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To find out whether we can control plant virus diseases by blocking viral RNA silencing suppressors (RSSs), we developed a strategy to screen inhibitors that block the association of RSSs with siRNAs using a surface plasmon resonance assay. The screened chemicals were tested in competition with RSSs for binding to siRNAs using a mobility shift assay. We then confirmed that tested chemicals actually inhibited the RSS activity in vivo using a protoplast assay which was developed for this purpose. This entire system can be adapted to screening inhibitors of not only plant viruses but also some animal viruses possessing RSSs.

Plants may activate posttranscriptional gene silencing (PTGS) as an immunity system when they are infected with viruses. Viruses may in turn interfere with this system by producing RNA silencing suppressor (RSS) proteins; most RSSs bind to viral small interfering RNAs (siRNAs). We previously reported that ascorbic acid (AsA) has the ability to interfere with the binding between viral siRNAs and viral RSSs in vitro. We thus expected that AsA-treated plants would show some tolerance to virus infection because the host PTGS will be strengthened by AsA. Brassica rapa subsp. rapa was inoculated with Turnip mosaic virus and treated with the AsA derivatives, l(+)-ascorbic acid 2-sulfate disodium salt dihydrate (AsA-SO), l(+)-ascorbyl palmitate (AsA-Pal) and dehydroascorbic acid (DHA) at 1 h postinoculation. The number of infection sites on inoculated leaves decreased by around 40 % after AsA-SO and AsA-Pal treatments and by 80 % after DHA treatment compared with the untreated control. As evidenced by an enzyme-linked immunosorbent assay, viral accumulation was significantly reduced after regular sprays with the AsA derivatives and DHA. In a detached leaf assay, AsA clearly functioned as a viral inhibitor in cells. Additionally, we confirmed that DHA also worked in the silencing pathway because its antiviral effect was not observed in the silencing-defective double mutant dcl2/dcl4 of Arabidopsis thaliana. On the basis of these results, we concluded that the AsA derivatives and DHA can significantly reduce viral infection and accumulation and that we can develop those compounds as a practical antiviral agent in the future. [ABSTRACT FROM AUTHOR]