BRI1 Research Articles

Evolutionarily conserved glycan signal to degrade aberrant brassinosteroid receptors in Arabidopsis

Asparagine-linked glycans (N-glycans) are crucial signals for protein folding, quality control, and endoplasmic reticulum (ER)-associated degradation (ERAD) in yeast and mammals. Although similar ERAD processes were reported in plants, little is known about their biochemical mechanisms, especially their relationships with N-glycans. Here, we show that a missense mutation in the Arabidopsis EMS-mutagenized bri1 suppressor 3 (EBS3) gene suppresses a dwarf mutant, bri1-9, the phenotypes of which are caused by ER retention and ERAD of a brassinosteroid receptor, BRASSINOSTEROID-INSENSITIVE 1 (BR1). EBS3 encodes the Arabidopsis ortholog of the yeast asparagine-linked glycosylation 9 (ALG9), which catalyzes the ER luminal addition of two terminal α1,2 mannose (Man) residues in assembling the three-branched N-glycan precursor [glucose(Glc)](3)(Man)(9)[N-acetylglucosamine(GlcNAc)](2). Consistent with recent discoveries revealing the importance of the Glc(3)Man(9)GlcNAc(2) C-branch in generating an ERAD signal, the ebs3-1 mutation prevents the Glc(3)Man(9)GlcNAc(2) assembly and inhibits the ERAD of bri1-9. By contrast, overexpression of EBS4 in ebs3-1 bri1-9, which encodes the Arabidopsis ortholog of the yeast ALG12 catalyzing the ER luminal α1,6 Man addition, adds an α1,6 Man to the truncated N-glycan precursor accumulated in ebs3-1 bri1-9, promotes the bri1-9 ERAD, and neutralizes the ebs3-1 suppressor phenotype. Furthermore, a transfer (T)-DNA insertional alg3-T2 mutation, which causes accumulation of an even smaller N-glycan precursor carrying a different exposed α1,6 Man, promotes the ERAD of bri1-9 and enhances its dwarfism. Taken together, our results strongly suggest that the glycan signal to mark an ERAD client in Arabidopsis is likely conserved to be an α1,6 Man-exposed N-glycan.

DWARF AND LOW-TILLERING acts as a direct downstream target of a GSK3/SHAGGY-like kinase to mediate brassinosteroid responses in rice.

In Arabidopsis thaliana, the GSK3/SHAGGY-like kinase BRASSINOSTEROID-INSENSITIVE2 (BIN2) plays a critical role in the brassinosteroid (BR) signaling pathway by negatively regulating the activities of bri1-EMS-SUPPRESSOR1/BRASSINAZOLE-RESISTANT1 family transcription factors that regulate the expression of downstream BR-responsive genes. In this study, we analyzed the function of a rice (Oryza sativa) GSK3/SHAGGY-like kinase (GSK2), which is one of the orthologs of BIN2. Overexpression of GSK2 (Go) led to plants with typical BR loss-of-function phenotypes, and suppression of GSK2 resulted in enhanced BR signaling phenotypes. DWARF AND LOW-TILLERING (DLT) is a positive regulator that mediates several BR responses in rice. Suppression of DLT can enhance the phenotypes of BR receptor mutant d61-1, and overexpression of DLT obviously suppressed the BR loss-of-function phenotypes of both d61-1 and Go, suggesting that DLT functions downstream of GSK2 to modulate BR responses. Indeed, GSK2 can interact with DLT and phosphorylate DLT. Moreover, brassinolide treatment can induce the dephosphorylation of DLT, leading to the accumulation of dephosphorylated DLT protein. In GSK2 transgenic plants, the DLT phosphorylation level is dictated by the GSK2 level. These results demonstrate that DLT is a GSK2 substrate, further reinforcing that the BIN2/GSK2 kinase has multiple substrates that carry out various BR responses.

Fluorescent castasterone reveals BRI1 signaling from the plasma membrane

Receptor-mediated endocytosis is an integral part of signal transduction as it mediates signal attenuation and provides spatial and temporal dimensions to signaling events. One of the best-studied leucine-rich repeat receptor-like kinases in plants, BRASSINOSTEROID INSENSITIVE 1 (BRI1), perceives its ligand, the brassinosteroid (BR) hormone, at the cell surface and is constitutively endocytosed. However, the importance of endocytosis for BR signaling remains unclear. Here we developed a bioactive, fluorescent BR analog, Alexa Fluor 647-castasterone (AFCS), and visualized the endocytosis of BRI1-AFCS complexes in living Arabidopsis thaliana cells. Impairment of endocytosis dependent on clathrin and the guanine nucleotide exchange factor for ARF GTPases (ARF-GEF) GNOM enhanced BR signaling by retaining active BRI1-ligand complexes at the plasma membrane. Increasing the trans-Golgi network/early endosome pool of BRI1-BR complexes did not affect BR signaling. Our findings provide what is to our knowledge the first visualization of receptor-ligand complexes in plants and reveal clathrin- and ARF-GEF-dependent endocytic regulation of BR signaling from the plasma membrane.

SPEECHLESS integrates brassinosteroid and stomata signalling pathways

Stomatal formation is regulated by multiple developmental and environmental signals, but how these signals are integrated to control this process is not fully understood. In Arabidopsis thaliana, the basic helix-loop-helix transcription factor SPEECHLESS (SPCH) regulates the entry, amplifying and spacing divisions that occur during stomatal lineage development. SPCH activity is negatively regulated by mitogen-activated protein kinase (MAPK)-mediated phosphorylation. Here, we show that in addition to MAPKs, SPCH activity is also modulated by brassinosteroid (BR) signalling. The GSK3/SHAGGY-like kinase BIN2 (BR INSENSITIVE2) phosphorylates residues overlapping those targeted by the MAPKs, as well as four residues in the amino-terminal region of the protein outside the MAPK target domain. These phosphorylation events antagonize SPCH activity and limit epidermal cell proliferation. Conversely, inhibition of BIN2 activity in vivo stabilizes SPCH and triggers excessive stomatal and non-stomatal cell formation. We demonstrate that through phosphorylation inputs from both MAPKs and BIN2, SPCH serves as an integration node for stomata and BR signalling pathways to control stomatal development in Arabidopsis.

Calcium/calmodulin inhibition of the Arabidopsis BRASSINOSTEROID-INSENSITIVE 1 receptor kinase provides a possible link between calcium and brassinosteroid signalling

The receptor kinase BRI1 (BRASSINOSTEROID-INSENSITIVE 1) is a key component in BR (brassinosteroid) perception and signal transduction, and has a broad impact on plant growth and development. In the present study, we demonstrate that Arabidopsis CaM (calmodulin) binds to the recombinant cytoplasmic domain of BRI1 in a Ca2+-dependent manner in vitro. In silico analysis predicted binding to Helix E of the BRI1 kinase subdomain VIa and a synthetic peptide based on this sequence interacted with Ca2+/CaM. Co-expression of CaM with the cytoplasmic domain of BRI1 in Escherichia coli strongly reduced autophosphorylation of BRI1, in particular on tyrosine residues, and also reduced the BRI1-mediated transphosphorylation of E. coli proteins on tyrosine, threonine and presumably serine residues. Several isoforms of CaM and CMLs (CaM-like proteins) were more effective (AtCaM6, AtCaM7 and AtCML8, where At is Arabidopsis thaliana) than others (AtCaM2, AtCaM4 and AtCML11) when co-expressed with BRI1 in E. coli. These results establish a novel assay for recombinant BRI1 transphosphorylation activity and collectively uncover a possible new link between Ca2+ and BR signalling.

Tyrosine Phosphorylation of the BRI1 Receptor Kinase Occurs via a Post-Translational Modification and is Activated by the Juxtamembrane Domain

In metazoans, receptor kinases control many essential processes related to growth and development and response to the environment. The receptor kinases in plants and animals are structurally similar but evolutionarily distinct and thus while most animal receptor kinases are tyrosine kinases the plant receptor kinases are classified as serine/threonine kinases. One of the best studied plant receptor kinases is Brassinosteroid Insensitive 1 (BRI1), which functions in brassinosteroid signaling. Consistent with its classification, BRI1 was shown in early studies to autophosphorylate in vitro exclusively on serine and threonine residues and subsequently numerous specific phosphoserine and phosphothreonine sites were identified. However, several sites of tyrosine autophosphorylation have recently been identified establishing that BRI1 is a dual-specificity kinase. This raises the paradox that BRI1 contains phosphotyrosine but was only observed to autophosphorylate on serine and threonine sites. In the present study, we demonstrate that autophosphorylation on threonine and tyrosine (and presumably serine) residues is a post-translational modification, ruling out a co-translational mechanism that could explain the paradox. Moreover, we show that in general, autophosphorylation of the recombinant protein appears to be hierarchical and proceeds in the order: phosphoserine > phosphothreonine > phosphotyrosine. This may explain why tyrosine autophosphorylation was not observed in some studies. Finally, we also show that the juxtamembrane domain of BRI1 is an activator of the kinase domain, and that kinase specificity (serine/threonine versus tyrosine) can be affected by residues outside of the kinase domain. This may have implications for identification of signature motifs that distinguish serine/threonine kinases from dual-specificity kinases.

Transphosphorylation of E. coli Proteins during Production of Recombinant Protein Kinases Provides a Robust System to Characterize Kinase Specificity

Protein kinase specificity is of fundamental importance to pathway regulation and signal transduction. Here, we report a convenient system to monitor the activity and specificity of recombinant protein kinases expressed in E. coli. We apply this to the study of the cytoplasmic domain of the plant receptor kinase BRASSINOSTEROID-INSENSITIVE 1 (BRI1), which functions in brassinosteroid (BR) signaling. Recombinant BRI1 is catalytically active and both autophosphorylates and transphosphorylates E. coli proteins in situ. Using enrichment approaches followed by LC-MS/MS, phosphosites were identified allowing motifs associated with auto- and transphosphorylation to be characterized. Four lines of evidence suggest that transphosphorylation of E. coli proteins by BRI1 is specific and therefore provides meaningful results: (1) phosphorylation is not correlated with bacterial protein abundance; (2) phosphosite stoichiometry, estimated by spectral counting, is also not related to protein abundance; (3) a transphosphorylation motif emerged with strong preference for basic residues both N- and C-terminal to the phosphosites; and (4) other protein kinases (BAK1, PEPR1, FLS2, and CDPKβ) phosphorylated a distinct set of E. coli proteins and phosphosites. The E. coli transphosphorylation assay can be applied broadly to protein kinases and provides a convenient and powerful system to elucidate kinase specificity.

Deactivation of the Arabidopsis BRASSINOSTEROID INSENSITIVE 1 (BRI1) receptor kinase by autophosphorylation within the glycine-rich loop

The activity of the dual-specificity receptor kinase, brassinosteroid insensitive 1 (BRI1), reflects the balance between phosphorylation-dependent activation and several potential mechanisms for deactivation of the receptor. In the present report, we elucidate a unique mechanism for deactivation that involves autophosphorylation of serine-891 in the ATP-binding domain. Serine-891 was identified previously as a potential site of autophosphorylation by mass spectrometry, and sequence-specific antibodies and mutagenesis studies now unambiguously establish phosphorylation of this residue. In vivo, phosphorylation of serine-891 increased slowly with time following application of brassinolide (BL) to Arabidopsis seedlings, whereas phosphorylation of threonine residues increased rapidly and then remained constant. Transgenic plants expressing the BRI1(S891A)-Flag-directed mutant have increased hypocotyl and petiole lengths, relative to wild-type BRI1-Flag (both in the bri1-5 background), and accumulate higher levels of the unphosphorylated form of the BES1 transcription factor in response to exogenous BL. In contrast, plants expressing the phosphomimetic S891D-directed mutant are severely dwarfed and do not accumulate unphosphorylated BES1 in response to BL. Collectively, these results suggest that autophosphorylation of serine-891 is one of the deactivation mechanisms that inhibit BRI1 activity and BR signaling in vivo. Many arginine-aspartate (RD)-type leucine-rich repeat receptor-like kinases have a phosphorylatable residue within the ATP-binding domain, suggesting that this mechanism may play a broad role in receptor kinase deactivation.

T-DNA mutagenesis in Brachypodium distachyon

During the past decade, Brachypodium distachyon has emerged as an attractive experimental system and genomics model for grass research. Numerous molecular tools and genomics resources have already been developed. Functional genomics resources, including mutant collections, expression/tiling microarray, mapping populations, and genome re-sequencing for natural accessions, are rapidly being developed and made available to the community. In this article, the focus is on the current status of systematic T-DNA mutagenesis in Brachypodium. Large collections of T-DNA-tagged lines are being generated by a community of laboratories in the context of the International Brachypodium Tagging Consortium. To date, >13 000 lines produced by the BrachyTAG programme and USDA-ARS Western Regional Research Center are available by online request. The utility of these mutant collections is illustrated with some examples from the BrachyTAG collection at the John Innes Centre-such as those in the eukaryotic initiation factor 4A (eIF4A) and brassinosteroid insensitive-1 (BRI1) genes. A series of other mutants exhibiting growth phenotypes is also presented. These examples highlight the value of Brachypodium as a model for grass functional genomics.

Dual Role of BKI1 and 14-3-3 s in Brassinosteroid Signaling to Link Receptor with Transcription Factors

The plasma membrane-localized plant steroid hormone receptor, BRASSINOSTEROID INSENSITIVE 1 (BRI1), is quiescent in the absence of steroids, largely due to a negative regulator, BRI1 KINASE INHIBITOR 1 (BKI1). Here, we report that the steroid-induced, plasma membrane-dissociated and phosphorylated BKI1 also plays positive roles in BR signaling by interacting with a subset of 14-3-3 proteins. The cytosolic fraction of BKI1 carboxyl terminal region enhances BR signaling. Mutations of two serine residues in this region lead to reduced phosphorylation by the BRI1 kinase and constitutive plasma membrane localization. The 14-3-3 proteins can interact with the phosphorylated BKI1 through a motif that contains the two phosphorylation sites to release inhibition of BRI1 by BKI1. Meanwhile, the cytosolic BKI1 antagonizes the 14-3-3s and enhances accumulation of BRI1 EMS SUPPRESSOR 1 (BES1)/BRASSINAZOLE RESISTANT 1 (BZR1) in the nucleus to regulate BR-responses.

Quantification of the brassinosteroid insensitive1 receptor in planta.

In plants, green fluorescent protein (GFP) is routinely used to determine the subcellular location of fusion proteins. Here, we show that confocal imaging can be employed to approximate the number of GFP-labeled protein molecules present in living Arabidopsis (Arabidopsis thaliana) root cells. The technique involves calibration with soluble GFP to provide a usable protein concentration range within the confocal volume of the microscope. As a proof of principle, we quantified the Brassinosteroid Insensitive1 (BRI1) receptor fused to GFP, under control of its own promoter. The number of BRI1-GFP molecules per root epidermal cell ranges from 22,000 in the meristem and 130,000 in the elongation zone to 80,000 in the maturation zone, indicating that up to 6-fold differences in BRI1 receptor content exist. In contrast, when taking into account differences in cell size, BRI1-GFP receptor density in the plasma membrane is kept constant at 12 receptors μm⁻² in all cells throughout the meristem and elongation zone. Only the quiescent center and columella cells deviate from this pattern and have 5 to 6 receptors μm⁻². Remarkably, root cell sensitivity toward brassinosteroids appears to coincide with uniform meristem receptor density.

Enhancing Arabidopsis Leaf Growth by Engineering the BRASSINOSTEROID INSENSITIVE1 Receptor Kinase

The BRASSINOSTEROID INSENSITIVE1 (BRI1) receptor kinase has recently been shown to possess tyrosine kinase activity, and preventing autophosphorylation of the tyrosine-831 regulatory site by site-directed mutagenesis enhances shoot growth. In this study, we characterized the increased leaf growth of Arabidopsis (Arabidopsis thaliana) plants expressing BRI1(Y831F)-Flag compared with BRI1-Flag (both driven by the native promoter and expressed in the bri1-5 weak allele background) and provide insights into the possible mechanisms involved. On average, relative leaf growth rate was increased 16% in the Y831F plants (in the bri1-5 background), and the gain of function of the Y831F-directed mutant was dominant in the wild-type background. Leaves were larger as a result of increased cell numbers and had substantially increased vascularization. Transcriptome analysis indicated that genes associated with brassinolide biosynthesis, secondary cell wall biosynthesis and vascular development, and regulation of growth were altered in expression and may contribute to the observed changes in leaf architecture and whole plant growth. Analysis of gas exchange and chlorophyll fluorescence indicated that Y831F mutant plants had higher rates of photosynthesis, and metabolite analysis documented enhanced accumulation of starch, sucrose, and several amino acids, most prominently glycine and proline. These results demonstrate that mutation of BRI1 can enhance photosynthesis and leaf growth/vascularization and may suggest new approaches to increase whole plant carbon assimilation and growth.

Structural insight into brassinosteroid perception by BRI1

Brassinosteroids are essential phytohormones that have crucial roles in plant growth and development. Perception of brassinosteroids requires an active complex of BRASSINOSTEROID-INSENSITIVE 1 (BRI1) and BRI1-ASSOCIATED KINASE 1 (BAK1). Recognized by the extracellular leucine-rich repeat (LRR) domain of BRI1, brassinosteroids induce a phosphorylation-mediated cascade to regulate gene expression. Here we present the crystal structures of BRI1(LRR) in free and brassinolide-bound forms. BRI1(LRR) exists as a monomer in crystals and solution independent of brassinolide. It comprises a helical solenoid structure that accommodates a separate insertion domain at its concave surface. Sandwiched between them, brassinolide binds to a hydrophobicity-dominating surface groove on BRI1(LRR). Brassinolide recognition by BRI1(LRR) is through an induced-fit mechanism involving stabilization of two interdomain loops that creates a pronounced non-polar surface groove for the hormone binding. Together, our results define the molecular mechanisms by which BRI1 recognizes brassinosteroids and provide insight into brassinosteroid-induced BRI1 activation.

Structural basis of steroid hormone perception by the receptor kinase BRI1.

Polyhydroxylated steroids are regulators of body shape and size in higher organisms. In metazoans, intracellular receptors recognize these molecules. Plants, however, perceive steroids at membranes, using the membrane-integral receptor kinase BRASSINOSTEROID INSENSITIVE 1 (BRI1). Here we report the structure of the Arabidopsis thaliana BRI1 ligand-binding domain, determined by X-ray diffraction at 2.5 Å resolution. We find a superhelix of 25 twisted leucine-rich repeats (LRRs), an architecture that is strikingly different from the assembly of LRRs in animal Toll-like receptors. A 70-amino-acid island domain between LRRs 21 and 22 folds back into the interior of the superhelix to create a surface pocket for binding the plant hormone brassinolide. Known loss- and gain-of-function mutations map closely to the hormone-binding site. We propose that steroid binding to BRI1 generates a docking platform for a co-receptor that is required for receptor activation. Our findings provide insight into the activation mechanism of this highly expanded family of plant receptors that have essential roles in hormone, developmental and innate immunity signalling.

Tyrosine phosphorylation controls brassinosteroid receptor activation by triggering membrane release of its kinase inhibitor

Receptor tyrosine kinases control many critical processes in metazoans, but these enzymes appear to be absent in plants. Recently, two Arabidopsis receptor kinases--BRASSINOSTEROID INSENSITIVE 1 (BRI1) and BRI1-ASSOCIATED KINASE1 (BAK1), the receptor and coreceptor for brassinosteroids--were shown to autophosphorylate on tyrosines. However, the cellular roles for tyrosine phosphorylation in plants remain poorly understood. Here, we report that the BRI1 KINASE INHIBITOR 1 (BKI1) is tyrosine phosphorylated in response to brassinosteroid perception. Phosphorylation occurs within a reiterated [KR][KR] membrane targeting motif, releasing BKI1 into the cytosol and enabling formation of an active signaling complex. Our work reveals that tyrosine phosphorylation is a conserved mechanism controlling protein localization in all higher organisms.

Characterization of cp3 reveals a new bri1 allele, bri1-120, and the importance of the LRR domain of BRI1 mediating BR signaling

BackgroundSince the identification of BRI1 (BRASSINOSTEROID-INSENSITIVE1), a brassinosteroids (BRs) receptor, most of the critical roles of BR in plant development have been assessed using various bri1 mutant alleles. The characterization of individual bri1 mutants has shown that both the extracellular and cytoplasmic domains of BRI1 are important to its proper functioning. Particularly, in the extracellular domain, regions near the 70-amino acid island are known to be critical to BR binding. In comparison, the exact function of the leucine rich-repeats (LRR) region located before the 70-amino acid island domain in the extracellular cellular portion of BRI1 has not yet been described, due to a lack of specific mutant alleles.ResultsAmong the mutants showing altered growth patterns compared to wild type, we further characterized cp3, which displayed defective growth and reduced BR sensitivity. We sequenced the genomic DNA spanning BRI1 in the cp3 and found that cp3 has a point mutation in the region encoding the 13th LRR of BRI1, resulting in a change from serine to phenylalanine (S399F). We renamed it bri1-120. We also showed that overexpression of the wild type BRI1 protein rescued the phenotype of bri1-120. Using a GFP-tagged bri1-120 construct, we detected the bri1-120 protein in the plasma membrane, and showed that the phenotypic defects in the rosette leaves of bri1-301, a kinase-inactive weak allele of BRI1, can be restored by the overexpression of the bri1-120 proteins in bri1-301. We also produced bri1-301 mutants that were wild type in appearance by performing a genetic cross between bri1-301 and bri1-120 plants.ConclusionsWe identified a new bri1 allele, bri1-120, whose mutation site has not yet been found or characterized. Our results indicated that the extracellular LRR regions before the 70-amino acid island domain of BRI1 are important for the appropriate cellular functioning of BRI1. Also, we confirmed that a successful interallelic complementation occurs between the extracellular domain mutant allele and the cytoplasmic kinase-inactive mutant allele of BRI1 in vivo.

Combining Enhanced Root and Shoot Growth Reveals Cross Talk between Pathways That Control Plant Organ Size in Arabidopsis

Functionally distinct Arabidopsis (Arabidopsis thaliana) genes that positively affect root or shoot growth when ectopically expressed were combined to explore the feasibility of enhanced biomass production. Enhanced root growth resulting from cytokinin deficiency was obtained by overexpressing CYTOKININ OXIDASE/DEHYDROGENASE3 (CKX3) under the control of the root-specific PYK10 promoter. Plants harboring the PYK10-CKX3 construct were crossed with four different transgenic lines showing enhanced leaf growth. For all combinations, the phenotypic traits of the individual lines could be combined, resulting in an overall growth increase. Unexpectedly, three out of four combinations had more than additive effects. Both leaf and root growth were synergistically enhanced in plants ectopically expressing CKX3 and BRASSINOSTEROID INSENSITIVE1, indicating cross talk between cytokinins and brassinosteroids. In agreement, treatment of PYK10-CKX3 plants with brassinolide resulted in a dramatic increase in lateral root growth that could not be observed in wild-type plants. Coexpression of CKX3 and the GROWTH-REGULATING FACTOR5 (GRF5) antagonized the effects of GRF5 overexpression, revealing an interplay between cytokinins and GRF5 during leaf cell proliferation. The combined overexpression of CKX3 and GIBBERELLIN 20-OXIDASE1 led to a synergistic increase in leaf growth, suggesting an antagonistic growth control by cytokinins and gibberellins. Only additive effects on root and shoot growth were visible in plants ectopically expressing both CKX3 and ARABIDOPSIS VACUOLAR PYROPHOSPHATASE1, hinting at an independent action mode. Our results show new interactions and contribute to the molecular and physiological understanding of biomass production at the whole plant level.

Latest news on Arabidopsis brassinosteroid perception and signaling

Brassinosteroids (BRs) are plant hormones regulating growth and development. In interaction with other hormones, they are involved in environmental cue responses. The present model of the BR response pathway in Arabidopsis includes the perception of the hormone by the plasma membrane (PM) receptor brassinosteroid insensitive 1 (BRI1) and its hetero-oligomerization with the co-receptor BRI1-associated receptor kinase 1 (BAK1), followed by the activation of a signaling-cascade finally resulting in the expression of BR-responsive genes. New findings have shed light on the receptor density in the PM and on the molecular mechanism of BR perception, which includes the hormone-induced formation of a platform in the BRI1 extracellular domain for interaction with BAK1. Furthermore, new knowledge on early, BRI1-initiated signaling events at the PM–cytoplasm interface has recently been gained. In addition, a fast BR response pathway that modifies the membrane potential and the expansion of the cell wall – both crucial processes preceding cell elongation growth – have been identified. In this review, these latest findings are summarized and discussed against the background of the present model of BRI1 signaling.

A genome-wide survey for Arabidopsis leucine-rich repeat receptor kinases implicated in plant immunity

Receptor-like kinases (RLK) are among the largest gene families encoded by plant genomes. Common structural features of plant RLKs are an extracellular ligand binding domain, a membrane spanning domain, and an intracellular protein kinase domain. The largest subfamily of plant RLKs is characterized by extracellular leucine-rich repeat (LRR-RLK) structures that are known biochemical modules for mediating ligand binding and protein–protein interactions. In the frame of the Arabidopsis Functional Genomics Network initiative of the German Research Foundation (DFG) we have conducted a comprehensive survey for and functional characterization of LRR-RLKs potentially implicated in Arabidopsis thaliana immunity to microbial infection. Arabidopsis gene expression patterns suggested an important role of this class of proteins in biotic stress adaptation. Detailed biochemical and physiological characterization of the brassinosteroid insensitive 1-associated receptor kinase 1 (BAK1) revealed brassinolide-independent roles of this protein in plant immunity, in addition to its well-established function in plant development. The LRR-RLK BAK1 has further been shown to form heteromeric complexes with various other LRR-RLKs in a ligand-dependent manner, suggesting a role as adapter or co-receptor in plant receptor complexes. Here, we review the current status of BAK1 and BAK1-interacting LRR-RLKs in plant immunity.

A Direct Docking Mechanism for a Plant GSK3-like Kinase to Phosphorylate Its Substrates

Glycogen synthase kinase 3 (GSK3) is a highly conserved serine/threonine protein kinase that plays important roles in a variety of physiological and developmental processes in animals. It is well known that the GSK3 kinase-catalyzed protein phosphorylation often requires a stable kinase-substrate docking interaction, which is achieved mainly by two mechanisms as follows: priming phosphorylation of a substrate by a distinct kinase to create a docking phosphate group and scaffold protein-mediated protein complex formation. Brassinosteroid-INsensitive 2 (BIN2) is an Arabidopsis GSK3-like kinase that negatively regulates brassinosteroid (BR) signaling by phosphorylating BES1 (bri1 EMS suppressor 1) and BZR1 (brassinazole-resistant 1), two highly similar transcription factors critical for bringing about characteristic BR responses. However, little is known about the biochemical mechanism by which BIN2 phosphorylates its substrates. Here, we show that BIN2 interacts directly with BZR1 through a 12-amino acid BIN2-docking motif adjacent to the C terminus of BZR1. Interestingly, this 12-amino acid motif is sufficient to allow a Drosophila GSK3 substrate Armadillo to be phosphorylated by BIN2 in vitro. Deletion of this motif inhibits the phosphorylation and subsequent degradation of BZR1 in vivo, resulting in phenotypic suppression of a hypermorphic bin2 mutation and enhanced resistance to a BR biosynthesis inhibitor. We thus concluded that BIN2 utilizes a direct kinase-substrate docking mechanism to phosphorylate BZR1 and regulate its protein stability.