Figure 5. The Rice stripe virus capsid protein competes with Ubc13 in binding GPS2.

(A) Western blotting showing the phosphorylated JNK (P-JNK) when dsUbc13-RNA and mouse TNF-α were injected into the non-viruliferous fourth instar nymphs of the planthopper. Injections of dsGFP–RNA or 20 mM Tris-HCl containing 0.1% Tween 20 were used as controls. Relative transcript levels of Ubc13 were compared after three days of treatment. **p<0.01. (B) and (C) His-tag pull-down assay for the competitive binding of GPS2 by CP and Ubc13 using recombinantly expressed proteins. Recombinantly expressed GPS2-His was bound to Ni Sepharose as a bait for hooking Ubc13-GST at a series of dilutions of CP-GST. (D) His-tag pull-down assay for the competitive binding of GPS2 by CP and in vivo Ubc13. In vitro expressed GPS2-His was bound to Ni Sepharose. CP-GST or GST was applied to the GPS2-His bound Sepharose. Then, the total proteins from non-viruliferous planthoppers were applied to the Sepharose. (E) His-tag pull-down assay for the competitive binding of GPS2 by in vivo CP and in vivo Ubc13. Recombinantly expressed GPS2-His was bound to Ni Sepharose. Then the total proteins from viruliferous or non-viruliferous planthoppers were applied to the Sepharose. (F) His-tag pull-down assay for the interaction between CP-GST and the N1-His (nucleotides 1–465), N2-His (1-657), or N3-His (1-903) fragment of GPS2. The expression products from His vector (pET28a) and GST vector (pGEX-3X) were applied as negative controls. (G) His-tag pull-down assay for the interaction between the N1-His, N2-His, or N3-His fragment of GPS2 and the CP from the total protein of viruliferous planthoppers. The expression product from His vector (pET28a) was applied as negative control. (H) His-tag pull-down assay for the interaction between the N1-His, N2-His, N3-His, or C-His (nucleotides 886–1296) fragment of GPS2 and Ubc13-GST. The expression product from His vector (pET28a) was applied as negative control. Anti-phospho-human JNK2 polyclonal antibody, anti-human JNK2 polyclonal antibody, anti-human tubulin monoclonal antibody, anti-human Ubc13 monoclonal antibody, anti-CP monoclonal, anti-His monoclonal, and anti-GST polyclonal antibody were used to detect proteins. From (B) to (H), three independent replicates were carried out for each experiment. We show one representative result for each experiment here.

DOI: http://dx.doi.org/10.7554/eLife.26591.018

Figure 5—figure supplement 1. Western blot of the phosphorylated JNK (P-JNK) in response to TNF-α treatment.

(A) Western blotting. The non-viruliferous fourth instar planthoppers were injected with 23 nL of 50 μM mouse TNF-α. The control groups were injected with 23 nL of 20 mM Tris-HCl containing 0.1% Tween 20. Activation or inhibition of JNK were checked after 3 d of treatment. P-JNK, total JNK, and tubulin were detected using an anti-phospho-human JNK2 antibody, an anti-human JNK2 polyclonal antibody, and an anti-human tubulin monoclonal antibody, respectively. (B) The relative densities of P-JNKwere normalized with those of tubulin and are presented as mean ± SE. *p<0.05.

DOI: http://dx.doi.org/10.7554/eLife.26591.020

Figure 5—figure supplement 2. Densitometry analysis for the phosphorylated JNK (P-JNK) image bands from Figure 5A.

The relative densities of P-JNK were normalized with those of tubulin and are presented as mean ± SE. **p<0.01.

DOI: http://dx.doi.org/10.7554/eLife.26591.021

Figure 5—figure supplement 3. Positions of the five fragments, N1, N2, N3, N4, and C, within GPS2.

DOI: http://dx.doi.org/10.7554/eLife.26591.022

Figure 5—figure supplement 4. His-tag pull down assay for the interactions of GPS2 fragments with CP or Ubc13.

(A) C-His fragment (nucleotides 886–1296) of GPS2 and CP-GST. (B) N4-GST fragment (466-885) of GPS2 and CP-His. (C) N4-GST and Ubc13-His. The expression products from His vector (pET28a) and GST vector (pGEX-3X) were used as negative controls. Anti-His and anti-GST antibodies were used to detect the proteins with corresponding tags. Three independent replicates were carried out for each experiment. Here we show one representative result.

DOI: http://dx.doi.org/10.7554/eLife.26591.023