Relocalization of nuclear ALY proteins to the cytoplasm by the tomato bushy stunt virus P19 pathogenicity protein

Abstract

The P19 protein of tomato bushy stunt virus (TBSV) is a multifunctional pathogenicity determinant involved in suppression of posttranscriptional gene silencing, virus movement, and symptom induction. Here, we report that P19 interacts with the conserved RNA-binding domain of an as yet uncharacterized family of plant ALY proteins that, in animals, are involved in export of RNAs from the nucleus and transcriptional coactivation. We show that the four ALY proteins encoded by the Arabidopsis genome and two ALY proteins from Nicotiana benthamiana are localized to the nucleus. Moreover, and in contrast to animal ALY, all but one of the proteins are also in the nucleolus, with distinct subnuclear localizations. Infection of plants by TBSV or expression of P19 from Agrobacterium results in relocation of three of the six ALY proteins from the nucleus to the cytoplasm demonstrating specific targeting of the ALY proteins by P19. The differential effects on subcellular localization indicate that, in plants, the various ALY proteins may have different functions. Interaction with and relocalization of ALY is prevented by mutation of P19 at residues previously shown to be important for P19 function in plants. Down-regulation of expression of two N. benthamiana ALY genes by virus-induced gene silencing did not interfere with posttranscriptional gene silencing. Targeting of ALY proteins during TBSV infection may therefore be related to functions of P19 in addition to its silencing suppression activity.

Figure 1.

Alignment of ALY homologs. Mouse (Mm), human (Hs), Drosophila (Dm), Arabidopsis (At), potato (St), tomato (Le), and N. benthamiana (Nb). Residues are shaded by degree of homology. Hatched boxes under the sequences indicate conserved N-terminal (N-cr) and C-terminal (C-cr) regions and RNA-binding domain (RRM).

Figure 2.

Yeast two-hybrid analysis of P19:ALY interactions. A, Yeast growth on double (SD-LW) and triple drop-out media (SD-LWH, supplemented with 20 mm 3-AT). AD is activation domain fusion, BD is DNA-binding domain fusion. The empty activation domain plasmid (pACT2) was used as a negative control. B, Semiquantitative α-galactosidase assay to measure interaction strength.

Figure 3.

P19 interacts in yeast with the RRM domain of AtALY2. Domain names as in Figure 1. N-vr and C-vr domains contain variable repeats of an RGG motif. Deleted regions indicated by hatched lines. l.s. is the most extensive N-terminal deletion obtained during the Arabidopsis cDNA library screen. HSΔ, RHΔ, RSΔ, and RRM of AtALY2 are described in the text. Interaction with P19 was scored by assessment of yeast growth on minimal medium and by semiquantitative α-galactosidase assay.

Figure 4.

Binding of P19 to ALY in vitro. Total proteins of E. coli not expressing recombinant protein (BL21), expressing GST fused with AtALY2 RSΔ (GST-ALY) or GST only (GST). A, Stained with Coomassie. B, Probed with in vitro translated P19 labeled with ³⁵S-Met. Arrows indicate positions of full-length GST-ALY and GST proteins.

Figure 5.

Nuclear localization of ALY proteins and delocalization by TBSV P19. Distribution of fluorescence in epidermal cell infiltrated with Agrobacterium expressing ALY proteins tagged with GFP is confined to the nucleus (A–G: AtALY1, AtALY2, AtALY2 HSΔ, AtALY3, AtALY4, NbALY615, and NbALY617, respectively). A, The nucleus is labeled as Nu and the nucleolus as No. Expression of TBSV P19 tagged with GFP results in fluorescence throughout the cell (H). Expression of AtALY2-GFP in the presence of TBSV, which expresses the P19 protein, resulted in relocalization of fluorescence out of the nuclei into the cytoplasm (close-up view I and field view K, compare with J in which AtALY2-GFP is expressed alone [field view]). By contrast, fluorescence remained nuclear when AtALY2 HSΔ was expressed in the presence of TBSV (L). As with AtALY2-GFP and AtALY4-GFP, NbALY617-GFP also was relocalized from the nucleus to the cytoplasm when coexpressed with P19 from Agrobacterium binary plasmids (M). However, in the presence of P19 mutants R72G and R85G, fluorescence from NbALY617-GFP remained nuclear (N and O, respectively). Bars = 10 μm, except J to L, bars = 100 μm.

Figure 6.

Expression of P19 in infiltrated tissue. Western blot of extracts of leaves infiltrated with Agrobacterium, probed with antibodies to the TBSV P19 protein. Mock is from a plant infiltrated with Agrobacterium carrying an empty binary plasmid. P19 wt are two separate plants infiltrated with Agrobacterium expressing wild-type P19 protein. P19 R72G and P19 R85G are plants infiltrated with Agrobacterium expressing either of the two mutant P19 proteins. Positions of molecular mass size markers appear at left. The location of the P19 protein is indicated at the right of the figure. ** indicates probable P19 dimers. * indicates a cross-reacting plant or Agrobacterium protein. Load indicates protein loading levels revealed by Ponceau Red staining of the blotted filter.

Figure 7.

Effect of inhibition of ALY expression on the local silencing of a transgene. A, VIGS of the endogenous NbALY615 and NbALY617 genes in N. benthamiana plants transgenic for GFP (line 16c) was achieved by inoculating them with TRV carrying fragments of both NbALY615 and NbALY617 (TRV-615/617). RT-PCR was used to show that after 3 weeks, NbALY615 and NbALY617 mRNAs were either absent or very significantly reduced in systemic tissue compared to control plants (last two lanes versus other lanes). Control plants were either nontransgenic, or 16c plants noninoculated (lanes Nt and Ni, respectively), or 16c plants mock-inoculated (lane M) or inoculated with TRV carrying part of the Arabidopsis PDS gene (lane TRV-PDS). Each lane represents a different individual plant. RT-PCR of ubiquitin mRNA was also performed for each plant as a control. B, Upper leaves of the same plants used in A were infiltrated with Agrobacterium carrying a 35SP/GFP/NosT T-DNA to induce silencing of the GFP transgene, which was monitored by northern-blot analysis of the GFP mRNA. Silencing of the GFP transgene (lanes M, TRV-PDS, and TRV 615/617) occurred irrespective of any preestablished silencing of NbALY615 and NbALY617 by VIGS. The normal level of GFP mRNA accumulation is indicated in lane Ni, a 16c plant that was not virus-inoculated and also not infiltrated with Agrobacterium. The ethidium bromide-stained rRNAs are shown below each lane, as loading controls.