Generation of mildew-resistant grapevine clones via genome editing

Pesticides-mostly fungicides- are massively used in viticulture to contain spreading of fungal and fungal-like diseases such as powdery mildew (PM) and downy mildew (DM), towards which cultivated grapevine (Vitis vinifera) is highly susceptible. Such consumption of fungicides is costly and deleterious both for human health and the environment. Alternatively, the successful control of such diseases was obtained by inactivation of plant susceptibility genes in other crops: in fact, knocking out of DMR6 genes was demonstrated very effective in controlling DM in Arabidopsis thaliana and other species. However their efficacy toward DM resistance has yet to be demonstrated in grapevine. In addition, silencing of a group of MLO genes resulted in resistance to PM in grapevine. Selection of non-functional copies of susceptibility genes can be achieved via traditional breeding; however crossing is not always desirable in grapevine, since maintenance of clonal genetic integrity is commercially important (i.e. in wine grapes). The revolutionary advent of genome editing now offers tools to edit and completely knock out susceptibility genes in many crops while maintaining their cultivar and clonal genetic backgrounds. CRISPR /Cas 9 technology was used in this work to edit DM and PM susceptibility genes in different grapevine clones. Several plants edited in DMR6 and MLO genes were obtained and are currently being screened for DM and PM resistance. This work will establish whether DMR6 is determinant in controlling resistance to DM in grapevine, whether a full knock-out of DMR6 and MLO genes provides resistance to DM and PM respectively, and will hopefully deliver mildew resistant grapevine plants.