Fig. 2: RARE is an E3 ubiquitin ligase that interacts directly with RRS1 and indirectly with RPS4 via RRS1. | Nature Communications

Fig. 2: RARE is an E3 ubiquitin ligase that interacts directly with RRS1 and indirectly with RPS4 via RRS1.

From: Reversible ubiquitination conferred by domain shuffling controls paired NLR immune receptor complex homeostasis in plant immunity

Fig. 2

A Assays of in vitro self-ubiquitination of RARE. GST-RARE and GST-RAREH213Y were assayed for E3 activity in the presence of E1 (UBA2), E2 (UBC10), ubiquitin (Ub) and ATP. “+” and “-” denote the presence or absence of the components of each reaction mixture. GST served as a negative control. The presence of RARE or RAREH213Y was detected using anti-GST antibody (upper panel). Protein ubiquitination bands generated by GST-RARE are indicated on the right. Ubiquitination results in a heterogeneous collection of higher molecular mass proteins that are detected with anti-ubiquitin antibody (lower panel). B Co-IP assays reveal that the interaction of RARE with RRS1/RPS4 complex is dependent on RRS1. Samples were harvested from Nb transiently coexpressing FLAG-tagged RRS1/RPS4 and HA-tagged RARE. Total extracts were immunoprecipitated with anti-HA antibody-coupled beads followed by immunoblotting with the indicated antibody. WRR4A served as a control. C Co-IP assays for the interaction of RARE with RPS4 in protoplasts from Col-0 and rrs1-3 plants. Protoplasts from Col-0 and rrs1-3 co-transfected with the RARE-HA and RPS4-FLAG constructs were incubated overnight, and total protein extracts were subjected to immunoprecipitation with anti-HA antibody-coupled beads followed by immunoblotting using either anti-HA or anti-FLAG antibody. D BiFC analyses for the interaction of RARE with RRS1/RPS4 complex in Col-0 protoplasts. Protoplasts were transformed with the indicated BiFC constructs and YFP fluorescence was visualized by confocal microscopy 16-20 h after transient expression. The positions of nuclei were shown by 4, 6-diamidino-2-phenylindole (DAPI) staining. Scale bar, 5 μm. E Co-IP assays to evaluate the association of RARE with individual domain of RRS1-R after transient co-expression in Nb. Total protein extracts were subjected to immunoprecipitation with agarose-conjugated anti-HA antibody followed by immunoblot analysis using either anti-HA or anti-FLAG antibody. F Pull-down assay for the interaction of RARE with WRKY domain of RRS1. FLAG-tagged WRKY was incubated with immobilized GST or GST-tagged RARE. After washing, bound proteins were eluted and subjected to immunoblot analysis using anti-FLAG antibody.

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