MG132 Treatment and In Vivo Ubiquitination Assay.

To verify whether RPA1a protein degradation is mediated by the 26S proteasome, A. tumefaciens strain GV3101 carrying 35S::RPA1a‐Myc was infiltrated into tobacco leaves, and samples were collected at different time points for immunoblot analysis. The leaves were treated with 50 µM MG132 (Sigma-Aldrich, M7449) for 12 h before being sampled.

The ubiquitinated form of the protein was detected as described previously (48, 49). The 35S::HEI10‐Flag, 35S::RPA1a‐Flag/Myc constructs were infiltrated separately or coinfiltrated into the tobacco leaves using A. tumefaciens GV3101. The primers used are listed in SI Appendix, Table S2. Total proteins were extracted by native extraction buffer (50 mM Tris–MES pH 8.0, 0.5 M sucrose, 1 mM MgCl2, 10 mM EDTA, 5 mM DTT and protease inhibitor cocktail) and incubated with anti‐Flag (Sigma, M8823) or anti‐Myc (Sigma, A7470-1ML) affinity beads 4 h for immunoprecipitation. The beads were then washed three times, and protein complexes were eluted by adding 1×SDS loading buffer for immunoblot analysis. The proteins were detected by anti‐Flag antibody (GNI, GNI4110-FG), anti‐Myc antibody (PROMOTER, P-MYC), and anti‐UBQ11 antibody (Agrisera, AS08 307A).

Tube2 agarose (Life Sensors, UM402) was used to enrich the polyubiquitin-modified proteins. The samples were treated by protein extraction buffer; then, the supernatant was incubated with Tube2 agarose for 2h at 4 °C. The agarose was washed three times before boiling in SDS loading buffer. Then, the ubiquitinated protein were detected with anti-Flag antibody.

To detect the endogenous RPA1a protein level, the anti-RPA1a antibody was generated by raising peptides CETDTEAQKTFSGTGNIPPPN in rabbits (GL Biochem Ltd, Shanghai, China) and used with 1:500 dilutions for western blot analysis.