Programmed Cell Death and Disease Resistance in Arabidopsis

拟南芥的程序性细胞死亡和抗病性

• 批准号: 7215540
• 负责人: Roger W. Innes
• 金额: $ 28.13万
• 依托单位: TRUSTEES OF INDIANA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-04-01 至 2010-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7215540
• 关键词: Abscisic Acid; Affect; Apoptosis; Arabidopsis; Binding; Biochemical; Biological Assay; Cell Death; Cells; Charge; Data; Disease Resistance; Dynamin; Enhancers; Fluorescent Dyes; Gel; Genes; Genetic; Grant; Green Fluorescent Proteins; Human; Immunoblotting; In Vitro; Infection; Isoelectric Focusing; Kinesin; Light; Link; Lipid Binding; Lipids; Localized; Maps; Mediating; Membrane Fusion; Membrane Potentials; Methods; Mitochondria; Molecular; Molecular Genetics; Monitor; Mouse-ear Cress; Mutate; Mutation; Numbers; Pathway interactions; Phenotype; Phosphorylation; Plant Growth Regulators; Plants; Play; Property; Protein Kinase; Proteins; Regulation; Reporting; Research Personnel; Resistance; Resistance to infection; Role; Signal Pathway; Signal Transduction; Standards of Weights and Measures; Structure; Time; Transgenic Plants; Vesicle; Work; defense response; disorder control; fungus; gene cloning; in vivo; insight; loss of function mutation; mildew; mitochondrial membrane; mutant; novel; pathogen; positional cloning; programs; response; senescence; transcription factor; yeast two hybrid system

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 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 in 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.

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。我们之前已经证明，拟南芥 EDR1 基因的功能缺失突变可增强对白粉病真菌白粉病感染的抵抗力。值得注意的是，这种抵抗力与包括 PCD 在内的多种防御反应的更快激活相关，表明 EDR1 是 PCD 的负调节因子。我们还表明，edr1 突变体对植物激素脱落酸 (ABA) 的敏感性增强，并且比野生型植物衰老得更快。因此，EDR1 代表了了解病原体感染和衰老过程中如何在分子水平上调节 PCD 诱导的绝佳切入点。该项目前三年的工作已经确定了 EDR1 的两种可能的底物，一种称为 KCA1 的驱动蛋白样蛋白和一种称为 AtMYC2 的 ABA 诱导转录因子。我们还发现了大量抑制 edr1 介导的耐药性的突变。最后，我们发现了另外两个基因，它们在突变时会赋予 edr1 样抗性表型：EDR2，它编码一种含有两个脂质结合域的新蛋白质；DRP1E，它编码一种动力相关蛋白质。 EDR2 和 DRP1E 似乎都定位于线粒体，提供了线粒体与植物中 PCD 调节之间的机制联系。我们在这一竞争性更新中的具体目标是 1) 克隆在我们的抑制筛选中识别出的基因； 2）确定EDR1在体内是否磷酸化AtMYC2和KCA1，如果是，磷酸化如何影响它们的功能和稳定性； 3) 确定 EDR2 和 DRP1E 的脂质结合特性，并评估脂质结合如何影响白粉病诱导的 PCD 和线粒体功能。具体目标1将通过标准的定位克隆方法来实现。完成这一目标将揭示 EDR1 途径中的其他基因，并揭示与 EDRL 相互作用的其他途径。 具体目标 2 将通过监测野生型与 edr1 突变体背景中 AtMYC2 和 KCA1 上的电荷差异、将 GFP 融合定位在转基因植物，并通过双突变体分析。具体目标 3 将通过使用 EDR2 和 DRP1E 的野生型和突变衍生物进行蛋白质-脂质重叠测定和脂质囊泡管化测定来实现。将使用报告线粒体膜电位的荧光染料来评估线粒体功能。总之，这些分析将为植物中病原体诱导的 PCD 的调控提供重要的新见解。这项工作还将揭示人类病原体防御和 PCD 的机制，因为本研究中鉴定的几种蛋白质与人类蛋白质具有显着的相似性，包括一些与人类细胞中的 PCD 和线粒体功能相关的蛋白质。