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The tombusviruses represent a group of small Icosahedral plant viruses that contain monopartite positive-sense RNA genome. Tombusviruses are able to generate small replicating deletion mutants of their genomes (i.e., defective interfering RNAs) during infections via RNA recombination and/or rearrangment. To further study the process of RNA recombination and to determine whether tombusviruses were capable of tran-recombination, protoplasts were coinoculated with in vitro-generated transcripts of a nonreplicating 3'- truncated genomic RNA of cucumber necrosis tombusvirus and either replicative or replication-defective DI RNAs of tomato bushy stunt tombusvirus. After a 48-hr incubation, two dominant replicative chimeric recombinant viral RNA populations were detected that contained various large contiguous 5' segments of the cucumber necrosis tombusvirus genomic RNA fused to 3'-terminal regions of the tomato bushy stunt tombusvirus defective interfering RNA. Some of the larger chimeric recombinants formed in protoplasts were able to systemically infect plants and induce wild-type symptoms. In addition, a functional chimeric genome was generated in planta after direct coinoculation of whole plants with the defective RNA components. These results indicate that (i) RNA recombination can occur relatively efficiently in single-cell infections, (ii) trans-recombination can occur with nonreplicating viral RNA components, and (iii) functional chimeric genomes can be generated via recombination. Possible mechaisms for the formation of the recombinants are proposed, and evolutionary implications are disussed.