Thioredoxins are involved in the activation of the PMK1...
Transcript of Thioredoxins are involved in the activation of the PMK1...
Thioredoxins are involved in the activation of thePMK1 MAP kinase pathway during appressoriumpenetration and invasive growth in Magnaportheoryzae
Shijie Zhang,1† Cong Jiang,1,2† Qiang Zhang,1
Linlu Qi,2 Chaohui Li1 and Jin-Rong Xu2*1State Key Laboratory of Crop Stress Biology for Arid
Areas, College of Plant Protection, Northwest A&F
University, Yangling, Shaanxi 712100, China.2Department of Botany and Plant Pathology, Purdue
University, West Lafayette, IN 47907, USA.
Summary
In Magnaporthe oryzae, the Mst11-Mst7-Pmk1 MAP
kinase pathway is essential for appressorium forma-
tion and invasive growth. To determine their roles in
Pmk1 activation and plant infection, we charac-
terized the two thioredoxin genes, TRX1 and TRX2,
in M. oryzae. Whereas the Dtrx1 mutants had no
detectable phenotypes, deletion of TRX2 caused
pleiotropic defects in growth, conidiation, light
sensing, responses to stresses and plant infection
progresses. The Dtrx1 Dtrx2 double mutant had
more severe defects than the Dtrx2 mutant and was
non-pathogenic in infection assays. The Dtrx2 and
Dtrx1 Dtrx2 mutant rarely formed appressoria on
hyphal tips and were defective in invasive growth
after penetration. Pmk1 phosphorylation was barely
detectable in the Dtrx2 and Dtrx1 Dtrx2 mutants.
Deletion of TRX2 affected proper folding or intra-/
inter-molecular interaction of Mst7 and expression
of the dominant active MST7 allele partially rescued
the defects of the Dtrx1 Dtrx2 mutant. Furthermore,
Cys305 is important for Mst7 function and Trx2
directly interacts with Mst7 in co-IP assays. Our data
indicated that thioredoxins play important roles in
intra-cellular ROS signalling and pathogenesis in M.
oryzae. As the predominant thioredoxin gene, TRX2
may regulate the activation of Pmk1 MAPK via its
effects on Mst7.
Introduction
Rice blast caused by Magnaporthe oryzae is one of the
most destructive fungal diseases of rice worldwide.
Although root infection has been observed under labora-
tory conditions, M. oryzae is a typical foliar pathogen that
initiates plant infection by the formation of a highly special-
ized infection structure known as an appressorium. The
pathogen then uses turgor pressure generated inside
heavily melanized appressoria for the penetration of plant
cuticle and cell wall (de Jong et al., 1997). After penetra-
tion, highly vacuolated, bulbous invasive hyphae are
enveloped by the extra-invasive-hyphal membrane (EIHM)
produced by the plant cells (Kankanala et al., 2007). Dur-
ing this biotrophic phase, M. oryzae delivers various
effectors into plant cells via the conventional ER to Golgi
secretory pathway or the Golgi-independent system involv-
ing the biotrophic interfacial complex (BIC) to avoid or
suppress plant defense responses (Khang et al., 2010;
Zhang and Xu, 2014). At later infection stages, fungal inva-
sive growth results in plant cell death, and conidia are
produced by M. oryzae on blast lesions to re-initiate the
infection cycle for disease spreading in the field.
In the past two decades, M. oryzae has been extensively
studied for infection-related morphogenesis and fungal-
plant interactions (Dean et al., 2005). Over a 100 genes
with diverse functions have been shown to be required for
plant infection in this model plant pathogen, including vari-
ous genes important for appressorium penetration and key
components of well-conserved signal transduction path-
ways (Jeon et al., 2007; Li et al., 2012). Whereas the
cAMP signalling pathway controls surface recognition and
appressorium initiation, late stages of appressorium forma-
tion is regulated by the Pmk1 MAP kinase (MAPK)
pathway, which also is required for appressorium penetra-
tion and invasive growth after penetration, two other critical
infection processes in the infection cycle of M. oryzae
(Zhao and Xu, 2007). Pmk1 is orthologous to Kss1 and
*For correspondence. E-mail [email protected]; Tel. 1-765-496-6918; Fax 765-494-0363. †These authors contributed equally tothis work.
VC 2016 Society for Applied Microbiology and John Wiley & Sons Ltd
Environmental Microbiology (2016) 18(11), 3768–3784 doi:10.1111/1462-2920.13315
the pmk1 deletion mutant is non-pathogenic (Xu and
Hamer, 1996). A number of genes functioning upstream
from Pmk1, including the MAPK kinase (MEK) Mst7 and
MEK kinase (MEKK) Mst11, and an adaptor protein Mst50
have been identified (Zhao et al., 2005; Park et al., 2006).
Several downstream targets regulated by Pmk1 also have
been functionally characterized, including MST12, SLF1,
GAS1, and GAS2 (Park et al., 2002; Xue et al., 2002; Li
et al., 2011). Mutants deleted of MST12, one of the down-
stream transcription factors of the PMK1 pathway, still form
melanized appressoria but are defective in appressorium
penetration and invasive growth (Park et al., 2004). In addi-
tion, the cell wall integrity Mps1 MAPK and calcium
signalling pathways also are known to be important for
appressorium morphogenesis, penetration, and invasive
growth in M. oryzae (Xu et al., 1998).
Reactive oxygen species (ROS) produced by NADPH
oxidases or in mitochondria serve as critical signalling mol-
ecules or secondary messengers in cell proliferation and
survival in eukaryotic organisms. In mammalian cells, ROS
can induce or mediate activation of the MAPK pathways
(McCubrey et al., 2006). The oxidative modification of sig-
nalling proteins by ROS (Thannickal and Fanburg, 2000) is
one of the possible mechanisms for the activation of
MAPK pathways. Apoptosis signal-regulated kinase 1
(ASK-1), an upstream MEK kinase that regulates the JNK
and p38 MAPK pathways (Ichijo et al., 1997), binds to
reduced thioredoxin in non-stressed cells. Under oxidative
stress, thioredoxins become oxidized and disassociated
from ASK-1, leading to activation of JNK and p38 path-
ways through oligomerization of ASK-1 (Nagai et al.,
2007). In mammalian cells, protein kinase A (PKA) was
also shown to be activated by ROS through formation of
intramolecular disulfide bonds. Cys199 in the catalytic sub-
unit of PKA was involved in the formation of a disulfide
bond when treated with H2O2, leading to dephosphoryl-
ation of Thr197 and thus resulted in PKA inactivation
(Humphries et al., 2005). Inhibition of phosphatases by
ROS also has been shown to regulate JNK (Kamata et al.,
2005) and p38 signalling (Liu et al., 2010a). In Arabidopsis,
H2O2 activates the MAP kinases MPK3 and MPK6 via
MEK or MEK kinase ANP1 (Kovtun et al., 2000). ROS-
induced activation of MAPKs appears to be central for
mediating cellular responses to multiple stresses, although
the mechanism for its activation of MAPK pathways is not
clear (Apel and Hirt, 2004).
An important redox system is formed by the thiore-
doxin (TRX) system. Thioredoxins are a family of small,
ubiquitous proteins with a conserved active site
sequence (Trp-Cys-Gly-Pro-Cys) that catalyzes a vari-
ety of redox reactions via reversible redox-active
disulfide/dithiol. Due to the redox-active cysteine pair at
the active site, thioredoxins can cycle between the oxi-
dized disulfide (Trx-S2) and reduced dithiol [Trx-(SH)2]
forms. They alter the redox state of target proteins via
the reversible oxidation of an active site dithiol present
in a CXXC motif. Thioredoxins are divided into two
groups. Whereas group I thioredoxins exclusively con-
tain the TRX domain, group II members have other
domains in addition to TRX (Sadek et al., 2003). The
budding yeast Saccharomyces cerevisiae contains
three group I thioredoxins. Two cytoplasmic ones, Trx1
and Trx2, regulate cellular redox homeostasis. The mito-
chondrial thioredoxin Trx3 protects against the oxidative
stress generated during respiratory metabolism. In the
fission yeast Schizosaccharomyces pombe, the cyto-
solic Trx1 and mitochondrial Trx2 thioredoxins have
similar amino acid sequences except in the C-terminal
region (Cho et al., 2001). Trx1 plays a major role in pro-
tecting S. pombe against various external stresses and
is required for sulfur metabolism (Song and Roe, 2008).
In living cells, TRX genes may be involved in the activa-
tion of MAPKs via regulating intracellular ROS levels or
directly modifying the structure or redox status of key sig-
nalling components (Laloi et al., 2004; Fujino et al., 2006).
However, to date, the role of thioredoxins in MAP kinase
signalling has not been determined in the two model
yeasts and Neurospora crassa, a model filamentous fun-
gus. In Aspergillus nidulans, deletion of the trxA
thioredoxin gene results in decreased growth, increased
catalase activities, and inability to form reproductive struc-
tures such as conidiophores and cleistothecia (Th€on et al.,
2007). In Candida albicans, the thioredoxin Trx1 regulates
three distinct regulatory proteins that are activated in
responses to H2O2 stress, which are the Cap1 transcrip-
tion factor, the Hog1 SAPK, and the Rad53 DNA
checkpoint kinase (da Silva Dantas et al., 2010). In Podo-
spora anserine, the PaMpk1 MAPK cascade was not
affected in the Patrx1 and Patrx3 mutants, although both
PaTrx1 and PaTrx3 are necessary for sexual reproduction
and development of the crippled growth cell degeneration,
which are two processes that involve the PaMpk1 pathway
(Malagnac et al., 2007).
To understand the role of TRX genes in activating the
PMK1 MAPK pathway, in this study we identified and
characterized TRX1 and TRX2 in M. oryzae. Whereas
the Dtrx1 mutants had no detectable phenotypes, dele-
tion of TRX2 caused pleiotropic defects in growth,
differentiation and pathogenesis. The Dtrx1 Dtrx2 double
mutant had more severe defects than the Dtrx2 mutant
and rarely formed appressoria on hyphal tips. Deletion
of TRX2 affected Pmk1 activation and proper folding or
intra-/inter-molecular interactions of the Mst7 MEK
kinase. We further showed that Cys305 is important for
Mst7 function, Trx2 directly interacts with Mst7 in co-IP
assays, and expression of the dominant active MST7
allele partially rescued the defects of the Dtrx1 Dtrx2
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mutant. Taken together, our data indicated that TRX2
may affect invasive growth via the Mst11-Mst7-Pmk1
pathway. It is likely that thioredoxins are important for
intra-cellular ROS signalling and pathogenesis in M.
oryzae.
Results
One of the M. oryzae TRX genes, TRX2, partially
rescues the yeast ~trx2 mutant
The M. oryzae genome has two group I TRX genes,
MGG_15004 and MGG_04236, which were named
TRX1 and TRX2, respectively, in this study. Both of
them have an N-terminal TRX domain and share the
same active site and catalytic residues (WCGPC) but
the predicted Trx1 protein is twice the size of Trx2 (Sup-
porting Information Fig. S1). In S. cerevisiae, Trx2 is the
dominant cytoplasmic thioredoxin involved in environ-
mental stress responses and the ~trx2 mutant has
increased sensitivity to hydrogen peroxide (Garrido and
Grant, 2002).
To determine their functions in S. cerevisiae, the full-
length ORFs of TRX1 and TRX2 were amplified from
cDNA and cloned into pYES2. The resulting constructs
were transformed into the yeast ~trx2 mutant in the
BY4741 background (Giaever et al., 2002). Expression of
TRX2 but not TRX1 of M. oryzae partially complemented
the increased sensitivity of ~trx2 mutant to 6 mM H2O2
and 0.8 mM tert-butyl hydroperoxide (t-BOOH) (Garrido
and Grant, 2002), although only data with the later was
shown (Fig. 1A).
TRX2 is important for vegetative growth
The TRX1 and TRX2 gene replacement constructs
were generated by double-joint PCR with the neomycin-
resistant and hygromycin-resistant selectable markers,
respectively, and transformed into protoplasts of Guy11.
The resulting neomycin-resistant Dtrx1 and hygromycin-
resistant Dtrx2 deletion mutants (Table 1) were identi-
fied by PCR and confirmed by Southern blot analysis
(Supporting Information Fig. S2). In comparison with
Guy11, the Dtrx1 mutant had no obvious defects in col-
ony morphology (Fig. 1B) and growth rate (Table 2).
However, the Dtrx2 mutant was reduced in growth rate
(Table 2). It rarely produced aerial hyphae but was
increased in colony pigmentation in comparison with
Guy11 (Fig. 1B).
We also generated the Dtrx1 and Dtrx2 deletion mutants
in the wild-type strain 70-15 (Table 1). The resulting
mutants had the same phenotypes with those of Guy11,
although only data from the Guy11 mutants were pre-
sented below.
Deletion of TRX2 but not TRX1 results in reduced
conidiation
Conidiation was assayed with 10-day-old oatmeal agar
(OTA) cultures. On average, the Dtrx1 mutant produced 3
x 107 conidia per plate (U9 cm), which was similar with
Guy11 (Table 2). Under the same conditions, the Dtrx2
mutant B9G was reduced approximately 15-fold in conidia-
tion (Table 2).
To determine whether the two TRX genes have overlap-
ping functions, we transformed the TRX2 knockout
construct into the Dtrx1 mutant A52G (Table 1). Transform-
ants resistant to both hygromycin and neomycin were
screened by PCR and analysed by Southern blot hybrid-
ization (Supporting Information Fig. S2). The resulting
Dtrx1 Dtrx2 double mutant D65 (Table 1) had similar
growth rate and colony morphology with the Dtrx2 mutant
(Fig. 1B). However, the double mutant was reduced
approximately 70% in conidiation in comparison with the
Dtrx2 mutant (Table 2).
To characterize the function of TRX2 deletion in conidio-
genesis, we assayed the expression levels in vegetative
hyphae of four transcription factor genes, CON1, CON7,
HTF1 and COM1 that are known to be related to different
stages of conidiation in M. oryzae (Odenbach et al., 2007;
Zhou et al., 2009; Liu et al., 2010b). The expression level of
COM1 that is important for conidiophore development
(Zhou et al., 2009) was reduced approximately eightfold in
the Dtrx2 mutant (Fig. 1C). Although CON1 expression was
not significantly affected, the trx2 mutant also was reduced
in the expression of the HTF1 and CON7 transcription factor
genes (Fig. 1C). Whereas the htf1 mutant produces conidio-
phores but not conidia, the con7 mutant forms conidia with
defective morphology (Odenbach et al., 2007; Liu et al.,
2010b). TRX2 may affect conidiation by altering the expres-
sion levels of these transcription factors in M. oryzae.
TRX2 plays an important role in responses to oxidative
and cell wall stresses
To determine the functions of thioredoxin genes in stress
responses, we assayed growth rates of the Dtrx1, Dtrx2,
and Dtrx1 Dtrx2 mutants (referred to as Dtrx mutants
below) on complete medium (CM) with 0.7 M KCl, 0.5 M
sorbitol, 300 lg ml21 Congo red, 200 lg ml21 Calcofluor
white, or 6 mM H2O2. Whereas deletion of TRX1 and/or
TRX2 had no obvious effect on tolerance to hyperosmotic
stress, the Dtrx2 and Dtrx1 Dtrx2 mutants had increased
sensitivity to Calcofluor white (CFW) and Congo red (Fig.
2A). Calcofluor white and Congo red are known to bind
with beta-glucans and nascent chitin chains. Reduced
growth in the presence of these compounds may be
related to the defect of the Dtrx2 mutants in cell wall integ-
rity. These results suggest that thioredoxins are not
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involved in osmoregulation but TRX2 is important for
responses to cell wall stress.
In the presence of H2O2, the Dtrx2 and Dtrx1 Dtrx2
mutants but not Dtrx1 mutant formed compact colonies with
limited growth (Fig. 2A), indicating that only TRX2 plays a
critical role in response to oxidative stress. Because the
yeast ~trx2 mutant is hypersensitive to H2O2 but has
increased tolerance to diamide (Muller, 1996), we also
assayed the effects of diamide on Dtrx mutants. In M. ory-
zae, the Dtrx2 but not Dtrx1 mutant was more tolerant to
diamide than the wild type (Fig. 2A). On CM plates treated
with or without 1.5 mM H2O2, colonies of Guy11 and Dtrx1
were stained dark-purple with 2, 20-azino-di-3-
ethylbenzthiazoline-6-sulfonate (ABTS), one indicator of per-
oxidase/laccase activity (Guo et al., 2011). ABTS staining
was not observed in the Dtrx2 and Dtrx1 Dtrx2 mutants (Fig.
2B), indicating that extracellular peroxidase/laccase activities
were significantly reduced in Dtrx2 and Dtrx1 Dtrx2 mutants,
which may be related to their increased sensitivity to H2O2.
The Dtrx2 mutants are defective in response to light/
dark transitions
When incubated under 12 h light/dark cycles, zones with
dark pigmentation were visible on CM cultures of Guy11
and Dtrx1 mutant cultures (Fig. 2C). Race tube cultures of
the Dtrx2 and Dtrx1 Dtrx2 mutants grown under the same
conditions lacked these daily zones (Fig. 2C). When incu-
bated under constant dark, daily zones were not observed
in any of these strains (Fig. 2C). These results indicate
that the TRX2 gene may play a role in light sensing or
light/dark transitions. Because conidiation is induced by
light in M. oryzae, failure of Dtrx2 mutants in response to
light/dark may be related to their defects in conidiation.
TRX2 is important for appressorium formation on hyphal
tips
On artificial hydrophobic surfaces, the Dtrx1 mutant was
normal in appressorium formation (Table 2). In contrast,
appressorium formation was slightly reduced in the Dtrx2
and Dtrx1 Dtrx2 mutants compared with the wild type
(Table 2). Nevertheless, appressoria formed by the Dtrx2
mutants were normal in morphology (Fig. 3A). Interest-
ingly, appressoria formed at hyphal tips were rarely
observed in the Dtrx2 and Dtrx1 Dtrx2 mutants after incu-
bation for 48 h (Fig. 3B). Less than 1% of hyphal tips of the
Dtrx2 mutants formed appressoria on hyphal tips. These
results showed that Dtrx2 mutants were defective in
appressorium formation on hyphal tips but not by germ
Fig. 1. Heterologous expression andmutant colony morphology of the TRX1and TRX2 genes.
A. Different concentrations of yeast cells
(1042106 cells ml21) of the wild type
strain BY4741, Dtrx2 mutant, and
transformants of the Dtrx2 mutant that
expressed TRX1 or TRX2 of M. oryzae
were cultured on YPG (Galactose)
medium with or without 0.8 mM tert-butyl
hydroperoxide (t-BOOH).
B. Ten-day-old OTA cultures of the wild
type (Guy11) and the Dtrx1, Dtrx2, and
Dtrx1 Dtrx2 double mutants. Deletion of
TRX2 reduced growth rate and aerial
hyphae but increased colony
pigmentation.
C. The expression levels of CON1, CON7,
HTF1, and COM1 in Guy11 and the Dtrx2
mutant were assayed by qRT-PCR. The
relative expression level in Guy11 was set
to 1. Mean and standard deviation were
calculated with data from three
independent replicates.
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tubes, which is similar to the con7 mutant (Kong et al.,
2013).
The Dtrx2 and Dtrx1 Dtrx2 mutants are defective in plant
infection
When 2-week-old rice seedlings of cultivar CO-39 were
sprayed with conidium suspensions of the wild type and
Dtrx1 mutant, numerous lesions were observed on inocu-
lated leaves at 7 dpi (Fig. 4A). Under the same conditions,
only few, small black or brownish spots (not typical blast
lesions) were observed on leaves inoculated with the Dtrx2
mutant (Fig. 4A). On leaves sprayed with conidia of the
double mutant, small black or brownish spots were rarely
observed in over five independent repeats (Fig. 4A). These
results indicate that TRX2 plays a more critical role than
TRX1 in plant infection. Nevertheless, the Dtrx1 Dtrx2
mutant had more severe defects than the Dtrx2 mutant in
virulence, suggesting that TRX1 plays a minor role, possi-
bly overlapping with TRX2, during plant infection.
Table 1. Wild-type and mutant strains of Magnaporthe oryzae used in this study.
Strains Genotype description Reference
Guy11 Wild-type (MAT1-2) Chao and Ellingboe, 1991
70-15 Wild-type (MAT1-1) Chao and Ellingboe, 1991
ZH32 mst7 deletion mutant of Guy11 Zhao et al., 2005
A44G Dtrx1 deletion mutant of Guy11 This study
A52G Dtrx1 deletion mutant of Guy11 This study
A1 Dtrx1 deletion mutant of 70-15 This study
A4 Dtrx1 deletion mutant of 70-15 This study
B9G Dtrx2 deletion mutant of Guy11 This study
B85 Dtrx2 deletion mutant of 70-15 This study
B110 Dtrx2 deletion mutant of 70-15 This study
D65 Dtrx1 Dtrx2 double mutant of Guy11 This study
D68 Dtrx1 Dtrx2 double mutant of Guy11 This study
GM7 MST723xFLAG transformant of Guy11 This study
TM7 MST723xFLAG transformant of Dtrx2 mutant B9G This study
MC1 MST723xFLAG transformant of mst7 mutant ZH32 This study
MD2 MST7C114A23xFLAG transformant of ZH32 This study
ME1 MST7C136A23xFLAG transformant of ZH32 This study
MF2 MST7C201A23xFLAG transformant of ZH32 This study
MG2 MST7C305A23xFLAG transformant of ZH32 This study
MDD1 MST7DA transformants of Dtrx1 Dtrx2 mutant D65 This study
MDD2 MST7DA transformants of Dtrx1 Dtrx2 mutant D65 This study
M7G MST7-GFP transformant of Guy11 This study
M7GF MST7-GFP MST723xFLAG transformant of Guy11 This study
M7T2 MST7-GFP TRX223xFLAG transformant of Guy11 This study
GP1 PMK1-GFP transformant of Guy11 This study
TP2 PMK1-GFP transformant of Dtrx2 mutant B9G This study
A52G-C Complemented strain of A52G (Dtrx1/TRX1-GFP) This study
B9G-C Complemented strain of B9G (Dtrx2/TRX2-GFP) This study
Ect1 Ectopic transformant of the TRX1 deletion construct This study
Ect2 Ectopic transformant for the TRX2 deletion construct This study
Table 2. Phenotype characterization of the trx mutants generated in this study.
Strain Growth rate (mm/d)a Conidia/plate (3106)b Appressorium formation (%)c
Guy11 (WT) 3.2 6 0.2A 32.4 6 4.5A 97.2 6 1.1A
A52G (Dtrx1) 3.2 6 0.2A 29.8 6 7.2A 96.6 6 1.6A
B9G (Dtrx2) 2.7 6 0.1B 2.2 6 0.6B 86.5 6 2.1B
D65 (Dtrx1 Dtrx2) 2.5 6 0.1C 0.7 6 0.3C 83.3 6 3.2B
A52G-C (Dtrx1/TRX1) 3.2 6 0.1A 30.2 6 3.3A 96.1 6 1.7A
B9G-C (Dtrx2/TRX2) 3.2 6 0.2A 32.2 6 2.6A 96.4 6 1.8A
a. Growth rate was measured as the average daily extension of colony radiometers after incubation at 25oC for 10 days.b. Conidiation was assayed with 12-day-old OTA cultures.c. Percentage of germ tubes that formed appressoria on hydrophobic surfaces.Means and standard deviations were calculated from at least three independent experiments. Data were analysed with Duncan’s pair-wisecomparison. Different letters mark statistically significant differences (P 5 0.05).
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To further characterize the Dtrx2 mutants, leaves of
10-day-old barley seedlings were wounded with a needle
and inoculated with culture blocks. On leaves inoculated
with the Dtrx2 and Dtrx1 Dtrx2 mutants, only limited
darkening at the wound site but not extensive spreading
of the necrosis zones was observed (Fig. 4B). When the
diseased segments of barley leaves were surface steri-
lized and incubated for 2 days in a moisture chamber
under constant light, abundant conidia and conidio-
phores were formed on lesions caused by Guy11 and
the Dtrx1 mutant. However, no conidia or hyphal growth
were observed on the sites inoculated with the Dtrx2 and
Dtrx1 Dtrx2 mutants (Fig. 4C), suggesting that TRX2 is
not only important for appressorium formation on hyphal
tips but also plays an important role in invasive growth
after penetration.
Thioredoxins are important for invasive growth
In penetration assays with onion epidermal cells, appres-
soria formed by Guy11 and the Dtrx1 mutant penetrated
and produced invasive hyphae inside plant cells by 48 h
post-inoculation (hpi) (Fig. 5A). Under the same condi-
tions, the Dtrx2 mutant had only limited invasive hyphae
in underlying plant cells (Fig. 5A), indicating that TRX2
is important for invasive growth in M. oryzae. Moreover,
less than 20% of appressoria formed by the Dtrx1 Dtrx2
mutant could penetrate underlying plant cells. Even for
those Dtrx1 Dtrx2 appressoria that were successful in
penetration, they had only limited infectious growth and
failed to differentiate branching invasive hyphae in
underlying plant cells (Fig. 5A), indicating that thioredox-
ins are important for invasive growth in M. oryzae,
Fig. 2. Defects of the Dtrx1 and Dtrx2mutants in responses to oxidative,hyperosmotic, and cell wall stresses.
A. The wild type (Guy11) and the Dtrx1,
Dtrx2, and Dtrx1 Dtrx2 mutants were
cultured for 7 days on CM with or without
200 lg ml21 Calcofluor white (CFW), 300
lg ml21 Congo red (CR), 0.7 M NaCl,
0.5 M Sorbitol, 6mM H2O2 or 2.0 mM
diamide.
B. ABTS staining with CM cultures of
Guy11 and the Dtrx mutants treated with
or without 1.5 mM H2O2.
C. Race tube cultures of Guy11 and the
Dtrxtrx1 Dtrx2 mutants cultured on CM for
30 days under a 12 h light/dark (L/D)
cycle or constant darkness (D/D).
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which is consistent with virulence defects of the Dtrx2
and Dtrx1 Dtrx2 mutants.
We also conducted penetration assays with barley
leaves inoculated with culture blocks. On barley leaves ino-
culated with Guy11 and Dtrx1 mutant, extensive growth of
invasive hyphae was observed inside plant cells by 48 hpi
(Fig. 5B). Rare appressoria formed on hyphal tips of the
Dtrx2 mutant could penetrate and develop un-branched
infectious hyphae inside epidermal cells (Fig. 5B). How-
ever, it was defective in further differentiation of secondary
branching invasive hyphae. Under the same conditions,
appressoria formed by the Dtrx1 Dtrx2 mutant failed to suc-
cessfully penetrate and develop invasive hyphae inside
barley epidermal cells (Fig. 5B). Similar results were
obtained in penetration assays with Brachypodium dis-
tachyon leaves (Fig. 5B). These results indicate that
thioredoxins are important for appressorium penetration
and invasive growth. TRX1 likely plays a minor role during
plant infection because the Dtrx1 Dtrx2 mutant had more
severe defects than the Dtrx2 mutant.
Trx2 is a cytoplasmic thioredoxin
The TRX1- and TRX2-GFP fusion constructs under the
control of their native promoters were generated and
transformed into Dtrx1 mutant A52G and Dtrx2 mutant
B9G respectively. The resulting Dtrx1/TRX1-GFP and
Dtrx2/TRX2-GFP transformants (Table 1) were identi-
fied by PCR and confirmed by examination for GFP
signals. The Dtrx1/TRX1-GFP transformant A52G-C
had weak GFP signals in conidia and appressoria (Fig.
6A). Although Trx1-GFP expression appeared to be
increased during appressorium formation, GFP signals
were barely detectable in invasive hyphae formed
inside onion epidermal cells (Fig. 6A).
In the Dtrx2/TRX2-GFP transformant B9G-C, defects of
the Dtrx2 mutant in conidiation, appressorium formation,
and penetration were rescued (Table 2). In infection
assays, the Dtrx2/TRX2-GFP transformant was as virulent
as the wild type (Fig. 4A). Relatively strong GFP signals
were observed in the cytoplasm and vacuoles in conidia,
appressoria, and invasive hyphae (Fig. 6B; Supporting
Information Fig. S3), indicating that Trx2 is a cytoplasmic
thioredoxin constitutively expressed in M. oryzae.
Unlike Trx2-GFP that was distributed through the cyto-
plasm, Trx1-GFP appeared to be restricted to certain
regions. To test whether the distribution of Trx1 is related
to mitochondria, we stained transformants A52G-C with
MitoTracker Red. In repeated experiments, the localization
of Trx1-GFP did not overlap with mitochondria (Fig. 6C),
indicating that Trx1 may not localize to mitochondria. It
appears that, unlike Trx3 in S. cerevisiae, Trx1 in M. oryzae
is not a mitochondrial thioredoxin.
TRX2 expression is up-regulated in the Dtrx1 mutant
Consistent with strong GFP signals observed in the
TRX2-GFP transformant, TRX2 had similar expression
levels in conidia, appressoria, and infected rice leaves
(Supporting Information Fig. S4A). In contrast, TRX1 dif-
fered significantly in expression levels at different stages
and had the highest expression level during appresso-
rium formation (Supporting Information Fig. S4A). In
comparison, TRX2 had a higher expression level than
TRX1 in different cell types examined (Supporting Infor-
mation Fig. S4B). These results indicate that TRX2 is
constitutively expressed in M. oryzae and has a higher
expression level than TRX1. We also assayed the effect
of oxidative stress on thioredoxin gene expression. In
vegetative hyphae harvested from CM cultures, the
expression levels of TRX1 and TRX2 were not
Fig. 3. Appressorium formation assays with conidia and hyphalblocks.
A. Conidia from Guy11, Dtrx1 mutant, Dtrx2 mutant, and Dtrx1
Dtrx2 mutant were incubated on hydrophobic surfaces for
appressorium formation.
B. Differentiation of appressoria at hyphal tips on hydrophobic
surfaces was observed in Guy11 and Dtrx1 mutant but not in the
Dtrx2 and Dtrx1 Dtrx2 mutants after 48 h. Bar 5 10 mm.
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significantly affected in samples treated with or without
6 mM H2O2 (Supporting Information Fig. S5).
To determine the effect of TRX2 deletion on TRX1
expression or vice versa, we assayed their expression levels
in vegetative hyphae of Guy11 and Dtrx1 or Dtrx2 mutants.
In the Dtrx1 mutant, TRX2 expression had a fourfold
increase (Supporting Information Fig. S6). In contrast, TRX1
expression was unaffected by deletion of TRX2. Therefore, it
is possible that increased expression of TRX2 may suppress
the defects caused by TRX1 deletion.
Fig. 4. Infection assays with rice andbarley leaves.
A. Two-week-old seedlings of rice cultivar
CO-39 were sprayed with conidia of
Guy11, Dtrx1 mutant, Dtrx2 mutant, Dtrx1
Dtrx2 mutant, Dtrx1/TRX1 transformant
and Dtrx2/TRX2 transformants. Inoculation
with 0.25% gelatin was the negative
control. Photos were taken 7 dpi.
B. Barley leaves were wounded with a
needle and inoculated at the wound sites
with culture blocks of the same set of
strains. Symptoms were examined 5 dpi.
Inoculation with OTA agar was the
negative control.
C. Leaf segments with the wound sites
were excised from wound-inoculated
barley leaves 7 dpi, surface sterilized, and
incubated in a moisture chamber under
constant light. After incubation for 2 days,
fungal growth and conidiation were directly
examined with a dissecting microscope.
Abundant hyphae and conidiophores were
observed on leaves inoculated with Guy11
and Dtrx1 mutant but not on leaves
inoculated with the Dtrx2 or Dtrx1 Dtrx2
mutant.
Fig. 5. Assays for penetration andinvasive growth with epidermal cells ofonion, barley, and B. distachyon.
A. Onion epidermal cells inoculated with
conidia of Guy11 and the Dtrx1, Dtrx2,
and Dtrx1 Dtrx2 mutants were examined
48 hpi.
B. Barley and B. distachyon leaves were
inoculated with hyphal blocks of the same
set of strains. Penetration and invasive
hyphae were examined 48 hpi after
discoloration with 94% alcohol for 12 h.
Bar 5 10 mm.
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TRX2 is involved in the activation of the Pmk1 MAPkinase
Because Pmk1 regulates appressorium formation, pene-
tration, and invasive growth (Xu and Hamer, 1996), we
assayed its activation in the hyphae of Dtrx mutants. When
detected with an anti-Pmk1 antibody, the 42 kD Pmk1-
band had similar intensities in the wild type and Dtrx1 or
Dtrx2 mutants (Fig. 7A). However, when detected with an
anti-pTEY antibody, an antibody specific for detection of
dual-phosphorylation of the TEY motif (Zhao et al., 2005;
Liu et al., 2011; Zheng et al., 2012), the levels of Pmk1
phosphorylation were significantly reduced in the Dtrx1
and Dtrx2 mutants (Fig. 7A). In the Dtrx1 Dtrx2 mutant,
phosphorylation of Pmk1 was not detectable (Fig. 7A).
These results indicate that Pmk1 phosphorylation was sig-
nificantly reduced in the double mutants. Meanwhile, the
46 kD Mps1-band had no obvious difference between the
wild type and mutants, suggesting that the activation of
Mps1 MAP kinase may be not affected by the deletion of
thioredoxins. Therefore, thioredoxins, especially TRX2,
may be involved in the activation of the Pmk1 MAPK
kinase pathway in M. oryzae.
Trx2 is required for the proper folding or dimerization of
Mst7
In M. oryzae, ROS signalling affects appressorium mor-
phogenesis and fungal-plant interactions (Ryder et al.,
2013). Because the Mst7 MEK that activates the down-
stream Pmk1 for appressorium formation (Zhao et al.,
2005) is known to interact with itself in yeast two-hybrid
assays (Zhao and Xu, 2007), thioredoxins may play a role
in Mst7 activation by affecting its possible dimerization or
formation of intramolecular disulfide bonds. To test
this hypothesis, we firstly generated PRP27-MST7-GFP
construct and co-transformed it into Guy11 with PRP27-
MST723xFLAG. Transformants resistant to both hygromy-
cin and neomycin were confirmed by PCR assays and
western blot analysis to contain both transforming con-
structs. In western blot analysis with the resulting MST7-
GFP MST7-3xFLAG transformant (Table 1), the 84-kD
Mst7-GFP band was detected with an anti-GFP antibody
in total proteins and proteins eluted from anti-FLAG beads
(Fig. 7B), indicating that Mst7-GFP may interact with Mst7-
3xFLAG to form dimers in vivo.
We then transformed the MST7-3xFLAG construct into
Guy11 and the Dtrx2 mutant B9G. The resulting transform-
ants GM7 (WT MST7-3xFLAG) and TM7 (Dtrx2 MST7-
3xFLAG) were confirmed by PCR and western blot analy-
sis with total proteins isolated from 2-day-old cultures was
separated on SDS-PAGE gels (Fig. 7C). On western blots
of the same proteins separated on native gels, both mono-
mers and likely homodimers of Mst7-3xFLAG proteins
were detected with an anti-FLAG antibody in the WT
MST723xFLAG transformant GM7 (Fig. 7C). However, no
Mst7-3xFLAG bands could be detected in proteins isolated
from the Dtrx2 MST723xFLAG transformant on western
blots of native gels (Fig. 7C). These results indicate that
deletion of TRX2 had no effect on MST7 expression but
affected the proper folding or intra-/inter-molecular
Fig. 6. Expression and localization of the Trx1-GFP and Trx2-GFPfusion proteins.
A. The Dtrx1/TRX1-GFP transformant A52G-C.
B. The Dtrx2/TRX2-GFP transformant B9G-C. Conidia were
harvested from 12-day-old OTA cultures. Appressorium formation
was assayed with glass coverslips after incubation for 12 h.
Penetration and invasive hyphae growth were assayed with onion
or barley epidermal cells 48 hpi. All samples were examined by
DIC and epifluorescence microscopy. GFP signals were stronger in
B9G-C than in A52G-C. Trx2-GFP localized to the cytoplasm, but
the distribution of Trx1-GFP was restricted to certain cytoplasmic
regions.
C. Germ tubes formed by conidia of Dtrx1/TRX1–GFP
transformants A52G-C were stained with MitoTracker Red and
examined by Difference Interference Contrast (DIC) and
epifluorescence microscopy. Bar 5 10 mm.
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interactions of Mst7, which may block the detection of the
FLAG-epitope tag.
The Cys305 residue is essential for Mst7 function
Mst7 contains 4 cysteine residues that are conserved
among its orthologs from Sordariomycetes and may be
involved in the formation of intra- or inter-molecular disul-
fide bonds. To test this hypothesis, we generated the
MST7C114A, MST7C136A, MST7C201A, and MST7C305A-
3xFLAG alleles by site-directed mutagenesis and trans-
formed them into the mst7 deletion mutant ZH32 (Zhao
et al., 2005) respectively. The resulting MST7C114A,
MST7C136A, and MST7C201A transformants (Table 1) were
normal in appressorium formation (Fig. 8A) and plant infec-
tion (Fig. 8B). However, the mst7/MST7C305A transformant
MG2, similar to the original mst7 mutant, failed to form
appressoria on hydrophobic surfaces (Fig. 8A) and was
non-pathogenic (Fig. 8B). Therefore, the C305A residue is
essential for Mst7 function.
We then conducted western blot analysis with total pro-
teins isolated from the MST7C305A-3xFLAG transformant.
When separated on SDS-PAGE gels, the 47-kD band of
the expected Mst7-3xFLAG fusion was detected with an
anti-3xFLAG antibody (Fig. 8C). However, when separated
on native gels, no Mst7-3xFLAG fusion bands were
detected in the mst7/MST7C305A-3xFLAG transformant
(Fig. 8C). In the MST7WT-3xFLAG transformant, 3xFLAG
fusion bands were detected in both native and SDS-PAGE
gels (Fig. 8C). These data indicate that residue C305 is
likely critical for the function and proper folding or intra-/
inter-molecular interaction of Mst7.
Expression of MST7DA partially rescues the defects of
the Dtrx1 Dtrx2 deletion mutant
To further investigate the relationship between Mst7 and
Trx2, we transformed the dominant active MST7DA allele
(Zhao et al., 2005) into the Dtrx1 Dtrx2 double mutant.
Although the resulting MST7DA transformants MDD1 had
similar colony morphology with the double mutant, it could
form appressoria on hyphal tips as efficiently as the wild
type (Fig. 9A), indicating a partial rescue of the Dtrx1 Dtrx2
mutant.
On barley leaves inoculated with culture blocks of the
Dtrx1 Dtrx2/MST7DA transformant, appressoria formed on
hyphal tips could penetrate and differentiate into invasive
hyphae in epidermal cells (Fig. 9B). Moreover, the Dtrx1
Dtrx2/MST7DA transformant caused typical blast lesions on
barley leaves (Fig. 9C). These results suggested that
expression of the dominant active MST7DA allele can par-
tially rescue the defects of the Dtrx1 Dtrx2 mutant.
To assay whether Trx2 regulates Mst7 through direct
interaction, the MST7-GFP and TRX223xFLAG con-
structs expressed under the control of their native
promoters were co-transformed into Guy11. In the
resulting transformants confirmed to express both trans-
forming fusion constructs, western blot analysis with
proteins isolated from 2-day-old CM/5xYEG cultures
showed that the 84-kD Mst7-GFP band was detectable
with an anti-GFP antibody in total proteins or proteins
eluted from anti-FLAG M2 beads (Fig. 9D). These
results indicate that Mst7 and Trx2 may interact with
each other in vegetative hyphae.
Discussion
Thioredoxins have the ability to reduce the disulfide bonds
in many other proteins and, together with glutathione
(GSH), to maintain a reduced cytoplasmic thiol redox bal-
ance. The rice blast fungus M. oryzae has two type I
thioredoxins. Unlike that two type I thioredoxins in the
Fig. 7. Assays for Pmk1 activation and expression of the Mst7-3xFLAG fusion in the wild-type and Dtrx2 mutant background.
A. Western blots were conducted with proteins isolated from
vegetative hyphae of the wild-type (Guy11) and the Dtrx1, Dtrx2,
and Dtrx1 Dtrx2 mutant strains. The anti-pTEY antibodies detected
the expression and phosphorylation levels of Pmk1 (42-kD) and
Mps1 (46-kD) respectively. Detection with the anti-Pmk1 antibody
was used as the control.
B. Western blot analysis with proteins isolated from transformants
of Guy11 expressing the MST7-GFP and MST7-GFP MST7-
3xFLAG constructs and separated on SDS-PAGE gels. In proteins
eluted from anti-FLAG beads, the Mst7-GFP band was detected in
the MST7-GFP MST7-3xFLAG transformant.
C. Western blots of proteins isolated from MST7-3xFLAG
transformants of Guy11 (GM7) and Dtrx2 mutant (TM7) were
separated on SDS-PAGE (upper) or native (lower) gels and
detected with an anti-FLAG antibody. The monomer and likely
dimer bands of Mst7 fusion proteins were detected in transformants
of Guy11 but not Dtrx2 mutant.
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budding yeast are similar in size, Trx1 is twice as large as
Trx2 in M. oryzae. Based on complementation assays and
mutant phenotype analyses, TRX2 is likely the ortholog of
yeast Trx2 and major cytoplasmic thioredoxin. For TRX1,
GFP signals did not overlap with mitochondria stained with
MitoTracker Red in the TRX1-GFP transformant and the
Dtrx1 Dtrx2 mutant had more severe defects than the
Dtrx1 mutant. Therefore, Trx1 may not function as a mito-
chondrial thioredoxin but plays a minor role as a
cytoplasmic thioredoxin in M. oryzae.
In M. oryzae, the Dtrx2 mutant had increased tolerance
to diamide but increased sensitivity to H2O2 or t-BOOH,
which is similar to the yeast trx2 mutant (Muller, 1996).
Like the trxA deletion mutant of A. nidulans (Th€on et al.,
2007), the Dtrx2 mutant of M. oryzae had increased cata-
lase activities. In the budding yeast, Yap1 is important for
regulating many oxidative stress response (OXR) genes,
including TRX2, TSA1, GPX2, and CTT1 (He and Fassler,
2005; Mulford and Fassler, 2011). Yap1 is activated upon
ROS treatment by the formation of intramolecular disulfide
bonds between C303 and C598 that disrupts the nuclear
export signal (NES) (Delaunay et al., 2000; Kuge et al.,
2001). Nuclear export of Yap1 is restored when disulfide
bonds are reduced by thioredoxin Trx2, whose expression
is controlled by Yap1. Therefore, YAP1 and TRX2 form a
negative feedback loop (Marinho et al., 2014). The
reduced form of Yap1 interacts with the nuclear export pro-
tein Crm1 in the nucleus, and with Ybp1 and peroxiredoxin
Hyr1 in the cytoplasm. In M. oryzae, MoAP1 may be nega-
tively regulated by Trx2 through some of the Cys residues,
such as C494, C518, and C527. Another major regulator
of responses to oxidative stress in S. cerevisiae is Skn7
(Raitt et al., 2000). Whereas the yap1 mutant is sensitive
to different ROS, the skn7 mutant has increased sensitivity
to t-BOOH and H2O2 but not superoxide-generating
reagents such as the thiol-oxidizing agent diamide
Fig. 8. Site-directed mutagenesis of fourCys residues in MST7.
A. Appressorium formation assays with
transformants of the mst7 mutant
expressing the MST7C114A-, MST7C136A -,
MST7C201A-, and MST7C305A23xFLAG
fusion constructs.
B. Rice leaves sprayed with conidia
of Guy11, mst7 mutant, mst7/
MST723xFLAG transformant, and mst7//
MST7C305A23xFLAG transformant.
C. Western blots of total proteins from the
mst7/MST723xFLAG (MC1) and mst7//
MST7C305A23xFLAG (MG2) transformants
separated on SDS-PAGE (upper) or native
(lower) gels were detected with an anti-
FLAG antibody. The Mst7-3xFLAG fusion
bands were detected in proteins isolated
from MC1 on western blots of SDS-PAGE
and native gels. In contrast, Mst7 fusion
proteins were detectable on western blots
of MG2 (MST7C305A-3xFLAG) proteins
separated on SDS-PAGE gels but not on
native gels.
Fig. 9. Expression of the MST7DA allele in the thioredoxin mutantand Co-IP assays for the Mst7-Trx2 interaction.
A. Appressorium formation assays with culture blocks of Guy11,
Dtrx1 Dtrx2 double mutant, and Dtrx1 Dtrx2 MST7DA transformant.
B. Penetration assays of the same set of strains with barley
epidermal cells were examined 48 hpi.
C. Barley leaves inoculated with culture blocks of Guy11, Dtrx1
Dtrx2 double mutant, and Dtrx1 Dtrx2 MST7DA transformant were
examined for blast symptoms 5 dpi.
D. Western blot analysis with proteins isolated from hyphae of
MST7-GFP transformant and MST7-GFP TRX2-3xFLAG
transformant construct and separated on SDS-PAGE gels. Gel
transfer and detection with anti-GFP or anti-FLAG antibody were
conducted under the same conditions for both transformants. In
proteins eluted from anti-FLAG beads, the Mst7-GFP band (84-kD)
was detected in the MST7-GFP TRX223xFLAG transformant.
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(Morgan et al., 1997). Therefore, the defects of the yeast
skn7 mutant in response to different oxidants are similar to
that of the Dtrx2 mutant in M. oryzae.
In M. oryzae, deletion of TRX1 and/or TRX2 had no
obvious effects on tolerance to hyperosmotic stress or the
activation of the Mps1 MAP kinase that regulates cell wall
integrity in M. oryzae and other fungi (Xu et al., 1998;
Zeng et al., 2012). However, the Dtrx2 deletion mutants
had increased sensitivity to Calcofluor white and Congo
red. Recently, it has been shown that deletion of the thiore-
doxin reductase or peroxidase gene also resulted in an
increased sensitivity to Congo red (Fernandez and Wilson,
2014). Therefore, TRX2 or intracellular ROS signalling
also is important for responses to cell wall stress, although
it is dispensable for osmoregulation. In M. oryzae, ROS
signalling plays important roles in both development and
virulence, and the CATB catalase gene is known to be
essential for both cell wall integrity and virulence (Skam-
nioti et al., 2007).
Although they were only slightly reduced in growth rate,
the Dtrx2 and the Dtrx1 Dtrx2 mutants were reduced in
conidiation In A. nidulans, the trxA deletion mutant also
was reduced in growth and failed to form reproductive
structures (Th€on et al., 2007). However, the conidiation
process differs between A. nidulans and M. oryzae. In M.
oryzae, TRX2 appeared to affect the expression of several
genes related to conidiation, including COM1 and HTF1,
that are important for conidiophore development and differ-
entiation of conidia (Liu et al., 2010b; Yang et al., 2010).
CON1 and CON7 are involved in the later stages of coni-
diogenesis (Shi et al., 1998; Odenbach et al., 2007).
Expression of CON7 also was reduced by deletion of
TRX2. For some of these transcription factors, thioredoxins
may be involved in keeping cysteine residues reduced,
which may be important for their DNA binding activities or
nuclear localization. Com1 (Cys328) and Htf1 (cys426)
have only one cysteine residue that may form intermolecu-
lar disulfide bonds. For three cysteine residues of Con7
(Cys72, 258, and 263), two of them flank the predicted
NLS (237GAQQHKRPRRRYE250).
In M. oryzae, the Pmk1 MAP kinase pathway regulates
three related but different infection processes: appresso-
rium formation, penetration, and invasive growth (Zhao
et al., 2005; Ding et al., 2009). Although the Dtrx2 and
Dtrx1 Dtrx2 mutant still formed appressoria by germ tubes,
thioredoxins were essential for appressorium penetration
and development of invasive hyphae in plant cells. In addi-
tion, the phosphorylation level of Pmk1 in vegetative
hyphae was significantly reduced in the Dtrx2 mutants and
barely detectable in the Dtrx1 Dtrx2 mutant. Therefore,
Trx2 may play a critical role in the activation of the Pmk1
pathway, which in turn may be responsible for the defects
of the Dtrx2 mutant in plant infection. Unfortunately, due
to the defects of Dnfort and Dnd 1 D t mutants in plant
infection, it is impossible to determine whether the
phosphorylation level of Pmk1 was reduced in invasive
hyphae. We attempted to determine the phosphorylation
level of Pmk1 during appressorium formation but the Dfoatt
and Dnd 1 D m mutants were significantly reduced in
conidiation and we failed to isolate sufficient high quality
proteins suitable for TEY phosphorylation assays from
appressoria formed by germ tubes. Mst12, one of the
downstream transcription factors of the Pmk1 pathway, is
essential for the regulation of appressorium penetration
and invasive growth by Pmk1 but it is dispensable for
appressorium formation (Park et al., 2002; Park et al.,
2004). Our data suggest that the involvement of Trx2 in the
Pmk1 pathway as an upstream component is only impor-
tant for appressorium penetration and invasive growth,
which is similar to that of Mst12 as a downstream tran-
scription factor. It is likely that phosphorylation of Mst12
may be affected during appressorium penetration and
invasive growth in the Dffect and Dnd 1 D t mutants. How-
ever, the Doweve and Dnd 1 D e mutants were defective in
invasive growth and there is no commercially available
antibody to detect the phosphorylation of Mst12. Neverthe-
less, it will be important to assay the phosphorylation
status of Mst12 in the Dn the and Dnd 1 D h mutants and
further characterize the functional relationship between
Trx2 and Mst12 in the future when the phosphorylation
sites of Mst12 by Pmk1 are clear and suitable detection
methods become available. The Pmk1 and its orthologous
MAPKs have been shown to be important for various plant
infection processes (Zhao et al., 2007; Li et al., 2012).
However, to our knowledge, there are no reports on the
involvement of thioredoxins and redox status in the activa-
tion of this well-conserved MAPK pathway in plant
pathogenic fungi. In C. albicans, Trx1 is involved in regulat-
ing the H2O2-induced activation of the Hog1 SAPK
pathway (da Silva Dantas et al., 2010).
Due to a redox-active cysteine pair in their conserved
active sites, thioredoxins can alter the redox state of target
proteins. In plants and animals, many cellular proteins are
directly regulated by the redox state via thioredoxins. In
Arabidopsis, MKK6 is a member of the MAP2K subfamily
that activates the p38 MAPKs. Upon oxidation, two cyste-
ine residues (Cys109 and Cys196) in MKK6 form an
intramolecular disulfide bond, which inhibits ATP binding
and inactivates its kinase activity (Diao et al., 2010). More-
over, PKGIa and MEKK1 also are regulated by cysteine
modifications (Cross and Templeton, 2006; Pantano et al.,
2006; Burgoyne et al., 2007; Leonberg and Chai, 2007). In
M. oryzae, Pmk1 is activated by the upstream MEK Mst7
and MEKK Mst11. In yeast two-hybrid assays, Mst7 has
been shown to interact with itself (Zhao et al., 2005). In
this study, we showed that Mst7 may form homodimers in
the wild type but not in the Dtrx2 mutant. In addition, proper
folding of Mst7 may be affected by TRX2 deletion because
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Mst7-3xFLAG or Mst7-GFP fusion proteins were detecta-
ble on western blots of SDS-PAGE gels but not western
blots of native gels. These results indicate that Trx2 plays a
critical role in the proper folding or protein-protein interac-
tions of Mst7 proteins, possibly by affecting the intra- or
inter-molecular disulfide bonds. However, it remains possi-
ble that deletion of TRX1 and TRX2 may affect Ap1 or
redox homeostasis in general, which may in turn indirectly
affect MAP kinase signalling in M. oryzae. Site-directed
mutagenesis showed that Cys305 of Mst7 is required for
its function. The C305A mutation had no impact on the
expression of Mst7-3xFLAG fusion proteins but affected
the detection of Mst7 on western blots of native gels.
Therefore, post-translational modifications that affected the
detection of Mst7-3xFLAG should be relatively specific to
the formation of disulfide bonds involving residue Cys305.
Interestingly, our data showed that the Dtrx1 Dtrx2
mutant still formed appressoria by germ tubes although it
was defective in appressorium penetration and differentia-
tion of invasive hyphae in plant cells. In M. oryzae, the
Mst11-Mst7-Pmk1 MAPK cascade regulates appressorium
penetration and invasive growth but it also plays an essen-
tial role in appressorium formation (Zhao et al., 2005; Ding
et al., 2009). These results indicate that Trx2 may be more
important for its role in the Pmk1 pathway during plant pen-
etration and invasive hyphal growth. Nevertheless,
appressorium formation on hyphal tips was affected in the
Dtrx2 and Dtrx1 Dtrx2 mutants, suggesting that thioredox-
ins displayed a hyphal-specific role in infection-related
morphogenesis. We noticed that the con7 and Dtrx2
mutants had the same defect in appressorium formation
on hyphal tips although both of them were normal in
appressorium formation by germ tubes on hydrophobic
surfaces (Kong et al., 2013). Therefore, it will be important
to further characterize the functional relationship between
CON7 and TRX2.
In S. cerevisiae, the cytosolic thioredoxin system con-
sists of the thioredoxin reductase Trr1 that catalyzes the
transfer of reducing equivalents from NADPH to thiore-
doxin (Yang and Ma, 2010). Thioredoxin reductase is
essential for viability in Cryptococcus neoformans and
affects the circadian conidiation rhythm in N. crassa (Onai
and Nakashima, 1997; Missall and Lodge, 2005; Th€on
et al., 2007). In M. oryzae, mutants with the thioredoxin
reductase or thioredoxin peroxidase-encoding gene dele-
tion were severely attenuated in their ability to grow in rice
cells and failed to produce spreading necrotic lesions on
leaf surfaces (Fernandez and Wilson, 2014). In this study,
we also generated the DMotrr1 deletion mutant. The
DMotrr1 mutant had less severe defects than the Dtrx2 or
Dtrx1 Dtrx2 mutants, suggesting that the DMotrr1 mutant
still had low levels of thioredoxin activities. It is possible
that other closely related enzymes have low capacity to
reduce thioredoxins in the presence of MoTrr1. On the
other hand, it could simply be because some of the thiore-
doxin functions are solely dependent on proteins but not
related to the reduced dithiol-form (Trx-SH). Overall, our
results showed that Trx2 is the major thioredoxin in M. ory-
zae. The Dtrx2 mutant had a more severe phenotype than
the Dtrx1 mutant in conidiation, appressorium formation,
appressorium penetration, and plant infection. Based on
the phenotypes of the Dtrx2 and Dtrx1 Dtrx2 mutants and
transformants of Dtrx1 Dtrx2 expressing the dominant
active MST7 allele, TRX2 may regulate the activation of
Pmk1 MAPK via its effects on Mst7. In M. oryzae, thiore-
doxins may be important for intracellular ROS signalling
and pathogenesis.
Experimental procedures
Strains and culture conditions
The wild-type, mutant strains, and different transformants of
M. oryzae used in this study are listed in Table 1. Conidiation
and growth rate were assayed with OTA cultures as previously
described (Xue et al., 2002; Li et al., 2004). Vegetative hyphae
harvested from 2-day-old 5xYEG (0.5% yeast extract and 2%
glucose) or CM were used for isolation of DNA, RNA, and pro-
teins (Zhou et al., 2011b). To assay for defects in stress
responses, growth rate was measured with cultures grown on
CM with 0.7 M KCl, 0.5 M sorbitol, 300 lg ml21 Congo red,
200 lg ml21 Calcofluor, 6 mM H2O2, or 2.0 mM diamide
(Zheng et al., 2012).
Generation of the TRX gene replacement constructsand mutants
The double-joint PCR approach (Kim et al., 2012) was used to
generate the TRX1 and TRX2 gene replacement constructs.
The upstream and downstream flanking sequences of TRX1
were amplified with the primer pairs F1/R2 and F3/R4 (Sup-
porting Information Table S1) respectively. The full-length
neomycin-resistance gene was amplified from pFL2 vector
(Zhou et al., 2011a). The resulting PCR products were used
as the templates for double-joint PCR to generate the TRX1
gene replacement construct, which was transformed into pro-
toplasts of wild-type strains Guy11 and 70-15. Putative Dtrx1
deletion mutants were identified by PCR and further confirmed
by Southern blot analysis (Supporting Information Fig. S2).
Similar strategies were used to generate the trx2 deletion
construct, with the hygromycin-phosphotransferase (hph)
gene as the selectable marker. Products from double-joint
PCR were then transformed into protoplasts of Guy11, 70-15,
or the Dtrx1 mutant A52G. Putative Dtrx2 deletion or Dtrx1
Dtrx2 double mutants identified by PCR were confirmed by
Southern analysis (Supporting Information Fig. S2).
Generation of the TRX1-, TRX2- and PMK1-GFP fusionconstructs
The full-length TRX1 gene, along with its 1.5-kb promoter
region was cloned into pDL2 that carries the hph cassette by
yeast gap repair as described (Zhou et al., 2011a). The
3780 S. Zhang et al.
VC 2016 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology, 18, 3768–3784
resulting TRX1–GFP fusion construct was confirmed by
sequencing analysis and transformed into protoplasts of
mutant A52G. Transformants resistant to both neomycin and
hygromycin were screened by PCR and examined for GFP
signals. Similar strategies were used to generate the TRX2-
GFP and PMK1-GFP construct with the neomycin-resistance
gene amplified from pFL2 (Zhou et al., 2011a) and transform-
ant of the Dtrx2 mutant B9G. The PMK1-GFP construct also
was transformed into protoplasts of Guy11 as a control.
Appressorium formation, penetration, and plant infectionassays
Conidia were harvested from 12-day-old OTA cultures and re-
suspended in sterile distilled water to 105 conidia ml21 (Liu
et al., 2011). For appressorium formation assays, drops of 50
ml of conidium suspensions were placed on cover slips or Gel-
Bond membranes (Cambrex, East Rutherford, NJ) and
incubated at 258C (Liu et al., 2011). Appressorium formation
assays with hyphal tips were performed as described (Liu
et al., 2010b). Two-week-old seedlings of rice cultivar LTH or
CO-39 and 8-day-old seedlings of barley cultivar Golden
Promise were used for infection assays with conidia (5 3 105
conidia ml21) re-suspended in 0.25% gelatin (Park et al.,
2002; Tucker et al., 2004; Kong et al., 2012). For assaying
hyphal growth and conidiation with wound-inoculated leaves,
segments of barley leaves with the wound sites were excised,
surface sterilized and incubated in a moisture chamber under
constant light as described (Xu and Hamer, 1996; Ding et al.,
2010). Penetration assays with onion epidermis and barley or
Brachypodium distachyon leaves were performed as
described (Tucker et al., 2004; Kankanala et al., 2007).
Complementation of the yeast ~trx2 mutant
The S. cerevisiae ~trx2 deletion mutant was derived from
strain BY4741 (Open Biosystems, Huntsville, AL). The full-
length TRX1 and TRX2 genes were amplified from first strand
cDNA of strain Guy11 and cloned into pYES2 (Invitrogen)
respectively. The resulting constructs were transformed into
the ~trx2 mutant with the alkali-cation yeast transformation
kit (MP Biomedicals, Solon, OH). Ura31 transformants were
confirmed by PCR and assayed for sensitivity to 0.8 mM tert-
butyl hydroperoxide as described (Garrido and Grant, 2002).
Generation of the MST7-3xFLAG and MST7-GFP fusionconstructs and mutant alleles of MST7
The MST7-33FLAG fusion construct (pM7F) was generated
by cloning the MST7 gene into pFL7 by yeast gap repair
(Zhou et al., 2011a). The same approach was used to gener-
ate the MST7-GFP construct. The QuickChange II site-
directed mutagenesis kit (Agilent Technologies, Santa Clara,
CA) was used to introduce the C114A, C136A, C201A, and
C305A mutations in the MST7-33FLAG vector pM7F. After
transformation of protoplasts of the mst7 mutant ZH32 (Zhao
et al., 2005), wild-type Guy11, or Dtrx2 mutant B9G, trans-
formants expressing individual MST7 fusion or mutant
constructs were identified by PCR and confirmed by western
blot analysis.
Quantitative RT-PCR assays
The TRIzol reagent (Invitrogen, San Diego, CA) was used to
isolate RNA from conidia collected from 12-day-old OTA cul-
tures, 24 h appressoria on artificial hydrophobic surfaces, and
infected rice leaves harvested 5 dpi. The DNA-free kit (Prom-
ega, Madison, USA) was used for RNA purification. All qRT-
PCR reactions were performed with the CFX96 Real Time
System machine (BIO-RAD, Munich, Germany) using RT2
Real-TimeTM SYBR Green (SABiosciences, MD). Primers
used for qRT-PCR assays were listed in Supporting Informa-
tion Table S1. The relative expression levels of each gene
were calculated by the 2-DDCT method (Livak and Schmittgen,
2001) with the MoACT1 actin gene as the internal control.
Mean and standard deviation were estimated with data from
three biological replicates.
Western blot analysis
Total proteins were isolated from vegetative hyphae as
described (Bruno et al., 2004) and separated on 10% SDS-
PAGE gels. After transfer to nitrocellulose membranes, west-
ern blot analysis was performed with the ECL Supersignal
system (Bruno et al., 2004; Liu et al., 2011). Phosphorylation
of the TEY dual phosphorylation site in Pmk1 and Mps1 was
detected with the PhophoPlus p44/42 MAP kinase antibody kit
(Cell Signaling Technology, Danvers, MA) following the manu-
facturer’s instructions. The same protein samples were used
for western blot analysis with native gels (Wu et al., 2013).
Unlike SDS-PAGE gels, for separating proteins on native gels,
b-mercaptoethanol was not added to the loading buffer and
SDS was not added to the polyacrylamide gel and electropho-
resis buffer in native gels. The electro-transfer buffer also
lacked SDS and was cooled to avoid overheating.
Acknowledgements
We thank Dr. Huiquan Liu for assistance with phylogenetic
analysis and Dr. Chenfang Wang and Chengkang Zhang for
fruitful discussions. We also thank Dr. Larry Dunkle for critical
reading of this manuscript. This work was supported by a
grant from the National Research Initiative of the USDA NIFA
to JRX (Award number: 2010-65110-20439) and a grant from
National Science Foundation of China to CJ (grant number:
31301607).
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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. Trx1 and Trx2 of Magnaporthe oryzae. A. A sche-
matic draw of the Trx1, Trx2, and MGG_08147 with the
TRX, DUF862, PUL (PLAP, Ufd3p and Lub1p), and PPPDEputative peptidase domains labelled. B. Phylogenetic rela-tionship of Trx1, Trx2, and their orthologs. The amino acidsequences of Trx1 from Magnaporthe oryzae (MGG_15004and MGG_04236), Saccharomyces cerevisiae (SCTRX1,SCTRX2 and SCTRX3), Schizosaccharomyces pombe(SPAC7D4.07c and SPBC12D12.07c), Aspergillus nidulans(ANID_00170 and ANID_08571), Botrytis cinerea(BC1G_14403), Sclerotinia sclerotiorum (SS1G_08534),Ustilago maydis (UM06521 and UM01370), Candida albi-cans (CAWG_02294 and CAWG_04484), and Neurosporacrassa (NCU05731, NCU06556 and NCU00598), were ana-lysed by the PhyML3.0 (http://www.atgc-montpellier.fr/phyml/).Fig. S2. The TRX1 and TRX2 deletion constructs andmutants. A. The TRX1 gene replacement event and South-ern blot analysis. Blots of KpnI-digested DNA of Guy11,Dtrx1 mutants (A44G, A52G), and an ectopic transformant(Ect1) were hybridized with a TRX1 fragment amplified withF5 and R6 (Probe 1) or a neoR neomycin resistance genefragment (Probe 2). B. The TRX2 gene replacement eventand Southern blot analysis. Blots of EcoRI-digested of DNAof Guy11, Dtrx2 mutant (B9G), Dtrx1 Dtrx2 double mutant(D65), an ectopic transformant (Ect2) of A52G were hybri-dized with a TRX2 fragment (Probe 3) or a hph fragmentamplified with H850 and H852 (Probe 4). The Dtrx2 andDtrx1 Dtrx2 mutants lacked the wild-type 8.0-kb band hybri-dized to probe 3 but had the expected 7.7-kb band hybri-dized to probe 4.
Fig. S3. Expression and localization of Trx2-GFP fusionproteins in appressoria formed on hyphal tips by the Dtrx2/TRX2-GFP transformant B9G-C.Fig. S4. Assays of TRX1 and TRX2 expression by qRT-PCR. A. The abundance of TRX1 and TRX2 transcripts inGuy11 was assayed with RNA isolated from conidia (con),12 h appressoria (ap), and infected rice leaves 7 dpi (in).Their relative expression levels in conidia were arbitrarilyset to 1. B. Comparison of the expression levels of TRX1and TRX2 in conidia, appressoria, and infected rice leaves.The relative expression level of TRX1 was set to 1 in all thesamples. Mean and standard deviation were calculated withdata from three independent replicates.
Fig. S5. The expression levels of TRX1 and TRX2 inGuy11 treated with or without 6 mM H2O2. Their relativeexpression level in Guy11 without H2O2 treatment was setto 1. Mean and standard deviation were calculated withdata from three independent replicates.Fig. S6. The expression levels of TRX1 and TRX2 in thewild type and Dtrx1 or Dtrx2 mutant was assayed with RNAisolated from vegetative hyphae. The relative expressionlevel in Guy11 was set to 1. Mean and standard deviationwere calculated with data from three independent replicates.
Table S1. PCR primers used in this study.
3784 S. Zhang et al.
VC 2016 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology, 18, 3768–3784