352 Kaveh EMAMI and Ethan HACK* Department of Biological and Nutritional Sciences, University of Newcastle, Newcastle upon Tyne NE1 7RU, UK. E-mail : ethan.hackncl.ac.uk Received 22 March 2000 ; accepted 31 August 2000. A xylanase gene, XYL2, was identified and characterised in Cochliobolus sativus (anamorph Bipolaris sorokiniana), a necrotrophic cereal pathogen that attacks both shoots and roots. The fungus was grown on a xylanase inducing medium containing mineral salts, oat spelt xylan, cellulose, and peptone, RNA was isolated, and a complementary DNA (cDNA) library constructed. The library was screened with a xylanase (XYL1) cDNA clone from the maize pathogen Cochliobolus carbonum. Xylanase cDNA clones, all representing a single gene, were identified. Corresponding genomic DNA was amplified by PCR. Sequencing of the cDNA and the PCR products gave a nucleotide sequence of 2211 bp containing two introns in an open reading frame of 693 bp that codes for a xylanase from glycosyl hydrolase family 11. The most similar sequences to this gene in nucleotide sequence databases are the XYL2 gene of C. carbonum and a xylanase (XYL1) cDNA from a saprophytic fungus, Humicola insolens. Northern blot analysis and reverse transcription PCR (RT-PCR) showed expression of the gene when the fungus was grown on xylan or cellulose, but not when peptone or sucrose was the only carbon source. Expression of XYL2 in inoculated barley seedlings was detected by RT-PCR. INTRODUCTION Cochliobolus sativus (anamorph Bipolaris sorokiniana, formerly called Helminthosporium sativum), is a plant pathogenic fungus with a wide host range, including both monocot and dicot plants (Tinline 1988). It is most common on grasses and can cause severe disease in barley and wheat. It occurs worldwide and is most serious in tropical regions, where complete loss of wheat yield has been reported because of its activity (Mehta 1997). C. sativus can attack many parts of its hosts, producing a broad spectrum of symptoms including foot and root rot, leaf blotch, and black point of seeds (Tinline 1988). Because of this unusually wide range of symptoms, cell wall degrading enzymes (CWDEs) are likely to have a diversity of important functions for the fungus – in penetration, in rotting, and in saprotrophic growth in dead tissue. Thus, C. sativus has the potential to provide an instructive system for analysing the contribution of CWDEs to pathogenesis. Fungi that can degrade plant cell walls, whether pathogens or saprophytes, typically produce a multiplicity of CWDEs with overlapping functions (Walton 1994), so that analysing the role of these enzymes in pathogenesis is a complex problem. Even a mutant of Cochliobolus carbonum lacking three xylanase genes can grow on xylan as the only carbon source and is fully pathogenic to maize (Apel-Birkhold & Walton * Corresponding author. 1996). Individual CWDEs can, however, make identifiable contributions to disease severity. In the case of fungi, specific polygalacturonase genes are important for full virulence, but not essential for pathogenicity, of Botrytis cinerea on apples and tomatoes (ten Have et al. 1998) and of Aspergillus flavus on cotton bolls (Shieh et al. 1997). Thus, to define the role of CWDEs in pathogenesis, it is important both to identify what enzyme activities are involved in wall degradation and to determine how many genes code for each kind of activity. Primary cell walls of plants in the family Poaceae consist of up to 60 % xylans, of which glucuronoarabinoxylan is the major component (Carpita 1996). Glucuronoarabinoxylans are complex polysaccharides containing heterogeneous side chains attached to a backbone that is largely β-1,4-linked - xylopyranose. Thus, xylanases are likely to be particularly important CWDEs in interactions between pathogens of gramineous plants and their hosts. Whereas dicot tissue can commonly be macerated by the action of pectolytic enzymes alone, xylanases are commonly required for efficient macer- ation of grasses (Ishii 1984 ; Braun & Rodrigues 1993). Xylanases are among the first CWDEs produced when the cereal pathogens Fusarium culmorum, Pseudocercosporella herpo- trichoides, and Rhizoctonia cerealis are grown on wheat cell walls, and these pathogens produce higher levels of xylanases and lower levels of pectolytic enzymes than is typical for pathogens of dicot plants (Cooper et al. 1988). Because of their expected importance, we are examining the role of xylanases as pathogenicity factors in C. sativus. C. Mycol. Res. 105 (3) : 352–359 (March 2001). Printed in the United Kingdom. Characterisation of a xylanase gene from Cochliobolus sativus and its expression