Fig. 3

PMMoV infection activates autophagy. Quantitative RT-PCR analyses of NbVPS15, NbATG3, NbATG5, NbBeclin1, NbATG7, NbATG8a, NbATG8f, and NbATG9 RNA transcript levels in the inoculated leaves at 5 dpi (a) and upper systemic leaves at 10 dpi (b) of Mock- and PMMoV-inoculated N. benthamiana plants, respectively. The expression level of NbActin was used as the internal control. c Confocal micrographs showing leaves of the PMMoV- and Mock-inoculated N. benthamiana plants. The leaves were infiltrated with an Agrobacterium culture harboring the pGD-GFP-NbATG8a plasmid and imaged at 48 h post infiltration. Numerous green fluorescence punctate were found in cells in a PMMoV-infected leaf. Bars = 30 μm. d Average number of green fluorescence GFP-NbATG8a punctate per 10 cells. This experiment was repeated three times and a total of 30 cells were counted for the punctate. The values represented are the mean punctate ± SD per 10 cells. e Total protein was isolated from young non-inoculated leaves harvested from the plants inoculated with PMMoV or PB at 3, 5 and 10 dpi, and then analyzed through Western blot using an anti-ATG8 or an anti-Rubisco antibody. The original data can be viewed from Additional file 11: Figure S4b-c. f Transmission electron microscopic analysis using leaf sections prepared from PMMoV- and PB (Mock)-inoculated N. benthamiana plants after 100 μM E64d treatment at 5 dpi. Autophagic structures (red arrows) were observed in a PMMoV-infected cell. Cp, chloroplast; CW, cell wall; S, starch; V, vacuole. Bars = 2 μm. g Average number of double-membrane autophagosomes ± SD in 15 cells per each treatment. The experiment was repeated twice