The signalling mucin msb2 in fusarium oxysporum: identification of putative interaction partners by yeast two-hybrid assay

Fungal Development and Pathogenesis
British Mycological Society - Annual Scientific Meeting
University of Exeter 13th-16th September 2011
ABSTRACTS from ARIADNE students

Establishment of molecular tools for monitoring the activation status of the Pathogenicity
MAPK cascade in Fusarium oxysporum

Mennat El Ghalid, David Turrà, Elena Pérez-Nadales, Antonio Di Pietro
Departamento de Genética, Universidad de Córdoba, Córdoba, Spain
In the vascular wilt fungus Fusarium oxysporum the mitogen-activated protein kinase (MAPK)
Fmk1, orthologous to the filamentous growth MAPK Kss1 in yeast, is required for root adhesion,
invasive growth and pathogenicity on tomato plants. At present, the signals that regulate this
MAPK cascade during infection are unknown. Our goal is to identify and characterize these stimuli
in the tomato-Fusarium system. To this aim, we are designing an experimental system that allows
rapid and accurate monitoring of the activation status of the Fmk1 pathway. First, we are generating
a strain in which MAPK activation can be transiently induced by expressing a dominant active
allele of ste7 encoding a conserved MAPK kinase. Second, we are establishing a pathway reporter
system by quantitatively measuring transcript levels of downstream effector genes that are
specifically down-regulated in the fmk1 mutant. One such gene is fpr1, encoding a homologue of
plant PR-1 proteins, which is dramatically down-regulated in the fmk1, msb2 and ste12 mutants
under conditions of invasive growth. We are searching for additional reporter genes that are
specifically controlled by the Fmk1 pathway. Once established, these tools will be applied to the
identification of upstream signals involved in the regulation of the Pathogenicity MAPK cascade.
The signalling mucin Msb2 in Fusarium oxysporum: Identification of putative interaction
partners by Yeast Two-Hybrid assay

Katja Schäfer, Elena Pérez-Nadales, Antonio Di Pietro
Departamento de Genética, Universidad de Córdoba, Córdoba, Spain
Fungal pathogenicity on plants requires a conserved mitogen-activated protein kinase (MAPK)
cascade homologous to the yeast filamentous growth pathway. In the soilborne vascular wilt
pathogen Fusarium oxysporum, the orthologous MAPK Fmk1 controls invasive growth and
virulence on tomato plants. We recently found that full phosphorylation of Fmk1 requires the
transmembrane protein Msb2, a member of the family of signalling mucins that have emerged as
novel virulence factors in fungal plant pathogens. F. oxysporum mutants lacking either msb2 or fmk1
share characteristic phenotypes related to invasive growth and virulence on tomato plants. Moreover,
Δmsb2 mutants also show fmk1-independent phenotypes suggesting additional roles of Msb2 in
distinct pathways.
The exact signalling mechanism of fungal transmembrane mucins is currently unknown. Our aim is
to find new Msb2 interaction partners with a role in the MAPK signalling cascade. A Yeast Two-
Hybrid screen against a cDNA library from F. oxysporum yielded eleven candidates interacting with
the Msb2 cytoplasmic tail. To test the role of these genes in MAPK signalling, a knockout approach
was designed, followed by phenotypic analysis of the mutants. Progress on this work will be
presented.
Immune recognition of Candida albicans influenced by defects in cell signal transduction
pathways
Pankaj Mehrotra, Rebecca A. Hall, Jeanette Wagener and Neil A.R. Gow
Aberdeen Fungal Group, Institute of Medical Sciences, University of Aberdeen, Foresterhill,
Aberdeen, AB25 2ZD, UK1. p.mehrotra@abdn.ac.uk
Candida albicans, is a major opportunistic fungal pathogen of humans which can cause superficial
skin infections as well as fatal systemic disease in immune-compromised individuals. During the
infection process C. albicans has to respond to stresses imposed by the host environment, like ROS
generation by phagocytic cells, which is achieved through the activation of multiple stress pathways
including the cAMP-PKA, several MAPK pathways and the Ca2+-calcineurin pathway. Mutations
in these pathways affect C. albicans cell surface chemistry and morphogenesis, both of which are
believed to be key factors in fungal immune recognition. In the context of this project, which is a
part of Marie Curie INT Network “ARIADNE”, we are investigating how the activation or
inhibition of these pathways influences myeloid cell immune recognition of C. albicans. To do this,
we have assembled and screened a library of mutants in each of the above pathways and assessed
them for their ability to activate human PBMCs, as determined by the secreted cytokine profile.
Results from this screen will be presented, which demonstrate that several of these pathways have a
marked influence on the immunological signature of the cell.

Candida albicans: signalling pathways and the regulation of cell wall biosynthesis under stress

Filomena Nogueira, Louise Walker, Keunsook Lee, Carol Munro, Neil Gow
Aberdeen Fungal Group, Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen,
AB25 2ZD, UK
The role of three signalling transduction pathways (MAPK, Ca2+/calcineurin and cAMP) in controlling
the Candida albicans cell wall stress responses are being investigated. A library of mutants lacking
receptors, signalling elements and transcription factors were screened for alterations in the ability to
respond to a range of cell wall stressing agents including Calcofluor White, Congo Red and caspofungin.
Pre-treatment of wild-type cells with CaCl2 and CFW, activated the Ca2+/calcineurin and PKC pathways,
leading to and increased chitin content, and reduced susceptibility to caspofungin. This pre-treatment
regimen also stimulated production of salvage septa in chs deletion mutants. These results suggest
possible alternative mechanisms of resistance to caspofungin. However, we show here that cells with
elevated chitin content are not always caspofungin resistant and, caspofungin resistant cells do not
always have high levels of cell wall chitin. The results show that elevation of chitin is a common
property of a range of mutants that are affected in coordinating cell wall stress pathways, but that
multiple parallel mechanisms are likely to operate in alerting the robustness of the C. albicans cell wall.
Functional analysis of the Mps1 MAP kinase pathway in the rice blast fungus Magnaporthe
grisea.
Elisabeth Grund (1), Cemile Ant (1), Marie-Joséphe Gagey (1), Carine Chaintreuil, (1), Christelle
Bonnet (1), Anne Lappartient (2), Roland Beffa (2), C Cartwright (3), Nick Talbot (3), Marc-Henri
Lebrun (1/4)
MAP CNRS-UCB-INSA-BCS, 14 impasse Pierre Baizet, 69009 Lyon, France (1), Bayer Cropscience,
Biochemistry department, Lyon, France (2) Biology, University of Exeter, UK (3). BIOGER INRA,
Thiverval-Grignon, France (4).
Cell wall integrity is crucial for fungal growth, development and stress survival. In yeast, Slt2 MAP
kinase and calcineurin signaling pathways monitor cell wall integrity. MPS1, the M. grisea SLT2
orthologue, is essential for cell wall repair and for appressorium mediated penetration into plants. In
yeast, Slt2 activates the transcription factors Rlm1, Swi4 and Swi6, while calcineurin activates Crz1.
Genes orthologous to yeast CRZ1, MPS1, RLM1, SWI4, and SWI6 genes were identified in M.
grisea. Swi4 and Swi6 interact with Mps1 in yeast two hybrid experiments. Δmps1 mutants
displayed an abnormal mycelial growth (no aerial hyphae), did not sporulate, and were non-
pathogenic on plants as reported (a). Δswi4 displayed phenotypes similar to Δmps1 with milder
growth and sporulation defects, while it was non pathogenic. Δcrz1 and Δswi6 mutants have a
normal mycelial growth and sporulation rates, while Δrlm1 is reduced in sporulation. Δcrz1 and
Δrlm1 were non pathogenic on barley and rice, while Δswi6 was pathogenic. These studies suggest
Swi4 is the major target of Mps1 during mycelial growth, while both Rlm1 and Swi4 are the targets
of Mps1 during sporulation and infection.
(a)
Xu, 2000. MAP kinases in fungal pathogens. Fungal Genet. Biol. 31:137–152.
Identification and Characterisation of novel transcription factor-encoding genes of the rice
blast fungus Magnaporthe oryzae

Miriam Oses-Ruiz, Darren M. Soanes, Apratim Charaborti, and Nicholas J. Talbot
School of Biosciences, University of Exeter, EX4 4QD, United Kingdom
To cause rice blast disease, the fungus Magnaporthe oryzae elaborates specialized infection
structures called appressoria, which use enormous turgor to rupture the tough outer cuticle of a rice
leaf and allow the fungus to invade living plant tissue. The M. oryzae PMK1 gene encodes a
functional homologue of the mitogen-activated protein kinases Fus3 and Kss1 from Saccharomyces
cerevisiae, and is essential for appressorium formation. The M. oryzae pmk1 MAP kinase mutant
forms long germ tubes but does not elaborate appressoria and is consequently non-pathogenic. The
requirement for the PMK1 MAP kinase signalling pathway in infection related development has
been shown to be conserved across a wide range of plant pathogenic fungi. The pathway leads to the
activation of signalling pathways, physiological changes and morphogenetic processes essential for
appressorium formation and function. We have recently carried out analysis of expression profiling
data for 233 putative transcription factor encoding genes that have been identified in the M. oryzae
genome, using both Super SAGE and RNA-seq and Illumina GAIIx sequencing. We have
subsequently identified 26 transcription factors which are up-regulated between 2 and 16 hours post
germination during initiation of appressorium development and maturation. Using this approach we
aim to learn more about the transcriptional regulatory circuits necessary for appressorium-mediate
plant infection by M. oryzae. This set of transcription factors have been selected for functional
characterization and results will be presented of their initial identification.

Pheromone-induced G2 cell cycle arrest in Ustilago maydis requires inhibitory phosphorylation
of Cdk1
Sónia Marisa Castanheira Dias and José Perez-Martín
Department of Microbial Biotechnology, Centro Nacional de Biotecnologia CSIC, Campus de
Cantoblanco Madrid E-28049, Spain
Both Saccharomyces cerevisiae and Ustilago maydis use a similar MAPK cascade to respond to
sexual pheromone and in both cases a morphogenetic response is provided (shmoo and conjugative
hypha, respectively). However, while S. cerevisiae arrests its cell cycle in G1 in response to
pheromone, U. maydis does this by arresting at G2. The mechanism involved in the
pheromone−induced cell cycle arrest in U. maydis is unknown. In this poster we would like to
introduce our first attempts to characterize the molecular mechanisms behind pheromone−induced
cell cycle arrest in U. maydis. Our preliminary results indicated that inhibitory phosphorylation of
Cdk1 is part of the mechanism, as expression of a mutant allele of Cdk1 refractory to inhibitory
phosphorylation impairs the cell cycle arrest. We analyzed the transcriptional pattern of cell cycle
related genes in response to overactivation of pheromone pathway (using a constitutively activated
allele of fuz7, the MAPKK of the cascade) and found that Hsl1, a kinase involved in G2/M transition
is downregulated.

Mating and more: The Ustilago maydis MAP Kinase signaling pathway
Soode Moghadas , Naik Vikram and Regine Kahmann
Max Planck Institute for Terrestrial Microbiology, Department of Organismic Interactions, Karl-
von-Frisch-Strasse 10, D-35043 Marburg, Germany.

The basidiomycete fungus Ustilago maydis is a plant pathogen that causes smut disease in maize. In
this pathogen a conserved mitogen-activated-protein-kinase (MAPK) module regulates distinct steps
of fungal development. We are interested in the identification of direct phosphorylation targets of the
related MAP kinase Kpp2 and Kpp6 which control discrete steps during mating and during plant
colonisation. Currently we use both phospho-proteomics and a chemical genetics approach for
detecting the phosphorylated targets of these MAP kinases. In phospho-proteomics approach we
generated strains by which MAP kinase signaling can be induced by expressing a constitutively
active version of the MAPKK Fuz 7 (Fuz7DD) under an inducible promoter. Detection of
phosphorylated proteins within complex mixtures will be done directly by using the phospho-
specific dye Pro-Q Diamond. Also we have engineered a mutant form of Kpp2 which contains a
larger ATP binding pocket. This mutant protein kinase is expected to utilize bulky ATP analogues.
To identify targets, we plan to use radiolabled ATP analogue in kinase reactions for labeling the
direct substrates of Kpp2 and Kpp6 in extracts. The current state of these experiments will be
described .We are hoping to identify interesting targets of Kpp2 and Kpp6 and study their role in
signaling and pathogenicity.
Functional analysis of the HOG-pathway in Ashbya gossypii
Therése Oscarsson, Lisa Wasserström, Klaus Lengeler, and Jürgen Wendland
Carlsberg Laboratory, Yeast Biology, Gamle Carlsberg Vej 10, DK-1799 Copenhagen V,
DENMARK
Fungal Mitogen-Activated Protein Kinase (MAPK) cascades receive their input from cell surface
receptors and are used to transduce and respond to environmental stimuli. Some of these cascades
are required for virulence in human- and plant pathogens. Here, we initiated studies of the HOG-
pathway in the filamentous fungi A. gossypii. Using PCR-based approaches we have generated an
A. gossypii deletion strain collection encompassing the major contributors to this pathway as
inferred from Saccharomyces cerevisiae. Phenotype characterization of the A. gossypii deletion
strains has been determined under different growth- and stress conditions (22 °C, 30 °C, 37 °C,
NaCl (0.1M), CaCl2 (0.1M), Menadione (50µM), SDS (0.001%), Calcofluor white (50µg/ml)
Caffeine (1mM), CuSO4 (1mM) and SD/Asn). Our results show, for example, that deletion of the
membrane receptor encoding genes MSB2 and SHO1 renders cells with an increased cold
resistance. Deletion of the PBS2 MAPKK or the HOG1 MAPK leads to severely crippled growth
and decreased NaCl resistance. On the other hand, disruption of the two-component-like SLN1 and
YPD1 genes results in lethality. This phenotype can be restored in AgΔsln1/Δssk1 and
AgΔypd1/Δssk1 double mutants indicating that SSK1 encodes a key downstream target of the
Sln1/Ypd1-branch.
Analysis of the pheromone cascade in Ashbya gossypii
Lisa Wasserström, Therése Oskarsson, Klaus B Lengeler and Jürgen Wendland
Carlsberg Laboratory, Yeast Biology, Gamle Carlsberg Vej 10, DK-1799 Copenhagen V,
DENMARK
MAP-kinase cascades are highly conserved among eukaryotes and numerous studies have shown
that MAPK cascades harbor key virulence factors in human- and plant pathogens. The pheromone-
signal transduction MAPK-cascade regulates mating and sporulation in sexually reproducing fungi.
Here, we study the pheromone response pathway in the filamentous fungus Ashbya gossypii. Its
genome contains homologs of all of the Saccharomyces cerevisiae genes involved in this pathway.
In spite of this high degree of conservation, however, A. gossypii is not known to have a sexual
cycle. We have used PCR-based approaches to delete essentially all components of the cascade in A.
gossypii starting with the pheromone receptor genes STE2, STE3, the G-protein components encoded
by STE4, GPA1, the MAP-kinase genes STE11, STE7, FUS3, KSS1 and also the transcription factors
STE12, TEC1. In S. cerevisiae deletion of any of these genes leads to sterility and in the case of
GPA1 it is lethal. In A. gossypii, however, all generated mutants grew with wild-type rates and were
able to sporulate. Strikingly, deletion of the major transcription factor activated by the cascade,
STE12, resulted in a hypersporulation phenotype similar to that of an Agtec1 mutant indicating that
the cascade might have a regulatory role in sporulation.
Role of MAP kinases pathways in the infection process of the wheat pathogen Mycosphaerella
graminicola

Elisabetta Marchegiani, Sian Deller, Thierry Marcel, Marc-Henri Lebrun
UR 1290 BIOGER-CPP, INRA, Thiverval-Grignon, France
Elisabetta.Marchegiani@versailles.inra.fr

Mitogen-activated protein kinases (MAPKs), are essential components of evolutionary conserved
signaling pathways in eukaryotic cells. Mycosphaerella graminicola, a worldwide pathogen causing
Septoria tritici leaf blotch (STB) on wheat, has three MAPK pathways that depend on one of the
three kinases MGFUS3, MGSLT2 and MGHOG1. These three signaling pathways are involved at
different stages of the infection process (Cousin et al., 2006; Mehrabi et al., 2006; Mehrabi et al.,
2006). To unraveling the infection mechanisms controlled by these signaling pathways, we are
developing a combination of molecular approaches. Comparative transcriptomics will be performed
on MgFUS3, MgHOG1, MgSLT2 null mutants and wild type strain using genome wide DNA
microarrays, thereby additional transcriptomic analyses will rely on conditional MAPKK mutants to
better control the activation/inactivation status of these pathway. Genes whose expression
(induction, repression) require an active MAPK will be further studied using reverse genetics and
biochemistry (phosphorylation status, protein-DNA interaction).
Cousin et al. (2006), Molecular Plant Pathology 7(4): 269-278.
Mehrabi et al. (2006), Molecular Plant-Microbe Interactions 19(4): 389-398. Mehrabi et al. (2006), Molecular Plant-Microbe Interactions 19(11): 1262-1269.

Source: http://www.carlsberglab.dk/professors/Wendland/collaborators/PublishingImages/BMS%202011%20students.pdf