DETECTION OF ARTHROPOD TRANSMITTED VIRUSES OF CEREAL CROPS AND RNAI INDUCED RESISTANCE TO WHEAT STREAK MOSAIC VIRUS By PATRICK RYDZAK Bachelor of Science in Marine Biology Texas A&M University Galveston, Texas 2012 Master of Science in Biology University of Texas at Tyler Tyler, Texas 2015 Submitted to the Faculty of the Graduate College of the Oklahoma State University in partial fulfillment of the requirements for the Degree of DOCTOR OF PHILOSOPHY July, 2019 DETECTION OF ARTHROPOD TRANSMITTED VIRUSES OF CEREAL CROPS AND RNAI INDUCED RESISTANCE TO WHEAT STREAK MOSAIC VIRUS Dissertation Approved: Astri Wayadande, Ph.D. _________________________________________ Dissertation Chair Francisco Ochoa Corona, Ph.D. _________________________________________ Co-Advisor Robert Hunger, Ph.D. __________________________________________ Committee Member Robert Burnap Ph.D. __________________________________________ Committee Member ii ACKNOWLEDGEMENTS On July 8, 2016, with the help of my parents I took the next step of my academic career by moving to Stillwater, Oklahoma to begin my doctorate at Oklahoma State University. Now in 2019, exactly three years to the day that I first moved to Stillwater, I will be defending my Ph.D. dissertation. This journey has been life changing for me and there are so many people that I would like to thank for their unwavering support. First, to my co-advisors Dr. Astri Wayadande and Dr. Franscisco Ochoa-Corona, I am honored to have had the privilege to be your student and witness you both be awarded the title of full professor. While working with you two over the years, I have never once been made to feel like ‘just your student’. Instead, every day you have treated me like a peer and a professional, always showing me respect even when I felt it was unearned. This has meant more to me than you can know, thank you. To my other committee members, Dr. Robert Hunger and Dr. Robert Burnap, thank you for your support, your willingness to teach me and your constant positivity. I do not believe I could have selected a better group of scientists to be on my thesis committee and I am grateful for everything you have done for me. I would also like to acknowledge the entire department of Entomology and Plant Pathology. Our department head, Dr. Phil Mulder. In my mind, Dr. Mulder is the quintessential example of what a leader should be, confident, attentive and gregarious, traits I hope to emulate in the future. To the faculty, staff and students of the EPP department, thank you for letting me be a part of your family over these three years. It has been an absolute pleasure to be a part of such a supportive environment where everyone you are surrounded by is pushing you to improve and succeed. A special thanks to NIMFFAB and the Virus Chasers, I have learned so much from working with all of you closely in the laboratory every day. It is exciting and fulfilling to watch you succeed and I can’t wait to see where you end up in your professional lives. Another special thanks to Dr. Peter Hoyt and everyone at the Core Facilities at Oklahoma State University for your assistance with my sequencing projects, I could not have done this work without you. Lastly, I would like to thank my parents Steve and Diane and my brother Andrew. Thank you for always believing in me. If any of you have ever had doubts about my ability to succeed you have never let them show. Once again, thank you to everyone mentioned above and to so many other who I do not have the space to mention by name, I am beyond grateful to you for being a part of this journey with me. iii Acknowledgements reflect the views of the author and are not endorsed by committee members or Oklahoma State University. Name: PATRICK RYDZAK Date of Degree: JULY, 2019 Title of Study: DETECTION OF ARTHROPOD TRANSMITTED VIRUSES OF CEREAL CROPS AND RNAI INDUCED RESISTANCE TO WHEAT STREAK MOSAIC VIRUS Major Field: PLANT PATHOLOGY Abstract: Bread wheat (Triticum aestivum) is one of the most important crops cultivated worldwide both in terms of nutrition and economic value. Some of the greatest challenges wheat growers face around the world are plant viruses, which may induce symptoms such as stunting or discoloration and can lead to yield losses, or in extreme cases, total crop failure. To identify potential solutions to the threat posed by plant viruses in wheat, one of the most important steps is to accurately and quickly detect and discriminate between viruses so the appropriate management strategy may be applied. Two of the most powerful technologies currently available for the detection and discrimination of plant viruses are PCR and massive parallel sequencing (MPS). The objectives of this study are to utilize multiplex PCR in combination with high- resolution melting (HRM), as well as the MPS based Electronic-Probe Diagnostic Nucleic Acid Analysis (EDNA), to develop new tools for the detection and discrimination of viruses of wheat such as Wheat streak mosaic virus (WSMV). Next, to test the efficacy of RNA interference (RNAi) as a potential treatment to induce resistance to WSMV in wheat. Primers were designed to simultaneously detect WSMV, Maize mosaic virus (MMV) and Barley yellow dwarf virus (BYDV) in multiplex, and to discriminate between the three species of BYDV using HRM. E- probes capable of detecting 21 different viruses of cereal in metagenomic data were designed and the theoretical limit of detection was assessed for WSMV using in silico predictive models. These models were validated by sequencing known ratios of WSMV to wheat nucleic acids in a series of dilutions, then analyzed using EDNA on the subsequent metagenomic data. Gallagher, a wheat variety susceptible to WSMV, was injected with an RNAi construct specific to WMSV, then inoculated with WSMV. Viral titer of WSMV was then monitored at 7, 14 and 21 days post inoculation with qPCR and results were compared to WSMV inoculated and uninoculated, susceptible and resistant wheat varieties. It was determined that susceptible wheat treated with RNAi specific to WSMV prior to inoculation with WSMV had significantly reduced viral expression. iv TABLE OF CONTENTS Chapter Page I. INTRODUCTION ......................................................................................................1 Objectives ..........................................................................................................8 II. REVIEW OF LITERATURE....................................................................................9 Introduction to Wheat ........................................................................................9 T. aestivum Life History .............................................................................10 T. aestivum Anatomy and Growth Stages ...................................................10 Management of Wheat Production Loss .....................................................11 Viruses of wheat ..............................................................................................12 Wheat streak mosaic virus ..........................................................................13 Barley yellow dwarf virus ...........................................................................16 Polymerase Chain Reaction .............................................................................18 High Resolution Melting..................................................................................20 Massive Parallel Sequencing ...........................................................................21 Metagenomics ..................................................................................................22 E-Probe Diagnostic Nucleic Acid Analysis .....................................................23 RNA Interference .............................................................................................24 Research Justification ......................................................................................27 References ........................................................................................................31 III. COMBINING MULTIPLEX PCR AND HIGH-RESOLUTION MELTING FOR THE DETECTION AND DISCRIMINATION OF ARTHROPOD TRANSMITTED VIRUSES OF CEREALS ................................................................................40 Abstract ............................................................................................................40 Introduction ......................................................................................................41 Materials and Methods .....................................................................................43 Results ..............................................................................................................46 Discussion ........................................................................................................51 References ........................................................................................................54 v Chapter Page IV. THE VALIDATION OF IN SILICO PREDICTIVE MODELS FO THE LIMIT OF DETECTION OF WHEAT STREAK MOSAIC VIRUS WITH ELECTRONIC PROBE DIAGNOSTICS ...............................................................................................58 Abstract ............................................................................................................58 Introduction ......................................................................................................59 Materials and Methods .....................................................................................62