Genome-wide analysis of the grapevine stilbene synthase multigenic family: genomic organization and expression profiles upon biotic and abiotic stresses

Plant stilbenes are a small group of phenylpropanoids, which have been detected in at least 72 unrelated plant species and accumulate in response to biotic and abiotic stresses such as infection, wounding, UV-C exposure and treatment with chemicals. Stilbenes are formed via the phenylalanine/polymalonate-route, the last step of which is catalyzed by the enzyme stilbene synthase (STS), a type III polyketide synthase (PKS). Stilbene synthases are closely related to chalcone synthases (CHS), the key enzymes of the flavonoid pathway, as illustrated by the fact that both enzymes share the same substrates. To date, STSs have been cloned from peanut, pine, sorghum and grapevine, the only stilbene-producing fruiting-plant for which the entire genome has been sequenced. Apart from sorghum, STS genes appear to exist as a family of closely related genes in these other plant species. RESULTS: In this study a complete characterization of the STS multigenic family in grapevine has been performed, commencing with the identification, annotation and phylogenetic analysis of all members and integration of this information with a comprehensive set of gene expression analyses including healthy tissues at differential developmental stages and in leaves exposed to both biotic (downy mildew infection) and abiotic (wounding and UV-C exposure) stresses. At least thirty-three full length sequences encoding VvSTS genes were identified, which, based on predicted amino acid sequences, cluster in 3 principal groups designated A, B and C. The majority of VvSTS genes cluster in groups B and C and are located on chr16 whereas the few gene family members in group A are found on chr10. Microarray and mRNA-seq expression analyses revealed different patterns of transcript accumulation between the different groups of VvSTS family members and between VvSTSs and VvCHSs. Indeed, under certain conditions the transcriptional response of VvSTS and VvCHS genes appears to be diametrically opposed suggesting that flow of carbon between these two competing metabolic pathways is tightly regulated at the transcriptional level. CONCLUSIONS: This study represents an overview of the expression pattern of each member of the STS gene family in grapevine under both constitutive and stress-induced conditions. The results strongly indicate the existence of a transcriptional subfunctionalization amongst VvSTSs and provide the foundation for further functional investigations about the role and evolution of this large gene family. Moreover, it represents the first study to clearly show the differential regulation of VvCHS and VvSTS genes, suggesting the involvement of transcription factors (TFs) in both the activation and repression of these genes

Dicarboximide resistance in field isolates of Alternaria alternata is mediated by a mutation in a two-component histidine kinase gene

Isolates of Alternaria alternata collected from a field site which had previously been treated with the dicarboximide fungicide iprodione were found to demonstrate a high level of resistance to iprodione and the phenylpyrrole fungicide, fludioxonil in plate assays. In order to determine the genetic basis for this fungicide resistance a partial length clone of a two-component histidine kinase (HK) was isolated from genomic DNA of a fungicide-sensitive A. alternata isolate using degenerate primers by PCR. Analysis of the AaHK1 gene structure indicates the presence of six 90 amino acid repeat domains upstream of a kinase domain as found in the homologous HK genes from other fungal species. Comparison of nucleic acid sequences from the fungicide-sensitive and fungicide-resistant A. alternata isolates confirmed the presence of mutations leading to premature termination of the translated HK protein. The possible role of the two-component HK in the development of dicarboximide resistance in A. alternata is discussed

Dicarboximide resistance in field isolates of Alternaria alternata is mediated by a mutation in a two-component histidine kinase gene

Isolates of Alternaria alternata collected from a field site which had previously been treated with the dicarboximide fungicide iprodione were found to demonstrate a high level of resistance to iprodione and the phenylpyrrole fungicide, fludioxonil in plate assays. In order to determine the genetic basis for this fungicide resistance a partial length clone of a two-component histidine kinase (HK) was isolated from genomic DNA of a fungicide-sensitive A. alternata isolate using degenerate primers by PCR. Analysis of the AaHK1 gene structure indicates the presence of six 90 amino acid repeat domains upstream of a kinase domain as found in the homologous HK genes from other fungal species. Comparison of nucleic acid sequences from the fungicide-sensitive and fungicide-resistant A. alternata isolates confirmed the presence of mutations leading to premature termination of the translated HK protein. The possible role of the two-component HK in the development of dicarboximide resistance in A. alternata is discussed

Identification of an R2R3 MYB transcription factor involved in the regulation of the stilbene synthase pathway in grapevine

none5siStilbene synthases (STSs) are a class of enzymes belonging to the general CHS type III polyketide synthase family involved in the last step of the biosynthesis of stilbenes. These enzymes, and their main products resveratrol or pynosylvin are detectable in only a limited number of unrelated plant species, including grape, and accumulate in response to biotic and abiotic stresses. Despite numerous studies that have been performed on the accumulation, metabolism and biological properties of resveratrol, little is known about the transcriptional regulation of this pathway. Based on mRNA-sequencing data obtained from grapevine leaf discs treated with biotic and abiotic stresses we identified a candidate R2R3 MYB transcription factor that shows an expression pattern similar to that observed for STS genes and could to be involved in their regulation in grape. This R2R3 MYB factor was designated VvMYB14, based on homology with the AtMYB14 R2R3 MYB factor. Neither gene has previously been functionally characterized in either plant species. Analysis of VvMYB14 expression in grape leaf discs treated with biotic (downy mildew infection) and abiotic stresses (wounding and UV-C exposure) known to be involved in the transcriptional activation of VvSTS genes, showed a close correlation between the pattern and timing of expression of selected VvSTS genes and VvMYB14. Using a Dual Luciferase Reporter Assay System in transiently transformed grapevine cells, VvMYB14 was also demonstrated to increase stilbene synthase promoter activity. Confirmation of the role of VvMYB14 in the trans-activation of VvSTS genes in-planta is currently being examined using a transgenic grapevine hairy root system for testing the effect of both silencing and over expression of VvMYB14 on the response of VvSTS expression.noneVannozzi, A.; Boss, P.K.; Walker, A.R.; Lucchin, M.; Dry, I.B.Vannozzi, A.; Boss, P. K.; Walker, A. R.; Lucchin, M.; Dry, I. B

Molecular strategies to enhance the genetic resistance of grapevines to powdery mildew

The Eurasian winegrape Vitis vinifera has little or no genetic resistance to the major fungal pathogens, powdery mildew (Erysiphe necator) and downy mildew (Plasmopora viticola). These pathogens were first introduced into French vineyards from North America in the 1800s before spreading to all major grape producing regions of the world. As a result, grape production is highly dependent on the use of fungicides. With the increasing financial and environmental costs of chemical application and the emergence of fungicide-resistant strains, the introduction of natural genetic resistance against these fungal pathogens is a high priority for viticultural industries worldwide. We are utilising a number of different molecular approaches to increase our understanding of the basis of resistance to these important major fungal pathogens and to identify potential new sources of genetic resistance. This review will outline the progress and the potential of each of these different molecular strategies to the generation of fungal-resistant grapevine germplasm

Molecular strategies to enhance the genetic resistance of grapevines to powdery mildew

International audienceThe Eurasian winegrape Vitis vinifera has little or no genetic resistance to the major fungal pathogens, powdery mildew (Erysiphe necator) and downy mildew (Plasmopora viticola). These pathogens were first introduced into French vineyards from North America in the 1800s before spreading to all major grape producing regions of the world. As a result, grape production is highly dependent on the use of fungicides. With the increasing financial and environmental costs of chemical application and the emergence of fungicide-resistant strains, the introduction of natural genetic resistance against these fungal pathogens is a high priority for viticultural industries worldwide. We are utilising a number of different molecular approaches to increase our understanding of the basis of resistance to these important major fungal pathogens and to identify potential new sources of genetic resistance. This review will outline the progress and the potential of each of these different molecular strategies to the generation of fungal-resistant grapevine germplasm

Combinatorial Regulation of Stilbene Synthase Genes by WRKY and MYB Transcription Factors in Grapevine ( Vitis vinifera L.)

Stilbene synthase (STS) is the key enzyme leading to the biosynthesis of resveratrol. Recently we reported two R2R3-MYB transcription factor (TF) genes that regulate the stilbene biosynthetic pathway in grapevine: VviMYB14 and VviMYB15. These genes are strongly co-expressed with STS genes under a range of stress and developmental conditions, in agreement with the specific activation of STS promoters by these TFs. Genome-wide gene co-expression analysis using two separate transcriptome compendia based on microarray and RNA sequencing data revealed that WRKY TFs were the top TF family correlated with STS genes. On the basis of correlation frequency, four WRKY genes, namely VviWRKY03, VviWRKY24, VviWRKY43 and VviWRKY53, were further shortlisted and functionally validated. Expression analyses under both unstressed and stressed conditions, together with promoter–luciferase reporter assays, suggested different hierarchies for these TFs in the regulation of the stilbene biosynthetic pathway. In particular, VviWRKY24 seems to act as a singular effector in the activation of the VviSTS29 promoter, while VviWRKY03 acts through a combinatorial effect with VviMYB14, suggesting that these two regulators may interact at the protein level as previously reported in other species.This work was supported by the Cariparo Foundation [Cariparo Starting Grants] and the Department of Agronomy, Food, Natural Resources, Animals, and Environment (DAFNAE), University of Padova

Genomics: the foundation for vine improvement

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Defence responses of grapevine cultivars to powdery mildew: Ontogenic resistance versus genetic resistance

International audienceThe most sustainable management of resistant varieties involves understanding which defence mechanisms the plant uses according to its resistance genes and stage of development and how effective these mechanisms are. Three grapevine varieties were compared for their susceptibility versus resistance to powdery mildew: Vitis vinifera 'Cabernet Sauvignon' (CS) and two hybrids, 'Artaban' and 'Prior'. Four different leaf ages were collected from 2-year-old grafted grapevines planted in pot or in vineyard, and several variables were assessed for each leaf: (a) morphological and physiological indicators, (b) pathogenicity traits: spore germination, infection ef?ciency, mycelial growth and sporulation, and (c) gene expression using a high-throughput quantitative reverse transcription PCR method to inform about plant defence status and functioning of primary metabolism. A significant decrease in germination and sporulation with increasing leaf age were observed for the three varieties: Prior showed a susceptibility similar to that of CS, whereas Artaban was fully resistant with no sporulation. Genes highlighted as markers of the variety were involved in defence (VvPR3, VvPR4 overexpressed in Artaban, VvPR4bis, VvAlli2 in Prior) or in primary metabolism (repression of VvCitS in Artaban, VvCHI in Prior, overexpression of VvCAD2, VvGST3 in Prior). After inoculation, more defence genes, especially PR proteins, were overexpressed in the oldest leaves as potential components of ontogenic resistance. PR proteins accounted for 25% of total disease variation. Phenylpropanoid and ethylene pathways were leaf age-dependent. The statistical method used highlighted the contributions of the genotypic expression involved in genetic (cultivar) and ontogenic (leaf age) resistances and of the metabolic pathways to the disease