ECFG 9 Poster Session 3

Fungal Biotechnology, Hyphal Growth and Morphogenesis, Evolutionary and Population Biology


Identification and phytotoxical properties of fungi genus Trichoderma from ancient lands and antagonistic activity by Trichoderma koningii against Fusarium oxysporum in experiences in vitro

Hector Alejandro Cabrera Fuentes, Farida Alimova, Rezeda Tukhbatova, Elina Rafailova

Kazan State University, Kazan, Tatarstan, Russian Federation

Different species of the genus Trichoderma are employed in different areas of human activity. We acquired more than 400 stems of fungi from the genus Trichoderma, from burial grounds dating 800-600 BC, on the territory of the Republic of Tatarstan, Russia. The objective of this study was to look for stems, perspective for the manufacture of biological products and evaluate Trichoderma species as potential biocontrol agents to reduce the impact of the Fusarium oxysporum. In order to reach this goal we identified, classified and systematized allocated stems with the use of morphological and molecular-genetic methods and investigated their physiological-biochemical properties. Descriptions of the appearance of colonies and their morphology have been recorded in a before-stated work developed by identifiers Samuels, Chaverri, Bissett. Molecular-genetic research was carried out with use of method PCR (RAPD-analysis). On the basis of the received results investigated stems have been related to species: Tr. longibrachiatum, Tr. koningii, Tr. harzianum, Tr. asperillum, Tr. citrinoviride, Tr. atroviride, Tr. oblongisporum, Tr. spirale. Classification of these stems was done with the help of an earlier specially developed program. Also have been investigated antagonistic activities and phytotoxical properties, enzymatic activity of the complex exohydrolase (xylanase, protease and cellulase activity) of allocated stems.Morphological analysis revealed the 2 types of colony (II and VI). Their kinetic parameters were also determined: slow growth isolates and fast growth isolates were calculated as 0.01-0.07 mm/h and 0.1 mm/h. We have also investigated antagonistic activities. As a result of our work, selected stems of Trichoderma koningii with a positive influence on the growth of rye and wheat with prospects in biotechnology have been identified.



Weak acid preservative resistance in Aspergillus niger

Malcolm Stratford, Andrew Plumridge, David Archer

University of Nottingham, Nottingham, United Kingdom

Sorbic acid is a weak acid, added to foods and beverages as a preservative. Certain moulds and yeasts are highly resistant to sorbic acid and therefore contaminate food products and cause spoilage. The mechanism of resistance employed by Aspergillus niger, a common food contaminant, to sorbic acid is mediated by PadA1. PadA1 (phenylacrylic acid decarboxylase, encoded by padA1) has been shown to decarboxylate and, thereby detoxify sorbic acid producing the volatile compound 1,3-pentadine. A. niger D padA1 mutants have been shown to be unable to decarboxylate sorbic acid and as a result are more sensitive to this preservative compared to the wild-type. The genome of A. niger contains two other genes (padA2 and padA3) encoding putative phenylacrylic acid decarboxylases (PadA2 and PadA3, respectively). Gene disruption studies showed that the absence of padA2 or padA3 did not increase the sensitivity of these strains to sorbic acid, nor did they reduce the ability to decarboxylate this preservative. Putative PadA proteins have been identified in a number of moulds whose genome sequences have been published and their ability to decarboxylate sorbic acid has been confirmed. Certain yeasts are also able to decarboxylate sorbic acid but to a much lesser extent than moulds. Pad1p from Saccharomyces cerevisiae, a common spoilage yeast, has been shown to facilitate the decarboxylation of sorbic acid, producing 1,3-pentadiene. The growth rate of pad1 mutants was retarded during sorbic acid-stress but the minimium inhibitory concentration of the preservative required to cause complete growth arrest was identical for the mutant and the wild-type strains. Thus, Pad1p-mediated detoxification of sorbic acid, is a genuine resistance mechanism in moulds but not in yeast.


Splicing activity of an artificially engineered mini-intein from Penicillium chrysogenum

Skander Elleuche1, Constantin Pelikan1, Nicole Nolting2, Kristin Döring2, Stefanie Pöggeler2

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

Inteins are selfish genetic elements that excise themselves from the host protein during post translational processing. Thereby they religate the host protein with a peptide bond. In contrast to mini-inteins, large inteins also contain an endonuclease domain that is important in intein propagation, called homing. The first PRP8 intein was found within the prp8 gene of the basidiomycete Cryptococcus neoformans. The PRP8 protein occupies a central position within the catalytic core of the spliceosome.

In our study, we selected several members of the ascomycetous genera Penicillium and Eupenicillium and investigated them for the presence of PRP8 inteins. We were able to identify mini-inteins in P. expansum, P. vulpinum, P. chrysogenum, E. baarnense and E. crustaceum but not in P. thomii, E. terrenum and E. meridianum. All of the PRP8 inteins identified are mini-inteins and undergo autocatalytic protein splicing when heterologously expressed in a model host protein in E. coli.

In a second approach, we tested the importance of conserved domains inside the P. chrysogenum intein and were able to produce split intein versions capable of protein splicing in trans, when theintein sequences encoding the N- and C-terminal moiety of the intein are transcribed as polycistronic mRNAs. The insertion of split sites in two different domains restored splicing activity. By deleting non-conserved amino acids at two different sites Furthermore, we produced the smallest functional eukaryotic intein known so far. This indicates that the mini-intein of P. chrysogenum might have a more robust structure than do many other inteins and that it might be of practical importance to engineer this intein for various applications.



A single amino acid change in the L-arabitol dehydrogenase (LadA) from Aspergillus niger broadens its substrate specificity and increases its activity

Blanca Trejo-Aguilar1, Lucy Rutten2, Cecile Ribot3, Manuela Pail1, Han A.B. Wösten1, Ronald P. de Vries1

1Microbiology, Utrecht University, Utrecht, Netherlands, 2Crystal and Structural Chemistry, Utrecht University, Utrecht, Netherlands, 3CNRS-UCB-INSA-Bayer CropScience, Lyon, France

L-arabitol dehydrogenase and xylitol dehydrogenase are part of the fungal pentose catabolic pathway. They are related to sorbitol dehydrogenases from higher eukaryotes.

Phylogenetic analysis of L-arabitol dehydrogenases (LAD), xylitol dehydrogenases (XDH) and sorbitol dehydrogenases (SDH) shows that XDHs are more similar to SDHs than LADs. By modeling A. niger LadA and XdhA on the structure of human SDH, we identified two residues in XdhA that were hypothesized to be important for the activity on D-sorbitol. Mutagenesis of one of these residues in LadA resulted in a nearly complete enzyme inactivation for reasons unknown. The other mutation resulted in increased affinity and activity for L-arabitol and sorbitol, demonstrating that this residue is not only important for activity on sorbitol, but also improves the general activity of the enzyme.



An improved & markerfreeÔ expression system for Aspergillus niger enzyme production

Ilse de Lange, Alrik Los, Janny Bakhuis, Noël van Peij, Herman Pel

DSM Food Specialties, Delft, Netherlands

For many decades, Aspergillus niger has been safely used in the commercial production of various food enzymes, such as glucose oxidase, pectinase, alpha-amylase and glucoamylase.

Genetically modified industrial strains of A. niger have been used as a host to over-express food and feed enzymes such as phytase and xylanase. Traditionally, the genes encoding these enzymes are integrated in a random fashion in the genome of the host organism.

New technological developments have enabled us to construct a new generation of A. niger CBS513-88 lineage strains according to a "design and build" concept, in which the genes of interest are targeted integrated in the host genome. A recombinant strain was derived from a glucoamylase production strain, in which the glucoamylase (glaA) genes were deleted, creating so-called ΔglaA loci. Each of these loci was designed in such a way that it individually can be detected by gel electrophoresis. Therefore, targeted integration of the gene of interest in all seven loci can be monitored, allowing selection of strains with multiple integrated expression units of the gene of interest. The homologous integration frequency was improved by disruption of the A. niger hdfA and/or hdfB genes, homologues of the human KU70 and KU80, that are essential for non-homologous end joining of DNA in double strand break repair. Deletion of hdf genes greatly reduces the frequency of non-homologous integration of transforming DNA fragments leading to dramatically improved gene targeting. The hdf knockout has contributed to a highly controlled strain construction process, with both increased targeting frequency and increased co-transformation percentage.


Automated screening for high-copy Aspergillus niger production strains

Ilse de Lange, Alrik Los, Janny Bakhuis, Noël van Peij, Herman Pel

DSM Food Specialties, P.O. Box 1, 2600MA Delft, Netherlands

The filamentous fungus Aspergillus niger has extensively been used for production of recombinant proteins. We have modified A. niger CBS513-88 lineage strains in order to enable highly controlled production strain construction, in which genes of interest are integrated at defined high expression loci. A recombinant strain was derived from a glucoamylase production strain, harboring 7 glucoamylase (glaA) genes. The glaA genes were deleted stepwise; the resulting Δ glaA-loci serve as tool for recombinant production strain construction according to the ‘design and build’ concept (see poster De Lange et al). Following integration of genes of interest at the target Δ glaA-locus, the gene copy number is subsequently amplified via gene conversion of the different Δ glaA-loci. This process can easily be monitored via PCR-screening and gel-electrophoresis. The throughput of this screening has significantly been increased by implementation of the LabChip 90 system (Caliper Life Sciences), thereby accelerating production strain construction.



Schizosaccharomyces pombe as host for high throughput screening of fungal proteins

Paulien Neefe, Lydia Dankmeyer, Pim van der Kley, Daan Meyer, Pauline Teunissen, Piet van Solingen

Genencor. A Danisco Division., Leiden, Netherlands

High Throughput Screening of libraries of enzymes from filamentous fungi cannot easily be carried out in the fungal host strains themselves for a number of reasons:

A limited number of stable transformants per transformation event

Instability of transformants because of multi nuclear character of mycelial protoplasts

Non-uniform growth at microtiterplate-scale leading to variable production yields

Difficulties in automated handling, e.g. inoculation and transfer of fungal colonies and cultures

High background of endogenous extracellular enzymes, co-expressed together with a protein of interest.

Other screening systems such as E.coli, Bacillus or Streptomyces are often not suitable either, as they are mainly limited to the expression of prokaryotic genes.

Therefore, the most obvious screening host is an unicellular yeast, for example Saccharomyces cerevisiae or Schizosaccharomyces pombe. One of the major advantages of S. pombe is high transformation efficiency and the absence of interfering background proteins. Additionally, it is easy to handle robotically.

It is rather unpredictable if enzymes from filamentous fungi can be expressed well in yeast host strains. S.pombe has proven to be an excellent host for cloning and expression of heterologous genes such as endoglucanase2 and cellobiohydrolase2 of Trichoderma reesei.

This poster describes the successful application of S.pombe as the screening host for HTS of protein engineered enzymes from the filamentous fungus T.reesei.


Intracellular protein production in Trichoderma reesei with the help of zera peptide technology

Ann Westerholm-Parvinen1, Kevin O'Connor2, Miriam Bastida2, Blanca Llompart2, Markku Saloheimo2

1VTT Technical Research Centre, Espoo, Finland, 2ERA Biotech, Barcelona, Spain

Several species of filamentous fungi, such as certain Aspergillus species and Trichoderma reesei, are utilised as cell factories for protein production, largely due to the superb secretion capacity of these organisms. However, for many heterologous proteins the production yields have been modest, due to various reasons like inefficient folding and transport along the secretory pathway and susceptibility to host proteases. In order to avoid these problems, we have developed an intracellular protein production system in T. reesei that utilises the Zera peptide technology commercialised by ERA Biotech. In this technology the foreign protein is fused with the Zera peptide, a fragment of the maize gamma-zein storage protein. In maize seeds this protein is localised into protein bodies that are derived from the ER membrane and work as protein storage organelles. When fused with a foreign protein of interest, the Zera peptide has been shown to recruit the foreign protein efficiently into protein bodies in plants, but also in mammalian and insect cells. The protein bodies have a high density and thus they are easy to isolate from the cells for product recovery.

We have tested whether Zera technology functions in filamentous fungi by expressing the eGFP fluorescent protein as a fusion with the Zera peptide in T. reesei from the strong cbh1 promoter. The transformants carrying the Zera-eGFP expression construct were observed to contain strongly fluorescent bodies in their cytoplasm, whereas a control construct expressing the eGFP alone with an ER localisation signal produced much lower and diffuse fluorescence. Western analysis with GFP antibodies showed that a greatly higher level of Zera-eGFP than eGFP alone was produced in the cells. The transformant cells were disrupted and the cell content was fractionated by gradient ultracentrifugation. Good yields of relatively pure Zera-eGFP protein could be isolated from the cells with the centrifugation as the single purification step. Our study demonstrates that the Zera peptide is fully functional in formation of protein bodies in T. reesei, and that the Zera technology can be successfully used in protein production in this fungus.



Improvement of the biocontrol of Heterobasidion root rot with antagonist fungus

Phlebiopsis gigantea

Hui Sun1, Kari Korhonen2, Risto Kasanen1, Jarkko Hantula2, Fred Asiegbu1

1Department of Forest Ecology, University of Helsinki, Helsinki, Finland, 2Finnish Forest Institute, Vantaa, Finland

Root and butt rot caused by Heterobasidion species is one of the most destructive diseases of conifers in the northern temperate regions of the world. Phlebiopsis gigantea (Fr.) Jül. is currently used as biocontrol agent. However, little is known about the physiological and molecular basis of interaction between P. gigantea and Heterobasidion spp.The hypothesis of our study is: the traits of P. gigantea related to biocontrol could be targets of breeding and could be used in developing the biocontrol abilities of P. gigantea. The aims of this investigation are: a) To study the importance of different traits (spore production ability, antagonism, and growth rate) related to biocontrol. b) To test whether the biocontrol abilities of P. gigantea can be enhanced with breeding of these traits. C) To study the differentially gene expression of P. gigantea to select candidate genes contributing to biocontrol.

64 Phlebiopsis gigantea isolates from different countries and hosts (Pinus sylvestris and Picea abies), including commercial Rotstop F and Rotstop S, were tested and P. gigantea strains with ‘good’ biocontrol ability were selected for homokaryon collection and breeding to produce heterokaryon progeny. The biocontrol ability of progeny was tested. Selected P. gigantea strains with ‘good’ and ‘bad’ biocontrol ability were studied for differentially gene expression using Macroarray method and quantitative reverse transcript Real-time PCR.

Preliminary result indicated that there was significant variation in each tested trait between different P. gigantea strains, but no correlation between three traits. P. gigantea progeny showed higher ability against H. annosum in three traits than that in parental P. gigantea. Moreover, there was significant difference between P. gigantea progeny and parental in mean efficacy with 88% and 78%, respectively. This indicated the biocontrol ability of P. gigantea against H. annosum could be enhanced through P. gigantea breeding. Future perspectives will be selecting candidate genes contributing to biocontrol by Macroarray and verifying candidate genes by quantitative RT-RT PCR.



Fast and efficient single step construction of replacement vectors by USER friendly cloning, for targeted gene replacement in fungi

Rasmus J.N. Frandsen, Jens A. Andersson, Henriette Giese

Institute for Ecology, Faculty of Life Sciences, University of Copenhagen, Frederiksberg C, Denmark

Functional genetics in filamentous fungi have always been dependent on the isolation or construction of mutant strains. The genome sequencing of over 40 fungi genomes has increased the need for faster and more efficient methods to construct targeted replacement and overexpression mutants. To accommodate this we have developed a new vector system that allows single step construction of vectors for targeted gene replacement, thereby cutting vector construction time from ten to only three days and removing half of the required work load. The vector system is dependent on the Uracil-Specific Excision Reagent cloning technology (USER FriendlyTM), which in its commercial version offers high efficient directional cloning of a single PCR amplicon. However, our research shows that USER friendlyTM cloning technology also can be used for the simultaneous directional cloning of several PCR amplicons and vector fragments, with a cloning efficiency of 85 %, thus allowing single-step construction of replacement vectors. In addition to the increased speed and reduced workload, the single-step construction strategy also offers greater freedom of operation with respect to the placing of the homologous recombination flanks, as it is independent of restriction enzymes.

The new vector system includes vectors for targeted gene replacement (pRF-HU2), promoter exchange (pRF-HU2E), ectopic overexpression (pRF-HUE) and general purpose cloning (pRF-HU). All are compatible with both protoplast and Agrobacterium tumefaciens mediated transformation technologies. The system has been used to analyse putative polyketide gene clusters in Fusarium graminearum.



Enhancement of the Trichoderma reesei expression system

Shingo Miyauchi1, Helena Nevalainen1, Peter Bergquist1, Junior Te'o2

1Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney / NSW, Australia, 2Applimex Systems Pty. Ltd, Sydney / NSW, Australia

Recent advances in fungal genomics and proteomics have facilitated further development of Trichoderma reesei as a production host. In most studies, the strongly inducible promoter of the cbh1 gene in T. reesei has been utilised for the expression of various gene products (1, 2) In addition to CBH1, T. reesei also produces significant amounts of other cellulases such as CBH2 and EG2, as shown by transcriptomic and proteomic studies (3). Therefore, the genes encoding these enzymes provide an alternative source of strong gene promoters.

We have constructed four expression vectors that feature either the cbh2 or egl2 promoter. The first pair of vectors, named pEG2/pCBH2-sigpro, feature a promoter sequence, secretion signal and pro-region derived from either the cbh2 or egl2 gene, whereas pEG2/pCBH2-cbmlin vectors contain a promoter sequence, secretion signal, pro-region, carbohydrate-binding module and a linker region. The xynB gene encoding xylanase B from the thermophilic bacterium Dictyoglomus thermophilum has been inserted into the vectors for heterologous expression. The codon usage of the xynB gene has been modified for the expression in T. reesei (4).

Expression of xynB from the pEG2-cbmlin and pCBH2-sigpro vectors was found to be greater than that using the EG2-sigpro and CBH2-cbmlin constructions based on zymogram analysis and liquid enzyme activity assays. In this presentation, we discuss the capability of the novel promoters to drive protein expression as influenced by the structural differences in the expression cassette motifs.

1 Ward et al (2004). Appl. Environ.Microbiol. 70: 2567–2576, 2 Punt et al (2002). Trends Biotechnol. 20: 200–206, 3 Foreman et al (2003). J. Biol. Chem. 278: 31988-31997, 4 Te’o et al (2000). FEMS Microbiol Lett 190: 13-19



Selection of microbial production hosts for real-life feedstock utilization

Karl Rumbold, Hugo van Buijsen, Johan van Groenestijn, Peter Punt, Mariët van der Werf

TNO Quality of Life, Zeist, Netherlands

Bulk chemicals produced by microbial production processes are becoming more competitive in comparison to those traditionally synthesized from crude oil. It is expected that products representing 10% of total sales in chemical industry by 2010 will originate from microbial production processes. Improvements in fermentation technology and down stream processing, as well as economies of scale, have significantly improved the competitiveness of bulk chemicals produced by biotechnology. Now, cost of goods, in particular substrate costs, has become a major parameter.

The use of lignocellulosic hydrolysates is gaining increasing attention as low-cost substrate feedstocks. These feedstocks contain, however, mixtures of different fermentable sugars, high salt concentrations as well as specific compounds that strongly inhibit microbial growth, depending on the feedstock pretreatment process applied. We have performed a benchmark study with six industrial microorganisms (i.e. Escherichia coli, Corynebacterium glutamicum, Saccharomyces cerevisiae, Pichia stipitis, Trichoderma reesei and Aspergillus niger) to evaluate their fermentation performance, versatility in feedstock use, and product producing capacities using ‘real-life’ feedstocks as the substrate. In this study, the performance of these microorganisms with respect to their substrate utilization potential and sensitivity towards inhibitors as well as extreme process conditions was evaluated. Moreover, these microorganisms were evaluated for their ability to ferment hydrolysates of "real-life" feedstocks, i.e. corn stover, wheat straw, sugar cane bagasse, willow wood and waste glycerol from biodiesel production.

Combining the data from the benchmarking test with existing knowledge, we were able to rank these industrial production hosts based on their suitability to utilize ‘real-life’ feedstocks for the production of bulk chemicals. A. niger and P. stipitis were found to perform the best. In general, all hydrolysates of "real-life" feedstocks were good substrates for fermentation.

The benchmark test developed in this study is a generic and can be applied to test the performance of other (industrial) production hosts and their ability to utilize different feedstocks as well their potential to produce specific chemicals.



EroA and oxidative protein folding in Aspergillus niger

Anna Harvey1, Andrew Plumridge1, Mick Ward2, Huaming Wang2, David Archer1

1University of Nottingham, Nottingham, United Kingdom, 2Genencor Intl, Palo Alto, California, United States

Disulphide bonds are recognised as an integral feature within the structure of many folded proteins. In the yeast model organism Saccharomyces cerevisiae, the formation of protein disulphide bonds occurs via an enzyme pathway involving the oxidoreductase Ero1p, and the foldase Pdi1p, which are encoded by the ERO1 and PDI1 genes respectively. In this pathway, oxidising equivalents are transferred between Ero1p, Pdi1p, and substrate proteins requiring disulphide bonds. In the filamentous fungus, Aspergillus niger, the presence of a PDI1 homologue, PDIA, has been demonstrated. PDIA has been shown to catalyse the refolding of denatured and reduced RNase A, and to complement Pdi1p function in a pdi1 strain of S. cerevisiae. We describe an A. niger homologue of ERO1, eroA, and show that it is transcriptionally up-regulated in the presence of agents that cause endoplasmic reticulum stress and induction of the unfolded protein response. We demonstrate that eroA is likely to be essential for viability, and are further characterising the functions through down-regulation, over-expression, and complementation studies.



Development of a new fungal RNA-silencing system in Acremonium chrysogenum and Penicillium chrysogenum

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

In this work we report the development and validation of a new RNA interference vector (pJL43-RNAi) containing a double stranded RNA expression cassette for gene silencing in the filamentous fungi Acremonium chrysogenum and Penicillium chrysogenum. Targeted gene disruption (knockout) in both microorganisms is too complicated, tedious and time-consuming due to the ectopic integration of exogenous DNA. As substitute, we propose a new system based in the RNA silencing machinery. We have tested this vector with two genes of the -lactam pathway; the pcbC gene for P. chrysogenum and the cefEF for A. chrysogenum. Antibiotic production and mRNA transcripts levels analysis showed gene knockdown in both pcbC and cefEF genes. This new RNAi vector is a new tool for exploring gene function into genomes of P. chrysogenum and A. chrysogenum.


Characterisation of the Trichoderma reesei proteasome

Liisa Kautto1, Liisa Kautto2, Jasmine Grinyer1, Peter Bergquist1, Peter Bergquist2, Peter Bergquist3, V.S.Junior Teo1, V.S.Junior Teo2, V.S.Junior Teo3, K.M.Helena Nevalainen1, K.M.Helena Nevalainen2

1Department of Chemistry and Biomolecular Sciences, Macquarie University, 2109 NSW Sydney, Australia, 2Macquarie University Biotechnology Research Institute, Macquarie University, 2109 NSW Sydney, Australia, 3Department of Molecular Medicine and Pathology, University of Auckland Medical School, Auckland, New Zealand

The broad aim of this work is to advance understanding of the role of the proteasome in cellular protein quality control in Trichoderma reesei, which provides an easy and cost-effective factory for protein production. Some proteins are synthesised with high yields, but others, especially foreign proteins suffer from low yields. This effect could be partly because of misfolding of the foreign proteins in the fungal host resulting in their targeting to the fungal proteasome for degradation.

We have isolated and characterised the 26S proteasome from T. reesei. From the 2D reference map, 12 of the 14 20S proteasome subunits, five of 19S subunits and several interactive proteins that were co-purified with the 26S proteasome have been identified.

We are now exploring the function of the proteasome in ERADication of misfolded proteins from the secretory pathway by using mutant forms of the main cellulase of T. reesei, CBHI. We are examining the CBHI mutant proteins by confocal microscopy so as to trace their secretion and presumed degradation by the proteasome of T. reesei. Also, we are in the process of analysing the transcriptional response of the cells to the production of mutant CBHI proteins by the application of genome-wide CombiMatrix oligonucleotide array analysis.



Artificial generation of large chromosomal deletions in Aspergillus oryzae and Aspergillus sojae by using ku70 deficiency

Tadashi Takahashi, Feng Jie Jin, Yukio Senou, Yasuji Koyama

Noda Institute for Scientific Research, Noda City, Chiba, Japan

By using the combination of a ku70-deficient strain and bidirectional markers of the koji molds Aspergillus oryzae and Aspergillus sojae, we established a fundamental technology for efficiently generating large chromosomal deletions. Previously, we have reported that the genomic deletion of a 60-kb region including the aflatoxin gene cluster was efficiently carried out in an A. sojae strain (1). In this study, we investigated the limitations of the possible deletion range in one deletion cycle and the efficiency of 2 deletion methods, the loop-out (resolution-type recombination) and direct (replacement-type recombination) deletions. The large genomic deletions generated by a loop-out deletion mechanism enable us to make multiple deletions by using marker recycling. As a result, over 300-kb regions of chromosome 3 including the aflatoxin biosynthetic gene cluster and those of chromosome 4 have been completely deleted from the koji molds. Moreover, no additional sequence remained in the resultant deletion strains, which makes this technique suitable for the breeding of food-grade microorganisms.

In addition, we recently discovered that the limitation of deletion range in the replacement-type recombination method was highly expanded in ku70 mutant strains. Thus far, replacement-type recombination between 2 homologous regions separated by a large nonhomologous sequence has been practically impossible due to the low frequency of recombination in wild-type koji mold strains. In this study, we have reported the successful deletion of regions larger than 200 kb in chromosome 3, including the aflatoxin gene cluster, and 470 kb in chromosome 8 with high efficiency by using ku-deficient strains of the koji molds A. sojae and A. oryzae. The results obtained in this study indicate that the ku70 mutation elevated not only the conventional gene-targeting frequency but also the frequency of recombination between 2 distant sites.

The technology described here is applicable for genomic engineering and molecular breeding of industrially used filamentous fungi by artificially generating large-scale genomic deletions and marker-free strains.

1 Takahashi et al. (2006) In: Abstracts of ECFG8, PP272.



A trial of the minimization of the chromosome 7 by large-scale chromosomal deletion in A. oryzae RIB40

Feng Jie Jin1, Toshi Furukido1, Masafumi Tokuoka1, Masahiro Ogawa1, Tadashi Takahashi1, Masayuki Machida2, Yasuji Koyama1

1Noda institute for scientific research, Noda city,Chiba pref., Japan, 2National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki, Japan

Chromosome engineering is a powerful technology with effectively allowing any chromosomal rearrangement to create desired strains by artificially introducing gene disruption and foreign gene insertion. In particular, large-scale DNA rearrangement is becoming an increasingly important method in this field. We have previously developed a method to efficiently construct large-scale deletion mutants by using a ku70-disrupted strain (1). In this study, using this method with pyrG-mediated transformation system, we attempted to construct a mutant with the minimum set of genes in chromosome 7, which is the smallest chromosome (2.93 Mb) in A. oryzae RIB40 strain. In this way, our final goal is to breed industrially favored strains by appropriately altering regulation of metabolite production.

We first constructed series of large-scale deletion mutants (16~150 kb) with a particular focus on the non-syntenic regions, which were characterized by the comparison of A. oryzae genome with those of other Aspergilli (2). Based on these results, we extrapolated that approximately a quarter of chromosome 7, at least 740 kb sequence, was non-essential and could be deleted. We then sequentially deleted these confirmed non-essential regions using the A. oryzae ku70 disruption strain. Until now, we have successfully constructed a mutant lacking as large as 685 kb genome sequence (23.4% of chromosome 7) by multiple deletions. In addition, we also observed and compared the growth phenotype of obtained large-scale deletion mutants and the parental strain. In one of deletion mutants, the number of formed conidia increased more than 1.6-fold compared with that of the parental strain. More interestingly, the number of metabolites of this mutant was markedly decreased. These results suggested that the large-scale deletion method not only could be useful to breed industrial strains, but also opened up a new avenue of research to analyze global genetic function on a large scale in chromosomes.

1 Takahashi et al. (2006) In: Abstracts of ECFG8, P. 272, 2 Tamano et al. (2008) Fungal Genet Biol. 45, 139-151


Extracellular beta-glucosidase activities in Zygomycetes

Csaba Vagvolgyi1, Miklos Tako1, Elvira Farkas1, Tamas Papp1, Judit Krisch2

1Department of Microbiology, Faculty of Sciences and Informatics, University of Szeged, Szeged, Hungary, 2Department of Food Engineering, Faculty of Engineering, University of Szeged, Szeged, Hungary

Introduction: Beta-glucosidases constitute a group of well characterized, biologically important enzymes that catalyze the transfer of the glycosyl group between oxygen nucleophiles. They play important roles in biology, including degradation of cellulose biomass by fungi and bacteria, degradation of glycolipids in mammalian lysosomes, and the cleavage of glycosylated flavonoids in plants. Several Zygomycetes fungi have biotechnological significance as producers of extracellular enzymes, e.g. proteases and lipases. However, this fungal group is poorly characterized from the aspect of beta-glucosidases. The aim of this work was to find new producer strains potentially applicable in further basic and biotechnological studies.

Methods: Detection of the extracellular beta-glucosidase activity of 71 strains was performed in minimal medium containing cellobiose as the sole carbon source. Ten isolates from the good producers were chosen for solid state fermentation studies, in which wheat or oat bran were used as carbon source. Enzyme activities were assayed spectrophotometrically by using p-nitrophenyl beta-D-glucopyranoside; isoenzyme analysis was also carried out with the tested strains. Temperature and pH optimum of some purified enzymes were determined also.

Results: Enzyme activities of the tested strains were found to be highly variable. On the basis of the liquid culture fermentation, ten isolates representing different Gilbertella, Rhizomucor and Rhizopus species showed high extracellular beta-glucosidase activity. In each case, the highest activity was detected on the sixth culturing day. In the solid state fermentation assay, an R. miehei isolate gave the highest activity, however culture filtrate from the same isolate growth in liquid medium showed the lowest activity among the selected producers. Extracellular enzyme activities of the other strains were also stronger in solid state fermentation than in liquid culture. Enzymes with different temperature optimum and stability could be isolated.

This research was supported by ETT grants (214/2006; 261/2006) and the J. Bolyai Research Scholarship of the Hungarian Academy of Sciences.



Carotenoid content of different species belonging to the class Zygomycetes

Arpad Csernetics1, Tamas Papp1, Andras Szekeres2, Csaba Vagvolgyi1

1Department of Microbiology, Faculty of Sciences and Informatics, University of Szeged, Szeged, Hungary, 2Analytical Laboratory of Cereal Research Non-profit Company, Szeged, Hungary

Introduction: Three closely related Zygomycetes fungi, Phycomyces blakesleeanus, Blakeslea trispora and Mucor circinelloides have been traditionally involved in the studies on the fungal carotene biosynthesis. The main industrial carotenoid producer fungus is even now the B. trispora, where high production level has been achieved by the selection of mutants and the optimization of the growth conditions. On the basis of morphological observations, several other Zygomycetes seems to be promising producer, but their carotenoid content has not been analysed.

Methods: The main objective of this study was to reveal the carotenoid composition and to characterize the carotenoid production of different fungi belonging to the order Mucorales. Twenty isolates representing several species of the genera Mucor, Backusella, Rhizopus and Gilbertella were involved in the study. After cultivation under continuous light, carotenoid content of the strains was analysed by HPLC and TLC. The effects of different carbon sources and the growth temperature on the carotenoid production were also examined.

Results: Some Mucor species produced the valuable hydroxylated derivatives of beta-carotene, e.g. beta-cryptoxanthin and zeaxanthin, in considerable quantities. Some strains belonging to the species M. bainieri and M. hiemalis produced two fold more amount of carotenoids than M. circinelloides and the wild-type B. trispora, model organisms of the carotenogenic studies. G. persicaria produced significant amount of pigments only if it was plated as a mixture of two strains with the opposite mating types.

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


A defensin-like protein from Aspergillus nidulans is a potent inhibitor of fungal growth

Andrea Eigentler1, István Pócsi2, Florentine Marx1

1Biocenter, Division of Molecular Biology, Innsbruck Medical University, Innsbruck, Austria, 2Department of Microbial Biotechnology and Cell Biology, Faculty of Science, University of Debrecen, Debrecen, Hungary

Antimicrobial proteins have great potential for the development of new therapeutic strategies. In the genome of Aspergillus nidulans we identified a small gene of 345 nt which we called defensin A (defA). The open reading frame of 225 nt encodes a preproprotein of 74 amino acids (aa). The mature protein has a molecular mass of 4 kDa and its primary sequence exhibits 51% identity and 83% homology with the aa sequence of the insect defensin AaDefA1 from the mosquito Aedes aegyptii. Northern blot analysis confirmed gene transcription and we found a strong induction of gene expression under stress conditions. By in silico analysis we identified four potential stress consensus sequences (STRE) in the promoter region of the defA gene, which correlates well with its expression pattern.

To study the putative antimicrobial activity of DefA we recombinantly expressed the protein by using the Pichia pastoris multicopy expression system (Invitrogen) to produce a high amount of protein for further analysis. The recombinant protein was purified from the supernatant of a 72 h culture by ion-exchange chromatography and the identity of the protein was confirmed by mass spectroscopy.

The purified protein showed potent growth inhibitory activity against filamentous fungi and yeasts as determined by growth inhibition assays. Further analyses of its species specificity, the MIC and its mode of action are currently in progress.

This work is in part supported by the D. Swarowski Forschungsfonds (FB2/06).



Prospecting of novel cold-active enzymes and their genes from Antarctic microfungi

Suja Mohammed1, Suja Mohammed2, V. S. Junior Te'o1, V. S. Junior Te'o2, Peter L. Bergquist1, Peter L. Bergquist2, Peter L. Bergquist3, K. M. Helena Nevalainen1,2

1Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, NSW, Australia, 2Applimex Systems Pty. Ltd., Sydney, NSW, Australia, 3Department of Molecular Medicine and Pathology, University of Auckland Medical School, Auckland, New Zealand

Cold-active enzymes have a high specific activity at low and moderate temperatures and are thermolabile (1). Their cold activity and thermolability makes them attractive as industrial catalysts to facilitate biotechnological processes at low temperatures and also as potential research tools. The aim of this study was to identify and isolate genes responsible for the synthesis of cold-active enzymes of interest from Antarctic microfungi. We have selected six Antarctic fungal strains to investigate the secretion of lipases, proteases and cellulases, which have major applications in food and detergent industry.

The initial study was carried out by plate assays with appropriate substrates for each enzyme. The fungal strains were grown in liquid culture and the enzyme activities in the culture supernatants were analyzed. Out of the six Antarctic strains, Geomyces pannorum and Phoma sp. were found to exhibit the highest activities for lipase and protease respectively, when compared to the other strains. These enzyme activities were confirmed by zymogram activity analysis. We have carried out modifications of the culture medium to improve enzyme production in the best strains. The pH and temperature profile of the enzyme activities were studied since these factors contribute to the use of enzymes for particular industrial purposes. Our results indicate the presence of a cold-active lipase and protease from the Antarctic strains Geomyces pannorum and Phoma sp. respectively.

1 Gerday, C., Aittaleb, M., Bentahir, M., Chessa, J., Claverie, P., Collins, T., D’Amico, S., Dumont, J., Garsoux, G., Georlette, D., Hoyoux, A., Lonhienne, T., Meuwis, M. and Feller, G. (2000). Trends Biotechnol. 18: 103-107.



Improved dominant selection markers for Coprinus cinereus: re-evaluation of hygromycin & phleomycin resistance vectors and a "new" carboxin resistance system

Sreedhar Kilaru, Catherine Collins, Amanda Hartley, Gary Foster, Andy Bailey

University of Bristol, Bristol, United Kingdom

Dominant selection systems are beneficial for transformation of many fungi. In this study, we used Coprinus cinereus as a model organism to develop vectors containing dominant selectable marker genes based on antibiotic selection. Plasmids previously used to establish hygromycin (hph) transformation systems of several basidiomycete species failed to give rise to hygromycin-resistant transformants of C. cinereus. Sequence analysis of these constructs showed that they had all been modified removing the two lysine residues adjacent to the N-terminal methionine. Replacement of the deleted 6 bp (AAA AAG) in the truncated hph gene led to generation of hygromycin-resistant transformants indicating the importance of these two residues in C. cinereus. Phleomycin-resistant (ble) transformants were also obtained but only with the intron-containing construct pblei004, not with intron-less construct pble004, showing that an intron is necessary for ble expression in C. cinereus. A carboxin (cbx) resistant allele of the C. cinereus sdi1 gene, encoding the iron-sulphur protein subunit of succinate dehydrogenase, was developed by introducing a suitable point mutation in the conserved histidine block. This modified gene was used to successfully confer carboxin resistance upon transformation of C. cinereus protoplasts. Based on the total number of transformants obtained, C. cinereus protoplasts were more efficiently transformed to carboxin resistance when compared to hygromycin and phleomycin resistance highlighting the potential use of this homologous marker gene. In order to investigate the expression of two different dominant selectable marker genes in a single transformation experiment, vectors pYES-hph-cbxgene and pYES-hph-icbxgene with dual marker genes were constructed with hph and sdiR cassettes in yeast through in vivo recombination. The unique restriction sites present in the sdiR and hph genes allow the replacement of one of the selectable marker genes with a gene of interest and consequently avoid the requirements for co-transformations. We replaced the sdiR gene in these vectors with the fluorescent marker gene Discosoma red (DsRed). Expression of DsRed was only observed in transformants containing vector with intron. These vectors should allow several transformations of same strain of C. cinereus and be of use in establishing transformation systems of other basidiomycete species.



Breeding of the hyper-producing strains for heterologous proteins by double proteinase disruption and mutagenesis in Aspergillus oryzae

Jun-Ichi Maruyama, Taisuke Watanabe, Takashi Nemoto, Katsuhiko Kitamoto

Department of Biotechnology, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan

Aspergillus oryzae has drawn much attention as one of the most excellent hosts for protein production due to its ability to secrete vast amounts of proteins and the safety guaranteed by the use in fermentative food industry. While the production level of homologous (fungal) proteins by A. oryzae can reach gram-per-liter scale, the production of heterologous (mammalian and plant) proteins yields only milligram-per-liter scale. Although hyper-producing strains are required for higher-level production of heterologous proteins, only a few attempts have been reported for breeding of an excellent production host in A. oryzae.

We previously reported that the double disruption of the proteinase genes (tppA and pepE) increased the production of human lysozyme (HLY) by 1.63-fold (15.6 mg/l to 25.4 mg/l).1) Moreover, by using the double proteinase disruptant, the production yield of bovine chymosin (CHY) was 65.1 mg/l, which was 1.93-fold increment from the control strain (33.8 mg/l). High productivity of the two heterologous proteins by the double disruptant raised the possibility that the strain showing a higher-level production of HLY might also produce other heterologous proteins in larger amounts. In order to test this hypothesis, hyper-producing mutants of HLY were screened from the double disruptant. For this purpose we developed an efficient screening system by employing halo assay with bacterial cells, the HLY substrate. Among 22 hyper-producing mutants obtained from ~80,000 colonies, the highest production level of HLY was 50.8 mg/l, which was 2.00-fold increment compared to that of parental strain (the double disruptant). Subsequently, the niaD-based plasmid for HLY production was cured from the mutants by positive selection using chlorate, which is metabolized into a cytotoxic compound by the nitrate assimilation pathway. The 8 strains obtained by the curing were named as AUT1~8 (A. oryzae hyper-producing strains bred in The University of Tokyo). The maximum production yield of CHY by the AUT strains was 107.9 mg/l, which was 1.66-fold increase from the double disruptant. In conclusion, we successfully increased heterologous protein production (3.26- and 3.19-folds for HLY and CHY, respectively) by double proteinase disruption and mutagenesis in A. oryzae. The AUT strains are expected as powerful hosts for higher-level production for many heterologous proteins.

1 Jin et al., Appl Microbiol Biotechnol. 2007; 76: 1059-1068.



Characterisation of novel oxalate decarboxylase from the white rot fungus Dichomitus squalens

Miia Mäkelä, Kristiina Hildén, Annele Hatakka, Taina Lundell

Department of Applied Chemistry and Microbiology, University of Helsinki, Helsinki, Finland

We have cloned a novel oxalate decarboxylase (OxDC) -encoding gene and partially characterised an OxDC protein from a basidiomycetous white rot fungus Dichomitus squalens. Certain bacteria and fungi produce OxDC (EC enzyme which catalyses the decarboxylation of oxalic acid into stoichiometric quantities of formic acid and carbon dioxide in a highly specific reaction. The regulation of organic acid production is an essential part of effective lignin degradation caused by white rot fungi which makes the specific oxalate decomposing enzymes, such as OxDC, important to study.

So far, only four sequences of OxDC-encoding genes from basidiomycetes are available: Flammulina (Collybia) velutipes, Flammulina sp., Phanerochaete chrysosporium, and Trametes versicolor (only cDNA). Recently, P. chrysosporium has been shown to produce the OxDC protein under growth conditions that promote wood degradation.

Because of their high specificity to oxalate, OxDCs have been used in clinical and food assays for the determination of oxalate. Transgenic crop plants expressing an oxdc-gene have been generated in order to increase the resistance of host plant to fungal infections. The use of OxDC for the degradation of oxalate deposits in breweries has been patented ( US4652452). The potential use of oxalate-degrading enzymes to prevent the formation of calcium oxalate deposits during the bleaching of pulp in paper making processes has also been noticed.

D. squalens produced oxalic acid during the growth in liquid cultures and on natural solid wood substrate. The fungus showed high intracellular OxDC activity after induction with oxalic acid. In order to find out the properties of D. squalens OxDC, the gene was cloned and the protein was partially characterised. The amino acid composition of the predicted Ds-OxDC has the highest pair wise identity of 56.9 % with P. chrysosporium OxDC. Two putative His-containing cupin motifs and four N-glycosylation sites are found in the translated Ds-OxDC aa-sequence. Ds-OxDC has a molecular weight of 72 kDa and in chromatofocusing it shows an isoelectric point of 4.2.



Transformation of Aspergillus oryzae RIB40 with the bleomycin acetyltransferase-encoding gene as a selection marker

Satoshi Suzuki1, Mari Fukuoka1, Hiroko Taketani1, Sawaki Tada1, Mayumi Matsushita1, Ken-Ichi Kusumoto1, Yutaka Kashiwagi1, Masanori Sugiyama2

1National Food Research Institute, Tsukuba, Ibaraki, Japan, 2Hiroshima University, Hiroshima, Hiroshima, Japan

Introduction: Aspergillus oryzae is one of the most important fungi in Japanese traditional fermentation industry. Since this microorganism also produces many-food processing enzymes, it is significant to establish a host-vector system for A. oryzae. In various filamentous fungi, the genes, which confer resistance to hygromycin B, aureobasidin, and G418, have been used as genetic markers for the gene manipulation. However, A. oryzae is resistant to these antibiotics. We notice that high concentration of bleomycin inhibits the growth of A. oryzae RIB40 which is known as a genome reading strain. We have recently found that some agents, such as tritonX100, increase the bleomycin-susceptibility of A. oryzae (data not shown). In the present study, we developed a transformation system for A. oryzae RIB40 by using bleomycin-resistance gene as a selective marker.

Methods: The codon of the bleomycin N-acetyltransferase gene from Streptomyces verticillus were optimized according to the codon usage of the fungus and inserted into the plasmid pPTRI under control of the histone H2B promoter. The resulting chimeric plasmid was introduced into the protoplasts from A. oryzae RIB40 by the polyethylene glycol method. The transformants were selected on the maltextract-peptone medium containing 27 microgram/ml of bleomycin and bleomycin potentiation agents. The integration of the plasmid into genomic DNA of transformants was confirmed by the colony PCR method using FTA card and the Southern blot analysis.

Results: We successfully selected the transformants, which exhibit bleomycin resistance, on the agar plate. The background outgrowth of non-transformants was clearly suppressed by bleomycin. The transformants were inoculated on the non-selective PDA plate and allowed to grow. The colony PCR analysis shows that the growing tip of colony inoculated on non-selective medium retains the DNA fragment carrying the bleomycin N-acetyltransferase gene.

Discussion: In the present study, we established the transformation system for A. oryzae by using the Streptomyces bleomycin N-acetyltransferase gene. We expect that the established system contribute to many research for A. oryzae.



Efficient production and partial characterization of one of metalloproteases, aspartyl aminopeptidase from Aspergillus oryzae

Ken-Ichi Kusumoto1, Mayumi Matsushita1, Ikuyo Furukawa1, Satoshi Suzuki1, Youhei Yamataga2, Yoshinao Koide3, Hiroki Ishida4, Michio Takeuchi5, Yutaka Kashiwagi1

1National Food Research Institute, Tsukuba, Ibaraki, Japan, 2Laboratory of Molecular Enzymology, Division of Life Science, Graduate School of Agricultural Science, Tohoku University, Sendai, Miyagi, Japan, 3Gifu R & D Center, Amano Enzyme Inc., Kagamihara, Gifu, Japan, 4Research Institute, Gekkeikan Sake Company Ltd., Fushimi-ku, Kyoto, Japan, 5Department of Agriscience and Bioscience, Tokyo University of Agriculture and Technology, Fuchu, Tokyo, Japan

Aspartyl aminopeptidase from Aspergillus oryzae has high substrate specificity, degrading only amino-terminal acidic amino acids from peptides, and may be suitable for processing physiological peptides. However, little is known about the biochemical properties of this enzyme and its efficient production in Aspergillus oryzae.

The gene encoding aspartyl aminopeptidase was overexpressed under a taka-amylase gene promoter, with His-tag linker in A. oryzae, during cultivation in a cobalt-containing medium. The enzyme was extracted from mycelia and purified with immobilized nickel ion absorption chromatography using a buffer containing cobalt ion and imidazole. The active fraction was further purified with gel filtration chromatography. The resultant, electrophoretically pure enzyme displayed a molecular mass of 520 kDa. This enzyme displayed high affinity to peptide substrate rather than synthetic substrates. Thus, recombinant A. oryzae aspartyl aminopeptidase was purified to homogeneity with an increased specific activity, when cultivated in a cobalt ion-rich medium. Moreover, the use of suitable metal ions in microbial cultivation and purification processes may help increase specific activity of other metalloproteases and their functional analysis.


Alternative oxidase is responsible for the oxidative defense in Aspergillus fumigatus

Taisa Magnani, Frederico Soriani, Anna Policarpo, Carlos Curti, Sergio Uyemura

Sao Paulo University, Ribeirão Preto/ São Paulo, Brazil

A. fumigatus is a human pathogenic fungus capable of inducing a range of disease states in immunocompromised patients. Fungal respiration exhibits peculiar features and involves an alternative oxidase pathway not yet fully elucidated. In this sense, we have silenced an aox gene in A. fumigatus (Afaox) by RNA interference. Inverted repeats of Afaox coding sequence were cloned in the pALB1 plasmid, which contain an alb1 gene cloned. In xylose medium, the Afaox/alb1 and alb1 transformants had the same phenotype, a green color, as the wild strain, indicating that the interference was not induced. However, in maltose medium the Afaox/alb1 transformant grew in white color indicating an entire silence and a Real-time PCR revealed a decrease of 95% in RNAm levels. It has been suggested that AOX may be important for fungal survival in macrophages, based on accumulating evidence for its antioxidant defense. ROS production was monitored using fluorescent probe CM-H2DCFDA. In Afaox/alb1 transformants, ROS production increased 12.5 and 2-fold when compared with wild-strain and alb1 transformants, respectively, suggesting that AOX can prevent ROS formation. The survival rate of silenced strain in macrophages was measured by MTT assay. Afaox, double silenced strain, revealed a highest survival when compared with the other strains. Conidia were incubated with different ROS donors and conidia viability was measured by flow cytometria with FUN-1 and PI uptakes.

These results indicate that the alternative oxidase has an important role in oxidative defense in Aspergillus fumigatus.

Supported by: FAPESP and CNPq



Overexpression of an E. coli phytase mutant in Trichoderma reesei and characterization of the enzyme

Dietrich Löbel, Tatiana Schwarz, Silvia Paladino, Anja Lingner, Gerhard Leiss, Oliver Schmidt, Astrid Pfeifer, Bruno Winter, Jari Vehmaanperä, Khanh Nguyen

AB Enzymes GmbH, Feldbergstrasse 78, 64293 Darmstadt, Germany

Phytases catalyze the sequential hydrolysis of phytic acid (IP6) to obtain less phosphory-lated myo-inositol derivatives plus inorganic phosphate. About 70% of phosphate in grain used as livestock feed exists in the form of phytic acid. However, monogastric animals such as pig, poultry and fish are unable to digest phytic acid efficiently, resulting in low phosphate availability. Phytases added to the diets of monogastric animals enhance the bioavailability of organic phosphate and reduce the need for inorganic phosphate supplementation.

Phytases belong to the family of histidine acid phosphatases and are found in several microorganisms and plants. E. coli phytase (EC is known to have a particularly high specific activity, paired with adequate gastric performance at low pH and strong pepsin resistance (Konietzny & Greiner, Int.J.Food Sci.Technol., 2002, 37:791). When expressed in Trichoderma reesei the E.coli phytase retains its favourable molecular characteristics and results in commercially feasible yields.

The aim of this study was to enhance E.coli phytase production through site-directed mutagenesis. The E. coli phytase gene used in this investigation is based on the amino acid sequence of acid phosphatase appA (Dassa et al., J.Bacteriol., 1990, 172(9):5497). The E.coli phytase encoding gene was designed and synthesized with the codon usage of T.reesei. The gene was placed under control of the T.reesei cbhI promoter and cbhI-terminator and the plasmid containing the WT-phytase sequence designated pKDa4. In another plasmid pKDa2 the amino acid Val200 (GTG) of the phytase gene was changed to Tyr200 (TAC).

The E.coli phytase product (spent medium) reached the highest activity after submerged fermentation in industrial medium for 6 days. The phytase mutant V200Y exhibited a 2.2-times higher expression level than the wild type.

The E.coli phytase produced in T. reesei is non-uniformly glycosylated and therefore present in 3 main bands in SDS-PAGE. Treatment of the phytase with PNGase F led to a shift in molecular weight towards a single band of 44.5 kDa. The pH dependence of the enzyme was investigated between pH 3.0 and pH 7.0 and the temperature activity profile of the purified enzyme was determined between 30°C and 70°C. The thermostability of the purified phytase was analyzed by differential scanning calorimetry (DSC).


Pcr-based early detection of Penicillium expansum in apple

Valentina Tolaini, Massimo Reverberi, Federico Punelli, Corrado Fanelli

Università La Sapienza, Roma, Italy

Penicillium expansum is one of the major post-harvest pathogens on apple fruit and its infection is associated with patulin production, a secondary metabolite with immunological, neurological and gastrointestinal toxic effects. Use of mouldy apples contaminated with P. expansum greatly increases the risk of patulin contamination in fruit juices. Early detection of this pathogen on apples is important for ensuring quality and safety of fruits and fruit juices. The objective of this study was to develop and optimize a PCR-based assay for rapid and specific detection of P. expansum in apple.

In order to discriminate P. expansum from other natural apple-contaminating fungi by PCR, specie-specific primers have been designed on the basis of the consensus conserved sequence of the Pepg1 gene of P. expansum, which encodes for the enzyme responsible of fruit tissue rot. Another set of primers was designed on the basis of the sequence of a PCR fragment of P. expansum obtained by amplification with M13 minisatellite primer. These primers were used for the amplification of DNA extracted by different fungi isolated from apples (cv Annurca and cv Golden) and grown in vitro on potato dextrose agar (PDA) and from apples contaminated and not contaminated with P. expansum.

The use of Pepg and Pem13 primers in optimal PCR conditions shows their ability to amplify a specific sequence of P. expansum DNA. None of the other species have been detected. These results indicate the specificity of this PCR system for P. expansum. Preliminary results obtained in contaminated apples confirm the specificity and sensitivity of the PCR-based assay.

Nucleic acid-based method as PCR could be used as a rapid tool for detecting P. expansum in apple. This assay can be a good alternative to the traditional diagnostic methods as cultural method.


Modified hydrolase profiles in Hypocrea jecorina by changing the expression of the general activator protein Xylanase regulator 1 (Xyr1)

Astrid R Stricker, Matthias G Steiger, Marion E Pucher, Robert L Mach

Vienna University of Technology, Vienna, Austria

The filamentous fungus Hypocrea jecorina (Trichoderma reesei) for a long time has been used for different important industrial applications in the areas food and feed, textile, as well as pulp and paper production. Its fundamental role in fermentation technologies remains unaltered, not only but in particular due to its high secretory capacity. Systematic strain improvement resulted in production of levels of extracellular protein of over 100 g/L. However, molecular approaches to change and/or improve the hydrolase cocktail composition remained rare. Here we present that the modification of the expression level of the major transcriptional activator Xyr1 significantly changes the expression profile of several genes coding for the hydrolase enzyme system of this fungus. Interestingly, an increase in the expression of xyr1 does not consequently increase the transcription of all hydrolytic enzyme-encoding genes subject to regulation by this factor, but changes the ratio of the respective mRNA levels. In addition, we can prove that a constitutive expression of xyr1 does not lead to the induction of the hydrolase expression, since the presence of an inducer molecule is strictly required. Finally, we provide evidence that a posttranslational modification, most probably a phosphorylation, is responsible for the conversion of Xyr1 into its active form.


PCR and spectral imaging based assays for the early detection of aflatoxigenic fungi on maize kernels

Antonella Del Fiore1, Silvia Serranti2, Alessandra Ricelli3, Massimo Reverberi2, Annadele Fabbri2, Giuseppe Bonifazi2, Corrado Fanelli2

1ENEA CR Casaccia, Roma, Italy, 2Univesità La Sapienza, Roma, Italy, 3CNR, Roma, Italy

Fungi are a real issue for the cereal industry. Some fungal species, such as Aspergillus flavus and A. parasiticus, can produce under suitable conditions, aflatoxins, secondary metabolites which are toxic for human and animals. Many methods have been utilized to measure fungal contamination on cereals. The aim of this work is to use a PCR based method and a spectral imaging based method to detect aflatoxigenic fungi on maize. In the experiments were used two aflatoxigenic strains, A. parasiticus NRRL 2999, A. flavus NRRL 3357, and one non aflatoxigenic strain of A. parasiticus NRRL 11096. An A. Niger strain and other strains of Aspergillus spp., Penicillium sp. and Fusarium sp. isolated from maize were also analyzed. Maize hybrids employed in food industry, were used in the PCR-based assay. Genomic DNA was extracted from pure fungal strains, not inoculated and inoculated maize kernels. A primer pair was designed on the coding portion of omt-1 gene which encodes for the enzyme O-methyltransferase II necessary for the last step of aflatoxin biosynthesis. The DNA extracted was used as template for PCR amplification with the primer pair selected. PCR amplification generate a fragment of 1254 bp length in A. flavus and A. parasiticus. Amplification operative conditions were optimized on pure fungal strains and confirmed on maize. DNA amplification was achieved only with DNA from fungal strains of A. parasiticus and A. flavus and from maize inoculated with A. flavus or A. parasiticus, never with DNA of other fungal strains. Specificity was confirmed with DNA extracted from different fungal strains. These results indicate that this PCR-based method could be able to discriminate maize kernels infected with A. flavus and A. parasiticus from un-infected ones; this method could be used like early detection system, for aflatoxigenic strains in maize. Several studies have tried non-destructive, spectral methods to detect fungal contamination on cereals. At this concern we present a method, based on the spectral imaging, to detect fungal contamination on maize. A desktop spectral imaging system, ImSpectorTM, V10 were used in the work; two aflatoxigenic species were used, A. parasiticus, and A. flavus. They are inoculated on maize and were imaged on day 7 of growth. Changes in reflectance of maize were observed during fungal growth. This approach could be a rapid method of detecting the aflatoxigenic fungi on cereals.



A molecular early detection method of Aspergillus carbonarius on grape and a novel analysis for Ochratoxin A in wine

Patrizia De Rossi1, Massimo Reverberi2, Alessandra Ricelli3, Domenico Caputo2, Giampiero De Cesare2, Augusto Nascetti2, Riccardo Scipinotti2, Corrado Fanelli2

1ENEA CR Casaccia, Roma, Italy, 2Università La Sapienza, Roma, Italy, 3CNR, Roma, Italy

Aspergillus carbonarius is an important ochratoxin A (OTA) producing fungus which is responsible for toxin contamination of grapes and wine. OTA is a secondary metabolite produced by fungi belonging to Aspergillus and Penicillium genera which has been shown to be nephrotoxic, nephrocarcinogenic, teratogenic and immunosuppressive.

The objective of this research was to investigate the presence of A. carbonarius in grapes and of OTA in wine.

Two PCR-based assays have been developed to detect the presence of A. carbonarius in grapes by designing specie-specific primers on the basis of Internal Transcribe Spacers of rDNA units (Ac-ITS) and of the polyketide synthases (Ac-PKS) sequences. These specific primers were used for A. carbonarius detection in grapes. For early detection of Ochratoxin A the performance of a system based on hydrogenated amorphous silicon photosensors was investigated. This device is based on the measurement of the photocurrent induced in a hydrogenated amorphous silicon (a-Si:H) photodiode by the fluorescence of the mycotoxin excited by a UV radiation.

The results of PCR using DNA extracted from various grape-contaminating fungi show that only A. carbonarius DNA was amplified with Ac-ITS and Ac-PKS primers. None of the other species gave a positive result with this PCR primers set used. Concerning OTA detection in wine the photocurrent measured by the sensor is due to the mycotoxin and the value is proportional to its quantity.

PCR-based methods that target DNA are considered a good alternative with respect traditional diagnostic methods for early detection because of their high specificity and sensitivity. This PCR analysis was also successfully employed to detect A. carbonarius in grape. Currently, the most of the methods available for the determination of OTA in wine are based on an extraction step, a clean-up passage and determination by High Performance Liquid Chromatografy (HPLC) with fluorescence detection. The sensor we used could contribute to early and rapid detection of potential presence of OTA in wine samples.


Production of full length antibody chains in Chrysosporium

Peter Jan Punt3, Margreet Heerikhuisen3, Eddy Motshagen3, Marian van Muijlwijk3, Cora van Zeijl3, Rich Burlingame2, Martijn Meens1, Jan Verdoes1

1Dyadic Nederland B.V, Wageningen, Netherlands, 2Dyadic Intl, Jupiter, Florida, United States, 3TNO Quality of Life, Zeist, Netherlands

Filamentous fungi are widely used for enzyme production for a wide variety of uses – for example food, feed, textile, paper and pulp, fuels and chemicals, detergents - due to the development of extremely productive strains and production processes. Levels in the range of 100 grams protein per liter have been reported, repeatedly. Therefore, these organisms have also been considered for the production of pharmaceutical proteins. In the pharmaceutical industry, the main production platforms are E. coli and mammalian cell lines. However, for those pharmaceutical proteins for which high yields and low production costs are important, filamentous fungi could provide a viable alternative.

We have explored the use of a highly productive fungal production platform (Chrysosporium C1) for the production of a very versatile class of pharmaceutical proteins, i.e., antibody molecules. Antibody molecules and molecules carrying antibody domains are currently the largest and fastest-growing class of biopharmaceuticals. Production of functional full-length human monoclonal antibodies has been accomplished using highly productive low protease mutant Chrysosporium host strains. High level expression was achieved using a glucoamylase-carrier approach, and recombinant strains expressing both heavy and light chains were obtained. Heterodimeric antibody molecules were formed efficiently, allowing simple purification of the protein from the culture fluid using protein A. Cell-based bio-assays performed on the culture supernatant and the purified samples revealed almost complete bioactivity.


Bioconversion of ginsenocides in red ginseng by fermentation with Monascus

Jee-Hwan Oh, Sung Yun Hong, Inhyung Lee

Kookmin University, Seoul, Republic of Korea

Red ginseng contains ginsenosides that have various healthy effects. Some of ginsenosides are transformed from its glycone to aglycone forms during processing of red ginseng, but lots of them still remain as glycone form. Because alycone ginsenosides are superior in bioavailability and some aglycone ginsenocides such as Rh1, Rh2 and Rg3, have excellent antitumor activities, it is important to transform glycone to aglycone gincenosides to benefit more healthy function of red ginseng. Here we converted glycones to aglycone ginsenosides in red ginseng by fermentation with Monascus. Because Monascus produces several bioactive compounds, Monascus fermented red ginseng may have additional healthy functions. Especially monacolin K, one of metabolites produced by Monascus, has effects of reducing cholesterol in blood. We isolated three Monascus sp., RC02, RK03 and RU03 that have high activities of β-D-glucosidase, α-L-rhamnosidase, and α-L-rhamnosidase with high monacolin K and without apparent citrinin production in 10% red ginseng medium. We scaled up to 50 L fermentation with 13% red ginseng, 2% glucose using two fermentation conditions of RC02, RK03, and RU03 mixed culture and RK03 single culture. Lyophilized powder of fermented product using mixed and RK03 cultures contained 693.04 mg/kg and 3,089 mg/kg of monacolin K, respectively. The total amount of ginsenosides, Rh1, Rh2, and Rg3 was 838.7 mg/kg in red ginseng and that increased up to 4,016 mg/kg and 4,117 mg/kg after fermentation with mixed culture and RK03 culture, respectively. In any case, citrinin was not detected. In addition total gensenocides was almost the same as in red ginseng after fermentation, indicating that fermentation with Monascus did not affect total amount of ginsenosides. The newly developed fermented red ginseng using Monascus would have not only better healthy effects of red ginseng but also additional functions by Monascus fermentation.



Approaching zero growth in retentostat cultures of Aspergillus niger

Thomas R. Jørgensen, Cees A.M.J.J. van den Hondel, Arthur F.J. Ram

1Institute of Biology Leiden University, Leiden, Netherlands, 2Kluyver Centre, Delft, Netherlands

Micro-organisms are frequently exposed to suboptimal growth conditions, in nature as well as in culture. The ideal industrial production culture is characterized by high product yield and minimal biomass formation. We study how cellular processes are affected by starvation in energy-source maintained cultures. For this purpose, the enzyme and organic acid producing fungus, Aspergillus niger, was cultivated in maltose-limited retentostat cultures. A newly developed filter, in stainless-steel, allowed efficient and durable retention of mycelia. The filtration efficiency exceeded 95% at any time. Retentostat cultivation was initiated in end-exponential growth phase of batch cultures and lasted for about 11-12 days. Cultivation was ended at this time, because of massive foaming.

Carbon- and energy-source starvation induced morphological and metabolic differentiation, which became apparent after 5-6 days. Simple asexual reproductive structures, resembling phialides, and reduced conidiophores were formed at tips of leading and branching hyphae. Concomitantly, the culture broth turned dark brown reflecting melanisation of mycelial structures and formation of conidia. Effects of maintenance energy requirement (1, 2) were apparent from biomass accumulation over time, although the growth rate never reached zero. Analyses of major cellular components revealed that accumulation of protein and lipid was time-proportional, and that the apparent maintenance effect reflected a decreasing content of carbohydrate. Changes in cell composition and biomass accumulation appeared to coincide with asexual differentiation and changes in hyphal dimensions. Product formation, in terms of secreted protein and glucoamylase activity, will be assessed for different strains in retentostat cultures. Thorough characterisation of A. niger N402 in retentostat cultures will be used for basal description of starvation-induced changes in cellular processes and to identify target pathways/elements for genetic engineering. In addition, these cultures will serve as reference for comparison of a glucoamylase hyper-producing strain and other engineered strains.

1 Pirt, S.J., Proc. Roy. Soc. B 163, 224-231. 1965, 2 van Verseveld, H.W., Metwally, M., el Sayed, M., Schrickx, J.M., A.H. Stouthamer, Antonie van Leeuwenhoek 60, 313-323. 1991.


The N-terminal region of Aspergillus oryzae hydrophobin RolA is important for RolA-cutinae CutL1 interaction

Toru Takahashi1, Kenji Uehara2, Yohei Yamagata2, Katsuya Gomi2, Fumihiko Hasegawa1, Keietsu Abe1

1New Industry Creation Hatchery Center, Tohoku University, Sendai, Miyagi, Japan, 2Graduate School of Agricultural Science, Tohoku University, Sendai, Miyagi, Japan

When fungi grow on plant or insect surfaces coated with wax polyesters that protect against pathogens, the fungi generally form aerial hyphae to contact the surfaces. Aerial structures such as hyphae and conidiophores are coated with hydrophobins, which are surface-active proteins involved in adhesion to hydrophobic surfaces. When the industrial fungus Aspergillus oryzae is cultivated in a liquid medium containing the biodegradable polyester polybutylene succinate-coadipate (PBSA), the rolA gene encoding a Type I hydrophobin RolA is highly transcribed. High levels of RolA are localized on the cell surface and also secreted into the liquid medium. Under these conditions, A. oryzae simultaneously produces the cutinase CutL1, which hydrolyzes PBSA. RolA adsorbed to the hydrophobic surface of PBSA particles in the medium recruits CutL1, resulting in condensation of CutL1 on the PBSA surface and consequent stimulation of PBSA hydrolysis (1, 2). The ability that RolA attached to the PBSA surfaces recruits esterase CutL1 is a newly discovered function in hydrophobin (1). In the present study, we studied amino acid residues involved in the RolA-CutL1 interaction by means of site-directed mutagenesis and chemical modification of RolA. Teflon particles were coated with RolA and its derivatives. The Teflon particles coated with RolA or its derivatives were incubated with soluble CutL1 for recruitment, and then the supernatant and particles were separated by centrifugation. Recruited CutL1 was extracted from the centrifuged particles with SDS and quantitatively measured by SDS-PAGE analyses. We found that the N-terminal region of RolA is important for RolA-CutL1 interaction. We discuss amino acid residues involved in the interaction.

1 Takahashi T. et al. Mol. Microbiol. 57:1780-1798 (2005), 2 Ohtaki S. et al. Appl. Environ. Microbiol. 72:2407-2413 (2006)


Improvement of homologous recombination in Penicillium chrysogenum: impact of the hdfA mutation on fitness and genome wide transcriptional response.

Ishtar Snoek1, Zita van der Krogt1, Hesselien Touw2, R Kerkman2, Jack Pronk1, Roel Bovenberg2, Marco van den Berg2, Jean-Marc Daran1

1TU Delft, Delft, Netherlands, 2DSM, Delft, Netherlands

Targeting a transformed DNA fragment to a specific site in the chromosome of an organism makes use of homologous recombination repair mechanisms. While integration of exogenous DNA occurs readily in Saccharomyces cerevisiae, this mechanism of DNA repair is not predominant in other fungi. However, the targeting efficiency can be increased by disabling the non homologous end joining (NHEJ) pathway. The deletion of the gene homologous to the human KU70 which encodes a protein that functions in the NHEJ pathway has been shown to improve homologous recombination successfully in many filamentous fungi, including Neurospora crassa, Kluyveromyces lactis and several Aspergilli.

Comparable to what was observed in other filamentous fungi, the deletion of hdfA, the Penicillium chrysogenum KU70 homolog, resulted in a significant improvement of gene targeting through homologous recombination; from 0.1% in WT to 50% in the mutant (using 1.8 kb flanking regions). Furthermore, the potential of such a high recombinant P. chrysogenum strain to serve as platform for gene targeting was studied. In order to characterize the impact of the hdfA deletion, the mutant was compared to its isogenic reference using chemostat cultures, transcriptome analysis and quantitative fitness profiling. Whereas little to no marked difference was recorded at both physiological and transcriptome levels, in a direct competition experiment, the isogenic reference strain had a clear advantage over the hfdA strain.


Functional analysis of essential genes in A. fumigatus by co-precipitation of associated proteins using an S-tag fusion peptide

Gethin Allen1, Mike Bromley2, Mike Birch2, Geoff Turner1

1The University Of Sheffield, Department of Molecular Biology and Biotechnology,, Sheffield, United Kingdom, 2F2G Ltd., Manchester, United Kingdom

In the opportunistic human pathogen A. fumigatus essential gene products are potential drug targets, however, because A. fumigatus is an asexual haploid organism traditional techniques used to identify and characterise genes are not always feasible. Essential genes can be identified using the MycoBank® screening method where genes are disrupted in a diploid background and essentiality of the disrupted gene is determined by loss of a marker when the diploids are broken down in to haploids.

Bioinformatics can sometimes indicate the function of genes by identifying previously characterised homologues or domains. This is not always fruitful as the homologues may be uncharacterised genes. In these cases functional analysis is required.

Genes can be characterised by analysing the phenotypes of mutants. Disruption of an essential gene is lethal so mutant strains are difficult to generate and the phenotypes are often uninformative.

Identifying previously characterised proteins that interact with uncharacterised proteins can provide clues to the function of the uncharacterised proteins. We used the S-tag fusion peptide as an affinity tag for co-precipitation experiments to identify proteins that interact with a selection of putative essential proteins identified by MycoBank® screening and analysis of clusters of orthologous genes.

The S-tag system was used because the purification procedure is simple and the tag is very small so that it will not significantly disrupt the structure or function of the tagged protein. The tagged protein must be functional otherwise strains containing S-tagged essential genes will be inviable. A 5X glycine-alanine polylinker was used to attach the tag to the target proteins to prevent the tag disrupting the native structure of the protein.

This approach has identified a novel component of the SWI/SNF ATP-dependent chromatin remodelling complex that is unique to fungi and conserved across many fungal species. This data correlates with data obtained from other approaches; GFP localisation had previously shown this protein to be present in the nucleus and probably closely associated with DNA.



Evaluation of different protein extraction protocols for the comparative analysis of Monilinia spp proteomes

Olja Bregar, Stanislav Mandelc, Tjasa Gril, Branka Javornik

Biotechnical Faculty, University of ljubljana, SI-1000 ljubljana, Slovenia

The brown rot fungi Monilinia laxa (Aderh. & Ruhl.) Honey, Monilinia fructigena (Aderh. & Ruhl.) Honey and Monilinia fructicola (Wint.) Honey are known as an important plant pathogens in stone and pome fruits. The detection and identification of these species is complex, so new target sequences or proteins could contribute to the development of efficient diagnostic markers. In order to achieve this goal we have started proteomic analysis of fungi and, as a first step, have evaluated protein extraction protocols in terms of protein yield, 2-D image quality and reproducibility. Sample preparation is one of the most crucial steps in proteom analysis and we therefore tested various extraction protocols. Three different lysis buffers were tested (a combination of CHAPS and Tris-HCl pH 8,0; 2-D lysis buffer with thiourea/urea and acidic extraction), followed by grinding, precipitation with TCA/acetone and purification. Fungal cell wall is very resistant to fragmentation, so special attention was devoted to thorough grinding and the removal of interfering compounds. Two isolates of M. laxa, one from apple and the other from apricot, were grown in malt extract medium and sampled for protein extraction after 3 and 7 days growth. All different isolations of total proteins were analyzed with 2-D electrophoresis (300µg load, pH 3-10 NL, 12,5% T), stained with CBB, and 2-D patterns were analyzed with ImageMaster Platinum software. Image comparison and statistical analysis showed that the three methods differ in the number of protein spots detected. The most effective was acidic protein extraction. A 2-D protein reference map of M. laxa was established and we are currently performing comparative analysis of M.laxa isolate proteomes showing host specificity.



Highly improved gene targeting in Penicillium chrysogenum

Paulo de Boer1, Hesselien Touw2, Jeroen Bastiaans1, Richard Kerkman2, Karolina Dukik1, Marco van den Berg2, Remko Offringa1

1Add2X Biosciences BV, Leiden, Netherlands, 2DSM Anti Infectives, Delft, Netherlands

Introduction: In most eukaryotes, including filamentous fungi, the predominant mode of DNA integration is via non-homologous recombination (NHR). For functional genomics studies and various other applications however, DNA integration via homologous recombination leading to gene targeting (GT) is the preferred pathway. The recent identification of key components of the NHR pathway, such as Ku70 and Ku80, has provided new tools for improving GT efficiencies. Knocking out the NHR pathway has resulted in efficient GT in a variety of eukaryotes, including several Aspergillus species. In the present study, we studied the effect of deletion of the Ku70 or Ku80 genes GT in the b -lactam producer Penicillium chrysogenum.

Methods: Deletion constructs consisting of 2.5 kb up- and downstream flanks of the target genes (ku70 and ku80), interrupted by the amdS marker driven by the gpdA-promoter were assembled using the Multisite Gateway system. These constructs were then used for transformation of protoplasts. Stable deletion mutants were subsequently analysed for GT at the niaD locus in comparison to the wildtype.

Results: Deletion of the ku70 or ku80 homologues of P. chrysogenum resulted in stable transformants. After the initial transformations, removing the marker over the direct repeat occurred spontaneously. Subesequent analysis of the D k70 mutant revealed a highly increased GT efficiency at the niaD locus compared to the wildtype (50% vs. 1% resp).

Discussion: GT efficiencies are highly improved in P. chrysogenum D ku70 or D ku80 mutants compared to the wildtype. The use of these deletion mutants opens up possibilities for efficient reverse genetics in this industrially important fungus. Recently, additional methods have been developed which enable GT efficiencies up to 100%.


Generation and characterisation of camelid antibodies against fungal melanin

Jeannette Schmaler1, Gernot Habicht2, Uwe Horn1, Axel Brakhage1

1Leibniz Institute for Natural Product Research and Infection Biology (HKI), Friedrich Schiller University, Jena, Germany, 2Darmstadt University of Technology, Darmstadt, Germany

Invasive aspergillosis is a leading cause of death in immunosuppressed patients. This is due to late diagnosis and limited therapies of the disease. Previously, we showed that Aspergillus fumigatus produces a specific melanin designated dihydroxynaphthalene (DHN)-melanin. This DHN-melanin might be produced during infection to protect the fungus from the host immune system. Our aim is to study melanisation as pathogenicity mechanism and to detect the fungus in tissue sections with the help of a melanin specific antibody.

To select antibodies against natural occuring DHN-melanin we chose conidia as target since they expose on their surface DHN-melanin. A competitive selection with conidia of a pksP mutant strain which lack DHN-melanin was performed to minimise the selection of unspecific antibodies. A recombinant library of camelid VHH-domains served as antibody pool. Selection was performed via a phage-display method. The antibody was produced as fusion to alkaline phosphatase and characterised in ELISA studies. Furthermore, we applied the biotinylated antibody to demonstrate the melanisation of conida and hyphae via an immunofluorescent staining method with streptavidin-Cy3 conjugate as detecting agent.

We successfully selected phages, that were presenting antibody domains, against melanised conidia. The camelid antibody domain was recombinantly produced as fusion to alkaline phosphatase in Escherichia coli via high cell density fermentation. The purified antibody possesses low molecular mass, good stability and binds with high affinity to melanised conidia of different Aspergillus species. A less stringent binding to synthetic DOPA-melanin and sepia-melanin was observed. The antibody also detects melanised hyphae and conidia but not pigmentless hyphae in in vitro grown cultures.

In future, we want to use this tool to study the pathogenicity mechanisms of A. fumigatus with regard to melanisation. We also intend to test the suitability of the antibody to detect the fungus in infected tissues.


Molecular characterization of the basidiomycete isolate Nematoloma frowardii b19: a novel corticioid species re-organized under the genus Phlebia?

Kristiina Hilden1, Ralf Bortfeldt3, Martin Hofrichter2, Annele Hatakka1, Taina Lundell1

1University of Helsinki, Department of Applied Chemistry and Microbiology, Helsinki, Finland, 2International Graduate School Zittau (IHI Zittau), Zittau, Germany, 3Friedrich-Schiller University Jena, Jena, Germany

The basidiomycete isolate b19, originally identified by morphological characteristics of the fruiting body as Nematoloma frowardii, efficiently produces manganese peroxidase (MNP) and is adopted for degradation of natural polymers such as wood lignin, soil humic acids and brown coal components. Basidiomycete MNPs are among the most promising metalloenzymes to be used as biocatalysts in industrial biotechnology (so called white chemistry). The N. frowardii MNP has also shown strength in conversion of xenobiotic compounds like polycyclic hydrocarbons (PAHs) and trinitrotoluene (TNT). Despite all of its potential, this biotechnologically interesting fungus has not been extensively studied at molecular biology level before.

According to the molecular characterization of its main MNP isozyme, Nf b19 MNP2, and partial sequencing of its MNP3, three LIP (lignin peroxidase) and two laccase encoding genes, and the ribosomal ssu RNA encoding 18S gene, we show here that the fungus reveals close phylogenetic relationship to the white-rot in wood causing corticioid basidiomycete Phlebia radiata (Fr.).

Comparison of the 390-amino acid long primary structure of Nf b19 MNP2 reveals 96% aa-identity to the MNP2 of Phlebia radiata 79 (gene Pr mnp2) with differences only within 15-aa that are apparently not involved in enzyme catalysis or heme coordination. Nf b19 MNP2 belongs to the classical long MNPs which we have designated the phylogenetic group B within the superfamily of fungal class II secreted heme peroxidases (see also the abstract of Hildén K. et al. for this meeting).

Phylogenetic analysis on the ITS sequence (ITS1+5.8S+ITS2) more precisely re-orders the fungus as a possible representative of a novel species under the genus Phlebia, nearest to the P. acerina and P. radiata clades. The genus Phlebia belongs to a completely different family (Corticiaceae) and order (Aphyllophorales) within the phylum Basidiomycota than the genus Nematoloma, which is classified to the family Strophariaceae under the order Agaricales. Our results thereby point to a need for traditional systematic re-identification and classification of the previously named N. frowardii isolate b19.


Evaluation of Coprinopsis cinerea genome annotations through proteomic analysis of the secretome

Andrzej Majcherczyk, Dorothea Fragner, Mojtaba Zomorrodi, Ursula Kües

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

The genome of the basidiomycete Coprinopsis cinerea has been established in 2003 and an annotation been published in the NCBI database by the Broad Institute. Other annotations - Twinscan, Snap, Glean, GleanMax300 and Augustus - were established by J. Stajich and M. Stanke with the help of EST libraries provided by A. Gathman, W. Lilly and co-workers. We combined the protein predictions of these six different annotations in a Mascot database used for searches of MS-data obtained from mass spectrometry analysis of C. cinerea proteins. C. cinerea secretes large number of proteins freely into its culture medium. Secreted proteins, beginning from the early exponential growth phase up to the starvation conditions, were separated by 2D-gel electrophoresis and ESI-LC-MS/MS analysis of proteins was either performed on single protein spots or by application of a shot-gun method on complex protein mixtures. Over 90% of single spots gave specific hits to one or to several predictions of protein sequence. The significance of protein identification and sequence coverage were compared with respect to the different annotation algorithms. Combining the annotations increased significantly the number of confidently identified proteins and demonstrated also an incompleteness of any single automated gene annotation method. Combining the Mascot results from repeated shot-gun analysis into SQL-databases increased further the number of confidently identified proteins. Several glucanases, proteins with sugar binding domains, various peptidases, redox-enzymes and some esterases/lipases have been identified. In addition, several low molecular weight proteins without known function and with low homology to known proteins from the NCBI database were detected. The secretome of C. cinerea changed significantly during growth and the highest complexity of the extracellular proteins was observed in the stationary growth phase. High amount of fungal polysaccharides and metabolites produced in the exponential growth phase hampered protein analysis by 2D-gel electrophoresis. However, this problem is solved by application of shot-gun proteomic methods.

We kindly appreciate sequencing and discussion inputs from members of C. cinerea annotation consortium.

This work is supported in frame of a Common Lower-Saxony-Israel Project (ZN 2043) by the Ministry of Science and Culture in Hannover, Germany.


Structurally and phylogenetically divergent manganese peroxidases from two lignin-degrading basidiomycetes

Kristiina Hildén, Miia Mäkelä, Terhi Hakala, Pekka Maijala, Annele Hatakka, Taina Lundell

University of Helsinki, Applied chemiatry and Microbiology, Helsinki, Finland

Class II extracellular heme peroxidases of filamentous fungi show diverse functions as biocatalysts (degradation of lignin, conversion of xenobiotics, bleaching of coloured compounds and polymeric dyes) but minor differences in protein structure. The lignin, manganese and versatile peroxidases (LiPs, MnPs and VPs) are involved in lignin brakedown thereby being described the lignin modifying enzymes.

Recently, we described structurally and phylogenetically divergent MnPs from two biotechnologically promising white-rot basidiomycetes, Phlebia radiata and Physisporinus rivulosus. Both species have two types of MnP that are functionally similar but differ significantly in protein and gene sequence and length, and gene intron-exon structure. The 3-D molecular models of the two divergent P. radiata MnPs confirm this diversity. MnP2 with a long C-terminal tail has the highest structural similarity with the crystal structure of Phanerochaete chrysosporium MnP, whereas the shorter MnP3 is structurally the most related to P. chrysosporium LiP.

Both P. radiata and P. rivulosus express the two divergent MnP isozymes when they are cultivated on milled wood. Expression levels of the two mnp genes of P. rivulosus were studied on defined media supplemented with Mn2+ ions and veratryl alcohol (VA), which is a non-phenolic aromatic compound biosynthesized and converted by some white rot fungi. Expression of the two mnp genes in agitated liquid cultures implicated differential regulation in response to the effectors. Transcription of mnpA gene was induced by the addition of VA but not by Mn2+. Induction of mnpA expression was noticeably observed in the cultures supplemented with milled wood. In contrast, the transcription of mnpB was induced in both cases, that is by addition of either VA or Mn2+. Our data on the two basidiomycetous species indicate that differential regulation of the two divergent MnP enzymes may in turn reflect their different functions for fungal wood and lignin degradation.



The fungal path for D-galacturonate catabolism

Satu Hilditch1, Suvi Berghäll2, Janis Liepins3, Merja Penttilä1, Peter Richard1

1VTT Technical Research Centre of Finland, Espoo, Finland, 2University of Helsinki, Department of Applied Chemistry and Microbiology, Helsinki, Finland, 3University of Latvia, Institute of Microbiology and Biotechnology, Riga, Latvia

Pectin is a major component of the primary cell wall of plants and mainly consists of D-galacturonate. D-galacturonate is an important carbon source for microorganisms living on decaying plant material. Bacterial catabolic pathways have been described but a eukaryotic pathway has remained unknown. We show that the D-galacturonate pathway in the mold Hypocrea jecorina (Trichoderma reesei) is different than the pathways in bacteria.

In the fungal catabolic pathway D-galacturonate is first reduced to L-galactonate by an NADPH-utilizing D-galacturonate reductase (GAR1). Then a water molecule is removed from L-galactonate by an L-galactonate dehydratase (LGD1) and L-threo-3-deoxy-hexulosonate is produced. Subsequently an L-threo-3-deoxy-hexulosonate aldolase (LGA1) splits L-threo-3-deoxy-hexulosonate to L-glyceraldehyde and pyruvate, and an NADPH-utilizing L-glyceraldehyde reductase (GLD1) converts L-glyceraldehyde to glycerol.

The activity of GAR1, LGD1 and LGA1 was induced when H. jecorina mycelia were grown on D-galacturonate but not when grown on other carbon sources, GLD1 was equally present on all the tested carbon sources. Deletion of the gar1, lgd1 or lga1 gene in H. jecorina resulted in a strain unable to grow on D-galacturonate. All the four genes of the pathway were identified, cloned from H. jecorina cDNA library, functionally expressed in the heterologous host S. cerevisiae, and the kinetic properties of the enzymes were determined.


Inhibitory effect of bamboo vinegar and wood vinegar towards sapstaining fungal growth

Natarajan Velmurugan1, Sang-Sub Han2, Dong-Min Sa3, Yang-Soo Lee1

1Department of Forest Science and Technology, Institute of Agriculture and Life Sciences, Chonbuk National University, Jeonju 561-756, Chonbuk, Republic of Korea, 2Institute of Agriculture and Life Sciences, Chonbuk National University, Jeonju 561-756, Chonbuk, Republic of Korea, 3College of Agriculture, Life & Environments Sciences, Chungbuk National Universsity, Chunbuk 361-763, Republic of Korea

The antifungal activity of bamboo vinegar and wood vinegar were investigated by radial mycelial growth measurement technique and characterisation of both extracts were performed my GC-MS analysis. Both bamboo and wood vinegar are natural resources and acidic dark brown liquid by-products of bamboo and broad leaved trees charcoal burner. Two percentage MEA plates were prepared amended with 0.001, 0.01, 0.1, 0.5 and 1.0 % of both bamboo vinegar and wood vinegar. Six mm plugs from the plates growing Ophiostoma flexuosum, Ophiostoma tetropii, Ophiostoma narcissi and Ceratocystis ips were transferred to 2 % MEA plates contain different concentrations of both extracts. The results revealed that both extracts were highly active at minimum concentrations (0.1-1.0 %) used. GC-MS analysis was performed to search for compounds with antifungal activity. The chloroform fractions of compounds were identified as 2,6-dimethoxy phenol, dehydroacetic acid, 2,3,5-trimethoxy toluene and 5-hydroxy-3-oxo-2-propionyl-1gamma-1-acetone-4-heptnoic acid in bamboo vinegar and 2-methoxy phenol, 2-methoxy-4-methyl phenol, 2,6-dimethoxy phenol, dehydroacetic acid and 2,3,5-trimethoxytoluene in wood vinegar by MS analysis. These phenolic compounds have powerful antifungal activity. Organic acids comprises 21.89 % in bamboo vinegar and 18.60 % in wood vinegar, the total phenolic content was 58.06 % in wood vinegar and 62.64 % in bamboo vinegar. The antifungal activity of bamboo vinegar and wood vinegar may arise from its phenolic content and pH. These extracts are easily available, inexpensive and non-toxic to environment, hence the applications of these extracts in wood industries are beneficial and uncomplicated.



Differential responses to cadmium in the ectomycorrhizal fungus Suillus luteus, a proteomic approach

Karen Verstraelen, Jan Colpaert, Jaco Vangronsveld

Center for Environmental Sciences, Environmental Biology Group, Hasselt University, Diepenbeek, Belgium

Heavy metals are important environmental pollutants and can cause serious problems to organisms. Still, some micro-organisms show a genetic adaptation to heavy metals and could be useful in the revegetation of contaminated sites. A promising organism is the ectomycorrhizal fungus Suillus luteus, a common root symbiont of Pinus species. S. luteus populations thriving in pioneer forests that colonize sites severely contaminated by Zn smelters in NE-Belgium, have undergone a genetic adaptation to toxic cadmium (Cd) concentrations. However, the exact mechanisms of metal tolerance in ectomycorrhizal fungi are largely unknown. The aim of this study was therefore to identify fungal proteins involved in the mechanism sustaining the adaptive cadmium tolerance in S. luteus. Using the high-tech 2D-DIGE technology, a comparison of the proteome under Cd stress was performed for two fungal strains, one sensitive and one tolerant strain. In this time-response experiment (0-48hrs), fungal proteins were extracted after Cd treatment, labelled with fluorescent CyDyes and analysed by two-dimensional gel electrophoresis. Proteins that showed statistically significant changes between both isolates will further be analysed with massaspectrometry.


Gene replacement by mutant versions without co-insertion of a selectable marker in Podospora anserina

Evelyne Coppin1, Riyad El-Khoury2, Carole, H. Sellem2, Antoine Boivin2, Marc Maas2, Annie Sainsard-Chanet2, Robert Debuchy1

1Univ Paris-Sud, Institut de Génétique et Microbiologie, CNRS UMR8621, Orsay, France, 2CNRS, Centre de Génétique Moléculaire, UPR2167, Gif sur Yvette, France

Testing the effect of targeted mutations, point mutations, deletions or promoter exchange, is a fundamental tool for the analysis of gene function. In particular, fusions to promoters that induce heterochronic expression of the gene of interest have proved highly valuable in investigating the function of developmental genes. However, in most cases, transgenes integrate in different ectopic positions, resulting in variations in their expression level from one transformant to another, and in possible and unpredictable alteration of the metabolism due to the structural change of the integration locus. These variations are a cause of concern with the expanding development of microarray analyses of transgenic strains, as this technology requires highly similar genetic context. It has been shown that the deletion of the KU70 orthologs in fungi is an efficient strategy to improve homologous recombination. By using a delta PaKU70 strain of P. anserina, we demonstrated that it is possible to reintroduce a mutated gene without co-insertion of a selectable marker and without any other modification of the target locus. Replacement of a gene of interest by versions in which it is fused to homologous or heterologous promoter sequences in a delta PaKU70 strain is in progress.

This work is funded by the Association Française contre les Myopathies, the European Commission (integrated Project MiMage LSHM –CT-2004-512020) and National Research Agency contract n° ANR-05-BLAN-0385-01


Cell wall-associated proteins of the basidiomycete Coprinopsis cinerea

Dorothea Fragner, Mojtaba Zomorrodi, Ursula Kües, Andrzej Majcherczyk

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

The fungal cell wall is a complex structure composed of a network of glucans, chitin and glycoproteins. To perform a comprehensive study on the largely unknown cell wall proteome in higher basidiomycetes, we use the model organism Coprinopsis cinerea grown in liquid medium to the early exponential phase. Secreted proteins were fractionated from the mycelium according to the nature of their binding to the fungal cell wall: proteins adsorbed to the outer hyphal sheath, extractable cell wall-associated proteins and non-extractable cell wall-bound proteins. 2D-gel-electrophoresis revealed a high diversity between the hyphal sheath proteins, the extractable cell wall-associated proteins and the free proteins in the culture supernatant, respectively. There is little overlap between the different protein fractions. By a mass spectrometry approach, we identified so far 62 proteins from the hyphal sheath, 86 extractable cell wall proteins and 41 proteins in the supernatant, adding up to a total of 141 different proteins. 10% of amounts of mycelium-isolated proteins are from the hyphal sheath and 90% from the cell walls. The amount of soluble proteins in the supernatant is equal to the total amount of extractable proteins from the mycelium. The protein complexity in the supernatant is thus comparably low with only 19% of all identified proteins, many of which are oxidases, glucanases (e.g. glyoxal and glucose oxidases) as well as numerous proteolytic enzymes. The protein variety with 53 % is much higher in the cell wall fraction and with 28% of the identified proteins highest in the hyphal sheath. Many of these proteins are new with so far no defined function.



The purification and characterisation of a low molecular weight xylanase from the hypercellulolytic filamentous fungus Trichoderma reesei

Mark Gaffney, Richard Murphy, Ronan Power

Alltech Biotechnology, Meath, Ireland

It has been well documented that arbinoxylans have anti-nutritional effects on monogastric animals. These arbinoxylans are a contributing factor to the solubility of non-starch polysaccharides (NSPs), which are found in the endosperm of cereal cell walls. NSPs attract large amounts of water, increasing the viscosity of the ingesta. The resulting effects are manifested as poor feed conversion, depressed weight gain and wet droppings. Incorporation of exogenous xylanase has been successfully proven to reduce gut viscosity and improve nutrient adsorption. The filamentous fungus Trichoderma reesei is not only an efficient producer of multiple xylanolytic enzymes, but also of cellulolytic and other hemicellulolytic enzymes when cultivated by solid state fermentation (SSF). The aim of this study was to produce, purify and characterise a low molecular weight endo-b -1,4-xylanase from a crude extract of T. reesei cultivated by SSF. One such xylanase was purified using a combination of ultrafiltration, cation exchange and gel filtration chromatographies. Purification was to a factor of 8.9 and resulted in a 2% final yield. When analysed by SDS-PAGE, the enzyme migrated as a 21 kDa band which appeared free of any contaminating proteins. The activity of the purified enzyme was strongly inhibited by SDS and by the divalent cations Fe2+ and Hg2+. Activity was largely unaffected by agents often found in animal feeds, such as calcium, manganese, potassium, sodium and zinc. The purified xylanase retained relatively high activity over a broad pH range, 3-10. The enzyme was also active when incubated up to 50°C, however a near complete loss in activity was observed when treated at 60°C and above. The enzyme was exclusively xylanolytic, displaying little specificity for other polysaccharide substrates including cellulose, glucan or dextran. These physiochemical traits are advantageous when considering the potential utilisation of the enzyme as an additive in animal feeds but also in other areas such as the bleaching of kraft pulps, the quality improvement of baked products and the recovery of fermentable sugars from hemicelluloses.



Enzymes of basidiomycetes in wood and plant litter degradation

Andrzej Majcherczyk1, Ravi Chandra Dwivedi1, Dorothea Fragner1, Mojtaba Zomorrodi1, Yitzhak Hadar2, Ursula Kües1

1Molecular Wood Biotechnology and Technical Mycology, Büsgen-Institute, Georg-August-University, Göttingen, Germany, 2Laboratory of Fungal Physiology, Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew University of Jerusalem, Rehovot, Israel

Higher basidiomycetes secrete numerous different proteins for the extracellular degradation of complex high molecular weight substrates. However, only few known enzymes of the complex fungal secretomes can be studied by direct detection of their activities and changes in substrate composition. To better understand and to follow the degradation processes, we developed special protocols for high-resolution separation of proteins from basidiomycetes by 2D-gel electrophoresis and in-gel-staining of oxidative enzymes. Various oxidative and hydrolytic enzymes from the white rots Pleurotus ostreatus and Trametes versicolor and the litter-degrading saprotroph Coprinopsis cinerea were identified by analyzing tryptic digests of separated proteins by mass spectrometry (ESI-LC-MS) and Mascot searching engine using a database with known protein sequences, annotated genomic sequences and ESTs. Secretion conditions and isoenzyme compositions were studied in different culture conditions with a focus on peroxidases and laccases. We are isolating selected proteins from culture supernatants and fungal cell walls for detailed biochemical characterization.

This work is supported in frame of a Common Lower-Saxony-Israel Project (ZN 2043) by the Ministry of Science and Culture in Hannover.



Sudies on the morphology of the filamentous basidiomycete Coprinopsis cinerea grown in liquid cultures

Martin Rühl, Max Richter, Sreedhar Kilaru, Karin Lange, Ursula Kües

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

In submerged fermentation, filamentous fungi can either grow in filaments or in pellet form which differentially can affect yields of fungal products. In our laboratory, we use the basidiomycete Coprinopsis cinerea for recombinant production of laccases and study the morphological effects of the fungal mycelium on laccase expression.

Several different laccase genes were subcloned in an expression vector under the control of the gpd promoter from Agaricus bisporus. In standard Coprinopsis medium at 37°C, enzyme activities in liquid shaken cultures of expressed laccases differed between genes, ranging from 0.1 U/ml to 10 U/ml. Medium optimisation and changing environmental conditions improved the yields of enzyme up to 30 U/ml. Along with increasing enzyme yields, a change in temperature had severe effects on the mycelial morphology. To characterise the morphology in submerged shaken flask cultures of C. cinerea laccase transformants during growth at different cultivation conditions, microscope techniques were used. Differences in pellet morphology of a lcc1 transformant were obvious at 25°C and 37°C with an even and a hairy pellet shape, respectively. At the lower temperature, pellets were more compact than at the usual cultivation temperature of 37°C. The outer layer of these pellets consists of small densely aggregated cells and the medulla of more loosely aggregated hyphae that during aging appear to degenerate. Laccase activity and gfp-expression tests are used to follow under the microscope the places of protein secretion.

In scaling up the volume for enzyme production, we also grow the transformants in stirred bioreactors and study the characteristics of C. cinerea morphology in relation to different fermentation parameters, such as pHs, amounts of inocula and agitation rates. Filamentous growth and pellet growth has been observed in the bioreactor and we are defining the fermentation parameters influencing these.


Comparative analysis of laccase-isozymes patterns of several Trametes species under different culture conditions and relationship with the family of genes that probably codify for these proteins

Cristina García, María C. Terrón, José M. Carbajo, Ainhoa Arana-Cuenca, Alejandro Téllez, Aldo E. González

1CIB-CSIC, Madrid, Spain, 2CIB-CSIC, Madrid, Spain, 3CIFOR-INIA, Madrid, Spain, 4Centro Biotecnologia, U. Politécnica Pachuca, Pachuca, Mexico, 5Centro Biotecnología, U.Politénica Pachuca, Pachuca, Mexico, 6CIB-CSIC, Madrid, Spain

The study of isozymes has to date been successfully applied to compare the activities of several Trametes species. Given that these strains produce a very characteristic laccase isozyme pattern, in the present work we compared these isozyme profiles with the profiles produced by other very closely phyllogenetically related fungi able to selectively degrade lignin and that  produce peroxidase activities in a N rich complete medium.

Extracellular laccase activity were detected in five fungi studied Trametes subectypus, T. pavonia, T. ochraceae, T. gallica and Trametes sp. I-62. In addition, the electrophoretic pattern showed differences between the species that correlated with the laccase genes that codify for these family enzymes. Moreover, laccase genes published up to date in these species presented differences between the different assayed strains. Given that phylogenetically related fungi growing in the same culture conditions exhibit different laccase patterns, these isozymes could be used as an additional criterion for fungal identification.



Production of laccases, peroxidases and enzymes that produce H2O2 in a population of monokaryons from Pleurotus ostreatus var. florida

Cristina García, María C. Terrón, Francisco Santoyo, José M. Carbajo, Antonio G. Pisabarro, Lucía Ramírez, Aldo E. González

1CIB-CSIC, Madrid, Spain, 2CIB-CSIC, Madrid, Spain, 3U. Pública de Navarra, Pamplona, Spain, 4CIFOR-INIA, Madrid, Spain, 5U. Pública de Navarra, Pamplona, Spain, 6U. Pública de Navarra, Pamplona, Spain, 7CIB-CSIC, Madrid, Spain

Once obtained the data of the enzymatic activities of 80 monokaryons, we tried the first approach to map them as quantitative traits with the aim to know whether they mapped in the linked map in the same chromosomes and in the same loci that the structural genes, until now sequenced and partially characterized, do. The results showed unexpected data, and it is highly probable that, due to their position, they could correspond to other genes still not detected or sequenced or with regions of the map where certain genes (regulating these activities or modulating the expression of pox, mnp and vpmnp genes), could be located (Santoyo et al. 2007).

On the other hand, the enzymes that up to now are known to produce H2O2, present activity at different time course during the experiments in the same way that the ligninolytic enzymes do. Nevertheless, this result is not enough to explain the peroxidase activity detected in some strains, due to the fact that aryl alcohol oxidase activity is detected after the day at which peroxidase activity is produced. This strongly suggest that other oxidases besides the six published in literature, and not determined  yet, should be implicated in the production of H2O2 required usually in excess by the peroxidases. All this facts have conducted us to conclude in the necessity of carrying out a more exhaustive study of the catalytic cycle of the peroxidases present in this population of monokaryons of P. ostreatus.

1 Santoyo, F., González, AE, Terrón,MC, Ramírez, L, Pisabarro, AG. (2007) Enz.. Microb. Technol. (in press).


Investigating the role of hydrolytic enzymes during the interaction between Trichoderma aggressivum and Agaricus bisporus

Kamal Abubaker, Alan Castle

Brock University, St. Catharines, Ontario, Canada

Green mould disease caused by the fungus Trichoderma aggressivum is considered to be one of the most serious problems of the commercial mushrooms Agaricus bisporus. The loss of millions of dollars in both Europe and North America has been caused by this disease. Since this parasitic interaction is not well understood, studying the mechanism leading to the development of Green mould disease will have a positive impact on the commercial mushroom industry. It has been reported that other Trichoderma spp. secretes hydrolytic enzymes such as chitinase, glucanase, and proteinase causing the damage to the cell wall of other fungi. In this study, we isolated and characterized the genes of three T. aggressivum f. aggressivum hydrolytic enzymes (endochitinase (ech42), β 1, 3 glucanase and alkaline proteinase (prb1)), and evaluated their expression profile over a period of seven days, throughout co-cultivation with mushroom white strain (U1) and brown strain (SB65) by qRT-PCR. The expression patterns of the three enzymes were found to be highly increased in the white strain compared to their expression in the T. aggressivum f. aggressivum in solitary culture. Whereas the expressions of the three enzymes were slightly expressed in the brown strain compared to their expression in the Trichoderma aggressivum in solitary culture. This investigation will serve to increase the understanding of the mechanism of disease development.


Phylogenetic analysis of some wood xylaraiaceae isolated from East Asian countries

Natarajan Velmurugan1, Cherdchai Phosrii2, Sang-Sub Han3, Dong-Min Sa4, Yang-Soo Lee1, Mi-Suk Choi1

1Department of Forest Science and Technology, Institute of Agricultural and Life Sciences, Chonbuk National University, Jeonju 561-756, Chonbuk, Republic of Korea, 2Department of Sciences, Faculty of Sciences & Technology, Piboonsongkram Rajabhat University, 156 Moo 5 Tambon Plychumpol Amphor Muang, Phitsanulok 65000, Thailand, 3Institute of Agricultural and Life Sciences, Chonbuk National University, Jeonju 561-756, Chonbuk, , Republic of Korea, 4College of Agriculture, LIfe & Environments Sciences, Chungbuk National University, Chungbuk 361-763, Republic of Korea

The nucleotide sequence of ITS 1, 5.8S rDNA and ITS 2 of 14 strains belong to Xylariaceae sp. were determined. On the analysis of sequences, a total number of 14 strains were identified and grouped in to 8. The XY-3008 and XY-3009 strains were belong to same group, and showed 89.96% identities with Xylaria banbusicora (Genebank accession no. EF026123). This results suggested that the XY-3008 and XY-3009 strains was clearly distinct with X. banbusicora sp. XY-3003 strain was high homologies with those of X. apiculata CBS 365(Genebank accession no. AF263027) and X. bambusicora. XY-3013 and XY-3014 were grouped with Daldinia sp. (Genebank accession no. AM749927) and Entonaema cinnabarium(Genebank accession no. AM292043) and XY-3005 was high identified with Hypoxyon monticulosm (Genebank accession no DQ223749). In group V, 97% similarity was observed between Hypoxyon stygium (Genebank accession no AJ390409), Annulohypoxyon stygium Genebank accession no DQ2237361) and XY-3004, XY-3012, XY-3012 strains. XY-3002, XY-3006 and XY-3007 strains were homologies with those of Xylaria multiplex (Genebank accession no DQ322155) and Rosellinia sp. ST2310 strains (Genebank accession no DQ322077). XY-3011 strain was clearly distinct those of deposited Xylariaceae in Genebank database, and only showed homologies 90% with Nemania illita (Genebank accession no EF026122). In this results indicated that the strains of XY-30008, XY-3009 and XY-3011, respectively, might represent a new group or subgroup into Xylariaceae.



Investigating global futures for regulation on ABS of genetic resources

Eun-Mi Kim1, Natarajan Velmurugan2, Dong-Min Sa3, Yang-Soo Lee2

1Department of International Trade, Chonbuk National University, Jeonju 561-756, Chonbuk, Republic of Korea, 2Department of Forest Science & Technology, Institute of Agriculture & Life Sciences, Chonbuk National University, Jeonju 561-756, Chonbuk, Republic of Korea, 3College of Agriculture, Life & Environments Sciences, Chungbuk National University, Chungbuk 361-763, Republic of Korea

Conservation and innovation are in conflict with one another because innovation cannot be made without a change. While the South part possessing genetic resources advocates conservation of genetic resource, the North part using the resources has a preference public use of genetic resources but need private property right for bio-industrial products. These contradict interests led to the negotiation and establishment of Convention on Biological Diversity (CBD) and the Trade Related Intellectual Property rights (TRIPs). The CBD negotiations originally focused on conservation alone. Soon, however, the negotiators included national sovereignty to genetic resources and a threefold objective- Conservation, Sustainable, Access and Benefit Sharing (ABS)- to reflect the worldwide concern to prevent unfair exploitation of the rich genetic wealth and traditional knowledge of the developing countries by the developed world. TRIPs agreement stimulates technological innovation through harmonization and strengthening of domestic patent legislation by all WTO members. The industrialized countries advocated it in order to ensure revenue from innovations in all technological fields, including biotechnology. Several scholars maintain that there is a potential conflict between CBD and TRIPs with regard to ABS because the two regimes operate with different approaches to property rights pertaining to genetic resources – hence, different objectives. This paper tries to anticipate the future for global regulation on ABS of genetic resources using a realist and ideational perspective. According to these theoretical frameworks implementation of the ABS of genetic resources would be hindered by the stronger regulatory force of the TRIPs for the following reasons. First while ABS advocates are generally politically weaker stakeholders the TRIPs is supported by stronger stakeholders including the US and the agribusiness and pharmaceutical industries. Second by industrialized member states pushed by strong multinational corporations, the WTO is stronger organization having capital and information necessary for negotiation. Third the WTO enforcement mechanism is strong enough to force member states to respect TRIPs.


Disruption of glucose repression in the industrially useful fungus Aspergillus oryzae

Adrian Hunter1, Joan Kelly1, Bo Jin1, Chris Saint2

1University of Adelaide, Adelaide, South Australia, Australia, 2SA Water, Adelaide, South Australia, Australia

The filamentous fungus Aspergillus oryzae has been used in Oriental food production for centuries, and its ability to secrete high levels of endogenous or heterologous enzymes facilitates its extensive use in the fermentation industry. We aim to utilise A. oryzae in a treatment process to convert glucose-rich winery wastes to protein-rich fungal biomass for use as livestock feed. In the model fungus Aspergillus nidulans, disruption of creB encoding a deubiquitinating enzyme leads to a relaxation of glucose repression, and elevated expression of a range of enzymes for the utilisation of non-preferred carbon sources. Here we present the deletion of creB in two strains of A. oryzae: RIB40, the strain that had its genome sequenced, and DAR3699, a strain with morphological properties well-suited to fermentation. We analyse the effects of creB deletion on the secretion of amylases, cellulases and other enzymes both in the presence and absence of glucose, and discuss implications for the use of A. oryzae in the bioconversion of wastes.


Utilization of a fusion protein to increase expression levels of a β-glucosidase in Trichoderma reesei

Sandy Merino, Suchindra Maiyuran

Novozymes Inc., Davis, CA, United States

Trichoderma reesei produces two cellobiohydrolases (CBH I and CBH II), five endoglucanases (EGI-V), and two β-glucosidases (BG). Cellulose is hydrolyzed to cellobiose, a water-soluble beta-1, 4-linked dimer of glucose, through synergistic action of cellobiohydrolases and endoglucanases. Cellobiose is then hydrolyzed to glucose by β-glucosidases. T. reesei secretes levels of β-glucosidase insufficient to hydrolyze all the cellobiose under conditions of high substrate concentration, resulting in product inhibition of CBH and EG enzymes and a reduced enzymatic hydrolysis of cellulose. Our goal is to increase the expression level of β-glucosidase was increased in order to alleviate product inhibition. Initially the BG from Aspergillus oryzae was recombinantly expressed in T. reesei, but expression levels were low. In order to improve expression the native signal sequence from the A. oryzae BG was replaced with a recombinant signal sequence from a Humicola insolens protein that is highly expressed in T. reesei. The swap in signal sequences increased BG expression and resulted in a 2-fold improvement in the conversion of cellulose to glucose at the substrate levels tested. Further improvement in BG expression levels were obtained by creating a fusion protein comprised of an endoglucanase catalytic domain fused at the N-terminus of BG. The fusion protein resulted in additional BG expression and further improvement of the cellulase activity in PCS hydrolysis assays.


Artificial cultivation of a wild species of Volvariella volvaceae

Victor Joseph Bella

University of YaoundeI, Yaounde, Cameroon

As the years go, edible mushrooms cultivation becomes familiar in the world. The total world production is increasing continuously. According to recent statistics, in spite of this progress, the African contribution abides the smallest. This delay is certainly due to many causes such as traditional believes, scarcity of local market, lack of technicity.

Studies show that varied and richest ecologies exist in tropical area where, in some countries, more than one hundred (100) mushrooms species can be found and sometimes ignored by the scientists. Contrary to this rich fungal abundance, all the fungi cultivated or suggested for cultivation are imported cultivars even if more adapted species could be found locally. According to empirical investigation, the Volvariella species, especially Volvariella volvaceae appears to be one of the more known edible mushrooms in tropical areas. It is also, appreciated by a lot of populations and its production should improve local mushrooms consumption. It cultivation involve, summary three mains stages. As following:

I Cell Culture

In this first stage, a cell culture is initiated from a piece of mushroom taken from a wild species sample.

II Seed production

In this second level, seed (mycelia) is prepared from the cell culture previously obtained for the third stage. Both stages take two weeks.

III Fruit body production

In the final phase, seed previously obtained is used inseminated in waste organic material in the goal to produce the final output: the fruit body.

Results: fruit body of wild Volvariella volvaceae grows as easily as imported species in less than two (2) months.

Discussion: the cultivation of local species of edible mushrooms must be encouraged because more adapted; many researchers should be occupied by local fungi because they certainly hide very highly efficient species; local species of mushrooms must be found to be popularize in tropical areas.



Chrysosporium lucknowense is a versatile fungal host for gene discovery and protein production

Hans Visser1, Peter Punt2, Jan Wery1, Dan Michalopoulos3, Jan Verdoes*1

1Dyadic Nederland BV, Wageningen, Netherlands, 2TNO Quality of Life, Zeist, Netherlands, 3Dyadic International Inc., Jupiter Florida, United States

Several filamentous fungi can secrete large amounts of protein into the growth medium and are therefore commonly used as hosts for the production of industrial enzymes. We have developed the ascomycetous fungus Chrysosporium lucknowense C1 for protein production on a commercial scale as an alternative to well known fungi like Aspergillus niger and Trichoderma reesei. Strain and process improvement strategies of the original C1 isolate resulted in strains that are able to secrete large amounts of a complex mixture of (hemi-) cellulases. Additionally, these strains show a strong reduction in culture viscosity (1) as a result of a morphology change. During fermentation the hyphae fragment in a structured way resulting in small fragments (propagules), which enables the application of C1 in high-throughput robotic screening systems (2). The low-viscosity property furthermore allows the use of richer media in fermentations. C1 can be cultivated in relatively short fermentation cycles within wide pH and temperature ranges. Strains having reduced protease activity and reduced protein background levels were designed in order to facilitate the production and down-stream processing of a protein of interest. Both homologous and heterologous genes were successfully expressed using a set of different expression signals and C1 host strains. As an integrated platform for gene discovery, gene expression, and protein production C1 provides benefits of reduced time lines and improved probabilities of success.

1 Burlingame and Verdoes (2006) BioPharm International (190), 40-47, 2 Verdoes et al (2007) Industrial Biotechnol. (3), 48-57.



Proteomic approach to study the interaction between wood decomposer fungus Stereum hirsutum with its competitor Coprinus disseminatus

Diluka Peiris, I Roy, M Dwek, John Hedger

University of Westminster, London, United Kingdom

During interspecific and intraspecific interactions, mycelia of wood decaying fungi release extra-cellular secondary metabolites. In a study of metabolite expression during mycelial interaction, we found that there is an up-regulation of metabolite production by wood decomposer fungus Stereum hirsutum when confronted with its competitor Coprinus disseminatus. This implies recognition of the presence of mycelia of other fungi and the switching on of a combative response. Two-dimensional electrophoresis was used to study the cellular protein patterns of self-paired and confronted mycelia of S. hirsutum. Protein profiles were compared using Progenesis SameSpots Software (Non-Linear Dynamics) to identify the S. hirsutum proteins up- or down-regulated in the presence of mycelia of C. disseminatus. In the confronted mycelia of S. hirsutum twelve proteins were detected as being up-regulated, and five were detected as being down-regulated, compared to self-paired mycelia. Six differentially expressed protein spots were identified using tandem mass spectrometry (MS-MS). Identified up-regulated proteins included NADH-quinone oxidoreductase, lytic transglycosylase catalytic, a heat shock protein and a hypothetical protein from Coprinus cinereus. Two down regulated proteins were identified as hypothetical proteins from C. cinereus. The results show proof of principle for the employment 2-dimensional electrophoresis for biological studies of mycelial interactions.


Building an efficient cell factory for Aspergillus oryzae

Wanwipa Vongsangnak1, Kim Hansen2, Peter Olsen2, Steen Krogsgaard2, Jens Nielsen3

1Technical University of Denmark, Lyngby, Denmark, 2Novozymes A/S, Bagsvaerd, Denmark, 3Chalmers University of Technology, Gothenburg, Sweden

An important filamentous fungus Aspergillus oryzae has been used in the fermentation industry for hundreds of years to produce a large number of industrial enzymes and fermented sauces. However, the cellular mechanisms governing these processes are poorly established and understood. We are combining an improved genome annotation through building stoichiometric metabolic model and transcriptomics level to reveal these mechanisms. Using a large set of assembled Expressed Sequence Tag (EST) data and different advanced bioinformatics tools for improved annotation we identified 1,046 newly predicted genes in the genome. Furthermore, it was possible to assign putative protein functions to 398 of the newly predicted genes. Moreover, our annotation strategy resulted in assignment of new putative functions to 1,469 hypothetical proteins already present in the A. oryzae genome database. Using the improved annotated genome with available biochemical pathway databases and literature, we reconstructed the metabolic network of A. oryzae. This network contains 729 enzymes, 1,314 enzyme-encoding genes (10% of the 13,120 total predicted genes), 1,073 metabolites and 1,846 (1,053 unique) biochemical reactions. The metabolic reactions are compartmentalized into the cytosol, the mitochondria, the peroxisome and the extracellular space. Transport steps between the compartments and the extracellular space represent 281 reactions, of which 161 are unique. The metabolic model was validated and shown to correctly describe the phenotypic behavior of A. oryzae grown on different carbon sources. In addition, the model was integrated with transcriptomics data to elucidate overall metabolic response of A. oryzae to alteration of carbon sources. The model serves as an important resource for gaining further insight into our understanding of cell factory A. oryzae.


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

Erzsebet Fekete1, Levente Karaffa1, Michel Flipphi2, Bernhard Seiboth3, Attila Szentirmai1, Christian Kubicek3

1University of Debrecen, Debrecen, Hungary, 2CSIC, Valencia, Spain, 3Technical University Vienna, Vienna, Austria

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

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

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


Modelling the conidial aggregation of filamentous fungi by population dynamics

P.-J. Lin , L.H. Grimm1, M. Wulkow2, D.C. Hempel1, R. Krull1

1TU Braunschweig, Braunschweig, Germany, 2Computing in Technology GmbH (CiT), Rastede, Germany

In industrial fermentation processes filamentous fungi are widely applied. Process design, however, is often hampered by the complex growth pattern of these organisms. Filamentous fungi were classified into non-coagulating and coagulating types according to their mechanism of pellet formation. For coagulating filamentous fungi like Aspergillus niger, conidial aggregation is the first step of filamentous morphogenesis and influence subsequently the development of pelleted morphology. For a proper description of this phenomenon it is therefore important to characterize conidial populations to assess the impact of the aggregation process. Conidial aggregation kinetics based on the time-dependent development of particle concentration studied with an in-line particle size analyzer (FBRM D600L, Lasentec) suggested that two separate stages and mechanisms of aggregation can be distinguished. The first step of conidial aggregation starts immediately after inoculation. Both the rate constants of formation and disintegration of aggregates have been determined by measuring of the concentration of conidia at the beginning of the cultivation and the concentration of particles at steady state during the first hours of cultivation. In contrast to the first aggregation step, where the collision of conidia is presumed to be responsible for the process, the second aggregation step is thought to be initiated by germination of conidia. Growing hyphae provide additional surface for the attachment of non-germinated conidia, which leads to a strong decrease in particle concentration. The specific hyphal length growth rate and the ratio of particle concentration to the growing adhesion hyphal surface are decisive matters of the second aggregation step. Both aggregation steps was comprehensively characterized, described by population dynamics which is the development of a particle collective over the time, e.g. the temporal change of the number distribution of particle sizes and the applied population balance is validated by comparison of experimental data and simulation with the program package PARSIVAL (PARticle SIze eVALution, CiT) which was successfully applied to the description of crystallization processes, aerosols and more. The population balance model presented here is meant to enhance process control by describing the basic aggregation behaviour of fungal conidia.



Ergosterol rich cap in membrane of germinating conidia

Richard van Leeuwen1, Wieger Smant2, Wietse de Boer2, Jan Dijksterhuis1

1Fungal Biodiversity Centre (CBS-KNAW), Utrecht, Netherlands, 2Centre for Terrestrial Ecology (NIOO-KNAW), Heteren, Netherlands

The polyene antibiotic filipin is commonly used as a fluorescent marker for sterol distribution in fungal cells, but it is also a potent polyene antibiotic. Here, we evaluate the reliability of filipin as a sterol marker in fungal cells. Our staining protocol identifies the appearance of a fluorescent cap on germinating conidia of the fungus Penicillium discolor at the location of germ tube formation. During germ tube emergence, the fluorescent intensity of the cap increases. This is confirmed by HPLC as an increase in ergosterol content. The fungal cell wall might display variable permeability for filipin and as a result might suggest certain filipin "hot spots" at, for instance, growing apices of fungal hyphae. We confirmed that membrane staining was evenly distributed as judged by FM4-64 staining of conidia. This shows that the conidial cell wall is permeable for similar molecules. To test if filipin binds to ergosterol in conidia, cells of mutants of Saccharomyces cerevisiae were quenched with natamycin, another polyene more specific for ergosterol. These erg deletion mutants contain an altered set of sterols and allowed us to determine the binding specificity of filipin in situ. Quenching of fluorescence in conidia was characteristic for high ergosterol levels. Taken together, these data suggest that filipin monitors ergosterol in germinating conidia. Furthermore, the sterol rich cap is affected by actin – and sphingolipid modulating compounds showing that membrane organization is connected to other cell processes and has an important role in morphogenesis in fungi.


Structure and function of the septal pore cap (SPC) in basidiomycetes

Arend F van Peer1, KGA van Driel3, T Boekhout3, WH Müller2, HAB Wösten1

1Utrecht University, Department of Microbiology, Utrecht, Netherlands, 2Utrecht University, Department of Cell Biology, Utrecht, Netherlands, 3Fungal Diversity Centre (CBS), Utrecht, Netherlands

Fungal septa are crucial for differentiation processes (e.g. sporulation) and damage control and they may have other functions as well. The septa of vegetative hyphae of ascomycetes and basidiomycetes are perforated. Those of ascomycetes are relatively simple structured, while those of basidiomycetes can be very complex. Complex septa contain a characteristic rim around their central pore, the dolipore, which in higher basidiomycetes is often covered by a septal pore cap (SPC) also called parenthesome. Despite extensive ultra-structural research in the past, their composition and function is still unknown.

To understand the function of the SPC of the model basidiomycete Schizophyllum commune, we purified these organelles using discontinuous sucrose gradients and filtration over a 450nm filter. Addition of detergent increased the buoyant density of SPCs, which indicates the removal of membranous material (ER) covering a proteinaceous core. The characteristic dome shape of the SPC was maintained during the whole process as observed by Transmission Electron Microscopy.

SDS-PAGE analysis of purified SPCs showed three proteins of 14 kDa, 33 kDa and 60 kDa of which the 14 kDa protein was particularly abundant. The N-terminal sequence and MS-MS patterns of the 14 kDa and 33 kDa proteins were determined and their corresponding genes isolated. Interestingly, homologues of both genes can only be found in fungi that possess SPCs. Immunogold labelling indicates that the 14 kDa protein indeed localizes to the SPC and over expression of the encoding gene results in an altered ultra- structural morphology of the SPC region. Currently we are studying the function of the SPC by gene deletions of both genes and fluorescence microscopy.



Exploring the transcriptome of Trametes versicolor during mycelial interactions with other saprotrophic basidiomycetes

Catherine Eyre, Jennifer Evans, Wafa Muftah, Lynne Boddy, Hilary Rogers

Cardiff University, Wales, United Kingdom

Saprotrophic basidiomycetes play a key role in decomposition and nutrient cycling within woodland ecosystems. Species compete for space and resources, resulting in interactions with a variety of outcomes, ranging from deadlock to replacement. Trametes versicolor was chosen to study changes in gene expression during these interactions. Suppression subtractive hybridisation libraries were constructed for the interaction of T. versicolor vs S. gausapatum, during which T. versicolor replaces S. gausapatum. This is one of the first studies to examine wide-scale transcriptional changes during interspecific interactions of saprotrophic basidiomycetes. 1824 expressed sequence tags (ESTs) were sequenced from these libraries, and compared to available databases. Microarray analysis was used to compare the expression profiles of these genes during three different types of interaction: replacement of S. gausapatum, deadlock with Bjerkandera adusta and replacement by Hypholoma fasciculare. Analysis revealed up-regulation of peroxidases, catalase, chaperone proteins and fungal cell wall enzymes, common to interactions. These genes may be employed to deal with an oxidative environment and intracellular damage generated during interactions, and responsible for changes in morphology. More genes were common to interactions in which T. versicolor deadlocked with, or replaced its competitor, than when it was replaced itself. Different mechanisms may be employed against different species resulting in the range of outcomes observed.


A spatial and temporal changes of Aspergillus niger cytosolic pH imaged using genetically encoded pH sensor

Mojca Bencina1, Tanja Bagar1, Kirsten Altenbach2, Nick Read2

1Department of Biotechnology, National Institute of Chemistry, Ljubljana, Slovenia, 22Fungal Cell Biology Group, Institute of Cell Biology, University of Edinburgh, Edinburgh, United Kingdom

A fundamental role of pH as signaling component of cell processes is not yet established. An accurate measurement of intracellular pH in unperturbed cells of filamentous fungi is fraught with difficulties, however, necessary for explaining its function.

To study spatial and temporal pH changes in vivo in hyphae of industrially important filamentous fungi Aspergillus niger, a genetically encoded pH probe was modified for expressing in fungi.

We showed that a pH sensitive derivative of green fluorescent probe, a ratiometric RaVe_C, which was codon optimized, could be used to image intracellular pH in vivo in fungal cells. RaVe_C has a ratiometric dual excitation and displays reversible emission ratio changes in the range from pH 5.5 to pH 8.0. With a pKa of 7.0 RaVe_C is ideal for monitoring pH changes in the cytosol.

In vivo calibration using nigericin proved that the RaVe_C probe is highly pH sensitive in living fungal hyphae, therefore RaVe_C can successfully be used as a noninvasive genetically encoded intracellular sensor.

Examination of cytoplasmic pH in growing cells was performed by simultaneous, dual excitation confocal ratio imaging of the ratiometric pH probe RaVe_C. Based on in vivo calibration, estimated value of mean cytoplasmic pH for cells expressing RaVe_C probe was 7.5. No significant cytoplasmic pH gradient was observed along growing hyphae. Sifts of external pH to acidic or alkaline pH values caused a temporal acidification or alkalization in cytoplasmic pH. Stimulation or inhibition of pH homeostasis by pharmacological agents resulted in rapid decrease of pH.



The nuclear migration protein NUDF/LIS1 forms a complex with NUDC and BNFA at spindle pole bodies

Kerstin Helmstaedt, Karen Laubinger, Katja Voßkuhl, Özgür Bayram, Silke Busch, Michael Hoppert, Oliver Valerius, Stephan Seiler, Gerhard H. Braus

Georg-August-Universität, Institut für Mikrobiologie und Genetik, Göttingen, Germany

Nuclear migration depends on microtubules, the dynein motor complex and regulatory components like LIS1 or NUDC. We sought to identify new binding partners of the fungal LIS1 homolog NUDF to clarify its function in dynein regulation. Therefore, we analysed the association between NUDF and NUDC in Aspergillus nidulans. NUDF and NUDC directly interacted in yeast two-hybrid experiments via NUDF's WD40 domain. NUDC-GFP was localized to immobile dots in the cytoplasm and at the hyphal cortex, some of which were spindle pole bodies (SPBs). We showed by bimolecular fluorescence complementation microscopy that NUDC directly interacted with NUDF at spindle pole bodies at different stages of the cell cycle. Applying tandem affinity purification, we isolated the NUDF-associated protein BNFA (binding to NUDF). BNFA was dispensable for growth and for nuclear migration. GFP-BNFA fusions localized to SPBs at different stages of the cell cycle. This localization depended on NUDF, since loss of NUDF resulted in cytoplasmic accumulation of BNFA. BNFA did not bind to NUDC in a yeast two-hybrid assay. These results show that the conserved NUDF and NUDC proteins play a concerted role at spindle pole bodies at different stages of the cell cycle and that NUDF recruits additional proteins specifically to the dynein complex at spindle pole bodies.


Autophagy in the filamentous ascomycete Sordaria macrospora

Nicole Nolting, Nicol Kruska, Stefanie Pöggeler

Georg-August-University, Göttingen, Germany

Autophagy is a highly conserved eukaryotic process in which cytoplasmic constituents are sequestered within a double membrane vesicle and delivered to the vacuole for the recycling of macromolecules. This well-known degradation system is involved in development and differentiation in eukaryotes. In filamentous ascomycetes, autophagy has been shown to participate in various processes such as conidiospore and aerial hyphae formation, as well as heterokaryon incompatibility. However, the impact of autophagy on fruiting-body development in filamentous ascomycetes is largely unknown. The purpose of this study was to determine the role of autophagy in the filamentous ascomycete Sordaria macrospora, especially on fruiting-body formation.

For this aim, in a first approach we transformed a construct containing the P. anserina atg8 gene fused to a gfp-tag which has previously been used as a cytological marker of the induction of autophagy (1). The localization of the GFP-ATG8 fusion protein and the detection of autophagic bodies inside the vacuoles of older hyphae provide first evidence of autophagy within developmental processes in S. macrospora.

In a second approach, we intend to investigate the role of conserved components of the autophagy machinery. We identified three genes already described to play an important role during different steps of autophagy in S. cerevisiae (atg7 and vps34) and P. anserine (idi4). These three genes are analyzed by means of knockout construction, overexpression and localization studies. Based on these experiments, detailed investigations concerning morphological changes in hyphal growth and sexual development are in process and will be presented.

1 Pinan-Lucarré B, Paoletti M, Dementhon K, Coulary-Salin-B, Clavé C (2003) Mol Microbiol 47: 321-333


Spatially segregated SNARE protein interactions in the filamentous fungus Trichoderma reesei

Mari Valkonen1, Markku Saloheimo1, Merja Penttilä1, Nick Read2, Rory Duncan2

1VTT, Espoo, Finland, 2University of Edinburgh, Edinburgh, United Kingdom

The machinery for trafficking proteins through the secretory pathway is well conserved in eukaryotes, but remains poorly characterized in filamentous fungi. Secretion is believed to be highly polarized in fungal hyphae and to mainly occur from hyphal tips. We describe the isolation of the snc1 and sso1 genes encoding exocytic SNARE proteins from Trichoderma reesei. The encoded SNCI protein can complement Snc protein depletion in S. cerevisiae whilst the T. reesei SSOI protein was unable to complement depletion of its yeast homologues. The localization and interactions of the T. reesei SNARE proteins were studied with advanced fluorescence imaging methods using fluorescent fusions of the SNARE proteins. The SSOI and SNCI proteins co-localized in sterol-independent clusters on the plasma membrane in sub-apical but not apical hyphal regions. The v-SNARE SNCI (but not the t-SNARE SSOI) localized to the apical vesicle cluster within the Spitzenkörper of the growing hyphal tips when expressed from the homologous T. reesei cbh1 promoter. Using fluorescence lifetime imaging microscopy (FLIM) and fluorescence energy transfer (FRET) analysis, we quantified the interactions between these proteins with high spatial resolution in living cells. Our data showed that the site of SNARE complex formation between these proteins is on the plasma membrane of non-growing hyphae in old sub-peripheral regions of the colony, but that there is no interaction between the proteins in growing hyphal tips in at the colony margin. These findings suggest spatially distinct sites of exocytosis within filamentous fungi and the existence of multiple exocytic SNAREs which are functionally and spatially segregated.



Analysis of the mating-type genes from the filamentous ascomycete Sordaria macrospora

Volker Klix, Stefanie Pöggeler

Georg-August University, Göttingen, Germany

The filamentous ascomycete Sordaria macrospora is homothallic and in contrast to heterothallic fungi needs no mating-partner for sexual reproduction. In the closely related, heterothallic ascomycete Neurospora crassa the mating type of the haploid mating-partners is genetically determined by the mating-type locus. In N. crassa the mat-locus is an allele pair, which can be present in two different forms (mat a and mat A). This mating system is referred to as a bipolar mating system.

The mating type of S. macrospora consists of four genes (SmtA-1, SmtA-2, SmtA-3 and Smta-1) which share similarities to mating-type genes from N. crassa mat A- and mat a-strains. The S. macrospora mating-type genes code for putative transcription factors (SmtA-1, Smta-1) or proteins without characteristic DNA-binding motifs (SmtA-2, SmtA-3).

Knockout mutants of the S. macrospora mating-type genes SmtA-1, SmtA-2 and SmtA-3 were generated by gene replacement. The same method was used to create over-expression strains of each gene. Fluorescence microscopy was used for localisation studies of GFP-tagged mating-type proteins.

In contrast to the N. crassa mat A-1 the S. macrospora SmtA-1 does not seem to be an essential regulator of the sexual development. D SmtA-1 knockout mutants showed reduced fertility but were still able to produce sexual ascospores. Instead, the knockout mutant of SmtA-2 was sterile and could no longer complete the sexual cycle. This phenotype is similar to the Smr1 knockout of Podospora anserina. Knockout mutants of SmtA-3 showed no phenotype.

The results of this study help to gain deeper insight into the role of mating-type genes in homothallic ascomycetes. The non-essential function of SmtA-1 for the sexual development contrasts the role of homologous genes from other homothallic or heterothallic ascomycetes, whereas the phenotype of the D SmtA-2 knockout mutant shows similiarities to P. anserine but not to the closely related N. crassa. These results provide evidence for functional loss or changes between mating-type genes in the process of evolution in homothallic and heterothallic fungi.


Spatial differentiation of mannitol dehydrogenase and mannitol-1-phosphate dehydrogenase explains the absence of a mannitol cycle in Aspergillus niger

Guillermo Aguilar1, Patricia vanKuyk2, Dirk Blom1, Arman Vinck1, Han Wosten1, Ronald de Vries1

1Microbiolgy, Utrecht University, Utrecht, Netherlands, 2Molecular Biology, Leiden University, Leiden, Netherlands

Mannitol is the most abundant polyol in spores from Aspergillus niger and disappears during early germination. The presence of a mannitol cycle has been suggested in several fungi in which mannitol is produced from fructose-6-phosphate by mannitol-1-phosphate dehydrogenase (MPD) and mannitol-1-phosphate phosphatase and converted back to fructose-6-phosphate by mannitol dehydrogenase (MTD) and hexokinase. Speculations into the function of such a cycle mainly focus on its role in the production of NADPH, based on the presence of all the enzymes from the pathway in fungal mycelium. However, no effects on NADPH production could be observed when either of the dehydrogenase-encoding genes was disrupted in several fungi. Here we demonstrate that spatial differentiation occurs with respect to the expression of mpdA and mtdA using GFP and dTomato fused to the promoters of these genes. While mpdA expression occurs in substrate hyphae, expression of mtdA is restricted to conidia. These data indicate that MPD is involved in mannitol sythesis and MTD in mannitol degradation, but that these processes do not occur in the same part of the colony.



Aspergillus nidulans conidiation inducing factors FlbB and FlbF are localised in association with the polar growth machinery

Oier Etxebeste1, Aitor Garzia1, Eduardo A. Espeso2, Unai Ugalde1

1University of The Basque Country, Donostia-San Sebastian, Gipuzkoa, Spain, 2Centro de Investigaciones Biológicas (CSIC), Madrid, Spain

Asexual reproduction (conidiation) in Aspergillus nidulans is a complex morphogenetic process that requires the activation of the transcriptional regulator BrlA as the fisrt and central element. This depends on the action of upstream acting elements, which define the early induction pathway.

This work presents new aspects on two of these upstream acting elements, FlbB and FlbF, and demonstrates that their action is also required beyond the upstream activation period. Both factors are probably located very close to or within the Spitzenkörper and backedby the endocytic machinery. Metabolic energy and an organised F-actin are required to maintain this location. FlbB is apically maintained throughout the cell cycle in vegetative cells. As yet unidentified processes provoke migration of FlbB to the most apical nucleus, where it purportedly binds DNA, as a bZIP transcription factor. On exposure to the air, FlbB is accumulated in all nuclei of the exposed hyphae, and during conidiophore development, it is located in elongating apex of the growing metulae.

We conclude that FlbB and FlbF are associated to polar extension growth machinery where they apparently exert no majour role. They appear rather to fulfill a reporter function on suitable conditions for the onset of conidiogenesis.



Endogenous bicarbonate levels regulate mycelial growth and morphology in Aspergillus nidulans

Ana Rodriguez, Aitor Garzia, Carlos Jimenez, Unai Ugalde

1University Of the Basque Country, Donostia-San Sebastian, Gipuzkoa, Spain, 2University of A Coruña, Faculty of Science, A Coruña, Spain

When Aspergillus nidulans was cultured on a 1 cm2 dialysis membrane squares overlaying minimal medium, the mycelium expanded beyond the membrane boundary after 48h at 37ºC. When the membrane was removed, the underlying clear patch was not recolonized despite nutrient supplementation. More detailed experiments showed that growth inhibition is proportional to the length of time at which pre-growth had taken place on the membrane, reaching 100% inhibition after 72h of pre-growth. Chemical analysis of the ‘spent’ medium, combined with an inhibition bioassay revealed that the inhibiting factor was a mixture of sodium and potassium bicarbonate. Under nitrate based growth, progressive medium alcalinisation occurs resulting in fixation of CO2 as bicarbonate beyond pH 6.3. At concentrations ranging between 0-50mM, bicarbonate exerts a progressive effect on extension rate (Kr) as well as the hyphal density of mycelia. The effect of bicarbonate is fungistatic, resulting in progressively higher number of cells arrested at G1. Their survival in this state is dependent on the provision of nutrients. Upon dilution of bicarbonate, viable cells resume growth normally. The results will be discussed in the context of bicarbonate as an endogenous regulator of within the mycelium.



Transcriptional regulation during the developmental programmes in the thermo-dimorphic fungal pathogen Penicillium marneffei

David Canovas1, Luke Pase2, Gordon Smyth2, Alex Andrianopoulos1

1Deptarment of Genetics - University of Melbourne, Melbourne, Vic, Australia, 2Walter and Eliza Hall Institute of Medical Research, Melbourne, Vic, Australia

Penicillium marneffei is a thermally dimorphic fungal pathogen of humans. At 37 ºC, the growth form is an elongated unicellular and uninucleated yeast, which divides by fission. At 25 ºC, growth resembles that of other Penicillium species where hyphal cells are separated by incomplete septa, and are usually multinucleated. Besides the dimorphic switch, P. marneffei can also undergo asexual development (conidiation) at 25 ºC. The different cell types produced during these developmental programmes are strictly controlled. In order to identify new genes involved in these two processes, we have screened for genes which are differentially expressed in the various stages of these developmental programmes by using DNA microarrays. We constructed slides containing an array of 5,000 clones originated from a random genomic DNA library. To identify genes expressed at the different stages of those developmental programmes, target cDNAs from vegetative hyphal growth at 25ºC, conidiation at 25ºC and yeast growth at 37ºC were made. Those clones that were differentially expressed were identified by DNA sequencing. A number of genes, including cell cycle regulators, transcriptional regulators, cytoskeletal proteins, proteins involved in signal transduction, and both primary and secondary metabolic genes were shown to be differentially expressed during conidiation and/or dimorphic switching in P. marneffei. Based on these results, a model for fungal dimorphism in P. marneffei is proposed.


The formin Dia1 is essential for septation during budding and filamentous growth in the pathogen Ustilago maydis

Susanne Rindt, Michael Bölker, Björn Sandrock

Philipps-University Marburg, Marburg, Germany

Formin proteins are conserved proteins, which are involved in the septation process during cytokinesis in fungal cells. In the basidiomycetous fungus Ustilago maydis septation occurs during budding and during filamentous growth. During budding a complex network composed of the guanine-nucleotide exchange factor Don1, the small GTPase Cdc42 and the germinal-centre kinase Don3 regulates the formation of the secondary septum.

In the U. maydis sequence database MUMDB we have identified two genes expressing the formins Dia1 and Dia2, whose properties and specificities we wanted to characterize. Interestingly, dia1 expression is up-regulated during filament development, whereas dia2 expression remains unchanged on a very low level.

In this study, we investigated the diaphanous-related formin Dia1. We found that deletion of dia1 results in a delay of secondary septum formation during budding. Co-inoculation of compatible dia1 cells results in normal filament formation. However, the tumour formation rate in infection assays on corn seedlings is dramatically reduced for dia1 cells. Detailed studies have revealed that dia1-filaments grow slower than comparable wt-filaments. Surprisingly, dia1-filaments do not contain retraction septa. In wt-filaments, these septa are built to separate the growing tip compartment from empty cytoplasmic sections.

Complementation with a dia1-expressing cassette restores retraction septa formation and tumour development.

This observation of the incapability to form a septum forced us to study retraction septa formation in filaments of don1, cdc42 and don3 cells. Interestingly, filaments of don1 and don3 cells lack such septa but can induce tumours whereas cdc42 cells do not form retraction septa and are unable to induce tumours suggesting an additional role of Cdc42 during plant infection. Using two-hybrid studies we could demonstrate that Dia1 interacts with active Cdc42 via the GTPase binding domain indicating that Dia1 is an effector of Cdc42.

We conclude that formation of the secondary septum during the budding process and of the retraction septum during filamentous growth in U. maydis is based on the same protein network.


Differentiation of the hyphae of Phlebiopsis gigantea from the conifer pathogen Heterobasidion annosum at barrage zones with confocal microscope

Fred Asiegbu

University of Helsinki, Helsinki, Finland

The saprophytic fungus Phlebiopsis gigantea has for several years been used as a biocontrol agent against the conifer pathogen (Heterobasidion annosum). However, very little is known about the mechanism behind P. gigantea mode of action. At cellular level, there have been suggestions that hyphal interference may be involved during the combative interaction. But distinguishing the hyphae of these two fungi within wood tissues poses a major challenge. The present study explored the feasibility of using a combined dual immunofluorescence labeling and confocal microscopy for detection of the two species in vivo. Over 48 monoclonal antibodies were screened using ELISA (Enzyme Linked Immunosorbent Assay) for their ability to specifically recognize hyphae of either P. gigantea or H. annosum. Two monoclonal antibodies met these criteria and were further investigated. The result revealed that with the aid of the dual immunofluoresence labeling technique it is possible to distinguish the hyphae of the two fungi within the barrage zone. Furthermore, at present, it is not clear what the antibody binds to, identifying the protein which the antibody binds to will hopefully assist studies on protein tagging and further exploitation of this technology in environmental monitoring.


Insights in growth and protein formation of Aspergillus niger on a molecular level

Katina Kiep, Kathrin Bohle, Yvonne Göcke, Rainer Krull, Dietmar C. Hempel

Technische Universität Braunschweig, Institute of Biochemical Engineering, Braunschweig, Germany

The filamentous fungus Aspergillus niger is known for its high secretion efficiency and capacity of homologous proteins and is therefore an important production strain in biotechnological processes, especially in the field of nutrition additives. Furthermore, its ability to carry out post-translational modifications results in an increasing usage of A. niger as production host for the formation of therapeutic agents. However, the obtainable yields of recombinant proteins are considerably lower than of homologous proteins. Hence, current research is focussed on genetically engineered strain improvement and the optimization of cultivation processes to increase the production of desired recombinant products.

Depending on the environmental parameters A. niger growth occurs either as distinct pellets or as freely dispersed mycelia in submerged cultivations, whereas the optimal type of morphology varies due to specific product properties. Protein secretion is mainly observed in the region of growing hyphal tips. In this study, the production of a recombinant protein is analysed in submerged cultivations of a genetically modified A. niger strain. Its genome comprises extra copies of the homologous beta-fructofuranosidase gene regulated by a constitutive promoter. The influences of different environmental parameters on morphology as well as the resulting productivity in regard to the recombinant product are investigated.

The transcription activity of genes involved in hyphal branching, septation and polarized growth and its alteration in course of the cultivation process are quantified by real-time PCR. Results are related to microscopic images. Furthermore, the corresponding gene expression of beta-fructofuranosidase as model product is monitored via real-time PCR. The resulting enzyme activities within the bulk phase of the cultivation and the biomass-linked product activity are determined.

As a result, the presented work intends to elucidate the influence of different environmental parameters on the morphological development even on the transcriptional level. This should allow a thorough characterization of protein formation as well as product secretion in dependency of morphological structures.



Conidiation and dimorphic switching in the human pathogen Penicillium marneffei

Kylie Boyce, Alex Andrianopoulos

The University of Melbourne, Parkville, Vic., Australia

Penicillium marneffei is an emerging human pathogenic fungus which causes a fatal systemic mycosis. Like many other fungal pathogens, P. marneffei exhibits dimorphic growth and hence can grow as two distinct cellular forms; unicellular yeast and multicellular hyphae. P. marneffei is the only known Penicillium species which is dimorphic and the switch between growth forms is regulated by temperature. At 25° C, in the saprophytic growth phase, P. marneffei grows as multinucleate, septate, branched hyphae. These hyphae produce conidia, the infectious agent, from specialised multicellular structures termed conidiophores. When switched to 37° C, P. marneffei undergoes arthroconidiation where cellular and nuclear division become coupled, double septa are laid down and hyphae fragment at these septation sites to liberate uninucleate yeast cells which consequently divide by fission. The yeast cells are the pathogenic form. The ability to produce infectious propagules such as asexual spores (conidiation) in the saprophytic growth state and the capacity upon infection to switch between a multicellular hyphal growth form and a unicellular yeast pathogenic form are both crucial for pathogenicity. We have identified a gene, pakB, which regulates both conidiation and dimorphic switching in P. marneffei. pakB encodes a serine/threonine kinase which is specifically expressed at 25° C. Deletion of pakB results in defects in ‘budding-like’ division such as during the formation of conidia from secondary sterigmata during conidiation. Conidia of the pakB deletion strain appear swollen and misshapen. In addition, the pakB deletion strain inappropriately produces yeast cells at 25° C. To investigate if the yeast cells produced by the pakB deletion are a result of defects in conidiation, a brlA pakB double deletion has been generated. In addition, forced expression of pakB constitutive alleles at 37° C results in swollen yeast cells, which in contrast to wildtype yeast cells which divide by fission, divide in a budding manner. These results suggest that the developmental pathways regulating conidiation at 25° C and yeast cell production at 37° C share a number of regulatory components and that the developmental outcomes of each pathway is regulated in part by the mode of cellular division.


Rapid tip-directed movement of Golgi-equivalents in growing Aspergillus nidulans hyphae suggests a mechanism for delivery of growth-related materials.

Michelle Hubbard, Susan Kaminskyj

Univ. Saskatchewan, Saskatoon SK, Canada

Fungal Golgi equivalents process and sort materials in the fungal secretory pathway. Despite the importance of localized secretion in fungal tip growth, Golgi behaviour in living hyphae has not previously been documented. We used an Aspergillus nidulans strain containing the predicted Golgi marker CopA:GFP in a hypA1 ts polarity-defective background (a gift of A. Breakspear and S. Assinder) to study Golgi distribution in growing wildtype and polarity-defective phenotype hyphae. CopA has been shown to have high homology to Saccharomyces cerevisiae alpha-COPI. Predominant Golgi localization of CopA:GFP was confirmed by inserting a tagged copy of the established Golgi marker alpha-2,6-sialyl transferase (ST-RFP) and finding a high degree of co-localization. As expected, the patterns of both markers became more diffuse following treatment with brefeldin A. We used CopA:GFP to study Golgi behaviour in growing A. nidulans hyphae using time-lapse confocal fluorescence microscopy. In wildtype A. nidulans hyphae, Golgi were more abundant near hyphal tips than subapically, as had previously been shown with CopA:GFP in fixed cells. A. nidulans Golgi move independently of each other, in all directions, but predominantly and more rapidly toward the hyphal tip. When hypA1 restrictive phenotype cells re-established polarity at 28 ºC, Golgi moved preferentially into the newly-formed branches. The average rate of tipward Golgi movement was positively correlated with but at least ten-fold faster than the hyphal growth rate in the same cells. This relationship held under all experimental conditions tested. At 5 μg/mL, the actin inhibitor latrunculin B significantly decreased tipward Golgi motility and tip growth rate, whereas at 1 μg/mL the microtubule (MT) inhibitor increased tipward Golgi motility despite decreased growth rate. The MT stabilizing drug taxol, which we had previously shown to increase cytoplasmic MT number without affecting growth rate, increased tipward Golgi movement but again without increased hyphal growth rate. The cytoskeletal basis for Golgi motility appears to be complex. Our data support a model for apical delivery of A. nidulans tip growth materials in which Golgi play a role in long-distance transport.


Sexual development and morphological consequences of mating partner recognition in Hypocrea jecorina

Verena Seidl, Christian P. Kubicek, Monika Schmoll

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

The term "anamorphic fungi" is traditionally used for fungi without a known teleomorph and which are incapable of sexual reproduction under laboratory conditions. However, the application of molecular phylogenetics has more recently identified an increasing number of previously unknown anamorph-teleomorph relationships. In addition, cryptosexuality was shown in several anamorphic fungi by the demonstration of a phylogenetic history of recombination and the presence of genes encoding components of the mating system.

The fungus Trichoderma reesei, widely used for large-scale production of cellulolytic and hemicellulolytic enzymes, has been previously identified to be an asexual clonal line of the tropical ascomycete Hypocrea jecorina. In the laboratory, however, sexual crossings could not be observed between T. reesei and teleomorphic H. jecorina strains.

We have applied a genomic approach to identify two pheromone precursor genes in T. reesei QM9414, and we obtained evidence that both of them, a standard alpha-type peptide pheromone precursor gene (ppg1) and an unusual a-type peptide pheromone precursor gene (min1), are transcribed during the life cycle of T. reesei QM9414. Further, we could show that contact of strain QM9414 with H. jecorina CBS999.97 leads to the formation of fruiting bodies in H. jecorina CBS999.97 and that gene deletion of min1 abolishes this process. Addition of the MIN1 peptide initiates growth arrest in this strain. These findings were complemented by a microscopical analysis of the morphological changes which take place after recognition of a mating partner and the initiation of fruiting body formation in H. jecorina.



Pseudomonas aeruginosa secreted factors dramatically reduce adherence and biofilm formation in Candida albicans

Gordon McAlester1, Carol Munro2, Chen Ding3, Brice Enjalbert2, Neil Gow2, Geraldine Butler3, Fergal O’Gara1, John Morrissey1

1University College Cork, Cork, Ireland, 2University of Aberdeen, Aberdeen, United Kingdom, 3University College Dublin, Dublin, Ireland

In human conditions such as cystic fibrosis (CF), prokaryotic and eukaryotic microorganisms co-exist and are likely to interact. The effect that this interkingdom communication has on disease progression remains largely unknown. Elaborate signaling interactions have recently been shown to exist between Candida albicans and Pseudomonas aeruginosa. Initially P. aeruginosa was shown to attach to and kill C. albicans hyphae, having no effect on the yeast form, and to block the yeast-hyphal transition using a secreted signal molecule called 3-oxo-C12HSL (3-o-C12). Furthermore, farnesol, a signal molecule produced by C. albicans, reduces production of some virulence factors in P. aeruginosa. We tested a number of P. aeruginosa CF clinical isolates for production of 3-o-C12 and found that levels of this signal varied significantly between isolates. We also tested the effects of these strains on C. albicans and confirmed that the P. aeruginosa effect is 3-o-C12-dependent. To determine global responses of C. albicans to P. aeruginosa signals, we carried out a transcriptome analysis using supernatants from two high and two low 3-o-C12 producing strains of P. aeruginosa. In addition to 3-o-C12-specific effects, we saw similar changes in expression profiles in response to all four supernatants, indicating that these changes were 3-o-C12-independent. Many of these changes in gene expression were consistent with a reduction in ability to adhere and form biofilms, so we tested this in phenotypic assays. Adherence to polystyrene was reduced by up to 50% after 24 hours upon addition of all four supernatants. Using confocal microscopy, we also found that biofilm formation on silicone is dramatically reduced. Control biofilms measured up to 180 μM thick, whereas addition of Pseudomonas supernatants led to reductions in thickness of 70-90%, depending on the strain. This suggests that adherence and biofilm formation in C. albicans is influenced, at least in part, by secreted factors other than 3-o-C12, and that the interaction between Pseudomonas and Candida can affect a number of different pathogenicity-associated traits.


Disruption of the Aopex11-1 gene involved in peroxisome proliferation resulted in immature differentiation of Woronin body in Aspergillus oryzae

Cristopher Escaño1, Praveen Rao Juvvadi1, Feng Jie Jin2, Tadashi Takahashi2, Yasuji Koyama2, Jun-Ichi Maruyama1, Katsuhiko Kitamoto1

1The University of Tokyo, Tokyo, Japan, 2Noda Institute of Scientific Research, Chiba, Japan

Woronin body is an organelle specifically found in Euascomycetes that plugs septal pore upon hyphal injury. HEX-1 protein is the major protein in Woronin body and has peroxisome targeting signal 1 (PTS1) at the C-terminus. This suggests that Woronin body is a specialized class of the peroxisome. However, not much is known at the molecular level how Woronin body is differentiated from the peroxisome. In this report, we investigated the role of AoPex11, a peroxisomal membrane protein required for peroxisome proliferation, in formation and function of Woronin body in Aspergillus oryzae. According to the genome database, A. oryzae contains two PEX11 homologous genes (named as Aopex11-1 and Aopex11-2). We deleted these Aopex11 genes in the A. oryzae Ku70-deficient strain, and only the Aopex11-1 deleted mutant showed reduced growth in the presence of oleic acid as carbon source, revealing a defect in peroxisome function. Expression of the EGFP-PTS1 construct visualizing the peroxisome in the Aopex11-1 deletion background showed a limited number of enlarged peroxisomes, while the control strain had various sizes of this organelle. Simultaneous expression of the mDsRed-AoHex1 fusion protein demonstrated that the deletion mutant contained fewer independent Woronin bodies. Moreover, distribution of AoHex1 was found to be peripheral in the enlarged peroxisome or junctional inside the dumbbell-shaped peroxisome, suggesting immature Woronin bodies inside the peroxisome. Consistently, hyphal tip bursting experiment induced by hypotonic shock revealed that only the Aopex11-1 deleted mutant partially lacked normal Woronin body function. These data indicate that AoPex11-1 involved in peroxisome proliferation plays a role in Woronin body differentiation from the peroxisome.


The evolutionary conserved BEM46-like protein from Neurospora crassa is targeted to the ER

Moritz Mercker, Silke Alves, Nancy Weiland, Frank Kempken

Botanisches Institut, Christian-Albrechts-Universität, Kiel, Germany

We present data regarding the expression and function of the Neurospora gene bem46. The deduced amino acid sequence of this gene appears to be conserved among eukaryotes. It was first identified in Schizosaccharomyces pombe, where it is involved in cell polarity and signal transduction. Recently the Arabidopsis bem46 homolog was found to have a function in root growth.

We identified a homologous sequence some years ago in the filamentous fungus Ascobolus immersus, which was located near a truncated transposable element. When the Neurospora crassa genome sequence became available, we decided to continue working with the bem46 gene of N. crassa. Mutations were introduced using the RIP technique. However, ascospores of strong RIP mutants of bem46 do not germinate or terminate germination at an early stage. We therefore established strains over expressing bem46, a bem46:gfp fusion, and a RNAi construct down regulating bem46 expression. In addition we recently obtained knock-out mutants. Strains over expressing bem46 and a RNAi construct exhibited abnormal ascospore germination, i.e. early arrest of germination. Vegetative hyphae, perithecia, and ascospores develop normally. Moreover, we found that the BEM46 protein is targeted to the ER, and localizes at or close to the plasma membrane. BEM46 thus being a new ER marker for filamentous fungi. Our data suggest BEM46 may play a role in a signal transduction. This finding also implies a higher degree of differentiation of fungal hyphae than currently expected. Finally, this work also has implications for higher eukaryotic cells with polarized growth, such as pollen tubes or neuronal cells.


Role of hyphal fusion in perithecium development

Kathryn Lord, Chris Jeffree, Nick Read

University of Edinburgh, Edinburgh, United Kingdom

Multicellular development in fungi is fundamentally different to that in plants or animals, and filamentous fungi achieve the multicellular state primarily by hyphal aggregation. We are studying perithecium development in two closely related ascomycetes, Neurospora crassa (heterothallic) and Sordaria macrospora (homothallic), using the perithecium as a model for fungal multicellular development. Live-cell imaging and low-temperature scanning electron microscopy are being used in our work. Mutants compromised in vegetative hyphal fusion are being analysed for defects in perithecial development and vice versa, mutants compromised in perithecial development are being analysed for defects in vegetative hyphal fusion.


High-spatial resolution whole-cell biochemical composition in single filamentous fungal hyphae using synchrotron FTIR spectromicroscopy

Susan Kaminskyj1, Adriana Szeghalmi2, Konstantin Jilkine2, Kathleen Gough2

1Univ. Saskatchewan, Saskatoon SK, Canada, 2Univ. Manitoba, Winnipeg MB, Canada

Cell function is related to cell composition. Hyphal composition can change substantially over a few tens of microns, and spores have different composition from hyphae. Most biochemical analyses are restricted to studying a few components at high spatial resolution (e.g. histochemistry) or many compounds at low spatial resolution (e.g. GC-MS). We are using synchrotron Fourier transform infrared (sFTIR) spectromicroscopy to study fungal cell composition by fingerprinting varieties of carbohydrate, protein and lipid at 3-10 μm spatial resolution. The diffraction limit depends on wavelength, which in the mid-IR spans 3-30 μm. We have previously shown (Szeghalmi et al 2007 Analyt Bioanalyt Chem 387:1779-89) that sFTIR can distinguish fungal species, near-isogenic strains, and apical vs subapical regions in single cells. Aspergillus and Neurospora hyphae had similar sFTIR profiles (rich in protein) which were different from Rhizopus (rich in carbohydrate). For all three fungi, the biochemical content near the tips was substantially less than at 50 μm or 100 μm subapically. Fungi grown under optimal vs mildly stressed (e.g. a change of 2 pH units or less, in either direction) differed in sFTIR profile despite typically exhibiting few morphological effects.

Here we compare hypha and spore composition of Neurospora and Rhizopus. There were biochemical profile changes when Neurospora hyphae committed to spore development, during spore maturation, and following germination. Many of these are consistent with results from molecular genetics, but had not been shown before at high spatial resolution. Notably, although Neurospora hyphal carbohydrate content was reduced by growth at sub-optimal pH, the spores formed at optimal and sub-optimal pH levels had remarkably similar composition. Germinated Neurospora spores retained a nutrient dowry even after the germ tube was >100 μm long, presumably to support the growth of additional germ tubes. Developing Rhizopus spores had similar protein content and significantly more carbohydrate than hyphae, and spores had different composition from the sporangial fluid that surrounds them during maturation.



RfxA is an essential regulator of cell cycle events during growth and morphogenesis of the dimorphic fungus Penicillium marneffei

Hayley Bugeja, Michael Hynes, Alex Andrianopoulos

University of Melbourne, Victoria, Australia

The imperfect ascomycete Penicillium marneffei is a haploid dimorphic fungus and an opportunistic pathogen of humans. Filamentous hyphal growth and conidiation occur at room temperature (25°C). During growth at body temperature (37°C) the dimorphic transition from hyphal to yeast growth occurs, via arthroconidiation. These yeast cells continue to divide by fission. We are interested in dissecting the regulatory networks controlling growth and development (conidiation and dimorphic switching) in response to environmental cues. The RFX proteins are a family of transcriptional regulators mediating appropriate differentiation of specialised cell types in eukaryotes. In fungi, RFX factors are involved in the response to DNA damage in Saccharomyces cerevisiae (Crt1) and cellular differentiation in Schizosaccharomyces pombe (Sak1). In Acremonium chrysogenum CPCR1 has dual roles in the regulation of Cephalosporin C biosynthesis and arthrosporulation, a process analogous to arthroconidiation in P. marneffei. The rfxA gene of P. marneffei, encoding the sole RFX protein family member, was chosen as a potential regulator of the transition from hyphal to yeast growth. Attempts to generate an rfxA deletion strain were unsuccessful and resulted in the isolation of an rfxA+/rfxAD diploid strain. The role of RfxA was assessed using overexpression, RNA-interference (RNAi) and the production of dominant interfering alleles. Reduced RfxA function resulted in defective mitoses resulting in mitotic catastrophe during growth at 25°C and 37°C. This was also observed for the heterozygous diploid strain during growth at 37°C. In contrast, overexpression of rfxA caused growth arrest during conidial germination. Therefore, the correct modulation of RfxA function appears to be essential for appropriate cell cycle progression. The identification of putative RfxA target genes suggests a role for this transcription factor as a novel component of a mitotic checkpoint pathway. Thus, reduced RfxA function may prevent this checkpoint and allow cells to prematurely exit an abnormal mitosis. It is also anticipated that the RfxA-dependent modulation of the cell cycle may contribute to the cellular differentiation events observed during conidiation and yeast growth, where the uninucleate state of these cell types necessitates tighter coupling of nuclear and cellular division than observed during multinucleate hyphal growth.



Physiological function of endocytosis in the filamentous fungus Aspergillus oryzae

Yujiro Higuchi, Jun-ya Shoji, Manabu Arioka, Katsuhiko Kitamoto

The University of Tokyo, Tokyo, Japan

In filamentous fungi, the existence of endocytosis had been elusive mainly because of lack of reliable indicators of endocytosis. Recently, however, we and others have shown that endocytic markers, fluorescent dye FM4-64 and AoUapC (uric acid-xanthine permease) fused with EGFP, are internalized from the plasma membrane by endocytosis. Although the occurrence of endocytosis was made clear, its physiological roles largely remain unaddressed in filamentous fungi. Thus, we have examined the physiological function of endocytosis in the filamentous fungus Aspergillus oryzae.

We cloned Aoend4, the A. oryzae homolog of Saccharomyces cerevisiae END4/SLA2 (synthetic lethal with ABP1), a gene related to endocytosis. End4p/Sla2p functions as an adaptor connecting actin cytoskeleton and invaginated plasma membrane. The Aoend4 disruptant was not obtained probably because Aoend4 is an essential gene for hyphal growth. Thus, we generated a strain, named TE4, in which Aoend4 was expressed under the control of thiA promoter from the Aoend4 locus. The growth of this strain was severely impaired when thiamine was added to the culture medium to shut off the expression of Aoend4. Furthermore, abnormal accumulation of the cell wall was observed by Calcofluor White staining and TEM analysis, indicating that the cell wall became thicker when the expression of Aoend4 was repressed. We next generated a strain named TESn1 which expresses EGFP-AoSnc1, a v-SNARE required for secretion fused with EGFP, in the conditional Aoend4 background. In TESn1, aberrant invagination-like structures labelled by EGFP-AoSnc1 were observed under Aoend4-repressed condition. These results suggest that endocytosis in filamentous fungi functions in recycling of cell wall-building enzymes and components required for the apical growth to the tip region.



Ras function in regulation of sexual development in Schizophyllum commune

Erika Kothe, Nicole Knabe, Florian Hennicke, Daniela Schuber

Friedrich-Schiller-Universität, Jena Thüringen, Germany

The white rot fungus Schizophyllum commune is used as a model to investigate sexual development in hymenomycetes. Th gene gap1 encoding a Ras GTPase-activating protein has been identified in a screen for sexual development. Disruption of gap1 could show three different phenotypes: (1) growth rate was reduced about 25 % in homokaryons, and 50 % in dikaryotic D gap1/D gap1 strains; (2) clamp formation was severly disturbed as hook cells failed to fuse with the penultimate cell; (3) fruitbody formation was altered and no spores produced. Similar, and overlapping phenotypes could also be observed in strains transformed with a constitutively active allele of Ras, mimicking the lack of deactivation in a D gap1 strain. In order to address fruitbody formation, fruitbodies at stages 1 through 5 of fruitbody development were analyzed morphologically and altered hymenium formation and basidia development could be seen. Similar phenotypes in fruitbody development had been previously described for elevated intracellular cAMP levels. Thus, the signalling of Ras is discussed with respect to cAMP signalling. The effect of Ras signalling on cAMP dependent protein kinase A was investigated by analysis of intracellular cAMP levels, use of caffeine and external cAMP, and phosphorylation studies on PkA.



The HET domain and the initiation of cell death during vegetative incompatibility in Podospora anserina

Mathieu Paoletti, Marie Aramendy, Frédérique Ness, Sven Saupe, Corinne Clavé

Laboratoire de Génétique Moléculaire des Champignons Filamenteux, IBGC, UMR 5095 Université de Bordeaux2 et CNRS, Bordeaux, France

In filamentous ascomycetes, vegetative incompatibility (VI) is a non self recognition process that decides the fate of heterokaryons formed after somatic fusion of genetically different cells. Fusion between compatible isolates results in viable heterokaryons while fusion between incompatible isolates triggers a programmed cell death reaction. Compatibility is defined by a set of heterokaryon incompatibility genes (het) and expression of incompatible alleles at any het locus is enough to trigger the death reaction.

VI is ubiquitous among filamentous fungi and het genes have been characterised in two species, Neurospora crassa and Podospora anserina. Genes involved in 6 different VI systems have been characterised from both species and two observations emerge:

-het genes, involved in the recognition process, are not conserved between species but in both species, alternative alleles are always highly polymorphic.

-Most VI systems involve at least one protein harbouring the fungal specific HET domain. This HET domain is either present on the recognition molecule itself, or is brought to the system by proteins whose alterations result in the loss of VI.

These observations lead to a modular conception of VI, whereby recognition is ensured by the polymorphic proteins, and the cell death is induced by the HET domain. We investigate elements of the death module of the het-C / het-E VI system in P. anserina. Over-expression of the HET domain results in a cell death reaction identical to VI, and dependant on the same MOD-A and MOD-B proteins. In addition, mutations in mod-C, encoding a transcription factor, suppress the het-C/het-E VI reaction. The roles of the HET domain and the MOD proteins in the regulation of the cell death reaction is being investigated.

It is interesting to note that the HET domain is found in variable numbers in fungal genomes (6 to over 150 copies), with 129 copies in P. anserina, and most HET domain containing proteins appear unique (no homologues in other species). These numbers are in contrast with the limited number of VI systems found in fungi (about 12 per species). What are these HET domains doing?


Cytokinesis and septation in the filamentous ascomycete Ashbya gossypii

Andreas Kaufmann1, Hans-Peter Schmitz2, Peter Philippsen1

1Biozentrum, University of Basel, BaBiozesel, Switzerland, 2University of Osnabrueck, Osnabrueck, Germany

Cells of unicellular organisms separate after cytokinesis. In contrast, cells of filamentous fungi do not separate but stay connected to form hyphae that are compartmentalised by septa. In the unicellular fungus Saccharomyces cerevisiae over eighty genes are involved in cytokinesis, septation, and cell separation. Interestingly, Ashbya gossypii grows filamentously although its genome encodes homologues of virtually all these genes. Here, we analysed by three-dimensional fluorescence live cell imaging the behaviour of several proteins important for cytokinesis and septation in A. gossypii.

Septins are observed constantly as thin cortical filaments in apical compartments of growing hyphae. To mark future septa they form a collar of cortical bars at the hyphal tip. Similarly, the novel PCH protein AgHof1, the landmark protein AgBud3, the IQGAP AgCyk1, and type II myosin AgMyo1 also form cortical bars. As the tip continues to grow, these proteins change their localisation to form continuous cortical rings that then colocalise with the contractile actin ring. This transition together with persistence of the rings can take several hours. Finally, ring contraction leads to the complete separation of the plasma membranes of the adjacent compartments. Concomitant with cytokinesis, a chitin-rich septum is formed between the hyphal compartments that remain connected to each other. An explanation why cell separation does not occur in A. gossypii might be that it lost its homologues of CTS1 and EGT2 during evolution. Each encodes a major enzyme essential for cell separation in S. cerevisiae. For example, the syntenic homologue of EGT2, encoding an endoglucanase, most likely got lost during evolution as a consequence of a translocation between chromosomes 4 and 7.

These studies allowed us to divide the process of septation in A. gossypii into five different stages: 1. septation site selection at the tip of the hypha, 2. bar-to-ring transition, 3. actin ring assembly and persistence, 4. cytokinesis and septation without cell separation, and 5. septum maturation.


Functional analysis of the MAP kinase gene mpkB that possibly contributes to cell wall integrity signaling pathway in Aspergillus nidulans

Akira Yoshimi1, Tomonori Fujioka2, Junichiro Marui1, Daisuke Hagiwara1, Osamu Mizutani3, Kentaro Furukawa4, Keietsu Abe1

1Tohoku University, Sendai, Japan, 2Kumiai Chemical Industry Co., Ltd, Tokyo, Japan, 3National Research Institute of Brewing, Higashihiroshima, Japan, 4Göteborg University, Göteborg, Sweden

The cell wall of filamentous fungi is a complex structure that is essential for the maintenance of cell’s shapes and integrity, for the prevention of cell lysis, and for protection against adverse environmental conditions. Because a mitogen activated protein kinase (MAPK) is a key enzyme of cell wall integrity signaling (CWIS), the transcriptional regulation of MAPK gene and of cell wall-related genes (CWGs) is important in fungal cell wall biogenesis. We previously reported that the transcriptional regulation of the MAPK gene mpkA and of CWGs in Aspergillus nidulans differs significantly from that in Saccharomyces cerevisiae (1). Except for alpha-1,3-glucan synthase genes, the transcription of most CWGs coding for beta-1,3-glucan synthase and chitin synthases were regulated by non-MpkA signaling pathway in A. nidulans (1). A. nidulans possesses a MAP kinase MpkB that has the putative phosphorylation motif TEY (same as that of MpkA), and of which amino acid sequence indicates similar to those of yeast Kss1p and Fus3p. To investigate the in vivo function of the mpkB gene and its possible contribution to CWIS pathway, we constructed A. nidulans mpkB gene disruptant (mpkB∆) strains. The mpkB∆ strains exhibited increased sensitivity to beta-1,3-glucan synthase inhibitor micafungin as well as the mpkA∆ strain, suggesting that the mpkB gene was also involved in cell wall integrity in A. nidulans. To confirm whether the regulation of CWGs depended on MpkB pathway, we analyzed levels of transcription of CWGs by quantitative RT-PCR. The results suggested that transcription of the agsA gene encoding an alpha-1,3-glucan synthase seemed to be regulated by MpkB-dependent signaling in A. nidulans. Additionally, the phosphorylation pattern of MpkA and MpkB induced by micafungin was assessed. Transcriptional analyses of mpkA∆ and mpkB∆ strains with DNA microarrays will be also discussed.

1 Fujioka et al. Eukaryotic Cell (2007) 6: 1497-1510


PprA is an essential pentatricopeptide family member required for mitochondria morphology, actin cytoskeleton organisation and nuclear distribution in Aspergillus nidulans

Lidia Araújo-Bazán1, Javier Fernández-Martínez2, Mark X. Caddick3, Miguel A. Peñalva1, Eduardo A. Espeso1

1Centro de Investigaciones Biológicas, Madrid, Spain, 2The Rockefeller University, New York, United States, 3The University of Liverpool, Liverpool, United Kingdom

In a mutagenic screening designed to obtain mutations altered in the nuclear transport of PacC we serendipitously isolated a conditional mutation leading to an accumulation of nuclei. The mutation mapped at a gene denoted as pprA because it encodes for a protein with several pentatricopeptide repeats (PPR). PPR proteins are abundant in plants and they play different functions; however, all of them appeared to be in a close relationship with mitochondria and chloroplasts. In A. nidulans, pprA is an essential gene and it is the first pentatricopeptide protein that has been characterized.

The thermosensitive mutation, pprA1, causes a strong growth defect. Mutant hyphae are much shorter and thicker than those of the wild-type strain. At the restrictive temperature nuclei accumulate and have not apparent organisation. Consistent with this the mitotic spindles are formed in anomalous orientations. The actin cytoskeleton is also affected. The actin accumulation at the cell tip is less abundant, although still prominent, and actin cables are not observed. In Aspergillus and budding yeast, mitochondria interact with the actin cytoskeleton and use it to move throughout the cell. As expected from the actin cytoskeleton disturbance, caused by the pprA1 mutation, mitochondria appeared completely fragmented.

We propose two different ways in which this essential protein could carry out its role. PprA could be acting either through the mitochondria integrity or directly, in the organisation of both cytoskeletons, which are required for hyphal growth and nuclear distribution maintenance.


Investigation of the role of RacA and CftA during polarized growth in Aspergillus niger

Min Jin Kwon, Mark Arentshorst, Cees AMJJ van den Hondel, Arthur FJ Ram

Institute of Biology Leiden, Leiden Wassenaarseweg 64, Netherlands

The establishment of cell polarity in filamentous fungi is critical for the control of many cellular and developmental processes, including polarized hyphal growth, intracellular movement of organelles, protein secretion, cell wall biosynthesis and the development of conidiophores. In yeasts, but also in other eukaryotic cells, Rho-related GTPases (Rho, Rac and Cdc42) are involved in controlling and organizing actin cytoskeleton and thereby controlling polarized cell growth. To assess the roles of the Rac1 and CDC forty-two homologs in Aspergillus niger (RacA and CftA), we have made racA and cftA deletion mutants. Deletion mutants were efficiently obtained using the A. niger kusA mutant strain (Meyer et al., 2007). We also attempted to construct a D racA, D cftA double mutant. However, the double knockout is most probably lethal as primary transformants containing both the racA and the cftA deletion were all heterokaryotic and spores derived from these transformants were never viable. The synthetic lethality between D racA and D cftA suggests an overlapping function of the two GTPases.

The phenotypic consequences of the loss of racA or cftA have been studied in detail. Deletion of racA resulted in abnormal germination and branching patterns, and in reduced conidiation. Deletion of cftA resulted in slower germination and a reduction in hyphal growth rate. The racA deletion phenotype was more severe than that of cftA. The localization of actin was examined using immunofluorescence in the wild type and the racA and cftA mutant strains. The localization of actin in a D racA showed condensation at the extreme apex of the hyphal tip, while in both the D cftA and wild type strains a gradient of actin patches towards the tip was observed. Overexpression of the dominant active form of RacA (RacAG12V) resulted in the loss of polarized hyphal growth and swollen germtubes. The polarized actin cytoskeleton in the swollen hyphae induced by RacAG12V overexpression is completely lost. To further assess the role of RacA and CftA in polar growth and their involvement in the organisation of the actin cytoskeleton, GFP-RacA and GFP-CftA fusion constructs will be made.


Sexual development of Schizophyllum commune – an interaction between mating pheromones and multi-specific pheromone receptors

Susann Jezewski, Erika Kothe

Institute of Microbiology - Microbial Phytopathology, Jena, Germany

The tetrapolar mating system of the homobasidiomycete Schizophyllum commune depends on two genetic complexes, the A and B genes. A pheromone/receptor system, encoded by the B genes, allows communication between two mating partners. Each B locus codes for one pheromone receptor, belonging to the family of G protein coupled seven transmembrane domain receptors, and several pheromones, small lipopeptide ligands. The importance of the pheromone/receptor system in S. commune is well-investigated, however there are little information about the expression of mating genes and the localization of the pheromone receptors. The functionality of the multi-specific pheromone receptors, to recognize pheromones of other S. commune strains and to distinguish between self and non-self, implies localization of receptor molecules in the tip of growing hyphae. The interaction of a pheromone receptor with non-self ligands results in clamp cell fusion, what presumes occurrence of pheromone receptors also in the membrane of building clamps. To investigate the pheromone receptor in vivo, the bar2 gene (= B α receptor gene of specificity 2) was integrated into a Bnull mutant strain. The visualization of the Bα2 receptor protein (Bar2) should be possible by fluorescent tag of Bar2 at the intracellular C-terminus with DsRed or GFP. The transient process of clamp cell formation and the low expression of mating type genes complicate the determination of receptor molecules. Nevertheless, by means of real time PCR the expression of bar2 could be detected. The quantification of mRNA will be performed in different mutant backgrounds under special conditions. The mating system of S. commune is not only a system for sexual reproduction, it also displays a model system for cellular development and cell communication with relevance for other organisms.


Impact of a golgi-located protein involved in secretion on morphogenesis of the filamentous fungus Aspergillus oryzae

Jun-ya Shoji, Ayako Taura, Manabu Arioka, Katsuhiko Kitamoto

Department of Biotechnology, The University of Tokyo, Tokyo, Japan

Continuous tip growth of filamentous fungi is achieved by establishment of polarity axis that modulates proper organization of cytoskeleton and subsequent polarized secretion. Polarized secretion of filamentous fungi is also crucial for progress of industrial production of useful proteins. However, our understanding of vesicular trafficking and secretion in filamentous fungi has been too fragmentary.

In our previous study, we observed intracellular distribution of enhanced green fluorescent protein (EGFP)-fused soluble NSF attachment protein receptors (SNAREs) in Aspergillus oryzae. Among the fusion proteins, overexpression of EGFP-fused AoSed5p, a Golgi target (t)-SNARE, resulted in pleiotropic defects in hyphal growth. These defects were not attributed to the fusion with EGFP, and overexpression of AoSed5p alone resulted in similar phenotype. Although overexpression of Sed5p in the budding yeast causes a growth defect as well, defects observed in A. oryzae were much more drastic. In the presence of excess AoSed5p, tip growth was severely impeded. Conidia and hyphal tips often swelled, and showed abnormal dichotomous-like branching. These results suggest that the overexpression of AoSed5p causes defective polarized growth. In addition, the overexpression of AoSed5p resulted in accumulation of nuclei, fragmentation of vacuoles, and accumulation of chitin at the cell wall. It also caused aberrant organization of actin and microtubules. In the budding yeast, overexpression of a phosphorylated state of Sed5p inhibits retrograde transport from Golgi to endoplasmic reticulum while a dephosphorylated state does not. We then overexpressed similar phosphorylated/dephosphorylated states of AoSed5p in A. oryzae and found that the phosphorylated state of AoSed5p is responsible for the above-mentioned growth defects. These results suggest that the Golgi t-SNARE AoSed5p, and probably secretion, play vital roles in hyphal morphogenesis, through feedback regulation on cellular polarity and cytoskeleton.


Comprehensive analysis of transcription factors involving conidiation in Aspergillus oryzae

Masahiro Ogawa1, Masafumi Tokuoka1, Ken-ichiro Matsushima1, Tadashi Takahashi1, Tomomi Toda2, Masayuki Machida2, Yasuji Koyama1

1Noda Institute for Scientific Research, Noda 399, Noda, Chiba 278-0037, Japan, Japan, 2National Institute of Advanced Industrial Science and Technology, Higashi 1-1-1, Tsukuba, Ibaraki 305-8566, Japan, Japan

Focused studies of the conidiation control in Aspergilli have been carried out in A. nidulans and A. fumigatus. Understanding of the conidiation control in koji-mold A. oryzae is important to improve productivity in the Japanese fermentation food, such as soy sauce, miso, and sake. However, little is known about the conidiation control in A. oryzae. Recently, it is reported that conidiation control in A. fumigatus is different from that of A. nidulans1). Thus, we are interested in examining whether the regulatory mechanism of conidiation is different among A. oryzae and other Aspergilus species.

We constructed a series of A. oryzae mutants in which each ortholog of the A. nidulans conidiation regulators genes (brlA, fluG, flbA, flbB, flbC, flbD, flbE, abaA or wetA) was disrupted. The fluG, brlA, abaA and wetA orthologs disruption mutants of A. oryzae showed the fluffy, bristle, abacus, and wet-white phenotypes, respectively, as has been shown in the corresponding disruption mutants of A. nidulans. Moreover, disruption of the flbA ortholog caused loss of conidiation and reduction of the aerial hyphal extension. The flbB, flbC, flbD, and flbE orthologs disruption mutants of A. oryzae exhibited the delayed conidiation phenotype. These results suggest that upstream regulation mechanism for conidiation in A. oryzae is similar to that in A. nidulans, which is shown to be different from that in A. fumigatus. In contrast, the brlA ortholog disruption mutant showed the identical phenotypes among A. oryzae, A nidulans and A. fumigatus, suggesting that the role of brlA in downstream conidiation regulation is conserved among these species. Additionally, the function of the abaA and wetA are likely conserved in A. oryzae and A. nidulans, given the similarity of mutant phenotypes between the two species. Results of DNA microarray analysis with these disruption mutants were consistent with the above-mentioned findings.

Finally, we obtained the novel conidiation mutants from a comprehensive disruption library of the transcription control genes in A. oryzae. We will also report results of analysis with these disruption mutants.

1 Jae-Hyung Mah and Jae-Hyuk Yu, Eukaryot. Cell, 5, 1585–1595 (2006)


Neurospora crassa GS-1, a protein involved in b -1,3-glucan synthesis, accumulates in the outer region of the spitzenkörper

Jorge Verdín, Salomón Bartnicki-García, Meritxell Riquelme

Department of Microbiology, Center for Scientific Research and Higher Education of Ensenada (CICESE), Ensenada, Baja California, Mexico

GS-1 is a protein necessary for b -1,3-glucan synthase activity and normal hyphal development of Neurospora crassa. GS-1 shares several sequence motifs with Knr4/Smi1 from Saccharomyces cerevisiae, a cell wall synthesis regulator that coordinates the action of proteins involved in cell wall maintenance and the establishment of cell polarity. To date, the role of GS-1 in b -1,3-glucan synthesis is unknown. In order to determine the localization, dynamics and possible function of GS-1, the N. crassa gene gs-1 (NCU04189.3) was endogenously fused to the green fluorescent protein gene sgfp. Total internal reflection fluorescence (TIRF) and laser scanning confocal microscopy (LSCM) were used to analyze the transformant strain. GS-1::sGFP arranges in an apical ring which corresponds to the outer layer of the Spitzenkörper (Spk). Such layer is composed of secretory vesicles that putatively contain b -glucan synthesizing enzymes. The annular structure highlighted by GS-1::sGFP is dynamic and pleomorphic. Its shape varies from a perfect ring to a rotating horseshoe-like form. Occasionally, the "Spitzenring" moves retrogradly and disintigrates while a new Spitzenring is assembled in the apex. In subapical regions, discrete fluorescent corpuscles move in anterograde fashion along paths that reach the Spitzenring, where GS-1::sGFP is recruited. Although GS-1 seems to be transported to sites of active growth, whether GS-1 interacts with its target protein(s) before or after arriving to the Spk remains an open question. Together with the previously reported localization of chitin synthases in the core of the Spk, these results suggest an architectural and functional stratification of the cell wall synthesis machinery within the Spk that should be considered in the models that describe hyphal growth.


Clp1 a novel regulator for cell cycle progression in Ustilago maydis

Kai Heimel1, Mario Scherer1, Jörg Kämper2

1Max-Planck Institute for Terrestrial Microbiology, Marburg, Germany, 2University of Karlsruhe, Karlsruhe, Germany

The regulation of pathogenic development in U. maydis is tightly connected to cell cycle control. Induction of the b-heterodimer leads to a G2 cell cycle arrest of the dikaryotic filament when growing on the leaf surface. b induces the expression of rbf1 encoding a C2H2 zinc finger transcription factor. Rbf1 is required and sufficient to induce the cell cycle arrest and controls more than 80% of the b-dependent genes, representing the major regulator downstream b. Clp1 is a b-regulated gene and required for pathogenic development of U. maydis. Deletion strains are not impaired in filamentous growth or appressorium formation, but development stops prior the first cell divison. Although the clp1-transcript is abundant rapidly after b-induction, the Clp1-protein is only observed when the hyphae penetrate the plant surface, coinciding with the time point when the cell cycle is released. In an unbiased Yeast Two-Hybrid approach we identified bW, Rbf1 and the bZip transcription factor Cib1 (Clp1 interacting bZIP) as Clp1 interacting proteins and confirmed the interaction in vivo using BiFC. Deletion of cib1 leads to a phenotype indistinguishable to that of clp1 mutant strains. Simultaneous induction of an active b-heterodimer and Clp1 blocks b-function as a transcriptional regulator globally. Preliminary results suggest a similar modulation of Rbf1 function by Clp1. Similar to Clp1, the Cib1 protein was found to be localized in the nucleus when the cell cycle is released; however, its function is still unclear. These findings add a new layer of complexity and place Clp1 in the centre of the regulatory circuits that connect cell cycle control and pathogenic development.


Study of the effect of the light in the development of eatable fungi (Pleurotus ostreatus)

Davinia Arjona, Leopoldo Palma, Lucía Ramírez, Gerardo Pisabarro

Public University of Navarre, Pamplona, Navarra, Spain

Pleurotus ostreatus is an edible basidiomycete with great importance in production, gastronomically as well because it has a medicinal value and plays a great role in the bioremediation. In this work we want to study with detail the molecular mechanisms responsible for the effect of the light on the development of P. ostreatus.

In this study we try to determine the effect of the continuous light and in regime of photoperiod on the vegetative growth and the morphogenesis in conditions of laboratory culture of P. ostreatus. The answer to the light requires a receptor that in the case of the oyster mushroom hasn’t been identified yet. After to have seen other homologous ones of con-6 in other fungi, we have determined in dicarionte N001 (var. Flowery) the structure of two alleles of MV-140 gene with introns and exons, in PC9 and PC15. In addition, this gene has been mapping, and it is in chromosome IV of P. ostreatus.

The effect of the continuous white light, continuous red fluorescent light and different photoperiods have been studied. Determining the necessity of photoperiod for the fructification, when observing that exposing to mycelium vegetative of dicarionte N001 to photoperiods of different times (6 and 12 hours) fruit bodies are obtained, and nevertheless, exposed to a pulse (12h) of light or the dark it does not take place the development of the mushroom. With red fluorescent light morphogenesis has been observed, reason why we have studied the spectro to verify what wavelengths composed the light. The wild type of N001 shows an important inhibition of the growth of mycelium vegetative if it is exposed to constant white light, reason why we have obtained a mutant protoclon photo insensible (it grows like in dark) that later we will localize in the map of ligamiento of P. ostreatus developed in the laboratory of Public University of Navarre (UPNa) and, when we cross it with other compatible protoclon, we will can see if it is also affected the fructification.

We have observed that it is possible to be accelerated the fructification if we modify the hours of photoperiod, getting to develop an experimental model to complete cycle of life of the fungus in only one week.


Characterisation of two unc104-related kinesin motor proteins in Aspergillus nidulans

Nadine Zekert, Reinhard Fischer

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

We have previously shown that in Aspergillus nidulans conventional kinesin (Kinesin-1), KinA, is required for fast hyphal growth (1) and the Kip2 family motor (Kinesin-7), KipA, for the maintenance of hyphal polarity (2). Here, we characterized UncA and UncB, two Unc-104 related motor proteins (Kinesin-3). UncA consists of 1631 amino acids and contains, in addition to the motor domain, a forkhead association domain and a PH domain. UncB in comparison is much smaller with only 671 amino acids. Homologues of those motors were shown to be required for mitochondrial motility along microtubules (MT) in Neurospora crassa (3), and for early endosome transportation in Ustilago maydis (4). Whereas deletion of uncB did not show any phenotype, deletion of uncA caused slower hyphal growth and a defect in hyphal polarity at higher temperature, suggesting a role in hyphal growth. UncA tagged with GFP was found in several spots in the cytoplasm, and showed rapid bidirectional movement along MT. The introduction of a point mutation at the ATPase site of the UncA motor domain inhibited its movement and caused binding of the motor to only one microtubule. UncB tagged with GFP localized to nuclei and septa at certain times and travelled from nuclei to septa along MTs. The introduction of a rigor point mutation at the ATPase site of the UncB motor domain inhibited its movement and showed accumulation of the protein in nuclei. These results suggest other functions for UncB than UncA, although creating an uncA uncB double deletion strain showed the same phenotype as an uncA-deletion strain. One important outcome of our analysis is that UncA apparently defines a certain class of MTs in the cells.

1 Requena, N., Alberti-Segui, C., Winzenburg, E., Horn, C., Schliwa, M., Philippsen, P., Liese, R. & Fischer, R. (2001). Mol. Microbiol. 42, 121-132, 2 Enke, C., Zekert, N., Veith, D. & Fischer, R. (2007). Eukaryot. Cell 6, 555-562, 3 Fuchs, F. & Westermann, B. (2005). Mol. Biol. Cell 16, 153-161, 4 Wedlich-Söldner, R., Straube, A., Friedrich, M.W. & Steinberg, G. (2002). EMBO J. 21, 2946-2957.


A new cell signaling mechanism in Neurospora crassa

Andre Fleissner1, M. Gabriela Roca3, Abigail C. Leeder2, Nick D. Read3, N. Louise Glass2

1Technische Universitaet Braunschweig, Braunschweig, Germany, 2University of California, Berkeley, United States, 3University of Edinburgh, Edinburgh, United Kingdom

In N. crassa germinating vegetative spores (conidia) show mutual attraction, directed growth towards each other and cell fusion. As a result an interconnected network of germlings is formed, which further develops into the mycelial fungal colony. Most cell fusion processes in other systems require genetic and/or physiological differences between the fusion partners. For example mating in Saccharomyces cerevisiae always requires two cells of opposite mating types. The cells secrete mating type specific pheromones and direct their growth towards each other by detecting the pheromone gradient. In contrast, in germling fusion in N. crassa the fusion partners are genetically (and probably physiologically) identical. Since both cells show attraction and growth towards each other, both individuals must function as signal sender and recipient at the same time. The formation of a signal substance gradient comparable to yeast mating seems impossible. Recently we obtained new data representing a first step towards the solution of this paradox. MAK-2, a MAP kinase, and SO, a protein of unknown molecular function, are both individually necessary for hyphal/germling fusion. Subcellular localization of GFP fusion constructs showed that both proteins localize to the tips of fusion cells and the point of cell fusion after cell-cell contact. Interestingly we found that the proteins localize to the fusion tip of only one fusion partner at the time. After a few minutes the signal disappears and the GFP fusion proteins accumulate at the tip of the second partner only to disappear again and accumulate in the first partner again. Based on these observations we hypothesize that the two fusion cells signal in an oscillating manner, in which the cells switch between sending and receiving the signal. Using a chemically inhibitable variant of MAK-2 we are now able to disturb the signaling process at different stages of the development. Our initial data show that functional MAK-2 is required for proper SO localization and oscillation and completion of cell-cell attraction and fusion.



Peroxisomal RING-finger complex and sexual development in Podospora anserina

Leonardo Peraza, Denise Zickler, Véronique Berteaux-Lecellier

Institut de Genetique et Microbiologie, Univ. Paris-Sud, Orsay, France

Peroxisomes are involved in numerous metabolic functions and modulate several developmental processes. We use the filamentous fungus P. anserina to study the role of peroxisomes in development. The sexual differentiation of this fungus is blocked in absence of PEX2, a peroxin that is part of a protein complex (RING-finger complex) required for peroxisomal protein import; however, this defect is not observed in absence of other peroxins also implicated in peroxisomal protein import (PEX5 and PEX7), suggesting an additional role for PEX2 during sexual development. To decipher this role, we decided to study the involvement of the RING-finger complex during development by deleting the genes coding for the reaming partners of the complex, PEX10 and PEX12. We show that these proteins are involved in peroxisome biogenesis in the same way as PEX2: they are required for the import of peroxisome matrix proteins containing the two known peroxisomal targeting sequences. In addition, we show that peroxisomal remnants are still present in absence of PEX10 and PEX12, indication that they are dispensable for peroxisomal membrane assembly. Absence of one, two or all three RING-finger proteins lead to the same developmental defect: inability to undergo karyogamy during sexual development. Subcellular localization of PEX2 during development discarded a role for the complex elsewhere in the cell since it was observed to be peroxisomal at all times, and its presence in the peroxisomal membranes in absence of PEX10 and PEX12, indicated that it is stably inserted into these membranes even in absence of the other members of the complex. As previously observed for pex2 mutants, developmental defects generated in absence of the entire RING-finger complex could be partially corrected by overproduction of the peroxisomal ABC transporter pABC1; however, no correction was observed by overproducing, in a given RING-finger mutant, another partner of the RING-finger complex, indicating that suppression by overproduction of pABC1 doesn’t occur via interaction with (nor stabilization of) the remaining members of this complex. Our results indicate: (i) that PEX2 involvement during sexual development can be extended to the entire RING-finger complex, (ii) that this involvement seems independent from its role in peroxisome biogenesis and (iii) that it can be bypassed by an unknown function of pABC1.


A new cell type produced by conidia that is photoregulated and involved in sexual reproduction in Neurospora crassa

Hsiao-Che Kuo, Karen Holliday, Nick Read

The University of Edinburgh, Edinburgh, United Kingdom

We have discovered, a new cell type, the conidial sex tube (CST), that can act as a male fertilizing agent in Neurospora crassa. It is morphologically and physiologically distinct from germ tubes and conidial anastomosis tubes (CATs) that also arise from conidia, and under separate genetic control. CSTs are characterized by being long, thin, straight and unbranched, and they do not avoid or grow towards each other. The surface texture and chemistry of CSTs is different to that of germ tubes and CATs. CSTs require the presence of the peptide sex pheromone of opposite mating type to be induced. Nuclear division in CSTs is arrested. Besides being stimulated by sex pheromone, CST induction is also regulated by red light and this is phytochrome-mediated. Neurospora crassa possesses two phytochromes: PHY-1 and PHY-2, and they play contrasting and mating type dependent roles in the photoregulation of CST induction.


Cell death caused by chitosan in filamentous fungi

Javier Palma-Guerrero1, Luis V. Lopez-LLorca1, Hans-Borje Jansson1, Jesus Salinas1, I-Ching Huang2, Nick D. Read2

1University of Alicante, Multidisciplinary Institute for Enviromental Studies (MIES) Ramón Margalef, Department of Marine Sciences and Applied Biology, Alicante, Spain, 2University of Edinburgh, Instute of Cell Biology, Edinburgh, United Kingdom

Chitosan, the deacetylated form of chitin, exhibits antibiotic activity against both bacteria and fungi. Fungal growth and spore germination have been found to be sensitive to chitosan in plant pathogenic fungi, and morphological changes associated with fungal growth inhibition by chitosan have been observed in fungal cells. Plasma membrane damage has previously been suggested to explain the fungicidal effects of chitosan on filamentous fungi, but clear evidence for this is lacking. Using Neurospora crassa as an experimental system we have characterized fungal cell death and lysis associated with plasma membrane permeabilization by chitosan. Rhodamine-labelled chitosan was used to study chitosan uptake by fungal cells. Sodium azide, low temperature (4ºC) and azoxystrobin were used to inhibit ATP production. The cell viability stains fluorescein diacetate and propidium iodide were used to study plasma membrane permeabilization and cell death following chitosan treatment. Plasma membrane permeabilization by chitosan was confirmed using a N. crassa transformant expressing aequorin in luminometer. We provide the first evidence of cell energy requirement by chitosan to permeabilize plasma membrane and kill fungal cells. Different sensitivity to chitosan on different cell stages has been demonstrated.



An oligo genome-wide DNA array approach to search for target genes of the mating-type transcription factors in the filamentous fungus Podospora anserina

Jinane Aitbenkhali1, Frédérique Bidard1, Sandrine Imbeaud2, Nancy Reymond2, Evelyne Coppin1, Hervé Delacroix2, Robert Debuchy1

1Univ Paris-Sud, Institut de Génétique et Microbiologie, CNRS UMR8621, Orsay, France, 2CNRS, Centre de Génétique Moléculaire, UPR 2167, Gif sur Yvette, France

The sexual cycle in Euascomycetes is characterized by a series of mitotic division of parental nuclei after fertilization, resulting in the formation of plurinucleate cells which give rise to dikaryotic ascogenous hyphae. Karyogamy, meiosis and ascospore formation take place in these ascogenous hyphae. In the heterothallic Sordariomycete Podospora anserina, the two mating-type idiomorphs, mat+ and mat- control fertilization as well as the recognition between nuclei of opposite mating type inside the plurinucleate cell. The unique mat+ gene FPR1 and two of the three mat- genes, FMR1 and SMR2, encode transcriptional regulators, which control intercellular and internuclear recognition. A full understanding of the mechanism of internuclear recognition clearly depends on the identification of the target genes of mating-type transcription factors. With the availability of the genomic sequence, it has been possible to develop oligos microarrays targeted against the 10,546 predicted CDS of P. anserina (see poster by Bidard et al). Using this technology, we are searching for target genes by monitoring changes in the transcription profile of vegetative cultures expressing either mat+ or mat- transcription factors. The experimental design has been established from microarray data, which demonstrated that FPR1 and FMR1 transcripts undetectable in 1 day cultures are abundant in 4 day cultures. By contrast, SMR2 is never transcribed vegetatively. To induce its expression, we introduced a SMR2 ectopic copy constitutively expressed into a mat- recipient (strain mat- SMR2ect). FPR1, FMR1 and SMR2 responsive genes should be identified by comparing the transcription profile of 4 day mycelium from mat+, mat- and mat- SMR2 ect strains. This first approach will be completed by analyses performed on reproducing cultures in an appropriate genetic system that we are now developing.

This project is funded by the National Research Agency contract n° ANR-05-BLAN-0385-01


PRO40 and PRO41, two organellar proteins involved in fruiting body development in Sordaria macrospora

Ines Engh, Minou Nowrousian, Ulrich Kück

Ruhr-University Bochum, Bochum, Germany

Fruiting body formation is a highly complex process in the life cycle of many fungi. The homothallic ascomycete Sordaria macrospora was used as a model system to gain a better understanding of the molecular mechanisms underlying fruiting body development. By conventional mutagenesis, several sterile mutants were obtained that can only form immature fruiting bodies. Two of these mutants, pro40 and pro41, were subjected to complementation analysis using an indexed cosmid library. This led to the identification of the pro40 gene encoding a 140 kDa WW domain protein and the pro41 gene encoding a 15 kDa membrane protein. Gene expression analysis using microarrays indicates an epistatic relationship of pro41 to the pro1 transcription factor gene previously identified during the analysis of another sterile mutant. The proteins were further analysed by means of fluorescence microscopy and sedimentation assays. Using different fluorescent proteins and organellar markers, PRO40 and PRO41 were found to be associated with the Woronin Body and the ER, respectively. However, both proteins are dispensable for the biogenesis and overall morphology of these organelles. Further analysis of these proteins such as protein protein interaction studies will improve our understanding of molecular mechanismsby which organelles promote developmental processes.



A mutation in a COG4 homologue affects polarity establishment in Aspergillus nidulans

Sara Gremillion, Darlene Loprete, Terry Hill, Kaddy Camara, Samuels Felicia, Sarah Mercer

Rhodes College, Memphis,TN, United States

We have identified a gene mutation in Aspergillus nidulans, designated swoP (for swollen cell phenotype), which causes a morphological defect during spore germination and hyphal growth at the restrictive temperature of 42°C. Conidia of mutants swell to approximately 1.5 times the diameter of a non-mutant strain of identical genetic background and establish multiple points of polarity. Most polarized points swell isotropically, giving a multiple-budding appearance, at which point growth arrests in most cells. Cell wall weakness is indicated by a strong tendency of swollen cells to burst when observed under a coverslip. At 42°C, a very small minority of cells eventually produce colonies and asexual spores. Growth at 30°C is essentially normal, though hyphae are slightly wider than wild type and exhibit a minor "steering" defect. Cells grown at 42°C for up to 18 hours are capable of recovering normal hyphal morphology upon transfer to 30°C. When mutant hyphae growing at 30°C are transferred to 42°C, terminal compartments swell irregularly and may produce multiple budding points; subapical compartments also may swell and produce buds. The growth defect of swoP is complemented by the wild type allele of AN7462, which shows strong sequence homology to COG4, a component of the Golgi tethering complex associated with retrograde transport of COPI-coated vesicles. Sequencing of the COG4 allele of the swoP mutant reveals an adenine-to-cytosine mutation at base pair 2672, which results in a tyrosine-to-STOP substitution at amino acid position 780, truncating the plasmid containing this mutant sequence (instead of wild type) produces a partial remediation of the phenotype, but hyphal compartments still exhibit severe swelling defects. Meiotic mapping produces a ca. 12% recombination frequency between the swoP and AcuK loci, which is consistent with the chromosomal location of AN7462. We are currently working to localize the putative COG4 of A. nidulans via GFP-tagging and immunolocalization, as well as to demonstrate interactions between A. nidulans COG4 and other proteins involved in retrograde vesicle transport in the Golgi apparatus.


Mutants in light regulation of sexual and asexual development in Coprinopsis cinerea

Wassana Chaisaena, Mónica Navarro-González, Ursula Kües

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

Fruiting body development in Coprinopsis cinerea is adapted to the light and dark phases of the normal day-night rhythm. If young developing structures (day 1 to day 4 of development) are transferred into constant darkness, normal cap and stipe differentiation arrests. Instead, the stipe base proliferates to give an elongated structure known as "etiolated stipe" or "dark stipe". Depending on the day of transfer into constant dark, etiolated stipes show differences in the stage of cap differentiation. Only at day 6 of normal fruiting body development a transfer from a day/night regime into constant darkness will not stop further normal stipe and cap development.

Rare mutations are known that affect light regulation of development. Recessive mutations in genes dst3 and dst4 cause an etiolated-stipe phenotype in the normal day-night regime. These genes are distinctive from dst1 and dst2 identified previously in Japan in mutants producing etiolated stipes in light. From morphological analysis of the cap tissues of the etiolated stipes, dst3 and dst4 appear to act later in development (at day 3 and day 4 of development, respectively) than dst1 and dst2 (defects at day 2 of development). Unlike dst3 and dst4, dst1 and dst2 also act in light-control of asexual sporulation. Only the dst2 mutant is totally blind and forms etiolated stipes also under constant light incubation. Normally in constant light, fruiting body development is totally repressed.



Microbial cell differentiation: a response to oxidative stress

Wilhelm Hansberg1, Leonardo Peraza1, Vanessa Vega1, Nallely Cano2, Jesús Aguirre2, Pablo Rangel1, María Chávez1

1Bioquímica, Inst. Fisiología Celular, Universidad Nacional Autónoma de México, México, D.F., Mexico, 2Genética Molecular, Inst. Fisiología Celular, Universidad Nacional Autónoma de México, México, D.F., Mexico

We have proposed that different stress conditions lead to an unstable state in which reactive oxygen species are formed above the antioxidant capacity of the cell, and that cell differentiation is a response to this inevitably transient hyperoxidant state. This hypothesis predicts that the inactivation of anti-oxidant enzymes should increase cell differentiation processes whereas the inactivation of pro-oxidant enzymes should inhibit these processes (1). Catalases are important antioxidant enzymes that we have studied extensively. According to our hypothesis, we found that in Neurospora cat-3 minus mutants show increased asexual and sexual sporulation and cat-2 mutants show increased submerged conidia formation. A superoxide dismutase sod-1 gene deleted strain generated a pattern of cyclic conidiation similar to the one observed in the bd strain, which contains a dominant mutation in ras-1. In contrast, the inactivation of NADPH oxidase gene nox-1 resulted in decreased asexual sporulation and prevented sexual development. The mutation of NADPH oxidase gene nox-2 resulted in lack of ascospore germination. Therefore, the mutation of antioxidant and pro-oxidant genes had opposite effects on different developmental processes in N. crassa and is in accordance with our hypothesis.

1 Aguirre J; Rios-Momberg M; Hewitt D; Hansberg W (2005) Trends in Microbiology 13:111-118

Acknowledgement: Grants CONACyT 50716-Q to WH and 49667 to JA; DGAPA PAPIIT IN228507 to WH and JA


Methylation of lysine 9 of histone 3 is important for normal growth and development in Aspergillus fumigatus

Jonathan M. Palmer, Robyn M. Perrin, Nancy P. Keller

University of Wisconsin, Madison, WI, United States

Several lines of evidence have shown that chromatin remodeling mediates regulation of secondary metabolism gene clusters in the aspergilli. Preliminary results in A. nidulans hint that LaeA functions through chromatin remodeling, in essence activating euchromatin or inhibiting heterochromatin. In addition, deletion of the heterochromatin associated histone deacetylase, HdaA, results in an increase in secondary metabolites, suggesting that chromatin remodeling may regulate many secondary metabolite gene clusters. Methylation of lysine 9 of histone 3 (H3K9) is a hallmark of heterochromatin formation and subsequent gene silencing. In Schizosaccharomyces pombe, Clr4 has been shown to be the sole methyltransferase of H3K9. Disruption of the Clr4 homolog in the pathogenic mold A. fumigatus (clrD) resulted in several growth abnormalities; however, the ∆clrD mutant did not show significant alterations in its secondary metabolite profile under the conditions tested. Developmental defects included a reduction in radial growth, delayed conidiation after developmental competence, and a reduction in conidial production. We confirmed by Western analysis that A. fumigatus ClrD methylates H3K9. In addition, heterologous complementation with the clrD homolog of A. nidulans rescued the phenotype, suggesting conserved H3K9 methylation machinery between Aspergillus species.


The role of BEM-1 during germling fusion In Neurospora crassa

Timo Schuerg, André Fleissner

Department of Genetics, Braunschweig, Germany

Cell-cell fusion is essential for a variety of biological processes such as fertilization, tumorigenesis or muscle and bone development. Although cell fusion is of such particular concern, the underlying molecular mechanisms are poorly understood. We are using the filamentous fungus Neurospora crassa as a model system to study cell fusion and related signaling mechanisms. Germinating N. crassa spores show chemotropic growth towards each other and fuse (germling fusion) to form a functional unit establishing the hyphal network. Recently we obtained evidence that BEM-1, a scaffolding protein involved in polarized growth, is essential for germling fusion. Spores of a temperature sensitive bem-1 mutant (Seiler and Plamann, 2003) form spherical cells instead of germ tubes under the restrictive temperature. In addition to this phenotype we identified defects in chemotropic growth which can be separated from the general polarity defect. To gain more insight into the role of BEM-1 in chemotropic interaction and germling fusion we determined its subcellular localization using GFP fusion constructs. Consistent with its role in polarized growth we detected BEM-1-GFP accumulation at every polar growing hyphal tip. In addition to this previously described localization (Leeder and Turner, 2008) we found two more regions of BEM-1-GFP accumulation. Once cell-cell contact is established in germling fusion pairs, BEM-1-GFP localizes to the fusion point. Deconvolution microscopy and subsequent 3-D reconstruction revealed BEM-1-GFP localization around the opening fusion pore, detectable as a bright ring. We also detected BEM-1-GFP localization at septa of germ tubes and mature hyphae. Together, our data suggest novel functions of BEM-1 in chemotropic growth, fusion pore formation and septa formation or maintenance. Our future studies will try to unravel and identify the distinct molecular functions of BEM-1 during Neurospora development.


In search for a role Of NADPH-oxidase generated reactive oxygen species In Aspergillus niger

Mark Arenthorst, Min Jin Kwon, Cees van den Hondel, Arthur Ram

Institute of Biology, Leiden, Netherlands

RacA encodes a small GTPase that belongs to the sub-family of Rho-like GTPases. Analysis of the phenotype of a RacA deletion strain revealed an effect on polarized growth as well as a strong conidiation defect. To identify proteins that interact with RacA, a yeast two hybrid screen was performed using an activated form of RacA (RacAG12V) protein as bait. This resulted in the identification of two RacAG12V interacting proteins. One of the two RacA interacting protein, shows a strong interaction with both RacAG12V and RacA, and is highly homologous to the mammalian p67phox protein and was named NoxR. In mammalian cells, p67phox is part of the NADPH oxidase complex which is regulated by Rac1 and is active during phagocytosis by producing reactive oxygen species (ROS). To study the possible role of RacA in activation of the NADPH-complex during conidiation, knock out strains of noxR and noxA (the catalytic subunit of the NADPH-complex) were made and phenotypically analyzed. Both knock out strains of noxR and noxA show a slightly reduced conidiation phenotype, whereas the D racA strain shows a severe reduction in conidiation. Apparently, the formation of ROS via the NADPH-complex is not necessary for a-sexual conidiation in A. niger.

To examine a possible role for ROS generation in polarized cells growth, the germination and further growth of the noxA and noxR mutants was examined in detail. No difference in germination and no morphological differences were observed in these strains when compared to a wild type strain.

We also showed (see also poster by Min Jin Kwon et al) that RacA and CftA are synthetically lethal which suggests an overlapping function of the two GTPases. To study the possible importance of RacA-NoxR mediated ROS production in a D cftA background, double knockouts of cftA with either noxR or noxA were constructed. Both double mutants do not show any additional phenotype compared to the D cftA strain. This indicates that ROS production via the NADPH oxidase complex is dispensable for polarized growth in A. niger, even in a D cftA strain.


Woronin body biogenesis by the membrane protein WSC

Fang Fang Liu, Seng Kah Ng, Yan Fen Lu, Wilson Low, Julian Loi, Gregory Jedd

National University of Singapore, Singapore, Singapore

Woronin bodies are dense-core organelles found exclusively in filamentous fungi, where they function to seal the septal pore in response to cellular wounding. These organelles consist of a membrane bound protein matrix comprised of the HEX protein, and although they are known to form from peroxisomes, little is known about their biogenesis. Through a forward genetic screen in the model filamentous fungus Neurospora crassa, we identify WSC (Woronin sorting complex), a PMP22 / MPV17- related membrane protein that executes a dual function in Woronin body biogenesis. WSC is localized to the membrane of large peroxisomes where it self assembles to form detergent-resistant oligomers that envelop HEX assemblies to produce asymmetric nascent Woronin bodies. These structures are delivered to the cell cortex in a reaction that also requires WSC and this permits partitioning of the nascent Woronin body and Woronin body inheritance. Together, our findings suggest that WSC and HEX collaborate and control distinct aspects of Woronin body biogenesis; cortical association depends critically on appropriate levels of WSC, which in turn depend on HEX. This dependency helps order events across the organellar membrane and permits the peroxisome to produce a second organelle with a distinct composition and intracellular distribution.



Apical expression of Fig1p during Candida albicans mating

Meng Yang, Neil Gow

University of Aberdeen, Aberdeen, United Kingdom

Candida albicans is a major human opportunistic fungal pathogen. Although classified as an asexual fungus, diploid yeast cells of C. albicans can mate to form a tetraploids. However subsequent meiosis has not so far been observed. The C. albicans pre-mating process includes homozygosis of the mating type locus and a unique white to opaque switching step. However, morphologically C. albicans mating is similar morphologically to that in Saccharomyces cerevisiae. ScFIG1 encodes a low affinity calcium influx system that is involved in S. cerevisiae mating and fusion of the gamete cells. Previous investigations in C. albicans showed that CaFIG1 plays an important role in hyphal apical contact sensing mechanism (Brand et al., Current Biology 2007 17, 347–352). However, the role of CaFIG1 and the distribution of the protein in C. albicans mating remains unknown. CaFig1p is similar to mammalian claudin protein which is located at cytoplasm membrane and involved in tight junctions function of epithelial cells. We constructed deletion mutant and reporter systems for CaFIG1. The C. albicans fig1Δ/fig1Δ null mutant was significantly attenuated in mating fusion efficiency. Similar to S. cerevisiae, the promoter of CaFIG1 was strongly activated during mating and by C. albicans alpha pheromone. However, CaFig1p-GFP was shown only detectable during shmoo formation - which is different to the pattern of expression in S. cerevisiae. Furthermore, the localization of CaFig1p was polarised at apices of shmoos, at the shmoos fusion sites and zygote daughter cells. This suggests that CaFig1p may be involved in shmoo tropism elongation and that it contributes to membrane fusion between mating partners. CaFig1p therefore plays important roles in hyphal tropisms, fusion and mating of C. albicans.


Where is the origin of the Cryptococcus gattii; Vancouver Island Outbreak?

Ferry Hagen, Eiko E. Kuramae, Marjan Bovers, Dave J.C. Gerits, Collin H.A. Gerritzen, Wieland Meyer, Teun Boekhout

1CBS Fungal Biodiversity Centre, Utrecht, Netherlands, 2Westmead Hospital, Molecular Mycology Laboratory, Sydney, Australia

The pathogenic basidiomycetous yeast Cryptococcus gattii may cause a life-threatening disease of the central nervous system, lungs and skin in humans and animals. C. gattii is found mainly in tropical and sub-tropical regions of South America, Africa, Asia and Australia where it is endemic. Recently, a cryptococcosis outbreak in both humans and animals occurred on Vancouver Island (British Columbia, Canada) (Kidd et al., PNAS 101, 2004). This outbreak was shown to be caused by a rare genotype of C. gattii (AFLP6A or RAPD VGIIa). The objective of this study was to find the origin of the outbreak isolates.

A selection of thirty-four C. gattii outbreak isolates and ninety C. gattii reference strains were analyzed by AFLP. The six different AFLP fingerprint analyses were carried out in duplicate. Reproducible marker fragments were used for population genetic analysis. In addition, polymorphic fragments from the AFLP analyses were used to develop a multilocus sequence typing (MLST) approach.

Fraser et al. (Nature 437, 2005) suggested that the Vancouver Island outbreak isolates originated from Australia. However, our results based on AFLP and MLST analyses show that the outbreak isolates originated from South America. South American isolates were found to be ancestral to Australian and Asian isolates as well.

1 Kidd, S.E., F. Hagen, R.L. Tscharke, M. Huynh, K.H. Bartlett, M. Fyfe, L. MacDougall, T, Boekhout, K.J. Kwon-Chung and W. Meyer: (2004) Proc Natl Acad Sci USA 101:17258-17263, 2 Fraser, J.A., S.S. Giles, E.C. Wenink, S.G. Geunes-Boyer, J.R. Wright, S. Diezmann, A. Allen, J.E. Stajich, F.S. Dietrich, J.R. Perfect and J. Heitman: (2005) Nature 437:1360-1364


The use of interfertility data and the internal transcribed spacer region 1 (ITS 1) of the rDNA in establishing the identity of Ganoderma boninense

Faridah Abdullah, Nelson Malik, Husrita Hussin

Universiti Putra Malaysia, Serdang, Selangor, Malaysia

The fungus Ganoderma boninense is of significance to Malaysia due to the damage it caused to the oil palm industry. The species is easily identified when in association with infected oil palms but its identification on other hosts is very difficult, compounded with the absence of a taxonomic key which is traditionally used for species identification. This study carried out interfertility studies using monokaryon testers developed from sibling spores of a local G. boninense specimen, based on its heterothallic and tetrapolar mating system type. The biologically-determined G. boninense could be delineated from the non-boninense. Results showed that G. boninense from India, Sri Lanka and Japan were conspecific with the local specimens; its hosts are mainly but not exclusively palms. Molecular studies using the Internal Transcribed Spacer region-1 (ITS 1) of the rDNA was performed on all of the Ganoderma isolates using non-boninense and non-Ganoderma mycelia as outgroup samples. The dendrogram supported the findings of the interfertility studies where all G. boninense samples grouped tightly into one cluster. A gene sequence analysis also showed the same nucleotide sequence among G. boninense from various geographical regions and hosts. This study concludes that for G. boninense, a combination of intefertility studies with the nucleotide sequence data using the ITS1 region of the rDNA has resolved the identity of the fungal species.



Genomic comparisons of mating type loci of Mycosphaerellla spp belonging to the sigatoka disease complex of banana

Mahdi Arzanlou, Pedro Crous, Lute-Harm Zwiers

CBS Fungal Biodiversity Centre, Utrecht, Netherlands

Introduction: The fungal genus Mycosphaerella represents several thousands of names and synonyms. Many are associated with leaf diseases on a wide range of plants including important food crops such as wheat, banana and citrus. Striking features of this diverse genus are the observed host-jumping and the co-occurrence of multiple species within a single leaf or even a single lesion of an infested host. This close co-occurrence of multiple species of Mycosphaerella both in time and space potentially leads to (sexual) interactions between these species which could result in the origin of new species with altered virulence patterns. Therefore, the mating type loci of three Mycosphaerella species (M. fijiensis, M.eumusae and M. musicola) belonging to the Sigatoka disease complex of banana were studied in detail.

Methods: The mating type loci of the Mycosphaerellae spp were cloned by genome walking and the sequences were compared to the idiomorphs of related species.

Results and discussion: The idiomorphs of the fungi belonging to the Sigatoka disease complex of banana are characterized by an expansion in size, and the presence of two additional Mycosphaerella specific genes (ORF1 and ORF2) which are present in an inverted orientation in both idiomorphs. Initial expression analysis indicated that these ORFs are expressed in a mating-type dependent fashion. Moreover, the interspecies homology of the proteins encoded by ORF1 and ORF2 is higher than the intraspecies homology, suggesting a recent shared evolutionary history of the banana pathogens. Finally, analysis of the M. fijiensis genome sequence revealed the presence of an additional mating type-like locus containing a fusion between a Mat1-1 and a Mat1-2 gene. This fused mating type gene (Mat1/2) is not physically linked to the idiomorph and is also present in the other two major banana pathogens. This unique additional locus might be a remnant of a homothallic ancestral state of Mycosphaerella spp. These results yield a better understanding of mating in these important plant-pathogens and might shed more light on the processes of (micro)evolution and (sympatric) speciation



Identity and phylogeny of yeasts inhabiting Mangrove sediments in 3 coastal regions of Tanzania based on RAPD – PCR profiles and D1/D2 domain of 26S rDNA

Eva Sosovele, Ken Hosea

University of Dar es Salaam, Dar es Salaam, United Republic of Tanzania

A total of 43 morphologically different yeast isolates (at 400x magnification) from various mangrove sediment samples from Dar es Salaam, lindi and tanga regions were investigated in this study. Genomic dna was isolated from all the yeast isolates and pcr amplification carried out using the rapd (randomly amplified polymorphic dna) approach using four different decamer rapd primers. The rapd patterns of 50% of lindi and tanga yeasts bore strong genetic affinities between each other with yeasts producing 4 dna fragments with sizes 300, 500, 600 and 1100 bp. The yeasts isolated from dar es salaam were almost identical to each other with 11 out of 13 isolates having the same rapd genotype with only two dna fragments [500 and 1100bp] regardless the fact that some of these were isolated at distances over 100km apart. Representatives from each rapd genetic profile were selected for full identification and phylogenetic analysis by sequencing the d1d2 domain of 26s lsu ribosomal dna.


Non-dermatophytic molds as a causative agents of onychomycosis in Tehran

Shahindokht Bassiri-Jahromi, Ali Asghar Khaksar, Golnar Sadeghi, Farnoosh Asghari, Ali Najafi

Pasteur Institute of Iran, Tehran, Islamic Republic of Iran

Back ground: Non dermatophytic onychomycosis is a fungal infection of fingernails or toenails caused by moulds and yeasts. Onychomycosis is an opportunistic fungal disease and is usually caused due to impaired barrier functions in healthy individuals, for example, trauma in nail. In the last few years the number of cases of non-dermatophytic onychomycosis has greatly increased, mainly in Europe, associated with cases of immunodepression and in immunocompetent subjects.

The aim of our study was to evaluate the incidence, the clinic characteristics and predisposing factors of non-dermatophytic onychomycosis.

Material and Methods: A total of 1268 patients’ samples including toe and finger nails clippings and subungual debris were collected at the Medical mycology Laboratory of Pasteur Institute of Iran, Tehran from March 2005 through March 2006. Specimens were obtained from clinically suspected fungal infections of toe and finger nail by mycosis, and primary care physicians. The etiologic agents of onychomycosis were established after repeated cultural examinations. All collected specimens were analyzed by direct microscopy and culture. Microscopic examination of these specimens was carried out in potassium hydroxide solution (20%) with demethyle sulfoxide (4%). These specimens were cultured on sabouraud glucose agar with chloramphenicol and sabouraud glucose agar with chloramphenicol and cyclohexamide. Cultures were incubated at 25ºc for up to 28 days and checked twice weekly for growth.

Results: The patients with onychomycosis were 410 cases , of whom 47 cases (11.5%) were non-dermatophytic onychomycosis. . We found that Aspergillus spp. were the more responsible etiologic agents of non dermatophytic agents of onychomycosis, resulting in a total of 28 patients (59.6%). The toe nail was more affected (72.3%). Non-dermatophytic onychomycosis were 11.5% of all ungual infections. In our study other causative agents were Acromonium spp., Fusarium spp., Trichosporum spp. Scopolariopsis spp. and Geotrichom spp.

Conclusion: In our opinion such studies should be performed in every country in order to determine the major fungal species responsible; such information is extremely useful in the treatment of nail onychomycosis. Early diagnosis and accurate therapy are important for the resolution of onychomycosis. Knowing the exact pathogens is important and has implications in therapy and prognosis.



Strain identification in Aspergillus fumigatus

Russell Poulter, Margi Butler

University of Otago, Dunedin, New Zealand

Purpose: Genome sequencing can be employed in species and strain identification. The use of PCR to target specific sequences can make the identification of fungal species or isolates more accurate and rapid. This would be valuable in situations such as the identification and monitoring of strains in a disease outbreak. This approach is, however, difficult in A. fumigatus because this species shows very little sequence variation among strains. Our study uses as a target the PCR and sequencing of specific orthologs of the repetitious Afut1 mobile elements that are present in the A. fumigatus genome.

Methods: We used standard end-point PCR and sequencing methods. Afut1 orthologs were selected using the published genome of strain Af293. The Afut1 elements of A. fumigatus show clear evidence of repeat-induced point mutation (RIP). This was noted when Afut1 was first described. Our study is the first attempt to compare an orthologous series of Afut1 elements derived from a group of strains. Orthologous elements are found at a specific site in the genomes of different strains and are related by vertical descent from an element that transposed into that site in the common ancestor of the group of strains.

Results: Orthologous Afut1 elements are highly variable in sequence between strains. This indicates that RIP has occurred in diverse strains since this species evolved. Some strains show minimal RIP, while others show high frequency of RIP at a particular ortholog. The sequence data permits strain recognition and the study of the intra-specific phylogeny.

Conclusions: The analysis of Afut1 sequences permits strain recognition in A. fumigatus. In other fungi, RIP occurs exclusively during sexual reproduction. The occurrence of RIP in diverse strains of A. fumigatus therefore suggests that there has been sexual reproduction within the species. In addition, sequence data from orthologous sites in multiple strains provides an indication of re-assortment between loci confirming sexual recombination.


RIP in Aspergillus niger and Penicillium chrysogenum

Ilka Braumann1, Marco van den Berg2, Frank Kempken1

1Botanisches Institut und Botanischer Garten, Christian-Albrechts-Universität zu Kiel, Olshausenstraße 40, 24098 Kiel, Germany, 2DSM Anti-Infectives (624-0270), Alexander Fleminglaan 1, 2613 AX, Delft, Netherlands

Transposons are ubiquitous genetic elements with the ability to move around in their hosts’ genomes. This mobility can induce mutations and therefore transposons most probably play an important role in creating genetic diversity in their hosts . But nevertheless transposons can have ditrimentrial effect on their hosts’ genomes: insertions into new genomic locations can also lead to gene disruption. The repetitive nature of transposons can enhance ectopic recombination followed by the removal of functional genes or exons . Hence the evolution of genome defence mechanisms against transposable elements is not surprising. A mechanism exclusively found in filamentous fungi is the "repeat induced point mutation" (RIP). This mechanism acts premeiotical and induces C:G to T:A transitions in both copies of a duplicated sequence .

Here we present an investigation for RIP in multiple copies of the Aspergillus niger retrotransposon AniTa1 and the Penicillium chrysogenum class II transposon PeTra2. RIP was identified in both elements, representing the first observation of RIP in these two fungi. For PeTra2 all sequences investigated seem to be effected by a moderate type of RIP in a varying extend. AniTa1 copies seem to be unRIPed with the exception of two copies in which 20% of all nucleotides have been altered due to RIP. These findings suggest a rather selective but very strong type of RIP in A. niger.


Analysis of the mating-type system of Ashbya gossypii

Jürgen Wendland, Alexander Dünkler, Andrea Walther, Sia Jorde

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

A prerequisite for classical genetic analysis in a fungus is the knowledge of its mating system to generate meiotic offspring. Ashbya gossypii is a filamentous ascomycete which is used to overproduce riboflavin at an industrial scale. The genome sequence revealed three mating-type loci on chromosomes IV, V, and VI. Interestingly, all loci bear the information of MATa and encode two divergently transcribed genes, MATa1 and MATa2. Analysis of the genome sequence furthermore shows that all classical elements of the Saccharomyces cerevisiae mating-pheromone response pathway are present. Starting from pheromone receptor genes homologous to ScSTE2 and ScSTE3, to the heterotrimeric G-protein (GPA1, STE4, STE18), the MAP-kinase cascade (STE11, STE7, FUS3/KSS1) and the transcription factor STE12. Mating pheromone genes are annotated as homologs to MFA2/YNL145w (ABL196c) and MFalpha1/YPL187w (AFL062w).

We re-sequenced AFL062w, however, this gene does not encode a mature alpha pheromone. We discovered a novel Ashbya gene encoding the A. gossypii alpha factor. By comparison with other alpha pheromones we deduced the sequence of the mature peptide. We present results on the use of the chemically synthesized peptide in growth inhibition assays of the MATa wild type strain. We also present the analysis of Ashbya strains deleted for BAR1, which encodes a protease homolog of S. cerevisiae Bar1 that cleaves alpha pheromone. Also we will show the use of the novel alpha pheromone in screens to isolate pheromone insensitive Ashbya strains that potentially are of MATalpha genotype. This research will promote the establishment of a mating system to allow classical genetics with A. gossypii.


Mitochondrial pAL2-1 plasmid homologues are independent senescence factors in Podospora anserina

Anne D. van Diepeningen, Alfons J.M. Debets, S. Marijke Slakhorst, Rolf F. Hoekstra

Dept. of Genetics, Wageningen University, Wageningen, Netherlands

In the over 25 years since the first description of a linear mitochondrial plasmid in Podospora anserina, pAL2-1 and homologous plasmids have gone from being considered beneficial longevity plasmids, via neutral genetic elements, towards mutator plasmids causing senescence. The plasmid has an invertron structure, with terminal inverted repeats and encodes a DNA and a RNA polymerase and can insert into the mitochondrial genome.

In our lab 112 wild-type P. anserina strains have been isolated between 1991 and 2001 in the surroundings of Wageningen, the Netherlands and these strains were characterised. Surprisingly, pAL2-1 homologues have a very common occurrence (40%) in them. Growth experiments showed that plasmid infection negatively influences the life span of the wild-type strains and largely negates any life span extending effect generally caused by Calorie Restriction (CR). In isogenic lines pAL2-1 homologues also negated calorie restriction effects and destabilized the mitochondrial genome via insertion. Moreover, pAL2-1 homologues interfered with other life span extending conditions as e.g. compounds interceding with mitochondrial functions like certain antibiotics or low incubation temperature. Even long-lived mutants have reduced life span when pAL2-1 homologues are introduced. Thus, pAL2-1 homologues act as senescence factors independent of other intrinsic life span determinants and external conditions.


ROS and ageing in Podopora anserina

Anne D. van Diepeningen, Daniël J. Goedbloed, Rolf F. Hoekstra, Alfons J.M. Debets

Dept. of Genetics, Wageningen University, Wageningen, Netherlands

In 1956 Harman proposed his so-called free-radical theory on ageing which has become the most widely accepted theory linking ageing with reactive oxygen species (ROS). Most of the ROS are formed as by-products of the cytochrome oxidase pathway generating energy in the mitochondria and it is there that ROS cause an accumulation of damage to nucleic acids, proteins and other compounds. Podospora anserina is one of the few filamentous fungi known to age in nature. It can be grown under controlled conditions on synthetic medium, has a life span of approximately 2-3 weeks and thanks to years of research quite a lot is known about ageing in this species, making it an ideal model organism. Though much is known about P. anserina and though longevity is often linked to a deficiency in the cytochrome oxidase pathway, nothing is known about the exact advance in ROS production during life.

We now have developed and adapted a method for the semi-quantified measurement of produced ROS to study the production during the life span of different P. anserina strains, under different growth conditions and with different mutant strains. This method is based on photospectrometry using diaminobenzidine tetrachloride (DAB) in a 0.1 M phosphate buffer at a pH of 7.4.

In this way we have monitored the normal ROS levels during ageing in strains under normal growth conditions: ROS levels are quite stable throughout most of the organism’s life span. There is however a strong increase in ROS concentration when the organism reaches the senescent stage announcing growth termination. We show that the pattern of stability and final increase in ROS levels is similar but the process takes less time in strains ageing more rapidly due to infection with linear mitochondrial plasmid pAL2-1homologues. Finally we show that ROS levels decrease when caloric reduced conditions are applied that extend life span two-fold or more.



Confirmation of identification of a population of Aspergillus fumigatus isolates using a polyphasic approach

Celine O'Gorman1, Hubert Fuller1, Paul Dyer2

1UCD School of Biology and Environmental Science, University College Dublin, Belfield, Dublin 4, Ireland, 2School of Biology, University of Nottingham, Nottingham, NG7 2RD, United Kingdom

A collection of 94 Aspergillus fumigatus sensu lato isolates was cultured from air samples taken at four outdoor locations during 2005 [1]. A polyphasic approach was employed for the identification of the isolates, using morphological, physiological and molecular data. Phenotypic characters were examined using established protocols [2] and included the macro- and micromorphology of the isolates, their maximal and minimal growth temperatures and their 25 °C/45 °C growth ratio. Results confirmed that all 94 isolates were A. fumigatus sensu stricto. Genotypic variation within the population was analysed by RAPD-PCR which revealed that the isolates are closely related but not identical. The distribution of the two complementary mating-type genotypes of the fungus (termed MAT1-1 and MAT1-2) was then determined using a multiplex mating-type PCR assay [3]. The population consisted of equal proportions of the MAT1-1 and MAT1-2 genotypes (49.4% MAT1-1, 50.6% MAT1-2), with no geographical clustering of mating-types evident. A representative sub-sample of 12 isolates from the population was chosen for further examination by multi-locus sequence typing of partial ß-tubulin and carboxypeptidase gene sequences. Phylogenetic analysis clearly grouped the 12 representative isolates with known Aspergillus fumigatus sensu stricto isolates. The results show that A. fumigatus isolates of complementary mating-type can be readily found in close proximity in nature, consistent with a sexually reproducing species.

1 O’Gorman, C.M., Fuller, H.T., 2008. Atmospheric Environment doi:10.1016/j.atmosenv.2008.01.009. 2 Hong, S.-B. et al., 2005. Mycologia 97: 1316-1329, 3 Paoletti, M. et al., 2005. Current Biology 15: 1242-1248.


Sequence-based identification, aggressiveness and fumonisin production of a population of Fusarium species causing bakanae disease of rice in the Philippines

Christian Joseph Cumagun1, Carlos Padilla1, Evelyn Gergon2, Francois Van Hove3, Maria Teresa Gonzàlez-Jaén4, Patricia Marin4

1Crop Protection Cluster, College of Agriculture, University of the Philippines Los Baños, Los Banos, College, Laguna, Philippines, 2Philippine Rice Research Institute, Munoz, Nueva Ecija, Philippines, 3Mycothèque de l’Université catholique de Louvain, Louvain-la Neuve, Belgium, 4Department of Genetics, Faculty of Biology, Universidad Complutense de Madrid, José Antonio Nováis, Madrid, Spain

Forty Fusarium strains were isolated from rice stems, shoots and grains in the Philippine provinces of Nueva Ecija and Laguna. All isolates were identified as Fusarium fujikuroi based on the elongation factor -1 α sequence except three isolates which were identified as F. proliferatum, F. sacchari and F. oxysporum.  Based on PCR amplification of MAT (mating type) specific sequences, the 37 F. fujikuroi segregated 10:27 of MAT-1 and MAT-2, respectively. Only five isolates produced fumonisins  in liquid culture; concentrations, estimated by Enzyme Linked Immunosorbent Assay (ELISA), ranged from 0.025 ppm to 0.238 ppm. High Performance Liquid Chromatography (HPLC) analysis of 20 isolates revealed seven isolates as fumonisin producers with production ranging from 0.86µg/g -210 µg/g. Amplification of a partial sequence of the fum1 gene (a key gene in fumonisin biosynthesis) is ongoing. No association between fumonisin production and aggressiveness of isolates under lab and greenhouse conditions were observed.The production of fumonisins of F. fujikuroi in rice implies the need to explore a larger population of this pathogen to promote food safety.


Aggressiveness, vegetative compatibility groups, mating type assessment and fumonisin production of Gibberella fujikuroi (Fusarium verticillioides) from corn in the Philippines

Christian Joseph Cumagun1, Mabel Vallena1, Ma. Leizel Vargas1, April Alviar1, Francoise Munaut2, Francois Van Hove2

1Crop Protection Cluster, College of Agriculture, University of the Philippines Los Baños, Los Baños, College, Laguna, Philippines, 2Mycothèque de l’Université catholique de Louvain (BCCMTM /MUCL), Université catholique de Louvain, Louvain-la Neuve, Belgium

Gibberella fujikuroi (Fusarium verticillioides) is one of the most important fungal pathogens of corn worldwide. The pathogen produces mycotoxin fumonisins that are potentially harmful to human and animal health. Twenty isolates of G. fujikuroi from Laguna, Philippines were characterized at the molecular level by mating population (MP) and mating type (MAT). The identity of the 12 isolates was confirmed by sexual crosses with standard tester strains. Eleven isolates were MATA-1 and one MATA-2. All the isolates were further characterized by fertility and vegetative compatibility group (VCG) in the laboratory and tested for their stalk rot aggressiveness on ‘Super Sweet’ corn variety under field conditions across two environments using the toothpick inoculation method. In two greenhouse trials, inhibition of seedling emergence, seedling height, fresh and dry weight were also determined. Fumonisin production was analysed by High Performance Liquid Chromatography (HPLC). Analysis of fertility revealed that six out of 12 MPA isolates were hermaphrodites. Significant (P = 0.05) differences in aggressiveness toward corn of some isolates were observed for both experimental locations while vegetative compatibility grouping by pairing nit mutants identified 19 vegetative compatibility groups for this population with a genotype diversity of 0.95. All isolates were pathogenic to corn seedlings and mature plants compared to non-inoculated control. Fumonisin production of the isolates was not associated with their fumonisin production. Majority of the MPA isolates, 9 out of 11 analyzed, were fumonisin producers whereas 3 out of 5 analyzed MPA isolates are non fumonisin producers.


Mating type genes and population structure of Paecilomyces variotii (teleomorph: Byssochlamys spectabilis)

Jos Houbraken, Janos Varga, Robert A. Samson

CBS Fungal Biodiversity Centre, Utrecht, Netherlands

Paecilomyces variotii is a common world-wide occurring species found in soils, indoor environments, plants, animals and foodstuffs. It is a fast growing thermo-tolerant fungus which is able to grow at low oxygen levels, in the presence of preservatives, able to form the mycotoxin viriditoxin and capable to survive heat treatments. In this study we examined the genetic variability and reproductive mode of this species by analyzing the nuclear ribosomal internal transcribed spacers (ITS1, ITS2) and the 5.8S rRNA gene, as well as partial b -tubulin, actin and calmodulin gene sequences. Phylogenetic analyses revealed that P. variotii is a highly variable species, and Byssochlamys spectabilis was found to be conspecific with it. Partition homogeneity tests revealed that P. variotii has a recombining population structure. Besides sequence analyses, mating experiments indicated that P. variotii is able to form ascomata and ascospores in culture in a heterothallic manner. The distribution of MAT1-1 and MAT1-2 genes showed a 1:1 ratio in the progeny of the mating experiments. From the sequence analyses and mating data we conclude that P. variotii is the anamorph of the heterothallic B. spectabilis and that it has a biallelic heterothallic mating system. Previously, examination of the population structure by phylogenetic methods revealed the presence of a recombining population structure in several species in the Trichocomaceae family which were considered previously to reproduce only asexually (clonally), including A. flavus, A. fumigatus, A. nomius, A. lentulus and Penicillium marneffei. However, the sexual stage of none of these anamorphs could be recovered so far. Additionally, this study is one of the few examples in the order Eurotiales where a teleomorph-anamorph relationship could be established based on both the phylogenetic and the biological species concept.



Geographic variation and positive diversifying selection in the host specific toxin SnToxA

Eva Stukenbrock, Bruce McDonald

ETH Zurich, Plant Pathology Group, Zurich, Switzerland

The host-specific toxin ToxA produced by the wheat pathogens Pyrenophora tritici-repentis and Phaeosphaeria nodorum interacts with the product of the dominant plant gene Tsn1 to induce necrosis. The ToxA gene is thought to have been acquired by P. tritici-repentis from P. nodorum through a recent horizontal gene transfer event. PCR and sequence analysis indicate that the level of ToxA variation, including gene deletion, in P. nodorum (SnToxA) is significantly higher than in P. tritici-repentis (PtrToxA).

We PCR-screened 788 isolates of P. nodorum originating from eight geographical regions to infer the pattern of SnToxA deletions. The frequency of deletions differed significantly among populations, ranging from 0% (Australia) to 98% (China). Sequence analysis of the SnToxA gene in 123 P. nodorum isolates revealed 13 distinct haplotypes. The distribution and diversity of haplotypes varied significantly among populations. The majority of SnToxA mutations were non-synonymous resulting in changes at the protein level. We applied different models of selection to infer the mode of evolution operating at the ToxA locus. Evidence for positive diversifying selection supports the hypothesis that evolution of the ToxA locus is driven by selection imposed by the host. The distribution of SnToxA alleles and deletions may reflect the distribution of different Tsn1 alleles in the corresponding host populations.


Frequency dependent selection acting on the MIP region in Serpula lacrymans

Ingeborg B. Svegården1, Ann-Kristin Hansen1, Timothy Y. James2, Håvard Kauserud1

1Dep. of Biology, University of Oslo, Oslo, Norway, 2Dep. of Evolutionary Biology, EBC, Uppsala University, Uppsala, Sweden

The dry rot fungus Serpula lacrymans (Basidiomycota) is the most damaging destroyer of wood construction materials in temperate regions. Employing various presumably neutral genetic markers, we have shown that the dry rot fungus is divided into two main lineages; one non-aggressive residing naturally in North America and Asia (var. shastensis), and another aggressive lineage including specimens from all continents (var. lacrymans). Genetic analyses pinpoint mainland Asia as the origin of the aggressive form var. lacrymans and only a few aggressive genotypes have migrated worldwide from Asia to Europe, North- and South America and Oceania followed by local population expansions. In contrast to the neutral genetic markers, high genetic variation was observed in the MIP region in the founder populations of var. lacrymans. The MIP region is tightly linked to one mating type gene and therefore probably governed by strong frequency dependent selection, leading to the high gene diversity. The genetically variable MIP region can probably be used as a target to distinguish between strains of var. lacrymans.


The supposedly asexual pathogen Aspergillus fumigatus expresses functional determinants of sexual development

Sven Krappmann

Research Center for Infectious Diseases, University of Wuerzburg, Wuerzburg, Germany

Pathogenic fungi have gained increasing attention over the last decades as being a menace especially for the immunocompromised individual. The outcome of infection of a susceptible host depends on an array of multiple factors, and from latest studies different patterns that determine fungal pathogenicity have emerged. Most recently, the role of sex is under discussion, based on the fact that the most common fungal pathogens responsible for systemic diseases in humans appear to have retained sexuality although with limited access to meiotic reproduction.

Among the aspergilli, A. fumigatus is by far the most frequent species to cause disease, and knowledge on the underlying cellular attributes for this is still scarce. Based on the working hypothesis that this pathogen, although appearing as asexual, has retained some cryptic sexuality, we aim at conserved key factors of sexual development. For that purpose, any conserved function of the A. fumigatus NsdD orthologue was examined in complementation and over-expression studies. When expressed in A. nidulans, the A. fumigatus gene is able to rescue the deletion phenotype of acleistothecial development. Moreover, forced expression of AfNsdD in A. nidulans results in predominant sexual development in this host. When either transcription factor, AfNsdD or AnNsdD, is expressed at high levels in the deuteromycete A. fumigatus, no formation of mature fruiting bodies is evident - however, unusual coiled hyphae are formed, and this is independent of the recipient’s mating-type.

In conclusion, first evidence is provided that that the genome of the presumably asexual pathogen A. fumigatus encodes a functionally conserved positive regulator of sexual development. This transcriptional activator is able to trigger the formation of morphological structures that may resemble first stages of sexual development.


Molecular characterization of Colletotrichum falcatum isolates from sugarcane in India

Palaniyandi Malathi1, Jaehyuk Choi2, Rasappa Viswanathan1, Purusothaman Padmanaban1, Yong-Hwan Lee2

1sugarcane Breeding Institute, Tamil Nadu, India, 2departmentof Agricultural Biotechnology, Seoul National University, Seoul, Korea, Republic of

Colletotrichum falcatum, the causal fungus of red rot, has far-reaching influence on the economy of sugarcane production in India. At present the newly emerging pathotypes are usually named based on the cultivars from which they are isolated and they are grouped by cultural characteristics, geographical origin, and the level of pathogenicity. However, little correlation exists between mycological characteristics and pathogenicity. Present study was undertaken to understand the correlation, if exists, between cultural, pathological and molecular characteristics with diverse collection of 68 isolates of 44 cultivars from different geographic origins for a period of 10 years prevalent all over India. All isolates were examined at molecular level using 5.8s rRNA-ITS, actin, calmodulin and glyceraldehydes-3 phosphate dehydrogenase gene sequences. Sequence analysis of ITS identified the genetic divergence with three distinct clades as 33, 32, and 3 isolates for clade I, II, and III, respectively. Among which, the isolates belonging to calde I is represented by most of the sub-tropical and less virulent isolates and the clade 2 has most of tropical and virulent types while clade 3 represents newer group similar to isolates of other countries. Representative 25 isolates from various locations were also studied for sequences of other three genes. The existence of clade III was highly implicated by all three gene sequences while clades I and II were not much supported. However actin gene sequences distinguished the isolates for virulence and sporulation in clade I and II. Comparative analysis with other species of Colletotrichum paved to design C. falcatum specific primer sets for its rapid identification through PCR amplification. These data would provide new strategies to identify the development of new pathotypes of C. falcatum, and to diagnosis infected sugarcane samples for screening genotypes and quarantine purpose.


Genetic heterogeneity versus biochemical similarity of mycocypins, fungal cysteine protease inhibitors

Jerica Sabotic, Tatjana Popovic, Joze Brzin

Jozef Stefan Institute, Ljubljana, Slovenia

Mycocypins, cysteine protease inhibitors from basidiomycetes, represent a new family of cysteine protease inhibitors. The first protein cysteine protease inhibitor characterized from higher fungi, clitocypin from Clitocybe nebularis, was on account of its unique characteristics assigned to a new family of cysteine protease inhibitors I48 of the MEROPS inhibitor classification. Genetic analysis revealed that clitocypin is encoded by a small family of closely related genes with more than 90% identity. Genes are composed of four exons and three short introns and heterogeneity is limited to 18 discreet positions distributed throughout the coding sequence. The second representative of mycocypin family, macrocypin, cysteine protease inhibitor isolated from Macrolepiota procera, is also encoded by a family of genes, composed of four exons and three short introns as clitocypin, however the heterogeneity is even higher compared to clitocypin gene family. Based on the presence of a cysteine residue we sorted macrocypin deduced amino acid sequences into three isoforms. Sequences that belong to one isoform share more than 95% sequence identity at the deduced amino acid sequence level, while sequence identity among isoforms is 80 – 86%. There are only 28 amino acid residues conserved in all mycocypin sequences that are probably important for their inhibitory activity. Despite low sequence identity of 21%, clitocypin and macrocypin show similar biochemical properties and inhibitory spectra. They both inhibited tested cysteine proteases to a similar extent, demonstrating an unusually broad inhibitory spectrum including distantly related proteases, papain and legumain. No sequence of significant similarity to that of any mycocypin gene was found in any of the databases searched, which is probably due to low sequence identity of mycocypins. Similarity of biochemical properties, however, points to a common physiological function of mycocypins, which was proposed to be a defensive role against viruses or parasitic organisms.


Some like it cold - root-endophytes on a Swiss permafrost site

Valentin Queloz, Christoph R. Gruenig

ETHZ, Zurich, Switzerland

Members of the Phialocephala fortinii s.l. – Acephala applanata species complex are among the most ubiquitous distributed root endophytes of conifer species. Community structures of these endophytes revealed that species occur sympatrically and quaternary ice ages were hypothesized to have triggered speciation within this species complex. One prerequisite would be that P. fortinii s.l. has a high ability to survive in cold environments. Therefore, the community structure of these root endophytes was studied on a permafrost site in the Swiss Jura (Creux du Van, Switzerland). The frequency of colonization of Picea abies and Vaccinium spp. roots by members of the P. fortinii s.l. – A. applanata species complex was similar as in subalpine spruce forests. In total, 247 strains were genotyped using single-copy RFLP, microsatellite and sequence markers. Interestingly, two new (cryptic) species were discovered beside some well-known recently described ones. In addition, significant levels of gene flow were detected between various populations of identical species in Switzerland. The results of the present study show that members of the P. fortinii s.l. – A. applanata species complex are well adapted to cold environments.


Philigenetic analysis and fumonisin production of Fusarium fujikuroi strains isolated from rice in the Philippines

Patricia Marín1, Christian Cumagum2, Alejandra Cruz1, Covadonga Vázquez3, Carmen Callejas1, Mª Teresa González-Jaén1

1Department of Genetics, University Complutense of Madrid, Jose Antonio Novais 2, 28040 Madrid, Spain, 2Crop Protection Cluster, University of the Philippines Los Baños, College, Laguna, 4031, Philippines, 3Department of Microbiology III, University Complutense of Madrid, Jose Antonio Novais 2, 28040 Madrid, Spain

Fumonisins are important mycotoxins which often contaminate several cereals such as maize, wheat or rice, although they can also occur in a wide variety of other commodities. Fumonisins are responsible of serious chronic and acute diseases in human and animals and their presence is under regulation in more than 100 countries. Fumonisins production is basically limited to the members of the formely so-called Gibberella fujikuroi species complex. Although Fusarium verticillioides and F. proliferatum are the main fumonisin producers, other related species have been also reported. F. fujikuroi has been described as a maize and rice pathogen causing important agricultural losses. However, little information is available about the phylogenetics of this species and its ability to produce fumonisins. In this work, we studied 23 strains isolated from rice in the Philippines and we performed a philogenetic analysis using the partial sequence of the Elongation Factor 1 alpha (EF-1a ) including isolates belonging to closely related species. Fumonisin producion was analysed in seven-day-old cultures grown in fumonisin-inducing medium by an ELISA-based method and by Real Time RT- PCR using primers for FUM1 gene, a key gene in fumonisin biosynthesis. The results indicated the ability of F. fujikuroi isolates to produce fumonisin at low levels in the conditions tested and a good agreement between results obtained by ELISA and Real Time RT-PCR.


Genetic diversity of clinical and environmental strains of Aspergillus fumigatus by microsatellite typing

Ricardo Araujo1, Leonor Gusmao1, Acacio G Rodrigues2, Cidalia Pina-Vaz2, Antonio Amorim3

1IPATIMUP, University of Porto, Porto, Portugal, 2Dept. Microbiology, Faculty of Medicine, University of Porto, Porto, Portugal, 3Faculty of Science, University of Porto, Porto, Portugal

De Valk et al. had previously developed a three multiplex panel with nine short tandem repeats (STR) showing a discriminatory power of 99.94% among A. fumigatus strains. We had improved this fingerprint reaction by developing a single multiplex with even higher discriminatory power (three new STR were included). The new method was tested in clinical and environmental strains of A. fumigatus and the genotype frequencies in both groups were compared. Ninety-two strains of A. fumigatus (47 clinical versus 45 environmental strains) were selected from a local collection. Following DNA extraction, a single multiplex with eight primer pairs (forward primers labelled with 6-FAM, PET, NED or VIC) was performed in a Genetic Analyzer ABI Prism 3100. Sequencing was further performed for the characterization of the different STR alleles structure. The multiplex was performed successfully and an excellent discrimination power was found (all strains were different). The markers resulted in high genetic diversity from 11 (marker 7) to 32 (marker 1) different genotypes. Tri- and pentanucleotide repeat motifs were responsible for higher diversity values when compared to tetranucleotide repeats. The association test between all pairs of markers showed that 3 out of a total of 28 comparisons were significantly deviated from randomness. No significant differences were found between the genotypes of clinical and environmental strains (99.3% of the variation was within populations). Thus, both populations show a probable similar pathogenic potential.

De Valk HA et al. J Clin Microbiol 2005; 43: 4112-20.


Horizontal transfer of the AC E1 secondary metabolite gene cluster between fungi

Nora Khaldi2, Jerome Collemare1, Marc-Henri Lebrun1, Kenneth Wolfe2

1UMR5240 CNRS/UCB/INSA/Bayer CropScience, Lyon, France, 2Smurfit Institute of Genetics, Trinity College Dublin, Dublin, Ireland

Filamentous fungi synthesize many secondary metabolites and are rich in genes encoding proteins involved in their biosynthesis. Genes from the same pathway are often clustered and co-expressed in particular conditions. Such secondary metabolism gene clusters evolve rapidly through multiple rearrangements, duplications and losses. It has long been suspected that clusters can be transferred horizontally between species, but few concrete examples have been described so far. In the rice blast fungus Magnaporthe grisea, the avirulence gene ACE1 that encodes for a hybrid PKS-NRPS belongs to a cluster of 15 genes involved in secondary metabolism. Additional related clusters were detected in the ascomycetes Chaetomium globosum, Stagonospora nodorum and Aspergillus clavatus. Gene-by-gene phylogenetic analysis showed that in C. globosum and M. grisea, the evolution of these ACE1-like clusters is characterized by successive complex duplication events including tandem duplication within the M. grisea cluster. The phylogenetic trees also present evidence that the homologous ACE1 gene cluster in Aspergillus clavatus originated by horizontal transfer of at least 5 genes from the M. grisea ACE1 cluster, which is a much simpler explanation of the observed data than the alternative of multiple events of duplication and losses of parts of the cluster.


Identification of Tapesia species causing cereal eyespot disease in Poland

Katarzyna Pieczul, Marek Korbas, Ilona Swierczynska

Institute of Plant Protection, Poznan, Poland

Eyespot is a widespread and economically important disease of cereals in Poland. Two species – Tapesia acuformis (R-type of Pseudocercosporella herpotrichoides) and Tapesia yallundae (W-type of P. herpotrichoides) were previously recognized as disease agent. Identification of the pathogens is usually possible by traditional tools like analysis of morphological features and differences in a host range as well as fungicide sensitivity. R-type isolates are equally pathogenic to rye and wheat and produce slow-growing colonies with uneven margin while W-type isolates are highly pathogenic only to wheat and form fast-growing, smooth-edged colonies. Both fungi – T. acuformis and T. yallundae exhibit two-allele heterothallic mating system, with mating types MAT-1 and MAT-2. Nowadays molecular tools like PCR reaction are frequently used to distinguish the Tapesia species and to identify their genetic features like mating types.

The aim of the study was the molecular identification of Tapesia species and their mating groups. One hundred isolates collected in years 2006-2007 from whole Poland were used for the genetic study. We used species specific PCR reaction to distinguish the Tapesia species and to estimate their frequency in Poland. Moreover multiplex PCR was used to determine the distribution of mating types (MAT-1 and MAT-2) of both pathogens.

We observed that both species - T. acuformis and T. yallundae are equally present in Poland. Also both mating types of each pathogen are present in the Polish population.

The presence of both mating types indicates the possibility of sexual crossing between isolates. On the basis of the performed analysis we estimate their frequency in Poland.


Functions and phylogeny of basidiomycete laccases

Miia Mäkelä, Kristiina Hilden, Terhi Hakala, Annele Hatakka, Taina Lundell

University of Helsinki, Department of Applied Chemistry and Microbiology, Helsinki, Finland

Laccases are phenol-oxidizing, generally four-Cu containing enzymes produced by fungi, plants, insects and bacteria. Laccases belong to the multicopper oxidase superfamily with so far over 350 cloned representatives with versatile functions. Due to their low specificity for reducing substrates, fungal laccases have been successfully utilized in a wide variety of industrial and commercial applications. e.g. for polymerization of phenolic compounds in wine and beverages, enhancement of dough properties in baking, treatment of wood and textile fibers, removal of toxic and dye compounds in industrial waste waters, biodegradation of xenobiotics in contaminated soils, and for conversion of lignin in wood pulp in biobleaching.

We recently described expression of laccase-encoding genes in the white rot basidiomycete Phlebia radiata 79, when the fungus was cultivated on softwood (Alnus incana) and hardwood (Picea abies). Clones for two laccase-encoding genes, the previously described Pr-lac1 and a new gene Pr-lac2 were characterized. Pr-lac2 coding region is interrupted by 12 introns and the deduced Lac2 protein displays a higher pI value (5.8) than Lac1 (pI 3.2-3.5). At protein level, these two laccases show structural differences within their Cu-1 site near the surface where the aromatic substrate molecules are bound.

Phylogenetic analysis indicates differential evolution for the two laccases, and Lac2 demonstrates the highest sequence identity with Trametes laccases (66%). Transcripts of Pr-lac1 were the most abundant both in solid-state softwood and semi-solid hardwood cultures, as analyzed by competitive RT-PCR and Northern hybridization. On spruce wood chips, Pr-lac1 and Pr-lac2 were expressed within 2-3 weeks of growth together with manganese and lignin peroxidase-encoding genes. Our results indicated time-dependent regulation of expression for the two, at protein and gene level distinct P. radiata laccases.

Biochemical properties and expression of laccases from several other wood-degrading basidiomycetes are described. One of the key questions is how extracellular laccases are involved in the white-rot fungal decay of wood and in particular, in coversion and degradation of wood lignin. This is discussed with focus on phylogenetic deviation of the fungal laccases.


Phylogenetic relationships of Colletotrichum acutatum isolates causing anthracnose in strawberry

Carlos Garrido, Inmaculada Vallejo, María Carbú, Francisco Javier Fernández-Acero, Jesús Manuel Cantoral

University of Cadiz, Puerto Real, Cadiz, Spain

Introduction: Colletotrichum acutatum is a major plant pathogen that infects a broad range of host plants. Extensive research has been carried out on C. acutatum populations affecting various hosts in different geographical locations, showing a considerable genotypic and phenotypic diversity. The purposes of the present study were: (i) to classify an extensive sample of C. acutatum strains collected from different strawberry cultivars around the world, within previously described molecular groups of the species, by using ITS sequence analyses; (ii) to establish phylogenetic relationships among the isolates.

Methods: Nucleotide sequences of the ITS1, 5.8S RNA gene, and ITS2 were used for 52 strains of Colletotrichum spp. isolated from 12 different countries. Forty four isolates were sequenced, whereas the sequence of the remainder 8 isolates had been previously reported. A consistent Neighbor-joining phylogenetic tree was produced using the 5.8S-ITS sequences. Five thousand bootstrap data sets and Kimura-2P model distance matrices were used. To allocate the isolates into 9 previously described C. acutatum molecular groups, additional 35 sequences of C. acutatum and 1 of C. lindemuthianum from the GenBank database were included in the phylogenetic tree.

Results and Discussion: Twenty nine isolates clustered in the molecular group A2, in which seventeen out of eighteen Spanish isolates were included, which may indicate that isolates of group A2 are the key pathogens in this country. The group A1 clustered all the C. acutatum isolates that were isolated from Lupin spp. The 5.8S-ITS sequences of five C. acutatum isolates were clustered with a bootstrap support of 79% within group A3. One isolate from Spain and one from United Kingdom clustered within group A4 with a high (94%) bootstrap support. One C. acutatum isolate from New Zealand clustered in the group A7 together with previously reported A7-isolates. Two isolates of C. acutatum from The Netherlands, isolated from Rose spp. and Phlox spp. respectively, were grouped within the group A5 with a bootstrap support of 62%. Finally, only one isolate, from Australia, was clustered within the molecular group A9. All differences found in the studied sequences of the isolates were located in the non-codificant internal transcriptional spacers (ITS1 or ITS2), so that these differences did not lead in amino acid changes.


Phylogenetic analysis of pheromone receptors of the Ustilaginaceae

Ronny Kellner, Dominik Begerow

Ruhr Universität Bochum, Bochum, Germany

The phytoparasitic smut fungi are an important model group to investigate principles of parasite evolution. They are characterized by a dimorphic life cycle with a haploid yeast and a dicaryotic parasitic stage. Successful infection is directly linked to mating in Ustilago maydis.

Mating in basidiomycetes is regulated by two loci one of which includes a pheromone/pheromone receptor (PR-locus) and the second includes two homeodomain transcription factors (HD-locus). In Ustilago maydis, the development of conjugation hyphae, their polar growth and fusion is triggered by the pheromone/pheromone receptor system. After perception of the pheromone the implemented signal transduction cascade activates transcription factors which regulate many genes responsible for mating and infection including the pheromones and pheromone receptors themself. So far the pheromone receptor genes were only known from U. maydis, U. hordei and Sporisorium reilianum. The investigation of their diversity could provide new molecular data for phylogenetic studies. Next to the presentation of pheromone receptor sequences of other Ustilaginaceae species we will compare the differently realized mating systems (bipolar vs. tetrapolar) with phylogenetic groupings based on ITS and LSU. Based on cDNA alignments we will discuss substitution rate patterns within the pheromone receptor genes.



Evolutionary patterns in smut fungi

Dominik Begerow

Ruhr-University Bochum, Bochum, Germany

Smut fungi have been of great interest for a long time for they include economically important plant pathogens. The phylogeny of the group has been under discussion and the combination of ultrastructural and molecular data provided insight into the evolution of this heterogeneous group. The hierachic level of the group has been updated several times and the former Ustilaginales are now treated as Ustilaginomycotina and include several new orders. These studies have also revealed the enormous influence of host plants in the evolution of these parasites.

The exclusion of Microbotryum and relatives from the Ustilaginomycotina highlights a number of convergences of many important fungal traits in two subphyla. The comparison of Microbotryales and Ustilaginales allows the discrimination between characters and traits, which are based on ecological or phylogenetic constraints. Thus, we will be able to study the evolutionary patterns of these plant parasites.

The use of functional genes for evolutionary studies provides deeper insights in the biology of the plant parasites. The comparison of substitution rates and gene arrangements together with functional analyses might result in new approaches to study interactions between plants and fungi.


Development of cost effective tools for assessing genetic diversity of Mycosphaerella fijiensis, the causal agent of black-Sigatoka disease in bananas (Musa spp.)

Claudia Ferreira, Theo A. J. Van der Lee, Olman Quiros, Bas L. Herrert, Jean Carlier, Caucasella D. Trujillo, Stephen B. Goodwin, Mauricio Guzman, Manoel Souza, Gert H.J. Kema

1PRI-Plant Research International, Wageningen, Netherlands, 2Embrapa Cassava and Tropical Fruits, Cruz das Almas/Bahia, Brazil

Mycosphaerella fijiensis, the causal agent of banana leaf streak disease, commonly known as Black Sigatoka disease, is one of the most devastating plant pathogens. M. fijiensis is a relatively new pathogen and still actively colonizes new areas where bananas are grown. Using the genome sequence of M. fijiensis isolate CIRAD86 ( we identified Variable Number of Tandem Repeat (VNTR) markers using a bioinformatics pipeline. The VNTR screening resulted in 1528 candidate VNTR loci of which many were imperfect mono- di- or tri- nucleotide repeats. Sixteen VNTR markers were selected based on repeat type, repeat length and the number of units of the repeat. Primers for these 16 VNTR markers were tested on a set of M. fijiensis isolates and five VNTR markers that showed multiple alleles were tested on progeny isolates and field isolates. The markers allowed robust scoring on agarose gels and proved useful for variability and population genetic studies using basic molecular instrumentation. We envisage that this set will allow more detailed analysis of the large scale migration pattern of this important pathogen and the small scale effect of fungicide treatments on the population diversity in banana plantations.



Characterization of toxigenic Fusarium species in China

Theo A.J. Van der Lee, X. Xu, C. Huang, L. Yang, D. Yu, Z. Zhang, J. Feng, C. Waalwijk

Plant Research International, Wageningen, Netherlands

The cereals wheat, maize, and barley are among the world’s most important food and feed crops, yet quantity and quality of the production is threatened by Fusarium Head Blight (FHB) caused by a complex of Fusarium species. The Chinese climate is very conducive for FHB and disease pressure in several regions in China is among the highest in the world. Consumption of food and feed contaminated with the secondary metabolites produced by Fusarium species poses a great threat as these mycotoxins can cause serious illnesses and immune-suppression in humans and animals. The EU has recently set limits for several of these mycotoxins. The Fusarium genus is represented by a large number of evolutionary related but relatively diverse groups of organisms. Molecular research has shown that isolates that are morphologically similar represent different species (O' Donnell, 2004). We characterized over 2500 single spore isolates collected from maize, wheat and barley from many different provinces in China using diagnostic primers for the different species and chemotype as well as SSR markers. We found F. asiaticum to be the dominant species collected along the Yangtze River from wheat and barley samples. In the Northeast (Liaoning, Jilin, Heilongjiang) Fusarium graminearum sensu stricto was the dominant species on maize, but more to the West (Inner Mongolia, Henan) F. boothii and F. meridionale were more prevalent. In addition to a clear distinction for the different species based on geographic origin and host, we also found that isolates collected from barley from the downstream valleys of the Yangtze River are mainly deoxynivalenol (DON) producers, while the majority of isolates collected in the isolated mountainous provinces of Sichuan and Yunnan produce nivalenol (NIV). Our data suggest a recent displacement of the Fusarium population in the valleys of the Yangtze River.

O' Donnell, K., T. J. Ward, et al. (2004). Fungal Genetics and Biology. 41(6): 600-623.



Phylogenetic analyses of diverse Corynespora cassiicola isolates indicate an evolutionary correlation with host not geography

Linley Smith, Lawrence Datnoff, Ken Pernezny, Jeff Rollins, Robert Schlub

University of Florida, Gainesville, FL, United States

The fungus Corynespora cassiicola (Berk. & Curt.) Wei is found as a pathogen, endophyte, and saprophyte primarily from tropical plants. It has been isolated from over 240 plant genera, and the host list is constantly growing. Understanding how the genetic diversity within this fungus correlates with host-specificity and geographic distribution would be ideal for developing disease management strategies of economically important plants such as tomato and papaya. Endophytic, pathogenic and saprophytic isolates of C. cassiicola were collected from over 80 host species. A multilocus phylogenetic analysis was performed using 125 isolates of Corynespora sp. from diverse hosts and locations in Brazil, Florida, Malaysia, Micronesia, and Samoa. Phylogenetic trees were congruent for three random hypervariable loci and the single copy nuclear gene, actin, indicating asexual propagation. Isolates had different pathogenicity profiles on seedlings of basil, bean, cowpea, cucumber, papaya, tomato, and soybean. Correlations also were observed between pathogenicity, growth rate, and genotype, although not geographic location. Common fungal genotypes were widely distributed geographically indicating long distance and global dispersal. This research reveals an abundance of previously unrecognized diversity within the species, provides evidence for redefining species distinctions within Corynespora, and may help characterize pathogenic isolates from endophytes and saprophytes leading to better disease control strategies.



Identification of the sex genes in the penicillin producer Penicillium chrysogenum

Birgit Hoff1, Stefanie Pöggeler2, Ulrich Kück1

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

Eighty years ago, Alexander Fleming discovered an anti-bacterial activity in the asexual mold Penicillium that later was replaced by an overproducing isolate still used for penicillin production today. Using a heterologous PCR approach, we identified the sex genes and show that these strains are of opposite mating types. Moreover, both have retained transcriptionally expressed pheromone and pheromone receptor genes required for sexual reproduction. The results of our transcriptional expression data suggest the existence of a heterothallic sexual cycle in P. chrysogenum. This discovery extends options for industrial strain improvement programs using conventional genetical approaches.



Diversity and functional roles of mycorrhizal fungal symbionts of Mediterranean orchids

Rossana Segreto1, Donata Cafasso2, Heiko Liebel3, Barbara Montanini4, Gerhard Gebauer3, Francis Martin4, Salvatore Cozzolino2, Mariangela Girlanda1, Silvia Perotto1

1University of Turin, Torino, Italy, 2University of Naples, Napoli, Italy, 3University of Bayreuth, Bayreuth, Germany, 4INRA Nancy, Champenoux, France

In the first stages of their development, all orchids share a heterotrophic phase and rely on symbiotic fungi for C supply ("mycoheterotrophy"). Whereas some orchids remain achlorophyllous in the adult phase, most species become green, although their actual photosynthetic efficiency may be reduced under certain conditions. The hypothesis of a correlation between actual photosynthetic efficiency (and hence dependency on external, fungus-derived C) and mycorrhizal diversity has been put forward. Achlorophyllous orchids exhibit narrow mycorrhizal specificity, behaving as highly specialized exploiters of the ectomycorrhizal symbiosis established by their fungal partners with surrounding tree species. In green orchids with reduced photosynthetic efficiency (such as forest species thriving in woodland shade), which feature a diverse range of mycorrhizal specificity, the fungus may, at least in part, subsidize the host plant with nutrients that include organic carbon. The diversity and functional roles of mycorrhizal fungi of fully photosynthetic terrestrial orchids living in open habitats are less understood.

We have been studying mycorrhizal strategies of Mediterranean orchids with different photosynthetic efficiency. Culture-dependent and culture-independent (ITS-based) identification of fungal partners of neighbouring individuals of four meadow green orchids (Serapias vomeracea, Ophrys fuciflora, Orchis purpurea and Anacamptis laxiflora) indicated that individual plants were colonized by either Ceratobasidium, Tulasnella and/or Sebacina species, with varying degrees of mycorrhizal specificity. The isotope signatures for 15N of the four orchids differed from non-orchid reference species collected close to the former, pointing to a significant N-gain from the fungi, whereas C-gain could only be traced in O. purpurea. A trancriptomic approach has been taken to investigate functional aspects of the mycorrhizal association in the genus Limodorum, that was found to be colonized by a single fungus belonging in the ECM genus Russula. A cDNA library was set up using mRNA extracted from mycorrhizal roots exhibiting intense fungal colonization. Sequences from both plant and fungus have already been identified, and further sequencing and bioinformatic analysis of the EST library is in progress.


Mitochondrial inheritance during outcrossing in substrate simultaneously inoculated with spores and mycelium of Agaricus bisporus

Philippe Callac1, Rana Kabalan1, Cyril Férandon2, Gérard Barroso1

1Inra, Bordeaux, France, 2université Victor Segalen, Bordeaux, France

Since two decennia, strains of A. bisporus, the button mushroom, have been isolated from several hundred of wild specimens but this biodiversity remains underexploited because it is is difficult to cross them. In the predominantly pseudohomothallic life cycle of A. bisporus, a low percentage of spores are homokaryotic (n), while most of them give rise to fertile heterokaryons (n+n) that cannot be easily crossed in vitro. In this context, the following method of hybridization in vivo was recently developed: compost trays are simultaneously inoculated with spores from one parent and with a homokaryotic mycelium from a second parent. It was shown that all the sporocarps produced on such trays were hybrids and that all or most of them resulted from Buller phenomenon, i.e. crosses between the inoculated homokaryon and heterokaryons issued from the inoculated spores. We propose here to analyse how mitochondria are inherited during this process. Firstly, we developed a new mitochondrial marker based on the presence/absence of certain introns of the COX1 gene, which allows us to distinguish the mitochondria of each parent. Secondly, analysing the genotypes of the hybrids sporocarps obtained with the new outcrossing method, we found that the mitochondrion of the parental homokaryon was systematically inherited in all the analyzed sporocarps. Thirdly, hybrid mycelia were also classically obtained in vitro, by confronting homokaryons issued from the two parents. In this case, genotype analysis revealed also a monoparental inheritance of the mitochondria, but, interestingly, the results were inverted: mitochondria were always inherited from the parent that never transmitted its mitochondrion in the ‘in vivo’ experiment. These data confirm that during the first experiment in vivo, (1) the rare homokaryotic spores did not participated to the crosses, and (2) during the outcrossing process, one nucleus migrates alone from a heterokaryotic spore or from the mycelium issued from it, towards the homokaryon.



Effect of chitosan a natural antifungal compound on yeast under experimental evolution conditions

Maria Jaime1, Luis Vicente Lopez-Llorca2, Corey Nislow3, Timothy Westwood1

1University of Toronto, Dept of Cell & Systems Biology, Mississauga, Ontario, Canada, 2Laboratory of Plant Pathology, Multidisciplinary Institute for Environmental Studies (MIES) Ramon Margalef, Department of Marine Sciences and Applied Biology, University of Alicante, Alicante, Spain, 3University of Toronto Donnelley Centre for Cellular and Biomolecular Research, Toronto, Ontario, Canada

Development of resistance to chemical fungicide in fungal pathogen populations is a major problem in disease control. Alternative compounds such as antifungal molecules from natural origin are been sought. Chitosan is a partially deacetylated form of chitin, and consists of polymers of β-1,4-glucosamine subunits. Chitosan and its derivatives exhibit antibiotic activity against bacteria and fungi. Little is known on the development of resistance to natural antifungal compounds, such as chitosan. To evaluate the long term effect of chitosan in fungi populations we are evolving yeast in the presence of chitosan. We have used the tagged deletion strains collection of Saccharomyces cerevisiae, (~6000 deletion strains) including heterozygous essential and homozygous non-essential deletion strains that have two "unique" barcodes. S. cerevisiae populations are evolving under two different selection regimes: i) a stepwise increase in benomyl concentration and ii) a single, supra-lethal concentration. An insight of the genes involved in chitosan resistance will be obtained from the universal barcode arrays that allow the analysis of thousands of deletion strains in parallel through the use of unique 20-base-pair DNA tags. We also plan to determine the gene expression profiles of S. cerevisiae populations that have been exposed to chitosan. To achieve a further understanding of the changes caused by chitosan a protein expression profiles analysis will be perform. Results of these experiments will be presented


Multi-locus phylogenetics and population genetics of Penicillium chrysogenum

Daniel Henk, Matthew Fisher

Imperial College London, London, United Kingdom

The antibacterial effect of the ubiquitous fungus Penicillium chrysogenum was noticed by Sir Alexander Fleming at St. Mary’s Hospital in a famously lucky set of circumstances. His observation ultimately led to penicillin production from P. chrysogenum and one of the largest single developments in the history of mankind. Despite its importance, many questions remain surrounding the ecology and evolution of P. chrysogenum, including whether it represents a single globally distributed species and if it is truly asexual.

Here we use phylogenetic and population genetic analyses to begin to address the unresolved questions about cryptic species, recombination, and geographic population structure in P. chrysogenum and to develop a functional multi-locus sequence typing (MLST) scheme for Penicillium section Chrysogena.

Primers were designed based on a phylogenetic analysis of genes from publicly available databases. This yielded a set of 30 alignments used in phylogenetic analyses. The analyses support the idea that P. chrysogenum is more closely related to Apergillus species than to Penicillium subgenus Biverticillium A set of potential MLST genes were chosen based on a series of criteria including a genetic architecture that enabled targeting of conserved coding regions around at least one intron. Sequences from a worldwide sample of over 120 isolates (largely based on historical collections from the USDA) showed that P. chrysogenum does contain at least three phylogenetically distinct groups which may represent cryptic species. Further, within each of these groups recombination is detected. However, rates of recombination are difficult to estimate because there are few mutations (nucleotide diversity ranges only between 0.002-0.01 among genes within P. chrysogenum) and without data on genome architecture it is not possible to distinguish between parasexual and sexual recombination. The historically important Fleming isolate has the most common genotype, one that is shared with isolates distributed from the North American arctic to New Guinea. Similarly, most genotypes show broad geographic distributions, and distinct clades overlap in distribution. This suggests that long distance dispersal is highly effective in this species and that either ecological isolation or genetic isolating mechanisms have evolved to maintain distinct lineages.


Differences in gene content related to variation in host preferences of the ectomycorrhizal fungus Paxillus involutus

Jenny Hedh, Tomas Johansson, Susanne Erland, Anders Tunlid

Lúnd University, Lund, Sweden

Gene gains and loss are together with sequence mutations, the major genomic mechanisms that could account for variation between phenotypes. We have compared the gene content in isolates of the ectomycorrhizal (EM) fungus Paxillus involutus that differ in host preferences. A majority of the examined isolates of this fungus are generalists and are compatible with many host species. A few isolates form EM with a limited number of tree species. Recent studies have shown that P. involutus consists of at least four genetically isolated lineages or phylogenetic species (PS I to IV). The aim of this study was to investigate whether variations in host preferences are found in several of the P. involutus lineages, and if so, whether the genomic mechanisms that could account for these phenotypic differences are similar or different. Thirty-six P. involutus isolates were collected in the south of Sweden and their capacities to infect the roots of birch (Betula pendula) and spruce (Picea abis) were examined in microcosms. Two isolates were identified that did not infect birch and spruce. These incompetent (-) isolates belonged to PS I and PS II, respectively. The remaining 34 isolates were compatible (+) and they were found in PS I and PS II. The gene content of the (-) isolates were compared with that of a closely related (+) isolate within the same PS using microarray-based comparative genomic hybridization (CGH).The cDNA array contained 4119 putative unique genes of P. involutus. Included in the study was also a third pair of (-)/(+) isolates from PS III. The proportion of genes that varied in copy numbers when comparing the (-)/(+) isolates within PS I, PS II and PS III was 3.4, 6.5 and 1.3%, respectively. Notably, only three genes showed similar patterns of variation in the three pairs. Our data suggests that incompetent isolates have arisen repeatedly and independently in several of the phylogenetic species within the P. involutus species group. The patterns of gains and loss of genes associated with the emergence of these phenotypes are different.


Colletotrichum spp. associated with coffee berry like disease in Vietnam

Phuong Nguyen1, Olga Vinnere Pettersson2, Erland Liljeroth1

1The Swedish University of Agricultural Sciences, Department of Plant Protection Biology, Alnarp, Sweden, 2Uppsala University, Department of Evolution, Genomics and Systematics, Uppsala, Sweden

Isolates of Colletotrichum associated with disease on coffee berries in Vietnam were identified and characterized morphologically and molecularly. Species-specific primers for amplification of the rDNA ITS1 (internal transcribed spacer) and the mtSSU (mitochondrial small subunit rRNA gene) regions were used for identification of C. gloeosporioides and C. acutatum. The genetic variation among isolates of C. gloeosporiodes was investigated with random amplified polymorphic DNA (RAPD)(38 isolates) and microsatellite-primered PCR (MpPCR)(52 isolates) markers. Cluster analysis showed that the isolates mainly grouped in accordance with geographical distributions. We found a higher genetic variation among isolates from the north compared to the south of the country and a moderate gene differentiation between populations from the north and the south. However, there was no differentiation among sublocations within the northern and the southern populations. A four gamete test indicated a high level of recombination, particularly in the south. The geographic differences may be explained by different histories of coffee cultivation in different parts of Vietnam.

Three isolates of Colletotrichum having falcate conidia, two isolates of C. acutaum and 34 isolates of C. gloeosporioies from Vietnam were further investigated by sequencing both the ITS and the mtSSU regions. Three African reference isolates of C. kahawae, causing coffee berry diseases (CBD), were also investigated as comparison. The results confirmed the identification of C. gloeosporioides and C. acutatum. The isolates with falcate conidia can be either C. dematium or C. capsici. All Vietnamese isolates, except two, could use tartrate as a sole carbon source. Two Vietnamese isolates and the CBD reference isolates could not utilize tartrate. However, according to the ITS1 sequence analysis and combination of ITS and mtSSU, the three reference CBD isolates fell separately from the clade of all Vietnamese C. gloeosporioides isolates. We also observed that the symptoms caused by the Vietnamese isolates on both hypocotyls and green berries were less severe than symptoms caused by the reference CBD isolates. This is indicative of the absence of C. kahwae on coffee in Vietnam. No significant correlation between pathogenicity and genotype was found among the Vietnamese isolates of C. gloeosporioides.


A marker-based re-interpretation of T.melanopsorum Vitt mating system and life cycle

Andrea Rubini, Claudia Riccioni, Beatrice Belfiori, Valentia Passeri, Sergio Arcioni, Francesco Paolocci

Consiglio Nazionale delle Recherche, Perugia, Italy

Tuber melanosporum is an ectomycorrhizal ascomycete producing edible and worldwide appreciated ascocarps, known as black truffles. The impossibility of mating Tuber species under controlled conditions prevents the direct assessment of their reproductive system. However, based on the assumption that in T. melanosporum and Tuber magnatum the hyphae of the fruiting bodies and mycorrhizae are diploid/dikaryotic, the lack of any heterozygotic profiles in these structures has been interpreted as a proof that these species strictly self (Bertault et al. Nature 1998 and Heredity 2001). By means of SSR markers and appropriate sampling strategies we have recently challenged the view that T. magnatum selfs and showed that its mycorrhizae indeed result from primary mycelia (Rubini et al. AEM 2005, Paolocci et al. AEM 2006). On these grounds, we sought to re-evaluate the life cycle and the reproductive system in T. melanosporum as well.

To this end, 7 polymorphic SSR loci have been isolated and used in combination with the sequence analysis of the nuclear rDNA ITS region to genotype separately the gleba and the asci from 32 ascocarps from natural populations. Our results show that the asci from more than 30% of the black truffles screened display additional SSR and/or ITS alleles with respect to the corresponding gleba that always exhibits a single allele. These findings let us to model that: 1) T. melanosporum outcrosses; 2) the fertilization step promotes and, likely, precedes the maternally-driven development of the gleba; 3) the diploid/dikaryotic phase is present only in the early stages of the truffle development; 4) its life cycle is prevalently haploid. This life cycle and reproductive model typify most of the ascomycetes, including T. magnatum (Rubini et al. New Phytol 2007).

The outcome of our study not only has an impact on the strategies for managing T.melanosporum man-made plantations, but is also relevant to properly design studies aiming to assess the distribution of the genetic variability at different geographical scales. Lastly, the ongoing T.melanosporum genome sequencing project will help us to unravel the organization of the mating type genes and understand whether or not this is an obligate outcrossing fungus.