Fig. 3

Ubiquitination and degradation of OsNRPD1a by both U-box- and RING-type E3 ligases. a Confirmation of the interaction between OsNRPD1aC and eight candidate E3 ligases in yeast. P3IP1 (indicated in blue) was identified as the cognate E3 of OsNRPD1a in a previous study. DDO, SD-Leu-Trp, QDO, SD-Leu-Trp-His-Ade. b Names and functions of the E3 ligases that interacted with OsNRPD1aC. The pink color line represents the identified interaction using the UbE3 library, and the blue color line represents the literature-reported interaction. c Co-IP assay of OsNRPD1aC with six candidate E3 ligases. N. benthamiana leaves were agro-infiltrated with the indicated plasmid combinations. Total protein was extracted and immunoprecipitated with anti-GFP/Agarose beads. Immunoblot detection was performed using anti-GFP or anti-HA antibody as indicated. d Degradation assay of OsNRPD1aC by P3IP1 and OsRFPH2-10 with or without 50 μM MG132 treatment. N. benthamiana leaves were agro-infiltrated with plasmids harboring OsNRPD1aC-GFP and two E3 ligase genes fused with HA tag. GUS-HA was used as a negative control. The protein abundance of OsNRPD1aC and the two E3 ligases were detected by immunoblotting using anti-GFP or anti-HA antibody, respectively. ACTIN was used as an internal control. OsNRPD1aC transcript level was measured by RT-PCR, and ACTIN was used as the internal control. e Ubiquitination assay of OsNRPD1aC by OsRFPH2-10. OsNRPD1aC protein purified from E. coli was incubated with MBP-OsRFPH2-10 and E1, E2, Ub, and ATP in the reactions. Immunoblot analysis was performed using anti-ubiquitin, anti-MBP, or anti-GST antibody