Two proteins encoded by beet necrotic yellow vein virus RNA 3 influence symptom phenotype on leaves

RNA 3 of the beet necrotic yellow vein virus (BNYVV) quadripartite RNA genome is not essential for virus multiplication on leaves of Tetragonia expansa but has dramatic effects on symptom expression. Virus isolates containing RNA 3 produce bright yellow local lesions while isolates lacking RNA 3 produce much milder symptoms. Using directed mutagenesis of cDNA clones followed by in vitro synthesis of biologically active transcripts, a 25 kDa open reading frame (ORF) of RNA 3 was shown to be responsible for the yellow local lesion phenotype. In addition, two deletion mutants of RNA 3 were found to elicit the appearance of severe necrotic local lesions. Analysis of one of these mutants revealed that necrosis was due to the overexpression of a second short ORF, N, overlapping the 3'-terminal portion of the 25 kDa ORF. As shown by gene fusion studies, gene N is not detectably expressed from full-length RNA 3 but is translationally activated by deletion of upstream sequences. Introduction of gene N into the genome of the unrelated DNA virus, cauliflower mosaic virus, elicits a necrotic response instead of the typical mosaic symptoms, demonstrating that gene N can induce necrosis outside of the context of a BNYVV infection.