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(Title of the Thesis)* Deciphering the Indoleacetic Acid Biosynthesis Pathways in the Rhizobacterium Pseudomonas sp. UW4 by Daiana Duca A thesis presented to the University of Waterloo in fulfillment of the thesis requirement for the degree of Doctor of Philosophy in Biology Waterloo, Ontario, Canada, 2017 ©Daiana Duca 2017 Examining Committee Membership The following served on the Examining Committee for this thesis. The decision of the Examining Committee is by majority vote. Dr. Bernard Glick Professor Ph. D. Supervisor Dr. David Rose Professor Ph. D Co-Supervisor Dr. Kirsten Muller Professor Ph. D. Thesis Examination Committee Member Dr. Trevor Charles Professor Ph. D. Thesis Examination Committee Member Dr. Raymond Legge Professor Ph. D. Internal External Thesis Examination Committee Member Department of Chemical Engineering University of Waterloo Dr. Manish Raizada Professor Ph. D External Thesis Examination Committee Member Department of Plant Agriculture University of Guelph Guelph, ON. ii Author’s Declaration I hereby declare that I am the sole author of this thesis. This is a true copy of the thesis, including any required final revisions, as accepted by my examiners. I understand that my thesis may be made electronically available to the public. iii Abstract Healthy plants host, within and on the surfaces of their tissues, diverse endophytic and epiphytic bacteria. Often, this interaction is mutualistic, leading to adaptive benefits for both partners. We refer to these beneficial microbes as plant growth-promoting bacteria (PGPB), as they can have a tremendous positive influence on plant health, yield and productivity. PGPBs can be used as natural biofertilizers to promote plant growth in an environmentally responsible manner. One of the main mechanisms used by PGPB to enhance plant growth is the production of indoleacetic acid (IAA). This compound is central to a plant’s lifecycle and overall functioning. Because of the indispensable role of IAA in plant-growth promotion, there is great interest in genetic manipulation of IAA biosynthesis to maximize phytostimulation. This is a cumbersome task, as the nature of IAA biosynthesis is convoluted; multiple, independent and inter-dependent pathways operate within a single bacterium. The research reported herein strived to decipher the IAA biosynthesis pathways at the genetic and biochemical level, in a particularly effective PGPB known as Pseudomonas sp. UW4. This remarkable rhizobacterium has been shown to enhance plant growth in the presence of flooding, heavy metals, cold, high levels of salt, and phytopathogens. The entire genome of strain UW4 was sequenced in our lab and seven genes were implicated to encode enzymes involved in the indole acetonitrile (IAN) and indoleacetamide (IAM) pathways of IAA biosynthesis. In this work, some of these enzymes were isolated and their catalytic activity was experimentally verified through biochemical assays. Transformants of strain UW4 with increased IAA biosynthetic capacity were created by introducing a second copy of the target IAA-genes, and canola seedlings inoculated with these transformants displayed enhanced root growth. Mutagenesis experiments were undertaken to create deletions in all seven IAA-genes, in order to delineate which of the genes/pathways contribute most to IAA production in this strain. Failure to produce mutants with a reduced ability to synthesize IAA, led to speculation of a third pathway. Biochemical evidence of this pathway is based on the detection of the IPyA metabolite. The IPyA pathway appears to be predominant in strain UW4 and can compensate for disruptions in the IAN/IAM pathways. Genomic screens identified several candidate IPyA pathway genes, however the functional roles of the encoded enzymes remain to be determined. Altogether, this work describes three interconnected IAA biosynthetic pathways in strain UW4. iv Acknowledgements Yu Gu and the Ron Johnson Lab at the University of Guelph are gratefully acknowledged for their technical support with the HPLC analyses. Richard Smith is also kindly acknowledged for LC-MS- MS training. I also thank Kirsten Muller at the University of Waterloo for her guidance with the phylogenetic analyses and all my colleagues who provided insight and expertise that greatly assisted the research. Finally, I wish to recognize and thank Janet Lorv and Merium Fernando for their help with mutant screens and enzyme assays. v Dedications To my parents and my brother - this work is dedicated to you. You have always been an unwavering source of support and encouragement during the challenges of grad school and throughout my entire life. There are no words to describe the love and immense grattitude that I feel for the three of you. Thank you for believing in me and loving me unconditionally. Thank you for the sacrifices you have made to give me every opportunity to succeed. This accomplishment would not have been possible without you. To Bernie, thank you for your wisdom, guidance and inspiration. You have taught me to be a scientist and if I become only half the thinker, half the teacher, half the person that you are, it will surely be one of my greatest accomplishments. To my friends, my home team, my middle-of-the-night, no-matter-what people; thank you for sticking with me all these years, for loving and understanding me throughout my sometimes weird, distant introvert phases. You know who you are- I love you. To Kas-it is entirely possible that I would not have made it through undergrad had you not been there with me every day, every class; and that I would have drowned in the sea of grad research had you not been there to keep my head above the water. You have helped me more than you know. Sometimes you get lucky and you find a soul that grooves with yours. To the Alien- you are a monument of mind. Thank you for the sustenance when I was overwhelmed with writing this thesis and for nourishing my mind when I was starved of insight. I may have never had a moment from myself had you not been as important a part of my life, my consciousness, as you are. Lace up your shoes, I’ll pack a bag and let’s get lost in a worldly adventure for a while… vi Table of Contents Examining Committee Membership ........................................................................................... ii Author’s Declaration .................................................................................................................... iii Abstract ......................................................................................................................................... iv Acknowledgements ........................................................................................................................v List of Figures ................................................................................................................................ x List of Tables ................................................................................................................................ xi Chapter 1 ....................................................................................................................................... 1 Introduction and Literature Review .............................................................................................. 1 1.1 Plant-Microbe Interactions ................................................................................................................ 1 1.2 Application of Plant-Growth-Promoting Bacteria .......................................................................... 2 1.3 Plant Indoleacetic Acid (IAA) ........................................................................................................... 5 1.4 Bacterial IAA in Plant-Microbe Interactions ................................................................................... 6 1.4.1 Bacterial IAA and Pathogenesis ................................................................................................. 7 1.4.2 Bacterial IAA and the Promotion of Plant Growth ............................................................... 11 1.5 The Biological Function of IAA in Bacteria .................................................................................. 14 1.6 IAA Biosynthesis Pathways ............................................................................................................. 20 1.6.1 The Indole-Pyruvic Acid (IPyA) Pathway............................................................................... 20 1.6.2 The Indoleacetamide (IAM) Pathway...................................................................................... 25 1.6.3 The Indoleacetaldoxime (IAOx)-Indoleacetonitrile (IAN) Pathway .................................... 29 1.7 Factors Modulating IAA Production in Bacteria........................................................................... 31 1.8 Genetic Factors involved in the regulation of IAA Biosynthesis................................................. 40 1.9 Introduction to Thesis ..................................................................................................................... 46 1.9.1 Research Goals .......................................................................................................................... 50 1.9.2 Organization of thesis ............................................................................................................... 52 Chapter 2 ...................................................................................................................................
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