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。我们以前已经表明，功能丧失突变 拟南芥的EDR1基因赋予了白粉病真菌对感染的耐药性增强 Erysiphe Cichoracearum。值得注意的是，这种电阻与几个更快的激活相关 包括PCD在内的防御反应表明EDR1是PCD的负调节器。我们也有 表明EDRL突变体对植物激素脱甲酸（ABA）具有增强的敏感性，并且它们 比野生型植物更快。因此，EDR1代表了理解的极好的主题 在病原体感染和衰老期间，如何在分子水平调节PCD的诱导。工作 在该项目的前三年中，已经确定了EDR1的两个可能的底物，一种驱动蛋白样蛋白 称为KCA1和一种称为ATMYC2的ABA诱导转录因子。我们还确定了一个大的 抑制EC/RF介导的电阻的突变数。最后，我们确定了两个 突变时赋予EC/RF样抗性表型的基因：EDR2，它编码一种新型蛋白 包含两个脂质结合域和DRP1E，它们编码了与动力学相关的蛋白质。两个EDR2 DRP1E似乎本地化为线粒体，在线粒体和 植物中PCD的调节。我们在这种竞争更新中的具体目标是1）克隆所鉴定的基因 在我们的抑制屏幕中； 2）确定EDR1是否磷酸化ATMYC2和KCA1IN VIVO，如果是，则 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 并评估脂质结合如何影响白粉病诱导的PCD和线粒体！功能。 特定的目标1将通过标准位置克隆方法来完成。这个目标的完成 发现EDR1途径中的其他基因，并揭示了其他途径与EDRL相互作用 特定的目标2将通过监视野生型ATMYC2和KCA1的电荷差异来实现 与EDRL突变体背景，转基因植物中的GFPFUSION和双突变体 分析。使用蛋白质脂质叠加测定和脂质囊泡将完成特定的目标3 EDR2和DRP1E的野生型和突变衍生物的管道测定。线粒体！功能将是 使用报告线粒体的荧光染料进行评估！膜电位。共同进行了这些分析 将提供有关病原体诱导的PCD在植物中如何调节PCD的重大见解。这项工作也将 阐明了人类病原体防御和PCD的机制，如在 这项研究与人类蛋白质具有显着相似之处，包括与PCD和线粒体相关的一些！ 在人类细胞中的功能。