NRT2.6 Antibody

What is NRT2.6 and why is it significant in plant molecular biology research?

NRT2.6 is a member of the high-affinity nitrate transporter (NRT2) family in Arabidopsis thaliana. Unlike other members of the NRT2 family that are primarily involved in nitrate uptake from soil, NRT2.6 plays a distinctive role in plant defense responses against pathogens.

The significance of NRT2.6 in research stems from its unique expression patterns and functions:

• It is weakly expressed in most plant organs, with higher expression in vegetative tissues than reproductive organs

• Unlike other NRT2 members, its expression is induced by high nitrogen levels rather than nitrogen limitation

• It shows strong induction after inoculation with phytopathogenic bacteria like Erwinia amylovora

• It appears to be involved in reactive oxygen species (ROS) accumulation during pathogen response

Methodologically, studying NRT2.6 requires specific approaches due to its weak baseline expression. Researchers typically use qRT-PCR with highly specific primers, reporter gene constructs (like ProNRT2.6:GFP), or antibody-based techniques to detect and quantify the protein in various plant tissues.

How can researchers distinguish between NRT2.6 and other closely related NRT family members?

Distinguishing between NRT2.6 and other NRT2 family members presents challenges due to sequence homology. For example, NRT2.4 shares 88% amino acid similarity with NRT2.1, while NRT2.6 also shows considerable sequence similarity to other family members .

Recommended methodological approaches include:

• Use of highly specific antibodies raised against unique epitopes in NRT2.6

• Careful primer design for qRT-PCR targeting unique regions of the NRT2.6 sequence

• Validation using knockout mutants (e.g., nrt2.6-1) as negative controls

• Expression pattern analysis under various conditions:

  • NRT2.6 is uniquely induced under high nitrogen conditions, while NRT2.1, NRT2.2, and NRT2.4 are induced under nitrogen limitation

  • NRT2.6 shows distinctive induction patterns after pathogen challenge

  • NRT2.4 is specifically expressed in lateral roots and younger parts of main roots under nitrogen starvation, whereas NRT2.6 has a different expression pattern

For immunological studies, pre-absorption of antibodies with recombinant proteins of related NRT2 family members can enhance specificity.

What are the standard protocols for NRT2.6 antibody validation in plant research?

When using or developing NRT2.6 antibodies, validation is critical to ensure specificity and reliability. A comprehensive validation protocol includes:

• Genetic validation:

  • Testing in nrt2.6-1 knockout mutants (negative control)

  • Testing in NRT2.6 overexpression lines (e.g., the complemented mutant lines CM1 and CM2 that overexpress NRT2.6 at 126 and 238 times higher levels than wild-type)

• Biochemical validation:

  • Western blot analysis showing specific band at expected molecular weight

  • Peptide competition assays to confirm epitope specificity

  • Cross-reactivity tests with recombinant proteins from related NRT family members

• Application-specific validation:

  • For immunolocalization: parallel staining with secondary antibody only

  • For immunoprecipitation: verification of pulled-down proteins by mass spectrometry

  • For ELISA/quantitative assays: generation of standard curves using recombinant NRT2.6

• Expression pattern consistency:

  • Antibody signal should increase after Erwinia amylovora infection, consistent with known transcript upregulation

  • Signal should be stronger in vegetative organs than reproductive organs

How can NRT2.6 antibodies be utilized to investigate pathogen-induced defense mechanisms?

NRT2.6 antibodies provide powerful tools for elucidating the role of this transporter in plant immunity. Research has established that NRT2.6 expression is rapidly induced following pathogen challenge, with expression peaking at 3 hours post-inoculation with Erwinia amylovora .

Advanced methodological approaches include:

• Temporal and spatial protein localization during infection:

  • Immunohistochemistry or immunofluorescence microscopy to track NRT2.6 protein localization before and after pathogen challenge

  • Co-localization studies with defense-related proteins to identify potential interaction partners

  • Live cell imaging with fluorescently-tagged antibodies to monitor dynamic changes

• Protein complex analysis:

  • Co-immunoprecipitation with NRT2.6 antibodies to identify interacting proteins during pathogen response

  • Chromatin immunoprecipitation (ChIP) if NRT2.6 may have DNA-binding activity

  • Blue native PAGE combined with immunoblotting to identify native protein complexes

• Functional assays:

  • Assessing ROS production in wild-type vs. nrt2.6-1 plants using both fluorescent probes and immunological detection of ROS-induced protein modifications

  • Correlating NRT2.6 protein levels with pathogen resistance phenotypes

  • Investigating changes in NRT2.6 post-translational modifications during infection

Research has shown that decreased NRT2.6 expression correlates with reduced ROS accumulation and increased susceptibility to E. amylovora infection . Antibody-based techniques can help quantify these relationships and elucidate the underlying mechanisms.

What experimental approaches can resolve contradictory data regarding NRT2.6 function in nitrogen transport versus pathogen response?

The dual nature of NRT2.6—a nitrate transporter family member involved in pathogen response rather than primary nitrogen transport—presents interesting research questions. Studies show that NRT2.6 overexpression failed to complement the nitrate uptake defect of nrt2.1-nrt2.2 double mutants, suggesting it may not function primarily in nitrogen uptake .

To resolve these seemingly contradictory functions, consider these methodological approaches:

• Dual-function analysis:

  • Simultaneous measurement of nitrate transport activity and defense responses in various genetic backgrounds

  • Use of split-ubiquitin or BiFC assays with antibody validation to identify different protein interaction networks in normal versus pathogen-challenged conditions

• Structure-function studies:

  • Site-directed mutagenesis of conserved domains followed by immunodetection to identify regions responsible for transport versus signaling

  • Domain-specific antibodies to detect potential proteolytic processing or conformational changes

• Comprehensive expression analysis:

  • Quantitative immunoblotting to compare protein levels across different conditions:

  Condition	NRT2.1 Protein	NRT2.6 Protein	ROS Accumulation
  High N	Low	High	Baseline
  Low N	High	Low	Baseline
  Pathogen	Variable	High	Elevated
  High N + Pathogen	Low	Very high	Highly elevated

• Cross-species comparison:

  • Immunological detection of NRT2.6 homologs in other plant species to determine evolutionary conservation of dual functionality

  • Heterologous expression systems with antibody-based detection to assess transport capability separated from plant defense context

How can researchers optimize NRT2.6 immunolocalization in plant tissues with low expression levels?

Given that NRT2.6 is weakly expressed in most plant organs , detecting the protein through immunolocalization presents technical challenges. Advanced protocols to enhance detection include:

• Signal amplification methods:

  • Tyramide signal amplification (TSA) to enhance fluorescence signal

  • Quantum dot-conjugated secondary antibodies for improved signal-to-noise ratio

  • Gold-enhanced immunohistochemistry for electron microscopy applications

• Sample preparation optimization:

  • Careful fixation protocols to preserve epitope accessibility (comparative analysis of formaldehyde, glutaraldehyde, and alternative fixatives)

  • Antigen retrieval methods adapted specifically for plant tissues

  • Ultra-thin sectioning techniques for improved antibody penetration

• Induction conditions for enhanced detection:

  • Samples taken at specific timepoints after Erwinia amylovora infection (optimally at 3 hours post-inoculation)

  • Growth under high nitrogen conditions to increase baseline expression

  • Methyl viologen treatment to stimulate ROS-related pathways, which may enhance NRT2.6 expression

• Control experiments:

  • Side-by-side comparison with ProNRT2.6:GFP reporter lines

  • Parallel processing of nrt2.6-1 knockout tissues and CM1/CM2 overexpression lines

  • Competitive binding with immunizing peptide to confirm specificity

What roles do post-translational modifications play in NRT2.6 function, and how can antibodies help investigate them?

While the search results don't specifically address post-translational modifications (PTMs) of NRT2.6, this represents an important research frontier. Custom antibodies recognizing specific PTMs could help elucidate regulatory mechanisms.

Advanced research approaches include:

• Phosphorylation-specific antibodies:

  • Generation of antibodies against predicted phosphorylation sites

  • Validation using phosphatase-treated samples as controls

  • Application in studying kinase signaling cascades during pathogen response

• Other PTM-specific detection:

  • Antibodies against ubiquitination, SUMOylation, or nitrosylation

  • Investigation of PTM dynamics following pathogen challenge

  • Correlation between PTMs and protein localization/function

• Multiplexed PTM analysis:

  • Sequential immunoprecipitation to enrich for different modified forms

  • Mass spectrometry validation of antibody-detected modifications

  • Development of antibody arrays for high-throughput PTM profiling

• Functional validation:

  • Site-directed mutagenesis of modified residues followed by immunodetection

  • In vitro modification assays with recombinant enzymes and subsequent antibody detection

  • Correlation between PTM status and association with ROS production machinery

How can NRT2.6 antibodies help investigate the relationship between nitrogen metabolism and ROS production during stress?

Research has established a correlation between NRT2.6 expression and ROS accumulation in response to bacterial infection and treatment with redox-active compounds like methyl viologen . NRT2.6 antibodies can help clarify this relationship through:

• Co-localization studies:

  • Dual immunolocalization of NRT2.6 and ROS-generating enzymes (e.g., NADPH oxidases)

  • Tracking cellular compartmentalization during stress responses

  • Investigation of membrane microdomain associations

• Protein interaction networks:

  • Immunoprecipitation of NRT2.6 from control and stressed plants

  • Identification of differential interaction partners under various conditions

  • Validation of interactions using reciprocal co-immunoprecipitation

• Temporal dynamics analysis:

  • Quantitative immunoblotting at multiple timepoints after stress induction

  • Correlation with ROS measurements and defense gene expression

  • Development of mathematical models describing the relationship between protein levels and physiological responses

• Cross-talk with hormonal pathways:

  • Investigation of how plant hormones affect NRT2.6 protein levels

  • Studies combining phytohormone quantification with NRT2.6 immunodetection

  • Exploration of signaling pathway integration points

Research has demonstrated that pathogen-induced expression of NRT2.6 correlates with ROS accumulation , making this protein a potential key mediator between nitrogen metabolism and plant defense responses.

What techniques can be combined with NRT2.6 immunodetection to study its role in different plant-microbe interactions?

NRT2.6 responds to various beneficial and pathogenic microorganisms, including Erwinia amylovora, Agrobacterium tumefaciens, and plant growth-promoting rhizobacteria (PGPR) . Comprehensive studies of its role in plant-microbe interactions can combine:

• Multi-organism experimental systems:

  • Side-by-side comparison of NRT2.6 protein dynamics during beneficial versus pathogenic interactions

  • Split-root experiments with localized microbial exposures and systemic response assessment

  • Development of microfluidic devices for controlled microbial exposure with real-time immunodetection

• Integrated omics approaches:

  • Correlation of NRT2.6 protein levels (via quantitative immunoblotting) with:

    • Transcriptome data (RNA-seq)

    • Metabolome changes (particularly nitrogen-related metabolites)

    • Proteome alterations (mass spectrometry)

• Functional imaging techniques:

  • FRET/FLIM with fluorescently labeled antibodies to detect protein-protein interactions in situ

  • Correlative light and electron microscopy to connect protein localization with ultrastructural changes

  • Super-resolution microscopy to investigate nanoscale organization during microbial interactions

• Genetic manipulation coupled with immunodetection:

  • CRISPR/Cas9-modified plants with tagged endogenous NRT2.6

  • Domain deletion constructs to identify regions essential for microbial response

  • Heterologous expression systems to study isolated component functions

What controls are essential when using NRT2.6 antibodies in different experimental contexts?

Rigorous experimental design requires appropriate controls to ensure valid interpretation of results. For NRT2.6 antibody applications, essential controls include:

• Genetic controls:

  • nrt2.6-1 knockout mutant (negative control)

  • CM1/CM2 complemented lines (overexpression positive controls)

  • Wild-type Columbia (baseline expression control)

• Technical controls for immunoblotting:

  • Loading controls (constitutively expressed proteins)

  • Recombinant NRT2.6 protein standards at known concentrations

  • Peptide competition assays to validate specificity

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

• Controls for immunolocalization:

  • Pre-immune serum application (background control)

  • Tissues with known expression patterns (e.g., roots vs. reproductive organs)

  • Parallel processing of samples with and without primary antibody

  • Samples from plants under conditions known to induce NRT2.6 (e.g., E. amylovora infection)

• Experimental treatment controls:

  • Time-course sampling to capture expression dynamics

  • Parallel analysis of related NRT2 family members

  • Stress-specific positive controls (e.g., defense marker proteins for pathogen studies)

Careful inclusion of these controls enables confident interpretation of results and helps reconcile any apparent contradictions in experimental outcomes.

How can researchers develop quantitative assays for NRT2.6 protein levels in different plant tissues?

Developing robust quantitative assays for NRT2.6 requires addressing its relatively low expression levels in most tissues . Advanced methodological approaches include:

• Optimized protein extraction protocols:

  • Membrane protein-specific extraction buffers

  • Detergent screening for optimal solubilization

  • Subcellular fractionation to enrich for NRT2.6-containing fractions

• Quantitative immunoassay development:

  • Sandwich ELISA with capture and detection antibodies against different epitopes

  • Automated capillary western systems for enhanced sensitivity

  • AlphaLISA or similar amplification technologies for ultra-sensitive detection

• Standard curve generation:

  • Expression and purification of recombinant NRT2.6 protein

  • Isotope-labeled internal standards for absolute quantification

  • Synthetic peptide standards for targeted mass spectrometry approaches

• Normalization strategies:

  • Identification of stable reference proteins across experimental conditions

  • Development of multiplexed detection systems for simultaneous measurement of target and reference proteins

  • Mathematical modeling to account for tissue-specific expression variations

A comprehensive approach might employ multiple complementary methods, each validated against genetic controls, to establish the most reliable quantification strategy for different experimental contexts.

How can researchers differentiate between NRT2.6 protein function in different subcellular compartments?

Understanding the subcellular distribution of NRT2.6 is critical for elucidating its role in both nitrogen transport and pathogen response. Advanced approaches include:

• Subcellular fractionation combined with immunodetection:

  • Differential centrifugation to separate membrane fractions

  • Immunoblotting with compartment-specific markers as controls

  • Enzyme activity assays to confirm fraction purity

• High-resolution immunolocalization:

  • Confocal microscopy with co-localization analysis

  • Immunogold electron microscopy for precise subcellular localization

  • Super-resolution techniques (STORM, PALM) for nanoscale distribution patterns

• Compartment-specific functional assays:

  • In vitro transport assays with isolated membrane fractions

  • Patch-clamp electrophysiology with immunolocalization validation

  • Reconstitution experiments in artificial membrane systems

• Targeted protein engineering approaches:

  • Creation of compartment-specific variants through addition of targeting sequences

  • Development of split-protein complementation assays across compartments

  • FRET-based sensors to detect conformational changes in different environments

Research has not definitively established the subcellular localization of NRT2.6, though other family members like NRT2.1 and NRT2.2 are located in the plasma membrane . Determining whether NRT2.6 shows differential localization during pathogen response could provide important insights into its dual functions.

What are the best approaches for studying NRT2.6 protein-protein interactions in plant defense contexts?

Investigating NRT2.6 protein interaction networks during defense responses requires specialized approaches:

• In vivo interaction detection:

  • Co-immunoprecipitation with NRT2.6 antibodies followed by mass spectrometry

  • Proximity labeling techniques (BioID, APEX) with NRT2.6 as the bait protein

  • Split-ubiquitin or split-GFP assays for membrane protein interactions

• Dynamic interaction studies:

  • Time-resolved immunoprecipitation after pathogen challenge

  • FRET/FLIM analysis with appropriately tagged proteins

  • Single-molecule tracking with quantum dot-conjugated antibodies

• Structural validation:

  • Cross-linking mass spectrometry to map interaction interfaces

  • Hydrogen-deuterium exchange mass spectrometry to identify conformational changes

  • Computational modeling validated by mutagenesis and immunodetection

• Functional validation of interactions:

  • Bimolecular fluorescence complementation in planta

  • Genetic analysis of candidate interactors (e.g., double mutant phenotyping)

  • In vitro reconstitution of minimal interacting components

These approaches can help identify whether NRT2.6 interacts directly with components of ROS-generating systems, defense signaling complexes, or other nitrogen transporters during pathogen challenge.