Location: Plant Gene Expression Center Albany_CA
2013 Annual Report
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.
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.
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.