Role of bacterial virulence proteins in Agrobacterium-mediated transformation of Aspergillus awamori

The Agrobacterium-mediated transformation of Aspergillus awamori was optimized using defined co-cultivation conditions, which resulted in a reproducible and efficient transformation system. Optimal co-cultivation conditions were used to study the role of Agrobacterium tumefaciens virulence proteins in T-DNA transfer. This study revealed that inactivation of either of the regulatory proteins (VirA, VirG), any of the transport pore proteins (VirB), proteins involved in generation of the T-strand (VirD, VirC) or T-strand protection and targeting (VirE2) abolishes or severely reduces the formation of transformants. The results indicate that the Agrobacterium-mediated transformation of A. awamori requires an intact T-DNA machinery for efficient transformation; however, the plant host range factors, like VirE3, VirH, and VirF, are not important.     

Agrobacterium-mediated transformation leads to improved gene replacement efficiency in Aspergillus awamori

In this study, the efficiency of gene replacement in Aspergillus awamori between Agrobacterium-mediated transformation and CaCl(2)/PEG-mediated transformation was compared. For the genes, pyrG and gfaA, it was found that the homologous recombination frequencies obtained by Agrobacterium-mediated transformation were 3- to 6-fold higher than the frequencies obtained with CaCl(2)/PEG protoplast transformation. For the pyrG gene, it was found that Agrobacterium-mediated transformation allowed an efficient homologous recombination with shorter DNA flanks than CaCl(2)/PEG protoplast transformation. Finally, the addition of the dominant amdS marker as a second selection marker to the gene replacement cassette led to a further 2-fold enrichment in transformants with gene replacement events, resulting in a gene replacement frequency of 55%. Based on the data it can be concluded that Agrobacterium-mediated transformation is an efficient tool for gene replacement and that the amdS gene can be successfully used as a second selection marker to select transformants with putative gene replacement.     

Agrobacterium-mediated insertional mutagenesis of the ochratoxigenic fungus Aspergillus westerdijkiae

Aspergillus westerdijkiae is a potent ochratoxin A (OTA) producer that has been found in coffee beans. OTA is known to have nephrotoxic effects and carcinogenic potential in animal species. Here we report for the first time the Agrobacterium-mediated transformation for Aspergillus westerdijkiae and the generation of ochratoxin-defective mutants. Conidia were transformed to hygromycin B resistance using strain AGL-1 of Agrobacterium tumefaciens. The obtained transformation frequency was up to 47 transformants per 10(6) target conidia. Among 600 transformants, approximately 5% showed morphological variations. Eight transformants with consistently reduced OTA production were obtained. Two of these transformants did not produce OTA (detection limit: 0.1 microg/kg); the other six mutants produced lower amounts of OTA (1%-32%) compared with the wild-type strain. By using thermal asymmetric interlaced polymerase chain reaction, we successfully identified a putative flavin adenine dinucleotide monooxygenase gene.     

Septum-directed secretion in the filamentous fungus Aspergillus oryzae

Although exocytosis in fungal cells takes place at hyphal tips, there also seems a line of circumstantial evidence suggesting the occurrence of exocytosis at other sites of cells, such as septa. To investigate whether exocytosis takes place at fungal septa, we monitored dynamics of EGFP‐fused α‐amylase (AmyB–EGFP), the representative secretory enzyme of the filamentous fungus Aspergillus oryzae. We found that AmyB–EGFP accumulates in Spitzenkörper at hyphal tips as well as septal periplasm between the plasma membrane and cell walls. The septal accumulation of AmyB–EGFP was a rapid process, and required microtubules but not F‐actin. Thus, this process is independent of exocytosis at hyphal tips that requires both microtubules and F‐actin. In addition, fluorescence recovery after photobleaching (FRAP) analysis of EGFP‐fused AoSnc1 revealed that secretory vesicles constitutively fuse with the septal plasma membrane. These results demonstrated that exocytosis takes place at septa in addition to hyphal tips. Analysis of two plasma membrane transporters, AoUapC and AoGap1, revealed that they preferentially accumulate at septa and the lateral plasma membrane with no clear accumulation at apical Spitzenkörper, suggesting that non‐tip directed exocytosis is important for delivery of these proteins.     

Genomic sequence of the aflatoxigenic filamentous fungus Aspergillus nomius

Background

Aspergillus nomius is an opportunistic pathogen and one of the three most important producers of aflatoxins in section Flavi. This fungus has been reported to contaminate agricultural commodities, but it has also been sampled in non-agricultural areas so the host range is not well known. Having a similar mycotoxin profile as A. parasiticus, isolates of A. nomius are capable of secreting B- and G- aflatoxins.

Results

In this study we discovered that the A. nomius type strain (NRRL 13137) has a genome size of approximately 36 Mb which is comparable to other Aspergilli whose genomes have been sequenced. Its genome encompasses 11,918 predicted genes, 72 % of which were assigned GO terms using BLAST2GO. More than 1,200 of those predicted genes were identified as unique to A. nomius, and the most significantly enriched GO category among the unique genes was oxidoreducatase activity. Phylogenomic inference shows NRRL 13137 as ancestral to the other aflatoxigenic species examined from section Flavi. This strain contains a single mating-type idiomorph designated as MAT1-1.

Conclusions

This study provides a preliminary analysis of the A. nomius genome. Given the recently discovered potential for A. nomius to undergo sexual recombination, and based on our findings, this genome sequence provides an additional evolutionary reference point for studying the genetics and biology of aflatoxin production.     

Genomic islands in the pathogenic filamentous fungus Aspergillus fumigatus

We present the genome sequences of a new clinical isolate of the important human pathogen, Aspergillus fumigatus, A1163, and two closely related but rarely pathogenic species, Neosartorya fischeri NRRL181 and Aspergillus clavatus NRRL1. Comparative genomic analysis of A1163 with the recently sequenced A. fumigatus isolate Af293 has identified core, variable and up to 2% unique genes in each genome. While the core genes are 99.8% identical at the nucleotide level, identity for variable genes can be as low 40%. The most divergent loci appear to contain heterokaryon incompatibility (het) genes associated with fungal programmed cell death such as developmental regulator rosA. Cross-species comparison has revealed that 8.5%, 13.5% and 12.6%, respectively, of A. fumigatus, N. fischeri and A. clavatus genes are species-specific. These genes are significantly smaller in size than core genes, contain fewer exons and exhibit a subtelomeric bias. Most of them cluster together in 13 chromosomal islands, which are enriched for pseudogenes, transposons and other repetitive elements. At least 20% of A. fumigatus-specific genes appear to be functional and involved in carbohydrate and chitin catabolism, transport, detoxification, secondary metabolism and other functions that may facilitate the adaptation to heterogeneous environments such as soil or a mammalian host. Contrary to what was suggested previously, their origin cannot be attributed to horizontal gene transfer (HGT), but instead is likely to involve duplication, diversification and differential gene loss (DDL). The role of duplication in the origin of lineage-specific genes is further underlined by the discovery of genomic islands that seem to function as designated "gene dumps" and, perhaps, simultaneously, as "gene factories".     

Vacuolar membrane dynamics in the filamentous fungus Aspergillus oryzae

Vacuoles in filamentous fungi are highly pleomorphic and some of them, e.g., tubular vacuoles, are implicated in intra- and intercellular transport. In this report, we isolated Aovam3, the homologue of the Saccharomyces cerevisiae VAM3 gene that encodes the vacuolar syntaxin, from Aspergillus oryzae. In yeast complementation analyses, the expression of Aovam3 restored the phenotypes of both Deltavam3 and Deltapep12 mutants, suggesting that AoVam3p is likely the vacuolar and/or endosomal syntaxin in A. oryzae. FM4-64 [N-(3-triethylammoniumpropyl)-4-(p-diethylaminophenyl-hexatrienyl)pyridinium dibromide] and CMAC (7-amino-4-chloromethylcoumarin) staining confirmed that the fusion protein of enhanced green fluorescent protein (EGFP) with AoVam3p (EGFP-AoVam3p) localized on the membrane of the pleomorphic vacuolar networks, including large spherical vacuoles, tubular vacuoles, and putative late endosomes/prevacuolar compartments. EGFP-AoVam3p-expressing strains allowed us to observe the dynamics of vacuoles with high resolutions, and moreover, led to the discovery of several new aspects of fungal vacuoles, which have not been discovered so far with conventional staining methods, during different developmental stages. In old hyphae, EGFP fluorescence was present in the entire lumen of large vacuoles, which occupied most of the cell, indicating that degradation of cytosolic materials had occurred in such hyphae via an autophagic process. In hyphae that were not in contact with nutrients, such as aerial hyphae and hyphae that grew on a glass surface, vacuoles were composed of small punctate structures and tubular elements that often formed reticulum-like networks. These observations imply the presence of so-far-unrecognized roles of vacuoles in the development of filamentous fungi.     

Osmotic adjustment in the filamentous fungus Aspergillus nidulans.

Aspergillus nidulans was shown to be xerotolerant, with optimal radial growth on basal medium amended with 0.5 M NaCl (osmotic potential [psi s] of medium, -3 MPa), 50% optimal growth on medium amended with 1.6 M NaCl (psi s of medium, -8.7 MPa), and little growth on medium amended with 3.4 M NaCl (psi s of medium, -21 MPa). The intracellular content of soluble carbohydrates and of selected cations was measured after growth on basal medium, on this medium osmotically amended with NaCl, KCl, glucose, or glycerol, and also after hyperosmotic and hypoosmotic transfer. The results implicate glycerol and erythritol as the major osmoregulatory solutes. They both accumulated during growth on osmotically amended media, as well as after hyperosmotic transfer, except on glycerol-amended media, in which erythritol did not accumulate. Furthermore, they both decreased in amount after hypoosmotic transfer. With the exception of glycerol, the extracellular osmotic solute did not accumulate intracellularly when mycelium was grown in osmotically amended media, but it accumulated after hyperosmotic transfer. It was concluded that the extracellular solute usually plays only a transient role in osmotic adaptation. The intracellular content of soluble carbohydrates and cations measured could reasonably account for the intracellular osmotic potential of mycelium growing on osmotically amended media.     

Farnesol-induced cell death in the filamentous fungus Aspergillus nidulans

FOH (farnesol), a non-sterol isoprenoid produced by dephosphorylation of farnesyl pyrophosphate, has been shown to inhibit proliferation and induce apoptosis. We have been using Aspergillus nidulans and FOH as a model system and cell death stimulus, respectively, aiming to understand by which means filamentous fungi are driven towards cell death. Here, we review some of our findings about FOH-induced cell death in A. nidulans.     

Metabolism of alkyl amines by the filamentous fungus Aspergillus versicolor

A variety of monoalkyl-substituted amines were able to act as nitrogen sources for heterotrophically growing cultures of Aspergillus versicolor. Only amines whose alkyl chains were at least five carbon atoms long were capable of supporting significant growth in the absence of a separate carbon substrate. However, biomass yields were significantly higher during growth on glucose-amine than on glucose-ammonia, indicating that some energy-generating dissimilation of the amine to CO2 took place.     

Intracellular pH homeostasis in the filamentous fungus Aspergillus niger.

Intracellular pH homeostasis in the filamentous fungus Aspergillus niger was measured in real time by 31P NMR during perfusion in the NMR tube of fungal biomass immobilized in Ca2+-alginate beads. The fungus maintained constant cytoplasmic pH (pH(cyt)) and vacuolar pH (pH(vac)) values of 7.6 and 6.2, respectively, when the extracellular pH (pH(ex)) was varied between 1.5 and 7.0 in the presence of citrate. Intracellular metabolism did not collapse until a Delta pH over the cytoplasmic membrane of 6.6-6.7 was reached (pH(ex) 0.7-0.8). Maintenance of these large pH differences was possible without increased respiration compared to pH(ex) 5.8. Perfusion in the presence of various hexoses and pentoses (pH(ex) 5.8) revealed that the magnitude of Delta pH values over the cytoplasmic and vacuolar membrane could be linked to the carbon catabolite repressing properties of the carbon source. Also, larger Delta pH values coincided with a higher degree of respiration and increased accumulation of polyphosphate. Addition of protonophore (carbonyl cyanide m-chlorophenylhydrazone, CCCP) to the perfusion buffer led to decreased ATP levels, increased respiration and a partial (1 microm CCCP), transient (2 microm CCCP) or permanent (10 microm CCCP) collapse of the vacuolar membrane Delta pH. Nonlethal levels of the metabolic inhibitor azide (N3-, 0.1 mm) caused a transient decrease in pH(cyt) that was closely paralleled by a transient vacuolar acidification. Vacuolar H+ influx in response to cytoplasmic acidification, also observed during extreme medium acidification, indicates a role in pH homeostasis for this organelle. Finally, 31P NMR spectra of citric acid producing A. niger mycelium showed that despite a combination of low pH(ex) (1.8) and a high acid-secreting capacity, pH(cyt) and pH(vac) values were still well maintained (pH 7.5 and 6.4, respectively).     

Lactose and D-galactose catabolism in the filamentous fungus Aspergillus nidulans

The disaccharide lactose is a byproduct of cheese production accumulating to amounts of 800,000 tons per year worldwide, of which 15% is used as a carbon source for various microbial fermentations. Nevertheless, little is known about the regulation of its metabolism in filamentous fungi. Lactose is metabolized slowly, and some important fungi such as A. niger cannot use it at all. A more detailed knowledge on the rate-limiting steps would be helpful to improve its industrial application. We have chosen A. nidulans as an object for investigating how lactose and galactose metabolism are regulated because it has long become a model system for biochemical and genetic research on fungi, and mutants in the lactose-metabolizing pathway of A. nidulans are available. In this paper, we will review the contributions of our research group achieved on this field.     

Isolation and characterisation of Aspergillus awamori BS05, a root-knot-nematode-trapping fungus

Nematode-trapping fungi are important biocontrol agents against parasitic nematodes through adhesive or mechanical hyphal traps. Aspergillus awamori, a root-knot-nematode-trapping fungus from tomato rhizosphere soil, was identified based on morphology and molecular characteristics of internal transcribed spacer DNA sequence. Conidial heads were white to black brown, loosely globose, and 72–127 μm in diameter. Conidiophores usually arose from the foot cell of basal mycelium, straight, and 960–1730 × 10.2–13.4 μm, hyaline to pale brown, not constricted below the vesicles; vesicles hemispherical to elongate, 43–56 μm in diameter, black brown, fertile over the upper half to two-thirds. Aspergilla were biseriate, and metulae were variable, 12–26 × 3.8–4.7 μm; phialides were 8.2–9.4 × 2.5–3 μm. Conidia were globose or subglobose, 3.6–4.8 μm in diameter, rough, grey brown and parallel in chains. A. awamori BS05 showed 44.9% control efficacy against Meloidogyne incogtina in pot experiments which suggests it as a potential biocontrol agent against Meloidogyne. This is the first report on A. awamori as nematode-trapping fungus.     

Metabolism of alkyl amines by the filamentous fungus Aspergillus versicolor.

A variety of monoalkyl-substituted amines were able to act as nitrogen sources for heterotrophically growing cultures of Aspergillus versicolor. Only amines whose alkyl chains were at least five carbon atoms long were capable of supporting significant growth in the absence of a separate carbon substrate. However, biomass yields were significantly higher during growth on glucose-amine than on glucose-ammonia, indicating that some energy-generating dissimilation of the amine to CO2 took place.