The serine proteinases of Fusarium grown on cereal proteins and in barley grain and their inhibition by barley proteins:Dissertation

Abstract

Fusarium head blight (FHB, scab) of wheat and barley is one of the most devastating diseases of cereals. Severe FHB epidemics have occurred all over the world, resulting in major yield and quality losses that cause problems to producers and to various industries that use grain as raw material. Scabby grain processes poorly and the toxins that are produced by the fungi cause potential health risks to humans and animals. The Fusarium fungi colonize cereal spikes and utilize the grain components for their own nutrition and reproduction. One of the interesting aspects of the infection mechanism is the question of how important is the hydrolysis of the host plant proteins by the invading fungus. Previous studies have indicated that protein degradation occurs in infected grains, implying that the fungi produce proteinases during the colonization of the kernel tissues. In addition, it has been proposed in the literature that host plants may use various proteinase inhibitors to defend themselves against pathogens. The purpose of this dissertation was to pinpoint and characterize the proteinases that are synthesized by Fusarium species to degrade grain proteins during infection and to identify and thoroughly examine any proteins in barley that can inhibit those enzymes. In this study, it was shown that species that cause FHB, F. culmorum, F. graminearum and F. poae, produced alkaline proteinases when grown in cereal protein media. Two proteinases were purified from a F. culmorum culture filtrate by using size-exclusion and ion exchange chromatographies. Both of the enzymes were maximally active at pH ~9 and 40-45 °C, but they were unstable under those conditions. The mechanistic classes of the enzymes were determined by measuring the effects of class-specific proteinase inhibitors on their activities and this indicated that they were subtilisin- and trypsin-like proteinases. In addition, portions of their amino acid sequences were homologous to those of other fungal proteinases that have been categorized into these classes. Both of the proteinases hydrolyzed C- and D hordeins (barley storage proteins) in vitro. The presence of these enzymes in field grown, FHB-infected barley was demonstrated by activity assays using N-succinyl-Ala-Ala-Pro-Phe pNA and N-benzoyl-Val-Gly-Arg pNA as substrates and by an immunoblotting method. These proteinases were inhibited by several barley proteins, which were then purified and identified. The subtilisin-like proteinase was inhibited by the barley a-amylase/subtilisin inhibitor (BASI) and by the chymotrypsin/subtilisin inhibitors 1A, 1B and 2A (CI-1A, -1B, -2A). The trypsin-like enzyme was only inhibited by the barley Bowman-Birk inhibitor (BBBI). The roles that these proteinases and their inhibitors may play during the Fusarium-infection are discussed