Fig. 1: Intact NcVg release from salivary gland to phloem for inhibiting H₂O₂ burst in rice plants.

a Domains and subunit composition of NcVg. b NcVg cleavage in organs as well as release to rice plant, as determined by western blot analysis. WB, whole body. Ov, ovary. Sg, salivary gland. Nf, non-feeding. c Y2H assays showing interaction of NcVg2 with NcRab5. +, positive control; –, negative control. QDO, SD/-Trp-Leu-His-Ade medium. d GST pull-down assays showing interaction of NcVg2 with NcRab5. e Distribution of NcVg and NcRab5 in cavities and cytoplasm of salivary gland, as determined by immunofluorescence microscopy. The schematic illustration of type III- to VI-cells in the salivary gland was shown. Panel i is the enlarged image of the boxed area. Panel showing green fluorescence (NcVg antigens), red fluorescence (NcRab5 antigens), or bright field is corresponding to panel i. APL, apical plasmalemma; Cv, cavity; SD, salivary duct. Bars, 10 μm. f Western blot assays of NcVg and NcRab5 in insect bodies and rice plants. g Distribution of NcVg and NcRab5 in rice phloem, as determined by immunofluorescence microscopy. Panels ii is enlarged image of the boxed area. AS, air space; BSC, Bundle sheath cell; EC, Epidermis cell; P, phloem; PV, pitted vessel; SC, Sclerenchyma cell; ST, sieve tube. Bars, 10 μm. h, i and j Knockdown of NcRab5 or NcVg expression reducing NcVg and NcRab5 accumulation in salivary glands and release to rice. Relative intensities of bands in western blot assays are shown. k Knockdown of NcVg expression inducing H₂O₂ burst and metabolism in rice plants, as determined by the content of H₂O₂ and MDA, as well as CAT and POD activity. l and m Knockdown of NcVg expression increasing the accumulation of H₂O₂ in feeding holes, as determined by DAB or H2DCFDA staining. The mean number of feeding holes per cm² of leaves are shown in m. Panels iii and iv are the enlarged images of the boxed areas. Bars, 200 μm. n Knockdown of NcVg expression increasing feeding difficulty of leafhoppers, as determined by EPG technique. Each dsNcVg- or dsGFP-treated leafhopper was continuously and electrically recorded during 3-hour feeding periods. Data in b, d, e, f, g, h, i, j, k, l and m represent at least 3 biological experiments. Data in n represent 13 valid biological replicates. Means ( ± SD) in i, k, m, and n are analyzed using two-tailed t-test. Ns, not significant.