p24G1 Encoded by Grapevine Leafroll-Associated Virus 1 Suppresses RNA Silencing and Elicits Hypersensitive Response-Like Necrosis in Nicotiana Species

Grapevine leafroll-associated virus 1 (GLRaV-1) is a major pathogen associated with grapevine leafroll disease. However, the molecular mechanisms underlying GLRaV-1 interactions with plant cells are unclear. Using Agrobacterium infiltration-mediated RNA-silencing assays, we demonstrated that GLRaV-1 p24 protein (p24G1) acts as an RNA-silencing suppressor (RSS), inhibiting local and systemic RNA silencing. Electrophoretic mobility shift assays showed that p24G1 binds double-stranded 21-nucleotide small interfering RNA (siRNA), and that siRNA binding is required but not sufficient for its RSS activity. p24G1 localizes in the nucleus and can self-interact through its amino acid 10 to 210 region. Dimerization is needed for p24G1 interaction with importin α1 before moving to the nucleus, but is not required for its siRNA binding and RSS activity. Expression of p24G1 from a binary pGD vector or potato virus X-based vector elicited a strong hypersensitive response in Nicotiana species, indicating that p24G1 may be a factor in pathogenesis. Furthermore, p24G1 function in pathogenesis required its RSS activity, dimerization and nuclear localization. In addition, the region of amino acids 122–139 played a crucial role in the nuclear import, siRNA binding, silencing suppression and pathogenic activity of p24G1. These results contribute to our understanding of the molecular mechanisms underlying GLRaV-1 infection.


Introduction
RNA silencing is considered to be the most effective antiviral mechanism in plants. Antiviral RNA silencing is triggered by double-stranded (ds) RNAs that are recognized and processed by the RNaseIII-type DICER enzymes into 21-24 nucleotide (nt) small interfering RNA (siRNA) duplexes. The siRNAs are protected from degradation by HUA enhancer 1-dependent methylation [1] and then recruited by argonaute (AGO) proteins to form an RNA-induced silencing complex (RISC), thereby initiating the sequence-specific degradation of target RNAs [2]. In plants, the RNA-silencing signal can spread systemically to trigger systemic antiviral responses. To counter these responses, most plant viruses encode at least one RNA-silencing suppressor (RSS). Viral RSSs act via two general mechanisms: sequestering siRNAs to prevent their entry into the RISC, or inhibiting the function of

Plant Materials and Growth Conditions, and Preparation of Plasmids
GFP-transgenic line 16c and wild type (wt) N. benthamiana plants were grown under controlled conditions at 23-25 • C with a 16-h light regime. Grapevine (Vitis vinifera) cv. Centennial Seedless plants naturally infected with GLRaV-1 were grown in the experimental fields.

Agroinfiltration and Fluorescence Imaging
The young fully expanded leaves of N. tabacum, and wt and line 16c N. benthamiana plants were used for agroinfiltration. Agrobacterium tumefaciens GV3101 was transformed with each plasmid and agroinfiltration was performed according to previously described methods [27]. For RSS activity and pathogenicity assays, agroinfiltrated plants were monitored every day. The leaf samples from the same treatment within each experiment were mixed for extraction of total RNA and protein. For BiFC and subcellular localization assays, 4',6-diamidino-2-phenylindole (DAPI) and fluorescence signals were visualized at 3 dpi using an Olympus FluoView 1000 confocal microscope equipped with Olympus FluoView FV10-ASW 3.1 Viewer Software, or an Olympus FluoView 3000 confocal microscope equipped with FV31S-SW Viewer Software.
For the RSS activity assay, plants were illuminated with a 100 W handheld longwave ultraviolet (UV) lamp (UV Products, Upland, CA, USA; Black Ray model B 100AP/R), and GFP images were taken with a digital camera. Each experiment was repeated three times.

Northern Blot
Sequences of digoxigenin (DIG)-labeled cDNA probes used for detection of the GFP-derived siRNAs and U6 small nuclear RNA have been previously described [33], and probes were generated by Sangon Biotech company (China).
Northern blotting and generation of DIG-labeled cDNA probes for detection of PVX RNA and GFP mRNA were performed using the DIG High Prime DNA Labeling and Detection Starter Kit II (Roche, Basel, Germany) according to the manufacturer's protocol. Sequences of probes for detection of PVX RNA or GFP mRNA corresponded to the PVX CP sequence or nt 62-673 of the GFP sequence, respectively. RNA samples of 15 or 30 µg were used to detect high-molecular weight RNA or siRNA. Total RNA was separated on 1% agarose-formaldehyde gels, transferred to Hybond-N+ membranes and hybridized with DIG-labeled probes.
Each assay was repeated in three independent experiments.

Protein Interaction Analysis in Yeast
The small-scale lithium acetate transformation method was performed according to the manufacturer's protocol (Clontech Laboratories).
A constant amount (10 ng) of probes was incubated with 0.05 or 0.5 µg of His-tagged fusion protein. Incubation with GFP-His was used as a negative control. Biotin-labeled duplex sRNA was detected by chemiluminescence. Each experiment was repeated three times.

Cell Death Analysis and H 2 O 2 Detection
For Trypan blue or 3,3 -diaminobenzidine (DAB) staining, the leaves were placed in the Trypan blue (10 mL lactic acid, 10 mL glycerol, 10 mL phenol, 10 mL double-distilled H 2 O and 15 mg Trypan blue) or DAB (1 mg/mL DAB, pH 5.7) staining solution for 5 h after slight vacuum infiltration, rinsed with double-distilled H 2 O and then boiled in a 95% (v/v) ethanol solution for 10 min. Each assay was conducted independently three times.

RT-PCR, qRT-PCR and Statistical Analysis
Total RNA was isolated from N. benthamiana leaves or grapevine petioles using the RNeasy Plant Mini Kit (Qiagen, Dusseldorf, North Rhine-Westphalia, Germany). Synthesis of cDNA (at 42 • C) was primed with a mix of random primers using 500 ng of total RNA. PCR amplification was conducted as follows: cDNA denaturation at 95 • C for 5 min; 35 cycles at 94 • C for 30 s, 52-55 • C (depending on the specific primer pair used) for 30 s and 72 • C for 45-60 s, and a final extension step at 72 • C for 10 min.
Data are presented as means and standard deviations. Significant differences between the treatments and the controls were determined using the statistical software package SPSS, which was also used for statistical analyses (SPSS Inc., Chicago, IL, USA, 2001).

p24 G1 Suppresses Local and Systemic RNA Silencing
Products of the ORFs in the 3' region of viruses of the Closteroviridae family are suggested to be involved in the suppression of RNA silencing [36]. Based on a comparison of the genome organization of GLRaV-1, GLRaV-2 and GLRaV-3, p24 G1 (GenBank Accession No. MN660142), encoded by the tenth ORF in the 3'-terminal region of the GLRaV-1 genome, was selected as a candidate RSS and cloned into the expression cassette of the binary vector pGD to explore its antisilencing activity. Leaves of GFP-expressing transgenic N. benthamiana line 16c [37] were infiltrated with cultures harboring pGD-GFP and pGD-p24 G1 . Coinfiltration of pGD-GFP with an empty vector (pGD), or with pGD-p19 [27] expressing p19 of TBSV, a well-characterized suppressor, was used as negative and positive controls, respectively. Leaf patches expressing p24 G1 showed strong GFP fluorescence three days post-infiltration (dpi), similar to that with expression of p19 ( Figure 1a). In contrast, GFP fluorescence was very weak in tissues coinfiltrated with pGD-GFP and pGD (Figure 1a). Moreover, Western blot and Northern blot analyses revealed that the strong GFP fluorescence in the sectors expressing p24 G1 was directly correlated with higher levels of GFP protein and mRNA, and lower accumulation of GFP-derived siRNA, as compared to the negative controls ( Figure 1b). These quantitative analyses confirmed the visual observations, indicating that p24 G1 can suppress local RNA silencing.
Viruses 2020, 12, x FOR PEER REVIEW 6 of 19 emerging leaves (Figure 1c, left panel). In contrast, about 94% or 95% of the upper noninfiltrated leaves in plants coinfiltrated with pGD-GFP and pGD-p24 G1 or pGD-p19 retained GFP fluorescence for 18 dpi (Figure 1c, left panel). Consistent with this, much higher levels of GFP protein and mRNA were observed in these leaves compared to negative control plants (Figure 1c, lower right panels), supporting the visual observation that silencing signals for the GFP transgene did not spread to the upper noninfiltrated leaves. Together, our results demonstrated that p24 G1 suppresses local and systemic RNA silencing.

p24 G1 is a Factor in Pathogenesis Eliciting HR-Like Necrosis in Nicotiana Species
During the Agrobacterium coinfiltration assay, we noticed that leaf patches of line 16c that coinfiltrated with pGD-p24 G1 and pGD-GFP displayed local necrosis at 4 dpi, whereas coinfiltration  Figure 1c. In 55% of the negative control plants (coinfiltrated with pGD-GFP and pGD), the upper newly emerging leaves started to lose GFP fluorescence between major veins as early as 8-10 dpi, and at 18 dpi, about 92% of the negative control plants displayed vein-proximal silencing of GFP in newly emerging leaves (Figure 1c, left panel). In contrast, about 94% or 95% of the upper noninfiltrated leaves in plants coinfiltrated with pGD-GFP and pGD-p24 G1 or pGD-p19 retained GFP fluorescence for 18 dpi (Figure 1c, left panel). Consistent with this, much higher levels of GFP protein and mRNA were observed in these leaves compared to negative control plants (Figure 1c, lower right panels), supporting the visual observation that silencing signals for the GFP transgene did not spread to the upper noninfiltrated leaves.
Together, our results demonstrated that p24 G1 suppresses local and systemic RNA silencing.

p24 G1 Is a Factor in Pathogenesis Eliciting HR-Like Necrosis in Nicotiana Species
During the Agrobacterium coinfiltration assay, we noticed that leaf patches of line 16c that coinfiltrated with pGD-p24 G1 and pGD-GFP displayed local necrosis at 4 dpi, whereas coinfiltration with pGD and pGD-GFP did not produce a necrotic response ( Figure S1), suggesting that p24 G1 plays a role in pathogenicity. To further evaluate this, leaves of wt N. benthamiana plants were infiltrated with pGD-p24 G1 alone. As expected, the agroinfiltrated tissues developed local necrosis at 4 dpi (Figure 2a), resembling the phenotype observed in the coinfiltration assay ( Figure S1). p24 G1 was then expressed from a PVX-based vector. As shown in Figure 2a, expression of p24 G1 substantially enhanced the virulence and pathogenicity of the recombinant virus PVX-p24 G1 : N. benthamiana plants infiltrated with PVX-p24 G1 showed local necrosis in infiltrated tissues at 4 dpi, and at 7 dpi, the plants showed typical apical necrosis that ultimately led to the death of the entire plant, at 10 dpi. In contrast, PVX-infected plants only showed mild mosaic symptoms. Western blot and RT-PCR results confirmed the expression of p24 G1 in the upper noninfiltrated N. benthamiana leaves before necrosis, at 4 dpi ( Figure S2), suggesting that p24 G1 was accurately maintained in the viral progeny. In addition, PVX-p24 G1 elicited local necrosis, covering the infiltrated patches of N. tabacum To investigate whether the necrosis triggered by p24 G1 shares HR characteristics, cell death, the accumulation of H 2 O 2 and induction of defense-related genes were analyzed. At 4 dpi, the PVX-p24 G1 -infiltrated N. benthamiana leaves were deeply stained by treatment with Trypan blue, while the PVX-infected leaves were only lightly stained (Figure 2c), suggesting the occurrence of cell death in response to p24 G1 . We also observed that, at 5 dpi, the upper noninfiltrated leaves of PVX-p24 G1 -infected plants produced a deep brown color after DAB staining, indicating strong accumulation of H 2 O 2 (Figure 2c).
Pathogenesis-related (PR) protein genes PR1, PR4 and PR10 have been reported to be involved in plant cell death and defense responses [38,39]. The qRT-PCR results revealed that transcript levels of NbPR1, NbPR4 and NbPR10 in the infiltrated leaves (3 dpi) and upper young leaves (4 dpi) of PVX-p24 G1 -infected plants, before necrosis, were all much higher than in PVX-infected plants (Figure 2d).
Taken together, our results indicated that p24 G1 is a factor in pathogenesis eliciting HR-like necrosis in Nicotiana species.

p24 G1 Self-Interacts in the Nucleus Through Its 10-210 aa Region
To assess whether p24 G1 can interact with itself, the p24 G1 sequence was fused with the GAL4 activation domain (AD) in pGADT7 or the DNA-binding domain (BD) in pGBKT7 in YTHS. Yeast transformants carrying pGBK-p24 G1 and pGAD-p24 G1 were able to grow on SD/-Leu-Trp-Ade-His medium, whereas negative controls (yeast transformants carrying pGBKT7/pGAD-p24 G1 ) could not (Figure 3a), indicating that p24 G1 can form a dimer in yeast cells.
The BiFC assay was used to investigate whether p24 G1 can self-interact in planta. The p24 G1 sequence was fused to YFP N (N terminus of YFP) in pSPYNE-35S and YFP C (C terminus of YFP) in pSPYCE-35S vectors, respectively. As shown in Figure 3b, strong YFP fluorescence was observed in the nucleus of N. benthamiana cells coexpressing p24 G1 -YFP N and p24 G1 -YFP C , which colocalized with nuclear DAPI staining. In contrast, no or negligible fluorescence was observed in the leaves coexpressing p24 G1 -YFP C /YFP N .

p24 G1 Self-Interacts in the Nucleus Through Its 10-210 aa Region
To assess whether p24 G1 can interact with itself, the p24 G1 sequence was fused with the GAL4 activation domain (AD) in pGADT7 or the DNA-binding domain (BD) in pGBKT7 in YTHS. Yeast transformants carrying pGBK-p24 G1 and pGAD-p24 G1 were able to grow on SD/-Leu-Trp-Ade-His medium, whereas negative controls (yeast transformants carrying pGBKT7/pGAD-p24 G1 ) could not (Figure 3a), indicating that p24 G1 can form a dimer in yeast cells.
The BiFC assay also showed that only ∆1-9 can self-interact and interact with wt p24 in the nucleus of plant cells (Figure 3d, right and upper left panels). Western blotting results demonstrated the expression of all YFP N -tagged mutants in plant cells (Figure 3d, lower left panel), suggesting that the lack of self-interaction is not due to loss of expression. However, the expression of these mutants was obviously weaker than that of wt p24 G1 , indicating that mutations affect the protein's stability.
Taken together, our results indicated that p24 G1 self-interacts in the nucleus, and that aa 10-210, a region that includes all putative α-helices and β-strands, is required for dimer formation.

Dimerization of p24 G1 is a Prerequisite for its Nuclear Targeting Mediated by Importin α1
Our observation that p24 G1 forms a dimer in the nucleus (Figure 3c) suggested its potential nuclear localization. To further clarify the subcellular localization of p24 G1 , p24 G1 -GFP and GFP-p24 G1 (GFP fused to the C or N terminus of p24 G1 , respectively) were expressed in N. benthamiana leaves via agroinfiltration. Confocal laser scanning microscopy of the leaves at 3 dpi revealed that GFP The BiFC assay was used to investigate whether p24 G1 can self-interact in planta. The p24 G1 sequence was fused to YFP N (N terminus of YFP) in pSPYNE-35S and YFP C (C terminus of YFP) in pSPYCE-35S vectors, respectively. As shown in Figure 3b, strong YFP fluorescence was observed in the nucleus of N. benthamiana cells coexpressing p24 G1 -YFP N and p24 G1 -YFP C , which colocalized with nuclear DAPI staining. In contrast, no or negligible fluorescence was observed in the leaves coexpressing p24 G1 -YFP C /YFP N .
The BiFC assay also showed that only ∆1-9 can self-interact and interact with wt p24 G1 in the nucleus of plant cells (Figure 3d, right and upper left panels). Western blotting results demonstrated the expression of all YFP N -tagged mutants in plant cells (Figure 3d, lower left panel), suggesting that the lack of self-interaction is not due to loss of expression. However, the expression of these mutants was obviously weaker than that of wt p24 G1 , indicating that mutations affect the protein's stability.
Taken together, our results indicated that p24 G1 self-interacts in the nucleus, and that aa 10-210, a region that includes all putative α-helices and β-strands, is required for dimer formation.

Dimerization of p24 G1 Is a Prerequisite for Its Nuclear Targeting Mediated by Importin α1
Our observation that p24 G1 forms a dimer in the nucleus (Figure 3c) suggested its potential nuclear localization. To further clarify the subcellular localization of p24 G1 , p24 G1 -GFP and GFP-p24 G1 (GFP fused to the C or N terminus of p24 G1 , respectively) were expressed in N. benthamiana leaves via agroinfiltration. Confocal laser scanning microscopy of the leaves at 3 dpi revealed that GFP fluorescence derived from both fusion proteins preferentially accumulates in the nucleus, with a very weak cytoplasmic distribution (Figure 4a). Within the former organelle, predominantly nucleolus localization was further observed for p24 G1 -GFP but not GFP-p24 G1 using a 100× oil objective (Figure 4a, lower middle and left panels). The distribution pattern of GFP-tagged p24 G1 differed from that of free GFP, which showed the typical cytoplasmic and nuclear distribution (Figure 4a).   Bioinformatics analysis (http://www.moseslab.csb.utoronto.ca/NLStradamus) predicted a possible nuclear localization signal (NLS) in the aa 122-139 region of p24 G1 ( Figure S4a, upper panel), which is rich in basic residues (122-RDRKKKGFSRTLLKRVTKA-139). Therefore, p24 G1 mutant ∆122-139 (deletion of aa 122-139; Figure S4a, lower panel) was generated and inserted into pCam35S-GFP for expression of ∆122-139-GFP. ∆122-139-GFP accumulated mainly in the cytoplasm, with only a very weak GFP signal in the nucleus of N. benthamiana cells (Figure 4a, lower right panel); this was further confirmed by DAPI staining (Figure 4b). The same approach was also employed to analyze the subcellular localization of other p24 G1 mutants: ∆1-9, ∆1-21, ∆1-39 and ∆194-210, which retained the predicted NLS. The subcellular localization of ∆1-9 with self-interaction ability was essentially identical to that of wt p24 G1 , whereas the other three mutants, which lacked dimerization ability, were almost evenly distributed in the cytoplasm and nucleus (Figure 4b). In addition, we observed that ∆122-139 lost homologous interactions in both yeast and plant cells, although the YFP N -tagged ∆122-139 was expressed at a level similar to that of YFP N -p24 G1 (Figure S4b,c). Therefore, our results confirmed the presence of an NLS in the region of aa 122-139 and indicated that the nuclear localization of p24 G1 requires its formation of a dimer.
The classical nuclear import pathway depends on importin α [40]. We investigated the possible interaction between p24 G1 and N. benthamiana importin α1 (NbIMPα1; No. EF137253.1) by BiFC. Reconstitution of YFP fluorescence was observed in the nucleus of cells in N. benthamiana leaves coexpressing p24 G1 -YFP N /IMPα1-YFP C or p24 G1 -YFP C /IMPα1-YFP N (Figure 4c). More specifically, the interaction was observed mainly in the nucleolus, which colocalized with the red fluorescence protein (RFP)-tagged nucleolar protein fibrillarin of N. benthamiana (Figure 4c). In contrast, no fluorescence was observed in the negative controls (leaves coinfiltrated with YFP N /p24 G1 -YFP C ). These results indicated that p24 G1 nuclear targeting is mediated by importin α.

p24 G1 Is Able to Bind ds siRNA
Viral RSSs generally adopt a ds siRNA-binding mechanism to block RNA silencing [7]. To assess whether p24 G1 shares this strategy, EMSA was conducted. His-tagged p24 G1 (p24 G1 -His) was incubated with 21-nt sRNA duplexes. The results showed that p24 G1 -His is able to bind sRNA duplexes, albeit very weakly ( Figure 5). In contrast, the negative control GFP-His protein failed to form complexes with sRNA duplexes. Mutants ∆1-9, ∆1-21, ∆1-39 and ∆194-210 were all able to bind to the 21-nt sRNA duplexes ( Figure 5), regardless of whether they could form dimers, suggesting that p24 G1 can bind ds siRNA as a monomer. The yield of purified His-tagged mutants obtained from the E. coli expression system was too low to be visualized by protein staining after SDS-PAGE ( Figure S5). However, they bound 21-nt sRNA duplexes more effectively than the wt p24 G1 : the binding of p24 G1 -His to probes was almost undetectable under the same experimental conditions ( Figure 5, left panel), and the shifted banding was still weak when a higher amount of p24 G1 -His (0.5 µg) was used (right panel).
In addition, ∆122-139-His without the predicted NLS failed to bind sRNA duplexes ( Figure 5), even though its purified yield obtained from the E. coli expression system was similar to that of wt p24 G1 ( Figure S5). These results indicated that the aa 122-139 region is involved in p24 G1 binding to ds siRNA. panel), and the shifted banding was still weak when a higher amount of p24 G1 -His (0.5 µg) was used (right panel).
In addition, ∆122-139-His without the predicted NLS failed to bind sRNA duplexes ( Figure 5), even though its purified yield obtained from the E. coli expression system was similar to that of wt p24 G1 ( Figure S5). These results indicated that the aa 122-139 region is involved in p24 G1 binding to ds siRNA.

Monomeric p24 G1 Can Suppress RNA Silencing, and siRNA Binding is Insufficient for its RSS Activity
To evaluate the relevance of the self-interaction for the suppression activity of p24 G1 , mutant ∆1-9 with dimerization ability, and the dimerization-defective mutants ∆1-21, ∆1-39, ∆194-210 and ∆122-139 were selected to assess their RSS activity. Leaf patches of line 16c expressing ∆1-9 or ∆1-21 displayed intense GFP fluorescence (Figure 6a), whereas only a weak GFP signal was observed on leaf patches expressing ∆122-139, ∆1-39 or ∆194-210, similar to the negative control (Figure 6a,b). Quantitative analysis of GFP protein by Western blot confirmed the visual observation (Figure 6b). Total RNA was extracted from leaf patches expressing GFP plus ∆1-9, ∆1-21 or ∆122-139 to further analyze the GFP mRNA level. Compared to the expression in ∆122-139-expressing leaf patches or negative controls, expression of ∆1-9 and ∆1-21 resulted in a high level of GFP mRNA (Figure 6c).

Monomeric p24 G1 Can Suppress RNA Silencing, and siRNA Binding Is Insufficient for Its RSS Activity
To evaluate the relevance of the self-interaction for the suppression activity of p24 G1 , mutant ∆1-9 with dimerization ability, and the dimerization-defective mutants ∆1-21, ∆1-39, ∆194-210 and ∆122-139 were selected to assess their RSS activity. Leaf patches of line 16c expressing ∆1-9 or ∆1-21 displayed intense GFP fluorescence (Figure 6a), whereas only a weak GFP signal was observed on leaf patches expressing ∆122-139, ∆1-39 or ∆194-210, similar to the negative control (Figure 6a,b). Quantitative analysis of GFP protein by Western blot confirmed the visual observation (Figure 6b). Total RNA was extracted from leaf patches expressing GFP plus ∆1-9, ∆1-21 or ∆122-139 to further analyze the GFP mRNA level. Compared to the expression in ∆122-139-expressing leaf patches or negative controls, expression of ∆1-9 and ∆1-21 resulted in a high level of GFP mRNA (Figure 6c). Thus, our results demonstrated that ∆1-9 and ∆1-21 retain RSS activity, indicating that the aa 22-210 region is responsible for RSS activity and that self-interaction is not required for p24 G1 suppression of RNA silencing. Since ∆1-39 and ∆194-210 can bind 21-nt sRNA duplexes ( Figure 5), our results also indicated that ds siRNA binding is required but not sufficient for suppression by p24 G1 . Thus, our results demonstrated that ∆1-9 and ∆1-21 retain RSS activity, indicating that the aa 22-210 region is responsible for RSS activity and that self-interaction is not required for p24 G1 suppression of RNA silencing. Since ∆1-39 and ∆194-210 can bind 21-nt sRNA duplexes ( Figure 5), our results also indicated that ds siRNA binding is required but not sufficient for suppression by p24 G1 .

Pathogenic Activity of p24 G1 Requires Both Its RSS Activity and Dimerization
∆1-9, with self-interaction ability and RSS activity, and dimerization-defective mutants with (∆1-21) or without (∆1-39, ∆122-139 and ∆194-210) RSS activity, were selected to assess the effects of RSS activity and dimerization on p24 G1 function in pathogenesis. Local necrosis was observed in N. benthamiana tissues expressing ∆1-9 from the pGD vector at 5 dpi, a one-day delay compared to p24 G1 -expressing leaves. However, the other mutants all lost their ability to elicit a necrotic response in N. benthamiana leaves (Figure 7a). These mutants were then expressed from the PVX vector to investigate their effects on the pathogenicity of recombinant PVX viruses. PVX-∆1-9 was able to elicit systemic necrosis, eventually resulting in plant death, but also with a one-day delay: necrosis in the infiltrated patches and apical necrosis were observed at 5 and 7 dpi, respectively, and the whole plant died at 11 dpi, compared to 4, 6 and 10 dpi, respectively, in PVX-p24 G1 -infected plants.  3.7. Pathogenic Activity of p24 G1 Requires Both Its RSS Activity and Dimerization ∆1-9, with self-interaction ability and RSS activity, and dimerization-defective mutants with (∆1-21) or without (∆1-39, ∆122-139 and ∆194-210) RSS activity, were selected to assess the effects of RSS activity and dimerization on p24 G1 function in pathogenesis. Local necrosis was observed in N. benthamiana tissues expressing ∆1-9 from the pGD vector at 5 dpi, a one-day delay compared to p24 G1 -expressing leaves. However, the other mutants all lost their ability to elicit a necrotic response in N. benthamiana leaves (Figure 7a). These mutants were then expressed from the PVX vector to investigate their effects on the pathogenicity of recombinant PVX viruses. PVX-∆1-9 was able to elicit systemic necrosis, eventually resulting in plant death, but also with a one-day delay: necrosis in the infiltrated patches and apical necrosis were observed at 5 and 7 dpi, respectively, and the whole plant died at 11 dpi, compared to 4, 6 and 10 dpi, respectively, in PVX-p24 G1 -infected plants. In contrast, N. benthamiana plants infected with PVX-∆122-139, PVX-∆1-21, PVX-∆1-39 or PVX-∆192-210 all displayed a phenotype similar to that caused by PVX infection (Figure 7b). These results indicated that both RSS activity and self-interaction are required for p24 G1 function in pathogenesis. expression of NbPR1, NbPR4 and NbPR10 in the infiltrated leaves at 4 dpi (middle panel), a one-day delay compared to the PVX-p24 G1 -infiltrated counterparts (left panel). In contrast, no significant difference in the expression of NbPR1, NbPR4 and NbPR10 was observed between PVX-∆1-21-and PVX-infiltrated leaves at 3 and 4 dpi. Thus, our results revealed a correlation between the pathogenic activity of p24 G1 and the upregulation of PR genes, supporting the notion that bioactive p24 G1 induces a HR-like response.

Discussion
Viral RSSs are key components of the counter-defense system, enabling viruses to overcome plant defenses. Therefore, identification of a viral RSS and elucidation of possible mechanisms involved in RNA-silencing suppression contribute to an understanding of the molecular basis In addition, mutants ∆1-9 and ∆1-21 were further selected to assess their effects on the transcript levels of PR genes. As shown in Figure 7c, similar to p24 G1 , ∆1-9 also greatly upregulated the expression of NbPR1, NbPR4 and NbPR10 in the infiltrated leaves at 4 dpi (middle panel), a one-day delay compared to the PVX-p24 G1 -infiltrated counterparts (left panel). In contrast, no significant difference in the expression of NbPR1, NbPR4 and NbPR10 was observed between PVX-∆1-21-and PVX-infiltrated leaves at 3 and 4 dpi. Thus, our results revealed a correlation between the pathogenic activity of p24 G1 and the upregulation of PR genes, supporting the notion that bioactive p24 G1 induces a HR-like response.

Discussion
Viral RSSs are key components of the counter-defense system, enabling viruses to overcome plant defenses. Therefore, identification of a viral RSS and elucidation of possible mechanisms involved in RNA-silencing suppression contribute to an understanding of the molecular basis underlying viral infection. Here, using an Agrobacterium coinfiltration assay, we showed that p24 G1 encoded by GLRaV-1 is an RSS, blocking local and systemic RNA silencing, and that its suppression activity is comparable to that of TBSV p19 (Figure 1). The finding that p24 G1 self-interacts in vitro and in vivo (Figure 3) supports the notion that oligomerization is a common phenomenon for unrelated RSSs encoded by a variety of plant viruses [9,[11][12][13]. Moreover, the fact that the aa 10-210 region, containing all of the α-helices and β-strands, was required for p24 G1 self-interaction (Figure 3c) also highlights the important role of α-helices and β-strands in dimer formation, as has been demonstrated or suggested for other viral RSSs [9,11,12]. However, in contrast to previous reports for viral RSSs such as CMV 2b and γb of barley stripe mosaic virus, which need to be homodimers to become functional [11,13], our results showed that the dimerization-defective mutant ∆1-21 retains RSS activity, suggesting that the p24 G1 monomer can suppress RNA silencing.
Viral RSSs have been reported to inhibit RNA silencing through ds siRNA binding [4,7,9,41]. We found that p24 G1 uses the same strategy; it showed recognition of 21-nt ds siRNA ( Figure 5), similar to that reported for Tombusvirus p19, HC-Pro of tobacco etch virus and BYV p21 [7]. Moreover, in contrast to crystal structure predictions that p19 [9] and 2b [12] proteins bind ds siRNA as dimers, the dimerization-defective mutants ∆1-21, ∆1-39 and ∆194-210 all bound 21-nt sRNA duplexes ( Figure 5), suggesting that monomeric p24 G1 is able to bind 21-nt ds siRNA. However, ds siRNA binding is not sufficient for the suppressive function of p24 G1 , because both ∆1-39 and ∆194-210 lost this function ( Figure 6). This phenomenon is similar to that reported for RNase3 of sweet potato chlorotic stunt virus [42]. Comparing the numbers of α-helixes and β-strands contained in ∆1-21, ∆1-39 and ∆194-210, it seems that secondary structural elements may also be involved in the RSS activity of p24 G1 . Interestingly, mutants ∆1-9, ∆1-21, ∆1-39 and ∆194-210 bound 21-nt sRNA duplexes much more effectively than wt p24 G1 (Figure 5), although their soluble expression was greatly affected by the mutations (Figure 3d, Figure S5). These results suggest that the deletion of aa 9-39 from the N terminus or aa 16 from the C terminus of p24 G1 leads to a more favorable conformation for its binding to ds siRNA.
The subcellular localization assay revealed that p24 G1 mainly accumulates in the nucleus, in agreement with the distribution pattern of some other viral RSSs [16][17][18][19]. Consistent with previous reports for CMV 2b [17], GVX p15 [18] and CVB p12 [19], our data also indicate that the nuclear distribution pattern of p24 G1 is not essential for its RSS activity, because ∆1-21 showed impaired nuclear localization (Figure 4) but could block RNA silencing ( Figure 6). Moreover, similar to the canonical importin α/β nuclear import pathway adopted by CMV 2b [43] and p6 of cauliflower mosaic virus (CaMV) [16], p24 G1 also interacted with NbIMPα1 (Figure 4b), suggesting that the nuclear import of p24 G1 is mediated by importin α1, although other importin α1-independent nuclear transport pathways cannot be excluded. However, in contrast to the report by Haas et al. [16], where it was shown that monomeric P6 of CaMV can be imported into the nucleus through the importin α pathway, p24 G1 homodimerization was required for its interaction with nuclear import receptor importin α1 and subsequent nuclear transport (Figures 3 and 4).
Expression of p24 G1 from the pGD-or PVX-based vectors triggered local necrosis and lethal systemic necrosis in the model plant N. benthamiana, respectively, and the enhanced symptoms caused by PVX-p24 G1 were not correlated with an increase in the titer of PVX ( Figure 2). Moreover, the systemic necrosis elicited by PVX-p24 G1 shared HR features (Figure 2), and there was a correlation between the pathogenic activity of p24 G1 and the upregulation of PR genes (Figure 7c). Therefore, our results demonstrate that biologically active p24 G1 is a factor in pathogenesis and can elicit a HR-like response in N. benthamiana. The HR response is commonly associated with specific recognition of a pathogen avirulence (avr) factor by a host R gene product [44], and some of viral RSSs have been reported to be elicitors of R gene-mediated HR [45,46]. Our finding warrants further investigation into whether p24 G1 is an avr gene encoded by GLRaV-1, which is recognized by an unknown host R gene, leading to HR. Our results also revealed that RSS activity is required but insufficient for p24 G1 to be a factor in pathogenesis, while self-interaction must be preserved, because dimerization-defective mutants ∆1-21, ∆1-39, ∆122-139 and ∆194-210 all failed to elicit HR-like necrosis in N. benthamiana (Figure 7), even though ∆1-21 retained RSS activity ( Figure 6). The importance of self-interaction for p24 G1 pathogenic activity is consistent with previous reports for CMV 2b [13] and βC1 of tomato yellow leaf curl China betasatellite [47].
Basic aa residues are critical for protein nuclear transport [40], as well as for viral RSS binding of siRNA and suppression of RNA silencing [10,12,27]. p24 G1 contains a predicted NLS in the aa 122-139 region ( Figure S4a) that is rich in basic residues ( Figure S3a), and our results showed that mutant ∆122-139 almost lost its nuclear localization, and failed to bind ds siRNA, block RNA silencing and induce a necrotic response in N. benthamiana (Figures 4-7). These results indicate that the region may act as an NLS, and is also crucial for p24 G1 binding to siRNA, suppressing RNA silencing and acting as a factor in pathogenesis. Analysis of single and multiple mutations is needed to further evaluate the role of the basic aa clusters in this region.
Viral suppressor proteins are diverse in sequence, structure and function. Our previous works showed that p24 encoded by GLRaV-2 belonging to the same family as GLRaV-1 functions as an RSS [27]. GLRaV-1 p24 G1 and GLRaV-2 p24 share only 7.62% sequence identity at the aa level, although they have similar molecular weight. Therefore, it is not surprising that the two suppressor proteins display distinct biological features: p24 G1 localizes in the nucleus ( Figure 4) and can suppress RNA silencing as a monomer (Figure 6), whereas p24 accumulates in the cytoplasm and self-interaction is important for p24 functionality [27].
In conclusion, our results demonstrate that p24 G1 of GLRaV-1 localizes in the nucleus and acts as a strong RSS and a factor in pathogenesis. p24 G1 is able to bind 21-nt ds siRNA, and siRNA binding is required but not sufficient for its suppressive function. p24 G1 interacts with itself, and dimerization is required for its pathogenic activity and importin α1-mediated nuclear targeting, but not for siRNA binding or RSS activity. The results presented here provide important insights into the molecular mechanisms of GLRaV-1 interactions with plant cells.
Supplementary Materials: The following are available online at http://www.mdpi.com/1999-4915/12/10/1111/s1, Figure S1: Local necrosis was observed in leaf patches of N. benthamiana line 16c coinfiltrated with pGD-p24 G1 /pGD-GFP at 4 dpi, but not in leaves coinfiltrated with pGD-GFP/pGD. Arrows indicate infiltrated leaves; Figure S2: Western blot and RT-PCR results confirm accurate maintenance of p24 G1 in the viral progeny. The upper noninfiltrated leaves of PVX-p24 G1 -or PVX-infected plants at 4 dpi were used for RNA and protein extraction. Anti-p24 G1 antiserum was used to detect p24 G1 accumulation in Western blot analysis. CBB staining served as a loading control. M, DNA marker; Figure S3: Predicted secondary structure of p24 G1 (a) and schematic representation of deletion mutants of p24 G1 (b); Figure S4. p24 G1 mutant ∆122-139 loses ability for homologous interaction. (a) Bioinformatics analysis prediction of an NLS in the aa 122-139 region of p24 G1 and schematic representation of deletion mutant ∆122-139. (b,c) ∆122-139 failed to self-interact and interact with wt p24 G1 in both yeast (b) and plant (c) cells. For YTHS, ∆122-139 was fused to GAL4 AD and BD. For the BiFC assay, ∆122-139 was fused to YFP N and YFP C . Bars = 50 µm. Expression of YFP N -tagged p24 G1 and ∆122-139 detected by Western blot using anti-p24 G1 antiserum is indicated at the bottom of the fluorescence images. CBB staining served as a loading control. Pairs of BD-p24 G1 /AD-p24 G1 (b) and p24 G1 -YFP N /p24 G1 -YFP C (c) served as positive controls; Figure S5: Yield of purified His-tagged p24 G1 and its mutants obtained from the E. coli expression system. M, protein marker. Arrows indicate positions of His-tagged fusion proteins; Table S1: Primers used in this study.