Nicotiana benthamiana plants were grown in a growth room at 28°C with 16 h light and 8 h dark. Three viruses were used for inoculation: BaMV strain S (Lin and Hsu, 1994), Potato virus X (PVX) strain Taiwan, and Cucumber mosaic virus (CMV) strain NT9 (Hsu et al., 1995).

The cDNA fragment of ACAC10-1 (fragment of NbCA gene) was cloned into the pGEM-T Easy vector (Promega, Madison, WI, United States) in a previous study (Cheng et al., 2010). To use ACAC10-1 in Tobacco rattle virus (TRV)-based VIGS (Liu et al., 2002) in N. benthamiana plants, ACAC10-1 in the pGEM-T Easy vector was digested with EcoRI and subcloned into the pTRV2 vector to generate pTRV2-NbCA and transformed into Agrobacterium C58C1 strain. Furthermore, Agrobacterium carrying a pTRV2-containing luciferase (Luc) gene or phytoene desaturase (PDS) gene were used as a negative or positive control, respectively. Agrobacterium containing pTRV1, pTRV2-NbCA, pTRV2-Luc, or pTRV2-PDS was cultured at 30°C to OD₆₀₀ 0.8∼1.0; the cells were collected by centrifugation at 5000 rpm, then suspended in the induction medium (10 mM MgCl_2, 10 mM MES pH5.6, and 150 μM acetosyringone) at 30°C for 1 h. After induction, equal volumes of both cultures (pTRV1 and pTRV2-NbCA, pTRV2-Luc or pTRV2-PDS) were mixed before agroinfiltration onto the second, third, and fourth leaf of true leaves of a 1-month old N. benthamiana. When the PDS-knockdown plants had a photo-bleach phenotype, 500 ng of BaMV virion was mechanically inoculated onto the fourth leaf above the infiltrated leaves.

The knockdown efficiency of NbCA was calculated by measuring the expression ratio normalized to the expression of actin between the NbCA-knockdown and control plants. Two sets of primers were used for the RT-PCR to amplify NbCA and actin gene expression: NbCA/F (5′-AGTGCATGTGGAGGTATCAAAGGT-3′)/NbCA/R (5′-GTCGACTACGGAAAGAGAAGG-3′) and actin/3′ (5′- GTGGTTTCATGAATGCCAGCA-3′)/actin/5′ (5′-GATGAAGATACTCACAGAAAGA-3′).

The preparation of protoplasts from N. benthamiana and viral RNA inoculation was described previously (Tsai et al., 1999). Approximately 2 g of agroinfiltrated leaf was collected from the knockdown N. benthamiana and digested with pectinase and cellulase at 25°C overnight. The mesophyll protoplasts were isolated from the interface zone between the Mannitol-MES buffer and the sucrose. After a few washes, protoplasts were stained with fluorescein diacetate to examine the quality of cells under a fluorescent microscope. Approximately 2.5 × 10⁵ protoplasts were inoculated with 1.5 μg BaMV, PVX, or CMV viral RNA with 40% polyethyleneglycol-6000. Total protein or RNA was extracted from protoplasts and detected by western blot analysis or real-time qRT-PCR, respectively.

The total protein of inoculated leaves or protoplasts was extracted with plant extraction buffer (50 mM Tris-HCl pH 6.8, 10% glycerol, and 2% SDS), boiled with the SDS sample buffer (10% glycerol, 12.5 μg/ml bromophenol blue, 10 mg/ml SDS, 125 mM Tris-HCl pH 6.8, 2.5% β-mercaptoethanol) for 5 min, separated on a 12% polyacrylamide gel containing 0.1% SDS, transferred onto a nitrocellulose membrane (PROTRAN BA 85 Schleicher and Schnell), and probed with primary antibody [anti-Orange fluorescent protein (OFP), -BaMV, -PVX, or -CMV] and with the secondary antibody [affinity purified anti-rabbit IgG conjugated IRDye 800 (ROCKLAND)]. Finally, membranes with fluorescent bands were scanned by using LI-COR Odyssey (LI-COR Biosciences). In addition, rbcL (RuBisCo large subunit) stained with Coomassie blue was used as a loading control.

Total RNA was extracted from leaves with STE buffer (400 mM Tris-HCl pH8.0, 400 mM NaCl, and 40 mM EDTA), 1% SDS, and 3.3 mg/ml bentonite and an equal volume of phenol/chloroform. After ethanol precipitation, the RNA was further precipitated with 3 M NH₄OAc, washed, dried, and dissolved in 30 μl de-ionized H₂O. For RNA extracted from protoplasts, cells were mixed with 200 μl protoplast RNA extraction buffer (100 mM Tris-HCl pH 8.0, 10 mM EDTA, 100 mM NaCl, 1% SDS, and 600 μg bentonite). After phenol/chloroform extraction and ethanol precipitation, RNA was further precipitated with 3 M NH₄OAc and dissolved in 13 μl de-ionized H₂O.

qRT-PCR was used to detect both BaMV plus- and minus-strand genomic RNA. The cDNA synthesis reaction involved use of ImProm-II Reverse Transcriptase (Promega, Carlsbad, CA, United States) as instructed with the primers for Oligo dT(25T) and BaMV+51 (5′-ACTGCCAATTGTCCCCTACA-3′) for the plus- and minus-strand, respectively. For quantifying the accumulation of BaMV genomic RNA or minus-strand RNA, primers for BaMV+51 and BaMV-282 (5′-TGTGCTGAACGGGTTATGAG-3′) or BaMV+1766 (5′-CACATCCGGCACTTACCA-3′) and BaMV-2002 (5′-ATGTATCACGGAAATAAGAGTT-3′) were used, respectively, in the reaction containing a 1000X dilution of SYBR green I (Cambrex Bio Science Rockland, ME, United States). qPCR was performed in 0.2-ml PCR tubes with 0.6 mM primer, 0.2 mM each deoxyribonucleoside triphosphate, 10 mM Tris-HCl pH 8.8, 1.5 mM MgCl₂, 50 mM KCl, 0.1% Triton X-100, 2 μl cDNA, 3 units of Taq DNA polymerase (Promega) and RNase-free water to a final volume of 20 μl. Cycling conditions began with an initial hold at 95°C for 5 min, followed by about 30 cycles of 94°C for 30 s, 56°C for 30 s and 72°C for 30 s. Reactions were carried out in a RotorGene 3000 (Corbett Research, Sydney, Australia) with data acquisition at 72°C on the channel, excitation at 470 nm and detection at 585 nm, by using a high-pass filter for both plus- and minus-strand. The reaction without template or reverse transcriptase was a negative control, and actin was detected for normalization. All samples were run at least three times.

The full-length CA cDNA of N. benthamiana was cloned into the pEpyon binary vector that carries the mOrange2 reporter gene (Shaner et al., 2008) to express the fusion protein NbCA-OFP. The ORF of the NbCA was amplified with NbCA/F, 5′-GGATCCATGTCAACTGCTTCCA-3′, and NbCA/R, 5′-GGTACCTACGGAAAGAGAAG-3′ (BamHI and KpnI underlined, respectively). The PCR product was first cloned into the pGEM-T easy vector (Promega, Madison, WI, United States), then sub-cloned into pEpyon with BamHI and KpnI after sequence verification.

Agrobacterium containing the binary vector with NbCA-OFP or vector alone was cultured and infiltrated into N. benthamiana plants. The fluorescent signals were detected at 3 days post-infiltration by confocal laser scanning microscope (FV1000, Olympus). To observe whether NbCA altered its localization after BaMV infection, pKBG, a plasmid containing an infectious cDNA of BaMV with a GFP reporter (Prasanth et al., 2011) was co-infiltrated with NbCA-OFP. The fluorescent signals were detected at 4 days post-infiltration by confocal laser scanning microscope.

NbCA-OFP was transiently expressed via agroinfiltration on a leaf for 1 day and the BaMV virion was inoculated on the same leaf for another 3 days. The expression of this fusion protein and accumulation of BaMV coat protein were detected by western blot analysis.

The coding sequence of NbCA without the predicted transit peptide (Slaymaker et al., 2002) was amplified with the primer set CAgene/F, 5′-GGATCCGAATTGCAATCATCAGATGG-3′, and CAgene/R, 5′-GCTCGAGTACGGAAAGAGAAGGAGAAA-3′ (BamHI and XhoI site underlined, respectively). The PCR product was cloned into the pGEM-T easy vector and the sequence was verified. Finally, NbCA was subcloned from the T-vector into the pET29a(+) expression vector (Invitrogen) and transformed into E. coli BL21(DE3). The resulting clone was designated pET29a(+)-NbCA.

Escherichia coli containing pET29a(+)-NbCA was cultured to OD₆₀₀ = 0.7 ∼ 1.2 (100 ml in total volume), the expression was induced with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 16°C for 1 day, then samples were centrifuged at 7000 rpm at 4°C for 7 min. The cell pellet was resuspended with 8 ml buffer A (50 mM NaH₂PO₄ pH 8.0, 300 mM NaCl) containing protease inhibitor cocktail (Roche, Germany) and subjected to the French Press to break cells: the sample was loaded into the French Press and squeezed out four times, then centrifuged at 12000 rpm at 4°C for 10 min. The supernatant was incubated with 1 ml complete His-tag Purification Resin (Roche, Germany) overnight, washed with 10 ml buffer A containing 50 mM imidazole, and eluted with buffer A containing 250 mM imidazole. Finally, the eluted protein was dialyzed with 150 ml buffer A four times to remove imidazole and stored at -80°C with the addition of final 10% glycerol. The vector-only construct was manipulated under the same condition as the negative control.

Bamboo mosaic virus-infected leaves were collected at 5 dpi and homogenized with polytron in replicase complex extraction buffer (50 mM Tris-HCl pH 7.6, 15 mM MgCl2, 120 mM KCl, 0.1% β-mercaptoethanol, 20% glycerol, 1 μM pepstatin A, 0.1 mM phenylmethylsulfonyl fluoride) with a 2 ml/g of buffer/leaf ratio. The leaf slur was filtrated through Miracloth (Calbiochem) and centrifuged at 500 × g for 10 min to remove the cell debris. The pellet was resuspended in suspension buffer (50 mM Tris-HCl pH 8.2, 10 mM MgCl2, 1 mM dithiothreitol, 1 μM pepstatin A, 1 μM leupeptin) after centrifugation at 30,000 × g for 35 min. Approximately 2 ml of the extract was loaded on 28 ml of 20 ∼ 60% continuous gradient of sucrose with the gradient buffer (50 mM Tris-HCl pH 8.0, 10 mM NaCl, 1 mM EDTA, 5% glycerol, 1 μM pepstatin A, 0.1 mM PMSF, 1 mM dithiothreitol) and centrifuged at 72,100 × g for 7.2 h. The 5th and 6th fractions of the 10 fractions (from top to bottom) with the highest RdRp activity were pooled and stirred with 1.5% NP-40 for 1 h to solubilize the membrane-associated RdRp.

For the in vitro replication assay with the endogenous RNA templates, 15 μl of the replicase complex preparation (pH 8.0) was added to a total 50 μl reaction containing 2 mM (A, C, and G) TP, 2 μM UTP, 3 mM MgCl2, 10 mM dithiothreitol, 50 mM Tris-HCl pH 8.2 (6.8, 7.4, 7.8, 8.8, or 9.0 was used in testing the proton concentration for the in vitro replication assays), 12 unit RNase OUT (Invitrogen, Carlsbad, CA, United States), 8 mg bentonite, 0.066 μM [α-³²P]UTP (3000 Ci mmol/1, Dupont-NEN) and 5.2 μg recombinant NbCA at 30°C for 1 h (the reaction made up of final pH is 8.1 when reaction buffer is 8.2). Therefore, the Tris-HCl buffer at different pH was used in the reaction to reach the final target pH as 7.1, 7.5, 7.8, 8.6, or 8.8, respectively. The reaction was stopped by adding 150 μl 5 mM EDTA, extracted with phenol/chloroform, and precipitated with ethanol. The radioactive RNA products were resolved on a 1% agarose gel and quantified by using the PhosphoImaging analyzer BAS-2500 (FUJIFILM).

For the exogenous RNA templates, 15 μl of the replicase complex preparation was first treated with 10 units of micrococcal ribonuclease containing 2.5 mM Ca(OAc)2 to remove endogenous RNA at 30°C for 30 min in a total volume of 11.5 μl reaction. Then the reaction was terminated by adding 16 mM EGTA and set on ice for 1 ∼ 2 min. An aliquot of 15 μl mixture was subjected to a total 50 μl reaction as in the endogenous RNA template reaction. The radioactive RNA products were resolved on a 5% polyacrylamide gel and quantified by using the PhosphoImaging analyzer BAS-2500 (FUJIFILM).

Ba-77 RNA (the 3′-end 77 nt of BaMV minus-strand RNA) and r138/40A RNA (the 3′ UTR of BaMV RNA) was prepared in an in vitro transcription with plasmids constructed previously (Cheng et al., 2001). The reaction was carried out in 100 μl containing 10 μg linearized plasmid (EcoRI and BamHI for Ba-77 and r138/40A, respectively, in pUC18), 40 mM Tris-HCl pH 8.0, 2 mM spermidine, 8 mM MgCl₂, 10 mM dithiothreitol, 0.4 mM NTP and 200 U T7 RNA polymerase at 37°C for 2 h. The RNA was then gel purified, quantified, and stored at -80°C.

The sequence of a gene, ACAC10-1, found downregulated in N. benthamiana plants by cDNA-amplified fragment polymorphism (AFLP) after BaMV inoculation (Cheng et al., 2010), showed 100% match with an EST clone 30F62 containing a nuclear-encoded chloroplast CA gene. The gene was designated NbCA. To gain a better understanding of the relation between NbCA and BaMV infection, we inoculated BaMV virion into leaves of N. benthamiana with Tobacco rattle virus (TRV)-based NbCA knockdown.

The reduced NbCA expression in N. benthamiana did not cause any morphological change as compared with control plants (infiltrated with a TRV vector-carrying luciferase gene) (Supplementary Figure S1). The mRNA level of NbCA in NbCA-knockdown plants was approximately 50% that in the control plants (Figure 1A). At 5 days post-inoculation (dpi), the accumulation of BaMV in NbCA-knockdown plants was reduced to 64% that in Luc-knockdown control plants (Figure 1B). Hence, NbCA could be a positive regulator for BaMV infection in N. benthamiana.

To determine whether the role of NbCA for BaMV accumulation is involved in virus replication or movement, cell wall-excluded protoplasts were prepared for viral RNA inoculation to eliminate the involvement of viral movement. The accumulation of BaMV coat protein in NbCA-knockdown protoplasts was reduced to 64 and 61% that of control protoplasts at 24 and 48 h post-inoculation (hpi), respectively (Figure 2A). To determine whether the involvement of NbCA is specific to the BaMV infection cycle, CMV and PVX were inoculated into NbCA-knockdown protoplasts. The accumulation of the coat protein of these two viruses in knockdown protoplasts did not differ from that in control protoplasts at 24 and 48 hpi (Figures 2B,C).

Furthermore, to elucidate whether this deficiency results from a defect in synthesizing the plus- or minus-strand viral RNA, qRT-PCR was used to quantify the accumulation of BaMV RNAs in knockdown protoplasts. At 24 hpi, the accumulation of the plus- and minus-strand of BaMV RNA in NbCA-knockdown protoplasts was reduced by approximately 30 and 44% as compared with control protoplasts. The similar reduced ratio in both plus- and minus-strand of BaMV RNA was observed at 48 hpi (42 and 47%, respectively). Therefore, the accumulation of plus- and minus-strand BaMV RNAs were similarly affected by the reduction in NbCA levels (Figure 3). These results suggest that NbCA is most likely involved in BaMV replication.

To clone the NbCA full-length gene, a primer was designed for the 3′ rapid amplification of cDNA ends (RACE) experiment to obtain the downstream sequence of ACAC10-1. The cDNA fragment derived from 3′ RACE contains the stop codon of NbCA. The upstream sequence of ACAC10-1 including the start codon of NbCA was retrieved from the transcriptome of the N. benthamiana draft genome (Hewett-Emmett and Tashian, 1996; Bombarely et al., 2012). Two specific primers were used to amplify the full-length NbCA coding region and cloned into the pEpyon binary vector (Chen et al., 2011), which carries the mOrange2 reporter gene (OFP), to produce the NbCA-OFP fusion protein. Furthermore, the amino acid sequence of NbCA (accession no.: MF346699) was aligned with those from N. tabacum (NtCA; accession no.: P27141), and Arabidopsis (AtCA; accession no.: NP_186799) (Supplementary Figure S2). The sequence of NbCA shared 97 and 68% identity with those of NtCA and AtCA, respectively.

To visualize the localization of NbCA in plant cells, NbCA-OFP was transiently expressed in N. benthamiana leaves by agroinfiltration to detect the fluorescent signal emitted from the OFP merged with the autofluorescence signal emitted from chloroplasts (Figure 4). NbCA was mainly localized in chloroplasts. Moreover, to observe whether the localization of NbCA was altered after BaMV inoculation, we co-infiltrated the infectious BaMV viral vector pKBG carrying green fluorescent protein (GFP) driven by subgenomic RNA promoter (Prasanth et al., 2011) with NbCA-OFP and found no re-localization of NbCA after BaMV inoculation (Figure 4B).

Since we found that the accumulation of BaMV coat protein and viral RNA was reduced in NbCA-knockdown plants (Figure 1) and protoplasts (Figures 2, 3), NbCA may assist viral RNA replication. To validate this hypothesis, we transiently expressed NbCA-OFP in N. benthamiana followed by BaMV inoculation. However, accumulation of BaMV coat protein was not enhanced at 3 dpi. The pool of NbCA in cells may be enough for BaMV replication and the addition of exogenous NbCA by transient expression might not provide additional help for BaMV accumulation. Hence, we used in vitro replication (Cheng et al., 2001; Lin et al., 2005b) to exclude the effect of sufficient amount of CA in chloroplasts. We cloned and expressed the full-length CA in E. coli to acquire the purified-NbCA for in vitro replication experiments. The E. coli BL21 (DE3)-expressed recombinant NbCA-His was purified through a Nickel-chelating resin column (Figure 5A).

First, we tested whether NbCA affects endogenous RNA template activity, which represents the elongation step of BaMV replication. Viral RNA synthesis did not differ with or without the addition of the E. coli-expressed NbCA in the replication assay (Figure 5B). Second, we tested whether NbCA is involved in the initiation of BaMV replication. In the in vitro replication, we tested the two RNA templates, r138/40A (the 3′ UTR of BaMV, the promoter for minus-strand RNA synthesis) (Cheng et al., 2001) and Ba-77 (the 3′-end 77 nt of the minus-strand genome, the promoter for plus-strand RNA synthesis) (Lin et al., 2005a). The addition of NbCA in the in vitro replication assay with the exogenous templates r138/40A and Ba-77 increased RNA synthesis to 150 and 120%, respectively, that with vector alone (Figures 5C,D).

Carbonic anhydrase activity condenses carbon dioxide with water to produce a free proton in the reaction. We wondered whether the proton concentration affects the viral RNA replication. In the in vitro replication assay, CA affected the exogenous but not the endogenous template activities. If the CA activity provides the free proton to change the micro-environment (reducing pH) such as the membrane-housed viral replication site, the condition for the re-initiation of the plus- or minus-strand RNA temples by BaMV replicase complex could be regulated. To test this hypothesis, we used various pH conditions for in vitro replication assays with endogenous and exogenous templates. The endogenous template (the viral RNAs already on the replicase complex and presumably at the elongation step) favored a higher pH condition (Figure 6). By contrast, the exogenous template (endogenous templates was removed by micrococcal nulcease and presumably at the re-initiation step) favored a lower pH condition. These results are implying that CA might be trapped into the viral replication site to produce free protons to create a more acidic microenvironment favoring the re-initiation of viral RNA replication.