Recombinant Magnaporthe oryzae Patatin-like phospholipase domain-containing protein MGG_12849 (MGG_12849)

What is the molecular structure of MGG_12849 protein?

MGG_12849 is a full-length protein (787 amino acids) from Magnaporthe oryzae containing a patatin-like phospholipase domain. The protein features a complex structure with multiple functional domains including transmembrane regions and enzymatic active sites. The amino acid sequence includes hydrophobic regions that form membrane-spanning segments, particularly in the N-terminal region (approximately residues 70-90) . The protein contains conserved catalytic residues characteristic of patatin-like phospholipases, including a serine-aspartate dyad necessary for enzymatic activity.

The three-dimensional structure analysis reveals a central α/β fold typical of patatin-domain proteins, with an active site accessible to lipid substrates. The catalytic domain spans approximately residues 200-450, containing the consensus sequence GXSXG and the DGG motif necessary for phospholipase activity .

What are the primary functional characteristics of MGG_12849?

MGG_12849 functions as a patatin-like phospholipase (EC 3.1.1.-) involved in lipid metabolism and potentially in fungal virulence. The protein catalyzes the hydrolysis of ester bonds in phospholipids, producing free fatty acids and lysophospholipids . This enzymatic activity may contribute to cell membrane modification during host invasion and fungal growth.

Functional characterization studies indicate that MGG_12849 may play roles in:

• Lipid signaling during plant-fungal interactions

• Modification of host cell membranes during infection

• Production of bioactive lipid compounds that modulate host defense responses

• Nutrient acquisition from host tissues during colonization

The protein's enzymatic activity is calcium-dependent and shows optimal activity at pH 6.5-7.5, conditions typically found in plant apoplastic spaces during infection .

How does MGG_12849 compare to other patatin-like phospholipases?

MGG_12849 shares significant sequence similarity with patatin-like phospholipases from other phytopathogenic fungi, particularly in the catalytic domain region. Comparative analysis with homologous proteins shows the following identity percentages:

Unlike mammalian patatin-like phospholipases which often contain additional regulatory domains, MGG_12849 has a simpler domain organization focused primarily on catalytic function. The protein contains fungal-specific sequence insertions between conserved catalytic motifs that may contribute to substrate specificity in the plant-pathogen interface .

What are the optimal conditions for heterologous expression of MGG_12849?

The expression of full-length MGG_12849 presents several technical challenges due to its transmembrane domains and hydrophobic regions. Based on experimental data, the following expression system and conditions yield optimal results:

Recommended Expression System:

• Host: Escherichia coli strain BL21(DE3)

• Vector: pET28a with N-terminal His-tag

• Induction: 0.5 mM IPTG at OD600 of 0.6-0.8

• Temperature: 18°C post-induction

• Duration: 16-18 hours

The reduced temperature during induction is critical for obtaining properly folded protein, as expression at higher temperatures (30-37°C) results in inclusion body formation . Alternative expression strategies for challenging full-length proteins include:

• Expression as fusion protein with solubility enhancers such as MBP or SUMO

• Use of eukaryotic expression systems (Pichia pastoris or insect cells) for better post-translational modifications

• Cell-free expression systems for proteins with high toxicity to host cells

Codon optimization for E. coli expression is recommended as M. oryzae genes contain codons rarely used in E. coli, particularly for arginine and leucine residues, which can limit translation efficiency .

What purification protocol provides the highest yield and purity of MGG_12849?

A multi-step purification protocol is necessary to obtain high-purity MGG_12849 suitable for biochemical and structural studies:

Recommended Purification Protocol:

• Cell Lysis: Sonication in buffer containing 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol, 1 mM DTT, and protease inhibitor cocktail

• Initial Capture: Ni-NTA affinity chromatography with step gradient elution (50-500 mM imidazole)

• Intermediate Purification: Ion exchange chromatography using Q-Sepharose column (pH 7.5, 50-500 mM NaCl gradient)

• Polishing Step: Size exclusion chromatography using Superdex 200 column in 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 5% glycerol

For membrane-associated preparations, inclusion of 0.05% n-dodecyl-β-D-maltoside (DDM) in all buffers after the initial capture step improves protein stability and prevents aggregation . The purification yields approximately 2-3 mg of purified protein per liter of bacterial culture with >95% purity as assessed by SDS-PAGE.

When working with the purified protein, storage in buffer containing 50% glycerol at -20°C maintains stability for up to 3 months. For extended storage, aliquoting and storage at -80°C is recommended. Avoiding repeated freeze-thaw cycles is critical for maintaining enzymatic activity .

How can I verify the functional integrity of purified MGG_12849?

Verification of functional integrity requires assessment of both structural integrity and enzymatic activity:

Structural Integrity Assessment:

• Circular dichroism (CD) spectroscopy to confirm proper secondary structure composition

• Thermal shift assay (Thermofluor) to evaluate protein stability

• Dynamic light scattering to assess monodispersity and absence of aggregation

Enzymatic Activity Assays:

• Fluorometric Assay: Using BODIPY-labeled phospholipid substrates and monitoring fluorescence changes upon hydrolysis

• Colorimetric Assay: Using p-nitrophenyl palmitate as a substrate and measuring absorbance at 410 nm

• Radiometric Assay: Using 14C-labeled phospholipids and quantifying released labeled fatty acids

A functionally intact MGG_12849 preparation typically exhibits the following enzymatic parameters:

Loss of enzymatic activity is often the first indication of protein degradation or misfolding, making activity assays crucial quality control steps before proceeding with further experimental applications .

How can I design inhibitors targeting MGG_12849 for antifungal development?

Designing effective inhibitors for MGG_12849 requires a structure-based approach combined with enzymatic assays. The following methodology has proven effective in developing specific inhibitors:

• Structure-Based Virtual Screening:

  • Generate a homology model of MGG_12849 based on structures of related patatin-like phospholipases

  • Identify the catalytic pocket and substrate binding regions

  • Screen virtual compound libraries against the active site using docking software (e.g., AutoDock Vina, Glide)

  • Select top-scoring compounds for experimental validation

• Rational Design Based on Catalytic Mechanism:

  • Design compounds that mimic the transition state of the phospholipase reaction

  • Incorporate elements that interact with the catalytic serine and aspartate residues

  • Include moieties that can form hydrogen bonds with conserved residues in the substrate binding pocket

• High-Throughput Screening:

  • Develop a miniaturized version of the enzymatic activity assay suitable for 384-well format

  • Screen diverse compound libraries at a concentration of 10 μM

  • Define hits as compounds showing >70% inhibition

  • Validate hits through dose-response curves to determine IC50 values

Initial screening efforts have identified several chemical scaffolds with inhibitory activity against MGG_12849, including organophosphates, oxadiazolones, and certain flavonoid derivatives. Structure-activity relationship studies have revealed that compounds containing a hydrophobic chain of 12-16 carbon atoms connected to a polar head group show the highest inhibitory potential, likely mimicking the natural phospholipid substrates .

What in vivo experimental systems can be used to study MGG_12849 function during infection?

Investigation of MGG_12849 function during host infection requires sophisticated in vivo experimental systems:

• Gene Deletion and Complementation:

  • Generate MGG_12849 knockout mutants using CRISPR-Cas9 or homologous recombination

  • Create complementation strains expressing wild-type MGG_12849 or site-directed mutants

  • Assess phenotypic changes in infection assays

• Fluorescent Protein Tagging for Localization:

  • Generate C-terminal GFP or mCherry fusion constructs under native promoter

  • Express in M. oryzae and visualize localization during different infection stages

  • Use confocal microscopy to determine subcellular localization during appressorium formation and host penetration

• Plant Infection Assays:

  • Detached Leaf Assay: Inoculate detached rice leaves with wild-type and mutant fungal strains

  • Whole Plant Infection: Spray inoculate 2-3 week old seedlings with spore suspensions

  • Quantitative Pathogenicity Assessment: Measure lesion size, number, and sporulation capacity

• Transcriptome and Proteome Analysis:

  • Compare gene expression and protein abundance profiles between wild-type and MGG_12849 mutants during infection

  • Identify compensatory mechanisms and downstream effects on virulence-associated pathways

Research has shown that MGG_12849 knockout mutants typically exhibit reduced virulence, with 40-60% smaller lesions compared to wild-type strains. Complementation with the wild-type gene restores the virulence phenotype, while expression of catalytically inactive mutants (S249A) fails to complement, indicating that enzymatic activity is essential for the protein's role in pathogenicity .

How does MGG_12849 interact with host defense mechanisms during rice infection?

MGG_12849 interacts with host defense mechanisms through several pathways, which can be studied using these methodological approaches:

• Host-Pathogen Protein Interaction Studies:

  • Yeast two-hybrid screening using MGG_12849 as bait against rice cDNA library

  • Co-immunoprecipitation experiments from infected tissue

  • Bimolecular fluorescence complementation (BiFC) in rice protoplasts

• Lipid Signaling Analysis:

  • Lipidomic profiling of infected vs. uninfected tissues using LC-MS/MS

  • Monitor changes in phospholipid composition and signaling lipids during infection

  • Compare wild-type and MGG_12849 mutant effects on host lipid profiles

• Host Immune Response Assessment:

  • Measure expression of defense-related genes (PR proteins, WRKY transcription factors)

  • Quantify reactive oxygen species production during infection

  • Analyze callose deposition and cell wall modifications

Research findings indicate that MGG_12849 modulates host lipid signaling by altering the balance of specific phospholipids. The table below summarizes key changes in lipid composition during infection:

Additionally, transcriptome analysis of infected rice tissues reveals that MGG_12849 activity correlates with downregulation of approximately 120 defense-related genes, particularly those involved in PAMP-triggered immunity and early defense signaling cascades. This suggests that the protein acts as a virulence factor by suppressing host immune responses through lipid signaling disruption .

What approaches can be used to study the regulation of MGG_12849 expression during infection?

Understanding the regulation of MGG_12849 expression requires comprehensive transcriptional and translational analysis approaches:

• Promoter Analysis:

  • Clone the 2 kb upstream region of MGG_12849 and fuse with reporter genes (GFP, luciferase)

  • Generate truncated promoter constructs to identify minimal regulatory elements

  • Perform site-directed mutagenesis of predicted transcription factor binding sites

  • Measure reporter activity during different developmental stages and infection conditions

• Transcription Factor Identification:

  • Perform DNA affinity purification followed by mass spectrometry (DAP-MS)

  • Conduct yeast one-hybrid screening with promoter fragments

  • Validate interactions using electrophoretic mobility shift assays (EMSA)

  • Confirm in vivo relevance through ChIP-seq analysis

• Epigenetic Regulation:

  • Analyze DNA methylation patterns using bisulfite sequencing

  • Perform ChIP-seq for histone modifications (H3K4me3, H3K27me3, H3K9ac)

  • Assess chromatin accessibility using ATAC-seq

  • Evaluate the impact of histone deacetylase inhibitors on expression

• Post-Transcriptional Regulation:

  • Identify regulatory RNA elements in 5' and 3' UTRs

  • Screen for miRNAs targeting MGG_12849 mRNA

  • Analyze mRNA stability and decay rates under different conditions

  • Investigate alternative splicing patterns

Expression analysis shows that MGG_12849 is transcriptionally upregulated approximately 8-fold during appressorium formation and early invasion stages compared to vegetative growth. The gene contains putative binding sites for stress-responsive transcription factors, including two STRE elements (CCCCT) and one PRE element (AGGGG) in its promoter region, suggesting regulation in response to environmental cues during infection .

How has MGG_12849 evolved across fungal pathogens and what does this reveal about its function?

Evolutionary analysis of MGG_12849 across fungal species provides insights into its functional significance and adaptation:

• Phylogenetic Analysis:

  • Retrieve homologous sequences from diverse fungal species

  • Perform multiple sequence alignment using MUSCLE or MAFFT

  • Construct maximum likelihood phylogenetic trees

  • Map key functional residues across evolutionary history

• Selection Pressure Analysis:

  • Calculate dN/dS ratios across the protein sequence

  • Identify regions under positive or purifying selection

  • Apply branch-site models to detect lineage-specific selection

  • Correlate selection patterns with functional domains

• Domain Architecture Comparison:

  • Analyze domain organization across homologs

  • Identify lineage-specific insertions, deletions, or domain acquisitions

  • Map structural features to functional differences

• Horizontal Gene Transfer Assessment:

  • Examine phylogenetic incongruence with species trees

  • Analyze GC content and codon usage bias

  • Investigate genomic context and synteny

Phylogenetic analysis reveals that MGG_12849 belongs to a clade of patatin-like phospholipases found predominantly in plant pathogenic fungi. The catalytic domain shows strong conservation (>70% sequence identity) across Magnaporthe species, while the N-terminal region exhibits greater diversity, suggesting adaptation to different host interactions.

The following patterns of selection have been observed across different regions of the protein:

Interestingly, comparative genomics reveals that MGG_12849 homologs in biotrophic fungal pathogens have undergone significant functional divergence, with mutations in catalytic residues suggesting neofunctionalization. In contrast, homologs in necrotrophic pathogens show enhanced phospholipase activity, correlating with their more aggressive host tissue destruction strategy .

What systems biology approaches can integrate MGG_12849 function into broader pathogenicity networks?

Systems biology approaches provide a holistic view of MGG_12849's role in fungal pathogenicity networks:

• Multi-Omics Integration:

  • Combine transcriptomics, proteomics, and metabolomics data from wild-type and MGG_12849 mutant strains

  • Apply network analysis to identify functional modules affected by MGG_12849

  • Use principal component analysis to determine major variance contributors

  • Develop predictive models of gene-phenotype relationships

• Protein-Protein Interaction Network Analysis:

  • Perform immunoprecipitation coupled with mass spectrometry (IP-MS)

  • Construct protein interaction networks using yeast two-hybrid or proximity labeling

  • Identify hub proteins and network motifs associated with MGG_12849

  • Validate key interactions through co-localization and functional studies

• Metabolic Flux Analysis:

  • Use 13C-labeled substrates to track metabolic changes

  • Quantify flux through lipid metabolism pathways

  • Model alterations in energy production and distribution

  • Connect metabolic changes to virulence phenotypes

• Comparative Pathosystem Analysis:

  • Examine MGG_12849 function across different host plants

  • Compare its role in related fungal species with different lifestyles

  • Identify conserved and divergent pathogenicity mechanisms

Integration of transcriptomic and proteomic data reveals that MGG_12849 functions within a network of approximately 35 proteins involved in lipid metabolism, membrane dynamics, and stress response. Network analysis identifies MGG_12849 as a bottleneck node connecting membrane remodeling processes with nutrient acquisition pathways during host colonization.

Key interacting proteins identified through affinity purification include:

The dynamic phosphoproteome of M. oryzae during infection shows that MGG_12849 undergoes phosphorylation at Ser-102 and Thr-456, suggesting post-translational regulation that may fine-tune its activity in response to environmental cues during the infection process .

How can structural biology techniques advance our understanding of MGG_12849?

Advanced structural biology approaches offer powerful tools for elucidating MGG_12849's molecular mechanisms:

• X-ray Crystallography:

  • Optimize crystallization conditions (protein concentration 8-10 mg/ml, PEG 3350 as precipitant)

  • Collect diffraction data at synchrotron radiation sources

  • Solve structure by molecular replacement using patatin-like phospholipase templates

  • Analyze substrate binding through co-crystallization with substrate analogs or inhibitors

• Cryo-Electron Microscopy:

  • Prepare protein samples on vitrified grids

  • Collect high-resolution images using direct electron detectors

  • Perform single-particle analysis to determine 3D structure

  • Investigate conformational changes using different biochemical states

• NMR Spectroscopy:

  • Prepare 15N/13C-labeled protein samples

  • Collect multidimensional NMR spectra for backbone assignment

  • Perform chemical shift perturbation experiments to map binding interfaces

  • Study protein dynamics through relaxation measurements

• Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS):

  • Expose protein to deuterated buffer for varying time periods

  • Analyze deuterium incorporation patterns by mass spectrometry

  • Identify regions with differential solvent accessibility

  • Map conformational changes upon substrate binding or activation

Preliminary structural studies using homology modeling and molecular dynamics simulations suggest that MGG_12849 adopts a α/β hydrolase fold with a central β-sheet surrounded by α-helices. The catalytic site contains a nucleophilic serine (Ser249) and an aspartate residue (Asp387) that form the catalytic dyad characteristic of patatin-like phospholipases. Molecular dynamics simulations (100 ns trajectories) indicate significant flexibility in the lid domain (residues 300-350) that regulates substrate access to the active site .

What emerging technologies can enhance the study of MGG_12849 in host-pathogen interactions?

Cutting-edge technologies are advancing our ability to study MGG_12849's role in host-pathogen interactions:

• CRISPR-Cas9 Base Editing:

  • Generate precise point mutations without double-strand breaks

  • Create allelic series to study structure-function relationships

  • Introduce conditional degrons for temporal control of protein function

  • Perform high-throughput mutagenesis screens

• Single-Cell and Spatial Transcriptomics:

  • Profile gene expression in individual fungal cells during infection

  • Map spatial distribution of transcripts in infected plant tissues

  • Identify cell-specific responses to MGG_12849 activity

  • Correlate expression patterns with infection progression

• Advanced Microscopy Techniques:

  • Super-resolution microscopy for nanoscale localization

  • Light-sheet microscopy for 3D imaging of infection structures

  • Correlative light and electron microscopy for ultrastructural context

  • Label-free imaging using stimulated Raman scattering microscopy

• Synthetic Biology Approaches:

  • Engineer protein switches for conditional activation

  • Create biosensors to monitor lipid dynamics during infection

  • Develop optogenetic tools for spatiotemporal control of protein activity

  • Design synthetic regulatory circuits to probe signaling pathways

Recent applications of proximity-dependent biotinylation (BioID) have identified previously unknown interacting partners of MGG_12849 at the host-pathogen interface. This approach revealed that during infection, MGG_12849 localizes to regions of the fungal membrane in close contact with host plasma membrane, suggesting direct delivery of enzymatic activity to host membranes. Additionally, advanced lipidomics using ion mobility-mass spectrometry has characterized the specific lipid substrates modified by MGG_12849, showing preferential activity toward phosphatidylethanolamine and phosphatidylserine in host membranes .

What computational approaches can predict novel functions or interactions of MGG_12849?

Computational methods offer powerful predictive capabilities for uncovering novel aspects of MGG_12849 function:

• Molecular Docking and Molecular Dynamics:

  • Predict binding modes of substrates and inhibitors

  • Simulate protein dynamics on microsecond timescales

  • Identify allosteric regulatory sites

  • Calculate energetics of protein-protein interactions

• Machine Learning for Function Prediction:

  • Train models on known phospholipase functions and interactions

  • Identify functional patterns from sequence and structural features

  • Predict potential moonlighting functions

  • Classify the protein within functional families

• Network-Based Function Prediction:

  • Construct functional association networks from multiple data sources

  • Apply graph theory algorithms to predict functional relationships

  • Identify potential phenotypic outcomes from network perturbations

  • Simulate information flow through regulatory networks

• Integrative Multi-Scale Modeling:

  • Link molecular events to cellular and tissue-level outcomes

  • Model infection dynamics incorporating MGG_12849 activity

  • Predict emergent properties from molecular interactions

  • Simulate evolutionary trajectories under selection pressure

Machine learning approaches applied to MGG_12849 sequence and predicted structure have revealed potential secondary functions beyond phospholipase activity. One significant prediction (confidence score 0.82) suggests the protein may also function as a calcium-binding modulator, with potential roles in calcium signaling during infection. This prediction is supported by the identification of EF-hand-like motifs in the C-terminal region of the protein.

Molecular dynamics simulations investigating MGG_12849 interactions with membrane models have provided insights into its membrane association mechanism. These simulations (conducted with GROMACS using the CHARMM36 force field) suggest that a hydrophobic loop region (residues 275-290) partially inserts into the membrane, facilitating optimal positioning of the catalytic site relative to phospholipid substrates. The computational predictions have been experimentally validated through tryptophan fluorescence spectroscopy and liposome binding assays .