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.

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.

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.

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').