Functional Characterization of Magnaporthe oryzae Effectors in the Infective Process of Rice Thesis Presented in Partial Fulfillment of the Requirement for the Degree Master of Science in the Graduate School of The Ohio State University By Oscar Burbano-Figueroa, B.Sc. Graduate Program in Plant Pathology The Ohio State University 2011 Thesis Committee: Dr. Thomas Mitchell, Advisor Dr. Anne E. Dorrance Dr. Guo-Liang Wang Copyright by Oscar Burbano-Figueroa 2011 ABSTRACT Rice is one of the three most important food crops of the world with an increasing worldwide production during the last decade. One of the major constraints in rice production is rice blast disease caused by the fungus Magnaporthe oryzae. During the last decade, the genomic sequence of rice and this pathogen were completed allowing the computational prediction of genes. The bio-molecular function of these predicted genes are largely unknown. M. oryzae genes MGG00194 and MGG03356 were identified as putative effectors of host defense using a protoplast transient expression system. Effectors are predicted to be secreted in the Blast Interfatial Complex in a specific spatiotemporal pattern, secreted during the development of the invasive hyphae or at least during the late stage of appresorium formation and penetration prior to interfacing with the plant cell. Consequently, the aim of this work was to determine how the putative effectors MGG_00194 and MGG_03356 are involved in the pathogenic process using in- planta secretion and functional analysis. M. oryzae strains that were transformed to constitutively express these genes were obtained and the effect of this condition on vegetative growth and pathogenicity was evaluated. Our results suggest that overexpression of MGG_03356 allows the pathogen to evade the host hypersensitive response possibly via degradation of pectin in the cell wall and consequently decreasing the mechanical sensing of the pathogen. Fluorescent expression patterns of these genes using GFP-tagged proteins in transformant strains were obtained in specific pathogen ii race-cultivar/species interactions. Even when two different strategies for homologous recombination were used, no deletion mutants were obtained. iii Dedication Dedicated to Maria del Carmen Figueroa, for her love and patience during all these years " Handeln ist leicht, Denken ist schwer, nach dem Gedanken handeln unbequem" — Johann Wolfgang von Goethe iv ACKNOWLEDGEMENTS I wish to thank my advisor, Dr. Thomas Mitchell, for giving me the opportunity, intellectual support, encouragement and freedom for working in my thesis during the last two years. I am very grateful to Dr. Anne Dorrance and Dr. Guo-Liang Wang, members of my Academic Committee, for his constructive critics during the development of my research and writing of this manuscript. I wish to thank my friends in Columbus and Athens for the unforgettable moments: Yuki, Sara, Marcela, Wanda, Aneta, Carola, Fiorela, Huaso, Jesus, Evelyn, Gautam, Norman, Fernanda, Amy, Veronica, Maria, Nun, Chan Ho. Thanks to Gautam, Nun and Chan Ho for their invaluable help, encourage and discussion during the development of my experiments. I would like to thank my family for all of their love and faith: Jazmin, Paola, Milena, Javier, John, Diego, Julia, Carlos, Juan Carlos, Patricia, Jonathan, Marcos and my grandparents. Thanks to Milena for all the time we spent together during the nights of winter. Finally, I like to thank to Fulbright-LASPAU and COLCIENCIAS for the financial and academic support during these years and Universidad de Nariño, the place where this adventure began. A special thank to Luz Stela Lagos Mora for her invaluable help and encouragement during the first beginning of this process. v VITA 1994…………………………………Colegio Centro de Integracion Popular, Pasto, Nariño, Colombia 2004………………………………....B.Sc. Biology minor Microbiology Universidad de Nariño 2006…………………………………Assistant Researcher Empresa Metropolitana de Aseo, EMAS SA ESP 2006-2008…………………………..Teaching/Researcher Assistant Department of Biology, University of Nariño 2008-present…………………………Graduate Research Associate Department of Plant Pathology, The Ohio State University FIELDS OF STUDY Major Field: Plant Pathology vi TABLE OF CONTENTS ABSTRACT........................................................................................................................ ii VITA .................................................................................................................................. vi LIST OF TABLES ............................................................................................................. ix LIST OF FIGURES ............................................................................................................ x CHAPTER 1: Literature Review ........................................................................................ 1 BACKGROUND ............................................................................................................ 1 Magnaporthe oryzae. .................................................................................................. 2 Effectors...................................................................................................................... 5 Defense responses of the plant.................................................................................... 8 Mechanosensing capacity of the plant cell wall.. ......................................................11 LIST OF REFERENCES.............................................................................................. 15 CHAPTER 2: Functional characterization of Magnaporthe oryzae effectors in the infective process of rice .................................................................................................... 22 INTRODUCTION ........................................................................................................ 22 MATERIALS AND METHODS .................................................................................. 25 Fungal strains, growth conditions and transformation............................................. 25 Plasmids and DNA manipulations.. .......................................................................... 25 Assays for growth, sporulation, appresoria formation and plant infection............... 26 RESULTS AND DISCUSSION.................................................................................... 27 M. oryzae transformation.......................................................................................... 27 Pathogenicity assays.. ............................................................................................... 28 Localization pattern.. ................................................................................................ 29 Knockout mutants.. ................................................................................................... 29 LIST OF REFERENCES.............................................................................................. 37 CHAPTER 3: Overexpression of MGG_03356 modifies virulence of Magnaporthe oryzae isolates................................................................................................................... 40 INTRODUCTION ........................................................................................................ 40 MATERIALS AND METHODS .................................................................................. 43 Fungal strains and transformation............................................................................. 43 Infection assays.)....................................................................................................... 43 RESULTS...................................................................................................................... 45 DISCUSION ................................................................................................................. 47 vii LIST OF REFERENCES.............................................................................................. 63 BIBLIOGRAPHY............................................................................................................. 66 Appendix A: Magnaporthe oryzae strains and transformants ......................................... 75 Appendix B: Rice Leaf Sheath Assay.............................................................................. 84 Appendix C: Distribution of Infection Stages for Overexpression and Native Expression of MGG_03356 in Different Host-Pathogen Interactions ................................................ 86 Appendix D: Distribution of Infection Stages for Overexpression and Native Expression of MGG_03356 in Different Host-Pathogen Interactions ................................................ 88 Appendix E: Evaluation of Pathogenicity of KJ201 and CHNOS Strains Expressing Constitutively MGG_03356 over Transgenic Rice Plants Expressing the Resistance Genes Pi2 and Pi9. ............................................................................................................ 93 viii LIST OF TABLES Table 2.1. Number of transformant colonies obtained from multiple transformation experiments using multiple knockout constructs. Only filamentous colonies were included while abortive small hyg resistantant colonies not were included. The split marker method and single insert construct strategy were used for 70-15 and KJ201, while only the single insert strategy was used for Guy11-Δku80………………………………32 Table 2.2. Sporulation intensity