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

Abstract The CRISPR/Cas9 technology has proven to be an effective tool for crop improvement. In tobacco (Nicotiana tabacum L.), the Mildew Locus O (MLO) genes, NtMLO1 and NtMLO2, are responsible for powdery mildew susceptibility. However, modification of multiple homoeoalleles is complex in polyploidy crops. Therefore, we employed the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats-associated protein 9) system containing a single-guide RNA (sgRNA) to enable targeted mutagenesis of both NtMLOs simultaneously and enhance the powdery mildew resistance of tobacco. The approach generated different mutants, of which two-thirds had both genes mutated. In the T1 lines, three types of mutations were identified for NtMLO1 and two types for NtMLO2 at the expected position based on Sanger sequencing. Meanwhile, four mutation events were identified in the mutants with both genes edited in T1 lines. These double mutants showed enhanced resistance to powdery mildew, whereas the mutants with only one edited gene showed no significant difference compared to wild-type plants. Our results suggest that CRISPR/Cas9-mediated targeted editing of NtMLO1 and NtMLO2 is a practical approach for improving the powdery mildew resistance of tobacco.