Functional characterization of pathogenicity genes in Fusarium circinatum

Abstract

The genus Fusarium includes some of the most destructive pathogens of agricultural and forestry crops. Within this genus, Fusarium circinatum is an important pathogen that causes severe disease on pine plants of all ages and is thus responsible for huge economic losses to forestry industries worldwide. In this thesis, the roles of putative pathogenicity genes (RAS2 and FUB1) were investigated in F. circinatum. This was done by generating ras2 and fub1 knockout mutants, and studying their phenotypes alongside F. circinatum wild type strain FSP34. The phenotypes included hyphal growth, conidiation, fertility, secondary metabolite production and virulence. Transcriptomes of these mutants and the wild type strain were also sequenced, and gene expression patterns were compared. Results from the phenotypic studies showed that deletion of RAS2 caused reduced growth of F. circinatum colonies as well as reduced virulence of the fungus on Pinus patula seedlings. The results also showed that the lack of the RAS2 gene in the knockout mutant strain slowed conidia germination, and resulted in fewer perithecia being produced in a sexual cross where this strain was a female. Phenotypic studies with the fub1 knockout mutant showed that disruption of the FUB1 gene caused an abolishment in the production of the fusaric acid secondary metabolite by F. circinatum. This disruption also caused the fub1 knockout mutant to produce smaller lesions on P. patula seedlings than the wild type strain. Analyses of gene expression patterns in the ras2 knockout mutant and wild type strain revealed that the deletion of RAS2 caused downregulation in numerous genes putatively involved in F. circinatum development and pathogenesis including transcriptional regulators and components of the mitogen-activated protein kinase (MAPK) signalling pathway. Results from analyses of gene expression patterns in the fub1 knockout mutant and the wild type strains also revealed a number of downregulated genes possibly involved in pathogenesis, and an upregulation genes of the bikaverin biosynthetic cluster. Collectively, the findings from this thesis show that RAS2 and FUB1 are virulence factor genes in F. circinatum. The findings also suggest that the Ras2 protein is a master regulator that controls growth, development and virulence in this fungus, and that it does so in a MAPK-dependent manner. This work will contribute to our knowledge of the molecular basis and mechanisms used by fungi in general to cause disease on their hosts.