Two-By-One model of cytoplasmic incompatibility: Synthetic recapitulation by transgenic expression of cifA and cifB in Drosophila

Wolbachia are maternally inherited bacteria that infect arthropod species worldwide and are deployed in vector control to curb arboviral spread using cytoplasmic incompatibility (CI). CI kills embryos when an infected male mates with an uninfected female, but the lethality is rescued if the female and her embryos are likewise infected. Two phage WO genes, cifA(wMel) and cifB(wMel) from the wMel Wolbachia deployed in vector control, transgenically recapitulate variably penetrant CI, and one of the same genes, cifA(wMel), rescues wild type CI. The proposed Two-by-One genetic model predicts that CI and rescue can be recapitulated by transgenic expression alone and that dual cifA(wMel) and cifB(wMel) expression can recapitulate strong CI. Here, we use hatch rate and gene expression analyses in transgenic Drosophila melanogaster to demonstrate that CI and rescue can be synthetically recapitulated in full, and strong, transgenic CI comparable to wild type CI is achievable. These data explicitly validate the Two-by-One model in wMel-infected D. melanogaster, establish a robust system for transgenic studies of CI in a model system, and represent the first case of completely engineering male and female animal reproduction to depend upon bacteriophage gene products. Author summary Releases of Wolbachia-infected mosquitos are underway worldwide because Wolbachia block replication of Zika and Dengue viruses and spread themselves maternally through arthropod populations via cytoplasmic incompatibility (CI). The CI drive system depends on a Wolbachia-induced sperm modification that results in embryonic lethality when an infected male mates with an uninfected female, but this lethality is rescued when the female and her embryos are likewise infected. We recently reported that the phage WO genes, cifA and cifB, cause the sperm modification and cifA rescues the embryonic lethality caused by the wMel Wolbachia strain deployed in vector control. These reports motivated proposal of the Two-by-One model of CI whereby two genes cause lethality and one gene rescues it. Here we provide unequivocal support for the model in the Wolbachia strain used in vector control via synthetic methods that recapitulate CI and rescue in the absence of a Wolbachia infections. Our results reveal the set of phage WO genes responsible for this powerful genetic drive system, act as a proof-of-concept that these genes alone can induce gene drive like crossing patterns, and establish methodologies and hypotheses for future studies of CI in Drosophila. We discuss the implications of the Two-by-One model towards functional mechanisms of CI, the emergence of incompatibility between Wolbachia strains, vector control applications, and CI gene nomenclature.