ECFG 9 Poster Session 1

Functional and Comparative Genomics, Plant-Fungal Interactions,



The rising tide of fungal genetics: a perspective from the FGSC

Kevin McCluskey, Aric Wiest, Michael Plamann

Fungal Genetics Stock Center, University of Missouri- Kansas City, Kansas City, United States

As we approach the post genomics age the number of fungal organisms which can call themselves genetic systems continues to grow. Because of this, the number of organisms in the Fungal Genetics Stock Center collection has expanded to include most strains that have had their genomes sequenced as well as genome associated libraries for a number of organisms.

As more fungal systems are amenable to genetic research new researchers have come into the field. This has led to an increase in the number of requests for materials from the FGSC including knock-out strains of Neurospora, cloning vectors and strains of sequenced organisms. These increases include numerous people who previously did not use filamentous fungi in their research. Overall we have seen over thirty seven percent increase in orders in 2007 as compared to the average of the eight previous years. This number does not include the nearly 32,000 knockout strains sent in 96-well arrays. Including these, the increase is on the order of 2,300 percent. While most of these are Neurospora, the FGSC has also received knockouts of Cryptococcus and tagged integration mutants of Magnaporthe. In total, we hold over 53,000 knockout or tagged mutants in 96-well format.

Beyond fungal strains, the FGSC holds and distributes gene libraries and individual clones from genome-mapped libraries for many of the main organisms used in genetic research. Using these resources, we have identified the underlying mutation for several temperature sensitive mutants in Neurospora crassa. We are endeavoring to develop useful applications of these findings in Neurospora and Aspergillus.



A novel polyketide biosynthesis gene cluster is involved in fruiting body morphogenesis in Sordaria macrospora and Neurospora crassa

Minou Nowrousian, Ulrich Kück

Ruhr-Universitaet Bochum, Bochum, Germany

During fungal fruiting body development, vegetative hyphae aggregate to form complex multicellular structures. Within the mature fruiting body, the sexual spores are formed. Using microarray analysis, we have identified a cluster of genes that are strongly upregulated during fruiting body development in the filamentous ascomycete Sordaria macrospora. Further analysis by quantitative real time PCR showed that the genes from the orthologous cluster in Neurospora crassa are upregulated during development, too. The genes occupy a region of ~50 kb in the genomes of both fungi and encode enzymes that are predicted to participate in polyketide biosynthesis, including a putative polyketide synthase. A partly conserved orthologous cluster is present in the genome of the rice pathogen Magnaporthe grisea. However, there are no functionally characterized orthologs from other organisms, thus, the product of the putative biosynthetic pathway remains to be elucidated. Analysis of both a N. crassa knockout strain as well as the corresponding S. macrospora knockout of one of the predicted dehydrogenase genes of the cluster demonstrated that this gene is involved fruiting body formation in both fungi. These data indicate that polyketides may play a role in sexual development in filamentous ascomycetes.



Localization and functional analysis of Penicillium chrysogenum ABC transporters

Andriy Kovalchuk, Marta Woszczynska, Arnold Driessen

Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Haren, Netherlands

The filamentous fungus Penicillium chrysogenum is well known as a major producer of β-lactam antibiotics, first of all penicillin G and V. Metabolic engineering approaches now open new ways for the industrial use of P. chrysogenum, as it allows efficient production of unnatural compounds not synthesized by natural strains. The availability of the genome sequence (van den Berg, M.A. et al., submitted) will stimulate the further improvement of P. chrysogenum as a versatile cellular factory. An important prerequisite for the successful introduction of novel metabolic pathway, however, is a characterization of transport processes and capacities within the cell.

One of the most prominent classes of transporter proteins are the ATP binding cassette (ABC) transporters. These systems use the energy of ATP to transport molecules across biological membranes, many of which are responsible for multidrug resistance. Some ABC transporters are also known to be involved in the secretion of secondary metabolites. Despite their importance, our knowledge about fungal ABC transporters is limited.

This project is focused on a genome-wide characterization of ABC transporters in P. chrysogenum. We use a combination of bioinformatics tools, localization studies and functional analyses to characterize several selected ABC transporters. GFP fusions were used to determine their intracellular localization. Analyzed transporters localized to the plasma membrane, vacuolar membrane, mitochondria, ER and microbodies. Further characterization of the expressed ABC transporters includes the construction of knock-out strains. The data obtained in this project will contribute to the better understanding of transport processes in P. chrysogenum cells.

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




Comparison of protein coding gene contents of fungal subphyla Pezizomycotina and Saccharomycotina

Mikko Arvas1, Teemu Kivioja2, Alex Mitchell3, Markku Saloheimo1, David Ussery4, Merja Penttilä1, Stephen Oliver5

1VTT, Espoo, Finland, 2University of Helsinki, Helsinki, Finland, 3EBI, Cambridge, Finland, 4CBS, Lyngby, Denmark, 5University of Manchester, Manchester, United Kingdom

Several dozens of fungi encompassing traditional model organisms, industrial production organisms and human and plant pathogens have been sequenced recently and their particular genomic features analysed in detail. In addition comparative genomics has been applied to make analyses within sub groups of fungi. Notably, analysis of the subphylum Saccharomycotina has revealed major events of evolution such as the recent genome duplication and subsequent gene loss. However, little has been done to gain a comprehensive comparative view to the fungal kingdom. We have carried out a computational genome wide comparison of protein coding gene content of the subphyla Saccharomycotina and Pezizomycotina, which include industrially important yeasts and filamentous fungi, respectively.

Our analysis shows that based on genome redundancy the traditional model organisms Saccharomyces cerevisiae and Neurospora crassa are exceptional among fungi. This can be explained by the recent genome duplication in S. cerevisiae and the repeat induced point mutation mechanism (RIP) in N. crassa. Interestingly, in Pezizomycotina a subset of protein families related to plant biomass degradation and secondary metabolism are the only ones showing signs of recent expansion. In addition, Pezizomycotina have a wealth of phylum-specific poorly characterised genes with a wide variety of predicted functions. These genes are well conserved in Pezizomycotina, but show no signs of recent expansion. The genes found in all fungi except Saccharomycotina are slightly better characterised and predicted to encode mainly enzymes. The gene classes specifically diverged in Saccharomycotina are enriched in transcription and mitochondrion related functions. Especially mitochondrial ribosomal proteins seem to have evolved from those of Pezizomycotina.

Our analysis predicts that all Pezizomycotina unlike Saccharomycotina can potentially produce a wide variety of secondary metabolites and secreted enzymes and that the respective genetic systems are likely to evolve fast. These systems allow Pezizomycotina to interact with their environment and therefore they are directly subjected to various evolutionary pressures due to changing environments.



Growth profiles of filamentous fungi on plant polysaccharides can largely be explained by their genome content

Birgit Gruben1, Evy Battaglia1, Han A.B. Wösten1, Pedro Coutinho2, Etienne Danchin2, Bernard Henrissat2, Ronald P. de Vries1

1Microbiology, Utrecht University, Utrecht, Netherlands, 2Architecture et Fonction des Macromolecules Biologiques, Universites Aix-Marseille I & II, Marseille, France

Plant polysaccharides are the most abundant carbon source in nature and are therefore commonly utilized by saprophytic and phytopathogenic fungi. These fungi produce a broad spectrum of extracellular enzymes to degrade the polysaccharides to monomeric components that can be taken up into the cell and used for carbon metabolism. The different enzyme activities can be divided into families based on their amino acid sequence and modular structure. A comprehensive database of these enzyme activities is available at the CAZy website ( The availability of fungal genome sequences has resulted in an exponential increase in the number of putative enzymes in the CAZy database and allows for fungal comparison with respect to these functions. We have initiated a study to link the genome content with respect to polysaccharide degrading functions to the growth profile of fungi. To this end we have obtained growth profiled of 15 fungi on 37 carbon sources (crude substrates, polysaccharides, oligosaccharides, monosaccharides). Improved or reduced growth on specific substrates has been linked to enriched or reduced numbers of relevant enzymes in the genome of these fungi. Initial results demonstrate a nearly perfect correlation between the genome content with respect to polysaccharide degrading enzymes and the ability to grow on specific sets of substrates.



Prevalence of transcriptional regulators across the fungal kingdom

Ronald P. de Vries1, Loek Visser1, Evy Battaglia1, Birgit Gruben1, Han A.B. Wösten1, Richard B. Todd2

1Microbiology, Utrecht University, Utrecht, Netherlands, 2Department of Genetics, The University of Melbourne, Melbourne, Australia

Transcriptional regulators are the major control points for all aspects of fungal life. Over the years, many transcriptional regulators have been identified and their biological function has been studied. Many of these regulators have been mainly studied in a relatively small number of model fungi, although some have also only been described for less commonly used fungal species. The availability of more than 40 fungal genomes allows an analysis on the prevalence of these regulators. We have selected 60 characterized fungal transcriptional regulators and analyzed their presence in the available fungal genomes. This demonstrated that some regulators are commonly found throughout the fungal kingdom (e.g. CreA), while others are restricted to a class (e.g. PrnA in the ascomycetes) or a genus (e.g. AraR). The presence or absence of a regulator has been linked to the natural habitat of the fungi to find correlations with respect to fungal physiology.



Annexin C4: in search of a role

Behrouz Vazir, Nahid Mortazavi, Bahareh Azarian, Vahid Kahl

Pasteur Institute of Iran, Tehran, Islamic Republic of Iran

Annexin C4 (ANXC4), the third member of fungal annexins, has been identified as a novel annexin with significant differences compare to other fungal and human annexins. To understand the ANXC4 function in human pathogen, A. fumigatus, a knock out strain has been developed using homologous recombination technique. Preliminary data on growth phenotype of anxC4 deletant confirmed a non-essential role in viability or sporulation of this fungus. For detailed analysis of ANXC4 loss effects and possible compensatory mechanisms, a proteomics approach was considered. In this sense, two dimensional protein profiles of anxC4 mutant and wild type strain were prepared. Comparison analysis of 1469 spots detected in the protein maps, showed expressional differences with nine over expressed and six under expressed spots in mutant type. Protein translation elongation factors were among those over expressed spots which identified by MALDI-TOF/TOF mass spectrometry. Further analysis of the proteomes is underway.




Comparative genomic hybridization as a tool to study novel genes involved in protein production in Trichoderma reesei

Marika Vitikainen, Mikko Arvas, Merja Penttilä, Markku Saloheimo

VTT Technical Research Centre of Finland, Espoo, Finland

Comparative Genomic Hybridization (CGH) allows detection of mutations and copy number differences between two genomic DNA samples. In high-density microarray CGH (array CGH), genomic DNA is hybridised to oligonucleotide arrays where the oligonucleotides cover the whole genomic sequence of the organism. Test and reference genomic DNA samples are independently labelled with fluorescent dyes and co-hybridized to a chip followed by scanning the spots and further data analysis, enabling detection of small genomic alterations.

Methodology has been developed for comparative genomic analysis of Trichoderma reesei, a fungus used widely in industrial protein production. The strains analyzed are high-cellulase producing strains generated by conventional mutagenesis and selection programs. The unique feature is that all the T. reesei strains used by industry and academic research groups are members of the same pedigree of cellulase production strains, each derived from a single natural isolate. Custom array design with 2,1 million isothermal overlapping oligonucleotide probes covering the entire T. reesei genome was made, and a number of strains from the T. reesei pedigree have been analysed.

Dozens of genomic changes, both intragenic and intergenic, in the T. reesei strains have been detected by array CGH. The number of changes in individual strains was proportional to the number of mutagenesis rounds to which each strain has been subjected. Most of the detected changes were deletions, yet some duplications were detected as well. Changes as small as two nucleotide substitution could be observed. Our results show that high-density array CGH is a very useful tool for genome-wide analysis of fungal strains to identify novel genes involved in industrially relevant strain qualities such as the efficacy in protein production.



Transposon mobility via true transposition and recombination in Aspergillus niger CBS513.88

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 mobile and mostly also repetitive sequences, which are found in all eukaryotic genomes. Basically transposons can move around their host genome in two different ways: via true transposition using a "copy and paste" or a "cut and paste" mechanism depending on which type of transposon is considered. But due to their repetitive nature transposons can also change their position via recombination resulting in genomic rearrangements of various extent.

We have analyzed transposon mobility in the A. niger CBS513.88 strain lineage both experimentally and in silico. A comparison of transposon distribution in different A. niger strains of this strain lineage showed that most transposons have remained at their genomic positions during the classical strain improvement programs. Still one non-autonomous element, the transposon Vader, was shown to be active. Vader mobility could also be shown in a transposon trap experiment. Due to its obvious activity during classical strain improvement and to its ability to insert into genes Vader can be used as a gene tagging tool. In addition to the described transposition events, recombination between two copies of the A. niger retrotransposon ANiTa1, resulting in the genomic rearrangement of an approximately 45 kb region, was observed. This genomic region contains nine hypothetical and furthermore eleven annotated genes. Many of them could be involved in plant cell wall degradation.



RNA silencing in Mucor circinelloides: genomic analysis of three argonaute genes

Maria Cervantes, Juan P. de Haro, Santiago Torres-Martinez, Rosa M. Ruiz Vazquez

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

RNA silencing is a regulatory mechanism that involves the suppression of gene expression through the sequence-specific degradation of the target mRNA, the repression of its translation or the inhibition of its transcription. This mechanism is triggered by dsRNA molecules, which are processed into small RNAs of 21-26 nt by the Dicer enzyme. The small RNAs are incorporated into RISC complexes (RNA-induced silencing complex) containing a protein from the Argonaute-Piwi family, which uses the antisense strand of the small RNAs to scan for complementary sequences in the mRNA, causing its degradation or preventing its translation. Although the machinery of RNA silencing is well conserved throughout the evolutionary scale, the number of paralogous silencing proteins differs considerably among species, as it does the specificity of each protein in different RNA silencing pathways. We have shown the existence of a silencing mechanism in the zygomycete Mucor circinelloides. Two dicer-like genes (dcl) have been identified, although only dcl-2 is essential for efficient gene silencing and production of the two size classes of siRNAs, 21- and 25-nt long, which are accumulated in silenced strains.

In order to characterize components of the RISC complex, we have cloned the M. circinelloides argonaute (ago) genes. A DNA fragment amplified with degenerated oligonucleotides was used to screen a lambda genomic library, and three different ago genes, ago-1, ago-2 and ago-3, were isolated. Genomic analysis of these genes demonstrated the presence of a premature stop codon in the ago-2 sequence, which would produce a truncated protein lacking the PIWI domain. This mutation is fixed in the parental wild type strain. However, the high level of similarity between the amino acid sequences of Ago-2 and the ago-1 and ago-3 gene products, together with the presence of all the conserved residues of the PAZ and PIWI domains in Ago-2 suggest that the mutation event occurred recently. Alternatively, a ribosome frameshift might occur during Ago-2 translation. Expression analysis indicated the three genes were expressed at different levels in the wild type strain and none of them increased their expression by induction of silencing by dsRNA. Construction of null mutants in each ago gene and analysis of their phenotype would allow us to clarify the role of these genes in different silencing pathways.



Sequencing the genome of the forest pathogen Heterobasidion annosum allow for tracking the evolution of pathogenicity factors

Jan Stenlid, Åke Olson, Kerstin Dalman, Magnus Karlsson, Mikael Brandström, Karl Lundén, Carl-Gunnar Fossdal, Matteo Garbelotto, Fred Asiegbu

1Department of Forest Mycology and Pathology, Swedish University of Agricultural Sciences, Uppsala, Sweden, 2Forest and Landscape, Ås, Norway, 3University of California, Berkeley, United States, 4University of Helsinki, Helsinki, Finland

Heterobasidion annosum s.l. causes a devastating root rot in conifer plantations and natural forests throughout the northern hemisphere. The genome sequencing of H. annosum will provide the first comprehensive genetic information on a plant pathogenic homobasidiomycete allowing for new insights into plant-microbe interactions with trees, in particular conifers. It is important to broaden the taxonomic base for understanding the mechanisms of plant-microbe interactions, studying genes and proteins involved, and identifying pathogenicity determinants. Comparative genomics of plant pathogens with a gradient of taxonomic relatedness to H. annosum will help to understand the evolution of such factors. The project involves resequencing of several species in the H. annosum species complex including pathogenic and non-pathogenic species. Comparisons can also be made with fungi with different nutritional relations to plants; necrotrophic, biotrophic and mycorrhizal mutualist. The project integrates genetic approaches e.g QTL mapping with comparative genomics. Data bases generated on genes under positive selection in the sequenced genomes will be compared with candidate genes derived from published QTL mapping and EST-projects but also known pathogenicity factors in other pathosystems.


Profiling of genes expressed under aflatoxin-producing conditions in Aspergillus flavus in comparison with A. oryzae

Jiujiang Yu1, William Nierman2, Natalie Fedorova2, Masayuki Machida3, Joan Bennett4, Bruce Campbell5, Deepak Bhatnagar1, Thomas Cleveland1, Gary Payne6

1USDA/ARS, Southern Regional Research Center, New Orleans, LA, United States, 2J. Craig Venter Institute, Rockville, MD, United States, 3National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki, Japan, 4Rutgers University, New Brunswick, NJ, United States, 5USDA/ARS, Western Regional Research Center, Albany, CA, United States, 6North Carolina State University, Raleigh, NC, United States

Aflatoxins produced by Aspergillus flavus and A. parasiticus are the most toxic and potent natural carcinogens known to date. A. flavus is also an opportunistic pathogen that is capable of infecting plants, animals and humans. Infection in preharvest crops such as corn, cotton, peanut and tree nuts by A. flavus not only reduces yield but also renders them unsafe for human and animal consumption due to aflatoxin contamination. The economic losses due to the contamination are staggering. The primary objectives of our A. flavus genomics program are to reduce and eliminate aflatoxin contamination in food and feed through validation of known genes involved in aflatoxin formation and fungal infection and identification of novel targets. To this end, we performed functional genomics studies in A. flavus and the non-toxigenic and non-pathogenic A. oryzae strain using three different types of microarrays (amplicon, oligo and Affymetrix arrays). Gene expression profiling demonstrated that genes involved in aflatoxin production are differentially expressed in A. flavus compared to A. oryzae under conditions that support aflatoxin formation and mimic infection. In addition, genes encoding cellulosic degrading enzymes and spore surface proteins are differentially expressed in A. flavus, but not in A. oryzae. This suggests that these proteins could play a key role in fungal infection. A. flavus is a saprobe having the ability to survive in the natural environment by extracting nutrition from plant debris and dead insects. The nature of its survivability could be related to the mechanisms of fungal pathogenicity. Functional characterization of these enzymes can help devise strategies to control fungal infection of agricultural crops.



Functional analysis of a novel fungal protein involved in cadmium resistance

Simona Abba, Marta Vallino, Roberto Borriello, Silvia Perotto

Department of Plant Biology, University of Turin, Italy, Turin, Italy

The ericoid fungal strain Oidiodendron maius Zn has been previously shown to tolerate high concentrations of heavy metal ions in the growth medium. A functional cloning approach using the cadmium-hypersensitive Saccharomyces cerevisiae mutant ∆yap1 was employed to isolate fungal genes involved in the tolerance to increasing concentrations of cadmium ions in the growth medium. A screening of an O. maius cDNA library on a cadmium gradient medium allowed to isolate a sequence coding a 179 amino acid-long Cys-rich protein, hereafter named OmFCR (Oidiodendron maius fungal cadmium resistance). The OmFCR-carrying yeast could grow at cadmium concentrations up to 120 µM. Database searches showed that this protein shared the ion binding motives C-X-X-C and C-P-X with metallothioneins and CPx-ATPases, but, except for this, sequence homologies were very weak. However, significant homologies were found with a group of fungal and plant proteins with an uncharacterized functional domain called DUF614. This is a Cys-rich domain of unknown function found in a number of eukaryotes, but only an Arabidopsis thaliana DUF614-containing protein has been characterized so far. As we demonstrated for OmFCR, a yeast complementation with this protein has been proved to cause an increase of the yeast cadmium resistance.

Sequence alignments with the closer OmFCR homologs revealed the presence of conserved amino acids which were primarily used as targets for site-directed mutagenesis. The multiple replacements of Cys with Ala in the N-terminus of the protein caused a reduction of the cadmium resistance which seemed to be dependent on the number of mutated cysteines. On the contrary, the replacement of both Cys142 and Cys145 with Ala in the C-terminus caused the yeast cadmium resistance to be doubled in respect to the wild-type gene. Moreover, the single replacement of the non-conserved Asp164 with Ala caused even an increase of the resistance to 320 µM. Thus, it appears that the cysteines in the N-terminus could be involved in cadmium sensing and in keeping the correct folding of the protein, while the amino acids in the C-terminus could be part of an inhibitory domain which reduces the protein activity.



The Tolypocladium inflatum CPA element encodes a RecQ relicase-like gene

Frank Kempken

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

In the past 15 years, a large number class I and class II transposons were identified in filamentous fungi. These classes contain retrotransposons and DNA transposons, respectively, some of which have been described on the molecular level almost three decades ago. However, in recent years new types of transposons have been discovered exhibiting clear differences to those previously known. A third class of transposons is called politons. These elements are widespread among protists, fungi and animals. They encode up to ten proteins and are the most complex transposable elements known. Transposition is coupled with their own DNA synthesis. Interestingly, politons share some characteristics with mitochondrial linear plasmids in fungi and plants. The fourth class of eukaryotic transposons consists of helitrons, which were first found in Arabidopsis thaliana and Caenorhabditis elegans. These elements are characterized by rolling-circle replication initiator and DNA helicase domains, which are part of a protein of about 1000-3000 amino acids.

Several years ago a repetitive sequence was discovered in the genome of the filamentous fungus Tolypocladium inflatum. This element was called CPA. When first detected it was found to be strain-specific, but no evidence for belonging to either transposon class I or class II was determined. Here the sequence of a large open reading frame encoded by the CPA element is described, which is most similar to a RecQ helicase-like protein from Metarhizium anisophila. As a consequence, the repetitive strain-specific CPA element appears to be a fungal member of the eukaryotic helitron class of transposable elements.



Post-genomic research on Aspergillus niger

Peter van de Vondervoort, Robbert Damveld, Noël van Peij, Hans Roubos, Denise Jacobs, Maurien Olsthoorn, Stefaan Breestraat, Hein Stam

DSM Food Specialties, Delft, Netherlands

For many decades, A. niger has been safely used in the commercial production of various food enzymes, such as glucose oxidase, pectinase, alpha-amylase and glucoamylase. DSM and the A. niger consortium published the genome of CBS 513.88, the ancestor of currently used enzyme production strains. A high level of synteny was observed with other aspergilli sequenced. Based on the sequence, Affymetrix TX chips were developed and used to study enzyme production in production strains. The knowledge obtained from this genomics effort enabled us to improve our "PluGbug" in a generic way, for example by using codon-optimization and to further develop proteomics. We will present important findings that led to a rational improvement of protein production in Aspergillus niger.



The invaded genome of the dothideomycete Leptosphaeria maculans

Thierry Rouxel1, Victoria Dominguez2, Stefano Torriani3, James Hane4, Barbara Howlett5, Steve Goodwin6, Richard Oliver4, Isabelle Fudal1, Michel Meyer1, Marie-Line Kuhn1, Maria Eckert1, Bruce McDonald3, Joelle Amselem2, Hadi Quesneville2, Marc-Henri Lebrun1, Patrick Wincker7, Arnaud Couloux7, Marie-Hélène Balesdent1

1INRA-Bioger, Versailles, France, 2INRA-URGI, Evry, France, 3ETH, Zurich, Switzerland, 4Murdoch University, Murdoch, WA, Australia, 5School of Botany, Melbourne University, Australia, 6USDA-ARS, West Lafayette, IN, United States, 7Genoscope, Evry, France

The genome of the Dothideomycete Leptosphaeria maculans ‘brassicae’ (Lmb), a pathogen of oilseed rape, was recently sequenced by Genoscope and a final assembly was provided in August 2007. Lmb estimated genome size is bigger (44.9 Mb) than those of the closely related species Stagonospora nodorum (37 Mb), or the related species Mycosphaerella graminicola (41.2 Mb). This increase in size is most likely due to an abundance of retrotransposons in the genome of Lmb, as compared to the other two species. These repeated elements were estimated to represent more than 30% of the Lmb genome, as compared to 4.25% for S. nodorum and less than 5% for M. graminicola. Nine of the Lmb retrotransposons already characterized are present in high copy number with the very abundant Pholy (Copia-like), Polly and Olly (Gypsy-like) being present in up to 1400 copies each. Retrotransposons consistently occurred as large clusters of nested repeats with many Repeat Induced Point mutation-degenerated and truncated intermingled copies. These regions are A+T-rich and mostly devoid of coding sequences, thus conferring to the genome of Lmb an isochore-like structure reminiscent of the heterochromatin organisation in upper Eukaryotes. Such an isochore-like genome structure was not observed in the related genomes of S. nodorum or M. graminicola, nor in other filamentous ascomycetes that have been sequenced, but may also occur in the 74 Mb genome of Mycosphaerella fijiensis. Surprisingly, the mitochondrial genome of Lmb (160 kb) is also larger than that of other filamentous fungi including M. graminicola (44 kb), S. nodorum (50 kb) and M. fijiensis (73 kb). Apart from a few small-sized duplications, this was attributable to the invasion of mitochondrial genes by mobile group I introns containing homing endonuclease genes of the LAGLIDADG and GIY-YIG families.

Compared to Lmb, other members of the L. maculans-L. biglobosa species complex have small genome sizes, e.g., L. maculans ‘lepidii’ (20-25 Mb) and L. biglobosa ‘brassicae’ (25-29 Mb), that both seem to lack the four most abundant Lmb retrotransposons. These two sub-species are weakly or non pathogenic on oilseed rape. These genomic data thus suggest that the retrotransposon invasion may be correlated with an increased efficiency of Lmb as a successful pathogen of oilseed rape and its world-wide geographic expansion.



Evolution of the oxidative phosphorylation (OXPHOS) metabolic pathway in fungi

Jose Luis Lavin, Lucia Ramirez, Jose Antonio Oguiza, Antonio Gerardo Pisabarro

Public University of Navarre, Pamplona, Navarra, Spain

Mitochondria are the energy producing organelles of the eukaryotic cell. The mitochondrial oxidative phosphorylation (OXPHOS) pathway is the primary energy-producing biological process in all aerobic organisms. In order to study the evolution of the OXPHOS pathway, we have carried out a detailed comparative analysis of proteins involved in OXPHOS among the mitochondrial and/or nuclear genome sequences of representative fungal species corresponding to the major taxonomic groups and exhibiting different lifestyles and evolutionary relationship. To identify OXPHOS proteins in the selected fungal genomes, the deduced proteomes were compared using BlastP to a set of Saccharomyces cerevisiae, Neurospora crassa, Cryptococcus neoformans and Yarrowia lipolytica OXPHOS proteins. Each fungal OXPHOS pathway was reconstructed, and their components and structures were compared. The comparative analysis revealed a high conservation of the OXPHOS pathway between fungal species, but significant differences were also observed in OXPHOS proteins suggesting that certain features of the OXPHOS pathway are not identical across fungi.



Transcriptome analysis reveals new insight into appressorium formation and function in the rice blast fungus, Magnaporthe oryzae

Ralph Dean5, Yeon Yee Oh5, Nicole Donofrio1, Hauqin Pan2, Sean Coughlan3, Douglas Brown5, Shaowu Meng5, Thomas Mitchell4

1University of Delaware, Newark, NJ, United States, 2RTI International, Research Triangle Park, NC, United States, 3Agilent Technologies, Little Falls, Delaware, United States, 4Ohio State University, Columbus, OH, United States, 5North Carolina State University, Raleigh, NC, United States

Rice blast disease is caused by the filamentous Ascomycetous fungus Magnaporthe oryzae and results in significant annual rice yield losses worldwide. Infection by M. oryzae and several other fungal plant pathogens requires the development of a specialized infection cell called an appressorium. To further investigate the molecular processes regulating appressorium formation, we analyzed genome-wide gene expression changes during spore germination and appressorium formation on a hydrophobic surface compared to induction by cAMP. During spore germination, 2,154 (~21%) genes showed differential expression, with the majority being up-regulated. Three hundred fifty seven genes were differentially expressed during appressorium formation in response to both induction stimuli. These genes, which we refer to as appressorium consensus genes, were functionally grouped into GO categories. Overall, we found a significant decrease in expression of genes involved in protein synthesis. Conversely, expression of genes associated with protein and amino acid degradation, lipid metabolism, secondary metabolism and cellular transportation exhibited a dramatic increase. We functionally characterized several differentially regulated genes, including a subtilisin protease (SPM1) and a NAD specific glutamate dehydrogenase (Mgd1), by targeted gene disruption. These studies revealed hitherto unknown findings that protein degradation and amino acid metabolism are essential for appressorium formation and subsequent infection.


Characterization of Colletotrichum gloeosporioides mutants affected in sexual reproduction

Francisco Luna-Martínez, Mariela Elizabeth Álvarez-Cruz, Raúl Rodríguez-Guerra, June Simpson

1CINVESTAV Campus Guanajuato, Irapuato, Guanajuato, Mexico, 2INIFAP Campo experimental General Terán, General Terán, Nuevo León, Mexico

Most heterothallic Ascomycetes carry a single mating-type (MAT) locus with two alternate idiomorphs (MAT1-1 and MAT1-2) and sexual reproduction results when strains carrying different idiomorphs come into contact. Homothallic strains where a single individual carries both MAT1-1 and MAT 1-2 have also been described. In the genus Glomerella mating pairs in which both individuals carry MAT 1-2 have been characterized but to date the corresponding MAT1-1 idiomorph has not been identified for this genus. Both heterothallic and homothallic isolates of Glomerella have been reported, with heterothallic strains showing significant differences in fertility levels during sexual reproduction. Several models have been proposed to explain the mating system in Glomerella including: a. unbalanced heterothallism, b. the presence of multiple alleles at MAT1-2, c. the presence of more than 1 MAT locus. Although many reports exist on classical genetic analysis of mating in Glomerella, little is known in regard to the genes involved and the regulation of their expression.

In order to identify and characterize genes involved in sexual reproduction in Glomerella we have generated mutants in this process for the phytopathogenic fungus Colletotrichum gloeosporioides (teleomorph, Glomerella cingulata). The mutants were obtained via Agrobacterium tumefaciens-mediated-transformation (ATMT) using ascospores of a homothallic strain of C. gloeosporioides and strain LBA4404 of A. tumefaciens carrying the binary vector pGFP-HPH (whose T-DNA contains both the green fluorescent protein (GFP) and the hygromycin B phosphotransferase (HPT) genes under the control of the Aspergillus nidulans glyceraldehyde-3-phosphate dehydrogenase promoter). ATMT of C. gloeosporioides resulted in a high transformation efficiency (1000 transformants per 106 ascospores). Putative transformants were confirmed by the detection of GFP and/or the presence of the HPT gene. This result is superior or similar to transformation efficiencies previously reported for conidia or mycelia in other Glomerella species. Screening of candidates affected in their capacity to undergo sexual reproduction is underway.


Metabolome analysis of Aspergillus oryzae: effect of environmental conditions and genetic backgrounds

Masafumi Tokuoka1, Masahiro Ogawa1, Tadashi Takahashi1, Toshi Furukido1, Hideaki Koike2, Yasunobu Terabayashi2, Noriko Yamane2, Masayuki Machida2, Yasuji Koyama1

1Noda Institute for Scientific Research, Noda, Chiba, Japan, 2National Institute Of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki, Japan

Aspergillus oryzae has been used as an advantageous fungus for the production of foods and enzymes because of its safety. The recent completion of A. oryzae genome sequencing revealed the presence of numerous genes for secondary metabolite. However, studies on metabolome of A. oryzae are scarce so far. Therefore, in this study, using liquid chromatography/mass spectrometry (LC/MS), we analyzed the metabolites of A. oryzae wild-type or mutant strains grown under various conditions to understand the effect of environmental conditions and genetic background on the metabolite profiles of A. oryzae.

At first, we investigated the metabolites of A. oryzae RIB40 strain cultured in various conditions including plate culture, submerged culture, and solid state culture for 0 to 7 days. Metabolite profiles obtained by LC/MS showed that the most of the metabolites produced by A. oryzae could be classified according to their temporal pattern. We also searched stage-specific metabolites by which we could evaluate the physiological state of A. oryzae. Next, we analyzed strains of gene-disruption library of A. oryzae. Metabolome analysis showed that the amount of metabolites was significantly decreased in the strains exhibiting aberrant conidiation, suggesting that a number of metabolites are related with conidiation process. In addition, using statistical analysis such as principal component analysis, we found that disruptants exhibiting aberrant conidiation could be grouped into at least three groups based on their metabolite profiles. Our study indicated that metabolome analysis has a high potential to find out a gene function related to not only metabolism but also morphological differentiation. Further analysis is in progress, and results will be discussed.


Transposons in biotechnologically relevant strains of 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, 22DSM Anti-Infectives, Alexander Fleminglaan 1, 2613 AX, Delft, Netherlands

Despite of the relatively late discovery of fungal transposons and despite of the small fraction of repetitive DNA in fungal genomes (1.4%, 2.9%, and 3% in the genomes of Aspergillus oryzae, A. fumigatus and A. nidulans (Galagan et al., 2005)), today virtually all types of eukaryotic transposons have been identified in fungi (reviewed in Pöggeler and Kempken 2004). However, the impact of transposons on their fungal hosts’ genomic development is still a matter of debate. Transposition might play a role in enhancing recombination in asexual fungi (Rep et al., 2006). However, it often failed to identify actively transposing mobile elements in the genomes of fungal laboratory strains, possibly because of ongoing selection for phenotypic stability (Daboussi and Capy, 2003) .

It is particularly interesting that relatively little is known about the role transposons have in industrial producing strains. We set out to investigate the transposon content in the genomes of biotechnologically important strains of the two fungi Aspergillus niger and Penicillium chrysogenum using the annotated genome sequences of strains CBS 513.88 and ATCC 28089.

A compilation of transposon-like sequences identified in the two genomes is given. Single sequence sets have been analysed in more detail: For example the P. chrysogenum class II element PeTraII was identified, to our knowledge the first transposon known in this fungus. We further compared the distribution of selected elements in a set of different fungal strains, all emanating from each other, to investigate transposon mobility in classical strain improvement programs. As an additional approach to test for transposon activity we performed transposon trap analysis for both fungi. Moreover we are currently developing a system which allows us to test the activity of putative transposable elements identified during database analysis.

References: Daboussi MJ, Capy P (2003) Ann Rev Microbiol 57, 275-99, Galagan JE, Calvo SE, Cuomo C et al. (2005), Nature 438, 1105-15, Pöggeler S, Kempken F (2004) In: The Mycota II, Genetics and Biotechnology, 2nd edition (ed. Kück U). Springer Verlag, Heidelberg, New York, Tokyo, Rep M, van der Does HC, Cornelissen BJC (2005) Fungal Genet Biol 42, 546-553


Comparative genomics of the transcription factors ftf1 and ftf2 in Fusarium

Brisa Ramos2, José J. de Vega-Bartol1, María A. García-Sánchez1, Noemí Martín-Rodrigues3, Arturo P. Eslava1, José M. Díaz-Mínguez1

1Centro Hispano-Luso de Investigaciones Agrarias (CIALE), Universidad de Salamanca, Salamanca, Spain, 2Centro de Biotecnología y Genómica de Plantas (CBGP), Universidad Politécnica de Madrid, Madrid, Spain, 3Instituto NEIKER, Centro de Arkaute, Vitoria, Spain

We have described a new transcription factor, called ftf1, which is likely a virulence factor in F. oxysporum, as it is found only in highly virulent strains and is drastically upregulated during infection of the host plant. We have also identified a highly homologous gene to ftf1, named ftf2, which can be found in F. oxysporum strains, either pathogenic and nonpathogenic.

Experimental results obtained in F. oxysporum f.sp. phaseoli and analyses of the genome sequences of F. graminearum, F. verticillioides and the recently available F. oxysporum f.sp. lycopersicy, show that ftf1 is a multiple copy gene only present in F. oxysporum. The four copies in F. oxysporum f.sp. phaseoli are located in a small chromosome, closely linked to copies of transposon marsu, while the nine copies found in the genome of the lycopersicy strain are scattered over different chromosomes of the optic map, but also linked to different kinds of transposons, including marsu and Fot types. On the other hand, ftf2 is a single copy gene present in the genomes of the three fusaria, the rice pathogen Magnaporthe grisea and the nonpathogenic fungi Neurospora crassa and Aspergillus nidulans. The genomic architecture of the ftf2 region is very similar in the three fusaria and shows syntheny with the M. grisea genome, but syntheny is lost in the nonpathogenic fungi A. nidulans and N. crassa.

The three Fusarium genus members show different host ranges, being F. oxysporum a very versatile pathogen with dozens of formae speciales, while F. verticillioides is restricted to maize and F. graminearum can only infect barley and wheat. The analyses of the genome sequences have the potential to enlighten our understanding of the genetic basis of host specificity. The results here presented show that the, comparatively, larger genome of F. oxysporum contains species specific genes (such as ftf1) involved in virulence. The linkage between ftf1 and transposable sequences and the high homology of this gene to ftf2 suggest that it may have evolved by gene duplication and the resulting copies become dispersed as part of genome reorganizations.



FTFD: an informatics pipeline supporting phylogenomic analysis of fungal transcription factors

Jongsun Park1, Jaejin Park1, Suwang Jang1, Seryun Kim1, Sunghyung Kong1, Jaeyoung Choi1, Kyohun Ahn1, Juhyeon Kim1, Seungmin Lee1, Sunggon Kim1, Bongsoo Park2, Kyoungyong Jung1, Soonok Kim1, Seogchan Kang2, Yong-Hwan Lee1

1Fungal Bioinformatics Laboratory, Department of Agricultural Biotechnology, Center for Fungal Genetic Resources and Center for Agricultural Biomaterials, Seoul National University, Seoul, Korea, Republic of, 2Penn State University, University Park, PA, United States

Transcription factors (TF) are proteins that regulate the activity of RNA polymerase by assembling on promoter regions and consequently modulate gene expression in organisms. In the fungal kingdom, TFs modulate not only conserved regulatory networks but also specialized functions like pathogenicity in certain fungi. Genomes of more than 60 fungal species have been sequenced to date, yet there has been no systematic approach to identify and analyze fungal TFs kingdom-wide. We developed a standardized two pipelines for annotating TFs in fungal genome sequences using the InterPro database. Resulting data have been archived in a new database termed the Fungal Transcription Factor Database (FTFD; In the FTFD, 31,175 putative fungal TFs, archived from 61 fungal and 3 Oomycete species, were classified into 61 TF families and phylogenetically analyzed. The proportions of TFs in the total number of proteins in fungi ranged from 2.29% to 7.15%, which were slightly lower than those in human and mouse. Via hierarchical clustering analysis, these TF families were grouped into seven clusters, one of which exhibited fungal-specific distribution. The FTFD will serve as a community resource supporting comparative analyses of the distribution of TFs and phylogenomic analyses of TFs within and across species.


Pathogenicity determinants of Fusarium graminearum on wheat ears

Andrew Beacham, John Antoniw, Martin Urban, Kim Hammond-Kosack

Rothamsted Research, Harpenden, United Kingdom

Using a novel bioinformatics approach, we have identified a micro-region of verified pathogenicity gene homologues in the important crop pathogen Fusarium graminearum. This region is now being analysed by a combination of bioinformatic and reverse genetics approaches to ascertain its role in the pathogenicity of this species.

Comparative genomics using FASTA alignment, protein identity and property prediction, together with nucleotide repeated element and gene expression analyses have been used to investigate the conservation and properties of the micro-region in other closely and less closely related species. Targeted deletion of single genes using the split marker technique has allowed the determination of the role of each micro-region gene in F. graminearum pathogenicity.

The micro-region, which contains 15 genes and spans 25kb in F. graminearum, appears highly conserved in the other Fusarium species but less so in more distantly related species. It is present in a region of the genome with a low recombination frequency and has a low content of repetitive sequence. The region appears, unlike other fungal gene clusters, not to exhibit coordinated gene expression either during in vitro growth or during plant infection. Deletion of the neutral trehalase gene NTH1 appears to slow infection of wheat ears, while deletion of the SNF1 kinase gene inhibits spread of the pathogen in wheat.

The micro-region is beginning to appear as a novel type of pathogenicity gene cluster, differing from the virulence-associated biosynthetic and secreted protein clusters identified so far in pathogenic fungi. Further investigation will reveal more about the nature of this unusual pathogenicity region.


Phylogenetic diversity of fungal stress signalling pathways

Elissavet Nikolaou1, Ian Stansfield1, Janet Quinn2, Alistair Brown1

1University of Aberdeen, Aberdeen, United Kingdom, 2University of Newcastle upon Tyne, Newcastle, United Kingdom

Microbes require robust stress responses to survive changing environments, and in particular pathogenic microbes need such responses to counter host defences. Cells sense changes in their environment (detection of external signals), activate signal transduction pathways (information routes) to trigger an appropriate response. These responses protect cells against the stress and repair damage caused by the stress. We have explored the sequence conservation of specific stress regulatory proteins across the fungal kingdom, including pathogenic and benign species that have evolved in divergent niches. These species are likely to have evolved stress responses appropriate to their diverse niches. The degree of conservation is measured relative to Saccharomyces cerevisiae because signalling pathways have been studied in detail in this organism using combination of genetic, molecular, biochemical and genomic tools. Comparative genomic analysis from our laboratory has been focused on the osmotic, oxidative and cell wall stress responses of pathogenic and benign fungi because, at least in the pathogen C. albicans, these pathways contribute to virulence, and they affect the sensitivity of cells to antifungal drugs. In parallel we have compared the resistance of the 18 fungal species under analysis to osmotic, oxidative and cell wall stresses. We have extended this work by developing a statistically robust approach to define the degree of conservation amongst stress regulators in the target fungi. Our approach, which allows direct analyses of proteins in functional peer groups, considers both the percentage identity between fungal orthologues and the percentage length of their sequence alignments. Using this approach we have shown significant differences in the rate of evolution of fungal MAPKs, MAPKKs and MAPKKKs, and in the rate of evolution of specific MAPKs. The implications of these data will be discussed.



Modulation of the unfolded protein response pathway in Aspergillus niger

Neuza Carvalho1, Theo Goosen2, Cees van den Hondel1, Arthur Ram1

1Institute of Biology Leiden, Leiden, Netherlands, 2Biocentre, HAN University, Nijmegen, Netherlands

The presence of unfolded proteins in the ER induces a cellular response called the Unfolded Protein Response (UPR). The HacA transcription factor plays a central role in the UPR pathway, resulting on the induction of a large number of genes, among them chaperones and foldases that assist in the protein folding in the ER.

We were interested in studying the effects of the hacA and ireA deletions, as well as the constitutive hacA expression on the growth and morphology of A. niger. For the knock-out mutants, deletion cassettes were made containing 500bp of the 5’ and 3’ flanking regions of the respective gene, and pyrG to be used as a selection marker. For generating the constitutive hacA mutant, a hacA gene construct was made that lacked the 20 nt intron sequence and transformed to replace the wild type hacA gene in the genome. For the construction of the deletion strains, an A. niger strain with kusA (ku70) gene deleted was used. In this background, not only the targeting efficiency is high, but also in the case of essential genes, heterokaryons are obtained as primary transformants, as ireA turn out to be. In S. cerevisiae, ire1 deletion is not lethal and results in inositol auxotrophy. However, supplementing growth media with inositol did not allow the growth of A. niger ireA deletion strains. Southern blot analysis confirmed the heterokaryotic nature of ireA transformants obtained. In A. niger, ΔhacA is not lethal, but the mutant shows a severe growth defect leading to a small and compact colony phenotype. Primary transformants displaying the hacA growth phenotype were never observed, but hacA deletion strains were efficiently obtained after purification of spores from primary transformants. These results show that gene deletions mutants with a strong defect in growth can easily be obtained via heterokaryons in the kusA background. The constitutive hacA mutant also shows a growth defect when compared with the wild type. Both on solid or liquid medium, this mutant grows slower then the wt. Morphologically, hyphae of the constitutive hacA mutant grow on a curly way, resembling mutants with defect in the tubulin cytoskeleton. The effect of constitutive hacA expression on tubulins is currently investigated.



Whole transcriptome analysis of Coprinus cinereus meiotic development

Claire Burns1, J.E. Stajich2, J.D. Lieb3, S.E. Hanlon3, W.W. Lilly4, A.C. Gathman4, S.K. Wilke3, M.E. Zolan1, P.J. Pukkila3

1Indiana University, Bloomington, IN, United States, 2UC Berkeley, Berkeley, CA, United States, 3University of North Carolina, Chapel Hill, NC, United States, 4SE Missouri State University, Cape Girardeau, MO, United States

Coprinus cinereus, a basidiomycete mushroom fungus ideally suited to meiotic studies due to its synchronous development, has a near-completed genome. The 13 chromosomes show an unusual distribution of repeated elements and regions of elevated recombination. Gene prediction algorithms indicate a total of approximately 12,500 genes. To analyze meiotic development at the transcriptional level, we designed and fabricated a 13,230 element 70-mer oligonucleotide microarray using ArrayOligoSelector. The tightly regulated light and temperature- dependent life cycle of the mushroom allows sampling of gill tissue in which cells are at a specific meiotic stage at a certain time. Meiosis occurs synchronously and takes ca. 12 hours. Our experiments concern a broad 15-hour timecourse through the meiotic process, describing events prior to fusion of dikaryotic nuclei, nuclear fusion, condensation and alignment of meiotic chromosomes, first meiotic division, and completion of the second division. Replicate microarrays were hybridized with cDNA from 3 hours prior to karyogamy, karyogamy, and 3, 6, 9 and 12 hours post-karyogamy, using a pooled mixture of cDNAs as the reference sample for each array. Analysis using materials from 4 independent fruitbody collections revealed that ~ 3000 genes were significantly differentially regulated during this timecourse, at a false discovery rate of 10%. Using a bifurcation strategy with K-means data clustering, we were able to identify temporally regulated waves of transcription throughout meiosis and mushroom development. Furthermore, clusters containing meiotic genes and predicted genes of meiotic function were found. These clusters also exhibit strong enrichment for Gene Ontology classes associated with meiosis. Array data concur with existing northern data. We conclude that our annotation pipeline produced informative gene predictions, which allowed design of informative 70-mer oligonucleotides, and our resultant microarrays provide an important tool for analysis of synchronous meiotic development in C. cinereus.

Supported by NSF, NIH, and by the Indiana METACyt Initiative of Indiana University, which is funded in part through a major grant from the Lilly Endowment, Inc.



Proteome map of the total cell and mitochondrial proteome of Aspergillus fumigatus

Martin Vödisch, Olaf Kniemeyer, Franziska Leßing, Daniela Albrecht, Robert Winkler, Axel A. Brakhage

Leibniz Institute for Natural Product Research and Infection Biology (HKI) / Friedrich-Schiller-Universität, Jena, Germany

Aspergillus fumigatus is a ubiquitously distributed saprophytic mould. In the last decade, A. fumigatus has become the most important airborne fungal pathogen causing life-threatening infections in immunosuppressed patients. Little is known about the pathogenicity determinants of A. fumigatus which enable this fungus to survive and grow within the host. In comparison to other fungi, A. fumigatus possesses obviously higher stress tolerance and better mechanisms to adapt to the host environment. As a basis for comparative proteomic studies, we established a reference proteome map. Using MALDI-TOF-MS/MS we identified 392 protein spots representing 344 proteins separated on 2D-gels. Proteins involved in primary metabolism, protein synthesis, cell cycle regulation, DNA processing and transcription, transport, cellular organisation and stress response were most abundant. Since mitochondria are important organelles involved in energy production, metabolism of amino acids, lipids and iron, we also established a protocol for the isolation of mitochondria of A. fumigatus. The mitochondrial protein fraction was separated by 2D-gel electrophoresis and a partially annotated mitochondrial proteome reference map was developed. 126 different proteins spots have been identified so far.



The genome of the zygomycete Phycomyces blakesleeanus, a model organism for sensory biology

Luis M. Corrochano1, Alan Kuo2, Asaf Salamov2, Jasmyn Pangilinan2, Erika Lindquist2, Harris Shapiro2, Scott Baker3, Jan-Fang Cheng2, Igor Grigoriev2, Phycomyces Genome Group1

1Universidad de Sevilla, Sevilla, Spain, 2DOE Joint Genome Institute, Walnut Creek, CA, United States, 3Pacific Northwest National Laboratory, Richland, WA, United States

The zygomycete Phycomyces blakesleeanus is a model microorganism for research on sensory biology. The giant fruiting bodies, sporangiophores, of Phycomyces change their speed and direction of growth in response to a variety of environmental signals, including light, gravity, wind, touch, and the presence of nearby objects. Additionally, light regulates sporangiophore development and induces the synthesis of the pigment beta-carotene. Phycomyces is an intensively studied, experimentally tractable model organism, and whole-genome analysis is expected to further elucidate the signaling pathways underlying its photoregulation. To this end the genome was sequenced to 7.49X depth and assembled into 475 scaffolds totaling 56Mbp, and 47847 ESTs were assembled from cDNAs of light and dark cultures. We combined into a single annotation pipeline a variety of gene modeling methods (homology-based, EST-based, and ab initio), and predicted 14792 protein-coding genes. Many of these gene predictions are supported by homology in nr (68%), by Pfam domains (44%), or by ESTs (35%). We next assigned GO terms to 41% of the proteins and EC numbers to 16%. We then distributed these annotations to the Phycomyces consortium, along with tools to curate them manually. We expect that the annotation will provide a solid platform for expression analysis. The initial characterization of the Phycomyces protein set has uncovered the presence of multiple photoreceptor proteins and large families of proteins involved in signal transduction. In addition, several new sets of repetitive DNA, including transposable elements, have been described in the genome of Phycomyces. Comparisons with genomes from Ascomycete, Basidiomycete, and Zygomycete fungi will help us to unravel the evolution of fungal photoreceptors and other sensory transduction proteins. We expect that the Phycomyces genome will help to understand the molecular mechanisms involved in the sensing of light, gravity and other environmental signals by this model fungus.


Proteome analysis of the response of Aspergillus fumigatus to iron limitation

André D. Schmidt1, Olaf Kniemeyer1, Hubertus Haas2, Axel A. Brakhage1

1Leibniz Institute for Natural Product Research and Infection Biology (HKI) / Friedrich-Schiller-University Jena, Jena, Germany, 2Division of Molecular Biology/Biocenter, Innsbruck Medical University, Innsbruck, Austria

The acquisition of iron is known to be an essential step in any microbial infection process due to iron-limiting conditions in the human host. This iron limitation is caused by high-affinity iron-binding proteins like transferrin or lactoferrin in the host. Since iron plays an essential role in key metabolic processes like DNA synthesis, oxidative phosphorylation or electron transport A. fumigatus has to overcome the iron deficiency by the synthesis of siderophores, which chelate iron. It was shown that an A. fumigatus strain unable to synthesise siderophores was attenuated in virulence in a murine infection model (Schrettl et al. 2004). Moreover, regulation of iron homeostasis via two transcription factors was recently described in filamentous fungi (Hortschansky et al. 2007). To understand the cellular processes induced by iron starvation, we analysed the proteome of A. fumigatus strain ATCC 46645 grown under iron-deficiency conditions. In total 98 spots, representing 88 different proteins, were upregulated under -Fe conditions and 88 spots, representing 78 different proteins, were downregulated under iron deficiency conditions. The major parts of the downregulated proteins belonged to proteins involved in primary metabolism (31 %), ribosomal protein synthesis (13 %) and chaperone activity (9 %). Many iron-sulphur cluster and heme-containing enzymes such as aconitase and cytochrome c peroxidase were found to be downregulated. On the other hand, proteins involved in the amino acid metabolism (16 %) represented the major group of upregulated proteins under iron starvation. In addition, proteins, which were related to siderophore biosynthesis (3%) or protein degradation (5 %) or proteins involved in antioxidative response like Cu, Zn superoxide dismutases (4%) that lack iron, showed also an upregulation under iron deficiency. Further proteins analysed under different non-linear pH-scales will be presented and their putative roles during iron depletion will be discussed.

Schrettl et al. (2004) J. Exp. Med. 200: 1213-1219, Hortschansky et al. (2007) EMBO J. 26, 3157-3168



Molecular characterization, location and isolation of telomeric and subtelomeric sequences of basidiomycete Pleurotus ostreatus

Gumer Pérez, Anotnio G. Pisabarro, Lucía Ramírez

Department of Agrarian Production, Public University of Navarre, 31006 Pamplona, Navarre, Spain

The white rot fungus P. ostreatus is an edible basidiomycete with increasing biotechnological interest and applications. Telomeres are specialized structures at the end of all eukaryotic chromosomes; they ensure chromosome stability and protect their ends from degradation and from fusing with other chromosomes. Telomeres sequences are extraordinary highly conserved in evolution. The loss of telomeric repeats triggers replicative senescence in cells. Telomeric DNA from several organisms has been identified and cloned by using telomeric repeats from non-related organisms as probes. Similarly, we have identified, mapped and cloned the P. ostreatus telomeres using the human telomeric repeat as probe. For mapping the telomeric, the genomic DNAs isolated form individuals of a segregating population was digested with restriction enzymes, electrophoresed and transferred to nylon membranes. The use of the heterologous human telomeric probe (TTAGGG)132 revealed numerous polymorphic bands. Some of them were mapped to the genetic linkage map using the segregation of the RFLP markers identified. The segregation of each telomeric restriction fragment was recorded as the presence vs. absence of a hibridizing band. Segregation data for seventy-three telomeric restriction fragments were used as an input table to be analysed using the MAPRF program software. Eighteen out of twenty two telomeres were identified and assigned to linkage map of this fungus. P. ostreatus telomere and subtelomere sequences were isolated using a modified version of single-specific-primer polymerase chain reaction technique. The results showed that P. ostreatus telomeres contain at least fifty-five tandem copies of the TTAGGG sequence. The analysis of their terminal restriction fragment showed that the range of variation in P. ostreatus telomere length ranges between 150 and 1500 bp. These results indicate that the telomeric hexanucleotide copy number ranges from 25 to 250 repetitions. We used fragments subtelomeric sequences identified with the SSP-PCR technique as probes to isolate the telomeric sequence of P. ostreatus in a lambda phage genomic library from vegetative mycelium of the dikaryotic strain N001. The results show that P. ostreatus contains a subtelomeric sequence in which it has found homology to a recQ helicase gene family, an ABC sugar transporter, and a short chain dehydrogenase.


Development of molecular markers for studying the genetic basis of natural phenotypic variation in Aspergillus nidulans

Julian Christians1, Cortney Watt2

1Simon Fraser University, Burnaby, BC, Canada, 2St. Francis Xavier University, Antigonish, NS, Canada

Introduction: Studying natural phenotypic variation within species will contribute to our understanding of the genes underlying traits of medical, industrial and evolutionary importance. Approaches for studying such variation, including quantitative trait loci (QTL) mapping, require many molecular markers. Microsatellites are a commonly-used marker in other organisms, but are relatively difficult to develop in fungi.

Methods: This study examined the relative polymorphism rates of mononucleotide, dinucleotide and trinucleotide repeats among four wild-type strains of Aspergillus nidulans.

Results: The probability of polymorphism increased with number of repeating units. Di- and trinucleotide repeats had higher polymorphism rates than mononucleotide repeats, but this was offset by the presence of numerous long mononucleotide repeats.

Discussion: The use of mononucleotide repeats will substantially increase the number of potential markers available for studies of Aspergillus nidulans. Given that mononucleotide repeats are more abundant than other repeats in fungal genomes in general, mononucleotide repeats are likely to be an important resource in other species as well.



Gene targeting in Chrysosporium lucknowense using a retrievable mutation in the KU70 gene

Cora van Zeijl1, Marian van Muijlwijk1, Margreet Heerikhuisen1, Jan Verdoes2, Peter Jan Punt1

1TNO Quality of Life, Zeist, Netherlands, 2Dyadic Nederland BV, Wageningen, Netherlands

Current approaches for the development of fungal industrial host strains have long time-lines and the resulting strains are genetically ill-defined. Moreover, available approaches to resolve the latter bottleneck with targeted gene replacement have even longer timelines. A new approach based on a KU70 mutant strain resulting in a non-homologous recombination deficient mutant strain is reported for several non-industrial host strains. We used the KU70 approach for actual commercial strains of Chrysosporium lucknowense. For industrial applications the presence of a KU70 mutation in a final production host strain is unwanted as the recombination deficient KU70 phenotype may have adverse effects in large scale fermentation. Therefore, we designed two different KU70 disruption vectors. One type of KU70 disruption vector was designed for strain development approaches, involving multiple gene disruptions at different loci. In the primary KU70::amdS disruption strain the amdS selection marker can easily be removed by fluoroacetamide selection leaving a KU70 mutant phenotype. The subsequent gene disruption could be carried out using a reusable amdS selection marker. The other KU70 disruption vector was designed to disrupt the KU70 gene in a strain background to be used for construction of genetically defined production strains, by introducing a desired expression cassette e.g. at the cbh1 locus. The wild type KU70 gene can easily be regenerated by fluroacetamide selection. The latter type of KU70 disruption vector can also be used to regenerate the WT KU70 locus in a multiple disrupted KU70 mutant host strain.

The integration properties of the Chrysosporium lucknowense KU70 mutant strains will be presented.



Analysis of the Trichoderma atroviride transcriptome during mycoparasitism

Barbara Reithner, Enrique Ibarra-Laclette, Alfredo Herrera-Estrella

Cinvestav Campus Guanajuato, Irapuato, Guanajuato, Mexico

Trichoderma spp. have been shown to attack and lyse various plant-pathogenic fungi using a complex mechanism. The way mycoparasitic Trichoderma strains recognize their hosts is to a large extent still unknown. But it has already been shown that they grow towards the target fungi, attach to and lyse the host cell wall by secreting antifungal enzymes and secondary metabolites. The importance of biocontrol and plant protection by Trichoderma result from the necessity to reduce the application of chemical fungicides to antagonize plant diseases caused by pathogenic fungi such as Rhizoctonia, Phythium, Phytophthora, Botrytis, and Fusarium. In the present work we concentrate on the mycoparasitic interaction of T. atroviride using plant-pathogens of different phylla to gain information about the host-specific response of Trichoderma. Therefore, we used a powerful sequencing system (pyrosequencing by 454 Life Science Technologies) in combination with a new concept of multiplex identifiers to pool genes which are differentially expressed during different stages of confrontation with R. solani, P. capsici and the non-pathogenic fungus Neurospora crassa. This technique allowed us to obtain approximately 250.000 reads in one run of a cDNA mixture of all samples. The assembled contigs and singletons were blasted against the available genomes of T. reesei, T. virens, F. graminiarum, and the genomes of the host fungi and compared between the conditions used. Differences were discovered between the EST's obtained from the plate-confrontationes assays with different plant-pathogenic fungi as well as for the different stages of interactions analyzed. In addition to the qualitative distinction, we can show that differences in gene-expression found with this method are extremely quantitative.

The present study displays an extensive analysis of genes involved in the crosstalk between T. atroviride and it's hosts.


A comparative protein profile of BLR mutants of Trichoderma reesei

Alberto Flores Martínez1, Sandra González Hernández1, Patricia Ponce Noyola1, Alfredo Herrera Estrella2

1Universidad de Guanajuato, IIBE Facultad de Química, Guanajuato, Guanajuato, Mexico, 2Langebio CINVESTAV Campus Guanajuato, Irapuato, Guanajuato, Mexico

Trichoderma atroviride, a fungus used in biological control, sporulates in a synchronized manner following a brief pulse of blue light. BLR-1 and BLR-2 are key regulatory elements of photoconidiation and mycelial growth in T. atroviride and T. reesei. We are using a proteomic approach, to study and analyze differences between wild type and brl1- and blr 2- mutants during the light response.

As a preliminiray step, the protein profile of soluble cellular extracts from three strains of Trichoderma reesei, wild type, brl 1and blr 2 mutants, exposed to light pulse has been compared by using two-dimensional gel electrophoresis. Up to 500 individual spots can be resolved in the 4 to 7 pI and 14 to 200 kDa MW ranges. The biological variability for 197 major spots was determined for each isolate. The isolates showed a similar 2-DE map, with both qualitative as well as quantitative differences in quite a number of spots, In silico analysis of some of the differential expressed spots (27 for wt, 7 for blr1 and 1 for blr2) have been identified. Such as Ornithine decarboxylase antizyme, Protein cwf17, Enolase, Serine/threonine phosphatase.

This study was supported by grant from the DINPO, Universidad de Guanajuato.


Molecular characterization of Sc1 protein from sclerotia of Sclerotium cepivorum Berk

Patricia Ponce-Noyola, Jorge Alegría-Torres, Edgar Alfaro-Rangel, Sandra González-Hernández, Arturo Flores-Carreón, Alberto Flores-Martínez

Universidad de Guanajuato, IIBE Facultad de Química, Guanajuato, Guanajuato, Mexico

Sclerotium cepivorum Berk is the etiological agent of the garlic disease known as "white rot", which is one of the major causes the low production of garlic in The Bajio region in Central Mexico. We are interested in learning more about the physiology and biochemistry of this microorganism, in order to define molecular markers that can be used to detect the fungus.

In this work, purification, location and molecular characterization of a protein (Sc1) from sclerotia that may be used as molecular markers to detect the fungus in soil and seed samples is presented. We have found that the Sc1 is the predominant protein representing up to 70% of the total Sclerotium protein. It exhibits a molecular weight of 36 kDa, a pI close to 5.0 and displays similar characteristics to other developmental proteins described in other fungi. Fungal growth kinetics and formation of sclerotia showed that this protein is sclerotia specific, i.e., it is not detected in mycelia but only at the onset of sclerotia formation where it can be immunodetected using specific antibodies. Reverse genetics based on the amino terminal sequence of the protein demonstrated that the Sc1 RNA expression starts 5 days before sclerotia formation during the growth period. The estimated mRNA size was near 1000b.

The study was supported by grant CONACyT-SAGARPA-CO1-11919 from Consejo Nacional de Ciencia y Tecnología, México.


Development of markers based on transposable element in Agaricus bisporus and their usefulness for genetic analysis

Marie Foulongne-Oriol, Jugurta Chedded, Cathy Spataro, Jean-Michel Savoie

INRA UR1264 Mycologie et Sécurité des Aliments, Villenave d'Ornon, France

Transposable elements constitute a significant fraction of fungal genome and contribute to genome plasticity and evolution. Each transposition event generates new variability and this property could be exploited in several molecular marker systems. We discuss here the identification of transposable elements in Agaricus bisporus and their potential use as tools for genetic analysis. Two different strategies were used. Firstly we performed PCR with degenerated primers designed in conserved domain of reverse transcriptase, using previously available sequence information from other fungi in order to isolate homologous genes in A. bisporus. From the sequences obtained we developed IRAP marker analysis, based on PCR amplification of genomic DNA fragments between two transposon insertion sites. Secondly, we exploited the SSAP technique. This approach similar to AFLP system required primer designed in sequence from terminal region. We used primers derived from LTR of other fungi retrotransposon such as Tricholoma matsutake and we used also A. bisporus. LTR sequence isolated in our lab. Either IRAP or SSAP techniques were tested on thirty genotypes of A. bisporus chosen in cultivated or wild strains. The usefulness of these marker systems is compared to others already used for this mushroom, and their potential application for genetic studies such as linkage mapping or diversity analysis is discussed.



FPC-DB: the fungal protein cluster database

Jaehee Jung1, Gangman Yi1, Serenella Sukno2, Michael Thon2

1Universidad de Salamanca, Centro Hispano-Luso de Investigaciones Agrarías (CIALE), Salamanca, Spain, 2Texas A&M University, College Station, TX, United States

Our ability to predict gene function for newly sequenced fungal genomes is based to a large extent on comparisons to other organisms. It is important when making these comparisons to take into account the large evolutionary distances, sometimes hundreds of millions of years, that separate many of the fungal taxa for which whole genome sequences exist. It is commonly accepted that orthologous proteins tend to have conserved function in different species, however numerous examples that demonstrate functional diversification among orthologous genes exist. The phylogenetic context of a gene family must be taken into account when transferring knowledge of gene function from other species. The Fungal Protein Cluster Database (FPC-DB) is designed to enable users to quickly locate information about the evolutionary history and the functions of fungal proteins and gene families and present the information in a number of comparative views. The proteins from fungal whole-genome sequencing projects are annotated with InterPro terms, SwissProt keywords, and Gene Ontology terms using an automated functional classification server previously developed in our group. The proteins are then clustered into putative gene families using the MCL application. Each protein cluster includes multiple sequence alignments, phylogenetic trees and a summary of functional categories found in the cluster. Users can perform queries using proteins IDs, protein functional categories, and species names as search terms. The results are displayed in a variety of forms that allow users to compare the occurrence of gene and their functional categories between species. Users can also view a detailed page for each protein cluster and simultaneously view a phylogenetic tree and a matrix of functional categories, enabling them to identify cases of lineage specific gains and losses of functional categories.


Genome sequence of the barley powdery mildew Blumeria graminis

Pietro Spanu1, Sarah Butcher1, James Abbot1, Timothy Burgis1, Marc-Henri Lebrun2, Joelle Amselem2, James Brown3, Christopher Ridout3, Nicholas Talbot4, Darren Soanes4, Sarah Gurr5

1Imperial College London, London, United Kingdom, 2INRA/CNRS, Paris, France, 3John Innes Centre, Norwich, United Kingdom, 4University of Exeter, Exeter, United Kingdom, 5University of Oxford, Oxford, United Kingdom

We have sequenced the genome of Blumeria graminis f sp hordei, strain DH14. A shotgun sequencing approach was employed using short insert (~2kb) plasmid libraries and large insert (~38kb) fosmid libraries made from purified conidial DNA. The resulting 460Mb have been assembled (Arachne) and are currently being analysed. Preliminary projections of genome size estimate a value of around 120Mb which is much higher than expected and needs to be taken with caution do to the possibility of a skew in the models due to the presence of large amounts of repetitive DNA (>60%) largely unique to Blumeria. We are currently analysing the repeat structure to evaluate this possibility. In collaboration with others we have access to 620Mb of Solexa generated sequence and we are combining the datasets by using de-novo and scaffold-based methodologies in the expectation of providing a reference genome for powdery mildews sequenced by high-throughput platforms. Comparison with existing published mapped genetic markers in Blumeria show that the two datasets are consistent and co-linear; we have been able to draw some of the linkage groups together and we expect many more to do so once the genomic assembly is improved.We have extended the Blumeria EST dataset by sequencing cDNA libraries from conidia, germlings isolated from infected barley (20hpi, 3dpi and 6dpi), cleistothecia and infected barley epidermis. The ESTs cluster into 3549 unisequences, 99% of these map to the current genome assembly. This data provides us with the training set for the gene finding software that will serve for the initial machine-based annotation of the genomic sequence. Of note is the fact that the cleistothecial cDNAs have an exceptionally high level of novel/unique sequences, indicating a highly differentiated state in which gene expression is very different from the fungal tissues analysed so far.

All this data is deposited in GenBank and publicly available through the website . In addition to sequence data it is intended that the site will be a general repository for information about powdery mildews in general and Blumeria in particular with links and access to relevant publications. All clones are also available for anyone requesting them through this website.



Analysis of Penicillium isolates belonging to the "Glabra group" in cork using fingerprinting and ITS-ARDRA methods

M Carmo Basilio1, Andreia Santos1, Susana Marcelino1, Rob Samson2, Rogério Tenreiro3, Vitória San Romão1, Vitória San Romão4

11 Instituto de Biologia Experimental e Tecnológica/Instituto de Tecnologia Química e Biológica,Universidade Nova de Lisboa, Oeiras, Portugal, 2Centraalbureau voor Schimmelcultures, P.O. Box 85167, NL-3508 AD, Utrecht, Netherlands, 3Universidade de Lisboa, Faculdade de Ciências, Centro de Genética e Biologia Molecular and Instituto de Ciência Aplicada e Tecnologia, Edificio ICAT, Campus da FCUL, Campo Grande, Lisboa, Portugal, 4LNRB/L-INIA (Ex-Estação Vitivinícola Nacional), Dois Portos, Portugal

Cork is the best sealing material for wine, champagne and sparkling drinks. 80 Penicillium strains were isolated from factory environments and from cork slabs in different stages of the manufacturing process of cork. More than half of those isolates belonging to the genus Penicillium were identified phenotypically as members of the "Glabra group". To study if members of the group, namely in P. glabrum and P. spinulosum, could be separated by molecular methods two approaches were established. The first fingerprinting method used was amplification of DNA using four different microsatellite primers. Clustering of profiles obtained with all results was performed using software that analyses gel band profiles and intensity. Strain/group discriminative bands were selected as potential molecular marker for species definition. Furthermore, restriction analysis of amplified ITS region (ITS-ARDRA) was also used. The combination of the two techniques contributes to distinguish the taxonomical position of P. glabrum and P. spinulosum.

Acknowledgments: FCT for PhD grant BD/19264/2004. Amorim & Irmãos (Coruche, Portugal). Jos Houbraken for helping in the phenotypical identification of the Glabra group.Paula Alves for technical analysis.



Functional analysis of a mitochondrial phosphopantetheinyl transferase (PPTase) gene pptB in Aspergillus fumigatus

Gethin Allen, David Keszenman-Pereyra, Geoff Turner

The University of Sheffield, Department of Molecular Biology and Biotechnology,, Sheffield, United Kingdom

Many secondary metabolism pathways of A. nidulans and A. fumigatus are dependent on the pptA gene product for post-translational modification of non-ribosomal peptide synthetases or polyketide synthases by addition of a 4’-phosphopantetheine cofactor. Inspection of the genome sequences of A. nidulans and A. fumigatus identified a second putative pantothenyl transferase termed pptB. Deletion of pptB from A. fumigatus resulted in stable heterokaryons, and permitted the identification of a deletion phenotype as microcolonies, capable of only very slow growth. When the gene was placed under control of the PalcA promoter, repression led to slow growing colonies. GFP localisation showed the presence of the gene product within the mitochondria.

Taken together, our data suggest that pptB is an orthologue of the yeast gene PPT2. PPT2 is known to encode a PPTase specific for a small mitochondrial acyltransferase Acp1p.

An orthologue of Acp1p has been previously investigated in N. crassa, and is a component of mitochondrial complex I. While yeast lacks complex I, Acp1p is involved in lipoic acid biosynthesis within the mitochondria. Putative orthologues of Acp1p are also present in A. fumigatus and A. nidulans.



Intraspecific variability in phytopathogenic fungus Monilinia laxa (Aderh. & Ruhland) honey

Tjasa Gril, Branka Javornik, Franci Celar, Jernej Jakse

University of Ljubljana, Biotechnical Faculty, Agronomy Department, Ljubljana, Slovenia

Brown rot fungi are found in most temperate regions, causing considerable losses to apples, pears and stone fruits. The group of rot fungi includes three species: Monilinia laxa (Aderh. & Ruhl.) Honey, Monilinia fructigena (Aderh. & Ruhl.) Honey and Monilinia fructicola (Wint.) Honey. A special form of the fungus, M. laxa f. sp. mali, is found only in apple, in which it causes blossom wilt, spur-kill and canker. It has not yet been clearly confirmed whether this is a specialized form of the fungus or merely a race.

The purpose of the present study was to analyse isolates of Monilinia laxa and Monilinia laxa f. sp. mali from different hosts by the AFLP method, in order to evaluate their genetic diversity and relationships and to detect possible intraspecific variability.

A total of sixty-seven fungi isolates of Monilinia laxa were acquired from a wide range of hosts comprising pome and stone fruit plants from different fruit growing areas in Slovenia and from abroad (Japan and three reference isolates from the Centraalbureau voor Schimmelcultures) were analysed. Genomic DNA from fungal mycelium was isolated with the standard CTAB procedure and fluorescent AFLP analysis was carried out using 20 different EcoRI/MspI primer combinations with two selective bases; electrophoregrams were analyzed by AlleleLocator 1.03. Distance-based clustering (Dice's coefficient of similarity, UPGMA and PCA method) and model-based clustering (Structure) were used to evaluate genetic similarities and relationships among the analysed samples.

A total of 1089 AFLP bands were scored, of which 354 or 32.5% were polymorphic. AFLP polymorphism thus showed high intraspecific variability. On average, 54 AFLP markers, ranging from 44 to 745 bp in fragment length, were amplified per combination. The highest number of fragments (96) was amplified with primer combination E-GA+M-AT and the lowest number of bands (33) with two different M-selective nucleotides (CG, GA). Some specific markers within the fingerprints of the individual isolates were also discovered. Clustering data analysis showed that Monilinia laxa isolates collected from apple trees form a well separated group of fungal isolates, which is clearly distinguished from the other group of host plant isolates.



RNA-mediated gene silencing as a tool to study gene function in the ascomycetous fungus Aspergillus niger

Jose Miguel Oliveira, Peter J Schaap, Leo H de Graaff

Microbiology, Wageningen University, Wageningen, Netherlands

Aspergillus niger is among the most important micro-organisms for the industrial production of organic acids and of extracellular enzymes used in the food and feed industry. Until recently, the most common approach for the study of gene function in A. niger was the construction of gene knock-out mutants. Despite its effectiveness, gene knock-out is a time-consuming and laborious method for silencing genes. Furthermore, this method cannot be applied to the study of essential genes. To address this problem, a system for gene knock-down through RNA interference (RNAi) in A. niger was created. As a first step, a destination vector for generation of RNAi clones by recombination reaction was constructed. To test this system, a segment of sequence coding for the xylanolytic regulator XlnR was recombined with the constructed destination vector to yield the corresponding silencing vector. After co-transformation in A. niger, strains were further selected by plate screening on the basis of low xylan-degrading activities. Compared to the wild type, these strains showed lower levels of xlnR transcripts and furthermore two tested genes regulated by XlnR (xyrA and xynB) also presented decreased transcript levels in these co-transformants. Since RNAi has not been applied or described in A. niger, these data show that RNAi constructs effectively work in this fungus. The newly developed system for RNA-mediated gene silencing in A. niger could therefore be efficiently used in functional genomics studies in this fungus.



Selection of optimal oligonucleotide probes for a whole genome microarray approach to decipher Podospora anserina sexual development

Frederique Bidard1, Sandrine Imbeaud2, Nancie Reymond2, Olivier Lespinet1, Philippe Silar1, Corinne Clave3, Veronique Berteaux-Lecellier1, Herve Delacroix2, Robert Debuchy1

1Institut de Genetique et Microbiologie, Univ. Paris-Sud, Orsay, France, 2Centre de Genetique Moleculaire, Godmap, Gif Sur Yvette, France, 3Institut de Biochimie et Genetique cellulaire, Univ. Bordeaux2, Bordeaux, France

The sexual development of the heterothallic euascomycete Podospora anserina has been analyzed for many years in terms of signal transduction pathways controlling the switch from vegetative to reproductive state, autophagy requirement for fruiting-body maturation, functions of MAT proteins and the developmental role of peroxisomal proteins. The completion of the P. anserina genome sequence along with developmental mutants disposal allow all these research areas to benefit now from a microarray approach.

A genome wide microarray, covering the 10546 presently known and predicted CDS, has been constructed with the Agilent in-situ synthesized 60-mers technology. A library of 10 oligonucleotides per CDS has been design by the manufacturer. To select only one oligonucleotide per CDS, a screening strategy has then been elaborated. 1) A computational step has been performed to determine 3’ position, intron position and cross-hybridization rate of all probes. These data have been compiled to attribute a score to each probe and thus, to select a first set of 4 oligonucleotides per CDS. 2) Microarrays, manufactured with this design, have been experimentally validated. 3) The last step of the selection procedure has been based on the use of transcriptomic experimental data generated with diverse culture conditions. An oligonucleotide (flag, signal on noise, coefficient of variation) and CDS (expression, intensity) scoring have been settled to select the best oligonucleotide per CDS.

To conclude :

1) A Podospora whole genome microarray containing (one 60-mers per CDS) X4 of high quality has been constructed.

2) A catalog of 10 probes per CDS with computational information and a sub-catalog of 4 probes per CDS with experimental data have been settled. This information would be useful, in the future, to create new microarray design in function of genome annotation evolution.

3) The powerful and highly stringent selection strategy elaborated in this work can be applied for any sequenced organism.

This project is funded by Agence Nationale de la Recherche, n° ANR-05-BLAN-0385-01.



Gene content and DNA polymorphism among six strains of A. flavus and A. oryzae show species-specific features

D.R. Georgianna1, J. Yu2, N. Khaldi3, D. Brown1, W.C. Nierman4, J.R. Wortman5, M. Machida6, R.A. Dean1, C.P. Woloshuk7, D. Bhatnagar2, T.E. Cleveland2, G.A. Payne1

1Department of Plant Pathology, North Carolina State University, Raleigh, NC, United States, 2USDA/ARS/SRRC, New Orleans, LA, United States, 3Department of Genetics, Trinity College, Dublin, Ireland, 4The J. Craig Venter Institute, Rockville, MD, United States, 5Institute for Genome Sciences, University of Maryland School of Medicine, Baltimore, MD, United States, 6National Institute of Advanced Industrial Science and Technology (AIST), Higashi, Tsukuba, Ibaraki, Japan, 7Department of Botany and Plant Pathology, Purdue University, West Lafayette, IN, United States

Aspergillus flavus (AFL) is an opportunistic plant and animal pathogen that produces aflatoxin, a potent carcinogen. In contrast, A. oryzae (AO) is used extensively in the food industry and thus is regarded as safe. An available genome sequence for AFL strain NRRL3357 and AO strain RIB40 provided the opportunity to carefully examine these fungi for subtle changes that may have resulted from adaptation to different ecological niches. To gain information on variation among strains within a species, genomic DNA from three diverse strains of AO and AFL was hybridized to a whole genome Affymetrix GeneChip prepared for AFL strain NRRL3357 and for genes unique to AO strain RIB40. This comparative genome hybridization (CGH) analysis revealed genes unique to each species and polymorphisms in genes common to each species. In the AFL species subset 43 genes were identified as unique to the species and 129 were unique to the AO species subset. Gene polymorphism was predicted for 1014 probes on the array representing species-specific polymorphism in 607 genes. These sites were verified by comparison of probe sequences to RIB40 and NRRL3357. Genes involved in metabolism and transport contained the greatest number of polymorphisms. Additionally, we examined gene expression between the two sequenced strains. We found 263 of 607 species-specific polymorphic genes to be differentially expressed. A total of 178 of these genes were more highly expressed in AFL, indicating that AO has become more selective in its metabolic processes. Our data show that while these two fungal species are highly similar, they can be distinguished from each other by species-specific features. This data set provides a snapshot of the genomic content changes specific to two ecologically different species.



Comparative genome hybridization demonstrates loss of chromosomes in Mycosphaerella graminicola isolates

Sarrah Ben M'Barek1, Theo A.J Van der Lee1, Alexander H.J Wittenberg1, Henk J. Schouten1, Manoel Souza2, Gert H.J Kema1

1Plant Research International B.V., Wageningen University and Research Centre (WUR), P.O. Box 16, 6700 AA, Wageningen, Netherlands, 2Embrapa-LABEX Europe - Wageningen University and Research Centre (WUR), P.O. Box 16, 6700 AA, Wageningen, Netherlands

Comparative Genomic Hybridization (CGH) arrays using a high density whole genome tilling NimbleGen platform ( were employed to further understand the genome plasticity of Mycosphaerella graminicola, the causal agent of septoria tritici blotch of wheat. The availability of the finished genome of M. graminicola IPO323 (, detailed genetic maps and karyotypes enables genome wide analysis for variation in DNA copy number and provides a new window to study chromosome polymorphism and translocations. In total, 387000 probes based on the IPO323 genome, were spotted on the array. These microarrays consist of 50 to 75–mer probes with a mean probe spacing of 118bp and a maximum stringency of 2 covering therefore the whole genome.

We have analyzed the parents and progeny isolates of the M. graminicola mapping population IPO94269 and the sequenced isolate IPO323. Hybridization of M. graminicola IPO94269 displayed substantial aneuploidy against the IPO323 reference genome.

Our results reveal widespread deletions between these two parental isolates and confirm the absence of two complete chromosomes in isolate IPO94269 previously demonstrated by karyotyping (Mehrabi et al. 2007) and genetic linkage analysis. Our data indicate CGH arrays can be used to detect aneuploidy, to predict the sites of chromosome breaks, and to identify recombination breakpoints. We currently exploit CGH to identify large genome differences such as deletions or translocations between isolates to understand genome plasticity and its relation with virulence, host specificity and speciation.

Mehrabi R., Masatoki T., Kema G.H.J. 2007. Electrophoretic and cytological karyotyping of the foliar wheat pathogen Mycosphaerella graminicola reveals many chromosomes with a large size range. Mycologia 99 (6): 804-812.

The authors thank the Joint Genome Institute (JGI) and the Stanford Human Genome Center for expert genome sequencing, and the members of the International Mycosphaerella Genomics consortium for discussions.

Sarrah Ben M’Barek is sponsored by an UNESCO L’ Oreal fellowship



Functional complementation in Saccharomyces cerevisiae: which promoter to use?

Libera Lo Presti1, Lorenzo Cerutti2, Michel Monod3, Philippe Hauser1

1Institute of Microbiology, Centre Hospitalier Universitaire Vaudois and University of Lausanne, Lausanne, Switzerland, 2Swiss Institute of Bioinformatics, Lausanne, Switzerland, 3Service of Dermatology, Centre Hospitalier Universitaire Vaudois and University of Lausanne, Lausanne, Switzerland

Introduction: Functional complementation of deletion mutants of Saccharomyces cerevisiae by expression of a gene on a plasmid is a common procedure for a number of genetic analyses. We tried to determine the best approach for successful complementation with heterologous genes.

Methods: We attempted complementation of four S. cerevisiae deletion mutants of essential genes involved in various processes with the homologous S. cerevisiae gene.

Results: Two mutants were rescued by expression under the control of constitutive S. cerevisiae  promoters commonly used for complementation, whereas the other two could be rescued only using the natural promoter. However, the two latter mutants could be complemented by their orthologs from Candida glabrata under the control of the constitutive S. cerevisiae  promoters.

Discussion: The use of constitutive S. cerevisiae promoter can be deleterious for homologous but not for heterologous complementation in S. cerevisiae. Consequently, the use of the homologous gene is useless to set up the conditions for heterologous complementation. An algorithm for successful heterologous complementation in S. cerevisiae is proposed.



Mycosphaerella graminicola sequencing heads towards the first finished genome of a filamentous plant pathogenic fungus

Theo A.J. Van der Lee1, Larry D. Dunkle2, Alice C.L. Churchill3, Jean Carlier4, Andy James5, Manoel T. Sousa Jr6, Pedro Crous7, Nicolas Roux8, Alexander Wittenberg1, Erika Erika Lindquist9, Igor Grigoriev9, Jim Bristow9, Jane Grimwood9, Stephen B. Goodwin2, Gerrit H.J. Kema1

1Plant Research International B.V, Wageningen, Netherlands, 2USDA-ARS Purdue University, West Lafayette, IN, United States, 33Department of Plant Pathology Cornell University, Ithaca, NY, United States, 4BGPI, CIRAD, Montpellier, France, 5CICY, Merida, Mexico, 6EMBRAPA Genetic Resources & Biotechnology, Brasília, Brazil, 7Fungal Biodiversity Centre, Utrecht, Netherlands, 8Bioversity, Montpellier, France, 99DOE-Joint Genome Institute, Walnut Creek, CA, United States

Mycosphaerella is one of the largest genera of plant pathogenic fungi with more than 1,000 named species, many of which are important pathogens causing leaf spotting diseases in a wide variety of crops including cereals, citrus, banana, eucalypts, soft fruits, and horticultural crops. An international project was initiated to sequence the genomes of M. graminicola and M. fijiensis, two of the most economically important pathogens of wheat and banana, respectively, along with 40,000 ESTs from M. fijiensis and the related maize pathogen Cercospora zeae-maydis, through the Community Sequencing Program sponsored by the U.S. DOE-Joint Genome Institute. The 9x M. graminicola sequencing is complete and was made public November 1, 2006 following automated and manual annotation. Due to the very good assembly statistics as well as a >2000-marker DArT linkage map that was aligned to the genome, JGI decided to finish the M. graminicola genome at the Stanford Human Genome Center. The majority of chromosomes have been sequenced completely including both telomeres. These data indicate that M. graminicola has both the largest chromosome number and the smallest chromosome sizes recorded among filamentous ascomycetes. Detailed analyses of progeny isolates showed the plasticity of the M. graminicola genome, as a significant number of chromosomes can be missed and potentially contain redundant information for pathogenicity and mating. The repetitive content of the individual chromosomes is disproportionally larger on the <2Mb chromosomes indicating that at least some may be considered supernumerary. Major factors for pathogenicity, however, are located on the larger chromosomes and mostly towards the telomeres. More than 1200 genes have been annotated manually and indicate that this pathogen contains smaller gene families compared to other well-studied filamentous plant pathogenic fungi, which may reflect its incognito life style. The M. fijiensis EST sequencing has resulted in more than 30,000 ESTs and the genome sequencing resulted in an approximately 70Mb genome size that contains a substantial amount of repetitions. Interestingly, the genome sequence showed a double CG-peak that is absent in the M. graminicola genome and appears to be specific for Mycosphaerella pathogens of banana. The current status of both sequencing projects will be discussed.



Functional gene analysis of Pneumocystis carinii by complementation of budding and fission yeast mutants

Philippe Hauser1, Libera Lo Presti1, Moira Cockell1, Lorenzo Cerutti2, Viesturs Simanis3

1Institute of Microbiology, Centre Hospitalier Universitaire Vaudois and University of Lausanne, Lausanne, Switzerland, 2Swiss Institute of Bioinformatics, Lausanne, Switzerland, 3Cell Cycle Control Laboratory, Swiss Institute for Experimental Cancer Research (ISREC), and School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne (EPFL), Epalinges, Switzerland

Pneumocystis jirovecii is a major cause of pneumonia in HIV infected patients. The only drugs available to manage infection target the folic acid biosynthetic pathway and selective pressure is giving rise to drug resistance. Development of new therapeutic agents is complicated by the fact that the organism cannot be cultured. The genome of the closely related species P. carinii has recently been sequenced. Approximately 4000 ORFs have been identified and ESTs corresponding to almost 1800 of them have been cloned. The fission yeast Schizosaccharomyces pombe is the closest relative of Pneumocystis that is amenable to classical and molecular analysis. To improve our understanding of Pneumocystis biology we use functional complementation of yeast mutants to study the function of P. carinii genes. We have focused upon genes for which there are sequence homologues in yeasts but not in higher eukaryotes, which may be useful drug targets. Our strategy is to transform a diploid heterozygous for a null mutation of the gene with a plasmid that expresses the P. carinii homologue. After meiosis, spores are germinated and colonies are analysed to determine whether cells carrying the null mutation can be rescued by the plasmid. We will present data for genes from S. pombe and Saccharomyces cerevisiae.



Heterologous expression of two novel zinc transporters of the ectomycorrhizal basidiomycete Suillus luteus (L.:Fr.)

Joske Ruytinx, Marc Lambaerts, Jaco Vangronsveld, Jan Colpaert

Centre for Environmental Sciences, Hasselt University, Diepenbeek, Belgium

Zinc is an essential micronutrient for all organisms. It is required for a wide range of cellular processes but becomes cytotoxic at elevated concentrations. Elevated concentrations of zinc in the environment may cause a selective pressure on exposed biota and can lead to the evolution of tolerant ecotypes. Zinc tolerant populations of the ectomycorrhizal basidiomycete Suillus luteus (L.:Fr.) have evolved in severely contaminated soils and have been shown to protect their host plant from zinc toxicity. Although the exact mechanism of zinc tolerance in S. luteus remains unclear, this trait is believed to be due to the adaptations of mechanisms involved in the general homeostasis of this essential element.

Zinc homeostasis in eukaryote cells is tightly controlled by transporters of different families: zinc-iron regulated protein (ZIP) family, cation diffusion facilitator (CDF) family, We isolated two S. luteus genes encoding transporters of the CDF-family by genome walking. Heterologous expression in yeast of the respective cDNA’s caused a slightly increased zinc tolerance and rescued the zinc sensitive zrt1 mutant. Both CDF-transporters seem to be zinc specific since Cd, Ni, Co and Mn sensitive strains could not be rescued by their expression. The increased zinc tolerance, zinc specificity and rescue of the zinc sensitive mutant indicate a role for the transporters in the zinc homeostasis of Suillus luteus. Further analysis (localisation, regulation of expression) of the transporters in the basidiomycete itself must reveal their involvement in the adaptive zinc tolerance.



Analysis of whole genome evolution of Aspergillus oryzae using DNA microarray

Kazuhiro Iwashita, Yasuhide Kobayashi, Kazutoshi Sakamoto, Osamu Yamada, Shigeaki Mikami

National research institute of brewing, Japan, Higashihiroshima, Hirosima, Japan

Aspergillus oryzae has been used for the several foods and alcohol beverages industries, such as sake and soy sauce making, for supplying hydroritic enzymes and vitamins et. al.. In the history of these industries, various strains were isolated from wild and selected for efficient fermentation and improvement of the product quality. Then some strains were further improved by several mutation and selection procedure. The genome structure of these strains will affect their characteristics and important for such nice adaptation. Further more, genome wide analysis of A. oryzae strains will important to understand the nature of this species. However, only little is known about their whole genome evolution. Recently, whole genome sequence of A. oryzae RIB40 strain, which isolated from surface of broad bean, was published. Using this information, we prepared A. oryzae DNA microarray (AO DNAchip) for their genetic study. The 8 strains for sake making, 6 strains for soy sauce making, 5 strains for miso paste making, 3 strains for soy sauce and miso past making, and 12 strains from other source were randomly selected from our strain library and their genomes were examined by AO DNAchip. As the result, in all analysed strains, about 10-1,000 genes were absent and most of them are clustered in their genome. However, only little number of the gene duplications was found. Moreover, these strains were classified into 7 groups containing two major groups by their signal intensity of all ORFs. One major group contained most of sake and miso past related strains and another major group contained most of soy sauce related strains. Among each groups, the pattern of absent genes are similar. These results suggested that the loss of genes will one of important driven force for the genome evolution and nice adaptation of A. oryzae.



Exploring the transcriptome of the ectomycorrhizal fungus Laccaria bicolor using expressed sequence tags and whole genome oligoarrays

Annegret Kohler1, Benoit Hilselberger1, Frédéric Duchaussoy1, Marie-Pierre Oudot-Le Secq1, Erika Lindquist2, Peter Brokstein2, Igor Grigoriev2, Arnaud Couloux3, Patrick Wincker3, Martina Peter4, Christine Delaruelle1, Aurélie Deveau1, Gopi Podila5, Francis Martin1

1INRA UMR1136, INRA-Nancy Université, Interactions Arbres/Microorganismes, 54280 Champenoux, France, 2) US DOE Joint Genome Institute, Walnut Creek, CA 94598, United States, 3GENOSCOPE, Centre National de Séquençage, 91057 EVRY Cedex, France, 4Swiss Federal Research Institute WSL, 8903 Birmensdorf, Switzerland, 5Department of Biological Sciences, Huntsville, United States

In the genome sequence of Laccaria bicolor, the first ectomycorrhizal fungus sequenced so far, 20,614 protein-encoding genes were predicted. Using these gene models, we constructed a whole-genome expression array (NimbleGen) containing in duplicates eight independent, non-identical, 60-mer probes per whole gene model. This oligoarray was used to validate and correct the first generation of annotated gene models. Expression of nearly 80% of the predicted genes could be detected in either free-living mycelium, fruiting bodies or ectomycorrhizal root tips. Further, the gene expression in these different tissues was compared to identify tissue-specific expression pattern. For example, transcripts of several small secreted proteins and an ammonium transporter, which both could play a role in the establishment of the symbiosis, were highly up-regulated in mycorrhizal root tips. Our analysis of the Laccaria bicolor transcriptome was completed by the investigation of 38,901 expressed sequence tags (ESTs) generated from different cDNA libraries and 186k reads assembled into 10,598 contigs derived from a 454 sequencing run, which were used to identify previously not detected gene models and to evaluate the proportion and ways of alternative splicing occurring in Laccaria bicolor.



Plasticity and genotype by environment interaction as tools to select for growth rate trait different strains of Pleurotus ostreatus var. Florida (N001)

Francisco Santoyo, Jordi Castellón, Verónica Pérez-Lanes, Gumer Pérez, Antonio G. Pisabarro, Lucía Ramírez

Department of Agrarian Production, Public University of Navarre, 31006 Pamplona, Navarre, Spain

Plasticiy (P) and Genotype by Environment Interactions (GEI) reveal the performance of genotypes in different environments. We have analysed the influence of different temperatures (18 ºC, 25 ºC and 32ºC) on the growth rate of dikaryons growing on Petri dishes. Dikaryons were arranged in families formed obtained after the mating of compatible full-sibs monokaryons. These monokaryons constitute part of the offspring meiotic-derived from Pleurotus ostreatus var. Florida (N001). Taking into account that the most prominent growth rate QTL has a R2 value of 20.7, and that it maps to chromosome VIII in the P. ostreatus map, three different dikaryon families were constructed: two formed by dikaryons carrying un-recombined chromosomes VIII (GrGr, fast growing; grgr, slow growing), and the other one formed by dikaryons heterozygous for chromosome VIII (fast and slow growing, Grgr). T-test analysis for the mean growth rate showed that no significant differences exist in the growth rate trait between GrGr and Grgr families at the three temperatures tested. Significant differences exist, however, between this group and that formed by dikaryons with the grgr genotype. Analysis of the results showed that the gene action governing the growth rate trait in full-sibs dikaryons is dominant with slight differences at different temperatures.

The effect of the environment on the growth rate trait (P) of the whole population as well as the identification of the genotypes with good performance in different environments (GEI) showed us that: high significant phenotypic plasticity (LOD score = 4,1) mapped to chromosome IV, just on in the same position where the second most important QTL controlling growth rate is located. This could be due to differences in allelic sensitivity of these loci to the temperatures. GEI was detected (with high significance) in several regions of the genome where QTLs were not previously identified. This could refute the gene regulation model that posits that specific loci may enhance (or suppress) expression of other genes in an environment-specific fashion.

These results will help us to select those genotypes with best performance in each environment to be used in future experiments dealing with pre-treatment culture conditions of straw aimed at bio-ethanol production



Whole genome sequencing of the fungal plant pathogens Botrytis cinerea and Sclerotinia sclerotiorum

Joelle Amselem2, Alain Billault3, Mathias Choquer8, Arnaud Couloux3, Christina Cuomo4, David DeCaprio4, Martin Dickman5, Sabine Fillinger1, Elisabeth Elisabeth1, James Galagan4, Corinne Giraud1, Chinnappa Kodira4, Linda Kohn6, Caroline Levis1, Evan Mauceli4, Cyril Pommier2, Jean-Marc Pradier1, Emmanuel Quevillon8, Jeffrey Rollins7, Béatrice Ségurens3, Adeline Simon1, Muriel Viaud1, Jean Weissenbach3, Patrick Wincker3, Marc-Henri Lebrun8

1UMR BIOGER, INRA AgroParisTech, Versailles, France, 2URGI, INRA, Evry, France, 3Génoscope - CNS, Evry, France, 4The Broad Institute of Genome Research, Cambridge, MA, United States, 5Institute for Plant Genomics and Biotech, College Station, United States, 6University of Toronto, Toronto, Mississauga, Canada, 7Plant Pathology, University of Florida, Gainesville, FL, United States, 8UMR 5240 CNRS-UCB-INSA-BCS, Lyon, France

Botrytis cinerea and Sclerotinia sclerotiorum are destructive fungal pathogens of many economically important crops provoking grey and white mould respectively. The Broad Institute has released the assembly of a 4-5 x genomic sequence from B. cinerea strain B05-10 (TMRI/Syngenta) as well as the 7-8 x genomic sequence of S. sclerotiorum (Broad Institute). In parallel, the French national sequencing center (Genoscope) has sequenced the genomes of grapevine and of two of its pathogens: Stolbur phytoplas nd B. cinerea strain T4. A large collection of ESTs (> 80 000) have been obtained for both B. cinerea and S. sclerotiorum. Automatic gene prediction with ab initio and similarity softwares have been applied to the three genomes revealing 14.000 to 16.000 potential gene calls for both species. Comparative analyses of both B. cinerea strains and of B. cinerea vs. S. sclerotiorum has revealed a common set of 8400 gene calls, 1200 of which are absent from other fungal genomes. In addition both species present a high degree of sequence similarity (80 % average protein sequence identity among orthologues), and of synteny at the contig level. These two species significantly differ in their repetitive DNA content, since S. sclerotiorum has more repetitive elements (7.7 %) than B. cinerea (3 - 4 %). This difference is likely related to the expansion of some families of transposable elements (TEs) in S. sclerotiorum. TEs seem to have been subject to RIP in both species although at higher levels in B. cinerea. The comparison between B. cinerea and S. sclerotiorum offers an interesting opportunity to compare the genomes of two closely related necrotrophic plant pathogens helping to understand evolutionary trends that have shaped their genomes. This comparison has highlighted differences between B. cinerea and S. sclerotiorum in secondary metabolism gene clusters, transcription factors and mating type loci, while sets of secreted proteins and metabolic enzymes are almost similar. These findings suggest that both species have evolved different strategies to behave as non specialized necrotrophic plant pathogens.



Sequencing of an emerging fungal pathogen, Penicillium marneffei, and its close saprophytic relative, Talaromyces stipitatus

Natalie Fedorova1, Paolo Amedeo1, Rama Maiti1, Vinita Joardar1, Jonathan Crabtree1, Jaideep Sundaram1, Elisabet Caler1, Matthew Fisher2, Alex Anrianopoulos3, William Nierman1

1The J. Craig Venter Institute, Rockville, MD, United States, 2Imperial College, London, United Kingdom, 3University of Melbourne, Victoria, Australia

Penicillium marneffei is an opportunistic human pathogenic fungus endemic to Southeast Asia where it typically infects patients with AIDS. In these regions, penicilliosis marneffei ranks as the third most common infection, following tuberculosis and cryptococcosis, in immunocompromised individuals. This fungal pathogen is unique among Penicillium and Aspergillus species as it is the only one with a known temperature-dependant dimorphic growth stage. The primary objective of this project was to obtain high quality complete genomic sequences of P. marneffei and its close sexual, non-pathogenic relative Talaromyces stipitatus for genome annotation and comparative analysis.

To this end, we employed a combination of Sanger ABI 3730xl sequencing of P. marneffei to 8X coverage and T. stipitatus to 5X coverage followed by autoclosure and finishing. Sequence data was assembled using Celera Assembler and the JCVI Eukaryotic Genome Control (EGC) pipeline was applied to assemblies (supercontigs) larger than 2 Kb. cDNA libraries were sequenced using 454 Biosciences GS-FLX. We used the PASA pipeline to leverage cDNA data, which resulted in improved gene models for ~10% of the genes. This was followed by transitive functional annotation using Sybil comparative software package and identification of secondary metabolism genes using SMURF.

The genome sequences of P. marneffei (28 Mb) and T. stipitatus (35 Mb) were assembled into 19 and 18 supercontigs containing 10,024 and 12,465 genes, respectively. Phylogenetic analysis based on 100 concatenated protein sequenced confirmed that these two species are indeed very closely related. They also differ considerably from another Penicillium species, P. chrysogenum, suggesting that the latter may represent a separate species. Despite phylogenetic proximity of P. marneffei and T. stipitatus, up to 40% of their genomes are represented by species-specific genes that have no orthologs in the other fungus. Furthermore, up to 50% of their genes are specific to the marneffei-stipitatus lineage including most genes involved in secondary metabolism. The results of this analysis are available at



Discovery of novel oomycete secreted proteins in a mycoparasite – plant pathogen interaction

Morten Nedergaard Grell, Lene Lange

Department of Biology, University of Copenhagen, Copenhagen, Denmark

Pythium oligandrum is an oomycete mycoparasite that holds a potential as biocontrol agent against various soil-borne, plant pathogenic oomycetes and fungi. A dual organism cDNA library was made from shake flask cultured hyphal biomass of the plant pathogenic Pythium ultimum, infected with and overgrown by Pythium oligandrum. The cDNA library was screened for secreted proteins, using the transposon assisted signal trapping technique (TAST, Becker et al., J. Microbial Methods, 2004, 57(1), 123-33). TAST allows for discovery of secreted proteins from organisms where limited or no sequence information is available. It also allows for discovery of novel enzymes where no activity assay is available. This represents the first example of using TAST for discovery of secreted pathogenesis related proteins and peptides of oomycete interactions. Details of the TAST screening technique (especially useful for studying host – pathogen interactions) will be presented. TAST screening led to the identification of a series of pathogenesis related proteins. The most prominent hits will be described, including a number of cellulases, a xylanase, a lipase, enzyme inhibitors, cellulose-binding elicitor lectins (CBELs) and elicitin-like proteins. Expression and characterization of selected proteins in yeast (Pichia pastoris) will be exemplified.



Identification of Fusarium graminearum proteins differentially expressed during mycotoxin synthesis

Linda Harris, Rebecca Taylor, Audrey Saparno, Barbara Blackwell, Valar Anoop, Steve Gleddie, Danielle Schneiderman, Nicholas Tinker

Eastern Cereal & Oilseed Research Centre, Agriculture & Agri-Food Canada, Ottawa, Ontario, Canada

Fusarium graminearum (Gibberella zeae [Schwein.] Petch) produces trichothecene mycotoxins early after plant contact to facilitate host invasion. We have attempted to identify genes, proteins, and pathways that may be induced under conditions that trigger mycotoxin synthesis. A shotgun proteomics approach was used to monitor protein expression changes under aseptic liquid culture conditions conducive to trichothecene production in the absence of contaminating plant proteins. The non-gel-based quantitative iTRAQ technology was used for proteomic profiling of three time course biological replicates. Statistical analysis of a filtered dataset of 435 proteins revealed 130 F. graminearum proteins that exhibited significant changes in expression. There was good agreement between up-regulated proteins identified by 2-D PAGE/tandem mass spectrometry and iTRAQ. Seventy-two proteins were significantly up-regulated relative to their level at the initial phase of the time course and this group included predicted secreted proteins, cellular transport proteins, homologs of other fungal virulence proteins, and many conserved hypothetical proteins. We are currently disrupting several genes encoding proteins identified in this study to explore function and contribute to our search for mechanisms of host invasion and novel antifungal targets.



Whole-genome gene expression and gene family analyses of Phycomyces blakesleeanus, a model photoresponsive zygomycete

Alan Kuo1, Asaf Salamov1, Alex Atkins1, Luis Corrochano2, Igor Grigoriev1

1DOE Joint Genome Institute, Walnut Creek, California, United States, 2Universidad de Sevilla, Sevilla, Spain

The photoresponsive zygomycete Phycomyces blakesleeanus is an experimentally tractable model system for elucidating the signalling pathways underlying photoregulation. Our recent sequencing, assembly, and annotation of the P. blakesleeanus genome allows us to complement classical molecular biological studies with genome-wide analyses of gene expression. We sequenced cDNAs from mycelia grown with and without light. The resulting 23410 ‘light’ ESTs and 24437 ‘dark’ ESTs were aligned with the genome and used to identify which of the 14792 genes could be described as transcriptionally active under either condition. We tagged 1538 genes (10%) as potentially light-specific, 1558 genes (11%) as potentially dark-specific, and 2431 genes (16%) as transcribed under both light and dark. In addition to its value as a model organism, P. blakesleeanus is one of only 2 zygomycetes with a sequenced genome, providing an opportunity to discover genes that are specific to or missing from zygomycetes, and gene families that are expanded or contracted in zygomycetes relative to the much better-sampled ascomycetes and basidiomycetes. We clustered P. blakesleeanus proteins with those of 13 other fungi and so far find 2 zygomycete-specific families of Zn-finger proteins, and a zygomycete-specific expansion of protein kinases. We also confirm the existence of a Phycomyces-specific family of F-box domain proteins.



Survey of mRNA isoforms in Fusarium verticillioides by ESTs: alternative splicing is part of the story

Daren Brown1, Robert Butchko1, Li-Jun Ma2

1USDA-ARS, Peoria, IL, United States, 2The Broad Institute of MIT, Cambridge, MA, United States

The filamentous fungus Fusarium verticillioides is a pathogen of maize and synthesizes a number of economically important toxins including fumonisins. Fumonisins cause a variety of animal diseases and have been shown to cause cancer in some animals. Contaminated maize and maize products lead to substantial losses to farmers each year. The transcriptional and posttranscriptional regulatory mechanisms controlling fungal pathogenesis and toxin production are poorly understood. Analysis of over 87,000 F. verticillioides ESTs identified a total of 578 genes with one or more mRNA isoforms and is equivalent to 6.3% of the genes represented. The most common isoform found correspond to alternatively spliced transcripts. Alternative splicing (AS) in higher eukaryotes play a critical role in expanding gene function by increasing protein diversity and by affecting mRNA stability. The different AS forms (ASF) include intron retention (73%), alternative donor or acceptor sites (25%) and exon skipping (2%). The percentage of genes with ASFs and the ratio of different ASFs is substantial different than that found in mammals and plants. Preliminary analysis of predicted open reading frames found that 28% of the AS events could generate altered proteins while 54% introduce frame shifts or stop codons and 16% were in 5’ or 3’ non-coding sequences. Transcript isoforms were also found that reflect multiple transcriptional initiation start as well as termination sites. And finally, we found sets of antisense RNA transcripts of which some have ASFs including retained introns and alternative donor and acceptor sites. Analysis of the Fusarium transcriptome indicate a complexity previously not described and the continued study of ASFs and their possible function will aid our understanding of fungal gene regulation.



A (semi) high-through-put pipeline for the functional characterization of putative sugar transporters

Marion Askin1, Douwe Doevendans1, Arthur Ram2, Jaap Visser3, Barbara Bakker4, Patricia vankuyk1

1Molecular Microbiology, IBL, Leiden University, Leiden, Netherlands, 2Kluyver Centre for Genomics of Industrial Fermentation, Delft, Netherlands, 3Fungal Genetics and Technology Consultancy, Wageningen, Netherlands, 4Molecular Cell Physiology, Vrije Universiteit Amsterdam, Amsterdam, Netherlands

In order to fully understand an organisms potential a better understanding of its components is required. Although thousands of Major Facilitator (MFS) Superfamily [TC# 2.A.1] members have been identified from genome sequence projects, a relatively small number have been functionally characterized. Sugar transport is an area of research traditionally not well studied, with the exceptions of Saccharomyces cerevisiae and Arabidopsis thaliana, in which the majority of sugar transporter encoding genes identified in their respective genomes have been functionally characterized. To date only 5 of the 34 S. cerevisiae sugar porters remain uncharacterized. All known eukaryotic sugar transporters belong to the Sugar Porter (SP) Family [TC#2.A.1.1.], the Oligosaccharide:H+ Symporter (OHS) Family [TC# 2.A.1.5.], the Fucose: H+ Symporter (FHS) Family [TC# 2.A.1.7.], and the Glycoside-Pentoside-Hexuronide (GPH):Cation Symporter Family [TC# 2.A.2], all of which are members of the MFS.

Genome comparisons show that MFS proteins constitute a large portion of the transporter capacity of eukaryotes. Due to the tendancy of MFS proteins of (distantly related) organisms to cluster based on organism, sequence comparison of uncharacterized MFS proteins to functionally characterized proteins is of limited assistance in indicating substrate specificity. Most MSF protein sequences from filamentous fungi, for example the Aspergilli, tend to form separate clusters from the (characterized) yeast transporters. Although members of the families within the MFS can be identified based on their aa sequence, individual members within each of these families can transport a variety of sugars. As yet the aa residues which confer transporter specificity remain a mystery.

With the goal of increasing our understanding of sugar transport we have developed a system for the rapid functional characterization of putative sugar transporter encoding genes. This includes a pipeline for the (relatively) rapid construction of cDNA expression constructs in a binary vector, and the use of a microtitre plate based system that allows us to screen our putative sugar transporter genes for the capacity to transport 13 different sugars and 2 polyols.



Regulatory network analysis of genome-wide transcription data from Aspergillus niger

Margarlita Salazar, Michael Nielsen, Jens Nielsen

Technical University of Denmark, Lyngby, Denmark

Carbon repression is a global regulatory mechanism in which the presence of glucose or other readily metabolized carbohydrates represses expression of genes involved in the utilization of less-favored carbon sources, respiration and gluconeogenesis. The carbon sources cause repression by affecting transcription of genes, a decrease in the translation rate of the proteins or its post-translational modification resulting in inactivation or degradation. In yeasts, the main effect of glucose takes place at the transcriptional level. The glucose repression signaling cascade in Saccharomyces cerevisiae has been widely studied, in contrast, not much is known in aspergilli. Mig1 is the major transcription factor responsible for carbon catabolite repression (CCR) in S. cerevisiae and its homologue, CreA is present in Aspergillus species. A central question is how glucose repression is triggered. Hexokinases which play an important role in yeast have not been identified as affecting glucose signaling cascade in any aspergilli. Even though one hexokinase and one glucokinase are present in A. niger. Therefore, it is not known if the transducing signal is linked to hexose phosphorylation and if a protein kinase, such as the Snf1 protein complex found in S. cerevisiae, is required for the expression of aspergillus glucose repressed genes. It is interesting to investigate whether CreA acts by itself or recruits other proteins to repress glucose-repressible promoters, like Mig1 interacting with Ssn6 and Tup1 and forming a complex. Comparative analysis of proteins involved in glucose sensing and repression pathways in S. cerevisiae, A. niger, A. nidulans and A. oryzae was performed. The analysis showed that several proteins involved in CCR in S. cerevisiae may also be present in these aspergilli. Specifically, the sensors Snf3 and Rgt2 seem to be present in A. niger, as well as homologues to the regulatory protein Grr1 and the phosphatase Glc7, involved in regulating the activity of the Snf1-complex. We grew an A. niger strain in batch fermentations using different carbon sources (glucose, xylose and glycerol). We applied DNA microarrays to conduct a transcription analysis using the Affymetrix platform with the objective of identifying global regulatory structures, mainly genes involved in CCR which are up or down regulated due to the presence of a repressing carbon source. DNA probe chips were designed by CMB staff and the analysis of the data obtained will be discussed.





Animal-Fungal Interactions, Secondary Metabolism, Regulation of Gene Expression



Control of Aspergillus flavus in corn using biological and natural materials

Fawzia Ibrahim1, Sobhy Mohsen2, Akila Hamza3, Gehan Moghazy4, Youssef Gibriel5

1Cairo University, Cairo, Egypt, 2Cairo University, Cairo, Egypt, 3Regional Lab for food and feed, Cairo, Egypt, 4Regional Lab for food and feed, Cairo, Egypt, 5Regional Lab for food and feed, Cairo, Egypt

It was found that Saccharomyces cerevisiae was not efficient in preventing the growth of Aspergillus flavus and aflatoxin production during storage in corn. Lactobacillus acidophilus was able to decrease the growth fungi and its production of aflatoxin in corn during storage. Bacillus subtilis was the strongest treatment among others which able to stop the growth fungi and the aflatoxin production when storage period reached 21 days. Addition of 0.05 ml per100 gram corn marjoram caused a reduction percentage of Aspergillus flavus count reached 62.5% at 21 days of storage. Raising the level of marjoram to 0.1 ml inhibited the growth Aspergillus flavus completely at 7 days and up to the end of storage also prevents aflatoxin production. This observation was noted clearly with other higher marjoram levels. The minimum inhibitory level of mint to be used against the development of Aspergillus flavus is 0.3 ml per 100 gram corn. This level was quite efficient not only to stop the growth of fungi but also to prevent aflatoxin production. The minimum inhibitory concentration of salt to prevent A. flavus growth in corn is 1 gram salt/100 gram corn. This concentration can be successfully used in the storage of corn up to 21 days. It will be also suitable to prevent aflatoxin production.



Insight into the molecular requirements for pathogenicity of Fusarium oxysporum f. sp. lycopersici through large-scale insertional mutagenesis

Caroline Michielse, Ringo van Wijk, Linda Reijnen, Ben Cornelissen, Martijn Rep

University of Amsterdam, Amsterdam, Netherlands

Fusarium oxysporum f. sp. lycopersici is a soil-born fungus that causes vascular wilt disease in tomato by penetrating the plant roots and colonizing the plant xylem vessels. In order to identify genes involved in pathogenicity 10,209 random insertion mutants were generated using T-DNA of Agrobacterium tumefaciens as an insertional mutagen.

All transformants were screened for loss of pathogenicity on tomato. This led to the identification of 20 non-pathogenic and 86 reduced-in-virulence mutants. The genomic regions flanking the T-DNA were isolated by TAIL-PCR. In total 129 potential pathogenicity genes were identified of which several known pathogenicity genes, such as class V chitin synthase, Zn(II)2Cys6 transcription factor FOW2, carbon catabolite derepressing protein kinase SNF1 and mannose-6-phosphate isomerase. Based on the putative function of the proteins identified, several general and specific processes seem to play a role in pathogenicity, e.g. certain metabolic pathways, peroxisome biogenesis and protein mannosylation. Complementation of three insertion mutants confirmed a role for peroxisomes and a probable cell wall mannosidase in pathogenicity.



A short chain dehydrogenase (Sch1) is required for development and toxin production in Stagonospora nodorum

Kar-Chun Tan1, Robert Trengove2, Garth Maker2, Richard Oliver1, Peter Solomon1

11Australian Centre for Necrotrophic Fungal Pathogens, SABC, Division of Health Sciences, Murdoch University, Murdoch, WA, Australia, 22School of Pharmacy, Division of Health Sciences, Murdoch University, Murdoch, WA, Australia

Two-dimensional gel electrophoresis (2DE) analysis of Stagonospora nodorum wild-type and signal transduction impaired mutants has led to the identification of a putative short-chain dehydrogenase (Sch1) that is subjected to positive regulation by Gα and MAPK signalling. Gene expression and transcriptional GFP fusion analyses of Sch1 indicates strong expression of during asexual development. Mutant strains of Stagonospora nodorum lacking Sch1 demonstrated poor growth on minimal media and exhibited a significant reduction in asexual sporulation on all growth media examined. Pathogenicity assays of the sch1 strains revealed the gene not to be required for lesion development although the mutants were unable sporulate. Detailed histological experiments demonstrated that Sch1 appears to be required for the differentiation of the sub-parietal layer of the asexual pycnidia. Non-targeted metabolomic analysis of the mutants identified the strong accumulation of a secondary metabolite whilst barely being detectable in the wild-type strains. Comparative analysis of the secondary metabolite and the postharvest mycotoxin alternariol confirmed the identity of the compound. This is the first report to confirm the presence of mycotoxins in S. nodorum and revolutionises our thinking on this disease.



GO annotation of the rice blast fungus, Magnaporthe grise

Shaowu Meng, Douglas Brown, Thomas K. Mitchell, Ralph A. Dean

Center for Integrated Fungal Research, North Carolina State University, Raleigh, NC, United States

Magnaporthe grisea is the causal agent of blast disease of rice and other agriculturally important cereals, which results in significant economic losses each year. The genome has been sequenced recently and an automatic annotation has been updated to the Version 5 release. However, a comprehensive manual curation remains to be performed. Gene ontology (GO) annotation has evolved into a reliable and rapid means of assigning functional information. Here, we report our GO annotation of M. grisea genome. Our GO annotation includes two parts. One is ortholog-based GO annotation, and the other is literature-based GO annotation. For the ortholog-based GO annotation, reciprocal best blastp hits were used to identify orthologs between predicted proteins of M. grisea and GO proteins from multiple organisms with published association to GO terms. After this, 29,126 GO terms, among which 1,911 were distinct, were assigned to 6,286 M. grisea proteins, which were manually reviewed. Eighty percent of the assignments were based on best blast hits with an e-value equal to zero. Then, reviewed data from different sources, such as NCBI RefSeq, NCBI CDD, curated secreted proteins, transcription factors and P450 proteins, were collected and checked for consistency between our GO annotations and these reviewed data. This resulted in 78% of GO annotations being assigned an evidence code ISS, 1% IC, 20% ND, and 1% IEA. Moreover, these manual reviews assigned functions to 2,732 hypothetical proteins, 125 predicted proteins, and 12 unknown proteins. The remaining 6,545 predicted proteins, where no GO orthologous proteins were found, were annotated using the three general GO terms, GO:0005575 (Cellular Component), GO:0003674 (Molecular Function), and GO:0008150 (Biological Process). In addition, 53 pathogenesis-related genes/proteins of M. grisea were manually annotated with existing GO terms as well as with newly derived plant-associated microbe (PAM) GO terms. Each of the 53 genes/proteins had been previously experimentally characterized, and had an ID in GenBank or EMBL-Bank. In summary, our GO annotation covers the entire genome of M. grisea, and each gene/protein is annotated by three GO categories of molecular function, cellular component and biological process. This GO annotation provides a foundation for further functional genomics analysis of M. grisea.



The role of the fungal f-box protein Frp1 in colonisation of tomato roots by Fusarium oxysporum f.sp. lycopersici

Wilfried Jonkers, Ben Cornelissen, Martijn Rep

University of Amsterdam, Amsterdam, Netherlands

Microbial pathogens have to break plant defence systems in order to colonize the plant. To investigate the genetic basis of this ability of pathogens, we use the interaction between the soil-borne fungus Fusarium oxysporum f.sp. lycopersici (Fol) and its host tomato as a model system. From an insertional mutagenesis screen, an F-box protein called Frp1 was found to be required for pathogenicity. It was shown that Frp1, like other F-box proteins, binds to Skp1, a subunit of E3 complexes. These complexes are involved in the ubiquitination of proteins recruited by F-box proteins.

We found that the ∆frp1 mutant has a defect in assimilation of certain carbon sources which might be related to the loss of pathogenicity. The mutant shows decreased growth on certain root exudate components, cell wall components and C2-carbon sources like ethanol and acetate, as well as fatty acids. The mutant is unable to induce expression of genes required for assimilation of these carbon sources: the Isocitrate lyase gene (ICL) encoding an enzyme of the glyoxylate cycle and the cell wall degrading enzyme genes polygalacturonase 1 (PG1), exo-polygalacturonase (PGX), and pectate lyase (PL). A gene knock out of ICL (∆icl) turned out to be not required for pathogenicity, but did resemble the phenotype of ∆frp1 strain on certain carbonn sources.

Microscopic studies with a wild type and ∆frp1 mutant strain transformed with GFP revealed the inability of the mutant to form a hyphal network around the roots of tomato seedlings.

We conclude that the F-box protein Frp1 is required for assimilation of the available nutrients from the plant and, therefore, for development of its pathogenic phase. Unravelling the biological and molecular role of Frp1 and its target proteins will provide more insight into the first stages of the infection process of pathogenic fungi.



Investigating the genetic basis of the corky root rot disease of tomato

Pierre-Henri Clergeot, Francisco Cabanero, Sophia Ekengren

Botaniska Institutionen - Stockholms Universitet, Stockholm, Sweden

Corky Root Rot (CRR) is a soil-borne disease caused by the filamentous ascomycete Pyrenochaeta lycopersici and affecting cultivated tomato (Solanum esculentum) grown under temperate conditions worldwide. This slow but debilitating disease is difficult to control by pesticides. Moreover, no efficient and sustainable resistance against CRR has been discovered so far, resulting in an increasing problem for tomato producers today. Besides the search for genetic resistance against CRR in wild tomato species, part of the work of our laboratory is focused on investigating the genetic basis of virulence of the pathogen, P. lycopersici. We aim at discovering the genes required for infection, as well as potential fungal effectors secreted during the interaction with tomato roots. We are currently using a forward genetic approach (insertional mutagenesis using plasmid or T-DNA transformation) to identify pathogenicity genes by random. We will also use reverse genetics (gene replacement or RNAi) to validate the role of candidate genes presumably important for infection. They will be chosen among a selection of genes encoding secreted proteins (using a yeast-trap system), or among genes expressed during infection (sequencing of an infection-specific cDNA library). Part of the problem faced by breeders to select for resistance against CRR arises from the difficulty to design a robust and reliable infection assay in the greenhouse. Therefore, we are also working on trying to improve the scoring of disease progression using cytology, both for the purpose of selecting non-pathogenic mutants of the fungus, but also for the purpose of selecting new tomato resistant lines. We will report on our progress in this project during the meeting.



Functional analysis of Botrytis cinerea NEP-like proteins

Eric Kalkman, Yaite Cuesta Arenas, Alexander Schouten, Peter Vredenbregt, Mirjam Dieho, Beatrice Uwumukiza, Ester Dekkers, Miriam Oses Ruiz, Jan van Kan

Wageningen University, Laboratory of Phytopathology, Wageningen, Netherlands

Necrosis and ethylene-inducing proteins (NEP) have been described in bacteria, oomycetes and fungi and have been proposed to act as phytotoxins in dicotyledons but not in monocotyledons. Their mode of action is unknown. Botrytis cinerea contains two NEP-like proteins, BcNEP1 and BcNEP2, which have been produced in Pichia pastoris. Infiltration of purified proteins into Nicotiana benthamiana leads to induction of ethylene in a dose-dependent manner. BcNEP2 is less active in inducing ethylene than BcNEP1. BcNEP1 and BcNEP2 contain a number of cysteines that may form disulfide bridges, as well as several potential posttranslational modification motifs. In order to study which amino acid residues are important for phytotoxic activity, site directed mutagenesis was performed and mutant proteins were expressed transiently in N. benthamiana through Agrobacterium tumefaciens (ATTA). Also through this approach, GFP was coupled to the C-terminus of BcNEP1 protein. The fusion protein retained phytotoxic activity and will be used for localization studies.

This research was funded by the Dutch Technology Foundation STW, with partial support of the Dutch Horticultural Product Board (PT).



Gene expression analysis of the response of sensitive and tolerant strains of Heterobasidion spp to natural antibiotics: implications for Norway spruce infection

Nina-Alexandra Lehr1, Aleksandra Adomas2, Frederick Asiegbu3, Ruediger Hampp1, Mika Tarkka4

1University of Tuebingen, Faculty of Biology, Institue of Botany, Tuebingen, Germany, 2Yale University, Department of Ecology and Evolutionary Biology, New Haven, United States, 3University of Helsinki, Faculty of Agriculture and Forestry, Department of Forest Ecology, Helsinki, Finland, 4UFZ, Helmholtz-Centre for Environmental Research, Department of Soil Ecology, Halle, Germany

The establishment of mycorrhizal symbiosis is positively influenced by certain bacterial isolates, called Mycorrhiza Helper Bacteria (MHB). Certain MHB can be used not only to promote mycorrhization but also to prevent attacks by phytopathogens [1] . Such interaction exists between Streptomyces sp. AcH 505, symbiotic Amanita muscaria, pathogenic Heterobasidion spp. and Norway spruce [2, 3]. The MHB Streptomyces sp. AcH 505 produces two substances that play a positive role in mycorrhiza establishment: the secondary metabolite auxofuran that promotes mycelial growth [4] and volatiles that facilitate plant colonisation [3]. However, as the bacterium promotes the growth of some fungal strains in co-culture, other strains are inhibited. This is due to the antifungal agent WS-5995 B [4]. We have studied the specificity of AcH 505-fungus interaction with the conifer pathogen Heterobasidion spp. Whereas most tested Heterobasidion strains were inhibited by the bacterium and 25 µM WS-5995 B, the F-type (Heterobasidion abietinum 331) was neither suppressed by 25 µM WS 5995 B nor in dual culture with AcH 505 [3]. In order to investigate the reasons behind the sensitivity of H.annosum to WS 5995-B, macroarray and qRT PCR analyses were performed. WS 5995-B induces the expression of genes related to detoxification and cell stress in H.annosum (glutathione S-transferase, mono-oxygenase, cyclophilin, cytochrome P450). To investigate the differences in responses between the sensitive H.annosum and the tolerant H.abietinum, comparative qRT PCR analyses of the detoxification and environmental stress related genes in H.abietinum are performed.

1Barea JM et al, 1998. Appl Environ Microbiol. 64:2304-2307, 2Schrey et al., 2005. New Phytol 168: 205-216, 3Lehr et al., 2007. New Phytol 174: 829-903, 4Riedlinger et al., 2006. Appl Environ Microbiol. 72:3550-3557



Antifungal proteins inhibit conidial anastomosis fusion and disrupt calcium signalling in Neurospora crassa

Peter Marris1, Anita K. Snyder2, Natalie Rolph1, M Gabriela Roca M.1, Mohammad Roghanian1, Patrick C. Hickey3, Dilip M. Shah2, Nick D. Read1

1University of Edinburgh, Edinburgh, Scotland, United Kingdom, 2Donald Danforth Plant Science Center, St. Louis, MO, United States, 3Lux Biotechnology Ltd, Edinburgh, Scotland, United Kingdom

The plant defensins MsDef1, MtDef2, MtDef4, RsAFP2 and the virally-encoded protein KP4 inhibited mycelial growth of Neurospora crassa from conidia with varying degrees of potency. All of these antifungal proteins also inhibited conidial anastomosis fusion between germlings and perturbed germ tube morphogenesis. These effects were similar to those observed when exogenous calcium was removed. Previous work has indicated that Ca2+ signalling may play a role in the mode of antifungal action of plant defensins and KP4, but that they have different cellular targets. We therefore investigated this by directly measuring cytosolic free Ca2+ ([Ca2+]c) in living conidial germlings of N. crassa using transgenic aequorin. The plant defensins, but not KP4, were each found to uniquely alter the Ca2+ signatures generated in response to mechanical perturbation and hypo-osmotic shock. Together, these results support the view that all of the antifungal proteins studied here have different modes of action, perturb germ tube morphogenesis, inhibit hyphal fusion at early stages of colony stablishment, and disrupt Ca2+ signalling and homeostasis.



Functional study of the Mps1 MAP Kinase pathway in the rice blast fungus Magnaporthe grisea

Cemile Ant, Phillipe Peret, Rolland Beffa, Marc-Henri LeBrun

Bayer Crop Science, Lyon, France

The M. grisea MAP Kinase Mps1 is orthologous to the yeast Slt2 kinase involved in the cell wall repair signaling pathway. This pathway controls the induction of the repair and biosynthesis of the cell wall in response to physical or chemical damages, stresses or developmental processes. In M. grisea this pathway is required for appressorium mediated penetration of the fungus into its host plant. This is reflected by the phenotype of the mps1 deletion mutant that is non pathogenic on rice and barley as a consequence of its inability to penetrate into its host plants (Xu et al. 1998 PNAS 95:12713–12718). These results suggest that the repair of the cell wall is essential for appressorium mediated penetration. This mutant has also developmental defects such as an abnormal mycelial growth rescued by high sorbitol and a very low sporulation rate. In Saccharomyces cerevisiae, the MAP kinase Slt2 activates the transcription factor Rlm1 and the SBF complex composed of the two transcription factors, Swi4 and Swi6. This last complex controls the expression of genes involved either in cell cycle control (G1/M transition) or in the maintenance and repair of the cell wall. M. grisea genes orthologous to Rlm1, Swi4 and Swi6 have been identified in the genome of M. grisea. The MgMPS1 deletion mutant was obtained in the P1.2 M. grisea background. The phenotypic analysis of mps1 mutants showed that it has the same phenotypic characteristics as those already described: (Xu et al. 1998 PNAS 95:12713–12718) abnormal mycelial growth rescued by high sorbitol, absence of sporulation, lack of pathogenicity on intact barley leaves, differentiation of non functional appressoria unable to penetrate into intact plant leaves.


Lichens from the Razavi Khorasan Province, Iran

Mahroo Haji Moniri1, Harrie J. M. Sipman2, Solmaz Jalili Mirasgari3, Nafiseh Noferesti3, Mahboobeh Tavakoli3, Mohammad Farsi4, Ali Gangali4

1Biology Department, Faculty of Science, Islamic Azad University, Mashad, Islamic Republic of Iran, 2Botanischer Garten und Botanisches Museum, Freie Universität Berlin, Berlin, Germany, 3M.S student, Faculty of Science, Islamic Azad University, Mashad, Islamic Republic of Iran, 4Research Institute for Plant Science, Ferdowsi University, Mashad, Islamic Republic of Iran

The lichen flora of the Khorasan Province (NE Iran), was investigated by the first author during 2002-2003. The Razavi Khorasan Province with an area of c. 127,432 Km2, is located in the Irano-Touranian region and between 33˚ 30́- 37˚ 41́ N and 56˚19́- 61˚18́ E. The area has semi desert climate with most altitude 1800 m from the sea level and annual precipitation 260 mm. The study was carried out in the two different parts with a total area of ca. 5,5 Km2 of the Binaloud mount slopes in the northwest of the Province. Morphological and anatomical identification of lichen has been undertaken using standard reagents and microscopical techniques. A total of 62 species belong to 28 genera are reported for the area. According to the lichen checklist of Iran (Sohrabi & Sipman, 2007) 12 of them are new to the country and 25 species are new to the Razavi Khorasan Province.


Evidence of siRNAs specific to three classes of retrotransposon elements in Phytophthora infestans

Ramesh Vetukuri, Christina Dixelius, Eugene Savenkov

Swedish University of Agricultural Sciences, Uppsala, Sweden

Eukaryotic genomes in general have repetitive DNA sequences, mainly derived from transposable elements. The genome size of Phytophthora infestans, P. ramorum and P. sojae are estimated to be 237 Mb, 65 Mb and 95 Mb, respectively. The large genome size of P. infestans is attributed to the presence of large number of mobile elements and repetitive sequences. There are three major classes of retroelements described in P. infestans - long terminal repeats (LTR), non-LTR (LINEs) and short interspersed elements (SINEs). Most eukaryotes control the frequency of transposition of retroelements by employing siRNAs. The purpose of our study is to see whether retroelements are controlled by siRNAs in P. infestans as seen in other eukaryote organisms. We adopted a strategy, where upon we used retrotransposons (SINEs, LTR and LINEs) to our advantage as a bait to fish out siRNAs.

To this end we cloned the most abundant members of the above mentioned three classes of mobile elements. The obtained clones were used as probes on northern blots for detecting siRNAs from the low molecular weight RNA fraction extracted from different strains of in P. infestans available in Sweden cultured under different growth conditions.

So far we have looked for siRNAs against SINE elements on northern blots using the probes described above and our data indicate significant accumulation of siRNAs specific to two SINE elements. The size of siRNAs falls within the range of 20-30 nucleotides. The exact size will be determined by both sequencing and northern blots. This confirms the fact that there are indeed siRNAs employed against transposable elements in P. infestans. We are in the process of finding siRNAs against other transposable elements under different growth conditions and their size determination.



Sensing and responding to plant surface contact by Ustilago maydis

Artemio Mendoza-Mendoza1, Patrick Berndt1, Daniel Lanver1, Armin Djamei1, Uwe Linne2, Miroslav Vranes1, Jörg Kämper1, Regine Kahmann1

1Max Planck Institute for terrestrial Microbiology, Marburg Lahn/ Hesse, Germany, 2Department of Biochemistry, Philipps University Marburg, Marburg Lahn/ Hesse, Germany

After contact with maize leaves the smut fungus Ustilago maydis switches to filamentous growth and develops appressoria. These structures are non-melanized and allow direct penetration of the plant cuticle in a process that is aided by lytic enzymes. We have identified the plant cues required for in vitro filamentation and appressorium development in U. maydis. These studies were aided by microarray-based identification of an appressorium specific gene from fungal material grown on the plant surface. We found that specific cutin monomers and hydrophobicity independently stimulated filament formation in U. maydis, but both were required for the induction of appresoria. To investigate the possibility that U. maydis is generating the second signal required for appressorium formation by secreting cutinases we have molecularly analysed the four putative cutinases encoding genes. One of these genes was up-regulated during fungal growth on the plant surface. Deletion of this gene reduced virulence of the U. maydis. Currently we are analyzing whether attenuated virulence of this mutant is due to a reduction in appressoria and investigate the possibility of redundant functions of the other cutinases. We have also shown that hydrophobicity sensing - and as consequence appressorium formation – depends on a transmembrane protein related to Sho1p from S. cerevisiae as well as on the presence of a second sensory protein with presumably redundant function.



Live cell imaging of migrating nuclei in the arbuscular mycorrhizal fungus Glomus intraradices

Stefanie Kiebart, Andreas Brachmann

Ludwig-Maximilians-Universität, München, Germany

For over 400 Mio years fungi of the phylum Glomeromycota has been forming a mutualistic symbiosis, the so-called arbuscular mycorrhiza (AM), with 80% of land plants. AM fungi are obligate biotrophs that complete their life cycle only in symbiosis with a host plant, to which they provide water and minerals. In spite of the importance, little is known about their genetic and cellular organization. No sexual stages have been observed during the life cycle of these coenocytic fungi. Plants are infected by hyphae that germinate from spores. This is the only host-independent stage (presymbiotic phase). Spores can contain up to a few hundred nuclei that, during germination, move out of the spore and are positioned along the aseptate hyphae. The point in the life cycle when nuclei multiply has not been observed, and it is not yet clear if all nuclei have the same genetic identity.

To elucidate the origin and fate of the many nuclei, we use the model system Glomus intraradices, and perform live cell imaging of nuclei migrating out of the spore into the germinating hyphae. Observation of nuclei is carried out in glass bottom dishes, which allow study of the movement in physically undisturbed hyphae. Nuclei are stained with the DNA-specific dye Hoechst 33342, whose quantitative uptake can be used for comparative determination of cell cycle phase. Thus far, quantitative image analyses show nuclei of similar DNA content, indicating absence of mitosis. Various modes of motion are displayed by the nuclei. Some are fixed to a certain position while others oscillate vigorously. A third mode of motion is a fast movement that enables some nuclei to overtake others and suggests a directional determination. These investigations provide insight into nuclear identity as well as triggers and mechanisms of nuclear motion in Glomus intraradices.


Population diversity and pathogenicity lifestyles in colletotrichum species

S. Sreenivasaprasad1, P. Talhinhas2, S. Muthumeenakshi1, J. Neves-Martins2, H. Oliveira2

1University of Warwick, Warwickshire, United Kingdom, 2Instituto Superior de Agronomia, Lisbon, Portugal

Colletotrichum species cause anthracnose diseases on a wide range of hosts including fruit, vegetable and cereal crops. Many of these pathogens exhibit considerable genotypic and phenotypic variability. We are using a range of molecular approaches to understand the population diversity and pathogenicity lifestyles in these pathogens. Based on ITS, tub2 and his4 sequences and PCR-based markers, C. acutatum isolates from olive within a single geographic location comprised five molecular groups A2-A6. A spatio-temporal survey over a 3 year period also enabled an understanding of the dynamics of these populations. Among the five groups, A2 was the dominant group mainly associated with intensive olive cultivation and heavy incidence of anthracnose. Isolates belonging to group A2 were also more aggressive than others, in pathogenicity assays. On a global scale, phylogenetic analysis of the ITS sequences of nearly 300 C. acutatum isolates from various hosts, revealed nine molecular groups A1 – A9 with varying bio-geographic association patterns. Diagnostic PCR based on ITS and tub2 sequences was also useful in understanding pathogen epidemiology, particularly with reference to olive anthracnose. C. acutatum exhibits different pathogenic strategies on various hosts, but the components regulating these processes are only beginning to be understood. We are using forward and reverse genetic approaches including a novel Colletotrichum-Arabidopsis model system to investigate the pathogenicity lifestyles. A collection of transformants generated by Agrobacterium T-DNA insertional mutagenesis is being tested for alternations in pathogenicity. Identification and characterisation of genes involved in these interactions is underway.


Molecular analysis of sclerotial mycoparasitism by Coniothyrium minitans

S. Sreenivasaprasad, S. Muthumeenakshi, C. Rogers, M.P. Challen, J.M. Whipps

University of Warwick, Warwickshire, United Kingdom

Coniothyrium minitans colonises and destroys the sclerotia of Sclerotinia sclerotiorum in nature exhibiting ecologically obligate mode of mycoparasitism as its spores remain dormant in soil and only grow actively in the presence of the sclerotia. The molecular mechanisms underlying this specialised host-parasite interaction are poorly defined. Suppression subtractive hybridisation was used to generate a cDNA library enriched for genes up-regulated during sclerotial mycoparasitism. Sequencing and bioinformatic analysis led to the identification of more than 250 unisequences and their assignment to various functional categories. Further, a set of mycoparasitism-deficient C. minitans mutants were identified from 4000 transformants generated by insertional mutagenesis. Molecular analysis of some of these mutants enabled the identification of putative genes responsible for the change in phenotype. Complementation and gene silencing technologies are being tested for functional analysis of some of the key genes. Comparative analysis identified genes associated with signalling (GPCRs), host degradation (glycosidases and peptidases), nutrient utilisation (MFS transporters), detoxification and stress response (ABC transporters and ROS scavengers) and DNA repair (PIF1 helicase) suggesting that C. minitans employs a number of key processes during host colonisation. Several of these genes including the PTH11-like GPCR, Secretin receptor-like GPCR and the TGF-beta signalling system are novel to fungal-fungal interactions. We have also identified the presence of an ETP gene cluster in C. minitans and the co-expression of these genes during sclerotial mycoparasitism. This work has led to a substantial increase in our molecular knowledge of this commercially important biocontrol agent and provides a platform for gene function analysis and comparative genomics in sclerotial mycoparasitism.



Siderophore biosynthesis in Epichloë festucae and its role in maintaining a mutualistic interaction with its host grass Lolium perenne

Melle Steringa1, Albert Koulman1, Michael Christensen1, Wayne Simpson1, Karl Fraser1, Richard Johnson1, Iain Lamont2, Linda Johnson1

1AgResearch Limited, Palmerston North, New Zealand, 2University of Otago, Dunedin, New Zealand

Epichloë and Neotyphodium fungal endophytes form mutualistic associations with cool-season grasses, considerably increasing the survival of their hosts during abiotic and biotic stress. Elimination of extracellular siderophore biosynthesis by disruption of the encoding nonribosomal peptide synthetase (NRPS) gene sidN from the obligate grass symbiont E. festucae has previously been shown to perturb symbiosis with its host Lolium perenne (perennial ryegrass). Plants inoculated with the sidN mutant were severely stunted and the fungus exhibited excessive hyphal branching. These phenotypes are likely to be due to a disturbance of iron homeostasis within the symbiotum as a result of loss of siderophore production. To further investigate the role of iron and siderophore biosynthesis in grass-endophyte interactions, other genes putatively involved in siderophore biosynthesis are functionally being characterised in this study.

The intracellular siderophore ferricrocin was previously identified from E. festucae mycelial extracts by LCMSMS, alongside a NRPS gene with high similarity to the ferricrocin NRPS genes NPS2 and sidC from Fusarium graminearum and Aspergillus nidulans respectively. Targeted gene replacement of this putative E. festucae sidC orthologue eliminated ferricrocin biosynthesis. However, plants inoculated with the sidC mutant were not stunted and hyphae in planta did not show excessive branching under light microscopy, showing that ferricrocin is less critical than the extracellular siderophore in maintaining symbiosis. Currently, experiments are under way to determine whether absence of ferricrocin has more subtle effects on grass-endophyte interactions. Studies include Transmission Electron Microscopy (TEM) to investigate hyphal ultrastructure of the sidC mutant in planta, and in vitro growth assays to explore the potential involvement of ferricrocin in resistance to oxidative stress. A double targeted gene replacement of both sidC and sidN is currently being created to determine how elimination of biosynthesis of both siderophores affects symbiosis. In addition, a targeted gene deletion of a putative E. festucae urbs1 orthologue from Ustilago maydis, encoding a GATA-type transcription factor that regulates siderophore biosynthesis, will further elucidate the role of siderophore biosynthesis and iron in maintaining these mutualistic grass-endophyte symbioses.


Mannitol: a main soluble carbon store in Botrytis cinerea

Thierry Dulermo1, Christine Rascle1, Richard Bligny1, Pascale Cotton2

1Bayer Cropscience, Lyon, France, 2Université Claude Bernard Lyon1, Lyon, France,

The wide range plant pathogen, Botrytis cinerea causes serious pre and post-harvey diseases. The disease cycle of the fungus is essentially based on production of ubiquitous conidia that land, attach to the plant surface and initiate penetration of the host. B. cinerea is a typical necrotroph inducing cell death through fungal toxins and an oxidative burst, and producing an efficient enzymatic degradation arsenal that allow plant tissue colonization and the release of nutrients. Then, to complete its life cycle, B. cinerea finally produces conidia on host plants. Thus, the dynamic nutritional challenges facing the pathogen through the infection cycle, and how the pathogen adapts are of considerable importance in our host pathogen study.

Exploration of the metabolic changes that occur during the pathogenic interaction of B. cinerea with sunflower cotyledons was performed by NMR spectroscopy. A rapid decrease in soluble sugars and amino acids was detected during the course of infection. Nutrients were essentially converted in a main carbon soluble compound, mannitol, that represented 65% of the total soluble carbon stores at the final stage of infection (sporulation of pathogen on host).

The metabolic pathway for mannitol biosynthesis and catabolism, well described in filamentous ascomycetes takes place through the mannitol cycle involving two pathways. The first is the direct reduction of fructose-6-phosphate to mannitol-1-phosphate. Mannitol biosynthesis occurs through the dephosphorylation of mannitol 1-phosphate via a mannitol-1-phosphate dehydrogenase (MPD). This last reaction was described as irreversible, consequently, mannitol degradation is supposed to occur through oxidation of mannitol in fructose via a reversible mannitol dehydrogenase (MTD).

Physiological, biochemical and molecular analysis were conducted in order to investigate the role of mannitol during B. cinerea development phases, stress response and infection process.

Whereas mannitol was accumulated in exponentially growing mycelium (in vitro and in planta) and spores, it was degraded during the early stages of germinating conidia and during the response to an osmotic shock. Transcriptional analyses were conducted to monitor mpd and mtd gene expression in order to evaluate the contribution of both pathways to mannitol metabolism. Q-PCR expression profiles revealed that if the expression of mpd was correlated to mannitol accumulation in developing spores, growing mycelium and infection, mpd was also majoritarily expressed during growth in the presence of mannitol as sole carbon source. Moreover, all tested conditions revealed a low mtd transcription level, except when mannitol degradation was correlated to an osmotic shock response, suggesting different transcriptional control networks for mtd and mpd pathways and their implication in different fungal cell responses .


Symbosis-expressed secreted proteins in the ectomycorrhizal basidiomycete Laccaria bicolor

Julien Gibon1, Veronica Pereda1, Balaji Rajishekar3, Dag Ahrén3, Yao-Cheng Li2, Annegret Kohler1, Marie-Pierre Oudot Le Secq1, Pierre Rouzé2, Anders Tunlid3, Francis Martin1

1INRA, Nancy, France, 2Ghent University, Ghent, Belgium, 3Lund University, Lund, Sweden

The ectomycorrhizal fungus Laccaria bicolor genome contains a large number of putative secreted proteins with unknown function. Of 2,931 proteins predicted to be secreted, 1,979 (67%) cannot be ascribed a function, and of these 82% (1,633) are specific for L. bicolor. Approx. 440 are cysteine-rich and have a size < 300 AA; several of these SSP being arranged in gene clusters. Genome-wide expression oligoarray profiling revealed that the expression of several SSP genes is induced in symbiotic tissues. Within the few symbiosis-specific transcripts (ectomycorrhizins) detected in both Populus and Douglas fir ectomycorrhizas, we found a 69 amino acid-SSP MISSP7 [Lacbi1:298595] and a 180 AA secreted protein with a CFEM domain [Lacbi1:332226]. The latter eight cysteine-containing domain was found in several fungal membrane proteins, such as Pth11 from M. grisea, and proposed to have important roles in fungal pathogenesis. In addition, a dozen of SSP showed a significant similarity to effector proteins and haustoria expressed secreted proteins (HESP) of Magnaporthe oryzea, Melampsora lini, and Uromyces fabae involved in pathogenesis. Immunolocalisation of MISSP7 was performed in symbiotic tissues and free living mycelium using confocal microscopy. Although MISSP7 was detected in the hyphal mantle layers ensheating the root tips, the protein mainly accumulated in the finger-like, labyrinthine branch hyphal system (Hartig net) which provides a very large area of contact between cells of the two symbionts. It accumulated in the cytosol and cell wall of the fungal cells. Although several SSP are likely involved in hyphal aggregation during fruit body and symbiotic mantle differentiation, it is tempting to speculate that this rich assortment of mycorrhiza-induced SSP may interact with or manipulate host plants during infection and subsequent symbiosis as suggested for rust fungi.


The yeast signal sequence trap identifies secreted proteins of the hemibiotrophic corn pathogen Colletotrichum graminicola

Jorrit-Jan Krijger, Ralf Horbach, Michael Behr, Holger B. Deising, Stefan G.R. Wirsel

Martin-Luther-Universität Halle, Institut für Agrar- und Ernährungswissenschaften, Halle, Germany

The hemibiotroph Colletotrichum graminicola is the causal agent of stem rot and leaf anthracnose on its host Zea mays. Following germination and penetration of epidermal cells, the fungus enters a short biotrophic phase that is followed by a destructive necrotrophic phase resulting in the production of conidia. During both phases, secreted fungal proteins are believed to determine the progress of pathogenesis. To identify genes encoding such proteins, we employed the Yeast Secretion Signal Trap (YSST) that takes advantage of a 5´-truncated invertase gene on a vector to identify cDNAs carrying a start codon that is followed by a signal sequence. A YSST-cDNA-library was constructed from RNA extracted from in vitro-grown mycelium that was induced with a corn leaf-extract. Of the 94 identified unigenes, 45 showed significant similarities to genes with a reported function, 24 sequences were similar to genes annotated by fungal genome projects and 27 sequences showed no similarity to the databases. Macroarray hybridisation confirmed that the vast majority of genes identified by the YSST screen are expressed in planta. Besides a set of genes that were constantly expressed, a larger set showed peaks of transcript abundances at certain times during pathogenesis. Another set exhibited two peaks and a minimum during the transition phase between biotrophy and necrotrophy. For each set, expression patterns from several genes were confirmed by Real-time RT-PCR. Equivalent transcript analyses for in vitro grown cultures revealed that several of the genes identified by the YSST screen were induced by addition of corn leaf extracts indicating that host-derived components have mimicked the host milieu when added to the culture medium. This work establishes a set of candidate genes that will be further on characterized by reverse genetic approaches.



Nitric oxide metabolism in Botrytis cinerea

Juan Luis Turrion-Gomez, Arturo Perez Eslava, Ernesto Perez Benito

Spaniard-Lusitanian Center for Agricultural Research (CIALE), University of Salamanca, Salamanca, Spain

Nitric Oxide (NO) is a gaseous signalling molecule known to play crucial roles in the establishment of plant-pathogen interactions. NO is toxic for microorganisms and essential for the activation of the plant hypersensitive response (HR). Although plants produce NO to reduce the pathogen invasion, these microorganisms have developed mechanisms to counteract the effects of NO, such as the production of flavohemoglobins. Botrytis cinerea has a single flavohemoglobin coding gene (Bcfhg1) that is expressed through all the developmental stages of the fungus, except in resting spores, with maximal expression values during germination of spores and decaying afterwards. Exogenously added NO enhances the expression of Bcfhg1 about 5-fold in germinating spores, however to a much lower extent in actively growing and branching mycelium. In planta expression level of Bcfgh1 is maxima at 8 hours post inoculation (-hpi-) decaying by 3-fold between 12 and 120 hpi.

Expression of Bcfhg1 in a Saccharomyces cerevisiae flavohemoglobin deficient mutant confirmed that the B. cinerea flavohemoglobin metabolize NO very efficiently and rapidly, revealing a high affinity for NO. ΔBcfhg1 mutants were completely unable to metabolize exogenously added NO; however the mutation did not determine any obvious alteration in the fungus, neither during saprophytic growth nor during infection. It could be reasoned that a reduction of NO degradation in the mutant strains could raise the NO levels at the infection sites and enhance the activation of the plant HR; nevertheless, B. cinerea development and plant HR were not affected. Interestingly, induced NO production in the ΔBcfhg1 and wild type strain was observed in developmental stages in which the expression of Bcfhg1 declines. Taken together these observations indicate that although the B. cinerea flavohemoglobin encoded by Bcfhg1 might play an important function during early developmental stages, it certainly does not seem to be essential for pathogenicity of this necrotroph. Hence during the spread of the lesion, the fungus not only reduces the NO degradation but increases the NO concentration to allow the enhancement of the HR. Thus, it is suggested that this necrotrophic pathogen exploits the defence system of the plant.



Role of the small GTPase Rheb in the development control of the phytopathogenic fungi Botrytis cinerea and Magnaporthe grisea

Heber Gamboa Melendez, Michel Droux, Geraldine Mey

Laboratoire de Génomique Fonctionnelle des Champignons Pathogènes des Plantes, UMR 5240 CNRS / UCBL / INSA /Bayer CropScience, Lyon, France

The ubiquitous monomeric GTPases constitute a large superfamily comprising more than 100 proteins involved in various cellular processes. The Ras family is the sole one which appeared conserved among animals and lower eukaryotes and absent in plants which, raised our interest in studying their role in the control of the development of phytopathogenic fungi. The GTPases Ras take part in essential cellular processes, which seem to vary according to the eukaryotic models. In particular, the Rheb proteins (Ras Homologue Enriched in Brain), which belong to the Ras family, were shown to be essential for the growth and the cell cycle in Drosophila melanogaster and Schizosaccharomyces pombe, but not in Saccharomyces cerevisiae and Aspergillus fumigatus. In addition, studies in lower eukaryotes suggested that Rheb contributes to the regulation of arginine and lysine uptake. This function could be ensured via the conserved signaling pathway depending on TOR: (1) in S. cerevisiae, TOR controls the synthesis of the general amino acid permease Gap1p and the degradation of the specific permeases Hip1p (histidine) and Tat2p (tryptophane) ; (2) in mammals, Rheb is the positive activator of TOR and the interaction between both proteins is released in amino acid starvation conditions. Thus Rheb might be involved in the control of the uptake of the plant metabolites and degradation products supporting the parasitic development of phytopathogenic fungi.

We performed a comparative analysis of the Rheb functions in phytopathogenic fungal models that develop different plant infection strategies: the necrotroph Botrytis cinerea and the hemibiotroph Magnaporthe grisea. The implication of the Rheb proteins in the control of amino acid uptake was appreciated using (1) complementation experiments of a mutant strain of S. cerevisiae, (2) analysis of amino acid uptake/metabolism by HPLC in B. cinerea and M. grisea mutant strains (3) quantification of the transcripts encoding amino acid transporters in these mutant strains. These results obtained will allow a better understanding of the mechanisms which control amino acid metabolism in pathogenic fungi.


RNAi silencing of the virulence gene ftf1 of Fusarium oxysporum causes a reduced pathogenicity

Jose Javier de Vega Bartol1, Bart P. H. J. Thomma2, Jose Maria Diaz-Minguez1

1Centro Hispano-Luso de Investigaciones Agrarias (CIALE), University of Salamanca, Salamanca, Spain, 2Laboratory of Phytopathology, Wageningen University, Wageningen, Netherlands

Fusarium oxysporum is a soil-born fungus that causes vascular wilt disease by penetrating the plant roots and colonizing the plant xylem vessels. We have isolated and described a new transcription factor (ftf1) that is likely a virulence factor, as its distribution is limited to highly virulent strains of several formae speciales of F. oxysporum, and its expression is drastically upregulated in planta. ftf1 is found in multiple copies, the number varying in different formae speciales (four copies in f.sp. phaseoli and up to nine in the sequenced strain of f.sp. lycopersici). This feature makes it very difficult to assess the role of ftf1 by standard knock-out methodology.

In order to check whether ftf1 is required for fully pathogenic F. oxysporum strains, an RNA interference (RNAi) vector was constructed to target expression of the virulence gene. Upon Agrobacterium tumefaciens-mediated transformation of this construct into F. oxysporum several transformants were obtained. We analyzed these transformants for a) growth in rich medium, b) virulence in infection essays conducted under greenhouse conditions, and c) ftf1 expression by means of quantitative RT-PCR. Preliminary results on tomato indicate that some transformants clearly showed reduced virulence in planta when compared to the wild type infection control, and a significative reduction of in planta expression of ftf1. We are currently investigating the effect of the silencing the ftf1 gene in strains of f.sp. phaseoli. The experimental confirmation of the power of gene silencing for abolishing the expression of multi-copy genes would make this tool very valuable for functional analysis in Fusarium.



Root infection and systemic, symptomless colonization of maize by Colletotrichum graminicola

Verónica M. García2, Michael R. Thon1, Serenella A Sukno1

1Universidad de Salamanca, Salamanca, Spain, 2Texas A&M University, College Station, Texas, United States

Anthracnose of maize is caused by the filamentous fungus, Colletotrichum graminicola, (Ces.) G. W. Wils. The disease can affect most plant tissues though the stalk rot and seedling blight forms of the disease are the most economically damaging. Previous reports indicate that C. graminicola can infect maize roots, though little is known about this important stage of the disease. Our hypothesis is that C. graminicola can infect roots and that such infections can lead to systemic infection of aerial plant tissues. We constructed a GFP-expressing strain of, C. graminicola, and used it to perform a detailed histological study of plant tissue colonization upon root infection. Structures produced by other root pathogenic fungi were observed, including runner hyphae, hyphopodia and microsclerotia. A mosaic pattern of infection resulted from specific epidermal and cortical cells becoming infected by intercellular hyphae while surrounding cells are uninfected. Interestingly, falcate conidia, normally restricted to acervuli, were also found filling epidermal cells and root hairs. We observed the development of several fungal structures that may serve as survival structures that enable the fungus to over-winter. The microsclerotia produced in culture were able to infect plant roots, demonstrating that the structures were viable and could serve as a source of inoculum. Twenty-eight percent of plants challenged with soil-borne inoculum became infected in above ground plant parts (stem and/or leaves) indicating that root infection can lead to asymptomatic systemic colonization of the plants. Many of the traits observed in C. graminicola have been previously reported in other root-pathogenic fungi, suggesting that these traits are evolutionally conserved in multiple fungal lineages. Continued study of tissue-specific infection processes by C. graminicola will help to reveal the molecular nature of tissue specificity, resistance and susceptibility.



Importance of the pH signalling transcription factor PacC in the infectiono process of Botrytis cinerea

Laetitia Muszkieta1, Vincent Girard2, Chrsitine Rascle2, Geneviève Billon-Grand1, Nathalie Poussereau1

1 Université Lyon I, Villeurbanne, France, 2CNRS, Villeurbanne, France, 3 Bayercropscience, Lyon, France

Botrytis cinerea has been described as one of the most devastating phytopathogenic fungi responsible for grey mould disease. This ascomycete is able to infect a wide range of crop species at any plant stage and at any plant structure (leaves, fruits, roots, flowers). The infection strategy of this necrotrophic fungi is based on the acidification of its ambient environment via the production of oxalic acid and on the secretion of a set of numerous Cell Wall Degrading Enzymes. The production of some of these enzymes has been shown to be under the transcriptional control of the ambient pH.

It has been established in fungi that the regulation of gene expression in response to changes in ambient pH is mediated by a conserved signal transduction pathway which has been well characterized by Penalva et al. in Aspergillus nidulans.

It involves six PAL proteins and the PacC transcription zinc finger transcription factor. Under neutral or alkaline conditions, the transcription factor PacC present in an inactive form in the cytoplasm of the cell is cleaved upon activation of the signalling pathway migrates to the nucleus where it activates alkaline genes and represses acidic genes. The orthologue of the PacC gene has been identified in B.cinerea and the deletion of the gene has been carried out. Characterization of the mutant has shown that PacC is implied in several essential functions such as growth rate, spore germination, oxalic acid production. Exploration of the secreted proteins revealed that the mutant presents proteins pattern modified. This approach revealed also that the production of several proteins is independent of PacC.

Through the characterization of the mutants, our work investigates the importance of the transcription factor PacC and the pH signalling in the infection process of this fungus.



A Botrytis cinerea gene differentially expressed in planta encodes a cyclin K

David Benito Pescador, Arturo Pérez Eslava, Ernesto Pérez Benito

Centro Hispano-Luso de Investigaciones Agrarias (CIALE) - University of Salamanca, Salamanca, Spain

Botrytis cinerea Pers. (teleomorph: Botrytinia fuckeliana (de Bary) Whetzel) is a filamentous fungus with a broad host range, causal agent of grey mould and responsible of important economic losses. In order to understand the molecular mechanisms involved in the infection process of B. cinerea, an experimental approach based on the analysis of differential gene expression during the plant/fungus interaction was applied. This analysis allowed us to detect different cDNA fragments derived from B. cinerea genes whose expression is induced in planta. The gene encoding one of these fragments, named Bde2, is being characterized in detail.

Using a cDNA fragment as a probe, Northern blot hybridization analysis showed that gene Bde2 is expressed only during late stages of the infection process (colonization and maceration of the infected tissue stages), and Southern analysis demonstrated it is a single copy gene.

The full-length genomic copy of Bde2 was cloned from a genomic library and its structure and sequence was determined. Sequence analysis and public databases searches revealed that it encoded a protein with significant homology to cyclin K. This gene, from the transcription cyclin family, may play a dual role in regulating Cdk9 and RNAPolimerase II activity.

To get deeper insights into the role of this new putative cyclin in the infection process of B. cinerea, a functional characterization approach based on the isolation and characterization of mutants specifically altered in gene Bde2 is in progress. After several gene replacement transformation experiments it has been impossible to obtain mutants lacking the Bde2 wild type allele. These observations might indicate that the Bde2 gene is essential for survival, making it impossible to generate mutants. The ability of this type of cyclin to rescue Saccharomyces cerevisiae from a conditional lethal mutation, which can show experimental evidences about the biological function of the Bde2 gene in B. cinerea, is being considered. Additionally, experiments to drive the overexpression of Bde2 and analyze possible alterations during saprophytic or pathogenic growth derived from it are being conducted.



Molecular bases of resistance to Qo-inhibitor fungicides in spanish isolates of the cucurbit pathogen Podosphaera fusca

Dolores Fernández-Ortuño1, Juan A. Torés1, Antonio de Vicente2, Alejandro Pérez-García2

1Estación Experimental La Mayora, Algarrobo-Costa, Málaga, Spain, 2Departamento de Microbiología, Universidad de Málaga, Málaga, Spain

Podosphaera fusca is the main causal agent of powdery mildew on cucurbits and one of the most important limiting factors for cucurbit production worldwide. Application of fungicides is presently the principal tool for managing powdery mildew in most cucurbit crops. Strobilurin fungicides, also known as Qo-inhibitors (QoI), have played an essential role in plant protection against powdery mildews since their introduction to the market in 1996. In a previous work, we have shown the current distribution of resistance to QoI fungicides in populations of P. fusca in south central Spain, evaluated by the traditional leaf disc assay, revealing worrying levels of resistance (32%). The aim of this study was to elucidate the mode of resistance to QoI fungicides in the Spanish populations of P. fusca as a first step to develop a DNA-based assay for rapid quantification of such resistance. Analysis of the Qo domains of cytochrome b in a collection of isolates revealed that none of the typical mutations conferring resistance to QoI in many phytopathogenic fungi, including the G143A and F129L substitutions, was present in the QoI-resistant isolates. Interestingly, sensitivity tests to a battery of respiration inhibitors revealed high levels of cross-resistance to all Qo-inhibitors tested but not to Qi-inhibitors, these features resembling those of a target-site-based resistance. Once the role of the Qo domains of cytochrome b in P. fusca QoI resistance has been analysed and it has been determined that no conserved modifications of the protein correlates with resistance, the hypothesis that a structural change in the mobile extrinsic domain of the Rieske-FeS protein (ISP), the other protein component of Qo site of cytochrome bc1 enzyme complex, could be responsible for resistance to QoI fungicides in P. fusca is being currently examined. To isolate the ISP encoding gene of P. fusca (RIP gene), we are carrying out a PCR approach using degenerate oligonucleotide primers based on conserved domains of the protein. A 750 bp fragment of this gene has been amplified and sequenced, revealing the predicted amino acid sequence of this PCR product a high homology with other ISP proteins. The sequence comparisons between QoI-sensitive and QoI-resistant isolates of P. fusca will help us to determine the presence of mutations in the ISP alleles that could explain the phenomenon of QoI resistance in the Spanish populations of P. fusca.


VeLB, a member of the velvet family, is a virulence factor in the vascular wilt fungus Fusarium oxysporum

Manuel Sánchez López-Berges, Concepción Hera, Antonio Di Pietro

University of Córdoba, Córdoba, Spain

Forward genetic screens are efficient tools to dissect complex biological processes such as fungal pathogenicity. A transposon tagging system was used to search for novel virulence genes in the vascular wilt fungus Fusarium oxysporum. A strain exhibiting reduced virulence on tomato plants carried a transposon insertion 80 bp upstream of the open reading frame FOXG_00016. The predicted gene product is a small protein with homology to Aspergillus nidulans VelB, a member of the velvet family involved in regulation of fungal development and secondary metabolism. Targeted knockout and functional complementation of the insertion mutant with the wild type velB allele confirmed its role in virulence of F. oxysporum. The velB strains exhibited developmental alterations such as reduced aerial mycelium and abnormally shaped microconidia. A search in the F. oxysporum genome database revealed the presence of two additional members of the velvet gene family, named veA and velC. These results suggest a novel role of velvet-type proteins in fungal virulence on plants.




Analysis of calcium dynamics in the plant pathogen Claviceps purpurea

Jörg Bormann, Paul Tudzynski

Westf. Wilhelms-Universität Münster, Institut für Botanik, Münster, Germany

The aim of our work is to reveal the molecular pathways involved in polarized growth of the phytopathogenic ascomycete Claviceps purpurea. The fungus penetrates the cuticle of stigmatic hairs, growths down the style and through the ovarian tissue. On one level with the micropyle the fungus leaves the pollen tube path and taps the vascular bundles. The fungus growths mainly intercellularly and therefore decomposes the middle lamella between the plant cells. This lamella predominantly consists of pectin which is in turn rich in calcium ions. On the other hand it is well known that the growing fungal hyphae possesses a tip high calcium gradient just as other tip growing cells like pollen tubes, axons and others do. One question we want to address is how this tip high calcium gradient is established. It is conceivable that the fungus follows the "calcium trail" that is set up by the degradation of pectin. For this purpose we have to postulate a calcium channel located in the tip region of the hyphae. We cloned a homologue of the yeast stretch activated, nonselective cation channel mid1 from our EST library and constructed a replacement vector. In Mid1 knockout mutants mycelial growth is significantly slower than in the wildtype. This growth defect can partially be complemented when growing on plates with a higher agar concentration which suggests that mechanical force plays a role in vegetative growth of C. purpurea.

In the past we generated various mutants that are impaired in polarized growth. Among them there is a knockout mutant of the small G-protein cdc42 which stops growing when reaching the ovarian tissue. It is of interest whether the actin cytoskeleton of this and other comparable defective mutants is battered in some way. To address this question we visualize the actin cytoskeleton in vivo. Therefore we designed a vector which contains an alcohol inducible promoter and the C-terminal f-actin binding domain of mouse talin coupled to YFP.

In order to get a global overview of calcium dynamics in growing hyphal cells of either the wildtype or several mutants, we want to ally FRET microscopy techniques using GFP based calcium sensors like Cameleon, forward as well as reverse genetic approaches for gene disruption and cytoskeleton labelling.


The role of NADPH-oxidase subunits in the biotrophic interaction of Claviceps purpurea and Secale cereale

Dagmar Buttermann, Sabine Giesbert, Timo Schürg, Sandra Scheele, Paul Tudzynski

Institute of Botany, WWU Münster, Münster, Germany

Reactive oxygen species (ROS) are of high importance in defence reactions against invading pathogens in many mammalian and plant systems, where the role of the superoxide generating NADPH-oxidase-complex within this oxidative burst is well established.

In fungi, the role of ROS is less well understood. Reactive oxygen molecules produced by phytopathogenic fungi induce oxidative stress on host organisms as they contribute to the ROS-status in planta but they also play a role in differentiation processes in fungal species.

In the genome of the phytopathogenic fungus Claviceps purpurea, an ecologically obligate biotroph on diverse monocotyledonous host plants, we identified several genes homologous to subunits of the NADPH-oxidase-complex.

As most other filamentous fungi, C. purpurea possesses two genes displaying homology to the mammalian gp91phox, named cpnox1 and cpnox2. While the deletion of cpnox2 did not lead to remarkable effects on pathogenicity during infection of rye, the knockout of the other nox gene, cpnox1, led to the consumption, that Cpnox1 is at least a virulence factor in C. purpurea: the knockout mutant is heavily impaired during colonization of C. purpurea´s host plant Secale cereale as it shows drastically reduced infection rates compared to the wild type. The symptom of honeydew appears strongly retarded and development of sclerotia, the typical fungal resting structures, has never been observed.

A recently identified gene from C. purpurea with homology to the regulatory subunit p67phox named noxR will be investigated soon. Experiments will show if the activation of NADPH-oxidase-components in C. purpurea is comparable to the mammalian phagocyte system.


Mining candidate effectors from the secretome of Colletotrichum higginsianum appressoria

Jochen Kleemann, Hiroyuki Takahara, Kurt Stueber, Richard O'Connell

Max-Planck-Institute for Plant Breeding Research, Department of Plant-Microbe Interactions, 50829 Köln, Germany

The hemibiotrophic ascomycete Colletotrichum higginsianum causes anthracnose disease on brassica crops and the model plant Arabidopsis. Melanized appressoria pierce the host cuticle and cell wall to form specialized biotrophic hyphae inside living epidermal cells. To identify developmentally-regulated proteins secreted by appressoria that may function as virulence effectors required for the establishment of the biotrophic interaction, a cDNA library was prepared from mature appressoria formed in vitro. ESTs derived from sequencing 980 clones were assembled into 518 unique sequences and searched for putative open reading frames ab initio. Biocomputational predictors, including SignalP, TMHMM and Phobius, were used to screen the predicted amino acid translations for N-terminal signal peptides and to differentiate between soluble secreted proteins and membrane proteins. Fifty-three unique sequences (10%) were predicted to encode proteins entering the secretory pathway, of which 26 were likely soluble secreted proteins, including hydrolases and oxidoreductases known to be secreted from other fungi. Seven genes encoding secreted proteins of unknown function, including two orphan sequences, were expressed early during plant infection and strongly upregulated or specifically expressed in mature appressoria and as such represent candidate effectors. Work is in progress to determine whether these proteins are higginsianum-specific and to evaluate their role in pathogenicity by means of targeted gene replacement and transient over-expression of the proteins in host epidermal cells.


Comparative study of tomato root colonization by Fusarium oxysporum f. sp. lycopersici wild type strain and the non-pathogenic knock-out mutants for class V and class VII chitin synthase genes

Magdalena Martin-Urdiroz1, M. Isabel G. Roncero1, Jose Antonio Gonzalez-Reyes2, Carmen Ruiz-Roldan1

1Dpto. Genetica, Edif. C5. Universidad de Cordoba, Cordoba, Spain, 2Dpto. Biologia Celular, Fisiologia e Inmunologia, Edif. C6. Universidad de Cordoba, Cordoba, Spain

Chitin synthases (CHSs) are the enzymes implicated in the synthesis of chitin, the main structural component of the fungal cell wall. These enzymes constitute a great family of isozymes grouped into seven classes. Furthermore, each fungal species contains a number of CHSs belonging to different classes. In the tomato pathogen Fusarium oxysporum f. sp. lycopersici the CHS genes of classes V (chsV) and VII (chsVb) have been shown to play an important role in the pathotypic behaviour towards tomato plants (Lycopersicon esculentum). The construction of single (ΔchsV and ΔchsVb) or double (ΔchsVΔchsVb) knock-out mutants showed that ChsV and ChsVb are required for full virulence of this pathogenic fungus.

To establish the differences in the infection process of the non-pathogenic strains of F. oxysporum compared to that of the wild-type strain, transmission electron microscopy (TEM) images were analyzed. TEM of sectioned tomato root samples inoculated with the wild-type strain, showed that all tissues were invaded by the pathogen 48 hours after inoculation. However, the non-pathogenic strains (ΔchsV, ΔchsVb and ΔchsVΔchsVb) were unable to colonize the cortex and the vascular bundles in the upper section of the root 48 hours after inoculation. This difference joined to the fact that the non-pathogenic strains did not induce host cell changes in the inoculated roots compared with roots inoculated with the wild-type strain, suggest that other mechanisms of plant defense are involved in the interaction between the non-pathogenic strains of F. oxysporum and tomato roots. Thus, complex responses protecting plants from pathogen involve intensive generation of reactive oxygen species (ROS) that can be used for rapid inhibition of pathogens. However, due to the damaging nature of ROS, plants deploy an enzymatic antioxidative system to prevent damage to host cells. Studies of the variation in the expression profile of the antioxidant enzymes ascorbate peroxidase, monodehydroascorbate reductase and dehydroascorbate reductase as well as determination of guaiacol peroxidase and ascorbate peroxidase activities, are currently being performed in tomato plants in response to inoculation with pathogenic and non-pathogenic strains of F. oxysporum.



The infection biology of Fusarium graminearum single gene deletion strains

Neil Brown1, Thomas Baldwin2, Martin Urban1, Allison van de Meene1, Kim Hammond-Kosack1

1Rothamsted Research, Harpenden, Hertfordshire, United Kingdom, 2INRA, Renne, France

F. graminearum causes a globally important disease on wheat that has both economic and health implications. Single gene knockout mutants are constantly being generated by the Fusarium community. However, the exact point of fungal arrest for each mutant at the cellular level remains to be determined. Therefore, the objective of this project is to create a fate map of the F. graminearum virulence gene mutants and to fully characterise their infection biology in planta.

Characterisation of the spread of the wild type or mutant pathogen throughout the ear was assessed using light, scanning electron and transmission electron microscopy. Histochemical staining and X-ray microanalysis was used to identify specific compounds or elements in infected and healthy tissue. The onset of DON mycotoxin production and its final distribution was determined via immuno-localisation.

A type I topoisomerase was the first mutant to be assessed. Initial observations suggested that the top1 gene deletion strain infection was blocked at the rachis node. A study of the anatomy of the healthy rachis node has shed light on this under-explored tissue type. In the case of the top1 mutant infection, there appears to be a plant defence response hindering hyphal progress into the rachis.

Observations from both the wild type and mutant infections may be correlated with gene expression profiles of the specific region via Laser Capture Microdissection. Comparisons between the transcriptome of the wild type and mutant infections should provide the detailed analysis required to assign function to these genes.


Differential abundance of proteins in pre-invasion stages of the downy mildew oomycete Peronospora viciae

Josiane Chuisseu Wandji, Judith Harrison, Mervyn Lewis, Heather Macdonald, Peter Spencer-Phillips

University of the West of England, Bristol, France

The plant pathogenic oomycete Peronospora viciae causes downy mildew of pea (Pisum sativum), resulting in up to 55% yield losses in the UK. Proteomics has been used to analyse the host-pathogen interaction, with the specific aim of identifying key marker and target proteins that may enable the development of novel methods for detection and control. We focus here on the application of two dimensional (2D) gel electrophoresis and mass spectrometry to proteins extracted from three pre-invasion developmental stages of the pathogen: 1) conidia from sporulating infections; 2) germinating conidia; 3) conidia with germ tubes and appressoria. Over 700 protein spots were present on 2D gels from each stage following staining with Coomassie blue. Data from MALDI-TOF peptide mass fingerprints and Q-TOF analysis of amino acid sequences has enabled identification of 11 proteins. These include 6 that matched other oomycete proteins: actin 1, actin 2, calmodulin, two glyceraldehyde-3-phosphate dehydrogenase (GAPDH) proteins and heat shock protein 70 (hsp70). A further 53 did not match any proteins in the Matrix Science (MASCOT) database. Calmodulin showed a similar abundance in all three pre-invasion stages, whilst actin 2, the two GAPDH proteins and hsp70 showed at least a two-fold decrease in abundance between the conidial and the appressorium stages. In contrast, actin 1 remained unchanged in abundance between un-germinated and germinated conidia, but decreased by two-fold at the appressorium stage. A further 9 unidentified proteins showed a two- to three-fold alteration in abundance between the different stages. These results suggest that the proteins with a differential abundance may play an important role in the pre-invasion stages of pathogen development, and therefore could have potential as novel targets for control. Proteins that did not alter in abundance (e.g. calmodulin and some of the unidentified proteins) have potential as markers for detection of early stages of infection using biosensors.


Tomatinase from Fusarium oxysporum f. sp. lycopersici is required for full virulence on tomato plants

Yolanda Pareja-Jaime, M. Isabel G. Roncero, Carmen Ruiz-Roldán

Universidad De Cordoba, Cordoba, Spain

Saponin detoxification enzymes from pathogenic fungi are involved in the infection process of their host plants. Fusarium oxysporum f. sp lycopersici, a tomato pathogen, produces the tomatinase enzyme Tom1, which degrades α-tomatine to non-toxic derivates. To study the role of tom1 gene in the virulence of F. oxysporum, we performed targeted disruption and overexpression of the gene. The infection process of tomato plants inoculated with transformants constitutively producing Tom1 resulted in an increase of symptom development. By contrast, tomato plants infected with the knockout mutants showed a delay in the disease process, indicating that Tom1, although not essential for pathogenicity, is required for the full virulence of F. oxysporum. Total tomatinase activity in the disrupted strains was reduced only 25%, being β2-tomatine the main hydrolysis product of the saponin in vitro. In silico analysis of F. oxysporum genome revealed the existence of four additional putative tomatinase genes showing identity to tomatinases belonging to family 3 of glycosyl hydrolases, which might be the responsible for the remaining tomatinase activity in the Δtom1 transformants. Our results indicate that detoxification of α-tomatine in F. oxysporum might be carried out by several tomatinase activities, suggesting the importance of these enzymes during the infection process.


Magnaporthe grisea cutinase 2 mediates appressorium differentiation and host penetration and is required for full virulence.

Pari Skamnioti, Sarah Gurr

Department of Plant Sciences, University of Oxford, South Parks Road, Oxford, OX1 3RB, United Kingdom

The rice blast fungus Magnaporthe grisea infects its host by forming a specialized infection structure, the appressorium, on the plant leaf. The enormous turgor pressure generated within the appressorium drives the emerging penetration peg forcefully through the plant cuticle. Hitherto, the involvement of cutinase(s) in this process has remained unproven. We identified a specific M. grisea cutinase CUT2, whose expression is dramatically upregulated during appressorium maturation and penetration1. The cut2 mutant has reduced extracellular cutin-degrading and serine esterase activity, when grown on cutin as the sole carbon source, compared with the wild-type strain. The cut2 mutant strain is severely less pathogenic than the wild-type or complemented cut2/CUT2 strain on rice and barley2. It displays reduced conidiation and anomalous germling morphology, forming multiple elongate germ tubes and aberrant appressoria on inductive surfaces. We show that Cut2 mediates the formation of the penetration peg but does not play a role in spore or appressorium adhesion, or in appressorial turgor generation. Morphological and pathogenicity defects in the cut2 mutant are fully restored with exogenous application of synthetic cutin monomers, cAMP, ΙΒΜΧ and diacylglycerol (DAG). We propose that Cut2 is an upstream activator of cAMP/PKA and DAG/PKC signaling pathways that direct appressorium formation and infectious growth in M. grisea. We also speculate on the role of rice blast hydrophobins as surface interactors facilitating fungal cutinase activity3. Cut2 is therefore required for surface sensing leading to correct germling differentiation, penetration and full virulence in this model fungus.

1Lees, Roberts, Skamnioti and Gurr. (2007). Journal of Computational Biology 14:1, 68-83, 2 Skamnioti and Gurr. (2007). Plant Cell, 19:8 2674-2689, 3 Skamnioti and Gurr. (2008) Plant Signaling and Behavior, 3:4, in press.



The link between anastomosis and sexual development in Epichloë endophytes

Nikki Charlton, Kelly Craven

The Samuel Roberts Noble Foundation, Ardmore, OK, United States

Vegetative hyphal fusion (anastomosis) plays an important role in vegetative growth and sexual development in filamentous fungi. A soft (so) mutant of Neurospora crassa was shown to be incapable of self-fusion. The inability to create interconnected networks of self-anastomosed hyphae may prohibit a fungus from producing structures of complex organization, such as sexual fruiting bodies. Epichloë endophytes are intimate fungal symbionts of cool-season grasses that mostly grow as sparse, unbranched hyphae in the intercellular spaces of all aboveground host tissues. However, these endophytes have an obligate sexual stage that occurs coincident with the onset of host inflorescence development, wherein the mycelium emerges as dense epiphytic growth that surrounds the host flower and causes its abortion. The synchronization of the sexual cycle of Epichloë endophytes with inflorescence development in the host may be associated with a switch from non-anastomosed to anastomosed tissue. In this study we are testing whether 1) the transition from the vegetative to the sexual stage of the Epichloë lifecycle is mediated by the so gene and 2) a signal of plant origin is involved in the initiation of this fungal lifestyle transition. We have sequenced the so gene from sexual and asexual non-hybrid endophytes and identified a putative transcription factor binding site present in both sexual and asexual isolates. The asexual Neotyphodium lolii isolates have a proliferation of additional copies of this putative binding site, which is absent in the sexual E. festucae isolates. A similar proliferation in S. cereviseae results in ADH2 gene repression, and we are working to determine the effects of this promoter proliferation on so gene expression. We are also using nitrate-nonuntilizing (nit) mutants to study the effects of plant signals on self-anastomosis. We have investigated the role of simple sugars and gibberellins (GAs), which have been shown to play a role in the control of flowering. Detailed studies are underway to examine additional plant signals to determine effects on anastomosis in Epichloë endophytes. Work is also in progress to elucidate the role of so in E. festucae and E. typhina by assessing the ability of knockout mutants to self-anastomose and express the sexual stage in planta.



Deletion of a fungal endophyte stress-activated MAP kinase disrupts host development

Carla Eaton, Barbara Ambrose, Jeremy Hyams, Barry Scott

Massey University, Palmerston North, New Zealand

The mutually beneficial association between the fungal endophyte Epichloë festucae and perennial ryegrass provides an excellent model system for studying the signalling involved in regulating plant-fungal interactions. In this association, fungal growth is highly regulated and co-ordinated with growth of the host. Production of reactive oxygen species (ROS) by the E. festucae NADPH oxidase (Nox) complex is essential for a normal association with perennial ryegrass (Tanaka et al. 2006; Takemoto et al. 2006). However, the exact nature of the signalling involved in maintaining this association is unclear. The aim of this project was to test whether the stress-activated MAP kinase, sakA, is involved in symbiotic maintenance. The sakA gene has been previously replaced and the mutant phenotype analysed in culture (Eaton et al. in press). To determine the role of sakA in the association with perennial ryegrass, seedlings were inoculated with the ∆sakA mutant and wild-type. The ∆sakA mutant had a drastically altered plant-interaction phenotype, displaying a significantly reduced ability to colonise perennial ryegrass seedlings. Where colonisation was established the fungus showed unregulated growth, with increased biomass and hyphal hyper-branching. Host growth was severely stunted and displayed premature senescence, possibly due to its poorly developed root system. The host also showed altered development, with the base of the tillers appearing swollen and bulbous and lacking anthocyanin pigments characteristic of plants infected with wild-type. This suggests that appropriate signalling by the E. festucae stress-activated MAP kinase is essential for normal development of the host. Electron microscopy revealed that hyphae of the ∆sakA mutant were frequently vacuolated and surrounded by an electron dense extracellular matrix, indicating a host incompatibility response. Taken together these results demonstrate that stress-activated MAP kinase signalling in E. festucae is essential for maintaining a mutually beneficial association with perennial ryegrass, and for normal host development.


Efficient genetargeting by Agrobacterium tumefaciens-mediated transformation in Colletotrichum higginsianum strains deficient for Neurospora crassa mus-51 gene homolog

Hiroshi Terada, Takuma Ushimaru, Kie Tsuboi, Gento Tsuji, Yasuyuki Kubo

Kyoto Prefectural University, Kyoto, Japan

Colletotrichum higginsianum is the causal agent of anthracnose disease of Brassicaceae plants. This fungus has become an excellent candidate for studying fungal-plant interactions because it is able to infect a model plant, Arabidopsis thaliana. The aim of our study is to establish a high throughput molecular genetic analysis of C. higginsianum by Agrobacterium tumefaciens-mediated transformation (AtMT). We previously developed AtMT as a method for random insertional mutagenesis in C. higginsianum. This method successfully allowed us to obtain a large number of transformants in a short period of time. Subsequently, we attempted to develop AtMT for targeted gene disruption through homologous recombination. We estimated the frequency of homologous integration for two pathogenicity-related genes, ChPKS1 coding for polyketide synthase and ChSSD1 coding for a putative regulator for cell wall integrity. Although AtMT provided high efficiency of transformation in C. higginsianum, homologous integration occurred only 2.4% for ChPKS1 and 0.6% for ChSSD1, respectively. This result suggested that it would be impractical to isolate homologous recombinants efficiently in C. higginsianum. Thus, we focused on the Neurospora crassa mus-51 gene encoding a protein that plays a role for non-homologous end-joining of DNA breaks. It was reported that the disruption strains of the gene showed a dramatic increase of homologous integration efficiency. We isolated an orthologue of the mus-51 gene from C. higginsianum, and named it as ChMUS51. Disruption strains of ChMUS51 were successfully isolated and used as recipients for targeted gene disruption. Frequency of homologous integration was dramatically increased up to 97.5% for ChPKS1 and 98.3% for ChSSD1, respectively. Next, we investigated the relationship between the length of the homologous sequence and the frequency of homologous integration. Longer than 1000 bp homology was sufficient for effective isolation of recombinants and the frequency was over 95%. However, shorter than 250bp homology was insufficient for practical isolation of recombinants and the frequency was less than 3%. The chmus51 mutants also showed no defect in hyphal growth, infectious morphogenesis, and pathogenicity. Thus, we concluded that the chmus51 disruption strain would be an efficient recipient for targeted gene disruption.



A kelch repeat protein claKEL1p associate with microtubule and is involved in appressorium morphogenesis in Colletotrichum lagenarium

Ayumu Sakaguchi, Toshihiko Miyaji, Gento Tsuji, Yasuyuki Kubo

Kyoto Prefectural University, Kyoto, Japan

C. lagenarium is the causal agent of cucumber anthracnose disease. The infection process involves a series of change in fungal morphology, including a specialized infection structure appressorium formation. We have revealed that Schizosaccharomyces pombe tea1 homolog ClaKEL2 gene encoding for a kelch motif protein was involved in appressorium morphogenesis in C. lagenarium. A database reference of the genome sequences of filamentous fungi revealed that these have a number of predicted kelch family proteins. However, the roles of these proteins are not well known.

We isolated novel kelch repeat encoding gene ClaKEL1 from C. lagenarium. ClaKEL1 was isolated as the tagged gene in a pathogenicity deficient mutant P24 generated by restriction enzyme-mediated integration. ClaKEL1 encodes a putative protein of 555 amino acids containing kelch repeat at the C teminal region. The deduced amino acid sequence of ClaKEL1 had high homology with the amino acid sequence of other filamentous fungal hypothetical proteins. To elucidate the cellular role of Clakel1p, GFP-ClaKEL1 fusion gene was constructed. The GFP-Clakel1p signal corresponded to mRFP1-a TUB1 signal localizing at cytosolic microtubule or spindle microtubule. This result suggested that Clakel1p is a microtubule-associated protein.

To define the function of ClaKEL1, we isolated clakel1 knock-out mutants by homologous recombination. The clakel1 mutant did not differ from the wild type in growth rate on PDA medium, but had reduced conidiation. Conidia from clakel1 mutants could germinate same as wild type on glass slide, but the approximately 50% of the appressoria produced by clakel1 mutants were irregular in form, showing elliptic shape, thick neck and lateral germination. On the host cucumber leaves, appressoria of clakel1 mutant formed very few infection hyphae. In the wild type, 27% of the appressoria formed infection hyphae into host surface, while only 3% of the clakel1 mutant appressoria formed infection hyphae. Accordingly, compared with lesions produced by the wild type, clakel1 mutants produced small lesions on intact host leaves. These results indicate that ClaKEL1 is involved in appressorium morphogenesis and pathogenicity.



Discovery of novel secreted effectors delivered by Magnaporthe oryzae during the fungal-plant interaction using a type III secretion system

Rita Galhano1, Andrew Steed1, David Studholme2, Paul Nicholson1, Ane Sesma1

1John Innes Centre, Norwich, United Kingdom, 2The Sainsbury Laboratory, Norwich, United Kingdom

The primary plant immune response has evolved to recognise common features of microbial pathogens (PAMP-triggered immunity). However, pathogens have acquired the ability to deliver effector proteins to the plant cell to suppress primary defence responses, allowing pathogen proliferation. The secretome of a pathogen largely determines its ability to grow and cause disease in its host. Magnaporthe oryzae is a haploid, filamentous, ascomycete fungus that infects rice and other cereals, causing a devastating disease known as rice blast. The role of M. oryzae effectors and their involvement during host colonisation is still largely unknown. In animal and plant bacterial pathogens, e.g. Pseudomonas and Xanthomonas spp., effectors are delivered through a specialised protein-delivery system, called type III. In order to evaluate the role of M. oryzae effectors individually, candidates derived from bioinformatic analyses are being cloned using a bacterial type III delivery vector and introduced into Pseudomonas spp. and Xanthomonas spp. isolates that can produce disease symptoms in rice, barley, and wheat. Preliminary results of the pathogenicity tests with Pseudomonas isolates containing AVR-Pita showed reduced susceptibility compared to Pseudomonas wild-type isolates on Nipponbare and CO39 rice leaves, suggesting that this approach may be useful for identification of novel M. oryzae effectors.


Colonisation of barley roots with DsRed expressing Fusarium avenaceum and GFP expressing Fusarium culmorum-competition and effect on toxin production

Thomas Johansen1, Morten Nedergaard Grell1, Johan Åhman1, Stefan Olsson1, Michael Hansen2, Henriette Giese1

1University of Copenhagen, LIFE, Department of Ecology, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Copenhagen, Denmark, 2University of Copenhagen, LIFE, Department of Plant Biology, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Copenhagen, Denmark

Fusarium culmorum and Fusarium avenaceum are infamous for causing root rot, seedling and head blight in cereal species. The latter disease has attracted the greater attention as the fungi are producers of mycotoxins that are deposited in the grain. The fungi are soil born and studies were carried out to register in real time the colonization process of these fungi in barley roots. Two reporter strains: F. culmorum expressing the green fluorescent protein (GFP) and F. avenaceum expressing the red fluorescent protein (DsRed) were generated. A sterile root system was used for the application of macroconidia from the fungi individually or together to determine any competitive effects. Infection of agar embedded barley roots allowed us for the first time to obtain Laser Scanning Confocal Microscopic recordings of F. culmorum and F. avenaceum hyphae growing inside root cells. The primary region of entry appears to be the root hair zone. Both fungi grow in the intercellular space and can directly enter living plant cells. The fungi do not appear to be antagonistic but F. culmorum appear to have a faster growth rate than F. avenaceum. Secondary metabolite profiles for F. culmorum, F. avenaceum and the combination of the two species under infection of barley roots were obtained by RP-HPLC. Aurofusarin were produced by F. culmorum under barley root infection while the same peak was missing in the F. avenaceum infection assay. The result was confirmed by qualitative RT-PCR on the PKS12 gene. In addition PKS6, PKS8, NPS2 and NPS6 were expressed in F. culmorum after 7 day of infection of barley roots.



Carbon uptake during Ustilago maydis infection: feeding on plant resources and hiding from plant defenses

Ramon Wahl1, Jörg Kämper2

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

Ustilago maydis is the causative agent of the common and ubiquitous maize smut disease. As its host plant maize is one of the most important crop plants worldwide, the disease gives reason to investigate the molecular interplay between fungus and host during the infection process. To investigate carbon acquisition by U. maydis in planta, we have chosen a candidate gene approach based on in silico identification of 20 U. maydis hexose transporters. Deletion analysis revealed that two transporters, Hxt1 and Ptr1, are both necessary for successful biotrophic development.

Hxt1 appears to be the main glucose transporter of U. maydis, verified by yeast complementation assays and growth analysis of ∆hxt1 strains. Furthermore, Hxt1 is involved in repression of growth on less important carbon sources like galactose and xylose. However, the inability of ∆hxt1 strains to take up glucose does not interfere with massive fungal proliferation in the carbon rich bundle sheath of the maize plant, which is probably due to the ability to take up and use sucrose as main energy source. Thus, the role of Hxt1 during biotrophic development of U. maydis is currently unclear.

Ptr1 is expressed most abundantly during the early infection stages, where cell wall degradation via fungal enzymes is of importance for pathogenic development of the fungus. Maize plants infected with ∆ptr1 strains show a strong defense reaction, leading to collapsing fungal hyphae covered with a thick layer of yet unknown composition. However, Ptr1 does not facilitate yeast growth on the most abundant plant -hexoses and –pentoses. Therefore, the observed in planta phenotype seems to be a result of the inability of the fungus to remove plant cell wall components which might serve as a trigger for plant defense reactions, rather than to promote fungal growth. Apparently, sugar transporters not only provide the substrates for in planta growth of U. maydis, but also interfere with the host defense to enable a pathogenic lifestyle.



Proteomics studies reveal induced defence responses in date palm by entomopathogenic fungi growing as endophytes

Sonia Gómez-Vidal1, Manuel Tena2, Jesús Salinas1, Luis V. Lopez-Llorca1

1University of Alicante, Multidisciplinary Institute for Environmental Studies (MIES) Ramón Margalef, Dept of Marine Sciences and Applied Biology, Alicante, Spain, 2University of Cordoba, Department of Biochemistry and Molecular Biology, ETSIAM, Cordoba, Spain

We have investigated by proteomic techniques the interaction between the entomopathogen fungi Beauveria bassiana, Lecanicillium dimorphum and L. cf. psalliotae and the date palm (Phoenix dactylifera L.). We have previously provided evidences of endophytic behaviour of these entomopathogenic fungi in date palm petioles leaves using laboratory and field bioassays, as well as light and scanning electron microscopy techniques.

In this study, entomopathogenic fungi were inoculated in 1-2 years old date palm in two independent bioassays (2004 and 2005). In field bioassays palm tissue was collected 15 and 30 days post-inoculation. Similar studies were carried out with in vitro date palm plants.

Analysis of two-dimensional electrophoresis (2-DE) gels revealed the existence of spots showing both qualitative and quantitative differences (presence and absence or higher and lower expression of determinate spots) between inoculated and uninoculated palms. Selected spots separated by 2-DE gels, were manually excised for mass spectrometry (MALDI/TOF-TOF) analysis. We identified by homology up-regulated proteins that are involved in plant defence: proteins codified by R genes, antioxidant enzymes (catalase and thioredoxin peroxidase), a galacturonase, an esterase, heat shock proteins, and a cyclophilin.

With this proteomic study, we have verified that the endophytic colonization by these fungi induce date palm defence responses. These entomopathogenic fungi could therefore be used as endophytes in the biocontrol against date palm pest or pathogens. Such studies are in progress in our laboratory.



Comparison of 2DE patterns of mild and lethal isolates of Verticillium albo-atrum from hop

Stanislav Mandelc1, Sebastjan Radisek2, Branka Javornik1

1University of Ljubljana, Biotechnical Faculty, Agronomy Department, Ljubljana, Slovenia, 2Slovenian Institute for Hop Research and Brewing, Zalec, Slovenia

Verticillium wilt, caused by the phytopathogenic fungus Verticillium albo-atrum, has been a serious threat to hop production in Slovenia since 1997, when a new, more virulent pathotype of V. albo-atrum was first observed. Previously, the lethal pathotype of V. albo-atrum was known only in England. Total proteins were isolated from cultured mycelia and separated with two-dimensional electrophoresis to determine differences between the pathotypes. 2DE patterns of mild and lethal pathotypes from Slovenia and England were analyzed and compared. Major differences were observed and identified by mass spectrometry (LC-MS/MS). Our results indicate that the main differences between mild and lethal pathotypes include proteins involved in interfering with plant defence, such as peroxiredoxine, and proteins which are the building blocks and regulating factors of the cytoskeleton. The cytoskeleton is thought to be of great importance in relation to the ability of fungus to penetrate the plant surface, hyphal growth inside the xylem vessels and, especially, the conidiation rate at trapping sites. Some proteins from carbohydrate and protein metabolism pathways were also up-regulated in lethal pathotypes. These results reveal some of the differences between the two pathotypes at molecular level, which could explain a considerable portion of the difference in virulence.



Investigating the role of calmodulin-dependent signal transduction in Magnaporthe grisea

Zaira Caracuel-Rios, N. J. Talbot

University of Exeter, Exeter, United Kingdom

M. grisea is an important pathogen of cultivated rice (Oryza sativa) and has emerged as an experimental model of the study of plant infection processes. M. grisea causes plant infection by means of specialized infection structures called appressoria. These dome-shaped cells differentiate from the end of fungal germ tube and generate mechanical force to bring about rupture of the plant cuticle and entry to internal tissues. Previous investigations in our laboratory implicated a cyclophilin in the control of cellular turgor and the generation of functional competent appressoria during plant infection (Viaud et al., 2002 Plant Cell 14: 917-30). We therefore recently set out to explore the role of calcineurin and calmodulin during appressorium-mediated plant infection. Calmodulin (CaM) is a calcium-binding protein that can bind to and regulate a multitude of different protein targets, thereby affecting many different cellular functions. Calcineurin is a heterodimer consisting of a catalytic A subunit and a Ca2+-binding regulatory B subunit, and the association of the two subunits is essential for activity. Ca2+/calmodulin binds to the C-terminal region of the A subunit, resulting in a conformational change that frees the active site from occlusion by an autoinhibitory domain. We found that calcium modulators and calmodulin antagonists inhibited appressorium formation, consistent with a role for calcium/calmodulin-dependent signalling during appressorium formation of M. grisea. We will present progress in analysing the cellular function of the calmodulin, calcineurin A, and calcineurin B in the pathogenesis of M. grisea.


Expansion of protein families and gene fate of paralogs in the ras and protein kinase families of the symbiotic fungus Laccaria bicolor

Balaji Rajashekar1, Annegret Kohler2, Tomas Johansson1, Jason Stajich3, Yao-Cheng Lin4, Pierre Rouzé4, Francis Martin2, Anders Tunlid1, Dag Ahrén1

1Lund University, Lund, Sweden, 2INRA-Nancy, Champenoux, France, 3University of California, Berkeley, United States, 4Flanders Interuniversity Institute for Biotechnology (VIB), Ghent, Belgium

Gene duplications and loss are major mechanisms generating evolutionary novelties and pruning specialized functions. Here, we have studied the role of duplication events for the evolution of ectomycorrhizae in Laccaria bicolor. Gene duplicates and gene families in the genomes of the L. bicolor, the saprophytes Coprinopsis cinerea and Phanerochaete chrysosporium, the human pathogen Cryptococcus neoformans and the plant pathogen Ustilago maydis were analyzed. Among these basidiomycetes, L. bicolor contained the highest number of, mostly young, gene duplicates. The differences in gene duplicates had a pronounced effect on the number and size of multigene families. In total, 7352 protein families were identified in the five basidiomycete genomes. L. bicolor contained the largest number of lineage-specific (1077) and expanded (1064) protein families. A large fraction (29%) of the young gene duplicates of L. bicolor were found within the 55 largest gene families, having more than 25 members. Phylogenetic analyses of two such families, protein kinases and small GTPases, showed that L. bicolor contained clusters of paralogs that have arisen though duplication events in the Laccaria lineage. The gene fate after duplications was determined in the two families. Analysis of protein motif structures indicated that in the majority of the cases the ancestral motif structures had been retained in the paralogs. However, a few cases of changes in motif structures were observed that are indicative of neofunctionalization. Divergence in function of paralogs was also suggested by analysis of their expression levels in different tissues of L. bicolor.



Localization of Magnaporthe grisea cell wall components during infection

Takashi Fujikawa1, Keietsu Abe2, Marie Nishimura1

1National Institute of Agrobiological Sciences, Tsukuba, Ibaraki, Japan, 2New Industry Creation Hatchery Center, Tohoku University, Sendai, Japan

Magnaporthe grisea, the causal agent of rice blast disease, invades into plant cells via appressoria which are formed on tips of germ tubes. Although the importance of polysaccharides, major components of the cell wall, in host-pathogen interactions was reported in some plant and mammalian systems, it is not well understood in M. grisea. To investigate the localization of the major M. grisea cell wall components such as chitin, chitosan, β-1,3 glucan, and α-1,3 glucan within infection structures, fluorescence-labeling and imaging techniques were used. Chitin, chitosan and β-1,3 glucan were observed in germ tubes and appressoria produced on plastic surfaces, while α-1,3 glucan was localized specifically in appressoria. Interestingly, α-1,3 glucan was clearly detected in germ tubes on rice sheath surfaces whereas β-1,3 glucan was hardly detected. Moreover, β-1,3 glucan and α-1,3 glucan were detected in incipient appressoria but not in mature appressoria formed on plant surfaces. The major cell wall components detected in infectious hyphae were α-1,3 glucan and chitosan. We used real-time RT-PCR to further examine the expression patterns of the genes encoding synthases required for biosynthesis of cell wall polysaccharides. In good agreement with our cytological observation, the transcription level of β-1,3 glucan synthase gene (MgFKS1) was decreased, while that of α-1,3 glucan synthase gene (MgAGS1) was increased during infectious growth in planta. Rice cells produce various enzymes that degrade fungal cell wall in response to fungal infection, however genes encoding chitosan and α-1,3 glucan degrading enzymes are not found in the rice genome.

Our results suggest that the fungus may protect itself from the enzymatic attack of the plant cells by means of modifying the localization and the composition of its cell wall polysaccharides during infection.



Towards functional analysis of transporters operating at the plant-fungal interface in Epichloë festucae-grass symbiotic association

Ranamalie Amarasinghe1, Uljana Hesse2, Haiquan Li1, Patrick Zhao1, Christopher Schardl2, Carolyn Young1

1The Samuel Roberts Noble Foundation, Ardmore, OK 73401, United States, 2University of Kentucky, Lexington, KY 40546, United States

Epichloë endophytes are mutualistic fungal symbionts of cool-season grasses (Poaceae), known to confer growth advantages to its host. However, the molecular basis of the interaction between the endophyte and the grass host remain largely unknown. Membrane transporters are key players in any plant–microbe interaction. As a step towards understanding Epichloë-grass interactions, we have identified and classified the membrane transporter complement that operates at the plant-fungal interface. Here we report the expression analysis of the E. festucae transporter genes and identify some of the potential key players in this symbiotic association.

The genome of Epichloë festucae, a model endophyte, has been sequenced and assembled resulting in 100% coverage, with 92% in supercontigs larger than 2kb. Using a bioinformatics approach based on nearest neighbor method to compare against the Transporter Classification Database (, we have identified the putative transporter complement in this Epichloë genome. The respective transporter classifications ( were also determined. Comparison of transporter complements in E. festucae with that of other organisms revealed interesting parallels between the composition of transporter proteins and their life style. We utilized a quantitative RT-PCR to compare the in planta expression levels of these genes to the levels detected in cultured mycelia. A number of transporters, including some members of the families POT, DMT, MFS, MIP, and Trk were found to be significantly upregulated in the plant host suggesting a role in the endophyte–plant association.

Expression analysis of different E. festucae transporter gene families provided valuable insights into the nutritional aspects of the interaction. Currently we are in the process of creating knockout mutants of candidate genes to understand their role in the association. Further characterization and functional analysis of these transporters in E. festucae will be a substantial contribution towards understanding plant-fungal interactions.



Identification of new components of the NADPH oxidase (Nox) complex in the endophytic fungus Epichloë festucae

Daigo Takemoto, Aiko Tanaka, Barry Scott

Massey University, Palmerston North, New Zealand

Endophytic fungi of the Epichloë group systemically colonise the intercellular spaces of grass plants to establish a symbiotic association. Maintenance of the symbiosis requires that the growth of the fungal endophyte be strictly controlled throughout the host plant. We have recently shown that reactive oxygen species (ROS) produced by a specific fungal NADPH oxidase isoform, NoxA, have a critical role in regulating hyphal growth in the mutualistic interaction between Epichloë festucae and perennial ryegrass. A fungal regulator of NoxA, designated NoxR, which has similar motifs to those of the vertebrate p67phox, has been shown to be essential for symbiosis maintenance. Perennial ryegrass containing a noxA or noxR mutant becomes severely stunted and undergoes precocious senescence. Hyphae in these symbiota are hyper-branched and the biomass dramatically increases, suggesting that ROS produced by NoxA/NoxR regulates the branching pattern of the endophyte in the host plant. NoxR has two protein-protein interaction domains, an N-terminal TPR motif domain and a C-terminal PB1 domain suggesting, NoxR acts as a scaffold for the Nox enzyme complex. Using yeast two-hybrid and pull-down assays NoxR was shown to interact with the small GTPase RacA via the N-terminal TPR motifs. A deletion mutant of racA had a very similar plant interaction phenotype to that previously reported for noxA and noxR mutants, indicating that RacA also regulates NoxA. Given PB1 domains are known to interact with other PB1 domains, fungal genome databases were screened for PB1-containing proteins to identify potential candidates that interact with NoxR. Proteins identified included NoxR itself, Cdc24, Bem1 and a protein of unknown function with CBS domains. Yeast two hybrid was used to test if these PB1 domains could interact. Three interactions were detected: the NoxR PB1 can interact with itself and Cdc24-PB1, and Cdc24-PB1 can interact with Bem1-PB1. Bem1 and Cdc24 are two components involved in the establishment of polarity in yeast, and localise to the plasma membrane. Our working model proposes that E. festucae hyphal branching in the host plant is controlled by localised bursts of ROS catalysed by NoxA, following recruitment of NoxR and RacA from the cytosol to the cell membrane, and Cdc24 and Bem1 could be involved in controlling the sub-cellular localisation of these Nox regulators.



A secreted arbuscular mycorrhizal fungal effector induces the plant gene MtMSBP1

Hannah Kuhn, Natalia Requena

University of Karlsruhe, plant-fungal interaction group, Karlsruhe, Germany

The arbuscular mycorrhizal fungi are obligate biotrophic organisms that rely on the mutualistic interaction with a plant root to fulfill their lifecycle. It is therefore of critical importance for them to recognize their symbiotic partner and undergo a functioning interaction. During early recognition processes as well as among later regulation events an extensive signal exchange is necessary to adjust the cellular program of the plant for the entry and establishment of the fungus in the cortex as well as for the fine-tuning of the further interaction. Despite of the importance of this molecular signals for the regulation of the symbiosis there are so far no fungal molecules described that obtain such a signalling function.

To identify fungal signal during the early recognition process of the symbiosis our aim is to identify Medicago truncatula marker genes that are able to indicate the recognition of fungal signals in the plant root and therefore help us to isolate fungal signal molecules.

Our search for plant marker genes involved in early recognition events led us to the identification of MtMSBP1, which was found to be upregulated during appressorium formation by Glomus intraradices. The induction was confirmed by Real Time RT-PCR, and analysis of the promoter activity pointed out to a possible role of the gene during initial communication between the symbionts. Time-lapse video microscopy showed that MtMSBP1 is induced in the vicinity of branched hyphae, but prior to hyphal contact, therefore suggesting the existence of a diffusible fungal effector molecule. The subcellular localization of the corresponding protein, a putative membrane steroid-binding polypeptide, was analyzed by GFP-fusion and was found to be located at the cytoplasmic membrane / ER and at the nuclear envelope. This, together with the fact that a putative orthologue of this protein has been shown to bind steroids and to regulate cell expansion, points to a function of MtMSBP1 in cell elongation during the AM symbiosis. Further experiments are being carried out to investigate the potential of the MSBP promoter as plant marker to identify fungal effector molecules received by the plant.


Isolation and characterization of candidate effectors from Blumeria graminis f. sp. hordei

Cristina Micali, Sarah Schmidt, Ralph Panstruga

Max-Planck Institute for Plant Breeding Research, Cologne, Germany

Powdery mildew diseases are caused by a diversity of pathogenic species under the broad umbrella of the order Erysiphales, class of Ascomycota. Among the best studied, Blumeria graminis belonging to various forma speciales cause economically important diseases on barley and wheat in Europe. After spore germination and appressorial formation, the fungus develops specialized feeding structures called haustoria, within infected epidermal cells. Through these structures, the fungus is able to highjack plant nutrients towards the growing mycelium. In addition, this intimate contact with the host is believed to be used to deliver, within the plant cell, effector proteins which manipulate host metabolism and presumably suppress defense responses.

We used bioinformatic data mining from existing fungal EST databases to identify candidate effector proteins from B. graminis f. sp. hordei (Bgh, pathogenic on barley). Putative genes qualify as effector candidates if they encode relatively small, secreted proteins of unknown function. Protein secretion is the most likely method of delivery of fungal proteins to the host cell. Herein we present the functional characterization of several candidate effectors from Bgh by transient expression in barley epidermal cells. Over-expression of several candidate effectors causes a statistically significant enhancement of penetration efficiency of the host fungus Bgh on barley. In addition, several effector candidates appear to partially suppress non-host resistance of barley to the non-adapted wheat powdery mildew fungus B. graminis f. sp. tritici.

A survey of allelic variability at candidate effector loci in five different isolates of Bgh indicate that some candidates display nucleotide polymorphisms that result in amino-acid substitutions. The functional implications of these amino acid changes remain to be addressed. With the exception of the barley AVRa10 and AVRk1 genes, which appear to be recognized and trigger an immune response in infected barley plants, no true effector has been isolated from Bgh. The study of these putative effectors will provide useful information regarding the mechanisms governing disease in this fungus.


The P-type ATPase activity of MgAPT2 is necessary for pathogenesis in Magnaporthe grisea

Ana Lilia Martinez Rocha, Nicholas J. Talbot

University of Exeter, Exeter, Devon, United Kingdom

Plant pathogenic fungi have evolved mechanisms to suppress plant defences during infection related development. They deliver proteins directly into plant cells to facilitate tissue invasion, but how the fungus delivers proteins during plant infection is currently unknown. Magnaporthe grisea is a plant pathogenic fungus which causes disease in a number of grasses, including the economically damaging rice blast disease. The MgAPT2 gene encodes a P-type ATPase in M. grisea, which has been shown to be involved in exocytosis during plant infection. MgAPT2 is required for both foliar and root infection by the fungus, and for the rapid induction of host defence responses in an incompatible reaction. Targeted gene replacement showed that ∆Mgapt2 mutants are non-pathogenic and are impaired in the secretion of a range of extracellular enzymes. However, the loss of MgAPT2 does not significantly affect hyphal growth or sporulation, indicating that the establishment of rice blast disease involves the use of MgApt2-dependent exocytotic processes that operate during plant infection. We have recently generated strains carrying ectopic copies of the MgAPT2D488N allele in a wild type (Guy11) and ∆Mgapt2 mutant background. Mutation of the aspartic acid (D488N) abolishes ATPase activity of MgAPT2. The ectopic expression of the MgAPT2D488N allele in ∆Mgapt2 mutant background does not restore pathogenicity. We have also explored the relationship between MgAPT2 and the yeast DRS2 gene in detail and investigated the role of MgAPT2 in protein delivery during pathogenesis. Progress towards deterining the precise biological function of MgAPT2 will be presented.



The role of autophagy in Fusarium graminearum during infection of cereals

Lone Josefsen, Jan Svensson, Stefan Olsson, Henriette Giese

1University of Copenhagen, Department of Ecology, Frederiksberg, Copenhagen, Denmark, 2University of Copenhagen, Department of Plant Biology, Frederiksberg, Copenhagen, Denmark

Fusarium graminearum is a fungus that infects cereal crops. The infection is a very rapid process that starts with spore germination and invasion of the plant tissue where it turns into a necrotrophic infection that reduces crop quality and cause yield losses.

Recycling of nutrients is a strategy for fungi to survive the stress and starvation it experiences prior to plant infection. Autophagy is one of the primary mechanisms for recycling of resources within fungal mycelia. It is dependent on the vesicular enclosement of portions of the cytosol resulting in the formation of autophagosomes (double layered vesicles). The autophagosomes in turn fuse with vacuoles, which contain digestive enzymes that degrade the contents of the autophagosome and make nutrients available for new production of proteins and organelles.

Autophagy has in other plant pathogens been shown to be essential for the infection process. It is thought that autophagy is necessary for turnover of organic matter and optimizing the energy needed for pathogenecity. Using homologous recombination and an Agrobacterium-mediated transformation technique we have produced a F. graminearum strain where the Atg8-gene essential for the autophagic process is interrupted. On nutrient rich media the ΔAtg8-mutant and the WT show identically growth patterns. However, on low nutrient media the mutant performs poorly compared to the WT, indicating that that the mutant has lost the ability to recycle internal resources. Plant infection assays show that the ΔAtg8-mutant is able to infect plant tissue; however, there is a delay in the time of infection and visible symptoms compared to the WT.



ROS signalling during early plant infection stages in Botrytis cinerea

Nora Temme, Paul Tudzynski

Westfälische Wilhelms-Universität, Münster, Germany

Reactive oxygen species (ROS) are present in all aerobic environments where they cause cell damages when interacting with cellular components. They are also part of normal cellular processes like signal transduction or pathogen defence.

In the interaction of the necrothrophic pathogen Botrytis cinerea that infects more than 200 different host species ROS are present during the early stages of infection and can even be detected 2 dpi in primary lesions. Attacked by the pathogen the host plant produces ROS in the so called oxidative burst to defend against the fungus. Therefore an effective fungal detoxification system with catalases and peroxidases should be essential for the survival on the host. However, expression of several H2O2 degrading enzymes is not induced in the primary lesions of B. cinerea on bean leaves and starts in axenic culture at very high H2O2 levels (2.5 - 5 mM) that might be not present on the plant tissue. Expression of these ROS degrading enzymes is mainly regulated by the transcription factor Bap1 (Botrytis activator protein): Macroarray analyses with the wild type B05.10 and the Δbap1 deletion mutant w/wo 10 mM H2O2 revealed more than 100 genes that are upregulated under ROS exposure in the wild type B05.10 with at least 12 genes directly involved in ROS detoxification. Induction of these genes could not be detected in the Δbap1 deletion mutant in these experiments. The transcription factor Bap1 as well as some of its target genes essential for the survival on H2O2 are of minor or even no importance for host invasion indicating that ROS degradation might be not essential for the infection.

On the plant B. cinerea produces ROS itself and might even depend on the host defence reaction to achieve full pathogenicity. So ROS generation rather than degradation seems to be essential for the infection. However, the stress-activated MAP kinase BcSak1 that provides amongst others ROS resistance is a pathogenicity factor. Characterisation of other proteins possibly downstream of BcSak1 or ulterior involved in the ROS regulation process is on its way and will enlighten the ROS signalling network during host invasion. Furthermore, studies during the very early stages of infection will reveal regulation of Bap1 dependent genes as well as the amount of H2O2 present in planta.



A reliable routine infection assay for black leaf streak disease of bananas caused by Mycosphaerella fijiensis

Caucasella Díaz-Trujillo1, Cláudia Ferreira Fortes1, Guy De Capdeville2, Henk Jalink1, Manoel Souza3, Gert H.J Kema1

1Plant Research International B.V., Wageningen University and Research Centre (WUR), P.O. Box 16, 6700 AA, Wageningen, Netherlands, 2Laboratory of Genetics. Wageningen University and Research Centre (WUR), P.O. Box 16, 6700 AA, Wageningen, Netherlands, 3Embrapa-LABEX Europe - Wageningen University and Research Centre (WUR), P.O. Box 16, 6700 AA, Wageningen, Netherlands

Mycosphaerella fijiensis (Paracercospora fijiensis), an ascomycete fungus, is the causal agent of Black Sigatoka (also known as black leaf streak disease), the most devastating foliar disease of banana and plantain (Musa spp.). This pathogen is a global threat to banana plantations, demanding a major input of fungicides for its control. In some banana production areas in South and Central America more than 50 sprays are required annually to control this disease.

The genome sequence of M. fijiensis CIRAD86 was recently made publicly accessible ( The availability of this genome database is essential to increase our understanding of this host-pathogen interaction. The molecular aspects of the interaction are largely unknown. In order to study these we developed a reliable infection protocol for routine phenotyping assays and specific molecular/expression studies. During the biotrophic stage, which can last for over three weeks, M. fijiensis does not trigger macroscopic symptoms. These only appear during the necrotrophic phase, which onset varies with pathogen virulence, nutrients availability, environmental conditions and susceptibility of the cultivar.


Determining the role of reactive oxygen species generation in Magnaporthe grisea

Martin Egan, Elise Lambeth, Nick Talbot

University of Exeter, Devon, United Kingdom

NADPH oxidases (Nox) are flavoenzymes used to generate reactive oxygen species (ROS). Until recently, research into Nox and ROS generation was focussed primarily on the oxidative burst associated with the plant defence response and leukocyte function. However, the discovery of new functional members of the Nox family in filamentous fungi has implicated ROS in many diverse processes including cellular differentiation and sexual development. Here, we investigate the role of NADPH oxidase-generated ROS in the infection-related development of the phytopathogenic ascomycete fungus Magnaporthe grisea. This fungus parasitizes more than fifty species of grasses, but is best known as the causal agent of rice blast disease. The formation of a specialised cell known as an appressorium allows the fungus to breach the host cuticle using mechanical force and subsequently cause plant infection. This project has focused on functional characterization of three NADPH oxidase homologues found within the M. grisea genome. We have demonstrated that Nox1 and Nox2 are independently required for pathogenicity of M. grisea and contribute to appressorium morphogenesis and function. Nox3 meanwhile plays a role in hyphal elongation and the regulation of conidiogenesis. When considered together these data suggest that Nox regulate cellular morphogenesis and virulence-associated development in filamentous fungi.



Determining the function and genetic regulation of autophagic programmed cell death during plant infection by Magnaporthe grisea

Michael.J Kershaw1, Claire Veneault-Fourrey2, Diane, O Saunders1, Nicholas, J Talbot1

1Unversity Of Exeter, Exeter, United Kingdom, 2University H. Poincaire, Nancy, France

The rice blast fungus Magnaporthe grisea causes the most debilitating disease of cultivated rice and is responsible for serious and recurrent epidemics throughout the world. To understand this disease we are studying the development of infection structures called appressoria, which are formed by the fungus to bring about cuticle penetration and plant infection. Rice blast is initiated when spores of the fungus land on the surface of a rice leaf. Germination of the conidium on the leaf surface is followed by a single round of mitosis during germ tube elongation, prior to appressorium formation. We discovered that blocking mitosis by using the DNA synthesis inhibitor hydroxyurea, or use of a temperature-sensitive MgNimA mutant, prevented appressorium morphogenesis. Significantly, we also observed that inhibition of mitosis prevented conidial cell collapse. MgATG8 encodes a ubiquitin-like protein associated with autophagosomes and is necessary for autophagy in M.grisea. we generated ∆mgatg8 mutants which were unable to undergo autophagy and these did not undergo conidial cell collapse during appressorium formation and were not able to cause rice blast disease. These results demonstrated that completion of mitosis and autophagic cell death are pre-requisites for plant infection. We are currently characterising all of the main components of the autophagy process as a means of understanding why autophagy is necessary for appressorium function and to learn more about how the process is spatially and temporally regulated. Analysis of the M.grisea genome has revealed a total of 17 autophagy-related genes including homologues of genes involved in initiation (ATG1, ATG17, ATG13, ATG24) nucleation (ATG6, VPS34, PI3K), expansion (ATG3, ATG8, ATG12, ATG16, ATG5, ATG4, ATG7) and autophagosome recycling (ATG2, ATG9, ATG18). Targeted deletions of all of these autophagy related genes has been carried out to generate an isogenic set of mutants affected during each stage of autophagy. We are currently characterising the role of each of these genes in autophagy in the rice blast fungus and defining how the process is utilised for recycling of the contents of the fungal spore during infection-related development and how autophagic cell death is spatially regulated in the fungus at the same time as cuticle penetration and fungal penetration of the host plant.



ClaNOX2 gene coding for NADPH oxidase of Colletotrichum lagenarium is involved in infection hyphae formation from appressoria

Takanori Oosawa, Gento Tsuji, Yasuyuki Kubo

1Kyoto Prefectural University, Kyoto, Japan, 2Kyoto Prefectural University, Kyoto, Japan, 3Kyoto Prefectural University, Kyoto, Japan

NADPH oxidases are enzymes that produce reactive oxygen species (ROS), which play a significant role in fungal morphogenesis and fungus-plant interaction. Recently, it was reported that NADPH oxidase encoding genes, NOX1 and NOX2 were involved in appressorium mediated cuticle penetration in Magnaporthe grisea. In this study, we isolated two NADPH oxidase genes, ClaNOX and ClaNOX2 from Colletotrichum lagenarium and investigated the phenotype of gene disrupted mutants. clanox1 disrupted mutants formed pale pigment colony with reduced conidia and abundant aerial hyphae, but did not differ from the wild type in conidial germination, appressorium formation and lesion formation on host cucumber cotyledons. Thus, ClaNOX1 was not involved in virulence. Next, we evaluated the function of ClaNOX2. The colony morphology of clanox2 mutants did not differ from the wild-type. And also, the clanox2 mutants showed no defect in conidial germination and appressorium formation. However, the clanox2 mutants showed reduced lesion formation on intact cucumber cotyledon compared with the wild type, but retained invasive growth ability forming lesions on wounded cotyledon. From this result it was suggested that ClaNOX2 is involved in virulence at the stage of appressorium mediated cuticle invasion. To evaluate the appressorial function, we made cellulose membrane penetration assay. The clanox2 mutants formed normal appressoria on the membranes but showed complete defectiveness for infectious hyphae formation into cellulose membranes. This result suggested that ClaNOX2 is involved in infectious hyphae morphogenesis from appressoria. But, surprisingly, the clanox2 mutants could partly form lesions on cucumber cotyledon. Lesions were observed at 7 of 100 spots inoculated with clanox2 mutants, while 98 of 100 with the wild-type. Microscopic observation of lesion sites revealed that the clanox2 mutants formed infectious hyphae from appressoria. From these results, it was suggested that infectious hyphae morphogenesis was controlled in part by plant specific signal transduction pathway independent of ClaNOX2.



Responses in metabolic pathways of the ectomycorrhizal fungus Paxillus involutus during assimilation of various nitrogen sources in soils

Tomas Johansson1, Derek P. Wright1, Carl Troein2, Carsten Peterson2, Anders Tunlid1

1Microbial Ecology, Lund University, Lund, Sweden, 2Complex Systems, Department of Theoretical Physics, Lund University, Lund, Sweden

The positive effects of ectomycorrhizal (ECM) fungi on plant nutrition have traditionally been attributed to the quantitative effect of the extramatrical mycelium on uptake of dissolved nutrients, such as nitrogen. In forest soils nitrogen is present either in inorganic (mainly as ammonium) or organic (amino acids, peptides and proteins) forms and nitrogen mobilization by hyphae from soil is directly linked to hyphae uptake capacities.

We are interested in mechanisms for assimilation of nitrogen and have analyzed transcriptional responses after providing different nitrogen sources representing various degrees of complexity, i.e. ammonium phosphate, ammonium sulphate, glutamine, chitin, and bovine serum albumin. These were provided as nutrient patches in a peat microcosm designed for extraamtrical mycelial in-growth using Paxillus involutus in an ectomycorrhizal association with Betula pendula (birch). After fungal establishment in these patches total RNA was isolated and used for global transcriptional analyses. We used a custom cDNA microarray containing approx. 4,900 gene representatives. Of the gene representatives tested we found 2,390 representatives to be significantly differentially regulated in response to one or several nutrient amendments. Among those we found 292 gene candidates being highly homologous to known enzymes, representing a uniset 187 different enzymes, and could be assigned into 67 different metabolic pathways.

To understand these results we developed an in-silico method to evaluate transcriptional data in a metabolic context. Transcriptional data were assigned metabolic pathways and responses were evaluated on a pathway level. We will show how genes involved common metabolism, are responding to the different amendments of nitrogen, which is indicating specific routes and mechanisms for the assimilation. Clearly, more complex nitrogen sources, such as BSA and chitin are triggering more extensive metabolic responses as compared to more simple sources. Although fine differences between treatments the BSA and chitin responses are very similar, as are the ammonium sulphate and the glutamine responses. Distant from the these patterns is the ammonium phosphate response



Determining the function of trehalose-6-phosphate synthase during plant infection by Magnaporthe grisea

Richard Wilson, Joanna Jenkinson, Nicholas Talbot

University of Exeter, Exeter, United Kingdom

Rice blast disease is a serious socio-economic problem in many parts of the world and is mediated by infection-competent appressoria of the causal agent, Magnaporthe grisea. The non-reducing disaccharide trehalose is present in conidia of M. grisea and is mobilised during appressorium formation. The first step in trehalose biosynthesis involves trehalose-6-phosphate synthase (Tps1), and deletion of TPS1 results in abolition of trehalose production, an inability to grow on glucose minimal media, and loss of the ability to cause rice blast disease. We have demonstrated that the Tps1 protein is necessary for the regulation of several diverse cellular processes such as NADPH production via regulation of the pentose phosphate pathway and the ability to utilise nitrate as sole nitrogen source. This regulation occurs at both the transcriptional and post-translational level, with loss of nitrate utilisation in the D tps1 strains being attributable to reduced glucose-6-phosphate dehydrogenase activity (resulting in reduced NADPH production), and misregulation of NMR1, a gene encoding an inhibitor of nitrogen metabolism. Moreover, although independent of trehalose biosynthesis, the regulatory roles of Tps1 are dependent on efficient binding of glucose-6-phosphate to the protein. Therefore, Tps1 integrates carbon and nitrogen signaling via glucose-6-phosphate sensing. We show here that a family of NMR-like genes - NMR1, NMR2 and NMR3- are present in the M. grisea genome and gene disruption of NMR2 in a D tps1 background results in remediation of growth on nitrate, identifying this gene as a downstream target of the Tps1-mediated signaling pathway.



Genome-wide analysis of secreted proteins encoding genes in Melampsora larici-populina, the causal agent of the poplar foliar rust disease

Sébastien Duplessis1, Stéphane Hacquard1, Hope Tice2, Eileen Dalin2, Susan Lucas2, Harris Shapiro2, Jasmyn Pangilinan2, Jeremy Schmutz3, Marie-Pierre Oudot-Le Secq1, Pascal Frey1, Paul Richardson2, Francis Martin1

1INRA, Champenoux, France, 2US DOE Joint Genome Institute, Walnut Creek, CA, United States, 3Stanford Human Genome Center, Palo Alto, CA, United States

The foliar rust caused by Melampsora larici-populina is the main disease affecting poplar plantation in northern Europe with severe economic losses. Epidemiology of the disease and plant defense response have been studied but very little is known about fungal molecular mechanisms during the infection process. It is crucial to increase our understanding of the biology of the pathogen in order to develop strategies to control the disease. In the wake of the Populus genome sequencing, the ~100 Mb genome of M. larici-populina have been sequenced (7X depth). The analysis of this genome is a great opportunity to identify loci coding for the arsenal developed by the rust fungus to penetrate and exploit its host.

It has emerged from different studies that secreted proteins are expressed by biotrophic fungi during the infection process and particularly during formation of the haustorial structure. Such secreted proteins may interact with or manipulate the host plant during the infection. Here we present a project conduced in the frame of the annotation of the genome sequence of M. larici-populina that aims at identifying secreted proteins in a genome-wide analysis. We identified homologs of Melampsora lini haustorially expressed secreted proteins and avirulence genes in M. larici-populina and studied their expression profiles during infection of poplar leaves. We showed that several secreted proteins encoding genes were specifically expressed after haustorium formation.



Molecular analysis of sterol C14α-demethylase gene (CYP51) from azole resistant and sensitive Podosphaera fusca isolates, causal agent of powdery mildew of cucurbits

Francisco Jose López Ruiz1, Christopher James Ridout3, Alejandro Pérez García2, Antonio de Vicente Moreno2, Laetitia Chartrain3, Juan Antonio Torés1

1Estación Experimental "La Mayora" (CSIC), Málaga, Spain, 2Grupo de Microbiología y Patología Vegetal-Unidad Asociada a CSIC, Departamento de Microbiología, Facultad de Ciencias, Universidad de Málaga, Málaga, Spain, 3Disease and Stress Biology Department, John Innes Centre, Norwich, United Kingdom

Introduction: Cucurbit powdery mildew caused by Podosphaera fusca is one of the most important limiting factors for cucurbit production worldwide. With the recent spread of resistance to strobilurins (QoIs), reliable control is mainly dependent upon inhibitors of sterol C14α-demethylase (DMIs). Strains with low sensitivity to DMIs are widespread and additional applications of these chemicals are needed to maintain control. DMIs target cytochrome P450 (CYP51), so variation in this protein could explain variation for DMI sensitivity.

Methods: Degenerate primers were used to amplify a fragment of P. fusca CYP51. A heterologous probe from B. graminis CYP51 was employed to identify the correct amplicons and RACE PCR was used to obtain 5´ and 3´ ends. A "step-down" PCR-based technique was applied to amplify the CYP51 upstream region. The CYP51 gene and the promoter region were isolated and sequenced from XX and XX isolates, respectively, which differed in their responses to three DMI fungicides (myclobutanil, triadimenol and fenarimol) in populations of P. fusca in Spain.

Results: The coding and the promoter sequences have been analyzed from 45 and 30 isolates respectively. A transposon-like element upstream of the promoter and 4 new non-synonymous amino acid substitutions in regions related with either substrate binding and catalysis were found. Also, a 400bp deletion in the promoter was found in a hypersensitive isolate. Based on these changes, it was possible to classify the isolates into DMI-resistant and sensitive classes, because of the correlation between mutations and phenotypes.

Discussion: No perfect correlation was found between changes in CYP51 and resistance. This result could be due to other mechanisms (e.g. enhanced gene expression and increased active efflux) present and acting at different resistance level, as previously described in other fungi. The relationship between these changes and gene expression is currently being investigated.



Differential regulation of the two high affinity iron uptake systems in Ustilago maydis

Britta Winterberg, Regine Kahmann, Jan Schirawski

Max Planck Institute for Terrestrial Microbiology, Marburg, Germany

The phytopathogenic basidiomycete Ustilago maydis has two high affinity iron uptake systems: a permease and a siderophore based system. The permease-based system is necessary for full virulence of U. maydis on its host plant maize since deletion mutants of the permease Fer2 are less pathogenic. In contrast, deletion of the two siderophore biosynthetic genes sid2 and fer3 did not affect virulence. Surprisingly, the progeny of germinated spores of sid2fer3 mutants were diploid, indicating a meiosis defect. This defect could be complemented by external supply of iron or siderophores during spore germination. Thus, siderophores are only essential at later stages of fungal development in planta and play a crucial role for storage of iron which is then needed during spore germination.

In several plant pathogens siderophores can act as elicitors of plant defense reactions. Therefore it could be advantageous for U. maydis to prevent siderophore secretion during early plant colonization. We could show that in axenic culture both high affinity iron uptake systems are down regulated by the iron responsive transcription factor Urbs1 in presence of iron. The plant constitutes an iron limiting environment to U. maydis. Therefore, if Urbs1 is the only transcription factor controlling transcription both iron uptake systems should be expressed during plant colonization. RT-PCR analysis showed that the permease based system is induced during all stages of the biotrophic phase while the siderophore biosynthetic genes are only induced at late stages of plant infection. This suggests the existence of an additional transcription factor specifically downregulating siderophore production in response to a plant derived signal. We are currently attempting to identify this transcription factor.



Poplar and pine response to potent pathogen Heterobasidion annosum sl.elicitors

Karl Lundén, Malin Elfstrand

Department of Forest Mycology and Patholgy, SLU, Uppsala, Sweden

Introduction: In an attempt to elucidate early interaction between the basidiomycete Heterobasidion annosum and deciduous and conifer trees, Populus tremula x Populus tremuloides clone T89 and Pinus sylvestris were challenged with spent medium of H.annosum.

Method: Pine and hybridaspen plantlets were inoculated in spent filtered MMN medium from the basidiomycetes H.parviporum FS6 and B1314 for 0, 3, 6, 12, 24, 48 and 72 hours. The saprotroph Phanerochaete chrysosporium RP78 and the mutualistic Paxillus involutus MAJ and NAU as well as Laccaria bicolor were also included in the study.



Poplar and pine response to elicitors of potent and impotent pathogenic and symbiotic basidiomycetes

Karl Lundén, Malin Elfstrand

Department of Forest Mycology and Patholgy, SLU, Uppsala, Sweden

Introduction: In an attempt to elucidate early interaction between compatible and incompatible pathogenic and symbiotic fungi and deciduous and conifer trees, Populus tremula x Populus tremuloides clone T89 and Pinus sylvestris were challenged with spent medium of basidiomycetes.

Method: Pine and hybridaspen plantlets were inoculated in spent filtered MMN medium from the basidiomycetes for 0,3,6,12,24,48 and 72 hours, before harvest.The pathogen H.annosum FS6 and B1314 The saprotroph Phanerochaete chrysosporium RP78 and the mutualistic Paxillus involutus MAJ and NAU aswell as Laccaria bicolor were included in the study. RNA was extracted and real time PCR was performed with defence and mycorrhizal specific primers as AMP, CAD,POX,PR10,Mt21b and ppC61.

Result: The preliminary results from the interaction with pine demonstrates different response patterns both at the trophic level and at the compatibility level of the interaction.

POX production is a clear example as it rapidly showed a high but transient increase with the symbionts P.involutus and L.bicolor between 6 and and 48 hours and a delayed reponse between 24 and 72 hours for H.annosum FS6 and a massive continuously increasing production for B1314 from 24 hour post inoculation. The B1314 isolate originates from poplar whereas FS6 from spruce.

Discussion: Specific patterns of response could be observed in some of the genes which demonstrates that the fungal elicitors alone were enough to make the study. The stability of the elicitors could affect the interpretation of the late time points in the case no production was detected with any of the fungi.



The impact of tillage practices and fertilizer regimens on arbuscular mycorrhizal fungal (AMF) diversity in crop fields investigated by ribosomal and mitochondrial markers

Roberto Borriello1, Erica Lumini1, Roberta Bergero3, Valeria Bianciotto2, Paola Bonfante1

1Università degli Studi di Torino, Dip. Biologia Vegetale, Torino, Italy, 2Consiglio Nazionale delle Ricerche (CNR), Istituto per la Protezione delle Piante, Torino, Italy, 3University of Edinburgh, Institute of Evolutionary Biology, School of Biological Sciences, Edinburgh, United Kingdom

The productivity and health of agricultural systems highly depend on the functional processes involving soil microbial communities. In most agrosystems, AMFs are a main component of soil microbiota and probably represent the most important terrestrial symbiosis.

The aims of this study are multiple. The first one is to describe AMF communities in agriculture fields located in Central Italy (Marche) focussing on the effects of agricultural management practices, such as intensive or moderate tillage, nutrient applications, crop types and rotation. The final goal is to identify agricultural practices that could enhance or cause decline of soil quality as indicated by the presence and diversity of AM fungi communities. The second aim is to develop molecular markers to be tested for the occurrence of genetic exchanges in AMFs. New markers are in fact required to infer ploidy level, segregation, nuclear exchange and also for species delimitation.

DNA extracted from soils and roots is analysed using AM fungal-specific primers to amplify sequences from partial SSU and LSU of ribosomal RNA genes. Cloned fragments obtained from each sample types are analysed by RFLP. We have found 14 and 12 RFLP types for the SSU and the LSU, respectively. Differences of number and composition of RFLP types are found in the different root/soil compartment and in the different tillage. Phylogenetic analysis revealed fungal types belonging to Glomus group A, group B and some member of Diversisporaceae and Gigasporaceae. The ordination analysis indicates that the composition of the AMF community is significantly affected by the tillage system more than by the fertilizer treatment. From our study we can conclude that agricultural practices can directly or indirectly influence AM biodiversity.

In the context of AMF genetic diversity, we looked for single-copy genes and with this purpose we have selected some putative single-copy genes by blasting EST sequences (developed from a G. rosea cDNA library) against several fungal genomes. As a first candidate gene, a mitochondrial gene, Cox1, has been successfully PCR-amplified from the species Gigaspora margarita.


Heterogeneous organization and expression of polyubiquitin genes in the oomycete plant pathogen Phytophthora parasitica

Franck Panabieres

Institut National de la Recherche Agronomique, Sophia Antipolis, France

Oomycetes contain some of the most devastating plant pathogens worldwide. These fungus-like eukaryotes belong to the Stramenopiles, closely related to photosynthetic organisms such as brown algae and diatoms. We investigated the mechanisms underlying pathogenicity of the broad host range oomycete Phytophthora parasitica through construction of cDNA libraries and generation of expressed sequence tags (EST).

Mining these libraries allowed identifying several genes encoding ubiquitin and ubiquitin-like proteins. We report here that P. parasitica ubiquitin genes constitute a multigene family that displays an unusual highly diverse intraspecific organization. We also show that these genes undergo differential expression during several stress conditions and during host infection. Intra- and interspecific comparisons show that distinct mechanisms have driven the evolution of Phytophthora polyubiquitin genes and monomer-encoding genes.


Discovery of pathogenicity genes in the crucifer anthracnose pathogen, Colletotrichum higginsianum, by random insertional mutagenesis

Aurelie Huser, Hiroyuki Takahara, Richard O'Connell

Max-Planck-Institute for Plant Breeding Research, Koeln, Germany

The ascomycete fungus Colletotrichum higginsianum invades Arabidopsis plants by means of a two-stage, hemibiotrophic infection process. Following initial penetration by melanised appressoria, specialised intracellular hyphae develop biotrophically in living epidermal cells before the fungus enters a destructive necrotrophic phase. To identify pathogenicity genes in C. higginsianum, we have taken the approach of random insertional mutagenesis. A library of 8,850 insertional mutants was generated by Agrobacterium tumefaciens - mediated transformation and screened for pathogenicity on Arabidopsis seedlings. Thirty-nine mutants (0.45%) showed reduced pathogenicity, of which five had defects in appressorial melanisation, 12 failed to penetrate without a visible host response, 13 induced papillae or hypersensitive cell death in host cells and three failed to make the transition to necrotrophy. Flanking sequences were recovered from 14 pathogenicity mutants using inverse PCR or TAIL-PCR and used to isolate the disrupted genes from a cosmid genomic library. Eleven of these mutants contained T-DNA insertions at a single position in the genome, of which eight were tandem insertions. Progress in the cytological and molecular characterisation of mutants with disruption in an MFS transporter, an importin and an endoribonuclease will be presented.


Identification of differentially expressed proteins of Verticillium longisporum during infection of Brassica napus (Rapeseed)

Seema Singh, Susanna Braus-Stromeyer, Christian Timpner, Oliver Valerius, Gerhard Braus

Georg-August-Universitaet Goettingen, Georg-August-Universitaet Goettingen, Germany

Verticillium longisporum is a devastating vascular pathogen on rapeseed crops causing wilt disease. It is host specific on the family Brassicaceae. V. longisporum is a soil-borne pathogen that infects through the roots and colonizes the vascular system of the plant. The complex communication between V. longisporum and rapeseed when it penetrates and colonizes the plant has not been elucidated fully yet. During growth of V. longisporum in the xylem vessels, it is likely that some proteins of V. longisporum may be differentially expressed on receiving possible defense signals from the host-plant. Such proteins could play an important role during fungal growth or pathogenesis.

This project aims at the identification of such differentially expressed intra- and extra-cellular proteins. It involved quantitative comparison of proteomes of cell extracts and secretomes of different culture conditions i.e. the fungus cultured with or without xylem sap from Brassica napus by 2D-PAGE. ESI-MS/MS identification of the digested 2-D spots was employed later. Some differentially expressed protein spots were successfully identified. Six protein spots are up-regulated and eight are down-regulated. These identified proteins serve as a basis for the cloning of corresponding coding genes. Reporter constructs of identified genes will be tagged with GFP and integrated in V. longisporum. These constructs will be tested on induction by xylem sap and in planta. This will establish a suitable system for the identification of the possible fungus-plant interaction by changed gene expression.



Characterization of an amino acid permease from the endomycorrhizal fungus Glomus mosseae

Luisa Lanfranco1, Gilda Cappellazzo1, Michael Fritz2, Daniel Wipf3, Paola Bonfante1

1Dipartimento di Biologia Vegetale, Università di Torino, Torino, Italy, 2Institut für Zelluläre und Molekulare Botanik Bonn University Bonn, Bonn, Germany, 3UMR INRA 1088/CNRS 5184/ Université de Bourgogne, Dijon, France

In many ecosystems amino acids in soils reach a considerable amount that could contribute significantly to nitrogen (N) nutrition of plants. To access and compete for this key nutrient with the microbial communities of the rhizosphere, plants have adapted different strategies. One of these is the establishment of symbiotic associations with mycorrhizal fungi.

Arbuscular mycorrhizal (AM), which under natural conditions colonise the majority of root systems, have been mainly considered for their role in phosphate uptake and translocation. AM fungi are also capable of exploiting organic N sources, but the molecular mechanisms controlling the uptake of organic N are largely unknown.

PCR-based approaches, bioinformatic tools and a heterologous expression system were used to characterize a sequence coding for an amino acid permease (GmosAAP1) from the AM fungus Glomus mosseae. GmosAAP1 shows primary and secondary structure similarity with other fungal amino acid permeases. Functional complementation and uptake experiments in a yeast mutant deficient for multiple amino acid uptake system demonstrated that GmosAAP1 is able to transport proline through a proton coupled, pH and energy dependent process. Competitive test showed that GmosAAP1 binds non polar and hydrophobic amino acids indicating a relatively specific substrate spectrum. GmosAAP1 mRNAs were detected in the extraradical fungal structures. Transcript abundance was increased upon exposure to organic N, in particular when supplied at 2 mM concentrations.

These findings suggest that GmosAAP1 may play a role in the first steps of amino acid acquisition, allowing the direct amino acid uptake from the soil.



Compatibility between green orchids and their symbiotic fungi in seed germination and protocorm development

Michele Rodda, Rossana Segreto, Nicola Figone, Mariangela Girlanda, Silvia Perotto

Dipartimento di Biologia Vegetale, Università di Torino, Torino, Italy

A key aspect of orchid biology is their symbiotic association with soil fungi, established at several stages of their life cycle, from seeds to adulthood. Since their seeds are minute and thus characterized by minimal reserves, orchid germination relies on symbiotic fungi to obtain nutrients, including organic carbon, a nutritional strategy known as mycoheterotrophy (MH). By contrast, in their adult stage members of the Orchidaceae develop strikingly different nutritional strategies, ranging from mycoheterotrophy to full photoautotrophy. In such a lifestage MH orchids exhibit very narrow specificity with their fungal partners (which simultaneously establish ectomycorrhiza with neighbouring trees), while the specificity between green orchids and symbiotic fungi is still contentious. Whether fungal partners of adult plants match protocorm symbionts is still under debate. Given the difficulties of cultivating their compatible fungi, MH orchids are less amenable than green orchids to in vitro studies aimed at assessing the genetic mechanisms underlaying the interdependence between orchid and fungi.

Therefore, we have focused on the compatibility between four green Mediterranean orchids and their fungal endosymbionts. We have been investigating the specificity of interaction between different Rhizoctonia-like fungi obtained from highly colonized roots of adult S. vomeracea, A. laxiflora, O. purpurea and A. morio plants testing their ability to germinate seeds of the same species. In preliminary assays with S. vomeracea seeds germination percentage, recorded after 30 days of co-culture was >70% for >85% of the fungi tested (21 strains), while in axenic culture germination was <35%. In spite of high germination percentage, only nine fungal isolates triggered the development of a promeristematic area in the protocorms, confirming their full compatibility with the orchid partner. Protocorms obtained in co-culture with these fungal partners were grown on in vitro till the green leaf stage, and successfully produced highly colonized roots. Assessment of the specificity pattern between S. vomeracea seeds and the fungal isolates tested allowed us to develop a quick and effective propagation protocol, that will permit to use such protocorms as a model system to study the molecular basis of the interaction in a fully mycotrophic stage.



Phases of fungal development and gene expression in Fusarium graminearum during crown rot disease development in wheat

Amber Stephens1, Donald Gardiner1, Kemal Kazan1, Rosemary White1, Alan Munn2, John Manners1

1CSIRO, Brisbane, Queensland, Australia, 2University of Queensland, Brisbane, Queensland, Australia

Fusarium graminearum (Fg) can cause head blight (FHB) and crown rot (CR) diseases in wheat. CR occurs at the stem base of the wheat plant, causing major reductions in grain yield and has been less intensively researched than FHB. Real-time quantitative PCR analysis of fungal genomic DNA relative to plant genomic DNA was used to estimate Fg biomass during CR disease development and using this method three distinct phases of infection were identified. Phase 1 was a statistically significant increase in fungal biomass that occurred in the first 2 days post-inoculation (dpi) of the stem base with Fg macroconidia. Phase 2 was a subsequent significant decrease in fungal biomass for a period of approximately 14 dpi, and this was followed by phase 3 where a massive increase in fungal biomass was observed for the period 14 - 45 dpi. Histological characterisation of Fg colonisation during these three phases of infection revealed that in phase 1 the spores germinated on the stem surface at the point of inoculation forming a superficial hyphal mat. In phase 2, infecting hyphae had moved down from the point of inoculation to the crown area of the wheat seedling and had penetrated and colonised the epidermis of the outer leaf sheath while in phase 3 a major colonisation of the internal tissues of the crown had occurred. Fungal gene expression at 2, 14 and 35 dpi was examined using the Affymetrix GeneChip system comprising 22,000 Fg gene probe sets. Results showed 1,839 Fg genes were significantly up regulated in planta compared to vegetative mycelia, including some known FHB virulence genes (e.g. TRI5 and TRI14). Fungal genes differentially regulated between the phases were identified. These results indicate that CR disease development requires a coordinated process that involves distinct phases of infection and associated fungal gene expression programs.



cell cycle and cell death are not necessary for appressorium formation and plant infection in the fungal plant pathogen Colletotrichum gloeosporioides

Iris Neasher, Sima Barhoom, Amir Sahron

Tel Aviv University, Tel Aviv, Israel

In order to initiate plant infection, fungal spores must germinate and penetrate into the host plant, usually with the aid of appressoria. In the model plant pathogen Magnaporthe grisea completion of mitosis and autophagy cell death of the spore are necessary for appressoria-mediated plant infection; blocking of mitosis prevents appressoria formation, and prevention of autophagy cell death result in non-functional appressoria. We found that in the related plant pathogen Colletotrichum gloeosporioides, blocking of the cell cycle did not prevent spore germination and appressoria formation. Nuclear division was arrested following appressorium formation and was resumed in mature appressoria after plant penetration. The cell cycle always lagged behind the morphogenetic changes that follow spore germination, including germ tube and appressorium formation, differentiation of the penetrating hypha, and in planta formation of primary hyphae. Blocking of mitosis had only marginal effect on appressoria formation; Normal numbers of fully developed mature appressoria were formed under conditions that support appressoria formation. Similar results were also observed in other Colletotrichum species. Spores, germ tubes, and appressoria retained intact nuclei and remained viable for several days post plant infection. Additionally, we could not detect cell death during the entire pathogenic development; Spores, germ tubes appressoria and primary hyphae remained viable throughout the infection cycle. These results show that control of basic cellular processes such as those coupling cell cycle and morphogenesis during fungal infection can be substantially different even between fungal species with similar life styles and pathogenic strategies.



Analysis of the Botrytis cinerea inhibitor of apoptosis gene BcBIR1

Neta Shlezinger, Elad Mochly, Amir Sharon

Tel Aviv University, Tel Aviv, Israel

Fungi undergo programmed cell death (PCD) with classical markers of metazoan apoptosis during various stages of development. PCD is observed in fungi during vegetative incompatibility, in sexual and asexual reproduction, at stationary phase, as well as in aged cultures. Homologs of mammalian apoptotic genes have been identified in fungi, supporting a conservation of the apoptotic machinery between mammals and fungi. However, only little is known on the role of apoptosis in fungi and even less is known on the regulation the associated genes. Our goal is to gain knowledge on the roles of apoptosis in fungi and particularly to determine possible involvement in pathogenic development. Towards this end we have searched the genome of the gray mold fungus Botrytis cinerea and identified a number of putative homologs of mammalian apoptotic genes. In this work we describe the isolation and initial characterization of BcBIR, a B. cinerea homolog of the yeast BIR1 gene (a homolog of mammalian IAP genes). IAPs (inhibitors of apoptosis proteins) are variable in structure and domain composition, but all members of the IAP family of proteins (eight in human) contain up to three baculoviral IAP repeats (BIR). IAPs prevent apoptosis in metazoan through binding to and inhibiting of caspases. BLAST Search of the B. cinerea genome with human IAP or the yeast BIR1 genes as queries identified a single homolog. Analysis of additional fungal genomes revealed a single homolog in most species. Comparison of the various fungal homologs with human IAPs classified them as type II IAP. Over 30 hygromycin-resistant transformants were obtained after transformation with a BcBIR1 knockout cassette. About 80% of them showed homologous integration however, all were heterokaryons. Attempts to obtain homokaryons were unsuccessful. We therefore concluded that BcBIR1 is an essential gene. Over expression reduced expression strains (using RNAi) were generated as well. Initial analysis of these isolates suggests possible involvement in oxidative stress response and pathogenesis.



Translocation of effector proteins from the oomycete Phytophthtora infestans into plant cells

Severine Grouffaud1, Petra Boevink2, Anna Avrova2, Pieter van West1, Paul Birch3, Stephen Whisson2

1University of Aberdeen, Aberdeen, United Kingdom, 2SCRI Invergowrie, Dundee, United Kingdom, 3University of Dundee at SCRI, Dundee, United Kingdom

Like bacteria and fungi, the potato blight pathogen Phytophthtora infestans translocates effector proteins into host plant cells during infection. Whereas bacteria possess the well characterized type III secretion system, the mechanism used by eukaryotic plant pathogens for delivering effector proteins into the host cell remains unclear. In oomycetes this process depends on a short conserved amino acid sequence (RxLR) located near the signal peptide of many secreted proteins. This motif, and its position within secreted proteins, is specific to oomycetes but resembles the host cell targeting-signal found in virulence proteins from the malaria parasite Plasmodium falciparum. Recently, it was shown in our laboratory that the RxLR, and a downstream conserved EER motif, are required for the translocation of the avirulence protein AVR3a into the host cell; AVR3a is recognised by the cognate R3a resistance gene in potato to trigger a hypersensitive resistance response.

A recent study showed that the RxLR motif from AVR3a was sufficient to export the green fluorescent protein (GFP) from P. falciparum to the erythrocyte, suggesting a conserved mechanism to deliver effector/virulence proteins into host cells. Supposedly, the host targeting signal used by the malaria parasite could function in P. infestans; moreover, we would expect that the RxLR motifs found in avirulence proteins from other oomycetes can also function in P. infestans, but these hypotheses still have to be demonstrated. We have used the AVR3a-R3a interaction as a reporter for translocation in P. infestans transformants and replaced the AVR3a RxLR-EER motif with the motifs from a related oomycete or from the malaria parasite. Transformation of a virulent P. infestans isolate with the various Avr3a constructs yielded avirulent transformants, implying that the alternative sequences are functionally similar to the native RxLR-EER.

Fluorescent protein fusions to translocated effectors to examine the host subcellular targeting of P. infestans effectors are also being performed and progress will be presented.




Three different cullins are neddylated in Aspergillus nidulans

Marcia Kress, Oliver Valerius, Gerhard Braus

Institute of Microbiology and Genetics, Georg August University, Göttingen, Germany

The NEDD8/RubA protein belongs to the Ubiquitin-like protein group and its conjugating process is mechanistically similar to ubiquitination. In this conjugating process NEDD8/Rub1 is activated by E1 enzyme (Uba3/Ula1) and conjugated by E2 enzyme (Ubc12). The known substrates of the NEDD8, in a process called neddylation, are the Cullin family proteins, and the activity of COP9 Signalosome Complex is required for the de-neddylation of the neddylated cullins. The neddylation pathway is essential in mammalian cells, C.elegans and fission yeast. NEDD8 is synthesized in a precursor form with a C-terminal extension peptide that ranges from 1 to 8 aminoacids depending on the species. These de novo synthesized precursors are accurately cleaved at the C-terminal site to form the mature NEDD8. The exposure of the C-terminal glycine (Gly) residue of NEDD8 is essential for its subsequent conjugation reaction. In this work, the filamentous ascomycete A. nidulans was used as a model organism for the identification of RubA-interacting proteins and RubA-substrates. The phenotype of the ΔrubA null allele was defined and has been confirmed by Heterokaryon rescue technique that rubA gene is essential in A. nidulans. Through the protein complex purification of TAP-tag::rubA and TAP-tag::cDNArubA strains of A. nidulans, some expected proteins were isolated and identified by Mass Spectrometry analysis. Among the detected proteins by Mass Spectrometry are all cullins described until now for A. nidulans (CulA, C and D), two E1 enzymes already described in the literature as partners of Nedd8/Rub1 (UlaA and UbaC), as well RubA and other proteins as ribosomal proteins, transcription facto protein, chaperonine and F-box proteins.



The Penicillium chrysogenum antifungal protein PAF – a signaling molecule?

Nikoletta Hegedüs1, 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

The filamentous fungus Penicillium chrysogenum secretes the low molecular weight, highly basic and cysteine-rich antifungal protein (PAF) that inhibits growth of numerous filamentous ascomycetes in vitro. The fact that PAF is abundantly secreted into the supernatant of pure stationary phase cultures and shows no growth inhibition against Penicillium itself allows us to speculate on a more basic function beside its cytotoxicity for other fungi.

Therefore, we started to study the function of PAF for P. chrysogenum. To this end we generated a paf deletion strain by employing the marker split method. P. chrysogenum protoplasts were sucessfully transformed at a frequency of 37 transformants per 10 μg DNA. Fifteen nourseothricine resistant clones were screened for single homologous integration of the transforming DNA by Southern blotting. Four positive clones were selected for further phenotype analysis.

We found that conidiation of the deletion strains is delayed under various growth conditions, e.g. induction of oxidative stress or nutrient starvation. This points out that PAF might be involved in cellular signaling in P. chrysogenum. Experiments are underway to prove our hypothesis and to characterize in more detail the effect of the deletion of paf on the physiology and the development of P. chrysogenum.

This work is supported by the Austrian Science Foundation (FWF 19970), the Austrian National Bank (OENB 9861) and the ÖAD (Austrian Exchange Service).



The Penicillium chrysogenum antifungal protein PAF disrupts calcium signalling in Neurospora crassa

Ulrike Binder1, Nick D. Read2, Florentine Marx1

1Biocenter, Division of Molecular Biology, Innsbruck Medical University, Innsbruck, Austria, 2Institute of Cell Biology, University of Edinburgh, Edinburgh, United Kingdom

The small, basic and cysteine-rich protein PAF is secreted by Penicillium chrysogenum and exhibits growth inhibitory activity against various members of filamentous ascomycetes including human and plant pathogens. We are interested in the elucidation of the mechanism of action of this antifungal protein. Previous investigations showed that PAF toxicity is related to plasma membrane hyperpolarization and the activation of ion channels. We hypothesize that PAF toxicity results, at least in part, in the perturbation of calcium homeostasis.

In this study we found that minimal concentrations of calcium neutralize PAF activity. Furthermore, we detected a PAF–induced elevation of the calcium resting level in Neurospora crassa expressing the calcium sensitive photoprotein aequorin. This increase in cytosolic free calcium is PAF-concentration dependent. High concentrations of PAF inhibited the amplitude of the calcium responses to mechanical perturbation and hypoosmotic shock. The addition of the extracellular calcium-selective chelator BAPTA abrogated the PAF induced elevation of the calcium, indicating that an influx of extracellular calcium is responsible for this effect.

This work is supported by the Austrian Science Foundation (FWF 19970), the Austrian National Bank (OENB 9861) and the Innsbruck Medical University (i-med Auslandsstipendium).



Transformation of progesterone by a progesterone-induced multienzyme hydroxylating system in Fusarium oxysporum

Anna Poli, Helena Lenasi

University of Ljubljana, Ljubljana, Slovenia

The ability to hydroxylate steroidal substrates including progesterone was demonstrated in several fungi including members of the genus Fusarium. Hydroxylated compounds are more water soluble, and easily removed from the mycelia, if surrounded by water: the meaning of the hydroxylation process is most likely to be detoxification. In the present study we investigated the progesterone-response in the vascular wilt fungus Fusarium oxysporum. Specifically, the hypothesis that a multienzyme hydroxylating system could be induced by the presence of the steroid was tested.

Progesterone was applied as inducer to the liquid growth medium for 120 min; following washes of the mycelium the steroid was supplied as a substrate for another 30 min. Steroids were extracted from mycelium and medium, respectively. The content of progesterone and progesterone transformed products was measured by HPLC and identified by gas chromatography/mass spectrometry.

Progesterone was shown to induce steroid hydroxylation: the content of hydroxylated product significantly increased from 1.5 ± 0.63% (control) to 9.42 ± 0.63% (induced). Progesterone was mainly transformed into 15α-hydroxyprogesterone, which was mostly present in the medium. In order to test whether mitogen activeted protein kinase MAPK, G proteins and histidine kinase were involved in the signling leading to the induction of the enzyme system the following mutants were studied: Δfmk1 (MAPK), Δfgb1 (G protein subunit β) , Δfh1 (histidine kinase) and Δfmk1/Δfgb1 (MAPK and G β double mutant). G β proteins seem to be involved in progesterone signaling: no steroid hydroxylation occurred in Δfgb1. Exogenous cAMP restored the ability of this mutant to transform progesterone, indicating the the signaling is mediated by cAMP-PKA pathway.

In the presence of progesterone fungal growth is inhibited, as revealed by toxicity tests; therefore, we can assume that the biological role of progesterone-induced enzymes is most likely transformation of toxic progesterone into a less toxic product.


The gene for the ubiquitin ligase regulator Cand1 is split into two genes in the filamentous fungus Aspergillus nidulans

Elke U. Schwier1, Martin Christmann1, Krystyna Nahlik1, Silke Busch2, Gerhard H. Braus1

1Georg-August University, Göttingen, Germany, 2Zentrum für Arzneimittelforschung, Entwicklung und Sicherheit, Frankfurt, Germany


Ubiquitin dependent proteolysis plays an important role in many cellular processes in eukaryotes. Cullin containing ubiquitin ligases like the SCF (Skp1-Cullin-F-box protein) complex mark proteins for degradation by ubiquitinylation. It has been shown that the protein Cand1 (cullin-associated Nedd8-dissociated protein 1) binds to cullins. The Cand1 C-terminus blocks the SKP1 binding site of the SCF complex component Cul1 and affects thereby the assembly/disassembly of the ubiquitin ligase. The N-terminus of Cand1 buries the neddylation site on Cul1, which prevents its modification by Nedd8, an ubiquitin-like protein and alters the activity of the complex.

In Aspergilli, the corresponding gene is split into two independent open reading frames encoding the N- and C-terminal part (candA-N, candA-C) of the mammalian homolog. Deletion of the single or both candA genes leads to mutants with identical phenotypes. They produce only few asexual spores, are blocked in early sexual development and appear dark red when grown under development inducing conditions. All defects of the candA deletion mutants can be complemented by a candA-N::C fusion construct indicating that the split is not crucial for protein functions. Only CandA-C, but not CandA-N, binds to cullins. Since both CandA proteins interact with each other, binding of CandA-N to cullin is presumably mediated by CandA-C. CandA-C is nuclear enriched and expressed in vegetative cultures but degraded at an early stage of sexual development indicating a role during onset of development. The split candA gene makes the fungus A. nidulans an attractive model organism for studying the putative different functions of the two parts of the Cand1 protein.



Palc, one of the two Bro1 domain proteins in the fungal signalling pathway, localizes to cortical structures and binds Vps32

Antonio Galindo1, América Hervás-Aguilar1, Olga Rodríguez-Galán1, Olvier Vincent2, Herbert N. Arst3, Joan Tilburn3, Miguel A. Peñalva1

1Centro de Investigaciones Biológicas-Csic, Madrid, Spain, 2Instituto de Investigaciones Biomédicas-CSIC, Madrid, Spain, 3Imperial College, London, United Kingdom

In the fungal pH signal transduction pathway six proteins are required for the proteolytic activation of the transcription factor PacC/Rim101 (denoted PalA, PalB, PalC, PalF, PalH and PalI in Aspergillus nidulans). Previous work suggested that the pH pathway is organized into two complexes, an "upstream" complex (involving PalH, PalI, and PalF) at the plasma membrane and a "downstream" complex on membranes of the endosomal system (involving PalA, which binds Vps32, PalB and PacC/Rim101). Prior to this work, PalC, which contains a Bro1 domain, was the only protein in the pH pathway which had not been placed in either of the pH signalling complexes and its orthologue in Saccharomyces cerevisae had not been identified. PalC-GFP is recruited to plasma membrane associated punctuate structures in an ambient pH- and 7-TMD receptor PalH dependent manner. PalC is a two hybrid interactor of the ESCRT-III Vps20/Vps32 subcomplex and binds Vps32.This binding is impaired by Pro439Phe, Arg442Ala and Arg442His substitutions in a conserved region mediating interaction of Bro1p with Vps32p, but these do not prevent cortical punctuate localization, indicating Vps32 independence. In contrast, Arg442∆ impairs Vps32 binding and prevents PalC-GFP recruitment to cortical structures. All these single residue substitutions result in a loss of function phenotype without dramatically affecting protein stability, which indicates that the PalC/Vps32 interaction has a physiological role.Vps32-GFP or Vp32-mRFP localizes to motile specks that resemble FM4-64 and GFP-Rab5 labelled early endosomes. In contrast, PalC-GFP structures are static. This work represents a significant advance in our understanding of the fungal pH pathway, because a key issue is how the two complexes above mentioned communicate to initiate the proteolysis of PacC. Evidence indicates that this connection involves endocytic trafficking. By means of its ability to localize to cortical structure and to bind Vps32, PalC has the expected features of a link between the two pH signalling complexes. We hypothesize that the role of PalC in its cortical location is determining identity on membranes and recruiting the endosomal Vps20/Vps32 ESCRT-III coat, which PalC binds. One working hypothesis is that maturation of these membranes following endocytosis would result in Vps32-mediated recruitment of PalA with the PalC release and the formation of the "downstream" complex.



On the involvement of ESCRT-III in pH signal transduction in Aspergillus nidulans

Olga Rodríguez-Galán1, Antonio Galindo1, Juan Francisco Abenza1, América Hervás-Aguilar1, Herbert N Jr Arst2, Miguel Ángel Peñalva1

1Centro de Investigaciones Biológicas, CSIC, Madrid, Spain, 2Imperial College London, London, United Kingdom

The mechanism of MVB (Multivesicular body) cargo sorting in endosomal membranes is conserved from fungi to mammals and mediated by ‘class E’ Vps proteins. ‘Class E’ proteins are functionally organised in four endosomal sorting complexes required for transport (ESCRTs 0, I, II, III). Work in Saccharomyces cerevisiae demonstrated that ESCRT-I, ESCRT-II and the Vps32/Vps20 subcomplex of ESCRT-III additionally play a key role in the fungal ambient pH signal transduction pathway. Vps32 interacts directly with two proteins in the pH signal transduction pathway, PalA and PalC. Promoter-replacement experiments strongly indicated that the ESCRT-III ‘core’ component Vps32 in essential in A. nidulans. Vps32-GFP, expressed at relatively low levels, localises to motile, cytosolic punctate structures resembling early endosomes as well as to larger, static structures associated with vacuoles. Vps32-GFP overexpression results in predominance of the fusion protein in the latter that, we propose, represent class E compartments, by analogy with the situation in mammalian cells, where overexpression of CHMP4A with a C-terminal GFP attachment causes a dominant-negative phenotype. Vps32-GFP overexpression results in alkaline pH sensitivity, which agrees with a role of ESCRT-III in pH regulation.

We have also analysed the role in pH signal transduction of the ESCRT-III peripheral component Did2/CHMP1 (DidB in A. nidulans), which had not yet been analysed in any organism. DidB-GFP, expressed at physiological levels, localizes to punctuate motile structures resembling early endosomes. DidB is a direct interactor of Vps4, the AAA ATPase that mediates the disassembly of ESCRT complexes from endosomal membranes. A didB∆ mutation is viable. didB∆ results in abnormally elevated plasma membrane levels of the endocytic tracer FM4-64 and of the transmembrane protein AgtA-GFP, an endocytic cargo which in the wild-type is efficiently sorted into the MVB pathway. didB∆ exacerbates the accumulation of Vps32-GFP in structures associated with the vacuole and, in agreement with the weak ‘class E’ phenotype resulting from the equivalent did2∆ mutation in yeast results in a slight sensitivity to alkaline pH.



Exploration of the role of the group III histidine kinase AbNIK1 in the necrotrophic seed-borne pathogen Alternaria brassicicola

Claire Campion, Thomas Guillemette, Nelly Bataillé-Simoneau, Anita Dongo, Beatrice Iacomi-Vasilescu, Bruno Hamon, Philippe Simoneau

University, Angers, France

In filamentous fungi, adaptation to high osmolarity conditions is regulated by a MAP kinase cascade, referred as the osmosensitivity (Os) pathway. Several studies have demonstrated the key role of group III histidine kinases (GIII-HK), as positive regulators of Os and Os-related pathways. It has also been suggested that GIII-HK might have essential functions in the response of fungi to oxidative stress and in developmental processes such as conidiation. In the present study, we explored the role of the GIII-HK AbNIK1p from the necrotrophic seed-borne pathogen Alternaria brassicicola using comparative phenotypic characterization of AbNIK1 mutant and wild-type isolates. AbNIK1 mutant isolates were either selected among dicarboximide fungicides resistant field or laboratory isolates or generated by targeted gene disruption. Phenotypic characterization was performed for different fitness components and for the sensitivity towards two synthetic antifungal drugs (fludioxonil and ambruticin) designed on the basis of natural bacterial antibiotics. The parameters measured to evaluate the saprophytic fitness were mycelial growth under standard and stress conditions, sporulation, and conidial germination. The effects of AbNIK1 mutations on the parasitic fitness were estimated under both controlled and field conditions after inoculation of host plants by individual isolates, to measure their aggressiveness, or by mixed conidial suspensions, to measure their competitive ability against wild-type isolates. Results showed that, unlike Botrytis cinerea, A. brassicicola-null mutants for GIII-HK had no significant reduction in characteristics that determine the saprophytic fitness, except for an increased sensitivity to the exposure to high concentrations of neutral sugars and high resistance levels towards fludioxonil and ambruticin. The cost for this phenotypic trait was a loss of competitiveness in non-selective environment that mainly affected the fungus during the seed colonization stage of the infection process, i.e. when the fungus had to overcome severe osmotic stress conditions consecutive to the gradual decrease in the water potential in maturing seeds. These results will be discussed with special reference to recently published data obtained with GIII-HK-null mutants from other fungal species.


The cAMP-protein kinase A pathway is dispensable for cellulase gene expression in Hypocrea jecorina (=Trichoderma reesei)

André Schuster, Doris Tisch, 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 cAMP-pathway represents a central signalling cascade with crucial functions in all organisms. In H. jecorina, cAMP is known to enhance cellulase gene expression if added in low concentrations to cultures induced by sophorose, whereas high concentrations have been reported to repress their synthesis. We were therefore interested, whether deletion of two components of the cAMP pathway, adenylyl cyclase (acy1) or protein kinase A (pka1; catalytic subunit) would have an impact on cellulase gene expression. While deletion of pka1 caused severely impaired growth with strongly reduced hyphal elongation, loss-of-function of acy1 resulted in an almost normal growth phenotype. However, closer inspection of this mutant by microscopy revealed very fragile hyphal tips, which tend to break even when handled cautiously during manipulation. Surprisingly, both mutations did not impair cellulase gene expression on cellulose or after induction by sophorose. Consequently, despite an enhancing action of exogenously added cyclic AMP, endogenous cAMP formation and thus the cAMP signalling pathway is not necessary for cellulase gene expression.



Approaching the morphogenetic program of Aspergillus niger by transcriptomics

Vera Meyer1, Mark Arentshorst2, Simon Flitter2, Ulf Stahl1, Benjamin Nitsche2, Cees van den Hondel2, Arthur Ram2

1Berlin University of Technology, Department Microbiology and Genetics, Berlin, Germany, 2Leiden University, Department Molecular Microbiology, Leiden, Netherlands

Aspergillus niger is used in industrial biotechnology as cell factory for the production of chemicals, pharmaceuticals and proteins. During the last years, however, it has become clear that the morphological features of A. niger in fermentation processes are critical to maximum product output. To accomplish a basis for rational genetic engineering of the morphology of A. niger, much more basic knowledge is required to obtain a deeper insight into the molecular networks regulating the morphology of A. niger.

Newly established tools such as the availability of the genome sequence of A. niger and of the corresponding Affymetrix DNA microarrays make it now feasible for the first time to study the morphogenetic program of A. niger in a genome-wide approach.

In order to get first insights into the regulatory networks involved in the morphogenetic program of A. niger, we made use of two transcriptomic approaches. The first one involved the treatment of young germlings of A. niger with sub-lethal concentrations of antifungal compounds (e.g. caspofungin, inhibitor of β-1,3 glucan synthesis and fenpropimorph, inhibitor of ergosterol synthesis). The rationale behind this approach was based on the observation that A. niger adapts to the inhibitory effect in such a way that it continues to grow by the establishment of new polarity axes and formation of new germ tubes. The second approach made use of the conditional mutant strain Ramosa-1. We found out that this strain represents an excellent and easy controllable model system to study different aspects of fungal polarity, as all critical steps of hyphal morphogenesis (establishment and maintenance of new polarity axes) can be altered in Ramosa-1 by controlling the growth temperature during cultivation.

Transcriptomic data from both approaches revealed that different processes seem to participate in the morphogenesis of A. niger, such as cell signalling (TOR, CWI, PIP2), cell wall biosynthesis, cytoskeletal organisation, oxidative stress response and others. The prospective elucidation of the corresponding differentially expressed genes will reveal to what extent they contribute to the morphogenesis of A. niger.



The role of an Aspergillus nidulans Rab5 homologue in endocytosis and membrane trafficking

Juan Francisco Abenza, Juan C Sanchez-Ferrero, Jose M Rodriguez, Miguel A Peñalva, Areti Pantazopoulou

Centro de Investigaciones Biologicas, CSIC, Madrid, Spain

Rab GTPases have been implicated as key regulatory molecules in all the events of membrane traffic in eucaryotic cells. The small GTPases of the Rab5 subgroup have been spatially connected to the early endosomal compartment and to clathrin coated vesicles pinching off the plasma membrane (PM) and have been implicated in the internalization of endocytic markers, the regulation of homotypic fusion and morphology of early endosomes and the biogenesis of endosomes and vacuoles.

Bioinformatic analysis of the A. nidulans proteome revealed the presence of three members of the Rab5 subgroup, one of which is a close homologue of animal cell Rab21/Rab22, whereas the other two are close relatives of human Rab5A/B/C and yeast Ypt51p/52p/53p. In this work we have studied AnRab5A, a protein presenting ~50% amino acid sequence identity to Ypt51p and human Rab5A/B/C. AnRab5A contains a conserved amino acid sequence motif in helix a2 which is characteristic of the Rab5 subgroup. The gene is expressed in hyphae. A deletion mutant is viable, grows like the wild type at 37ºC or below on several carbon and nitrogen sources and in a range of ambient pH from acidic to alkaline values, but its growth is impaired at 42°C. Bulk membrane internalization, visualized with FM4-64 at 25ºC, is unaffected in the null mutant. We have studied the degradative endocytic pathway and the PM-early endosome-Golgi-PM recycling pathway in the wild-type vs. the rab5A∆ mutant, using the AgtA amino acid transporter and the v-snare SynA/Snc1p, respectively, as prototypic cargoes. AnRab5A fused to GFP is localized in cytosolic punctate structures (~0.5 μm in size), that move bidirectionally on microtubules with a speed of 2-2.4μm/sec and resemble the highly motile structures that are labelled with FM4-64. This punctate localization is dependent on the Ser29, as in the S29N mutant form (GDP-locked -inactive- conformation) the protein is cytosolic. Overexpression of GFP-AnRab5A driven by the inducible alcA promoter does not seem to affect the morphology or the size of the punctate structures. This study is a starting point for dissecting the early steps of endocytosis in A. nidulans.



Protein kinase a regulates growth, sporulation and pigmentation in Aspergillus fumigatus

Thorsten Heinekamp, Christina Große, Olaf Kniemeyer, Axel A. Brakhage

Leibniz Institute for Natural Product Research and Infection Biology – Hans-Knöll-Institute (HKI), Jena, Germany

Aspergillus fumigatus is an opportunistic human pathogenic fungus, causing severe infections with invasive growth in immunocompromised patients. Cyclic AMP signal transduction plays an important role for virulence. A central component of this signaling cascade is the protein kinase A (PKA), which regulates cellular processes by phosphorylation of specific target proteins. Here, we describe the generation and analysis of A. fumigatus mutants expressing the gene encoding the catalytic subunit of PKA, pkaC1, under control of an inducible promoter. Overexpression of pkaC1 resulted in high PKA activity. Mutants showed reduced growth, sporulation deficiency and formation of a dark pigment in the mycelium, indicating that cAMP/PKA signaling is involved in the regulation of important processes like growth, asexual reproduction and biosynthesis of secondary metabolites. Furthermore, deletion of the gene pkaR, encoding the regulatory subunit of PKA, resulted in elevated PKA activity and induced expression of the pksP gene. The polyketide synthase, encoded by pksP, is an essential enzyme for production of dihydroxynaphthalene (DHN) melanin in A. fumigatus. Our results suggest, that increased pksP expression is responsible for pigment formation in the mycelium. Comparative proteome analysis of the pkaC1 overexpressing strain and the wild-type strain led to the identification of proteins regulated by the cAMP/PKA signal transduction pathway. We showed, that elevated PKA activity resulted in activation of stress-associated proteins and of enzymes involved in protein biosynthesis and glucose catabolism. By contrast, proteins which were involved in nucleotide and amino acid biosynthesis are downregulated as well as enzymes involved in catabolism of C-sources other than glucose.



Light-dependent cellulase regulation in Hypocrea jecorina (anamorph Trichoderma reesei) and the influence of heterotrimeric G-protein signaling on this process

Monika Schmoll, Gabriela Gremel, Marcel Dorrer, Roberto do Nascimento Silva, Christian Kubicek

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

H. jecorina is one of the most potent cellulase producers known to biotechnology and also a powerful host for heterologous protein production. We therefore intend to elucidate signal transduction pathways with respect to their impact on regulation of cellulase gene expression. After the finding that the light regulatory protein ENVOY is involved in this process, the light dependence of cellulase gene regulation initiated investigation of this process from a new perspective. Our analysis of the G-alpha subunits GNA1 and GNA3 revealed that both are involved in modulation of cellulase regulation, but not responsible for induction. Interestingly, the observed effects are dependent on the light status in both cases. Deletion of gna1 causes strongly increased cellulase transcription in darkness, while constitutive activation of GNA3 causes a comparable effect in light. Recently, during investigation of sulphur utilization of H. jecorina a further influencing variable of cellulase gene expression became obvious. H. jecorina needs sulphate for growth on cellulose in light, irrespective of the presence of methionine. Moreover, addition of methionine considerably enhanced cellulase transcription in darkness, but abolished it in light. Since methionine-sensing G-protein coupled receptors have been detected in fungi, we conclude that methionine could represent one of the signals transduced by the heterotrimeric G-protein pathway targeting cellulase gene regulation. Due to the opposite effect of this signal in light and darkness, its significance most likely reaches beyond detection of a favourable sulphur source.


Trichoderma atroviride G protein-coupled receptors of the cAMP receptor-like class: isolation, transcriptional regulation and involvement in host sensing

Kurt Brunner, Marion Pucher, Marizela Delic, Patrick Domnanich, Markus Omann, Susanne Zeilinger

Vienna University of Technology, Vienna, Austria

G alpha α subunits affect host recognition and activation of the mycoparasitic response in Trichoderma atroviride. To extend our knowledge on G protein signalling during Trichoderma biocontrol, we aimed to analyse G protein-coupled receptors (GPCRs). As the genome sequence of T. atroviride is not publicly available yet, we carried out an in silico exploration of the genome of Trichoderma reesei, a close relative. 18 genes encoding putative GPCRs distributed over eight classes and additional 31 proteins similar to the Magnaporthe grisea PTH11 receptor were identified in the T. reesei genome.

Four T. atroviride genes were obtained by a PCR-based approach aiming at the isolation of cAMP receptor-like GPCRs and their membership confirmed by phylogenetic and topological analyses. All four receptor-encoding genes showed lowest expression on glycerol and N-acetyl-glucosamine and highest mRNA levels when transferring the fungus to medium without any carbon source. In addition, transcription of gpr3 and gpr4 responded to exogenously added cAMP and the shift from liquid to solid media. gpr3 mRNA levels also responded to the presence of fungal hyphae or cellulose membranes, while gpr1, gpr2 and gpr4 only showed moderate transcriptional regulation under these conditions.

Transcription of gpr1 was silenced and resulted in avirulent mutants unable to attack and overgrow respective host fungi, whereas gpr2-silenced mutants behaved like the parental strain and showed typical mycoparasitism of phytopathogenic fungi.


Involvement of three G Protein alpha subunits and an adenylyl cyclase in sexual development of the homothallic fungus Sordaria macrospora

Jens Kamerewerd1, Malin Jansson1, Minou Nowrousian1, Stefanie Pöggeler2, Ulrich Kück1

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

In the past years, an involvement of G Protein α subunits and adenylyl cyclases in sexual development of some filamentous fungus was demonstrated. However, most of the studies were done with heterothallic fungi. As a homothallic fungus, S. macrospora has not to be fertilized to enter the sexual lifecycle. Nevertheless, previous work has shown, that S. macrospora expresses a functional pheromone/receptor system within a single cell. While in heterothallic fungi mutations affecting fertilization can not be analyzed further regarding fruiting body development, this is not the case for S. macrospora. Besides, S. macrospora lacks any asexual cycle which might interfere with components of the sexual cycle, making this fungus an ideal model to study sexual development. We generated knock-out strains for all three Gα subunit genes (gsa1-3) of S. macrospora as well as all combinations of double mutants. Phenotypic analysis of single and double mutants showed that the genes for Gα subunits have distinct roles in the sexual life cycle. While single mutants show some reduction of fertility, double mutants ∆gsa1∆gsa2 and ∆gsa1∆gsa3 are completely sterile. To test whether the pheromone receptors PRE1 and PRE2 mediate signalling via distinct Gα subunits, two recently generated ∆pre strains were crossed with all ∆gsa strains. Analysis of the corresponding double mutants revealed that compared to GSA2, GSA1 is a more predominant regulator of a signal transduction cascade downstream of the pheromone receptors and that GSA3 is involved in another signaling pathway which also contributes to fruiting body development and fertility. We further isolated the gene encoding the adenylyl cyclase (sac1) for construction of a knock-out strain. Analysis of the three ∆gsa∆sac1 double mutants and a ∆gsa2∆gsa3∆sac1 triple mutant indicate that SAC1 acts downstream of GSA3, parallel to a GSA1-GSA2 mediated pathway. Here we propose a model for how different Gα subunits interact with pheromone receptors or adenylyl cyclase and thus cooperatively regulate sexual development in S. macrospora.


Expression of the red fluorescent protein mCherry during the circadian rhythm of Neurospora crassa

Ernestina Castro-Longoria1, Michael Ferry2, Stuart Brody3, Salomon Bartnicki1

1Department of Microbiology, CICESE, Ensenada, Baja California, Mexico, 2Department of Bioengineering, UCSD, San Diego, CA., United States, 3Division of Biology/Molecular Biology, UCSD, San Diego, CA., United States

The circadian rhythm in Neurospora crassa is expressed by the production of conidiation bands alternated by interbands with low or null conidiation. The rhythm is regulated by a transcription/translation clock that revolves around a feedback loop involving the central frequency (frq) gene. In N. crassa, a number of clock-controlled genes (ccgs) have been identified. The ccgs accumulate mRNA rhythmically and ccg-1 and ccg-2 have been shown to be regulated by the clock at the level of transcription. In this work, we describe the behavior of a band (bd) strain of N. crassa containing the ccg-2 promoter driving the production of the red fluorescent protein (RFP) mCherry (Abs/Em 587/610 nm). Colonies were grown in special chambers and scanned from the edge inward by confocal microscopy. In fully grown colonies scanned at low magnification (10x), we noticed a strong accumulation of mCherry in the conidiation bands, i.e. aerial hyphae and conidia, but not in the interbands. To trace the expression or accumulation of mCherry we scanned growing colonies at higher magnification (100x) every hour for an entire circadian cycle. During development of the interband, mCherry was detected in vegetative hyphae at the growing edge of the colony. The level of expression increased gradually and reached maximal intensity during the first 3h. The intensity remained high for the following 7h and then decreased rapidly during the next 2 h. During development of the conidiation band, the expression of mCherry in vegetative peripheral hyphae was very low during the entire phase (11h), but a strong fluorescence developed in aerial hyphae and conidia. It remains to be seen whether the observed differences in mCherry accumulation in the colony represent circadian control of gene expression or are due to differential transport of the RFP from vegetative hyphae to aerial hyphae and conidia, or both. If differential transport is excluded, do differences in mCherry expression suggest the existence of spatially different morphogenetic control of the promoter? Clearly, the Pccg-2 - mCherryNC construct is a useful tool for studying gene dynamics in aerial hyphae development and conidiogenesis of fungi and may provide a new tool for labeling proteins involved in the morphogenesis and apical growth of N. crassa.


Opening a window for understanding the cell signaling processes in the filamentous fungus Tuber borchii: RhoGdi, another element in the molecular network

Antonella Amicucci1, Michele Menotta1, Giorgio Basili1, Emanuela Polidori2, Paola Ceccaroli1, Vilberto Stocchi1, Francisco Rivero3

1Istituto di Chimica Biologica "G. Fornaini", Università degli Studi di Urbino "Carlo Bo", 61029 Urbino (PU), Italy, 2Istituto di Ricerca sull’Attività Motoria, Università degli Studi di Urbino "Carlo Bo", 61029 Urbino (PU), Italy, 3Zentrum für Biochemie, Medizinische Fakultät, Universität zu Köln, 50931 Köln, Germany

Little is known regarding the cell signaling processes underlying apical growth in fungi belonging to the genus Tuber. Several genes and proteins mainly involved in the hyphal membrane and cell wall development were previously identified. However, very limited information is available on the signaling pathways that regulate cytoskeleton reorganization during the life cycle of ascomycetous ectomycorrhizal fungi. Previously we showed that T. borchii Cdc42, a Rho GTPase, is involved in the polarized growth and that it has a fundamental role in the cytoskeleton organization. Rho GTPases are regulated by Guanine nucleotide exchange factors (GEFs), GTPase-activating proteins (GAPs) and by GDP-dissociation inhibitors (GDIs), whose function is not only to inhibit the spontaneous dissociation of bound guanine nucleotide (usually GDP) from their partner GTPases, but they can actively contribute to the delivery of Rho proteins to specific subcellular membranes and signaling pathways. Herein we report a study on the RhoGDI homolog of Tuber borchii Vittad. The Tbgdi gene is present in two copies in the T. borchii genome. The predicted amino acid sequence shows high similarity to other known RhoGDIs. Real time PCR analyses revealed an increased expression of Tbgdi during the pre-symbiotc phase, in particular after stimulation with root exudates extracts, that correlates with expression of Tbcdc42. Using a yeast two-hybrid approach TbRhoGDI was found to interact with TbCdc42 but surprisingly not with S. cerevisiae Cdc42, precluding the use of yeast as a surrogate model for functional studies. To study the role of TbRhoGDI we performed complementation experiments using a RhoGDI null strain of Dictyostelium discoideum, a model organism where the roles of Rho signaling pathways are well established. For comparison, complementation with mammalian RhoGDI1 and LyGDI was also studied in the null strain. Although interacting with Rac1 isoforms, TbRhoGDI was not able to revert the defects of the D. discoideum RhoGDI null strain, but displayed an additional negative effect on the cAMP-stimulated actin polymerization response. TbRhoGDI appears as an important modulator of cytoskeleton reorganization during polarized apical growth. These results permit to broaden our knowledge of the cell signaling at the basis of cytoskeleton reorganization, necessary for apical growth and ectomycorrhizal development.



The COP9 signalosome influences cell wall degradation and development of Aspergillus nidulans

Krystyna Nahlik1, Silke Busch2, Elke U. Schwier1, Birte Könnecke1, Reiner Hitt3, Gerhard H. Braus1

1Institut für Mikrobiologie und Genetik, Göttingen, Germany, 2Zentrum für Arzneimittelforschung, Entwicklung und Sicherheit, Frankfurt, Germany, 3Transkriptom Analyse Labor - Georg-August-Universität, Göttingen, Germany

Fruit body formation in the filamentous fungus Aspergillus nidulans requires complex genetic regulation and mobilization of energy, stored mainly in cell wall glucans. The COP9 signalosome (CSN), a conserved eukaryotic multiprotein complex playing a role in the regulation of protein degradation by the ubiquitin system, is necessary for sexual fruit body formation and establishment of balance between sexual and asexual development in this fungus.

We have analysed the mechanism of CSN action during development by genome-wide transcriptome profiling and proteome analyses of a csnE deletion strain. We show that the fungal CSN affects the regulation of distinct groups of genes, including oxidoreductases, genes involved in secondary metabolite biosynthesis and cell wall glucan degradation. Furthermore, CSN is required for expression of a hormone synthesis involved in regulation of developmental balance and necessary for developmentally induced b -glucanase activity, which might be prerequisite for fruit body formation.


The heme pathway in Aspergillus niger

A.C.W. Franken1, B.C. Lokman2, A.F.J. Ram1, C.A.M.J.J. van den Hondel1, S. de Weert1

1Leiden University, Institute of Biology Leiden, Leiden, Netherlands, 2HAN Biocentre, Nijmegen, Netherlands, 3Kluyver Centre for Genomics of Industrial Fermentation, Delft, Netherlands

The production of enzymes by microorganisms as "biofriendly" replacements for chemical and polluting industrial processes has gained increasing attention over the last years. Due to their broad range of substrates, oxidoreductases like peroxidases are potent enzymes to be used for these purposes, but production in their natural host is far from optimal for industrial application. For the overproduction of oxidoreductases, filamentous fungi like Aspergillus sp. are preferred host organisms due to their high capacity of producing homologous and heterologous proteins. However, only limited success has been obtained so far with the overproduction of fungal peroxidases in Aspergillus sp.

Incorporation of heme as a cofactor, essential for enzyme activity, is one of the limiting factors in the overproduction of fungal peroxidases which require heme.

Heme is an essential molecule for the cell due to its involvement in many essential processes. On the other hand, free heme and accumulation of its intermediates is toxic when present in high concentrations. This is caused by the generation of reactive oxygen species (ROS) and photosensitivity of the porphyrins. As a consequence, in order to increase heme availability for the overproduction of peroxidases, insight in the complete heme pathway and its regulation is necessary. With the recent publication of the Aspergillus niger genome, a first approach to determine the heme biosynthetic pathway in A. niger is performed by in silicio analysis. The results show a putative conserved heme biosynthesis pathway in which all enzymes are represented although subtle differences also become apparent in comparison with various organisms. After analysis and identification of specific motifs within the enzymes belonging to the heme pathway, these enzymes appear to be more closely related to their mammalian counterparts than to Saccharomyces cerevisiae.


Systematic deletion and fluorescent tagging analysis of the importin beta like karyopherins in Aspergillus nidulans

Ane Marquina-Iñarrairaegui1, Lidia Araújo-Bazán1, Jairo Flores-Sereno1, Javier Fernandez-Martinez2, Stephen A. Osmani3, Eduardo A. Espeso1

1centro De Investigaciones Biologicas, Madrid, Spain, 2the Rockefeller University, New York, United States, 3ohio State University, Columbus, United States

In eukaryotic cells the presence of a nuclear envelope necessitates the existence of a specific machinery to mediate the transport of macromolecules between the nucleoplasm and the cytoplasm in both directions. Transport into (import) or out (export) of the nucleus occurs through the nuclear pore complex (NPC), a massive structure comprising at least 30 proteins (nucleoporins) in Aspergillus nidulans. Nucleoporins are the so-called "non-soluble" components of the transport machinery, while karyopherins are the dynamic components (soluble components). Most karyopherins belong to the Importin beta-like super family involved in the transport of proteins and RNAs. Mononucleated cells have been the main model to study this machinery. However, we are using A. nidulans as a model organism to study how the elements of the nuclear transport machinery are organised and work in a multinucleated cell. We have two main objectives: 1) Characterise which of these transporters are essential. 2) Determine the subcellular localisation of these carriers during interphase and mitosis, to understand how these systems/pathways might be acting at different stages of the cell cycle.

We have identified, in agreement with studies done by others, 14 importin beta like karyopherins. We have characterised components from the two main nuclear import and export pathways: KapA the importin alpha and KapB the importin beta 1 mediate one of the best known nuclear import pathways, and KapK (orthologue of the general Exportin 1) that mediates the principal nuclear export pathway. To identify essential genes we are using gene replacement techniques together with heterokaryon rescue. At least 5 A. nidulans karyopherins have been found to be essential, consistent with data from Saccharomyces cerevisae. Live cell imaging of endogenously GFP-tagged versions of the studied karyopherins reflect a preferential nuclear localisation during interphase. However, most of these transporters are released to the cytoplasm during mitosis, when the NPC is known to be partially disassembled. Our results reveal differences in the localisation of these nuclear carriers during the cell cycle compared with the yeast system. Our recent progress and future perspectives shall be presented.


The use of the exo-inulinase promoter (PinuE) as a new inducible promoter for heterologous protein production in Aspergillus niger

Sandra de Weert1, Xiao-Lian Yuan1, B. Christien Lokman2, Angelique C.W. Franken1, Cees. A.M.J.J van den Hondel1, Arthur F.J. Ram1

1IBL, Leiden University, Leiden, Netherlands, 2Biocentre, HAN University, Nijmegen, Netherlands

The replacement of harsh, toxic chemicals by enzymes in industrial processes related to lignin degradation is highly desirable. To produce these enzymes in the high amounts needed for industrial applications, Aspergillus niger is a promising host for the production of these enzymes because of its high secretion capacity (Punt et al., 2002).

As an alternative for the well established Glucoamylase (GlaA) based protein expression system in A. niger we have used an another inducible expression system. We previously identified the inuE gene, encoding for the exo-inulinase protein in A. niger, as the most strongly induced gene in the presence of inulin and sucrose (Yuan et al., 2006; Yuan et al., 2007). To further study the expression of the inuE gene, a PinuE-eGFP reporter strain was constructed. The reporter strain confirmed previous Northern blot analyses (Yuan et al., 2006) and showed that the inuE gene is highly expressed on inulin and sucrose. Analyses of GFP fluorescence of the reporter strain revealed no expression from the inuE promoter on glucose, fructose and xylose, indicating a tight control of the regulation of the inuE promoter in relation to the different carbon source. Advantages of the use of the inuE expression system compared to the glaA expression include: (i) the inuE system allows production of enzymes on substrates containing sucrose and inulin as the expression of the glaA gene is low at these carbon sources (ii) it allows tight control of the expression of protein of interest which might be advantageous if the protein of interest hampers the growth of the fungus. We have used the inuE expression system for the successful expression of Coprinus cinereus peroxidases in A. niger.



pH signalling in the rice blast fungus Magnaporthe grisea

Patricia Landraud, Christophe Bedetti, Christophe Bruel

CNRS - Université Lyon 1 - BayerCropscience - INSA, Lyon, France

Perception of external environment is important for successful interactions between plants and fungi. In fungi, a specific and conserved pathway is responsible for pH signalling. In the rice blast fungus Magnaporthe grisea, the role of this pathway in cell physiology and development of the fungus in planta is still unknown. Seven proteins, including the putative transmembrane sensor protein PalH and the transcription factor PacC, are part of the signal transduction machinery for pH sensing. Alkaline pH leads to PacC activation and its transfer into the nucleus where it activates transcription of a specific set of genes expressed in alkaline conditions. In order to analyse the role of the two proteins PalH and PacC in M. grisea, the deletion of the corresponding genes has been performed. Several phenotypes were studied in the two mutant strains, including growth rate, adhesion on plant leaves, penetration and ability to infect host plants. This enabled to investigate the involvement of the pH sensing in the M. grisea development cycle. Furthermore, a gene expression profiling analysis of the ΔpacC mutant has been initiated. Taken all together, the results indicate that this pathway is important for the fungus adaptation to an alkaline environment and that it plays a role in the fungus pathogenicity when tested on barley plants.


Characterization of the osmotic signal transduction cascade in the grey mould agent Botrytis cinerea

Weiwei Liu, Pierre Leroux, Sabine Fillinger

INRA, Versailles, France

Hog1-like fungal signal transduction cascades are involved in diverse cellular functions, such as adaptation to various stresses, fungicide resistance, development and, in some cases, virulence. In this work we characterized the homologous pathway of the plant pathogenic ascomycete Botrytis cinerea via the inactivation of the sensor histidine kinase (HK) Bos1, its relationship to the downstream MAP kinase (MAPK) Sak1, and the regulation of target genes.

Western-blot analyses show that, without any external stimulus, Bos1 inhibits Sak1 phosphorylation. Under stress conditions, this inhibition is released, leading to Sak1 phosphorylation, which is involved in the adaptation to high ionic and peroxide stress, macroconidia development, plant penetration and necrosis development. Through an epistasis test, we demonstrate that Bos1 regulates certain functions, independently of Sak1. They include melanin production, but also susceptibility to three families of fungicides (dicarboximides, phenylpyrroles and aromatic hydrocarbons) in contrast to the other homologous fungal MAPK cascades. Cell wall integrity, appressoria-, and sclerotia development are probably controlled by two parallel signalling cascades both regulated by the Bos1 HK.

Real-time RT-PCR analysis was realised on selected sets of genes based on the different mutant phenotypes. Some but not all phenotypes can be related to differential gene expressions. Expression pattern of most Bos1-Sak1 controlled genes under standard conditions corroborates the negative control of Sak1 phosphorylation.

Our results show a complex regulation of diverse cellular functions in this agronomically important pathogen by the osmosensing cascade which probably interacts with other MAPKs. Genomic approaches shall unravel those steps that differ from other fungal systems.



Cellular localization of Neurospora crassa FK506-binding proteins

Débora Pinto, Margarida Duarte, Susana Soares, Arnaldo Videira

IBMC - Instituto de Biologia Molecular e Celular, Porto, Portugal

Immunophilins are intracellular receptors of immunosuppressive drugs, carrying peptidyl prolyl cis-trans isomerase activity, with a general role in protein folding but also involved in specific regulatory mechanisms. Four immunophilins belonging to the FKBP family, named FKBP11, FKBP13, FKBP22 and FKBP50 were identified in the genome of Neurospora crassa. The FKBP22 has been previously characterized as a homodimeric protein located in the lumen of the endoplasmic reticulum with a PPIase and a novel chaperone activity and the FKBP13 was found to be located both in the cytoplasm and mitochondria. The FKBP11 is localized in the cytoplasm and it is not expressed during normal development of the fungus although its expression can be induced by high amounts of calcium. The overexpression of the FKBP11 appears to give an advantage to the strain in media containing high levels of CaCl2 or ZnCl2, since the strain displays a larger cell proliferation. The FKBP50 is a nuclear protein homologous to the Saccharomyces cerevisiae nucleolar protein Fpr4 involved in rDNA silencing. The localization in defined cellular compartments suggests specific functions for the different FKBPs.


Characterization of FKBP mutants from Neurospora crassa

Débora Pinto, Margarida Duarte, Arnaldo Videira

IBMC - Instituto de Biologia Molecular e Celular, Porto, Portugal

The FK506-binding proteins are the intracellular receptors of the immunosuppressive drugs FK506 and rapamycin. Besides their role in protein folding they were also found to be involved in other important cellular processes as signal transduction, protein trafficking and cell-cycle regulation. In a search for the physiological role of the FKBPs identified in N. crassa we have obtained the ∆fkbp11, ∆fkbp13, fkbp22RIP and ∆fkbp50 mutants. The ∆fkbp11, ∆fkbp13 and fkbp22RIP show no phenotypic differences when compared to the wild type strain in what concerns to growth, temperature-sensitivity, response to oxidative stress or protein misfolding. In contrast, the ∆fkbp50 mutant presented a notorious growth defect when grown at 26ºC. Interestingly, this growth deficiency was almost completely reversed when the strain was grown at 37ºC. Disruption of all four immunophilins did not affect the fungus viability, showing that all these proteins are dispensable for N. crassa survival. Cellular and molecular characterization of these mutants might provide us new insights of fundamental cellular processes common to all eukaryotes.


Stimulation of chitin synthesis via a cell wall compensatory response reduces susceptibility to caspofungin and induces the formation of novel salvage septa in Candida albicans

Carol Munro, Louise Walker, Megan Lenardon, Alastair McKinnon, Neil Gow

University of Aberdeen, Aberdeen, United Kingdom

Echinocandins are a new class of antifungal agent which affect fungal cell wall integrity by inhibiting b (1,3) glucan synthesis. Assembly of the fungal wall is a dynamic process and cell wall defects activate compensatory mechanisms that upregulate chitin biosynthesis. Although chitin represents a minor component of the fungal cell wall, it is essential for viability especially under conditions of cell wall damage. C. albicans has four chitin synthases Chs1, 2, 3 and 8. We found that treatment with echinocandins such as caspofungin results in a compensatory increase in chitin synthesis through activation of the PKC, Ca2+/calcineurin and HOG signalling pathways. Pre-treating wild-type cells with CaCl2 and Calcofluor white (CFW) increased chitin content, leading to reduced susceptibility to caspofungin. Reciprocally, synergism was observed between echinocandins and inhibitors of chitin synthesis. These findings indicate a possible alternative mechanism of resistance to caspofungin and suggest the potential benefits of combination therapies that use glucan synthase and chitin synthase inhibitors.

C. albicans septum formation involves two chitin synthases: Chs3 forms the chitinous septal ring and Chs1 synthesises the primary septal chitin. Caspofungin pre-treatment of Chs1/Chs3-deficient C. albicans stimulated production of a novel proximally-offset septum. This salvage septum was able to restore cell division and viability by preventing the formation of septum-less chains of cells and the resulting cell lysis that is associated with loss of Chs1. Formation of the salvage septum was dependent on chitin synthesis and addition of nikkomycin Z, which inhibits the activity of Chs2 and Chs8, prevented its formation. In the absence of Chs1 and Chs3, pre-treatment with CaCl2 and CFW stimulates Chs2 and Chs8 to form a novel salvage septum. Similarly, in the absence of the other chitin synthases, Chs3 alone could be stimulated by pre-treatment with CaCl2 and CFW to form a second type of salvage septum. The thick, amorphous salvage septum synthesised by Chs3 was also able to restore viability, overcoming the lethal absence of Chs1. Co-stimulation of the PKC and Ca2+ signalling pathways therefore restores cell division and viability by inducing chitin synthases to make two different novel forms of salvage septa.



Monitoring of the status of the unfolded protein response pathway in Aspergillus niger by fluorescent protein based reporters

Chika E. Ogbuadike, Theo Goosen, Cees A.M.J.J. van den Hondel, Arthur F.J. Ram

Institute of Biology, Leiden, Netherlands

Aspergillus niger is known to secrete large amounts of native (homologous) proteins and is widely utilized as a cell factory for protein production (1). Inefficiency in the production of heterologous proteins by A. niger is often caused by an inefficient folding of the protein of interest in the endoplasmic reticulum (ER). A specific mechanism, referred to as the Unfolded Protein Response (UPR), is activated upon the buildup of unfolded proteins in the ER. The UPR directs adaptive responses to the stress condition by activating the expression of molecular chaperones and protein foldases to boost the protein folding capacity of the ER. The UPR also stimulates other cellular processes like the induction of phospholipid synthesis and the reduction of general translation of genes encoding secreted enzymes. Prolonged exposure to severe ER stress can induce apoptosis.

Here we present a reporter system to monitor the status of the UPR pathway in A. niger in vivo. The cypB gene, one of the UPR target genes that we have earlier shown to be induced in response to ER stress (2), was used as the UPR-responsive promoter. A strain was constructed that contained two reporter genes controlled by the cypB promoter. The first reporter consists of a fusion between the cypB promoter and the Cyan Fluorescent Protein-Histon2B protein (H2B-CFP). We show that the reporter protein is induced in response to UPR stress, resulting in increased CFP fluorescence in nuclei of living cells. The second reporter consists of the metabolic and fluorescent AmdS-YFP fusion gene (PcypB-amdS-YFP). The A. nidulans amdS gene encodes an acetamidase and high expression of the amdS gene in A. niger allows A. niger to grow on acetamide as the sole nitrogen source. Analysis of the reporter strains containing the two reporters showed that both CFP and YFP fluorescence could be induced in response to UPR stress. Besides having a system to monitor the level of UPR stress in the living cell, this reporter system can also be used as a positive selection system for the isolation of mutants with a constitutive activation of the UPR response pathway by selecting for mutants growing on acetamide. Such mutants might be interesting in relation to the production of heterologous proteins in A. niger.