Recombinant Magnaporthe oryzae Enolase-phosphatase E1 (UTR4)

What is Magnaporthe oryzae Enolase-phosphatase E1 (UTR4) and what is its role in the pathogen?

Enolase-phosphatase E1 (UTR4) is an enzyme from Magnaporthe oryzae (rice blast fungus, also known as Pyricularia oryzae), with the UniProt accession number A4RM80. Its recommended name is Enolase-phosphatase E1 (EC 3.1.3.77), alternatively known as 2,3-diketo-5-methylthio-1-phosphopentane phosphatase . While the specific role of UTR4 in M. oryzae pathogenicity hasn't been fully characterized in the provided research, the enzyme belongs to a class of proteins involved in metabolic pathways that may contribute to fungal survival and potentially to its virulence mechanisms during rice infection.

M. oryzae is known to secrete numerous effector proteins that facilitate infection of host plants, with most still awaiting functional characterization . The rice blast fungus forms specialized structures such as the biotrophic interfacial complex (BIC) during infection, which is essential for effector translocation into host cells . Understanding enzymes like UTR4 provides insight into the metabolic processes that may support these infection mechanisms.

What expression systems are available for producing recombinant UTR4?

Recombinant UTR4 can be produced using different expression systems, with the two most common being:

• E. coli expression system - Allows for high-yield bacterial expression of the protein, with product codes such as CSB-EP007674MPM .

• Baculovirus expression system - Enables expression in insect cells, potentially offering different post-translational modifications, with product codes such as CSB-BP007674MPM .

Both expression systems produce the full-length protein (amino acids 1-231) with >85% purity as determined by SDS-PAGE . The choice between expression systems depends on your specific research requirements, particularly regarding protein folding, post-translational modifications, and downstream applications.

What are the optimal storage conditions for maintaining UTR4 stability?

For optimal stability of recombinant UTR4, the following storage conditions are recommended:

It is strongly advised to avoid repeated freeze-thaw cycles as they can significantly reduce protein activity . For working with the protein, it's recommended to create small aliquots upon initial reconstitution to minimize the need for repeated thawing of the entire stock.

What is the recommended protocol for reconstituting recombinant UTR4?

The recommended reconstitution protocol for recombinant UTR4 involves the following steps:

• Briefly centrifuge the vial containing lyophilized protein to bring contents to the bottom.

• Reconstitute the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL.

• Add glycerol to a final concentration of 5-50% (with 50% being the standard recommendation) for storage stability.

• Prepare small working aliquots to avoid repeated freeze-thaw cycles.

• Store reconstituted aliquots according to the recommended temperature guidelines (-20°C to -80°C for long-term storage) .

This procedure ensures optimal protein stability and activity for downstream applications. The addition of glycerol serves as a cryoprotectant to prevent protein denaturation during freezing.

How can UTR4 be integrated into protoplast transient expression systems for functional studies?

For functional studies of fungal proteins like UTR4, protoplast transient expression systems have proven effective. Based on methodologies used for similar M. oryzae proteins, the following approach can be adapted:

• Clone the UTR4 gene from M. oryzae cDNA library using appropriate primers.

• Sub-clone into an entry vector such as pENTR™1A (Invitrogen).

• Transfer to a gateway-compatible expression vector like pIPKb002 for transient expression.

• Prepare plant protoplasts - For rice, cut 14-day-old etiolated seedling stems and sheaths into 1.5 mm strips and soak in protoplast isolation buffer for 3 hours in the dark with gentle shaking.

• Transfect recombinant vectors into the prepared protoplasts suspended in transfection buffer (concentration: 3.5 × 10^5 cells/mL).

• Detect expression using appropriate assays, such as the Luciferase Assay System if using a luciferase reporter .

This transient expression system allows for rapid functional analysis of UTR4 without the need for stable transformation, making it valuable for initial characterization studies.

What methods can be used to assess the enzymatic activity of recombinant UTR4?

To assess the enzymatic activity of recombinant UTR4 (Enolase-phosphatase E1), researchers can employ several methodologies:

• Phosphatase activity assay: Measure the release of inorganic phosphate from the substrate 2,3-diketo-5-methylthio-1-phosphopentane using colorimetric methods such as malachite green assay.

• Coupled enzyme assays: Design assays where the product of UTR4 activity serves as a substrate for a secondary enzyme reaction that produces a detectable signal.

• LC-MS/MS analysis: Monitor substrate depletion and product formation using liquid chromatography coupled with tandem mass spectrometry for precise quantification.

• Radiometric assays: Use radiolabeled substrates to track the conversion to products with high sensitivity.

When designing these assays, it's important to consider optimal buffer conditions, including pH (typically 6.5-7.5), temperature (typically 25-37°C), and potential cofactor requirements based on similar enolase-phosphatases characterized in other organisms.

How does UTR4 compare functionally to effector proteins in M. oryzae's pathogenicity mechanisms?

While UTR4 (Enolase-phosphatase E1) is not classified as a typical effector protein, understanding its relationship to M. oryzae's effector-mediated pathogenicity provides valuable context:

M. oryzae secretes numerous effector proteins to facilitate infection, with two main categories identified:

• Cytoplasmic effectors: Proteins like Bas1 and Pwl2 are first secreted into specialized structures called biotrophic interfacial complexes (BICs), then packaged into membranous effector compartments (MECs) before being translocated into host cells via clathrin-mediated endocytosis (CME) .

• Apoplastic effectors: Proteins like Bas4 remain in the extracellular space between the fungal cell wall and host plasma membrane .

Recent research has identified additional effector proteins that either induce cell death in rice (GAS1, BAS2, MoCEP1, MoCEP2) or suppress host immune responses (MoCEP3 to MoCEP8) .

What comparative analysis methods can differentiate UTR4 function across different M. oryzae strains?

To differentiate UTR4 function across different M. oryzae strains, researchers can employ several comparative analysis methods:

• Sequence analysis and phylogenetics:

  • Compare UTR4 sequences from different strains (e.g., laboratory strain 70-15 vs. field isolates like P131)

  • Construct phylogenetic trees to evaluate evolutionary relationships

  • Identify potential strain-specific variants that might correlate with virulence differences

• Expression profiling:

  • Analyze UTR4 expression levels during infection using RT-qPCR or RNA-Seq

  • Compare expression patterns between strains with different host specificities

  • Examine temporal expression during different infection stages

• Functional genomics approaches:

  • Generate UTR4 knockout mutants in different M. oryzae strains following methods similar to those used for other genes:
    a. Construct replacement vectors using ~1.4kb upstream/downstream flanking sequences
    b. Transform into protoplasts via PEG-mediated transformation
    c. Confirm deletions by Southern blot hybridization

  • Compare phenotypes and pathogenicity of these mutants on different host plants

• Biochemical characterization:

  • Purify recombinant UTR4 from different strains

  • Compare enzyme kinetics and substrate specificity

  • Analyze potential strain-specific post-translational modifications

How might UTR4 contribute to M. oryzae's differential infection mechanisms between rice and Arabidopsis?

The differential infection mechanisms of M. oryzae between rice (its natural host) and Arabidopsis (a model plant) may involve metabolic enzymes like UTR4 in several potential ways:

Research has demonstrated that M. oryzae infects Arabidopsis via mechanisms distinct from those required for rice infection . These differences might involve:

• Metabolic adaptation: UTR4, as an enzyme involved in metabolic pathways, could contribute to the fungus's ability to utilize different nutrient sources available in rice versus Arabidopsis tissues.

• Host-specific signaling: The metabolites processed by UTR4 might participate in signaling cascades that regulate the expression of host-specific virulence factors.

• Stress response modulation: UTR4 might be involved in pathways that help the fungus adapt to different defense responses mounted by rice versus Arabidopsis.

• Interaction with host-specific factors: The enzyme might directly or indirectly interact with host proteins or metabolites that are differentially present in rice and Arabidopsis.

Testing these hypotheses would require comparative analysis of UTR4 expression, activity, and the effects of UTR4 deletion on M. oryzae pathogenicity in both host systems. Techniques similar to those used to study candidate effector genes could be employed, including gene knockout followed by pathogenicity assays on both rice and Arabidopsis .

What role might UTR4 play in fungal adaptation to host immune responses?

While direct evidence for UTR4's role in immune response adaptation is not detailed in the provided sources, several hypotheses can be formulated based on knowledge of fungal pathogenicity mechanisms:

• Metabolic flexibility: UTR4, as an enolase-phosphatase, likely participates in metabolic pathways that may need to be reconfigured when the fungus encounters host immune responses. This metabolic flexibility could be crucial for survival under immune pressure.

• Potential moonlighting functions: Beyond its primary enzymatic role, UTR4 might have secondary functions related to immune evasion. Some metabolic enzymes in pathogens have been shown to moonlight as virulence factors.

• Support for effector production: The metabolic pathways involving UTR4 might be essential for producing or secreting the various effector proteins that M. oryzae uses to suppress host immunity. Recent research has identified multiple candidate effector proteins that suppress immune responses such as the flg22-induced reactive oxygen species (ROS) burst .

• Adaptation to different infection stages: The expression of UTR4 might be regulated differently during various stages of infection, especially as the fungus transitions from penetration to biotrophic growth phases, where immune evasion becomes critical.

To investigate these hypotheses, researchers could analyze UTR4 expression during infection of susceptible versus resistant plant varieties, or study how UTR4 knockout affects the fungus's ability to cope with various immune challenges.

What are common pitfalls in working with recombinant UTR4 and how can they be avoided?

Common pitfalls when working with recombinant UTR4 include:

Proper planning of experiments and adherence to recommended handling protocols can significantly reduce these challenges and improve research outcomes.

How can researchers optimize gene knockout studies to investigate UTR4 function in M. oryzae pathogenicity?

To optimize gene knockout studies for investigating UTR4 function in M. oryzae pathogenicity, researchers should consider the following methodological approach:

• Vector design optimization:

  • Use ~1.4kb upstream and downstream flanking sequences of UTR4 for efficient homologous recombination

  • Clone these fragments into a suitable vector like pKOV21 using one-step cloning kits

  • Confirm vector construction by sequencing before transformation

• Transformation protocol refinement:

  • Use PEG-mediated transformation of M. oryzae protoplasts

  • Select an appropriate M. oryzae strain (laboratory strain 70-15 or field isolate like P131)

  • Optimize protoplast preparation conditions for maximum transformation efficiency

• Rigorous mutant validation:

  • Confirm gene deletion using both PCR and Southern blot hybridization

  • Verify the absence of UTR4 expression using RT-PCR or Western blotting

  • Check for potential compensatory changes in related genes

• Comprehensive phenotypic analysis:

  • Assess growth, sporulation, and morphology of knockout mutants

  • Compare pathogenicity on multiple host plants using both spray and drop inoculation methods

  • Quantify lesion area and numbers at 5 days post-inoculation

  • Document infection process using microscopy to identify specific stages affected

• Controls and complementation:

  • Include wild-type control in all experiments

  • Generate complementation strains by reintroducing the UTR4 gene to confirm phenotype restoration

  • Consider creating point mutations in catalytic residues to distinguish enzymatic from structural roles

What experimental approaches can address the technical challenges of studying UTR4 in the context of plant-fungal interactions?

Studying UTR4 in the context of plant-fungal interactions presents several technical challenges that can be addressed through innovative experimental approaches:

• Real-time visualization of infection process:

  • Develop fluorescently tagged UTR4 to track its localization during infection

  • Use confocal microscopy to observe interactions with host structures

  • Implement time-lapse imaging to capture dynamic changes during infection progression

• Host-specific expression analysis:

  • Employ laser capture microdissection to isolate fungal structures from infected plant tissue

  • Use RNA-seq or qRT-PCR to measure UTR4 expression in different infection structures

  • Compare expression patterns between compatible and incompatible interactions

• Protein-protein interaction studies:

  • Use yeast two-hybrid or co-immunoprecipitation to identify host or fungal proteins interacting with UTR4

  • Verify interactions using bimolecular fluorescence complementation (BiFC) in planta

  • Investigate whether these interactions differ between susceptible and resistant plant varieties

• Metabolomic approaches:

  • Compare metabolite profiles between wild-type and UTR4 knockout mutants

  • Identify substrates and products of UTR4 activity during infection

  • Trace metabolic fluxes using stable isotope labeling

• Alternative host systems:

  • Develop simplified infection models using alternative hosts like Arabidopsis

  • Compare UTR4 function during infection of different hosts

  • Use model systems to overcome challenges associated with rice experimentation

• CRISPR-Cas9 for precise genome editing:

  • Implement CRISPR-Cas9 for more efficient generation of UTR4 mutants

  • Create promoter replacements to control UTR4 expression levels

  • Develop conditional knockout systems to study UTR4 function at specific infection stages

How might structural analysis of UTR4 contribute to understanding its function and potential inhibition?

Structural analysis of UTR4 could significantly advance our understanding of this enzyme through several avenues:

• Structure-function relationships:

  • Determine the crystal structure of UTR4 to identify catalytic residues and binding pockets

  • Compare with homologous structures from other organisms to identify conserved functional domains

  • Analyze potential conformational changes upon substrate binding

• Rational inhibitor design:

  • Use structural information to design specific inhibitors that target UTR4

  • Develop structure-activity relationships (SAR) for potential antifungal compounds

  • Implement virtual screening approaches to identify lead compounds from chemical libraries

• Protein engineering opportunities:

  • Identify sites for mutation to alter substrate specificity or catalytic efficiency

  • Design fusion proteins for specific research applications

  • Create reporter constructs based on structural insights

• Mechanistic insights:

  • Elucidate the catalytic mechanism of the enolase-phosphatase reaction

  • Understand how substrate recognition occurs

  • Investigate the role of potential cofactors or regulatory molecules

• Comparative analysis across strains:

  • Compare UTR4 structures from different M. oryzae strains to identify strain-specific features

  • Correlate structural variations with differences in pathogenicity

  • Identify conserved epitopes for potential broad-spectrum targeting

What potential exists for UTR4 as a target for novel fungicide development?

The potential of UTR4 as a target for novel fungicide development can be evaluated from several perspectives:

• Target validation criteria:

  • Essentiality: If UTR4 knockout studies demonstrate reduced pathogenicity , this would suggest its importance for fungal virulence

  • Selectivity: Structural and sequence differences between fungal and plant/human homologs would enable selective targeting

  • Accessibility: As a cytoplasmic enzyme rather than a secreted effector, delivery systems for inhibitors would need consideration

• Screening approaches:

  • Develop high-throughput enzymatic assays to screen chemical libraries

  • Implement fragment-based drug discovery approaches

  • Use computational docking studies based on structural information

• Resistance management strategy:

  • Assess the likelihood of resistance development through mutation

  • Consider dual-targeting approaches with other fungal enzymes

  • Evaluate combination treatments with existing fungicides

• Delivery and formulation considerations:

  • Develop formulations that can penetrate the fungal cell wall

  • Consider systemic vs. contact fungicide approaches

  • Evaluate environmental stability and persistence

• Translational research pathway:

  • Initial proof-of-concept studies using purified enzyme

  • Testing in fungal cultures under controlled conditions

  • Greenhouse and field trials with formulated compounds

  • Regulatory considerations for agricultural application

How might systems biology approaches integrate UTR4 into broader metabolic networks in M. oryzae?

Systems biology approaches offer powerful frameworks for understanding UTR4's role within broader metabolic networks in M. oryzae:

The integration of UTR4 into systems-level models would provide a more comprehensive understanding of how this enzyme contributes to M. oryzae's metabolic flexibility during infection and could identify unexpected connections to virulence mechanisms.