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

Abstract 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. Competing Interest Statement The authors have declared no competing interest.