Application of recyclable CRISPR/Cas9 tools for targeted genome editing in the postharvest pathogenic fungi Penicillium digitatum and Penicillium expansum.

Penicillium digitatum and Penicillium expansum are plant pathogenic fungi that cause the green and blue mold diseases, respectively, leading to serious postharvest economic losses worldwide. Moreover, P. expansum can produce mycotoxins, which are hazardous compounds to human and animal health. The development of tools that allow multiple and precise genetic manipulation of these species is crucial for the functional characterization of their genes. In this sense, CRISPR/Cas9 represents an excellent opportunity for genome editing due to its efficiency, accuracy and versatility. In this study, we developed protoplast generation and transformation protocols and applied them to implement the CRISPR/Cas9 technology in both species for the first time. For this, we used a self-replicative, recyclable AMA1-based plasmid which allows unlimited number of genomic modifications without the limitation of integrative selection markers. As test case, we successfully targeted the wetA gene, which encodes a regulator of conidiophore development. Finally, CRISPR/Cas9-derived ΔwetA strains were analyzed. Mutants showed reduced axenic growth, differential pathogenicity and altered conidiogenesis and germination. Additionally, P. digitatum and P. expansum ΔwetA mutants showed distinct sensitivity to fungal antifungal proteins (AFPs), which are small, cationic, cysteine-rich proteins that have become interesting antifungals to be applied in agriculture, medicine and in the food industry. With this work, we demonstrate the feasibility of the CRISPR/Cas9 system, expanding the repertoire of genetic engineering tools available for these two important postharvest pathogens and open up the possibility to adapt them to other economically relevant phytopathogenic fungi, for which toolkits for genetic modifications are often limited.