Comparative genomics of MAP kinase and calcium-calcineurin signalling components in plant and human pathogenic fungi.

Abstract

Mitogen-activated protein kinase (MAPK) cascades and the calcium-calcineurin pathway control fundamental aspects of fungal growth, development and reproduction. Core elements of these signalling pathways are required for virulence in a wide array of fungal pathogens of plants and mammals. In this review, we have used the available genome databases to explore the structural conservation of three MAPK cascades and the calcium-calcineurin pathway in ten different fungal species, including model organisms, plant pathogens and human pathogens. While most known pathway components from the model yeast Saccharomyces cerevisiae appear to be widely conserved among taxonomically and biologically diverse fungi, some of them were found to be restricted to the Saccharomycotina. The presence of multiple paralogues in certain species such as the zygomycete Rhizopus oryzae and the incorporation of new functional domains that are lacking in S. cerevisiae signalling proteins, most likely reflect functional diversification or adaptation as filamentous fungi have evolved to occupy distinct ecological niches.

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Review
Comparative genomics of MAP kinase and calcium–calcineurin signalling
components in plant and human pathogenic fungi
Nicolas Rispaila, Darren M. Soanesb, Cemile Antc, Robert Czajkowskid, Anke Grünlere, Romain Huguetb,
Elena Perez-Nadalesa, Anna Polif, Elodie Sartorelg, Vito Valianteh, Meng Yangi, Roland Beffac,
Axel A. Brakhageh, Neil A.R. Gowi, Regine Kahmannd, Marc-Henri Lebrunc, Helena Lenasif,
José Perez-Marting, Nicholas J. Talbotb, Jürgen Wendlande, Antonio Di Pietroa,*
aDepartamento de Genética, Universidad de Córdoba, 14071 Córdoba, Spain
bSchool of Biosciences, Geoffrey Pope Building, University of Exeter, Exeter EX4 4QD, United Kingdom
cUMR2847 Centre National de la Recherche Scientifique/Bayer CropScience, 14 Rue Pierre Baizet, 69263 Lyon Cedex 09, France
dMax-Planck-Institute for Terrestrial Microbiology, Department of Organismic Interactions, Karl-von-Frisch-Strasse, D-35043 Marburg, Germany
eCarlsberg Laboratory, Yeast Biology, Gamle Carlsberg Vej 10, DK-2500 Valby, Copenhagen, Denmark
fInstitute of Biochemistry, Faculty of Medicine, University of Ljubljana, Vrazov trg 2, Ljubljana, Slovenia
gDepartamento de Biotecnología Microbiana, Centro Nacional de Biotecnología-CSIC, 28049 Madrid, Spain
hDepartment of Molecular and Applied Microbiology, Leibniz Institute for Natural Product Research and Infection Biology – Hans Knöll Institute (HKI), 07745 Jena, Germany
iAberdeen Fungal Group, School of Medical Sciences, Institute of Medical Sciences, University of Aberdeen, Aberdeen, UK
a r t i c l ei n f o
Article history:
Received 25 November 2008
Accepted 17 January 2009
Available online 7 February 2009
Keywords:
Calcium
MAPK
Signalling
Stress
Virulence
a b s t r a c t
Mitogen-activated protein kinase (MAPK) cascades and the calcium–calcineurin pathway control funda-
mental aspects of fungal growth, development and reproduction. Core elements of these signalling path-
ways are required for virulence in a wide array of fungal pathogens of plants and mammals. In this
review, we have used the available genome databases to explore the structural conservation of three
MAPK cascades and the calcium–calcineurin pathway in ten different fungal species, including model
organisms, plant pathogens and human pathogens. While most known pathway components from the
model yeast Saccharomyces cerevisiae appear to be widely conserved among taxonomically and biologi-
cally diverse fungi, some of them were found to be restricted to the Saccharomycotina. The presence of
multiple paralogues in certain species such as the zygomycete Rhizopus oryzae and the incorporation
of new functional domains that are lacking in S. cerevisiae signalling proteins, most likely reflect func-
tional diversification or adaptation as filamentous fungi have evolved to occupy distinct ecological niches.
? 2009 Elsevier Inc. All rights reserved.
1. Introduction
Adaptation to changes in the environment is crucial for viability
of all organisms. In fungi, conserved signal transduction pathways
control fundamental aspects of growth, development and repro-
duction. Two important classes of fungal signalling pathways are
mitogen-activated protein kinase (MAPK) cascades and the cal-
cium–calcineurin pathway. MAPK cascades are characterized by a
three-tiered module comprising a MAP kinase kinase kinase (MAP-
KKK), a MAP kinase kinase (MAPKK) and the MAPK which is acti-
vated by dual phosphorylation of conserved threonine and
tyrosine residues within the activation loop (Chang and Karin,
2001). The calcium–calcineurin pathway functions via the Ca2+-
binding protein calmodulin and the calmodulin-dependent ser-
ine–threonine phosphatase, calcineurin (Chin and Means, 2000).
There is evidence for crosstalk between the MAPK and the cal-
cium–calcineurin pathways, since the mating MAPK cascade regu-
lates certain upstream components of the calcium–calcineurin
pathway (Muller et al., 2003). Fungal MAPK and calcium signalling
cascades are triggered by an array of stimuli and target a broad
range of downstream effectors such as transcription factors,
cytoskeletal proteins, protein kinases and other enzymes, thereby
regulating processes such as the cell cycle, reproduction, morpho-
genesis and stress response (Cyert, 2003; Kraus and Heitman,
2003; Qi and Elion, 2005).
Core elements of MAPK and calcium signalling pathways are re-
quired for virulence in a wide array of fungal pathogens of plants
and mammals (Kraus and Heitman, 2003; Lee et al., 2003; Lengeler
et al., 2000; Zhao et al., 2007). Such a degree of functional conser-
vation is remarkable, considering the taxonomic and biological
diversity among these pathogens, but also raises a number of ques-
tions regarding the specific role of these pathways in fungal infec-
tion. Are virulence defects in signalling mutants simply caused by
1087-1845/$ - see front matter ? 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.fgb.2009.01.002
* Corresponding author. Fax: +34 957212072.
E-mail address: ge2dipia@uco.es (A. Di Pietro).
Fungal Genetics and Biology 46 (2009) 287–298
Contents lists available at ScienceDirect
Fungal Genetics and Biology
journal homepage: www.elsevier.com/locate/yfgbi

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perturbation of general metabolic and developmental processes, or
are they related to ‘‘true” pathogenicity mechanisms that are spe-
cific for host infection? If the latter is true, what are these specific
pathogenicity functions and which are the upstream and down-
stream signalling components that regulate their activity?
The availability of complete genome sequences from an increas-
ing number of pathogenic fungi allows us to approach these ques-
tions at the genomic level. Comparative analysis of complete
genome sequences from different yeasts and fungi has provided
valuable insight into the evolution of genome organisation (Die-
trich et al., 2004; Dujon et al., 2004; Kellis et al., 2004), facilitated
the identification of regulatory sequences (Cliften et al., 2003) and
assisted genome annotation (Dujon et al., 2004). Genome se-
quences are also valuable tools for the functional analysis of pro-
teins and cellular pathways. At the protein level, comparison of
orthologous sequences allows predictions on putative functional
domains or key residues, whereas at the pathway level it provides
the opportunity to assess the level of evolutionary conservation of
specific pathways and to generate new hypotheses for their func-
tional analysis.
In this review, we have explored the structural conservation of
MAPK cascades and the calcium–calcineurin pathway in ten differ-
ent fungi, including the model organisms Saccharomyces cerevisiae,
Ashbya gossypii, Neurospora crassa and Schizosaccharomyces pombe,
as well as three plant pathogens, Fusarium graminearum, Magna-
porthe grisea and Ustilago maydis, the two human pathogens Asper-
gillus fumigatus, Candida albicans, and the opportunistic pathogen
Rhizopus oryzae. The study included four species of filamentous
ascomycetes from the subphylum Pezizomycotina (euascomycetes),
one from the subphylum Taphrinomycotina (archiascomycetes) and
three from the subphylum Saccharomycotina, as well as one basid-
iomycete (U. maydis) and one zygomycete (R. oryzae), thus covering
a broad taxonomic range separated by nearly a billion years of evo-
lution. The analysis addresses the conservation of signalling com-
ponents beyond the core pathway modules, as well as the
existence of paralogues in different organisms and the degree of
sequence conservation among the components. Besides compari-
son of primary sequence, analysis of domain composition and pre-
dicted protein size was carried out to assess the quality of
annotation in the genome databases.
2. Results
2.1. Pathway components included in the analysis
The following signalling pathways were included in the analy-
sis: the Fus3 and Kss1 mating/filamentation MAPK cascade, the
Mpk1 cell integrity MAPK cascade, the osmostress Hog1 MAPK cas-
cade, and the calcium–calcineurin pathway. Database resources
and bioinformatic analysis tools used in this study are indicated
in Supplementary Table 1. Whenever possible, sequences were re-
trieved by BLAST (Altschul et al., 1997), using the S. cerevisiae se-
quence for query. Where blast searches with the S. cerevisiae
sequence failed to retrieve a hit in a given species, orthologues
from another species included in the analysis were used for blast
analysis. Candidate genes were systematically validated by reci-
procal blast, and only those that identified the original protein
when used in a blast search of the S. cerevisiae genome were con-
sidered for further analysis. Multiple alignments, as well as calcu-
lations of identity scores, were performed with ClustalW at default
settings. Validated candidate sequences were examined for poten-
tial annotation errors, and if required the annotation was corrected
using the prediction software outlined in Supplementary Table 1.
For several signalling components, orthologues from the basidio-
mycete human pathogen Cryptococcus neoformans were included
in the analysis to confirm and extend results obtained in U. maydis.
Fig. 1 presents a schematic overview of the signalling pathways
and their components in S. cerevisiae. Table 1 shows the number of
orthologues for each component identified in the different fungal
species. Identity scores of the S. cerevisiae protein with the closest
orthologue of each species are provided in Supplementary Table 2.
For a number of pathway components, reliable orthologues could
not be detected in certain species, either because sequence conser-
vation was too low or because they apparently do not exist. In the
following sections, the results of the analysis are summarized for
each of the pathways studied.
2.2. The Fus3 and Kss1 MAPK pathways
The Fus3 MAPK cascade mating pathway in S. cerevisiae has
been characterized in detail (Elion, 2000; Gustin et al., 1998; Kur-
jan, 1993; Wang and Dohlman, 2004). Signalling is initiated when
pheromone binds to the cognate cell surface receptors Ste2 or Ste3.
Orthologues of Ste2 and Ste3 were identified in all ascomycetes
tested in the study, including putative asexual species. Structural
domains, such as the seven transmembrane regions, were well
conserved although there were considerable variations in protein
size. As reported previously (Bolker et al., 1992), the basidiomycete
U. maydis has no Ste2 orthologues, but instead has two Ste3 ortho-
logues, Pra1 and Pra2, reflecting the fact that basidiomycetes only
have type a, but not type a pheromones. Analysis in C. neoformans
provided similar results, suggesting that the duplication of Ste3-
like receptors occurred early in the basidiomycete clade. Neither
Ste2 or Ste3 orthologues could be detected in the zygomycete R.
oryzae. While this may be due to low sequence homology, an alter-
native explanation is that this type of receptors is not present in
the zygomycetes, which employ a structurally distinct type of
pheromones, trisporic acid derivatives, for sexual reproduction
(Schimek and Wöstemeyer, 2006).
Once pheromone binds to its cognate receptor, it triggers disso-
ciation of the G protein a subunit Gpa1 from the G protein bc sub-
units Ste4 and Ste18. Fungal Ga proteins are divided into three
groups according to their structure. S. cerevisiae Gpa1 and its ortho-
logues in filamentous fungi belong to class I whereas Gpa2 belongs
to class III (Li et al., 2007). In contrast, the two Ga proteins in S.
pombe, Gpa1 and Gpa2, belong to classes II and III, respectively.
Due to its close homology with S. cerevisiae Gpa1, S. pombe Gpa1
was nevertheless included in the analysis. Gpa1 orthologues from
Saccharomycotina contain a region of approximately 100 amino
acids which is absent in the rest of the fungal species studied. All
Gpa1 orthologues are predicted to be prenylated on the N-terminal
cysteine residue and myristoylated on an N-terminal glycine resi-
due required for the lipid-anchor to the plasma membrane.
According to a recent study, R. oryzae has four class I Ga pro-
teins, RO3G_01120, RO3G_09475, RO3G_0005 and RO3G_00875
(Li et al., 2007). We detected a new member of class I,
RO3G_06003, whose original predicted sequence lacked the char-
acteristic N-terminal region of Ga proteins. The sequence annota-
tion was manually corrected using Fgenesh+ to include the
sequence of a predicted overlapping EST, resulting in a predicted
polypeptide of 353 amino acids containing all typical features of
Ga proteins. In contrast, RO3G_00875 was found to be closer to
S. cerevisiae Gpa2 and was therefore excluded from this analysis.
We also identified new class II and class III Ga proteins
RO3G_16598 and RO3G_15639, respectively, both of which had
not been described previously.
The heterotrimeric bc subunits Ste4 and Ste18 dissociate from
Ga to transmit the signal to the downstream pathway components
(Wang and Dohlman, 2004). Only one orthologue of Ste4 and Ste18
was detected in most fungal species, except for R. oryzae, in which
four orthologues of each subunit were identified (Table 1). For two
putative Gb subunits, RO3G_06062 and RO3G_08023, the pre-
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dicted sequence in the database lacked the first exon and was cor-
rected manually. Similar to Ga proteins, Gb subunits from the Sac-
charomycotina differ from those of filamentous fungi in size, due to
the presence of additional regions throughout the length of the
protein. All Ste18 orthologues contain a predicted prenylation site
for lipid-anchoring to the membrane. No clear Ste18 orthologue
was found in S. pombe, although its annotated genome contains a
predicted G protein gamma subunit, Git11, which was included
in the analysis.
Downstream of the bc subunits, the signal is transmitted to the
guanine nucleotide exchange factor Cdc24 which activates the G
protein Cdc42. Both proteins have one clear, and highly conserved
orthologue in all fungal species, except for R. oryzae which contains
two orthologues of each component. Cdc42 activates the PAK-like
protein kinase Ste20 and the adaptor protein Ste50, which cooper-
ate in activating the downstream MAPK module. Ste20 orthologues
display considerable divergence in size between species. The pre-
dicted S. pombe and R. oryzae proteins are about 300 amino acids
shorter than S. cerevisiae Ste20, whereas the orthologues from M.
grisea, F. graminearum, A. fumigatus and U. maydis are approxi-
mately 100 residues longer, and that of C. albicans is 200 amino
acids longer than the S. cerevisiae protein. The Ste20 orthologue
Smu1 from U. maydis which contains a N-terminal Cdc42-binding
domain and a C-terminal kinase domain, was previously found to
be non-essential for mating and plant infection (Smith et al., 2004).
Orthologues of S. cerevisiae Ste50 were detected in all fungal
species studied except for R. oryzae. Ste50 is an adaptor that links
G protein-associated Cdc42–Ste20 complex to the MAPKKK Ste11
through the presence of a Sterile Alpha Motif (SAM) and a Ras
Association (RA) domain (Wu et al., 1999). The SAM domain of
the M. grisea orthologue Mst50 was previously shown to be essen-
tial for its interaction with Mst11 and for appressorium formation
(Zhao et al., 2005). Interestingly, Ste50 orthologues of the basidio-
mycetes U. maydis (Ubc2) and C. neoformans are approximately
double in size and contain a Src Homology 3 (SH3) domain which
is lacking in the ascomycete Ste50 proteins. Deletion of the U. may-
dis orthologue Ubc2 was found to impair pheromone responses
and virulence. Interestingly, the SH3 domains of Ubc2 were appar-
ently not involved in morphogenesis, but clearly required for path-
ogenicity, suggesting that they are required for some, but not all
signalling outputs of the pathway (Mayorga and Gold, 2001).
Bem1 is a SH3-domain protein that links the Ste5–MAPK cas-
cade complex to upstream activators and specific downstream
substrates, thus enabling efficient circuitry for G1 arrest and mat-
ing (Lyons et al., 1996). Bem1 orthologues are well conserved in
the fungal species studied. R. oryzae has two Bem1 orthologues,
one of which (RO3G_02285) contains a Rho-GDI domain that is
lacking in the other Bem1 proteins. On the other hand, a conserved
PB1 domain associated with heterodimer formation is lacking in
the Bem1 orthologues of C. albicans, R. oryzae and U. maydis.
The MAPK module of the S. cerevisiae pheromone response
pathway is composed of MAPKKK Ste11, MAPKK Ste7 and MAPK
Fus3 (Wang and Dohlman, 2004). Ste11 functions in the Fus3
and Kss1 cascade, as well as in the Hog1 pathway by phosphorylat-
ing MAPKKs Ste7 and Pbs2, respectively. It contains a sterile alpha
motif (SAM) domain involved in interaction with Ste50 (Grimshaw
et al., 2004), which is conserved in all fungal orthologues except
that of A. gossypii. In addition, Ste11 proteins from F. graminearum,
M. grisea, N. crassa and R. oryzae contain a Ras association (RA) do-
main which is lacking in the other Ste11 orthologues. In S. cerevisi-
ae, the RA domain of Ste50, an interaction partner of Ste11, is
essential for tethering Ste11 to the plasma membrane through
Fig. 1. Schematic view of signalling components included in the study. Colors indicate different degrees of conservation among the fungal species Saccharomyces cerevisiae,
Ashbya gossypii and Candida albicans (hemiascomycetes), Schizosaccharomyces pombe (archiascomycetes), Aspergillus fumigatus, Fusarium graminearum, Magnaporthe grisea and
Neurospora crassa (euascomycetes), Rhizopus oryzae (zygomycetes) and Ustilago maydis (basidiomycetes): blue, components detected in all species studied; green, all except
zygomycetes; orange, all except basidiomycetes; cyan, all except zygomycetes and archiascomycetes; pink, all except zygomycetes, basidiomycetes and archiascomycetes;
red, all except euascomycetes; purple, only ascomycetes; yellow: only hemiascomycetes.
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Table 1
Signalling pathway components included in the study and number of orthologues identified in different fungal species.
ProteinFunction
Saccharomyces
cerevisiae
Ashbya
gossypii
Candida
albicans
Schizosaccharomyces
pombe
Aspergillus
fumigatus
Fusarium
graminearum
Magnaporthe
grisea
Neurosapora
crassa
Rhizopus
oryzae
Ustilago
maydis
Fus3 and Kss1 MAPK pathway
Ste2
a-Factor pheromone receptor
Ste3a-Factor receptor
Gpa1 Guanine nucleotide-binding protein a subunit
Ste4Guanine nucleotide-binding protein b subunit
Ste18 Guanine nucleotide-binding protein c subunit
Cdc24 Guanine nucleotide exchange factor
Cdc42 Small rho-like GTPase
Bem1SH3-domain protein
Ras2 GTP-binding protein
Ste20PAK (p21-activated kinase)
Ste50Protein kinase regulator
Ste11MAP kinase kinase kinase
Ste7 MAP kinase kinase
Ste5 Pheromone-response scaffold protein
Fus3/
Kss1a
Ste12 Transcription factor
Bni1 Formin
Tec1TEA/ATTS DNA-binding domain transcription
factor
Sst2Regulator of G protein signalling
Far1Cyclin-dependent kinase inhibitor
Msg5/
Sdp1a
Dig1Regulatory protein
Dig2 Regulatory protein
Ptp2/3a
Tyrosine-protein phosphatase 3
1
1
1
1
1
1
1
1
1
1
1
1
1
1
2
1
1
1
1
1
1
1
1
1
1
1
1
1
1
2
1
1
1
1
1
1
1
1
1
1
1
1
1
0
2
1
1
1
1
1
1
1
1
1
1
1
1
1
0
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0
1
0
0
4
4
4
2
2
2
1
2
0
1
1
0
2
0
2
1
1
1
1
1
1
1
1
1
1
1
0
2MAP Kinase
1
1
1
1
1
1
1
1
1
0
1
0
1
1
1
1
1
0
1
1
0
1
1
0
1
1
2
0
1
1
1
1
2
1
1
1
1
1
1
1
0
1
1
1
1
1
1
1
1
1
0
1
1
1
2
0
2
1
1
2 MAPK phosphatase
1
1
2
1
0
2
0
0
2
0
0
2
0
0
1
0
0
1
0
0
1
0
0
1
0
0
2
0
0
1
Hog1 MAPK pathway
Msb2/
Hkr1a
Sho1
Sln1
Ypd1
Cdc42
Ste20
Cla4
Ssk1
Ste50
Ste11
Ssk2/22a
Pbs2
Hog1
Rck1/2a
Sko1
Msn2/4a
Hot1
Smp1/
Rlm1a
Mcm1
Mucin family member2210111101
Transmembrane osmosensor
Osmosensing histidine protein kinase
Phosphorelay intermediate protein
Small rho-like GTPase
PAK (p21-activated kinase)
PAK (p21-activated kinase)
Cytoplasmic response regulator
Protein kinase regulator
MAP kinase kinase kinase
MAP kinase kinase kinase
MAP kinase kinase
MAP kinase
Serine–threonine protein kinase
Basic leucine zipper (bZIP) transcription factor 1
Zinc finger transcription factor
Transcription factor
MADS-box transcription factor
1
1
1
1
1
1
1
1
1
2
1
1
2
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0
0
1
1
1
1
1
1
1
2
1
1
2
1
1
0
1
1
1
1
1
1
1
1
1
1
1
1
2
1
1
1
0
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0
1
0
0
1
2
2
1
1
0
1
1
1
1
1
1
1
0
2
1
0
1
1
1
1
1
1
1
1
1
1
1
1
1
0
1
2
1
2
MADS-box transcription factor1111111121
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Ptc1
Ptc2/3a
Protein phosphatase 2C homolog 1
Protein phosphatase 2C homolog 2
1
2
1
1
1
1
1
2
1
1
1
1
1
1
1
1
1
1
1
1
Mpk1 MAPK pathway
Wsc1
Wsc2/3a
Mid2/
Mtl1*
Zeo1
Rom1/2a
Tus1
Sac7
Bem2
Rho1
Pkc1
Sit4
Bck1
Bck2
Mkk1/2a
Mpk1
Swi4/
Mbp1a
Swi6
Smp1/
Rlm1a
Fks2
Pst1
Ppz1/2a
Pir3
Msg5/
Sdp1a
Ptp2/3a
Plasma membrane sensor
Plasma membrane sensor
Plasma membrane sensor
1
2
2
1
1
1
1
1
2
1
1
0
1
1
0
1
1
0
1
1
0
0
1
0
0
0
0
0
0
0
Peripheral membrane protein
Guanine nucleotide exchange factor
Guanine nucleotide exchange factor
GTPase activating protein
GTPase activating protein
GTP-binding protein
Protein kinase C
Type 2A-related serine–threonine phosphatase 1
MAP kinase kinase kinase
MAP kinase kinase kinase
MAP kinase kinase
MAP kinase
DNA-binding component of the SBF complex
1
2
1
1
1
1
1
0
1
1
1
1
1
1
1
1
1
1
1
2
0
1
1
1
1
1
1
1
1
0
1
1
1
1
2
1
1
0
2
2
1
1
0
1
1
2
0
1
1
1
0
1
1
1
1
0
1
1
1
0
1
1
1
0
1
1
1
1
0
1
1
1
0
1
1
1
0
1
1
1
1
0
1
1
1
0
1
1
1
0
1
1
1
1
0
1
1
1
0
2
1
1
2
1
2
2
1
0
1
1
2
0
1
0
1
1
1
1
1
1
0
1
1
1
1
1
2
1
2
Transcription cofactor
MADS-box transcription factor
1
2
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
2
1
1
Catalytic subunit of b-1,3-glucan synthase
Cell wall protein
Protein Phosphatase Z
Protein containing internal repeats
MAPK phosphatase
1
1
2
1
2
1
1
1
1
1
1
1
1
1
1
4
2
1
0
1
1
1
1
0
1
1
1
1
0
1
1
1
1
0
0
1
1
1
0
1
1
1
2
0
2
1
0
1
0
2
Tyrosine-protein phosphatase 32222111121
Ca2+–calmodulin–calcineurin pathway
Cch1Probable calcium-channel protein
Mid1Putative stretch-activated Ca2+channel
component
Fig1Integral membrane protein required for efficient
mating
Cmd1 Calmodulin
Cna1/
Cmp2a
Cnb1 Calcineurin subunit B
Pmc1Calcium-transporting ATPase 2
Pmr1 Calcium-transporting ATPase 1
Vcx1 Vacuolar calcium ion transporter
Yvc1Vacuolar cation channel
Fpr1 Peptidyl–prolyl cis–trans isomerase
Cpr1Peptidyl–prolyl cis–trans isomerase
Rcn1Calcineurin inhibitor
Crz1Transcriptional regulator
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
2
1
1
1
1111111100
1
2
1
1
1
1
2
1
1
1
1
1
1
1
1
1
4
2
1
1 Calcineurin subunit A
1
1
1
1
1
1
1
1
1
1
1
1
1
0
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0
1
1
1
2
1
3
2
4
1
1
1
1
1
1
5
2
4
1
1
1
1
1
1
3
2
5
1
1
1
1
1
1
2
2
5
1
1
1
1
1
1
5
2
5
0
1
1
0
2
1
1
2
2
1
1
1
1
1
aTwo paralogues present in Saccharomyces cerevisiae.
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association of Ste50 with Cdc42 (Truckses et al., 2006). The pres-
ence of a RA domain in Ste11 in filamentous species suggests, that
the MAPKKK in these fungi could localize to the plasma membrane
by directly binding Cdc42. Orthologues of the MAPKK Ste7 were
detected in all species. Fuz7 and Mst7 were previously shown to
be required for mating and virulence in U. maydis (Banuett and
Herskowitz, 1994) and M. grisea (Zhao et al., 2005).
In S. cerevisiae, two MAPKs regulate distinct signalling outputs
downstream of Ste7. One of them, Fus3, is essential for mating,
whereas the other, Kss1, controls invasive growth and pseudohy-
phal development (Madhani et al., 1997). In contrast to Fus3,
Kss1 can also be activated by Ste7 that is not bound to the Ste5
scaffold (Elion, 1998). Fus3 and Kss1 orthologues play crucial roles
during infection in many plant pathogenic fungi including the
three phytopathogens surveyed in this study, M. grisea (Xu and
Hamer, 1996), F. graminearum (Jenczmionka et al., 2003) and U.
maydis (Brachmann et al., 2003; Mayorga and Gold, 1999; Muller
et al., 1999). Several species analyzed here have two orthologues
of Fus3 and Kss1, including the close relative of S. cerevisiae, A. gos-
sypii. In C. albicans, one of the two MAPKs, Cek2, clusters close to
the Saccharomycotina sequences whereas the second MAPK, Cek1,
which is involved in yeast-hyphal switching, mating efficiency
and virulence (Csank et al., 1998; Chen et al., 2002), is more closely
related to MAPK orthologues from filamentous Ascomycetes. In U.
maydis, Kpp2 (Ubc3) and Kpp6 are two orthologues with overlap-
ping functions in mating and plant infection, but Kpp6, which con-
tains an unusual N-terminal domain, appears to be more specific
for host penetration (Brachmann et al., 2003). The zygomycete R.
oryzae also has two orthologues of Fus3 and Kss1 whose functions
remain to be determined.
The scaffold protein Ste5 plays an essential role in S. cerevisiae
pheromone signalling by recruiting the Ste11–Ste7–Fus3 complex
to the plasma membrane (Pryciak and Huntress, 1998) and stimu-
lating phosphorelay by proximity effects, oligomerization, and
conformational changes (Qi and Elion, 2005). Our analysis failed
to detect Ste5 orthologues in any of the fungal species studied ex-
cept A. gossypii. It is possible that other, hitherto unknown, signal-
ling components may carry out the scaffold function in this MAPK
pathway.
Phosphorylated Fus3 in S. cerevisiae activates downstream
effectors such as Ste12, Far1 or Sst2, leading to cell cycle arrest,
polarized growth and formation of specialized fusion tubes called
shmoos (Elion et al., 1993). Ste12 is a key transcription factor
downstream of the pheromone-response cascade, which binds to
pheromone response elements (PREs) in the upstream activating
sequences of its target genes and, in cooperation with Tec1, also
regulates genes involved in invasive growth (Madhani and Fink,
1997). A single Ste12 orthologue was detected in all fungal species
examined, except S. pombe and U. maydis. Lack of Ste12 in U. may-
dis appears to be characteristic for this species rather than for the
basidiomycete group, since C. neoformans does contain mating
type-specific Ste12 orthologues (Wickes et al., 1997). In addition
to the characteristic Ste-like homeodomain in the N-terminal re-
gion of the protein, Ste12 orthologues from filamentous fungi con-
tain two C-terminal C2H2zinc finger motifs which are lacking in
the Saccharomycotina (Fig. 2A). The role of the zinc finger domain
in Ste12 function is poorly understood. In M. grisea, both the Ste-
like region and the zinc finger region of Mst12 were required for
invasive growth and virulence on rice plants (Park et al., 2004).
Far1 mediates the cell cycle arrest in response to pheromone
(Peter et al., 1993), and specifies direction of polarized growth dur-
ing mating by linking the heterotrimeric G bc subunits to the
polarity establishment machinery (Butty et al., 1998). The Far1
protein contains a C3HC4-type ring zinc finger domain with a pre-
dicted role in the ubiquitination pathway. Far1 was recently shown
to act as a dosage-dependent regulator of the pheromone response
during mating in C. albicans (Cote and Whiteway, 2008). Far1
orthologues were found in all species except S. pombe and R. ory-
zae. In spite of sharing a low degree of sequence conservation
(10–20%), all predicted Far1 proteins have the characteristic ring
zinc finger domain. In addition, Far1 orthologues from filamentous
fungi, but not from Saccharomycotina, contain a pleckstrin homol-
ogy domain and a von Willebrand factor type A (VWA) domain,
indicative of a possible involvement in multiprotein complexes
(Fig. 2B). The role of Far1 proteins in fungal pathogenicity has
not been addressed experimentally so far.
Sst2 is a GTPase-activating regulator of G protein signalling
(RGS) for Gpa1, which regulates pheromone desensitization and
prevents receptor-independent signalling of the mating pathway
(Dohlman et al., 1996). Orthologues of Sst2 were identified in all
species examined, including two orthologues in R. oryzae. Annota-
tion errors in the predicted protein database sequences of F. grami-
nearum and M. grisea were corrected manually. S. pombe Sst2 is
significantly shorter (480 aa) than the rest of the orthologues
(650–780 aa). All Sst2 proteins share the RGS domain, but the pre-
dicted A. gossypii and C. albicans proteins lack a conserved DEP-like
segment (residues 50–135) which is required for binding to the
cognate G protein-coupled receptor Ste2 (Ballon et al., 2006).
The filamentation and pseudohyphal growth pathway in S. cere-
visiae is activated by a mucin-like protein, Msb2, consisting of a N-
terminal signal peptide, an extracellular serine–threonine-rich re-
peat region predicted to be highly O-glycosylated, a single trans-
membrane domain and a short cytoplasmic tail interacting with
the downstream component Cdc42 (Cullen et al., 2004). Recently
a second mucin-like protein, Hkr1, was shown to function together
with Msb2 as an osmosensor in the S. cerevisiae Hog1 pathway
(Tatebayashi et al., 2007). We detected single orthologues of
Msb2, but not Hkr1, in all species studied except for S. pombe
and R. oryzae. Only A. gossypii has also an orthologue of Hkr1. All
predicted Msb2 proteins contain a putative signal peptide and
transmembrane region. While the exact amino acid repeats in
the extracellular region are not present in some of the fungal
orthologues, the high content of putatively glycosylated serine
and threonine residues in this region is maintained, suggesting a
conserved role of O-glycosylation in Msb2 function. Similarly, the
amino acid sequence of the short cytoplasmatic tail is well con-
served among filamentous ascomycetes, indicating an important
role of this domain in intracellular signalling.
Another component required specifically for the filamentation
and invasive growth pathway upstream of Cdc42, Ste20 and Kss1
is the small GTP-binding protein Ras2 (Mosch et al., 1996). In C.
albicans, Ras links cellular morphogenesis to virulence by regulat-
ing the MAPK and cAMP signalling pathways (Leberer et al.,
2001). In U. maydis, expression of a dominant active allele of the
ras2 orthologue promoted pseudohyphal growth in a manner
dependent on the pheromone-response MAPK cascade (Lee and
Kronstad, 2002). Likewise, expression of a dominant active ras2 al-
lele of M. grisea stimulated appressorium formation on non-induc-
tive surfaces in the wild-type strain, but not in the pmk1 mutant,
suggesting that Ras2 functions upstream of the Mst11–Mst7–
Pmk1 cascade (Park et al., 2006). A similar signalling role for
Ras2 was proposed in F. graminearum (Bluhm et al., 2007). In this
study, Ras2 orthologues containing predicted palmitoyl and far-
nesyl groups at the C-terminus for membrane localization were
identified in all fungal species studied.
In S. cerevisiae, two nuclear protein substrates of Kss1, Dig1 and
Dig2, negatively regulate the invasive growth pathway by repress-
ing Ste12 action (Cook et al., 1996). We failed to detect Dig1 and
Dig2 orthologues in any of the species analyzed except for the close
relative A. gossypii, suggesting that a regulatory mechanism other
than that mediated by Dig1 and Dig2 must be operating in filamen-
tous fungi.
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For activation of genes involved in filamentous and invasive
growth, Ste12 forms a heterodimer with the TEA/ATTS family tran-
scription factor Tec1 to bind filamentation response elements
(FREs) (Madhani and Fink, 1997). It has been suggested that Tec1
orthologues of pathogenic fungi could be of interest due to their
possible implication in virulence (Madhani and Fink, 1998). Indeed,
the Tec1 orthologue of C. albicans regulates hyphal development
and virulence (Schweizer et al., 2000). However, in our survey
we failed to detect clear Tec1 orthologues in S. pombe and the fila-
mentous ascomycetes F. graminearum, M. grisea and N. crassa.
Interestingly, the human pathogen A. fumigatus contains a Tec1
orthologue that is highly similar to A. nidulans AbaA, a transcription
factor with an ATTS DNA-binding motif required for conidiophore
development (Andrianopoulos and Timberlake, 1994). The role of
Tec1 in virulence of A. fumigatus has not been explored so far.
In summary, most components of the Fus3 and Kss1 MAPK cas-
cades are well conserved among the fungal species studied, includ-
ing basidiomycetesandzygomycetes.
pheromone-response scaffold protein Ste5 and the two Ste12 reg-
ulators Dig1 and Dig2. A noteworthy finding is the multiplicity of
heterotrimeric G protein subunits in the zygomycete R. oryzae.
Exceptions arethe
2.3. The Hog1 MAPK pathway
Thehighosmolarityglycerol(HOG)pathwaymediatesresponses
to hyperosmoticshockand to other stresses (Hohmann et al., 2007).
In S. cerevisiae, the Hog1 pathway has two upstream branches that
converge on the MAPKK Pbs2 (see Fig. 1). One branch consists of a
phosphorelay system composed of the sensor histidine kinase
Sln1, the phosphotransfer protein Ypd1 and the response regulator
Ssk1.HyperosmoticshockdeactivatesSln1,leadingtoenhancedlev-
els of dephospho-Ssk1 and sequential phosphorylation of the MAP-
KKKs Ssk2 and Ssk22, the MAPKK Pbs2 and the MAPK Hog1 (Posas
et al., 1996). Whereas Sln1 is the only histidine kinase present in S.
cerevisiae, other fungi contain multiple histidine kinases which can
be classified into different groups according to their topology (Cat-
lett et al., 2003). Members of group VI, which includes Sln1, contain
two transmembrane domains in addition to the characteristic
phosphoacceptor, ATP-binding and response regulator receiver do-
mains. The C. albicans orthologue CaSLN1 was shown to be involved
in hyphal formation and virulence (Nagahashi et al., 1998), whereas
deletion of the A. fumigatus orthologue TcsB produced no clear phe-
notype (Du et al., 2006). Orthologues, in which the critical Sln1 do-
mains are conserved, were identified in most species studied.
However, S. pombe, U. maydis and R. oryzae genomes do not contain
anymemberofgroupVI.Bycontrast,asingleorthologueofthephos-
phorelay protein Ypd1 and of the cytoplasmic response regulator
Ssk1 was identified in all species. C. albicans mutants lacking Ssk1
are avirulent in an invasive murine model and fail to adhere to hu-
man cells (Calera et al., 2000). The orthologous RRG-1 response reg-
ulatorfromN.crassawasrecentlyshowntofunctionupstreamofthe
osmoresponse MAPK pathway, and to regulate asexual develop-
STE Homeodomain
STE Homeodomain
C2H2Zinc Finger
S. cerevisiae S. cerevisiaeS. cerevisiae
A. gossypii
C. albicansC. albicans C. albicans
A. fumigatus A. fumigatusA. fumigatus
F. graminearum
M. grisea M. griseaM. grisea
N. crassaN. crassaN. crassa
R. oryzae R. oryzaeR. oryzae
100 bp100 bp 100 bp
S. cerevisiae S. cerevisiae
A. gossypii
C. albicansC. albicans
A. fumigatus A. fumigatus
F. graminearum
M. griseaM. grisea
N. crassaN. crassa
U. maydisU. maydis
C3HC4Zinc Finger, Ring type
es a l onEesa l onE
Plecktrin Homology Domain Plecktrin Homology Domain
n i amn i amooDD AAeepp y t r o t ca fy t r o t c a fddnna r b a r b e l l i e l l iWWnnoovv
100 bp 100 bp100 bp
C2H2Zinc Finger
A. gossypii A. gossypii
F. graminearum F. graminearum
A
B
A. gossypii
F. graminearum
C3HC4Zinc Finger, Ring type
Fig. 2. Scaled cartoon of the domain structure of Ste12 and Far1 orthologues from different fungal species. (A) Ste12 orthologues all contain a characteristic STE
homeodomain in the N-terminal region. In addition, filamentous ascomycetes and the zygomycete R. oryzae have a C2H2zinc finger domain in the C-terminal region. (B) Far1
orthologues all contain a C3HC4ring type zinc finger domain in the N-terminal region. In addition, filamentous ascomycetes and the basidiomycete U. maydis have a pleckstrin
homology domain and a von Willebrand factor type A domain, whereas A. gossypii has an enolase domain.
N. Rispail et al./Fungal Genetics and Biology 46 (2009) 287–298
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ment,femalefertility,osmoticstressandfungicideresistance(Jones
et al., 2007).
The MAPKKKs Ssk2 and Ssk22 function downstream of the Sln1
branch to activate the MAPKK Pbs2 and the MAPK Hog1. All fungi
surveyed including, A. gossypii, contain a single orthologue of Ssk2/
Ssk22, except for S. pombe which has two paralogues clustering in a
separate branch with the basidiomycetes and zygomycetes.
In the second osmosensing branch, the plasma membrane pro-
teinSho1recruitstheMAPKKKSte11andtheMAPKKPbs2tothecell
surface. Orthologues of Sho1 displaying conserved structural fea-
turesweredetectedinallspeciesstudied,exceptS.pombeandR.ory-
zae. Sho1 orthologues were found to link oxidative stress to
morphogenesis and cell wall biosynthesis in C. albicans (Bermejo
etal.,2008;Romanetal.,2005)andtoregulatehyphalgrowth,mor-
phologyand oxidantadaptationinA. fumigatus(Ma et al., 2008),but
weredispensableforvirulenceinbothhumanpathogens.Theroleof
Sho1infungalpathogenicityonplantshasnotbeendeterminedyet.
Similar to the Fus3 and Kss1 pathway, activation of Ste11 by the
Sho1 branch of the osmoresponse pathway requires the small G
protein Cdc42, the adaptor protein Ste50 and the PAK kinase
Ste20 (Raitt et al., 2000). A second PAK kinase, Cla4, functions in
parallel with Ste20 (Tatebayashi et al., 2006). In this study we de-
tected a single Cla4 orthologue in all the fungal species studied.
The MAPKK Pbs2 serves as a scaffold for several components of
the HOG pathway and integrates the two upper branches of the
pathway. Phosphorylation of Pbs2 via Ssk2 and Ssk22 occurs under
severe osmotic stress (Posas et al., 1996), whereas its activation by
Ste11 takes place under less severe hyperosmotic conditions,
whereby Pbs2 acts as a scaffold for Sho1, Ste11 and Hog1 (Posas
and Saito, 1997). Single Pbs2 orthologues showing a conserved do-
main composition were detected in all the species studied.
All fungi surveyed contain a single orthologue of the osmore-
sponse MAPK Hog1, except for A. fumigatus which has two ortho-
logues, similar to other Aspergilli (Miskei et al., 2009). The role of
Hog1 orthologues has been studied in different fungal pathogens.
C. albicans hog1 mutants are de-repressed in serum-induced hyphal
formation and show reduced virulence (Alonso-Monge et al., 1999).
In A. fumigatus, two Hog1 orthologues, SakA and MpkC play distinct
rolesintheresponsetooxidativeandnutritionalstressesbutarenot
requiredfor virulence(Reyes et al., 2006;Xue et al., 2004). Likewise,
M. grisea mutantslacking the Hog1 orthologue Osm1 were sensitive
to osmotic stress, but formed functional appressoria and were fully
virulent on rice plants (Dixon et al., 1999). In F. graminearum, dele-
tion mutants of MAPKKK FgOs4, MAPKK FgOs5 and MAPK FgOs2
showedmarkedly enhancedpigmentation andfailed to produce tri-
chothecenes in aerial hyphae, although their virulence phenotype
has yet to be determined (Ochiai et al., 2007).
Downstream targets of Hog1 in S. cerevisiae include the MAPK-
dependent protein kinases Rck1 and Rck2 (Bilsland et al., 2004), as
well as the transcription factors Sko1 (Rep et al., 2001), Msn2 and
Msn4 (Martinez-Pastor et al., 1996), Hot1 (Rep et al., 2000), Smp1
and Rlm1 (de Nadal et al., 2003), and Mcm1 (Yu et al., 1995). Our
study indicates that all these downstream components are well
conserved across the fungal phyla, except for Hot1 whose presence
is limited to the Saccharomycotina. In most cases, single ortho-
logues of each component were detected, although R. oryzae has
two orthologues for the MADS-box transcription factors Smp1,
Rlm1 and Mcm1. In summary, the components of the Hog MAPK
pathway are very well conserved throughout the fungal kingdoms,
with the exception of the transcription factor Hot1 which is spe-
cific for the Saccharomycotina.
2.4. The Mpk1 cell integrity pathway
The Mpk1 cell integrity cascade is responsible for orchestrat-
ing changes in the cell wall through the cell cycle and in re-
sponse to various forms of stress (Levin, 2005). This pathway
is activated by the integrin-like proteins Wsc1,2,3 which share
a conserved extracellular motif of eight cysteines (Verna et al.,
1997). A second activator of the Mpk1 pathway is Mid2, an O-
glycosylated plasma membrane protein that interacts with
Rom2, the guanine nucleotide exchange factor for Rho1, and
with the cell integrity pathway protein Zeo1 (Philip and Levin,
2001). Orthologues of the Wsc1 and Wsc2 and 3 proteins are
present in most ascomycetes, whereas Mid2 and Zeo1 are re-
stricted to the Saccharomycotina. Wsc1 and Mid2 are linked to
the guanine nucleotide exchange factors (GEFs) Rom1 and 2
which activate the GTPase Rho1 (Ozaki et al., 1996). Similar to
S. cerevisiae, Rho1 is required for cell viability in C. albicans
(Smith et al., 2002). In contrast, rho1 knockout mutants of the
soilborne pathogen Fusarium oxysporum were viable and showed
drastically reduced virulence on plants, but retained full viru-
lence on immunodepressed mice (Martinez-Rocha et al., 2008).
Both Rom1 and 2, as well as Rho1, are widely conserved in fun-
gi, with a single orthologue of the two GEFs present in almost all
species studied. In addition, Rho1 activity in S. cerevisiae is reg-
ulated by the GEF Tus1 and the GTPase activating proteins Sac7
and Bem2 (Levin, 2005). Both Tus1 and Sac7 are widely con-
served among the fungal species studied. By contrast, Bem2
was detected in the Saccharomycotina, U. maydis and R. oryzae,
but not in the other ascomycetes. The evolutionary and func-
tional implications of this interesting differential distribution
are currently unknown.
Rho1 activates protein kinase C (PKC) 1 which, in turn, activates
a three-tiered kinase module composed of the MAPKKK Bck1, the
MAPKKs Mkk1 and Mkk2 and the MAPK Mpk1 (Levin, 2005). In
contrast to most fungi surveyed in this study, R. oryzae has two
Pkc1 orthologues, as previously described for S. pombe (Kobori
et al., 1994). Single orthologues were detected for each of the three
components of the Mpk1 MAPK module. The role of Mpk1 ortho-
logues has been determined in a number of fungal pathogens.
Mps1 is essential for conidiation, appressorial penetration, and
plant infection in M. grisea (Xu et al., 1998). In F. graminearum,
Mgv1 is required for hyphal fusion and heterokaryon formation
(Hou et al., 2002). In C. albicans, Mkc1 regulates cell wall integrity,
growth at high temperatures, morphological transition and patho-
genesis (Diez-Orejas et al., 1997). Recently, it was shown that the A.
fumigatus orthologue MpkA controls cell wall signalling and oxida-
tive stress response, but is dispensable for virulence (Valiante et al.,
2008).
Mpk1 regulates multiple nuclear targets, including the SBF
complex which is formed by DNA-binding component Swi4,
Mbp1 and co-factor Swi6 and acts as a transcriptional activator
of cell cycle-dependent genes (Nasmyth and Dirick, 1991). Two
Swi4 and Mbp1 orthologues were found in A. gossypii, S. pombe
and R. oryzae, opposed to only one in the other fungi. By contrast,
all species studied have a single Swi6 orthologue.
A second nuclear target of Mpk1 is the MADS-box transcription
factor Rlm1 which regulates the expression of at least 25 genes in
S. cerevisiae, most of which have been implicated in cell wall bio-
genesis and function (Jung et al., 2002). These include the glycosyl-
phosphatidylinositol (GPI)-anchored
glycosylated protein Pir3 (Jung and Levin, 1999) and the glucan
synthase catalytic subunit Fks2. Conserved orthologues were de-
tected for Rlm1 itself, Pst1 (except in U. maydis) and Fks2 (includ-
ing four orthologues in S. pombe). By contrast, no Pir3 orthologues
were found outside of the Saccharomycotina.
The serine/threonine protein phosphatases Ppz1 and Ppz2 are
key regulators of K+and pH homeostasis, thus determining salt tol-
erance, cell wall integrity and cell cycle progression (Yenush et al.,
2002). In contrast to S. cerevisiae, all fungal species studied with the
exception of R. oryzae contain only a single Ppz1 and 2 orthologue.
protein Pst1,the O-
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In summary, a high conservation of the Mpk1 MAPK cascade
components was detected throughout the species studied, except
for certain plasma membrane sensors such as Mid2, and for the
downstream effector protein Pir3.
2.5. MAPK-regulatory protein phosphatases
Tyrosine, serine/threonine and dual-specificity phosphatases
co-ordinately dephosphorylate and thereby inactivate different
MAPKs in S. cerevisiae (Martin et al., 2005). The dual-specificity
protein phosphatase Msg5 and the tyrosine phosphatase Ptp3
dephosphorylate Fus3, thereby regulating the adaptive response
to pheromone (Zhan et al., 1997). Ptp2 and 3, as well as the type
2C protein phosphatase (PP2C) Ptc1 antagonize the osmosensing
MAPK cascade by dephosphorylating Hog1 (Warmka et al.,
2001; Wurgler-Murphy et al., 1997). The stress-inducible dual-
specificity MAPK phosphatase Sdp1 negatively regulates the cell
integrity pathway by dephosphorylating Mpk1 (Hahn and Thiele,
2002). In general, orthologues of these protein phosphatases were
detected in all fungal species surveyed, suggesting that the mech-
anisms of regulating MAPK activity via dephosphorylation are
broadly conserved in fungi. We detected mostly single ortho-
logues, except for the Saccharomycotina and, in some cases, U.
maydis and R. oryzae. The M. grisea genome database lacked an
annotated Msg5 orthologue, but inspection of a genomic region
from the excluded reads of annotated strain 70-15 and subse-
quent analysis of a genomic fragment from a field isolate (Y34)
revealed the presence of a reliable Msg5 orthologue which was
included in the analysis.
2.6. The calcium/calcineurin signalling pathway
Calcium signalling through the Ca2+-binding protein calmodulin
and the Ca2+–calmodulin-dependent phosphatase calcineurin has
been implicated in a multitude of processes, including stress re-
sponse, mating, budding, and actin-based processes (Cyert, 2001)
as well as in determining tolerance to antifungal drugs (Cruz
et al., 2002; Del Poeta et al., 2000; Kontoyiannis et al., 2003; Sang-
lard et al., 2003; Steinbach et al., 2007; Walker et al., 2008). Cellu-
lar calcium levels in S. cerevisiae are regulated by multiple channels
and transporters, including the voltage-gated high-affinity calcium
channel Cch1 which functions together with the stretch-activated
cation channel Mid1 (Locke et al., 2000). In contrast to the trans-
membrane protein Cch1, Mid1 is anchored to the membrane by a
GPI-anchor.
A third player in calcium regulation is Fig1, an integral mem-
brane protein required for efficient mating which may participate
in the low affinity Ca2+influx system, affecting intracellular signal-
ling and cell–cell fusion (Muller et al., 2003). Cch1, Mid1 and Fig. 1
have single orthologues in all fungal species studied, except for the
presence of two putative Cch1 orthologues in R. oryzae, and for the
absence of Fig. 1 orthologues in U. maydis and R. oryzae. Both R. ory-
zae Cch1 sequences in the database contained annotation errors
that were corrected manually. The role of these upstream sensors
in filamentous fungi is largely unknown. Recently, a N. crassa mu-
tant lacking a Mid1 orthologue was found to be affected in calcium
homeostasis and vegetative growth (Lew et al., 2008). Moreover,
deletion of a Cch1 orthologue affected ascospore discharge and
mycelial growth in F. graminearum (Hallen and Trail, 2008).
In addition to plasma membrane channels, the calcium-trans-
porting ATPases Pmr1 and Pmc1, the vacuolar ion exchanger
Vcx1 and the vacuolar cation channel Yvc1 also contribute to reg-
ulation of cellular calcium levels and calcium signalling. Strikingly,
while S. cerevisiae only has one of each of these components, fila-
mentous fungi consistently contain two Pmr1 orthologues and
most of them have between three and five orthologues of Pmc1
and Vcx1. Whether these multiple components have distinct or
redundant functions is currently an open question.
S. cerevisiae calmodulin is a small, essential Ca2+-binding pro-
tein encoded by CMD1, which has both Ca2+-dependent and
independent targets. One of the major Ca2+-dependent targets
is calcineurin, a Ca2+and calmodulin dependent phosphatase. S.
cerevisiae calcineurin is composed of a heterodimer of a catalytic
A subunit encoded by CMP2 and CNA1, and of a regulatory B
subunit encoded by CNB1. In the presence of stimulatory levels
of Ca2+, calmodulin binds to the A subunit of calcineurin, displac-
ing an autoinhibitory domain. Calmodulin–calcineurin-activated
gene expression is triggered by multiple external cues, including
high temperature, high concentrations of ions, cell wall stress
and exposure to mating pheromone (Cyert, 2003; Kraus and
Heitman, 2003). In our survey we detected single orthologues
for Cmd1, Cnb1, Cna1 and Cmp2, respectively, except for R. ory-
zae which has two Cna1 and Cmp2 orthologues. All these com-
ponents showed a high degree of sequence identity (40–90%)
among the fungal species studied. In the human pathogens C.
albicans, C. neoformans and A. fumigatus, calcineurin was required
for survival in serum and for virulence (Bader et al., 2003; Blan-
kenship et al., 2003; Da Silva Ferreira et al., 2007; Fox et al.,
2001; Odom et al., 1997; Steinbach et al., 2006, 2007). In the
plant pathogen Sclerotinia sclerotiorum, the calcineurin ortho-
loguecontrols sclerotialdevelopment
et al., 2006).
A key target of calcineurin is the zinc finger transcription factor
Crz1, whose nuclear localization is positively regulated by calci-
neurin-mediated dephosphorylation (Cyert, 2001). Orthologues of
Crz1 were recently shown to act as virulence factors in the human
pathogen A. fumigatus (Cramer et al., 2008; Soriani et al., 2008) and
the plant pathogen Botrytis cinerea (Schumacher et al., 2008). The
fungal species surveyed in this study all contain a single ortho-
logue of Crz1, except for S. pombe and R. oryzae which have two
orthologues.
Fpr1 and Cpr1, two peptidyl–prolyl cis–trans isomerases that
catalyze the cis–trans isomerization of peptide bonds N-terminal
to proline residues, are the cellular targets of the drugs Cyclosporin
A and FK506 and function as inhibitors of calcineurin through for-
mation of a ternary complex (Wang and Heitman, 2005). Ortho-
logues of these two proteins were detected in all fungal species
studied, although their role in calcineurin regulation has not been
determined. Similarly, orthologues of the Rcn1 protein, which is in-
volved in regulation of calcineurin during calcium signalling
(Kingsbury and Cunningham, 2000), were found in all species ex-
cept R. oryzae.
In summary, all the components of the calcium–calcineurin
pathway were highly conserved throughout the different fungal
kingdoms. A noteworthy feature, whose biological significance is
unclear, is the multiplicity of the calcium-transporting ATPases
and the vacuolar calcium channels in filamentous fungal species.
andinfection (Harel
3. Conclusions
Three conserved MAPK cascades and the calcium–calcineurin
pathway play crucial roles in fungal pathogenicity. Here we have
taken advantage of the availability of complete fungal genome se-
quences to survey the inventory of predicted MAPK and calcium–
calcineurin signalling components in ten fungal species, including
several plant and human pathogens, covering a wide array of taxo-
nomical and biological diversity (Fig. 1, Table 1). While most com-
ponents were found to be conserved among the model yeast S.
cerevisiae and filamentous fungi, some components such as the
scaffold protein Ste5, the regulatory proteins Dig1 and Dig2 and
the transcription factor Hot1 are limited to the Saccharomycotina.
The incorporation of new domains which are lacking in S. cerevisi-
N. Rispail et al./Fungal Genetics and Biology 46 (2009) 287–298
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ae, such as the RA domain in Ste11, the zinc finger in Ste12, or the
pleckstrin homology domain and VWA domains in Far1 (Fig. 2),
might reflect functional adaptations as filamentous fungi have
evolved to occupy different ecological niches, including their roles
as pathogenic agents. The presence of multiple paralogues of many
signalling components in the zygomycete R. oryzae is striking,
although the evolutionary and functional significance of this find-
ing is currently unknown. Thus, while the model yeast S. cerevisiae
has provided an excellent roadmap of the components of MAPK
and calcium–calcineurin pathways, functional analysis in patho-
genic species represents the only way to understand the role of
these signalling cascades in fungal virulence. So far, most studies
have focused on a few core pathway components. While this has
provided a useful overview of the general implication of these
pathways in fungal virulence, a detailed analysis of the upstream
and downstream factors that interact with these core signalling
cascades is now clearly necessary. Such an approach should allow
a more careful and critical evaluation of the specific role of each
signalling pathway in infection-associated functions, and extend
our understanding regarding how these conserved signalling cas-
cades have been recruited by fungal pathogens to infect their
eukaryotic hosts.
Acknowledgments
The authors acknowledge access to the genome data generated
by the Broad Institute/Fungal Genome Initiative. We are grateful
for the valuable suggestions of the anonymous reviewers. This
analysis was carried out by members of the SIGNALPATH Marie
Curie training network (MRTN-CT-2005-019277), which provided
financial support for N.R., C.A., R.C., A.G., R.H., E.P.N., A.P., E.S.,
V.V. and M.Y.
Appendix A. Supplementary material
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.fgb.2009.01.002.
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