Efficient Disruption and Replacement of an Effector Gene in the Oomycete Phytophthora sojae using CRISPR/Cas9

Phytophthora sojae is a pathogenic oomycete that infects soybean seedlings as well as stems and roots of established plants, costing growers $1-2 billion per year. Due to its economic importance, P. sojae has become a model for the study of oomycete genetics, physiology and pathology. Despite the availability of several genome sequences, the lack of efficient techniques for targeted mutagenesis and gene replacement have long hampered genetic studies of pathogenicity in Phytophthora species. Here, we describe a CRISPR/Cas9 system enabling rapid and efficient genome editing in P. sojae. Using the RXLR effector gene Avr4/6 as target, we observed that in the absence of a homologous template, the repair of Cas9-induced double-strand breaks (DSBs) in P. sojae was mediated by non-homologous end joining (NHEJ), primarily resulting in short indels. Most mutants were homozygous, presumably due to gene conversion triggered by Cas9-mediated cleavage of non-mutant alleles. When donor DNA was present, homology directed repair (HDR) was observed, which resulted in the replacement of the target gene with the donor DNA. By testing the specific virulence of several NHEJ mutants and HDR -mediated gene replacements on soybeans, we have validated the contribution of Avr4/6 to recognition by soybean R gene loci, Rps4 and Rps6, but also uncovered additional contributions to resistance by these two loci. Our results establish a powerful tool for studying functional genomics in Phytophthora, which provides new avenues for better control of this pathogen.