Fig. 2: ATG proteins associate with SGs components in vivo under HS.

a BiFC assays demonstrate the association of ATG proteins and UBP1a in vivo under HS. ATG13a-YN, ATG6-YN, ATG5-YN, and UBP1a-YC were co-expressed in tobacco leaves. mCherry-RBP47b was co-expressed as SGs marker. For HS treatment, the isolated tobacco leaves were placed in a 38 °C incubator for 1 h. Scale bar = 10 μm. The value represents the Pearson Correlation Coefficient. Data represent mean ± SEM, n = 3. Three different merged images are used for the calculation. b–d Co-IP assays demonstrate associations between ATG proteins and SGs components (UBP1a) in planta. Total proteins were extracted from plant cells co-expressing GUS-GFP/ATG-FLAG and UBP1a-GFP/ATG-FLAG with 22 °C (control) and 38 °C (HS) treatment, followed by immunoprecipitation using GFP-Trap magnetic beads. The immunoprecipitated protein was detected by WB using anti-GFP or anti-FLAG antibody. e Schematic diagram of separate SGs by differential centrifugal method. f, g Arabidopsis cells co-expressing mCherry-RBP47b and UBP1a-GFP, ATG13a-GFP, ATG6-GFP or ATG5-GFP were subjected to HS treatment at 38 °C for 1 h, followed by differential centrifugation to separate SGs. Precipitate was resuspended in a few of the wash buffers, followed by observation using confocal microscopy. Scale bar = 10 μm. h Arabidopsis cells co-expressing UBP1a-FLAG/ATG13a-GFP were subjected to treatment at 22 °C (control) or 38 °C (HS) for 1 h, followed by differential centrifugation to separate SGs. An equal proportion of denatured samples (T: total protein. S: supernatant. P: pellet) were separated by 10% SDS-PAGE, followed by immunoblotting with anti-GFP, and anti-FLAG antibodies. Ponceau staining represents the Rubisco large subunit, which was used as the reference for loading control. The value represents the S or P vs. T. Data represent mean ± SEM; n = 2. Source data are provided as a Source Data file.