Genome Editing and Protoplast Regeneration to Study Plant-Pathogen Interactions in the Model Plant .

Biotic diseases cause substantial agricultural losses annually, spurring research into plant pathogens and strategies to mitigate them. is a commonly used model plant for studying plant-pathogen interactions because it is host to numerous plant pathogens and because many research tools are available for this species. The clustered regularly interspaced short palindromic repeats (CRISPR) system is one of several powerful tools available for targeted gene editing, a crucial strategy for analyzing gene function. Here, we demonstrate the use of various CRISPR-associated (Cas) proteins for gene editing of protoplasts, including Cas9 (SaCas9), Cas9 (SpCas9), Cas12a (FnCas12a), and nCas9-activation-induced cytidine deaminase (nCas9-Target-AID). We successfully mutated () and () and the disease-associated genes (), and (), and confirmed that the mutated alleles were transmitted to progeny. mutants showed the expected phenotype, including absence of () siRNA and abundant expression of the GFP reporter. Progeny of both and null mutants were sterile. Our analysis of the phenotypes of the regenerated progeny indicated that except for the predicted phenotypes, they grew normally, with no unexpected traits. These results confirmed the utility of gene editing followed by protoplast regeneration in . We also developed a method for flowering and seed production in , allowing the regenerants to produce progeny without environmental constraints.