Fungi and Cytokinins: Investigating the Impact Of
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FUNGI AND CYTOKININS: INVESTIGATING THE IMPACT OF CYTOKININS ON FUNGAL DEVELOPMENT AND DISEASE PROGRESSION IN THE Ustilago maydis- Zea mays PATHOSYSTEM A dissertation submitted to the Committee of Graduate Studies in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the faculty of Arts and Science Trent University Peterborough, Ontario, Canada © Copyright by Erin Nicole Morrison, 2016 Environmental & Life Sciences Ph. D. Graduate Program May 2016 ABSTRACT Fungi and Cytokinins: Investigating the impact of cytokinins on fungal development and disease progression in the Ustilago maydis- Zea mays pathosystem Erin Nicole Morrison Cytokinin biosynthesis in organisms aside from plant species has often been viewed as a byproduct of tRNA degradation. Recent evidence suggests that these tRNA degradation products may actually have a role in the development of these organisms, particularly fungi. This thesis examines the importance of cytokinins, a group of phytohormones involved in plant cell division and differentiation as well as the phytohormone abscisic acid, involved in plant response to environmental factors, and their presence and role in fungi. An initial survey was conducted on 20 temperate forest fungi of differing nutritional modes. Using HPLC-ESI MS/MS, cytokinin and abscisic acid were detected in all fungi regardless of their mode of nutrition or phylogeny. The detection of the same seven CKs across all fungi suggested the existence of a common CK biosynthetic pathway and dominance of the tRNA pathway in fungi. Further, the corn smut fungus Ustilago maydis is capable of producing CKs separate from its host and different U. maydis strains induce disease symptoms of differing severity. To determine if CK production during infection alters disease development a disease time course was conducted on cob tissue infected with U. maydis dikaryotic and solopathogenic strains. ii Dramatic changes in phytohormones including an increase in ABA followed by increases in cisZCKs were detected in tumour tissue particularity in the more virulent dikaryon infection, suggesting a role for CKs in strain virulence. Mining of the U. maydis genome identified a sole tRNA-isopentenyltransferase, a key enzyme in CK biosynthesis. Targeted gene deletion mutants were created in U. maydis which halted U. maydis CK production and decreased pathogenesis and virulence in seedling and cob infections. CK and ABA profiling carried out during disease development found that key changes in these hormones were not found in deletion mutant infections and cob tumour development was severely impaired. These findings suggested that U. maydis CK production is necessary for tumour development in this pathosystem. The research presented in this thesis highlights the importance of fungal CKs, outlines the dominant CK pathway in fungi, identifies a key enzyme in U. maydis CK biosynthesis and reveals the necessity of CK production by U. maydis in the development of cob tumours. KEYWORDS: Ustilago maydis, Zea mays, abscisic acid, cytokinins, fungi, high performance liquid chromatography-electrospray ionization tandem mass spectrometry, tRNA degradation pathway. iii PREFACE This thesis is presented in manuscript format. Each chapter is either published or has been submitted for publishing. Co-authors and their contributions are listed in the preface of each chapter. Permission from copyright holders for each chapter is presented in Appendix I. Additional publication not included in thesis Morrison EN, Donaldson ME, Saville BJ. 2012. Identification and analysis of genes expressed in the Ustilago maydis dikaryon: uncovering a novel class of pathogenesis genes. Canadian Journal of Plant Pathology 34:417-435. iv ACKNOWLEDGEMENTS As all great stories go, a seemingly inconsequential switch to a plant biology course during my undergraduate studies put me on the path that has led me here today. There have been many people who have helped me along that path and I thank you all for your encouragement and support. I would first like to thank my co-supervisors Dr. Neil Emery and Dr. Barry Saville for giving me the opportunity to pursue combined research in their labs. You have both helped to shape me into the researcher I am today, and it has been an honour and a privilege to learn from and work with you both. I would also like to thank my committee member Dr. Jim Sutcliffe for his valued advice. Thank you to Linda Cardwell, Mary-Lynn Scriver, Jane Rennie and Erin Davidson, for their much appreciated administrative direction over the years. Thank you to the Emery and Saville labs and their members, both past and present. You are a ‘wonderfully-kooky’ bunch that have made my academic journey all the better. I would particularly like to thank Dr. Mike Donaldson. Your guidance, instruction, friendship and encouragement helped me grow as a researcher. Special thanks to my office mates Colleen Doyle and Kitty Cheung for your support and help over the years. Thank you to my family and friends for your continued presence, encouragement, and prayers. Specifically, thank you to my parents Grant and Debra Morrison, who have inspired, directed, supported, and prayed me through this chapter of my life, as well as the ones that came before. Without your love and support, this road would have looked v very different. To my sister Lizzy I thank you for being such an amazing sister and best friend. Thank you for always being there for me. Thank you to Dustin Bowers, your love and support has helped me through. Also, I think people would be disappointed if I did not mention the cups that fill my recycling bin and the fuel that started my days: Tim Horton's tea (I will be buying shares in the company soon). Importantly, I thank my Lord and Saviour Jesus Christ who has seen me through this and taught me many things along the way. When I am weak, He is strong. Finally, thank you to Trent University as a whole. It is here where I was given the space to grow and learn not only as a scientist but also as a person. I have been truly blessed. vi TABLE OF CONTENTS PAGE TITLE PAGE i ABSTRACT ii KEYWORDS iii PREFACE iv ACKNOWLEDGEMENTS v TABLE OF CONTENTS vii LIST OF TABLES x LIST OF FIGURES xi LIST OF ABBREVIATIONS xiii CHAPTER 1 General Introduction. ABSCISIC ACID 1 CYTOKININS 3 tRNA MODIFICATION 5 FUNGAL PLANT-PATHOGENS AND PHYTOHORMONES 6 USTILAGO MAYDIS 8 RESEARCH OBJECTIVES 10 FIGURES 13 REFERENCES 15 CHAPTER 2 Detection of phytohormones in temperate forest fungi predicts consistent abscisic acid production and a common pathway for cytokinin biosynthesis. PREFACE 20 ABSTRACT 21 INTRODUCTION 22 MATERIALS AND METHODS 25 RESULTS 29 DISCUSSION 32 TABLES AND FIGURES 42 SUPPLEMENTARY MATERIAL 47 ACKNOWLEDGEMENTS 48 vii REFERENCES 49 CHAPTER 3 Phytohormone involvement in the Ustilago maydis– Zea mays pathosystem: relationships between abscisic acid and cytokinin levels and strain virulence in infected cob tissue. PREFACE 55 ABSTRACT 56 INTRODUCTION 58 MATERIALS AND METHODS 60 RESULTS 67 DISCUSSION 72 CONCLUSIONS 82 TABLES AND FIGURES 84 SUPPLEMENTARY MATERIAL 95 ACKNOWLEDGEMENTS 96 REFERENCES 97 CHAPTER 4 Interplay between fungal and plant derived cytokinins is necessary for normal Ustilago maydis infection of corn. PREFACE 103 ABSTRACT 104 INTRODUCTION 106 MATERIALS AND METHODS 110 RESULTS 122 DISCUSSION 131 CONCLUSIONS 139 TABLES AND FIGURES 141 SUPPLEMENTARY MATERIAL 154 ACKNOWLEDGEMENTS 180 REFERENCES 181 CHAPTER 5 General discussion. INTRODUCTION 188 PHYTOHORMONE DETECTION IN FOREST FUNGI 189 PHYTOHORMONES AND DISEASE DEVELOPMENT 190 CONTROL OF CK BIOSYNTHESIS IN U. MAYDIS 192 FURTHER CHARACTERIZATION OF tRNA-IPT 194 THE ROLE OF CKS AT DIFFERENT FUNGAL LIFESTAGES 195 PLANT AND PATHOGEN GENE EXPRESSION 196 ABA AND CK INTERACTION 198 CONCLUSIONS 200 FIGURES 201 REFERENCES 203 viii APPENDIX I Permission from copyright holders. CHAPTER 2 206 CHAPTER 3 208 ix LIST OF TABLES PAGE Table 2.1. Fungi used in this study. 42 Table 3.1. Tissue sampling for phytohormone analysis. 84 Table 3.2. Cytokinin concentrations (pmol g -1 FW) in mock-infected Z. mays cob tissue, during the U. maydis- Z. mays infection time course. 85 Table 3.3. Cytokinin concentrations (pmol g -1 FW) in U. maydis dikaryon infected Z. mays cob tissue, during the U. maydis- Z. mays infection time course. 86 Table 3.4. Cytokinin concentrations (pmol g -1 FW) in U. maydis solopathogen infected Z. mays cob tissue, during the U. maydis- Z. mays infection time course. 87 Table 4.1. Cytokinin concentrations (pmol g -1 FW) for SG200 filamentous tissue grown on minimal medium (CK negative) and double complete medium (CK positive). 141 Table 4.2. Cytokinin concentrations (pmol g -1 FW) in mock-infected Z. mays cob tissue, during the U. maydis- Z. mays infection time course. 142 Table 4.3. Cytokinin concentrations (pmol g -1 FW) in SG200∆ipt1 infected Z. mays cob tissue, during the U. maydis- Z. mays infection time course. 143 Table 4.4. Cytokinin concentrations (pmol g -1 FW) in SG200 infected Z. mays cob tissue, during the U. maydis- Z. mays infection time course. 144 Table 4.5. Putative candidate CK signaling and biosynthesis orthologs identified in U. maydis through blastp analysis and reciprocal best blast hits. 145 x LIST OF FIGURES PAGE Fig. 1.1. CK biosynthesis pathway. 13 Fig. 1.2. Ustilago maydis life cycle. 14 Fig. 2.1. Abscisic acid concentration (pmol g-1 FW) in ectomycorrhizal, wood-rot and saprotrophic fungi. 43 Fig. 2.2. Total cytokinin concentration (pmol g-1 FW) in ectomycorrhizal, wood-rot and saprotrophic fungi. 44 Fig. 2.3. Cytokinin types (A) isopentenyl CKs (iPRP, iPR and iP), (B) cis-zeatin CKs (cisZRP and cisZR) and (C) methylthiol CKs (2MeSZR and 2MeSZ) reported as percentage of total cytokinin in ectomycorrhizal, wood-rot and saprotrophic fungi. 45 Fig. 2.4. Proposed tRNA degradation pathway. 46 Fig. 3.1. Representative time course of disease progression for Zea mays- U.