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Fungal Genetics and Biology 45 (2008) 310–318

www.elsevier.com/locate/yfgbi

The Aspergillus nidulans esdC (early sexual development) gene

is necessary for sexual development and is controlled by veA
and a heterotrimeric G protein
Kap-Hoon Han b,1, Jong Hwa Kim b,1, Hosun Moon a, Serha Kim a, Sung-Suk Lee a,
Dong-Min Han c, Kwang-Yeop Jahng a, Keon-Sang Chae a,*
a
Division of Biological Sciences, Basic Science Research Institute, Chonbuk National University, Chonju, Chonbuk 561-756, Republic of Korea
b
Department of Pharmaceutical Engineering, Woosuk University, Wanju, Chonbuk 575-701, Republic of Korea
c
Division of Life Science, Wonkwang University, Iksan, Chonbuk 570-749, Republic of Korea

Received 24 May 2007; accepted 10 September 2007

Available online 25 September 2007

Abstract

The esdC (early sexual development) gene was isolated by using an expressed sequence tag (EST) as a probe from a genomic library of
the early sexual developmental stage mycelia of Aspergillus nidulans. The sequence analysis revealed that the esdC gene contains a 59 bp
intron and encodes a 266 amino acid polypeptide with a calculated molecular weight of 29.4 kDa. The EsdC protein is conserved among
filamentous fungi and has a domain with similarity to a glycogen binding domain conserved in the b subunit of the AMP-activated pro-
tein kinase (AMPK) complex. Although the esdD gene was expressed during asexual development, the expression reached its maximum
at 10 h and decreased thereafter up to 50 h after the end of the induction of sexual development. In an esdC-null mutant under a veA+
background, no sexual structures were formed at any condition examined. However, esdC overexpression did not lead to an induction of
sexual development. In addition, to the effect of the esdC mutation on the sexual development, more conidiophores were formed in the
esdC-null mutant than in a wild type. These results indicate that the esdC gene is necessary for sexual structure formation but its over-
expression is not sufficient to enhance this process. Expression of the esdC gene throughout development was positively regulated by the
veA gene. In addition, very little and no esdC transcript, respectively, was observed in an flbA-null mutant and in a fadAG42R mutant, and
the esdC transcript level was higher in a fadA-null mutant and in a sfaD-null mutant than in a wild type, indicating that inactivation of
FadA is necessary for positive regulation of esdC expression.
Ó 2007 Elsevier Inc. All rights reserved.

Keywords: Sexual development; Aspergillus nidulans; esdC; Glycogen binding domain; FadA

1. Introduction (Adams et al., 1998). In contrast, the sexual development

A homothallic filamentous fungus, Aspergillus nidulans
has two distinctive reproductive developmental processes,
sexual and asexual development. The step-wise formation
of asexual structures such as vesicle, phialides, and conidia
are well dissected, and many genes involved in asexual
development have been identified and characterized
* Among them, the nsdD gene has been isolated and charac-
Corresponding author. Fax: +82 63 270 3345.
E-mail address: chaeks@chonbuk.ac.kr (K.-S. Chae).
1
These authors are contributed equally to this work.
process has not been thoroughly investigated. Although
several genes including veA, nsdD, stuA, steC, noxA, rosA,
and nosA have been known as being involved in sexual
development (Kim et al., 2002; Han et al., 2001; Miller
et al., 1991; Dutton et al., 1997; Wu and Miller, 1997; Val-
lim et al., 2000; Lara-Ortiz et al., 2003; Vienken et al., 2005;
Vienken and Fischer, 2006), the interaction between these
genes and the signaling cascade are not well elucidated.
terized to reveal that it encodes a GATA type zinc-finger
domain protein and functions as a positive regulator of sex-

1087-1845/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved.
doi:10.1016/j.fgb.2007.09.008
 K.-H. Han et al. / Fungal Genetics and Biology 45 (2008) 310–318
ual development (Han et al., 2001). Expression of the nsdD
gene is affected by the flbA gene, which encodes a regulator
of G protein signaling (RGS) protein for antagonizing a
growth signal transmitted by a heterotrimeric G protein,
FadA, SfaD, and GpgA, the Ga subunit, the Gb subunit
and the Gc subunit, respectively (Han et al., 2001; Yu,
2006). The FadA and SfaD are required for normal vegeta-
tive growth and a loss-of-function mutation of the negative
regulator flbA or the dominant active mutation, fadAG42R,
results in improper vegetative growth without development
and eventual autolysis (Yu et al., 1996; Rosen et al., 1999;
Adams et al., 1998; Yu, 2006).
The veA gene has been proposed to be a negative regu-
lator of asexual development, since the veA1 mutant, first
isolated and characterized by Käfer (1965), forms more
conidia than a wild type, and sexual development of the
veA1 mutant is generally retarded and reduced while asex-
ual development is promoted and increased. Furthermore,
the veA1 mutation can bypass the requirement of light illu-
mination, necessary for inducing asexual development in a
wild type (Champe et al., 1981; Mooney and Yager, 1990;
Yager, 1992). The veA gene has been isolated and charac-
terized as being required for sexual development as a posi-
tive regulator and also acts as a negative regulator of
asexual development (Kim et al., 2002). A veA deletion
mutant never forms sexual structures even under condi-
tions where a lot of sexual structures are formed in a wild
type, but forms more conidial heads. Consistently, overex-
pression of the veA gene causes formation of sexual struc-
tures even under conditions where a wild type does not
form sexual structures. Recent studies provided some clues
about the cellular function of the veA gene not only in A.
nidulans but also in related fungal species. In A. nidulans,
secondary metabolite production as well as sexual develop-
ment is controlled by the veA gene (Kato et al., 2003; Spro-
te and Brakhage, 2007). Also, consistent with its
phenotype, the subcellular localization of the VeA is con-
trolled by light (Stinnett et al., 2007). Similarly, the veA
orthologs in other fungi, including Aspergillus parasiticus
and Fusarium verticillioides, also play an important role
in development and secondary metabolism (Calvo et al.,
2004; Dreyer et al., 2007; Li et al., 2006). To understand
the functions of the veA gene in more details, proteins
interacting with the VeA protein and genes of which
expression is controlled by the veA gene need to be identi-
fied and characterized.
Sequencing of randomly selected cDNA clones in a 3 0 -
directed cDNA library is one of the most important meth-
ods to identify cell-type specific or tissue-specific genes
(Okubo et al., 1992). Genes expressed highly in a specific
cell-type or a tissue can be easily identified, because it
can be assumed that the more frequently an expressed
sequence tag (EST) appears in a library, the higher the
expression level of a gene having the EST (Okubo et al.,
1992). About 300 randomly selected clones in 3 0 -directed
cDNA libraries constructed from mycelia at the early sex-
ual developmental (ESD) stage, at the late sexual develop-
311
mental stage and from vegetative mycelia of A. nidulans
have been sequenced, followed by sequence comparison
and Northern hybridization, and genes of 17 ESTs have
been identified as being expressed more highly at the
ESD stage than the other two stages (Jeong et al., 2000).
During the studies on genes expressed abundantly at the
ESD stage, it has been found that expression of a gene hav-
ing one of the ESTs, esd0543 (GenBank Accession No.
AJ237173), is possibly affected by the presence of the veA
gene (Jeong et al., 2000). In this study, a gene containing
the esd0543 EST, named esdC, was isolated and character-
ized. The gene structure, the expression pattern during
development, and the function of the gene in development
were investigated. Relationships between the esdC gene and
others involved in development and growth control, such
as veA, nsdD, flbA, sfaD, and fadA were also analyzed.
2. Materials and methods
2.1. Strains, media, and DNAs
All A. nidulans strains used in this study were shown in
Table 1 and cultured on complex medium (CM) or minimal
medium (MM) supplemented appropriately (Pontecorvo
et al., 1953; Lee et al., 2001). When necessary, 0.3% casami-
no acid was added to MM (MMCA). Genotypes of the
strains The VDEC72-1 strain was obtained from VER7
by replacing a part of the esdC gene with the argB gene
(see below). The KHH22 strain was a segregant of a cross
between NSD219 and G34 (Han et al., 2001). An overex-
pressor of the esdC gene under the veA1 background,
ROEC11, was constructed by introduction of the pRB2-
esdC into RMS011.
2.2. Nucleic acid manipulations
Standard methods were used for construction, isolation
of plasmids, and hybridizations (Sambrook et al., 1989).
Total RNA and genomic DNA were isolated from A. nidu-
lans as previously described (Jeong et al., 2000). The nucle-
otide sequence was determined by using an automatic
DNA sequencer ABI 377 (Perkin-Elmer, CT). Oligonucleo-
tide primers used for sequencing or PCR were synthesized
from Genotech, Co. (Daejeon, Korea).
The pRB2-esdC was constructed by the insertion of the
esdC coding region into the pRB2 to express the esdC gene
under the control of the niiA promoter (Punt et al., 1995).
The plasmid pJYargB, constructed by the transfer of the A.
nidulans argB gene to pBluescript II KS(+) from pDC1,
was a kind gift of Dr. Yu at University of Wisconsin–Mad-
ison (Madison, WI). The esdC-specific probe for colony
hybridization and Northern hybridization was the 682 bp
BamHI/NcoI-digested fragment of the esdC gene, unless
indicated otherwise. The fadA-, flbA-, and nsdD-specific
probes were a 200 bp BglII-digested fragment of a fadA
cDNA (Yu et al., 1996), a 2.5 kb EcoRI-digested fragment
of pBN30 containing the flbA gene (Lee and Adams, 1994),
312 K.-H. Han et al. / Fungal Genetics and Biology 45 (2008) 310–318

Table 1
Genotypes of strains used in this study
Strain Genotype Source
+
FGSC A4 Wild type, veA FGSCa
FGSC A26 biA1; veA1 FGSC
VER7 pabaA1, yA2; DargB::trpC; trpC801, veA+ Han et al. (2001)
VER7 (+pJYargB) pabaA1, yA2; DargB::trpC; trpC801, veA+; argB+ This study
RMS011 pabaA1, yA2; DargB::trpC; trpC801, veA1 This study
RMS011 (+pJYargB) pabaA1, yA2; DargB::trpC; trpC801, veA1; argB+ This study
VDEC72-1 pabaA1, yA2; DargB::trpC; trpC801, veA+ ; DesdC::argB This study
ROEC11 pabaA1, yA2; DargB::trpC; trpC801, veA1; niiA(p)::esdC, argB+ This study
NSD219 biA1; nsdD19; sB3, chaA1 Han et al. (2001)
KHH22 argB2, methH2; nsdD19, sB3; veA1 Han et al. (2001)
KHH22 (+pRB2-esdC) argB2, methH2; nsdD19, sB3; veA1; niiA(p)::esdC, argB+ This study
FGSC A1036 biA1; methG1; argB2; DfadA::argB, veA1 FGSC
H1FAD4 biA1; fadAG42R; veA1 Yu et al. (1996)
TRSB1.16 biA1; methG1; argB2; DsfaD::argB, veA1 Rosen et al. (1999)
TBN39.5 biA1, DflbA::argB; methG1; veA1 Lee and Adams (1994)
DVAR1 pabaA1, yA2; DargB::trpC; trpC801, DveA::argB Kim et al. (2002)
a
Fungal Genetics Stock Center.

and a 2.6 kb NotI-digested nsdD cDNA (Han et al., 2001),
respectively. The sfaD-specific probe was a 970 bp PCR-
amplified product of the sfaD gene with a primer pair, of
which the sequences were 5 0 -CGG GAT CCC ACT ACG
AAC AAA GTC CA-3 0 and 5 0 -CCA AGC TTG TGA
TTT TAT TAT TCT TT-3 0 . All probe were labeled with
[a-32P]-dCTP.
2.3. Manipulation of A. nidulans
DNA mediated transformation of A. nidulans was per-
formed as described by Yelton et al. (1984). Sexual devel-
opment of A. nidulans was induced according to the
method described previously (Jeong et al., 2000). Mycelial
balls of about 106 conidia obtained from the 14 h culture in
liquid CM at 37 °C were transferred onto solid MMCA.
Then, the plate was wrapped with parafilm and aluminum
foil to protect it from light and gas exchange, and the plate
was incubated for 20 h at 37 °C to induce sexual develop-
ment. After release of sealing, the plate was incubated for
a given time.
2.4. Isolation of the esdC gene
The esdC gene containing the EST number esd0543 was
isolated by using Southern hybridization of genomic DNA
library, which was constructed by the digestion of genomic
DNA with EcoRI followed by the insertion of the digested
genomic DNA into pBluescript SK(+) prelinearized with
the same enzyme. The pESD0543-5.5 was isolated from
the constructed library by colony hybridization using the
EST DNA as a probe.
To isolate the esdC cDNA, a cDNA library was con-
structed from mRNA isolated from mycelia at the end of
the induction of sexual development, using CytoTrapä
XR Library Construction Kit according to the manufac-
turer’s instruction (Stratagene, CA). From the cDNA
library, a clone having the esdC cDNA was isolated by col-
ony hybridization using the 1.1 kb EcoRV-digested frag-
ment of pESD0543-4.2 as an esdC-specific probe.
2.5. Analysis of the 5 0 end of the esdC mRNA
To verify the 5 0 end of the esdC mRNA, the 5 0 RACE
(rapid amplification of cDNA end) by inverse PCR was
carried out according to the procedure described previously
(Maruyama et al., 1995). cDNAs were synthesized by using
a reverse-transcriptase, SuperScript II (Invitrogen) and the
primer esdCRT of which the sequence was 5 0 -ACC AGT
ACC GAC CAC CAA-3 0 . The synthesized cDNA was cir-
cularized with RNA ligase, followed by inverse PCR with a
primer pair, esdRT-up and esdRT-low, of which the
sequences were 5 0 -CTA CTC TCG CCA GAT CCC C-3 0
and 5 0 -AAG CAG ATG CAC GGT CTT G-3 0 , respec-
tively. The amplified DNA fragment was subcloned into
pBluescript II KS(+) and the nucleotide sequence near
the 5 0 end was determined by using universal primers.
2.6. Disruption of the esdC gene
After subcloning of the 3.7 kb XhoI/PstI fragment con-
taining the entire esdC coding region into pESD0543-3.7,
the 1.8 kb XhoI/BamHI-digested fragment having the argB
gene from pJYargB was inserted in place of the 1.3 kb SalI/
BglII fragment of the esdC gene in pESD0543-3.7, generat-
ing pESD0543-4.2. A 2.5 kb DNA fragment having the 5 0
flanking region of the esdC gene, the argB gene, and the
3 0 flanking region was amplified from pESD0543-4.2 with
a primer pair, of which the sequences were 5 0 -CTA TCA
AGT CAA AAA AGC GG-3 0 (543U1) and 5 0 -AGT
CGT TAT TCA TCA CCA TC-3 0 (543L7), and introduced
into VER7. Genomic DNAs of argB+ transformants were
isolated, digested with EcoRV, and subjected to Southern
hybridization by using the 1.1 kb EcoRV-digested frag-
 K.-H. Han et al. / Fungal Genetics and Biology 45 (2008) 310–318 313

ment of pESD0543-4.2 as a probe. Upon Southern hybrid-

EcoRV

EcoRV
BamHI
SmaI

NcoI
XhoI

BglII
SalI
ization, a desired disruptant would generate a 1.7 kb band,
while a wild type generates a 1.1 kb fragment. One
(VDEC72) out of 77 argB+ transformants of VER7 gener-
ated the 1.7 kb fragment upon Southern hybridization. To
confirm further whether VDEC72 has only a deletion 0.5 kb EsdC
mutation of the esdC gene and no other mutations that
affect developments, the strain was crossed with VER7.
Approximately 50% of the segregants from the cross argB

EcoRV
XhoI

BamHI
showed same phenotype of VDEC72, supporting that the
VDEC72 phenotype came from the esdC deletion. A segre-
gant of VDEC72 (VDEC72-1) was selected as a desired
esdC deletion mutant under the veA+ background. A
An 1 M S A V Q L K F N L R T S S N V K T V H L L G SW D N Y S R Q I P L S R D - - S S K
Northern hybridization result that the esdC transcript Af 1 M T A V Q L K F S L R T S S N V K T V H L V G SW D N Y S R Q I P L S R D - - D S K
Ao 1 M S A V Q L K F T L R T S S N V K T V H L L G SW D N Y S R Q I P L S R D - - E G K
was not detected in VDEC72-1 but detected in a wild type Gz
Mg
1
1
M
M
S
S
A
-
I
T
Q
Q
L
L
S
S
F
F
S
S
L
L
R
R
V
V
S
S
A
S
G
G
V
V
K
K
T
S
V
V
H
H
L
L
L
L
G
G
SW
SW
D
D
G
N
Y
Y
V
Q
G
G
Q
Q
L
L
P
P
L
L
S
S
K
K
D
D
K
K
S
T
S
S
S
S
K
K
clearly indicated that VDEC72-1 is a esdC deletion mutant Nc 1 M S A I Q L S F S L R V S S G V K T V H L L G SW D N Y A G Q L P L S K D K T S S K

An 41 P G SW V G K F R F Q T S M L K L G G R YW Y Y Y I LD G Y H V S H D P A A D Y T V
and does not have any other mutation affecting Af 41 P G SW V G K F R F Q T S M L K L G G R YW Y Y Y I MD G Y H V S H D P A V E Y T V
Ao 41 P G SW V G K F R F Q T S M L K L G G R YW Y Y Y I MD G Y H V S H D P A V E Y T I
development. Gz
Mg
43
42
S
S
G
G
SW
SW
K
K
G
G
T
T
F
F
R
R
F
F
Q
P
N
G
S
S
T
T
L
L
E
E
A
S
G
G
Q
Q
R
R
YW
YW
Y
Y
Y
Y
Y
Y
I
I
I D
I D
G
G
Y
Y
H
H
V
C
A
A
H
H
N
N
P
P
S
S
V
E
T
A
S
S
T
T
V
V
Nc 43 S G SW K G T F R F Q P A L V Q P G Q R YW Y Y Y I I D G Y H V S H N P S E E F T V

An 83 E P T T N R K L N I L D V P G G - K E S S S S - - - - - - - - - - - - - - - - - - -
Af 83 E P T T G R K L N I L D V P G G - - K S S N P - - - - - - - - - - - - - - - - - - -
Ao 83 E P T T G R K L N I L D V P G G S K K S S S S - - - - - - - - - - - - - - - - - - -
3. Results Gz
Mg
85
84
E
E
P
P
T
T
T
T
G
G
R
R
E
S
L
L
N
N
V
I
L
L
D
D
V
V
P
A
T
K
D
S
S
S
H
S
K
S
S
S
-
K
-
S
-
S
-
S
-
K
-
S
-
S
-
S
S
S
S
S
R
K
S
S
S
S
S
S
S
R
K
H
S
S
S
S
S
S
K
S
S
K
S
S
Nc 85 E P T T G R S L N I L D V P K S S S S P - - - - - - - - - S S S S S S K S S S R H S

An 105 - - - - - A R R P R R G S S D I A T G R A L S P S K I H H P K P SK P Y A S R Q I R
3.1. The esdC gene encodes a putative glycogen binding Af 104 - - - - - A P K T R R G S N D V V K G R A L S P S K I H H P K P SK P Y A S R Q I R
Ao 106 - - - - - A Q K P R R T S D E V V K G R A P S P S K I H H P K P SK P Y A S R Q I R
domain protein Gz
Mg
119
126
S
S
S
S
S
K
S
S
K
S
S
S
S
S
SN
S S
S
S
K
K
L
R
S
S
V
S
D
D
I
V
P
A
K
K
G
G
R
R
P
P
L
L
S
S
I
V
S
S
Q
Q
I
I
Q
K
A
A
P
P
K
K
P
P
M S
MA
P
P
H
H
A
A
T
T
K
R
H
H
I
I
L
L
Nc 118 R D S V K S S MR E S L S V D I P K G R P L S I S Q I K A P K P M S P H A T R H I L

An 142 E A D F A P T H A MD E L S RR F G S S R L S D D G Y S D L S N S P P S S V G - - -
Southern hybridization of fragments generated by diges- Af 141 E T D F A P - - T M E D L T RR F A G S R M S D E Y S - - Y S N S P P S S V G - - -
Ao 143 E T D F A P - - T M E D L S MR F A G S R L S D E Y S - - L S N S P P S S V G - - -
tion of the 5.5 kb fragment in pESD0543-5.5 with several Gz
Mg
161
168
D
D
A
G
S
S
Y
Y
Y
D
D
D
N
E
G
-
E
E
LD
VA
E
E
L
L
A
A
DR
ER
L
F
G
G
S
Y
A
A
N
T
I
F
E
E
D
D
E
E
F
Y
I
N
T
E
D
D
F
L
S
V
T
T
S
D
P
F
V
G
S S
S S
-
S
-
P
-
V
-
S
-
S
restriction enzymes and partial sequencing of those frag- Nc 160 D A D Y N I D - - - - E L S TR F A S A G I Y D E Y D E D V I T D F D MR S - - - S

An 181 - - - S S L S S R S S - - - G S T S P S S L S S M S D P - - - P S V C H C E R Y G I
ments indicated that a 4.2 kb EcoRI/PstI-digested frag- Af 176 - - - S S L S S R S S - - - G S T S P S S L S S M S D P P - - T P I C R C E R Y G I
Ao 178 - - - S S L S S R S S R S S G S T S P S S L S S M S D P - - - A S V C R C E R Y G I
ment in pESD0543-5.5 contains the entire esdC gene. The Gz
Mg
198
209
-
V
-
G
-
S
-
G
S
S
G
D
S
L
S
S
L
S
S
Y
Y
Y
R
R
S
S
D
D
S
S
S
S
S
S
P
P
N
N
S
S
S
S
L
M
S
S
G
G
Y
Y
S
S
T
T
P
P
G
S
S
S
D
D
V
C
S
S
S
S
C
C
T
T
C
C
E
E
R
R
Y
Y
G
G
I
I
4.2 kb EcoRI/PstI-digested fragment was subcloned into Nc 195 P V S S T G S S V S Y R S D S S S P S S S L S G Y S T P A S D C S S C I C E R Y G I

An 214 T R K G D R V K L D C G G S R C G Y L S E T S G D S C S E D S D S D E E Y R R A R R
pBluescript SK(+), yielding pESD0543-4.2, and the nucle- Af 210 T R K G D R V K L D C G G S R C G Y V T E S S D A S C S E - S D S D E E Y R R A R T
Ao 214 T R K G D R V K L D C G G S R C G Y V T E S S E A S C S E - S D S D E E Y R R A R R
otide sequence of the 3346 bp starting from the EcoRI site Gz
Mg
236
251
T
T
R
R
K
K
G
G
E
D
R
R
V
V
K
R
L
L
D
D
C
C
G
G
G
G
S
S
R
R
C
C
G
G
F
Y
D
D
D
N
D
E
S
S
D
E
S
-
C
C
S
S
S
S
G
G
S
S
E
E
D
D
E
-
Y
-
E
-
Y
-
-
-
E
E
A
Y
V
V
S
S
P
S
A
R
in pESD0543-4.2 was determined completely. Also, a Nc 237 T R K G E R V R L D C G G S R C G Y E D S C S S E D E D S Y V E - - - - - - - - - R

An 256 G V R R Q G I V V R R
1799 bp esdC cDNA was isolated from a cDNA library, Af 251 A V R R Q G I V V R R
Ao 255 G V R R Q G I V V R R
and its nucleotide sequence was determined. Furthermore, Gz
Mg
277
287
S
S
S
S
R
R
R
R
H
N
G
G
V
M
V
V
A
V
-
R
-
A
the 5 0 end of mRNA was identified by 5 0 RACE. The tran- Nc 270 S S R R N G I V V R A

scription start site was mapped 435 bp upstream of the
start codon. The complete nucleotide sequences of the
genomic DNA and its cDNA revealed that the esdC open
reading frame (ORF) encodes a 266 amino acid polypep-
tide, interrupted by one 59 bp intron, and its calculated
molecular mass was 29.4 kDa as shown in Fig. 1 (GenBank
Accession No. AF532169). This structure was in agreement
with the facts that the esdC transcript size is 2.0 kb and that
there is no initiation codon upstream of the putative start
codon. The genomic locus of esdC is AN0859.3. Blast
search of a database from National Center for Biotechnol-
ogy Information (NCBI) (http://www.ncbi.nih.nlm.gov/
BLAST) showed that the putative EsdC protein is well con-
served within various ascomycetes, including Aspergillus
fumigatus, Aspergillus oryzae, Neurospora crassa, Gibber-
ella zeae, and Magnaporthe grisea (Fig. 1b). However, none
of them has been characterized experimentally so far. Fur-
thermore, a domain analysis revealed that the putative
EsdC protein contains a glycogen binding domain con-
served in the b subunit of the AMP-activated protein
Fig. 1. Restriction map of the esdC gene, construction of the esdC-null
mutant and multiple alignment of EsdC homologous proteins found in
different fungi. (a) On the basis of a restriction map of the 2.5 kb SmaI–
EcoRV fragment that contains the esdC gene, the esdC gene was disrupted
as represented schematically. The argB gene obtained by digestion of
pJYargB with XhoI and BamHI was inserted into the pESD0543-3.7 pre-
digested with SalI and BglII, generating pESD0543-4.2. The open box
with an arrow on the restriction map indicates the EsdC ORF. The
hatched box and the vertical line show locations of glycogen binding
domain and an intron, respectively. The open box at the lower part with
an arrow represents the argB gene and restriction enzymes used for
deletion of the esdC gene are also shown. Arrows indicate the orientation
of genes. (b) Multiple alignment of homologous proteins found in various
filamentous fungi. Gray boxes and outlines indicate identical and similar
amino acid residues, respectively. The thick line over the aligned sequence
is a putative glycogen binding domain typically found in the AMPK b
subunit. An, Aspergillus nidulans EsdC; Af, A. fumigatus EsdC
(XP_746739); Ao, A. oryzae AO090038000569 (BAE64337); Gz, Gibber-
ella zeae FG10132.1 (XP_390308); Mg, Magnaporthe grisea MGG_12316
(EDK03697); Nc, Neurospore crassa NCU03600.1 (XP_961327).
kinase (AMPK) complex which can be found from yeast
to human (Machovic and Janecek, 2006).
314 K.-H. Han et al. / Fungal Genetics and Biology 45 (2008) 310–318

3.2. The esdC gene plays an essential role in development

Because the esdC gene was isolated as a gene expressed

early in sexual development, it was anticipated that it plays
a role during this developmental process. To analyze such a
role of the esdC gene, an esdC deletion mutant was con-
structed under a veA+ background (see Section 2). North-
ern analysis of total RNA isolated from VDEC72-1
(DesdC, veA+) showed that no esdC transcript was synthe-
sized (data not shown). Phenotypes of this DesdC mutant
were examined under various conditions as shown in Table
2. VDEC72-1 did not form cleistothecia on CM or on MM
containing casamino acid (MMCA) even after the induc-
tion of sexual development by plate-sealing which induces
formation of plenty of sexual structures in a wild type
(Han et al., 2001; Fig. 2). Simultaneously, VEDC72-1
formed much more conidiophores than a control strain,
VER7 (+pJYargB), under these two conditions. Further-
more, VDEC72-1 did not produce sexual structures and
formed plenty of conidiophores under all conditions exam-
ined so far (Table 2). These results indicated that the esdC
gene promotes sexual development and negatively regulates
conidiophore formation similar to the veA gene. Because
overexpression of veA induces precocious sexual develop-
ment we examined whether overexpression of esdC would
cause a similar effect. However, overexpression of the esdC Fig. 2. Phenotypes of the DesdC mutant. About 106 conidia were
inoculated on 40 ml of CM and incubated for 5 days at 37 °C (a), or on
gene under a veA1 background did not show any effect one
MMCA with the induction of sexual development followed by further
sexual and asexual development (Table 2). Taken together, incubation for 3 days (b). VER7 (+pJYargB) and VDEC72-1 are a wild
these results suggest that the esdC gene is required but type control strain and a DesdC mutant, respectively (Table 1). Arrows
alone is not sufficient for sexual structure formation and indicate mature cleistothecia. The length of the scale bar is 200 lm.
for inhibition of conidiophore formation.
in mycelia cultured for 14 h in liquid medium (indicated by
3.3. The esdC gene expression is regulated during Asexual 0), but present from 6 to 24 h after the transfer of
development the 14 h submerge-cultured mycelial balls onto solid med-
ium. In contrast, the esdC transcript, which appeared in a
To confirm the previous study (Jeong et al., 2000) that small amount at the start of sexual development (indicated
the esdC gene appears to be expressed highly at the ESD by Sexual 0), was increased to a very large amount from 10
stage, esdC expression was analyzed during development. to 20 h after the induction of sexual development, and was
As shown in Fig. 3a, the esdC transcript was not detectable decreased thereafter. This result indicated that esdC expres-

Table 2
Phenotypes of A. nidulans strains on various media
Strain Induction with NaNO3 CM MM MMKa MMCAb
c d
AS S AS S AS S AS S
VER7 (+pJYargB) ++ ++ +++ + +++ + + +++
VDEC72-1 +++  +++  +++  +++ 
RMS011 (+pJYargB)  +++  +++  +++  +++ +
+ +++  +++  +++  +++ +
ROEC11  +++  +++  +++  +++ +
+ +++  +++  +++  +++ +
Approximately 106 conidia were inoculated onto 30 ml of solid medium and incubated at 37 °C for 4 days without induction of sexual development. The
number of conidial heads was determined per 1 mm2 area: +, <50; ++, 100–200; +++, >200. The number of cleistothecial initials was determined per
1 mm2 area: , 0; +, <5; ++, 5–10; +++, >10.
a
MM containing 0.6 M KCl.
b
Sexual development was induced as described in Section 2.
c
Asexual development.
d
Sexual development.
 K.-H. Han et al. / Fungal Genetics and Biology 45 (2008) 310–318
Asexual Sexual
a mutant (data not shown), this result suggested that VeA
0 6 12 18 24 0 2 10 20 30 40 50 h
esdC
rRNA
b
eA
D
eA
-v
sd
WT
OE
Δn
Δv
esdC
rRNA
Fig. 3. esdC gene expression during developmental processes and effect of
the veA and nsdD genes on esdC expression. (a) Total RNA was isolated
from mycelia at different developmental stages. Vegetative mycelia
cultured for 14 h (0) were transferred onto MM and incubated for a
given time as indicated above each lane (Asexual). The same mycelia were
transferred onto MMCA, sealed with parafilm and aluminum foil,
incubated for 24 h to induce sexual development, and incubated further
for a given time as indicated above each lane after removal of sealing
(Sexual). The probe was the 682 bp BamHI/NcoI-digested fragment of the
esdC gene. (b) Total RNA was isolated from ESD stage mycelia (at 2 h
after sexual induction), and hybridized with an esdC-specific probe. WT,
wild type [VER7 (+pJYargB)]; DnsdD, deletion mutant (KHH22) of the
nsdD gene; DveA, deletion mutant (DVAR1) of the veA gene; OE-veA,
overexpressor of the veA gene.
sion is regulated developmentally. Furthermore, this
expression profile was different from that of a house-keep-
ing gene, such as c-actin, in that expression of a house-
keeping gene is very high at the ESD stage, much reduced
thereafter, and is increased again at the late sexual develop-
mental stage such as 40–50 h after the induction of sexual
development (Jeong et al., 2000, 2001). Therefore, the esdC
gene expression profile supported the role of esdC in sexual
development.
3.4. The esdC gene expression is induced by the veA gene
The esdC gene was identified as being required for sex-
ual development and its expression was suggested to be
dependent on the veA gene (Jeong et al., 2000). Like a reg-
ulatory cascade consisting of brlA, abaA and wetA in asex-
ual development, the esdC gene possibly form a regulation
network in sexual development with genes involved in sex-
ual development such as veA and nsdD (Adams et al., 1998;
Han et al., 2001; Kim et al., 2002). To analyze the relation-
ships between esdC and veA as well as nsdD, the effect of
veA and/or nsdD on esdC expression was analyzed. Total
RNA was isolated from ESD stage mycelia of a veA dele-
tion mutant (DveA) and an nsdD deletion mutant (DnsdD),
and hybridized with an esdC-specific probe. As shown in
Fig. 3b, the esdC transcript level was very low in a DveA
mutant and was also reduced in a DnsdD mutant when
compared to that in a wild type. Since the nsdD transcript
315
level was equal in both of the wild type and the veA1
induces esdC expression. To confirm further the depen-
dence of esdC expression on the veA gene, total RNA
was isolated from mycelia of VER7 (+pJYargB; veA+)
and DVAR1 (DveA; Kim et al., 2002) at different stages
of development on MM containing 2% glucose instead of
1% glucose and hybridized with an esdC-specific probe.
As shown in Fig. 4a, the esdC transcript level was higher
at any stage in VER7 than in DVAR1. In addition, the
esdC transcript level was equal in both of the veA+
[VER7 (+pJYargB)] and the veA1 mutant [RMS011
(+pJYargB)] at 30 °C, but higher in the wild type than in
the veA1 mutant at 42 °C (Fig. 4b). This result was consis-
tent with the temperature-sensitivity of the veA1 mutation
(Champe et al., 1981). All of these results more clearly indi-
cated that accumulation of the esdC transcript is dependent
on the presence of the intact veA gene at any stage. Fur-
thermore, it was also examined whether the esdC gene
can suppress the veA1 mutation or the nsdD19 mutation
(Kim et al., 2002; Han et al., 2001). When the esdC gene
was overexpressed in a strain having a veA1 mutation or
an nsdD19 mutation, the overexpressor showed identical
phenotypes to a control strain under conditions shown in
Table 2 (data not shown). These results showed that the
esdC gene does not rescue the veA1 mutation or the nsdD19
mutation, and, therefore, esdC expression is not sufficient
veA+
a Asexual Sexual
0 12 24 0 2 20
esdC
rRNA
ΔveA
Asexual Sexual
0 12 24 0 2 20
esdC
rRNA
b 30oC 42 oC
A+
A1
A+
A1
ve
ve
ve
ve
esdC
rRNA
Fig. 4. Effect of the different veA alleles on esdC expression with various
developmental time points as well as different temperatures. (a) All
experiments were carried out as in Fig. 3 except 2% glucose was added
instead of 1%. (b) Total RNA was isolated from ESD stage mycelia of
VER7 (+pJYargB; veA+) and RMS011 (+pJYargB; veA1) cultured at
30 °C or 42 °C, and hybridized with an esdC-specific probe.
316 K.-H. Han et al. / Fungal Genetics and Biology 45 (2008) 310–318
to direct sexual development in veA or nsdD
background.
3.5. FadA–SfaD G protein is involved in the esdC gene
expression
Upon reception of environmental and/or genetic signals,
a key regulator of asexual development, brlA, is induced
and then in turn brlA induces its downstream genes includ-
ing abaA and wetA (Boylan et al., 1987; Mirabito et al.,
1989; Lee and Adams, 1994). Furthermore, FlbA having
an RGS domain causes inactivation of the GTP-bound
activated FadA, which is a Ga subunit of a heterotrimeric
G protein system, by enhancing the intrinsic GTPase activ-
ity of FadA and results in induction of asexual develop-
ment (Yu et al., 1996). On the other hand, FlbA is also
required for accumulation of mRNA of the nsdD gene that
has been identified as one of genes required for sexual
development and to encode a transcription factor having
a GATA-type zinc-finger motif (Han et al., 2001). In addi-
tion, the nsdD gene is necessary for the accumulation of the
veA gene (unpublished result). Therefore, it was of interest
to examine relationships between the esdC gene and genes
for growth and/or asexual development such as flbA, fadA,
and sfaD which encodes a Gb subunit of a heterotrimeric G
protein complex. To examine the relationships between
those genes, total RNA was isolated from strains having
different mutations in those genes as shown in Fig. 5. Those
mutations included a DfadA, a fadAG42R, a DsfaD, and a
DflbA. The fadAG42R is known as a dominant activating
mutation of the fadA gene (Yu et al., 1996; Wieser et al.,
1997). The esdC transcript level was not detectable in a
fadAG42R mutant and in a DflbA mutant, but higher in a
DfadA and a DsfaD mutant than in a wild type. However,
the fadA, sfaD, and flbA transcript levels were not different
in strains examined in Fig. 5 except for the null mutant of
each gene used as a probe. These results suggested that the
flbA gene is also necessary for esdC expression, but the acti-
vated FadA and SfaD inhibit esdC expression. Since the
amount of the esdC transcript was much lower in fadAG42R
strain, in which a wild type flbA gene is present, and higher
in a DfadA mutant than in a wild type, FlbA may induce
esdC expression by inactivation of the activated FadA
and SfaD protein as shown in Fig. 6.
R
A G 42
dA
faD
bA
WT
Δfa
fad
Δs
Δfl
esdC
rRNA
Fig. 5. Effects of various genetic backgrounds on esdC expression. Total
RNA was isolated from mycelia of various genetic backgrounds, such as
DfadA, DsfaD, and DflbA as well as fadAG42R, cultured on the surface of
liquid MM at 14 h, and hybridized with the esdC-specific probe.
Signal?
Extracellular area
GprD
Cell membrane
Cytoplasm
Gα? Gβγ?
nsdD
Sexual
veA esdC
Development
GpgA FlbA
? ?
SfaD
GPCR?
FadA FadA + SfaD
GDP GTP
GpgA
Growth
Fig. 6. Proposed model of regulation of esdC expression. VeA is needed
for the normal expression of the esdC gene that is necessary for sexual
development. FlbA inactivates the activated FadA and SfaD and each of
the activated FadA and SfaD represses esdC expression. However, it is still
uncertain whether the repression is direct or indirect.
4. Discussion
The esdC gene was originally identified from the EST
library constructed at 2 h after induction of sexual develop-
ment. Since the esdC transcript was supposed to be highly
accumulated at the ESD stage, some positive functions for
sexual development were expected. Indeed, deletion of the
esdC gene caused a block of procession of sexual develop-
ment under standard conditions, implying that the esdC
gene is required for normal sexual development. However,
overexpression of the esdC gene did not induce forced or
unscheduled fruiting body formation as found in the nsdD
or veA overexpressors (Han et al., 2001; Kim et al., 2002).
These results lead us to propose that the esdC gene plays an
important role in sexual development and is necessary but
not sufficient for sexual development.
The putative EsdC protein contains a putative glycogen
binding domain which is found in AMPK b subunit. The
AMPK complex is a multi-substrate enzyme with a, b,
and c subunits, which is activated by a high AMP concen-
tration, and plays a pivotal role in responding to the cellu-
lar energy status by sensing to the cellular AMP/ATP ratio
(Hardie et al., 2006). In yeast, the Snf1 kinase complex,
consisting of Snf1 (a subunit), Sip1/Sip2/Gal83 (b subunit),
and Snf4 (c subunit), is a homolog of the AMPK system
and is required for the cellular response to a metabolic
stress such as carbon source fluctuations (Sanz, 2003).
Although the EsdC protein contains a glycogen binding
domain found in the AMPK b subunit, it is apparent that
EsdC is not a homolog of the b subunit of the AMPK com-
plex. Blast search and domain analysis showed that
AN6570.3 (gi:67540800) is a putative b subunit of the
AMPK complex, because the similarity is higher than the
EsdC and it contains AMPKBI (AMPK beta subunit com-
 K.-H. Han et al. / Fungal Genetics and Biology 45 (2008) 310–318
plex-interacting region) domain at the C-terminus found in
all AMPK b subunits. Rather, as shown in Fig. 1b, the
EsdC protein is highly conserved only in fungi, suggesting
that the esdC gene encodes a fungal specific glycogen bind-
ing protein for regulating developmental processes accord-
ing to the metabolic status. This expectation could be
possible because many different carbon sources affect sex-
ual development in A. nidulans positively or negatively
(Han et al., 2003).
The veA gene was identified as an activator of sexual
development and simultaneously as an inhibitor of asexual
development (Kim et al., 2002). Accordingly, the veA1
mutants form fewer sexual structures than wild type, and
do not form cleistothecia at 42 °C but form at 30 °C
(Champe et al., 1981). Such a temperature-sensitivity in
the sexual structure formation is probably due to a point
mutation in the veA1 allele (Kim et al., 2002). In addition,
VeA is needed for normal level expression of esdC
(Fig. 4a). The esdC transcript level was equal in the wild
type and in the veA1 mutant at 30 °C, but higher in the wild
type than in the veA1 mutant at 42 °C (Fig. 4b). These
results suggested that possible reason of the fact that the
veA1 mutant forms fewer sexual structures than wild type
would be the consequences of reduced amount of the esdC
transcript. However, an overexpressor of the veA gene
forms more sexual structures than the wild type (Kim
et al., 2002), but the esdC transcript level was not higher
in the veA-overexpressor than in the wild type (Fig. 3b),
implying that the increased formation of sexual structures
in the veA-overexpressor is not due to the induction of esdC
expression. Consistently, an esdC-overexpressor in a veA1
background formed almost as an equal amount of cleisto-
thecia as the wild type (Table 2). Taken together, it is sug-
gested that the esdC gene is necessary but not sufficient for
sexual development and its expression is dependent on
VeA.
The flbA gene is necessary for sexual development as
well as for asexual development, and the necessity can be
explained by the requirement of the flbA gene for nsdD
expression (Han et al., 2001). Beside for nsdD expression,
the flbA gene is also necessary for esdC expression as
shown in Fig. 5, indicating that the failure of sexual struc-
ture formation in the DflbA mutant is probably due to the
absence of the esdC transcript as well as the nsdD transcript
in the mutant. Therefore, the relationship between the esdC
gene and the nsdD gene was investigated and found that the
esdC transcript level was lower in the DnsdD mutant than
in the wild type (Fig. 3b), suggesting that NsdD, as a tran-
scription factor, induces esdC expression. Furthermore,
FlbA can regulate esdC and nsdD expression probably
through the FadA and SfaD G protein subunits. When
FlbA is unavailable in the flbA-null mutant, the activated
FadA and SfaD are present that lead to constant signaling
to growth and inhibition of asexual and sexual develop-
ment. In addition, each of the activated FadA and SfaD
represses esdC expression, directly or indirectly, since the
increased amount of the esdC transcripts was present in
317
the DfadA mutant and in the DsfaD mutant than in the wild
type (Fig. 5). When FlbA is available in the wild type, the
reduced amount of the activated FadA and SfaD, com-
pared to that in the DflbA mutant, can not efficiently
repress esdC and nsdD expression, and thus, growth and
sexual development as well as asexual development are bal-
anced. Consistently with this explanation, the dominant-
activating fadAG42R mutant also does not form sexual
structures (data not shown). Therefore, it seems obvious
that the growth signaling from the activated FadA and
SfaD inhibits sexual development, as well as asexual devel-
opment, by repressing esdC and nsdD expression. Also, it
can be suggested that developmental signals act to cause
cessation of growth signaling from the active FadA and
SfaD, and then esdC and nsdD expression is derepressed
to lead to sexual development.
For the genetically programmed development, the
growth signaling from the activated FadA and SfaD
should be ceased by FlbA that inactivates the activated
FadA and SfaD. Upon the inactivation of them, FlbA
causes asexual development and sexual development by
the activation of the brlA gene and the derepression of
the nsdD gene and/or the esdC gene, respectively. Taken
together, the preliminary genetic model for the veA gene,
the esdC gene and sexual development as well as a hetero-
trimeric G protein was proposed in Fig. 6.
Acknowledgments
This work was supported by the Korea Science and
Engineering Foundation (KOSEF) Grants R01-1998-
00053 and R01-2003-000-10162-0 funded to K.S.C. and
J.H.K., respectively.
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