TYLCCNV betasatellite-encoded βC1 up-regulates the expression of a host calmodulin-like protein

To gain more insights into the mode of action of TYLCCNB βC1 in PTGS suppression, whole genome tiling microarrays were used to probe the changes of transcriptome profile in response to TYLCCNB. The global gene expression patterns of N. benthamiana infected with TYLCCNV isolate Y10 (hereafter referred to 10A) were compared to infections with TYLCCNV and the associated TYLCCNB betasatellite (hereafter referred to 10Aβ). The most pronounced of the differentially expressed genes, was an N. benthamiana ortholog of tobacco calmodulin-like protein, which has been previously identified as an endogenous regulator of gene silencing (rgs-CaM) that is induced by the potyvirus-encoded VSR HC-Pro [27]. Relative transcript quantification by reverse transcription quantitative real-time PCR (RT-qPCR) verified that N. benthamiana rgs-CaM (Nbrgs-CaM) is substantially up-regulated in 10Aβ-infected plants as compared to plants infected by 10A alone or mock-treated plants (Fig. 1A). Northern blots analyses indicated that the levels of Nbrgs-CaM mRNA are low in mature leaves of mock-treated plants and of 10A-infected plants, but are enhanced greatly by 10Aβ infections (Fig. 1B). To determine whether βC1, the only protein known to be encoded by TYLCCNB, is responsible for the enhanced expression of Nbrgs-CaM, we measured the mRNA levels of Nbrgs-CaM in transgenic N. benthamiana plants expressing βC1 under the control of the constitutive CaMV 35S promoter [39]. Levels of Nbrgs-CaM mRNA in the βC1 transgenic plants were over 20-fold higher than those of wild-type (wt) plants (Fig. 1A–B). In addition, in other Solanaceae hosts, TYLCCNV infections containing the betasatellite also exhibited up-regulated expression of the rgs-CaM orthologs in tobacco (Ntrgs-CaM) and tomato (Slrgs-CaM) (Fig. S1).

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Figure 1. Induction of Nicotiana benthamiana calmodulin-like protein Nbrgs-CaM by TYLCCNB βC1.

(A) RT-qPCR analyses of Nbrgs-CaM mRNA levels in N. benthamiana plants infected by TYLCCNV (10A) alone or together with betasatellite (10Aβ), or with agrobacterium carrying an empty vector (Mock) (left bar graph) at 21 dpi, and in βC1 transgenic plants (βC1) and nontransgenic control plants (wt) at 6 to 7-leaf period (right bar graph). Each mean value was derived from three independent experiments (n = 9) and normalized to mRNA of NbGAPDH that served as an internal standard. The relative mRNA levels of Nbrgs-CaM in mock plants (left bar graph) or wt plants (right bar graph) is arbitrarily set as 1. Values are means ± SD. The data were analyzed using one-way ANOVA followed by Student's t test. Asterisk indicates significant differences between genotypes or treatments (**P≤0.01). (B) Northern blot analyses of the Nbrgs-CaM and βC1 mRNA levels in plants described in (A). Ethidium bromide staining of 25S ribosomal RNA (rRNA) was used to confirm equal RNA loading. (C) Pairwise alignment of rgs-CaMs from N. benthamiana (Nbrgs-CaM), N. tabacum (Ntrgs-CaM) and Solanum lycopersicum (Slrgs-CaM) using ClustalX2 software. The N-terminal extension specific for rgs-CaM is denoted by a solid line above the sequences, whereas the three EF-hand motifs characteristic of calmodulins are illustrated by the dotted lines. (D) Phylogenetic tree showing the relationship between the three rgs-CaMs and selective calcium-binding proteins from Arabidopsis (At) and tobacco (Nt). Numbers represent systemic names or GenBank accession numbers for the corresponding genes. Sequence alignments were performed in ClustalX2 and tree construction was conducted in MEGA5.1 using the Neighbour Joining method. The bootstrap values are shown near the internal nodes. CaM: calmodulin; CML: calmodulin-like protein; CBL: calcineurin B-like protein. The scale bar represents the number of changes per site.

https://doi.org/10.1371/journal.ppat.1003921.g001

EST sequences of Nbrgs-CaM were aligned to Ntrgs-CaM and Slrgs-CaM sequences obtained from the Genbank database, and primers were designed to amplify the full-length genes encoding the proteins. Sequencing revealed that the Nbrgs-CaM open reading frame (ORF) is 573-nt (GenBank accession number JX402081), and Blast searches against the N. benthamiana transcriptome database (http://benth-web-pro-1.ucc.usyd.edu.au/blast/blast.php) demonstrated that the ORF had a 100% identity to the Nbv3K725607008 unigene transcript. Sequence analysis also revealed that Nbrgs-CaM encodes a 188-amino acid (aa) calcium-binding protein, which has the highest similarity (94% and 80% aa identities) to Ntrgs-CaM and Slrgs-CaM ORF, respectively. These rgs-CaM orthologs contain a ∼50-aa N-terminal domain, in addition to a calmodulin (CaM) domain in the C-terminal region that has three characteristic EF-hand calcium binding motifs (EF I, II and IV) (Fig. 1C). A phylogenetic tree was constructed to compare the evolutionary relationships between the rgs-CaMs and representative calmodulins (CaM), calmodulin-like (CML) proteins and calcineurin B-like (CBL) proteins from Arabidopsis and Nicotiana spp. The Rgs-CaMs are clustered with Arabidopsis CMLs, with the closest relationships to CML38 and CML39. The three Rgs-CaMs are only distantly related to two CaMs from tobacco (NtCaM1 and NtCaM2), which formed a separate branch with CaMs from Arabidopsis (Fig. 1D).

Nbrgs-CaM-overexpression in N. benthamiana plants phenocopies βC1-transgenic plants

To investigate functional links between Nbrgs-CaM and βC1, we generated transgenic N. benthamiana lines over-expressing Nbrgs-CaM under the control of the CaMV 35S promoter (35S:CaM). Many of the Nbrgs-CaM primary transgenic plants displayed developmental abnormities that could be grouped into three types based on phenotypic severity. Type I plants developed mild morphological alterations with slightly upward curled leaves. Type II plants showed moderate upward leaf curling and small interveinal outgrowths from abaxial leaf surfaces (Fig. 2A). The upper leaves of the type I and II plants usually appeared normal (Fig. 2A), and these plants produced fertile inflorescence. Type III plants had severely distorted, upward curling leaves and more extensive tissues outgrowth (Fig. 2A). The upper leaves of Type III plants maintained the severe curling syndrome and most of these plants failed to produce inflorescences or produced only sterile flowers. Overall the phenotypes of 35S:CaM plants were strikingly similar to those of N. benthamiana transgenic plants expressing βC1 (35S:βC1) [39]. Both the 35S:CaM and 35S:βC1 transgenic plants developed similar abnormities including upward leaf curling, vein swelling and outgrowth of tissues (compare left panels with right panels in Fig. 2A), albeit several 35S:CaM plants lines showed a chlorotic mottling on leaves that was not routinely observed in 35S:βC1 plants (Fig. 2A, middle panel). Three independent transgenic lines #1.1, #2.3 and #3.1, which represented type I, II and III phenotype plants, respectively, were analyzed for transgene expression by Northern and Western blotting. High levels of Nbrgs-CaM accumulated in these transgenic lines compared to undetectable levels in nontransgenic (wt) plants (Fig. 2B). Although plants with severer phenotypes appeared to have slightly higher levels of Nbrgs-CaM mRNA and protein (Fig. 2B), a direct correlation between transgene expression and symptom severity was not evident. Nevertheless, the phenotypic resemblance between 35S:CaM and 35S:βC1 plants suggests that both genes may have functional similarities. For subsequent studies, line #1.1 was used unless otherwise stated due to its high levels of transgene expression and lack of severe phenotype in the plants.

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Figure 2. Ecotopic expression of Nbrgs-CaM in Nicotiana benthamiana phenocopys βC1 transgenic plants.

(A) Parallel comparisons of phenotypes in Nbrgs-CaM and βC1 transgenic N. benthamiana plants. Representative leaves of type I, type II and type III 35S:CaM transgenic plants with varied phenotypic severities were shown on the left panel with the close-up views of the adaxial and abaxial side of upward-curled leaves. βC1 transgenic plants were shown in parallel on the right panels. The arrows indicate outgrowth tissues on the abaxial leaf surfaces of Nbrgs-CaM and βC1 transgenic plants. (B) Analysis of Nbrgs-CaM mRNA and protein levels in nontransgenic (wt) and three Nbrgs-CaM transgenic lines (35S:CaM). Equal loading of proteins and total RNA were confirmed by Coomassie blue staining of Rubisco large subunit and ethidium bromide staining of 25S rRNA, respectively. (C and D) Phenotype of 35S:dsCaM transgenic N. benthamiana (C) and levels of Nbrgs-CaM analyzed by RT-qPCR in 35S:dsCaM-1, 35S:dsCaM-2 transgenic lines at 6–7 leaf period (D). Values are means ± SD. Nine individual plants per genotype were used in each of the measurements. The asterisk indicates significant differences between genotypes analyzed by Student t test (**P≤0.01).

https://doi.org/10.1371/journal.ppat.1003921.g002

Transgenic plants with reduced expression of endogenous Nbrgs-CaM were also generated by transforming N. benthamiana plants with a hairpin RNA interference (RNAi) construct under the control of CaMV 35S promoter (35S:dsCaM). These 35S:dsCaM plants appeared to be normal without discernible developmental defects (Fig. 2C). Moreover, Nbrgs-CaM transcripts in wt and 35S:dsCaM plants were low in abundance and difficult to detect by Northern blotting (data not shown), although RT-qPCR showed that RNAi had reduced the Nbrgs-CaM mRNAs to approximately 20% of wt levels (Fig. 2D).

Nbrgs-CaM suppresses PTGS and blocks geminivirus-induced gene silencing

An earlier study has shown that Ntrgs-CaM suppresses PTGS, representing the first identified ESR in plants [27]. To investigate whether Nbrgs-CaM and its orthologs can suppress PTGS, we employed a transient silencing suppression assay based on the GFP transgenic N. benthamiana line 16c [50], [51] in the study. In this assay, Agrobacterium tumefaciens cultures harboring a binary vector designed to transiently express sense GFP mRNA (35S:GFP) and Agrobacterium derivative harboring a candidate suppressor gene were co-infiltrated into leaves of 16c plants. Agroinfiltration of 16c leaves with 35S:GFP and an empty vector (negative control) triggered GFP RNA silencing and resulted in weak GFP fluorescence under long-wavelength UV light at 4-days post infiltration (dpi) (Fig. 3A). As expected, co-infiltration of 35S:GFP with βC1 of TYLCCNB or p19 of Tomato bushy stunt virus (TBSV) elicited strong green fluorescence as a consequence of suppression of GFP RNA silencing (Fig. 3A). Expression of Nbrgs-CaM, Ntrgs-CaM and Slrgs-CaM also suppressed GFP silencing as indicated by appearance of green fluorescence in co-infiltrated regions (Fig. 3A). The GFP fluorescence imaging shown in Fig. 3A was also correlated with the accumulation of GFP protein and mRNA as assessed by immunoblot and RNA blot analyses (Fig. 3B). High levels of expression of Nbrgs-CaM, Ntrgs-CaM and Slrgs-CaM in agroinfiltrated leaves were also verified by RNA blotting with rgs-CaM-specific probes (Fig. 3B). Notably, transient expression of βC1 also led to increased accumulation of Nbrgs-CaM mRNA compared to undetectable basal levels in vector-infiltrated leaves. By contrast, an unrelated VSR p19 failed to induce Nbrgs-CaM expression (Fig. 3B), suggesting that induction of Nbrgs-CaM is unlikely a common feature associated with VSRs. Small RNA gel blot analysis revealed that high levels of 21, 22 and 24 nt GFP-specific siRNAs accumulated in the vector control-treated leaves as a result of GFP RNA silencing. In contrast, expressions of the three rgs-CaMs and βC1 markedly decreased the amount of GFP siRNAs (Fig. 3B). Similarly, p19 also inhibited the accumulation of the siRNAs (Fig. 3B) as previously reported [26]. Rgs-CaMs infiltrated leaves had comparable suppression activity to those of βC1 infiltrations, but were weaker than that elicited by p19 (Fig. 3A–B). Overall, these data demonstrate that, as with βC1, all three rgs-CaMs exhibit PTGS suppression activity and inhibit siRNA biogenesis.

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Figure 3. Nbrgs-CaM suppresses PTGS of GFP and VIGS of an endogenous gene.

(A) Suppression of GFP silencing in Nicotiana benthamiana line 16c. Leaf patches were co-infiltrated with Agrobacterium tumefaciens cultures expressing GFP (35S:GFP) and vector control (Vec), Nbrgs-CaM, Ntrgs-CaM, Slrgs-CaM, TYLCCNB βC1 or TBSV p19, and GFP fluorescence were photographed under UV light at 4 dpi. (B) Protein and RNA gel blot analyses of GFP silencing in agroinfiltrated leaf samples. GFP or βC1 specific monoclonal antibody was used in immunoblottings. [α-³²P]-labeled GFP- or Nbrgs-CaM-specific probe was used in the large RNA blot. Note that the Nbrgs-CaM-specific probes also cross-reacts to Ntrgs-CaM and Slrgs-CaM mRNAs in Northern blots owing to their high sequence similarities. [γ-³²P] ATP-labeled GFP or U6 oligonucleotides were used as probes in the small RNA blots. The sizes of the 21-, 22- and 24-nt RNAs are indicated to the right of the small RNA panel. Ethidium bromide staining of 25S rRNA and Coomassie blue staining of the large subunit of Rubisco served as loading controls for RNA and protein gels, respectively. (C) Phenotypes of Su VIGS. Wt, 35S:CaM or 35S:dsCaM N. benthamiana plants were infected with the TYLCCNV-derived VIGS vector carrying a portion of the endogenous Su gene (10A+2mDNA1-NbSu) and photographed at 30 dpi. (D) RT-qPCR analysis of Su silencing. The relative levels of Su mRNA in plants shown in (C) were normalized to mRNA of NbGAPDH that served as an internal standard. Mock represents relative Su mRNA in wt plants infected by the VIGS vector without Su insertion (10A+2mDNA1). The relative level of Su mRNA in mock plants is arbitrarily set as 1. Error bars represent SD of nine individual plants. Asterisks indicate P values compared with mock-treated wild type plants: *P≤0.05, **P≤0.01 (Student's t test). (E) Southern blot analysis of viral DNAs in the VIGS vector-infected plants shown in (C). Total DNAs were blotted with α-³²P-labeled probes specific for TYLCCNV (10A) and alphasatellite (DNA1). The migrations of viral supercoiled DNA (scDNA) are indicated to the right of the panel. Total DNA agarose gel was stained with ethidium bromide to show the equal loading.

https://doi.org/10.1371/journal.ppat.1003921.g003

To further examine the function of Nbrgs-CaM in RNA silencing suppression in the context of a virus infection, we tested whether Nbrgs-CaM could suppress VIGS by using a previously established VIGS vector based on 10A helper virus in association with a geminivirus alphasatellite derivative (2mDNA1) [52], [53]. The alphasatellite vector (2mDNA1-NbSu) was designed to express a fragment of the N. benthamiana sulfur (Su) reporter gene that encodes a component required for chlorophyll II biosynthesis. The recombinant viral vector (10A+2mDNA1-NbSu) triggered Su silencing in leaves of infected plants as evidenced by yellow-white leaf spots [52], [53]. In wt N. benthamiana infected by the VIGS vector, the typical Su silencing phenotype first appeared along the main veins of the newly expanded leaves at 10 dpi, and later expand to the lateral veins to produce a white lattice-like phenotype at 30 dpi (Fig. 3C). However, infected 35S:CaM transgenic line #1.1 failed to develop an Su silencing by 30 dpi (Fig. 2C). Even at late stages of infection (40 dpi), only sporadic white spots were observed in some of the newly emerged leaves in 35S:CaM plants (data not shown). On the other hand, Su silencing appeared 4 to 5 days earlier in infected Nbrgs-CaM RNAi line plants (35S:dsCaM) than in wt plants, and more extensive Su silencing was noted at 30 dpi (Fig. 3C). When additional Nbrgs-CaM transgenic lines were analyzed, similar lack of or delayed Su silencing phenotypes were observed in 35S:CaM lines, in contrast to more extensive silencing in 35S:dsCaM lines (Fig. S2). Analysis of Su mRNA levels in these plants by RT-qPCR also supported the observed silencing phenotypes (Fig. 3D). When compared to endogenous Su mRNA level in mock-silenced plants (infection with an empty VIGS vector 10A+2mDNA1), VIGS reduced the Su mRNA levels by ∼4-fold in wt plants, and ∼5-fold in 35S:dsCaM plants but less than 2-flod in 35S:CaM plants (Fig. 3D, Fig. S2). Notably, both the 10A helper virus and alphasatellite accumulated to higher levels in 35S:CaM plants and to lower levels in 35S:dsCaM plants, relative to wt plants (Fig. 3E). Therefore, the altered VIGS efficiencies in Nbrgs-CaM transgenic lines are not a consequence of differential virus accumulations. These data indicate that the cellular levels of Nbrgs-CaM are negatively correlated with the effectiveness of VIGS in N. benthamiana, and support a model that Nbrgs-CaM functions as an endogenous negative regulator of RNA silencing.

Rgs-CaMs and βC1 target a step upstream of dsRNA generation in PTGS

In plants, PTGS can be triggered by sense RNA (S-PTGS) or inverted repeat RNA (IR-PTGS). Gene silencing triggered by sense RNA trigger requires the host RNA copy machinery to convert target RNA into dsRNA in the initiation step, whereas inverted repeat RNA or hairpin RNA could directly trigger PTGS [3], [4]. To pinpoint the specific steps targeted by βC1 and Nbrgs-CaM in RNA silencing pathway, we investigated their abilities to suppress S-PTGS and IR-PTGS. For S-PTGS, N. benthamiana leaves were co-infiltrated with Agrobacterium cultures expressing the suppressors, GFP reporter (35S:GFP) and a C-terminal 400 bp-fragment of sense GFP RNA (35S:FP). The procedure of IR-PTGS suppression assay is similar to that of S-PTGS, except that a fragment of GFP dsRNA was expressed from binary vector (35S:dsFP) to serve as a silencing inducer. As shown in Fig. 4A–B, in the absence of a suppressor (vector control), GFP expression was efficiently silenced by simultaneous expression of either the sense RNA trigger (35:FP) or the dsRNA trigger (35S:dsFP) as indicated by red fluorescence in the infiltrated leaves (top row). Similar to the experiments in Fig. 3A, both βC1 and Nbrgs-CaM suppressed GFP silencing triggered by 35S:FP, resulting in bright green fluorescence in co-infiltrated areas of leaves (Fig. 4A, second and third row, respectively). However, neither βC1 nor Nbrgs-CaM suppressed GFP silencing in IR-PTGS, as the infiltrated areas showed red autofluorescence similar to the vector-infiltrated leaves (Fig. 4B, compare second and third rows with top row). As a positive control, p19 suppressed both S-PTGS and IR-PTGS of GFP (Fig. 4A–B, bottom row; Fig. 4C–D), which is consistent with the model that p19 suppresses RNA silencing by sequestering 21-nt siRNA [20]. The GFP imaging data were further confirmed by immunoblot and RNA blot analyses of the accumulation levels of GFP protein and mRNA in infiltrated leaf patches (Fig. 4C–D). The siRNA blots also confirmed that βC1, Nbrgs-CaM and p19 drastically reduced the GFP siRNA during S-PTGS (Fig. 4C). For detection of GFP siRNAs in IR-PTGS assays, we designed two different probes corresponding to the FP portion and G portion of GFP mRNA. The “FP siRNAs” were presumably produced from DCLs-processing of dsRNA precursors transcribed from the 35:dsFP binary vector, and thus represents primary siRNAs. The “G siRNAs” were likely generated from GFP mRNA templates by activities of plant RDRs followed by DCLs-cleavages, and hence should be regarded as secondary siRNAs. Small RNA hybridization showed that both βC1 and Nbrgs-CaM strongly inhibited G siRNA levels without obvious effect on FP siRNAs (Fig. 4D), suggesting that the two proteins suppressed secondary siRNA production. Likewise, Ntrgs-CaM and Slrgs-CaM were also found to suppress S-PTGS but not IR-PTGS of GFP (Fig. S3). Therefore, our data suggest that both βC1 and rgs-CaMs interfere with a step upstream of dsRNA generation in the PTGS pathway.

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Figure 4. Nbrgs-CaM and βC1 suppress sense-PTGS and the production of secondary siRNA.

(A) and (B) GFP fluorescence in leaves of Nicotiana benthamiana plants co-infiltrated Agrobacterium cultures expressing the indicated suppressors and GFP reporter (35S:GFP), together with either a sense RNA fragment of GFP (35S:FP) (A) or dsRNA fragment of GFP (35S:dsFP) (B) as indicated on the top of each panel. The infiltrated leaves were photographed at 3 and 5 dpi under UV light. (C) and (D) Protein and RNA gel blot analyses of GFP silencing in agroinfiltrated leaf samples shown in (A) and (B), respectively. GFP or βC1 specific monoclonal antibody was used in immunoblotting. Coomassie blue staining of the large subunit of Rubisco served as loading controls. In the large RNA blots, [α-³²P]-labeled DNA fragments of GFP and Nbrgs-CaM were used as probes and ethidium bromide staining of 25S rRNA confirmed the equal loading. For small RNA blots, [γ-³²P] ATP-labeled GFP oligonucleotides annealed to different region of GFP mRNA were mixed and used as probes in (C), whereas labeled GFP oligonucleotides corresponding to “FP” region and “G” regions were used to detect primary FP siRNA (1^st siRNA) and secondary G siRNA (2^nd siRNA), respectively in (D). The U6 RNA blot was used as a loading control of small RNA blot. The sizes of the 21-, 22- and 24-nt RNAs are indicated to the right of the small RNA panel.

https://doi.org/10.1371/journal.ppat.1003921.g004

Nbrgs-CaM mediates βC1 functions in PTGS suppression and symptom induction

The up-regulation of Nbrgs-CaM by βC1 (Fig. 1), together with their overlapping functions in PTGS suppression and symptom modulation (Fig. 2, 3 and 4), led us to develop a model in which βC1 functions are mediated by Nbrgs-CaM. To test this hypothesis, we first analyzed the ability of βC1 to suppress S-PTGS in wt and 35S:dsCaM plants using a transient agroinfiltration assay. In wt plants, suppression of S-PTGS of GFP by βC1 was confirmed by the presence of strong GFP fluorescence in infiltrated leaf patches under UV light (Fig. 5A), the accumulation of GFP protein and mRNA and the absence of GFP siRNA (Fig. 5B). However, in 35S:dsCaM plants, βC1 failed to suppress GFP silencing (Fig. 5A–B), even though comparable amounts of βC1 were expressed in wt and 35S:dsCaM plants (Fig. 5B). As a positive control, p19 suppressed GFP silencing in both wt and 35S:dsCaM plants (Fig. 5A–B).

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Figure 5. Nbrgs-CaM is genetically required for the functions of βC1 in PTGS suppression and symptom modulation.

(A) GFP fluorescence in leaves of wt and 35S:dsCaM Nicotiana benthamiana plants co-infiltrated with 35S:GFP and 35S:FP, together with indicated suppressors. Photographs were taken under UV light at 3 and 5 dpi. (B) Protein and RNA gel blot analyses of GFP silencing in agroinfiltrated leaf samples shown in (A) at 5 dpi. GFP or βC1 specific monoclonal antibody was used in immunoblotting. Coomassie blue staining of the large subunit of Rubisco served as loading controls. In large RNA blot, [α-³²P]-labeled DNA fragments of GFP and Nbrgs-CaM were used as probes and ethidium bromide staining of 25S rRNA confirmed the equal loading. In the small RNA blot, [γ-³²P] ATP-labeled GFP or U6 oligonucleotides were used as probes. The sizes of the 21-, 22- and 24-nt marker RNAs are indicated to the right of the small RNA panel. (C) Symptoms of wt and dsCaM transgenic N. benthamiana plants infected with PVX-βC1 at 15 dpi. (D and E) Analyses of the RNA and protein levels of PVX coat protein (CP) and βC1 in wt and dsCaM transgenic plants infected with PVX-βC1. Total RNAs were extracted from infected plants at 15 dpi and blotted with probes specific for PVX CP or βC1 (D), and total protein samples were blotted with specific PVX-CP and βC1 antibodies in a Western blot (E). Ethidium bromide staining of 25S rRNA and Coomassie staining are shown to indicate equal loading. The migrations of genomic RNA (gRNA), subgenomic RNA1 (sgRNA1), CP subgenomic RNA (CPsgRNA) and βC1 subgenomic RNA (βC1sgRNA) were indicated.

https://doi.org/10.1371/journal.ppat.1003921.g005

To exclude any unspecific defect of the 35S:dsCaM transgenic plants in the βC1 VSR function, we used N. benthamiana 16c plants in which the expression of Nbrgs-CaM was silenced by the well-characterized Tobacco rattle virus (TRV)-based VIGS vector [54], which decreased Nbrgs-CaM mRNA levels to 20% of mock-treated plants at 7 dpi (Fig. S4B). Systemically silenced leaves of these plants were then used for Agrobacterium co-infiltration in the transient PTGS suppression assay. Again, the abilities of βC1 to suppress GFP silencing and decreased GFP siRNA levels were compromised in Nbrgs-CaM-silenced plants, but not in mock-silenced plants (Fig. S4A and S4C). These data collectively suggest that Nbrgs-CaM is required for the ability of βC1 to suppress PTGS and to inhibit siRNA generation.

To further investigate whether βC1-induced developmental abnormities are mediated by Nbrgs-CaM, we infected wt and 35S:dsCaM N. benthamiana plants at 6–7 leaf stage with a recombinant PVX vector carrying the βC1 gene (PVX-βC1). As shown in Fig. 5C, PVX-βC1-infected wt plants developed typical βC1-associated phenotypes such as upward leaf curling and enation, in addition to the PVX mosaic symptoms. However, in PVX-βC1-infected 35S:dsCaM plants, symptoms characteristic of βC1 failed to appear, and only slightly downward leaf curling and mosaic symptoms indicative of PVX infection were observed. Comparable levels of the recombinant PVX genomic and subgenomic RNAs were verified by RNA blot analyses with probes specific to PVX coat protein (CP) and βC1 (Fig. 5D). Furthermore, immunoblotting also confirmed similar expression levels of βC1 and PVX CP in wt and 35S:dsCaM plants (Fig. 5E). Taken together, we conclude that βC1 functions as a VSR and symptom determinant are mediated by Nbrgs-CaM.

Rgs-CaMs are required for TYLCCNV virulence and genome amplification in Solanaceae hosts

Given the important roles of βC1 in TYLCCNV pathogenesis [38], [39], and the involvement of Nbrgs-CaM in these processes (Fig. 5), we anticipated that Nbrgs-CaM has a major function in TYLCCNV infection. To this end, we compared the susceptibilities of Nbrgs-CaM transgenic plants and wt plants to 10Aβ infection. After infection, the 35S:CaM plants showed more extensive leaf curling and greater numbers of curled leaves than wt plants. In contrast, the symptoms in infected 35S:dsCaM plants were much milder than those in wt plants (Fig. 6A). The onset of disease symptoms was also advanced by 2 days in 35S:CaM plants (5 dpi compared to 7 dpi in wt plants), but was delayed to 9 to 10 days in 35S:dsCaM plants. Because the #1.1 line of the 35S:CaM plants had only minor abnormality in upper leaves prior to infection (Fig. 2A), the exacerbated symptoms observed in 35S:CaM plants were most likely due to enhanced virulence of 10Aβ in the plants. Accordingly, Southern blot analysis indicated an increased viral DNA accumulations of both helper (10A) and the betasatellite (10β) in 35S:CaM plants. This finding, plus decreased viral DNA accumulations in 35S:dsCaM plants (Fig. 6B), further supports a role of Nbrgs-CaM in TYLCCNV infection. We also tested the sensitivities of these Nbrgs-CaM transgenic plants to infection with CMV, an unrelated RNA virus. As with the case with 10A and 10Aβ, 35S:CaM plants were more prone to CMV infection whereas 35S:dsCaM plants were more recalcitrant, as judged from symptom severities and viral CP accumulations (Fig. S5). Thus, it appears that Nbrgs-CaM-mediated negative regulation of RNA silencing in plants may confer general susceptibilities to virus infections.

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Figure 6. Nbrgs-CaM positively regulated the symptom and accumulation of 10Aβ.

(A) Symptoms of wild type (wt), overexpression line (35S:CaM) and RNAi line (35S:dsCaM) of Nbrgs-CaM transgenic Nicotiana benthamiana plants infected with 10Aβ at 30 dpi. (B) Southern blots of 10Aβ accumulation in systemic leaves of wt, 35S:CaM, 35S:dsCaM N. benthamiana infected with 10Aβ at 30 dpi. The DNA agarose gel was stained with ethidium bromide as loading control and then blotted using probes specific for 10A and betasatellite (10β). (C) Symptoms of rgs-CaM-silenced plants infected by 10Aβ. Partial fragments of Nbrgs-CaM, Ntrgs-CaM and Slrgs-CaM were cloned into the RNA2 of the TRV VIGS vector. N. benthamiana (N.b), N. tabacum (N.t), Solanum lycopersicum (S.l) plants at 4–5 leaf stage were infiltrated with Agrobacterium cultures carrying an empty vector (Mock), or pTRV1 with pTRV2-GFP, or with pTRV1 and respective pTRV2-CaM. Rgs-CaM silencing was confirmed in newly emerged leaves 7 days after TRV infiltration by RT-qPCR (Fig. S6). The systemic leaves of these plants were superinfected with 10Aβ and photographs were taken 20 dpi. (D) Southern blots of 10Aβ accumulation in systemic leaves of plants shown in (C) at 20 dpi. The DNA agrose gel was stained with ethidium bromide as a loading control. Viral single-stranded DNA (ssDNA) and supercoiled DNA (scDNA) are indicated.

https://doi.org/10.1371/journal.ppat.1003921.g006

Given that expressions of Ntrgs-CaM and Slrgs-CaM were also induced by TYLCCNB in tobacco and tomato plants, respectively (Fig. S1), and the three rgs-CaMs possessed comparable activity in suppressing PTGS (Fig. 3A–B), we next explored whether Ntrgs-CaM and Slrgs-CaM have similar roles in 10Aβ infection. To this end, N. benthamiana, tobacco and tomato seedlings were agro-infiltrated with recombinant TRV vectors which carry partial fragments of Nbrgs-CaM, Ntrgs-CaM and Slrgs-CaM, respectively. Knowdowns of the three rgs-CaMs were verified by RT-qPCR, which showed an approximately 80% reduction in their mRNA levels as compared to TRV-GFP-treated plants or mock plants (no TRV infection) at 7 dpi (Fig. S6). Rgs-CaM-silenced N. benthamiana, tobacco and tomato plants had indistinguishable symptoms from TRV-GFP-infected plants, indicating reductions in the abundance of rgs-CaM do not cause discernible phenotype in these host plants (data not shown). The rgs-CaM-silenced plants were infected with 10Aβ and monitored for symptom appearance. As shown in Fig. 6C–D, at 20 days after 10Aβ infection, rgs-CaM-silenced plants developed much milder symptoms and accumulated lower amounts viral DNAs than TRV-GFP-treated or mock-silenced plants, suggesting similar roles of these rgs-CaMs in TYLCCNV infection in Solanaceae hosts. Overall, our data are consistent with a model that Nbrgs-CaM and its orthologs in Solanaceae hosts function as negative cellular regulators of RNA silencing that are induced by βC1 to potentiate TYLCCNV infections.

Induction of Nbrgs-CaM by βC1 represses NbRDR6 expression

In plants, calmodulins or calmodulin-like proteins regulate a variety of cellular processes by controlling the expression of genes encoding downstream effectors [29], [30]. To investigate whether βC1-induced Nbrgs-CaM altered the expression of components in the RNA silencing pathway, specific primers for RT-qPCR detection were designed to analyze the transcription levels of N. benthamiana homologs of DICER 1 (NbDCL1), DICER 2 (NbDCL2), DICER 3 (NbDCL3), DICER 4 (NbDCL4), ARGONAUTE 1-1 (NbAGO1-1), ARGONAUTE 4-1 (NbAGO4-1), SGS3 (NbSGS3), RDR1 (NbRDR1), RDR2 (NbRDR2) and RDR6 (NbRDR6). For this purpose, individual Agrobacterium cultures expressing GFP (35S:GFP), Nbrgs-CaM (35S:CaM) or βC1 (35S:βC1) were infiltrated into N. benthamiana leaves and the transcription levels of RNAi components were measured by RT-qPCR. As compared with the GFP control, expression of βC1 or Nbrgs-CaM at 48 hours post infiltration (hpi) resulted in only moderate changes (60∼150%) in mRNA levels of the RNAi components in repeated experiments (Fig. 7A), although up-regulation of NbDCL3, NbDCL4 and NbRDR1, and down-regulation of NbDCL1, NbAGO4 or NbSGS3 expression by either βC1 or Nbrgs-CaM appeared to be significant (p≤0.05 or p≤0.01). However, the NbRDR6 mRNA level was consistently reduced by 2-fold by both Nbrgs-CaM and βC1 (Fig. 7A). The inhibitory effect was more evident when βC1 and Nbrgs-CaM were expressed from a PVX vector or from a stable transgene, and in these cases, NbRDR6 mRNA was reduced to 10∼20% of that of control plants (Fig. 7B). Infections with 10Aβ also decreased the NbRDR6 mRNA levels to ∼40% of that in 10A-infected or mock-infected plant at 7 dpi (Fig. 7D).

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Figure 7. Nbrgs-CaM is genetically required for βC1 to repress NbRDR6 expression.

(A) Effects of βC1 and Nbrgs-CaM on the expression of RNAi components. Nicotiana benthamiana leaves were infiltrated with Agrobacterium cultures expressing 35S:GFP (negative control), 35S:βC1 and 35S: CaM. Total RNAs were extracted from infiltrated zones from three pools of plants at 48 hpi. Relative mRNA levels of NbDCL1, NbDCL2, NbDCL3, NbDCL4, NbAGO1-1, NbAGO4-1, NbSGS3, NbRDR1, NbRDR2 and NbRDR6 were analyzed by RT-qPCR with specific primers. The value obtained from 35S:GFP-infiltrated plants was arbitrarily set as 1. (B) Levels of NbRDR6 mRNA in plants infected by a recombinant PVX vector expressing βC1 (PVX-βC1) and Nbrgs-CaM (PVX-NbCaM) versus the vector control (PVX) at 10 dpi (left columns), or in transgenic N. benthamiana plants expressing βC1 and Nbrgs-CaM driven by CaMV 35S promoter versus wt plants (right columns). The value in PVX-infected plants or wt plants was arbitrarily set as 1, respectively. (C) Levels of NbRDR6 mRNA in wt and 35S:dsCaM transgenic plants agroinfiltrated with binary plasmids expressing βC1 and GFP at 48 hpi. The value in GFP-infiltrated wt plants was set as 1. (D) Levels of NbRDR6 mRNA in wt and 35S:dsCaM plants infected by TYLCCNV without (10A) or with betasatellite (10Aβ), or mock inoculated plants at 7 dpi. The value in mock plants was set as 1. The mRNA levels of the analyzed genes were normalized to NbGAPDH mRNA that served as an internal standard. Each mean value was derived from three independent experiments. Values are means ± SD (n = 9). The data were analyzed using Student's t test and asterisks denote significant differences between treatments (*P≤0.05, **P≤0.01).

https://doi.org/10.1371/journal.ppat.1003921.g007

The parallel functions of Nbrgs-CaM and βC1 in PTGS suppression and NbRDR6 down-regulation led us to postulate that Nbrgs-CaM is required for βC1 to repress NbRDR6 expression. To test this possibility, Agrobacterium cultures harboring expression cassettes of the GFP (control) or βC1 were infiltrated into leaves of wt and 35S:dsCaM plants, and the effects on NbRDR6 transcription were measured by RT-qPCR. As compared to expression of GFP, expression of βC1 reduced the NbRDR6 mRNA levels by more than 2-fold in wt plants. However, upon transient expression of GFP and βC1 in 35S:dsCaM plants, the NbRDR6 mRNA levels were comparable (Fig. 7C). Likewise, 10Aβ infection also led to reduced NbRDR6 expression in wt plants but not in in 35S:dsCaM plants, when compared to that of 10A-infected or mock plants (Fig. 7D). These results indicate that the requirement of Nbrgs-CaM for repression of NbRDR6 transcription is likely to be biologically relevant. It is worth noting that higher basal levels of NbRDR6 mRNA (∼1.5-fold) were observed in 35S:dsCaM plants than in wt plants (Fig. 7D, compare mock-treatment in dsCaM and wt plants), suggesting that endogenous Nbrgs-CaM suppresses NbRDR6 expression. Taken together, our data suggest that Nbrgs-CaM acts downstream of βC1 to suppress NbRDR6 expression.

RDR6 mediates RNA silencing-based antiviral defense against TYLCCNV infection in N. benthamiana and Arabidopsis

To verify the role of NbRDR6 in host antiviral defense against TYLCCNV infection, we used a N. benthamiana NbRDR6 RNAi line (dsRDR6) described earlier [13]. We first compared the efficiency of Su silencing induced by the 10A-derived VIGS vector in wt and dsRDR6 N. benthamiana plants. At 30 dpi, typical Su silencing phenotypes were developed on wt plants, but not on dsRDR6 plants (Fig. 8A). RT-qPCR analysis revealed a 5-fold reduction of Su mRNA in wt plants compared to mock-silenced plants (infected by 10A+2mDNA1), whereas in dsRDR6 plants Su mRNA was reduced less than 2-fold (Fig. 8B), confirming the defective Su silencing in dsRDR6 plants (Fig. 8A). Interestingly, in dsRDR6 plants, both DNA components of the viral vector accumulated to higher levels than in wt plants (Fig. 8C), suggesting that the compromised Su silencing in dsRDR6 plants is due to an inability to mount RNA silencing response upon TYLCCNV infection, rather than a failure to support robust virus replication.

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Figure 8. Compromised VIGS efficiency and enhanced susceptibility to TYLCCNV infection in RDR6-deficient Nicotiana benthamiana plants.

(A) Phenotype of Su silencing in the wt and NbRDR6 RNAi line (dsRDR6) induced by the 10A+2mDNA1-NbSu VIGS vector at 30 dpi. (B) RT-qPCR analysis of levels of Su mRNA in silenced plants shown in (A). Mock indicates wt plants infected by the empty VIGS vector (10A+2mDNA1) and the relative Su mRNA value in mock plants was set as 1. Error bars: ± SD. Asterisk indicates P value compared with mock-treated wild type plants: *P≤0.05 (Student's t test). (C) Southern blot analysis of 10A and DNA1 accumulation in systemic leaves of Su-silenced plants shown in (A) at 30 dpi. (D) Symptoms of wt and dsRDR6 N. benthamiana plants infected with 10A or 10Aβ at 30 dpi. (E) Southern blot analysis of viral DNA accumulation in systemically infected leaves of wt and dsRDR6 at 30 dpi shown in (D). Total DNA agarose gels were stained with ethidium bromide to show the equal loading. Viral single-stranded DNA (ssDNA) and supercoiled DNA (scDNA) forms are indicated. (F) RNA blot hybridization analysis of viral small RNA accumulations in VIGS vector-infected plants shown in (A) and wt virus-infected plants shown in (D). The blots were hybridized to single-stranded DNA oligonucleotide probes specific for 10A, 10β, NuSu and U6 small RNA.

https://doi.org/10.1371/journal.ppat.1003921.g008

The functions of NbRDR6 in RNA silencing-based defense were further tested by infection of 10A and 10Aβ. As described in earlier studies [38], infection of wt plants with 10A elicited very mild symptoms, which were greatly exacerbated when the pathogenesis-enhancing satellite was present (10Aβ) (Fig. 8D). Interestingly, in dsRDR6 plants, infection with the 10A helper alone resulted in severe downward leaf curling. However, the phenotypes characteristic of the betasatellite infection, including shoot bending, leaf distortion, vein thickening and enations, were not observed (Fig. 8D). Infection of dsRDR6 plants with 10Aβ produced slightly severer symptoms than those in wt plants (Fig. 8D), and the disease onset was also advanced in dsRDR6 plants (3∼4 dpi) as compared to those in wt plants (6∼7 dpi). Southern blot hybridization analysis of 10A-infections also revealed that total viral DNA accumulated at higher levels in dsRDR6 plants than in wt plants (Fig. 8E). Similarly, betasatellite co-infections also resulted in increased helper viral DNA accumulation. In particularly, the single-stranded viral DNA forms (ssDNA) were barely visible in 10A-infected wt plants, but were greatly enhanced in dsRDR6 plants and in betasatellite co-infected plants (Fig. 8E). Previously, it was thought that disease phenotypes induced by 10Aβ could be solely attributed to the expression of the pathogenesis factor βC1, because infections with 10A alone elicited negligible symptoms (Fig. 8D). However, our data here suggest that betasatellite-mediated amplification of 10A, likely as a result of repression of NbRDR6, also contributes to disease manifestation. Small RNA blot analyses also showed that betasatellite suppressed viral siRNA (vsiRNA) production (Fig. 8F, compare 10Aβ-infectd plants with 10A-infected plants). Interestingly, vsiRNAs accumulated at higher levels in dsRDR6 plants when compared with wt plants, regardless of the presence or absence of the betasatellite, so is also the case with the NbSu-derived siRNAs (Fig. 8F). Nevertheless, the abundant siRNAs observed in dsRDR6 plants apparently failed to efficiently silence their targets (Fig. 8A–C) or to confer effective antiviral defense (Fig. 8D–E).

A. thaliana is known as a susceptible host for only a few geminiviruses including CaLCuV and Beet curly top virus. TYLCCNV, either alone (10A) or in association with betasatellite (10Aβ), has consistently failed to systemically infect Arabidopsis ecotype Columbia (Col-0) (Fig. 9 A–B). Remarkably, Arabidopsis rdr6 mutant plants were susceptible to infection of 10Aβ, showing typical downward leaf curling on new leaves starting at 7 dpi (Fig. 9A). The narrow curled leaves and cotyledons of infected plants were reminiscent of the phenotypes induced by transgenic expression of βC1 in Arabidopsis [55]. After 30 dpi, the infected plants began to develop necrosis on newly expanded leaves and shoots, and some of the shoots eventually died. However, in contrast to 10Aβ, 10A alone failed to systemically infect rdr6 plants (Fig. 9A). Consistent with the observed symptoms, Southern blot analysis showed high levels of helper and satellite DNA accumulations in leaves of rdr6 plants systemically infected with 10Aβ, whereas viral DNAs were absent in inoculated wt plants or 10A-inoculated rdr6 plants (Fig. 9B). These data strongly suggest a major role of RDR6 in host antiviral defense against TYLCCNV infection and underscore the function of RDR6-mediated RNA silencing in restricting the host range of a geminivirus.

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Figure 9. RDR6 mediates nonhost defense against TYLCCNV in Arabidopsis.

(A) Symptoms of wt (Col-0) and rdr6-11 Arabidopsis plants infected by 10A and 10Aβ at 30 dpi. (B) Southern blot analysis of viral DNAs of 10A and 10β in systemically infected leaves of Col-0 and rdr6-11 plants at 30 dpi. The total DNA agarose gel was stained with ethidium bromide to show the equal loading. Viral single-stranded DNA (ssDNA) and supercoiled DNA (scDNA) forms are indicated.

https://doi.org/10.1371/journal.ppat.1003921.g009

Plant materials and growth conditions

N. benthamiana seedlings were potted in soil and placed in an insect-free growth chamber at 25°C and 60% relative humidity under a 16 h light/8 h dark photoperiod. 35:βC1 transgenic N. benthamiana lines were generated in a previous study [36], and the transgenic GFP 16c and dsRDR6 lines were generous gifts of David C. Baulcombe. The A. thaliana ecotype Columbia (Col-0) and rdr6-11 mutant were used for this study. Seeds were surface sterilized with 75% ethanol and 50% bleach, and then washed three times with sterile water. Sterile seeds were suspended in 0.05% agarose and plated on Murashige and Skoog (Duchefa Biochemie, Haarlem, Netherlands) medium plus 2.0% sucrose. Plates were stratified in darkness for 3 d at 4°C and then transferred to a tissue culture room at 22°C under an 8-h-light/16-h-dark photoperiod. After 2 weeks, seedlings were potted in soil and placed in a growth chamber at 22°C and with 70% relative humidity under an 8-h-light/16-h-dark photoperiod.

Cloning full-length rgs-CaM cDNAs and plasmid constructs

The tobacco rgs-CaM sequence was used to identify orthologous sequences from available N. benthamiana ESTs. BLAST searches revealed high homology between rgs-CaM from tobacco and N. benthamiana. Primers designed to anneal to conserved sequences in the 5′ and 3′ untranslated regions of tobacco rgs-CaM were used to amplify the coding region of N. benthamiana rgs-CaM by reverse transcription PCR (RT-PCR). Amplification with primer pairs CaM-cds-F/CaM-cds-R yielded a specific product of approximately 600-bp, which was cloned into pMD18-T (TaKaRa, Dalian, China) and sequenced. Detailed primer information is listed in Table S1. The full-length coding sequence of Nbrgs-CaM was deposited in GenBank under the accession number JX402081. To construct the plant Nbrgs-CaM expression vector for transgenic over-expression or transient agroinfiltration assays, a primer pair CaM-F/CaM-R was designed to amplify the full-length coding region of Nbrgs-CaM. The Nbrgs-CaM coding sequence was subcloned into the binary vector pCHF3 downstream of a CaMV 35S promoter to produce pCHF3-35S-CaM, or into the PVX-based vector (a kind gift of David Baulcombe) between the ClaI and SalI sites to produce PVX-Nbrgs-CaM.

An RNAi construct containing an Nbrgs-CaM inverted repeat sequence of spaced by a soybean intron was produced by overlapping PCR. The fragment of Nbrgs-CaM sense sequence was amplified with A-CaM-F/A-CaM-intron-R primer pair and overlapped with intron sequence amplified by B-CaM-intron-F/B-Intron-R primers. The overlapping products were cloned into pCHF3 between the SacI and BamHI sites to produce pCHF3-35S-CaM-intron. The corresponding antisense Nbrgs-CaM fragment was amplified with the primer pair C-CaM-F/C-CaM-R and subsequently cloned into pCHF3-35S-sCaM-intron between BamHI and PstI sites to produce the RNAi construct pCHF3-35S-dsCaM. To construct a TRV-based recombinant VIGS vector of Nbrgs-CaM, a partial fragment of Nbrgs-CaM was generated by PCR amplification with the primer pair TRV-CaM-F/TRV-CaM-R and then cloned into the pTRV2 vector (a kind gift of Yule Liu) between the BamHI and XhoI sites [54].

The coding sequences of Ntrgs-CaM and Slrgs-CaM were PCR-amplified from N. tabacum and S. lycopersicum and then subcloned to the pCHF3 binary vector or the TRV vectors using different primers and restriction enzyme sites listed in Table S1. The pCHF3-based vectors were used for the transient expression of rgs-CaMs in N. benthamiana leaves, and the TRV-based VIGS vectors were used to silence the expression of rgs-CaMs in N. tabacum and S. lycopersicum, respectively.

Binary vectors for the PTGS suppression assay, such as pCHF3-35S-GFP and pCHF3-35S-dsFP, were constructed previously [82]. The pCHF3-35S-FP vector for transcription of a partial sense sequence of C-terminal fragment of 400 bps of GFP was constructed by amplification with the primer pair S-FP-F/S-FP-R and cloned into pCHF3.

Plant transformation

Transgenic lines over-expressing or down-regulating Nbrgs-CaM were generated by transforming N. benthamiana with the pCHF3-CaM and pCHF3-dsCaM constructs, respectively. The binary vectors were mobilized into A. tumefaciens EHA105 strain, and then used to transformed N. benthamiana leaf discs. Selection of transformants was performed in media containing 200 µg/ml kanamycin. Kanamycin-resistant shootlets were collected, placed on rooting media, grown to a height of 5–6 cm, and then transferred to soil. Transgenic plants were first screened by PCR with specific primers targeted to promoter or intron sequences and then confirmed by Southern blot hybridization. Alterations in Nbrgs-CaM mRNA levels in transgenic plants were confirmed by RT-qPCR and Northern blot analyses.

Viral inoculation and agroinfiltration

For virus agroinoculation, equal volumes of individual A. tumefaciens cultures at an OD₆₀₀ of 1 were mixed prior to inoculations. Viral infectious clones, including TYLCCNV (pBinPLUS-Y10-1.7A), and TYLCCNV/TYLCCNB (pBinPLUS-Y10-1.7A+Y10β), were described previously [38]. The TYLCCNV-derived VIGS vectors 2mDNA1 (pBinPLUS-2mDNA1) and 2mDNA1-NbSu (pBinPLUS-2mDNA1-NbSu) were constructed previously [52], [53]. The recombinant PVX vector expressing βC1 gene (PVX-βC1) was described previously [66]. Agrobacterium cultures carrying viral infectious clone(s) were infiltrated into N. benthamiana leaves and inoculated plants were photographed with a Canon 400D digital camera at different periods.

For CMV rub-inoculation, 1 g of CMV-infected N. glutinosa leaves were ground in 1 mL of 5 mM phosphate buffer, pH 7.2. N. benthamiana plants at the 6–7 leaf stages were inoculated by rubbing leaves with freshly prepared sap. Inoculated plants were grown in an insect-free growth chamber at 25°C and monitored for symptom appearance.

For PTGS experiments, transient silencing suppression assays were performed as described previously [50], [51]. Classic two-component transient PTGS assays were performed by agroinfiltration of 35S:GFP with control or suppressor vectors into leaves of N. benthamiana 16c plants at the 6–7 leaf stages. For S-PTGS experiments, Agrobacterium cultures harboring the pCHF3-35S-GFP, pCHF3-35S-FP and VSRs-expressing vectors were mixed in equal proportions and infiltrated into N. benthamiana leaves. For IR-PTGS experiments, the pCHF3-35S-dsFP was used as an RNA silencing trigger instead of the pCHF3-35S-GF as a RNA silencing trigger. After 3 dpi, GFP fluorescence was monitored with a 100-W handheld long-wavelength UV lamp (Black Ray Model B 100A; UV products) and the infiltrated leaves were photographed with a Canon 400D digital camera with a 58-mm yellow filter. Exposures were 3 to 6 seconds long, depending on the fluorescence intensity and distance from the leaf.

DNA extraction and Southern blot analysis

Total DNA was extracted from infected plant leaves using the CTAB method (35). DNA agarose gels were stained with ethidium bromide to provide a loading control. After denaturation and neutralization, total DNA was transferred to Hybond N⁺ nylon membranes (GE Healthcare, Pittsburgh, PA) by capillary transfer. Membranes were hybridized at 55°C to specific probes labeled with [α-³²P] dCTP.

RNA extraction, northern blot, siRNA blot and RT-qPCR analysis

Total RNAs were extracted from plants with Trizol reagent (Invitrogen, Carlsbad, CA) as recommended by the manufacturer. Total RNA was stained with ethidium bromide as a loading control, and then transferred to Hybond N⁺ nylon membranes by upward capillary transfer in 20×SSC buffer. Membranes were hybridized to specific probes for rgs-CaM, βC1, GFP or PVX CP labeled with [α-³²P] dCTP using random primed labeling System (Promega, Madison, WI). The hybridization signals were detected by phosphorimaging with a Typhoon 9200 imager (GE Healthcare).

To analyze the production siRNAs, low-molecular-mass RNAs were enriched from total RNA as described previously [3]. The enriched small RNAs (15 µg) were fractionated on a 15% denaturing polyacrylamide–7 M urea gel in 0.5× Tris–borate–EDTA (TBE) buffer. The RNA was transferred to Hybond N⁺ membranes (GE Healthcare) by electroblotting in 0.5× TBE at 400 mA for 1 h. The transferred RNAs was UV crosslinked to the membrane 4 times at 1200 µJ in a UV Stratalinker 1800 (Stratagene, La Jolla, CA). Membranes were stored at 4°C until probing. One DNA oligonucleotides complementary to N. benthamiana U6 RNA and a mixture of oligonucleotides corresponding to G, F and P regions of GFP mRNA sequences were synthesized and used as probes for siRNA hybridization (Table S1). The oligos were end-labelled with [γ-³²P] ATP in 50 µL reactions containing 1 µM DNA oligo and 7 U T4 polynucleotide kinase. Hybridizations were performed overnight at 42°C and the membranes were subsequently washed three times (10 min each) at 40°C with 1× SSC (0.15 M NaCl and 0.015 M sodium citrate) supplemented with 0.1% SDS. Hybridization signals were detected as described above for Northern blot analysis.

For RT-qPCR analysis, 10 µg of total RNA was treated with DNase I (Takara) and reverse transcribed according to manufacturer's instructions. Specific primer pairs, which annealed to GFP, βC1, Nbrgs-CaM, Su or genes encoding known components in RNA silencing pathway (Table S1), were designed by Primer Premier 5 software. The GenBank accession numbers of genes analyzed in this study are as follows: Ntrgs-CaM (AF329729), Slrgs-CaM (AY642285), NbDCL1 (FM986780), NbDCL2 (FM986781), NbDCL3 (FM986782), NbDCL4 (FM986783), NbAGO1-1 (DQ321488), NbAGO4-1 (DQ321490), NtSGS3a (AB690269), NbRDR1m (AY574374), NbRDR2 (AY722009), NbRDR6 (AY722008). The lengths of amplification products were between 180–250 bp, and the Tm for each primer pair was between 55–65°C. RT-qPCR was performed using a LightCycler 480 (Roche Diagnostics, Rotkreuz, Switzerland) for 45 cycles, and NbGAPDH was used an internal control unless otherwise stated. Each experiment was performed in triplicate and repeated three times, and the results were analyzed by software supplied by the manufacturer.

Immunoblotting and antibodies

Total proteins were extracted from N. benthamiana leaves as described previously [66]. Immunoblotting was performed with primary mouse monoclonal or rabbit polyclonal antibodies, followed by goat anti-mouse or anti-rabbit secondary antibody conjugated to horseradish peroxidase (Bio-Rad, Hercules, CA). The GFP monoclonal antibody was obtained from Hua An Company, China, and the monoclonal antibodies against PVX CP, CMV CP and βC1 were generated in house. The rabbit polyclonal antibody against CMV 2b was a generous gift from Dr. Huishan Guo. Blotted membranes were washed thoroughly and visualized using chemiluminescence according to the manufacturer's manual (GE Healthcare). Nbrgs-CaM antibody was produced in rabbit with recombinant protein expressed in E. coli. In brief, we expressed Nbrgs-CaM in E. coli cells as a 6× Histidine and Maltose binding protein (MBP) fusion. The Nbrgs-CaM-MBP-His fusion protein was purified over a nickle column (Merck, Darmstadt, Germany) and eluted with a buffer containing 200 mM imidazole according to the manufacturer's manual. The purified protein was used to immunize rabbits for polyclonal antiserum. The Nbrgs-CaM-specific immunoglubin was given an additional purification step on an affinity column filled with Cyanogen bromide-activated agarose beads conjugated with the Nbrgs-CaM-MBP-His fusion proteins.