The Cif proteins from Wolbachia prophage WO modify sperm genome integrity to establish cytoplasmic incompatibility
Fig 4
A nuclear localization signal in CifA is necessary for CI, rescue, and protamine levels.
(A) Schematic representation of CifA annotation shows the annotated bNLS with engineered amino acid substitutions and deletions. (B, C) Hatch rate assays assessed both CI (B) and rescue (C) in flies expressing wild-type, transgenic, and mutant cifA. Each dot represents the percent of embryos that hatched from a single male and female pair. Sample size is listed in parentheses. Horizontal bars represent the median. Letters to the right indicate significant differences determined by a Kruskal–Wallis test and Dunn multiple comparison tests. All the p-values are reported in S2 Table. (D) Antibody labeling (green) and DAPI staining of onion stage spermatids in the testes of the bNLS mutant line (cifAΔbNLS) reveals that the deletion ablates CifA’s localization to the nucleus, and CifA thus remains in the surrounding cytoplasm. The imaging experiment was conducted in parallel to nos;cifAB line shown in Fig 1. (E) Mature sperms isolated from seminal vesicles (n = 15) of <8-hour-old males of transgenic cifAΔbnlsB line shows reduced fluorescence indicative of less Protamine deficiency compared to cifAB. To control for any background confounding effects of nos-Gal4VP16 driver line, wMel− fathers were prior crossed to nos- mothers to generate males with nos;wMel− genotype. CMA3 fluorescence levels of sperms isolated from nos;wMel− males were similar to wMel− wild-type lines used in previous assays in this study. Vertical bars represent mean, and error bars represent standard deviation. Letters indicate statistically significant (p < 0.05) differences as determined by multiple comparisons based on a Kruskal–Wallis test and Dunn multiple test correction. All of the p-values are reported in S1 Table, and raw data underlying this figure can be found in S1 Data file. bNLS, bipartite nuclear localization signal; CI, cytoplasmic incompatibility.