title: Construction of an infectious clone of a plant RNA virus in a binary vector using one-step Gibson Assembly.
abstract: The construction of full-length infectious clones of RNA viruses is often laborious due to the many cloning steps required and the DNA exclusion within the plasmid during Escherichia coli transformation. We demonstrate single-step cloning procedure of an infectious cDNA of the tomato blistering mosaic virus (ToBMV) using Gibson Assembly (GA), which drastically reduces the number of cloning steps. By agro-inoculation with the construct obtained by this procedure, ToBMV was recovered six days post-inoculation in Nicotiana benthamiana plants. The symptoms induced by the recovered virus were indistinguishable from those caused by the wild-type virus. We conclude that the GA is very useful method particularly to construct a full-length cDNA clone of a plant RNA virus in a binary vector.
authors.affiliation: Universidade de Brasília, Department of Cell Biology, Campus Universitário Darcy Ribeiro, 70910-900 Brasília, DF, Brazil.
authors.forename: Rosana
authors.initials: R
authors.lastname: Blawid
authors.affiliation: Universidade de Brasília, Department of Cell Biology, Campus Universitário Darcy Ribeiro, 70910-900 Brasília, DF, Brazil. Electronic address: tatsuya@unb.br.
authors.forename: Tatsuya
authors.initials: T
authors.lastname: Nagata
mesh_terms: D003001:Cloning, Molecular / Q000379:methods*; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D010957:Plasmids; D012367:RNA, Viral / Q000235:genetics; D059386:Reverse Genetics / Q000379:methods*; D014026:Nicotiana / Q000821:virology; D014170:Transformation, Genetic; D017806:Tymovirus / Q000235:genetics*
chemical_list: D018076:DNA, Complementary; D012367:RNA, Viral
keywords: Full-length cDNA clone; Gibson Assembly; Seamless cloning; Tymovirus