Fig. 2: Binding of PbrRALF2 to PbrCrRLK1L13.

A PbrRALF2 interacts with the extracellular domain of PbrCrRLK1L13 in yeast. PbrRALF2 was fused to the pGADT7 vector, and the extracellular domain of PbrCrRLK1L13 was fused to the pGBKT7 vector. Growth in the medium that lacked Trp, Leu, His, and Ade indicated protein–protein interactions. B Extracellular juxtamembrane (exJM) region of PbrCrRLK1L13 is required for the PbrRALF2 interaction. The predicted structure of PbrCrRLK1L13, including the extracellular domain (ECD), exJM, transmembrane domain (TM), and intracellular kinase domain (IKD). Dissection of the exJM region of PbrCrRLK1L13 is required for the interaction with PbrRALF2 in a yeast two-hybrid assay. The interaction strength was quantified through β-galactosidase activity. Different letters indicate significant differences, as determined by one-way ANOVA (P < 0.05, n = 3). C Binding of four concentrations of PbrRALF2 to PbrCrRLK1L13 in the surface plasmon resonance (SPR) assay. Different colored lines represent the titration of different concentrations of PbrRALF2 protein in the SPR assay. The light blue line indicates the 120 nM PbrRALF2 assay, the pink line indicates the 60 nM PbrRALF2 assay, the dark blue line indicates the 30 nM PbrRALF2 assay, and the green line indicates the 15 nM PbrRALF2 assay. The black lines represent the fitting line. The solid line is a fit with Michaelis–Menten kinetics, yielding an apparent dissociation constant of Kd = 73 ± 17 nM. D Quantitative binding analysis using PbrRALF2 and the extracellular domain of PbrCrRLK1L13 using isothermal titration calorimetry (ITC). A representative thermogram was obtained from 200 μM PbrRALF2 titrations into 20 μM PbrCrRLK1L13. Nonlinear regression of the PbrRALF2 vs. PbrCrRLK1L13 dosage yielded an apparent dissociation constant of Kd = 512 ± 21 nM