Defining Transgene Insertion Sites and Off-Target Effects of Homology-Based Gene Silencing Informs the Application of Functional Genomics Tools in Phytophthora infestans.
Identifieur interne : 000546 ( Main/Exploration ); précédent : 000545; suivant : 000547Defining Transgene Insertion Sites and Off-Target Effects of Homology-Based Gene Silencing Informs the Application of Functional Genomics Tools in Phytophthora infestans.
Auteurs : Andrea L. Vu [États-Unis] ; Wiphawee Leesutthiphonchai [États-Unis] ; Audrey M V. Ah-Fong [États-Unis] ; Howard S. Judelson [États-Unis]Source :
- Molecular plant-microbe interactions : MPMI [ 0894-0282 ] ; 2019.
Descripteurs français
- KwdFr :
- MESH :
English descriptors
- KwdEn :
- MESH :
- genetics : Phytophthora infestans, Transgenes.
- Gene Silencing, Genomics.
Abstract
DNA transformation and homology-based transcriptional silencing are frequently used to assess gene function in Phytophthora spp. Since unplanned side-effects of these tools are not well-characterized, we used P. infestans to study plasmid integration sites and whether knockdowns caused by homology-dependent silencing spread to other genes. Insertions occurred both in gene-dense and gene-sparse regions but disproportionately near the 5' ends of genes, which disrupted native coding sequences. Microhomology at the recombination site between plasmid and chromosome was common. Studies of transformants silenced for 12 different gene targets indicated that neighbors within 500 nt were often cosilenced, regardless of whether hairpin or sense constructs were employed and the direction of transcription of the target. However, this cis spreading of silencing did not occur in all transformants obtained with the same plasmid. Genome-wide studies indicated that unlinked genes with partial complementarity with the silencing-inducing transgene were not usually down-regulated. We learned that hairpin or sense transgenes were not cosilenced with the target in all transformants, which informs how screens for silencing should be performed. We conclude that transformation and gene silencing can be reliable tools for functional genomics in Phytophthora spp. but must be used carefully, especially by testing for the spread of silencing to genes flanking the target.
DOI: 10.1094/MPMI-09-18-0265-TA
PubMed: 30811313
Affiliations:
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Ah-Fong</name><affiliation wicri:level="2"><nlm:affiliation>Department of Microbiology and Plant Pathology, University of California, Riverside, CA 92521, U.S.A.</nlm:affiliation><country xml:lang="fr">États-Unis</country><wicri:regionArea>Department of Microbiology and Plant Pathology, University of California, Riverside, CA 92521</wicri:regionArea><placeName><region type="state">Californie</region></placeName></affiliation></author><author><name sortKey="Judelson, Howard S" sort="Judelson, Howard S" uniqKey="Judelson H" first="Howard S" last="Judelson">Howard S. 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Insertions occurred both in gene-dense and gene-sparse regions but disproportionately near the 5' ends of genes, which disrupted native coding sequences. Microhomology at the recombination site between plasmid and chromosome was common. Studies of transformants silenced for 12 different gene targets indicated that neighbors within 500 nt were often cosilenced, regardless of whether hairpin or sense constructs were employed and the direction of transcription of the target. However, this <i>cis</i> spreading of silencing did not occur in all transformants obtained with the same plasmid. Genome-wide studies indicated that unlinked genes with partial complementarity with the silencing-inducing transgene were not usually down-regulated. We learned that hairpin or sense transgenes were not cosilenced with the target in all transformants, which informs how screens for silencing should be performed. We conclude that transformation and gene silencing can be reliable tools for functional genomics in <i>Phytophthora</i> spp. but must be used carefully, especially by testing for the spread of silencing to genes flanking the target.</div></front></TEI><pubmed><MedlineCitation Status="MEDLINE" IndexingMethod="Curated" Owner="NLM"><PMID Version="1">30811313</PMID><DateCompleted><Year>2019</Year><Month>08</Month><Day>01</Day></DateCompleted><DateRevised><Year>2020</Year><Month>05</Month><Day>02</Day></DateRevised><Article PubModel="Print-Electronic"><Journal><ISSN IssnType="Print">0894-0282</ISSN><JournalIssue CitedMedium="Print"><Volume>32</Volume><Issue>8</Issue><PubDate><Year>2019</Year><Month>Aug</Month></PubDate></JournalIssue><Title>Molecular plant-microbe interactions : MPMI</Title><ISOAbbreviation>Mol Plant Microbe Interact</ISOAbbreviation></Journal><ArticleTitle>Defining Transgene Insertion Sites and Off-Target Effects of Homology-Based Gene Silencing Informs the Application of Functional Genomics Tools in <i>Phytophthora infestans</i>.</ArticleTitle><Pagination><MedlinePgn>915-927</MedlinePgn></Pagination><ELocationID EIdType="doi" ValidYN="Y">10.1094/MPMI-09-18-0265-TA</ELocationID><Abstract><AbstractText>DNA transformation and homology-based transcriptional silencing are frequently used to assess gene function in <i>Phytophthora</i> spp. Since unplanned side-effects of these tools are not well-characterized, we used <i>P. infestans</i> to study plasmid integration sites and whether knockdowns caused by homology-dependent silencing spread to other genes. Insertions occurred both in gene-dense and gene-sparse regions but disproportionately near the 5' ends of genes, which disrupted native coding sequences. Microhomology at the recombination site between plasmid and chromosome was common. Studies of transformants silenced for 12 different gene targets indicated that neighbors within 500 nt were often cosilenced, regardless of whether hairpin or sense constructs were employed and the direction of transcription of the target. However, this <i>cis</i> spreading of silencing did not occur in all transformants obtained with the same plasmid. Genome-wide studies indicated that unlinked genes with partial complementarity with the silencing-inducing transgene were not usually down-regulated. We learned that hairpin or sense transgenes were not cosilenced with the target in all transformants, which informs how screens for silencing should be performed. We conclude that transformation and gene silencing can be reliable tools for functional genomics in <i>Phytophthora</i> spp. but must be used carefully, especially by testing for the spread of silencing to genes flanking the target.</AbstractText></Abstract><AuthorList CompleteYN="Y"><Author ValidYN="Y"><LastName>Vu</LastName><ForeName>Andrea L</ForeName><Initials>AL</Initials><AffiliationInfo><Affiliation>Department of Microbiology and Plant Pathology, University of California, Riverside, CA 92521, U.S.A.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Leesutthiphonchai</LastName><ForeName>Wiphawee</ForeName><Initials>W</Initials><AffiliationInfo><Affiliation>Department of Microbiology and Plant Pathology, University of California, Riverside, CA 92521, U.S.A.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Ah-Fong</LastName><ForeName>Audrey M V</ForeName><Initials>AMV</Initials><AffiliationInfo><Affiliation>Department of Microbiology and Plant Pathology, University of California, Riverside, CA 92521, U.S.A.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Judelson</LastName><ForeName>Howard S</ForeName><Initials>HS</Initials><Identifier Source="ORCID">http://orcid.org/0000-0001-7865-6235</Identifier><AffiliationInfo><Affiliation>Department of Microbiology and Plant Pathology, University of California, Riverside, CA 92521, U.S.A.</Affiliation></AffiliationInfo></Author></AuthorList><Language>eng</Language><GrantList CompleteYN="Y"><Grant><GrantID>S10 OD016290</GrantID><Acronym>OD</Acronym><Agency>NIH HHS</Agency><Country>United States</Country></Grant></GrantList><PublicationTypeList><PublicationType UI="D016428">Journal Article</PublicationType></PublicationTypeList><ArticleDate DateType="Electronic"><Year>2019</Year><Month>06</Month><Day>25</Day></ArticleDate></Article><MedlineJournalInfo><Country>United States</Country><MedlineTA>Mol Plant Microbe Interact</MedlineTA><NlmUniqueID>9107902</NlmUniqueID><ISSNLinking>0894-0282</ISSNLinking></MedlineJournalInfo><CitationSubset>IM</CitationSubset><MeshHeadingList><MeshHeading><DescriptorName UI="D020868" MajorTopicYN="Y">Gene Silencing</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D023281" MajorTopicYN="Y">Genomics</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D055750" MajorTopicYN="Y">Phytophthora infestans</DescriptorName><QualifierName UI="Q000235" MajorTopicYN="N">genetics</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D019076" MajorTopicYN="Y">Transgenes</DescriptorName><QualifierName UI="Q000235" MajorTopicYN="N">genetics</QualifierName></MeshHeading></MeshHeadingList><KeywordList Owner="NOTNLM"><Keyword MajorTopicYN="N"> diseases</Keyword><Keyword MajorTopicYN="N">genomics</Keyword><Keyword MajorTopicYN="N">oomycete-plant interactions</Keyword></KeywordList></MedlineCitation><PubmedData><History><PubMedPubDate PubStatus="pubmed"><Year>2019</Year><Month>2</Month><Day>28</Day><Hour>6</Hour><Minute>0</Minute></PubMedPubDate><PubMedPubDate PubStatus="medline"><Year>2019</Year><Month>8</Month><Day>2</Day><Hour>6</Hour><Minute>0</Minute></PubMedPubDate><PubMedPubDate PubStatus="entrez"><Year>2019</Year><Month>2</Month><Day>28</Day><Hour>6</Hour><Minute>0</Minute></PubMedPubDate></History><PublicationStatus>ppublish</PublicationStatus><ArticleIdList><ArticleId IdType="pubmed">30811313</ArticleId><ArticleId IdType="doi">10.1094/MPMI-09-18-0265-TA</ArticleId></ArticleIdList></PubmedData></pubmed><affiliations><list><country><li>États-Unis</li></country><region><li>Californie</li></region></list><tree><country name="États-Unis"><region name="Californie"><name sortKey="Vu, Andrea L" sort="Vu, Andrea L" uniqKey="Vu A" first="Andrea L" last="Vu">Andrea L. Vu</name></region><name sortKey="Ah Fong, Audrey M V" sort="Ah Fong, Audrey M V" uniqKey="Ah Fong A" first="Audrey M V" last="Ah-Fong">Audrey M V. Ah-Fong</name><name sortKey="Judelson, Howard S" sort="Judelson, Howard S" uniqKey="Judelson H" first="Howard S" last="Judelson">Howard S. Judelson</name><name sortKey="Leesutthiphonchai, Wiphawee" sort="Leesutthiphonchai, Wiphawee" uniqKey="Leesutthiphonchai W" first="Wiphawee" last="Leesutthiphonchai">Wiphawee Leesutthiphonchai</name></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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