Recombinant Chrysanthemum coronarium Thaumatin-like protein 5

What is Chrysanthemum coronarium thaumatin-like protein 5 and how is it classified?

Chrysanthemum coronarium thaumatin-like protein 5 (CTLP-5) is a pathogenesis-related (PR) protein belonging to the PR-5 family. It is characterized by a molecular weight of approximately 29.9 kD and an isoelectric point (pI) of 4.75, as determined by two-dimensional gel electrophoresis and molecular weight calibration. CTLP-5 is one of six thaumatin-like proteins identified in garland chrysanthemum (Chrysanthemum coronarium) that accumulate specifically in response to phytoplasma infection. These proteins share high sequence similarities with other known PR-5 proteins, such as those found in Arabidopsis thaliana and other plant species, suggesting evolutionary conservation of this protein family across plant taxa .

How does phytoplasmal infection induce CTLP-5 expression in Chrysanthemum coronarium?

Phytoplasmal infection, particularly by onion yellows phytoplasma, triggers a defense response in Chrysanthemum coronarium that leads to the specific accumulation of CTLP-5. The induction mechanism appears to be time-dependent, with significant accumulation observed approximately 45 days post-infection, coinciding with the development of severe symptoms including stunting, yellowing, and proliferation in infected plants. The induction pathway likely involves plant defense signaling cascades similar to those activated during other pathogen infections, though the exact signaling molecules and transcriptional regulators involved remain to be fully characterized. Notably, this accumulation is observed across multiple plant tissues including leaves, apical shoots, axillary shoots, and stems, indicating a systemic response to infection .

What methods are typically used to isolate and purify recombinant CTLP-5 from Chrysanthemum coronarium?

The isolation and purification of recombinant CTLP-5 typically involves a multi-step process:

• Protein Extraction: Fresh plant tissue is homogenized with chilled phosphate buffer (pH 8.0 containing 37 mM K₂HPO₄, 1.7 mM KH₂PO₄, and 400 mM NaCl) followed by centrifugation at 12,000 g for 10 minutes.

• Precipitation: Trichloroacetic acid is added to the supernatant to achieve a final concentration of 10%, followed by incubation at 4°C for 20 minutes and centrifugation.

• Solubilization: The precipitate is suspended in lysis buffer, sonicated, and pH-adjusted to 7.0 with NaOH.

• Separation: Two-dimensional gel electrophoresis with isoelectric focusing in the first dimension and SDS-PAGE in the second dimension is used to separate proteins.

• Identification: Protein spots are visualized using Coomassie Brilliant Blue staining and analyzed using software such as ImageMaster 2D elite.

• Sequence Analysis: For N-terminal sequencing, proteins are electroblotted onto PVDF membranes, excised, and subjected to automated Edman degradation using a gas phase protein sequencer .

For recombinant production, expression systems similar to those used for other thaumatin-like proteins may be employed, such as Komagataella phaffii (formerly Pichia pastoris), which has been successfully used for the expression of similar proteins .

What are the structural and functional differences between naturally occurring and recombinant CTLP-5, and how do these differences impact experimental applications?

Recombinant CTLP-5 may exhibit structural and functional differences compared to its naturally occurring counterpart due to several factors:

Structural Considerations:

• Post-translational modifications: Recombinant systems may not reproduce the same glycosylation patterns found in planta. Natural CTLP-5 likely contains specific glycosylation patterns that could affect protein folding, stability, and activity.

• Disulfide bond formation: PR-5 proteins typically contain multiple disulfide bonds that are essential for their structural integrity. The correct formation of these bonds in recombinant systems depends on the expression host's capacity for proper protein folding.

• N-terminal processing: As observed in studies of similar proteins, the N-terminal amino acid sequences of CTLPs share high similarity with other PR-5 proteins, suggesting conserved signal peptide cleavage sites that must be properly processed in recombinant systems .

Functional Implications:

• Antifungal activity: Any variations in structure could affect the protein's antifungal properties, which are characteristic of many PR-5 proteins.

• Thermal stability: Recombinant proteins may display different melting points compared to native proteins, similar to observations in wine-related TLPs where thermal stability is crucial for functionality .

• Aggregation potential: Structural differences may alter the protein's tendency to form aggregates, which can affect both experimental applications and functional assays.

These differences should be carefully considered when designing experiments using recombinant CTLP-5, potentially requiring validation of the recombinant protein against naturally induced proteins extracted from phytoplasma-infected plants.

How can researchers optimize heterologous expression systems for high-yield production of functionally active recombinant CTLP-5?

Optimizing heterologous expression of recombinant CTLP-5 requires careful consideration of several factors:

Expression System Selection:

• Yeast-based systems like Komagataella phaffii have proven effective for similar proteins, offering eukaryotic post-translational modifications while maintaining high expression levels .

• Plant-based expression systems may provide more authentic post-translational modifications but often with lower yields.

• Bacterial systems like E. coli offer high yields but may struggle with proper folding and disulfide bond formation of plant PR proteins.

Expression Optimization Parameters:

• Codon optimization: Adapting the CTLP-5 coding sequence to the preferred codon usage of the expression host can significantly improve translation efficiency.

• Signal peptide selection: Replacing the native signal peptide with one optimized for the expression host can enhance secretion and proper processing.

• Induction conditions: Temperature, pH, inducer concentration, and induction timing should be systematically optimized to balance protein yield with proper folding.

• Growth media formulation: Supplementation with specific ions or precursors may enhance expression and proper folding.

Purification Strategy:

• Affinity tags: Strategic placement of tags (His, GST, etc.) that do not interfere with protein folding or function can facilitate purification.

• Chromatography sequence: A multi-step purification strategy typically involving initial capture by affinity chromatography followed by polishing steps using ion exchange and size exclusion chromatography.

Functional Validation:

• Activity assays comparing the recombinant protein to naturally occurring CTLP-5 should be performed to confirm proper folding and functionality.

• Structural analyses including circular dichroism spectroscopy and thermal stability assessments can provide insights into protein conformation.

What is the relationship between the amino acid sequence, tertiary structure, and antifungal activity of CTLP-5 compared to other PR-5 proteins?

The relationship between sequence, structure, and function of CTLP-5 involves several key elements:

Sequence-Structure Relationships:

• N-terminal sequence analysis has revealed high similarity between CTLP-5 and other PR-5 proteins, particularly in regions associated with the characteristic thaumatin fold .

• PR-5 proteins typically contain 16 conserved cysteine residues that form 8 disulfide bridges, creating a highly stable tertiary structure resistant to proteolysis and thermal denaturation.

• Three-dimensional modeling based on sequence homology would likely reveal the characteristic three-domain architecture seen in other PR-5 proteins, with a prominent central domain containing a β-sandwich motif.

Structure-Function Correlations:

• The antifungal activity of PR-5 proteins is often attributed to specific acidic clefts formed by the tertiary structure, which can disrupt fungal cell membranes through osmotic mechanisms.

• Variations in surface charge distribution, particularly in the acidic cleft region, may account for differences in antifungal specificity between CTLP-5 and other PR-5 proteins.

• The thermal stability of PR-5 proteins, including CTLP-5, is a critical functional property related to their compact disulfide-rich structure. This property is particularly relevant in environments like wine where protein stability affects product quality .

Evolutionary Implications:

• Sequence alignment between CTLP-5 and PR-5 proteins from other species reveals evolutionary conservation of functional domains, suggesting fundamental importance to plant defense mechanisms.

• The accumulation of multiple isoforms of thaumatin-like proteins (CTLP-1 through CTLP-6) in infected Chrysanthemum coronarium suggests possible functional specialization or redundancy, potentially providing broader protection against various pathogens .

A comprehensive understanding of these structure-function relationships would require experimental approaches including site-directed mutagenesis, crystallographic analysis, and functional assays against various fungal pathogens.

What are the most effective protocols for analyzing CTLP-5 expression patterns in response to different phytoplasma strains?

Effective analysis of CTLP-5 expression patterns in response to different phytoplasma strains requires a multi-faceted approach:

Infection and Sampling Design:

• Standardized inoculation procedures using leafhopper vectors carrying different phytoplasma strains should be established to ensure consistent infection.

• Time-course sampling is critical, as research has shown that significant CTLP accumulation occurs approximately 45 days post-infection, coinciding with symptom development .

• Multiple tissue types (leaves, apical shoots, axillary shoots, and stems) should be sampled separately to assess tissue-specific expression patterns.

Protein Expression Analysis:

• Two-dimensional gel electrophoresis remains the gold standard for separating and visualizing CTLPs, allowing detection of post-translational modifications and protein isoforms .

• Western blotting using antibodies raised against conserved PR-5 epitopes or CTLP-5-specific peptides can provide quantitative data on protein accumulation.

• Mass spectrometry approaches, particularly liquid chromatography-tandem mass spectrometry (LC-MS/MS), offer higher sensitivity and the ability to distinguish between highly similar protein isoforms.

Transcriptional Analysis:

• Quantitative RT-PCR targeting CTLP-5 mRNA provides data on transcriptional regulation.

• RNA-Seq analysis offers a broader view of the transcriptional landscape, revealing potential co-regulated genes and regulatory networks involved in the defense response.

Data Integration and Analysis:

• Statistical methods should be employed to assess the significance of observed changes across different phytoplasma strains.

• Multivariate analysis techniques can help identify patterns in protein expression across different tissues, time points, and phytoplasma strains.

• Correlation analyses between symptom severity, phytoplasma titer, and CTLP-5 levels can provide insights into the biological significance of CTLP-5 induction.

How can researchers accurately differentiate between the six identified CTLP isoforms (CTLP-1 through CTLP-6) in experimental samples?

Differentiating between the six CTLP isoforms requires specialized techniques due to their structural similarities:

Two-Dimensional Electrophoresis:

• 2D-E separation based on both isoelectric point and molecular weight provides initial differentiation of the six isoforms, as they have distinct pI/MW coordinates: CTLP-1 (pI 7.35, MW 29.6 kD), CTLP-2 (pI 7.35, MW 21.0 kD), CTLP-3 (pI 6.26, MW 28.8 kD), CTLP-4 (pI 5.40, MW 29.0 kD), CTLP-5 (pI 4.75, MW 29.9 kD), and CTLP-6 (pI 4.75, MW 27.6 kD) .

• Differential staining methods, including phosphoprotein-specific and glycoprotein-specific stains, can help identify post-translational modifications that distinguish between isoforms.

Mass Spectrometry Approaches:

• Peptide mass fingerprinting following tryptic digestion can identify unique peptide signatures for each isoform.

• Multiple reaction monitoring (MRM) mass spectrometry using isoform-specific peptides allows for targeted quantification of individual CTLPs in complex samples.

• Top-down proteomics approaches that analyze intact proteins can resolve subtle differences between isoforms that share high sequence similarity.

Immunological Methods:

• Development of isoform-specific antibodies targeting unique epitopes in each CTLP isoform, particularly in regions where N-terminal sequences diverge.

• Epitope mapping to identify unique surface-exposed regions for each isoform that could serve as targets for specific antibodies.

Chromatographic Separation:

• Ion exchange chromatography exploiting the different isoelectric points of the CTLP isoforms.

• Hydrophobic interaction chromatography may reveal subtle differences in surface hydrophobicity between isoforms.

• Affinity chromatography using ligands that interact differentially with the various isoforms based on structural variations.

A combination of these approaches would provide the most reliable differentiation between CTLP isoforms in experimental samples.

What experimental approaches best elucidate the role of CTLP-5 in plant defense against phytoplasma infection?

To elucidate the role of CTLP-5 in plant defense against phytoplasma, researchers should consider the following experimental approaches:

In vivo Functional Studies:

• RNA interference (RNAi) or CRISPR/Cas9-mediated gene silencing/knockout of CTLP-5 in Chrysanthemum coronarium, followed by phytoplasma challenge to assess susceptibility changes.

• Overexpression of CTLP-5 in transgenic plants to evaluate enhanced resistance potential.

• Virus-induced gene silencing (VIGS) as a rapid approach for functional assessment in non-model plants like chrysanthemum.

In vitro Antimicrobial Activity Assessment:

• While direct testing against phytoplasma is challenging due to their uncultivable nature, recombinant CTLP-5 can be tested against surrogate microorganisms.

• Membrane permeabilization assays using artificial liposomes to assess the membrane-disrupting potential of CTLP-5.

• Growth inhibition assays against fungi and bacteria that may co-infect with phytoplasma to evaluate broader antimicrobial activity.

Localization and Interaction Studies:

• Immunolocalization of CTLP-5 in infected tissues to determine its spatial distribution relative to phytoplasma presence.

• Co-immunoprecipitation and yeast two-hybrid assays to identify potential protein-protein interactions with host or pathogen factors.

• Bimolecular fluorescence complementation (BiFC) to visualize protein interactions in planta.

Physiological and Biochemical Analyses:

• Evaluation of oxidative stress markers, callose deposition, and other defense responses in relation to CTLP-5 expression.

• Analysis of phytohormone levels (salicylic acid, jasmonic acid, ethylene) to understand the signaling pathways involved in CTLP-5 induction.

• Metabolomic profiling to identify changes in plant metabolism associated with CTLP-5 accumulation during infection.

These complementary approaches would provide a comprehensive understanding of CTLP-5's role in plant defense against phytoplasma infection.

How do post-translational modifications affect the functional properties of recombinant CTLP-5, and how can these modifications be characterized?

Post-translational modifications (PTMs) significantly influence the functional properties of recombinant CTLP-5, and their characterization requires specialized analytical techniques:

Common PTMs in PR-5 Proteins:

• Glycosylation: N-linked and O-linked glycans can affect protein folding, stability, and immunogenicity.

• Disulfide bond formation: The pattern of disulfide bonding is critical for maintaining the characteristic PR-5 protein tertiary structure.

• Proteolytic processing: Proper cleavage of signal peptides and potential pro-domains is essential for protein activation.

• Phosphorylation: May regulate protein activity or interaction with other defense components.

Impact on Functional Properties:

• Thermal stability: Glycosylation patterns can significantly affect the melting temperature and aggregation properties of thaumatin-like proteins, similar to observations in wine-related TLPs .

• Antifungal activity: The specific pattern of PTMs may modulate the strength and specificity of antimicrobial activity.

• Protein-protein interactions: Surface modifications can alter interaction with other defense components or potential receptors.

• Subcellular localization: PTMs may contain targeting information directing the protein to specific cellular compartments.

Characterization Methods:

Strategies for Controlling PTMs in Recombinant Systems:

• Selection of appropriate expression hosts (mammalian, insect, yeast, plant) based on desired PTM patterns.

• Genetic modification of expression hosts to humanize glycosylation or eliminate undesired modifications.

• Culture condition optimization to influence PTM enzyme activity.

• In vitro enzymatic modification post-purification to achieve desired PTM profiles.

What comparative analyses reveal about the evolutionary relationships between Chrysanthemum coronarium TLPs and other plant PR-5 proteins?

Comparative analyses of Chrysanthemum coronarium TLPs and other plant PR-5 proteins reveal important evolutionary insights:

Sequence-Based Phylogenetic Analysis:

• N-terminal amino acid sequences of CTLP-1–4 share significant similarity with PR-5 proteins from diverse plant species including Arabidopsis thaliana and Prunus avium, suggesting conservation of core functional domains .

• Phylogenetic tree construction using full-length sequences would likely place CTLPs within distinct clades corresponding to their functional specialization (e.g., antifungal, membrane-permeabilizing, or signaling functions).

• Analysis of synonymous vs. non-synonymous substitution rates in coding sequences can identify regions under positive selection, potentially indicating adaptation to specific pathogen pressures.

Structural Comparisons:

• Homology modeling based on crystallized PR-5 proteins would likely reveal conservation of the characteristic thaumatin fold across plant families.

• Surface electrostatic potential mapping could identify conserved charged regions associated with antimicrobial activity.

• Analysis of conservation patterns in the acidic cleft region, which is associated with fungal membrane permeabilization in many PR-5 proteins.

Functional Domain Conservation:

• The presence of multiple CTLP isoforms (CTLP-1 through CTLP-6) with varying isoelectric points suggests functional diversification within Chrysanthemum coronarium, potentially providing broader defense capabilities .

• Comparison with functionally characterized PR-5 proteins from model plants could reveal conservation of specific activity-related motifs or domains.

Genomic Organization and Evolution:

• Analysis of gene structure (exon-intron organization) across species can provide insights into evolutionary mechanisms such as gene duplication, domain shuffling, or horizontal gene transfer.

• Promoter region analysis could reveal conservation of pathogen-responsive elements regulating expression.

• Investigation of synteny relationships might identify blocks of genes that have been conserved throughout evolution, suggesting functional interdependence.

These comparative analyses would not only illuminate the evolutionary history of PR-5 proteins but also provide insights into their adaptation to specific pathogen pressures, potentially guiding the development of improved plant defense strategies.

How might CTLP-5 be engineered to enhance its antifungal properties while maintaining stability and expression efficiency?

Engineering CTLP-5 for enhanced antifungal properties requires strategic modifications based on structure-function relationships:

Structure-Guided Rational Design:

• Acidic cleft enhancement: Modifications to increase negative charge density in the acidic cleft region, which is associated with membrane permeabilization in PR-5 proteins.

• Hydrophobic patch modifications: Alterations to surface-exposed hydrophobic residues that interact with fungal membranes to increase membrane affinity.

• Disulfide bond engineering: Introduction of additional disulfide bonds to enhance thermal stability while preserving the active conformation.

• Loop region optimization: Modification of flexible loop regions to improve interaction with target fungal components while preserving the core fold.

Directed Evolution Approaches:

• Error-prone PCR to generate libraries of CTLP-5 variants followed by screening for enhanced antifungal activity.

• DNA shuffling between CTLP-5 and other potent antifungal PR-5 proteins to create chimeric proteins with improved properties.

• Phage display selection to identify variants with enhanced binding to fungal cell wall components.

Fusion Protein Strategies:

• Creation of bifunctional proteins by fusing CTLP-5 with complementary antifungal peptides or proteins targeting different fungal structures.

• Addition of cell-penetrating peptides to enhance delivery to the site of action.

• Incorporation of controlled-release domains that activate the protein under specific conditions encountered during fungal infection.

Stability and Expression Optimization:

• Codon optimization for the expression host while avoiding rare codons in critical structural regions.

• Removal of protease recognition sites without affecting functional domains.

• Engineering glycosylation sites in non-critical regions to enhance solubility and stability.

• Surface charge distribution optimization to reduce aggregation propensity.

These engineering strategies should be implemented with careful consideration of their effects on protein folding, stability, and immunogenicity, particularly if the engineered proteins are intended for agricultural applications.

What research gaps exist in understanding the interactions between CTLP-5 and phytoplasma, and how might these be addressed?

Significant research gaps exist in understanding CTLP-5-phytoplasma interactions, primarily due to the uncultivable nature of phytoplasmas. These gaps and potential approaches to address them include:

Gap: Direct interaction mechanisms between CTLP-5 and phytoplasma components

• Approach: Heterologous expression of phytoplasma membrane proteins in surrogate systems (e.g., bacterial ghosts or liposomes) for interaction studies with purified CTLP-5.

• Approach: Yeast two-hybrid or bacterial two-hybrid screening using CTLP-5 as bait against a library of phytoplasma-encoded proteins.

• Approach: In silico molecular docking and simulation studies to predict potential interactions between CTLP-5 and known phytoplasma surface proteins.

Gap: Subcellular localization of CTLP-5 in relation to phytoplasma colonization sites

• Approach: Immunogold electron microscopy using CTLP-5-specific antibodies to visualize protein distribution in infected phloem tissue.

• Approach: Super-resolution microscopy of fluorescently tagged CTLP-5 in relation to phytoplasma cells in infected tissue sections.

• Approach: Laser capture microdissection of infected vs. uninfected phloem cells followed by proteomics to quantify CTLP-5 enrichment.

Gap: Temporal dynamics of CTLP-5 induction relative to phytoplasma multiplication

• Approach: Time-course studies correlating phytoplasma titer (determined by qPCR) with CTLP-5 levels (determined by Western blot or ELISA).

• Approach: Transcriptomics at different infection stages to identify regulatory networks controlling CTLP-5 expression.

• Approach: Reporter gene constructs driven by the CTLP-5 promoter to visualize induction dynamics in real-time during infection.

Gap: Functional significance of multiple CTLP isoforms during phytoplasma infection

• Approach: Selective silencing of individual CTLP genes to determine their specific contributions to defense.

• Approach: Biochemical characterization of each CTLP isoform's activity against surrogate membrane systems.

• Approach: Comparative analysis of CTLP isoform induction patterns in response to different phytoplasma strains.

Gap: Systemic signaling leading to CTLP-5 induction in uninfected tissues

• Approach: Grafting experiments between infected and uninfected plant parts to study systemic CTLP-5 induction.

• Approach: Analysis of phloem exudate composition from infected plants to identify potential mobile signals.

• Approach: Transcriptomic and metabolomic profiling along the infection-to-systemic response continuum.

Addressing these gaps would significantly advance our understanding of the role of CTLP-5 in plant defense against phytoplasma infection and potentially inform new strategies for disease management.

What are the critical factors to consider when designing experiments to compare naturally induced versus recombinant CTLP-5 activity?

When comparing naturally induced versus recombinant CTLP-5 activity, researchers should consider these critical factors:

Protein Source and Preparation:

• Natural CTLP-5 isolation: Standardized extraction methods from phytoplasma-infected Chrysanthemum coronarium plants at consistent infection stages (approximately 45 days post-infection when protein accumulation is significant) .

• Recombinant production: Selection of appropriate expression systems (yeast, insect, plant) that can reproduce key post-translational modifications, particularly glycosylation patterns and disulfide bond formation .

• Purification strategy: Comparable purification protocols that maintain native conformation and activity, avoiding harsh conditions that might differentially affect natural versus recombinant proteins.

Protein Characterization:

• Structural integrity: Circular dichroism spectroscopy and thermal stability analysis to confirm comparable secondary structure elements and melting temperatures.

• Post-translational modification analysis: Glycoprotein staining, mass spectrometry, and other PTM-specific analyses to document differences in modification patterns.

• Oligomerization state: Size exclusion chromatography and analytical ultracentrifugation to determine if proteins form similar quaternary structures.

Activity Assays:

• Fungal growth inhibition: Standardized assays against a panel of fungal species using multiple metrics (growth rate, hyphal morphology, membrane integrity).

• Membrane permeabilization: Liposome leakage assays using membrane compositions relevant to target organisms.

• Thermal stability assays: Heat treatment followed by activity measurements to compare functional resilience.

• pH response profiles: Activity measurements across a range of pH values relevant to the plant apoplast and phytoplasma environment.

Experimental Controls:

• Concentration normalization: Careful protein quantification to ensure equivalent molar concentrations in activity comparisons.

• Storage stability: Assessment of activity retention during storage to ensure differences aren't artifacts of differential stability.

• Unrelated control proteins: Inclusion of non-PR proteins produced in the same systems to control for system-specific effects.

Data Analysis and Reporting:

• Statistical rigor: Appropriate statistical methods to determine the significance of observed differences.

• Multiple batch testing: Analysis of proteins from multiple purification batches to account for batch-to-batch variability.

• Comprehensive reporting: Documentation of all relevant experimental parameters to enable reproducibility.

By carefully controlling these factors, researchers can make valid comparisons between naturally induced and recombinant CTLP-5, identifying any functional differences that might impact experimental applications or theoretical understanding.