Programmed Cell Death and Disease Resistance in Arabidopsis

拟南芥的程序性细胞死亡和抗病性

基本信息

批准号：
8290576
负责人：
Roger W. Innes
金额：
$ 32.1万
依托单位：
TRUSTEES OF INDIANA UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2002
资助国家：
美国
起止时间：
2002-04-01 至 2014-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8290576
关键词：
Abscisic Acid Animal Experiments Animals Apoptosis Arabidopsis Area Autoimmune Diseases Autophagocytosis Autophagosome Biological Biological Assay Biological Testing Cancer Biology Cell Death Cell Survival Cell physiology Cells Cloning Cytoplasm DNA Resequencing Defect Disease Disease Resistance Eukaryotic Cell Genes Genetic Genome Homeostasis Human Immunity Immunology Infection Investigation Knock-out Life Luciferases Malignant Neoplasms Mass Spectrum Analysis Mediating Membrane Modeling Molecular Mouse-ear Cress Muramidase Mutation Organelles Pathway interactions Phenotype Phosphorylation Phosphotransferases Plant Growth Regulators Plants Play Process Protein Kinase Proteins Resistance Resistance to infection Role Screening procedure Stress Suppressor Mutations Testing Time Ubiquitin Ubiquitination Vacuole Vesicle Yeasts base defense response fungus insight loss of function mutation mildew mutant novel pathogen plant fungi protein complex protein degradation public health relevance response senescence transcription factor ubiquitin ligase ubiquitin-protein ligase yeast two hybrid system

项目摘要

DESCRIPTION (provided by applicant): 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.

描述（由申请人提供）：该项目重点研究介导植物程序性细胞死亡（PCD）的分子机制，特别是响应病原体感染的分子机制。我们之前已经表明，拟南芥 EDR1 基因的功能缺失突变可增强对病原体感染的抵抗力。值得注意的是，这种抗性与 PCD 和防御基因的激活、对植物激素脱落酸的敏感性增强以及对生物和非生物胁迫的响应增强衰老相关。所有这些表型都可以通过称为 KEG 的 E3 泛素连接酶的突变来抑制。泛素连接酶催化将泛素添加到底物蛋白上，然后底物蛋白靶向这些蛋白质进行降解。抑制 edr1 的 keg 突变不会阻止 KEG 功能，但似乎会导致 KEG 被组成型激活。因此，我们假设 EDR1 和 KEG 可能调节对防御反应、衰老和 PCD 至关重要的蛋白质水平。在此模型下，EDR1 的磷酸化将增强 EDR1 靶蛋白与 KEG 的相互作用，导致其泛素化和降解。因此，EDR1 和 KEG 代表了理解病原体感染和衰老期间如何在分子水平上调节 PCD 诱导的绝佳切入点。我们的具体目标是 1) 鉴定 KEG 蛋白的底物并确定它们是否有助于 edr1 介导的表型； 2) 鉴定与 EDR1 相关的蛋白质并确定它们是否有助于 edr1 介导的表型； 3) 鉴定抑制 edr1-突变表型的其他突变； 4)确定自噬在edr1介导的PCD中的作用。具体目标 1 和 2 将使用三种不同的方法来实现：酵母双杂交筛选、直接测试 edr1 突变植物中上调的转录因子以及纯化 KEG 和 EDR1 含有的蛋白质复合物，然后进行质谱分析。将使用分裂荧光素酶测定在植物中确认候选相互作用，然后通过将这些基因中的敲除系与 edr1 交叉并测定 edr1 介导的表型的抑制或增强来测试生物学相关性。对于特定目标 3，将通过专门筛选 edr1 早期衰老表型的抑制来鉴定新的抑制突变体。然后，将使用新型全基因组重测序方法快速识别因果突变。包括具体目标 4 是因为自噬最近被认为在压力下维持细胞稳态中发挥着核心作用，而自噬缺陷会导致压力诱导的 PCD 激活。 edr1 表型都可以用自噬缺陷来解释，如果属实，则表明 EDR1 是自噬途径的重要调节因子。由于大多数自噬基因在植物、真菌和动物之间是保守的，这些实验将阐明我们对人类如何调节自噬的理解，这一过程在免疫学和癌症生物学中具有广泛的影响。与具体目标 1-3 一起，这些分析将为了解 PCD 和免疫的调节方式提供重要的新见解。公共卫生相关性：该项目将研究控制细胞存活的分子机制，特别是在病原体感染的情况下。了解细胞如何决定生存或死亡对于我们了解癌症和自身免疫性疾病至关重要。