Biochemical and molecular biological studies have shown that a variety of responses are associated with plant disease resistance. The goal of the proposed research it to extend these studies by applying genetic strategies to identify genes involved in a resistance reaction using infection of Arabidopsis thaliana with the bacterial phytopathogen Pseudomonas syringae as a model system. This model system offers the significant advantage that both the host and the pathogen are amenable to genetic analyses, including the ability to clone genes that have been identified by mutation. The long term goals of these studies are to determine the mechanisms whereby potential pathogens are perceived, and how the recognition signals are transduced to the genome, causing the transcriptional activation of specific defense genes. Two genetic approaches will be used to identify specific genes involved in plant disease resistance; 1) screens for mutant A. thaliana that have become susceptible to avirulent P. syringae strains or have become resistance to virulent P. syringae strains, and 2) screens of transgenic A. thaliana for aberrant expression of gene fusions constructed from the promoter of the putative defense gene, phenylalanine ammonia-lyase, and the reporter gene beta-glucuronidase. These screens will identify mutations in genes that are critically involved in microbial recognition, recognition signal transduction pathways, and the production of defense compounds. Two approaches will be used to characterize mutants identified by these two screens. First, the expression of previously described defense-related genes will be examined in the mutants. This analysis will demonstrate whether or not the mutation affects specific defense-related genes and will provide some direct genetic evidence concerning the role of these genes in disease resistance. The second approach will be to identify and characterize the gene affected by the mutation. The gene can be cloned by determining the map position of the mutation relative to the currently available morphological and restriction fragment length polymorphism (RFLP) maps and walking from the closest RFLP to the map position of the mutation. Specific clones containing wild-type DNA from this region can be used to complement the mutation and identify the gene of interest. Alternatively, if the mutation maps to """"""""contig"""""""" clones contained in a completed region of the available physical maps, these clones can be used directly for complementation experiments. Sequence analysis may provide some insight into the function of these genes and provide the basis of further studies of their regulation. The major significance of the proposed studies is that they will identify genes that are truly involved in plant disease resistance and provide significant new insights into how eukaryotic cells recognize bacterial pathogens and mount a successful defense.
