An Investigation of The Role of Amino Acids in Plant-Plant Parasitic Nematode Chemotaxis and Infestation DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Timothy S. Frey Graduate Program in Plant Pathology The Ohio State University 2019 Dissertation Committee; Professor Christopher G. Taylor, Advisor Professor Soledad M. Benitez Ponce Professor Pierluigi Bonello Professor Joshua Blakelslee Copyright by Timothy S. Frey 2019 Abstract Plant parasitic nematodes are economically devastating crop pests. They are responsible for billions of dollars in crop loss in all crop growing regions of the world. Management of these pests is difficult and involves many laborious, toxic or marginally effective measures that in the best of circumstances do not lead to complete control. Plant-parasitic nematodes are obligate biotrophic parasites and must obtain all of their nutrition from a living host. Plant parasitic nematodes lack the metabolic enzymes to synthesize certain amino acids, thus it is essential for them to obtain them from a plant. Because of the essential nature of amino acids for plant- parasitic nematodes the general aim of this study was to investigate their impact throughout nematode life cycles. This investigation examined the role of amino acids throughout the lifecycle of root-knot nematode, Meloidogyne incognita, and as a factor for chemotaxis of the sugar beet cyst nematode, Heterodera schactii, and the soybean cyst nematode, Heterodera glycines as well as the model nematode, Caenorhabditis elegans. The role of amino acids in M. incognita infestation was investigated using amino acid homeostasis knockouts and overexpression lines. Changes in threonine homeostasis, particularly in the threonine catabolism overexpression mutant 35s omr1-7 were found to lead to decreased early infestation, late infestation, fecundity and increased male production in M. incognita. These effects were particularly pronounced at higher competition levels. Threonine homeostasis is important for successful nematode infestation and manipulation of host amino acid homeostasis should be further explored for its impacts on M. incognita parasitism ii The role of amino acids in chemotaxis was investigated using choice assays. Threonine, aspartic acid, proline, and histidine were found to be the most attractive amino acids for M. incognita. Cysteine, phenylalanine and serine were the most repellant amino acids. The model nematode C. elegans showed different preferences for amino acids. Amino acids were present in soybean (Glycine max) root exudates, and a reconstructed blend of amino acids present in soybean root exudates was attractive to nematode species that can utilized soybean as a host, including M. incognita and H. glycines. These data could be utilized to create new integrated pest management strategies for nematode control. iii Acknowledgements In order to make this research happen a lot of support, encouragement and assistance was required. First, the Ohio State Plant Pathology Department has an amazing atmosphere conducive to learning and collaboration. The wonderful logistical work put in by Monica Lewandowski, Ken Nanes, Lori Jones and Lynn West, was crucial to my success. The Plant Pathology Department faculty was warm, friendly, and always willing to give advice. Particularly, I would like to thank Larry Madden, Mike Ellis, Peg Redinbaugh, Anne Dorrance, Pierce Paul, Sally Miller and Soledad Benitez Ponce for their support. I would also like to thank the friendly people of Selby hall – Bob, Lee, Mark Jones, Jane Todd, Tea and many others for making this a wonderful work environment. I would like to thank the wonderful Post. Docs who I have had a chance to work with, including Sawsan Elateek, Gina Pengue, and Wenshuang Xie. I have gleaned much from your technical and life wisdom. I would like to acknowledge my fellow Taylor Lab graduate students – Ellie Walsh, Rebecca Kimmelfield, Brittany Nauth, Dee Marty, Rachel Kauffman, Cecilia Chagas de Freitas, Krystel Navarro Acevedo, Marlia Bosques Martinez, Donald Gillis, and Edwin Navarro – Your countless readings and criticism of my various ideas, presentations, and manuscripts were important for my success. Your support and friendship kept me sane while over the course of the degree. I iv would particularly like to thank Ellie for teaching me many things in the lab over the years. Finally, a huge thanks is owed to Rebecca for countless hours editing my thesis chapters. I would like to acknowledge all of the interns that I have had the pleasure of working directly or indirectly with over the years – Allison Grenell, Krystel Navarro Acevedo, Guillermo Valero David, Madeline Horvath, Amanda Lietz, Gilbert Chen. You taught me so much. Your Curiosity inspires me and I am proud to see where you are now. This work would not have been the same without you. I must also acknowledge the following lab members –Leslie Taylor, for propagation of thousands of Arabidopsis plants, and for allowing me to fiddle around in your garden which helped keep me mentally sharp during grad school. Therese Miller, for the maintenance of the greenhouse nematode collections, and for your many contributions to making the Taylor Lab a fun place to work. I would like to acknowledge OSU SROP program which I had the pleasure of working with for 2 summers. The leadership of this program Cyndi Freeman, Pamela Thomas, Ana’ Brown and Jesse Goliath for inspiration and encouragement in my professional pursuits. All of my inspiring students in the program, I am overwhelmed with pride to see what you have achieved. I would like to acknowledge PPGSA, and my fellow graduate student in Plant Pathology. Thanks for all of the opportunities for leadership and to build relationships among fellow graduate students. I would like to acknowledge the amazing leadership the year I was President – particularly the thoughtful guidance provided by Emile Gluck-Thaler and Coralie Farinas. v Another debt is owed to my current and former committee members, Dr. Ana Alonso, Dr. Soledad Benitez Ponce, Dr. Pierluigi (Enrico) Bonello, and Dr. Joshua Blakeslee. Your questions, guidance and patience have been critical for the success of this dissertation. A huge debt of gratitude is owed to my advisor Dr. Christopher G. Taylor – your deep patience, wise guidance, consistent cheerleading, bubbly passion, and enduring curiosity, made this Ph.D. one of the best experiences that I have ever had in my life. I would like to acknowledge my parents, Charles and Susan Frey, for imbuing me with a love of learning and for always supporting me in all the crazy directions that life has taken me. Finally, I would like to acknowledge my wife, Alessandra Frey, for all of your support, patience and curiosity about the research contained within; I could not have done it without you. vi Vita 2011……………………………………………….B.S. Plant Health Management, minor in Horticulture, The Ohio State University 2011-2012……………………………………….Research Assistant, Taylor Lab, The Ohio State University 2014…………………………………………………..Teaching Assistant, Plant Pathology 5040/41, Mycology, The Ohio State University 2014,2015,2016,2018………………………..…………Guest Lecturer, Plant Disease Diagnostics Course, The Ohio State university 2015-2016…………………………………………..Graduate Teaching Assistant, Summer Research Opportunity Program, The Ohio State University 2015-2018………………………………………………..Guest Lecturer, Plant Pathology 5030, Nematology, The Ohio State University 2012-Present……………………………………….Graduate Research Assistant, The Department of Plant Pathology, The Ohio State University Fields of Study Major Field: Plant Pathology vii Table of Contents Abstract……………………………………………………………………………………………………………………………………ii Acknowledgements………………………………………………………………………………………………………………….iv Vita…………………………………………………………………………………………………………………………………………vii Table of Contents……………………………………………………………………………………………………………………viii List of Tables……………………………………………………………………………………………………………………………xi List of Figures…………………………………………………………………………………………………………………………xiii Chapter 1: Literature Review…………………………………………………………………………………………….….……1 1.1 The Impact of Plant-Parasitic Nematodes on Agricultural Production……………………….…….……1 1.2 Lifecycles of Important Nematodes………………………………………………………………………………..……5 1.3 Current Options for Nematode Control………………………………………………………………………………10 1.4 Amino Acids; Their Influence on Nematode Parasitism………………………………………………………25 1.5 References…………………………………………………………………………………………………………………………35 Chapter 2 – Threonine Homeostasis Plays a Role in Successful Root-knot Nematode Infestation of Arabidopsis ……………………………………………………………………………………………………………….…..76 2.1Abstract………………………………………………………………………………….……………………………………….…76 2.2 Introduction………………………………………………………………………………………………………………………77 viii 2.3 Materials and Methods…………………………………………………………………………………………………….83 2.4 Results…………………………………………………………………………………………………………….………………..90 2.5 Discussion…………………………………………………………………………………………………….……….………….94 2.6 Acknowledgments………………………………………………………………………………………..…………………..99 2.7 References…………………………………………………………………………………………………..…….…………….100 Chapter 3 – Amino Acids Play a Role in Plant-Parasitic Nematode Chemotaxis……….…….……….128 3.1 Abstract…………………………………………………………………………………….........................................128 3.2 Introduction…………………………………………………………………………………………………..…….…………..130 3.3 Materials and Methods……………………………………………………………………….………..……….……...…134 3.4 Results…………………………………………………………………………………………….………..………….………….145