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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM063761-11
Application #
8290576
Study Section
Host Interactions with Bacterial Pathogens Study Section (HIBP)
Program Officer
Sledjeski, Darren D
Project Start
2002-04-01
Project End
2014-06-30
Budget Start
2012-07-01
Budget End
2013-06-30
Support Year
11
Fiscal Year
2012
Total Cost
$320,962
Indirect Cost
$109,349
Name
Indiana University Bloomington
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
006046700
City
Bloomington
State
IN
Country
United States
Zip Code
47401
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