FUNCTIONAL CHARACTERIZATION OF EXTRACELLULAR PROTEASE INHIBITORS OF PHYTOPHTHORA INFESTANS DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Miaoying Tian, M.S. * * * * * The Ohio State University 2005 Dissertation Committee: Dr. Sophien Kamoun, Adviser Dr. Terrence L. Graham Approved by Dr. Saskia A. Hogenhout Dr. Margaret G. Redinbaugh Adviser Dr. Guo-Liang Wang Graduate Program in Plant Pathology ABSTRACT The oomycetes form one of several lineages within the eukaryotes that independently evolved a parasitic lifestyle and are thought to have developed unique mechanisms of pathogenicity. The devastating oomycete plant pathogen Phytophthora infestans causes late blight, a ravaging disease of potato and tomato. Little is known about processes associated with P. infestans pathogenesis, particularly the suppression of host defense responses. We used data mining of P. infestans sequence databases to identify 18 extracellular protease inhibitors belonging to two major structural classes: (i) Kazal-like serine protease inhibitors (EPI1 to EPI14) and (ii) cystatin-like cysteine protease inhibitors (EPIC1 to EPIC4). A variety of molecular, biochemical and bioinformatic approaches were employed to functionally characterize these genes and investigate their roles in pathogen virulence. The 14 EPI proteins form a diverse family and appear to have evolved by domain shuffling, gene duplication, and diversifying selection to target a diverse array of serine proteases. Recombinant EPI1 and EPI10 proteins inhibited subtilisin A among major serine proteases, and inhibited and interacted with tomato P69B subtilase, a pathogenesis-related protein belonging to PR7 class. The recombinant cystatin-like cysteine protease inhibitor EPIC2B interacted with a novel tomato papain-like extracellular cysteine protease PIP1 with an implicated role in plant defense. PIP1 is closely related to Rcr3, an apoplastic cysteine protease required for ii tomato Cf-2 and Cladosporium fulvm Avr2-dependent defense response. Both EPIC1 and EPIC2B interacted with Rcr3. Interactions with plant defense-related proteases suggest a counterdefense role of these extracellular protease inhibitors. Interestingly, EPIC1 and EPIC2B were degraded by tomato pathogenesis-related P69B subtilase and EPI1 protected both proteins from degradation, indicating that EPI1 contributes to virulence by protecting pathogen proteins from degradation by defense-related proteases. In addition, our overall results suggest that complex cascades of inhibition of host proteases by diverse extracellular protease inhibitors of P. infestans might occur in the plant apoplast during infection, thus leading to multifaceted suppression of plant defense responses. Both Kazal-like and cystatin-like inhibitors are widespread in the oomycetes, but have not been reported in other microbial plant pathogens. Inhibition of host proteases by P. infestans protease inhibitors is proposed to be a novel mechanism of pathogen suppression of plant defense. iii Dedicated to my parents and husband iv ACKNOWLEDGMENTS When I started writing this section, my brain started to gather memories during my Ph. D study. I felt that I had improved a lot in many aspects, including experimental skills, oral presentations, writing and personal confidence in my future career. All of these are not possible without my adviser Dr. Sophien Kamoun’s great contributions. He guided me with his great intelligence, enthusiasm, patience, encouragement and support. I would like to express my special appreciation and respect to him. I am also greatly thankful to my other Student Advisory Committee members: Dr. Terrence Graham, Dr. Margaret Redinbaugh, Dr. Guoliang Wang and Dr. Saskia Hogenhout for their stimulating discussions and constructive advices. I would like to thank all the previous and current Kamoun lab members, who I have worked together, especially Diane Kinney, Shujing Dong, Joe Win, Edgar Huitema, Trudy Torto-Alalibo, Luis da Cunha, Zhenyu Liu, Jorunn Bos, Nicolas Champouret, Jing Song, Thirumala-devi Kanneganti and Cahid Cakir for all kinds of help and discussion. I am grateful to MCIC staff and Maize Virology Group at USDA for the convenience they provided for me to use all kinds of equipments. Finally, I would like to say “ Thank you so much” to my husband Dongliang Wu. Without his support and understanding, my Ph.D dream wouldn’t come true. v VITA 1990 - 1994…………………. B.S. Plant Protection, Agricultural University of Hebei, P. R.China 1994 - 1997………………….. M.S., Plant Pathology, The Graduate School of Chinese Academy of Agricultural Sciences, P. R. China 1997 - 2000………………….. Research associate, Institute of Vegetables and Flowers, Chinese Academy of Agricultural Science, P.R. China 2000 - present ……………….. Graduate Research Associate, The Ohio State University PUBLICATIONS Research Publication 1. Tian, M., Champouret, N., and Kamoun, S. 2004. Extracellular protease inhibitors of Phytophthora infestans determine a novel counterdefense mechanism. Phytopathology. 94: S136. 2. Tian, M., Huitema, E., da Cunha, L., Torto, T., and Kamoun, S. 2004. A Kazal- like extracellular serine protease inhibitor from Phytophthora infestans targets the tomato pathogenesis-related protease P69B. Journal of Biological Chemistry. 279(25), 26370- 26377. 3. Huitema, E., Bos, J. I. B., Tian, M., Win, J., Waugh, M. E. and Kamoun, S. 2004. Linking sequence to phenotype in Phytophthora-plant interactions. Trends in Microbiology. 12(4), 193-200. 4. Tian, M., and Feng, L. (2000) Identification of a molecular marker linked with ToMV resistance gene in tomato using Randomly Amplified Polymorphic DNA. Acta Phytopathologica Sinica 30(2):158-161. vi 5. Tian, M., Wu, M., and Cheng, Z. (1999) Construction of plant expression vector harboring defective replicase gene of Barley Yellow Dwarf Virus and gaining of transgenic wheat plants Scientia Agricultura Sinica 32(5): 49-54. FIELDS OF STUDY Major Field: Plant Pathology Specialty: Molecular Plant-Microbe Interactions vii TABLE OF CONTENTS Page Abstract………………………………………………………………………………….. ii Dedication……………………………………………………………………………….. iv Acknowledgments……………………………………………………………………….. v Vita………………………………………………………………………………………. vi List of Tables…………………………………………………………………………….. x List of Figures…………………………………………………………………………… xi Chapters: 1. Introduction……………………………………………………………………... 1 References………………………………………………………………………. 10 2. A Kazal-like extracellular serine protease inhibitor from Phytophthora infestans targets the tomato pathogenesis-related protease P69B………………………… 17 2.1 Abstract……………………………………………………………………... 17 2.2 Introduction…………………………………………………………………. 18 2.3 Materials and Methods……………………………………………………… 20 2.4 Results………………………………………………………………………. 28 2.5 Discussion…………………………………………………………………... 34 2.6 Acknowledgments…………………………………………………………... 37 2.7 References……………………………………………………………………38 3. Evolution of Kazal-like protease inhibitors in Phytophthora was driven by gene duplication, domain shuffling, and diversifying selection……………...51 3.1 Abstract………………………………………………………………………51 3.2 Introduction…………………………………………………………………..52 3.3 Materials and Methods……………………………………………………….56 3.4 Results………………………………………………………………………..58 3.5 Discussion……………………………………………………………………65 3.6 Acknowledgments……………………………………………………………70 3.7 References……………………………………………………………………70 viii 4. A second Kazal-like protease inhibitor from Phytophthora infestans inhibits and interacts with the tomato pathogenesis-related protease P69B……………...87 4.1 Abstract………………………………………………………………………87 4.2 Introduction…………………………………………………………………..88 4.3 Materials and Methods……………………………………………………….91 4.4 Results………………………………………………………………………..96 4.5 Discussion…………………………………………………………………..101 4.6 Acknowledgments…………………………………………………………..105 4.7 References…………………………………………………………………..105 5. An atypical two disulfide bridge Kazal domain from Phytophthora exhibits stable inhibitory activity against serine proteases of the subtilisin family……..117 5.1 Abstract……………………………………………………………………..117 5.2 Introduction…………………………………………………………………118 5.3 Materials and Methods……………………………………………………...123 5.4 Results………………………………………………………………………126 5.5 Discussion…………………………………………………………………..131 5.6 Acknowledgments…………………………………………………………..134 5.7 References…………………………………………………………………..134 6. Kazal-like serine protease inhibitor EPI1 from Phytophthora infestans is involved in virulence by initiating cascades of inhibition of plant defense-related proteases…………………………………………………143 6.1 Abstract……………………………………………………………………..143 6.2 Introduction…………………………………………………………………144 6.3 Materials and Methods……………………………………………………...148 6.4 Results………………………………………………………………………154 6.5 Discussion…………………………………………………………………..163 6.6 Acknowledgments…………………………………………………………..170 6.7 References…………………………………………………………………..170 Bibliography……………………………………………………………………………189 ix LIST OF TABLES Table Page 2.1 Predicted Kazal-like proteins from the oomycete plant pathogens Phytophthora infestans, Phytophthora sojae, Phytophthora ramorum, Phytophthora brassicae, and Plasmopara halstedii…………………………….50 3.1 Primers used for RT-PCR amplifications of epi genes from P. infestans……… 85 3.2 Putative orthologous epi genes in P. infestans, P. sojae and P. ramorum………86 6.1 Primers used in Chapter 6………………………………………………………187 6.2 Predicted cystatin-like extracellular protease inhibitors from the oomycete plant pathogens Phytophthora infestans, Phytophthora sojae and Phytophthora ramorum…………………………………………………………188