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. 2019 Feb 27;10:955. doi: 10.1038/s41467-019-08860-4

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© The Author(s) 2019

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 4 — Interaction of RGDV P8 with HSPG. a Live sperms were incubated with purified virions for 5, 30, or 90 min, then stained with virus-rhodamine (red) and DAPI (blue). b Structure of RGDV virion. c Live sperms were incubated with purified virions, P8 or P2, then stained with virus-, P8-, or P2-rhodamine (red) and DAPI (blue). d Live sperms were incubated with purified virions and RGDV-, P8-, or P2-specific antibodies, then stained with virus-rhodamine (red) and DAPI (blue). e Schematic representation of R. dorsalis HSPG gene. f Interactions between different domains of HSPG and RGDV P8 in the yeast two-hybrid system. SD-2: SD-Trp-Leu, SD-4: SD-Trp-Leu-His-Ade. g Pull-down analysis of RGDV P8 interaction with HSPG DoIII. HSPG DoIII was fused with GST as a bait protein. P8 was fused with His as a prey protein. P2 and GFP were fused with His as controls. h, i The transcript h or protein i levels of HSPG in reproductive systems of V⁻ or V⁺ male adults. j Confocal micrographs showing colocalization (white triangles) of RGDV (red) and HSPG (green) on sperm head (blue) surface. k Live sperms were incubated with purified virions and 3% BSA, pre-immune serum, purified HSPG DoIII, or HSPG-specific antibodies, then stained with virus-rhodamine (red) and DAPI (blue). l The reduced colocalization (white triangles) of RGDV (red) and HSPG (green) on the surface of sperm heads (blue) after dsHSPG treatment. m, n The transcript m or protein n levels of HSPG and RGDV P8 in male reproductive systems after dsGFP or dsHSPG treatments. o Paternal transmission rates of RGDV from crosses between dsHSPG- or dsGFP-treated V⁺ males with V⁻ females. Eggs were collected for tracing RGDV. p Proposed model for interaction of RGDV P8 with HSPG mediating viral attachment on the surface of sperm heads and subsequent paternal transmission. All images are representative of at least tree replicates. h, m, and o Data are presented as mean ± SD from three independent experiments (Student’s t test, two tailed); ^*P < 0.05, ^**P < 0.01. V⁺, viruliferous. V⁻, nonviruliferous. Ab, antibodies. PM, plasma membrane. N, nucleus. Bars, 5 μm