Interleukin-13 (IL-13) functions as a key cytokine mediating type 2 inflammation and serves as an important pathogenic factor in various inflammatory conditions. This cytokine has emerged as a significant contributor to the pathophysiology of atopic dermatitis, where it drives inflammatory processes and tissue damage . The biological importance of IL-13 has made it a prime target for therapeutic intervention, particularly in conditions characterized by type 2 inflammatory responses. Understanding the role of IL-13 in these pathways has led to the development of specific monoclonal antibodies designed to neutralize its activity and thereby reduce inflammatory burden in affected tissues. This targeting approach represents a more precise intervention compared to broader immunosuppressive strategies.
The pharmacokinetic properties of anrukinzumab have been systematically investigated across different patient populations. Studies by Hua et al. (2015) conducted comparative pharmacokinetic analyses among healthy volunteers, asthma patients, and ulcerative colitis patients to characterize how different disease states affect drug disposition . These investigations provide crucial information about how the antibody behaves in different patient populations.
In clinical investigations, anrukinzumab has been administered intravenously at doses of 200 mg to ulcerative colitis patients . Concentration-time profiles show the pharmacokinetic behavior following multiple intravenous administrations of 200 mg doses, with comparisons between ulcerative colitis patients and non-UC subjects . These profiles help determine parameters such as maximum concentration (Cmax), area under the curve (AUC), elimination half-life, and clearance rates, which are essential for establishing appropriate dosing regimens.
Systematic pharmacokinetic modeling has been employed to predict antibody behavior across different doses, as evidenced by visual predictive check analyses for different dosing strategies . This modeling approach allows for optimization of dosing regimens and helps predict antibody behavior in different clinical scenarios.
Anrukinzumab has undergone clinical evaluation for inflammatory conditions, particularly ulcerative colitis. Research by Reinisch et al. (2015) assessed the efficacy and safety profile of anrukinzumab in active ulcerative colitis through a phase IIa randomized multicentre study . This study represents a significant step in establishing the clinical utility of anti-IL-13 therapy for inflammatory bowel conditions.
One important biomarker evaluated was faecal calprotectin, a sensitive indicator of intestinal inflammation. Studies have measured the effect of anrukinzumab compared with placebo on fold changes in this marker, providing objective assessment of anti-inflammatory effects . Reduction in faecal calprotectin levels would suggest decreased neutrophil migration to the intestinal mucosa, indicating reduced inflammatory activity.
Pharmacodynamic effects were monitored through measurement of total IL-13 levels over time, with median IL-13 level versus time profiles compared between treatment groups . These measurements help establish the relationship between antibody administration, target engagement, and subsequent clinical effects. The temporal relationship between antibody administration and changes in IL-13 levels provides insight into the duration of effect and optimal dosing intervals.
Beyond clinical applications, anrukinzumab derivatives serve important functions in laboratory research. The IL-13 Antibody (anrukinzumab) conjugated with FITC (fluorescein isothiocyanate) is utilized in multiple research applications including ELISA, flow cytometry, and functional assays . This laboratory-grade antibody enables investigation of IL-13 biology and potential therapeutic approaches.
The technical specifications for research-grade anrukinzumab include:
| Property | Specification |
|---|---|
| Species Reactivity | Human |
| Applications | ELISA, Flow Cytometry, Functional assays |
| Fluorescent Label | FITC (Excitation = 495 nm, Emission = 519 nm) |
| Antibody Source | Recombinant Monoclonal Human IgG |
| Clone | anrukinzumab |
| Immunogen | IL-13 |
| Clonality | Monoclonal |
| Host | Human |
| Isotype | IgG |
| Purification Method | Protein A purified |
| Formulation | PBS with 0.05% Sodium Azide |
| Storage Conditions | 4°C in the dark |
These research tools are specified for laboratory use only and are not approved for human administration or clinical diagnosis . The availability of such research reagents facilitates investigation into IL-13 biology and therapeutic development.
Lebrikizumab represents another important anti-IL-13 monoclonal antibody that has been extensively studied for inflammatory conditions, particularly atopic dermatitis . Comparing anrukinzumab with lebrikizumab provides important insights into the therapeutic potential of IL-13 targeting across different inflammatory conditions.
A phase 2 clinical study evaluated lebrikizumab as an adjunctive therapy to topical corticosteroids (TCS) in patients with moderate-to-severe atopic dermatitis . This study employed a rigorous methodology:
Randomized, placebo-controlled, double-blind design
Four treatment arms with 1:1:1:1 randomization:
Lebrikizumab 125 mg single dose
Lebrikizumab 250 mg single dose
Lebrikizumab 125 mg every 4 weeks for 12 weeks
Placebo every 4 weeks for 12 weeks
Initial 2-week TCS run-in period before randomization
Primary endpoint: percentage of patients achieving at least 50% improvement in Eczema Area and Severity Index (EASI-50) at week 12
The clinical outcomes demonstrated significant therapeutic benefit:
| Treatment Group | EASI-50 Achievement | P-value vs Placebo |
|---|---|---|
| Lebrikizumab 125 mg Q4W | 82.4% | 0.026 |
| Placebo Q4W | 62.3% | - |
| Single dose lebrikizumab groups | No statistically significant difference | >0.05 |
Safety analysis revealed comparable adverse event profiles between lebrikizumab and placebo groups (66.7% for all lebrikizumab groups vs. 66.0% for placebo), with most events being mild or moderate in severity . This favorable safety profile, combined with efficacy data, supports further investigation of lebrikizumab for inflammatory skin conditions.
The development of anti-IL-13 antibodies represents an evolving field with significant therapeutic potential. Current research limitations highlight important areas for future investigation :
Understanding efficacy as monotherapy remains a crucial research gap, as most studies have evaluated these antibodies as add-on therapies to treatments like topical corticosteroids. Establishing their effectiveness as standalone treatments would clarify their therapeutic value and potentially expand their clinical applications. The optimal dosing regimens, including dose amount and frequency, continue to be refined through ongoing clinical investigations.
Long-term efficacy and safety evaluations are needed beyond the relatively short duration of existing studies . Given that many inflammatory conditions requiring these therapies are chronic in nature, understanding the sustained benefits and potential long-term risks is essential for clinical decision-making. Extended follow-up studies will help establish the durability of response and identify any delayed adverse effects that might not be apparent in shorter trials.
Additional inflammatory conditions mediated by IL-13 beyond the currently studied indications represent potential therapeutic opportunities. As our understanding of IL-13 biology expands, new applications may emerge for anti-IL-13 antibodies in various inflammatory and allergic conditions where this cytokine plays a pathogenic role.
IAN13 (Immune-Associated Nucleotide-binding protein 13) belongs to the GTPase of immunity-associated proteins (GIMAP) family in Arabidopsis thaliana. This protein family plays crucial roles in plant immune responses, development, and stress signaling pathways. IAN13 (UniProt ID: Q9T0F4) is particularly important in studying plant immunity mechanisms. The IAN13 Antibody enables researchers to investigate protein localization, expression patterns, and protein-protein interactions under various experimental conditions. Studies of IAN13 contribute to our understanding of how plants regulate immune responses at the molecular level, potentially leading to applications in crop improvement and disease resistance .
Proper storage of IAN13 Antibody is critical for maintaining its activity and specificity. Based on standard antibody handling protocols:
| Storage Duration | Temperature | Additives | Notes |
|---|---|---|---|
| Long-term (>1 month) | -20°C | 50% glycerol | Aliquot to avoid freeze-thaw cycles |
| Short-term (<1 month) | 4°C | 0.02% sodium azide | Protect from light |
| Working solution | 4°C | 1% BSA, 0.02% sodium azide | Use within 7 days |
Repeated freeze-thaw cycles significantly reduce antibody activity. For optimal results, dividing the stock into small aliquots upon receipt is strongly recommended. When preparing working dilutions, use sterile techniques and high-quality buffers to prevent microbial contamination that could degrade the antibody .
IAN13 Antibody (such as CSB-PA154892XA01DOA) is designed with high specificity for the IAN13 protein in Arabidopsis thaliana. When compared to antibodies targeting related immune proteins:
| Antibody | Target Protein | Cross-Reactivity | Application Strength |
|---|---|---|---|
| IAN13 Antibody | IAN13 (Q9T0F4) | Minimal with IAN family | Strong in Western blot, IHC |
| IAN12 Antibody | IAN12 (Q9T0F3) | May cross-react with IAN13 | Similar applications |
| IAN5 Antibody | IAN5 (Q9C8U8) | Low cross-reactivity | Good for differential studies |
| IAN9 Antibody | IAN9 (F4HT21) | Distinct epitope recognition | Complementary validation |
Researchers should validate specificity through appropriate controls, especially when studying multiple IAN family members simultaneously. Using antibodies raised against different epitopes can provide confirmatory evidence for protein identification .
For successful Western blot analysis with IAN13 Antibody:
Sample Preparation:
Extract total protein from Arabidopsis tissues using buffer containing 50mM Tris-HCl (pH 7.5), 150mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, and protease inhibitor cocktail
Homogenize tissue thoroughly while keeping samples cold
Centrifuge at 12,000g for 15 minutes at 4°C and collect supernatant
Gel Electrophoresis and Transfer:
Separate 20-40μg protein on 10-12% SDS-PAGE
Transfer to PVDF membrane (100V for 60 minutes or 30V overnight)
Verify transfer efficiency with reversible staining
Antibody Incubation:
Block membrane with 5% non-fat milk in TBST for 1 hour at room temperature
Incubate with IAN13 Antibody (1:1000 dilution) in blocking buffer overnight at 4°C
Wash 3×15 minutes with TBST
Incubate with HRP-conjugated secondary antibody (1:5000) for 1 hour
Wash 3×15 minutes with TBST
Develop using ECL substrate and image
Troubleshooting Guide:
| Issue | Possible Cause | Solution |
|---|---|---|
| No signal | Protein degradation | Use fresh samples, increase protease inhibitors |
| Multiple bands | Cross-reactivity | Increase antibody dilution, extend washing |
| High background | Insufficient blocking | Optimize blocking conditions, try 3% BSA alternative |
| Weak signal | Low protein abundance | Increase protein loading, reduce antibody dilution |
Including both wild-type and ian13 mutant samples as controls is essential for confirming signal specificity .
Rigorous validation of IAN13 Antibody specificity is crucial for reliable experimental outcomes. A comprehensive validation approach includes:
Genetic Controls Testing:
Compare signal between wild-type Arabidopsis and ian13 knockout/knockdown mutants
Examine IAN13 overexpression lines for increased signal intensity
Use CRISPR-edited plants with epitope modifications
Biochemical Validation:
Perform peptide competition assays by pre-incubating antibody with immunizing peptide
Conduct immunoprecipitation followed by mass spectrometry to confirm target identity
Compare results with alternative antibodies targeting different IAN13 epitopes
Cross-Reactivity Assessment:
Test against recombinant IAN family proteins to evaluate potential cross-reactivity
Examine tissues with known differential expression of IAN family members
Perform Western blots under high-stringency conditions
The most convincing validation combines multiple approaches, particularly the absence of signal in genetic knockout lines coupled with specific recognition of the target protein at the expected molecular weight in wild-type samples .
For robust immunoprecipitation (IP) experiments with IAN13 Antibody, implement these essential controls:
Mandatory Controls:
| Control Type | Purpose | Implementation |
|---|---|---|
| Input sample | Verify starting material | Set aside 5-10% of pre-IP lysate |
| Isotype control | Detect non-specific binding | Use same concentration of irrelevant antibody |
| Negative genetic control | Confirm specificity | Process ian13 mutant samples identically |
| Beads-only control | Identify matrix binding | Perform IP without antibody |
| Pre-immune serum control | Establish baseline binding | If using polyclonal antibodies |
Additional Controls for Co-IP Experiments:
Binding condition controls:
Compare native versus denaturing conditions to identify specific interactions
Include RNase/DNase treatments to exclude nucleic acid-mediated associations
Reciprocal co-IP:
Perform reverse experiment using antibodies against suspected interaction partners
Validate interactions through orthogonal methods (Y2H, BiFC)
Specificity controls:
Use competitive elution with excess IAN13 peptide
Include predicted non-interacting proteins as negative controls
These controls help distinguish genuine interactions from experimental artifacts and are particularly important when studying novel protein-protein interactions involving IAN13 .
Discrepancies between antibody-based protein detection and gene expression analysis are common in plant research and may reflect important biological phenomena rather than technical errors:
Post-transcriptional Regulation Assessment:
mRNA abundance frequently does not correspond directly to protein levels due to translation efficiency differences
Investigate protein stability through cycloheximide chase experiments
Examine potential miRNA regulation of IAN13 mRNA
Protein Modification Considerations:
Post-translational modifications may mask antibody epitopes
Treatment with phosphatases or deubiquitinating enzymes before analysis
Test multiple antibodies targeting different regions of IAN13
Methodological Approach to Resolve Discrepancies:
| Observation | Possible Biological Explanation | Validation Method |
|---|---|---|
| High mRNA, low protein | Rapid protein turnover | Proteasome inhibitor treatment (MG132) |
| Inefficient translation | Polysome fractionation analysis | |
| Post-translational regulation | Phosphorylation or ubiquitination analysis | |
| Low mRNA, high protein | High protein stability | Cycloheximide chase with timepoints |
| Regulated protein degradation | Compare stress vs. normal conditions | |
| Antibody cross-reactivity | Immunoprecipitation with MS verification |
The integration of multiple techniques (RT-qPCR, Western blot, mass spectrometry) provides complementary data for a more complete understanding of IAN13 regulation .
Immunofluorescence microscopy in plant tissues presents unique challenges when using antibodies like IAN13 Antibody:
Common Artifacts and Solutions:
Plant-Specific Autofluorescence:
Chlorophyll autofluorescence in green tissues (especially problematic in red channels)
Cell wall components (lignin, suberin) create background signal
Solution: Use appropriate filter sets, spectral unmixing, or chemical treatments to quench autofluorescence
Fixation-Related Artifacts:
Overfixation can mask epitopes and create false negatives
Insufficient fixation compromises cellular architecture
Solution: Optimize fixation protocols specifically for IAN13 detection (typically 2-4% paraformaldehyde for 1-2 hours)
Cell Wall Penetration Issues:
Plant cell walls hinder antibody penetration
Solution: Include appropriate permeabilization steps (0.1-0.3% Triton X-100) and consider enzymatic digestion with cell wall-degrading enzymes
Non-Specific Binding:
Plant tissues often show high background due to hydrophobic interactions
Solution: Use specialized blocking reagents containing BSA, normal serum, and plant-specific blockers
Artifact Identification Guide:
| Artifact Type | Distinguishing Features | Critical Controls |
|---|---|---|
| Autofluorescence | Present in untreated samples | Image unlabeled tissue with identical settings |
| Fixation artifact | Variable pattern between fixation methods | Compare multiple fixation protocols |
| Cell wall binding | Uniform signal at cell periphery | Include pre-immune serum control |
| Vacuolar trapping | Signal accumulation in vacuoles | Test shorter incubation times, different detergents |
Always include negative controls (pre-immune serum, secondary antibody only) and positive controls (proteins known to co-localize with IAN13) .
IAN13 Antibody offers several approaches for investigating protein-protein interactions in plant immunity contexts:
Co-Immunoprecipitation (Co-IP):
Use IAN13 Antibody to pull down IAN13 protein complexes from plant lysates
Identify interaction partners through mass spectrometry analysis
Validate interactions using reciprocal Co-IP with antibodies against putative partners
Consider mild crosslinking to capture transient interactions in immune signaling cascades
Proximity Labeling Approaches:
Combine with BioID or TurboID systems for proximity-dependent biotinylation
Create fusion proteins with IAN13 and proximity labeling enzymes
Use IAN13 Antibody to verify expression and localization of fusion proteins
Identify proximal proteins through streptavidin pulldown and mass spectrometry
Immunofluorescence Co-Localization:
Perform dual labeling with IAN13 Antibody and antibodies against suspected partners
Analyze co-localization using quantitative methods (Pearson's correlation, Manders' coefficients)
Examine dynamic changes in localization during immune responses
Methodological Workflow:
Initial screening via Co-IP/MS to identify potential interaction partners
Validation using orthogonal methods (Y2H, BiFC)
Functional characterization through mutant analysis
Spatiotemporal dynamics using advanced microscopy
These approaches can uncover novel components of plant immune signaling networks and provide mechanistic insights into IAN13 function .
Quantifying IAN13 expression levels requires careful consideration of methodological limitations and appropriate controls:
Western Blot Quantification:
Semi-quantitative assessment with densitometry
Requires carefully optimized loading controls (ACTIN, TUBULIN, or total protein stains)
Standard curve using recombinant protein recommended for absolute quantification
Maintain samples within linear dynamic range of detection system
Include biological and technical replicates (minimum n=3)
ELISA-Based Quantification:
Develop sandwich ELISA using IAN13 Antibody and a second antibody targeting different epitope
Create standard curve using purified recombinant IAN13 protein
Higher throughput for multiple samples and conditions
More suitable for absolute quantification than Western blot
Flow Cytometry (for Protoplasts):
Quantify IAN13 expression at single-cell level
Require permeabilization for intracellular IAN13 detection
Use fluorophore-conjugated secondary antibodies
Include calibration beads for standardization
Quantification Method Comparison:
| Method | Quantitation Precision | Dynamic Range | Sample Throughput | Key Considerations |
|---|---|---|---|---|
| Western blot | Moderate | ~10-fold | Low | Best for relative comparisons |
| ELISA | High | ~1000-fold | High | Requires purified standards |
| Flow cytometry | High | ~10,000-fold | Medium | Requires protoplast preparation |
| Immunohistochemistry | Low-Moderate | Variable | Low | Provides spatial information |
The choice of method should be guided by experimental requirements for sensitivity, throughput, and whether spatial information is needed .
IAN13 Antibody is a valuable tool for investigating post-translational modifications (PTMs) that regulate IAN13 function during immune responses:
Phosphorylation Analysis:
Combine IAN13 Antibody immunoprecipitation with phospho-specific staining
Compare phosphorylation status before and after pathogen treatment
Use phosphatase treatments to confirm modification
Phospho-specific antibodies can be developed if modification sites are known
Ubiquitination Studies:
Perform IAN13 immunoprecipitation followed by ubiquitin Western blot
Include proteasome inhibitors (MG132) to stabilize ubiquitinated forms
Examine changes in ubiquitination patterns during immune activation
Consider tandem ubiquitin binding entity (TUBE) pulldowns to enrich ubiquitinated proteins
Other Modifications:
SUMOylation can be assessed through similar immunoprecipitation approaches
Redox modifications may be investigated using redox-sensitive probes
Glycosylation can be examined using glycosidase treatments
Experimental Workflow for PTM Analysis:
Immunoprecipitate IAN13 from control and treated samples
Divide precipitated material for different analyses:
Direct Western blot for total IAN13
Modification-specific antibody detection
Mass spectrometry analysis for unbiased PTM mapping
Validate findings with site-directed mutagenesis of modified residues
Assess functional consequences of mutations in plant immunity assays
This approach can reveal regulatory mechanisms controlling IAN13 activity during plant immune responses and stress adaptation .
Optimizing IAN13 Antibody concentration for plant immunohistochemistry requires systematic titration and protocol refinement:
Antibody Titration Strategy:
Initial Concentration Range Testing:
Prepare serial dilutions (1:50, 1:100, 1:200, 1:500, 1:1000)
Use consistent tissue samples with known IAN13 expression
Process all samples identically except for antibody concentration
Fixation Method Optimization:
Test multiple fixatives (4% paraformaldehyde, 2% glutaraldehyde, acetone)
Evaluate different fixation durations (30 min, 1 hour, 2 hours, overnight)
Assess requirement for antigen retrieval methods
Signal-to-Background Evaluation:
Systematically document signal intensity versus background at each concentration
Include ian13 mutant tissues as negative controls
Test secondary antibody alone to assess non-specific binding
Optimization Results Template:
| Dilution | Fixative | Antigen Retrieval | Signal Strength | Background | Specificity |
|---|---|---|---|---|---|
| 1:50 | 4% PFA, 1h | None | ++++ | ++ | Medium |
| 1:100 | 4% PFA, 1h | None | +++ | + | High |
| 1:200 | 4% PFA, 1h | None | ++ | +/- | Very High |
| 1:100 | Acetone, 10m | None | +++ | + | High |
| 1:100 | 2% Glut., 1h | Citrate buffer | + | +++ | Low |
The optimal dilution provides clear specific signal with minimal background. For most applications with IAN13 Antibody in Arabidopsis tissues, a 1:100-1:200 dilution with PFA fixation typically yields the best results .
When facing weak signal issues with IAN13 Antibody in Western blot analysis, consider these targeted optimization approaches:
Sample Preparation Enhancement:
Use fresh tissue and maintain cold chain throughout extraction
Include additional protease inhibitors (PMSF, aprotinin, leupeptin)
Optimize buffer composition for IAN13 solubilization
Consider using specialized plant protein extraction kits designed for low-abundance proteins
Protocol Modifications for Signal Amplification:
Increase protein loading (40-60μg per lane)
Reduce antibody dilution incrementally (1:1000 to 1:500 to 1:250)
Extend primary antibody incubation time (overnight at 4°C)
Use high-sensitivity detection reagents (enhanced chemiluminescence plus)
Consider biotin-streptavidin amplification systems
Technical Optimization:
Switch membrane type (PVDF typically better than nitrocellulose for weak signals)
Reduce washing stringency slightly (use 0.05% instead of 0.1% Tween-20)
Try different blocking agents (BSA vs. milk vs. commercial blockers)
Use film exposure for very weak signals (more sensitive than digital imaging)
Systematic Troubleshooting Approach:
| Issue | Diagnostic Test | Solution |
|---|---|---|
| Protein degradation | Run sample immediately after extraction vs. stored sample | Use fresh samples, add more protease inhibitors |
| Poor transfer | Stain membrane for total protein after transfer | Optimize transfer conditions, reduce methanol % |
| Inactive antibody | Dot blot test with recombinant protein | Use new antibody aliquot |
| Low abundance target | Verify expression with RT-PCR | Enrich target through immunoprecipitation first |
| Epitope masking | Test multiple extraction buffers | Try denaturing conditions, heat samples longer |
If IAN13 is consistently difficult to detect, consider creating transgenic plants expressing epitope-tagged versions (HA, FLAG, GFP) which can be detected with highly optimized commercial antibodies .
Advanced multiplexed approaches enable simultaneous analysis of IAN13 and other immune-related proteins:
Multiplex Immunofluorescence Strategies:
Use IAN13 Antibody alongside antibodies against defense-related proteins (NPR1, EDS1, PAD4)
Combine with subcellular markers to track localization changes during immune responses
Employ spectrally distinct fluorophores for each target protein
Utilize automated image analysis for colocalization quantification
Sequential Immunoblotting Approaches:
Strip and reprobe membranes to detect multiple proteins on the same blot
Use differently sized proteins with same-species antibodies
Employ fluorescently-labeled secondary antibodies with different emission spectra
Conduct quantitative analysis of relative protein levels
Advanced Multiplex Technologies:
Mass cytometry (CyTOF) adapted for plant protoplasts with metal-conjugated antibodies
Proximity extension assays for protein interaction networks
Micro-western arrays for high-throughput, low-volume analysis
These multiplexed approaches are particularly valuable for studying signaling networks, as they preserve the relationships between different immune components within the same sample and eliminate variation that might occur between separate analyses .
Emerging spatial biology technologies are revolutionizing our ability to study the distribution and interactions of proteins like IAN13 in plant tissues:
Advanced Microscopy Techniques:
Super-resolution microscopy (STORM, PALM, SIM) to visualize IAN13 distribution beyond diffraction limit
Expansion microscopy for physical magnification of plant tissues while preserving protein epitopes
Light-sheet microscopy for rapid 3D imaging with reduced phototoxicity
Spatial Transcriptomics Integration:
Combined antibody detection with in situ RNA analysis
Correlation of protein localization with transcriptomic data
Multi-modal data integration for comprehensive pathway analysis
Emerging Antibody-Based Spatial Technologies:
Imaging mass cytometry for highly multiplexed protein detection in plant tissues
Digital spatial profiling platforms adapted for plant tissue architecture
Proximity ligation assays for in situ protein interaction detection
Future Directions and Applications:
| Technology | Resolution | Multiplexing Capacity | Application to IAN13 Research |
|---|---|---|---|
| Expansion Microscopy | ~70nm | 4-5 proteins | Subcellular localization in relation to cellular structures |
| CODEX | Cell-level | 40+ proteins | Comprehensive immune protein networks |
| Imaging Mass Cytometry | 1μm | 40+ proteins | Tissue-specific expression patterns during infection |
| Spatial Transcriptomics | 10-100μm | Whole transcriptome + proteins | Integration of protein and RNA regulation |
These technologies promise to provide unprecedented insights into how IAN13 functions within the spatial context of plant tissues during immune responses .