Development of a CRISPR/Cas9-Mediated Gene Knockout Method for Functional Genomics of the Barley Spot Blotch Pathogen Bipolaris sorokiniana

Bipolaris sorokiniana (=Cochliobolus sativus) is an important fungal pathogen that causes spot blotch, common root rot, and kernel blight in barley and wheat. In this study, we aimed to develop a CRISPR/Cas9-mediated approach for efficiently knocking out genes of B. sorokiniana. We first assessed the efficiency of a split-marker system combined with Cas9 and single guide RNA (sgRNA) for gene replacement. We designed sgRNAs to target a polyketide synthase gene (PKS1) that is required for melanin biosynthesis of the fungus. When the preassembled Cas9/sgRNA ribonucleoproteins (RNPs) and the split hygromycin B resistance gene (HygB) fragments that each harbored 449-bp-upstream and 595-bp-downstream flanking sequences of PKS1 were cotransformed into the protoplasts of B. sorokiniana, the number of transformants with PKS1 replaced by the selection marker were significantly increased compared to the control without RNPs. We then used the RNPs and PCR-amplified HygB gene cassette carrying 40- or 60-bp arms homologous to the flanking sequences of the PKS1 target site for fungal transformation and showed that the RNPs significantly enhanced gene disruption efficiency through the short-homology recombination and that more gene knockout mutants were generated with longer (60-bp) homologous arms than with the shorter (40-bp) ones. Finally, we disrupted an uncharacterized nonribosomal peptide synthetase (NRPS) gene (NPSx) in B. sorokiniana strain ND90Pr using the RNP-mediated gene knockout approach and showed that the mutants lost virulence on barley cultivar Bowman. The Cas9/sgRNA-mediated gene knockout method developed will facilitate large-scale functional genomics studies of B. sorokiniana.