De novo generation of Lettuce infectious yellows virus defective RNAs in protoplasts

Summary Lettuce infectious yellows virus (LIYV)-infected plants contain a heterogeneous population of defective RNAs (D RNAs) derived from LIYV genomic RNA 2. To partly address how LIYV D RNAs are generated, in vitro synthesized transcripts corresponding to the LIYV genomic RNAs 1 and 2 were inoculated to protoplasts, and these were analysed for genomic and D RNAs. De novo generated D RNAs were readily detected by 48 h post-inoculation. Furthermore, when separate aliquots from the same protoplast preparation were separately inoculated with aliquots of the same LIYV RNA 1 and RNA 2 transcript preparations, different D RNA populations were detected in each. Thus, different D RNAs arose de novo within separate protoplast samples. Nucleotide sequence analysis of some de novo LIYV D RNAs revealed that they have a similar structure to the LIYV D RNAs described previously from whole plants, and to those of other plant viruses, consisting of one large internal deletion of the LIYV genomic RNA 2, but retaining 5' and 3' terminal sequences. However, one of the LIYV D RNAs had two non-contiguous internal deletions.