CBP60G Antibody

What is CBP60G and why is it important in plant immunity research?

CBP60G (Calmodulin-Binding Protein 60G) is a plant-specific transcription factor that functions as a master regulator in plant immunity. It plays a critical role in MAMP-triggered salicylic acid (SA) accumulation and is required for resistance to bacterial pathogens such as Pseudomonas syringae . CBP60G expression is strongly induced in response to pathogen infection and MAMP treatments, with significant upregulation observed between 3-6 hours after Pseudomonas syringae infection . The protein is particularly important because it provides a calcium-dependent link between MAMP recognition and SA accumulation, making it a key component in the plant immune signaling network . Studies with cbp60g mutants have demonstrated enhanced susceptibility to bacterial pathogens, confirming its significant role in plant defense mechanisms .

How does CBP60G antibody detection correlate with defense responses in plants?

CBP60G antibody detection can serve as an excellent marker for monitoring defense activation in plants. Expression profiling studies have shown that CBP60G is rapidly induced after pathogen infection or MAMP treatment, with expression remaining elevated for at least 24 hours . When using CBP60G antibodies for immunodetection, researchers should observe increased protein accumulation in the nucleus following pathogen challenge or MAMP treatment such as flg22 application . This protein accumulation pattern directly correlates with the activation of defense-related genes, particularly those involved in salicylic acid biosynthesis pathway like ICS1 (Isochorismate Synthase 1) and FMO1 (Flavin-containing Monooxygenase 1) . The localization pattern of CBP60G is also informative - while some basal levels may be detected in untreated plants, pathogen challenge triggers significant nuclear accumulation, which can be visualized using CBP60G antibodies in immunofluorescence studies .

What are the key structural domains of CBP60G that antibodies typically target?

CBP60G contains several distinct functional domains that are important to consider when designing or selecting antibodies. The protein contains:

• An N-terminal calmodulin-binding domain essential for its function in defense signaling

• A DNA-binding domain located in the N-terminal portion (amino acids 1-361)

• A C-terminal transcription activation domain (amino acids 211-440) that is required for gene induction activity
  When developing or selecting CBP60G antibodies, researchers should consider which domain they wish to target based on their experimental goals. Antibodies targeting the calmodulin-binding domain are useful for studying CBP60G activation mechanisms, while those targeting the DNA-binding domain may interfere with its transcription factor activity. Notably, the C-terminal portion (CBP60gC) is particularly important as it harbors transcription activator activity and is the target of pathogen effectors like VdSCP41 . Therefore, antibodies recognizing this region can be valuable for studying pathogen interference with CBP60G function.

How is CBP60G gene expression regulated during pathogen infection?

CBP60G expression exhibits a distinct temporal pattern during pathogen infection. Real-time quantitative PCR analysis has revealed that:

• CBP60G is induced between 3-6 hours after Pseudomonas syringae pv. maculicola ES4326 infection and remains highly expressed for at least 24 hours

• Expression is also induced after infection with P. syringae pv. tomato DC3000, though to a lesser extent compared to Psm ES4326

• MAMP treatments, particularly flg22 peptide application, strongly induce CBP60G expression within 3 hours

• The type III secretion system-defective bacterial strain P. syringae pv. tomato DC3000 hrcC also triggers CBP60G expression, confirming that MAMP recognition is sufficient for induction
  When monitoring CBP60G induction using antibodies, researchers should design their experiments to capture these temporal dynamics, collecting samples at multiple time points (3, 6, 9, and 24 hours post-infection) to properly document the expression pattern. This information is valuable for correlating CBP60G expression with downstream defense responses and provides insight into the optimal timing for CBP60G antibody detection in experimental settings.

How does CBP60G interact with calmodulin and why is this interaction critical for antibody-based studies?

CBP60G contains a calmodulin-binding domain located near its N-terminus that interacts with calmodulin in a calcium-dependent manner . This interaction is essential for CBP60G's function in defense signaling, as mutations that abolish calmodulin binding prevent complementation of the SA production and bacterial growth defects in cbp60g mutants . When designing experimental approaches using CBP60G antibodies, researchers must consider how this calmodulin interaction might affect epitope accessibility.
The calcium-dependent nature of this interaction suggests that buffer conditions during immunoprecipitation or immunoblotting procedures can significantly impact results. For optimal detection of the CBP60G-calmodulin complex, buffers should contain appropriate calcium concentrations (typically 1-2 mM CaCl₂). Conversely, if the goal is to detect CBP60G independent of its calmodulin-bound state, EGTA or EDTA may be included in buffers to chelate calcium and disrupt the interaction. This consideration is particularly important when using CBP60G antibodies to study how pathogen effectors like VdSCP41 might interfere with the calmodulin-CBP60G interaction as part of their virulence strategy .

What is the relationship between CBP60G and SARD1 in plant defense signaling?

• Both CBP60G and SARD1 are targeted by the Verticillium dahliae effector VdSCP41, suggesting their importance in defense against this pathogen

• CBP60G and SARD1 bind to similar DNA motifs and regulate an overlapping set of defense genes, including ICS1, ALD1, and SARD4

• Despite their functional overlap, they do not appear to physically interact with each other according to luciferase complementation assays

• While both proteins contribute to pathogen-induced SA accumulation, they may have different temporal expression patterns or tissue specificity
  When using antibodies to study these proteins, researchers should ensure specificity between CBP60G and SARD1 due to potential structural similarities. Cross-reactivity testing is essential, and epitope selection should target regions with the least sequence homology. For co-immunoprecipitation studies investigating whether CBP60G and SARD1 participate in the same protein complexes, appropriate controls should be included to rule out non-specific interactions. Additionally, researchers may need to generate phospho-specific antibodies if post-translational modifications differentially regulate these proteins during immune responses.

How does pathogen effector VdSCP41 target and inhibit CBP60G function?

The Verticillium dahliae effector VdSCP41 directly targets CBP60G to suppress plant immunity, providing a fascinating system for studying pathogen manipulation of host defenses. Research using co-immunoprecipitation and localization studies has revealed that:

• VdSCP41 binds specifically to the C-terminal portion of CBP60G (CBP60gC)

• The C-terminal region of VdSCP41 (VdSCP41C) is sufficient for interaction with CBP60G

• VdSCP41 co-localizes with CBP60G in the nucleus and significantly increases CBP60G nuclear accumulation

• Despite increasing nuclear accumulation, VdSCP41 inhibits CBP60G's transcription factor activity, preventing induction of defense genes like ICS1 and FMO1
  When using CBP60G antibodies to study this interaction, researchers should design experiments that can distinguish between protein accumulation and protein activity. For example, ChIP (Chromatin Immunoprecipitation) assays using CBP60G antibodies can determine whether VdSCP41 interferes with CBP60G binding to target promoters. Additionally, co-immunoprecipitation experiments with fragments of CBP60G can map the precise interaction domains. The finding that VdSCP41 binds to the transcription activation domain (amino acids 211-440) suggests that this effector directly interferes with CBP60G's ability to recruit transcriptional machinery rather than preventing DNA binding .

What are the downstream targets of CBP60G in defense signaling pathways?

CBP60G functions as a master transcription regulator controlling the expression of numerous defense-related genes. ChIP-seq and expression profiling studies have identified several key downstream targets:

• ICS1 (Isochorismate Synthase 1): A critical enzyme in SA biosynthesis whose expression is directly regulated by CBP60G . Dual reporter analyses have demonstrated that CBP60G significantly enhances the expression of ICS1::LUC .

• FMO1 (Flavin-containing Monooxygenase 1): An important component of systemic acquired resistance whose expression is activated by CBP60G .

• ALD1 and SARD4: Key enzymes involved in pipecolic acid (Pip) biosynthesis, which contributes to systemic immunity .
  When using CBP60G antibodies for ChIP experiments to identify binding sites, researchers should design primers for these known targets as positive controls. For immunoblotting experiments examining CBP60G protein levels in response to different treatments, parallel RT-qPCR analysis of these downstream genes can provide functional correlation. Additionally, when comparing wild-type and cbp60g mutant plants, researchers should examine not only SA levels but also Pip accumulation, as CBP60G directly regulates genes involved in both pathways . This comprehensive approach allows for a more complete understanding of CBP60G's role in coordinating various branches of the plant immune response.

What are the optimal methods for detecting CBP60G expression using antibodies?

For effective detection of CBP60G using antibodies, researchers should consider the following methodological approaches:

• Immunoblotting (Western Blot): When performing western blots, nuclear protein extraction is recommended since CBP60G accumulates primarily in the nucleus upon pathogen challenge . Standard SDS-PAGE with 10% acrylamide gels typically provides good resolution for CBP60G (expected size ~60 kDa). For loading controls, nuclear proteins like histone H3 are preferable to cytoplasmic markers. Importantly, researchers should include appropriate positive controls (e.g., plants overexpressing tagged CBP60G) and negative controls (cbp60g knockout mutants) to validate antibody specificity .

• Immunoprecipitation: For co-IP studies investigating CBP60G protein interactions, use of mild non-ionic detergents (0.5-1% NP-40 or Triton X-100) helps preserve protein-protein interactions. When studying calmodulin binding, buffer conditions should include 1-2 mM CaCl₂ to maintain the interaction . For interactions with pathogen effectors like VdSCP41, expression of tagged proteins in Arabidopsis protoplasts followed by co-IP has proven effective .

• Immunofluorescence: For subcellular localization studies, fixation with 4% paraformaldehyde followed by permeabilization is recommended. CBP60G typically shows nuclear localization that increases upon pathogen challenge or co-expression with effectors like VdSCP41 . DAPI co-staining confirms nuclear localization. Comparison of signal intensity between treated and untreated samples provides quantitative data on nuclear accumulation.

• ChIP (Chromatin Immunoprecipitation): When performing ChIP to identify CBP60G binding sites, formaldehyde cross-linking followed by sonication to generate 200-500 bp DNA fragments works well. Enrichment at known targets like ICS1, FMO1, and ALD1 promoters should be used as positive controls .

How can researchers effectively evaluate CBP60G-protein interactions?

To effectively study CBP60G interactions with other proteins, researchers can employ several complementary approaches:

• Co-immunoprecipitation (Co-IP): This is the gold standard for confirming protein-protein interactions. When studying CBP60G, researchers have successfully used HA-tagged CBP60G for reverse Co-IP analysis to verify interactions with proteins like VdSCP41 . For these experiments, transient expression in Arabidopsis protoplasts provides a convenient system . Buffer conditions should be optimized based on the specific interaction being studied - for calmodulin binding, calcium should be present; for other interactions, standard IP buffers with mild detergents work well.

• Luciferase Complementation Assays: This approach can be used to study protein interactions in planta. CBP60G and its potential interacting partner are fused to complementary fragments of luciferase (CLuc and NLuc). Interaction brings the fragments together, restoring luciferase activity that can be measured by luminometer . This approach has successfully demonstrated that CBP60G and SARD1 do not directly interact despite their functional overlap .

• Fluorescence Co-localization: By co-expressing fluorescently tagged CBP60G (e.g., GFP-CBP60G) with potential interacting partners tagged with a different fluorophore (e.g., mCherry), researchers can assess co-localization by confocal microscopy. This approach successfully demonstrated co-localization of CBP60G with VdSCP41 in the nucleus of Nicotiana benthamiana cells .

• Domain Mapping: To identify specific interaction domains, researchers should generate truncated versions of CBP60G (such as CBP60gN and CBP60gC) and test their ability to interact with proteins of interest. This approach revealed that VdSCP41 specifically binds to CBP60gC but not CBP60gN, and further narrowed the interaction to amino acids 211-440 of CBP60G .

What are optimal protocols for studying CBP60G function in planta?

For functional studies of CBP60G in planta, researchers should consider the following approaches:

• Genetic Analysis: Comparison of cbp60g mutants, wild-type plants, and complementation lines is essential for establishing gene function. The cbp60g-1 and cbp60g-2 mutants have been well-characterized and show enhanced susceptibility to Pseudomonas syringae . When generating complementation lines, use of the native promoter (approximately 1 kb upstream of the start codon) ensures physiologically relevant expression .

• Bacterial Growth Assays: To assess the impact of CBP60G on disease resistance, standard bacterial growth assays using pathogens like Pseudomonas syringae pv. maculicola ES4326 or P. syringae pv. tomato DC3000 provide quantitative data. Bacterial titers should be measured 3 days post-infection, and pad4 mutants can be included as highly susceptible controls .

• SA Measurement: Since CBP60G regulates SA accumulation, measurement of free SA levels after MAMP treatment (e.g., flg22) or bacterial infection (e.g., Pst DC3000 hrcC) provides important functional data. High-performance liquid chromatography (HPLC) or gas chromatography-mass spectrometry (GC-MS) methods can be used for this purpose .

• Transcriptional Reporter Assays: Dual luciferase reporter assays in Arabidopsis protoplasts effectively demonstrate CBP60G's transcriptional activation capacity. By co-expressing CBP60G with reporter constructs containing promoters of interest (such as ICS1::LUC or FMO1::LUC), researchers can quantify its transcriptional activity and how this is affected by interacting proteins like VdSCP41 .

How should experimental controls be designed when studying CBP60G?

Proper experimental controls are crucial for CBP60G research. Researchers should incorporate the following controls:

• Genetic Controls:

  • Wild-type plants (Col-0 for Arabidopsis) serve as positive controls

  • Multiple independent cbp60g mutant alleles (e.g., cbp60g-1 and cbp60g-2) to confirm phenotypes are caused by the mutation

  • Complementation lines expressing the wild-type gene under its native promoter to verify phenotype rescue

  • For redundancy studies, sard1 single mutants and cbp60g sard1 double mutants should be included

• Antibody Controls:

  • Preimmune serum for immunoprecipitation and immunoblotting

  • Known quantities of recombinant protein for calibration

  • Extracts from cbp60g knockout plants as negative controls

  • Samples from plants overexpressing tagged CBP60G as positive controls

• Treatment Controls:

  • Mock-treated plants alongside pathogen or MAMP treatments

  • Time-course sampling to capture the dynamics of CBP60G induction (3, 6, 9, and 24 hours)

  • For MAMP responses, both purified MAMPs (e.g., flg22) and bacterial strains lacking effector delivery (e.g., Pst DC3000 hrcC) should be used

• Interaction Controls:

  • For co-IP experiments, non-interacting proteins as negative controls

  • For domain mapping, truncated proteins should have overlapping regions to ensure complete coverage

  • For calmodulin-binding studies, parallel experiments with and without calcium

  • For effector studies, non-functional effector mutants as controls

How to address inconsistent results in CBP60G-related experiments?

Inconsistent results in CBP60G experiments may stem from several factors. Researchers should consider the following troubleshooting approaches:

• Temporal Dynamics: CBP60G expression and activity follow specific temporal patterns after pathogen challenge. If results are inconsistent, ensure samples are collected at appropriate time points. Expression begins increasing 3-6 hours post-infection and remains elevated for at least 24 hours . Sampling too early or too late may miss peak activity.

• Environmental Conditions: Plant age, growth conditions, and circadian rhythms can significantly affect immune responses. Standardize plant age (typically 4-5 weeks for Arabidopsis), growth conditions (temperature, light cycle, humidity), and treatment time of day. Document these parameters meticulously to ensure reproducibility.

• Bacterial Inoculation Variability: For pathogen assays, bacterial concentration and infiltration technique can introduce variability. Use standardized inoculum (typically OD₆₀₀ = 0.0001-0.0002 for growth assays) and ensure consistent infiltration technique. Include multiple biological replicates (minimum n=8) per experiment and repeat experiments at least three times .

• Protein Extraction Efficiency: For immunoblotting, inconsistent extraction efficiency can affect results. Use standardized nuclear extraction protocols, include protease inhibitors, and verify equal loading with appropriate controls. For CBP60G, nuclear extraction is particularly important as it accumulates primarily in the nucleus upon activation .

• Antibody Specificity Issues: Cross-reactivity with related proteins (particularly SARD1) can confound results. Validate antibody specificity using extracts from cbp60g mutants and consider using epitope-tagged versions of CBP60G for critical experiments .

What are common pitfalls when working with CBP60G antibodies?

Researchers should be aware of several common pitfalls when working with CBP60G antibodies:

• Cross-Reactivity with SARD1: CBP60G and SARD1 share functional similarity and potentially structural features . Antibodies may cross-react unless carefully designed against unique epitopes. Always validate specificity using extracts from both cbp60g and sard1 single mutants.

• Detection of Protein Complexes: Since CBP60G interacts with calmodulin in a calcium-dependent manner , immunoprecipitation conditions can affect complex integrity. If studying the CBP60G-calmodulin interaction, buffers should contain calcium; if studying CBP60G independently, consider including chelating agents.

• Post-Translational Modifications: CBP60G may undergo phosphorylation or other modifications during immune activation. These modifications could affect antibody recognition or protein mobility on SDS-PAGE. Consider using phosphatase treatment of samples to determine if mobility shifts are due to phosphorylation.

• Nuclear Localization: Since CBP60G functions primarily in the nucleus , cytoplasmic extraction protocols may yield poor results. Nuclear extraction protocols are recommended for maximum recovery of the protein of interest.

• Epitope Masking: When CBP60G interacts with partners like VdSCP41 or calmodulin, epitopes may be masked. If antibody recognition seems inconsistent, consider using multiple antibodies targeting different regions of the protein. The C-terminal region (amino acids 211-440) is particularly important for protein interactions , so antibodies targeting other regions may be preferable for some applications.

How to interpret CBP60G phenotypes in different genetic backgrounds?

Interpreting CBP60G phenotypes requires careful consideration of genetic context:

What statistical approaches are recommended for CBP60G functional studies?

Appropriate statistical analysis is crucial for interpreting CBP60G functional data:

• Bacterial Growth Assays: For comparing bacterial titers between genotypes, log-transformation of colony-forming unit (CFU) data is standard practice since bacterial growth is exponential. Analysis of variance (ANOVA) followed by appropriate post-hoc tests (such as Tukey's HSD) is recommended for comparing multiple genotypes . A minimum of 8 biological replicates per genotype should be included, and experiments should be repeated at least three times.

• Gene Expression Analysis: For qRT-PCR data analyzing CBP60G expression or its target genes, the 2^(-ΔΔCt) method with appropriate reference genes (such as ACTIN2) is commonly used . Statistical analysis should be performed on the ΔCt values rather than the fold-change values, as the former follow a normal distribution. For comparing multiple treatments or time points, two-way ANOVA with appropriate post-hoc tests is recommended.

• Reporter Assays: For dual luciferase reporter assays measuring CBP60G transcriptional activity, normalization to an internal control (such as Renilla luciferase) corrects for transformation efficiency variations. Data from at least three independent experiments, each with multiple technical replicates, should be analyzed by ANOVA .

• Protein Interaction Studies: For co-immunoprecipitation and other interaction studies, quantification of band intensities from multiple biological replicates allows statistical comparison. Input-normalized data can be analyzed by t-tests or ANOVA depending on the number of comparisons.

• Multiple Testing Correction: When performing genome-wide analyses (such as microarray or RNA-seq) to identify CBP60G-regulated genes, appropriate multiple testing correction is essential. The false discovery rate (FDR) approach with q-value cutoffs (typically q < 0.05) has been successfully applied in CBP60G studies .