Molecular analysis of the early interaction between the grapevine flower and Botrytis cinerea reveals that prompt activation of specific host pathways leads to fungus quiescence

Grape quality and yield can be impaired by bunch rot, caused by the necrotrophic fungus Botrytis cinerea. Infection often occurs at flowering, and the pathogen stays quiescent until fruit maturity. Here, we report a molecular analysis of the early interaction between B. cinerea and Vitis vinifera flowers, using a controlled infection system, confocal microscopy and integrated transcriptomic and metabolic analysis of the host and the pathogen. Flowers from fruiting cuttings of the cultivar Pinot Noir were infected with green fluorescent protein (GFP)-labelled B. cinerea and studied at 24 and 96 hours post-inoculation (h.p.i.). We observed that penetration of the epidermis by B. cinerea coincided with increased expression of genes encoding cell-wall-degrading enzymes, phytotoxins and proteases. Grapevine responded with a rapid defence reaction involving 1193 genes associated with the accumulation of antimicrobial proteins, polyphenols, reactive oxygen species and cell wall reinforcement. At 96 h.p.i., the reaction appears largely diminished both in the host and in the pathogen. Our data indicate that the defence responses of the grapevine flower collectively are able to restrict invasive fungal growth into the underlying tissues, thereby forcing the fungus to enter quiescence until the conditions become more favourable to resume pathogenic development

Development and Use of Biotechnology Tools for Grape Functional Analysis

The aim of this chapter is to provide a description of the latest scientific advances in the field of gene functional analysis in grapevine. It provides general information about the studies conducted during the past decade to understand the natural variation of this plant and how this information has been exploited for the understanding of traits of interest. Likewise, it is exposed how the use of biotechnology tools have helped to characterize the mechanisms of gene expression and its regulation, as well as the subcellular localization of proteins and their interactions with other molecules. Finally, an approximation to the new technologies of gene editing and their potential application in the functional study of grapevine has been carried out

Malbec, genética y cambio climático

La evolución de la temperatura en los últimos 20 años muestra una tendencia de calentamiento global en el hemisferio Norte que se acentuará en los próximos 50 años.Según una publicación de Hans R. Schultz and Gregory Jones (2010) en Journal of Wine Research, esto cambiaría los márgenes de adecuación para el crecimiento de la uva, afectando los límites del cultivo de la vid y la distribución de las variedades. En el hemisferio Sur, adicionalmente, es posible se que sumen progresivos cambios hacia un clima de carácter subtropical. En áreas de bajo riego como Cuyo y otros valles la escasez de agua sería el escenario más probable.El aumento de la temperatura acelera el desarrollo de la vid, alterando la interacción entre los ciclos vegetativos y reproductivos. Tiene efectos en el desarrollo de la uva,acelera la caída de ácidos orgánicos e induce un incremento en la concentración de azúcares, desacoplando ambos procesos, de la acumulación de antocianos y de la maduración fenólica, que no responden de igual manera al incremento de temperatura.Como consecuencia, estas uvas dan lugar a vinos que pueden presentar un grado alcohólico excesivo, baja acidez, baja intensidad de color y caracteres fenólicos verdes.Mientras que las estrategias de producción o elaboración de vinos pueden permitir una aplicación a corto plazo, para revertir esta situación en el medio plazo resulta más atractivo generar soluciones con un mayor recorrido de su aplicación y que no impliquen un incremento en los costes de producción. En este sentido, la selección de variantes somáticas (clones) puede suponer una posibilidad de identificar genotipos más adaptados para las nuevas condiciones ambientales manteniendo el genotipo básico de la variedad, sus características productivas y enológicas y su denominación. Todo ello resulta muy importante en la elaboración de vinos de calidad al mantener el mismo producto final que es reconocido por productores, entes reguladores y consumidores.Fil: Lijavetzky, Diego Claudio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Biología Agrícola de Mendoza. Universidad Nacional de Cuyo. Facultad de Ciencias Agrarias. Instituto de Biología Agrícola de Mendoza; Argentin

Identification and functional prediction of anthocyanin biosynthesis regulatory long non-coding RNAs (lncRNAs) in carrot

Carrot (Daucus carota L.) is one of the most cultivated vegetable in the world and of great importance in the human diet. Its storage organs can accumulate large quantities of anthocyanins, metabolites that confer the purple pigmentation to carrot tissues and whose biosynthesis is well characterized. Long non-coding RNAs (lncRNAs) play critical roles in regulating gene expression of various biological processes in plants. In this study, we used a high throughput stranded RNA-seq to identify and analyze the expression profiles of lncRNAs in phloem and xylem root samples using two genotypes with a strong difference in anthocyanin production. We identified 639 differentially expressed lncRNAs between genotypes, and certain were specifically associated with a particular tissue. We then established regulatory correlations between lncRNAs and anthocyanin biosynthesis genes in order to identify a molecular framework for the differential expression of the pathway between genotypes. A specific natural antisense transcript (NAT) linked to the DcMYB7 key anthocyanin biosynthetic transcription factor suggested how the regulation of this pathway may have evolved between genotypes.Competing Interest StatementThe authors have declared no competing interest.Fil: Chialva, Constanza Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Biología Agrícola de Mendoza. Universidad Nacional de Cuyo. Facultad de Ciencias Agrarias. Instituto de Biología Agrícola de Mendoza; ArgentinaFil: Blein, Thomas. Université de Paris; FranciaFil: Crespi, Martin. Université de Paris; FranciaFil: Lijavetzky, Diego Claudio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Biología Agrícola de Mendoza. Universidad Nacional de Cuyo. Facultad de Ciencias Agrarias. Instituto de Biología Agrícola de Mendoza; Argentin

PHC9 COST-EFFECTIVENESS OF RIVAROXABANVERSUS ENOXAPARIN FORTHROMBOPROPHYLAXIS AFTER TOTAL HIP REPLACEMENT IN THE UK

Vitis vinifera cell cultures respond to pathogens and elicitors by synthesizing and extracellularly accumulating stilbenoid phytoalexins. Large amounts of trans-resveratrol (t-R) are produced when a cell culture is elicited with methylated cyclodextrins (MBCD), either alone or combined with methyl jasmonate (MeJA). t-R transport to the extracellular medium, which represents the apoplastic space, would place this antifungal defense right in the battlefield to efficiently fight against pathogen attack. Yet despite their physiological relevance, these transport pathways are mostly unknown. A broad hypothesis-free DIGE-based proteomic experiment of a temporal series of elicited grapevine cell cultures was performed to explore the expression profiles of t-R biosynthetic proteins and other co-expressing proteins potentially involved in such a cell response. A correlation between two tau class glutathione-S-transferases (GSTs) with several stilbene synthase and phenylalanine ammonia-lyase isoforms, and with the t-R metabolite itself, was found and further assessed by a qRT-PCR gene expression analysis. The best candidate, GSTU-2, was cloned from the cDNA of the MBCD + MeJA-elicited grapevine cells and used for Agrobacterium-mediated grapevine cell transformation. The non-elicited lines that overexpressed GSTU-2 displayed an extracellular t-R accumulating phenotype, but stabilization of t-R required the addition to culture medium of adsorbent compounds, e.g., PVP or β-cyclodextrin. The wild-type cell cultures accumulated no t-R, not even in the presence of adsorbents. The transient expression of the GSTU-2-GFP fusion proteins in grapevine cells showed localisation in the plasma membrane, and the immunoprecipitation of HA-tagged GSTU-2 revealed its interaction with HIR, a plasma membrane-bound protein. These findings are consistent with a functional role in transport. This is the first report providing several pieces of experimental evidence for the involvement of a specific tau class GST in t-R transport to the extracellular medium

Identification and relationship of the autochthonous ‘Romé’ and ‘Rome Tinto’ grapevine cultivars

The ‘Romé’ variety is considered an Andalusian (southern region in Spain) autochthonous black grape cultivar. However, several white and black grapevine accessions are known by this name, according to Vitis International Variety Catalogue. The aim of the present work was to clarify the identity of the ‘Romé’ and ‘Rome Tinto’ as black grapevine cultivar. Eight accessions known as ‘Romé’ and two as ‘Rome Tinto’ were analyzed using 30 OIV descriptors and 22 SSR loci. The morphologic and genetic analysis showed that all accessions studied presented the same genotype and phenotype and grouped with South Spanish cultivars. This study helps to clarify the confusion over the identity of ‘Romé’ grapevine cultivar, and provides a solid basis to develop a germplasm collection to protect grapevine diversity and to recover cultivars that may be in danger of extinction

Plasticity of the Berry Ripening Program in a White Grape Variety

Grapevine (Vitis vinifera L) is considered one of the most environmentally sensitive crops and is characterized by broad phenotypic plasticity, offering important advantages such as the large range of different wines that can be produced from the same cultivar, and the adaptation of existing cultivars to diverse growing regions. The uniqueness of berry quality traits reflects complex interactions between the grapevine plant and the combination of natural factors and human cultural practices, defined as terroir, which leads to the expression of wine typicity. Despite the scientific and commercial importance of genotype interactions with growing conditions, few studies have characterized the genes and metabolites directly involved in this phenomenon. Here we used two large-scale analytical approaches to explore the metabolomic and transcriptomic basis of the broad phenotypic plasticity of Garganega, a white berry variety grown at four sites characterized by different pedoclimatic conditions (altitudes, soil texture and composition). These conditions determine berry ripening dynamics in terms of sugar accumulation and the abundance of phenolic compounds. Multivariate analysis unraveled a highly plastic metabolomic response to different environments, especially the accumulation of hydroxycinnamic and hydroxybenzoic acids and flavonols. Principal component analysis revealed that the four sites strongly affected the berry transcriptome allowing the identification of environmentally-modulated genes and the plasticity of commonly-modulated transcripts at different sites. Many genes that control transcription, translation, transport and carbohydrate metabolism showed different expression depending on the environmental conditions, indicating a key role in the observed transcriptomic plasticity of Garganega berries. Interestingly, genes representing the phenylpropanoid/flavonoid pathway showed plastic responses to the environment mirroring the accumulation of the corresponding metabolites. The comparison of Garganega and Corvina berries showed that the metabolism of phenolic compounds is more plastic in ripening Garganega berries under different pedoclimatic conditions

High throughput SNP discovery and genotyping in grapevine (Vitis vinifera L.) by combining a re-sequencing approach and SNPlex technology

Background: Single-nucleotide polymorphisms (SNPs) are the most abundant type of DNA sequence polymorphisms. Their higher availability and stability when compared to simple sequence repeats (SSRs) provide enhanced possibilities for genetic and breeding applications such as cultivar identification, construction of genetic maps, the assessment of genetic diversity, the detection of genotype/phenotype associations, or marker-assisted breeding. In addition, the efficiency of these activities can be improved thanks to the ease with which SNP genotyping can be automated. Expressed sequence tags (EST) sequencing projects in grapevine are allowing for the in silico detection of multiple putative sequence polymorphisms within and among a reduced number of cultivars. In parallel, the sequence of the grapevine cultivar Pinot Noir is also providing thousands of polymorphisms present in this highly heterozygous genome. Still the general application of those SNPs requires further validation since their use could be restricted to those specific genotypes. Results: In order to develop a large SNP set of wide application in grapevine we followed a systematic re-sequencing approach in a group of 11 grape genotypes corresponding to ancient unrelated cultivars as well as wild plants. Using this approach, we have sequenced 230 gene fragments, what represents the analysis of over 1 Mb of grape DNA sequence. This analysis has allowed the discovery of 1573 SNPs with an average of one SNP every 64 bp (one SNP every 47 bp in non-coding regions and every 69 bp in coding regions). Nucleotide diversity in grape ( = 0.0051) was found to be similar to values observed in highly polymorphic plant species such as maize. The average number of haplotypes per gene sequence was estimated as six, with three haplotypes representing over 83% of the analyzed sequences. Short-range linkage disequilibrium (LD) studies within the analyzed sequences indicate the existence of a rapid decay of LD within the selected grapevine genotypes. To validate the use of the detected polymorphisms in genetic mapping, cultivar identification and genetic diversity studies we have used the SNPlex genotyping technology in a sample of grapevine genotypes and segregating progenies. Conclusion: These results provide accurate values for nucleotide diversity in coding sequences and a first estimate of short-range LD in grapevine. Using SNPlex genotyping we have shown the application of a set of discovered SNPs as molecular markers for cultivar identification, linkage mapping and genetic diversity studies. Thus, the combination a highly efficient re-sequencing approach and the SNPlex high throughput genotyping technology provide a powerful tool for grapevine genetic analysis. © 2007 Lijavetzky et al; licensee BioMed Central Ltd

Development and evaluation of a 9K SNP array for peach by internationally coordinated SNP detection and validation in breeding germplasm

Although a large number of single nucleotide polymorphism (SNP) markers covering the entire genome are needed to enable molecular breeding efforts such as genome wide association studies, fine mapping, genomic selection and marker-assisted selection in peach [Prunus persica (L.) Batsch] and related Prunus species, only a limited number of genetic markers, including simple sequence repeats (SSRs), have been available to date. To address this need, an international consortium (The International Peach SNP Consortium; IPSC) has pursued a coordinated effort to perform genome-scale SNP discovery in peach using next generation sequencing platforms to develop and characterize a high-throughput Illumina Infinium® SNP genotyping array platform. We performed whole genome re-sequencing of 56 peach breeding accessions using the Illumina and Roche/454 sequencing technologies. Polymorphism detection algorithms identified a total of 1,022,354 SNPs. Validation with the Illumina GoldenGate® assay was performed on a subset of the predicted SNPs, verifying ∼75% of genic (exonic and intronic) SNPs, whereas only about a third of intergenic SNPs were verified. Conservative filtering was applied to arrive at a set of 8,144 SNPs that were included on the IPSC peach SNP array v1, distributed over all eight peach chromosomes with an average spacing of 26.7 kb between SNPs. Use of this platform to screen a total of 709 accessions of peach in two separate evaluation panels identified a total of 6,869 (84.3%) polymorphic SNPs.The almost 7,000 SNPs verified as polymorphic through extensive empirical evaluation represent an excellent source of markers for future studies in genetic relatedness, genetic mapping, and dissecting the genetic architecture of complex agricultural traits. The IPSC peach SNP array v1 is commercially available and we expect that it will be used worldwide for genetic studies in peach and related stone fruit and nut species