Characterisation of a xylanase gene from Cochliobolus sativus and its expression

Abstract

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.