Characterization and disruption of a gene in the maize pathogen Cochliobolus carbonum encoding a cellulase lacking a cellulose binding domain and hinge region

Abstract

A gene, CEL1, in the maize pathogen Cochliobolus carbonum was identified using the cbh1-3 gene of Phanerochaete chrysosporium as a heterologous probe. The predicted product of CEL1, Cel1, is 62% identical and 71% similar to the product of cbh1-3 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 CEL1. The transcriptional start site was determined by rapid amplification of cDNA ends (RACE) to be 39 bp upstream of the putative translational start site. CEL1 mRNA abundance is high when C. carbonum is grown on cellulose or maize cell walls but is undetectable when grown on 2% sucrose or cellulose plus sucrose. Cel1 has a predicted signal peptide of 18 amino acids and therefore a mature size of 46.4 kDa. Like the product of cbh1-1 of P. chrysosporium, but unlike most other endoglucanases and cellobiohydrolases (including the predicted product of cbh1-3), Cel1 does not have a putative cellulose binding domain or associated hinge region. The codon bias of CEL1 is stronger than the bias of cbh1-1 and comparable to that of cbh1-3 and that of the C. carbonum genes PGN1 and XYL1, (encoding endopolygalacturonase and endo-xylanase, respectively). A strain of C. carbonum specifically mutated at CEL1 was produced by transformation with a truncated copy of CEL1. Integration and disruption of CEL1 in the mutant was confirmed by DNA and RNA blotting. Pathogenicity of the CEL1 mutant was indistinguishable from the wild-type, indicating that CEL1 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 beta-glucosidase activities that were separable by chromatofocusing, hydrophobic interaction, or ion-exchange high-performance liquid chromatography.(ABSTRACT TRUNCATED AT 250 WORDS)