Structure-Function Analysis of the Plant NB-LRR Disease Resistance Protein, Rx

植物NB-LRR抗病蛋白的结构-功能分析，RxPeter MoffettBoyce Thompson植物研究所项目摘要植物已经进化出先天免疫系统，其中抗病性取决于植物和病原体的基因型。这被称为基因对基因抗性，因为植物抗性（R）基因赋予对病原体的抗性，并与无毒（Avr）基因相匹配。R基因中最丰富的类型是NB-LRR类基因;如此命名是因为编码的蛋白质具有类似的预测结构，包括核苷酸结合和富含亮氨酸的重复基序。植物基因组编码数百个NB-LRR基因，通常具有多个等位基因或拷贝。这类基因已被证明赋予对非常不同类型的病原体包括细菌、病毒、真菌、线虫和昆虫的抗性。这些蛋白质被认为在识别Avr决定簇后启动信号转导级联。植物R基因代表了一种重要的农艺学资源，但可能具有局限性，如非持久性和缺乏物种间的可转移性。这些情况可能会得到改善，但只有深入了解这类蛋白质在分子水平上的功能。尽管克隆了许多具有已知特异性的NB-LRR基因和数百个具有未知特异性的NB-LRR基因，但直到最近，对这些蛋白质将识别事件翻译成信号事件的物理机制知之甚少。这些相互作用在其Avr决定簇，马铃薯病毒X（PVX）外壳蛋白（CP）的存在下被破坏。这些结果使研究人员能够制定NB-LRR蛋白如何发挥作用的模型，这些模型可以使用本塞姆烟草（烟草的野生亲戚）叶子中的瞬时表达系统进行快速评估。该方法是将Rx活性分成许多离散的功能，包括分子内和分子间相互作用的精细映射。使用随机和定点诱变，将确定Rx的哪些基序参与不同的功能。然后将评估这些功能如何相互关联，并制定Rx活动的综合模型。此外，使用大小分离技术，Rx与细胞组分的相互作用以及它们如何与分子内相互作用相关将被表征。这些结果将使研究人员能够开发一种模型，说明Rx蛋白如何作为分子开关诱导细胞处于不适合病原体的状态。从植物中纯化Rx的计划也将进行，以确定相互作用的蛋白质使用N。携带亲和标记的Rx转基因的本氏植物。这些蛋白质将被预测参与使Rx有能力发挥作用，在Avr决定簇的情况下保持其无活性，或参与信号传导过程。鉴于R基因结构的相似性，Rx的工作将深入了解抗病性的一般机制。 这项工作还将为本科生、研究生和博士后研究员提供培训机会。

Structure-function analysis of the plant NB-LRR disease resistance protein, RxPeter MoffettBoyce Thompson Institute for Plant ResearchProject summaryPlants have evolved an innate immune system whereby disease resistance is dependent on the genotypes of both the plant and the pathogen. This is known as gene-for-gene resistance, in that plant resistance (R) genes confer resistance to pathogens with a matching Avirulence (Avr) gene. The most abundant type of R gene is the NB-LRR class of genes; so named because the encoded proteins have a similar predicted structure including nucleotide binding and leucine rich repeat motifs. Plant genomes encode for hundreds of NB-LRR genes, often with multiple alleles or copies. Genes of this class have been shown to confer resistance to very different types of pathogens including bacteria, viruses, fungi, nematodes and insects. These proteins are thought to initiate a signal transduction cascade upon recognition of an Avr determinant. Plant R genes represent a resource of great agronomic importance but can have limitations such as non-durability and lack of transferability between species. These situations may be improved upon, but only with an in-depth knowledge of how this class of proteins functions at the molecular level. Despite the cloning of many NB-LRR genes with known specificities and hundreds more with unknown specificities there has, until recently, been very little known about the physical mechanisms by which these proteins translate a recognition event into a signaling event.It has been demonstrated that the potato NB-LRR protein Rx undergoes at least two intra-molecular protein-protein interactions between different domains of the protein. These interactions are disrupted in the presence of its Avr determinant, the potato virus X (PVX) coat protein (CP). These results have allowed the investigators to formulate models of how NB-LRR proteins function that can be rapidly assessed using a transient expression system in Nicotiana benthamiana (a wild relative of tobacco) leaves. The approach is to divide Rx activity into a number of discrete functions including fine- mapping of intra- and inter-molecular interactions. Using random- and site-directed mutagenesis, it will be determined which motifs of Rx are involved in the different functions. How these functions relate to each other will then be assessed, and a comprehensive model of Rx activity will be formulated. Additionally, using size separation techniques, the interactions of Rx with cellular components and how they relate to the intra-molecular interactions will be characterized. These results will allow the investigators to develop a model of how the Rx protein acts as a molecular switch to induce a cellular state inhospitable to pathogens. A program to purify Rx from plants will also be undertaken to identify interacting proteins using N. benthamiana plants carrying affinity tagged Rx transgenes. Such proteins would be predicted to be involved in making Rx competent to function, in retaining it inactive in the absence of Avr determinants, or in the signaling process. Given the similarity in structure of R genes, work on Rx will yield insights into the general mechanisms of disease resistance. This work will also provide training opportunities for undergraduates, graduate students and postdoctoral fellows.