Figure 1.
Cortical punctate structures that contain RCNMV MP are sites of viral RNA replication.
Nicotiana benthamiana protoplasts were inoculated with recombinant RCNMV RNAs that expressed the MP-mCherry fusion protein (Figure S1B) and subjected to immunostaining with anti-dsRNA primary antibody followed by Alexa Fluor 488-conjugated secondary antibody at an early stage of infection (16 hpi, left 2 rows of panels) and at a late stage of infection (24 hpi, center 2 rows of panels). The right-most panels show the results for mock-inoculated protoplasts treated with the same antibodies. Images present confocal projections of five optical sections at 1 µm intervals, which range from the surface to the middle of the protoplasts. DIC: differential interference contrast, N: nucleus. Scale bar = 20 µm.
Figure 2.
Identification of proteins that were copurified with RCNMV MP.
Protein extracts prepared from Agrobacterium-infiltrated leaves that expressed RCNMV RNA1 plus RNA2 (WT, pBICR12, Figure S1C), or RNA1 plus recombinant RNA2 encoding MP tagged with TAP tag sequences (MP-TAP, pBICR12/MP-TAP, Figure S1D), were subjected to two-step affinity purification using an anti-HA antibody followed by an anti-FLAG antibody. (A) One fifth of the affinity-purified fractions eluted from the anti-FLAG beads (Eluate) and the proteins contained with the beads after elution (Beads) were subjected to Western blotting using anti-FLAG antibody. (B) Four fifths of the affinity-purified fractions were subjected to SDS-PAGE and stained using MS-compatible silver staining. The protein bands of interests were excised, subjected to in-gel digestion, and analyzed by tandem mass spectrometry. Proteins with Mascot search scores >50, which were absent from the control protein bands, and proteins with significantly higher scores than the control proteins are indicated on the right-hand sides of the panels. The NCBI accession numbers of the identified proteins are also indicated. (C) The GAPDH-A gene of N. benthamiana was cloned and the deduced amino acid sequence was aligned using GENETYX-Mac ver. 14.0.1 with its closest homolog from N. tabacum (accession number P09043) and that from Arabidopsis thaliana (accession number AAA32793). The blue lines show the peptide sequences detected.
Figure 3.
Multiplication of RCNMV is inhibited in NbGAPDH-A-silenced N. benthamiana leaves.
(A) Gene silencing of NbGAPDH-A was induced in N. benthamiana plants inoculated with the ALSV vector, which harbored a 294 bp partial fragment (nucleotides 352–645 from start codon) of NbGAPDH-A (ALSV/gsGAP), via Agrobacterium. The empty ALSV vector (ALSV) was used as a control. Total RNA was prepared from two ALSV-infected and three ALSV/gsGAP-infected plants. NbGAPDH-A mRNA levels were determined by real time PCR using primers specific to NbGAPDH-A (nucleotides 755–954 from start codon). The real time PCR results for the EF-1 mRNA (closed column) were used to adjust the relative accumulation levels of NbGAPDH-A mRNA (gray column). (B) In vitro transcripts of a recombinant RCNMV that expressed GFP from its subgenomic RNA (RCNMV-GFP, Figure S1E) were inoculated mechanically onto ALSV- or ALSV/gsGAP-infected N. benthamiana plants. At 20 hpi, the percentages of fluorescent foci that comprised multiple cells were measured using epifluorescence microscopy. ‘n’ represents the total number of fluorescent foci in 4 inoculated leaves (about 25 square centimeters). (C) An Agrobacterium culture that contained the pBICR1sG2 plasmid, which expressed RCNMV-GFP (Figure S1F), was diluted to OD600 = 0.03 and infiltrated into ALSV- or ALSV/gsGAP-infected N. benthamiana plants. At 35 hpi, protein was extracted from the infiltrated leaves and subjected to Western blotting using anti-GFP antibody. RbcL is a Coomassie brilliant blue-stained gel image, which shows the large subunit of Rubisco proteins. The accumulated levels of GFP from three separate experiments were quantified using the Image Gauge program and plotted in the graph. The lowest two panels show representative epifluorescence microscopy images of the infiltrated leaves at 35 hpi. Scale bar = 50 µm.
Figure 4.
NbGAPDH-A does not affect the accumulations of RCNMV RNA and MP at the single-cell level.
(A) An Agrobacterium culture that contained the pBICR12fsMP plasmid, that expressed movement-deficient RCNMV (Figure S1G), was diluted to OD600 = 0.8 and infiltrated into ALSV- or ALSV/gsGAP-infected N. benthamiana plants (two plants for each). At 26 hpi and 43 hpi, the total RNA was extracted from the infiltrated leaves and subjected to Northern blotting using DIG-labeled riboprobes specific for the plus-strand (+) RNA1 or RNA2 of RCNMV. rRNA is an ethidium bromide-stained agarose gel image of 1 µg total RNA, which was used as the loading control. The numbers below the images represent the relative accumulation levels (means ± SE) of viral RNAs using the Image Gauge program (Fuji Film), which were calculated based on two independent experiments. Asterisk indicates a not significant (P>0.05; Student's t-test) difference compared with the viral RNA accumulation level in the protoplasts from ALSV-infected N. benthamiana. (B) Protoplasts prepared from ALSV- or ALSV/gsGAP-infected N. benthamiana plants (each two plants) were inoculated with a mixture of in vitro transcripts of the recombinant RCNMV RNA1, which expressed GFP from subgenomic RNA; and RNA2, which expressed MP tagged with HA (Figure S1H). Proteins extracted from 2×104 protoplasts were loaded in each lane. GFP and MP-HA were detected using a rabbit polyclonal antibody against GFP and a rat polyclonal antibody against HA, respectively. RbcL is a Coomassie brilliant blue-stained gel image of proteins extracted from 2×104 protoplasts, which shows the large subunit of Rubisco proteins. The numbers below the images represent the relative accumulation levels (means ± SE) of the proteins using the Image Gauge program (Fuji Film), which were calculated from two independent experiments. Double asterisk indicates a not significant (P>0.05; Student's t-test) difference compared with the protein accumulation level in the protoplasts from ALSV-infected N. benthamiana.
Figure 5.
NbGAPDH-A changes its subcellular localization in association with RCNMV RNA replication, but not with viral proteins.
Representative CLSM images of agroinfiltrated N. benthamiana epidermal cells. Each Agrobacterium culture was diluted to OD600 = 0.8 and equal volume of cultures were mixed for infiltration. Confocal microscopy images were taken at 40 h after infiltration. Images present confocal projections composed of 15 optical sections taken at 1 µm intervals, from the surface to the middle of epidermal cells. Chloroplast autofluorescence was detected in all the RFP-channel images by the use of 610IF emission filter. Scale bar = 20 µm. (A) Epidermal cells transiently expressing NbGAPDH-A-GFP (left two panels), and NbGAPDH-A-GFP with MP-mCherry (right four panels). Arrows represent PD-localized MP-mCherry signals. (B) Epidermal cells, which transiently expressed NbGAPDH-A-GFP (panels 1 and 2), or NbGAPDH-A-GFP with RCNMV RNA1 and ER marker (ER-mCherry) (panels 3 and 4), or NbGAPDH-A-GFP with viral replicase component proteins p27 and p88 (panels 5 and 6). Arrowheads represent chloroplast-localized NbGAPDH-A-GFP signals and arrows represent cortical ER-localized NbGAPDH-A-GFP signals.
Figure 6.
Subcellular localization of free GFP and GFP with chloroplast targeting signal is not affected by coexpression of RCNMV RNA1.
Representative CLSM images of agroinfiltrated N. benthamiana epidermal cells. Epidermal cells transiently expressing RCNMV RNA1 together with GFP (left panels), or with GFP with chloroplast targeting signal peptide of Rubisco small subunit (RbcSTP-GFP, right panels). Scale bar = 20 µm. Conditions for infiltration, plant incubation, and CLSM were similar to those in Figure 5.
Figure 7.
Bimolecular fluorescence complementation analyses of the interaction between RCNMV MP and NbGAPDH-A.
NbGAPDH-A fused to the C-terminal half of YFP at the C-terminus (NbGAPDH-A-cYFP), or C-terminal half of YFP (cYFP) as the negative control, was expressed with TBSV silencing suppressor p19 in N. benthamiana leaves via Agrobacterium infiltration. 18 h after infiltration, in vitro transcripts of the recombinant RCNMV that expressed fusion protein of the MP and N-terminal half of YFP at the C-terminus (R1-MnY+R2fsMP, Figure S1I), or the recombinant virus that expressed N-terminal half of YFP (R1-nYFP+R2fsMP, Figure S1J) as the negative control, was mechanically inoculated. At 28 hpi of the recombinant virus, reconstructed YFP signal was visualized using CLSM. (A) Reconstituted YFP signals were detected as foci composed of 5–10 cells in the leaves that expressed NbGAPDH-A-cYFP and inoculated with R1-MnY+R2fsMP (left panel). No YFP signals were detected in the leaves that expressed unfusedcYFP (center panel), or those inoculated with R1-nYFP+R2fsMP (right panel). Scale bar = 50 µm (B) Large magnification images of the reconstituted YFP in the cortical (left panel) and inner cell wall region (right panel). Images are from optical sections taken at upper part for cortical region and middle part for cell wall region in the same site of the leaf and mergers of DIC and GFP channel. Scale bar = 10 µm (C) Reconstituted YFP signals as cortical punctates (left panel), ER-mCherry signals (center panel) and overlapped (right panel). Most YFP-punctates are larger than those in (B), because this cell is closer to the center of infection and that shows the cell is at a later stage of virus infection. Scale bar = 10 µm.
Figure 8.
NbGAPDH-A interacts with both RCNMV MP and p27 in vitro.
Glutathione resin-bound GST, GST-fused MP (GST-MP-HA) and GST-fused p27 (GST-p27) was incubated with the purified recombinant NbGAPDH-A (His-GAP-myc). After washing, the pulled-down complexes were subjected to SDS-PAGE and analyzed by Western blotting (Wb) using anti-GST and anti-myc antibodies. Same samples were subjected to SDS-PAGE and stained with Coomasie brilliant blue CBB). The bands that appeared in GST-MP-HA lanes (*) with almost similar mobility as GST-p27 are probably the degradation product of GST-MP-HA.
Figure 9.
Gene silencing of NbGAPDH-A inhibits the localization of RCNMV MP to the cortical VRCs.
(A) Representative CLSM images of ALSV- or ALSV/gsGAP-infected N. benthamiana mesophyll (upper panels) and epidermal cells (lower panels) infiltrated with Agrobacterium cultures that contained pBICR1/MsG2fsMP, which expressed recombinant RCNMV RNAs encoding MP-GFP (Figure S1K). The images were obtained at 38 h after infiltration. Scale bar = 30 µm. The images include the merged DIC and GFP channels, and they represent confocal projections of 20 optical sections at 1 µm intervals, ranging from the surface to the middle of the cells. (B) The leaf samples in (A) were subjected to epifluorescence microscopy and the percentage of fluorescent cells containing cortical punctates was determined. The data shown are the totals from three replicate assays. (C) Protoplasts prepared from ALSV- or ALSV/gsGAP-infected N. benthamiana plants were inoculated with a mixture of in vitro transcripts of pUCR1-MsG and pRNA2fsMP (Figure S1L) [10]. Representative CLSM images of the protoplasts at 12 hpi. Scale bar = 30 µm. The images include the merged DIC and GFP channels (panels 1 and 2), or GFP channel (panel 3). They represent confocal projections of 15 optical sections at 2 µm intervals, ranging from the surface to the center of the cells. (D) The protoplast samples in (C) were subjected to Western blotting. Proteins extracted from 2×104 protoplasts were loaded in each lane. MP-GFP was detected using a rabbit polyclonal antibody against GFP. The numbers below the image represent the relative accumulation levels (means ± SE) of the proteins obtained using the Image Gauge program (Fuji Film), which were calculated from two independent experiments. Asterisk indicates a not significant (P>0.05; Student's t-test) difference compared with the accumulation of proteins in the protoplasts from ALSV-infected N. benthamiana. RbcL is a Coomassie brilliant blue-stained gel image, which shows the large subunit of Rubisco proteins.
Figure 10.
Cortical VRC formation in the NbGAPDH-A-silenced N. benthamiana protoplasts.
Protoplasts prepared from ALSV- or ALSV/gsGAP-infected N. benthamiana plants were inoculated with recombinant RCNMV RNAs that expressed the MP-GFP fusion protein (Figure S1L) and subjected to immunostaining with anti-dsRNA primary antibody followed by Alexa Fluor 594-conjugated secondary antibody at an early stage of infection (16 hpi). The left-most panels show the results for mock-inoculated protoplasts treated with the same antibodies. The right-most panels show the results for protoplasts prepared from ALSV-infected plants treated with only secondary antibody. Images present confocal projections of five optical sections at 1 µm intervals, which range from the surface to the middle of the protoplasts. N: nucleus. Scale bar = 20 µm.