Recombinant Arabidopsis thaliana Probable receptor-like protein kinase At2g42960 (At2g42960)

What is At2g42960 and what protein family does it belong to?

At2g42960 is a protein-coding gene in Arabidopsis thaliana (thale cress) that encodes a member of the protein kinase superfamily. Specifically, it belongs to the receptor-like cytoplasmic kinase subfamily XI (RLCK XI) in plants. The gene has several synonyms including F7D19.4 and F7D19_4, and is found on chromosome 2 of A. thaliana. Several transcript variants have been identified, including NM_129858.2, NM_001337003.1, NM_001337005.1, NM_001337002.1, and NM_001337004.1, all encoding protein kinase superfamily proteins .

What are the key structural domains of the At2g42960 protein?

The At2g42960 protein contains several key structural domains characteristic of receptor-like cytoplasmic kinases in subfamily XI. These include:

• A transmembrane domain (TM) at the N-terminus

• A protein kinase (PK) domain

• A kinase insertion domain (KID)

• Multiple nuclear localization signals (NLS) within the KID and C-terminal regions

The protein kinase domain can be identified and annotated using the Conserved Domain Database (CDD), while the nuclear localization signals can be predicted using tools such as the Eukaryotic Linear Motif (ELM) resource .

What is the subcellular localization pattern of At2g42960?

Studies using GFP-tagged full-length At2g42960 protein reveal a dual localization pattern. The protein localizes both to the plasma membrane and the nucleus, with some signal also detected in the cytosol. This dual localization is directly observable in both transient expression systems (Arabidopsis protoplasts) and in stable transgenic Arabidopsis plants. The transmembrane domain directs the protein to the plasma membrane, while the nuclear localization signals within the KID domain are responsible for nuclear targeting. When the transmembrane domain is deleted (ΔTM), the protein localizes exclusively to the nucleus, demonstrating the functional importance of both domains in determining the protein's subcellular distribution pattern .

How can I clone the full-length At2g42960 gene for recombinant expression?

For cloning the full-length At2g42960 gene, a gateway cloning strategy has proven effective. PCR amplify the protein-coding sequence from Arabidopsis cDNA using gene-specific primers with appropriate attB sites. The amplified product can be subcloned into the pCR8/GW/TOPO entry vector following the manufacturer's protocol. From this entry clone, the sequence can be transferred to various destination vectors through LR recombination reactions. For protein expression and localization studies, binary vectors carrying fluorescent protein tags (such as GFP) are recommended. Note that cloning the full-length transcript may present challenges, as researchers have reported difficulties with At2g42960 homologs (At2g42960-1 and -4) . Alternatively, commercial cDNA ORF clones are available starting from $99.00 .

What expression systems are suitable for recombinant At2g42960 production?

Two principal expression systems have been successfully employed for At2g42960 expression:

• Transient expression in Arabidopsis protoplasts:

  • Transfect the construct into Arabidopsis protoplasts using polyethylene glycol

  • Observe expression after 16 hours

  • Ideal for rapid subcellular localization studies and protein domain analysis

  • Allows testing of multiple construct variants simultaneously

• Stable transgenic Arabidopsis plants:

  • Transform constructs into Arabidopsis Columbia (Col-0) using Agrobacterium tumefaciens (GV3101) floral dipping

  • For controlled expression, use inducible promoter systems (e.g., estradiol-inducible pMDC7 vector)

  • Grow transgenic T2 lines on agar plates with appropriate selection

  • Induce expression with 10 μM β-estradiol for 2 days before observation

  • Allows in vivo analysis in intact plant tissues

How can I visualize the subcellular localization of At2g42960?

The subcellular localization of At2g42960 can be visualized using confocal microscopy techniques applied to either protoplasts or intact plant tissues. This table summarizes the effective visualization methods:

For optimal results, C-terminal GFP tagging is recommended as N-terminal tagging has been shown to cause protein aggregation in the cytosol. When designing constructs, consider that different protein domains influence localization: full-length protein (membrane + nucleus), ΔTM variants (nuclear only), and ΔKID variants (membrane + cytosolic aggregates) .

What approaches can be used to investigate the role of At2g42960 in signaling pathways?

To investigate the signaling role of At2g42960, a multi-faceted approach is recommended:

• Expression profiling:

  • Use cDNA microarray analysis to examine expression changes of At2g42960 in response to pathogens and defense-related signaling molecules (salicylic acid, methyl jasmonate, ethylene)

  • Compare expression profiles across treatments to identify pathway associations

• Domain-specific functional analysis:

  • Generate constructs with specific domain deletions (ΔTM, ΔKID, ΔTMΔKID)

  • Assess effects on protein localization, interaction partners, and downstream responses

  • The dual localization of At2g42960 suggests possible roles in transmembrane signal perception and nuclear regulation

• Pathway integration analysis:

  • Examine co-expression patterns with known defense-related genes

  • Approximately 5% of Arabidopsis defense genes show induction by multiple treatments, suggesting integrated signaling networks

  • Look for correlation between At2g42960 expression and these co-regulated gene patterns

How does the kinase insertion domain (KID) influence At2g42960 function?

The kinase insertion domain (KID) plays a critical role in determining the functional properties of At2g42960. Experimental data reveals several key aspects:

• The KID contains functional nuclear localization signals (NLS) that are essential for directing the protein to the nucleus, as demonstrated by the exclusive nuclear localization of GFP-tagged KID expressed in Arabidopsis protoplasts.

• Deletion of the KID (ΔKID) results in plasma membrane and cytosolic localization with formation of protein aggregates, indicating the KID is necessary for proper protein folding and/or trafficking.

• The presence of the KID in conjunction with the deletion of the transmembrane domain (ΔTMΔKID) results in punctate signals in the nucleus or cytosol, further confirming its role in proper subcellular targeting.

• The conservation of the KID across RLCK XI members from different plant species suggests evolutionary preservation of this functional domain, implying its fundamental importance in receptor-like kinase signaling mechanisms .

What experimental approaches can determine if At2g42960 participates in plant defense responses?

To investigate At2g42960's role in plant defense responses, several experimental strategies can be employed:

• Pathogen challenge experiments:

  • Inoculate wild-type and At2g42960 mutant/overexpression lines with incompatible pathogens (e.g., Alternaria brassicicola)

  • Measure disease progression, lesion development, and molecular markers of defense activation

  • Compare results with established defense pathway mutants

• Treatment with defense signaling molecules:

  • Apply salicylic acid (SA), methyl jasmonate (MJ), or ethylene to plants

  • Monitor At2g42960 expression changes using RT-qPCR or microarray analysis

  • Evaluate whether At2g42960 responds to specific defense pathways or shows integrated response patterns

  • Look for both induction and repression patterns, as some genes show antagonistic regulation by different defense signals

• Protein interaction studies:

  • Perform co-immunoprecipitation or yeast two-hybrid screens to identify interaction partners

  • Focus on known defense signaling components and other receptor-like kinases

  • Map interactions to specific protein domains using truncation constructs

How can I design experiments to resolve the dual localization mechanism of At2g42960?

The dual localization of At2g42960 to both the plasma membrane and nucleus presents an intriguing research question. To investigate the mechanism governing this dual localization, consider the following experimental design:

• Time-course imaging with dual fluorescent tagging:

  • Generate constructs with different fluorescent proteins positioned at strategic locations

  • Add a fluorescent protein tag downstream of the transmembrane domain but upstream of the kinase domain

  • Retain C-terminal GFP tagging

  • Use live-cell imaging to track potential translocation between compartments

  • Employ photoconvertible fluorescent proteins to trace protein movement

• Stimulus-dependent localization analysis:

  • Test whether pathogen challenge or defense hormone treatments alter the distribution ratio between membrane and nuclear pools

  • Quantify fluorescence intensity in different compartments before and after treatment

  • Correlate localization changes with defense gene expression

• Phosphorylation-dependent regulation:

  • Identify potential phosphorylation sites using computational prediction

  • Generate phospho-mimetic and phospho-dead mutants

  • Assess effects on localization pattern

  • Determine if kinase activity is required for proper localization

What statistical approaches are appropriate for analyzing At2g42960 expression data from multiple treatments?

When analyzing At2g42960 expression data across multiple treatments and experimental conditions, robust statistical approaches are essential. Consider the following analytical framework:

• Experimental design considerations:

  • Ensure proper replication (biological and technical)

  • Include appropriate controls for each treatment

  • Consider factorial designs to test interactions between treatments

• Expression data normalization:

  • Apply appropriate normalization methods for microarray or RNA-seq data

  • Consider using multiple reference genes for RT-qPCR validation

  • Test for homogeneity of variance and apply transformations if necessary

• Statistical testing framework:

  • For comparison across multiple treatments, use ANOVA followed by appropriate post-hoc tests

  • Control for multiple testing using methods like Benjamini-Hochberg FDR

  • For complex datasets, consider multivariate approaches like principal component analysis (PCA) or hierarchical clustering

  • When examining co-expression patterns, use correlation analyses with significance testing

How can contradictory findings about At2g42960 function be reconciled using advanced experimental approaches?

When faced with contradictory findings regarding At2g42960 function, several advanced approaches can help reconcile discrepancies:

• Context-dependent functional analysis:

  • Test function under varied environmental conditions

  • Examine effects in different genetic backgrounds

  • Consider tissue-specific or developmental stage-specific effects

  • Use inducible expression systems to control timing and level of expression

• Integration of multiple data types:

  • Combine transcriptomics, proteomics, and metabolomics data

  • Look for consistent patterns across different experimental platforms

  • Employ machine learning approaches to identify subtle relationships

  • Develop mathematical models that can account for apparently contradictory observations

• Single-cell resolution studies:

  • Examine At2g42960 function at the individual cell level

  • Use cell-specific promoters or sorting techniques

  • Consider that whole-tissue studies may mask cell-type specific responses

  • Compare results across different cell types within the same plant

What are common challenges in studying At2g42960 localization and how can they be addressed?

Several technical challenges have been reported when studying At2g42960 localization:

• N-terminal tagging issues:

  • Problem: N-terminal GFP-tagged protein results in irregular aggregates in the cytosol

  • Solution: Use C-terminal GFP tagging to avoid disrupting proper targeting

  • Rationale: N-terminal tagging likely interferes with transmembrane domain function

• Transcript amplification difficulties:

  • Problem: Unable to clone full-length transcripts of some RLCK XI members

  • Solution: Use cDNA synthesis methods optimized for long transcripts; consider using commercial clones as starting material

  • Alternative: Design overlapping PCR strategies to assemble the complete sequence

• Fluorescence signal interpretation:

  • Problem: Distinguishing true localization from artifacts

  • Solution: Include appropriate controls (free GFP, known membrane and nuclear markers)

  • Quantification: Measure signal ratios between compartments using imaging software

How should contradictory data on At2g42960 expression in different defense pathways be analyzed?

When confronting contradictory data regarding At2g42960 expression in different defense pathways:

• Evaluate methodological differences:

  • Compare experimental conditions, plant ages, and tissue types

  • Assess timing of measurements relative to treatment application

  • Consider whether different ecotypes or accessions were used

  • Examine sensitivity and dynamic range of detection methods

• Analyze pathway interactions:

  • Some genes show both co-induction and antagonistic regulation patterns between pathways

  • Example: 55 genes are co-induced by SA and MJ treatments, while 8 genes are induced by SA but repressed by MJ

  • Determine if At2g42960 exhibits treatment-specific or signal-integrated responses

• Consider threshold effects:

  • Establish dose-response relationships for different treatments

  • Determine whether contradictory results reflect different positions on the same response curve

  • Apply mathematical modeling to predict response patterns under different conditions

What experimental controls are essential when studying At2g42960 function in transgenic systems?

When studying At2g42960 function in transgenic systems, the following controls are essential:

• Expression level controls:

  • Include wild-type plants as negative controls

  • Use empty vector transformants to control for transformation effects

  • Generate multiple independent transgenic lines with varying expression levels

  • Quantify transgene expression by RT-qPCR relative to endogenous reference genes

• Localization verification controls:

  • Include free GFP to establish baseline distribution patterns

  • Use known subcellular markers (membrane, nuclear, cytosolic)

  • Perform domain-deletion studies as internal controls

  • For inducible systems, include non-induced samples

• Phenotypic analysis controls:

  • Compare multiple independent transgenic lines

  • Include appropriate wild-type and negative controls

  • Use complementation tests with knockout mutants

  • For defense studies, include known defense pathway mutants as references

  • Test multiple pathogens or elicitors to establish specificity