Intercellular communication of vegetative cells and their subsequent cell fusion is vital for different aspects of growth, fitness, and differentiation of filamentous fungi. Cell fusion between germinating spores is important for early colony establishment, while hyphal fusion in the mature colony facilitates the movement of resources and organelles throughout an established colony. Approximately 50 proteins have been shown to be important for somatic cell-cell communication and fusion in the model filamentous fungus Neurospora crassa. Genetic, biochemical, and microscopic techniques were used to characterize the functions of seven previously poorly characterized cell fusion proteins. HAM-6, HAM-7 and HAM-8 share functional characteristics and are proposed to function in the same signaling network. Our data suggest that these proteins may form a sensor complex at the cell wall/plasma membrane for the MAK-1 cell wall integrity mitogen-activated protein kinase (MAPK) pathway. We also demonstrate that HAM-9, HAM-10, AMPH-1 and WHI-2 have more general functions and are required for normal growth and development. The activation status of the MAK-1 and MAK-2 MAPK pathways are altered in mutants lacking these proteins. We propose that these proteins may function to coordinate the activities of the two MAPK modules with other signaling pathways during cell fusion.

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.

The Neurospora crassa genome encodes five GH72 family transglycosylases, and four of these enzymes (GEL-1, GEL-2, GEL-3 and GEL-5) have been found to be present in the cell wall proteome. We carried out an extensive genetic analysis on the role of these four transglycosylases in cell wall biogenesis and demonstrated that the transglycosylases are required for the formation of a normal cell wall. As suggested by the proteomic analysis, we found that multiple transglycosylases were being expressed in N. crassa cells and that different combinations of the enzymes are required in different cell types. The combination of GEL-1, GEL-2 and GEL-5 is required for the growth of vegetative hyphae, while the GEL-1, GEL-2, GEL-3 combination is needed for the production of aerial hyphae and conidia. Our data demonstrates that the enzymes are redundant with partially overlapping enzymatic activities, which provides the fungus with a robust cell wall biosynthetic system. Characterization of the transglycosylase-deficient mutants demonstrated that the incorporation of cell wall proteins was severely compromised. Interestingly, we found that the transglycosylase-deficient mutant cell walls contained more β-1,3-glucan than the wild type cell wall. Our results demonstrate that the GH72 transglycosylases are not needed for the incorporation of β-1,3-glucan into the cell wall, but they are required for the incorporation of cell wall glycoprotein into the cell wall.

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A biochemical pathway for the incorporation of cell wall protein into the cell wall of Neurospora crassa was recently proposed. In this pathway, the DFG-5 and DCW-1 endo-␣-1,6-mannanases function to covalently cross-link cell wall protein-associated N-linked galactomannans, which are structurally related to the yeast outer chain mannans, into the cell wall glucan-chitin matrix. In this report, we demonstrate that the mannosyltransferase enzyme Och1p, which is needed for the synthesis of the N-linked outer chain mannan, is essential for the incorporation of cell wall glycoproteins into the Candida albicans cell wall. Using endoglycosidases, we show that C. albicans cell wall proteins are cross-linked into the cell wall via their N-linked outer chain mannans. We further demonstrate that the Dfg5p and Dcw1p ␣-1,6-mannanases are needed for the incorporation of cell wall glycoproteins into the C. albicans cell wall. Our results support the hypothesis that the Dfg5p and Dcw1p ␣-1,6-mannanases incorporate cell wall glycoproteins into the C. albicans cell wall by cross-linking outer chain mannans into the cell wall glucan-chitin matrix.T he fungal cell wall plays a critical role in fungal survival, growth, and morphology. The fungal cell wall is generated by the cross-linking of glucans, chitin, and cell wall proteins in the cell wall space to create a three-dimensional matrix (1-6). In Candida albicans and Saccharomyces cerevisiae, the major glucans in the cell wall are ␤-1,3-glucan and ␤-1,6-glucan (1-8). The ␤-1,3-glucan and chitin elements are synthesized as linear polymers and extruded into the cell wall space during their synthesis by glucan synthases and chitin synthases. These enzymes use UDP-glucose and UDP-N-acetylglucosamine as substrates and add glucose and N-acetylglucosamine to the nonreducing ends of the nascent glucan and chitin polymers. As the glucan and chitin polymers are extruded into the cell wall space, they are cross-linked by groups of cell wall enzymes with glucanase and glucan transferase (transglycosylase) activities. ␤-1,6-Glucan has been implicated in crosslinking the other cell wall elements (1-3, 6). The enzymes involved in cross-linking the glucan and chitin polymers are encoded by multigene families which produce enzymes with overlapping specificities.Fungal cell walls have a characteristic array of integral cell wall proteins which are cross-linked into the glucan-chitin matrix. These include the glucanase/glucan transferase enzymes involved in cross-linking the wall polymers, adhesins, signal pathway receptors, and structural proteins (8)(9)(10)(11)(12)(13)(14). Some of these cell wall proteins have been shown to be critical for virulence and for the formation and functionality of the cell wall. These proteins contain signal peptide sequences at their N termini and travel through the canonical secretory pathway. As these proteins pass through the C. albicans endoplasmic reticulum (ER) and Golgi apparatus, they become heavily glycosylated with O-linked and N-linked oligosaccharides. Th...