Class I and class II chitin synthases are involved in septum formation in the filamentous fungus Aspergillus nidulans

The class II and class I chitin synthases of the filamentous fungus Aspergillus nidulans are encoded by chsA and chsC, respectively. Previously, we presented several lines of evidence suggesting that ChsA and ChsC have overlapping functions in maintaining cell wall integrity. In order to determine the functions of these chitin synthases, we employed electron and fluorescence microscopy and investigated in detail the cell wall of a DeltachsA DeltachsC double mutant (DeltaAC mutant) along with the localization of ChsA and ChsC. In the lateral cell wall of the DeltaAC mutant, electron-transparent regions were thickened. Septa of the DeltaAC mutant were aberrantly thick and had a large pore. Some septa were located abnormally close to adjacent septa. A functional hemagglutinin (HA)-tagged ChsA (HA-ChsA) and a functional FLAG-tagged ChsC (FLAG-ChsC) were each localized to a subset of septation sites. Comparison with the localization pattern of actin, which is known to localize at forming septa, suggested that ChsA and ChsC transiently exist at the septation sites during and shortly after septum formation. Double staining of HA-ChsA and FLAG-ChsC indicated that their localizations were not identical but partly overlapped at the septation sites. Fluorescence of FLAG-ChsC, but not of HA-ChsA, was also observed at hyphal tips. These data indicate that ChsA and ChsC share overlapping roles in septum formation.     

Hyphal walls of isolated lichen fungi

The hyphal walls of three mycobionts, isolated from the lichens Xanthoria parietina, Tornabenia intricata and Sarcogyne sp. were investigated by two techniques: microautoradiography of fungal colonies exposed to radioactive carbohydrate precursors; and binding, in vivo, of fluorescein conjugated lectins to hyphal walls of such colonies.N-[³H] acetylglucosamine was readily incorporated into tips, young hyphal walls and septa of the three mycobionts and the free-living fungus Trichoderma viride, but not into Phytophthora citrophthora, indicating that chitin is a major component of the mycobionts' hyphal walls. All three mycobionts, but neither of the free-living fungi, incorporated [³H] mannose and [³H] mannitol into their hyphal walls.Fluorescein-conjugated wheat germ agglutinin was bound to the hyphal walls of the three mycobionts and T. viride, but not to the walls of P. citrophthora; the binding pattern was similar to the grain pattern obtained in autoradiographs after short N-[³H] acetylglucosamine labelling. As wheat germ agglutinin binds specifically to chitin oligomers, the lectin binding tests further confirmed that chitin is a mycobiont hyphal wall component.Binding characteristics of several fluorescein-conjugated lectins to the three mycobionts indicated that this technique can yield useful information concerning the chemical composition of hyphal wall surfaces.List of abbreviations FITC fluorescein isothiocyanate - WGA wheat germ agglutinin - TCA trichloroacetic acid - PNA peanut agglutinin - LA lotus agglutinin - Glc NAc N-acetylglucosamine - ConA concanavalin A - SBA soybean agglutinin - WBA waxbean agglutinin Part of an M.Sc. thesis submitted by A. Braun to the Department of Botany, Tel Aviv University.     

Evidence that the Aspergillus nidulans class I and class II chitin synthase genes, chsC and chsA, share critical roles in hyphal wall integrity and conidiophore development

Although many chitin synthase genes have been identified in a broad range of fungal species, there have been only a few reports about their role in fungal morphogenesis. In most cases, single gene disruption or replacement did not reveal their function, possibly because of functional redundancy among them. We obtained null mutants of Aspergillus nidulans chsA and chsC genes encoding non-essential class II and class I chitin synthases, respectively. The DeltachsA DeltachsC mutant exhibited growth defects on media supplemented with sodium dodecyl sulfate (SDS), high concentration of salts, chitin-binding dyes, or chitin synthase competitive inhibitors, suggesting loss of integrity of hyphal wall. Moreover, remarkable abnormalities of the double mutant were observed microscopically during its asexual development. The conidiophore population was drastically reduced. Interestingly, secondary conidiophores were occasionally produced from vesicles of the primary ones. The morphology of these conidiophores was similar to those of the A. nidulans developmental mutants, medusa (medA), abacus (abaA), and some kinds of bristle (brlA). In situ staining patterns suggested that chsA was mainly expressed in the metulae, phialides, and conidia, whereas chsC was expressed in hyphae as well as conidiophores. These results suggest that ChsA and ChsC share critical functions in hyphal wall integrity and differentiation.     

TheAspergillus nidulans geneschsA andchsD encode chitin synthases which have redundant functions in conidia formation

We previously isolated three chitin synthase genes (chsA, chsB, andchsC) fromAspergillus nidulans. In the present work, we describe the isolation and characterization of another chitin synthase gene, namedchsD, fromA. nidulans. Its deduced amino acid sequence shows 56.7% and 55.9% amino acid identity, respectively, with Cal1 ofSaccharomyces cerevisiae and Chs3 ofCandida albicans. Disruption ofchsD caused no defect in cell growth or morphology during the asexual cycle and caused no decrease in chitin content in hyphae. However, double disruption ofchsA andchsD caused a remarkable decrease in the efficiency of conidia formation, while double disruption ofchsC andchsD caused no defect. Thus it appears thatchsA andchsD serve redundant functions in conidia formation.     

The Aspergillus nidulans genes chsA and chsD encode chitin synthases which have redundant functions in conidia formation

 We previously isolated three chitin synthase genes (chsA, chsB, and chsC) from Aspergillus nidulans. In the present work, we describe the isolation and characterization of another chitin synthase gene, named chsD, from A. nidulans. Its deduced amino acid sequence shows 56.7% and 55.9% amino acid identity, respectively, with Cal1 of Saccharomyces cerevisiae and Chs3 of Candida albicans. Disruption of chsD caused no defect in cell growth or morphology during the asexual cycle and caused no decrease in chitin content in hyphae. However, double disruption of chsA and chsD caused a remarkable decrease in the efficiency of conidia formation, while double disruption of chsC and chsD caused no defect. Thus it appears that chsA and chsD serve redundant functions in conidia formation.     

Wall Structure and spore Germination in Fusarium culmorum

The conidial and germ-tube walls of Fusarium culmorum (W. G.Smith) Sacc. have been examined by various chemical and electron-microscopetechniques. On the basis of these results and hypothesis isproposed for the organization of these walls. Microchemicaltests indicate the presence of chitin in the walls and suggestthat the mucilaginous layer around the conidium is mainly composedof xylan. Chemical analyses of isolated wall material confirmthe presence of chitin constituents in the wall, and a rylanlayer around the conidium. Furthermore, the wall contains apolypeptide moiety which has a different amino acid compositionfrom the rest of the protein of the cell. Electron microscopestudies of replicas and sections of conidia, germ tubes, andhyphae reveal a layered structure for the wall. The centrallayer is non-microfibrillar and is overlaid on both sides witha layer of randomly orientated microfibrils. The mucilaginouslayer of the conidium obscures the microfibrillar structurebeneath it unless the mucilage is removed by hydrolysis. Theproblem of hyphal growth is discussed on the basis of the structureof germ-tube tips and mature hyphae observed.     

Biochemical and Histochemical Localization of Invertase in Neurospora crassa During Conidial Germination and Hyphal Growth

The intracellular localization of Neurospora invertase, an enzyme partially secreted and partially retained by Neurospora at the cell periphery, was investigated. A cell wall fraction was isolated, to which 24% of the cell-bound invertase was firmly attached. A sensitive osmiophilic stain for invertase was developed and used in conjunction with the technique of indirect immunofluorescence to follow the pattern of invertase localization during the development of Neurospora from the germination of conidia to the mature hypha. These studies revealed that: (i) conidial invertase was uniformly distributed along the cell periphery; (ii) growing hyphal tips of germinating conidia showed pronounced invertase activity as the rest of the conidial cell wall lost its peripheral activity; (iii) hyphae in early log-phase growth had strong enzyme activity associated with the cell wall, and in late log phase the activity became associated with the plasma membrane and points where new hyphal branches were being formed; and (iv) hyphae in early stationary phase had strong fluorescence at incipient branching points, in "dots" close to the plasma membrane, and in the cytoplasm.     

Energy Value Evaluation of Hydrogenated Resistant Maltodextrin

Two chitin synthase genes, designated chsA and chsB, were isolated from Aspergillus nidulans with the Saccharomyces cerevisiae CHS2 gene as the hybridization probe. Nucleotide sequencing showed that chsA and chsB encoded polypeptides consisting of 1013 and 916 amino acid residues, respectively; the hydropathy profiles of the enzymes were similar to those of other fungal chitin synthases. Northern analysis indicated that both genes were transcribed, suggesting that cellular chitin in A. nidulans is synthesized by at least two chitin synthases. For examination of the roles of the chitin synthase genes in cell growth, gene disruption experiments were done. The chsA disruptant grew as well as the wild-type strain, but the chsB disruptant had severe growth defects that could not be overcome by the addition of 1.2 m sorbitol as an osmotic stabilizer. These findings suggested that chsB but not chsA is essential for hyphal growth.     

Characterization of <Emphasis Type="Italic">chsA,</Emphasis> a new gene controlling the chemotactic response in <Emphasis Type="Italic">Azospirillum brasilense</Emphasis> Sp7

We report, here, the characterization of a mutant strain of Azospirillum brasilense Sp7 impaired in surface motility and chemotactic response. Presence of flagella in the mutant strain was confirmed by western blot analysis, using antisera raised against the polar and lateral flagellins, and by electron microscopy. Genetic complementation and nucleotide sequencing led to the identification of a new gene, named chsA. The deduced translation product, ChsA protein, contained a PAS sensory domain and an EAL domain. As ChsA displayed characteristic signaling protein architecture, it is thought that this protein is a component of the signaling pathway controlling chemotaxis in Azospirillum.     

Septum-directed secretion in the filamentous fungus Aspergillus oryzae

Although exocytosis in fungal cells takes place at hyphal tips, there also seems a line of circumstantial evidence suggesting the occurrence of exocytosis at other sites of cells, such as septa. To investigate whether exocytosis takes place at fungal septa, we monitored dynamics of EGFP‐fused α‐amylase (AmyB–EGFP), the representative secretory enzyme of the filamentous fungus Aspergillus oryzae. We found that AmyB–EGFP accumulates in Spitzenkörper at hyphal tips as well as septal periplasm between the plasma membrane and cell walls. The septal accumulation of AmyB–EGFP was a rapid process, and required microtubules but not F‐actin. Thus, this process is independent of exocytosis at hyphal tips that requires both microtubules and F‐actin. In addition, fluorescence recovery after photobleaching (FRAP) analysis of EGFP‐fused AoSnc1 revealed that secretory vesicles constitutively fuse with the septal plasma membrane. These results demonstrated that exocytosis takes place at septa in addition to hyphal tips. Analysis of two plasma membrane transporters, AoUapC and AoGap1, revealed that they preferentially accumulate at septa and the lateral plasma membrane with no clear accumulation at apical Spitzenkörper, suggesting that non‐tip directed exocytosis is important for delivery of these proteins.     

Transportation of Aspergillus nidulans Class III and V Chitin Synthases to the Hyphal Tips Depends on Conventional Kinesin

Cell wall formation and maintenance are crucial for hyphal morphogenesis. In many filamentous fungi, chitin is one of the main structural components of the cell wall. Aspergillus nidulans ChsB, a chitin synthase, and CsmA, a chitin synthase with a myosin motor-like domain (MMD) at its N-terminus, both localize predominantly at the hyphal tip regions and at forming septa. ChsB and CsmA play crucial roles in polarized hyphal growth in A. nidulans. In this study, we investigated the mechanism by which CsmA and ChsB accumulate at the hyphal tip in living hyphae. Deletion of kinA, a gene encoding conventional kinesin (kinesin-1), impaired the localization of GFP-CsmA and GFP-ChsB at the hyphal tips. The transport frequency of GFP-CsmA and GFP-ChsB in both anterograde and retrograde direction appeared lower in the kinA-deletion strain compared to wild type, although the velocities of the movements were comparable. Co-localization of GFP-ChsB and GFP-CsmA with mRFP1-KinA^rigor, a KinA mutant that binds to microtubules but does not move along them, was observed in the posterior of the hyphal tip regions. KinA co-immunoprecipitated with ChsB and CsmA. Co-localization and association of CsmA with KinA did not depend on the MMD. These findings indicate that ChsB and CsmA are transported along microtubules to the subapical region by KinA.     

The Aspergillus nidulans phsB4 mutation alters colonial growth and development of the mould at acidic pH

The phsB4 mutant of the mould Aspergillus nidulans, identified as showing increased sensitivity to acid pH, is mitotically unstable and its conidia swell and lyse, forming protoplasts during germination and early development in shaken liquid cultures. On solid medium, we observed balloon-shaped hyphal swellings, a phenotype also exhibited by the chitin synthase gene (chsD) disruptants. We also observed that lysis was osmotically remediable with 0.5 M NaCl, but the balloon-shaped hyphal swelling was remedied in a pH-dependent way i.e., this phenotype was remedied only at pH values above 6.5. Based on the nature of our mutant selection, the pH sensitive phenotype of the selected strains, the known occurrence of hyphal swelling in cell wall mutants of A. nidulans, and the transformation with cosmids that hybridize to chsD gene, the phsB and chsD genes are possibly alleles.     

ChsVb, a class VII chitin synthase involved in septation, is critical for pathogenicity in Fusarium oxysporum

A new myosin motor-like chitin synthase gene, chsVb, has been identified in the vascular wilt fungus Fusarium oxysporum f. sp. lycopersici. Phylogenetic analysis of the deduced amino acid sequence of the chsVb chitin synthase 2 domain (CS2) revealed that ChsVb belongs to class VII chitin synthases. The ChsVb myosin motor-like domain (MMD) is shorter than the MMD of class V chitin synthases and does not contain typical ATP-binding motifs. Targeted disrupted single (DeltachsVb) and double (DeltachsV DeltachsVb) mutants were unable to infect and colonize tomato plants or grow invasively on tomato fruit tissue. These strains were hypersensitive to compounds that interfere with fungal cell wall assembly, produced lemon-like shaped conidia, and showed swollen balloon-like structures in hyphal subapical regions, thickened walls, aberrant septa, and intrahyphal hyphae. Our results suggest that the chsVb gene is likely to function in polarized growth and confirm the critical importance of cell wall integrity in the complex infection process of this fungus.     

Scanning electron microscopy of the conidia produced by the mycelial form of Paracoccidioides brasiliensis

The conidia produced by the mycelial form of Paracoccidioides brasiliensis were examined by scanning electron microscopy for the first time. Several different conidial types were characterized. These included intercalary arthroconidia, several types of septate conidia that are formed from other conidia, pedunculate conidia, and terminal hyphal conidia. In addition, the ultrastructure of the supporting pedestal of the pedunculate conidium was found to be separated from the mother conidium by a septum in some instances, and at other times it was not.     

Bronchoalveolar lavage in an immunosuppressed rabbit model of invasive pulmonary aspergillosis

A rabbit model of invasive aspergillosis has been used to investigate the pathogenesis of Aspergillus infection in the immunosuppressed host. The animals received hydrocortisone daily and a single dose of cyclophosphamide 2 days prior to intratracheal instillation of conidia from Aspergillus fumigatus. Bronchoalveolar lavage (BAL) was performed in 3 infected and 2 control saline treated animals sacrificed on days 1, 2, 4, 7 and 10 following inoculation. Infective load within the lung was quantified using an assay for chitin which is an important component of fungal cell walls (in particular the hyphal cell wall) and is not present in vertebrate tissue. The total BAL white cell count did not discriminate between infected and saline treated animals and Aspergillus was cultured from one lavage specimen only. Infected animals developed a marked neutrophil alveolitis by day 2 in contrast to a near total absence of neutrophils in the lavages of the control animals. Phagocytosis of conidia by alveolar macrophages was prominent but did not prevent progressive infection as confirmed by measurement of lung chitin. This pattern of cellular response within the alveolar airspace reflects the complex nature of the response to Aspergillus infection in the immunosuppressed host.     

The Guanine Nucleotide Exchange Factor RIC8 Regulates Conidial Germination through Gα Proteins in Neurospora crassa

Heterotrimeric G protein signaling is essential for normal hyphal growth in the filamentous fungus Neurospora crassa. We have previously demonstrated that the non-receptor guanine nucleotide exchange factor RIC8 acts upstream of the Gα proteins GNA-1 and GNA-3 to regulate hyphal extension. Here we demonstrate that regulation of hyphal extension results at least in part, from an important role in control of asexual spore (conidia) germination. Loss of GNA-3 leads to a drastic reduction in conidial germination, which is exacerbated in the absence of GNA-1. Mutation of RIC8 leads to a reduction in germination similar to that in the Δgna-1, Δgna-3 double mutant, suggesting that RIC8 regulates conidial germination through both GNA-1 and GNA-3. Support for a more significant role for GNA-3 is indicated by the observation that expression of a GTPase-deficient, constitutively active gna-3 allele in the Δric8 mutant leads to a significant increase in conidial germination. Localization of the three Gα proteins during conidial germination was probed through analysis of cells expressing fluorescently tagged proteins. Functional TagRFP fusions of each of the three Gα subunits were constructed through insertion of TagRFP in a conserved loop region of the Gα subunits. The results demonstrated that GNA-1 localizes to the plasma membrane and vacuoles, and also to septa throughout conidial germination. GNA-2 and GNA-3 localize to both the plasma membrane and vacuoles during early germination, but are then found in intracellular vacuoles later during hyphal outgrowth.     

Morphological anomalies induced by Congo red in Aspergillus niger

Aspergillus niger germ tubes were exposed for 6 h to 0.15 mg/ml of Congo red, a stain which prevents chitin microfibril assembly. The most evident alterations, detected under ultraviolet light and by transmission and scanning electron microscopy, concerned the hyphal tips which burst or, most frequently, expanded into bulges. In the latter structures, new hyphal tips appeared which, after giving rise to more or less developed hyphae, were themselves converted into new bulges. Therefore, segments derived from isotropic and polarized growth alternated in the organisms exposed to the dye.An interpretation of these abnormalities is advanced based upon the assumption that the maintainance of a regular gradient of wall viscosity in the hyphal extension zone depends primarily on the capability of glycan chains to form crystalline aggregates of increasing complexity.     

The Aspergillus nidulans genes chsA and chsD encode chitin synthases which have redundant functions in conidia formation

We previously isolated three chitin synthase genes (chsA, chsB, and chsC) from Aspergillus nidulans. In the present work, we describe the isolation and characterization of another chitin synthase gene, named chsD, from A. nidulans. Its deduced amino acid sequence shows 56.7% and 55.9% amino acid identity, respectively, with Cal1 of Saccharomyces cerevisiae and Chs3 of Candida albicans. Disruption of chsD caused no defect in cell growth or morphology during the asexual cycle and caused no decrease in chitin content in hyphae. However, double disruption of chsA and chsD caused a remarkable decrease in the efficiency of conidia formation, while double disruption of chsC and chsD caused no defect. Thus it appears that chsA and chsD serve redundant functions in conidia formation.