Recombination, truncation and horizontal transfer shape the diversity of cytoplasmic incompatibility patterns

Abstract

Wolbachia are endosymbiotic bacteria inducing various reproductive manipulations of which cytoplasmic incompatibility (CI) is the most common. CI leads to reduced embryo viability in crosses between males carrying Wolbachia and uninfected females or those carrying an incompatible symbiont strain. In the mosquito Culex pipiens, the Wolbachia wPip causes highly complex crossing patterns. This complexity is linked to the amplification and diversification of the CI causal genes, cidA and cidB, with polymorphism located in the CidA-CidB interaction regions. We previously showed correlations between the identity of gene variants and CI patterns. However, these correlations were limited to specific crosses, and it is still unknown whether cid gene polymorphism in males' and females' Wolbachia can explain and predict the wide range of crossing types observed in C. pipiens. Taking advantage of a new method enabling full-gene acquisition, we sequenced complete cid repertoires from 45 wPip strains collected worldwide. We demonstrated that the extensive diversity of cid genes arises from recombination and horizontal transfers. We uncovered further cidB polymorphism outside the interface regions and strongly correlated with CI patterns. Most importantly, we showed that in every wPip genome, all but one cidB variant are truncated. Truncated cidBs located in palindromes are partially or completely deprived of their deubiquitinase domain, crucial for CI. The identity of the sole full-length cidB variant seems to dictate CI patterns, irrespective of the truncated cidBs present. Truncated CidBs exhibit reduced toxicity and stability in Drosophila cells, which potentially hinders their loading into sperm, essential for CI induction.

Figure 1.. Recombinant cidA variants with common architecture.

Monomorphic gene regions are shown in black. Polymorphic regions are composed of several recombination blocks, named A to 0. These blocks are always in the same order, with each having two distinct alternative versions, shown here in blue and red. A variant is the assembly of different versions of each block. The lowest panel shows the repartition of block versions among wPip groups. If the two versions of a block are found in a given group, the block contains triangles of both colors, whereas if a single block version is found, the cell shows a single color. Colors are arbitrarily determined, and versions of a given color from different blocks are not more likely to be found together.

Figure 2.. Palindromes observed in the genome long reads of the strains Tunis (wPip-I) and Harash (wPip-IV).

The complete cidB described in the wPip-Pel genome is given here for comparative purposes. Two distinct palindromes P1 and P2 were found with a cidA-cidB-cidB-cidA structure, where the first two genes are sense-orientated and the following two are in opposite directions. For both palindromes, the two cidB are truncated. They share a common cidB, cidB-TrA, truncated at 2,466 base pairs (bp). cidB-TrB (found in P1) and cidB-TrC (found in P2) are chimeric open reading frames (ORFs) that contain part of the “classic” cidB (2,381 bp and 2,958 bp for cidB-TrB and cidB-TrC, respectively), an insert (551 bp and 25 bp, respectively) and part of the cidB-TrA (83 bp for both). Overall, the cidB-TrB ORF is 3,015 bp, while that of cidB-TrC is 3,066 bp.

Figure 3.. Evolutionary scenario for the evolution of palindromes in wPip.

A three-step evolutionary scenario could explain the current genomic architecture of the cid genes for the wPip and wNaev genomes: all wPip repertoires acquired so far include at least a complete cidB along with a palindromic structure containing two truncated cidB. All sequenced genomes also have two further cidB modules, the WP_1291 and WP_1292 open reading frames (ORFs). In wNaev genomes, 10 cidB were recovered (Fig S2), one of which belongs to pair 4 and is represented here in light green. wPip cid architecture could result from an ancestral genome with two complete cidB copies, which underwent a horizontal transfer of a cidB from wNaev or a genome with a similar cidB, followed by a recombination into a palindromic structure.

Figure 4.. Truncated CidB variants less toxic and less stable than the full-length CidB toxin.

(A) Mitosis frequency over mitosis and apoptosis observed for each construct after the transfection of S2R+ cells over 48h of time-lapse microscopy. Each transfection experiment was performed twice independently, and the number of transfected cells observed is >1000. (B) Flow cytometry analyses of S2R+ cell growth from day 2 to day 4 after transfection in three independent transfection experiments. The blue and pink dots represent log2(sfGTP) and log2(mKate2) for each construct, while the associated measures are linked for each biological replicate.

Figure 5.. Only long cidB plays a role in CI.

Wolbachia is eliminated over the course of sperm maturation. One hypothetical explanation for the role played by the sole long CidB in CI is that truncated CidB, which are unstable, disappear as sperm matures. In mature sperm, the only CidB remaining is thus the long CidB.