This application describes genetic experiments designed to help elucidate the molecular mechanism of the plant defense response to bacterial pathogens. To carry out these experiments, our laboratory has developed a new model system that involves infection of the small flowering plant, Arabidopsis thaliana, with pathogenic pseudomonads. We have cloned individual Pseudomonas syringae avirulence (avr) genes that elicit defense responses and we have cloned several Arabidopsis defense-related genes for use in monitoring the defense response including PAL1 (phenylalanine ammonia lyase), BGL1, BGL2, BGL3 (beta-1,3-glucanases), and GST1 (glutathione-S-transferase). Utilizing the power of the Arabidopsis genetic system, we propose to isolate, map, and characterize the following classes of Arabidopsis mutants that respond aberrantly to pathogen attack: 1) Mutants that cannot respond to specific cloned avirulence genes by mounting a hypersensitive response. 2) Mutants incapable of mounting a hypersensitive response irrespective of the signal. 3) Mutants that fail to activate specific defense-related target genes such as those encoding phenylalanine ammonia lyase (PAL1) and glutathione-S-transferase (GST1) that are at the ends of signal transduction pathways. 4) Mutants that fail to synthesize the Arabidopsis-specific indole-based phytoalexin, camalexin. Our overall strategic approach is to isolate many mutants that belong to each of these categories so that all possible genes that affect the phenotype being examined are identified. Although any particular mutant can be informative, genetic approaches work best when many mutant alleles of each gene of interest are available. The goal is to let the mutant phenotypes give an overall picture of the plant defense response. Mutants will be characterized phenotypically by examining the effects of the mutation(s) they contain on a variety of parameters associated with an active defense response, including restriction of bacterial growth, induction of plant defense-related genes, and pH and ion flux changes. The mutants will be sorted into complementation groups, mapped, and the genes corresponding to selected mutants will be cloned. Analysis of the data so obtained should allow us to: 1) determine the number of signal transduction pathways leading to defense responses, 2) identify the components of each signal transduction pathway, and 3) determine the significance of particular defense responses and/or specific defense- related genes for conferring resistance to a particular pathogen.
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