This project uses the interaction between tomato leaves and Pseudomonas syringae as a model system to investigate the molecular basis of bacterial pathogenesis and host responses to bacterial infection. To understand bacterial pathogenesis, we focus on a key virulence protein, AvrPtoB, that is secreted into host cells by the bacterial type III secretion system. To explore the host response, we focus on the rapid programmed cell death (PCD) that is associated with plant immunity. We have found that AvrPtoB acts to suppress PCD in both plants and in yeast indicating that AvrPtoB likely targets a highly conserved eukaryotic process. AvrPtoB is a modular protein: the N-terminal domain interacts with the host kinase Pto to elicit immunity, whereas the C-terminus is required for PCD inhibition. In preliminary work, we have discovered that in the host cell AvrPtoB is likely ubiquitinated, cleaved, and the cleavage products are localized to different subcellular compartments. As part of a collaboration, the structure of the AvrPtoB anti-PCD domain has been solved and shown to have homology to E3 ubiquitin ligases; determination of the N-terminal structure is underway. Possible host targets of AvrPtoB anti-PCD activity, including MEK1, a MAPKK, have been identified. Finally, we have developed a gene silencing screen for use in identifying genes that play a role in host PCD. We propose to: 1) Investigate the role in pathogenesis of AvrPtoB post-translational modifications; 2) Determine if AvrPtoB is an E3 ubiquitin ligase; 3) Characterize the interaction of AvrPtoB with MEK1; 4) Examine the role of differential subcellular localization of AvrPtoB cleavage products; and 5) Use AvrPtoB to identify novel components of host PCD pathways. Our research takes advantage of two experimentally tractable organisms to elucidate host PCD and an important pathogen virulence mechanism, PCD-suppression, - processes which impact both plant agriculture and human health. Our studies are relevant to U.S. biosecurity concerns because type III effector proteins, such as AvrPtoB, occur in many potentially weaponizable human pathogens such as Yersinia and understanding their virulence mechanisms could lead to specific intervention strategies. ? ? ?
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