EXPRESSION OF ORF-69 OF TURNIP YELLOW MOSAIC-VIRUS IS NECESSARY FOR VIRAL SPREAD IN PLANTS

Turnip yellow mosaic virus (TYMV) RNA has two extensively overlapping open reading frames (ORFs) encoding noncapsid proteins. The longest of these, ORF-206, is essential for RNA replication. We have investigated the expression and role of ORF-69 (encoding a protein with a MW of 69K) using specific antiserum and mutant genomic RNAs designed to interrupt ORF-69 expression. TYC69 antiserum immunoprecipitated a protein with an apparent MW of 75K (p69) from in vitro translations of TYMV RNA. Mutant RNAs with stop codons inserted at nucleotides 139, 178, and 178/224 in ORF-69 expressed ORF-69 at very low levels in vitro. These mutants replicated to wild-type levels in turnip or Chinese cabbage protoplasts, but were not recovered from any leaves of inoculated plants. These results suggest that ORF-69 products are dispensible for replication, but are required for viral spread. Revenant and pseudorevertant viruses with uninterrupted ORF-69 were recovered from plants showing delayed onset of symptoms after inoculation with two of the above mutants, indicating the importance of ORF-69 expression in establishing a systemic infection in plants. ORF-69 expression could be detected using Western blots in extracts from young, symptomatic leaves of infected plants. A single band with an apparent MW of 75-80K was detected in leaves infected with cDNA-derived viral RNAs, while a doublet was detected after infection with type strain viral RNA, suggesting the presence of two ORF-69 alleles or differential post-translational modification. © 1992.