Fig 1.
Pdc1 fermentation enzyme is an essential host factor for tombusvirus replication in yeast.
(A) Depletion of pyruvate decarboxylase (Pdc1p) in combination with deletion of the homologous PDC5 inhibits TBSV replicon (rep)RNA replication in yeast. Top panels: northern blot analyses of TBSV repRNA using a 3’ end specific probe demonstrates reduced accumulation of repRNA in GAL::PDC1 pdc5Δ yeast strain with depleted Pdc1p (raffinose-containing media) in comparison with the WT yeast strain or GAL::PDC1 pdc5Δ yeast strain with induced Pdc1p (galactose-containing media). Viral proteins His6-p33 and His6-p92 of TBSV were expressed from plasmids from the copper-inducible CUP1 promoter, while DI-72(+) repRNA was expressed from the constitutive TET1 promoter. Second panel: northern blot with an 18S ribosomal RNA specific probe was used as a loading control. Bottom images: western blot analysis of the level of His6-tagged p33 protein with anti-His antibody. Coomassie blue-stained SDS-PAGE was used for protein loading control. The down-regulation of Pdc1 mRNA was confirmed with RT-PCR. Each experiment was repeated three times. (B-D) Expression of Pdc1p from a plasmid increases tombusvirus replication in pdc1Δ yeast strain. His6-p33 and His6-p92 were expressed from the GAL1 promoter, whereas (+)repRNA was expressed from the GAL10 promoter. For panel C, Flag-p36 and Flag-p95 were expressed from the CUP1 promoter, whereas the repRNA from the GAL10 promoter. The untagged or His6-tagged Pdc1 were expressed from the TET promoter in all these experiments. See further details in panel A above.
Fig 2.
Interaction between tombusvirus replication proteins and Pdc1 fermentation enzyme.
(A) The split ubiquitin-based MYTH assay was used to test binding between the TBSV p33 and the yeast Pdc1p and Arabidopsis Pdc1 proteins in yeast. The bait p33 was co-expressed with the shown prey proteins. The Ssa1p heat shock protein 70 (Hsp70) and the empty prey vector (NubG) were used as the positive and the negative controls, respectively. The right panel shows p33: Pdc1 interactions, the left panel demonstrates that comparable amounts of yeasts were used for these experiments. (B-C) Co-purification of the yeast His6-Pdc1p and His6-Pdc1S455F mutant with TBSV Flag-p33 and Flag-p92pol or the carnation Italian ringspot virus (CIRV) Flag-p36/Flag-p95 replication proteins from subcellular membranes. Top two panels: western blot analysis of the co-purified WT His6-Pdc1p and His6-Pdc1S455F mutant with the Flag-affinity purified replication proteins. The His6-tagged proteins were detected with an anti-His antibody, while Flag-p33 and Flag-p36 were detected with an anti-Flag antibody. The negative control was from yeast expressing His6-p33 and His6-p92pol purified using a Flag-affinity column (lane 1). Samples were cross-linked with formaldehyde in intact yeast cells. Bottom two panels: western blot of total His6-Pdc1p and Flag-p33 and Flag-p36 in the total yeast extracts. (D-E) Co-purification of the Arabidopsis His6-Pdc1 with the TBSV Flag-p33 and Flag-p92pol or the CIRV Flag-p36/Flag-p95 replication proteins from subcellular membranes of yeast. Top two panels: western blot analysis of the co-purified His6-AtPdc1 with Flag-affinity purified replication proteins. The His6-tagged proteins were detected with anti-His antibody, while the Flag-p33 and Flag-p36 were detected with an anti-Flag antibody. The negative control was from yeast expressing the His6-tagged replication proteins purified using a Flag-affinity column (lane 1). Samples were cross-linked with formaldehyde. Bottom two panels: western blot of the total Flag-p33 or Flag-p36 and the His6-AtPdc1 and in the total yeast extracts (F) Co-purification of HA-AtPdc1 with the TBSV p33-Flag replication protein from N. benthamiana. Top two panels: western blot analysis of the co-purified HA-tagged AtPdc1 (lane 2) with the Flag-affinity purified Flag-p33. HA-Pdc1 was detected with an anti-HA antibody, while the p33-Flag was detected with an anti-Flag antibody as shown. Bottom two panels: western blot of the total plant extracts. (G) Pull-down assay including the GST-His6-p33 replication protein and the MBP-tagged yeast Pdc1p or the MBP-AtPdc1. Note that we used the soluble C-terminal region of the TBSV p33 replication protein, which lacked the N-terminal sequence, including the trans-membrane TM domain. Top panel: western blot analysis of the captured His6-p33 with the MBP-affinity purified MBP-Pdc1 was performed with an anti-His antibody. The negative control was MBP (lane 1). Middle panel: Coomassie-blue stained SDS-PAGE of the captured yeast MBP-Pdc1p or MBP-AtPdc1 and MBP. Bottom panels: western blot analysis of the His6-p33 in the total extracts. Coomassie-blue stained SDS-PAGE of the MBP-Pdc1p or MBP-AtPdc1 and MBP in the total extracts. Each experiment was repeated three times. (H) Pull-down assay including the GST-His6-AtPdc1 and the MBP-tagged p33 or the CIRV MBP-p36 replication proteins. Please see further details in panel G. (I) Decreasing level of co-purification of His6-Pdc1p with the Flag-tagged viral replicase after blocking new VRC assembly. The yeast samples were collected at the shown time points after the addition of cycloheximide (blocks cellular translation, thus new VRC formation) to the yeast culture. Note that samples were from yeasts replicating TBSV repRNA. Top panel: western blot analysis of the co-purified His6-Pdc1with the Flag-affinity purified Flag-p33 and Flag-p92pol from membrane fraction of yeast. The His6-Pdc1p was detected with an anti-His antibody. The negative control was the His6-p33 and His6-p92pol purified from yeast extracts using a Flag-affinity column. Middle panel: western blot of the purified Flag-p33 detected with an anti-Flag antibody. Bottom panels: western blots of His6-Pdc1p and Flag-p33 proteins in the total yeast extracts using an anti-His and an anti-Flag antibodies. The graph shows the % of co-purified His6-Pdc1p with the tombusviral replication proteins with standard deviation. Each experiment was repeated three times.
Fig 3.
Interaction between the tombusvirus replication proteins and Adh1 fermentation enzyme.
(A) The split ubiquitin-based MYTH assay was used to test binding between the TBSV p33 and the yeast alcohol dehydrogenase Adh1-5p and the Arabidopsis Adh1 proteins in yeast. The bait p33 was co-expressed with the shown prey proteins. The Ssa1p Hsp70 and the empty prey vector (NubG) were used as the positive and the negative controls, respectively. The right panel shows p33: Adh1-5 interactions, the left panel demonstrates that comparable amounts of yeasts were used for these experiments. (B) Co-purification of the yeast His6-Adh1, 2, 3p with the TBSV Flag-p33 and Flag-p92pol replication proteins from subcellular membranes. Top two panels: western blot analysis of the co-purified His6-Adh1-3p with the Flag-affinity purified replication proteins. His6-tagged proteins were detected with an anti-His antibody, while Flag-p33 was detected with an anti-Flag antibody. The negative control was from yeast expressing His6-p33 and His6-p92pol purified using a Flag-affinity column (lane 1). Samples were cross-linked with formaldehyde. Bottom two panels: western blot of the total His6-Adh1-3p and Flag-p33 in the total yeast extracts. (C) Co-purification of the yeast His6-Adh1p with the CIRV Flag-p36 and Flag-p95pol replication proteins from subcellular membranes. See further details in panel B. (D) Co-purification of His6-AtAdh1 with either the TBSV or the CIRV replicase from yeast subcellular membranes. Top two panels: western blot analysis of the co-purified His6-Adh1p (lanes 2–3) with the Flag-affinity purified Flag-p33 or CIRV Flag-p36. His6-Adh1p was detected with an anti-His antibody, while the Flag-p33 or CIRV Flag-p36 replication proteins were detected with an anti-Flag antibody as shown. Bottom two panels: western blot of the total plant protein extracts. (E-F) Pull-down assay including GST-His6-Adh1p or GST-His6-AtAdh1 with the TBSV MBP-p33C or the CIRV MBP-p36C replication proteins and MBP. See further details in panel B. Each experiment was repeated three times.
Fig 4.
Tombusvirus infection induces the expression of Pdc1 and Adh1 mRNAs in N. benthamiana.
(A) Top panels: semi-quantitative RT-PCR analysis of the NbPdc1 mRNA level at 1.5 dpi in N. benthamiana leaves infected with either TBSV or mock-infected. (B) Semi-quantitative RT-PCR analysis of the NbPdc1 mRNA level at 3 dpi in N. benthamiana leaves infected with either CIRV or mock-inoculated. The samples were taken 3 days after tombusvirus inoculation. Second panel: RT-PCR analysis of the tubulin mRNA level in the same plants. Each experiment was repeated three times. Bottom panels: Ethidium-bromide-stained agarose gels show the comparable amounts of RNA loading, as shown for the ribosomal RNA. (C) Top panel: semi-quantitative RT-PCR analysis of the NbPdc1 mRNA level at 3 days post agroinfiltration in N. benthamiana leaves agro-infiltrated to express TBSV p33, CIRV p36 or no-expression control. See further details in panels A-B. (D-F) Semi-quantitative RT-PCR analysis of the NbAdh1 mRNA level at 1.5 dpi in N. benthamiana leaves infected with either TBSV or mock-infected or CNV- or mock-infected at 3 dpi. See further details in panels A-B.
Fig 5.
Knockdown of Pdc1 mRNA level inhibits tombusvirus replication in N. benthamiana plants.
(A) Top panel: Accumulation of the TBSV genomic (g)RNA in Pdc1-silenced N. benthamiana plants 1.5 days post-inoculation (dpi) in the inoculated leaves was measured by northern blot analysis. Inoculation of TBSV gRNA was done 12 days after silencing of Pdc1 expression. Agroinfiltration of tobacco rattle virus (TRV) vector carrying NbPdc1 or 3’-terminal GFP (as a control) sequences was used to achieve virus-induced gene silencing (VIGS). Second panel: Ribosomal RNA is shown as a loading control in an ethidium-bromide stained agarose gel. Third panel: RT-PCR analysis of NbPdc1 mRNA level in the silenced and control plants. Fourth panel: RT-PCR analysis of tubulin mRNA level in the silenced and control plants. Each experiment was repeated three times. Delayed development of TBSV-induced symptoms is observed in the Pdc1-silenced N. benthamiana plants as compared with the control plants. Note the lack of phenotype in the Pdc1-silenced and mock-inoculated N. benthamiana plants. Note the severe wilting and beginning stage of necrosis in the control TBSV-infected plant versus the lack of those symptoms in the Pdc1-silenced N. benthamiana plants. The pictures were taken at 8 dpi. (B) Top panel: Accumulation of the TBSV gRNA in protoplasts isolated from Pdc1-silenced N. benthamiana was measured by northern blot analysis 16 hours after virus transfection. Protoplasts were isolated 12 days after silencing of Pdc1 expression. Agroinfiltration of TRV-NbPdc1 or TRV-cGFP (as a control) was used to induce VIGS. Second panel: Ribosomal RNA is shown as a loading control in an ethidium-bromide stained agarose gel. Third panel: RT-PCR analysis of NbPdc1 mRNA level in the silenced and the control protoplasts. Fourth panel: RT-PCR analysis of tubulin mRNA level in the silenced and the control protoplasts. Each experiment was repeated three times. (C) Accumulation of the CIRV gRNA in the Pdc1-silenced N. benthamiana plants 3 dpi in the inoculated leaves and at 5 dpi in the systemically-infected leaves was measured by northern blot analysis. See further details in panel A. (D) Accumulation of the TCV gRNA in the Pdc1-silenced N. benthamiana plants 6 dpi in the inoculated leaves was measured by northern blot analysis. See further details in panel A.
Fig 6.
Knockdown of Adh1 mRNA level inhibits tombusvirus replication in N. benthamiana plants.
(A-C) Accumulation of the TBSV, CNV and CIRV gRNA in the Adh1-silenced N. benthamiana plants. The experimental data are presented as in Fig 5.
Fig 7.
Recruitment of Pdc1 and Adh1 fermentation enzymes by the tombusvirus replication protein into the viral replication compartment in N. benthamiana.
(A) Confocal microscopy images show efficient co-localization of the TBSV p33-BFP replication protein and the RFP-AtPdc1 within the viral replication compartment, marked by GFP-SKL peroxisomal luminal marker in N. benthamiana leaves. Expression of these proteins from the 35S promoter was done after co-agroinfiltration into N. benthamiana leaves. The plant leaves were either TBSV-infected or mock-inoculated as shown. The images were taken 1.5 days after TBSV inoculation of plant leaves. Scale bars represent 10 μm. (B) Recruitment of Pdc1 by the CIRV p36 replication protein into the mitochondria-derived viral replication compartment in N. benthamiana. Confocal microscopy images show efficient co-localization of CIRV p36-BFP replication protein and the RFP-AtPdc1 within the viral replication compartment, marked by GFP-AtTim21 mitochondrial marker in N. benthamiana leaves. The images were taken 1.5 days after agro-infiltration of plant leaves. See further details in panel A. (C) Confocal microscopy imaging shows the cytosolic localization of RFP-AtPdc1 in the absence of viral components. See further details in panel A. (D) Confocal microscopy images show efficient co-localization of TBSV p33-RFP replication protein and the BFP-AtAdh1 within the viral replication compartment, marked by GFP-SKL peroxisomal luminal marker in N. benthamiana leaves. See further details in panel A. (E) Recruitment of Adh1 by the CIRV p36 replication protein into the mitochondria-derived viral replication compartment in N. benthamiana. Confocal microscopy images show efficient co-localization of CIRV p36-RFP replication protein and the BFP-AtAdh1 within the viral replication compartment, marked by GFP-AtTim21 mitochondrial marker in N. benthamiana leaves. See further details in panel A. (F) Confocal microscopy imaging shows the cytosolic localization of BFP-AtAdh1 in the absence of viral components. See further details in panel A.
Fig 8.
Confocal microscopy shows co-localization of the co-opted fermention enzymes with the viral repRNAs in whole plants infected with CNV.
(A-B) Most of GFP-AtPdc1 is re-targeted into the replication compartment where the viral RNA synthesis takes place. The viral (+)repRNA carried six copies of the MS2 bacteriophage RNA hairpin (MS2hp), which is recognized by the MS2 coat protein (RFP-MS2-CP). The replication compartment was marked by the BFP-tagged p33 replication protein in N. benthamiana. Panel B shows images from plants mock-inoculated (no viral RNA replication). Note that RFP-MS2-CP contains a week nuclear localization signal, therefore this protein ends up in the nucleus in the absence of replicating (+)repRNA-MS2hp in the cytosol. Expression of the above proteins from the 35S promoter was done after co-agroinfiltration into N. benthamiana leaves. The images were taken 3.5 days after agro-infiltration of plant leaves. Scale bars represent 10 μm. Each experiment was repeated three times. (C-D) Similar experimental set-up as in panel A-B, except the six MS2hps form the suitable structures on the viral (-)repRNA-MS2hp, which is recognized by RFP-MS2-CP. See further details in Panel A. (E-H) Most of GFP-AtAdh1 is re-targeted into the replication compartment where the viral RNA synthesis takes place. See further details in Panel A and C.
Fig 9.
Interactions between TBSV p33/p92 or CIRV p36 replication proteins and the AtPdc1 or AtAdh1 proteins were detected by BiFC.
The TBSV p33-cYFP or p92-cYFP or CIRV p36-cYFP replication proteins and the nYFP-AtPdc1 (panel A) or nYFP-AtAdh1 (panel B) proteins and the marker proteins were expressed via agroinfiltration. The merged images show the efficient co-localization of the peroxisomal RFP-SKL or the mitochondrial RFP-AtTim21 with the bimolecular fluorescence complementation (BiFC) signals, indicating that the interactions between the tombusvirus replication proteins and the co-opted AtPdc1 or AtAdh1 proteins occur in the large viral replication compartments, which consist of either aggregated peroxisomes or aggregated mitochondria. Scale bars represent 10 μm.
Fig 10.
Dependence of TBSV repRNA accumulation on Pdc1/5 in an in vitro replicase reconstitution assay based on CFE obtained from yeast with depleted Pdc1/5.
(A) Top: A scheme of the in vitro replicase reconstitution assay based on yeast cell-free extracts (CFEs). The purified recombinant TBSV p33 and p92pol replication proteins from E. coli were added in combination with the (+)repRNA template to program the in vitro tombusvirus replication assay. The CFEs were prepared from yeast strains cultured in the shown media prior to CFE preparation. Bottom: Non-denaturing PAGE shows the accumulation of 32P-labeled (+)repRNAs and the dsRNA replication intermediate products made by the reconstituted replicases in the shown CFE preparations. All the samples shown were loaded on the same PAGE gel. Each experiment was repeated. (B) RdRp assay with Flag-affinity purified tombusvirus replicase preparations. The shown yeast strains expressing Flag-p33 and Flag-p92pol from the CUP1 promoter and (+)repRNA from the TET promoter were cultured in the shown media prior to preparation of the purified replicase preps. The replicase preparations containing the same amount of p33 replication protein were programmed with the shown (+) or (-)RNA templates. The denaturing PAGE gels show the produced complementary RNA products by the given replicase preparations. All the samples shown were loaded on the same PAGE gel. Each experiment was repeated. (C) The in vitro RdRp activation assay is based on (+)repRNA and p92-Δ167N RdRp protein in the presence of the soluble fraction of yeast CFE. The CFEs were prepared from yeast strains cultured in the shown media prior to CFE preparation. Denaturing PAGE analysis of the 32P-labeled RNA products obtained in an in vitro assay with recombinant p92-Δ167N RdRp. Each experiment was repeated three times.
Fig 11.
The co-opted cytosolic Pdc1 fermentation enzyme affects ATP accumulation within the tombusvirus replication compartment in yeast.
(A) A scheme of the FRET-based detection of ATP within the tombusvirus replication compartment. The enhanced ATP biosensor, ATeamYEMK was fused to TBSV p92pol replication protein. See further details in the main text. (B) Comparison of the ATP level produced within the tombusvirus replication compartment in pdc1Δ yeast strain expressing WT Pdc1p, Pdc1S455F mutant or without Pdc1 expression using ATeamYEMK -p92pol. The more intense FRET signals are white and red (between 0.5 to 1.0 ratio), whereas the low FRET signals (0.1 and below) are light blue and dark blue. We show the quantitative FRET values (obtained with ImageJ) for a number of samples in the graph. Note that we also used a reduced ATP-sensitive version of ATeamRK-p92 (bottom panel) to demonstrate that the FRET signal is due to ATP-sensing. Scale bars represent 5 μm. Each experiment was repeated three-four times.
Fig 12.
Knockdown of the cellular Pdc1 fermentation enzyme inhibits ATP accumulation within the tombusvirus replication compartment in N. benthamiana.
(A) A scheme of the FRET-based detection of cellular ATP within the replication compartment. The enhanced ATP biosensor, ATeamYEMK was fused to TBSV p33 replication protein. (B) Knock-down of Pdc1 mRNA level by VIGS in N. benthamiana was done using a TRV vector. Twelve days latter, expression of p33-ATeamYEMK was done in upper N. benthamiana leaves by agroinfiltration. The YFP signal was generated by mVenus in p33-ATeamYEMK via FRET 1.5 days after agro-infiltration. The FRET signal ratio is shown in the right panels. The more intense FRET signals are white and red (between 0.5 to 1.0 ratio), whereas the low FRET signals (0.1 and below) are light blue and dark blue. We also show the average quantitative FRET values (obtained with ImageJ) for 10–20 samples on the graph. Note that N. benthamiana plants were mock-inoculated. (C-D) Comparable experiments with p33-ATeamYEMK in the Pdc1 knockdown N. benthamiana plants infected with the peroxisomal TBSV and CNV tombusviruses. See further details in panel B.
Fig 13.
The co-opted cellular Pdc1 fermentation enzyme affects ATP accumulation within the CIRV replication compartment in N. benthamiana.
(A) Knock-down of Pdc1 mRNA level by VIGS in N. benthamiana was done using a TRV vector. Twelve days later, expression of the CIRV p36-ATeamYEMK was done in upper N. benthamiana leaves by agroinfiltration. The FRET-based confocal microscopy analysis was performed 1.5 days after agro-infiltration. The FRET signal ratio is shown in the right panels. We show the average quantitative FRET values for 10–20 samples on the graph. Note that N. benthamiana plants were mock-inoculated. (B) Comparable experiments to those in panel A, except CIRV supported repRNA replication in the cells. See further details in panel A.
Fig 14.
The co-opted cytosolic Adh1 is needed for ATP generation within the tombusvirus replication compartment in plants.
(A-D) Knock-down of Adh1 mRNA level by VIGS in N. benthamiana was done using a TRV vector. Twelve days latter, expression of TBSV p33-ATeamYEMK (panels A-B) or the CIRV p36-ATeamYEMK (panels C-D) was done in upper N. benthamiana leaves by agroinfiltration. The YFP signal was generated via FRET 1.5 days after agro-infiltration. The FRET signal ratio is shown in the right panels. We show the average quantitative FRET values for 10–20 samples on the graph. Note that N. benthamiana plants were mock-inoculated or the plants supported TBSV and CIRV repRNA replication as shown. See further details in Fig 12B. Scale bars represent 10 μm. Each experiment was repeated three or four times.
Fig 15.
Dependence of BaMV replication on Pdc1 and Adh1 proteins in N. benthamiana.
(A) Top panels: semi-quantitative RT-PCR analysis of NbPdc1 and NbAdh1 mRNA levels at 3 dpi in N. benthamiana leaves infected with bamboo mosaic virus (BaMV) or mock-inoculated. Third panel: RT-PCR analysis of tubulin mRNA level in the same plants. Bottom panel: RT-PCR detection of the BaMV CP subgenomic RNA. (B) Semi-quantitative RT-PCR analysis of NbPdc1 and NbAdh1 mRNA levels at 7 dpi in N. benthamiana leaves infected with BaMV or mock-inoculated. Third panel: RT-PCR analysis of tubulin mRNA level in the same plants. Bottom panel: RT-PCR detection of the BaMV CP subgenomic RNA. Each experiment was repeated three times. (C) Knockdown of Pdc1 or Adh1 mRNA levels inhibits BaMV replication in N. benthamiana plants. Top panel: Accumulation of the BaMV genomic (g)RNA in the Pdc1-silenced N. benthamiana plants 2.5 days post-inoculation (dpi) in the inoculated leaves was measured by quantitative RT-PCR. Inoculation of BaMV gRNA was done 12 days after silencing of Pdc1 or Adh1 expression. Agroinfiltration of tobacco rattle virus (TRV) vector carrying NbPdc1 or NbAdh1 or luciferase (LUC, as a control) sequences was used to induce VIGS. Second panel: RT-PCR analysis of tubulin mRNA level in the silenced and control plants. Each experiment was repeated three times. (D-E) The BaMV ORF1-capping-cYFP or ORF1-helicase-cYFP or ORF1-RdRp-cYFP domains of the replicase protein and the AtPdc1-nYFP (panel D) or AtAdh1-nYFP (panel E) proteins were expressed via agroinfiltration. The DIC and the merged images are also shown. The BiFC signals were detected via confocal microscopy 2 days after agroinfiltration to N. benthamiana plants. Scale bars represent 25 μm.