Identification and applicability of azg1 as counter-selectable marker in Trichoderma species

Background The widespread use of pesticides and antibiotics in agriculture and clinical settings has accelerated the emergence of antimicrobial resistance across diverse organisms. Although this phenomenon is widely recognized in pathogenic microorganisms, much less is known about the effect on beneficial fungi that are playing a role in sustainable agriculture, such as Trichoderma . Many Trichoderma isolates, especially those originating from intensively treated soils, exhibit low sensitivity to commonly used laboratory antifungals, complicating their genetic manipulation. Results In this study, we developed new genetic tools to facilitate the characterization and engineering of Trichoderma species, exploiting endogenous counter-selectable markers. Using Trichoderma atroviride as a model species, we first assessed fungal susceptibility to the nucleobase analogs 8-azaguanine, 5-fluorouracil and 5-fluorocytosine. While the fungus was sensitive to the first two compounds, 5-fluorocytosine showed no inhibitory effect. Using CRISPR/Cas9-mediated protoplast transformation, we generated mutants lacking a putative nucleobase analog transporter and identified the gene responsible for 8-azaguanine uptake. Deletion of this gene conferred resistance to 8-azaguanine without affecting the overall phenotype or growth behavior of T. atroviride under a range of tested conditions. This locus was subsequently employed as a counter-selectable marker for targeted integration events in T. atroviride and additional Trichoderma species. Furthermore, heterologous expression of the Penicillium chrysogenum cytidine deaminase gene, which catalyzes the intracellular metabolization of 5-fluorocytosine to 5-fluorouracil, rendered T. atroviride sensitive to this compound. Conclusion Together, our findings establish azg1 as a multifunctional genomic platform and expand the genetic toolkit available for Trichoderma research, providing the foundation for advanced strain engineering in agricultural and biotechnological applications.