Development of a CRISPR/Cas9 gene editing system for Fusarium oxysporum and characterization of an extracellular superoxide dismutase and its contribution to pathogenicity on cotton

Abstract

The Fusarium oxysporum species complex (FOSC) is an important group of filamentous fungi that are able to infect both animals and plants. Based on its economic importance, an effort has been made to better understand the genetics and pathogenicity of members of the FOSC; however, efficient reverse genetic techniques including gene disruption/deletion methods have been limited. One aim of this research was to establish an efficient, stable CRISPR/Cas9 mediated genome editing system for investigating fungal genetics and pathogenicity. In Chapter 2, a transformation method based on Cas9 ribonucleoproteins was developed and used to obtain URA3 and URA5 mutants, proving that the Cas9 RNPs could be transferred into fungal protoplasts and function. In addition, the FoBIK1 gene from a secondary metabolite biosynthetic cluster was mutated using this system and the maximum efficiency of this gene disruption was ~50%. In Chapter 3, the CRISPR/Cas9 system was used to carry out endogenous gene tagging (EGT) for the study of protein subcellular localization using two different integration strategies, homology-independent targeted integration (HITI) and homology-dependent recombination integration (HDRI). The HITI strategy was able to facilitate integration of a large DNA fragment, ~8 kb in length, into the genome of F. oxysporum at the sgRNA cleavage site, and was used to insert a C-terminal 3×sGFP tag to the FoCHS5 gene and a N-terminal mCherry tag to the FoSSO2 gene. The HDRI strategy was used to tag the paralogous gene, FoSSO1, with a C-terminal mCherry marker. In Chapter 4, an extracellular SOD protein (FoSOD5) from F. oxysporum was characterized. Expression of FoSOD5 was dependent on external environmental stimuli and the protein was localized to various cellular structures in different environments, and a lacZ expression reporter strain indicated FoSOD5 was induced in alkaline conditions. Importantly, a ΔFoSOD5 strain was more sensitive to reactive oxygen species and has attenuated virulence on cotton. Above all, a stable and efficient CRISPR/Cas9 system has been developed for F. oxysporum genome editing and was used for investigating gene regulation and protein subcellular localization.