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

Abstract

During sclerotial infection of Sclerotinia sclerotiorum the mycoparasite Coniothyrium minitans penetrates through the host cell wall, which contains beta-1,3-glucan as its major component. A PCR-based strategy was used to clone a beta-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-beta-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 approximately 100-kDa beta-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 microg x 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.

FIG. 1

Alignment of CMG1 of C. minitans with EXGA of A. quisqualis, LAM1.3 (LAM1) of T. harzianum, EXG1 of C. carbonum, and BGN13.1 (BGN13) of T. harzianum. Identical and similar residues are indicated by black and grey backgrounds, respectively. The C terminus of LAM1.3 of T. harzianum shows no homology to the other sequences presented and therefore is not shown. The arrow indicates the predicted signal peptide cleavage site. Asterisks indicate the N-terminal residues of the yeast-expressed enzyme, as determined by protein sequencing. The dashed lines indicate the putative substrate binding regions.

FIG. 2

Detection of β-1,3-glucanase activity in polyacrylamide gels. S. cerevisiae INVSc1 transformed either with pYES2 (lanes Y) or with pYGL (lanes G) was induced by galactose, and 100 μg of intracellular protein (IP) or 15 μl of 10×-concentrated culture supernatant (extracellular protein [EP]) was separated by SDS-PAGE, renatured, and screened for β-1,3-glucanase activity. The positions of molecular mass markers are indicated on the left.

FIG. 3

Purification of the recombinant glucanase. (A) Ion-exchange chromatography. (B) Silver-stained protein in SDS-PAGE gel. Lane 1, molecular mass standards; lane 2, concentrated culture supernatant of S. cerevisiae INVScl(pYGL); lanes 3 and 4, samples from the glucanase active fraction.

FIG. 4

Northern blot analysis of cmg1. C. minitans Cm-2 was grown as a shaken culture in SM supplemented with 2% glucose (lanes 1 and 4), 0.1% glucose (lanes 2 and 5), or 0.5% freeze-dried, ground sclerotia of S. sclerotiorum (lanes 3 and 6). Five micrograms of total RNA extracted after 2 days (2d) (lanes 1 to 3) or 6 days (6d) (lanes 4 to 6) of growth was electrophoresed on a formaldehyde gel, blotted, and hybridized to the radiolabelled cmg1 gene (upper panels). The middle panels show control hybridizations with a ribosomal DNA (rDNA) probe. The bottom panels show ethidium bromide-stained rRNA. Representative results of two independent experiments are shown.

FIG. 5

Expression of cmg1 during sclerotial parasitism. RNA was extracted from dual cultures of C. minitans and S. sclerotiorum (P) and from mycelium of S. sclerotiorum (S) or C. minitans (C) grown alone, as controls. Five micrograms of RNA was loaded onto a formaldehyde gel and hybridized to the radiolabelled cmg1 gene. The cultures were 3, 5, and 10 days old (3d, 5d, and 10d, respectively) when samples were removed. The bottom panel shows ethidium bromide-stained rRNA as an indication of the amounts of RNA loaded. Representative results of three independent experiments are shown.