Fig. 3: Pelo-Hbs1 promotes Pns11 tubule-mediated RGDV paternal transmission.

a Reduced Pns11-sperm binding by pre-treatment with Pelo antibody. Live sperms dissected from nonviruliferous testes were incubated with purified Pns11 proteins and pre-immune antibody or Pelo antibody, then stained with Pns11-rhodamine (red) and DAPI (blue). Bars, 10 μm. b The number of sperms associated with Pns11 was calculated as 100 sperms. c Paternal transmission rates of RGDV determined by mating nonviruliferous females with Pelo or pre-immune antibody-treated viruliferous males. Ten pairs of mating combinations were performed for three biological replicates. d, e The effects of dsPelo on the expression of Pelo, Hbs1, Pns11 and P8 in viruliferous males, as determined by RT-qPCR and western blot assays. f, g The effects of dsHbs1 treatment on the expression of Hbs1, Pelo, Pns11 and P8 in viruliferous males, as determined by RT-qPCR and western blot assays. Relative protein levels in (e) and (g) are quantified using ImageJ with H3 as a reference protein, and the data represent three replicates, with each replicate containing 30 testes. h The effects of dsPelo or dsHbs1 on the expression of Pelo, Hbs1, and Pns11 in viruliferous males, as determined by immunofluorescence microscopy. The dissected testes were immunostained with Pelo-Alexa Fluor 647 (blue), Hbs1-FITC (green), and Pns11-rhodamine (red). The right three panels are the enlarged images of the boxed areas in the left panels. Bars, 10 μm. i The mean percentages of sperms (n = 100) associated with Pns11 in testes. j Proposed mechanism of Pelo-Hbs1 complex-mediating paternal transmission of RGDV in testis. Pns11-Pelo interaction mediates the spread of virus-induced tubules from the testes epithelium into the sperm lumen for targeting sperm surfaces. Means (± SD) in (b, c, d, f), and (i) represent three replicates and are analysed using a two-tailed t test. Te testes, AP apical plasmalemma, BL basal lamina, EC epithelial cell, N nucleus, S sperm.