GENERATION OF DEFECTIVE INTERFERING RNA DIMERS OF CYMBIDIUM RINGSPOT TOMBUSVIRUS

Inoculation of Nicotiana clevelandii and N. benthamiana plants with in vitro transcripts of both genomic and defective interfering (DI) RNAs of cymbidium ringspot tombusvirus resulted in a rapid accumulation of new DI-like RNA species which were demonstrated by cloning and sequen cing to be head-to-tail dimers of unit length DI RNAs. The junction re gions of dimers were represented by sequences derived precisely from t he 5' and 3' termini of DI RNAs. Only infection with DI RNAs of smalle r size (DI-2 and DI-3, 402 and 482 nt, respectively) produced detectab le amount of dimers; in contrast, infection with the largest DI RNA (D I-13, 679 nt) was unable to accumulate dimers during viral infection. Analysis of mutant DI RNAs containing deletions or insertions revealed that the size of the monomer molecule is a major factor in the accumu lation of dimers. Monomeric DI RNAs were formed in both plants and pro toplasts inoculated with in vitro-transcribed dimers. No heterodimers were found in plants inoculated simultaneously with DI-2 and DI-3 RNA molecules, which may indicate that replicase is not released from the template during synthesis of dimer molecules. However, the occurrence of a recombinant DI RNA dimer molecule derived from the two DI RNAs su ggests that simultaneous infection of the same cells with two DI RNAs did indeed take place and that absence of heterodimers did not depend on compartmentalization. (C) 1995 Academic Press, Inc.