Generation of non-transgenic mildew-resistant grapevine clones via gene-editing: potentials and hurdles

Grapevine (Vitis vinifera) is among the most valuable crops cultivated in Europe for both economic and cultural reasons, but it is also highly susceptible to fungal diseases such as powdery mildew (PM) and downy mildew (DM). The control of these diseases’ accounts for over 50% of the fungicides that are used every year in Europe, raising the urgent need for sustainable viticulture practices with a lower impact on human health and the environment. Such practices include, but are not limited to, the introduction of PM- and DM-resistant varieties. The recent advent of New Breeding Techniques (NBTs), and in particular of gene-editing, offered a great opportunity to obtain resistant plants either by the introduction of known resistance genes, or by knocking out susceptibility genes in commercial cultivars. As compared to traditional breeding, this approach has the advantage of maintaining the integrity of the parental variety by introducing very specific and small mutations, and of being faster because it doesn't require several cycles of backcrossing. Some hurdles need to be overcome before the creation of non-transgenic grape cultivars by NBTs becomes of practice: i) the identification of appropriate target genes to generate resistant cultivars, ii) the development of efficient protocols to deliver the CRISPRCas machinery as protein/RNA complex into single cells, and iii) plant regeneration. In addition to these technical problems, the products of NBTs are considered GMOs under the current European regulation. In the last years, we tackled these challenges by acting along two lines. On one hand, we characterized susceptibility genes of the MLO and DMR6 gene families by generation of knock-out mutants, in order to identify which genes are required for the establishment of the DM- and PM-diseases. Embryogenic calli were transformed via Agrobacterium tumefaciens with CRISPR/Cas9 vectors designed to specifically edit candidate susceptibility genes. Highly efficient targeted-mutagenesis in one or two genes was obtained, and several edited lines were regenerated from transformed calli. Edited plants grown in soil were challenged with the DM and PM pathogens, and preliminary results highlighted a role of these genes in grapevine susceptibility. In parallel, we also developed a new DNA-free methodology to obtain nontransgenic edited grapevine plants regenerated from protoplasts derived from embryogenic calli. These plants were regenerated from a single edited-cell, and therefore do not show chimerism