Trichothecene transport in the phytopathogen Fusarium graminearum and trichothecene production in the entomopathogen Beauveria bassiana.

Abstract

This dissertation explores the transport mechanisms involved in the export of the trichothecene mycotoxin deoxynivalenol (DON) in the plant pathogenic fungus Fusarium graminearum, and the potential expression of a trichothecene biosynthetic gene homolog in the insect pathogen Beauveria bassiana. First, our current understanding of the biosynthesis and transport of fungal secondary metabolites is reviewed. The major classes of fungal secondary metabolites and the biosynthetic enzymes and gene clusters which produce them are covered, and notable examples of each secondary metabolite class are highlighted. This review emphasizes how our understanding of the biosynthesis of many secondary metabolites is far more complete than our understanding of the mechanisms which export or sequester the final bioactive molecule. Then, the role of the F. graminearum vesicular t-SNARE Sso2 in the export of DON is investigated. A knockout mutant of Sso2 is generated and shown to have an essential role in DON accumulation, virulence, and xenobiotic resistance. A double mutant, generated by crossing the Δsso2 mutant with the membrane-bound transporter mutant Δabc1, was shown to be further reduced in DON accumulation and virulence, indicating potentially interconnected, but independent, modes of DON export. Deletion of Sso2 is shown to down-regulate the expression of a number of F. graminearum secondary metabolite gene clusters and up-regulate many cellular repair gene categories. Subsequently, the role of Abc1 and other membrane-bound transporters in DON accumulation, virulence, and xenobiotic defense is investigated in further detail. Through in vitro, in planta, and heterologous expression studies it is shown that Abc1 plays the most substantial role in these processes, and other membrane-bound transporters may function primarily in defense and as accessory DON exporters. Afterwards, this dissertation examines the whether other Hypocreales fungi contain homologs for the trichothecene biosynthetic enzymes and identifies a putative trichothecene biosynthetic cluster in B. bassiana. Deletion attempts of the B. bassiana Tri5 homolog are undertaken but are unsuccessful, and further work suggests that a new antibiotic selection agent may be necessary. Expression studies indicate, however, that the BbTri5 homolog is not expressed in conditions known to induce trichothecene production in F. graminearum, bringing into question whether the B. bassiana trichothecene cluster is expressed in unknown conditions or not at all. This dissertation concludes by revisiting the major findings of each chapter and proposing future perspectives and experiments which have arisen throughout this body of research. Further examination of unused F. graminearum deletion mutants and heterologous expression of B. bassiana Tri homologs are emphasized for future consideration.