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 that this effector disrupts host endocytosis. We developed a live assay for visualizing Wolbachia infection and showed that the actin cytoskeleton must be intact for this to occur. We identified important Wolbachia effectors upregulated during infection using proteomics and show that the T4SS is upregulated upon host internalization. In this proposed renewal, we will continue or work to identify the Wolbachia secretome across strains, within both Drosophila and Aedes, and identify pathways important for Wolbachia infection of both host species. We will also further identify how the important effector WalE1 functions across strains and determine the conservation and function of important domains for this protein. Guided by strong preliminary data, we propose to pursue three Specific Aims to identify and characterize Wolbachia effectors, host pathways important for infection by the microbe, and WalE1 function. We will (1) Determine and characterize the Wolbachia secretome, across strains, (2) Identify host pathways important for colonization of hosts by different Wolbachia strains, and (3) Characterize the molecular biology and evolution of Wolbachia’s WalE1. 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 effectors to establish infection, a necessary prerequisite to pathogen blocking.