IDD14 Antibody

What is IDD14 and why is it important in plant research?

IDD14 (INDETERMINATE DOMAIN 14) is a plant-specific transcription factor belonging to the highly conserved IDD/BIRD protein family. IDD14 plays crucial roles in multiple biological processes, most notably in regulating starch metabolism in response to environmental conditions like cold . Research has shown that IDD14 functions through alternative splicing, generating a self-controlled regulatory loop that modulates starch accumulation. In rice, IDD14 works alongside IDD12 and IDD13 to activate MDPK (Malectin Domain Protein Kinase), enhancing resistance to Sheath Blight (ShB) disease . Understanding IDD14 is essential for researchers investigating plant stress responses, developmental regulation, and crop improvement strategies.

What are the known splice variants of IDD14 and how do they functionally differ?

Two primary splice variants of IDD14 have been identified:

• IDD14α - The full-length functional form that contains an intact DNA-binding domain and can directly bind to target gene promoters, particularly the Qua-Quine Starch (QQS) gene that regulates starch accumulation .

• IDD14β - An alternatively spliced form produced predominantly under cold conditions. This variant lacks the functional DNA-binding domain but retains the ability to form heterodimers with IDD14α .

The functional difference is significant: IDD14α-IDD14β heterodimers have reduced binding activity to target promoters compared to IDD14α homodimers. This creates a mechanism where IDD14β acts as a competitive inhibitor of IDD14α, forming a self-regulatory loop that modulates starch metabolism in response to environmental signals . This splice variant-based regulation represents an elegant example of how alternative splicing can generate competitive inhibitors that fine-tune transcriptional networks.

What experimental approaches are most effective for detecting IDD14 protein-protein interactions?

Based on published research, multiple complementary techniques have proven effective for studying IDD14 protein-protein interactions:

• Yeast Two-Hybrid (Y2H) Assays: Successfully used to demonstrate interactions between IDD14 and other IDD family proteins (IDD12 and IDD13). The system utilizes GAL4-DNA binding domains and activation domains to validate interactions through growth on selective media .

• Bimolecular Fluorescence Complementation (BiFC): This technique allows visualization of protein interactions in plant cells. For IDD14, researchers have used fusion proteins with split YFP fragments (IDD14-nYFP with partner proteins fused to cYFP) transiently expressed in rice protoplasts. Reconstituted fluorescence indicates interaction .

• Co-Immunoprecipitation (Co-IP): Effective for confirming interactions in planta. For example, IDD14-GFP and IDD12-MYC or IDD13-MYC co-expressed in tobacco leaves have been immunoprecipitated with anti-GFP antibodies and analyzed by Western blot using anti-MYC antibodies .

For optimal results, researchers should consider using at least two of these methods for validation, as each has different strengths and limitations for detecting direct versus indirect interactions.

How can researchers effectively validate the specificity of an IDD14 antibody?

Validating IDD14 antibody specificity requires multiple approaches:

• Western Blot Analysis with Proper Controls:

  • Use tissue from IDD14 knockout/knockdown plants as negative controls

  • Include recombinant IDD14 protein as a positive control

  • Test cross-reactivity with closely related IDD family proteins (especially IDD12 and IDD13)

• Immunoprecipitation Followed by Mass Spectrometry:

  • Confirm that the immunoprecipitated protein is indeed IDD14

  • Analyze any co-precipitated proteins to avoid misinterpretation of results

• Immunocytochemistry Comparison:

  • Compare antibody staining patterns in wild-type versus IDD14 mutant tissues

  • Perform peptide competition assays to confirm binding specificity

• Validation in Multiple Plant Species/Tissues:

  • Test antibody performance across different plant tissues and species if cross-species reactivity is claimed

  • Document tissue-specific expression patterns that match known IDD14 expression data

When validating antibodies against IDD14, particular attention should be paid to distinguishing between splice variants (IDD14α vs. IDD14β), as this can significantly impact experimental interpretations .

What are the best techniques to study IDD14 DNA-binding properties?

Based on successful approaches in IDD protein research, the following techniques are recommended for studying IDD14 DNA-binding properties:

• Electrophoretic Mobility Shift Assay (EMSA): This has been effectively used to demonstrate that IDD proteins bind to specific motifs within target gene promoters. For example, researchers used biotin-labeled probes containing putative IDD-binding motifs from the MDPK promoter to confirm binding specificity .

• Yeast One-Hybrid (Y1H) Assays: This approach has successfully identified IDD proteins (including IDD14) that bind to specific promoter regions. The system uses reporter constructs with the promoter region of interest driving expression of a selectable marker like HIS3 .

• Chromatin Immunoprecipitation followed by qPCR (ChIP-qPCR): This technique allows quantification of enrichment at specific genomic regions. For IDD proteins, anti-GFP antibodies have been used with GFP-tagged IDD fusion proteins to measure enrichment at target promoters .

• Chromatin Immunoprecipitation followed by Sequencing (ChIP-seq): For genome-wide identification of binding sites, ChIP-seq provides comprehensive binding profiles. This approach requires validation with multiple independent transgenic lines expressing tagged versions of the protein, as demonstrated for related IDD proteins .

When designing these experiments, researchers should consider that DNA-binding properties may be affected by post-translational modifications, as has been demonstrated for the related IDD4 protein, which shows altered binding activity following phosphorylation .

How does phosphorylation affect IDD protein function and what methods can detect this modification?

While specific phosphorylation data for IDD14 is limited in the provided search results, insights from the related family member IDD4 reveal important principles:

IDD4 is a phospho-protein that interacts with and becomes phosphorylated by the MAP kinase MPK6 on two conserved sites . This phosphorylation significantly affects DNA-binding affinity to ID1 motif-containing promoters, altering its function as a transcriptional regulator. Phosphorylation status also influences susceptibility to pathogens, salicylic acid levels, and transcriptome reprogramming .

For detecting and studying IDD protein phosphorylation:

• Mass Spectrometry:

  • Phosphopeptide enrichment followed by LC-MS/MS analysis

  • Enables identification of specific phosphorylation sites

• Phospho-specific Antibodies:

  • Can be developed against known phosphorylation sites

  • Useful for Western blotting and immunoprecipitation

• Phospho-mimetic and Phospho-dead Mutants:

  • Create protein variants where phosphorylation sites are mutated to either mimic phosphorylation (e.g., S→D or S→E) or prevent it (S→A)

  • Compare functional outcomes to determine phosphorylation effects

• In vitro Kinase Assays:

  • Test direct phosphorylation by candidate kinases (such as MPK6)

  • Use recombinant proteins and radioactive or fluorescent ATP analogs

Given the importance of phosphorylation in IDD4 function, researchers studying IDD14 should investigate potential phosphorylation events and their functional consequences.

What controls should be included when studying IDD14 using antibody-based techniques?

When using antibody-based techniques to study IDD14, the following controls are essential:

• Genetic Controls:

  • IDD14 knockout/knockdown lines as negative controls

  • Overexpression lines as positive controls

  • Complementation lines to verify functional rescue

• Protein Controls:

  • Recombinant IDD14 protein as positive control

  • Related IDD family proteins (especially IDD12 and IDD13) to test cross-reactivity

  • Both splice variants (IDD14α and IDD14β) to ensure detection specificity

• Experimental Controls for Western Blot:

  • Loading controls (tubulin, actin) to normalize protein amounts

  • Molecular weight markers to confirm band size

  • Secondary antibody-only controls to detect non-specific binding

• Experimental Controls for Immunoprecipitation:

  • IgG control to identify non-specific binding

  • Input samples to confirm protein presence before IP

  • Protein A/G beads alone to detect non-specific binding to beads

• Controls for ChIP Experiments:

  • Input DNA control for normalization

  • IgG control for non-specific binding

  • Positive control regions known to bind IDD14

  • Negative control regions not expected to bind IDD14

Including these comprehensive controls will significantly improve data reliability and interpretability when studying IDD14 using antibody-based techniques.

How can researchers distinguish between IDD14α and IDD14β in experimental settings?

Distinguishing between IDD14 splice variants requires careful experimental design:

When designing experiments:

• Consider using epitope-tagged constructs of each variant for easier distinction

• Remember that IDD14β increases under cold conditions, so temperature treatments can be used to modulate relative abundance

• Focus on both molecular weight differences and functional differences (DNA-binding capacity) for comprehensive distinction

What are the recommended protocols for IDD14 immunoprecipitation?

Based on successful protocols with IDD proteins, the following optimized immunoprecipitation approach is recommended:

• Tissue Preparation:

  • Harvest 1-2g fresh plant tissue (preferably young leaves/seedlings)

  • Flash freeze in liquid nitrogen and grind to fine powder

  • Extract proteins in buffer containing:

    • 50mM Tris-HCl pH 7.5

    • 150mM NaCl

    • 1% Triton X-100

    • 1mM EDTA

    • Protease inhibitor cocktail

    • Phosphatase inhibitors (if studying phosphorylation)

  • Centrifuge at 14,000g for 15 minutes at 4°C

• Immunoprecipitation:

  • Pre-clear lysate with Protein A/G beads for 1 hour at 4°C

  • Incubate cleared lysate with anti-IDD14 antibody (or anti-tag antibody for tagged constructs) overnight at 4°C

  • Add pre-washed Protein A/G beads and incubate for 2-3 hours at 4°C

  • Wash beads 4-5 times with wash buffer (extraction buffer with reduced detergent)

  • Elute proteins by boiling in SDS sample buffer

• Analysis:

  • Separate proteins by SDS-PAGE

  • Perform Western blot using appropriate antibodies

  • For protein interaction studies, probe with antibodies against suspected interaction partners

This protocol has been successful for related IDD proteins, as demonstrated in the Co-IP assays performed to analyze interactions between IDD14 and IDD12/IDD13 in tobacco leaves .

How should researchers approach studying interactions between IDD14 and other transcription factors?

To comprehensively study interactions between IDD14 and other transcription factors, researchers should implement a multi-layered strategy:

• Identification of Potential Interactors:

  • Yeast two-hybrid screening using IDD14 as bait

  • Affinity purification followed by mass spectrometry (AP-MS)

  • Candidate approach focusing on known IDD14 family interactors (start with IDD12 and IDD13)

• Validation of Direct Interactions:

  • In vitro pull-down assays with recombinant proteins

  • Bimolecular Fluorescence Complementation (BiFC) in plant cells

  • Co-immunoprecipitation from plant tissues expressing tagged proteins

• Functional Characterization:

  • DNA-binding assays (EMSA) to determine how interactions affect binding to target promoters

  • Transactivation assays to assess transcriptional outcomes

  • Analysis of target gene expression in single and double mutants

• Structural Studies:

  • Domain mapping to identify interaction interfaces

  • Mutagenesis of key residues to disrupt specific interactions

  • Structural modeling based on related IDD proteins

Research with IDD14 has already demonstrated that it interacts with IDD12 and IDD13, forming complexes that can activate MDPK to enhance disease resistance in rice . These interactions were confirmed through yeast two-hybrid, BiFC, and Co-IP approaches, providing a methodological foundation for studying additional potential interaction partners.

What experimental systems are most effective for producing recombinant IDD14 protein?

While the search results don't provide specific information about IDD14 recombinant protein production, best practices for plant transcription factors suggest the following approaches:

• Bacterial Expression Systems:

  • E. coli BL21(DE3): Optimize with reduced temperature (16-18°C), lower IPTG concentration (0.1-0.5mM), and specialized tags (MBP, GST, SUMO) to enhance solubility

  • E. coli Arctic Express: Consider for difficult-to-express proteins as it contains cold-adapted chaperonins

  • Expression constructs: Focus on functional domains rather than full-length protein if solubility issues arise

• Eukaryotic Expression Systems:

  • Insect cells (Sf9, Sf21): Baculovirus expression system provides post-translational modifications

  • Plant-based expression: Transient expression in Nicotiana benthamiana using Agrobacterium infiltration

• Cell-free Protein Synthesis:

  • Wheat germ extract systems have shown success with plant transcription factors

  • Allows expression of toxic proteins and rapid optimization

• Purification Strategy:

  • Two-step purification (affinity followed by size exclusion chromatography)

  • Include protease inhibitors and phosphatase inhibitors if studying phosphorylation

  • Consider native versus denaturing conditions based on downstream applications

When producing IDD14 recombinant protein, researchers should be mindful of splice variants and consider creating constructs for both IDD14α and IDD14β to enable comparative studies .

How can researchers use antibodies to study the dynamics of IDD14 in response to environmental stresses?

To effectively study IDD14 dynamics during stress responses:

• Time-Course Experiments:

  • Expose plants to relevant stresses (particularly cold treatment, which affects IDD14 splicing)

  • Collect samples at multiple time points (0, 1, 3, 6, 12, 24, 48 hours)

  • Process for both protein (Western blot) and RNA (RT-PCR) analyses to track both transcriptional and post-transcriptional changes

• Protein Localization Studies:

  • Perform nuclear/cytoplasmic fractionation followed by Western blotting

  • Alternatively, use immunofluorescence microscopy to track subcellular localization changes

  • Consider live-cell imaging with fluorescently-tagged IDD14 constructs

• Protein Modification Analysis:

  • Use phospho-specific antibodies or Phos-tag gels to detect phosphorylation changes

  • Perform immunoprecipitation followed by mass spectrometry to identify stress-induced modifications

  • Compare results with phospho-mimetic and phospho-dead mutants

• Protein-Protein Interaction Dynamics:

  • Use co-immunoprecipitation at different stress time points to track changes in interaction partners

  • Apply BiFC or FRET techniques to visualize interaction dynamics in living cells

• DNA-Binding Dynamics:

  • Perform ChIP-qPCR across stress time points to track binding to target promoters

  • Compare binding patterns of IDD14α and IDD14β during stress

Alternative splicing of IDD14 increases production of the IDD14β form under cold conditions, creating a regulatory mechanism that modulates starch accumulation . This system provides an excellent model for studying how environmental signals trigger post-transcriptional regulation to fine-tune plant responses.

What approaches are recommended for studying the role of IDD14 in plant disease resistance?

Building on findings that IDD proteins enhance disease resistance in rice , researchers investigating IDD14's role in plant immunity should consider:

• Genetic Manipulation and Phenotyping:

  • Generate and characterize IDD14 knockout/knockdown lines, overexpression lines, and splice variant-specific manipulations

  • Challenge plants with various pathogens and quantify disease progression

  • Measure physiological responses (ROS production, callose deposition, etc.)

• Transcriptional Network Analysis:

  • Perform RNA-seq on wild-type versus IDD14-modified plants during pathogen infection

  • Identify defense-related genes regulated by IDD14

  • Use ChIP-seq to map direct binding targets during defense responses

• Hormonal Crosstalk Investigation:

  • Quantify defense-related hormones (SA, JA, ET) in IDD14 mutants

  • Test sensitivity to exogenous hormone application

  • Examine expression of hormone biosynthesis and signaling genes

  • Create double mutants with key hormone pathway components

• Protein Complex Characterization:

  • Identify defense-specific interaction partners using Co-IP during infection

  • Investigate potential phosphorylation of IDD14 during defense responses

  • Test interactions with known immunity regulators

• Evolutionary Analysis:

  • Compare IDD14 function across different plant species

  • Examine conservation of regulatory mechanisms and target genes

Research has shown that IDD12, IDD13, and IDD14 activate MDPK to enhance ShB resistance in rice . Similar mechanisms might exist in other plant species, making this a promising research direction with potential agricultural applications.

How can CRISPR/Cas9 technology be optimized for studying IDD14 function?

For researchers applying CRISPR/Cas9 to study IDD14, consider these optimized approaches:

• Strategic Guide RNA Design:

  • Target conserved functional domains for complete loss-of-function

  • Design splice variant-specific guides to selectively disrupt IDD14α or IDD14β

  • Use multiple guides simultaneously to increase editing efficiency

  • Verify guide RNA specificity against related IDD family members

• Advanced Editing Strategies:

  • Gene Knockout: Complete deletion of IDD14 coding sequence

  • Domain Editing: Targeted modification of DNA-binding or protein interaction domains

  • Promoter Editing: Modification of regulatory regions to alter expression patterns

  • Base Editing: Introduce precise amino acid changes at functionally important sites

• Validation Approaches:

  • Confirm edits by sequencing at both DNA and RNA levels

  • Verify protein expression changes by Western blot

  • Test functional consequences through target gene expression analysis

  • Assess phenotypic effects under relevant conditions (e.g., cold stress)

• Applications Beyond Knockouts:

  • Create endogenously tagged IDD14 (GFP, FLAG, etc.) for native expression level studies

  • Introduce specific mutations mimicking or preventing phosphorylation

  • Generate promoter reporter fusions to study IDD14 expression patterns

This technology enables precise manipulation of IDD14 to dissect its various functions in starch metabolism regulation and plant stress responses with unprecedented specificity.

What computational tools can help predict and analyze IDD14 binding sites?

For computational prediction and analysis of IDD14 binding sites:

• Motif Discovery and Scanning Tools:

  • MEME Suite: Identify enriched motifs in ChIP-seq data

  • FIMO/MAST: Scan genome for occurrences of identified motifs

  • RSAT: Regulatory Sequence Analysis Tools for comprehensive motif analysis

  • Focus on known IDD-binding motifs, particularly those containing the ID1 element

• Integrated ChIP-seq Analysis Pipelines:

  • HOMER: Peak calling, motif discovery, and genomic feature annotation

  • ChIPseeker: Comprehensive ChIP peak annotation and visualization

  • ChIP-Atlas: Compare binding profiles with other transcription factors

• Comparative Genomics Approaches:

  • Examine conservation of binding sites across species

  • Identify co-occurring motifs of potential cooperation partners

  • Search for enrichment of binding sites near co-regulated genes

• Machine Learning Applications:

  • Implement DyAb or similar models to predict binding affinity changes with sequence variations

  • Use deep learning approaches to integrate multiple data types for binding prediction

  • Apply CNN-based models to capture complex binding patterns from ChIP-seq data

• Structural Modeling:

  • Use AlphaFold or similar tools to predict IDD14 structure

  • Model protein-DNA interactions based on known structures of related proteins

  • Simulate effects of mutations or phosphorylation on binding properties