Heterotrimeric G Protein Signaling in Filamentous Fungi *

Department of Plant Pathology and Microbiology, University of California, Riverside, California 92521, USA.
Annual Review of Microbiology (Impact Factor: 12.18). 02/2007; 61(1):423-52. DOI: 10.1146/annurev.micro.61.080706.093432
Source: PubMed
Filamentous fungi are multicellular eukaryotic organisms known for nutrient recycling as well as for antibiotic and food production. This group of organisms also contains the most devastating plant pathogens and several important human pathogens. Since the first report of heterotrimeric G proteins in filamentous fungi in 1993, it has been demonstrated that G proteins are essential for growth, asexual and sexual development, and virulence in both animal and plant pathogenic filamentous species. Numerous G protein subunit and G protein-coupled receptor genes have been identified, many from whole-genome sequences. Several regulatory pathways have now been delineated, including those for nutrient sensing, pheromone response and mating, and pathogenesis. This review provides a comparative analysis of G protein pathways in several filamentous species, with discussion of both unifying themes and important unique signaling paradigms.


Available from: Gyungsoon Park, Sep 11, 2014
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Heterotrimeric G Protein
Signaling in Filamentous
Liande Li, Sara J. Wright, Svetlana Krystofova,
Gyungsoon Park, and Katherine A. Borkovich
Department of Plant Pathology and Microbiology, University of California, Riverside,
California 92521; email:
Annu. Rev. Microbiol. 2007. 61:423–52
The Annual Review of Microbiology is online at
This article’s doi:
2007 by Annual Reviews.
All rights reserved
These four authors contributed equally to this
Key Words
G protein–coupled receptor, cAMP, carbon sensing, pheromone
response, pathogenesis
Filamentous fungi are multicellular eukaryotic organisms known
for nutrient recycling as well as for antibiotic and food produc-
tion. This group of organisms also contains the most devastating
plant pathogens and several important human pathogens. Since the
first report of heterotrimeric G proteins in filamentous fungi in
1993, it has been demonstrated that G proteins are essential for
growth, asexual and sexual development, and virulence in both ani-
mal and plant pathogenic filamentous species. Numerous G protein
subunit and G protein–coupled receptor genes have been identi-
fied, many from whole-genome sequences. Several regulatory path-
ways have now been delineated, including those for nutrient sens-
ing, pheromone response and mating, and pathogenesis. This review
provides a comparative analysis of G protein pathways in several
filamentous species, with discussion of both unifying themes and
important unique signaling paradigms.
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Filamentous fungi:
a group of
comprising most
species in the
Kingdom Fungi that
grow by extension of
tubular cellular
structures called
INTRODUCTION................. 424
COMPONENTS ................ 425
Gα Proteins...................... 425
Gβ and Gγ Subunits ............. 427
G Protein–Coupled Receptors .... 427
Regulatory Proteins .............. 435
Output Pathways ................. 436
NUTRIENT SENSING ............ 437
Carbon Sensing and Conidial
Germination in Aspergillus
nidulans and Botrytis cinerea ..... 437
Gpa1 and Detection of Glucose in
Cryptococcus neoformans ......... 437
Methionine-Regulated System
in Cryptococcus neoformans....... 438
Carbon-Sensing Pathway in
Neurospora crassa ............... 438
DEVELOPMENT............... 438
Ascomycete Fungi ................ 439
Basidiomycete Fungi ............. 440
PATHOGENESIS .................. 441
Plant Pathogens .................. 441
Human Pathogens................ 442
Mycotoxin Biosynthesis ........... 443
FUTURE DIRECTIONS........... 444
Fungi are crown eukaryotes that originated
approximately 1 billion years ago. It is esti-
mated that 1.5 million species of fungi may
exist in nature and that most of these are fil-
amentous (38). Filamentous fungi are multi-
cellular organisms that grow through elabo-
ration of tube-like structures termed hyphae
(13). Filamentous fungi are critical for nu-
trient recycling in the biosphere and provide
food, antibiotics, and other substances for hu-
man use. Filamentous fungi are the most dam-
aging pathogens of plants (1). Many fungi,
including both yeast and filamentous species,
are also important human pathogens, partic-
ularly in immunocompromised patients (66).
Numerous contributions to our knowledge of
genetics, metabolism, cell biology, and mul-
ticellular development have been made us-
ing filamentous fungi. For example, the semi-
nal one-gene one-polypeptide hypothesis that
gave the Nobel Prize to George Beadle and
Edward Tatum was proven using the geneti-
cally and biochemically tractable filamentous
fungus Neurospora crassa (6).
Heterotrimeric G proteins (G proteins)
were first cloned in filamentous fungi in the
early 1990s (114). G proteins had previously
been characterized in several eukaryotic sys-
tems, most notably the rhodopsin-transducin
visual system in mammals (128) and more re-
cently for their role during the pheromone re-
sponse in the yeast fungus Saccharomyces cere-
visiae (5). However, possible functions for G
proteins in filamentous fungi were unknown
largely because of the absence of sequence in-
formation or characterized G protein subunit
mutants in these species.
G proteins have apparently universal roles
as signaling proteins in eukaryotes. Each G
protein heterotrimer is composed of α, β, and
γ subunits that are associated with the plasma
membrane (85) (Figure 1). The Gα subunit
binds GTP and GDP and hydrolyzes GTP
to GDP, and the Gβ and Gγ subunits form a
dimer. In the inactive state, the three subunits
are present in a complex, in association with a
G protein–coupled receptor (GPCR). Ligand
binding to the GPCR leads to exchange
of GTP for GDP on the Gα protein and
dissociation of the Gα and Gβγ dimer. Both
the Gα-GTP and Gβγ moieties regulate
downstream effector proteins in various
systems, including ion channels, adenylyl
cyclases, phosphodiesterases, and phospholi-
pases (85). GTP hydrolysis on the Gα subunit
allows the GDP-bound Gα to reassociate
with the Gβγ dimer and the GPCR at the
membrane, ready to reinitiate the signaling
424 Li et al.
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Figure 1
The G protein cycle. Ligand binding to the G
protein–coupled receptor (GPCR) leads to
GDP-GTP exchange on the Gα protein and
dissociation of the Gα and Gβγ dimer. Both
Gα-GTP and Gβγ may regulate downstream
effectors. GTP hydrolysis by the Gα subunit
results in reassociation of GDP-bound Gα with
the Gβγ dimer and the GPCR, thus completing
the cycle. RGS (regulator of G protein signaling)
proteins accelerate the rate of GTP hydrolysis by
Gα proteins (blue arrow).
This review provides examples of several
G protein signaling pathways in filamentous
fungi. Because of space limitations, we are un-
able to reference all the work in this rapidly
expanding field. We focus on three phyla for
which significant information is available: As-
comycota, Basidiomycota, and Zygomycota
(35) (Table 1). The Ascomycota include ap-
proximately 75% of known fungal species
(35). Ascomycete fungi are characterized by
production of an ascus (sac) that encloses
the sexual spores (ascospores) produced af-
ter meiosis (35). This phylum contains the
model filamentous organisms N. crassa, As-
pergillus nidulans, and Magnaporthe grisea,as
Yeast: species in the
Kingdom Fungi that
can grow and divide
as single-celled
G protein: protein
that binds guanine
nucleotides (GTP
and GDP) and can
hydrolyze GTP to
GDP and inorganic
G protein–coupled
receptor (GPCR):
a protein with 7-TM
helices, with the N
terminus external to
the cell and the C
terminus extending
into the cytoplasm
that interacts with
heterotrimeric G
Ascomycetes: one
division in the
Kingdom Fungi so
named because of
the presence of an
ascus (sac) enclosing
the meiotic products
RGS: regulator of G
protein signaling
well as the well-studied yeasts S. cerevisiae and
Schizosaccharomyces pombe. Genome sequences
are available for numerous filamentous as-
comycetes, and many of these species, espe-
cially N. crassa and A. nidulans, are particularly
amenable to genetic and molecular analysis.
The Basidiomycota comprise 25% of known
fungal species (35). Basidiomycete fungi pro-
duce basidia, specialized hyphal tips that are
the site of nuclear fusion and meiosis af-
ter mating. The sexual spores, basidiospores,
are displayed on the outside of the basidia.
Basidiomycete fungi include both mushroom
(e.g., Coprinus cinereus and Schizophyllum com-
mune) and non-mushroom (e.g., Ustilago may-
dis and Cryptococcus neoformans) members. Sev-
eral Basidiomycete genome sequences have
been completed and this phylum contains
many species that have been used for both
classical and molecular-genetic investigations
of mating and sporulation. Zygomycota con-
tain species that differentiate asexual spores in
structures called sporangia or that form ben-
eficial associations with plant roots (1, 35).
Currently, there are fewer genome sequences
available for this phylum than for the Ascomy-
cota and Basidiomycota. However, this is ex-
pected to change in the future, as transforma-
tion and other molecular techniques continue
to be developed for Zygomycete species. For
treatment of G protein signal transduction
pathways in the yeasts S. cerevisiae, Candida
albicans, and S. pombe, the reader is directed to
several excellent reviews (5, 41, 69).
Gα Proteins
In contrast to yeasts, which contain two
Gα proteins, most characterized filamentous
fungi possess three Gα proteins that are mem-
bers of distinct groups (10, 53) (Table 2).
Group I Gα proteins are highly conserved in
most filamentous fungi, and accumulating ev-
idence suggests that individual Group I Gα
proteins regulate multiple pathways. The first
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Table 1 Taxonomic classification of species covered in this review
Phylum Class Representative species
(Subphylum Pezizomycotina)
Sordariomycetes Neurospora crassa
Chaetomium globosum
Cryphonectria parasitica
Magnaporthe grisea
Fusarium graminearum (Gibberella zeae)
Fusarium oxysporum
Colletotrichum trifolii
Rosellinia necatrix
Sporothrix schenckii
Eurotiomycetes Aspergillus fumigatus
Aspergillus nidulans
Aspergillus terreus
Coccidioides immitis
Histoplasma capsulatum (Ajellomyces capsulatus)
Paracoccidioides brasiliensis
Penicillium marneffei
Uncinocarpus reesii
Leotiomycetes Botrytis cinerea (Botryotinia fuckeliana)
Sclerotinia sclerotiorum
Dothideomycetes Phaeosphaeria nodorum (Stagonospora nodorum)
Alternaria alternata
Cochliobolus heterostrophus
Homobasidiomycetes Coprinus cinereus
Coprinus congregatus (Coprinellus congregatus)
Lentinula edodes (Lentinus edodes)
Schizophyllum commune
Heterobasidiomycetes Cryptococcus neoformans
Ustilaginomycetes Ustilago maydis
Zygomycota/Zygomycetes Zygomycetes Rhizopus oryzae
one division in the
Kingdom Fungi that
is characterized by
production of basidia
Group I Gα identified in filamentous fungi
was N. crassa GNA-1, which is 55% identi-
cal to mammalian Gα
superfamily proteins
(114). GNA-1 contains a consensus sequence
for myristoylation (MGXXXS) at the amino
terminus (14) and a site for ADP-ribosylation
by pertussis toxin (CAAX) at the carboxy ter-
minus (123) that is conserved in Gα
family proteins. Most of the characterized
filamentous fungi possess a single Group I
Gα protein (53); however, the Rhizopus oryzae
genome contains four (Table 2).
Group II Gα proteins are not as well con-
served as Group I or III Gα proteins (53)
(Table 2). Of the sequenced yeasts, only S.
pombe contains a Gα protein (Gpa1) that is
similar to Group II subunits (53). The func-
tions of Group II Gα proteins are less ob-
vious than those of Group I and III in most
organisms. For example, deletion of magC in
M. grisea leads to reduced conidiation (74) and
Botrytis cinerea bcg-2 mutants have slightly re-
duced pathogenicity (36). In N. crassa, GNA-2
was originally thought to be compensatory to
GNA-1 and GNA-3 (a Group III Gα) (4, 53),
but recent work has demonstrated a function
in mass accumulation on poor carbon sources
for this Gα protein (70). The Group III Gα
proteins are highly conserved and most pos-
sess a myristolyation sequence at the amino
terminus (53) (Table 2). Group III has been
designated the Gα
-analogous group because
426 Li et al.
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many Group III proteins positively influence
cAMP levels (10). However, the Group III
Gα GNA-3 from N. crassa is more simi-
lar to human Gα
(49%) than to Gα
teins, suggesting a functional but not close
evolutionary relationship with Gα
. In addi-
tion, various Group III subunits, including N.
crassa GNA-3, possess both cAMP-dependent
and cAMP-independent functions. Group III
proteins have been well studied in yeasts,
e.g., S. cerevisiae Gpa2p and S. pombe Gpa2
Gβ and Gγ Subunits
Gβ proteins from filamentous fungi are
highly conserved (66–92% identity)
(Table 3), but they share significantly
lower homology with those from the yeasts S.
cerevisiae and S. pombe (38% and 45% identity,
respectively) (127). Most fungi have only a
single predicted Gβ protein, although the R.
oryzae genome may have four Gβ subunits
(Table 3). Mutational inactivation of Gβ
genes leads to numerous defects in the sexual
and asexual life cycles of filamentous fungi.
Results from studies in Cryphonectria parasitica
and N. crassa suggest that the Gβ is essential
for complex formation and/or stability of Gα
and Gγ proteins in filamentous fungi (50, 61,
Gγ subunits form a large family of small
proteins containing 68 to 110 amino acids
(27). In contrast to mammals, most charac-
terized filamentous fungi have a single Gγ
protein that is conserved (39–92% identity)
(61) (Table 3). To date, only C. neoformans,
C. cinereus, Podospora anserina, and R. oryzae
possess more than one Gγ subunit in their
genome (2–5 genes) (90) (Table 3). Deletion
of the N. crassa Gγ gene gng-1 leads to phe-
notypes identical to those observed in gnb-1
mutants (55, 127).
Accumulating evidence suggests that
proteins other than canonical Gβ and Gγ
subunits can form complexes with G protein
subunits to regulate signal transduction from
receptors in fungi. A Gβ-like RACK1 (re-
cAMP: cyclic AMP
ceptor for activated C-kinase 1) homologue,
Gib2, interacts with Gpa1 (Group III Gα),
Gpg1 (Gγ), and Gpg2 (Gγ) to regulate
cAMP signaling in C. neoformans (90). Gib2
also binds to downstream targets of the Gpa1-
cAMP signaling pathway (90). RACK1-like
proteins are conserved in filamentous fungi (S.
Krystofova & K.A. Borkovich, unpublished
data), and the N. crassa RACK1-like/cpc-2
gene (cross pathway control-2; NCU05810)
encodes a protein involved in regulation of
amino acid biosynthetic genes and formation
of female reproductive structures (83).
In S. pombe, the Gβ-like protein Gnr1p
interacts with the Gpa1p Gα and functions
as a negative regulator of the pheromone-
response pathway (34). Several filamentous
fungal genomes contain a protein with
24–38% identity to Gnr1p (S. Krystofova &
K.A. Borkovich, unpublished data). In con-
trast, filamentous fungi do not possess homo-
logues of the kelch repeat Gβ mimic proteins
Gbp1p and Gpb2p, or the Gγ mimic Gpg1p,
that interact with the Gpa2p Gα protein in S.
cerevisiae (13, 64).
G Protein–Coupled Receptors
GPCRs are plasma-membrane-localized
proteins that communicate changes in the
environment to intracellular heterotrimeric
G proteins. GPCRs contain seven trans-
membrane (7-TM) helices connected by
intracellular and extracellular loops, with an
extracellular amino terminus and the carboxy
terminus extending into the cytoplasm. The
GPCR family is the largest TM receptor
group, with more than 600 members in the
human genome (117). Ten GPCRs were
predicted during the initial annotation of the
N. crassa genome sequence (the first available
for a filamentous fungus) and placed into five
classes (13, 31) (Table 4) (see below). Addi-
tional predicted GPCRs that were reported
subsequent to the release and annotation
of recent genome sequences are listed in
Table 5. Some proteins with similarity to
GPCRs do not possess 7-TM helices; in some
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Table 2 Gα protein subunits in several filamentous fungi
Species Class Name Broad #
% Identity
Species Class Name Broad #
% Identity
I GNA-1 06493.3 XP 957133 100 Rhizopus
I 00875.1 73
II GNA-2 06729.3 Q05424 100 I 01120.1 64
III GNA-3 05206.3 XP 962205 100 I 09475.1 62
I FadA 0651.3 XP 658255 93 I 00005.1 66
II GanA 3090.3 XP 660694 52 Rosellinia
I RGA2 BAB20820 96
III GanB 1016.3 XP 658620 78 II RGA3 BAB20821 78
Botrytis cinerea
I BCG1 01681.1 CAC19871 79
III RGA1 BAB20819 82
II BCG2 08985.1 CAC19872 68 Sclerotinia
I 12343.1 96
III BCG3 03006.1 BAD93277 72
II 10286.1 68
I 03321.1 XP 001229837 97 III SPG1 07597.1 72
II 07125.1 XP 001224781 82 Stagonospora
I GAP1 10086.1 AAQ94737 94
III 03816.1 XP 001223030 84 II 10733.1 EAT82127 58
I 06818.2 XP 001242922 93 III 13299.1 EAT79183 53
II 09597.2 XP 001239976 52 Sporothrix
I SSG-1 O74259 96
III 05912.2 XP 001242016 77 II SSG-2 AAR37394 81
Cochliobolus I CGA1 AAC23576 94 III SSG-3 AAL57853 85
heterostrophus Trichoderma
I Tga1 AAK74191 96
I CPG-1 Q00580 98 III Tga3 AAM69919 85
II CPG-3 AAM14395 79
III CPG-2 AAA67707 86
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Colletotrichum I Ctg1 O42784 97 Uncinocarpus
I 03656.1 73
trifolii II 04559.1 51
(Gibberella zeae)
I GBA1 05535.1 XP 385711 97 III 04434.1 76
II 09988.1 XP 390164 74 Ustilago maydis I Gpa1 05123.1 XP 761270 69
III GBA3 09614.1 XP 389790 85 II Gpa2 02517.1 XP 758664 51
I 00742.1 91 III Gpa3 04474.1 XP 760621 68
II 04995.1 51 IV
Gpa4 05385.1 XP 761532
I MAGB 00365.5 XP 368879 98 Cryptococcus
I Cga1 00179.1 AAQ62550 62
II MAGC 04204.5 AAB65427 50 II Gpa3 02090.1 AAQ74379 46
III MAGA 01818.5 XP 363892 86 III Gpa1 04505.1 XP 572793 54
I GPA1 AAT40562 91 Schizophyllum
I ScGP-B BAB18736 71
II GPA2 AAT40564 52 III ScGP-C BAB18737 63
III GPA3 AAT40563 78 III SCGPα2 AAG27721 63
I GasA AAM64110 92 III ScGP-A BAB78537 65
II GasB AAQ24336 56 III SCGPα1 AAD45318 63
III GasC AAO41857 77 Coprinus
I 09275 EAU90798 46
Podospora anserina I Pa 7 7970
98 II 02876 EAU93646 43
II Pa 2 10260
81 Coprinus I CGP1 P30675 67
III MOD-D AAC24766 86 congregatus
I Ga1 AAP13579 68
Broad #, Broad Institute accession number; NCBI #, National Center for Biotechnology Information accession number.
% identity based on amino acid sequence.
Broad and NCBI sequences differ; % identity is based on the Broad sequence.
Sequence information from the Podospora sequencing project (
Novel Gα, does not belong to group I-III (99).
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Table 3 Gβ and Gγ subunits in filamentous fungal species
Gβ Gγ
Species Name Broad #
Name Broad #
Neurospora crassa GNB-1 NCU00440.3 AAM53552 100 GNG-1 NCU000041.3 542AAV83 100
Aspergillus nidulans SfaD AN0081.3 EAA65259 86 GpgA AN2742.3 EAA63176 65
Botrytis cinerea BC1G 15399.1 AL114303 87
CHGG03114.1 EAQ91179 91 CHGG05752.1 EAQ89133 87
Coccidioides immitis CIMG 09237.2 79
Cgb1 AA025585 76
Coprinus cinereus CC1G 00488.1 EAU92269 72 CC1G 01215.1
CPGB1 AAC49838 91 CB688576 92
GPB1 CNAG 01262 AAW43317 67 Gpg1 CNAG 05889.1 54
Gpg2 CNAG 05890.1 63
(Gibberella zeae)
FG04104.1 EAA72306 80 FG07235.1 EAA77833 90
HCAG 02608.1 76 HCAG 03570.1 44
Magnaporthe grisea MGB1 MGG 05201.5 MGG 10193.5 ABD14415 86
ABH09720 81 ABH09719 67
Podospora anserina Pa 7 6570
85 Pa 7 9770
Pa 7 9800
Rhizopus oryzae RO3G 06400.1 70 RO3G 05661.1 45
RO3G 00046.1 66 RO3G 02502.1 46
RO3G 06062.1 71 RO3G 06343.1 47
RO3G 08023.1 66 RO3G 01472.1 43
RO3G 02606.1 39
SS1G 03482.1 86 SS1G 12567.1 69
Sporothrix schenckii AF484341 92
SNOG 16044.1 72 SNOG 00288 58
CF875833 86
AA091808 92
Lentinula edodes AAT74567 72 Gpg1 Q870G5 52
Ustilago maydis Bpp1 UM00703.1 AAN33051 71 UM06109.1 EAK86991 55
Broad #, Broad Institute accession number; NCBI #, National Center for Biotechnology Information accession number.
% identity based on amino acid sequence.
Information was gathered from the Podospora sequencing project:
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cases this appears to result from annotation
or sequence errors (Table 4 and Table 5).
Pheromone receptors. S. cerevisiae Ste2p
and Ste3p were the first pheromone recep-
tors (and GPCRs) characterized in fungi (26).
Ste2-like and Ste3-like pheromone receptors
are nearly universally present in ascomycete
filamentous fungi (Table 4). In contrast, ba-
sidiomycete pheromone receptors are all of
the Ste3-like type (15) (Table 4). No clear ho-
mologue of either pheromone receptor could
be identified in the annotated genome of the
Zygomycete R. oryzae (Table 4).
cAMP receptor-like GPCRs. The cAMP
receptor-like (CRL) class of GPCRs is not
present in the yeasts S. cerevisiae and S.
pombe. The number of CRLs varies in fil-
amentous fungal species (Table 4). CRLs
are distantly related to the four cAMP re-
ceptors (cAR1-cAR4) (58) and three cAMP
receptor-like proteins (CrlA-CrlC) (93) from
Dictyostelium discoideum, as well as Arabidopsis
thaliana GCR1 (91). N. crassa GPR-1 was the
first CRL GPCR characterized in ascomycete
fungi (62).
Carbon-sensing proteins. S. cerevisiae
Gpr1p was the first fungal GPCR demon-
strated to have a carbon-sensing function (68,
75), and N. crassa GPR-4 was the first such
protein characterized in filamentous fungi
(70). Homologues of Gpr1p and GPR-4 are
present in the genomes of most filamentous
fungi (Table 4).
Stm1-related proteins. N. crassa GPR-5
and GPR-6 (13) are homologous to Stm1, a
predicted GPCR involved in sensing nitro-
gen availability, possibly through coupling to
the Gα Gpa2 in S. pombe (19). This class of
putative GPCRs, all containing PQ loop re-
peats, is well conserved and widely present in
filamentous fungi, including all the annotated
genomes from Zygomycetes, Ascomycetes,
and Basidiomycetes (Table 4). No functional
receptor-like protein
characterization of a protein from this group
has been reported in filamentous fungi.
Microbial opsins. The microbial opsin class
of GPCRs, with similarity to archaeal opsins,
is represented by the NOP-1 opsin and an
opsin-related protein, ORP-1, in N. crassa.
NOP-1 was the first opsin identified in eu-
karyotic microbes and has been demonstrated
to bind all-trans retinal in vitro and may
have signaling functions in vivo (7, 8). No
proteins similar to microbial opsins were
identified in the annotated genomes of the
Basidiomycete C. cinereus and the Zygomycete
R. oryzae (Table 4). To date, there is no data
linking microbial opsins to heterotrimeric G
PTH11, mPR, and rat growth hormone
releasing factor-related proteins. Subse-
quent to release of the M. grisea genome,
Kulkarni et al. (63) annotated three new
classes of putative GPCRs in filamentous
fungi. The PTH11-related group exhibits
similarity to M. grisea PTH11, previously im-
plicated in pathogenesis (24, 63). The second
class includes proteins related to the Homo
sapiens mPR steroid receptor. The third group
was represented by a single protein with weak
homology to rat growth hormone releasing
factor (MG00532). No PTH11-related 7-
TM proteins are present in the current an-
notated genomes of the Basidiomycetes U.
maydis and C. neoformans, as reported previ-
ously (63). Homologues of H. sapiens mPR-
like proteins are present in both Ascomycetes
and Basidiomycetes (Table 5). However, no
protein similar to MG00532 is present in the
two Basidiomycetes examined (Table 5). Fun-
gal mPR and MG00532 homologues have not
been functionally characterized in any fila-
mentous species.
AtRGS1 homologues. Lafon et al. (64)
identified a unique 7-TM protein (GprK)
with an RGS (regulator of G protein sig-
naling) domain in the cytoplasmic region in
Aspergillus sp. GprK is similar to A. thaliana
Heterotrimeric G Protein Signaling in Filamentous Fungi 431
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Table 4 Five originally annotated GPCR groups in filamentous fungi
Species Pheromone receptors cAMP receptor-like proteins Carbon sensors
Putative nitrogen
Microbial opsins
PRE-1 PRE-2 GPR-1, GPR-2, GPR-3 GPR-4 GPR-5, GPR-6 NOP-1, ORP-1
Afu5g07880 Afu3g14330 Afu3g01750, Afu5g04140,
(GprC), AN8262 AN5720, AN10166 AN3361
ATEG 08338 ATEG 03500 ATEG 8000 ATEG 04369
ATEG 08003,
ATEG 08691 (5),
Botrytis cinerea BC1G 07387 BCIG 13582 BC1G 02359, BC1G 04448,
BC1G 03450
BC1G 02874
BC1G 02456,
CHG05819 CHGG 08469 CHG01216, CHG07294,
CHG04030, CHG05145,
CHGG 04337 CHGG 03233,
CHGG 10008
immitis RS
CIMG 06159
CIMG 00128
CIMG 00505 CIMG 05768
CIMG 06648,
CIMG 08103
CC1G 02137
CC1G 02136
CC1G 02129
CC1G 02288
, CC1G 02310
CC1G 07132 (6)
Serotype A
CNAG 06808
CNAG 01855 (6)
03846 (6)
CNAG 03572
FG07270 FG02655 FG09693, FG05239, FG07716,
FG01861, FG03023
FG05579, FG08496 FG07554,
432 Li et al.
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HCAG 02974 HCAG 01152 HCAG 0212 HCAG 05534 (6),
01299 (4)
HCAG 07963
MGG 06452 MGG 04711 MGG 11962, MGG 06257,
MGG 08803,
MGG 04698,
MGG 09015
Rhizopus oryzae RO3G 03874,
15181 (5),
13115 (4),
13187 (4),
10064 (4)
SS1G 10310 SS1G 08113, SS1G 09369,
SS1G 08243
SS1G 07709,
SS1G 01614,
SNOG 09060 SNOG 10426 SNOG 00659 SNOG 14046 (4)
SNOG 00993
SNOG 11116 (6),
SNOG 00807
UREG 04230
UREG 00233 UREG 04530
UREG 04530
UREG 03789 (4TMs),
01945 (4)
UREG 02372
Ustilago maydis UM02383 UM03423
UM06006, UM01546 UM00371 (6),
CNC03390 (6),
CNB03360, CNB02600
BLASTp was performed on the annotated genomes of filamentous fungi at the Fungal Genome Initiative ( or TIGR
( databases using the indicated N. crassa protein as the query. All proteins have 7-TMs with the amino terminus outside and the carboxyl terminus on the
cytoplasmic side as verified by Hmtmm (, Tmpred (
form.html), Phobius
(, and Toppred (; except as noted in parentheses (number of predicted TMs). Deviations from
7-TM structure may be due to misannotation or sequencing errors. The cutoff E value is e-09 except for those that are footnoted as
(see below). –, no hit at cutoff E value of 1e-03.
E-value is between 1e-09 and 1e-03.
Intron has been misannotated in BC1G 02874.1, CIMG 04019.2, and UM03423.1.
Low number of TMs may result from sequencing errors or a large number of gapped nucleotides.
For intron correction, see Reference 70.
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Table 5 Additional predicted GPCRs in filamentous fungi
Species Homologues of Magnaporthe grisea PTH11
Neurospora crassa NCU06531, NCU00700, NCU08624, NCU05854, NCU07649, NCU07591,
NCU09201, NCU07538, NCU05189, NCU05307, NCU08447, NCU02903,
NCU09823, NCU09796, NCU04106, NCU07769, NCU04931, NCU08718,
NCU09022, NCU05101, NCU08431, NCU08429, NCU06891, NCU05187.1,
Magnaporthe grisea MG05871, MG10473, MG07553, MG06755, MG09022, MG07565, MG07946,
MG11006, MG09070, MG07806, MG03584, MG05214, MG09863, MG10407,
MG10571, MG01867, MG09455, MG10050, MG05352, MG07420, MG10442,
MG02160, MG02001, MG10257, MG01905, MG07987, MG10438, MG06171,
MG07851, MG04935, MG05386, MG09865, MG05514, MG06535, MG01190,
MG10581, MG03009, MG10747, MG03935, MG04682, MG09416, MG02692,
MG07857, MG00826, MG06624, MG00435, MG08653, MG10706, MG04170,
MG08525, MG00277, MG02365, MG06595, MG06084, MG09437, MG01890,
MG01871, MG03794, MG09667, MG09061
Aspergillus nidulans AN5639, AN1930, AN2587, AN7774, AN0178 (4), AN7232, AN7395, AN5059,
AN2249, AN9444, AN9036, AN5664, AN11159, AN8328 (4), AN1738, AN3395 (5),
AN6415 (6), AN8951, AN1540 (5), AN9306, AN8971, AN10886, AN10369 (5),
AN2386 (6), AN10357, AN7400, AN4452 (5), AN6413, AN8943, AN4378 (16),
AN2575 (5), AN5069, AN5312, AN2683 (6), AN9387 (5), AN0011 (5), AN4213 (4),
AN7270 (5), AN7523 (6), AN9266 (4), AN1557 (4), AN3349 (8), AN2649, AN2726
(6), AN0171, AN3257, AN5943, AN7406 (3), AN3241, AN2044, AN3348, AN3886
(5), AN8548, AN11079 (3), AN4642, AN8512, AN8984, AN0857, AN8151,
AN6924, AN6946 (5), AN2108, AN2389 (3), AN8661, AN1317 (5), AN8727,
AN0500 (5), AN0751, AN6622, AN6458 (6)
Fusarium graminearum FG03707, FG02155, FG05821, FG03897 (6), FG07839, FG00994, FG08408,
FG04529, FG11381, FG07663, FG04731, FG02374, FG05039, FG03464, FG07757,
FG03005, FG04825 (5), FG04159, FG08189, FG06541, FG03800, FG02614,
FG07136, FG03237, FG11256, FG00101 (6), FG07489, FG10028 (17), FG03504,
FG03268, FG09370, FG07655 (4), FG03932 (6), FG10085 (6), FG11343, FG03688,
FG01989 (6), FG07601, FG11161 (6), FG03164, FG03501, FG00057 (11), FG04693
(5), FG03151 (6), FG03409, FG11529, FG03009 (5), FG09352, FG07841, FG02134,
FG05722, FG10630 (5), FG04749 (6), FG07792, FG02981, FG02417, FG05793,
FG05754, FG03588, FG04664, FG07994, FG02401, FG03959, FG03823,
FG11598, FG01833, FG03277, FG11385, FG03336 (6), FG00966, FG10946,
FG02853, FG03200, FG02569, FG01806, FG04500, FG04865, FG11390 (19),
FG03561 (6), FG03215, FG11351 (6), FG11080 (5), FG03091, FG00201, FG10983,
FG02334 (6), FG03192, FG03962, FG04815, FG10958, FG02844, FG03310,
FG08222, FG10023, FG02907, FG06439, FG02274, FG02402, FG02818 (5),
FG03104, FG03476, FG03377 (5), FG09749, FG02405, FG06440, FG01994
Cryptococcus neoformans Serotype A CNAG 06323
Ustilago maydis
Homologues of Homo sapiens mPR-like GPCRs
N. crassa NCU04987 (6), NCU03238
M. grisea MG05072, MG09091, MG04679
A. nidulans AN4932, AN10630, AN5151
F. graminearum FG04051 (6)
, FG01064
C. neoformans Serotype A CNAG 05370, CNAG 05344
U. maydis UM01737 (10), UM00006
(Continued )
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Table 5 (Continued )
Species M. grisea MG00532-like GPCR
N. crassa NCU03253 (5)
M. grisea MG00532
A. nidulans AN6680, AN5508, AN3567
F. graminearum FG00527 (5)
C. neoformans Serotype A
U. maydis
Aspergillus GprK-like GPCRs
N. crassa NCU09883
M. grisea MGG 13926, MGG 11693 (6)
A. nidulans AN7795
F. graminearum FG04628
C. neoformans Serotype A
U. maydis
Modified from Reference 63. The sequences are from the Fungal Genome Initiative ( The proteins
are listed as the best hits using the M. grisea/N. crassa protein or AN7795/ NCU09883 in BLASTp (E-value cutoff: 1e-09). TM analysis was as in
Table 4 . If the number of predicted TMs is not seven, the actual number is indicated in the parentheses.
Reference 63.
Reference 64.
The presence of 17 predicted transmembrane helices may result from misannotation.
The absence of a seventh TM helix at the carboxy terminus of FG04051 may due to a sequencing or annotation error.
7-TM low probability.
AtRGS1, which negatively regulates the Gpa1
Gα subunit and associated cellular prolif-
eration (17). GprK homologues, containing
both 7-TMs and a RGS domain (see be-
low), are present in filamentous Ascomycetes,
but not in the two Basidiomycetes (64)
(Table 5).
Regulatory Proteins
RGS proteins act as GTPase-activating pro-
teins to negatively control Gα protein signal-
ing (Figure 1). RGS proteins accelerate the
GTPase activity of Gα proteins by binding
to the transition state during GTP hydrolysis
(98). All RGS proteins contain a 130-residue
motif (RGS box) important for Gα interac-
tion and may also possess additional domains,
such as Gβ binding, membrane targeting, Ras
binding, and GoLoco sequences. RGS pro-
teins also bind and phosphorylate a GPCR
in order to deactivate the signal (79). In the
yeast S. cerevisiae, the RGS proteins Sst1p and
Rgs2p regulate the two Gα proteins, Gpa1p
and Gpa2p, respectively (40).
A. nidulans contains four RGS proteins
(37) and A. nidulans FlbA was the first
RGS protein characterized in filamentous
fungi (67). FlbA promotes asexual sporulation
through negative regulation of the Group I
Gα subunit FadA (129). A second A. nidulans
RGS, RgsA, modulates colony growth, aerial
hyphae, and pigment formation by negatively
regulating the Group III Gα subunit GanB
(37). Two additional RGS proteins, RgsB and
RgsC, have been identified but not charac-
terized in A. nidulans (37). RGS proteins also
regulate G protein–regulated processes in
C. parasitica (101), C. neoformans (118), and
S. commune (29).
Phosducin and phosducin-like proteins
(PhLPs) participate in G protein signaling
through regulation of Gβγ subunits. It was
originally postulated that PhLPs block the
ability of Gβγ to interact with Gα and effec-
tors by binding to the Gβγ dimer (99). This
Heterotrimeric G Protein Signaling in Filamentous Fungi 435
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protein kinase
model was in opposition to work in the fila-
mentous fungus C. parasitica, which showed
that a PhLP acts as a positive regulator of
Gβγ subunit function (51). Similar findings
have been reported for a PhLP (PhnA) from
A. nidulans (102). However, results from sev-
eral recent studies in mammals now support
the results in filamentous fungi by showing
that PhLPs act as chaperones during Gβγ
dimer formation by stabilizing the nascent Gβ
polypeptide until it can associate with the Gγ
(76, 77).
Output Pathways
Accumulating evidence implicates cAMP-
dependent and mitogen-activated protein ki-
nase (MAPK) signaling cascades downstream
of G proteins in filamentous fungi (Figure 2).
cAMP produced by adenylyl cyclase can bind
CnGpr4 NcGPR-4
Amino acids
Levels of
Gα proteins
Carbon sources
Gα Ι
Pathogenesis Mating
Peptide pheromones Ligand
Gα Ι
Pheromone sensing/mating
Fruiting body formation
Figure 2
Nutrient-sensing and pheromone response/mating pathways. (a) G protein–mediated nutrient sensing.
AC catalyzes the production of cAMP from ATP. Binding of cAMP to PKA-R results in release of
PKA-C, which can then phosphorylate downstream protein targets. In N. crassa, the GPR-4 GPCR
(NcGPR-4) senses carbon sources by coupling to the Group I Gα GNA-1, which operates upstream of
the cAMP signaling pathway to regulate growth and asexual development. In other fungi, Group III Gα
proteins, including A. nidulans GanB and C. neoformans Gpa1, are implicated in carbon sensing and
regulation of cAMP signaling; however, the GPCR has not been identified (see text for details). The
amino acid–sensing GPCR Gpr4 in C. neoformans (CnGpr4) functions upstream of the Group III Gα
Gpa1 and cAMP signaling to regulate growth, asexual development, and mating. (b) Pheromone response
and mating. Peptide pheromones bind to pheromone receptors (GPCRs) at the PM. The number of
pheromone receptors varies from one to three in filamentous fungi, while the number of pheromone
precursor genes can be as much as seven. In various systems, Group I Gα proteins and/or the Gβ subunit
function downstream of pheromone receptors. These subunits are thought to activate a MAPK cascade,
leading to expression of genes required for mating. Group III Gα subunits modulate mating through
control of cAMP levels and associated regulated expression of genes involved in mating. Other GPCR
types, such as N. crassa GPR-1 (a cAMP receptor-like GPCR), also regulate mating and fruiting body
formation. Abbreviations: AC, adenylyl cyclase; Gα I/II/III, Group I/II/III Gα; PKA-C, PKA control
subunit; PKA-R, PKA regulatory subunit; PM, plasma membrane.
436 Li et al.
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to the regulatory subunit of cAMP-dependent
protein kinase (PKA, or protein kinase A),
leading to activation of the catalytic PKA sub-
unit and phosphorylation of downstream tar-
gets (69). Mutation of Group I or III Gα,Gβ,
or Gγ genes can lead to changes in the intra-
cellular cAMP level and addition of exogenous
cAMP often rescues defects observed in these
mutants (69, 71, 84, 86, 127), suggesting that
the Gα and/or Gβγ dimer can mediate sig-
nal transfer to the cAMP pathway. However,
as mentioned above, effects due to loss of the
Gβγ dimer may result from lower levels of
Gα proteins.
MAPK cascades consist of three kinases
that are sequentially phosphorylated, from
MAPK kinase kinase, to MAPK kinase, to the
terminal MAPK. The activated MAPK phos-
phorylates various cellular proteins, including
transcription factors and other regulatory
proteins (5). Annotation of filamentous
fungal genomes has revealed the existence
of three major MAPK cascades containing
components homologous to those used for
the pheromone response, osmosensing, and
cell integrity in S. cerevisiae (13). Results
from genetic analyses in filamentous fungi
suggest that Gβ proteins can transfer signals
to the MAPK pathway most similar to the
S. cerevisiae pheromone response cascade in
C. neoformans (119) and M. grisea (86).
Nutrients supply energy and provide sub-
strates for biosynthesis and catabolism, and
they also have important regulatory effects
in fungi (42). Evidence from multiple species
supports regulation of a downstream cAMP
signaling pathway by the GPCR-Gα during
nutrient sensing in both filamentous fungi
and yeasts (Figure 2a) (see below). In S.
cerevisiae, Gpr1p, Gpa2p, and a downstream
cAMP pathway are involved in sensing glu-
cose and sucrose (agonists) and mannose (an-
tagonist) (68, 75). In S. pombe and C. albi-
cans, Gpr1p-homologous GPCRs participate
in glucose (both species) and amino acid (C.
PKA: protein kinase
A or cAMP-
dependent protein
albicans) sensing in a cAMP-dependent man-
ner (78, 82, 121). Below, we discuss several
nutrient-sensing pathways mediated by het-
erotrimeric G proteins in filamentous fungi.
Carbon Sensing and Conidial
Germination in Aspergillus nidulans
and Botrytis cinerea
Many species of filamentous fungi produce
asexual spores, known as conidia, that germi-
nate in the presence of water and adequate
nutrition. The requirement for an external
carbon source to induce spore germination
in A. nidulans and B. cinerea has been demon-
strated (23, 25). A. nidulans GanB is a Group
III Gα that controls a rapid and transient
cAMP increase in response to glucose during
early germination (65). Mutation of the G
protein subunit genes ganB (Gα), sfaD (Gβ),
or gpgA (Gγ) led to altered germination kinet-
ics and defects in trehalose mobilization (65);
the latter phenomenon has been previously
linked to cAMP signaling during early coni-
dial germination (28). The observation that
germination of ganB conidia is impaired in
the presence of glycerol, ethanol, galactose,
and fructose (65) indicates that GanB may
sense additional carbon sources in A. nidulans.
Induction of conidial germination by carbon
sources in B. cinerea requires the Group III
Gα protein BCG3 and the adenylyl cyclase
BAC, suggesting that a BCG3/BAC/cAMP
pathway is required for carbon sensing during
conidial germination in B. cinerea (25). In
contrast, although the Group III Gα GasC is
crucial for conidial germination in Penicillium
marneffei, this protein is not involved in
carbon or nitrogen sensing (130).
Gpa1 and Detection of Glucose in
Cryptococcus neoformans
Mutation of the gpa1 (Group III Gα)orcac1
(adenylyl cyclase) gene blocks synthesis of the
virulence factor melanin in response to glu-
cose starvation in C. neoformans, and cAMP
supplementation restores melanin production
Heterotrimeric G Protein Signaling in Filamentous Fungi 437
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in both mutants (3). After addition of glu-
cose to glucose-starved cells, wild-type, but
not gpa-1, strains exhibit a transient increase
in cAMP levels, suggesting that Gpa1 medi-
ates cAMP production in response to glucose
(3, 124). These results are consistent with
Gpa1 functioning in a glucose-sensing path-
way upstream of cAMP signaling.
Methionine-Regulated System
in Cryptococcus neoformans
C. neoformans Gpr4 is a 7-TM protein re-
ported to share low sequence identity with D.
discoideum cAR1 and A. nidulans GprH (124).
However, our analysis shows that C. neofor-
mans Gpr4 is more similar to N. crassa GPR-4
and other Gpr1p-related proteins (Table 4).
In spite of its homology to carbon-sensing
GPCRs, C. neoformans Gpr4 is not required
for glucose sensing (124). C. neoformans gpr4
mutants have defects in capsule production
and mating similar to those in strains lacking
the Gpa1 Gα (although the defects of gpa1
mutants are more severe). Both genetic epis-
tasis and split ubiquitin yeast two-hybrid anal-
ysis support interaction between Gpr4 and
Gpa1, and the defects of both mutants are sup-
pressed by exogenous cAMP (124). Addition
of methionine to cells starved for amino acids
leads to a transient increase in cAMP levels
in wild type, but not gpa1 or gpr4 mutants;
a fluorescently tagged Gpr4 fusion protein is
rapidly internalized in response to methion-
ine, and methionine addition stimulates mat-
ing filamentation in wild type, but not gpr4
or gpa1 mutants. These observations indicate
that methionine is a potential ligand for Gpr4
Carbon-Sensing Pathway
in Neurospora crassa
N. crassa GPR-4 is similar to putative carbon-
sensing GPCRs from yeasts, including S. cere-
visiae Gpr1p, S. pombe Git3, and C. albicans
Gpr1 (70). When grown on poor carbon
sources such as glycerol, gpr-4 strains ac-
cumulate much less mass than does the wild
type. GPR-4 is also required for a carbon-
source-dependent transient increase in cAMP
levels after addition of glucose to glycerol-
grown cultures (70). Mutants lacking the Gα
subunit genes gna-1, gna-2, and gna-3 have
mass accumulation defects on glycerol, sug-
gesting that all three Gα proteins may be in-
volved in carbon sensing (70). Results from
genetic epistasis and yeast two-hybrid assays
were consistent with coupling of GPR-4 to
GNA-1. The data support a GPR-4/GNA-
1/cAMP signaling pathway that regulates car-
bon source sensing in N. crassa (70). But
the coupling between GPR-4 and GNA-1
(a Group I Gα) also reveals an important vari-
ation. In S. cerevisiae and S. pombe, the Gα
proteins coupled to the carbon sensory re-
ceptors belong to Group III. Results from
other species mentioned above also support
regulation of a downstream cAMP signaling
pathway by Group III Gα proteins during
carbon sensing in filamentous fungi. Like-
wise, the methionine-responsive Gpr4/Gpa1
system in C. neoformans also involves regu-
lation of cAMP levels. The GPCRs coupled
to GNA-2 and GNA-3 during carbon sens-
ing are currently unknown; however, previ-
ous work has revealed a role for the Group III
Gα GNA-3 in regulation of cAMP levels and
PKA signaling in N. crassa (52). Thus, both
Group I and Group III Gα proteins are impli-
cated in cAMP-dependent carbon sensing in
N. crassa. Along these lines, it is of interest that
Group I (GNA-1) and Group III (GNA-3)
Gα proteins modulate adenylyl cyclase in N.
crassa through regulation of adenylyl cyclase
activity and protein levels, respectively (44,
The receptors and pheromone ligands that
regulate recognition of different mating types
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are similar in yeasts and in characterized fil-
amentous fungal species. However, in con-
trast to the mating process in S. cerevisiae,
which takes place between two morphologi-
cally identical cells, mating often involves dif-
ferentiation of specialized multicellular struc-
tures and/or fusion of different cell types
(males and females) in filamentous fungi.
cAMP signaling pathways can positively or
negatively regulate sexual development, and
there is evidence for involvement of a
MAPK cascade in the pheromone response in
C. neoformans.
Ascomycete Fungi
The pheromone response has been well-
studied in the yeast S. cerevisiae. This system
includes the peptide pheromones α-factor
and a-factor, pheromone receptors Ste2p
and Ste3p, and a heterotrimeric G protein
consisting of Gpa1p (Gα) and Ste4p/Ste18p
(Gβγ dimer) operating upstream of a MAPK
pathway (5). Conserved components in the
genomes of numerous filamentous fungi have
now been identified (13, 31, 64), and examples
of pathways from Ascomycetes and Basid-
iomycetes are presented below (Figure 2b).
Neurospora crassa. N. crassa is a heterothallic
(self-sterile) filamentous fungus with two
mating types, mat a and mat A. Nitrogen
starvation induces differentiation of female
reproductive structures (protoperithecia)
that elaborate chemotropic hyphae, termed
trichogynes (13). Mating is accomplished by
growth of trichogynes toward males (often
conidia) of opposite mating type. The N. crassa
genome contains two pheromone receptor
( pre-1 and pre-2) and two pheromone (ccg-4
and mfa-1) genes. pre-1 and ccg-4 are expressed
in mat A cells, and pre-2 and mfa-1 are pro-
duced by mat a strains (9, 55–57, 92). Genetic
studies indicate that pheromones are essential
for male fertility in a mating-type-specific
manner (56, 57). Accordingly, pheromone
receptors are required for chemotropism of
trichogynes toward opposite mating type
Heterothallic: a
fungus that is
self-sterile (cannot
mate with itself)
because it produces
gametes that cannot
combine with one
another during
Fruiting body:
structure (organ)
produced during the
reproductive phase
of the fungal life
cycle that encases the
meiotic products
(sexual spores)
flask-shaped fruiting
bodies with pores
through which
mature sexual spores
are ejected
Homothallic: a
fungus that is
self-fertile (can mate
with itself) due to
elaboration of
gametes on the same
strain that can
combine during
closed fruiting
bodies that lack
pores for release of
sexual spores
males (55). gna-1 (Group I Gα), gnb-1
(Gβ), and gng-1 (Gγ) strains are female-
sterile (but male-fertile) in both mating types
(43, 55, 61, 127), consistent with the GNA-
1/GNB-1/GNG-1 heterotrimer operating
downstream of both pheromone receptors.
The events following mating, including
meiosis and fruiting body (perithecial) devel-
opment, are also influenced by G proteins
in N. crassa. Loss of the CRL GPCR gene
gpr-1 leads to defects in perithecial devel-
opment and sexual spore (ascospore) ejec-
tion (62), and crosses involving two gna-3
(Group III Gα) parents produce ascospores
with reduced viability (54).
Magnaporthe grisea. In the heterothallic
fungus M. grisea, the sexual cycle is initiated
during growth on minimal medium supple-
mented with plant extracts. Mating occurs be-
tween cells of opposite mating types, MAT1-1
and MAT1-2 (49, 106). The M. grisea genome
contains two putative pheromone receptors
(63) (Table 4). The M. grisea Gα subunits
MagA, MagB, and MagC and the Gβ sub-
unit Mgb1 are involved in sexual reproduction
(74, 86). Deletion of MAGB and MGB1 re-
sulted in female sterility with a block in
perithecial formation (74, 86). Conversely,
magA and magC strains produce perithe-
cia containing immature asci (74), suggesting
possible involvement of MagA and MagC in
postfertilization events.
Aspergillus nidulans. A. nidulans is a fila-
mentous fungus that can participate in both
homothallic (self-fertile) and heterothallic
crosses to form sexual fruiting bodies termed
cleistothecia. Similar to N. crassa, A. nidu-
lans has two pheromone receptors, GprA and
GprB (103). In contrast to N. crassa, deletion
of either gprA or gprB results in the production
of fewer and smaller cleistothecia that con-
tain reduced numbers of ascospores, and loss
of both genes eliminates fruiting body for-
mation in homothallic crosses (103). These
data indicate that GprA and GprB are posi-
tive regulators of cleistothecial development
Heterotrimeric G Protein Signaling in Filamentous Fungi 439
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and that both proteins are necessary for full
sexual fertility in self-crosses. No Gα protein
has been linked to this pathway; however, the
Gβγ dimer SfaD-GpgA and PhnA phosducin
are required for sexual fruiting body forma-
tion (97, 104, 105). The phnA deletion mutant
exhibits defects identical to those of sfaD and
gpgA mutants and is not able to form cleis-
tothecia under self-fertilized conditions or in
outcrosses (105).
Basidiomycete Fungi
Basidiomycetes are primarily heterothallic,
requiring each parent to be a different mating
type. However, in contrast to Ascomycetes
with two mating types, the number of mating
types ranges from two to the thousands in
basidiomycete fungi. In general, meiosis
occurs in a specialized structure called the
basidium, in which basidiospores eventually
develop (15). The sexual phase of the life
cycle involves pheromone receptors and
heterotrimeric G proteins (15, 69).
Ustilago maydis. U. maydis utilizes two un-
linked mating type loci, a and b. Each of
the two alleles of the a locus contains genes
for a peptide pheromone similar to yeast
a-factor (mfa1 or mfa2) and a pheromone
receptor of the Ste3p class ( pra1 or pra2)
(12, 115). Studies using various wild-type and
mutant strains and purified pheromone show
that pheromones and pheromone receptors
are required for yeast cells to produce con-
jugation tubes and to form dikaryons in U.
maydis (108, 110). gpa3 (Group III Gα) and
bbp1 (Gβ) mutants grow filamentously and are
impaired in conjugation tube formation and
pheromone gene expression (84, 95). Epistasis
analysis supports Bbp1 acting downstream of
Gpa3, and addition of cAMP to gpa3 and bpp1
mutants restores budding growth, allowing
production of conjugation tubes (84). Taken
together, these results suggest that Gpa3 and
Bpp1 are not coupled to pheromone recep-
tors, but instead operate upstream of the
cAMP signaling cascade in U. maydis (60, 84).
Cryptococcus neoformans. In C. neoformans,
two pheromone receptor genes, CPRα and
CPRa, were identified in the MATα and MATa
mating type strains, respectively. Deletion of
CPRα decreases mating efficiency and the
ability to sense the MATa pheromone, but
it does not completely abolish mating (20).
Loss of CPRa negatively influences mating
and virulence (16). Deletion of the Group III
Gα gene gpa1 leads to scarce hyphal growth
and decreased basidiospore production after
cell fusion; these defects can be remedied
by cAMP supplementation (2). In contrast,
gpb1 Gβ deletion mutants are completely de-
ficient in mating, failing to form conjugation
tubes, and addition of cAMP does not sup-
press this phenotype (119). The gpb1 pheno-
type is, however, corrected by overexpression
of the cpk1 MAPK gene, and conjugation tube
formation is stimulated by overexpression of
gpb1 in wild-type cells (119). This suggests
that C. neoformans Gpb1 regulates a MAPK
cascade during pheromone signaling, and that
Gpa1 is upstream of a cAMP signaling path-
way important for sexual development.
Basidiomycete mushroom fungi. The
mushroom fungi S. commune and C. cinereus
possess two different mating type loci, A and
B (15). In S. commune, there are functionally
redundant subloci of A and B, termed Aα
Aβ,Bα, and Bβ. For fully compatible mating,
strains must differ at both Aα or Aβ and
Bα or Bβ (116, 122). The Bα and Bβ loci
each have nine different specificities (94)
and contain pheromone and pheromone
receptor genes. For example, Bα1 encodes a
pheromone receptor gene (bar1) and three
pheromone genes (bap1-1, -2, -3) (122).
The presence of multiple pheromone genes
suggests some redundancy, and studies show
that a single pheromone can interact with
multiple receptors and vice versa (116, 122).
Evidence from a heterologous S. cerevisiae
system indicates that several S. commune
pheromone receptors can couple to yeast
Gpa1, supporting the presence of a Gα
protein downstream of pheromone receptors
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in S. commune (30). Studies using constitu-
tively activated Gα alleles demonstrate that
SCGP-A and SCGP-C (both Group III) are
negative regulators of mating in S. commune
In C. cinereus, there are two mating type
loci, A and B. The B locus is composed of
three groups of genes, each of which contains
one pheromone receptor and one to three
pheromone genes (89, 96). Similar to S. com-
mune, individual C. cinereus pheromone re-
ceptors can couple to downstream signaling
pathways in S. cerevisiae strains that express
a Gpa1/Gα
chimeric Gα (87, 88). Although
this indicates that C. cinereus pheromone re-
ceptors can activate Gα subunits, coupling to
specific C. cinereus Gα proteins has not yet
been demonstrated.
Many filamentous fungi are pathogenic to
plants, animals, and humans. Diseases caused
by fungal pathogens have often been re-
sponsible for enormous economic losses in
agriculture and are also threats to human
health. In addition, toxins produced by cer-
tain plant pathogenic fungi (mycotoxins) can
cause disease in humans. The pathogenic
process is correlated with infection-related
morphogenesis and development in many
pathogenic fungi. Connections between mat-
ing and pathogenesis have been demonstrated
in several systems, but unshared components
and regulatory interactions highlight the dif-
ferences in signal networking and output
in these two processes. Heterotrimeric G
protein–mediated signaling plays an impor-
tant role in both fungal pathogenesis and my-
cotoxin synthesis.
Plant Pathogens
Pathogenicity in plant fungal pathogens is
closely associated with morphogenesis and
development. Some fungi form special struc-
tures that are essential for host infection (21).
Functional studies show that Group I or III
Gα proteins and the cAMP pathway together
Pathogenic morphogenesis/development
Mycotoxin biosynthesis
Environmental signals
Figure 3
G protein signaling pathways involved in
pathogenesis in filamentous fungi. G protein
signaling during fungal pathogenesis can be
initiated by various environmental stimuli,
including nutrients, pheromones, host signals, and
other factors. Although the involvement of Group
I and III Gα proteins in the control of
pathogenesis has been established, the identity of
coupled GPCRs is not yet clear. Gα subunits
regulate downstream pathways (cAMP and
MAPK) that control fungal pathogenic
morphogenesis and mycotoxin production.
play an essential role in fungal pathogenesis
(69) (Figure 3).
Cryphonectria parasitica. In the chestnut
blight fungus C. parasitica, infection with
viruses of the genus Hypovirus affects the
level of fungal virulence (18). Virus infection
downregulates levels of the Group I Gα pro-
tein Cpg-1 and delays activation of a MAPK
kinase during development (18, 113). Ge-
netic analyses demonstrate that the require-
ment for Cpg-1 during pathogenesis stems
from a role in regulating vegetative growth
and conidiation and that Cpg-1 possesses
some Gβγ-independent functions in control
of pathogenicity (18, 33, 100). In contrast, the
Gβ (Cpgb-1) positively regulates pathogenic-
ity but is not required for vegetative growth
(50). G protein signaling is also modulated
Heterotrimeric G Protein Signaling in Filamentous Fungi 441
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by the coordinated actions of the PhLP
Bdm-1 (as described above) and a RGS
(Cprgs-1) (51, 101).
Magnaporthe grisea. M. grisea forms a spe-
cialized structure (appressorium) that pene-
trates into the host plant epidermis by forming
a penetration peg, with physical force supplied
by turgor pressure inside the appressorium
(21). MagB (Group I Gα ), Mgb1 (Gβ), and
Mgg1 (putative Gγ) regulate appressorium
formation (71, 74, 86). MagB and Mgb1 are
required in a cAMP-dependent manner for
recognition of host surface hydrophobic-
ity during appressorium formation (74, 86).
However, cAMP does not rescue defects in ap-
pressorial penetration and infectious growth
in mgb1 and mgg1 strains, suggesting
these processes utilize another downstream
pathway, perhaps involving MAPK signaling
(71, 86). In addition, the putative GPCR
Pth11 also regulates appressorium forma-
tion, presumably through sensing surface
hydrophobicity and plant cutin monomers
(24). Thus, Pth11/MagB/Mgb1/Mgg1 could
comprise a GPCR/G protein system that
controls appressorium differentiation in
response to plant surface molecules.
Ustilago maydis. The maize smut fungus
U. maydis has been studied as a model or-
ganism for phytopathogenic fungi that re-
quire a dimorphic switch from a yeast-like
form to multicellular filaments in order to
become pathogenic (11). Only filamentous
forms resulting from a dimorphic switch dur-
ing mating are able to infect maize tissues.
Heterotrimeric G proteins and coupled re-
ceptors play essential roles in regulating mat-
ing and pathogenicity in U. maydis through
pheromone response and cAMP-dependent
signaling pathways (84, 95). Of the identi-
fied G protein subunits, Gpa3 (Group III)
and Bpp1 (Gβ) are required for mating
and mediation of cAMP-dependent signal-
ing, but only Gpa3 functions directly in con-
trolling pathogenicity (60, 84). Gpa3 trans-
duces signals to a cAMP signaling pathway
that includes Uac1 (adenylyl cyclase) and
Adr1 (cAMP-dependent protein kinase A)
Other species. G protein involvement in
plant pathogenesis has been demonstrated
in many other filamentous fungi. Group I
Gα proteins identified in B. cinerea (Bcg1),
Fusarium oxysporum (Fga1), Colletotrichum
trifolii (Ctg-1), Stagonospora nodorum (Gna1),
and Alternaria alternata (Aga1) play essential
roles in pathogenicity (36, 47, 109, 112, 125).
Of these, Bcg1, Fga1, and Aga1 are associated
with cAMP-dependent signaling (36, 47, 125).
Group III Gα subunits in B. cinerea (Bcg3)
and F. oxysporum (Fga2) are also required for
virulence (25, 48). However, Bcg3 is involved
in formation of primary lesions, but not
infectious growth in expanding lesions (25).
Cgb1 and Fgb1, Gβ genes from Cochliobolus
heterostrophus and F. oxysporum, respectively,
are required for virulence (22, 32).
Human Pathogens
Human diseases caused by fungal infections
are an emerging problem for public health,
especially in immunocompromised patients.
Fungal development and resistance to host
defense systems are key factors determining
pathogenicity in human fungal pathogens. G
protein signaling plays an important role in
regulating pathogenesis and production of
virulence factors in various species.
Cryptococcus neoformans. In C. neoformans,
two factors, extracellular polysaccharide cap-
sule and melanin, play an essential role in
establishing pathogenicity (59, 120). The
polysaccharide capsule confers resistance to
phagocytosis by macrophages and the melanin
pigment protects the fungus against oxidants
produced by the host and antifungal agents
(59, 120). Gpa1 (Group III Gα) and adeny-
lyl cyclase (Cac1) positively control the for-
mation of capsule and melanin and virulence
in C. neoformans (2, 3, 124). Mutation of the
RGS gene, crg1, results in greatly increased
442 Li et al.
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virulence (118). On the other hand, the
Gpb1 Gβ is dispensable for capsule forma-
tion, melanin synthesis, and virulence (119).
Although gpr4 GPCR mutants are deficient
in capsule production, these strains produce
melanin and are virulent (124). These results
suggest a model for virulence in which an as
yet unknown GPCR acts upstream of Gpa1
and adenylyl cyclase and in which Crg1 may
function as a negative regulator of Gpa1 in C.
neoformans (118).
Aspergillus fumigatus. A. fumigatus is a com-
mon air-borne fungal pathogen that causes
invasive aspergillosis and pneumonia in im-
munocompromised patients (45). The fun-
gus becomes pathogenic once the inhaled
conidia overcome the primary defense sys-
tems of the host. Pigmentation of conidia
is an important virulence factor because it
provides resistance against reactive oxygen
species produced by human cells (45). Another
pathogenic determinant, PksP, functions in
conferring resistance to the antimicrobial ac-
tivity of macrophages (46). The Group III
Gα subunit GpaB positively influences coni-
dial survival in macrophages and pksP ex-
pression in A. fumigatus (72). gpaB mu-
tants produce conidia, but the survival rate
of such conidia after ingestion by human
monocyte-derived macrophages is greatly re-
duced (72). In addition, pksP gene expression
is greatly reduced in gpaB strains (72). The
phenotypes of gpaB and adenylyl cyclase
(acyA) mutants are similar and cAMP de-
pendent, leading to the hypothesis that GpaB
mediates a cAMP-dependent signaling path-
way to regulate pathogenesis in A. fumigatus
(72, 73).
Mycotoxin Biosynthesis
Mycotoxins produced and secreted by fila-
mentous fungi are virulence factors typically
associated with postharvest fungal diseases of
commodity crops. Contamination of fruits,
vegetables, small grains, cereals, and seeds by
fungal mycotoxins can have harmful effects on
plants, humans, and animals, and cause enor-
mous economic losses in the food and feed
Although A. nidulans does not normally
infect plant hosts, a mycotoxin produced by
this fungus, sterigmatocystin (ST), is carcino-
genic and toxic to humans. Two Gα sub-
units, FadA (Group I) and GanB (Group III),
regulate ST biosynthesis in A. nidulans, with
coordinated action of their RGS regulators,
FlbA and RgsA, respectively (37, 39). Because
FadA negatively regulates ST biosynthesis,
inhibition of FadA-mediated signaling by the
RGS protein FlbA is required for ST pro-
duction (39, 129). Activated FadA positively
regulates PkaA (cAMP-dependent protein ki-
nase A), leading to lower expression of aflR,
a transcription factor necessary for ST pro-
duction (107). Another RGS, RgsA, enhances
ST biosynthesis by inhibiting its counterpart
Gα subunit, GanB (37). Although the Gβγ
dimer regulates sporulation, which is strongly
correlated with ST biosynthesis, involvement
of Gβγ in ST biosynthesis is less clear in A.
nidulans (97, 105). Mutants lacking sfaD (Gβ),
gpgA (Gγ), or both did not produce ST, sug-
gesting that these two genes are essential for
ST biosynthesis (105). However, deletion of
sfaD and gpgA in the flbA mutant was not able
to rescue ST biosynthesis, in contrast to the
case for fadA (105).
Aflatoxin, a potent mycotoxin synthesized
from precursor ST, is more of a concern
for human safety than ST because of its
production by the more common species
Aspergillus parasiticus and Aspergillus flavus.
FadA-mediated signaling is apparently con-
served in regulation of aflatoxin biosynthe-
sis. Constitutive activation of fadA inhibited
aflatoxin biosynthesis in these two aflatoxin-
producing species (39, 80).
Trichothecenes and fumonisins are myco-
toxins produced by many Fusarium species.
In Fusarium sporotrichioides, introduction of
a dominant constitutively activated allele of
fadA (from A. nidulans) stimulated T-2 toxin
biosynthesis and altered trichothecene gene
expression (111). This result was opposite to
Heterotrimeric G Protein Signaling in Filamentous Fungi 443
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that observed in A. nidulans, in which acti-
vated fadA inhibited ST biosynthesis (111).
These findings demonstrate that the same G
protein signaling pathway can mediate oppos-
ing modes of toxin biosynthesis in different
The past 10 years have yielded much new
knowledge regarding G protein signaling
in filamentous fungi. Accumulating evidence
implicates heterotrimeric G proteins in sens-
ing of nutrients (carbon and amino acids),
peptide pheromones, and host signals that
are important for pathogenesis. Thus, het-
erotrimeric G proteins are crucial for detec-
tion of major environmental stimuli for food
acquisition, asexual sporulation and spore ger-
mination, mating, and host infection. The im-
portance of G proteins to the filamentous
fungal lifestyle is also underscored by the ob-
servation that a N. crassa mutant lacking all
Gα subunits is severely restricted in growth
and exhibits major defects in all known devel-
opmental pathways (52).
The availability of numerous complete
genome sequences and improved gene-
knockout and associated molecular genetic
techniques has greatly speeded the analysis
of this important group of organisms. The
results have revealed significant differences
in signaling between yeasts and filamentous
fungi, including an expanded number of G
protein subunits and GPCRs in filamentous
species, as well as diversification of the cell
types and structures whose differentiation is
controlled by G proteins.
Future investigations will focus on high-
throughput functional and expression studies,
analysis of protein-protein interactions, and
discovery of downstream effectors. Identifica-
tion of the ligands and GPCR(s) for each path-
way is a challenging but essential pursuit, if
new control practices are to be developed us-
ing small molecules as antifungal agents. Such
studies can benefit from the rich history of
pharmacological approaches used for analysis
of G proteins in mammalian systems. Finally,
because filamentous fungi are closely related
to animals, but genetically more tractable,
functional studies in filamentous fungi will
advance G protein studies in mammals. Such
investigations will shed light on understand-
ing and controlling human diseases associated
with G protein signal transduction pathways.
1. Most filamentous fungi possess three conserved Gα subunits, one Gβ protein, and
one Gγ protein. The number of predicted GPCRs varies widely, with a larger number
identified in ascomycetes than in basidiomycetes. This may result from a lower number
of GPCRs or the presence of novel GPCR classes in basidiomycete fungi.
2. In several examples, nutrient sensing involves a GPCR and a heterotrimeric G protein
operating upstream of a cAMP signaling pathway.
3. The pheromone response requires recognition of peptide pheromones by cell surface
GPCRs coupled to heterotrimeric G proteins. Roles for G proteins in regulation
of downstream MAPK cascades have been indicated in a few species, and future
work will focus on this area. Functions for both Gα and Gβ proteins in control
of cAMP signaling and mating-specific gene expression have been demonstrated in
several systems.
4. Pathogenesis can be accompanied by cell differentiation, virulence factor production,
and/or toxin biosynthesis in fungi. Heterotrimeric G protein, cAMP, and MAPK
signaling pathways have been implicated in fungal pathogenesis.
444 Li et al.
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We acknowledge receipt of National Institutes of Health Grant GM48626, “G proteins and
signal transduction in Neurospora crassa,” to KAB for the past 15 years.
The authors wish to acknowledge fungal genome sequence databases at the Broad Institute at
MIT, the Institute for Genome Research and the Podospora anserina Genome Project in France.
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