Preliminary steps of a protocol to isolate transcription factors bound to a specific DNA locus in grapevine using CRISPR-dCas9 system

The Matrix Assisted Laser Desorption/Ionization–Time-Of-Flight Mass Spectrometry (MALDI-TOF MS) technology is commonly used in food and medical sector to identify yeast or bacteria species isolated from a nutritive culture media. Since a decade, brewery and oenology industries have been attracted to this method which combines fast analysis times, reliability and low cost of analysis. Briefly, this method is based on the comparison of the MALDI-TOF/MS protein spectra of an isolated colony of yeast or bacteria with those contain in a manufacturer’s reference protein spectra database. Initiated in 2015, the creation of the first oenological mass spectra database has proved to be essential for increase quality of species identification.

Polyphenols, bioactive secondary metabolites abundantly found in various grapevine components such as stalks, skins, and seeds, have attracted considerable attention in recent decades due to their potential health benefits. These compounds, including flavan-3-ols, flavanols, flavones, and stilbenes, are known for their antioxidant and anti-inflammatory properties.

The unique characteristics of terroir play a fundamental role in shaping the identity and quality of wines, influencing the aromatic complexity of young wines and their long-term aging potential. The volatile compounds responsible for these aromas are crucial to identifying and appreciating a given wine.

Soil is a reservoir of microorganisms playing important roles in biogeochemical cycles and interacting with plants whether in the rhizosphere or in the root endosphere. The composition of the microbial communities thus impacts the plant health. Rhizodeposits (such as sugar, organic and amino acids, secondary metabolites, dead root cells …) are released by the roots and influence the communities of rhizospheric microorganisms, acting as signaling compounds or carbon sources for microbes. The composition of root exudates varies depending on several factors including genotypes. As most of the cultivated grapevines worldwide are grafted plants, the aim of this study was to explore the influence of rootstock and scion genotypes on the microbial communities of the rhizosphere and the root endosphere. The work was conducted in the GreffAdapt plot (55 rootstocks x 5 scions), in which the 275 combinations have been planted into 3 blocks designed according to the soil resistivity. Samples of roots and rhizosphere of 10 scion x rootstock combinations were first collected in May among the blocks 2 and 3. The quantities of bacteria, fungi and archaea have been assessed in the rhizosphere by quantitative PCR, and by cultivable methods for bacteria and fungi. The communities of bacteria, fungi and arbuscular mycorrhizal fungi (AMF) was analyzed by Illumina sequencing of 16S rRNA gene, ITS and 28S rRNA gene, respectively. The level of mycorrhization was also evaluated using black ink coloration of newly formed roots harvested in October. The level of bacteria, fungi and archaea was dependent on rootstock and scion genotypes. A block effect was observed, suggesting that the soil characteristics strongly influenced the microorganisms from the rhizosphere and root endosphere. High-throughput sequencing of the different target genes showed different communities of bacteria, fungi and AMF associated with the scion x rootstock combinations. Finally, all the combinations were naturally mycorrhized. The root mycorrhization intensity was influenced by the rootstock genotype, but not by the scion one. Altogether, these results suggest that both rootstock and scion genotypes influence the rhizosphere and root endophytic microbiomes. It would be interesting to analyze the biochemical composition of the rhizodeposition of these genotypes for a better understanding of the processes involved in the modulation of these microbiomes. Moreover, crossing our data with the plant agronomic characteristics could provide insights into their roles on plant fitness.

The work aims to characterise the colour features of the wines of two new resistant varieties breeeded at the Edmund Mach Foundation (Italy) and recently inscribed in the Italian National Registriy of Vine Varieties.