P6 forms punctate, cytoplasmic viroplasm-like structures in vivo and self-interacts in YTH system
To determine the subcellular localization of P6, the plasmid expressing P6 fused with green fluorescent protein (GFP) at its C terminus (P6-GFP) was introduced into onion epidermal cells by particle bombardment. Confocal fluorescence microscopy analysis indicated that abundant, punctate viroplasm-like fluorescent foci were observed in the cytoplasm of the onion cells. The bright discrete foci were of different sizes and scattered in the cytoplasm. No apparent fluorescence was visualized in the nuclei. As a negative control, free GFP resulted in a diffuse pattern of fluorescence that was both nuclear and cytoplasmic, which indicated that the moiety GFP does not affect the localization of P6-GFP (Figure 1A). Identical results were observed when the proteins were expressed in the protoplasts of Nicotiana. benthamiana (Additional file 1, Figure S1). This demonstrated that P6 tends to aggregate to form structures that resemble the matrix of the viroplasm when expressed in the absence of other RBSDV proteins, and led us to speculate that P6 might self-associate and be involved in the formation of the viroplasm.
Subsequently, a YTH assay was performed to find out whether P6 had an intrinsic ability to self-interact in vivo. Combinations of plasmids expressing bait protein BD-P6 and prey protein AD-P6 were transformed into Y187 and AH109 strains, respectively. Making sure there was no transcriptional activation or toxicity of BD-P6 for yeast strains, western blot analysis was carried out to verify that both BD-P6 and AD-P6 were expressed in the yeast (data not shown). Cotransformation and yeast mating assays showed that independent yeast colonies containing pGADT7-P6 and pGBKT7-P6 grew well and turned blue in the β-galactosidase colony-lift filter assay (data not shown), indicating that there were strong interactions between P6 molecules. In contrast, no growth was observed for the negative controls (Figure 1B). This suggested that P6 has an inherent ability to self-interact and is able to form VLS when expressed alone in plant cells.
YTH assays indicate the centrally located region spanning residues 395 to 659 is necessary for P6 self-interaction
As there was not much information available from the literature about P6, protein sequence analysis was performed. BLAST searches indicated that the region approximately inclusive of residues 400 to 675 exhibited limited conservation of amino-acid sequence with the ATPase domain of structural maintenance of chromosomes proteins (SMCs), which play an essential role in chromosome segregation, condensation and organization [18].
In order to determine the region necessary for P6-P6 self-interaction, we sequentially constructed a collection of truncation derivatives that express BD-P698-792, BD-P6274-792, BD-P6274-703, BD-P6395-703, BD-P6395-659, AD-P61-449, AD-P6341-792, AD-P6271-703, AD-P6274-703, AD-P6395-703 and AD-P6395-659, based on the protein sequence analysis results. Homologous binding capabilities between P6 and these deletions were investigated via the YTH assay. Schematic representation of the different P6 truncations is shown in Figure 2A.
The YTH analysis indicated that a centrally located domain between positions 395 and 659 was required for P6-P6 interaction. All truncations harbouring this region were able to interact with intact P6. However, as their N and C termini approached this region, the abilities of the P6 mutants to associate with intact P6 decreased. Varying interaction abilities were indicated by the rates of yeast growth on the selective medium. When the deletion comprised exactly the region from positions 395 to 659, the interaction with P6 was very weak, and the colonies transformed with pGADT7-P6395-659/pGBKT7-P6 or pGADT7-P6/pGBKT7-P6395-659 showed obvious growth inhibition and the streaks turned dark red. Mutant P61-449, in which most of the central and C-terminal region was deleted, showed complete inability to interact with P6 (Figure 2B). Binding capabilities between these deletions were also investigated, and the results demonstrated that, even when both the N and C termini were absent, the deletions had some ability to associate with each other (data not shown). The results suggested that the region from residues 395 to 659 is necessary to sustain the P6 self-interaction and that further truncation might abolish this interaction.
Transient expression experiments of P6 derivatives indicate residues 395 to 659 are important for P6 self-interaction
Recombinant plasmids that can express P6274-792, P6395-703 and P6395-659, fused in-frame to the N terminus of GFP (P6mutant-GFP) or the C terminus of DsRed2 (DsRed-P6mutant), were constructed and their subcellular localization was determined. Plasmids expressing P6mutant-GFP were delivered into onion epidermal cells via biolistic bombardment, whereas those expressing DsRed-P6mutant were introduced into epidermal cells of N. benthamiana leaves by agroinfiltration assay [19].
Biolistic bombardment experiments indicated that P6274-792-GFP mostly formed large bright discrete foci in the cytoplasm of onion cells, but low levels of diffuse cytoplasmic fluorescence were also observed. P6395-703-GFP expression resulted in the formation of irregular aggregate-like structures, and minor levels of diffuse GFP signals were also observed at the peripheries of the nuclei, P6395-659-GFP resulted in very few (generally less than five) discrete and bright foci in the cytoplasm (Figure 3A). Similar results were obtained when these mutants fused with DsRed2 were expressed in the epidermal cells of tobacco leaves (Figure 3B) or tobacco protoplasts (Additional file 2, Figure S2). Numerous dispersed punctate VLS were detected in the tobacco cells expressing DsRed-P6274-792, and the expression of DsRed-P6395-703 and DsRed-P6395-659 resulted in amounts of irregular aggregate-like foci. Weak and uniform red fluorescence signals were present in the cells expressing free DsRed2.
Generally, the fluorescence distribution patterns of the three mutants (P6274-792, P6395-703 and P6395-659) indicated that the 395-659 region is important for P6 localization and that self-assembly is possible outside of the P6 native environment. The results also suggested that residues on both sides of the 395-659 region might be engaged in the process, based on the numbers and the size of the fluorescent foci.
Bimolecular fluorescence complementation assay confirms that P6 molecules self-interact in planta
In order to determine whether P6 molecules self-interact in planta, bimolecular fluorescence complementation assays were carried out (Figure 4). One pair of combinations that can express P6274-703 fused either to YN or YC was constructed and then delivered into N. benthamiana leaves via agroinfiltration. As expected, co-expression of P6274-703-YN and P6274-703-YC induced strong recovered YFP signals, which formed numerous tiny fluorescent sites or irregular aggregate-like structures in the cytoplasm. No YFP signals were detected for the negative controls following the co-expression of P6274-703-YN/YC or P6274-703-YC/YN. The BiFC assay provided strong evidence that the truncated mutant P6274-703 participates in self-interaction so that recovered YFP signals are detected easily in the tobacco cells. From these results, we can confirm that P6 molecules have the ability to self-interact in planta.
Polypeptides consisting of residues 580 to 620 and 615 to 655 are involved in VLS formation
In light of the results above, it is evident that P6395-659, which only constitutes one-third of the entire P6 protein, is essential to P6 self-interaction. It is possible that some specific elements in this fragment are responsible for the VLS formation. A P6 motif prediction using My-Hits scan http://www.expasy.cn showed that three putative motifs might have relatedness to this interacting region. These three putative motifs are designated pumilio RNA-binding repeat profile, sialic-acid binding micronemal adhesive repeat and intra-flagellar transport protein 57, and they correspond to P6 residues 401-439, 584-608 and 624-654, respectively. In addition, the secondary structure prediction demonstrated that a putative coiled-coil motif might reside in the region from residues 550 to 640. To determine which motifs might be involved in VLS formation, corresponding derivatives that express P6△403-440-GFP, P6△580-620-GFP, P6△615-655-GFP, DsRed-P6C△403-440, DsRed-P6C△580-620 and DsRed-P6C△615-655 were constructed and their subcellular localization was investigated. It is noteworthy that we did create several plasmids aiming to express intact P6 fused with DsRed2 but failed to detect the fused protein for unknown reasons. Previous results showed that DsRed-P6274-792 was sufficient to induce inclusion bodies, so we created the corresponding mutants (DsRed-P6C△403-440, DsRed-P6C△580-620 and DsRed-P6C△615-655) based on this abridged construction. Schematic representation of the different P6 deletion derivatives is shown in Figure 5. As described earlier, plasmids expressing P6mutant-GFP were bombarded into onion cells, while those expressing DsRed-P6mutant were introduced into tobacco leaves by agroinfiltration assay.
Confocal fluorescence microscopy showed that P6△403-440-GFP accumulated to form numerous punctate bright foci in the cytoplasm, indistinguishable from those induced by P6-GFP. In contrast, P6△580-620-GFP and P6△615-655-GFP distributed throughout the cytoplasm displaying a weaker fluorescence pattern, compared to free GFP, and the fluorescence signals were always visualized at the periphery of the nuclei. Similar results were obtained when P6 mutants were fused with DsRed2. Numerous dispersed punctate aggregates were detected in the tobacco cells expressing DsRed-P6C△403-440, whereas weak and uniform DsRed2 signals were present in the cells expressing either DsRed-P6C△580-620 or DsRed-P6C△615-655. The results are shown in Figure 6.
To sum up, two polypeptide chains, comprising residues 580 to 620 and 615 to 655, are implicated in VLS formation, and loss of them alters the subcellular localization of P6.
YTH assays demonstrate P6 interacts with P9-1
Immunoelectron microscopy revealed that antibodies against P9-1 reacted with viroplasm in infected cells [8]. Based on our findings above, P6 likely participates in viroplasm formation. This prompted us to further explore the relationship between P6 and P9-1 via a YTH assay. A plasmid that can express BD-P9-1 was constructed and transformed into Y187 strain. Interestingly, the results showed that there is an intimate association between P9-1 and P6 (Figure 7A). Yeast colonies containing both pGBKT7-P9-1 and pGADT7-P6 grew well on the selective medium, whereas yeast transformed with pGBKT7-P9-1 and pGADT7, which was used as a negative control, was unable to grow. This result indicated that P6 interacts with P9-1 in vivo.
P9-1 cannot form inclusion-like structures when expressed alone
Two plasmids that express P9-1-GFP and DsRed-P9-1 were constructed and bombarded into onion epidermal cells to determine P9-1 subcellular localization. Fluorescence microscopy indicated that both P9-1-GFP and DsRed-P9-1 resulted in a pattern of diffuse and uniform fluorescence distribution in the cytoplasm and nuclei of onion cells, which was a little weaker than that of free GFP or DsRed2 controls (Figure 8A). Our results are inconsistent with the conclusion of Zhang et al that P9-1 alone aggregates to form inclusion bodies [16]. The same results were obtained when the plasmids were delivered into tobacco protoplasts via polyethylene glycol (PEG) transfection method or introduced into epidermal cells of tobacco leaves by agroinfiltration assay (Additional file 3, Figure S3). Therefore, we consider that P9-1 has a widespread distribution but no ability to aggregate in the cytoplasm when expressed in plant cells on its own.
Colocalization experiments indicate P6 relocalizes the distribution of P9-1 and recruits P9-1 to VLS
Co-expression experiments were developed to investigate potential P6-P9-1 interactions (Figure 8B). We introduced two plasmids expressing P6-GFP and DsRed-P9-1 into onion cells by cobombardment. Contrary to the case when DsRed-P9-1 was expressed alone, when P6-GFP and DsRed-P9-1 were co-expressed, a striking relocalization of red fluorescence emerged. DsRed-P9-1 displayed a nearly complete coincidence with the intracellular distribution of P6-GFP. The two proteins were colocalized and exclusively presented in discrete punctate VLS, identical to those formed by P6-GFP alone, and no diffuse green or red fluorescent signals were observed in the cytoplasm or the nuclei. Control combinations were also investigated to rule out the possibility that GFP or DsRed2 expression might have some aberrant effects on the DsRed-P9-1 or P6-GFP distribution. The colocalization of P6-GFP and DsRed-P9-1 confirmed that P6 has a dramatic effect on the distribution of P9-1 and that it is caused by the direct association between these two proteins.
YTH assays confirm residues 395 to 659 of P6 are necessary for P6-P9-1 heterologous interaction
Further YTH analyses were performed to examine the regions of P6 crucial for P6-P9-1 heterologous interaction. P6 AD-fused deletions, including AD-P61-449, AD-P6341-792, AD-P6274-703, AD-P6271-703, AD-P6395-703 and AD-P6395-659, were tested and all P6 deletions except AD-P61-449 were able to interact with P9-1. Transformants expressing BD-P9-1 and AD-P61-449 showed no growth on the selective medium, whereas those containing other combinations grew well (Figure 7B). The results indicated that the region located between amino acids 395 and 659 is indispensable for P6-P9-1 interaction.
YTH assays indicate deletion mutants of P9-1 do not interact with P6
We also investigated P9-1 regions crucial for P6-P9-1 interaction. A dozen P9-1 BD-fused deletions that express fusions BD-P9-11-197, BD-P9-11-207, BD-P9-11-248, BD-P9-176-347, BD-P9-1167-347, BD-P9-1198-347, BD-P9-1208-347 and BD-P9-176-207 were constructed. YTH results indicated that all deletions completely lost the ability to interact with P6 (Figure 7C). It is supposed that minor changes in the protein sequence might affect the properties and protein structure of P9-1 and thereby abrogate P6-P9-1 interaction.