Fig 1.
Cytoplasmic incompatibility, the Two-by-One genetic model, and Cif protein architecture.
(A) CI is caused when Wolbachia-infected males expressing cifA and cifB mate with uninfected females. If females are infected and express cifA then offspring are rescued. (B) The Two-by-One genetic model indicates that males expressing cifA and cifB, even in the absence of an infection, can cause CI that can be rescued by uninfected females expressing cifA [21]. (C) Schematic showing the protein architecture of CifA and CifB from the wMel Wolbachia of D. melanogaster. Annotations are based on a prior structural homology-based analyses [27].
Fig 2.
cifA1 and cifA2 fail to cause or rescue CI, and cifA3 can rescue but fails to cause CI.
(A) schematic showing the location of amino acid mutations in CifA relative to previously predicted domains [27]. (B) Hatch rate experiment testing if cifA mutants can induce CI when dual expressed with cifB in uninfected males. (C) Hatch rate experiment testing if expressing cifA mutants can rescue transgenic CI when expressed in uninfected females. (B/C) Each dot represents the percent of embryos that hatched from a single male and female pair. Expressed genes are noted to the right of the corresponding sex. Gray bars represent median hatch rates for each cross and letters to the right indicate significant differences based on α = 0.05 calculated by Kruskal-Wallis and Dunn’s test for multiple comparisons between all groups. Panel B was conducted three times and C was conducted twice. P-values are reported in S1 Table.
Fig 3.
All cifB mutants fail to contribute to CI.
(A) schematic showing the location of mutations in CifB relative to previously predicted domains [23, 27]. (B) Hatch rate experiment testing if cifB mutants can induce CI when dual expressed with cifA in uninfected males. Each dot represents the percent of embryos that hatched from a single male and female pair. Expressed genes are noted to the right of the corresponding sex. Gray bars represent median hatch rates for each cross and letters to the right indicate significant differences based on α = 0.05 calculated by Kruskal-Wallis and Dunn’s test for multiple comparisons between all groups. Panel B was conducted three times. P-values are reported in S1 Table.
Fig 4.
Sliding window analyses reveal selective pressures on regions surrounding evolution-guided mutagenesis.
Analyses are based on pairwise alignments of cifA and cifB variants of wMel and wHa where the ratio of non-synonymous to synonymous substitutions (Ka/Ks) are calculated along a 25 amino acid sliding window. The analysis of CifA was previously reported [24]. The horizontal dotted line represents a Ka/Ks value of 1, indicating neutrality. Residues with Ka/Ks values above the dotted line are under positive selection and those under the line are under purifying selection. Both proteins are largely under strong purifying selection, but CifB has two regions of positive selection. Sites mutated in CifA1, CifA2, CifA4, CifB2, CifB3, and CifB4 are in regions of purifying selection. Sites mutated in CifA3 and CifB1 appear to be under neutrality or slight purifying selection. Triangles on protein schematics represent a selection of mutated amino acids. The specific amino acids mutated are further illustrated in Figs 2A and 3A for CifA and CifB respectively. Annotations are from prior studies and based on structural homology based analyses [27].