Fig. 5: ATG8-positive vesicle formation is not dependent on the ESCRT or the cytoskeleton.

a Analysis of the subcellular location of GFP-FREE1 before and after monensin treatment. Scale bar, 20 μm. b Time-series analysis of the invagination of ATG8-positive vesicles in free1(-/-) mutant. The magenta arrows indicated a vesicle that adhered inside the vacuolar membrane; white arrows indicated invaginated vesicles. Scale bar, 10 μm. c Confirmation of the validity of the dexamethasone (DEX)-inducible dominant-negative mutants, DEX:SNF7.1(L32W) and DEX:VPS4(E232Q). The transgenic seeds were germinated on agar plates containing 10 μM DEX. Representative images were captured after 5 days of growth under light. Scale bar, 1 cm. d A schematic diagram showing the treatment flow of DEX-inducible dominant-negative mutants. e, f Time-series analysis of the invaginated vesicles in YFP-ATG8e/DEX:SNF7.1(L32W) (e) and YFP-ATG8e/DEX:VPS4(E232Q) (f) plants after DEX induction. The magenta arrows indicated vesicles that adhered inside the tonoplast. Scale bars, 20 μm. g TEM analysis of vacuolar morphology in free1(-/-) mutant and DEX:VPS4(E232Q) transgenic plants. The magenta arrows indicate invaginating vesicles. Scale bars, 1 μm. h, i Analysis of the effects of cytoskeletal disruption on the formation of ATG8-positive vesicles. Microtubules were visualized using a mCherry-TUA5 fusion protein (h), while microfilaments were labeled with ABD2-GFP (i). Oryzalin (10 μM) and latrunculin B (10 μM) were employed to disrupt microtubule and microfilament structures, respectively. The invaginated vesicles were denoted by yellow arrowheads. Scale bars, 20 μm. j Confocal time-lapse analysis the effects of microfilament disruption on the dynamics of ATG8-positive vesicle invagination. White arrowheads indicated the invaginated vesicles. Scale bar, 20 μm. Similar confocal imaging results were obtained in at least six individual roots with three replicates.