At1g61310 Antibody

What is At1g61310 and why is it significant in plant immunity research?

At1g61310 is a probable disease resistance protein in Arabidopsis thaliana (Mouse-ear cress) containing LRR (Leucine-Rich Repeat) and NB-ARC domains. It is classified as a CC-type NLR (Nucleotide-binding Leucine-rich Repeat) protein that functions in plant immune signaling pathways . The significance of At1g61310 stems from its involvement in TNL-mediated immunity, where it appears to function as a negative regulator. Research has shown that At1g61310 (also known as NRG1C) is significantly upregulated (approximately 200-fold in snc1 mutants and 7,000-fold in chs3-2D mutants) in plant autoimmune backgrounds, suggesting its importance in immune regulation mechanisms . Understanding this protein's function provides critical insights into plant defense mechanisms and potential applications in crop protection strategies.

What detection methods are available for At1g61310 protein in plant tissues?

Several detection methods are available for At1g61310 protein characterization:

• Western blotting (immunoblotting): Using polyclonal antibodies specific to At1g61310, researchers can detect the native protein in plant lysates . The commercially available rabbit anti-At1g61310 polyclonal antibody has been validated for Western blot applications, allowing for identification of the protein based on molecular weight.

• ELISA (Enzyme-Linked Immunosorbent Assay): This method enables quantitative detection of At1g61310 protein levels in plant samples using the specific antibody .

• Immunoprecipitation: Experiments using tagged versions of the protein have been successful, as evidenced by studies using HA-TurboID-tagged proteins detected with anti-HA antibodies .

• Expression analysis: While not directly detecting the protein, qRT-PCR analysis of At1g61310 transcripts provides valuable information about expression patterns in different genetic backgrounds or under pathogen challenge .

For optimal results, protein extraction should be performed using specialized plant protein extraction buffers that account for the high levels of interfering compounds in plant tissues.

How should researchers interpret variations in At1g61310 sequence across plant species?

When analyzing At1g61310 across different plant species, researchers should focus on the domain-specific variability profiles. According to comprehensive intraspecies diversity analysis, At1g61310.1 exhibits a distinct pattern of highly variable amino acid residues that are not evenly distributed across its structure :

This pattern indicates that the LRR domain contains the highest concentration of variable residues (9.9%), which is consistent with its role in pathogen recognition specificity. When performing cross-species comparisons, researchers should pay particular attention to these LRR domain variations as they likely reflect adaptation to different pathogen pressures . The NB-ARC domain shows moderate variability (2.0%), suggesting some functional constraints while maintaining species-specific adaptations. The conservation patterns observed in the linker and postLRR regions indicate functionally critical, less adaptable regions.

How can At1g61310 antibodies be effectively used to investigate TNL-mediated immunity signaling?

At1g61310 (NRG1C) antibodies can be strategically employed to investigate TNL-mediated immunity through several advanced approaches:

• Co-immunoprecipitation studies: Using At1g61310 antibodies to pull down protein complexes can reveal interaction partners in the TNL signaling network. This approach has revealed that NRG1C functions in TNL-mediated immune pathways with different strengths for distinct TNLs, similar to phenotypes observed in nrg1a nrg1b or sag101 studies .

• Chromatin immunoprecipitation (ChIP): For studying whether At1g61310 influences transcriptional regulation during immunity, researchers can employ ChIP experiments using anti-At1g61310 antibodies followed by sequencing.

• Immunolocalization: Determining the subcellular localization of At1g61310 during infection or in autoimmune backgrounds (like snc1 or chs3-2D) provides insights into protein trafficking during immune responses.

• Phosphorylation state analysis: Combined with phospho-specific antibodies, researchers can investigate whether At1g61310's activity is regulated through post-translational modifications during immune signaling.

• Proximity labeling: Building on the demonstrated HA-TurboID tagging approach, researchers can combine antibody detection with proximity labeling to identify proteins that transiently interact with At1g61310 during immune activation .

For such studies, it's critical to validate antibody specificity using knockout lines (such as nrg1c) as negative controls to ensure that detected signals are genuine.

What experimental approaches can resolve contradictions between At1g61310 knockout and overexpression phenotypes?

Current research reveals an interesting paradox: while nrg1c knockout does not exhibit severe immune-related defects, overexpression of NRG1C (At1g61310) surprisingly suppresses TNL-mediated autoimmunity in the chs3-2D and snc1 backgrounds . To resolve these contradictions, researchers should consider these experimental approaches:

• Genetic suppressor screens: Performing forward genetic screens in NRG1C overexpression backgrounds can identify components essential for its immune-suppressive function.

• Domain swap/mutation analysis: Creating chimeric constructs between NRG1C and related proteins (like NRG1A/B) can help identify which domains are responsible for the differential activities.

• Temporal expression analysis: Using inducible expression systems to control when NRG1C is expressed during infection can determine whether timing is critical for phenotypic outcomes.

• Proteomics comparisons: Compare protein interaction networks in knockout versus overexpression lines to identify differentially recruited signaling components.

• Biochemical activity assays: Investigate whether NRG1C possesses enzymatic activities (like ATPase activity often associated with NB-ARC domains) and how these activities correlate with immunity phenotypes.

• Transcriptome analysis: RNA-seq comparisons between wild-type, knockout, and overexpression lines can reveal changes in downstream defense gene activation, potentially explaining the seemingly contradictory phenotypes .

Importantly, researchers should examine pathogen-specific effects, as NRG1C overexpression suppressed immunity against oomycete pathogen Hyaloperonospora arabidopsidis (H.a. Noco2) but showed no altered resistance against Pseudomonas syringae expressing AvrRps4 or AvrRpt2 .

How does the structural analysis of At1g61310 inform antibody design and experimental application?

Structural insights into At1g61310 reveal important considerations for antibody design and experimental applications:

• Domain-targeted antibodies: The table from the search results indicates that At1g61310.1 has distinctly different variability patterns across domains. The LRR domain contains 35 highly variable amino acids (9.9% of total), while the NB-ARC domain has 7 variable residues (2.0%). For maximum specificity, antibodies targeting conserved epitopes in the NB-ARC domain would provide better cross-reactivity across accessions, while LRR-targeting antibodies might be more protein-specific but less consistent across variants.

• Conformational considerations: NLR proteins like At1g61310 undergo conformational changes during activation. Antibodies recognizing only certain conformational states may provide biased results in experiments. Researchers should consider using multiple antibodies targeting different epitopes to capture all protein states.

• Post-translational modification awareness: When designing antibodies, researchers should avoid epitopes containing potential phosphorylation sites that might be modified during immune signaling, as this could interfere with antibody binding.

• Cross-reactivity assessment: At1g61310 shares sequence similarity with the adjacent CNL AT1G61300 , potentially leading to cross-reactivity. Epitope selection should account for unique regions to ensure specificity.

• Functional domain preservation: When using antibodies for functional studies (e.g., neutralization experiments), target regions should be selected that don't interfere with critical functional domains unless such interference is the experimental goal.

For optimal results, researchers should validate antibodies using both recombinant At1g61310 protein and natural protein from plant extracts, including extracts from knockout lines as negative controls.

What controls are essential when using At1g61310 antibodies in plant immunity studies?

When designing experiments with At1g61310 antibodies, several critical controls must be included:

• Genetic knockout controls: Include nrg1c knockout lines as negative controls to confirm antibody specificity . This is particularly important given the sequence similarity between At1g61310 and other NLR proteins.

• Expression level controls: Since At1g61310 expression is dramatically upregulated in autoimmune backgrounds (200-fold in snc1 and 7,000-fold in chs3-2D compared to wild-type) , researchers must account for this variation when comparing protein detection across different genetic backgrounds.

• Recombinant protein standards: Include purified recombinant At1g61310 protein (available commercially with ≥85% purity) as a positive control and for quantification purposes.

• Cross-reactivity assessment: Test antibody reactivity against closely related proteins, particularly AT1G61300, which is adjacently located in the genome and shares structural features .

• Non-specific binding controls: Include pre-immune serum controls or isotype-matched control antibodies to distinguish specific from non-specific signals.

• Tissue-specific expression controls: As expression levels may vary across tissues, include controls appropriate for the tissue being studied.

• Treatment timing controls: When studying pathogen-induced changes, include appropriate time-course sampling as At1g61310 expression changes dynamically during infection .

• Antibody validation across applications: If using the antibody in multiple applications (Western blot, ELISA, immunoprecipitation), validate specificity for each method separately as performance can vary between applications .

How should researchers optimize protein extraction for At1g61310 detection in different plant tissues?

Optimizing protein extraction for At1g61310 detection requires careful consideration of plant tissue characteristics and protein properties:

• Buffer composition: Use extraction buffers containing:

  • EDTA (1-5 mM) to inhibit metalloproteases

  • Protease inhibitor cocktail specific for plant tissues

  • Reducing agents (DTT or β-mercaptoethanol) to maintain protein structure

  • PVPP (polyvinylpolypyrrolidone) to absorb phenolic compounds

  • Appropriate detergents (0.1-1% Triton X-100 or NP-40) for membrane-associated proteins

• Tissue-specific considerations:

  • For leaf tissue: Include higher concentrations of PVPP (2-5%) to address high phenolic content

  • For roots: Add additional wash steps to remove soil contaminants that may interfere with antibody binding

  • For floral tissues: Adjust pH to 7.5-8.0 to counteract naturally acidic environments

• Physical disruption methods:

  • Flash-freeze tissues in liquid nitrogen before grinding

  • Use mechanical disruption (bead beater or mortar and pestle) to ensure complete homogenization

  • For recalcitrant tissues, consider multiple freeze-thaw cycles

• Subcellular fractionation: Since At1g61310 is involved in immune signaling, consider separate extraction of cytosolic, membrane, and nuclear fractions to determine protein localization and ensure complete extraction.

• Sample concentration: For tissues with low At1g61310 expression (such as wild-type Col-0), concentrate proteins using TCA precipitation or commercial protein concentration kits before Western blot analysis.

• Protein quantification: Use Bradford or BCA assays compatible with plant extracts for accurate protein quantification before loading samples for Western blotting.

• Storage considerations: Add glycerol (10-15%) for samples intended for long-term storage to prevent freeze-thaw damage.

These optimizations are particularly important when comparing At1g61310 levels between wild-type plants and autoimmune mutants where expression differences can be several thousand-fold .

What experimental design best addresses the specificity of At1g61310 in different TNL-mediated immune pathways?

To effectively investigate At1g61310's specificity in different TNL-mediated immune pathways, researchers should implement a comprehensive experimental design:

• Genetic background matrix: Create a matrix of different genetic backgrounds including:

  • Wild-type (Col-0)

  • Single TNL mutants (snc1, chs3-2D)

  • Double mutants (combining At1g61310 overexpression or knockout with various TNL mutations)

  • Triple mutants incorporating known downstream components (e.g., nrg1a nrg1b, sag101)

• Pathogen diversity panel: Challenge plants with multiple pathogens that trigger different TNL sensors:

  • Bacterial pathogens: P. syringae pv. tomato DC3000 with various effectors (AvrRps4, AvrRpt2)

  • Oomycete pathogens: H. arabidopsidis Noco2

  • Viral pathogens to test broader immunity pathways

• Protein-protein interaction analysis: Perform co-immunoprecipitation or yeast two-hybrid assays to test direct interactions between At1g61310 and:

  • Different TNL proteins (SNC1, CHS3, RPS4)

  • Known downstream signaling components (NRG1A/B, SAG101, EDS1)

  • Potential novel interactors identified through proteomics

• Quantitative phenotyping: Measure multiple immunity outputs:

  • Growth phenotypes (fresh weight, rosette diameter)

  • Pathogen growth curves

  • Cell death assays (trypan blue staining, electrolyte leakage)

  • ROS production measurements

  • Defense gene expression profiles using qRT-PCR or RNA-seq

• Domain swap experiments: Create chimeric constructs exchanging domains between At1g61310 and related proteins to determine domain-specific contributions to TNL pathway specificity.

• Temporal analysis: Utilize inducible expression systems to activate or suppress At1g61310 at different timepoints during infection to determine temporal requirements.

• Biochemical activity assays: Assess whether At1g61310 has different biochemical activities (such as ATP hydrolysis rates) in the presence of components from different TNL pathways.

This comprehensive approach would address the observation that NRG1C (At1g61310) shows varying degrees of involvement in different TNL pathways, fully suppressing chs3-2D-mediated dwarfism while only partially suppressing snc1-mediated autoimmunity .

How should researchers interpret contradictory results when At1g61310 antibodies show different patterns across detection methods?

When faced with contradictory results across different detection methods using At1g61310 antibodies, researchers should systematically evaluate several factors:

• Epitope accessibility differences: At1g61310's conformation may differ between native conditions (immunofluorescence, IP) and denatured conditions (Western blot), affecting epitope exposure. Compare results using antibodies targeting different regions of the protein.

• Post-translational modifications: Phosphorylation, ubiquitination, or other modifications may occur differentially across experimental conditions, affecting antibody recognition. Consider using phosphatase treatment or ubiquitin-targeted analyses to assess this possibility.

• Cross-reactivity assessment: The highly conserved NB-ARC domain (showing only 2.0% variable residues) may lead to cross-reactivity with related NLRs . Validate specificity by:

  • Testing antibody reactivity in nrg1c knockout lines

  • Performing peptide competition assays

  • Using multiple antibodies targeting different epitopes

• Expression level variability: Consider that baseline expression of At1g61310 is low in wild-type plants but dramatically increases in autoimmune backgrounds (200-7,000 fold higher) . This dynamic range may exceed the linear detection range of some methods but not others.

• Method-specific artifacts: Each detection method has specific limitations:

  • Western blot: Issues with transfer efficiency or blocking

  • ELISA: Non-specific binding to plate surfaces

  • Immunofluorescence: Autofluorescence from plant tissues

  • IP: Pull-down of interacting proteins rather than direct targets

• Sample preparation variations: Different extraction methods may preferentially isolate certain protein pools or conformational states. Compare results using multiple extraction protocols.

• Antibody batch variation: Different antibody production lots may have varying specificities and affinities. Include positive controls with known reactivity for each new antibody batch.

When publishing contradictory results, researchers should transparently report all methods tried and the specific conditions under which each result was obtained.

What are the most common challenges in reproducing At1g61310 antibody-based experimental results?

Researchers face several significant challenges when attempting to reproduce At1g61310 antibody-based experimental results:

• Antibody lot variability: Polyclonal antibody production inherently yields batch-to-batch variation. Even slight differences in epitope recognition can significantly impact results, particularly when studying proteins like At1g61310 that have highly conserved domains shared with related NLRs .

• Expression level dynamics: At1g61310 shows dramatic expression differences across genetic backgrounds and in response to pathogens . Variations in:

  • Plant growth conditions (light intensity, photoperiod, temperature)

  • Plant developmental stage

  • Stress conditions
    Can all influence baseline expression, making standardization challenging.

• Genetic background effects: Small differences in the genetic background of Arabidopsis lines can influence At1g61310 expression. Even "identical" Columbia-0 lines maintained in different laboratories can accumulate genetic differences over time.

• Protein extraction efficiency: Plant tissues contain numerous compounds that interfere with protein extraction and detection. Differences in extraction protocols can significantly impact yield and quality of At1g61310 protein recovery.

• Post-translational modifications: Variations in plant growth conditions can alter the phosphorylation state and other modifications of At1g61310, affecting antibody recognition.

• Technical differences in detection methods:

  • Western blot: Variations in transfer efficiency, blocking agents, and detection systems

  • ELISA: Differences in coating buffers, blocking solutions, and washing stringency

  • Immunoprecipitation: Variations in bead type, binding conditions, and elution methods

• Pathogen strain variations: When studying At1g61310 in the context of pathogen challenge, even minor variations in pathogen strains (such as P. syringae or H. arabidopsidis) can affect results .

To maximize reproducibility, researchers should thoroughly document all experimental conditions, validate antibodies with appropriate controls, and consider sharing standardized protocols and materials within the research community.

How can researchers distinguish between direct and indirect effects of At1g61310 in immune signaling using antibody-based approaches?

Distinguishing between direct and indirect effects of At1g61310 in immune signaling requires sophisticated antibody-based experimental designs:

• Proximity-based interaction assays:

  • Implement proximity ligation assays (PLA) using At1g61310 antibodies paired with antibodies against suspected interaction partners

  • Apply FRET/FLIM imaging with fluorescently-labeled antibodies to detect direct protein-protein interactions in planta

  • Use split-complementation systems (like split-GFP) combined with antibody validation to confirm direct interactions

• Temporal resolution approaches:

  • Employ time-course experiments with high temporal resolution sampling

  • Use synchronizable systems (like DEX-inducible promoters controlling At1g61310 expression) coupled with antibody detection

  • Apply rapid subcellular fractionation followed by immunoblotting to track protein movement during signaling

• In vitro reconstitution:

  • Purify recombinant At1g61310 protein and potential interaction partners

  • Perform in vitro binding assays with purified components

  • Use antibodies to detect complexes formed exclusively from purified components

• Domain-specific antibodies and mutants:

  • Generate antibodies targeting specific functional domains of At1g61310

  • Create domain deletion/mutation constructs of At1g61310

  • Compare antibody detection patterns between wild-type and domain mutants to map interaction interfaces

• Chemical crosslinking with immunoprecipitation:

  • Apply membrane-permeable crosslinkers to stabilize transient protein interactions

  • Use At1g61310 antibodies for immunoprecipitation

  • Analyze crosslinked complexes by mass spectrometry to identify direct binding partners

• Comparative analysis across genetic backgrounds:

  • Compare At1g61310 interaction networks in wild-type versus signaling-deficient backgrounds (e.g., eds1, pad4, or sag101 mutants)

  • Use immunoprecipitation followed by quantitative proteomics to identify differentially associated proteins

• Heterologous expression systems:

  • Express At1g61310 in non-plant systems lacking plant immune components

  • Test for reconstitution of specific immune outputs

  • Use antibodies to confirm protein expression and localization

By combining these approaches, researchers can build a comprehensive understanding of whether At1g61310 directly interacts with other immune components or influences their activity through indirect mechanisms, similar to how studies have revealed its differential contributions to distinct TNL-mediated immune pathways .

How might At1g61310 antibodies be used to study crosstalk between disease resistance and abiotic stress responses?

At1g61310 antibodies offer powerful tools for investigating the intersection between disease resistance and abiotic stress responses:

• Stress-induced protein modifications: Use At1g61310 antibodies in combination with phospho-specific antibodies to track how different abiotic stressors (drought, salt, temperature extremes) affect post-translational modifications of At1g61310. This can reveal how environmental signals might modulate immune function through this protein.

• Protein complex remodeling: Employ immunoprecipitation with At1g61310 antibodies followed by mass spectrometry to identify how interaction partners change under combined biotic and abiotic stress conditions. This approach can reveal stress-specific shifts in immune signaling complexes.

• Subcellular relocalization tracking: Use immunofluorescence with At1g61310 antibodies to monitor protein relocalization under different stress combinations. Changes in localization patterns may indicate functional shifts in response to environmental conditions.

• Chromatin association dynamics: Apply ChIP (Chromatin Immunoprecipitation) using At1g61310 antibodies to determine if this immune regulator associates with chromatin under specific stress conditions, potentially identifying direct gene regulation roles.

• Hormone response integration: Combine At1g61310 antibody-based detection with treatments of different plant hormones (salicylic acid, jasmonic acid, abscisic acid) to map how hormone signaling networks interface with At1g61310 function during stress responses.

• Protein stability assessment: Track At1g61310 protein levels using antibodies under different stress conditions to determine if protein stability is regulated as part of stress responses, particularly important given that overexpression studies have revealed antagonistic effects on immunity .

• Interspecies conservation mapping: Apply At1g61310 antibodies across diverse plant species to examine how this immune component's function is conserved or diversified in species with different stress adaptation strategies.

These approaches would build upon observations that At1g61310 (NRG1C) is dynamically regulated during pathogen challenge and would explore whether this regulation extends to or is modified by concurrent abiotic stresses.

What new insights might At1g61310 antibodies provide about the evolution of plant immune systems?

At1g61310 antibodies can serve as valuable tools for evolutionary studies of plant immune systems:

• Cross-species immunoprecipitation: Using antibodies raised against conserved epitopes of At1g61310, researchers can perform immunoprecipitation in diverse plant species to identify interacting partners. This comparative approach can reveal how immune complexes have evolved across plant lineages.

• Protein domain conservation analysis: Apply domain-specific antibodies to detect At1g61310 homologs across plant species, determining which domains are most conserved. This approach complements the existing data showing that At1g61310's LRR domain contains the highest percentage (9.9%) of highly variable amino acids, while other domains show greater conservation .

• Functional conservation testing: Use antibodies to detect and quantify At1g61310 homologs in different plant species challenged with the same pathogens. This can reveal whether expression patterns and protein accumulation responses are conserved.

• Ancient protein reconstruction: Generate antibodies against reconstructed ancestral versions of At1g61310 to test functional properties of evolutionary intermediates and track the acquisition of novel immune functions.

• Comparative post-translational modification profiling: Apply antibodies specific to modified forms of At1g61310 across species to determine if regulatory mechanisms are conserved or have diverged.

• Paralog-specific detection: Develop antibodies that can distinguish between closely related NLR paralogs (like At1g61310 and At1g61300) to track expression patterns and determine subfunctionalization or neofunctionalization events .

• Co-evolutionary analysis: Combine antibody detection of At1g61310 with pathogen effector studies to identify potential co-evolutionary relationships between plant immune components and pathogen virulence factors.

• Diversification mapping: Apply antibodies to natural accessions of Arabidopsis to correlate protein variations with known highly variable residues in the LRR domain (35 highly variable residues accounting for 9.9% of total amino acids) .

These approaches would build upon the intraspecies diversity analysis data showing domain-specific patterns of conservation and variation in At1g61310 , potentially revealing how these patterns translate to functional diversity across plant lineages.

How might advanced microscopy techniques combined with At1g61310 antibodies reveal new aspects of immune complex formation?

Combining cutting-edge microscopy techniques with At1g61310 antibodies opens new frontiers for visualizing immune complex dynamics:

• Super-resolution microscopy applications:

  • STORM/PALM imaging with fluorophore-conjugated At1g61310 antibodies can visualize immune complexes at nanometer resolution

  • SIM (Structured Illumination Microscopy) can reveal spatial relationships between At1g61310 and other immune components during signaling events

  • Expansion microscopy combined with immunolabeling can physically magnify subcellular structures for enhanced visualization of immune complex architecture

• Live-cell imaging approaches:

  • Antibody fragments (Fab, nanobodies) against At1g61310 labeled with cell-permeable fluorophores can track protein dynamics in living cells

  • Single-molecule tracking using quantum dot-conjugated antibodies can follow individual At1g61310 molecules during immune signaling

  • FRAP (Fluorescence Recovery After Photobleaching) with fluorescent antibodies can measure At1g61310 mobility during immune activation

• Multi-protein complex visualization:

  • Multiplex immunofluorescence with combinatorial antibody labeling can simultaneously track At1g61310 and its interaction partners (like components of the nrg1a nrg1b or sag101 pathways)

  • Multi-color STORM imaging can determine precise spatial arrangements within immune complexes

  • Correlative Light and Electron Microscopy (CLEM) with immunogold labeling can place At1g61310 in ultrastructural context

• Functional imaging approaches:

  • FRET sensors based on antibody fragments can detect conformational changes in At1g61310 during activation

  • Optogenetic tools combined with antibody detection can link light-induced activation with complex formation visualization

  • Calcium imaging paired with At1g61310 immunodetection can correlate immune signaling with calcium flux events

• In vivo applications:

  • Intravital microscopy with minimally invasive antibody delivery can visualize At1g61310 dynamics in intact plant tissues

  • Light-sheet microscopy with cleared tissues and antibody labeling can provide 3D visualization of immune structures

  • Two-photon microscopy with immunolabeling can achieve deeper tissue penetration for in situ visualization

These advanced imaging approaches would provide unprecedented insights into the dynamic behavior of At1g61310 during immune responses, potentially explaining the observed differences in its contributions to different TNL-mediated immune pathways by revealing spatial and temporal patterns of complex formation that cannot be detected by biochemical methods alone.