Development of a CRISPR/Cas9-mediated gene knockout method for functional genomics of the barley spot blotch pathogen Bipolaris sorokiniana

Abstract

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 Cas9/sgRNA combined with the split marker system 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/gRNA ribonucleoproteins (RNPs) and the split hygromycin B resistance gene (HygB) fragments each harboring 449 bp upstream and 595 bp downstream flanking sequences of PKS1 were co-transformed into the protoplasts of B. sorokiniana, the number of transformants with the PKS1 gene replaced by the selection marker were significantly increased compared to the control without RNPs. We then used the RNPs with PCR-amplified HygB gene cassette carrying 40 bp 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 more gene knockout mutants were obtained with longer (60 bp) homologous arms than the shorter (40 bp) ones. Finally, we disrupted an uncharacterized non-ribosomal peptide synthetase (NRPS) gene (NPSx) in B. sorokiniana strain ND90Pr using the RNP-mediated gene knockout approach and showed the mutants lost virulence on barley cv. Bowman. The Cas9/sgRNA-mediated gene knockout method developed will facilitate large-scale functional genomics studies of B. sorokiniana.