CRISPR/Cas9‐driven double modification of grapevine MLO6‐7 imparts powdery mildew resistance, while editing of NPR3 augments powdery and downy mildew tolerance

SUMMARY The implementation of genome editing strategies in grapevine is the easiest way to improve sustainability and resilience while preserving the original genotype. Among others, the Mildew Locus‐O ( MLO ) genes have already been reported as good candidates to develop powdery mildew‐immune plants. A never‐explored grapevine target is NPR3 , a negative regulator of the systemic acquired resistance. We report the exploitation of a cisgenic approach with the Cre‐lox recombinase technology to generate grapevine‐edited plants with the potential to be transgene‐free while preserving their original genetic background. The characterization of three edited lines for each target demonstrated immunity development against Erysiphe necator in MLO6‐7 ‐edited plants. Concomitantly, a significant improvement of resilience, associated with increased leaf thickness and specific biochemical responses, was observed in defective NPR3 lines against E. necator and Plasmopara viticola . Transcriptomic analysis revealed that both MLO6‐7 and NPR3 defective lines modulated their gene expression profiles, pointing to distinct though partially overlapping responses. Furthermore, targeted metabolite analysis highlighted an overaccumulation of stilbenes coupled with an improved oxidative scavenging potential in both editing targets, likely protecting the MLO6‐7 mutants from detrimental pleiotropic effects. Finally, the Cre‐loxP approach allowed the recovery of one MLO6‐7 edited plant with the complete removal of transgene. Taken together, our achievements provide a comprehensive understanding of the molecular and biochemical adjustments occurring in double MLO ‐defective grape plants. In parallel, the potential of NPR3 mutants for multiple purposes has been demonstrated, raising new questions on its wide role in orchestrating biotic stress responses.