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 bacterioph age T7 RNA polymerase promoter. Capped-RNA transcripts produced in vit ro were highly infectious when mechanically inoculated onto seedlings of Nicotiana benthamiana and Oncidium Gower Ramsey. A representative c lone, designated pOT2, caused a disease phenotype identical to that pr oduced by parental viral RNA. ELISA, Western blot analysis, Northern b lot 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 tha t the coat protein was dispensable for RNA replication and for movemen t. This is believed to be the first report of an ORSV infectious clone driven by a T7 RNA polymerase promoter.