CRISPR/Cas9-mediated editing of PhMLO1 confers powdery mildew resistance in petunia

Mildew Locus O (MLO), which encodes a seven-transmembrane domain protein, is responsible for the development of powdery mildew disease symptoms in many plant species. Hence, we knocked out PhMLO1 from Petunia hybrida cv. Mirage Rose at the genomic level using the Clustered regularly interspaced short palindromic repeat/CRISPR-associated nuclease 9 (CRISPR/Cas9) system and investigated involvement of the gene in powdery mildew disease development in the petunia. The MLO1 transcript levels observed in all mlo1 mutants were significantly lower than that in the wild type (WT). Specifically, the transcript level was the lowest in the mutants (line no. 1, 25, 33, and 85), followed by the mutants (line no. 14, 17, and 81), and line no. 6. Disease development was positively linked to the higher transcript levels of PhMLO1, because the disease severity observed in WT was greater than that observed in the mutants (line no. 6,14, 17, and 81), and disease symptoms were almost not observed in the mutants (line no. 1, 25, 33, and 85). In addition, the transmission of the edited alleles from the T-0 to T-1 generation was also confirmed, and the disease severity results were identical to those observed in the T-0 mutant lines. Overall, the editing of PhMLO1 completely or partially prevented the development of this disease in this petunia, depending on MLO1 transcript level of the mutants. This study highlights the role of PhMLO1 in the development of powdery mildew disease in petunia and points to the need to edit the MLO genes in other ornamental plants to improve ornamental quality against powdery mildew disease.