The NHAl gene of Saccharomyces cerevisiae, transcribed into a 3-5 kb mRNA, encodes a protein mediating Na+ and K+ efflux through the plasma membrane that is required for alkali cation tolerance at acidic pH. Deletion of the gene in a wild-type strain resulted in higher sensitivity to both K+ and Na+ at acidic pH.Measurements of cation loss in strains carrying deleted or overexpressed alleles of NHAl demonstrated its role in K+ and Na+ efflux. In addition, high K+ and Na+ efflux observed upon alkalinization of the cytoplasm implies a role of Nhalp in the regulation of intracellular pH. Moreover, the overexpression of ENA1 and NHAl genes in an enal-46-nhalb strain showed that the Nhal alkalication antiporter is responsible for growth on high concentrations of KCI and NaCl at acidic pH, and Ena alkali-cation ATPases are necessary at higher pH values. Both systems have a complementary action to maintain the intracellular steadystate concentration of K+ and Na+.

BackgroundIn the model organism Saccharomyces cerevisiae, the transposable elements (TEs) consist of LTR (Long Terminal Repeat) retrotransposons called Ty elements belonging to five families, Ty1 to Ty5. They take the form of either full-length coding elements or non-coding solo-LTRs corresponding to remnants of former transposition events. Although the biological features of Ty elements have been studied in detail in S. cerevisiae and the Ty content of the reference strain (S288c) was accurately annotated, the Ty-related intra-specific diversity has not been closely investigated so far.ResultsIn this study, we investigated the Ty contents of 41 available genomes of isolated S. cerevisiae strains of diverse geographical and ecological origins. The strains were compared in terms of the number of Ty copies, the content of the potential transpositionally active elements and the genomic insertion maps. The strain repertoires were also investigated in the closely related Ty1 and Ty2 families and subfamilies.ConclusionsThis is the first genome-wide analysis of the diversity associated to the Ty elements, carried out for a large set of S. cerevisiae strains. The results of the present analyses suggest that the current Ty-related polymorphism has resulted from multiple causes such as differences between strains, between Ty families and over time, in the recent transpositional activity of Ty elements. Some new Ty1 variants were also identified, and we have established that Ty1 variants have different patterns of distribution among strains, which further contributes to the strain diversity.

The RNA3 species of the beet necrotic yellow vein virus (BNYVV), a multipartite positive-stranded RNA phytovirus, contains the ‘core’ nucleotide sequence required for its systemic movement in Beta macrocarpa. Within this ‘core’ sequence resides a conserved “coremin” motif of 20 nucleotides that is absolutely essential for long-distance movement. RNA3 undergoes processing steps to yield a noncoding RNA3 (ncRNA3) possessing “coremin” at its 5′ end, a mandatory element for ncRNA3 accumulation. Expression of wild-type (wt) or mutated RNA3 in Saccharomyces cerevisiae allows for the accumulation of ncRNA3 species. Screening of S. cerevisiae ribonuclease mutants identified the 5′-to-3′ exoribonuclease Xrn1 as a key enzyme in RNA3 processing that was recapitulated both in vitro and in insect cell extracts. Xrn1 stalled on ncRNA3-containing RNA substrates in these decay assays in a similar fashion as the flavivirus Xrn1-resistant structure (sfRNA). Substitution of the BNYVV-RNA3 ‘core’ sequence by the sfRNA sequence led to the accumulation of an ncRNA species in yeast in vitro but not in planta and no viral long distance occurred. Interestingly, XRN4 knockdown reduced BNYVV RNA accumulation suggesting a dual role for the ribonuclease in the viral cycle.

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Transposable elements (TE) are an important source of genetic variation with a dynamic and content that greatly differ in a wide range of species. The origin of the intraspecific content variation is not always clear and little is known about the precise nature of it. Here, we surveyed the species-wide content of the Ty LTR-retrotransposons in a broad collection of 1,011 Saccharomyces cerevisiae natural isolates to understand what can stand behind the variation of the repertoire, i.e. the type and number of Ty elements. We have compiled an exhaustive catalog of all the TE sequence variants present in the S. cerevisiae species by identifying a large set of new sequence variants. The characterization of the TE content in each isolate clearly highlighted that each subpopulation exhibits a unique and specific repertoire, retracing the evolutionary history of the species. Most interestingly, we have shown that ancient interspecific hybridization events had a major impact in the birth of new sequence variants and therefore in the shaping of the TE repertoires. We also investigated the transpositional activity of these elements in a large set of natural isolates, and we found a broad variability related to the level of ploidy as well as the genetic background. Overall, our results pointed out that the evolution of the Ty content is deeply impacted by clade-specific events such as introgressions and therefore follows the population structure. In addition, our study lays the foundation for future investigations to better understand the transpositional regulation and more broadly the TE-host interactions.

Cell-to-cell movement of beet necrotic yellow vein furovirus is controlled by three slightly overlapping genes on RNA 2 called the triple gene block (TGB) encoding, in order, P42, P13, and P15. Synthesis of P42 is directed by subgenomic RNA 2suba while synthesis of both P13 and P15 is probably directed by a dicistronic subgenomic RNA, 2subb. For complementation experiments, each TGB protein gene was inserted into a “replicon” derived from viral RNA 3. In mixed infections, the replicons expressing P42 and P13 complemented RNA 2 mutants defective in the corresponding gene. A P15-containing replicon did not complement a P15-defective RNA 2 but complementation was observed with a dicistronic replicon containing the P15 gene placed behind the P13 gene. In mixed infections with wild-type viral RNAs, the P15-containing replicon did not inhibit viral RNA replication in protoplasts but blocked local lesion formation on leaves. Infection of leaves was also inhibited by an RNA3-derived replicon containing the third TGB gene from another furovirus, peanut clump virus. The results are consistent with a model in which viral cell-to-cell movement requires production of appropriate relative amounts of P13 and P15, and their expression from a dicistronic subgenomic RNA provides a mechanism for coordinating their synthesis.

Cell-to-cell movement of beet necrotic yellow vein virus (BNYVV) requires three proteins encoded by a triple gene block (TGB) on viral RNA 2. A BNYVV RNA 3-derived replicon was used to express movement proteins to functionally substitute for the BNYVV TGB proteins was tested by coinoculation of TGB-defective BNYVV with the various replicons to Chenopodium quinoa. Trans-heterocomplementation was successful with the movement protein (P30) of tobacco mosaic virus but not with the tubule-forming movement proteins of alfalfa mosaic virus and grapevine fanleaf virus. Trans-complementation of BNYVV movement was also observed when all three TGB proteins of the distantly related peanut clump virus were supplied together but not when they were substituted for their BNYVV counterparts one by one. When P30 was used to drive BNYVV movement in trans, accumulation of the first TGB protein of BNYVV was adversely affected by null mutations in the second and third TGB proteins. Taken together, these results suggest that highly specific interactions among cognate TGB proteins are important for their function and/or stability in planta.

Transposable elements (TEs) are widespread in eukaryotes but uncommon in yeasts of the Saccharomycotina subphylum, in terms of both host species and genome fraction. The class II elements are especially scarce, but the hAT element Rover is a noteworthy exception that deserves further investigation.Here, we conducted a genome-wide analysis of hAT elements in 40 ascomycota. A novel family, Roamer, was found in three species, whereas Rover was detected in 15 preduplicated species from Kluyveromyces, Eremothecium, and Lachancea genera, with up to 41 copies per genome. Rover acquisition seems to have occurred by horizontal transfer in a common ancestor of these genera. The detection of remote Rover copies in Naumovozyma dairenensis and in the sole Saccharomyces cerevisiae strain AWRI1631, without synteny, suggests that two additional independent horizontal transfers took place toward these genomes. Such patchy distribution of elements prevents any anticipation of TE presence in incoming sequenced genomes, even closely related ones.The presence of both putative autonomous and defective Rover copies, as well as their diversification into five families, indicate particular dynamics of Rover elements in the Lachancea genus. Especially, we discovered the first miniature inverted-repeat transposable elements (MITEs) to be described in yeasts, together with their parental autonomous copies. Evidence of MITE insertion polymorphism among Lachancea waltii strains suggests their recent activity. Moreover, 40% of Rover copies appeared to be involved in chromosome rearrangements, showing the large structural impact of TEs on yeast genome and opening the door to further investigations to understand their functional and evolutionary consequences.