Source: Board of Trustees of the Leland Stanford Junior University submitted to
IDENTIFYING BIOCHEMICAL MECHANISMS BY WHICH A NOVEL XANTHOMONAS T3S EFFECTOR SUPPRESSES IMMUNITY AND PROMOTES PATHOGENESIS IN TOMATO
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
0229300
Grant No.
2012-67011-19669
Project No.
CALW-2012-01166
Proposal No.
2012-01166
Multistate No.
(N/A)
Program Code
A7101
Project Start Date
Sep 1, 2012
Project End Date
Mar 31, 2015
Grant Year
2012
Project Director
Stork, W.
Recipient Organization
Board of Trustees of the Leland Stanford Junior University
340 Panama Street
Stanford,CA 94305-6203
Performing Department
Office of Sponsored Research
Non Technical Summary
Xanthomonas bacteria are the causal agents of disease of major crop plants worldwide, including rice, tomato, pepper, citrus, banana, cassava, sugarcane, and crucifers. Xanthomonas infection can be especially damaging in developing countries that rely heavily on staple crops like rice and cassava, but it also has severe consequences for farmers in the United States. I study Xanthomonas campestris vesicatoria (Xcv), the causal agent of bacterial spot disease in tomato. Xcv colonizes tomato leaves and fruit during infection and negatively affects tomato quality and crop yield worldwide. Like many bacterial pathogens of plants and animals, Xcv can inject proteins (called type III secretion effectors, or T3Es) directly into host cells during infection. A major problem scientists face in improving plant health is a limited knowledge of the biochemical details of immune signaling and plant defense in important crop plants. My long-term goals are (1) to elucidate the plant cellular signaling networks that regulate immunity to microbial pathogens in important crops and (2) to determine how bacterial pathogens use T3Es to suppress immunity and cause disease. Achieving these goals is essential in order to engineer more robust and healthy crops. To work toward these goals, this project specifically will identify unknown components of the cellular immune signaling network in tomato that are targeted by Xcv during infection and will determine how the novel, broadly conserved Xanthomonas T3E, XopX, contributes to Xcv pathogenesis. Identifying the biochemical activity of Xcv T3Es in plant cells will help scientists understand and combat plant disease. Tomato is part of the plant family Solanaceae, one of the most valuable crop families in the world. In addition to being a representative crop species, there are many molecular and genetic tools that make tomato an excellent model for study in the laboratory, including a fully sequenced genome, high genotypic diversity from a number of cultivated and wild accessions, and amenability to genetic manipulation through virus-induced gene silencing (VIGS) and Agrobacterium-mediated transformation. These tools make tomato an excellent plant to study the molecular and genetic basis of plant immunity in valuable crops.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2121460100040%
2121460103030%
2121460108030%
Knowledge Area
212 - Pathogens and Nematodes Affecting Plants;

Subject Of Investigation
1460 - Tomato;

Field Of Science
1080 - Genetics; 1030 - Cellular biology; 1000 - Biochemistry and biophysics;
Goals / Objectives
This project will test the hypothesis that the Xanthomonas bacterial type III secretion effector protein XopX targets immune signaling pathway(s) in tomato during infection in order to suppress effector-triggered immunity and promote bacterial pathogenesis. I plan to (1) determine the range of plant defense responses suppressed by XopX, (2) identify biochemical characteristics of XopX required for suppression activity, and (3) identify cellular host targets of XopX activity. Major milestones in this project are identifying host proteins that directly interact with XopX, generating transgenic tomato lines expressing XopX, and demonstrating the role of XopX host targets in regulating plant immunity. Outputs from this project will be discovery of new model(s) of plant immunity, collaboration with international scientists, and dissemination of research findings in published manuscript(s) and in presentations at relevant conferences. Aspects of this project will be used to mentor undergraduate and high school students in plant biology research. This project will advance our understanding of diseases affecting important crop plants and help us improve plant health and increase food security.
Project Methods
During the first year, I will study the defense suppression activity of XopX by using Agrobacterium-mediated transient expression of protein constructs in tobacco leaves. This approach will allow me to rapidly assess the range of XopX suppression activity in response to a number of known plant defense elicitors. It will also allow me to identify biochemical characteristics of XopX required for suppression activity by testing the ability of natural and synthetic XopX variants to suppress plant defense. I will also identify host proteins that interact with XopX in a large-scale yeast two-hybrid screen, and I will begin to generate transgenic tomato lines expressing XopX. During the second year, I will confirm the importance of initial findings during natural infection of tomato by Xanthomonas. I will also confirm that direct XopX-host protein interactions are important for Xanthomonas virulence in tomato and that XopX host targets are central components of the plant immune response. Through this project, I will complete a mentorship effort that will introduce undergraduate and high school students to basic research in plant biology and help them complete independent research projects leading to honors research. This project will be evaluated through hands-on interaction and weekly one-on-one meetings with my mentor, Dr. Mary Beth Mudgett, an expert in biochemistry and plant-microbe interactions. Additionally, I will present my progress every six months during the fellowship period to a committee of three independent faculty members at Stanford University for evaluation and assistance. Key project milestones are identifying host proteins that directly interact with XopX, generating transgenic tomato lines expressing XopX, and demonstrating the role of XopX host targets in regulating plant immunity.

Progress 09/01/12 to 03/31/15

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Final experiments for Objectives 1-3 were performed to satisfy requirements for conferral of my Ph.D. degree and to report findings in a peer-reviewed journal article for Molecular Plant-Microbe Interactions. Experiments, data, reagents, and products from this project were appropriately summarized and stored for access by future researchers.

Publications


    Progress 09/01/13 to 08/31/14

    Outputs
    Target Audience: My efforts during this period included: 1) laboratory instruction for an undergraduate student at Stanford University, 2) materials produced for 1 high school, 2 undergraduate, 1 graduate, and 2 post-doctoral students (data and instruction for using computational biology tools), 3) science-based knowledge shared through oral presentation to undergraduate, graduate, and postdoctoral students and professional scientists in weekly meetings, 4) science-based knowledge shared with Stanford University scientists at committee meetings, 5) science-based knowledge shared with a large group of U.S. and international scientists from academia and industry through poster presentation and personal interactions at the International Society for Molecular Plant-Microbe Interactions in Rhodes, Greece, and 6) science-based knowledge shared with scientists in the field through publication of my research in a journal article in Molecular Plant-Microbe Interactions. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? This project has provided training opportunities in my field through one-on-one work with my advisor, Mary Beth Mudgett, and a senior researcher in our lab, Jung-Gun Kim. I continue to develop advanced research skills through this training, including genetic manipulation and analysis of bacteria and plants for research, tools for biochemical analysis of gene products, tools for analyizing cell biology including fluorescence microscopy for subcellular protein localization. I have used this project to learn computational biology skills related to analyzing gene expression patterns at a genome-wide scale. I have networked with international scientists at a major research conference. I have developed leadership, teaching, and mentorship skills by training personnel and other students in lab techniques, experimental design and execution, data analysis, and conceptual thinking. I have further improved my ability to critically review the literature in my field, evaluate new research, and write scientifically by serving as a journal referee with my mentor, helping to prepare research grants, and developing manuscripts for publication. This project has also provided the opportunity for professional development through group meetings, workshops, retreats, and seminars, and through career exploration seminars and career fairs organized by Stanford University and the Stanford School of Medicine Career Center. How have the results been disseminated to communities of interest? Results from this work were distributed to the scientific community at a major international research conference and in a journal article publication.Aspects of this research were used to mentor an undergraduate student in plant biology research. Results were also disseminated and used for education of high school, undergraduate, graduate, and post-doctoral students. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

    Impacts
    What was accomplished under these goals? I have determined the suppression activity of XopX against a range of plant defense elicitors, which has provided insight into its potential roles in the plant cell during infection (Aim 1, Change in Knowledge). I have identified a plant protein that interacts directly with XopX and have provided evidence for a role for this protein in plant immunity (Aim 2-3, Change in Knowledge). I have identified plant responses associated with XopX and have performed a global transcriptional analysis of tomato genes that have altered expression patterns in the presence of XopX (Aim 3, Change in Knowledge). Results from this work were distributed to the scientific community at a major international research conference and in a journal article publication. Aspects of this research were used to mentor an undergraduate student in plant biology research.

    Publications

    • Type: Journal Articles Status: Published Year Published: 2014 Citation: Stork W, Kim JG, Mudgett MB (2014) Functional analysis of plant defense suppression and activation by the Xanthomonas core type III effector XopX. Molecular Plant-Microbe Interactions. Advanced online publication. doi:10.1094/MPMI-09-14-0263-R


    Progress 09/01/12 to 08/31/13

    Outputs
    Target Audience: My efforts during this period included: 1) laboratory instruction for an undergraduate student, 2) laboratory instruction for an international graduate student, 3) materials produced for science education for high school students from an ethnically diverse high school in Fremont, CA, 4) science-based knowledge shared through oral presentation to undergraduate, graduate, and postdoctoral students and professional scientists in weekly meetings, 5) science-based knowledge shared through oral presentation to scientists at the Carnegie Institute for Science, Department of Plant Biology, 6) science-based knowledge shared with Stanford University scientists at committee meetings, and 7) science-based knowledge shared with a large group of U.S. and international scientists from academia and industry at the New Phytologist Symposium in Fallen Leaf Lake, CA. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? This project has provided training opportunities in my field through one-on-one work with my advisor, Mary Beth Mudgett, and a senior researcher in our lab, Jung-Gun Kim. I continue to develop advanced research skills through this training, including genetic manipulation and analysis of bacteria and plants for research, tools for biochemical analysis of gene products, tools for analyizing cell biology including fluorescence microscopy for subcellular protein localization. I have developed leadership, teaching, and mentorship skills by training personnel and other students in lab techniques, experimental design and execution, data analysis, and conceptual thinking. I have further improved my ability to critically review the literature in my field, evaluate new research, and write scientifically by serving as a journal referee with my mentor, helping to prepare research grants, and developing manuscripts for publication. This project has also provided the opportunity for professional development through group meetings, workshops, retreats, and seminars, and through career exploration seminars and career fairs organized by Stanford University and the Stanford School of Medicine Career Center. How have the results been disseminated to communities of interest? I have disseminated these results through oral and poster presentations to undergraduate student, graduate students, postdoctoral students, and professional scientists from academia and research. I have collaborated with a laboratory at Tel Aviv University in Israel and communicated with scientists from other countries to reach the international scientific community. Materials generated from this research have also been shared for science-based education of high school students. What do you plan to do during the next reporting period to accomplish the goals? I have demonstrated that XopX is required for Xanthomonas growth and disease symptom development in tomato and I have identified a plant protein that directly interacts with XopX. I have determined that XopX can suppress immune signaling elicited by multiple elicitors of plant immunity (Objective 1), suggesting that XopX acts downstream of major immune signaling pathways or that XopX acts in a general way to suppress the effects of immune signaling. Major research goals that remain in the project are identifying specific features of XopX that are required for its activity (Objective 2), and confirming the XopX-plant protein interaction with multiple techniques and silencing the expression of this protein and its homologs in tomato to determine the role it plays during Xanthomonas infection (Objective 3). Progress in Objective 3 will yield the most valuable new insight into plant immunity and is prioritized. I have already generated the necessary constructs to complete these goals and developed a research pipeline for completing the necessary experiments, which are currently underway. Potential limitations of this approach are difficulties in achieving sufficient silencing of targeted genes or failure to detect a role for the target in plant immunity. To address this concerns, I am (1) working one-on-one with an expert in gene silencing to address technical issues, and (2) using constructs and markers from established components of plant immunity to test the role that XopX plays in modulating known components of immune signaling (i.e., hormone biosynthesis mutants and markers, silencing known components of tomato immunity using established constructs). The combination of these two approaches will yield new knowledge of how Xanthomonas uses XopX to promote infection. In parallel, I expect to make progress on Objective 2 (identifying essential XopX domains). I have identified a potential conserved activity in the C-terminus of XopX and generated truncation mutants of XopX for transgenic expression in plants and E. coli as well as expression in Xanthomonas. I will use these truncation mutants to test the interaction of XopX with the target interacting protein as well as test the ability of XopX truncations to rescue wild-type growth in a xopX mutant. I have achieved the goals of using this research to mentor other students and collaborate and communicate with international scientists in academia and community. I will continue to complete this goal of the reserach in the next reporting period. Finally, I plan to disseminate this reserach through submission of a first-author journal article in the next reporting period.

    Impacts
    What was accomplished under these goals? I have made significant progress towards the objectives of this project. Specifically, I have tested XopX suppression activity against multiple elicitors of plant immunity to determine potential sites of action for XopX (Change in Knowledge). I have generated XopX truncation mutants in several backgrounds and generated preliminary data on domains required for XopX function (Change in Knowledge). I have identified a plant protein that directly interacts with XopX and am pursuing this protein and homologous proteins as new components of plant immune signaling (Change in Knowledge). I have collaborated with international agricultural scientists from Israel to complete this research and disseminated preliminary findings in oral and poster presentations with other scientists, including an international research conference. I have used this project to mentor undergraduate and graduate students and provided research materials for science-based education of high school students (Change in Knowledge, Change in Action). To accomplish my stated goals not yet met, I am testing the role of the XopX interacting protein in plant immune signaling as well as testing other established plant immune signaling components to identify and demnostrate the role of XopX host targets in regulating immunity. I am continuing to test biochemical characteristics of XopX required for XopX suppression activity and I plan to generate transgenic tomato lines expressing XopX as a community tool to study plant immunity. Results from this next phase of research will be used to develop new model(s) of plant immunity.

    Publications

    • Type: Journal Articles Status: Published Year Published: 2013 Citation: Kim, Jung-Gun, William Stork, Mary Beth Mudgett. Xanthomonas Type III Effector XopD Desumoylates Tomato Transcription Factor SlERF4 to Suppress Ethylene Responses and Promote Pathogen Growth. Cell Host & Microbe - 13 February 2013 (Vol. 13, Issue 2, pp. 143-154).