Autophagy pathway induced by a plant virus facilitates viral spread and transmission by its insect vector
(A, B) At 48 hpi, ATG8 in mock- or virus-infected VCMs treated with (+) and without (-) 3-MA or BFA was detected by western blot assay using ATG8-specifc IgG. Insect ACTB was detected with ACTB-specific IgG as a control. (C-E) Autophagosomes in mock (D) and virus-infected (E) VCMs were detected by immunofluorescence microscopy. At 48 hpi, VCMs were immunolabeled for autophagosomes with ATG8-FITC (green), for viral particles with P8-rhodamine (red), and for viroplasms with Pns9-Alexa Fluor 647 (blue), and then processed for immunofluorescence microscopy. Viral antigens surrounding the spherical structures composed of Pns9, a component of viroplasms, are indicated by arrowheads. Arrows indicate the colocalization of ATG8-specific autophagosomes and viral particles. The average number of ATG8-specific puncta per R. dorsalis cell and a minimum of 100 cells were counted (C), and data points presented are the averages from six different fields (D, E). *P < 0.05. Bars, 15 μm. (F-L) Transmission electron micrographs for virus-induced autophagosomes in VCMs. Representative images were shown for mock VCMs (F), and for virus-containing autophagosomes in the cytoplasm (G) or at the periphery of viroplasm at 48 hpi (H). Panel II is an enlargement of the boxed area in panel I. Red arrows in panel G indicate double-membrane of autophagosomes. Red arrow in panel H indicates single-membrane of autophagosomes. (I) Virus-infected VCMs at 48 hpi were 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. Panel II is an enlargement of the boxed area in panel I. Arrows indicate gold particles. (J-L) At 48 hpi, virus-containing autophagosomes inside (J), at the periphery (K), or outside (L) of infected cells. (M) The quantification of the average number of autophagosome-like vesicles per R. dorsalis cell and a minimum of 30 cells were counted. *P < 0.05. M, mitochondrion. N, nucleus. Vi, virion. VP, viroplasm. Bars in panel F (I) and (II), 500 nm. Bars in panels G-L, 200 nm.