Semester

Summer

Date of Graduation

2011

Document Type

Dissertation

Degree Type

PhD

College

Davis College of Agriculture, Natural Resources and Design

Department

Biochemistry

Committee Chair

Daniel G. Panaccione.

Abstract

Ergot alkaloids are mycotoxins that negatively affect humans and animals but also have useful pharmaceutical properties. Ergot alkaloids have been extensively studied in Claviceps purpurea, and also have been reported in other fungi, such as certain Penicillium spp., Aspergillus fumigatus, and several Neotyphodium spp. that are endophytes of grasses. There is a high degree of variation in the production of ergot alkaloids within an individual species and among producers from different taxa, which means different fungi accumulate different profiles of ergot alkaloids rather than a single pathway end product. These varied profiles result, in part, from inefficiency in the pathway which leads to increased accumulation of certain pathway intermediates in addition to the end product of the pathway. These intermediates have different biological activities and provide different benefits to the host than the pathway end products. I conducted a series of experiments to investigate the potential mechanisms by which fungi accumulate different profiles of intermediates and end products. In A. fumigatus, efficiency in completing the pathway varied developmentally. Wild type conidiating cultures containing all cell types involved in conidiation (hyphae, vesicle of conidiophore, phialides of conidiophore, and conidia) accumulated more of the pathway intermediate festuclavine and less of the pathway end product fumigaclavine C than did isolated mature conidia. Such differences also were indicated by studies with a medA mutant, which proliferates phialides. The medA mutant accumulated more pathway intermediates than did the wild type or a complemented medA mutant. A stuA mutant, lacking recognizable conidiophores, produced no detectable ergot alkaloids. Although this variation in alkaloid accumulation indicated a difference in distribution or activity of ergot pathway enzymes, fluorescence associated with the expression of five tested alkaloid pathway genes indicated a similar pattern of expression of each gene in the different cell types. Fluorescence was not observed in young hyphae but was detected at the apices of hyphae as they swelled to form the vesicle of the conidiophore. More fluorescence was observed in mature vesicles, phialides, and conidia. No qualitative differences were observed in cell type-specific expression of different pathway genes, indicating that observed differences in accumulation of intermediates versus end product must be due to quantitative differences in expression or differences in activity of pathway enzymes. In Neotyphodium lolii x Epichloe typhina isolate Lp1, an endophytic symbiont of perennial ryegrass, different ergot alkaloid profiles were observed in different tissues of the host plant. RNA interference (RNAi) and overexpression of the easA gene genetically manipulated the flux of metabolites down the pathway. When compared to wild type, knock-down of easA gene resulted in higher accumulation of a pathway intermediate and a shunt product in comparison to pathway end product. In contrast, over-expression of easA resulted in higher accumulation of the pathway end product and no significant difference in the accumulation of intermediates and shunt products of the pathway. Collectively, my results indicated that the efficiency of the ergot pathway varies at cellular level in A. fumigatus and at the tissue level in Neotyphodium sp. Lp1. Also, in Neotyphodium sp. Lp1, efficiency in the flow of intermediates down the ergot alkaloid pathway can be manipulated by altering the expression of easA. .

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