Recombinant Laccaria bicolor Protein SEY1 (SEY1)

What is the SEY1 protein in Laccaria bicolor and what is its biological significance?

SEY1 (UniProt No. B0D0N9) is a protein expressed in Laccaria bicolor, an ectomycorrhizal basidiomycete fungus that forms symbiotic relationships with plant roots. According to enzyme classification, SEY1 belongs to enzyme class EC 3.6.5.-, suggesting its involvement in hydrolytic activity, potentially related to GTP binding and hydrolysis . Its biological significance is likely related to the establishment and maintenance of symbiotic relationships, as many proteins in L. bicolor are specialized for facilitating mutualistic interactions with host plants. Similar to how the endoglucanase LbGH5-CBM1 plays a key role in cell wall remodeling during symbiosis formation , SEY1 may have specialized functions in the symbiotic relationship between L. bicolor and its plant hosts.

How does SEY1 differ from other characterized proteins in Laccaria bicolor?

Unlike the well-characterized LbGH5-CBM1 endoglucanase that has a demonstrated role in cell wall remodeling during the formation of the Hartig net , SEY1's precise function remains less thoroughly documented in the scientific literature. While LbGH5-CBM1 contains a cellulose-binding module (CBM1) linked to a glycoside hydrolase domain and shows highest activity toward cellulose and galactomannans, SEY1 belongs to a different enzyme classification (EC 3.6.5.-) . This suggests that SEY1 likely participates in different biochemical pathways within the fungus compared to cell-wall-modifying enzymes. Additionally, while proteins like those involved in the PtSSP1-LbGAL4-like interaction have been shown to regulate symbiosis between poplar and L. bicolor , the regulatory networks involving SEY1 require further investigation.

What are the optimal conditions for storage and handling of Recombinant Laccaria bicolor Protein SEY1?

For optimal storage and handling of Recombinant Laccaria bicolor Protein SEY1, follow these research-validated protocols:

• Storage Conditions:

  • Liquid form: Stable for approximately 6 months at -20°C/-80°C

  • Lyophilized form: Maintains stability for 12 months at -20°C/-80°C

• Pre-Use Preparation:

  • Briefly centrifuge the vial prior to opening to ensure contents settle at the bottom

  • For reconstitution, use deionized sterile water to achieve a concentration of 0.1-1.0 mg/mL

• Long-Term Stability:

  • Add glycerol to a final concentration of 5-50% (standard recommendation is 50%)

  • Aliquot in appropriate volumes to minimize freeze-thaw cycles

  • Avoid repeated freezing and thawing as this significantly reduces protein activity

• Working Conditions:

  • For short-term experiments, working aliquots can be stored at 4°C for up to one week

  • Return to -20°C/-80°C promptly after use to maintain protein integrity

Following these precise handling protocols will ensure experimental reproducibility and optimal protein activity for research applications.

How should researchers design experiments to study potential enzymatic activities of SEY1?

To effectively design experiments investigating the enzymatic activities of SEY1, researchers should implement the following methodological framework:

• Enzyme Activity Screening:

  • Based on the EC classification (3.6.5.-) , design initial screening assays focusing on hydrolytic activities

  • Test substrate specificity using a panel of potential substrates, including GTP and other nucleotides

  • Monitor reaction products using chromatographic techniques (HPLC or LC-MS)

• Reaction Condition Optimization:

  • Systematically evaluate optimal pH (range 5.0-8.0), temperature (20-37°C), and buffer compositions

  • Assess divalent cation requirements (Mg²⁺, Mn²⁺, Ca²⁺) as these frequently influence hydrolase activities

  • Develop a standardized activity assay with high reproducibility

• Kinetic Parameter Determination:

  • Perform substrate concentration gradients to determine Km, Vmax, and kcat values

  • Analyze data using appropriate enzyme kinetics software and models

  • Compare kinetic parameters with related enzymes from other organisms

• Inhibition Studies:

  • Test potential inhibitors based on structural similarity to substrates

  • Determine IC₅₀ values and inhibition mechanisms (competitive, non-competitive, uncompetitive)

  • Use inhibition patterns to further characterize the active site architecture

• Structure-Function Analysis:

  • Correlate enzymatic activity with protein structural elements

  • Consider employing site-directed mutagenesis of conserved residues to validate catalytic mechanisms

  • Combine with computational modeling if structural data becomes available

This comprehensive experimental design approach will systematically characterize SEY1's enzymatic properties while minimizing experimental artifacts and ensuring reproducibility.

What protocols should be used to study potential interactions between SEY1 and plant host proteins?

When investigating potential interactions between SEY1 and plant host proteins, researchers should implement a multi-layered approach combining both in vitro and in planta methodologies:

• In Vitro Interaction Screening:

  • Yeast Two-Hybrid (Y2H) Assays: Construct a bait plasmid containing SEY1 and screen against a prey library of plant host proteins, similar to the approach used to identify the interaction between PtSSP1 and LbGAL4-like

  • Pull-Down Assays: Use purified recombinant SEY1 with appropriate tags (His, GST) as bait to capture interacting plant proteins from host extracts

  • Surface Plasmon Resonance (SPR): Quantify binding kinetics (kon, koff) and affinity constants (KD) for identified interactions

• Cellular Localization and Co-Localization:

  • Bimolecular Fluorescence Complementation (BiFC): Validate protein-protein interactions in plant cells, as demonstrated with PtSSP1 and LbGAL4-like

  • Confocal Microscopy: Track fluorescently tagged SEY1 in symbiotic structures to determine its spatial distribution

  • Subcellular Fractionation: Biochemically separate cellular compartments to confirm co-localization of SEY1 with plant proteins

• Functional Validation:

  • Co-Immunoprecipitation (Co-IP): Isolate protein complexes from mycorrhizal tissues using SEY1-specific antibodies

  • RNA Interference (RNAi): Generate SEY1-knockdown mutants to assess phenotypic effects on symbiosis, similar to approaches used with LbGH5-CBM1

  • Protein-Protein Interaction Interface Mapping: Identify critical residues involved in interactions through alanine scanning mutagenesis

• Systems-Level Analysis:

  • Transcriptomics: Compare gene expression profiles in wildtype and SEY1-modified systems to identify downstream effectors

  • Proteomics: Use quantitative proteomics to identify changes in protein abundance and post-translational modifications

  • Network Analysis: Construct interaction networks to position SEY1 within the broader context of symbiotic signaling

This comprehensive protocol system offers methodological redundancy while providing complementary data types that together can establish the biological significance of SEY1 interactions with host proteins.

How might SEY1 be involved in the establishment or maintenance of ectomycorrhizal symbiosis?

Based on current understanding of ectomycorrhizal symbiosis mechanisms, SEY1 may contribute to this complex process through several potential mechanisms:

• Symbiotic Interface Development:
  Similar to LbGH5-CBM1, which accumulates at the periphery of hyphae forming the Hartig net and mantle , SEY1 could participate in specialized structures at the fungus-plant interface. Its enzymatic activity may modify cell wall components or contribute to signaling events that coordinate hyphal development during colonization.

• Signal Transduction:
  If SEY1 functions as a GTPase (based on its EC classification 3.6.5.-) , it may participate in critical signal transduction pathways regulating symbiotic development. GTPases often function as molecular switches in cellular signaling, potentially controlling fungal responses to plant-derived signals. This would be analogous to how the plant-derived effector PtSSP1 interacts with the fungal transcription factor LbGAL4-like to regulate symbiosis .

• Nutrient Exchange Regulation:
  The symbiotic relationship between L. bicolor and host plants involves bidirectional nutrient exchange. SEY1 could potentially regulate transporters or channels involved in this process, perhaps through energy-dependent conformational changes facilitated by its putative GTPase activity.

• Hartig Net Formation:
  The Hartig net is a critical interface structure in ectomycorrhizal symbiosis. As demonstrated with LbGH5-CBM1, which plays a key role in cell wall remodeling during Hartig net formation , SEY1 might similarly contribute to the structural modifications necessary for establishing this specialized interface, though potentially through different biochemical mechanisms.

• Host Immune Response Modulation:
  Successful symbiosis requires suppression or modulation of host defense responses. SEY1 could potentially function as an effector protein that influences plant immune signaling pathways, similar to how some fungal effectors have been shown to manipulate host responses.

Future research using knockout/knockdown approaches combined with detailed phenotypic analysis of symbiotic structures would be essential to elucidate SEY1's precise contributions to ectomycorrhizal symbiosis.

What techniques can be used to investigate the influence of SEY1 on fungal growth and morphology?

To comprehensively investigate SEY1's influence on fungal growth and morphology, researchers should implement the following advanced technical approaches:

• Genetic Manipulation Strategies:

  • RNAi-Mediated Knockdown: Develop RNAi constructs targeting SEY1 mRNA, similar to approaches used for LbGH5-CBM1

  • CRISPR/Cas9 Gene Editing: Generate precise knockout mutants to completely eliminate SEY1 function

  • Controlled Overexpression: Create fungal strains with SEY1 under inducible promoters to assess dose-dependent effects

• High-Resolution Microscopy Techniques:

  • Confocal Microscopy: Analyze hyphal morphology using fluorescent markers for cell walls and organelles

  • Transmission Electron Microscopy (TEM): Examine ultrastructural changes in SEY1-modified strains

  • Live-Cell Imaging: Monitor dynamic growth processes in real-time using fluorescently-tagged cellular components

• Quantitative Growth and Morphological Analysis:

  • Automated Image Analysis: Implement computer vision algorithms to quantify morphological parameters (hyphal diameter, branching frequency, septation patterns)

  • Microfluidic Cultivation: Use microfluidic devices to precisely control growth environments and monitor single-hypha dynamics

  • Biomass Accumulation Assays: Measure dry weight, protein content, and ergosterol levels under standardized conditions

• Symbiotic Competence Assessment:

  • In Vitro Mycorrhization Assays: Quantify colonization rates of mutants with host plants

  • Hartig Net Development Analysis: Section mycorrhizal roots to measure Hartig net formation similar to analyses performed with PtSSP1

  • Competitive Colonization Experiments: Co-inoculate wildtype and SEY1-modified strains to assess relative fitness

• Molecular Response Monitoring:

  • Transcriptomics: Compare gene expression profiles between wildtype and SEY1-modified strains

  • Proteomics: Analyze changes in protein abundance, especially those involved in cell wall biogenesis and hyphal morphogenesis

  • Metabolomics: Identify alterations in metabolic pathways that may contribute to growth and morphological phenotypes

This multi-faceted approach provides complementary datasets that together can establish causal relationships between SEY1 function and specific aspects of fungal growth and morphology.

How can bioinformatic approaches be used to predict SEY1 function and evolutionary significance?

Employing sophisticated bioinformatic methodologies can yield valuable insights into SEY1's function and evolutionary significance. The following comprehensive analytical framework is recommended:

• Sequence-Based Functional Prediction:

  • Homology Detection: Utilize sensitive profile-based methods (HHpred, HMMER) to detect remote homologs beyond standard BLAST searches

  • Domain Architecture Analysis: Map functional domains and compare with experimentally characterized proteins with similar domain compositions

  • Motif Identification: Search for catalytic signatures consistent with its EC classification (3.6.5.-)

  • Post-translational Modification Prediction: Identify potential regulatory sites (phosphorylation, glycosylation) that may influence function

• Structural Bioinformatics Approaches:

  • Homology Modeling: Generate 3D structural models based on related proteins with solved structures

  • Molecular Dynamics Simulations: Analyze conformational dynamics to identify potential substrate binding pockets

  • Protein-Protein Docking: Predict interactions with potential partners identified in experimental studies

  • Active Site Analysis: Characterize catalytic residues and substrate specificity determinants

• Evolutionary Analysis:

  • Phylogenetic Profiling: Compare SEY1 distribution across fungal lineages, correlating with symbiotic lifestyle

  • Selection Pressure Analysis: Calculate dN/dS ratios to identify regions under purifying or positive selection

  • Synteny Analysis: Examine conservation of genomic context to identify functionally linked genes

  • Horizontal Gene Transfer Assessment: Evaluate evidence for potential acquisition from non-fungal sources

• Comparative Genomics:

  • Pan-Genome Analysis: Compare SEY1 presence/absence and sequence conservation across multiple Laccaria species

  • Expression Correlation Networks: Identify genes with similar expression patterns across different conditions

  • Mycorrhizal vs. Saprotrophic Fungi Comparison: Determine if SEY1 represents a symbiosis-specific innovation

• Integration with Experimental Data:

  • Metaproteomic Data Mining: Search for SEY1 expression evidence in field-collected ectomycorrhizal samples

  • Transcriptomic Data Integration: Analyze SEY1 expression patterns across developmental stages and symbiotic states

  • Network Inference: Position SEY1 within predicted functional interaction networks

This multi-layered bioinformatic analysis provides a comprehensive framework for generating testable hypotheses about SEY1 function that can guide experimental design.

What are common challenges in working with recombinant SEY1 protein and how can they be addressed?

Researchers working with recombinant SEY1 protein may encounter several technical challenges. The following troubleshooting guide provides methodological solutions:

These methodological approaches provide systematic solutions to common technical challenges, ensuring robust and reproducible research outcomes when working with recombinant SEY1 protein.

How can researchers distinguish between direct and indirect effects of SEY1 in functional studies?

Distinguishing between direct and indirect effects is crucial for accurate interpretation of SEY1 function. Implement these methodological approaches to establish causality:

• Temporal Resolution Studies:

  • Time-Course Experiments: Monitor phenotypic changes at fine time intervals after SEY1 perturbation

  • Inducible Expression Systems: Utilize promoters that allow precise temporal control of SEY1 expression

  • Rapid Protein Inactivation: Employ techniques such as auxin-inducible degron tags for acute protein depletion

  • Analytical Framework: Plot primary responses (direct) that appear rapidly versus secondary responses (indirect) that emerge later

• Genetic Interaction Analysis:

  • Epistasis Testing: Create double mutants with genes in putative SEY1 pathways to determine hierarchical relationships

  • Suppressor Screens: Identify mutations that rescue SEY1 loss-of-function phenotypes

  • Synthetic Lethality Mapping: Identify genes that become essential only in SEY1-deficient backgrounds

  • Interpretation Guidelines: Direct interactions typically show strong genetic interactions

• Biochemical Evidence for Direct Effects:

  • In Vitro Reconstitution: Demonstrate SEY1 activity with purified components

  • Substrate Modification Analysis: Directly measure changes to putative substrates using mass spectrometry

  • Structure-Function Correlations: Design mutations that specifically disrupt particular activities

  • Analytical Controls: Include catalytically inactive SEY1 variants as negative controls

• Proximity-Based Methods:

  • BioID or TurboID: Identify proteins in close proximity to SEY1 in living cells

  • Proximity Ligation Assays: Visualize and quantify molecular proximities in situ

  • FRET-Based Sensors: Develop sensors to detect direct SEY1-substrate interactions

  • Data Integration: Combine with interaction data from yeast two-hybrid or co-immunoprecipitation studies

• Pharmacological Approaches:

  • Specific Inhibitors: Apply selective inhibitors of SEY1 activity and monitor rapid responses

  • Bypass Experiments: Artificially activate downstream pathways in SEY1-deficient backgrounds

  • Chemical-Genetic Profiling: Compare profiles of SEY1 mutants with chemical perturbations

  • Control Recommendations: Include dose-response analyses to strengthen causal inferences

This comprehensive methodological framework provides multiple lines of evidence to differentiate direct SEY1 effects from downstream consequences, enabling more precise functional characterization.

What are important considerations for experimental design when studying SEY1 in the context of symbiotic interactions?

When investigating SEY1 within symbiotic contexts, researchers must address several critical experimental design considerations:

• Physiologically Relevant Conditions:

  • Growth Media Composition: Use defined media that mimics the rhizosphere environment rather than rich laboratory media

  • Temperature and pH Control: Maintain conditions that reflect natural soil environments (typically 18-22°C, pH 4.5-6.5)

  • Light Cycle Regulation: Implement appropriate photoperiods for plant partners (16h light/8h dark)

  • Experimental Timeline: Allow sufficient time for complete symbiotic development (typically 3-4 weeks for ectomycorrhiza formation)

• Appropriate Controls and Comparisons:

  • Multiple Fungal Genotypes: Include both wildtype and at least two independent SEY1-modified lines

  • Plant Genotype Variation: Test multiple plant genotypes/species to avoid host-specific artifacts

  • Non-Symbiotic Controls: Include free-living fungal cultures to differentiate symbiosis-specific responses

  • Mock Inoculations: Perform equivalent handling procedures without fungal partners for plant-only controls

• Comprehensive Phenotypic Analysis:

  • Quantitative Metrics: Measure percentage of colonized root tips , Hartig net depth, and mantle thickness

  • Multi-Scale Imaging: Combine macroscopic observation with microscopic analysis of symbiotic interfaces

  • Physiological Parameters: Assess nutrient transfer efficiency using isotope labeling techniques

  • Molecular Markers: Monitor expression of established symbiosis marker genes in both partners

• Statistical Robustness:

  • Biological Replicates: Use minimum n=5 biological replicates per condition

  • Technical Replicates: Include 3+ technical replicates for molecular analyses

  • Power Analysis: Pre-determine sample sizes needed to detect biologically relevant effect sizes

  • Appropriate Statistical Tests: Apply mixed-effects models to account for technical and biological variation

• Multi-Organism Interactions:

  • Axenic Systems: Initially establish SEY1 function in sterile, controlled systems

  • Synthetic Communities: Gradually increase complexity by adding defined microbial community members

  • Natural Soil Testing: Validate findings in more complex, natural substrates

  • Field Validation: When possible, confirm laboratory findings in natural settings

This systematic approach ensures experimental designs that yield robust, reproducible findings about SEY1's role in symbiotic interactions while minimizing confounding variables and artifacts.

What emerging technologies could advance our understanding of SEY1 function in Laccaria bicolor?

Several cutting-edge technologies hold promise for elucidating SEY1 function in greater detail:

• CRISPR-Based Technologies:

  • Base Editing: Introduce precise amino acid substitutions without double-strand breaks

  • CRISPRi/CRISPRa: Deploy systems for tunable gene repression or activation

  • Prime Editing: Enable precise genome modifications for detailed structure-function studies

  • Application Potential: Generate comprehensive allelic series to map functional domains with unprecedented precision

• Single-Cell Technologies:

  • Single-Cell RNA-Seq: Profile transcriptional heterogeneity within fungal colonies

  • Spatial Transcriptomics: Map gene expression patterns across symbiotic interfaces

  • Single-Cell Proteomics: Detect cell-specific protein abundance patterns

  • Research Impact: Reveal cell-specific roles of SEY1 in specialized hyphal types during symbiosis

• Advanced Imaging Technologies:

  • Super-Resolution Microscopy: Visualize subcellular localization beyond diffraction limits

  • Light Sheet Microscopy: Perform long-term, low-phototoxicity imaging of living symbiotic structures

  • Correlative Light and Electron Microscopy (CLEM): Connect molecular localization with ultrastructural context

  • Implementation Strategy: Track SEY1 dynamics during key developmental transitions in symbiosis formation

• Protein Structure Determination:

  • Cryo-Electron Microscopy: Resolve SEY1 structure in near-native states

  • AlphaFold2 and RoseTTAFold: Apply AI-based structure prediction with increasing accuracy

  • Hydrogen-Deuterium Exchange Mass Spectrometry: Map protein interaction surfaces

  • Research Applications: Inform structure-guided mutagenesis and inhibitor design

• Systems Biology Approaches:

  • Multi-Omics Integration: Combine transcriptomics, proteomics, and metabolomics data

  • Network Inference Algorithms: Construct regulatory networks involving SEY1

  • Constraint-Based Metabolic Modeling: Predict metabolic consequences of SEY1 perturbation

  • Implementation Framework: Position SEY1 within the broader context of symbiotic signaling networks

These emerging technologies, when applied systematically to SEY1 research, promise to resolve current knowledge gaps and provide unprecedented insights into its molecular function and biological significance.

How might advances in our understanding of SEY1 contribute to broader ecological and agricultural applications?

Advances in SEY1 research could translate into significant ecological and agricultural applications through several mechanistic pathways:

• Enhanced Mycorrhizal Inoculation Technologies:

  • If SEY1 proves critical for symbiotic establishment, manipulating its expression could potentially enhance colonization efficiency in forestry and agriculture

  • Understanding SEY1 regulation might enable development of environmental triggers that promote mycorrhization under field conditions

  • This knowledge could parallel findings with LbGH5-CBM1, where expression levels directly impact colonization success

• Improved Plant Growth and Stress Resistance:

  • Similar to how PtSSP1 overexpression increased ectomycorrhiza formation with L. bicolor , SEY1 modulation might enhance symbiotic benefits

  • Enhanced nutrient acquisition through optimized mycorrhizal associations could reduce fertilizer requirements

  • Symbiosis-mediated stress tolerance mechanisms might be strengthened through targeted SEY1 manipulation

• Forest Ecosystem Restoration:

  • Optimized mycorrhizal inoculum incorporating SEY1 knowledge could improve seedling establishment in degraded soils

  • Enhanced understanding of host-specificity determinants might allow tailored inoculation strategies for different forest types

  • Monitoring SEY1 expression could potentially serve as a molecular indicator of successful mycorrhizal establishment in restoration projects

• Climate Change Adaptation Strategies:

  • If SEY1 functions in stress response pathways, this knowledge could inform selection of fungal strains with enhanced climate resilience

  • Carbon sequestration efforts could benefit from optimized mycorrhizal associations that maximize belowground carbon allocation

  • Understanding SEY1's role in nutrient cycling could inform models of forest ecosystem responses to changing climatic conditions

• Biotechnological Applications:

  • If SEY1 exhibits unique enzymatic properties, it could potentially be harnessed for industrial applications

  • Protein engineering based on structure-function relationships could yield optimized variants for specific applications

  • Knowledge of SEY1 regulation might inform development of biosensors for monitoring environmental conditions relevant to forest health

These potential applications highlight how fundamental research on SEY1 could translate into practical solutions for pressing ecological and agricultural challenges, similar to applications being developed from other L. bicolor proteins like LbGH5-CBM1 and the PtSSP1-LbGAL4-like interaction system .