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

Mireya Martínez-Pérez; José A Navarro; Vicente Pallás; Jesús A Sánchez-Navarro

doi:10.1016/j.virusres.2019.197733

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

Virus Res. 2019 Oct 15:272:197733. doi: 10.1016/j.virusres.2019.197733. Epub 2019 Aug 25.

Authors

Mireya Martínez-Pérez¹, José A Navarro², Vicente Pallás³, Jesús A Sánchez-Navarro⁴

Affiliations

¹ Department of Molecular and Evolutionary Plant Virology, Instituto de Biología Molecular y Celular de Plantas (IBMCP) (UPV-CSIC), Universitat Politècnica de Valencia, Ingeniero Fausto Elio s/n, 46022 Valencia, Spain. Electronic address: mimarpe6@etsid.upv.es.
² Department of Molecular and Evolutionary Plant Virology, Instituto de Biología Molecular y Celular de Plantas (IBMCP) (UPV-CSIC), Universitat Politècnica de Valencia, Ingeniero Fausto Elio s/n, 46022 Valencia, Spain. Electronic address: janavarr@ibmcp.upv.es.
³ Department of Molecular and Evolutionary Plant Virology, Instituto de Biología Molecular y Celular de Plantas (IBMCP) (UPV-CSIC), Universitat Politècnica de Valencia, Ingeniero Fausto Elio s/n, 46022 Valencia, Spain. Electronic address: vpallas@ibmcp.upv.es.
⁴ Department of Molecular and Evolutionary Plant Virology, Instituto de Biología Molecular y Celular de Plantas (IBMCP) (UPV-CSIC), Universitat Politècnica de Valencia, Ingeniero Fausto Elio s/n, 46022 Valencia, Spain. Electronic address: jesanche@ibmcp.upv.es.

PMID: 31461660
DOI: 10.1016/j.virusres.2019.197733

Abstract

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.

Keywords: Alfamovirus; P12 system; RSS assay.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Alfalfa mosaic virus / genetics*
Gene Expression*
Gene Order
Gene Silencing*
Genetic Vectors / genetics*
Phenotype
Plant Diseases / virology
Plant Viruses / genetics
RNA Interference*
RNA, Viral
Sensitivity and Specificity

Substances

RNA, Viral