Volatile metabolites produced by different flor yeast strains during wine biological ageing.

Sherry white wine called Fino is produced by dynamic biological ageing under the action of flor yeasts using traditional practices aimed at ensuring uniform quality and characteristics over time. These kinds of yeasts provide typical sensory properties to Fino wines. Although there are studies of the volatile composition of these wines submitted to biological ageing in wood barrels, there is a lack of knowledge on the particular volatile profile produced by different flor yeast strains from Sherry zone wineries. For this reason, the aim of this study was to analyse the volatile profiles produced by 15 pure culture flor velum yeasts, with the goal of observing their suitability for obtaining high quality Fino sherry wines. Volatile composition was determined by dual sequential stir bar sorptive extraction, followed by GC-MS analysis. All yeast strains studied produced the increase of most acetals, highlighting acetaldehyde diethylacetal which was the compound that most increased. Among terpenes, nerolidol and farnesol underwent remarkable increases. However, results showed that in a month of biological ageing, significant differences were observed among the volatile metabolites produced by flor yeast strains studied. Only some of them stood out for their high production of volatile compounds characteristic of Sherry Fino wines, which are good candidates for producing starter cultures.

A plant defense response effector induces microbial apoptosis.

Osmotin is a tobacco PR-5 protein that has antifungal activity and is implicated in host-plant defense. We show here that osmotin induces apoptosis in Saccharomyces cerevisiae. Induction of apoptosis was correlated with intracellular accumulation of reactive oxygen species and was mediated by RAS2, but not RAS1. Osmotin treatment resulted in suppression of transcription of stress-responsive genes via the RAS2/cAMP pathway. It was therefore concluded that osmotin induced proapoptotic signaling in yeast. The results indicate that the ability of antimicrobial proteins to induce microbial apoptosis could be an important factor in determining a pathogen's virulence and could therefore be targeted for the design of new antifungal drugs.

Resistance to the plant PR-5 protein osmotin in the model fungus Saccharomyces cerevisiae is mediated by the regulatory effects of SSD1 on cell wall composition.

The capacity of plants to counter the challenge of pathogenic fungal attack depends in part on the ability of plant defense proteins to overcome fungal resistance by being able to recognize and eradicate the invading fungi. Fungal genes that control resistance to plant defense proteins are therefore important determinants that define the range of fungi from which an induced defense protein can protect the plant. Resistance of the model fungus Saccharomyces cerevisiae to osmotin, a plant defense PR-5 protein, is strongly dependent on the natural polymorphism of the SSD1 gene. Expression of the SSD1-v allele afforded resistance to the antifungal protein. Conversely, yeast strains carrying the SSD1-d allele or a null ssd1Delta mutation displayed high sensitivity to osmotin. The SSD1-v protein mediates osmotin resistance in a cell wall-dependent manner. Deletion of SSD1-v or SSD1-d impeded sorting of the PIR proteins (osmotin-resistance factors) to the cell wall without affecting mRNA levels, indicating that SSD1 functions in post-transcriptional regulation of gene expression. The sensitivity of ssd1Delta cells to osmotin was only partially suppressed by over-accumulation of PIR proteins in the cell wall, suggesting an additional function for SSD1 in cell wall-mediated resistance. Accordingly, cells carrying a null ssd1 mutation also displayed aberrant cell-wall morphology and lower levels of alkali-insoluble cell-wall glucans. Therefore SSD1 is an important regulator of fungal cell-wall biogenesis and composition, including the deposition of PIR proteins which block the action of plant antifungal PR-5 proteins.

Fungal cell wall phosphomannans facilitate the toxic activity of a plant PR-5 protein.

Osmotin is a plant PR-5 protein. It has a broad spectrum of antifungal activity, yet also exhibits specificity for certain fungal targets. The structural bases for this specificity remain unknown. We show here that full sensitivity of Saccharomyces cerevisiae cells to the PR-5 protein osmotin is dependent on the function of MNN2, MNN4 and MNN6. MNN2 is an alpha-1, 2-mannosyltransferase catalyzing the addition of the first mannose to the branches on the poly l,6-mannose backbone of the outer chain of cell wall N-linked mannans. MNN4 and MNN6 are required for the transfer of mannosylphosphate to cell wall mannans. Null mnn2, mnn4 or mnn6 mutants lack phosphomannans and are defective in binding osmotin to the fungal cell wall. Both antimannoprotein antibody and the cationic dye alcian blue protect cells against osmotin cytotoxicity. MNN1 is an alpha-1,3-mannosyltransferase that adds the terminal mannose to the outer chain branches of N-linked mannan, masking mannosylphosphate. Null mnn1 cells exhibit enhanced osmotin binding and sensitivity. Several cell wall mannoproteins can bind to immobilized osmotin, suggesting that their polysaccharide constituent determines osmotin binding. Our results demonstrating a causal relationship between cell surface phosphomannan and the susceptibility of a yeast strain to osmotin suggest that cell surface polysaccharides of invading pathogens control target specificity of plant PR-5 proteins.

Osmotin, a plant antifungal protein, subverts signal transduction to enhance fungal cell susceptibility.

The plant pathogenesis-related protein osmotin is an antifungal cytotoxic agent that causes rapid cell death in the yeast S. cerevisiae. We show here that osmotin uses a signal transduction pathway to weaken defensive cell wall barriers and increase its cytotoxic efficacy. The pathway activated by osmotin includes the regulatory elements of the mating pheromone response STE4, STE18, STE20, STE5, STE11, STE7, FUS3, KSS1, and STE12. Neither the pheromone receptor nor its associated G protein alpha subunit GPA1 are required for osmotin action. However, mutation of SST2, a negative regulator of G alpha proteins, resulted in supersensitivity to osmotin. Phosphorylation of STE7 was rapidly stimulated by osmotin preceding any changes in cell vitality or morphology. These results demonstrate that osmotin subverts target cell signal transduction as part of its mechanism of action.

Mitochondrial DNA loss caused by ethanol in Saccharomyces flor yeasts.

Saccharomyces flor yeasts proliferate at the surface of sherry wine, which contains over 15% (vol) ethanol. Since ethanol is a powerful inducer of respiration-deficient mutants, this alcohol has been proposed to be the source of the high diversity found in the mitochondrial genomes of flor yeasts and other wine yeasts. Southern blot analysis suggests that mitochondrial DNA (mtDNA) polymorphic changes are due to minor lesions in the mitochondrial genome. As determined in this work by pulsed-field gel electrophoresis, restriction analysis, and Southern blot analysis, ethanol-induced petite mutants completely lack mtDNA (rho zero). Ethanol-induced changes in the mitochondrial genome that could explain the observed mtDNA polymorphism in flor yeasts were not found. The transfer of two different mtDNA variants from flor yeasts to a laboratory strain conferred in both cases an increase in ethanol tolerance in the recipient strain, suggesting that mtDNAs are probably subjected to positive selection pressure concerning their ability to confer ethanol tolerance.

Genomic complexity and chromosomal rearrangements in wine-laboratory yeast hybrids.

In this report we describe the genomic complexity of a number of Saccharomyces yeast strains isolated from sherry wine (flor yeasts), and the genomic stability of a yeast hybrid derived from one of these and a laboratory strain. Flor yeast strains largely differed in their DNA content, but showed very few variations their molecular karyotype. These strains contained a large number of Ty2 sequences, but lacking the Ty1 elements commonly found in laboratory strains. The genetic analysis of a flor-laboratory hybrid indicated that flor yeasts were aneuploid. Hybridization patterns obtained with Ty1 and Ty2 probes in the meiotic progeny of this hybrid suggested that recombination may occur not only among homologous chromosomes of similar length, but also among polymorphic partners with different sizes. New chromosomal variants were frequently observed in the meiotic products, suggesting that polymorphism in chromosome length may itself be a major source of karyotypic variation. The genetic analysis of such variants indicated that recombinational events leading to new chromosomal forms may occur both mitotically and meiotically.

Detection of Dekkera-Brettanomyces strains in sherry by a nested PCR method.

Brettanomyces sp. and its ascosporogenous sexual state, Dekkera sp., have been well documented as spoilage microorganisms, usually associated with barrel-aged red wines. In this report, we describe the genetic characterization, on the basis of DNA content per cell, electrophoretic karyotyping, and mitochondrial DNA restriction patterns, of a Dekkera yeast strain isolated from sherries and of a number of other Brettanomyces and Dekkera strains. By using a genomic DNA fragment of the isolated Dekkera strain, we developed a two-step PCR method which directs the specific amplification of target DNA from this strain and from other Brettanomyces-Dekkera strains. The method efficiently amplified the target DNA from intact cells, obviating DNA isolation, and yielded a detection limit of fewer than 10 yeast cells in contaminated samples of sherry.