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.

Genetic and biochemical characterization of the GH72 family of cell wall transglycosylases in Neurospora crassa

“…Our previous proteomic analyses have identified four of the encoded gene products (GEL-1 (NCU08909), GEL-2 (NCU07253), GEL-3 (NCU01162), and GEL-5 (NCU06781)) as being cell wall proteins (Maddi et al, 2009, Maddi and Free, 2010, Maddi et al, 2012, Ao et al, 2016). We decided to focus on these four GH-72 enzymes and determine if they were important for cell wall biosynthesis in N. crassa .…”

“…It was particularly interesting that GEL-3 was among the cross-linking enzymes needed to make cell wall during conidiation. We have previously shown that GEL-3 is found in the conidia cell wall and has a conidia-specific expression pattern (Ao et al, 2016). What the two most severely affected triple mutants have in common is the loss of both gel-1 and gel-2 , a tight colonial morphology in liquid growth conditions, and a restoration of normal morphology in the presence of the osmotic stabilizer 1.5 M sorbitol.…”

“…We have characterized mutants affected in all possible combinations of the four GH72 transglycosylases found in the N. crassa cell wall proteome (Ao et al, 2016, Maddi et al, 2009, Maddi and Free, 2010, Maddi et al, 2012). Our results demonstrate that different combinations of these transglycosylases are being used in different cell types.…”

“…We have previously carried out proteomic analyses of Neurospora crassa vegetative hyphae and conidia cell walls and demonstrated the existence of cross-linking enzymes from all five families of commonly found cross-linking enzymes (Ao et al, 2016, Maddi et al, 2009, Maddi and Free, 2010, Maddi et al, 2012). The N. crassa genome has 13 genes from the GH16 family, 3 genes from the GH17 family, 10 genes from the GH18 family, 5 genes from the GH72 family and 9 genes from the GH76 family (Colot et al, 2006).…”

Hyphal ontogeny in Neurospora crassa: a model organism for all seasons

“…Additional proteomic analysis indicated compositional differences between cell walls of conidia and hyphae, suggesting differential regulation of polysaccharide biosynthesis through development 48 . Mannan and galactomannan are found as glycoconjugates on cell walls.…”