Characterization of the cell wall of the filamentous fungus, Neurospora crassa
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The cell wall is a critical organelle for a fungus. Being the outermost structure it bears the brunt of the environmental stresses, and most importantly is involved in generating intracellular signals in response to environmental changes. Some of these signals regulate the phenotypic changes that occur in fungal life cycles. The cell wall is a three dimensional matrix composed of two major components, the polysaccharides and the cell wall proteins. A large number of cell wall studies have been focused on the yeast form fungi, Saccharromyces cerevisiae and Candida albicans . Information regarding the cell walls of filamentous fungi has been particularly lacking. The work done for this thesis comprises a detailed characterization of the cell wall of the model filamentous fungus, Neurospora crassa . An in depth analysis of the cell wall proteome was performed using a novel cell wall solubulization technique that uses trifluoromethanesulfonic acid (TFMS). TFMS cleaves the glycosidic linkages found in the chitin and glucan cell wall polymers and releases the cell wall proteins. Following TFMS digestion, the cell wall proteins can be isolated and identified by mass spectrometry (MS). Using this analysis, a total of 26 integral cell wall proteins have been identified in the cell walls of vegetative hyphae and conidia, the asexual phases of the N. crassa life cycle. Nine secreted proteins were also identified in the culture media that included cell wall proteins. NCBI BLAST searches of the aminoacid sequences of the identified cell wall proteins indicated that many were unique to the filamentous fungi and highly conserved in filamentous fungi. To characterize the functions of individual N. crassa cell wall proteins, an extensive screening of knockout mutants of 65 cell wall proteins was performed. Knockout mutants lacking six cell wall proteins were found to have a cell wall phenotype or morphological phenotype that genetically co-segregated with the knockout mutation. Three of these cell wall proteins GEL-1, GEL-3 and WSC-2 have homologs in yeast, but the other three proteins, CCG-6, ACW-13 and HAM-7, are unique to filamentous fungi and their functions are yet to be characterized. This mutational analysis, the first for filamentous fungi, showed that some cell wall proteins play critical roles in the life cycles of filamentous fungi. Though a substantial amount of information is available on the structures of the cell wall polysaccharides, very little is known about how the cell wall is synthesized and how the various components, especially the proteins, are covalently cross-linked into the cell wall matrix. To this end, a characterization of the N. crassa OCH-1 knockout mutant was carried out. OCH-1 is a Golgi based enzyme that catalyzes the first step in the addition of the N-linked galactomannan modification to cell wall proteins. The cell wall from the och-1 knockout mutant had a reduction in the amount of cell wall-associated galactomannan. The mutant was also found to release large amounts of cell wall proteins into the medium and the cell wall was deficient in integrated cell wall protein. The results demonstrated that the N-linked galactomannan modification on cell wall proteins was critical for cell wall protein incorporation into the cell wall matrix.