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Pseudomonas/Brachypodium As a Model System for Studying Rhizosphere Plant Microbe Interactions Under Water Stress

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Pseudomonas/Brachypodium As a Model System for Studying Rhizosphere Plant Microbe Interactions Under Water Stress The University of Southern Mississippi The Aquila Digital Community Master's Theses Fall 12-1-2018 Pseudomonas/Brachypodium as a Model System for Studying Rhizosphere Plant Microbe Interactions Under Water Stress Janiece McWilliams University of Southern Mississippi Follow this and additional works at: https://aquila.usm.edu/masters_theses Recommended Citation McWilliams, Janiece, "Pseudomonas/Brachypodium as a Model System for Studying Rhizosphere Plant Microbe Interactions Under Water Stress" (2018). Master's Theses. 602. https://aquila.usm.edu/masters_theses/602 This Masters Thesis is brought to you for free and open access by The Aquila Digital Community. It has been accepted for inclusion in Master's Theses by an authorized administrator of The Aquila Digital Community. For more information, please contact [email protected]. PSEUDOMONAS/BRACHYPODIUM AS A MODEL SYSTEM FOR STUDYING RHIZOSPHERE PLANT-MICROBE INTERACTIONS UNDER WATER STRESS by Janiece Hutchins McWilliams A Thesis Submitted to the Graduate School, the College of Arts and Sciences and the School of Biological, Environmental, and Earth Sciences at The University of Southern Mississippi in Partial Fulfillment of the Requirements for the Degree of Master of Science Approved by: Dr. Dmitri Mavrodi, Committee Chair Dr. Kevin Kuehn Dr. Micheal Davis ____________________ ____________________ ____________________ Dr. Dmitri Mavrodi Dr. Jake Schaefer Dr. Karen S. Coats Committee Chair Director of School Dean of the Graduate School December 2018 ABSTRACT In contrast to well-studied mechanisms of drought tolerance in plants, the interactions between plants and their microbiome during water stress are still poorly understood. This missing knowledge is crucial for the exploitation of beneficial microbial communities to improve the sustainability of agriculture under changing climatic conditions. The research described here bridged this gap by focusing on the molecular interactions between beneficial rhizobacterium Pseudomonas synxantha 2-79 and annual grass Brachypodium distachyon Bd21. 2-79 exemplifies a group of rhizobacteria associated with dryland wheat, while B. distachyon originates from the Middle East and has emerged as a model for valuable biomass, food, forage, and turf crops. The experimental system was used to test the hypothesis that the adaptation of rhizobacteria to water stress is mediated by the exchange of metabolites between the host plant and its microbiota. Results revealed that Brachypodium root exudates contain a mixture of plant metabolites that serve as carbon and energy sources for rhizobacteria and include compounds that act as osmoprotectants and may help rhizobacteria maintain physiological activity and mutualistic interactions with their plant host in dry soils. The genome of P. synxantha 2-79 encodes numerous pathways involved in de novo synthesis and uptake of osmoprotectants and formation of biofilms for protection from desiccation. Some of these pathways are conserved across many taxa, while others are strain-specific and may contribute to the differential affinity toward plants growing in under different soil water regimens. Future studies will inactivate these pathways and test resultant mutants for rhizosphere fitness under drought stress. ii ACKNOWLEDGMENTS I would like to thank Drs. Dmitri Mavrodi and Olga Mavrodi; if it not been for their persistence and encouragement, I could not have finished. The standards, knowledge and discipline they instilled made me a better scientist, researcher and person. Thanks to my committee member Dr. Kevin Kuehn for accepting me into graduate school, supporting my transition to Dr. Mavrodi’s lab, and teaching me how to use the lyophilizer. Thanks also to my committee member Dr. Micheal Davis for his help and lab space for experiments. Thanks to Dr. Mac Alford for his interest in my research and teaching me about monocot seed embryos. I cannot leave out my friend and unofficial committee member Dr. Glenn Shearer, whom I thank for all the advice and support through this journey. I also thank Dr. Anna Berim from the WSU Laboratory for Cellular Metabolism and Engineering for the metabolomic analysis of root exudates. Thanks to Bio Soil Enhancers, Inc. for enabling me to pursue a graduate degree and to Wayne Wade and Dr. Lalithakumari Janarthanum for igniting my interest and excitement in plant- microbe relationships. Thank you to all my family and friends; I could not have done this without their personal support and prayers. Many thanks to my Mavrodi lab family for all their help. Special thanks to my husband, Chris McWilliams, who endured all my tears and anxiety through the challenges and rejoiced with me through the accomplishments. This work was supported by NSF grant IOS-1656872 and by the Mississippi INBRE, funded by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant P20GM103476. iii TABLE OF CONTENTS ABSTRACT ........................................................................................................................ ii ACKNOWLEDGMENTS ................................................................................................. iii LIST OF TABLES ............................................................................................................ vii LIST OF ILLUSTRATIONS ........................................................................................... viii LIST OF SCHEMES........................................................................................................... x LIST OF ABBREVIATIONS ............................................................................................ xi CHAPTER I - INTRODUCTION ...................................................................................... 1 1.1 Plant-microbe interactions under water stress .......................................................... 1 1.2 Root exudates and their role in the selection of rhizosphere microbiome ................ 3 1.3 Microbial response to water stress ............................................................................ 4 1.4 Brachypodium distachyon and Pseudomonas synxantha as a model to study rhizosphere plant-microbe interactions ........................................................................... 6 1.5 Aims of this study ..................................................................................................... 9 CHAPTER II - MATERIALS AND METHODS ............................................................ 11 2.1 Establishment and propagation of B. distachyon Bd21 at USM ............................ 11 2.2 Collection and analysis of sterile root exudates ...................................................... 11 2.3 Annotation and analysis of P. synxantha 2-79 genome .......................................... 13 2.4 Bacterial strains and plasmids ................................................................................. 14 2.5 Osmoprotection assays............................................................................................ 14 iv 2.6 Rhizosphere colonization assays............................................................................. 15 2.7 Construction of 2-79 reporter derivatives ............................................................... 16 2.8 Bioreporter GFP assays........................................................................................... 17 2.9 Microscopic observations ....................................................................................... 17 2.10 Flow cytometry ..................................................................................................... 18 CHAPTER III - RESULTS ............................................................................................... 20 3.1 Collection and processing of root exudates of B. distachyon Bd21 ....................... 20 3.2 Analytical profiling of root exudates for the presence of osmoprotectant compounds .................................................................................................................... 21 3.3 P. synxantha 2-79 is a competent colonizer of the rhizosphere of B. distachyon Bd21 .............................................................................................................................. 25 3.4 Analysis of water stress response pathways in the P. synxantha 2-79 genome ...... 26 3.5 The protective effect of root exudates and their constituents on P. synxantha 2-79 under conditions of water stress .................................................................................... 35 3.6 The effect of water stress on the expression of defense pathways present in the 2-79 genome .......................................................................................................................... 36 CHAPTER IV – DISCUSSION........................................................................................ 41 APPENDIX A - Metabolites in root exudates from B. distachyon Bd21 ......................... 50 APPENDIX B - Amino Acid in root exudates of B. distachyon Bd21 ............................. 53 APPENDIX C - Putative pathways and homologs ........................................................... 54 v APPENDIX D - Bacterial strains and plasmids used in this study ................................... 62 APPENDIX E – Oligonucleotides used in this study ....................................................... 64 REFERENCES ................................................................................................................
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