Recombinant Sclerotinia sclerotiorum DNA mismatch repair protein msh3 (msh3), partial

What is the role of MSH3 in DNA repair mechanisms?

MSH3 is a critical component of the mismatch repair (MMR) system that participates in maintaining genomic integrity. It typically forms a heterodimer (MutSβ) with MSH2 to correct insertion/deletion loops and base-base mispairs in microsatellites during DNA synthesis . The MSH3-MSH2 complex focuses specifically on recognizing and initiating repair of longer insertion/deletion loops, whereas the MSH2-MSH6 complex (MutSα) handles base-base mispairs and smaller insertion/deletion loops . This specialized function makes MSH3 particularly important for maintaining stability in repetitive DNA sequences.

How is MSH3 gene expression regulated in fungal systems?

In fungal systems, MSH3 expression patterns likely parallel those observed in other organisms, where it is typically expressed at low levels across different tissues and cell types . Based on studies in Aspergillus species, expression of DNA repair proteins may be influenced by environmental factors and developmental stages, including sclerotial development . The genetic control networks governing sclerotia formation, which involve transcription factors such as SclR and SclB, might indirectly affect MSH3 expression during different morphological transitions . Understanding these regulatory patterns is crucial for predicting how MSH3 functions during different phases of the fungal life cycle.

How do variations in the MSH3 gene influence pathogenicity in Sclerotinia sclerotiorum?

Variations in MSH3 may significantly impact S. sclerotiorum pathogenicity through several mechanisms:

• Genomic stability: MSH3 variants with altered function might increase mutation rates in the pathogen, potentially accelerating adaptation to host defenses.

• Sclerotial development: Given the relationship between DNA repair mechanisms and cellular differentiation, MSH3 may influence sclerotial formation - critical survival structures for this pathogen .

• Stress response: Pathogens encounter various stresses during host invasion; MSH3 variants could affect how well the fungus maintains genomic integrity under these conditions.

To investigate these relationships, researchers should consider:

• Creating MSH3 knockout or knockdown strains using CRISPR-Cas9

• Performing pathogenicity assays with mutant strains on various host plants

• Assessing sclerotial formation efficiency in strains with different MSH3 variants

• Measuring mutation rates in repetitive genomic regions that may influence virulence factors

What experimental approaches are most effective for expressing and purifying recombinant S. sclerotiorum MSH3?

Recommended expression systems:

Purification strategy:

• Clone the S. sclerotiorum MSH3 gene (full-length or partial) into an appropriate vector with an affinity tag (His6, GST, or MBP)

• Express in your chosen system under optimized conditions

• Lyse cells in buffer containing protease inhibitors

• Perform initial purification using affinity chromatography

• Apply secondary purification via ion exchange chromatography

• Finalize with size exclusion chromatography for highest purity

• Verify protein integrity by SDS-PAGE and Western blotting

• Assess functionality through DNA binding and ATPase activity assays

How does MSH3 influence repeat sequence stability in fungal genomes?

Based on findings in human systems, MSH3 plays a crucial role in the stability of repetitive DNA sequences, particularly in CAG·CTG repeat regions . In Huntington's disease and myotonic dystrophy type 1, specific variants of MSH3 are associated with reduced somatic expansion of disease-causing repeats . This suggests that in fungal systems like S. sclerotiorum, MSH3 may similarly impact the stability of repetitive elements.

Research approaches to investigate this function in fungi should include:

• Generating MSH3-deficient strains and measuring microsatellite stability over multiple generations

• Introducing reporter constructs with repetitive sequences to quantify expansion/contraction rates

• Comparing whole-genome sequences of wild-type and MSH3 mutant strains after growth under stress conditions

• Analyzing whether repeat instability in specific genomic regions correlates with phenotypic changes

The results from such studies may reveal whether MSH3-dependent repeat stability contributes to fungal adaptation or virulence, similar to how MSH3 variants affect disease progression in human trinucleotide repeat disorders .

What are the most reliable methods for assessing functional activity of recombinant S. sclerotiorum MSH3?

Table: Protocols for Functional Assessment of Recombinant MSH3

When performing these assays, researchers should consider:

• Testing multiple substrate types to determine specificity (different mismatches and loop sizes)

• Analyzing kinetic parameters under varying conditions (salt concentration, pH, temperature)

• Comparing activity of full-length versus partial/domain constructs to map functional regions

• Assessing the impact of site-directed mutations at conserved residues

How might MSH3-targeting strategies be developed for potential antifungal applications?

Building on insights from human studies, where MSH3 suppression shows therapeutic potential for Huntington's disease , researchers could explore similar approaches for antifungal development:

• Target identification: Recent findings show that lowering MSH3 levels by 41% halved CAG expansion rates, while 83% reduction completely halted expansion in human cells . This dose-dependency relationship could be investigated in fungal systems.

• Delivery methods: Antisense oligonucleotides (ASOs) have successfully targeted MSH3 in human cells . For fungi, modified delivery systems would be needed, potentially using:

  • Cell-penetrating peptides conjugated to nucleic acid therapeutics

  • Nanoparticle formulations optimized for fungal cell wall penetration

  • RNA interference constructs expressed from plant hosts

• Specificity considerations: Design strategies that specifically target fungal MSH3 without affecting plant or human homologs by focusing on divergent sequence regions.

• Resistance management: Since DNA repair processes influence mutation rates, monitor for potential resistance development through genomic instability.

What role does MSH3 play in the sclerotial development of S. sclerotiorum?

Sclerotia are melanized survival structures critical for the life cycle of S. sclerotiorum. While direct links between MSH3 and sclerotial formation have not been established, several connections can be investigated:

• Developmental transitions: Sclerotial formation involves dramatic cellular differentiation , which may require precise regulation of genomic stability where MSH3 plays a role.

• Research approach:

  • Generate MSH3 knockout or knockdown strains and assess their ability to form sclerotia

  • Analyze MSH3 expression patterns during different stages of sclerotial development

  • Investigate potential interactions between MSH3 and known sclerotial development regulators like SclR and SclB

  • Examine whether environmental factors that influence sclerotial formation (light, temperature, nutrients) also affect MSH3 expression

• Potential mechanism: MSH3's role in maintaining repetitive sequence stability may influence the expression of genes containing microsatellites in their regulatory regions, potentially affecting developmental pathways.

How do MSH3 interaction networks compare between S. sclerotiorum and other organisms?

MSH3 typically interacts with several proteins as part of its function in DNA repair. In humans, MSH3 has been shown to interact with MSH2, PCNA, and BRCA1 . A comparative analysis of MSH3 interaction networks could reveal:

• Core conserved interactions: The MSH3-MSH2 interaction is likely conserved across species as the formation of the MutSβ complex is fundamental to MSH3 function .

• Fungal-specific interactions: S. sclerotiorum MSH3 may interact with unique proteins involved in fungal-specific processes, such as those related to sclerotial development or plant infection.

• Experimental approaches:

  • Yeast two-hybrid screening using S. sclerotiorum MSH3 as bait

  • Co-immunoprecipitation followed by mass spectrometry

  • Proximity-dependent biotin identification (BioID)

  • Computational prediction and comparative analysis with known interactors from other species

Understanding these interaction networks could reveal potential targets for antifungal strategies or explain species-specific aspects of DNA repair in fungi.

What are the key considerations for designing experiments to study MSH3 in S. sclerotiorum?

When designing experiments to study MSH3 in S. sclerotiorum, researchers should consider:

• Genetic manipulation strategies:

  • CRISPR-Cas9 systems optimized for filamentous fungi

  • Homologous recombination-based approaches for gene replacement

  • Inducible expression systems to study dosage effects

• Phenotypic assays:

  • Standardized methods for measuring sclerotial formation

  • Protocols for assessing pathogenicity on different host plants

  • Mutation rate analysis using reporter genes or whole-genome sequencing

• Environmental variables:

  • Growth conditions that might affect MSH3 expression (temperature, pH, nutrients)

  • Oxidative stress conditions to challenge DNA repair systems

  • Light conditions, which can impact sclerotial development

• Controls:

  • Include positive controls with known DNA repair deficiencies

  • Use complementation studies to confirm phenotype specificity

  • Compare results across multiple independently generated mutant lines

How can researchers effectively measure MSH3-dependent mutation rates in fungi?

To accurately measure MSH3-dependent mutation rates in S. sclerotiorum, consider these methodological approaches:

• Reporter gene systems:

  • Integrate microsatellite sequences within reporter genes (e.g., URA3 or GFP)

  • Measure frequency of loss-of-function or frameshifts

  • Compare rates between wild-type and MSH3-deficient strains

• Whole-genome approaches:

  • Perform long-term evolution experiments with wild-type and MSH3 mutant strains

  • Use whole-genome sequencing to identify accumulated mutations

  • Focus analysis on repetitive regions most likely to be affected by MSH3 deficiency

• Specific locus analysis:

  • Design PCR-based assays targeting known microsatellite regions

  • Use fragment analysis to detect length changes in repetitive sequences

  • Sequence amplicons to confirm expansions or contractions

• Data analysis considerations:

  • Apply appropriate statistical models for mutation rate calculation

  • Account for selection bias in your experimental system

  • Consider using fluctuation analysis (Luria-Delbrück method) to distinguish between mutation rates and selection effects

What technology platforms are most suitable for structural studies of S. sclerotiorum MSH3?

For structural characterization of S. sclerotiorum MSH3, researchers should consider these complementary approaches:

What are the most promising future research directions for S. sclerotiorum MSH3 studies?

Based on current knowledge and gaps, several promising research directions emerge:

• Comparative genomics: Analyzing MSH3 sequence and function across fungal pathogens with different lifestyles could reveal adaptations specific to plant pathogenicity.

• Gene-environment interactions: Investigating how environmental conditions affect MSH3 function could explain aspects of fungal adaptation to different hosts or environmental niches.

• Translational applications: Developing MSH3-targeting antifungal strategies based on the proven efficacy of MSH3 suppression in human disease models .

• Systems biology approaches: Integrating MSH3 function into broader networks of DNA repair, stress response, and developmental pathways in S. sclerotiorum.

• Evolutionary studies: Examining how MSH3 variants contribute to genetic diversity and adaptation in fungal populations, potentially influencing host range and virulence.

These research directions offer opportunities to not only understand fundamental aspects of DNA repair in fungal pathogens but also to develop novel strategies for controlling economically important plant diseases caused by S. sclerotiorum.

How might findings from human MSH3 studies inform research on fungal MSH3?

Recent discoveries about MSH3 in human disease contexts provide valuable insights that could guide fungal research:

• Dosage sensitivity: Studies in Huntington's disease models have established precise relationships between MSH3 levels and repeat expansion rates, with a 41% reduction halving expansion rates and an 83% reduction completely halting expansion . This dose-dependency could be investigated in fungal systems.

• Genetic variants: The identification of a three-repeat allele in human MSH3 exon 1 associated with reduced somatic expansion and disease modification suggests that searching for natural variants in fungal MSH3 might reveal functional differences.

• Interaction networks: Human MSH3 interacts with several proteins including MSH2, PCNA, and BRCA1 . Comparative analysis of these interactions in fungi could reveal conserved and divergent aspects of MMR function.

• Expression regulation: Understanding how MSH3 expression is regulated in different organisms might reveal fungal-specific control mechanisms that could be targeted.