New improvements in grapevine genome editing: high efficiency biallelic homozygous knock-out from regenerated plantlets by using an optimized zCas9i.

BACKGROUND: For ten years, CRISPR/cas9 system has become a very useful tool for obtaining site-specific mutations on targeted genes in many plant organisms. This technology opens up a wide range of possibilities for improved plant breeding in the future. In plants, the CRISPR/Cas9 system is mostly used through stable transformation with constructs that allow for the expression of the Cas9 gene and sgRNA. Numerous studies have shown that site-specific mutation efficiency can vary greatly between different plant species due to factors such as plant transformation efficiency, Cas9 expression, Cas9 nucleotide sequence, the addition of intronic sequences, and many other parameters. Since 2016, when the first edited grapevine was created, the number of studies using functional genomic approaches in grapevine has remained low due to difficulties with plant transformation and gene editing efficiency. In this study, we optimized the process to obtain site-specific mutations and generate knock-out mutants of grapevine (Vitis vinifera cv. 'Chardonnay'). Building on existing methods of grapevine transformation, we improved the method for selecting transformed plants at chosen steps of the developing process using fluorescence microscopy. RESULTS: By comparison of two different Cas9 gene and two different promoters, we increased site-specific mutation efficiency using a maize-codon optimized Cas9 containing 13 introns (zCas9i), achieving up to 100% biallelic mutation in grapevine plantlets cv. 'Chardonnay'. These results are directly correlated with Cas9 expression level. CONCLUSIONS: Taken together, our results highlight a complete methodology for obtaining a wide range of homozygous knock-out mutants for functional genomic studies and future breeding programs in grapevine.

Increasing vineyard sustainability: innovating a targeted chitosan-derived biocontrol solution to induce grapevine resistance against downy and powdery mildews.

The European Green Deal aims to reduce the pesticide use, notably by developing biocontrol products to protect crops from diseases. Indeed, the use of significant amounts of chemicals negatively impact the environment such as soil microbial biodiversity or groundwater quality, and human health. Grapevine (Vitis vinifera) was selected as one of the first targeted crop due to its economic importance and its dependence on fungicides to control the main damaging diseases worldwide: grey mold, downy and powdery mildews. Chitosan, a biopolymer extracted from crustacean exoskeletons, has been used as a biocontrol agent in many plant species, including grapevine, against a variety of cryptogamic diseases such as downy mildew (Plasmopara viticola), powdery mildew (Erysiphe necator) and grey mold (Botrytis cinerea). However, the precise molecular mechanisms underlying its mode of action remain unclear: is it a direct biopesticide effect or an indirect elicitation activity, or both? In this study, we investigated six chitosans with diverse degrees of polymerization (DP) ranging from low to high DP (12, 25, 33, 44, 100, and 470). We scrutinized their biological activities by evaluating both their antifungal properties and their abilities to induce grapevine immune responses. To investigate their elicitor activity, we analyzed their ability to induce MAPKs phosphorylation, the activation of defense genes and metabolite changes in grapevine. Our results indicate that the chitosans with a low DP are more effective in inducing grapevine defenses and possess the strongest biopesticide effect against B. cinerea and P. viticola. We identified chitosan with DP12 as the most efficient resistance inducer. Then, chitosan DP12 has been tested against downy and powdery mildews in the vineyard trials performed during the last three years. Results obtained indicated that a chitosan-based biocontrol product could be sufficiently efficient when the amount of pathogen inoculum is quite low and could be combined with only two fungicide treatments during whole season programs to obtain a good protection efficiency. On the whole, a chitosan-based biocontrol product could become an interesting alternative to meet the chemicals reduction targeted in sustainable viticulture.

Grapevine Potassium Nutrition and Fruit Quality in the Context of Climate Change.

Potassium (K+) nutrition is of relevant interest for winegrowers because it influences grapevine growth, berry composition, as well as must and wine quality. Indeed, wine quality strongly depends on berry composition at harvest. However, K+ content of grape berries increased steadily over the last decades, in part due to climate change. Currently, the properties and qualities of many fruits are also impacted by environment. In grapevine, this disturbs berry properties resulting in unbalanced wines with poor organoleptic quality and low acidity. This requires a better understanding of the molecular basis of K+ accumulation and its control along grape berry development. This mini-review summarizes our current knowledge on K+ nutrition in relation with fruit quality in the context of a changing environment.

Unique features of the grapevine VvK5.1 channel support novel functions for outward K+ channels in plants.

Grapevine (Vitis vinifera L.), one of the most important fruit crops, is a model plant for studying the physiology of fleshy fruits. Here, we report on the characterization of a new grapevine Shaker-type K+ channel, VvK5.1. Phylogenetic analysis revealed that VvK5.1 belongs to the SKOR-like subfamily. Our functional characterization of VvK5.1 in Xenopus oocytes confirms that it is an outwardly rectifying K+ channel that displays strict K+ selectivity. Gene expression level analyses by real-time quantitative PCR showed that VvK5.1 expression was detected in berries, roots, and flowers. In contrast to its Arabidopsis thaliana counterpart that is involved in K+ secretion in the root pericycle, allowing root to shoot K+ translocation, VvK5.1 expression territory is greatly enlarged. Using in situ hybridization we showed that VvK5.1 is expressed in the phloem and perivascular cells of berries and in flower pistil. In the root, in addition to being expressed in the root pericycle like AtSKOR, a strong expression of VvK5.1 is detected in small cells facing the xylem that are involved in lateral root formation. This fine and selective expression pattern of VvK5.1 at the early stage of lateral root primordia supports a role for outward channels to switch on cell division initiation.