Wolbachia manipulates host reproduction through cytoplasmic incompatibility (CI). CI results in embryonic lethality when sperm from a Wolbachia-infected male fertilizes an uninfected egg. Thus, CI is exploitable in pest management. The genes mediating CI are in two-gene operons, with cinA (or cidA) upstream of cinB (or cidB). Previously, we demonstrated that the CinB nuclease from wNo Wolbachia is sufficient to induce CI and female-expressed CinAwNo can block CI, suggesting a toxin-antidote (TA) relationship. TA-like behavior of various cognate Wolbachia CI proteins has been demonstrated in mosquitoes, Drosophila, and yeast. Yet in some transgenic Drosophila systems, co-expression of both proteins is necessary to induce CI, suggesting host modification. An understanding of sperm modification timing and location is thus critical. We have employed tissue immunohistochemistry, transfected insect cell lines, and proteomics to probe CI mechanism. In testes, CinB associates with mature spermatids whereas CinA was not detected. CinA localizes in the oocyte of the last stage egg chamber, placing it where it could later antagonize CinB in male pronucleus. CinA binds CinB tightly in vitro. Here, we show that both proteins localize in the cytoplasm of Drosophila S2 cells, indicating potential in vivo interaction. Since CinB is a nuclease, we wondered whether any host factors promote its activity. Proteomics data revealed CinB associates with importins and nuclear assembly proteins in testes and ovaries. Together, these findings corroborate the toxin-antidote hypothesis, enhance our understanding of the molecular mechanisms of CI, and highlight promising targets for further research to extend CI for pest management.
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