An Overdose of the Arabidopsis Coreceptor BRASSINOSTEROID INSENSITIVE1-ASSOCIATED RECEPTOR KINASE1 or Its Ectodomain Causes Autoimmunity in a SUPPRESSOR OF BIR1-1-Dependent Manner

The membrane-bound BRASSINOSTEROID INSENSITIVE1-ASSOCIATED RECEPTOR KINASE1 (BAK1) is a common coreceptor in plants and regulates distinct cellular programs ranging from growth and development to defense against pathogens. BAK1 functions through binding to ligand-stimulated transmembrane receptors and activating their kinase domains via transphosphorylation. In the absence of microbes, BAK1 activity may be suppressed by different mechanisms, like interaction with the regulatory BIR (for BAK1-INTERACTING RECEPTOR-LIKE KINASE) proteins. Here, we demonstrated that BAK1 overexpression in Arabidopsis (Arabidopsis thaliana) could cause detrimental effects on plant development, including growth arrest, leaf necrosis, and reduced seed production. Further analysis using an inducible expression system showed that BAK1 accumulation quickly stimulated immune responses, even under axenic conditions, and led to increased resistance to pathogenic Pseudomonas syringae pv tomato DC3000. Intriguingly, our study also revealed that the plasma membrane-associated BAK1 ectodomain was sufficient to induce autoimmunity, indicating a novel mode of action for BAK1 in immunity control. We postulate that an excess of BAK1 or its ectodomain could trigger immune receptor activation in the absence of microbes through unbalancing regulatory interactions, including those with BIRs. Consistently, mutation of SUPPRESSOR OF BIR1-1, which encodes an emerging positive regulator of transmembrane receptors in plants, suppressed the effects of BAK1 overexpression. In conclusion, our findings unravel a new role for the BAK1 ectodomain in the tight regulation of Arabidopsis immune receptors necessary to avoid inappropriate activation of immunity.Plants rely on their innate immune system to detect microbes and mount an active defense against pathogens. The plant immune system is traditionally considered to be composed of two layers (Jones and Dangl, 2006). The first one is based on the activity of pattern-recognition receptors (PRRs) that can detect microbe-associated molecular patterns (MAMPs) and trigger what is termed pattern-triggered immunity (PTI; Boller and Felix, 2009). Many plant pathogens can suppress this basal defense response using virulence factors termed effectors. In a second layer of defense, plants can make use of resistance (R) proteins to recognize the presence of pathogen effectors resulting in effector-triggered immunity (ETI), which resembles an accelerated and amplified PTI response (Jones and Dangl, 2006).Plants utilize plasma membrane-associated receptor-like proteins (RLPs) or receptor-like kinases (RLKs) as PRRs to sense specific signals through their ectodomains (Böhm et al., 2014). RLPs and RLKs require the function of additional RLKs to form active receptor complexes and transfer the external signal to the inside of the cells (Zhang and Thomma, 2013; Cao et al., 2014; Liebrand et al., 2014). The best-known coreceptor is the leucine-rich repeat (LRR)-RLK BRASSINOSTEROID INSENSITIVE1-ASSOCIATED RECEPTOR KINASE1 (BAK1), which was originally identified as a positive regulator and partner for the brassinosteroid (BR) receptor BRASSINOSTEROID INSENSITIVE1 (BRI1; Li et al., 2002; Nam and Li, 2002). BRs refer to phytohormones that promote plant growth and development (Fujioka and Yokota, 2003). Thus, loss-of-function mutations in BAK1 negatively impact Arabidopsis (Arabidopsis thaliana) growth due to improper cell elongation. In short, bak1 mutants display compact rosettes with round-shaped leaves and shorter petioles and phenocopy weak bri1 mutations (Li et al., 2002; Nam and Li, 2002). Conversely, certain mutants affected in the BAK1 ectodomain show increased activity in the BR signaling pathway and share phenotypic similarities with BRI1-overexpressing lines (Wang et al., 2001), including elongated hypocotyls, petioles, and leaf blades and an overall increase in height (Jaillais et al., 2011; Chung et al., 2012).Furthermore, BAK1 is involved in the containment of cell death, independently of its function in BR signaling. Arabidopsis bak1 knockout mutants exhibit extensive cell death spreading after microbial infection (Kemmerling et al., 2007). In addition, spontaneous cell death develops in Arabidopsis double mutant plants lacking both BAK1 (also named SOMATIC EMBRYOGENESIS RECEPTOR KINASE3 SERK3]) and its closest homolog BAK1-LIKE1 (BKK1)/SERK4, causing seedling lethality even in the absence of microbes (He et al., 2007). Similar phenotypes are observed in Arabidopsis, rice (Oryza sativa), and Nicotiana benthamiana by lowering the expression of BAK1 and its homologs (Heese et al., 2007; Jeong et al., 2010; Park et al., 2011). Interestingly, typical defense responses, like the production of reactive oxygen species and constitutive callose deposition, are also detected in those plants, although the basis for this phenomenon remains poorly understood (He et al., 2007; Kemmerling et al., 2007; Park et al., 2011; Gao et al., 2013).On the other hand, BAK1 is widely studied as a key component of immune signaling pathways due to its known association with different PRRs, including RLKs and RLPs (Kim et al., 2013; Böhm et al., 2014). Upon MAMP perception, PRRs induce signaling and physiological defense responses like mitogen-activated protein kinase (MAPK) activation, reactive oxygen species and ethylene production, and modifications in gene expression, all of which contribute to PTI. Among the best-studied examples of BAK1-regulated PRRs are two LRR-receptor kinases, ELONGATION FACTOR Tu RECEPTOR (EFR), which senses the active epitope elf18 of the bacterial elongation factor Tu, and the flagellin receptor FLAGELLIN SENSING2 (FLS2), which senses the active epitope flg22 of bacterial flagellin (Gómez-Gómez and Boller, 2000; Chinchilla et al., 2006; Zipfel et al., 2006). Immediately after flg22 binding to its LRR ectodomain, FLS2 forms a tight complex with BAK1 (Chinchilla et al., 2007; Sun et al., 2013). This heteromerization step may bring the two kinase domains closer and thereby induce, within seconds, the phosphorylation of BAK1 and FLS2 (Schulze et al., 2010; Schwessinger et al., 2011). These steps are sufficient to initiate the immune signaling pathway, even if the ectodomains and kinase domains are switched between FLS2 and BAK1 (Albert et al., 2013).While PRRs, such as FLS2 and EFR, are extremely sensitive to even subnanomolar concentrations of their ligands, a tight control of these receptors is expected, since constitutive activation of defense responses in plants dramatically impairs fitness and growth (Tian et al., 2003; Korves and Bergelson, 2004). However, the mechanisms that underlie the attenuation of PRR activation or prevent these receptors from signaling constitutively remain largely unknown (Macho and Zipfel, 2014). Several independent observations indicate that BAK1 and FLS2 are present in close spatial proximity in preformed complexes at the plasma membrane (Chinchilla et al., 2007; Schulze et al., 2010; Roux et al., 2011). Negative regulation of immune signaling prior to ligand perception could happen within the PRR complex and depend on conformational changes following the association of FLS2 with flg22 (Meindl et al., 2000; Schulze et al., 2010; Mueller et al., 2012). Additionally, other partners might prevent the constitutive interaction of BAK1 with FLS2. Such could be the case for the LRR-RLK BAK1-INTERACTING RECEPTOR-LIKE KINASEs (BIRs): BIR2 was recently discovered as a substrate and negative regulator for BAK1, while the absence of BIR1 leads to the activation of defense induction and strong dwarfism (Gao et al., 2009; Halter et al., 2014b). Furthermore, MAMP signaling may be constrained by phosphatases, as suggested in earlier studies (Felix et al., 1994; Gómez-Gómez et al., 2001) and recently shown for the protein phosphatase 2A, which controls PRR activation likely by modulating the BAK1 phosphostatus (Segonzac et al., 2014). These examples illustrate the variety of mechanisms that may tightly control BAK1 activity.In this work, we show that regulation of BAK1 accumulation is crucial for Arabidopsis fitness, as its overexpression leads to dwarfism and premature death. The phenotype differs from BR mutants and is very reminiscent of or even identical to the autoimmune phenotype of plants showing constitutive activation of R proteins (Oldroyd and Staskawicz, 1998; Bendahmane et al., 2002; Zhang et al., 2003). BAK1 overexpression is associated with constitutive activation of defense pathway(s) involving the general coregulator of RLPs, SUPPRESSOR OF BIR1-1 (SOBIR1; Liebrand et al., 2013, 2014). To our knowledge, this is the first report and comprehensive characterization of such an autoimmunity phenotype for Arabidopsis plants overexpressing BAK1, and it highlights the importance of the regulation of PTI overactivation.