Mutations introduced in susceptibility genes through CRISPR/Cas9 genome editing confer increased late blight resistance in potatoes

The use of pathogen-resistant cultivars is expected to increase yield and decrease fungicide use in agriculture. However, in potato breeding, increased resistance obtained via resistance genes (R-genes) is hampered because R-gene(s) are often specific for a pathogen race and can be quickly overcome by the evolution of the pathogen. In parallel, susceptibility genes (S-genes) are important for pathogenesis, and loss of S-gene function confers increased resistance in several plants, such as rice, wheat, citrus and tomatoes. In this article, we present the mutation and screening of seven putative S-genes in potatoes, including two DMR6 potato homologues. Using a CRISPR/Cas9 system, which conferred co-expression of two guide RNAs, tetra-allelic deletion mutants were generated and resistance against late blight was assayed in the plants. Functional knockouts of StDND1 , StCHL1, and DMG400000582 ( StDMR6-1 ) generated potatoes with increased resistance against late blight. Plants mutated in StDND1 showed pleiotropic effects, whereas StDMR6-1 and StCHL1 mutated plants did not exhibit any growth phenotype, making them good candidates for further agricultural studies. Additionally, we showed that DMG401026923 (here denoted StDMR6-2 ) knockout mutants did not demonstrate any increased late blight resistance, but exhibited a growth phenotype, indicating that StDMR6-1 and StDMR6-2 have different functions. To the best of our knowledge, this is the first report on the mutation and screening of putative S-genes in potatoes, including two DMR6 potato homologues.