Genome editing of a recalcitrant wine grape genotype by lipofectamine-mediated delivery of CRISPR/Cas9 ribonucleoproteins to protoplasts.

The main bottleneck in the application of biotechnological breeding methods to woody species is due to the in vitro regeneration recalcitrance shown by several genotypes. On the other side, woody species, especially grapevine (Vitis vinifera L.), use most of the pesticides and other expensive inputs in agriculture, making the development of efficient approaches of genetic improvement absolutely urgent. Genome editing is an extremely promising technique particularly for wine grape genotypes, as it allows to modify the desired gene in a single step, preserving all the quality traits selected and appreciated in elite varieties. A genome editing and regeneration protocol for the production of transgene-free grapevine plants, exploiting the lipofectamine-mediated direct delivery of CRISPR-Cas9 ribonucleoproteins (RNPs) to target the phytoene desaturase gene, is reported. We focused on Nebbiolo (V. vinifera), an extremely in vitro recalcitrant wine genotype used to produce outstanding wines, such as Barolo and Barbaresco. The use of the PEG-mediated editing method available in literature and employed for highly embryogenic grapevine genotypes did not allow the proper embryo development in the recalcitrant Nebbiolo. Lipofectamines, on the contrary, did not have a negative impact on protoplast viability and plant regeneration, leading to the obtainment of fully developed edited plants after about 5 months from the transfection. Our work represents one of the first examples of lipofectamine use for delivering editing reagents in plant protoplasts. The important result achieved for the wine grape genotype breeding could be extended to other important wine grape varieties and recalcitrant woody species.

TaqMan® and HRM approaches for SNP genotyping in genetic traceability of musts and wines.

The fight against fraud in the wine sector requires continuous improvements and validations of new technologies applicable to musts and wines. Starting from published data from the Vitis18kSNP array, a series of new specific single nucleotide polymorphism (SNP) markers have been identified for some important north-western Italian cultivars, such as Barbera, Dolcetto and Arneis (Vitis vinifera L.), used in the production of high-quality wines under Protected Denomination of Origin. A pair of new SNP markers for each grape variety were selected and validated using two real-time PCR techniques: TaqMan® genotyping assays and high-resolution melting analysis (HRM). The TaqMan® assay has proven to be more reliable and repeatable than HRM analysis because despite being an economical and versatile technique for the detection of different types of genomic mutations (SNPs, insertions or deletions), HRM has shown limitations in the presence of poor-quality DNA extracted from musts and wines. TaqMan® assays have successfully identified Barbera, Dolcetto and Arneis in their respective musts and experimental wines, and with good efficiency in commercial wines. Marked differences between genotypes were observed, varietal identification in Dolcetto-based musts/wines was more efficient than that in Arneis-based wines. Therefore, the TaqMan® assay has considerable potential for varietal identification in wines and the procedure described in the present work can be easily adapted to all wines with adequate setup of DNA extraction methods that should be adapted to different wines.

Grapevine virome and production of healthy plants by somatic embryogenesis.

Grapevine (Vitis spp.) is a widespread fruit tree hosting many viral entities that interact with the plant modifying its responses to the environment. The production of virus-free plants is becoming increasingly crucial for the use of grapevine as a model species in different studies. Using high-throughput RNA sequencing, the viromes of seven mother plants grown in a germplasm collection vineyard were sequenced. In addition to the viruses and viroids already detected in grapevine, we identified 13 putative new mycoviruses. The different spread among grapevine tissues collected in vineyard, greenhouse and in vitro conditions suggested a clear distinction between viruses/viroids and mycoviruses that can successfully be exploited for their identification. Mycoviruses were absent in in vitro cultures, while plant viruses and viroids were particularly accumulated in these plantlets. Somatic embryogenesis applied to the seven mother plants was effective in the elimination of the complete virome, including mycoviruses. However, different sanitization efficiencies for viroids and grapevine pinot gris virus were observed among genotypes. The absence of mycoviruses in in vitro plantlets, associated with the absence of all viral entities in somaclones, suggested that this regeneration technique is also effective to eradicate endophytic/epiphytic fungi, resulting in gnotobiotic or pseudo-gnotobiotic plants.

Impact of oenological processing aids and additives on the genetic traceability of 'Nebbiolo' wine produced with withered grapes.

'Nebbiolo' is a well-known grapevine variety used to produce prestigious monovarietal Italian red wines. Genetic traceability is an important tool used to protect the authenticity of high-quality wines. SNP-based assays are an effective method to reach this aim in wines, but several issues have been reported for the authentication of commercial wines. In this study, the impact of the most common commercial additives and processing aids used in winemaking was analysed in 'Nebbiolo' wine using SNP-based traceability. Gelatine and bentonite had the strongest impact on the turbidity, colour and phenolic composition of wines and on residual grapevine DNA. The DNA reduction associated with the use of bentonite and gelatine (>99% compared to the untreated control) caused issues in the SNP-based assay, especially when the DNA concentration was below 0.5 pg/mL of wine. This study contributed to explaining the causes of the reduced varietal identification efficiency in commercial wines.

Stress responses and epigenomic instability mark the loss of somatic embryogenesis competence in grapevine.

Somatic embryogenesis (SE) represents the most appropriate tool for next-generation breeding methods in woody plants such as grapevine (Vitis vinifera L.). However, in this species, the SE competence is strongly genotype-dependent and the molecular basis of this phenomenon is poorly understood. We explored the genetic and epigenetic basis of SE in grapevine by profiling the transcriptome, epigenome, and small RNAome of undifferentiated, embryogenic, and non-embryogenic callus tissues derived from two genotypes differing in competence for SE, Sangiovese and Cabernet Sauvignon. During the successful formation of embryonic callus, we observed the upregulation of epigenetic-related transcripts and short interfering RNAs in association with DNA hypermethylation at transposable elements in both varieties. Nevertheless, the switch to nonembryonic development matched the incomplete reinforcement of transposon silencing, and the evidence of such effect was more apparent in the recalcitrant Cabernet Sauvignon. Transcriptomic differences between the two genotypes were maximized already at early stage of culture where the recalcitrant variety expressed a broad panel of genes related to stress responses and secondary metabolism. Our data provide a different angle on the SE molecular dynamics that can be exploited to leverage SE as a biotechnological tool for fruit crop breeding.

Secondary Metabolism and Defense Responses Are Differently Regulated in Two Grapevine Cultivars during Ripening.

Vitis vinifera 'Nebbiolo' is one of the most important wine grape cultivars used to produce prestigious high-quality wines known throughout the world, such as Barolo and Barbaresco. 'Nebbiolo' is a distinctive genotype characterized by medium/high vigor, long vegetative and ripening cycles, and limited berry skin color rich in 3'-hydroxylated anthocyanins. To investigate the molecular basis of these characteristics, 'Nebbiolo' berries collected at three different stages of ripening (berry pea size, véraison, and harvest) were compared with V. vinifera 'Barbera' berries, which are rich in 3',5'-hydroxylated anthocyanins, using transcriptomic and analytical approaches. In two consecutive seasons, the two genotypes confirmed their characteristic anthocyanin profiles associated with a different modulation of their transcriptomes during ripening. Secondary metabolism and response to stress were the functional categories that most differentially changed between 'Nebbiolo' and 'Barbera'. The profile rich in 3'-hydroxylated anthocyanins of 'Nebbiolo' was likely linked to a transcriptional downregulation of key genes of anthocyanin biosynthesis. In addition, at berry pea size, the defense metabolism was more active in 'Nebbiolo' than 'Barbera' in absence of biotic attacks. Accordingly, several pathogenesis-related proteins, WRKY transcription factors, and stilbene synthase genes were overexpressed in 'Nebbiolo', suggesting an interesting specific regulation of defense pathways in this genotype that deserves to be further explored.

Somatic embryogenesis is an effective strategy for dissecting chimerism phenomena in Vitis vinifera cv Nebbiolo.

KEY MESSAGE: The tendency of somatic embryogenesis to regenerate plants only from the L1 layer, associated with the spread of chimerism in grapevine, must be carefully considered in the framework of biotechnological improvement programmes. Grapevine is an important fruit crop with a high economic value linked to traditional genotypes that have been multiplied for centuries by vegetative propagation. In this way, somatic variations that can spontaneously occur within the shoot apical meristem are fixed in the whole plant and represent a source of intra-varietal variability. Previously identified inconsistencies in the allelic calls of single nucleotide variants (SNVs) suggested that the Vitis vinifera 'Nebbiolo' CVT185 clone is a potential periclinal chimera. We adopted the somatic embryogenesis technique to separate the two genotypes putatively associated with the L1 and L2 layers of CVT185 into different somaclones. Despite the recalcitrance of 'Nebbiolo' to the embryogenic process, 58 somaclones were regenerated and SNV genotyping assays attested that the genotype of all them differed from that of the mother plant and was only attributable to L1. The results confirmed that L2 has low or no competence for differentiating somatic embryos. After one year in the greenhouse, the somaclones showed no phenotypic alterations in comparison with the mother plant; however further analyses are needed to identify potential endogenous sources of variation. The tendency of somatic embryogenesis to regenerate plants only from L1 must be carefully considered in the framework of biotechnological improvement programmes in this species.

Biological and molecular interplay between two viruses and powdery and downy mildews in two grapevine cultivars.

Grapevine may be affected simultaneously by several pathogens whose complex interplay is largely unknown. We studied the effects of infection by two grapevine viruses on powdery mildew and downy mildew development and the molecular modifications induced in grapevines by their multiple interactions. Grapevine fanleaf virus (GFLV) and grapevine rupestris stem pitting-associated virus (GRSPaV) were transmitted by in vitro-grafting to Vitis vinifera cv Nebbiolo and Chardonnay virus-free plantlets regenerated by somatic embryogenesis. Grapevines were then artificially inoculated in the greenhouse with either Plasmopara viticola or Erysiphe necator spores. GFLV-infected plants showed a reduction in severity of the diseases caused by powdery and downy mildews in comparison to virus-free plants. GFLV induced the overexpression of stilbene synthase genes, pathogenesis-related proteins, and influenced the genes involved in carbohydrate metabolism in grapevine. These transcriptional changes suggest improved innate plant immunity, which makes the GFLV-infected grapevines less susceptible to other biotic attacks. This, however, cannot be extrapolated to GRSPaV as it was unable to promote protection against the fungal/oomycete pathogens. In these multiple interactions, the grapevine genotype seemed to have a crucial role: in 'Nebbiolo', the virus-induced molecular changes were different from those observed in 'Chardonnay', suggesting that different metabolic pathways may be involved in protection against fungal/oomycete pathogens. These results indicate that complex interactions do exist between grapevine and its different pathogens and represent the first study on a topic that still is largely unexplored.

DNA-based genealogy reconstruction of Nebbiolo, Barbera and other ancient grapevine cultivars from northwestern Italy.

Northwestern Italy is a wine region of the world with the highest of reputations, where top quality wines of remarkable economic value are produced from traditional, long-cultivated varieties. Kinship analyses were performed using 32 microsatellite loci and more than 10 K single-nucleotide polymorphism markers on 227 traditional grapes mostly from Northwestern Italy-including those that have been neglected or are threatened. This was done to better understand the genetic grapevine origins and history of this reputable wine producing area, thus enhancing its cultural value and the marketing appeal of its wines. The work revealed a complex network of genetic relationships among varieties, with little contribution of genotypes from other areas. It revealed the major role played by a few ancient grape varieties as parents of numerous offspring, including some that are endangered today. The ancestry of many cultivars is proposed. Among these are Dolcetto, Barbera and Riesling italico. Through the inference of parent-offspring and sibling relations, marker profiles of ungenotyped putative parents were reconstructed, suggesting kinship relations and a possible parentage for Nebbiolo, one of the most ancient wine grapes worldwide. Historic and geographic implications from the resulting kinships are discussed.

Single-nucleotide polymorphism (SNP) genotyping assays for the varietal authentication of 'Nebbiolo' musts and wines.

'Nebbiolo' (Vitis vinifera L.) is renowned for its use in producing monovarietal high-quality red wines, such Barolo and Barbaresco. The fight against fraud to safeguard high-quality productions requires an effective varietal identification system applicable in musts and wines. 'Nebbiolo'-specific single-nucleotide polymorphisms (SNPs) were identified starting from available databases and 260 genotypes analysed by Vitis18kSNP array. Two SNPs were sufficient to identify 'Nebbiolo' from 1157 genotypes. The SNP TaqMan® genotyping assays developed in this work successfully identified 'Nebbiolo' in all musts and wines collected at different experimental wine-making steps. The high sensitivity of the assays allowed identification of must mixtures at 1% and wine mixtures at 10-20% with non-'Nebbiolo' genotypes. In commercial wines, the amplification efficiency was limited by the low amount of grapevine DNA and the presence of PCR inhibitors. The TaqMan® genotyping assay is a rapid, highly sensitive and specific methodology with remarkable potential for varietal identification in wines.

Distinct Metabolic Signals Underlie Clone by Environment Interplay in "Nebbiolo" Grapes Over Ripening.

Several research studies were focused to understand how grapevine cultivars respond to environment; nevertheless, the biological mechanisms tuning this phenomenon need to be further deepened. Particularly, the molecular processes underlying the interplay between clones of the same cultivar and environment were poorly investigated. To address this issue, we analyzed the transcriptome of berries from three "Nebbiolo" clones grown in different vineyards, during two ripening seasons. RNA-sequencing data were implemented with analyses of candidate genes, secondary metabolites, and agronomical parameters. This multidisciplinary approach helped to dissect the complexity of clone × environment interactions, by identifying the molecular responses controlled by genotype, vineyard, phenological phase, or a combination of these factors. Transcripts associated to sugar signalling, anthocyanin biosynthesis, and transport were differently modulated among clones, according to changes in berry agronomical features. Conversely, genes involved in defense response, such as stilbene synthase genes, were significantly affected by vineyard, consistently with stilbenoid accumulation. Thus, besides at the cultivar level, clone-specific molecular responses also contribute to shape the agronomic features of grapes in different environments. This reveals a further level of complexity in the regulation of genotype × environment interactions that has to be considered for orienting viticultural practices aimed at enhancing the quality of grape productions.

miRVIT: A Novel miRNA Database and Its Application to Uncover Vitis Responses to Flavescence dorée Infection.

Micro(mi)RNAs play crucial roles in plant developmental processes and in defense responses to biotic and abiotic stresses. In the last years, many works on small RNAs in grapevine (Vitis spp.) were published, and several conserved and putative novel grapevine-specific miRNAs were identified. In order to reorganize the high quantity of available data, we produced "miRVIT," the first database of all novel grapevine miRNA candidates characterized so far, and still not deposited in miRBase. To this aim, each miRNA accession was renamed, repositioned in the last version of the grapevine genome, and compared with all the novel and conserved miRNAs detected in grapevine. Conserved and novel miRNAs cataloged in miRVIT were then used for analyzing Vitis vinifera plants infected by Flavescence dorée (FD), one of the most severe phytoplasma diseases affecting grapevine. The analysis of small RNAs from healthy, recovered (plants showing spontaneous and stable remission of symptoms), and FD-infected "Barbera" grapevines showed that FD altered the expression profiles of several miRNAs, including those involved in cell development and photosynthesis, jasmonate signaling, and disease resistance response. The application of miRVIT in a biological context confirmed the effectiveness of the followed approach, especially for the identification of novel miRNA candidates in grapevine. miRVIT database is available at http://mirvit.ipsp.cnr.it. Highlights: The application of the newly produced database of grapevine novel miRNAs to the analysis of plants infected by Flavescence dorée reveals key roles of miRNAs in photosynthesis and jasmonate signaling.

Dissecting interplays between Vitis vinifera L. and grapevine virus B (GVB) under field conditions.

Plant virus infections are often difficult to characterize as they result from a complex molecular and physiological interplay between a pathogen and its host. In this study, the impact of the phloem-limited grapevine virus B (GVB) on the Vitis vinifera L. wine-red cultivar Albarossa was analysed under field conditions. Trials were carried out over two growing seasons by combining agronomic, molecular, biochemical and ecophysiological approaches. The data showed that GVB did not induce macroscopic symptoms on 'Albarossa', but affected the ecophysiological performances of vines in terms of assimilation rates, particularly at the end of the season, without compromising yield and vigour. In GVB-infected plants, the accumulation of soluble carbohydrates in the leaves and transcriptional changes in sugar- and photosynthetic-related genes seemed to trigger defence responses similar to those observed in plants infected by phytoplasmas, although to a lesser extent. In addition, GVB activated berry secondary metabolism. In particular, total anthocyanins and their acetylated forms accumulated at higher levels in GVB-infected than in GVB-free berries, consistent with the expression profiles of the related biosynthetic genes. These results contribute to improve our understanding of the multifaceted grapevine-virus interaction.

Whole-genome sequencing and SNV genotyping of 'Nebbiolo' (Vitis vinifera L.) clones.

'Nebbiolo' (Vitis vinifera) is among the most ancient and prestigious wine grape varieties characterised by a wide genetic variability exhibited by a high number of clones (vegetatively propagated lines of selected mother plants). However, limited information is available for this cultivar at the molecular and genomic levels. The whole-genomes of three 'Nebbiolo' clones (CVT 71, CVT 185 and CVT 423) were re-sequenced and a de novo transcriptome assembly was produced. Important remarks about the genetic peculiarities of 'Nebbiolo' and its intra-varietal variability useful for clonal identification were reported. In particular, several varietal transcripts identified for the first time in 'Nebbiolo' were disease resistance genes and single-nucleotide variants (SNVs) identified in 'Nebbiolo', but not in other cultivars, were associated with genes involved in the stress response. Ten newly discovered SNVs were successfully employed to identify some periclinal chimeras and to classify 98 'Nebbiolo' clones in seven main genotypes, which resulted to be linked to the geographical origin of accessions. In addition, for the first time it was possible to discriminate some 'Nebbiolo' clones from the others.

Ozone Improves the Aromatic Fingerprint of White Grapes.

Ozone, a powerful oxidative stressor, has been recently used in wine industry as sanitizing agent to reduce spoilage microflora on grapes. In this study, we evaluated ozone-induced metabolic and molecular responses during postharvest grape dehydration. Ozone increased the contents of total volatile organic compounds (VOCs), which have a great impact on the organoleptic properties of grapes and wines. Among terpenes, responsible for floral and fruity aroma, linalool, geraniol and nerol were the major aromatic markers of Moscato bianco grapes. They were significantly affected by the long-term ozone treatment, increasing their concentration in the last phases of dehydration (>20% weight loss). At molecular level, our results demonstrated that both postharvest dehydration and ozone exposure induce the biosynthesis of monoterpenes via methylerythritol phosphate (MEP) pathway and of aldehydes from lipoxygenase-hydroperoxide lyase (LOX-HPL) pathway. Therefore, transcriptional changes occurred and promoted the over-production of many important volatile compounds for the quality of white grapes.

Grapevine Grafting: Scion Transcript Profiling and Defense-Related Metabolites Induced by Rootstocks.

Rootstocks are among the main factors that influence grape development as well as fruit and wine composition. In this work, rootstock/scion interactions were studied using transcriptomic and metabolic approaches on leaves of the "Gaglioppo" variety, grafted onto 13 different rootstocks growing in the same vineyard. The whole leaf transcriptome of "Gaglioppo" grafted onto five selected rootstocks showed high variability in gene expression. In particular, significant modulation of transcripts linked to primary and secondary metabolism was observed. Interestingly, genes and metabolites involved in defense responses (e.g., stilbenes and defense genes) were strongly activated particularly in the GAG-41B combination, characterized in addition by the down-regulation of abscisic acid (ABA) metabolism. On the contrary, the leaves of "Gaglioppo" grafted onto 1103 Paulsen showed an opposite regulations of those transcripts and metabolites, together with the greater sensitivity to downy mildew in a preliminary in vitro assay. This study carried out an extensive transcriptomic analysis of rootstock effects on scion leaves, helping to unravel this complex interaction, and suggesting an interesting correlation among constitutive stilbenes, ABA compound, and disease susceptibility to a fungal pathogen.

Cultivar-specific gene modulation in Vitis vinifera: analysis of the promoters regulating the expression of WOX transcription factors.

The family of Wuschel-related Homeobox (WOX) genes is a class of transcription factors involved in the early stages of embryogenesis and organ development in plants. Some of these genes have shown different transcription levels in embryogenic tissues and mature organs in two different cultivars of Vitis vinifera: 'Chardonnay' (CH) and 'Cabernet Sauvignon' (CS). Therefore, we investigated the genetic basis responsible for these differences by cloning and sequencing in both the cultivars the promoter regions (~2000 bp) proximal to the transcription start site of five VvWOX genes. We then introduced these promoters into Arabidopsis thaliana for expression pattern characterisation using the GUS reporter gene. In the transgenic Arabidopsis, two promoters isolated from CS (pVvWOX13C_CS and pVvWOX6_CS) induced increased expression compared to the sequence isolated in CH, confirming the data obtained in grapevine tissues. These results were corroborated by transient expression assays using the agroinfiltration approach in grapevine somatic embryos. Truncated versions of pVvWOX13C demonstrated that few nucleotide differences between the sequences isolated from CH and CS are pivotal for the transcriptional regulation of VvWOX13C. Analysis of promoters using heterologous and homologous systems appear to be effective for exploring gene modulation linked with intervarietal sequence variation in grapevine.

Novel functional microRNAs from virus-free and infected Vitis vinifera plants under water stress.

MicroRNAs (miRNAs) are small non-coding RNAs that regulate the post-transcriptional control of several pathway intermediates, thus playing pivotal roles in plant growth, development and response to biotic and abiotic stresses. In recent years, the grapevine genome release, small(s)-RNAseq and degradome-RNAseq together has allowed the discovery and characterisation of many miRNA species, thus rendering the discovery of additional miRNAs difficult and uncertain. Taking advantage of the miRNA responsiveness to stresses and the availability of virus-free Vitis vinifera plants and those infected only by a latent virus, we have analysed grapevines subjected to drought in greenhouse conditions. The sRNA-seq and other sequence-specific molecular analyses have allowed us to characterise conserved miRNA expression profiles in association with specific eco-physiological parameters. In addition, we here report 12 novel grapevine-specific miRNA candidates and describe their expression profile. We show that latent viral infection can influence the miRNA profiles of V. vinifera in response to drought. Moreover, study of eco-physiological parameters showed that photosynthetic rate, stomatal conductance and hydraulic resistance to water transport were significantly influenced by drought and viral infection. Although no unequivocal cause-effect explanation could be attributed to each miRNA target, their contribution to the drought response is discussed.

Hydrogen peroxide accumulation and transcriptional changes in grapevines recovered from flavescence dorée disease.

Flavescence dorée (FD) is considered one of the most severe phytoplasma diseases affecting grapevine. The spontaneous, complete, and stable remission of the symptoms of FD (recovery) is a phenomenon that may occur in infected grapevines. The molecular bases of this phenomenon are still unclear, although some works suggest that recovery could be linked to the accumulation of hydrogen peroxide (H2O2). Several genes coding for enzymes involved in H2O2 metabolism, in the ascorbate-glutathione cycle, defense responses, and the biosynthesis of hormones were identified. The H2O2 content was biochemically determined and the expression levels of 44 genes were analyzed through quantitative real-time reverse-transcription polymerase chain reaction in healthy (H), infected by FD-associated phytoplasma (I), and 2-years-recovered (R) plants of Vitis vinifera 'Barbera'. In tissues of R plants, large amounts of H2O2 were detected, essentially linked to an upregulation of genes involved in the production of H2O2 (germin-like protein and glycolate oxidase); whereas, in I grapevines, the overexpression of some scavenging genes reduced the quantity of H2O2. The recovery state was characterized by the activation of ethylene biosynthesis and of defense genes not linked to salicylic acid (SA) signaling, such as the WRKY2 transcription factor. Conversely, I plants reacted to phytoplasma with SA-mediated signaling, even though this response does not appear to be effective against the pathogen.