A member of the OSCA/TMEM63 family of mechanosensitive calcium channels participates in cell wall integrity maintenance in Aspergillus nidulans.

The calF7 mutation in Aspergillus nidulans causes hypersensitivity to the cell wall compromising agents Calcofluor White (CFW) and Congo Red. In this research we demonstrate that the calF7 mutation resides in gene AN2880, encoding a predicted member of the OSCA/TMEM63 family of transmembrane glycoproteins. Those members of the family whose physiological functions have been investigated have been shown to act as mechanosensitive calcium transport channels. Deletion of AN2880 replicates the CFW hypersensitivity phenotype. Separately, we show that CFW hypersensitivity of calF deletion strains can be overcome by inclusion of elevated levels of extracellular calcium ions in the growth medium, and, correspondingly, wild type strains grown in media deficient in calcium ions are no longer resistant to CFW. These observations support a model in which accommodation to at least some forms of cell wall stress is mediated by a calcium ion signaling system in which the AN2880 gene product plays a role. The genetic lesion in calF7 is predicted to result in a glycine-to-arginine substitution at position 638 of the 945-residue CalF protein in a region of the RSN1_7TM domain that is highly conserved amongst filamentous fungi. Homology modeling predicts that the consequence of a G638R substitution is to structurally occlude the principal conductance pore in the protein. GFP-tagged wild type CalF localizes principally to the Spitzenkörper and the plasma membrane at growing tips and forming septa. However, both septation and hyphal morphology appear to be normal in calF7 and AN2880 deletion strains, indicating that any role played by CalF in normal hyphal growth and cytokinesis is dispensable.

Two amino acid sequences direct Aspergillus nidulans protein kinase C (PkcA) localization to hyphal apices and septation sites.

The Aspergillus nidulans ortholog of protein kinase C (pkcA) is involved in the organism's putative cell wall integrity (CWI) pathway, and PkcA also is highly localized at growing tips and forming septa. In the present work we identify the regions within PkcA that are responsible for its localization to hyphal tips and septation sites. To this end, we used serially truncated pkcA constructs and expressed them as green fluorescent protein (GFP) chimeras and identified two regions that direct PkcA localization. The first region is a 10 amino-acid sequence near the carboxyl end of the C2 domain that is required for localization to hyphal tips. Proteins containing this sequence also localize to septation sites. A second region between C2 and C1B (encompassing C1A) is sufficient for localization to septation sites but not to hyphal tips. We also report that localization to hyphal tips and septation sites alone is not sufficient for truncated constructs to complement hypersensitivity to the cell wall compromising agent calcofluor white in a strain bearing a mutation in the pkcA gene. Taken together, these results suggest that localization and stress response might be independent.

GDP-mannose transporter paralogues play distinct roles in polarized growth of Aspergillus nidulans.

GDP-mannose transporters (GMT) carry GDP-mannose nucleotide sugars from the cytosol across the Golgi apparatus membrane for use as substrates in protein glycosylation in plants, animals and fungi. Genomes of some fungal species, such as the yeast Saccharomyces cerevisiae, contain only one gene encoding a GMT, while others, including Aspergillus nidulans, contain two (gmtA and gmtB). We previously showed that cell wall integrity and normal hyphal morphogenesis in A. nidulans depend upon the function of GmtA and that GmtA localizes to a Golgi-like compartment. Cells bearing the calI11 mutation in gmtA also have reduced cell surface mannosylation. Here we show that GmtB colocalizes with GmtA, suggesting that the role of GmtB is similar to that of GmtA, although the respective transcript levels differ during spore germination and early development. Transcript levels of gmtB are high in ungerminated spores and remain so throughout the first 16 h of germination. In contrast, transcript levels of gmrtA are negligible in ungerminated spores but increase to levels comparable to those of gmtB during germination. These observations suggest that although GmtA and GmtB reside within the same subcellular compartments, they nevertheless perform distinct functions at different stages of development.

Two GDP-mannose transporters contribute to hyphal form and cell wall integrity in Aspergillus nidulans.

In order to identify novel genes affecting cell wall integrity, we have generated mutant strains of the filamentous fungus Aspergillus nidulans that show hypersensitivity to the chitin-binding agent Calcofluor White (CFW). Affected loci are designated cal loci. The phenotype of one of these alleles, calI11, also includes shortened hyphal compartments and increased density of branching in the absence of CFW, as well as reduced staining of cell walls by the lectin FITC-Concanavalin A (ConA), which has strong binding affinity for mannosyl residues. We have identified two A. nidulans genes (AN8848.3 and AN9298.3, designated gmtA and gmtB, respectively) that complement all aspects of the phenotype. Both genes show strong sequence similarity to GDP-mannose transporters (GMTs) of Saccharomyces and other yeasts. Sequencing of gmtA from the calI11 mutant strain reveals a G to C mutation at position 943, resulting in a predicted alanine to proline substitution at amino acid position 315 within a region that is highly conserved among other fungi. No mutations were observed in the mutant strain's allele of gmtB. Meiotic mapping demonstrated a recombination frequency of under 1 % between the calI locus and the phenA locus (located approximately 9.5 kb from AN8848.3), confirming that gmtA and calI are identical. A GmtA-GFP chimera exhibits a punctate distribution pattern, consistent with that shown by putative Golgi markers in A. nidulans. However, this distribution did not overlap with that of the putative Golgi equivalent marker CopA-monomeric red fluorescent protein (mRFP), which may indicate that the physically separated Golgi-equivalent organelles of A. nidulans represent physiologically distinct counterparts of the stacked cisternae of plants and animals. These findings demonstrate that gmtA and gmtB play roles in cell wall metabolism in A. nidulans similar to those previously reported for GMTs in yeasts.

The protein kinase C orthologue PkcA plays a role in cell wall integrity and polarized growth in Aspergillus nidulans.

The calC2 mutation in Aspergillus nidulans causes hypersensitivity to Calcofluor White, along with other drug sensitivities that indicate a defect in cell wall integrity. We have cloned CalC by complementation, isolating the A. nidulans orthologue of protein kinase C (PkcA). The pkcA allele of the calC2 strain contains a mutation predicted to introduce a charged arginine residue in place of neutral glycine at a conserved site located immediately beside the C1B regulatory domain. Both PkcA and calC2 map to the same region of chromosome VIII. A PkcA::GFP chimera localizes to hyphal apices and growing septa, as well as to the conidiogenous apices of phialides, indicating a role for PkcA in polarized cell wall growth. These observations support the hypothesis that the role of PkcA in A. nidulans, is comparable to that played by Pkc1p in the Saccharomyces cerevisiae cell wall integrity pathway.

Isolation of cell wall mutants in Aspergillus nidulans by screening for hypersensitivity to Calcofluor White.

As a first step toward identifying novel genes of wall metabolism in filamentous fungi, we have screened a collection of Aspergillus nidulans mutants for strains exhibiting hypersensitivity toward the chitin binding agent Calcofluor White (CFW). This strategy has been used previously to identify cell wall mutants in Saccharomyces cerevisiae. We have identified 10 mutants representing eight loci, designated calA through calH, for Calcofluor hypersensitivity. All cal mutants are impaired for sporulation at 30 C or 42 C or both, and in eight of the 10 mutations this sporulation defect shows at least partial osmotic remediability. All cal mutants show elevated sensitivity to one or more of the following agents: Caspofungin, Nikkomycin, Tunicamycin, Congo red and SDS, which are recognized wall-compromising agents or have been shown to be inhibitory to wall integrity mutants in yeast. Seven of the 10 cal mutants show swelling at elevated temperature, which in most cases is osmotically remediable. Spore swelling also can be induced at 30 C in all but one of the cal mutants by germination in the presence of one or more of the following: Caspofungin, Nikkomycin or Tunicamycin. Analysis of wall sugars showed no major changes in mutant strains. We also report that the chitin synthase inhibitor Nikkomycin induces excessive spore swelling during germination in all tested strains that have wild type cell wall metabolism (GR5, A4, A28 and AH12) at 42 C but not at 30 C. This effect mimics that of certain temperature-sensitive swollen cell (swo) mutations.

Proteolytic release of membrane-bound endo-(1,4)-beta-glucanase activity associated with cell wall softening in Achlya ambisexualis.

Branching and other cell wall softening events in fungi and oomycetes are thought to involve the activity of secreted enzymes, which are packaged in membrane vesicles and delivered to sites of cell expansion, there to work in a carefully regulated manner upon the structure of the wall. Here we demonstrate a latent endo-(1,4)-beta-glucanase activity in a mixed membrane fraction of the oomycete Achlya ambisexualis, which can be released by cysteine proteases with an increase of apparent activity. In addition, a similar endogenous process is strongly inhibited by the cysteine protease inhibitor iodoacetamide, while inhibitors of other types of proteases have a much smaller effect. Detergent treatment of membranes releases two glucanases detectable by electrophoretic activity staining, with apparent molecular masses of about 164 and 35 kDa. Proteolysis produces several activity bands, with major species having apparent molecular masses of about 149, 133, 48, 35, and 25 kDa. The ca. 35- and 25-kDa bands migrate in parallel with glucanases secreted during wall softening in vivo. We propose that the initiation of wall softening in Achlya involves the proteolytic processing and solubilization of at least some secreted endoglucanases. We also propose that the solubilization component of this process functions not just to provide the enzymes with access to wall matrix substrates but also may provide a mechanism for the eventual termination of their biological function.

Isolation and characterization of an endo-(1,4)-{beta}-glucanase secreted by Achlya ambisexualis.

Models of wall loosening in fungi and other walled eukaryotes require the action of proteins able to reduce the degree of linkage between components of the wall. In the oomycete Achlya ambisexualis, such a role has been proposed for a suite of endoglucanases that are secreted during branching and during the measurable wall softening associated with osmotic stress. We report here the isolation and characterization of one of these isoenzymes. The enzyme has a molecular weight of 32 kDa, a pH optimum of 6.75, a pI of 4.5, and a temperature optimum of 35 C. It is partially inhibited by sulfhydryl-binding reagents and completely inhibited by the tryptophan-binding reagent NBS. The enzyme has an endohydrolytic mode of action with substrate specificity towards glucans that contain ß-(1,4) linkages, either alone (carboxymethyl cellulose) or as mixed linkage (1,4-1,3)-ß-glucans (e.g., Avena glucan). It does not, however, degrade amorphous insoluble (phosphoric acid swollen) cellulose. Most significantly, the enzyme can also hydrolyze linkages in an Achlya cell wall fraction previously shown to consist of a mixed-linkage (1,4-1,3)-ß-glucan. This property is consistent with the long-standing hypothesis that the branching-related endoglucanases of oomycetes play a role in cell wall loosening.