Source: UNIVERSITY OF NEBRASKA submitted to
PSEUDOMONAS SYRINGAE TYPE III EFFECTORS THAT SUPPRESS HOST IMMUNITY
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
HATCH
Reporting Frequency
Annual
Accession No.
0226657
Grant No.
(N/A)
Project No.
NEB-35-120
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Aug 1, 2011
Project End Date
Jul 31, 2016
Grant Year
(N/A)
Project Director
Alfano, JA.
Recipient Organization
UNIVERSITY OF NEBRASKA
(N/A)
LINCOLN,NE 68583
Performing Department
Plant Pathology
Non Technical Summary
Pseudomonas syringae pv. tomato DC3000 causes the economically significant bacterial speck disease of tomato and infects the model plant Arabidopsis thaliana. One of the primary strategies that DC3000 uses to be a pathogen is the injection of type III effector (T3E) proteins into host cells. The majority of DC3000 T3Es act to suppress plant immunity. However, the activities and mechanisms T3Es use to suppress innate immunity are not well understood. The study of their functions and targets is a valuable tool for the identification of novel components of plant immunity. We have found that two DC3000 T3Es localize to plant organelles and act as strong suppressors of innate immunity. These data indicate that mitochondria and chloroplasts have important immune functions and this provides a unique opportunity to dissect the involvement of these organelles in plant immunity. The experiments outlined in this proposal will allow us to explore this understudied aspect of plant pathogenesis. The specific aims of this proposal are the following: (1) Analyze the targeting signals for organelle-targeted effectors and determine if they are required for their roles in virulence; (2) Identify innate immunity-related phenotypes associated with organelle-targeted effectors; (3) Identify the organelle-targeted effectors plant targets and evaluate the targets' effect on innate immunity. Our experiments address program area priorities because they seek to elucidate the molecular mechanisms used by microbes to interact with plant hosts and they are agriculturally relevant because they may lead to the development of crops more resistant to biotic stress.
Animal Health Component
(N/A)
Research Effort Categories
Basic
(N/A)
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2062499104010%
2062499110010%
2064010104010%
2064010110010%
2122499104020%
2122499110010%
2124010104010%
2124010110020%
Knowledge Area
212 - Pathogens and Nematodes Affecting Plants; 206 - Basic Plant Biology;

Subject Of Investigation
4010 - Bacteria; 2499 - Plant research, general;

Field Of Science
1040 - Molecular biology; 1100 - Bacteriology;
Goals / Objectives
The specific aims of this proposal are the following: (1) Analyze the targeting signals for HopG1/HopK1 and determine if they are required for their roles in virulence; (2) Identify innate immunity-related phenotypes associated with HopG1/HopK1; (3) Identify HopG1/HopK1 plant targets and evaluate the targets' effect on innate immunity.
Project Methods
1. Analyze targeting signals for organelle-targeted effectors and determine if they are required for their roles in virulence. DNA corresponding to targeting sequence truncations will be amplified using PCR and fused to GFP DNA in pK7FWG2, a Gateway Destination binary vector that constitutively expresses C-terminal GFP gene fusions. Ultimately, the important residues will be substituted in the full length proteins to confirm that the identified residues are important for the organelle targeting of the native proteins. Once we have mutations that disrupt the localization to organelles in the full length proteins, we will test the extent that these proteins are capable of suppressing innate immunity (using assays are described in Specific Aim 2). Another important objective of this aim is to establish the organelle localization for the virulence and immune suppression activities of organelle-targeted effectors. 2. Identify innate immunity-related phenotypes associated with organelle-targeted effectors.In this sub-aim we will determine to what extent the physical outputs of innate immunity are suppressed in tomato and Arabidopsis plants expressing organelle-targeted effectors. We have many assays and variations of these assays to rely on to evaluate the immune-suppressing activity of these T3Es. Bacterial assays. To determine how each transgenic plant responds to different P. syringae strains we will perform pathogenicity assays. Callose assays. One assay that we will continue to use is the callose deposition assay, which stains callose in the plant cell wall that is deposited after challenge with either a PAMP or a T3E that induces ETI (i.e., an Avr protein). We will also assess transgenic plants expressing these effectors to determine if they have reduced or wild type amounts of callose when chitin or elf18, the active peptide from the PAMP EF-Tu, are used as PTI inducers. ROS assays. Another assay we will use to evaluate PTI and ETI suppression in organelle-targeted effector expressing transgenic plants (and plants generated in Specific Aims 1 and 3) is the production of ROS. These assays will be done on transgenic and wild type plants with similar treatments to those described above. 3. Identify plant targets and evaluate the targets' effect on innate immunity. Yeast two hybrid assays. To identify additional Arabidopsis proteins we will use a BD Matchmaker yeast two hybrid system (Clontech). The cDNA libraries that we will use in these experiments were made from untreated wild type plants, P. syringae infected plants, and plants infected with an avirulent P. syringae strain. Putative interactors will then be tested in GST pull-down or co-immunoprecipitation assays. We will be especially interested in interactors that are associated with plant organelles, known innate immunity components, or plant genes known to be induced by biotic stress. However, since little is known about the function of these T3Es in plants we will pursue any strong interactors and determine the phenotypes of plants lacking (or reduced in) these proteins.

Progress 08/01/11 to 07/31/16

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? N/A

Publications


    Progress 10/01/11 to 09/30/12

    Outputs
    OUTPUTS: Global changes in Arabidopsis histone modifications after exposure to wild type (DC3000) or the type III defective hrcC mutant. To determine the extent that P. syringae induces alterations in histone modifications in Arabidopsis, we vacuum infiltrated 4 week old plants with a buffer control, wild type P. syringae DC3000, or a DC3000 hrcC mutant, a strain incapable of injecting type III effectors into plant cells, at an OD600 of 0.05. Immunoblot analyses of leaf tissue harvested 15 hours post infiltration with antibodies that could detect five different types of histone modifications revealed a significant decrease in histone H3K9 acetylation (H3K9ac) in plants treated with DC3000 and an increase of H3K9ac in plants treated with the hrcC mutant compared to a buffer control. Although we also detected changes in other histone modifications in bacterial-treated plants, the H3K9ac modification was the most robust in these experiments and we decided to focus our initial efforts exploring the relevance of this modification in plant immunity and disease. In three independent experiments we found that the level of H3K9ac was reduced in plant tissue treated with DC3000 and increased in plant tissue treated with the hrcC mutant when compared to plants treated with the buffer control. Reduced H3K9ac correlates with a reduction in immunity-related gene expression. Because H3K9ac is commonly found in actively transcribed regions of the genome, we hypothesized that the DC3000-induced reduction in H3K9ac would occur in promoter regions of Arabidopsis immunity-related genes and that this reduction would correlate with a reduction in their expression. Using chromatin immunoprecipitation (ChIP) assays combined with quantitative-PCR (qPCR) on DC3000, hrcC, and buffer-treated plants, we found a significant reduction of H3K9ac in promoter regions of a number of immunity-related genes (i.e., known to be induced by biotic stress) in plants treated with DC3000 compared to hrcC or buffer treated plants. Furthermore, we found this reduced acetylation correlated with reduced mRNA transcripts. This reduction in both H3K9ac and transcript abundance was absent in hrcC treated plants. In fact, we detected an increase in H3K9ac associated with a subset of the immunity-related genes. Type III effectors are required for H3K9ac reduction caused by DC3000. To begin to analyze which type III effectors are involved in eliciting histone deacetylation we analyzed how H3K9ac global levels changed in Arabidopsis plants treated with a variety of P. syringae poly-effector mutants. Most of the DC3000 type III effector genes are located in 6 different DNA clusters in the genome. We made use of our own collection of single cluster deletion strains and two different strains deleted for all 6 type III effector gene clusters, DC3000D28E and UNL275, which has these type III effector cluster mutations and one additional type III effector gene deleted. We found that H3K9ac levels of plants treated with the DC3000D28E or UNL275 strains were similar to those treated with the type III defective hrcC mutant. PARTICIPANTS: Guangyong Li, a research assistant professor in my research group worked on the acetylation project. We collaborated with Karin van Dijk's research group at Creighton University on this project. Emerson Crabill, a graduate student that graduated with a PhD in August 2012, worked on the translocator project. Two research assistant professors in my lab, Ming Guo and Anna Block, worked on the catalase project. Anna Block recently left my research group. Other research members helped out on these projects. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

    Impacts
    We found that plants change histone acetylation in response to P. syringae. We have further developed the targets of HopU1 effector and how this target is involved in plant immunity. We have discovered how the type III translocators are secreted via the type III secretion system. We have assessed the involvement of catalases as virulence factors for P. syringae.

    Publications

    • Guo, M., A. Block, C.D. Bryan, D.F. Becker, and J.R. Alfano. 2012. Pseudomonas syringae catalases are collectively required for plant pathogenesis. J. Bacteriol. 194: 5054-5064.
    • Crabill, E., A. Karpisek, and J.R. Alfano. 2012. The Pseudomonas syringae HrpJ protein controls the secretion of type III translocator proteins and has a virulence role inside plant cells. Mol. Microbiol. 85: 224-238.
    • Jeong, B.-r., Y. Lin, A. Joe, M. Guo, C. Korneli, H. Yang, P. Wang, M. Yu, R.L. Cerny, D. Staiger, J.R. Alfano*, and Yanhui Xu*. 2011. Structure function analysis of an ADP-ribosyltransferase type III effector and its RNA-binding target in plant immunity. J. Biol. Chem. 286:43272-43281. *Co-corresponding authors