Phosphorylation of BIK1 is critical for interaction of downstream signaling components

Abstract

Botrytis-induced Kinase 1 (BIK1) is a receptor-like cytoplasmic kinase (RLCK) involved in the defense, growth, and development of higher plants. It interacts with various receptor-like kinases (RLKs) such as Brassinosteroid Insensitive 1 (BRI1), Flagellin Sensitive 2 (FLS2), and Perception of the Arabidopsis Danger Signal Peptide 1 (PEPR1), but little is known about signaling downstream of BIK1. Interestingly, Arabidopsis thaliana BIK1 (AtBIK1) displays strong autophosphorylation kinase activity on tyrosine and threonine residues, whereas Brassica rapa BIK1 (BrBIK1) does not exhibit autophosphorylation kinase activity in vitro. Herein, we demonstrated that four proteins (RGP2, PATL2, PP7, and SULTR4.1) interact with BrBIK1 but not AtBIK1 in a yeast two-hybrid (Y2H) system. We subsequently employed bimolecular fluorescence complementation (BiFC) to confirm interactions between BIK1 and candidates in Nicotiana benthamiana, and found that only BrBIK1 bound the three proteins tested. We selected three phosphosites, T90, T362, and T368, based on amino acid sequence alignment between AtBIK1 and BrBIK1, and performed site-directed mutagenesis (SDM) on AtBIK1 and BrBIK. S90T, P362T, and A368T mutations in BrBIK1 restored autophosphorylation kinase activity on threonine residues comparable with AtBIK1. However, T90A, T362P, and T368A mutations in AtBIK1 did not alter autophosphorylation kinase activity on threonine residues compared with wild-type AtBIK1. Interestingly, BiFC results showed that BIK1 mutations restored kinase activity but not binding to RGP2, PATL2, or PP7 proteins. Our results suggest that phospho-BIK1 might be involved in plant innate immunity, while non-phospho BIK1 may regulate plant growth and development through interactions with RGP2, PATL2, and PP7.