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.     

Contribution to the morphology of the productive strains ofPenicillium chrysogenum thom from the Wisconsin family

The morphology of four productive strains ofPenicillium chrysogenum Thom from the Wisconsin family was studied. The strains Q-176, 47–1564, 49–133, 51–20Z, which were naturally or artificially obtained mutants of thePenicillium chrysogenum NRRL 1951 strain were very variable as to the colony structure and the character of conidiophores. The present study is concerned with the evaluation of their taxonomic position. The macrohabitus of the colonies was not remarkably changed. All different types of colonies (U, D, C, B, rarely A) described by Backus and Stauffer, were found on Czapek agar; they were not recognized on malt agar. Deviations from the normal asymmetric conidiophore were found with every type of colonies, most often with the more floccose or lanose ones showing a higher and a sparser overgrowth. These deviations or changes in the microstructure were divided into three degrees according to their quality and occurrence: (1) A strongly divaricate conidiophore where only metulae and phialides were developed; (2) monoverticilate conidiophore or single phialides on the conidiophore filament; (3) degeneration of phialides or metulae to sterile globose cells or an ultimate reduction of conidiophore to dichotomically branched stump-like hypha. The investigated strains can be involved in the taxonPenicillium chrysogenum Thom; it is necessary, however, to include some additional traits into the characteristics of the taxon: Colonies of the naturally or artificially obtained mutants often have lanose overgrowths sporulating sparsely. Formation of the yellow pigment and the exudate was not obligatory. conidiophores of these strains had a tendency to be more simple. They were scarcer, divaricately open, characterized sometimes by the formation of monoverticilate penicilli. A degeneration was frequently found of the ends of conidiophores (phialides and metulae) to globose enlarged sterile cells as well as the formation of giant cells in the mycelium or reduction of conidiophore to dichotomically branched hypha with stump-like ends.     

The Aspergillus nidulans putative kinase, KfsA (kinase for septation), plays a role in septation and is required for efficient asexual spore formation

In Aspergillus nidulans nuclear division and cytokinesis are coupled processes during asexual sporulation. Metulae, phialides and conidia contain a single nucleus. Here we describe the role of a putative Saccharomyces cerevisiae Kin4-related kinase, KfsA (kinase for septation) in the control of septum formation in A. nidulans. The kfsA deletion caused an increase in the number of conidiophores with septa in their stalks from 20% in wild type to 60% in the mutant strain. Interestingly, 7% of metulae contained two nuclei and the corresponding phialides remained anucleate, suggesting septum formation before proper segregation of nuclei. This points to a checkpoint control of KfsA, which prevents septum formation before nuclear separation. KfsA localized to the cortex and septa in hyphae and in conidiophores but not to the spindle-pole bodies, as it was shown for Kin4 in yeast. KfsA appeared at septa after actin disappeared, suggesting an additional role of KfsA late during septum formation.     

RacA-Mediated ROS Signaling Is Required for Polarized Cell Differentiation in Conidiogenesis of Aspergillus fumigatus

Conidiophore development of fungi belonging to the genus Aspergillus involves dynamic changes in cellular polarity and morphogenesis. Synchronized differentiation of phialides from the subtending conidiophore vesicle is a good example of the transition from isotropic to multi-directional polarized growth. Here we report a small GTPase, RacA, which is essential for reactive oxygen species (ROS) production in the vesicle as well as differentiation of phialides in Aspergillus fumigatus. We found that wild type A. fumigatus accumulates ROS in these conidiophore vesicles and that null mutants of racA did not, resulting in the termination of conidiophore development in this early vesicle stage. Further, we found that stress conditions resulting in atypical ROS accumulation coincide with partial recovery of phialide emergence but not subsequent apical dominance of the phialides in the racA null mutant, suggesting alternative means of ROS generation for the former process that are lacking in the latter. Elongation of phialides was also suppressed by inhibition of NADPH-oxidase activity. Our findings provide not only insights into role of ROS in fungal cell polarity and morphogenesis but also an improved model for the developmental regulatory pathway of conidiogenesis in A. fumigatus.     

Fine Structure of Phialide and Conidiospore Development in Aspergillus giganteus 'Wehmer'

The conidiophore vesicle is composed of a peripheral regionwhich contains many nuclei and mitochondria and a central regionwhich is densely packed with glycogen granules but containsvery few organelles. The phialides, which arise as sphericalprotuberances from the surface of the vesicle, are bounded bya much thinner wall than that of the vesicle. Nuclei migrateinto the phialides at a comparatively early stage in their development.Septa are present at the proximal ends of mature phialides andalthough pores are present in these septa it is suggested thatthey are too small to permit the ingress of such organellesas nuclei and mitochondria from the vesicle. The developmentof adjacent phialides is well synchronized but the developmentof the vesicle as a whole is not precisely co-ordinated. Eachmature phialide contains a single nucleus, as do the conidiosporeswhich they produce. The first conidiospore produced by a phialidearises as a spherical protrusion from the tapered, thickenedapex of the phialide. The conidiospores are delimited from thephialide by the formation of a cross wall in a manner reminiscentof the process of septation in hyphae. When first formed theconidiospores have a cylindrical shape and they only assumetheir characteristic spherical shape at a later stage in development.     

The Spitzenkörper: a signalling hub for the control of fungal development?

In Aspergillus nidulans , asexual development culminates in the formation of a multicellular conidiophore that bears spores known as conidia. Although several factors required for conidiophore formation have been characterized, the mechanisms underlying the transition from vegetative growth to development remain obscure. However, the recent characterization of two of these factors, FlbB and FlbE, provides important new insight. Notably, these studies suggest that the Spitzenkörper (i.e. apical body), which is known to regulate hyphal morphogenesis, might also serve as a signalling hub that co-ordinates development transitions.     

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.     

Molecular characterization of HymA, an evolutionarily highly conserved and highly expressed protein of Aspergillus nidulans

Aspergillus nidulans reproduces asexually via uninucleate, haploid spores, which are produced on morphologically differentiated aerial structures, called conidiophores. These consist of four distinct cell types, a foot with a terminally swollen stalk, metulae, phialides and conidiospores. The molecular mechanisms underlying the morphological changes that occur during conidiophore development have been studied by mutant analysis. We have isolated the hymA mutant, in which conidiophore development is affected at the metula stage. In the mutant metulae do not differentiate properly but come to resemble hyphae (hym?=?hypha-like metulae). In this paper we have analyzed the corresponding gene. It encodes a highly expressed 44?kDa protein which resides in the cytoplasm and has homologues in yeast, plants, fly, worm, fish, mice and man. We constructed hym deletion strains of Saccharomyces cerevisiae and of A. nidulans and found that the gene is essential in S. cerevisiae but is dispensable in the filamentous fungus. A cellular function for the Hym protein has not yet been defined in any organism. To demonstrate functional conservation we constructed a chimeric protein comprised of the N-terminal half of the A.?nidulans and the C-terminal half of the mouse homologue MO25. This hybrid protein could fully substitute for HymA function in A. nidulans. In addition, the mouse protein itself partially rescued the hymA mutation in the fungus. HymA is thus highly conserved in evolution and probably serves similar functions. The fact that hymA is required for conidiophore development in A. nidulans suggests that homologous genes in other organisms might also be involved in morphogenesis.     

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.     

Calcineurin localizes to the hyphal septum in Aspergillus fumigatus: implications for septum formation and conidiophore development

A functional calcineurin A fusion to enhanced green fluorescent protein (EGFP), CnaA-EGFP, was expressed in the Aspergillus fumigatus DeltacnaA mutant. CnaA-EGFP localized in actively growing hyphal tips, at the septa, and at junctions between the vesicle and phialides in an actin-dependent manner. This is the first study to implicate calcineurin in septum formation and conidiophore development of a filamentous fungus.     

Molecular characterization of HymA, an evolutionarily highly conserved and highly expressed protein of Aspergillus nidulans

Aspergillus nidulans reproduces asexually via uninucleate, haploid spores, which are produced on morphologically differentiated aerial structures, called conidiophores. These consist of four distinct cell types, a foot with a terminally swollen stalk, metulae, phialides and conidiospores. The molecular mechanisms underlying the morphological changes that occur during conidiophore development have been studied by mutant analysis. We have isolated the hymA mutant, in which conidiophore development is affected at the metula stage. In the mutant metulae do not differentiate properly but come to resemble hyphae (hym = hypha-like metulae). In this paper we have analyzed the corresponding gene. It encodes a highly expressed 44 kDa protein which resides in the cytoplasm and has homologues in yeast, plants, fly, worm, fish, mice and man. We constructed hym deletion strains of Saccharomyces cerevisiae and of A. nidulans and found that the gene is essential in S. cerevisiae but is dispensable in the filamentous fungus. A cellular function for the Hym protein has not yet been defined in any organism. To demonstrate functional conservation we constructed a chimeric protein comprised of the N-terminal half of the A.␣nidulans and the C-terminal half of the mouse homologue MO25. This hybrid protein could fully substitute for HymA function in A. nidulans. In addition, the mouse protein itself partially rescued the hymA mutation in the fungus. HymA is thus highly conserved in evolution and probably serves similar functions. The fact that hymA is required for conidiophore development in A. nidulans suggests that homologous genes in other organisms might also be involved in morphogenesis. Received: 11 February 1998 / Accepted: 14 September 1998     

Aspergillus nidulans asexual development: making the most of cellular modules

Asexual development in Aspergillus nidulans begins in superficial hyphae as the programmed emergence of successive pseudohyphal modules, collectively known as the conidiophore, and is completed by a layer of specialized cells (phialides) giving rise to chains of aerial spores. A discrete number of regulatory factors present in hyphae play different stage-specific roles in pseudohyphal modules, depending on their cellular localization and protein-protein interactions. Their multiple roles include the timely activation of a sporulation-specific pathway that governs phialide and spore formation. Such functional versatility provides for a new outlook on morphogenetic change and the ways we should study it.     

Ultrastructural analysis of conidiophore development in the fungusAspergillus nidulans using freeze-substitution

Summary Conidiation inAspergillus nidulans can be divided conveniently into five morphologically distinct stages. These are development of the conidiophore stalk, formation of the conidiophore vesicle, differentiation of metulae, differentiation of phialides, and production of conidia. The results presented here demonstrate that freeze-substitution fixation greatly facilitates the study of most of these stages. Ultrastructural features of vesicles, mitochondria, microtubules and nuclei were more easily resolved in freeze-substituted samples than in chemically fixed samples. In addition, certain structures and events simply not visible in chemically fixed samples were found routinely in freeze-substituted samples. Examples include Golgi bodies and multivesicular bodies and mitotic divisions associated with various stages of conidiation.Abbreviations C conidium - CI conidium initial - CV conidiophore vesicle - FC foot cell - GB Golgi body - M mitochondrion - ME metula - MT microtubule - MVB multivesicular body - N nucleus - PM plasma membrane - P phialide - RER rough endoplasmic reticulum - S spindle apparatus - SPB spindle pole body - V vacuole - W fungal wall - WB Woronin body