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: 7596345
• 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 2010-07-08
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7596345
• 关键词: Abscisic Acid; Affect; Apoptosis; Arabidopsis; Binding; Biochemical; Biological Assay; Cells; Charge; Data; Disease Resistance; Dynamin; Enhancers; Fluorescent Dyes; Gel; Genes; Genetic; Grant; Human; Immunoblotting; In Vitro; Infection; Isoelectric Focusing; Kinesin; Light; Link; Lipid Binding; Lipids; Maps; Mediating; Membrane Fusion; Membrane Potentials; Methods; Mitochondria; Molecular; Molecular Genetics; Monitor; Mouse-ear Cress; Mutate; Mutation; 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; Structure; Time; Transgenic Plants; Vesicle; Work; defense response; fungus; gene cloning; in vivo; insight; loss of function mutation; mildew; mutant; novel; pathogen; positional cloning; programs; response; senescence; transcription factor; yeast two hybrid system

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 edrl 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 KCA1and an ABA-inducible transcription factor called AtMYC2. We have also identified a large number of mutations that suppress ec/rf-mediated resistance. Finally, we have identified two additional genes that confer ec/rf-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 KCA1in vivo, and if so, how phosphorylation affects their function and stability; and 3) determine the lipid binding properties of EDR2 and DRP1Eand assess how lipid binding affects powdery mildew-induced PCD and mitochondria! function. Specific aim 1will be accomplished by standard positional cloning methods. Completion of this aim will uncover additional genes in the EDR1 pathway,as well as reveal other pathwaysthat interact with EDRL Specific aim 2 will be accomplished by monitoring charge differences on AtMYC2 and KCA1 in wild-type versus edrl mutant backgrounds, localizing GFPfusions 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. Mitochondria! function will be assessed using a fluorescent dye that reports mitochondria! 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 mitochondria! function in human cells.

该项目重点研究介导植物程序性细胞死亡（PCD）的分子机制， 特别是针对病原体感染的反应。我们之前已经证明，功能丧失突变 拟南芥的 EDR1 基因增强了对白粉病真菌感染的抵抗力 菊苣白粉菌。值得注意的是，这种抵抗力与多种细胞的更快激活有关。 防御反应，包括 PCD，表明 EDR1 是 PCD 的负调节因子。我们还有 研究表明，edrl 突变体对植物激素脱落酸 (ABA) 的敏感性增强，并且它们 比野生型植物衰老得更快。因此，EDR1 代表了理解的绝佳切入点 在病原体感染和衰老过程中如何在分子水平上调节 PCD 的诱导。工作 在该项目的前三年中，我们已经确定了 EDR1 的两种可能的底物，EDR1 是一种类似驱动蛋白的蛋白 称为 KCA1 和 ABA 诱导转录因子称为 AtMYC2。我们还发现了一个大 抑制 ec/rf 介导的耐药性的突变数量。最后，我们还确定了另外两个 突变时赋予 ec/rf 样抗性表型的基因：EDR2，编码一种新蛋白质 含有两个脂质结合域和 DRP1E，编码动力相关蛋白。两者都是 EDR2 DRP1E 似乎定位于线粒体，在线粒体和 植物中 PCD 的调控。我们在这一竞争性更新中的具体目标是 1) 克隆已识别的基因 在我们的抑制器屏幕中； 2)确定EDR1在体内是否磷酸化AtMYC2和KCA1，如果是， 磷酸化如何影响其功能和稳定性； 3) 确定 EDR2 的脂质结合特性 和 DRP1E 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。这一目标的完成将 发现 EDR1 通路中的其他基因，以及与 EDRL 相互作用的其他通路 具体目标 2 将通过监测野生型中 AtMYC2 和 KCA1 的电荷差异来实现 与 edrl 突变体背景相比，在转基因植物中定位 GFP 融合，并通过双突变体 分析。具体目标 3 将使用蛋白质-脂质叠加测定和脂质囊泡来实现 使用 EDR2 和 DRP1E 的野生型和突变体衍生物进行管化测定。线粒体！函数将是 使用报告线粒体的荧光染料进行评估！膜电位。综合起来，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。这部作品也将 揭示了人类病原体防御和 PCD 的机制，因为在 这项研究与人类蛋白质具有显着的相似性，包括一些与 PCD 和线粒体相关的蛋白质！ 在人体细胞中发挥作用。