Cloning and characterization of two endoxylanases from the cereal phytopathogen Fusarium graminearum and their inhibition profile against endoxylanase inhibitors from wheat Tim Belie ¨n, Steven Van Campenhout * , Maarten Van Acker, Guido Volckaert Laboratory of Gene Technology, Katholieke Universiteit Leuven, Kasteelpark Arenberg 21, B-3001 Leuven, Belgium Received 29 November 2004 Available online 15 December 2004 Abstract Two genes encoding family 11 endo-b-1,4-xylanases (XylA, XylB) from Fusarium graminearum were cloned and expressed in Escherichia coli. The amount of active endoxylanase in the cytoplasmic soluble fraction was considerably improved by varying dif- ferent expression parameters, including host strain and temperature during induction. Both recombinant endoxylanases showed a temperature optimum around 35 °C and neutral pH optima (around pH 7 and 8 for XylB and XylA, respectively). For the first time this allowed one to test endoxylanases of a phytopathogenic organism for inhibition by proteinaceous endoxylanase inhibitors TAXI and XIP. Whereas XylA and XylB were inhibited by TAXI-I, no inhibition activity could be detected upon incubation with XIP-I. The insensitivity of both F. graminearum endoxylanases towards XIP is surprising, since the latter is typically active against endoxylanases produced by (aerobic) fungi. As F. graminearum is an important phytopathogen, these findings have implications for the role of endoxylanase inhibitors in plant defence. Ó 2004 Elsevier Inc. All rights reserved. Keywords: Endoxylanase; Endoxylanase inhibitor; Fusarium graminearum; Plant defence Fusarium graminearum (teleomorph Gibberella zeae) is an important pathogen of cereal crops, causing head blight of wheat and barley, and producing a range of toxins [1]. Like most phytopathogenic microorganisms, Fusarium spp. secrete a number of hydrolytic enzymes capable of degrading cell wall polymers to invade the plant tissue and feed on the released nutrients [2]. Among these, pectic enzymes are extensively studied and established (pectate lyases) or considered (polygal- acturonases) to be virulence factors for phytopathogenic Fusarium spp. [2,3]. However, unlike dicotyledonous plants, pectin is only a minor constituent of gramina- ceous cell walls, which consist mainly of hemicellulose [4]. Since especially arabinoxylans represent a large pro- portion of the hemicellulosic fraction of cereal cell wall matrices, arabinoxylan degrading enzymes are expected to be important components of the offence arsenal of cereal pathogens [5,6] and may have a role similar to that of pectic enzymes in infection of dicotyledons [7]. Endo-b-1,4-xylanases (EC 3.2.1.8, further referred to as endoxylanases) are crucial for arabinoxylan depoly- merization as they break down the xylan backbone by catalysing the hydrolysis of b-1,4-xylan linkages. They have been classified mainly into two classes, family 10 and family 11 glycoside hydrolases, based on amino acid sequence similarities [8]. The only Fusarium endoxylan- ase genes studied to date belong to the tomato vascular wilt fungus Fusarium oxysporum f. sp. lycopersici [9–11]. Their differential expression pattern during infection of tomato plants (throughout the whole disease cycle or 0006-291X/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.12.036 * Corresponding author. Fax: +32 16 321965. E-mail address: steven.vancampenhout@agr.kuleuven.ac.be (S. Van Campenhout). www.elsevier.com/locate/ybbrc Biochemical and Biophysical Research Communications 327 (2005) 407–414 BBRC