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）：该项目着重于介导植物中介导编程细胞死亡（PCD）的分子机制，特别是针对病原体感染的响应。我们先前已经表明，拟南芥的EDR1基因中的功能丧失突变赋予病原体对感染的抗性增强。值得注意的是，这种耐药性与PCD和防御基因的激活相关，对植物激素脱离的脱甲酸的敏感性增强，以及对生物和非生物胁迫的响应增强的衰老。所有这些表型可以通过称为桶盖的E3泛素连接酶中的突变来抑制。泛素连接酶会催化在底物蛋白中添加泛素，然后将这些蛋白靶向降解。抑制EDR1的小桶突变不会阻止桶函数，而是导致小桶被组成式激活。因此，我们假设EDR1和KEG可能调节对防御反应，衰老和PCD核心的蛋白质水平。在该模型下，EDR1的磷酸化将增强EDR1靶蛋白与小桶的相互作用，从而导致其泛素化和降解。因此，EDR1和KEG代表了理解在病原体感染和衰老过程中如何在分子水平调节PCD的诱导方式的出色条目。我们的具体目的是1）确定桶蛋白的底物，并确定它们是否有助于EDR1介导的表型； 2）确定与EDR1关联的蛋白质，并确定它们是否有助于EDR1介导的表型； 3）确定抑制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。具体目的1和2将使用三种不同的方法完成：酵母两杂化筛选，在EDR1突变植物中上调的转录因子的直接测试，以及含有KEG和EDR1的蛋白质复合物的纯化，然后进行质谱法。候选相互作用将在Planta中使用分裂的荧光素酶测定，然后通过将这些基因的基因敲除线与EDR1交叉敲除线进行测试，并分析抑制或增强EDR1介导的表型。对于特定的目标3，将通过专门筛选以抑制EDR1早期衰老表型来识别新的抑制突变体。然后，将使用一种新型的整个基因组重新配置方法来快速识别因果突变。包括特定的目标4是因为自噬最近被认为在压力时保持细胞稳态方面发挥了核心作用，并且自噬的缺陷导致激活压力诱导的PCD。 EDR1表型都可以通过自噬中的缺陷来解释，如果为真，则将EDR1视为自噬途径的重要调节剂。由于大多数自噬基因在植物，真菌和动物之间都保存下来，因此这些实验将阐明我们对人类自动噬菌体如何调节的理解，这一过程对免疫学和癌症生物学具有广泛的影响。这些分析将与特定的目标1-3一起，对如何调节PCD和免疫力提供重大的新见解。公共卫生相关性：该项目将研究控制细胞存活的分子机制，尤其是在病原体感染的背景下。了解细胞如何决定生活或死亡对于我们对癌症和自身免疫性疾病的理解至关重要。