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

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

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

Volker Klix, Stefanie Pöggeler

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

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

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

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

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

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

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

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

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

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

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

Ana Rodriguez, Aitor Garzia, Carlos Jimenez, Unai Ugalde

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

PR8.13

David Canovas¹, Luke Pase², Gordon Smyth², Alex Andrianopoulos¹

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

Susanne Rindt, Michael Bölker, Björn Sandrock

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

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

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

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

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

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

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

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

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

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

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

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

Kylie Boyce, Alex Andrianopoulos

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

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

Verena Seidl, Christian P. Kubicek, Monika Schmoll

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

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

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

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

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

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

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

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

Kathryn Lord, Chris Jeffree, Nick Read

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

Susan Kaminskyj¹, Adriana Szeghalmi², Konstantin Jilkine², Kathleen Gough²

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

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

Hayley Bugeja, Michael Hynes, Alex Andrianopoulos

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

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

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

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

Erika Kothe, Nicole Knabe, Florian Hennicke, Daniela Schuber

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

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

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

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

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

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

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

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

Andreas Kaufmann¹, Hans-Peter Schmitz², Peter Philippsen¹

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

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

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

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

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