Branching is coordinated with mitosis in growing hyphae of Aspergillus nidulans

Filamentous fungi like Aspergillus nidulans can effectively colonize their surroundings by the formation of new branches along the existing hyphae. While growth conditions, chemical perturbations, and mutations affecting branch formation have received great attention during the last decades, the mechanisms that regulates branching is still poorly understood. In this study, a possible relation between cell cycle progression and branching was studied by testing the effect of a nuclei distribution mutation, cell cycle inhibitors, and conditional cell cycle mutations in combination with tip-growth inhibitors and varying substrate concentrations on branch initiation. Formation of branches was blocked after inhibition of nuclear division, which was not caused by a reduced growth rate. In hyphae of a nuclei distribution mutant branching was severely reduced in anucleated hyphae whereas the number of branches per hyphal length was linearly correlated to the concentration of nuclei, in the nucleated hyphae. In wild type cells, branching intensity was increased when the tip extension was reduced, and reduced when growing on poor substrates. In these situations, the hyphal concentration of nuclei was maintained and it is suggested that branching is correlated to cell cycle progression in order to maintain a minimum required cytoplasmic volume per nucleus and to avoid the formation of anucleated hyphae in the absence of nuclear divisions. The presented results further suggest the hyphal diameter as a key point through which the hyphal element regulates its branching intensity in response to the surrounding substrate concentrations.     

hyp loci control cell pattern formation in the vegetative mycelium of Aspergillus nidulans.

Aspergillus nidulans grows by apical extension of multinucleate cells called hyphae that are subdivided by the insertion of crosswalls called septa. Apical cells vary in length and number of nuclei, whereas subapical cells are typically 40 microm long with three to four nuclei. Apical cells have active mitotic cycles, whereas subapical cells are arrested for growth and mitosis until branch formation reinitiates tip growth and nuclear divisions. This multicellular growth pattern requires coordination between localized growth, nuclear division, and septation. We searched a temperature-sensitive mutant collection for strains with conditional defects in growth patterning and identified six mutants (designated hyp for hypercellular). The identified hyp mutations are nonlethal, recessive defects in five unlinked genes (hypA-hypE). Phenotypic analyses showed that these hyp mutants have aberrant patterns of septation and show defects in polarity establishment and tip growth, but they have normal nuclear division cycles and can complete the asexual growth cycle at restrictive temperature. Temperature shift analysis revealed that hypD and hypE play general roles in hyphal morphogenesis, since inactivation of these genes resulted in a general widening of apical and subapical cells. Interestingly, loss of hypA or hypB function lead to a cessation of apical cell growth but activated isotropic growth and mitosis in subapical cells. The inferred functions of hypA and hypB suggest a mechanism for coordinating apical growth, subapical cell arrest, and mitosis in A. nidulans.     

The Polo-like kinase PLKA in Aspergillus nidulans is not essential but plays important roles during vegetative growth and development

The Polo-like kinases (Plks) are conserved, multifunctional cell cycle regulators that are induced in many forms of cancer and play additional roles in metazoan development. We previously identified plkA in Aspergillus nidulans, the only Plk investigated in filamentous fungi to date, and partially characterized its function through overexpression. Here, we report the plkA null phenotype. Surprisingly, plkA was not essential, unlike Plks in other organisms that contain a single homologue. A subset of cells lacking PLKA contained defects in spindle formation and chromosome organization, supporting some conservation in cell cycle function. However, septa were present, suggesting that PLKA, unlike other Plks, is not a central regulator of septation. Colonies lacking PLKA were compact with multibranched hyphae, implying a role for this factor in aspects of hyphal morphogenesis. These defects were suppressed by high temperature or low concentrations of benomyl, suggesting that PLKA may function during vegetative growth by influencing microtubule dynamics. However, the colonies also showed reduced conidiation and precocious formation of sexual Hülle cells in a benomyl- and temperature-insensitive manner. This result suggests that PLKA may influence reproduction through distinct mechanisms and represents the first example of a link between Plk function and development in fungi. Finally, filamentous fungal Plks have distinct features, and phylogenetic analyses reveal that they may group more closely with metazoan PLK4. In contrast, yeast Plks are more similar to metazoan proteins PLK1 to PLK3. Thus, A. nidulans PLKA shows some conservation in cell cycle function but may also play novel roles during hyphal morphogenesis and development.     

Inhibitory effect of 2-Deoxyglucose on cell wall alpha-1,3-glucan synthesis and cleistothecium development in Aspergillus nidulans

2-Deoxy-d-glucose (2-DG) (0.1% w/v) added at inoculation time to cultures of Aspergillus prevents α-1,3-glucan, α-1,3-glucanase, and cleistothecium formation. 2-DG given after α-1,3-glucan synthesis, inhibits α-1,3-glucanase and glucan breakdown partially, and cleistothecium formation completely, perhaps because 2-DG, a toxic analog, is the only sugar available to the organism. When 2-DG is provided after about half the α-1,3-glucan is already made, about half the amount of α-1,3-glucanase is synthesized. Cleistothecia do develop, probably because at the time of addition of 2-DG glucose is still available in the medium. Experiments designed to test whether 2-DG acts primarily on α-1,3-glucan, α-1,3-glucanase, or both show that extra glucose appears mainly in an alkali-insoluble fraction and only little is synthesized into α-1,3-glucan. This is the reverse of what happens in the absence of 2-DG. These results indicate that both α-1,3-glucan and α-1,3-glucanase are indispensable for fructification in Aspergillus. Moreover, 2-DG primarily influences the synthesis of α-1,3-glucan, and the inhibition of α-1,3-glucanase is either a secondary effect or a consequence of disturbing the cell's metabolism.     

stcS, a putative P-450 monooxygenase, is required for the conversion of versicolorin A to sterigmatocystin in Aspergillus nidulans.

Sterigmatocystin (ST) and aflatoxin are carcinogenic end point metabolites derived from the same biochemical pathway, which is found in several Aspergillus spp. Recently, an ST gene cluster, containing approximately 25 distinct genes that are each proposed to function specifically in ST biosynthesis, has been identified in Aspergillus nidulans. Each of these structural genes is named stc (sterigmatocystin) followed by a consecutive letter of the alphabet. We have previously described stcU (formerly verA) as encoding a keto-reductase required for the conversion of versicolorin A to ST. We now describe a second A. nidulans gene, stcS (formerly verB), that is located within 2 kb of stcU in the ST gene cluster. An stcS-disrupted strain of A. nidulans, TSS17, was unable to produce ST and converted ST/aflatoxin precursors to versicolorin A rather than ST, indicating that stcS functions at the same point in the pathway as stcU. Genomic sequence analysis of stcS shows that it encodes a cytochrome P-450 monooxygenase and constitutes a novel P-450 family, CYP59. Assuming that StcU activity mimics that of similar P-450s, it is likely that StcU catalyzes one of the proposed oxidation steps necessary to convert versicolorin A to ST. These results constitute the first genetic proof that the conversion of versicolorin A to ST requires more than one enzymatic activity.     

Expression of the Aspergillus fumigatus rheb homologue,rhbA, is induced by nitrogen starvation

A gene encoding a ras protein with homology to the rheb family was cloned from Aspergillus fumigatus. Although conserved ras domains are present, the predicted RhbA protein sequence deviates from the ras consensus in a manner that is characteristic of rheb proteins. The invariant Gly-Gly in the first GTP-binding domain of ras proteins is replaced by Arg-Ser in RhbA, and a conserved Asp in the effector region of ras proteins is replaced by Asn in RhbA. The rhbA mRNA was detected throughout the A. fumigatus asexual developmental cycle, and accumulated over 5-fold in response to nitrogen starvation. The rhbA gene was able to complement the canavanine hypersensitivity of Saccharomyces cerevisiae Deltarhb1 mutants, suggesting that the two proteins share overlapping function.     

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.     

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.     

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.