This project focuses on the molecular mechanisms that mediate programmed cell death (PCD) in plants, specifically in response to pathogen infection. We have previously shown that loss-of-function mutations in the EDR1 gene of Arabidopsis confer enhanced resistance to infection by pathogens. Significantly, this resistance is correlated with activation of PCD and defense genes, enhanced sensitivity to the plant hormone abscisic acid, and enhanced senescence in response to biotic and abiotic stresses. The edr1 mutant appears to be deficient in autophagy, an intracellular recycling system responsible for breakdown of damaged organelles such as mitochondria and chloroplasts. EDR1 localizes to endomembrane vesicles and the endoplasmic reticulum, thus we hypothesize that EDR1 may be involved in regulation of vesicle trafficking and/or formation and processing of auotphagosomes. We are currently testing this hypothesis using genetic and cell biological approaches. All edr1 mutant phenotypes can be suppressed by specific missense mutations in an E3 ubiquitin ligase known as KEG. These mutations do not block KEG function, but instead appear to cause KEG to be constitutively activated. KEG appears to largely co-localize with EDR1, thus we hypothesize that it regulates vesicle trafficking/autophagosome function at a step downstream of EDR1. We are currently identifying potential substrates of both EDR1 and KEG using yeast two-hybrid analysis and by purification of protein complexes containing KEG and/or EDR1.