Animals and microbes interact in intricate ways. Wolbachia, a common intracellular insect symbiont, can manipulate reproduction and protect hosts from viruses. Thus, Wolbachia is an asset in the control of insect-borne diseases. However, as Wolbachia cannot be cultured outside of host cells or genetically manipulated, the mechanisms of its antiviral phenotype remain poorly understood, and this inhibits wider exploitation. I have been working to remedy these deficiencies, and now stand poised to discover the mechanisms of Wolbachia-conferred antiviral protection by answering the following questions: 1) Where does the protection originate? Up to now, mechanisms of protection have been studied in whole organisms, often lacking resolution, or in cultured cells, which lack emergent properties. I will identify tissues and cell types of the host where protection starts. To do this, I will: a) quantify titers of Wolbachia and virus at early time points post-viral infection in insect tissues, b) measure gene expression of host and microbes to identify candidates for further molecular characterisation, and c) test the extent of the utility of widely adopted, yet unvalidated, cell-culture models of antiviral protection. 2) Which Wolbachia genes effect protection? Wolbachia research has historically been impeded by a lack of tools to study gene function. Here, I will deploy antisense technology, which I have recently developed, to interrogate function of candidate Wolbachia genes in the native system. I will also engineer new methods to target Wolbachia genes and proteins, based on my data on cell-penetrating peptide-mediated delivery of bioactive cargo to Wolbachia. This project has two major outcomes: it will uncover Wolbachia factors responsible for Wolbachia-conferred antiviral protection, and it will transform Wolbachia and symbiosis research by creating tools to study symbiont gene function.