CRISPR/Cas9-targeted mutagenesis of the tomato susceptibility gene PMR4 for resistance against powdery mildew

Background The development of CRISPR/Cas9 technology has facilitated targeted mutagenesis in an efficient and precise way. Previously, RNAi silencing of the susceptibility (S) genePowderyMildewResistance 4(PMR4) in tomato has been shown to enhance resistance against the powdery mildew pathogenOidium neolycopersici(On). Results To study whether full knock-out of the tomatoPMR4gene would result in a higher level of resistance than in the RNAi-silenced transgenic plants we generated tomatoPMR4CRISPR mutants. We used a CRISPR/Cas9 construct containing four single-guide RNAs (sgRNAs) targeting the tomatoPMR4gene to increase the possibility of large deletions in the mutants. After PCR-based selection and sequencing of transformants, we identified five different mutation events, including deletions from 4 to 900-bp, a 1-bp insertion and a 892-bp inversion. These mutants all showed reduced susceptibility toOnbased on visual scoring of disease symptoms and quantification of relative fungal biomass. Histological observations revealed a significantly higher occurrence of hypersensitive response-like cell death at sites of fungal infection in thepmr4mutants compared to wild-type plants. Both haustorial formation and hyphal growth were diminished but not completely inhibited in the mutants. Conclusion CRISPR/Cas-9 targeted mutagenesis of the tomatoPMR4gene resulted in mutants with reduced but not complete loss of susceptibility to the PM pathogenOn.Our study demonstrates the efficiency and versatility of the CRISPR/Cas9 system as a powerful tool to study and characterizeS-genes by generating different types of mutations.