title: The benyvirus RNA silencing suppressor is essential for long-distance movement, requires both zinc-finger and NoLS basic residues but not a nucleolar localization for its silencing-suppression activity.
abstract: The RNA silencing-suppression properties of Beet necrotic yellow vein virus (BNYVV) and Beet soil-borne mosaic virus (BSBMV) cysteine-rich p14 proteins have been investigated. Suppression of RNA silencing activities were made evident using viral infection of silenced Nicotiana benthamiana 16C, N. benthamiana agroinfiltrated with green fluorescent protein (GFP), and GF-FG hairpin triggers supplemented with viral suppressor of RNA silencing (VSR) constructs or using complementation of a silencing-suppressor-defective BNYVV virus in Chenopodium quinoa. Northern blot analyses of small-interfering RNAs (siRNAs) in agroinfiltration tests revealed reduced amounts of siRNA, especially secondary siRNA, suggesting that benyvirus VSR act downstream of the siRNA production. Using confocal laser-scanning microscopy imaging of infected protoplasts expressing functional p14 protein fused to an enhanced GFP reporter, we showed that benyvirus p14 accumulated in the nucleolus and the cytoplasm independently of other viral factors. Site-directed mutagenesis showed the importance of the nucleolar localization signal embedded in a C4 zinc-finger domain in the VSR function and intrinsic stability of the p14 protein. Conversely, RNA silencing suppression appeared independent of the nucleolar localization of the protein, and a correlation between BNYVV VSR expression and long-distance movement was established.
authors.affiliation: Institut de Biologie Moléculaire des Plantes, Laboratoire Propre du CNRS (UPR 2357) Conventionné avec l'Université de Strasbourg, 12 rue de Générale Zimmer, 67084 Strasbourg, France.
authors.forename: Sotaro
authors.initials: S
authors.lastname: Chiba
authors.forename: Kamal
authors.initials: K
authors.lastname: Hleibieh
authors.forename: Alice
authors.initials: A
authors.lastname: Delbianco
authors.forename: Elodie
authors.initials: E
authors.lastname: Klein
authors.forename: Claudio
authors.initials: C
authors.lastname: Ratti
authors.forename: Véronique
authors.initials: V
authors.lastname: Ziegler-Graff
authors.forename: Salah
authors.initials: S
authors.lastname: Bouzoubaa
authors.forename: David
authors.initials: D
authors.lastname: Gilmer
mesh_terms: D000595:Amino Acid Sequence; D027461:Beta vulgaris / Q000821:virology; D002466:Cell Nucleolus / Q000378:metabolism; D027465:Chenopodium quinoa / Q000821:virology; D003593:Cytoplasm / Q000378:metabolism; D018506:Gene Expression Regulation, Plant; D015967:Gene Expression Regulation, Viral; D049452:Green Fluorescent Proteins; D016297:Mutagenesis, Site-Directed; D010935:Plant Diseases / Q000821:virology*; D018515:Plant Leaves / Q000648:ultrastructure / Q000821:virology; D010942:Plant Viruses / Q000235:genetics / Q000502:physiology*; D055550:Protein Stability; D021381:Protein Transport; D034622:RNA Interference; D012328:RNA Viruses / Q000235:genetics / Q000502:physiology; D034741:RNA, Small Interfering; D012367:RNA, Viral / Q000235:genetics; D011993:Recombinant Fusion Proteins; D014026:Nicotiana / Q000648:ultrastructure / Q000821:virology*; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D016335:Zinc Fingers
chemical_list: D034741:RNA, Small Interfering; D012367:RNA, Viral; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; C094216:small cysteine-rich protein P14, plant virus; D049452:Green Fluorescent Proteins