A proteomic and genetic analysis of the Neurospora crassa conidia cell wall proteins identifies two glycosyl hydrolases involved in cell wall remodeling
Introduction
The formation of asexual spores is an important part of the life cycles of many fungi. The asexual spore is a differentiated cell type adapted for dissemination and for the establishment of a new vegetative hyphal network. Asexual spores are generated in very large numbers, and can be rapidly and widely disseminated in the environment, to allow the fungi to colonize new sites and substrates. The formation of asexual spores has been examined in many fungi, and the details of how asexual spores are generated varies widely. In Neurospora crassa the process of conidiation has been extensively studied (Springer, 1993, Springer and Yanofsky, 1989). A number of proteins have been shown to be expressed during conidiation, and transcription factors needed for conidial development have been identified (Greenwald et al., 2010, Lee and Ebbole, 1998, Sachs and Yanofsky, 1991, Springer and Yanofsky, 1992). Our analysis of conidiation focused on the question of how the cell wall proteome changes as cells go through conidial development. We have previously demonstrated that the conidial cell wall contains α-1,3-glucan, a glucan not found in the vegetative cell wall (Fu et al., 2014).
The cell wall plays a critical role in fungal cell biology. Not only does the wall protein protect the cell from environmental factors, such as osmotic stress, desiccation, heat, freezing, and attacks from other microbes, the cell wall also allows the fungus to assess its environment and activate signaling pathways in response to a changing environment (Free, 2013). The wall consists of a cross-linked matrix of glucans, chitins, and cell wall proteins. The proteins are particularly important in allowing the fungus to assess and respond to its environment. Cell wall proteins function in cross-linking cell wall components together, and are therefore essential for cell wall biogenesis.
In order to do a proteomic analysis of fungal cell wall proteins, the proteins, (or peptides derived from them) need to be obtained free of the cell wall glucans and chitin to which they are covalently attached. Several different approaches for obtaining cell wall proteins for proteomic analyses have been used over the years (Chaffin, 2008, Free, 2013, Klis et al., 2006, Latge, 2007, Lesage and Bussey, 2006, Ruiz-Herrera et al., 2006). Cell wall proteins have been isolated from among the proteins being secreted by sphaeroplast that are regenerating their cell walls. Some cell wall proteins have been isolated from among the proteins being released into the medium, suggesting that either the incorporation of the proteins into the wall isn’t 100% efficient or that some proteins that have been incorporated into the wall are subsequently released into the medium. Some cell wall proteins can be released from the wall by chemical treatment of the wall or by digesting the wall with glucanase and chitinase preparations, and these can then be isolated and subjected to proteomic analysis. Yet another approach has been to treat the cell wall with trypsin, and to use the released tryptic peptides for proteomic analyses. Recently we introduced the use of trifluoromethanesulfonic acid (TFMS), which digests the glycosidic bonds found in the glucan and chitin cell wall components and releases deglycosylated cell wall proteins for proteomic analysis (Birkaya et al., 2009, Bowman et al., 2006, Liu and Free, 2015, Maddi et al., 2009, Maddi et al., 2012, Maddi and Free, 2010).
In this study, we have used TFMS to release cell wall proteins from the cell wall of N. crassa asexual spores (macroconidia or more simply referred to as conidia) and compared the cell wall proteome with the cell wall proteins found in vegetative hyphae. After identifying the proteins in the conidia cell wall, we then used a genetic analysis to determine whether these proteins were important for conidia formation, and to determine if the genes encoding these proteins were transcribed in a conidia-specific manner. We report that the conidia cell wall contains 15 conidia-specific proteins, and that the genes encoding these proteins are transcribed in a conidia-specific manner. We demonstrate that two of these conidia-specific cell wall proteins, the NGA-1 exochitinase and the CGL-1 β-1,3-glucanase, play critical roles in remodeling the cell walls between adjacent conidia to allow the conidia to separate.
Section snippets
Proteomic analysis of the conidia cell wall
Conidial cell walls were obtained by growing a wild type isolate (FGSC #2400) on six 3 ml slants of Vogel’s sucrose medium and collecting the conidia in water. The conidia were passed through 4 layers of cheese cloth to remove hyphae. The conidial cells were broken by subjecting them to two 45 s treatments in a Fast Prep machine with glass beads (1.0 mm in diameter) at a setting of 6.6. The cell walls were collected by a 10 min centrifugation at 5000g, and washed two times with sterile distilled
Proteomic analysis to identify cell wall proteins
Integral cell wall proteins are covalently attached to the glucan/chitin cell wall matrix. To release these cell wall proteins from the wall and to deglycosylate the proteins, we used the trifluoromethanesulfonic acid (TFMS) technique described in Section 2. TFMS cleaves glycosidic bonds, but leaves peptide bonds intact. The proteins released from the cell wall can then be directly used for proteomic analysis. Using this approach, we have been able to identify 35 cell wall proteins from the
Discussion
Using a combination of proteomic and genetic approaches, we have characterized the N. crassa conidia cell wall. We found that like other fungal cell walls, the conidia cell wall contains a number of commonly-found structural proteins and cross-linking enzymes. These structural and cross-linking proteins are encoded by multi-gene families in the N. crassa genome and in the genomes of other fungi (Free, 2013). These cross-linking and structural proteins are found in virtually all cell wall
Acknowledgments
Funding for this study was provided by grant RO3-Al103897 from the National Institutes of Health, by the UB Foundation, and by Kuwait University. We thank Alan Segal for help with the confocal microscope imaging and Jim Stamos for help with manuscript preparation.
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