Kinetics of the Inhibition of Fusarium Serine Proteinases by Barley (Hordeum vulgare L.) Inhibitors

Abstract

Fungal infections of barley and wheat cause devastating losses of these food crops. The endogenous proteinase inhibitors produced by plant seeds probably defend the plants from pathogens by inhibiting the degradation of their proteins by the pathogen proteases. We have studied the interactions of barley grain inhibitors with the subtilisin-like and trypsinlike proteinases of Fusarium culmorum. The inhibition kinetics of three inhibitor proteins, chymotrypsin/subtilisin inhibitor 2 (CI-2), barley alpha-amylase/subtilisin inhibitor (BASI), and Bowman-Birk trypsin inhibitor (BBBI), have been studied in detail for the first time using fungal enzymes. The kinetic studies were performed at physiological pH values to mimic in vivo conditions. Numerical approaches to kinetic analyses were used to calculate the inhibition constants, because the data analyses were complicated by some inhibitor turnover and the instability of enzymes and substrates. All were slow, tight-binding inhibitors that followed either a two-step mechanism (CI-2 and BASI) or a single-step mechanism (BBBI) under the conditions investigated. The overall Ki values derived were approximately 50 pM, 1 nM, and 0.1 nM for CI-2, BASI, and BBBI, respectively. The main difference between the CI-2 and the BASI inhibitions was accounted for by the stabilities of their final complexes and the rate constants for their second dissociation steps (9 x 10(-6)/s and 3 x 10(-4)/s, respectively). Understanding the inhibition mechanisms will be valuable in designing improved strategies for increasing the resistance of the grains to fungal infections.

The influence of Fusarium infection and growing location on the quantitative protein composition of (Part II) naked barley (Hordeum vulgare nudum)

“…The reasons for this lower degree of degradation of barley storage proteins in comparison to that found with wheat [11] may be (1) the localized infection that occurred only on selected single grains in barley [29], or perhaps (2) the occurrence of proteinase inhibition by microbial proteinase inhibitors such as barley BowmanBirk inhibitor (BBI) or serine protease inhibitor [30,31], or (3) the synthesis of pathogenesis-related proteins (chitinase), defence-response proteins (oxidative burst response), protein-synthesis-related proteins and proteins involved in the phenylpropanoid biosynthesis (phenole and indole derivates) pathway as a reaction to the Fusarium infection [13][14][15][16].…”

The influence of Fusarium infection and growing location on the quantitative protein composition of (part I) emmer (Triticum dicoccum)

“…The reasons for this eVect may be the existence or higher expression of protease inhibitors (serine proteinase inhibitors) in emmer than in wheat [27].…”

Jasmonate and ethylene dependent defence gene expression and suppression of fungal virulence factors: two essential mechanisms of Fusarium head blight resistance in wheat?

“…During the rachis colonization when hyphae grow vertically [17], the toxin may inhibit the onset of various cell wall reinforcement processes in the vicinity of invading hyphae [18]. At the same time, fungal proteases are likely to participate in the suppression of plant defences by degrading pathogenesis-related (PR) proteins or defence-signalling compounds according to their property to cause proteolytic protein digestion [19-21]. In the spikes of the resistant landrace Wangshuibai the down-regulation of different housekeeping proteins was reported already 6 to 24 h after F. graminearum inoculation as a consequence of the secretion of fungal hydrolytic enzymes and toxins [22].…”

“…Both virulence factors are probably essential for the penetration and mortification of host cells, as Fusarium pathogens use cell wall digestion to enter living host cells [30] and DON, in particular, is known to activate plant programmed cell death [31,32]. In summary, DON and proteases have a significant impact on cell wall digestion, protein matrix reduction and damage to starch granules, typically seen in Fusarium -infected wheat kernels rendering grain yield unsuitable and unsafe for food, feed or malting purposes [19,33-36]. …”