Autophagy pathway induced by a plant virus facilitates viral spread and transmission by its insect vector
(A, B) At 48 hpi, mock- or virus-infected VCMs were immunolabeled for autophagosomes with ATG8-FITC (green), for viral particles with P8-rhodamine (red), and for viroplasms with Pns12-Alexa Fluor 647 (blue), and then processed for confocal microscopy. Red arrows indicate colocalization of ATG8-specific autophagosomes and P8 antigens of RDV. Data points presented are the averages from six different fields. Bars, 10 μm. (C) The average number of ATG8-specific puncta per N. cincticeps cell and a minimum of 100 cells were counted. *P < 0.05. (D-F) Transmission electron micrographs for virus-induced autophagosomes in VCMs. (D) Representative images were shown for mock VCMs. Panel II was an enlargement of the boxed area in panel I. Bars, 500 nm. (E) Electron microscopy showed the presence of virus-containing autophagosomes in the cytoplasm. (F) Immunoelectron micrographs of virus-containing autophagosomes positive for ATG8. VCMs were immunolabeled with ATG8-specific IgG as the primary antibody, then treated with 15-nm gold particle-conjugated goat antibodies against rabbit IgG as secondary antibodies. Red arrows indicate gold particles. Bars in panels E-F, 200 nm. (G) The quantification of the average number of autophagosome-like vesicles per N. cincticeps cell and a minimum of 30 cells were counted. *P < 0.05. (H) At 48 hpi, ATG8 and SQSTM1 in VCMs treated with (+) and without (-) 3-MA were detected by western blot assay, respectively. Insect ACTB was detected with ACTB-specific IgG as a control. (I) Autophagy induced by viral infection increased the extracellular viral RNA levels. VCMs were transfected for 8 h with 3-MA or rapamycin, then inoculated with RDV at a MOI of 10 for 2 h. At 48 hpi, culture supernatant was collected to measure the viral titers detected by RT-qPCR assay. Means (±SD) from three biological replicates are shown. *P < 0.05. (J) At 48 hpi, the accumulation levels of ATG8 or SQSTM1 in 3-MA- or rapamycin-treated VCMs were analyzed by western blot assay. Insect ACTB was detected with ACTB-specific IgG as a control. M, mitochadria. N, nucleus. Vi, virion.