Cell-wall-degrading enzymes are expected to be important inpathogenesis by Cochliobolus sativus (Bipolaris sorokiniana). The xylanase activity of an isolate from barley was assayed in five different culture media. The highest activity was in Fries' medium containing 2% oat spelt xylan, 3% cellulose, and 4% peptone. To investigate genes coding for cell-wall-degrading enzymes in C. sativus, total DNA was digested with restriction enzymes and probed with C. carbonum XYL1 and PGN1 cDNAs, coding for a xylanase and an endo- polygalacturonase. The hybridization patterns indicate that C. sativus has genes resembling both. A cDNA library was constructed from mRNA of C. sativus grown on xylanase-inducing medium, and screened with C. carbonum XYL1 cDNA. All xylanase-encoding clones identified in this way were more similar to a second C. carbonum xylanase gene, XYL2, than to XYL1. Part of the C. sativus xylanase gene corresponding to the clones was amplified by the polymerase chain reaction (PCR), and found to have two introns like C. carbonum XYL2. Amplification with reduced stringency gave a second PCR product; DNA sequencing showed that it is derived from the C. sativus homologue of the C. carbonum XYL1 gene, with a single intron. The 5¢ and 3¢ flanking regions of the C. sativus XYL2 homologue were amplified by inverse PCR. The resemblance between the C. sativus and C. carbonum XYL2 sequences extends to the ends of the reported C. carbonum sequence.

English

9806|AGRIS 9802

Jose Juan Caballero

CIMMYT Publications Collection