Fig. 4: Molecular basis of tonoplast ATG8ylation.
From: ATG8ylation of vacuolar membrane protects plants against cell wall damage
a, Proximity-dependent biotin labelling of ATG8A-interacting proteins during cell-wall-damage-induced tonoplast ATG8ylation. TID, TurboID. b, Venn diagram summarizing the results of TurboID-based proximity-labelling proteomics of TurboID–ATG8A under Torin (4 hs, 9 µM) and ES20-1 (8 h, 100 µM) treatments, highlighting overlap and unique proteins. TurboID alone is used as a negative control. c, Volcano plot of TurboID–ATG8A ES20-1 treatment versus Torin (n = 3 replicates, 348 total proteins). The yellow dots indicate proteins enriched in ES20-1 (Student’s t-test, P < 0.05, n = 81); the blue dots indicate those enriched in Torin (P < 0.05, n = 34). d, Proteins in five categories shown as dot plots. Circle colour represents log fold change; edge colour indicates confidence (Student’s t-test with false discovery rate (FDR) correction, FDR < 0.05, grey; FDR < 0.01, black). e, Confocal micrographs showing the localization of TBC/RabGAP1–GFP and mCh–ATG8E in A. thaliana root cells, under mock or ES20-1 (8 h, 100 µM) treatments. A single optical slice, a maximum projection, a merged image and a bright field image are shown. Representative images from ten seedlings were analysed under each treatment. Scale bars, 10 µm. f, PS biosensor mCITRINE–2xPH–EVECTIN2 changes localization upon cell wall damage. Confocal micrographs of A. thaliana root cells under mock, Torin (1.5 h, 9 µM) and ES20-1 (8 h, 100 µM) treatments are shown. Maximum-intensity projections are shown in green and inverted greyscale. Representative images from ten seedlings were analysed under each treatment. Scale bars, 10 µm. g, Confocal micrographs depicting the localization of six different PIP sensors in A. thaliana root cells, under mock and ES20-1 (8 h, 100 µM) treatments. 1xFYVEHRS and 1xPXP40 target phosphatidylinositol 3-phosphate (PI3P), 1xPHFAPP1 and 1xPHOSBP target phosphatidylinositol 4-phosphate (PI4P) and 1xPHPLδ1 and 1xTUBBY-C target phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). All images are represented as inverted greyscale. Representative images from ten seedlings were analysed under each treatment. Scale bars, 10 µm. h, Cell wall damage increases vacuolar pH. Confocal images of Col0 roots treated with LysoSensor Yellow/Blue DND-160 under mock or ES20-1 (8 h, 100 µM) conditions are presented, showing dual emission (Em) in yellow and blue on the basis of pH. Representative images from ten seedlings were analysed under each treatment. Scale bars, 10 µm.
