Plants have developed a complex immune system to detect and respond to challenge by potential pathogens. In many ways these pathogen detection methods resemble the innate immunity and adaptive immunity mechanisms utilized by vertebrate animals. One of the best characterized plant proteins involved in the plant immune system is RPS5. RPS5 belongs to the NOD-LRR class of resistance proteins, which mediate pathogen recognition in both plants and animals. How NOD-LRR proteins detect pathogen molecules is poorly understood. RPS5 recognizes Pseudomonas syringae expressing the pathogen effector, AvrPphB, through an indirect mechanism. Upon infection, AvrPphB is injected into host cells and utilizes its cysteine protease activity to cleave the Arabidopsis protein PBS1. The state of PBS1 is monitored by RPS5. Upon PBS1 cleavage, RPS5 becomes activated, triggering downstream signal transduction. This results in localized cell death and termination of pathogen spread. The biochemical and cellular mechanisms involved in RPS5 activation and downstream signal transduction are unknown. This proposal aims to characterize the molecular and cellular events that lead to RPS5 activation as well as identify and characterize downstream interacting partners of RPS5. Several methods will be used to achieve these goals. The dynamics of RPS5 activation in response to pathogen challenge will be determined in two ways. The intramolecular interactions of RPS5 domains will be examined in the presence or absence of PBS1 and/or AvrPphB using co-immunoprecipitation. Additionally, the dynamics of RPS5, PBS1, and AvrPphB cellular localization in response to pathogen recognition will be determined using fluorescence microscopy techniques. Finally, downstream interacting partners will be identified using yeast two-hybrid analysis and protein complex purification. Mutant alleles of these partners will be examined to determine their role in RPS5-mediated disease resistance by monitoring pathogen growth and development of disease symptoms. The range of function of these partner proteins will be examined by determining their requirement in other well characterized bacterial and fungal disease resistance pathways. Both animal and plant NOD-LRR proteins are involved in disease resistance and mutations in NOD-LRR proteins are associated with a number of human diseases such as Crohn's disease and Blau syndrome. Characterization of plant NOD-LRR proteins, such as RPS5, will lead to a better understanding of disease resistance in both plants and animals. In addition, these experiments may identify disease resistance pathways previously unknown in animal systems, which could lead to novel treatments for human disease. [unreadable] [unreadable] [unreadable] [unreadable]