A proteomic and genetic analysis of the Neurospora crassa conidia cell wall proteins identifies two glycosyl hydrolases involved in cell wall remodeling

Abstract

A proteomic analysis of the conidial cell wall identified 35 cell wall proteins. A comparison with the proteome of the vegetative hyphae showed that 16 cell wall proteins were shared, and that these shared cell wall proteins were cell wall biosynthetic proteins or cell wall structural proteins. Deletion mutants for 34 of the genes were analyzed for phenotypes indicative of conidial cell wall defects. Mutants for two cell wall glycosyl hydrolases, the CGL-1 β-1,3-glucanase (NCU07523) and the NAG-1 exochitinase (NCU10852), were found to have a conidial separation phenotype. These two enzymes function in remodeling the cell wall between adjacent conidia to facilitate conidia formation and dissemination. Using promoter::RFP and promoter::GFP constructs, we demonstrated that the promoters for 15 of the conidia-specific cell wall genes, including cgl-1 and nag-1, provided for conidia-specific gene expression or for a significant increase in their expression during conidiation.

Keywords: Asexual development; Chitinase; Conidia; Fungal cell wall; Glucanase; Glycosyl hydrolase.

Fig. 1

The NAG-1 exochitinase and CGL-1 β-glucanase are required for conidia cell separation. DIC images of 7 day old conidia from wild type cells, the Δ-cgl-1 mutant, the Δnag-1 mutant, and a Δnag-1 mutant that has been transformed with a wild type copy of the nag-1 gene showing that the wild type gene complements the mutant allele. The black bars represent a distance of 40 μm.

Fig. 2

Cell type-specific expression of the rds-1 gene (NCU05143). The promoter region preceding the rds-1 gene was cloned in front of the GFP coding region and the expression of the gene assessed in developing conidia. The left panel shows the GFP image, the middle panel a DIC image, and the right panel shows a merged image. The promoter directs expression in the maturing conidia, but not in vegetative hyphae or in early stage conidial development. The black bars represent a distance of 40 μm.

Fig. 3

Cell type-specific expression of the cgl-1 gene (NCU07523). The promoter region preceding the cgl-1 gene was cloned in front of the RFP coding region and the expression of the gene assessed in developing conidia. The left panel shows the RFP image, the middle panel a DIC image, and the right panel shows a merged image. The promoter directs high level expression in the maturing conidia, but not in vegetative hyphae or in early stage conidial development. The black bars represent a distance of 40 μm.