Expression of cmg1 , an Exo-β-1,3-Glucanase Gene from Coniothyrium minitans , Increases during Sclerotial Parasitism

Abstract

ABSTRACT During sclerotial infection of Sclerotinia sclerotiorum the mycoparasite Coniothyrium minitans penetrates through the host cell wall, which contains β-1,3-glucan as its major component. A PCR-based strategy was used to clone a β-1,3-glucanase-encoding gene, designated cmg1 , from a cDNA library of the fungus. The nucleotide and deduced amino acid sequences of this gene showed high levels of similarity to the sequences of other fungal exo-β-1,3-glucanase genes. The calculated molecular mass of the deduced protein (without the predicted 24-amino-acid N-terminal secretion signal peptide) was 83,346 Da, and the estimated pI was 4.73. Saccharomyces cerevisiae INVSc1 expressing the cmg1 gene secreted a ∼100-kDa β-1,3-glucanase enzyme (as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) into the culture medium. N-terminal sequence analysis of the purified recombinant enzyme revealed that the secreted enzyme starts at Ala-32, seven amino acids downstream from the predicted signal peptidase cleavage site. The purified recombinant glucanase inhibited in vitro mycelial growth of S. sclerotiorum by 35 and 85% at concentrations of 300 and 600 μg ml −1 , respectively. A single copy of the cmg1 gene is present in the genome of C. minitans . Northern analyses indicated increases in the transcript levels of cmg1 due to both carbon starvation and the presence of ground sclerotia of S. sclerotiorum ; only slight repression was observed in the presence of 2% glucose. Expression of cmg1 increased during parasitic interaction with S. sclerotiorum .