Molecular characterization of GDSL lipase mutants of Fusarium graminearum and evaluation of their virulence in planta.

Fusarium graminearum is a well-known pathogen infecting various cereals and causing the Fusarium Head Blight (FHB) disease. Its capacity to infect the spike tissue during anthesis causes a reduction of crop yields and quality, mostly because F. graminearum contaminates grains with mycotoxins, particularly deoxynivalenol (DON), which represents a threat for food safety and human health. Understanding the molecular mechanisms that explain the infection process of this pathogen is essential in order to develop practical preventive techniques. Research has been made on the F. graminearum genome showing more than 15 genes encoding for GDSL lipases, which is a class of catalytic enzymes widely found in plants and microorganisms. This class of lipases has a unique mechanism of catalysis, represented by a flexible substrate-binding site as well as a conserved amino acid sequence motif (Gly-Asp-Ser-Leu), which makes it structurally and functionally different from classic lipases, which have a typical triad catalytic core (Ser, His, Asp). Although there is a lot of research about the role of GDSL lipases in plants, their role in fungi has not been ascertained yet. A gene expression study performed by qPCR identified that some of the 15 F. graminearum GDSL lipase encoding genes show a remarkably high expression during the early stages of plant infection. Targeted knock-out mutants of the most expressed genes (Lip2, Lip4, Lip12 and Lip14) were produced aiming to understand their function by observing the effects of gene loss. Preliminary, two steps of molecular characterization of the mutant strains obtained were performed. A first PCR screening to identify gene disruption in the knocked-out mutants and discriminate the ectopic mutants where the insertion of the construct occurs elsewhere in the fungal genome; a second PCR screening with primers targeting the flanking regions of the construct to confirm its integration in the right gene locus and verify possible undesirable mutations occurring during the knocking out process. Finally, the GDSL lipase mutants characterized by PCR were used in inoculation tests on soybean to check whether the loss of these GDSL lipase encoding gene may affect fungal virulence.