Fig. 3: PBL13-interacting RING type E3 ubiquitin ligase (PIRE) interacts with PBL13 and RBOHD.
a Schematic representation of the Arabidopsis protein microarray. The PATH protein microarray with ~5000 Arabidopsis proteins was probed once with V5-tagged PBL13. b Quantification of PIRE and control protein (BSA)-binding intensity on the PATH array. c RBOHD associates with both PIRE and PBL13. YFP-LTI6b, YFP-RBOHD, PBL13-3xHA, and 3xHA-PIRE were transiently expressed in N. benthamiana in the indicated combinations. YFP-tagged proteins were subjected to anti-GFP immunoprecipitation and associations were detected by immunoblotting. d PIRE associates with RBOHD in Arabidopsis. Microsomal fractions were isolated from Col-0 or an Arabidopsis transgenic line expressing YFP-PIRE and subjected to co-immunoprecipitation using anti-GFP. e PIRE interacts with RBOHD-C and PBL13 by yeast two-hybrid assay. Serial dilution of yeast cells containing the indicated plasmids were spotted on -UTHL medium plates. The catalytic dead mutant of PIREC244S/C247S was in the pTBS1 (bait) plasmid. PBL13, RBOHD-C, and RBOHD-N were screened for interaction with pTBS1-PIREC244S/C247S. -UTHL, SD medium lacking uracil, tryptophan, histidine, leucine with galactose (gal) and raffinose (raf) as a sugar source. f PIRE directly interacts with PBL13 and RBOHD-C. Purified recombinant MBP-RBOHD-C, MBP-BIK1, or MBP-PBL13 were incubated with GST-PBL13 and pulled-down with GST agarose. Protein input and pulled-down products were analyzed by immunoblotting.
