The prevalence of killer yeasts and double-stranded RNAs in the budding yeast Saccharomyces cerevisiae.

Killer toxins are antifungal proteins produced by many species of "killer" yeasts, including the brewer's and baker's yeast Saccharomyces cerevisiae. Screening 1270 strains of S. cerevisiae for killer toxin production found that 50% are killer yeasts, with a higher prevalence of yeasts isolated from human clinical samples and winemaking processes. Since many killer toxins are encoded by satellite double-stranded RNAs (dsRNAs) associated with mycoviruses, S. cerevisiae strains were also assayed for the presence of dsRNAs. This screen identified that 51% of strains contained dsRNAs from the mycovirus families Totiviridae and Partitiviridae, as well as satellite dsRNAs. Killer toxin production was correlated with the presence of satellite dsRNAs but not mycoviruses. However, in most killer yeasts, whole genome analysis identified the killer toxin gene KHS1 as significantly associated with killer toxin production. Most killer yeasts had unique spectrums of antifungal activities compared to canonical killer toxins, and sequence analysis identified mutations that altered their antifungal activities. The prevalence of mycoviruses and killer toxins in S. cerevisiae is important because of their known impact on yeast fitness, with implications for academic research and industrial application of this yeast species.

Novel viruses of the family Partitiviridae discovered in Saccharomyces cerevisiae.

It has been 49 years since the last discovery of a new virus family in the model yeast Saccharomyces cerevisiae. A large-scale screen to determine the diversity of double-stranded RNA (dsRNA) viruses in S. cerevisiae has identified multiple novel viruses from the family Partitiviridae that have been previously shown to infect plants, fungi, protozoans, and insects. Most S. cerevisiae partitiviruses (ScPVs) are associated with strains of yeasts isolated from coffee and cacao beans. The presence of partitiviruses was confirmed by sequencing the viral dsRNAs and purifying and visualizing isometric, non-enveloped viral particles. ScPVs have a typical bipartite genome encoding an RNA-dependent RNA polymerase (RdRP) and a coat protein (CP). Phylogenetic analysis of ScPVs identified three species of ScPV, which are most closely related to viruses of the genus Cryspovirus from the mammalian pathogenic protozoan Cryptosporidium parvum. Molecular modeling of the ScPV RdRP revealed a conserved tertiary structure and catalytic site organization when compared to the RdRPs of the Picornaviridae. The ScPV CP is the smallest so far identified in the Partitiviridae and has structural homology with the CP of other partitiviruses but likely lacks a protrusion domain that is a conspicuous feature of other partitivirus particles. ScPVs were stably maintained during laboratory growth and were successfully transferred to haploid progeny after sporulation, which provides future opportunities to study partitivirus-host interactions using the powerful genetic tools available for the model organism S. cerevisiae.

The Characterization of a Novel Virus Discovered in the Yeast Pichia membranifaciens.

Mycoviruses are widely distributed across fungi, including the yeasts of the Saccharomycotina subphylum. This manuscript reports the first double-stranded RNA (dsRNA) virus isolated from Pichia membranifaciens. This novel virus has been named Pichia membranifaciens virus L-A (PmV-L-A) and is a member of the Totiviridae. PmV-L-A is 4579 bp in length, with RNA secondary structures similar to the packaging, replication, and frameshift signals of totiviruses that infect Saccharomycotina yeasts. PmV-L-A was found to be part of a monophyletic group within the I-A totiviruses, implying a shared ancestry between mycoviruses isolated from the Pichiaceae and Saccharomycetaceae yeasts. Energy-minimized AlphaFold2 molecular models of the PmV-L-A Gag protein revealed structural conservation with the Gag protein of Saccharomyces cerevisiae virus L-A (ScV-L-A). The predicted tertiary structure of the PmV-L-A Pol and other homologs provided a possible mechanism for totivirus RNA replication due to structural similarities with the RNA-dependent RNA polymerases of mammalian dsRNA viruses. Insights into the structure, function, and evolution of totiviruses gained from yeasts are essential because of their emerging role in animal disease and their parallels with mammalian viruses.

The Species-Specific Acquisition and Diversification of a K1-like Family of Killer Toxins in Budding Yeasts of the Saccharomycotina.

Killer toxins are extracellular antifungal proteins that are produced by a wide variety of fungi, including Saccharomyces yeasts. Although many Saccharomyces killer toxins have been previously identified, their evolutionary origins remain uncertain given that many of these genes have been mobilized by double-stranded RNA (dsRNA) viruses. A survey of yeasts from the Saccharomyces genus has identified a novel killer toxin with a unique spectrum of activity produced by Saccharomyces paradoxus. The expression of this killer toxin is associated with the presence of a dsRNA totivirus and a satellite dsRNA. Genetic sequencing of the satellite dsRNA confirmed that it encodes a killer toxin with homology to the canonical ionophoric K1 toxin from Saccharomyces cerevisiae and has been named K1-like (K1L). Genomic homologs of K1L were identified in six non-Saccharomyces yeast species of the Saccharomycotina subphylum, predominantly in subtelomeric regions of the genome. When ectopically expressed in S. cerevisiae from cloned cDNAs, both K1L and its homologs can inhibit the growth of competing yeast species, confirming the discovery of a family of biologically active K1-like killer toxins. The sporadic distribution of these genes supports their acquisition by horizontal gene transfer followed by diversification. The phylogenetic relationship between K1L and its genomic homologs suggests a common ancestry and gene flow via dsRNAs and DNAs across taxonomic divisions. This appears to enable the acquisition of a diverse arsenal of killer toxins by different yeast species for potential use in niche competition.

The design and implementation of restraint devices for the injection of pathogenic microorganisms into Galleria mellonella.

The injection of laboratory animals with pathogenic microorganisms poses a significant safety risk because of the potential for injury by accidental needlestick. This is especially true for researchers using invertebrate models of disease due to the required precision and accuracy of the injection. The restraint of the greater wax moth larvae (Galleria mellonella) is often achieved by grasping a larva firmly between finger and thumb. Needle resistant gloves or forceps can be used to reduce the risk of a needlestick but can result in animal injury, a loss of throughput, and inconsistencies in experimental data. Restraint devices are commonly used for the manipulation of small mammals, and in this manuscript, we describe the construction of two devices that can be used to entrap and restrain G. mellonella larvae prior to injection with pathogenic microbes. These devices reduce the manual handling of larvae and provide an engineering control to protect against accidental needlestick injury while maintaining a high rate of injection.

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

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.