Identifying and Characterizing Phage Genes Involved in Wolbachia-Induced Cytoplasmic Incompatibility
Shropshire, J. Dylan
0000-0003-4221-2178
:
2020-04-02
Abstract
Wolbachia are maternally transmitted bacteria that infect ~40-65% of insect species, and commonly hijack their host’s reproduction to favor their spread. Cytoplasmic incompatibility (CI) is Wolbachia’s most common manipulation and is characterized by embryonic death when infected males mate with uninfected females. Importantly, the offspring of infected females are rescued from this lethality, increasing their relative fitness and Wolbachia’s transmission. This dissertation aims to identify the genetic basis of CI and rescue, and characterize how sequence variation impacts these phenotypes.
A comparative ‘omic analysis identified two genes from Wolbachia’s prophage WO that were present in CI-inducing Wolbachia and absent in non-parasitic Wolbachia. Transgenic expression of both genes in uninfected Drosophila melanogaster males recapitulated partial CI-like hatching and cytological defects that could be rescued by infected females. We named these genes CI factors A (cifA) and B (cifB). Next, transgenic expression of cifA in uninfected females was found to rescue CI induced by infected males, establishing a Two-by-One genetic model for CI where cifA;B causes CI and cifA rescued CI. This model was validated by optimizing the transgenic system to recapitulate complete embryonic death with cifA;B-expressing males and rescuing that transgenic lethality with cifA-expressing females.
We then aimed to investigate the relationship between sequence variation and phenotypic expression. First, we determined that while cifA homologs are generally able to contribute to both CI and rescue, cifB homologs failed to contribute to strong transgenic CI. Since these homologs are not native to D. melanogaster we predict that cifB is optimized for expression in specific host backgrounds, but this remains to be tested. Finally, a mutation analysis of conserved residues across Cif proteins revealed that mutating conserved sites in CifB prevented CI. However, sites mutated in CifA’s N-terminus impacted both CI and rescue, a central domain prevented CI, and mutations in the C-terminus had no impact. These assays suggest that CifA has multiple biochemical functions and that some of these functions are important in both phenotypes. More work will be necessary to determine the biochemical properties of each region of these proteins.