Identification and Functional Analysis of in vitro/in vivo Phosphorylation Sites of the Arabidopsis BAK1 Interacting Receptor Kinase BIR2

Abstract

The recognition of plant pathogens by plants is primarily mediated by pattern recognition receptors (PRRs), acting as watchdogs at the plant surface. The leucine-rich repeat receptor-like kinase (LRR-RLK) BRI1-ASSOCIATED KINASE 1 (BAK1), and other SOMATIC EMBRYOGENESIS RECEPTOR KINASES (SERKs) play crucial roles as co-receptors of LRR-RLKs in diverse signaling pathways. Signaling has to be tightly controlled, thus the discovery of BAK1-INTERACTING RLK 2 (BIR2) as a negative regulator of BAK1, has added novel aspects to the understanding of regulatory processes in plant immunity. BIR2 can be transphosphorylated by BAK1 in vitro, and kinase complex formations are often mediated by phosphorylation-dependent mechanisms. Subject of this work is the identification of in vivo BIR2 phosphorylation sites (P-sites) by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS). Selected BIR2 P-sites have been mutated to alanine or aspartate to prevent or mimic phosphorylation. Functional and interaction studies with bir2 mutant lines, complemented with these constructs have revealed the potential action of these BIR2 P-sites. BIR2 transphosphorylation could have a dual function; strengthening and weakening the protein-protein interaction to regulate receptor complex formation and downstream signaling. Nevertheless, BAK1-unrelated BIR2 P-sites were detected, pointing to an involvement of other kinases in BIR2-BAK1 complex action. These findings underline the importance of phosphorylation as a key protein modification for the regulation of receptor complex dynamics.