Recombinant Botryotinia fuckeliana V-type proton ATPase subunit F (vma7)

What is the V-type proton ATPase subunit F (vma7) in Botryotinia fuckeliana?

The V-type proton ATPase subunit F (vma7) is a critical component of the vacuolar ATPase complex in Botryotinia fuckeliana (teleomorph of Botrytis cinerea), a haploid, filamentous, heterothallic ascomycete that causes "grey mould" on over 200 crops worldwide . V-ATPase functions as a proton pump that acidifies intracellular compartments, playing essential roles in cellular pH homeostasis. The vma7 subunit specifically functions as part of the central shaft of the V-ATPase complex, connecting the cytoplasmic V1 domain with the membrane-embedded V0 domain. Based on homology with other fungal V-ATPases, the B. fuckeliana vma7 is likely a protein of approximately 14 kDa, similar in size to the V-ATPase subunit F characterized in other fungi .

How does genetic variation in Botryotinia fuckeliana populations affect vma7 studies?

Botryotinia fuckeliana exhibits significant intrapopulation genetic variation, with molecular markers revealing two unexpected sympatric populations in the Champagne region of France . One group (transposa) contains the transposable elements Boty and Flipper, while the other (vacuma) lacks these elements . RFLP markers demonstrate genetic recombination occurs in both populations. This genetic diversity necessitates careful consideration when working with recombinant vma7, as isolates from different populations may exhibit variations in gene sequence and regulation. Researchers should document the source isolate and its genetic background when expressing recombinant vma7, as these variations could impact functional assays and comparative studies.

What methods are used to verify V-ATPase functionality in recombinant vma7 studies?

Several complementary approaches can verify functional activity of recombinant vma7:

• Complementation assays - Expression of recombinant vma7 in V-ATPase null mutants should restore:

  • Growth ability on media containing high calcium (100 mM CaCl₂)

  • Growth at elevated pH (pH 7.0)

  • Vacuolar acidification

• Fluorescence-based acidification assays - pH-sensitive fluorescent proteins like pHluorin can quantitatively measure vacuolar acidification when fused to membrane proteins. Studies have shown that V-ATPase mutants expressing recombinant subunits show reduced fluorescence (40-60% decrease) compared to wild type when expressed from native promoters, with stronger expression (from the TPI1 promoter) resulting in near-complete restoration of acidification (>90% reduction in fluorescence) .

• Localization studies - Fluorescently tagged V-ATPase subunits should properly localize to the vacuolar membrane. Interestingly, studies in yeast have shown that in vma7Δ mutants, the V0 subunit Vph1p is mislocalized to the ER rather than the vacuolar membrane, suggesting vma7 plays a crucial role in proper complex assembly .

How can functional complementation be used to study evolutionary conservation of vma7?

Functional complementation provides powerful insights into evolutionary conservation of V-ATPase subunits. Research methodology typically involves:

• Generation of vma7-deficient mutants in B. fuckeliana using gene deletion techniques

• Transformation with vectors expressing:

  • Native B. fuckeliana vma7 (positive control)

  • vma7 homologs from other species (experimental)

  • Mutated versions of vma7 to identify critical domains

Experimental data from studies of V-ATPase complementation in yeast provide a methodological framework. For example, when human V-ATPase subunits were expressed in corresponding yeast mutants, varying degrees of functional recovery were observed:

Data adapted from functional complementation studies in yeast

This approach allows researchers to determine the degree of functional conservation between species and identify domains critical for V-ATPase function.

What experimental approaches can assess vma7's role in Botryotinia fuckeliana pathogenicity?

The role of vma7 in B. fuckeliana pathogenicity can be investigated through:

• Genetic manipulation:

  • Creation of vma7 knockout mutants

  • Development of conditionally expressed vma7 variants

  • Site-directed mutagenesis of functional domains

• Pathogenicity assays:

  • Infection of host plants under controlled conditions

  • Quantification of lesion development and spread

  • Analysis of host tissue colonization efficiency

• Toxin production analysis:

  • Quantification of sesquiterpene botryanes (particularly botrydial) production

  • Measurement of polyketide botcinins synthesis

  • Expression analysis of toxin biosynthetic genes

The potential relationship between V-ATPase function and virulence mechanisms is particularly relevant as B. fuckeliana produces multiple toxins that induce chlorosis and cell collapse during plant infection . V-ATPase function may impact the production, transport or secretion of these compounds, affecting the fungus's ability to establish infection and overcome host defenses.

How can researchers optimize expression systems for recombinant B. fuckeliana vma7?

Optimization of expression systems for recombinant vma7 requires consideration of multiple factors:

• E. coli expression systems:

  • Advantages: High yield, simplicity, cost-effectiveness

  • Methodology: Clone vma7 into expression vectors with appropriate tags (His, GST)

  • Considerations: May require codon optimization for efficient expression

  • Purification: Affinity chromatography followed by size exclusion chromatography

  • Expected purity: >85% as assessed by SDS-PAGE

• Yeast expression systems:

  • Advantages: Eukaryotic post-translational modifications, functional assessment

  • Methodology: Express in vma7-deficient strains to assess complementation

  • Promoter selection: Native promoters provide physiological expression levels while stronger promoters (TPI1) can maximize protein yield

  • Assessment: Growth recovery and vacuolar acidification assays

• Insect cell expression:

  • Advantages: Higher eukaryotic processing, suitable for structural studies

  • Methodology: Baculovirus expression vectors with secretion signals

  • Applications: When large quantities of correctly folded protein are required

Storage recommendations for purified recombinant V-ATPase subunit F include maintaining at -20°C, with extended storage at -20°C or -80°C, potentially with the addition of 5-50% glycerol for long-term stability .

What approaches can investigate interactions between vma7 and other V-ATPase subunits?

Investigation of vma7's interactions within the V-ATPase complex can employ complementary techniques:

• Epitope tagging strategies:

  • HA, FLAG or Myc tags can be fused to vma7 for immunoprecipitation studies

  • Tag placement (N- or C-terminal) should be optimized to maintain function

  • Functionality verification through complementation assays is essential

• Crosslinking coupled with mass spectrometry:

  • Chemical crosslinkers can capture transient interactions

  • Crosslinked complexes can be analyzed by mass spectrometry to identify interaction interfaces

  • Zero-length crosslinkers reveal direct protein-protein contacts

• Yeast two-hybrid or split-GFP assays:

  • Systematic testing of interactions with other V-ATPase subunits

  • Identification of specific domains involved in interactions

  • Validation in vivo through co-localization studies

Experimental evidence from V-ATPase studies in yeast demonstrated that antibodies against HA-tagged Vma7p affected enzyme activity, confirming accessibility of this subunit within the complex . Additionally, in vma7Δ yeast mutants, the Vo subunit Vph1p mislocalized to the ER instead of the vacuolar membrane, indicating vma7's critical role in proper complex assembly or trafficking .

What purification strategies yield highest activity for recombinant B. fuckeliana vma7?

Multi-step purification strategies can optimize both yield and activity:

• Initial capture:

  • Immobilized metal affinity chromatography (IMAC) for His-tagged vma7

  • Buffer optimization: 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10-250 mM imidazole gradient

  • Alternative: GST affinity purification if GST-tagged constructs are used

• Intermediate purification:

  • Ion exchange chromatography based on theoretical isoelectric point

  • Buffer considerations: low salt for binding, gradient elution

• Polishing step:

  • Size exclusion chromatography (Superdex 75 or similar)

  • Buffer: 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 5% glycerol

• Quality control:

  • SDS-PAGE with target purity >85%

  • Western blot verification with anti-tag antibodies

  • Mass spectrometry confirmation of protein identity

  • Activity assays to ensure functional protein

Tag removal considerations: If the presence of tags interferes with functional studies, tags can be removed using specific proteases (TEV, PreScission) followed by a second affinity step to separate the cleaved protein.

What assays most effectively measure vacuolar acidification in vma7 functional studies?

Multiple complementary assays can effectively measure vacuolar acidification:

• Fluorescence-based in vivo assays:

  • pH-sensitive fluorescent proteins (pHluorin) fused to vacuolar membrane proteins

  • Quantification: Decreased fluorescence indicates successful acidification

  • Data interpretation: Wild-type cells show minimal fluorescence, while V-ATPase mutants show increased fluorescence

  • Complemented strains expressing recombinant vma7 should show restoration of acidification

• Fluorescent dye-based assays:

  • Quinacrine accumulation in acidic compartments

  • BCECF or LysoSensor dyes to measure vacuolar pH

  • Flow cytometry quantification of fluorescence intensity

• Biochemical assays with isolated vacuoles:

  • Proton pumping assays using pH-sensitive dyes (ACMA)

  • ATP hydrolysis measurements using malachite green assay

  • Inhibitor sensitivity tests (bafilomycin A1, concanamycin A)

Quantitative analysis from yeast complementation studies demonstrated that when V₁ subunits were replaced with human homologs, fluorescence of pH-sensitive reporters decreased to varying degrees depending on expression level . For example, with the V₁C subunit, fluorescence decreased to ~56.7% when expressed from the native promoter but to ~0.4% when expressed from the stronger TPI1 promoter, indicating almost complete restoration of function with higher expression .

How can researchers analyze the effects of vma7 mutations on V-ATPase assembly?

Analysis of V-ATPase assembly with mutated vma7 requires multiple approaches:

• Localization studies:

  • Fluorescently tagged V-ATPase subunits can reveal assembly defects

  • Co-localization with compartment markers (ER, Golgi, vacuole)

  • Quantitative analysis categorizing localization patterns

  • Expected outcomes: Properly assembled V-ATPase localizes to vacuolar membrane, while assembly defects may cause ER retention

• Biochemical analysis:

  • Blue native PAGE to visualize intact complexes

  • Size exclusion chromatography to analyze complex formation

  • Immunoprecipitation to identify interaction partners

• Structural approaches:

  • Homology modeling based on related V-ATPase structures

  • Molecular dynamics simulations to predict mutation effects

  • Cryo-EM of purified complexes to visualize structural changes

• Functional assessment:

  • Growth complementation assays under stress conditions (high calcium, elevated pH)

  • Vacuolar acidification measurements

  • ATP hydrolysis activity tests

Studies in yeast have shown that in the absence of vma7, other V-ATPase subunits like Vph1p mislocalize to the ER rather than the vacuolar membrane . Quantitative analysis revealed that in vma7Δ yeast mutants, the localization pattern of fluorescently tagged Vph1 shifted dramatically from exclusively vacuolar membrane to predominantly ER localization, demonstrating vma7's essential role in proper V-ATPase assembly or trafficking .

What strategies can identify species-specific features of B. fuckeliana vma7?

Identifying species-specific features of B. fuckeliana vma7 requires comparative analyses:

• Sequence analysis approaches:

  • Multiple sequence alignment with vma7 from diverse fungi

  • Identification of B. fuckeliana-specific residues or motifs

  • Evolutionary rate analysis to identify rapidly evolving regions

  • Structural predictions to map species-specific features

• Functional complementation studies:

  • Cross-species complementation using vma7 from different fungi

  • Chimeric proteins with domains swapped between species

  • Site-directed mutagenesis of species-specific residues

• Interaction mapping:

  • Yeast two-hybrid or pull-down assays comparing interactions across species

  • Competition assays to identify binding preferences

  • In vitro reconstitution of partial complexes

• Pathogenicity correlation:

  • Comparison of vma7 sequences from isolates with varying virulence

  • Association studies between sequence polymorphisms and host range

  • Genetic manipulation to introduce B. fuckeliana-specific features into non-pathogenic fungi

Genetic analysis has shown that B. fuckeliana populations contain significant genetic variation, with two distinct sympatric populations (transposa and vacuma) differing in their complement of transposable elements and other genetic markers . This genetic diversity could extend to vma7, potentially revealing adaptations specific to this pathogenic fungus that might correlate with its ability to infect multiple host species or produce specialized virulence factors .