The GUL-1 Protein Binds Multiple RNAs Involved in Cell Wall Remodeling and Affects the MAK-1 Pathway in Neurospora crassa.

The Neurospora crassa GUL-1 is part of the COT-1 pathway, which plays key roles in regulating polar hyphal growth and cell wall remodeling. We show that GUL-1 is a bona fide RNA-binding protein (RBP) that can associate with 828 "core" mRNA species. When cell wall integrity (CWI) is challenged, expression of over 25% of genomic RNA species are modulated (2,628 mRNAs, including the GUL-1 mRNA). GUL-1 binds mRNAs of genes related to translation, cell wall remodeling, circadian clock, endoplasmic reticulum (ER), as well as CWI and MAPK pathway components. GUL-1 interacts with over 100 different proteins, including stress-granule and P-body proteins, ER components and components of the MAPK, COT-1, and STRIPAK complexes. Several additional RBPs were also shown to physically interact with GUL-1. Under stress conditions, GUL-1 can localize to the ER and affect the CWI pathway-evident via altered phosphorylation levels of MAK-1, interaction with mak-1 transcript, and involvement in the expression level of the transcription factor adv-1. We conclude that GUL-1 functions in multiple cellular processes, including the regulation of cell wall remodeling, via a mechanism associated with the MAK-1 pathway and stress-response.

Transcriptional profiling and localization of GUL-1, a COT-1 pathway component, in Neurospora crassa.

Impairment of theNeurospora crassaCOT-1 kinase results in defects in hyphal polarity. Some of these effects are partially suppressed by inactivation of gul-1 (encoding an mRNA-binding protein involved in translational regulation). Here, we report on the transcriptional profiling 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. The GUL-1 protein itself was found to be distributed within the entire hyphal cell, along with a clear presence of aggregates that traffic within the cytoplasm. Live imaging of GUL-1-GFP demonstrated that GUL-1 transport is microtubule-dependent. Cellular stress, as imposed by the presence of the cell wall biosynthesis inhibitor Nikkomycin Z or by nitrogen limitation, resulted in a 2-3-fold increase of GUL-1 aggregate association with nuclei. Taken together, this study demonstrates that GUL-1 affects multiple processes, its function is stress-related and linked with cellular traffic and nuclear association.

Regulation of Neurospora crassa cell wall remodeling via the cot-1 pathway is mediated by gul-1.

Impairment of the Neurospora crassa Nuclear DBF2-related kinase-encoding gene cot-1 results in pleiotropic effects, including abnormally thick hyphal cell walls and septa. An increase in the transcript abundance of genes encoding chitin and glucan synthases and the chitinase gh18-5, but not the cell wall integrity pathway transcription factor rlm-1, accompany the phenotypic changes observed. Deletion of chs-5 or chs-7 in a cot-1 background results in a reduction of hyperbranching frequency characteristic of the cot-1 parent. gul-1 (a homologue of the yeast SSD1 gene) encodes a translational regulator and has been shown to partially suppress cot-1. We demonstrate that the high expression levels of the cell wall remodeling genes analyzed is curbed, and reaches near wild type levels, when gul-1 is inactivated. This is accompanied by morphological changes that include reduced cell wall thickness and restoration of normal chitin levels. We conclude that gul-1 is a mediator of cell wall remodeling within the cot-1 pathway.

Sensitivity of Neurospora crassa to a marine-derived Aspergillus tubingensis anhydride exhibiting antifungal activity that is mediated by the MAS1 protein.

The fungus Aspergillus tubingensis (strain OY907) was isolated from the Mediterranean marine sponge Ircinia variabilis. Extracellular extracts produced by this strain were found to inhibit the growth of several fungi. Among the secreted extract components, a novel anhydride metabolite, tubingenoic anhydride A (1) as well as the known 2-carboxymethyl-3-hexylmaleic acid anhydride, asperic acid, and campyrone A and C were purified and their structure elucidated. Compound 1 and 2-carboxymethyl-3-hexylmaleic acid anhydride inhibited Neurospora crassa growth (MIC = 330 and 207 µM, respectively) and affected hyphal morphology. We produced a N. crassa mutant exhibiting tolerance to 1 and found that a yet-uncharacterized gene, designated mas-1, whose product is a cytosolic protein, confers sensitivity to this compound. The ∆mas-1 strain showed increased tolerance to sublethal concentrations of the chitin synthase inhibitor polyoxin D, when compared to the wild type. In addition, the expression of chitin synthase genes was highly elevated in the ∆mas-1 strain, suggesting the gene product is involved in cell wall biosynthesis and the novel anhydride interferes with its function.