Gu Y, Innes RW

Loss-of-function mutations in the Arabidopsis thaliana ENHANCED DISEASE RESISTANCE 1 (EDR1) gene confer enhanced resistance to powdery mildew infection, enhanced senescence, and enhanced programmed cell death (PCD) under both abiotic and biotic stress conditions. All edr1-mediated phenotypes can be suppressed by a specific missense mutation (keg-4) in the KEEP ON ...

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GOING (KEG) gene, which encodes a multi-domain protein that includes a RING E3 ligase domain, a kinase domain, Ankyrin repeats, and HERC2-like repeats. The molecular and cellular mechanisms underlying this suppression are poorly understood. Using confocal laser scanning microscopy and fluorescent protein fusions, we determined that KEG localizes to trans-Golgi network/early endosome vesicles (TGN/EE). Both the keg-4 mutation, which is located in the C-terminal HERC2-like repeats, and deletion of the entire HERC2-like repeats reduced endosomal localization of KEG and increased localization to the endoplasmic reticulum and cytosol, indicating that the HERC2-like repeats facilitate the TGN/EE targeting of KEG. EDR1 co-localized with KEG to the TGN/EE when co-expressed, but localized primarily to the endoplasmic reticulum when expressed alone. Yeast two-hybrid and co-immunoprecipitation analyses revealed that EDR1 and KEG physically interact. Deletion of the HERC2-like repeats abolished the interaction between KEG and EDR1, as well as the KEG-induced TGN/EE localization of EDR1, indicating that the recruitment of EDR1 to the TGN/EE is based on a direct interaction between EDR1 and KEG mediated by the HERC2-like repeats. Collectively, these data suggest that EDR1 and KEG function together to regulate endocytic trafficking and/or formation of signaling complexes on TGN/EE vesicles during stress responses.

Date: Feb. 22, 2011

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