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.

Vaginal Isolates of Candida glabrata are Uniquely Susceptible to Ionophoric Killer Toxins Produced by Saccharomyces cerevisiae.

Compared to other species of Candida yeasts, the growth of Candida glabrata was inhibited by many different strains of Saccharomyces killer yeasts. The ionophoric K1 and K2 killer toxins were broadly inhibitory to all clinical isolates of C. glabrata from patients with recurrent vulvovaginal candidiasis, despite high levels of resistance to clinically relevant antifungal therapeutics.

Scent of a killer: How killer yeast boost its dispersal

Vector-borne parasites often manipulate hosts to attract uninfected vectors. For example, parasites causing malaria alter host odor to attract mosquitoes. Here we discuss the ecology and evolution of fruit-colonizing yeast in a tripartite symbiosis – the so-called “killer yeast” system. “Killer yeast” consists of Saccharomyces cerevisiae yeast hosting two double stranded RNA viruses (M satellite dsRNAs, L-A dsRNA helper virus). When both dsRNA viruses occur in a yeast cell, the yeast converts to lethal toxin‑producing “killer yeast” phenotype that kills uninfected yeasts. Yeasts on ephemeral fruits attract insect vectors to colonize new habitats. As the viruses have no extracellular stage, they depend on the same insect vectors as yeast for their dispersal. Viruses also benefit from yeast dispersal as this promotes yeast to reproduce sexually, which is how viruses can transmit to uninfected yeast strains. We tested whether insect vectors are more attracted to killer yeasts than to non‑killer yeasts. In our field experiment, we found that killer yeasts were more attractive to Drosophila than non-killer yeasts. This suggests that vectors foraging on yeast are more likely to transmit yeast with a killer phenotype, allowing the viruses to colonize those uninfected yeast strains that engage in sexual reproduction with the killer yeast. Beyond insights into the basic ecology of the killer yeast system, our results suggest that viruses could increase transmission success by manipulating the insect vectors of their host.

Killer Yeasts for the Biological Control of Postharvest Fungal Crop Diseases

Every year and all over the world the fungal decay of fresh fruit and vegetables frequently generates substantial economic losses. Synthetic fungicides, traditionally used to efficiently combat the putrefactive agents, emerged, however, as the cause of environmental and human health issues. Given the need to seek for alternatives, several biological approaches were followed, among which those with killer yeasts stand out. Here, after the elaboration of the complex of problems, we explain the hitherto known yeast killer mechanisms and present the implementation of yeasts displaying such phenotype in biocontrol strategies for pre- or postharvest treatments to be aimed at combating postharvest fungal decay in numerous agricultural products.

A Novel Report on Killer Yeast Strains Identification Methods

This study was conducted to isolate and identify killer yeasts from soil samples that collected from different locations in Basrah and Dhi-Qar provinces. Seventy-five soil samples were collected from different areas, including sandy, arable, surface sediment and uncultivated soil, using dilution methods to cultivate a serial dilution of each soil sample. The results showed that a 112 isolates were identified biochemically using VITEK system and molecularly using internal transcribed spacer (ITS1- 5.8S-ITS2) marker. The molecular identification provided fast and precise identification results for the 112 isolates, whereas the VITEK test resulted low identification efficiency (8.2% were accurate and 91.8 % were not). The Diazonium blue B salts produced a good colour reaction in distinguishing between ascomycetes and basidiomycetes. The PCR was more accurate in identification of killer yeasts compared to the VITEK system.

Dynamic modelling of the killing mechanism of action by virus-infected yeasts

Killer yeasts are microorganisms, which can produce and secrete proteinaceous toxins, a characteristic gained via infection by a virus. These toxins are able to kill sensitive cells of the same or a related species. From a biotechnological perspective, killer yeasts are beneficial due to their antifungal/antimicrobial activity, but also regarded as problematic for large-scale fermentation processes, whereby those yeasts would kill starter cultures species and lead to stuck fermentations. Here, we propose a mechanistic model of the toxin-binding kinetics pertaining to the killer population coupled with the toxin-induced death kinetics of the sensitive population to study toxic action. The dynamic model captured the transient toxic activity starting from the introduction of killer cells into the culture at the time of inoculation through to induced cell death. The kinetics of K1/K2 activity via its primary pathway of toxicity was 5.5 times faster than its activity at low concentration inducing the apoptotic pathway in sensitive cells. Conversely, we showed that the primary pathway for K28 was approximately three times slower than its equivalent apoptotic pathway, indicating the particular relevance of K28 in biotechnological applications where the toxin concentration is rarely above those limits to trigger the primary pathway of killer activity.