CNGC9 Antibody

What is CNGC9 and what role does it play in plant immunity?

CNGC9 is a cyclic nucleotide-gated channel protein that functions as a calcium-permeable channel in plants. In rice, OsCNGC9 (also known as CDS1 - CELL DEATH and SUSCEPTIBLE to BLAST 1) positively regulates resistance to rice blast disease by mediating pathogen-associated molecular pattern (PAMP)-induced Ca²⁺ influx . This calcium influx is critical for the PAMP-triggered reactive oxygen species (ROS) burst and induction of defense gene expression, establishing CNGC9 as an essential component of pattern-triggered immunity (PTI) . The channel forms part of a signaling cascade that links pattern recognition to calcium channel activation, which is required for initiating PTI and disease resistance.

What are the established mechanisms of CNGC9 regulation in immune signaling?

CNGC9 activity is primarily regulated through phosphorylation by receptor-like cytoplasmic kinases. In rice, OsRLCK185 physically interacts with and phosphorylates OsCNGC9 to activate its channel activity following PAMP perception . This phosphorylation event can be detected using phos-tag assays, which reveal a band-shift of the CNGC9 C-terminal fragment after chitin treatment in wild-type plant cells . Similarly, other CNGCs like CNGC5/6/9/12 are regulated by OST1 (OPEN STOMATA 1) phosphorylation at conserved residues in their N-termini . Additionally, the cyclic nucleotide-binding domain and the calmodulin-binding domain play crucial roles in channel regulation, as seen with related CNGCs, where these domains control channel opening and closing in response to cellular signals.

How can contradictory findings on CNGC9 localization and interaction partners be reconciled in experimental designs?

Contradictory findings regarding CNGC9 localization and interaction partners can be reconciled through comprehensive experimental approaches:

• Multiple localization techniques: Combine fluorescence microscopy (using CNGC9-GFP fusions) with subcellular fractionation and immunolocalization using CNGC9 antibodies to verify localization patterns across different tissues and conditions.

• Dynamic interaction studies: Utilize BiFC (Bimolecular Fluorescence Complementation) assays similar to those used for CNGC12-CaM1 interactions , supplemented with co-immunoprecipitation at different time points after immune elicitation.

• Tissue-specific analysis: Differentiate between cell types and developmental stages, as CNGC localization and interactions may vary contextually.

• Stimulus-specific responses: Evaluate interactions under different immune challenges (bacterial PAMPs, fungal elicitors, etc.) as interaction dynamics may differ based on stimulus type.

• Post-translational modification mapping: Systematically analyze the phosphorylation status of CNGC9 following immune activation, as modifications like the OsRLCK185-mediated phosphorylation of OsCNGC9 can significantly affect channel activity and interaction profiles .

This multi-faceted approach will help reconcile apparently contradictory findings by revealing condition-specific regulation mechanisms.

What are the implications of CNGC9 mutations on calcium flux patterns and downstream immune responses?

CNGC9 mutations can significantly alter calcium flux patterns and downstream immune responses, with complex implications:

• Altered calcium signatures: Mutations in transmembrane domains, similar to the cngc20-4 L371F exchange that leads to increased Ca²⁺ accumulation , may cause abnormal calcium influx patterns that disrupt the temporal and spatial calcium signatures required for proper immune activation.

• Dysregulated defense responses: Mutations can lead to either compromised immunity (as seen in cds1 rice mutants with increased susceptibility to blast disease ) or autoimmunity phenotypes (as observed in cngc20-4 which exhibits enhanced PTI responses and ETI hypersensitive cell death ).

• Modified oxidative burst profiles: Since CNGC9-mediated calcium influx is critical for PAMP-triggered ROS production , mutations can alter the intensity and timing of oxidative burst, affecting antimicrobial activity and cell death regulation.

• Disrupted gene expression patterns: Calcium signaling influences defense gene expression networks; thus, CNGC9 mutations can reprogram transcriptional responses, potentially activating salicylic acid (SA) pathways inappropriately, similar to what occurs in other CNGC mutants .

• Altered phosphorylation cascades: Mutations may affect recognition by regulatory kinases, changing the activation threshold of the channel and subsequent defense signaling cascades.

These complex effects highlight the importance of analyzing multiple aspects of immune responses when characterizing CNGC9 mutants.

How can researchers distinguish between direct and indirect effects of CNGC9 inhibition in their experimental systems?

Distinguishing between direct and indirect effects of CNGC9 inhibition requires sophisticated experimental approaches:

• Temporal analysis of calcium responses: Use real-time calcium imaging with genetically encoded calcium indicators to determine the immediate effects of CNGC9 inhibition on calcium influx within seconds to minutes after PAMP perception.

• Pharmacological dissection: Apply calcium channel blockers (La³⁺ and Gd³⁺) that have been shown to abolish CNGC-mediated inward currents alongside targeted CNGC9 inhibition to differentiate between specific and general calcium channel blockade effects.

• Genetic complementation strategies: Utilize CNGC9 variants with differing sensitivities to inhibitors to establish dose-dependent relationships between channel activity and downstream responses.

• Phosphorylation site mutants: Create phosphomimetic (S→D) and non-phosphorylatable (S→A) versions of CNGC9 at key regulatory residues, similar to the approach with CNGC6 , to distinguish between channel activity effects and scaffold protein functions.

• Direct target validation: Implement CNGC9 antibodies in ChIP-seq or RIP-seq approaches to identify direct targets of calcium-dependent transcription factors or RNA-binding proteins that respond to CNGC9-mediated calcium signals.

• Heterologous expression systems: Express CNGC9 in systems like HEK293T cells or Xenopus oocytes, as done with CNGC11/12 , to isolate channel activity from plant-specific regulatory networks.

These approaches collectively allow researchers to establish causal relationships between CNGC9 inhibition and downstream immune responses.

What are the optimal conditions for CNGC9 antibody validation to ensure specificity in immunological assays?

Optimal CNGC9 antibody validation requires a multi-step approach to ensure specificity:

• Western blot validation against recombinant protein: Test antibody against purified recombinant CNGC9 protein alongside negative controls to establish basic recognition capability.

• Knockout/knockdown controls: Compare antibody reactivity between wild-type samples and CNGC9 knockout or knockdown lines to confirm specific recognition of the endogenous protein.

• Cross-reactivity assessment: Test against related CNGC family members (particularly close homologs) to determine potential cross-reactivity.

• Multiple epitope targeting: Validate antibodies targeting different regions of CNGC9 (N-terminus, C-terminus, and extracellular loops) to ensure consistent detection.

• Immunoprecipitation efficiency evaluation: Assess the ability of the antibody to immunoprecipitate CNGC9 from plant lysates, confirming with mass spectrometry analysis of the pulled-down proteins.

• Native vs. denatured conditions: Test antibody recognition under both native conditions (for immunoprecipitation and immunolocalization) and denatured conditions (for western blotting).

• Signal inhibition by antigen: Demonstrate that pre-incubation of the antibody with purified CNGC9 antigen blocks signal detection in immunoassays.

These validation steps ensure that the antibody is specifically recognizing CNGC9 across multiple experimental platforms and conditions.

What experimental setup is recommended for distinguishing CNGC9 activity from other calcium channels in plant immunity?

The recommended experimental setup for distinguishing CNGC9 activity from other calcium channels includes:

• Electrophysiological characterization: Employ whole-cell patch-clamp recordings of CNGC9-expressing cells (either in planta or heterologous systems) to determine its distinct ion conductance properties, similar to approaches used for CNGC11/12 .

• Ion selectivity profiling: Systematically test channel permeability to various cations (Ca²⁺, Mg²⁺, K⁺, Na⁺, Ba²⁺) under controlled conditions to establish a CNGC9-specific conductance fingerprint.

• Pharmacological toolkit: Apply channel-specific blockers and activators, including La³⁺ and Gd³⁺ , at various concentrations to identify differential sensitivity patterns.

• Kinetic analysis: Measure activation and inactivation kinetics of CNGC9 channels under physiological stimuli, as channel kinetics often differ between channel types.

• Stimulus-specific activation: Compare calcium responses to different PAMPs/DAMPs between wild-type and CNGC9-deficient plants to identify CNGC9-specific immune elicitors.

• Subcellular resolution imaging: Employ high-resolution calcium imaging with targeted calcium indicators to visualize CNGC9-specific calcium microdomains within the cell.

• Regulatory protein interactions: Characterize specific regulatory proteins that interact with CNGC9 (such as receptor-like cytoplasmic kinases similar to OsRLCK185 ) but not with other calcium channels.

This comprehensive approach enables researchers to distinguish CNGC9-mediated calcium influx from that of other channels in plant immune responses.

What are the best practices for using CNGC9 antibodies in co-immunoprecipitation experiments to identify novel interaction partners?

Best practices for using CNGC9 antibodies in co-immunoprecipitation (co-IP) experiments include:

• Membrane protein extraction optimization: Use specialized buffers containing appropriate detergents (typically 0.5-1% NP-40, Triton X-100, or digitonin) to efficiently solubilize membrane-bound CNGC9 while maintaining protein-protein interactions.

• Crosslinking considerations: Implement reversible crosslinkers like DSP (dithiobis(succinimidyl propionate)) to stabilize transient interactions before cell lysis, particularly important for capturing dynamic immune signaling complexes.

• Antibody immobilization: Covalently couple purified CNGC9 antibodies to solid support (Protein A/G beads or activated agarose) to prevent antibody contamination in the eluted samples.

• Sequential elution strategy: Employ increasingly stringent elution conditions to differentiate between strong and weak interactors of CNGC9.

• Stimulus-dependent interactions: Perform co-IPs at different time points after immune elicitation to capture dynamic interaction partners, as demonstrated in studies of CNGC-kinase interactions .

• Reciprocal confirmation: Validate key interactions through reverse co-IP using antibodies against the identified partners, similar to the OsRLCK185-OsCNGC9 interaction confirmation .

• Negative controls: Include isotype control antibodies and samples from CNGC9-deficient plants to identify non-specific binding.

• Sample preparation for mass spectrometry: Use special consideration for membrane protein complexes when preparing samples for MS-based interactor identification, including specific digestion and peptide preparation protocols.

• Validation through alternative methods: Confirm key interactions identified by co-IP using yeast two-hybrid or BiFC assays as performed for other CNGC interactions .

This methodical approach maximizes the chances of identifying genuine CNGC9 interaction partners while minimizing false positives.

What are the most reliable methods for measuring CNGC9-mediated calcium influx in plant cells?

The most reliable methods for measuring CNGC9-mediated calcium influx in plant cells include:

• Genetically encoded calcium indicators (GECIs): Transform plants with aequorin or GCaMP variants targeted to specific cellular compartments to measure real-time changes in calcium concentrations with high temporal resolution.

• Electrophysiological approaches: Employ whole-cell patch-clamp recordings to directly measure CNGC9-mediated calcium currents, similar to methods used for characterizing CNGC12 . This technique provides direct evidence of channel activity and ion selectivity.

• Fluorescent calcium dyes: Load plant cells with ratiometric calcium indicators like Fura-2 or Fluo-4 to measure calcium dynamics across different cellular regions.

• Heterologous expression systems: Express CNGC9 in HEK293T cells or Xenopus oocytes to isolate channel function from other plant components, allowing precise measurement of channel properties in response to various stimuli, as demonstrated with other CNGCs .

• Calcium channel blockers: Use specific inhibitors like La³⁺ or Gd³⁺ as experimental controls to validate that the observed calcium influx is channel-mediated .

• Phos-tag mobility shift assays: Monitor the phosphorylation status of CNGC9 in parallel with calcium measurements to correlate channel activation with calcium influx, similar to the approach used for OsCNGC9 .

• CRISPR-edited channel variants: Generate plants expressing CNGC9 variants with altered conductance properties to establish causality between channel activity and measured calcium signals.

These complementary approaches provide robust evidence for CNGC9-specific calcium signaling events in plant immunity.

How should researchers optimize immunohistochemistry protocols for detecting CNGC9 in different plant tissues?

Optimizing immunohistochemistry protocols for CNGC9 detection requires attention to several key factors:

• Tissue fixation optimization: Test multiple fixatives (4% paraformaldehyde, glutaraldehyde, or combinations) and fixation times to preserve CNGC9 epitopes while maintaining tissue architecture.

• Antigen retrieval methods: Implement heat-induced or enzymatic antigen retrieval to expose epitopes that may be masked during fixation, particularly important for membrane proteins like CNGC9.

• Membrane permeabilization: Carefully titrate detergent concentrations (0.1-0.5% Triton X-100 or Tween-20) to allow antibody access to transmembrane proteins without disrupting membrane structure.

• Blocking optimization: Test different blocking reagents (BSA, normal serum, casein) at various concentrations to minimize background while maintaining specific signal.

• Antibody dilution series: Perform systematic dilution tests of primary CNGC9 antibodies to determine optimal concentration for specific signal with minimal background.

• Signal amplification systems: For low-abundance CNGC9 detection, employ tyramide signal amplification or quantum dot-conjugated secondary antibodies to enhance sensitivity.

• Multiple detection systems: Compare chromogenic (like DAB staining used for CA9 ) versus fluorescent detection methods to determine which best visualizes CNGC9 distribution.

• Tissue-specific controls: Include CNGC9 knockout/knockdown tissues and pre-immune serum controls for each tissue type to verify signal specificity.

• Co-localization markers: Include markers for plasma membrane, endoplasmic reticulum, and other cellular compartments to precisely determine CNGC9 subcellular localization.

• Developmental stage considerations: Adjust protocols for different plant organs and developmental stages as protein abundance and accessibility may vary significantly.

These optimizations ensure reliable visualization of CNGC9 across different plant tissues and experimental conditions.

What approaches are most effective for studying CNGC9 phosphorylation dynamics during immune activation?

Effective approaches for studying CNGC9 phosphorylation dynamics include:

• Phos-tag SDS-PAGE: Implement Phos-tag gel electrophoresis to detect mobility shifts of CNGC9 after immune elicitation, as demonstrated for OsCNGC9 after chitin treatment .

• Phospho-specific antibodies: Develop antibodies recognizing specific phosphorylated residues in CNGC9, particularly targeting predicted kinase substrates sites in the N- and C-terminal domains.

• Mass spectrometry-based phosphoproteomics: Apply targeted MS approaches to identify and quantify CNGC9 phosphorylation sites using techniques like parallel reaction monitoring (PRM) or selected reaction monitoring (SRM).

• In vitro kinase assays: Conduct kinase assays using purified kinases (like receptor-like cytoplasmic kinases) and CNGC9 protein domains to map phosphorylation sites and kinetics, similar to the OsRLCK185-OsCNGC9 phosphorylation study .

• Phosphomimetic and phospho-dead mutants: Generate CNGC9 variants with S/T→D (phosphomimetic) or S/T→A (phospho-dead) mutations at key regulatory sites, as done with CNGC6 , to study the functional consequences of phosphorylation.

• Temporal phosphorylation mapping: Sample at multiple time points after immune elicitation (30 seconds to several hours) to track the dynamic phosphorylation pattern of CNGC9.

• Kinase inhibitor studies: Use specific kinase inhibitors to block particular phosphorylation pathways and determine their effects on CNGC9 activity and immune responses.

• FRET-based phosphorylation sensors: Develop and express FRET sensors incorporating CNGC9 domains to monitor phosphorylation events in real-time in living cells.

• Cellular fractionation: Track changes in CNGC9 localization concurrent with its phosphorylation status to understand how phosphorylation affects channel trafficking and membrane insertion.

These approaches collectively provide a comprehensive view of the phosphorylation events regulating CNGC9 during immune responses.

How can researchers properly normalize and analyze CNGC9 expression data across different experimental conditions?

Proper normalization and analysis of CNGC9 expression data requires:

• Reference gene selection: Use multiple, experimentally validated reference genes specific to the plant species, tissue type, and experimental condition being studied.

• Normalization algorithm selection: Apply geometric averaging of multiple reference genes (using methods like geNorm or NormFinder) rather than relying on a single housekeeping gene.

• Technical vs. biological variation: Distinguish between technical replicates (assay variation) and biological replicates (sample variation) when designing experiments and analyzing data.

• Standard curve validation: For absolute quantification, verify that standard curves for CNGC9 and reference genes have similar efficiencies (90-110%) and R² values (>0.98).

• Statistical model selection: Choose appropriate statistical tests based on data distribution, with non-parametric tests for non-normally distributed data.

• Time-course analysis: For immune response studies, use time-series statistical methods rather than comparing individual time points independently.

• Multi-factorial analysis: When studying CNGC9 expression across treatments, genotypes, and time points, use appropriate multi-factorial statistical approaches (ANOVA, mixed models).

• Tissue-specific normalization: When comparing CNGC9 expression across different tissues, consider tissue-specific normalization factors due to varying cellular composition.

• Data visualization standards: Present data with appropriate error bars (standard deviation for descriptive statistics, standard error or confidence intervals for inferential statistics).

• Effect size reporting: Report not only statistical significance but also effect sizes to indicate biological relevance of CNGC9 expression changes.

These practices ensure that CNGC9 expression data accurately reflects biological realities rather than technical artifacts.

What criteria should be used to evaluate the specificity and sensitivity of new CNGC9 antibodies?

The evaluation of new CNGC9 antibodies should follow these specific criteria:

This systematic evaluation ensures that new CNGC9 antibodies meet rigorous standards for research applications.

How should researchers interpret conflicting results between different methods of detecting CNGC9-mediated calcium signaling?

When facing conflicting results between different methods of detecting CNGC9-mediated calcium signaling, researchers should implement this interpretative framework:

• Method-specific limitations assessment: Evaluate each method's temporal and spatial resolution, sensitivity thresholds, and potential artifacts. For example, aequorin provides excellent temporal resolution but limited spatial information, while fluorescent indicators offer spatial resolution but may be compartmentalized or buffered differently than endogenous calcium.

• Signal localization discrepancies: Consider that whole-cell calcium measurements (like aequorin luminescence) may obscure localized CNGC9-specific calcium microdomains that could be detected by high-resolution imaging techniques.

• Kinetic differences reconciliation: Compare the time resolution of different methods—electrophysiological recordings of CNGC9 (as done with CNGC12 ) detect immediate channel opening, while calcium-responsive reporters may reflect downstream amplification events.

• Genetic background influences: Assess how different genetic backgrounds, particularly in calcium signaling components, might affect measurements. CNGC9 activity in knockout vs. knockdown plants may reveal different phenotypes.

• Physiological state considerations: Evaluate how plant growth conditions, developmental stage, and immune priming state affect CNGC9 responsiveness across different detection methods.

• Method integration strategy: When possible, apply multiple detection methods simultaneously on the same samples (e.g., patch-clamp with calcium imaging) to directly correlate measurements.

• Independent validation approaches: Implement calcium channel blockers (La³⁺, Gd³⁺) alongside genetic approaches (CNGC9 mutants) to establish causality between channel activity and calcium signatures.

• Recapitulation in heterologous systems: Express CNGC9 in controlled systems like Xenopus oocytes or HEK293T cells to isolate channel properties from plant-specific regulatory mechanisms, as done with other CNGCs .

This systematic approach helps researchers integrate seemingly contradictory data into a coherent model of CNGC9 function in calcium signaling.

What are the most promising approaches for developing CNGC9-specific modulators for research applications?

The most promising approaches for developing CNGC9-specific modulators include:

• Structure-guided rational design: Using homology modeling based on related channel structures to design small molecules that interact with unique features of CNGC9, particularly focusing on regions that differ from other CNGCs.

• High-throughput screening platforms: Developing cell-based assays using CNGC9-expressing heterologous systems coupled with calcium-sensitive reporters to screen chemical libraries for CNGC9-specific modulators.

• Peptide-based inhibitors: Designing peptides based on the sequences of natural CNGC9 regulators, such as the binding domains of interacting kinases like those in the RLCKs family that regulate OsCNGC9 .

• Nucleic acid aptamers: Selecting RNA or DNA aptamers that specifically bind to extracellular or cytoplasmic domains of CNGC9 to modulate its function.

• Cyclic nucleotide analogs: Creating modified cyclic nucleotides that selectively activate or inhibit CNGC9 based on the unique properties of its cyclic nucleotide-binding domain.

• Allosteric modulators: Identifying compounds that bind to regulatory sites rather than the channel pore to modify CNGC9 gating properties without directly blocking ion flow.

• Phosphomimetic peptides: Developing cell-permeable peptides that mimic phosphorylated regions of CNGC9 to competitively interfere with regulatory protein interactions.

• PROTAC approach: Creating proteolysis-targeting chimeras that selectively degrade CNGC9 protein as a chemical genetics alternative to genetic knockouts.

These approaches, particularly when used in combination, hold promise for developing the selective tools needed to dissect CNGC9-specific functions in plant immunity.

How might researchers effectively integrate CNGC9 studies with broader calcium signaling networks in plant immunity?

Effective integration of CNGC9 studies with broader calcium signaling networks requires:

• Multi-omics integration platforms: Combine transcriptomics, proteomics, and metabolomics data from CNGC9-modified plants to construct comprehensive signaling networks that position CNGC9 within the immunity signalosome.

• Spatiotemporal calcium signature mapping: Employ high-resolution calcium imaging in CNGC9 wildtype, mutant, and overexpression lines to define CNGC9-specific calcium signatures and how they integrate with signals from other calcium channels.

• Calcium decoder identification: Identify and characterize calcium-binding proteins (CaMs, CMLs, CDPKs) that specifically respond to CNGC9-mediated calcium signals versus those activated by other channels.

• Dynamic interactome analysis: Use proximity labeling techniques like BioID or TurboID fused to CNGC9 to capture transient interactions within signaling complexes during immune activation.

• Mathematical modeling approaches: Develop computational models that integrate CNGC9 activity with other calcium channels, buffers, and transporters to predict calcium dynamics under different immune scenarios.

• Single-cell analysis techniques: Apply single-cell transcriptomics or proteomics to understand cell-specific CNGC9 functions within heterogeneous plant tissues responding to pathogens.

• Synthetic biology circuits: Engineer minimal calcium signaling networks incorporating CNGC9 to test hypotheses about network architecture and signal processing.

• Cross-species comparative studies: Compare CNGC9 function across plant species with different immune strategies to identify conserved and diversified aspects of calcium signaling networks.

This integrative approach will help establish CNGC9's precise role within the complex calcium signaling networks mediating plant immunity.

What technological advancements would most benefit CNGC9 research in the coming decade?

Key technological advancements that would significantly benefit CNGC9 research include:

• Cryo-EM structures of plant CNGCs: High-resolution structural data of CNGC9 in different conformational states would revolutionize understanding of gating mechanisms and guide rational design of specific modulators.

• Optogenetic tools for CNGC control: Development of light-controlled CNGC9 variants would allow precise spatiotemporal manipulation of channel activity in specific cell types.

• Genetically encoded calcium sensors with enhanced properties: Next-generation calcium indicators with improved signal-to-noise ratios, faster kinetics, and expanded dynamic ranges would better capture CNGC9-mediated calcium signatures.

• Plant-optimized proximity labeling techniques: Refined BioID or TurboID systems functioning efficiently at plant growth temperatures would improve identification of transient CNGC9 interaction partners during immune signaling.

• Single-molecule tracking in plant membranes: Advanced super-resolution microscopy capable of tracking individual CNGC9 channels in living plant membranes would reveal dynamic regulation of channel clustering and activity.

• Plant tissue-specific gene editing: Precise CRISPR-based editing technologies allowing tissue-specific or condition-specific CNGC9 modification would help delineate local versus systemic roles.

• Improved heterologous expression systems: Development of plant membrane protein expression platforms that better replicate native lipid environments would enhance functional studies of CNGC9.

• Computational prediction of calcium signatures: Advanced algorithms capable of predicting calcium dynamics from channel properties would accelerate hypothesis testing about CNGC9 function.

• Nanobody-based probes: Development of CNGC9-specific nanobodies for tracking, modulating, or precipitating the channel in living cells would provide powerful new research tools.