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

Thioredoxins and infection-related morphogenesis 3769

VC 2016 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology, 18, 3768–3784

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

3770 S. Zhang et al.


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.



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).



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).



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.



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.



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.



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.



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.



(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



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



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.



http://www.atgc-montpellier.fr/phyml

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