Fig. 3: TaHAKAI^R interacts with P2 and mediates its degradation via the UPS.

Verification of the interaction between TaHAKAI^R and P2 through LCI assay (A), pulldown assay (B), microscale thermophoresis assay, the values are the means ± SDs, n = 3 biologically independent experiments (C) and BiFc (Bimolecular Fluorescence Complementation), bar = 100 μm (D). E A semi-in vivo assay was used to determine the stability of the P2 protein. Total protein was extracted from wheat protoplasts at 48 h, and protein levels were analysed at different time points after 100 μm CHX treatment with a GFP antibody in the absence or presence of 20 mM ATP. F, H The relative abundance of P2-GFP. The values are the means ± SDs (ANOVA with two-sided t-test; n = 3 biologically independent experiments, ‘*’ p < 0.05, ‘**’ p < 0.01). The p value results in F are as follows. ‘<0.0001’ in 15 min; ‘0.0005’ in 30 min and ‘0.0028’ in 45 min. The p value results in H are as follows. ‘<0.0067’ in 15 min; ‘0.0138’ in 30 min and ‘0.0166’ in 45 min. G A semi-in vivo assay was used to determine the effect of MG132 on the stability of the P2 protein. Total protein was extracted from wheat protoplasts at 48 h, and the protein levels were analysed at different time points after treatment with 100 μM CHX and 20 mM ATP with a GFP antibody in the absence or presence of MG132. Equal amounts of dimethyl sulfoxide (DMSO) were used as a control. I TaHAKAI^R promotes P2 degradation via the 26S proteasome pathway. P2-Flag was coexpressed with TaHAKAI^R-GFP or TaHAKAIR^(H111Y)-GFP. The 26S proteasome inhibitor MG132 (50 μm) was added 12 h before sampling, and an equal volume of DMSO was used as a control. The relative transcript levels of NbUBC and P2 were determined via RT‒PCR. Ponceau staining in (E, G, I) shows the protein loaded in the assays.