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Author summaryRNA silencing, a small RNA (sRNA)-mediated sequence-specific mechanism, is a primary antiviral response of host plants against viral infection. Argonaute 1 (AGO1), an effector protein of RNA silencing, associates with sRNAs to degrade viral RNAs or silence the genes involved in biotic stresses in cytoplasm; such functions are promoted by its translocation between the nucleus and cytoplasm. To survive host RNA silencing, cucumber mosaic virus (CMV) encodes an RNA silencing suppressor (RSS), 2b protein (2b). 2b plays multiple roles in suppressing RNA silencing by binding to either sRNAs or AGOs, in the nucleus and the cytoplasm. However, several previous reports suggest that 2b exerts its RSS activity mainly when it is in the cytoplasm. Here, we found that the serine residue at position 28 is phosphorylated, and its phosphorylation negatively regulated the RSS activity of 2b by regulating 2b's nuclear/nucleolar localization. Furthermore, we also found that 2bs were dephosphorylated in the nucleus and exported to the cytoplasm in an exportin-dependent manner. Because 2b's transport between the nucleus and cytoplasm is similar to that of AGOs, 2b seems to work in cells as if it is manipulating its nucleocytoplasmic trafficking to track down its targets in the RNA silencing pathway. The 2b protein (2b) of cucumber mosaic virus (CMV), an RNA-silencing suppressor (RSS), is a major pathogenicity determinant of CMV. 2b is localized in the nucleus and cytoplasm, and its nuclear import is determined by two nuclear localization signals (NLSs); a carrier protein (importin [IMP alpha]) is predicted to be involved in 2b's nuclear transport. Cytoplasmic 2bs play a role in suppression of RNA silencing by binding to small RNAs and AGO proteins. A putative nuclear export signal (NES) motif was also found in 2b, but has not been proved to function. Here, we identified a leucine-rich motif in 2b's C-terminal half as an NES. We then showed that NES-deficient 2b accumulated

Symptoms on virus-infected plants are often very specific to the given virus. The molecular mechanisms involved in viral symptom induction have been extensively studied, but are still poorly understood. Cucumber mosaic virus (CMV) Y satellite RNA (Y-sat) is a non-coding subviral RNA and modifies the typical symptom induced by CMV in specific hosts; Y-sat causes a bright yellow mosaic on its natural host Nicotiana tabacum. The Y-sat-induced yellow mosaic failed to develop in the infected Arabidopsis and tomato plants suggesting a very specific interaction between Y-sat and its host. In this study, we revealed that Y-sat produces specific short interfering RNAs (siRNAs), which interfere with a host gene, thus inducing the specific symptom. We found that the mRNA of tobacco magnesium protoporphyrin chelatase subunit I (ChlI, the key gene involved in chlorophyll synthesis) had a 22-nt sequence that was complementary to the Y-sat sequence, including four G-U pairs, and that the Y-sat-derived siRNAs in the virus-infected plant downregulate the mRNA of ChlI by targeting the complementary sequence. ChlI mRNA was also downregulated in the transgenic lines that express Y-sat inverted repeats. Strikingly, modifying the Y-sat sequence in order to restore the 22-nt complementarity to Arabidopsis and tomato ChlI mRNA resulted in yellowing symptoms in Y-sat-infected Arabidopsis and tomato, respectively. In 5'-RACE experiments, the ChlI transcript was cleaved at the expected middle position of the 22-nt complementary sequence. In GFP sensor experiments using agroinfiltration, we further demonstrated that Y-sat specifically targeted the sensor mRNA containing the 22-nt complementary sequence of ChlI. Our findings provide direct evidence that the identified siRNAs derived from viral satellite RNA directly modulate the viral disease symptom by RNA silencing-based regulation of a host gene.