Analysis of phosphorylation of the BRI1/BAK1 complex in arabidopsis reveals amino acid residues critical for receptor formation and activation of BR signaling

The plasma membrane-localized BRASSINOSTEROID-INSENSITIVE1 (BRI1) and BRI1-ASSOCIATED KINASE1 (BAK1) are a well-known receptor pair involved in brassinosteroids (BR) signaling in Arabidposis. The formation of a receptor complex in response to BRs and the subsequent activation of cytoplasmic domain kinase activity share mechanistic characteristics with animal receptor kinases. Here, we demonstrate that BRI1 and BAK1 are BR-dependently phosphorylated, and that phosphorylated forms of the two proteins persist for different lengths of time. Mutations of either protein abolished phosphorylation of the counterpart protein, implying transphosphorylation of the receptor kinases. To investigate the specific amino acids critical for formation of the receptor complex and activation of BAK1 kinase activity, we expressed several versions of BAK1 in yeast and plants. L32E and L46E substitutions resulted in a loss of binding of BAK1 to BRI1, and threonine T455 was essential for the kinase activity of BAK1 in yeast. Transgenic bri1 mutant plants overexpressing BAK1(L46E) displayed reduced apical dominance and seed development. In addition, transgenic wild type plants overexpressing BAK1(T455A) lost the phosphorylation activity normally exhibited in response to BL, leading to semi-dwarfism. These results suggest that BAK1 is a critical component regulating the duration of BR efficacy, even though it cannot directly bind BRs in plants.     

BRI1/BAK1, a receptor kinase pair mediating brassinosteroid signaling

The Arabidopsis BAK1 (BRI1 Associated receptor Kinase 1) was identified by a yeast two-hybrid screen as a specific interactor for BRI1, a critical component of a membrane brassinosteroid (BR) receptor. In yeast, BAK1/BRI1 interaction activates their kinase activities through transphosphorylation. BAK1 and BRI1 share similar gene expression and subcellular localization patterns and physically associate with each other in plants. Overexpression of the BAK1 gene leads to a phenotype reminiscent of BRI1-overexpression transgenic plants and rescues a weak bri1 mutant. In contrast, a bak1 knockout mutation gives rise to a weak bri1-like phenotype and enhances a weak bri1 mutation. We propose that BAK1 and BRI1 function together to mediate plant steroid signaling.     

Transgenic rice plants ectopically expressing AtBAK1 are semi-dwarfed and hypersensitive to 24-epibrassinolide

Brassinosteroids (BRs) are endogenous plant hormones essential for plant growth and development. Brassinosteroid insensitive1 (BRI1)-assocaiated receptor kinase (BAK1) is one of the key components in the BR signal transduction pathway due to its direct association with the BR receptor, BRI1. Although BRI1 and its orthologs have been identified from both dicotyledonous and monocotyledonous plants, less is known about BAK1 and its orthologs in higher plants other than Arabidopsis. This article provides the first piece of evidence that AtBAK1 can greatly affect growth and development of rice plants when ectopically expressed, suggesting that rice may share similar BR perception mechanism via BRI1/BAK1 complex. Interestingly, transgenic rice plants displayed semi-dwarfism and shortened primary roots. Physiological analysis and cell morphology assay demonstrated that the observed phenotypes in transgenic plants were presumably caused by hypersensitivity to endogenous levels of BRs, different from BR insensitive and deficient rice mutants. Consistently, several known BR inducible genes were also upregulated in transgenic rice plants, further suggesting that BAK1 was able to affect BR signaling in rice. On the other hand, the transgenic plants generated by overproducing AtBAK1 may potentially have agricultural applications because the dwarfed phenotype is generally resistant to lodging, while the fertility remains unaffected.     

The multifaceted function of BAK1/SERK3: Plant immunity to pathogens and responses to insect herbivores

Almost a decade ago BRI1-associated kinase 1 (BAK1) was identified as a co-receptor of brassinosteroid (BR) insensitive 1 (BRI1), the receptor for BRs, which plays an essential role in transducing BR signaling to regulate plant development. BAK1 is also critical in resistance to various pathogens. BAK1 rapidly binds to certain receptors for pathogen/microbe-associated molecular patterns (PAMPs/MAMPs) after the perception of pathogen elicitors and is required for the full elicitation of pathogen-induced defense responses, such as the activation of the mitogen-activated protein kinase 6 (MPK6) and production of reactive oxygen species. Thus, BAK1 functions in both BR signaling and PAMP-triggered immunity (PTI). Recently BAK1 was also found to play an important role in mediating defense responses against an insect herbivore (Manduca sexta) of Nicotiana attenuata. In this interaction, BAK1 positively modulates wound- or herbivore feeding-induced accumulation of jasmonic acid (JA) and JA-isoleucine (JA-Ile). This mini-review summarizes recent advances in our understanding of the functions of BAK1 in resistance to pathogens and herbivores.Key words: BAK1, defense, herbivore, immunity, insect, jasmonate, pathogen, wounding     

Sequential transphosphorylation of the BRI1/BAK1 receptor kinase complex impacts early events in brassinosteroid signaling

Brassinosteroids (BRs) regulate plant development through a signal transduction pathway involving the BRI1 and BAK1 transmembrane receptor kinases. The detailed molecular mechanisms of phosphorylation, kinase activation, and oligomerization of the BRI1/BAK1 complex in response to BRs are uncertain. We demonstrate that BR-dependent activation of BRI1 precedes association with BAK1 in planta, and that BRI1 positively regulates BAK1 phosphorylation levels in vivo. BRI1 transphosphorylates BAK1 in vitro on specific kinase-domain residues critical for BAK1 function. BAK1 also transphosphorylates BRI1, thereby quantitatively increasing BRI1 kinase activity toward a specific substrate. We propose a sequential transphosphorylation model in which BRI1 controls signaling specificity by direct BR binding followed by substrate phosphorylation. The coreceptor BAK1 is then activated by BRI1-dependent transphosphorylation and subsequently enhances signaling output through reciprocal BRI1 transphosphorylation. This model suggests both conservation and distinct differences between the molecular mechanisms regulating phosphorylation-dependent kinase activation in plant and animal receptor kinases.     

BAK1, an Arabidopsis LRR receptor-like protein kinase,interacts with BRI1 and modulates brassinosteroid signaling

Brassinosteroids regulate plant growth and development through a protein complex that includes the leucine-rich repeat receptor-like protein kinase (LRR-RLK) brassinosteroid-insensitive 1 (BRI1). Activation tagging was used to identify a dominant genetic suppressor of bri1, bak1-1D (bri1-associated receptor kinase 1-1Dominant), which encodes an LRR-RLK, distinct from BRI1. Overexpression of BAK1 results in elongated organ phenotypes, while a null allele of BAK1 displays a semidwarfed phenotype and has reduced sensitivity to brassinosteroids (BRs). BAK1 is a serine/threonine protein kinase, and BRI1 and BAK1 interact in vitro and in vivo. Expression of a dominant-negative mutant allele of BAK1 causes a severe dwarf phenotype, resembling the phenotype of null bri1 alleles. These results indicate BAK1 is a component of BR signaling.     

Specifying the role of BAK1‐interacting receptor‐like kinase 3 in brassinosteroid signaling

Brassinosteroids (BR) are involved in the control of several developmental processes ranging from root elongation to senescence and adaptation to environmental cues. Thus, BR perception and signaling have to be precisely regulated. One regulator is BRI1‐associated kinase 1 (BAK1)‐interacting receptor‐like kinase 3 (BIR3). In the absence of BR, BIR3 forms complexes with BR insensitive 1 (BRI1) and BAK1. However, the biophysical and energetic requirements for complex formation in the absence of the ligand have yet to be determined. Using computational modeling, we simulated the potential complexes between the cytoplasmic domains of BAK1, BRI1 and BIR3. Our calculations and experimental data confirm the interaction of BIR3 with BAK1 and BRI1, with the BAK1 BIR3 interaction clearly favored. Furthermore, we demonstrate that BIR3 and BRI1 share the same interaction site with BAK1. This suggests a competition between BIR3 and BRI1 for binding to BAK1, which results in preferential binding of BIR3 to BAK1 in the absence of the ligand thereby preventing the active participation of BAK1 in BR signaling. Our model also suggests that BAK1 and BRI1 can interact even while BAK1 is in complex with BIR3 at an additional binding site of BAK1 that does not allow active BR signaling.     

BAK1 plays contrasting roles in regulating abscisic acid-induced stomatal closure and abscisic acid-inhibited primary root growth in Arabidopsis

The mechanisms that balance plant growth and stress responses are poorly understood, but they appear to involve abscisic acid (ABA) signaling mediated by protein kinases. Here, to explore these mechanisms, we examined the responses of Arabidopsis thaliana protein kinase mutants to ABA treatment. We found that mutants of BRASSINOSTEROID INSENSITIVE 1-ASSOCIATED RECEPTOR KINASE 1 (BAK1) were hypersensitive to the effects of ABA on both seed germination and primary root growth. The kinase OPEN STOMATA 1 (OST1) was more highly activated by ABA in bak1 mutant than the wild type. BAK1 was not activated by ABA treatment in the dominant negative mutant abi1-1 or the pyr1 pyl4 pyl5 pyl8 quadruple mutant, but it was more highly activated by this treatment in the abi1-2 abi2-2 hab1-1 loss-of-function triple mutant than the wild type. BAK1 phosphorylates OST1 T146 and inhibits its activity. Genetic analyses suggested that BAK1 acts at or upstream of core components in the ABA signaling pathway, including PYLs, PP2Cs, and SnRK2s, during seed germination and primary root growth. Although the upstream brassinosteroid (BR) signaling components BAK1 and BR INSENSITIVE 1 (BRI1) positively regulate ABA-induced stomatal closure, mutations affecting downstream components of BR signaling, including BRASSINOSTEROID-SIGNALING KINASEs (BSKs) and BRASSINOSTEROID-INSENSITIVE 2 (BIN2), did not affect ABA-mediated stomatal movement. Thus, our study uncovered an important role of BAK1 in negatively regulating ABA signaling during seed germination and primary root growth, but positively modulating ABA-induced stomatal closure, thus optimizing the plant growth under drought stress.     

Overexpression of miR172 suppresses the brassinosteroid signaling defects of bak1 in Arabidopsis

BRI1-Associated Receptor Kinase 1 (BAK1) is a leucine-rich repeat serine/threonine receptor-like kinase (LRR-RLK) that is involved in multiple developmental pathways, such as brassinosteroid (BR) signaling, plant immunity and cell death control in plants. Because the roundish and compact rosette leaves of bak1 mutant plants are characteristic phenotypes for deficient BR signaling, we screened genetic suppressors of bak1 according to changes in leaf shape to identify new components that may be involved in BAK1-mediated BR signaling using the activation-tagging method. Here, we report bak1-SUP1, which exhibited longer and narrower rosette leaves and an increased BR sensitivity compared with those of bak1. Analyses of the T-DNA insertional site and the gene expression that was affected by the T-DNA insertion revealed that a microRNA, namely, miR172, over-accumulates in bak1-SUP1. Detailed phenotypic analyses of bak1-SUP1 and a single mutant in which the bak1 mutation was segregated out (miR172-D) revealed that the overexpression of miR172 promotes leaf length elongation in adult plants and increases the root and hypocotyl growth during the seedling stage compared with that of wild type plants. Taken together with its increased BR sensitivity, these results suggest that miR172 regulates vegetative growth patterns by modulating BR sensitivity as well as by the previously identified developmental phase transition.     

Phosphorylation-dependent differential regulation of plant growth, cell death, and innate immunity by the regulatory receptor-like kinase BAK1

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.     

Tyrosine phosphorylation in brassinosteroid signaling

Brassinosteroids (BRs) regulate plant growth and development through a complex signal transduction pathway involving BRASSINOSTEROID INSENSITIVE 1 (BRI1), which is the BR receptor, and its co-receptor BRI1-ASSOCIATED KINASE 1 (BAK1). Both proteins are classified as Ser/Thr protein kinases. Recently, we reported that recombinant cytoplasmic domains (CD) of BRI1 and BAK1 also autophosphorylate on tyrosine residues and thus are dual-specificity kinases.¹ Two sites of Tyr autophosphorylation were identified that appear to have different effects on BRI1 function. Tyr-831 in the juxtamembrane domain is not essential for kinase activity but has a regulatory role, with phosphorylation of Tyr-831 causing inhibition of growth and delay of flowering. In contrast, Tyr-956 is located in subdomain IV of the kinase domain and is essential for kinase activity, and we are speculating that the free hydroxyl group at this position is essential and thus phosphorylation of Tyr-956 would inhibit BRI1 kinase activity. Expression of BRI1(Y831F)-Flag in the weak allele bri1-5 rescued the dwarf phenotype but plants had rounder leaves, increased shoot biomass, and flowered earlier than plants expressing the BRI1(wild type)-Flag in the bri1-5 background. To further elaborate on earlier results, we present additional phenotypic analysis of transgenic Arabidopsis plants expressing BRI1(Y831F)-Flag or site-directed mutants of other Tyr residues within the kinase domain. The results highlight the unique role of Tyr-831 in regulation of BR signaling in vivo. Elucidating the molecular basis for increased biomass accumulation in plants expressing BRI1(Y831F)-Flag may have applications for agriculture.Key words: brassinosteroids, LRR-RLK, autophosphorylation, tyrosine phosphorylation, signal transduction     

Genetic evidence for an indispensable role of somatic embryogenesis receptor kinases in brassinosteroid signaling

The Arabidopsis thaliana somatic embryogenesis receptor kinases (SERKs) consist of five members, SERK1 to SERK5, of the leucine-rich repeat receptor-like kinase subfamily II (LRR-RLK II). SERK3 was named BRI1-Associated Receptor Kinase 1 (BAK1) due to its direct interaction with the brassinosteroid (BR) receptor BRI1 in vivo, while SERK4 has also been designated as BAK1-Like 1 (BKK1) for its functionally redundant role with BAK1. Here we provide genetic and biochemical evidence to demonstrate that SERKs are absolutely required for early steps in BR signaling. Overexpression of four of the five SERKs-SERK1, SERK2, SERK3/BAK1, and SERK4/BKK1-suppressed the phenotypes of an intermediate BRI1 mutant, bri1-5. Overexpression of the kinase-dead versions of these four genes in the bri1-5 background, on the other hand, resulted in typical dominant negative phenotypes, resembling those of null BRI1 mutants. We isolated and generated single, double, triple, and quadruple mutants and analyzed their phenotypes in detail. While the quadruple mutant is embryo-lethal, the serk1 bak1 bkk1 triple null mutant exhibits an extreme de-etiolated phenotype similar to a null bri1 mutant. While overexpression of BRI1 can drastically increase hypocotyl growth of wild-type plants, overexpression of BRI1 does not alter hypocotyl growth of the serk1 bak1 bkk1 triple mutant. Biochemical analysis indicated that the phosphorylation level of BRI1 in serk1 bak1 bkk1 is incapable of sensing exogenously applied BR. As a result, the unphosphorylated level of BES1 has lost its sensitivity to the BR treatment in the triple mutant, indicating that the BR signaling pathway has been completely abolished in the triple mutant. These data clearly demonstrate that SERKs are essential to the early events of BR signaling.     

The Pseudomonas syringae effector HopF2 suppresses Arabidopsis immunity by targeting BAK1

Pseudomonas syringae delivers a plethora of effector proteins into host cells to sabotage immune responses and modulate physiology to favor infection. The P. syringae pv. tomato DC3000 effector HopF2 suppresses Arabidopsis innate immunity triggered by multiple microbe‐associated molecular patterns (MAMP) at the plasma membrane. We show here that HopF2 possesses distinct mechanisms for suppression of two branches of MAMP‐activated MAP kinase (MAPK) cascades. In addition to blocking MKK5 (MAPK kinase 5) activation in the MEKK1 (MAPK kinase kinase 1)/MEKKs–MKK4/5–MPK3/6 cascade, HopF2 targets additional component(s) upstream of MEKK1 in the MEKK1–MKK1/2–MPK4 cascade and the plasma membrane‐localized receptor‐like cytoplasmic kinase BIK1 and its homologs. We further show that HopF2 directly targets BAK1, a plasma membrane‐localized receptor‐like kinase that is involved in multiple MAMP signaling. The interaction between BAK1 and HopF2 and between two other P. syringae effectors, AvrPto and AvrPtoB, was confirmed in vivo and in vitro. Consistent with BAK1 as a physiological target of AvrPto, AvrPtoB and HopF2, the strong growth defects or lethality associated with ectopic expression of these effectors in wild‐type Arabidopsis transgenic plants were largely alleviated in bak1 mutant plants. Thus, our results provide genetic evidence to show that BAK1 is a physiological target of AvrPto, AvrPtoB and HopF2. Identification of BAK1 as an additional target of HopF2 virulence not only explains HopF2 suppression of multiple MAMP signaling at the plasma membrane, but also supports the notion that pathogen virulence effectors act through multiple targets in host cells.     

BAK1 and BKK1 regulate brassinosteroid-dependent growth and brassinosteroid-independent cell-death pathways

Brassinosteroids (BRs) are phytosteroid hormones controlling various physiological processes critical for normal growth and development. BRs are perceived by a protein complex containing two transmembrane receptor kinases, BRASSINOSTEROID INSENSITIVE 1 (BRI1) and BRI1-ASSOCIATED RECEPTOR KINASE 1 (BAK1) [1-3]. BRI1 null mutants exhibit a dwarfed stature with epinastic leaves, delayed senescence, reduced male fertility, and altered light responses. BAK1 null mutants, however, only show a subtle phenotype, suggesting that functionally redundant proteins might be present in the Arabidopsis genome. Here we report that BAK1-LIKE 1 (BKK1) functions redundantly with BAK1 in regulating BR signaling. Surprisingly, rather than the expected bri1-like phenotype, bak1 bkk1 double mutants exhibit a seedling-lethality phenotype due to constitutive defense-gene expression, callose deposition, reactive oxygen species (ROS) accumulation, and spontaneous cell death even under sterile growing conditions. Our detailed analyses demonstrate that BAK1 and BKK1 have dual physiological roles: positively regulating a BR-dependent plant growth pathway, and negatively regulating a BR-independent cell-death pathway. Both BR signaling and developmentally controlled cell death are critical to optimal plant growth and development, but the mechanisms regulating early events in these pathways are poorly understood. This study provides novel insights into the initiation and crosstalk of the two signaling cascades.     

Brassinosteroid-independent functions of the BRI1-associated kinase BAK1/SERK3

Eukaryotes have evolved programmed cell death (PCD) mechanisms that play important roles in both, development and immunity.^1–3 We demonstrated a requirement for the Arabidopsis thaliana leucine-rich repeat receptor-like kinase (LRR-RLK), BAK1/SERK3 (BRI1-Associated receptor Kinase 1/Somatic Embryogenesis Receptor Kinase 3) in regulating the containment of microbial infection-induced necrosis. BAK1-deficient plants showed constitutive expression of defense-related genes and developed spreading cell death upon infection by necrotizing pathogens that result in enhanced susceptibility to necrotrophic pathogens. This reaction was not inducible by exposition of bak1 mutants to general stresses but appeared to be solely inducible by necrotizing pathogen infection. BAK1 is known to interact with the brassinosteroid receptor, BRI1, and thereby facilitates plant growth and development in a brassinolide (BL)-dependent manner.^4,5 Surprisingly, the cell death-related phenotype in bak1 mutants is brassinolide-independent. In this addendum we want to present recent new data on BAK1 and discuss its role as a general regulator in plant processes being as diverse as brassinosteroid signaling in development, perception of pathogen associated molecular patterns (PAMPs), and cell-death control in innate immunity.Key words: LRR-RLK, cell-death control, immunity, brassinosteroids, BAK1, SERK3, BRI1, FLS2     

受体激酶介导的油菜素内酯信号转导途径

油菜素内酯（brassinosteroids,BRs）是一类重要的类固醇激素,参与调控植物生长发育的许多过程。结合应用遗传学、生物化学以及蛋白质组学等研究手段现已基本阐明了BR信号转导的主要过程。BRI1作为受体在细胞表面感知BR,BRI1抑制子BKI1从质膜上解离下来,使BRI1与其共受体BAK1结合。BRI1和BAK1通过顺序磷酸化将BR信号完全激活。活化的BRI1将BSK磷酸化激活,BSK活化BSU1,BSU1将BIN2去磷酸化使其失活,解除BIN2对BES1/BZR1的抑制功能。PP2A可以将BES1/BZR1去磷酸化激活,又可以将受体BRI1去磷酸化促使其降解。BR信号的传递最终使去磷酸化状态的BES1/BZR1在细胞内累积,激活BR信号通路下游的转录调控。     

Identification and functional analysis of in vivo phosphorylation sites of the Arabidopsis BRASSINOSTEROID-INSENSITIVE1 receptor kinase

Brassinosteroids (BRs) regulate multiple aspects of plant growth and development and require an active BRASSINOSTEROID-INSENSITIVE1 (BRI1) and BRI1-ASSOCIATED RECEPTOR KINASE1 (BAK1) for hormone perception and signal transduction. Many animal receptor kinases exhibit ligand-dependent oligomerization followed by autophosphorylation and activation of the intracellular kinase domain. To determine if early events in BR signaling share this mechanism, we used coimmunoprecipitation of epitope-tagged proteins to show that in vivo association of BRI1 and BAK1 was affected by endogenous and exogenous BR levels and that phosphorylation of both BRI1 and BAK1 on Thr residues was BR dependent. Immunoprecipitation of epitope-tagged BRI1 from Arabidopsis thaliana followed by liquid chromatography-tandem mass spectrometry (LC/MS/MS) identified S-838, S-858, T-872, and T-880 in the juxtamembrane region, T-982 in the kinase domain, and S-1168 in C-terminal region as in vivo phosphorylation sites of BRI1. MS analysis also strongly suggested that an additional two residues in the juxtamembrane region and three sites in the activation loop of kinase subdomain VII/VIII were phosphorylated in vivo. We also identified four specific BAK1 autophosphorylation sites in vitro using LC/MS/MS. Site-directed mutagenesis of identified and predicted BRI1 phosphorylation sites revealed that the highly conserved activation loop residue T-1049 and either S-1044 or T-1045 were essential for kinase function in vitro and normal BRI1 signaling in planta. Mutations in the juxtamembrane or C-terminal regions had only small observable effects on autophosphorylation and in planta signaling but dramatically affected phosphorylation of a peptide substrate in vitro. These findings are consistent with many aspects of the animal receptor kinase model in which ligand-dependent autophosphorylation of the activation loop generates a functional kinase, whereas phosphorylation of noncatalytic intracellular domains is required for recognition and/or phosphorylation of downstream substrates.