Prophage WO genes recapitulate and enhance Wolbachia-induced cytoplasmic incompatibility

Abstract

The genus Wolbachia is an archetype of maternally inherited intracellular bacteria that infect the germline of numerous invertebrate species worldwide. They can selfishly alter arthropod sex ratios and reproductive strategies to increase the proportion of the infected matriline in the population. The most common reproductive manipulation is cytoplasmic incompatibility, which results in embryonic lethality in crosses between infected males and uninfected females. Females infected with the same Wolbachia strain rescue this lethality. Despite more than 40 years of research and relevance to symbiont-induced speciation, as well as control of arbovirus vectors and agricultural pests, the bacterial genes underlying cytoplasmic incompatibility remain unknown. Here we use comparative and transgenic approaches to demonstrate that two differentially transcribed, co-diverging genes in the eukaryotic association module of prophage WO from Wolbachia strain wMel recapitulate and enhance cytoplasmic incompatibility. Dual expression in transgenic, uninfected males of Drosophila melanogaster crossed to uninfected females causes embryonic lethality. Each gene additively augments embryonic lethality in crosses between infected males and uninfected females. Lethality associates with embryonic defects that parallel those of wild-type cytoplasmic incompatibility and is notably rescued by wMel-infected embryos in all cases. The discovery of cytoplasmic incompatibility factor genes cifA and cifB pioneers genetic studies of prophage WO-induced reproductive manipulations and informs the continuing use of Wolbachia to control dengue and Zika virus transmission to humans.

Extended Data Figure 1. CI and the evolution of Wolbachia and prophage WO genes

(a) Diagram shows the effect of parental Wolbachia infection on progeny viability and infection status. CI (embryonic inviability) occurs in crosses between Wolbachia-infected males and uninfected females. Wolbachia-infected females mated to infected males rescue the inviability. (b) Bayesian phylogenies based on a 393-aa alignment of WD0723, the wMel ftsZ gene, and its homologs and (c) a 70-aa alignment of WD0640, the phage WO gpW gene, and its homologs. Trees are based on JTT+G and CpRev+I models of evolution, respectively, and are unrooted. Consensus support values are shown at the nodes. (*) indicates that the CI genes are not included in Figure 1. The WOPip5 homolog is truncated while the WOPip2 and second wAlbB homologs are highly divergent from WD0632.

Extended Data Figure 2. WD0631/WD0632 homologs always associate with the eukaryotic association module in prophage WO regions

CI gene homologs are labeled and colored pink. Structural modules are labeled as baseplate, head or tail. The WD0611-WD0621 label highlights a conserved gene cluster that is often associated with the CI genes. Only one phage haplotype is shown per Wolbachia strain when multiple copies of the same type are present.

Extended Data Figure 3. Wolbachia CI patterns correlate with WD0631/WD0632 homolog similarity and copy number

(a) The % amino acid (aa) identity between each WD0631/WD0632 homolog correlates with Wolbachia compatibility patterns. The only compatible cross, wMel males × wRi females, features close homology between WOMelB and WORiB. All other crosses are greater than 30% divergent and are bidirectionally incompatible. Each “% aa identity” value is based on the region of query coverage in a 1:1 BLASTp analysis. (b) CI strength, protein architecture and clade type are listed for each of the Wolbachia strains shown in Figure 1d. (*) indicates the proteins are disrupted and not included in comparison analyses.

Extended Data Figure 4. Wolbachia titers, the male age effect, and the younger brother effect

(a) Relative Wolbachia titers in wild type lines do not decrease with age. DNA copy number of the wMel groEL gene is shown normalized to D. melanogaster rp49 gene copy number in testes at the indicated ages. (b) Absolute Wolbachia titers do not decrease with male age. (c, d) In wMel-infected males, WD0631 gene expression is equal between older (first day of emergence) and younger brothers (fifth day of emergence) while WD0632 gene expression is slightly higher in early emerging brothers. (e) There is no statistical difference in CI penetrance between older and younger brothers. Bars show mean and standard deviation. * = P<0.05, *** = P<0.001, **** = P<0.0001 by ANOVA with Kruskal-Wallis test and Dunn’s multiple test correction for (a), (b), and (e), and two-tailed Mann-Whitney U test used for (c), and (d). Exact p-values are provided in Supplementary Table 7. These experiments have been performed once.

Extended Data Figure 5. WD0625 transgene expression does not induce CI-like defects

Expression of control gene WD0625 in one-day-old uninfected males does not affect (a) embryo hatch rates or (b) sex ratios. Infection status is designated with filled in symbols for a wMel-infected parent or open symbols for an uninfected parent. Transgenic flies are labeled with their transgene to the right of their male/female symbol. Unlabeled symbols represent wild type flies. Data points are colored according to the type of cross, with blue indicating no CI, red indicating a CI cross, and purple indicating a rescue cross with wMel-infected females. Bars indicate mean and standard deviation. * = P<0.05, *** = P<0.001 by ANOVA with Kruskal-Wallis test and Dunn’s multiple test correction. Exact p-values are provided in Supplementary Table 7. This experiment has been replicated three times.

Extended Data Figure 6. Expression of transgenes does not alter sex ratios

Graphs correspond to the same crosses as Figure 3. Infection status is designated with filled in symbols for a wMel-infected parent or open symbols for an uninfected parent. Transgenic flies are labeled with their transgene to the right of their gender symbol. Unlabeled gender symbols represent WT flies. Data points are colored according to the type of cross, with blue indicating no CI, red indicating a CI cross, and purple indicating a rescue cross with wMel-infected females. Bars indicate mean and standard deviation. Statistics include a Kruskal-Wallis tests and Dunn’s multiple test corrections. Extended Data Figures 6a and 6c have been performed once, while Extended Data Figure 6b has been performed twice.

Extended Data Figure 7. Transgenes are expressed in testes

(a, b) WD0508 and WD0625 transgenes are expressed in testes as evident by PCR performed against cDNA generated from dissected males utilized in Figure 3a and Extended Data Fig. 5a, respectively. (c, d) WD0631 and WD0632 transgenes are expressed in the testes from transgenic males specifically inducing high CI, no CI, or rescued CI. Testes were removed from males used in a replicate of Figure 3b. This experiment has been performed once.

Extended Data Figure 8. Transgenic expression of WD0508, WD0625, and WD0625/WD0632 does not enhance or induce CI

(a) The WD0508 transgene alone does not enhance CI in two- to four-day-old infected males. (b) The WD0625 transgene alone does not enhance CI either; conversely, WD0632 does enhance CI as previously shown in Figure 3C. The WD0625 transgene together with WD0632 does not enhance CI further than WD0632 alone. (c) WD0625/WD0632 dual expression cannot induce CI in uninfected one-day-old males. Infection status is designated with filled in symbols for a wMel-infected parent or open symbols for an uninfected parent. Transgenic flies are labeled with their transgene to the right of their male/female symbol. Unlabeled symbols represent WT flies. Data points are colored according to the type of cross, with blue indicating no CI, red indicating a CI cross, and purple indicating a rescue cross with wMel-infected females. Bars indicate mean and standard deviation. ** = P<0.01, *** = P<0.001, **** = P<0.0001 by ANOVA with Kruskal-Wallis test and Dunn’s multiple test correction. Exact p-values are provided in Supplementary Table 7. These experiments have been done twice (8a, 8c), three times (8b, WD0625, WD0632), or once (8b, WD0625/WD0632).

Extended Data Figure 9. Transgenic expression of control genes does not affect sex ratios

All flies are from same crosses shown in Extended Data Figure 8, except for 9c which comes from a replicate experiment. Infection status is designated with filled in symbols for a wMel-infected parent or open symbols for an uninfected parent. Transgenic flies are labeled with their transgene to the right of their male/female symbol. Unlabeled symbols represent WT flies. Data points are colored according to the type of cross, with blue indicating no CI, red indicating a CI cross, and purple indicating a rescue cross with wMel-infected females. Bars indicate mean and standard deviation. Statistics performed by ANOVA with Kruskal-Wallis test and Dunn’s multiple test correction. These experiments have been done twice (9b) or once (9a,c).

Extended Data Figure 10. There is variation in Wolbachia titers in transgenic lines

(a–c) Relative Wolbachia titers are higher in WD0508, WD0631, and WD0632 transgenic lines in comparison to wild type lines. This does not occur in the WD0625 transgenic line nor does there appear to be an additive effect. DNA copy number of wMel groEL gene is shown normalized to D. melanogaster rp49 gene copy number in testes of the indicated strains. Bars show mean and standard deviation. * = P<0.05, *** = P<0.001, **** = P<0.0001 for two-tailed Mann-Whitney U test (a) and Kruskal-Wallis test with Dunn’s multiple test correction (b–c). Exact p-values are provided in Supplementary Table 7. These experiments have been done once.

Figure 1. Comparative analyses reveal WD0631 and WD0632 in the eukaryotic association module of prophage WO as candidate CI genes

(a) Venn diagram illustrating the number of unique and shared gene families from four CI-inducing Wolbachia strains. (b) Venn diagram illustrating the number of unique and shared wMel genes matching each criteria combination. Bayesian phylogenies of (c) WD0631 (e) and WD0632 and their homologs, based on a core 256-aa alignment of WD0631 reciprocal BLASTp hits and a core 462-aa alignment of WD0632 reciprocal BLASTp hits. When multiple similar copies exist in the same strain, only one copy is shown. Consensus support values are shown at the nodes. Both trees are based on the JTT+G model of evolution and are unrooted. (d) CI patterns correlate with WD0631/WD0632 sequence homology. wRi rescues wMel and both share a similar set of homologs (*). The inability of wMel to rescue wRi correlates with a type (^†) that is present in wRi but absent in wMel. Likewise, bidirectional incompatibility of all other crosses correlates to divergent homologs. This diagram was adapted from Bossan et. al. (f) Protein architecture of the WD0631 and WD0632 types is conserved for each clade and is classified according to the WD0632-like domain. TM = transmembrane. Dotted shading represents the region of shared homology used to construct phylogenetic trees. For (c) and (e), the WO-prefix indicates a specific phage WO haplotype and the w-prefix refers to a “WO-like island,” a small subset of conserved phage genes, within that specific Wolbachia strain.

Figure 2. Relative expression of CI candidate and prophage WO genes decreases as males age

RNA expression in one-day-old (1d) versus seven-day-old (7d) testes, normalized to expression of groEL in wMel-infected D. melanogaster testes from the fastest developing males. Values denote 2^{−(Delta Ct)}. Bars indicate mean and standard deviation. * = P<0.05, ** = P<0.01 by Mann-Whitney U test. This experiment has been performed once. Exact p-values are provided in Supplementary Table 7.

Figure 3. Dual expression of WD0631 (cifA) and WD0632 (cifB) is necessary to induce CI-like defects

Hatch rate assays used the fastest developing males that were aged either (a, b) one-day or (c) two- to four-days in parental crosses. Parental infection status is designated with filled symbols for wMel-infected parents or open symbols for uninfected parents. Transgenic flies are labeled with their transgene to the right of their male/female symbol. Unlabeled symbols represent WT flies. Data points are colored according to the type of cross, with blue indicating no CI, red for CI crosses, and purple for rescue crosses with wMel-infected females. Bars indicate mean and standard deviation. * = P<0.05, ** = P<0.01, *** = P<0.001, **** = P<0.0001 by ANOVA with Kruskal-Wallis test and Dunn’s multiple test correction. Statistical comparisons are between all groups (panels a and b); or between CI crosses only (panel c). All experiments were performed at least twice, except for the increase of wild-type CI by WD0631, which has been performed once. Exact p-values are provided in Supplementary Table 7.

Figure 4. Dual expression of WD0631 (cifA) and WD0632 (cifB) recapitulates CI-associated embryonic defects

Representative embryo cytology is shown for (a) unfertilized embryos, (b) normal multi-nucleated embryos at one hour of development, (c) normal embryos near two hours of development in which nuclei begin to migrate to the periphery of the cytoplasm, and three different mitotic abnormalities: (d) failure of nuclear division after two to three mitoses, (e) chromatin bridging, and (f) regional mitotic failure. (g) The number of embryos with each cytological phenotype resulting from the indicated crosses. Infection status is designated with filled symbols for wMel-infected parents or open symbols for uninfected parents. Transgenic flies are labeled with their transgene to the right of their male/female symbol. Unlabeled symbols represent WT flies. Black lines on each graph indicates mean hatch rate for the cross. *** = P<0.001, **** = P<0.0001 by two-tailed Fisher’s exact test comparing normal (phenotypes b and c) to abnormal (phenotypes a, d, e, and f) for each cross. (h) Quantitation of cytological defects in control crosses. Cytology for (g) has been performed twice and cytology for (h) has been performed once. Exact p-values are provided in Supplementary Table 7.