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.

Mohammed Abdo Elgabbar, Elham Sulima, Wisal Abdallah

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.

Martin Eisendle, Beate Abt, Hubertus Haas

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.

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.

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

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.

Claudia Kragl, Markus Schrettl, Martin Eisendle, Hubertus Haas

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.

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

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.

Ulrike Fohgrub, Monika Trienens, Marko Rohlfs, Frank Kempken

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.

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.

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.

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

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.

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

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.

Katrin Volling, Axel Brakhage, Hans Saluz

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.

Nancy Alexander, Susan McCormick

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.

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.

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.

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.

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.

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.

Ken Oda, Motoaki Sano, Akiko Kobayashi, Shinichi Ohashi

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.

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.

Abdelhamid Abbas, Alan Dobson

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.

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

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.

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.

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 (www.softberry.com) 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.

Claudia Maerker, Matthias Brock

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.

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

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.

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

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.

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

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.

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.

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 (www.b-basic.nl) through B-Basic, a public-private NWO-ACTS programme (ACTS = Advanced Chemical Technologies for Sustainability).

Patricia Marín¹, Miguel Jurado¹, Naresh Magan², Covadonga Vázquez³, Mª 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.

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

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.

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

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.

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

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.

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.

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.

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.

Birgit Hoff, Ulrich Kück

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.

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.

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.

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

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

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

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.

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

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.

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

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.

Doris Tisch, Gabriela Gremel, Christian Kubicek, Monika Schmoll

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.

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

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.

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

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

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.

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

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.