A sensitive and rapid RNA silencing suppressor activity assay based on alfalfa mosaic virus expression vector

• Published in: Virus research 2019-10, Vol.272, p.197733-197733, Article 197733
• Main Authors: Martínez-Pérez, Mireya, Navarro, José A., Pallás, Vicente, Sánchez-Navarro, Jesús A.
• Format: Article
• Language: English
• Subjects: Alfamovirus; P12 system; RSS assay
• ISSN: 0168-1702; 1872-7492
• DOI: 10.1016/j.virusres.2019.197733

[Display omitted] •Alfalfa mosaic virus (AMV) and P12 plants can be used for the RSS screening.•The AMV-P12 RSS assay is simple, sensitive, rapid and quantitative.•The coat protein (CP) of carnation mottle virus (CarMV) is a RSS.•The CarMV CP blocks the accumulation of small RNAs. Plant viruses express RNA silencing suppressor (RSS) proteins to counteract plant defence mechanisms. Here, we describe a method to assess the RSS activity based on an alfalfa mosaic virus (AMV) RNA 3 expression vector and transgenic Nicotiana tabacum plants that express the P1 and P2 subunits of the AMV replicase (P12 plants). Inoculation of P12 plants with different AMV RNA 3 constructs expressing different HC-Pro mutants that differ in their RSS capabilities, revealed a perfect correlation between necrotic lesions on inoculated leaves and RSS activity. Protoplast analysis showed that the RSS activity correlated with the accumulation of the AMV RNAs. A direct comparison between three RSS activity assays and the AMV-P12 system revealed that the coat protein of carnation mottle virus displays RSS activity with the four assays and reduced the accumulation of the siRNAs.