Annual Progress Report on Malting Barley Research March, 2002

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STUDIES ON THE PROTEINASES THAT ARE PRODUCED WHEN FUSARIUM
GROWS ON CEREAL PROTEINS AND BARLEY PROTEINS THAT INHIBIT THEM
Anja I. Pekkarinen and Berne L. Jones
Cereal Crops Research Unit, USDA, ARS, Department of Agronomy,
University of Wisconsin, Madison, and VTT Biotechnology, Espoo, Finland
Objectives
The protein matrix of the wheat grain endosperm is degraded when the kernels are
attacked by the disease Fusarium head blight (FHB) or ‘scab’. It seems likely that the
same phenomenon also occurs in heavily infected barley kernels and that it may be
related to the method whereby Fusarium attacks the barley plant. We have shown that
two alkaline serine proteinases are synthesized by F. culmorum when it is cultured on
cereal storage proteins, and it seems likely that these enzymes are the ones that degrade
the proteins of infected barley grains. The proteinases are not produced when the
Fusarium is grown in media that lack protein. If, as seems probable, one function of these
proteinases is to help the fungus attack the barley head, then it seemed possible that the
grain might have evolved to produce compounds that could inactivate these enzymes and
thus retard its infection by the Fusarium. This study was carried out to purify and
characterize the Fusarium proteinases, to use the purified enzymes to test for the presence
of inhibitors in the barley and to determine whether it might be possible to manipulate the
inhibitors to increase the resistance of barleys to Fusarium attack.
<strong>Progress</strong> in studies on Fusarium culmorum endoproteinases
In the 2001 AMBA <strong>Annual</strong> Research <strong>Progress</strong> <strong>Report</strong>, we described how two alkaline F.
culmorum serine proteinases were purified from a culture medium and characterized.
These enzymes were denominated proteinase C (chymotrypsin-like) and proteinase T
(trypsin-like). At that time, we already had analyzed the N-terminal amino acid sequence
of the proteinase T, but our attempts to sequence the N-terminal amino acids of the other
protein had failed because the N-terminus of the enzyme was blocked and couldn’t be
sequenced by the Edman degradation. To get around this problem, the purified enzyme
has been digested into small peptides with a modified trypsin. These peptides were
separated and their amino acids were sequenced at the Protein Chemistry Laboratory in
the University of Texas Medical Branch (UTMB), Galveston, TX. The amino acids of the
peptides were highly homologous with those of several fungal subtilisins, but no
completely identical matches were found among the proteins that are listed in the various
amino acid sequence databases, indicating that it was a novel enzyme. Hence, it was
renamed proteinase S (subtilisin-like). In addition, we demonstrated that both proteinase
S and T hydrolyzed some of the barley storage proteins, the C- and D hordeins, in vitro.
The description of the purification and characterization of the proteinase S has recently
been published in the European Journal of Biochemistry (2002, Vol. 269, p. 798-807). A
manuscript covering the characterization of the second enzyme, proteinase T, has been
submitted to the Journal of Agricultural and Food Chemistry.
Recently, we discovered that the F. culmorum culture medium contained a third
proteinase enzyme. Preliminary studies have indicated that it is very different from the