Knock-out of lipase-encoding genes in F. graminearum using homologous recombination and CRISPR-Cas9 system

Fusarium graminearum is the most prevalent Fusarium species that causes head blight disease in wheat, barley and other small grain cereals, resulting in major crop losses worldwide. This pathogen is also associated with other diseases, such as root rot, seedling blight, and foot rot, and with the production of mycotoxins, which lead to the reduction of quality and safety of the harvested grain. During the infection process, F. graminearum produces several hydrolytic enzymes, including lipases, which have been proposed as virulence factors in head blight disease. Nevertheless, fungal lipases have a broad range of functions and substrates, and the precise role of each is not yet completely understood. To test if two lipases of F. graminearum are involved in the infection process of the plant, two lipase-encoding genes, namely FGSG_02140 (Fgl2) and FGSG_03583 (Fgl5), have been addressed. Two approaches have been developed for the knock-out of Fgl5: homologous recombination and CRISPR-Cas9. The knock-out has been performed respectively by gene replacement and by active site disruption driven by specifically designed sgRNAs of the CRISPRCas9 system. The efficacy of all designed sgRNAs has been tested on their target region using PCRamplified DNA fragment as substrate. The main outputs of this project are the overall improvement of the optimization of the bioinformatic and wet lab pipeline of fungal transformation, especially for the design of sgRNAs for CRISPR-Cas9. This thesis work brings about the need for further studies on the optimization of a protocol to exploit the CRISPR-Cas approach in F. graminearum and represents a small step towards a better understanding of the molecular basis of the infection process of a fungal pathogen that is increasingly concerning.