Expression of cmg1, an exo-beta-1,3-glucanase gene from Coniothyrium minitans, increases during sclerotial parasitism

During sclerotial infection of Sclerotinia sclerotiorum the mycoparasite Co niothyrium minitans penetrates through the host cell wall, which contains b eta -1,3-glucan as its major component. A PCR-based strategy was used to cl one a beta -1,3-glucanase-encoding gene, designated cmg1, from a cDNA libra ry of the fungus, The nucleotide and deduced amino acid sequences of this g ene showed high levels of similarity to the sequences of other fungal exo-b eta -1,3-glucanase genes. The calculated molecular mass of the deduced prot ein (without the predicted 24-amino-acid N-terminal secretion signal peptid e) was 83,346 Da, and the estimated pI was 4.73, Saccharomyces cerevisiae I NVSc1 expressing the cmg1 gene secreted a similar to 100-kDa beta -1,3-gluc anase enzyme (as determined by sodium dodecyl sulfate-polyacrylamide gel el ectrophoresis) into the culture medium. N-terminal sequence analysis of the purified recombinant enzyme revealed that the secreted enzyme starts at Al a-32, seven amino acids downstream from the predicted signal peptidase clea vage site. The purified recombinant glucanase inhibited in vitro mycelial g rowth of S. sclerotiorum by 35 and 85% at concentrations of 300 and 600 mug ml(-l), respectively. A single copy of the cmg1 gene is present in the gen ome of C. minitans, Northern analyses indicated increases in the transcript levels of cmg1 due to both carbon starvation and the presence of ground sc lerotia of S. sclerotiorum; only slight repression was observed in the pres ence of 2% glucose, Expression of cmg1 increased during parasitic interacti on with S. sclerotiorum.