title: Construction of a full-length infectious cDNA clone of Cowpea mild mottle virus.
abstract: Infectious cDNA clones are an important tool to study the molecular and cellular process of RNA virus infection. In vitro and in vivo transcription systems are the two main strategies used in the generation of infectious cDNA clones for RNA viruses. This study describes the first generation of a full-length infectious cDNA clone of Cowpea mild mottle virus (CPMMV), a Carlavirus. The full-length genome was synthesized by Overlap Extension PCR of two overlapping fragments and cloned in a pUC-based vector under control of the SP6 RNA polymerase promoter. After in vitro run-off transcription, the produced RNA was mechanically inoculated into soybean plants cv. CD206. The systemic infection was confirmed by RT-PCR and further sequencing of amplified cDNA fragments. To simplify the transfection process, the complete genome was subcloned into a binary vector under control of the 35S promoter of cauliflower mosaic virus by the Gibson Assembly protocol. The resulting clones were inoculated by particle bombardment onto soybean seedlings and the recovery of the virus was confirmed 2 weeks later by RT-PCR. Our results indicate the constructs of the full-length cDNA of CPMMV are fully infectious in both in vitro and in vivo transcription strategies.
authors.affiliation: Department of Phytopathology, Universidade Federal de Viçosa, Av. P. H. Rolfs, s/n Campus Universitário, Viçosa, MG, 36570-900, Brazil.
authors.forename: Silvia L
authors.initials: SL
authors.lastname: Carvalho
authors.affiliation: Department of Cell Biology, Universidade de Brasília, Campus Universitário Darcy Ribeiro, Brasília, DF, 70910-900, Brazil.
authors.forename: Tatsuya
authors.initials: T
authors.lastname: Nagata
authors.affiliation: Department of Cell Biology, Universidade de Brasília, Campus Universitário Darcy Ribeiro, Brasília, DF, 70910-900, Brazil.
authors.forename: Bruna R
authors.initials: BR
authors.lastname: Junqueira
authors.affiliation: Department of Phytopathology, Universidade Federal de Viçosa, Av. P. H. Rolfs, s/n Campus Universitário, Viçosa, MG, 36570-900, Brazil.
authors.forename: Larissa G
authors.initials: LG
authors.lastname: Zanardo
authors.affiliation: Department of Phytopathology, Universidade Federal de Viçosa, Av. P. H. Rolfs, s/n Campus Universitário, Viçosa, MG, 36570-900, Brazil.
authors.forename: Ana C S
authors.initials: AC
authors.lastname: Paiva
authors.affiliation: Department of Phytopathology, Universidade Federal de Viçosa, Av. P. H. Rolfs, s/n Campus Universitário, Viçosa, MG, 36570-900, Brazil. claudine.carvalho@ufv.br.
authors.forename: Claudine M
authors.initials: CM
authors.lastname: Carvalho
mesh_terms: D017788:Carlavirus / Q000235:genetics*; D003001:Cloning, Molecular; D018076:DNA, Complementary; D023061:Gene Order; D016679:Genome, Viral; D016366:Open Reading Frames; D010935:Plant Diseases / Q000821:virology; D013025:Glycine max / Q000821:virology
chemical_list: D018076:DNA, Complementary
keywords: Carlavirus; Cowpea mild mottle virus; Gibson assembly; Infectious clone; Overlap extension PCR