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. 2000 Apr 3;19(7):1672-80.
doi: 10.1093/emboj/19.7.1672.

Suppression of post-transcriptional gene silencing by a plant viral protein localized in the nucleus

A P Lucy  1 , H S Guo, W X Li, S W Ding
Affiliations

Suppression of post-transcriptional gene silencing by a plant viral protein localized in the nucleus

A P Lucy et al. EMBO J. .

Abstract

Post-transcriptional gene silencing (PTGS) is a homology-dependent RNA degradation process that may target RNA exclusively in the cytoplasm. In plants, PTGS functions as a natural defense mechanism against viruses. We reported previously that the 2b protein encoded by cucumber mosaic cucumovirus (CMV) is a virulence determinant and a suppressor of PTGS initiation in transgenic Nicotiana benthamiana. By fusion with the green fluorescent protein, we now show that the CMV 2b protein localizes to the nuclei of tobacco suspension cells and whole plants via an arginine-rich nuclear localization signal, (22)KRRRRR(27). We further demonstrate that the nuclear targeting of the 2b protein is required for the efficient suppression of PTGS, indicating that PTGS may be blocked in the nucleus. In addition, our data indicate that the PTGS suppressor activity is important, but not sufficient, for virulence determination by the 2b protein.

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Figures

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Fig. 1. Transient expression of GFP or GFP fusion proteins in BY2 cells. Free GFP (A), 2bGFP (B), 2b6AGFP (C), 2bQAQGFP (D), 2bQGFP (E) or 2b6A–nlsGFP (F). Micrographs on the left show bright field images of the cells to identify the nuclei. The central images show GFP fluorescence alone. Overlays of the fluorescence and bright field images are shown in the right panel. Bar, 10 μm.
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Fig. 2. Optical sections through BY2 cell nuclei. Near-consecutive, serial confocal sections through BY2 cell nuclei transiently expressing 2bGFP (A) or 2b6AGFP (B). Bar, 10 μm; total distance, ∼6 μm.
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Fig. 3. Distribution of GFP and GFP fusion proteins in N.glutinosa leaf epidermal cells. Confocal micrographs showing transient expression of free GFP (A), 2bGFP (B) and 2b6AGFP (C). Bar, 10 μm.
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Fig. 4. Nuclear targeting and silencing suppression. Completely silenced (+) N.benthamiana plants were infected with in vitro RNA transcripts (A) or sap derived from transcript-inoculated plants (B). Total RNAs were extracted from the new leaves that had emerged after systemic infection was established at the days post-inoculation indicated above each lane, and analyzed by Northern blotting. The specificities of the 32P-labeled riboprobes used are indicated next to the panels. Equivalent loading [5 μg for (A) and 4 μg for (B)] of the total plant RNA for each lane was determined by methylene blue staining and densitometry of the 28S rRNA. (–), RNA extracted from an unsilenced GFP transgenic plant.
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Fig. 5. Silencing suppression and virulence determination. Symptoms on N.benthamiana plants inoculated with in vitro transcripts of, from left to right, pPVX–C2b, pPVX–CΔ2b, pPVX–2b6A or pPVX–2b6A–nls.
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Fig. 6. Nuclear localization signals in the N–terminal region of the cucumoviral 2b proteins. Monopartite NLSs are identified with bold lettering and predicted NESs are in bold italics. Shaded residues indicate potential CKII phosphorylation sites. The TAV and PSV 2b proteins also encode putative bipartite NLS in the N–terminal half of the protein (boxed regions).
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