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.

Secondary metabolites in grapevine: crosstalk of transcriptional, metabolic and hormonal signals controlling stress defence responses in berries and vegetative organs.

Abiotic stresses, such as temperature, heat waves, water limitation, solar radiation and the increase in atmospheric CO2 concentration, significantly influence the accumulation of secondary metabolites in grapevine berries at different developmental stages, and in vegetative organs. Transcriptional reprogramming, miRNAs, epigenetic marks and hormonal crosstalk regulate the secondary metabolism of berries, mainly the accumulation of phenylpropanoids and of volatile organic compounds (VOCs). Currently, the biological mechanisms that control the plastic response of grapevine cultivars to environmental stress or that occur during berry ripening have been extensively studied in many worlds viticultural areas, in different cultivars and in vines grown under various agronomic managements. A novel frontier in the study of these mechanisms is the involvement of miRNAs whose target transcripts encode enzymes of the flavonoid biosynthetic pathway. Some miRNA-mediated regulatory cascades, post-transcriptionally control key MYB transcription factors, showing, for example, a role in influencing the anthocyanin accumulation in response to UV-B light during berry ripening. DNA methylation profiles partially affect the berry transcriptome plasticity of different grapevine cultivars, contributing to the modulation of berry qualitative traits. Numerous hormones (such as abscisic and jasmomic acids, strigolactones, gibberellins, auxins, cytokynins and ethylene) are involved in triggering the vine response to abiotic and biotic stress factors. Through specific signaling cascades, hormones mediate the accumulation of antioxidants that contribute to the quality of the berry and that intervene in the grapevine defense processes, highlighting that the grapevine response to stressors can be similar in different grapevine organs. The expression of genes responsible for hormone biosynthesis is largely modulated by stress conditions, thus resulting in the numeourous interactions between grapevine and the surrounding environment.

Effects of fungicide application on physiological and molecular responses of grapevine (Vitis vinifera L.): a comparison between copper and sulfur fungicides applied alone and in combination with novel fungicides.

BACKGROUND: Chemical products against fungi and oomycetes pose serious environmental issues. In the last decade, the use of less impacting active ingredients was encouraged to reduce chemical inputs in viticulture. In this study, the effect of different antifungal compounds on grapevine agronomic, physiological, and molecular responses in the vineyard was evaluated in addition to protection against powdery and downy mildews. RESULTS: In 2 years and in two Vitis vinifera cultivars (Nebbiolo and Arneis), a conventional crop protection approach, based on traditional fungicides (sulfur and copper), was compared to combined strategies. A well-known resistance inducer (potassium phosphonate), Bacillus pumilus strain QST 2808 and calcium oxide, both active ingredients whose biological interaction with grapevine is poorly characterized, were applied in the combined strategies in association with chemical fungicides. Despite a genotype effect occurred, all treatments optimally controlled powdery and downy mildews, with minimal variations in physiological and molecular responses. Gas exchange, chlorophyll content and photosystem II efficiency increased in treated plants at the end of season, along with a slight improvement in the agronomic performances, and an activation of molecular defense processes linked to stilbene and jasmonate pathways. CONCLUSION: The disease control strategies based on potassium phosphonate, Bacillus pumilus strain QST 2808 or calcium oxide combined with traditional chemical compounds did not cause severe limitations in plant ecophysiology, grape quality, and productive yields. The combination of potassium phosphonate and calcium oxide with traditional fungicides can represent a valuable strategy for reducing copper and sulfur inputs in the vineyards, including those organically managed. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Tomato Yellow Leaf Curl Sardinia Virus Increases Drought Tolerance of Tomato.

Drought stress is one of the major physiological stress factors that adversely affect agricultural production, altering critical features of plant growth and metabolism. Plants can be subjected simultaneously to abiotic and biotic stresses, such as drought and viral infections. Rewarding effects provided by viruses on the ability of host plants to endure abiotic stresses have been reported. Recently, begomoviruses causing the tomato yellow leaf curl disease in tomatoes were shown to increase heat and drought tolerance. However, biological bases underlying the induced drought tolerance need further elucidation, particularly in the case of tomato plants. In this work, tomato plants infected by the tomato yellow leaf curl Sardinia virus (TYLCSV) were subjected to severe drought stress, followed by recovery. Morphological traits, water potential, and hormone contents were measured in leaves together with molecular analysis of stress-responsive and hormone metabolism-related genes. Wilting symptoms appeared three days later in TYLCSV-infected plants compared to healthy controls and post-rehydration recovery was faster (2 vs. 4 days, respectively). Our study contributes new insights into the impact of viruses on the plant's adaptability to environmental stresses. On a broader perspective, such information could have important practical implications for managing the effects of climate change on agroecosystems.

The C4 protein of tomato yellow leaf curl Sardinia virus primes drought tolerance in tomato through morphological adjustments.

Viruses can interfere with the ability of plants to overcome abiotic stresses, indicating the existence of common molecular networks that regulate stress responses. A begomovirus causing the tomato yellow leaf curl disease was recently shown to enhance heat tolerance in tomato and drought tolerance in tomato and Nicotiana benthamiana and experimental evidence suggested that the virus-encoded protein C4 is the main trigger of drought responses. However, the physiological and molecular events underlying C4-induced drought tolerance need further elucidation. In this study, transgenic tomato plants expressing the tomato yellow leaf curl Sardinia virus (TYLCSV) C4 protein were subjected to severe drought stress, followed by recovery. Morphometric parameters, water potential, gas exchanges, and hormone contents in leaves were measured, in combination with molecular analysis of candidate genes involved in stress response and hormone metabolism. Collected data proved that the expression of TYLCSV C4 positively affected the ability of transgenic plants to tolerate water stress, by delaying the onset of stress-related features, improving the plant water use efficiency and facilitating a rapid post-rehydration recovery. In addition, we demonstrated that specific anatomical and hydraulic traits, rather than biochemical signals, are the keynote of the C4-associated stress resilience. Our results provide novel insights into the biology underpinning drought tolerance in TYLCSV C4-expressing tomato plants, paving the way for further deepening the mechanism through which such proteins tune the plant-virus interaction.

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.

Spray-induced gene silencing targeting a glutathione S-transferase gene improves resilience to drought in grapevine.

Along with the ongoing climate change, drought events are predicted to become more severe. In this context, the spray-induced gene silencing (SIGS) technique could represent a useful strategy to improve crop stress resilience. A previous study demonstrated that the Arabidopsis mutants for a glutathione S-transferase (GST) gene had increased abscisic acid (ABA) levels and a more activated antioxidant system, both features that improved drought resilience. Here, we used SIGS to target a putative grape GST gene (VvGST40). Then, ecophysiological, biochemical and molecular responses of 'Chardonnay' cuttings were analysed during a drought and recovery time-course. Gas exchange, ABA and t-resveratrol concentration as well as expression of stress-related genes were monitored in not treated controls, dsRNA-VvGST40- and dsRNA-GFP- (negative control of the technique) treated plants, either submitted or not to drought. VvGST40-treated plants revealed increased resilience to severe drought as attested by the ecophysiological data. Analysis of target metabolites and antioxidant- and ABA-related transcripts confirmed that VvGST40-treated plants were in a priming status compared with controls. SIGS targeting an endogenous gene was successfully applied in grapevine, confirming the ability of this technique to be exploited not only for plant protection issues but also for functional genomic studies.

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.

Chemical inhibition of xylem cellular activity impedes the removal of drought-induced embolisms in poplar stems - new insights from micro-CT analysis.

In drought-stressed plants a coordinated cascade of chemical and transcriptional adjustments occurs at the same time as embolism formation. While these processes do not affect embolism formation during stress, they may prime stems for recovery during rehydration by modifying apoplast pH and increasing sugar concentration in the xylem sap. Here we show that in vivo treatments modifying apoplastic pH (stem infiltration with a pH buffer) or reducing stem metabolic activity (infiltration with sodium vanadate and sodium cyanide; plant exposure to carbon monoxide) can reduce sugar accumulation, thus disrupting or delaying the recovery process. Application of the vanadate treatment (NaVO3, an inhibitor of many ATPases) completely halted recovery from drought-induced embolism for up to 24 h after re-irrigation, while partial recovery was observed in vivo in control plants using X-ray microcomputed tomography. Our results suggest that stem hydraulic recovery in poplar is a biological, energy-dependent process that coincides with accumulation of sugars in the apoplast during stress. Recovery and damage are spatially coordinated, with embolism formation occurring from the inside out and refilling from the outside in. The outside-in pattern highlights the importance of xylem proximity to the sugars within the phloem to the embolism recovery process.

The Molecular Priming of Defense Responses is Differently Regulated in Grapevine Genotypes Following Elicitor Application against Powdery Mildew.

Molecular changes associated with response to powdery mildew (PM) caused by Erysiphe necator have been largely explored in Vitis vinifera cultivars, but little is known on transcriptional and metabolic modifications following application of resistance elicitors against this disease. In this study, the whole transcriptome sequencing, and hormone and metabolite analyses were combined to dissect long-term defense mechanisms induced by molecular reprogramming events in PM-infected 'Moscato' and 'Nebbiolo' leaves treated with three resistance inducers: acibenzolar-S-methyl, potassium phosphonate, and laminarin. Although all compounds were effective in counteracting the disease, acibenzolar-S-methyl caused the most intense transcriptional modifications in both cultivars. These involved a strong down-regulation of photosynthesis and energy metabolism and changes in carbohydrate accumulation and partitioning that most likely shifted the plant growth-defense trade-off towards the establishment of disease resistance processes. It was also shown that genotype-associated metabolic signals significantly affected the cultivar defense machinery. Indeed, 'Nebbiolo' and 'Moscato' built up different defense strategies, often enhanced by the application of a specific elicitor, which resulted in either reinforcement of early defense mechanisms (e.g., epicuticular wax deposition and overexpression of pathogenesis-related genes in 'Nebbiolo'), or accumulation of endogenous hormones and antimicrobial compounds (e.g., high content of abscisic acid, jasmonic acid, and viniferin in 'Moscato').

Molecular memory of Flavescence dorée phytoplasma in recovering grapevines.

Flavescence dorée (FD) is a destructive phytoplasma disease of European grapevines. Spontaneous and cultivar-dependent recovery (REC) may occur in the field in FD-infected vines starting the year following the first symptoms. However, the biological underpinnings of this process are still largely unexplored. In this study, transcriptome sequencing (RNAseq), whole-genome bisulphite sequencing (WGBS) and metabolite analysis were combined to dissect molecular and metabolic changes associated to FD and REC in leaf veins collected in the field from healthy (H), FD and REC plants of the highly susceptible Vitis vinifera 'Barbera'. Genes involved in flavonoid biosynthesis, carbohydrate metabolism and stress responses were overexpressed in FD conditions, whereas transcripts linked to hormone and stilbene metabolisms were upregulated in REC vines. Accumulation patterns of abscisic acid and stilbenoid compounds analysed in the same samples confirmed the RNAseq data. In recovery conditions, we also observed the persistence of some FD-induced expression changes concerning inhibition of photosynthetic processes and stress responses. Several differentially expressed genes tied to those pathways also underwent post-transcriptional regulation by microRNAs, as outlined by merging our transcriptomic data set with a previously conducted smallRNAseq analysis. Investigations by WGBS analysis also revealed different DNA methylation marks between REC and H leaves, occurring within the promoters of genes tied to photosynthesis and secondary metabolism. The results allowed us to advance the existence of a "molecular memory" of FDp infection, involving alterations in the DNA methylation status of REC plants potentially related to transcriptional reprogramming events, in turn triggering changes in hormonal and secondary metabolite profiles.

A novel strigolactone-miR156 module controls stomatal behaviour during drought recovery.

miR156 is a conserved microRNA whose role and induction mechanisms under stress are poorly known. Strigolactones are phytohormones needed in shoots for drought acclimation. They promote stomatal closure ABA-dependently and independently; however, downstream effectors for the former have not been identified. Linkage between miR156 and strigolactones under stress has not been reported. We compared ABA accumulation and sensitivity as well as performances of wt and miR156-overexpressing (miR156-oe) tomato plants during drought. We also quantified miR156 levels in wt, strigolactone-depleted and strigolactone-treated plants, exposed to drought stress. Under irrigated conditions, miR156 overexpression and strigolactone treatment led to lower stomatal conductance and higher ABA sensitivity. Exogenous strigolactones were sufficient for miR156 accumulation in leaves, while endogenous strigolactones were required for miR156 induction by drought. The "after-effect" of drought, by which stomata do not completely re-open after rewatering, was enhanced by both strigolactones and miR156. The transcript profiles of several miR156 targets were altered in strigolactone-depleted plants. Our results show that strigolactones act as a molecular link between drought and miR156 in tomato, and identify miR156 as a mediator of ABA-dependent effect of strigolactones on the after-effect of drought on stomata. Thus, we provide insights into both strigolactone and miR156 action on stomata.

Grapevine Phyllosphere Community Analysis in Response to Elicitor Application against Powdery Mildew.

The reduction of antimicrobial treatments and mainly the application of environmentally friendly compounds, such as resistance elicitors, is an impelling challenge to undertake more sustainable agriculture. We performed this research to study the effectiveness of non-conventional compounds in reducing leaf fungal attack and to investigate whether they influence the grape phyllosphere. Pathogenicity tests were conducted on potted Vitis vinifera "Nebbiolo" and "Moscato" cultivars infected with the powdery mildew agent (Erysiphe necator) and treated with three elicitors. Differences in the foliar microbial community were then evaluated by community-level physiological profiling by using BiologTM EcoPlates, high throughput sequencing of the Internal Transcribed Spacer (ITS) region, and RNA sequencing for the viral community. In both cultivars, all products were effective as they significantly reduced pathogen development. EcoPlate analysis and ITS sequencing showed that the microbial communities were not influenced by the alternative compound application, confirming their specific activity as plant defense elicitors. Nevertheless, "Moscato" plants were less susceptible to the disease and presented different phyllosphere composition, resulting in a richer viral community, when compared with the "Nebbiolo" plants. The observed effect on microbial communities pointed to the existence of distinct genotype-specific defense mechanisms independently of the elicitor application.

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.

Small RNA Mobility: Spread of RNA Silencing Effectors and its Effect on Developmental Processes and Stress Adaptation in Plants.

Plants are exposed every day to multiple environmental cues, and tight transcriptome reprogramming is necessary to control the balance between responses to stress and processes of plant growth. In this context, the silencing phenomena mediated by small RNAs can drive transcriptional and epigenetic regulatory modifications, in turn shaping plant development and adaptation to the surrounding environment. Mounting experimental evidence has recently pointed to small noncoding RNAs as fundamental players in molecular signalling cascades activated upon exposure to abiotic and biotic stresses. Although, in the last decade, studies on stress responsive small RNAs increased significantly in many plant species, the physiological responses triggered by these molecules in the presence of environmental stresses need to be further explored. It is noteworthy that small RNAs can move either cell-to-cell or systemically, thus acting as mobile silencing effectors within the plant. This aspect has great importance when physiological changes, as well as epigenetic regulatory marks, are inspected in light of plant environmental adaptation. In this review, we provide an overview of the categories of mobile small RNAs in plants, particularly focusing on the biological implications of non-cell autonomous RNA silencing in the stress adaptive response and epigenetic modifications.

Priming xylem for stress recovery depends on coordinated activity of sugar metabolic pathways and changes in xylem sap pH.

Some plant species are capable of significant reduction of xylem embolism during recovery from drought despite stem water potential remains negative. However, the functional biology underlying this process is elusive. We subjected poplar trees to drought stress followed by a period of recovery. Water potential, hydraulic conductivity, gas exchange, xylem sap pH, and carbohydrate content in sap and woody stems were monitored in combination with an analysis of carbohydrate metabolism, enzyme activity, and expression of genes involved in sugar metabolic and transport pathways. Drought resulted in an alteration of differential partitioning between starch and soluble sugars. Upon stress, an increase in the starch degradation rate and the overexpression of sugar symporter genes promoted the efflux of disaccharides (mostly maltose and sucrose) to the apoplast. In turn, the efflux activity of the sugar-proton cotransporters caused a drop in xylem pH. The newly acidic environment induced the activity of apoplastic invertases leading to the accumulation of monosaccharides in the apoplast, thus providing the main osmoticum necessary for recovery. During drought and recovery, a complex network of coordinated molecular and biochemical signals was activated at the interface between xylem and parenchyma cells that appeared to prime the xylem for hydraulic recovery.

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.