ECFG 9 Poster Session 2 Animal-Fungal Interactions, Secondary Metabolism, Regulation of Gene Expression


Expression of Trichophyton rubrum genes during ex vivo human skin infection

Nalu Teixeira de Aguiar Peres1, Diana Ester Gras1, Pablo Rodrigo Sanches1, Juliana Pfrimer Falcão2, Lenaldo Branco Rocha3, Marcos Antonio Rossi3, Mendelson Mazucato1, Antônio Rossi4, Rolf Alexander Prade5, Nilce Maria Martinez-Rossi1

1Departamento de Genética, FMRP, Universidade de São Paulo, Ribeirão Preto, São Paulo, Brazil, 2FCFRP, Universidade de São Paulo, Ribeirão Preto, São Paulo, Brazil, 3Departamento de Patologia, FMRP, Universidade de São Paulo, Ribeirão Preto, São Paulo, Brazil, 4Departamento de Bioquímica e Imunologia, FMRP, Universidade de São Paulo, Ribeirão Preto, São Paulo, Brazil, 5Department of Microbiology and Molecular Genetics, Oklahoma State University, Stillwater, Oklahoma, United States

Trichophyton rubrum is a pathogenic fungus commonly isolated from human nails and skin lesions worldwide, causing infection in both healthy and immunocompromised hosts. Usually, during the pathogenic process in dermatophytosis no living tissue is invaded, with the keratinized stratum corneum being simply colonized. However, the presence of the fungus and its metabolic products may induce allergic and inflammatory eczematous responses in the host, and the infection may be invasive in immunodeficient hosts. Although it is a wide spread infection, little is known about the pathogenic process in dermatophytosis. So, the analysis of T. rubrum responses during infection could reveal the molecular alterations required for the organism to survive in the host milieu. Here, we used the subtractive suppressive hybridization (SSH) methodology to identify genes potentially related to fungal–host interaction, analyzing the transcriptome of T. rubrum during an ex vivo human skin infection. The up-regulated genes during skin infection provide insights into metabolic adaptations performed by T. rubrum during host infection, and these findings encourage further studies about T. rubrum pathogenesis.

Financial Support: CNPq, FAPESP, CAPES and FAEPA.



Isolation and identification of three Candida spp isolated from bovine mastitis in Sharg Alneel district-Khartoum state

Mohammed Abdo Elgabbar, Elham Sulima, Wisal Abdallah

Central Veterinary Research Laboratories (CVRL), Khartoum, Sudan

Bovine mastitis is a multi-etiological ailment that accounts for an immense economic loss due to decrease in milk production and poor quality milk as well as therapeutic expenditures. High rates of bovine mastitis in Sharg Alneel district- Khartoum state were primarily due to high prevalence of bacterial infection, fungal invasion through contaminated teat siphons during the bacterial infection, and/or from the gastrointestinal tract through the blood. We investigated the causative organisms of non responsive mastitic cases for local (Neomycin and cefaloxin), and systemic (Penicillin) antibacterial agents in the study area. Milk samples from thirty four non responsive mastitic cows were collected and cultured for fungi. Three of the investigated cows (8.8%) showed the presence of three Candida species which were identified as Candida albicans 2, Candida parapsilosis and Candida guilliermondii.



CCCA mediates vacuolar iron storage in Aspergillus fumigatus

Martin Eisendle, Beate Abt, Hubertus Haas

Innsbruck Medical University, Innsbruck, Austria

The opportunistic fungal pathogen Aspergillus fumigatus synthesizes three structurally different siderophores (low molecular mass, ferric iron-specific chelators): extracellular triacetylfusarinine C for iron uptake, intracellular ferricrocin for hyphal iron storage and intracellular hydroxyferricrocin for conidial iron storage. Siderophore biosynthesis is repressed by iron via the GATA-transcription factor SreA and, consequently, inactivation of SreA results in excessive iron uptake during iron-replete conditions. Siderophore biosynthesis is essential for virulence of A. fumigatus. Here we show that A. fumigatus employs a second mechanism for hyphal iron storage. Transcription of cccA, a homolog of the Saccharomyces cerevisiae vacuolar iron transporter CCC1, is SreA-independent induced by iron. A ∆cccA mutant displays wild type-like growth during iron depleted and high-iron conditions but reduced growth and increased ferricrocin accumulation after a shift from iron-depleted to high-iron conditions. Deletion of cccA in a ∆sreA background impairs growth in direct correlation to the ambient iron availability. In contrast, inactivation of ferricrocin biosynthesis impairs growth during iron depleted but not iron replete conditions. These results suggest that vacuolar iron storage is more important for detoxification of iron excess than ferricrocin-mediated iron storage.



Evidence for a lectin-mediated defense of mushrooms against predators and parasites

Martin Wälti1, Mattia Garbani1, Silvia Bleuler1, Alex Butschi2, Michael Hengartner2, Tiemeyer Michael3, Markus Aebi1, Markus Künzler1

1Institute of Microbiology, ETH Zürich, Zürich, Switzerland, 2Institute of Molecular Biology, University of Zürich, Zürich, Switzerland, 3Complex Carbohydrate Research Center, University of Georgia, Athens, Georgia, United States

The galectins CGL1 and CGL2 as well as the homologous lectin CGL3 of the homobasidiomycete Coprinopsis cinerea are strongly induced during sexual development and highly enriched in the fruiting body. Neither ectopic expression during vegetative growth nor silencing of the respective genes had any obvious effect on fruiting. These results make a role of these lectins in mushroom development unlikely. In search of an alternative function, we found a pronounced toxicity of the galectin CGL2 towards the nematode Caenorhabditis elegans and two different insect larvae (Aedes aegypti, Drosophila melanogaster). The phenotype of the toxicity resembles the one of nematotoxic and insecticidal crystal toxins from Bacillus thuringiensis. These results, which were obtained by feeding E. coli cells that expressed CGL2 in the cytoplasm, suggest, that mushrooms and probably also other multicellular fungi contain, analogous to plants, a lectin-mediated defense system against predators and parasites.

In order to test this hypothesis, we are currently testing a panel of characterized fungal lectins for nematotoxic and insecticidal activity. In parallel, we are screening C. cinerea for novel fruiting body-specific lectins in order to get an idea of the complexity of the "lectinome" of this organism. In order to test the physiological significance of the putative defense system, we are currently setting up laboratory cultures of the fungivore nematode Aphelenchus avenae. This experimental setup will also allow us to check whether the C. cinerea lectinome, besides being developmentally regulated, can be induced by challenge of the fungus with the predator and, if yes, to identify additional components of this defense system. Finally, we are exploiting C. elegans and D. melanogaster genetics to dissect the the mechanism of CGL2-mediated nematotoxic and insecticidal activity. We will present a progress report of these studies.


Abstract does not use the template

Christian Seibel, Laszlo Kredics, Christina Kratzer, Zsusanna Antal, Christian Peter Kubicek, Monika Schmoll

1Research Area Gene Technology and Applied Biochemistry, Institute of Chemical Engineering, Vienna University of Technology, Getreidemarkt 9-1665, A-1060 Vienna, Austria, 2Microbiological Research Group, Hungarian Academy of Sciences and University of Szeged, P. O. Box 533, H-6701 Szeged, Hungary, 3Department of Internal Medicine I, Division of Infectious Diseases and Chemotherapy, Medical University of Vienna, Währinger Gürtel 18-20, 1090 Vienna, Austria

Filamentous fungi represent an emerging class of human pathogens. Due to an increasing number of immunosuppressed patients new opportunistic pathogens are emerging from a backround formerly considered as harmless saprophytes. While established opportunistic pathogens like Aspergillus fumigatus or Cryptococcus neoformans have been subject to intense investigation other pathogens like Trichoderma longibrachiatus remained poorly understood. To elucidate pathogenicy mechanisms of T. longibrachiatum we developed an experimental system of simulated infection and applied Rapid Substraction Hybridisation to detect genes differentially expressed upon contact with host cells. For a simulated infection a clinical T. longibrachiatum isolate was grown either in the presence of human bronchial epithelial cells or in BEGM cell culture medium alone. Amongst 500 independent and putatively positive clones we identified an extracellular DNase and a mannosyl transferase both of which have homologues known to promote virulence in other pathogens. Furthermore a cyanovirdin-N-homologue and a putative polyketide synthase were detected as well as genes being involved in ATP or ferric ion uptake, respectively. The results were confirmed using a quantitative PCR approach and hint at a certain adaption or of the originally saprophytic fungus T. longibrachiatum towards pathogenicity.



The Aspergillus fumigatus ABC transporter AbcB is involved in excretion of siderophore breakdown product

Claudia Kragl, Markus Schrettl, Martin Eisendle, Hubertus Haas

Division of Molecular Biology/Biocenter, Innsbruck Medical University, Innsbruck, Austria

Aspergillus fumigatus is a ubiquitous fungal saprophyte and one of the most important opportunistic pathogens in immunocompromised patients. Recently, siderophore biosynthesis was shown to be essential for virulence of A. fumigatus in a mouse model of invasive aspergillosis. A. fumigatus produces two major siderophores: it excretes desferri-triacetylfusarinine C (DF-TafC) to mobilize extracellular iron and accumulates desferri-ferricrocin (DF-FC) for iron storage. Subsequent to uptake of DF-TafC-chelated iron, DF-TafC is hydrolyzed and the resulting fusarinines are excreted.

The A. fumigatus B-type ABC transporter-encoding abcB is located within an iron-regulated gene cluster, indicating a role in iron metabolism. Enhanced green fluorescent protein (EGFP)-tagging localized AbcB in the plasma membrane. Deletion of abcB caused increased intracellular and decreased extracellular accumulation of fusarinines. Consistently, AbcB-deficiency impaired the growth rate during iron depleted but not iron-replete conditions. Phylogenetic analysis suggested that AbcB is a prototype of a clade of the ABC transporter superfamily that is involved in siderophore metabolism with members in siderophore-producing but not siderophore-lacking fungal species. Most ABC transporters are implicated in multidrug resistance. AbcB is one of few members of this protein family with a known physiologically relevant function in the absence of xenobiotics and the first eukaryotic ABC-transporter that is involved in siderophore metabolism.



Co-evolution of enzyme function in the attine ant-fungus symbiosis

Henrik H. De Fine Licht, Morten Schiøtt, Jacobus J. Boomsma

Centre for Social Evolution, Department of Biology, University of Copenhagen, Copenhagen, Denmark

Introduction: Fungus-growing ants cultivate specialized fungi in the tribe Leucocoprineae (Lepiotaceae: Basidiomycota) inside their nests. The conspicuous leaf-cutting ants in the genus Atta build huge nests displacing several cubic meters of soil, whereas lower attine genera such as Cyphomyrmex have small nests with a fungus garden the size of a table-tennis ball. Only the leaf-cutting ants are specialized on using fresh leaves as substrate for their fungus gardens, whereas the more basal attine genera use substrates such as dry plant material (leaf litter and small twigs) and also insect feces and insect carcasses. This diverse array of fungal substrates across the attine lineage implies that the symbiotic fungus needs different enzymes to break down the plant material that the ants provide or different efficiencies of enzyme function.

Methods: (1.) We made a literature survey of substrate use in all extant fungus-growing ant genera to know the range of substrates used for any particular ant genus. (2.) Field assays of enzyme activity in fungus gardens of five candidate enzymes (Amylase, proteinase, pectinase, cellulose and xylanase) to indicate differences in enzyme activity between ant groups. (3.) Phylogenetic comparison and divergence estimates of nuclear ribosomal sequences and sequences coding for candidate enzyme genes (work in progress).

Results: Enzyme activity assays showed significant differences in enzyme activity across major fungus-growing ant groups. Notably the fresh leaf feeding species had a higher activity of amylase. The group of higher attines had a higher activity of proteinase and pectinase. In contrast the lower genera had a non-significant trend towards a higher activity of xylanase compared to the higher attine genera. Cellulase activity was uniform across all tested genera.

Discussion: In this study we document that there are differences in fungus garden enzyme activity between the different ant genera. These different enzyme activity profiles can be partially explained by the difference in substrates brought back by the ants to manure the fungus garden. This system can be viewed as ant induced crop optimization similar to human agricultural practices.



Functional analysis of evolutionary and ecological interactions between saprophage insects and moulds

Ulrike Fohgrub, Monika Trienens, Marko Rohlfs, Frank Kempken

Botanisches Institut und Botanischer Garten, Christian Albrechts-Universität zu Kiel, Kiel, Germany

Filamentous fungi and saprophage insects’ are suspected to be competitors on decaying organic matter. Both organisms have equal requirements considering habitat and nutrition. Insect larvae negatively influence mould development (1), but filamentous fungi can be an important cause of mortality of insect larvae (2). These competitions in insect-mould interactions have largely been ignored. First investigations suggest a role of genes for fungal secondary metabolism (3). Using a combination of experimental ecology and functional genomic techniques the function of moulds secondary metabolites (e.g. Mycotoxine) as a chemical defence in insects-moulds-interactions as well as the influence of these competitors at trophic interaction between insects ought to be investigated. For our research the vinegar fly Drosophila melanogaster and its natural mould antagonist Aspergillus were used as a form of ecology model system.

Microarrays of secondary metabolism genes of Aspergillus species are being used to identify fungal target genes up- or downregulated when interacting on festered matter with the antagonistic Drosophila larvae. Specific down- or upregulation of these target genes will be performed to analyse their importance for competitions in insect-mould interactions. The consequence on evolutionary fitness of the fungi and insects will be analysed.

1Rohlfs M (2005) Mycologia 97:996-1001, 2 Rohlfs M (2005) Frontiers in Zoology 2:2, 3 Rohlfs M, Albert M, Keller NP, Kempken F (2007) Biol Lett, doi:10.1098/rsbl.2007.0338


Paralysis of nematodes: shifts in the transcriptome of the nematode-trapping fungus Monacrosporium haptotylum during infection of Caenorhabditis elegans

Anders Tunlid1, Csaba Fekete2, Margareta Tholander1, Balaji Rajashekar1, Dag Ahrén1, Eva Friman1, Tomas Johansson1

1Lund University, Lund, Sweden, 2University of Pécs, Pécs, Hungary

Soils contain a diverse range of fungi that are parasites on nematodes. These fungi include the nematode-trapping and the endoparasitic fungi that attack free-living nematodes by using specialized structures, and the egg- and cyst- parasitic fungi that infect these stages with their hyphal tips. The transcriptional response in the nematode-trapping fungus Monacrosporium haptotylum and its nematode host Caenorhabditis elegans were analyzed during infection using cDNA microarrays. M. haptotylum traps nematodes using a spherical structure called knob, which develops on the apex of a hyphal branch. The advantage of using this species is that the unicellular knobs retain the function as infection structures even after separation from the mycelium. The cDNA array contained 2,684 fungal and 372 worm gene reporters. Dramatic shifts occurred in the transcriptome of M. haptotylum during the different stages of the infection. An initial transcriptional response was recorded after 1h of infection when the traps adhered to the cuticle, but before immobilization of the captured nematodes. Among the differentially expressed genes were two serine proteases (spr1 and spr2), and several homologues to genes known to be regulated in other pathogenic fungi. After 4 hours, when approximately 40 % of the nematodes were paralyzed, we identified an up-regulated cluster of 372 fungal genes which were not regulated during the other phases of the infection. This cohort contained a large proportion (79%) of genes that appear to be specific for M. haptotylum and closely related species. These genes were of two different classes; those translating into presumably functional peptides and those with no apparent protein coding potential (noncoding RNAs). Among the infection-induced C. elegans genes were those encoding antimicrobial peptides, protease inhibitors and lectins. This is the first study examining the global patterns of gene regulation occurring during the interaction between nematophagous fungi and nematodes.



Fungus-growing termites maximize harvest and minimize host-symbiont conflict by growing Termitomyces fungi in monoculture

Duur Aanen

Laboratory of Genetics, Wageningen, Netherlands

Although mutualistic symbiosis per definition provides a net benefit to the partners involved, these partners’ interests are usually not completely identical. Also in the mutualistic symbiosis between fungus-growing termites and Termitomyces fungi, such conflicts of interest exist, for example over reproduction and the degree of symbiont mixing. Recently, a proximate mechanism on how these conflicts of interest are regulated has been proposed. The key to the proposed mechanism is the within-nest propagation mode of fungal symbionts by termites. The termites suppress horizontal fungal transmission by consuming modified unripe mushrooms (nodules) for food. However, these nodules provide asexual gut-resistant spores that form the inoculum of new substrate. This within-nest propagation implies that harvesting and inoculation are coupled. Furthermore, the inoculation of spores in high density ensures the fast establishment of a fungal colony in the new substrate. In this talk, I describe in vitro experiments to test the hypothesis that fusion between clonally related subcolonies, but not between non-clonally related subcolonies, leads to synergism. We compared the ‘harvest’ (number of nodules) of five monocultures (heterokaryons belonging to a single biological species of Termitomyces associated with Macrotermes natalensis) with the harvest of varying degrees of mixed cultures of these five strains (with two, three or five heterokaryons in all combinations) in a high density. In line with the prediction, we found a highly significant correlation between harvest and ‘relatedness’ (the chance to meet a clonally related subcolony in a mixture). The results of these experiments imply that i) monoculture maximizes the harvest; ii) monoculture is self-maintaining as there is positive frequency-dependent selection; and iii) the termites and the resident fungus share the same short-term interest as the introduction of an unrelated fungus will initially lead to a lower harvest.


Fungivorous mites promote inbreeding in Coprinopsis cinerea by distribution of basidiospores in faecal pellets

Mónica Navarro-González, Wassana Chaisaena, Olivia Sánchez-Hernández, Pavel Plašil, Stefan Schütz, Ursula Kües

Molecular Wood Biotechnology and Technical Mycology, Büsgen-Institute, Georg-August-University, Göttingen, Germany

Mites such as Tyrophagus putrescentiae are attracted likely by volatile organic compounds to fungal cultures of Coprinopsis cinerea in order to graze on the mycelium. Both monokaryotic and dikaryotic mycelium are eaten by the mites including asexual spores (oidia). In vegetative cultures, only the melanized sclerotia are left, supporting a function in nature for duration: on fresh media, these sclerotia germinate. Upon fruiting body induction, primordia are also refused by the mites. When fruiting bodies mature, one can observe mites to climb up the stipe to feed on the autolysing cap tissues with the basidiospores. Stipes are refused as the sclerotia. Basidiospores are ingested but not digested. Compact faecal pellets are formed containing around 400 basidiospores. These spores germinate on suitable substrate. Germinated spores can easily form a dikaryon since the high number of spores ensures that all four possible mating types arising from meiosis in a mushroom are present. The fast moving mites thus contribute to the distribution of basidiospores in the environment with inbreeding effects on the otherwise outbreeding fungus. Mites lay eggs in close vicinity to spore pellets. Eggs hatch after about 5 days, whilst germinated spores and form already a considerably sized colony. This raises the idea that there might be a form of symbiotic interaction between mites and the fungus since germinated fungal colonies will serve as food for the hatching larvae. We present a scheme with the interlinked life cycles of the two organisms.


Molecular studies of the Saprolegnia-fish interaction

Andrew Phillips, Emma Robertson, Vicky Anderson, Chris Secombes, Pieter van West

University of Aberdeen, Aberdeen, United Kingdom

Oomycetes of the genus Saprolegnia are responsible for devastating infections of fish. The disease (Saprolegniosis) is characterized by visible white or grey patches of filamentous mycelium on the body or fins of freshwater fish and is of particular problem to aqua-cultural businesses. We are investigating the molecular mechanisms which enable Saprolegnia to successfully infect fish, the molecular processes that suppress host defenses during infection, and the nature of the pathogen/host interaction. To enable us to study the fish-pathogen interaction we have developed an in-vitro infection model. In this model system a cultured-monolayer of a primary fish cell-line (RTG-2) is infected with cysts of S. parasitica. This model has enabled us to harvested material from several stages of the interaction between fish and Saprolegnia, allowing us to investigate the kinetics of the infection using a range of molecular, microscopic and biochemical techniques. We are particularly interested in the early time-points of the interaction, and are studying the mechanisms, which allow Saprolegnia to establish an infection, and the defensive mechanisms employed by the host. We are currently addressing the latter by conducting microarray studies to detect changes in the host-transcriptome in response to infection by S. parasitica. Our latest findings will be presented.



Apoptosis inhibition of alveolar macrophages upon interaction with conidia of Aspergillus fumigatus

Katrin Volling, Axel Brakhage, Hans Saluz

Leibniz Institute for Natural Product Research, Jena, Germany

The opportunistic pathogen Aspergillus fumigatus (Af) causes the majority of cases of invasive aspergillosis. Because Af enters the body through inhalation of air-borne conidia, the interaction of conidia with the innate immune system (alveolar macrophages) plays a key role in the aetiology of invasive aspergillosis. The mechanisms underlying the anticonidial activity of macrophages and the relative resistance of conidia against the respective effector processes are a matter of debate. Modulation of host cell apoptosis has been reported to be one of the mechanisms pathogens employ to overcome host cell defences.

Hence, we focused our attention on the influence of Af conidia on staurosporine (STS)-induced apoptosis of murine alveolar macrophages (MH-S). MH-S cells exposed to Af conidia and treated with STS showed a decreased number of apoptotic cells compared to STS-induced control cells examined by flow cytometry analysis, DNA fragmentation and immunoblotting. The observed anti-apoptotic effect of Af conidia on MH-S cells was found to be associated with a significant reduction of active caspase-3, -6, -7, -8 and -9, which are critical mediators of receptor as well as mitochondria-mediated events of apoptosis. Furthermore, inhibition of apoptosis in MH-S cells exposed to Af conidia correlated with phosphorylation of the proapoptotic Bcl-2 family member Bad and a diminished cytochrome c release from mitochondria. An Af mutant strain lacking a specific enzyme in the dihydroxynapthol (DHN)-melanin biosynthesis pathway, which is required for the biosynthesis of the gray-green pigment, showed no inhibitory effect on STS-induced apoptosis in MH-S cells. Thus, it seems likely that an intermediate or derivative of DHN melanin functions as a virulence factor by enhancing the resistance of fungal cells against attack by host effector mechanisms and also has the ability to modulate host cell apoptosis.



Quantification of trichothecene biosynthetic genes during the growth cycle of Fusarium sporotrichioides in culture

Nancy Alexander, Susan McCormick

National Center for Agricultural Utilization Research, Peoria, IL, United States

Trichothecene mycotoxins are secondary metabolites produced by several species of phytopathogenic fungi and are potent inhibitors of protein biosynthesis. The genes involved in the biosynthetic pathway of T-2 toxin in Fusarium sporotrichioides have been characterized and are located in four identified loci. We wished to determine when these genes were turned on in the metabolic cycle and in what quantity. RNA was harvested at 8, 12, 16, and 24 h after inoculation with spores of F. sporotrichioides in a defined, liquid medium. Reverse transcriptase was used to form cDNA and quantitative PCR was performed using primers specific to selected trichothecene biosynthetic genes. Trichothecene genes were transcribed between 12 and 16 h post inoculation, typical of secondary metabolite genes, while basic metabolic genes were detected earlier at 8 h. As the expression of the trichothecene genes rise, the expression of the basic housekeeping genes declines. FsTri4 and FsTri5 are the most highly expressed trichothecene genes, with 7-fold greater expression than FsTri1, FsTri13, and FsTri15. FsTri15, a gene involved in the negative regulation of the pathway, has a delayed profile. Together, these and other data indicate that the level of expression of each of the trichothecene pathway genes is unique. This may lead to a novel method for controlling the expression of this pathway with the ultimate goal of reducing the presence of these mycotoxins in our food and feed supply.


Carbonic anhydrases in the filamentous fungus Sordaria macrospora

Skander Elleuche1, Stefanie Pöggeler2

1Ruhr-University, Bochum, Germany, 2Georg-August University, Göttingen, Germany

Fungi are able to sense and respond to gases such as ammonia or carbon dioxide. Metabolic cascades that mediate CO2/HCO3- homeostasis have been recently elucidated. The appropriate reaction is spontaneously balanced and can be accelerated up to 10.000 times by enzymes called carbonic anhydrases. This ubiquitous group of enzymes comprised five groups, which exhibit common structural features, referred to as α-, β-, γ-, δ- and ε-carbonic anhydrase.

It has been shown that carbonic anhydrases are conserved among fungal species. All fungal carbonic anhydrases belong to the β-group. The carbonic anhydrases of Saccharomyces cerevisiae, Cryptococcus neoformans and Candida albicans have been characterized. In contrast to humans, where 15 isoforms of carbonic anhydrases are encoded within the genome, hemiascomycetous yeasts encode only one carbonic anhydrase, whereas basidiomycetes contain two carbonic anhydrase genes. In this study, we present the analysis of three carbonic anhydrase genes (cynT1, cynT2 and cynT3) of the filamentous ascomycete Sordaria macrospora.

To investigate the physiological role of the three carbonic anhydrases in S. macrospora, we deleted each gene, by replacing parts of the coding region by the hph resistance cassette. Phenotypes of single and double knockout mutants will be presented.

Since, bicarbonate is an essential factor for several enzymes we investigated the role of the cyanase encoding gene cyn1. This enzyme catalyzes a bicarbonate dependent reaction: the degradation of toxic cyanate into two molecules of carbon dioxide and one molecule of ammonia. We showed that, that S. macrospora CYN1 is highly active in a heterologous system in E. coli and decomposates cyanate in vivo.



Discovery of a putative peptaibiotic from Clonostachys rogersoniana

Chanikul Chutrakul1, Sarocha Panchanawaporn2, Suttipun Kaewsompong2, Kanyawim Kirtikara1

1BIOTEC, Pathumthani, Thailand, 2Kasetsart University, Bangkok, Thailand

Peptaibiotics, a group of polypeptide antibiotics with α-aminoisobutyric acid (Aib) as a major amino acid component, have been discovered and identified chemically together with reports of their biological activities. The biosynthesis of peptaibiotic compounds involves a (non-ribosomal) protein complex. A signature sequence for each adenylation (A) domain determines specific amino acid incorporation into the peptaibiotic chain. This study aimed to identify peptaibiotic producing fungi based on a screening regime. This involved the use of PCR to determine signature sequences for Aib residues in the fungal genomes, the detection of Aib in isolated metabolites and the use of biological assays. A trichotoxin-peptaibiotic producing strain, Trichoderma asperellum BCC12530, used as a control presented Aib A-domain gene fragments, anti-bacterial and fungal activities, and free Aib residue as performed by PCR, microdilution bioassays and TLC analysis of amino acid hydrolysis, respectively. The presence of Aib in components of the T. asperellum extracts was confirmed by HPLC. These protocols were followed to screen 27 strains of filamentous fungi from the BIOTEC Culture Collection (BCC), for peptaibiotic producers. From this, a fungus of interest, Clonostachys rogersoniana BCC4862, indicated Aib related to peptaibiotic production and anti-microbial activities, however, the Aib A-domain gene could not be detected from the genome. Culture broth from 14 days fermentation gave the highest production yield. Putative peptaibiotic(s) from this fungus have been elucidated. This may be the first report of the genus Chlonostachys as a new peptaibiotic producer.



The velvet complex coordinates light, fungal development and secondary metabolism

Özgür Bayram1, Sven Krappmann1, Min Ni3, Kerstin Helmstaedt2, Oliver Valerius1, Susanna Braus-Stromeyer1, Nak-Jung Kwon3, Jin Woo Bok4, Nancy P. Keller4, Jae-Hyuk Yu3, Gerhard H. Braus1

1Institute of Microbiology and Genetics, Georg August University, Göttingen, Germany, 2DFG Research Center for Molecular Physiology of the Brain (CMPB), Göttingen, Germany, 3Department of Bacteriology, and Department of Genetics, University of Wisconsin, Madison, United States, 4Department of Plant Pathology and Department of Medical Microbiology and Immunology, University of Wisconsin, Madison, United States

Differentiation and secondary metabolism are correlated processes in fungi and respond to light. In Aspergillus nidulans, light inhibits sexual reproduction as well as secondary metabolism. We identified the heterotrimeric velvet complex as link between light-responding developmental regulation and control of secondary metabolism. VeA, which is primarily expressed in the dark, physically interacts with VelB that is expressed during sexual development. VeA bridges VelB to the nuclear master regulator of secondary metabolism LaeA. Deletion of either velB or veA results in defects in both sexual fruiting body formation and production of secondary metabolites.



The Ppt-encoded phosphopantetheinyl transferase of Penicillium chrysogenum is required for pigment, lysine and antibiotic biosynthesis, but not for roquefortine or fatty acids formation

Carlos Garcia-Estrada1, Ricardo V. Ullan1, Tania Velasco-Conde1, Ramiro P. Godio1, Fernando Teijeira2, Inmaculada Vaca1, Raul Feltrer1, Katarina Kosalkova1, Elba Mauriz1, Juan Francisco Martin1

1Instituto de Biotecnologia (INBIOTEC), Leon, Spain, 2Area de Microbiologia, Departamento de Biologia Molecular, Facultad de CC. Biologicas y Ambientales, Universidad de Leon, Leon, Spain

Introduction: Some secondary metabolites such as antibiotics, pigments or mycotoxins are produced by filamentous fungi and other organisms. Nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs) are multidomain enzymes in charge of the biosynthesis of these compounds. NRPSs and PKSs require a post-translational modification to become active through the covalent attachment of the 4'-phosphopantetheine moiety, which is derived from coenzyme A. This reaction is catalysed by the 4'-phosphopantetheinyl transferase (PPTase). It is important to identify the gene encoding the PPTase of P. chrysogenum (ppt gene) to elucidate the relevance of the gene and protein in primary and secondary metabolism.

Methods: We have carried out the functional characterization of the gene encoding the PPTase of P. chrysogenum using strong heterologous promoters for gene overexpression and gene attenuation. HPLC has been used to determine some of the secondary metabolites produced by this microorganism.

Results: We have isolated and characterizated the gene encoding the single-domain large-type PPTase (ppt gene) from the filamentous fungus P. chrysogenum. The predicted protein encoded by this gene shared 50% homology with the stand-alone large PPTases (class III) of A. nidulans and A. fumigatus. Amplification of the ppt gene in the Wis54-1255 strain produced increases in both the IPN and benzylpenicillin production. One mutant defective in PPTase activity (Wis54-PPT—) was obtained. This mutant lacked penicillin production, was a lysine auxotroph and showed growth defects regardless the addition of fatty acids. The biosynthesis of roquefortine was not prevented in the Wis54-PPT mutant and sporulation was not affected. However, conidia were white, lacking the green pigment.

Discussion: Complementation of the Wis54-PPT— strain with the ppt gene, restored the lysine prototrophy, pigmentation, full growth rate and penicillin production, which suggests that a single broad-range PPTase controls lysine biosynthesis, pigments and penicillin (but not roquefortine) formation through the activation of PKSs and NRPSs in P. chrysgonenum. It is likely that siderophores formation is also controlled by the ppt gene, but modification of the fatty acid synthetase occurs by a different FAS-integrated PPTase.



Compartmentalization of the cephalosporin C biosynthetic pathway in Acremonium chrysogenum

Fernando Teijeira1, Ricardo Vicente Ullán2, Carlos García-Estrada2, Inmaculada Vaca2, Ramiro Pedro Godio2, Juan Francisco Martín2

1Area of Microbiology, Faculty of Biological and Environmental Sciences, University of León, Leon, Spain, 2Institute of Biotechnology (INBIOTEC), Science Park of León, Leon, Spain

Acremonium chrysogenum is a filamentous fungi widely used for the industrial cephalosporin production. Although the biosynthetic pathway of the cephalosporin C has been characterized, with the exception of the cefT gene, nothing is known about the exportation of the end product (cephalosporin C) and intermediates of the pathway. Overexpression of the cefT gene encoding a transmembrane protein resulted in a two-fold increase in cephalosporin production but the target inactivation of the cefT gene did not change this secretion indicating the present of redundant systems of cephalosporin exportation. The cefT2 gene encodes a membrane transport protein that contains 12 transmembrane domains and includes the A, B, C, D2 and G characteristic motifs of Drug: H+ antiporter 12-TMS family of the MFS superfamily. The target inactivation of the cefT2 gene by means of the double marker technique showed a drastic reduction in the cephalosporin and penicillin N secretion. HPLC analysis revealed a reduction in the secretion and an important intracellular penicillin N accumulation. In trans complementation of the disrupted mutant restored completely the cephalosporin and penicillin production to wild-type levels. Fluorescence microscopy analysis the CefT2-GFP hybrid protein showed an organelle fluorescence indicating a probable microbody membrane location of the CefT2 protein. Taken together these results indicate that the last steps of the biosynthetic pathway of cephalosporin C could take place in an organelle locations.



Aspergillus oryzae laeA affects production of kojic acid

Ken Oda, Motoaki Sano, Akiko Kobayashi, Shinichi Ohashi

Kanazawa Institute of Technology, Hakusan, Ishikawa, Japan

Filamentous fungi produce a large variety of secondary metabolites, and are used as medicine provider. Recently secondary metabolite regulating gene laeA was found in Aspergillus nidulans, and the regulation of metabolites production has begun to be clarified in filamentous fungi1). It was reported that laeA affects the production of secondary metabolites differently depending on species. For the purpose of elucidating the regulation mechanism of laeA on secondary metabolites production in A. oryzae, we cloned A. oryzae laeA gene and produce a disruption mutant. We used deltaligD deltapyrG strain as a host, of which homologous recombination efficiency was increased by ligD gene deletion. We disrupted laeA by deleting putative S-adenosylmethionine binding site with pyrG marker. The laeA disruptant (delta LaeA) showed pigment decreasing phenotype similar to other species. Delta LaeA strain exhibited extreme reduction of conidia production, which did not change in A. nidulans.

We examined the effect of laeA deletion on production of kojic acid, one of the most famous secondary metabolites in A. oryzae. In delta LaeA strain production of kojic acid was greatly reduced in kojic acid production medium. While in laeA complement strain (delta LaeAcomp) kojic acid production was recovered as much as control strain. These suggested that LaeA affects kojic acid production in A. oryzae. In addition, analysis of LaeA-regulated gene by DNA microarray will be also discussed.

1 Jin Woo Bok and Nancy P. Keller, Eucaryotic Cell, 3, p.527-535 (2004)



Expression of CefT of Acremonium chrysogenum increases cephalosporin secretion in Penicillium chrysogenum

Jeroen Nijland, Marco van der Berg2, Roel Bovenberg2, Arnold Driessen1

1University of Groningen, Groningen, Netherlands, 2DSM anti-infectives, Delft, Netherlands

Antibiotic production in fungi in the last decade has shifted from penicillins to cephalosporins. To fulfill the increased demand cephalosporins e.g. 7-ADCA have been produced in the high penicillin producing fungus Penicillium chrysogenum via the heterologous expression of CefE (expandase) of Streptomyces clavuligerus. However cephalosporins with a carbamoyl side chain at position 3 cannot be readily produced using 7-ADCA therefore recently CefEF of Acremonium chrysogenum and CmcH of Streptomyces clavuligerus were expressed in P. chrysogenum yielding a strain producing adipoyl-7-amino-3-carbamoyloxymethyl-3-cephem-4-carbooxylic acid (adACCA). The adACCA producing strain was used in a second round of transformation with the CefT gene of A. chrysogenum (AcCefT) belonging to the multidrug efflux pump subfamily of MFS transporters. AdACCA production was increased on average 1.7 fold after 8 days of growth in adACCA producing medium containing adipic acid as side chain. Also adAHCA is increased in these transformants by 1.3 fold while the 5 membered penam precursors of adACCA e.g. IPN and ad6APA levels are decreased. This suggests that the substrate specificity of AcCefT is limited to cepalosporins, and demonstrates that specific export can be used to increase the productivity of an engineered cephalosporin-producing P. chrysogenum strain.



Identification of the biosynthetic pathway of cyclopiazonic acid in Aspergillus oryzae

Masafumi Tokuoka1, Tadashi Takahashi1, Yasuyo Seshime2, Isao Fujii2, Katsuhiko Kitamoto3, Yasuji Koyama1

1Noda Institute for Scientific Research, Noda, Chiba, Japan, 2Iwate Medical University, Shiba-gun, Iwate, Japan, 3University of Tokyo, Bunkyo-ku, Tokyo, Japan

Cyclopiazonic acid (CPA) is a specific inhibitor of Ca2+-ATPase which is produced by several strains of Penicillium and Aspergillus species. Whereas A. oryzae strains used for production of fermented foods are confirmed not to produce CPA, several wild type A. oryzae strains are known to produce CPA. Therefore, to strengthen the safety of A. oryzae, it is important to clarify the biosynthetic pathway of CPA. Putative biosynthetic pathway was proposed in Penicillium species. However, little is known about the genes involved in the CPA synthesis. In this study, we identified a gene cluster responsible for CPA synthesis in CPA-producing A. oryzae strains by systematic gene disruption based on a gene targeting technique.

We first compared the sequence around dimethylallyl-cyclo-acetoacetyl-L-tryptophan synthase (DCAT-S) gene of the CPA non-producing strain with that of the CPA producing strain because DCAT-S gene is the only known gene involved in CPA synthesis. As a result, it was found that polyketide synthase/non-ribosomal peptide synthetase (PKS/NRPS) gene adjacent to DCAT-S gene is truncated by telomere in the CPA non-producing strain while the CPA producing strain has the intact gene. Further sequence analysis revealed presence of a predicted gene cluster consisting of genes encoding transcription factor, PKS/NRPS, DCAT-S, amine oxidase, cytochrome P450, methyltransferase, and multidrug-resistant protein. To investigate the involvement of the predicted genes in the CPA synthesis, we next constructed a series of knockout strains of each gene using a host strain lacking Ku70 gene to facilitate gene targeting. Culture extracts of the knockout strains were subjected to LC/MS to determine the amount of intermediates of the proposed CPA synthetic pathway. The knockout mutants for PKS/NRPS, DCAT-S and amine oxidase lost the CPA productivity, but accumulated the expected intermediates in the proposed pathway, indicating that the CPA synthesis pathway of A. oryzae is identical to the proposed pathway in P. cyclopium. In addition, MS/MS analysis showed that chromatographic peaks missing from the cytochrome P450 gene knockout mutant were presumed to be mono- and di-hydroxy CPA. These metabolites are novel derivatives of CPA that have not been described.



Identification and transcriptional analysis of a pks gene putatively involved in ochratoxin A biosynthesis in P. verrucosum

Abdelhamid Abbas, Alan Dobson

Microbiology Department, National University of Ireland (UCC), Cork, Ireland

Penicillium verrucosum is an important producer of Ochratoxin A (OTA) and is one of the main contaminants of barley which is an important cereal crop in Ireland. Knowing the factors that influence the production of OTA by P. verrucosum in the field and in storage is of a major concern for food safety and economic. However, this would not be possible without the knowledge of the nucleotide sequence and the expression of the gene/s responsible for the production of this mycotoxin. Using genome walker technology we succeeded in identifying the sequence of a gene cluster likely to be responsible for the production of OTA. The gene cluster contains a full-length polyketide synthase (PKS) gene (otapks) and two flanking genes. The otapks encodes a type I PKS of 2553 amino acids with the known ancestral enzymatic domain structure, namely ketoacyl synthase (KS), acyl transferase (AT), and acyl carrier protein (ACP). The PKS also contains a methyltransferase domain but lacks the reducing domains such as dehydratase and enoyl reductase. A putative ketoreductase (KR) domain is suspected to be present close to the C-terminal region of the PKS. However, no thioesterase domain was found downstream of the ACP domain. We analysed the otapks gene expression under permissive and restrictive conditions for OTA production and found that transcription of the otapks gene correlated with OTA production. This data strongly suggest that the cloned pks gene is involved in OTA production in Penicillium verrucosum.


Correlation between ochratoxin A biosynthesis and oxidant/antioxidant modulation in Aspergillus ochraceus

Federico Punelli, Massimo Reverberi, Katia Gazzetti, Slaven Zjalic, Alessandra Ricelli, Corrado Fanelli, Annadele Fabbri

1Università La Sapienza, Roma, Italy, 2CNR, Roma, Italy

Reactive Oxygen Species (ROS) and lipoperoxides in Aspergillus parasiticus stimulated aflatoxin production and the expression of transcription factors related to oxidative stress. These factors promoted the transcription of genes encoding antioxidant-related enzymes. In A. parasiticus cells oxidative stress, induced by ROS and lipoperoxides, appeared to be perceived and transduced by Apyap1 (Yap1 orthologue) to primes antioxidant defense activities. Δapyap1 mutant had an altered perception of oxidative stress and presented a precocious and increased production of aflatoxin in comparison to wild type (WT). Also in A. ochraceus (MPV 2037 strain - OTA producer) hydroperoxides of linoleic acid are able to modulate ochratoxin A (OTA) production (Punelli et al., 2007). The correlation between lipoperoxides and OTA biosynthesis was analyzed in previous works (Punelli et al., 2007) and a Δaolox-like (lipoxygenase mutant) presented a delay in the sporulation and OTA production. In this study a correlation among lipoperoxidation, activity of antioxidant defence system and OTA biosynthesis was more deeply evidenced and new approaches were used to analyze the function of oxidative stress in fungal cells. Oxidant inducers like Menadione 0.1 and 0.5 mM, CCl4 0.5% v/v, H2O2 10 mM, tBOOH 100 mM were used to test their effect on OTA production by WT. Furthermore the construction of Δaoyap-like mutant was perfomed to investigate the correlation between oxidative stress and OTA biosynthesis. To analyze the A. ochraceus genome, several primers were used (Aoyap, Aocat and Aosod for e rev). The transcription level in WT and mutant was studied by reverse PCR analysis. In A. ochraceus, OTA producer, a 657 bp sequence of aoyap-like (accession number EU045307 - 82% of aa homology with A. fumigatus yap1), a 309 bp Aocat-like (95-100% of aa homology with A. clavatus CatA) and 233 bp Cu-Zn Aosod-like (77% of aa homology with A. fumigatus Cu,Zn SOD) were characterized and partially cloned and the activity and expression of these enzymes were analyzed in mycelia. The results obtained show that a delay in the sporulation was observed in the mutant and the best antioxidant enzymes stimulator were Menadione 0.5 mM and CCl4 0.5% v/v, also the OTA production was affected differently by the oxidant inducers.



Identification and characterisation of aatB, a novel penicillin biosynthesis gene of Aspergillus nidulans

Petra Sproete1, Michael Hynes2, Axel A. Brakhage1

1Leibniz Institute for Natural Product Research and Infection Biology (HKI) / Friedrich-Schiller-University Jena, Jena, Germany, 2Department of Genetics, University of Melbourne, Melbourne, Australia

The acyl coenzyme A:isopenicillin N acyltransferase (IAT) of A. nidulans, which is encoded by the aatA gene, is located within the microbodies and catalyses the final step of the penicillin biosynthesis, i.e., the exchange of the hydrophilic L-alpha-aminoadipic acid side chain of isopenicillin N for a hydrophobic acyl group. Surprisingly, an aatA disruption strain is still able to produce small amounts of penicillin. Therefore, the A. nidulans database was searched for a putative redundant protein. A gene displaying a very similar exon distribution and a 58% similarity with the aatA gene but lacking a sequence for peroxisomal targeting was named aatB. Analyses of strains carrying an aatB disruption and an aatA/aatB double disruption, respectively, indicated a participitation of AatB in penicillin biosynthesis of A. nidulans. Time and media dependent expression of the aatB gene as well as the influence of transcription factors that also regulate aatA expression were investigated. Furthermore, the capability of aatB and variants of the aatB gene to complement an aatA disruption strain was analysed. Since comparative database analyses revealed homologues of the aatB gene in both Aspergilli and other filamentous fungi including non-penicillin-producers, an ancestral role of this gene during evolution of the penicillin biosynthesis gene cluster is conceivable.


Cell redox balance, lipid metabolism and aflatoxin biosynthesis in Aspergillus sect. flavi

Massimo Reverberi1, Marta Punelli1, Carrie Smith2, Federico Punelli1, Slaven Zjalic1, Alessandra Ricelli3, Gary Payne2, Annadele Fabbri1, Corrado Fanelli1

1Università La Sapienza, Roma, Italy, 2North Carolina State University, Raleigh, United States, 3CNR, Roma, Italy

Oxidative stress represents a trigger for different metabolic events in all organisms and occurs during oxidative processes in the cell such as glucose oxidation and -oxidation of fatty acids. In aflatoxigenic fungi these processes are able to supply the aflatoxin building blocks i.e. the acetyl-CoA and an oxidative cell environment, which is necessary for the formation of precursors of aflatoxins. In fact among the several enzyme-catalysed reactions in aflatoxin biosynthesis, at least 6 are represented by oxidative transformations.

In order to demonstrate a correlation between cell redox balance, lipid metabolism and aflatoxin synthesis we used WT and mutant strains of Aspergillus parasiticus (NRRL 2999) and of A. flavus (NRRL 3357), aflatoxin producers. A null mutant approach was used for studying the involvement of the oxidative stress related transcription factor ApyapA (Yap1 ortholog) in toxin biosynthesis in A. parasiticus. To study how lipid metabolism is related to aflatoxin formation, a gene (controlled by the Cu,Zn sod promoter of A. flavus) encoding for a viral (Cymbidium Ringspot Virus) protein, P33, which is able to induce peroxisome proliferation, was inserted in A. flavus. Further, in order to find a more direct link among oxidative stress perception, lipid metabolism and aflatoxin biosynthesis, an in silico analysis of the promoter of the aflatoxin gene regulator AflR using the N_SITE tool of softberry software ( was carried out.

In A. parasiticus, oxidative stress, via the activation of the oxidative stress related transcription factor ApyapA, regulates conidiogenesis and aflatoxin synthesis whereas an up-regulation of the lipid metabolism (FFA -oxidation and TG accumulation) induces a hyperoxidant status and aflatoxin biosynthesis enhancement in A. flavus. In silico N_SITE analysis indicates the presence of regulatory elements (RE) responsive to CREB (cAMP), SREBP-ADD1 (lipid and glucose metabolism), AP1 (human ortholog of Yap1 and ApyapA), PPAR (lipid metabolism) binding factors.

It emerges a scenario in which carbon source availability, the turnover of lipids and the cell redox balance can modulate aflatoxin synthesis by affecting aflR transcription, furnishing acetate blocks and creating an oxidative cell environment.


Oxidation of fungal aconitases, a regulatory function?

Claudia Maerker, Matthias Brock

1Leibniz University of Hanover, Hannover, Germany, 2Leibniz Institute for Natural Product Research and Infection Biology -Hans-Knöll-Institute, Jena, Germany

The aconitase AcoA from Aspergillus nidulans reveals high similarity to mammalian aconitases (68% identity, pig heart), whereby all catalytic active residues are conserved. Besides playing an essential role in the Krebs’ cycle, AcoA also acts in the fungal methylcitrate cycle, which is required for the degradation of propionate. In this pathway AcoA catalyzes the reversible addition of water to 2-methyl-cis-aconitate to form 2-methylisocitrate (and thus comprises methylisocitrate dehydratase activity). Since both, aconitase and methylisocitrate dehydratase activity, are required during growth on propionate, we studied the catalytic mechanism of the A. nidulans aconitase in more detail. Reduced AcoA contains a [4Fe-4S]2+-cluster as a cofactor, which is essential for aconitase activity. Oxidative degeneration of the [4Fe-4S]2+-cluster into the [3Fe-4S]+-cluster strongly reduced aconitase but, interestingly, increased methylisocitrate dehydratase activity. Nevertheless, dialysis against 2mM EDTA, which destroys the cluster, abolishes both activities. Due to this unique conversion of the mitochondrial aconitase into a methylisocitrate dehydratase in A. nidulans, a role in balancing of propionate metabolism in filamentous fungi is discussed.



Development of a qPCR assay for Aspergillus ochraceus detection in grapes and green coffee beans

Jéssica Gil-Serna, Belén Patiño, Covadonga Vázquez, María Teresa González-Jaén

Complutense University of Madrid, Madrid, Madrid, Spain

Ochratoxin A is a secondary metabolite with nephrotoxic and carcinogenic properties produced by several Aspergillus species. OTA is one of the main mycotoxin due to its high toxicity to humans and animals. Moreover, the maximum OTA limits allowed in food and raw agroproducts are under legal regulation. Aspergillus ochraceus is an important OTA producer species and it is considered the main source in coffee.

Early detection of OTA producer species is a critical point to prevent that mycotoxin entering the food chain. Molecular techniques based in DNA are rapid, sensitive and specific and make possible to obtain relevant information on fungal strains.

The aim of this work was to develop a method based in qPCR assays to detect A. ochraceus strains in two of its more common substrates: grapes and green coffee.

Extraction of fungal and green coffee DNA was carried out with DNeasy Plant Mini Kit (QIAgen). This protocol was modified for DNA extraction from grapes adding a hydrosoluble polymer, polyvinylpirrolidone, in cell lyses and precipitation steps. qPCR reactions were performed using an ABI PRISM 7900HT Sequence Detection System and SYBR® Green PCR Master Mix (Applied Biosystems). The pair of primers used were located in ITS region and were specific for A. ochraceus strains. We have confirmed negative amplification with DNA of grapes and green coffee and that the presence of this exogenous DNA did not interfere in qPCR assay designed.

This method was efficient to detect A. ochraceus strains in samples of grapes and green coffee artificially contaminated with a fungal spore suspension and using genomic DNA extracted after 0, 8, 16 and 24 hours of incubation.

In conclusion, the results obtained in the present work indicate that this method is suitable to detect Aspergillus ochraceus in contaminated samples even without sample incubation.

This work was supported by the Spanish Ministery of Education and Science (AGL2004-07549-C05-05 and AGL2007-66416-C05-02) and UCM-CM-961014.


A light regulation complex controls development and mycotoxin formation in Aspergillus nidulans

Christian Kastner Kastner, Sylvia Mueller, Janina Purschwitz, Reinhard Fischer

Department of Applied Microbiology, University of Karlsruhe, Karlsruhe, Germany

It was discovered recently, that plant-like phytochrome is involved in light sensing in the filamentous fungus Aspergillus nidulans. We could show that phytochrome (FphA) is part of a protein complex containing the white-collar homologues LreA and LreB, the two central components of the Neurospora crassa blue-light sensing system. We found that FphA represses sexual development and mycotoxin formation, whereas LreA and LreB stimulate both. Surprisingly, FphA also interacted with VeA, another regulator involved in light sensing and mycotoxin biosynthesis. All protein interactions occurred in the nucleus, despite cytoplasmic subfractions of the proteins. According to the developmental effects we asked whether there are phase specific transcriptions factors that are part of the light regulation complex (LRC) or interact with it. We tested a number of candidate proteins and found that NosA (=number of sexual spores) and NsdD (=never sexual development) are part of the LRC. The interaction between NosA and phytochrome was restricted to the nucleus, whereas NsdD and FphA interacted in the cytoplasm.

To learn more about the light dependent regulation of the mycotoxin formation, we analysed the production of sterigmatocystin under different light and nutritional conditions. We found that with 1 % glucose in the medium white and blue light inhibited the synthesis in comparison to the synthesis under red light or dark conditions. Interestingly, with 2 % glucose, light stimulated sterigmatocystin formation compared to the production in the dark.

Blumenstein, A., Vienken, K., Tasler, R., Purschwitz, J., Veith, D., Frankenberg-Dinkel, N. & Fischer, R. (2005). Curr Biol 15, 1833-1838, Purschwitz, J., Müller, S., Kastner, C. & Fischer, R. (2006). Curr Opin Microbiol 9, 566-571, Purschwitz, J., Müller, S., Kastner, C., Schöser, M., Haas, H., Espeso, E. A., Atoui, A., Calvo, A. M. & Fischer, R. (2008). Curr Biol in press.


Cloning and characterization of the gene encoding the squalene epoxidase from Hypholoma sublateritium

Ramiro P. Godio1, Ana García Guerra1, Carlos García-Estrada1, Ricardo V. Ullán1, Fernando Teijeira2, Juan F. Martín2

1Instituto de Biotecnología de León (INBIOTEC), León, Spain, 2Área de Microbiología, Departamento de Biología Molecular, Facultad de Biología y Ciencias Ambientales, Universidad de León, León, Spain

The basidiomycete Hypholoma sublateritium produces the triterpenoid antitumor clavaric acid, an inhibitor of the human Ras-farnesyl transferase. The H. sublateritium squalene epoxidase gene (erg1) has been cloned and shown to encode a flavoprotein monooxygenase that requires FAD, NADPH and P450 cofactors. Basidiomycetes are very difficult to manipulate at the molecular level. Genetic modification of Hypholoma has been achieved by Agrobacterium tumefaciens-mediated transformation. Silencing of the erg1 gene in H. sublateritium using constructions with the gdh gene promoter of Agaricus bisporus showed that the squalene epoxidase is involved in clavaric acid formation and in ergosterol biosynthesis; silenced expression of erg1 resulted in an ergosterol-dependent phenotype for full growth. Overexpression of erg1 gene in H. sublateritium resulted in up to 32-to-97% increment of clavaric acid production confirming its involvement in the biosynthesis of this antitumor product. Oxidosqualene (or dioxidosqualene) appears to be the branching point for primary metabolism (sterols) and secondary metabolites in basidiomycetes.

This study evidences the involvement of the squalene epoxidase encoded by the erg1 gene in the biosynthesis of the antitumor compound clavaric acid in the producer basidiomycete H sublateritium.



Cloning and disruption of the obp1 and eph1 genes in Xanthopyllomyces dendrorhous

Ramiro P. Godio1, Ana García Guerra1, Eduardo J. Gudiña1, Ricardo V. Ullán1, Fernando Teijeira2, Carlos García-Estrada1, Juan F. Martín2

1Instituto de Biotecnología de León (INBIOTEC), León, Spain, 2Área de Microbiología, Departamento de Biología Molecular, Facultad de Biología y Ciencias Ambientales, Universidad de León, León, Spain

The conversion of β-carotene into xanthophylls is a subject of great scientific and industrial interest. In a previous work, we cloned the crtS gene involved in astaxanthin biosynthesis from Xanthophyllomyces dendrorhous ATCC 24203. The ORF of this gene has a length of 3166 bp, including 17 introns, and codes a protein of 62.6kDa with similarity to cytochrome-P450 hydroxylases.

To study the genes adjacent to crtS, a phage containing the region around crtS was isolated by screening a genomic library of X. dendrorhous ATCC 24203 in the EMBL3 phage vector, by the PCR procedure of Vaiman. Phage fcrtS160, including the crtS gene in the center of the insert, was selected and sequenced in both DNA strands.

The crtS gene was flanked upstream by a truncated ORF that encodes a putative epoxide hydrolase (eph1) and downstream by a putative oxysterol binding protein (obp1).

In order to elucidate the function of these two genes, constructions for obp1 and eph1 gene disruption by gene replacement were made (pBS-OBPi and pBS-EPHi). The only phenotype detected for the strains disrupted in the obp1 or eph1 genes is the normal accumulation of astaxanthin. The obp1 and eph1 gene expression and astaxanthin production was determined in the ∆obp1 or ∆eph1 strains. They showed 98-100% of the normal astaxanthin production levels. Northern analysis of the expression of obp1 and eph1 in transformants XDOBPi93 (∆obp1) and XDEPHi187 (∆eph1) showed a total reduction of the steady-state levels of obp1 and eph1 transcripts.

The nule expression of obp1 or eph1 genes in transformants XDOBPi93 and XDEPHi187, correlated with a normal astaxanthin production in both transformants (compared to the wild-type strain), support the conclusion that the OBP1 and EPH1 encoded by obp1 and eph1 genes are not involved in the astaxanthin biosynthesis.



The microbody proteome of penicillin-producing Penicillium chrysogenum cells

Jan A.K.W. Kiel1, Marco van den Berg2, Wim Huibers3, Bert Poolman3, Roel A.L. Bovenberg2, Marten Veenhuis1, Ida J. van der Klei1

1Molecular Cell Biology, GBB, University of Groningen, Haren, Netherlands, 2DSM Anti-Infectives, DSM Gist, Delft, Netherlands, 3Department of Biochemistry, GBB & ZIAM, University of Groningen, Groningen, Netherlands

Microbodies are versatile organelles that control various important metabolic processes. In filamentous fungi, microbodies are among others required for the β-oxidation of fatty acids as well as the biosynthesis of β-lactam antibiotics like penicillin. It was demonstrated that enhanced production of penicillin by the filamentous fungus Penicillium chrysogenum was paralleled by an enhanced number and volume fraction of microbodies in the fungal cells. The importance of microbodies in penicillin production was further emphasized by the finding that an increase in the number of microbodies by genetic manipulation of the level of the peroxin Pex11p enhanced penicillin production.

To better understand the role of the microbody in penicillin production, we set out to make a catalogue of proteins related to microbody homeostasis and function. Knowledge of the microbody proteome and of the processes that govern formation, proliferation and degradation of microbodies allow enhancing the efficiency of beta lactam production.

We first performed a proteome-wide identification of P. chrysogenum microbody matrix proteins. To this end we purified microbodies from penicillin-producing P. chrysogenum hyphae via classical sucrose gradients and analyzed their matrix protein contents by Mass Spec analysis. This has resulted in the identification of approximately 150 proteins of which 36 contain a putative microbody (peroxisomal) targeting signal (PTS). This proteomics approach is complemented by a novel genomics approach to screen the P. chrysogenum genome for proteins with PTSs, which has resulted in the identification of 187 putative PTS1 and 10 putative PTS2 proteins. In addition to this, we have screened the P. chrysogenum genome for proteins involved in microbody homeostasis. We have identified 20 novel P. chrysogenum proteins involved in peroxisome biogenesis and proliferation (so-called peroxins) and 30 novel P. chrysogenum Atg proteins involved in autophagy and autophagy-related pathways, like selective microbody degradation.

This project is financially supported by the Netherlands Ministry of Economic Affairs and the B-Basic partner organizations ( through B-Basic, a public-private NWO-ACTS programme (ACTS = Advanced Chemical Technologies for Sustainability).



Effects of solute and matric potential stress, growth and FUM1 gene expression in Fusarium proliferatum

Patricia Marín1, Miguel Jurado1, Naresh Magan2, Covadonga Vázquez3, MŞ Teresa González-Jaén1

1Department of Genetics, University Complutense of Madrid, Jose Antonio Novais 2, 28040 Madrid, Spain, 2Applied Mycology Group, Cranfield Health, Cranfield University, Silsoe, Bedford MK45 4DT, United Kingdom, 3Department of Microbioloy III, University Complutense of Madrid, Jose Antonio Novais 2, 28040 Madrid, Spain

Fusarium proliferatum is considered one of the most important fungal species capable to produce fumonisins during host plant colonization besides F. verticillioides. These compounds are toxic to human and animals and represent a source of risk for health especially in corn-based commodities. Both species belong to the so-called Gibberella fujikuroi species complex and, although they may occur simultaneously in maize, they have differences in host range. The occurrence of fungal species and their mycotoxin production are influenced by ecophysiological factors, in particular water stress. The objective of this work was to study the effect of water stress, solute and matric potential, on growth rate and fumonisin gene expression in two strains of F. proliferatum isolated from a maize field in Spain. The effect of ionic and non-ionic solute water stress and matric potential, which indicates the water stress during growing in soils, was analysed by measuring in vitro mycelial growth rates. The expression of FUM1 gene, involved in fumonisin biosynthesis, was quantified by a species specific Real Time RT-PCR protocol. The results obtained indicated a reduction in growth, in particular in response to matric potencial stress, while FUM1 gene expression was enhanced, in particular when caused by non-ionic solute in both species. The temporal kinetic performed to examine the effect of ionic solute stress on growth and FUM1 expression suggested that water stress might be a critical factor affecting fumonisin accumulation by F. proliferatum under natural conditions, in particular when water stress is progressively increasing during kernel maturation. On the other hand, the results also indicated that F. proliferatum may represent a similar risk for fumonisin production than F. verticillioides in natural conditions. The gene expression studies by Real Time RT-PCR have proven to be a valuable tool to gain knowledge of the ecophysiological basis for fumonisin gene expression and enable better control strategies to be developed during the life cycle of these toxigenic fungi.


Real-time PCR assay for detection and quantification of Aspergillus carbonarius in grapes: comparison between SYBR®Green and TaqMan®

Amaia González, Belén Patiño, Jéssica Gil-Serna, Covadonga Vázquez, María Teresa González-Jaén

University Complutense of Madrid, Madrid, Spain

Aspergillus carbonarius is the main species responsible for the production of Ochratoxin A (OTA) in grapes and wine. This mycotoxin is one of the most common naturally occurring contamination in agroproducts and is toxic to both humans and animals. The purpose of this work was to develop a specific qPCR assay for identifying and quantifying A. carbonarius genomic DNA occurring on contaminated grapes in order to predict the potential OTA risk.

Nucleotide sequences obtained in our lab and from the GeneBank were aligned to design a specific primer pair QCARBOF/QCARBOR from the Internal Transcribed Region ITS. A FAM labelled TaqMan® probe was also designed for the TaqMan® assays. The specificity and sensibility of the primer/probe combinations were tested on a number of A. carbonarius strains and on mixes in different proportions of genomic DNA from A. carbonarius and other Aspergillus species commonly found on grapes, Fusarium sp., Penicillium sp. and Alternaria sp. None of the other species alone gave a positive result with this PCR primer set either using Sybr®Green or TaqMan®. No inhibition was even observed in the mixes of different genomic DNAs.

To test the ability of the designed primers to detect A. carbonarius in grapes, the QPCR assays were coupled with a fungal enrichment and a DNA extraction method for grapes. Different lots of commercially grapes were contaminated with a spore suspension (102 and 106 spores/ml) and DNA was extracted after 0, 8, 16 and 24 hours of incubation. The results indicated that the critical qPCR amplification product was clearly observed for grapes contaminated by 106 spores without incubation using either SYBR®Green or TaqMan®.

These quantitative analyses demonstrated that both reporters SYBR®Green or TaqMan® are equally valid, but we recommend SYBR®Green as is less expensive, easy to use and gave more sensibility than TaqMan®.The qPCR assays presented in this work are based on ITS sequence and therefore are more sensitive than primers based on single copy sequences.

Supported by AGL2004-07549-C05-05, AGL2007-66416-C05-02 and UCM-CM-961014.



Regulation of secondary metabolism in Fusarium fujikuroi: alteration of nitrogen-regulated pathways in carotenoid overproducing mutants

L. Roberto Rodríguez-Ortiz, M. Carmen Limón, Javier Avalos

Department of Genetics, University of Sevilla, Sevilla, Spain

The ability of Fusarium fujikuroi (Gibberella fujikuroi MP-C) to produce gibberellins, growth-stimulating plant hormones, makes this fungus an outstanding model in secondary meta-bolism. Additionally, F. fujikuroi produces bikaverins, reddish polyketides with antibiotic properties, and carotenoids, health-promoting terpenoid pigments. Gibberellins and bikaverins are produced upon exhaustion of the nitrogen source, a regulation mediated by the AreA protein. In contrast, carotenoids are primarily regulated by light, which induces the transcription of the structural genes of the pathway by a WC-independent mechanism (Estrada et al, FGB, in press), currently under investigation. Little information is available on the regulatory genes for carotenogenesis. We have investigated the effect of nitrogen availability on the biosynthesis of carotenoids and the expression of structural genes of the pathway and we have compared it with the effect on bikaverin and gibberellin production and the expression of key genes for each pathway. The experiments were extended to four independent carotenoid overproducing mutants (abbreviated carS), a phenotype attributed to the loss of a key-repressor protein. As expected, the mutants produced higher amounts of carotenoids under the culture conditions tested, but nitrogen starvation produced a partial derepression of carotenogenesis in the wild type. Expression of gene ggs1, putatively responsible for the synthesis of geranylgeranyl pyrophosphate (GGP) for carotenoid production, was derepressed in the carS mutants. Two of them produced less gibberellic acid and three produced less bikaverins than the wild type. These results correlated with reduced mRNA levels of key genes for both pathways in the mutants (ggs2 and cps/KS, coding for GGPP and kaurene synthases for gibberellin biosynthesis, and pks4, coding for bikaverin polyketide synthase). Unexpectedly, expression of the gene niaD (nitrate reductase) was very high in minimal medium with an organic nitrogen source (asparagine) in the four carS mutants compared to the wild type. However, the expression of the partner gene in nitrate assimilation, niiA, was not affected. Taken together, our results suggest a regulatory connection between the function of the putative CarS protein and AreA-mediated processes in F. fujikuroi. The effect of the carS mutations on other nitrogen-regulated processes, such as conidiation, will be also analyzed.


Regulation of bikaverin and fusarin production in Fusarium fujikuroi

M. Carmen Limón, Violeta Díaz-Sánchez, Bina J. Mehta, Javier Avalos

Department of Genetics, University of Sevilla, Sevilla, Spain

Fusarium fujikuroi (Gibberella fujikuroi MP-C) is used for the production of gibberellins, terpenoid hormones that induce growth and regulate various stages of development in plants. This fungus synthesizes many other secondary metabolites, mainly mycotoxins and pigments. Among them are some polyketides, such as bikaverins and fusarins. Regulation of bikaverin and gibberellin biosynthesis by nitrogen has been thoroughly investigated in Fusarium but little is known about their regulation by carbon source. On the other hand, little information is available on the control of fusarin production. Our main interest is to understand the regulation of the biosynthesis of these compounds in F. fujikuroi.

Gibberellin production was not affected significantly by different concentrations of a variety of carbon sources. However, bikaverin production was enhanced when sucrose was the only carbon source. The high production in sucrose required a minimal amount of the sugar, but did not change appreciably above this threshold along a wide range of concentrations. Bikaverin synthesis was repressed when glucose coexisted with sucrose in the medium. The former identification of the gene coding for the polyketide synthase of bikaverin in F. fujikuroi, pks4, allows studying the regulation of the pathway at mRNA level. The stimulation of bikaverin production by sucrose was not paralleled by an induction of pKS4 mRNA.

We have obtained a collection of mutants affected in the production of bikaverins. Mutants are different with regards to their regulation, morphology and secretion of these polyketides. Some mutants produced bikaverins under any culture condition whereas others produced them only under nitrogen starvation. Some mutants were affected morphologically, being the best producers the most affected. Outstandingly, some mutants excreted most of these pigments into the culture media meanwhile others accumulated them mainly in the mycelium.

Fusarin biosynthesis depended on incubation temperature and nitrogen availability but it was not affected by light. The effect of these regulatory signals is being also investigated in mutant SG62, formerly described as a fusarin overproducer. Two different wild type strains, IMI58289 and FKMC1995, strongly differed in their capacity to produce fusarins, indicating a variability of this trait in this species.


Fungal secondary metabolism is an essential component of the complex interplay between Magnaporthe grisea and rice

Jerome Collemare1, Alexis Billard1, Mikael Pianfetti1, Zhongshu Song2, Russell Cox2, Didier Tharreau3, Marc-Henri Lebrun1

1UMR5240 CNRS/UCB/INSA/BayerCropScience, Lyon, France, 2School of Chemistry, University of Bristol, Bristol, United Kingdom, 3UMR BGPI CIRAD-INRA-AGROM, Montpellier, France

Functional analyses of fungal genomes are expanding our view of the metabolic pathways involved in the production of secondary metabolites. These genomes contains a significant number of genes encoding key biosynthetic enzymes such as PKS (Polyketide Synthase) and hybrids PKS-NRPS involved in the biosynthesis of polyketides, NRPS (Non Ribosomal Peptide Synthetase) responsible for peptides synthesis, as well as TS (Terpen cyclase) and DMATS (DimethylAllyl Tryptophan Synthase) that synthesize terpens and alkaloids, respectively. Magnaporthe grisea has a high number of such key enzymes (22 PKS, 8 NRPS, 10 PKS-NRPS, 5 TS and 3 DMATS), suggesting that this fungal species produce a large number of diverse secondary metabolites. Most of these key genes belong to gene clusters that could reflect 36 distinct biosynthetic pathways of secondary metabolites. In particular, M. grisea has the highest number (10) of PKS-NRPS in fungi. Among them, 4 are expressed during penetration of the fungus into host leaves (ACE1, SYN2, SYN8) or during colonization (SYN6), suggesting that they could be involved in the biosynthesis of secondary metabolites involved in pathogenicity. ACE1 and SYN2 genes belong to the same gene cluster that comprise 15 genes specifically expressed during the appressorium-mediated penetration of the fungus likely involved in the biosynthesis of a single polyketide. This secondary metabolite would be recognized by resistant rice cultivars that carry the Pi33 resistance gene because the D ace1 null mutant is virulent towards such cultivars unlike corresponding wild type. However, this mutant is as pathogenic as wild type on sensitive rice or barley cultivars. D syn2 and D syn6 null mutants displayed no visible phenotype related to avirulence or pathogenicity. These results suggest that these genes are not required for plant infection. However, this could be due to a functional redundancy between the different metabolites produced, which could be assessed by constructing double and triple mutants D ace1, D syn2 and D syn6. Identifying the metabolites produced by these enzymes and characterizing their biological activities should give us new clues to better understand their role in pathogenicity. In order to characterize the metabolite produced by ACE1, this enzyme is currently constitutively expressed in M. grisea and under the control of an inducible promoter in Fusarium venenatum.


Cloning and heterologous expression of fungal type I polyketide synthases in Saccharomyces cerevisiae

Michaela Peruci1, Angela Cszifersky1, Franz Berthiller2, Rainer Schuhmacher2, Rudolf Mitterbauer1, Gerhard Adam1

1Institute of Applied Genetics and Cell Biology, Department of Applied Plant Sciences and Plant Biotechnology, University of Natural Resources and Applied Life Sciences, Vienna, Austria, 2Center for Analytical Chemistry, Department for Agrobiotechnology (IFA-Tulln), University of Natural Resources and Applied Life Sciences, Tulln, Austria

Fungal polyketides constitute a large family of secondary metabolites endowed with a high degree of structural diversity and various biological activities. Despite their apparent structural diversity, polyketides share a common mechanism of biosynthesis. Polyketides are synthesized by sequential reactions catalysed by enzymes called polyketide synthases (PKSs) by repetitive Claisen condensations of an acyl-coenzyme A (CoA) starter with malonyl-CoA elongation units in a fashion reminiscent of fatty acid biosynthesis. Both the extender unit of malonyl-CoA and the growing poly-b-ketone intermediates are covalently tethered to the ACP subunit of PKSs in an acyl thioester linkage to the phosphopantetheinyl moiety during polyketide biosynthesis. The latter prosthetic group is introduced posttranslationally by the phosphopantetheinyl transferase (PPTase), which transfers the 4'-phosphopantetheine moiety of CoA to the highly conserved Ser residue of apo-ACP, converting the enzyme into holo-ACP. Phosphopantetheinylation is absolutely necessary for polyketide biosynthesis, without which the PKS is nonfunctional.

It was shown, that the 4‘ phosphopantetheinyl transferease of F. gramineaum (FgPPT1) is necessary for full virulence on wheat (Peruci et. al. in preparation). The aim of our study was to establish a heterologous expression system for polyketide synthases which allows easy screening for 4'-phosphopantetheinyl transferases inhibitors. We expressed type I PKSs in a genetically modified yeast strain allowing detection systems of pigment formation or the estrogenic activity of b-ZOL. For that purpose we have coexpressed FgPPT1 with the PKSs responsible for nor-rubrofusarin production FgPKS12 or the two PKSs required for ZON biosynthesis, FgPKS4 and FgPKS13. In case of FgPKS12, we also had to coexpress the structural gene for a putative methyltransferase aurJ to obtain nor-rubrofusarin.

Obviously, all three heterologous PKS genes are expressed and posttranlationally activated. Detectable amounts of nor-rubrofusarin and ß-ZOL were produced in liquid culture. Efforts to increase the expression levels and to establish suitable screening methods for PPTase inhibitors are ongoing.


PclaeA acts as a regulator of penicillin biosynthesis and asexual differentiation in Penicillium chrysogenum

Birgit Hoff, Ulrich Kück

Ruhr-University Bochum, Bochum, Germany

The filamentous fungus Penicillium chrysogenum is the main industrial producer of the pharmaceutical relevant beta-lactam antibiotic penicillin. All three biosynthesis genes are found in a single cluster and the expression of these genes is known to be controlled by a complex network of global regulators. One candidate is the LaeA protein which was identified first in Aspergillus nidulans as a protein methyltransferase. This factor is required for the transcription of several secondary metabolite gene clusters in Aspergillus species.

In a first attempt, we have clearly identified a LaeA homologue from P. chrysogenum by in silico and PCR approaches. Using a Pcku70 deletion strain with an improved gene targeting efficiency, we have constructed a PclaeA knockout strain for functional gene analysis. Expression and HPLC analyses have clearly demonstrated that PclaeA acts as a regulator of penicillin biosynthesis. Interestingly, the P. chrysogenum PclaeA deletion mutant also shows a significant defect in sporulation even on solid medium. From the sum of our investigations, we can conclude that PclaeA seems to be a global regulator coordinating not only secondary metabolite synthesis but also morphological differentiation in the industrially used fungus P. chrysogenum.


Bioluminescent fungi

Patrick Hickey

1University of Edinburgh, Edinburgh, United Kingdom, 2NIPHT Ltd., Edinburgh, United Kingdom

The aims of this study were to characterise features of five different species of wood-loving bioluminescent fungi. The following Basidiomycetes were studied: Armillaria mellea, Lampteromyces japonicus, Omphalotus nidiformis, Omphalotus olearius and Panellus stipticus. Mycelium was grown on a variety of substrates including cereal grain, wood chips, paper and cardboard. Bioluminescence was imaged using a variety of digital cameras. There was considerable variation in growth characteristics and luminescence between different species and also depending on the substrate. The brightest mycelium tended to be hyphal aggregations, primordia and fruiting bodies. Techniques for fruiting are described and the possible roles of bioluminescence are discussed.



Gene silencing by RNA interference in the white-rot fungus Phanerochaete chrysosporium

Avi Matityahu2, Yitzhak Hadar2, Paula A. Belinky1

1MIGAL – Galilee Technology Center, Kiryat Shmona, Israel, 2Department of Plant Pathology and Microbiology, Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew University of Jerusalem, Rehovot, Israel

RNA interference (RNAi) has not been widely used or analyzed in white-rot fungi. In this work, we proposed using this technique with the white-rot fungus Phanerochaete chrysosporium. The orthologous genes required for RNAi were found in the P. chrysosporium genome database, and a plasmid for gene silencing was constructed. Manganese-containing superoxide dismutase (MnSOD) was used as the target gene for RNAi. The construct contained a transcriptional unit for hairpin RNA expression, which consisted of inverted repeats of the target gene. Significantly lower MnSOD expression at either mRNA or protein levels were detected in RNAi transformants. Furthermore, even though P. chrysosporium possesses three copies of the MnSOD gene, this RNAi construct was sufficient to decrease the enzymatic activity by as much as 70% relative to control levels. Implementation of RNAi technique in P. chrysosporium provides an alternative genetic tool for studies of gene function, particularly those of essential genes or gene families, by enabling simple investigations via reduced expression of a gene of interest in this fungus.


Regulation of Neurospora crassa MAP Kinase genes in response to exogenous phosphate levels

Diana Ester Gras1, Henrique César Santejo Silveira1, Juliana Leal1, Nalu Teixeira de Aguiar Peres1, Pablo Rodrigo Sanches1, Nilce Maria Martinez-Rossi1, Antônio Rossi2

1Departamento de Genética, FMRP, Universidade de São Paulo, Ribeirão Preto, São Paulo, Brazil, 2Departamento de Bioquímica e Imunologia, FMRP, Universidade de São Paulo, Ribeirão Preto, São Paulo, Brazil

Nutrient uptake has important regulatory effects on fungal metabolism, physiology and growth. Although many proteins that function in inorganic phosphate (Pi) acquisition, storage and utilization have been identified, the molecular details of their regulation are not well understood. Under the action of regulatory genes nuc-2, preg, pgov and nuc-1, Neurospora crassa synthesizes a number of phosphatases to scavenge Pi from the environment in response to Pi starvation. Under limiting Pi, NUC-2 inhibits the action of PREG/PGOV complex, allowing the translocation of transcription factor NUC-1 into the nucleus and expression of the Pi-repressible phosphatases. Subtractive hybridizations allowed the identification of two transcripts coding for MAP Kinases (NCU02393 and NCU07024) overexpressed in a strain of N. crassa carrying a loss-of-function mutation in the nuc-2 gene. To further understand the role of these MAP Kinases in the acquisition of Pi, we also carried out a series of Northern blot and semi-quantitative RT-PCR experiments with various strains carrying mutations in genes involved in the phosphate acquisition pathway, which revealed novel aspects of this regulatory network.

Financial support: FAPESP, CNPq, FAEPA and CAPES


Subtractive hybridizations revealed genes down-regulated in the palA1 mutant strain of Aspergillus nidulans grown at acid pH

Janaina Silva Freitas1, Emiliana Mandarano Silva1, Juliana Leal2, Diana Ester Gras2, Nilce Maria Martinez-Rossi2, Antônio Rossi1

1Departamento de Bioquímica e Imunologia, FMRP, Universidade de São Paulo, São Paulo, Brazil, 2Departamento de Genética, FMRP, Universidade de São Paulo, São Paulo, Brazil

The extracellular pH signaling is mediated in A. nidulans by at least seven genes: the six members of the pal activating cascade (pal A, B, C, F, H and I) and pacC, which codes for the transcription factor PacC. The proteolized form of PacC, which occurs by the action of the pal genes at alkaline pH, activates the transcription of alkaline-expressed genes and represses at alkaline pH the transcription of acid-expressed genes. The PalA protein interacts with the YPXL/I motifs in PacC promoting the action of PalB, a calpain-like protease. Thus, whatever the growth pH is, palA1 mutation should cause an acidic-mimicking growth phenotype. This model implies that loss-of-function mutation in any of the six pal genes shall lead to a wild-type acidic growth phenotype regardless of the ambient pH, an effect not observed in the palA1 mutant strain. Employing subtractive hybridizations, we identified genes down-regulated in the palA1 mutant strain of A. nidulans grown in low-Pi medium, at pH 5.0, whose expression should occur only at alkaline pH. Thus, the overexpression of genes at pH 5.0 suggests that palA gene has a function at acid pH and, therefore, the palA1 mutation did not cause an acidic-mimicking growth phenotype.

Financial support: FAPESP, CNPq, CAPES and FAEPA



Coordinated control of pentose release from polysaccharides, the pentose catabolic pathway by two transcriptional activators in Aspergillus niger

Evy Battaglia, Loek Visser, Anita Nijssen, Han Wösten, Ronald de Vries

Microbiology, Utrecht University, Utrecht, Netherlands

During growth on plant polysaccharides Aspergillus niger produces enzymes to release L-arabinose and D-xylose. These monosaccharides are then taken up by the fungal cell and converted through the pentose catabolic pathway to D-xylulose-5-phosphate that enters the pentose phosphate pathway. The pentose catabolic pathway (PCP) is controlled by two transcriptional activators, AraR and XlnR, which respond to the presence of L-arabinose and D-xylose, respectively.

Deletion of one of the regulators results in compensatory regulation by the other regulator, which ensures that growth on L-arabinose and D-xylose still occurs. However, growth on the polymeric substrate (xylan for XlnR and arabinan for AraR) is inhibited, demonstrating that the regulators do not compensate for each other with respect to pentose release. Deletion of both regulators fully inhibits growth on D-xylose and L-arabinose and related polysaccharides.

Gene expression analysis and enzyme activity measurements were performed as well and we observed that members of the PCP are controlled either by AraR (L-arabinose specific steps) or XlnR (D-xylose specific steps) or by both (common steps). In addition, both direct and indirect control was observed on genes of other metabolic pathways.


Integration of additional copies of the Trichoderma reesei gene encoding protein O-mannosyltransferase I results in decrease of the enzyme activity and alteration of cell wall composition

Wioletta Górka-Niec, Anna Kania, Urszula Perlinska-Lenart, Grazyna Palamarczyk, Joanna Kruszewska

Institute of Biochemistry and Biophysics Polish Academy of Sciences, Warsaw, Poland

In fungi transfer of the first mannosyl residue to proteins during their O-glycosylation is catalyzed by protein O-mannosyltransferases.

Using semiquantitative RTPCR method to estimate pmt1 expression we found that integration of an additional copy of the pmt1 gene to the genome of Trichoderma reesei unexpectedly resulted in the silencing of pmt1 expression. Strains exhibited a decrease of total activity of protein O-mannosyltransferases to a level even lower than that obtained by pmt1 disruption. Such a result suggests that in the strains carrying an additional copy of the pmt1 gene not only expression of the pmt1 gene is silenced but also expression of the other pmt genes. Additional evidence that integration of additional copy of pmt1 silenced expression of the other pmt genes was limited O- and N-glycosylation of secreted proteins not found in the pmt1 disruptant. Our study showed also that the strains characterized with decrease of the amount of -(1,3) glucan and simultaneous increase of both, alkali soluble and alkali insoluble -(1,6) glucans in their cell wall. .

We also found alteration of hyphae morphology, limited growth of the strains on the agar plates and sensitivity to Calcofluor white.

Since very often fungi with deficient cell wall are cultivated with osmotic stabilizers added to the cultivation media we showed how 1M sorbitol added to the medium could influence our results.

Our results showed that sorbitol limited growth of the strains, protein secretion, O-glycosylation of secreted proteins and synthesis of cell wall components. Activity of protein O-mannosyltransferases was not altered while activity of N-acetylglucosamine transferase was limited by sorbitol. In addition the morphology of hyphae of the strains cultivated with sorbitol was changed giving narrower hyphae and smaller distances between septa.



Disruption of the Trichoderma reesei gene encoding protein O-mannosyltransferase I results in decrease of the enzyme activity and alteration of cell wall composition

Wioletta Górka-Niec, Michal Pniewski, Anna Kania, Urszula Perlinska-lenart, Grazyna Palamarczyk, Joanna Kruszewska

Institute of Biochemistry and Biophysics Polish Academy of Sciences, Warsaw, Poland

In fungi transfer of the first mannosyl residue to proteins during their O-glycosylation is catalyzed by protein O-mannosyltransferases encoded by pmt genes.

Up to now only one pmt gene encoding protein O-mannosyltransferase in Trichoderma has been cloned. Analysis of the predicted protein sequence of the Trichoderma PMTI protein showed the highest 51 % identity with S.cerevisiae Pmt4p. On the other hand, expression of the Trichoderma PMTI protein in S.cerevisiae pmt mutants revealed its functional similarity to the yeast Pmt2 protein.

We made disruption of the pmt1 gene in T.reesei TU-6, a pyr4 mutant of T.reesei QM9414 using a DNA fragment of about 5.5kb containing T.reesei pyr4 gene flanked with pmt1 sequences. Disruption of the gene caused significant decrease in the total activity of protein O-mannosyltransferases and resulted in osmotic sensitivity of the strain indicating essential role of the PMTI protein activity for cell wall synthesis. At the same time, the strain revealed changes in septa formation resulted in the twice less septa per length of the mycelium compared to the control strain.

The disruptant was able to grow very slowly on agar plates without sorbitol, and the growth was promoted by osmotic stabilizers (1M sorbitol or 0.6M KCl). On the other hand, osmotic stabilizers disturbed growth of the control strain.

Disruption of the pmt1 gene resulted also in a decrease of protein secretion and gave no effect on the glycosylation of secreted proteins. Since protein O-mannosyltransferases are substrate-specific and lack of the PMTI activity did not change O-mannosylation of the secretory proteins we could conclude that PMTI protein is not taking part in the O-glycosylation of these proteins.


ENVOY is a circuit breaker between light response and heterotrimeric G-protein signaling in Hypocrea jecorina (anamorph Trichoderma reesei)

Doris Tisch, Gabriela Gremel, Christian Kubicek, Monika Schmoll

Research Area Gene Technology and Applied Biochemistry, Institute of Chemical Engineering, Vienna University of Technology, Vienna, Austria

Light is a key factor in the environment of many species, also of fungi. The complex signaling pathway of light responsiveness via heterotrimeric G-proteins has been dealt with only in few studies in fungi. Our investigations are focused on the influence of light on G-protein dependent cellulase gene transcription in Hypocrea jecorina. The research into G-alpha subunit GNA1 and GNA3 revealed a connection between G-protein signaling, adaption to a certain nutritional condition and light response in fungi. One of the possible key regulators in this signaling machinery is ENVOY (encoded by env1). This light regulatory protein is involved in cellulase regulation and influences numerous cellular processes both in light and darkness. Therefore we investigated the role of ENVOY in the light response of gna1 and gna3 and found out that ENVOY is influenced by GNA1 and itself influences gna3. Expression of constitutively activated GNA1 and GNA3 lead to an enhanced transcription of cbh1. Intriguingly, ENVOY obviously overrules these effects of GNA1 and GNA3. Double mutants with constitutively activated GNA1 or GNA3, respectively and lacking ENVOY showed similar cbh1 expression compared to a deletion mutant of env1. These results support the idea that ENVOY is a circuit breaker between light response and heterotrimeric G-protein signaling.



Production of oxygenated beta-carotene derivatives by modified Mucor circinelloides strains

Tamás Papp, Enrique A. Iturriaga, Árpád Cserneti, Szekeres András, Eslava Arturo P., Vágvölgyi Csaba

1University of Salamanca, Salamanca, Spain, 2University of Szeged, Szeged, Hungary, 3Analytical Laboratory of Cereal Research Non-profit Company, Szeged, Hungary

Introduction: Carotenoids are yellow to orange-red natural pigments with a broad range of biological functions. Recently, they are attracting an increasing attention, due to their beneficial effects on health. Mucor circinelloides is a beta-carotene-accumulating Zygomycetes fungus, which has been used to study the fungal carotene biosynthesis. In a previous study (1), heterologous expression vectors based on the Agrobacterium aurantiacum b-carotene ketolase gene (crtW) were constructed to achieve the production of xanthophylls in Mucor. In the present work, transformations of a M. circinelloides strain have been performed with combinations of these heterologous vectors and vectors containing Mucor genes encoding the rate-limiting steps of the isoprenoid pathway.

Methods: The genes of isopentenyl pyrophosphate (IPP) isomerase; farnezyl pyrophosphate (FPP) synthase and geranylgeranyl pyrophosphate (GGPP) synthase were introduced via PEG mediated protoplast transformation into M. circinelloides in combinations with the crtW gene. Carotenoid production of the transformants was analysed by TLC and HPLC.

Results: Increased carotenoid production was observed in the new transformants: they produced astaxanthin and canthaxanthin in higher amounts than the strain transformed just with the crtW. In general, co-transformants produced about two times more carotenoids than the original strain.

Conclusion: Strains able to produce oxygenated beta-carotene derivatives were constructed by heterologous genetic transformation. Carotenoid synthesis also could be increased by the over-expression of the early isoprenoid steps via the gene dosis effect.The highest carotenoid content was observed in the transformant harbouring crtW together with extra copies of carG. The product of this gene determines the last isoprenoid step before the carotenoid-specific biosynthetic steps.

The research was supported by ETT grants (214/2006; 261/2006). T. P. is a grantee of the J. Bolyai Research Scholarship.

1 Papp, Velayos, Bartók, Eslava, Vágvölgyi, Iturriaga 2006. Applied Microbiol. Biotechnol. 69: 526-531.


Transcription factor PacC also has metabolic functions specific to acid pH sensing by the dermatophyte Trichophyton rubrum

Henrique C S Silveira1, Diana Gras1, Pablo Sanches1, Antônio Rossi2, Nilce Martinez-Rossi1

1Universidade de São Paulo, Departamento de Genética, Ribeirão Preto, SP, Brazil, 2Universidade de São Paulo, Departamento de Bioquimica e Imunologia, Ribeirão Preto, SP, Brazil

Dermatophytosis is commonly caused by fungi that parasite human skin and nails. Although several factors contribute to their pathogenicity, the successful initiation of infection depends on the capacity of the infecting dermatophyte to sense and overcome the acid pH of the skin. Thus, it is important to understand the metabolic responses that govern the acid pH signaling in dermatophytes. To identify genes supposedly expressed in the initial steps of infection, a suppression subtractive hybridization (SSH) was used between RNA isolated from the H6 strain of the dermatophyte T. rubrum exposed to pH 5.0 and to pH 8.0. This study reveled genes involved in diverse cellular processes as, for example, defense and virulence, protein synthesis and cell transport, which are significantly up-regulated in the H6 strain exposed to pH 5.0. Among these, we confirmed by Northern blot analyses the over-expression of the genes coding for carboxipeptidase S1, acetamidase, aconitase, fatty acid desaturase, TINA, amino acid permease, elongation factor 1-alpha, 60S ribosomal protein L10 and an hypothetical protein. It is worth noting that the expression of at least three of these transcripts is lowered in the pacC-1 mutant strain of T. rubrum (pacC gene disrupted) exposed to pH 5.0, indicating that protein PacC may also have metabolic functions specific to acidic growth pH. The pacC gene encodes a protein homologous to the PacC/Rim101p transcriptional regulator of the conserved route of pH signaling, whose functionality was previously described as exclusive to alkaline pH. We also showed previously that disruption of gene pacC was correlated with a decreased ability of the pacC-1 mutant strain to grow on human nail fragments as the only source of nutrition, which is consistent with the fact that keratinolytic proteases are under the regulation of gene pacC.

Financial support: FAPESP, CNPq, FAEPA and CAPES.



The importance of being nitrite. Functional investigation of NitA, the nitrite transporter of A. nidulans

Vicki F. Symington

St Andrews University, St Andrews, United Kingdom

Aspergillus nidulans has been used extensively as a model organism in the study the nitrate transport protein NrtA, which contributes to the process of nitrate assimilation. Recently, nitrite transporter NitA has been identified in A. nidulans. Nitrite, while not the principal source of nitrogen to organisms, can be used by many bacteria, algae, fungi and plants in certain conditions. The NitA protein is structurally a very different protein when compared directly with the NrtA and NrtB nitrate transporters in A. nidulans; it is half the size with only 6 putative trans-membrane spanning regions. Net nitrite assays using 13N has revealed the kinetic profile for nitrite transport of the WT strain as Km=4.2 ±1 µM, Vmax = 168±21 nmol mg -1 DW-1 h-1. My work concerns the characterisation of the NitA protein in an attempt to assign function to specific amino acid regions using a barrage of site specific mutagenesis; regions have been identified which may be important in substrate translocation.



Identification of cis-acting elements controlling xylanase III gene expression in Trichoderma reesei PC-3-7

Wataru Ogasawara1, Takanori Furukawa1, Yosuke Shida1, Masashi Kato2, Tetsuo Kobayashi2, Hirofumi Okada1, Yasushi Morikawa1

1Nagaoka University of Technology, Nagaoka, Niigata, Japan, 2Nagoya University, Nagoya, Aichi, Japan

The xylanase III gene (xyn3) from the filamentous fungus Trichoderma reesei PC-3-7 is induced only by cellulose, its derivatives and L-sorbose, and not by xylan. In this study, the xyn3 gene from the filamentous mesophilic fungus T. reesei PC-3-7 was cloned and sequenced. The sequence of the 5’-upstream region of the gene in the parent strain QM9414 is identical to that of PC-3-7, although the expression level of xyn3 in PC-3-7 has been reported to be at least 1000 times greater than in QM9414. These results suggest that xyn3 expression in T. reesei QM9414 is silenced. The consensus sequences for ACEI, ACEII, Xyr1, CREI and the Hap2/3/5 protein complex are all present in the upstream region of xyn3. We define three cis-activating elements in the region upstream of xyn3 by using detailed deletion and mutation analysis. In addition to the Xyr1/ACEII binding motif (5’GGCTAA3’), the two analogous motifs, presented as an inverted repeat spaced by 16-bp internal sequences, are identified as the elements essential for xyn3 expression. An electrophoretic mobility shift assay using heterologously expressed Xyr1 demonstrates that each of the three cis-acting elements can interact with Xyr1. Furthermore, no xyn3 transcript was expressed in the xyr1 disruptant upon induction by sophorose and L-sorbose. These results indicate that xyn3 expression is transcriptionally regulated by Xyr1, and suggest that the two analogous motifs play roles in Xyr1-mediated cellulase and xylanase gene expression in T. reesei.


Role of an RNA-dependent RNA polymerase in induction and amplification of RNA silencing in Mucor circinelloides

Silvia Calo, Santiago Torres-Martinez, Rosa M. Ruiz-Vazquez

University of Murcia, Dept. Genetics and Microbiology, Murcia, Spain

RNA silencing is a complex regulatory mechanism that has challenged the central dogma of molecular biology and highlighted the regulatory role of small RNA molecules. Triggered by double-stranded RNA (dsRNA), it acts through transcriptional or post-transcriptional gene silencing of homologous sequences, preventing the expression of exogenous nucleic acids or regulating endogenous pathways. We have demonstrated the existence of a gene silencing mechanism triggered by non-integrative sense transgenes in the zygomycete Mucor circinelloides. Induction of silencing by these transgenes must require the activity of an RNA-dependent RNA polymerase (RdRP), which converts aberrant RNA transcripts derived from the transgene into dsRNA molecules. As special feature, gene silencing in Mucor is associated with two size classes of siRNA, 25-nt and 21-nt long, which are differentially accumulated during the vegetative growth. Amplification of the silencing signal, a process in which an RdRP enzyme produces secondary siRNAs using target mRNAs as a template, has also been demonstrated in M. circinelloides.

Using degenerated oligonucleotides, we have cloned an rdrp-1 gene of Mucor and have obtained a null rdrp-1 mutant by gene disruption. To investigate the role of this RdRP enzyme in the induction and amplification of silencing, we have compared the efficiency and stability of gene silencing triggered by sense transgenes with that obtained using hairpin RNA expressing constructs as triggers, both in the wild type strain and in mutants affected in the rdrp-1 gene. Hairpin constructs containing an intron are highly efficient silencing triggers in both strains, the expression of hairpin RNA being associated with the accumulation of the two size classes of siRNA. However, phenotypic analysis of the null rdrp-1 mutant demonstrated that the rdrp-1 gene is essential for silencing induced by sense transgenes. Isolation of siRNAs in silenced rdrp-1- strains indicates that this gene is not required for amplification of silencing, since production of secondary siRNAs in the rdrp-1 mutant is similar to that of the wild type strain. This suggests the existence of a second rdrp gene in Mucor. Recent advances in M. circinelloides genomic sequencing has allowed us to clone an rdrp-2 gene. The role of this gene in amplification of silencing will be discussed.



Localisation of gene expression in developmental stages of Schizophyllum commune using the fluorescent marker dTomato

R.A. Ohm, S.N. Sharif, T.C.D. Huijzer, T.E.F. Quax, J.G.E. van Leeuwen, J.F. de Jong, H.A.B. Wösten, L.G. Lugones

Microbiology, Institute of Biomembranes, Utrecht University, Utrecht, Netherlands

The basidiomycete Schizophyllum commune is used as a model system to study mushroom formation. This process proceeds through five stages. After aggregation of hyphae (stage I) light induces further development into primordia (stage II). Finally, in stages III-V gills are formed and the hymenial surface expanses.

Several genes have been isolated, which are specifically expressed in the dikaryon. Among these genes are the laccase gene lccA, SC7 which encodes a hydrophilic cell wall protein and the hydrophobin genes SC1 and SC4. We have cloned the promoters of these genes in front of the red fluorescent reporter gene dTomato to study their spatial expression during fruiting body development. Expression of the constitutive promoter GPD served as a control.

GPD - driven dTomato expression resulted in clear fluorescence in monokaryotic and dikaryotic cultures. In the dikaryon, the reporter was detected in both vegetative hyphae and fruiting structures. These results indicate that the method can be used to analyse gene expression in S. commune.

In monokaryotic cultures, no expression of SC1, SC4, SC7 or lccA could be detected. In contrast, in stage I fluorescence was observed in aggregates, indicating that these promoters are fruiting structure specific. In stage II structures, SC1, SC4 and SC7, but not lccA, were still expressed. In mature fruiting bodies (stage V) fluorescence driven by the promoter of SC7 was restricted to the outer surface of the aerial structure. Localisation of expression of the other genes in mature fruiting bodies is currently under investigation.

Similar spatial and temporal expression of genes, suggests that these genes are co-regulated. We determined that the shortest active promoter of SC4, SC7 and lccA are 400 bp, 425 bp and 650 bp upstream of the translation start site, respectively. When these promoters were compared, several putative transcription factor binding sites were found, some of which are currently being mutagenised.



Regulation by blue light of carotenoid regulatory genes, crg, in the fungus Phycomyces blakesleeanus

Victor G. Tagua, Julio Rodriguez-Romero, Luis Corrochano

Universidad de Sevilla, Sevilla, Spain

The biosynthesis of beta-carotene and its regulation by environmental factors, including blue light, have been investigated in detail in the Zygomycete fungus Phycomyces blakesleeanus. Several Phycomyces mutants show altered regulation of beta-carotene biosynthesis by environmental signals. Strains with mutations in the genes madA, similar to the Neurospora photoreceptor gene wc-1, and madB show reduced photocarotenogenesis, and other responses to blue light. In addition, strains with mutations in genes carS, carF, or carD overproduce beta-carotene in the micelium, presumably due to regulatory mutations. Mutants in gene crgA of the Zygomycete Mucor circinelloides overproduce beta-carotene. The CRGA protein contains a RING finger protein motif that is present in proteins involved in ubiquitination of target proteins. It is possible that CRGA may regulate beta-carotene biosynthesis by regulation of protein degradation in Mucor, and it is possible that similar proteins will play similar functions in the regulation of carotenogenesis in Phycomyces.

The genome of Phycomyces contains four genes similar to Mucor crgA that we have named crgA, crgB, crgC, and crgD. Blue light can activate transcription of the four crg genes but to different levels, from 1000 fold to five fold. We have determined the sensitivity of gene photoactivation for each crg gene, and the effect of light exposures of different durations, and we have found that each crg gene responds to blue light in different ways. The photoactivation of crgC and crgD shows a very low threshold and a sustained mRNA accumulation after long light exposures, and the mRNAs for crgA and crgB are only detected after short light exposures. As expected, crg photoactivation requires the photoreceptor MADA, but other mad mutations reduce crg photoactivation. Beta-carotene overproducer strains (carS, carD, and carF) do not show changes in crg mRNA accumulation, but they may carry mutations in any crg gene. We are currently sequencing the crg genes in each beta-carotene overproducer strain to identify a possible Phycomyces crg mutant. Our results suggest that each crg gene may play especialized roles during Phycomyces photocarotenogenesis.


The MAL cluster in Aspergillus oryzae is involved in production of amylolytic enzymes at early stage of maltose induction

Sachiko Hasegawa, Takahiro Shintani, Katsuya Gomi

Grad. Sch. Agric. Sci.,Tohoku Univ., aoba-ku, Sendai, Japan

Aspergillus oryzae has been used in Japanese traditional foods industry, and it produces a copious amount of amylolytic enzymes such as alpha-amylase, glucoamylase, and alpha-glucosidase. These amylolytic genes are regulated by a transcriptional activator, AmyR, in the presence of starch or malto-oligosaccharides including maltose. The amyR gene disruptant showed significantly poor growth on starch medium but showed normal growth on maltose medium, suggesting the existence of alternative maltose-utilizing enzymes whose expression might not be regulated by AmyR. We have found a gene cluster highly homologous to the yeast maltose-utilizing MAL cluster by gene mining in A. oryzae EST and genome databases. This cluster consists of MAL61 homolog (designated malP), MAL62 homolog (designated malT), and MAL63 homolog (designated malR), and thus is designated MAL cluster in A. oryzae. Complementation of yeast mal61 showed that malP encodes a functional maltose permease. The purpose of this study is to elucidate the expression and function of MAL cluster genes.

Northern analyses showed that malP and malT were expressed at high level in the presence of maltose but malR was expressed constitutively. Disruption of malR resulted in the loss of expression of malP and malT. These results indicated that MalR is required for gene expression of malP and malT at post-translational level. The malP or malR disruptant showed poor growth on maltose as well as on starch, comparable to the amyR disruptant, suggesting that malP and malR are somehow involved in assimilating starch. Alpha–amylase activities of both disruptants were significantly low compared with those of wild type at early stage of maltose induction, and they were increasing gradually at late stages of induction. The result suggested that maltose transported into cell mainly by maltose permease encoded by malP is required for AmyR activation and resulting induction of amylolytic enzymes. Expression profiles of the malP and malR disruptants using A. oryzae microarrays will be reported.



Genome-wide analyses of fungicide-response genes regulated via his-asp phosphorelay system and hogA MAPK cascade in Aspergillus nidulans

Daisuke Hagiwara1, Junichiro Marui1, Akira Yoshimi1, Takeshi Mizuno2, Keietsu Abe1

1New Industry Creation Hatchery Center, Tohoku University, Sendai, Japan, 2Graduate School of Bioagricultural Sciences, Nagoya university, Nagoya, Japan

His-Asp phosphorelay (or two-component) systems are common signal transduction mechanisms implicated in a wide variety of cellular responses to environmental stimuli in both prokaryotes and eukaryotes. We have revealed that His-Asp phosphorelay system of a model filamentous fungus Aspergillus nidulans plays a role for response to the fungicide as well as to osmotic and oxidative stresses. Indeed, a mutant strain of which NikA(Histidine kinase) gene was deleted and a mutant strain in which both SskA and SrrA(Response regulators) genes were deleted show resistance to the fungicide (fludioxonil, iprodione). The growth inhibitory effect of the fungicide is seemed to be dependent on His-Asp phosphorelay signaling system. To understand molecular mechanism of growth inhibitory effect, here we conducted DNA microarray analyses, in which we identified the fungicide-response genes (FRGs) dependent on the His-Asp phosphorelay system. Further analyses showed that gene expression of most FRGs are independent on SrrA-pathway, but exclusively dependent on SskA-HogA MAPK pathway. Since HogA MAPK cascade is activated by osmotic stress and plays a pivotal role for osmotic stress response, then we confirmed whether the FRGs identified here are also regulated by treatment with osmotic stress. The transcriptional analyses with DNA microarrays and quantitative RT-PCR revealed that most of FRGs also responded to osmotic stress. Moreover, we found that gene expression of some FRGs was fully induced during asexual development process, especially in conidia. Taking these results together, fungicide treatment is predicted to stimulate SskA-HogA MAPK pathway without physiological cues such as osmotic stress and asexual developmental events. Such abnormal expression of osmotic stress-induced genes and conidia-specific genes might cause the growth inhibition by treatment with fungicides. Finally, we will provide the expression profiles of FRGs under several conditions, which get insight of the diversity and complexity of regulatory mechanism of HogA MAPK.



Investigating the link between TORC1 signalling and nitrogen metabolism in Aspergillus fumigatus

Alan Dobson, Tracy Power, John Morrissey

University College Cork, Cork, Ireland

The ability of a pathogen to sense and respond to its environment is important. In particular, because nitrogen sources available during infection are likely to be sub-optimal, the capacity to reprogramme metabolism in response to different nitrogen sources is known to be a crucial virulence determinant for Aspergillus fumigatus and other fungal pathogens. Although, the genetic response to different nitrogen sources, nitrogen catabolite repression (NCR), is very well understood in Saccharomyces cerevisiae, less in known in filamentous fungi, and some differences are apparent between different fungal species. A key area of interest is in determining whether the rapamycin-sensitive TORC1 complex is involved in NCR. TORC1 is a conserved kinase complex that regulates a broad range of genes and phenotypes in cells in response to growth and nutritional status. We investigated the link between TORC1 and regulation of genes involved in nitrogen metabolism in A. fumigatus. First, we carried out simple growth inhibition assays on agar plates to determine whether rapamycin inhibited the growth of an A. fumigatus colony is the same way as it did the non-pathogen A. nidulans and found that this was the case, confirming that TORC1 activity is required for growth of A. fumigatus. Second, we investigated whether S. cerevisiae could be used as a surrogate host to study the function of A. fumigatus genes. Using the A. fumigatus genome sequence, we identified TORC1/ NCR genes and cloned selected genes into yeast vectors to test whether they would complement yeast mutants. In this way, we showed that AfareA, Afure2, Afgap1, AfprA could all complement S. cerevisiae mutants lacking the orthologous genes. Third, we used RT-PCR to look at expression of TORC1 and NCR regulated genes in A. fumigatus and A. nidulans when growing under different nitrogen conditions and when TORC1 was inhibited by rapamycin. In agreement with previous workers, we did not see a link between TORC1 and NCR in A. nidulans, but we did find that TORC1 regulated, at least to some extent, NCR genes such as areA, mepA and gap1 in A. fumigatus. This indicates a significant difference in genetic and metabolic wiring between the non-pathogenic and pathogenic Aspergilli. Currently, we are carrying out whole genome analysis using A. fumigatus gene arrays to determine the full-spectrum of genes regulated by the TORC1 and NCR systems.



Unravelling the complex transcriptional regulation of the afp gene encoding the antifungal protein AFP of Aspergillus giganteus

Anja Spielvogel1, Ulf Stahl1, Eduardo A. Espeso2, Vera Meyer1

1Technische Universität Berlin, Institut für Biotechnologie, Fachgebiet Mikrobiologie und Genetik, 13355 Berlin, Germany, 2Departamento de Microbiología Molecular, Centro de Investigaciones Biológicas CSIC, 28040 Madrid, Spain

Aspergillus giganteus is known to secrete a small basic protein named AFP with highly selective antifungal activities against numerous plant- and human-pathogenic fungi. To study the biological function of AFP, we have among other things focussed on the identification of signals involved in regulating transcription of the corresponding afp gene.

We have previously shown that transcription of the afp gene is strictly coupled to the asexual development of A. giganteus and strongly up-regulated under several environmental stress conditions such as alkaline pH, osmotic stress, heat shock and phosphate limitation. Interestingly, alkaline pH-induced increase in afp transcription is not dependent on the Pal/PacC signalling cascade, pointing to the involvement of another pH-dependent regulatory system so far not described for filamentous fungi.

We here show that afp gene expression is also up-regulated under cell wall stress provoked by Congo Red and the presence of high external calcium concentrations, suggesting that expression of the afp gene is under control of multiple regulatory systems.

To unravel these networks, we have screened the afp promoter sequence and have identified single or multiple putative binding sites for transcription factors such as StuA (asexual development), RlmA (cell wall integrity), PalcA (phosphate regulation), CrzA (calcium signalling) and SltA (salt stress). Results obtained from different approaches, such as determination of reporter activities in a heterologous system (afp::lacZ reporter in wild-type and different mutant strains of A. nidulans), phosphate uptake measurements and gel shift assays, let us postulate that StuA, PalcA, SltA and CrzA might indeed be involved in afp gene regulation. Furthermore, our data indicate that pH-dependent and phosphate-dependent regulation of the afp gene are linked to each other.

The afp gene is thus under control of different regulatory systems some of which might even be interconnected. Hence, the afp promoter could serve as an excellent model promoter to study the interaction of important signalling pathways in Aspergillus.



Global players involved in nitrogen regulation in Fusarium fujikuroi

Bettina Tudzynski, Birgit Schönig, Dominik Wagner, Sabine Teichert

Institut für Botanik, Westfälische Wilhelms-Universität Münster, Schlossgarten 3, D-48149 Münster, Germany

In F. fujikuroi, the global nitrogen regulator AreA does not only control the utilization of alternative nitrogen sources, but also the expression of gibberellin (GA) biosynthetic genes. Using microarrays of the closely related species F. verticillioides, we identified a set of genes down-or up-regulated in the areA mutant, but also a set of genes which are nitrogen-regulated in an AreA-independent manner. However, not much is known about regulatory proteins, affecting AreA activity. In contrast to A. nidulans, the AreA binding protein NMR does not play a major role in regulating AreA activity in F. fujikuroi. The deletion of nmr did not result in a significant deregulation of AreA target genes, but only in a slightly earlier expression of these genes suggesting that other regulators must exist which inhibit AreA activity under nitrogen sufficient conditions. One candidate is the F. fujikuroi homologue of the A. nidulans meaB gene. Deletion of meaB led to a slight up-regulation of several nitrogen-regulated genes such as the GA- and bikaverin biosynthesis genes and the GS-encoding gene glnA demonstrating that MeaB negatively affects the expression of these genes. A differential hybridization of macroarrays with cDNA from the wild-type and the meaB mutant revealed a set of MeaB target genes. A MeaB-GFP fusion clearly showed that MeaB is translocated to the nucleus under nitrogen-sufficient conditions. To find out if MeaB and NMR act in the same pathway as it has been shown in A. nidulans, or if both factors act independently from each other, we generated nmr meaB double knock-out mutants and compared the expression of nitrogen-regulated genes in the wild-type with that in all mutants. In addition to these regulators, we are searching for signalling components acting upstream of AreA. We show that MepB, one of three ammonium permeases, seems to be involved in sensing and transduction of the nitrogen signal. ΔmepB mutants revealed severe growth defect on low ammonium concentrations suggesting that MepB is the major permease. AreA target genes such as GA- biosynthetic genes or several permease genes, which are normally repressed by ammonium, are de-repressed in ΔmepB mutants. Overexpression of mepC, encoding a second ammonium permease, in ΔmepB restored the growth but not the regulation defect. Based on our recent results, we propose a model for the nitrogen regulation network in F. fujikuroi.




Sulphur nutrition of the rice blast fungus Magnaporthe grisea

Oceane Frelin, Marie-Josephe Gagey, Philippe Perret, Roland Beffa, Geraldine Mey, Michel Droux

CNRS / UCBL / INSA / Bayer CropScience UMR 5240, Lyon, France

Nutritional needs of pathogenic fungi during the interaction with their host remain poorly documented. The very low concentrations of several amino acids (cysteine, methionine, proline, tryptophan, histidine and arginine) in the plant leaf apoplast may not fulfil the pool required for the parasitic growth. Thus phytopathogenic fungi may first mobilize their storage compounds to synthesize their own amino acids during the early stages of the infection process. This hypothesis is supported by genetic studies since mutants auxotrophic for histidine or methionine are non pathogenic. The amino acid metabolism in parasitic fungi may thus be redirected and adapted to the different stages of plant infection.

Our studies focus on the characterization of the sulphur compound metabolism by the hemibiotrophic rice blast fungus Magnaporthe grisea. The genes encoding sulphur metabolism enzymes were identified using M. grisea genome sequence and null mutants were generated by gene replacement. The deleted strain for methionine synthase gene was auxotrophic for methionine and unable to penetrate its host. The methionine synthase mutant was used to assess the role of methionine biosynthesis on fungal development using transcriptomic and biochemical (reversed chromatography by HPLC) approaches.

The deletion led to strong modifications in sulphur metabolites and amino acid contents. Using DNA microarray, we determined the transcriptional profiles of M. grisea using the methionine synthase mutant grown at low and high methionine conditions. Among those genes whose expression in affected, we identified genes involved in methionine, methyl cycle, folate and nitrogen biosynthetic pathways. The decrease or increase in gene expression was correlated to modifications in metabolite contents.

The results strongly suggest the occurrence of complex interactions between metabolic and regulatory network. Taken together, these complementary approaches will allow a better understanding of the methionine metabolism in pathogenic fungi.



Expression of ribosomal protein genes is depressed by the CCAAT-binding complex in Aspergillus species

Akemi Takahashi1, Junya Sugiyama1, Motoaki Sano2, Tetsuo Kobayashi1, Masashi Kato1

1Nagoya University, Nagoya, Aichi, Japan, 2Kanazawa Institute of Technology, Hakusan, Ishikawa, Japan

Introduction: The CCAAT sequence is one of cis-elements present in the promoter regions of numerous eukaryotic genes. A statistical analysis revealed that the CCAAT sequence is one of the most common elements that are present in approximately 30% in the eukaryotic promoters. In filamentous fungi, the CCAAT-binding complex (Hap complex), which is homologous to the yeast Hap complex has been characterized extensively. Although the many genes were reported to be regulated by the Hap complex, it is still unclear what category of genes is regulated by the Hap complex. To elucidate the over all picture of the regulatory network, we carried out DNA microarray analyses using Aspergillus oryzae oligonucleotide arrays.

Methods: A dominant-negative type mutant hapB gene, which encodes a mutant HapB subunit possessing a lesion in the putative DNA binding domain (hapB-1M) was used, since no deletion mutants of Hap subunit genes of A. oryzae were obtained so far. The transformants of A. oryzae with the mutant gene showed a similar phenotype to the hap deletion mutants of A. nidulans such as poor conidiation. Messenger RNAs from the transformant (designated HapB-1M ) and wild-type A. oryzae strains were prepared and used for the DNA microarray analysis.

Results and Discussion: Microarray analysis revealed that not many genes were down-regulated in the HapB-1M strain, unexpectedly. Most of them were categorized as unannotated genes. Together with the fact that CCAAT-boxes are present in promoter regions at a high frequency, it is suggested that the Hap complex mainly enhances the transcription of genes, which are induced only when the genes are under specific conditions. On the other hand, many genes that were up-regulated in the HapB-1M strain were identified. The most remarkable group of genes was that of ribosomal protein genes. Northern analysis with A. oryzae HapB-1M also supported that the Hap complex decrease the amounts of transcripts of ribosomal protein genes. Furthermore, similar results were obtained with an A. nidulans deletion mutant of a Hap subunit gene, indicating that the negative effect of the Hap complex on the ribosomal protein genes is a more general phenomenon, not specific to A. oryzae.



A molecular mechanism for light-dependent conidiation: the fluffy gene, a major regulator of conidiation in Neurospora, is activated by light

Maria Olmedo, Luis M Corrochano

Universidad de Sevilla, Sevilla, Spain

Light stimulates the development of conidia in Neurospora crassa. The products of the wc genes, wc-1 and wc-2, are required for all the Neurospora responses to light, including the activation by light of conidiation. WC-1 is a blue-light photoreceptor and transcription factor that interacts with WC-2 to form a complex (WCC) that binds to the promoters of light-regulated genes. Several strains with mutations that block conidiation have been identified. The fluffy gene is a major regulator of conidiation. The fluffy gene product, FLUFFY (FL), is an 88-kDa polypeptide containing a Zn2Cys6 binuclear zinc cluster domain that is necessary and sufficient to induce conidiophore development in Neurospora. The major role of FL in conidiation prompted us to investigate the possible regulation by light of fluffy gene transcription. The transcription of gene fluffy is activated by light. Wild type mycelia exposed to 15 min of light increased fluffy mRNA accumulation four-fold, but longer light exposures reduced fluffy photoactivation due to photoadaptation. The photoactivation of fluffy is observed with blue light, but not with red light, as in other light responses of Neurospora, including photoconidiation. The threshold for fluffy photoactivation is similar to the threshold for other Neurospora light activated genes. The activation of fluffy by light was observed in other conidiation mutants of Neurospora, suggesting that light-dependent activation of fluffy does not require an active conidiation pathway. As expected, strains with mutations in wc-1 or wc-2 did not accumulate fluffy mRNA after light exposure, an indication that WC-1 and WC-2 are required for fluffy activation by light. The promoter of fluffy contains a putative site for WCC binding at position -560 (from the transcription initiation site). We propose that light-dependent binding of WCC to the promoter of fluffy will trigger light-dependent gene transcription. The increased accumulation of FL will induce conidiophore development resulting in an increased production of conidia after light exposure. Since genes similar to wc-1, wc-2, and fluffy have been described in other Ascomycete genomes, we can speculate that this simplified model for light-dependent conidiation could be useful to understand light-dependent conidiation in other Ascomycetes fungi.


Dissecting molecular steps of cooperativity between the wide-domain GATA factor and the pathway-specific Zn-cluster activator in the Aspergillus nidulans nitrate cluster

Harald Berger1, Asjad Basheer1, Thomas Dalik1, Sandra Boeck1, Joseph Strauss2

1BOKU-University, Vienna, Austria, 2Austrian Research Centers, Vienna, Austria

In Aspergillus nidulans, the transcriptional activation of nitrate assimilating genes (niiA, niaD) is associated with chromatin remodelling processes which depend on the cooperativity between a general nitrogen status-sensing regulator (the GATA-factor AreA) and a pathway-specific activator (the Zn-cluster regulator NirA). In addition to chromatin restructuring, AreA is required for efficient NirA in vivo DNA-binding and both proteins are required for the synergistic transcriptional activation process.

Here, we further dissect NirA and AreA functions in vivo in relation to different nitrogen forms supplied during growth, including nitrogen starvation conditions. We show that AreA activity is strictly controlled exclusively by the level of intracellular free glutamine. Although AreA has been shown to hyperaccumulate in the nucleus during nitrogen starvation, but not during nitrate induction, the level of DNA-bound AreA at the nitrate promoter is not different between these two conditions. This suggests that active NirA promotes AreA chromatin occupancy during nitrate induction. We further show that AreA mediates chromatin remodelling independently of transcription by increasing histone H3 acetylation. NirA mediates additional chromatin restructuring, but this process is not associated with high histone H3ac levels, thus functioning through a different mechanism. NirA function in chromatin remodelling of the nitrate promoters can only be replaced by strongly elevated AreA chromatin occupancy and subsequent hyperacetylation of histone H3, molecular events that are established during nitrogen starvation conditions of the culture.



The Phycomyces blakesleeanus madB gene is a member of the White Collar 2 family

Catalina Sanz1, Julio Rodriguez-Romero2, Alex Idnurm3, M Isabel Alvarez1, Luis M Corrochano2, Joseph Heitman3, Arturo P Eslava1

1Centro Hispano-Luso de Investigaciones Agrarias (CIALE), Departamento de Microbiología y Genética. Universidad de Salamanca., Salamanca, Spain, 2Departamento de Genética. Universidad de Sevilla., Sevilla, Spain, 3Department of Molecular Genetics and Biotecnology. Duke University Medical Centre., Durham, N.C., United States

Introduction: The P. blakesleeanus genome sequence has allowed the identification of three genes from the White Collar 1 family of blue-light photoreceptors, and four wct genes (A - D) from the White Collar 2 family. In a recent work, we have found that mutations in one of those White Collar 1 homologous genes are responsible for the blind phenotype observed in the madA strains, and this gene has been named madA (Idnurm et al., 2006).In the present work we have analyzed the wct genes in several P. blakesleeanus strains that exhibit an abnormal sensitivity to light (madA- madJ) in order to detect possible mutations.

Methods: We have amplified by PCR and sequenced the wct genes in several mad strains and performed Real time PCR to study the expression levels of the madA, wcoA, wcoB and wct genes. We have tested the putative interaction of these genes in yeast-two hybrid assays, and in vitro in co-expression experiments in E. coli.

Results and Discussion: All the analyzed madB strains were affected in the wctA gene and present the same G+906A mutation. We have amplified the cDNA copy of the wctA gene in the madB mutants by PCR and have observed that these cDNAs are partially unspliced. The cDNAs from the madB strains C111, C112 and C109 have been cloned and sequenced to ensure that the mutation does indeed prevent the splicing of the first intron, and to show that there’s no secondary splicing events. We have also analyzed genetic crosses of the wild type strain with madB strains and have observed a close linkage between the phototropic phenotype and the mutation in the wctA gene. All the wild type recombinants do have the wild type copy of the wctA while the madB recombinants have the G to A change, therefore we have decided to name the wctA gene madB. The expression levels of madA and madB genes are modestly repressed by light whereas the expression levels of wcoA, wcoB, wctB and wctD are highly induced by light. The expression of wcoA, wcoB, wctB and wctD is altered in madA and/or madB mutants, thus madA and madB genes contribute to light regulation of the wcoA, wcoB, wctB and wctD genes expression. We have observed that the products of genes madA and madB interact in vivo and in vitro in E. coli. The Phycomyces MAD complex may serve as a photoresponsive transcription factor similar to the WC complex in Neurospora crassa.


Carbon signaling by hexose phosphorylating enzymes in Hypocrea jecorina

Lukas Hartl, Christian Kubicek, Bernhard Seiboth

Vienna University of Technology, Wien, Austria

The role of hexose phosphorylating enzymes in the assimilation and signaling of carbon compounds was investigated in the ascomycete Hypocrea jecorina. The fungus features two hexokinases including one glucokinase (GLK1) and one hexokinase (HXK1) responsible for d-glucose respectively d-fructose phosphorylation and growth on these carbon sources. Deletion of the hxk1 or glk1 gene results in slower growth on different carbohydrates including d-xylose, l-arabinose or glycerol whose catabolism is independent of the catalytic function of the encoded hexokinases. Growth is further reduced in double Δglk1Δhxk1 strains which are also unable to sporulate. Three genetic systems, diagnostic for carbon catabolite (de)repression (cellulase formation, cbh1; xylanase formation, xyn1; and β-galactosidase formation, bga1) were analyzed: Carbon catabolite repression by d-glucose and d-fructose is retained in both single deletion strains and only Δglk1Δhxk1 strains are derepressed. The level of derepression in Δglk1Δhxk1 strains was higher compared to a cre1 (encoding the carbon catabolite repressor) mutant. Induction by specific carbon compounds is differently influenced: induction of cbh1 by sophorose and of bga1 by d-galactose is impaired in Δglk1Δhxk1 strains, whereas induction of xyn1 by d-xylose is increased in all deletions strains being highest in a Δglk1Δhxk1 strain. Our data show that the two hexose phosphorylating enzymes of H. jecorina exert a general influence on carbohydrate metabolism and suggest that the two hexokinases have acquired additional non-enzymatic functions in carbon signaling.



Master transcriptional control of morphogenesis in Ustilago maydis by an APSES domain regulatory protein

Maria D. Garcia-Pedrajas1, Scott E. Gold2

1Estacion Experimental "La Mayora", CSIC, Algarrobo-Costa, Malaga, Spain, 2University of Georgia, Athens, Georgia, United States

In Ustilago maydis, the causal agent of corn smut, the morphological transition from yeast to filamentous growth is inextricably linked to pathogenicity; budding haploid cells are saprobic and upon mating of compatible strains the fungus converts to dikaryotic filamentous growth and obligate parasitism. The filamentous dikaryon proliferates in the host plant inducing tumour formation and finally undergoes another morphological change that results in the production of melanised diploid teliospores. In an attempt to identified new trans-acting factors that regulate morphogenesis in U. maydis, we searched for the presence of common binding sequences in the promoter region of a set of 37 genes whose expression is downregulated in the filamentous form. Putative cis-acting regulatory sequences fitting the consensus binding site for the Aspergillus nidulans transcription factor StuA were identified in 13 of these genes. StuA is a member of the APSES transcription factors which contain a highly conserved DNA-binding domain with a basic helix-loop-helix (bHLH)-like structure. This class of proteins comprises critical regulators of developmental processes in fungi. A search for StuA orthologs in the U. maydis genome identified a single closely related protein that we designated Ust1. Deletion of ust1 in haploid wild type and solopathogenic strains led to filamentous growth, and abolished mating, gall induction and consequently in planta teliosporogenesis. Furthermore, cultures of ust1 null mutants produced abundant thick-walled highly pigmented cells resembling teliospores which are normally produced only in planta. We showed that ssp1, a gene highly induced in teliospores produced in the host is also abundantly expressed in cultures of ust1 null mutants containing these pigmented cells. Modification of a conserved putative PKA target in Ust1 to mimic constitutive phosphorylation did not significantly affect morphology but resulted in absence of galls in inoculated plants. This phenotype resembles constitutive activation of the cAMP/PKA signal transduction pathway. We are investigating further the putative link between Ust1 and cAMP signalling by production of double mutants. Our results indicate that Ust1 is a major regulator of dimorphism, virulence and the sporulation program in U. maydis. We are currently determining if this regulatory protein functions at least in part as a downstream effector of PKA.


Identification and isolation of the xylanase repressor protein (Xrp1) of Hypocrea jecorina (Trichoderma reesei)

Marion E Pucher, Marcio J Pocas-Fonseca, Robert L Mach, Astrid R Stricker

Vienna University of Technology, Vienna, Austria

Xylan which is a predominantly formed renewable plant biomass component comprises of heteropolysaccarides with a backbone of β-1,4-linked xylopyranosyl residues. Enzymes capable of degrading the xylan backbone of Hypocrea jecorina (anamorph Trichoderma reesei) have received strong attention, because of their application in food and feed, and pulp and paper industry. Thus, the filamentous ascomycete H. jecorina is an industrial important organism for production of xylanases. Recently, it has been demonstrated that aside to the general activator protein Xyr1, the expression of the xylanase system in this fungus is regulated by additional fine tuning transcription factors. Not only wide domain regulators (e.g. Cre1 or the Hap-complex), but also narrow domain regulators, such as Ace1, Ace 2 and a yet only postulated Xylanase repressor protein (Xrp1) are involved in the regulation of differential expression of the major xylanolytic enzyme-encoding genes of H. jecorina. Here we report on the isolation and characterization of Xrp1. After characterization of the respective DNA binding element in the xyn2 promoter, DNA binding proteins were enriched via affinity chromatography and thereafter identified by nano HPLC tandem MS analysis. The candidate genes were subjected to transcript analysis and respective electrophoretic mobility shift assays. Three potential regulator proteins were identified and further characterized via in silico analysis.



Role of Fusarium oxysporum white collar 1 photoreceptor on carotenogenesis, UV resistance, hydrophobicity and virulence on mammalian hosts

Carmen Ruiz-Roldán1, Victoriano Garre2, Josep Guarro3, Marçal Mariné3, M. Isabel G. Roncero1

1Universidad de Córdoba, Córdoba, Spain, 2Universidad de Murcia, Murcia, Spain, 3Universidad Rovira y Virgili, Reus, Tarragona, Spain

Light regulates developmental and physiological processes in a wide range of organisms, including filamentous fungi. Fusarium oxysporum, the causal agent of the vascular wilt disease in a wide variety of crops and an emerging human pathogen, contains a putative photoreceptor Wc1, the orthologe to WC-1 of Neurospora crassa. Isolation and characterisation of the wc1 gene revealed that the predicted protein contained all the characteristic domains present in other fungal photoreceptors, including a conserved LOV domain, two PAS dimerization domains, a nuclear localization sequence and a Zn-finger DNA binding domain. Wc1 is expressed constitutively at very low levels and does not regulate expression of wc2 gene. Targeted disrupted mutants showed that this gene is involved in formation of aerial hyphae when grown on solid medium under white light. Defects in aerial hyphae development could be related to altered hydrophobocity, since ∆wc1 mutants showed altered colony surface hydrophobicity under white light, as well as differential expression pattern of the hydrophobin gene hyd1. ∆wc1 mutants were also affected in induction of carotenogenesis by light, indicating that this gene is involved in this process. Additionally, Wc1 contributes to photoreactivation after UV treatment and controls light induced up-regulation of the photolyase gene phr1. Pathotypic behaviour of ∆wc1 mutants on tomato plants was unaltered, indicating that this gene is dispensable for pathogenicity on host plants. By contrast, mutation of wc1 impairs virulence on immunodepressed mice. These results demonstrate that light perception in F. oxysporum plays important roles in the development and behaviour of this ascomycete fungus.



A distinct sequence motif in RRPD3-type histone deacetylases of filamentous fungi is essential for growth and development of Aspergillus nidulans

Stefan Graessle Graessle, Martin Tribus, Johannes Galehr, Gerald Brosch, Ingo Bauer

Medical University Innsbruck, Innsbruck, Tirol, Austria

Acetylation of the N-terminal tails of core histones is crucial for the functional properties of chromatin and plays an important role in the regulation of a large number of genes in eukaryotic organisms.

Although filamentous fungi have contributed significantly to our understanding of gene regulatory mechanisms, only little is known about the enzymes, histone acetyltransferases (HATs) and histone deacetylases (HDACs), responsible for a balanced acetylation status of the histone tails in these organisms. However, it becomes more and more clear today that HDACs, together with other chromatin modifying enzymes, affect the regulation of genes involved in pathogenicity, stress defence, and production of secondary metabolites (such as antibiotics or toxins) of filamentous fungi.

Despite the impact of HDACs for a variety of cellular processes in fungi, all HDAC deletion strains generated so far were viable. Here we show that depletion of RpdA, a classical RPD3-type HDAC of Aspergillus nidulans, leads to a drastic reduction of growth and sporulation of the fungus.

Functional studies of RpdA revealed that a distinct motif specific for the C-terminal tail of RPD3-type proteins in filamentous fungi is absolutely essential for the catalytic activity of the enzyme and cannot be deleted without affecting the viability not just of A. nidulans but most likely also of other filamentous fungi, like the human pathogen Aspergillus fumigatus or the plant pathogenic species Cochliobolus carbonum. Therefore the C-terminal extension of RpdA-type proteins represents a possible target for fungal specific HDAC-inhibitors that might have potential as new antimycotic compounds with clinical and agricultural applications.



Mitochondrial aspect of the oxidative stress in Phanerochaete chrysosporium

Mélanie Morel1, Loïc Diss1, Michel Droux2, Claire Fourrey1, Michel Chalot1, Jean-Pierre Jacquot1, Eric Gelhaye1

1UMR 1136 Interaction arbre Microorganismes Université Nancy I, Vandoeuvre les Nancy, France, 2UMR 5240 CNRS-UCB-INSA-Bayer CropScience, Lyon, France

Phanerochaete chrysosporium produces unique extracellular oxidative enzymes that degrade lignin, as well as related compounds found in explosive-contaminated materials, pesticides, and toxic wastes. The genome of this white rot fungus was the first basidiomycete genome to be sequenced. P. chrysosporium secretes a large range of peroxidases and oxidases that act non-specifically via the generation of lignin free radicals, which then undergo spontaneous cleavage reactions. The non-specific nature and exceptional oxidation potential of these enzymes led to considerable interest for application in bioprocesses such as organopollutants degradation and fibbers bleaching.

To better understand the link between oxidative stress and lignin peroxidase production, we have focused our study on the mitochondrial properties of Phanerochaete into these stress conditions. We analyzed the expression of some genes coding for mitochondrial enzymes putatively involved in the metabolism of ROS, and tested the activity of the mitochondrial complexes. We used a culture medium without manganese to low-down the activity of the Mn-superoxide dismutase and increase the level of intracellular reactive oxygen species (ROS). This culture condition led to the excretion of lignin peroxidases in the culture medium, a modification of mitochondrial properties and gene expression of some enzymes involved in oxidative stress.



RrmA, an RNA binding protein with key roles in amino acid biosynthesis, nitrogen metabolism and oxidative stress

Kinga Krol2, Igor Morozov1, Meriel G. Jones1, Agnieszka Dzikowska2, Mark X. Caddick1

1The University of Liverpool, School of Biological Sciences, Biosciences, Liverpool, L69 7ZB, United Kingdom, 2Institute of Genetics and Biotechnology, Warsaw University,, 02-106 Warsaw, Poland

The protein RrmA is involved in posttranscriptional regulation of gene expression in Aspergillus nidulans. rrmA mutations were first selected on the basis of altered arginine/proline metabolism, leading to suppression of proline auxotrophy in a proA- strain. Independently of this, the protein was identified as specifically binding the 3’ UTR of areA mRNA. The areA transcript encodes a transcription factor involved in nitrogen metabolite repression and is subject to regulated degradation by glutamine (Gln), being highly unstable under conditions of nitrogen sufficiency. Phenotypic analysis of rrmA mutants indicated a significantly increased sensitivity to H2O2, suggesting that RrmA is also involved in the oxidative stress response.

To investigate the role of RrmA protein in the regulation of transcript stability we have utilised Northern hybridisation analysis. Results show increased stability for agaA, otaA and areA transcripts in the ∆rrmA mutant when comparison with wild type. agaA and otaA encode enzymes of arginine catabolism. However, not all transcripts subject to Gln signalled degradation are affected by this mutation. These observations indicate that the RrmA protein plays a direct role in the destabilisation of a specific subset of transcripts subject to degradation in response to a specific signal (eg Gln). The consequences of rrmA deletion on transcript levels and stability during oxidative stress are currently being investigated.

An RrmA:GFP fusion was generated to investigate intracellular localisation, and possible changes that may occur in response to shifts in nitrogen regimes or oxidative stress. From this we have determined that RrmA is a highly expressed cytoplasmic protein. In the presence of ammonium (ie a rich nitrogen source) small aggregates appear which may be linked to P-bodies, the RNA-protein complexes associated with mRNA storage and degradation. Oxidative stress leads to an increase the amount of RrmA in the cell of A. nidulans.



The novel transcription activator AtrR regulates gene expression of ABC transporters and contributes to azole resistance in filamentous fungi

Daisuke Miura1, Ayumi Ooba1, Takahiro Shintani1, Kiminori Shimizu2, Marie Nishimura3, Katsuya Gomi1

1Graduate School of Agriculturral Science, Tohoku University, Sendai, Japan, 2Medical Mycology Research Center, Chiba University, Chiba, Japan, 3National Institute of Agrobiological Sciences, Tsukuba, Japan

Infections by human pathogenic fungi such as Aspergillus fumigatus are commonly treated with the azole fungicides that inhibit the ergosterol biosynthesis in the fungal cell membrane. During the long-term usage of the drug, sometimes emerge azole resistant isolates of A. fumigatus. A major mechanism of azole resistance in A. fumigatus is the upregulation of genes encoding drug efflux pumps, mainly belonging to ABC transporters.

We have already isolated a spontaneous resistant mutant of Aspergillus oryzae for azole drugs and found that at least three ABC transporters were upregulated in the mutant compared to the wild type. Since the expression of several ABC transporter genes was upregulated simultaneously in the mutant, we assumed that azole resistance is caused by mutation of a common transcription factor that controls these gene expressions. Overexpression analyses of transcription factor genes found in A. oryzae genome revealed that upregulation of a zinc cluster gene, designated atrR, resulted in increased drug resistance and also induced the gene expression of ABC transporters in A. oryzae. Deletion of the atrR reduced the expression level of the three ABC transporter genes and consequently resulted in significant increase in azole drug susceptibility, especially the atrR mutant was also susceptible to fluconazole (~5 ppm). Orthologous genes of the A. oryzae atrR have been found widely in genomes of filamentous fungi, including Aspergillus nidulans, A. fumigatus, and Magnaporthe grisea. Both strains with deletion of these orthologs in A. nidulans and A. fumigatus were also hypersensitive to azole drugs. These results indicate that the novel transcription factor, AtrR, regulates gene expression of ABC transporters that would function as drug efflux pumps and contributes to the azole resistance in Aspergillus fungi. In addition, a deletion mutant of the atrR ortholog has been constructed in M. grisea and is now being examined for azole susceptibility.


Oxidative stress response in Ashbya gossypii depends on YAP1

Andrea Walther, Jürgen Wendland

Carlsberg Laboratory, Yeast Biology, DK-2500 Valby, Copenhagen, Denmark

Ashbya gossypii is a filamentous ascomycete with a close relationship to Saccharomyces cerevisiae. In the Ashbya genome there is one ortholog of the S. cerevisiae YAP1 gene that is one of the major regulators of the oxidative stress response yeast. In S. cerevisiae and other organisms Yap1 is suggested to act as a redox sensor. Intramolecular disulphide bonds can activate these proteins by inhibiting nuclear export. The A. gossypii Yap1 seems to be unique since it lacks the conserved Cysteine-rich domains. The Ashbya YAP1 encodes a protein of only 297aa while ScYap1 is 650aa in length (ScYap2/Cad1 is 409aa). In order to understand the differences in Yap1 function and regulation we initiated the functional analysis of AgYAP1/2. To this end we generated complete yap1-ORF deletion mutants by PCR-based gene targeting methods. Agyap1 strains exhibit an increased sensitivity to H2O2 compared to wild type cells, which was tested by measuring growth inhibition in agar diffusion assays using H2O2. This indicates that AgYap1/2 indeed plays a role in regulating the oxidative stress response in this organism.

We will present our ongoing analyses on YAP1/2 considering the following aspects:

Heterologous complementation of the S. cerevisiae yap1 mutant with AgYAP1;

Elucidating the subcellular localization of AgYap1 in Ashbya using a chromosomally integrated GFP-tagged YAP1 allele;

Analysis of the Yap1-regulon in Ashbya gossypii.



Identification of yeast SKI genes orthologs and their involvement in nonstop mRNA decay in Aspergillus oryzae

Mizuki Tanaka1, Masafumi Tokuoka2, Takahiro Shintani1, Katsuya Gomi1

1Bioindustrial Genomics, Bioscience and biotechnology for future bioindustries, Graduate School of Agricultural Science, Tohoku University, 1-1 Amamiya-machi, Tsutsumidori, Aoba-ku, Sendai, Japan, 2Noda institute for scientific research, 399 Noda, Noda city, Japan

Aspergillus oryzae has been paid an attention as a favorable host organism for heterologous protein production. However, despite using a strong expression promoter, heterologous gene expression sometimes results in low mRNA level. We showed previously, using a mite allergen protein, Der f 7, as a model, that premature polyadenylation within ORF of the heterologous gene occurred but was prevented by codon optimization in A.oryzae. In addition, insertion of an internal stop codon into the glucoamylase-Der f 7 (native) fusion gene resulted in the increase in the amount of the transcript. These observations suggested that low mRNA level of the heterologous gene is caused by the degradation process of mRNA lacking translation termination codon, which is called ‘nonstop mRNA decay’ pathway. In this study, we analyze the mechanism of nonstop mRNA decay pathway in A.oryzae.

Nonstop mRNA decay mechanism has been studied mainly in Saccharomyces cerevisiae, and Ski7p-exosome-dependent 3’à 5’ degradation and Ski7p-independent 5’à 3’ degradation models have been proposed. To elucidate the nonstop mRNA decay mechanism in A. oryzae, we searched for yeast Ski protein homologs in the published Aspergillus genome databases. Interestingly, there are no homologues of Ski7p that is known to be most important for nonstop mRNA decay in yeast, suggesting that nonstop mRNA decay mechanism in filamentous fungi differs from that in yeast. On the other hand, except for Ski7p there are other corresponding Ski protein homologs in Aspergillus genomes, especially Ski1p (5’à 3’ exoribonuclease involved in mRNA decay), Ski2p (one of the components of Ski complex required for 3’à 5’ decay by exosome), and Ski4p (subunit of the exosome) homologs share high identity with those of yeast. Gene disruptants of ski1, ski2, and ski4 were constructed in A. oryzae to examine their involvement in nonstop mRNA decay. Whereas the expression level of nonstop mRNA significantly reduced compared with that of normal mRNA in wild-type and ∆ski1, this reduction was depressed clearly in ∆ski4 and also slightly in ∆ski2. This suggested that nonstop mRNA is degraded by 3’à 5’ decay pathway in A. oryzae.



Towards an understanding of how the PacC/Pal-mediated alkaline ambient pH response transcriptionally regulates itself in Aspergillus nidulans

Tatiana Múnera-Huertas1, América Hervás-Aguilar2, Eduardo Espeso2, Miguel Peñalva2, Herbert Arst1, Joan Tilburn1

1Imperial College London, London, United Kingdom, 2Centro de Investigaciones Biologicas, Consejo Superior de Investigaciones Cientificas, Madrid, Spain

pH regulation in A. nidulans is mediated by the PacC transcription factor whose full-length form (PacC72) undergoes signalling proteolysis at alkaline pH to yield an ~500 residue form PacC53 in response to Pal pathway signalling. This is followed by a second proteasomal, pH-independent cleavage to give PacC27, an activator of alkaline–expressed genes and a repressor of acid-expressed genes (reviewed in [1]). Western blot analysis of MYC3-tagged PacC demonstrates that on shifting from acidic to alkaline growth media PacC72, which predominates under acidic conditions, undergoes two-step proteolytic processing via PacC53 to PacC27. After 60’ exposure to alkalinity PacC72 is undetectable and, at least in part, has been converted to PacC53 and PacC27. After two hours PacC72 accumulates again, indicating de novo synthesis. After four hours PacC72 and PacC27 are the predominant forms with PacC53 being barely detectable, indicating that pH signal transduction has become rate-limiting in PacC processing [2]. This is suggestive of a negative feed back loop in the alkaline pH response. Here we shall focus on two genes specifying components of the PacC/Pal mediated alkaline pH response which are themselves pH responsive. pH signal transduction component PalF is encoded by an acid-expressed gene which is negatively regulated in a PacC27 dependent manner. Thus low PalF levels are a potential bottleneck in pH signalling. pacC is an alkaline-expressed gene and behaves to a certain extent as a PacC27 positively regulated gene. However, under acidic conditions pacC transcript levels are elevated in a pacC null strain relative to those in a wildtype strain suggesting that there is, at least to some extent, negative autogenous regulation by PacC72 or PacC53. Our results suggest that transcriptional regulation of the pH responsive machinery is mediated by a finely tuned rheostat involving at least two interdependent feedback loops which are crucial for the wide range of pH response observed in A. nidulans. We shall discuss our recent progress and future perspectives.

1) Peñalva MA and Arst HNJr, 2004. Ann. Rev. Microbiol. 2004. 58:425-51. 2) Hervás-Aguilar A, Rodríguez, JM, Tilburn, J, Arst HNJr. Peñalva MA, J. Biol. Chem. 282:34735-47.



The role of Aspergillus nidulans’s pumilio family (PUF) RNA binding proteins implicated in mRNA stability during nitrogen metabolism

Sunthorn Chooluck, Igor Morozov, Mariel Jones, Mark Caddick

University of Liverpool, Liverpool, United Kingdom

The stability of any given transcript plays an important role in determining the level of the mRNA and subsequent gene expression. Highly stable transcripts are optimal for achieving high levels of gene expression. As a consequence RNA stability varies significantly among genes, and in some cases this is directly regulated as a means of controlling gene expression. We have previously demonstrated a differential rate of decay for the areA transcript, which encodes the major transcription factor mediating nitrogen metabolite repression. The areA mRNA is destabilised by the presence of intracellular Gln, a signal of nitrogen sufficiency. The same mechanism is important to the expression of various structural genes involved in nitrogen metabolism, including niiA and niaD. Intriguingly, some of these genes are also subject to an additional regulatory mechanism at the level of RNA stability that stabilises the transcripts in the presence of the respective protein’s substrates. Bioinformatics analysis of the untranslated regions of Aspergillus genes revealed the presence of a number of conserved motifs, including many likely to be involved in interactions with the Pumilio homology domain (Puf) RNA-binding proteins. In other eukaryotes, including Saccharomyces cerevisiae and Drosophila melanogaster, these proteins have been found to coordinate expression of specific groups of genes. In our laboratory, we have identified a number of proteins whose orthologues are shown to be involved in RNA degradation. Amongst these are the five Puf proteins, pufA, pufB, pufC, pufD and pufE. All five A.nidulans’s puf genes have successfully been deleted. Deletion of pufD results in a dramatically reduced response to Gln with respect to destabilisation of areA and meaA transcripts, and the areA transcript also reveals significantly reduced basal degradation. Deletion of pufA appears to disrupt the sexual cycle. The pufB, pufC and pufE deleted strains are currently being characterised.


Identification of a putative class of transcription factors in the rice blast fungus

Maria Besi, Ane Sesma

John Innes Centre, Norwich, United Kingdom

Fungi belonging to the Magnaporthe grisea species-complex infect a wide range of cereals and grasses, including economically important crops such as rice, barley, wheat, and millet. Magnaporthe oryzae is a member of this species-complex and causes rice-blast disease which is responsible for the loss of 10-30% of the annual rice harvest worldwide. M. grisea has been regarded as a typical foliar pathogen, but recently it has been shown that it can also infect cereal roots, undergoing developmental steps typical for root-infecting pathogens.

A forward-genetics screen using in vitro tests on hydrophilic and hydrophobic surfaces as well as in planta assays on roots and leaves has enabled the identification of additional tissue-specific and general determinants involved in pathogenicity of M. grisea. A transformant of the fungus identified during the in vitro screen on polystyrene hydrophilic surfaces has been found to be compromised in root and leaf pathogenesis. The disrupted gene was identified by recovery of the sequence flanking the T-DNA insertion site and encodes a putative transcription factor (rfeG), previously identified in Aspergillus terreus. Targeted gene replacement mutants of this gene have been generated, via Agrobacterium tumefaciens-mediated transformation. The DNA-binding function of the rfeG protein has been shown by yeast-one-hybrid experiments. The coding sequences of other two putative transcription factors, related to rfeG, have also been selected and disruption mutants are being generated.



A transcription factor cascade involving creA and xlnR regulates the expression of the Aspergillus nidulans genes encoding the xylanolytic complex

Elsy Tamayo2, Adela Villanueva1, Daniel Ramón1, Margarita Orejas1

1Instituto de Agroquímica y Tecnología de Alimentos. CSIC, Valencia, Spain, 2Instituto Tecnológico de Mérida. División de Estudios Avanzados, Mérida, Yucatán, Mexico

The A. nidulans xlnR gene encodes a Zn2Cys6 transcription activator necessary for the synthesis of the main xylanolytic enzymes, i.e. endo-xylanases X22, X24 and X34, and β-xilosidase XlnD. In previous work it has been established that the wide domain carbon catabolite repressor CreA indirectly represses the xlnA (encodes X22) and xlnB (encodes X24) genes as well as exerting direct repression on xlnA. In this study we show that that CreA-mediated indirect repression occurs through repression of xlnR: (i) the xlnR gene promoter is repressed by glucose and this repression is abolished in creAd30 mutant strains and (ii) deregulated expression of xlnR completely relieves glucose repression of xlnA and xlnB. Thus, part of the carbon catabolite repression of the A. nidulans genes comprising the xylanolytic complex occurs via a transcription factor cascade in which the wide domain repressor CreA controls the expression of the xylanolytic transactivator xlnR.



HapX, a novel member of the iron metabolom in A. fumigatus

Markus Schrettl1, Martin Eisendle2, Hubertus Haas2

1Department of Biology, National University of Ireland Maynooth, Maynooth, Ireland, 2Division of Molecular Biology, Biocenter, Innsbruck Medical University, Innsbruck, Austria

Aspergillus fumigatus represents an important opportunistic pathogen in immunocompromised patients. Under conditions of low iron availability this filamentous ascomycete produces, like the closely related A. nidulans, two major siderophores: it excretes triacetylfusarinine C (TafC) to capture iron and contains ferricrocin as an intracellular iron storage compound. Recently it was shown, that this siderophor system is absolutely required for virulence in A. fumigatus and therefore regulation of the iron metabolism is of great importance. Here we report the functional characterization of a bZIP transcription factor, encoded by hapX. Its deletion is deleterious under iron limiting conditions and under oxidative stress inducing conditions, leading to reduced growth rates and inefficient conidiation. Moreover HapX seems to act in an activating mode on TafC and ferricrocin production and remarkably, hapX deletion resulted in an increased accumulation of protoporphyrin IX and in de-repression of iron-dependent pathways during iron shortage.

This work was supported by Austrian Science Foundation Grant FWF-P15959-B07.



Functional characterization of protein arginine methyltransferases in Aspergillus nidulans

Ingo Bauer, Stefan Graessle, Gerald Brosch, Peter Loidl

Biocenter – Division of Molecular Biology, Innsbruck Medical University, Tyrol, Austria

The importance of protein arginine methylation in the regulation of cell function is currently emerging. Among the multitude of substrates of protein arginine methyltransferases (PRMTs) are proteins involved in RNA processing, regulation of transcription, translation, signal transduction, apoptosis, and subcellular transport. It has also been shown that core histones are methylated by PRMTs in vivo and that histone arginine methylation takes part in transcriptional regulation.

In the filamentous fungus Aspergillus nidulans three PRMTs (RmtA, RmtB, and RmtC) are present. In order to study PRMT-dependent processes we generated deletion mutants via a gene targeting strategy. Deletion strains could be achieved for all PRMT genes, thus none of these genes alone is essential for the viability of A. nidulans.

Phenotypic analysis revealed that growth of rmtA and rmtC mutants was significantly reduced when acetate was provided as sole carbon source, or when strains were grown under oxidative stress inducing conditions. We further could demonstrate that catalytic activity of RmtA is crucial for the oxidative stress defense in A. nidulans. Moreover, rmtC mutants exhibited decreased ability to grow at elevated temperatures.

Biochemical analysis revealed substrate specificity of RmtA for histone H4. Further, methylation of H3 was dependent on the presence of both, RmtB and RmtC, indicating a cooperative role of these enzymes in establishing this modification in vitro. Interestingly, also H2B methylation could be detected, however, the methylation status was not affected by either deletion, indicating the presence of an additional yet unidentified enzymatic activity.

Taken together, our results substantiate the physiological significance of protein arginine methylation in A. nidulans. Generated deletion mutants will be valuable tools for further biochemical and genetic studies on the function of this modification in A. nidulans, and may also improve our understanding of the role of protein arginine methylation in higher eukaryotes.



Facultative heterochromatin is involved in switching from primary to secondary metabolism in Aspergillus nidulans

Yazmid Reyes-Dominguez1, Jin Woo Bok2, Asjad Basher1, Elliot K Schwab2, Harald Berger1, Andreas Gallmetzer1, Nancy P Keller2, Claudio Scazzocchio3, Joseph Strauss1

1University of Natural Resources and Applied Life Sciences, Vienna, Austria, 2University of Wisconsin, Madison, United States, 3Université Paris Sud, Orsay, France

Heterochromatin is a repressive chromatin structure involved in gene silencing phenomena. One of the principal components of heterochromatin formation and maintenance is the heterochromatin protein 1, HP1 (or SWI6 in S .pombe) which requires lysine 9 methylation marks on histone H3 (H3K9met) to bind to chromatin and induce a repressive heterochromatin structure.

The deletion of the unique HP1 homologous gene, hepA in A. nidulans, causes the deregulation of several genes related to secondary metabolism, including genes belonging to the sterigmatocystin (ST) biosynthetic cluster. The deletion of hepA leads to an earlier production and higher accumulation of ST.

We show by chromatin immunoprecipitation (ChIP) analysis on the promoter region of aflR, the gene encoding the specific ST cluster transcriptional activator that HepA occupies the aflR locus during transcriptional silencing of the cluster but dissociates from the promoter during production of ST. The amount of HepA present at the aflR promoter directly correlates with the level of lysine-9 trimethylation of histone H3.

In A. nidulans, LaeA was shown to be required for transcription of ST and penicillin clusters. We show that deletion of hepA or the H3K9 methylase clrD suppresses the laeAΔ phenotype and that production of secondary metabolites is restored in a double laeAΔ/hepAΔ or laeAΔ/clrDΔ strain. Our ChIP results using the single and double mutant strains suggest that LaeA counteracts binding of HepA to H3K9me in the aflR promoter region under conditions of ST production.

We show by Micrococcal nuclease (MNase) chromatin analysis that, in a wild type strain, MNase accessibility of the ST locus is restricted during conditions of primary metabolism suggesting the formation of heterochromatic structures. In contrast, at the onset of ST cluster activation MNase access increases indicating a reversal of silencing by euchromatization of the ST locus. Additionally, the effect of the deletion of hepA and laeA on heterochromatin/euchromatin switching of ST locus is presented. Our results indicate that facultative heterochromatin structures regulate the timing of expression and overall activity of the ST-gene locus in A. nidulans and that H3K9me and subsequent HepA binding determines locus silencing during primary metabolism. LaeA, the general positive regulator of secondary metabolism is directly involved in reversing this repressive chromatin structure thus contributing to cluster activation at the onset of secondary metabolism.



Lactose metabolism in Aspergillus nidulans requires a lactose permease and an intracellular beta-galactosidase

Erzsébet Fekete1, Levente Karaffa1, Bernhard Seiboth2, Attila Szentirmai1, Christian P. Kubicek2

1Department of Genetics and Applied Microbiology, Faculty of Science and Technology, University of Debrecen, Debrecen, Hungary, 2Research Area Gene Technology and Applied Biochemistry, Institute of Chemical Engineering, TU Wien, Vienna, Austria

The heterodisaccharide lactose (1,4-O-β-D-galactopyranosyl-D-glucose) occurs mainly in mammalian milk where it makes up 2-8 % of the dry weight. Although fungi usually do not encounter lactose in their native habitats, some yeast and most multicellular fungi can utilize lactose as a carbon source, although often at only low rates. For this reason, lactose has been used as a preferred carbon source for penicillin biosynthesis by Penicillium chrysogenum in the 60’s and 70’s to bypass carbon catabolite repression. It is currently also the only soluble carbon source for cellulase and recombinant protein production under cellulase expression signals in the fungus Trichoderma reesei.

In the yeast Kluyveromyces lactis, the LAC12 locus encoding a lactose permease is located immediately upstream of a GH2 family ß-galactosidase-encoding gene (LAC4), with which its shares a bidirectional promoter. In Aspergillus nidulans, genetic analysis of lactose utilization has detected eight loci (lacA-lacH) to be involved, including two putatively encoding intracellular ß-galactosidases (lacC, lacG). In agreement with this, we only had detected intracellular ß-galactosidases in A. nidulans during growth on lactose. However, a lactose permease – the essential prerequisite for this pathway – has not yet been identified from A. nidulans, and also not from any other filamentous fungus.

In this study, we will describe a lactose permease of A. nidulans named lacA. By disruption of this locus we demonstrate that it is heavily involved but not essential for growth on lactose. We will also demonstrate that this lactose permease is the orthologue of the K. lactis LAC12, and that this locus has differently evolved in different filamentous fungi. We will also provide evidence that the clustering of LacA and LacG (the latter encoding an intracellular ß-galactosidase) has been maintained during evolution in A. nidulans. Consequently, the expression of lacA and lacG is coregulated with one example being the CreA-dependent carbon catabolite repression.



Transcriptional regulation of genes encoding extracellular enzymes produced by strains of Aspergillus niger and Trichoderma reesei using solid state fermentation

Sheena Fagan, Richard Murphy

Alltech, Dunboyne, Co. Meath, Ireland

Over the past decades, many cellulolytic fungi have been isolated and their relevant enzymes characterised. However, compared with the number of isolates, very little research was dedicated to understanding the induction and repression mechanisms of cellulolytic enzymes produced using solid state fermentation (SSF) techniques. The biotechnological importance of filamentous fungi and associated enzymes has accelerated an interest in understanding the regulation of gene expression and the characterisation of the transcription factors involved.

The ability to regulate gene expression and to adapt to environmental changes helps living organisms to survive. Study of regulation phenomena by genetic and molecular methods are therefore of growing interest, not only because of biotechnological importance, but also from an evolutionary point of view. Most of the published results are related to growth of filamentous fungi under submerged fermentation conditions and very little is known about gene expression under solid state conditions. In this study, the transcript expression levels of a number of candidate regulatory elements including ace1, ace2, creA, cre1, hapB, pacC, xlnR, xyrI, and areA were examined after cultivation of Aspergillus niger and Trichoderma reesei in solid state fermentations supplemented with different carbon and nitrogen sources. It was anticipated that this type of analysis would enable a direct evaluation of effect of nutrient supplementation on gene expression and facilitate identification of possible co-regulation mechanisms, which would assist in the optimisation of culture conditions for enzyme production.

In the present study, the analysis of gene expression revealed noticeable differences not only between solid state and submerged fermentations, but also between the two strains. It was concluded that certain carbon sources induced high levels of expression of one gene or a set of genes, such as those induced in T. reesei cultures, whereas the effect on expression of other genes was weak or insignificant, as was evident with A. niger supplemented cultures.


Biz1 is a key player during plant penetration and in planta development of Ustilago maydis

Miroslav Vraneš, Florian Finkernagel, Mario Scherer, Jörg Kämper

Max-Planck-Institute for Terrestrial Microbiology, Marburg, Germany

In the corn smut fungus Ustilago maydis, sexual development is initiated by fusion of two haploid sporidia, generating a filamentous dikaryon that is capable to infect the plant. To get insight into the processes that precede plant infection, we performed microarray analysis of U. maydis cells grown on the plant surface. One of the genes specifically induced in a pathogenic strain is the C2H2 zinc finger transcription factor biz1. biz1 cells are severely affected in appressoria formation and plant penetration. Biz1 regulates about 30% of all genes induced on the plant surface; for 19 of these genes, Biz1 is both required and sufficient for induction. Systematic deletion analysis of these genes led to the identification of pst1 and pst2, encoding potentially secreted U. maydis specific proteins. ∆pst1/pst2 cells are still able to penetrate the plant surface, but subsequently fail to invade and colonize the plant, resembling the biz1 deletion phenotype.

Microarray analysis revealed that 76 of the biz1-dependent genes are induced during various stages of pathogenic development. An integrative in silico motif search combining several algorithms (such as Weeder [2] and ANN-Spec[3]) and metrices was established. The feasibility of the algorithm was proven by use of several test data sets with known regulatory motifs. We identified an enriched CCCAC motif in the 5´ regions of 95% of the Biz1 induced genes. 96% of the Biz1 regulated genes that are induced on planta harbour the CCCAC consensus within 500 bp from the start codon. Our data suggest that Biz1 is not only a regulator of genes required for plant penetration, but also for genes with impact on pathogenicity at later stages.

1 Vraneš et al., Plant Cell Sep 2006;18(9): 2369-2387, 2 Pavesi et al., Nucleic Acid Res 2004;32: W199-W203, 3 Workman et al., Pac Symp Biocomput 2000;5: 467--478



An arginine-riboswitch regulates alternative splicing of Aspergillus nidulans arginase mRNA

Piotr Borsuk1, Karina Solecka2, Anna Przykorska2, Jerzy Pawlowicz2, Malgorzata Pekala1, Michal Koper1, Piotr Weglenski1

1University of Warsaw, Warsaw, Poland, 2Polish Academy of Science, Warsaw, Poland

Expression of the arginase structural gene (agaA) in Aspergillus nidulans is subject to complex transcriptional and post-transcriptional regulation. This regulation is dependent on growth conditions, particularly the presence of arginine and various nitrogen and carbon sources. Arginase mRNA has a long 5'UTR sequence. Analysis of this sequence in silico revealed its putative complex secondary structure, the presence of arginine-binding motifs (arginine aptamers) and a short intron with two potential 3' splicing sites. The arginine-5’UTR interaction was confirmed both by 5’UTR structure mapping and FPLC chromatography on arginine-sepharose column. The arginine biding site was minimized to 79 nucleotides localized in the 5’ terminal region. The specific arginine-mRNA interaction was not influenced by any analyzed specific mutation introduced into putative main aptamer sequence. In this report we present evidence that L-arginine binds directly to the arginase 5'UTR, invokes drastic changes in the secondary structure of the 5'UTR, unlike several other L-amino acids and D-arginine and forces the selection of one of two 3' splice sites of an intron present in the 5'-UTR. We postulate that expression of the eukaryotic structural gene coding for arginase in A. nidulans is regulated at the level of mRNA stability, depending on riboswitch-mediated alternative splicing of the 5'-UTR intron.



A cross-talk of the XYR1 regulator, the XYL1 aldose reductase and L-arabinitol regulates arabinan and L-arabinose metabolism in Hypocrea jecorina (Trichoderma reesei)

Eda Akel1, Bernhard Seiboth2, Christian P. Kubicek2

1University of Istanbul, Faculty of Pharmacy, Department of Biochemistry, Istanbul, Turkey, 2Research Area Gene Technology and Applied Biochemistry, Institute of Chemical Engineering, Vienna, Austria

Arabinan is a polysaccharide found in plant cell walls as a side chain of pectin. H. jecorina is also able to degrade arabinan to L-arabinose by an arabinanolytic system. To date two arabinanases have been characterized: an α-L-arabinofuranosidases (ABF1) and a ß-xylosidase which has a separate α–arabinofuranosidase domain and activity. L-arabinose can be used by the fungus as a carbon source via the L-arabinose catabolic pathway, which converts L-arabinose to D-xylulose 5-phosphate. Little is known about the regulation of the arabinanase system in H. jecorina, however. The Zn(II)-Cys6-protein XYR1/XlnR has been shown to be a transcriptional activator of cellulase and xylanase gene transcription in different Aspergillus spp. and in H. jecorina. XlnR does not regulate arabinan and L-arabinose metabolism in the Aspergilli. In H. jecorina, however, the aldose reductase XYL1 (which is also involved in L-arabinose catabolism) is controlled by XYR1, thus implying that XYR1 would also regulate L-arabinose metabolism. However, Stricker et al. (2006) reported that a knock out in xyr1 in H. jecorina did not affect growth on D-arabinose. These conflicting data prompted us to perform a more detailed investigation on the possibility of arabinan and L-arabinose metabolism by XYR1. In this paper, we will provide evidence that XYR1 is indeed essential for expression of the enzymes of arabinan and D-arabinose degradation. Consistent with a role of L-arabinitol as inducer of the arabinolytic system, L-arabinitol dehydrogenase-knock out mutants display strongly increased levels of arabinofuranosidase gene expression. However, L-arabinitol cannot rescue gene expression in delta-xyr1 and delta-xyl1 strains, indicating a cross-talk of XYR1, XYL1 and L-arabinitol in the induction of these genes.



Transcript profiling during the reproductive phase change in Agaricus bisporus

Daniel Eastwood, Bram Herman, Surapareddy Sreenivasaprasad, Andreja Dobrovin-Pennington, Ralph Noble, Kerry Burton

University of Warwick, Warwickshire, United Kingdom

The phase change from vegetative mycelium to reproductive growth in the basidiomycete Agaricus bisporus is regulated by environmental triggers. Fruiting of higher fungi in the environment is generally seasonal and it is assumed that the environmental stimuli control the timing of reproduction in such fungi. Changes in temperature, levels of carbon dioxide and the concentration of 8-carbon volatiles cause A. bisporus mycelia to aggregate and form fruiting bodies. It is not known how these stimuli interact or influence the fungus at a molecular level to cause a change in growth phase and morphology.

A suppression subtractive hybridisation and custom oligo-microarray screening approach was employed to investigate the transcriptional changes in A. bisporus to environmental and morphogenetic change. The initiation of fruiting under standard growing conditions was compared with experiments where one stimulus was perturbed. The data has been used to profile the response of A. bisporus genes during fruiting and to identify the genetic response to individual stimuli. This work has relevance not only to the mushroom production industry, but to understanding the role of the environment in the control of reproduction in higher fungi.