Fig. 4: TaCLV1 interacts with, and phosphorylates TaSG-D1.

a Phos-tag assay shows the kinase domain (KD) of TaCLV1-mediated phosphorylation of TaSG-D1. The phosphorylation status of TaSG-D1 was examined in a phos-tag gel assay (anti-MBP). The levels of TaCLV1 (anti-GST) and TaSG-D1 (anti-MBP) proteins are shown in the SDS-PAGE gel. Phosphorylated TaSG-D1 band is indicated by arrow and red asterisks. (n = 3 biologically independent experiments). b TaCLV1 reduces TaSG-D1 stability in N. benthamiana leaves. TaSG-D1 abundance was examined with anti-GFP antibody. The kinase domain (KD) of TaCLV1 and TaCEPR1 were detected with anti-Flag antibody. Actin was detected with anti-β-Actin antibody as a loading control. TaCEPR1-KD was used as negative control. The relative abundance of TaSG-D1 was quantified using ImageJ software. (n = 3 biologically independent experiments). c TaCLV1 promotes TaSG-D1 degradation in a cell-free degradation assay. TaSG-D1 abundance was examined with anti-MBP antibody. RuBisCO was detected by Ponceau S staining as a loading control. The relative protein abundance of TaSG-D1 was quantified using ImageJ software. The relative protein level of TaSG-D1 at 0 min was defined as 1.00. (n = 3 biologically independent experiments). d TaCLE24b accelerates TaSG-D1 degradation. TaSG-D1 was detected with anti-GFP antibody. The full-length cDNA sequence of TaCLV1 was detected with anti-Flag antibody. Actin was detected with anti-β-Actin antibody as a loading control. The relative abundance of TaSG-D1 was quantified using ImageJ software. The relative protein levels of TaSG-D1 without TaCLV1 and TaCLE24b were defined as 1.00. TaCLE24b: synthetic TaCLE24b peptides (0.1 μM). (n = 3 biologically independent experiments). e A working model for the role of the TaCLE24b–TaCLV1–TaSG-D1 signaling pathway during lateral root development in wheat. In the presence of TaCLE24b peptide, TaCLE24b enhances the interaction between TaCLV1 and TaSG-D1, leading to increased phosphorylation and degradation of TaSG-D1, and consequently results in an increase in the density of lateral root and lateral root primordium.