CRISPR/Cas9-mediated targeted mutagenesis of two homoeoalleles in tobacco confers resistance to powdery mildew

Clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 (CRISPR-associated protein 9) technology has emerged as an effective tool for crop improvement. In tobacco (Nicotiana tabacum L.), the Mildew Locus O (MLO) genes, NtMLO1 and NtMLO2, are responsible for susceptibility to powdery mildew. However, the modification of multiple homoeoalleles in polyploid crops has inherent complexities. We employed the CRISPR/Cas9 system with single-guide RNA for targeted simultaneous mutagenesis of both NtMLO genes to enhance powdery mildew resistance in tobacco. This approach generated mutants, of which two-thirds had mutations in both genes. Among the 20 T0 transgenic plants, the mutation rates of NtMLO1 and NtMLO2 were 65% and 60%, respectively. The rate of simultaneous mutations in NtMLO1 and NtMLO2 was approximately 66.7%, indicating that the CRISPR/Cas9 system was efficient. In the T1 lines, three types of mutations (1 or 2 bp deletion and 1 bp insertion) were identified for NtMLO1, and two types (1 bp insertion and 11 bp deletion) for NtMLO2 at the positions expected based on Sanger sequencing. Four mutation events were identified in mutants with both genes edited in T1 lines. These double mutants exhibited enhanced resistance to powdery mildew. Our results suggest that CRISPR/Cas9-mediated targeted editing of NtMLO1 and NtMLO2 is a practical approach for improving powdery mildew resistance in tobacco.