Botrytis cinerea causes gray mold, decreasing the quality of table grapes. The berry response to B. cinerea infection was explored in present study, focusing on the relationship between presence of autophagy and programmed cell death (PCD). Results demonstrated B. cinerea infection decreased cell viability, triggering cell death, possibly resulting in PCD occurrence. It was further verified by increased terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL)-positive nuclei, heightened caspase 3-like and caspase 9-like protease activity, and elevated expression of metacaspase genes. Additionally, autophagy was indicated by the increased VvATG expression and autophagosome formation. Notably, the autophagy activator rapamycin reduced TUNEL-positive nuclei, whereas the autophagy inhibitor 3-methyladenine increased caspase 9-like protease activity. The PCD activator C2-ceramide inhibited autophagy, whereas the PCD inhibitor Acetyl-Asp-Glu-Val-Asp-aldehyde (Ac-DEVD-CHO) enhanced autophagy gene expression. Autophagy and B. cinerea-induced PCD in berry cells are reciprocally negatively regulated; and the rapamycin and Ac-DEVD-CHO could potentially maintain table grape edible quality.

Long-distance transfer of genetic material and metabolites between rootstock and scions is well documented in homo-grafted hybrids but has rarely been reported in genetically-distant grafts where the rootstock and scion belong to different families. In this study, we grafted Vitis vinifera scions onto Schisandra chinensis stocks and obtained 20 vegetative hybrids, Vitis vinifera/Schisandra chinensis (Vs). After 25 years of growth, we found that the phenotypes of the leaves, internodes, and fruits of the Vs hybrids above the graft union resembled an intermediate phenotype between V. vinifera and S. chinensis, and the new traits were stable when propagated vegetatively. We further analyzed genetic differences between Vv plants and Vs hybrids using high-throughput sequencing, while metabolomes were analyzed by liquid chromatography-mass spectrometry (LC-MS). We found a total of 2113 differentially expressed genes (DEGs). GO annotation and KEGG pathway enrichment analysis showed that these DEGs enriched mainly in oxidation-reduction and metabolic processes. Seventy-nine differentially expressed miRNAs (DEMs) containing 27 known miRNAs and 52 novel miRNAs were identified. A degradation analysis detected 840 target genes corresponding to 252 miRNAs, of which 12 DEMs and their corresponding target gene expression levels were mostly negatively correlated. Furthermore, 1188 differential metabolic compounds were identified. In particular, in Vs hybrids, the abundance of the metabolites schizandrin and gomisin as the main medicinal ingredients in S. chinensis were down-regulated and up-regulated, respectively. Our data demonstrated the effects of interfamily grafts on the phenotype, transcript profile and metabolites of the scion, and also provided new insight into the genetic, phenotypic, and metabolic plasticity associated with genetically distant grafted hybrids.

Haplotype-resolved genome assemblies were produced for Chasselas and Ugni Blanc, two heterozygous Vitis vinifera cultivars by combining high-fidelity long-read sequencing and high-throughput chromosome conformation capture (Hi-C). The telomere-to-telomere full coverage of the chromosomes allowed us to assemble separately the two haplo-genomes of both cultivars and revealed structural variations between the two haplotypes of a given cultivar. The deletions/insertions, inversions, translocations, and duplications provide insight into the evolutionary history and parental relationship among grape varieties. Integration of de novo single long-read sequencing of full-length transcript isoforms (Iso-Seq) yielded a highly improved genome annotation. Given its higher contiguity, and the robustness of the IsoSeq-based annotation, the Chasselas assembly meets the standard to become the annotated reference genome for V. vinifera. Building on these resources, we developed VitExpress, an open interactive transcriptomic platform, that provides a genome browser and integrated web tools for expression profiling, and a set of statistical tools (StatTools) for the identification of highly correlated genes. Implementation of the correlation finder tool for MybA1, a major regulator of the anthocyanin pathway, identified candidate genes associated with anthocyanin metabolism, whose expression patterns were experimentally validated as discriminating between black and white grapes. These resources and innovative tools for mining genome-related data are anticipated to foster advances in several areas of grapevine research.

A phenomenon of pathogenicity attenuation of Plasmopara viticola was consistently observed during its subculture on grape. To clarify the causes of attenuated pathogenicity of P. viticola, culturable microbes were isolated from the P. viticola mass (mycelia, sporangiophores, and sporangia) in each generation and tested for their biocontrol efficacies on grape downy mildew (GDM). The results showed that the incidence of GDM decreased with the increase in the number of subculture times on both vineyard-collected leaves and grape leaves from in vitro-grown seedlings. The number of culturable microbial taxa on the surface of P. viticola decreased, whereas the population densities of four specific strains (i.e., K2, K7, P1, and P5) increased significantly with the increase in subculture times. Compared with the control, the biocontrol efficacies of the bacterial strain K2 reached 87.5%, and those of both fungal strains P1 and P5 reached 100.0%. Based on morphological characteristics and molecular sequences, strains K2, P1, and P5 were identified as Curtobacterium herbarum, Thecaphora amaranthi, and Acremonium sclerotigenum, respectively, and these three strains survived very well and multiplied on the surface of P. viticola. As the number of times P. viticola was subcultured increased, all three of these strains became the predominant strains, leading to greater P. viticola inhibition, attenuated P. viticola pathogenicity, and effective GDM biological control. To the best of our knowledge, this is the first report of C. herbarum and T. amaranthi having biological control activity against GDM.

Clustered regularly interspaced short palindromic repeats (CRISPR) /Cas12a system, also known as CRISPR/Cpf1, has been successfully harnessed for genome engineering in many plants, but not in grapevine yet. Here we developed and demonstrated the efficacy of CRISPR/Cas12a from Lachnospiraceae bacterium ND2006 (LbCas12a) in inducing targeted mutagenesis by targeting the tonoplastic monosaccharide transporter1 (TMT1) and dihydroflavonol-4-reductase 1 (DFR1) genes in 41B cells. Knockout of DFR1 gene altered flavonoid accumulation in dfr1 mutant cells. Heat treatment (34℃) improved the editing efficiencies of CRISPR/LbCas12a system, and the editing efficiencies of TMT1-crRNA1 and TMT1-crRNA2 increased from 35.3% to 44.6% and 29.9% to 37.3% after heat treatment, respectively. Moreover, the sequences of crRNAs were found to be predominant factor affecting editing efficiencies irrespective of the positions within the crRNA array designed for multiplex genome editing. In addition, genome editing with truncated crRNAs (trucrRNAs) showed that trucrRNAs with 20 nt guide sequences were as effective as original crRNAs with 24 nt guides in generating targeted mutagenesis, whereas trucrRNAs with shorter regions of target complementarity ≤ 18 nt in length may not induce detectable mutations in 41B cells. All these results provide evidence for further applications of CRISPR/LbCas12a system in grapevine as a powerful tool for genome engineering.

Nitrogen (N) fertilization is important for grape growth and wine quality. Unreasonable N fertilizer application affects wine growth and has a negative impact on wine quality. Therefore, it is essential to address the mismatch between N application and wine composition. To regulate vine growth and improve grape and wine quality, Cabernet Gernischt (Vitis vinifera L.) grapevines were subjected to lower levels of N, compared to normal N supply treatments, during the grape growing seasons of 2019 and 2020 in the wine region of Yantai, China. The effects of reduced N application from pre-boom to pre-veraison on vine growth, yield and composition of grapes, and dry red wine anthocyanin and non-anthocyanin phenolic compound content were studied. We found that reduced N application significantly decreased dormant shoot fresh mass and yield. However, the effect of N application on fruit ripening depended on the season. Nitrogen-reduction treatment significantly improved wine phenolic parameters, including total phenolics, tannins, and anthocyanins, and enhanced most of the individual anthocyanins and some non-anthocyanin phenolics, especially stilbenes, including piceatannol, trans-resveratrol, and polydatin, regardless of the season. Overall, our findings highlight the importance of reducing N application during the grape growing season in order to modify the wine phenolic profiles.

Stilbene and flavonoid are phytochemicals in plants and play an important role in plant disease resistance and human health. The regulation of stilbene and flavonoid synthesis in plants has been extensively studied at the transcriptional level, but translational and post-translational controls of stilbene and flavonoid biosynthesis are still poorly understood. In this study, a grape F-box E3 ubiquitin ligase VviKFB07 associated with the metabolism of stilbene and flavonoid was screened out with transcriptome. Overexpression of VviKFB07 in the Nicotiana tabacum resulted in a decrease in flavonol and anthocyanin content in corolla, and stable overexpression assays of VviKFB07 in grape callus promoted the accumulation of resveratrol. Subsequently, Yeast two-hybrid and bimolecular fluorescence complementation assays identified the physical interaction between VviKFB07 and VviCHSs proteins. In vivo experiments verified that VviKFB07 was involved in the ubiquitination and degradation of VviCHSs protein. Taken together, our findings clarify the role of ubiquitin ligase VviKFB07 in the synthesis of stilbene and flavonoid in grapes.

Grape gray mold disease (Botrytis cinerea) is widespread during grape production especially in Vitis vinifera and causes enormous losses to the grape industry. In nature, the grapevine cultivar \'Beta \' (Vitis riparia × Vitis labrusca) showed high resistance to grape gray mold. Until now, the candidate genes and their mechanism of gray mold resistance were poorly understood. In this study, we firstly conducted quantitative trait locus (QTL) mapping for grape gray mold resistance based on two hybrid offspring populations that showed wide separation in gray mold resistance. Notably, two stable QTL related to gray mold resistance were detected and located on linkage groups LG2 and LG7. The phenotypic variance ranged from 6.86% to 13.70% on LG2 and 4.40% to 11.40% on LG7. Combined with RNA sequencing (RNA-seq), one structural gene VlEDR2 (Vitvi02g00982) and three transcription factors VlERF039 (Vitvi00g00859), VlNAC047 (Vitvi08g01843), and VlWRKY51 (Vitvi07g01847) that may be involved in VlEDR2 expression and grape gray mold resistance were selected. This discovery of candidate gray mold resistance genes will provide an important theoretical reference for grape gray mold resistance mechanisms, research, and gray mold-resistant grape cultivar breeding in the future.

UNASSIGNED: Pathogen infection influences the post-harvest shelf life of grape berries. In a preliminary study, metabolites produced by Bacillus velezensis significantly inhibited the growth of the grape postharvest pathogen Penicillium olsonii.
UNASSIGNED: To investigate the mechanism of interaction between B. velezensis and P. olsonii, a draft genome was generated for P. olsonii WHG5 using the Illumina NovaSeq platform, and the transcriptomic changes in WHG5 were analyzed in response to the exposure to B. velezensis metabolites (10% v/v).
UNASSIGNED: The expression levels of genes associated with sporulation, including GCY1, brlA, and abaA, were down-regulated compared with those of the control. In addition, spore deformation and abnormal swelling of the conidiophore were observed. The expression of crucial enzymes, including fructose 2,6-bisphosphate and mannitol-2-dehydrogenase, was down-regulated, indicating that the glycolytic pathway of WHG5 was adversely affected by B. velezensis metabolites. The KEGG pathway enrichment analysis revealed that glutathione metabolism and the antioxidant enzyme system were involved in the response to B. velezensis metabolites. The down-regulation of the pathogenesis-related genes, PG1 and POT1, suggested that B. velezensis metabolites decreased the pathogenicity of P. olsonii. B. velezensis metabolites disrupted the homeostasis of reactive oxygen species in P. olsonii by affecting glucose metabolism, resulting in spore deformation and disruption of growth. In addition, the expression of key pathogenesis-related genes was down-regulated, thereby reducing the pathogenicity of P. olsonii.
UNASSIGNED: This study provides insights into the responses of P. olsonii to B. velezensis metabolites and identifies potential target genes that may be useful in biocontrol strategies for the suppression of post-harvest spoilage in grapes.

Phenolic compounds and their derivatives play a major role in the intensity and characteristics of grape (Vitis vinifera) astringency. The present study investigated the relationship between phenolic composition and astringency of six commercial table grape varieties (two of each white-, red-, and black-skinned). Qualitative and quantitative liquid chromatography-mass spectrometry analysis was used to identify the variety-specific phenolic profiles in the skins and total astringency intensity was assessed and described by a trained sensory panel. Thirty phenolic compounds were identified among the six varieties. Principal component analysis of the phenolic profiles revealed that the intensity of astringency of grape skin was positively correlated with catechin, epicatechin, epicatechin-3-O-gallate, and proanthocyanidin dimers B1, B2, and B3. A further orthogonal partial least-squares discrimination analysis of these compounds showed that catechin was the substance most strongly and positively correlated (R = 0.904) with grape skin astringency. PRACTICAL APPLICATION: This study provided a better understanding of the relationships between phenolic composition and table grape astringency and highlighted a potential metabolic marker that could be used as a predictor for the complex astringency sensory attributes of table grape berries.