Functional characterization and subcellular localization of the 16K cysteine-rich suppressor of gene silencing protein of tobacco rattle virus.
The 16 kDa cysteine-rich protein (16K) of tobacco rattle virus (TRV) is known to partially suppress RNA silencing in Drosophila cells. In this study, we show that 16K suppresses RNA silencing in green fluorescent protein (GFP)-transgenic Nicotiana benthamiana plants using an Agrobacterium-mediated transient assay. 16K slightly reduced the accumulation of short interfering RNAs (siRNA) of GFP, suggesting that the protein may interfere with the initiation and/or maintenance of RNA silencing. Deletion of either the N- or C-terminal part of 16K indicated that the entire 16K open reading frame (ORF) is necessary for its silencing suppression function. Pentapeptide insertion scanning mutagenesis (PSM) revealed that only two short regions of 16K tolerated five extra amino acid insertions without considerable reduction in its silencing suppression function. The tolerant regions coincide with sequence variability between tobravirus cysteine-rich proteins, indicating a strong functional and/or structural conservation of TRV 16K. Confocal laser scanning microscopy of transiently expressed 16K fusions to red fluorescent protein (RFP) revealed a predominant cytoplasmic localization and, in addition, a nuclear localization. In contrast, fusions of RFP with the N-terminal region of 16K localized exclusively to the cytoplasm, whereas fusions between RFP and the C-terminal region of 16K displayed an exclusive nuclear localization. Further analysis of 16K-derived peptide fusions demonstrated that the 16K C-terminal region contained at least two functional bipartite nuclear localization signals which were independently capable of nuclear targeting.
Ghazala
Walid
W
Department of Crop Sciences, Section Plant Virology, Universität Göttingen, Grisebachstraße 6, D-37077 Göttingen, Germany.
Waltermann
Angelika
A
Institute of Plant Diseases and Plant Protection, Leibniz Universität Hannover, Herrenhaeuser Str. 2, D-30419 Hannover, Germany.
Pilot
Ruth
R
Department of Crop Sciences, Section Plant Virology, Universität Göttingen, Grisebachstraße 6, D-37077 Göttingen, Germany.
Winter
Stephan
S
Plant Virus Department, German Collection of Microorganisms and Cell Cultures (DSMZ), Mascheroder Weg 1b, D-38124 Braunschweig, Germany.
Varrelmann
Mark
M
Department of Crop Sciences, Section Plant Virology, Universität Göttingen, Grisebachstraße 6, D-37077 Göttingen, Germany.
D000595:Amino Acid Sequence; D019677:Artificial Gene Fusion; D002460:Cell Line; D002467:Cell Nucleus / Q000737:chemistry; D003593:Cytoplasm / Q000737:chemistry; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000235:genetics; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D019913:Nuclear Localization Signals; D010942:Plant Viruses / Q000502:physiology*; D034622:RNA Interference; D012328:RNA Viruses / Q000502:physiology*; D034741:RNA, Small Interfering / Q000037:antagonists & inhibitors*; D012231:Rhizobium / Q000235:genetics; D016415:Sequence Alignment; D017384:Sequence Deletion; D014026:Nicotiana / Q000821:virology; D014764:Viral Proteins / Q000032:analysis*; D000097573:Red Fluorescent Protein
D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D019913:Nuclear Localization Signals; D034741:RNA, Small Interfering; D014764:Viral Proteins; D000097573:Red Fluorescent Protein; C485184:enhanced green fluorescent protein