Fig. 5: Identification of the leafhopper sperm protein ubiquitin ligase E3 interacting with and negatively regulating Pelo expression.

a The accumulation levels of Pelo, Hbs1 and Pns11 in the testes of viruliferous male adults treated with MG132, as determined by western blot assay. b Y2H assays showing the interaction relationship for E3, Pelo, Hbs1, P8 or Pns11. Transformants were plated on QDO. QDO, SD/-Trp-Leu-His-Ade medium. c Pull-down assay showing the interaction between E3 and Pelo. Proteins were detected using GST or His antibody. d, e RT-qPCR and western blot assays showing the relative transcript (d) and accumulation (e) levels of E3 in different leafhopper organs. Means (± SD) in d represent three biological replicates. Different letters indicate a significant difference (P < 0.05, Tukey’s HSD multiple test). f, g The relative transcript (f) and accumulation (g) levels of E3 in nonviruliferous or viruliferous leafhopper testes, as determined by RT-qPCR and western blot assays. h, i Distribution of E3, Pelo, or Pns11 in the testes of nonviruliferous or viruliferous leafhoppers, as determined by immunofluorescence microscopy. The testes were immunolabelled with E3-FITC, E3-rhodamine, Pelo-FITC, Pelo-Alexa Fluor 647, or Pns11-rhodamine. Bars, 10 μm. j, k Localization of E3 on the sperms in the testes from nonviruliferous (j) or viruliferous (k) males, as determined by immunoelectron microscopy. Leafhopper testes were immunolabelled with E3-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. Bars, 100 nm. l, m RT-qPCR and western blot assays showing the relative transcript and accumulation levels of E3, Pelo and Hbs1 in the testes from nonviruliferous (l) or viruliferous (m) leafhoppers treated with dsE3 or dsGFP. Relative protein levels in (a, e, g, l), and (m) are quantified using ImageJ with H3 as a reference protein, and the data present three replicates, with each replicate containing 30 testes. Means (± SD) in (f, l), and (m) represent three replicates and are analysed using a two-tailed t test. Wb whole body, Sg salivary gland, Mg midgut, Ov ovary, S sperm, Te testis, Ts tubular structures, V⁻ nonviruliferous, V⁺ viruliferous.