The chloroplast ribosomal protein large subunit 1 interacts with viral polymerase and promotes virus infection

Abstract

Chloroplasts play an indispensable role in the arms race between plant viruses and hosts. Chloroplast proteins are often recruited by plant viruses to support viral replication and movement. However, the mechanism by which chloroplast proteins regulate potyvirus infection remains largely unknown. In this study, we observed that Nicotiana benthamiana ribosomal protein large subunit 1 (NbRPL1), a chloroplast ribosomal protein, localized to the chloroplasts via its N-terminal 61 amino acids (transit peptide), and interacted with tobacco vein banding mosaic virus (TVBMV) nuclear inclusion protein b (NIb), an RNA-dependent RNA polymerase. Upon TVBMV infection, NbRPL1 was recruited into the 6K2-induced viral replication complexes in chloroplasts. Silencing of NbRPL1 expression reduced TVBMV replication. NbRPL1 competed with NbBeclin1 to bind NIb, and reduced the NbBeclin1-mediated degradation of NIb. Therefore, our results suggest that NbRPL1 interacts with NIb in the chloroplasts, reduces NbBeclin1-mediated NIb degradation, and enhances TVBMV infection.

Figure 1

NbRPL1 physically interacts with TVBMV NIb in vitro and in vivo. A, Determination of interactions between HIS:eGFP:NbRPL1, HIS:eGFP:NbEF-Tu, and HIS:MBP:MYC:NIb using in vitro pull-down assay. The combination of HIS:eGFP and HIS:MBP:MYC:NIb was used as the negative control. Pull-down assays were performed using GFP-Trap_A beads followed by Western blotting analyses using an MYC-specific or a GFP-specific antibody. B, BiFC analysis of the interaction between NIb:YN and NbRPL1:YC in N. benthamiana leaf cells. Co-expression of NIb:YN and YC or YN and NbRPL1:YC were used as negative controls. C, Confocal micrographs of N. benthamiana leaves agroinfiltrated with plasmids expressing NbRPL1:eGFP or NbRPL1:DsRed. Images of N. benthamiana leaf cells were taken at 36 hpai. Scale bars = 10 μm.

Figure 2

Identification of the cTP domain, and its effects on the subcellular distribution of NbRPL1 and the interaction between NIb and NbRPL1. A, Prediction of cTP in NbRPL1. B, SDS–PAGE analysis of NbRPL1:DsRed expressed in the noninfected and TVBMV-infected N. benthamiana leaves. Sizes of the bands of the molecular weight marker are shown on the left. C, Schematic representations of NbRPL1 and its deletion mutants. D and E, Confocal micrographs of N. benthamiana leaf cells expressing NbRPL1^ΔcTP(N61aa):DsRed, NbRPL1^ΔN41aa:DsRed, NbRPL1^ΔN20aa:DsRed, NbRPL1^ΔN42–61aa:DsRed, NbRPL1^ΔN21–61aa:DsRed, and NbRPL1^ΔN21–42aa:DsRed (D), and NbRPL1^1–280aa:DsRed and NbRPL1^281–340aa:DsRed (E). F, BiFC analysis of the interaction between NIb:YN and NbRPL1^ΔcTP:YC. Co-expression of YN and NbRPL1^ΔcTP:YC was used as a negative control. Images of N. benthamiana leaf cells were captured under a confocal microscope at 36 hpai. Scale bars = 10 μm.

Figure 3

The NbRPL1/NIb complex co-localizes with TVBMV 6K2 on chloroplasts upon TVBMV infection. A, Co-localization of NIb:YN, NbRPL1:YC, and 6K2:DsRed in N. benthamiana leaf cells. Co-expressions of NIb:YN, YC, and 6K2:DsRed or YN, NbRPL1:YC, and 6K2:DsRed were used as negative controls. White arrowheads indicate the co-localization of the NIb:YN/NbRPL1:YC complex with 6K2:DsRed. B, Subcellular distributions of NIb:YN, NbRPL1:YC, and H2B:mCherry or NIb:YN, NbRPL1:YC, and AtPDLP1:DsRed in N. benthamiana leaf cells. H2B:mCherry is a nuclear marker and AtPDLP1:DsRed is a plasmodesmata marker. C, Co-localization of NIb:YN, NbRPL1:YC, and 6K2:DsRed in the TVBMV-infected N. benthamiana leaf cells. White arrowheads indicate the co-localization of the NIb:YN/NbRPL1:YC complex with 6K2:DsRed. Images were taken under a confocal microscope at 36 hpai. Scale bars = 10 μm.

Figure 4

Silencing of NbRPL1 expression affects TVBMV infection and the subcellular distribution of NIb in N. benthamiana plants. A, Phenotypes of the NbRPL1-silenced (TRV-NbRPL1) and nonsilenced (TRV-GUS) N. benthamiana plants at 8 dpai. B, Relative expression of NbRPL1 in the systemic leaves of the NbRPL1-silenced and nonsilenced N. benthamiana plants was determined through RT-qPCR. Error bars indicate the standard deviations of three biological replicates per treatment. Statistical significance was determined using a two-tailed Student’s t test (**P < 0.01). C, Phenotypes of the NbRPL1-silenced and nonsilenced N. benthamiana plants at 6 d post TVBMV-GFP inoculation (dpi). Plants were photographed under UV illumination. D, Western blotting analysis of TVBMV CP accumulation in the systemic leaves of the TVBMV-GFP-inoculated NbRPL1-silenced or nonsilenced N. benthamiana plants at 6 d post TVBMV-GFP inoculation. The Coomassie Brilliant Blue R-250 (CBB)-stained gel shows sample loadings. E, RT-PCR detection of TVBMV RNA accumulation in the systemic leaves of the TVBMV-GFP-inoculated NbRPL1-silenced or nonsilenced N. benthamiana plants at 6 dpi using TVBMV CP gene specific primers. F, GFP fluorescence in the NbRPL1-silenced N. benthamiana leaves. Images of TVBMV-GFP-inoculated nonsilenced or NbRPL1-silenced leaves were taken under normal light or UV illumination at 72 hpai. G, Western blotting analysis of TVBMV CP accumulation in the TVBMV-GFP-inoculated NbRPL1-silenced or nonsilenced N. benthamiana leaves at 72 hpai. The CBB-stained gel shows sample loadings. H, RT-PCR detection of TVBMV RNA accumulation in the TVBMV-GFP-inoculated NbRPL1-silenced or nonsilenced N. benthamiana leaves at 72 hpai using the TVBMV CP gene specific primers. I, RT-qPCR analysis of TVBMV^ΔP3NPIPO-GFP RNA accumulation in the TVBMV-GFP-inoculated NbRPL1-silenced or nonsilenced N. benthamiana leaves at 36 hpai using the TVBMV CP gene specific primers. Error bars indicate the standard deviation of three biological replicates per treatment. Statistical significance was determined using a two-tailed Student’s t test (**P < 0.01). J, Localization of 6K2:DsRed in the NbRPL1-silenced or nonsilenced N. benthamiana leaves. K, Co-localization of NIb:mCherry and TVBMV-6K2:GFP in the NbRPL1-silenced or nonsilenced N. benthamiana leaves. White arrowheads indicate the co-localization of 6K2:GFP and NIb:mCherry. Scale bars = 10 μm.

Figure 5

The overexpression of NbRPL1 in N. benthamiana leaves increases TVBMV cell-to-cell movement and RNA accumulation, and increases the accumulation of TVBMV NIb. A, Micrographs of TVBMV-GFP cell-to-cell movement in the control and NbRPL1-overexpressing N. benthamiana leaves. Images were taken at 96 hpai. Green fluorescence represents the GFP-tagged TVBMV. Scale bars = 500 μm. B, The mean number of TVBMV-GFP-infected cells in each examined field. The number of TVBMV-GFP-infected cells in each locus was counted under the stereo fluorescence microscope. For each treatment, 30 infection loci were counted. In the NbRPL1-overexpressing experiment, the boxplots represent the data dispersion around the median with the position of the lower limit value (18), the lower quartile (28.5), the median (45.5), the upper quartile (58.75), and the upper limit value (91). In the control, the boxplots represent the data dispersion around the median with the position of the lower limit value (10), the lower quartile (23), the median (28), the upper quartile (33), and the upper limit value (46). Statistical significance was calculated using a two-tailed Student’s t test (**P < 0.01). C, Effect of NbRPL1 overexpression on viral protein translation efficiency in N. benthamiana leaves. Relative activities of RLUC and FLUC in the pCB301TVBMV^ΔGDD-RLUC//FLUC-inoculated leaves were determined at 60 hpai. Error bars indicate the standard deviation of three biological replicates per treatment. D, RT-qPCR detection of TVBMV genomic RNA in the NbRPL1-overexpressing and control N. benthamiana protoplasts using TVBMV CP gene-specific primers. Expression of NbEF1α was used as an internal control. Error bars indicate the standard deviation of three biological replicates per treatment. Statistical significance was determined using a two-tailed Student’s t test (*P < 0.05). E, RT-qPCR detection of TVBMV^ΔP3NPIPO-GFP RNA in the NbRPL1-overexpressing or control N. benthamiana leaves using TVBMV CP gene specific primers. Expression of NbEF1α was used as an internal control. Error bars indicate the standard deviation of three biological replicates per treatment. Statistical significance was calculated using a two-tailed Student’s t test (*P < 0.05). F, Western blotting detection of MYC:NIb co-expressed with NbRPL1:DsRed or DsRed in the TVBMV-infected N. benthamiana leaves at 2 dpai using MYC-specific and mCherry-specific antibodies, respectively. Band intensities were measured using the ImageJ software. The CBB-stained gel shows sample loadings.

Figure 6

NbRPL1 competes with NbBeclin1 for NIb. A, Co-IP analysis of NbBeclin1:eGFP and MYC:NIb in vivo interaction. Combination of eGFP and MYC:NIb was used as the negative control. The Co-IP analysis was performed with GFP-Trap_A beads followed by western blotting detection using an MYC-specific antibody. B, Determination of NIb and NbBeclin1 interaction through Y2H assay. Yeast cells were co-transformed with BD-NIb and AD-NbBeclin1 or AD-NIb and BD-NbBeclin1. The transformed cells were grown on the SD/-Trp/-Leu/-His/-Ade selection medium supplemented with X-α-Gal for 4 d. Y2H cells co-transformed with AD-T-ant and BD-p53 were used as the positive control, whereas Y2H cells co-transformed with AD-T-ant and BD-lam, BD-NIb and AD, AD-NIb and BD, BD and AD-NbBeclin1, or AD and BD-NbBeclin1 were used as negative controls. BD, pGBKT7; AD, pGADT7. C, BiFC analysis of NIb:YN and NbBeclin1:YC interaction in N. benthamiana leaves. NIb:YN and YC or YN and NbBeclin1:YC co-expressing N. benthamiana leaves were used as negative controls. Confocal images of leaf cells were taken at 36 hpai. Scale bars = 10 μm. D, Western blotting assay of total protein extracts from N. benthamiana leaves co-expressing NbBeclin1:eGFP and MYC:NIb using a GFP specific or a MYC specific antibody at 2 dpai. A N. benthamiana leaf sample co-expressing NbBeclin1:eGFP and MYC:NIb was used as the negative control. The CBB-stained gel shows sample loadings. E, RT-qPCR detection of TVBMV RNA in the N. benthamiana leaves co-expressing TVBMV and NbBeclin1:eGFP at 2 dpai. Co-expression of TVBMV and eGFP in N. benthamiana leaves was used as the negative control. Expression of NbEF1α was used as an internal control. Error bars indicate the standard deviation of three biological replicates per treatment. Statistical significance was determined using a two-tailed Student’s t test (**P < 0.01). F, Western blotting analysis of total protein extracts from N. benthamiana leaves co-expressing HA:NbRPL1, NbBeclin1:eGFP, and MYC:NIb using a HA specific, a GFP specific or a MYC-specific antibody at 2 dpai. Co-expression of NbBeclin1:eGFP, MYC:NIb, and the empty vector in N. benthamiana leaves was used as a negative control. The CBB-stained gel shows sample loadings. G, RT-qPCR detection of TVBMV RNA in the N. benthamiana leaves co-expressing NbBeclin1:eGFP and HA:NbRPL1 at 2 dpai. Co-expression of NbBeclin1:eGFP and empty vector in N. benthamiana leaves was used as the negative control. Expression of NbEF1α was used as an internal control. Error bars indicate the standard deviation of three biological replicates per treatment. Statistical significance was determined using a two-tailed Student’s t test (*P < 0.05). H, Competitive binding of NbRPL1 and NbBeclin1 to NIb in vitro. The mixed protein samples of HIS:MBP:MYC:NIb, HIS:eGFP:NbBeclin1, and HIS:HA:NbRPL1 with different concentrations were adsorbed with GFP-Trap_A beads followed by western blotting detection using a HA specific, a GFP specific or a MYC specific antibody. Band intensities shown in (D), (F), and (H) were quantified with ImageJ software.