Cucumber mosaic virus coat protein modulates the accumulation of 2b protein and antiviral silencing that causes symptom recovery in planta

Shoot apical meristems (SAM) are resistant to most plant viruses due to RNA silencing, which is restrained by viral suppressors of RNA silencing (VSRs) to facilitate transient viral invasion of the SAM. In many cases chronic symptoms and long-term virus recovery occur, but the underlying mechanisms are poorly understood. Here, we found that wild-type Cucumber mosaic virus (CMVWT) invaded the SAM transiently, but was subsequently eliminated from the meristems. Unexpectedly, a CMV mutant, designated CMVRA that harbors an alanine substitution in the N-terminal arginine-rich region of the coat protein (CP) persistently invaded the SAM and resulted in visible reductions in apical dominance. Notably, the CMVWT virus elicited more potent antiviral silencing than CMVRA in newly emerging leaves of infected plants. However, both viruses caused severe symptoms with minimal antiviral silencing effects in the Arabidopsis mutants lacking host RNA-DEPENDENT RNA POLYMERASE 6 (RDR6) or SUPPRESSOR OF GENE SILENCING 3 (SGS3), indicating that CMVWT induced host RDR6/SGS3-dependent antiviral silencing. We also showed that reduced accumulation of the 2b protein is elicited in the CMVWT infection and consequently rescues potent antiviral RNA silencing. Indeed, co-infiltration assays showed that the suppression of posttranscriptional gene silencing mediated by 2b is more severely compromised by co-expression of CPWT than by CPRA. We further demonstrated that CPWT had high RNA binding activity leading to translation inhibition in wheat germ systems, and CPWT was associated with SGS3 into punctate granules in vivo. Thus, we propose that the RNAs bound and protected by CPWT possibly serve as templates of RDR6/SGS3 complexes for siRNA amplification. Together, these findings suggest that the CMV CP acts as a central hub that modulates antiviral silencing and VSRs activity, and mediates viral self-attenuation and long-term symptom recovery.

Introduction RNA silencing is a well-established plant antiviral response triggered by viral double-stranded RNAs or highly structured single-stranded RNAs in host plants. Host Dicer-like (DCL) enzymes cleave both RNA types into 21 to 24 nucleotide (nt) small interfering RNAs (siRNAs) that are subsequently sorted into Argonaute-containing RNA-induced silencing complexes (RISC) to guide specific cleavage of the cognate viral RNAs [1][2][3][4][5][6][7]. Some cleavage products serve as templates for host RNA-directed RNA polymerase (RDR) 1, or RDR6, to synthesize abundant de novo dsRNAs that are processed by DCLs into secondary siRNAs that enhance antiviral RNA silencing [5,[7][8][9][10][11]. In addition, a plant-specific RNA binding protein, Suppressor of Gene Silencing 3 (SGS3), is required for siRNA amplification through forming complexes with RDR6 [12]. RNA silencing, as a major defense mechanism, occurs in all virusinfected tissues and is an extensive feature in newly emerging tissues [11,[13][14][15]. Recent studies have revealed that symptom recovery from viral infection is generally concomitant with induction of RNA silencing in the shoot apices of infected plants and this depends in part on RDR activity. For example, Potato virus X (PVX), Turnip crinkle virus (TCV), and Potato spindle tuber viroid (PSTVd) transiently invade the meristems of plant mutants defective in host RDR6 [11,13,14].
As a counter-defense against host RNA silencing, many plant viruses have evolved VSRs to block various RNA silencing steps [16][17][18]. Some VSRs are also viral pathogenicity factors during systemic infections. For instance, the 2b protein of CMV and the 16K protein of Tobacco rattle virus (TRV) facilitate shoot apical meristem (SAM) invasion by blocking antiviral RNA silencing [19][20][21]. Ectopic expression of VSRs, like the potyvirus HC-Pro protein, enhance viral RNA accumulation of two distinct nepoviruses and prevent symptom recovery [22,23]. Nonetheless, viral meristem invasion is transient and is followed by long-term meristem exclusion [19,20]. Hence, the mechanisms of long-term recovery from transient SAM invasion remain to be elucidated.
CMV is the type virus of the genus Cucumovirus in the family Bromoviridae. The CMV genome is composed of three positive-stranded RNAs [24]. RNA1 and RNA2 encode 1a and 2a proteins, respectively, which comprise the viral RNA-dependent RNA polymerase subunits [24]. RNA2-derived subgenomic RNA4A encodes 2b protein, a well-known VSR and determinant factor in viral virulence and systemic infection [5,25,26]. RNA3 and its subgenomic RNA4 encode movement protein (MP) and coat protein (CP) that are required for viral cellto-cell and systemic movements, respectively [24].
The CMV CP is a multifunctional factor that has roles in viral systemic movement, host range and aphid transmission [27][28][29][30][31]. For the Pepo and MY17 CMV strains, CP-mediated cell-to-cell movement is implicated in SAM invasion of host plants [32]. In addition, the CMV CP is an aphid transmission determinant that mediates viral spread between host plants [28,31]. These properties and studies of different CMV strains suggest that CMV CPs are key host range determinants [30]. Previous studies have also shown that some amino acid residues of CMV CP contribute to various symptoms induction. For instance, CMV pepper strain mutants, in which proline 129 is replaced by 19 other amino acids, induce various systemic symptoms in plants [33]. Moreover, CMV CP amino acid 129 also determines viral invasion of the SAM in tobacco plants [32]. Although CMV CPs have been extensively studied as factors involved in positive regulation of viral spread and symptom induction, their negative roles in SAM infections have not been described.
In the current study, we found that the N-terminal R-rich region (R 13 RRRPRR 19 ) of the CMV CP has a negative role in persistent viral SAM invasion. Our data indicate that elevated expression levels of the CMV CP induces potent antiviral RNA silencing by down-regulating the accumulation level of 2b VSRs and inducing siRNA amplification. Thus, we propose a novel self-attenuation mechanism, in which the CMV CP antagonizes the suppression effects of 2b protein and plays a pivotal role in regulating compatible interactions between CMV and host plants to prevent viral over-accumulation and persistent viral invasion of the SAM.

Results
The N-terminal R-rich region of CMV CP negatively regulates viral virulence in shoot apices of N. benthamiana plants Plant virus-encoded CPs mainly participate in encapsidation and movement [34][35][36][37][38], and are increasingly appreciated as an important regulator of viral RNA replication and translation that are associated with CP RNA binding affinity [15,39,40]. Protein sequence analyses revealed that the N-terminal region (R 13 RRRPRR 19 ) of the CMV CP is enriched in basic and positively charged amino acid residues that contribute to functional RNA binding activities. To explore the requirements of this R-rich region in viral infection, the basic amino acid residues were substituted by alanine (R13-19: A), and the resulted mutant was designated as CP RA (Fig 1A). To compare the functions of CP WT and CP RA in the context of viral sequences, we first developed an Agrobacterium tumefaciens-mediated CMV infection system in N. benthamiana plants. The cDNAs of the three CMV genomic RNAs were engineered into pCass4-Rz to generate pCass-RNA1, -RNA2, and -RNA3 ( Fig 1A). The CP WT -and CP RA -containing viruses are named CMV WT and CMV RA , respectively. Infections were carried out by co-infiltration of equal concentrations of A. tumefaciens EHA105 mixtures harboring pCass-RNA1, -RNA2, and -RNA3 into N. benthamiana leaves.
We first explored whether CMV RA could form normal viral particles during viral infections. The CMV WT and CMV RA -infected leaves were homogenized for virions purification, and the purified virions were observed by transmission electron microscopy, which showed that CMV WT and CMV RA formed viral particles with similar appearance (S1A Fig). Nonetheless, the CMV RA particles were less stable than CMV WT virions in an RNase assay (S1B Fig). These results indicate that the CP R-rich motif is not essential for virion assembly or systemic infection in N. benthamiana plants.
The susceptibility of N. benthamiana was examined to determine the function of the Rrich region in systemic infections. At 7 dpi, CMV WT induced mosaic symptoms in the fully expanded leaves, but only elicited limited or recovered symptoms in shoot apices of infected N. benthamiana plants (Fig 1B, right panel). In sharp contrast, CMV RA infections resulted in severely distorted newly emerging leaves (Fig 1B, middle panel). Subsequently, CMV RAinfected plants exhibited extremely short internodes and petioles at shoot apices to produce a rosette appearance combined with substantial stunting between 14 and 21 dpi (Fig 1C). At 42 dpi, CMV WT -infected plants developed mosaic symptoms in leaves present in the central parts of the stems, and exhibited substantial reductions in growth compared to mock-infected plants ( Fig 1D). However, symptoms in the shoot apices of CMV WT -infected plants were modulated and the apices maintained apical dominance (Fig 1D). In contrast, all of the CMV RA -infected plants developed several lateral shoots without distinguishable primary stems (Fig 1D). These symptoms suggested that the shoot apices were severely infected and that apical dominance was disturbed leading to production of lateral bud outgrowths [41].
Since the 2b protein is a strong silencing suppressor [5,25,26], we next examined whether the persistent SAM invasion by CMV RA depends on the 2b protein. A 2b-deleted mutation (CMV-Δ2b) was engineered into pCass-RNA2 by point mutations as described previously [6], and co-infiltrated into N. benthamiana with pCass-RNA1 and wild-type pCass-RNA3 (CMV WT -Δ2b), or mutated pCass-RNA3-CP RA (CMV RA -Δ2b). CMV WT -Δ2b caused mild mosaic symptoms in the systemic leaves, whereas CMV RA -Δ2b did not induce any obvious symptoms (S2A Fig). To explore the replication of CMV WT -Δ2b and CMV RA -Δ2b at 7 dpi, the infiltrated leaves were sampled and the CP accumulation was detected by Western blotting at 7 dpi. Both CMV WT -Δ2b and CMV RA -Δ2b CPs had accumulated to similar levels (S2B Fig), suggesting that the 2b deletion did not affect virus proliferation in infiltrated leaves. In contrast, CMV WT -Δ2b CP was present in the upper uninfiltrated leaves at 7 dpi, but CMV RA -Δ2b CP was not detected (S2C Fig). RT-PCR was performed to detect viral RNA accumulation in the upper leaves with primers corresponding to the RNA3 CP region. CP specific bands were detected in plants inoculated with CMV WT -Δ2b, but not with CMV RA -Δ2b (S2D Fig). Collectively, these data demonstrate that CMV CP WT exerts a negative role in shoot apex infections, and 2b protein is required for systemic infection of the CMV RA mutant.
CMV RA , but not CMV WT , persistently invades the shoot apices of infected plants Because apical dominance was abolished in CMV RA infections, we proposed that CMV RA invaded meristems and altered the meristematic activity of the infected plants. To explore this possibility, longitudinal sections of the topmost flowers and shoot apices from mock-, CMV WT -, and CMV RA -infected plants were examined by in situ hybridization with digoxigenin-labeled CMV RNA3 probes. CMV WT invaded most shoot meristems and floral primordia by 7 dpi, but subsequently disappeared between 14 and 21 dpi, despite of some detectable signals below the SAM and floral primordia (Fig 2, middle panels). In contrast, CMV RA was abundant below the meristems at 7 dpi, and partially moved into the meristems by 14 dpi (Fig  2, right panels). CMV RA invaded the meristems of all the infected plants by 21 dpi (Fig 2, right panels), but as expected were absence in meristems from mock inoculated plants (Fig 2, left panels). These results demonstrate that CMV RA accumulated to high levels and resulted in severe stunting and abolished apical dominance, whereas CMV WT -infected plants recovered from meristem infection.
CMV WT induces more potent antiviral silencing than CMV RA in emerging N. benthamiana tissues Because RNA silencing is a key antiviral mechanism in meristems infections [11,13,14,[19][20][21], we analyzed levels of viral RNA and siRNAs by Northern blotting at 7 dpi. The newly grown tissues were collected for the Northern blotting analysis of viral RNA and siRNA at 7 dpi. A markedly increased accumulation of viral RNA of CMV RA was detected in the newly grown tissues compared with those of CMV WT (  To explore whether CMV WT induction of potent antiviral silencing depends on host RDRs, we inoculated A. thaliana rdr6 and sgs3 mutants with CMV WT or CMV RA . The rdr6 and sgs3 Only a low signal density was present in CMV WT infected meristems at 7 dpi and these disappeared from the meristem by 14 dpi (middle panels). In contrast, CMV RA infection contained abundant signal densities beneath the meristems at 7 dpi, and indicated partially invaded meristems by 14 dpi, and completely invaded meristems at 21 dpi (right panels). Bars = 100 μm.
https://doi.org/10.1371/journal.ppat.1006522.g002 mutants infected with CMV RA displayed severe symptoms similar to those of wild-type plants and whole plant development was restrained ( Fig 4A and Fig 4C). The rdr6 and sgs3 mutant plants infected with CMV WT exhibited more severe disease symptoms in the newly emerging tissues compared with wild-type plants ( Fig 4A and Fig 4C). In addition, Northern RNA hybridizations revealed that the accumulation level of viral RNA in CMV WT -infected wildtype plants was dramatically lower than those of infected rdr6 or sgs3 plants, whereas higher 7 dpi with CMV RA and CMV WT . Loading controls for the high and low molecular weight RNAs were rRNA and U6 RNAs, respectively. (C) Ratios of accumulation levels of CMV gRNA3 and RNA3-vsiRNAs calculated from signal intensities in three independent hybridization experiments. The vsiRNAs/RNA3 values refer to the relative ratios of RNA3-vsiRNAs versus viral genomic RNA3. The values of RNA3 and RNA3-vsiRNAs in CMV RA -infected leaves were set as 1. ** P-value < 0.01; *** P-value < 0.001.
https://doi.org/10.1371/journal.ppat.1006522.g003 . In contrast, high levels of viral RNA and low levels of vsiRNAs were detected in the emerging tissues of Col-0, rdr6 and sgs3 mutants infected with CMV RA (Fig 4B, compare lanes 2, 4, and 6). Collectively, these results demonstrate that RDR6 and SGS3-dependent amplification of vsiRNAs is required for the CMV WT -induced potent antiviral RNA silencing in emerging tissues of CMV WT infected plants.
We next investigated the accumulation of the CP and 2b protein in emerging tissues of CMV infected Col-0, rdr6, and sgs3 mutants. Accumulation of CMV WT CP in the rdr6 and sgs3 mutants increased to level similar to those of CMV RA CP ( Fig 4D, top panel). Notably, accumulation of CMV WT 2b protein was lower level in the rdr6 and sgs3 mutants compared with CMV RA infection, indicating that the accumulation of 2b protein was significantly downregulated in CMV WT -infected plants (Fig 4D, middle panel). Collectively, these results demonstrate that the down-regulated 2b protein cannot efficiently inhibit RDR6/SGS3-dependent antiviral silencing that restricts elevated accumulation of CMV WT in emerging tissues.

CMV CP attenuates 2b-mediated suppression of local GFP silencing in a dose-dependent manner
Our findings have demonstrated that CP WT exerts negative effects during viral SAMs infection. To independently verify the antagonistic roles of CP and 2b, we next examined the effects of CP WT and CP RA on VSR activities of 2b by co-infiltration assays in N. benthamiana plants [42]. Green fluorescence occurring early during transient co-expression of GFP disappeared completely at 5 dpi, indicating that potent silencing was induced (Fig 5A). In contrast, high intensity of GFP fluorescence in the regions of leaves co-expressing 2b and GFP suggested that GFP silencing was efficiently suppressed by the 2b protein ( Fig 5A). We next compared local GFP silencing suppression by 2b co-expressed with the empty vector (V), CP WT , or CP RA , respectively. The CP WT , unlike V or CP RA , significantly attenuated the 2b-mediated suppression of GFP silencing (Fig 5A). The observations were further verified by Western blotting showing that co-expression of CP WT markedly reduced the expression levels of GFP protein expression compared to co-expression of V and CP RA (Fig 5B, top panel). Simultaneously, the CP and 2b protein also accumulated to lower level during co-expression with CP WT than coexpression with V and CP RA (Fig 5B, middle and bottom panels).
To examine the silencing potency in infiltrated regions of N. benthamiana leaves, the accumulation of GFP mRNA and siRNAs was compared by Northern blotting analyses, and hybridization signal densities were measured to determine the relative accumulation of mRNA and siRNAs. The values from leaf samples co-expressing GFP, 2b and V were set as one unit. The GFP mRNA expression level in the sample co-expressing 2b protein and CP WT significantly decreased compared with those of V or CP RA (Fig 5C, top panel and RA1 values, compare lane 4 with lanes 3 and 5), but all patches accumulated similar levels of GFP-derived siRNAs (Fig 5C, middle panel and RA2 values, compare lane 4 with lanes 3 and 5). The relative accumulated levels of GFP siRNAs versus GFP mRNA (RA2/RA1) were compared. The GFP siRNAs/mRNA ratios in the patches co-expressing 2b and CP WT were at least four-fold higher than those co-expressing 2b with V or CP RA (Fig 5C, RA2/RA1 values, compare lane 4 with lanes 3 and 5), demonstrating that CP WT enhances the potency of GFP silencing.
During CMV infection, the accumulation of the CP increases gradually during viral propagation. Therefore, we wondered whether CP affected VSR activities of 2b in a dose-dependent manner. To answer this question, we compared the GFP fluorescence from the different patches infiltrated with a CP concentration gradient (OD 600 = 0, 0.1, 0.2, 0.4, and 0.8) and 2b (OD 600 = 0.2) (Fig 5D, middle panel). A low concentration of infiltrated CP (OD 600 = 0.1) had negligible effects on 2b-mediated suppression of GFP silencing, whereas increasing CP concentrations gradually compromised the inhibitory effects as the increasing concentration of CP, and the highest CP concentration (OD 600 = 0.8) significantly down-regulated the 2b suppression ( Fig 5D, left panel). Additionally, Western blotting analyses revealed that the accumulation of GFP and 2b decreased in proportion to the increasing concentrations of co-expressed CP (Fig 5D, right panel). Thus, our results indicate that low CMV CP levels do not affect 2b suppressive activities, whereas highly abundant CMV CP concentrations down-regulate 2b protein accumulation and attenuate 2b-mediated silencing suppression. The R-rich region of CP is highly conserved in many subgroup I and subgroup II CMV strains, as well as in In the field, synergistic viral diseases are usually caused by interactions of different viruses that result in dramatically increased viruses titer and symptom induction, which are mainly dependent on VSRs activities [43]. With regard to the co-infection of CMV with other plant viruses in the field, we postulated that CMV CP compromises the suppression activity of other VSRs in co-infected plants. To test this hypothesis, we co-infiltrated CP with P19, P38, or HC-Pro in N. benthamiana leaves. In agreement with 2b, the suppressions mediated by the VSRs were attenuated by coinfiltration with CP WT , but this effect was not observed with the CP RA (S5A Fig). The results were also confirmed by Western blotting which revealed a substantial reduction of GFP accumulation in the patches co-expressing of VSRs and CP WT compared with VSRs and CP RA (S5B Fig). At the same time, the co-expression of CP WT rather than CP RA also decreased the accumulation of the VSRs (S5B Fig). We further demonstrated that the compromising effect of the CMV CP on P19-mediated suppression was also dependent on CP WT concentrations (S5C Fig). In conclusion, highly abundant CMV CP concentrations compromise various VSRs suppression activities in patch assays, implying that the CMV CP modulates the synergistic viral disease by regulating silencing interactions and VSRs in the co-infected plants.

CMV CP WT has strong RNA-binding affinity and mediates translation inhibition
The decreased accumulation of VSRs by high-abundant CMV CP might be due to the strong RNA binding activity of CP WT and resulting in translation inhibition. To test this possibility, we carried out North-Western blotting assays to compare the RNA binding affinity of CP WT and CP RA . GST-tagged CP WT , CP RA , and untagged GST were expressed and purified from E. coli expression systems. Different amounts (1 μg, 2 μg, and 4 μg) of GST-CP WT and GST-CP RA were separated in SDS-PAGE gels, transferred to nitrocellulose membranes, renatured, and exposed to digoxigenin-labeled CMV RNA4 or luciferase (Luc) mRNA. High amounts (4 μg) of GST served as a negative control and failed to binding the RNAs (Fig 6A, lane 7), confirming that the GST tag does not have RNA-binding affinity. The GST-CP WT bound much higher levels of CMV RNA4 and Luc mRNA than the GST-CP RA protein (Fig 6A, compare lanes 2, 4  and 6 with lanes 1, 3 and 5). Together, these results clearly indicate that the R-rich region is important for the high RNA binding affinity of CMV CP WT .
We further examined whether the high unspecific RNA binding activity of CP WT resulted in translation inhibition by comparing in vitro translation efficiency in the wheat germ system. The results showed that high concentrations of GST tag did not affect the translation of Luc mRNA (Fig 6B, black line). The highest concentration (66.7 μg/ml) of GST-CP RA had a~20% translation reduction compared with the empty control (P-value < 0.01) (Fig 6B, blue line), indicating that the weak RNA binding of the GST-CP RA protein partially affected mRNA translation. However, more dramatic reductions in the luciferase translation effects were observed as the GST-CP WT concentration was gradually increased, and finally the highest concentration (66.7 μg/ml), was~1% of the empty control (P-value < 0.001) (Fig 6B, red line). These results are in agreement with previous studies, in which the Potato virus A (PVA) CP was found to be involved in inhibition of viral RNA translation [44,45]. Therefore, it appears that the strong RNA-binding affinity and resulting translation inhibition by the CMV CP WT contributes to reduced VSRs accumulation in CMV WT virus infections.

CMV CP associates with SGS3 and RDR6 protein in punctate granules in vivo
In previous studies, high CP concentrations of several positive-sense RNA viruses repressed RNA translation and facilitated virion assembly [44,46,47]. Similarly, our results show that CMV CP also efficiently inhibits translation of Luc mRNA in wheat germ system (Fig 6B). In addition to initiating viral encapsidation and/or CMV ribonucleoprotein (RNP) formation, translation inhibition also contributes to elevated accumulation of aborted mRNA transcripts without translation that might be recognized as aberrant RNA by host RDR to initiate siRNAs amplification. SGS3, acting as co-factor of RDR6, mainly binds to and stabilizes RNA substrates to amplify secondary siRNAs [48][49][50]. To visualize potential CP-RDR6/SGS3 protein associations in living cells and their subcellular occurrence, we conducted bimolecular fluorescence complementation (BiFC) assays with Agrobacterial-infiltrated leaves of N. benthamiana. For this assay, the SGS3 and RuBisco proteins (Rub) were fused with the N-terminal half of sYFP, and the tagged CP WT , CP RA and Rub proteins were fused with the C-terminal half of sYFP. YFP C -CP WT and YFP C -CP RA could be associated with YFP N -SGS3 in the cytoplasm, and formed punctate granules that co-localized with RFP-tagged RDR6 (Fig 7A, top two panels). BiFC fluorescence was not detected in the Rub control samples that were co-expressed with CP or SGS3 (Fig 7A, three bottom panels). To further evaluate the CP-SGS3 protein association in vivo, we performed co-immunoprecipitation (co-IP) assays in planta. In these experiments, Flag-CP WT , Flag-CP RA proteins were co-Luciferase in vitro was measured with a luminometer. Luciferase activities from mRNA without GST, GST-CP RA , or GST-CP WT was set as 100%. Error bars represent the standard error of the mean. Data points are the mean value of three independent experiments. *P-value < 0.05; ** P-value < 0.01; *** Pvalue < 0.001. https://doi.org/10.1371/journal.ppat.1006522.g006 expressed with GFP-SGS3 or GFP proteins, and extracts from infiltrated leaves were used in co-IP assays with anti-Flag beads. Both the Flag-CP WT and Flag-CP RA , immunoprecipitated GFP-SGS3 efficiently, but the GFP protein did not (Fig 7B). Nevertheless, we could not detect the interaction of CPs and SGS3 through a yeast-two-hybrid assay (S6 Fig), hence the association of CPs and SGS3 in N. benthamiana might be indirect. Collectively, both CP WT and CP RA appear to colocalize with SGS3 in punctate granules in vivo and RDR6 also is present in these granules. However, in contrast with CP RA , the CP WT protein has a high affinity with RNA to result in general inhibition of host RNA and viral RNA translation. This inhibition results in production of aberrant RNAs that in turn appear to be associated with SGS3 and RDR6 for siRNA amplification.
In summary, we conclude that the CMV CP WT protein RNA binding contributes indirectly to high potency RNA silencing that presumably results in reduced accumulation of the CMV 2b protein. Reductions in 2b VSRs activities in turn result in a cycle in which increased siRNAs production by RDR6/SGS3 dependent amplification leads to reductions in CMV RNAs, elevated virus attenuation, and protracted symptom recovery in newly emerging leaves of infected plants.

Discussion
Symptom recovery represents an extreme virus attenuation effect, in which, infected plants initially develop sever leaf symptoms, but subsequently newly emerging leaves exhibit a drastically reduced virus accumulation due to induction of antiviral RNA silencing [15,51,52]. In previous studies, host RNA silencing effects and interactions with the CMV Pepo strain 2b protein were shown to be involved in transient appearance of CMV in meristems at 7 dpi, and decreases in virus concentration as new leaves emerged and disappearance in recovered tissues [19,21]. In agreement with these results, we found that CMV Fny strain also infected SAM transiently and then was excluded from the shoot apices leading to symptom recovery (Fig 1  and Fig 4). Our studies unexpectedly found that a mutant harboring an alanine substitution in the N-terminal R-rich region of the CMV CP could persistently invade meristems and block the growth of apical shoots, implying that CMV CP WT facilitates long-term SAM exclusion at late infection stages (Fig 1 and Fig 2). The previous studies have shown that the amino acid 129 of the Pepo CMV CP affects the cell-to-cell movement and determines successful SAM invasion in tobacco plants at 6-8 dpi, but the shoot meristems recovered from the Pepo infection at 21 dpi [21,32]. Combined with previous studies, our results indicate that CMV CP is not only required for successful SAM invasion at the early infection, but also modulates viral exclusion from SAM later in infection. Thus, we propose that the CMV CP plays a critical role in compatible interactions between CMV and host plants.
Plant viral CPs have a primary function involving viral genome encapsidation, but also have been implicated in viral translation and/or replication. At low concentrations, viral CPs usually facilitate RNA replication and/or translation, whereas at higher concentrations, they may inhibit these processes in favor of virion assembly [39,40,[44][45][46]. For instance, the Brome mosaic virus (BMV) CP binds to an RNA element within the 5´UTRs of the viral genome and suppresses the translation of RNA replication proteins [46]. In addition, the Hepatitis C virus (HCV) core protein binds specifically to the internal ribosome entry site (IRES) in the 5´UTR of the viral genome [47,[53][54][55]. This binding requires positively-charged residues in the N-terminal portion of the core protein and results in suppression of translation of downstream genes. In addition, lysine-to-alanine mutations in the N-terminal region of Red clover necrotic mosaic dianthovirus (RCNMV) CP induced more severe symptoms than wild-type virus in N.benthamiana, indicating that the lysine-rich N-terminus of RCNMV CP modulates symptomatology, independently of its role in virion assembly [56]. In line with these examples, we have shown that the CMV CP has high unspecific RNA binding activities and inhibits the translation of Luc mRNA protein in the wheat germ system (Fig 7). The mutant CP RA harboring an alanine substitution in the N-terminal R-rich region was significantly compromised in translation inhibition, implying that the basic and positively charged amino acid residues are required for translation repression by the CMV CP (Fig 7). The high concentrations of CMV CP in the wheat germ system resemble CP concentrations at late stages of infection when the CP is among the most prominent proteins in the cell. Additional experiments are required to explore the binding regions of CMV CP in the mRNA and the viral genomic/subgenomic RNA interactions. Given the wellknown functions of 2b as viral virulence determinants and silencing suppression [6,9,26,[57][58][59][60][61][62][63], we postulate that decreased accumulation of 2b protein by saturated CP directly or indirectly contributes to potent antiviral RNA silencing that resulting in virus exclusion from SAM and symptom recovery. However, we cannot exclude that other host and/or viral components possibly affected by the CP are involved in symptom recovery. For example, non-specifically binding of RNAs by the CP WT protein might disturb metabolic processes of the host and lead to low virus accumulation in the SAM. Another possibility is that unstable particles assembled by the CP RA mutant permit re-infections in the same cells and therefore lead to higher RNA accumulation. Future studies are anticipated to provide exciting insights into symptom recovery processes.
In the process of RNA silencing, host RDRs are required to initiate or amplify RNA silencing via dsRNA synthesis, and the substrates for dsRNA synthesis in vivo are aberrant RNA lacking a cap structure or poly(A) tails. Arabidopsis ETHYLENE-INSENSITIVE5 (EIN5)/ EXORIBONUCLEASE4 (XRN4) encodes a cytoplasmic 5 0 -3 0 exoribonuclease that degrades RNA intermediates derived during mRNA decay and/or RISC slicing, and regulates RDR6-dependent production of siRNAs [64,65]. Arabidopsis Super-Killer2 (SKI2) functions as a cytoplasmic SKI complex to unwind RNAs into the 3´to 5´exoribonuclease complex for decay, and acts as a repressor of endogenous PTGS [66]. Therefore, both 5´to 3´and 3´to 5´cytoplasmic RNA decay pathways function in RDR-dependent silencing. Here, we propose that the high binding activity of CP WT with RNA protects viral RNA intermediates from RNA decay, which increases the substrate concentration of RDR/SGS3 complex and subsequently improves host antiviral silencing. We consistently found that CMV WT -induced antiviral silencing is significantly compromised in emerging leaves of sgs3 and rdr6 Arabidopsis mutants compared with those of wild-type Arabidopsis plants (Fig 4).
As the result of our comparative analyses of CP WT and CP RA in the virus infection and GFP co-infiltration assays, we propose the schematic model shown in Fig 8. At early stages of infection, a low level of CP fails to efficiently inhibit 2b protein accumulation or to induce siRNA amplification, which facilitates high-speed replication of viral RNA. Later in infection, abundant CP binds to viral RNA to initiate virion packaging, inhibit RNA translation and facilitate ribonucleoprotein formation for viral movement, as is consistent with the CP functions of other plant viruses, such as BMV and PVA [44,46]. Simultaneously, saturated CP results in decreased accumulation of 2b protein, which interferes with inhibition of host antiviral RNA silencing. In our experiments, the CP bound RNAs failed to participate in translation and the mRNAs likely were recognized as aberrant RNAs by the RDR/SGS3 complex, and used as substrates for siRNA amplification. Accordingly, the high amounts of CP found at late infection stages reduces synthesis and accumulation of the 2b protein and/or induces siRNA amplification to culminate viral clearance from the shoot apices of infected plants (Fig 8). By comparison, the much lower affinity of CP RA with viral RNA fail to reduce the accumulation of 2b protein efficiently, or induce siRNA amplification. Therefore, we propose that the VSR activities of 2b facilitate continuous infection of CMV RA in SAM regions. Although, it must be noted here that the proposed model is based only on N.benthamiana and Arabidopsis as the host plants, but since CMV infects more than 1000 species, our proposed model provides the basis for a variety of other experiments in a diverse assay of host plants.
Since a relative healthy host provides a better environment for multiplication within-host and between-host transmission, many plant viruses down-regulate viral virulence to avoid severe disease in order to promote the survival of their hosts [67]. For instance, the strong suppressor P0 suppressor encoded by poleroviruses does not accumulate to detectable levels because of suboptimal translation initiation, which leads to low suppressor activity and reduced viral pathogenicity [68]. The Tobacco mosaic virus movement protein also regulates the spread of RNA silencing to self-control viral propagation [69]. Here, our study has revealed a novel self-attenuation mechanism in which the suppression effects of the 2b protein are downregulated by saturated CP.
Collectively, the roles of the interactions between RNA silencing and virus-encoded suppressors in the co-evolution of hosts and pathogens have been extensively investigated. Our results demonstrate that the CMV CP serves as a central hub to facilitate regulation of dynamically integrated connections between antiviral silencing and VSR activities.

Plant materials and virus inoculation
N. benthamiana plants were grown in a growth room with a controlled environmental climate programmed for 16 hours (hrs) of light at 24˚C and 8 hrs in the dark at 21˚C. Seedlings with six to eight fully expanded leaves were used for virus inoculations. For Agrobacterium tumefactions constructions, the cDNAs of RNA1, RNA2, and RNA3, as well as RNA2-Δ2b and RNA3-CP RA were amplified, digested with Stu I and BamH I, introduced into pCass4-Rz, and transformed into A. tumefaciens strain EHA105 [70]. Equal amount of agrobacteria harboring CMV plasmid derivatives were mixed and infiltrated into N. benthamiana leaves as described previously [71]. All the experiments were repeated at least three times with reproducible results. Arabidopsis thaliana rdr6-15 (SAIL_617_H07) and sgs3-1 in Columbia (Col) ecotype were described previously [6]. After vernalized in the dark at 4˚C, the seeds were transferred into a growth room with the condition of 10 hrs in light and 14 hrs in dark at 22˚C. CMV WT and CMV RA virions propagated in N. benthamiana leaves were purified and used as a inocula at 100 μg/mL.

In situ hybridization
Shoot and floral apices of infected plants were collected from infected N. benthamiana, embedded in wax, sectioned, and in situ hybridized as described previously [14]. CMV RNA was detected with the digoxigenin (Roche Diagnostics GmbH) labelled vitro-transcribed RNA fragment corresponding to 3 0 terminal 200 nucleotides of CMV RNA3, and then detected with antibody anti-digoxigenin conjugated to alkaline phosphatase (Roche Diagnostics GmbH) with nitroblue tetrazolium (NBT) and 5-Bromo-4-Chloro-3-Indolyl Phosphate (BCIP, Sigma). Stained samples were examined with a bright-field microscope (DP72, Olympus) for visualization and photography.

Agro-infiltration and local suppression of GFP silencing in patch assays
For transient protein expression in N. benthamiana leaves, CMV CP WT , CP RA and 2b cDNAs were introduced into the pGD binary vector [72]. Leaves of 4-week-old N. benthamiana plants were co-infiltrated with mixed Agrobacterium cultures harboring the positive sense GFP (sGFP) expression plasmid with different combinations of empty vector (V), 2b, and CP plasmids. At 5 dpi, GFP fluorescence in infiltrated leaves was recorded under a long wavelength UV lamp (UVP, California, USA) using a 600D Cannon digital camera [62]. Local suppression assays were independently performed at least three times with reproducible results.

RNA analysis by Northern blotting and RT-PCR
The topmost infected leaves of 10 to 15 plants were pooled for RNA extraction with Trizol reagent according to the manufacturer instruction (Invitrogen, USA). As described previously [6], 5 μg and 10 μg total RNAs were used for detection of viral RNA and vsiRNAs, respectively. CMV genomic and sgRNAs cDNA detection probes from the 3 0 terminal 240 nt of Fny-CMV RNA2 was randomly labeled with [α-32 P] dCTP. VsiRNAs were detected by the labeled DNA oligonucleotides corresponding to CMV gRNA3 as described previously [6]. The upper uninoculated leaves inoculated with CMV WT -Δ2b or CMV RA -Δ2b were collected for RNA extraction and detection at 7 dpi. Total RNA was treated with RNase free-DNase I, and used as a template for first-strand cDNA synthesis with M-MLV reverse transcriptase (Promega, USA) as described previously [73]. Viral infections were monitored by RT-PCR using primers corresponding to the RNA3 CP region. Protein phosphatase 2A (PP2A) was used as an RT-PCR control. For local silencing suppression assay, 5 μg and 10 μg total RNA from infiltrated leaves were used for GFP mRNA and siRNA detections, respectively. The randomly-labeled cDNA probe corresponding to GF region (nt 1-400) of GFP cDNA was used for GFP mRNA detection. The GFP-derived siRNA was detected by [α-32 P] UTP-labeled RNA probe corresponding to GF region of GFP.

Protein expression and RNA binding assay
Firstly, amplified CP WT and CP RA cDNAs were introduced into pGEX-KG vectors respectively, and the recombinant plasmids were transformed into BL21. After induction with 0.2mM isopropyl β-D-thiogalactoside(IPTG) at 18˚C for 18 h, the resulting GST-tagged fusion proteins and GST tags were purified over Glutathione Sepharose 4B (GE Healthcare) affinity columns according to the manufacturer's instructions. The RNA binding assays were performed via a described North-Western blot procedure as described [74]. Briefly, 5 μg of purified GST, GST-CP WT , or GST-CP RA were separated by 12.5% SDS-PAGE and transferred to nitrocellulose membranes. The membranes were incubated with renaturation buffer (50 mM Tris-HCl, pH 7.5, 0.1% TritonX-100, 10% glycerol, 0.1 mM ZnCl 2 and 250 mM KCl) overnight at 4˚C. Then, the membranes were transferred into binding buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 100 mM NaCl, 0.05% Triton X-100, and 1X Denhardt's reagent) containing digoxigenin-11-UTP-labelled (Roche) RNA probe corresponding to CMV RNA 4 or Luc RNA. The bound RNA was blotted with the anti-digoxigen conjugated alkaline phosphatase (1:3000 dilution, Roche) in a NBT/BCIP solution.

In vitro translation assays
The in vitro translation assay were performed as described previously [75]. First, the full-length luciferase (Luc) cDNA was introduced into the pMD19-T vector. Then, the bacteriophage T7 promoter and a poly(A) tail were inserted at the Luc cDNA N-and C-termini, respectively, and the resulting Xba I-linearized plasmid was used as a template for in vitro transcription by the mMESSAGE T7 kit (Ambion, USA). Two micrograms (μg) of Luc mRNA and different concentrations of purified GST-CP WT , GST-CP RA or GST were translated in the Wheat Germ Extract Plus kit (Promega, USA) for 2 hours at 25˚C. Then, luciferase activity of translated products was determined with a 20/20 luminometer (Promega, USA) as described previously [76].

Bimolecular fluorescence complementation (BiFC) assays
BiFC assays were performed with minor modifications as described previously [78]. AtSGS3 and CP WT/RA cDNA fragments, and were the RuBisco control protein (Rub) were cloned into the BiFC vectors pSPYNE-35S and pSPYCE-35S, respectively. A. tumefaciens EHA105 strains containing the recombinant BiFC plasmids and the tomato bushy stunt virus P19 plasmid were co-infiltrated into N. benthamiana leaves at a final ratio of 0.5:0.5:0.3 (OD 600 ) and epidermal cells of infiltrated leaves were observed for fluorescence analysis (YFP) at 2 dpi using confocal laser scanning microscopy (CLSM) (Olympus FV1000). Full-length cDNAs of SGS3 and Impα1 clones was fused to the GAL4 DNA binding domain in pGBKT7 vector, and CP WT and CP RA cloned cDNAs were fused to the GAL4 activation domain of the pGADT7 vector, respectively. Combination of plasmids were co-transformed into the yeast stain AH109 (Left panel). All transformants were grown at 30˚C on media lacking Trp and Leu, and then transferred to media lacking Trp, Leu, His and Ade. (B) Western blotting analyses were performed to determine expression of the SGS3 and CPs proteins. (TIF)