EFR Antibody

What is EFR and what is its role in plant immunity?

EFR (ELONGATION FACTOR TU RECEPTOR) is a pattern recognition receptor (PRR) that plays a crucial role in plant immunity by perceiving the pathogen-associated molecular pattern (PAMP) ELONGATION FACTOR TU, or its active peptide epitope elf18. This recognition triggers plant pattern-triggered immunity (PTI), a first line of defense against pathogens. When activated, EFR initiates heterodimerization with the co-receptor BAK1, resulting in phosphorylation of the EFR intracellular kinase domain, which subsequently relays signals to cytoplasmic kinases such as BIK1 and PBL1 . The activated immune signaling cascade elicits various cellular responses including oxidative burst, Ca²⁺-influx, callose deposition, MAPK activation, and transcriptional reprogramming, all critical components of the plant's defense mechanism .

How does EFR interact with co-receptors to initiate immune signaling?

Upon recognition of its ligand elf18, EFR undergoes conformational changes that enable it to interact with co-receptors, particularly members of the somatic embryogenesis receptor like kinase (SERK) family, including SERK1, SERK3/BAK1, and SERK4/BKK1 . These interactions form the basis for signal transduction. Research indicates that EFR can interact in a ligand-inducible manner with most SERKs except SERK5 . The interaction between EFR and these co-receptors is crucial for the activation of downstream signaling components. Recent studies have revealed that rather than requiring its kinase activity to transactivate BAK1, EFR employs non-catalytic activation mechanisms to form the EFR-BAK1 complex , highlighting the sophisticated nature of these molecular interactions in plant immunity.

What are the primary techniques used to identify EFR-interacting proteins?

The identification of EFR-interacting proteins (EIPs) primarily relies on immunoprecipitation followed by mass spectrometry. This approach has been successfully employed in both Nicotiana benthamiana and Arabidopsis expressing functional GFP-tagged EFR . The methodology involves:

• Generation of transgenic plants expressing tagged EFR proteins

• Verification of functionality through complementation assays

• Immunoprecipitation using antibodies against the tag

• Mass spectrometry analysis of co-immunoprecipitated proteins

• Validation of interactions through independent techniques

This approach has led to the identification of several chaperones responsible for EFR receptor maturation, as well as receptor-like kinases (RLKs) from different subfamilies, termed receptor kinases associated with EFR (RAEs) . The success of these techniques depends significantly on the quality of antibodies used and the preservation of protein complexes during extraction and purification processes.

How can researchers functionally validate EFR signaling pathways?

Functional validation of EFR signaling pathways involves multiple complementary approaches:

• Seedling growth inhibition (SGI) assays: These assays measure plant sensitivity to elf18 treatment, where reduced growth indicates functional EFR signaling. For example, studies have shown that EFR Y836F and EFR SSAA lines exhibit decreased sensitivity to elf18 treatment compared to wild-type EFR in SGI assays .

• MAPK activation analysis: Western blotting is used to detect the phosphorylation status of MAPKs following elf18 perception, providing insights into downstream signaling events .

• Bacterial resistance assays: Functional EFR signaling confers resistance against certain bacteria, such as Agrobacterium tumefaciens. GUS activity measurements following bacterial infection with A. tumefaciens carrying a GUS reporter gene can quantify infection success and, indirectly, EFR functionality .

• Mutant complementation studies: Introducing wild-type or mutated EFR into efr knockout plants helps determine which domains and residues are essential for receptor function. For instance, the EFR F761H mutation has been shown to restore full signaling function in otherwise impaired EFR Y836F and EFR SSAA variants .

These methodologies collectively provide robust insights into the functional aspects of EFR signaling pathways and how they contribute to plant immunity.

What mechanisms govern EFR receptor maturation and quality control?

EFR receptor maturation involves a sophisticated quality control system in the endoplasmic reticulum. Research has identified that EFR requires a specific chaperone complex consisting of SDF2, ERdj3B, and BiP for proper maturation . This process involves:

• N-glycosylation: As a nascent polypeptide, EFR undergoes N-glycosylation in the endoplasmic reticulum, which is crucial for its proper folding and function .

• Chaperone-assisted folding: The identified chaperone complex assists in the correct folding of EFR, ensuring that only properly folded receptors progress through the secretory pathway .

• Quality control checkpoints: Misfolded or improperly glycosylated EFR proteins are retained in the ER and targeted for degradation, preventing non-functional receptors from reaching the plasma membrane.

Understanding these processes is essential for researchers working with EFR, as mutations or perturbations in these pathways can significantly impact receptor functionality and experimental outcomes.

How do allosteric mechanisms contribute to EFR-BAK1 complex activation?

Recent research has revealed that allosteric mechanisms play a critical role in the activation of the EFR-BAK1 complex. Rather than relying solely on its kinase activity to transactivate BAK1, EFR employs non-catalytic activation mechanisms . This allosteric activation involves:

• Conformational changes: Binding of elf18 to EFR induces conformational changes that enable interaction with BAK1.

• Key residue interactions: Specific amino acid residues, such as F761 in EFR, have been identified as crucial for this allosteric activation. Mutation studies show that the EFR F761H modification can restore signaling function in otherwise impaired EFR variants .

• Signal amplification: These allosteric mechanisms likely serve as signal amplifiers, allowing for robust immune responses even with relatively low levels of receptor activation.

This understanding of allosteric regulation provides new opportunities for engineering enhanced plant immunity through targeted modifications of the EFR-BAK1 interaction interface.

What is the relationship between extrafollicular responses (EFRs) and antibody production?

Extrafollicular plasmablast responses (EFRs) represent a critical pathway for rapid antibody production during immune challenges. Contrary to the traditional view that EFRs generate only low-affinity antibodies with limited protective capacity, recent research indicates that EFRs can produce protective antibodies through Toll-like receptor (TLR)-mediated mechanisms . The process involves:

• B cell activation: Antigen-specific B cells are activated through B cell receptor engagement and inflammatory signals.

• Fate determination: TLR-mediated inflammatory signals direct B cells towards becoming antibody-secreting cells (ASCs) through EFRs rather than entering germinal centers (GCs) .

• Rapid protection: EFRs generate antibodies more rapidly than GC responses, providing early protection during infections such as influenza .

Understanding this pathway is particularly relevant for researchers studying immune responses to viral infections, as EFRs appear to contribute significantly to early protection before GC-derived antibodies develop.

How do Toll-like receptor (TLR) signals influence the development of EFRs?

TLR-mediated signals play a dual role in directing B cells towards EFRs through both B cell-intrinsic and extrinsic mechanisms :

• B cell-intrinsic effects: TLR signals within B cells support antigen-stimulated B cell survival, clonal expansion, and differentiation by inducing IRF4 (the master regulator of B cell differentiation) through activation of NF-kB c-Rel .

• Inflammatory environment: TLR activation creates an inflammatory milieu that further promotes B cell differentiation toward the EFR pathway.

• Fate switching: Sustained TLR4 stimulation after immunization shifts the fate of virus-specific B cells towards EFRs instead of GCs, resulting in rapid antibody production and improved protective capacity compared to antigen/alum administration alone .

These findings suggest that strategic manipulation of TLR signaling could enhance vaccine efficacy by promoting rapid protective antibody responses through the EFR pathway.

What are optimal approaches for detecting and studying EFR in different experimental systems?

Detecting and studying EFR presents several technical challenges that researchers should consider:

• Antibody limitations: Endogenous EFR detection can be difficult, as anti-EFR antibodies may not effectively detect native Arabidopsis EFR . Alternative approaches include:

  • Epitope tagging (e.g., GFP-tagged EFR)

  • Overexpression systems for enhanced detection

  • Transgenic complementation of efr mutant lines

• Expression systems: For heterologous expression, Nicotiana benthamiana has proven effective for transient expression of functional EFR that localizes to the cell periphery . Transgenic Arabidopsis lines expressing EFR under either native or constitutive promoters have also been successfully used to complement efr-1 mutants .

• Functionality verification: When working with tagged or modified EFR variants, researchers should always verify functionality through complementation assays, including:

  • Elf18 response assays

  • Oxidative burst measurements

  • MAPK activation analysis

  • Resistance to relevant pathogens

These technical considerations are crucial for ensuring the reliability and reproducibility of EFR-related research.

What are the key considerations for mass spectrometry analysis of EFR and its interacting proteins?

Mass spectrometry (MS) analysis of EFR and its interacting proteins requires careful attention to several aspects:

• Sample preparation: Thorough immunoprecipitation protocols are essential, with attention to buffer compositions that preserve protein-protein interactions while minimizing non-specific binding.

• Post-translational modification analysis: MS analysis can identify both glycosylated EFR peptides and phosphopeptides, providing insights into receptor maturation and activation states . Tables of identified glycosylated peptides and phosphopeptides have been documented in previous studies .

• Interactome validation: MS-identified interactions should be validated through independent techniques such as co-immunoprecipitation, FRET-FLIM, or split-luciferase assays.

• Data analysis considerations: When analyzing MS data:

  • Establish appropriate significance thresholds

  • Consider the number of unique peptides identified for each protein

  • Take into account the coverage percentage of the identified proteins

  • Compare results with appropriate negative controls

These methodological considerations ensure robust identification of true EFR-interacting proteins while minimizing false positives.

How can EFR-mediated immunity be exploited to enhance disease resistance in plants?

EFR-mediated immunity offers several avenues for enhancing disease resistance in plants:

• Transgenic approaches: Introducing EFR into plant species that naturally lack this receptor can confer novel recognition capabilities. For example, transfer of Arabidopsis EFR to crops that don't naturally express this receptor could provide protection against a broader range of pathogens.

• Receptor engineering: Modifications of the EFR receptor itself, such as the F761H mutation that enhances signaling capability , can potentially strengthen immune responses. This approach leverages the understanding of allosteric activation mechanisms to design more effective receptors.

• Co-receptor manipulation: Optimizing the interaction between EFR and its co-receptors like BAK1 could enhance signal transduction efficiency and amplify immune responses.

• Integration with other immune pathways: Combining EFR-mediated recognition with other defense mechanisms could provide more comprehensive protection against diverse pathogens.

These strategies represent promising directions for translational research aimed at improving crop protection through enhanced innate immunity.

What are the current limitations in translating EFR research to practical applications?

Despite the potential, several limitations currently affect the translation of EFR research to practical applications:

• Species specificity: The functionality of EFR may vary across plant species due to differences in downstream signaling components and co-receptor availability.

• Metabolic costs: Constitutive activation of immune responses can impose significant metabolic costs on plants, potentially reducing growth and yield.

• Pathogen adaptation: Pathogens may evolve mechanisms to evade recognition or suppress EFR-mediated signaling, limiting long-term effectiveness of resistance strategies.

• Integration challenges: Effective integration of EFR-based resistance with other disease management practices requires further research and development.

Addressing these limitations requires continued basic research on EFR function across different plant species and environmental conditions, as well as field testing of EFR-based resistance strategies.

What emerging technologies might advance our understanding of EFR biology?

Several emerging technologies hold promise for advancing EFR research:

• Cryo-electron microscopy: This technology could provide detailed structural insights into the EFR-BAK1 complex and how it changes upon ligand binding, illuminating the molecular basis of allosteric activation.

• Single-molecule techniques: Methods such as single-molecule FRET could reveal the dynamics of EFR interactions with co-receptors and downstream signaling components in real-time.

• Genome editing: CRISPR/Cas9 and related technologies enable precise modification of EFR and its signaling components, facilitating functional studies of specific domains and residues.

• Advanced imaging: Super-resolution microscopy and other advanced imaging techniques could reveal the spatial organization of EFR signaling complexes at the plasma membrane and during endocytosis.

• Systems biology approaches: Integration of transcriptomics, proteomics, and metabolomics data could provide a more comprehensive understanding of how EFR signaling reshapes cellular physiology during immune responses.

These technologies, applied individually or in combination, have the potential to resolve current knowledge gaps and open new avenues for EFR research.

What are the key unresolved questions in EFR research?

Despite significant progress, several key questions remain unresolved in EFR research:

• Structural determinants: What are the precise structural changes that occur in EFR upon elf18 binding, and how do these changes facilitate co-receptor recruitment?

• Signaling specificity: How does EFR achieve signaling specificity despite sharing many downstream components with other immune receptors?

• Crosstalk mechanisms: How does EFR-mediated signaling integrate with other immune and developmental pathways?

• Evolutionary aspects: How has EFR evolved across different plant species, and what can this tell us about the co-evolution of plants and their pathogens?

• Receptor dynamics: What are the dynamics of EFR synthesis, trafficking, activation, and turnover during immune responses?

Addressing these questions will require interdisciplinary approaches and continued technological innovation in the field of plant immunity research.