Identification of the amino acid residues and domains in the cysteine-rich protein of Chinese wheat mosaic virus that are important for RNA silencing suppression and subcellular localization

Abstract

Cysteine-rich proteins (CRPs) encoded by some plant viruses in diverse genera function as RNA silencing suppressors. Within the N-terminal portion of CRPs encoded by furoviruses, there are six conserved cysteine residues and a Cys-Gly-X-X-His motif (Cys, cysteine; Gly, glycine; His, histidine; X, any amino acid residue) with unknown function. The central domains contain coiled-coil heptad amino acid repeats that usually mediate protein dimerization. Here, we present evidence that the conserved cysteine residues and Cys-Gly-X-X-His motif in the CRP of Chinese wheat mosaic virus (CWMV) are critical for protein stability and silencing suppression activity. Mutation of a leucine residue in the third coiled-coil heptad impaired CWMV CRP activity for suppression of local silencing, but not for the promotion of cell-to-cell movement of Potato virus X (PVX). In planta and in vitro analysis of wild-type and mutant proteins indicated that the ability of the CRP to self-interact was correlated with its suppression activity. Deletion of up to 40 amino acids at the C-terminus did not abolish suppression activity, but disrupted the association of CRP with endoplasmic reticulum (ER), and reduced its activity in the enhancement of PVX symptom severity. Interestingly, a short region in the C-terminal domain, predicted to form an amphipathic α-helical structure, was responsible for the association of CWMV CRP with ER. Overall, our results demonstrate that the N-terminal and central regions are the functional domains for suppression activity, whereas the C-terminal region primarily functions to target CWMV CRP to the ER.

Figure 1

Silencing suppression activity of Chinese wheat mosaic virus (CWMV) cysteine‐rich protein (CRP) in Agrobacterium co‐infiltration assay. (A) Green fluorescent protein (GFP)‐transgenic Nicotiana benthamiana line 16c leaves were infiltrated with mixtures of Agrobacterium culture harbouring pBin‐GFP plus Agrobacterium harbouring the construct indicated [CWMV CRP, Potato virus Y (PVY) HC‐Pro or Tomato bushy stunt virus (TBSV) p19]. GFP fluorescence was visualized under long‐wavelength UV light and photographed 3, 4 and 5 days after inoculation (dai). (B) Northern blot analysis of GFP mRNA accumulation in agroinfiltrated patches of leaves shown in (A). The RNA gel was stained with ethidium bromide and 28S rRNA is shown as a loading control (bottom panel). (C) Northern blot analysis of GFP siRNA accumulation in agroinfiltrated patches of leaves shown in (A). The RNA gel was stained with ethidium bromide and 5S rRNA and tRNA are shown as loading control (bottom panel).

Figure 2

Silencing suppression activity of Chinese wheat mosaic virus (CWMV) cysteine‐rich protein (CRP) in a viral movement complementation assay. (A) Schematic representation of recombinant Potato virus X‐green fluorescent protein (PVX‐GFP) and P25 mutants used in trans‐complementation assay (not to scale). Broken lines represent the deleted region of the P25 gene (ΔP25). The P25 mutant (P25/T117A) which contains the substitution of tyrosine‐117 by alanine (marked with an asterisk) is defective in silencing suppression, but retains its virus movement function. 35S and Nos represent cauliflower mosaic virus (CaMV) 35S promoter and nopaline synthase terminator sequences, respectively. (B–D) Leaves of Nicotiana benthamiana plants infiltrated with mixtures of Agrobacterium cultures (1 : 1 : 1) harbouring pGR106:PVX(ΔP25)‐GFP (diluted 10 000‐fold), pBin‐P25/T117A and pBin‐p19, pBin‐HC‐Pro, pBin‐CRP or CRP mutants (see Fig. 3A). GFP fluorescence was observed using long‐wavelength UV light, and photographed (B) or observed using confocal laser scanning microscopy (C, D) at 5 days after inoculation (dai). Bars, 200 μm.

Figure 3

Effects of mutations in conserved amino acid residues and deletion of C‐terminal amino acids on silencing suppression activity and protein stability of Chinese wheat mosaic virus (CWMV) cysteine‐rich protein (CRP). (A) Amino acid sequence alignment of the CRPs encoded by furoviruses. The amino acid positions of CWMV CRP are indicated above the alignment. Dots represent gaps. Dark brown or light brown boxes indicate that amino acids are identical or chemically similar, respectively. Broken red rectangles indicate the predicted heptad repeats. Highly hydrophobic residues at positions a and d of the heptad repeat (abcdefg)_n are marked with asterisks. Amino acids that were substituted by alanine are marked with arrows and the C‐terminal ends of the deletion mutants are marked with arrowheads. (B) RNA silencing suppression activity of CWMV mutants in an Agrobacterium co‐infiltration assay. Leaves of Nicotiana benthamiana line 16c plants were infiltrated with mixtures of Agrobacterium harbouring pBin‐GFP plus Agrobacterium harbouring the binary vector containing the CWMV CRP mutants indicated in the images. Green fluorescent protein (GFP) fluorescence was visualized under UV light and photographed at 3 days after inoculation (dai). (C) Northern blot analysis of GFP mRNA accumulation in agroinfiltrated patches of leaves shown in (B). (D) Western blot analysis to detect the accumulation of wild‐type and mutants of CWMV CRP in insect cells. Coomassie blue (CB)‐stained total cell proteins are shown as loading controls (bottom panel). Antibody‐reactive bands of unknown origin are marked with asterisks.

Figure 4

Self‐interaction of wild‐type and mutants of Chinese wheat mosaic virus (CWMV) cysteine‐rich protein (CRP). (A, C) Bimolecular fluorescence complementation (BiFC) assay: leaves of Nicotiana benthamiana plants were infiltrated with mixtures of Agrobacterium harbouring constructs indicated above and on the left side of the images. Yellow fluorescent protein (YFP) fluorescence was observed using confocal laser scanning microscopy (CLSM) at 3 days after inoculation (dai). Bars, 30 μm. (B, D) Maltose‐binding protein (MBP) pull‐down assay: MBP‐CRP, MBP‐C20A, MBP‐L105A or free MBP (MBP) was mixed with glutathione S‐transferase (GST)‐CRP or unfused GST (GST) and an MBP pull‐down was performed (B). MBP‐ΔC40 was mixed with GST‐ΔC40 or unfused GST (GST) and an MBP pull‐down was performed (D). The purified proteins were analysed by Western blot using GST‐ and MBP‐specific antisera. An asterisk marks the signal from the degraded protein.

Figure 5

Subcellular distribution of wild‐type and deletion mutants of Chinese wheat mosaic virus (CWMV) cysteine‐rich protein (CRP). (A, B) Epidermal cells of Nicotiana benthamiana plants transiently expressing enhanced green fluorescent protein (EGFP) fused with CWMV CRP wild‐type or mutants. Panels in (B) show the perinuclear areas. (C) Subcellular distribution of red fluorescent protein (RFP) fused with CWMV CRP in epidermal cells of transgenic N. benthamiana line 16c expressing GFP. Top and bottom panels show the cytoplasmic and perinuclear areas, respectively. Fluorescent proteins and 4′,6‐diamidino‐2‐phenylindole (DAPI) staining were observed using confocal laser scanning microscopy (CLSM) at 3 days after inoculation (dai). Images are derived from single confocal sections. Bars: 20 μm (A); 10 μm (B, C).

Figure 6

Role of the C‐terminal amphipathic α‐helical region in the association of Chinese wheat mosaic virus (CWMV) cysteine‐rich protein (CRP) with endoplasmic reticulum (ER). (A) Secondary structure predictions of the C‐terminal 40 amino acids of CWMV CRP using psipred and Jpred3. The predicted structures are indicated as helical (h), strand (e) or undetermined (c, coil). (B) Linear and helical wheel projections of CWMV CRP amino acids 141–158. Colour coding indicates amino acid characteristics. Amino acids that were substituted by alanine are indicated by arrows. (C) A helical net projection of CWMV CRP amino acids 138–163. The potential hydrophobic patch is shaded. (D) Subcellular localization of enhanced green fluorescent protein (EGFP) fused to the C‐terminal 40 amino acids (134–173aa), the predicted α‐helical region (141–158aa) or its substitution mutant (141–158aa M154A/V157A) of CWMV CRP. GFP fluorescence was observed using confocal laser scanning microscopy (CLSM) at 3 days after inoculation (dai). Bars, 25 μm.

Figure 7

Effects of the expression of wild‐type and mutants of Chinese wheat mosaic virus (CWMV) cysteine‐rich protein (CRP) on Potato virus X (PVX) symptoms and accumulation. (A) Schematic representation of a PVX vector (pGR106 base) chimera carrying CWMV CRP or mutant genes (not to scale). (B) Nicotiana benthamiana plants infected with PVX vector chimeras carrying the wild‐type, antisense (as) or mutants (C20A, ΔC9, ΔC19 and ΔC40) of CWMV CRP. Plants were photographed at 14 days after inoculation (dai). (C) Western blot analysis of PVX coat protein (CP) accumulation in the upper systemically infected leaves of N. benthamiana. Total protein was extracted from leaves at 10 dai. Coomassie blue (CB)‐stained total cell proteins are shown as loading controls (bottom panel).