Fig. 2: PsAF5 interacts with and co-locates with PsATG8 through an AIM1 motif.

a PsAF5 deletion reduces H₂O₂-induced intracellular ATG8 activation. The proteins detected by the ATG8 antibody were isolated using urea SDS–PAGE. b The ratio of ATG8-PE to ATG8 after 1 mM H₂O₂ treatment over time; n = 3 biologically independent samples. c Interaction of PsAF5 and PsATG8 in vivo detected by a Co-IP assay. H₂O₂ treatment was 1 mM for 1 h. d Interaction of PsAF5 and PsATG8 in vitro detected by a pull-down assay. e Schematic diagram illustrating PsAF5 tagged in tandem with mCherry and acid-sensitive GFP while ATG8 is tagged on the N-terminus with a BFP (blue fluorescent protein) tag. The enlarged area shows the four AIM motifs of PsAF5 and their respective mutation sites. f PsAF5 and ATG8 colocalize into puncta during infection (2 h post inoculation) or H₂O₂ treatment (1 mM 1 h). g Co-localization of BFP-PsATG8 with different PsAF5 domain and AIM motif mutant proteins, observed by fluorescence microscopy, without any H₂O₂ or infection treatment. Scale bar, 5 μm (f, g). h The distribution of fluorescence colors in the experiments shown in (g), as described in the Methods and illustrated in Fig. 2e. Experiments in (a, c, d, g, f) were independently repeated 3 times with similar results. i Interaction of PsATG8 with different PsAF5 domain and AIM motif mutant proteins in vitro, detected by a pull-down assay. j Interaction of PsATG8 with different PsAF5 domain and AIM motif mutant proteins in vivo, detected by a Co-IP assay. H₂O₂ treatment was 1 mM for 1 h. Experiments in (i, j) were independently repeated twice with similar results. In (b) and (h), error bars in graphs represent means ± SEM. Source data are provided as a Source Data file.