Phosphorylation-Dependent Differential Regulation of Plant Growth, Cell Death, and Innate Immunity by the Regulatory Receptor-Like Kinase BAK1

Abstract

Plants rely heavily on receptor-like kinases (RLKs) for perception and integration of external and internal stimuli. The Arabidopsis regulatory leucine-rich repeat RLK (LRR-RLK) BAK1 is involved in steroid hormone responses, innate immunity, and cell death control. Here, we describe the differential regulation of three different BAK1-dependent signaling pathways by a novel allele of BAK1, bak1-5. Innate immune signaling mediated by the BAK1-dependent RKs FLS2 and EFR is severely compromised in bak1-5 mutant plants. However, bak1-5 mutants are not impaired in BR signaling or cell death control. We also show that, in contrast to the RD kinase BRI1, the non-RD kinases FLS2 and EFR have very low kinase activity, and we show that neither was able to trans-phosphorylate BAK1 in vitro. Furthermore, kinase activity for all partners is completely dispensable for the ligand-induced heteromerization of FLS2 or EFR with BAK1 in planta, revealing another pathway specific mechanistic difference. The specific suppression of FLS2- and EFR-dependent signaling in bak1-5 is not due to a differential interaction of BAK1-5 with the respective ligand-binding RK but requires BAK1-5 kinase activity. Overall our results demonstrate a phosphorylation-dependent differential control of plant growth, innate immunity, and cell death by the regulatory RLK BAK1, which may reveal key differences in the molecular mechanisms underlying the regulation of ligand-binding RD and non-RD RKs.

Cautionary Notes on the Use of C-Terminal BAK1 Fusion Proteins for Functional Studies

“…In all assays, we compared the ability of these BAK1 variants to complement the phenotypes of bak1-4 or bak1-4 bkk1-1 in comparison to wild-type Columbia-0 (Col-0) and to bak1-4 complemented with untagged BAK1. Arabidopsis thaliana mutants impaired in BR perception or signaling, including bak1-4, display an altered rosette morphology and size under short-day conditions and are hyposensitive to exogenous BR treatments Nam and Li, 2002;Wang et al, 2008;Schwessinger et al, 2011).…”

“…C-terminal BAK1 fusion proteins are often used in Arabidopsis and Nicotiana benthamiana to study the ability of BAK1 or derived variants to form a ligand-induced complex with FLS2 (Chinchilla et al, 2007;Heese et al, 2007;Shan et al, 2008;Lu et al, 2010Lu et al, , 2011Zhang et al, 2010;Roux et al, 2011;Schwessinger et al, 2011;Xiang et al, 2011). Using the transgenic lines described in Figure 1, we tested the ability of the different BAK1-tagged proteins to associate with FLS2 in response to flg22 treatment in coimmunoprecipitation experiments.…”

“…We tested the ability of the different BAK1 variants to complement the hyporesponsive phenotype of bak1-4 during FLS2-and EFR-mediated innate immune responses (Chinchilla et al, 2007;Heese et al, 2007;Shan et al, 2008;Ranf et al, 2011;Roux et al, 2011;Schwessinger et al, 2011). An early response triggered by flg22 and elf18 perception is the rapid production of reactive oxygen species (ROS) in an oxidative burst.…”

“…The LRR-RLK FLAGELLIN SENSING2 (FLS2) perceives bacterial flagellin (or the conserved peptide flg22) and forms, almost instantaneously, a ligand-induced complex with BAK1 (Chinchilla et al, 2007;Heese et al, 2007;Schulze et al, 2010). Similarly, the LRR-RLK EF-TU RECEPTOR (EFR) that perceives bacterial elongation factor Tu (or the conserved peptide elf18) forms a ligand-induced complex with BAK1 Schwessinger et al, 2011). FLS2 and EFR heterooligomerize with other SERKs in addition to BAK1, including BKK1, which appears to act redundantly with BAK1 in FLS2-and EFR-mediated PTI signaling .…”

“…FLS2 and EFR heterooligomerize with other SERKs in addition to BAK1, including BKK1, which appears to act redundantly with BAK1 in FLS2-and EFR-mediated PTI signaling . Notably, the functional role of BAK1 seems more critical for PTI signaling than for BRI1-mediated responses, as bak1 mutants are strongly impaired in most flg22-and elf18-induced responses (Chinchilla et al, 2007;Heese et al, 2007;Shan et al, 2008;Ranf et al, 2011;Roux et al, 2011;Schwessinger et al, 2011), while the single mutants display only mild defects in BR-induced responses Nam and Li, 2002;Karlova et al, 2006;Albrecht et al, 2008).…”