title: Gibson assembly: an easy way to clone potyviral full-length infectious cDNA clones expressing an ectopic VPg.
abstract: 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.
authors.affiliation: INRA, UMR 1332 de Biologie du Fruit et Pathologie, CS 20032, 33882, Villenave d'Ornon, France. amandine.bordat@bordeaux.inra.fr.|Université de Bordeaux, UMR 1332 de Biologie du Fruit et Pathologie, CS 20032, 33882, Villenave d'Ornon, France. amandine.bordat@bordeaux.inra.fr.
authors.forename: Amandine
authors.initials: A
authors.lastname: Bordat
authors.affiliation: INRA, UMR 1332 de Biologie du Fruit et Pathologie, CS 20032, 33882, Villenave d'Ornon, France. Marie-Christine.Houvenaghel@bordeaux.inra.fr.|Université de Bordeaux, UMR 1332 de Biologie du Fruit et Pathologie, CS 20032, 33882, Villenave d'Ornon, France. Marie-Christine.Houvenaghel@bordeaux.inra.fr.
authors.forename: Marie-Christine
authors.initials: MC
authors.lastname: Houvenaghel
authors.affiliation: INRA, UMR 1332 de Biologie du Fruit et Pathologie, CS 20032, 33882, Villenave d'Ornon, France. german@bordeaux.inra.fr.|Université de Bordeaux, UMR 1332 de Biologie du Fruit et Pathologie, CS 20032, 33882, Villenave d'Ornon, France. german@bordeaux.inra.fr.
authors.forename: Sylvie
authors.initials: S
authors.lastname: German-Retana
mesh_terms: D003001:Cloning, Molecular / Q000379:methods*; D018076:DNA, Complementary; D015870:Gene Expression; D023061:Gene Order; D005822:Genetic Vectors / Q000235:genetics; D016679:Genome, Viral; D018545:Lactuca; D018515:Plant Leaves / Q000235:genetics / Q000378:metabolism / Q000821:virology; D017800:Potyvirus / Q000235:genetics*; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D018076:DNA, Complementary; D011993:Recombinant Fusion Proteins