Recombinant Sclerotinia sclerotiorum Nucleolar protein 58 (NOP58), partial

How do expression patterns of NOP58 compare with other virulence factors during infection?

Expression analysis would likely reveal temporal patterns similar to other S. sclerotiorum virulence factors. For example, SsNEP2 is abundantly induced during infection, suggesting its importance in pathogenicity . Similarly, the monooxygenase gene SsMNO1 plays pivotal roles in hyphal growth, sclerotial development, and virulence of S. sclerotiorum .

Methodological approach: Researchers should perform qRT-PCR analysis at various infection stages to determine NOP58 expression patterns. Dual RNA sequencing, as demonstrated in Bacillus/Sclerotinia interaction studies, can provide comprehensive insights into gene expression changes during infection .

What are effective approaches for producing recombinant S. sclerotiorum NOP58?

Recombinant NOP58 production requires careful consideration of expression systems and purification strategies.

Methodological approach:

• Expression system selection: E. coli BL21(DE3) is commonly used for fungal protein expression due to its high yield and simplicity.

• Vector design: Include a 6×His-tag or GST-tag for purification.

• Induction conditions: Optimize IPTG concentration (typically 0.1-1.0 mM) and temperature (16-37°C).

• Solubility enhancement: Consider fusion partners (SUMO, MBP) if NOP58 shows poor solubility.

• Purification: Use immobilized metal affinity chromatography followed by size exclusion chromatography.

Similar approaches have been successfully used for other S. sclerotiorum proteins, including the production of dsRNA targeting SsMNO1, which demonstrated inhibitory effects on sclerotial development .

What gene manipulation techniques are most effective for studying NOP58 function?

RNA interference and homologous recombination have proven highly effective for studying S. sclerotiorum gene functions.

Methodological approach:

• RNA interference: Design dsRNA or siRNA targeting NOP58 conserved regions, similar to the approach used for SsMNO1 .

  • External application of dsRNA demonstrated persistent inhibitory activity for over one week on Brassica napus .

  • For in planta expression, create hairpin RNAi constructs to generate transgenic plants with enhanced resistance .

• Gene knockout: Use homologous recombination with split marker method as demonstrated for SsNEP2 .

  • Verify knockout by PCR and RT-PCR to confirm complete deletion and absence of transcription .

  • Create complementation strains to validate phenotype restoration .

• CRISPR-Cas9: Design guide RNAs targeting NOP58 exons, followed by homology-directed repair to introduce specific mutations.

How can NOP58 research contribute to understanding S. sclerotiorum resistance mechanisms?

Studying NOP58 may reveal novel interactions between pathogen nucleolar proteins and host defense responses.

Methodological approach:

• Comparative transcriptomics: Perform dual RNA sequencing of wild-type and NOP58-deficient strains during infection, similar to the approach used in B. amyloliquefaciens and S. sclerotiorum interaction studies .

  • In such studies, the average FPKM of S. sclerotiorum transcripts in treatments with antagonistic bacteria decreased from 117.82 to 50.79, showing significant inhibition of pathogen gene expression .

• Protein-protein interaction studies: Use yeast two-hybrid or co-immunoprecipitation to identify host proteins interacting with NOP58.

• Host-induced gene silencing (HIGS): Develop transgenic plants expressing RNAi constructs targeting NOP58, similar to successful approaches with SsMNO1 that increased plant resistance .

What role might NOP58 play in S. sclerotiorum stress responses?

NOP58 could be involved in adaptation to environmental stresses during infection.

Methodological approach:

• Stress exposure experiments: Subject wild-type and NOP58-deficient strains to oxidative stress, temperature fluctuations, and plant defense compounds.

  • For oxidative stress tests, use H₂O₂ at varying concentrations (1-10 mM) and measure growth inhibition.

  • Compare with findings on other virulence factors like SsNEP2, which did not affect fungal sensitivity to oxidative stress but decreased ROS accumulation in S. sclerotiorum .

• Measure sclerotial development under stress conditions in wild-type and NOP58-manipulated strains, using protocols similar to those that revealed SsMNO1's role in sclerotial development .

How should researchers address functional redundancy when studying NOP58?

Nucleolar proteins often have redundant functions, complicating phenotype analysis of single gene manipulations.

Methodological approach:

• Multiple gene knockdowns: Design experiments targeting NOP58 alongside related nucleolar proteins.

• Domain-specific mutations: Instead of complete knockout, introduce mutations in specific functional domains to dissect roles.

• Conditional expression systems: Use inducible promoters to control NOP58 expression at specific developmental stages.

• Careful phenotypic analysis: Examine multiple parameters (growth rate, sclerotia formation, virulence) as done for SsNEP2, where knockout affected virulence but not mycelium morphology, sclerotial formation, or growth rate .

What are the best practices for analyzing NOP58 roles in pathogen-host interactions?

Comprehensive analysis requires multiple approaches to connect molecular function with pathogenicity.

Methodological approach:

• Infection assays on multiple host plants: Test virulence on diverse hosts as done with SsNEP2 on both A. thaliana and N. benthamiana .

  • Include trypan blue staining to visualize dead cells in infected tissues .

• ROS detection assays: Measure hydrogen peroxide and superoxide levels using fluorescent probes like DCFH-DA.

• Transcriptional profiling of host defense genes: Monitor expression of defense-related genes in plants infected with wild-type versus NOP58-deficient strains.

• Combine in vitro and in planta studies: Verify that observed effects in plate confrontation experiments translate to actual plant infections, as demonstrated in B. amyloliquefaciens antagonism studies .

How might NOP58 be exploited for developing novel control strategies against S. sclerotiorum?

NOP58 could serve as a target for RNA interference-based control strategies.

Methodological approach:

• External RNAi application: Test topical application of dsRNA targeting NOP58, similar to successful approaches with SsMNO1 where inhibitory activity persisted for over one week on Brassica napus surfaces .

• Transgenic resistance: Develop plants expressing hairpin RNAi constructs targeting NOP58, potentially conferring increased resistance to S. sclerotiorum infection .

• Biocontrol approaches: Investigate antagonistic microorganisms that might interfere with NOP58 function or expression, similar to how B. amyloliquefaciens inhibits S. sclerotiorum growth and gene expression .

What techniques are most effective for studying NOP58 interactions with the plant immune system?

Understanding how pathogen proteins interact with host immunity requires specialized approaches.

Methodological approach:

• PAMP-triggered immunity assays: Test if NOP58-derived peptides trigger immune responses, similar to studies showing nlp24 peptide from SsNEP2 triggered host MAPK activation and enhanced defense gene expression .

• Heterologous expression in model plants: Express NOP58 in N. benthamiana via agroinfiltration to observe potential necrosis-inducing activity.

• Co-immunoprecipitation with plant defense proteins: Identify potential interactions between NOP58 and host immunity components.

• Plant immune suppression assays: Determine if NOP58 can suppress immune responses triggered by known PAMPs.