ABSTRACTSECRETED PROTEINS FROM THE PLANT PATHOGENIC FUNGUS FUSARIUM GRAMINEARUM: IDENTIFICATION, ROLE IN DISEASE, AND BIOTECHNOLOGICAL APPLICATIONSByJanet Marie Paper Fusarium graminearum, the causal agent of wheat head blight, is a necrotrophic pathogen of cereal crops worldwide, including wheat, maize and barley. Like other fungi, it acquires nutrients by secreting a large number of degradative enzymes into its environment. During pathogenesis, these proteins represent one of the first contacts between the pathogen and its host. In this dissertation, the secreted proteins of F. gramineariumwere identified and studied for their role in pathogenesis. The proteins secreted by F. graminearum during growth in vitro on 13 media containing different carbon sources and in planta during the infection of wheat heads were identified using mass spectrometry based proteomics. Out of a total of 256 fungal proteins identified, forty-nine were found in in planta growth but in none of the in vitro conditions. These proteins included secreted glycosyl hydrolases, unknown proteins, and a surprisingly large number of housekeeping proteins. The role of 38 of the in planta-specific proteins in pathogenesis was investigated by creating gene replacement mutants. None of the mutants had a strong virulence phenotype, probably due to genetic redundancy. One mutant in a gene encoding a putative alpha-arabinofuranosidase showed a small but statistically significant decrease in virulence. The secreted proteins were also investigated for their possible activity as elicitors, i.e., triggers of defense responses in Arabidopsis. The same culture filtrates used for proteomics analysis were assayed on Arabidopsis tissue for initiation of classic defense responses such as stunting of growth and induction of reactive oxygen species (ROS). The culture filtrates did inhibit the growth of Arabidopsis seedlings but did not induce ROS. One of the proteins that F. graminearum secretes in vitro on various carbon sources, including corn cell walls, is a putative &alpha-fucosidase, but this functional assignment had never been proven biochemically. The gene was expressed in Pichia pastoris and demonstrated to have activity on fucosylated pea xyloglucan but not on the model substrate p-nitrophenyl-fucoside. An &alpha- fucosidase from F. oxysporum that was previously biochemically identified but whose gene had never been cloned was purified and its gene cloned. This protein has activity on pNP-fucoside but not pea xyloglucan. The F. graminearum and F. oxysporum fucosidases are the first fungal fucosidases to be completely characterized from activity to protein to gene. They have possible applications in the conversion of biomass to fermentable sugars.
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