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

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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. • Protocol for the generation of protoplasts from Trichoderma koningiopsis • Transformation techniques (PEG- and Agrobacterium -mediated) of T. koningiopsis • Use of electroporation and lipofection as transformation techniques for T. koningiopsis Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. 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.