Characterization of Fusarium graminearum mutants with disruption of genes encoding GDSL lipases highly expressed during wheat spike infection

Wheat (Triticum aestivum) is the world’s second most significant staple crop. Global yields (typically up to 3 tonnes/ha) are severely threatened by the Fusarium Head Blight (FHB) disease, predominantly caused by the fungal pathogen Fusarium graminearum (Fg). This fungus impairs wheat grains quality contaminating them with hazardous mycotoxins like deoxynivalenol (DON), posing risks to both human and animal health. Given the escalating global demand for wheat and the increasing incidence of FHB outbreaks attributed to climate change, understanding the molecular mechanisms underlying Fg virulence is crucial for developing effective defence strategies to protect wheat. Previous studies have established a role for common GxSxG lipases, such as FGL1, as essential virulence factor for full Fg pathogenicity. However, the GDSL lipase/esterase family, an equally important class of hydrolytic enzymes, has been substantially overlooked in the context of Fg virulence. The GDSL lipases/esterases are a large family of hydrolytic enzymes with conserved GDSL catalytic domain and SGNH motif, usually present at the protein’s N terminus and in four conserved blocks (Ser-Gly-Asp-His). These enzymes are characterized by a remarkable versatility and a flexible active site which allow them to catalyse a broad range of reactions. Sixteen putative GDSL lipase genes have been identified in the Fg genome, yet their precise roles in the fungal molecular mechanisms and pathogenicity remain largely uncharacterised. This study aimed to characterize the 16 Fg GDSL lipase genes. An in-silico analysis confirmed their family classification and predicted enzymatic activities associated with the degradation of plant lipids and cell walls. Based on their high expression levels during wheat spike infection and the presence of a signal peptide, five GDSL genes were knocked out to produce mutant strains. The knockout mutants were characterized by growth under various stress conditions (osmotic, oxidative, alkaline, acidic/basic or fungicidal) and by pathogenicity assay on wheat spikes. The most significant discovery of this research pertains to the FGSG_04848 gene, one of the most upregulated during wheat spike infection. The corresponding FGSG_04848 knockout mutant was the sole to exhibit a 25% reduction in virulence during infection of wheat spikes. However, the experiments performed in this thesis did not clarify the possible mechanisms underlying the reduced virulence displayed by the FGSG_04848 knockout mutant, nor have detected possible defects caused by the knock-out of the five GDSL lipase genes.