Evolution-guided mutagenesis of the cytoplasmic incompatibility proteins: Identifying CifA’s complex functional repertoire and new essential regions in CifB

Abstract

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...

The Biochemistry of Cytoplasmic Incompatibility Caused by Endosymbiotic Bacteria

“…In the mis-timing and sink models, on the other hand, CifA and CifB together may function as a complex in the sperm to induce the respective host modification while CifA alone somehow acts as the rescue factor to reverse this. An alternative explanation, as in a more recent version of the two-by-one scheme, is that CifA functions as the major CI-inducing protein as well as the rescue factor, with CifB only having an "adjunct" function in CI induction [50]. Intriguingly, the proposal of the sink model was partly based on the observation that Wolbachia likely interact with histones.…”

“…The authors of the two-by-one/HM model, by contrast, argue that CI induction is most consistent with CifA as the major inducer because a complex mutation of the putative Puf domain in CifA abrogates CI induction without affecting rescue [50]. If CifA were antagonizing CifB to prevent premature defects in the testes by the same mechanism it uses against CifB for rescue in the embryo (i.e., through direct physical binding), then such a separation-of-function mutation should not be possible.…”

Symbiont-Mediated Cytoplasmic Incompatibility: What have we Learned in 50 Years?

“…However, interpretation of these results is significantly complicated by the absence of rescue data for any cifB-associated reduction in embryonic hatching in insects. Moreover, mutagenesis analyses, described in further detail below, indicate that changing conserved residues across CifA can crucially prevent CI-induction, lending additional support for CifA's important role as a CI-inducing factor (Shropshire et al, 2020). More functional genetic analyses will be necessary to provide evidence for an alternative to the Two-by-One genetic model of CI.…”

“…Thus, a single mutation in CifA may shift both CI and rescue phenotypes, yield bidirectional CI relative to an ancestor, and maintain self-compatibility (Shropshire et al, 2018). Indeed, mutagenesis of highly conserved amino acids across CifA reveal that sites within CifA's N-terminal region are crucially important for the expression of both CI and rescue, suggesting that residues in this single region are coopted for both phenotypes (Shropshire et al, 2020). Notably while this one-step model of bidirectional CI avoids the maladaptive valley, it may only spread if transferred into a new aposymbiotic (sub)population since emergence of a new incompatibility type within a symbiontbearing population would be immediately incompatible with the more common symbiont in the population.…”

“…On the other hand, sliding window analyses of selection for CifA[T1] suggest that while the full protein is under purifying selection, the catalaserel domain and the unannotated N-terminal region are under the strongest selection (Shropshire et al, 2018). Indeed, CifA cannot contribute to transgenic CI or rescue when conserved amino acids are mutated within the unannotated N-terminal region or in the putative catalase-rel domain of CifAwMel[T1] (Shropshire et al, 2020). Conversely, when sites are mutated in CifA's DUF domain, it maintains the ability to contribute to rescue, but loses CI-capability (Shropshire et al, 2020).…”