An in planta, Agrobacterium-mediated transient gene expression method for inducing gene silencing in rice (Oryza sativa L.) leaves.
Identifieur interne : 000535 ( Main/Exploration ); précédent : 000534; suivant : 000536An in planta, Agrobacterium-mediated transient gene expression method for inducing gene silencing in rice (Oryza sativa L.) leaves.
Auteurs : Aurélie Andrieu [France] ; Jean Christophe Breitler ; Christelle Siré ; Donaldo Meynard ; Pascal Gantet ; Emmanuel GuiderdoniSource :
- Rice (New York, N.Y.) [ 1939-8425 ] ; 2012.
Abstract
BACKGROUND
Localized introduction and transient expression of T-DNA constructs mediated by agro-infiltration of leaf tissues has been largely used in dicot plants for analyzing the transitivity and the cell-to cell movement of the RNAi signal. In cereals, however, the morphology of the leaf and particularly the structure of the leaf epidermis, prevent infiltration of a bacterial suspension in cells by simple pressure, a method otherwise successful in dicots leaves. This study aimed at establishing a rapid method for the functional analysis of rice genes based on the triggering of RNA interference (RNAi) following Agrobacterium-mediated transient transformation of leaves.
RESULTS
Using an agro-infection protocol combining a wound treatment and a surfactant, we were able to obtain in a reliable manner transient expression of a T-DNA-borne uidA gene in leaf cells of japonica and indica rice cultivars. Using this protocol to transiently inhibit gene expression in leaf cells, we introduced hairpin RNA (hpRNA) T-DNA constructs containing gene specific tags of the phytoene desaturase (OsPDS) and of the SLENDER 1 (OsSLR1) genes previously proven to trigger RNAi of target genes in stable transformants. SiRNA accumulation was observed in the agro-infected leaf area for both constructs indicating successful triggering of the silencing signal. Accumulation of secondary siRNA was observed in both stably and transiently transformed leaf tissues expressing the HpRNA OsSLR1 construct. Gene silencing signalling was investigated in monitoring the parallel time course of OsPDS-derived mRNA and siRNA accumulation in the agro-infiltrated leaf area and adjacent systemic sectors. The sensitive RT-Q-PCR method evidenced a consistent, parallel decrease of OsPDS transcripts in both the agroinfiltred and adjacent tissues, with a time lag for the latter.
CONCLUSIONS
These results indicate that the method is efficient at inducing gene silencing in the agro-infected leaf area. The transfer of low amounts of siRNA, probably occurring passively through the symplastic pathway from the agro-infected area, seemed sufficient to trigger degradation of target transcripts in the adjacent tissues. This method is therefore well suited to study the cell-to-cell movement of the silencing signal in a monocot plant and further test the functionality of natural and artificial miRNA expression constructs.
DOI: 10.1186/1939-8433-5-23
PubMed: 24279881
PubMed Central: PMC4883685
Affiliations:
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In cereals, however, the morphology of the leaf and particularly the structure of the leaf epidermis, prevent infiltration of a bacterial suspension in cells by simple pressure, a method otherwise successful in dicots leaves. This study aimed at establishing a rapid method for the functional analysis of rice genes based on the triggering of RNA interference (RNAi) following Agrobacterium-mediated transient transformation of leaves.</p></div><div type="abstract" xml:lang="en"><p><b>RESULTS</b></p><p>Using an agro-infection protocol combining a wound treatment and a surfactant, we were able to obtain in a reliable manner transient expression of a T-DNA-borne uidA gene in leaf cells of japonica and indica rice cultivars. Using this protocol to transiently inhibit gene expression in leaf cells, we introduced hairpin RNA (hpRNA) T-DNA constructs containing gene specific tags of the phytoene desaturase (OsPDS) and of the SLENDER 1 (OsSLR1) genes previously proven to trigger RNAi of target genes in stable transformants. SiRNA accumulation was observed in the agro-infected leaf area for both constructs indicating successful triggering of the silencing signal. Accumulation of secondary siRNA was observed in both stably and transiently transformed leaf tissues expressing the HpRNA OsSLR1 construct. Gene silencing signalling was investigated in monitoring the parallel time course of OsPDS-derived mRNA and siRNA accumulation in the agro-infiltrated leaf area and adjacent systemic sectors. The sensitive RT-Q-PCR method evidenced a consistent, parallel decrease of OsPDS transcripts in both the agroinfiltred and adjacent tissues, with a time lag for the latter.</p></div><div type="abstract" xml:lang="en"><p><b>CONCLUSIONS</b></p><p>These results indicate that the method is efficient at inducing gene silencing in the agro-infected leaf area. The transfer of low amounts of siRNA, probably occurring passively through the symplastic pathway from the agro-infected area, seemed sufficient to trigger degradation of target transcripts in the adjacent tissues. This method is therefore well suited to study the cell-to-cell movement of the silencing signal in a monocot plant and further test the functionality of natural and artificial miRNA expression constructs.</p></div></front></TEI><pubmed><MedlineCitation Status="PubMed-not-MEDLINE" Owner="NLM"><PMID Version="1">24279881</PMID><DateCompleted><Year>2013</Year><Month>11</Month><Day>27</Day></DateCompleted><DateRevised><Year>2020</Year><Month>09</Month><Day>30</Day></DateRevised><Article PubModel="Electronic"><Journal><ISSN IssnType="Print">1939-8425</ISSN><JournalIssue CitedMedium="Print"><Volume>5</Volume><Issue>1</Issue><PubDate><Year>2012</Year><Month>Aug</Month><Day>31</Day></PubDate></JournalIssue><Title>Rice (New York, N.Y.)</Title><ISOAbbreviation>Rice (N Y)</ISOAbbreviation></Journal><ArticleTitle>An in planta, Agrobacterium-mediated transient gene expression method for inducing gene silencing in rice (Oryza sativa L.) leaves.</ArticleTitle><Pagination><MedlinePgn>23</MedlinePgn></Pagination><ELocationID EIdType="doi" ValidYN="Y">10.1186/1939-8433-5-23</ELocationID><Abstract><AbstractText Label="BACKGROUND" NlmCategory="BACKGROUND">Localized introduction and transient expression of T-DNA constructs mediated by agro-infiltration of leaf tissues has been largely used in dicot plants for analyzing the transitivity and the cell-to cell movement of the RNAi signal. In cereals, however, the morphology of the leaf and particularly the structure of the leaf epidermis, prevent infiltration of a bacterial suspension in cells by simple pressure, a method otherwise successful in dicots leaves. This study aimed at establishing a rapid method for the functional analysis of rice genes based on the triggering of RNA interference (RNAi) following Agrobacterium-mediated transient transformation of leaves.</AbstractText><AbstractText Label="RESULTS" NlmCategory="RESULTS">Using an agro-infection protocol combining a wound treatment and a surfactant, we were able to obtain in a reliable manner transient expression of a T-DNA-borne uidA gene in leaf cells of japonica and indica rice cultivars. Using this protocol to transiently inhibit gene expression in leaf cells, we introduced hairpin RNA (hpRNA) T-DNA constructs containing gene specific tags of the phytoene desaturase (OsPDS) and of the SLENDER 1 (OsSLR1) genes previously proven to trigger RNAi of target genes in stable transformants. SiRNA accumulation was observed in the agro-infected leaf area for both constructs indicating successful triggering of the silencing signal. Accumulation of secondary siRNA was observed in both stably and transiently transformed leaf tissues expressing the HpRNA OsSLR1 construct. Gene silencing signalling was investigated in monitoring the parallel time course of OsPDS-derived mRNA and siRNA accumulation in the agro-infiltrated leaf area and adjacent systemic sectors. The sensitive RT-Q-PCR method evidenced a consistent, parallel decrease of OsPDS transcripts in both the agroinfiltred and adjacent tissues, with a time lag for the latter.</AbstractText><AbstractText Label="CONCLUSIONS" NlmCategory="CONCLUSIONS">These results indicate that the method is efficient at inducing gene silencing in the agro-infected leaf area. The transfer of low amounts of siRNA, probably occurring passively through the symplastic pathway from the agro-infected area, seemed sufficient to trigger degradation of target transcripts in the adjacent tissues. This method is therefore well suited to study the cell-to-cell movement of the silencing signal in a monocot plant and further test the functionality of natural and artificial miRNA expression constructs.</AbstractText></Abstract><AuthorList CompleteYN="Y"><Author ValidYN="Y"><LastName>Andrieu</LastName><ForeName>Aurélie</ForeName><Initials>A</Initials><AffiliationInfo><Affiliation>CIRAD, UMR AGAP, TAA108/03, Av Agropolis, F-34398 Montpellier Cedex 05, France. emmanuel.guiderdoni@cirad.fr.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Breitler</LastName><ForeName>Jean Christophe</ForeName><Initials>JC</Initials></Author><Author ValidYN="Y"><LastName>Siré</LastName><ForeName>Christelle</ForeName><Initials>C</Initials></Author><Author ValidYN="Y"><LastName>Meynard</LastName><ForeName>Donaldo</ForeName><Initials>D</Initials></Author><Author ValidYN="Y"><LastName>Gantet</LastName><ForeName>Pascal</ForeName><Initials>P</Initials></Author><Author 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