Studies were initiated to monitor generation and accumulation of defective interfering (DI) RNAs associated with tomato bushy stunt virus (TBSV) in the absence of serial, high multiplicity of infection passage. Infections were initiated in Nicotiana clevelandii host plants and protoplast cell suspensions by inoculation with in vitro-synthesized infectious TBSV RNA transcripts containing a genomic marker. The infections were then assayed for DI-size RNAs by both Northern blot analysis and reverse transcription coupled with PCR amplification. DI-size RNAs could not be detected by Northern blot analysis in either plants or protoplasts after an evident viral infection. However, RT-PCR amplification permitted the isolation of DI-size cDNAs (600-700 nt) from plant, but not protoplast, infections as early as 8 days postinoculation. Sequence analysis of these DI-size cDNA clones revealed that they contained the four conserved regions found in all previously identified competent DI RNAs. Several DI RNA clones contained the genomic marker which confirmed their de novo generation from the input transcript inoculum. A comparison of the nucleotide sequence of these clones to previously sequenced DI RNAs, isolated from plants after multiple passages, showed that differences existed at the junctions between regions. These results demonstrate that a heterogeneous population of DI RNAs accumulated in plants in the absence of serial host passage. In addition, the similarity of these DI RNAs to previously characterized DI RNAs that accumulate upon passage indicates that evolution can occur very rapidly within the initially inoculated plant.
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