Fig. 7: SidK expression induces tonoplast ATG8ylation in A. thaliana.
From: ATG8ylation of vacuolar membrane protects plants against cell wall damage
a, Western blot analysis from immunoprecipitation (IP) experiments using Flag–GFP, Flag–SidK or Flag–SidK-F62A to pull down the VHA-A subunit of V-ATPase. The blots were probed with anti-Flag and anti-VHA-A antibodies to detect the presence of VHA-A in the pull-down from each bait protein. b, IP followed by mass spectrometry (IP–MS) results, presenting the identification of V-ATPase subunits co-immunoprecipitated with Flag–SidK and Flag–SidK-F62A. A schematic representation of the V-ATPase complex is also shown, detailing all its subunits. The ones marked with bold letters were detected in the SidK IP–MS experiment. c, Inducible expression of SidK as a tool to probe V-ATPase function in Arabidopsis cells. Western blot analysis of GFP–ATG8A lines expressing either mCh–SidK or mCh–SidK-F62A under a DEX-inducible promoter is presented, showing protein levels with and without DEX induction. The blots were probed for anti-GFP and mCh to detect the fusion proteins. d, SidK expression changes vacuolar pH. Confocal micrographs of mCh–SidK or mCh–SidK-F62A expressing lines treated with LysoSensor Yellow/Blue DND-160 are shown, displaying blue, yellow and red emissions, alongside bright field images, with and without DEX induction. Representative images from ten seedlings were analysed under each treatment. Scale bars, 10 µm. e, SidK expression induces tonoplast ATG8ylation. Confocal micrographs of GFP–ATG8A co-expressing mCh–SidK or mCh–SidK-F62A, treated with the tonoplast marker MDY64 displayed in magenta, are shown. Images show the GFP channel, a merge of the magenta and green channels, red emissions and bright field, with and without DEX induction. Representative images from ten seedlings were analysed under each treatment. Scale bars, 10 µm.
