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. 2017 Oct 11;9(10):313.
doi: 10.3390/toxins9100313.

Variation and Distribution of L-A Helper Totiviruses in Saccharomyces sensu stricto Yeasts Producing Different Killer Toxins

Nieves Rodríguez-Cousiño  1   2 , Pilar Gómez  3 , Rosa Esteban  4
Affiliations

Variation and Distribution of L-A Helper Totiviruses in Saccharomyces sensu stricto Yeasts Producing Different Killer Toxins

Nieves Rodríguez-Cousiño et al. Toxins (Basel). .

Abstract

Yeasts within the Saccharomyces sensu stricto cluster can produce different killer toxins. Each toxin is encoded by a medium size (1.5-2.4 Kb) M dsRNA virus, maintained by a larger helper virus generally called L-A (4.6 Kb). Different types of L-A are found associated to specific Ms: L-A in K1 strains and L-A-2 in K2 strains. Here, we extend the analysis of L-A helper viruses to yeasts other than S. cerevisiae, namely S. paradoxus, S. uvarum and S. kudriavzevii. Our sequencing data from nine new L-A variants confirm the specific association of each toxin-producing M and its helper virus, suggesting co-evolution. Their nucleotide sequences vary from 10% to 30% and the variation seems to depend on the geographical location of the hosts, suggesting cross-species transmission between species in the same habitat. Finally, we transferred by genetic methods different killer viruses from S. paradoxus into S. cerevisiae or viruses from S. cerevisiae into S. uvarum or S. kudriavzevii. In the foster hosts, we observed no impairment for their stable transmission and maintenance, indicating that the requirements for virus amplification in these species are essentially the same. We also characterized new killer toxins from S. paradoxus and constructed "superkiller" strains expressing them.

Keywords: L-A totivirus; Saccharomyces sensu stricto; double-stranded RNA virus; yeast killer toxins.

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Conflict of interest statement

The authors declare no conflict of interest. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.

Figures

Figure 1
Figure 1
(A) Presence (or absence) of M1 or M28 virus in Killer strains of S. paradoxus. RNAs from a number of killer toxin-producing S. paradoxus (Sp) strains (lanes 2 to 9) and the S. cerevisiae (Sc) laboratory strain SK1 (lane 1) were separated on an agarose gel and transferred to a nylon membrane for Northern hybridization. The upper panel shows the ethidium bromide-stained gel (EtBr) with indications to the mobility of main nucleic acids. Below panels are autoradiograms of the Northern blot hybridized with a mixture of L-A- and M1-specific probes (middle) or L-A-28- and M28-specific probes (lower). (B) Killer activity of strains Sp T21.4 (left panels) and Sc SK1 (right panels) over lawns of a sensitive K-o strain (1) or four S. cerevisiae strains producing the indicated killer toxins: K1 (2), K21 (3), K28 (4) or K74 (5). Clear halos of growth inhibition in the lawn surrounding the central spots of cells indicate toxin activity. (C) Killer activity of the three S. paradoxus strains carrying M28 dsRNA variants analyzed in lanes 2, 3 and 4 of panel (A). The lawn is the same K-o strain as in (B).
Figure 2
Figure 2
(A) Schematic diagram of L-A helper virus (+) strand genomic organization with two cis signals involved in a -1 translational frame shift and encapsidation (conserved in all the L-A helper viruses of S. sensu stricto species). The two ORFs for Gag and Pol are shown below. Oligos used for RT-PCR amplification and sequencing (see Methods) are indicated. (B) Dendrogram of the Gag-Pol amino acid sequences of 14 L-A variants analyzed in this work. The names and GenBank accession numbers are described in Methods. Each Saccharomyces species is indicated by distinct colored dots: S. paradoxus (Sp) red, S. cerevisiae (Sc) green, S. kudriavzevii (Sk), black and S. uvarum (Su), blue. In brackets their geographical location are indicated: European (E), or Far East (FE). Two subgroups of S. paradoxus European strains are indicated as E-1 or E-2 on the right. We used as outgroup the tuber aestivum (Tav-1) virus (GenBank HQ158596.1). The evolutionary history was inferred using the Neighbor-Joining method [45]. The optimal tree with the sum of branch length = 1.92844968 is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches [46]. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method [47] and are in the units of the number of amino acid substitutions per site. The analysis involved 15 amino acid sequences. All positions containing gaps and missing data were eliminated. There were a total of 1504 positions in the final dataset. Evolutionary analyses were conducted in MEGA6 [48].
Figure 3
Figure 3
(A) Diagram of the experimental approach followed to introduce killer viruses from sporulating cells of a S. paradoxus killer strain into the haploid S. cerevisiae 2405 K-o strain carrying the kar1 mutation and expressing the geneticin resistance marker from a vector (open circles in the nucleus). After growing both strains together in a rich medium agar-plate for 24 h, cells were streaked for single colony isolation on a plate with geneticin. Three types of cells can grow: (1) the original recipient K-o cells; (2) cytoductants that carry the nucleus of 2405 but they are killers (K+); and (3) hybrid diploids produced by mating S. paradoxus and S. cerevisiae, which are also killers (K+). (B) The panel shows an example of the three types of clones after replica plating on an MB plate seeded with the sensitive strain 5×47. Halos of growth inhibition surrounding certain colonies indicate successful transmission of viruses. The black arrowhead indicates a hybrid clone and the white arrowhead indicates a cytoductant (S. cerevisiae cells carrying viruses from S. paradoxus).
Figure 4
Figure 4
(A) Four S. paradoxus strains (upper panel) and their diploid S. cerevisiae derivatives (lower panel) were tested for killer activity on a lawn of the K-o strain 5×47. Similar amounts of each killer strain (ca. 5 × 105 cells) were spotted. The size and aspect of the halos vary among the strains. (B) Two independent colonies from each strain (with the exception of S28 with only one) were grown for 2 days in rich media and RNA was extracted. After treatment with RNase A in the presence of 0.5 M NaCl the remaining nucleic acids (dsRNAs and DNA) were separated on an agarose gel and stained with ethidium bromide. The sizes of M dsRNAs vary among the strains. (C) M dsRNAs producing different killer toxins in S. paradoxus are maintained in S. cerevisiae by L-A coat proteins (different from their original helpers) expressed from a vector. The L-A coats exclude the helper viruses present in the donor strains. The minor dsRNA band with a size of 4.6 Kb is L-BC dsRNA, the genome of another totivirus in S. cerevisiae unrelated with the killer phenotype. Note that the amounts of each M dsRNA in these strains are at least 20-fold higher than those in S. paradoxus (B). After being transferred to a nylon membrane, dsRNAs were hybridized with four specific probes that recognized M28, M21, M45 or M74, respectively. The autoradiograms are shown below the ethidium bromide-stained gel. (D) All the M dsRNAs carry in their 3′ non-coding regions putative encapsidation signals similar to that of L-A. A prediction of their secondary structures by MFOLD is depicted. In yellow color, the two features in the signals necessary and sufficient for encapsidation, the protruding A and the GC pair close to the loop are shown. Below appears a diagram of M (+) strands showing the polyA internal region (wavy line) and the location of the putative encapsidation signals.
Figure 5
Figure 5
(A) RFLP mitochondrial analysis of S. paradoxus and their S. cerevisiae derivative killer strains. Viruses from four S. paradoxus (Sp) strains: S28 (lane 1), T21.4 (lane 2), Q74.4 (lane 6), or N-45 (lane 7) were introduced by cytoduction into S. cerevisiae (Sc) strain 2405 (lanes 5 and 10). Cytoductants are indicated by two names separated by an arrow (the first name is the donor and the second the recipient). DNA digested with HinfI was separated in agarose gels and stained with Ethidium bromide. λ, lambda DNA digested with HindIII used as mobility markers. (B) A similar analysis was done with S. kudriavzevii (Sk) strain 1802 (lane 12) and a cytoductant that received K1 viruses from Sc strain 2403 (lane 14). In the gel, we also show the mitochondrial pattern of one hybrid diploid strain between Sk and Sc parental strains (lane 13). (C) L-A-lus or L-A-2 viruses were cytoduced from Sc strains 1333 or 1337, respectively, into S. uvarum (Su) haploid strain 1241.
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