A Rapid Method for Sequencing Double-Stranded RNAs Purified from Yeasts and the Identification of a Potent K1 Killer Toxin Isolated from Saccharomyces cerevisiae

Viruses. 2019 Jan 16;11(1):70. doi: 10.3390/v11010070.

Abstract

Mycoviruses infect a large number of diverse fungal species, but considering their prevalence, relatively few high-quality genome sequences have been determined. Many mycoviruses have linear double-stranded RNA genomes, which makes it technically challenging to ascertain their nucleotide sequence using conventional sequencing methods. Different specialist methodologies have been developed for the extraction of double-stranded RNAs from fungi and the subsequent synthesis of cDNAs for cloning and sequencing. However, these methods are often labor-intensive, time-consuming, and can require several days to produce cDNAs from double-stranded RNAs. Here, we describe a comprehensive method for the rapid extraction and sequencing of dsRNAs derived from yeasts, using short-read next generation sequencing. This method optimizes the extraction of high-quality double-stranded RNAs from yeasts and 3' polyadenylation for the initiation of cDNA synthesis for next-generation sequencing. We have used this method to determine the sequence of two mycoviruses and a double-stranded RNA satellite present within a single strain of the model yeast Saccharomyces cerevisiae. The quality and depth of coverage was sufficient to detect fixed and polymorphic mutations within viral populations extracted from a clonal yeast population. This method was also able to identify two fixed mutations within the alpha-domain of a variant K1 killer toxin encoded on a satellite double-stranded RNA. Relative to the canonical K1 toxin, these newly reported mutations increased the cytotoxicity of the K1 toxin against a specific species of yeast.

Keywords: dsRNA; killer toxin; mycovirus; sequencing; totivirus.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Cloning, Molecular
  • DNA, Complementary
  • High-Throughput Nucleotide Sequencing / methods*
  • Killer Factors, Yeast / genetics*
  • Mutation
  • RNA, Double-Stranded / genetics*
  • RNA, Double-Stranded / isolation & purification
  • RNA, Viral / genetics*
  • RNA, Viral / isolation & purification
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae / virology*

Substances

  • DNA, Complementary
  • K1 killer toxin
  • Killer Factors, Yeast
  • RNA, Double-Stranded
  • RNA, Viral