Fibrillar structures induced by a plant reovirus target mitochondria to activate typical apoptotic response and promote viral infection in insect vectors
Fig 4
Pns11 of RGDV specifically interacted with VDAC on the outer membrane of mitochondria.
(A) RGDV Pns11 specifically interacted with VDAC in Y2H assays. For the Gal-4 based Y2H assay, transformants on plate of SD/Trp-Leu-His-Ade medium are labeled as follows: +, Positive control, i.e., pGBKT7-53/ pGADT7-T;–, negative control, i.e., pGBKT7-Lam/pGADT7-T; Pns11+VDAC, pGBKT7-Pns11/pGADT7-VDAC. Serially diluted yeast cultures appeared blue in the β-galactosidase assay. For the DUALmembrane Y2H assay, transformants on plate of SD/Trp-Leu-His-Ade medium are labeled as follows: +, positive control (pTSU2-APP/pNubG-Fe65);–, negative control (pTSU2-APP/pPR3-N); Pns11+VDAC, pBT-STE-Pns11/ pBT3-N-VDAC. Serially diluted yeast cultures appeared blue in the β-galactosidase assay. (B) GST pull-down assay demonstrated the interaction of Pns11 with VDAC. Protein GST and recombinant protein GST-Pns11 were respectively incubated with cell lysate expressing protein His-VDAC. Pull-down products were detected using Western blotting. An antibody against GST was used to detect GST and GST-Pns11; an antibody against His was used to detect bound proteins. (C) RT-qPCR assay of relative expression of VDAC and RGDV Pns11 genes in R. dorsalis cells after dsRNA treatment. The dsRNA-treated cells were inoculated with purified RGDV (MOI of 0.1), then harvested at 48 hpi. Means (±SD) from three biological replicates are shown. *P < 0.05, **P < 0.005. Data were analyzed with a two-tailed t-test in GraphPad Prism 7. (D) Immunofluorescence assay showing that knockdown of VDAC expression decreased the number of Pns11 fibrillar structures at 48 hpi. Mock- or RGDV-infected cells after treatment with dsGFP or dsVDAC were immunostained with Pns11-FITC (green) and MitoTracker (red). His-Mg buffer-treated cells served as mock controls. Bars, 10 μm. (E) Immunogold labeling of Pns11 on fibrillar structures (red arrows) in dsGFP- or dsVDAC-treated infected cultured cells of R. dorsalis at 48 hpi. Yellow arrows mark gold particles. Mit, mitochondria. Bar, 200 nm.