Novel insights into the infection mechanism of oomycete Pythium spp. in the host Arabidopsis thaliana. A Thesis Submitted to the College of Graduate and Postdoctoral Studies in Partial Fulfillment of the Requirements for the Degree of Master of Science in the Department of Biology University of Saskatchewan Saskatoon, Canada By: Wilhelmina Gerarda Hendrica Maria Hendriks (Anouk Hendriks) 2020 © Copyright Wilhelmina Gerarda Hendrica Maria Hendriks, July 2020. All rights reserved. PERMISSION TO USE In presenting this thesis in partial fulfilment 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 in any manner, in whole or in part, for scholarly purposes may be granted by the professor or professors who supervised my thesis 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. 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 College of Graduate and Postdoctoral Studies University of Saskatchewan University of Saskatchewan 112 Science Place OR 116 Thorvaldson Building, Saskatoon, Saskatchewan S7N5E2 Saskatoon, Saskatchewan S7N 5C9 Canada Canada i ABSTRACT Phytopathogenic Pythium spp. cause seedling damping-off of a wide range of plant species worldwide and are traditionally considered necrotrophs. In this study, novel pathogenicity was discovered involving the oomycete Pythium cryptoirregulare and the model plant Arabidopsis thaliana. This pathogen was isolated from A. thaliana seedlings that were showing damping-off symptoms and was later identified as Pythium cryptoirregulare based on morphological and molecular characterization alongside reference species P. irregulare and P. ultimum var. ultimum. To examine its infection strategy, A. thaliana was inoculated with P. cryptoirregulare and studied using a microscopy approach. Viable colonized cells were observed based on neutral red uptake and the ability to undergo cell plasmolysis after infection. This biotrophic interaction contradicts the previously reported necrotrophic lifestyle of Pythium spp., which is characterized by killing the host cells prior to colonization. In addition, inhibition of root growth was detected prior to colonization by P. cryptoirregulare, suggesting that P. cryptoirregulare secreted growth inhibitors. Potentially, these inhibiting metabolites facilitate infection by delaying plant development and, thereby, extending the seedling stage that is targeted by this pathogen. Notably, P. cryptoirregulare culture filtrates disturbed transport and distribution of auxins, indicated by altered GFP expression in the A. thaliana lines PIN1-GFP, PIN2-GFP, PIN3-GFP, PIN7-GFP and DR5::GFP which visualize the auxin efflux. This disturbance was further confirmed by a reduced inhibitory effect on the auxin-insensitive A. thaliana mutants axr1-3, axr4-2, and aux1-7. Metabolic activity assay results suggested that P. cryptoirregulare secretes auxin-related metabolites that are involved in reprogramming plant growth. Overall, the characterization of P. cryptoirregulare as a novel pathogen on A. thaliana gives new insights into understanding the pathogenic mechanisms and interactions between oomycetes and plants. Keywords: Pythium (cryptoirregulare), infection strategy, hemibiotrophy, metabolic activity, auxin homeostasis ii ACKNOWLEDGEMENTS First, I would like to thank my supervisor, Dr. Yangdou Wei, for his guidance, support, and knowledge in my journey to obtaining an M.Sc degree. He was and still is a very knowledgeable mentor, and I am very thankful for sharing his knowledge with me during my project. Secondly, I would like to thank my advisory committee, Drs. Sabine Banniza and Art Davis for their guidance and advice in my course work, my research project, and other related matters. Also, I would like to thank Dr. Zamir Punja for acting as an external examiner during my thesis defence. Confocal microscopy techniques were taught by Dr. Guosheng Liu, who taught me how to use the Confocal 510 and Confocal 880 microscope, and Dr. Chris Ambrose, who taught me how to use the Confocal 880 and how to analyse the pictures with the imaging software “ImageJ”. They are therefore greatly thanked for their support. I would also like to thank my lab members Dr. Lijiang Lui and Li Qin, who practically helped whenever and wherever this was requested. The National Research Council (NRC) Canada is also appreciated for supporting this project financially and Agriculture and Agri-Food Canada (AAFC) for providing the two Pythium reference strains from their Canadian Collection of Fungal Cultures (DAOMC). Finally, I would like to thank my family, friends, colleagues, and remaining Biology faculty and staff for all their care, support, and patience throughout my graduate studies by creating a home away from home. Of this list, special thanks go to my parents and other family members who were my greatest motivators to complete this degree at the University of Saskatchewan in Canada. iii TABLE OF CONTENTS PERMISSION TO USE ................................................................................................................. i ABSTRACT ................................................................................................................................... ii ACKNOWLEDGEMENTS ........................................................................................................ iii TABLE OF CONTENTS ............................................................................................................ iv LIST OF FIGURES .................................................................................................................... vii LIST OF TABLES ........................................................................................................................ x LIST OF ABBREVIATIONS .................................................................................................... xii CHAPTER 1 GENERAL INTRODUCTION ...................................................................... 1 1.1 Research objectives .......................................................................................................... 2 CHAPTER 2 LITERATURE REVIEW ............................................................................... 4 2.1 An overview of plant-pathogen interactions .................................................................... 4 2.1.1 Biotrophy .................................................................................................................. 5 2.1.2 Necrotrophy .............................................................................................................. 6 2.1.3 Hemibiotrophy .......................................................................................................... 6 2.2 Infection mechanism and host-response .......................................................................... 8 2.3 Plant-oomycete pathosystems .......................................................................................... 9 2.4 Plant-Pythium interactions ............................................................................................. 10 2.4.1 The genus Pythium .................................................................................................. 11 2.4.2 The life cycle of Pythium species ........................................................................... 15 2.4.3 Infection strategy of Pythium species ..................................................................... 16 2.4.4 Pythium interaction with the host Arabidopsis thaliana ......................................... 17 2.4.5 The importance of damping-off and root rot disease caused by Pythium species .. 23 2.4.6 Management strategy .............................................................................................. 24 CHAPTER 3 ISOLATION, GROWTH, AND IDENTIFICATION OF PYTHIUM CRYPTOIRREGULARE ............................................................................................................. 26 3.1 Introduction .................................................................................................................... 26 iv 3.2 Materials and methods ................................................................................................... 28 3.2.1 Plant and pathogen materials .................................................................................. 28 3.2.2 Isolation of Pythium cryptoirregulare from Arabidopsis thaliana ......................... 31 3.2.3 Media optimization to induce Pythium structure development .............................. 31 3.2.4 Morphological characterization and identification ................................................. 32 3.2.5 Molecular characterization and identification ........................................................ 33 3.3 Results ...........................................................................................................................