26Wolbachia are the world's most common, maternally-inherited, arthropod endosymbionts. Their 27 worldwide distribution is due in part to a selfish drive system termed cytoplasmic incompatibility 28 (CI) that confers a relative fitness advantage to females that transmit Wolbachia to their offspring. 29 CI results in embryonic death when infected males mate with uninfected females but not infected 30females. Under the Two-by-One genetic model of CI, males expressing the two phage WO 31 proteins CifA and CifB cause CI, and females expressing CifA rescue CI. While each protein is 32 predicted to harbor three functional domains, there is no knowledge on how sites across these 33Cif domains, rather than in any one particular domain, contribute to CI and rescue. Here, we use 34 evolution-guided, substitution mutagenesis of conserved amino acids across the Cif proteins, 35 coupled with transgenic expression in uninfected Drosophila melanogaster, to determine the 36 functional impacts of conserved residues evolving mostly under purifying selection. We report that 37 amino acids in CifA's N-terminal unannotated region and annotated catalase-related domain are 38 important for both complete CI and rescue, whereas C-terminal residues in CifA's putative domain 39 of unknown function are solely important for CI. Moreover, conserved CifB amino acids in the 40 predicted nucleases, peptidase, and unannotated regions are essential for CI. Taken together, 41 these findings indicate that (i) all CifA amino acids determined to be involved in rescue are 42 correspondingly involved in CI, (ii) an additional set of CifA amino acids are uniquely important 43 in CI, and (iii) CifB amino acids across the protein, rather than in one particular domain, are all 44 essential for CI. We discuss how these findings advance an expanded view of Cif protein evolution 45 and function, inform the mechanistic and biochemical bases of Cif-induced CI/rescue, and 46 continue to substantiate the Two-by-One genetic model of CI. 47 48 49 Article summary 50Wolbachia are maternally-transmitted, intracellular bacteria that occur in approximately half of arthropod 51 species worldwide. They can spread rapidly though host populations via the cytoplasmic incompatibility 52 (CI) drive system. CI causes embryonic death when infected males mate with infected females, but 53 offspring of infected females are rescued. Two proteins, CifA and CifB, underlie the genetic basis of CI and 54 rescue, but how amino acid sites across these proteins contribute to CI and/or rescue remain unknown. 55Here, we employed evolution-guided, combinatorial mutagenesis on conserved amino acids to 56 understand their relative contributions to CI and rescue. The results of this study reveal a phenotypic 57 complexity underlying the expression of these proteins and provide relevance to the biochemical and 58 mechanistic bases of CI and rescue. 59 60 61 129 130 131 Figure 1. Cytoplasmic incompatibility, the Two-by-One genetic model, and Cif protein architecture. (A) CI is caused when 132 Wolb...

Wolbachia are the world's most common, maternally-inherited, arthropod endosymbionts. Their worldwide distribution is due, in part, to a selfish drive system termed cytoplasmic incompatibility (CI) that confers a relative fitness advantage to females that transmit Wolbachia to their offspring. CI results in embryonic death when infected males mate with uninfected females but not infected females. Under the Two-by-One genetic model of CI, males expressing the two phage WO proteins CifA and CifB cause CI, and females expressing CifA rescue CI. While each protein is predicted to harbor three functional domains, there is no knowledge on how sites across these Cif domains, rather than in any one particular domain, contribute to CI and rescue. Here, we use evolution-guided, substitution mutagenesis of conserved amino acids across the Cif proteins, coupled with transgenic expression in uninfected Drosophila melanogaster, to determine the functional impacts of conserved residues evolving mostly under purifying selection. We report that amino acids in CifA's N-terminal unannotated region and annotated catalase-related domain are important for both complete CI and rescue, whereas C-terminal residues in CifA's putative domain of unknown function are solely important for CI. Moreover, conserved CifB amino acids in the predicted nucleases, peptidase, and unannotated regions are essential for CI. Taken together, these findings indicate that (i) all CifA amino acids determined to be crucial in rescue are correspondingly crucial in CI, (ii) an additional set of CifA amino acids are uniquely important in CI, and (iii) CifB amino acids across the protein, rather than in one particular domain, are all crucial for CI. We discuss how these findings advance an expanded view of Cif protein evolution and function, inform the mechanistic and biochemical bases of Cif-induced CI/rescue, and continue to substantiate the Two-by-One genetic model of CI.