Figure 7.

OsHRZ1 is required for ubiquitination and degradation of OsPRI1. A, Ubiquitination of OsPRI1 by OsHRZ1. E. coli-purified GST-tagged OsHRZ1 and His-tagged OsPRI1 proteins were subjected to in vitro ubiquitination assay containing ATP, biotin-tagged ubiquitin (Ub), and human E1 and E2 (UbcH5c). Proteins were immunodetected using the HRP-Streptavidin detection system. B, Degradation of OsPRI1 by OsHRZ1. Degradation of OsPRI1 was carried out by detecting the OsPRI1-GFP protein level in coinfiltration experiments with increasing amounts of OsHRZ1-GFP. GFP antibody was used. Numbers indicate the ratio of the concentrations of agrobacteria used in coinfiltration. OsPRI1-GFP mRNA expression levels were analyzed by RT-PCR. The asterisk indicates a nonspecific signal. C, Cell-free degradation. 7-d-old roots grown in Fe-sufficient media were harvested and used for protein extraction. Incubation with or without MG132 was performed over the indicated time course. WT, Wild type.