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Investigating the Role of the Mediator Complex in Plant Defence

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Investigating the Role of the Mediator Complex in Plant Defence Investigating the role of the Mediator complex in plant defence Thorya A Fallath Master of Biotechnology Bachelor of biology A thesis submitted for the degree of Doctor of Philosophy at The University of Queensland in 2016 School of Agriculture & Food Sciences Abstract The Mediator complex is an evolutionarily conserved protein complex involved in transcriptional regulation. In plants, the Mediator complex plays an important role in regulating development as well as the response to abiotic and biotic stress. In this thesis, I investigated three subunits of the Arabidopsis thaliana Mediator complex MEDIATOR25, MEDIATOR18 and MEDIATOR20 (MED25, MED18 and MED20) in plant defence. MED25 has been shown to interact with several transcription factors (TFs) to control jasmonate (JA)-associated transcription. Here, I investigated these interactions further and characterized the minimum protein domains of the TFs needed for interaction and identified new proteins that interact with MED25. The results showed that the protein sequence corresponding to amino acids 138-218 of ETHYLENE RESPONSE FACTOR1 (ERF1) appears to be the minimum protein length for interaction with MED25 and contains the conserved motif (CMIX). Although the protein motif has been found in different proteins in A. thaliana that belong to a variety of families, only members from the IXc AP2/ERF family were found to interact with the MED25 ACID domain. Apart from MED25, I found that the MED18 and MED20 subunits interact within the Mediator complex and infection of the corresponding mutants with Fusarium oxysporum revealed that they display a high level of resistance. RNAseq analysis of F. oxysporum infected med18 and med20 roots showed that salicylic acid (SA)-associated PATHOGENESIS RELATED genes and (ROS) reactive oxygen producing genes were up-regulated while some JA-regulated genes were repressed. We also noticed induction and repression of genes involved in glucosinolate production. This may suggest that ROS and antifungal metabolites are induced in med18 and med20 mutants and could potentially contribute to the reduced colonization of the mutants by F. oxysporum. Overall, the results reveal a new role for MED18 and MED20 in susceptibility to F. oxysporum, most likely via their involvement in JA signalling whose up-regulation would compromise SA and ROS signalling pathways that can confer resistance against this hemibiotrophic pathogen. i Declaration by author This thesis is composed of my original work, and contains no material previously published or written by another person except where due reference has been made in the text. I have clearly stated the contribution by others to jointly-authored works that I have included in my thesis. I have clearly stated the contribution of others to my thesis as a whole, including statistical assistance, survey design, data analysis, significant technical procedures, professional editorial advice, and any other original research work used or reported in my thesis. The content of my thesis is the result of work I have carried out since the commencement of my research higher degree candidature and does not include a substantial part of work that has been submitted to qualify for the award of any other degree or diploma in any university or other tertiary institution. I have clearly stated which parts of my thesis, if any, have been submitted to qualify for another award. I acknowledge that an electronic copy of my thesis must be lodged with the University Library and, subject to the policy and procedures of The University of Queensland, the thesis be made available for research and study in accordance with the Copyright Act 1968 unless a period of embargo has been approved by the Dean of the Graduate School. I acknowledge that copyright of all material contained in my thesis resides with the copyright holder(s) of that material. Where appropriate I have obtained copyright permission from the copyright holder to reproduce material in this thesis. ii Publications during candidature 1- Journal papers Fallath TA, Kidd BN, Stiller J, Manners J, Kazan K and Schenk PM (2016). Mediator head domain subunits, MEDIATOR18 and MEDIATOR20 control susceptibility to Fusarium oxysporum in Arabidopsis thaliana (submitted to PLoS ONE) 2-Book chapters Fallath TA, Bin Rosli A, Kidd BN, C. Carvalhais L and Schenk PM (2016). Towards plant defense mechanisms against root pathogens. In Agriculturally important microbes for sustainable agriculture. (submitted to Springer) 3- Conference papers Fallath TA, Kidd BN, Stiller J, Kazan K, Schenk PM (2015). Investigating the role of Mediator complex in plant defence. The 11 Annual SCMB Research Student Symposiums, Brisbane, Australia, 26 November. Fallath TA, Kidd BN, Stiller J, Kazan K, Schenk PM (2015). Investigating the role of Mediator complex in plant defence. ComBio Conference, Melbourne, Australia, 27 September-1 October. Schenk PM, Rincon-Florez V, Rosli A, Chen YC, Liu H, Pretorius LS, Fallath T, Mirzaee H, Shuey L, Al-Amery A, Dennis P, Iram S, Kidd B, Carvalhais LC (2014) Metagenomics and transcriptomics to elucidate new plant-microbe interactions at the rhizosphere. 15th International Symposium on Microbial Ecology, Seoul, South Korea, August 24-29. Publications included in this thesis None iii Contributions by others to the thesis RNA sequencing was carried out by the Australian Genome Research Facility (AGRF). The initial analysis of RNAseq data was assisted by Dr. Jiri Stiller (CSIRO). The conception and design of this project was guided by Dr. Brendan Kidd and Prof. Peer Schenk. Statement of parts of the thesis submitted to qualify for the award of another degree None iv Acknowledgements First and foremost, I would like to express my gratitude to Allah “God” (subhana wa taala) for endowing me with health, patience, and knowledge to complete this thesis. Secondly, I would like to thank my supervisor Prof Peer Schenk who gave me the opportunity to work in a wonderfully vibrant and hardworking lab and for his guidance, advice and invaluable help during my project. My deepest gratitude and thanks go to Dr Brendan Kidd; he was a wonderful source of knowledge and whose assistance and advice was greatly appreciated throughout my project. He also was never too busy to help with all my ridiculous inquiries and problems and who provided a greatly deal of support and guidance. Without them this project would not have been possible. I would like to thanks Dr. Max Chen, Dr. Lilia Carvalhais, Dr. Ahmad Radhzlan Rosli, Dr. Lara Pretorius and Dr. Anaheed Al-Ameir, Dr. Hongwei Liu, Asmaa Ali and Amreen Kwaja for helping me and providing a great advice and knowledge since the beginning of my work in the lab. I would like to thank all the members of the Schenk Lab for the very educational and entertaining lab meetings, making Mondays easier to handle and more importantly for their friendship. Also, my gratitude and thanks goes to the Saudi Arabian government in particular The Ministry Of High Education for providing me with this great opportunity to study overseas at the University of Queensland and all the financial support during my studies. My greatest thanks go to my dearest darling mum for all her night prayers and moral support and blessings. I would like to express a deep sense of gratitude to my wonderful husband Sami Howsawi for his endless patience, support, encouragement and sacrifices to achieve my dreams and I simply could not have done it without him. Special thanks are due to my loving brothers and sisters for all their emotional and financial support. Sweet thank is also due to my lovely sons Mohammed, Abdulrahman and Ahmad for their puerile support. Last but not the least I would like to gift all of my work to my dad may God keep his soul in peace in heaven. Finally, I am also thankful to my friends here in Brisbane who helped make my stay in Australia a very pleasant one. v Keywords Arabidopsis thaliana, Mediator complex, med18, med20, Fusarium oxysporum, root defence, disease resistance, gene expression, jasmonate, susceptibility Australian and New Zealand Standard Research Classifications (ANZSRC) ANZSRC code: 060702 PLANT CELL AND MOLECULAR BIOLOGY 50%, 060704 PLANT PATHOLOGY 50% Fields of Research (FoR) Classification FoR code: 0607, Plant Biology, 100% vi Table of Contents LIST OF FIGURES .................................................................................................................................................. IX LIST OF TABLES ...................................................................................................................................................... X LIST OF ABBREVIATIONS ................................................................................................................................. XI CHAPTER I .................................................................................................................................................................. 1 1.1 INTRODUCTION ................................................................................................................................................................... 2 1.2 REVIEW OF LITERATURE ................................................................................................................................................. 3 1.2.1 Plant Disease and Resistance .................................................................................................................................. 3 1.2.2 The role of Mediator complex in plant defence
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