UC San Diego

Using a myriad of genetic, biochemical, and cell biology based approaches, the interactions between pathogens and their hosts have been intensely examined since microbes were first described by Hooke and van Leeuwenhoek in the 17th century. Despite these efforts, the molecular mechanisms that underlie the basis of pathogenesis and host defense are still only partially unraveled. Our work has focused on the interaction between bacterial phytopathogens, specifically Pseudomonas syringae, and the reference plant Arabidopsis thaliana. Here, we examined the sub-cellular localization strategies utilized by a subset of P. syringae virulence factors, the AvrPphB-like family of type III effector proteins. Remarkably, some members of the AvrPphB family of effector proteins utilize their own cysteine protease activities to direct their localization within the plant cell. We have demonstrated that, following delivery through the type III secretion system, these effectors undergo self-proteolytic processing to reveal a novel amino terminus containing consensus sites for eukaryotic fatty acylation. We show that these effectors hijack the eukaryotic acylation machinery to ensure lipid modification, and are consequently delivered to the host plasma membrane. Additionally, we found that acylation of AvrPphB by the host lipidation machinery is absolutely required for successful cleavage of its in planta substrate. Finally, we have demonstrated that additional AvrPphB family members, surprisingly, employ acylation-independent localization strategies. Nonetheless, these effectors also localize to the host plasma membrane, underscoring the plant plasma membrane as a critical site for type III effector function. In addition, we have also investigated specific aspects of the plant defense system. Although the signaling pathways that encode innate immunity against invading microbes have been well studied, epigenetic regulation of plant defenses through modification of heritable DNA methylation patterns represents an unexplored regulatory mechanism of plant defense. Here, we show that Arabidopsis mutants deficient in cytosine methyltranserase activity are markedly more resistant to P. syringae, a phenotype that strongly correlates with up- regulation of known plant defense genes. Remarkably, we have also demonstrated that wild-type plants utilize transgenerational memory of P. syringae infection to encode enhanced pathogen resistance in their progeny, a mechanism that requires the activity of the DRM1, DRM2, and CMT3 DNA methyltransferases. Furthermore, using high- throughput sequencing technology, we mapped methylcytosines across the genome of Arabidopsis plants infected with P. syringae, and found multiple examples of pathogen-induced transient DNA methylation changes that correlate with transcriptional changes of proximal genes. Together, our results have revealed that cytosine methylation contributes to regulation of plant defense genes during infection, and that transient alterations in DNA methylation patterns may encode resistance to pathogens in subsequent generations