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The Xylanolytic System of Claviceps purpurea: Cytological Evidence for Secretion of Xylanases in Infected Rye Tissue and Molecular Characterization of Two Xylanase Genes

Claviceps purpurea is a common phytopathogenic fungus that colonizes ovarian tissue of grasses. A concerted approach involving cytological and molecular techniques was initiated to investigate the role of the fungus' xylanolytic system in the interaction. Using enzyme-gold and immuno-gold electron-microscopic techniques, the presence of arabinoxylans in cell walls of rye ovarian tissues (i.e., along the usual path of infection of C. purpurea) was confirmed; tissue-print and immunostaining analyses indicated the presence of xylanase(s) exclusively in ovaries infected with C. purpurea. This strongly suggests that C. purpurea secretes xylanase while colonizing its host. Two xylanase genes (cpxyl1 and cpxyl2) were isolated from a genomic library of C. purpurea using genes from Cochliobolus carbonum (xyl1) and Magnaporthe grisea (xyn33) as heterologous probes. cpxyl1 of C. purpurea had an open reading frame (ORF) of 832 bp interrupted by a 181-bp intron. The derived gene product (CPXYL1) had a molecular mass of 21.5 kDa and an pI of 8.88; it showed significant homology to family G endo-β-1,4-xylanases. The cpxyl2 ORF (1,144 bp) contained two introns (76 and 90 bp) and coded for a polypeptide of 33.8 kDa with an pI of 7.01; CPXYL2 belonged to family F xylanases. Southern analyses with genomic DNA demonstrated that both genes were single-copy genes. Using reverse transcription polymerase chain reaction, it could be shown that both genes were expressed in vitro and in planta (during all infection stages). Inactivation of cpxyl1 was achieved by a gene-replacement approach. The mutant strain (Δcpxyl1) had significantly reduced xylanase activity; Western analyses confirmed that it lacked a polypeptide of approximately 23 kDa.