Recombinant Neosartorya fumigata Exocyst complex component exo84 (exo84), partial

Basic Research Questions

• What is the fundamental role of Exo84 in the exocyst complex of Neosartorya fumigata?

  Exo84 serves as a crucial scaffolding component of the octameric exocyst complex in N. fumigata, likely facilitating polarized secretion and hyphal growth similar to its homologs in other fungi. Based on studies in yeast, Exo84 participates in the organization and polarization of the exocyst complex at sites of active secretion . Methodologically, its function can be investigated through gene deletion or mutation approaches combined with phenotypic analysis of growth, morphology, and secretion. Temperature-sensitive mutants, as generated for yeast Exo84p, can serve as valuable tools to conditionally disrupt function and observe resulting phenotypes .

• How can researchers generate and characterize temperature-sensitive mutations in N. fumigata Exo84?

  Temperature-sensitive mutants provide valuable tools for studying essential exocyst components. The methodological approach includes:

  • PCR-based random mutagenesis of the EXO84 gene

  • Integration of mutant alleles into the native genomic locus

  • Selection of transformants on appropriate media

  • Screening for temperature-dependent growth phenotypes

  • Complementation testing to confirm mutation effects

  • PCR verification of correct integration

  This approach parallels the successful generation of temperature-sensitive yeast exo84 mutants, where linearized DNA fragments containing mutated exo84 sequences were integrated into strains with the EXO84 locus disrupted . The resulting mutants should be tested for secretion defects using appropriate assays at both permissive and restrictive temperatures.

• What experimental approaches can determine protein-protein interactions involving N. fumigata Exo84?

  Multiple complementary techniques can verify interactions between Exo84 and other proteins:

  • Yeast two-hybrid (Y2H) screening for identifying potential interaction partners

  • Bimolecular fluorescence complementation (BiFC) for in vivo visualization of interactions

  • Co-immunoprecipitation (co-IP) using epitope-tagged versions

  • Size exclusion chromatography to observe complex formation in vitro

  These approaches have been successfully applied to study Exo84 interactions in other systems. For example, Y2H assays revealed that the N-terminal domains of Exo84 and its interaction partners are often critical for binding . Similarly, BiFC assays demonstrated specific interactions between plant Exo84c and VAP27 proteins at punctate structures in vivo, which was confirmed by immunoblot analysis .

• How should researchers design fluorescent protein fusions to study N. fumigata Exo84 localization?

  GFP-tagging is a powerful approach to study Exo84 localization. Consider the following methodological aspects:

  • Express Exo84-GFP under control of its native promoter to maintain physiological expression levels

  • C-terminal GFP fusions are often preferable to avoid disrupting N-terminal interaction domains

  • Confirm functionality of the fusion protein by complementation of exo84 mutant phenotypes

  • Use confocal microscopy to visualize localization patterns during different growth stages

  • Compare localization under various stress conditions and temperatures

  In yeast studies, Exo84-GFP expressed under its endogenous promoter localized to a bright crescent at the tips of small buds during polarized growth . This approach can be adapted to study the dynamic localization of N. fumigata Exo84 during hyphal growth and development.

• What assays can measure the impact of Exo84 mutations on secretion in N. fumigata?

  Secretion defects can be quantified through several approaches:

  • Invertase secretion assays comparing internal versus external activity

  • Quantitative proteomics of secreted proteins

  • Tracking fluorescently tagged secretory cargo proteins

  • Measurement of cell wall biosynthetic enzyme secretion

  • Electron microscopy to visualize vesicle accumulation

  The invertase assay has been particularly informative in yeast exo84 mutants, where cells are grown to early log phase, shifted to appropriate conditions, and then assessed for both internal and external invertase activity . Similar approaches can be adapted for N. fumigata to assess secretory defects in Exo84 mutants.

Advanced Research Questions

• How does N. fumigata Exo84 interact with vesicle trafficking machinery during hyphal growth?

  Investigating the relationship between Exo84 and vesicle trafficking involves sophisticated approaches:

  • Generate conditional mutants in various trafficking pathways (ER-to-Golgi, post-Golgi)

  • Perform epistasis analysis between exo84 and sec mutants affecting different trafficking steps

  • Use live-cell imaging with dual fluorescent markers to track vesicle movement in relation to Exo84

  • Implement super-resolution microscopy to visualize nanoscale organization at growth sites

  • Apply correlative light and electron microscopy to connect protein localization with ultrastructural features

  Studies in yeast demonstrated that Exo84-GFP localization was affected in mutants disrupting various stages of vesicle trafficking (sec18-1, sec22-3, sec19-1, and sec7-1), suggesting that proper vesicle trafficking is required for Exo84 polarization . Similar dependencies may exist in N. fumigata and could reveal important aspects of hyphal growth regulation.

• What role might N. fumigata Exo84 play in immune evasion during host infection?

  Exploring the immunological aspects of Exo84 function requires:

  • Generation of conditional exo84 mutants for infection studies

  • Transcriptomic analysis comparing wild-type and mutant strains during host interaction

  • Quantification of immunogenic surface component exposure

  • Assessment of host immune cell responses to mutant versus wild-type strains

  • Measurement of inflammatory cytokine production in response to fungal challenge

  Research on plant pathogens has shown that bacterial effectors can target host exocyst components to suppress immunity. For example, the XopP effector from Xanthomonas interacts with host EXO70B1, inhibiting exocyst-dependent exocytosis of immunity-promoting molecules . This suggests that fungal exocyst components might similarly be involved in immune interaction, either as targets of host immunity or as regulators of fungal immune evasion mechanisms.

• How can researchers investigate potential connections between N. fumigata Exo84 and autophagy pathways?

  Recent discoveries suggest connections between exocyst components and autophagy that warrant investigation:

  • Co-localization studies of Exo84 with autophagy markers (e.g., ATG8)

  • Analysis of autophagy flux in exo84 mutants

  • Identification of potential interactions between Exo84 and autophagy machinery

  • Assessment of Exo84 protein turnover under stress conditions

  • Electron microscopy to visualize autophagosomes in relation to Exo84-positive structures

  In plants, Exo84c interacts with VAP27 proteins at the ER membrane and ER-derived autophagosomes labeled with ATG8, facilitating the degradation of exocytosis vesicles through the autophagy pathway . Similar mechanisms might exist in filamentous fungi, potentially connecting secretion and autophagy during stress responses or developmental transitions.

• What structural approaches can elucidate the molecular architecture of N. fumigata Exo84?

  Structural characterization requires sophisticated techniques:

  • X-ray crystallography of purified recombinant Exo84 domains

  • Cryo-electron microscopy of the assembled exocyst complex

  • Hydrogen-deuterium exchange mass spectrometry to map interaction surfaces

  • Small-angle X-ray scattering to determine solution structure

  • NMR spectroscopy for dynamic studies of smaller domains

  Domain truncation experiments in yeast and plants have shown that Exo84 proteins typically interact with binding partners through their N-terminal domains . Expressing and purifying these domains individually can facilitate structural studies while avoiding the challenges associated with full-length protein crystallization.

• How do post-translational modifications regulate N. fumigata Exo84 function?

  Post-translational modification (PTM) analysis requires:

  • Phosphoproteomic analysis under different growth conditions

  • Site-directed mutagenesis of modified residues to mimic or prevent modifications

  • In vitro kinase assays to identify regulatory enzymes

  • Quantitative analysis of modification dynamics during hyphal development

  • Correlation of modification patterns with phenotypic outcomes

  While specific PTMs of N. fumigata Exo84 have not been well-characterized, studies in other systems suggest that phosphorylation and other modifications likely play important roles in regulating exocyst assembly and function during polarized growth.

• What experimental approaches can determine whether N. fumigata Exo84 forms a spatial landmark for polarized secretion?

  Investigating Exo84's role in establishing polarity landmarks requires:

  • Tracking Exo84 dynamics during polarity establishment using fluorescence recovery after photobleaching (FRAP)

  • Disrupting the actin cytoskeleton and assessing effects on Exo84 localization

  • Creating deletion mutants of potential upstream regulators and examining Exo84 localization

  • Generating chimeric proteins with domains from other fungal Exo84 homologs

  • Implementing optogenetic approaches to acutely mislocalize Exo84

  Studies in yeast showed that unlike Sec3p, which acts as a spatial landmark independent of secretory function, Exo84p polarization depends on functional secretory pathways . Determining whether N. fumigata Exo84 behaves similarly or functions more like a spatial landmark would provide important insights into hyphal growth regulation.

• How can researchers develop high-throughput screening approaches to identify small molecule inhibitors of N. fumigata Exo84?

  Development of targeted screening approaches includes:

  • Establishing in vitro binding assays with purified Exo84 and interaction partners

  • Designing split-reporter systems for monitoring Exo84 interactions in yeast

  • Creating fungal strains with growth dependent on Exo84 function

  • Implementing image-based screening for compounds that disrupt Exo84 localization

  • Developing biochemical assays for Exo84-dependent activities

  Given Exo84's essential role in fungal growth and potential involvement in virulence, compounds that specifically disrupt its function could represent novel antifungal strategies for treating aspergillosis.

Table 1: Predicted Functional Domains in N. fumigata Exo84 Based on Homology

Table 2: Comparison of Experimental Approaches for Studying Exo84 Function

Table 3: Potential Effects of Temperature on N. fumigata Exo84 Function Based on Yeast Studies