Regulation and cell biology of secondary metabolite production in Fusarium graminearum and Fusarium pseudograminearum

Abstract

Ascomycete fungi have the potential to produce a vast array of secondary metabolites, which are metabolites not required for normal growth and development. In any one species there is typically the potential for production of upward of fifty of these compounds. Many fungal secondary metabolites are used as medicines (e.g. penicillin or cyclosporine), or are of importance due to their toxicity in humans or animals (e.g. aflatoxins), or because they are virulence factors during crop plant infection (e.g. deoxynivalenol). Despite the vast potential encoded in ascomycete genomes, the vast majority of fungal secondary metabolites are yet to be discovered. This is in part due to our lack of understanding of how the production of these compounds is regulated. Moreover, even for the compounds we do know, we are just beginning to understand the complex interaction of external stimuli, signalling pathways, cellular and developmental biology that leads to their production. In this thesis, biological insights into these aspects of two fungal secondary metabolites, deoxynivalenol (DON) and fusatin are provided. In chapter II a high-throughput fluorescence activated cell sorting (FACS) based mutant screen was developed and the regulation of DON production in Fusarium graminearum was analysed. DON is an economically very important mycotoxin, as it is the most common contaminant of wheat, barley and corn worldwide. It is produced by plant pathogenic filamentous fungi of the Fusarium family, which can cause Fusarium head blight as well as Fusarium crown rot. During plant infection, DON is an important virulence factor and it can be toxic for humans and animals. The regulation of DON production is not yet fully understood. To identify regulators, a mutant screen was developed using FACS to allow high throughput screening, coupled with whole genome sequencing to identify mutations. Segregation analyses were performed to determine the mutation causing the phenotype. The mutant screen identified an adenylyl cyclase allele, which resulted in production of DON under normally repressive conditions. In addition, the mutant developed cellular structures associated with toxin production under repressive conditions. The levels of the fundamental second messenger cAMP, which adenylyl cyclases produce, were increased in the identified mutant, suggesting it might be a gain-of-function adenylyl cyclase mutant. This mutant screening methodology can be adapted to identify regulators of different secondary metabolites as well as to identify mutants overproducing yet unknown secondary metabolites. In chapter III the cell biology of DON production in Fusarium graminearum was elucidated with quantitative super resolution microscopy. During the last five years the first insights into the cell biology of DON production were gained, indicating complex cell biological mechanisms. During toxin production, F. graminearum develops endoplasmic reticulum proliferations, called toxisomes, at which two DON biosynthesis enzymes, TRI1 and TRI4, are localized. However, TRI5 which catalyses the first DON biosynthesis step is localized in the cytosol. Thus, the question arose, how DON biosynthesis is coordinated between the cytosol and toxisomes. The subcellular localization of TRI5-GFP was analysed with structured illumination super resolution microscopy. This indicated a potential accumulation of the cytosolic TRI5 around the toxisomes. Accordingly, a quantification system was developed which confirmed a significant enrichment of TRI5-GFP around the toxisomes in comparison to the general cytosol. It is not understood yet, how the cytosolic TRI5 could be recruited to the toxisomes, but it could be part of a potential DON biosynthesis multi-enzyme complex at the toxisomes. In chapter IV the regulation of fusatin production was analysed in Fusarium pseudograminearum. F. pseudograminearum is another member of the Fusarium family and causes Fusarium crown rot of wheat and barley. Recently it was shown that F. pseudograminearum is able to produce cytokinins. Here, the regulation of cytokinin production was analysed in vitro and in planta. This revealed that cytokinin production in F. pseudograminearum can be induced by specific nitrogen sources similar to DON production in F. graminearum. DON and cytokinin quantifications showed that their production seems to be partially co-regulated. Fluorescence microscopy revealed that cytokinin production is induced during wheat seedling infection in hyphae in close association with the plant, indicating a potential role of cytokinin production during plant infection. In summary, this thesis presents a novel tool for high throughput mutant screens, as well as novel insights into the regulation and cell biology of DON and fusatin production in F. graminearum and F. pseudograminearum. Using these two secondary metabolites as examples, the results presented here show how complex the regulation of the production of these compounds can be and that it often also involves complex developmental and cell biological programs.