Investigation of the Plasmodiophora brassicae life cycle and its interactions with host plants A Thesis Submitted to the College of Graduate and Postdoctoral Studies In Partial Fulfillment of the Requirements For the Degree of Doctor of Philosophy In the Department of Biology University of Saskatchewan Saskatoon, Saskachewan By Jiangying Tu © Copyright Jiangying Tu, October 2018. All rights reserved. Permission to Use In presenting this thesis/dissertation in partial fulfillment of the requirements for a Postgraduate degree from the University of Saskatchewan, I agree that the Libraries of this University may make it freely available for inspection. I further agree that permission for copying of this thesis/dissertation in any manner, in whole or in part, for scholarly purposes may be granted by the professor or professors who supervised my thesis/dissertation work or, in their absence, by the Head of the Department or the Dean of the College in which my thesis work was done. It is understood that any copying or publication or use of this thesis/dissertation or parts thereof for financial gain shall not be allowed without my written permission. It is also understood that due recognition shall be given to me and to the University of Saskatchewan in any scholarly use which may be made of any material in my thesis/dissertation. Disclaimer Reference in this thesis/dissertation to any specific commercial products, process, or service by trade name, trademark, manufacturer, or otherwise, does not constitute or imply its endorsement, recommendation, or favoring by the University of Saskatchewan. The views and opinions of the author expressed herein do not state or reflect those of the University of Saskatchewan, and shall not be used for advertising or product endorsement purposes. Requests for permission to copy or to make other uses of materials in this thesis/dissertation in whole or part should be addressed to: Head of the Department of Biology University of Saskatchewan 112 Science Place Saskatoon, Saskatchewan S7N 5E2 Canada OR Dean College of Graduate and Postdoctoral Studies University of Saskatchewan 116 Thorvaldson Building, 110 Science Saskatoon, Saskatchewan S7N 5C9 Canada i Abstract Plasmodiophora brassicae is a soilborne parasitic protist and the causal agent of clubroot disease in the family Brassicaceae. The formation of characteristic galls on the roots of susceptible hosts disrupts the uptake of water and nutrients by the plant roots, causing stunting, wilting and yellowing of above-ground parts and even premature plant death, resulting in large yield losses and lower oil quality. As an intracellular pathogen, the life cycle of P. brassicae that displays various developmental stages during pathogenesis, is complex. This dissertation describes my efforts during my graduate studies to coordinate the P. brassicae cellular study, investigating the P. brassicae infection process, with the recent published genome sequence and develop tools with which to help in answering the burning question of what happens in resistant cultivars and non- host cultivars, in relation to their susceptible relatives, to prevent cortical infection and terminate disease initiation. I have established a new protocol based on a two-step axenic culture of P. brassicae with its host tissues, for easy and in-planta observation of cellular interactions between P. brassicae and host plants. The double staining of P. brassicae with fluorescent dyes, as established in this study, provides a promising technique for observing the infection process in-planta. Using the live-cell imaging protocols established in the axenic culture, together with transmission electron microscopy, the life cycle of P. brassicae was investigate in its natural pathosystem in soil. P. brassicae infecting a root har or epidermal cell underwent zoosporogenesis between five and 10 days post inoculation (dpi) and completed its life cycle with the production of resting spores, after meiosis and cytoplasmic cleavage of sporulating plasmodia in cortical cells, around 28 dpi. From all the genes involved in meiosis and flagella production, from a diverse array of sequenced eukaryotes, I have identified novel stage specific molecular markers for meiosis and flagella development during the P. brassicae infection cycle. Auxin accumulation in infected root tissues, resulting in cell enlargement to form root galls, could be due to auxin directly secreted by P. brassicae or could be a result of redirected auxin transport towards the site of infection. I carried out a study of the role of the auxin transport system governed by the AUX1 influx protein and the PIN FORMED (PIN)-family efflux proteins during clubroot development in A. thaliana. Transcript analysis of infected tissue showed that host auxin transport was upregulated in early infection stages and down-regulated at the later stages of gall ii formation. Mutants in auxin eflux proteins PIN1 and PIN3 bear smaller galls, suggesting that auxin import into the infected cell is needed for gall formation. Understanding the P. brassicae life cycle and the role of auxin flow in gall development will benefit plant pathologists and plant breeders in developing clubroot disease resistance in canola and other cruciferae vegetables worldwide. iii Preface This thesis is based on the findings presented in the following papers and manuscripts: I. Irani, S., Trost, B., Waldner, M., Nayidu, N., Tu, J., Kusalik, A. J., Todd, C. D., Wei, Y. and Bonham-Smith, P. C. (2018). Transcriptome analysis of response to Plasmodiophora brassicae infection in the Arabidopsis shoot and root. BMC genomics 19:23. (not included in the thesis but parts of results from my Ph.D. project) II. Tu, J., Bush, J., Wei, Y. and Bonham-Smith, P. C. Live cell imaging of Plasmodiophora brassicae - host plants interactions based on a two-step axenic culture system. Microbiology Open. Accepted on October 18, 2018. Manuscript ID: MBO3-2018-08- 0279R2 (Chapter 3) III. Pérez-López, E., Tu, J., Waldner, M., Kusalik, A. J., Wei, Y., Todd, C. D., and Bonham- Smith, P. C. Genome-wide transcriptomic analysis identified putative small-secreted Plasmodiophora brassicae proteins. In preparation. To be submitted to Frontiers in Microbiology. (not included in the thesis but parts of results from my Ph.D. project) IV. Tu, J., Wei, Y. and Bonham-Smith, P. C. Cellular tracking and gene profiling of Plasmodiophora brassicae during clubroot disease development elucidate life stage specific markers. In preparation. To be submitted to Molecular Plant Pathology. (Chapter 4) iv Acknowledgements This dissertation is a milestone in my academic career path at University of Saskatchewan. I have been fortunate to learn theories, concepts and theniques which would have been impossible if I had not extensively carried out the needed research and teaching in Department of Biology. I am grateful to a number of people who have guided and supported me throughout my Ph.D. study and provided assistance for my venture. The persons I have interacted with the most during my study and who I am deeply indebted to and owe my heartfelt gratitude to are easily my supervisors, Dr. Peta Bonham-Smith and Dr. Yangdou Wei. For all the time, encouragement and patience you invested in me, be it personal or professional, influenced my thoughts and guided my roads on science, a big thank you. I would like to express my deep appreciation to my advisory committee, Dr. Carlos Egydio Carvalho, Dr. Aaron Beattie and Dr. Pierre Forbert for their support, guidance and helpful suggestions. I would like to thank Dr. Stephen Strelkov for serving as my external examiner. Members of Dr. Bonham-Smith and Dr. Wei lab also deserve my sincerest thanks, their friendship and assistance has meant more to me than I could ever express. I also would like to thank Dr. Guosheng Liu for his technique support in Department of Biology. A very special thanks to Mrs. LaRhonda Sobchishin (Western College of Veterinary Medicine, University of Saskatchewan) for her help and support with transimission electron microsopy techniques. I would like to acknowledge the financial support provided by Saskatchewan Agriculture Development Fund, Canola Coucil of Canada, University of Saskatchewan and China Scholarship Council for funding my projects. I consider myself very lucky to be one of the few individuals who have had the freedom to pursue what they want in life. I understand now, that this privilege is largely due to my fanily and my freedom is the end result of their struggles and persistence. I would like to express my great appreciations to my parents, who gave me life and brought me up. My husband, Luyu Liu, has always supported my decisions and shared my enthusiasm for my research work. And my daughter, Raven Liu, who made me understand the true meaning of life. I also want to thank my parents-in- law for their unconditional support and love. v Table of Contents Permission to Use .............................................................................................................................. i Abstract ........................................................................................................................................... ii Preface ............................................................................................................................................. iv Acknowledgements .......................................................................................................................... v Table of Contents