Investigating Root-Knot and Soybean Cyst Nematode Parasitic Interactions through Transcriptomic Analyses of the Host and Parasite DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Ellie Walsh Graduate Program in Plant Pathology The Ohio State University 2016 Dissertation Committee: Professor Christopher G. Taylor, Advisor Professor Thomas Mitchell Professor Terry Niblack Professor Margaret Redinbaugh Copyrighted by Ellie Kathleen Walsh 2016 Abstract Plant-parasitic nematodes are a major threat to global agricultural production. Root-knot nematodes (RKN, Meloidogyne spp.) are arguably the biggest threat, capable of parasitizing virtually every crop. Soybean cyst nematode (SCN; Heterodera glycines) has a narrow host range, but is the most destructive pathogen of a particularly important crop, soybean (Glycine max). RKN’s wide host range makes crop rotation often inadequate for management, and host resistance is unavailable in many crops. Effective resistance is available against SCN however populations have adapted to the most frequently used sources of resistance. RKN and SCN both induce elaborate feeding sites. In addition to being the sole source of nutrition, the feeding sites are the primary targets of nematode secretions to manipulate host cellular functions; consequently, they are very important interfaces of the interaction. The general aim of this research was to elucidate changes in the transcriptome underlying the successful interaction between these nematodes and their hosts. Although the use of RNA interference (RNAi) to knockdown nematode genes is actively being pursued as a new strategy for nematode control, little is known about the effects of general RNAi mechanisms during parasitism. As the suppression of RNAi has been characterized in other pathosystems, I hypothesized that parasitic nematodes may also be influencing these pathways. Tanscriptomic analysis of genes associated with RNAi machinery and target genes indicates that RNAi-regulated pathways are altered during the parasitic interaction. Using a silenced reporter gene, I found the disturbance to be specific to the nematode feeding site. Furthermore, disrupting these pathways with viral suppressors of RNAi renders the host more susceptible to nematode parasitism. Transcriptomic analysis indicates that this effect extends into later stages in parasitism, making the adult female stage of particular interest for further analyses. ii I performed a transcriptomic analysis of adult female M. incognita to address the hypothesis that transcriptional patterns in this later stage of parasitism will reveal new candidate genes encoding proteins that regulate the parasitic process, such as proteins that interact with RNAi among other plant pathways. Results from RNA-Seq analysis and reverse transcriptase PCR indicate that cell wall modifiers likely continue to play an important role in the parasitic interaction. Results from transcriptomic analysis including the putative secretome have highlighted new candidates for functional analysis to determine their role in the interaction. The later stage in parasitism is similarly of interest in SCN. SCN populations are adapting to the most commonly planted host resistance available in soybean, derived from Plant Introduction (PI) 88788 and “Peking.” The resistance response in PI 88788 appears to be longer-lasting than that in Peking, which impacts nematodes’ early development. Due to my focus on the adult female stage, I chose to investigate parasitism on PI 88788. I hypothesize that transcriptional differences between females from populations avirulent and virulent on PI 88788 may play a role in their adaptation to resistance. Results from this study indicate that the expression of an effector-like gene may have been lost in virulent populations, presumably allowing them to evade host detection and subsequent defense responses. iii Acknowledgements The research detailed herein would not have been possible without a lot of guidance and support. I have been very fortunate to work in the Department of Plant Pathology at The Ohio State University, which houses amazing faculty, fellow students, and staff. I would like to specifically acknowledge those individuals that have directly impacted this body of work. Therese Miller – the “mother of nematodes,” and Bob James – the greenhouse keeper contributed significantly to the success of experiments. My College of Wooster advisee, Allison Grenell, both executed experiments, and was instrumental to my own growth as a scientist and as a teacher; it is difficult to say who mentored whom. Numerous postdoctoral scientists have shared their time to teach me, namely Drs. Bryan Cassone, Gina Pengue, Phanikanth Turlapati, Yuhong Li, and Linjian Jiang. I am also indebted for the tremendous efforts of Monica Lewandowski, Lynn West, Ken Nanes, Niqui Beckrum, and Ramona Powell in handling graduate school logistics, likely more so than I will ever fully realize. I would also like to thank all of my committee members for their support, unwavering confidence in me, and tremendous patience. Finally, I would like to express my gratitude and admiration for my major advisor, Dr. Christopher G. Taylor. His ability to convey his passion for research inspired me to pursue this degree and his guidance and relentless encouragement have allowed me to see it to completion. iv Vita 2008....................................................B.S. Plant Science, International Agriculture & Rural Development, Cornell University 2010....................................................M.S.Ed. Science Education, CUNY Lehman College 2008 – 2010........................................Science Teacher, 7th grade, The Melrose School 2010 – 2012........................................Graduate Fellow, Plant Molecular Biology & Biotechnology, The Ohio State University 2012....................................................M.S. Plant Pathology, The Ohio State University 2013....................................................Graduate Teaching Assistant, Plant Disease Diagnosis, The Ohio State University 2014....................................................Instructor, OSU International Diagnostics Short Course Program, The Ohio State University 2015....................................................Instructor, Workshop entitled “Investigating Fungi in the Environment,” University of Puerto Rico, Mayaguez 2012 to present ..................................Graduate Research Associate, Department of Plant Pathology, The Ohio State University v Publications Testen AL, Walsh EK, Taylor CG, Miller SA, Lopez-Nicora H. (2014) First report of bloat nematode (Ditylenchus dipsaci) infecting garlic in Ohio. Plant Disease 98(6): 859- 859. Veturi Y, Kump K, Walsh EK, Ott O, Poland J, Kolkman JM, Nelson RJ, Balint-Kurti P, Holland J, Wisser R. (2012) The longitudinal mixed linear model: an information- rich statistical technique for analyzing disease resistance data. Phytopathology 102(11):1016- 25. Chung C, Poland JA, Kump K, Benson J, Longfellow JM, Walsh EK, Balint-Kurti PJ, Nelson RJ. (2011) Targeted discovery of quantitative trait loci for resistance to northern leaf blight and other diseases of maize. Theoretical and Applied Genetics 123(2):307- 326. Chung C, Longfellow J, Walsh EK, Kerdieh Z, Esbroek GV, Balint-Kurti P, Nelson R. (2010) Resistance loci affecting distinct stages of fungal pathogenesis in maize: use of introgression lines for QTL mapping and characterization. BMC Plant Biology 10:103. Field of Study Major Field: Plant Pathology vi Table of Contents Abstract .................................................................................................................. ii Acknowledgments ................................................................................................. iv Vita ..........................................................................................................................v Table of Contents ................................................................................................. vii List of Tables ........................................................................................................ ix List of Figures ...................................................................................................... xii Chapter 1: Literature Review 1.1. Plant Parasitism in the Phylum Nematoda: Root-Knot and Soybean Cyst Nematode Pathogenesis .......................1 1.2. Plant-Parasitic Nematodes: Effects on Agricultural Production ..........6 1.3. Root-Knot Nematode Parasitism .......................................................11 1.4. Host Resistance to Root-Knot Nematodes .........................................15 1.5. Soybean Cyst Nematode Parasitism ..................................................16 1.6. Host Resistance to Soybean Cyst Nematodes ....................................18 1.7. Host Response to RKN & SCN Parasitism ........................................22 1.8. Role of RNA Silencing in Plant-Parasitic Nematode Research .........24 1.9. References ..........................................................................................28 Chapter 2: Root-Knot Nematode Parasitism Suppresses RNA Silencing 2.1. Abstract ..............................................................................................39 2.2. Introduction ........................................................................................40 2.3. Materials