A DNA clone encoding the full-length infectious genome of odontoglossum ringspot tobamovirus and mutagenesis of its coat protein gene.
A full-length DNA clone encoding the genome of odontoglossum ringspot tobamovirus (ORSV) was synthesized and placed adjacent to a bacteriophage T7 RNA polymerase promoter. Capped-RNA transcripts produced in vitro were highly infectious when mechanically inoculated onto seedlings of Nicotiana benthamiana and Oncidium Gower Ramsey. A representative clone, designated pOT2, caused a disease phenotype identical to that produced by parental viral RNA. ELISA, Western blot analysis, Northern blot hybridization and electron microscopy verified the infectivity of pOT2. A coat protein deficient mutant of the clone, pO delta CP1, was produced with the initiation codon of the coat protein cistron of ORSV abolished. Transcripts from pO delta CP1 were infective, able to move in N. benthamiana but produced no coat protein. This demonstrates that the coat protein was dispensable for RNA replication and for movement. This is believed to be the first report of an ORSV infectious clone driven by a T7 RNA polymerase promoter.
Yu
H H
HH
School of Biological Sciences, National University of Singapore, Kent Ridge, Singapore.
Wong
S M
SM
D015152:Blotting, Northern; D002213:Capsid / Q000235:genetics*; D004274:DNA, Recombinant / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay; D005814:Genes, Viral / Q000235:genetics*; D005818:Genetic Engineering; D016679:Genome, Viral; D016297:Mutagenesis, Site-Directed; D018515:Plant Leaves / Q000821:virology; D010944:Plants / Q000821:virology; D010947:Plants, Toxic; D012327:RNA Virus Infections / Q000235:genetics* / Q000821:virology; D012367:RNA, Viral / Q000235:genetics; D014026:Nicotiana / Q000821:virology; D017898:Tobamovirus / Q000235:genetics* / Q000254:growth & development; D014158:Transcription, Genetic / Q000235:genetics; D015678:Viral Structural Proteins / Q000235:genetics*; D014779:Virus Replication / Q000235:genetics
D004274:DNA, Recombinant; D012367:RNA, Viral; D015678:Viral Structural Proteins