Generation of mildew-resistant grapevine clones via genome editing

Pesticides, mostly fungicides, are used in large quantities in viticulture to contain the spread of fungal and fungal-like diseases such as powdery mildew (PM) and downy mildew (DM), toward which cultivated grapevine (Vitis vinifera) is highly susceptible. Such consumption of fungicides is costly and deleterious for human health and environment. As an alternative, such diseases have been successfully controlled by inactivation of plant susceptibility genes in crops. The knocking out of DMR6 genes was demonstrated to be very effective in controlling DM in Arabidopsis thaliana and some cultivated species, but its efficacy toward DM resistance has yet to be demonstrated in grapevine. In addition, silencing of MLO genes resulted in resistance to PM in grapevine. Introgression of nonfunctional copies of susceptibility genes can be achieved via traditional breeding, but crossing is not always desirable in grapevine, since maintenance of clonal genetic integrity is commercially important for wine grapes. The advent of genome editing now offers revolutionary tools to edit and completely knock out susceptibility genes in many crops while maintaining their cultivar and clonal genetic backgrounds. In the present work, CRISPR/Cas9 technology was used 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 full knockout of DMR6 and MLO genes provides durable resistance to DM and PM, respectively, and will hopefully deliver mildew-resistant grapevine plants