Figure 4

Phenotypes of programmed cell death. A Representative light micrographs of the abaxial leaf epidermis of TRV control and TRV:NAG lines (20 DAI). Scale bars: 100 μm. B Nuclear degradation. Fluorescence micrographs of abaxial leaf epidermal cells from VIGS lines (20 DAI) after nuclear staining with 4',6-diamidino-2-phenylindole (DAPI; 100 μg/ml). C Oligonucleosomal DNA fragmentation. Genomic Southern blotting was performed using total genomic DNA of N. benthamiana as a probe. D-F Mitochondrial membrane integrity. Leaf protoplasts from VIGS lines (20 DAI) were observed after staining with TMRM (200 nM) (D). TMRM fluorescence (E) and chlorophyll autofluorescence (F) were quantified. Data points represent means ± SD of 20 individual protoplasts. Significant differences between control and other samples were indicated by one (P≤0.05) or two (P≤0.01) asterisks. PIV, pixel intensity values. G and H ROS production. Leaf protoplasts were incubated with a ROS indicator H₂DCFDA (2 μM) (G). Fluorescence of protoplasts from the VIGS lines was quantified by pixel intensity (H). Data points represent means ± SD of 30 individual protoplasts. Scale bars: 50μm.