Extended Data Fig. 4: Identification of a specific E3 ligase that targets acetylated ADA2 in rice.
(a) Network view of predicted E3-substrate interactions in UbiBrowser web services. In order to found the candidate E3 ligase we used ADA2 homologs gene TADA2b (Homo sapiens) as queried substrate to predicted potential E3 ligase. In network view, the central node is the queried substrate, and the surrounding nodes are the predicted E3 ligases. The width of the edge reflects the confidence of the interaction. (b) Predicted high confidence (green background) and middle confidence (yellow background) interacting E3 proteins in Homo sapiens, and their homologs in rice. (c) Co-localization of E3-2 (fused to RFP) with wild type and mutant ADA2 proteins (fused to GFP) in transiently transfected rice protoplasts. (d) Visualization of the ZDOCK predicted ADA2 and E3-2 interacting residues. The distances between E3-2 and ADA2 or ADA2 K263Q mutant residues were measured using pymol software. (e) Production of rice E3-2 CRISPR/Cas9 mutants. sgRNA position in the gene and decoded sequence mutations of the E3-2 gene are shown. (f) Phenotypes of e3-2-1 and e3-2-2 plants in comparison with a control line (with null mutation) at seedling stage. (g) ADA2 protein levels in e3-2 mutants relative to the control line. Nuclear proteins were tested by immunoblotting with anti-ADA2 and anti-H3 antibodies. Band intensities were quantified using ImageJ software, relative to the control line, and indicated below the blots.
