Poster Category 6:


Fungal Way of Living; Cell Biology



The antifungal activity of the Penicillium chrysogenum antifungal protein PAF disrupts calcium signalling and homeostasis in Neurospora crassa

Ulrike Binder[2] Meiling Chu[1] Nick D. Read[1] Florentine Marx[2]

1Institute of Cell Biology, University of Edinburgh, Edinburgh, EH9 3JH, UK
2Biocenter, Division of Molecular Biology, Innsbruck Medical University, Fritz-Pregl Strasse 3, A-6020 Innsbruck, Austria

The antifungal protein PAF from Penicillium chrysogenum exhibits growth inhibitory activity against a broad range of filamentous fungi. Recent evidence has suggested that calcium (Ca2+) signalling may play an important role in the mechanistic basis of PAF as a growth inhibitor. Supplementation of the growth medium with high Ca2+ concentrations counteracted PAF toxicity towards sensitive moulds. By using a transgenic Neurospora crassa strain expressing codon optimized aequorin, PAF was found to cause a significant increase in the [Ca2+]c resting level. The Ca2+ signatures in response to stimulation by mechanical perturbation or hypo-osmotic shock were significantly changed in the presence of PAF. BAPTA, a Ca2+ selective chelator, ameliorated the PAF toxicity in growth inhibition assays and counteracted the PAF induced perturbation of Ca2+ homeostasis. These results indicate that extracellular Ca2+ was the major source of these PAF-induced effects. The L-type Ca2+ channel blocker diltiazem disrupted Ca2+ homeostasis in a similar manner to PAF. Diltiazem in combination with PAF acted additively in enhancing growth inhibition and aggravating the change in Ca2+ signatures. Notably, both substances, PAF and diltiazem increased the [Ca2+]c resting level possibly by blocking Ca2+ channel activity. However, examination of a N. crassa Δcch1 deletion strain excluded the L-type Ca2+ channel cch1 to be the major target of PAF.





Multivesicular body-ESCRT components and pH response regulation in Aspergillus nidulans

Ana Maria Calcagno[2] Peñalva Miguel[1] Hervás-Aguilar America[1] Bignell Elaine[2] Scazzocchio Caludio[2] Arst Herb[2]

1Centro de investigaciones Biológicas, Madrid, 2Imperial College, London

Involvement of MVB class E components in pH signalling is well characterised in yeasts. In S. cerevisiae, all components, of ESCRT-I, -II and Vps32p-Vps20p of ESCRT-III are required for pH signalling. Regulation of gene expression by ambient pH in Aspergillus nidulans is mediated by the transcription factor PacC. The ESCRT –III  components Vps32 and Vps24 interact with the pH signalling components PalA and PalB respectively, thus participating in pH signalling.

We report that the deletion of genes encoding Vps20, Vps32 and Vps36 in A. nidulans is nearly lethal and nearly always accompanied by selection of suppressor mutations greatly improving growth.  These (partial) suppressors occur in two genes, supA and supB.  SupA is a transcription factor and SupB is a putative protein kinase. The suppressor mutations do not affect pH regulation or trafficking but they do alter the volume and quantity of vacuoles, even in vps+ strains.  vps20, -32 and -36 deletions prevent pH signalling, consistent with results in yeast. These deletions also impair trafficking of FM4-64 and of the dicarboxylic amino acid transporter to the vacuolar membrane. We conclude that at least ESCRT-II and -III components are required for pH signalling.




Control of organelle differentiation and copy number by positive feedback to import competence

FANGFANG LIU, Yanfen Lu, Tajaswini Dhavale, Gregory Jedd

Temasek Life Sciences Laboratory


The differentiation of organelles is fundamental to the growth and development of eukaryotic cells. Woronin bodies are low -copy fungal organelles produced from a high-copy number organelle – the peroxisome. Here, we show that Woronin body producing peroxisomes differentiate de novo and are hyper-competent for matrix protein import.  This produces a few dominant organelles that receive the majority of nascent matrix protein import.  Differentiation depends on a key oligomeric contact in the Woronin body core protein HEX.  Mutational disruption of this oligomer abolishes the differentiation of peroxisomes and interaction with two proteins that are enriched in the membrane of WB producing peroxisomes.  Our results are consistent with a model where the HEX oligomer promotes peroxisome differentiation through positive feedback to the targeting of key membrane proteins.





Penicillium chrysogenum Pex14/17p: a novel component of the peroxisomal membrane that is important for penicillin production

Lukasz Opalinski[1] Jan A.K.W. Kiel[1] Tim Homan[2] Marten Veenhuis[1] Ida J. van der Klei[1]

1Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, Kluyver Centre for Genomics of Industrial Fermentation
2Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen

By genome analysis, we identified Pex14/17p as a putative novel peroxin of Penicillium chrysogenum. Here we show that Pex14/17p is a component of the peroxisomal membrane that is essential for efficient PTS1 and PTS2 matrix protein import, implying that the protein is indeed a bona fide peroxin. Additionally, a PEX14/17 deletion strain is affected in conidiospore formation. Pex14/17p has properties of both Pex14p and Pex17p in that the N-terminus of this protein is similar to the highly conserved Pex5p binding region present in the N-termini of Pex14p’s, whereas its C-terminus shows weak similarity to yeast Pex17p’s. We have identified a novel motif in both Pex17p and Pex14/17p that is absent in Pex14p.


We show that an N-terminally truncated, but not a C-terminally truncated Pex14/17 protein is able to complement both the matrix protein import and sporulation defects of a Δpex14/17 strain, implying that it is the Pex17p-related portion of the protein that is crucial for its functioning as a peroxin. Possibly, this compensates for the fact that P. chrysogenum lacks a Pex17 protein. Finally, we show that in P. chrysogenum Pex14/17p plays a role in the efficiency of the penicillin biosynthesis process.




Comparative gene expression analyses reveal genes with functional roles during fruiting body development of filamentous ascomycetes

Stefan Gesing, Minou Nowrousian

Lehrstuhl für Allgemeine und Molekulare Botanik, Ruhr-Universität Bochum

Many filamentous ascomycetes form fruiting bodies during a highly complex differentiation process. Four major morphological types (apothecia, perithecia, pseudothecia and cleistothecia) are differentiated that derive from an ancestral fruiting body. Thus, fruiting body differentiation most likely is controlled by a set of common core genes. One way to identify such genes is to search for genes with evolutionary conserved expression patterns, which is a powerful criterion for functional importance.

Using “Suppression Subtractive Hybridization”, we selected differentially expressed transcripts during fruiting body development in Pyronema confluens (Pezizales). By real time PCR, expression patterns were shown to be conserved in members of the Sordariales (Sordaria macrospora and Neurospora crassa), a derived group of ascomycetes. Knockout studies with correlated N. crassa orthologues, revealed a functional role during fruiting body development for NCU05079, a putative MFS peptide transporter.

Additionally we verified a conserved expression pattern for the homologues of yeast asf1 (anti-silencing function protein 1) during fruiting body development of four distantly related ascomycetes (N. crassa, S. macrospora, Fusarium graminearum and P. confluens) by microarray and real time PCR analysis. Asf1 is a highly conserved histone chaperone involved in the balance of nucleosome assembly and disassembly. Knockout and complementation analysis with S. macrospora Asf1 indicate a functional role of the protein during sexual development of the fungus.

These data indicate conserved gene expression patterns and a functional role of the corresponding genes during fruiting body development, which are candidates of choice for further analysis.





The Aspergillus nidulans cortical marker teaa mediates microtubule-cortex interactions through an interaction with the XMAP215 family protein AlpA

Norio Takeshita, Daniel Mania, Saturnino Herrero de Vega, Reinhard Fischer

Karlsruhe Institute of Technology


In eukaryotic cells microtubule (MT) length is determined by polymerization and depolymerization phases. One important parameter for length determination is the contact with the cell cortex. Here, we show in the model organism Aspergillus nidulans that the contact of MT plus ends with the cortex is mediated through interaction between a putative MT polymerase (XMAP215, A. nidulans homologue AlpA), and a cortical cell end marker protein, TeaA. Although both proteins localized to MT plus ends during MT growth, AlpA-TeaA interaction was observed in a bimolecular fluorescence complementation assay only after MT plus ends contacted the cortex. In the absence of TeaA, MT plus ends contacted random places along the tip cortex and occasionally continued to grow after reaching the cortex. In the absence of AlpA, the microtubule array was laregely affected and MTs grew very slowly. In an in vitro MT polymerase assay we show that fast MT polymerization depends on the presence of AlpA and is inhibited by TeaA. Further investigation with truncated variants of TeaA suggests that the interaction of TeaA with AlpA is necessary not only for the proper behavior of MTs at tips but also proper polarity establishment and maintenance.




Signaling through the Aspergillus nidulans orthologue of PKC mediates septum formation

Loretta Jackson-Hayes, Terry W. Hill, Darlene M. Loprete, Britany Chavez, Chassidy Groover, Erinn Ogburn, Michael Pluta

Rhodes College

We have shown that the Aspergillus nidulans orthologue of protein kinase C (PkcA) participates in regulating cell wall integrity (CWI) and localizes at sites of cell wall synthesis, including growing hyphal tips and septa.  PkcA’s role in CWI is regulated independently of its capacity to target to sites of wall growth, as shown by the ability of the calC2 mutation to inhibit resistance to wall-damaging compounds without affecting growth or cytokinesis.  To better understand the mechanisms by which PkcA localizes to tips and septa, we have observed the formation of cortical rings at sites of septation by fluorescently tagged PkcA in hyphae defective in expression of other proteins necessary for septum formation, using either temperature-sensitive mutants or regulation under the AlcA promoter.  In addition, we have co-imaged PkcA and other septation proteins bearing complementary tags.  Here we report that localization of PkcA to septa lies “downstream” of the functions performed by MobA (Mob1p orthologue), TpmA (tropomyosin), SepA (formin), SepD, SepG, and proteins encoded by two other not-yet-cloned Sep loci.  In the absence of function of these proteins, PkcA cortical rings were not observed.  PkcA localization lies “upstream” of MyoB (myosin II orthologue), the A. nidulans orthologue of Bud4p (in yeast, a bud site selection marker), and a protein encoded by a third uncloned Sep locus.  PkcA cortical rings still form in the absence of function of these proteins, though septa do not develop.  SepA, TpmA, MyoB, and MobA all appear to colocalize with PkcA during normal septum formation.  While PkcA localizes to the very apex of hyphal tips and to the leading edge of growing septa, the protein phosphatase BimG localizes to sites lateral to the most active sites of growth.  Studies with other septation-related proteins are ongoing.





Calcium and pH homeostasis in Aspergillus: small molecules under control

Mojca Bencina[1] Tanja Bagar[1] Nada Kraševec[2]

1Department of Biotechnology, National institute of Chemistry, 2Department for Biosynthesis and Biotransformations, National institute of Chemistry

The well tuned orchestra of enzymes is precondition for any cell to function. A coordination of enzymes’ activities is manly a responsibility of wide variety of secondary messengers. Some of them like cAMP, PIPs are locally synthesized. Others like free calcium ions are constantly present, but stored in organelles and the concentration is rigorously controlled by homeostatic machinery. And then there is a pH, which might serve as a mechanism by which cells co-ordinate the regulation of various processes that lack any other common regulating factors and may provide a link between metabolic state and physiological responses. However, not lot is known how calcium and pH homeostatic machinery are working which is most likely due to lack of easy methods to monitor changes in intracellular free calcium ions concentration and pH in vivo. The methods for tracking changes in intracellular calcium concentration and pH will be explained and evidence for calcium and pH homeostasis will be given. An examination of cytoplasmic pH in growing cells of Aspergillus niger was performed by simultaneous, dual excitation confocal ratio imaging of the ratiometric pH probe RaVC. For calcium homeostasis studies a genetically encoded calcium probe aequorin was used. The role of PMRA, Golgi P-type ATPase and PMCA, plasma membrane P-type ATPase in maintaining calcium homeostasis of filamentous fungus Aspergillus niger will be demonstrated.





Fusarium graminearum as a model for human Niemann-Pick Type C disease

H Corby Kistler[1] Matias Pasquali[2] Andrew Breakspear[1]

1University of Minnesota, 2Département Environnement et Agro-biotechnologies (EVA), Centre de Recherche Public-Gabriel Lippmann, Luxembourg

Niemann-Pick Type C (NPC) disease is a fatal autosomal recessive lipid storage disorder in humans.  An accumulation of endocytosed cholesterol in lysosomes/late endosomes of spleen, liver and brain cells causes progressive dementia, usually resulting in death by the age of twenty.  NPC is caused by defects in two genes which function in cholesterol transport, npc1 and npc2, with mutations in the former being responsible for 95% of documented cases.  Filamentous fungi make attractive models to study the endomembrane system, growing rapidly in a highly polarized manor.  Here, we explore the potential of the ascomycete Fusarium graminearum as a tool to study NPC.  A BLAST search revealed the presence of a putative F. graminearum homolog of npc1, possessing 34% sequence identity (51% positives) at the amino acid level.  Deletion of F. graminearum npc1 produced a viable mutant which displayed defects in ergosterol localization.  A combination of fluorescence and differential interference contrast microscopy revealed an accumulation of ergosterol in vacuoles of mutant cells.  Furthermore, a GFP tagged version of NPC1 was found to localize to the vacuolar membrane, analogous to lysosomal membrane anchored human NPC1p.  Our results suggest that filamentous fungi may provide good model systems to study NPC.





Functional analyses of α-1,3-glucan synthase genes, agsA and agsB, in Aspergillus nidulans

Akira Yoshimi[3] Motoaki sano[1] Tomonori Fujioka[2] Yuko Kokubun[3] Osamu Mizutani[4] Daisuke Hagiwara[3] Takashi Fujikawa[5] Marie Nishimura[5] Fumihiko Hasegawa[3] Keietsu Abe[3]

1KIT, 2Kumiai Chemical Industry Co., Ltd., 3Tohoku Univ. NICHe, 4NRIB, 5NIAS

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. We previously reported that the transcriptional regulation of a MAP kinase gene mpkA and of cell wall-related genes (CWGs) in Aspergillus nidulans differs significantly from that in Saccharomyces cerevisiae. The transcription of two α-1,3-glucan syntase genes, agsA and agsB, were regulated by MpkA pathway, but most CWGs were not. Recently, the importance of α-1,3-glucan in host-parasite interactions has been studied in both mammalian and plant pathogenic fungi. In this study, to understand the role of α-1,3-glucan in A. nidulans, functional analyses of the agsA and agsB genes were performed. The deletion mutants of agsA gene did not show any significant phenotypes under normal growth conditions. In contrast, the disruptants of agsB gene could not be obtained, suggesting that AgsB seems to play a crucial role in α-1,3-glucan synthesis of A. nidulans. To assess this issue, we constructed the conditional agsB strain whose agsB expression is conditionally regulated under the control of alcA promoter. The transcription of many CWGs coding for β-1,3-glucan synthase and chitin synthase were induced under agsB repressed conditions. This suggests that the decrease of α-1,3-glucan content was counterbalanced by an increase in other cell wall components. The results of the sensitivities to cell wall stress compounds such as micafungin, CFW and Congo Red and the susceptibility test for cell wall degrading enzymes will be presented.





Reduced expression of SccA increases sensitivity to wall stress

Darlene Loprete, Loretta Jackson-Hayes, Terry Hill, Erinn Ogburn, John Musgrove

Rhodes College


Decreased expression of the extragenic suppressor, designated SccA, affects cell wall integrity in the filamentous fungus Aspergillus nidulans.  Overexpression of SccA suppresses the phenotype of the calC2 mutation in the A. nidulans orthologue of protein kinase C (PkcA), which results in hypersensitivity to the chitin-binding agent Calcofluor White (CFW).  In addition, we have shown that SccA rescues 6 wall-sensitive strains.  In filamentous fungi, as in yeasts, hypersensitivity to CFW correlates with defects in cell wall integrity.  SccA is predicted to have a single transmembrane domain with 42% of its amino acids residues being serine or threonine, which indicates it is bound to carbohydrates in the cell wall.  Homologues exist in the genomes of other filamentous fungi, but not in yeasts or other organisms.  A SccA-GFP hybrid localizes to the plasma membrane and septa of vegetative hyphae.  When SccA is placed under the control of the regulatable AlcA promoter and grown under low expression conditions (glucose), we observed a sensitivity to CFW, indicating it plays an important role in cell wall integrity. Taking into consideration the protein’s cell surface location and its influence on the function of PkcA, we hypothesize that SccA plays a role in signal transduction as part of a cell wall integrity pathway.





Essentiality of RNA exosome subunit encoding genes in Aspergillus oryzae

Mizuki Tanaka, Takahiro Shintani, Katsuya Gomi

Grad. Sch. Agric. Sci., Tohoku Univ., Japan

The exosome is a multi-subunit 3’→5’ exonucleolytic complex that is conserved in eukaryotes. The ring-shaped core structure of the exosome is constituted of nine subunits. In yeast, all of nine exosome subunits are essential for viability. On the other hand, in plant, Csl4 is dispensable for growth and development, whereas Rrp41 and Rrp4 are essential for the development of female gametophytes and embryogenesis. These results suggest that the function of individual subunit of the exosome is different in each eukaryotic cell. Since there has been no report on the exosome itself in filamentous fungi, we attempted to construct the disruptants of genes encoding exosome subunits in Aspergillus oryzae.

We have chosen two orthologous genes for csl4 and rrp4 as targets for disruption, and successfully obtained a csl4 disruptant but not an rrp4 disruptant. The disruption of csl4 gene had no apparent defect on growth in A. oryzae. Since the rrp4 disruptant could not been obtained, this gene would be essential for cell viability. Thus, we generated the conditional rrp4 expression mutant strain by using the promoter of nmtA, expression level of which is regulated by riboswitch existed within its 5’UTR and is repressed considerably in the presence of thiamine. The resultant conditional rrp4 expression strain displayed a remarkable growth defect when thiamine was added to the medium. These results suggested that Rrp4 is essential but Csl4 is not for cell growth in A. oryzae and that function of individual exosome components in A. oryzae is similar to that in plant.





Directed growth during germling fusion in Neurospora crassa requires bem-1, a protein which is dispensable for general polar growth

Timo Schürg, Ulrike Brandt, André Fleißner

Institute of Genetics, Technische Universität Braunschweig

We are using Neurospora crassa as a model system to study the molecular mechanisms of chemotropic growth and cell fusion. When spores of N. crassa germinate, they attract each other to undergo cell fusion and to form the mycelial colony. Earlier studies have shown that the MAP kinase MAK-2 is an essential part of a signaling cascade involved in chemotropic growth during germling fusion (Pandey et al. 2004; Fleissner et al. 2009). To further characterize this signaling pathway we analyzed the role of BEM-1. The homologous protein in Saccharomyces cerevisiae is interacting with several upstream factors of the Fus3 cascade, which is homologous to the MAK-2 pathway in N. crassa. In contrast to yeast, where BEM-1 is involved in polarity establishment, a N. crassa delta bem-1 knock out mutant is not significantly impaired in spore germination and polar hyphal growth. However,  delta bem-1 germlings do not interact chemotropically. Complementation with a bem delta PB-1 construct did not complement the knock out mutant phenotype, suggesting that the PB-1 domain is essential for BEM-1 function. Subcellular localization of BEM-1 using GFP fusion constructs showed that BEM-1-GFP accumulates at every growing hyphal tip. In germling fusion pairs, BEM‑1‑GFP concentrates at the fusion point and localizes around the opening fusion pore. We also detected BEM-1-GFP at septa of germ tubes and mature hyphae. Taken together, our data suggest novel functions of BEM-1 in chemotropic growth, fusion pore formation and at the septa. In our further studies we will try to unravel and identify the distinct molecular functions of BEM-1 during Neurospora development.





Heterogeneity in micro-colonies of Aspergillus niger in liquid shaken cultures

Charissa de Bekker, G. Jerre van Veluw, Arman Vinck, L. Ad Wiebenga, Han A.B. Wösten

Microbiology and Kluyver Centre for Genomics of Industrial Fermentations, Institute of Biomembranes, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands

Colonies of the filamentous fungus Aspergillus niger secrete large amounts of proteins. Previously, it has been shown that on solid media only part of the colony participates in secretion. We assessed whether heterogeneity can also be found between and within micro-colonies of A. niger from submerged cultures.

Strains expressing GFP from the glucoamylase (glaA) or ferulic acid esterase (faeA) promoters were grown. The Complex Object Parametric Analyzer and Sorter (COPAS) was used to analyze the diameter and fluorescence of micro-colonies. At least two populations were found in these cultures, one population of small (25%) and one of large micro-colonies (75%). Fluorescence correlated with volume. 27% and 73% of the micro-colonies expressing GFP from the faeA promoter were lowly and highly fluorescent, respectively. This implies that heterogeneity in this strain depends on the volume only. In contrast, lowly fluorescent micro-colonies of the glaA::GFP strain comprised about 79% of the culture; this implies an additional factor besides volume.

To assess heterogeneity within a micro-colony, central and peripheral parts were isolated by laser microdissection and pressure catapulting (LMPC). QPCR showed that glaA and faeA expression is similar in both zones. However, RNA content per hypha was 50 times higher in the periphery.

Here, COPAS and LMPC were used for the first time to assess gene expression in a microbial system. Our results show heterogeneity between micro-colonies from a submerged culture. Moreover, it is shown that zones within a pellet are also heterogenic. Peripheral hyphae have about 50 times more RNA than central hyphae.



RacA is required for actin distribution and affects the localization of the exocytosis machinery in the filamentous fungus Aspergillus niger

Min Jin Kwon, Mark Arentshorst, Cees A.M.J.J. van den Hondel, Vera Meyer, Arthur F.J. Ram

Leiden University, IBL, Molecular Microbiology, Kluyver Centre for Genomics of Industrial Fermentation, Sylviusweg 72, 2333 BE Leiden, The Netherlands

Tip growth in filamentous fungus requires coordination of basic cellular processes in the cell such as exocytosis, polarity maintenance, endocytosis and cell wall biosynthesis. The small GTPase RacA, the fungal orthologue of the human Rac1 protein is important for fungal growth. In the absence of RacA, apical dominance of tip growth is lost resulting in a hyperbranching phenotype. Actin patches in the racA deletion mutant were found to be hyperpolarized at the extreme apex, while in the wild type strain a smoother gradient of actin patches towards the tip was observed. GFP-RacA localizes to the plasma membrane at the extreme apex of growing hyphae probably, marking at the site of exocytosis. To understand the function of RacA in relation to exocytosis, secretory vesicles were visualized by tagging the V-SNARE (SynA) with GFP (GFP-SynA). In both ΔracA and wild type cells, GFP-SynA is present on intracellular structures representing secretory vesicles and/or endocytic vesicles. High levels of GFP-SynA are also present in the Spitzenkörper, a filamentous fungal specific structures that is thought to act as a vesicle supplying center. In ΔracA the intensity of signal was less. The tips of wild type hyphae display a ~10-15 mm gradient of GFP-SynA protein, whereas tips in ΔracA show a much shorter (<5 mm) gradient of GFP-SynA. We conclude that the A. niger RacA protein is necessary for precise actin localization and distribution in hyphal tip cells and that the gradient of GFP-SynA towards the tip is actin dependent.





PBS (Phenotype Based Screening) System: an effective strategy for bidirectional genetic approach in the rice blast fungus, Magnaporthe oryzae

Jaejin Park, Jeil Hong, Yong-Hwan Lee

Department of Agricultural Biotechnology, Center for Fungal Genetic Resources, and Center for Fungal Pathogenesis, Seoul National University, Seoul 151-921, Korea

The most straightforward strategy of reverse genetic approach includes targeted gene deletion process. It requires homologous recombination (HR) events and efficiency of gene deletion is subject to frequency of HR. In many filamentous fungi, however, HR occurs at low frequency due to the dominance of non-homologous end joining (NHEJ) and in case of the rice blast fungus Magnaporthe oryzae, frequency of HR is locus-dependent and very low that has been reported less than 10%. It causes inefficiency of experimental progress and generates lots of undesirable ectopic transformants. For the improved efficiency of genetic study, we designed an add-on system named PBS (Phenotype Based Screening). This system is added to the targeted gene deletion process and makes efficient use of ectopic transformants previously abandoned. As a forward genetic approach, ectopic transformants showing gene disruptions probably in random manner are screened for various defectives in several phenotypes such as growth rate, pigmentation, colony morphology, and conidiation. Location of ectopic integration is achieved by inverse-PCR and sequencing. With aid of PBS system, two growth-retarded ectopic transformants were confirmed as related to disruption of two loci MGG_00839.6 and MGG_04395.6. Consequently, coupling of PBS system and targeted gene deletion process can be regarded as the union of forward and reverse genetic approaches. This bidirectional approach will advance the functional genomic studies of filamentous fungi with high efficiency.




Comparative Proteomic Analysis of Colletotrichum higginsianum Infection Structures

Jochen Kleemann, Bleddyn Hughes, Tom Colby, Anne Harzen, Jürgen Schmidt, Richard J. O'Connell

Max Planck Institute for Plant Breeding Research

The hemibiotrophic ascomycete Colletotrichum higginsianum causes anthracnose disease of crucifers, including the model plant Arabidopsis. Following conidial germination, the pathogen differentiates a specialised infection structure called an appressorium that is essential for initial host invasion. We used a comparative proteomics approach to uncover changes in the total and secreted proteome during germination and appressorium formation, taking advantage of the ability to mass-produce Colletotrichum appressoria in vitro on polystyrene substrata. Proteins harvested at 5h, corresponding to early appressorium formation, and 22h, representing mature melanized appressoria, were compared to the proteome of undifferentiated mycelia. A total of 677 protein spots were identified using MALDI-TOF-MS and MS/MS by reference to the C. higginsianum genome sequence. 27 secreted proteins were identified in the germination liquid surrounding mature appressoria.  Of the proteins that were co-regulated during appressorium formation, enzymes involved in fungal wall modification and melanin biosynthesis were represented during early morphogenesis, while enzymes potentially involved in plant cell wall degradation and toxin biosynthesis were represented in mature appressoria. The identified peptides provide experimental support for annotation of the C. higginsianum genome sequence.





Mutations in two Golgi Apparatus COG proteins affect growth and glycosylation in Aspergillus nidulans

Sara Gremillion[2] Darlene Loprete[1] Terry Hill[1]

1Rhodes College, 2Armstrong Atlantic State University

The swoP1 (swollen cell) and podB1 (polarity defective) mutations in Aspergillus nidulans interfere with establishment and maintenance of polarity. At restrictive temperatures, conidia of both mutants swell to approximately 1.5 times the normal diameter. Conidia of swoP1 also produce abnormally wide hyphae and establish multiple points of polarity, which grow isotropically before arrest. Genes complementing the mutations of swoP1 and podB1 have strong sequence homology to COG4 (ANID7462.1) and COG2 (ANID8226.1), respectively.  In mammals and yeast, COG2 and COG4 are part of a multi-protein structure called the COG (conserved oligomeric Golgi) complex associated with retrograde transport within the Golgi apparatus. A GFP-tagged COG2 displayed a punctuate distribution within fungal hyphae, a pattern consistent with other Golgi protein localization. COG4 was not successfully GFP tagged. Protein overexpression studies provided evidence of intra-complex interactions between COG2 and COG4 as well as between COG2 and COG3. To study the role of these proteins in growth of filamentous fungi, an AlcA promoter replacement strategy was performed. When grown on AlcA-suppressive media, the COG4 AlcA-replaced promoter strain displayed normal growth, while the COG2 AlcA-replaced promoter strain displayed abnormally wide hyphae.  A lectin blot using concanavalin A revealed significant differences in protein glycosylation patterns between the swoP1 and podB1 mutants when compared to wild type when grown at restrictive temperatures. The glycosylation patterns of the two mutants were indistinguishable under these conditions.




How coupled oscillators shape daily timing

Marc Maas, Martha Merrow

Groningen University

The circadian clock is a complex trait, involving a network of interlocked molecular loops that depend on oscillatory feedback. There are many indications that also in the fungus Neurospora crassa the clock consists of multiple oscillators. One molecular oscillator system that has been described in great detail is the frequency-white collar (FRQ-WC) transcription-translation feedback loop (FWC-TTFL).  In the absence of a functional FWC loop, strains are still able to entrain systematically to temperature, they show oscillations in nitrate reductase activity and, under particular conditions, rhythmic conidiation is within the circadian range. There thus is ample evidence for one or more FRQ-less oscillators (FLOs). Microarrays have revealed several candidate components of these FLOs. We have studied the circadian behaviour of one of these: cpc-1 (for cross pathway control), an orthologue of the gcn4 gene from yeast. Cpc-1 encodes a transcription factor involved in the coordination of many amino acid biosynthetic pathways. It is therefore a candidate node in several metabolic feedback loops. We show that coupling oscillations of FWC and CPC-1-dependent feedback loops control fundamental clock attributes like amplitude, precision and robustness.





Regulation of the SNARE complex formation in Trichoderma reesei
Mari Valkonen1, Markku Saloheimo1, Merja Penttilä1, and Rory R. Duncan2
1 VTT Biotechnology, P.O. Box 1000, FIN02044 VTT, Finland
2Membrane Biology Group, University of Edinburgh, George Square, EH8 9XD, UK

Eukaryotic cells contain membrane-bound compartments that are connected by trafficking of vesicular intermediates. Merging of the donor and acceptor membranes accomplishes three tasks: 1) Surface of the plasma membrane increases (by the surface of the fused vesicle), which is important for cell growth. 2) The substances within the vesicle are released into the exterior. 3) Proteins embedded in the vesicle membrane become part of the plasma membrane.  

To maintain compartmental organization, proper targeting of transport vesicles is required. In the process of exocytosis, where Golgi derived secretory vesicles fuse to the plasma membrane, three major classes of proteins are needed SNARE, Sec1 and Rab proteins. Although t- and v-SNAREs are sufficient to drive the fusion of membranes in vitro, it is clear that other proteins are important regulators of this event in the cell.

It has been shown that the formation of SNARE complex is very strictly regulated in mammalian and yeast cells. The NH2-terminal domain from isolated mammalian t-SNARE, syntaxin, has been shown to fold back onto the SNARE motif, and this closed conformation is stabilized by the binding of members of the Sec1-munc (SM) family, apparently preventing SNARE complex formation. Two reports have demonstrated that key neuronal mammalian SM protein, munc18-1, can interact with the assembled SNARE complex, via an N-terminal peptide in syntaxin. This motif is conserved in some fungal SSO homologs and in mammalian syntaxin1. The function of the SM family of proteins in membrane fusion is still controversial, but it has been shown to be essential regulator of the membrane fusion and in yeast has been shown to concentrate on the sites of secretion.

To address the question of the control of membrane fusion, we have cloned the Sec1 homologue of T. reesei and expressed it as a fusion with a yellow fluorescent marker protein (Venus). The localisation of the fusion protein is cytoplasmic. Interactions of the SECI protein with two plasma membrane t-SNARE proteins SSOI and SSOII has been studied using multi-dimensional time-correlated single photon counting (TCSPC) fluorescence lifetime imaging microscopy (FLIM) to detect FRET. It was shown that there is FRET occurring between mCer::SSOI and the SECI::Venus fusion proteins indicating that there’s a protein-protein interaction between these two proteins. The major site of interaction is at the plasma membrane. The finding was further verified by immunoprecipitation (IP) studies showing that SSOI and SECI can be isolated as a complex. Both SSOI and SECI proteins seem to be essential for growth as deletion of either is lethal to the cells. The FLIM/FRET results from the mCer::SSOII and SECI::Venus interaction were inconclusive and in IP studies, no interaction has been seen between the SECI and SSOII proteins.





Characterisation of functional domains of the iron-dependent transcription factor HapX

Daniel H. Scharf[1] Peter Hortschansky[1] Hubertus Haas[2] Axel A. Brakhage[1]

1Department of Molecular and Applied Microbiology, Leibniz-Institute for Natural Product Research and Infection Biology (HKI) and Friedrich-Schiller-University Jena, Beutenbergstrasse 11a, D-07745 Jena
2Division of Molecular Biology, Innsbruck Medical University, Fritz-Pregl-Str. 3, A-6020 Innsbruck

Recently, a putative fourth CCAAT-binding complex (CBC) subunit with an unknown function was identified in A. nidulans and designated HapX. hapX expression is repressed by iron via the GATA-factor SreA. Various iron-dependent pathways (e.g., heme biosynthesis) are repressed during iron starvation by the interaction of HapX with the CBC. These data suggest a model, in which HapX/CBC interaction is regulated at both transcriptional and post-translational levels. Iron starvation causes expression of hapX. Subsequent binding of HapX to the CBC results in transcriptional repression of iron-dependent pathways. During iron-replete conditions, hapX is repressed and, therefore, iron-dependent pathways are derepressed. Moreover, HapX/CBC interaction is inhibited by increased iron concentrations. This post-translational mechanism allows rapid adjustment to iron availability by disruption of the HapX/CBC complex. Mutual transcriptional control of hapX and sreA coordinates iron acquisition and iron-dependent pathways, thereby serving for both iron supply and prevention of iron toxicity. These data indicate that the CBC has a general role and that HapX function is confined to iron depleted conditions.


Here, we describe the domain architecture of the HapX protein. Phylogenetic analysis revealed the conservation of certain domains, which were characterised further with the help of surface-plasmon-resonance and complementation experiments.





Regulated silencing of the spindle assembly checkpoint without mitotic spindles

Colin P. DeSouza, Stephen A. Osmani

Ohio State University

The spindle assembly checkpoint (SAC) is a universal mechanism which arrests mitosis if a bipolar spindle cannot be formed. Although higher eukaryotic cells that are unable to satisfy the SAC eventually die, such a mechanism would not confer an obvious advantage to filamentous fungi encountering environmental conditions that interfere with spindle formation. Following the status of nuclear pore complex and SAC proteins we show that Aspergillus nidulans cells treated with the spindle poison benomyl activate the SAC and arrest in mitosis. However after a defined period of time, the SAC is actively turned off and cells exit mitosis without chromosomal segregation. Most remarkably we find that cells which have undergone one such failed mitosis surprisingly transit interphase and enter a second mitosis in which the SAC is re-activated before again being inactivated. This cyclic activation then inactivation of the SAC can occur for at least three cell cycles in which the nucleus completes all aspects of mitosis except those depending on spindle function. Further, following one or more cell cycles without spindle function, if cells are allowed to reform microtubules they assemble spindles upon mitotic entry and can undergo successful mitosis. Therefore, we propose that inactivation of the SAC allows filamentous fungi to continue growth under environmental conditions which prevent spindle formation and then periodically test the environment for conditions which are compatible with mitosis. We conclude that the SAC can be silenced in a cyclic regulated manner independent of spindle formation.





Tip-focused Rho GTPase activity and the actin cytoskeleton regulate directional growth of Neurospora crassa germlings

Alexander Lichius, Nick D. Read

University of Edinburgh

The ability to establish and maintain cell polarity is a prerequisite for spore germination and cell fusion in Neurospora crassa. Without it spores are limited to isotropic growth and unable to protrude germ tubes and conidial anstomosis tubes (CATs). Germ tubes avoid each other, whilst CATs attract each other. During CAT homing, tip orientation follows a chemoattractant gradient to establish cell-cell contact and fusion. Using strains co-expressing Lifeact-TagRFP and β-tubulin-GFP we recently analyzed the interrelated localization pattern and dynamic rearrangement of F-actin and microtubules during conidial germination and cell fusion. We found that recruitment of both cytoskeletal elements occurs in a distinct but coordinated manner that might influence which protrusion is being formed and maintained at any point in time. Molecular mechanisms involving cortical markers whichlocally and specifically recruit the cytoskeleton in order to achieve re-orientation during germ tube avoidance and CAT homing, are virtually unknown in Neurospora. Focused activation of Rho GTPases, such as CDC42 and RAC-1, at the cell cortex is a key determinant of polarized F-actin organization, and thought to occur in sterol-rich microdomains within the plasma membrane. Displacement of these microdomains in response to external stimuli might regulate directional growth. Interestingly, ∆rac-1 mutants of Neurospora still show germ tube avoidance but are unable to undergo CAT-mediated cell fusion. To study the spatial and temporal relationship between GTPase activity and F-actin rearrangement during tip orientation in more detail, we engineered a green fluorescent Cdc42-Rac-interactive binding (CRIB) biosensor for activated GTPases and co-expressed it with Lifeact-TagRFP-T.




Novel peroxin, Fam1p of Colletotrichum orbiculare is essential for pathogenesis and associates with Woronin bodies

Yasuyuki Kubo[1] Ayumu Sakaguchi[1] Naoki Fujihara[1] Gento Tsuji[1] Ulla Neumann[2] Richard O’Connell[2]

1Laboratory of Plant Pathology, Graduate School of Life and Environmental Sciences, Kyoto Prefectural University, Kyoto 606-8522, Japan
2Department of Plant Microbe Interactions, Max Planck Institute for Plant Breeding Research, Carl von Linné Weg 10 D-50829 Köln, Germany

The cucumber anthracnose fungus Colletotrichum orbiculare forms an infection structure called an appressorium. Melanization of appressoria is essential for host penetration and requires the β-oxidation of fatty acids in peroxisomes. Here, we identified and characterized a protein specific to filamentous ascomycete fungi, Fam1, that is essential for peroxisome function and which associates with Woronin bodies. The FAM1 gene was isolated by screening random insertional mutants for deficiency in fatty acid metabolism. The fam1 disrupted mutants were unable to grow on medium containing oleic acids as the sole carbon source. Green fluorescent protein carrying the peroxisomal targeting signal 1 (PTS1) or PTS2 were not imported into peroxisomes of fam1 mutants, suggesting that FAM1 is a novel peroxisomal biogenesis gene (peroxin). Accordingly, fam1 mutants were defective in both appressorium melanization and host penetration. Microscopy showed that the Fam1p-GFP fusion protein localized to small punctate structures in the apical region of hyphae, near septa and adjacent to peroxisomes. This resembled the distribution of Woronin bodies, which are peroxisome-derived organelles involved in septal pore plugging. After cloning CoHEX1, a homolog of the HEX1 Woronin body structural gene, we generated a Cohex1p-mRFP1 fusion protein, which co-localised with Fam1p-GFP in Woronin bodies. Furthermore, the apical and septum localization of Fam1p was impaired in cohex1 mutants. Our results indicate that Fam1p is a novel Woronin body-associated protein and raise the possibility that filamentous ascomycete fungi coordinate peroxisome function via Woronin bodies.



NOX genes of Cochliobolus heterostrophus: role in ROS production, development and virulence

Mordechai Ronen, Benjamin A. Horwitz

Department of Biology, Technion, Haifa 32000, Israel


Reactive oxygen species (ROS) are synthesized by specific NADPH oxidases (NOX), enzymes inserted in the plasma membrane. NOX enzymes use cytoplasmic NADPH to produce superoxide. ROS can provide both defense and differentiation signaling roles in animals and plants. Fungal NADPH oxidases have a structure very similar to the human gp91phox. Specific isoforms of fungal NOX have been reported to be required for various physiological processes and cellular differentiation events, including development of sexual fruiting bodies, ascospore germination, and hyphal growth in both mutualistic and antagonistic plant–fungal interactions. We identified three NADPH oxidase homologues in the necrotrophic filamentous fungus Cochliobolus heterostrophus, and we set out to investigate their function and importance in the fungal life cycle through study of loss-of-function mutants in genes encoding catalytic and regulatory subunits of NADPH oxidases. Mutants in noxA, noxC and the predicted regulatory subunit gene noxR have decreased pigmentation and delayed conidiation. noxA and noxR show decreased virulence on the host plant, maize. All nox mutants produce superoxide as detected by nitro blue tetrazolium (NBT) staining. We are investigating ROS production in single and multiple nox mutants under different conditions.




The RmsA protein – a hub in the protein interaction networks of Aspergillus niger?

Vera Meyer, Susann Minkwitz, Tabea Schütze, Cees A.M.J.J van den Hondel, Arthur F.J. Ram,  

Leiden University, Institute of Biology Leiden, Department Molecular Microbiology and Biotechnology, Sylviusweg 72, 2333 BE Leiden, The Netherlands & Kluyver Centre for Genomics of Industrial Fermentation, P.O. Box 5057, 2600 GA Delft, The Netherland

Many cells and organisms go through polarized growth phases during their life. Cell polarization is achieved by local accumulation of signaling molecules which guide the cytoskeleton and vesicular trafficking to specific parts of the cell and thus ensure polarity establishment and maintenance. Polarization of signaling molecules is also fundamental for the lifestyle of filamentous fungi such as Aspergillus niger and essential for their morphogenesis, development and survival under environmental stress conditions. Considerable advances in our understanding on the protagonists and processes mediating polarized growth in filamentous fungi has been made over the past years. However, how the interplay of different signaling pathways is coordinated has yet to be determined. We found recently that the A. niger RmsA protein is central for the polarization of actin at the hyphal tip (1). However, we show here that RmsA is also of vital importance for the metabolism, viability and stress resistance of A. niger. This suggests that RmsA could occupy an important position in the global network of pathways that balance growth, morphogenesis and survival of A. niger.

Meyer V, Arentshorst M, Flitter SJ, Nitsche BM, Kwon MJ, Reynaga-Pena CG, Bartnicki-Garcia S, van den Hondel CA, Ram AF (2009) Reconstruction of signalling networks regulating fungal morphogenesis by transcriptomics. Eukaryot Cell 8: 1677-1691 


Subcellular localisation of AreA and MeaB is driven by different nitrogen sources.

Magdalena Mos1 Dominik Wagner2, Bettina Tudzynski2, Igor Y Morozov1, Meriel G Jones1, and Mark Caddick1

1The University of Liverpool, School of Biological Sciences, Bioscience Building, Crown Street, Liverpool L69 7ZB, United Kingdom

2Institut für Botanik der Westfälischen Wilhelms-Universität Münster, Schloßgarten 3,D-48149 Münster, Germany.


In A. nidulans the utilisation of nitrogen sources other than ammonium or glutamine is dependent on the transcriptional factor AreA, which enables transcription of a broad range of genes specifically under nitrogen limitation. A second transcription factor, MeaB, also plays a role in the cellular response to nitrogen availability. Although the function of MeaB is less well characterised it appears, at least in part, to act in opposition to AreA, being most active under nitrogen sufficiency. Both AreA and MeaB have been tagged with GFP and their intracellular localisation monitored in response to changes in nitrogen regime using confocal microscopy. As controls histone H1:RFP was used to identify the nuclei and mitochondria were stained with MitoTracker. The subcellular localisation of MeaB and AreA both respond to changes in nitrogen regime. In the case of AreA, under conditions of nitrogen repression (eg Gln or NH4+) the protein appears to be distributed throughout the cell. Surprisingly under nitrogen derepression conditions (eg NO3- or –N) AreA is primarily localised to the mitochondria and not the nuclei. In the case of MeaB, growth on Gln or NH4+ results in localisation in and around the nuclei and within the mitochondria. In the presence of NO3- or during nitrogen starvation MeaB accumulates exclusively in mitochondria, although this is not the case with all deprepressing nitrogen sources (eg proline) where MeaB is evenly distributed throughout the cell.  In Fusarium fujikuori subcellular localisation of MeaB is also subject to nitrogen regulation and, as in A. nidulans, it is located in the nucleus under nitrogen repressing conditions. However, in contrast to the A. nidulans, there was no obvious accumulation of MeaB within the mitochondria under the regimes tested.  In summary, as expected AreA and MeaB both show very distinct response to nitrogen availability. Unexpectedly, in A. nidulans both transcription factors localise to the mitochondria under nitrogen regimes.



ColA, a white spore color mutant in Aspergillus niger, identifies the phosphopantetheinyl transferase (PptA) protein which is required for melanin biosynthesis.

Joohae Park, Thomas R. Jørgensen, Mark Arentshorst, Patricia A. vanKuyk, Robbert A. Damveld, C.A.M.J.J. van den Hondel, Arthur F.J. Ram

Institute of Biology Leiden, Leiden University, Molecular Microbiology and Biotechnology, Kluyver Centre for Genomics of Industrial Fermentation, Sylviusweg 72, 2333 BE, Leiden, The Netherlands


A characteristic hallmark of Aspergillus niger is the formation of black conidiospores. In this study, we report the characterization of a color mutant, colA, which was isolated because of a complete loss of pigmentation resulting in white conidia. Pigmentation of the colA mutant was restored by a gene that encodes the A. niger ortholog of the 4’-phosphopantetheinyl transferase protein (PptA). 4’phosphopantetheinyl transferase activity is required for the activation of Polyketide synthases (PKSs) and Non-Ribosomal Peptide Synthases (NRPSs) (1). Complementation analysis showed that the colA mutant is allelic to a previously isolated color mutant, gryA. Sequencing of the colA and gryA loci and the targeted deletion of the pptA gene further confirmed that the colA /gryA mutants are mutated in the pptA gene. Spores from the ∆pptA deletion are paler in color that spores of an A. niger strain disrupted in the pksA gene. PksA encodes the polyketide synthase required for melanin biosynthesis (2) and spores from the pksA disruption strain become fawn colored. Spores from both the ∆pksA and the ∆pptA were hypersensitive to UV-radiation indicating that melanin is required for resistance against UV-radiation. The ∆pksA strain was equally sensitive to hydrogen peroxide as the parental strain, but spores from the ∆pptA strain showed increased sensitivity. The results suggest the involvement of PKS or NRPS-derived metabolites that confer resistance towards oxidative stress conditions.



(1) Walsh C.T., Gehring A.M., Weinreb P.H., Quadri L.E., Flugel R.S., Curr. Opin. Chem. Biol. 1, 309-315. 1997.

(2) Shuster J.R., Bindel Connelley M., MGG 262, 27-34. 1999.





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