Fig. 2: Sperm-specific HongrES1 of R. dorsalis interacts with RdFV CP or RGDV P8. | Nature Communications

Fig. 2: Sperm-specific HongrES1 of R. dorsalis interacts with RdFV CP or RGDV P8.

From: Arboviruses and symbiotic viruses cooperatively hijack insect sperm-specific proteins for paternal transmission

Fig. 2

a Y2H assays showing interaction of HongrES1 with RdFV CP or RGDV P8. Transformants on QDO plates are labeled as follows: HongrES1, pPR3-N-HongrES1 / pDHB1-CP or -P8; vector, pPR3-N / pDHB1-CP or -P8; + , positive control, i.e., pLargeT/p53; –, negative control, i.e., pDHB1 / pRR3N. β-galactosidase assay was detected in Z buffer with X-Gal. QDO, SD/-Trp-Leu-His-Ade medium. b GST pull-down assays showing interaction of HongrES1 with RdFV CP or RGDV P8. c, d HongrES1 expression levels in different organs of 30 RdFV-positive leafhoppers, as determined by RT-qPCR (c) and western blot (d) assays, respectively. Means (± SD) are shown from three biological replicates (two-tailed t-test). Ns, not significant. WB, whole bodies. ♂RO, male reproductive organs. ♀RO, female reproductive organs. AC, alimentary canals. SG, salivary glands. Res, residues. e Localization of HongrES1 on sperm surface in male testis, as determined by immunoelectron microscopy. Testes of leafhoppers were immunolabeled with HongrES1-specific IgG as the primary antibody, followed by treatment with 15-nm gold particle-conjugated IgG as the secondary antibody. Red arrows indicate gold particles. Bar, 100 nm. Effect of RdFV or RGDV infection on the expression of HongrES1 in male reproductive organs from 30 RdFV-free, RdFV-positive, or RdFV and RGDV co-positive leafhoppers, as determined by RT-qPCR (f) and western blot (g) assays. Means (±SD) are shown from at least three biological replicates (two-tailed t-test). The proteins were detected by using HongrES1 or histone H3 antibody in western blot assays. Data in d and g represent three biological replicates. h Schematic illustration of male reproductive system of R. dorsalis. Ag, accessory gland. S, sperm. Sv, seminal vesicle. Te, testis. Vd, vas deferens. i Distribution of HongrES1 in RdFV-free or positive testes, as determined by immunofluorecense microscopy. The testes dissected from 30 RdFV-free or positive males were immunostained with HongrES1-FITC (green). S, sperm. Te, testis. Bars, 10 μm.

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