Transient Expression of CRISPR/Cas9 Machinery Targeting TcNPR3 Enhances Defense Response in Theobroma cacao.
Identifieur interne : 000628 ( Main/Exploration ); précédent : 000627; suivant : 000629Transient Expression of CRISPR/Cas9 Machinery Targeting TcNPR3 Enhances Defense Response in Theobroma cacao.
Auteurs : Andrew S. Fister [États-Unis] ; Lena Landherr [États-Unis] ; Siela N. Maximova [États-Unis] ; Mark J. Guiltinan [États-Unis]Source :
- Frontiers in plant science [ 1664-462X ] ; 2018.
Abstract
Theobroma cacao, the source of cocoa, suffers significant losses to a variety of pathogens resulting in reduced incomes for millions of farmers in developing countries. Development of disease resistant cacao varieties is an essential strategy to combat this threat, but is limited by sources of genetic resistance and the slow generation time of this tropical tree crop. In this study, we present the first application of genome editing technology in cacao, using Agrobacterium-mediated transient transformation to introduce CRISPR/Cas9 components into cacao leaves and cotyledon cells. As a first proof of concept, we targeted the cacao Non-Expressor of Pathogenesis-Related 3 (TcNPR3) gene, a suppressor of the defense response. After demonstrating activity of designed single-guide RNAs (sgRNA) in vitro, we used Agrobacterium to introduce a CRISPR/Cas9 system into leaf tissue, and identified the presence of deletions in 27% of TcNPR3 copies in the treated tissues. The edited tissue exhibited an increased resistance to infection with the cacao pathogen Phytophthora tropicalis and elevated expression of downstream defense genes. Analysis of off-target mutagenesis in sequences similar to sgRNA target sites using high-throughput sequencing did not reveal mutations above background sequencing error rates. These results confirm the function of NPR3 as a repressor of the cacao immune system and demonstrate the application of CRISPR/Cas9 as a powerful functional genomics tool for cacao. Several stably transformed and genome edited somatic embryos were obtained via Agrobacterium-mediated transformation, and ongoing work will test the effectiveness of this approach at a whole plant level.
DOI: 10.3389/fpls.2018.00268
PubMed: 29552023
PubMed Central: PMC5841092
Affiliations:
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Guiltinan</name><affiliation wicri:level="4"><nlm:affiliation>Department of Plant Science, Pennsylvania State University, University Park, PA, United States.</nlm:affiliation><country xml:lang="fr">États-Unis</country><wicri:regionArea>Department of Plant Science, Pennsylvania State University, University Park, PA</wicri:regionArea><placeName><region type="state">Pennsylvanie</region><settlement type="city">University Park (Pennsylvanie)</settlement></placeName><orgName type="university">Université d'État de Pennsylvanie</orgName></affiliation><affiliation wicri:level="4"><nlm:affiliation>Huck Institutes of the Life Sciences, Pennsylvania State University, University Park, PA, United States.</nlm:affiliation><country xml:lang="fr">États-Unis</country><wicri:regionArea>Huck Institutes of the Life Sciences, Pennsylvania State University, University Park, PA</wicri:regionArea><placeName><region type="state">Pennsylvanie</region><settlement type="city">University Park (Pennsylvanie)</settlement></placeName><orgName type="university">Université d'État de Pennsylvanie</orgName></affiliation></author></analytic><series><title level="j">Frontiers in plant science</title><idno type="ISSN">1664-462X</idno><imprint><date when="2018" type="published">2018</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><i>Theobroma cacao</i>, the source of cocoa, suffers significant losses to a variety of pathogens resulting in reduced incomes for millions of farmers in developing countries. Development of disease resistant cacao varieties is an essential strategy to combat this threat, but is limited by sources of genetic resistance and the slow generation time of this tropical tree crop. In this study, we present the first application of genome editing technology in cacao, using Agrobacterium-mediated transient transformation to introduce CRISPR/Cas9 components into cacao leaves and cotyledon cells. As a first proof of concept, we targeted the cacao <i>Non-Expressor of Pathogenesis-Related 3 (TcNPR3)</i> gene, a suppressor of the defense response. After demonstrating activity of designed single-guide RNAs (sgRNA) <i>in vitro</i>, we used <i>Agrobacterium</i> to introduce a CRISPR/Cas9 system into leaf tissue, and identified the presence of deletions in 27% of <i>TcNPR3</i> copies in the treated tissues. The edited tissue exhibited an increased resistance to infection with the cacao pathogen <i>Phytophthora tropicalis</i> and elevated expression of downstream defense genes. Analysis of off-target mutagenesis in sequences similar to sgRNA target sites using high-throughput sequencing did not reveal mutations above background sequencing error rates. These results confirm the function of NPR3 as a repressor of the cacao immune system and demonstrate the application of CRISPR/Cas9 as a powerful functional genomics tool for cacao. Several stably transformed and genome edited somatic embryos were obtained via <i>Agrobacterium</i>-mediated transformation, and ongoing work will test the effectiveness of this approach at a whole plant level.</div></front></TEI><pubmed><MedlineCitation Status="PubMed-not-MEDLINE" Owner="NLM"><PMID Version="1">29552023</PMID><DateRevised><Year>2020</Year><Month>10</Month><Day>01</Day></DateRevised><Article PubModel="Electronic-eCollection"><Journal><ISSN IssnType="Print">1664-462X</ISSN><JournalIssue CitedMedium="Print"><Volume>9</Volume><PubDate><Year>2018</Year></PubDate></JournalIssue><Title>Frontiers in plant science</Title><ISOAbbreviation>Front Plant Sci</ISOAbbreviation></Journal><ArticleTitle>Transient Expression of CRISPR/Cas9 Machinery Targeting <i>TcNPR3</i> Enhances Defense Response in <i>Theobroma cacao</i>.</ArticleTitle><Pagination><MedlinePgn>268</MedlinePgn></Pagination><ELocationID EIdType="doi" ValidYN="Y">10.3389/fpls.2018.00268</ELocationID><Abstract><AbstractText><i>Theobroma cacao</i>, the source of cocoa, suffers significant losses to a variety of pathogens resulting in reduced incomes for millions of farmers in developing countries. Development of disease resistant cacao varieties is an essential strategy to combat this threat, but is limited by sources of genetic resistance and the slow generation time of this tropical tree crop. In this study, we present the first application of genome editing technology in cacao, using Agrobacterium-mediated transient transformation to introduce CRISPR/Cas9 components into cacao leaves and cotyledon cells. As a first proof of concept, we targeted the cacao <i>Non-Expressor of Pathogenesis-Related 3 (TcNPR3)</i> gene, a suppressor of the defense response. After demonstrating activity of designed single-guide RNAs (sgRNA) <i>in vitro</i>, we used <i>Agrobacterium</i> to introduce a CRISPR/Cas9 system into leaf tissue, and identified the presence of deletions in 27% of <i>TcNPR3</i> copies in the treated tissues. The edited tissue exhibited an increased resistance to infection with the cacao pathogen <i>Phytophthora tropicalis</i> and elevated expression of downstream defense genes. Analysis of off-target mutagenesis in sequences similar to sgRNA target sites using high-throughput sequencing did not reveal mutations above background sequencing error rates. These results confirm the function of NPR3 as a repressor of the cacao immune system and demonstrate the application of CRISPR/Cas9 as a powerful functional genomics tool for cacao. Several stably transformed and genome edited somatic embryos were obtained via <i>Agrobacterium</i>-mediated transformation, and ongoing work will test the effectiveness of this approach at a whole plant level.</AbstractText></Abstract><AuthorList CompleteYN="Y"><Author ValidYN="Y"><LastName>Fister</LastName><ForeName>Andrew S</ForeName><Initials>AS</Initials><AffiliationInfo><Affiliation>Department of Plant Science, Pennsylvania State University, University Park, PA, United States.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Landherr</LastName><ForeName>Lena</ForeName><Initials>L</Initials><AffiliationInfo><Affiliation>Department of Plant Science, Pennsylvania State University, University Park, PA, United States.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Maximova</LastName><ForeName>Siela N</ForeName><Initials>SN</Initials><AffiliationInfo><Affiliation>Department of Plant Science, Pennsylvania State University, University Park, PA, 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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="Guiltinan, Mark J" sort="Guiltinan, Mark J" uniqKey="Guiltinan M" first="Mark J" last="Guiltinan">Mark J. Guiltinan</name><name sortKey="Landherr, Lena" sort="Landherr, Lena" uniqKey="Landherr L" first="Lena" last="Landherr">Lena Landherr</name><name sortKey="Maximova, Siela N" sort="Maximova, Siela N" uniqKey="Maximova S" first="Siela N" last="Maximova">Siela N. Maximova</name><name sortKey="Maximova, Siela N" sort="Maximova, Siela N" uniqKey="Maximova S" first="Siela N" last="Maximova">Siela N. Maximova</name></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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