(A) Schematic representation of the structures of RDV particle and P2 protein. P2 is composed of an L-shaped ﬂexible structure with a 15 nm domain (aa 1–688, P2N) toward the exterior, and a 10 nm domain (aa 689–1149, P2C) on the viral envelope. (B) RDV P2N, but not RDV P2C or P8, specifically interacted with Rab5 in a yeast two-hybrid assay. Transformants on SD/-Trp-Leu-Ade-His plates are labeled as follows: +, positive control, i.e., pGBKT7-53/pGADT7-T; –, negative control, i.e., pGBKT7-Lam/pGADT7-T; Rab5 +P8, pGADT7-Rab5/pGBKT7-P8; Rab5 +P2C, pGADT7-Rab5/pGBKT7-P2C; Rab5 +P2N, pGADT7-Rab5/pGBKT7-P2N. Yeast cultures appeared blue in the β-galactosidase assay. (C) GST pull-down assay demonstrating the interaction of P2N with Rab5. Rab5 or RDV P8 fused with GST served as the bait, and GST alone served as the control. P2N or Rab5 fused with His served as the prey. The bait protein or the GST control were incubated with cell lysate expressing the His-fused protein. Input and pull-down samples were probed with antibodies against GST or His for western blot assays. (D) Immunogold labeling of Rab5 on exosomes in the salivary cavities. The salivary glands from nonviruliferous (panel I) or viruliferous (panel II) leafhoppers were immunostained with Rab5-specific IgG as primary antibody, followed by treatment with 15 nm gold particle-conjugated goat antibody against rabbit IgG as secondary antibody. Arrows indicate gold particles. Ex, exosome; VEx, virus-containing exosome. Bars, 100 nm. (E) Immunoﬂuorescence assay showing the distribution of Rab5 during virus infection in the salivary glands of N. cincticeps. Virus-infected or uninfected salivary glands of N. cincticeps were fixed, immunostained with virus-FITC (green), Rab5-rhodamine (red), and actin dye phalloidin-Alexa Fluor 647 carboxylic acid (blue). Immunostained salivary glands were then processed for immunofluorescence microscopy. Arrows show the colocalization puncta in the salivary cavities. APL, apical plasmalemma; Cv, cavity. Bars, 10 μm. (F) The mean number of Rab5-positive puncta in infected or uninfected salivary glands. Twenty random 70 × 70 μm fields of type III cell samples from infected or uninfected salivary glands were examined. (G) RT-qPCR assay showing the transcript levels of Rab5 in the salivary glands of viruliferous or nonviruliferous leafhoppers. RT-qPCR results were normalized against the actin expression level, and the transcript levels of Rab5 in the salivary glands of nonviruliferous leafhopper were normalized as 1. Means (± SD) from three biological replicates are shown in F and G. *p<0.05; **p<0.01. (H) Western blot assay showing the expression levels of Rab5 in the salivary glands of viruliferous or nonviruliferous leafhoppers. The relative intensities of bands in the analyses of Rab5 and RDV P8 are shown below. Coomassie-blue-stained gels demonstrated the loading of equal amounts of proteins. Data shown here are representative of three biological replicates. (I) RT-qPCR assay showing the transcript levels of RDV P8 and Rab5 in the salivary glands of dsRab5- or dsGFP-treated insects. Results are normalized against the actin expression level. The relative expression levels of the genes in individual insects are shown in a dot plot, with the middle line representing the mean value and the top and bottom lines representing the SD. *p<0.05. (J) Western blot assay showing the expression levels of RDV P8 and Rab5 in the salivary glands of dsRab5- or dsGFP-treated insects. The relative intensities of bands are shown below. Coomassie-blue-stained gels demonstrated the loading of equal amounts of protein. Data shown here are representative of three replicates. V-, nonviruliferous insects; V+, viruliferous insects.