Subviral RNAs of TCV: Symptom Induction and the Generation of Discontinous RNAs

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Turnip crinkle virus (TCV) will be used as a model to study symptom manifestation, defective interfering (DI) RNA formation and RNA recombination. The TCV system is uniquely suited for these studies; it is the only plant or animal virus associated with avirulent and virulent sat-RNAs which can recombine their genomes and DI RNAs which can be generated de novo from in vitro synthesized TCV transcripts. Furthermore, two isolates of TCV have distinct biological properties: subviral RNAs only produce symptoms when associated with the TCV-M isolate and DI RNAs have only been generated from the TCV-B isolate. Chimeric TCV genomic RNAs will be constructed to ascertain which region of TCV RNA is involved in differential symptom production and DI formation. The mechanism by which DI RNAs and sat-RNA recombinants are formed will also be investigated. The putative internal replicase recognition sequence in sat-RNA C, found at the right hand junction of all recombinant sat-RNAs, will be deleted, altered and/or moved from its current location to determine the importance of the sequence in recombination. A copy choice model versus a breakage and ligation model for the formation of descendance RNAs will also be tested. Sat-RNA transcripts with defined mutations, allowing differential replication of plus or minus strands, will be added to both protoplasts and plant extracts. RNA recombination will be detected by polymerase chain reaction techniques.***//