A simple RT-multiplex PCR method for diagnosis of L-A and M totiviruses in Saccharomyces cerevisiae

Killer yeasts and their toxins have many potential applications in environmental, medical and industrial biotechnology. The killer phenotype in Saccharomyces cerevisiae relies on the cytoplasmic persistence of two dsRNA viruses, L-A and M. M encodes the toxin, and L-A provides proteins for expression, replication, and capsids for both viruses. Yeast screening and characterization of this trait is usually performed phenotypically, on the basis of their toxin production and immunity. In this study, we describe a simple and specific RT-multiplex PCR assay for direct diagnosis of the dsRNA totivirus genomes associated to the killer trait in the S. cerevisiae yeast. This method obviates RNA purification steps and primers addition to the RT reaction. Using a mixture of specific primers at the PCR step, this RT-multiplex PCR protocol provides accurate diagnosis of both L-A and M totivirus in all its known variants L-A-1/M1, L-A-2/M2, L-A-28/M28 and L-A-lus/Mlus to be found in infected killer yeasts. By means of this method, expected L-A-2/M2 totivirus associations in natural wine yeasts cells were identified, but importantly, asymptomatic L-A-2/M2 infected cells, as well as unexpected L-A-lus/M2 totiviral associations, were also found. Importance The killer phenomenon in S. cerevisiae yeast cells provides the opportunity to study host-virus interactions in a eukaryotic model. Therefore, development of simple methods for their detection significantly facilitates their study. The simplified RT-multiplex PCR protocol described here provides a useful and accurate tool for the genotypic characterization of yeast totiviruses in killer yeast cells. The killer trait depends on two dsRNA totiviruses, L-A and M. Each M dsRNA depends on a specific helper L-A virus. Thus, direct genotyping by the described method also provides valuable insights into L-A/M viral associations and their coadaptional events in nature.