Protocol for generating protoplasts of Trichoderma koningiopsis and transformation of plasmid DNA

Trichoderma koningiopsis is a filamentous fungus that produces numerous bioactive molecules and has the potential to be used as a biological control and plant growth-promoting agent. Here, we present a protocol for generating protoplasts of T. koningiopsis. We then describe four techniques for the stable transformation of plasmid DNA: polyethylene glycol (PEG)-mediated transformation of protoplasts, Agrobacterium-mediated transformation, electroporation of spores, and lipofection of mycelia. We then detail procedures for confirming the expression of a spCas9 gene using fluorescence microscopy.