Recombinant Ustilago maydis Altered inheritance of mitochondria protein 31, mitochondrial (AIM31)

What is AIM31 protein in Ustilago maydis?

AIM31 (Altered inheritance of mitochondria protein 31) is a mitochondrial protein in Ustilago maydis, also known as RCF1 (Respiratory supercomplex factor 1). It is a 214 amino acid protein with the sequence: MSGMPNAELVREQQQPGDPMGSSAHPNAYVPEVGGLGSELPEAPRDKFFRKMREQPLVPIGSLLTCGALIAASNHLRSGNRDQFNKALRWRVGFQGLTVLAALVGSFYYGQQAAATIPAPASSSADAPLQQGAVTTLPGRAPTVWQQTRADERANKGRNEFEGRVGQALDRELNDDKRLEEALLGKEEEINLEQLKKTATKPRPVIGQDARRQV . The protein has a UniProt ID of Q4PIK6 and is associated with the ORF name UMAG_00057. AIM31 is believed to play roles in both mitochondrial function and potentially in the regulation of mitochondrial inheritance during cell division, which is particularly important during the complex life cycle of U. maydis.

How does AIM31 relate to fungal pathogenicity mechanisms?

While direct evidence linking AIM31 to pathogenicity is not fully elucidated in the available research, its function must be understood within the context of U. maydis pathogenic development. In U. maydis, pathogenic development is tightly controlled by the b-mating type locus, which encodes the heterodimeric bE/bW transcription factor complex . This transcription factor complex is essential for filament formation, G2 cell cycle arrest, and triggering pathogenicity . Proper mitochondrial function and inheritance, potentially mediated by AIM31, would be critical supporting processes for the energy-intensive morphological changes that occur during pathogenic development. The b-mating type pathway involves a network of transcription factors, with Rbf1 serving as a master regulator for many genes involved in pathogenic development . Understanding AIM31's position within this regulatory network could provide insights into how mitochondrial functions support pathogenicity.

What is the genetic location and structure of the AIM31 gene?

The AIM31 gene in Ustilago maydis is identified by the ORF name UM00057 or UMAG_00057 . The gene encodes a protein of 214 amino acids that appears to have characteristics consistent with mitochondrial localization. The gene's specific chromosomal location within the U. maydis genome can be determined through its ORF designation. The protein sequence suggests it contains regions that facilitate its targeting to and function within mitochondria. Detailed structural analysis would be necessary to fully understand the functional domains of the protein and how they contribute to its role in mitochondrial processes.

What experimental systems are most suitable for studying AIM31 function?

Several experimental approaches are suitable for investigating AIM31 function:

• Recombinant protein expression systems - Both E. coli and eukaryotic expression systems have been used to produce recombinant AIM31 for biochemical studies . The recombinant protein can be produced with tags (such as His-tags) to facilitate purification and analysis.

• U. maydis genetic manipulation systems - Gene deletion, mutation, or fluorescent tagging of AIM31 in its native context allows for functional studies in vivo.

• Protein-protein interaction assays - Methods such as co-immunoprecipitation, yeast two-hybrid analysis, or bimolecular fluorescence complementation could identify interaction partners, similar to approaches used to study other U. maydis proteins like Clp1 .

• Microscopy-based approaches - Fluorescence microscopy can be used to visualize mitochondrial distribution and AIM31 localization during different developmental stages of U. maydis.

• Plant infection models - The maize-U. maydis pathosystem provides an excellent model to study the impact of AIM31 mutations on pathogenic development.

How is AIM31 conserved across fungal species?

While specific conservation data for AIM31 across fungal species is not detailed in the provided search results, addressing this question requires comparative genomic analysis. As a protein involved in mitochondrial function (and specifically as a respiratory supercomplex factor based on its RCF1 synonym ), AIM31 likely has homologs in other fungi, particularly basidiomycetes. The degree of sequence conservation would reflect the evolutionary importance of its function. Conservation analysis would be particularly interesting in the context of other plant pathogenic fungi to determine whether AIM31's potential role in pathogenicity represents a conserved mechanism. Sequence alignment tools and phylogenetic analysis would help determine the evolutionary relationships between AIM31 homologs and potentially identify functionally important domains that are conserved across species.

How does AIM31 interact with the b-mating type pathway in U. maydis?

The potential interaction between AIM31 and the b-mating type pathway represents a sophisticated research question. In U. maydis, the b-mating type locus encodes the bE/bW heterodimeric transcription factor, which controls both sexual development and pathogenicity . This heterodimer activates a complex regulatory cascade, with the zinc finger transcription factor Rbf1 serving as a master regulator that controls 90% of the genes responding to bE/bW activation .

The search results also identify Clp1 as a protein that physically interacts with both bW and Rbf1, affecting cell cycle regulation and pathogenic development . The potential interactions between AIM31 and these key regulatory proteins could reveal how mitochondrial inheritance is coordinated with sexual and pathogenic development. The complex regulatory network in U. maydis allows for multiple potential points of interaction:

• Direct interaction with b-mating type proteins

• Regulation by downstream transcription factors like Rbf1

• Coordination with cell cycle regulators during the G2 arrest phase

• Interaction with Clp1 during nuclear distribution in the dikaryon

Testing these possibilities would require techniques such as co-immunoprecipitation, yeast two-hybrid analysis, or bimolecular fluorescence complementation to identify direct protein-protein interactions.

What is the role of AIM31 in mitochondrial dynamics during the dimorphic transition?

U. maydis undergoes a dramatic morphological transition from yeast-like growth to filamentous form during its pathogenic development, triggered by the formation of the active bE/bW heterodimer . This transition is accompanied by a G2 cell cycle arrest and likely requires significant reorganization of cellular components, including mitochondria. AIM31's role in mitochondrial inheritance suggests it may be critically involved in ensuring proper mitochondrial distribution during this transition.

Research questions in this area could explore:

• How mitochondrial morphology and distribution change during the yeast-to-filament transition

• Whether AIM31 expression or localization changes during this transition

• If AIM31 mutations affect mitochondrial dynamics specifically during filamentous growth

• How mitochondrial inheritance mechanisms differ between yeast-like budding and filamentous growth

These questions would require advanced live-cell imaging techniques to visualize mitochondria during the morphological transition, coupled with genetic manipulation of AIM31 to observe the consequences of its absence or alteration.

How does the structure of AIM31 contribute to its function?

Understanding the structure-function relationship of AIM31 requires detailed structural biology approaches. Based on the amino acid sequence provided , several structural features could be analyzed:

• Potential transmembrane domains that might anchor the protein to mitochondrial membranes

• Conserved motifs that could mediate protein-protein interactions

• Regions that facilitate its mitochondrial localization

• Structural elements that could interact with the respiratory chain complexes (given its RCF1 synonym)

Techniques such as X-ray crystallography, nuclear magnetic resonance spectroscopy, or cryo-electron microscopy could be employed to determine the three-dimensional structure of AIM31. Site-directed mutagenesis of key residues followed by functional assays would help identify critical regions of the protein. Comparative structural analysis with similar proteins in other fungi could provide evolutionary insights into conserved mechanisms of mitochondrial inheritance and respiratory complex formation.

What is the impact of AIM31 mutations on U. maydis pathogenicity?

Investigating the effects of AIM31 mutations on U. maydis pathogenicity would provide direct evidence of its role in the infection process. This research direction could involve:

• Creating AIM31 knockout strains and assessing their ability to infect maize plants

• Generating point mutations in specific domains of AIM31 to identify regions critical for pathogenicity

• Analyzing the formation of infection structures in AIM31 mutant strains

• Examining the progression of infection over time, from penetration to spore formation

The assessment should consider multiple aspects of pathogenicity:

These studies would place AIM31 within the broader context of U. maydis pathogenic development and reveal the importance of mitochondrial inheritance for successful plant infection.

How does AIM31 function coordinate with cell cycle regulation during pathogenic development?

In U. maydis, activation of the b-mating type locus triggers a G2 cell cycle arrest that is essential for the formation of the infectious dikaryon; this arrest is only released after penetration of the host plant . The coordination between AIM31's function in mitochondrial inheritance and this cell cycle regulation represents a complex research question.

Key aspects to investigate include:

• Whether AIM31 expression or activity is cell cycle-dependent

• How mitochondrial inheritance is coordinated with the G2 arrest

• Whether AIM31 interacts with cell cycle regulators

• If AIM31 mutations affect the timing or maintenance of the G2 arrest

The search results indicate that Clp1 interacts with key regulatory proteins (bW, Rbf1, and Cib1) and is required for proper nuclear distribution during cell division in the dikaryon . Investigating potential interactions between AIM31 and Clp1 could reveal mechanisms that coordinate mitochondrial inheritance with nuclear distribution during the unique cell divisions that occur during U. maydis pathogenic development.

What are the optimal conditions for working with recombinant AIM31 protein?

Based on the product information for recombinant AIM31 , several key considerations emerge for working with this protein:

For recombinant AIM31 with tags (such as His-tags) , researchers should consider whether the tag might interfere with the protein's native function or interactions in their specific experimental context. If necessary, tag removal using appropriate proteases can be considered, though this should be balanced against potential yield reduction.

How can researchers effectively study AIM31 protein-protein interactions?

Investigating protein-protein interactions involving AIM31 requires careful experimental design. Based on the interaction studies described for other proteins in U. maydis , several approaches could be effective:

• Co-immunoprecipitation (Co-IP): This technique could identify proteins that physically interact with AIM31 in vivo. The recombinant AIM31 with appropriate tags can facilitate pull-down experiments.

• Bimolecular Fluorescence Complementation (BiFC): As demonstrated with Clp1 interactions , BiFC can visualize protein interactions in vivo. The search results describe how "coexpression of Clp1-NG either with Cib1-CG, bW1-CG, or Rbf1-CG led to a focused fluorescent signal in the nucleus, indicating protein–protein interactions" .

• Yeast Two-Hybrid (Y2H) Screening: This approach could identify novel interaction partners of AIM31.

• In vitro binding assays: Using purified recombinant AIM31, direct binding to candidate interactors can be assessed.

When designing these experiments, researchers should consider:

• The mitochondrial localization of AIM31 and how this compartmentalization affects interaction studies

• The need for appropriate controls to distinguish specific from non-specific interactions

• Validation of identified interactions through multiple independent methods

What experimental approaches can elucidate AIM31's role in mitochondrial inheritance?

To investigate AIM31's specific role in mitochondrial inheritance during U. maydis development, several experimental approaches are recommended:

• Gene deletion/mutation studies: Creating AIM31 knockout or mutant strains would allow observation of phenotypic effects on mitochondrial inheritance.

• Fluorescent protein tagging: Generating AIM31-GFP fusion constructs would enable visualization of protein localization and dynamics during different developmental stages.

• Mitochondrial labeling: Using mitochondrial-specific dyes or fluorescent proteins to track mitochondrial distribution in wild-type versus AIM31 mutant strains.

• Live-cell imaging: Time-lapse microscopy to observe mitochondrial movement and distribution during cell division in different genetic backgrounds.

• Electron microscopy: Ultrastructural analysis of mitochondrial morphology and distribution in wild-type versus mutant strains.

A comprehensive experimental design would combine these approaches to address questions such as:

• How are mitochondria distributed during different types of cell division in U. maydis (budding, filamentous growth, dikaryon formation)?

• How does AIM31 disruption affect these distribution patterns?

• Are there specific cell cycle phases or developmental stages where AIM31 function is most critical?

How can researchers design experiments to investigate AIM31's role in pathogenic development?

Investigating AIM31's role in pathogenic development requires experimental designs that span the infection cycle of U. maydis:

• Plant infection assays: Comparing the virulence of wild-type and AIM31 mutant strains on maize plants would provide direct evidence of its role in pathogenicity.

• Filament formation assays: Assessing the ability of AIM31 mutants to form filaments in response to appropriate stimuli could reveal its role in morphological transitions.

• Cell cycle analysis: Given the importance of G2 arrest in pathogenic development , analyzing cell cycle progression in AIM31 mutants during mating and early infection could be informative.

• Gene expression profiling: RNA-seq analysis comparing wild-type and AIM31 mutant strains during key developmental transitions could identify affected pathways.

• Epistasis analysis: Creating double mutants with known pathogenicity factors (e.g., components of the b-mating type pathway) could place AIM31 within the regulatory network.

A comprehensive experimental design would include multiple timepoints spanning the infection process:

What controls should be included when studying AIM31 in mitochondrial inheritance experiments?

Rigorous experimental design for studying AIM31's role in mitochondrial inheritance requires comprehensive controls:

• Wild-type controls: Include wild-type U. maydis strains processed identically to mutant strains for direct comparison.

• Complemented mutant controls: Reintroduce wild-type AIM31 into knockout strains to confirm that observed phenotypes are specifically due to the absence of AIM31.

• Mitochondrial visualization controls: Include established mitochondrial markers (e.g., MitoTracker dyes) to validate observations made with fluorescently tagged proteins.

• Cell cycle markers: Use established markers of cell cycle phases to correlate mitochondrial inheritance patterns with cell cycle progression.

• Environmental controls: Maintain consistent growth conditions across experiments, as mitochondrial dynamics can be affected by environmental factors.

• Technical controls: For microscopy-based studies, include controls for photostability, spectral overlap, and background fluorescence.

• Positive and negative controls: Include strains with known mitochondrial inheritance defects as positive controls and strains with mutations in unrelated processes as negative controls.

By incorporating these controls, researchers can ensure that observed phenotypes are specifically related to AIM31's function in mitochondrial inheritance rather than secondary effects or experimental artifacts.