Response of Vitis vinifera cell cultures to Eutypa lata and Trichoderma atroviride culture filtrates: expression of defence-related genes and phenotypes.

Cell suspension cultures of Vitis vinifera cv. Dauphine berries were used to study the response to the vascular pathogen, Eutypa lata, in comparison with a biological control agent, Trichoderma atroviride, that was previously shown to be effective in pruning wound protection. The expression of genes coding for enzymes of the phenylpropanoid pathway and pathogenesis-related (PR) proteins was profiled over a 48-h period using quantitative reverse transcriptase PCR. The cell cultures responded to elicitors of both fungi with a hypersensitive-like response that lead to a decrease in cell viability. Similar genes were triggered by both the pathogen and biocontrol agent, but the timing patterns and magnitude of expression was dependent on the specific fungal elicitor. Culture filtrates of both fungi caused upregulation of phenylalanine ammonia-lyase (PAL), 4-coumaroyl Co-A ligase (CCo-A) and stilbene synthase (STS), and a downregulation of chalcone synthase (CHS) genes. The pathogen filtrate caused a biphasic pattern in the upregulation of PAL and STS genes which was not observed in cells treated with filtrates of the biocontrol agent. Analytical assays showed significantly higher total phenolic content and chitinolytic enzyme activity in the cell cultures treated with the T. atroviride filtrate compared to the pathogen filtrate. These results corresponded well to the higher expression of PAL and chitinase class IV genes. The response of the cell cultures to T. atroviride filtrate provides support for the notion that the wound protection by the biocontrol agent at least partially relies on the induction of grapevine resistance mechanisms.

Towards an open grapevine information system.

Viticulture, like other fields of agriculture, is currently facing important challenges that will be addressed only through sustained, dedicated and coordinated research. Although the methods used in biology have evolved tremendously in recent years and now involve the routine production of large data sets of varied nature, in many domains of study, including grapevine research, there is a need to improve the findability, accessibility, interoperability and reusability (FAIR-ness) of these data. Considering the heterogeneous nature of the data produced, the transnational nature of the scientific community and the experience gained elsewhere, we have formed an open working group, in the framework of the International Grapevine Genome Program (www.vitaceae.org), to construct a coordinated federation of information systems holding grapevine data distributed around the world, providing an integrated set of interfaces supporting advanced data modeling, rich semantic integration and the next generation of data mining tools. To achieve this goal, it will be critical to develop, implement and adopt appropriate standards for data annotation and formatting. The development of this system, the GrapeIS, linking genotypes to phenotypes, and scientific research to agronomical and oeneological data, should provide new insights into grape biology, and allow the development of new varieties to meet the challenges of biotic and abiotic stress, environmental change, and consumer demand.

Identification of a functional TATA element in the STA2 glucoamylase gene promoter from Saccharomyces cerevisiae.

Accurate transcription by RNA polymerase II is usually dependent on the presence of a TATA element, and/or an initiator element, in the promoters of protein-encoding genes. The STA1-3 genes, encoding three glucoamylase isozymes (Sta1p, Sta2p and Sta3p, respectively) responsible for starch hydrolysis in the yeast Saccharomyces cerevisiae, have been shown to contain long and complex promoters with several regulatory regions. These promoters are also virtually identical to the yeast MUC1 gene promoter; this gene encodes a mucin-like protein and is evolutionary linked to, and transcriptionally co-regulated with, STA1-3. The STA1-3 genes contain two putative TATA sequences; one conforming to the typical TATA box sequence, TATAAA, and another with the sequence of TATAAT. Here we present a study into the functional relevance of these putative TATA sequences and their effects on the transcription of the STA2 gene (as a representative model of the STA1-3 multigene family) and, by analogy, the MUC1 gene. We show that the TATAAA motif is the functional TATA box for STA2 and influences transcript levels, transcript initiation sites, and glucoamylase activities.

The Saccharomyces cerevisiae chitinase, encoded by the CTS1-2 gene, confers antifungal activity against Botrytis cinerea to transgenic tobacco.

The Saccharomyces cerevisiae chitinase, encoded by the CTS1-2 gene has recently been confirmed by in vitro tests to possess antifungal abilities. In this study, the CTS1-2 gene has been evaluated for its in planta antifungal activity by constitutive overexpression in tobacco plants to assess its potential to increase the plant's defence against fungal pathogens. Transgenic tobacco plants, generated by Agrobacterium-mediated transformation, showed stable integration and inheritance of the transgene. Northern blot analyses conducted on the transgenic tobacco plants confirmed transgene expression. Leaf extracts from the transgenic lines inhibited Botrytis cinerea spore germination and hyphal growth by up to 70% in a quantitative in vitro assay, leading to severe physical damage on the hyphae. Several of the F1 progeny lines were challenged with the fungal pathogen, B. cinerea, in a detached leaf infection assay, showing a decrease in susceptibility ranging from 50 to 70%. The plant lines that showed increased disease tolerance were also shown to have higher chitinase activities.

Coregulation of starch degradation and dimorphism in the yeast Saccharomyces cerevisiae.

Saccharomyces cerevisiae, the exemplar unicellular eukaryote, can only survive and proliferate in its natural habitats through constant adaptation within the constraints of a dynamic ecosystem. In every cell cycle of S. cerevisiae, there is a short period in the G1 phase of the cell cycle where "sensing" transpires; if a sufficient amount of fermentable sugars is available, the cells will initiate another round of vegetative cell division. When fermentable sugars become limiting, the yeast can execute the diauxic shift, where it reprograms its metabolism to utilize nonfermentable carbon sources. S. cerevisiae can also initiate the developmental program of pseudohyphal formation and invasive growth response, when essential nutrients become limiting. S. cerevisiae shares this growth form-switching ability with important pathogens such as the human pathogen, Candida albicans, and the corn smut pathogen Ustilago maydis. The pseudohyphal growth response of S. cerevisiae has mainly been implicated as a means for the yeast to search for nutrients. An important observation made was that starch-degrading S. cerevisiae strains have the added ability to form pseudohyphae and grow invasively into a starch-containing medium. More significantly, it was also shown that the STA1-3 genes encoding three glucoamylase isozymes responsible for starch hydrolysis in S. cerevisiae are coregulated with a gene, MUC1, essential for pseudohyphal and invasive growth. At least two putative transcriptional activators, Mss10p and Mss11p, are involved in this regulation. The Muc1p is a putative integral membrane-bound protein similar to mammalian mucin-like proteins that have been implicated in the ability of cancer cells to invade other tissues. This provided us with an excellent example of integrative control between nutrient sensing, signaling, and differential development.

Functional analysis of multiple AUG codons in the transcripts of the STA2 glucoamylase gene from Saccharomyces cerevisiae.

A scanning ribosome will usually initiate translation as soon as it encounters the first favourable AUG codon and only a few eukaryotic transcripts have more complex arrangements. These relatively few complex transcripts are normally characterized by structural features such as multiple AUGs and significant secondary structure. However, the functional relevance of these features has rarely been established. We present here a study of the functional significance of the multiple AUGs in the leader of STA2 transcripts of the budding yeast Saccharomyces cerevisiae, and extrapolate, where applicable, these results to a co-regulated gene, MUC1. The STA2 gene (a representative member of the polymorphic STA1-3 gene family), encodes an extracellular glucoamylase, and is evolutionarily linked to, and transcriptionally co-regulated with, the MUC1 gene, which encodes a mucin-like protein essential for pseudohyphal/invasive growth and cell-adhesion in S. cerevisiae. Each of these genes contains a putative upstream ORF, while STA2 has two additional in-frame AUG codons 5' to the major cistron. We show that utilization of the alternative translational start-sites of STA2 results in glucoamylases that differ at their N-termini, which are associated with differences in their localization patterns. Analysis of mutants revealed the presence of a putative secretion-enhancing signal that might prove to be relevant to the alternative targeting mechanism recently uncovered in S. cerevisiae. We show that a short up-stream ORF present in the leaders of STA1-3 and MUC1 is probably bypassed by a process of leaky scanning.

The development of bactericidal yeast strains by expressing the Pediococcus acidilactici pediocin gene (pedA) in Saccharomyces cerevisiae.

The excessive use of sulphur dioxide and other chemical preservatives in wine, beer and other fermented food and beverage products to prevent the growth of unwanted microbes holds various disadvantages for the quality of the end-products and is confronted by mounting consumer resistance. The objective of this study was to investigate the feasibility of controlling spoilage bacteria during yeast-based fermentations by engineering bactericidal strains of Saccharomyces cerevisiae. To test this novel concept, we have successfully expressed a bacteriocin gene in yeast. The pediocin operon of Pediococcus acidilactici PAC1.0 consists of four clustered genes, namely pedA (encoding a 62 amino acid precursor of the PA-1 pediocin), pedB (encoding an immunity factor), pedC (encoding a PA-1 transport protein) and pedD (encoding a protein involved in the transport and processing of PA-1). The pedA gene was inserted into a yeast expression/secretion cassette and introduced as a multicopy episomal plasmid into a laboratory strain (Y294) of S. cerevisiae. Northern blot analysis confirmed that the pedA structural gene in this construct (ADH1P-MFa1S-pedA-ADH1T, designated PED1), was efficiently expressed under the control of the yeast alcohol dehydrogenase I gene promoter (ADH1P) and terminator (ADH1T). Secretion of the PED1-encoded pediocin PA-1 was directed by the yeast mating pheromone alpha-factor's secretion signal (MFa1S). The presence of biologically active antimicrobial peptides produced by the yeast transformants was indicated by agar diffusion assays against sensitive indicator bacteria (e.g. Listeria monocytogenes B73). Protein analysis indicated the secreted heterologous peptide to be approximately 4.6 kDa, which conforms to the expected size. The heterologous peptide was present at relatively low levels in the yeast supernatant but pediocin activity was readily detected when intact yeast colonies were used in sensitive strain overlays. This study could lead to the development of bactericidal yeast strains where S. cerevisiae starter cultures not only conduct the fermentations in the wine, brewing and baking industries but also act as biological control agents to inhibit the growth of spoilage bacteria.