Analysis on the BAK1-interacting RLKs BIR2 and BIR3, two proteins that differentially regulate BAK1-dependent pathways

BAK1 is a multifunctional co-receptor that positively regulates multiple plant signaling pathways by interacting with the corresponding ligand-binding receptors, e.g. BRI1, FLS2, EFR, PEPR1 and PEPR2. Moreover, BAK1 plays a role in cell death control. In order to investigate how BAK1 is regulated we purified in vivo BAK1 complexes and identified two new BAK1 interactors, the BAK1 interacting RLKs 2 and 3 (BIR2 and BIR3). Functional analysis of bir2 mutants showed that they are hyperresponsive to MAMP treatment, show cell death spreading after pathogen infection but are not impaired in BL signaling. In this study the molecular mechanism how BIR2 regulates MAMP responses was further investigated. With Co-IP analysis in Arabidopsis wildtype plants it was shown that treatment with flg22 leads to a partial release of BAK1 from BIR2. Treatment with a cocktail of flg22, elf18, BL and Atpep1 leads to increased release of BAK1 from BIR2 compared to single treatments, indicating that BAK1 exists in preformed complexes with different ligand-binding receptors. Functional analyses of 35S-BIR3 plants have shown that BIR3 is a negative regulator of BL responses, MAMP responses and cell death. In this study the role of BIR3 in BL responses was further investigated and it could be shown that BIR3 not only interacts with BAK1 but also shows direct interaction with the ligand-binding receptor BRI1. The involvement of BIR3 in MAMP responses was confirmed with different functional assays. On the molecular level it was shown that BIR3 interacts with BAK1 and FLS2 and regulates BAK1-FLS2 complex formation. bak1 bir3 double mutants show a strong growth phenotype with spontaneous lesion formation and strong cell death spreading after A. brassicicola infections, confirming the involvement of BIR3 in cell death regulation. To investigate the relationship of the closely related proteins BIR2 and BIR3, bir2-1 35S-BIR3 plants were used. It was shown that BIR2 and BIR3 are partially redundant but BIR2 seems to masks the BIR3 function in the wildtype situation and to have a stronger impact on the MAMP and cell death pathway