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 is also highly susceptible to fungal (-like) diseases such as powdery mildew (PM), and downy mildew (DM). These diseases – impacting grapevine production worldwide – are controlled every year at the expenses of a great consumption of pesticides, especially fungicides. Therefore, the introduction of PM- and DM-resistant cultivars is an urgent need for the foreseeable sustainability of viticulture worldwide. 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, because it allows to introduce very specific and small mutations. Before the creation of non-transgenic grape cultivars by NBTs becomes common practice, some hurdles need to be overcome: i) the poor knowledge on appropriate target genes to generate resistant cultivars, ii) the lack of efficient protocols to deliver the CRISPR-Cas9 machinery as protein/RNA complex into single cells, and iii) the low efficiency of plant regeneration. 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 knockout 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. High 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 developed a novel DNA-free methodology to obtain edited transgene-free grapevine plants. These plants were regenerated from a single edited-cell, and therefore do not show chimerism.