DISSECTION OF PLANT VIRUS RESISTANCE SIGNALING PATHWAYS

植物通过系统表现出对疾病的天然抵抗力 基因对基因的相互作用。病原体表达被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。其中一个例子是 这种相互作用是在编码抗病的烟草N基因之间 烟草花叶病毒(TMV)。另一个是拟南芥的RCY1基因，它 编码对黄瓜花叶病毒Y(CMV-Y)的抗性。事实上，许多无毒的 近些年来，抗性基因已经被发现，但知之甚少。 关于抵抗力的机制。从病理上讲，耐药的特征是 通过导致大量细胞死亡的过敏性反应，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Rcy1/CMV-Y系统。AvR蛋白在拟南芥中的表达 N表达细胞的TMV(p50解旋酶)或CMV-Y外壳蛋白(CP) 在幼苗萌发过程中表达Rcy1的细胞导致植物迅速死亡 发展中的植物。这为失败的植物突变体提供了基因选择 对AVR蛋白做出反应，潜在地识别AVR下游的蛋白质 抗性基因。 该提案有7个具体目标。前三个解决的身份是 构成对这两种病原体的反应系统的细胞组件。目标 1将尝试识别影响该途径组件的突变， 功能丧失突变和功能获得突变。此外，酵母菌二号和 三杂交方法和生化方法(共纯化、亲和 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的物质。目标2将识别其基因 在对病原体的反应过程中，表达会发生变化，目标3将测试 影响其他基因对基因反应的已知基因也会影响这两种生物 在这里学习过。 最后四个具体目标涉及到生物化学特征 抗性基因。目标4将确定Rcy1基因产物是否与 通过分离和测序RCy1基因，获得N蛋白。目标5将进一步 鉴定Rcy1反应的激发子，CMV-Y外壳蛋白。目标6 将测试在N中发现的一个假定的核苷酸结合位点的功能 蛋白。核苷酸及其类似物与蛋白质的结合将是 这样做是为了确定相互作用的特异性。Dinesh-Kumar博士将 确定N蛋白是NTPase还是激酶，包括测试它是否是 被自身或其他胞质蛋白磷酸化的。AIM 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。

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.