Functional Characterization of Extracellular Protease Inhibitors of Phytophthora Spp and Their Targets Tomato Proteases

FUNCTIONAL CHARACTERIZATION OF EXTRACELLULAR PROTEASE INHIBITORS OF PHYTOPHTHORA SPP AND THEIR TARGETS TOMATO PROTEASES DISSERTATION Presented in Partial Fulfillment of the Requirements for The Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Jing Song, M.S. * * * * The Ohio State University 2007 Dissertation Committee: Dr. Sophien Kamoun, Adviser Approved by Dr. Anne E. Dorrance Dr. Terrence L. Graham Adviser Dr. Margaret G. Redinbaugh ______________________ Dr. Eric J. Stockinger Graduate Program in Plant Pathology ABSTRACT The interplay between proteases and protease inhibitors during plant- pathogen interaction represents a common strategy for defense and counterdefense. The plant pathogens Phytophthora infestans and Phytophthora mirabilis secrete effectors such as protease inhibitors that facilitate host colonization through a defense-counterdefense mechanism. The P. infestans serine protease inhibitors EPI1 and EPI10 physically bind and inhibit the tomato serine protease P69B. On the other hand, the P. infestans cysteine protease inhibitor EPIC2B targets PIP1, a papain-like protease that has close similarity to another tomato cysteine protease Rcr3, which is required for the fungal resistance and Avr2 hypersensitivity in Cf-2 tomato. The objective of this research is to characterize these protease inhibitors and their association with specific targets in the host. We studied the structure and activities of these protease inhibitors and their target proteases using recombinant proteins expressed in Escherichia coli and Nicotiana benthamiana. PIP1-His was pulled down using coimmunoprecipitation with anti-FLAG resin from N. benthamiana apoplast by recombinant protein FLAG-EPIC2B, suggesting physical interaction of EPIC2B and PIP1. Similarly, tomato protease Rcr3pim was shown to be a common target for both the ii Cladosporium fulvum effector Avr2 and P. infestans effector EPIC2B using pull down assays and DCG-04 activity profiling. However, unlike Avr2, EPIC2B is a reversible inhibitor of Rcr3pim and does not trigger hypersensitivity on Cf-2/Rcr3pim tomato. We also found that the rcr3-3 mutant of tomato that carries a premature stop codon in the Rcr3 gene exhibits enhanced susceptibility to P. infestans, suggesting a role for Rcr3pim in basal defense. These findings are consistent with the predictions of the guard model and suggest that effectors from unrelated pathogens can target the same tomato defense protease Rcr3pim. It appears that relative to C. fulvum, P. infestans evolved a cunning effector that carries virulence activity without triggering plant innate immunity. Like EPIC1, PmEPIC1, an EPIC1 homolog from P. mirabilis, was also found to bind Rcr3pim but not PIP1. Unlike EPIC2B, neither EPIC1 nor PmEPIC1 binds or inhibits PIP1. This work was possible by the ability to express and purify proteins in the apoplast of N. benthamiana. Three tomato proteases with C-terminal 6XHistidine tag were successfully expressed in N. benthamiana apoplast. Our findings suggest that C- terminal His-tagging of proteins in N. benthamiana apoplast is efficient enough to enable purification of functional proteins. Further studies will focus on the three dimensional structural of the protease-protease inhibitor complexes, identifying the interactors of EPIC1 and PmEPIC1 in their respective host cells, and further characterizing the biochemical activities of these protease inhibitors. iii Dedicated to my grandfather, my mother, my father and my love iv ACKNOWLEDGMENTS I would like to give my greatest appreciation to my advisor, Dr. Sophien Kamoun for his intelligence, encouragement and full support. He gave me the opportunities not only to conduct the designed project but also to explore what I have learned to the application and the extension of our research design. With all his advices and support, I learned how to input and integrate our thoughts into the experimental performance, into the critical analysis of the designs and the results, and into the effective exclusion of the obstacles that we encountered. I also want to thank all my other Student Advisory Committee members Dr. Anne Dorrance, Dr. Eric Stockinger, Dr. Margaret Redinbaugh and Dr. Terrence Graham for their stimulating discussion, technical support and all the suggestions and encouragement that they gave me during my graduate study. Kamoun lab is my second home, where I met talented people, made many friends and shared my happiness and sadness with them. I wish to give my appreciation to our previous lab members Diane Kinney, Nicolas Champouret, Miaoying Tian, Joe Win, Cahid Cakir, Edgar Huitema, Thirumala-devi Kanneganti, Zhenyu Liu, Karen Liu, William Morgan, and current lab members Kerilynn Jagger, Jorunn Bos, Sang-Keun Oh, Liliana Cano, Angela Chaparro, Carla Garzon, Cristian Quispe, Tolga Bozkurt, for their help and discussion. v I am grateful to those who helped me in providing the technical support and experimental recourses for my research, especially MCIC staff and Maize Virology Group at USDA for the convenience that they provided. Finally, I want to give my great appreciation to my family for their full support during my study. vi VITA Aug. 31, 1978 ........................... Born in Xi’an, P. R. China 1996-2000 ........................... B.S. Department of Biochemistry and Molecular Biology, Beijing Normal University, Beijing, P. R. China 2000-2003 ........................... M.S. Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, P. R. China 2003-present ........................... Graduate Research Associate Department of Plant Pathology, The Ohio State University, OH, USA PUBLICATIONS Research Publications 1. Tian, M., Win, J., Song, J., van der Hoorn, R., van der Knaap, E., and Kamoun, S. (2007). A Phytophthora infestans cystatin-like protein targets a novel tomato papain-like apoplastic protease. Plant Physiol. 143, 364-377. 2. Jing Song, Yongru Sun, Liming Zhang, and Wenbin Li. 2003. Construction of indole dioxygenase vector and expression in Escherichia coli. High Technology Letters 9, 39-42. 3. Jing Song, Hengyao Niu, Yongru Sun, Liming Zhang, and Wenbin Li. 2002. Research of Changing the Color of Cotton Fiber by Gene Engineering. High Technology Letters 7, 103-107. vii 4. Wenbin Li, Jing Song, Yongru Sun, Liming Zhang, Yiqin Wang, Hengyao Niu. 2002. Cotton fiber specific expression vector containing bec gene. Patent 02103787.6, Chinese National Patent Office 5. Ning Liu, Jing Song, Chapter 2, << Plant Biology >>of textbook series for 21st century (ISBN7-4-007747-7), 1999, Higher Education Press, Beijing, China FIELDS OF STUDY Major Field: Plant Pathology Specialty: Molecular Plant-Microbe Interactions viii TABLE OF CONTENTS Page Abstract ............................................................................................................ ii Dedication ........................................................................................................ iv Acknowledgments ............................................................................................ v Vita ................................................................................................................... vii List of Tables .................................................................................................... xi List of Figures ................................................................................................... xii List of Abbreviations ......................................................................................... xiv Chapters: 1. Protease inhibitors in host-pathogen interactions .................................. 1 1.1 Introduction ................................................................................... 1 1.2 Overview of protease inhibitors ..................................................... 4 1.2.1 What are protease inhibitors? ........................................ 4 1.2.2 Classification of protease inhibitors ................................ 5 1.3 Examples of protease inhibitors involved in host- pathogen interactions .................................................................... 9 1.3.1 Protease inhibitors in hosts (plants, animals and human) ............................................................................. 9 1.3.2 Protease inhibitors in pathogens .................................... 13 1.4 Summary and prospective research on PIs ................................... 18 1.5 Research objectives ...................................................................... 20 1.6 References .................................................................................... 22 2. Two effectors, EPIC2B and Avr2, from unrelated pathogens target tomato defense protease Rcr3pim ................................................. 34 2.1 Abstract ......................................................................................... 34 2.2 Introduction ................................................................................... 36 2.3 Materials and Methods .................................................................. 41 2.4 Results .......................................................................................... 47 2.5 Discussion ..................................................................................... 51 ix 2.6 Acknowledgments ......................................................................... 56 2.7 References ...................................................................................

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