Poster Category
2:
Fungal-Host Biology
Michael Price,
Marisol Betancourt-Quiroz, Anthony Lee, Dena L. Toffaletti, John R.
Perfect
michael.price@duke.edu
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.
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.
PR2.3
Caroline Michielse,
Matthias Becker, Jens Heller,
Paul Tudzynski
Westf. Wilhelms Universität Münster
c.b.michielse@uni-muenster.de
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.
Andrea Herrmann,
Yvonne Rolke, Julia von Sengbusch, Janine Schürmann, Paul Tudzynski
andrea.herrmann@uni-muenster.de
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.
References
[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.
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
nalu@usp.br
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.
Linda Harris,
Margaret Balcerzak, Danielle Schneiderman, Anne Johnston, Thérèse Ouellet
Agriculture &
Linda.Harris@agr.gc.ca
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.
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.
2URGI. Route
bsalim@versailles.inra.fr
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.
PR2.8
Susanna Braus-Stromeyer[2]
Seema Singh[1] Christian Timpner[2] Gerhard Braus[2]
1Maine
Medical Center Research Institute, 2Georg-August-University
Goettingen
sbraus@gwdg.de
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.
PR2.9
Stefan Wirsel,
Michael Behr, Klaus Humbeck, Gerd Hause, Holger B. Deising
Martin-Luther-Universität Halle
stefan.wirsel@landw.uni-halle.de
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.
PR2.10
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,
hubertus.haas@i-med.ac.at
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
PR2.11
Sean Walkowiak,
Winnie Leung, Anne Johnston, Linda Harris and Gopal Subramaniam
Agriculture and
swalkowi@connect.carleton.ca
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
Janine Schürmann
and Paul Tudzynski
Westfälische Wilhelms-Universität,
Institut für Botanik, Schlossgarten 3, D-48149 Münster, Germany
j_schu51@uni-muenster.de
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.
PR2.13
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
amandine.levan@angers.inra.fr
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.
John Manners,
Louise Thatcher, Donald Gardiner, Kemal Kazan
CSIRO
john.manners@csiro.au
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.
PR2.15
Susanne Zeilinger
and Markus Omann
szeiling@mail.tuwien.ac.at
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.
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),
christoph.heddergott@hki-jena.de
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.
PR2.17
Benoît Calmes[1]
Guillemette Thomas[1] Lemoine Remi[2] Simoneau Philippe[1]
1Université
d'Angers, 2Université de Poitiers
calmes.b@gmail.com
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∆mpd∆mdh
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.
PR2.18
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
jens.heller@uni-muenster.de
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
B. cinerea
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.
PR2.19
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
ebel@mvp.uni-muenchen.de
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.
PR2.20
Mariarosaria Vergara,
Moncini Lorenzo, Vannacci Giovanni, Baroncelli R.
Dept. of Tree Science, Entomology and Plant Pathology, Section of Plant
Pathology,
rvergara@agr.unipi.it
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.
Li-Jun Ma,
Fusarium comparative genomics working group
The Broad Institute
lijun@broadinstitute.org
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.
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
yloveit@gmail.com
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
Buiate, E.[1] Torres, M.[1] Park, E.[1] Thon,
M.[2] Vaillancourt, L.J.[1]
1Department
of Plant Pathology,
vaillan@uky.edu
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.
Kulshrethsa, S., Dotson, P., Venugopal, S., Chandra, B.,
Kachroo, A.,
Kachroo, P.,
Vaillancourt, L.J.
Department of Plant Pathology,
vaillan@uky.edu
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.
Marina Franceschetti, Emilio Bueno, Ane Sesma
Department of Disease and Stress Biology, John Innes Centre,
ane.sesma@bbsrc.ac.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.
PR2.26
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
Lindetore@gmail.com
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,
*Corresponding author: marc_cubeta@ncsu.edu
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.
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
fkempken@bot.uni-kiel.de
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.
p.spanu@imperial.ac.uk
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.