The eukaryotic innate immune system represents an important barrier that pathogens need to circumvent in order to cause disease. Several components of this system are conserved in eukaryotes. Recently, bacterial pathogen effectors that are injected into host cells by type III protein secretion systems (TTSSs) have been shown to be capable of suppressing innate immunity in eukaryotes. The bacterial plant pathogen Pseudomonas syringae is dependent on a TTSS to cause disease on plants. The P. s. pv. tomato DC3000 effector gene hopU1 resembles ADP ribosyltransferases (ADP-RTs) genes. These genes encode some of the best understood toxins in bacterial pathogens of animals (e. g., cholera toxin). Preliminary data within this proposal show that HopU1 is an active ADP-RT and that it ADP-riboslylates several plant proteins. Mass spectrometry determined that chloroplast and glycine-rich RNA-binding proteins acted as in vitro substrates for HopU1. These are novel substrates for ADP-RTs. Moreover, HopU1 has the ability to suppress several responses of the plant innate immune system in a manner that is dependent on its ADP-RT active site. An Arabidopsis mutant lacking one HopU1 substrate, AtGRP7, displayed enhanced susceptibility to P. syringae suggesting that it is a component of innate immunity. AtGRP7 is a glycine-rich RNA-binding protein, which suggests this pathogen targets proteins involved in RNA metabolism to suppress innate immunity. The central hypothesis of the proposed experiments is that AtGRP7 and perhaps other targets of the HopU1 ADP-RT type III effector are components of innate immunity. Several of the experiments seek to elucidate the role AtGRP7 plays in innate immunity using biochemical and molecular biological approaches. The P. syringae-Arabidopsis pathosystem is an excellent model to study the innate immune system because of the resources available, the similarities between innate immune systems between eukaryotes, and the cost efficient research. These experiments will contribute to a fundamental understanding of the molecular mechanism of bacterial pathogenesis and innate immunity. The Specific Aims are the following: (1) Determine the molecular consequence of ADP- ribosylation on the function of AtGRP7 and elucidate the role this protein plays in innate immunity;(2) Identify additional substrates of HopU1 and verify their involvement in innate immunity;(3) Analyze the affect that HopU1 has on host-microbe interactions. Project Narrative: Identifying the eukaryotic targets for the P. syringae HopU1 ADP-ribosyltransferase will contribute to our understanding of bacterial pathogenesis and will likely reveal important components of the innate immune system. One HopU1 target belongs to a large group of proteins called glycine-rich RNA binding proteins, which are not well understood, and this research will likely increase our understanding of these proteins. Because there are considerable similarities between the innate immune systems in plants and mammals we expect that our findings will be relevant to the mission of the NIH and be broadly interesting to researchers studying molecular mechanisms of bacterial pathogenesis and innate immunity.