Visualization of nuclei in Aspergillus oryzae with EGFP and analysis of the number of nuclei in each conidium by FACS

Aspergillus oryzae has been reported to form conidia with multinuclei. In order to analyze nuclei in living cells, we developed an expression system of the A. nidutans histone H2B protein tagged by EGFP (H2B::EGFP). In both A. oryzae niaD300 and A. nidulans FGSC89 transformants expressing H2B::EGFP, fluorescence was detected in nuclear regions of hyphae and conidia. While a conidium contained only one fluorescent spot in the A. nidulans transformant, approximately 66% of conidia had two, 24% had one, and 10% had three or more in the A. oryzae transformant. The conidia expressing H2B::EGFP were put through FACS (fluorescence-activated cell sorting) analysis and two sharp peaks, corresponding to one and two nuclei in each conidium, were noted in the A. oryzae transformant. In addition, the A. oryzae uninucleate conidia that were successfully isolated by FACS reproduced conidia with almost the same number distribution of nuclei as that of the original. Conidia of five A. oryzae strains used in sake brewing were scored for the number of nuclei, showing that a varied number of nuclei existed in each conidium and some strains had a small number of uninucleate conidia.     

The Formation, General Characteristics and Production of α-Amylase in Heterocaryons and Diploids of Aspergillus oryzae

S ummary . Heterocaryons and diploids from Aspergillus oryzae were investigated with respect to nuclear number/conidium and to conidial size. Heterocaryons usually had larger conidia and more nuclei/conidium than diploids and the haploid parent mutants. Diploids contained significantly fewer nuclei/conidium than haploids. However, they could not be distinguished from haploids by measurement of conidial size. The strains were examined for the production of α-amylase. All auxotrophic mutants produced less α-amylase than the prototrophic wild type. Heterocaryons gave yields which were intermediate between that of their parent mutants or the same as the best producing parent. Diploids which produced more α-amylase than the best producing parent strain were synthesized. The highest yield from a diploid was of the same order of magnitude as the yield from the wild type.     

Successful induction and recognition of conidiation,conidial germination and chlamydospore formation in pure culture of Morchella

Morels, fungi from the genus Morchella, are popular edible mushrooms. However, little knowledge of their asexual reproduction and inaccessible pure mitospores hamper illumination of their life cycle. Herein, we successfully induced conidiation, conidial germination and chlamydospore formation in pure culture of Morchella sextelata. Conidiation proceeded via four morphologically distinct stages: development of the conidiophore stalk, stalk branching, phialide differentiation, and conidium production. Terminal and intercalary chlamydospores were formed on conidial hyphae. The development of conidiophores occurred earlier, with more conidia produced, in cross-mating cultures than in single-spore cultures. Mature conidia were spherical and 2.5–8 μm in diameter, with a vast majority (nearly 99%) 2.5–5 μm in diameter. Each conidium contained one to three nuclei (80.2% conidia contained one nucleus, 19.1% contained two nuclei, and 0.7% contained three nuclei). The conidial nucleus diameter was 1–2 μm. The nuclear mitosis in detached conidia that was observed may benefit their colony initiation. Additionally, morel conidia formed conidial anastomosis tubes. Conidia (mitospores) likely not only function as spermatia, but also as reproductive propagules in Morchella. Further research is imperative to elucidate the relationship between the conidia and chlamydospores, and their unique function in the morel life cycle.     

EglD, a putative endoglucanase, with an expansin like domain is localized in the conidial cell wall of Aspergillus nidulans

Although the process of conidial germination in filamentous fungi has been extensively studied, many aspects remain to be elucidated since the asexual spore or conidium is vital in their life cycle. Breakage and reformation of cell wall polymer bonds along with the maintenance of cell wall plasticity during conidia germination depend upon a range of hydrolytic enzymes whose activity is analogous to that of expansins, a highly conserved group of plant cell wall proteins with characteristic wall loosening activity. In the current study, we identified and characterized the eglD gene in Aspergillus nidulans, an expansin-like gene the product of which shows strong similarities with bacterial and fungal endo-beta1,4-glucanases. However, we failed to show such activity in vitro. The eglD gene is constitutively expressed in all developmental stages and compartments of A. nidulans asexual life cycle. However, the EglD protein is exclusively present in conidial cell walls. The role of the EglD protein in morphogenesis, growth and germination rate of conidia was investigated. Our results show that EglD is a conidial cell wall localized expansin-like protein, which could be involved in cell wall remodeling during germination.     

Observation of EGFP-visualized nuclei and distribution of vacuoles in Aspergillus oryzae arpA null mutant

The arpA gene encoding Arp1 (actin-related protein) was previously cloned and characterized from Aspergillus oryzae. Phenotypes of the arpA null mutant indicate its requirement for normal nuclear distribution and morphology of conidiophores. In this study, we further characterized the function of the arpA gene in distribution of organelles. For further analysis of nuclear migration in living cells, an expression system consisting of a fusion protein of Aspergillus nidulans histone H2B and EGFP (H2B::EGFP) was used. This demonstrated diminished hyphal-tip growth rate and inefficient nuclear transport to apical regions in the arpA null mutant. Expression of H2B::EGFP also revealed an increase in the nuclear number of each conidium in the arpA null mutant, implicating a role for the arpA gene in controlling the nuclear movement into conidia. Furthermore, staining of vacuoles of the arpA null mutant with CMAC (cell tracker blue) suggested that the arpA gene is required for proper vacuolar distribution in addition to its role in normal nuclear distribution.     

The Aspergillus nidulans bncA1 mutation causes defects in the cell division cycle, nuclear movement and developmental morphogenesis

Wild-type Aspergillus nidulans conidia are uninucleate. The mutation bncA1 (binucleated conidia) was first described as a single mutation located on chromosome IV that caused formation of approximately 25% binucleate and 1% trinucleate conidia. In this study, we show that bncA1 conidia exit G1 arrest earlier than the wild type. Germlings have hyphal elements with abnormal morphology, elevated numbers of randomly distributed nuclei and an irregular septation pattern. Older hyphal elements undergo mitotic catastrophe, suggesting the nuclear division cycle of internal (nonterminal) elements is not arrested. The bncA1 mutation also causes aberrant morphogenesis of the asexual reproductive structure, the conidiophore. Metulae and phialides are elongated and have incorrect numbers of nuclei. Phialides also have internal septation that appears to delineate hyphal-like elements. Heterokaryon analysis using strains with contrasting auxotrophic markers showed that the bncA1 mutation resulted in a higher frequency of diploid and multinucleated prototrophic conidia than control heterokaryons. These results suggest that in bncA1 strains multiple nuclei can move from the conidiophore vesicle to the metulae and/or from the phialide to the conidium. The bncA1 mutant also showed hypersensitivity to the anti-microtubule drugs thiabendazole and nocodazole, which is consistent with the defects in cell cycle regulation and nuclear movement. We propose that bncA has an important role in correctly regulating both the cell division cycle and nuclear movement.     

Microcycle conidiation in Penicillium urticae: an ultrastructural investigation of conidiogenesis.

A cultivation system has been developed for Penicillium urticae which yields 'microcycle' conidiation in submerged culture. Spherical growth of spores was initiated by incubation at 37 degrees C in a growth-favoring medium. Transfer of these enlarged spores to a nitrogen-poor medium at 35 degrees C results in synchronous germination and limited outgrowth followed by roughly synchronous conidiation. A study of the conidiation stage showed that a phialide and an immature conidium began to form at the tip of all germ tubes 18 h after the temperature shift. By 24 h additional phialides commonly appeared as a branch near the tip of the germ tube and the more mature conidia exhibited increasing refractility. The earliest ultrastructural signs of conidiation were various round invaginations in the plasma membrane and a thickening and rounding of the new spore wall which appeared as an inner extension of the phialide cell wall. Upon segregation of the conidium from the phialide cell by conidial wall formation, 'trench-like' invaginations gradually appeared in the plasma membrane and a disorganized rodlet pattern was formed on the outer surface of the maturing conidial wall. Continued maturation involved the formation of chains of conidia and phialide senescence which was characterized by a general degradation of intracellular structure. A comparison with standard surface and submerged culture conidiation indicated that 'microcycle' conidiation, while less prolific, was essentially identical.     

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.     

A simple method for enrichment of uninucleate conidia of Aspergillus oryzae

Aspergillus oryzae produces multinucleate conidia, which makes the obtaining of homokaryons labor-intensive. Analysis of conidia by flow cytometry clarified the relationship that conidia of lower nuclear number were smaller in size. Based on this, we have developed a simple way to enrich uninucleate conidia with a membrane filter. Our results also suggest that the method is useful for elimination of heterokaryons.     

Trehalase in conidia of Aspergillus oryzae

Horikoshi, Koki (The Institute of Physical and Chemical Research, Bunkyo-ku, Tokyo, Japan), and Yonosuke Ikeda. Trehalase in conidia of Aspergillus oryzae. J. Bacteriol. 91:1883-1887. 1966.-Trehalases (soluble trehalase and coat-bound trehalase) were found in the conidia of Aspergillus oryzae, and the total activity of the trehalases increased during the germination process. The soluble trehalase was purified by diethylaminoethyl-cellulose column chromatography; its optimal pH, Michaelis constant, and heat stability were studied. In vitro, the trehalases were competitively inhibited by d-mannitol, which was also contained in the conidia. Since the trehalose content in the conidia decreased at an early stage of germination, it was assumed that trehalase might begin to hydrolyze trehalose after the inhibitory effect of d-mannitol decreased.     

Characterization of an Aspergillus nidulans mutant with abnormal distribution of nuclei in hyphae, metulae, phialides and conidia

The V10 deteriorated variant of Aspergillus nidulans has hyphae, metulae, phialides and conidia with abnormal nuclear distributions. The alterations observed were: increase in the number of nuclei in hyphae, metulae and phialides, presence of anucleate, uninucleate and multinucleate conidia, abnormal vegetative growth and defective conidiation. When 0.5 M NaCl was added to the medium, an increase in the number of conidia was observed but their morphology and number of nuclei were not modified. The gene responsible for these alterations was named anuA1. The anuA1 gene is located on linkage group VII and is possibly involved in nuclear migration to hyphae, metulae, phialides and conidia.     

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.     

Protoplast fusion between Aspergillus oryzae and Aspergillus niger in relation to their multinuclear nature

Protoplasts of cycloheximide-resistant strains from Aspergillus oryzae IFO 5239 were fused with those of kabicidin-resistant strains from Aspergillus niger IFO 4407. By nuclear staining in conidia, it appeared that all of the fusant conidia had two kinds of nuclei. Small nuclei seemed to be derived from A. oryzae and large nuclei seemed to be derived from A. niger. However, three types of antibiotic resistance were shown among the conidia of fusants. Almost all were kabicidin resistant. Conidia of fusants were multinuclear and had various DNA contents and various sizes. By the comparison with the growth rates of parental strains, the growth rates of A. niger were superior to those of A. oryzae. The inclination in the distribution of antibiotic resistance of fusant conidia seemed to owe more to the differences of growth rates between parental strains than the influence of the multinucleate nature of a parental strain.     

Novel role of cytoplasmic dynein motor in maintenance of the nuclear number in conidia through organized conidiation in Aspergillus oryzae

Cytoplasmic dynein is a minus-end-directed, microtubule-dependent motor protein complex. DhcA, cytoplasmic dynein heavy chain in Aspergillus oryzae, contained four P-loops involved in ATP binding which were conserved as in cytoplasmic dynein heavy chains of other organisms. The amino acid sequence of A. oryzae DhcA was similar to cytoplasmic dynein heavy chains from other organisms except for the N-terminus of Saccharomyces cerevisiae Dyn1. Disruption of dhcA gene in the region encoding four P-loop motifs resulted in a defective growth and perturbed distribution of nuclei and vacuoles. The dhcA disruptant exhibited an abnormal morphology of conidial heads and conidia with an increased nuclear number. The present study implicates a novel role of cytoplasmic dynein in maintenance of the nuclear number in conidia through an organized conidiation.     

On the role of microtubules, cell end markers, and septal microtubule organizing centres on site selection for polar growth in Aspergillus nidulans

Tip growth of filamentous fungi depends on continuous polarized growth and requires the actin and microtubule (MT) cytoskeleton. Cortical proteins at polarity sites, also known as cell end markers, play important roles in polarity maintenance. Deletion of the cell end marker teaA results in zigzag hyphal morphologies, which is contrary to the normal rectilinear growth pattern. Here we studied the role of TeaA and MTs in the establishment of polarity during tip growth of Aspergillus nidulans, including conidia germination, second germtube formation, hyphal branching and conidiophore development. TeaA is delivered to the cortex by growing MTs. In conidia TeaA appeared at the germination site prior to germtube formation, and deletion of teaA resulted in germination at multiple sites, increased branching and abnormal conidiophores. The formation of a second germtube opposite the first conidial germtube depended on the presence of a septum at the base of the first germtube. An MT-organizing centre, associated to the septum, produced microtubules, which delivered TeaA towards the opposite side of the conidium. These results suggest a new function for TeaA in polarity establishment. It can be a positive function, but TeaA could also suppress polarity sites in the vicinity of the first germtube.     

cAMP and ras signalling independently control spore germination in the filamentous fungus Aspergillus nidulans

The role of cAMP signalling during germination of asexual spores (conidia) of the filamentous fungus Aspergillus nidulans was investigated. A. nidulans strains defective for adenylate cyclase (CyaA) or for the functionally overlapping cAMP-dependent protein kinase (PkaA) and newly characterized SchA protein kinase, homologous to Saccharomyces cerevisiae Sch9, show altered trehalose mobilization and kinetics of germ tube outgrowth, in addition to other defects in colony formation. cAMP-dependent trehalose breakdown is triggered by the addition of a carbon source independently of further catabolism, suggesting that cAMP signalling controls early events of conidial germination in response to carbon source sensing. Additional results suggest that cAMP has targets other than PkaA and SchA and that PkaA retains activity in the absence of cAMP. Conversely, PkaA regulates cAMP levels in A. nidulans because these are elevated by approximately 250-fold in a strain that lacks PkaA. Furthermore, analysis of mutant strains impaired in both adenylate cyclase and RasA GTPase previously implicated in the control of A. nidulans spore germination suggested that RasA and cAMP signalling proceed independently during germination in A. nidulans.     

PHIALIDE AND CONIDIUM DEVELOPMENT IN ASPERGILLUS CLAVATUS

Phialide formation in Aspergillus clavatus begins with the formation of thin areas in the vesicle wall. These thin-walled regions and adjacent cytoplasm then push out synchronously to produce the phialides. Mature phialides are broadly oval with an attenuated base and tapered apex. A secondary wall forms inside the phialide apex. The entire phialide apex pushes out to form the first conidium which is delimited by formation of a septum inside the mouth of the phialide. No collarette is present as the first conidium forms, but as the second conidium begins to develop, the outer wall breaks at the mouth of the phialide, leaving a collarette. The walls of the second and subsequent conidia are continuous with the inner wall of the phialide apex, from which they form. Conidia are held in chains by a connective which is a greatly thickened septum.