Project description:NACs (NAM/ATAF/CUC) proteins are plant-specific transcription factors with key roles in plant development and stress responses. Here, Vitis vinifera NAC61 was selected for its functional characterization as one of the key candidates of the grape post-harvest withering process. VviNAC61 is particularly expressed at veraison and post-veraison berry stages and also shows a high expression in lignified tissues such as roots, tendrils, seeds and rachis. In leaves, VviNAC61 achieves its highest expression at senescence stage. In line with this observation, a NAC family multispecies phylogenetic tree revealed that VviNAC61 clusters with VviNAC33 and AtANAC046, both involved in regulating senescence processes. VviNAC61 was transiently overexpressed in N. benthamiana leaves, showing wilting/water-soaking symptoms correlated to low stomatal conductance, high leaf temperature, increased loss of the membrane integrity and accumulation of hydrogen peroxide and water. Transcriptomic analysis of cultivar ‘Thompson Seedless’ leaves transiently overexpressing VviNAC61 and the search of TF-bound genes by DAP-seq was allowed us to narrow down a list of VviNAC61-regulated genes. The ‘secondary metabolism’, ‘transcription factor regulation’ and ‘oxidative stress’ functional categories were found to be the most enriched among the candidate target genes. More in detail, several genes encoding for the phenylpropanoid pathway-related enzymes, several MYBs and NAC genes (including VviNAC61), were bound in their promoter region by this TF and were among the upregulated genes. Direct regulation of the stilbene synthase regulator VviMYB14, the abiotic stress-related gene DHN1b, the Botrytis cinerea susceptibility gene VviWRKY52 and the VviNAC61 self-regulation were validated through dual luciferase assays. Lastly, the upstream regulation mechanism of VviNAC61 was further investigated demonstrating that VviNAC61 is controlled by and interacts with VviNAC60, a proposed master regulator of grapevine organ maturation.

Project description:Downy mildew, caused by the oomycete Plasmopara viticola, is a serious disease in Vitis vinifera, the most commonly cultivated grapevine species. Several wild Vitis species have instead shown resistance to this pathogen and they have been used as a source to introgress resistance into a V. vinifera background. Stilbenoids represent the major phytoalexins in grapevine, and their toxicity has been strictly related to the specific compound. The aim of this study was to assess the resistance response to P. viticola of the Merzling x Teroldego cross by profiling the stilbenoid content of the leaves of the entire population and the transcriptome of resistant and susceptible individuals following infection. A three-year analysis of the population?s response to artificial inoculation showed that individuals were distributed in nine classes ranging from total resistance to total susceptibility. In addition, quantitative metabolite profiling of stilbenoids in the population, carried out using HPLC-DAD-MS, identified three distinct groups differing according to the concentrations present and the complexity of their profiles. The high producers were characterized by the presence of trans-resveratrol, trans-piceid, trans-pterostilbene and up to thirteen different viniferins, nine of them new in grapevine. Accumulation of these compounds is consistent with a resistant phenotype and suggests that they may contribute to the resistance response. A preliminary transcriptional study using cDNA-AFLP selected a set of genes modulated by the oomycete in a resistant genotype. The expression of this set of genes in resistant and susceptible genotypes of the progeny population was then assessed by comparative microarray analysis. A group of 57 genes was found to be exclusively modulated in the resistant genotype suggesting that they are involved in the grapevine-P. viticola incompatible interaction. Functional annotation of these transcripts revealed that they belong to the categories defense response, photosynthesis, primary and secondary metabolism, signal transduction and transport.This set of genes should be taken into account in future breeding programs. Leaves from one resistant (F1 21/66) and two susceptible (F1 22/73 and Teroldego) genotypes were infected with Plasmopara viticola, and transcriptome changes were investigated at 12h and 96h after infection (treated samples) and at 0h after spraying with water (control samples) Two biological replicates were considered and each hybridization was performed three times. One color labeling was performed.

Project description:Downy mildew, caused by the oomycete Plasmopara viticola, is a serious disease in Vitis vinifera, the most commonly cultivated grapevine species. Several wild Vitis species have instead shown resistance to this pathogen and they have been used as a source to introgress resistance into a V. vinifera background. Stilbenoids represent the major phytoalexins in grapevine, and their toxicity has been strictly related to the specific compound. The aim of this study was to assess the resistance response to P. viticola of the Merzling x Teroldego cross by profiling the stilbenoid content of the leaves of the entire population and the transcriptome of resistant and susceptible individuals following infection. A three-year analysis of the population?s response to artificial inoculation showed that individuals were distributed in nine classes ranging from total resistance to total susceptibility. In addition, quantitative metabolite profiling of stilbenoids in the population, carried out using HPLC-DAD-MS, identified three distinct groups differing according to the concentrations present and the complexity of their profiles. The high producers were characterized by the presence of trans-resveratrol, trans-piceid, trans-pterostilbene and up to thirteen different viniferins, nine of them new in grapevine. Accumulation of these compounds is consistent with a resistant phenotype and suggests that they may contribute to the resistance response. A preliminary transcriptional study using cDNA-AFLP selected a set of genes modulated by the oomycete in a resistant genotype. The expression of this set of genes in resistant and susceptible genotypes of the progeny population was then assessed by comparative microarray analysis. A group of 57 genes was found to be exclusively modulated in the resistant genotype suggesting that they are involved in the grapevine-P. viticola incompatible interaction. Functional annotation of these transcripts revealed that they belong to the categories defense response, photosynthesis, primary and secondary metabolism, signal transduction and transport.This set of genes should be taken into account in future breeding programs.

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Direct regulation of shikimate, early phenylpropanoid and stilbenoid pathways by Subgroup 2 R2R3-MYBs in grapevine

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