CRISPR-Cas9 editing of grapevine clade V-related mlo genes revealed new insights into the contribution of each mlo gene to grapevine powdery mildew resistance (Erysiphe necator) and plant development.

MLO proteins are a family of transmembrane proteins that play an essential role in plant immunity, host-pathogen interactions, and a wide range of other developmental processes in land plants. More specifically, clade V-MLOs are associated with powdery mildew susceptibility in several plants. In grapevine, four MLO genes, MLO3, 4, 13, and 17, belong to clade V and have been studied to determine their contribution to the susceptibility to Erysiphe necator (Grape Powdery Mildew - GPM). Using somatic embryogenesis to generate knockout mutants in grapevine, a series of multiplex gene-editing-mediated transformations was performed to determine the contribution of each clade V-related MLO to GPM resistance. A hierarchy of contributions from the four MLOs to GPM was characterized, with MLO17 having the most significant role, followed by MLOs 3 and 13. In addition, MLO4 was shown to be a non-essential modifier of GPM resistance: its loss-of-function, in combination with other MLOs, conferred a modest but consistent improvement in GPM resistance. However, defects in flower development in the majority of mlo4 mutant lines suggest a role for this gene in reproductive tissues. Our observation also indicated functional redundancy within the clade V-MLO, 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 relatively complete resistance to GPM without major growth defects or yield penalties.