title: A simplified approach to construct infectious cDNA clones of a tobamovirus in a binary vector.
abstract: Infectious cDNA clones of RNA viruses are important tools to study molecular processes such as replication and host-virus interactions. However, the cloning steps necessary for construction of cDNAs of viral RNA genomes in binary vectors are generally laborious. In this study, a simplified method of producing an agro-infectious Pepper mild mottle virus (PMMoV) clone is described in detail. Initially, the complete genome of PMMoV was amplified by a single-step RT-PCR, cloned, and subcloned into a small plasmid vector under the T7 RNA polymerase promoter to confirm the infectivity of the cDNA clone through transcript inoculation. The complete genome was then transferred to a binary vector using a single-step, overlap-extension PCR. The selected clones were agro-infiltrated to Nicotiana benthamiana plants and showed to be infectious, causing typical PMMoV symptoms. No differences in host responses were observed when the wild-type PMMoV isolate, the T7 RNA polymerase-derived transcripts and the agroinfiltration-derived viruses were inoculated to N. benthamiana, Capsicum chinense PI 159236 and Capsicum annuum plants.
authors.affiliation: Pós-graduação em Biologia Molecular, Universidade de Brasília, 70910-900 Brasília, DF, Brazil.
authors.forename: Bruna Rayane Teodoro
authors.initials: BR
authors.lastname: Junqueira
authors.affiliation: Departamento de Biologia Celular, Universidade de Brasília, 70910-900 Brasília, DF, Brazil.
authors.forename: Cícero
authors.initials: C
authors.lastname: Nicolini
authors.affiliation: Departamento de Biologia Celular, Universidade de Brasília, 70910-900 Brasília, DF, Brazil.
authors.forename: Natalia
authors.initials: N
authors.lastname: Lucinda
authors.affiliation: Departamento de Biologia Celular, Universidade de Brasília, 70910-900 Brasília, DF, Brazil.
authors.forename: Anelise Franco
authors.initials: AF
authors.lastname: Orílio
authors.affiliation: Pós-graduação em Biologia Molecular, Universidade de Brasília, 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*; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics*; D016679:Genome, Viral / Q000235:genetics; D010935:Plant Diseases / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D012328:RNA Viruses / Q000235:genetics*; D012367:RNA, Viral / Q000235:genetics; D014026:Nicotiana / Q000821:virology; D017898:Tobamovirus / Q000235:genetics*; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D018076:DNA, Complementary; D012367:RNA, Viral; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases
keywords: Full-length cDNA clone; Overlap-extension PCR; PMMoV; Tobamovirus