At4g14610 Antibody

What is AT4G14610 and what is its role in plant immunity?

AT4G14610 is a coiled-coil nucleotide-binding leucine-rich repeat (CC-NLR) receptor in plants, specifically belonging to Group D of CNL receptors as classified in Arabidopsis thaliana. It functions in plant innate immunity, participating in pathogen recognition and defense response pathways. The extended coiled-coil (ECC) domain of AT4G14610 is capable of inducing necrosis when expressed in various plant species, suggesting its role in programmed cell death associated with plant immune responses . This protein belongs to the larger family of NLR receptors that form a critical component of the plant's surveillance system against pathogens.

How is AT4G14610 structurally organized?

AT4G14610 contains several key structural domains typical of CC-NLR proteins:

• An N-terminal extended coiled-coil (ECC) domain

• A nucleotide-binding (NB-ARC) domain

• A C-terminal leucine-rich repeat (LRR) domain

The ECC domain of AT4G14610 contains predicted α-helices that are important for its function in signaling. The protein likely shares structural similarities with other Group D CNLs, which contain the conserved EDVID motif and specific hydrophobicity patterns in their N-terminal regions . The structural organization follows patterns observed in other plant NLRs where the CC domain is involved in downstream signaling activities following pathogen recognition.

How does the ECC domain of AT4G14610 induce necrosis?

The ECC domain of AT4G14610 induces moderate necrosis when transiently expressed in plants such as Arabidopsis thaliana (Col-0) and lettuce cultivar Ninja. This necrosis-inducing activity appears to require interaction with endogenous plant proteins, specifically members of the RGC21 CNL family in lettuce, as demonstrated through genetic mapping and post-transcriptional gene silencing (PTGS) experiments . The signaling mechanism likely involves homodimeric or monomeric interactions between the CC domains and downstream signaling partners, similar to what has been observed with other plant NLRs like Mla10 and Sr33 .

What plant species respond to AT4G14610-ECC expression?

Research has demonstrated that transient expression of AT4G14610-ECC induces responses in multiple plant species:

• Arabidopsis thaliana (Col-0): Moderate necrosis/chlorosis response

• Nicotiana benthamiana: Variable response

• Lettuce cultivar Ninja: Moderate necrosis response

What specific amino acid residues in AT4G14610-ECC are critical for its function?

Critical amino acid residues in the ECC domain of AT4G14610 and related CNLs have been identified through structure-function analysis. Four conserved amino acid residues in Group D CCNLs (to which AT4G14610 belongs) are located on the opposite side of the H2a-b turn in predicted structural models based on crystallographic data from Mla10-CC and Sr33-CC. These residues are likely important for intramolecular interactions and downstream signaling .

Additionally, specific regions within the ECC have been analyzed through deletion and swap mutations to identify areas required for cell death induction. The areas following the α-helix H2b contain variable amino acid residues (CCVX) that influence cell death-inducing activity. The sequence alignment of this area in AT4G14610 compared to other CNLs reveals important structural features that determine functional specificity .

How does AT4G14610-ECC interact with other plant proteins?

Research indicates that AT4G14610-ECC interacts with other plant proteins, particularly members of the RGC21 family in lettuce. These interactions are essential for the necrosis-inducing activity of AT4G14610-ECC, as silencing of RGC21 family members compromised the necrosis response in lettuce .

The interactome of ECCs representing the CNL repertoire in Arabidopsis shows that certain ECCs frequently interact with each other, forming a network of interactions. While AT4G14610-ECC's specific interactions within this network aren't fully detailed in the available research, it likely participates in similar protein-protein interactions that are critical for its signaling function .

What methodologies are most effective for studying AT4G14610 antibody interactions in immunoprecipitation experiments?

For effective immunoprecipitation studies of AT4G14610 interactions, researchers should consider the following methodological approach:

• Express epitope-tagged versions of AT4G14610 (such as HA-tagged constructs) in plant tissues

• Use anti-epitope antibodies for immunoprecipitation (similar to the approach shown for other ECCs using anti-HA antibodies)

• Perform co-immunoprecipitation assays followed by western blotting to detect interacting partners

• Consider crosslinking approaches to stabilize transient protein interactions

• Use appropriate negative controls including non-specific antibodies and unrelated protein constructs

When designing experiments, researchers should account for potential protein degradation and optimize extraction buffers to maintain protein stability and native interactions. Based on the immunoblotting approaches used for other ECC proteins, detection using primary rat anti-HA and secondary goat anti-rat antibody fused to HRP has been effective for detecting ECC-domain proteins expressed in plant tissues .

How can genetic mapping approaches identify host components required for AT4G14610-mediated immunity?

Genetic mapping to identify host components required for AT4G14610-mediated immunity can follow the methodology used in the referenced studies:

• Develop mapping populations by crossing plants with differential responses to AT4G14610-ECC expression (e.g., similar to the cross between lettuce cultivars Ninja and Valmaine)

• Phenotype F2 individuals for necrosis response following AT4G14610-ECC expression

• Perform QTL analysis to identify genomic regions associated with the response variation

• Focus on regions containing NLR-encoding sequences, such as the 22-cM interval in linkage group 3 (LG3) of the lettuce genome that explained nearly all variation in necrosis induction

• Validate candidate genes through posttranscriptional gene silencing (PTGS) or other functional approaches

The research with lettuce demonstrated that this approach successfully identified members of the RGC21 CNL family as required for AT4G14610-ECC-induced necrosis, providing a template for similar studies in other plant species .

What are the optimal conditions for expressing AT4G14610 in heterologous systems?

Based on research methodologies for similar plant NLRs, the optimal conditions for expressing AT4G14610 in heterologous systems include:

• Transient expression systems:

  • Agrobacterium-mediated transient expression in Nicotiana benthamiana leaves

  • Use of Tobacco Rattle Virus (TRV) vectors for viral-mediated expression

  • Optimal optical density (OD) of Agrobacterium cultures: 0.4-0.6

  • Incubation period: 2-8 days post-infection for observing phenotypes

• Expression constructs:

  • Use of epitope tags (HA, FLAG) for detection and purification

  • Inclusion of appropriate plant promoters (35S CaMV)

  • Consideration of codon optimization for the expression system

• Protein extraction and detection:

  • Extraction 48 hours post-infiltration for optimal protein levels

  • Use of appropriate extraction buffers to maintain protein stability

  • Western blotting using anti-epitope antibodies for detection

How can researchers differentiate between specific and non-specific binding when using AT4G14610 antibodies?

To differentiate between specific and non-specific binding when using AT4G14610 antibodies, researchers should implement the following controls and approaches:

• Negative controls:

  • Include samples from knockout or silenced lines lacking AT4G14610

  • Use pre-immune serum or isotype control antibodies

  • Perform competition assays with purified antigen

• Validation approaches:

  • Confirm antibody specificity using recombinant AT4G14610 protein

  • Test antibody reactivity against truncated versions of the protein

  • Perform peptide competition assays

  • Compare results from multiple antibodies targeting different epitopes of AT4G14610

• Technical considerations:

  • Optimize antibody concentrations through titration experiments

  • Include appropriate blocking agents to reduce background

  • Use stringent washing conditions to remove weakly bound antibodies

  • Compare signal intensity across multiple sample dilutions

What are the key considerations for developing effective AT4G14610 antibodies for research applications?

Developing effective antibodies against AT4G14610 requires several key considerations:

• Epitope selection:

  • Choose unique, exposed regions of AT4G14610 that distinguish it from other CNLs

  • Avoid highly conserved regions to prevent cross-reactivity

  • Consider using the variable regions outside the conserved motifs

  • Target regions with high antigenicity and surface accessibility

• Antibody format selection:

  • Monoclonal antibodies: For high specificity and reproducibility

  • Polyclonal antibodies: For robust detection across multiple epitopes

  • Recombinant antibody fragments: For specialized applications

• Validation strategies:

  • Test reactivity against recombinant full-length and truncated versions of AT4G14610

  • Evaluate cross-reactivity with related CNL proteins

  • Confirm specificity using tissues from knockout/knockdown plants

  • Validate across multiple applications (Western blot, immunoprecipitation, immunohistochemistry)

• Application-specific optimization:

  • Optimize fixation and extraction methods for maintaining epitope accessibility

  • Determine ideal antibody concentrations for each application

  • Develop appropriate blocking and washing protocols

How can researchers analyze complex AT4G14610 interaction networks?

Analyzing complex interaction networks involving AT4G14610 requires sophisticated approaches:

• Network visualization and analysis:

  • Use interaction data to construct networks similar to those shown for other ECCs

  • Apply network analysis tools to identify hub proteins and key interaction modules

  • Visualize networks using software such as Cytoscape with nodes representing proteins and edges representing interactions

• Functional analysis of interactions:

  • Categorize interacting partners by function, localization, and expression pattern

  • Perform GO term enrichment analysis for interacting partners

  • Identify biological pathways enriched among interacting proteins

• Validation of key interactions:

  • Prioritize interactions for validation based on network metrics

  • Confirm direct interactions using multiple methods (Y2H, BiFC, FRET)

  • Assess the biological significance of interactions through genetic approaches

• Comparative analysis:

  • Compare AT4G14610 interaction networks across different plant species or conditions

  • Identify conserved interactions that may represent core functional complexes

  • Analyze species-specific interactions that may explain differential responses

What statistical approaches are recommended for analyzing AT4G14610 antibody-based immunoprecipitation data?

For analyzing AT4G14610 antibody-based immunoprecipitation data, the following statistical approaches are recommended:

• For protein identification in mass spectrometry data:

  • Apply appropriate false discovery rate (FDR) controls (typically 1-5%)

  • Use statistical models that account for the specific characteristics of mass spectrometry data

  • Implement tools like MaxQuant or Proteome Discoverer with built-in statistical frameworks

• For quantitative comparisons:

  • Use replicate experiments (minimum 3 biological replicates)

  • Apply appropriate normalization methods for the data type

  • Utilize statistical tests like t-tests for pairwise comparisons or ANOVA for multiple comparisons

  • Consider non-parametric alternatives for data that doesn't meet normality assumptions

• For differential interaction analysis:

  • Apply statistical frameworks such as SAINT or CompPASS for scoring confidence in protein-protein interactions

  • Use fold-change thresholds combined with statistical significance measures

  • Consider Bayesian approaches for more robust interaction scoring

• For handling missing values:

  • Distinguish between missing at random and missing not at random

  • Apply appropriate imputation methods based on the nature of the missing data

  • Consider specialized approaches for dealing with sparse data in interaction proteomics

What are common challenges in AT4G14610 antibody experiments and how can they be addressed?

Common challenges in AT4G14610 antibody experiments and their solutions include:

• Low antibody specificity:

  • Solution: Use epitope tags (HA, FLAG) for detection when studying recombinant proteins

  • Alternative: Develop monoclonal antibodies targeting unique regions of AT4G14610

  • Approach: Validate antibody specificity using knockout/knockdown lines

• Low protein abundance:

  • Solution: Use enrichment methods before detection (immunoprecipitation)

  • Alternative: Implement more sensitive detection methods like proximity ligation assays

  • Approach: Consider plant-optimized expression systems for recombinant studies

• Protein degradation:

  • Solution: Optimize extraction buffers with appropriate protease inhibitors

  • Alternative: Use shorter extraction protocols at lower temperatures

  • Approach: Consider native extraction conditions that maintain protein stability

• Cross-reactivity with related CNLs:

  • Solution: Pre-absorb antibodies with recombinant proteins from related CNLs

  • Alternative: Perform parallel detection with antibodies against related proteins

  • Approach: Use highly specific monoclonal antibodies or targeted proteomics approaches

• Inconsistent results between experiments:

  • Solution: Standardize protocols and use consistent biological materials

  • Alternative: Include internal controls for normalization

  • Approach: Implement rigorous statistical analyses with appropriate replicates

How can advanced imaging techniques be combined with AT4G14610 antibodies for subcellular localization studies?

Advanced imaging techniques can be combined with AT4G14610 antibodies for subcellular localization studies using the following approaches:

• Super-resolution microscopy:

  • Implementation: Use techniques like STORM, PALM, or SIM for nanoscale resolution

  • Advantage: Resolves protein distribution beyond the diffraction limit

  • Application: Detect AT4G14610 clustering during immune activation

• Live-cell imaging:

  • Implementation: Create fluorescent protein fusions with AT4G14610

  • Advantage: Tracks dynamic changes in protein localization

  • Application: Monitor relocalization during pathogen challenge

• Multi-channel co-localization:

  • Implementation: Combine AT4G14610 antibodies with markers for cellular compartments

  • Advantage: Precisely defines subcellular localization

  • Application: Determine association with specific organelles or structures

• FRET/FLIM analysis:

  • Implementation: Use fluorescently labeled antibodies or fusion proteins

  • Advantage: Detects protein-protein interactions in situ

  • Application: Visualize AT4G14610 interactions with signaling partners

• Correlative light and electron microscopy (CLEM):

  • Implementation: Combine fluorescence microscopy with electron microscopy

  • Advantage: Provides both molecular specificity and ultrastructural context

  • Application: Resolve protein localization at the ultrastructural level

What cutting-edge approaches can advance our understanding of AT4G14610 structure-function relationships?

Cutting-edge approaches to advance our understanding of AT4G14610 structure-function relationships include:

• Cryo-electron microscopy:

  • Application: Determine high-resolution structures of AT4G14610 alone or in complex with interacting partners

  • Advantage: Can capture different conformational states

  • Similar approach was used for other plant NLRs like Mla10 and Sr33

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS):

  • Application: Map dynamic protein regions and conformational changes upon activation

  • Advantage: Provides information about protein dynamics in solution

  • Can identify regions involved in protein-protein interactions

• Cross-linking mass spectrometry (XL-MS):

  • Application: Identify interaction interfaces between AT4G14610 and partners

  • Advantage: Captures transient and stable interactions

  • Provides distance constraints for structural modeling

• AlphaFold2 and other AI-based structure prediction:

  • Application: Generate high-confidence structural models of AT4G14610

  • Advantage: Can model protein structures without experimental data

  • Useful for structure-guided mutagenesis studies

• CRISPR-based structure-function screening:

  • Application: Systematically mutate AT4G14610 to identify functional residues

  • Advantage: High-throughput assessment of structure-function relationships

  • Can be combined with phenotypic screening for immune function