DOI: 10.5061/dryad.x0k6djhm8
title: Infection by dsRNA viruses is associated with enhanced sporulation efficiency in Saccharoymces cerevisiae

description: Upon starvation, diploid cells of the facultative sexual yeast
 Saccharoymces cerevisiae undergo sporulation, forming four metabolically
 quiescent and robust haploid spores encased in a degradable ascus. All
 endosymbionts, whether they provide net benefits or costs, utilise host
 resources; in yeast, this should induce an earlier onset of sporulation.
 Here, we tested whether the presence of endosymbiotic dsRNA viruses (M
 satellite and L-A helper) correspond with higher sporulation rate of their
 host, S. cerevisiae. We find that S. cerevisiae hosting both the M and L-A
 viruses (so-called “killer yeasts”) have significantly higher sporulation
 efficiency than those without. We also found that the removal of the M
 virus did not reduce sporulation frequency, possibly because the L-A virus
 still utilises host resources with and without the M virus. Our findings
 indicate that either virulent resource use by endosymbionts induces
 sporulation, or that viruses are spread more frequently to sporulating
 strains. Further exploration is required to distinguish cause from effect.
description: <strong>Study
 system</strong> In total, 11 <em>S.
 cerevisiae</em> strains – five non-killers (DBVPG1985; DBVPG 6302,
 DBVPG4410, DBVPG4460, DBVPG6223) and six killers (NCYC 190, Y-2429,
 YJM1307, NCYC1001, Ca7, Ex198) – were used (Rodriguez-Cousino et al. 2013,
 Peter et al. 2018, Fredericks et al. 2021). Of the killer strains, three
 belonged to the K1 killer-type, two to K2 and one to Klus. For each killer
 strain sample, a corresponding M virus-free strain was created from
 treatment with elevated temperature (Wickner 1974), anisomycin (Fredericks
 et al. 2021) or cycloheximide (Fink and Styles 1972) to destroy genetic
 material of the M virus. This procedure is commonly referred to as
 “curing” the strains. Killer strains and their cured equivalent were
 donated and cured with the aforementioned methods by the groups of Paul
 Rowley and Nieves Rodríguez-Cousiño (Fredericks et al. 2021;
 Rodríguez-Cousiño et al. 2013). For a control, all non-killer strains were
 additionally independently exposed to elevated temperature (a widely
 utilised curing method) prior to the experiment (Wickner 1974), as this
 would allow us to establish whether curing of the killer factor per se or
 simply a curing process can influence sporulation efficiency. This also
 leads to a full factorial design of cured non-killers, non-killers,
 cured-killers and killers. Background information on each strain can be
 found in the respective collections of each strain. <strong>Sporulation</strong> Three independent replicates per <em>S. cerevisiae </em>strain were cultivated on standard YPD media (1% Yeast Extract, 2% Peptone, 10% Glucose) at 25°C overnight, and subsequently the optical densities (OD) of the growing yeast cells were measured (SpectraMax 19, software: SoftMax Pro 6.2.2). When the OD was between 0.6-0.8, 50 ml of yeast were washed according to the protocol of Knight and Goddard (2016), with the following modifications: washed twice with 3 ml of sterile water. 0.5 ml of each yeast strain was plated on sporulation plates (40 mm plates, sporulation media: 1% Potassium Acetate, 0.1% Yeast Extract, 0.05% Glucose, 2% Agar, 25 mg/L Zinc Sulphate). Yeast plates were inoculated for 7 days at 25° C. Finally, we evaluated 100 cells for each strain replicate under the microscope and classified their sporulation status to calculate the sporulation efficiency, defined as the proportion of cells in a culture that have sporulated (Knight and Goddard 2016). <strong>References</strong> Fink, G., and C. A. Styles. 1972. Curing of a killer factor in <em>Saccharomyces cerevisiae</em>. Proceedings of the National Academy of Sciences <strong>69</strong>:2846-2849. Fredericks, L. R., M. D. Lee, A. M. Crabtree, J. M. Boyer, E. A. Kizer, N. T. Taggart, C. R. Roslund, S. S. Hunter, C. B. Kennedy, C. G. Willmore, N. M. Tebbe, J. S. Harris, S. N. Brocke, and P. A. Rowley. 2021. The species-specific acquisition and diversification of a K1-like family of killer toxins in budding yeasts of the Saccharomycotina. PLoS Genet 17:e1009341. Knight, S. J., and M. R. Goddard. 2016. Sporulation in soil as an overwinter survival strategy in <em>Saccharomyces cerevisiae</em>. FEMS Yeast Res <strong>16</strong>:fov102. Peter, J., M. De Chiara, A. Friedrich, J.-X. Yue, D. Pflieger, A. Bergström, A. Sigwalt, B. Barre, K. Freel, A. Llored, C. Cruaud, K. Labadie, J.-M. Aury, B. Istace, K. Lebrigand, P. Barbry, S. Engelen, A. Lemainque, P. Wincker, G. Liti, and J. Schacherer. 2018. Genome evolution across 1,011 <em>Saccharomyces cerevisiae</em> isolates. Nature <strong>556</strong>:339-344. Rodriguez-Cousino, N., P. Gomez, and R. Esteban. 2013. L-A-lus, a new variant of the L-A totivirus found in wine yeasts with Klus killer toxin-encoding Mlus double-stranded RNA: possible role of killer toxin-encoding satellite RNAs in the evolution of their helper viruses. Appl Environ Microbiol <strong>79</strong>:4661-4674. Wickner, R. B. 1974. “Killer character” of <em>Saccharomyces cerevisiae</em>: curing by growth at elevated temperature. Journal of Bacteriology <strong>117</strong>:1356-1357.