Characterization and Disruption of A Gene in the Maize Pathogen Cochliobolus carbonum Encoding a Cellulase Lacking a Cellulose Binding Domain and Hinge Region. Paola Sposato . DOE-Plant Research Laboratory, Michigan State University, E. Lansing, Ml 48824. Joong-Hoon Ahn, and Jonathan D.Walton. DOE-Plant Research Laboratory, Michigan State University, E. Lansing, Ml 48824. MPMI 8:602-609. Accepted 18 April 1995. Copyright 1995 The American Phytopathological Society.

A gene, CEL1, in the maize pathogen Cochliobolus carbonum was identified using the cbhl-3 gene of Phanero-chaete chrysosporium as a heterologous probe. The predicted product of CELl, Cell, is 62% identical and 71% similar to the product of cbhl-3 and 54 to 62% identical to five cellobiohydrolases from other filamentous fungi. The location of the polyadenylation site 221 bp downstream of the stop codon and the location of a single intron of 55 bp were identified by comparison of the sequences of genomic and cDNA copies of CELl. The transcriptional start site was determined by rapid amplification of cDNA ends (RACE) to be 39 bp upstream of the putative translational start site. CELl mRNA abundance is high when C. carbonum is grown on cellulose or maize cell walls but is unde-tectable when grown on 2% sucrose or cellulose plus sucrose. Cell has a predicted signal peptide of 18 amino acids and therefore a mature size of 46.4 kDa. Like the product of cbhl-l of P. chrysosporium, but unlike most other endoglucanases and cellobiohydrolases (including the predicted product of cbhl-3), Cell does not have a putative cellulose binding domain or associated hinge region. The codon bias of CELl is stronger than the bias of cbhl-1 and comparable to that of cbhl-3 and that of the C. carbonum genes PGN1 and XYL1, (encoding endo-polygalacturonase and endo-xylanase, respectively). A strain of C. carbonum specifically mutated at CELl was produced by transformation with a truncated copy of CELl. Integration and disruption of CELl in the mutant was confirmed by DNA and RNA blotting. Pathogenicity of the CELl mutant was indistinguishable from the wild-type, indicating that CELl by itself is not a critical disease determinant. Culture filtrates of C. carbonum grown on cellulose or maize cell walls had several cellobiohydrolase, endoglucanase, and (-glucosidase activities that were separable by chromatofocusing, hydrophobic interaction, or ion-exchange high-performance liquid chromatography. However, all of the activities that were found were present in both the wild type and the CELl mutant and therefore are not Cell.