Poster Category 3:


Regulation of Gene Expression at the Genome Level



Intracellular NADP and ATP regulate transcription of the yeast GAL regulon

Tianhong Wang, Li Yan, Guanjun Chen

State Key Laboratory of Microbial Technology, Shandong University, P.R.China,

The interplay between the yeast prototypical transcriptional activator Gal4p and the inhibitor protein Gal80p determines the transcriptional status of the genes needed for galactose utilization in Saccharomyces cerevisiae. In this study, we showed that deletion of components responsible for mitochondria and cytocytoplasmic synthesis of NADP including pos5 and utr1 impaired the induction of GAL genes. The malfunction of respiratory oxidative phosphorylation was also found to affect the induced transcription of GAL genes. Increase in intracellular level of NADP or ATP in these mutants restored the normal induction process with the recovered occupancy of Ser5 phosphorylated RNA polymerase II on the GAL promoter. On the other hand, deletion of dsg1 coding for an F box protein impaired the early-onset expression of a GAL1-LacZ reporter. However, the induced transcription of GAL mRNA was not affected although a decreased occupancy of RNA polymerase II on the 5’ coding region of GAL  genes occurred. The Dsg1-mediated induction defect was partly complemented by increase in intracellular level of NADP but not by that of ATP. Nonetheless all the induction defects were abrogated by the absence of transcription inhibitor Gal80p. In vitro analysis revealed that NADP synergizes with ATP to destabilize the interaction between Gal80 and Gal4. Taken together, these results suggest that interaction status between Gal4p and Gal80p not only determines the on-off of transcription of GAL genes, but may also be involved in the fine tuning of the whole transcription process including the formation of mature mRNAs.




Blue- and red-light photoreceptors regulate the activation by light of conidiation genes in Aspergillus nidulans

CARMEN RUGER-HERREROS[1] Raúl Fernández-Barranco[1] Maria Olmedo[2] Luis M. Corrochano[1] David Cánovas[1]

1Department of Genetics, University of Sevilla, Spain, 2Department of Chronobiology, University of Groningen, The Netherlands

The ascomycete Aspergillus nidulans is a model organism to study fungal development. Conidiation is controlled by the product of the brlA gene. Many gene products act upstream of brlA, probably allowing the synthesis of chemicals or allowing the transduction of environmental signals to trigger brlA transcription. The A. nidulans genome contains genes for a phytochrome (fphA), two homologs of N.crassa WC-1 and WC-2 (lreA and lreB) and a veA gene. Red and blue light stimulate conidiation in A. nidulans, but mutations in the veA gene allow conidiation in the dark. Recently, it has been shown that the phytocrome FphA interacts with VeA and LreA and LreB. However, the mechanism that the photoreceptors employ to activate conidiation remains unknown. We have found that the expression of several conidiation genes, including brlA, fluG flbA, flbB and flbC, was regulated by light. The photoactivation of these genes showed a quick reponse with mRNA accumulation increasing after 5 minutes of illumination. brlA mRNA accumulation after illumination increased with time showing maximum values between 30 to 60 minutes and revealed a two-component activation. Deletion of the photoreceptor genes fphA, lreA and lreB reduced the activation by light of the studied genes. None of these genes are essential for gene photoactivation since we observed light-dependent mRNA accumulation in strains with single deletion of photoreceptor genes. On-going experiments provides a model for the light-dependent activation of conidiation.


RRMA, an RNA binding protein involved in regulated mRNA degradation

Kinga Krol[1] Igor Y. Morozov[2] Piotr Weglenski[1] Massimo Reverberi[3] Mark X. Caddick[2] Agnieszka Dzikowska[1]

1Institute of Genetics and Biotechnology, University of Warsaw, Poland, 2School of Biological Sciences, The University of Liverpool, UK, 3Plant Biology, Universita La Sapienza, Roma, Italy


RRMA is the RNA binding protein involved in posttranscriptional regulation of gene expression in Aspergillus nidulans. rrmA gene was identified as a suppressor of mutations in arginine/proline catabolic pathway. Independently RRMA protein was shown to bind to the 3’UTR of areA transcript (nitrogen positive regulator). ΔrrmA mutation results in slow growth phenotype and higher sensitivity to oxidative stress. Analysis of main antioxidant enzymes revealed different activity pattern during early development stages in ΔrrmA strain comparing to the control strain. Transcriptional analysis has shown that ΔrrmA mutation results in higher stability of specific transcripts under conditions of oxidative stress and nitrogen starvation.  Our results indicate that RRMA plays important role in metabolism of A. nidulans and can be involved in the mechanism of regulated degradation of specific mRNAs in response to environmental signals.





D-xylose: repressor or inducer of xylanase expression in Hypocrea jecorina (Trichoderma reesei)

Marion E. Pucher, Astrid R. Mach-Aigner, Robert L. Mach

Vienna University of Technology, Institute of Chemical Engineering, Department of Gene Technology and Applied Biochemistry

The saprophytic fungus Trichoderma reesei (teleomorph Hypocrea jecorina) is able to degrade a huge variety of biopolymers such as xylan and cellulose, the predominant compound of plant materials. The produced hydrolytic enzymes have received great industrially importance (e.g. food and feed industry, pulp and paper industry). Before we started our investigations, it was already known that D-xylose is an inducer for hydrolytic-enzymes encoding genes. In this study we show that the degree of induction is dependent on the applied D-xylose concentration. We could demonstrate that high induction of hydrolytic-enzymes encoding genes can be observed using 0.5 or 1 mM D-xylose for 3 hours of cultivation. D-xylose causes never glucose-like repression of transcription of xylanase-encoding genes, even not at high concentrations (66 mM). The investigations show that the transcription factor Carbon catabolite repressor 1 (Cre1) reduces the expression of the Xylanase regulator 1 (Xyr1), the main activator of many hydrolytic enzymes encoding genes, and as a consequence, lower amounts of hydrolytic enzymes are expressed. In this study we can demonstrate that D-xylose has to be metabolized via the Xylose reductase (Xyl1) to achieve induction of xylanase expression. Finally, we show that a strain bearing a constitutively expressed xyr1 could partly overcome the negative influence of Cre1.






Time course microarray analysis of ΔPcvelA and ΔPclaeA mutants of Penicillium chrysogenum

Jens Kamerewerd[1] Birgit Hoff[1] Tim Dahlmann[1] Ivo Zadra[2] Thomas Specht[2] Hubert Kürnsteiner[2] Ulrich Kück[1]

1Christian Doppler Laboratory for “Fungal Biotechnology”, Lehrstuhl für Allgemeine und Molekulare Botanik, Ruhr-Universität Bochum, Universitätsstraße 150, 44780 Bochum, Germany
2Sandoz GmbH, Biochemiestraße 10, 6250 Kundl, Austria

The filamentous fungus Penicillium chrysogenum is the main industrial producer of the β-lactam antibiotic penicillin. Recently the genome sequence of P. chrysogenum was published, thus strain improvement by reverse genetics became feasible. For efficient gene replacements, a P. chrysogenum strain lacking non-homologous end joining (NHEJ) was constructed by deletion of the Pcku70 gene, a homologue of human Ku70 gene essential for NHEJ. Although ΔPcku70 strain lacks any obvious phenotype under native conditions, we were able to identify a stress-related molecular phenotype by conducting a whole genome microarray time course analysis. Strain ΔPcku70 was used to generate the mutants ΔPcvelA and ΔPclaeA. The proteins PcvelA and PclaeA, homologues of the VeA and LaeA proteins from Aspergillus nidulans, are part of a multiprotein complex and positively regulate penicillin biosynthesis in P. chrysogenum. In order to identify common target genes of PcvelA and PclaeA, a comparative microarray analysis was conducted with transcripts from the deletion mutants ΔPcvelA and ΔPclaeA isolated after 48, 60 and 96 hours of growth. For comparison, we used the data from the time course analysis of the ΔPcku70 strain. Our results clearly demonstrate common as well as distinct functions of PcvelA and PclaeA as regulators of secondary metabolism and fungal development in P. chrysogenum.





Components of a velvet-like complex in Fusarium fujikuroi affect differentiation, secondary metabolism and virulence

Philipp Wiemann[5] Daren W. Brown[1] Karin Kleigrewe[2] Jin Woo Bok[3] Nancy P. Keller[4] Hans-Ulrich Humpf[2] Bettina Tudzynski[5]

1Mycotoxin Research Unit, USDA, ARS, NCAUR, 2Institut für Lebensmittelchemie, WWU Münster
3Department of Medical Microbiology and Immunology, University of Wisconsin
4Department of Medical Microbiology and Immunology / Department of Bacteriology, University of Wisconsin, 5Institut für Botanik, WWU Münster

Besides industrially produced gibberellins (GAs), Fusarium fujikuroi is able to produce additional secondary metabolites such as the pigment bikaverin and the mycotoxins fumonisins and fusarins. The global regulation of these biosynthetic pathways is only poorly understood. Recently, the Velvet complex containing VeA and several other regulatory proteins was shown to be involved in global regulation of secondary metabolism and differentiation in Aspergillus nidulans. Here we report on the characterization of two components of the F. fujikuroi Velvet-like complex, FfVel1 and FfLae1. The gene encoding this first reported LaeA ortholog outside the class of Eurotiomycetidae is upregulated in the ΔFfvel1 mutant (shown by microarray-studies) and FfLae1 interacts with FfVel1 in the nucleus (shown by BiFC). Deletion of Ffvel1 and Fflae1 revealed for the first time that Velvet can simultaneously act as positive (GAs, fumonisins and fusarins) and negative (bikaverin) regulator of secondary metabolism, most likely by interconnecting with the nitrogen regulation network. Furthermore, we show that both components affect conidiation and virulence of F. fujikuroi on rice plants. Cross genus complementation studies of Velvet complex component mutants between Fusarium and Aspergillus support an ancient origin for this complex which has undergone a divergence in specific functions mediating development and secondary metabolism.



Two GATA factors AREA and AREB negatively regulate arginine catabolism genes in Aspergillus nidulans in response to nitrogen and carbon source.

Maria Macios, Piotr Weglenski, Agnieszka Dzikowska[1]

Institute of Genetics and Biotechnology, Warsaw University, Poland

In A. nidulans arginine is utilised as a nitrogen and carbon source. It is a good system for investigation the connection between the two global carbon and nitrogen repression regulatory systems. Utilization of arginine depends on the presence and inducibility of two arginine catabolic enzymes arginase and ornithine aminotransferase (OAT) encoded by agaA and otaA genes, respectively. Analysis of different single and double areA and areB mutants have shown that two GATA factors AREA and AREB negatively regulate the expression of arginine catabolism genes under nitrogen repression conditions. AREA and AREB activities depend on carbon source. AREA regulator is necessary for the ammonium repression of arginine catabolism genes under carbon repressing conditions while AREB - under carbon limited, non-repressing conditions. AREA activity was shown to be modulated by a direct protein – protein interaction with NMRA protein which is proposed to bind to the C terminus of AREA and repress its activity in the presence of glutamine (Platt et al., 1996; Andrianopoulos et al., 1998; Lamb 2003 et al., Lamb et al., 2004; Wong et al., 2007). We have shown that these interactions are also important in nitrogen metabolite repression of arginine catabolism genes.





Trichophyton rubrum gene expression kinetics during growth on keratin as the sole nutrient source.

Rodrigo Anselmo Cazzaniga, Silveira HCS, Marques MMC, Evangelista AF, Sanches PR, Passos GAS, Rossi A, Martinez-Rossi, NM

University of São Paulo

Trichophyton rubrum is a filamentous fungus that infects human skin and nails, being the most prevalent dermatophyte worldwide. During its growth on keratin a shift on the extracellular pH from acidic to alkaline occurs, which may be an efficient strategy for its successful infection and maintenance in the host. T. rubrum responds to environmental pH by derepressing nonspecific proteolytic enzymes and keratinases with optimal activity at acidic pH during the initial stages of infection, probably in response to the acidic pH of human skin. Although several factors contribute to its pathogenicity, successful infection depends on the adherence capacity of the infecting dermatophyte and its ability to sense and overcome the acidic pH of the skin, and usage of the molecules from the host tissue as nutrient source. In this work T. rubrum gene expression kinetics during keratin degradation was evaluated by cDNA microarrays to gain a better comprehension of the adaptive responses to molecules presented in the host microenvironment. During T. rubrum growth on keratin medium, we observed that germination of the conidia and hyphal formation were accompanied by a gradual increase in the extracellular pH, ranging values from pH 6.5 to pH 8.5. After hybridization of the microarrays, and SAM analysis, 124 genes were found to be differentially expressed throughout the cultivation times. In 24h of cultivation, the transcript enconding a glutamate carboxypeptidase 2 was up-regulated, which must be important for protease release contributing to the clivage of keratin. At 48h, in which the extracellular pH was 8.0, we observed an up-regulation of the genes encoding PalA and PalB proteins, showing the activation of the environmental pH sensing pathway during keratin utilization, suggesting its role during T. rubrum pathogenic process. The genes encountered here are involved in several cellular processes, and their regulation during keratin degradation and pH sensing may be important in the initial stages of dermatophyte infection or in its maintenance in the host tissue.





A novel CUCU modification of poly(A) tail signals the end of mRNA

Igor Morozov[1] Meriel G. Jones[1] Dave G. Spiller[1] Joseph Strauss[2] Mark X Caddick[1]

1School of Biological Sciences, The University of Liverpool, Crown Street, Liverpool L69 7ZB, UK
2Fungal Genetics and Genomics Unit, Austrian Institute of Technology and BOKU University Vienna, Muthgasse 18 A-1190 Vienna, Austria

In eukaryotes differential transcript degradation represents an integral component of gene regulation. Generally the limiting step in mRNA turnover is deadenylation, which triggers translational repression and 5’ decapping once the length of poly(A) tail reached about 15 A residues.  We have recently shown that in Aspergillus nidulans both the Caf1 and Ccr4 orthologues are functionally distinct deadenylases: Ccr4 is responsible for basal degradation while Caf1 is required for the regulated degradation of specific transcripts and the variation in Processing (P) body formation, which occurs in response to a wide range of stimuli. Disruption of the Ccr4-Caf1-Not complex leads to premature, poly(A) independent decapping.  We have shown that decapping is correlated with a novel transcript modification, the addition of a CUCU sequence.  This 3’ modification of mRNA occurs precisely at the point decapping is triggered.  The addition of the CUCU based sequence tag requires a nucleotidyltransferase CutA, the disruption of which significantly stabilises mRNA and blocks the formation of P-bodies.  Intriguingly, the key enzyme complex responsible for deadenylation and therefore degradation, Ccr4-Caf1-Not, also  protects mRNA from premature modification and deadenylation independent decapping.  We propose that 3’ modification of adenylated mRNA, which is likely to represent a common eukaryotic process, primes the transcript for dissociation from ribosomes, decapping and efficient degradation (1). 

1. Morozov IY, Jones MG, Razak AA, Rigden DJ and Caddick MX. CUCU modification of mRNA promotes decapping and transcript degradation in Aspergillus nidulans. Mol Cell Biol, 2010, V.30 (4) in press.




Agaricus bisporus as a model to study effects of chromosomes on complex traits

Wei Gao, Johan J.P Baars, Anton S.M. Sonnenberg

Wageningen UR

The button mushroom Agaricus bisporus has a typical life cycle in that after meiosis-II, most basidia form two spores and each spore receives non-sister nuclei. Only a small portion of the basidia form four spores, each receiving one haploid nucleus. There are indications that most of the present-day commercial hybrids of the button mushroom are derived from Horst U1 via fertile single spore cultures. We have isolated both parental nuclei from one of the “new” commercial lines and compared the genetic make-up with that of the parental lines of Horst U1 using 580 SNPs. Only three recombinations between homologous chromosomes were found and both were completely reciprocal. The redistribution of homologues over both nuclei was also completely reciprocal when compared to Horst U1. Both strains thus have an identical allelic constitution but show, nevertheless, clear phenotypic differences. It indicates that the redistribution of homologues has an influence on phenotypes and this offers an interesting tool for breeding.

SNP markers were also used for segregation analysis using homokaryotic single spore cultures of Horst U1. This particular set of offspring showed hardly any recombination (18 recombinants in 143 individuals). Since all homologues are segregated independently, this offers an opportunity to generate chromosome substitution lines within a limited number of generations.

Agaricus bisporus offers the opportunity to study the effect of individual chromosomes on complex traits either by chromosome substitution or redistribution of homologous chromosomes over the parental nuclei.


Genome sequence of Shiitake mushroom Lentinula edodes

Hoi Shan Kwan; AU, Chun Hang; WONG, Man Chun; QIN, Jing; KWOK, Iris Sze Wai; CHUM, Winnie Wing Yan; YIP, Pui Ying; WONG, Kin Sing; CHENG, Chi Keung

The Chinese University of Hong Kong

Understanding the genomics and functional genomics of a mushroom allows us to improve its cultivation and quality. Sequencing the genome provides a comprehensive understanding of the biology of the mushroom. We can also develop many molecular genetic markers for breeding and genetic manipulation. We can identify genes encoding various bioactive proteins and pathways leading to bioactive compounds. Our laboratory sequenced the genome of Lentinula edodes (Shiitake, Xianggu). The genome size is about 60 Mbases. We are annotating the genes and analyzing the metabolic pathways. In addition, we have been using a battery of molecular techniques to study Lentinula edodes development. We used RNA arbitrarily primed-PCR, SAGE, LongSAGE, EST sequencing and cDNA microarray to analyze genes differentially expressed along the developmental stages. We are learning more about the molecular biology and genetics of this important mushroom. We will also know more about mushroom fruiting body development through comparative genomics among the genome-sequenced mushrooms.





Mycoparasitic response of Trichoderma atroviride to host fungi of different phyla on the transcriptome level

Barbara Reithner, Enrique Ibarra-Laclette,Verenice Ramírez-Rodríguez, Alfredo Herrera-Estrella

Laboratorio Nacional de Genómica para la Biodiversidad, Cinvestav, Unidad Irapuato

Trichoderma spp. are capable to recognize and attack plant-pathogenic fungi of distinct phyla like Rhizoctonia solani (Basidiomycete), Botrytis cinerea, Fusarium graminearum (both Ascomycetes), Phytophthora spp, and Pythium spp (both Oomycetes). The scope of action involving recognition, secretion on cell wall lysing enzymes, secretion of secondary metabolites, formation of penetration structures, and lysis of the host fungus is commonly summarized as mycoparasitism. In the present work the transcriptome of different stages of interaction of T. atroviride IMI206040 with P. capsici and R. solani were sequenced using pyrosequencing by 454 Life Science Technologies. Due to the distinct cell wall composition of the host-fungi chosen, the aim was to obtain differences in gene expression depending on the presence or absence of chitin in the host cell wall and genes which may be involved in host specificity. A total number of almost 300.000 reads represented more than 2000 and up to 6500 different Trichoderma transcripts, depending on interaction stage and plant-pathogenic fungus. Statistical analysis revealed that out of 353 differentially expressed genes only 6 were common during mycoparasitism of T. atroviride with P. capsici or R. solani. One of them was identified as epl1, coding for an elucidating plant response-like protein in Trichoderma. The remaining 347 transcripts are currently analyzed due to their possible involvement in the host-specific response of Trichoderma.





High throughput sequencing of the A. nidulans transcriptome

Mark Caddick, Christopher Sibthorp, Huihai Wu, Prudence Wong, Neil Hall

University of Liverpool

Next generation sequencing is transforming the way in which we can analyse the transcriptome.  It potentially provides quantitative information which is superior to microarray data, whilst also providing information about differential use of promoters, splicing and transcript 3’ ends.  We are applying this approach to A. nidulans.  In the first instance we have specifically investigated transcript start site location, by sequencing mRNA 5’ ends.  This has provided genome wide data with over ~3500 transcript start sites being localised to within two nucleotide and over 7000 localised to within four nucleotides.  From this we have been able to investigate DNA motifs and features associated with transcription start sites.  We will present these data and current work focused on characterising the full transcriptome.  Our aim is to provide a community resource that will significantly improve genome annotation and greatly increase our understanding of its flexibility and regulation.





RNA-Seq analysis of aflatoxin gene expression in Aspergillus flavus

 Jiujiang Yu[1] Natalie Fedorova[2] Thomas E. Cleveland[1] Deepak Bhatnagar[1] William C. Nierman[2]

1USDA/ARS, Southern Regional Research Center, 2J. Craig Venter Institute

A. flavus is the major producer of aflatoxin, which is responsible for millions of dollars in losses in the world and for significant health issues in developing countries, and is the second leading cause of aspergillosis in immunocompromised individuals.  Sequencing of A. flavus NRRL3357 showed that its 36-Mb genome contains 13,488 genes including the aflatoxin gene cluster.  Here we describe our efforts to use the RNA-Seq technology to characterize the entire transcriptome of the species under conditions conducive to aflatoxin production.  To that end, we sequenced cDNA fragments obtained from Poly(A)-enriched total RNA samples extracted from fungal mycelium grown under 3 conditions: (i) PMS medium, 29 C, 24h, no toxin; (ii) GMS medium, 29 C, 24h, make toxin; and (iii) GMS medium, 37 C, 24h, no toxin.  Two cDNA libraries from each treatment were sequenced using the Illumina (SOLEXA) short-read technology.  Over 5 Million 100 nt reads were sequenced for each cDNA prep, which were combined to generate a powerful high resolution map of the A. flavus transcriptome.  In addition, we used the RPKM analysis to determine transcript abundance in the 3 mRNA samples.  The analysis detected expression in at least 50 % of the genes for each condition and contributed to our understanding of the genetic basis of the aflatoxin regulation.





Transcriptional analysis of the response to extracellular pH changes in Fusarium graminearum Pac1 mutants and effect on trichothecenes B accumulation

Jawad Merhej, Christian Barreau, Florence Ricahrd-Forget


Fusarium graminearum infects wheat and maize and produces type B trichothecenes. These mycotoxins cause serious problems when consumed via contaminated cereals. Tri genes, located in the “Tri cluster”, are responsible for the biosynthesis of trichothecenes B. In vitro, Tri genes of F. graminearum strain CBS 185.32 are expressed at day 3 with the toxin starting to accumulate one day later. Strikingly, the induction of Tri genes expression always seems concomitant with a sharp pH drop in the media. Acidic pH seems a determinant factor for induction, as neither the toxin nor the Tri genes are detectable at neutral pH. The pH regulation of gene expression in fungi is mediated by the Pac1 transcription factor involved in various secondary metabolites regulation. An Fg∆Pac1 deletion mutant and a strain expressing a constitutively active form (FgPac1C) were constructed in F. graminearum. Expression of this constitutive Pac1C factor strongly reduces expression of Tri genes and toxin accumulation at acidic pH. Unexpectedly, deletion of Pac1 does not induce toxin production at neutral pH. However, it causes an earlier Tri5 induction and toxin accumulation at acidic pH. In order to determine the interference with other Tri genes regulatory mechanisms, exploring general transcriptional response to pH variation for mutants and wild-type strains were also performed using microarrays. Preliminary results will be presented.





Molecular analysis of the role of the HOG MAP kinase gene in the response and adaption of the basidiomycete Heterobasidion annosum to stress

Raffaello Tommaso, Fred O. Asiegbu

University of Helsinki

The basidiomycete Heterobasidion annosum (Fr.) Bref. is a filamentous white rot fungus, considered to be the most economical important pathogen of conifer trees. Very little is known about molecular and biochemical aspects related to this fungus pathogenicity. The role of some signal transduction genes (MAP kinases) as well as their importance in the stress response and adaptation of the conifer pathogen was investigated. To date, no MAP kinases have been characterized in this white rot fungus H. annosum. The role of the stress related HOG MAP kinase which is thought to be involved in the fungal osmotic tolerance was studied under different osmotic stress conditions. In the bioinformatic analysis the HOG gene shows a typical MAP kinase domain with high level of similarity among basidiomycetes. Phylogenetic revealed the basidiomycete HOG genes group together in a clade quite separated from the ascomycetes. To assay for functional relevance of the gene during osmotic stress, total RNA was extracted and the expression level of the HOG transcript quantified by qPCR. In a parallel study, the full-length HOG gene was cloned and used for a functional complementation assay in the S. cerevisiae hog1∆ mutant strain. The result showed that the fungus displayed a decreased growth when exposed to an increased salt osmolarity conditions. Increased levels of the MAPK HOG gene transcript was observed in high stress conditions.  Complementation functional study with full length gene in the yeast hog1∆ mutant strain is ongoing and the results will be presented. Taken together these results show the putative role of the HOG gene in the basidiomycete Heterobasidion annosum and its importance in the capacity of the fungus to overcome osmolarity stress conditions in the natural environment.




Dynamic and functional interactions between the components of the trimeric velvet complex in the filamentous fungus Aspergillus nidulans

Oezlem Sarikaya Bayram[1] Oezguer Bayram[1] Oliver Valerius[1] Jennifer Gerke[1] Stefan Irniger[1] Kap-Hoon Han[2] Gerhard H. Braus[1]

1Georg-August-University, Institute of Microbiology & Genetics
2Pharmaceutical Engineering, Woosuk University, Korea

We have recently discovered the trimerinc velvet complex which is comprised of the light-dependent regulator VeA, Velvet-like protein VelB, and master regulator of secondary metabolism LaeA. The velvet complex coordinates development and secondary metabolism upon light signal in Aspergillus nidulans. VeA protein serves as a light-dependent bridging function between VelB and LaeA proteins. We are currently analysing the functional as well as physical relationships between the components of the velvet complex. First data suggest that there might be some subcomplexes regulating development. We have new insights into the function of the complex. The current state of the ongoing studies will be presented.





Up-regulation of ABC transporter genes stimulated by azole drugs dependent on the transcription activator AtrR in Aspergillus oryzae

Ayumi Ohba, Takahiro Shintani, Katsuya Gomi

Graduate School of Agricultural Science, Tohoku university, Japan

During the long-term usage of the azole drug for antifungal therapy in aspergillosis, emergence of azole resistant isolates of Aspergillus fumigatus has been recently documented. One of the possible mechanisms of azole resistance is the up-regulation of genes encoding drug efflux pumps, mainly belonging to ABC transporters. However, the mechanism that regulates ABC transporter gene expression has not been elucidated in filamentous fungi including Aspergillus species. In Aspergillus oryzae, until now we have shown that overexpression of a transcription regulator gene atrR positively regulates expression of ABC transporter genes (atrA, atrF, and atrG) leading to azole drug resistance. In this study, expression of atrR and the three ABC transporter genes was induced by azole drug added in the wild-type strain. Furthermore, addition of azole drug significantly enhanced the expression level of the ABC transporter genes in an overexpression strain of atrR, although equal amount of the atrR transcript was accumulated in the strain irrespective of addition of azole drug. In contrast, in an atrR deletion mutant, no expression of the above mentioned ABC transporter genes was observed regardless of the presence of azole. In Saccharomyces cerevisiae and Candida glabrata, xenobiotic substrates such as azole drugs have recently been shown to directly bind and activate the transcription regulator Pdr1p via a nuclear-receptor-like pathway. Also in A. oryzae, AtrR protein may be activated by azole drugs through a similar mechanism to accelerate the gene expression of ABC transporter genes to survive in such an environment.





Complementation experiments in histone deacetylase-deficient Aspergillus strains

Divyavaradhi VARADARAJAN, Ingo Bauer, Martin Tribus, Peter Loidl, Stefan Graessle

Division of Molecular Biology, Biocenter, Innsbruck Medical University, Fritz Pregl Strasse 3, A-6020 Innsbruck, Austria

Modifications on the N-terminal tails of core histones highly impact the regulation of many genes in eukaryotic organisms. In filamentous fungi, however, only little is known about the enzymes that modify histones. Nevertheless, it has become increasingly evident that histone acetyltransferases and histone deacetylases (HDACs), which are responsible for a balanced acetylation status of the histone tails, are crucial for the regulation of genes involved in fungal pathogenicity, stress response, production of antibiotics or mycotoxins, and resistance against antifungal drugs. Our work focuses on RpdA, a class 1 HDAC of the model organism Aspergillus nidulans. We recently could demonstrate that RpdA is essential for growth and development of the fungus. Now we investigate the ability of complementation of class 1 HDACs from different filamentous fungi, yeasts and human in an Aspergillus strain with depleted RpdA activity. The RpdA orthologs of filamentous fungi like A. fumigatus or Cochliobolus carbonum were able to complement RpdA minus strains, whereas orthologs of yeasts (RPD3/Clr6) or human (HDAC1/HDAC2) were not. These results confirm that RpdA-type enzymes of filamentous fungi comprise distinct motifs that are essential for fungal growth and development. These characteristic features and their significance turn RpdA-type enzymes into promising targets for (fungal specific) HDAC inhibitors (HDACi) with impact on the vitality of these organisms. Several HDACi were recently approved (or are under evaluation) as therapeutic and chemo-preventive agents against cancer, neurodegenerative disorders and transplantation intolerance. In the future, these molecules may also represent attractive canditates for the development of novel antifungal agents, or may have potential as compounds used in combination with drugs administered in classical antifungal therapies. 





Investigating the regulation of glia expression in Aspergillus fumigatus

Taylor Schoberle, Gregory S May

UT MD Anderson Cancer Center

Gliotoxin, a member of the epipolythiodioxopiperazine (ETP) class of toxins, is important to virulence in certain host models.  Most genes involved in gliotoxin production and transport are located on a gene cluster, which is co-transcribed.  Deletion of an essential gene within the gliotoxin biosynthetic pathway, gliP, led to a lack of gliotoxin production within A. fumigatus, as well as a significant reduction in virulence in a steroid treated host.  Reduced virulence was a result of the presence of neutrophils within the host, as other labs using neutropenic mouse models did not see this trend.  In microarray studies, gliA, the gliotoxin efflux pump, is induced over 30-fold in the presence of neutrophils.  Growth of A. fumigatus in medium containing sodium nitrate also significantly induces expression of gliA.  Although many studies have been done to elucidate the effects of gliotoxin on host cells, little is known about the expression of the gliotoxin cluster.  To identify cis-acting regulatory elements in the gliA promoter, we examined expression from promoter deletion mutants fused to lacZ.  Several positive and negative regulatory elements were identified that altered expression in a nitrogen source dependent manner.  Using a gliA promoter lacZ fusion reporter construct, we screened for activators of gliA expression and identified candidate plasmids that activate the reporter.  We are conducting additional experiments to investigate this regulation.  Elucidating the genes that are responsible for the regulation of gliA will lead to a greater understanding of gliotoxin synthesis and transport, which is important to the pathogenesis of A. fumigatus.




Genome of Schizophyllum commune yields insight into regulation of mushroom formation

Robin A. Ohm, Jan F. de Jong, Luis G. Lugones, Han A.B. Wösten

Department of Microbiology and Kluyver Centre for Genomics of Industrial Fermentations


Genome Of Schizophyllum Commune Yields Insight Into Regulation Of Mushroom Formation
The wood degrading fungus Schizophyllum commune is a model system for mushroom development. The recently sequenced 38.5 Mb genome contains 13,210 genes. We performed whole genome expression analysis using the sequencing-based technique MPSS (Massively Parallel Signature Sequencing) on 4 developmental stages: monokaryon, stage I aggregates, stage II primordia and mushrooms. These data yield insight into the process of mushroom formation in high detail. For example, compared to other stages of development protein production is up-regulated in stage I, gene regulation is up-regulated in stage II and metabolism is down-regulated in mushrooms. Interestingly, 43% of the genes were shown to be also expressed in anti-sense direction, suggesting a role for anti-sense gene regulation during development.

We identified 471 transcription factor genes in the genome, of which 27 were evolutionary restricted to mushroom forming fungi. Moreover, about one third of all transcription factor genes were differentially regulated during development. Eleven transcription factor genes were deleted using recently developed and efficient gene deletion techniques. Inactivation of reg1, reg2 or reg3 resulted in colonies with only vegetative mycelium. Development stopped in stage I after inactivation of reg4, whereas inactivation of reg5, reg6 or reg7 resulted in more but smaller mushrooms than wild-type. These are the first strains with a targeted gene inactivation that show an effect on mushroom formation.

Taken together, we conclude that the genome sequence and genetic amenability of S. commune strongly contribute to our understanding of mushroom formation.





A novel motif in fungal Class 1 Histone deacetylases is essential for growth and development of Aspergillus

Ingo Bauer, Martin Tribus, Johannes Galehr, Birgit Faber, Divyavaradhi Varadarajan, Gerald Brosch, Stefan Graessle

Biocenter – Division of Molecular Biology, Innsbruck Medical University

ct" class="mceAdvanced">In eukaryotic organisms DNA is compacted into an elaborate structure called chromatin, thus enabling regulation of transcription by controlling the accessibility of the genetic information for transcription factors. Among the key players involved in the regulation of chromatin structure are histone acetyltransferases and histone deacetylases (HDACs) —enzymes establishing distinct acetylation patterns in the N- terminal tails of core histones. In filamentous fungi only little is known about the biological functions of these enzymes; nevertheless recent studies have shown that class 2 HDACs affect the regulation of genes involved in stress response and secondary metabolite production. Here we report that depletion of RpdA, a class 1 HDAC of Aspergillus nidulans, leads to a drastic reduction of growth and sporulation. Functional studies revealed that a short C-terminal motif unique for RpdA-type proteins of filamentous fungi is required for catalytic activity and consequently cannot be deleted without affecting the viability of A. nidulans. Thus, the C-terminal extension of RpdA-type proteins represents a promising target for fungal specific HDAC-inhibitors that might have potential as new antifungal compounds with clinical and agricultural applications.





On the presence of N6-methyl adenine in fungal genomic DNA

Miguel Corral González[1], David Lara Martínez[1], Arturo Pérez Eslava[2], Enrique A. Iturriaga[1]

1 Área de Genética, Departamento de Microbiología y Genética, Facultad de Biología, Universidad de Salamanca.

2 Centro Hispano-Luso de Investigaciones Agrarias (CIALE), Universidad de Salamanca


The presence of modified bases in the DNA of almost every organism analysed is known for more than fifty years now. While m5C (5-methyl-citosine) methylation has been shown and broadly studied in bacteria, fungi, other lower eukaryotes, plants and animals, the investigation on DNA m6A (6-methyl-adenine) methylation in eukaryotes has been relatively ignored, probably by the thinking of it being absent, irrelevant, or difficult to accomplish. The idea that m6A has a modest (or not at all), role in the regulation of eukaryotic genomic structure, organisation, and regulation contrasts with what it happens in bacteria, in which more than one important cellular and “epigenetic” mechanisms of regulation are mediated by m6A methylation.

Anyhow, several lower and higher (human, rat and plant) eukaryotes have been investigated in relation to their m6A presence and content, with some interesting results. A gap in these studies is the fungal kingdom, which probably could give mixed (and probably relevant), information on what happened from prokaryotes to eukaryotes in this matter. Our first approach to answer this question was based on m6A sensitive digestion with restriction enzymes, and in silico analyses of DNA from more then ten fully-sequenced fungi.

High-molecular weight DNA was obtained from the fungi to be analysed and digested with DpnI, an enzyme that cuts the sequence GA↓TC only when the A in this sequence is methylated. What we have found is that several groups of fungi have in fact an m6A DNA-methylation system in their genomes. Why? How? What for? These are today questions to be resolved.

These preliminary results only show what happens at the GATC sequence. So, we also analysed in silico the fungal genomes looking for N6-methyl adenine transferases. In some of them, which do not show any significant digestion with DpnI, we have been able to find sequences more than similar to m6A DNA-methyl-transferases.

The data obtained in these two kind of experiments give us new ideas to find out the presence, importance, and possible function of m6A DNA-methylation in fungi.



Mutualism versus pathogenesis: fungal endophyte friend or foe?

Linda Johnson, Stuart Card, Wayne Simpson, Anar Khan, Anouck de Bonth, Christine Voisey, Richard Johnson

AgResearch Limited, Grasslands Research Centre, Palmerston North, New Zealand

We are investigating the molecular mechanisms behind how a mutualistic symbiotic endophyte in its natural host grass, Elymus spp. (tribe Triticeae) has become pathogenic on Triticum spp. To unravel the switch from mutualism to pathogenesis, we have applied a mycological approach along with the latest transcriptomic technology. We obtained a low (5-10%) systemic infection rate in a New Zealand wheat cultivar with resulting plants becoming severely stunted and eventually dying. To specifically perform a high-throughput analysis of fungal differential gene expression, the new generation sequencing method, SOLiDTM is being utilised to compare systemic infections of endophyte with Elymus spp. (mutualistic interaction) versus Triticum spp. (pathogenic interaction). The Affymetrix 61k Wheat GeneChip has been employed to focus on wheat differentially expressed genes in mock-infected versus endophyte-infected wheat plants at various stages of infection. Initial wheat GeneChip experiments have shown that the low systemic infection rate is a consequence of activation of plant defence responses, along with perturbations in hormonal homeostasis. In conjunction, we have observed meristem re-organisation resulting in trapping of fungal hyphae so that colonisation of the auxiliary buds is impossible.



Transcriptome sequencing and comparative transcriptome analysis of the scleroglucan producer Sclerotium rolfsii

Jochen Schmid1,3 Dirk Müller-Hagen3 Thomas Bekel2 Laura Funk3 Ulf Stahl3 Volker Sieber1 Vera Meyer3, 4

1Chemistry of Biogenic Resources, Straubing Centre of Science, Technische Universität München, Straubing, Germany, 2Computational Genomics, Center for Biotechnology (CeBiTec), Bielefeld University, Bielefeld, Germany,3Department of Microbiology and Genetics, Berlin University of Technology, Berlin, Germany, 4Leiden University, Institute of Biology, Molecular Microbiology and Biotechnology, Leiden, The Netherlands

The plant pathogenic basidiomycete Sclerotium rolfsii produces the industrially exploited exopolysaccharide scleroglucan, a polymer that consists of β-(1→3) linked glucose with a β-(1→6) glycosyl branch on every third unit. Although the physicochemical properties of scleroglucan are well understood, almost nothing is known about the genetics of scleroglucan biosynthesis. Similarly, the biosynthetic pathway of oxalate, the main by-product during scleroglucan production, has not been elucidated yet. In order to provide a basis for genetic and metabolic engineering approaches, we studied scleroglucan and oxalate biosynthesis in S. rolfsii using different transcriptomic approaches.

Two S. rolfsii transcriptomes obtained from scleroglucan-producing and scleroglucan-nonproducing conditions were pooled and sequenced using the 454 pyrosequencing technique yielding ~ 350,000 reads. These could be assembled into 21,937 contigs and 171,833 singletons, for which 6,951 had significant matches in public protein data bases. Sequence data were used to obtain first insights into the genomics of scleroglucan and oxalate production and to predict putative proteins involved in the synthesis of both metabolites. Using comparative transcriptomics, namely Agilent microarray hybridization and suppression subtractive hybridization, we identified ~ 800 unigenes which are differently expressed under scleroglucan-producing and non-producing conditions. From these, candidate genes were identified which could represent potential leads for targeted modification of the S. rolfsii metabolism for increased scleroglucan yields.

The results provide for the first time genomic and transcriptomic data about S. rolfsii and demonstrate the power and usefulness of combined transcriptome sequencing and comparative microarray analysis. The data obtained allowed us to predict the biosynthetic pathways of scleroglucan and oxalate synthesis and to identify important genes putatively involved in determining scleroglucan yields. Moreover, our data establish the first sequence database for S. rolfsii, which allows research into other biological processes of S. rolfsii, such as host-pathogen interaction.


The black truffle of perigord responds to cold stress with an extensive reprogramming of its transcriptional activity

Elisa Zampieri[3] Raffaella Balestrini[1] Annegret Kohler[2] Simona Abbà[3] Francis Martin[2] Paola Bonfante[4]

1Istituto per la Protezione delle Piante del CNR, Sezione di Torino,Torino, Italy;
2UMR INRA-UHP ‘Interaction Arbres/ Micro-Organismes’, Centre INRA de Nancy, Champenoux, France.
3Dipartimento di Biologia Vegetale dell’Università di Torino, Torino, Italy
4Dipartimento di Biologia Vegetale dell’Università di Torino, Torino, Italy; Istituto per la Protezione delle Piante del CNR, Sezione di Torino,Torino, Italy; 2Istituto per la Protezione delle Piante del CNR, Sezione di Torino,Torino, Italy; 2Istitu


Free-living fungi often encounter different kinds of environmental stresses, including changes in temperature, osmolarity, pH, humidity, availability of O2 and nutrients, exposure to toxins, UV or heavy metals, as well as competition with other organisms. To understand the cell adaptation and the survivor in non-ideal conditions, a better comprehension of many basic events is required. Tuber melanosporum can be subjected to different stress conditions, considering its life cycle. In this work, the genome sequence of the ectomycorrhizal ascomycete Tuber melanosporum was analysed with the aim to identify and characterize genes involved in environmental stress response. As a second step whole genome arrays were used to verify the transcriptional profiling in the presence of a cold shock (4°C for 7 days). In a whole genome microarray (7496 genes/probe), 423 genes resulted significantly differentially expressed (> 2.5 fold; p-value < 0.05) in stressed mycelia compared to the control ones. After 4°C exposure for 7 days the number of up-regulated genes was 187; the down-regulated genes were 236. The 50-60% of the up- or down-regulated transcripts had no KOG classification and were clustered as unclassified proteins, which represent the most abundant category both in up- and down-regulated genes. A gene subset, concerning a range of biological functions, was chosen to validate the microarray experiment using qRT-PCR. Sixteen out of 22 considered genes confirmed the array data. At our knowledge this is the first work, which considers the global gene expression profiling in a filamentous fungus under cold stress condition.





Screening and sequence analysis of zhd101 (zearalenone lactonohydrolase) homologues in Trichoderma/Clonostachys sp.

Delfina Popiel, Grzegorz Koczyk, Lidia Błaszczyk, Jerzy Chełkowski

Institute of Plant Genetics, Polish Academy of Sciences, Poland


Zearalenones are economically important group of Fusarium sp. mycotoxins, exhibiting estrogenic activity and chemical strucure consisting of a resorcinol moiety fused to a 14-membered macrocyclic lactone. These compounds are converted into a far less estrogenic product by incubation with Clonostachys rosea IFO 7063 expressing zhd101 zearalenone lactonohydrolase gene. In the present study we described screening of Trichoderma/Clonostachys combined collection for new strains with functional lactonohydrolase homologues. In the screened samples, we observed degradation reactions in 10 of 79 total Trichoderma sp. and Clonostachys sp. isolates and have been able to determine new lactonohydrolase homologue sequences with average sequence identity of 90%.




Light-dependent gene regulation in Aspergillus nidulans is mediated through binding of a phytochrome white-collar light regulator complex

Julio Rodriguez-Romero, Maren Hedtke, Reinhard Fischer

Department of Microbiology. Karlsruhe Institute of Technolgy (KIT), Karlsruhe, Germany

Light serves as one important environmental signal to regulate development and metabolism in many fungi. The light response has been studied to great detail in Neurospora crassa (1) and A. nidulans. A. nidulans develops mainly asexually in the light and mainly sexually in the dark. We have discovered phytochrome (FphA) as a red-light sensor (3), and found that the blue-light receptor system proteins LreA (WC-1) and LreB (WC-2) along with VeA form a light-regulator complex (LRC)(2). We propose a functional relationship and a cross-talk between both photoreceptors.

In order to identify light-regulated genes, we used a two-colour microarray system. After 30 minutes of illumination about 260 genes (approx. 2.5% of the whole genome) were differentially regulated. 209 genes were up- and 51 down-regulated. Some of those genes display homology to other photo-inducible genes identified previously in N. crassa like ccg-1, con-10, con-6, etc. Among the light-inducible genes were also transcription factors probably implicated in secondary metabolism regulation and genes encoding enzymes of the secondary metabolism. Many genes encode proteins involved in stress responses, and a large group represents uncharacterized genes.

The regulation of conJcon-10 and ccgAccg-1 has been studied in detail. Differential expression of conJ and ccgA was confirmed by real time PCR. Using chromatin IP (ChIP), we found that the LRC binds directly to the promoters of conJ and ccgA. We do have evidence that not only the WC homologues are able to bind DNA but also phytochrome. Phytochrome-DNA interaction occurs probably through the response regulator domain at the carboxy terminus of FphA.

1. C. H. Chen, et al., EMBO J 28, 1029-42 (2009).
2. J. Purschwitz, et al., Mol Genet Genomics  (2008).
3. A. Blumenstein, et al., Curr Biol 15, 1833-8 (2005).


Transcriptional response is a limiting factor in cellulase overproduction by Trichoderma reesei

Thomas Portnoy1,3,+, Antoine Margeot1,+, Verena Seidl2, Stéphane Le Crom3, Fadhel Ben Chaabane1, Bernhard Seiboth2, Christian P. Kubicek2


1 IFP, Département Biotechnologie, 1-4 Avenue de Bois-Préau, 92852 Rueil-Malmaison Cedex, France

2Research Area Gene Technology and Applied Biochemistry, Institute of Chemical Engineering, Technische Universität Wien, Getreidemarkt 9/166, A-1060 Vienna, Austria

3 Institute of Biology of the École Normale Supérieure, CNRS UMR8197, INSERM U1024, 46 rue d’Ulm, 75230 Paris Cedex 05, France


+ Both authors equally contributed to this work.


Due to its capacity to produce large amounts of cellulases, the tropical ascomycete Trichoderma reesei is increasingly being investigated in second generation biofuel production from lignocellulosic biomass. The induction mechanisms of T. reesei cellulases have recently been described in some details, but the regulation of the genes involved in their transcription has not been studied thoroughly. Our work reports the regulation of expression of the two activator genes xyr1 and ace2, and the corepressor gene ace1 during induction of cellulase biosynthesis by the inducer lactose in a low producing strain T. reesei QM 9414. We show that all three genes are induced by lactose. Xyr1 is also induced by D-galactose, but this induction was independent of lactose metabolism. Moreover, xyr1 and ace1, but not ace2, were carbon catabolite repressed. For xyr1, this repression operated both on the basal and also the induced expression level, while ace1 was mostly affected at the basal level of expression. Significant differences in these regulatory patterns were observed in the hyperproducer strain T. reesei CL847. This suggests that a strongly elevated basal transcription level of xyr1 and reduced upregulation of ace1 by lactose may have been important for generating the hyperproducer strain, and are thus major control elements of cellulase production.





Functions for the Magnaporthe oryzae Flb3p and Flb4p transcription factors in the regulation of conidiation

Matheis, S., Thines, E., Scheps, D. Andresen, K. and Foster A.J.

Institut für Biotechnologie und Wirkstoff-Forschung, Germany


The M. oryzae genes FLB3 and FLB4, orthologues of the A. nidulans regulators of conidiation FlbC and FlbD respectively were deleted. Analysis of resultant mutants demonstrated that Flb4p is essential for spore formation and that strains lacking this gene have a ‘fluffy’ colony morphology due to an inability to complete conidiophore formation. Meanwhile Flb3p is required for normal levels of aerial mycelium formation. Using microarray analysis we identified genes dependent on both transcription factors. This analysis revealed that the transcription of several genes encoding proteins previously implicated in sporulation in Magnaporthe or in other filamentous fungi are affected by FLB3 and/or FLB4 deletion. Additionally the microarray analysis revealed Flb3p may effectively metabolically reprogramme the cell by repressing transcription of genes encoding biosynthetic enzymes and inducing transcription of genes encoding catabolic enzymes. From seven genes whose transcription is controlled by one or both of these transcription factors we identified the Flb4p dependent gene CON11 as being required for normal levels of sporulation in M. oryzae. CON11-deleted mutants also exhibited a reduced growth rate and virulence.


Constructed Aspergillus niger gene co-expression networks relate to biological processes

Robert A. van den Berg1, Machtelt Braaksma2, Douwe van der Veen3, Mariet J. van der Werf2, Peter J. Punt2, John van der Oost3, Leo H. de Graaff3.

1 SymBioSys, Katholieke Universiteit Leuven, Leuven, Belgium

2 TNO Quality of Life, Zeist, The Netherlands

3 Laboratory of Microbiology, Wageningen University, Wageningen, The Netherlands


The fungus Aspergillus niger has been studied in considerable detail with respect to various industrial applications. Although its central metabolic pathways are relatively well understood, the mechanisms that control the adaptation of its metabolism are understood poorly. In this study, clustering of coexpressed genes has been performed on the basis of DNA microarray data sets from two experimental approaches. In one approach, low amounts of inducer caused a relatively mild perturbation, while in the other approach the imposed environmental conditions including carbon source starvation caused severe perturbed stress. Evolutionary conserved genes were used to construct gene co-expression networks for both the individual and combined data sets.


Comparative analysis revealed the existence of modules, some of which are present in all three networks. In addition, experimental condition-specific modules were identified. Module-derived consensus expression profiles enabled the integration of all protein-coding A. niger genes to the coexpression analysis, including hypothetical and poorly conserved genes. Conserved sequence motifs were detected in the upstream region of genes that cluster in some modules, e.g., the binding site for the amino acid metabolism-related transcription factor CpcA as well as for the fatty acid metabolism related transcription factors, FarA and FarB. Moreover, not previously described putative transcription factor binding sites were discovered for two modules: the motif 5'-CGACAA is overrepresented in the module containing genes encoding cytosolic ribosomal proteins, while the motif 5'-GGCCGCG is overrepresented in genes related to 'gene expression', such as RNA helicases and  translation initiation factors.




Aspergillus niger: Mapping fungal specific zinc-finger transcription factors to gene co-expression networks

Benjamin M Nitsche1, Vera Meyer1, Arthur FJ Ram2

1Leiden Universitiy, Institute of Biology, Section of Molecular Microbiology and Biotechnology, Sylviusweg 72, 2333 BE Leiden, The Netherlands

2Kluyver Centre for Genomics of Industrial Fermentation, Sylviusweg 72, 2333 BE Leiden, The Netherlands


Aspergillus niger is a saprophytic filamentous fungus with a long history in industrial production of organic acids such as citric and gluconic acid as well as various enzymes like glycoamylases and pectinases. Furthermore, A. niger is an important multi-cellular model organism to study for example the establishment of cell polarity or protein secretion and it is used for the discovery of new anti-fungal drugs. A. niger can easily be genetically modified and cultivated under defined growth conditions in bioreactors. DSM published its 33.9 million base pair genome in February 2007 and an Affymetrix DNA microarray platform with more than 14.000 annotated open reading frames (ORFs) and genetic elements was designed. Among the predicted ORFs, over 300 fungal specific zinc-finger transcription factors (TFs) have been annotated. The function of most of these TFs remains unknown.

In the current study we, have built gene co-expression networks from a dataset of about 100 Affymetrix microarrays covering more than 30 different growth conditions including those which induce stress related to secretion, maintenance of polarity, cell wall integrity, carbon-source utilization and starvation. Exemplarily for a wide range of applications, we show mapping of putative TFs with unknown functions to these networks. Allocation of TFs to functionally enriched gene clusters can serve as an indication for their regulatory role and thereby give valuable leads for further experimental studies.



The flbF gene encoding a putative C2H2-type transcription factor is involved in the expression of sterigmatocystin genes and asexual development in Aspergillus nidulans

Yong Jin Kim, Yeong Man Yu, Sang Eun An, Sun-Ho Kim and Pil Jae Maeng

Department of Microbiology & Molecular Biology, College of Biological Sciences and Biotechnology, Chungnam National University, Daejeon, Republic of Korea


Asexual reproduction cycle is a common mode of reproduction for variety of filamentous fungi. The asexual reproductive cycle in Aspergillus nidulans can be divided into two distinct phases : vegetative growth and development. In spite of the extensive efforts and prominent progress in addressing the functions of the genes involved in the fungal differentiation, large numbers of the genes responsible for the developmental process in A. nidulans remain to be elucidated. Thus we performed transcriptome profiling during the entire process from spore germination to asexual differentiation using the 70-base-oligomer microarrays and ANURR. The array revealed stage-dependent expression of distinct genes set. The most significantly regulated genes (p<0.05) were grouped in five and six clusters based on their expression profiles during vegetative growth and asexual differentiation, respectively. More than 51 genes encoding putative transcription factors were found to be included in the stage-specific clusters. To figure out the function of the genes for the putative transcription factors, we are performing construction of knock-out mutants of the genes.


Here, we report the functional analysis of the flbF gene which was expected to encode a potential transcription factor with a C2H2 zinc finger DNA binding motif, bipartite nuclear localization signal, and glutamine rich region. Deletion of flbF resulted in delay of asexual reproduction and partial fluffy phenotype, also accumulation of brown pigment on media. From these observations, we propose that flbF is a putative regulator for initiation of asexual reproduction and secondary metabolism.


This work was supported by grant from Korea Research Foundation




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