DISSERTATION BIOLOGY, COMPARATIVE GENOMICS AND MOLECULAR DIAGNOSTICS OF XANTHOMONAS SPECIES INFECTING RICE AND CORN Submitted by Jillian M. Lang Graduate Degree Program in Cell and Molecular Biology In partial fulfillment of the requirements For the Degree of Doctor of Philosophy Colorado State University Fort Collins, Colorado Fall 2017 Doctoral Committee: Advisor: Jan E. Leach Daniel Bush Anireddy Reddy Valérie Verdier Copyright by Jillian M. Lang 2017 All Rights Reserved ABSTRACT BIOLOGY, COMPARATIVE GENOMICS AND MOLECULAR DIAGNOSTICS OF XANTHOMONAS SPECIES INFECTING RICE AND CORN Emerging bacterial diseases on staple and economically important crops can pose critical threats to food security. Accurate identification of bacterial plant pathogens is the foundation of effective management for growers. This work advances the application of genomics to identify and characterize bacterial plant pathogens in the genus Xanthomonas that can cause destructive diseases on most agricultural crops, including rice and corn. In this thesis, taxonomy, host range, disease phenotypes and basic biology of the following pathogens were established: X. oryzae pv. oryzae, X. o. pv. oryzicola, X. o. pv. leersiae and X. vasicola pv. vasculorum. X. o. pv. oryzae and X. o. pv. oryzicola infect rice and cause bacterial blight and bacterial leaf streak, respectively. X. o. pv. leersiae infects cutgrass (Leersia sp.), weedy grasses that can serve as alternative hosts to X. oryzae and are endemic in all rice growing regions. X. vasicola pv. vasculorum was identified as the causal agent of bacterial leaf streak of corn, an emerging and now wide-spread disease in the United States, that was reported for the first time in 2017. This work established that X. vasicola pv. vasculorum can also infect sorghum and sugarcane and that the US strain is 99% similar to strains isolated over 20 years ago in S. Africa. To develop robust molecular diagnostic tools for these pathogens, unique features needed to be first identified. Using comparative genomics that included closely related bacteria and distant relatives, PCR-based diagnostic tools were developed, then validated using isolated cultures and field grown plant materials. Comparative genomics also contributed to elucidation of the taxonomy and phylogeny of X. o. pv. leersiae and X. v. pv. vasculorum. Characterization of X. o. pv. leersiae revealed adaptations to both the weedy grass hosts and rice. These features include virulence proteins that target homologous host genes (transcription activator like effectors, TALEs) to influence host gene expression. I conclude that X. oryzae is a complex that includes X. oryzae pv. oryzae, X. o. pv. oryzicola and X. o. pv. leersiae and that ii this complex can provide a unique window into pathogen evolution. By better understanding how pathogens adapt to their environments including new hosts, growers can manage surrounding ecosystems more effectively to minimize yield losses and therefore, contribute to food security. iii ACKNOWLEDGEMENTS ‘Alone, all alone Nobody, but nobody Can make it out here alone.’ - From ‘Alone’ in Oh Pray My Wings Are Gonna Fit Me Well, Maya Angelou, 1975 This long journey has been alongside a village of generous, kind humans. I would not have come this far alone. Below is a small attempt to acknowledge them, though no amount of words can fully articulate the gratitude and humility I feel. Dr. Jan Leach: your role in my life is so multi-faceted. You are my mentor, advisor, boss, friend and my family. I love you dearly. You inspire me with your brilliance, strength and immense generosity. Thank you for supporting me, challenging me and giving me more opportunities than I ever thought possible in my life. You have let me see the world and subsequently, grow as a scientist and a person. Dr. Valérie Verdier: Thank you for your generous support both while Owen and I were in France and for the invigorating science while you were in the US. I admire your perseverance, breadth of knowledge and ability to manage and balance so many things. I am incredibly fortunate to have two women who are amazing scientists, leaders and friends. My remaining committee members, Dr. Dan Bush and Dr. Anireddy Reddy: Thank you for serving on my committee and for having patience as my research shifted, turned, and ultimately culminated in this dissertation. Thank you for challenging me with questions of science and career, and the time you spent with me. There are far too many people to thank in the lab for being fun, charismatic co-workers and integral members of my research. I thank you all past and present for your patience as I tackled this challenge while balancing my job duties. Particularly, these last two months as I have driven to completion I know my absence was significant. Thank you for giving me the support and space to complete this massive endeavor. A few people specifically I would like to acknowledge here: Emily Luna and John Long – you both accepted and relieved me of duties while I traveled abroad and as I turned my focus to this document, I am indebted to your willingness to help our group and myself. Your quiet generosity and iv kindness is inspiring. Elysa DuCharme – I still argue you have the ability to read my mind. I am so proud of the scientist you have become and thankful for all the time you worked on all my crazy ideas with grace and calm. This data would not have been generated without you. Paul Langlois – your contributions to optimizing LAMP were no small feat and I admire your organization and patience. Robin Mom and Rémi Pelissier – two excellent interns who traveled from abroad to join me in research. It was a privilege to mentor you. Thank you for your excitement, flexibility and dedication to helping through several projects and making your summers in the lab fun and productive. Drs. Kirk Broders, Tamra Jackson- Ziems, Kevin Korus, Jeff Hoy and Terra Hartman: thank you for coordinating corn and sugarcane samples and facilitating the challenging progress of reporting bacterial leaf streak of corn in the USA for the first time. Lastly, to all the wonderful undergraduate and high school students who have contributed in some form to this work from growing countless plants in the greenhouse to washing dishes to pouring mountains of media, I thank each of you for your bright energy, hard work, excitement about plant pathology and smiles. Funding for this work and many miles traveled came from the Embassy of France, USDA, C2B2, BecA, IS-MPMI and APS. My dear friends and colleagues at IRD (past and present) – Drs. Alvaro Pérez-Quintero, Ralf Koebnik, Mathilde Hutin, Céline Pesce, Tu Tran and Boris Szurek – thank you for hosting me and continuing to engage in collaborative science and friendship, I am grateful to know such an intelligent, lovely group of people. Drs. Ned Tisserat and Howard Schwartz while you have both retired, you hold a dear place in my scientific journey. I admire your immense knowledge of plant pathology and dedication to it. Thank you for mentoring me and giving me the foundation to build this degree on. Drs. Scott Fulbright and Stephen Chisholm– you provided me with so much encouragement when I was deciding to complete this degree and throughout its duration. Thank you for having faith in me and pushing me to take this on. Dr. Jonathan Jacobs – you are my scientific compliment and have become a beloved friend. Thank you for your neverending enthusiasm, support at the bench and beyond and so much laughter. Dr. Federico Martin – you hold a special place in my heart. Thank you for always v encouraging and supporting me. You taught me to be paitient and confident and to strive for balance when I needed it most. To Kate and Mitch Schneider – you are such dear, generous friends. I am incredibly grateful to you for the shelter, love and support these past ten years, you are my family. To Paul Covey – our ability to tackle and balance the diverse challenges of life humbles and amazes me. Thank you for your support scientifically and personally over so many years. To my mother (Kath): you have donated hundreds of hours caring for my son, my dog, preparing meals, offering refuge and support endlessly and selflessly. You have been my biggest cheerleader my entire life and I never take that for granted. Jamie, my dear sister and lifelong friend, you have been my rock and I know the three letters that will soon follow my name are because of you both. Thank you, I love you dearly. Dr. Christine Battista – you are my beacon of light, laughter, wisdom, strength and comfort. You hold such a large place in my life and I am certain I would not have survived this journey without you. Finally, I dedicate this work to my son, Owen Jude Covey. You are my favorite human. You have given me a reason to pursue science for a greater good and have been so patient through this big adventure. I hope you can see that hard work and dedication can culminate in a bountiful basket of friends and family, not just results and pride. My love for you is infinite. vi TABLE OF CONTENTS ABSTRACT .................................................................................................................................................. ii ACKNOWLEDGMENTS ........................................................................................................................... iv CHAPTER 1. INTRODUCTION ............................................................................................................... 1 1.1 RICE AND CORN: STAPLE CROPS AND MODEL