Tobacco rattle virus 16K silencing suppressor binds ARGONAUTE 4 and inhibits formation of RNA silencing complexes.
The cysteine-rich 16K protein of tobacco rattle virus (TRV), the type member of the genus Tobravirus, is known to suppress RNA silencing. However, the mechanism of action of the 16K suppressor is not well understood. In this study, we used a GFP-based sensor strategy and an Agrobacterium-mediated transient assay in Nicotiana benthamiana to show that 16K was unable to inhibit the activity of existing small interfering RNA (siRNA)- and microRNA (miRNA)-programmed RNA-induced silencing effector complexes (RISCs). In contrast, 16K efficiently interfered with de novo formation of miRNA- and siRNA-guided RISCs, thus preventing cleavage of target RNA. Interestingly, we found that transiently expressed endogenous miR399 and miR172 directed sequence-specific silencing of complementary sequences of viral origin. 16K failed to bind small RNAs, although it interacted with ARGONAUTE 4, as revealed by bimolecular fluorescence complementation and immunoprecipitation assays. Site-directed mutagenesis demonstrated that highly conserved cysteine residues within the N-terminal and central regions of the 16K protein are required for protein stability and/or RNA silencing suppression.
Department of Environmental Biology, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Ramiro de Maeztu 9, 28040 Madrid, Spain.
Lourdes
L
Fernández-Calvino
Department of Environmental Biology, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Ramiro de Maeztu 9, 28040 Madrid, Spain.
Llúcia
L
Martínez-Priego
Department of Dermatology and Allergology, University of Szeged, H-6720 Szeged, Koranyi str. 6, Hungary.
Edit Z
EZ
Szabo
Department of Environmental Biology, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Ramiro de Maeztu 9, 28040 Madrid, Spain.
Irene
I
Guzmán-Benito
Department of Environmental Biology, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Ramiro de Maeztu 9, 28040 Madrid, Spain.
Inmaculada
I
González
Department of Environmental Biology, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Ramiro de Maeztu 9, 28040 Madrid, Spain.
Tomás
T
Canto
Department of Dermatology and Allergology, University of Szeged, H-6720 Szeged, Koranyi str. 6, Hungary.|MTA-SZTE Dermatological Research Group, Hungary.
Lóránt
L
Lakatos
Department of Environmental Biology, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Ramiro de Maeztu 9, 28040 Madrid, Spain.
César
C
Llave
D054884:Host-Pathogen Interactions; D010942:Plant Viruses / Q000502:physiology*; D011485:Protein Binding; D034622:RNA Interference; D012328:RNA Viruses / Q000276:immunology / Q000502:physiology*; D016601:RNA-Binding Proteins / Q000378:metabolism*; D014026:Nicotiana / Q000276:immunology / Q000821:virology; D014764:Viral Proteins / Q000378:metabolism*
D016601:RNA-Binding Proteins; D014764:Viral Proteins