Autophagy pathway induced by a plant virus facilitates viral spread and transmission by its insect vector
(A) At 4 days padp, ATG8 and SQSTM1 in nonviruliferous or viruliferous insects were detected by western blot assay using ATG8- and SQSTM1-specifc IgG, respectively. Insect ACTB was detected with ACTB-specific IgG as a control. ATG8-I was detected in both nonvirulifeorus and viruliferous insects, and ATG8-II was only detected in viruliferous insects. (B-D) Autophagosomes in nonviruliferous (B) or viruliferous (C, D) insects were detected by confocal microscopy. At 4 days padp, insect intestines were immunolabeled for autophagosomes with ATG8-FITC (green), for viral particles with P8-rhodamine (red), and for ACTB with ACTB dye phalloidin-Alexa Fluor 647 (blue), then examined with confocal microscopy. Panels II are enlargements of boxed areas in panels I. Single optical sections of epithelial side (C) and muscle side (D) of the intestines from viruliferous R. dorsalis are shown from a confocal Z-stack at 100 μm depth. Red arrows indicate colocalization of ATG8-specific autophagosomes and P8-specific viral particles. Bars, 30 μm. (E-J) RGDV infection induced autophagosome formation as measured by electron microscopy. (E) Representative images were shown for nonviruliferous intestinal epithelium. Panel II was an enlargement of the boxed area in panel I. (F) Viruliferous intestinal epithelium was immunolabeled with ATG8-specific IgG as the primary antibody, followed by treatment with 10-nm gold particle-conjugated goat antibodies against rabbit IgG as secondary antibodies. Red arrows indicate gold particles. (G-J) Transmission electron micrographs of virus-containing autophagosomes within the epithelial cytoplasm (G), along the microvilli (H), in the gut lumen (I), or in the visceral muscles tissues (J) of insect intestines. Insect in panel H was the enlargement of the boxed area. Red arrow indicated the double-membrane of virus-containing autophagosome. Vi, virion. CM, circular muscle. LM, longitudinal muscle. BL, basal lamina. Mv, microvilli. M, mitochondrion. N, nucleus. EC, epithelial cell. GL, gut lumen. Bar in panel E (I), 2 μm. Bars in panels E (II)-J, 500 nm. Bar in inset of panel F, 200 nm.