Poster Category 2:


Fungal-Host Biology



Glycolytic function is necessary for Cryptococcus neoformans virulence

Michael Price,  Marisol Betancourt-Quiroz, Anthony Lee, Dena L. Toffaletti, John R. Perfect

Duke University Medical Center


Cryptococcus neoformans is an important fungal pathogen of immunocompromised individuals, with a close relative - C. gattii - emerging as a serious threat for the immunocompetent.  During active infection, C. neoformans colonizes the airspaces of the lungs resulting in pneumonia, and subsequently migrates to the central nervous system (CNS).  The fungus persists in the cerebrospinal fluid (CSF), and causes meningoencephalitis that is fatal if untreated.  Since C. neoformans colonizes these fundamentally different niches within the host, we sought to understand fungal carbon utilization during infection, and in particular the role of glycolysis in this model fungal pathogen.  We created mutants at either end of the glycolytic metabolic pathway, which are restricted for growth on glucose.  A pyruvate kinase mutant (pyk1∆) and a hexose kinase I & II double mutant (hxk1∆/hxk2∆) were made and evaluated for virulence in both rabbit-CSF and murine-inhalation models of cryptococcosis.  Results show that both mutations blocking glucose utilization result in complete attenuation of disease in both animal models.  Since the pyk1∆ mutant cannot utilize lactate for growth when glucose is present, we made a pyk1∆/mig1∆ double mutant lacking the glucose catabolite repressor MIG1.  This double mutant should be able to utilize all carbon sources available, yet it exhibited greater reduction in CSF persistence in the rabbit model compared to the pyk1∆ single mutant and was also attenuated in the mouse model.  These data suggest that energy production from glucose in various host contexts is crucial for virulence in C. neoformans.




Insights into calcium signal transduction – functional characterization of calcineurin, calcipressin and two putative calcium channels in B. cinerea

Karin Harren, Julia Schumacher, Bettina Tudzynski

Westf. Wilhelms-Universität Münster


For gaining more insights into the host-pathogen interaction and cell differentiation of the phytopathogen B. cinerea, we are interested in studying the role of the Ca2+-signal transduction pathway and its arrangement in the intracellular signalling network.

The Ca2+/calmodulin-dependent phosphatase calcineurin is a conserved protein that plays a critical role in Ca2+ signalling and stress response. Contrary to our previous expectation that the deletion of calcineurin is lethal, we recently succeeded with the generation of calcineurin A (bccnA) deletion mutants. They are strongly impaired in growth and non-pathogenic, as strains grow very slowly as small, compact colonies. However, improved growth was observed on media containing high sugar concentrations or sodium chloride.

                Recently, a new class of conserved calcineurin regulators, named calcipressins, has been identified in yeast. The deletion of the single calcipressin homologue in B. cinerea (bcrcn1) affects vegetative growth on all tested media and showed reduced virulence on living bean plants.

                In addition to these components of the Ca2+ signalling pathway, the cellular impact of external Ca2+ has been investigated by deletion of two genes encoding the putative Ca2+ channels BcCCH1 and BcMID1. Both mutants and the double knockout mutant exhibit similar phenotypes, as there are generally not impaired in growth, morphology or virulence. However, under high salt conditions and with the Ca2+-chelator agent EGTA vegetative growth is affected.


The role of Botrytis cinerea Ryp1-like protein (Rlp), a putative transcriptional regulator, during pathogenesis

Caroline Michielse,  Matthias Becker,  Jens Heller, Paul Tudzynski

Westf. Wilhelms Universität Münster


Botrytis cinerea, which causes gray-mold rot, attacks a wide range of plant species. Due to this wide host range and the severe damage to agriculture, efforts have been made to understand the infection process and to identify genes involved in pathogenicity. In this study, the role of BcRLP, which shows similarity to the morphological switch regulators Candida albicans WOR1 and Histoplasma capsulatum RYP1, in pathogenicity was analyzed. Gene knock-out and complementation studies revealed that RLP is required for pathogenicity. The rlp mutant is able to penetrate plant tissue, but is not able to cause necrotrophy. In addition, the mutant is blocked in conidia formation, is sensitive to oxidative stress and is severely reduced in the production of the toxin botrydal. We speculate that Rlp is required for the necrotrophic growth phase.





The role of small GTPases in growth and virulence of Claviceps purpurea

Andrea Herrmann, Yvonne Rolke, Julia von Sengbusch, Janine Schürmann, Paul Tudzynski

Institute of Botany, Westf. Wilhelms-Universität Münster


The biotrophic plant pathogen Claviceps purpurea which infects monocotyledonous plants, among them important crops like rye, is an interesting model organism for research in plant-pathogen interactions. The strict polar growth in early infection stages of C. purpurea  in rye ovaries is of particular interest, as the fungus is not recognized as a pathogen possibly due to its pollen tube-like growth. Small GTPases and their downstream effectors are known to be involved in polarity. Therefore the investigation of the effects of these factors is crucial for a better understanding of polar growth in filamentous fungi.

Knockout strains of the small GTPases Rac and Cdc42 and the p21 activated kinase Cla4 as a GTPase downstream effector have been generated. Deletion of Cdc42 lead to a hyperbranching phenotype with increased sporulation[1] whereas deletion of Rac and Cla4 respectively caused a phenotype with severe impairment in sporulation, branching and growth[2]. Thus, an antagonistic relationship between the GTPases Rac and Cdc42 as well as an involvement of Rac and Cla4 in the same pathway can be concluded. Furthermore the effects of introduced constitutive active forms of Cla4 and Rac in C. purpurea have been investigated. Additionally, functional studies of the guanine nucleotide exchange factor Cdc24 were started.

[1] J. Scheffer et al. (2005) Eukaryot Cell 2005 4(7): 1228-38.
[2] Y. Rolke, P. Tudzynski (2008) Mol Microbiol.2008 68(2):405-23.





Treatment of Trichophyton rubrum ex vivo skin infection with a new antifungal agent, ambruticin, leads to the activation or repression of several genes, including a putative mitochondrial protein and a subtilisin protease

Nalu T A Peres[1] Gras, DE[1] Sanches, PR[1] Falcao, JP[1] Rocha, L[1] Mazucato, M[1] Rossi, A[1] Prade, RA[2] Martinez-Rossi, NM[1]

1University of Sao Paulo, 2Oklahoma State University


During host infection and treatment with cytotoxic agents, microorganisms must be able to remodel metabolic pathways to scavenge nutrients and to respond to the stress in order to survive in the host milieu. The identification of the mechanisms involved in fungal adaptation to host microenvironment and stress response is essential to understand the pathogenic process of fungal infection, enabling the establishment of new therapeutic measures. The anthropophilic dermatophyte Trichophyton rubrum is one of the most prevalent fungi isolated from cutaneous infections; however, the molecular mechanisms involved in its pathogenicity remain unclear. Moreover, antifungal drugs commonly used in the treatment of fungal infections act upon a limited number of cellular targets, presenting numerous side effects. In this work, an ex vivo human skin infection model was used to identify T. rubrum expressed genes during interaction with human epidermal cells and treatment with the antifungal agent ambruticin, which supposedly interfere with the osmoregulation system. The results show that this drug presents antifungal activities against this dermatophyte, leading to a swelling of the hyphal tip and consequently fungal death, also modulating the expression of several genes. The ex vivo human skin infection model followed by suppressive subtraction hybridization allowed the identification of genes involved in cellular transport and biogenesis of cellular components that were activated or repressed in response to ambruticin. The activation of the gene coding for a mitochondrial protein with unknown function, homolog to the Saccharomyces cerevisiae Tar1p, or repression of the gene coding for a subtilisin protease 5, during treatment of T. rubrum ex vivo skin infection suggests that they might be important for the maintenance of this dermatophyte in the host tissue, especially in the presence of an inhibitory agent.





Comparing Fusarium graminearum gene expression during infection of three monocot hosts

Linda Harris,  Margaret Balcerzak, Danielle Schneiderman, Anne Johnston, Thérèse Ouellet

Agriculture & Agri-Food Canada


Fusarium graminearum is a broad host pathogen which has a major impact on cereal crops worldwide, causing fusarium head blight in small grain cereals (wheat, barley, oats) and gibberella ear rot in maize. To examine whether F. graminearum responds differently to diverse hosts, we compared the Fusarium gene expression profile during early infection of maize kernels and wheat and barley heads under the same environmental conditions. A single F. graminearum strain was used for the inoculation of all three hosts. Custom 4X44K Agilent microarrays designed to represent the F. graminearum gene set (13,918 predicted ORFs) were used to obtain transcriptome profiles of tissues 1d, 2d and 4d post-inoculation. Cross-hybridizing plant sequences were eliminated by conducting duo-dye hybridizations, comparing mock-inoculated and Fusarium-inoculated tissues directly.  An analysis of the gene expression profiling will be presented.





What large-scale T-DNA insertional mutagenesis tells us about pathogenicity? a functional genomics analysis in the dothideomycete Leptosphaeria maculans

Salim A. Bourras[1] Meyer Michel[1] Grandaubert Jonathan[1] Lapalu Nicolas[2] Balesdent Marie-Hélène[1] Rouxel Thierry[1]

1INRA-Bioger. Campus AgroParisTech. Avenue Lucien Bretignieres, 78 850 Thiverval Grignon. France
2URGI. Route de Saint Cyr, 78026 Versailles. France.


The Dothideomycete phytopathogen Leptosphaeria maculans is capable to alternate saprophytic, hemibiotrophic, endophytic and necrotrophic life styles during a single infectious cycle on its host plant, Brassica napus. However, little is known about the determinants of such plasticity. A reverse genetic strategy was developed, and a large-scale T-DNA insertional mutagenesis project was conducted resulting in: i) a collection of 5000 transformants phenotyped when interacting with plants, ii) a collection of 170 pathogenicity altered mutants, and iii) a set of 400 T-DNA flanking sequences. In addition, the L. maculans genome was sequenced and annotated, and whole genome Gene Ontology (GO) analysis was performed. Here we present combined analyses of the genomic pattern of 318 T-DNA insertion events (T-IEs), and the functional pattern of 279 T-DNA targeted genes (T-TGs). T-IEs analyses showed that: i) T-IEs favoured regulatory regions of gene-rich euchromatic genomic regions, and ii) T-IEs density correlated with CG skew near the transcription initiation site. These results are consistent with the T-DNA intranuclear targeting model, relying on gene expression machinery. T-TGs analyses showed that: i) T-IEs targeted 48.9% of the biological processes that were identified by whole-genome GO analysis, and that ii) T-IEs favoured biological processes that are consistent with the cellular state of a germinating conidia. Functional analysis of T-TGs in pathogenicity altered mutants will be presented.





Host-parasite interaction in the Brassica napus/Verticillium longisporum system

Susanna Braus-Stromeyer[2] Seema Singh[1] Christian Timpner[2] Gerhard Braus[2]

1Maine Medical Center Research Institute, 2Georg-August-University Goettingen


The first leaky auxotrophic mutant for aromatic amino acids of the near-diploid fungal plant pathogen Verticillium longisporum (VL) has been generated. VL enters its host Brassica napus through the roots and colonizes the xylem vessels. The xylem contains little nutrients including low concentrations of amino acids. We isolated the gene Vlaro2 encoding chorismate synthase by complementation of the corresponding yeast mutant strain. Chorismate synthase produces the first branch point intermediate of aromatic amino acid biosynthesis. A novel RNA-mediated gene silencing method reduced gene expression of both isogenes by 80% and resulted in a bradytrophic mutant, which is a leaky auxotroph due to impaired expression of chorismate synthase. In contrast to the wild type, silencing resulted in increased expression of the cross-pathway regulatory gene VlcpcA (similar to cpcA/ GCN4) during saprotrophic life. The mutant fungus is still able to infect the host plant B. napus and the model Arabidopsis thaliana with reduced efficiency. VlcpcA expression is increased in planta in the mutant and the wild-type fungus. We assume that xylem colonization requires induction of the cross-pathway control, presumably because the fungus has to overcome imbalanced amino acid supply in the xylem.





Colletotrichum graminicola globally accelerates senescence but locally induces photosynthetically active green islands on aging maize leaves

Stefan Wirsel, Michael Behr, Klaus Humbeck, Gerd Hause, Holger B. Deising

Martin-Luther-Universität Halle


Typically, pathogenesis of the hemibiotrophic fungus Colletotrichum graminicola and defense responses of its host, Zea mays, are studied by inoculation of young leaves. Equivalent studies have not been performed with leaves undergoing senescence, a situation which may be epidemiologically relevant in the field. We compare the infection of C. graminicola on senescing and mature leaves. We discovered that in contrast to the anthracnose symptom known from young and mature leaves green islands, reminiscent to those from obligate biotrophs, are formed on senescing leaves. Microscopy revealed that the fungus grows in both symptoms from the epidermal layer towards the bundle sheath that was used for longitudinal spreading. In green islands host tissues remained intact for a more extended period. Imaging PAM fluorescence analyses at high spatial resolution revealed that photosynthesis is transiently maintained at green islands but declined in tissue surrounding the infection site. In contrast, in younger green leaves photosynthesis is reduced only at the infection sites. Further support for the local modification of host physiology comes from qRT-PCR experiments analyzing gene expression at high spatial resolution. Decreased transcript levels of senescence markers corroborated a pathogen-induced delay of senescence. Expression of several genes encoding proteins involved in photosynthesis was strongly reduced after fungal infection. In contrast, the transcript levels of a cell wall invertase were strongly increased at green islands suggesting that C. graminicola induced a new carbon sink in the senescing tissue.


Hapx is involved in maintenance of iron homeostasis and virulence of Aspergillus fumigatus

Markus Schrettl[1] Martin Eisendle[1] Thorsten Heinekamp[2] Ernst R. Werner[1] Ilse Jacobsen[2] Axel A. Brakhage[2] Hubertus Haas[1]

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


Consistent with iron playing a crucial role in virulence, we have previously shown that biosynthesis of siderophores (iron chelators involved in uptake, storage and intracellular distribution of iron) is essential for virulence of A. fumigatus. The characterization of the fungal iron metabolism might aid improvement of diagnosis and treatment of fungal infections.

During iron-replete conditions, siderophore biosynthesis is repressed by the GATA factor SreA. Here we report the characterization of a second iron regulator, the bZIP transcription factor HapX. SreA and HapX are interconnected by a regulatory feedback loop: SreA repressed expression of hapX during iron sufficiency and, vice versa, HapX repressed expression of sreA during iron starvation. During iron starvation, inactivation of HapX resulted in derepression of iron-dependent pathways (e.g. the mutant strain displayed accumulation of the iron-free heme precursor protoporphyine IX) but reduced production of extra- and intracellular siderophores. Moreover, the hapX deletion mutant displayed significantly reduced virulence in a murine model of aspergillosis.

This study demonstrates the crucial role of HapX in iron regulation and virulence of A. fumigatus. Deleterious consequences of inactivation of SreA and HapX are strictly confined to iron replete and -depleted conditions, respectively. Consequently, attenuation of virulence by inactivation of HapX, but not of SreA, underlines that A. fumigatus faces iron-limited conditions during mammalian infection.

Acknowledgement: This work was supported by the Austrian Science Foundation grant FWF P18606-B11 to HH


Characterization of a novel regulatory gene involved in virulence in the phytopathogen Fusarium graminearum

Sean Walkowiak, Winnie Leung, Anne Johnston, Linda Harris and Gopal Subramaniam

Agriculture and Agri-Food Canada


F. graminearum is a pathogenic fungus that infects cereal crops. It produces a trichothecene mycotoxin, deoxynivalenol (DON), which is positively associated with virulence in the fungus and causes toxicity in humans and livestock. A study performed by Alexander et al. suggested Tri15 may be negatively regulating some of the genes in the trichothecene biosynthetic pathway in F. sporotrichioides. In contrast, disruption of Tri15 in F. graminearum, neither affected its ability to synthesize 15-ADON nor its pathogenicity. This study explores the role of Tri15alt, a homologue of Tri15. Tri15alt encodes for a protein that has three zinc fingers, two of which are highly homologous to the zinc fingers found in Tri15. Targeted disruption of Tri15alt in F. graminearum did not compromise the biosynthesis of 15-ADON. However, pathology studies performed on a susceptible variety of wheat (Roblin) revealed that Tri15alt disrupted strain is more virulent than the wildtype strain. We have performed microarray analyses on this mutant and results will be presented to identify genes involved in virulence.

Alexander N.J., S.P. McCormick, T.M. Larson and J.E. Jurgenson. 2004. Expression of Tri15 in Fusarium sporotrichioides. Curr Genet 45: 157-162





Crosstalk between Claviceps purpurea and rye during early stages of pathogenesis

Janine Schürmann and Paul Tudzynski

 Westfälische Wilhelms-Universität, Institut für Botanik, Schlossgarten 3, D-48149 Münster, Germany


The ergot fungus Claviceps purpurea is a phytopathogenic fungus with a broad host range including economically important species like wheat, rye, barley and rice. Nevertheless the infection with C. purpurea is highly conserved and organ specific as only young ovaries of flowering grasses are infected on a defined pathway probably mimicking pollen tube growth. During infection C. purpurea shows directed and polar growth comparable to axon differentiation, pollen tube growth and root hair formation (Tudzynski and Scheffer 2004). Thus we are currently comparing in planta growth of C. purpurea during infection with pollen tube growth. Apart from striking differences in growth rates the fungus deviates from the pollen tube pathway after early infection stages. To investigate the host reaction in detail a microarray analysis is underway. The deletion of the two known small GTPases Cdc42 and Rac have different but severe effects on the polar and directed growth of C. purpurea. In other fungi, their temporal and spatial regulation has shown to be of major importance for their functions. Therefore we are analyzing the functions of the scaffold protein Bem1 in C. purpurea. A bem1 homolog was detected in Claviceps and completely sequenced. Protein interaction studies and a gene replacement analysis of bem1 are in progress.




Selection pressures exerted by apple scab resistance QTL on Venturia inaequalis co-inoculated strains

Amandine Lê Van[1] Caffier Valérie[1] Le Cam Bruno[1] Pauline Lasserre[2] Durel Charles-Eric[2]

1INRA Angers-Nantes UMR 077 PaVé, 2INRA Angers-Nantes UMR 1259 Genhort


Pathogen adaptation to major resistance genes has often been reported. Partial resistance conferred by QTL is expected to be more durable. However adaptation to this type of resistance is not well documented. Partial resistance erosion can be seen as a multiple step breakdown if several QTL are successively overcome by the pathogen. This process can be driven by the differential selection exerted by the QTL on the pathogen population. In this study we will evaluate selection pressures exerted by QTL on a mixed inoculum of V. inaequalis, the apple scab causal pathogen. Broad-spectrum and isolate-specific scab resistant QTL were previously identified in a F1, partially resistant, apple progeny independently challenged with 6 contrasted monoconidial strains. In this study, we equally mixed these 6 strains, inoculated them on the same F1 progeny, scored sporulation severity and then collected leaves on each individual in order to identify sporulating strains. A QTL analysis performed on the AUDPC revealed that only broad spectrum QTL were detected, underlying the usefulness of mixed inoculum for broad spectrum QTL detection. Quantification of the relative proportion of each strain on each individual by pyrosequencing to evaluate selection pressures exerted by QTL alone or in combination is underway. A QTL analysis will be performed on these genotyping data and compared to the previous QTL analyses. This study should help us to better understand how partial resistance filters a mixture of contrasted strains according to the number, effect, and spectrum of action of the involved QTL.





Fungal virulence and host susceptibility genes in the Fusarium oxyporum-Arabidopsis interaction

John Manners, Louise Thatcher, Donald Gardiner, Kemal Kazan



The interaction of the root infecting fungal pathogen Fusarium oxysporum with Arabidopsis is a highly tractable system for a molecular analysis of fungal virulence and host susceptibility and immunity. We have completed a rigorous analysis of 6868 T-DNA insertion mutants of Arabidopsis Col-0 ecotype, selected lines with altered disease phenotype (P=<0.01) and retested these to identify mutants with significant & reproducible increased resistance or susceptibility. Second allele insertions are currently being tested to provide certainty on specific gene functions. These studies have identified ~100 novel genes with previously unidentified roles in immunity and susceptibility to this pathogen. To complement this we have identified a small range of fungal mutants with altered pathogenicity and virulence. One of these includes mutants in the SIX4 gene which is required for full virulence. Experiments are underway to attempt to match putative functions in the host that are necessary for susceptibility with functions in the pathogen required for virulence. Initial experiments are focusing on the role of host jasmonate signalling in susceptibility and how the pathogen may intervene in this.



The G protein-coupled receptor Gpr1 is required for host recognition and mycoparasitism in the biocontrol agent Trichoderma atroviride

Susanne Zeilinger and Markus Omann

Vienna University of Technology


Mycoparasitic Trichoderma species are applied as biocontrol agents in agriculture to guard plants against fungal diseases. During mycoparasitism, Trichoderma interacts with phytopathogenic fungi which is preceded by recognition of the host and results in its disarming. In various fungal pathogens including mycoparasites, G protein signaling regulates pathogenicity-related functions. Nevertheless, the corresponding G protein-coupled receptors (GPCRs) involved in the recognition of host-derived signals are largely unknown. Functional characterization of Gpr1 from T. atroviride revealed a prominent role of this GPCR in host recognition and regulation of mycoparasitism-associated processes. gpr1 silencing led to altered vegetative growth, conidiation and antifungal metabolite production. When confronted with host fungi, the mutants exhibited an avirulent phenotype resulting from their inability to recognize and attach to host hyphae and undergo mycoparasitism-related infection structure formation. In addition, mutants were unable to respond to living host fungi with the expression of chitinase- and protease-encoding genes. Interestingly, their ability to produce chitinases was not impaired in liquid culture in the presence of colloidal chitin or N-acetyl-glucosamine, ruling out that these are ligands of Gpr1. Exogenous cAMP was able to restore infection structure formation in the mutants but had no effect on mycoparasitic overgrowth. A search for targets of the signaling pathway(s) involving Gpr1 resulted, amongst others, in the isolation of a gene encoding a member of the cyclin-like superfamily. Additionally we found evidence for Gpr1 to be constitutively active. Our data highlight the fundamental role of Gpr1 during sensing of environmental signals and transduction to intracellular regulatory targets during the antagonist-host interaction. 





Arthroderma benhamiae secretome analysis and pathogenicity factor characterisation

Christoph Heddergott[1] Olaf Kniemeyer[1] Johannes Wöstemeyer[2] Axel A. Brakhage[1,2]

1Leibniz Institute for Natural Product Research and Infection Biology e.V. - Hans-Knöll-Institute (HKI), Jena, 2Friedrich-Schiller-University (FSU), Jena


Arthroderma benhamiae, a zoophilic pathogen causing inflammatory Dermatophytosis in humans and rodents, was selected to serve as a model organism for elucidation of general pathogenicity mechanisms of Dermatophytes. Proteins secreted by the fungus are believed to have important functions during infection. After growth on keratin, a major component of the host’s skin, proteins secreted by the fungus were analysed using 2D-PAGE and mass spectrometry for protein separation and identification, respectively. A. benhamiae secretes a large number of proteolytic enzymes from different protease families. Beside these, numerous proteins of carbon hydrate and lipid metabolism as well as functionally uncharacterised proteins lacking conserved domains have been found. This work presents the first comprehensive secretome analysis for A. benhamiae. In extracts of purified mycelial cell walls two major proteins were identified. The hydrophobin Hyp1 is released by hydrogen fluoride/pyridine treatment whereas a secreted but functional uncharacterised protein was also present in SDS extracts. Its appearance was highest on keratin, it was absent upon growth on casamino acids and therefore termed keratin-induced protein 1 (Kip1). These two proteins are located at the cell surface, where they could directly contact host structures during infection. To elucidate the putative role of these proteins in pathogenicity, gene deletion mutants (Δkip1, Δhyp1) as well as a strain constitutively expressing Kip1 were constructed and are currently analysed.




Role of mannitol in pathogenic determinism in Alternaria brassicicola

Benoît Calmes[1] Guillemette Thomas[1] Lemoine Remi[2] Simoneau Philippe[1]

1Université d'Angers, 2Université de Poitiers


Mannitol is the most abundant polyols occurring in nature and is usually the most abundant of all the soluble carbohydrates within the mycelium. This compound is known to play different roles in the cell including carbohydrate storage and stress tolerance. The ability of mannitol to quench reactive oxygen species (ROS) was demonstrated (Voegele et al., 2005), leading to the hypothesis that this polyol could play an antioxydant role in host-pathogen interactions. This study focused on the role of mannitol in the infectious process of the nécrotrophic pathogen Alternaria brassicicola responsible for the black spot disease of Brassicacae. The production and secretion of mannitol by A. brassicicola were induced in the presence of host-plant extract suggesting its involvement in the pathogenic process. Genes coding two essential enzymes of the mannitol metabolism, mannitol dehydrogenase (MDH) and mannitol-1-phosphate dehydrogenase (MPD), were characterized and were separately or additionally disrupted by insertional mutagenesis. Enzyme assays confirmed the lack of enzymatic activity for each enzyme in corresponding null mutants. Double-mutant, mutants deficient in MPD or MDH alone produced 15%, 50% and 100% respectively, of the mannitol synthesized by the wild type. Pathogenicity assays revealed the disruption of mannitol metabolism genes affected aggressiveness of A. brassicicola. All mutants showed increased sensitivity to hydrogen peroxide compared to wild type. Ab∆mpd and Ab∆mpdmdh also exhibited a reduced growth at lower matric potential whereas Ab43∆mdh was more tolerant. Data throw new light on specifics functions of  mannitol metabolism in necrotrophic metabolic strategy of A. brassicicola.





The use of redox-sensitive GFP2 as a biosensor of the redox status in Botrytis cinerea

 Jens Heller[1] Andreas Meyer[2] Julia Schumacher[1] Paul Tudzynski[1]

1Institut für Botanik, Westf. Wilhelms-Universität, Schlossgarten 3, D-48149 Münster, Germany
2Heidelberg Institute for Plant Science, Ruprecht-Karls-Universität Heidelberg, Im Neuenheimer Feld 360, D-69120 Heidelberg, Germany


The necrotrophic plant pathogen Botrytis cinerea is the causal agent of grey mould disease in more than 200 plant species. There has been evidence that Bcinerea has to cope with oxidative stress during the infection process caused by an early plant defence reaction, the oxidative burst. However, it was shown recently that the AP1-transcription factor Bap1 as main transcriptional regulator of H2O2-scavenging proteins is not essential for the virulence of B. cinerea. In fact Botrytis even seems to enhance the ROS level in planta by producing its own ROS during infection. To answer the rising question if Botrytis really suffers oxidative stress during the infection process, we are in need of tools to measure its redox status in vivo. The ratiometric redox-sensitive GFP2 (roGFP2) reversibly responds to redox changes induced by incubation with H2O2 or DTT in vitro and specifically senses the glutathione redox potential (EGSH) after expression in Arabidopsis thaliana. Our aim is to establish roGFP2 in the filamentous fungus B. cinerea to investigate the redox status of Botrytis during infection. Here we show that roGFP2 is functional in Botrytis hyphae and can be used to determine changes in EGSH in vivo. Since we also want to compare the stress-induced redox changes of different signalling mutants in real time using roGFP2, we need a defined integration site of the reporter gene construct. Analyses of the gene bcniaD, encoding for a nitrate reductase, show that the bcniaD-locus is suitable as site of integration for reporter gene constructs in B. cinerea.





Characterization of mannosyltransferase mutants of the pathogenic mold Aspergillus fumigatus

Andrea Kotz[1] Johannes Wagener[1] Françoise Routier[2] Bernd Echtenacher[3] Manfred Rohde[4] Frank Ebel[1]

1Max-von-Pettenkofer-Institut, LMU Munich, Germany
2Department of Cellular Chemistry, Hannover Medical School, Germany
3Institute for Immunology, University of Regensburg, Germany
4Department of Microbial Pathogenesis, Helmholtz Center for Infection Research, Braunschweig, Germany


The fungal cell wall comprises different carbohydrates either as part of glycoconjugates, like glycolipids and glycoproteins, or as constituents of its core structure. During infection the cell wall of pathogenic fungi has to be robust enough to withstand the stress applied by the host immune response. Moreover, distinct cell wall carbohydrates are likely to be recognized by host pattern recognition receptors, a key event in the innate immune response to fungal infections. We currently analyze a set of mannosyltransferases of the pathogenic mold Aspergillus fumigatus that by homology to their Saccharomyces counterparts are supposed to catalyze key steps in the formation of glycoconjugates. GDP-mannose:inositol-phosphorylceramide (MIPC)-derived glycosphingolipids are important pathogen-associated molecular patterns (PAMP) of Candida albicans and have recently been discussed as relevant PAMPs of A. fumigatus. We identified MitA as the only MIPC transferase in A. fumigatus. A ΔmitA mutant lacks MIPC and MIPC-derived glycosphingolipids and accumulates the precursor IPC. The mutant grows normally, shows no defects in cell wall or membrane organization and a normal resistance to various stressors. However, it is delayed in germination and sensitive to high Ca2+ concentrations. The ΔmitA mutant is not significantly impaired in its virulence or ability to trigger a cytokine response in macrophages, arguing against a role of MIPC-derived glycosphingolipids as important A. fumigatus PAMPs. We have also analyzed two mutants that lack key enzymes in the synthesis of O- and N-linked glycans, namely Mnt1 and OchA. Data on their fitness, pathogenicity and interaction with innate immune cells will be presented.





Molecular interaction between Trichoderma virens and tomato roots

Mariarosaria Vergara, Moncini Lorenzo, Vannacci Giovanni, Baroncelli R.

Dept. of Tree Science, Entomology and Plant Pathology, Section of Plant Pathology, University of Pisa


The ability of Trichoderma spp. to antagonise plant pathogens, to induce plant resistance and to promote growth in plants has been widely described. Endo-PG produced by Trichoderma spp. can assist root penetration and play a pre-eliciting role in systemic resistance (SIR), a beneficial effect detected in plants colonised by Trichoderma.

Two endo-PG genes (eptv1 and eptv2) have been identified and partially characterised in a T. virens isolate, I10, previously investigated for its antagonistic ability in several biological systems.

A GFP transformant, I10GFP, derived from the same isolate was exploited to monitor the tomato root colonisation process and to define suitable timing for further experiments.

An expression analysis has pointed out a different regulation of those genes. The eptv1 gene resulted induced when the fungal isolate was grown in liquid cultures supplemented by pectin or plant cell walls and when it was applied to tomato roots in growth chamber. Expression times were comparable in both systems. The eptv2 gene resulted constitutively expressed in all conditions tested, including controls.

Root exudates showed a promoting effect both on mycelial growth and on conidia germination of T. virens I10.

Further mechanisms of the molecular communication between tomato roots and T. virens mycelium are under investigation.


Comparative genomics reveals mobile pathogenicity chromosomes in Fusarium oxysporum

Li-Jun Ma, Fusarium comparative genomics working group

The Broad Institute


Fusarium species are among the most important phytopathogenic fungi, having significant impact on crop production and animal health. Distinctively, strains of F. oxysporum exhibit wide host range and are pathogenic to both plant and animal species, reflecting remarkable genetic adaptability. To understand the mechanism underlying such genetic plasticity and rapid pathogenic development, we compared the genomes of three economically important and phylogenetically related, yet phenotypically distinct phytopathogenic species, F. graminearum, F. verticillioides and F. oxysporum f. sp. lycopersici. Comparative analysis revealed greatly expanded lineage-specific (LS) genomic regions in F. oxysporum that include four entire chromosomes and account for more than one-quarter of the genome. These regions are rich in transposons and genes involved in host-pathogen interactions, including known effectors, enzymes targeting plant substrates or processes, and genes involved in lipid signalling and gene silencing. In addition, we studied the LS chromosomes among 10 selected F. oxysporum strains using both high throughput sequence and optical mapping technologies and confirmed the wide existence of these LS chromosomes in F. oxysporum species complex. We also found evidence for the acquisition of the LS chromosomes through horizontal transfer, which may explain the polyphyletic origins of host specificity in F. oxysporum and the rapid emergence of new pathogenic lineages in distinct genetic backgrounds.





Loss-of-function of the avirulence gene, SIX4, by transposon-insertion in tomato wilt pathogen Fusarium oxysporum f. sp. lycopersici

KEIGO INAMI[2] Yasuaki MORITA[1] Chizu YOSHIOKA[2] Tohru TERAOKA[2] Tsutomu ARIE[2]

1Kochi Agricultural Research Center, Kochi, Japan
2Department of Biological Production Science, Tokyo University of Agriculture and Technology (TUAT), 1838509, Tokyo, Japan


Fusarium oxysporum f. sp. lycopersici (FOL) is the soilborne pathogen of tomato wilt.  In the pathogen, three races 1, 2, and 3 have been determined based on the specific pathogenicity to tomato varieties.  The compatible or incompatible relationships between races and varieties can be explained by the interactions between the avirulence genes carried by FOL and resistance genes carried by tomato varieties according to gene-for-gene theory (Flor, 1956).  For example, race 1 carrying AVR1 is avirulent to tomato cultivars with a resistance gene I, and races 2 and 3 carrying no AVR1 is virulent to the tomato cultivars with I.  Houterman et al. (2008) reported SIX4 corresponding to AVR1 in FOL race 1.

In 2008 a strain of FOL (KoChi-1), overcoming I-mediated resistance, emerged in Japan.  Although KoChi-1 is not race 1, PCR revealed that KoChi-1 carried SIX4.  Sequence analysis showed that SIX4 ORF in KoChi-1 was truncated by a transposon (759 bp).  The inserted transposon is non-autonomous and belongs to hAT family (Hua-Van et al., 2000).  According to the Genome Databases of Broad Institute, 72 copies of the identical transposon exist in F. oxysporum.  Integration of an intact SIX4 derived from a race 1 isolate into KoChi-1 genome complemented avirulence to a tomato cultivar possessing I.  This is the first report of an avirulence gene truncated by transposon-insertion in F. oxysporum.




The role of a signal peptidase component in pathogenicity of Colletotrichum graminicola to maize.

Buiate, E.[1] Torres, M.[1] Park, E.[1] Thon, M.[2] Vaillancourt, L.J.[1]

1Department of Plant Pathology, University of Kentucky, USA, 2University of Salamanca, Spain


Fungal stalk rot is one of the most economically important diseases of maize, estimated to result in losses worldwide of approximately 5% annually. Colletotrichum graminicola causes anthracnose, one of the most common and damaging of the fungal stalk rots. C. graminicola also causes a leaf blight disease on maize. We used a random mutagenesis technique (REMI) to identify several genes that are required for pathogenicity of C. graminicola to maize stalks and leaves. The sequence of one tagged gene (Cpr1) identified it as a conserved component of the endoplasmic reticulum (ER)-localized signal peptidase.  Given the essential function of that enzyme complex in protein transport it was surprising that, except for a slightly reduced growth rate, the REMI mutant was almost normal in culture. However, it was completely non-pathogenic to maize leaves and stalks. The REMI mutant was able to colonize senescing or paraquat-treated maize tissues normally.  It also infected host cells normally if it was co-inoculated with the wild type. The REMI mutant was more sensitive in vitro to thermal stress and to chemically-induced secretion stress. We are testing the hypothesis that Cpr1 functions specifically in adaptation to secretion stress during establishment of the biotroph, and to facilitate the secretion of effectors that induce susceptibility of host cells.





The role of glycerol metabolism in the Colletotrichum higginsianum-Arabidopsis interaction

Kulshrethsa, S., Dotson, P., Venugopal, S., Chandra, B.,  Kachroo, A.,  Kachroo, P.,  Vaillancourt, L.J.

Department of Plant Pathology, University of Kentucky, USA


Glycerol-3-phosphate (G3P) is an important component of carbohydrate and lipid metabolic processes. Inoculation of Arabidopsis with the hemibiotrophic fungal pathogen Colletotrichum higginsianum was associated with an increase in G3P levels in the host.  A mutation in the Arabidopsis G3P generating G3P dehydrogenase (G3Pdh, GLY1) resulted in reduced levels of G3P and enhanced susceptibility to C. higginsianum.  Correspondingly, overexpression of GLY1 increased G3P levels and enhanced resistance to the pathogen.  Manipulating endogenous G3P levels by genetic mutations, or by overexpression in transgenic plants of other genes affecting G3P biosynthesis, demonstrated that higher amounts of G3P were always associated with higher levels of resistance. Interestingly, there was a similar effect of G3P levels on pathogenicity in the fungus: a knock out of the C. higginsianum G3Pdh resulted in reduced levels of G3P, and a significant reduction in pathogenicity to Arabidopsis. Most intriguing of all, the mutant strain regained its normal pathogenicity to mutant plants containing reduced levels of endogenous G3P.  Together, these results suggest a novel and specific link between G3P metabolism in the host and pathogen during pathogenesis.


A shuttling RNA-Binding protein involved in synthesis of natural products regulates fungal development and pathogenicity

Marina Franceschetti, Emilio Bueno, Ane Sesma

Department of Disease and Stress Biology, John Innes Centre, Colney Lane, Norwich, NR4 7UH, UK.


Very little is known about the function of RNA-binding proteins during plant infection in filamentous fungi. The Magnaporthe oryzae T-DNA mutant M35 was identified as being defective in plant colonisation. M35 has undergone insertional inactivation of a gene encoding a novel RNA-binding protein (RBP35) with two protein modules, an RRM domain (RNA Recognition Motif) and the Arg-Gly-Gly rich region. The RBP35 homologues are found only in filamentous fungi. The full length cDNA has been isolated of the RBP35 gene. It contains an unusually large 5’UTR (~930 bp) with several regulatory regions including a conserved intron (211 bp) and several uATGs. A his-tagged version of the RBP35 protein binds specifically poly(G)30 RNA homopolymers and not ssDNA or dsDNA.  Amino and carboxy RPB35-cherry protein fusion constructs localise both in the nucleus and cytoplasm. Western blots carried out with a specific antibody raised against RBP35 revealed the presence of two protein isoforms, the full length protein (~44 kDa, RBP35a), and a smaller protein (~31 kDa, RBP35b) derived from the proteolytic cleavage of the carboxy end of RBP35a.  The RBP35b protein lacks the Arg-Gly-Gly rich region and partially restores the altered phenotype of Drbp35. A proteomics comparison between the Drbp35 mutant and the corresponding wild-type strain showed that several enzymes required for flavonoid and melanin synthesis were up- and down-regulated in Drbp35.  Proteomic results correlated with the metabolic profiles from the wild-type strain and Drbp35. Currently, we are optimising methods to identify mRNAs and proteins interacting with RBP35a and RBP35b. This will allow us to identify the post-transcriptional network regulated by RBP35 and in which step of the RNA processing RBP35 is participating.





Comparative studies of the transcriptome of the leafcutter ant fungal symbiont

Tore Linde[1] Morten Nedergaard Grell[1] Morten Schiøtt[2] Sanne Nygaard[2] Kåre Lehmann[3] Jacobus J. Boomsma[2] Lene Lange[1]

1Section for Biotechnology and Bioenergy, Copenhagen Institute of Technology, Aalborg University, Denmark, 2Department of Biology, University of Copenhagen, Denmark, 3Center for Biotechnology, Department of Biotechnology, Chemistry and Environmental Engineering, Aalborg University, Denmark


It has been possible by using the method “Serial Analysis of Gene Expression” (SAGE) to show quantitative differences in the enzyme expression in the vertical profile of the fungus Leucoagaricus gongylophorus, who lives in symbiosis with the leafcutter ant Acromyrmex echinatior. The ants live and nest inside the fungal garden and feed the fungus fresh leaves harvested from the surroundings. The fresh leaves are placed on top of the fungal garden, and the fungus starts degrading the plant material while growing upwards, in the end releasing glucose and nutrients for the fungus and the ants. Due to the slow degradation of plant material the fungal garden is different in structure and in the enzyme production profile in a vertical direction from top to bottom. In this study, the fungus was divided into three layers, top, middle and bottom, and enzyme production in the different layers was investigated at the transcriptome level using SAGE combined with cDNA tag and genome sequencing. The results reflected major differences in plant cell wall degradation among layers, revealed specifically by big differences in the production of laccases, endoglucanases, cellobiohydrolases and xylanases.




Unraveling the complex dikaryotic genome sequence of the soil fungus Rhizoctonia solani.

Marc A. Cubeta 1*, Ralph A. Dean1, Elizabeth Thomas1, Suman Pakala1, Doug Brown1, Paul Bayman2, Suha Jabaji3, Stephen Neate4, David Schwartz5, Shiguo Zhou5, Stellos M. Tavantzis6, Takeshi Toda7, Rytas Vilgalys8, Paulo Ceresini9, Natalie Fedorova10 and William C. Nierman10

1Dept. Plant Pathology, North Carolina State University, Raleigh, NC 27695 7616, USA, 2Dept. Biology, University of Puerto Rico, San Juan, PR 00931-3360, USA, 3Dept. Agricultural and Environmental Sciences, McGill University, Quebec  H9x 3V9, Canada, 4Dept. Primary Industries and Fisheries, Leslie Research Centre, Queensland 4350 Australia, 5Dept. of Chemistry, Laboratory of Genetics, UW-Biotechnology Center,    University of Wisconsin, Madison, USA, 6Dept. of Biological Sciences, University of Maine, Orono, ME 04469-5722, USA 7Dept. Bioresource Sciences, Akita Prefectural University, Akita, 010-0195, Japan, 8Dept. Biology, Duke University, Durham, NC 27708, USA 9Dept. Plant Pathology, ETH Institute of Integrative Biology LFW B28 8092 Zurich, Switzerland, 10J. Craig Venter Institute, Rockville, MD 20850, USA

*Corresponding author:


A consortium consisting of North American and international scientists in the Rhizoctonia community is actively involved in a collaborative project to obtain a high quality complete genome sequence of the soil fungus R. solani anastomosis group 3 (teleomorph=Thanatephorus cucumeris), strain Rhs1AP. This fungus is a competitive saprobe and an important pathogen of plants in the family Solanaceae. In addition to its economic importance as a plant pathogen, the fungus and its closely related species can often function as beneficial endomycorrhizal symbionts that promote the germination of orchid seeds and growth of orchid seedlings. Sanger, 454 Titanium FLX pyrosequencing, and Illumina (Solexa) methods have been used to generate approximately 2.5 Gb of sequence data that represents approximately 22X coverage of the estimated 90 Mb genome. An optical restriction map of the fungal chromosomes has been developed and there are 23 contigs with both telomeric regions and seven unfinished contigs with one or no telomeric regions. The size of the contigs ranged from 1.85 to 5.85 Mb and the rDNA repeats were identified on the latter contig. A method to generate haploid components of the genome has been developed and will be used to complement the optical map and cDNA library data to better assemble and annotate the dikaryotic genome sequence. The genome sequence of R. solani will provide a basis for comparative studies to increase our understanding of the evolution of fruiting body development and modes of trophic behavior in important basal and transitional group of basidiomycete fungi in the Agaricomycotina.





Fungal secondary metabolism and its impact on ecological interactions with insects

Frank Kempken[1] Ulrike Fohgrub[1] Monika Trienens[2] Marko Rohlfs[2]

1Botanisches Institut und Botanischer Garten, Christian-Albrechts-Universität, Olshausenstraße 40, 24098 Kiel, Germany, 2Zoologisches Institut, Christian-Albrechts-Universität


Filamentous fungi and saprophage insects are suspected to be competitors on decaying organic matter (1, 2). Both organisms have equal requirements considering habitat and nutrition. Insect larvae negatively influence mould development (3), but filamentous fungi can be an important cause of mortality of insect larvae (4). These competitions in insect-mould interactions have largely been ignored. First investigations suggest a role of genes for fungal secondary metabolism (1). Using a combination of experimental ecology and functional genomic techniques the function of secondary metabolites (e.g. mycotoxine) as a chemical defence in insect-fungal interactions as well as the influence of these competitors at trophic interactions between insects ought to be investigated. For our research the vinegar fly Drosophila melanogaster and its natural antagonist Aspergillus were used as a form of ecology model system. Microarrays of Aspergillus nidulans have been used to identify fungal target genes up- or downregulated when interacting on festered matter with the antagonistic Drosophila larvae. Preliminary test with quantitative RT-PCR of RNA from A. nidulans confronted with D. melanogaster larvae indicates upregulation of the global regulator laeA, as well as aflR. Specific down- or upregulation of these target genes will be performed to analyse their importance for competitions in insect-fungal interactions. The consequence on evolutionary fitness of the fungi and insects will be analysed. (1) Rohlfs M, Trienens M, Fohgrub U, Kempken F (2009) In: The Mycota XV, pp131-151 (2) Kempken F, Rohlfs M (2009) Fungal Ecol, doi:10.1016/j.funeco. 2009.08.001 (3) Rohlfs M (2005) Mycologia 97:996-1001 (4) Rohlfs M (2005) Frontiers in Zoology 2:2





Genome expansion in powdery mildews is caused by loss of immunity against genomic parasites

Pietro D. Spanu  and the Blumeria Genome sequencing Consortium.

Department of Life Sciences, Imperial College London, UK; Exeter University, UK; University of Oxford, UK; John Innes Centre, UK; URGI/INRA, France.


We report that the genome of the powdery mildew Blumeria graminis f.sp. hordei is ~120Mb,  which is much larger than other closely related Ascolycetes. This is due to an extraordinary amplification of retro-transposons which results in > 70% repetitive DNA in the genome. All the three genes encoding enzymes known to be necessary for Repeat Induced Point mutation (RIP) are absent from all powdery mildews analysed. A comparative genome-wide analysis of repetitive DNA in B. graminis and other Ascomycetes reveals an absence of RIP-ing in the repetitive DNA. This suggests that the evolution of a larger genome in the Erysiphales has been driven by a massive proliferation of genomic parasites (retro-transposons) allowed by the loss of one of the mechanisms to keep these elements under control. The fact that B. graminis, like all powdery mildews, is an obligate biotrophic pathogen whose significant trophic stage is intracellular suggests that genome miniaturisation is not a necessary accompaniment of parasitism observed elsewhere. Indeed other plant pathogenic fungi such as the rusts (Basidiomycetes), Phytophthora infestans (Oomycetes) and mycorrhizal fungi such as truffles and the Glomales show similar genome expansion suggests that the phenomenon is connected to their common life style. We speculate that genome expansion driven by retro-transposition may provide a selective advantage for fungi and fungal-like organisms interacting with plants.




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