Simultaneous editing of two DMR6 genes in grapevine results in reduced susceptibility to downy mildew

Abstract

The reduction of pesticide treatments is of paramount importance for the sustainability of viticulture, and it can be achieved through a combination of strategies, including the cultivation of vines (Vitis vinifera) that are resistant or tolerant to diseases such as downy mildew (DM). In many crops, the knock-out of Downy Mildew Resistant 6 (DMR6) proved successful in controlling DM-resistance, but the effect of mutations in DMR6 genes is not yet known in grapevine. Today, gene editing serves crop improvement with small and specific mutations while maintaining the genetic background of commercially important clones. Moreover, recent technological advances allowed to produce non-transgenic grapevine clones by regeneration of protoplasts edited with the CRISPR/Cas9 ribonucleoprotein. This approach may revolutionize the production of new grapevine varieties and clones, but it requires knowledge about the targets and the impact of editing on plant phenotype and fitness in different cultivars. In this work we generated single and double knock-out mutants by editing DMR6 susceptibility (S) genes using CRISPR/Cas9, and showed that only the combined mutations in VviDMR6-1 and VviDMR6-2 are effective in reducing susceptibility to DM in two table-grape cultivars by increasing the levels of endogenous salicylic acid. Therefore, editing both genes may be necessary for effective DM control in real-world agricultural settings, which could potentially lead to unwanted phenotypes. Additional research, including trials conducted in experimental vineyards, is required to gain a deeper understanding of DMR6-based resistance.

Keywords: DMR6; Plasmopara viticola; Vitis vinifera; downy mildew; gene editing; salicylic acid; susceptibility gene.

Figure 1

Genotype of selected edited plants. (A) Grouping of edited plants based on the fraction of edited reads in NGS sequencing. Completely edited plants were defined as those with more than 99% of edited reads. (B) Editing types of plants selected for phenotypic analysis. Color coding: PAM sequence (gray); Sugraone (blue), Crimson seedless (red), Thompson seedless (black). Non-transgenic dmr6-2 mutants obtained from edited protoplasts are underlined.

Figure 2

Images of mutants in two genetic backgrounds: Greenhouse-adapted plants of cv. Crimson seedless and Sugraone. From right to left are the non-edited wild type, and lines of dmr6-1, dmr6-2, and double dmr6-1_2 mutants. Pictures were taken six weeks after pruning, at the time of DM-inoculation.

Figure 3

(A) DM-assay on detached leaf-discs of young plants. Bullets represent the relative severity (logit transformed) of leaf discs at 7 dpi in Crimson seedless and Sugraone collected in three experiments. (B) In planta DM-assay. Boxplots show average severity (logit transformed, colored bullets) of individual greenhouse-adapted plants collected from two inoculation experiments per cultivar. Each experiment included multiple lines per genotype for a total of 85 individual plants, as described in the methods. P values of significant differences are indicated.

Figure 4

Quantification of free SA in the two cultivars. Data points represent the SA content in different biological replicates (10^th-11^th leaf of individual plants) of wt (blue), dmr6-1 (red), dmr6-2 (green) and dmr6-1_2 (yellow) mutants. Different lines within each genotype were analyzed as described in the legend: D39, D56 (dmr6-1), H1D, H1C1 (dmr6-2), O26, P21, O79, M42 (double mutants, Crimson seedless), and M57 and N12 (double mutants, Sugraone). Significantly different groups are indicated by asterisks (**P<0.01).