THE XYLANOLYTIC SYSTEM OF CLAVICEPS-PURPUREA - CYTOLOGICAL EVIDENCE FOR SECRETION OF XYLANASES IN INFECTED RYE TISSUE AND MOLECULAR CHARACTERIZATION OF 2 XYLANASE GENES

Claviceps purpurea is a common phytopathogenic fungus that colonizes o varian tissue of grasses. A concerted approach involving cytological a nd molecular techniques was initiated to investigate the role of the f ungus' xylanolytic system in the interaction. Using enzyme-gold and im munogold electron-microscopic techniques, the presence of arabinoxylan s in cell walls of rye ovarian tissues (i.e., along the usual path df infection of C. purpurea) was confirmed; tissue-print and immunostaini ng analyses indicated the presence of xylanase(s) exclusively in ovari es infected with C. purpurea. This strongly suggests that C. purpurea secretes xylanase while colonizing its host. Two xylanase genes (cpxyl 1 and cpxyl2) were isolated from a genomic library of C. purpurea usin g genes from Cochliobolus carbonum (xyl1) and Magnaporthe grisea (xyn3 3) as heterologous probes. cpxl1 of C. purpurea had an open reading fr ame (ORF) of 832 bp interrupted by a 181-bp intron. The derived gene p roduct (CPXYL1) had a molecular mass of 21.5 kDa and an pI of 8.88; it showed significant homology to family G endo-beta-1,4-xylanases. The cpxyl2 ORF (1,144 bp) contained two introns (76 and 90 bp) and coded f or a polypeptide of 33.8 kDa with an pI of 7.01; CPXYL2 belonged to fa mily F xylanases. Southern analyses with genomic DNA demonstrated that both genes were single-copy genes. Using reverse transcription polyme rase 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 (Delta cpxyl1) had significantly reduced xylanase activity; Western an alyses confirmed that it lacked a polypeptide of approximately 23 kDa.