Providing a toolbox for genomic engineering of Trichoderma aggressivum

Area of Science:

• Mycology
• Molecular Biology
• Biotechnology

Background:

• Trichoderma aggressivum causes green mold disease in Agaricus bisporus, impacting edible fungi yield.
• Despite its pathogenic role, T. aggressivum has potential as a biocontrol agent and plant growth promoter.
• Efficient molecular manipulation tools are needed to study T. aggressivum's biology.

Purpose of the Study:

• To develop a transformation protocol for Trichoderma aggressivum using plasmid DNA for ectopic integration.
• To establish a Cas9-ribonucleoprotein (RNP)-based approach for genome editing in T. aggressivum.
• To validate hph and pyr4 genes from Trichoderma reesei as effective selection markers.

Main Methods:

• Ectopic integration of the hph gene to determine transformation efficiency.
• CRISPR-Cas9 mediated disruption of the ornithine-5' phosphate decarboxylase gene.
• Sequencing of genomic loci around CRISPR target sites to confirm gene disruption.
• Validation of the pyr4 gene as a selection marker in generated auxotrophic strains.

Main Results:

• Successful transformation of T. aggressivum using plasmid DNA.
• Demonstrated genome editing capability via CRISPR-Cas9 RNP system.
• Identified four candidate strains with uridine auxotrophy and 5-FOA resistance.
• Confirmed the suitability of both hph and pyr4 genes as selection markers.

Conclusions:

• A robust genetic transformation and genome editing protocol for T. aggressivum has been established.
• The developed methods provide essential tools for researchers studying T. aggressivum's mycoparasitism and secondary metabolites.
• These advancements facilitate deeper investigation into T. aggressivum's biological mechanisms and biotechnological potential.