Functional Gene Analysis of Resistance QTL towards Phytophthora sojae on Soybean Chromosome 19 Dissertation Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Anna K. Stasko Graduate Program in Plant Pathology The Ohio State University 2018 Dissertation Committee Anne E. Dorrance, Advisor Joshua J. Blakeslee Leah K. McHale Christopher G. Taylor Feng Qu Copyrighted by Anna Kathryn Stasko 2018 Abstract Phytophthora sojae is the causal agent of Phytophthora root and stem rot of soybean. One of the most effective disease management strategies against this pathogen is the use of resistant cultivars, primarily through single gene, Rps-mediated resistance. However, numerous populations of P. sojae have adapted to most Rps genes that are deployed in modern soybean cultivars, rendering them susceptible to this pathogen. Quantitative resistance, conferred by quantitative disease resistance loci (QDRL), offers an alternative to Rps-based resistance. Previous studies mapped two QDRL to chromosome 19 in the soybean cultivar Conrad, which has a high level of quantitative resistance. A recombinant inbred line (RIL) population derived from a cross of Conrad by Sloan (a moderately susceptible cultivar) used for mapping these QDRL was advanced to the F9:11 generation. This population was used to map/re-map the QDRL towards three isolates of P. sojae, and one isolate each of Pythium irregulare and Fusarium graminearum, using the SoySNP6K BeadChip for high-density marker genotyping. A total of ten, two, and three QDRL and suggestive QDRL were found that confer resistance to P. sojae, Py. irregulare, and F. graminearum, respectively. Individual QDRL explained 2-13.6% of the phenotypic variance (PV). One QDRL for both Py. irregulare and F. graminearum co-localized on chromosome 19. This resistance was contributed by Sloan and was juxtaposed to a QDRL for P. sojae with resistance ii contributed from Conrad. Alleles for resistance to different pathogens contributed from different parents in the same region, the number of unique QDRL for each pathogen, and the lack of correlation of resistance suggest that different mechanisms are involved in resistance towards these three pathogens. Interestingly, the QDRL located on chromosome 19 contained several genes related to auxin processes, which are known to contribute to susceptibility to several pathogens in Arabidopsis and may contribute to susceptibility of soybean to P. sojae. In this study, auxin metabolites were measured in P. sojae mycelia, media from P. sojae liquid cultures, and inoculated soybean roots. Auxin precursors were detected in the mycelia of P. sojae as well as the synthetic media. More importantly, auxin levels were significantly higher in inoculated roots than the mock controls in both resistant and susceptible genotypes at 48 hours after inoculation (hai). To examine the role of auxin transport in susceptibility to P. sojae, the nucleotide sequences and expression of root-related soybean auxin efflux transporters, GmPINs, were compared between Conrad and Sloan. There were sequence differences between the two cultivars; however, experimental variability prevented accurate detection of expression differences through a quantitative PCR approach. An auxin transport inhibitor and a synthetic auxin were applied to Conrad and Sloan to assess changes in infection of these cultivars with chemically altered auxin processes. As with the gene expression analysis, experimental variation prevented us from determining the exact effect of these treatments. Finally, several different approaches were used to begin developing a system for functional gene analysis, including composite plant-based hairy roots, cotyledon- based hairy roots, and virus-induced gene silencing (VIGS). Composite plant-based hairy iii roots were difficult to inoculate with P. sojae, Py. irregulare, and F. graminearum. Cotyledon-based hairy roots allowed for more consistent inoculation with P. sojae and expedited experimental testing of RNAi constructs targeting candidate genes. One of these constructs was able to reduce the expression of its target gene in three soybean genetic backgrounds. A Bean pod mottle virus (BPMV) VIGS vector used here moved systemically into soybean roots but was not effective at silencing candidate gene targets in this tissue. Future studies should continue to refine environmental/experimental conditions to reduce variation and develop a reliable method of assessing change in quantitative disease resistance to define the roles of candidate genes. iv Dedication To Great Uncle Michael Stasko, who laid the foundation for this work, and to Our Lady of Perpetual Help, who championed it. v Acknowledgments If it takes a village to raise a child, it certainly takes a department to train a PhD. This journey would not have been possible without the help and support of many people. I would like to thank Anne Dorrance, my advisor, for all of her help, guidance, and patience during this process. I also thank my committee members for their time, advice, and lab space. To all past and present members of the Dorrance lab, especially Chrissy Balk, Damitha Wickramasinghe, Clifton Martin, Cassidy Gedling, Deloris Veney, and Jonell Wenger, thanks for all your help will all of my experiments, no matter how tedious they were. I also extend a special thank you to Leslie Taylor, Shuang Xie, Brittany Tangevald, Dee Marty, Junping Han, Yun Lin, and Ella Lin for their help and expertise. To the unsung heroes of Selby Hall, Lynn West, Ken Nanes, Lee Wilson, and Bob James, you take care of many details that make all of this work possible. Thanks for making life a little easier for the rest of us. Thanks to plant pathology graduate students and members of the SoyRes team, especially Ellie Walsh, Anna Testen, Tim Frey, and Rebecca Kimmelfield. Thanks also to the faculty at Ohio State and Concordia College, especially Mark Jensen and Laura Aldrich-Wolfe, who helped to build the foundation for this this journey. Thanks also to the Center for Applied Plant Sciences, SEEDS, the Ohio Soybean Council, and the United Soybean Board for funding this project and my fellowship. vi Finally, I would like to thank my family and friends. Maren, your friendship means so much to me. Thanks for being a bright presence in my life. To my biking partners Susan and Joanne, and the members of my extended “family” at St. Mary’s, Gina, Joe, Barb, Rich, Bernie, Tencha, MaryAnn, Marjorie, Kaalyn, and Caitlyn, thanks for helping me lead a somewhat balanced life. To Nick, Mom, and Dad, thank you for all of the phone calls, support, advice, and other countless help. Thanks be to God, who makes all things possible. Soli Deo Gloria. vii Vita February 1990 Born-San Antonio, TX May 2012 B.A. in Biology and Chemistry, Concordia College August 2012- August 2014 Graduate Research Associate, The Ohio State University May 2016 M.S. Plant Pathology, The Ohio State University August 2014-present Graduate Research Fellow, The Ohio State University Publications Stasko, A.K., Wickramasinghe, D., Nauth, B.J., Acharya, B., Ellis, M.L., Taylor, C.G., McHale, L.K., and Dorrance, A.E. 2016. High density mapping of resistance QTL towards Phytophthora sojae, Pythium irregulare, and Fusarium graminearum in the same soybean population. Crop Sci. 56:2476-2492 Liu, Z.H., Zhong, S., Stasko A.K., Edwards M.C., and Friesen, T.L. 2012. Virulence profile and genetic structure of a North Dakota population of Pyrenophora teres f. teres, the causal agent of net form net blotch of barley. Phytopathology 102:539-546. Fields of Study Major Field: Plant Pathology viii Table of Contents Abstract ............................................................................................................................... ii Dedication ........................................................................................................................... v Acknowledgments.............................................................................................................. vi Vita ................................................................................................................................... viii Table of Contents ............................................................................................................... ix List of Tables ................................................................................................................... xiii List of Figures ................................................................................................................... xv Chapter 1: Introduction ....................................................................................................... 1 Soybean and Phytophthora sojae.................................................................................... 1 Quantitative resistance .................................................................................................... 3 Plant susceptibility factors .............................................................................................. 6 Auxin’s role in plant roots .............................................................................................. 8 Auxin’s role in plant-microbe interactions ................................................................... 12 Research Objectives ...................................................................................................... 16 Hypothesis: ..............................................................................................................