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.
Original language | English |
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Qualification | Doctor Degree |
Awarding Institution |
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Supervisors/Advisors |
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Award date | 19 Feb 2003 |
Place of Publication | Espoo |
Publisher | |
Print ISBNs | 951-38-6027-2 |
Electronic ISBNs | 951-38-6433-2 |
Publication status | Published - 2003 |
MoE publication type | G5 Doctoral dissertation (article) |
Keywords
- cereals
- barley
- Fusarium
- fungi
- plant pathogens
- proteinases
- proteinase inhibitors
- proteolytic enzymes