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Downy mildew of grapevine (Vitis vinifera), caused by the oomycete Plasmopara viticola, is an important disease that is present in cultivation areas worldwide, and using resistant varieties provides an environmentally friendly alternative to fungicides. DOWNY MILDEW RESISTANT 6 (DMR6) from Arabidopsis is a negative regulator of plant immunity and its loss of function confers resistance to downy mildew. In grapevine, DMR6 is present in two copies, named VvDMR6-1 and VvDMR6-2. Here, we describe the editing of VvDMR6-1 in embryogenic calli using CRISPR/Cas9 and the regeneration of the edited plants. All edited plants were found to be biallelic and chimeric, and whilst they all showed reduced growth compared with non-transformed control plants, they also had reduced susceptibility to P. viticola. Comparison between mock-inoculated genotypes showed that all edited lines presented higher levels of salicylic acid than controls, and lines subjected to transformation presented higher levels of cis-resveratrol than controls. Our results identify VvDMR6-1 as a promising target for breeding grapevine cultivars with improved resistance to downy mildew. [ABSTRACT FROM AUTHOR]

Abstract Background Natural disease resistance is a cost-effective and environmentally friendly way of controlling plant disease. Breeding programmes need to make sure that the resistance deployed is effective and durable. Grapevine downy mildew, caused by the Oomycete Plasmopara viticola, affects viticulture and it is controlled with pesticides. Downy mildew resistant grapevine varieties are a promising strategy to control the disease, but their use is currently restricted to very limited acreages. The arising of resistance-breaking isolates under such restricted deployment of resistant varieties would provide valuable information to design breeding strategies for the deployment of resistance genes over large acreages whilst reducing the risks of the resistance being defeated. The observation of heavy downy mildew symptoms on a plant of the resistant variety Bianca, whose resistance is conferred by a major gene, provided us with a putative example of emergence of a resistance-breaking isolate in the interaction between grapevine and P. viticola. Results In this paper we describe the emergence of a P. viticola isolate (isolate SL) that specifically overcomes Rpv3, the major resistance gene carried by Bianca at chromosome 18. We show that isolate SL has the same behaviour as two P. viticola isolates avirulent on Bianca (isolates SC and SU) when inoculated on susceptible plants or on resistant plants carrying resistances derived from other sources, suggesting there is no fitness cost associated to the virulence. Molecular analysis shows that all three isolates are genetically closely related. Conclusions Our results are the first description of a resistance-breaking isolate in the grapevine/P. viticola interaction, and show that, despite the reduced genetic variability of P. viticola in Europe compared to its basin of origin and the restricted use of natural resistance in European viticulture, resistance-breaking isolates overcoming monogenic resistances may arise even in cases where deployment of the resistant varieties is limited to small acreages. Our findings represent a warning call for the use of resistant varieties and an incentive to design breeding programmes aiming to optimize durability of the resistances.

Abstract Background Grapevine is subjected to numerous pests and diseases resulting in the use of phytochemicals in large quantities. The will to decrease the use of phytochemicals leads to attempts to find alternative strategies, implying knowledge of defence mechanisms. Numerous studies have led to the identification of signalling pathways and regulatory elements involved in defence in various plant species. Nonexpressor of Pathogenesis Related 1 (NPR1) is an important regulatory component of systemic acquired resistance (SAR) in Arabidopsis thaliana. Results Two putative homologs of NPR1 gene were found in the two sequenced grapevine genomes available in the Genoscope database for line 40024 and in the IASMA database for Pinot noir ENTAV 115. We named these two NPR1 genes of Vitis vinifera : VvNPR1.1 and VvNPR1.2. A PCR-based strategy with primers designed on exons was used to successfully amplify NPR1 gene fragments from different Vitaceae accessions. Sequence analyses show that NPR1.1 and NPR1.2 are highly conserved among the different accessions not only V. vinifera cultivars but also other species. We report nucleotide polymorphisms in NPR1.1 and NPR1.2 from fifteen accessions belonging to the Vitaceae family. The ratio of nonsynonymous to synonymous nucleotide substitutions determines the evolutionary pressures acting on the Vitaceae NPR1 genes. These genes appear to be experiencing purifying selection. In some of the species we have analysed one of the two alleles of NPR1.1 contains a premature stop codon. The deduced amino acid sequences share structural features with known NPR1-like proteins: ankyrin repeats, BTB/POZ domains, nuclear localization signature and cysteines. Phylogenetic analyses of deduced amino acid sequences show that VvNPR1.1 belongs to a first group of NPR1 proteins known as positive regulators of SAR and VvNPR1.2 belongs to a second group of NPR1 proteins whose principal members are AtNPR3 and AtNPR4 defined as negative regulators of SAR. Conclusion Our study shows that NPR1.1 and NPR1.2 are highly conserved among different accessions in the Vitaceae family. VvNPR1.1 and VvNPR1.2 are phylogenetically closer to the group of positive or negative SAR regulators respectively. Reviewers This article was reviewed by Fyodor Kondrashov, Purificación López-García and George V. Shpakovski.

Geographical distribution and diversity of current plant species have been strongly shaped by climatic oscillations during the Quaternary. Analysing the resulting divergence among species and differentiation within species is crucial to understand the evolution of taxa like the Vitis genus, which provides very useful genetic resources for grapevine improvement and might reveal original recolonization patterns due to growth habit and dispersal mode. Here, we studied the genetic structure in natural populations of three species from eastern North America: Vitis aestivalis, V. cinerea and V. riparia using different marker types. Vitis aestivalis and V. cinerea showed higher diversity than V. riparia. The two former species are less differentiated, confirming an earlier divergence of V. riparia. V. aestivalis and V. riparia exhibited different genetic groups on both sides of the Appalachian Mountains that could mirror different recolonization routes from southern refugia. Genetic structure was stronger in V. cinerea, for which two varieties (var. berlandieri and var. cinerea) are morphologically recognized. Our results confirm this distinction and suggest the existence of three other lineages within var. cinerea. These discontinuities appear linked to adaptation of var. berlandieri to dry and limy areas of Texas and partially to the Mississippi River Valley. Rapid range expansions from refugia upon climate warming are also suggested by the low linkage disequilibrium values observed. Furthermore, large variation for downy mildew resistance was observed in the three species. Our findings appeared consistent with the vegetation history of eastern North America. [ABSTRACT FROM AUTHOR]

Abstract Background Grapevine can be a periclinal chimera plant which is composed at least of two distinct cell layers (L1, L2). When the cell layers of this plant are separated by passage through somatic embryogenesis, regenerated plants could show distinct DNA profiles and a novel phenotype which proved different from that of the parent plant. Results Genetically Chardonnay clone 96 is a periclinal chimera plant in which is L1 and L2 cell layers are distinct. Plants obtained via organogenesis through meristematic bulks are shown to be composed of both cell layers. However, plants regenerated through somatic embryogenesis starting from anthers or nodal explants are composed only of L1 cells. These somaclones do not show phenotypic differences to the parental clone up to three years after regeneration. Interestingly, the only somaclone showing an atypical phenotype (asymmetric leave) shows a genotypic modification. Conclusion These results suggest that the phenotype of Chardonnay 96 does not result from an interaction between the two distinct cell layers L1 and L2. If phenotype conformity is further confirmed, somatic embryogenesis will result in true-to-type somaclones of Chardonnay 96 and would be well suitable for gene transfer.

Inducible plant defences against pathogens are stimulated by infections and comprise several classes of pathogenesis-related (PR) proteins. Endo-β-1,3-glucanases (EGases) belong to the PR-2 class and their expression is induced by many pathogenic fungi and oomycetes, suggesting that EGases play a role in the hydrolysis of pathogen cell walls. However, reports of a direct effect of EGases on cell walls of plant pathogens are scarce. Here, we characterized three EGases from Vitis vinifera whose expression is induced during infection by Plasmopara viticola, the causal agent of downy mildew. Recombinant proteins were expressed in Escherichia coli. The enzymatic characteristics of these three enzymes were measured in vitro and in planta. A functional assay performed in vitro on germinated P. viticola spores revealed a strong anti-P. viticola activity for EGase3, which strikingly was that with the lowest in vitro catalytic efficiency. To our knowledge, this work shows, for the first time, the direct effect against downy mildew of EGases of the PR-2 family from Vitis. [ABSTRACT FROM AUTHOR]

Clonal polymorphism mainly results from somatic mutations that occur naturally during plant growth. In grapevine, arrays of clones have been selected within varieties as a valuable source of diversity, among them clones showing berry color polymorphism. To identify mutations responsible for this color polymorphism, we studied a collection of 33 clones of Pinot noir, Pinot gris, and Pinot blanc. Haplotypes of the L2 cell layer of nine clones were resolved by genotyping self-progenies with molecular markers along a 10.07 Mb region of chromosome 2, including the color locus. We demonstrated that at least six haplotypes could account for the loss of anthocyanin biosynthesis. Four of them resulted from the replacement of sections of the ‘colored’ haplotype, sized from 31 kb to 4.4 Mb, by the homologous sections of the ‘white’ haplotype mutated at the color locus. This transfer of information between the two homologous sequences resulted in the partial homozygosity of chromosome 2, associated in one case with a large deletion of 108 kb-long. Moreover, we showed that, in most cases, somatic mutations do not affect the whole plant; instead, they affect only one cell layer, leading to periclinal chimeras associating two genotypes. Analysis of bud sports of Pinot gris support the hypothesis that cell layer rearrangements in the chimera lead to the homogenization of the genotype in the whole plant. Our findings shed new light on the way molecular and cellular mechanisms shape the grapevine genotypes during vegetative propagation, and enable us to propose a scheme of evolutionary mechanism of the Pinot clones. [ABSTRACT FROM AUTHOR]

To investigate the in-situ response to a stress, grapevine leaves have been subjected to mass spectrometry imaging (MSI) experiments. The Matrix Assisted Laser Desorption/Ionisation (MALDI) approach using different matrices has been evaluated. Among all the tested matrices, the 2,5-dihydroxybenzoic acid (DHB) was found to be the most efficient matrix allowing a broader range of detected stilbene phytoalexins. Resveratrol, but also more toxic compounds against fungi such as pterostilbene and viniferins, were identified and mapped. Their spatial distributions on grapevine leaves irradiated by UV show their specific colocation around the veins. Moreover, MALDI MSI reveals that resveratrol (and piceids) and viniferins are not specifically located on the same area when leaves are infected by Plasmopara viticola. Results obtained by MALDI mass spectrometry imaging demonstrate that this technique would be essential to improve the level of knowledge concerning the role of the stilbene phytoalexins involved in a stress event. [ABSTRACT FROM AUTHOR]

Grapevine breeding requires renewed efforts to meet the challenge of the ongoing climate change and the societal demand for low input viticulture. A breeding program supported by Bordeaux wine professional committee (CIVB) aims at generating new cultivars that should meet these two objectives by improving traits such as disease resistance (downy and powdery mildew, black rot), while retaining the typicality of Bordeaux wines. As part of this program, two V. vinifera bi-parental mapping populations were generated by crossing, on the one hand, 'Petit Verdot' (PV) and 'Cabernet Franc' (CF) (n = 202) and, on the other hand, 'Ugni blanc' (UB) and 'Sauvignon blanc' (SB) (n = 214). These two mapping populations were investigated for quantitative trait loci (QTLs) detection of relevant segregating traits such as phenology or aromatic potential with GC-MS and LC-MS analyses. Genotyping by sequencing (GBS) by Illumina HiSeq 3000 of both mapping populations was performed on GET-plage platform (Toulouse, France). After bioinformatics treatments, the construction of the related genetic maps was achieved using JoinMap 4.1. High-density genetic maps, based on single nucleotide polymorphism (SNPs), allowed the detection of 25 QTLs related with phenology, berry quality, aroma composition and aroma precursors, as well as the identification of some relevant candidate genes. The QTLs identified typically accounted for 7-27% of the trait variation. The molecular markers identified in this project will be used for marker-assisted selection in current and future grapevine breeding programs. The project will provide the basis of the genetic architecture of useful traits for the production of red and white wines with emblematic Bordeaux typicality and will thus contribute to the design of new ideotypes. [ABSTRACT FROM AUTHOR]

Estimating growth traits of grapevine plants is common practice to describe the effects of cultural practices, training systems, environmental conditions, or genotypic effects, both for rootstocks and scions varieties. Among these traits, the simple pruning weight is one of the most popular, while being time-consuming when hundreds of genotypes are to be characterized. To face the challenge of adaptation to climate change and the global demand for grapevine varieties resistant to disease, there is an increasing need for high throughput phenotyping methods. We studied in the vineyard the variability of plant vigor in the progeny of the cross between two genotypes carrying resistance genes for powdery and downy mildew. The pruning weight was measured for more than 200 genotypes and, in parallel, we used a newly developed high throughput LiDAR acquisition system to characterize the plants both in summer and in winter. We will present the LiDAR system and the comparison between LiDAR data, pruning weights and leaf area. Using "genotyping by sequencing" technology to describe the genetic heritage of the offspring, we performed quantitative trait loci (QTL) detection and identifieds everal genomic regions associated with the genetic variability both for pruning weights; leaf area and LiDAR data. [ABSTRACT FROM AUTHOR]

A major aim in modern grapevine (Vitis vinifera L.) breeding programs is the introgression of resistance genes along with desired cultural and oenological traits. Understanding the genetic links between resistance genes and agro-oenological traits is an important issue for grapevine breeders. In this work, we studied the genetic determinism of yield components and berry quality in the progeny of a cross between two grapevine hybrids carrying each several known quantitative trait loci (QTL) of resistance to fungal diseases. Several yield descriptors like number of inflorescences per shoot, berry weight and berries sugar content were recorded during three successive seasons for 209 genotypes in the vineyard. High density parental and consensus genetic maps were built with 'Genotyping by sequencing' technology using 239 individuals. Many QTL were detected for all studied traits. We found co-localisations between resistance genes and two of the studied traits: berry weight and berries sugar content. Detailed will be presented. [ABSTRACT FROM AUTHOR]

Genomic prediction (GP) of breeding values has developed widely both in animal and plant breeding programs for its ability to accelerate genetic gains, saving cost and time while handling complex traits. However, its routine deployment in most of the major plant species is still not effective, mainly because of the many factors that are likely to influence prediction accuracies. In grapevine, very few studies have evaluated potential usefulness of GP in breeding contexts. Although the French grapevine breeding program for durable resistance to downy and powdery mildew (INRAE-ResDur) has now proved to be effective with about ten new varieties already officially registered, the duration of each breeding cycle remains long (about 15 years). GP can potentially help us to accelerate and diversify our breeding schemes by targeting complex traits involved in the determination of the optimal productivity and quality of the berries and wines. The main objective is to reduce as much as possible, the long intermediate selection phase of the breeding scheme where every genotype is evaluated in the yard during six years. Here we evaluated GP across four different breeding populations for main phenological and agronomic traits to identify main factor affecting accuracies. This study brings additional information to the grapevine community regarding the implementation of GP to accelerate breeding programs. [ABSTRACT FROM AUTHOR]

Grapevine fanleaf disease, caused by grapevine fanleaf virus (GFLV) and transmitted by the soilborne nematode Xiphinema index, is the most detrimental of the grapevine viral diseases. It provokes severe symptoms and economic losses, threatening vineyards worldwide. Many strategies of control based on prophylaxis, biotechnology, biocontrol have been explored. So far, no effective and environmentally friendly solution has been reached. Natural resistance to GFLV has equally been examined but, in the most comprehensive screening study, none of the tested accessions were found to be resistant. As recessive genetic determinisms have often been described for plant virus resistance, the challenges of finding a source of natural resistance to GFLV may be explained by the high rate of heterozygosity in grapevine. We investigated the presence of recessive resistance to GFLV in grapevine genetic resources through the screening of the progenies from self-fertilization of various varieties and species. We discovered that the Riesling variety displays resistance to GFLV, although it is susceptible to X. index. This resistance is determined by a single recessive factor located on grapevine chromosome 1, which we have named resistance to grapevine fanleaf virus 1 (rgflv1). rgflv1 is located in a 5.7 cM interval which represents a physical distance of ~1.1 Mb. Analysis of recombinant allowed us to narrow the interval to a region of 600 kb encompassing 57 genes. To our knowledge, this is the first and only instance of resistance to grapevine fanleaf virus identified in grapevine so far. This finding represents strong basement to go further in the identification and the characterization of the gene underlying the rgflv1 resistance locus. In terms of innovation, it paves the way for the design of new ideotypes of multi-resistant grape varieties that combine resistance to root and aerial major diseases. [ABSTRACT FROM AUTHOR]

Background: Natural disease resistance is a cost-effective and environmentally friendly way of controlling plant disease. Breeding programmes need to make sure that the resistance deployed is effective and durable. Grapevine downy mildew, caused by the Oomycete Plasmopara viticola, affects viticulture and it is controlled with pesticides. Downy mildew resistant grapevine varieties are a promising strategy to control the disease, but their use is currently restricted to very limited acreages. The arising of resistance-breaking isolates under such restricted deployment of resistant varieties would provide valuable information to design breeding strategies for the deployment of resistance genes over large acreages whilst reducing the risks of the resistance being defeated. The observation of heavy downy mildew symptoms on a plant of the resistant variety Bianca, whose resistance is conferred by a major gene, provided us with a putative example of emergence of a resistance-breaking isolate in the interaction between grapevine and P. viticola. Results: In this paper we describe the emergence of a P. viticola isolate (isolate SL) that specifically overcomes Rpv3, the major resistance gene carried by Bianca at chromosome 18. We show that isolate SL has the same behaviour as two P. viticola isolates avirulent on Bianca (isolates SC and SU) when inoculated on susceptible plants or on resistant plants carrying resistances derived from other sources, suggesting there is no fitness cost associated to the virulence. Molecular analysis shows that all three isolates are genetically closely related. Conclusions: Our results are the first description of a resistance-breaking isolate in the grapevine/P. viticola interaction, and show that, despite the reduced genetic variability of P. viticola in Europe compared to its basin of origin and the restricted use of natural resistance in European viticulture, resistance-breaking isolates overcoming monogenic resistances may arise even in cases where deployment of the resistant varieties is limited to small acreages. Our findings represent a warning call for the use of resistant varieties and an incentive to design breeding programmes aiming to optimize durability of the resistances. [ABSTRACT FROM AUTHOR]

Background: Grapevine is subjected to numerous pests and diseases resulting in the use of phytochemicals in large quantities. The will to decrease the use of phytochemicals leads to attempts to find alternative strategies, implying knowledge of defence mechanisms. Numerous studies have led to the identification of signalling pathways and regulatory elements involved in defence in various plant species. Nonexpressor of Pathogenesis Related 1 (NPR1) is an important regulatory component of systemic acquired resistance (SAR) in Arabidopsis thaliana. Results: Two putative homologs of NPR1 gene were found in the two sequenced grapevine genomes available in the Genoscope database for line 40024 and in the IASMA database for Pinot noir ENTAV 115. We named these two NPR1 genes of Vitis vinifera : VvNPR1.1 and VvNPR1.2. A PCR-based strategy with primers designed on exons was used to successfully amplify NPR1 gene fragments from different Vitaceae accessions. Sequence analyses show that NPR1.1 and NPR1.2 are highly conserved among the different accessions not only V. vinifera cultivars but also other species. We report nucleotide polymorphisms in NPR1.1 and NPR1.2 from fifteen accessions belonging to the Vitaceae family. The ratio of nonsynonymous to synonymous nucleotide substitutions determines the evolutionary pressures acting on the Vitaceae NPR1 genes. These genes appear to be experiencing purifying selection. In some of the species we have analysed one of the two alleles of NPR1.1 contains a premature stop codon. The deduced amino acid sequences share structural features with known NPR1-like proteins: ankyrin repeats, BTB/POZ domains, nuclear localization signature and cysteines. Phylogenetic analyses of deduced amino acid sequences show that VvNPR1.1 belongs to a first group of NPR1 proteins known as positive regulators of SAR and VvNPR1.2 belongs to a second group of NPR1 proteins whose principal members are AtNPR3 and AtNPR4 defined as negative regulators of SAR. Conclusion: Our study shows that NPR1.1 and NPR1.2 are highly conserved among different accessions in the Vitaceae family. VvNPR1.1 and VvNPR1.2 are phylogenetically closer to the group of positive or negative SAR regulators respectively. Reviewers: This article was reviewed by Fyodor Kondrashov, Purificación López-García and George V. Shpakovski. [ABSTRACT FROM AUTHOR]

Stilbenes are considered the most important phytoalexin group in grapevine ( Vitis vinifera) and they are known to contribute to the protection against various pathogens. The main stilbenes in grapevine are resveratrol and its derivatives and, among these, pterostilbene has recently attracted much attention due both to its antifungal and pharmacological properties. Indeed, pterostilbene is 5 to 10 times more fungitoxic than resveratrol in vitro and recent studies have shown that pterostilbene exhibits anticancer, hypolipidemic, and antidiabetic properties. A candidate gene approach was used to identify a grapevine resveratrol O-methyltransferase (ROMT) cDNA and the activity of the corresponding protein was characterized after expression in Escherichia coli. Transient coexpression of ROMT and grapevine stilbene synthase in tobacco (Nicotiana benthamiana) using the agroinfiltration technique resulted in the accumulation of pterostilbene in tobacco tissues. Taken together, these results showed that ROMT was able to catalyze the biosynthesis of pterostilbene from resveratrol both in vitro and in planta. ROMT gene expression in grapevine leaves was induced by different stresses, including downy mildew (Plasmapara viticola) infection, ultraviolet light, and AlCl3 treatment. [ABSTRACT FROM AUTHOR]