Extended Data Fig. 6: Brachypodium distachyon WAKL20 ortholog (BdWAKL20) interacts with and phosphorylates BSR1.
a, Confocal microscopy analysis revealed the colocalization of BdWAKL20 with BSR1. b, BiFC assay demonstrating the interaction between BdWAKL20 and BSR1. YFPc-fused bsr1, a null allele of BSR1, served as a negative control. The cell wall (dashed lines), the retracted plasma membrane (solid arrowhead), and Hechtian strands (hollow arrowhead) between the plasma membrane and the cell wall after plasmolysis are indicated. Scale bars, 20 μm. c, Immunoblot analysis validating the protein expression shown in b. Asterisk represents a nonspecific band. d, e, BdWAKL20 phosphorylates BSR1 (d) and its NB-ARC domain (e) in vitro. Full-length BSR1 and the NB-ARC domain were incubated with purified BdWAKL20 kinase domain. The kinase-dead mutant BdWAKL20K246E served as the negative control. f, BdWAKL20 predominantly phosphorylates the Ser 470 (S470) site within the NB-ARC domain. For (d–f), autoradiographic signals (upper) and the CBB-stained gel used as a loading control (lower) are shown. The experiments were independently repeated twice with similar results.
