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 o...

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Bibliographic Details
Published inMolecular plant pathology Vol. 17; no. 1; pp. 127 - 139
Main Authors Fang, Yufeng, Tyler, Brett M.
Format Journal Article
LanguageEnglish
Published England Blackwell Science in collaboration with the British Society of Plant Pathology 2016
Blackwell Publishing Ltd
John Wiley & Sons, Inc
Subjects
Amino Acid Sequence
Avr4/6
Base Sequence
CRISPR-Cas Systems - genetics
CRISPR/Cas9
DNA repair
gene replacement
Genes
Genetic Loci
Genetic Techniques
Genome
genome editing
Glycine max - genetics
Glycine max - microbiology
Molecular Sequence Data
Mutagenesis - genetics
Mutation - genetics
Oomycetes
Open Reading Frames - genetics
Phenotype
Phytophthora - genetics
Phytophthora sojae
RNA Editing
RNA, Guide, CRISPR-Cas Systems - metabolism
RXLR effector
Avr4/6
Phytophthora sojae
genome editing
CRISPR/Cas9
gene replacement
oomycetes
RXLR effector
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Summary: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.
Bibliography:http://dx.doi.org/10.1111/mpp.12318
ArticleID:MPP12318
US Department of Agriculture (USDA) - No. 2011-68004-30104
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Fig. S1 Phytophthora U6 promoter evaluation. (A) Alignment of selected oomycete U6 genes, showing that U6 transcripts are highly conserved. The numbers of U6 genes were variable in different oomycete species: eight in P. sojae P6497 (PsojP6497), 127 in P. infestans (PinfT30-4), five in P. capsici LT1534 (PcapLT1534), one in P. parasitica (Ppar INRA-310) and one in Hyaloperonospora arabidopsidis (HaraEmoy2). Genome data were obtained from the fungidb.org website. (B) Alignment of the eight annotated P. sojae U6 genes. PsU6-1 was used to test promoter activity. The red lines indicate the border of the upstream and downstream tRNAs. (C) One of the 127 P. infestans U6 genes cloned to test U6 promoter activity. Top: position of the PiU6 gene on P. infestans T30-4 Supercontig 65. Bottom: PiU6 sequence used for promoter activity test. The putative U6 coding region is underlined; a putative TATA-box is indicated in red. (D) The plasmid used for testing the functions of the PsU6-1 and PiU6 promoters in P. sojae. Residues 1-150 bp of eGFP (enhanced green fluorescent protein) were used as a transcription detection marker. Arrows indicate the primer pair U6GFP_F and U6GFP_R used for cloning the eGFP fragment and also for the detection of U6 transcripts by reverse transcription-polymerase chain reaction (RT-PCR). The EcoNI restriction enzyme site used for insertion of the GFP detection marker is underlined in (A) and (B) and double underlined in (C).Fig. S2 Representative sequencing chromatograms of the Avr4/6 mutations in the single zoospore purified mutants. Regions in the box show the single guide RNA (sgRNA) target sites within the Avr4/6 gene. The unambiguous sequencing profiles indicate that these mutant lines are all homozygous.Fig. S3 Sanger sequencing profiles revealing that the subcultured Cas9:sgRNA transformant T47 (NHEJ-T47) and homology-directed repair (HDR) mutant T29 (HDR-T29-2) had non-homologous end-joining (NHEJ) mutations (1-bp insertion and deletion, respectively). Red triangles indicate the differences between wild-type (WT) and mutants.Fig. S4 Plasmid backbones used for the expression of hSpCas9 and single guide RNA (sgRNA) in Phytophthora sojae. (A) pYF2-2XGFP is used for tracking the subcellular localization and expression of PsNLS-fused hSpCas9. PsNLS is inserted into SacII and SpeI sites. hSpCas9 is inserted into SpeI and AflII sites for subcellular localization examination, and SpeI and ApaI sites for CRISPR expression. (B) pYF2.2-GFP is used for the expression of sgRNA (inserted into NheI and AgeI sites).Table S1 Sequences of the oligonucleotides used in this study.Appendix S1 Supplemental sequences. Sequences of plasmids used in this study.Methods S1 Generation of Phytophthora sojae CRISPR/Cas9 plasmids.
ObjectType-Article-1
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content type line 23
ISSN:1464-6722
1364-3703
DOI:10.1111/mpp.12318