Fig. 2: RDV hijacking NcVg then releasing together from salivary glands to rice phloem via exosomal pathway.

a, b RDV infection inducing NcVg, NcRab5, and NcRab27a expression in salivary glands and release to rice plants, as determined by RT-qPCR a and western blot b assays. c RDV colocalizing with NcVg and inducing colocalization of NcVg with NcRab5 or NcRab27a in cavities and cytoplasm of salivary gland, as determined by immunofluorescence microscopy. APL, apical plasmalemma. Cv, cavity. Bars, 10 μm. The mean number of NcVg colocalizing with NcRab5 or NcRab27a in uninfected or infected salivary gland are shown in c-ii. Fifteen random 20 × 20 μm fields of samples from infected or uninfected salivary glands were examined. V + , infected salivary glands. V-, uninfected salivary glands. d Immunoelectron microscopy showing the localization of NcVg at RDV-packaging exosomes within the cytoplasm and cavities of salivary glands. Salivary glands of leafhoppers were immunolabeled with NcVg-specific IgG as the primary antibody, followed by treatment with 15-nm gold particle-conjugated IgG as the secondary antibody. Panels iii to iv are the enlarged images of the boxed areas in d-i. Red arrows indicate gold particles. Bar, 200 nm. The mean number of vesicles associated with NcVg antibody in cavities per section of one type III-cell of one uninfected or infected salivary gland are shown in d-ii. Fifteen random samples from infected or uninfected salivary glands were examined. e Colocalization of NcVg with RDV, NcRab5, and NcRab27a in rice phloem, as determined by immunofluorescence microscopy. Panels v to viii are the enlarged images of the boxed areas in e. P, phloem; AS, air space; PV, pitted vessel; ST, sieve tube. Bars, 10 μm. f, g Knockdown of NcRab27a expression f or treatment of GW4869 g reducing NcVg and NcRab27a accumulation in and release from salivary glands of nonviruliferous leafhoppers. Relative intensities of bands in western blot assays are shown. Data in a, b, c-ii, e, d-ii, f and g represent at least 3 biological replicates. Means ( ± SD) in a, c-ii and d-ii are analyzed using two-tailed t-test.