CNGC19 Antibody

Here’s a structured collection of advanced FAQs for researchers studying CNGC19 antibodies in plant immunity research, synthesized from the provided literature:

What is the primary functional role of CNGC19 in plant defense mechanisms?

CNGC19 is a Ca²⁺-permeable cyclic nucleotide-gated channel critical for herbivory-induced cytosolic Ca²⁺ influx. It activates downstream jasmonate (JA) signaling and aliphatic glucosinolate biosynthesis, which are essential for defense against insects like Spodoptera litura .
Methodological Insight: Use Arabidopsis cngc19 mutants in feeding assays with herbivores. Measure Ca²⁺ fluxes via fluorescent dyes (e.g., Fluo-4 AM) and validate JA levels using LC-MS.

Which experimental models are optimal for studying CNGC19 antibody specificity?

• Plant Systems: Arabidopsis thaliana (Col-0 ecotype) mutants (cngc19-1, SALK_027306) for phenotypic comparisons .

• Heterologous Systems: Xenopus oocytes expressing CNGC19 to assay Ca²⁺ currents under hyperpolarization with cAMP/Ca²⁺ .
  Validation: Combine Western blotting (anti-CNGC19 antibody) with electrophysiology to confirm channel activity.

How does CNGC19 interact with immune signaling components?

CNGC19 phosphorylates and stabilizes BIK1 (Botrytis-Induced Kinase 1), linking Ca²⁺ signaling to PTI/ETI responses. It also binds calmodulin 2 (CaM2) post-herbivory, modulating defense gene expression .
Method: Co-immunoprecipitation (Co-IP) with anti-CNGC19 antibodies in Arabidopsis extracts treated with insect oral secretions.

How do genetic interactions between CNGC19 and parallel immune pathways affect experimental design?

CNGC19-dependent autoimmunity in cngc20-4 mutants requires EDS1 and salicylic acid (SA) pathways .
Experimental Design:

• Use double mutants (cngc19 eds1-2, cngc19 sid2-1) to decouple SA/JA crosstalk.

• Measure glucosinolate profiles via HPLC and transcript levels of BCAT4 (key for aliphatic glucosinolates) .

What contradictions exist in CNGC19 subcellular localization data, and how can they be resolved?

• Conflict: Plasma membrane ( ) vs. vacuolar membrane localization ( ).
  Resolution Strategies:

  • Perform co-localization assays with tonoplast (e.g., VHA-a1) and plasma membrane (e.g., PIP2A) markers in stable transgenic lines.

  • Use detergent-free fractionation protocols to minimize artifactual membrane redistribution .

What are the limitations of current CNGC19 antibody-based detection methods?

• Cross-reactivity: CNGC19 shares 67% homology with CNGC20; validate antibodies using cngc19/cngc20 double mutants .

• Low Abundance: CNGC19 is weakly expressed; combine immunoprecipitation with tandem mass spectrometry (IP-MS/MS) for detection .

Table 1: Phenotypic Comparison of cngc19 Mutants Under Stress

Table 2: Localization Studies of CNGC19

Methodological Recommendations

• CRISPR-Cas9 Controls: Use cngc19/cngc20 double mutants to rule out functional redundancy .

• Calcium Imaging: Combine cameleon sensors (e.g., YC3.6) with confocal microscopy to map Ca²⁺ fluxes in vasculature .

• Antibody Validation: Include cngc19 KO lines as negative controls in Western blots .

Integration with Omics Data

• Transcriptomics: CNGC19 is upregulated >4-fold within 15 min of herbivory (microarray data) .

• Metabolomics: Hyperaccumulation of methionine in cngc19 mutants suggests disrupted glucosinolate chain elongation .