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，Boyce Thompson植物研究所项目概述植物已经进化出一种先天免疫系统，由此抗病取决于植物和病原菌的基因类型。这被称为基因对基因抗性，因为植物抗性(R)基因与匹配的无毒(AVR)基因赋予对病原菌的抗性。最丰富的R基因类型是Nb-LRR类基因；之所以这样命名，是因为编码的蛋白质具有相似的预测结构，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，通常带有多个等位基因或副本。这类基因已被证明对非常不同类型的病原体具有抵抗力，包括细菌、病毒、真菌、线虫和昆虫。这些蛋白质被认为在识别AVR决定簇后启动了信号转导级联反应。植物R基因是一种具有重要农学意义的资源，但也有其局限性，如非持久性和物种间缺乏可转移性。这些情况可能会得到改善，但前提是对这类蛋白质如何在分子水平上发挥作用有深入的了解。尽管已经克隆了许多已知特异性的NB-LRR基因，还有数百个特异性未知的基因，但直到最近，这些蛋白质将识别事件翻译成信号事件的物理机制还知之甚少。已有研究表明，马铃薯NB-LRR蛋白Rx在蛋白质的不同结构域之间至少经历了两次分子内蛋白质-蛋白质相互作用。这些相互作用在其AVR决定簇--马铃薯X病毒(PVX)外壳蛋白(CP)的存在下被破坏。这些结果使研究人员能够建立NB-LRR蛋白如何发挥作用的模型，这些模型可以使用烟草(烟草的野生近亲)叶片中的瞬时表达系统来快速评估。方法是将Rx活性划分为一些离散的功能，包括分子内和分子间相互作用的精细映射。利用随机和定点突变，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，并将制定一个全面的Rx活动模型。此外，使用大小分离技术，将表征Rx与细胞组分的相互作用以及它们与分子内相互作用的关系。这些结果将使研究人员能够开发一个模型，说明Rx蛋白如何作为分子开关来诱导不适合病原体的细胞状态。一项从植物中提纯Rx的计划也将被实施，以利用携带亲和力标记的Rx转基因的N.benthamiana植物来鉴定相互作用的蛋白质。据预测，这些蛋白质参与使Rx有能力发挥作用，在没有AVR决定簇的情况下保持其不活跃，或参与信号传递过程。鉴于R基因在结构上的相似性，对Rx的研究将有助于深入了解抗病的一般机制。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.