University of Alberta Characterization of the Brassica napus -fungal pathogen interaction by Bo Yang A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Doctor of Philosophy In Plant Science Department of Agricultural, Food and Nutritional Science © Bo Yang Fall 2009 Edmonton, Alberta Permission is hereby granted to the University of Alberta Libraries to reproduce single copies of this thesis and to lend or sell such copies for private, scholarly or scientific research purposes only. Where the thesis is converted to, or otherwise made available in digital form, the University of Alberta will advise potential users of the thesis of these terms. The author reserves all other publication and other rights in association with the copyright in the thesis and, except as herein before provided, neither the thesis nor any substantial portion thereof may be printed or otherwise reproduced in any material form whatsoever without the author's prior written permission. Examining Committee Dr. Nat Kav (Supervisor) Dr. Stephen Strelkov Dr. Michael Deyholos Dr. Walter Dixon Dr. Mark Gijzen (External Examiner) Abstract Sclerotinia sclerotiorum is a devastating pathogen causing stem rot in Brassica napus (canola). Microarray analysis was performed to investigate pathogen- induced transcript profiling in B. napus responses to S. sclerotiorum . Several genes were identified, which included defensins, those encoding proteins involved in oxidative burst, the biosynthesis of jasmonic acid (JA), and several transcription factors (TFs), but not salicylic acid (SA)-related genes. To further characterize the roles of SA, JA and ethylene (ET) in the response of B. napus and Brassica carinata (tolerant species) to S. sclerotiorum , the expression of five genes known to respond to these phytohormones were investigated. We observed that S. sclerotiorum triggered JA/ET signaling in B. napus . Furthermore, the heterologous expression of 1-aminocyclopropane- 1-carboxylate (ACC) deaminase, which reduced ET levels, enhanced the susceptibility of B. napus to S. sclerotiorum . Our microarray analysis also revealed the importance of TFs in mediating responses of B. napus to S. sclerotiorum . To probe the involvement of one such TF family in B. napus , the WRKYs, public sequence databases were mined. Three groups of B. napus WRKYs were indentified from a phylogenetic tree and four selected ones were shown to localize to the nucleus using GFP fusions. Sclerotinia sclerotiorum and Alternaria brassicae (another necrotrophic pathogen affecting canola) and phytohormone-induced expression of representative WRKYs from each clade was also investigated using quantitative real-time PCR. In another aspect of our study, two recombinant single chain variable fragment (ScFvs) antibodies specific for a S. sclerotiorum endopoly- galacturonase (SSPG1d) were isolated and characterized. Our results indicated that these antibodies may have utility in the detection of this pathogen when used with other S. sclerotiorum -specific antibodies in a panel format assay. Transgenic Arabidopsis expressing the ScFvs were evaluated for tolerance to S. sclerotiorum and it was observed that the heterologous expression of the cDNAs encoding these ScFvs did not enhance the tolerance of Arabidopsis. However, additional research aimed at stabilization of the ScFvs and/or their localization must be conducted together with research into their usefulness in imparting tolerance in B. napus to this pathogen, since the observed effects in this species may be different from A. thaliana . Acknowledgements I would like to acknowledge my supervisor Dr. Nat Kav for not only giving me a chance to study in his laboratory under his supervision, but also for his instruction, and encouragement throughout my graduate study and research. I want to express my appreciation to my supervisory committee members: Drs. Michael Deyholos and Stephen Strelkov for providing valuable suggestions, critical evaluation and help on my proposal and thesis. I very much appreciate the valuable instruction and suggestions from Drs. Walter Dixon and Mavanur Suresh during my candidacy examination and to Drs. Walter Dixon and Mark Gijzen for being examination committee members. This thesis project could not have been completed without the help of lots of people. I appreciate Dr. Anthony Cornish and Troy Locke in MBSU for help in microarray and quantitative PCR, Lisa Ostafichuk, Pat Murray and Cheryl Nargang for DNA sequencing service. I gratefully acknowledge the technical help and kind cooperation provided received from Dr. Muhammad Rahman and Bill Yajima. I also sincerely appreciate the support from Yue Liang, Dr. Dipankar Das, Dr. Sanjeeva Srivastava, Dr. Nidhi Sharma, Dr. Shiv Verma, Sowmya Krishaswamy, Dr . Moshen Mohammadi, Shiv Ganesh, members of Dr. Mike Deyholos’ and Dr. Stephen Strelkov’s laboratories. I really enjoyed and appreciated the available facilities and the smooth operation of those would not have been possible without our managers: Dr. Urmila Basu, Bruce Alexanda, Lynn Elmes, Will Cornet and Laura Smith. I also would like to thank Jody Forslund, our graduate programs assistant, for her help during my PhD program. Financial support from AARI (NNKV) is also gratefully acknowledged. Finally, I would like to thank my grandparents, my parents, my sister and especially my husband Yuanqing Jiang, father-in-law, grandfather-in-law for their understanding, support, and encouragement throughout my doctoral research. Without them, I could not have gone this far. To both my maternal and paternal grandmas! Table of contents Chapter 1 Literature Review ....................................................................................1 Introduction ......................................................................................................... 1 Mechanisms of fungal infection.......................................................................... 2 Plant defense systems ......................................................................................... 4 Molecular events triggered in plants during infection ........................................ 5 Signal transduction in plant defense ............................................................... 6 SA-dependent signaling .............................................................................. 7 JA-dependent signaling ............................................................................... 8 ET-dependent signaling .............................................................................. 9 Other defense mechanisms of plants............................................................. 11 Fungal pathogens of canola .............................................................................. 12 Sclerotinia sclerotiorum ................................................................................ 12 Recent progress in the study of S. sclerotiorum (development and infection cycle).......................................................................................... 14 Alternaria brassicae (Berk.) Sacc ............................................................. 18 Recent progress on the study of A. brassicae ........................................... 19 Traditional approaches to managing the two fungi....................................... 20 Current studies on plant responses to S. sclerotiorum ...................................... 23 Interaction of OA and oxalate oxidase (OXO) ............................................. 23 Interaction of PGs and polygalacturonase-inhibiting proteins (PGIPs) ........ 24 Current studies on responses in plants to A. brassicae ..................................... 24 Recombinant antibodies and their application in plant pathology .................... 25 Objectives and significance of this research ..................................................... 28 References ......................................................................................................... 30 Chapter 2 Transcriptional profiling of canola ( Brassica napus L.) responses to the fungal pathogen Sclerotinia sclerotiorum ..............................................................59 Introduction ....................................................................................................... 59 Materials and methods ...................................................................................... 61 Fungal and plant materials ............................................................................ 61 RNA isolation, microarray slide preparation and hybridization conditions . 62 Microarray data analysis ............................................................................... 62 Quantitative RT-PCR (qRT-PCR) ................................................................ 63 Results and discussion ...................................................................................... 66 Whole plant response to S. sclerotiorum ...................................................... 66 Transcriptional profiling using microarrays ................................................. 66 Temporal expression patterns of Sclerotinia responsive genes .................... 68 Functional classification of S. sclerotiorum -responsive genes ..................... 71 Cell rescue and defense related transcriptional ......................................... 73 Reactive oxygen responses ......................................................................