Construction of tobacco mosaic virus subgenomic replicons that are replicated and spread systemically in tobacco plants

Two tobacco mosaic virus (TMV)derived replicons, created by deletion of most of the 126/183-kDa open reading frame (ORF), replicated and systemically invaded tobacco plants when supported by wild type TMV. One RNA replicon contained an internal direct repeat of 476 nucleotides from the 3′ end of the 30-kDa ORF. Although this RNA was replicated, most of the progeny were heterogeneous in size and smaller than the original transcript. A second TMV-derived RNA replicon, without any internally repeated sequences and containing a deletion of the 5′ portion of the 30-kDa ORF as well as most of the 126/183-kDa ORF, was created and coinoculated with wild type TMV as helper. This RNA also was replicated efficiently and systemically invaded tobacco plants. An examination of the sequences of cDNA clones obtained after PCR amplification of the progeny population of this RNA replicon demonstrated that the observed size heterogeneity was due to deletions and insertions adjacent to the artificially created deletion junction. These data demonstrate that a TMV infection is capable of supporting an artificially created RNA replicon, similar to defective interfering RNAs or satellites. However, these dependent RNAs were replicated without noticeably interfering with wild type TMV symptoms or replication.