A Versatile Assay for the Identification of RNA Silencing Suppressors Based on Complementation of Viral Movement

Abstract

The cell-to-cell movement of Turnip crinkle virus (TCV) in Nicotiana benthamiana requires the presence of its coat protein (CP), a known suppressor of RNA silencing. RNA transcripts of a TCV construct containing a reporter gene (green fluorescent protein) (TCV-sGFP) in place of the CP open reading frame generated foci of three to five cells. TCV CP delivered in trans by Agrobacterium tumefaciens infiltration potentiated movement of TCV-sGFP and increased foci diameter, on average, by a factor of four. Deletion of the TCV movement proteins in TCV-sGFP (construct TCVDelta92-sGFP) abolished the movement complementation ability of TCV CP. Other known suppressors of RNA silencing from a wide spectrum of viruses also complemented the movement of TCV-sGFP when delivered in trans by Agrobacterium tumefaciens. These include suppressors from nonplant viruses with no known plant movement function, demonstrating that this assay is based solely on RNA silencing suppression. While the TCV-sGFP construct is primarily used as an infectious RNA transcript, it was also subcloned for direct expression from Agrobacterium tumefaciens for simple quantification of suppressor activity based on fluorescence levels in whole leaves. Thus, this system provides the flexibility to assay for suppressor activity in either the cytoplasm or nucleus, depending on the construct employed.

A Dual Strategy for the Suppression of Host Antiviral Silencing: Two Distinct Suppressors for Viral Replication and Viral Movement Encoded by Potato Virus M

“…The promotion of movement of PVX-GFP by p19, pvmTGBp1, and pvmCRP can be interpreted as supporting the idea that silencing suppression has a role in viral cell-to-cell movement, as shown in previous studies (5,37). To test this, we developed a viral movement complementation assay (VMCA) using mutant variants of p25 encoded by PVX (5).…”

“…Although the classical GFP transient-coexpression assay is convenient and has been widely used to identify silencing suppressors, this assay is unable to identify spread-specific silencing suppressors such as pvmTGBp1. Accordingly, a VMCA such as the p25 mutant assay performed in the present study or the turnip crinkle virus (TCV) vector-based assay (which utilizes recombinant TCV expressing GFP instead of CP [37]) will help to identify such suppressors.…”

Phytophthora infestans effector Pi14054 is a novel candidate suppressor of host silencing mechanisms

“…Thus, TCV CP prevents the degradation of viral transcripts by host silencing machinery. For the TCV-sGFP complementation assay, s wapping the TCV CP gene with a reporter gene for a synthetic GFP (sGFP) blocks the movement of TCV, restricting the virus to single cells or foci consisting of two or three cells (visualized by fluorescence) due to induction of antiviral silencing (Powers et al 2008a). Complementation with potential suppressors of silencing in trans by Agrobacterium tumefaciens infiltration enables movement of TCV-sGFP and increases the foci (fluorescence) diameter.…”

Análisis Funcional De Proteínas Codificadas Por El Virus De La Rotura Del Color De La Flor Del Pelargonium

“…presentar actividad supresora no detectable mediante el ensayo que hemos empleado [Powers et al, 2008b], los resultados concuerdan con los obtenidos con varios carmovirus, que indican que el gen situado en posición 3´-proximal codifica un polipéptido con una función dual como CP y como inhibidor del PTGS [Qu et al, 2003;Thomas et al, 2003;Meng et al, 2006;Genovés et al, 2006]. Al igual que otros supresores relacionados o no filogenéticamente [Voinnet et al, 1999;Thomas et al, 2003;Te et al, 2005;Meng et al, 2006;Zhou et al, 2006b], la proteína p37 del PFBV es capaz de acentuar dramáticamente los síntomas de una infección viral, como se ha comprobado mediante su expresión a partir de un vector derivado de PVX.…”