CBP60B Antibody

What is the function of CBP60B in plant immunity?

CBP60B serves as a central transcriptional activator in plant immune responses. Unlike its family members CBP60g and SARD1 which are pathogen-induced, CBP60B is constitutively and highly expressed in unchallenged plants. It positively regulates immunity through direct activation of defense-related genes and plays a distinct role in basal defense, partially through direct regulation of CBP60g and SARD1 . CBP60B acts downstream of calcium signaling as its function relies on DNA-binding domains (DBDs) and calmodulin-binding domains . Research demonstrates that CBP60B is a sequence-specific DNA-binding protein capable of binding to promoter regions of immunity genes to activate their expression .

How does CBP60B differ from other members of the CBP60 family?

The CBP60 family consists of members with varied roles in immunity. While CBP60g and SARD1 positively regulate immunity and are pathogen-induced, CBP60a negatively regulates immunity . CBP60B is unique in that it is constitutively expressed at high levels even in unchallenged plants, unlike the pathogen-induced expression patterns of CBP60g and SARD1 . Furthermore, CBP60B plays both positive and negative regulatory roles in plant immunity . Studies reveal that the CBP60b clade contains the prototype transcription factors of the CBP60 family, as demonstrated by functional homology between CBP60B proteins from early land plants and Arabidopsis .

What experimental methods can be used to detect CBP60B protein expression?

Several methodologies are effective for detecting CBP60B:

• Western blotting with CBP60B-specific antibodies: Useful for quantifying protein levels in plant tissues under different conditions.

• Immunoprecipitation (IP): Research shows successful IP of CBP60B using HA-tagged constructs, as demonstrated in studies examining interactions between CBP60g and VdSCP41 .

• Chromatin immunoprecipitation (ChIP): This technique has been effectively used to demonstrate CBP60B's function as a transcriptional activator by showing its binding to promoters of immunity genes .

• Fusion with reporter tags: Studies have successfully used CBP60B fused with HA or FLAG tags for detection and interaction studies .

How does the dual positive/negative regulatory role of CBP60B in plant immunity function at the molecular level?

The seemingly contradictory dual role of CBP60B represents a sophisticated regulatory mechanism in plant immunity. Experimental evidence shows that both loss-of-function and overexpression of CBP60B result in similar autoimmune phenotypes, including dwarfism, over-accumulation of reactive oxygen species and salicylic acid, highly induced expression of PR genes, and enhanced resistance to pathogens .
This paradox is explained through the guard/decoy hypothesis: CBP60B likely serves as a guardee, with its protein levels monitored by the nucleotide-binding leucine-rich repeat receptor (NLR) surveillance system. The absence of CBP60B can be detected by NLRs, leading to the activation of effector-triggered immunity (ETI) . Specifically:

• Loss-of-function autoimmunity: When CBP60B is absent, the ETI pathway is activated through EDS1 and PAD4-dependent mechanisms, which can be fully rescued by EDS1 or PAD4 loss-of-function mutations .

• Overexpression autoimmunity: This occurs through an EDS1 and PAD4-independent pathway, as the autoimmunity resulting from CBP60B overexpression cannot be rescued by the loss of EDS1 or PAD4 function .
  Research supports that CBP60B may be required for the expression of an unknown gene encoding a guardee/decoy or a negative regulator of TNLs, and in its absence, either the absence of this unknown protein is detected by NLRs, or the inhibition of NLRs is released, activating ETI .

What are the critical considerations when designing chromatin immunoprecipitation experiments using CBP60B antibodies?

When designing ChIP experiments with CBP60B antibodies, researchers should consider:

• Antibody specificity: CBP60B shares sequence similarity with other CBP60 family members. Validate antibody specificity against recombinant protein and in CBP60B knockout lines to prevent cross-reactivity with CBP60g or SARD1.

• Constitutive expression patterns: Unlike pathogen-induced CBP60g and SARD1, CBP60B is constitutively expressed, allowing ChIP experiments in both unchallenged and pathogen-challenged conditions .

• DNA binding specificity: When analyzing ChIP data, focus on the presence of known binding motifs. CBP60g and SARD1 bind to the sequence GAAATTTTGG, with GAAATTT being overrepresented in their target promoters . CBP60B may have overlapping but distinct binding preferences.

• Controls and normalization: Include appropriate controls such as input DNA and IgG control, and consider using CBP60B knockout plants as negative controls. When comparing binding across conditions, normalize to account for potential changes in CBP60B expression levels.

• Cross-linking optimization: As a transcription factor, CBP60B's interaction with DNA may be influenced by its interactions with calmodulin and other proteins. Optimize formaldehyde cross-linking time to capture these protein-DNA interactions effectively.

How can researchers properly interpret seemingly contradictory results in CBP60B functional studies?

Interpreting contradictory results in CBP60B research requires careful consideration of several factors:

• Genetic background effects: The autoimmune phenotype of CBP60B knockout mutants can vary depending on genetic background. For example, mutations in the EDS1-PAD4-dependent ETI pathway fully suppress the defects of CBP60B loss-of-function but not CBP60B gain-of-function .

• Redundancy with other family members: CBP60B displays functional redundancy with CBP60g and SARD1, but also has distinct roles. The enhanced autoimmunity observed in cbp60b cbp60g-1 double mutant compared to either single mutant demonstrates this complex relationship .

• Evolutionary conservation versus species-specific adaptations: While the function of CBP60B is broadly conserved across plant species from early land plants to flowering plants, species-specific differences exist. For example, GmCBP60b.1/2 in soybean shows similar but not identical functionality to Arabidopsis CBP60B .

• Context-dependent function: The role of CBP60B may change depending on the pathogen challenge, developmental stage, or environmental conditions, requiring careful experimental design to control these variables.

• Technical considerations: Antibody specificity issues or differences in protein expression systems can lead to apparently contradictory results, necessitating thorough validation of reagents and methodologies.

What are the optimal conditions for immunoprecipitation of CBP60B complexes?

For successful immunoprecipitation of CBP60B protein complexes:

• Sample preparation: Extract nuclear proteins from plant tissues using a buffer containing 50mM Tris-HCl (pH 7.5), 150mM NaCl, 5mM EDTA, 0.1% Triton X-100, 0.2% NP-40, 10% glycerol, and protease inhibitor cocktail. This buffer composition has proven effective in previous CBP60 family studies .

• Antibody selection: Use either commercial anti-CBP60B antibodies (if available and validated) or epitope-tagged CBP60B (HA or FLAG tags work well as demonstrated in published studies) .

• Cross-linking considerations: For protein-protein interaction studies, mild cross-linking with 0.5-1% formaldehyde for 10 minutes can help preserve transient interactions, particularly important for studying CBP60B's interaction with calmodulin.

• Washing conditions: Use stringent washing conditions (high salt concentration) to reduce background, but be cautious as CBP60B interactions with DNA or other proteins might be sensitive to high salt.

• Calcium dependency: Since CBP60B function depends on calmodulin binding, consider the calcium concentration in your buffers. Including calcium (1-2mM CaCl₂) in some buffers might help preserve calmodulin-dependent interactions, while chelating agents like EGTA can be used to study calcium-independent interactions.

• Controls: Include appropriate negative controls such as IgG control immunoprecipitations and samples from CBP60B knockout plants to identify specific interactions.

How should researchers design gene expression studies when investigating CBP60B-regulated genes?

When designing gene expression studies to investigate CBP60B-regulated genes:

• Experimental design considerations:

  • Include appropriate genotypes: wild-type, CBP60B knockout mutants, and CBP60B overexpression lines

  • Consider double or triple mutants with other CBP60 family members to account for redundancy

  • Include time-course experiments after pathogen challenge to capture both early and late responses

• Target gene selection:

  • Focus on known immunity genes such as SID2, EDS1, PAD4, and PR genes

  • Include CBP60g and SARD1 to assess regulatory relationships

  • Consider genes with the GAAATTT motif in their promoters, as this sequence is bound by CBP60 family members

• Normalization strategy:

  • Use multiple reference genes that are stable under your experimental conditions

  • Consider normalizing to plant size/developmental stage, as CBP60B mutants often display growth phenotypes

• Data analysis approach:

  • Use appropriate statistical methods to account for biological variation

  • Consider systems biology approaches to identify gene regulatory networks

  • Validate key findings with ChIP assays to confirm direct regulation

• Validation experiments:

  • Confirm key findings using luciferase reporter assays with promoters of putative target genes

  • Use electrophoretic mobility shift assays (EMSA) to confirm direct binding to specific promoter regions

What are the key considerations when comparing CBP60B function across different plant species?

When conducting comparative studies of CBP60B function across plant species:

• Sequence homology analysis:

  • Perform comprehensive phylogenetic analysis to identify true orthologs

  • Pay special attention to conservation of DNA-binding domains and calmodulin-binding domains

  • Note that soybeans, being paleopolyploid, typically have two homologous genes for CBP60B (GmCBP60b.1 and GmCBP60b.2 share 97% identity at the nucleotide level)

• Functional complementation experiments:

  • Test whether CBP60B genes from other species can rescue Arabidopsis cbp60b mutant phenotypes

  • Research shows that tomato and cucumber CBP60b-like genes can rescue the defects of Arabidopsis cbp60b and activate the expression of tomato and cucumber SID2 and EDS1 genes

• Species-specific considerations:

  • Account for differences in pathogen susceptibility between species

  • Consider species-specific immune response pathways

  • Develop species-appropriate phenotyping assays (CBP60B mutants show stunted growth and enhanced pathogen resistance across species, but the magnitude may vary)

• Technical adaptations for different plant systems:

  • Modify extraction protocols based on species-specific tissue composition

  • Adapt transformation methods for functional studies in recalcitrant species (as demonstrated in the use of tobacco to study soybean GmCBP60b.1 function)

  • For species where genetic transformation is challenging, consider virus-induced gene silencing approaches (BPMV-VIGS was successfully used to silence GmCBP60b.1/2 in soybean)

How can researchers validate the specificity of anti-CBP60B antibodies?

A thorough validation of anti-CBP60B antibodies is crucial due to potential cross-reactivity with other CBP60 family members:

• Western blot analysis using recombinant proteins:

  • Express and purify recombinant CBP60B, CBP60g, SARD1, and CBP60a proteins

  • Perform Western blots to check for cross-reactivity

  • Expected result: A specific antibody should show strong signal for CBP60B and minimal or no signal for other family members

• Genetic validation:

  • Test antibody on protein extracts from wild-type plants, CBP60B overexpression lines, and CBP60B knockout mutants

  • Expected result: Signal should increase in overexpression lines and be absent in knockout lines

• Peptide competition assay:

  • Pre-incubate the antibody with the peptide/protein used for immunization

  • This should block specific binding and eliminate the signal in Western blots or immunostaining

• Immunoprecipitation followed by mass spectrometry:

  • Perform IP with the anti-CBP60B antibody

  • Analyze the precipitated proteins by mass spectrometry

  • Expected result: CBP60B should be among the most abundant proteins identified

• Cross-species reactivity testing:

  • If using the antibody across plant species, test on protein extracts from different species

  • Compare with sequence alignment data to predict cross-reactivity

What strategies can overcome technical challenges when working with CBP60B in chromatin immunoprecipitation?

Several strategies can help overcome common challenges in CBP60B ChIP experiments:

• Low ChIP signal:

  • Use epitope-tagged CBP60B (HA or FLAG) if antibody efficiency is low

  • Optimize cross-linking conditions (1% formaldehyde for 10-15 minutes at room temperature works well for many transcription factors)

  • Increase the amount of starting material (5-10g of tissue may be needed)

  • Use a two-step cross-linking approach with DSG (disuccinimidyl glutarate) followed by formaldehyde

• High background signal:

  • Implement more stringent washing conditions (increase salt concentration in wash buffers)

  • Pre-clear chromatin with protein A/G beads before adding antibody

  • Use highly specific antibodies validated for ChIP applications

  • Include appropriate negative controls (IgG, non-target regions)

• Target identification challenges:

  • Focus on regions containing the known binding motif (GAAATTT) similar to that bound by CBP60g and SARD1

  • Design primers for ChIP-qPCR to cover regions both with and without this motif

  • For genome-wide studies, consider using ChIP-seq rather than ChIP-chip for better resolution

• Data analysis complexities:

  • Use peak calling algorithms suitable for transcription factors

  • Perform motif enrichment analysis to identify CBP60B binding consensus

  • Integrate with transcriptome data from CBP60B mutants to link binding with gene regulation

• Technical variability:

  • Pool material from multiple plants

  • Include biological replicates (at least three)

  • Use spike-in controls for normalization across samples

How can researchers effectively use CBP60B antibodies to study its interactions with other proteins during immune responses?

To effectively study CBP60B protein interactions during immune responses:

• Co-immunoprecipitation approaches:

  • Perform reverse co-IP experiments using antibodies against both CBP60B and potential interacting partners

  • When investigating calmodulin interactions, conduct experiments in buffers both with calcium (1-2mM CaCl₂) and without calcium (containing EGTA)

  • Consider mild cross-linking to stabilize transient interactions that may occur during immune signaling

• Proximity-based interaction techniques:

  • Implement bimolecular fluorescence complementation (BiFC) by fusing CBP60B and potential interactors to complementary fragments of a fluorescent protein

  • Use split-luciferase assays for quantitative measurement of interactions in planta

  • Consider proximity-dependent labeling methods like BioID or TurboID fused to CBP60B to identify the interaction landscape

• Mass spectrometry-based approaches:

  • Perform immunoprecipitation of CBP60B followed by mass spectrometry before and after pathogen challenge

  • Use quantitative proteomics to identify differentially associated proteins during immune responses

  • Implement crosslinking mass spectrometry (XL-MS) to capture transient interactions

• Yeast-based interaction assays:

  • Use yeast two-hybrid screens with CBP60B as bait to identify novel interactors

  • Consider split-ubiquitin systems for studying membrane-associated interactors

• Domain-specific interaction mapping:

  • Create truncated versions of CBP60B to map interaction domains

  • Focus particularly on the DNA-binding domain and calmodulin-binding domain

  • Evidence suggests that both domains are essential for CBP60B function in immunity

How should researchers design experiments to distinguish direct versus indirect targets of CBP60B transcriptional regulation?

Distinguishing direct from indirect targets requires a multi-faceted approach:

• Integrated genomic approaches:

  • Combine ChIP-seq to identify genome-wide binding sites with RNA-seq from CBP60B mutants

  • Direct targets should show both binding and expression changes

  • Consider using inducible systems (such as estradiol-inducible CBP60B) combined with transcriptome analysis to identify rapid expression changes that are more likely to be direct targets

• Motif analysis and validation:

  • Identify enriched motifs in CBP60B ChIP-seq peaks

  • Test binding to these motifs using electrophoretic mobility shift assays (EMSA)

  • Create reporter constructs with native and mutated motifs to validate functionality

• Time-course experiments:

  • Direct targets typically show more rapid expression changes after CBP60B activation

  • Design time-course experiments with fine temporal resolution (0, 1, 3, 6, 12, 24 hours) after pathogen challenge or inducible CBP60B activation

• Transcriptional inhibition studies:

  • Use transcriptional inhibitors like cordycepin or actinomycin D to block de novo transcription

  • Direct targets should still show CBP60B-dependent changes even with blocked secondary transcription

• In vitro transcription assays:

  • Develop in vitro transcription systems with purified components to test direct activation

  • Include recombinant CBP60B protein, target promoter DNA, and basic transcriptional machinery

What experimental approaches can be used to investigate the evolutionary conservation of CBP60B function across plant lineages?

To investigate evolutionary conservation of CBP60B:

• Phylogenetic and structural analysis:

  • Construct comprehensive phylogenetic trees of CBP60 family members across plant lineages

  • Identify conserved domains and motifs, particularly focusing on DNA-binding domains and calmodulin-binding domains

  • Use homology modeling to predict structural conservation when crystal structures are unavailable

• Complementation experiments:

  • Express CBP60B genes from diverse plant species in Arabidopsis cbp60b mutants

  • Evidence shows that CBP60B-like genes from earliest land plant lineages (Physcomitrium patens and Selaginella moellendorffii) are functionally homologous to Arabidopsis CBP60B

  • Similarly, tomato and cucumber CBP60B-like genes can rescue Arabidopsis cbp60b defects

• Binding site conservation analysis:

  • Compare the promoter sequences of putative CBP60B target genes across species

  • Focus on conservation of known binding motifs (such as GAAATTT)

  • Test binding of CBP60B proteins from different species to these motifs

• Cross-species gene regulation studies:

  • Express Arabidopsis CBP60B in other plant species and assess its ability to regulate orthologous target genes

  • Create reporter constructs with promoters from different species to test cross-species functionality

• Comparative response to pathogens:

  • Compare immune responses in CBP60B mutants across different plant species when challenged with the same pathogen

  • Assess whether downstream signaling pathways are conserved

How can CRISPR-Cas9 genome editing technologies be optimized for studying CBP60B function?

CRISPR-Cas9 offers powerful approaches for studying CBP60B:

• Knockout strategy optimization:

  • Design gRNAs targeting conserved functional domains (DNA-binding domain or calmodulin-binding domain) rather than just early coding regions

  • When studying highly similar family members like CBP60B and CBP60g, carefully design gRNAs that avoid off-target effects on related genes

  • Consider creating knockout collections of multiple CBP60 family members to address redundancy

• Base editing approaches:

  • Use cytosine or adenine base editors to create specific amino acid substitutions in key functional residues

  • Target conserved residues in the calmodulin-binding domain to specifically disrupt calcium/calmodulin regulation while maintaining protein expression

• Promoter editing:

  • Modify the CBP60B promoter to alter its expression pattern, making it pathogen-inducible like CBP60g/SARD1 to assess the importance of constitutive expression

  • Create synthetic promoter variants with different strengths to titrate CBP60B expression levels

• Domain swapping via precise editing:

  • Use CRISPR-mediated homology-directed repair to swap domains between CBP60B and other family members

  • Create chimeric proteins (e.g., CBP60B with the DNA-binding domain of SARD1) to dissect domain-specific functions

• Tagged endogenous CBP60B:

  • Insert epitope tags or fluorescent proteins at the C-terminus of the endogenous CBP60B gene

  • This maintains natural expression patterns while facilitating protein detection, localization studies, and ChIP experiments