CIPK10 Antibody

What is CIPK10 and why is it significant for plant research?

CIPK10 (Calcineurin B-like interacting protein kinase 10) is a serine/threonine kinase that plays crucial roles in plant immunity and stress responses. It functions as part of the CBL-CIPK signaling network, which helps plants respond to various environmental stresses, particularly biotic stresses caused by pathogens. In wheat, TaCIPK10 has been shown to positively regulate resistance to Puccinia striiformis f. sp. tritici (Pst), the causal agent of stripe rust disease, by mediating defense responses including hypersensitive cell death, reactive oxygen species accumulation, and pathogenesis-related gene expression . The significance of CIPK10 lies in its position as a molecular link between calcium signaling and downstream defense responses, making it an important target for understanding plant immunity mechanisms.

How does CIPK10 differ structurally and functionally from other CIPKs?

CIPK10 contains two main domains typical of the CIPK family: a kinase catalytic domain and a regulatory domain. The kinase domain includes an activation loop with three conserved phosphorylatable residues (threonine, serine, and tyrosine) that are essential for its activity. The regulatory domain contains the NAF/FISL motif, which is crucial for interaction with Calcineurin B-like (CBL) proteins . What distinguishes CIPK10 functionally is its specific interaction profile with CBL proteins and its particular role in biotic stress responses. In wheat, TaCIPK10 interacts with six TaCBLs (TaCBL1.1, TaCBL1.2, TaCBL2, TaCBL3, TaCBL4, and TaCBL9) but not with TaCBL6, with the strongest interaction occurring with TaCBL4 . This interaction specificity determines its unique signaling functions and downstream targets.

What are the typical applications for CIPK10 antibodies in plant research?

CIPK10 antibodies are essential tools for studying CIPK10 expression, localization, and functional interactions. Typical applications include:

• Western blotting to detect protein expression levels and post-translational modifications

• Immunoprecipitation to study protein-protein interactions

• Immunolocalization to determine subcellular distribution

• Chromatin immunoprecipitation if CIPK10 is associated with transcriptional complexes

• Phosphorylation assays to study kinase activity

When selecting antibodies for CIPK10 research, specificity is crucial as CIPK family members share high sequence similarity. Polyclonal antibodies against unique peptide sequences, similar to the approach used for CDK antibodies, are often preferred for ensuring specificity .

How is CIPK10 kinase activity regulated in response to pathogen perception?

CIPK10 kinase activity is tightly regulated through multiple mechanisms. Research has shown that TaCIPK10 kinase activity is calcium-dependent and modulated by CBL proteins. Specifically, TaCIPK10's kinase activity significantly increases only in the presence of both TaCBL4 and Ca²⁺, suggesting a calcium-dependent regulatory mechanism .

The regulation involves several steps:

• Pathogen perception triggers calcium influx

• Increased cytosolic Ca²⁺ activates CBL proteins, particularly TaCBL4

• TaCBL4 binds to the NAF/FISL motif in TaCIPK10

• This interaction releases the auto-inhibition of TaCIPK10

• TaCIPK10 becomes activated and phosphorylates downstream targets like TaNH2

Mutations in the NAF motif that prevent interaction with CBLs result in constitutively active CIPK10, demonstrating the auto-inhibitory function of this motif . This complex regulation ensures that CIPK10 activity is precisely controlled during pathogen responses.

What is the relationship between CIPK10 and the salicylic acid (SA) signaling pathway?

TaCIPK10 appears to be intricately connected to salicylic acid (SA) signaling. In wheat, TaCIPK10 transcript levels were significantly enhanced (approximately fivefold) at 2 hours post-treatment with exogenous SA, while other tested CIPKs were not significantly induced . This suggests a specific relationship between TaCIPK10 and SA signaling.

Additionally, during wheat resistance responses to Pst infection, SA concentrations were significantly increased, indicating activation of the SA signaling pathway . The fact that TaCIPK10 interacts with and phosphorylates TaNH2, a homolog of Arabidopsis NPR3/4 (known components of SA signaling), further strengthens the connection between CIPK10 and SA-mediated defense responses .

This relationship positions CIPK10 as a potential molecular link between calcium signaling and SA-dependent immunity, suggesting that antibodies against CIPK10 could be valuable tools for studying crosstalk between these signaling pathways.

How does phosphorylation by CIPK10 affect the function of its target proteins?

CIPK10 functions as a serine/threonine kinase that phosphorylates specific target proteins to modulate their activity. In wheat, TaCIPK10 has been shown to interact with and phosphorylate TaNH2, a homolog of Arabidopsis NPR3/4 . This phosphorylation is likely to alter TaNH2's activity or interactions with other proteins, subsequently affecting downstream defense responses.

Using phospho-specific antibodies designed to recognize CIPK10-specific phosphorylation sites on target proteins would be a valuable approach to further elucidate these regulatory mechanisms.

What criteria should be considered when selecting a CIPK10 antibody for research?

When selecting a CIPK10 antibody for research, several critical factors should be considered:

• Specificity: Due to high sequence similarity among CIPK family members, antibodies should be raised against unique epitopes, preferably from the variable regions outside the conserved kinase domain.

• Cross-reactivity assessment: Pre-test the antibody against recombinant proteins of closely related CIPKs to ensure specificity. This is particularly important in plant species with multiple CIPK paralogs.

• Host species compatibility: Consider the experimental design and species compatibility. For co-immunoprecipitation studies involving multiple proteins, antibodies raised in different host species may be required.

• Application suitability: Verify that the antibody has been validated for your specific application (Western blotting, immunoprecipitation, immunolocalization).

• Recognizes post-translational modifications: If studying phosphorylated forms of CIPK10, select phospho-specific antibodies that recognize specific phosphorylation states.

• Epitope location: Consider whether the epitope is in a functionally significant region that might be masked by protein-protein interactions or conformational changes.

• Validation in your plant species: Antibodies raised against CIPK10 from one plant species may not recognize CIPK10 from distantly related species due to sequence divergence.

What are the optimal protocols for using CIPK10 antibodies in Western blotting experiments?

For optimal Western blotting with CIPK10 antibodies, the following protocol is recommended:

Sample Preparation:

• Extract total proteins from plant tissues using a buffer containing phosphatase inhibitors to preserve phosphorylation states.

• Quantify protein concentration using Bradford or BCA assay.

• Add loading buffer and denature samples at 70-95°C for 5-10 minutes.

Gel Electrophoresis and Transfer:

• Load 20-40 μg of total protein per lane.

• Separate proteins on a 10-12% SDS-PAGE gel.

• Transfer to a PVDF membrane (recommended over nitrocellulose for phosphorylated proteins).

Antibody Incubation:

• Block membrane with 5% non-fat dry milk or BSA in TBST for 1 hour at room temperature.

• Incubate with CIPK10 primary antibody at an optimized dilution (typically starting at 0.5-3 μg/ml) in blocking buffer overnight at 4°C.

• Wash 3x with TBST for 10 minutes each.

• Incubate with appropriate HRP-conjugated secondary antibody (e.g., for a goat primary antibody, use rabbit anti-goat IgG-HRP) at recommended dilution for 1 hour at room temperature .

• Wash 3x with TBST for 10 minutes each.

Detection:

• Apply ECL substrate and image using a chemiluminescence imaging system.

• CIPK10 typically appears as a band of approximately 49-50 kDa (TaCIPK10 from wheat is 49.9 kDa) .

Controls:

• Include a positive control (recombinant CIPK10 protein if available).

• Include a negative control (extract from CIPK10-knockout or silenced plants).

• Consider including a loading control (housekeeping protein) on the same membrane.

How can CIPK10 antibodies be used to detect protein-protein interactions in plant immunity research?

CIPK10 antibodies can be powerful tools for detecting protein-protein interactions using the following methodologies:

Co-immunoprecipitation (Co-IP):

• Prepare plant protein extracts under native conditions using a mild lysis buffer.

• Pre-clear the lysate with protein A/G beads to reduce non-specific binding.

• Incubate cleared lysate with CIPK10 antibody (or antibody against the putative interacting protein).

• Add protein A/G beads to capture antibody-protein complexes.

• Wash beads thoroughly to remove non-specifically bound proteins.

• Elute protein complexes and analyze by Western blotting.

• Probe the membrane with antibodies against suspected interaction partners.

For example, this approach can be used to confirm the interaction between TaCIPK10 and TaCBL4 or TaNH2 in plant tissues under various conditions such as pathogen infection or SA treatment .

Bimolecular Fluorescence Complementation (BiFC):
While not directly using antibodies, BiFC can complement antibody-based approaches:

• Fuse CIPK10 and its potential partner with complementary fragments of a fluorescent protein.

• Express constructs in plant cells.

• Analyze fluorescence signal indicating protein-protein interaction.

• Validate results with Co-IP using CIPK10 antibodies.

Proximity Ligation Assay (PLA):

• Fix and permeabilize plant cells.

• Add primary antibodies against CIPK10 and its potential partner (from different host species).

• Add species-specific secondary antibodies linked to complementary oligonucleotides.

• If proteins are in close proximity, oligonucleotides can be ligated and amplified.

• Detect amplification products by fluorescence microscopy.

These methods can be particularly valuable for studying dynamics of CIPK10 interactions with CBLs and downstream targets during pathogen infection.

How can researchers distinguish between different phosphorylation states of CIPK10?

Distinguishing between different phosphorylation states of CIPK10 requires specialized approaches:

Phospho-specific antibodies:

• Use antibodies specifically raised against phosphorylated activation loop residues of CIPK10.

• Run parallel Western blots with phospho-specific and total CIPK10 antibodies.

• Calculate the ratio of phosphorylated to total CIPK10 to quantify activation levels.

Phos-tag SDS-PAGE:

• Incorporate Phos-tag reagent into polyacrylamide gels.

• This causes mobility shifts based on phosphorylation status.

• Different phosphorylation states of CIPK10 will migrate as distinct bands.

• Detect with standard CIPK10 antibodies.

2D gel electrophoresis:

• Separate proteins by isoelectric focusing followed by SDS-PAGE.

• Phosphorylated forms of CIPK10 will appear as spots with more acidic pI.

• Detect with CIPK10 antibodies.

Mass spectrometry following immunoprecipitation:

• Immunoprecipitate CIPK10 using specific antibodies.

• Digest purified protein and analyze by LC-MS/MS.

• Identify specific phosphorylation sites and quantify their occupancy.

These approaches are particularly useful for monitoring CIPK10 activation during pathogen response, as the activation loop containing three conserved phosphorylatable residues (threonine, serine, and tyrosine) is critical for CIPK10 kinase activity .

What are common pitfalls when investigating CIPK10 in knockout or silenced plant lines?

Researchers investigating CIPK10 function through knockout or silencing approaches should be aware of several potential pitfalls:

Genetic redundancy:

• Multiple CIPK homologs or paralogs may compensate for CIPK10 loss.

• For example, wheat contains multiple CIPK genes that could have overlapping functions .

• Consider simultaneous knockdown of closely related CIPKs or comprehensive analysis of the CIPK family.

Incomplete silencing:

• VIGS or RNAi approaches often result in partial knockdown rather than complete knockout.

• Quantify silencing efficiency using both qRT-PCR and Western blotting with CIPK10 antibodies.

• In wheat, where three copies of TaCIPK10 exist on chromosomes 4A, 4B, and 4D, ensure all copies are targeted .

Pleiotropic effects:

• CIPKs participate in multiple signaling pathways, so their loss may cause developmental phenotypes.

• Separate developmental effects from specific immunity phenotypes using inducible or tissue-specific silencing systems.

Specificity of silencing constructs:

• Ensure silencing constructs target only the intended CIPK and not related family members.

• For example, verify that VIGS fragments do not contain consecutive 21- to 24-nucleotide sequences matching other CIPKs .

Verification of knockout/knockdown:

• Confirm protein absence using specific CIPK10 antibodies, not just mRNA reduction.

• Assay kinase activity loss to verify functional knockdown.

In the study of TaCIPK10 in wheat, researchers successfully used three specific fragments of TaCIPK10 cDNA for VIGS and confirmed silencing efficiency by qRT-PCR, showing significantly reduced transcript levels compared to controls .

How can researchers overcome cross-reactivity issues with CIPK10 antibodies?

Cross-reactivity is a significant challenge when working with CIPK10 antibodies due to high sequence conservation among CIPK family members. Strategies to overcome this include:

Pre-absorption techniques:

• Pre-incubate the antibody with recombinant proteins of closely related CIPKs.

• This captures antibodies that cross-react with those proteins.

• Use the remaining antibody solution, which should be more specific for CIPK10.

Peptide competition assays:

• Perform parallel experiments with antibody pre-incubated with the immunizing peptide.

• Signals that disappear in the peptide-blocked experiment represent specific binding.

Validation with genetic controls:

• Include samples from CIPK10 knockout/knockdown plants.

• Specific bands should be absent or reduced in these samples.

Epitope selection for custom antibodies:

• Design antibodies against unique regions of CIPK10, particularly in the C-terminal regulatory domain.

• Avoid the highly conserved kinase domain when possible.

• Use peptide immunogens that are specific to CIPK10 and not conserved in other family members.

Sequential immunoprecipitation:

• For complex samples, first deplete cross-reactive proteins by immunoprecipitation with antibodies against related CIPKs.

• Then immunoprecipitate CIPK10 from the depleted sample.

Western blot optimization:

• Use higher antibody dilutions to reduce non-specific binding.

• Optimize blocking conditions (BSA may work better than milk for phospho-epitopes).

• Increase washing stringency with higher detergent concentrations.

How should researchers interpret changes in CIPK10 expression versus activity in pathogen response experiments?

Proper interpretation of CIPK10 data requires distinguishing between expression levels and kinase activity:

Expression versus activity correlation:

• Increased CIPK10 expression (measured by qRT-PCR and Western blotting) does not necessarily indicate increased kinase activity.

• In wheat, TaCIPK10 transcript levels are rapidly induced by Pst inoculation and SA treatment , but this upregulation must be distinguished from activation.

• Wild-type TaCIPK10 shows minimal kinase activity in vitro unless activated by TaCBL4 and Ca²⁺ .

Multi-level analysis approach:

• Measure transcript levels by qRT-PCR.

• Quantify protein levels by Western blotting with CIPK10 antibodies.

• Assess phosphorylation status using phospho-specific antibodies.

• Measure kinase activity using in vitro kinase assays with immunoprecipitated CIPK10.

• Evaluate downstream effects by monitoring substrate phosphorylation (e.g., TaNH2).

Temporal considerations:

• Expression changes may precede activity changes.

• Different timepoints may reveal distinct regulatory mechanisms.

• In wheat-Pst interactions, TaCIPK10 induction occurs early, suggesting it functions in initial defense responses .

Context interpretation:

• Compare CIPK10 data with other defense markers.

• Correlate with physiological responses like reactive oxygen species production.

• Consider data in light of calcium signaling dynamics, as CIPK10 activity is Ca²⁺-dependent .

A comprehensive experimental design should include measurements at multiple levels of regulation to distinguish between increased expression and enhanced activity of CIPK10 during pathogen responses.

What experimental controls are essential when studying CIPK10 phosphorylation of target proteins?

When studying CIPK10-mediated phosphorylation of target proteins, several essential controls must be included:

Kinase activity controls:

• Kinase-dead mutant: Include a kinase-inactive version of CIPK10 (e.g., K42N mutation) to demonstrate that phosphorylation is specifically due to CIPK10 kinase activity.

• Constitutively active mutant: Include a CIPK10 variant lacking the auto-inhibitory NAF motif to serve as a positive control .

• Phosphatase treatment: Pre-treat samples with lambda phosphatase to remove existing phosphorylation before kinase assays.

Substrate specificity controls:

• Non-substrate protein: Include a protein not expected to be phosphorylated by CIPK10.

• Mutated substrate: Use versions of the substrate with potential phosphorylation sites mutated (Ser/Thr to Ala).

• Competing substrate: Add excess non-labeled substrate to competition assays.

Buffer condition controls:

• Ca²⁺ dependence: Perform parallel reactions with and without Ca²⁺ to demonstrate CBL-dependent activation .

• CBL specificity: Include reactions with different CBL proteins to demonstrate specificity of the CBL-CIPK10 interaction .

In vivo validation controls:

• CIPK10-silenced plants: Verify reduced phosphorylation of the target in CIPK10-silenced plants.

• Phospho-mimetic substrate: Express phospho-mimetic (Ser/Thr to Asp/Glu) versions of the substrate to test functional consequences.

In the study of TaCIPK10, researchers demonstrated that TaCIPK10 interacts with and phosphorylates TaNH2 in vitro and showed that silencing of either gene affected wheat resistance to Pst, providing both biochemical and genetic evidence for the functional relationship .

How can researchers integrate CIPK10 antibody data with functional studies to establish causality in plant immunity?

Establishing causality between CIPK10 activity and plant immunity outcomes requires integration of multiple experimental approaches:

Correlation to causation approach:

• Temporal sequence: Use CIPK10 antibodies to demonstrate that CIPK10 accumulation or phosphorylation precedes defense responses.

• Dose-response relationship: Show that levels of active CIPK10 (determined by immunoblotting) correlate with immunity strength.

• Genetic manipulation: Compare CIPK10 protein levels, phosphorylation states, and kinase activity in wild-type, overexpression, and silenced plants.

• Pharmacological intervention: Use kinase inhibitors to specifically block CIPK10 activity and observe effects on immunity.

Multi-level evidence integration:

• Biochemical evidence: Use CIPK10 antibodies to immunoprecipitate and identify interacting partners by mass spectrometry.

• Genetic evidence: Demonstrate altered pathogen susceptibility in CIPK10-silenced plants .

• Phenotypic evidence: Show that TaCIPK10 overexpression enhances resistance through specific defense responses like hypersensitive cell death and ROS accumulation .

• Substrate function: Demonstrate that substrate (e.g., TaNH2) silencing produces similar phenotypes to CIPK10 silencing .

Mechanistic pathway reconstruction:

• Use CIPK10 antibodies to track:

  • CIPK10 localization changes during infection

  • CIPK10-CBL complex formation dynamics

  • CIPK10-mediated phosphorylation of substrates

• Map the complete pathway from pathogen perception to defense outputs with CIPK10 as a central node.

In the case of TaCIPK10, researchers combined:

• Expression analysis showing TaCIPK10 induction by Pst and SA

• In vitro biochemical studies demonstrating Ca²⁺/CBL-dependent activation

• VIGS functional studies showing compromised resistance in TaCIPK10-silenced plants

• Identification and validation of TaNH2 as a substrate

This integrated approach established TaCIPK10 as a positive regulator of wheat resistance to stripe rust .

What are emerging technologies for studying CIPK10 protein dynamics in living cells?

Several cutting-edge technologies are emerging for studying CIPK10 dynamics in living cells:

Optogenetic approaches:

• Light-controllable CIPK10 variants to temporally activate kinase function

• Optogenetic control of Ca²⁺ influx to study CIPK10 activation dynamics

• Light-induced dissociation of CIPK10 from regulatory proteins

Live-cell imaging with fluorescent protein fusions:

• FRET-based biosensors to monitor CIPK10-substrate interactions in real-time

• Split fluorescent protein complementation to visualize CIPK10-CBL complex formation

• Single-molecule tracking of CIPK10 during immune responses

Proximity labeling methods:

• APEX2 or BioID fused to CIPK10 to identify transient interaction partners

• Temporal analysis of the CIPK10 interactome during infection

• Spatial mapping of CIPK10 complexes in different subcellular compartments

CRISPR-based technologies:

• Base editing to introduce specific mutations in CIPK10 phosphorylation sites

• CRISPRa/CRISPRi for temporal control of CIPK10 expression

• CRISPR-mediated tagging of endogenous CIPK10 with fluorescent or affinity tags

Nanobody-based detection:

• Development of CIPK10-specific nanobodies for live-cell imaging

• Nanobody-based sensors for detecting conformational changes in CIPK10

• Intrabodies to manipulate CIPK10 function in specific cellular compartments

These technologies, combined with traditional antibody-based approaches, will provide unprecedented insights into CIPK10 dynamics during plant immunity responses.

How might comparative studies using CIPK10 antibodies across different plant species advance our understanding of plant immunity evolution?

Comparative studies using CIPK10 antibodies across different plant species can provide valuable insights into the evolution of plant immunity:

Evolutionary conservation analysis:

• Use CIPK10 antibodies to compare expression patterns and protein levels across monocots and dicots

• Identify conserved vs. species-specific post-translational modifications

• Compare CIPK10-CBL interaction specificity across evolutionary distance

Functional conservation testing:

• Determine if CIPK10 in different species phosphorylates homologous substrates

• Compare kinase activity regulation mechanisms across species

• Test cross-species complementation of CIPK10 mutants

Pathogen specificity investigation:

• Compare CIPK10 activation in response to host-adapted vs. non-host pathogens

• Analyze CIPK10 phosphorylation patterns during compatible vs. incompatible interactions

• Determine if CIPK10 is targeted by pathogen effectors across different plant-pathogen systems

Signaling network evolution:

• Map CIPK10 position in Ca²⁺ and SA signaling networks across species

• Identify lineage-specific CIPK10 interactors and substrates

• Compare upstream regulators and downstream targets of CIPK10

Practical implementation:

• Develop broadly cross-reactive CIPK10 antibodies targeting conserved epitopes

• Create species-specific antibodies for divergent regions

• Use epitope tagging for comparative immunoprecipitation studies

TaCIPK10 shares the same phylogenetic clade with CIPK10 proteins from other monocot plants , suggesting functional conservation that could be explored using comparative antibody-based studies.

What methodological advances are needed to better understand the quantitative aspects of CIPK10-mediated phosphorylation in plant immunity?

Several methodological advances are needed to better understand quantitative aspects of CIPK10-mediated phosphorylation:

Improved phospho-proteomics approaches:

• Development of antibodies that specifically recognize CIPK10 phosphorylation motifs

• Enhanced sensitivity for detecting low-abundance phosphorylation events

• Better temporal resolution to capture rapid phosphorylation dynamics during immune responses

Quantitative biochemical assays:

• Advanced in vitro kinase assays with precise stoichiometry measurements

• Determination of kinetic parameters (Km, Vmax) for CIPK10 with various substrates

• Real-time monitoring of phosphorylation reactions using fluorescent sensors

Single-cell analysis:

• Methods to measure CIPK10 activity in individual cells during defense responses

• Spatial mapping of phosphorylation gradients within tissues

• Correlation of CIPK10 activity with cell-specific immunity outcomes

Mathematical modeling:

• Integration of CIPK10 kinetics into computational models of plant immunity

• Prediction of phosphorylation network behavior under various conditions

• Simulation of feedback loops in CIPK10-mediated signaling

Structural biology approaches:

• Determination of CIPK10 structure in various activation states

• Analysis of CIPK10-substrate complex structures

• Understanding how phosphorylation alters target protein conformation and function

Antibody engineering:

• Development of antibodies with greater specificity for distinct phosphorylated forms of CIPK10

• Creation of intrabodies that specifically recognize active CIPK10 in living cells

• Engineering of degradation-inducing antibodies to achieve temporal control of CIPK10 function

Advancements in these areas would provide a more comprehensive understanding of how CIPK10-mediated phosphorylation quantitatively contributes to plant immunity outcomes.