Fig. 5: DAY directly interacts with BRI1.
From: Chaperone-like protein DAY plays critical roles in photomorphogenesis
a DAY interacts with BRI1 in TGN/EE and plasma membrane. BiFC between BRI1-nYFP and DAY-cYFP results in yellow fluorescence, suggesting protein interaction in vivo (middle, lower panel). BiFC fluorescence was compared with SYP61-mRFP fluorescence, which marks the TGN/EE (right panel). N. benthamiana epidermal cells were observed two days after agroinfiltration. Enlarged views of the boxed areas are shown (En). Scale bar, 50 µm. b DAY directly interacts with the BRI1 cytosolic domain. Yeast two-hybrid assays suggest that both DAY deletion variants (∆cTP/TM and ∆TM) interact with the cytosolic domain of BRI1. The p53/T-antigen and Lambda/T-antigen combinations were used as positive and negative controls, respectively. CD, the cytosolic domain; ∆TM, deletions of all four TMs. c Extracellular domain interaction assays show that neither of the DAY deletion variants, ΔcTP/TM and ΔcTP, interact with the extracellular domain of BRI1. DAY variants and the extracellular domain of BRI1 are expressed as prey and bait, respectively. The prey was fused with alkaline phosphatase (AP) and the bait with the fragment crystallizable region (Fc) from the tail region of the antibody. BAK1/BRI1 with brassinolide (+BL) was used as a positive control. (n = 2 technical replicates) d Co-immunoprecipitation shows that DAY interacts with BRI1. Total Arabidopsis seedling proteins were immunoprecipitated with GFP-Trap Agarose, and co-immunoprecipitated proteins were detected using the anti-BRI1 antibody. Two isoforms of DAY protein, DAY iso1, and iso2, are indicated. e Deletion of the N-terminal lobe in the BRI1 kinase domain reduces its binding affinity for DAY. Upper panel; diagram of the cytosolic domain of BRI1. JM juxta-membrane domain, KD kinase domain, CT C-terminal tail, N-lobe N-terminal lobe, C-lobe C-terminal lobe, AC activation loop. Amino acid residue numbers are indicated. Lower panel; lysates of E. coli cells expressing the Flag-tagged KD of BRI1 (Flag-KD) or the Flag-tagged KD of BRI1 lacking the N-terminal lobe (Flag-∆N-lobe) were incubated with MBP or MBP-∆cTP DAY (MBP-∆cTP) immobilized on amylose agarose resins. Eluates were analyzed by immunoblotting with anti-Flag antibody or Coomassie staining.
