University of Nebraska - Lincoln DigitalCommons@University of Nebraska - Lincoln Dissertations and Theses in Biological Sciences Biological Sciences, School of 7-2012 THE PSEUDOMONAS SYRINGAE TYPE III SECRETION SYSTEM: THE TRANSLOCATOR PROTEINS, THEIR SECRETION, AND THE RESTRICTION OF TRANSLOCATION BY THE PLANT IMMUNE SYSTEM Emerson Crabill University of Nebraska-Lincoln, [email protected] Follow this and additional works at: https://digitalcommons.unl.edu/bioscidiss Part of the Pathogenic Microbiology Commons, and the Plant Pathology Commons Crabill, Emerson, "THE PSEUDOMONAS SYRINGAE TYPE III SECRETION SYSTEM: THE TRANSLOCATOR PROTEINS, THEIR SECRETION, AND THE RESTRICTION OF TRANSLOCATION BY THE PLANT IMMUNE SYSTEM" (2012). Dissertations and Theses in Biological Sciences. 44. https://digitalcommons.unl.edu/bioscidiss/44 This Article is brought to you for free and open access by the Biological Sciences, School of at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Dissertations and Theses in Biological Sciences by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. THE PSEUDOMONAS SYRINGAE TYPE III SECRETION SYSTEM: THE TRANSLOCATOR PROTEINS, THEIR SECRETION, AND THE RESTRICTION OF TRANSLOCATION BY THE PLANT IMMUNE SYSTEM By Emerson Crabill A DISSERTATION Presented to the Faculty of The Graduate College at the University of Nebraska In Partial Fulfillment of Requirements For the Degree of Doctor of Philosophy Major: Biological Sciences Under the Supervision of Professor James R. Alfano Lincoln, Nebraska July, 2012 THE PSEUDOMONAS SYRINGAE TYPE III SECRETION SYSTEM: THE TRANSLOCATOR PROTEINS, THEIR SECRETION, AND THE RESTRICTION OF TRANSLOCATION BY THE PLANT IMMUNE SYSTEM Emerson Crabill, Ph.D. University of Nebraska, 2012 Adviser: James R. Alfano Pseudomonas syringae is a Gram-negative plant pathogen whose virulence is dependent upon its type III secretion system (T3SS), a nanosyringe that facilitates translocation, or injection, of type III effector (T3E) proteins into eukaryotic cells. The primary function of P. syringae T3E proteins is suppression of plant immunity. Bacterial proteins called translocators form a translocon that forms a pore in the host plasma membrane which is traversed by T3Es. HrpK1, a putative P. syringae translocator, is a type III-secreted protein important for virulence and T3E injection, but not secretion of T3Es. Harpins are a group of proteins specific to plant pathogens that are also important for T3E translocation. P. syringae pv. tomato DC3000 has 4 harpins – HrpZ1, HrpW1, HopAK1, and HopP1. Here, HrpK1 is confirmed to be a translocator. HrpK1 had a greater impact on T3E translocation than the harpins. HrpK1 and HrpZ1 disrupted liposomes. Both proteins interacted with phosphatidic acid which interfered with T3E translocation. HrpJ, a type III-secreted protein required for HrpZ1 secretion, was also required for secretion of HrpK1, HrpW1, and HopAK1. A hrpJ mutant secreted elevated levels of the Hrp pilus protein HrpA1. HrpJ appears to control transition from Hrp pilus secretion to translocator secretion. Secretion was complemented by secretion incompetent HrpJ derivatives indicating that HrpJ controls secretion from inside the bacteria. The hrpJ mutant expressing secretion incompetent HrpJ was reduced in virulence but was complemented by HrpJ expressed inside plant cells. Additionally, transgenic Arabidopsis plants expressing HrpJ were reduced in their immune responses indicating that HrpJ can suppress plant immunity. Plants pretreated with an inducer of pathogen- associated molecular pattern-triggered immunity are unable to produce an HR. Plants, as an immune response, have evolved the ability to block T3E translocation when plant immunity has been induced prior to bacterial inoculation. This is especially true in non-host interactions whereas virulent bacteria appear to be able to attenuate injection restriction in host plants via T3E activity. v Acknowledgements There are very few important things that one does in life completely on his own. In the more than six years I have spent as a graduate student I have come to understand that making it through graduate school requires help from a number of other people. I would like to take this opportunity to thank some of those people who helped me throughout my graduate studies. First, I would like to thank my adviser, Dr. Jim Alfano, for taking a chance on me when I had little or no real research experience. Jim let me work on projects that I was interested in and pushed me to work hard and constantly try new things. I want to thank him not only for his direction and his advice but also for allowing me to take ownership of my research projects and giving me freedom to explore things, which may have delayed progress in some ways, but in the end made me a much more creative and independent researcher. Jim was supportive outside the lab as well, providing career advice, sending me to different regional, national, and international meetings, and introducing me to many faculty members in the field of microbiology. I have learned a lot from him and from working in his lab. I would also like to thank the members of my committee, Drs. Tom Clemente, Ken Nickerson, Andrew Benson, and Heriberto Cerutti. All of whom were very helpful and responsive whenever I asked for anything and I really appreciate them for that. I interacted with Dr. Clemente most, likely due to his proximity to the Alfano lab and I want to thank him specifically for constantly vi keeping me on my toes and frequently providing a certain amount of levity to the time spent at the bench. I was very fortunate to be in a lab that was so supportive and collaborative and I would like to thank some of the members of the Alfano lab specifically. First, thanks to Dr. Ming Guo, who took me under his wing when I first joined the lab and taught me all the techniques and everything I needed to know about working in the lab. He was incredibly selfless and was always willing to take time to show or explain something to me or answer my questions. Without Ming’s help I would have been lost early in graduate school and he remained a huge help to me throughout my time in the lab. Many of the projects I worked on, some of which are presented in this dissertation, involved several people. Andrew Karpisek, a former technician in the lab, initiated the HrpJ project and I want to thank him for his work getting the project going. Also since our projects were so similar, we had many fruitful discussions about techniques or ideas for experiments. We continue to collaborate on other projects even as he has moved to a lab at Creighton University. Anna Joe and Dr. Anna Block were involved with the injection restriction project, which was a time-sensitive endeavor, and when they joined in they made it a priority and working with them was a fun experience. I want to thank Jennifer van Rooyen, who made so many poly-effector mutants, a project which I then took over after she showed me the techniques involved. Many of the mutants we made have proven useful in a number of different experiments. There are several other current and former lab members who made working in the lab very enjoyable. Zhengqing Fu helped me become acclimated vii in the lab and understand what was expected of me as a graduate student. Dr. B.J. Jeong helped me a lot with technical issues, and could always be counted on to explain the finer details of any experiment. Fang “Tina” Tian sat next to me at the bench and helped in many ways. Dr. Guangyong Li helped make the lab more fun and taught me the best strategies for buying lottery tickets. I enjoyed getting to know Tania Toruño, a graduate student with whom I had many discussions about experimental protocols. Zhengxiang Ge helped make the lab environment pleasant. All of these people contributed to my research through various discussions and interactions and many became more friends than just colleagues. I was given the opportunity to mentor three undergraduate students which was very beneficial to me both for the experience it provided and because they are such great people. Additionally those students, Ashley Gutwein, Nicole Staton, and Will Becker worked very hard for me and proved to be very helpful and productive. I also want to thank Drs. Bill and Wendy Picking from Oklahoma State University who taught me the liposome assay that was a critical component of the HrpK1 project. They were great collaborators and very helpful with ideas and advice. Last, but most importantly, I want to thank my family who were always supportive of me during graduate school. I want to thank especially my wife, Elizabeth, who more than anyone believed in me and supported me during my time at the University of Nebraska. Her love and patience were paramount to my viii success as a graduate student, and without her I would not have been able to do it. During the most trying times she was the person I turned to for help and she always provided it for me. I cannot thank her enough. ix Table of Contents Abstract…………………………………………………………………………………..iii Acknowledgements……………………………………………………………………..v Table of Contents ................................................................................................ ix List of Figures and Tables .................................................................................. xii Chapter 1 Background ........................................................................................................