A furoviral replicase recruits host HSP70 to membranes for viral RNA replication

Abstract

Many host factors have been identified to be involved in viral infection. However, although furoviruses cause important diseases of cereals worldwide, no host factors have yet been identified that interact with furoviral genes or participate in the viral infection cycle. In this study, both TaHSP70 and NbHSP70 were up-regulated in Chinese wheat mosaic furovirus (CWMV)-infected plants. Their overexpression and inhibition were correlated with the accumulation of viral genomic RNAs, suggesting that the HSP70 genes could be necessary for CWMV infection. The subcellular distributions of TaHSP70 and NbHSP70 were significantly affected by CWMV infection or by infiltration of RNA1 alone. Further assays showed that the viral replicase encoded by CWMV RNA1 interacts with both TaHSP70 and NbHSP70 in vivo and vitro and that its region aa167-333 was responsible for the interaction. Subcellular assays showed that the viral replicase could recruit both TaHSP70 and NbHSP70 from the cytoplasm or nucleus to the granular aggregations or inclusion-like structures on the intracellular membrane system, suggesting that both HSP70s may be recruited into the viral replication complex (VRC) to promote furoviral replication. This is the first host factor identified to be involved in furoviral infection, which extends the list and functional scope of HSP70 chaperones.

Figure 1

CWMV infection induced the accumulation of heat shock protein 70s (HSP70s) (A) and inhibition of HSP70s by quercetin treatment reduced the accumulation of CWMV RNAs in plants (B). (A) Western-blotting analysis of HSP70 in CWMV-infected Nicotiana benthamiana (top line) and Triticum aestivum (bottom line) using an anti-HSP70 antibody. Rubisco stained with Coomassie brilliant blue (CBB) was used for the loading controls. (B) Northern blotting assay of CWMV RNA accumulation in N. benthamiana plants treated with various concentrations of quercetin. Ethidium bromide (EtBr)-stained rRNAs are shown as the loading controls. Treatment with DMSO was chosen as the control. sgRNA: subgenomic RNA. (C) Symptoms developed at 4 dpi on quercetin- and DMSO-treated N. benthamiana plants.

Figure 2

Expression levels of heat shock protein 70 (HSP70) transcripts in CWMV-infected Triticum aestivum (A) and Nicotiana benthamiana (B) plants. Experiments were repeated at least three times. Bars represent the standard errors of the means. Three sample unequal variance directional t-test was used to test the significance of the difference (*p < 0.05).

Figure 3. Over-expression of TaHSP70 enhanced the accumulation of CWMV RNAs in N. benthamiana plants.

Total RNAs were extracted from the inoculated leaves 4 dpi after co-inoculation with 35S:TaHSP70 and CWMV RNAs by Agrobacterium infiltration. Accumulation of CWMV RNAs was analyzed by Northern blotting. Ethidium bromide (EtBr)-stained rRNAs are shown as loading controls. Inoculation with water and empty vector (35S:00) was used as the negative control. Three replicate plants were used for each treatment.

Figure 4. Expression levels of NbHSP70 are associated with the accumulation of CWMV RNAs in N.benthamiana plants.

(A) Symptoms in N. benthamiana plants infiltrated with the tobacco rattle virus (TRV) vector harboring a partial fragment of N. benthamiana HSP70 (TRV:NbHSP70). The empty vector (TRV:00) was infiltrated and used as a control. Pictures were taken at 7 dpi. (B) qPCR analysis showing the expression levels of NbHSP70 in the N. benthamiana infiltrated with TRV:NbHSP70 compared with the control (TRV:00). Experiments were repeated three times. Bars represent the standard errors of the means. (C) Accumulation of CWMV RNAs analyzed by Northern blotting. Total RNAs were extracted from the inoculated leaves at 4 dpi. Ethidium bromide (EtBr)-stained rRNAs are shown as loading controls. (D) Accumulation of CWMV RNA1 analyzed by Northern blotting using an RNA1-specific probe. Lane 1, the total RNA extracted from a plant inoculated with CWMV RNA 1 and RNA 2. Lane 2, the total RNA extracted from the mock inoculated N. benthamiana. Lane 3, the total RNA extracted from the silencing plants co-inoculated with CWMV RNA1 and 35S:NbHSP70. Lane 4, the total RNA extracted from the control plants inoculated with TRV:00 and CWMV RNA1. Lane 5, the total RNA extracted from the NbHSP70 silenced plants inoculated with CWMV RNA1. Lane 6, the total RNA extracted from the plants inoculated with CWMV RNA1 only. Total RNAs were extracted from the inoculated leaves 4 dpi after inoculation. Ethidium bromide (EtBr)-stained rRNAs are shown as loading controls.

Figure 5. Localization of TaHSP70 or NbHSP70 was affected by CWMV infection.

GFP fluorescence in healthy (Mock) or CWMV-infected N. benthamiana leaf epidermal cells agroinfiltrated with pCV-GFP-N1 (A), pCV-TaHSP70-GFP (B) and pCV-NbHSP70-GFP (C), respectively. The results were observed 72 h after infiltration. Scale bar, 50 μm.

Figure 6. Interactions of TaHSP70 or NbHSP70 with CWMV replicase or MP in the yeast two hybrid system.

(A) Seven truncated mutants, covering the three conserved domains (Met, Hel and RdRp) of the CWMV replicase, were designed for assays. The numbers denote CWMV replicase amino acid positions. The ability of CWMV replicase fragments to interact with TaHSP70 or NbHSP70 in YTH assays is shown on the right (+, positive; −, negative). (B) Yeast colonies expressing BD-TaHSP70 with AD-Rep^1–670 and AD-Rep^167–333 grew well on the selective medium, but those expressing AD-Rep^670–1430, AD-Rep^1430–1840, AD-Rep^1–167, AD-Rep^333–670 or AD-MP with BD-TaHSP70 did not. (C) Yeast colonies expressing BD-NbHSP70 with AD-Rep^1–670 and AD-Rep^167–333 grew well on the selective medium, but those expressing AD-Rep^670–1430, AD-Rep^1430–1840, AD-Rep^1–167, AD-Rep^333–670 or AD-MP with BD-NbHSP70d did not. Yeast co-transformed with BD-53 and AD-T, and BD-Lam and AD-T were used as the positive and negative controls, respectively.

Figure 7. Interaction between the N-terminus of CWMV replicase and TaHSP70 or NbHSP70 in vivo and vitro.

(A) Visualization of the interaction between Rep^1–333 and TaHSP70 or NbHSP70 in N. benthamiana epidermal cells by BiFC assay. N. benthamiana leaves were co-infiltrated with recombinant BiFC vectors containing the constructs indicated above the images. The results were observed 48 h after infiltration. Scale bar, 50 μm. The fluorescent and merged images are depicted in the upper and lower panels, respectively. (B,C) Interaction of CWMV Rep^1–333 with TaHSP70 and NbHSP70 proteins in ELISA-based binding assays. Wells of a microtiter plate were coated with 25 pmol of E. coli purified GST-tagged Rep^1–333 protein and incubated with increasing amounts of E. coli purified 6×-histidine-tagged TaHSP70 protein (▲) or 6×-histidine-tagged NbHSP70 protein (▼), respectively. Retention of the complex was detected with polyclonal anti-His antibodies. The recombinant 6×-histidine tag was used alone as a control (■). Experiments were repeated three times. Bars represent the standard errors of the means.

Figure 8. Localization of TaHSP70 or NbHSP70 was affected by expression of the N-terminus of CWMV Rep^1–1350.

(A) Sub-cellular localization of CWMV Rep^1–1350 fused with GFP or mCherry in N. benthamiana leaf epidermal cells. (B) Epidermal cells of N. benthamiana transiently co-expressing Rep^1–1350-mCherry and TaHSP70-GFP or NbHSP70-GFP. The results were observed 72 h after infiltration. Scale bar, 25 μm.

Figure 9

Subcellular fractionation assays of TaHSP70 and CWMV replicase or its truncated mutants expressed alone (A) or together (B) in N. benthamiana leaf epidermal cells. C, cytoplasm fraction; N, nuclear fraction; M, membrane fraction.