The interaction between endogenous 30S ribosomal subunit protein S11 and Cucumber mosaic virus LS2b protein affects viral replication, infection and gene silencing suppressor activity

Abstract

Cucumber mosaic virus (CMV) is a model virus for plant-virus protein interaction and mechanism research because of its wide distribution, high-level of replication and simple genome structure. The 2b protein is a multifunctional protein encoded by CMV that suppresses RNA silencing-based antiviral defense and contributes to CMV virulence in host plants. In this report, 12 host proteins were identified as CMV LS2b binding partners using the yeast two-hybrid screen system from the Arabidopsis thaliana cDNA library. Among the host proteins, 30S ribosomal subunit protein S11 (RPS11) was selected for further studies. The interaction between LS2b and full-length RPS11 was confirmed using the yeast two-hybrid system. Bimolecular fluorescence complementation (BIFC) assays observed by confocal laser microscopy and Glutathione S-transferase (GST) pull-down assays were used to verify the interaction between endogenous NbRPS11 and viral CMVLS2b both in vivo and in vitro. TRV-based gene silencing vector was used to knockdown NbRPS11 transcription, and immunoblot analysis revealed a decline in infectious viral RNA replication and a decrease in CMV infection in RPS11 down-regulated Nicotiana benthamiana plants. Thus, the knockdown of RPS11 likely inhibited CMV replication and accumulation. The gene silencing suppressor activity of CMV2b protein was reduced by the RPS11 knockdown. This study demonstrated that the function of viral LS2b protein was remarkably affected by the interaction with host RPS11 protein.

Fig 1. Interaction between bait LS2b protein and full-length prey protein was re-examined using yeast two-hybrid screening by DDO and QDO selection.

Gradient dilutions of transformation mix were spread respectively on DDO/X and QDO/X/A plates which were cultivated for 5 days at 30°C. Yeast colony contained vectors of pGADT7-RPS11 with pGBKT7-LS2b and pGADT7-2bBP19 with pGBKT7- LS2b were showed on QDO/X/A plates. Vector pGADT7-T with pGBKT7-p53 and pGADT7 with pGBKT7-Lamda were used as positive and negative controls respectively.

Fig 2. GST pull-down assay.

GST and GST-LS2b fusion proteins incubated with cell lysates expressed His-RPS11 in tubes were the put-in samples transformed onto each right side of the membrane. Glutathione beads added to bind GST and GST-LS2b were washed down, proteins eluted from the beads were the pull-down samples transformed onto each left side of the membrane. The presence of RPS11 was detected by immunoblot with anti-HIS antibody. The presence and expression of GST and GST-LS2b was confirmed by immunoblotting with anti-GST antibody.

Fig 3. Bimolecular fluorescence complementation (BiFC) assay to test interaction of RPS11 with LS2b proteins in vivo. N. benthamiana was co-agroinfiltrated to transiently express combination of pSP-YCE-RPS11 and pSP-YNE-LS2b, pSP-YNE-RPS11 and pSP-YCE-LS2b.

co-agroinfiltrated to transiently express pSP-YNE-RPS11 and pSP-YCE-RPS11 as self-interaction control. Also shown as controls in which the unfused YFP fragments (YFP^C and YFP^N) were co-expressed with other of the RPS11-YFP fusion proteins. Cells in infiltrated tissues were imaged at 3 days post agroinfiltration by confocal scanning laser microscopy for YFP fluorescence and under bright field microscopy. The bar represented 25μm and 100μm.

Fig 4

Knockdown of NbRPS11 decreased LS-CMV infectious viral RNA replication (A) Northern blot hybridization analysis of infectious viral RNA agroinfiltrated N. benthamiana 7 days after TRV2-RPS11 inoculation plants (lane 1–3) and TRV2 inoculation control plants (lane 4–6). Ethidium bromide stained rRNA from the same volume of each sample is shown below each lane. (B) Northern blot hybridization analysis of CMVΔ2b agroinfiltrated N. benthamiana 7 days after TRV2-RPS11 inoculation plants (lane 4–6) and TRV2 inoculation control plants (lane 1–3). (C) Western blot hybridization analysis of GFP accumulation 3 days post-inoculation of TRV2-RPS11 plants and conrol. Infectious viral RNA accumulation was analyzed by immunoblotting of testing accumulation of LS309-GFP of RPS11 downregulated plants (lane 1–3) and control plants (lane 4–6) using anti-GFP antibody. (D) GFP accumulation of PVX-GFP inoculation on RPS11 downregulated plants as comparative control showed at down panel. Ponceaus staining of rubisco protein was used to monitor equivalence of protein loading.

Fig 5. Viral symptoms of LS-CMV infected on systemic leaves at 7 dpi TRV2-RPS11 N. benthamiana plants and TRV2 controls.

Symptom photos were taken at respectively 5 days, 7days, 15days after CMV mechanically infection. Mock was wild N. benthamiana plant 7 dpi TRV2-RPS11 without CMV infection.

Fig 6

Northern blot hybridization analysis of 7 days post-infection of CMV(A) and ToMV(B) on RPS11 downregulated plants. CMV and ToMV were mechanically infected on N. benthamiana 7 days after TRV2-RPS11 inoculation plants (lane 1–3) and TRV2 inoculation control plants (lane 4–6). Equal loading of lanes of the northern blot was checked by ethidium bromide staining revealing rRNA bands showed as lower panel.

Fig 7. The gene silencing suppressor activity of CMV2b protein was reduced by the RPS11 knockdown.

(A) Western blot hybridization analysis of gene silencing suppressor activity of CMVLS2b compared to P19 protein on RPS11 knockdown plants. Total protein samples were extracted 3 days post inoculation of gene silencing suppressor. (B) Further GFP silencing suppressor activity test of LS2b protein was performed with comparison of both P19 and homologous Fny2b proteins. Ponceaus staining of rubisco protein was used to monitor equivalence of protein loading. (C) Quantification for relative GFP accumulation of gene silencing suppressor affected on RPS11 knockdown plants and controls. (D) Comparison of each gene silencing suppressing activity on RPS11 knockdown plants and control. Each value is the mean of three replicates and vertical bars are SD. For a given parameter, means with different letters in C are statistically different at P<0.05. Asterisks in D indicate a significant difference between treatments and the control at P<0.01 (**) according to Student–Newman–Keuls test.