The EDR1 gene of Arabidopsis encodes a MAP Kinase Kinase Kinase that functions as a negative regulator of defense responses and leaf senescence. An overlap between plant defense responses and senescence at the molecular level has been well documented; however, the mechanistic basis of this overlap is not understood. The research proposed here is aimed at answering three fundamental questions regarding the EDR1 signal transduction pathway: 1) What environmental and cellular signals regulate activity of the EDR1 MAPKKK? 2) What MAP kinase(s) are activated by EDR1? and 3) What plant responses are directly regulated by this (these) MAP kinases? Because EDR1 is homologous to CTR1, which is regulated by histidine kinase response regulators, we predict that EDR1 may also be regulated by a histidine kinase. The recent identification of a cytokinin receptor that belongs to this family, along with the classic observation that cytokinins inhibit leaf senescence, has led us to hypothesize that EDR1 may function in a cytokinin signal transduction pathway that negatively regulates both age-induced and pathogen-induced leaf senescence.
Five specific aims will be pursued to test this hypothesis. First, the edr1 mutant will be assessed for resistance to cytokinins, and cytokinin receptor mutants will be assessed for resistance to pathogens and to dark-induced senescence. Second, a genetic screen will be performed to identify mutations that suppress or enhance the edr1 mutant phenotype. These screens will uncover additional genes in the EDR1 pathway, as well as identify other pathways that interact with the EDR1 pathway. Third, we will evaluate global gene expression in Arabidopsis plants expressing constitutively activated forms of EDR1. The phenotypic responses of these plants to pathogens will also be investigated. These analyses will reveal specific genes regulated by EDR1. Fourth, proteins that physically interact with EDR1 will be identified using yeast two-hybrid analyses and in vitro kinase assays. Fifth, Arabidopsis MAP kinases that are activated by EDR1 will be identified using a myelin basic protein (MBP) phosphorylation assay. This assay involves coexpression of EDR1 and candidate MAP kinases in Arabidopsis protoplasts. The candidate MAPKs are then immunoprecipitated and assayed for their ability to phosphorylate MBP, a universal substrate of MAPKs. These analyses, together, will enable us to assemble the EDR1 MAPKKK pathway and identify signal inputs and outputs. This information, in turn, may provide new insights into the regulation of programmed cell death in plants and the overlap between normal senescence processes and pathogen defense.
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