Fig. 3: RARE ubiquitinates RRS1 through its integrated WRKY domain.

A Detection of ubiquitination of RRS1-R by RARE in Nb leaves. HA-tagged RRS1-R was co-expressed with GFP-tagged RARE or its mutant form RARE (H213Y) in the presence or absence of FLAG-Ub in Nb leaves. The Nb leaves were pretreated with 50 μM MG132 for 6 h before harvesting. Total ubiquitinated proteins were immunoprecipitated at 36 h post-infiltration with anti-FLAG antibody, and ubiquitinated RRS1-R proteins were detected by immunoblotting with anti-HA antibody. B Reduced in vivo ubiquitination level of RRS1 in rare mutant compared to wild-type background. Total protein extracts from FLAG-tagged RRS1 plants in wild-type or rare background were subjected to immunoprecipitation using anti-FLAG antibody. Following immunoprecipitation with anti-FLAG antibody, the ubiquitination of RRS1 was detected by immunoblot analysis using anti-Ubiquitin antibody. Immunoblots were probed with anti-ubiquitin and anti-FLAG antibody, respectively. C Ubiquitination of WRKY domain of RRS1 by RARE in vitro. FLAG-tagged WRKY domain of RRS1 was incubated with GST-RARE in ubiquitination assay buffer. Samples were resolved by SDS-PAGE and subjected to immunoblot analysis with anti-FLAG (top panel) or anti-Ub (bottom panel) antibody. Direct ubiquitination of WRKY domain was evident by higher molecular laddering detected by immunoblotting with anti-FLAG antibody. D WRKY domain is required for RRS1 ubiquitination by RARE. HA-tagged RRS1-R or its WRKY domain deletion variant was co-expressed with GFP-tagged RARE in the presence of FLAG-Ub in Nb leaves. The Nb leaves were pretreated with 50 μM MG132 for 6 h before harvesting. Total ubiquitinated proteins were immunoprecipitated at 36 h post-infiltration with anti-FLAG antibody, and ubiquitinated RRS1-R proteins were detected by immunoblotting with anti-HA antibody.