Submitted to: European Journal of Biochemistry
Publication Type: Peer reviewed journal
Publication Acceptance Date: 8/20/2003
Publication Date: 8/20/2003
Citation: PEKKARINEN, A.I., JONES, B.L. Purification and identification of barley (Hordeum vulgare L.) proteins that inhibit the alkaline serine proteinases of Fusarium culmorum. JOURNAL OF AGRICULTURAL FOOD CHEMISTRY. 2003. v. 51. p. 1710-1717. Interpretive Summary: In recent years the fungus Fusarium has caused great damage to barley crops in the Upper Midwest. This pest causes various biochemical changes in the barley that make it unusable for malting. This hurts producers by robbing them of the bonus they normally get for selling malting quality barley and consumers (maltsters and brewers) by imperiling their supply of acceptable raw product. When growing on barley, the Fusarium fungus produces two protein-degrading enzymes that apparently help it to attack and colonize the grain. In turn, the barley has evidently evolved to produce compounds, called `proteinase inhibitors', that can inactivate these enzymes. This study was carried out to ascertain how many of these inhibitors are present in barley grains and to determine their biochemical structures. The inhibitor proteins were purified from contaminants, separated from each other, and identified. Four major proteins (together with some partially degraded forms) were found that inhibited the Fusarium subtilisin-like proteinase and a single inhibitor of the trypsin-like proteinase was identified. These inhibitors had each been individually studied previously by other researchers, but this is the first time anyone has shown that, in a clearly defined pathogen-cereal system, the pathogen synthesizes enzymes to aid it in attacking the grain and that, in response, the grain makes inhibitors to counter these fungal enzymes. The import of this study is that the knowledge gained can be used by researchers to develope barley lines that are more resistant to attack by Fusarium species. They can increase the amounts of the inhibitors in the grain or target them to interact better with the fungus during infestation, thus enhancing the resistance of the grain. This in turn will provide resistant grain for producers to grow, which will ensure that there is sufficient malting barley to supply the malting and brewing industries.
Technical Abstract: It has been proposed that microbial proteinase inhibitors, which are present in abundance in cereal grains, protect the seed against plant pathogens. So far, however, very little is known about the interactions of those inhibitors with the proteinases of phytopathogenic microbes. The increased alkaline proteinase activities of Fusarium head blight (FHB) diseased wheat and barley grain imply that the Fusarium fungi synthesize those enzymes during colonization of the kernel. To study which barley proteins can inhibit Fusarium proteinases, and hence, possibly protect the seed from FHB, proteins of a grain extract have been separated and tested for their abilities to inhibit two alkaline serine proteinases that we previously isolated from F. culmorum. The proteins were separated by size exclusion, ion exchange and reversed phase-HPLC chromatographies. The purified inhibitors were identified by their molecular masses and N-terminal amino acid sequences. The proteins that inhibited the subtilisin-like Fusarium proteinase were the chymotrypsin/subtilisin (CI) inhibitor forms 1A, 1B and 2A and barley a-amylase/subtilisin inhibitor (BASI). Only one protein inhibited the trypsin-like proteinase. It was identical with the barley Bowman-Birk inhibitor (BBBI). The potential roles of these inhibitors in defending plants against diseases are discussed.