Poster Category
5:
Fungal Way of Living; Sex and Other Encounters
Identification of natural αAAα diploid hybrids in the
Cryptococcus neoformans
population
Mojgan Aminnejad and Wieland Meyer
Molecular Mycology Research Laboratory, Centre for Infectious Diseases and
Microbiology, University of Sydney, Sydney Medical School-Western at Westmead
Hospital, Westmead Millennium Institute, Westmead, NSW, Australia
mami3842@uni.sydney.edu.au
Cryptococcus neoformans is an opportunistic encapsulated pathogenic
basidiomycetous yeast that causes meningoencephalitis in immunocompromised (e.g.
AIDS) patients world-wide. The fungus is typically haploid, and sexual
reproduction occurs normally between two individuals with opposite mating types,
α and
a.
C. neoformans occurs in two serotypes (A and D), which correspond to
C. neoformans var.
grubii (molecular type: VNI & VNII) and
C. n. var.
neoformans (molecular type: VNIV). In addition, rare isolates with
AD serotype have been reported and suggested to be diploid or aneuploid. In this
study, several
C. neoformans isolates were collected globally and their molecular
type was assigned by
URA5-RFLP analysis with
HhaI and
Sau96I in a double digest. The results showed unusual combination
patterns of VNI and VNII for 8 isolates. These isolates were tested for ploidy
by fluorescence flow cytometry analysis and were found to be diploid. PCR
analysis with primers specific for serotype A or D genes and serotype- and
mating-type genes revealed that the isolates were serotype A and mating type α.
Also, the serotype of the isolates was further confirmed by
CAP59-RFLP analysis with
BsmFI and
HpaII. This study confirmed the presence of same sex αAAα hybrids in
nature, indicating that diploids can also occur naturally as a result of
intra-varietal crossings. The hybrids identified, were isolated from different
geographic locations indicating independent non-clonal origins. The fact that
the majority of those hybrids are clinical strains possibly indicates that
hybridization events may have an impact on
C. neoformans virulence and subsequent infection. Moreover, this
finding provides insights on the origin and impact of unisexual hybridization on
the
Cryptococcus neoformans population.
Takehito Nakazawa,
Hirofumi Kondoh, Kiyoshi Nakahori, Takashi Kamada
tnakazaw@cc.okayama-u.ac.jp
We isolated and characterized a REMI mutant (B28) defective in sexual
development (fruiting initiation) from a
Coprinopsis cinerea homokaryotic fruiting strain 326 (Amut
Bmut pab1-1). Genetic analysis strongly suggested that the defect in
fruiting directly results from insertion of plasmid pPHT1. A plasmid rescue
followed by complementation experiments revealed that the gene disrupted and
responsible for the phenotype in strain B28 encodes a homologue of Ustilago
maydis Ubc2. We designated this gene
Cc.ubc2.
The
U. maydis ubc2 gene has been shown to be required for pheromone
responses as well as for pathogenicity. Also, Ubc2 has been proposed to act as
an adaptor for a pheromone responsive MAPK cascade. On the basis of these
findings in
U. maydis, we performed further analyses, which revealed that strain
B28 is defective in two pheromone responses in
C. cinerea, clamp cell fusion and nuclear migration for dikaryosis.
We then carried out immunoblot analyses, which demonstrated that the
Cc.ubc2-1 mutation inhibits phosphorylation of a MAP kinase similar
to Ubc3/Kpp2 and that the phosphorylation is not under the control of the B
mating type genes encoding pheromone and their conjugate receptors.
PR5.3
Stefanie Pöggeler[1]
Skander Elleuche[2] Yasmine Bernhards[3] Christian
Schäfers[4] Jans Manjali Varghese[3] Nicole Nolting[5]
1Georg-August-University
Göttingen, 2Institut für Technische Mikrobiologie, Technische
Universität Hamburg-Harburg, Hamburg, Germany, 3Institut für
Mikrobiologie und Genetik, Abteilung Genetik eukaryotischer Mikroorganismen,
Georg-August Universität Göttingen, Göttingen, Germany, 4Lehrstuhl
für Allgemeine und Molekulare Botanik, Ruhr-Universität Bochum, Bochum, Germany
5Heinrich-Pette-Institut für Experimentelle Virologie und Immunologie
an der Universität Hamburg, Hamburg, Germany
spoegge@gwdg.de
In fungi, the homoeodomain protein STE12 controls diverse developmental
processes and derive its regulatory specificity from a wide range of different
protein interactions. We have recently shown that in the homothallic ascomycete
Sordaria macrospora STE12 is essential for ascospore development and
able to interact with the alpha-domain mating-type protein SMTA-1 and the MADS
box protein MCM1. To further evaluate the functional roles of STE12, we used the
yeast two-hybrid approach to identify new STE12-interacting partners. Using
ste12 as bait, a small, serine-threonine rich protein (designated
STE12 interacting protein 2, SIP2) was identified. SIP2 is
conserved among members of the fungal class
Sordariomycetes.
In vivo localization studies revealed that SIP2 was targeted to
nuclei and the cytoplasm. The STE12/SIP2 interaction was further confirmed in
vivo by biomolecular fluorescence complementation (BiFC). Nuclear localization
of SIP2 was apparently mediated by STE12. Unlike to the deletion (D) of
ste12, deletion of
sip2 in
S. macrospora led only to a slight decrease in ascospore
germination, but no other obvious morphological phenotype. In comparison to the
Dste12 single knock-out strain, ascospore germination was significantly
increased in a Dsip2/Dste12 double knock-out strain. Our data provide evidence
for a regulatory role of the novel fungal protein SIP2 in ascospore germination.
Céline O'Gorman[1]
Birgit Hoff[1] Julia Boehm[1] Stefanie Poeggeler[2]
Ulrich Kueck[1]
1Christian
Doppler Laboratory for “Fungal Biotechnology”, Lehrstuhl fuer Allgemeine und
Molekulare Botanik, Ruhr-Universitaet
2Abteilung Genetik eukaryotischer Mikroorganismen, Institut fuer
Mikrobiologie und Genetik, Georg-August Universitaet Goettingen, 37077
Goettingen, Germany
celine.ogorman@rub.de
Penicillium chrysogenum
is the most ubiquitous filamentous fungus worldwide; however, it is best-known
as the major industrial producer of penicillin. It is classified as an asexual
ascomycete because there has never been any direct observation of mating or
meiosis in the fungus. Significantly, despite its apparent asexuality there is
increasing evidence for a latent sexual cycle in
P. chrysogenum, similar to what was previously observed for the
cryptically-sexual human pathogen
Aspergillus fumigatus. Isolates of opposite mating type are found in
near-equal proportion in nature and transcriptionally express pheromone and
pheromone receptor genes. Furthermore, analysis of the genome sequence has
revealed the presence of other key genes required for completion of the sexual
cycle. Experiments are in progress to both characterise the functions of the
‘master regulator’ mating-type genes and induce a sexual cycle under laboratory
conditions. As a first step, a
MAT1-1 knockout strain was generated by gene replacement with a
phleomycin resistance cassette in a ΔPcku70 background. Asexual conidiophore
development in the mutant strain was affected; it produces significantly higher
quantities of conidia relative to the recipient strain P2niaD18, although the
ratio of light-to-dark conidial production remains the same. The
MAT1-1 gene was also found to play a role in hyphal morphogenesis,
with implications for pellet formation in industrial fermentation systems. These
results highlight the diverse regulatory roles of the
MAT1-1 gene in developmental processes other than sexual
reproduction.
PR5.5
Francine Hiromi Ishikawa[1] Elaine Aparecida de Souza[1]
Nick D. Read[2] Maria Gabriela Roca[2]
1Universidade
Federal de Lavras, Brazil, 2University of Edinburgh, UK
Fusion of conidia and conidial germlings by means of conidial anastomosis tubes
(CATs) is a common phenomenon in filamentous fungi, including many plant
pathogens. It has a number of different roles, and has been speculated to
facilitate parasexual recombination and horizontal gene transfer between
species.
The bean pathogen
Colletotrichum lindemuthianum naturally undergoes CAT fusion on the
host surface and within asexual fruiting bodies in anthracnose lesions on its
host. It has not been previously possible to analyze the whole process of CAT
fusion in this or any other pathogen using live-cell imaging techniques. Here we
report the development of a robust protocol for doing this with
C. lindemuthianum
in vitro. The percentage of conidial germination and CAT fusion was
found to be dependent on culture age, media and the fungal strain used.
Increased CAT fusion was correlated with reduced germ tube formation. We show
time-lapse imaging of the whole process of CAT fusion in
C. lindemuthianum for the first time and monitored nuclear migration
through fused CATs using nuclei labelled with GFP. CAT fusion in this pathogen
was found to exhibit significant differences to that in the model system
Neurospora crassa. In contrast to
N. crassa, CAT fusion in
C. lindemuthianum is inhibited by nutrients (it only occurs in
water) and the process takes considerably longer.
Financial support: CNPq, Capes e Fapemig
Jan van Kan[3]
Gavin Eyres[1] Linda Kohn[2] Paul Dyer[1]
1University
of
jan.vankan@wur.nl
Botrytis cinerea
is a heterothallic ascomycete with two mating types, MAT1-1 and MAT1-2.
B. cinerea
MAT loci have novel features. Fragments of the
MAT1-2-1 and
MAT1-1-1 genes were detected bordering idiomorphs of the MAT1-1 and
MAT1-2 isolates, respectively. Both of these fragments encode truncated,
non-functional proteins.
B. cinerea has probably evolved from a homothallic ancestor
containing complete
MAT1-1-1 and
MAT1-2-1 genes at the same locus, with MAT1-1 and MAT1-2
arising from the loss of HMG and alpha-domain sequences, leaving the
disabled gene fragments seen in current
MAT loci. Two ORFs, designated
MAT1-1-5 and
MAT1-2-3, have not previously been reported from other fungi.
Homologs of
MAT1-1-5 are present in other leotiomycetes, whereas the
MAT1-2-3 gene is exclusively present within the genus
Botrytis. Knockout mutants in
MAT1-1-5 are sterile, due to the inability of the dikaryon to
develop a cap structure.
B. cinerea is unusual in that some isolates are capable of ‘dual
mating’. This refers to the observation that most isolates act in a standard
heterothallic fashion (MAT1-1 or MAT-1-2), but some isolates can mate with both
MAT1-1 ánd MAT1-2 isolates. Some dual mater isolates can self-fertilize and are
truly homothallic. The
MAT locus of five homothallic
B. cinerea isolates was analysed. Four of those contain a MAT1-2
locus, without any sequence of the MAT1-1 locus being detected. Remarkably, one
homothallic isolate contains a MAT1-1 locus, without any sequence of the MAT1-2
locus being detected. We conclude that dual mating and homothallism is
controlled by sequences outside the
MAT locus.
Silvia Bleuler-Martinez[1] Alex Butschi[2] Mario Schubert[3]
Martin A. Wälti[1] Pascal Egloff[1] Fred Allain[3]
Michael O. Hengartner[2] Markus Aebi[1] Markus Künzler[1]
1Institute
of Microbiology, ETH Zürich, 2Institute of Molecular Biology,
University of Zürich, 3Institute of Molecular Biology and Biophysics,
ETH Zürich
markus.kuenzler@micro.biol.ethz.ch
We identified a novel lectin, termed RedA, from the saprobic mushroom
Coprinopsis cinerea by virtue of its binding to the plant
glycoprotein horseradish peroxidase (HRP). Like many fungal lectins, RedA is
highly soluble, lacks a signal sequence for classical secretion and is
specifically produced in the fruiting body. Homologous proteins are encoded in
the genomes of
C. cinerea and the symbiotic mushroom
Laccaria bicolor. Glycan array analysis of recombinant RedA revealed
a pronounced carbohydrate-specificity for the disaccharide Fucα1,3GlcNAc.
Structural studies by NMR showed that the lectin adopts a beta-trefoil (R-type)
fold and coordinates one ligand molecule per monomer at a site different from
the canonical carbohydrate-binding site of R-type lectins. The glycoepitope
recognized by RedA is found at the non-reducing ends of animal glycans and at
the core of N-linked glycans of plants, insects and nematodes but not of fungi.
Toxicity bioassays with model organisms including
Aedes aegypti (insect),
Caenorhabditis elegans (nematode) and
Acanthamoeba castellanii (amoeba) showed an exclusive toxicity of
RedA towards nematodes. Resistance of
C. elegans mutants defective in the biosynthesis of the α1,3-core
fucoside revealed that this nematotoxicity is dependent on the binding of this
specific glycoepitope. Feeding
C. elegans with the dTomato-RedA fusion protein showed that this
binding occurs at the intestinal epithelium of the nematode. Our results are in
agreement with the existence of a protein-mediated defense system of fungi
against predators and parasites and suggest that the fruiting body lectin RedA
is an effector protein against predatory nematodes.
Edyta Szewczyk,
Sven Krappmann
Julius-Maximilians-University Wuerzburg
Edyta.Szewczyk@uni-wuerzburg.de
Sexual reproduction of the human pathogen
Aspergillus fumigatus (teleomorph:
Neosartorya fumigata) was assumed to be absent or cryptic until
recently, when fertile crosses among geographically restricted environmental
isolates were described. Here, we provide evidence for mating, fruiting body
development, and ascosporogenesis accompanied by genetic recombination between
unrelated, clinical isolates of
A. fumigatus, which demonstrates the generality and reproducibility
of this long undisclosed phase in the life cycle of this heterothallic fungus.
Successful mating requires the presence of both mating type idiomorphs
MAT1-1 and
MAT1-2, as does expression of genes encoding putative factors
involved in this process. Moreover, analysis of an
A. fumigatus mutant deleted for the
nsdD gene suggests a role of this conserved regulator of
cleistothecia development in hyphal fusion and hence heterokaryon formation.
PR5.9
B Gillian Turgeon
Dept. of Plant Pathology & Plant-Microbe Biology,
btg1@cornell.edu
Two-component histidine kinase (HK) phosphorelay signaling systems are a major
mechanism by which fungi sense and adapt to their environment. In response to a
signal, the HK autophosphorylates a conserved histidine residue, then the
phosphate is transferred to a conserved aspartic acid residue in a response
regulator (RR) protein, resulting in an output. Nearly all eukaryotic HKs are
hybrid with the HK and the RR domains in a single polypeptide; most
characterized hybrids require an additional phosphorelay step through a
histidine phosphotransfer (HPt) domain protein and a second RR protein. This
additional phosphorelay step may allow the organism to integrate multiple input
signals into a single output. The maize pathogen,
Cochliobolus heterostrophus has 21 HKs, 4 RRs (SSK1,
SKN7, RIM15, REC1) and one HPt gene. Because all HKs signal
through the 4 RRs, we chose the latter to make gene deletion mutants and then
screened for altered phenotypes (virulence, asexual and sexual development,
stress responses, drug resistance). No altered phenotypes were detected
for
rim15 and
rec1 mutants.
C. heterostrophus and
Gibberella zeae Ssk1p are required for virulence to maize and wheat,
respectively. Lack of Ssk1p affects fertility of heterothallic
C. heterostrophus, self-fertility and ascospore maturation in
homothallic
G. zeae, and proper timing of sexual development in both fungi.
Pseudothecia from crosses involving
C. heterostrophus
ssk1 mutants ooze masses of single ascospores unlike those from WT
crosses, which do not ooze, and tetrads cannot be found. Ssk1p also controls
asexual spore proliferation, and represses asexual spore production in the
sexual reproductive phase of both fungi. Double
C. heterostrophus
ssk1skn7-deletion mutants are more sensitive to oxidative and
osmotic stress, and more exaggerated in their spore-type balance phenotype than
single
ssk1 and
skn7-deletion mutants.
ssk1-deletion mutant phenotypes largely overlap with
hog1-deletion mutant phenotypes, allowing us to place
SSK1 upstream of the
HOG1 MAPK pathway.
Sandra Bloemendal[1]
Ines Engh[1] Kathryn M Lord[2] Nick D Read[2]
Ulrich Kück[1]
1Department
of General and Molecular Botany, Ruhr-University Bochum, Universitaetsstrasse
150, D-44780 Bochum, Germany
2Institute of Cell Biology, The University of Edinburgh, Rutherford
Building, King's Buildings, Edinburgh EH9 3JH, UK
sandra.bloemendal@rub.de
The formation of fruiting bodies in filamentous fungi is a multicellular
differentiation process and controlled by many developmentally regulated genes
[1]. The filamentous ascomycete
Sordaria macrospora represents an excellent model system for cell
differentiation during fruiting body development, because this homothallic
fungus is self-fertile.
For
Sordaria macrospora, several developmental mutants are described.
Though these mutants can build young protoperithecia, they are not able to form
mature perithecia. These mutants, named “pro”-mutants, include the mutant pro22,
which shows a point mutation in the
pro22 ORF, leading to undifferentiated protoperithecia consisting of
a loose hyphal coil. By means of forced-heterokaryon tests using auxotrophic
strains and fluorescence microscopic investigations with nuclear labeled strains
it could be demonstrated that pro22 is restricted in hyphal anastomosis in
homozygous crosses [2].
The PRO22 protein possesses two highly conserved domains A and B and homologs
can be found in many other eukaryotes from yeast to humans. Fluorescence
microscopy of PRO22 tagged with GFP and mRFP1 provides evidence for a
localization in vacuoles. Our aim is to extend the functional analysis of the
pro22 mutant via fluorescence microscopy and to identify interaction partners of
PRO22
in vitro via yeast two-hybrid and
in vivo via tandem-affinity purification.
[1] Kück U et al. (2009) In: Anke T & Weber D (ed) The Mycota XV,
Springer-Verlag, Heidelberg
[2] Rech C et al.
(2007) Curr Genet 52:259-266
Monika Schmoll,
Christian Seibel, Christian P. Kubicek
monika.schmoll@tuwien.ac.at
Sexual development is one of the most important achievements in evolution. The
case of sexuality in the industrial workhorse
Trichoderma
reesei (anamorph of
Hypocrea
jecorina) is special in so far as both research as well as
industrial strain improvement is solely based on the single isolate QM6a and its
(asexual) progeny. As with all other filamentous fungi applied in industry, the
possibility of crossing was not available for
T. reesei, although in silico data had identified
H. jecorina as its teleomorph more than a decade ago. Recently, we
described the two mating type loci of
T. reesei (MAT1-1 and MAT1-2) and we were for the first time able to
induce sexual reproduction of
T. reesei QM6a (MAT1-2) in crossings with a MAT1-1 wild-type isolate
of
H. jecorina, and we obtained fertilized stromata and mature
ascospores . However, a serious issue with our findings was, that while male
fertile,
T. reesei QM6a is female sterile. We now investigated the role of
the peptide pheromone precursor genes, the pheromone receptors as well as of the
known photoreceptors in sexual development in T. reesei. Additionally we provide
first insights into the relevance of RIP for crossing in this organism.
PR5.12
Erika Kothe,
S. Erdmann, M. Wirth, E.-M. Jung, D. Freihorst, D. Senftleben, N. Knabe
Friedrich-Schiller-University, Dept. Microbiology, Microbial Phytopathology
erika.kothe@uni-jena.de
The recent genome sequence of the wood-rotting homobasidiomycete
Schizophyllum commune, in comparison to genomes of other
Agaricomycotina like
Coprinopsis cinerea, Laccaria bicolor, Phanerochaete chrysosporium , Postia
placenta and the heterobasidiomycetes
Ustilago maydis and
Cryptococcus neoformans now allow comparative “omics” studies
on mating interactions, pheromone response pathways. Linking the identified
genes to phenotypes in sexual development and mushroom formation can provide
insight into development of structures typical for the kingdom of the mycota. In
mushroom forming homobasidiomycetes, the two mating type loci
A, coding for homeodomain (HD) type transcription factors, and
B encoding a pheromone/receptor system, regulate mating and sexual
development. We could localize the pheromone receptor solving the old question
of whether pheromone signalling is involved in cell-cell recognition or rather,
after mycelial fusion, recognition of nuclei in a dikaryon. In addition to the
mating type loci,
in silico analyses now permit the identification of putative
components of the signaling pathways leading to dikaryotic growth, mushroom
formation and meiotic spore production. The involvement of G proteins like
Ras could be shown for nuclear migration, nuclear positioning, clamp formation
and fruitbody morphogenesis, involving cAMP-dependent protein kinase A
signaling. Nuclear migration has been investigated looking into dynein function.
The heavy chain of dynein in S. commune, like in other basidiomycetes, is
encoded by two separate genes which are investigated by performing knock-out
analyses. The resulting strain is viable, with phenotypes which, again, can be
linked to sexual development, fruitbody formation and spore production as well
as to apical hyphal growth. With availability of the fully sequenced genome,
thus, many of the long-established pathways of signalling and morphogenesis can
be revisited and surprises from the genome sequence provide insight into
long-standing questions.
Christine Schimek,
Rudigier Yvonne, Park Min-Jeong, Sarkar Sarbani, Wetzel Jana, Wöstemeyer
Johannes
General Microbiology and Microbial Genetics, Institute for Microbiology,
Friedrich-Schiller-University
Christine.Schimek@uni-jena.de
Sexual development within the class
Zygomycetes is regulated by retinoid-like beta-carotene derivatives,
the trisporoids. Trisporoids serve as pheromones in partner recognition and as
internal transcription regulators. Numerous studies deal with their complex and
co-operative synthesis, some of the biosynthesis enzymes have been studied into
molecular detail, but their mode of action, molecular reaction mechanisms and
signal perception events are completely unknown. We are researching the putative
reaction partners involved in recognition and signal perception events. Our main
goal is to identify putative trisporoid receptors and binding proteins, but we
also include the search for proteins undergoing regulatory interactions with the
trisporoid biosynthesis enzymes. 2D-PAGE reveals only a small number of
deviating proteins between the (+) and the (-) mating type in cell
surface-attached proteins. Several receptor candidates have been selected and
are currently under analysis. Using antibodies raised against animal retinoid
signaling and developmental regulation proteins, we identified a putative
retinoid binding protein, cross-reacting with an antibody against cellular
retinaldehyde-binding protein CRALBP, and a putative homeobox protein. Direct
transcriptional modulation of effector genes may occur via trisporoid binding to
specific transcription factors and trisporoid synthesis enzymes themselves might
act as transcription factors during sexual development. Based on the observation
that both the trisporoid biosynthesis enzymes 4-dihydrotrisporin dehydrogenase
and 4-dihydromethyltrisporate dehydrogenase are inactivated at certain
developmental stages by binding to other proteins forming multimeric complexes,
we are using heterologously expressed 4-dihydrotrisporin dehydrogenase as bait
for the purification of such binding proteins from cytoplasmatic protein
fractions from
Mucor mucedo.
Min-Jeong Park,
John Jasmine, Wetzel Jana, Schimek Christine, Burmester Anke, Wöstemeyer
Johannes
General Microbiology and Microbial Genetics, Institute for Microbiology,
Friedrich-Schiller-University
pmj4842@gmail.com
Genetic analysis of
Mucor mucedo, a zygomycete model organism for studying sexual
communication and action of the trisporoid system, is hampered by a strong
tendency towards autonomous replication of introduced plasmids instead of stable
integration. We are working at developing a tool for genetic manipulation based
on interference with the DNA double strand break-repair system.
Targeted gene disruption via homologous recombination is routinely used for
analysis of gene function. Although integration of exogenous DNA at homologous
sites in the genome occurs easily in
Saccharomyces cerevisiae, it is rare in many filamentous fungi where
DNA integration occurs predominantly through non-homologous end joining (NHEJ)
and exogenous DNA can be integrated at ectopic sites in the genome. Direct
ligation of DNA strands in NHEJ is mediated by a DNA-dependent protein kinase, a
DNA ligase complex, and the Ku70-Ku80 heterodimer. Homologues of Ku70 and Ku80
have been identified in many organisms. Recent studies in filamentous fungi have
shown increased gene targeting frequencies in
KU deficient mutants, indicating that
KU disruption strains are efficient recipients for gene targeting.
We identified and cloned a
M. mucedo homologoue to the human
KU70 gene using PCR, inverse PCR, and Southern hybridization.
Genetic manipulation will be done using an RNA silencing approach with
Morpholino antisense constructs directed against
KU70 mRNA, thus resulting in the functional knock-down of the
KU70 gene product. The suitability of this approach is shown by
morpholino-mediated down-regulation of crgA, a protein involved in the
regulation of carotene synthesis in zygomycete fungi.
Björn Sandrock,
Johannes Freitag, Daniel Lanver, Stefan Arens, Michael Bölker
Philipps-University Marburg, Department
of Biology - Genetics Karl-von-Frisch-Str.8, 35032
The basidiomycetes
Ustilago maydis is a tumour-causing agent on corn plants. Tumour
development can occur in all green parts of the plant and is initiated by the
invasion of dicaryotic hyphae from the plant surface. These hyphae are formed
after fusion of two compatible haploid sporidia. Hyphal growth is uni-polar and
the cytoplasmic part of elongating filaments is restricted by the sequential
formation of retraction septa leaving empty sections behind.
We have identified a regulatory network essential for the formation of the
secondary septum in budding cells composed of the guanine-nucleotide exchange
factor Don1, the corresponding small GTPase Cdc42 and the germinal-centre kinase
Don3.
We will demonstrate our recent data that the diaphanous related formin Drf1 is
an effector of the Cdc42-module for secondary septum formation. Formins are
proteins that polymerize and bundle actin cables.
Furthermore we will show that among these proteins only Cdc42 is essential for
the morphological switch from yeast-like to hyphal growth. We will present data
how Cdc42 regulates the MAP kinase cascade.
The complete Don1-Cdc42-Drf1 module organizes the retraction septa formation in
filaments. In addition, this cascade is also necessary for appressoria
formation. This also indicates that actin nucleation is important for these
processes.
Currently we are investigating the role of the Don3 kinase during filamentous
growth to elucidate its interplay with the Cdc42 module.
Genetic and epigenetic control of a nonself recognition complex in
N. crassa
Lafontaine, D.L.
and M.L. Smith
Heterokaryon incompatibility (HI) in filamentous fungi is a form of nonself
recognition that operates during the vegetative phase of the life cycle. One HI
complex in Neurospora crassa, the
het-6 locus, comprises two tightly
linked incompatibility genes, het-6
and un-24,
each having two allelic variants, Oak Ridge (OR) and Panama (PA). The
un-24 gene also encodes the large
subunit of ribonuclotide reductase while
het-6 appears to be a member of a repetitive gene family with no other known
function aside from HI. These two genes are in severe linkage disequilibrium
such that only un-24OR
het-6OR and
un-24PA
het-6PA haplotypes occur
in nature. In this study we constructed novel
un-24PA
het-6OR strains and
het-6 deletion strains. The
un-24PA
het-6OR strains are
initially self-incompatible, but escape from incompatibility in a predictable
manner that is associated with loss of
het-6OR incompatibility function. HI tests using
het-6 deletion strains reveals an
allelic incompatibility interaction between OR and PA forms of
un-24, which is asymmetrically
enhanced by the presence of het-6OR
or het-6PA. Mutations in
vib-1 suppress HI associated with
allelic differences at un-24 or at
het-6, but only partially suppress
incompatibility reactions that occur when both
un-24 and
het-6 differ. We have also identified
a second genetic suppressor that acts synergistically with
vib-1 and will present evidence that
this suppressor compromises epigenetic silencing of HI.
PR5.17
The fungal immune response: from detection of the pathogen to the cellular
response
Paoletti M., Clavé C., Breton A., Ness F., Chevanne D., Sabourin M., Cescau S.,
Daskalov A. and Saupe S.
Institut de Biochimie et genetique cellulaire, CNRS, 43 rue Camille Saint Saëns,
Mathieu.Paoletti@ibgc.u-bordeaux2.fr
Living organisms have developed complexe processes to fight off pathogens.
Plants and animals initiate an immune response upon detection of Pathogen
Associated Molecular Patterns (PAMPS) by dedicated Pattern Recognition Receptors
(PRRs) that belong to the STAND class of proteins involved in signal
transduction. Pathogen driven evolution of the PRR encoding genes can lead to
auto-immune diseases. No such fungal immune systems have been described so far.
In Podospora anserina vegetative
incompatibility (VI) is under the control the
NWD gene family that interacts with
incompatible alleles of the het-c
locus encoding for a glycolipid transfer protein (GLTP). The
NWD gene family evolves extremely
rapidly and new variants in the WD repeat domain involved in protein protein
interactions are permanently being generated. NWD proteins also belong to the
STAND class of proteins. We have hypothesized that these proteins function as
PRRs and that the VI reaction corresponds to a pathological manifestation of an
auto-immune disease. We have discovered a fungal species that initiates a strong
reaction in P. anserina that is
dependant on het-c and all known VI
suppressors. We have undertaken the task of describing the fungal immune
response from pathogen recognition to the cellular response.
Genetic relatedness of multi-azole resistant
Aspergillus fumigatus isolates
S.M.T. Camps,
P.E. Verweij, W.J.G. Melchers
Department of Medical microbiology,
S.Camps@mmb.umcn.nl
Objectives
In the last few years multi-azole resistant
Aspergillus fumigatus seem to emerge
in the
Methods
Resistant isolates containing the TR/L98H resistance mechanism (n=57) were
subjected to CSP, microsatellite and mating type analysis. As a control, both
susceptible (n=57) and resistant isolates without the TR/L98H resistance
mechanism (n=17) were analyzed.
Results
The TR/L98H isolates clustered together in microsatellite analysis and were
grouped into three CSP types (2, 4B and 11). Susceptible and non-TR/L98H
resistant control isolates were classified to other CSP types than the TR/L98H
isolates, except for several isolates belonging to CSP type 2. Mating type genes
MAT1-1 and
MAT1-2 were almost equally
represented in each group.
Conclusions
1.
Clinical and environmental TR/L98H isolates were grouped to the same CSP types
and clustered together using microsatellite typing, indicating that the TR/L98H
resistance mechanism has only recently developed and might not be induced in the
patient but might be acquired from the environment.
2.
As the TR/L98H isolates grouped to different CSP types and clustered apart from
the control isolates, the TR/L98H isolates might belong to a separate genetic
A. fumigatus lineage.
3.
Although microsatellite and CSP analysis indicate that the TR/L98H isolates are
rather homogeneous, segregation of the mating type locus is equal to the control
isolates. This might suggest that sexual reproduction played a role in
resistance development.
PR5.19
The fungal immune response: from detection of the pathogen to the cellular
response
Paoletti M.,
Clavé C., Breton A., Ness F., Chevanne D., Sabourin M., Cescau S., Daskalov A.
and Saupe S.
Institut de Biochimie et genetique cellulaire
mathieu.paoletti@ibgc.cnrs.fr
Living organisms have developed complexe processes to fight off pathogens.
Plants and animals initiate an immune response upon detection of Pathogen
Associated Molecular Patterns (PAMPS) by dedicated Pattern Recognition Receptors
(PRRs) that belong to the STAND class of proteins involved in signal
transduction. Pathogen driven evolution of the PRR encoding genes can lead to
auto-immune diseases. No such fungal immune systems have been described so far.