DISSECTION OF PLANT VIRUS RESISTANCE SIGNALING PATHWAYS

植物病毒抗性信号通路的剖析

• 批准号: 6481509
• 负责人: SAVITHRAMMA P DINESH-KUMAR
• 金额: $ 5万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-02-01 至 2006-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6481509
• 关键词: Arabidopsis; affinity chromatography; antiviral agents; apoptosis; biological signal transduction; cell component structure /function; gene expression; gene mutation; genes; immunity; microarray technology; molecular genetics; molecular site; plant diseases; plant ecology; plant genetics; plant physiology; plant virus; plants; replicase; suppressor mutations; tobacco mosaic virus; yeast two hybrid system

Plants exhibit natural resistance to diseases through systems of gene-for-gene interactions. Pathogens express genes called avirulence genes (Avr) that interact with plant encoded resistance genes (R). One example of such an interaction is between the tobacco N gene that encodes resistance to tobacco mosaic virus (TMV). Another is the RCY1 gene of Arabidopsis, which encodes resistance to cucumber mosaic virus-Y (CMV-Y). In fact, many avirulence and resistance genes have been identified in recent years, yet little is known about the mechanism of resistance. Pathologically, resistance is characterized by a hypersensitive response that results in massive cell death, but restricts propagation of the virus. The structure of many of resistance proteins suggests that the responses may activate signaling pathways. This proposal will use an Arabidopsis thaliana genetic system to study both the N/TMV and Rcy1/CMV-Y systems. Expression in Arabidopsis of the Avr protein of TMV (p50 helicase) in N- expressing cells or CMV-Y coat protein (CP) in Rcy1-expressing cells during germination of seedlings causes rapid death of the developing plant. This provides a genetic selection for plant mutants that fail to respond to the Avr protein, potentially identifying proteins downstream of the resistance genes. The proposal has 7 Specific Aims. The first three address the identity of cellular components that make up the response system to the two pathogens. Aim 1 will attempt to identify mutations that affect components of the pathway, both loss and gain-of- function mutations. In addition, yeast two and three-hybrid approaches and biochemical approaches (co-purification, affinity chromatography or interaction cloning) will also be used to identify proteins that interact with the resistance proteins. Aim 2 will identify genes whose expression is altered during response to pathogens and Aim 3 will test whether known genes affecting other gene-for-gene responses also affect the two being studied here. The last four Specific Aims concern the biochemical characterization of resistance genes. Aim 4 will determine whether the Rcy1 gene product resembles the N protein by isolating and sequencing the RCY1 gene. Aim 5 will further characterize the elicitor of the Rcy1 response, the CMV-Y coat protein. Aim 6 will test the function of a putative nucleotide binding site found in the N protein. Binding of nucleotides and nucleotide analogs to the protein will be done to determine the specificity of the interaction. Dr. Dinesh-Kumar will determine if the N protein is an NTPase or kinase, including testing if it is phosphorylated by itself or other cytosolic proteins. Aim 7 will determine whether N protein directly or indirectly interacts with its elicitor, TMV replicase.

植物通过免疫系统表现出对疾病的天然抵抗力。 基因间的相互作用病原体表达称为无毒基因的基因 (Avr)与植物编码的抗性基因（R）相互作用。的一个示例 这种相互作用是在编码对烟草病害的抗性的烟草N基因之间， 烟草花叶病毒（TMV）。另一个是拟南芥的RCY 1基因， 编码对黄瓜花叶病毒-Y（CMV-Y）的抗性。事实上，许多无毒的 近年来，人们已经确定了抗性基因，但对它们知之甚少。 关于抗药性的机制。在病理学上，耐药性的特征是 过敏反应导致大量细胞死亡，但限制了 病毒的传播。许多抗性蛋白的结构表明 这些反应可能会激活信号通路。 这项提议将使用拟南芥遗传系统来研究 N/TMV和Rcy 1/CMV-Y系统。拟南芥中Avr蛋白的表达 N表达细胞中的TMV（p50解旋酶）或N表达细胞中的CMV-Y外壳蛋白（CP）。 在幼苗萌发过程中表达Rcy 1的细胞导致细胞的快速死亡。 发展中的植物这为失败的植物突变体提供了遗传选择 对Avr蛋白做出反应，可能识别出 抗性基因。 该计划有7个具体目标。前三个地址的身份 构成对这两种病原体的反应系统的细胞成分。目的 1将试图识别影响通路组分的突变， 功能丧失和功能获得突变。此外，酵母二和 三杂交方法和生物化学方法（共纯化，亲和纯化， 层析或相互作用克隆）也将用于鉴定蛋白质 与抗性蛋白相互作用。目标2将确定基因， 在对病原体的反应过程中表达发生改变，Aim 3将测试是否 影响其他基因对基因反应的已知基因也会影响这两个基因， 在这里学习。 最后四个具体目标涉及的生物化学特性， 抗性基因目的4将确定Rcy 1基因产物是否类似于 通过分离和测序RCY 1基因来获得N蛋白。第五，将进一步 表征Rcy 1应答的激发子，CMV-Y外壳蛋白。目标6 将测试在N中发现的假定核苷酸结合位点的功能， 蛋白核苷酸和核苷酸类似物与蛋白质的结合将是可能的。 以确定相互作用的特异性。迪尼斯·库马尔博士 确定N蛋白是否是NTR或激酶，包括测试它是否是 自身或其他胞质蛋白磷酸化。目标7将决定 N蛋白是否直接或间接与其激发子TMV相互作用 复制酶