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

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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.