Differentially regulated, vegetative-mycelium-specific hydrophobins of the edible basidiomycete Pleurotus ostreatus

Abstract

Three different hydrophobins (Vmh1, Vmh2, and Vmh3) were isolated from monokaryotic and dikaryotic vegetative cultures of the edible fungus Pleurotus ostreatus. Their corresponding genes have a number of introns different from those of other P. ostreatus hydrophobins previously described. Two genes (vmh1 and vmh2) were expressed only at the vegetative stage, whereas vmh3 expression was also found in the fruit bodies. Furthermore, the expression of the three hydrophobins varied significantly with culture time and nutritional conditions. The three genes were mapped in the genomic linkage map of P. ostreatus, and evidence is presented for the allelic nature of vmh2 and POH3 and for the different locations of the genes coding for the glycosylated hydrophobins Vmh3 and POH2. The glycosylated nature of Vmh3 and its expression during vegetative growth and in fruit bodies suggest that it should play a role in development similar to that proposed for SC3 in Schizophyllum commune.

FIG. 1.

SDS-PAGE of hydrophobins purified from P. ostreatus var. florida fruit bodies (lanes 1), monokaryotic vegetative mycelium (lanes 2), and dikaryotic vegetative mycelium (lanes 3) and SC3 hydrophobin purified from S. commune (lanes 4). (A) Silver staining; (B) staining with Schiff reagent; (C) immunoreaction with antibodies raised against S. commune hydrophobin SC3.

FIG. 2.

Northern analysis showing vmh1, vmh2, and vmh3 expression at different development stages. Total RNA was purified from fruit bodies (lane 1), dikaryotic vegetative mycelium of monokaryon M001 (lane 2), and vegetative mycelium of monokaryons M001 (lane 3) and M005 (lane 4). Shown are hybridization using vmh1 cDNA (A), vmh2 cDNA (B), vmh3 cDNA (C), and RNA amount shown as ethidium bromide fluorescence (D). All the cDNA probes corresponded to the mature proteins (i.e., without leader peptide).

FIG. 3.

Northern analysis showing time course of vmh1, vmh2, and vmh3 expression. Total RNA was purified from dikaryotic mycelia growing after 5 days (lane 1), 7 days (lane 2), 10 days (lane 3), 15 days (lane 4), and 20 days (lane 5) of vegetative growth. Shown are hybridizations with vmh1 cDNA (A), vmh2 cDNA (B), vmh3 cDNA (C), and P. ostreatus 5.8S rRNA as a control (D). All the cDNA probes corresponded to the mature proteins (i.e., without leader peptide).

FIG. 4.

SDS-PAGE of hydrophobins secreted to the medium by the dikaryotic mycelium of P. ostreatus N001 after 5 (lane 1), 10 (lane 2), 15 (lane 3), and 20 (lane 4) days of submerged culture.

FIG. 5.

RFLP analysis of vmh1, vmh2, and vmh3 genes. Shown are XhoI digestions (A and B) and EcoRI digestion (C) of DNA purified from dikaryotic strain N001 (lane 1) and from 13 monokaryons derived from it (lanes 2 to 14). DNA was probed with vmh1 cDNA (A), vmh2 cDNA (B), and vmh3 cDNA (C). All the cDNA probes corresponded to the mature proteins (i.e., without leader peptide).

FIG. 6.

RFLP analysis of vmh2 and POH3 alleles. XhoI digestion of DNA purified from dikaryotic strain N001 (lane 1) and from 10 monokaryons derived from it (lanes 2 to 11) were probed with a cDNA sequence corresponding to the vmh2 gene (leader peptide plus mature protein). Identical results were obtained with the POH3 probe.

FIG. 7.

Schematic representation of the structures of the vegetative mycelium hydrophobin genes from of P. ostreatus. ▨, peptide signals; gray boxes, exons; _, introns; ▮, cysteines.