CRISPR/Cas9-based generation of mlo mutants for allelic complementation experiments to elucidate MLO function in barley

Barley ( Hordeum vulgare ) mildew locus o ( mlo ) mutants exhibit strong resistance to penetration by the powdery mildew fungus Blumeria graminis f. sp. hordei . MLO, a seven-transmembrane protein localized at the plasma membrane is thought to be involved in intracellular calcium signaling. However, its molecular function and the mechanism by which mlo mutations confer resistance to penetration by the fungus remain poorly understood. A large number of mlo alleles with different amino acid substitutions at each intracellular loop have been found in various cultivars. However, it is difficult to analyze how each amino acid is involved in penetration resistance by comparing these cultivars because they differ substantially in their genetic background and in the presence or absence of resistance genes recognizing avirulence factors from the pathogen. In this study, we used a CRISPR/Cas9-mediated genetic modification system to generate mlo mutants in the transformable cultivar Golden Promise to enable complementation experiments with the aim of elucidating the molecular function of MLO. An mlo mutant with a thymine insertion in the second exon and penetration resistance to B . graminis f. sp. hordei was obtained. Susceptibility was restored in cells in which the MLO-mCherry gene was introduced using particle bombardment, indicating that this mlo mutant could be a useful genetic tool for complementation experiments using transgenes expressing a variety of mlo alleles.