Increased nuclear traffic chaos in hyphae of Aspergillus nidulans: molecular characterization of apsB and in vivo observation of nuclear behaviour

Filamentous fungi are model microorganisms for studying nuclear migration in eukaryotic cells. Two genes, apsA and apsB (=anucleate primary sterigmata), were identified in Aspergillus nidulans that affect nuclear distribution in hyphae and specifically block conidiophore development at the metula stage when mutant. Here we describe the cloning, sequencing and molecular analysis of apsB. The gene encodes a 121 kDa coiled-coil, hydrophilic protein that was localized in the cytoplasm. No protein-protein interaction was detected between ApsB and ApsA, a membrane-associated, previously identified protein. An apsB null mutant was characterized by video epifluorescence microscopy using strains that express green fluorescent protein (GFP) in nuclei. With this novel approach, we have discovered a new mutant phenotype and have found that nuclei display an increased chaotic movement in older hyphal compartments that results in clustering and an uneven distribution of these organelles. These results suggest a regulatory role of ApsB in nuclear migration.     

Isolation of Two Apsa Suppressor Strains in Aspergillus Nidulans

Aspergillus nidulans reproduces asexually with single nucleated conidia. In apsA (anucleate primary sterigmata) strains, nuclear positioning is affected and conidiation is greatly reduced. To get further insights into the cellular functions of apsA, aconidial apsA strains were mutagenized and conidiating suppressor strains were isolated. The suppressors fell into two complementation groups, samA and samB (suppressor of anucleate metulae). samA mapped on linkage group I close to pyrG. The mutant allele was dominant in diploids homozygous for apsA. Viability of conidia of samA suppressor strains (samA(-); apsA(-)) was reduced to 50% in comparison to wild-type conidia. Eighty percent of viable spores produced small size colonies that were temperature- and benomyl-sensitive. samB mapped to chromosome VIII and was recessive. Viability of conidia from samB suppressor strains (apsA(-); samB(-)) was also affected but no small size colonies were observed. Both suppressors produced partial defects in sexual reproduction and both suppressed an apsA deletion mutation. In wild-type background the mutant loci affected hyphal growth rate (samA) or changed the colony morphology (samB) and inhibited sexual spore formation (samA and samB). Only subtle effects on conidiation were found. We conclude that both suppressor genes bypass the apsA function and are involved in microtubule-dependent processes.     

abaA controls phialide differentiation in Aspergillus nidulans.

Aspergillus nidulans is an ascomycetous fungus that reproduces asexually by forming multicellular conidiophores and uninucleate spores called conidia. Loss of function mutations in the abacus A (abaA) regulatory locus result in formation of aberrant conidiophores that fail to produce conidia. Wild-type conidiophores form two tiers of sterigmata. The first tier, metulae, divide to produce the second tier, phialides. Phialides are sporogenous cells that produce conidia through a specialized apical budding process. We have examined conidiophore development in an abaA- strain at the ultrastructural level. The results showed that in the mutant metulae produce supernumerary tiers of cells with metula-like, rather than phialide-like, properties. Temperature shift experiments with an abaA14ts strain demonstrated that abaA+ function induced phialide formation by the aberrant abacus cells and was continuously required for maintenance of phialide function. In the absence of abaA+ activity, metulae simply proliferated and later developmental steps never occurred. We conclude that abaA+ directs the differentiation of phialides and is continuously required for maintenance of their function.     

Autophagy during conidiation and conidial germination in filamentous fungi

Filamentous fungi form aerial hyphae on solid medium, and some of these differentiate into conidiophores for asexual sporulation (conidiation). In the filamentous deuteromycete, Aspergillus oryzae, aerial hyphae are formed from the foot cells and some differentiate into conidiophores, which are composed of vesicles, phialides and conidia. Recently, we isolated the yeast ATG8 gene homologue Aoatg8 from A. oryzae, and visualized autophagy by the expression of an EGFP (enhanced green fluorescent protein)-AoAtg8 fusion protein and DsRed2 protein in this fungus. Furthermore, by constructing the Aoatg8 deletion and conditional mutants, we demonstrated that autophagy functions during the process of differentiation of aerial hyphae, conidiation and conidial germination in A. oryzae. Here, we discuss the contribution of autophagy towards the differentiation and germination processes in filamentous fungi.     

Surface Ultrastructural Studies on the Germination,Penetration and Conidial Development of Aspergillus Flavus Link : Fries Infecting Silkworm,Bombyx Mori Linn

Aspergillosis is a common disease of the silkworm Bombyx mori Linn., caused by an insect mycopathogen Aspergillus flavus Link:Fries. The present study reveals the germination, penetration and conidial development of A. flavus on the larval integument of B. mori under SEM. Four different strains (NB18, KA, NB4D2 and NB7) of B. mori was surface inoculated with ca. of 1 x 10(6) conidia/ml. Each conidium germinated on the cuticle approximately 6 h after inoculation, forming a humpy or suctorial appressoria within 24 h. The hyphae which entered into haemocoel 2 day post-inoculation, grew and multiplied extensively, forming a mycelial complex, causing death of the host larva in about 4-5 days. This occurred with minimal breakdown of the internal tissues. Death of the host was followed by ramification of the fungus through the mesodermal and epidermal tissues, leading to larval mummification about 5-6 days after inoculation. Extensive fungal growths on the entire larval body followed, consisting of aerial hyphae, which developed branched conidiophores. The aerial hyphae with abundant conidiophores formed a confluent yellowish green fungal mat over the entire larval body in 6-7 days of post-inoculation. The tip of each emerging conidiophores gradually dilated and developed to become a bulbous head known as the vesicle. A large number of conidiogenous cells were produced over the entire surface of vesicle, which later developed into finger-like projections termed as sterigmata or phialides. The phialides matured within 2 days after the aerial hyphae emerged as evidenced by chains of conidia at their tips. The conidia were globose with externally roughened walls. The life cycle of the fungus on B. mori was completed in six to seven days.     

Isolation of Aspergillus nidulans Mutants That Overcome brlA-Induced Growth Arrest

The Aspergillus nidulans brlA gene is a primary regulator of development-specific gene expression during conidiation. Forced activation of brlA in vegetative cells leads to inappropriate induction of conidiophore formation and causes growth to stop. In fact, when conidia containing a nutritionally inducible brlA gene fusion are placed on inducing medium, they fail to germinate. We used this phenotype to select 174 mutants that continue growing following such forced brlA activation. Forty-six of these mutants also produced abnormal developmental structures during air-induced conidiation as expected if the mutations resulted in an altered response to BrlA (designated sbr mutants for suppressors of brlA response). The predominant mutant class identified was defective in a known developmental regulatory gene, abaA. We also identified mutants with defects in the previously characterized early acting developmental regulatory genes flbB and flbD and in four previously undescribed loci designated sbrA-D. sbrA mutants represent the second largest group and are characterized by production of conidiophore stalks that lack a normal vesicle and form branching sterigmata that rarely make spores. Because abaA expression could not be detected in sbrA mutants following brlA activation we propose that sbrA functions as a developmental modifier, participating in brlA-dependent activation of other developmental regulators.     

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.     

ami1, an orthologue of the Aspergillus nidulans apsA gene, is involved in nuclear migration events throughout the life cycle of Podospora anserina

The Podospora anserina ami1-1 mutant was identified as a male-sterile strain. Microconidia (which act as male gametes) form, but are anucleate. Paraphysae from the perithecium beaks are also anucleate when ami1-1 is used as the female partner in a cross. Furthermore, in crosses heterozygous for ami1-1, some crozier cells are uninucleate rather than binucleate. In addition to these nuclear migration defects, which occur at the transition between syncytial and cellular states, ami1-1 causes abnormal distribution of the nuclei in both mycelial filaments and asci. Finally, an ami1-1 strain bearing information for both mating types is unable to self-fertilize. The ami1 gene is an orthologue of the Aspergillus nidulans apsA gene, which controls nuclear positioning in filaments and during conidiogenesis (at the syncytial/cellular transition). The ApsA and AMI1 proteins display 42% identity and share structural features. The apsA gene complements some ami1-1 defects: it increases the percentage of nucleate microconidia and restores self-fertility in an ami1-1 mat+ (mat-) strain. The latter effect is puzzling, since in apsA null mutants sexual reproduction is quite normal. The functional differences between the two genes are discussed with respect to their possible history in these two fungi, which are very distant in terms of evolution.     

Identification of Developmental Regulatory Genes in Aspergillus Nidulans by Overexpression

Overexpression of several Aspergillus nidulans developmental regulatory genes has been shown to cause growth inhibition and development at inappropriate times. We set out to identify previously unknown developmental regulators by constructing a nutritionally inducible A. nidulans expression library containing small, random genomic DNA fragments inserted next to the alcA promoter [ alcA (p) ] in an A. nidulans transformation vector. Among 20,000 transformants containing random alcA (p) genomic DNA fusion constructs, we identified 66 distinct mutant strains in which alcA (p) induction resulted in growth inhibition as well as causing other detectable phenotypic changes. These growth inhibited mutants were divided into 52 FIG (Forced expression Inhibition of Growth) and 14 FAB (Forced expression Activation of brlA) mutants based on whether or not alcA (p) induction resulted in accumulation of mRNA for the developmental regulatory gene brlA. In four FAB mutants, alcA (p) induction not only activated brlA expression but also caused hyphae to differentiate into reduced conidiophores that produced viable spores from the tips as is observed after alcA (p) :: brlA induction. Sequence analyses of the DNA fragments under alcA (p) control in three of these four sporulating strains showed that in two cases developmental activation resulted from overexpression of previously uncharacterized genes, whereas in the third strain, the alcA (p) was fused to brlA. The potential uses for this strategy in identifying genes whose overexpression results in specific phenotypic changes like developmental induction are discussed.     

Accumulation of Ergot Alkaloids During Conidiophore Development in Aspergillus fumigatus

Production of ergot alkaloids in the opportunistic fungal pathogen Aspergillus fumigatus is restricted to conidiating cultures. These cultures typically accumulate several pathway intermediates at concentrations comparable to that of the pathway end product. We investigated the contribution of different cell types that constitute the multicellular conidiophore of A. fumigatus to the production of ergot alkaloid pathway intermediates versus the pathway end product, fumigaclavine C. A relatively minor share (11 %) of the ergot alkaloid yield on a molar basis was secreted into the medium, whereas the remainder was associated with the conidiating colonies. Entire conidiating cultures (containing hyphae, vesicle of conidiophore, phialides of conidiophore, and conidia) accumulated higher levels of the pathway intermediate festuclavine and lower levels of the pathway end product fumigaclavine C than did isolated, abscised conidia, indicating that conidiophores and/or hyphae have a quantitatively different ergot alkaloid profile compared to that of conidia. Differences in alkaloid accumulation among cell types also were indicated by studies with conidiophore development mutants. A ?medA mutant, in which conidiophores are numerous but develop poorly, accumulated higher levels of pathway intermediates than did the wildtype or a complemented ?medA mutant. A ?stuA mutant, which grows mainly as hyphae and produces very few, abnormal conidiophores, produced no detectable ergot alkaloids. The data indicated heterogeneous spatial distribution of ergot alkaloid pathway intermediates versus pathway end product in conidiating cultures of A. fumigatus. This skewed distribution may reflect differences in abundance or activity of pathway enzymes among cell types of those conidiating cultures.     

Genetic and cytological characterization of a developmental mutant of Aspergillus nidulans induced by 5-azacytidine.

An analysis of a new medusa mutant of Aspergillus nidulans obtained by 5-azacytidine-treatment and named B116 is provided. The B116 mutant was phenotypically characterized by the production of conidiophores with reduced pigmentation and vesicles bearing multiple tiers of sterigmata. A single nuclear gene located on chromosome I is responsible for phenotypical changes in the mutant. The 5-azacytidine-altered locus, designated medA102, is recessive in heterozygous diploid and the medusa mutant is a Dp(II,I) duplication bearer that renders the strain mitotically unstable.     

Spatial control of developmental regulatory genes inAspergillus nidulans

ThebrlA andabaA genes ofAspergillus nidulans regulate stages of conidiophore development and are themselves regulated during development.brlA⁻ mutants produce conidiophore stalks devoid of vesicles, sterigmata, and spores.abaA⁻ mutants produce most of the conidiophore structures but fail to form conidia. To assess the spatial expression of these two genes, we fused the 5′ flanking region ofbrlA orabaA to theEscherichia coli lacZ gene.A. nidulans transformants with a single copy of either fusion gene integrated at a defined heterologus locus (argB) expressedβ-galactosidase during conidiophore development, parallelingbrlA andabaA mRNA accumulation. Controls lacking the fusion genes produced little or noβ-galactosidase activity. A method forin situ detection ofβ-galactosidase was devised. Hyphae or conidiophores were permeabilized by treatment with chloroform vapors and stained with 5-bromo-4-chloroindolyl-β-d-galactoside.β-Galactosidase activity was detected in specific conidiophore cell types.brlA- andabaA-directedβ-galactosidase accumulated in vesicles, sterigmata, and immature conidia. This procedure should be applicable for determining cellular specificities of gene expression in fungi for which transformation systems exist.     

Morphogenetic and developmental functions of the Aspergillus nidulans homologues of the yeast bud site selection proteins Bud4 and Axl2

The yeast bud site selection system represents a paradigm for understanding how fungal cells regulate the formation of a polarity axis. In Saccharomyces cerevisiae, Bud4 and Axl2 are components of the axial bud site marker. To address the possibility that these proteins regulate cellular morphogenesis in filamentous fungi, we have characterized homologues of Bud4 and Axl2 in Aspergillus nidulans. Our results show that Bud4 is involved in septum formation in both hyphae and developing conidiophores. Whereas Axl2 appears to have no obvious role in hyphal growth, it is required for the regulation of phialide morphogenesis during conidiation. In particular, Axl2 localizes to the phialide-spore junction, where it appears to promote the recruitment of septins. Furthermore, the developmental regulators BrlA and AbaA control the expression of Axl2. Additional studies indicate that Axl2 is also involved in the regulation of sexual development, not only in A. nidulans, but also in the phylogenetically unrelated fungus Fusarium graminearum. Our results suggest that Axl2 plays a key role in phialide morphogenesis and/or function during conidiation in the aspergilli.     

A NEW NEMATODE-DESTROYING HYPHOMYCETE OF THE GENUS HARPOSPORIUM

Drechsler , Charles . (USDA, Plant Industry Sta., Beltsville, Md.) A new nematode-destroying hyphomycete of the genus Harposporium. Amer. Jour. Bot. 50(8): 839–842. Illus. 1963.—A mucedinaceous parasite that destroyed a largish nematode in a maize-meal-agar plate culture to which had been added some leaf mold from an oak wood in central Maryland is newly described as Harposporium dicorymbum. It permeates the host animal with colorless septate assimilative hyphae, 2.5-8.5 μ wide, that soon put forth procumbent septate conidiophores bearing mostly subglobose phialides with 1-3 sterigmata. Its very distinctive conidia are composed of a minutely pedicellate globose part, 4-7 μ in diameter, together with a cylindrical outgrowth, 3-9 μ long and 2-3 μ wide, which is furnished distally with a short posterior and a longer anterior beak.     

Autophagy During Conidiation and Conidial Germination in Filamentous Fungi

Filamentous fungi form aerial hyphae on solid medium, and some of these differentiate into conidiophores for asexual sporulation (conidiation). In the filamentous deuteromycete, Aspergillus oryzae, aerial hyphae are formed from the foot cells and some differentiate into conidiophores, which are composed of vesicles, phialides and conidia. Recently, we isolated the yeast ATG8 gene homologue Aoatg8 from A. oryzae, and visualized autophagy by the expression of an EGFP (enhanced green fluorescent protein)–AoAtg8 fusion protein and DsRed2 protein in this fungus. Furthermore, by constructing the Aoatg8 deletion and conditional mutants, we demonstrated that autophagy functions during the process of differentiation of aerial hyphae, conidiation and conidial germination in A. oryzae. Here, we discuss the contribution of autophagy towards the differentiation and germination processes in filamentous fungi.

Addendum to:

Functional Analysis of the ATG8 Homologue Aoatg8 and Role of Autophagy in Differentiation and Germination in Aspergillus oryzae

T. Kikuma, M. Ohneda, M. Arioka and K. Kitamoto

Eukaryot Cell 2006; 5:1328-36     

Functional analysis of the ATG8 homologue Aoatg8 and role of autophagy in differentiation and germination in Aspergillus oryzae

Autophagy is a well-known degradation system, induced by nutrient starvation, in which cytoplasmic components and organelles are digested via vacuoles/lysosomes. Recently, it was reported that autophagy is involved in the turnover of cellular components, development, differentiation, immune responses, protection against pathogens, and cell death. In this study, we isolated the ATG8 gene homologue Aoatg8 from the filamentous fungus Aspergillus oryzae and visualized autophagy by the expression of DsRed2-AoAtg8 and enhanced green fluorescent protein-AoAtg8 fusion proteins in this fungus. While the fusion proteins were localized in dot structures which are preautophagosomal structure-like structures under normal growth conditions, starvation or rapamycin treatment caused their accumulation in vacuoles. DsRed2 expressed in the cytoplasm was also taken up into vacuoles under starvation conditions or during the differentiation of conidiophores and conidial germination. Deletion mutants of Aoatg8 did not form aerial hyphae and conidia, and DsRed2 was not localized in vacuoles under starvation conditions, indicating that Aoatg8 is essential for autophagy. Furthermore, Aoatg8 conditional mutants showed delayed conidial germination in the absence of nitrogen sources. These results suggest that autophagy functions in both the differentiation of aerial hyphae and in conidial germination in A. oryzae.