PROJECT SUMMARY/ABSTRACT Wolbachia pipientis is an obligate intracellular alpha-proteobacterium that infects 40-60% of insect species on the planet. Wolbachia infection inhibits RNA virus replication in insects, a phenomenon known as pathogen blocking. Therefore, Wolbachia infected mosquitos are being released in many parts of the world to control the spread of human diseases. Importantly, although the mechanism behind Wolbachia’s virus inhibition is not known, Wolbachia must colonize the host and be efficiently maternally transmitted in order for pathogen blocking to work. Our long-term goals are to identify the mechanisms used by Wolbachia to establish infection. To that end, we focus on the type IV secretion system (T4SS), a molecular nanomachine used by Wolbachia to inject proteins, termed effectors, into the host cellular environment. Via these secreted effectors, the host cell is modified, allowing Wolbachia to invade and persist. Our previous work identified and characterized the first secreted effector in Wolbachia (WalE1) and established an important correlation between T4SS gene expression and expression of secreted effectors. Wolbachia effectors and the T4SS are upregulated during host pupation. Strikingly, overexpression of WalE1 in Wolbachia-infected flies facilitated colonization of the developing oocyte and maternal transmission. These results led to our central hypothesis that Wolbachia uses secreted effectors to establish and maintain host colonization. Towards this hypothesis, we have identified Wolbachia effectors across the genus, and used a large-scale growth screen to further identify Wolbachia effectors. We have discovered that many Wolbachia effectors are clade specific, but many are shared between the insect associated strains. In collaboration with Peter Christie (UTH), we developed a heterologous secretion assay for Wolbachia effectors, allowing us to confirm interactions between the type IV coupling protein VirD4 and candidate Wolbachia effectors. Guided by strong preliminary data, we propose to pursue three Specific Aims to identify and characterize Wolbachia effectors, and their host targets, using the power of the Drosophila system. We will (1) determine the Wolbachia secretome, (2) predict effectors and their conservation across Wolbachia infecting insects, and (3) identify host targets and effectors that facilitate infection by Wolbachia. Studies of Wolbachia - host interactions are still in their infancy despite the recognized contributions of endosymbiotic associations to insect reproduction and evolution, and the ability to alter vector competence. These proposed studies will significantly advance our understanding of how Wolbachia employs its T4SS to establish infection, a necessary prerequisite to pathogen blocking.