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

作者：Teodoro Junqueira Bruna Rayane; Nicolini Cicero; Lucinda Natalia; Orilio Anelise Franco; Nagata Tatsuya^*

来源：Journal of Virological Methods, 2014, 198: 32-36.

DOI：10.1016/j.jviromet.2013.12.024

• Tobamovirus
• PMMoV
• Full-length cDNA clone
• Overlap-extension PCR

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 P1159236 and Capsicum annuum plants.

• 出版日期2014-3-15