Recombinant Arabidopsis thaliana Probable inactive receptor kinase At1g48480 (RKL1)
Description
Introduction to RKL1
RKL1 (receptor-like kinase 1) is a protein encoded by the At1g48480 gene in Arabidopsis thaliana, commonly known as mouse-ear cress, a model organism in plant molecular biology. The gene is also identified by alternative locus identifiers such as T1N15.9 and T1N15_9 . RKL1 belongs to the large family of leucine-rich repeat receptor-like kinases (LRR-RLKs), which represent one of the largest gene families in plants, with more than 400 members identified in Arabidopsis alone . These proteins play crucial roles in perceiving extracellular signals and transducing them into intracellular responses, thereby regulating various aspects of plant growth, development, and defense.
The full name "Probable inactive receptor kinase At1g48480" suggests that while the protein contains kinase domain structures, it may lack phosphorylation activity typical of active kinases . This classification as "probable inactive" indicates that the protein may function through mechanisms other than direct phosphorylation of substrates, possibly serving as a scaffold for protein complexes or exhibiting alternative modes of signaling regulation.
Protein Structure and Domains
RKL1 is synthesized as a precursor protein that undergoes processing to form the mature functional protein. The mature form spans amino acids 33-655, indicating the first 32 amino acids likely constitute a signal peptide that is cleaved during processing . As a receptor-like kinase, RKL1 exhibits a modular structure consisting of three main domains:
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An extracellular domain containing leucine-rich repeats (LRRs) that likely function in ligand binding and recognition
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A transmembrane domain that anchors the protein in the cell membrane
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An intracellular kinase domain that mediates downstream signaling events
The protein contains specific structural motifs including leucine-rich repeats in the extracellular domain, which are typically involved in protein-protein interactions and ligand recognition . These structural features are characteristic of LRR-RLKs and essential for their function in signal perception and transduction.
Protein Identifiers and Classification
RKL1 is cataloged in various biological databases with specific identifiers that facilitate tracking and research:
| Database | Identifier | Description |
|---|---|---|
| UniProt | Q9LP77 | Primary protein database identifier |
| UniProt ID | Y1848_ARATH | Alternative identifier |
| PRO ID | PR:Q9LP77 | Protein Ontology identifier |
| TAIR | AT1G48480 | The Arabidopsis Information Resource identifier |
The protein is classified as "probable inactive receptor kinase At1g48480 precursor" in the UniProt database and categorized under the organism-gene category in the Protein Ontology system . The "probable inactive" designation suggests that despite having kinase-like structures, the protein may lack conventional kinase activity, possibly functioning through alternative mechanisms.
Expression Systems
Recombinant RKL1 is typically produced using bacterial expression systems, primarily Escherichia coli. This approach allows for efficient production of substantial quantities of the protein for research purposes . The expression construct typically includes:
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The coding sequence for the mature RKL1 protein (amino acids 33-655)
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A fusion tag (commonly a polyhistidine tag) to facilitate purification
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Appropriate bacterial promoters and regulatory elements for controlled expression
The recombinant protein is produced as a fusion protein with an N-terminal 10× histidine tag, which facilitates one-step purification using immobilized metal affinity chromatography . This approach yields protein suitable for various biochemical and structural studies.
Signal Transduction Pathways
Although classified as a "probable inactive receptor kinase," RKL1 appears to play significant roles in signal transduction pathways. Research indicates that RKL1 shares high homology (75% identity at the amino acid level) with another leucine-rich repeat receptor-like kinase, RLK902 . This structural similarity suggests potential functional overlap or redundancy between these proteins.
Yeast two-hybrid screening has identified three proteins (referred to as Y-1, Y-2, and Y-3) that interact specifically with the kinase domains of both RLK902 and RKL1, but not with BRI1 (brassinosteroid insensitive 1), another LRR-RLK . This specificity suggests that RKL1 and RLK902 may participate in common downstream signaling pathways distinct from those mediated by other receptor-like kinases.
Response to Environmental Stresses
Experimental evidence suggests that RKL1 may be involved in plant responses to various stress conditions. Analysis of RKL1 expression patterns indicates responsiveness to:
These findings suggest that RKL1 may function in plant defense responses against biotic and abiotic stresses. The presence of W-box elements in the promoter region, which are binding sites for WRKY transcription factors associated with stress responses, further supports this hypothesis .
Protein Interactions
The BioGRID database reports 28 protein interactors forming 30 interactions with RKL1, highlighting its potential involvement in complex protein interaction networks . These interactions may be crucial for RKL1's function in signal transduction and cellular responses to environmental cues. The identification of specific proteins that interact with RKL1's kinase domain provides insights into the downstream components of its signaling pathway.
Genetic Resources for Functional Studies
To facilitate functional studies of RKL1, several genetic resources have been developed, including T-DNA insertion lines. One significant resource is the homozygous T-DNA insertion line (CS2107837) for the root-expressed RKL1 gene, derived from SAIL_772_B09 . This line carries the following characteristics:
| Parameter | Specification |
|---|---|
| Background | Columbia (Col-0) |
| Marker | BASTA (for selection) |
| Insertion Location | RKL1 (At1g48480) |
| Expression Pattern | Root-expressed |
Such genetic resources are valuable for investigating the physiological roles of RKL1 through loss-of-function approaches. By analyzing phenotypic changes in plants lacking functional RKL1, researchers can deduce its biological functions.
Applications in Proteomic Studies
Recombinant RKL1 serves as an important tool for proteomic studies, particularly for:
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Identification of interaction partners using pull-down assays
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Analysis of protein complex formation
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Investigation of signaling mechanisms
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Development of antibodies against RKL1 for expression studies
The availability of purified recombinant protein facilitates these applications and accelerates research into the molecular mechanisms underlying RKL1's functions.
Evolutionary Conservation
Comparative genomic analyses suggest evolutionary conservation of RKL1-like proteins across plant species. For example, homologs have been identified in Prunus avium (sweet cherry), where a gene (Pav_sc0000293.1_g160.1.mk) on chromosome 3 shows significant similarity to the Arabidopsis RKL1 . This conservation across different plant lineages suggests important biological functions that have been maintained throughout plant evolution.
Product Specs
Note: While we prioritize shipping the format currently in stock, we are happy to accommodate your specific requirements. Please indicate your desired format in the order notes, and we will prepare the product accordingly.
Note: All protein shipments are standardly packaged with blue ice packs. If you require dry ice shipping, please inform us in advance as additional charges will apply.
Generally, liquid forms have a shelf life of 6 months at -20°C/-80°C, while lyophilized forms have a shelf life of 12 months at -20°C/-80°C.
Target Background
- Research suggests that RLK902 and RKL1 share common biochemical functions, particularly in signal transduction. They are implicated in various stress responses, including mechanical wounding, salicylic acid treatment, and pathogen infection. [RKL1] PMID: 15618630
Q&A
What is RKL1 and what is its structural characterization?
RKL1 (Receptor Kinase-Like 1) is encoded by the At1g48480 gene in Arabidopsis thaliana and belongs to the extensive receptor-like kinase (RLK) family. It is classified as a "probable inactive receptor kinase" with the following structural features:
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Contains a signal sequence, a single transmembrane region, and a cytoplasmic kinase domain
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Features a leucine-rich repeat (LRR) extracellular domain for potential ligand perception
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Shares 75% amino acid sequence identity with another RLK, RLK902 (At3g17840)
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Full protein consists of 655 amino acids with the functional expression region spanning residues 33-655
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Possesses an LRR-containing N-terminal domain, transmembrane domain, and a C-terminal kinase domain
The complete amino acid sequence is available and reveals conserved domains characteristic of plant RLKs, with the kinase domain showing the subdomain signatures typical of eukaryotic kinases .
What is the expression pattern of RKL1 in Arabidopsis tissues?
RKL1 shows a distinct tissue-specific expression pattern that provides clues to its potential physiological roles:
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Dominant expression in stomata cells of leaves
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Strong expression in hydathodes and trichomes of young rosette leaves
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Notable expression in floral organ abscission zones
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Expression pattern differs from its close homolog RLK902, which shows strong expression in root tips, lateral root primordia, and stipules
These expression patterns were determined using promoter-reporter fusion studies (RKL1 promoter::GUS transgenic lines) and suggest potential roles in specialized cell types related to water regulation, defense, or developmental processes.
What genetic resources are available for studying RKL1 function?
Several genetic resources have been developed for RKL1 research:
These resources enable diverse experimental approaches, from protein-level studies to genetic analysis in planta.
How does RKL1 participate in plant-pathogen interactions, particularly with Ralstonia solanacearum?
Recent genome-wide association studies (GWAS) identified RKL1 as a susceptibility factor in Arabidopsis thaliana's response to Ralstonia solanacearum, specifically the hpaP mutant strain:
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RKL1 was identified alongside three other genes (IRE3, RACK1B, and PEX3) as potential susceptibility factors
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Validation experiments confirmed RKL1 plays a role in susceptibility to bacterial wilt caused by R. solanacearum
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The study examined natural diversity in both worldwide and local A. thaliana populations, revealing different genetic architectures in response to the pathogen
The experimental approach used included:
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Phenotyping of A. thaliana accessions in response to R. solanacearum GMI1000 wild-type strain and hpaP mutant
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GWAS analysis to identify genetic loci associated with differential responses
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Reverse genetic validation of candidate genes
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Expression analysis in resistant and susceptible accessions
Quantitative data from the study showed significant broad-sense heritability estimates (H²) for response to the hpaP mutant:
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Worldwide collection: H² = 0.47 at 8 days after inoculation (p < 0.0001)
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Local population (TOU-A): H² = 0.44 at 7 days after inoculation
What are the current hypotheses about RKL1's signaling mechanisms in plant immunity?
Research on RLK signaling mechanisms provides several hypotheses about how RKL1 may function:
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Co-receptor model: RKL1 may function similarly to other LRR-RLKs by partnering with co-receptors like BAK1/SERK3 and SERK1, which have been shown to be pivotal in plant-microbe interactions
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Redundant signaling: The lack of phenotype in rkl1 and rlk902/rkl1 mutants suggests functional redundancy, where "at least one other complementary signaling pathway to these two RLKs might exist"
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Negative regulation hypothesis: Unlike the cytoplasmic receptor kinase BIK1, which is a positive regulator of RK signaling but a negative regulator of RP-type immune receptors , RKL1 may act as a susceptibility factor by negatively regulating defense responses
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Potential interplay with immunity-related RLKs: Research on Caulobacter RHG1-Arabidopsis interactions identified four RLKs (BAK1, SERK1, EFR, and AT3G28040) involved in plant growth promotion , suggesting RKL1 might function in related pathways
To investigate these hypotheses, researchers could employ:
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Co-immunoprecipitation studies to identify interaction partners
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Phosphorylation assays to examine kinase activity
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Transcriptomic analysis to identify downstream signaling components
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Genetic epistasis experiments with known immunity regulators
What experimental design approaches are recommended for studying RKL1 function?
When designing experiments to study RKL1 function, researchers should consider:
Step 1: Define variables relevant to RKL1 research
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Independent variables: RKL1 expression levels, pathogen challenge, environmental conditions
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Dependent variables: Disease symptoms, defense gene expression, reactive oxygen species production
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Control for extraneous variables: Growth conditions, plant age, genetic background
Step 2: Formulate testable hypotheses
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Null hypothesis (H0): "RKL1 disruption has no effect on plant susceptibility to R. solanacearum"
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Alternative hypothesis (H1): "Plants lacking functional RKL1 show altered susceptibility to R. solanacearum"
Step 4: Implement appropriate controls and measurements
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Use multiple T-DNA insertion lines to confirm phenotypes
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Include RKL1-complemented lines to verify gene-specific effects
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Measure various immune responses: ROS burst, callose deposition, defense gene expression
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Consider time-course experiments to capture dynamic responses
How can researchers address challenges in studying functionally redundant RLKs?
Studying RKL1 presents challenges due to potential functional redundancy, as demonstrated by the lack of obvious phenotypes in single and double mutants . To overcome these challenges:
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Generate higher-order mutants:
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Create triple or quadruple mutants targeting closely related RLKs
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Use CRISPR/Cas9 to generate multiple gene knockouts simultaneously
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Employ conditional expression systems:
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Use inducible promoters to control RKL1 expression
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Create dominant-negative versions of RKL1 to interfere with redundant proteins
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Analyze natural variation:
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Consider experimental design modifications:
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Test plants under various stress conditions rather than normal growth
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Examine subtle phenotypes using sensitive measurement techniques
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Focus on specific tissues where RKL1 is highly expressed
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Utilize systems biology approaches:
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Perform transcriptome analysis of mutants under specific conditions
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Conduct protein-protein interaction studies to identify functional partners
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Map phosphorylation networks to position RKL1 in signaling cascades
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What are the contradictions in the literature regarding RKL1 function, and how can researchers address them?
Several apparent contradictions exist in the current understanding of RKL1:
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Phenotypic contradictions:
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Functional categorization:
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Evolutionary considerations:
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High conservation across plant species suggests important function
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Lack of obvious phenotype suggests redundancy or specialized role
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To address these contradictions, researchers could:
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Design contradiction-resolving experiments:
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Test rkl1 mutants under diverse stress conditions beyond normal growth
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Perform detailed biochemical characterization of RKL1 kinase activity
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Examine subtle phenotypes in specific tissues where RKL1 is expressed
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Apply methodological approaches from contradiction literature:
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Consider contextual factors:
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Evaluate experimental conditions across studies (temperature, humidity, light)
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Examine genetic background effects on RKL1 phenotypes
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Consider developmental timing of observations
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How can genomic and transcriptomic approaches be used to further understand RKL1 function?
Advanced genomic and transcriptomic approaches offer powerful ways to investigate RKL1 function:
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Genome-wide association studies (GWAS):
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RNA-Seq analysis:
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Compare transcriptomes of wild-type and rkl1 mutants under various conditions
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Identify genes differentially expressed in response to RKL1 disruption
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Map transcriptional networks regulated by RKL1
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ChIP-Seq and DNA-binding studies:
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Identify potential transcription factors downstream of RKL1 signaling
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Map chromatin modifications associated with RKL1-dependent responses
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Proteomics approaches:
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Phosphoproteomics to identify RKL1 substrates and signaling partners
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Interactome mapping to position RKL1 in protein networks
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Quantitative proteomics to measure changes in protein abundance
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Comparative genomics:
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Analyze RKL1 evolution across plant species
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Identify conserved regulatory elements in the RKL1 promoter
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Compare function with RLK homologs in crop species
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Using these approaches in combination with traditional genetic and molecular biology techniques will provide a comprehensive understanding of RKL1 function in plant immunity and development.
