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Published on: September 23, 2021
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An efficient transformation system for Trichoderma atroviride using the pyr4 gene as a selectable marker
Gabriela Calcáneo-Hernández1, Erick Rojas-Espinosa1, Fidel Landeros-Jaime1
1Unit for Basic and Applied Microbiology, School of Natural Sciences, Autonomous University of Queretaro, 76140, Queretaro, Mexico.
Summary
Uracil auxotrophic mutants in Trichoderma atroviride were created for genetic studies. This uracil metabolism system serves as a novel tool for gene knockouts, aiding in understanding fungal stress responses and light signaling.
Area of Science:
- Fungal Genetics and Molecular Biology
- Microbial Physiology
- Biotechnology
Background:
- Efficient genetic transformation systems are crucial for understanding Trichoderma atroviride.
- Developing novel selectable markers is essential for advancing genetic manipulation in fungi.
Purpose of the Study:
- To establish a uracil auxotrophy-based transformation system in Trichoderma atroviride.
- To utilize this system for gene knockout studies, specifically targeting MAPKK Pbs2, MAPK Tmk3, and blue light receptors Blr1/Blr2.
- To investigate the role of uracil metabolism as a selectable marker in fungal genetics.
Main Methods:
- Generation of uracil auxotrophic mutants by replacing the pyr4 gene with a hygromycin phosphotransferase (hph) marker.
- Selection of auxotrophs using 5-fluoroorotic acid (5-FOA) resistance.
- Knockout of target genes (Pbs2, Tmk3, Blr1, Blr2) using a short pyr4 version as a homologous marker.
- Phenotypic analysis of generated mutants, including stress sensitivity and photoconidiation.
Main Results:
- Uracil auxotrophs were successfully generated and characterized, with specific uracil concentrations and 5-FOA doses determined for growth restoration and lethality.
- Two distinct pyr4 deletions were identified in some auxotrophs, while others suggested potential mutagenic activity of 5-FOA.
- Knockout mutants for tmk3 and pbs2 exhibited high sensitivity to stressors and impaired photoconidiation.
- Mutants lacking blr1 and blr2 were unresponsive to light, with uracil complementation not affecting blue light-inducible gene expression.
Conclusions:
- Uracil metabolism provides a viable and versatile homologous selectable marker system for genetic manipulation in Trichoderma atroviride.
- This system facilitates the study of essential genes involved in stress response and light signaling pathways in fungi.
- The findings open new avenues for genetic engineering and functional genomics in Trichoderma species.
