Zymocin-like killer plasmids were present in the common ancestor of terrestrial fungi

Some budding yeasts secrete killer toxins made by linear dsDNA plasmids located in the cytosol. The best-known example is the Kluyveromyces lactis toxin zymocin, which is encoded by a 9-kb killer plasmid assisted by a 13-kb helper plasmid. These plasmids are distantly related to eukaryotic dsDNA viruses and have been called Virus-Like Elements (VLEs) but they do not produce virus particles. Their evolutionary origin is unclear because VLEs have been found only in budding yeasts (subphylum Saccharomycotina of phylum Ascomycota) and not in any other fungi. Here, we show that similar VLEs are present in two deeply divergent phyla of terrestrial fungi, Zoopagomycota and Mucoromycota. In Zoopagomycota, some isolates of Coemansia harbor more than 20 different linear dsDNA plasmids simultaneously, many of which encode their own DNA polymerases (DNAPs). We did not identify any functional killer toxins encoded by the new VLEs, but phylogenetic analysis of the chitinase genes present on some of them suggests that they are orthologs of the chitinase subunits of killer toxins encoded by Saccharomycotina VLEs. Phylogenetic analysis of DNAPs shows that the diversity of VLEs in Zoopagomycota greatly exceeds that in Saccharomycotina, and that Saccharomycotina killer and helper plasmids are related to two lineages of VLEs present in Zoopagomycota. Our results indicate that VLEs were present in the common ancestor of all terrestrial fungi about 650 Mya, and that they were already subdivided into killer and helper types by this time.