Protocol: transient expression system for functional genomics in the tropical tree Theobroma cacao L.
Identifieur interne : 000279 ( Main/Exploration ); précédent : 000278; suivant : 000280Protocol: transient expression system for functional genomics in the tropical tree Theobroma cacao L.
Auteurs : Andrew S. Fister [États-Unis] ; Zi Shi [États-Unis] ; Yufan Zhang [États-Unis] ; Emily E. Helliwell [États-Unis] ; Siela N. Maximova [États-Unis] ; Mark J. Guiltinan [États-Unis]Source :
- Plant methods [ 1746-4811 ] ; 2016.
Abstract
BACKGROUND
Theobroma cacao L., the source of cocoa, is a crop of significant economic value around the world. To facilitate the study of gene function in cacao we have developed a rapid Agrobacterium-mediated transient genetic transformation protocol. Here we present a detailed methodology for our transformation assay, as well as an assay for inoculation of cacao leaves with pathogens.
RESULTS
Agrobacterium tumefaciens cultures are induced then vacuum-infiltrated into cacao leaves. Transformation success can be gauged 48 h after infiltration by observation of green fluorescent protein and by qRT-PCR. We clarify the characteristics of cacao leaf stages and demonstrate that our strategy efficiently transforms leaves of developmental stage C. The transformation protocol has high efficacy in stage C leaves of four of eight tested genotypes. We also present the functional analysis of cacao chitinase overexpression using the transient transformation system, which resulted in decreased pathogen biomass and lesion size after infection with Phytophthora tropicalis.
CONCLUSIONS
Leaves expressing transgenes of interest can be used in subsequent functional genetic assays such as pathogen bioassay, metabolic analysis, gene expression analysis etc. This transformation protocol can be carried out in 1 day, and the transgenes expressing leaf tissue can be maintained in petri dishes for 5-7 days, allowing sufficient time for performance of additional downstream gene functional analysis. Application of these methods greatly increases the rapidity with which candidate genes with roles in defense can be tested.
DOI: 10.1186/s13007-016-0119-5
PubMed: 26973706
PubMed Central: PMC4788949
Affiliations:
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Fister</name><affiliation wicri:level="2"><nlm:affiliation>The Huck Institutes of the Life Sciences, The Pennsylvania State University, 422 Life Sciences Building, University Park, PA 16802 USA.</nlm:affiliation><country xml:lang="fr">États-Unis</country><placeName><region type="state">Pennsylvanie</region></placeName><wicri:cityArea>The Huck Institutes of the Life Sciences, The Pennsylvania State University, 422 Life Sciences Building, University Park</wicri:cityArea></affiliation></author><author><name sortKey="Shi, Zi" sort="Shi, Zi" uniqKey="Shi Z" first="Zi" last="Shi">Zi Shi</name><affiliation wicri:level="2"><nlm:affiliation>Center for Applied Genetic Technologies, University of Georgia, Athens, GA 30602 USA.</nlm:affiliation><country xml:lang="fr">États-Unis</country><placeName><region type="state">Géorgie (États-Unis)</region></placeName><wicri:cityArea>Center for Applied Genetic Technologies, University of Georgia, Athens</wicri:cityArea></affiliation></author><author><name sortKey="Zhang, Yufan" sort="Zhang, Yufan" uniqKey="Zhang Y" first="Yufan" last="Zhang">Yufan Zhang</name><affiliation wicri:level="2"><nlm:affiliation>Department of Electrical Engineering, Princeton University, Princeton, NJ 08544 USA.</nlm:affiliation><country xml:lang="fr">États-Unis</country><placeName><region type="state">New Jersey</region></placeName><wicri:cityArea>Department of Electrical Engineering, Princeton University, Princeton</wicri:cityArea></affiliation></author><author><name sortKey="Helliwell, Emily E" sort="Helliwell, Emily E" uniqKey="Helliwell E" first="Emily E" last="Helliwell">Emily E. Helliwell</name><affiliation wicri:level="2"><nlm:affiliation>Department of Botany and Plant Pathology, Center for Genome Research and Biocomputing, Oregon State University, Corvallis, OR 97331 USA.</nlm:affiliation><country xml:lang="fr">États-Unis</country><placeName><region type="state">Oregon</region></placeName><wicri:cityArea>Department of Botany and Plant Pathology, Center for Genome Research and Biocomputing, Oregon State University, Corvallis</wicri:cityArea></affiliation></author><author><name sortKey="Maximova, Siela N" sort="Maximova, Siela N" uniqKey="Maximova S" first="Siela N" last="Maximova">Siela N. Maximova</name><affiliation wicri:level="2"><nlm:affiliation>The Huck Institutes of the Life Sciences, The Pennsylvania State University, 422 Life Sciences Building, University Park, PA 16802 USA ; The Department of Plant Science, The Pennsylvania State University, University Park, PA 16802 USA.</nlm:affiliation><country xml:lang="fr">États-Unis</country><placeName><region type="state">Pennsylvanie</region></placeName><wicri:cityArea>The Huck Institutes of the Life Sciences, The Pennsylvania State University, 422 Life Sciences Building, University Park, PA 16802 USA ; The Department of Plant Science, The Pennsylvania State University, University Park</wicri:cityArea></affiliation></author><author><name sortKey="Guiltinan, Mark J" sort="Guiltinan, Mark J" uniqKey="Guiltinan M" first="Mark J" last="Guiltinan">Mark J. Guiltinan</name><affiliation wicri:level="2"><nlm:affiliation>The Huck Institutes of the Life Sciences, The Pennsylvania State University, 422 Life Sciences Building, University Park, PA 16802 USA ; The Department of Plant Science, The Pennsylvania State University, University Park, PA 16802 USA.</nlm:affiliation><country xml:lang="fr">États-Unis</country><placeName><region type="state">Pennsylvanie</region></placeName><wicri:cityArea>The Huck Institutes of the Life Sciences, The Pennsylvania State University, 422 Life Sciences Building, University Park, PA 16802 USA ; The Department of Plant Science, The Pennsylvania State University, University Park</wicri:cityArea></affiliation></author></analytic><series><title level="j">Plant methods</title><idno type="ISSN">1746-4811</idno><imprint><date when="2016" type="published">2016</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p><b>BACKGROUND</b></p><p>Theobroma cacao L., the source of cocoa, is a crop of significant economic value around the world. To facilitate the study of gene function in cacao we have developed a rapid Agrobacterium-mediated transient genetic transformation protocol. Here we present a detailed methodology for our transformation assay, as well as an assay for inoculation of cacao leaves with pathogens.</p></div><div type="abstract" xml:lang="en"><p><b>RESULTS</b></p><p>Agrobacterium tumefaciens cultures are induced then vacuum-infiltrated into cacao leaves. Transformation success can be gauged 48 h after infiltration by observation of green fluorescent protein and by qRT-PCR. We clarify the characteristics of cacao leaf stages and demonstrate that our strategy efficiently transforms leaves of developmental stage C. The transformation protocol has high efficacy in stage C leaves of four of eight tested genotypes. We also present the functional analysis of cacao chitinase overexpression using the transient transformation system, which resulted in decreased pathogen biomass and lesion size after infection with Phytophthora tropicalis.</p></div><div type="abstract" xml:lang="en"><p><b>CONCLUSIONS</b></p><p>Leaves expressing transgenes of interest can be used in subsequent functional genetic assays such as pathogen bioassay, metabolic analysis, gene expression analysis etc. This transformation protocol can be carried out in 1 day, and the transgenes expressing leaf tissue can be maintained in petri dishes for 5-7 days, allowing sufficient time for performance of additional downstream gene functional analysis. Application of these methods greatly increases the rapidity with which candidate genes with roles in defense can be tested.</p></div></front></TEI><pubmed><MedlineCitation Status="PubMed-not-MEDLINE" Owner="NLM"><PMID Version="1">26973706</PMID><DateCompleted><Year>2016</Year><Month>03</Month><Day>14</Day></DateCompleted><DateRevised><Year>2020</Year><Month>09</Month><Day>30</Day></DateRevised><Article PubModel="Electronic-eCollection"><Journal><ISSN IssnType="Print">1746-4811</ISSN><JournalIssue CitedMedium="Print"><Volume>12</Volume><PubDate><Year>2016</Year></PubDate></JournalIssue><Title>Plant methods</Title><ISOAbbreviation>Plant Methods</ISOAbbreviation></Journal><ArticleTitle>Protocol: transient expression system for functional genomics in the tropical tree Theobroma cacao L.</ArticleTitle><Pagination><MedlinePgn>19</MedlinePgn></Pagination><ELocationID EIdType="doi" ValidYN="Y">10.1186/s13007-016-0119-5</ELocationID><Abstract><AbstractText Label="BACKGROUND" NlmCategory="BACKGROUND">Theobroma cacao L., the source of cocoa, is a crop of significant economic value around the world. To facilitate the study of gene function in cacao we have developed a rapid Agrobacterium-mediated transient genetic transformation protocol. Here we present a detailed methodology for our transformation assay, as well as an assay for inoculation of cacao leaves with pathogens.</AbstractText><AbstractText Label="RESULTS" NlmCategory="RESULTS">Agrobacterium tumefaciens cultures are induced then vacuum-infiltrated into cacao leaves. Transformation success can be gauged 48 h after infiltration by observation of green fluorescent protein and by qRT-PCR. We clarify the characteristics of cacao leaf stages and demonstrate that our strategy efficiently transforms leaves of developmental stage C. The transformation protocol has high efficacy in stage C leaves of four of eight tested genotypes. We also present the functional analysis of cacao chitinase overexpression using the transient transformation system, which resulted in decreased pathogen biomass and lesion size after infection with Phytophthora tropicalis.</AbstractText><AbstractText Label="CONCLUSIONS" NlmCategory="CONCLUSIONS">Leaves expressing transgenes of interest can be used in subsequent functional genetic assays such as pathogen bioassay, metabolic analysis, gene expression analysis etc. This transformation protocol can be carried out in 1 day, and the transgenes expressing leaf tissue can be maintained in petri dishes for 5-7 days, allowing sufficient time for performance of additional downstream gene functional analysis. Application of these methods greatly increases the rapidity with which candidate genes with roles in defense can be tested.</AbstractText></Abstract><AuthorList CompleteYN="Y"><Author ValidYN="Y"><LastName>Fister</LastName><ForeName>Andrew S</ForeName><Initials>AS</Initials><AffiliationInfo><Affiliation>The Huck Institutes of the Life Sciences, The Pennsylvania State University, 422 Life Sciences Building, University Park, PA 16802 USA.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Shi</LastName><ForeName>Zi</ForeName><Initials>Z</Initials><AffiliationInfo><Affiliation>Center for Applied Genetic Technologies, University of Georgia, Athens, GA 30602 USA.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Zhang</LastName><ForeName>Yufan</ForeName><Initials>Y</Initials><AffiliationInfo><Affiliation>Department of Electrical Engineering, Princeton University, Princeton, NJ 08544 USA.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Helliwell</LastName><ForeName>Emily E</ForeName><Initials>EE</Initials><AffiliationInfo><Affiliation>Department of Botany and Plant Pathology, Center for Genome Research and Biocomputing, Oregon State University, Corvallis, OR 97331 USA.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Maximova</LastName><ForeName>Siela N</ForeName><Initials>SN</Initials><AffiliationInfo><Affiliation>The Huck Institutes of the Life Sciences, The Pennsylvania State University, 422 Life Sciences Building, University Park, PA 16802 USA ; The Department of Plant Science, The Pennsylvania State University, University Park, PA 16802 USA.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Guiltinan</LastName><ForeName>Mark J</ForeName><Initials>MJ</Initials><Identifier Source="ORCID">0000-0003-0018-9846</Identifier><AffiliationInfo><Affiliation>The Huck Institutes of the Life Sciences, The Pennsylvania State University, 422 Life Sciences Building, University Park, PA 16802 USA ; The Department of Plant Science, The Pennsylvania State University, University Park, PA 16802 USA.</Affiliation></AffiliationInfo></Author></AuthorList><Language>eng</Language><PublicationTypeList><PublicationType UI="D016428">Journal Article</PublicationType></PublicationTypeList><ArticleDate DateType="Electronic"><Year>2016</Year><Month>03</Month><Day>11</Day></ArticleDate></Article><MedlineJournalInfo><Country>England</Country><MedlineTA>Plant Methods</MedlineTA><NlmUniqueID>101245798</NlmUniqueID><ISSNLinking>1746-4811</ISSNLinking></MedlineJournalInfo></MedlineCitation><PubmedData><History><PubMedPubDate PubStatus="received"><Year>2015</Year><Month>12</Month><Day>01</Day></PubMedPubDate><PubMedPubDate PubStatus="accepted"><Year>2016</Year><Month>03</Month><Day>01</Day></PubMedPubDate><PubMedPubDate PubStatus="entrez"><Year>2016</Year><Month>3</Month><Day>15</Day><Hour>6</Hour><Minute>0</Minute></PubMedPubDate><PubMedPubDate PubStatus="pubmed"><Year>2016</Year><Month>3</Month><Day>15</Day><Hour>6</Hour><Minute>0</Minute></PubMedPubDate><PubMedPubDate PubStatus="medline"><Year>2016</Year><Month>3</Month><Day>15</Day><Hour>6</Hour><Minute>1</Minute></PubMedPubDate></History><PublicationStatus>epublish</PublicationStatus><ArticleIdList><ArticleId IdType="pubmed">26973706</ArticleId><ArticleId IdType="doi">10.1186/s13007-016-0119-5</ArticleId><ArticleId IdType="pii">119</ArticleId><ArticleId IdType="pmc">PMC4788949</ArticleId></ArticleIdList><ReferenceList><Reference><Citation>Science. 1987 Sep 4;237(4819):1176-83</Citation><ArticleIdList><ArticleId IdType="pubmed">17801640</ArticleId></ArticleIdList></Reference><Reference><Citation>BMC Plant Biol. 2013 Dec 06;13:204</Citation><ArticleIdList><ArticleId IdType="pubmed">24314063</ArticleId></ArticleIdList></Reference><Reference><Citation>Genome Biol. 2013 Jun 03;14(6):r53</Citation><ArticleIdList><ArticleId 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Jersey</li><li>Oregon</li><li>Pennsylvanie</li></region></list><tree><country name="États-Unis"><region name="Pennsylvanie"><name sortKey="Fister, Andrew S" sort="Fister, Andrew S" uniqKey="Fister A" first="Andrew S" last="Fister">Andrew S. Fister</name></region><name sortKey="Guiltinan, Mark J" sort="Guiltinan, Mark J" uniqKey="Guiltinan M" first="Mark J" last="Guiltinan">Mark J. Guiltinan</name><name sortKey="Helliwell, Emily E" sort="Helliwell, Emily E" uniqKey="Helliwell E" first="Emily E" last="Helliwell">Emily E. Helliwell</name><name sortKey="Maximova, Siela N" sort="Maximova, Siela N" uniqKey="Maximova S" first="Siela N" last="Maximova">Siela N. Maximova</name><name sortKey="Shi, Zi" sort="Shi, Zi" uniqKey="Shi Z" first="Zi" last="Shi">Zi Shi</name><name sortKey="Zhang, Yufan" sort="Zhang, Yufan" uniqKey="Zhang Y" first="Yufan" last="Zhang">Yufan Zhang</name></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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