The cloning, comparison, and expression of three family G endo beta-1,4-xylanase genes of the maize fungal pathogen Cochliobolus carbonum and analysis of their importance for pathogenicity on maiz

The filamentous fungus Cochliobolus carbonum, a pathogen of maize, makes three xylanases when grown in culture. A degenerate oligonucleotide based on the sequence of a tryptic peptide of the major xylanase, Xyl1, was used to clone the corresponding gene, XYL1. The oligonucleotide aLso hybridized to another xylanase gene, XYL3. A third xylanase gene, XYL2, was cloned by using XYL1 as a heterologous probe. All three xylanase genes encode family G endo-$beta$1,4-xylanases with basic pI's and predicted Mr's of approximately 22,000. At the amino acid level Xyl2 and Xyl3 are 60% and 42% identical to Xyl1, Xyl2 and Xyl3 are 39% identical. XYL1 and XYL2 but not XYL3 are expressed at the mRNA level in fungus grown in culture. XYL1 and XYL3 but not XYL2 are expressed in infected plants. Transformation-mediated gene disruption was used to create strains mutated in all three xylanase genes. In the XYL1 mutant, total xylanase activity decreased by 85% to 94% and two of the three previously characterized xylanase enzymes were gone. The XYL 2 mutant lacked XYL2 mRNA but no enzyme activity or major protein disappeared. By immunoblotting using an antibody raised against a 22-kDa xylanase from Trichoderma viride, a minor protein of 22 kDa could be observed to disappear in the XYL2 mutant. Since in all three xylanase mutants the third peak of xylanase activity remained in culture filtrates, this third xylanase must be encoded by yet another xylanase gene. The single xylanase mutants were crossed to each other to obtain multiple xylanase disruptions within the same strain. Strains disrupted in combinations of two and in all three xylanases were obtained. The triple mutant grew at the same rate as the wild type on xylane and maize cell walls. Additionally, the triple mutant was still fully pathogenic on maize as determined by lesion size, morphology, and rate of lesion development.