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Autophagy pathway induced by a plant virus facilitates viral spread and transmission by its insect vector

Fig 3

Autophagy pathway induced by RGDV infection facilitated viral nonlytic release from VCMs of R. dorsalis.

(A) Percentage of insect vector cells infected with RGDV. The VCMs were transfected for 8 h with rapamycin or 3-MA (panels I) or dsAtg5 or dsTorc1 (panels II), then inoculated with RGDV at a MOI of 0.1 for 2 h. At 48 hpi, cells were immunolabeled with ATG8-FITC (green) and P8-rhodamine (red), then examined with confocal microscopy. Arrows indicate the colocalization of ATG8-specific autophagosomes and viral particles. Data points presented are the averages from six different fields (A). Bars, 20 μm. (B) At 48 and 72 hpi, effects of rapamycin or 3-MA (panels I) and dsAtg5 or dsTorc1 (panels II) on transcript levels of RGDV P8 gene in VCMs as revealed by RT-qPCR assay. Means (±standard deviation [SD]) from three biological replicates are shown. *P < 0.05. (C) At 48 hpi, effects of rapamycin or 3-MA (panels I) and dsAtg5 or dsTorc1 (panels II) on transcript levels of Atg8 gene in VCMs as revealed by RT-qPCR assay. Means (±SD) from three biological replicates are shown. *P < 0.05. (D) The accumulation levels of ATG8 and SQSTM1 were detected by western blot assay using ATG8- and SQSTM1-specifc IgG, respectively. Insect ACTB was detected with ACTB-specific IgG as a control. (E) Autophagy induced by viral infection increased the extracellular viral RNA levels. VCMs were transfected for 8 h with dsRNAs, then inoculated with RGDV at a MOI of 10 for 2 h. At 48 and 72 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.

Fig 3

doi: https://doi.org/10.1371/journal.ppat.1006727.g003