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  • Partial suppression of the cot ts phenotype

    2019-11-08

    Partial suppression of the cot-1 (ts) phenotype by deletion of gul-1 has been well documented (Seiler et al., 2006). This is now also very clearly evident in the transcriptional profile of the various strains (Fig. 2). Furthermore, the comparative analysis of the single and double mutants revealed, for the first time that GUL-1 is likely to be involved in processes yet not associated with this protein, such as nitrogen metabolism, amino hydroxycarboxylic acid receptors syntheses and various transporter activities. A significant change in the expression of a number of hypothetical proteins was also observed. These data suggest that GUL-1 function may involve multiple pathways. The role GUL-1 plays in these additional processes has yet to be elucidated. Under standard growth conditions, deletion of gul-1 results in only slight changes in morphology, when compared to the wild type (Seiler et al., 2006, Terenzi and Reissig, 1967). Nonetheless, Lin et al. (2018) showed that when cultured in Avicel medium, Δgul-1 grew as pellets, showed a low viscosity phenotype and changes in cell wall structure. Despite the morphological differences observed under Avicel versus Vogel’s media, the expression levels of several cell wall related genes was similarly altered in Δgul-1. This was exemplified by the fact that in both cases among the 22 cell wall proteins that were identified in N. crassa hyphae (Maddi et al., 2009), 8 common genes were significantly altered in Δgul-1. Among them, four GPI-anchored glycoside hydrolase genes (NCU09175, NCU01353, NCU05974 and NCU06781) and three anchored cell wall genes (NCU08936, NCU05667 and NCU06185) showed a significant decrease in expression levels. Nevertheless, we found the non-anchored cell wall gene NCU07817 to be down-regulated in Δgul-1, while Lin et al (2018) observed upregulation of this gene in cultures grown on avicel. In addition, we found two vegetative cell wall proteins, β-Glucosidase (NCU09326) and NCW-2 (NCU01752) which were uniquely altered only under our experimental conditions. One of the genes whose expression was down regulated was bgt-2 (NCU09175), encoding a β-1,3-endoglucanase. Deletion of this gene was shown to result in changes in cell wall architecture suggesting that it is involved in remodeling of the cell wall (Martínez-Núñez and Riquelme, 2015). Among the other genes whose expression was altered, some have been analyzed in other fungi. These include NCU05974, whose S. cerevisiae homologue crh1 is hyper sensitive to Congo Red and Calcofluor White (Cabib et al., 2007), while the NCU06781 homologue in Aspergillus fumigatus homologue, gel2, showed altered cell wall composition (Mouyna et al., 2005). In A. fumigatus, AfuEcm33, a homologue of the N. crassa acw-1 (NCU08936) was found to be involved in maintaining cell wall integrity (Romano, 2006). A major down regulated gene, NCU05404, was annotated as a glycosyl hydrolase unclassified family-2. This gene and corresponding protein was described as a secreted endoglucanase by Maddi et al. (2010). The protein also contains an “Auxiliary Activity Family 11″ domain which implies it has monooxygenase activity (http://www.cazypedia.org/index.php). In addition to those mentioned above, we also identified a representative of the “SUN” family, NCU02668, which is homologous to the S. cerevisiae Sun4, a cell wall degrading protein that was shown to be regulated by the yeast Ssd1 (Jansen et al., 2009). Taken together, our data show a considerable change in cell wall remodeling-related transcripts as a result of inactivation of gul-1. It is thus surprising that this does not appear to have a morphological effect on the fungus. One possible explanation is that there are additional proteins and other pathways involved in the stability of the cell wall which can compensate for the transcriptional changes observed. Nonetheless, Δgul-1 was shown to be hypersensitive to the chitin synthase inhibitor Nikkomycin Z (Herold and Yarden, 2017). The significance of GUL-1 in cell wall remodeling was further supported by analysis of changes in transcript abundance occurring in the presence of the drug (Fig. 4).