Regulation of conidiation by light in Aspergillus nidulans

Light regulates several aspects of the biology of many organisms, including the balance between asexual and sexual development in some fungi. To understand how light regulates fungal development at the molecular level we have used Aspergillus nidulans as a model. We have performed a genome-wide expression analysis that has allowed us to identify >400 genes upregulated and >100 genes downregulated by light in developmentally competent mycelium. Among the upregulated genes were genes required for the regulation of asexual development, one of the major biological responses to light in A. nidulans, which is a pathway controlled by the master regulatory gene brlA. The expression of brlA, like conidiation, is induced by light. A detailed analysis of brlA light regulation revealed increased expression after short exposures with a maximum after 60 min of light followed by photoadaptation with longer light exposures. In addition to brlA, genes flbA-C and fluG are also light regulated, and flbA-C are required for the correct light-dependent regulation of the upstream regulator fluG. We have found that light induction of brlA required the photoreceptor complex composed of a phytochrome FphA, and the white-collar homologs LreA and LreB, and the fluffy genes flbA-C. We propose that the activation of regulatory genes by light is the key event in the activation of asexual development by light in A. nidulans.     

Effect of citrate on radial growth and conidiation of the mould Aspergillus nidulans

A mutation of the ctsA locus of Aspergillus nidulans affects both the radial growth and conidiation of the mould when grown in the presence of citrate. The ctsA locus was allocated to linkage group IV but it recombines freely with inoB2 and pyroA4 (which are also in linkage group IV). It is recessive in heterozygous diploids. A possible role for this gene in maintaining membrane integrity is discussed.The authors are with the Departamento de Genética, FMRP-USP, Av. Bandeirantes, 3900, 14049 Ribeirão Preto, SP., Brazil.     

Aspergillus nidulans UDP-galactopyranose mutase, encoded by ugmA plays key roles in colony growth, hyphal morphogenesis, and conidiation

Growing resistance to current anti-fungal drugs is spurring investigation of new targets, including those in fungal wall metabolism. Galactofuranose (Galf) is found in the cell walls of many fungi including Aspergillus fumigatus, which is currently the most prevalent opportunistic fungal pathogen in developed countries, and A. nidulans, a closely-related, tractable model system. UDP-galactopyranose mutase (UGM) converts UDP-galactopyranose into UDP-Galf prior to incorporation into the fungal wall. We deleted the single-copy UGM sequence (AN3112.4, which we call ugmA) from an A. nidulans nkuADelta strain, creating ugmADelta. Haploid ugmADelta strains were able to complete their asexual life cycle, showing that ugmA is not essential. However, ugmADelta strains had compact colonial growth, which was associated with substantially delayed and abnormal conidiation. Compared to a wildtype morphology strain, ugmADelta strains had aberrant hyphal morphology, producing wide, uneven, highly-branched hyphae, with thick, relatively electron-dense walls as visualized by transmission electron microscopy. These effects were partially remediated by growth on high osmolarity medium, or on medium containing 10 microg/mL Calcofluor, consistent with Galf being important in cell wall structure and/or function.     

A spindle pole body-associated protein, SNAD, affects septation and conidiation in Aspergillus nidulans

The nudA1 mutation in the cytoplasmic dynein heavy chain gene inhibits nuclear migration, colony growth and asexual sporulation (conidiation) in the filamentous fungus Aspergillus nidulans. It also alters the location of the first cell division event (septation) and prevents nucleation of tip cells. We showed previously that a suppressor of nudA1, snaD290, partially reversed the nuclear migration defect and partially restored colony growth. We have now demonstrated that the snaD290 mutation also delays septation and restores the septum to its normal position, allowing tip cells to be nucleated. Although snaD290 does not affect nuclear migration or vegetative hyphal growth, it almost completely inhibits conidiation. We propose that the SNAD protein participates in septation, and is essential for asexual spore formation. SnaD encodes a novel 76-kDa coiled-coil protein (SNAD) that is located at the spindle pole body throughout the cell cycle. Therefore, our results suggest that proteins at the spindle pole body are likely to be involved in temporal regulation of septation in A. nidulans. Received: 5 October 1999 / Accepted: 28 December 1999     

The septin AspB in Aspergillus nidulans forms bars and filaments and plays roles in growth emergence and conidiation

In yeast, septins form rings at the mother-bud neck and function as diffusion barriers. In animals, septins form filaments that can colocalize with other cytoskeletal elements. In the filamentous fungus Aspergillus nidulans there are five septin genes, aspA (an ortholog of Saccharomyces cerevisiae CDC11), aspB (an ortholog of S. cerevisiae CDC3), aspC (an ortholog of S. cerevisiae CDC12), aspD (an ortholog of S. cerevisiae CDC10), and aspE (found only in filamentous fungi). The aspB gene was previously reported to be the most highly expressed Aspergillus nidulans septin and to be essential. Using improved gene targeting techniques, we found that deletion of aspB is not lethal but results in delayed septation, increased emergence of germ tubes and branches, and greatly reduced conidiation. We also found that AspB-green fluorescent protein (GFP) localizes as rings and collars at septa, branches, and emerging layers of the conidiophore and as bars and filaments in conidia and hyphae. Bars are found in dormant and isotropically expanding conidia and in subapical nongrowing regions of hyphae and display fast movements. Filaments form as the germ tube emerges, localize to hyphal and branch tips, and display slower movements. All visible AspB-GFP structures are retained in ΔaspD and lost in ΔaspA and ΔaspC strains. Interestingly, in the ΔaspE mutant, AspB-GFP rings, bars, and filaments are visible in early growth, but AspB-GFP rods and filaments disappear after septum formation. AspE orthologs are only found in filamentous fungi, suggesting that this class of septins might be required for stability of septin bars and filaments in highly polar cells.     

Isolation and physical characterization of three essential conidiation genes from Aspergillus nidulans

We cloned and characterized three genes from Aspergillus nidulans, designated brlA, abaA and wetA, whose activities are required to complete different stages of conidiophore development. Inactivation of these genes causes major abnormalities in conidiophore morphology and prevents expression of many unrelated, developmentally regulated genes, without affecting expression of nonregulated genes. The three genes code for poly(A)⁺RNAs that begin to accumulate at different times during conidiation. The brlA-and abaA-encoded RNAs accumulate specifically in cells of the conidiophore. The wetA-encoded RNA accumulates in mature conidia. Inactivation of the brlA gene prevents expression of the abaA and wetA genes, whereas inactivation of the abaA gene prevents expression of the wetA gene. Our results confirm genetic predictions as to the temporal and spatial patterns of expression of these genes and demonstrate that these patterns are specified at the level of RNA accumulation.     

Generation and phenotypic characterization of Aspergillus nidulans methylisocitrate lyase deletion mutants: methylisocitrate inhibits growth and conidiation

Propionate is a very abundant carbon source in soil, and many microorganisms are able to use this as the sole carbon source. Nevertheless, propionate not only serves as a carbon source for filamentous fungi but also acts as a preservative when added to glucose containing media. To solve this contradiction between carbon source and preservative effect, propionate metabolism of Aspergillus nidulans was studied and revealed the methylcitrate cycle as the responsible pathway. Methylisocitrate lyase is one of the key enzymes of that cycle. It catalyzes the cleavage of methylisocitrate into succinate and pyruvate and completes the alpha-oxidation of propionate. Previously, methylisocitrate lyase was shown to be highly specific for the substrate (2R,3S)-2-methylisocitrate. Here, the identification of the genomic sequence of the corresponding gene and the generation of deletion mutants is reported. Deletion mutants did not grow on propionate as sole carbon and energy source and were severely inhibited during growth on alternative carbon sources, when propionate was present. The strongest inhibitory effect was observed, when glycerol was the main carbon source, followed by glucose and acetate. In addition, asexual conidiation was strongly impaired in the presence of propionate. These effects might be caused by competitive inhibition of the NADP-dependent isocitrate dehydrogenase, because the K(i) of (2R,3S)-2-methylisocitrate, the product of the methylcitrate cycle, on NADP-dependent isocitrate dehydrogenase was determined as 1.55 microM. Other isomers had no effect on enzymatic activity. Therefore, methylisocitrate was identified as a potential toxic compound for cellular metabolism.     

A phosphoglucose isomerase mutant in Aspergillus nidulans is defective in hyphal polarity and conidiation

Upon germination Aspergillus nidulans swoM1 exhibits abnormal development by extending a primary germ tube that quickly reverts to isotropic growth and results in an enlarged, swollen apex with pronounced wall thickenings. Apical lysis occurs in 38% of the germlings. A point mutation in the AN6037.3 gene encoding the only phosphoglucose isomerase in A. nidulans is responsible for the defect. Loss of polarity is bypassed when glucose is replaced with alternate carbon sources but in all cases the mutant is unable to conidiate due to a block in conidiophore development at vesicle formation. In conidiophores SwoM::GFP localizes to multiple punctate, foci within each actively growing cell type, and to multiple foci in mature dormant conidia. In hyphae SwoM::GFP localized to two rings spanning the center of mature septa. In hyphae localization is concentrated at actively growing hyphal tips.     

Isolation and physical characterization of three essential conidiation genes from Aspergillus nidulans.

We cloned and characterized three genes from Aspergillus nidulans, designated brlA, abaA, and wetA, whose activities are required to complete different stages of conidiophore development. Inactivation of these genes causes major abnormalities in conidiophore morphology and prevents expression of many unrelated, developmentally regulated genes, without affecting the expression of nonregulated genes. The three genes code for poly(A)+ RNAs that begin to accumulate at different times during conidiation. The brlA- and abaA-encoded RNAs accumulate specifically in cells of the conidiophore. The wetA-encoded RNA accumulates in mature conidia. Inactivation of the brlA gene prevents expression of the abaA and wetA genes, whereas inactivation of the abaA gene prevents expression of the wetA gene. Our results confirm genetic predictions as to the temporal and spatial patterns of expression of these genes and demonstrate that these patterns are specified at the level of RNA accumulation.     

Roles of the Aspergillus nidulans UDP-galactofuranose transporter, UgtA in hyphal morphogenesis, cell wall architecture, conidiation, and drug sensitivity

Galactofuranose (Galf) is the 5-member-ring form of galactose found in the walls of fungi including Aspergillus, but not in mammals. UDP-galactofuranose mutase (UgmA, ANID_3112.1) generates UDP-Galf from UDP-galactopyranose (6-member ring form). UgmA-GFP is cytoplasmic, so the UDP-Galf residues it produces must be transported into an endomembrane compartment prior to incorporation into cell wall components. ANID_3113.1 (which we call UgtA) was identified as being likely to encode the A. nidulans UDP-Galf transporter, based on its high amino acid sequence identity with A. fumigatus GlfB. The ugtAΔ phenotype resembled that of ugmAΔ, which had compact colonies, wide, highly branched hyphae, and reduced sporulation. Like ugmAΔ, the ugtAΔ hyphal walls were threefold thicker than wild type strains (but different in appearance in TEM), and accumulated exogenous material in liquid culture. AfglfB restored wild type growth in the ugtAΔ strain, showing that these genes have homologous function. Immunostaining with EBA2 showed that ugtAΔ hyphae and conidiophores lacked Galf, which was restored in the AfglfB-complemented strain. Unlike wild type and ugmAΔ strains, some ugtAΔ metulae produced triplets of phialides, rather than pairs. Compared to wild type strains, spore production for ugtAΔ was reduced to 1%, and spore germination was reduced to half. UgtA-GFP had a punctate distribution in hyphae, phialides, and young spores. Notably, the ugtAΔ strain was significantly more sensitive than wild type to Caspofungin, which inhibits beta-glucan synthesis, suggesting that drugs that could be developed to target UgtA function would be useful in combination antifungal therapy.     

Flavonols stimulate development, germination, and tube growth of tobacco pollen

The effect of anther-derived substances on pollen function was studied using pollen produced by in vitro culture of immature pollen of tobacco (Nicotiana tabacum L.) and petunia (Petunia hybrida). Addition of conditioned medium consisting of diffusates from in situ matured pollen strongly increased pollen germination frequency and pollen tube growth, as well as seed set after in situ pollination. Thin-layer chromatography and depletion of phenolic substances by Dowex treatment indicated that flavonols are present in the diffusate and may be the active compounds. When added to the germination medium, flavonols (quercetin, kaempferol, myricetin) but not other flavonoids strongly promoted pollen germination frequency and pollen tube growth in vitro. The best results were obtained at very low concentrations of the flavonols (0.15-1.5 μm), indicating a signaling function. The same compounds were also effective when added during pollen development in vitro.     

脂肪酸及其氧合物对曲霉属真菌菌丝生长、产孢和黄曲霉毒素合成的影响

黄曲霉毒素是由黄曲霉菌合成的一类毒性极高、致癌性极强的次生代谢物。一般认为,高油脂含量的作物种子被曲霉属真菌感染后容易产生黄曲霉毒素,但是,脂肪酸的处理实验结果表明不同类型的脂肪酸对曲霉属真菌毒素合成的作用不同,有的促进合成,有的抑制合成。最近研究结果显示所有脂肪酸都促进黄曲霉毒素合成,但是多不饱和脂肪酸在暴露空气之后对毒素合成有抑制作用。这种抑制产毒的作用似乎是由多不饱和脂肪酸氧化所产生的脂氧合物所介导。本文结合我们的研究结果,综合评述了脂肪酸和脂氧合物调控曲霉属真菌菌丝生长、产孢和毒素合成研究的最新进展。