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. 2006 Sep;80(18):9064-72.
doi: 10.1128/JVI.00953-06.

Translocation of Tomato bushy stunt virus P19 protein into the nucleus by ALY proteins compromises its silencing suppressor activity

Tomas Canto  1 , Joachim F Uhrig, Maud Swanson, Kathryn M Wright, Stuart A MacFarlane
Affiliations

Translocation of Tomato bushy stunt virus P19 protein into the nucleus by ALY proteins compromises its silencing suppressor activity

Tomas Canto et al. J Virol. 2006 Sep.

Abstract

The P19 protein of Tomato bushy stunt virus is a potent suppressor of RNA silencing and, depending on the host species, is required for short- and long-distance virus movement and symptom production. P19 interacts with plant ALY proteins and relocalizes a subset of these proteins from the nucleus to the cytoplasm. Here we showed that coexpression by agroinfiltration in Nicotiana benthamiana of P19 and the subset of ALY proteins that are not relocalized from the nucleus interfered with the ability of P19 to suppress RNA silencing. We demonstrated that this interference correlates with the relocation of P19 from the cytoplasm into the nucleus, and by constructing and analyzing chimeric ALY genes, we showed that the C-terminal part of the central, RNA recognition motif of ALY is responsible for interaction with P19, relocalization or nonrelocalization of ALY, and inhibition of silencing suppression by P19. We studied the interaction of ALY and P19 by using the technique of bimolecular fluorescence complementation to show that these proteins associate physically in the nucleus but not detectably in the cytoplasm, and we present a model to explain the dynamics of this interaction.

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Figures

FIG. 1.
FIG. 1.
Expression of a subset of ALY proteins inhibits P19 silencing suppressor function. Northern blot analysis of RNA extracted from plants probed for GUS. The blots show GUS mRNA and GUS siRNAs, with ethidium bromide (EtBr) staining as a loading control. All plants infiltrated with a mixture of three Agrobacterium cultures containing binary vectors expressing the GUS gene, together with empty binary vector only (lane 1), empty binary vector plus ALY (lanes 2 to 5), P19 plus empty binary vector (lane 6), and P19 plus ALY (lanes 7 to 10). (A) Arabidopsis ALYs; (B) N. benthamiana ALYs. The presence (+) or absence (−) of GUS, P19, and ALY proteins (AtALY1 to AtALY4 and NbALY615, NbALY617, NbALY916, and NbALY1693) is shown above the blots.
FIG. 2.
FIG. 2.
Tagging of P19 with mRFP does not alter its silencing suppression activity. Northern blots of GUS mRNA and GUS siRNAs extracted from plants infiltrated with the GUS gene together with the empty binary vector (lane 1), native P19 (lane 2), P19-GFP (lane 3), and P19-mRFP (lane 4). RNA loading controls are stained with ethidium bromide (EtBr).
FIG. 3.
FIG. 3.
(A) Fluorescence derived from the suppressor P19-mRFP is mostly cytoplasmic (the right panel shows a view of a field of N. benthamiana epidermal cells) and nuclear but not nucleolar (the left panel shows a view of a nucleus). (B) Fluorescence from the two newly identified N. benthamiana ALY proteins tagged with GFP (NbALY916-GFP and NbALY1693-GFP) accumulated in the nucleus (the left and central panels show views of individual nuclei and of fields of epidermal cells, respectively). However, in the presence of the suppressor P19, NbALY916-GFP relocated to the cytoplasm, whereas NbALY1693-GFP remained nuclear (right panels). (C) Fluorescence from the suppressor P19-mRFP accumulated in the nucleus/nucleolus when coexpressed with ALY-GFPs that remain nuclear in its presence (top row, panels with AtALY1 and AtALY3). P19-mRFP remained mainly cytoplasmic when coexpressed with ALY-GFPs that delocalize to the cytoplasm in the presence of the suppressor (top row, AtALY2 and AtALY4). The images show mRFP- and GFP-derived fluorescence as red and green, respectively. In places where both fluorescences are present, the resulting color is yellow-orange. The panels in the bottom row show a nucleus from an epidermal cell in which both AtALY3-GFP and P19-mRFP are being expressed. The left and central panels show GFP- and mRFP-derived fluorescence, respectively. The rightmost panel is an overlay of both images. Fluorescence from P19-mRFP accumulates strongly in the nucleolus in the presence of AtALY3-GFP. The bars are 10 μm for nucleus panels and 100 μm for field panels. Nu, nucleolus.
FIG. 4.
FIG. 4.
Chimeras between AtALY2 and AtALY3 reveal domains required for relocalization by P19 and nucleolar localization. The left panel of each pair shows the nuclear distribution of GFP-tagged ALY chimeras expressed in the absence of P19. The right panel of each pair shows a field view of GFP-derived fluorescence from coexpression of ALY-GFP and P19-mRFP. The chimera name denotes the origin of the three domains (N-terminal, RRM, and C-terminal domains in that order). The relocation of ALY-GFP to the cytoplasm by coexpression with P19-mRFP (+) and the absence of relocalization (−) are indicated. The bars are 10 μm for the nucleus panel and 100 μm for the field panel.
FIG. 5.
FIG. 5.
(A) The localization of ALY to the nucleus or cytoplasm in the presence of P19 maps to the C-terminal part of the central RRM motif. An AtALY3 chimera carrying the N-terminal RRM portion from AtALY2 (ALY3-23-3-GFP) remained nuclear in the presence of P19 or P19-mRFP (top and middle left panels). In contrast, an AtALY3 chimera carrying the C-terminal RRM fragment from AtAL2 (ALY3-32-3-GFP) relocalized to the cytoplasm in the presence of either P19 or P19-mRFP (top and middle right panels). Bar, 100 μm. (B) The subcellular location of ALY affects the suppressor activity of P19. Northern blot analysis of RNAs extracted from plants probed for GUS coinfiltrated with the intra-RRM chimeras shown in panel A. The blots show GUS mRNA and GUS siRNAs, with ethidium bromide (EtBr) staining as a loading control. All plants infiltrated with a mixture of Agrobacterium cultures containing the GUS gene together with empty binary vector alone (lane 1) or empty binary vector plus AtALY3-23-3-GFP (lanes 2, 4, and 6) or AtALY3-32-3-GFP (lanes 3, 5, and 7). Lanes 1 to 3, no P19; lanes 4 and 5, P19, lanes 6 and 7, P19-mRFP. The suppressor activity of P19 decreased in the presence of the chimera (AtALY3-23-3-GFP) that remained nuclear (lanes 4 and 6 versus lanes 5 and 7).
FIG. 6.
FIG. 6.
BiFC images of ALY-P19 interaction in nuclei. Nuclei are DAPI stained and appear blue, and chloroplasts appear yellow. Pink areas show interaction between the split-mRFP-tagged ALY and P19 proteins. (a) AtALY1; (b) AtALY2; (c) AtALY3; (d) AtALY4; (e) lack of mRFP complementation between P19 and an Arabidopsis proteosome subunit protein. Bars, 5 μm.
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