Location: Plant Gene Expression Center Albany_CA
Project Number: 5335-21000-046-00
Start Date: Jun 26, 2013
End Date: Jun 25, 2018
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.