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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

February 2013 , Volume 26 , Number  2
Pages  168 - 181

Sotaro Chiba,1,2 Kamal Hleibieh,1 Alice Delbianco,1,3 Elodie Klein,1 Claudio Ratti,3 Véronique Ziegler-Graff,1 Salah Bouzoubaa,1 and David Gilmer1

1Institut de Biologie Moléculaire des Plantes, Laboratoire Propre du CNRS (UPR 2357) Conventionné avec l'Université de Strasbourg, 12 rue du Général Zimmer, 67084 Strasbourg, France; 2Institute of Plant Science and Resources, Okayama University, 2-20-1 Kurashiki, Japan; 3Università di Bologna, Dipartimento di Scienze e Tecnologie Agroambientali Area Patologia Vegetale, Viale G. Fanin, 40, II piano 40127, Bologna, Italy

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Accepted 20 September 2012.

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.

© 2013 The American Phytopathological Society