CRISPR screens in Drosophila cells identify Vsg as a Tc toxin receptor

Entomopathogenic nematodes are widely used as biopesticides(1,2). Their insecticidal activity depends on symbiotic bacteria such as Photorhabdus luminescens, which produces toxin complex (Tc) toxins as major virulence factors(3–6). No protein receptors are known for any Tc toxins, limiting our understanding of their specificity and pathogenesis. Here, we use genome-wide CRISPR-Cas9-mediated knockout screening in Drosophila melanogaster S2R+ cells and identify Visgun (Vsg) as a receptor for an archetypal P. luminescens Tc toxin (pTc), which recognizes the extracellular O-glycosylated mucin-like domain of Vsg containing high density repeats of proline, threonine, and serine (HD-PTS). Vsg orthologs in mosquitoes and beetles contain HD-PTS and can function as pTc receptors, whereas orthologs without HD-PTS such as moth and human versions are not pTc receptors. Vsg is expressed in immune cells including hemocytes and fat body cells. Hemocytes from Vsg knockout (KO) Drosophila are resistant to pTc and maintain phagocytosis in the presence of pTc, and their sensitivity to pTc is restored by transgenic expression of mosquito Vsg. Lastly, Vsg KO Drosophila showed reduced bacterial loads and lethality from P. luminescens infection. Our findings identify a proteinaceous Tc toxin receptor, reveal how Tc toxins contribute to P. luminescens pathogenesis, and establish a genome-wide CRISPR screening approach for investigating insecticidal toxins and pathogens.