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Development of a molecular genetics and cell biology toolbox for the filamentous fungus Diplodia sapinea

Summary

Scientists have developed new tools to study a fungus called Diplodia sapinea that damages pine trees around the world. They created a method to genetically modify this fungus and tag its cell nuclei with a red fluorescent marker so they can track the infection process. They also developed a simple way to test infections using young pine seedlings in the laboratory instead of large greenhouse setups. Using these new tools together, researchers can now watch in real-time how the fungus grows inside infected pine plants, which will help develop better ways to protect forests.

Background

Diplodia sapinea is a widespread fungal pathogen affecting conifers globally, causing shoot blight, canker, and tree death, with increasing impact due to climate change. Despite extensive research on this pathogen, fundamental questions about its biology and plant-associated lifestyle remain unanswered. The development of molecular genetics and cell biology tools is necessary to advance understanding of D. sapinea infection mechanisms.

Objective

To establish an Agrobacterium-mediated genetic transformation protocol for D. sapinea enabling targeted mutagenesis and heterologous gene expression. To develop a fluorescent protein tagging system for subcellular protein localization and to create a laboratory-scale infection assay using young Pinus sylvestris seedlings for studying host-pathogen interactions.

Results

Agrobacterium-mediated transformation achieved 20 transformants per 1×10⁵ spores with high rates of homologous integration at the niaD locus. The H2B-mCherry fusion protein successfully labeled fungal nuclei, demonstrating mCherry’s suitability for subcellular localization studies. Young P. sylvestris seedlings showed 77-100% infection success when wounded and inoculated with spore mass, and confocal microscopy revealed hyphal growth in non-necrotic plant tissue adjacent to infection sites.

Conclusion

The established transformation protocol, fluorescent protein labeling system, and laboratory-scale infection assay provide a comprehensive toolbox for D. sapinea research. These tools enable molecular and cellular analysis of infection processes, including visualization of pathogen-host interactions. The simplified experimental system facilitates future studies on virulence mechanisms, gene function, and infection dynamics.
  • Published in:PLoS One,
  • Study Type:Methodology Development Study,
  • Source: PMID: 39729429, DOI: 10.1371/journal.pone.0308794
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