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 the powdery mildew fungus Erysiphe cichoracearum. Significantly, this resistance is correlated with a more rapid activation of several defense responses, including PCD, indicating that EDR1 is a negative regulator of PCD. We have also shown that edr1 mutants have enhanced sensitivity to the plant hormone abscisic acid (ABA) and that they senesce more rapidly than wild-type plants. EDR1 thus represents an excellent entree into understanding how induction of PCD is regulated at a molecular level during pathogen infection and senescence. Work during the first three years of this project has identified two likely substrates of EDR1, a kinesin-like protein known as KCA1 and an ABA-inducible transcription factor called AtMYC2. We have also identified a large number of mutations that suppress edr1-mediated resistance. Finally, we have identified two additional genes that confer edr1-like resistance phenotypes when mutated: EDR2, which encodes a novel protein containing two lipid-binding domains, and DRP1E, which encodes a dynamin-related protein. Both EDR2 and DRP1E appear to be localized to mitochondria, providing a mechanistic link between mitochondria and regulation of PCD in plants.
Our specific aims i n this competing renewal are to 1) clone the genes identified in our suppressor screen; 2) determine whether EDR1 phosphorylates AtMYC2 and KCA1 in vivo, and if so, how phosphorylation affects their function and stability; and 3) determine the lipid binding properties of EDR2 and DRP1E and assess how lipid binding affects powdery mildew-induced PCD and mitochondrial function.
Specific aim 1 will be accomplished by standard positional cloning methods. Completion of this aim will uncover additional genes in the EDR1 pathway, as well as reveal other pathways that interact with EDRL Specific aim 2 will be accomplished by monitoring charge differences on AtMYC2 and KCA1 in wild-type versus edr1 mutant backgrounds, localizing GFP fusions in transgenic plants, and by double mutant analysis.
Specific aim 3 will be accomplished using a protein-lipid overlay assay and a lipid vesicle tubulation assay with wild-type and mutant derivatives of EDR2 and DRP1E. Mitochondrial function will be assessed using a fluorescent dye that reports mitochondrial membrane potential. Together, these analyses will provide significant new insight into how pathogen-induced PCD is regulated in plants. This work will also shed light on mechanisms of pathogen defense and PCD in humans, as several of the proteins identified in this study share significant similarity with human proteins, including some linked to PCD and mitochondrial function in human cells. ? ? ?
|Rutter, Brian D; Innes, Roger W (2017) Extracellular Vesicles Isolated from the Leaf Apoplast Carry Stress-Response Proteins. Plant Physiol 173:728-741|
|Qi, Dong; Innes, Roger W (2014) In vitro Detection of S-acylation on Recombinant Proteins via the Biotin-Switch Technique. Bio Protoc 4:|
|Serrano, Irene; Gu, Yangnan; Qi, Dong et al. (2014) The Arabidopsis EDR1 protein kinase negatively regulates the ATL1 E3 ubiquitin ligase to suppress cell death. Plant Cell 26:4532-46|
|Ashfield, Tom; Redditt, Thomas; Russell, Andrew et al. (2014) Evolutionary relationship of disease resistance genes in soybean and Arabidopsis specific for the Pseudomonas syringae effectors AvrB and AvrRpm1. Plant Physiol 166:235-51|
|Gu, Yangnan; Innes, Roger W (2012) The KEEP ON GOING protein of Arabidopsis regulates intracellular protein trafficking and is degraded during fungal infection. Plant Cell 24:4717-30|
|Gu, Yangnan; Innes, Roger W (2011) The KEEP ON GOING protein of Arabidopsis recruits the ENHANCED DISEASE RESISTANCE1 protein to trans-Golgi network/early endosome vesicles. Plant Physiol 155:1827-38|
|Christiansen, Katy M; Gu, Yangnan; Rodibaugh, Natalie et al. (2011) Negative regulation of defence signalling pathways by the EDR1 protein kinase. Mol Plant Pathol 12:746-58|
|Wawrzynska, Anna; Rodibaugh, Natalie L; Innes, Roger W (2010) Synergistic activation of defense responses in Arabidopsis by simultaneous loss of the GSL5 callose synthase and the EDR1 protein kinase. Mol Plant Microbe Interact 23:578-84|
|Wawrzynska, Anna; Christiansen, Katy M; Lan, Yinan et al. (2008) Powdery mildew resistance conferred by loss of the ENHANCED DISEASE RESISTANCE1 protein kinase is suppressed by a missense mutation in KEEP ON GOING, a regulator of abscisic acid signaling. Plant Physiol 148:1510-22|
|Tang, Dingzhong; Ade, Jules; Frye, Catherine A et al. (2006) A mutation in the GTP hydrolysis site of Arabidopsis dynamin-related protein 1E confers enhanced cell death in response to powdery mildew infection. Plant J 47:75-84|
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