Arrows, activation; bars, repression. SUMOylation machinery consists of the SUMO-activating enzyme, the SUMO-conjugating enzyme, and the SUMO E3 and E4 ligases. In protoplasts and leaves, respectively. Total proteins were extracted and precipitated Toltrazuril sulfone with an anti-HA or anti-GFP antibody and then immunoblotted with different antibodies. (D)SUMOylation of SEU. Plants were grown under light (80?mol/m2/s) for 5 Toltrazuril sulfone d. Proteins were precipitated with an anti-GFP monoclonal antibody conjugated to agarose beads. (E)SUMOylation of SEU. SUMOylated MBP-SEU-FLAG was detected with an DDPAC anti-SUMO1 antibody. To test this possibility, we performed transient and SUMOylation experiments. Firstly, we transiently coexpressed SEU-3HA with FLAG-SUMO1GG (wild type) or FLAG-SUMO1AA (a conjugation-deficient mutant) in protoplasts. We then immunoprecipitated SEU-3HA with an anti-HA antibody and detected the immunoprecipitated proteins with an anti-FLAG antibody. Higher-molecular-weight?SUMOylated SEU-3HA bands (SUMO1-SEU) were detected when SEU-3HA was coexpressed with FLAG-SUMO1GG, but not with FLAG-SUMO1AA (Figure?1B). Secondly, we performed a transient SUMOylation assay in leaves?and obtained similar results (Figure?1C). Thirdly, Toltrazuril sulfone we generated transgenic plants, where was fused with GFP and driven by its native promoter in the background. We extracted proteins from and plants and immunoprecipitated them with anti-SEU and anti-SUMO1 antibodies. The anti-SUMO1 antibody detected higher-molecular-weight bands of SEU-GFP in plants (Figure?1D). There were two higher-molecular-weight bands whose exact weights differed from those in protoplast and assays, indicating that SEU may be multi- or poly-SUMOylated at one or more conjugating sites in different plants. Fourthly, we incubated affinity-purified recombinant proteins MBP-SEU-FLAG (substrate), SUMO E1 (His-SAE1b and His-SAE2), and SUMO E2 (His-SCE1) with His-SUMO1GG or His-SUMO1AA in the presence or absence of SUMO E3 ligase (MBP-SIZ1-Myc). Anti-FLAG and anti-SUMO1 antibodies detected slowly migrating bands above the original SEU protein only in the reaction containing His-SUMO1GG and SIZ1, but not in the other reactions (Figure?1E), suggesting that SEU is SUMOylated pull-down assay in leaves. (C)pull-down assay. (D) Co-IP assay. Plants were grown under light (80?mol/m2/s) for 5 d. Proteins were precipitated with an anti-GFP antibody conjugated to agarose beads. (E) Detection of SUMOylated proteins expression. Relative gene expression was normalized to the level of pull-down assay in leaves, in which MBP-SEU-FLAG was incubated with SIZ1–FLAG. Precipitation with MBP beads revealed that SEU interacts with the truncated version of SIZ1 (Figure?2B). The pull-down assay of MBP-SEU-FLAG incubated with?MBP-SIZ1-Myc demonstrates that SEU directly interacts with full-length SIZ1 (Figure?2C). fully complemented the short-hypocotyl phenotype of (see Figure?3A in detail; Huai et?al., 2018). An co-immunoprecipitation (Co-IP) assay showed that the anti-GFP antibody immunoprecipitated SIZ1 from plants, but not mutant plants (Figure?2D). These results demonstrate that SEU interacts with SIZ1 and and was normalized to the level of (WT) using Student’s double mutant. The hypocotyl length of plants was slightly but significantly shorter than that of and single mutants (Supplemental Figure?2A). largely complemented the phenotype, but this effect was partly suppressed in the background (Supplemental Figure?2A). Similarly, at the adult stage, plants showed a severe dwarf phenotype, much more extreme?than the phenotypes of the parental single mutants. The complementary effect of on was largely inhibited by the mutation (Supplemental Figure?2B and 2C). These observations suggest that SEU and SIZ1 coordinate? plant growth and development, and that the function of SEU largely requires SIZ1. Furthermore, the immunoblotting experiment showed that the SUMOylated SEU-GFP bands seen in were almost abolished in (Figure?2E). Moreover, SEU protein levels were partially reduced in the mutant in comparison with those in Col (Figure?2F), likely due to the transcriptional regulation by SIZ1 (Figure?2G). These results demonstrate that SIZ1 directly mediates the SUMOylation of SEU. SUMOylation of SEU Is Required.