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hal.structure.identifierBiologie du fruit et pathologie [BFP]
dc.contributor.authorBORDAT, Amandine
hal.structure.identifierBiologie du fruit et pathologie [BFP]
dc.contributor.authorHOUVENAGHEL, Marie-Christine
hal.structure.identifierBiologie du fruit et pathologie [BFP]
dc.contributor.authorGERMAN-RETANA, Sylvie
dc.date.issued2015
dc.identifier.issn1743-422X
dc.description.abstractEnAbstract Background Approaches to simplify and accelerate the construction of full-length infectious cDNA clones for plant potyviruses have been described, based on cloning strategies involving in vitro ligation or homologous recombination in yeast. In the present study, we developed a faster and more efficient in vitro recombination system using Gibson assembly (GA), to engineer a Lettuce mosaic virus (LMV) infectious clone expressing an ectopic mcherry-tagged VPg (Viral protein genome-linked) for in planta subcellular localization of the viral protein in an infection context. Methods Three overlapping long distance PCR fragments were amplified and assembled in a singlestep process based on in vitro recombination (Gibson assembly). The resulting 17.5 kbp recombinant plasmids (LMVmchVPg_Ec) were inoculated by biolistic on lettuce plants and then propagated mechanically on Nicotiana benthamiana. Confocal microscopy was used to analyze the subcellular localization of the ectopically expressed mcherry-VPg fusion protein. Results The Gibson assembly allowed the cloning of the expected plasmids without any deletion. All the inoculated plants displayed symptoms characteristic of LMV infection. The majority of the mcherry fluorescent signal observed using confocal microscopy was located in the nucleus and nucleolus as expected for a potyviral VPg. Conclusions This is the first report of the use of the Gibson assembly method to construct full-length infectious cDNA clones of a potyvirus genome. This is also the first description of the ectopic expression of a tagged version of a potyviral VPg without affecting the viability of the recombinant potyvirus.
dc.language.isoen
dc.publisherBioMed Central
dc.subject.enFull-length infectious clone
dc.subject.enGibson assembly
dc.subject.enPotyvirus
dc.subject.enVPg
dc.subject.enImproved cloning
dc.title.enGibson assembly : an easy way to clone potyviral full-length infectious cDNA clones ex pressing an ectopic VPg
dc.typeArticle de revue
dc.identifier.doi10.1186/s12985-015-0315-3
dc.subject.halSciences du Vivant [q-bio]/Sciences agricoles
dc.subject.halSciences du Vivant [q-bio]/Biotechnologies
dc.subject.halSciences du Vivant [q-bio]/Microbiologie et Parasitologie
dc.subject.halSciences du Vivant [q-bio]/Microbiologie et Parasitologie/Virologie
bordeaux.journalVirology Journal
bordeaux.volume12
bordeaux.peerReviewedoui
hal.identifierhal-02636073
hal.version1
hal.popularnon
hal.audienceInternationale
hal.origin.linkhttps://hal.archives-ouvertes.fr//hal-02636073v1
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