Efficient disruption and replacement of an effector gene in the oomycete Phytophthora sojae using CRISPR/Cas9.
Phytophthora sojae is an oomycete pathogen of soybean. As a result of its economic importance, P. sojae has become a model for the study of oomycete genetics, physiology and pathology. 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 a target, we observed that, in the absence of a homologous template, the repair of Cas9-induced DNA 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 as a result of 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 Avr4/6 with the NPT II gene. By testing the specific virulence of several NHEJ mutants and HDR-mediated gene replacements in soybean, 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 the study of functional genomics in Phytophthora, which provides new avenues for better control of this pathogen.
Interdisciplinary PhD Program in Genetics, Bioinformatics & Computational Biology, Virginia Tech, Blacksburg, VA, 24061, USA.|Center for Genome Research and Biocomputing and Department of Botany and Plant Pathology, Oregon State University, Corvallis, OR, 97331, USA.
Yufeng
Y
Fang
Interdisciplinary PhD Program in Genetics, Bioinformatics & Computational Biology, Virginia Tech, Blacksburg, VA, 24061, USA.|Center for Genome Research and Biocomputing and Department of Botany and Plant Pathology, Oregon State University, Corvallis, OR, 97331, USA.
Brett M
http://orcid.org/0000-0003-1549-2987
BM
Tyler
D000595:Amino Acid Sequence; D001483:Base Sequence; D064113:CRISPR-Cas Systems / Q000235:genetics*; D056426:Genetic Loci; D005821:Genetic Techniques; D016678:Genome; D008969:Molecular Sequence Data; D016296:Mutagenesis / Q000235:genetics*; D009154:Mutation / Q000235:genetics; D016366:Open Reading Frames / Q000235:genetics; D010641:Phenotype; D010838:Phytophthora / Q000235:genetics*; D017393:RNA Editing; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000378:metabolism; D013025:Glycine max / Q000235:genetics / Q000382:microbiology
D000094704:RNA, Guide, CRISPR-Cas Systems
Avr4/6; CRISPR/Cas9; Phytophthora sojae; RXLR effector; gene replacement; genome editing; oomycetes