BRI1 Research Articles

Genome-wide identification and expression profiling analysis of brassinolide signal transduction genes regulating apple tree architecture

Brassinolide (BR) is crucial for regulating plant architecture. Apple dwarfing rootstocks are used to control apple tree size. However, information regarding the effects of BR on apple trees is limited. In addition, the molecular mechanism underlying the dwarfing of apple rootstocks is poorly understood. To elucidate the role of BR signal transduction genes in controlling apple tree architecture, five BR receptor kinase 1 (BRI1), nine BR-signaling kinase 1 (BSK1), two BRI1 KINASE INHIBITOR 1 (BKI1), and seven BR-insensitive 2 (BIN2) genes were analyzed. Bioinformatic analyses revealed that gene duplication events likely contributed to the expansion and evolution of the identified genes. Nine homologs between apple and Arabidopsis thaliana were also identified, and their expression patterns in different tissues were characterized. Exogenous BR treatments increased the primary shoot length and altered the expression of BR signal transduction genes (MdBRI1-5, MdBSK3-8, MdBKI1–2, MdBIN1–4, and MdBIN6/7). The scion of Fuji/Malling 9 (M.9) trees exhibited inhibited growth compared with that of Fuji/Fuji trees. The Fuji/M.9 trees had lower levels of the positive regulators of BR signaling (MdBRI1-5,MdBSK1, MdBSK4/7, and MdBSK6) and higher levels of the negative regulators (MdBIN5-7) compared with the Fuji/Fuji trees. Thus, the above-mentioned genes may help to regulate apple tree size in response to BR. In addition, MdBRI1–5, MdBSK1, MdBSK4/7, MdBSK6, and MdBIN5–7 have important roles in different grafting combinations. Our results may provide the basis for future analyses of BR signal transduction genes regarding their potential involvement in the regulation of plant architecture.

Molecular dynamics simulations reveal the conformational dynamics of Arabidopsis thaliana BRI1 and BAK1 receptor-like kinases

The structural motifs responsible for activation and regulation of eukaryotic protein kinases in animals have been studied extensively in recent years, and a coherent picture of their activation mechanisms has begun to emerge. In contrast, non-animal eukaryotic protein kinases are not as well understood from a structural perspective, representing a large knowledge gap. To this end, we investigated the conformational dynamics of two key Arabidopsis thaliana receptor-like kinases, brassinosteroid-insensitive 1 (BRI1) and BRI1-associated kinase 1 (BAK1), through extensive molecular dynamics simulations of their fully phosphorylated kinase domains. Molecular dynamics simulations calculate the motion of each atom in a protein based on classical approximations of interatomic forces, giving researchers insight into protein function at unparalleled spatial and temporal resolutions. We found that in an otherwise "active" BAK1 the αC helix is highly disordered, a hallmark of deactivation, whereas the BRI1 αC helix is moderately disordered and displays swinging behavior similar to numerous animal kinases. An analysis of all known sequences in the A. thaliana kinome found that αC helix disorder may be a common feature of plant kinases.

Phosphosite charge rather than shootward localization determines OCTOPUS activity in root protophloem

Polar cellular localization of proteins is often associated with their function and activity. In plants, relatively few polar-localized factors have been described. Among them, the plasma membrane-associated Arabidopsis proteins OCTOPUS (OPS) and BREVIS RADIX (BRX) display shootward and rootward polar localization, respectively, in developing root protophloem cells. Both ops and brx null mutants exhibit defects in protophloem differentiation. Here we show that OPS and BRX act genetically in parallel in this process, although OPS dosage increase mends defects caused by brx loss-of-function. OPS protein function is ancient and conserved in the most basal angiosperms; however, many highly conserved structural OPS features are not strictly required for OPS function. They include a BRASSINOSTEROID INSENSITIVE 2 (BIN2) interaction domain, which supposedly mediates gain-of-function effects obtained through ectopic OPS overexpression. However, engineering an increasingly positive charge in a critical phosphorylation site, S318, progressively amplifies OPS activity. Such hyperactive OPS versions can even complement the severe phenotype of brx ops double mutants, and the most active variants eventually trigger gain-of-function phenotypes. Finally, BRX-OPS as well as OPS-BRX fusion proteins localize to the rootward end of developing protophloem cells, but complement ops mutants as efficiently as shootward localized OPS. Thus, our results suggest that S318 phosphorylation status, rather than a predominantly shootward polar localization, is a primary determinant of OPS activity.

BRL3 and AtRGS1 cooperate to fine tune growth inhibition and ROS activation.

Plasma membrane-localized leucine-rich repeat receptor-like kinases directly activates G protein complex via interaction with seven transmembrane domain Regulator of G-protein Signaling 1 (AtRGS1) protein. Brassinosteroid insensitive 1 (BRI1) LIKE3 (BRL3) phosphorylates AtRGS1 in vitro. FRET analysis showed that BRL3 and AtRGS1 interaction dynamics change in response to glucose and flg22. Both BRL3 and AtRGS1 function in glucose sensing and brl3 and rgs1-2 single mutants are hyposensitive to high glucose as well as the brl3/rgs1 double mutant. BRL3 and AtRGS1 function in the same pathway linked to high glucose sensing. Hypocotyl elongation, another sugar-mediated pathway, is also implicated to be partially mediated by BRL3 and AtRGS1 because rgs1-2, brl3-2 and brl3-2/ rgs1-2 mutants share the long hypocotyl phenotype. BRL3 and AtRGS1 modulate the flg22-induced ROS burst and block one or more components positively regulating ROS production because the brl3/rgs1 double mutant has ~60% more ROS production than wild type while rgs1-2 has a partial ROS burst impairment and brl3 has slightly more ROS production. Here, we proposed a simple model where both BRL3 and AtRGS1 are part of a fine-tuning mechanism sensing glucose and flg22 to prevent excess ROS burst and control growth inhibition.

Arabidopsis glycosylphosphatidylinositol-anchored protein LLG1 associates with and modulates FLS2 to regulate innate immunity

Plants detect and respond to pathogen invasion with membrane-localized pattern recognition receptors (PRRs), which recognize pathogen-associated molecular patterns (PAMPs) and activate downstream immune responses. Here we report that Arabidopsis thaliana LORELEI-LIKE GPI-ANCHORED PROTEIN 1 (LLG1), a coreceptor of the receptor-like kinase FERONIA, regulates PRR signaling. In a forward genetic screen for suppressors of enhanced disease resistance 1 (edr1), we identified the point mutation llg1-3, which suppresses edr1 disease resistance but does not affect plant growth and development. The llg1 mutants show enhanced susceptibility to various virulent pathogens, indicating that LLG1 has an important role in plant immunity. LLG1 constitutively associates with the PAMP receptor FLAGELLIN SENSING 2 (FLS2) and the elongation factor-Tu receptor, and forms a complex with BRASSINOSTEROID INSENSITIVE 1-ASSOCIATED RECEPTOR KINASE 1 in a ligand-dependent manner, indicating that LLG1 functions as a key component of PAMP-recognition immune complexes. Moreover, LLG1 contributes to accumulation and ligand-induced degradation of FLS2, and is required for downstream innate immunity responses, including ligand-induced phosphorylation of BOTRYTIS-INDUCED KINASE 1 and production of reactive oxygen species. Taken together, our findings reveal that LLG1 associates with PAMP receptors and modulates their function to regulate disease responses. As LLG1 functions as a coreceptor of FERONIA and plays central roles in plant growth and development, our findings indicate that LLG1 participates in separate pathways, and may suggest a potential connection between development and innate immunity in plants.

Scanning for New BRI1 Mutations via TILLING Analysis.

The identification and characterization of a mutational spectrum for a specific protein can help to elucidate its detailed cellular functions. BRASSINOSTEROID INSENSITIVE1 (BRI1), a multidomain transmembrane receptor-like kinase, is a major receptor of brassinosteroids in Arabidopsis (Arabidopsis thaliana). Within the last two decades, over 20 different bri1 mutant alleles have been identified, which helped to determine the significance of each domain within BRI1. To further understand the molecular mechanisms of BRI1, we tried to identify additional alleles via targeted induced local lesions in genomes. Here, we report our identification of 83 new point mutations in BRI1, including nine mutations that exhibit an allelic series of typical bri1 phenotypes, from subtle to severe morphological alterations. We carried out biochemical analyses to investigate possible mechanisms of these mutations in affecting brassinosteroid signaling. A number of interesting mutations have been isolated via this study. For example, bri1-702, the only weak allele identified so far with a mutation in the activation loop, showed reduced autophosphorylation activity. bri1-705, a subtle allele with a mutation in the extracellular portion, disrupts the interaction of BRI1 with its ligand brassinolide and coreceptor BRI1-ASSOCIATED RECEPTOR KINASE1. bri1-706, with a mutation in the extracellular portion, is a subtle defective mutant. Surprisingly, root inhibition analysis indicated that it is largely insensitive to exogenous brassinolide treatment. In this study, we found that bri1-301 possesses kinase activity in vivo, clarifying a previous report arguing that kinase activity may not be necessary for the function of BRI1. These data provide additional insights into our understanding of the early events in the brassinosteroid signaling pathway.

Crop Seed Size: BR Matters

Sufficient food supply is critically important to humans, and food security will be particularly challenging in view of the ever-growing world population, decreasing arable cropland, and global warming. To meet the challenges, most crop geneticists think it will be necessary to enrich the cultivated gene pool for breeding programs, and thus equally important to increase our understanding of the underlying genetic and molecular mechanism for grain yield. However, crop yield is a complex trait that is controlled simultaneously by multiple genes (i.e., quantitative trait loci [QTLs]) and heavily influenced by the surrounding environment; hence, to dissect such a complex trait into component contributory traits is necessary.

GW5 acts in the brassinosteroid signalling pathway to regulate grain width and weight in rice.

Grain size is a major determinant of grain yield in cereal crops. qSW5/GW5, which exerts the greatest effect on rice grain width and weight, was fine-mapped to a 2,263-bp/21-kb genomic region containing a 1,212-bp deletion, respectively. Here, we show that a gene encoding a calmodulin binding protein, located ∼5 kb downstream of the 1,212-bp deletion, corresponds to qSW5/GW5. GW5 is expressed in various rice organs, with highest expression level detected in young panicles. We provide evidence that the 1,212-bp deletion affects grain width most likely through influencing the expression levels of GW5. GW5 protein is localized to the plasma membrane and can physically interact with and repress the kinase activity of rice GSK2 (glycogen synthase kinase 2), a homologue of Arabidopsis BIN2 (BRASSINOSTEROID INSENSITIVE2) kinase, resulting in accumulation of unphosphorylated OsBZR1 (Oryza sativa BRASSINAZOLE RESISTANT1) and DLT (DWARF AND LOW-TILLERING) proteins in the nucleus to mediate brassinosteroid (BR)-responsive gene expression and growth responses (including grain width and weight). Our results suggest that GW5 is a novel positive regulator of BR signalling and a viable target for genetic manipulation to improve grain yield in rice and perhaps in other cereal crops as well.

Molecular actions of two synthetic brassinosteroids, iso-carbaBL and 6-deoxoBL, which cause altered physiological activities between Arabidopsis and rice.

Brassinosteroid (BR) is an important plant hormone that is perceived by the BRASSINOSTEROID INSENSITIVE 1 (BRI1) receptor. BRI1 is conserved among dicot and monocot species; however, the molecular mechanism underlying BR perception in monocots is not fully understood. We synthesised two BRs, iso-carbabrassinolide (iso-carbaBL) and 6-deoxoBL, which have different BR activities in Arabidopsis thaliana (Arabidopsis) and rice. Our bioassay indicated that iso-carbaBL has relatively strong BR activity in Arabidopsis, but is inactive in rice and competitively inhibits BR activity. The bioactivity of 6-deoxoBL was similar to that of BL in Arabidopsis, but was much lower in rice. Binding experiments using recombinant Arabidopsis and rice BRI1 protein fragments suggested that iso-carbaBL and 6-deoxoBL bind to both receptors. These results showed that iso-carbaBL and 6-deoxoBL act as an antagonist and agonist, respectively, of BRs in rice. A docking simulation analysis suggested that iso-carbaBL fits deeper in the binding pocket to block the binding of active BR to rice BRI1. The simulated binding energy of 6-deoxoBL with rice BRI1 is much lower than that with Arabidopsis BRI1. The possible structural characteristics of rice BRI1 were determined based on the difference in the BR activities of iso-carbaBL and 6-deoxoBL in Arabidopsis and rice.

Noncanonical role of Arabidopsis COP1/SPA complex in repressing BIN2-mediated PIF3 phosphorylation and degradation in darkness

The E3 ligase CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1) has been known to mediate key signaling factors for degradation via the ubiquitin/26S proteasome pathway in both plants and animals. Here, we report a noncanonical function of Arabidopsis COP1, the central repressor of photomorphogenesis, in the form of a COP1/ SUPPRESSOR of phyA-105 (SPA) complex. We show that the COP1/SPA complex associates with and stabilizes PHYTOCHROME INTERACTING FACTOR 3 (PIF3) to repress photomorphogenesis in the dark. We identify the GSK3-like kinase BRASSINOSTEROID-INSENSITIVE 2 (BIN2) as a kinase of PIF3, which induces PIF3 degradation via 26S proteasome during skotomorphogenesis. Mutations on two typical BIN2 phosphorylation motifs of PIF3 lead to a strong stabilization of the protein in the dark. We further show that the COP1/SPA complex promotes PIF3 stability by repressing BIN2 activity. Intriguingly, without affecting BIN2 expression, the COP1/SPA complex modulates BIN2 activity through interfering with BIN2-PIF3 interaction, thereby inhibiting BIN2-mediated PIF3 phosphorylation and degradation. Taken together, our results suggest another paradigm for COP1/SPA complex action in the precise control of skotomorphogenesis.

BRASSINOSTEROID INSENSITIVE2 negatively regulates cellulose synthesis in Arabidopsis by phosphorylating cellulose synthase 1

The deposition of cellulose is a defining aspect of plant growth and development, but regulation of this process is poorly understood. Here, we demonstrate that the protein kinase BRASSINOSTEROID INSENSITIVE2 (BIN2), a key negative regulator of brassinosteroid (BR) signaling, can phosphorylate Arabidopsis cellulose synthase A1 (CESA1), a subunit of the primary cell wall cellulose synthase complex, and thereby negatively regulate cellulose biosynthesis. Accordingly, point mutations of the BIN2-mediated CESA1 phosphorylation site abolished BIN2-dependent regulation of cellulose synthase activity. Hence, we have uncovered a mechanism for how BR signaling can modulate cellulose synthesis in plants.

Sorghum DW1 positively regulates brassinosteroid signaling by inhibiting the nuclear localization of BRASSINOSTEROID INSENSITIVE 2

Semi-dwarf traits have been widely introgressed into cereal crops to improve lodging resistance. In sorghum (Sorghum bicolor L. Moench), four major unlinked dwarfing genes, Dw1-Dw4, have been introduced to reduce plant height, and among them, Dw3 and Dw1 have been cloned. Dw3 encodes a gene involved in auxin transport, whereas, Dw1 was recently isolated and identified as a gene encoding a protein of unknown function. In this study, we show that DW1 is a novel component of brassinosteroid (BR) signaling. Sorghum possessing the mutated allele of Dw1 (dw1), showed similar phenotypes to rice BR-deficient mutants, such as reduced lamina joint bending, attenuated skotomorphogenesis, and insensitivity against feedback regulation of BR-related genes. Furthermore, DW1 interacted with a negative regulator of BR signaling, BRASSINOSTEROID INSENSITIVE 2 (BIN2), and inhibited its nuclear localization, indicating that DW1 positively regulates BR signaling by inhibiting the function of BIN2. In contrast to rice and wheat breeding which used gibberellin (GA) deficiency to reduce plant height, sorghum breeding modified auxin and BR signaling. This difference may result from GA deficiency in rice and wheat does not cause deleterious side effects on plant morphology, whereas in sorghum it leads to abnormal culm bending.

NBS-LRR Protein Pik-H4 Interacts with OsBIHD1 to Balance Rice Blast Resistance and Growth by Coordinating Ethylene-Brassinosteroid Pathway.

The regulation of innate immunity and plant growth, along with the trade-off between them, affects the defense and recovery mechanisms of the plant after it is attacked by pathogens. Although it is known that hormonal crosstalk plays a major role in regulating interaction of plant growth and PAMP-triggered immunity, the relationship between plant growth and effector-triggered immunity (ETI) remains unclear. In a large-scale yeast two-hybrid screening for Pik-H4-interacting proteins, a homeodomain transcription factor OsBIHD1 was identified, which is previously known to function in biotic and abiotic stress responses. The knockout of OsBIHD1 in rice lines carrying Pik-H4 largely compromised the resistance of the rice lines to Magnaporthe oryzae, the fungus that causes rice blast. While overexpression of OsBIHD1 resulted in enhanced expression of the pathogenesis-related (PR) and ethylene (ET) synthesis genes. Moreover, OsBIHD1 was also found to directly bind to the promoter region of ethylene-synthesis enzyme OsACO3. In addition, OsBIHD1 overexpression or deficiency provoked dwarfism and reduced brassinosteroid (BR) insensitivity through repressing the expression of several critical genes involved in BR biosynthesis and BR signaling. During M. oryzae infection, transcript levels of the crucial BR catabolic genes (CYP734A2, CYP734A4, and CYP734A6) were significantly up-regulated in OsBIHD1-OX plants. Furthermore, OsBIHD1 was found to be capable of binding to the sequence-specific cis-elements on the promoters of CYP734A2 to suppress the plant growth under fungal invasion. Our results collectively suggest a model that OsBIHD1 is required for Pik-H4-mediated blast resistance through modulating the trade-off between resistance and growth by coordinating brassinosteroid-ethylene pathway.

Biological Activity of Brassinosteroids – Direct Comparison of Known and New Analogs in planta

A systematic investigation of structural modifications of brassinosteroids is presented. We describe in detail their synthetic preparation, which includes significant improvements of previously reported protocols as well as access to new analogs with functional modifications of the steroid skeleton and of the C(17)‐attached side chain. We report the biological potency of the prepared brassinosteroid analogs as plant hormones, which were carefully established in the French bean second internode elongation assay and discuss our observations in light of the recently reported structural data detailing the molecular interactions between brassinolide in the trimeric complex with the protein receptors kinases BRASSINOSTEROID INSENSITIVE 1 (BRI1) and SOMATIC EMBRYOGENESIS RECEPTOR KINASE 1 (SERK1). In a further part of this work we describe the preparation of H2O‐soluble pro‐forms of 24‐epicastasterone and we discuss their physical properties, hydrolytic stabilities and biological activity.

Visualization of BRI1 and SERK3/BAK1 Nanoclusters in Arabidopsis Roots.

Brassinosteroids (BRs) are plant hormones that are perceived at the plasma membrane (PM) by the ligand binding receptor BRASSINOSTEROID-INSENSITIVE1 (BRI1) and the co-receptor SOMATIC EMBRYOGENESIS RECEPTOR LIKE KINASE 3/BRI1 ASSOCIATED KINASE 1 (SERK3/BAK1). To visualize BRI1-GFP and SERK3/BAK1-mCherry in the plane of the PM, variable-angle epifluorescence microscopy (VAEM) was employed, which allows selective illumination of a thin surface layer. VAEM revealed an inhomogeneous distribution of BRI1-GFP and SERK3/BAK1-mCherry at the PM, which we attribute to the presence of distinct nanoclusters. Neither the BRI1 nor the SERK3/BAK1 nanocluster density is affected by depletion of endogenous ligands or application of exogenous ligands. To reveal interacting populations of receptor complexes, we utilized selective-surface observation—fluorescence lifetime imaging microscopy (SSO-FLIM) for the detection of Förster resonance energy transfer (FRET). Using this approach, we observed hetero-oligomerisation of BRI1 and SERK3 in the nanoclusters, which did not change upon depletion of endogenous ligand or signal activation. Upon ligand application, however, the number of BRI1-SERK3 /BAK1 hetero-oligomers was reduced, possibly due to endocytosis of active signalling units of BRI1-SERK3/BAK1 residing in the PM. We propose that formation of nanoclusters in the plant PM is subjected to biophysical restraints, while the stoichiometry of receptors inside these nanoclusters is variable and important for signal transduction.

Brassinosteroid signaling directs formative cell divisions and protophloem differentiation in Arabidopsis root meristems.

ABSTRACTBrassinosteroids (BRs) trigger an intracellular signaling cascade through its receptors BR INSENSITIVE 1 (BRI1), BRI1-LIKE 1 (BRL1) and BRL3. Recent studies suggest that BR-independent inputs related to vascular differentiation, for instance root protophloem development, modulate downstream BR signaling components. Here, we report that protophloem sieve element differentiation is indeed impaired in bri1 brl1 brl3 mutants, although this effect might not be mediated by canonical downstream BR signaling components. We also found that their small meristem size is entirely explained by reduced cell elongation, which is, however, accompanied by supernumerary formative cell divisions in the radial dimension. Thus, reduced cell expansion in conjunction with growth retardation, because of the need to accommodate supernumerary formative divisions, can account for the overall short root phenotype of BR signaling mutants. Tissue-specific re-addition of BRI1 activity partially rescued subsets of these defects through partly cell-autonomous, partly non-cell-autonomous effects. However, protophloem-specific BRI1 expression essentially rescued all major bri1 brl1 brl3 root meristem phenotypes. Our data suggest that BR perception in the protophloem is sufficient to systemically convey BR action in the root meristem context.

Brassinosteroid-Induced Transcriptional Repression and Dephosphorylation-Dependent Protein Degradation Negatively Regulate BIN2-Interacting AIF2 (a BR Signaling-Negative Regulator) bHLH Transcription Factor.

Brassinosteroids (BRs) are plant polyhydroxy-steroids that play important roles in plant growth and development via extensive signal integration through direct interactions between regulatory components of different signaling pathways. Recent studies have shown that diverse helix-loop-helix/basic helix-loop-helix (HLH/bHLH) family proteins are actively involved in control of BR signaling pathways and interact with other signaling pathways. In this study, we show that ATBS1-INTERACTING FACTOR 2 (AIF2), a nuclear-localized atypical bHLH transcription factor, specifically interacts with BRASSINOSTEROID-INSENSITIVE 2 (BIN2) among other BR signaling molecules. Overexpression of AIF2 down-regulated transcript expression of growth-promoting genes, thus resulting in retardation of growth. AIF2 renders plants hyposensitive to BR-induced root growth inhibition, but shows little effects on BR-promoted hypocotyl elongation. Notably, AIF2 was dephosphorylated by BR, and the dephosphorylated AIF2 was subject to proteasome-mediated degradation. AIF2 degradation was greatly induced by BR and ABA, but relatively slightly by other hormones such as auxin, gibberellin, cytokinin and ethylene. Moreover, AIF2 transcription was significantly suppressed by a BRI1/BZR1-mediated BR signaling pathway through a direct binding of BRASSINAZOLE RESISTANT 1 (BZR1) to the BR response element (BRRE) region of the AIF2 promoter. In conclusion, our study suggests that BIN2-driven AIF2 phosphorylation could augment the BIN2/AIF2-mediated negative circuit of BR signaling pathways, and the BR-induced transcriptional repression and protein degradation negatively regulate AIF2 transcription factor, reinforcing the BZR1/BES1-mediated positive BR signaling pathway.

Identification of Brassinosteroid Signaling Complexes by Coimmunoprecipitation and Mass Spectrometry.

A combination of coimmunoprecipitation (coIP) of tagged proteins followed by protein identification and quantitation using Liquid Chromatography Mass Spectrometry/Mass Spectrometry (LCMS/MS) has proven to be a reliable method to qualitatively characterize membrane-bound receptor complexes from plants. Success depends on a range of parameters, such as abundance and stability of the complex and functionality of the tagged receptors, efficiency of the protein complex isolation procedure, MS equipment, and analysis software in use. In this Chapter, we focus on the use of one of the green fluorescent protein-tagged receptors of the SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE (SERK) family, of which SERK3, also known as BRASSINOSTEROID INSENSITIVE1 (BRI1) ASSOCIATED KINASE1 (BAK1), is a coreceptor of BRI1. Like BRI1 itself, SERK3 is a leucine-rich repeat receptor kinase (LRR RK) with a single-pass transmembrane domain. The latest updated laboratory protocol is presented as well as examples of data analysis and typical results obtained. Potential drawbacks of the procedure employed for plant membrane proteins will be pointed out.

A Technical Framework for Studying the Signaling Nexus of Brassinosteroids and Immunity.

Pathway cross-communication cannot be simply tackled by studying isolated signaling systems. Yet understanding how signal transduction pathways attenuate or reinforce each other in vivo is a challenging task. In plants, biosynthesis and signaling of brassinosteroids (BRs) finely regulate growth and defense programs through a complex array of mechanistic and physiological interactions. Conversely, induction of defenses also impacts on the BR biosynthesis at the transcriptional level. In this chapter, we present an experimental framework to study the physiological connection between BR-controlled growth and defenses. We focus on the signaling pathways regulated by the two archetypal cell surface receptors, BRASSINOSTEROID INSENSITIVE1 (BRI1) and FLAGELLIN-SENSITIVE2 (FLS2), to illustrate the signaling nexus of BRs and plant immunity. In Arabidopsis thaliana, these pathways provide one of the very few systems in which the tools and mechanistic details exist to study cross talk at the molecular level.

Probing Activation and Deactivation of the BRASSINOSTEROID INSENSITIVE1 Receptor Kinase by Immunoprecipitation.

Brassinosteroids (BRs) are sterol-derived hormones that control plant growth and development. The BR receptor complex is encoded by the BRASSINOSTEROID INSENSITIVE1 (BRI1) and members of the SOMATIC EMBRYOGENESIS RECEPTOR KINASE family. For activation and deactivation, the BR receptor complex uses different posttranslational modifications and recruits various partner proteins. Here, we describe optimized immunoprecipitation protocols and variants for biochemical analyses of posttranslational modifications of BRI1 and of protein-protein interactions.