Several extragenic cot ts suppressors have been identified A
Several extragenic cot-1 (ts) suppressors have been identified. Among them, cytoplasmic dynein, dynactin, and nuclear distribution mutants of N. crassa, as well as an additional gene gul-1 (NCU01197), have been shown to have genetic interactions with cot-1 (Bruno et al., 1996, Seiler et al., 2006, Terenzi and Reissig, 1967). A mutation ingul-1partially suppresses the severity of thecot-1(ts) phenotype and is accompanied by altered gene expression of the Calcipotriol remodeling machinery and, subsequently, cell wall composition, (Herold and Yarden, 2017).
Similarly, SSD1, which is the gul-1 homologue in Saccharomyces cerevisiae and was determined to encode a translational regulator of cell wall remodeling genes, can suppress the phenotype of the yeast cot-1 homologue, CBK1 (Hogan et al., 2008, Jansen et al., 2009, Kurischko et al., 2011a). Ssd1 has been shown to be an mRNA-binding protein whose function has an impact on maintenance of cell integrity, mRNA processing, stress signaling and cellular aging (Kurischko et al., 2011b). Ssd1 was also shown to migrate between the cytoplasm and the nucleus and has been suggested to be involved in shuttling mRNAs destined for polarized localization and translation, including mRNAs encoding cell wall proteins (Hogan et al., 2008, Jansen et al., 2009, Kurischko et al., 2011a, Kurischko et al., 2011b, Kurischko and Broach, 2017, Mitchell et al., 2013, Uesono et al., 1997). Under stress conditions, the protein-RNA complex was shown to physically interact with processing bodies (P bodies) and stress granules in the nucleus (Kurischko et al., 2011a, Richardson et al., 2012, Tarassov et al., 2008, Zhang et al., 2014). These were subsequently exported to the cytoplasm for mRNA storage or decay (Kurischko and Broach, 2017).
Ssd1 function has been demonstrated to be negatively regulated via phosphorylation by Cbk1 (Jansen et al., 2009, Kurischko et al., 2011a). Cbk1 inhibition and cellular stress lead to translational repression along with the localization of Ssd1 to cytoplasmic puncta (Kurischko et al., 2011a, Kurischko et al., 2011b).
One explanation for the pleiotropic nature of the cot-1 (ts) mutant is the fact that COT-1 is an upstream component in a hierarchal pathway that can effect different processes in the cell. In this study, we describe the transcriptional profile of cot-1 inactivation and demonstrate that gul-1 affects transcript abundance of multiple genes in the COT-1 pathway, including processes such as cell wall remodeling, nitrogen and amino acid metabolism as well as 298 genes of yet unknown function. Furthermore, we show that GUL-1 transport in the cell is tubulin-dependent and the extent of its association with nuclei is stress-dependent.
Materials and methods
Discussion Homologs of the N. crassa Ndr kinase COT-1 are important for cell differentiation and polar morphogenesis in various fungi (Buhr et al., 1996, Dürrenberger and Kronstad, 1999, Johns et al., 2006, Kodama et al., 2017, McNemar and Fonzi, 2002, Scheffer et al., 2005, Yarden et al., 1992, Ziv et al., 2009). At least part of COT-1 and its homologs function is mediated by its effector GUL-1 or appropriate counterparts in other fungi (Herold and Yarden, 2017, Jansen et al., 2009, Kurischko et al., 2011a, Lee et al., 2015, Nuñez et al., 2016). In this study, we demonstrate that the morphological consequences of impaired cot-1 (ts) function are accompanied by major changes in the transcriptome. In fact, almost 10% of N. crassa transcriptome was found to be differently expressed in a cot-1 (ts) background. Among the different genes, we identified enrichment in the expression of oxidation signaling and catalytic activity-encoding genes. These can explain the fact that in the cot-1 (ts) mutant cellular stress responses are activated. This is in line with a previous observation showing that in a cot-1 (ts) strain, a variety of stress-related physiological responses are evident, even when external stress was not applied (Gorovits and Yarden, 2003). Thus, our current results support the hypothesis that at least part of the phenotypic suppression by environmental stresses that cot-1 (ts) undergoes, is related to the transcriptional and subsequent physiological state imposed by impaired COT-1 function.