At3g14460 Antibody

What is At3g14460 and what role does it play in plant immunity?

At3g14460 encodes a putative disease resistance protein in Arabidopsis thaliana that functions within the plant's immune system. The protein belongs to the nucleotide-binding site-leucine-rich repeat (NLR) family of immune receptors that constitute a core component of effector-triggered immunity (ETI) . These NLR proteins recognize pathogen effectors often indirectly through association with effector targets and become activated through oligomerization to form resistosomes when host factors are altered . At3g14460 specifically has been implicated in defense responses against viral pathogens, working alongside DICER-LIKE ribonucleases in the plant's antiviral immunity system .

What are the key structural characteristics of the At3g14460 protein?

The At3g14460 protein contains typical structural domains associated with plant disease resistance proteins, including nucleotide-binding (NB) and leucine-rich repeat (LRR) domains. These domains are critical for pathogen recognition and subsequent immune response activation. The protein sequence has been characterized and is available in reference databases, with a coding sequence of approximately 2094 base pairs . The protein functions in conjunction with other disease resistance proteins and may undergo conformational changes upon activation, similar to other characterized NLRs that oligomerize to form functional immune complexes .

How does At3g14460 integrate into plant immune signaling networks?

At3g14460 functions within a complex network of plant defense mechanisms, particularly in relation to DICER-LIKE (DCL) ribonuclease-dependent immunity. Research suggests that it may play a role similar to other NLRs like L5 (AT1G12290) and RPP9/RAC1 (AT1G31540) that contribute to DCL2-dependent activation of immune responses . The protein likely interacts with components of the plant immune signaling pathway, including potential association with WRKY transcription factors, as evidenced by the enrichment of W-box elements ((T)TGACY) in the promoters of genes differentially expressed during immune responses .

What methods are most effective for producing antibodies against At3g14460?

For producing antibodies against At3g14460, recombinant protein-based immunization strategies have demonstrated significantly higher success rates compared to peptide-based approaches. According to comprehensive antibody production studies for Arabidopsis proteins, the success rate with peptide antibodies was notably low, while using recombinant proteins yielded more reliable antibodies . The optimal approach involves expressing the full-length protein or specific domains (particularly unique regions that distinguish it from other NLR proteins) as recombinant antigens for immunization, followed by affinity purification of the resulting antibodies to enhance specificity and reduce background signals .

What validation methods should be employed to confirm At3g14460 antibody specificity?

Thorough validation of At3g14460 antibodies should include multiple complementary approaches:

• Western blot analysis using:

  • Wild-type Arabidopsis tissues

  • At3g14460 knockout/mutant lines as negative controls

  • Tissues overexpressing tagged versions of At3g14460

• Immunoprecipitation followed by mass spectrometry to confirm target binding

• Immunohistochemistry comparing wild-type and knockout tissues

• Pre-absorption controls with the immunizing antigen

Notably, affinity purification of antibodies has been shown to massively improve detection rates, with studies demonstrating that properly purified antibodies can achieve up to 55% detection success with high confidence for plant proteins .

How can cross-reactivity with other NLR proteins be minimized in At3g14460 antibody development?

Minimizing cross-reactivity requires careful antigen design focusing on unique regions of At3g14460 that differ from other NLR family proteins. Bioinformatic analysis should identify protein segments with minimal sequence homology to related proteins. Subsequently, rigorous cross-validation testing against closely related proteins is essential, particularly testing against L5 (AT1G12290) and RPP9/RAC1 (AT1G31540), which function in similar immune pathways . Additionally, antibody preparations should undergo exhaustive pre-absorption with proteins from knockout lines lacking At3g14460 but containing other NLR family members to remove antibodies with cross-reactivity potential.

What are the optimal conditions for using At3g14460 antibodies in Western blot applications?

The optimal Western blot conditions for At3g14460 antibodies typically involve:

For enhanced results, including an overnight incubation with the primary antibody at 4°C similar to protocols used in related studies can substantially improve detection sensitivity .

How can At3g14460 antibodies be effectively used in immunolocalization studies?

For effective immunolocalization of At3g14460:

• Tissue fixation: Use 4% paraformaldehyde in PBS for 1-2 hours, followed by careful washing.

• Embedding and sectioning: Embed in paraffin or resin and create 5-10 μm sections, or use a whole-mount approach for root tissues.

• Antigen retrieval: Perform citrate buffer (pH 6.0) heat-mediated antigen retrieval to expose epitopes.

• Blocking: Block with 3% BSA, 0.1% Triton X-100 in PBS for 1-2 hours.

• Primary antibody incubation: Apply affinity-purified At3g14460 antibody (1:100-1:500 dilution) and incubate overnight at 4°C.

• Secondary antibody: Use fluorophore-conjugated secondary antibodies appropriate for the detection system.

Importantly, include appropriate controls including tissues from At3g14460 knockout plants and pre-immune serum controls. Research indicates that properly affinity-purified antibodies significantly increase the likelihood of successful immunocytochemistry, with approximately 30% of carefully produced plant protein antibodies being suitable for immunolocalization studies .

What approaches can be used to study At3g14460 protein interactions with other immune system components?

Several complementary approaches can effectively investigate At3g14460 protein interactions:

• Co-immunoprecipitation (Co-IP): Use At3g14460 antibodies to pull down the protein complex from plant tissues, particularly following pathogen challenge, then identify interacting partners through mass spectrometry.

• Proximity labeling: Employ BioID or APEX2 fusions with At3g14460 to identify proximal proteins in living cells.

• Yeast two-hybrid screening: Identify direct protein interactions using At3g14460 domains as bait.

• Split-GFP or FRET-based approaches: Visualize interactions in planta through bimolecular fluorescence complementation.

• In vitro pull-down assays: Validate specific interactions using recombinant proteins.

When investigating At3g14460 interactions, particular attention should be paid to potential associations with components of the DCL-dependent immune pathway, especially considering its potential functional relationship with L5 and RPP9/RAC1 NLRs in DCL2-dependent activation of immune responses .

How can researchers differentiate between constitutive and pathogen-induced activation of At3g14460?

Differentiating between constitutive and pathogen-induced activation requires multi-faceted approaches:

• Temporal protein analysis: Monitor At3g14460 protein levels and potential post-translational modifications at different timepoints following pathogen challenge using the antibody in Western blot analysis.

• Subcellular localization dynamics: Track potential relocalization of At3g14460 following pathogen exposure using immunofluorescence or live-cell imaging with fluorescent protein fusions.

• Conformational change assessment: Utilize limited proteolysis followed by Western blot to detect structural changes in At3g14460 following activation.

• Oligomerization analysis: Employ native PAGE or size exclusion chromatography with Western blot detection to monitor formation of higher-order complexes upon activation.

• Transcriptional reporter systems: Create reporter lines where promoters of At3g14460-dependent defense genes drive fluorescent protein expression.

These approaches should be performed in wild-type plants as well as in mutant backgrounds affecting known upstream components of the immune signaling pathway, such as dcl4 mutants, which exhibit autoimmune phenotypes associated with DCL2-dependent activation of defense responses .

What is the relationship between At3g14460 and the DCL-dependent antiviral immunity pathway?

The relationship between At3g14460 and DCL-dependent immunity appears complex based on current research. At3g14460 likely functions similarly to other characterized NLRs like L5 and RPP9/RAC1 that have been implicated in DCL2-dependent immune activation . The current model suggests a two-tiered antiviral defense system where DCL4 mediates the primary defense layer (RNA interference), while DCL2 activates NLR-mediated immunity when RNAi is defeated and cytoplasmic dsRNA concentrations reach high levels .

In this pathway, At3g14460 may serve as one of the NLR sensors that becomes activated when DCL2 processes viral dsRNA, potentially generating specific small RNA fragments that trigger immune activation. This relationship is particularly evident in dcl4 mutants, which exhibit autoimmune phenotypes characterized by activation of defense-related genes bearing WRKY transcription factor binding sites (W-box elements) in their promoters .

How does phosphorylation status affect At3g14460 function in immune signaling?

While specific phosphorylation data for At3g14460 is limited in the current search results, research on related NLR proteins suggests that phosphorylation likely plays a critical role in regulating At3g14460 function. Researchers investigating this question should:

• Identify potential phosphorylation sites through bioinformatic prediction tools and phosphoproteomics data from Arabidopsis immunity studies.

• Generate phospho-specific antibodies against predicted sites or use general phospho-antibodies alongside At3g14460 antibodies to detect phosphorylation events.

• Create phospho-mimetic (Ser/Thr to Asp/Glu) and phospho-null (Ser/Thr to Ala) mutants to assess functional consequences.

• Examine effects of kinase inhibitors on At3g14460-mediated immune responses.

• Investigate potential interacting kinases and phosphatases through co-immunoprecipitation and mass spectrometry.

Phosphorylation likely influences key aspects of At3g14460 function including protein stability, subcellular localization, protein-protein interactions, and conformational changes associated with activation—patterns observed in other plant NLR proteins involved in immune signaling.

What are the common challenges in detecting At3g14460 in plant tissues and how can they be overcome?

Several challenges may arise when detecting At3g14460 in plant tissues:

Research on Arabidopsis antibodies indicates that affinity purification of antibodies substantially improves detection success rates from very low to approximately 55% for high-confidence detection . For particularly problematic detection, consider using epitope-tagged versions of At3g14460 expressed in the at3g14460 mutant background.

How can researchers distinguish between specific At3g14460 signal and cross-reactivity with other NLR proteins?

Distinguishing specific signal from cross-reactivity requires rigorous controls:

• Genetic controls: Always include samples from at3g14460 knockout/knockdown plants processed identically to wild-type samples.

• Competitive inhibition: Pre-incubate the antibody with excess purified antigen before applying to samples; specific signals should disappear.

• Multiple antibodies: When possible, use antibodies raised against different regions of At3g14460 and confirm concordant results.

• Correlation with transcript levels: Verify that protein detection correlates with transcript abundance across treatments or genotypes.

• Mass spectrometry validation: Confirm protein identity in immunoprecipitated samples using mass spectrometry.

• Specificity testing: Test the antibody against recombinant proteins of closely related NLRs, particularly those with significant sequence homology.

These approaches collectively provide strong evidence for signal specificity and help avoid misinterpretation of experimental results due to antibody cross-reactivity.

What are the best methods for quantifying At3g14460 protein levels in comparative studies?

For accurate quantification of At3g14460 protein levels:

• Western blot quantification:

  • Use infrared fluorescence-based detection systems (e.g., LI-COR Odyssey) for wider linear range

  • Include calibration curves with recombinant At3g14460 protein

  • Normalize to multiple loading controls (ideally both structural and processed proteins)

  • Perform technical replicates and biological replicates

• ELISA-based quantification:

  • Develop sandwich ELISA using two antibodies targeting different epitopes

  • Generate standard curves with purified recombinant protein

  • Validate with samples from knockout plants

• Mass spectrometry approaches:

  • Use targeted proteomics (MRM/PRM) with isotopically labeled peptide standards

  • Select proteotypic peptides unique to At3g14460

  • Process multiple biological replicates

• Statistical analysis:

  • Apply appropriate statistical tests based on experimental design

  • Account for biological variation between samples

  • Consider using mixed-effects models for complex experimental designs

Careful consideration of these methodological approaches enables reliable comparative analysis of At3g14460 protein levels across different experimental conditions, genotypes, or treatments.

How can At3g14460 antibodies be used to study the evolution of plant immune responses across species?

At3g14460 antibodies can facilitate evolutionary studies of plant immunity through:

• Cross-species reactivity testing: Evaluate whether the antibody recognizes homologous proteins in related plant species, establishing evolutionary conservation of epitopes.

• Comparative proteomics: Use the antibody for immunoprecipitation across diverse plant species followed by mass spectrometry to identify co-evolving interaction partners.

• Functional conservation analysis: Compare subcellular localization, expression patterns, and activation triggers of At3g14460 homologs across plant lineages.

• Heterologous expression studies: Express At3g14460 homologs from different species in Arabidopsis at3g14460 mutants to assess functional complementation.

• Molecular archaeology: Examine how sequence divergence in At3g14460 homologs correlates with changes in antibody recognition, providing insights into selective pressures on immune receptor evolution.

These approaches can reveal the evolutionary trajectories of plant immune systems, particularly regarding the relationship between DCL-dependent pathways and NLR-mediated immunity across plant species .

What insights can At3g14460 antibodies provide about the coordination between RNA silencing and NLR-mediated immunity?

At3g14460 antibodies can help elucidate the mechanistic links between RNA silencing and NLR-mediated immunity:

• Immunity activation studies: Investigate whether At3g14460 is activated in various RNA silencing mutants (dcl2, dcl4, rdr6) following viral infection, using the antibody to detect protein modifications or relocalization.

• RNA-protein interaction analysis: Combine immunoprecipitation with the At3g14460 antibody and RNA sequencing to identify potential RNA molecules associated with At3g14460 during immune responses.

• Subcellular co-localization: Use immunofluorescence to determine whether At3g14460 co-localizes with components of the RNA silencing machinery during infection.

• Temporal analysis of pathway activation: Monitor the kinetics of At3g14460 activation relative to RNA silencing responses during pathogen invasion.

These investigations may support emerging models suggesting that DCL2-dependent processing of viral dsRNA can trigger NLR-mediated immunity when the primary DCL4-dependent RNAi defense is compromised . At3g14460 likely plays a role similar to the characterized NLRs L5 and RPP9/RAC1 in this integrated defense system.

How might At3g14460 contribute to broad-spectrum disease resistance in crops?

Understanding At3g14460's contribution to disease resistance has significant translational potential:

• Comparative functional analysis: Use At3g14460 antibodies to study the expression, localization, and activation of homologous proteins in crop species like Beta vulgaris (sugar beet), which contains the homologous gene LOC104887559 encoding a putative disease resistance protein .

• Resistance mechanism characterization: Determine whether At3g14460 and its homologs recognize conserved pathogen features that could provide broad-spectrum resistance.

• Transgenic approaches: Express optimized versions of At3g14460 in crops and use the antibody to monitor protein accumulation and function.

• CRISPR-based genome editing: Modify endogenous At3g14460 homologs in crops to enhance their function, then verify modifications at the protein level.

• Protein engineering: Design synthetic immune receptors incorporating functional domains from At3g14460 and validate their expression and activity using antibodies.

These translational applications could ultimately contribute to developing crops with enhanced disease resistance, particularly against viral pathogens, potentially reducing reliance on chemical controls and improving agricultural sustainability.