Four yeast strains have been used in this work, S. cerevisiae FX10 (Laffort, SA), a widely used industrial wine yeast strain, T. delbrueckii CECT 11199 (CBS 1146), C. sake CECT 11909 (CBS 159), and H. uvarum CECT 10389 (MCYC 1857). Synthetic must contained (per liter): glucose: 100 g; fructose: 100 g; malic acid: 6 g; citric acid 6 g; YNB w/o aa; w/o (NH₄)₂SO₄ 1.7 g; nitrogen sources (Asp 29 mg; Glu 80 mg; Ser 52 mg; Gln 333 mg; Hys 31 mg; Gly 12 mg; Thr 50 mg; Arg 296.28 mg; Ala 97 mg; Tyr 13 mg; Cys 18.2 mg; Val 29 mg; Met 21 mg; Trp 116 mg; Phe 25 mg; Ile 22 mg; Leu 32 mg; Lys 13.72 mg; Pro 400 mg; NH₄Cl 306 mg); anaerobic factors (ergosterol 15 mg; oleic acid 5 mg; tween 80 0.5 mL); inositol 18 mg; pH adjusted at 3.5 with NaOH.

Pre-cultures were grown in YPD (1% yeast extract, 2% peptone, and 2% glucose) medium for 48 h at 25°C and 150 rpm. Before co-inoculation, pre-cultures were centrifuged at 2200 × g, for 15 min at room temperature, and washed twice with distilled water. Mixed cultures constituted by S. cerevisiae and one of the assayed strains were inoculated to a total initial optical density (OD_600nm) of 0.2 (0.1 for each strain) in 200 mL of synthetic must medium contained in 250 mL flasks with wide aluminum foil caps allowing aeration. Single culture of S. cerevisiae strain was inoculated to an OD_600nm of 0.2 to match conditions in mixed cultures. Flasks were incubated at 25°C under agitation (250 rpm) during 3 h. Experiments were performed in triplicate. The viability of the different populations in the mixed cultures was confirmed after 24 h of co-cultivation. Cells from the mixed cultures were plated at 25°C at different dilutions to ensure individual colony growth and then re-plated at 37°C were S. cerevisiae cells can be differentiated from the other yeast species that do not growth at this temperature. This confirmed that after 24 h both yeast species were present in the media.

After 3 h of cultivation, total flasks volumes were centrifuged and collected cells washed twice with distilled water before samples were submerged in liquid nitrogen and stored at -80°C for total RNA isolation. Total RNA from the biological triplicates was extracted using RNeasy^® mini kit (QIAGEN) and subjected to DNAase treatment using the Ambion DNA-free^TM kit according to the manufacturers’ instructions. Concentration, purity, and integrity of RNA samples were determined by spectrophotometric analysis considering the absorbance ratio at 260/280 nm and at 230/260 nm. Library preparation and sequencing of RNA was performed at the Genomics Core Facility in the Center for Biomedical Research of La Rioja (CIBIR). After poly-A filtering, libraries were generated for the different conditions, triplicates of S. cerevisiae single cultures and triplicates of S. cerevisiae co-cultivated with T. delbrueckii, C. sake, and H. uvarum. From these libraries, 100-bp pair-end sequence reads were produced with Illumina HiSeq 2000. All raw RNA-Seq data have been deposited in NCBI under Sequence Read Archive SRR5422019 (BioProject PRJNA381847) accession number.

Alignment of reads to the S288c R64 S. cerevisiae yeast reference genome assembly was carried out using TopHat2 v.2.0.13 (Kim et al., 2013). Only uniquely mapped single copy, ≤1 polymorphism per 25 bp reads with quality≥ 20 were kept for further analysis. The htseq-count tool (v.0.5.4p5) from HTSeq (Anders et al., 2015) was used to estimate unambiguous read count per genome assembly annotated transcript. Normalization following the trimmed mean of M-values (TMM) method (Robinson and Oshlack, 2010), as well as a time-points DEGs searches (adjusted Benjamini–Hochberg P ≤ 0.05 and ≥twofold change) were performed in edgeR v.2.2.6 (Robinson et al., 2010). Finally, fragments per kb of exon per million fragments mapped (FPKM) was calculated using Cuffdiff v.2.2.1 (Trapnell et al., 2013) and low-expressed transcripts were filtered out when FPKM was <1 in both samples. In order to confirm that there was no cross mapping from the co-cultivation, a quimeric genome from S. cerevisiae and the non-Saccharomyces species was created when the genome was available (T. delbrueckii and H. uvarum). Almost the same genes (98%) appeared DE comparing both strategies, quimeric and regular mapping.

Different sets of genes were considered for analysis purposes. We refer to significantly up- or down-regulated genes for those that have a log fold change (LogFC) ≥ 0.5 or ≤-0.5 and an adjusted p-value ≤ 0.05. When we refer to highly up- or down-regulated genes (a more restrictive category), showing a LogFC ≥ 1 or ≤-1 and an adjusted p-value ≤ 0.05. The response of S. cerevisiae to each different strain has been analyzed using the more restrictive dataset, including gene ontology analysis. For comparative analysis among the different datasets the broader database has been used (Venn Diagram). Gene expression values showing higher adjusted p-values were never taken into consideration for data analysis or discussion, independently of the associated LogFC.

Principal component analysis (PCA) was done using AltAnalyze software (2.1.0) (Emig et al., 2010). AltAnalyze was feeded with the normalized RNAseq data transcripts per million (TPM). The remaining statistical analyses were done using STATA-SE. Venn diagram was done by using Venny 2.1 on-line tool software (Oliveros, 2007–2015). GO term analysis was performed using YeastMine (Balakrishnan et al., 2012). The p-values were corrected for multiple testing by the Bonferroni test for functional associations and GO analyses. The statistical level of significance was set at p-value ≤ 0.05. Then, GO terms were grouped in biomodules by GO/Module (Yang et al., 2011) to prioritize Gene Ontology.

In this work, we have analyzed the effect of three different yeast species over the S. cerevisiae transcriptome when grown together at early stages of an aerobic synthetic must fermentation. T. delbrueckii, C. sake, and H. uvarum were chosen to be co-inoculated with S. cerevisiae at equal cell density. After 3 h, cells were sampled and the transcriptome of the S. cerevisiae cells from mixed and single cultures was compared by RNAseq analysis. These three yeast species are often isolated from grape must at early stages of fermentation, and are hence natural competitors of S. cerevisiae (Fleet, 2003; Jolly et al., 2014). In a previous work the transcriptome of S. cerevisiae and T. delbrueckii was analyzed after 2 and 12 h of anaerobic co-cultivation. Comparison of single and mixed cultures showed that genes from “Glucose Fermentation Pathway” were overexpressed in both species due to the presence of the other yeast in the media. Overexpression in S. cerevisiae is noticed in the first sampling point and in T. delbrueckii in the second one. Even though T. delbrueckii shows good fermentative fitness in pure culture, it is quickly overtaken by S. cerevisiae, perhaps because the earlier reply of S. cerevisiae. This made us wonder if the observed quick response of S. cerevisiae to the presence of T. delbrueckii was specific for this yeast species or similar responses could be obtained with different yeasts. For this reason, other yeast species present at early stages of grape must fermentation but phylogenetically more distant than T. delbrueckii (Masneuf-Pomarede et al., 2016) were chosen.

T. delbrueckii was used to keep a reference to our published work (Tronchoni et al., 2017) but based on our previous results, the selection of an early time point was set to 3 instead of 2 h. The number of overexpressed genes was similar, with only 44 genes being highly up-regulated (Supplementary File S1). Several genes from the “Glucose Fermentation Pathway” appear significantly up-regulated as well as several genes encoding for glucose transporter proteins, as previously described (Supplementary File S1). Under standard conditions, genes related to fermentation of glucose and its transport into the cell are tightly regulated by the extracellular concentration of glucose through carbon catabolite repression (CCR) (Gancedo, 1998). For instance, high-affinity glucose transporters are only expressed when the concentration of this sugar is low (Ozcan, 2002; Kayikci and Nielsen, 2015). Interestingly, seven out of the eight genes involved in glucose uptake and metabolism are under the control of this carbon source that repress its expression when sugar concentration is high. Based on the glucose concentration of the media, around 200 g/L, these genes should be down-regulated. This result points to a partial relieve of CCR in S. cerevisiae by the presence of T. delbrueckii.

In addition, Gene Ontology categories enriched for S. cerevisiae genes up-regulated in the presence of T. delbrueckii were mostly related with nitrogen metabolism, specifically allantoin catabolism (Table 1). Actually, most genes induced by co-cultivation with T. delbrueckii were involved in utilization of alternative nitrogen sources; and are under nitrogen catabolite repression (NCR) control. The relevance of the activation of these genes is such that five out of the main up-regulated and even half of the highly up-regulated genes were under the control of this transcription factor GLN3 (Supplementary File S1), required for the expression of genes involved in the use of non-preferred nitrogen sources (Magasanik and Kaiser, 2002). Among them, genes showing the highest over-expression values belong to the DAL family. Indeed, the entire pathway for allantoin catabolism was overexpressed.

Like the CCR and NCR dependent genes mentioned above, expression of other genes expected to show low activity in rich medium, especially after only 3 h of incubation (i.e., before actual consumption of carbon or nitrogen sources might be observed), was also highly induced in S. cerevisiae by co-cultivation with T. delbrueckii. Among them, we found other NCR dependent genes like those coding for proline permease, PUT4; proline oxidase, PUT1; general amino acid permease, GAP1; GABA permease, UGA4; or a putative allantoate permease; as well as some high affinity permeases and metal transporters like those for inorganic phosphate, PHO84; sulfate, SUL1; copper, CTR3; or cysteine, YCT1 (Supplementary File S1).

No significant gene ontology enrichment was found for the only 20 genes showing a high overexpression in S. cerevisiae when co-cultivated with C. sake. About half of them were shared with the list of highly overexpressed genes coming from T. delbrueckii co-cultivation, including genes already discussed above, related with the allantoin pathway, nitrogen uptake or non-preferred nitrogen sources, and genes involved in glucose uptake and metabolism (Supplementary File S1). Some of these genes in common showed higher expression compared to the T. delbrueckii experiment, like CHA1, involved in the use of nitrogen sources (serine or threonine). Other highly overexpressed genes shared between C. sake and T. delbrueckii co-cultivation are all involved in replication (specially RNA helicases, but also rRNA and ribosome biogenesis, or Start checkpoint), or related with cell wall (TIP1), and membrane lipid composition (OLE1, ERG5, ERG3, ERG11, ERG1, and ERG25) (Supplementary File S1). The up-regulation of these genes suggests another possible strategy of S. cerevisiae to improve competitiveness in grape must, besides or complementary to the activation of genes required for sugar and nitrogen consumption. This would consist of an increase in relative membrane surface (through increased cell numbers), which will help accelerate nutrient uptake in detriment of other yeasts.

In contrast to the T. delbrueckii experiment, in C. sake competition, there are more genes highly down-regulated than up-regulated (34 genes). These genes are summarized in the GO term categories “carboxylic acid transmembrane transport” and “sulfur compound metabolic process” (Supplementary File S1). Some of the genes under these two categories are MET1, MET2, MET8, MET32, ISU2, or SUL2. They encode methionine and sulfur permeases, and are involved in methionine synthesis, or the synthesis of iron-sulfur proteins. Thus, the transcriptional response of S. cerevisiae to co-cultivation with T. delbrueckii or C. sake is similar, considering overexpressed genes. However, there are clear differences among the down-regulated genes. Actually, from the 34 genes highly down-regulated in the C. sake experiment, only four appear in the T. delbrueckii down-regulated dataset, while other two genes appear as up-regulated (Supplementary File S1).

Co-cultivation with H. uvarum resulted in a low gene expression profile as seen with T. delbrueckii and C. sake, with 29 genes showing high overexpression, 6 genes in common with T. delbrueckii, and only 3 with C. sake (Supplementary File S1). Among the 29 genes significantly highly up-regulated in these cultures 12 out of them belong to the GO categories “response to stimulus” and/or “response to stress.” Thus, many of the most overexpressed genes are involved in resistance to several stresses, for instance, pleiotropic drug resistance (2 genes), heat (4 genes), DNA replication stress or DNA damage (6 genes) or osmotic stress (Hog1 dependent, 2 genes). It is also worth mentioning three genes coding for cell wall mannoproteins (TIR1, TIP1, and DAN1), among the 10 most overexpressed genes. These mannoproteins belong to the Srp1/Tip1 family and have been described to respond to different stresses like cold stress and to be stimulated in the adaptation to hypoxia (Sertil et al., 1997; ter Linde et al., 1999; Abramova N. et al., 2001; Abramova N.E. et al., 2001; Tai et al., 2005). The results obtained for H. uvarum co-cultivation suggest that some cell wall proteins might be also important for the adaptation of S. cerevisiae to biotic stress.

As seen with C. sake mixed cultures, transcriptional response to co-cultivation with H. uvarum results in the repression of a great number of genes in S. cerevisiae, 31 in this case highly down-regulated (Supplementary File S1). This response is similar to that observed for C. sake, with several genes showing a reverse behavior, as compared to the T. delbrueckii experiment (7 genes). Some of these genes were important in the discussion of the effect of T. delbrueckii over S. cerevisiae in mixed cultures made above (HSP12 and PDC5) and in our previous work (Tronchoni et al., 2017), like HSP12 (described by us and others in S. cerevisiae – S. cerevisiae interactions).

Total LogFC datasets without threshold restrictions were used to perform a PCA to compare the responses of S. cerevisiae to the different yeasts in co-cultivation (Figure 1). Results cluster each independent replicate together for each yeast, although there is a higher dispersion of T. delbrueckii and H. uvarum compared with C. sake. Principal Component 1 (PC1) explaining 62.1% of the variance separates T. delbrueckii and C. sake from H. uvarum, and PC2 explaining 15.2% of the variance, T. delbrueckii and H. uvarum from C. sake. Thus, each yeast ends up in a different section of the PCA. Highlighting a particular response of S. cerevisiae when co-cultivated with different yeast species.

Although PCA results define a different response depending on the yeast mixed culture, the co-cultivation experiments shared global transcriptomic characteristics as well as genes behaving in a similar manner. One of the main characteristics shared by all the experiments carried out so far is that it is a moderate response, gene expression fold changes are low as well as the number of significant genes. Venn diagram showing significant up-regulated genes (LogFC ≥ 0.5; p-adjusted ≤ 0.05) for the three species tested shows the degree of similarity described (Figure 2). Although there are genes in common among them, the percentage varies from the 35% of the genes being in common between T. delbrueckii and H. uvarum, both with similar number of up-regulated genes to the much lower number of genes shared with C. sake or among the three of them. On the other hand, Venn diagram for down-regulated genes (LogFC ≤-0.5; p-adjusted ≤ 0.05) shows a much more heterogeneous response to co-cultivation depending on the competing yeast species.

A general picture can be drawn from the short list of genes equally up- or down-regulated in S. cerevisiae in response to co-cultivation among the three experiments (Figure 2). From the 12 genes commonly up-regulated, 3 are involved in glucose uptake and glycolysis, and according to literature should be repressed by high levels of glucose (after 3 h of co-cultivation in synthetic must, glucose concentration is close to 200 g/L). HXT12, a high-affinity glucose transporter and both cytoplasmic and mitochondrial aldehyde dehydrogenases; other three involved in membrane lipid metabolism, OLE1 (monounsaturated fatty acid synthesis), FAA4 (long-chain fatty acyl-CoA synthetase), and ERG5 (ergosterol biosynthesis pathway); also, the major cell wall mannoprotein TIP1; two more related with the nitrogen sources available, CHA1 (serine or threonine) and PUT1 (proline). The three remaining genes codified for a protein required for antifungal drug resistance (COS111), a membrane protein involved in zinc ion homeostasis (IZH1) and INA1 a putative protein of unknown function which paralog is FAT3, a protein required for fatty acid uptake. Therefore, as has been described previously for each individual yeast co-cultivation, this set of genes can be summarized in glucose uptake, membrane and cell wall biogenesis, and nitrogen utilization. Thus, despite the clean separation of the three yeast species co-cultivated by the PCA there are some trends common to all experiments. This can be seen in the expression of the DAL family of metabolic genes. Plotting LogFC for the different co-cultivation vs. single S. cerevisiae cultures of chromosome IX reveals the induction of this gene cluster (Figure 3), showing higher overexpression values for T. delbrueckii, lower values for C. sake despite a clear trend is observed, and just one gene significative for H. uvarum.