Recombinant Ashbya gossypii Serine/threonine-protein phosphatase 4 catalytic subunit (PPH3)

What is Ashbya gossypii and why is it significant in research?

Ashbya gossypii is a filamentous fungus that has become an important model organism for studying fungal developmental biology. It is closely related to unicellular yeasts such as Saccharomyces cerevisiae but grows exclusively as multinucleated hyphae with lateral branching and tip-splitting . A. gossypii was first isolated from diseased cotton bolls nearly 100 years ago and is associated with specific insects of the suborder Heteroptera, particularly cotton stainers .

Research significance:

• Genome analysis revealed extensive synteny (>90%) with S. cerevisiae, making it valuable for comparative genomics

• Used to understand the evolution of filamentous growth versus yeast growth patterns

• Commercially exploited as a natural overproducer of riboflavin (vitamin B2)

• Model for studying hyphal growth regulation in filamentous fungi

What is the function of PPH3 in Ashbya gossypii?

PPH3 in A. gossypii functions as the catalytic subunit of serine/threonine-protein phosphatase 4. Based on available research:

• Involved in the dephosphorylation and activation of the transcription factor GLN3 in response to nutrient availability

• Forms histone H2A phosphatase activity

• May participate in phosphorylation/dephosphorylation regulatory networks affecting hyphal growth and development

• Likely contributes to nutrient sensing pathways, which are critical for riboflavin production in A. gossypii

The PPH3 gene has been identified in the A. gossypii genome as AGOS_AGR136W according to databases .

How is A. gossypii cultured and maintained in laboratory settings?

To successfully culture A. gossypii for research purposes:

Media compositions:

• AFM (Ashbya Full Medium) – used for general cultivation

• AMM (Ashbya Minimal Medium) – used for selection experiments with auxotrophic strains

• SPA (Sporulation Agar) – used to induce sporulation for generating homokaryotic strains

Growth conditions:

• Optimal growth temperature: 28-30°C

• For strain preservation: Incubation at 16°C, 30°C, and 37°C is used to analyze radial growth speeds

• Solid media typically contains ampicillin (100 μg/mL) and tetracycline (100 μg/mL) to limit bacterial growth

Strain maintenance protocol:

• Grow A. gossypii on appropriate solid medium

• Collect mycelium and store at -80°C for long-term preservation

• For genetic studies, isolate homokaryotic spores using zymolyase treatment (1 mg/ml at 37°C for 50 min)

• Verify strain identity using PCR or microscopic examination of growth patterns

What methods are available for genetic manipulation of PPH3 in A. gossypii?

A robust genetic toolbox exists for manipulating the PPH3 gene in A. gossypii:

Traditional Gene Targeting:

• Homologous recombination with selection markers (GEN3, ScLEU2)

• Verification through PCR and Southern blotting

CRISPR/Cas9 System:
A one-vector CRISPR/Cas9 editing system has been adapted for A. gossypii containing:

• CAS9 expression module under TEF1 promoter control

• sgRNA expression module controlled by A. gossypii SNR52 promoter

• dDNA module for double-strand break repair

Implementation protocol for CRISPR/Cas9 editing of PPH3:

• Design sgRNA targeting PPH3 with appropriate PAM site (5'-NGG-3')

• Generate donor DNA for precise mutation introduction

• Assemble CRISPR/Cas9 vector with specific sgRNA-dDNA using directional cloning

• Transform A. gossypii via electroporation

• Select transformants using G418 resistance

• Induce sporulation to isolate homokaryotic clones

This approach enables marker-free gene deletions, insertions, and nucleotide substitutions in the PPH3 gene.

How can interactions between PPH3 and other proteins be characterized in A. gossypii?

To characterize PPH3 protein interactions in A. gossypii:

Co-immunoprecipitation (Co-IP):

• Express epitope-tagged PPH3 (e.g., GFP-PPH3) in A. gossypii using available plasmids like pYCP111

• Prepare cell lysates under non-denaturing conditions

• Immunoprecipitate using anti-tag antibodies

• Identify interacting partners via mass spectrometry

Fluorescence microscopy for co-localization:

• Generate strains expressing fluorescently-tagged PPH3 and potential interacting partners

• Perform live-cell imaging using available GFP-tagging protocols

• Analyze co-localization patterns in different growth phases and conditions

Yeast two-hybrid analysis:
Due to the evolutionary closeness of A. gossypii to S. cerevisiae, hybrid systems can be employed:

• Clone PPH3 into bait vector

• Create or obtain A. gossypii cDNA library in prey vector

• Transform into S. cerevisiae

• Screen for interactions using appropriate reporter systems

Bimolecular Fluorescence Complementation (BiFC):

• Fuse PPH3 to N-terminal fragment of fluorescent protein

• Fuse candidate interacting proteins to C-terminal fragment

• Co-express in A. gossypii

• Analyze reconstituted fluorescence signals

What are the optimal conditions for expressing and purifying recombinant A. gossypii PPH3?

Expression systems:

E. coli expression protocol:

• Clone A. gossypii PPH3 gene (AGOS_AGR136W) into a suitable expression vector with an affinity tag

• Transform into an E. coli expression strain (BL21 derivatives recommended)

• Induce expression at lower temperatures (16-20°C) to improve folding

• Harvest cells and lyse using appropriate buffer systems

• Purify using affinity chromatography based on the chosen tag system

• Further purify using size exclusion chromatography

Buffer considerations for phosphatase activity preservation:

• Include metal ions (Mn²⁺ or Mg²⁺) in purification buffers

• Add reducing agents (DTT or β-mercaptoethanol) to prevent oxidation of catalytic site

• Consider phosphatase inhibitor-free buffers during final purification steps

• Maintain pH between 7.0-7.5

Quality control:

• SDS-PAGE for purity assessment

• Western blot for identity confirmation

• Enzymatic activity assay using phosphatase substrates

• Mass spectrometry for intact mass verification

What assays can be used to measure PPH3 phosphatase activity?

In vitro assays:

• Colorimetric phosphatase assays:

  • pNPP (para-Nitrophenyl phosphate) hydrolysis

  • Malachite green assay for released phosphate

  • Conditions: 30°C, pH 7.0-7.5, presence of Mn²⁺ or Mg²⁺

• Synthetic phosphopeptide substrates:

  • Design based on known PPH3 substrates (GLN3 phosphorylation sites)

  • Monitor dephosphorylation via mass spectrometry or phospho-specific antibodies

• Protein substrate assays:

  • Use phosphorylated histone H2A as substrate (given PPH3's role as H2A phosphatase)

  • Detect dephosphorylation using phospho-specific antibodies

In vivo approaches:

• Reporter systems:

  • Create GLN3-dependent reporter constructs in A. gossypii

  • Compare reporter activity in wild-type vs. PPH3 mutant strains

• Phosphorylation state analysis:

  • Western blots with phospho-specific antibodies against known substrates

  • Phosphoproteomic analysis comparing wild-type and PPH3 mutant strains

• Growth phenotype assessment:

  • Monitor hyphal growth patterns and branching in response to nutrient availability

  • Compare riboflavin production levels as this is affected by phosphorylation-dependent pathways

How should researchers design experiments to study PPH3 function in A. gossypii metabolism?

To effectively study PPH3's role in A. gossypii metabolism:

Experimental design framework:

• Generate genetic tools:

  • Create PPH3 deletion mutant (Δpph3) using CRISPR/Cas9 system

  • Develop complementation strains with wild-type and mutant PPH3 alleles

  • Generate catalytic-dead PPH3 mutants by site-directed mutagenesis of key residues

• Phenotypic characterization:

  • Growth rate measurements under various nutrient conditions

  • Hyphal morphology assessment (branching patterns, septation)

  • Riboflavin production quantification

  • Stress response profiling (oxidative, nutritional, temperature)

• Molecular analysis:

  • Transcriptome analysis (RNA-seq) comparing wild-type and mutant strains

  • Phosphoproteomic profiling to identify differentially phosphorylated proteins

  • Metabolomic analysis focusing on pathways linked to riboflavin production

• Integration with known pathways:

  • Combine PPH3 mutations with mutations in PRPP synthetase genes (AGR371C, AGL080C)

  • Analyze double mutants with components of the Ras GTPase pathway (e.g., Rsr1p/Bud1p)

  • Investigate interactions with polarisome components (e.g., AgSpa2p)

Controls and validation:

• Include appropriate genetic controls (empty vector, catalytic-dead mutants)

• Perform rescue experiments with wild-type PPH3 to confirm specificity

• Validate key findings using multiple methodological approaches

How do I troubleshoot issues with recombinant PPH3 expression and activity?

Common expression issues and solutions:

Activity troubleshooting:

• No detectable activity:

  • Verify protein folding using circular dichroism or thermal shift assays

  • Ensure correct metal cofactors are present (Mn²⁺, Mg²⁺)

  • Test multiple buffer conditions and pH ranges (pH 6.5-8.0)

  • Examine potential inhibitors in your buffer components

• Variable activity:

  • Standardize protein concentration determination methods

  • Use internal controls for normalization

  • Ensure consistent substrate quality

  • Maintain consistent reaction temperature

• High background in assays:

  • Include appropriate phosphatase inhibitor controls

  • Use higher purity substrate preparations

  • Include enzyme-free and substrate-free controls

  • Consider alternative detection methods

How do I interpret conflicting results from PPH3 functional studies?

When faced with conflicting data regarding PPH3 function:

Systematic interpretation approach:

Review case:
In A. gossypii, PRPP synthetase (Agl080cp) deletion causes altered hyphal morphology with increased branching . If PPH3 studies show variable effects on hyphal morphology, consider whether PPH3 might regulate PRPP synthetase activity directly or indirectly through shared signaling pathways.

What are the most promising research directions for studying PPH3 in A. gossypii?

Based on current knowledge, several promising research directions emerge:

• PPH3's role in metabolic regulation:

  • Investigate how PPH3 influences riboflavin biosynthesis pathways

  • Study PPH3's contribution to PRPP synthetase regulation, as PRPP is critical for riboflavin production

  • Explore connections between nutrient sensing and metabolic output

• Developmental functions:

  • Characterize PPH3's impact on hyphal growth patterns and branching

  • Investigate nuclear division and distribution in multinucleated hyphae

  • Study PPH3's role in sporulation and spore germination processes

• Stress response regulation:

  • Examine PPH3's function during nutrient limitation

  • Investigate potential roles in oxidative stress responses (relevant to riboflavin production)

  • Study adaptation to environmental changes through phosphorylation dynamics

• Evolutionary comparisons:

  • Compare PPH3 function between A. gossypii and S. cerevisiae to understand evolutionary divergence

  • Study how phosphatase networks have adapted for filamentous growth versus yeast-like growth

  • Investigate PPH3 roles in other industrial filamentous fungi

• Biotechnological applications:

  • Engineer PPH3 activity to optimize riboflavin production

  • Develop PPH3 modulators that could enhance strain performance

  • Apply PPH3 knowledge to optimize growth in industrial fermentation

Methodological innovations:

• Develop phospho-specific sensors to monitor PPH3 activity in real-time

• Apply single-nucleus transcriptomics to address multinuclearity challenges

• Implement optogenetic control of PPH3 activity for temporal studies

What key reagents and tools are available for studying A. gossypii PPH3?

Commercial reagents:

• Recombinant A. gossypii PPH3 protein (available from suppliers like Cusabio)

• PPH3 antibodies specific for A. gossypii strain ATCC 10895

• Expression vectors optimized for A. gossypii

Genetic resources:

• A. gossypii strain collection including ATCC 10895 (reference strain)

• Agleu2Δthr4Δ strain (commonly used for genetic manipulation)

• CRISPR/Cas9 vector system adapted for A. gossypii

Plasmids and constructs:

• pGEN3 and pScLEU2 for deletion cassette generation

• pYCP111 (CEN-ARS plasmid with S. cerevisiae LEU2 gene) for expression studies

• Plasmids for N-terminal GFP fusions (e.g., pHPS250)

Protocols:

• Transformation protocols using modified electroporation methods

• Sporulation and homokaryotic strain isolation procedures

• Hyphal morphology and branching pattern analysis methods

Where can I find A. gossypii genomic data and PPH3 sequence information?

Genome resources:

• Complete genomic sequence of A. gossypii strain ATCC 10895 is available in RefSeq (NC_005788)

• A. gossypii gene annotations are accessible through KEGG (ago:AGOS_AGR136W)

• STRING database for protein interaction networks (33169.AAS54626)

PPH3 specific information:

• UniProtKB entry: Q74ZR2 contains curated information about PPH3

• ENTREZ Gene ID: 4623104 (AGOS_AGR136W)

• Genomic coordinates and structural information available through UniProt

Database tools:

• AGD (A. gossypii Database) contains annotated gene information

• Comparative genomics resources for analyzing synteny with S. cerevisiae

• Phosphorylation site prediction tools like NetPhorest can be applied to A. gossypii proteins