The Cucumber mosaic virus (CMV) Y-satellite RNA (Y-Sat) has a small non-protein-coding RNA genome that induces yellowing symptoms in infected Nicotiana tabacum (tobacco). How this RNA pathogen induces such symptoms has been a longstanding question. We show that the yellowing symptoms are a result of small interfering RNA (siRNA)-directed RNA silencing of the chlorophyll biosynthetic gene, CHLI. The CHLI mRNA contains a 22-nucleotide (nt) complementary sequence to the Y-Sat genome, and in Y-Sat-infected plants, CHLI expression is dramatically down-regulated. Small RNA sequencing and 5′ RACE analyses confirmed that this 22-nt sequence was targeted for mRNA cleavage by Y-Sat-derived siRNAs. Transformation of tobacco with a RNA interference (RNAi) vector targeting CHLI induced Y-Sat-like symptoms. In addition, the symptoms of Y-Sat infection can be completely prevented by transforming tobacco with a silencing-resistant variant of the CHLI gene. These results suggest that siRNA-directed silencing of CHLI is solely responsible for the Y-Sat-induced symptoms. Furthermore, we demonstrate that two Nicotiana species, which do not develop yellowing symptoms upon Y-Sat infection, contain a single nucleotide polymorphism within the siRNA-targeted CHLI sequence. This suggests that the previously observed species specificity of Y-Sat-induced symptoms is due to natural sequence variation in the CHLI gene, preventing CHLI silencing in species with a mismatch to the Y-Sat siRNA. Taken together, these findings provide the first demonstration of small RNA-mediated viral disease symptom production and offer an explanation of the species specificity of the viral disease.
Viral infections result in a variety of disease symptoms that vary in character and severity depending on the type of viral infection and individual host factors. Despite extensive research, the molecular basis of viral disease development has remained poorly understood. Both plant and animal viruses express 20–25 nucleotide siRNAs or microRNAs (miRNAs) in their host. These virus-specific small RNAs (sRNAs) direct RNA silencing of homologous viral genes to restrict virus replication forming part of the host's antiviral defense mechanism. Using a plant viral satellite RNA as a model system, we demonstrate here a new function for virus-derived sRNAs: induction of disease symptoms by silencing of a physiologically important host gene. Furthermore, we demonstrate that such viral-derived sRNA-induced disease symptoms can be prevented by the expression of a either naturally evolved, or artificially introduced, silencing-resistant sequence variant of the viral siRNA-targeted gene. These findings not only provide the first demonstration of a sRNA-mediated viral disease mechanism, but also offer an alternate strategy to prevent the onset of such viral diseases.