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. All of these phenotypes can be suppressed by mutations in an E3 ubiquitin ligase known as KEG. Ubiquitin ligases catalyze the addition of ubiquitin to substrate proteins, which then targets these proteins for degradation. The keg mutations that suppress edr1 do not block KEG function, but instead appear to cause KEG to be constitutively activated. We thus hypothesize that EDR1 and KEG may regulate levels of proteins that are central to defense responses, senescence and PCD. Under this model, phosphorylation by EDR1 would enhance interaction of EDR1 target proteins with KEG, leading to their ubiquitylation and degradation. EDR1 and KEG thus represent excellent entries into understanding how induction of PCD is regulated at a molecular level during pathogen infection and senescence.
Our Specific Aims are to 1) Identify substrates of the KEG protein and determine whether they contribute to edr1-mediated phenotypes;2) Identify proteins that associate with EDR1 and determine whether they contribute to edr1-mediated phenotypes;3) Identify additional mutations that suppress the edr1- mutant phenotype;4) Determine the role of autophagy in edr1-mediated PCD.
Specific Aims 1 and 2 will be accomplished using three different approaches: yeast two-hybrid screening, direct testing of transcription factors upregulated in edr1 mutant plants, and purification of KEG and EDR1-containing protein complexes followed by mass spectrometry. Candidate interactions will be confirmed in planta using a split luciferase assay, then tested for biological relevance by crossing knockout lines in these genes to edr1 and assaying for suppression or enhancement of edr1-mediated phenotypes.
For Specific Aim 3, new suppressor mutants will be identified by screening specifically for suppression of the edr1 early senescence phenotype. The causal mutations will then be rapidly identified using a novel whole genome resequencing approach.
Specific Aim 4 is included because autophagy has recently been recognized as playing a central role in maintaining cellular homeostasis under times of stress, with defects in autophagy leading to activation of stress-induced PCD. The edr1 phenotypes could all be explained by defects in autophagy, and if true, would implicate EDR1 as an important regulator of the autophagy pathway. As the majority of autophagy genes are conserved between plants, fungi and animals, these experiments will illuminate our understanding of how autophagy is regulated in humans, a process that has broad implications in both immunology and cancer biology. Together with Specific Aims 1-3, these analyses will provide significant new insight into how PCD and immunity are regulated.

Public Health Relevance

This project will investigate the molecular mechanisms that control cell survival, particularly in the context of pathogen infection. Understanding how cells decide to live or die is critical to our understanding of cancer and autoimmune diseases.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Host Interactions with Bacterial Pathogens Study Section (HIBP)
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Singh, Shiva P
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Indiana University Bloomington
Schools of Arts and Sciences
United States
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Rutter, Brian D; Innes, Roger W (2017) Extracellular Vesicles Isolated from the Leaf Apoplast Carry Stress-Response Proteins. Plant Physiol 173:728-741
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
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
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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