Recombinant Sclerotinia sclerotiorum Assembly factor cbp4 (cbp4)

What expression systems are commonly used for recombinant production of cbp4?

The most commonly used expression system for recombinant production of S. sclerotiorum cbp4 is Escherichia coli. According to available data, the full-length protein (amino acids 1-125) has been successfully expressed in E. coli with an N-terminal His tag . This bacterial expression system provides several advantages for research applications:

• Rapid growth and high protein yields

• Well-established protocols for transformation and protein induction

• Relatively low cost compared to eukaryotic expression systems

• Compatibility with various affinity tags for purification

The recombinant cbp4 is typically expressed with a His tag to facilitate purification through nickel affinity chromatography . While E. coli is the predominant system, researchers working on specific applications might consider alternative expression systems if:

• Post-translational modifications are required

• Protein solubility issues are encountered

• Functional studies demand a eukaryotic cellular environment

What are the optimal storage conditions for recombinant cbp4 protein?

Proper storage of recombinant cbp4 protein is critical for maintaining its stability and biological activity. Based on supplier recommendations, the following storage guidelines should be followed :

It is strongly recommended to avoid repeated freeze-thaw cycles as they can lead to protein denaturation and loss of activity . When receiving lyophilized protein, it should be briefly centrifuged prior to opening to bring the contents to the bottom of the vial .

How can researchers verify the functional activity of recombinant cbp4 protein in experimental settings?

Verifying the functional activity of recombinant cbp4 requires understanding its native role in cytochrome b mRNA processing and designing appropriate activity assays. While specific activity assays for cbp4 are not directly described in the provided search results, researchers can employ several approaches to assess functionality:

• RNA-binding assays: Since cbp4 is involved in mRNA processing, electrophoretic mobility shift assays (EMSA) can be used to detect protein-RNA interactions using cytochrome b mRNA sequences.

• In vitro mRNA processing assays: Researchers can develop assays using mitochondrial extracts supplemented with recombinant cbp4 to observe changes in cytochrome b mRNA processing efficiency.

• Complementation studies: Expression of recombinant cbp4 in cbp4-deficient fungal mutants to assess restoration of cytochrome b mRNA processing and mitochondrial function.

• Proteomic interaction studies: Identification of cbp4 binding partners through co-immunoprecipitation or pull-down assays using the His-tagged recombinant protein.

• Comparative activity studies: Measure activity differences between wild-type and mutated versions of recombinant cbp4 to identify critical functional residues.

For all functional assays, proper controls must be included to distinguish between specific activity and non-specific effects. These might include:

• Heat-denatured cbp4 protein

• Unrelated proteins of similar size and charge

• Buffer-only controls

What approaches can be used to investigate the role of cbp4 in Sclerotinia sclerotiorum pathogenicity?

Investigating the role of cbp4 in S. sclerotiorum pathogenicity requires a multidisciplinary approach combining molecular genetics, biochemistry, and plant pathology techniques:

• Gene knockout or knockdown studies: Generate cbp4 deletion or silenced mutants in S. sclerotiorum and assess changes in:

  • Virulence on host plants

  • Sclerotia formation (fungal survival structures)

  • Apothecia development (reproductive structures)

  • Mycelial growth and morphology

• Overexpression studies: Create cbp4 overexpression strains to observe phenotypic changes related to pathogenicity.

• Transcriptome analysis: Compare gene expression profiles of wild-type and cbp4 mutant strains during infection to identify downstream pathways affected by cbp4.

• Plant infection assays: Conduct controlled infections of host plants (e.g., soybean, canola) with wild-type and cbp4-modified strains. The stem test protocol described for canola can be adapted for such studies .

• Mitochondrial function analysis: Since cbp4 is involved in cytochrome b mRNA processing, investigate the relationship between mitochondrial function and virulence by measuring:

  • Oxygen consumption rates

  • ATP production

  • ROS generation

  • Expression of other mitochondrial genes

When designing plant infection studies, researchers should follow established protocols similar to those used for evaluating disease resistance. For example, the stem test protocol involves:

• Proper experimental design with adequate replication

• Uniform plant establishment

• Appropriate growth conditions

• Randomized complete block design

• Standardized disease assessment methods

How does the structure-function relationship of cbp4 compare with homologous proteins in other fungal species?

Understanding the structure-function relationship of cbp4 requires comparative analysis with homologous proteins from related fungal species. Although the search results don't provide explicit structural data for cbp4, researchers can adopt the following approaches:

• Sequence alignment and phylogenetic analysis: Compare cbp4 sequences across different fungal species to identify:

  • Conserved domains

  • Variable regions

  • Evolutionary relationships

  Particular attention should be paid to:

  • The N-terminal region (amino acids 1-40), which may contain targeting sequences

  • The central region (amino acids 41-100), likely containing functional domains

  • The C-terminal region (amino acids 101-125), which may be involved in protein-protein interactions

• Structural prediction and modeling: Use bioinformatics tools to predict secondary and tertiary structures, then compare these with solved structures of homologous proteins.

• Domain mapping: Create truncated or chimeric proteins to identify functional domains through complementation studies.

• Mutational analysis: Introduce point mutations at conserved residues to assess their impact on protein function.

• Comparative functional assays: Test complementation of cbp4 function across species by expressing homologs from different fungi in a cbp4-deficient S. sclerotiorum strain.

This comparative approach can reveal insights into how cbp4 function has evolved across fungal species and potentially identify structural features that correlate with pathogenicity in plant pathogens.

What purification strategies yield the highest purity and activity for recombinant cbp4?

Purification of recombinant cbp4 requires careful optimization to maintain protein integrity and activity. Based on available information, the following purification strategy is recommended:

• Expression optimization:

  • Express in E. coli with an N-terminal His tag

  • Use optimal induction conditions (temperature, IPTG concentration, induction time)

  • Consider codon optimization for the E. coli expression system

• Cell lysis and initial clarification:

  • Use gentle lysis methods to preserve protein activity

  • Include protease inhibitors in lysis buffers

  • Clarify lysate by centrifugation (15,000-20,000 × g for 30 minutes)

• Affinity chromatography (primary purification):

  • Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin

  • Use imidazole gradient elution (20-250 mM) to reduce non-specific binding

  • Collect fractions and analyze by SDS-PAGE

• Secondary purification (if higher purity is required):

  • Size exclusion chromatography

  • Ion exchange chromatography

• Buffer exchange and concentration:

  • Exchange into storage buffer (Tris/PBS-based buffer, pH 8.0)

  • Add stabilizers (6% trehalose or 50% glycerol)

• Quality control:

  • SDS-PAGE analysis (purity should be >90%)

  • Western blot confirmation

  • Activity assays

  • Mass spectrometry verification

The purified protein should be aliquoted into small volumes for single use and stored at -20°C or -80°C for long-term storage . Working aliquots can be kept at 4°C for up to one week to avoid freeze-thaw cycles.

What are the most effective experimental designs for studying cbp4's role in fungal mitochondrial function?

Designing effective experiments to study cbp4's role in fungal mitochondrial function requires careful consideration of both in vitro and in vivo approaches:

• In vitro mitochondrial assays:

  • Isolated mitochondrial studies: Prepare mitochondria from wild-type and cbp4 mutant strains to compare:

    • Oxygen consumption rates

    • ATP synthesis efficiency

    • Membrane potential

    • Cytochrome c oxidase activity

  • RNA processing assays: Develop in vitro systems to study cytochrome b mRNA processing using:

    • Mitochondrial extracts

    • Purified recombinant cbp4

    • Synthetic or in vitro transcribed cytochrome b mRNA substrates

• In vivo approaches:

  • Genetic manipulation strategies:

    • Gene deletion (knockout)

    • Conditional expression (using inducible promoters)

    • Site-directed mutagenesis of key residues

    • Fluorescent protein tagging for localization studies

  • Phenotypic characterization:

    • Growth rate under different carbon sources

    • Resistance to mitochondrial stress inducers

    • Morphological changes in mitochondria (using fluorescence microscopy)

    • Transcriptome analysis focusing on mitochondrial genes

• Recommended experimental design:

  • Include appropriate controls (wild-type, empty vector, unrelated protein)

  • Use multiple independent transformants/mutants

  • Perform biological replicates (minimum 3)

  • Blind scoring of phenotypic data when possible

  • Use statistical analysis to validate findings (ANOVA, t-tests)

  • Validate key findings using complementary techniques

By combining these approaches, researchers can establish the specific role of cbp4 in mitochondrial function and determine how this relates to the biology and pathogenicity of S. sclerotiorum.

How can researchers effectively use recombinant cbp4 protein as a tool for studying Sclerotinia sclerotiorum pathogenesis?

Recombinant cbp4 protein can serve as a valuable research tool for investigating S. sclerotiorum pathogenesis through several experimental approaches:

• Antibody production and immunolocalization:

  • Generate anti-cbp4 antibodies using purified recombinant protein

  • Use these antibodies for:

    • Immunolocalization during different stages of infection

    • Western blot analysis of cbp4 expression levels

    • Immunoprecipitation to identify interacting partners

• Protein-protein interaction studies:

  • Identify fungal or plant proteins that interact with cbp4 using:

    • Yeast two-hybrid screening

    • Pull-down assays with His-tagged recombinant cbp4

    • Surface plasmon resonance for measuring binding kinetics

  • Characterize these interactions to understand pathogenesis mechanisms

• Functional complementation assays:

  • Introduce recombinant cbp4 into cbp4-deficient mutants via:

    • Transformation

    • Protein delivery systems

  • Assess restoration of wild-type phenotypes

• Development of inhibitors or targeting molecules:

  • Screen for compounds that specifically bind to cbp4

  • Evaluate these compounds for antifungal activity

  • This approach aligns with research on novel fungicides, similar to the exploration of peptide-based biofungicides mentioned for white mold management

• Experimental design considerations:

  • Include proper controls (denatured protein, buffer-only, unrelated proteins)

  • Use standardized infection models like the stem test protocol

  • Maintain consistent experimental conditions

  • Employ statistical analysis to evaluate significance of results

When using recombinant cbp4 for these applications, researchers should verify that the protein maintains its native conformation and activity. The addition of tags (such as His tags) should be considered when interpreting results, as they may occasionally affect protein behavior in certain assays.

What are common challenges in recombinant cbp4 expression and how can they be addressed?

Researchers working with recombinant cbp4 may encounter several challenges during protein expression and purification. The following table outlines common issues and recommended solutions:

When troubleshooting expression issues, a systematic approach is recommended:

• First, optimize expression conditions using small-scale cultures

• Verify protein expression by SDS-PAGE and Western blot

• Test solubility using different lysis methods

• Optimize purification conditions before scaling up

• Validate protein quality through activity assays

For proteins that remain challenging to express in E. coli, alternative systems such as yeast, insect cells, or cell-free expression systems could be considered, although these may require additional optimization.

How can researchers design effective controls for experiments investigating cbp4 function in pathogenicity?

Designing appropriate controls is critical for experiments investigating cbp4 function in pathogenicity. The following control strategies should be implemented:

• Genetic Controls:

  • Wild-type strain: Essential baseline control for all experiments

  • Empty vector transformants: Control for transformation effects

  • Complemented mutants: cbp4 mutants with the wild-type gene reintroduced

  • Point mutants: Strains with specific amino acid changes to identify functional residues

• Experimental Controls:

  • Multiple independent transformants/mutants: To control for position effects

  • Environmental controls: Standardized growth conditions and infection protocols

  • Host plant controls: Include known susceptible and resistant varieties

  • Timing controls: Sample collection at consistent time points post-infection

• Biochemical Controls:

  • Protein controls: For recombinant protein experiments:

    • Heat-inactivated protein

    • Unrelated proteins of similar size/structure

    • Wild-type protein vs. mutated versions

  • Antibody controls: For immunological studies:

    • Pre-immune serum

    • Isotype controls

    • Blocking peptide controls

• Control Design for Plant Infection Studies:

  • Follow established protocols like those used in the stem test procedure

  • Include susceptible check lines (e.g., 45H29 for canola)

  • Use a randomized complete block design with sufficient replication (minimum 6 replications)

  • Implement consistent disease rating systems

  • Consider environmental factors that may affect disease development

What are promising research areas for further understanding cbp4's role in fungal biology and plant disease?

Several promising research directions could significantly advance our understanding of cbp4's role in fungal biology and plant disease:

• Structural Biology Approaches:

  • Determine the three-dimensional structure of cbp4 using X-ray crystallography or cryo-EM

  • Identify RNA-binding domains and characterize protein-RNA interactions

  • Investigate structural changes during protein function

• Systems Biology Integration:

  • Conduct comprehensive multi-omics studies (transcriptomics, proteomics, metabolomics) in wild-type vs. cbp4 mutants

  • Develop predictive models of metabolic networks affected by cbp4 function

  • Map the position of cbp4 in cellular signaling pathways

• Comparative Genomics and Evolution:

  • Compare cbp4 function across diverse fungal species

  • Investigate evolutionary relationships between cbp4 and virulence

  • Identify selective pressures on cbp4 in plant pathogens

• Translational Research Applications:

  • Develop cbp4-targeting antifungal compounds

  • Explore cbp4 as a biomarker for early detection of Sclerotinia infection

  • Investigate potential for generating plant resistance through interference with cbp4 function

• Technological Innovations:

  • Apply CRISPR-Cas9 genome editing for precise modification of cbp4

  • Develop biosensors based on cbp4 for monitoring fungal metabolism

  • Utilize single-cell approaches to understand cbp4 expression heterogeneity

These research areas could be especially valuable when integrated with current efforts to develop control strategies for Sclerotinia diseases, such as the development of peptide-based biofungicides with novel multi-site modes of action mentioned in the search results .