Identification of Parthenogenesis-Inducing Effector Proteins in Wolbachia

Abstract

Bacteria in the genus Wolbachia have evolved numerous strategies to manipulate arthropod sex, including the conversion of would-be male offspring to asexually reproducing females. This so-called "parthenogenesis induction" phenotype can be found in a number of Wolbachia strains that infect arthropods with haplodiploid sex determination systems, including parasitoid wasps. Despite the discovery of microbe-mediated parthenogenesis more than 30 yr ago, the underlying genetic mechanisms have remained elusive. We used a suite of genomic, computational, and molecular tools to identify and characterize two proteins that are uniquely found in parthenogenesis-inducing Wolbachia and have strong signatures of host-associated bacterial effector proteins. These putative parthenogenesis-inducing proteins have structural homology to eukaryotic protein domains including nucleoporins, the key insect sex determining factor Transformer, and a eukaryotic-like serine-threonine kinase with leucine-rich repeats. Furthermore, these proteins significantly impact eukaryotic cell biology in the model Saccharomyces cerevisiae. We suggest that these proteins are parthenogenesis-inducing factors and our results indicate that this would be made possible by a novel mechanism of bacterial-host interaction.

Keywords: Wolbachia; mitosis; parasitoid; parthenogenesis; sex; symbiosis.

Fig. 1.

Wolbachia-mediated parthenogenesis and putative PI factors. A) Haplodiploid sex determination in the absence of Wolbachia. Haploid males develop from unfertilized eggs, and diploid females develop from fertilized eggs. In some sexual hymenopterans, a paternal factor is required for driving female development in the offspring on top of fertilization (+pff). B) Parthenogenesis-inducing wTpre Wolbachia causes gamete duplication via a failed anaphase during the first mitotic division in Trichogramma embryos in the absence of fertilization. C) Candidate PI factors (pifs) are located within a degraded EAM. The schematic shows coding regions from base pairs 1,084,989 to 1,122,316 of the wTpre reference (National Center for Biotechnology Information (NCBI) Reference Sequence: NZ_CM003641.1; Lindsey et al 2016). D) Exon use of transformer and E) double-sex in female T. pretiosum with and without Wolbachia infections. Exons that are differentially skipped between Wolbachia-infected and -uninfected wasps are indicated with gray boxes. Schematics created with BioRender.com. Trichogramma photo taken by A.R.I.L.

Fig. 2.

Candidate parthenogenesis-inducing factors PifA and PifB. A) Domain prediction indicates PifA and PifB contain eukaryotic-like structures. Colored coordinates indicate amino acid positions of the relevant domain in the wTpre ortholog. B and B′) Two views of the predicted structure of the PifA protein from wTpre. B″ view is rotated 90° clockwise around the vertical axis as compared to the view in (B). C) Predicted structure of the PifB protein from wTpre. D) pif expression (relative to a bacterial housekeeping gene) increases across wasp development in T. pretiosum.

Fig. 3.

PifA is conserved and repetitive in the Tra-like region. Dotplots showing amino acid similarity, where each point represents a window of five identical amino acids. A) wTpre PifA versus wLcla PifA, showing high conservation between the orthologs in the ∼320–420 amino acid region. B) wTpre PifA versus self, showing a series of repeated regions in the Tra-like region. C) wLcla PifA versus self, showing a series of repeated regions in the Tra-like region.

Fig. 4.

PifA and PifB impact eukaryotic cell biology. PifA and PifB cause growth defects in yeast relative to empty vector and no induction controls. Yeast strain W303 MATa was transformed with galactose-inducible 2-μm expression vectors that were either empty or contained a Pif coding sequence. Transformants were grown up in selective non-inducing media, normalized to OD600 = 1, and serial 1:10 dilutions were spotted on selective inducing or non-inducing plates and grown at 30 °C for 48 h. Single vector conditions leveraged the pAG425 backbone, and double expression conditions leveraged both pAG424 and pAG425 (see Supplementary material online).

Fig. 5.

PifA associates with dividing nuclei. To localize PifA proteins, yeast W303 MATa were transformed with galactose-inducible pFUS expression vectors (with an N-terminal GFP tag) and grown in selective inducing media (with 2% galactose as a carbon source) for 6 h, and stained with NucBlue. Scale bars represent 10 μm. A) Yeast with empty pFus, expressing GFP alone. B–D) Yeast with pFus-PifA, expressing GFP-PifA.