Poster Category 5:


Fungal Way of Living; Sex and Other Encounters



Identification of natural αAAα diploid hybrids in the Cryptococcus neoformans population
Mojgan Aminnejad and Wieland Meyer
Molecular Mycology Research Laboratory, Centre for Infectious Diseases and Microbiology, University of Sydney, Sydney Medical School-Western at Westmead Hospital, Westmead Millennium Institute, Westmead, NSW, Australia

Cryptococcus neoformans is an opportunistic encapsulated pathogenic basidiomycetous yeast that causes meningoencephalitis in immunocompromised (e.g. AIDS) patients world-wide. The fungus is typically haploid, and sexual reproduction occurs normally between two individuals with opposite mating types, α and a. C. neoformans occurs in two serotypes (A and D), which correspond to C. neoformans var. grubii (molecular type: VNI & VNII) and C. n. var. neoformans (molecular type: VNIV). In addition, rare isolates with AD serotype have been reported and suggested to be diploid or aneuploid. In this study, several C. neoformans isolates were collected globally and their molecular type was assigned by URA5-RFLP analysis with HhaI and Sau96I in a double digest. The results showed unusual combination patterns of VNI and VNII for 8 isolates. These isolates were tested for ploidy by fluorescence flow cytometry analysis and were found to be diploid. PCR analysis with primers specific for serotype A or D genes and serotype- and mating-type genes revealed that the isolates were serotype A and mating type α. Also, the serotype of the isolates was further confirmed by CAP59-RFLP analysis with BsmFI and HpaII. This study confirmed the presence of same sex αAAα hybrids in nature, indicating that diploids can also occur naturally as a result of intra-varietal crossings. The hybrids identified, were isolated from different geographic locations indicating independent non-clonal origins. The fact that the majority of those hybrids are clinical strains possibly indicates that hybridization events may have an impact on C. neoformans virulence and subsequent infection. Moreover, this finding provides insights on the origin and impact of unisexual hybridization on the Cryptococcus neoformans population.




A mutation in Coprinopsis cinerea Ubc2 affects multiple cellular processes required for dikaryosis and sexual development

Takehito Nakazawa, Hirofumi Kondoh, Kiyoshi Nakahori, Takashi Kamada

Okayama University

We isolated and characterized a REMI mutant (B28) defective in sexual development (fruiting initiation) from a Coprinopsis cinerea homokaryotic fruiting strain 326 (Amut Bmut pab1-1). Genetic analysis strongly suggested that the defect in fruiting directly results from insertion of plasmid pPHT1. A plasmid rescue followed by complementation experiments revealed that the gene disrupted and responsible for the phenotype in strain B28 encodes a homologue of Ustilago maydis Ubc2. We designated this gene Cc.ubc2.

The U. maydis ubc2 gene has been shown to be required for pheromone responses as well as for pathogenicity. Also, Ubc2 has been proposed to act as an adaptor for a pheromone responsive MAPK cascade. On the basis of these findings in U. maydis, we performed further analyses, which revealed that strain B28 is defective in two pheromone responses in C. cinerea, clamp cell fusion and nuclear migration for dikaryosis. We then carried out immunoblot analyses, which demonstrated that the Cc.ubc2-1 mutation inhibits phosphorylation of a MAP kinase similar to Ubc3/Kpp2 and that the phosphorylation is not under the control of the B mating type genes encoding pheromone and their conjugate receptors.



The novel 9.5-kDa protein SIP2 specifically interacts with STE12 in Sordaria macrospora

Stefanie Pöggeler[1] Skander Elleuche[2] Yasmine Bernhards[3] Christian Schäfers[4] Jans Manjali Varghese[3] Nicole Nolting[5]

1Georg-August-University Göttingen, 2Institut für Technische Mikrobiologie, Technische Universität Hamburg-Harburg, Hamburg, Germany, 3Institut für Mikrobiologie und Genetik, Abteilung Genetik eukaryotischer Mikroorganismen, Georg-August Universität Göttingen, Göttingen, Germany, 4Lehrstuhl für Allgemeine und Molekulare Botanik, Ruhr-Universität Bochum, Bochum, Germany 5Heinrich-Pette-Institut für Experimentelle Virologie und Immunologie an der Universität Hamburg, Hamburg, Germany

In fungi, the homoeodomain protein STE12 controls diverse developmental processes and derive its regulatory specificity from a wide range of different protein interactions. We have recently shown that in the homothallic ascomycete Sordaria macrospora STE12 is essential for ascospore development and able to interact with the alpha-domain mating-type protein SMTA-1 and the MADS box protein MCM1. To further evaluate the functional roles of STE12, we used the yeast two-hybrid approach to identify new STE12-interacting partners. Using ste12 as bait, a small, serine-threonine rich protein (designated STE12 interacting protein 2, SIP2) was identified. SIP2 is conserved among members of the fungal class Sordariomycetes. In vivo localization studies revealed that SIP2 was targeted to nuclei and the cytoplasm. The STE12/SIP2 interaction was further confirmed in vivo by biomolecular fluorescence complementation (BiFC). Nuclear localization of SIP2 was apparently mediated by STE12. Unlike to the deletion (D) of ste12, deletion of sip2 in S. macrospora led only to a slight decrease in ascospore germination, but no other obvious morphological phenotype. In comparison to the Dste12 single knock-out strain, ascospore germination was significantly increased in a Dsip2/Dste12 double knock-out strain. Our data provide evidence for a regulatory role of the novel fungal protein SIP2 in ascospore germination.





Penicillium chrysogenum – is it another shy sexual ascomycete?

Céline O'Gorman[1] Birgit Hoff[1] Julia Boehm[1] Stefanie Poeggeler[2] Ulrich Kueck[1]

1Christian Doppler Laboratory for “Fungal Biotechnology”, Lehrstuhl fuer Allgemeine und Molekulare Botanik, Ruhr-Universitaet Bochum, Universitaetsstr. 150, 44780 Bochum, Germany
2Abteilung Genetik eukaryotischer Mikroorganismen, Institut fuer Mikrobiologie und Genetik, Georg-August Universitaet Goettingen, 37077 Goettingen, Germany

Penicillium chrysogenum is the most ubiquitous filamentous fungus worldwide; however, it is best-known as the major industrial producer of penicillin. It is classified as an asexual ascomycete because there has never been any direct observation of mating or meiosis in the fungus. Significantly, despite its apparent asexuality there is increasing evidence for a latent sexual cycle in P. chrysogenum, similar to what was previously observed for the cryptically-sexual human pathogen Aspergillus fumigatus. Isolates of opposite mating type are found in near-equal proportion in nature and transcriptionally express pheromone and pheromone receptor genes. Furthermore, analysis of the genome sequence has revealed the presence of other key genes required for completion of the sexual cycle. Experiments are in progress to both characterise the functions of the ‘master regulator’ mating-type genes and induce a sexual cycle under laboratory conditions. As a first step, a MAT1-1 knockout strain was generated by gene replacement with a phleomycin resistance cassette in a ΔPcku70 background. Asexual conidiophore development in the mutant strain was affected; it produces significantly higher quantities of conidia relative to the recipient strain P2niaD18, although the ratio of light-to-dark conidial production remains the same. The MAT1-1 gene was also found to play a role in hyphal morphogenesis, with implications for pellet formation in industrial fermentation systems. These results highlight the diverse regulatory roles of the MAT1-1 gene in developmental processes other than sexual reproduction.


Live-cell imaging of conidial fusion in the bean pathogen, Colletotrichum lindemuthianum

Francine Hiromi Ishikawa[1] Elaine Aparecida de Souza[1] Nick D. Read[2] Maria Gabriela Roca[2]

1Universidade Federal de Lavras, Brazil, 2University of Edinburgh, UK

Fusion of conidia and conidial germlings by means of conidial anastomosis tubes (CATs) is a common phenomenon in filamentous fungi, including many plant pathogens. It has a number of different roles, and has been speculated to facilitate parasexual recombination and horizontal gene transfer between species. The bean pathogen Colletotrichum lindemuthianum naturally undergoes CAT fusion on the host surface and within asexual fruiting bodies in anthracnose lesions on its host. It has not been previously possible to analyze the whole process of CAT fusion in this or any other pathogen using live-cell imaging techniques. Here we report the development of a robust protocol for doing this with C. lindemuthianum in vitro. The percentage of conidial germination and CAT fusion was found to be dependent on culture age, media and the fungal strain used. Increased CAT fusion was correlated with reduced germ tube formation. We show time-lapse imaging of the whole process of CAT fusion in C. lindemuthianum for the first time and monitored nuclear migration through fused CATs using nuclei labelled with GFP. CAT fusion in this pathogen was found to exhibit significant differences to that in the model system Neurospora crassa. In contrast to N. crassa, CAT fusion in C. lindemuthianum is inhibited by nutrients (it only occurs in water) and the process takes considerably longer.


Financial support: CNPq, Capes e Fapemig





Mating type loci of Botrytis cinerea

Jan van Kan[3] Gavin Eyres[1] Linda Kohn[2] Paul Dyer[1]

1University of Nottingham, UK, 2University of Toronto, Canada, 3 Wageningen University, The Netherlands

Botrytis cinerea is a heterothallic ascomycete with two mating types, MAT1-1 and MAT1-2. B. cinerea MAT loci have novel features. Fragments of the MAT1-2-1 and MAT1-1-1 genes were detected bordering idiomorphs of the MAT1-1 and MAT1-2 isolates, respectively. Both of these fragments encode truncated, non-functional proteins. B. cinerea has probably evolved from a homothallic ancestor containing complete MAT1-1-1 and MAT1-2-1 genes at the same locus, with MAT1-1 and MAT1-2 arising from the loss of HMG and alpha-domain sequences, leaving the disabled gene fragments seen in current MAT loci. Two ORFs, designated MAT1-1-5 and MAT1-2-3, have not previously been reported from other fungi. Homologs of MAT1-1-5 are present in other leotiomycetes, whereas the MAT1-2-3 gene is exclusively present within the genus Botrytis. Knockout mutants in MAT1-1-5 are sterile, due to the inability of the dikaryon to develop a cap structure.

B. cinerea is unusual in that some isolates are capable of ‘dual mating’. This refers to the observation that most isolates act in a standard heterothallic fashion (MAT1-1 or MAT-1-2), but some isolates can mate with both MAT1-1 ánd MAT1-2 isolates. Some dual mater isolates can self-fertilize and are truly homothallic. The MAT locus of five homothallic B. cinerea isolates was analysed. Four of those contain a MAT1-2 locus, without any sequence of the MAT1-1 locus being detected. Remarkably, one homothallic isolate contains a MAT1-1 locus, without any sequence of the MAT1-2 locus being detected. We conclude that dual mating and homothallism is controlled by sequences outside the MAT locus.




A novel effector protein in the defense of mushrooms against predatory nematodes

Silvia Bleuler-Martinez[1] Alex Butschi[2] Mario Schubert[3] Martin A. Wälti[1] Pascal Egloff[1] Fred Allain[3] Michael O. Hengartner[2] Markus Aebi[1] Markus Künzler[1]

1Institute of Microbiology, ETH Zürich, 2Institute of Molecular Biology, University of Zürich, 3Institute of Molecular Biology and Biophysics, ETH Zürich

We identified a novel lectin, termed RedA, from the saprobic mushroom Coprinopsis cinerea by virtue of its binding to the plant glycoprotein horseradish peroxidase (HRP). Like many fungal lectins, RedA is highly soluble, lacks a signal sequence for classical secretion and is specifically produced in the fruiting body. Homologous proteins are encoded in the genomes of C. cinerea and the symbiotic mushroom Laccaria bicolor. Glycan array analysis of recombinant RedA revealed a pronounced carbohydrate-specificity for the disaccharide Fucα1,3GlcNAc. Structural studies by NMR showed that the lectin adopts a beta-trefoil (R-type) fold and coordinates one ligand molecule per monomer at a site different from the canonical carbohydrate-binding site of R-type lectins. The glycoepitope recognized by RedA is found at the non-reducing ends of animal glycans and at the core of N-linked glycans of plants, insects and nematodes but not of fungi. Toxicity bioassays with model organisms including Aedes aegypti (insect), Caenorhabditis elegans (nematode) and Acanthamoeba castellanii (amoeba) showed an exclusive toxicity of RedA towards nematodes. Resistance of C. elegans mutants defective in the biosynthesis of the α1,3-core fucoside revealed that this nematotoxicity is dependent on the binding of this specific glycoepitope. Feeding C. elegans with the dTomato-RedA fusion protein showed that this binding occurs at the intestinal epithelium of the nematode. Our results are in agreement with the existence of a protein-mediated defense system of fungi against predators and parasites and suggest that the fruiting body lectin RedA is an effector protein against predatory nematodes.





Determinants of Aspergillus fumigatus mating

Edyta Szewczyk, Sven Krappmann

Julius-Maximilians-University Wuerzburg

Sexual reproduction of the human pathogen Aspergillus fumigatus (teleomorph: Neosartorya fumigata) was assumed to be absent or cryptic until recently, when fertile crosses among geographically restricted environmental isolates were described. Here, we provide evidence for mating, fruiting body development, and ascosporogenesis accompanied by genetic recombination between unrelated, clinical isolates of A. fumigatus, which demonstrates the generality and reproducibility of this long undisclosed phase in the life cycle of this heterothallic fungus. Successful mating requires the presence of both mating type idiomorphs MAT1-1 and MAT1-2, as does expression of genes encoding putative factors involved in this process. Moreover, analysis of an A. fumigatus mutant deleted for the nsdD gene suggests a role of this conserved regulator of cleistothecia development in hyphal fusion and hence heterokaryon formation.




Sex, virulence, stress, and histidine kinase response regulator proteins

B Gillian Turgeon

Dept. of Plant Pathology & Plant-Microbe Biology, Cornell University

Two-component histidine kinase (HK) phosphorelay signaling systems are a major mechanism by which fungi sense and adapt to their environment. In response to a signal, the HK autophosphorylates a conserved histidine residue, then the phosphate is transferred to a conserved aspartic acid residue in a response regulator (RR) protein, resulting in an output. Nearly all eukaryotic HKs are hybrid with the HK and the RR domains in a single polypeptide; most characterized hybrids require an additional phosphorelay step through a histidine phosphotransfer (HPt) domain protein and a second RR protein. This additional phosphorelay step may allow the organism to integrate multiple input signals into a single output.  The maize pathogen, Cochliobolus heterostrophus has 21 HKs, 4 RRs (SSK1, SKN7, RIM15, REC1) and one HPt gene.  Because all HKs signal through the 4 RRs, we chose the latter to make gene deletion mutants and then screened for altered phenotypes (virulence, asexual and sexual development, stress responses, drug resistance).  No altered phenotypes were detected for rim15 and rec1 mutants. C. heterostrophus and Gibberella zeae Ssk1p are required for virulence to maize and wheat, respectively. Lack of Ssk1p affects fertility of heterothallic C. heterostrophus, self-fertility and ascospore maturation in homothallic G. zeae, and proper timing of sexual development in both fungi.  Pseudothecia from crosses involving C. heterostrophus ssk1 mutants ooze masses of single ascospores unlike those from WT crosses, which do not ooze, and tetrads cannot be found. Ssk1p also controls asexual spore proliferation, and represses asexual spore production in the sexual reproductive phase of both fungi.  Double C. heterostrophus ssk1skn7-deletion mutants are more sensitive to oxidative and osmotic stress, and more exaggerated in their spore-type balance phenotype than single ssk1 and skn7-deletion mutants.  ssk1-deletion mutant phenotypes largely overlap with hog1-deletion mutant phenotypes, allowing us to place SSK1 upstream of the HOG1 MAPK pathway. 





Localization and protein-protein interaction studies of pro22, a protein involved in hyphal fusion and sexual development in Sordaria macrospora

Sandra Bloemendal[1] Ines Engh[1] Kathryn M Lord[2] Nick D Read[2] Ulrich Kück[1]

1Department of General and Molecular Botany, Ruhr-University Bochum, Universitaetsstrasse 150, D-44780 Bochum, Germany
2Institute of Cell Biology, The University of Edinburgh, Rutherford Building, King's Buildings, Edinburgh EH9 3JH, UK

The formation of fruiting bodies in filamentous fungi is a multicellular differentiation process and controlled by many developmentally regulated genes [1]. The filamentous ascomycete Sordaria macrospora represents an excellent model system for cell differentiation during fruiting body development, because this homothallic fungus is self-fertile.

For Sordaria macrospora, several developmental mutants are described. Though these mutants can build young protoperithecia, they are not able to form mature perithecia. These mutants, named “pro”-mutants, include the mutant pro22, which shows a point mutation in the pro22 ORF, leading to undifferentiated protoperithecia consisting of a loose hyphal coil. By means of forced-heterokaryon tests using auxotrophic strains and fluorescence microscopic investigations with nuclear labeled strains it could be demonstrated that pro22 is restricted in hyphal anastomosis in homozygous crosses [2].

The PRO22 protein possesses two highly conserved domains A and B and homologs can be found in many other eukaryotes from yeast to humans. Fluorescence microscopy of PRO22 tagged with GFP and mRFP1 provides evidence for a localization in vacuoles. Our aim is to extend the functional analysis of the pro22 mutant via fluorescence microscopy and to identify interaction partners of PRO22 in vitro via yeast two-hybrid and in vivo via tandem-affinity purification.

[1] Kück U et al. (2009) In: Anke T & Weber D (ed) The Mycota XV, Springer-Verlag, Heidelberg
[2] Rech C et al.
(2007) Curr Genet 52:259-266





Applications of sexual development of Trichoderma reesei in research and industry: Perspectives and challenges

Monika Schmoll, Christian Seibel, Christian P. Kubicek

Vienna University of Technology, Research Area Gene Technology and Applied Biochemistry, Getreidemarkt 9, 1060 Wien

Sexual development is one of the most important achievements in evolution. The case of sexuality in the industrial workhorse Trichoderma reesei (anamorph of Hypocrea jecorina) is special in so far as both research as well as industrial strain improvement is solely based on the single isolate QM6a and its (asexual) progeny. As with all other filamentous fungi applied in industry, the possibility of crossing was not available for T. reesei, although in silico data had identified H. jecorina as its teleomorph more than a decade ago. Recently, we described the two mating type loci of T. reesei (MAT1-1 and MAT1-2) and we were for the first time able to induce sexual reproduction of T. reesei QM6a (MAT1-2) in crossings with a MAT1-1 wild-type isolate of H. jecorina, and we obtained fertilized stromata and mature ascospores . However, a serious issue with our findings was, that while male fertile, T. reesei QM6a is female sterile. We now investigated the role of the peptide pheromone precursor genes, the pheromone receptors as well as of the known photoreceptors in sexual development in T. reesei. Additionally we provide first insights into the relevance of RIP for crossing in this organism.




Mushroom forming basidiomycetes: Mating and more

Erika Kothe, S. Erdmann, M. Wirth, E.-M. Jung, D. Freihorst, D. Senftleben, N. Knabe

Friedrich-Schiller-University, Dept. Microbiology, Microbial Phytopathology

The recent genome sequence of the wood-rotting homobasidiomycete Schizophyllum commune, in comparison to genomes of other Agaricomycotina like Coprinopsis cinerea, Laccaria bicolor, Phanerochaete chrysosporium , Postia placenta and the heterobasidiomycetes Ustilago maydis and Cryptococcus neoformans  now allow comparative “omics” studies on mating interactions, pheromone response pathways. Linking the identified genes to phenotypes in sexual development and mushroom formation can provide insight into development of structures typical for the kingdom of the mycota. In mushroom forming homobasidiomycetes, the two mating type loci A, coding for homeodomain (HD) type transcription factors, and B encoding a pheromone/receptor system, regulate mating and sexual development. We could localize the pheromone receptor solving the old question of whether pheromone signalling is involved in cell-cell recognition or rather, after mycelial fusion, recognition of nuclei in a dikaryon. In addition to the mating type loci, in silico analyses now permit the identification of putative components of the signaling pathways leading to dikaryotic growth, mushroom formation and meiotic spore production. The  involvement of G proteins like Ras could be shown for nuclear migration, nuclear positioning, clamp formation and fruitbody morphogenesis, involving cAMP-dependent protein kinase A signaling. Nuclear migration has been investigated looking into dynein function. The heavy chain of dynein in S. commune, like in other basidiomycetes, is encoded by two separate genes which are investigated by performing knock-out analyses. The resulting strain is viable, with phenotypes which, again, can be linked to sexual development, fruitbody formation and spore production as well as to apical hyphal growth. With availability of the fully sequenced genome, thus, many of the long-established pathways of signalling and morphogenesis can be revisited and surprises from the genome sequence provide insight into long-standing questions.


Analysis of proteins involved in sexual development in zygomycetes

Christine Schimek, Rudigier Yvonne, Park Min-Jeong, Sarkar Sarbani, Wetzel Jana, Wöstemeyer Johannes

General Microbiology and Microbial Genetics, Institute for Microbiology, Friedrich-Schiller-University Jena, Neugasse 24, 07743 Jena, Germany

Sexual development within the class Zygomycetes is regulated by retinoid-like beta-carotene derivatives, the trisporoids. Trisporoids serve as pheromones in partner recognition and as internal transcription regulators. Numerous studies deal with their complex and co-operative synthesis, some of the biosynthesis enzymes have been studied into molecular detail, but their mode of action, molecular reaction mechanisms and signal perception events are completely unknown. We are researching the putative reaction partners involved in recognition and signal perception events. Our main goal is to identify putative trisporoid receptors and binding proteins, but we also include the search for proteins undergoing regulatory interactions with the trisporoid biosynthesis enzymes. 2D-PAGE reveals only a small number of deviating proteins between the (+) and the (-) mating type in cell surface-attached proteins. Several receptor candidates have been selected and are currently under analysis. Using antibodies raised against animal retinoid signaling and developmental regulation proteins, we identified a putative retinoid binding protein, cross-reacting with an antibody against cellular retinaldehyde-binding protein CRALBP, and a putative homeobox protein. Direct transcriptional modulation of effector genes may occur via trisporoid binding to specific transcription factors and trisporoid synthesis enzymes themselves might act as transcription factors during sexual development. Based on the observation that both the trisporoid biosynthesis enzymes 4-dihydrotrisporin dehydrogenase and 4-dihydromethyltrisporate dehydrogenase are inactivated at certain developmental stages by binding to other proteins forming multimeric complexes, we are using heterologously expressed 4-dihydrotrisporin dehydrogenase as bait for the purification of such binding proteins from cytoplasmatic protein fractions from Mucor mucedo.



Development of a tool for genetic manipulation of the zygomycete Mucor mucedo

Min-Jeong Park, John Jasmine, Wetzel Jana, Schimek Christine, Burmester Anke, Wöstemeyer Johannes

General Microbiology and Microbial Genetics, Institute for Microbiology, Friedrich-Schiller-University Jena, Neugasse 24, 07743 Jena, Germany

Genetic analysis of Mucor mucedo, a zygomycete model organism for studying sexual communication and action of the trisporoid system, is hampered by a strong tendency towards autonomous replication of introduced plasmids instead of stable integration. We are working at developing a tool for genetic manipulation based on interference with the DNA double strand break-repair system.

Targeted gene disruption via homologous recombination is routinely used for analysis of gene function. Although integration of exogenous DNA at homologous sites in the genome occurs easily in Saccharomyces cerevisiae, it is rare in many filamentous fungi where DNA integration occurs predominantly through non-homologous end joining (NHEJ) and exogenous DNA can be integrated at ectopic sites in the genome. Direct ligation of DNA strands in NHEJ is mediated by a DNA-dependent protein kinase, a DNA ligase complex, and the Ku70-Ku80 heterodimer. Homologues of Ku70 and Ku80 have been identified in many organisms. Recent studies in filamentous fungi have shown increased gene targeting frequencies in KU deficient mutants, indicating that KU disruption strains are efficient recipients for gene targeting. We identified and cloned a M. mucedo homologoue to the human KU70 gene using PCR, inverse PCR, and Southern hybridization. Genetic manipulation will be done using an RNA silencing approach with Morpholino antisense constructs directed against KU70 mRNA, thus resulting in the functional knock-down of the KU70 gene product. The suitability of this approach is shown by morpholino-mediated down-regulation of crgA, a protein involved in the regulation of carotene synthesis in zygomycete fungi.



The diaphanous-related formin Drf1 is an effector of the rho-GTPase Cdc42 in the basidiomycetous fungus Ustilago maydis

Björn Sandrock, Johannes Freitag, Daniel Lanver, Stefan Arens, Michael Bölker

 Philipps-University Marburg, Department of Biology - Genetics Karl-von-Frisch-Str.8, 35032 Marburg, Germany


The basidiomycetes Ustilago maydis is a tumour-causing agent on corn plants. Tumour development can occur in all green parts of the plant and is initiated by the invasion of dicaryotic hyphae from the plant surface. These hyphae are formed after fusion of two compatible haploid sporidia. Hyphal growth is uni-polar and the cytoplasmic part of elongating filaments is restricted by the sequential formation of retraction septa leaving empty sections behind.

We have identified a regulatory network essential for the formation of the secondary septum in budding cells composed of the guanine-nucleotide exchange factor Don1, the corresponding small GTPase Cdc42 and the germinal-centre kinase Don3.

We will demonstrate our recent data that the diaphanous related formin Drf1 is an effector of the Cdc42-module for secondary septum formation. Formins are proteins that polymerize and bundle actin cables.
Furthermore we will show that among these proteins only Cdc42 is essential for the morphological switch from yeast-like to hyphal growth. We will present data how Cdc42 regulates the MAP kinase cascade.
The complete Don1-Cdc42-Drf1 module organizes the retraction septa formation in filaments. In addition, this cascade is also necessary for appressoria formation. This also indicates that actin nucleation is important for these processes.
Currently we are investigating the role of the Don3 kinase during filamentous growth to elucidate its interplay with the Cdc42 module.



Genetic and epigenetic control of a nonself recognition complex in N. crassa

Lafontaine, D.L. and M.L. Smith

Carleton University, Canada


Heterokaryon incompatibility (HI) in filamentous fungi is a form of nonself recognition that operates during the vegetative phase of the life cycle. One HI complex in Neurospora crassa, the het-6 locus, comprises two tightly linked incompatibility genes, het-6 and un-24, each having two allelic variants, Oak Ridge (OR) and Panama (PA). The un-24 gene also encodes the large subunit of ribonuclotide reductase while het-6 appears to be a member of a repetitive gene family with no other known function aside from HI. These two genes are in severe linkage disequilibrium such that only un-24OR het-6OR and un-24PA het-6PA haplotypes occur in nature. In this study we constructed novel un-24PA het-6OR strains and het-6 deletion strains. The un-24PA het-6OR strains are initially self-incompatible, but escape from incompatibility in a predictable manner that is associated with loss of het-6OR incompatibility function. HI tests using het-6 deletion strains reveals an allelic incompatibility interaction between OR and PA forms of un-24, which is asymmetrically enhanced by the presence of het-6OR or het-6PA. Mutations in vib-1 suppress HI associated with allelic differences at un-24 or at het-6, but only partially suppress incompatibility reactions that occur when both un-24 and het-6 differ. We have also identified a second genetic suppressor that acts synergistically with vib-1 and will present evidence that this suppressor compromises epigenetic silencing of HI.




The fungal immune response: from detection of the pathogen to the cellular response

Paoletti M., Clavé C., Breton A., Ness F., Chevanne D., Sabourin M., Cescau S., Daskalov A. and Saupe S.

Institut de Biochimie et genetique cellulaire, CNRS, 43 rue Camille Saint Saëns, France


Living organisms have developed complexe processes to fight off pathogens. Plants and animals initiate an immune response upon detection of Pathogen Associated Molecular Patterns (PAMPS) by dedicated Pattern Recognition Receptors (PRRs) that belong to the STAND class of proteins involved in signal transduction. Pathogen driven evolution of the PRR encoding genes can lead to auto-immune diseases. No such fungal immune systems have been described so far.


In Podospora anserina vegetative incompatibility (VI) is under the control the NWD gene family that interacts with incompatible alleles of the het-c locus encoding for a glycolipid transfer protein (GLTP). The NWD gene family evolves extremely rapidly and new variants in the WD repeat domain involved in protein protein interactions are permanently being generated. NWD proteins also belong to the STAND class of proteins. We have hypothesized that these proteins function as PRRs and that the VI reaction corresponds to a pathological manifestation of an auto-immune disease. We have discovered a fungal species that initiates a strong reaction in P. anserina that is dependant on het-c and all known VI suppressors. We have undertaken the task of describing the fungal immune response from pathogen recognition to the cellular response.




Genetic relatedness of multi-azole resistant Aspergillus fumigatus isolates

S.M.T. Camps, P.E. Verweij, W.J.G. Melchers

Department of Medical microbiology, Radboud University Nijmegen Medical Center, Nijmegen, The Netherlands



In the last few years multi-azole resistant Aspergillus fumigatus seem to emerge in the Netherlands. The dominant resistance mechanism is a point mutation in the cyp51A gene resulting in the substitution of leucine to histidine at codon 98 (L98H) combined with a 34 bp tandem repeat (TR) in the promoter region of this gene. The emergence of this single resistance mechanism suggests that the resistant isolates have a common evolutionary origin. We analyzed the CSP gene, microsatellites and the mating type locus to study azole susceptible and resistant A. fumigatus isolates of clinical and environmental origin to investigate the genetic relatedness of TR/L98H isolates.


Resistant isolates containing the TR/L98H resistance mechanism (n=57) were subjected to CSP, microsatellite and mating type analysis. As a control, both susceptible (n=57) and resistant isolates without the TR/L98H resistance mechanism (n=17) were analyzed.


The TR/L98H isolates clustered together in microsatellite analysis and were grouped into three CSP types (2, 4B and 11). Susceptible and non-TR/L98H resistant control isolates were classified to other CSP types than the TR/L98H isolates, except for several isolates belonging to CSP type 2. Mating type genes MAT1-1 and MAT1-2 were almost equally represented in each group.


1.       Clinical and environmental TR/L98H isolates were grouped to the same CSP types and clustered together using microsatellite typing, indicating that the TR/L98H resistance mechanism has only recently developed and might not be induced in the patient but might be acquired from the environment.

2.       As the TR/L98H isolates grouped to different CSP types and clustered apart from the control isolates, the TR/L98H isolates might belong to a separate genetic A. fumigatus lineage.

3.       Although microsatellite and CSP analysis indicate that the TR/L98H isolates are rather homogeneous, segregation of the mating type locus is equal to the control isolates. This might suggest that sexual reproduction played a role in resistance development.



The fungal immune response: from detection of the pathogen to the cellular response

Paoletti M., Clavé C., Breton A., Ness F., Chevanne D., Sabourin M., Cescau S., Daskalov A. and Saupe S.

Institut de Biochimie et genetique cellulaire


Living organisms have developed complexe processes to fight off pathogens. Plants and animals initiate an immune response upon detection of Pathogen Associated Molecular Patterns (PAMPS) by dedicated Pattern Recognition Receptors (PRRs) that belong to the STAND class of proteins involved in signal transduction. Pathogen driven evolution of the PRR encoding genes can lead to auto-immune diseases. No such fungal immune systems have been described so far.


In Podospora anserina vegetative incompatibility (VI) is under the control the NWD gene family that interacts with incompatible alleles of the het-c locus encoding for a glycolipid transfer protein (GLTP). The NWD gene family evolves extremely rapidly and new variants in the WD repeat domain involved in protein protein interactions are permanently being generated. NWD proteins also belong to the STAND class of proteins. We have hypothesized that these proteins function as PRRs and that the VI reaction corresponds to a pathological manifestation of an auto-immune disease. We have discovered a fungal species that initiates a strong reaction in P. anserina that is dependant on het-c and all known VI suppressors. We have undertaken the task of describing the fungal immune response from pathogen recognition to the cellular response.



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