A simplified approach to construct infectious cDNA clones of a tobamovirus in a binary vector

• Published in: Journal of virological methods Vol. 198; pp. 32 - 36
• Main Authors: Junqueira, Bruna Rayane Teodoro, Nicolini, Cícero, Lucinda, Natalia, Orílio, Anelise Franco, Nagata, Tatsuya
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 15.03.2014
• Subjects: Cloning, Molecular - methods; DNA, Complementary - genetics; DNA-Directed RNA Polymerases - genetics; DNA-Directed RNA Polymerases - metabolism; Full-length cDNA clone; Genetic Vectors - genetics; Genome, Viral - genetics; Nicotiana - virology; Overlap-extension PCR; Plant Diseases - genetics; PMMoV; Promoter Regions, Genetic - genetics; RNA Viruses - genetics; RNA, Viral - genetics; Tobamovirus; Tobamovirus - genetics; Viral Proteins - genetics; Viral Proteins - metabolism; Full-length cDNA clone; Tobamovirus; Overlap-extension PCR; PMMoV
• ISSN: 0166-0934; 1879-0984
• DOI: 10.1016/j.jviromet.2013.12.024

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.