Location: Plant Gene Expression Center2013 Annual Report
1a. Objectives (from AD-416):
Effective defense responses are essential for plants to protect themselves against pathogens. Despite much research, many determinants of host resistance and their roles in signaling remain unknown. Pseudomonas syringae, a plant pathogenic bacterium, causes disease in more than 150 plant species. ZAR1 is the R protein responsible for recognizing the acetyltransferase HopZ1a, a P. syringae type III secreted effector protein (T3SE). ZAR1 is found in many plant species, including the Solanaceae, castor bean, and rice. However ZAR1 defense signaling does not depend on any components previously implicated in plant resistance, including salicylic acid. Further investigation of ZAR1 signaling gives us a unique opportunity to identify novel genes involved in plant resistance and potential targets for improving plant immunity. This project plan will address three major questions: 1) What other genes are genetically implicated in resistance to HopZ1a? Preliminary data from a forward genetics screen has identified several mutants that lack HopZ1a-mediated defense responses (hopz-effector-triggered-immunity-deficient or zed). The proposed work will map these mutants and characterize their roles in resistance. 2) What is the mechanism of ZAR1-mediated resistance? The proposed work will identify other proteins that contribute to ZAR1-mediated resistance. 3) Is ZAR1 resistance conserved in other plant species? The proposed work will examine ZAR1 resistance in wild and domesticated tomato. Using a multi-pronged approach, this work will identify and characterize new sources of resistance to protect plants from disease. The specific objectives are: Objective 1: Conduct genetic screens coupled with next-generation DNA sequencing to identify genes that contribute to pathogen resistance in plants. Objective 2: Characterize the mechanism of ZAR1-mediated resistance using molecular, genetic, and proteomic approaches. Objective 3: Evaluate susceptibility and resistance of plant species to bacterial pathogens to determine the conservation of resistance.
1b. Approach (from AD-416):
Objective 1: Hypothesis: Using HopZ1a as a probe of immune pathways in plants, we will identify novel genes (ZED1 and ZED2). We hypothesize that ZED1 and ZED2 will not be affected in basal defenses or responses to other T3SEs, and will be specifically involved in HopZ1a recognition. Further, we hypothesize that ZED1 or ZED2 will act as the guardee, and will be acetylated by HopZ1a. Experimental design: Screen F2 population of zed1 (or zed2) cross to Ler for a loss of the HopZ1a-induced macroscopic HR, when pressure-infiltrated with P. syringae carrying hopz1a. Identify single-nucleotide polymorphisms by Illumina sequencing of whole genomes from zed1 or zed2 populations that lack defenses. Characterize the roles of the zed mutants in defense and virulence, and their molecular functions. Contingencies: We can also conduct a genetic suppressor screen for restoration of HopZ1a-mediated immunity in zar1 mutants. Objective 2: Hypothesis: Plasma membrane-localized complexes of ZAR1, HopZ1a and other plant proteins will contribute to immunity. Defense-related host proteins will interact specifically with ZAR1. Epitope-tagged ZAR1 will be functional, complement the null zar1 mutant, and interact with other host proteins. Loss of function alleles of unknown (non-zed) plant genes will result in a loss of HopZ1a recognition and increased bacterial growth. Experimental Design: Identify ZAR1-interacting proteins using membrane-based high-throughput yeast two-hybrid approaches and biochemical approaches. Test whether the interacting protein affects defense responses. Contingencies: If the gene of a single interacting protein is part of a gene family, we will silence the gene family by RNAi and test for changes in defense responses. Objective 3: Hypothesis: As ZAR1 appears to be an ancient R gene, I hypothesize that HopZ1a will be recognized by a complex of proteins homologous to ZAR1 in tomato. Pto and Prf, known resistance-related genes in tomato, will not be needed for HopZ1a recognition. I further hypothesize that we will observe natural diversity for HopZ1a recognition. Silencing of ZAR1 homologs will result in the loss of HopZ1a resistance. Experimental Design: Infiltrate P. syringae carrying hopz1a into tomato. Test for induction of defense responses by conductivity assays, which measure rapid ion leakage upon recognition of a pathogen, or by bacterial growth assays, which quantitate bacterial growth over time. Silence homologs of ZAR1 to determine if they are necessary for HopZ1a recognition. Contingencies: If our study of natural diversity in tomato progresses more quickly than anticipated or if we fail to identify accessions that are resistant to HopZ1a, we can test additional accessions of tomato. HopZ1a can also be delivered to tomato by Agrobacterium-mediated transient expression.
3. Progress Report:
This report documetns progress for Project Number 5335-21000-046-00D, which started on June 2013 and continues research from Project Number 5335-21000-040-00D, entitled "Transgene Management Through Site-Specific Recombination." Objective 1. Identify genes involved in resistance to HopZ1a. Objective 1. Identify genes involved in resistance to HopZ1a. We screened a segregating population from a cross between one mutant and Ler, and are in the process of mapping the mutant from our forward genetic screen. Objective 2. Determine the mechanism of ZAR1-mediated resistance. We are in the process of constructing epitope-tagged constructs for ZAR1 expression in plants. Objective 3. Determine whether ZAR1 resistance is conserved in other species. We have shown that HopZ1a is expressed in Pseudomonas syringae pv. maculicola ES4326, and are testing additional strains of Pseudomonas syringae for their suitability in tomato infections.
Lee, A., Hurley, B., Felsensteiner, C., Yea, C., Ckurshumova, W., Bartetzko, V., Wang, P.W., Quach, V., Lewis, J.D., Liu, Y.C., Bornke, F., Angers, S., Wilde, A., Guttman, D.S., Desveaux, D. 2012. A bacterial acetyltransferase destroys plant microtubule networks and blocks secretion. PLoS Pathogens. 8(2): e1002523.