Clade V-MLOs are associated with powdery mildew susceptibility in several plants. In grapevine, four MLO genes (MLO3, MLO4, MLO13, and MLO17) belong to clade V and have been studied to determine their contribution to susceptibility to Erysiphe necator (Grape Powdery Mildew-GPM). Using somatic embryogenesis to generate knockout mutants in grapevine, we performed a series of multiplex gene-editing-mediated transformations to determine each clade V-related MLO's contribution to GPM resistance. We characterized a hierarchy of contributions from the four MLOs to GPM, with MLO17 playing the largest role, followed by MLO3 and MLO13. In addition, MLO4 was a less essential modifier of GPM resistance: its loss-of-function, in combination with other MLOs, conferred a modest but consistent improvement in GPM resistance, and mlo mutants with a fraction of WT MLO4 allele chimerism lacked the mlo-associated developmental pleiotropic effects. Defects in flower development in most mlo4 mutant lines suggest a role for this gene in reproductive tissues. Our data also indicated functional redundancy within the clade V-MLOs, particularly between MLO13 and MLO17. Ultimately, our results show that a certain degree of chimerism (wild-type background in the mutants) is necessary to achieve resistance to GPM without major growth defects or yield penalties. Ultimately, this approach offers a viable strategy for breeding powdery mildew-resistant grapevines while maintaining agronomic fitness.
