Recombinant Botryotinia fuckeliana Protein rot1 (rot1)

What is Protein rot1 and what organism does it originate from?

Protein rot1 is a fungal protein originally identified in Botryotinia fuckeliana (strain B05.10), commonly known as the Noble rot fungus or by its anamorph name Botrytis cinerea . This pathogenic fungus is widely studied due to its significant impact on numerous economically important plant species, particularly in temperate regions . The rot1 protein (UniProt accession: A6S3W1) is encoded by the rot1 gene (ORF name: BC1G_07283) and represents a full-length protein with the mature expression region spanning amino acids 23-264 .

What expression systems are suitable for recombinant rot1 production?

Based on available research data, E. coli represents the predominant expression system for recombinant rot1 protein production across multiple fungal species . The bacterial expression system provides several advantages for research applications, including high protein yields, well-established purification protocols, and compatibility with His-tag fusion constructs. The recombinant Botryotinia fuckeliana rot1 protein is typically expressed as the full-length mature protein (amino acids 23-264) with an attached His-tag to facilitate purification .

What are the known or predicted functional domains of rot1 protein?

While the search results do not provide comprehensive information about the specific functional domains of rot1, the protein appears to contain several structurally important regions. Based on its sequence, researchers can employ bioinformatic approaches to predict potential functional domains through comparison with homologous proteins in related fungal species such as Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Ashbya gossypii, which also express rot1 proteins .

Common methodological approaches to identify functional domains include:

• Multiple sequence alignment with homologous proteins

• Secondary structure prediction algorithms

• Conserved domain database searches

• Structural modeling based on known protein structures

How can researchers design experiments to investigate rot1 protein interactions in fungal systems?

When investigating protein-protein interactions involving rot1, researchers should consider several methodological approaches:

• Co-immunoprecipitation (Co-IP) assays using anti-His antibodies for tagged recombinant rot1

• Yeast two-hybrid screening to identify potential binding partners

• Proximity-dependent biotin labeling (BioID) to capture transient interactions

• Surface plasmon resonance (SPR) to quantify binding kinetics

• Pull-down assays using recombinant rot1 as bait

For fungal-specific interactions, researchers should account for the native cellular environment of rot1 in Botryotinia fuckeliana. This may involve studies in heterologous fungal expression systems or directly in B. fuckeliana using genetic manipulation approaches similar to those described for other genes in this organism .

What genetic tools are available for studying rot1 gene function in Botryotinia fuckeliana?

Based on research with Botryotinia fuckeliana, several genetic approaches can be applied to study rot1 gene function:

• Transformation systems: DNA-mediated transformation methods have been developed for B. fuckeliana, as evidenced by work with benomyl resistance as a selectable marker . These systems can be adapted for rot1 gene manipulation.

• Gene disruption/replacement: Homologous recombination approaches can be employed to create rot1 knockout or modified strains to assess gene function.

• Expression analysis: Standard molecular techniques including RT-PCR, RNA sequencing, and Northern blotting can be applied to study rot1 expression patterns.

• Sexual crossing systems: The well-characterized sexual reproduction system of B. fuckeliana provides opportunities for genetic studies through controlled crosses and progeny analysis . The fungus exhibits a single mating type gene (MAT1) with two alleles (MAT1-1 and MAT1-2), which can be exploited for genetic manipulation .

How does genetic variation in rot1 compare across different fungal species?

Comparative analysis of rot1 proteins across fungal species reveals important evolutionary and functional insights. The following table summarizes key characteristics of recombinant rot1 proteins from various fungal species :

This comparative data indicates conservation of the general rot1 protein structure across diverse fungal lineages, with variations in protein length that may reflect species-specific functional adaptations . Researchers studying rot1 should consider these cross-species variations when designing experiments and interpreting results.

What methods are optimal for determining the three-dimensional structure of rot1 protein?

For structural determination of recombinant rot1 protein, researchers should consider several complementary approaches:

• X-ray crystallography: Requires high-purity protein samples and successful crystallization conditions. The His-tagged recombinant rot1 proteins available from expression systems would provide a starting point for purification and crystallization trials .

• NMR spectroscopy: Suitable for smaller protein domains if the full-length rot1 proves challenging for structural studies.

• Cryo-electron microscopy: Increasingly useful for proteins resistant to crystallization.

• Computational modeling: In the absence of experimental structures, homology modeling based on related proteins with known structures can provide structural insights.

The storage recommendations for rot1 proteins (in Tris-based buffer with 50% glycerol at -20°C or -80°C for extended storage) suggest potential challenges with protein stability that may need to be addressed during structural studies .

What are the key biochemical properties of rot1 protein that researchers should consider for experimental design?

When designing experiments involving recombinant rot1 protein, researchers should account for several biochemical properties:

• Storage stability: The protein is recommended to be stored in Tris-based buffer with 50% glycerol at -20°C for regular use or -80°C for long-term storage. Repeated freeze-thaw cycles should be avoided, and working aliquots can be maintained at 4°C for up to one week .

• Tag considerations: The His-tag commonly used in recombinant rot1 proteins may influence certain biochemical assays and should be considered when interpreting results .

• Buffer compatibility: The optimal Tris-based buffer system suggests potential sensitivity to buffer conditions that should be evaluated when designing biochemical assays .

• Protein regions: The mature protein region (e.g., amino acids 23-264 for B. fuckeliana rot1) represents the biologically relevant portion for most studies, though the specific function of different regions within this sequence requires further investigation .

How can rot1 protein be used in studying fungal pathogenicity mechanisms?

As Botryotinia fuckeliana (Botrytis cinerea) is a significant plant pathogen, rot1 protein may play roles in pathogenicity mechanisms that could be investigated through several research approaches:

• Comparative expression analysis: Examining rot1 expression levels during different stages of plant infection to correlate with pathogenicity events.

• Knockout/knockdown studies: Creating rot1-deficient strains to assess impacts on virulence in plant infection models.

• Protein-protein interaction studies: Identifying plant host proteins that may interact with rot1 during infection processes.

• Localization studies: Determining the subcellular localization of rot1 during host colonization using fluorescently tagged constructs.

Related research on B. fuckeliana has demonstrated sophisticated genetic approaches for studying pathogenicity factors, including histidine kinase mechanisms involved in fungicide resistance , which could serve as methodological models for rot1 studies.

What approaches can researchers use to investigate potential roles of rot1 in fungicide resistance mechanisms?

Building on established research with B. fuckeliana regarding fungicide resistance mechanisms , several approaches could be applied to investigate potential rot1 involvement:

• Comparative gene expression analysis: Examining rot1 expression levels in fungicide-resistant versus sensitive strains.

• Sequence analysis: Identifying potential polymorphisms in the rot1 gene between resistant and sensitive isolates, similar to approaches used for the beta-tubulin gene in benomyl resistance studies .

• Functional complementation: Introducing wild-type or mutated rot1 genes into sensitive strains to assess impact on fungicide sensitivity.

• Protein interaction studies: Investigating whether rot1 interacts with known fungicide resistance factors such as osmosensing histidine kinases implicated in dicarboximide resistance .

The evolution of fungicide resistance in B. fuckeliana field populations has been well-documented , providing a framework for similar investigations involving rot1 protein.

What are common challenges in working with recombinant rot1 protein and how can they be addressed?

Researchers working with recombinant rot1 protein may encounter several challenges:

• Protein stability: The storage recommendations (Tris-based buffer with 50% glycerol at -20°C/−80°C) suggest potential stability issues . Researchers should prepare small working aliquots to avoid repeated freeze-thaw cycles and consider adding protease inhibitors during experimental manipulations.

• Solubility concerns: As with many recombinant proteins, solubility may be limited under certain buffer conditions. Optimization of buffer components (salt concentration, pH, additives) may be necessary for specific applications.

• Activity preservation: The functional activity of rot1 may be sensitive to experimental conditions. When designing activity assays, researchers should control temperature, pH, and exposure to potential inhibitors.

• Tag interference: The His-tag commonly used with recombinant rot1 may interfere with certain functional studies. Where appropriate, researchers should consider tag removal using specific proteases, or comparing results with alternatively tagged constructs.

How can researchers validate the authenticity and activity of recombinant rot1 proteins?

Validation of recombinant rot1 protein quality and activity should include multiple approaches:

• Sequence verification: Confirming the expected amino acid sequence through mass spectrometry or N-terminal sequencing.

• Purity assessment: SDS-PAGE analysis with appropriate staining methods to verify size and purity.

• Western blot analysis: Using anti-His antibodies or rot1-specific antibodies to confirm identity.

• Structural integrity: Circular dichroism (CD) spectroscopy to assess secondary structure elements.

• Functional assays: Development of specific activity assays based on predicted functions or comparison with well-characterized homologs from model organisms.

• Thermal stability analysis: Differential scanning fluorimetry (DSF) to assess protein stability under various buffer conditions to optimize experimental protocols.