Efficient disruption and replacement of an effector gene in the oomycete Phytophthora sojae using CRISPR/Cas9.
Identifieur interne : 000C50 ( Main/Exploration ); précédent : 000C49; suivant : 000C51Efficient disruption and replacement of an effector gene in the oomycete Phytophthora sojae using CRISPR/Cas9.
Auteurs : Yufeng Fang [États-Unis] ; Brett M. Tyler [États-Unis]Source :
- Molecular plant pathology [ 1364-3703 ] ; 2016.
Descripteurs français
- KwdFr :
- ARN guide (métabolisme), Cadres ouverts de lecture (génétique), Données de séquences moléculaires (MeSH), Génome (MeSH), Locus génétiques (MeSH), Mutagenèse (génétique), Mutation (génétique), Phytophthora (génétique), Phénotype (MeSH), Soja (génétique), Soja (microbiologie), Systèmes CRISPR-Cas (génétique), Séquence d'acides aminés (MeSH), Séquence nucléotidique (MeSH), Techniques génétiques (MeSH), Édition des ARN (MeSH).
- MESH :
- génétique : Cadres ouverts de lecture, Mutagenèse, Mutation, Phytophthora, Soja, Systèmes CRISPR-Cas.
- microbiologie : Soja.
- métabolisme : ARN guide.
- Données de séquences moléculaires, Génome, Locus génétiques, Phénotype, Séquence d'acides aminés, Séquence nucléotidique, Techniques génétiques, Édition des ARN.
English descriptors
- KwdEn :
- Amino Acid Sequence (MeSH), Base Sequence (MeSH), CRISPR-Cas Systems (genetics), Genetic Loci (MeSH), Genetic Techniques (MeSH), Genome (MeSH), Molecular Sequence Data (MeSH), Mutagenesis (genetics), Mutation (genetics), Open Reading Frames (genetics), Phenotype (MeSH), Phytophthora (genetics), RNA Editing (MeSH), RNA, Guide (metabolism), Soybeans (genetics), Soybeans (microbiology).
- MESH :
- chemical , metabolism : RNA, Guide.
- genetics : CRISPR-Cas Systems, Mutagenesis, Mutation, Open Reading Frames, Phytophthora, Soybeans.
- microbiology : Soybeans.
- Amino Acid Sequence, Base Sequence, Genetic Loci, Genetic Techniques, Genome, Molecular Sequence Data, Phenotype, RNA Editing.
Abstract
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.
DOI: 10.1111/mpp.12318
PubMed: 26507366
PubMed Central: PMC6638440
Affiliations:
Links toward previous steps (curation, corpus...)
Le document en format XML
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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. </div></front></TEI><pubmed><MedlineCitation Status="MEDLINE" Owner="NLM"><PMID Version="1">26507366</PMID><DateCompleted><Year>2016</Year><Month>10</Month><Day>14</Day></DateCompleted><DateRevised><Year>2020</Year><Month>02</Month><Day>25</Day></DateRevised><Article PubModel="Print-Electronic"><Journal><ISSN IssnType="Electronic">1364-3703</ISSN><JournalIssue CitedMedium="Internet"><Volume>17</Volume><Issue>1</Issue><PubDate><Year>2016</Year><Month>Jan</Month></PubDate></JournalIssue><Title>Molecular plant pathology</Title><ISOAbbreviation>Mol Plant Pathol</ISOAbbreviation></Journal><ArticleTitle>Efficient disruption and replacement of an effector gene in the oomycete Phytophthora sojae using CRISPR/Cas9.</ArticleTitle><Pagination><MedlinePgn>127-39</MedlinePgn></Pagination><ELocationID EIdType="doi" ValidYN="Y">10.1111/mpp.12318</ELocationID><Abstract><AbstractText>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. </AbstractText><CopyrightInformation>© 2015 THE AUTHORS. MOLECULAR PLANT PATHOLOGY PUBLISHED BY JOHN WILEY & SONS LTD AND BSPP.</CopyrightInformation></Abstract><AuthorList CompleteYN="Y"><Author ValidYN="Y"><LastName>Fang</LastName><ForeName>Yufeng</ForeName><Initials>Y</Initials><AffiliationInfo><Affiliation>Interdisciplinary PhD Program in Genetics, Bioinformatics & Computational Biology, Virginia Tech, Blacksburg, VA, 24061, USA.</Affiliation></AffiliationInfo><AffiliationInfo><Affiliation>Center for Genome Research and Biocomputing and Department of Botany and Plant Pathology, Oregon State University, Corvallis, OR, 97331, USA.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Tyler</LastName><ForeName>Brett M</ForeName><Initials>BM</Initials><Identifier Source="ORCID">http://orcid.org/0000-0003-1549-2987</Identifier><AffiliationInfo><Affiliation>Interdisciplinary PhD Program in Genetics, Bioinformatics & Computational Biology, Virginia Tech, Blacksburg, VA, 24061, USA.</Affiliation></AffiliationInfo><AffiliationInfo><Affiliation>Center for Genome Research and Biocomputing and Department of Botany and Plant Pathology, Oregon State University, Corvallis, OR, 97331, USA.</Affiliation></AffiliationInfo></Author></AuthorList><Language>eng</Language><PublicationTypeList><PublicationType UI="D016428">Journal Article</PublicationType><PublicationType UI="D013486">Research Support, U.S. Gov't, Non-P.H.S.</PublicationType></PublicationTypeList><ArticleDate DateType="Electronic"><Year>2015</Year><Month>11</Month><Day>11</Day></ArticleDate></Article><MedlineJournalInfo><Country>England</Country><MedlineTA>Mol Plant Pathol</MedlineTA><NlmUniqueID>100954969</NlmUniqueID><ISSNLinking>1364-3703</ISSNLinking></MedlineJournalInfo><ChemicalList><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="D017394">RNA, Guide</NameOfSubstance></Chemical></ChemicalList><CitationSubset>IM</CitationSubset><MeshHeadingList><MeshHeading><DescriptorName UI="D000595" 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MajorTopicYN="N">Mutation</DescriptorName><QualifierName UI="Q000235" MajorTopicYN="N">genetics</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D016366" MajorTopicYN="N">Open Reading Frames</DescriptorName><QualifierName UI="Q000235" MajorTopicYN="N">genetics</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D010641" MajorTopicYN="N">Phenotype</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D010838" MajorTopicYN="N">Phytophthora</DescriptorName><QualifierName UI="Q000235" MajorTopicYN="Y">genetics</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D017393" MajorTopicYN="N">RNA Editing</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D017394" MajorTopicYN="N">RNA, Guide</DescriptorName><QualifierName UI="Q000378" MajorTopicYN="N">metabolism</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D013025" MajorTopicYN="N">Soybeans</DescriptorName><QualifierName UI="Q000235" MajorTopicYN="N">genetics</QualifierName><QualifierName UI="Q000382" MajorTopicYN="N">microbiology</QualifierName></MeshHeading></MeshHeadingList><KeywordList Owner="NOTNLM"><Keyword MajorTopicYN="N">Avr4/6</Keyword><Keyword MajorTopicYN="N">CRISPR/Cas9</Keyword><Keyword MajorTopicYN="N">Phytophthora sojae</Keyword><Keyword MajorTopicYN="N">RXLR effector</Keyword><Keyword MajorTopicYN="N">gene replacement</Keyword><Keyword MajorTopicYN="N">genome editing</Keyword><Keyword MajorTopicYN="N">oomycetes</Keyword></KeywordList></MedlineCitation><PubmedData><History><PubMedPubDate PubStatus="entrez"><Year>2015</Year><Month>10</Month><Day>29</Day><Hour>6</Hour><Minute>0</Minute></PubMedPubDate><PubMedPubDate PubStatus="pubmed"><Year>2015</Year><Month>10</Month><Day>29</Day><Hour>6</Hour><Minute>0</Minute></PubMedPubDate><PubMedPubDate 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