) Ta.673.1.S1_at up 8 2.4 0.006 OSJNBb0070J16.14 [Oryza sativa (japonica cultivar-group)] gi|32482945|emb|CAE02349.1| OSJNBb0072M01.10 [Oryza sativa (japonica cultivar-group)] gi|38345698|emb|CAE01918.2| OSJNBb0070J16.14 [Oryza sativa (japoni Ta.681.2.S1_at up 8 2.6 0.049 ferritin [Triticum monococcum] Ta.6896.1.A1_x_at up 8 2.4 0.042 unknown Ta.7129.2.S1_at up 8 2.2 0.007 Transcribed locus, weakly similar to XP_463207.1 putative auxin response factor [Oryza sativa (japonica cultivar-group)] Ta.7378.18.S1_x_at up 8 2.3 0.028 LOC543387 Alpha-tubulin Ta.7378.37.S1_at up 8 2.3 0.045 LOC543387 Alpha-tubulin Ta.7378.6.S1_at up 8 2.4 0.017 LOC543387 Alpha-tubulin Ta.7671.1.S1_at up 8 2.0 0.046 myosin-like protein [Oryza sativa (japonica cultivar-group)] gi|38175471|dbj|BAD01168.1| myosin-like protein [Oryza sativa (japonica cultivar-group)] 240 Probe Set ID regulation hai FD p-val Gene Symbol Gene Title Ta.7791.1.A1_at up 8 2.2 0.014 Hordeum vulgare BAC 184G9, complete sequece Ta.7835.3.S1_x_at up 8 2.2 0.019 unknown Ta.8448.1.A1_at up 8 2.1 0.005 plant viral-response family protein-like [Oryza sativa (japonica cultivar-group)] Ta.8726.1.S1_s_at up 8 2.4 0.044 1-phosphatidylinositol-4,5-bisphosphate phosphodiesterase (PLC) (Phosphoinositide phospholipase Ta.8834.1.S1_x_at up 8 2.2 0.050 P0470A12.42 [Oryza sativa (japonica cultivar- group)] gi|20161390|dbj|BAB90314.1| P0470A12.42 [Oryza sativa (japonica cultivar- group)] gi|20804983|dbj|BAB92659.1| P0004D12.3 [Oryza sativa (japonica cultivar- grou Ta.9117.3.A1_x_at up 8 2.4 0.045 unknown Ta.9220.3.S1_x_at up 8 2.1 0.003 Phenylalanine ammonia-lyase Ta.9347.2.A1_x_at up 8 2.2 0.014 membrane protein [Oryza sativa (japonica cultivar-group)] Ta.9347.3.S1_at up 8 2.0 0.040 putative membrane protein [Oryza sativa (japonica cultivar-group)] Ta.9401.3.S1_x_at up 8 2.1 0.049 B12D-like protein [Phaseolus vulgaris] Ta.9534.1.A1_at up 8 2.4 0.043 OSJNBb0017I01.27 [Oryza sativa (japonica cultivar-group)] gi|32488629|emb|CAE03422.1| OSJNBa0032F06.5 [Oryza sativa (japonica cultivar-group)] gi|32487413|emb|CAE05747.1| OSJNBb0017I01.27 [Oryza sativa (japonic TaAffx.105165.1.S1_at up 8 2.0 0.007 unknown TaAffx.105769.1.S1_at up 8 2.0 0.021 unknown TaAffx.107897.1.S1_at up 8 2.2 0.039 Chloroplast 30S ribosomal protein S18 TaAffx.107939.1.S1_at up 8 2.0 0.047 cytochrome P450 monooxygenase [Hordeum vulgare subsp. vulgare] TaAffx.108735.1.S1_at up 8 2.1 0.008 unknown 241 Probe Set ID regulation hai FD p-val Gene Symbol Gene Title TaAffx.108931.1.S1_at up 8 2.2 0.020 unknown TaAffx.110614.3.S1_at up 8 2.0 0.037 similar to Oxalate oxidase / germin TaAffx.111293.1.S1_at up 8 2.2 0.026 OSJNBa0061G20.3 [Oryza sativa (japonica cultivar-group)] TaAffx.111446.1.S1_at up 8 2.3 0.033 PDR-type ABC transporter-like [Oryza sativa (japonica cultivar-group)] TaAffx.113263.16.S1_at up 8 2.7 0.041 Corn histone H3 (H3C3) gene, complete cds TaAffx.114370.1.S1_at up 8 2.2 0.037 unknown TaAffx.118564.1.A1_at up 8 2.1 0.038 unknown TaAffx.119097.1.S1_at up 8 2.1 0.036 Oryza sativa (japonica cultivar-group) chromosome 5 clone OSJNOa0048I04, complete sequence TaAffx.12368.2.S1_at up 8 2.2 0.012 unknown TaAffx.124056.1.S1_at up 8 3.0 0.015 NADH dehydrogenase (ubiquinone) (intron left in?) TaAffx.128541.69.S1_at up 8 9.2 0.031 TaAffx.128576.1.S1_at up 8 2.0 0.019 ethylene-forming enzyme [Oryza sativa (japonica cultivar-group)] TaAffx.12867.1.S1_at up 8 2.7 0.020 Oryza sativa genomic DNA, chromosome 4, BAC clone: OSJNBa0073L04, complete sequence TaAffx.128686.3.A1_at up 8 2.1 0.009 Zea mays PCO123562 mRNA sequence TaAffx.128740.2.S1_s_at up 8 2.4 0.026 expb11 expansin EXPB11 protein precursor TaAffx.128795.21.S1_at up 8 2.7 0.032 Cytochrome b6-f complex subunit V (Cytochrome b6f complex subunit petG) TaAffx.128795.21.S1_s_at up 8 3.4 0.019 Cytochrome b6-f complex subunit V (Cytochrome b6f complex subunit petG) TaAffx.128828.1.A1_at up 8 2.4 0.030 Histone H2A TaAffx.129824.5.S1_x_at up 8 2.4 0.041 ribosomal protein S11 [Triticum aestivum] TaAffx.134856.1.S1_x_at up 8 2.6 0.015 Maturase K (Intron maturase) TaAffx.134856.3.S1_s_at up 8 3.2 0.012 maturase [Triticum aestivum] 242 Probe Set ID regulation hai FD p-val Gene Symbol Gene Title TaAffx.16988.1.A1_at up 8 2.2 0.012 putative Rieske iron-sulfur protein Tic55 [Oryza sativa (japonica cultivar-group)] gi|51964474|ref|XP_507022.1| PREDICTED OJ1249_F12.25 gene product [Oryza sativa (japonica TaAffx.18131.1.S1_at up 8 2.1 0.048 Putative phytosulfokine receptor precursor (Phytosulfokine LRR receptor kinase) TaAffx.20799.1.A1_at up 8 4.1 0.025 Fasciclin-like arabinogalactan protein 1 precursor TaAffx.21659.1.A1_at up 8 3.3 0.046 envelope membrane protein [Triticum aestivum] TaAffx.21983.2.S1_at up 8 2.1 0.025 unknown TaAffx.25228.1.S1_at up 8 2.7 0.036 unknown TaAffx.25375.1.S1_at up 8 2.1 0.023 putative H1 gene protein [Oryza sativa (japonica cultivar-group)] gi|53791799|dbj|BAD53744.1| putative H1 gene protein [Oryza sativa (japonica cultivar- group)] TaAffx.25602.1.S1_s_at up 8 3.3 0.003 tandem repeat sequence specific for chromosome arm 4AS [Triticum monococcum] TaAffx.25679.1.S1_at up 8 2.0 0.017 unknown TaAffx.25679.1.S1_x_at up 8 2.3 0.001 unknown TaAffx.29059.1.S1_x_at up 8 2.1 0.011 unknown TaAffx.29732.1.S1_at up 8 2.1 0.036 unknown TaAffx.30052.1.S1_at up 8 2.1 0.004 unknown TaAffx.31447.1.S1_at up 8 2.2 0.034 unknown TaAffx.31627.1.S1_at up 8 2.2 0.006 unknown TaAffx.3194.2.S1_at up 8 2.0 0.026 unknown TaAffx.3194.2.S1_x_at up 8 2.1 0.028 unknown TaAffx.37109.1.S1_at up 8 2.4 0.003 similar to receptor protein kinase [Arabidopsis thaliana] TaAffx.37517.1.A1_at up 8 2.0 0.004 unknown 243 Probe Set ID regulation hai FD p-val Gene Symbol Gene Title TaAffx.38852.1.S1_at up 8 2.7 0.037 SWIRM domain containing protein [Oryza sativa (japonica cultivar-group)] (contig annotation) TaAffx.39148.1.S1_x_at up 8 2.0 0.014 Transcription factor MYB86 (Myb-related protein 86) (AtMYB86) (Myb homolog 4) (AtMyb4) TaAffx.40608.1.A1_at up 8 2.3 0.044 unknown TaAffx.4142.1.S1_at up 8 2.3 0.026 rps16 TaAffx.4142.3.S1_at up 8 2.2 0.030 Triticum aestivum chloroplast DNA, complete genome TaAffx.4244.1.A1_at up 8 2.2 0.036 unknown TaAffx.43361.1.S1_at up 8 2.1 0.049 Triticum aestivum clone wkm2n.pk004.a19:fis, full insert mRNA sequence TaAffx.44362.1.A1_at up 8 2.0 0.037 Histone H3 TaAffx.48020.1.S1_x_at up 8 4.4 0.012 unknown TaAffx.5088.1.S1_at up 8 2.1 0.041 unknown TaAffx.52266.1.S1_at up 8 2.4 0.001 tuber-specific and sucrose-responsive element binding factor [Solanum tuberosum] TaAffx.52710.1.S1_at up 8 2.1 0.005 unknown TaAffx.52879.1.S1_at up 8 2.0 0.009 unknown TaAffx.53260.1.S1_at up 8 2.3 0.021 DNA-directed RNA polymerase alpha chain (PEP) (Plastid-encoded RNA polymerase alpha subunit) (RNA polymerase alpha subunit) TaAffx.54035.1.S1_at up 8 2.3 0.000 unknown protein [Arabidopsis thaliana] gi|9280642|gb|AAF86511.1| F21B7.6 [Arabidopsis thaliana] TaAffx.54203.1.S1_at up 8 2.1 0.000 unknown TaAffx.54368.1.S1_at up 8 2.0 0.029 Retinoid-inducible serine carboxypeptidase precursor (Serine carboxypeptidase 1) TaAffx.5526.1.S1_at up 8 3.3 0.013 unknown TaAffx.55413.1.S1_at up 8 2.3 0.003 unknown TaAffx.55587.1.S1_at up 8 2.8 0.017 unknown 244 Probe Set ID regulation hai FD p-val Gene Symbol Gene Title TaAffx.55812.1.S1_at up 8 2.1 0.013 Sugar carrier protein A TaAffx.56717.1.S1_x_at up 8 2.3 0.009 similar to integral membrane protein-like [Oryza sativa (japonica cultivar-group)] (contig annotation) TaAffx.57428.2.S1_at up 8 2.2 0.001 Aegilops tauschii strain TA1649 cosmid 69-7- 1 Lr21 gene, complete cds; and unknown gene TaAffx.57451.1.S1_at up 8 2.0 0.022 unknown TaAffx.5802.2.S1_s_at up 8 2.1 0.027 chloroplast rRNA-operon [Zea mays] TaAffx.58864.1.S1_x_at up 8 2.2 0.048 PEPTIDE TRANSPORTER PTR2-B (HISTIDINE TRANSPORTING PROTEIN) TaAffx.58892.1.S1_at up 8 2.1 0.004 unknown TaAffx.59265.1.S1_at up 8 2.9 0.038 unknown TaAffx.61558.1.S1_at up 8 2.3 0.034 alpha/beta hydrolase-like [Oryza sativa (japonica cultivar-group)] TaAffx.65868.1.A1_at up 8 2.0 0.010 unknown TaAffx.69933.4.A1_at up 8 2.1 0.005 Triticum monococcum BAC clone 453N11, complete sequence TaAffx.7031.1.S1_at up 8 2.2 0.046 NBS-type putative resistance protein [Triticum aestivum/Thinopyrum intermedium alien addition line] TaAffx.71647.1.A1_at up 8 2.3 0.039 unknown TaAffx.7336.1.S1_at up 8 2.1 0.044 similar to patatin-like protein [Oryza sativa (japonica cultivar-group)] (contig annotation) TaAffx.75901.1.S1_s_at up 8 2.2 0.021 unknown TaAffx.78382.1.S1_at up 8 2.3 0.009 unknown TaAffx.79634.1.S1_at up 8 2.2 0.026 Oryza sativa (japonica cultivar-group) cDNA clone:002-108-D06, full insert sequence 245 Probe Set ID regulation hai FD p-val Gene Symbol Gene Title TaAffx.80022.1.S1_at up 8 2.2 0.002 OSJNBa0083I11.14 [Oryza sativa (japonica cultivar-group)] gi|38346739|emb|CAE04304.2| OSJNBa0083I11.14 [Oryza sativa (japonica cultivar-group)] gi|32489422|emb|CAE03707.1| OSJNBa0021F22.1 [Oryza sativa (japonic TaAffx.80312.1.S1_x_at up 8 2.2 0.047 Triticum monococcum DV92 BAC 109N23, complete sequence TaAffx.83311.1.S1_x_at up 8 2.2 0.048 isoamylase [Aegilops tauschii] TaAffx.83543.2.S1_at up 8 2.1 0.047 LOC543460 SET domain protein TaAffx.83861.1.S1_at up 8 2.0 0.011 putative PrMC3 [Oryza sativa (japonica cultivar-group)] TaAffx.84198.1.S1_at up 8 2.1 0.015 Zinc transporter 9 (ZRT/IRT-like protein 9) TaAffx.84244.1.S1_at up 8 2.3 0.024 Triticum monococcum phosphatidylserine decarboxylase, ZCCT2, ZCCT1, and SNF2P genes, complete cds; nucellin pseudogene, complete sequence; putative TaAffx.84550.1.S1_at up 8 2.1 0.010 unknown TaAffx.86266.1.S1_at up 8 2.1 0.038 BETA-GLUCOSIDASE HOMOLOG PRECURSOR TaAffx.86601.1.S1_x_at up 8 2.1 0.017 unknown TaAffx.86919.1.S1_at up 8 2.5 0.000 unknown TaAffx.9121.1.S1_at up 8 2.3 0.036 unknown TaAffx.92061.1.S1_at up 8 2.1 0.027 unknown TaAffx.92741.1.A1_at up 8 2.2 0.024 unknown TaAffx.93296.1.S1_at up 8 2.1 0.005 Retrotransposable element Tf2 155 kDa protein type 2 TaAffx.97879.1.S1_at up 8 2.2 0.030 OSJNBa0011J08.2 [Oryza sativa (japonica cultivar-group)] Ta.27926.3.S1_a_at up 24 2.1 0.008 5'-3' exoribonuclease 2 (Dhm1 protein) Ta.28519.1.S1_s_at up 24 2.3 0.009 pectate lyase [Oryza sativa (japonica cultivar- group)] 246 Probe Set ID regulation hai FD p-val Gene Symbol Gene Title Ta.3868.2.S1_at up 24 2.3 0.038 putative sugar transporter [Arabidopsis thaliana] Ta.6567.3.A1_x_at up 24 2.3 0.007 fiber protein Fb2-like [Oryza sativa (japonica cultivar-group)] TaAffx.117285.1.S1_at up 24 2.1 0.020 similar to harpin-induced protein-related-like [Oryza sativa (japonica cultivar-group)] TaAffx.30005.1.S1_at up 24 2.0 0.017 unknown TaAffx.55987.1.S1_at up 24 2.1 0.025 DNA repair helicase XPB2 (XPB homolog 2) (ERCC3 homolog 2) (RAD25 homolog 2) (AtXPB2) TaAffx.56334.3.S1_s_at up 24 2.1 0.012 Probable mitochondrial processing peptidase alpha subunit 2, mitochondrial precursor (Alpha-MPP 2) TaAffx.65385.1.S1_at up 24 3.6 0.006 mucin-like protein [Oryza sativa (japonica cultivar-group)] TaAffx.84474.1.S1_at up 24 2.1 0.001 unknown Tri5- vs water Ta.11124.1.A1_at down 3 2.3 0.034 glucan endo-1,3-beta-D-glucosidase [Hordeum vulgare subsp. vulgare] (contig annotation) Ta.11412.1.A1_at down 3 2.2 0.049 putative Ap21 [Oryza sativa (japonica cultivar- group)] Ta.1155.1.S1_s_at down 3 2.3 0.011 unknown Ta.11598.1.A1_at down 3 2.0 0.008 signal peptide peptidase [Oryza sativa (japonica cultivar-group)] Ta.1196.1.A1_at down 3 2.0 0.008 unknown Ta.12238.1.A1_at down 3 2.0 0.011 unknown Ta.12810.1.S1_x_at down 3 2.3 0.043 quinolinate synthetase A-related [Arabidopsis thaliana] (contig annotation) Ta.16851.1.S1_at down 3 2.0 0.042 unknown Ta.18916.1.S1_x_at down 3 2.1 0.013 unknown Ta.19013.1.S1_at down 3 2.3 0.025 epoxide hydrolase [Oryza sativa (japonica cultivar-group)] 247 Probe Set ID regulation hai FD p-val Gene Symbol Gene Title Ta.1989.3.S1_s_at down 3 2.4 0.008 auxin-induced putative CP12 domain- containing protein [Arachis hypogaea] (contig annotation) Ta.20399.1.A1_at down 3 2.2 0.004 pectinesterase-like protein [Arabidopsis thaliana] Ta.23376.2.S1_s_at down 3 2.1 0.018 Peroxidase 47 precursor (Atperox P47) (ATP32) Ta.24988.2.S1_x_at down 3 2.0 0.002 putative finger transcription factor [Oryza sativa (japonica cultivar-group)] Ta.28775.3.S1_at down 3 2.3 0.004 Transcribed locus, strongly similar to NP_176968.1 HPR; oxidoreductase, acting on the CH-OH group of donors, NAD or NADP as acceptor [Arabidopsis thaliana] Ta.3064.2.S1_x_at down 3 2.6 0.016 VIP2 protein [Avena fatua] Ta.30733.2.S1_at down 3 2.2 0.035 Ubiquitin-fold modifier 1 [Oryza sativa (japonica cultivar-group)] Ta.3322.3.S1_x_at down 3 2.2 0.023 Ankyrin protein kinase-like [Poa pratensis] Ta.3495.2.S1_x_at down 3 2.3 0.025 Oryza sativa (japonica cultivar-group) cDNA clone:J033070O19, full insert sequence Ta.5420.2.S1_x_at down 3 2.6 0.007 Oryza sativa (japonica cultivar-group) cDNA clone:J013096G23, full insert sequence Ta.6312.1.S1_at down 3 2.1 0.024 unknown Ta.6663.2.S1_a_at down 3 2.1 0.017 Transcribed locus, strongly similar to NP_910307.1 unknown protein [Oryza sativa (japonica cultivar-group)] Ta.6954.3.S1_x_at down 3 2.4 0.037 similar to choline kinase [Oryza sativa (japonica cultivar-group)] TaAffx.100076.1.S1_x_at down 3 2.0 0.010 protein phosphatase 2C [Oryza sativa (japonica cultivar-group)] TaAffx.105798.1.S1_s_at down 3 2.5 0.049 zinc finger protein [Oryza sativa (japonica cultivar-group)] (contig annotation) TaAffx.107614.1.S1_at down 3 2.1 0.019 unknown TaAffx.110801.1.S1_at down 3 2.7 0.024 unknown TaAffx.116106.1.S1_at down 3 2.2 0.045 PrMC3 [Oryza sativa (japonica cultivar-group)] 248 Probe Set ID regulation hai FD p-val Gene Symbol Gene Title TaAffx.118607.1.S1_s_at down 3 2.2 0.033 farnesylated protein [Oryza sativa Japonica Group] TaAffx.118887.1.S1_at down 3 2.1 0.044 YME1 PROTEIN (TAT-BINDING HOMOLOG 11) (OSD1 PROTEIN) TaAffx.119070.1.S1_at down 3 2.1 0.001 gene_id:K19E20.14~unknown protein [Arabidopsis thaliana] TaAffx.123400.1.S1_at down 3 2.3 0.021 SPS1 sucrose-phosphate synthase TaAffx.128643.6.S1_at down 3 2.1 0.018 similar to proline-rich protein [Oryza sativa (japonica cultivar-group)] (contig annotation) TaAffx.129134.2.S1_at down 3 2.7 0.021 a2b Glycosyltransferase TaAffx.19589.1.S1_at down 3 2.1 0.026 putative hematopoietic-specific IL-2 deubiquitinating enzyme [Oryza sativa (japonica cultivar-group)] gi|47497222|dbj|BAD19267.1| putative hematopoietic-specific IL-2 deubiquitinating enzyme [Oryza TaAffx.23269.1.A1_at down 3 2.1 0.002 ABC1 family protein [Arabidopsis thaliana] TaAffx.23746.1.S1_at down 3 2.4 0.022 OSJNBa0011J08.14 [Oryza sativa (japonica cultivar-group)] gi|32488507|emb|CAE03259.1| OSJNBa0011J08.14 [Oryza sativa (japonica cultivar-group)] TaAffx.24722.1.S1_at down 3 2.1 0.005 PrMC3 [Oryza sativa (japonica cultivar-group)] TaAffx.38017.1.A1_at down 3 2.0 0.011 receptor-type protein kinase LRK1 [Oryza sativa (japonica cultivar-group)] TaAffx.4633.1.S1_at down 3 2.3 0.030 unknown TaAffx.50698.1.S1_at down 3 2.1 0.039 putative CTV.22 [Oryza sativa (japonica cultivar-group)] gi|45735914|dbj|BAD12946.1| putative CTV.22 [Oryza sativa (japonica cultivar-group)] gi|42409072|dbj|BAD10323.1| putative CTV.22 [Oryza sativa (japonica TaAffx.52324.1.S1_at down 3 2.3 0.010 unknown TaAffx.5401.1.S1_x_at down 3 2.2 0.019 unknown TaAffx.5570.1.S1_at down 3 2.1 0.000 ARGONAUTE-LIKE PROTEIN 249 Probe Set ID regulation hai FD p-val Gene Symbol Gene Title TaAffx.57101.1.S1_at down 3 2.2 0.001 similar to ternary complex factor-like [Oryza sativa (japonica cultivar-group)] TaAffx.57201.1.S1_at down 3 2.0 0.018 unknown TaAffx.64857.1.S1_at down 3 2.1 0.025 unknown TaAffx.65385.1.S1_at down 3 2.9 0.049 mucin-like protein [Oryza sativa (japonica cultivar-group)] TaAffx.7773.1.S1_at down 3 2.2 0.007 isochorismatase hydrolase-like [Oryza sativa (japonica cultivar-group)] gi|50252495|dbj|BAD28672.1| isochorismatase hydrolase-like [Oryza sativa (japonica TaAffx.79407.1.S1_at down 3 2.1 0.006 unknown TaAffx.80108.1.S1_at down 3 3.2 0.033 unknown TaAffx.80718.1.S1_at down 3 2.1 0.030 similar to Arabidopsis thaliana putative chloroplast outer envelope 86-like protein (AC002330) [Oryza sativa] TaAffx.98037.1.S1_at down 3 2.0 0.041 unknown Ta.12713.1.S1_at down 24 2.4 0.028 unknown Ta.9834.1.S1_x_at down 24 2.1 0.010 unknown TaAffx.107388.1.S1_at down 24 2.3 0.037 cysteine protease [Triticum aestivum] TaAffx.128414.24.A1_s_at down 24 2.6 0.039 ribulose 1,5 bisphosphate carboxylase/oxygenase, large subunit [Limonium gibertii] TaAffx.23038.1.S1_at down 24 2.0 0.045 similar to F-box domain containing protein [Oryza sativa (japonica cultivar-group)] TaAffx.9604.1.S1_at down 24 2.1 0.018 unknown TaAffx.53736.1.S1_at up 3 3.5 0.050 unknown TaAffx.54339.1.S1_at up 3 2.1 0.008 MITOGEN-ACTIVATED PROTEIN KINASE HOMOLOG MMK2 TaAffx.5802.2.S1_s_at up 3 2.1 0.018 chloroplast rRNA-operon [Zea mays] Ta.10574.1.S1_a_at up 8 2.0 0.013 Transcribed locus, moderately similar to XP_470664.1 Hypothetical protein [Oryza sativa (japonica cultivar-group)] 250 Probe Set ID regulation hai FD p-val Gene Symbol Gene Title Ta.13041.1.S1_at up 8 2.1 0.002 putative nucleoid DNA-binding protein cnd41, chloroplast [Oryza sativa (japonica cultivar- group)] gi|55296886|dbj|BAD68338.1| putative nucleoid DNA-binding protein cnd41, chloroplast [Oryza Ta.14005.2.S1_s_at up 8 2.2 0.030 chloroplast 16S rRNA [Aegilops speltoides] Ta.16315.1.A1_at up 8 2.2 0.044 putative AT-hook DNA-binding protein [Oryza sativa (japonica cultivar-group)] gi|41053039|dbj|BAD07970.
UBC Theses and Dissertations
Investigating the molecular mechanisms of Fusarium Head Blight resistance in wheat Foroud, Nora Afsaneh 2011
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