This proposal investigates the molecular basis of disease resistance in plants, utilizing the molecular-genetic resources of the model plant Arabidopsis thaliana. A critical aspect of plant disease resistance is pathogen recognition. The PI has recently isolated the plant disease resistance gene RPM1, which mediates recognition of the bacterial pathogen Pseudomonas syringae. RPM1 likely functions as a receptor that upon binding a signal molecule secreted by P. syringae activates a suite of physiologic; responses. However, the signal transduction pathway between RPM1 and activation of these downstream responses is completely unknown. In this proposal, the PI presents two complementary approaches for identifying and isolating genes that encode components of this signal transduction pathway: a classical genetic approach and a yeast two-hybrid approach. The PI has already initiated the classical genetic work, and has identified at least seven different genes in Arabidopsis that when mutated, block or impair the function of RPM1 and/or two other plant disease resistance genes. RPS2 and RPS5. One of these seven genes is NDR1, which was identified independently in another lab. The six remaining genes are named SMA1 - SMA6, for Symptomatic to Multiple Avr genotypes. These seven genes may encode components of the signal transduction pathway.
The specific aims are to: 1) Determine the specificity of the sma mutants. The PI will examine how the sma mutations affect the resistance mediated by several different R genes. Preliminary studies suggest some sma mutants affect only a subset of R genes. 2) Characterize the physiological defects of the sma mutants. The PI will evaluate the sma mutants for defense responses such as hypersensitive response, electrolyte leakage, phytoalexin biosynthesis, callose production, induction of defense genes and induction of systemic acquired resistance. These analysis may allow the PI to assign specific SMA genes to distinct response pathways. 3). Use the yeast two-hybrid system to identify Arabidopsis proteins that interact with RPM1 and RPS5 receptor proteins. Determine if any clones isolated using the two-hybrid screen map to any of the 6 sma mutants. 4) Isolate at least two SMA genes by positional cloning or be cosegregation of sma mutant with gene cloned by two-hybrid. 5) Analyze interactions between the products of R genes and the products of putative signal transduction genes. The PI proposes to specifically examine interactions between RPM1 or RPS5 gene products and the NDR1 gene, the six SMA genes and the clones isolated by two hybrid, using both in vitro and in vivo assays for these analyses. Two hybrid clones that do not correspond to any known gene will be assayed for biological activity in Arabidopsis using a genetic suppression assay.
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