Fig. 2: RGDV Pns11 interacts with Pelo to mediate the spread of virus-induced tubules to target sperms.

a–d Electron microscopy showing the sperms associated with RGDV virions or virus-containing tubular structures in virus-infected testes., Testes in (b–d) were immunolabelled with Pns11-specific IgG as the primary antibody, followed by treatment with 15- nm gold particle-conjugated IgG as the secondary antibody. Panel (b–i) shows the enlarged image of the boxed area in panel (b). Red arrows indicate gold particles. Bars, 100 nm. e–h Immunoelectron microscopy showing the association of Pelo (e, f) or Hbs1 (g, h) with virus-induced tubules and sperms. Testes were immunolabelled with Pelo- or Hbs1-specific IgG as the primary antibody and treated with 15- nm gold particle-conjugated IgG as the secondary antibody. Red arrows indicate gold particles. Bars, 100 nm. i–m Immunofluorescence microscopy showing the association of RGDV Pns11 with Pelo or Hbs1 in infected male vas deferens (i–k) and testes (l and m). Dissected male reproductive organs from virus-free or positive males were immunostained with Pelo-FITC, Hbs1-FITC, or Pns11-rhodamine. Panels (j) and (k) are the enlarged images of the boxed areas in panel (i). Arrows in (j) and (k) indicate the colocalization of Pns11 and Pelo to the puncta structures. Ec, epithelial cells; Te, testes; Vd, vas deferens; S, sperm; AP, apical plasmalemma; Vi, virions; Ts, tubular structure. V⁻, nonviruliferous; V⁺, viruliferous. Bars, 10 μm. n Y2H assay showing the interactions between Pelo and Pns11 or P8, and between Hbs1 and Pns11 or P8. Transformants were plated on QDO. QDO, SD/-Trp-Leu-His-Ade medium. o Interaction between Pelo and Pns11 was detected by GST Pull-down assay. Pns11-GST was incubated with glutathione-Sepharose beads, and Pelo-His was then added to the beads, followed by a western blot assay to detect the Pelo-His bound to Pns11-GST.