Environmental Microbiology (2016) 18(11), 3689–3701
doi:10.1111/1462-2920.13279
TRI6 and TRI10 play different roles in the regulation of
deoxynivalenol (DON) production by cAMP signalling
in Fusarium graminearum
Cong Jiang,1,2 Chengkang Zhang,2 Chunlan Wu,1
Panpan Sun,1 Rui Hou,1 Huiquan Liu,1
Chenfang Wang1* and Jin-Rong Xu1,2**
1
State Key Laboratory of Crop Stress Biology for Arid
Areas, Northwestern A&F University, Yangling, Shaanxi
712100, China.
2
Department of Botany and Plant Pathology, Purdue
University, West Lafayette, IN 47907, USA.
Summary
The biosynthesis of mycotoxin deoxynivalenol (DON)
in Fusarium graminearum is regulated by two
pathway-specific transcription factors Tri6 and Tri10
and affected by various host and environmental factors. In this study, we showed that cyclic adenosine
monophosphate (cAMP) treatment induced DON production by stimulating TRI gene expression and
DON-associated cellular differentiation in F. graminearum. Interestingly, exogenous cAMP had no
effects on the tri6 mutant but partially recovered the
defect of tri10 mutant in DON biosynthesis. Although
the two cAMP phosphodiesterase genes PDE1 and
PDE2 had overlapping functions in vegetative
growth, conidiation, sexual reproduction and plant
infection, deletion of PDE2 but not PDE1 activated
intracellular PKA activities and increased DON production. Whereas the tri6 pde2 mutant failed to
produce DON, the tri10 pde2 double mutant produced
a significantly higher level of DON than the tri10
mutant. Cellular differentiation associated with DON
production was stimulated by exogenous cAMP or
deletion of PDE2 in both tri10 and tri6 mutants. These
data indicate that TRI6 is essential for the regulation
of DON biosynthesis by cAMP signalling but elevated
PKA activities could partially bypass the requirement
of TRI10 for TRI gene-expression and DON production, and Pde2 is the major cAMP phosphodiesterase
Received 17 January, 2016; Revised: 18 February, 2016; accepted
19 February, 2016. For correspondence. *E-mail wangchenfang@
nwsuaf.edu.cn; Fax. 86-29-8708-1270. Tel. 86-29-8708-1270. **E-mail
[email protected]; Fax. 1-765-494-5896; Tel. 1-765-496-6918.
C 2016 Society for Applied Microbiology and John Wiley & Sons Ltd
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to negatively regulate DON biosynthesis in F.
graminearum.
Introduction
Fusarium head blight (FHB) caused by Fusarium graminearum is a major threat to global wheat production
(Goswami and Kistler, 2004). In addition to severe yield
losses and grain quality reduction, F. graminearum produces trichothecene mycotoxin deoxynivalenol (DON) and
other toxic secondary metabolites in infested grains. DON
is a potent inhibitor of protein synthesis in eukaryotic
organisms. It is harmful to human and animal health and
plays a critical role in plant infection (De Walle et al., 2010;
Audenaert et al., 2014).
All the TRI genes involved in trichothecene biosynthesis
have been identified in F. graminearum and Fusarium sporotrichioides (Brown et al., 2004; Alexander et al., 2009).
Except TRI1, TRI16 and TRI101, the other TRI genes are
in the main TRI gene cluster. Among them, TRI6 and
TRI10 are two major transcriptional regulators of TRI gene
expression (Seong et al., 2009). The binding site of Tri6 in
the promoters of TRI genes have been identified and characterized in previous studies (Seong et al., 2009; Nasmith
et al., 2011). Although the exact function of Tri10 is not
clear, deletion of TRI10 in F. graminearum also significantly
reduces TRI gene expression (Seong et al., 2009). Nevertheless, it is not clear how the Tri6 and Tri10 transcription
factors are activated to regulate the expression of other
TRI genes. The functional relationship between Tri6 and
Tri10 also is not clear. Interestingly, cyclic adenosine
monophosphate (cAMP) signalling and all three MAP
kinase pathways have been shown to be important for
DON biosynthesis in F. graminearum (Hou et al., 2002;
Jenczmionka and Schafer, 2005; Zheng et al., 2012; Hu
et al., 2014). It is likely that some of these key signalling
pathways are involved in transducing extracellular signals
to regulate TRI gene expression via Tri6 and Tri10 activation. Various environmental factors, such as nitrogen
sources, pH and ROS, are known to affect DON biosynthesis in F. graminearum (Merhej et al., 2011; Hou et al.,
2015; Jiang et al., 2015).
3690 C. Jiang et al.
As a key secondary messenger, cAMP synthesized by
adenylate cyclase plays a central role in the transduction
of environmental stimuli to its downstream target cAMP
protein kinase A (PKA). In the budding yeast Saccharomyces cerevisiae and fission yeast Schizosaccharomyces
pombe, cAMP signalling regulates nutrient sensing, spore
germination and mating processes. The adenylate cyclase
gene and genes encoding regulatory and catalytic subunits
of PKA have been functionally characterized in a number
of fungal pathogens (Kronstad et al., 1998; BorgesWalmsley and Walmsley, 2000; Lee et al., 2003; Choi and
Xu, 2010). In addition to its conserved roles in pathogenesis, the cAMP-PKA pathway is important for various
growth and developmental processes in different fungi,
such dimorphic switch and mating in Ustilago maydis and
surface recognition in Magnaporthe oryzae (Durrenberger
et al., 1998; Ramanujam and Naqvi, 2010). In addition,
cAMP signalling has been implicated in regulating secondary metabolisms in several fungi, including F. graminearum
and Aspergillus species (Brakhage and Liebmann, 2005;
Hu et al., 2014). In F. graminearum, functional characterizations of the FAC1, CPK1 and CPK2 genes have
showed that the cAMP-PKA pathway plays a role in morphogenetic switch from vegetative growth to pathogenic
lifestyle, DON production, and sexual reproduction (Bormann et al., 2014; Hu et al., 2014).
Like in other organisms, cAMP is hydrolyzed to 50 -AMP
by phosphodiesterases (PDEs) (Moorthy et al., 2011). In
S. cerevisiae, whereas the high affinity phosphodiesterase
Pde2 controls the basal level of cAMP and protects the
cells from extracellular cAMP, the low affinity phosphodiesterase Pde1 regulates cAMP level induced by glucose
stimulation or acidification (Ma et al., 1999). Deletion of
PDE2 but not PDE1 led to an increased cell size, abnormal
cell wall morphology and increased sensitivities to osmotic
and oxidative stresses (Park et al., 2005). However, S.
pombe has only one phosphodiesterase that functions to
modulate intracellular cAMP level (Matviw et al., 1993).
Like the budding yeast, most filamentous fungal pathogens
have two PDE genes. In M. oryzae, deletion of PDEH, an
ortholog of yeast PDE2, results in an elevated intracellular
cAMP level and defects in appressorium development but
deletion of PDEL has no obvious phenotypes (Ramanujam
and Naqvi, 2010; Zhang et al., 2011). In Botrytis cinerea,
the pde2 deletion mutant is defective in vegetative growth,
differentiation and virulence although it has a reduced
intracellular cAMP level (Harren et al., 2013). In human
pathogen Candida albicans, PDE2 deletion mutant also
has an increased intracellular cAMP level and it is defective in nutrient sensing, filamentation and cell wall and
membrane integrity (Jung et al., 2005). Nevertheless,
Pde1 but not Pde2 regulates the basal cAMP level in Cryptococcus neoformans (Hicks et al., 2005).
To further characterize the relationship between intracellular cAMP and DON biosynthesis, in this study, we
determined the effects of exogenous cAMP on DON production and cellular differentiation associated with DON
production in the wild type and tri6 and tri10 deletion
mutants. Interestingly, we found that cAMP treatment could
partially suppress the defects of tri10 but not tri6 mutant in
DON biosynthesis. We then functionally characterized the
two phosphodiesterase genes, PDE1 and PDE2, in F. graminearum. Although they have overlapping functions in
growth, sexual and asexual reproduction and plant infection,
PDE2 plays a major role in negatively regulating DON production. Deletion of PDE2 also could partially recovered
DON production of tri10 but not tri6 mutant. Our results
showed that the cAMP-PKA pathway interacts differently
with TRI6 and TRI10, and two PDE genes have distinct and
overlapping functions in regulating growth, development,
infection and secondary metabolism in F. graminearum.
Results
Exogenous cAMP increases DON production in F.
graminearum
To determine the effect of cAMP on trichothecene synthesis, the wild-type strain PH-1 (Table 1) was treated with 1,
2 or 4 mM cAMP in Liquid trichothecene biosynthesis
(LTB) medium (Gardiner et al., 2009) and assayed for
DON production after incubation for 7 days (Fig. 1A).
Although treatment with 1 mM cAMP had only a slight
effect, DON production was increased approximately 40folds in cultures treated with 4 mM cAMP (Fig. 1A). As the
negative control, exogenous cAMP had no effect on the
cpk1 cpk2 mutant (Hu et al., 2014) for DON production
(Supporting Information Fig. S1). When assayed by
quantitative reverse transcription polymerase chain
reaction (qRT-PCR), all the TRI genes assayed had
increased expression levels in cultures treated with 4 mM
cAMP (Fig. 1B). The expression level of TRI5 that encodes
the key trichodiene synthase for DON biosynthesis was
increased approximately 45-folds.
Because intracellular cAMP is degraded by cAMP phosphodiesterases (Moorthy et al., 2011), we also assayed
the effects of phosphodiesterase inhibitors on DON production in PH-1. In the presence of 5 mM IBMX, DON
production in 7-day-old LTB cultures was increased 8.5fold in comparison with that of normal PH-1 cultures (Fig.
1C). Caffeine treatment also significantly increased DON
production in F. graminearum (Fig. 1C).
cAMP treatment stimulates cellular differentiation
associated with DON production
It has been reported that DON production is associated
with the formation of intercalary swollen hyphal
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cAMP induced DON production 3691
Table 1. Wild-type and mutant strains of F. graminearum used in this study.
Strain
Genotype description
Reference
PH-1
PH1Dtri6
PH1Dtri10
DM-1
L1
L2
H2
H3
HL4
HL5
DH61
DH62
DH101
DH103
HC1
LC3
Wild-type
tri6 deletion mutant of PH-1
tri10 deletion mutant of PH-1
cpk1 cpk2 deletion mutant of PH-1
pde1 deletion mutant of PH-1
pde1 deletion mutant of PH-1
pde2 deletion mutant of PH-1
pde2 deletion mutant of PH-1
pde1 pde2 double mutant
pde1 pde2 double mutant
tri6 pde2 double mutant
tri6 pde2 double mutant
tri10 pde2 double mutant
tri10 pde2 double mutant
PDE2-GFP complemented transformant of H3
PDE1-RFP complemented transformant of L2
(Cuomo et al., 2007)
(Seong et al., 2009)
(Seong et al., 2009)
(Hu et al., 2014)
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
compartments (Jonkers et al., 2012; Menke et al., 2013).
In PH-1 cultures treated with 4 mM cAMP, more abundant
bulbous structures were observed than the untreated control after incubation for 3 days (Fig. 2A). In fact, hyphal
bulbous structures became visible in cAMP-treated cultures as early as in two days (Fig. 2A). In the wild type,
hyphal bulbous structures were observed only after incubation more than 2 days.
We then assayed DON production in LTB cultures of
PH-1 treated with 4 mM cAMP. Production of DON was
detected in 3-day-old cultures (Fig. 2B). In the untreated
controls, DON was only detectable in 5-day-old cultures.
After incubation for 5 days, DON production in cAMPtreated cultures was 11.3-times higher than untreated cultures (Fig. 2B). These results indicate that cAMP
treatment stimulated cellular differentiation and DON production in juvenile cultures.
Exogenous cAMP partially bypasses the function of
TRI10 in DON regulation
In F. graminearum, TRI6 and TRI10 are two pathwayspecific transcriptional regulators of TRI gene expression
and DON production. To determine the effects of cAMP
treatment on TRI gene regulation, we treated argininecontaining LTB cultures of the tri6 and tri10 mutants (Table 1)
with 4 mM cAMP. Although exogenous cAMP had no effects
on the tri6 mutant, DON production was partially recovered
in the tri10 mutant (Fig. 3A). When assayed by qRT-PCR,
the expression level of TRI5 was increased threefold in the
tri10 mutant in cultures treated with cAMP in comparison
with untreated controls (Fig. 3B). In the tri6 mutant, the
expression level of TRI5 was not affected by exogenous
cAMP (Fig. 3B). These results indicate that cAMP treatment
could partially suppress the effects of TRI10 deletion on TRI
gene expression. However, TRI6 is essential for the regulation of DON biosynthesis by the cAMP-PKA pathway.
Deletion of PDE1 or PDE2 has no significant effects on
conidiation, sexual reproduction and plant infection
Like other eukaryotes, the intracellular cAMP level is regulated by adenylyl synthase and cAMP phosphodiesterase
(PDE) in fungi. The F. graminearum genome contains two
putative phosphodiesterase genes, FGSG_06633 and
FGSG_06914 that are named PDE1 and PDE2 respectively, in this study. PDE1 encodes a 502-aa polypeptide
that has a conserved class II PDE consensus and is orthologous to the low-affinity phosphodiesterase Pde1 of S.
cerevisiae and M. oryzae (Ma et al., 1999; Zhang et al.,
2011). In contrast, PDE2 encodes an 879-aa protein with a
conserved class I PDE consensus and it is orthologous to
high-affinity phosphodiesterase Pde2 of the budding yeast
and M. oryzae (Park et al., 2005; Ramanujam and Naqvi,
2010) (Supporting Information Fig. S2).
To determine the functions of these two PDE genes
and their roles in cAMP signalling in F. graminearum, we
generated the pde1 and pde2 deletion mutants in the
wide-type strain PH-1 by the split-marker approach (Catlett et al., 2003) (Table 1). In comparison with PH-1, the
pde1 mutant had no obvious defects in vegetative
growth and colony morphology (Fig. 4A). The pde2
mutant formed compact colonies that was slightly
reduced in growth rate but the reduction was not statistically significant (Fig. 4A; Table 2). Nevertheless, both
pde1 and pde2 mutants were normal in sexual reproduction (Fig. 4B), conidiation (Table 2), and plant infection
(Fig. 4C). These results indicate that, unlike the importance of PDE2 in asexual development and
pathogenicity in M. oryzae and B. cinerea (Ramanujam
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Fig. 2. Stimulation of cellular differentiation associated with DON
biosynthesis by cAMP.
A. LTB cultures of the wild-type strain PH-1 treated with or without
4 mM cAMP were examined for bulbous structures (marked with
arrows) after incubation for 1, 2 and 3 days. Bar 5 10 lm.
B. Assays for DON production in LTB cultures treated with 0 (black
circles) or 4 mM (white circles) cAMP after incubation for 1, 3 and 5
days.
Deletion of PDE2 significantly increases DON
production and TRI gene expression
Fig. 1. Effects of exogenous cAMP on DON production and TRI
gene expression in the wild-type strain.
A. DON production in LTB cultures treated with 0 (CK), 1, 2 and
4 mM cAMP after incubation for 7 days.
B. TRI gene expression assayed by qRT-PCR with RNA samples
isolated from cultures incubated in the presence of 0 or 4 mM
cAMP for 3 days. The relative expression level of each gene in
cultures without exogenous cAMP was arbitrarily set to 1.
C. 7-day-old LTB cultures with or without 10 mg/ml caffeine or
5 mM IBMX. Mean and standard error were calculated from three
independent biological replicates.
and Naqvi, 2010; Harren et al., 2013), deletion of either
PDE2 or PDE1 had no significant effects on virulence
and sexual reproduction in F. graminearum.
When assayed with LTB cultures, the pde1 mutant was
only slightly increased in DON production in comparison
with PH-1. However, DON production was increased over
91.3-fold in the pde2 mutant (Fig. 5A). Furthermore, the
expression level of TRI5 was increased 84.5-fold in the
pde2 mutant in comparison with PH-1 (Fig. 5B). These
data indicate that PDE2 but not PDE1 plays a negative
role in the regulation of DON biosynthesis. We also
assayed DON production with rice grain cultures and inoculated wheat kernels that developed FHB symptoms by
14 dpi. Similar to results obtained with LTB cultures, DON
production was 9.8- and 9.4-folds higher in rice grain cultures of pde2 mutant than those of PH-1 or pde1 mutant
(Table 2). Although it was reduced in virulence, the pde2
mutant produced a higher concentration of DON in the diseased wheat kernels than the wild type and pde1 mutant
(Table 2).
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Fig. 3. Exogenous cAMP partially rescues the defects of tri10
mutant in DON regulation.
A. DON production in 7-day-old arginine-containing LTB cultures of
the wide type PH-1 and the tri6 and tri10 mutants treated with or
without 4 mM cAMP. nd, not detectable.
B. The expression of TRI5 was assayed by qRT-PCR with RNA
samples isolated from cultures of the tri6 and tri10 mutants treated with
or without 4 mM cAMP after incubation for 3 days. The relative
expression level of TRI5 in untreated samples was arbitrarily set to 1.
When assayed for their expressions in YEPD and LTB
cultures, the expression of PDE2 but not PDE1 was downregulated under DON-producing conditions (Fig. 5C),
which is consistent with a negative role of PDE2 in DON
regulation. After being incubated in LTB cultures for 3
days, bulbous structures were rarely observed in PH-1 and
the pde1 mutant. Under the same conditions, abundant
bulbous hyphal structures were observed in the pde2
mutant (Fig. 5D), which is consistent with increased DON
production.
For complementation assays, we generated the PDE1RFP and PDE2-GFP constructs under the control of their
native promoters and transformed them into the pde1 and
pde2 mutants respectively. In the resulting pde1/PDE1RFP or pde2/PDE2-GFP transformants, no or only faint
Fig. 4. Defects of the pde1 pde2 double mutant in vegetative
growth, sexual production, conidiation and plant infection.
A. Three-day-old cultures of the wide type PH-1, pde1 mutant,
pde2 mutant and pde1 pde2 mutant.
B. Mating cultures of the same set of strains were examined for
perithecia and cirrhi 2-week post-fertilization. Arrows point to cirrhi.
C. Flowering wheat heads of cultivar Norm were drop-inoculated
with conidia from the same set of strains. Spikelets with typical
symptoms were photographed 14 days post-inoculation (dpi).
GFP signals were observed. Like the pde1 mutant, the
pde1/PDE1-RFP transformant had the wild-type growth
rate and colony morphology. However, the pde2/PDE2GFP transformant was normal in colony growth and DON
production (Supporting Information Fig. S3), indicating that
deletion of PDE2 was responsible for the mutant phenotypes observed.
PDE1 and PDE2 have overlapping functions in growth,
development and pathogenesis
We then generated the pde1 pde2 double mutant by deletion of PDE2 in the pde1 mutant (Table 1). The resulting
pde1 pde2 double mutant had a significant reduction in
growth rate than the pde2 single mutant (Table 2) and produced compact colonies with limited aerial hyphae (Fig.
4A). In the presence of 2 mM cAMP, the growth rate of
pde2 mutant was significantly reduced in comparison with
that of the wide-type PH-1 or pde1 mutant. However,
growth of the double mutant was completely blocked
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Fig. 6. Assays for PKA activities and MAP kinase phosphorylation
in PDE deletion mutants.
A. PKA activities were assayed with proteins isolated from hyphae
of the wild type PH-1, pde1 mutant, pde2 mutant and pde1 pde2
double mutant using the PepTag A1 PKA substrate peptide.
Whereas phosphorylated peptides migrated towards the anode (1),
un-phosphorylated peptides migrated towards the cathode (-) on a
0.8% agarose gel. N, non-phosphorylated sample control; P,
phosphorylated sample control.
B. Western blots of total proteins isolated from vegetative hyphae
of the same set of strains were detected with an anti-TpEY antibody
for the phosphorylation of Mgv1 (46-kDa) and Gpmk1 (42-kDa)
bands. The expression level of Pmk1 was detected with the antiPmk1 antibody.
Fig. 5. PDE2 negatively regulates DON production and TRI gene
expression.
A. DON production in 7-day-old LTB cultures of the wide type PH-1,
pde1 mutant, and pde2 mutant.
B. TRI5 expression assayed by qRT-PCR with RNA isolated
from 3-day-old cultures of PH-1 and pde1 or pde2 mutant. Its
relative expression level in PH-1 was arbitrarily set to 1. nd, not
detectable.
C. The relative expression level of PDE1 and PDE2 was assayed
with RNA isolated from 3-day-old YEPD (arbitrarily set to 1) and
LTB (DON-producing) cultures.
D. Three-day-old LTB cultures of the wild type PH-1 and the pde1
and pde2 mutants were examined for bulbous structures (labelled
with arrows). Bar 5 10 lm.
(Supporting Information Fig. S4), indicating that exogenous cAMP may be inhibitory to fungal growth when the
cAMP degradation system is abolished.
Although the pde1 and pde2 mutants were normal in
perithecium development and ascospore release, the pde1
pde2 double mutant was sterile and failed to develop perithecia 2 weeks post-fertilization (Fig. 4B) or after longer
incubation. The double mutant also was significantly
reduced in conidiation and virulence (Table 2). It rarely produced conidia and conidia of the double mutant tended to
have abnormal morphology (Supporting Information Fig.
S5). In infection assays with wheat heads, the pde1 pde2
double mutant has a disease index of less than 1 (Table 2)
and caused FHB symptoms only on the inoculated kernels
(Fig. 4C), indicating that it was defective in spreading from
the inoculation site to nearby spikelets. Therefore, PDE1
and PDE2 must have overlapping functions in hyphal
growth, conidiation, sexual development and plant infection
in F. graminearum.
We also assayed DON production with the pde1 pde2
double mutant. In comparison with the pde2 mutant, the
pde1 pde2 double mutant was increased in DON production in rice grain cultures and diseased wheat kernels
(Table 2). These results suggest that, although Pde2 is the
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cAMP induced DON production 3695
although PKA activity was normal in the pde1 mutant, the
double mutant had a higher PKA activity than the pde2
mutant, indicating that Pde1 also negatively regulates PKA
activities, particularly in the absence of Pde2 (Fig. 6A).
To determine the relationship between cAMP signalling
and MAP kinase pathways, we assayed the activation of
Gpmk1 and Mgv1 in the wild type and PDE deletion
mutants. When detected with an anti-MAPK antibody, the
42-kD band had similar intensities in all the strains
assayed. However, when detected with an anti-TpEY antibody, phosphorylation of Gpmk1 was significantly reduced
in the pde2 and pde1 pde2 double mutants (Fig. 6B).
Phosphorylation of the 46-kD Mgv1 band also was
reduced in the pde2 and pde1 pde2 mutants (Fig. 6B).
These results indicate that an elevated level of intracellular
cAMP may have a negative impact on the activation of
Gpmk1 and Mgv1 MAP kinases, which may be related to
the defect of pde2 mutant in plant infection although it was
increased in DON production.
The tri10 pde2 but not tri6 pde2 mutant is partially
recovered in DON regulation
Fig. 7. Assays for DON production and TRI gene expression in the
tri10 pde2 and tri6 pde2 mutants.
A. DON production in the wide type PH-1, tri6 mutant, tri6 pde2
mutant, tri10 mutant and tri10 pde2 mutant in 7-day-old argininecontaining LTB cultures. nd, not detectable.
B. TRI5 expression in 3-day-old cultures of PH-1 and the tri6, tri6
pde2, tri10 and tri10 pde2 mutants. Its relative expression level in
PH-1 was arbitrarily set to 1.
C. Three-day-old LTB cultures of PH-1, pde2, tri6 pde2, and tri10
pde2 strains were examined for bulbous structures after incubation
for 3 days. Bar 5 10 lm.
major regulator, Pde1 also plays a minor role in negatively
regulating DON production in F. graminearum.
Deletion of PDE2 increases PKA activities but reduces
MAP kinase activation
To determine the effect of PDE deletion on the activation of
PKA, we assayed PKA activities with proteins isolated
from hyphae of YEPD cultures. In comparison with PH-1,
the pde2 mutant had a higher PKA activity. Interestingly,
To confirm different effects of exogenous cAMP on DON
production in the tri6 and tri10 mutants, we generate the
tri6 pde2 and tri10 pde2 double mutants (Table 1). Same
to the tri6 mutant, the tri6 pde2 double mutant failed to produce DON in arginine-containing LTB cultures. In contrast,
the tri10 pde2 double mutant produced significantly more
DON than the tri10 mutant, although not as much as the
wild type in 7-day-old arginine-containing LTB cultures
(Fig. 7A).
When assayed for TRI gene expression, TRI5 was significantly down-regulated in the tri6 pde2 and tri6 mutant in
comparison with PH-1 (Fig. 7B). Therefore, deletion of
PDE2 could not recover DON production or TRI gene
expression in the tri6 mutant. In contrast, TRI5 expression
was significantly increased in the tri10 pde2 mutant compared to the tri10 mutant (Fig. 7B). These results further
indicate that over-stimulation of the cAMP-PKA pathway
could partially suppress the defects of the tri10 but not tri6
mutant.
The tri6 pde2 mutant has increased bulbous structures
Interestingly, although DON biosynthesis was blocked in
the tri6 pde2 mutant, it produced abundant bulbous structures in 3-day-old LTB cultures (Fig. 7C), which is similar to
the pde2 single mutant. Under the same conditions, the
wild type strain rarely had bulbous structures (Fig. 7C).
Similar to the pde2 mutant, the tri10 pde2 double mutant
also was increased in the production of intercalary bulbous
structures in comparison with the wild type strain (Fig. 7C).
These results indicate that cellular differentiation
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3696 C. Jiang et al.
Fig. 8. Proposed model for DON regulation by cAMP signalling in
F. graminearum.
Both exogenous cAMP and intracellular cAMP synthesized by the
Fac1 adenylate cyclase directly bind with regulatory subunits (R) to
release the catalytic subunits (C) of PKA for activation and
induction of DON production in F. graminearum. The two cAMP
phosphodiesterase genes PDE1 and PDE2 have overlapping
function in modulating the basal intracellular level of cAMP during
vegetative growth and asexual reproduction but PDE2 plays a
major role in DON regulation. Overstimulation of the cAMP-PKA
pathway may release the self-inhibition of TRI6 and up-regulate TRI
gene expression. TRI10, another key transcription regulator of DON
production, may be indirectly regulated by PKA via its interaction
with AreA that is likely phosphorylated by PKA. The regulation of
cellular differentiation associated with DON biosynthesis is
independent of TRI6 or TRI10. FGP1, a WOR1 ortholog in F.
graminearum, may function as one of the transcription factors
regulating cellular differentiation stimulated by cAMP during DON
production.
stimulated by deletion of PDE2 is independent of TRI6 or
TRI10. Therefore, TRI6 may function downstream from the
cAMP signalling pathway to regulate DON production but it
is dispensable for DON production-related cellular
differentiation.
Discussion
As a second messenger, cAMP plays a key role in the regulation of morphogenesis, nutrient sensing, secondary
metabolism and virulence in various fungal pathogens (Xu
and Hamer, 1996; Freitas et al., 2010; Hu et al., 2014). In
this study, we showed that DON production was induced
by treatment with exogenous cAMP or IBMX that is stimulatory to intracellular cAMP signalling (Fig. 8). Treatment
with 4 mM cAMP significantly increased DON production
and TRI gene expression. Although the cAMP-PKA pathway has been implicated in the regulation of secondary
metabolism in fungal pathogens (Freitas et al., 2010; Studt
et al., 2013), to our knowledge significant stimulation of
mycotoxin production by exogenous cAMP has not been
reported in plant pathogenic fungi. A report on the effect of
exogenous cAMP on secondary metabolism is in Monascus species, in which treatment with 10 mM cAMP
represses the production of lovastatin, red pigments and
citrinin (Miyake et al., 2006). Results from our pharmacological studies with cAMP and IBMX are consistent with
earlier publications on the positive regulatory role of the
FAC1, CPK1 and CPK2 genes in F. graminearum
(Bormann et al., 2014; Hu et al., 2014). However, the
cAMP-PKA pathway is known to negatively regulate the
biosynthesis of fusarubins and positively regulate bikaverin
biosynthesis in Fusarium fujikuroi (Studt et al., 2013).
Interestingly, we observed that exogenous cAMP stimulated the differentiation of bulbous hyphae or intercalary
bulbous structures associated with DON production in F.
graminearum. In plant pathogenic fungi, cAMP signalling is
known to regulate pathogenesis-related morphogenesis
(Borges-Walmsley and Walmsley, 2000; D’Souza and Heitman, 2001). In M. oryzae, the cAMP-PKA pathway is
important for surface recognition and initiation of appressorium formation (Xu et al., 1997). Exogenous cAMP
induces appressorium formation on hydrophobic surfaces
(Lee and Dean, 1993). Disruption of cAMP signalling also
results in defects in appressorium formation in Colletotrichum lagenarium and Colletotrichum trifolii (Yang and
Dickman, 1999; Yamauchi et al., 2004). In F. graminearum,
DON is one of the best characterized pathogenicity factors
and DON production during plant infection is affected by
various environmental and plant factors (Boutigny et al.,
2010; Schreiber et al., 2011; Hou et al., 2015; Jiang et al.,
2015). It is likely that the regulation of cellular differentiations associated with DON production by cAMP signalling
in F. graminearum may involve similar mechanisms regulating pathogenesis-related morphogenesis in other fungal
pathogens in response to the physical or chemical signals
of host plants.
Although their functional relationship is not clear, TRI6
and TRI10 are two pathway-specific transcriptional regulators of trichothecene biosynthesis. TRI genes expression
was blocked or significantly reduced in both tri6 and tri10
deletion mutants (Seong et al., 2009). Interestingly, we
found that treatment with exogenous cAMP had different
effects on the tri6 and tri10 mutants in DON production
(Fig. 8). In the tri10 mutant, cAMP treatment stimulated
TRI5 gene expression and DON production, suggesting
that overstimulation of the cAMP-PKA pathway could
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cAMP induced DON production 3697
partially suppress its defects. This observation was further
confirmed by increased DON production in the tri10 pde2
mutant. Deletion of PDE2 resulted in an increase in the
intracellular cAMP level, which likely had similar effects as
exogenous cAMP in the tri10 mutant background. These
results indicate that the regulatory function of TRI10 in trichothecene biosynthesis could be somehow partially
bypassed by elevated PKA activities. Therefore, TRI10
may only play a non-essential or indirect role in the regulation of trichothecene biosynthesis by cAMP signalling in F.
graminearum. It is likely that TRI10 is not directly activated
by PKA or its normal activation involves PKA and other signalling pathways. In F. graminearum, Tri10 has been
shown to interact with AreA, a transcription factor that positively regulates TRI gene expression and DON regulation
(Hou et al., 2015). AreA is the key regulator of nitrogen
metabolism in filamentous fungi. In F. graminearum, the
conserved PKA phosphorylation site S874 is important for
AreA function and the areA deletion mutant is significantly
reduced in DON production, PKA activities, and ammonium permease (MEP) gene expression (Hou et al., 2015).
The interaction between Tri10 and AreA may be affected
by exogenous cAMP or deletion of PDE2 (Fig. 8). It is also
possible that elevated PKA activities may allow AreA to
function in the absence of Tri10 in growth or developmental
processes that normally require the Tri10-AreA protein
complex.
Unlike the tri10 mutant, exogenous cAMP had no effects
on the tri6 mutant for DON biosynthesis. DON production
also was not detected in the tri6 pde2 mutant. Exogenous
cAMP or deletion of PDE2 failed to induce the expression
of TRI5 and TRI12 in the tri6 deletion background. Therefore, it is likely that regulation of DON production by cAMP
signalling directly involves TRI6. When analysed with NetPhosK 1.0 (Blom et al., 2004), two conserved PKA
phosphorylation sites, S61 and S117, were identified in the
predicted Tri6 protein. To determine whether TRI6 indeed
functions as a direct downstream target of the cAMP-PKA
pathway, it will be important to experimentally characterize
the PKA-phosphorylation sites in Tri6 and determine their
roles in the regulation of DON biosynthesis in F. graminearum. Interestingly, the expression of TRI6 itself was upregulated when treated with 4 mM cAMP. Binding of Tri6 to
its own promoter has been shown to negatively regulate
TRI6 expression (Nasmith et al., 2011). It is possible that
exogenous cAMP or deletion of PDE2 results in the hyperphosphorylation of Tri6, which may release its selfinhibitory binding to the TRI6 promoter.
Hydrolysis of cAMP by PDEs plays an important role in
regulating the intracellular concentration of this second
messenger. In most fungi, the high-affinity PDE play a
major role in regulating the basal cAMP level and PKA
activities (Ramanujam and Naqvi, 2010; Harren et al.,
2013) although the low-affinity PDE appears to be more
important in C. neoformans (Hicks et al., 2005). Whereas
mutants deleted of the yeast PDE2 ortholog are defective
in sexual reproduction and pathogenesis, deletion of the
PDE1 ortholog usually had no obvious phenotypes in plant
pathogenic fungi M. oryzae and B. cinerea (Ramanujam
and Naqvi, 2010; Harren et al., 2013). In F. graminearum,
PDE2 plays a more important role than PDE1 in the regulation of trichothecene biosynthesis (Fig. 8). However,
PDE1 and PDE2 have redundant functions in conidiation,
sexual reproduction and plant infection. Whereas the pde1
and pde2 deletion mutants had no obvious defects, the
pde1 pde2 double mutant was significantly reduced in conidiation and virulence and blocked in sexual reproduction
in F. graminearum, suggesting that PDE1 and PDE2 are
equally important for cAMP phosphodiesterase activities
during sexual/asexual differentiation and plant infection.
This observation is different from earlier studies with PDE
genes in other fungal pathogens (Ramanujam and Naqvi,
2010; Harren et al., 2013). It will be important to determine
the molecular mechanisms responsible stage-specific
functional redundancy and differences between Pde1 and
Pde2 during conidiation, sexual reproduction, DON production and plant infection in F. graminearum.
Deletion of PDE1 had no obvious effects on hyphal
growth but the pde2 mutant deletion mutant formed compact colonies and had a slight reduction in growth rate.
Therefore, similar to its role in DON biosynthesis, PDE2
likely is more important and PDE1 in vegetative hyphae.
Hyphal growth of the pde2 but not pde1 mutant was hypersensitive to exogenous cAMP, further proving that Pde2
plays a major role in cAMP phosphodiesterase activities
during vegetative growth. These results indicate that the
functional relationship between Pde1 and Pde2 varies in
different growth and developmental stages in F. graminearum. It will be important to determine what factors
determine when Pde1 and Pde2 have similar functions
during conidiation, sexual reproduction and plant infection
or when Pde2 plays a major role in cAMP phosphodiesterase activities during vegetative growth and DON
production in F. graminearum. One possibility is that the
intracellular cAMP level may differ among various cell
types, affecting the requirement of Pde1 and Pde2 cAMP
phosphodiesterase.
Like treatment with exogenous cAMP, deletion of PDE2
stimulated the formation of intercalary bulbous structures.
In F. graminearum, this type of hyphal bulbous structures
developed before TRI gene transcription is tightly related
to DON production (Jonkers et al., 2012). Similar to the
pde2 mutant, the tri6 pde2 and tri10 pde2 double mutants
also produced abundant intercalary hyphal bulbous structures. These results indicated that the formation of hyphal
bulbous structures stimulated by cAMP or deletion of
PDE2 is independent of TRI6 or TRI10. Although it
remains possible that TRI6 or TRI10 have redundant roles
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V
3698 C. Jiang et al.
Table 2. Defects of pde mutants in growth, conidiation, DON production, and virulence.
DON production (ppm)c
Strains
PH-1
pde1 mutant
pde2 mutant
pde1 pde2 mutant
Growth rate (mm/day)a
A
12.7 6 0.1
12.5 6 0.2A
11.5 6 0.2A
6.3 6 0.2B
Conidiation (105/ml)b
A
12.7 6 0.8
13.1 6 1.3A
11.0 6 0.9A
0.2 6 0.0B
Rice grains
Wheat kernels
A
885.3 6 76.1
922.3 6 180.8A
8700.0 6 722.7B
10962.7 6 301.2B
A
753.3 6 110.3
822.4 6 124.0A
2536.7 6 285.1B
2838.1 6 334.6B
Disease Indexd
8.3 6 0.6A
8.0 6 1.0A
6.3 6 0.5B
0.8 6 0.4C
a. Average daily extension in colony radius on PDA plates.
b. Conidiation in 5-day-old CMC cultures.
c. DON production assayed with rice grain cultures and diseased wheat kernels.
d. Disease index was estimated as the number of diseased spikelets on each inoculated wheat heads 14 days post-inoculation (dpi).
*Mean and standard deviation were calculated with results from three replicates for growth rate and conidiation assays and five replicates for
DON and infection assays. Data were analyzed with Duncan’s pair wise comparison. Different letters mark statistically significant differences
(P 5 0.05).
in this cellular differentiation, other transcription factors
may be involved in regulating the development of hyphal
bulbous structures associated with DON production. One
candidate transcription factor is Fgp1, the Wor1 ortholog,
that functions as a positive regulator of DON synthesis in
F. graminearum (Jonkers et al., 2012). The fgp1 deletion
mutant rarely forms hyphal bulbous structures associated
with DON production. In C. albicans, Wor1 is phosphorylated by Tpk2, a subunit of PKA, for activation (Huang
et al., 2010). The Fgp1 protein has a conserved PKA phosphorylation site and it may be phosphorylated by PKA in F.
graminearum as a downstream transcription factor (Fig. 8).
Experimental procedures
Strains and phenotype observation
All the wild-type and mutant F. graminearum strains and transformants generated in this study were listed in Table 1. Potato
dextrose agar (PDA) cultures grown at 258C were used for
assaying growth rate and colony morphology. Conidiation was
assayed with 5-day-old carboxymethylcellulose (CMC) cultures as described (Hou et al., 2002). For mating assays,
aerial hyphae of 7-day-old carrot agar cultures were pressed
down with 0.1% Tween 20 and further incubated at 258C
under black light as described (Bowden and Leslie, 1999).
Perithecium formation, production of cirrhi, and ascospore
release were examined 10–14 days post-fertilization (dpf). CM
medium with 2 mM cAMP was used to assay for sensitivities
of the pde mutants to exogenous cAMP.
Generation of the pde1, pde2 and pde1 pde2 deletion
mutants
To generate the gene replacement construct for PDE1 with
the split-marker approach (Catlett et al., 2003), the 0.9-kb
upstream and 0.9-kb downstream flanking sequences were
amplified by polymerase chain reaction (PCR), connected
with the hygromycin phosphotransferase (hph) cassette by
overlapping PCR, and transformed into protoplasts of PH-1 as
described (Hou et al., 2015; Jiang et al., 2015). Hygromycinresistant transformants were screened by PCR and putative
pde1 deletion mutants were confirmed by Southern blot analy-
sis. The same approach was used to generate the PDE2
deletion mutants. To generate the pde1 pde2 double mutants,
the PDE2 gene replacement construct was generated with the
neomycin resistance gene (Jiang et al., 2015) and transformed into the pde1 mutant. Transformants resistant to both
hygromycin and G418 were confirmed by PCR.
For complementation assays, the PDE2-GFP fusion construct was generated by the gap repair approach by cotransformation of the PDE1 gene fragment and XhoI-digested
pXY201 (Zhou et al., 2011) into yeast strain XK1-25 as
described (Bruno et al., 2004). The resulting PDE2-GFP
fusion construct recovered from Trp1 yeast transformants was
confirmed by sequencing analysis. The same approach was
used to generate the PDE2-GFP fusion construct with the
pDL2 vector (Zhou et al., 2011). The PDE1-RFP and PDE2GFP constructs were then transformed into protoplasts of the
pde1 and pde2 mutants respectively. Transformants resistant
to both hygromycin and bleomycin were screened by PCR
and examined for GFP and RFP signals as described (Zhou
et al., 2012).
Plant infection
Conidia were harvested from 5-day-old CMC cultures and resuspended to a final concentration of 105 spores per milliliter
in sterile distilled water as described (Zhou et al., 2010; Jiang
et al., 2015). Flowering wheat heads of cultivar Norm were
drop-inoculated at the fifth spikelet from the bottom with 10 ll
of conidium suspensions as described (Gale et al., 2002). To
keep the moisture, inoculated wheat heads were capped with
a plastic bag for 48 h. After removing plastic bags, inoculated
wheat plants were grown under normal conditions in a greenhouse. Scab symptoms were examined 14 days postinoculation (dpi) and infected wheat kernels were harvested
and assayed for DON production as described (Bluhm et al.,
2007).
Assays for stimulatory effects of cAMP on DON
production and TRI gene expression
For assaying its stimulatory effects on DON production, cAMP
(Sigma, USA), were added to a final concentration of 1, 2 and
4 mM to LTB culture as described (Gardiner et al., 2009).
IBMX (Sigma, USA) and caffeine (Sigma, USA) were added
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V
cAMP induced DON production 3699
to LTB to the final concentration of 5 mM and 10 mg/ml
respectively. DON production in LTB cultures was assayed
with a competitive ELISA based DON detection plate kit (Beacon Analytical Systems, Inc, USA) after incubation at 258C for
1, 3 or 5 days as described (Gardiner et al., 2009).
For assaying TRI gene expression, hyphae were harvested
from 3-day-old LTB cultures and used for RNA isolation with
the TRIzol reagent (Invitrogen, USA). First-strand cDNA was
synthesized by iScriptTM cDNA Synthesis Kit (BIO-RAD, USA)
R Green
and used for qPCR with an iTaqTM Universal SYBRV
Supermix (BIO-RAD, USA) following manufacturer’s instructions. The beta-tubulin gene of F. graminearum was used as
the internal control. Relative expression levels of each gene
were calculated with the 2–䉭䉭Ct method (Livak and Schmittgen, 2001). The mean and standard deviation were calculated
with data from three independent biological replicates.
Assays for TEY phosphorylation and PKA activities
Hyphae were harvested from 48 h YEPD (Yeast extract peptone dextrose) cultures (Zheng et al., 2012) by filtration
through two layers of Miracloth (Sigma, USA) and washed
with sterile distilled water. Proteins were isolated from vegetative hyphae as described (Zheng et al., 2012). PKA activities
were assayed with the PepTag nonradioactive PKA assay kit
(Promega, Madison, WI) as described (Adachi and Hamer,
1998). For western blot analyses, total proteins were separated on 10% SDS-PAGE gels and transferred to
nitrocellulose membranes (Zhou et al., 2012). Phosphorylation
of Gpmk1 and Mgv1 was detected with the PhophoPlus p44/
42 MAP kinase antibody kit (Cell Signaling Technology, USA)
(Ding et al., 2009). Detection with an anti-Pmk1 antibody
(Bruno et al., 2004) was used as the expression control.
Acknowledgements
We thank Dr. Shijie Zhang, Mr. Qiang Zhang, and Mr. Tao Yin
for assistance with DON measurement. We also thank Dr.
Jianhua Wang and Dr. Qinhu Wang for and bioinformatics
analysis. This work was supported by the National Major
Project of Breeding for New Transgenic Organisms
(2012ZX08009003), Program for New Century Excellent Talents in University (NCET-12-0472), National Natural Science
Foundation of China (No. 31271989 and 31301607), The Special Fund for Agro-scientific Research in the Public Interest
(201303016) and Youth Science & Technology star of Shaanxi
(2014KJXX-41). The authors have no conflict of interest to
declare.
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Supporting information
Additional Supporting Information may be found in the
online version of this article at the publisher’s web-site:
Fig. S1. Effect of exogenous cAMP on DON production in
the cpk1 cpk2 mutant.
DON production was assayed with arginine-containing LTB
cultures of the cpk1 cpk2 double mutant in the presence of
0 or 4 mM cAMP. The wild-type strain PH-1 was used as
the control. nd, not detectable.
Fig. S2. Identification of two phosphodiesterase genes in F.
graminearum.
A. Sequence alignment of the predicted active sites of the
low-affinity cAMP phosphodiesterase from Neurospora
crassa, B. cinerea, Aspergillus nidulans, M. oryzae, Verticillium dahlia, C. albicans, S. cerevisiae, and F. graminearum.
The lower panel shows the class II PDE consensus
sequence.
B. Sequence alignment of the predicted active sites of the
high-affinity cAMP phosphodiesterase from marked fungi.
The lower panel shows the class I PDE consensus
sequence.
Fig. S3. Colony growth and DON production of the pde2/
PDE2-GFP transformant.
A. Cultures of PH-1 and pde2/PDE2-GFP transformant
grown on CM for 4 days.
B. DON production in 7-day-old LTB cultures of PH-1 and
pde2/PDE2-GFP transformant.
Fig. S4. Increased sensitivity of the pde2 and pde1 pde2
mutants to exogenous cAMP. Cultures of PH-1 (Upper),
pde1 mutant (Left), pde2 mutant (Right) and pde1 pde2
double mutant (Lower) grown on CM medium with 2 mM
cAMP. Photos were taken after incubation for 4 days.
Fig. S5. Conidia of PH-1 and the pde1, pde2 and pde1
pde2 mutants. Conidium morphology was examined with
conidia harvested from 5-day-old CMC cultures. Bar 5 10
lm.
C 2016 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology, 18, 3689–3701
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