Biomaterial Database

Efficient disruption and replacement of an effector gene in the oomycete Phytophthora sojae using CRISPR/Cas9. 2016

Yufeng Fang, and Brett M Tyler

Interdisciplinary PhD Program in Genetics, Bioinformatics & Computational Biology, Virginia Tech, Blacksburg, VA, 24061, USA.

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.

UI	MeSH Term	Description	Entries
D008969	Molecular Sequence Data	Descriptions of specific amino acid, carbohydrate, or nucleotide sequences which have appeared in the published literature and/or are deposited in and maintained by databanks such as GENBANK, European Molecular Biology Laboratory (EMBL), National Biomedical Research Foundation (NBRF), or other sequence repositories.	Sequence Data, Molecular,Molecular Sequencing Data,Data, Molecular Sequence,Data, Molecular Sequencing,Sequencing Data, Molecular
D009154	Mutation	Any detectable and heritable change in the genetic material that causes a change in the GENOTYPE and which is transmitted to daughter cells and to succeeding generations.	Mutations
D010641	Phenotype	The outward appearance of the individual. It is the product of interactions between genes, and between the GENOTYPE and the environment.	Phenotypes
D010838	Phytophthora	A genus of destructive parasitic OOMYCETES in the family Peronosporaceae, order Peronosporales, affecting numerous fruit, vegetable, and other crops. Differentiation of zoospores usually takes place in the sporangium and no vesicle is formed. It was previously considered a fungus.	Phytophthora sp. ex Parrotia persica,Sudden Oak Death Agent,Phytophthora cinnamomi,Phytophthora parasitica,Phytophthora ramorum,Phytophthora sojae
D005821	Genetic Techniques	Chromosomal, biochemical, intracellular, and other methods used in the study of genetics.	Genetic Technic,Genetic Technics,Genetic Technique,Technic, Genetic,Technics, Genetic,Technique, Genetic,Techniques, Genetic
D000094704	RNA, Guide, CRISPR-Cas Systems	A component of CRISPR-Cas SYSTEMS. Cas endodeoxyribonucleases assemble with a guide RNA complex, a hybrid of CRISPR RNA (crRNA) and transactivating crRNA (tracrRNA) molecules, to form an active complex that cleaves DNA. crRNA and tracrRNA can be synthetically fused into a single RNA molecule, namely single guide RNA. Synthetic sgRNA is used with CRISPR-Cas SYSTEMS for targeted GENE EDITING.	CRISPR Guide RNA,CRISPR-Cas Systems sgRNA (Single Guide RNA),Guide RNA (CRISPR-Cas Systems),Guide RNA, CRISPR-Cas Systems,RNA, CRISPR Guide,RNA, Guide (CRISPR-Cas Systems),RNA, Single Guide,RNA, Single-Guide,Single Guide RNA,Single-Guide RNA,Transactivating crRNA,crRNA,crRNA, Transactivating,sgRNA (CRISPR-Cas Systems),sgRNA (Single-Guide RNA),tracrRNA,Guide RNA, CRISPR,Guide RNA, CRISPR Cas Systems,Guide RNA, Single
D000595	Amino Acid Sequence	The order of amino acids as they occur in a polypeptide chain. This is referred to as the primary structure of proteins. It is of fundamental importance in determining PROTEIN CONFORMATION.	Protein Structure, Primary,Amino Acid Sequences,Sequence, Amino Acid,Sequences, Amino Acid,Primary Protein Structure,Primary Protein Structures,Protein Structures, Primary,Structure, Primary Protein,Structures, Primary Protein
D001483	Base Sequence	The sequence of PURINES and PYRIMIDINES in nucleic acids and polynucleotides. It is also called nucleotide sequence.	DNA Sequence,Nucleotide Sequence,RNA Sequence,DNA Sequences,Base Sequences,Nucleotide Sequences,RNA Sequences,Sequence, Base,Sequence, DNA,Sequence, Nucleotide,Sequence, RNA,Sequences, Base,Sequences, DNA,Sequences, Nucleotide,Sequences, RNA
D013025	Glycine max	An annual legume. The SEEDS of this plant are edible and used to produce a variety of SOY FOODS.	Soy Beans,Soybeans,Bean, Soy,Beans, Soy,Soy Bean,Soybean
D016296	Mutagenesis	Process of generating a genetic MUTATION. It may occur spontaneously or be induced by MUTAGENS.	Mutageneses