Characterization of the macronuclear and micronuclear pheromone genes of Euplotes raikovi reveals the origin of the mating type genetic diversity

Ciliates produce diffusible, cell-type-specific pheromones to regulate growth and mating. In Euplotes, these signaling molecules belong to species-specific families of disulfide-rich and structurally homologous proteins. Pheromones are co-dominantly expressed by genes in the somatic macronucleus (MAC), whereas their allelic diversity originates from the mating type locus in the germline micronucleus (MIC). During MAC development in sexual process, the MIC-derived diversity of specific alleles is rearranged via macronucleus-destined sequences (MDSs) assembly. While many MAC pheromones are well characterized, their MIC precursors and rearrangement process remain unknown. Here, we identified two MAC pheromone genes (mac-er-13/14) of E. raikovi, and two MIC regions (19 kb in total) containing 10 MDSs that assemble into mac-er-13. These MDSs are separated by internal eliminated sequences (234-3345 bp). The shortest MDSs (9-36 bp) encode the secreted region of pheromone, while longer MDSs (44-419 bp) encode other regions. Considering that the secreted regions show a higher sequence variation and the shorter MDSs have higher probability of alternative processing or imprecise assembly, we hypothesize that the high sequence variability of the macronuclear pheromone genes, which underlies the large number of mating types in E. raikovi, may result from alternative processing or imprecise assembly of these short MDSs.