Recombinant Acremonium coenophialum Tubulin beta chain (TUB2)

What is the biological significance of Tubulin beta chain (TUB2) in Acremonium coenophialum?

The Tubulin beta chain (TUB2) in Acremonium coenophialum is a critical cytoskeletal protein that forms microtubules essential for various cellular activities, including cell division, intracellular transport, and maintenance of cell structure. In A. coenophialum, a mutualistic mycosymbiont of tall fescue grass (Festuca arundinacea), TUB2 plays a vital role in fungal growth, development, and the establishment of symbiotic relationships with host plants. Additionally, β-tubulins are significant targets for benzimidazole fungicides, making TUB2 an important protein for both basic research and applied studies in agricultural science and fungal biology .

How does TUB2 in A. coenophialum compare structurally to orthologs in related fungi?

TUB2 in A. coenophialum shares structural similarities with orthologs in related ascomycetes, particularly with the closely related Epichloë typhina. Molecular evolutionary analyses have revealed that β-tubulin genes underwent multiple independent duplications and losses throughout fungal evolution. The last common ancestor of basidiomycetes and ascomycetes likely possessed two paralogs of β-tubulin (β1/β2), but most ascomycetes lost the β2-tubulin genes during evolution. The remaining β-tubulin genes show evidence of divergent selection and adaptive positive selection at key functional sites, suggesting functional specialization across fungal lineages. This structural comparison is vital for understanding the evolutionary trajectory and functional divergence of TUB2 in A. coenophialum within the broader context of fungal tubulin evolution .

What are the known regulatory mechanisms controlling TUB2 expression in A. coenophialum?

The regulation of TUB2 expression in A. coenophialum involves several mechanisms, including elements in the 5' region of the gene. Research has shown that DNA segments 5' to the beta-tubulin gene can effectively drive expression of heterologous genes (such as hph conferring hygromycin resistance) when used in transformation systems. Additionally, the incorporation of terminator elements, such as the Emericella nidulans trpC terminator, significantly improves expression efficiency. This suggests a complex regulatory landscape involving both promoter elements and terminator sequences that control TUB2 expression under different conditions. Understanding these regulatory mechanisms is crucial for developing effective genetic manipulation systems in A. coenophialum and for exploring the physiological roles of TUB2 in this fungal symbiont .

What methods have proven most effective for recombinant expression of A. coenophialum TUB2?

The most effective method for recombinant expression of A. coenophialum TUB2 has been electroporative transformation of fungal protoplasts. This approach has been successfully used to introduce DNA constructs containing beta-tubulin gene elements into A. coenophialum. The protocol involves:

• Preparation of high-quality protoplasts from A. coenophialum cultures

• Introduction of DNA constructs via electroporation

• Selection of transformants on appropriate media (typically containing hygromycin when using the hph marker)

• Verification of successful transformation through molecular techniques such as Southern blotting

The efficiency of this method is significantly improved by incorporating specific genetic elements, particularly the Emericella nidulans trpC terminator, which can increase transformation efficiencies by 30-200% depending on the plasmid construct used. Vectors such as pCSN43, which incorporate trpC controlling elements for expression of selection markers, have also proven effective for A. coenophialum transformation, although they may integrate randomly into the genome .

How can one establish a marker recycling system for sequential genetic manipulation of TUB2 and related genes in Acremonium species?

Establishing a marker recycling system for sequential genetic manipulation of TUB2 and related genes in Acremonium species can be achieved through a uridine auxotrophy-based scarless gene deletion method, as demonstrated in Acremonium sp. HDN16-126. The detailed methodology involves:

• Identification and deletion of the native pyrG gene (encoding orotidine-5'-phosphate decarboxylase) to create a uridine auxotrophic strain

• Construction of gene disruption cassettes containing:

  • The target gene's upstream homologous region

  • The pyrG marker gene

  • The target gene's downstream homologous region

• Transformation of the auxotrophic strain with the disruption cassette

• Selection of transformants on uridine-free medium where pyrG complements the auxotrophy

• Second-round selection on medium containing 5-fluoroorotic acid (5-FOA) to counter-select against pyrG, allowing for marker recycling

• Verification of successful gene deletion through PCR and phenotypic analysis

This system allows for the recycling of the same selection marker in successive transformations, enabling multiple gene manipulations with limited markers. For TUB2 and related genes, this approach offers an efficient way to study gene function through sequential deletions or replacements. The method has shown high efficiency (66.7-83.3% for gene disruption) and creates stable multiple knock-out mutants without leaving residual sequences in the genome .

What factors influence the integration and expression stability of recombinant TUB2 in A. coenophialum?

Several factors influence the integration and expression stability of recombinant TUB2 in A. coenophialum:

Southern blot analysis of transformants has indicated the possibility of random integration of vectors like pCSN43 into the A. coenophialum genome, which can lead to variability in expression levels. For stable and predictable expression, targeted integration through homologous recombination is generally preferred. Additionally, the incorporation of species-appropriate regulatory elements, such as promoters and terminators, significantly enhances expression stability .

What experimental approaches best elucidate the functional role of TUB2 in A. coenophialum-grass symbiosis?

To elucidate the functional role of TUB2 in A. coenophialum-grass symbiosis, a multi-faceted experimental approach is most effective:

• Gene disruption and complementation studies: Create TUB2 knockout mutants using marker recycling systems, then reintroduce wild-type or modified TUB2 to assess rescue of phenotypes. This approach can reveal the necessity of TUB2 for symbiosis establishment and maintenance.

• Symbiosis establishment assays: Compare the ability of wild-type and TUB2-modified strains to colonize Festuca arundinacea seedlings, assessing colonization efficiency through microscopy and quantitative PCR.

• Transcriptomic analysis: Perform RNA-Seq on both fungal partners and host plants during different stages of symbiosis to identify co-regulated gene networks involving TUB2.

• Protein localization studies: Utilize fluorescent protein tagging (GFP-TUB2 fusion proteins) to visualize TUB2 distribution during different stages of fungal growth and plant colonization.

• Proteomic interaction studies: Employ co-immunoprecipitation followed by mass spectrometry to identify proteins that interact with TUB2 during symbiosis.

• Physiological assays: Assess the impact of TUB2 modifications on known symbiosis outputs, such as host protection against herbivory, drought tolerance, and alkaloid production.

These complementary approaches provide a comprehensive understanding of TUB2's role in the complex symbiotic relationship between A. coenophialum and tall fescue grass, potentially revealing new targets for agricultural applications .

How can researchers differentiate between the structural and regulatory functions of TUB2 in A. coenophialum?

Differentiating between the structural and regulatory functions of TUB2 in A. coenophialum requires sophisticated experimental designs that selectively target different aspects of TUB2 biology:

• Domain-specific mutations: Create precise mutations in different functional domains of TUB2 (GTP-binding, microtubule assembly, or protein interaction domains) to selectively disrupt specific functions while preserving others.

• Conditionally regulated expression systems: Develop temperature-sensitive or chemically-inducible TUB2 expression systems to temporally control TUB2 levels, allowing for the observation of immediate effects (likely structural) versus delayed effects (potentially regulatory).

• Interactome analysis: Compare the protein interaction networks of wild-type TUB2 versus mutant variants using techniques such as BioID or proximity labeling, followed by proteomics to identify regulatory partners.

• Chromatin immunoprecipitation (ChIP) studies: If TUB2 has potential nuclear functions, ChIP assays can identify any DNA-binding activities that would suggest direct regulatory roles.

• Phosphoproteomics: Identify post-translational modifications of TUB2 under different conditions to understand how its functions might be regulated and how it might regulate other cellular processes.

• Comparative studies with paralogs: Compare the functions of TUB2 with other tubulin paralogs in A. coenophialum or related species to identify specialized roles that have evolved through functional divergence.

This methodological framework allows researchers to disentangle the direct structural functions of TUB2 (microtubule formation and cytoskeletal organization) from its potential indirect regulatory roles in cellular signaling, gene expression, or symbiotic interactions .

What are the best methods for analyzing TUB2 protein-protein interactions in A. coenophialum?

The analysis of TUB2 protein-protein interactions in A. coenophialum requires specialized techniques adapted for filamentous fungi. The most effective methods include:

• Yeast two-hybrid (Y2H) screening: While not performed in A. coenophialum itself, Y2H can be used to screen for potential interaction partners when TUB2 is expressed in yeast along with a cDNA library from A. coenophialum.

• Co-immunoprecipitation (Co-IP): This technique involves:

  • Expression of tagged TUB2 (His, FLAG, or HA tags) in A. coenophialum

  • Cell lysis under conditions that preserve protein-protein interactions

  • Immunoprecipitation using antibodies against the tag

  • Mass spectrometry analysis of co-precipitated proteins

• Proximity-dependent biotin identification (BioID): TUB2 is fused to a biotin ligase, which biotinylates nearby proteins that can then be purified and identified by mass spectrometry.

• Fluorescence resonance energy transfer (FRET): TUB2 and potential interaction partners are tagged with appropriate fluorophores to detect interactions in living cells through energy transfer.

• Split-GFP complementation: TUB2 and candidate interacting proteins are fused to complementary fragments of GFP, which fluoresce only when brought together by protein interaction.

• Crosslinking mass spectrometry (XL-MS): Proteins are crosslinked in vivo or in vitro, digested, and analyzed by mass spectrometry to identify interaction interfaces.

When applying these methods to A. coenophialum, researchers must consider challenges specific to filamentous fungi, including cell wall digestion for protein extraction, potential interference from fungal secondary metabolites, and optimization of transformation protocols for expression of tagged proteins. The experimental design should also account for the dynamic nature of TUB2 interactions during different growth stages and symbiotic states .

How has TUB2 evolved in A. coenophialum compared to other fungal species, and what are the implications for functional studies?

TUB2 in A. coenophialum has undergone significant evolutionary trajectories shaped by both functional constraints and adaptation. Comparative genomic analyses across fungal lineages reveal several key evolutionary patterns with important implications for functional studies:

• Paralogous evolution: The last common ancestor of basidiomycetes and ascomycetes (which includes A. coenophialum) likely possessed two paralogs of β-tubulin (β1/β2). While most ascomycetes lost the β2-tubulin genes during evolution, the retention of specific paralogs in certain lineages suggests functional specialization. When designing functional studies, researchers must consider which paralog of TUB2 they are working with and be careful about extrapolating functions between paralogs .

• Positive selection: Molecular evolutionary analyses have identified sites in β-tubulins that have been under positive selection during fungal evolution. Many of these positively selected sites are at or adjacent to important functional sites and likely contribute to functional diversification. For functional studies, these sites represent high-priority targets for site-directed mutagenesis experiments .

• Functional divergence: Experimental confirmation of functional divergence between β-tubulin paralogs in Fusarium (another ascomycete) provides a blueprint for similar studies in A. coenophialum. Researchers identified type II variations responsible for functional shifts, which can guide the search for similar functional determinants in A. coenophialum TUB2 .

• Symbiosis-related adaptation: As a symbiotic fungus, A. coenophialum likely experienced selection pressures related to its association with grass hosts, potentially leading to unique adaptations in TUB2 not found in non-symbiotic fungi. Functional studies should incorporate comparative analyses with both closely related symbiotic fungi (e.g., other Epichloë species) and non-symbiotic relatives to identify symbiosis-specific adaptations.

These evolutionary insights provide crucial context for designing functional studies of TUB2 in A. coenophialum and interpreting their results within a broader evolutionary framework .

What are the key functional sites in A. coenophialum TUB2 that have experienced positive selection during evolution?

Molecular evolutionary analyses have identified several key functional sites in fungal β-tubulins that have experienced positive selection during evolution. While the specific sites in A. coenophialum TUB2 have not been directly enumerated in the provided search results, comparative analyses of β-tubulins across fungal lineages reveal patterns that likely apply to A. coenophialum TUB2. These positively selected sites include:

Many of these positively selected sites are at or adjacent to important functional sites and likely contribute to functional diversification of β-tubulins in different fungal lineages. For A. coenophialum TUB2, these sites represent prime candidates for site-directed mutagenesis to investigate functional specialization, especially in the context of symbiotic interactions with host plants. Molecular modeling approaches combined with experimental validation would be valuable for confirming the functional significance of these sites in A. coenophialum specifically .

How do differences between β1 and β2-tubulin paralogs inform our understanding of TUB2 function in A. coenophialum?

The differences between β1 and β2-tubulin paralogs provide critical insights into TUB2 function in A. coenophialum, particularly through comparative functional analysis:

• Paralog-specific functions: Molecular evolutionary analyses indicate that β1 and β2-tubulins have been under different selective pressures, with β2-tubulins showing evidence of strong divergent selection and adaptive positive selection. This suggests functional specialization, where each paralog may have evolved to perform distinct cellular roles. Although most ascomycetes lost β2-tubulin genes during evolution, understanding which paralog is represented by TUB2 in A. coenophialum is crucial for predicting its function .

• Experimental evidence from model systems: Functional divergence between β-tubulin paralogs has been experimentally confirmed in Fusarium, where researchers identified specific type II variations in FgTub2 responsible for functional shifts. These findings provide a template for investigating similar functional specialization in A. coenophialum TUB2 .

• Expression patterns: Different β-tubulin paralogs often show distinct expression patterns across developmental stages or environmental conditions. Analysis of when and where TUB2 is expressed in A. coenophialum, particularly during symbiotic interactions with host plants, can provide clues about its specialized functions.

• Protein interaction profiles: β1 and β2-tubulin paralogs may interact with different sets of proteins, reflecting their distinct cellular roles. Identifying the specific interaction partners of TUB2 in A. coenophialum can help determine whether it functions more like a β1 or β2 paralog from other fungi.

• Structural differences: Comparative structural analysis between β1 and β2-tubulins can highlight key amino acid differences that may account for functional specialization. These differences often cluster in regions involved in protein-protein interactions or post-translational modifications.

Understanding these paralog-specific features helps researchers develop more targeted hypotheses about TUB2 function in A. coenophialum, particularly in the context of its symbiotic lifestyle .

What are the most effective protocols for site-directed mutagenesis of A. coenophialum TUB2 to study structure-function relationships?

Site-directed mutagenesis of A. coenophialum TUB2 requires specialized protocols optimized for filamentous fungi. The most effective approach combines molecular techniques with the established transformation system for A. coenophialum. A comprehensive protocol includes:

• Target site selection:

  • Identify evolutionarily conserved or variable residues using sequence alignments

  • Focus on sites under positive selection as identified by molecular evolutionary analyses

  • Target functional domains (GTP-binding, lateral interactions, MAP-binding sites)

• Mutagenesis strategy:

  • For single amino acid substitutions: Use overlap extension PCR with mutagenic primers

  • For multiple mutations: Consider gene synthesis with all desired mutations

  • Design silent mutations to create restriction sites for screening

• Vector construction:

  • Clone the mutated TUB2 into a vector containing:

    • Native TUB2 promoter and terminator regions

    • Selectable marker (e.g., hygromycin resistance)

    • Flanking sequences for homologous recombination

  • Include a tag (e.g., HA, FLAG) if protein detection is needed

• Transformation:

  • Prepare protoplasts from A. coenophialum using cell wall-degrading enzymes

  • Transform using electroporation, which has proven effective for A. coenophialum

  • Select transformants on hygromycin-containing medium

• Verification:

  • PCR and sequencing to confirm the presence of the mutation

  • Western blotting to verify protein expression

  • RT-qPCR to assess expression levels

• Functional analysis:

  • Assess microtubule dynamics using fluorescence microscopy

  • Evaluate sensitivity to microtubule-targeting drugs

  • Test symbiotic capabilities with host plants

  • Analyze growth, development, and phenotypic changes

When implementing this protocol, the incorporation of the Emericella nidulans trpC terminator is recommended, as it has been shown to significantly improve transformation efficiencies in A. coenophialum. Additionally, for comprehensive structure-function studies, a complementation system using a TUB2-deleted background strain would be ideal, though this may require the marker recycling system described for other Acremonium species .

How can researchers effectively analyze the impact of TUB2 mutations on microtubule dynamics in A. coenophialum?

Analyzing the impact of TUB2 mutations on microtubule dynamics in A. coenophialum requires sophisticated imaging and biochemical techniques adapted for filamentous fungi. An effective analytical framework includes:

• Live-cell imaging approaches:

  • Express fluorescently-tagged (GFP or mCherry) α-tubulin in TUB2 mutant backgrounds to visualize intact microtubules

  • Use spinning disk confocal microscopy with high temporal resolution (1-5 seconds between frames) to capture dynamic instability

  • Employ photoactivatable or photoconvertible tubulin constructs for single-microtubule tracking

  • Analyze parameters including growth rate, shrinkage rate, catastrophe frequency, and rescue frequency

• Fluorescence recovery after photobleaching (FRAP):

  • Bleach a section of fluorescently-labeled microtubules and measure recovery rate

  • Calculate tubulin turnover rates in different TUB2 mutant backgrounds

  • Compare recovery half-times to quantify impacts on microtubule stability

• Biochemical assays:

  • Purify recombinant wild-type and mutant TUB2 proteins from heterologous expression systems

  • Perform in vitro polymerization assays to measure assembly kinetics

  • Use light scattering or turbidity measurements to quantify polymerization rates

  • Assess GTPase activity of purified TUB2 variants

• Electron microscopy:

  • Examine microtubule ultrastructure in TUB2 mutants using transmission electron microscopy

  • Quantify protofilament number and arrangement

  • Analyze microtubule-associated protein decoration patterns

• Drug sensitivity profiling:

  • Test sensitivity of TUB2 mutants to microtubule-targeting drugs (e.g., benomyl, nocodazole)

  • Generate dose-response curves to quantify resistance or hypersensitivity

  • Correlate structural changes with altered drug binding

• Computational modeling:

  • Build molecular dynamics simulations of wild-type and mutant TUB2

  • Predict effects of mutations on protein stability and interactions

  • Correlate computational predictions with experimental observations

• In vivo phenotypic analysis:

  • Quantify growth rates, morphology, and nuclear distribution in TUB2 mutants

  • Assess hyphal growth directionality and branching patterns

  • Evaluate effects on symbiotic capability with host plants

This multi-faceted approach provides complementary data on how specific TUB2 mutations affect microtubule dynamics at molecular, cellular, and organismal levels, enabling comprehensive structure-function analyses .

What are the challenges and solutions for integrating TUB2 studies with whole-genome functional analysis in A. coenophialum?

Integrating TUB2 studies with whole-genome functional analysis in A. coenophialum presents several significant challenges along with potential solutions:

Effective solutions for these challenges include:

• Adapt CRISPR-Cas9 for Acremonium: Optimize CRISPR-Cas9 systems for efficient genome editing in A. coenophialum, potentially using the electroporative transformation system that has proven effective for this organism.

• Develop marker recycling systems: Implement the uridine auxotrophy-based marker recycling system demonstrated in other Acremonium species for sequential gene deletions and modifications.

• Establish transcriptomic resources: Generate comprehensive RNA-Seq data across different growth conditions and symbiotic states to identify co-expressed gene networks involving TUB2.

• Create protein interaction maps: Develop systematic protein interaction networks centered on TUB2 and other cytoskeletal components to understand their functional relationships.

• Implement comparative genomic approaches: Leverage evolutionary insights from tubulin studies across fungal lineages to predict functional relationships in A. coenophialum.

• Develop integrative computational tools: Create bioinformatic pipelines specifically designed for integrating multi-omics data from symbiotic fungi to facilitate systems-level analysis.

By addressing these challenges with appropriate methodological and computational approaches, researchers can effectively integrate TUB2 studies with whole-genome functional analysis to gain comprehensive insights into the biology of A. coenophialum and its symbiotic relationships .

How can understanding TUB2 function in A. coenophialum contribute to enhancing grass-endophyte symbiosis for agricultural applications?

Understanding TUB2 function in A. coenophialum can significantly contribute to enhancing grass-endophyte symbiosis for agricultural applications through several research pathways:

• Optimizing colonization efficiency: TUB2 plays a crucial role in fungal growth and hyphal development, which are essential for successful plant colonization. By characterizing how specific TUB2 variants affect colonization patterns and efficiency, researchers can develop A. coenophialum strains with enhanced colonization capabilities, potentially leading to more robust symbiotic relationships with host grasses.

• Enhancing stress tolerance transfer: A. coenophialum confers significant drought and pest resistance to tall fescue. The cytoskeleton, including TUB2-containing microtubules, is likely involved in the production, transport, and possibly secretion of protective compounds. Understanding how TUB2 contributes to these processes could lead to optimized endophyte strains that provide enhanced stress protection to host plants.

• Regulating alkaloid production: A. coenophialum produces alkaloids that protect host plants from herbivory but can be toxic to livestock. The cytoskeleton likely plays a role in the cellular processes required for alkaloid biosynthesis. Engineered modifications to TUB2 could potentially modulate these pathways, allowing for the development of endophyte strains that maintain protective capabilities while reducing toxicity to grazing animals.

• Improving nutrient exchange: The mutualistic relationship between A. coenophialum and tall fescue involves bidirectional nutrient exchange. TUB2-containing microtubules likely facilitate intracellular transport involved in this exchange. A deeper understanding of TUB2's role could lead to optimized nutrient dynamics between the symbionts, enhancing plant growth and yield.

• Developing targeted fungicides: As β-tubulins are common targets for benzimidazole fungicides, detailed structural and functional analysis of A. coenophialum TUB2 could facilitate the development of compounds that selectively affect pathogenic fungi while preserving beneficial endophytes.

• Creating genetic tools for endophyte engineering: The transformation systems developed for A. coenophialum using TUB2-related elements provide valuable tools for genetic manipulation. Refining these tools based on deeper understanding of TUB2 biology could facilitate more sophisticated endophyte engineering for agricultural applications.

By pursuing these research directions, scientists can translate fundamental knowledge about TUB2 function into practical applications that enhance the agricultural benefits of grass-endophyte symbiosis .

What are the emerging research directions for studying the relationship between TUB2 evolution and functional adaptation in fungal endophytes?

Several emerging research directions show promise for elucidating the relationship between TUB2 evolution and functional adaptation in fungal endophytes like A. coenophialum:

• Comparative genomics across symbiotic gradients: Comparing TUB2 sequences and genomic contexts across closely related fungi with varying degrees of symbiotic dependence (from free-living to obligate symbionts) can reveal how TUB2 has evolved during the transition to symbiotic lifestyles. This approach can identify genomic signatures of adaptation specific to endophytic relationships.

• Ancestral sequence reconstruction: Reconstructing the evolutionary history of TUB2 through ancestral sequence reconstruction allows researchers to synthesize and test hypothetical ancestral TUB2 proteins. This can reveal which specific amino acid changes were critical for developing functions related to symbiosis.

• Experimental evolution: Subjecting A. coenophialum to controlled evolutionary pressures in laboratory settings, either in symbiosis with plants or in axenic culture, followed by sequencing to identify adaptive changes in TUB2, can provide direct evidence of how this protein evolves under different selective regimes.

• Structural biology of TUB2 variants: Using cryo-electron microscopy and X-ray crystallography to determine the structures of different TUB2 variants can reveal how evolutionary changes affect protein structure and function. This is particularly valuable for understanding how positively selected sites affect microtubule dynamics and interactions with other proteins.

• Ecological genomics: Field-based studies examining TUB2 sequence variation in A. coenophialum populations across different ecological gradients can link genetic variation to ecological adaptation, revealing how environmental factors drive TUB2 evolution.

• Systems biology of cytoskeletal evolution: Integrating data on TUB2 with other cytoskeletal components and regulatory networks can provide a systems-level understanding of how the entire cytoskeletal system co-evolves during adaptation to symbiotic lifestyles.

• Horizontal gene transfer analysis: Investigating the possibility of horizontal gene transfer of TUB2 or related genes between fungi, which could facilitate rapid acquisition of adaptive traits related to symbiosis.

• Correlating molecular evolution with symbiotic outcomes: Linking specific evolutionary changes in TUB2 with measurable parameters of symbiotic success (colonization efficiency, metabolite production, stress protection) can reveal which molecular changes have been most important for symbiotic adaptation.

These research directions represent frontier areas that combine evolutionary biology, structural biology, systems biology, and ecology to understand how TUB2 has evolved to support the endophytic lifestyle of A. coenophialum .

How might advances in understanding A. coenophialum TUB2 inform broader questions in cytoskeletal biology across eukaryotes?

Advances in understanding A. coenophialum TUB2 have the potential to inform fundamental questions in cytoskeletal biology across eukaryotes in several significant ways:

• Evolutionary plasticity of essential proteins: TUB2 is part of a highly conserved protein family that performs essential cellular functions, yet shows remarkable evolutionary plasticity in fungi. Studies in A. coenophialum can reveal how an essential cytoskeletal protein can undergo adaptive evolution without compromising its core functions, providing insights into the balance between conservation and innovation in essential proteins across eukaryotes.

• Specialized cytoskeletal functions in symbiosis: A. coenophialum's symbiotic lifestyle likely requires specialized cytoskeletal adaptations. Understanding how TUB2 has been modified to support symbiosis can reveal broader principles about cytoskeletal adaptations in other symbiotic systems, from lichens to mycorrhizal fungi to animal-microbe symbioses.

• Microtubule dynamics in non-model organisms: Most of our understanding of microtubule dynamics comes from a small number of model organisms. A. coenophialum represents a phylogenetically and ecologically distinct lineage whose study can broaden our understanding of the diversity of microtubule behaviors across eukaryotes.

• Environmental adaptation of the cytoskeleton: As a symbiont of plants growing in variable environments, A. coenophialum must adapt its cytoskeleton to diverse conditions. Studies of how TUB2 responds to environmental stressors can inform broader questions about cytoskeletal environmental adaptation in eukaryotes.

• Drug resistance mechanisms: β-tubulins are targets for various anti-fungal, anti-helminthic, and anti-cancer drugs. Understanding the molecular basis of naturally occurring variations in A. coenophialum TUB2 can provide insights into potential resistance mechanisms relevant to medical and agricultural applications across eukaryotes.

• Cytoskeletal role in specialized cellular trafficking: The symbiotic lifestyle of A. coenophialum likely involves specialized cellular trafficking pathways for exchanging signals and nutrients with host plants. Insights into how TUB2-containing microtubules support these pathways can inform our understanding of specialized trafficking in other systems, including polarized cells in animals.

• Post-translational regulation networks: Comparative studies of TUB2 regulation in A. coenophialum versus other fungi can reveal conserved and divergent aspects of cytoskeletal regulation networks, with potential implications for understanding these networks across eukaryotes.

By positioning research on A. coenophialum TUB2 within these broader contexts, findings from this specific system can contribute to fundamental questions in cytoskeletal biology with relevance across the eukaryotic domain .