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. 2020 Nov 10;11(6):e01142-20. doi: 10.1128/mBio.01142-20

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Copyright © 2020 Mao et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.

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FIG 4 — Interaction of NcVg with Nasuia porin. (A) Interactions between NcVg1, NcVg2, NcVg3, or NcVg4 with Nasuia porin or Sulcia OMP in the yeast two-hybrid system. DDO, SD-Trp-Leu; QDO, SD-Trp-Leu-His-Ade. (B) In vitro pulldown analysis of interaction of NcVg2 with Nasuia porin or Sulcia OMP. Nasuia porin or Sulcia OMP was fused with GST as the bait protein. NcVg2 was fused with His as a prey protein. GST or purified GST proteins were used as controls. (C) Ncprp expression in different tissue parts (head, thorax, abdomen, or bacteriome) of N. cincticeps as revealed by Western blot analysis. (D to F) RT-qPCR assay showed that dsNcprp treatment significantly decreased the expression level of Ncprp (D) and reduced the abundance of Nasuia (E) but had no significant effect on the abundance of Sulcia in N. cincticep (F). (G) The reduction of Nasuia movement into the epithelial plug of adult female leafhoppers after dsNcprp treatment. The female ovaries from 50 dsNcprp- or dsgfp-treated adult female leafhoppers were stained with Sulcia-cy5, Nasuia-cy3, and NcVg-FITC and observed by confocal microscopy. The number of positive cy3 fluorescent spots in each ovary was calculated to measure the population of Nasuia. (H) NcVg and Nasuia porin protein levels decreased but Sulcia OMP levels remained constant in dsNcprp-treated female leafhoppers as revealed by Western blot assay. The protein accumulation levels of NcVg, Nasuia porin, and Sulcia OMP in N. cincticep that received dsgfp were taken to be 1.00. (I) Confocal microscopy showed the reduced accumulation of Nasuia and NcVg but the normal accumulation of Sulcia in the epithelial plug of the oocyte in dsNcprp-treated female leafhoppers. The female ovarioles were stained with Sulcia-cy5 (blue), Nasuia-cy3 (red), and NcVg-FITC (green). Bars, 50 μm. All images are representative of at least three replicates. Data in panels D to G are presented as means ± SD of three independent experiments. The significance of any differences was tested using independent t test. *, P < 0.05; **, P < 0.01; NS, not significant.