Fig. 6
The GDD motif of NIb is required for the NbBeclin1–NIb interaction and NbBeclin1, via its AIM, interacts with NbATG8a to facilitate the formation of autophagosomes. a Y2H assays to detect possible interactions between NbBeclin1 truncated proteins (NbBeclin1-N and NbBeclin1-C) and NIb and between NbBeclin1 and NIb truncated proteins (NIb-N, NIb-M, and NIb-C). b Y2H assays to detect possible interactions between NbBeclin1 and NbATG8f or NbATG8a and between NbATG8a and NbBeclin1-N, NbBeclin1-C, or NbBeclin1-N AIM mutant (NbBeclin1- NΔAIM). c BiFC assays in H2B-RFP (red) transgenic N. benthamiana leaves at 48 hpi. Yellow fluorescence (green) was observed as a consequence of the complementation of the YN and YC tagged with NbBeclin1 and NbATG8a or NbBeclin1-N and NbATG8a. Bars, 50 μm. d Representative TEM images from N. benthamiana leaf cells agroinfiltrated with buffer (mock), NbBeclin1, NbBeclin1ΔAIM, NbBeclin1-N, NbBeclin1-NΔAIM, or NbBeclin1-C at 60 hpi. Typical autophagic structures (red arrows) were observed in NbBeclin1- or NbBeclin1-N-expressing leaves in the cytoplasm. Cp chloroplast, CW cell wall, S starch, V vacuole. Bars mean 1 μm or 2 μm as indicated. e The number of typical double-membrane autophagic structures in mock, NbBeclin1-, NbBeclin1ΔAIM-, NbBeclin1-N-, NbBeclin1-NΔAIM-, or NbBeclin1-C-infiltrated leaves. Experiments were repeated three times and typical autophagic structures were counted in 20 cells in each treatment. Values represent the mean number of autophagosomes ±SD per 10 cells. Single asterisk indicates statistically significant difference (P < 0.05) between mock and NbBeclin1-infiltrated leaves, and double asterisks indicate P < 0.01 between mock and NbBeclin1-N (Student’s t-test, two-sided). f Confocal micrographs showing N. benthamiana leaf cells co-infiltrated with Agrobacterium harboring a YFP-NbATG8a expression construct and Agrobacterium carrying NbBeclin1, NbBeclin1ΔAIM, NbBeclin1-N, NbBeclin1-NΔAIM or NbBeclin1-C at 48 hpi. Bars, 25 μm
