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Gibson assembly: an easy way to clone potyviral full-length infectious cDNA clones expressing an ectopic VPg

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 effic...

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Autores principales: Bordat, Amandine, Houvenaghel, Marie-Christine, German-Retana, Sylvie
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2015
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4475333/
https://www.ncbi.nlm.nih.gov/pubmed/26070311
http://dx.doi.org/10.1186/s12985-015-0315-3
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author Bordat, Amandine
Houvenaghel, Marie-Christine
German-Retana, Sylvie
author_facet Bordat, Amandine
Houvenaghel, Marie-Christine
German-Retana, Sylvie
author_sort Bordat, Amandine
collection PubMed
description 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 single-step 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. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12985-015-0315-3) contains supplementary material, which is available to authorized users.
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spelling pubmed-44753332015-06-21 Gibson assembly: an easy way to clone potyviral full-length infectious cDNA clones expressing an ectopic VPg Bordat, Amandine Houvenaghel, Marie-Christine German-Retana, Sylvie Virol J Methodology 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 single-step 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. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12985-015-0315-3) contains supplementary material, which is available to authorized users. BioMed Central 2015-06-14 /pmc/articles/PMC4475333/ /pubmed/26070311 http://dx.doi.org/10.1186/s12985-015-0315-3 Text en © Bordat et al. 2015 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
spellingShingle Methodology
Bordat, Amandine
Houvenaghel, Marie-Christine
German-Retana, Sylvie
Gibson assembly: an easy way to clone potyviral full-length infectious cDNA clones expressing an ectopic VPg
title Gibson assembly: an easy way to clone potyviral full-length infectious cDNA clones expressing an ectopic VPg
title_full Gibson assembly: an easy way to clone potyviral full-length infectious cDNA clones expressing an ectopic VPg
title_fullStr Gibson assembly: an easy way to clone potyviral full-length infectious cDNA clones expressing an ectopic VPg
title_full_unstemmed Gibson assembly: an easy way to clone potyviral full-length infectious cDNA clones expressing an ectopic VPg
title_short Gibson assembly: an easy way to clone potyviral full-length infectious cDNA clones expressing an ectopic VPg
title_sort gibson assembly: an easy way to clone potyviral full-length infectious cdna clones expressing an ectopic vpg
topic Methodology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4475333/
https://www.ncbi.nlm.nih.gov/pubmed/26070311
http://dx.doi.org/10.1186/s12985-015-0315-3
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