Comprehensive analysis of both long and short read transcriptomes of a clonal and a seed-propagated model plant species reveal the prerequisites for transcriptional activation of autonomous and non-autonomous transposons in plants

Background Transposable element (TE) transcription is a precursor to its mobilisation in host genomes. However, the characteristics of expressed TE loci, the identification of self-competent transposon loci contributing to new insertions, and the genomic conditions permitting their mobilisation remain largely unknown. Results Using Vitis vinifera embryogenic callus, we explored the impact of biotic stressors on transposon transcription through the exposure of the callus to live cultures of an endemic grapevine yeast, Hanseniaspora uvarum. We found that only 1.7%-2.5% of total annotated TE loci were transcribed, of which 5%-10% of these were full-length, and the expressed TE loci exhibited a strong location bias towards expressed genes. These tendencies were also observed in Arabidopsis thaliana wild-type and ibm2, but not in ddm1 mutant. Moreover, differentially expressed TE loci in the grapevine model tended to share expression patterns with co-localised differentially expressed genes. Utilising nanopore cDNA sequencing, we found a strong correlation between the inclusion of intronic TEs in gene transcripts and the presence of premature termination codons in these transcripts. Finally, we identified low levels of full-length transcripts deriving from structurally autonomous TE loci in the grapevine model. Conclusion Our observations in two disparate plant models representing clonally and seed propagated plant species reveal a closely connected transcriptional relationship between TEs and co-localised genes, particularly when the epigenetic silencing system is intact. We found that the stress treatment alone was insufficient to induce large-scale full-length transcription from structurally competent TE loci, a necessity for non-autonomous and autonomous mobilisation.

In Vitro Biological Control of Aspergillus flavus by Hanseniaspora opuntiae L479 and Hanseniaspora uvarum L793, Producers of Antifungal Volatile Organic Compounds

Aspergillus flavus is a toxigenic fungal colonizer of fruits and cereals and may produce one of the most important mycotoxins from a food safety perspective, aflatoxins. Therefore, its growth and mycotoxin production should be effectively avoided to protect consumers’ health. Among the safe and green antifungal strategies that can be applied in the field, biocontrol is a recent and emerging strategy that needs to be explored. Yeasts are normally good biocontrol candidates to minimize mold-related hazards and their modes of action are numerous, one of them being the production of volatile organic compounds (VOCs). To this end, the influence of VOCs produced by Hanseniaspora opuntiae L479 and Hanseniaspora uvarum L793 on growth, expression of the regulatory gene of the aflatoxin pathway (aflR) and mycotoxin production by A. flavus for 21 days was assessed. The results showed that both yeasts, despite producing different kinds of VOCs, had a similar effect on inhibiting growth, mycotoxin biosynthetic gene expression and phenotypic toxin production overall at the mid-incubation period when their synthesis was the greatest. Based on the results, both yeast strains, H. opuntiae L479 and H. uvarum L793, are potentially suitable as a biopreservative agents for inhibiting the growth of A. flavus and reducing aflatoxin accumulation.

Diversity of Mycobiota in Spanish Grape Berries and Selection of Hanseniaspora uvarum U1 to Prevent Mycotoxin Contamination

The occurrence of mycotoxins on grapes poses a high risk for food safety; thus, it is necessary to implement effective prevention methods. In this work, a metagenomic approach revealed the presence of important mycotoxigenic fungi in grape berries, including Aspergillus flavus, Aspergillus niger aggregate species, or Aspergillus section Circumdati. However, A. carbonarius was not detected in any sample. One of the samples was not contaminated by any mycotoxigenic species, and, therefore, it was selected for the isolation of potential biocontrol agents. In this context, Hanseniaspora uvarum U1 was selected for biocontrol in vitro assays. The results showed that this yeast is able to reduce the growth rate of the main ochratoxigenic and aflatoxigenic Aspergillus spp. occurring on grapes. Moreover, H. uvarum U1 seems to be an effective detoxifying agent for aflatoxin B1 and ochratoxin A, probably mediated by the mechanisms of adsorption to the cell wall and other active mechanisms. Therefore, H. uvarum U1 should be considered in an integrated approach to preventing AFB1 and OTA in grapes due to its potential as a biocontrol and detoxifying agent.

Immunostimulatory activity of Pichia kluyveri, Hanseniaspora uvarum, Candida intermedia, Saccharomyces boulardii and their derivatives on RAW 264.7 macrophages

Yeasts are a group of microorganisms with structural and metabolic characteristics that influence their recognition by immune cells resulting in a species-specific response. Although Saccharomyces boulardii is a widely studied probiotic yeast, immunostimulation by non-Saccharomyces yeasts still underexplored. Therefore, the aim of this study was to characterize the response induced in macrophages stimulated by yeasts Pichia kluyveri, Hanseniaspora uvarum, Candida intermedia and their derivatives: heat-killed cells, supernatant and DNA. RAW 264.7 murine macrophages were stimulated in vitro for 24 h and the response generated was evaluated by analyzing mRNA transcription of cytokines (IL2, IL4, IL10, IL13, IL23, TNF-α), transcription factors (Bcl6, NFκβ, STAT3), Toll-like receptor 2 (TLR2) and YM1 protein. Viable and heat-killed cells of P. kluyveri and H. uvarum were responsible for high levels of relative mRNA transcription of transcription factors and TLR2 (between 2 – 8-fold increase), however were able to induce only low transcription levels for analyzed cytokines (≤ 2-fold increase). Viable cells of C. intermedia were able to stimulate a significant transcription of IL4 (7.6-fold increase) and Bcl6 (4-fold increase), while heat-killed cells stimulated the highest level of TNF-α (2.4-fold increase) among yeasts and their derivatives. Furthermore, supernatant from C. intermedia culture induced significant (p < 0.05) levels of TLR2 (4.4-fold increase), being the only one among supernatants to present high levels of relative mRNA transcription of TLR2. Data found in this work arouse interest in further studies on interaction between non-Saccharomyces yeasts and immune system cells, mainly referring to immunomodulatory capacity.

Wine Aroma Characterization of the Two Main Fermentation Yeast Species of the Apiculate Genus Hanseniaspora

Hanseniaspora species are the main yeasts isolated from grapes and grape musts. Regarding genetic and phenotypical characterization, especially fermentative behavior, they can be classified in two technological clusters: the fruit group and the fermentation group. Among the species belonging to the last group, Hanseniaspora osmophila and Hanseniaspora vineae have been previously isolated in spontaneous fermentations of grape must. In this work, the oenological aptitudes of the two species of the fermentation group were compared with Saccharomyces cerevisiae and the main species of the fruit group, Hanseniaspora uvarum. Both H. osmophila and H. vineae conferred a positive aroma to final wines and no sensory defects were detected. Wines fermented with H. vineae presented significantly higher concentrations of 2-phenylethyl, tryptophol and tyrosol acetates, acetoin, mevalonolactone, and benzyl alcohol compared to H. osmophila. Sensorial analysis showed increased intensity of fruity and flowery notes in wines vinificated with H. vineae. In an evolutionary context, the detoxification of alcohols through a highly acetylation capacity might explain an adaption to fermentative environments. It was concluded that, although H. vineae show close alcohol fermentation adaptations to H. osmophila, the increased activation of phenylpropanoid metabolic pathway is a particular characteristic of H. vineae within this important apiculate genus.

Brewing and Probiotic Potential Activity of Wild Yeasts Hanseniaspora Uvarum PIT001, Pichia Kluyveri LAR001 and Candida Intermedia ORQ001

Non-conventional yeasts can be isolated from a wide range of environmental sources, often found in beverage industry in mixed fermentations, in which the microorganisms’ inoculum usually is not fully known. It is important to know starter cultures, since in addition to favoring reproducibility, other properties can be discovered. Thus, the objective of this work was to identify and characterize yeasts isolated from environment, evaluating their probiotic potential and possible use in brewery. Isolates were obtained from flowers, fruits, leaves and mixed-fermentation beers, being identified by PCR. Yeasts with promising activity were evaluated regarding their growth under different pHs, temperature and presence of organic acids. To explore probiotic potential, in vitro tests were performed of antimicrobial activity and co-aggregation with food pathogens, auto-aggregation, and survival in simulated gastrointestinal tract conditions. In our study, Pichia kluyveri (LAR001), Hanseniaspora uvarum (PIT001) and Candida intermedia (ORQ001) were selected among 20 isolates. P. kluyveri was the only one that tolerated pH 2.5. Lactic acid was not inhibitory, while acetic acid and incubation at 37 °C had a partially inhibitory effect on yeasts growth. All yeasts tolerated α-acids from hops and NaCl up to 1%. It is suggested that isolates are able to adhere to intestinal cells and influence positively the organism in combating pathogens, as they showed auto-aggregation rates above 99% and antagonistic activity to pathogenic bacteria. The yeasts tolerated gastric environment conditions, however were more sensitive to pancreatic conditions. We conclude that isolated non-conventional yeasts showed probiotic potential and promising application in beer fermentation.

Effects of Simultaneous Co-Fermentation of Five Indigenous Non-Saccharomyces Strains with S. cerevisiae on Vidal Icewine Aroma Quality

In this study, Vidal grape must was fermented using commercial Saccharomyces cerevisiae F33 in pure culture as a control and in mixed culture with five indigenous non-Saccharomyces yeast strains (Hanseniaspora uvarum QTX22, Saccharomycopsis crataegensis YC30, Pichia kluyveri HSP14, Metschnikowia pulcherrima YC12, and Rhodosporidiobolus lusitaniae QTX15) through simultaneous fermentation in a 1:1 ratio. Simultaneous fermentation inhibited the growth of S. cerevisiae F33 and delayed the time to reach the maximum biomass. Compared with pure fermentation, the contents of polyphenols, acetic esters, ethyl esters, other esters, and terpenes were increased by R. lusitaniae QTX15, S. crataegensis YC30, and P. kluyveri HSP14 through simultaneous fermentation. S. crataegensis YC30 produced the highest total aroma activity and the most abundant aroma substances of all the wine samples. The odor activity values of 1 C13-norisoprenoid, 3 terpenes, 6 acetic esters, and 10 ethyl esters improved significantly, and three lactones (δ-decalactone, γ-nonalactone, and γ-decalactone) related to coconut and creamy flavor were only found in this wine. Moreover, this sample showed obvious “floral” and “fruity” note odor due to having the highest amount of ethyl ester aromatic substances and cinnamene, linalool, citronellol, β-damascenone, isoamyl ethanoate, benzylcarbinyl acetate, isobutyl acetate, etc. We suggest that simultaneous fermentation of S. crataegensis YC30 with S. cerevisiae might represent a novel strategy for the future production of Vidal icewine.