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Prophage WO genes recapitulate and enhance Wolbachia-induced cytoplasmic incompatibility

Nature volume 543, pages 243–247 (2017)Cite this article

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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 research1 and relevance to symbiont-induced speciation2,3, as well as control of arbovirus vectors4,5,6 and agricultural pests7, 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 WO8 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.

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Figure 1: Comparative analyses reveal WD0631 and WD0632 in the eukaryotic association module of prophage WO as candidate CI genes.
Figure 2: Relative expression of CI candidate and prophage WO genes decreases as males age.
Figure 3: Dual expression of WD0631 (cifA) and WD0632 (cifB) is necessary to induce CI-like defects.
Figure 4: Dual expression of WD0631 (cifA) and WD0632 (cifB) recapitulates CI-associated embryonic defects.

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Acknowledgements

This work was supported by National Institutes of Health (NIH) R21 HD086833 and National Science Foundation IOS 1456778 to Seth R.B., National Science Foundation DEB-1501398 and NIH 5T32GM008554 training grant support to D.P.L., NIH T32GM07347 training grant support for J.A.M. to the Vanderbilt Medical Scientist Training Program, and NIH AI081322 to A.M.F. Imaging was performed in part through the use of the Vanderbilt University Medical Center Cell Imaging Shared Resource (supported by NIH grants CA68485, DK20593, DK58404, DK59637, and EY08126). We thank K. Jernigan and P. Snider for help with preliminary studies, and A. Brooks for assistance with figure preparation.

Author information

Author notes

  1. Daniel P. LePage and Jason A. Metcalf: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Biological Sciences, Vanderbilt University, Nashville, 37235, Tennessee, USA

    Daniel P. LePage, Jason A. Metcalf, Sarah R. Bordenstein, Jungmin On, Jessamyn I. Perlmutter, J. Dylan Shropshire, Emily M. Layton, Lisa J. Funkhouser-Jones & Seth R. Bordenstein

  2. Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, 06520, Connecticut, USA

    John F. Beckmann

  3. Department of Pathology, Microbiology, and Immunology, Vanderbilt University, Nashville, 37235, Tennessee, USA

    Seth R. Bordenstein

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Contributions

D.P.L. performed gene expression and hatch rate assays, embryo cytology, and assayed for transgene and infection status of flies. J.A.M. performed comparative genomics analyses, generated transgenic flies, and drafted the manuscript. Sarah R.B. performed evolutionary and bioinformatic analyses. J.O. performed hatch rates, assayed sex ratios, collected flies for all experiments, and assayed for transgene and infection status of flies. J.I.P. conducted younger brother effect experiments and performed embryo cytology. J.D.S. performed hatch rate assays, collected flies for parallel embryo cytology, and assayed for transgene and infection status of flies. E.M.L. collected flies and performed hatch rate assays. L.J.F.-J. obtained the wVitA transcriptome. J.F.B. obtained the wPip proteome. Seth R.B. supervised the work and contributed to all experimental designs, data analysis, and data interpretation. All authors participated in manuscript preparation, editing, and final approval.

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Correspondence to Seth R. Bordenstein.

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Reviewer Information Nature thanks S. L. O’Neill, W. Sullivan and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Extended data figures and tables

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

a, 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 homologues, and (c) a 70-aa alignment of WD0640, the phage WO gpW gene, and its homologues. Trees are based on JTT+G and CpRev+I models of evolution, respectively, and are unrooted. Consensus support values are shown at the nodes. Asterisk indicates that the CI genes are not included in Fig. 1. The WOPip5 homologue is truncated while the WOPip2 and second wAlbB homologues are highly divergent from WD0632.

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

CI gene homologues are labelled and coloured pink. Structural modules are labelled 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 homologue similarity and copy number.

a, The percentage aa identity between each WD0631/WD0632 homologue 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 Fig. 1d. Asterisk indicates the proteins are disrupted and not included in comparison analyses.

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

a, Relative Wolbachia titres in WT 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 titres do not decrease from day 1 to day 7 males. c, d, In wMel-infected males, WD0631 gene expression is equal between older (first day of emergence) and younger (fifth day of emergence) brothers 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. n = 8 for each group in a–d; n = 19–25 for each group in e. Bars, mean ± s.d. *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 for c and d. Exact P values are provided in Supplementary Table 7. These experiments were performed once.

Source data

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

Expression of control gene WD0625 in 1-day-old uninfected males does not affect (a) embryo hatch rates or (b) sex ratios. Infection status is designated with filled symbols for a wMel-infected parent or open symbols for an uninfected parent. Transgenic flies are labelled with their transgene to the right of their male/female symbol. Unlabelled symbols represent WT flies. Data points are coloured according to the type of cross: blue, no CI; red, a CI cross; purple, a rescue cross with wMel-infected females. n = 18–47 for each group in a; n = 7–8 for b. Bars, mean ± s.d. *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 was replicated three times.

Source data

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

Graphs correspond to the same crosses as in Fig. 3. Infection status is designated with filled symbols for a wMel-infected parent or open symbols for an uninfected parent. Transgenic flies are labelled with their transgene to the right of their gender symbol. Unlabelled gender symbols represent WT flies. Data points are coloured according to the type of cross: blue, no CI; red, a CI cross; purple, a rescue cross with wMel-infected females. n = 10–36 for each group. Bars, mean ± s.d. Statistics include a Kruskal–Wallis tests and Dunn’s multiple test corrections. The experiment in Extended Data Fig. 6a, c was performed once, while that in Extended Data Fig. 6b was performed twice.

Source data

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 used in Fig. 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 Fig. 3b. n = six pools of six pairs of testes, with representative image shown. This experiment was performed once.

Source data

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

a, The WD0508 transgene alone does not enhance CI in 2- to 4-day-old infected males. b, The WD0625 transgene alone does not enhance CI either; conversely, WD0632 does enhance CI as previously shown in Fig. 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 1-day-old males. Infection status is designated with filled symbols for a wMel-infected parent or open symbols for an uninfected parent. Transgenic flies are labelled with their transgene to the right of their male/female symbol. Unlabelled symbols represent WT flies. Data points are coloured according to the type of cross: blue, no CI; red, a CI cross; purple, a rescue cross with wMel-infected females. n = 12–44 for each group. Bars, mean ± s.d. **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 were done twice (a, c), three times (b, WD0625, WD0632), or once (b, WD0625/WD0632).

Source data

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

All flies are from same crosses shown in Extended Data Fig. 8, except for c, which comes from a replicate experiment. Infection status is designated with filled symbols for a wMel-infected parent or open symbols for an uninfected parent. Transgenic flies are labelled with their transgene to the right of their male/female symbol. Unlabelled symbols represent WT flies. Data points are coloured according to the type of cross: blue, no CI; red, a CI cross; purple, a rescue cross with wMel-infected females. n = 4–27 for each group. Bars, mean ± s.d. Statistics performed by ANOVA with Kruskal–Wallis test and Dunn’s multiple test correction. These experiments were done twice (b) or once (a, c).

Source data

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

a–c, Relative Wolbachia titres are higher in WD0508, WD0631, and WD0632 (cifB) transgenic lines than in WT lines. This does not occur in the WD0625 transgenic line, nor does there appear to be an additive effect. DNA copy number of the wMel groEL gene is shown normalized to D. melanogaster rp49 gene copy number in testes of the indicated strains. n = 8 independent pools of 15 pairs of testes for each group. Bars, mean ± s.d. *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 were done once.

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LePage, D., Metcalf, J., Bordenstein, S. et al. Prophage WO genes recapitulate and enhance Wolbachia-induced cytoplasmic incompatibility. Nature 543, 243–247 (2017). https://doi.org/10.1038/nature21391

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