Asano et al. Proteome Science 2012, 10:61
http://www.proteomesci.com/content/10/1/61

RESEARCH

Open Access

The defense response in Arabidopsis thaliana
against Fusarium sporotrichioides
Tomoya Asano1,2, Makoto Kimura3 and Takumi Nishiuchi1,4*Abstract
Background: Certain graminaceous plants such as Zea mays and Triticum aestivum serve as hosts for Fusarium
sporotrichioides; however, molecular interactions between the host plants and F. sporotrichioides remain unknown.
It is also not known whether any interaction between Arabidopsis thaliana and F. sporotrichioides can occur.
To understand these interactions, we performed proteomic analysis.
Results: Arabidopsis leaves and flowers were inoculated with F. sporotrichioides. Accumulation of PLANT
DEFENSIN1.2 (PDF1.2) and PATHOGENESIS RELATED1 (PR1) mRNA in Arabidopsis were increased by inoculation of
F. sporotrichioides. Furthermore, mitogen-activated protein kinase 3 (MPK3) and mitogen-activated protein kinase
6 (MPK6), which represent MAP kinases in Arabidopsis, were activated by inoculation of F. sporotrichioides.
Proteomic analysis revealed that some defense-related proteins were upregulated, while the expression ofphotosynthesis- and metabolism-related proteins was down regulated, by inoculation with F. sporotrichioides. We
carried out the proteomic analysis about upregulated proteins by inoculation with Fusarium graminearum. The
glutathione S-transferases (GSTs), such as GSTF4 and GSTF7 were upregulated, by inoculation with F. graminearuminfected Arabidopsis leaves. On the other hand, GSTF3 and GSTF9 were uniquely upregulated, by inoculation with
F. sporotrichioides.
Conclusions: These results indicate that Arabidopsis is a host plant for F. sporotrichioides. We revealed that defense
response of Arabidopsis is initiated by infection with F. sporotrichioides.
Keywords: Arabidopsis thaliana, Defense response, Fusarium sporotrichioides, T-2 toxin, MAP kinase, GSTs,
Superoxide dismutase, Ascorbate peroxidase

BackgroundFusarium head blight (FHB) is a severe disease that
affects cereal crops worldwide [1,2]. This disease iscaused by Fusarium species, such as Fusarium sporotrichioides, F. graminearum, and F. culmorum. The soilborne pathogen F. sporotrichioides is often observed incold climates, such as in northern Japan, the northernUSA, northern Europe, and Russia [3]. F. sporotrichioides
was first isolated from corn in France and was identified
as F. tricinctum NRRL 3229 [3]; it was later determinedthat this strain included both F. sporotrichioides andF. tricinctum. Similarly, the F. sporotrichioides IFO 9955
* Correspondence: tnish9@staff.kanazawa-u.ac.jp
1
Division of Functional Genomics, Advanced Science Research Center,
Kanazawa University, 13-1 Takaramachi, Kanazawa 920-0934, Japan
4
Division of Life Science, Graduate School of Natural Science andTechnology, Kanazawa University, Kanazawa 920-1192, Japan
Full list of author information is available at the end of the article

strain was identified from a bean hull by Ueno et al. [3],although it had previously been misidentified as F. solani[3]. It has been reported that overwintered cereals
colonized by F. sporotrichioides caused the deaths ofapproximately 1,100 people in the erstwhile USSR
during World War II [3].
Some Fusarium species also produce trichothecene
mycotoxins, which are known to inhibit protein synthesisin eukaryotes [4,5]. Trichothecenes encompass many
molecular species, which can be classified into 4 majorgroups (types A–D). Among these species, the type A(i.e., T-2 toxin) and type B (i.e., deoxynivalenol [DON])
trichothecenes are distinguished by the absence orpresence of a ketone group at the C-8 position, respectively [6]. Trichothecenes are often found in cereal grains
and cereal-derived commodities [6]. T-2 toxin has beenreported to be approximately 10 times more toxic tomammals and plants than the type B trichothecenes,© 2012 Asano et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, andreproduction in any medium, provided the original work is properly cited.

Asano et al. Proteome Science 2012, 10:61
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such as DON [7]. The tri5 mutant of F. graminearum,
which cannot produce DON, can infect wheat florets andspikes, but it has decreased virulence against host plants[8]. Bai et al. suggested that DON supports the infectionof F. graminearum in wheat [8]. On the other hand, theexpression of PLANT DEFENSIN1.2 (PDF1.2) andPATHOGENESIS RELATED1 (PR1) mRNAs in Arabidopsis were induced by T-2 toxin [9]. Mitogen-activatedprotein kinase 3 (MPK3) and mitogen-activated protein
kinase 6 (MPK6) in Arabidopsis have also been shown tobe activated by T-2 toxin [9]. Nishiuchi et al. suggested
that T-2 toxin possesses an elicitor-like activity [9].
The elicitors from Fusarium species induce the defense
response of Arabidopsis cell suspension cultures [10,11].
The chitosan from F. moniliforme induced the expressionof glutathione S-transferases (GSTs) in Arabidopsis,
which is marker protein of pathogen defense response[11]. Moreover, some Fusarium species produce the
mycotoxins, such as fumonisins [12]. The fumonisins areproduced by F. moniliforme, [12] F. proliferatum, F.
anthophilum, F. dlamini, F. napiforme and Alternaria
alternata f. sp. lycopersici [12]. The fumonisins acts as
an inhibitor of sphingosine N-acetyltransfearase [12],and the fumonisin B1 (FB1) causes the program cell
death (PCD) in plants and animals [13]. Furthermore,
the expression of PR genes and PDF1.2 were induced
by FB1 [13].
The upregulation of GSTs has been reported in typeB-producing F. graminearum-infected wheat flowers [14].
GSTs function in the detoxification of both endogenous
and xenobiotic compounds [15]. Gardiner et al. foundevidence for nonenzymatic formation of DON-GSH conjugates in vitro using both liquid chromatography-mass
spectrometry and nuclear magnetic resonance analysis[16] and suggested that GST is involved in DON detoxification [16]. Furthermore, disease symptoms manifested
by Arabidopsis in response to F. graminearum are
related to ethylene signaling [17]; other interactions between host plants and some Fusarium species have beenreported. However, it is unknown whether there is aninteraction between A. thaliana and F. sporotrichioides.
Proteomics is a tool to gain information on the protein
levels, and it has frequently been used to study plantdiseases [18,19]. Mukherjee et al. suggested that proteomic analysis of the defense response to Alternaria brassicicola can be compared to other types of plant–
pathogen interactions and leaf senescence in Arabidopsis[20]. The interaction of F. graminearum with some hostplants has also previously been investigated by proteomic
analysis. For instance, Triticum aestivum interacts with
F. graminearum [21]. In this regard, Zhou et al. suggested that proteins related to jasmonic acid signaling
pathways, PR protein, amino acid synthesis, and nitrogenmetabolism were upregulated by inoculation of the plant

Page 2 of 10with F. graminearum [21]. The defense response of
Hordeum vulgare is also elicited by infection with
F. graminearum [22]. Geddes et al. suggested that FHB
caused increases in proteins associated with the oxidative
burst and oxidative stress response, such as malate dehydrogenase, peroxidases, and PR protein [22]. However,
although F. sporotrichioides produces the strong T-2
toxin, the molecular interaction between host plants andF. sporotrichioides is not understood.
In this paper, we report that Arabidopsis can act as ahost to F. sporotrichioides. We demonstrate the defense
response in Arabidopsis leaves caused by inoculation
with F. sporotrichioides; furthermore, a proteomic analysis revealed induction of some defense response proteins by inoculation with F. sporotrichioides.

Results and discussion
In this study, we first examined the virulence of F.
sporotrichioides in Arabidopsis rosette leaves and flower
buds. Makandar et al. revealed that efficient infection
and disease by F. graminearum occurs when the fungus
is infiltrated into Arabidopsis leaves [23]. F. graminearum
H3 and F. graminearum ZEA-1 strains [24] werereported to show the pathogenicity to cereals in a farm
(unpublished data). F. graminearum H3 produces DON,
and F. graminearum ZEA-1 produces zearalenone (ZEA)
and DON [24]. We infiltrated with highly concentrated
conidial suspensions (1 × 105 conidia/mL) of plantfungal F. graminearum H3 or ZEA-1 into Arabidopsis
leaves. The both infectious hyphae of F. graminearum
ZEA-1 and F. graminearum H3 were observed at a rateof about 50% in inoculated Arabidopsis leaves at 2 days
post inoculation (dpi). These results indicate thatF. graminearum ZEA-1 and F. graminearum H3 arephytopathogens to Arabidopsis. Next, we inoculated theArabidopsis leaves with highly concentrated conidial
suspensions (1 × 106 conidia/mL) of F. sporotrichioides(Figure 1). At 2 dpi, normal leaves were observed inabout 72% of the Arabidopsis leaves that had been infiltrated with F. sporotrichioides (Figures 1B, F) comparedwith mock treatment leaves (Figures 1A, E). However,transparent regions were observed in about 21% of
those leaves (Figures 1C, G), and infectious hyphae wereobserved in about 7% of those leaves (Figures 1D, H). On
the other hand, hyphae of F. sporotrichioides were also
observed in all the inoculated Arabidopsis flower buds
(Figures 1O, P). Trypan blue staining showed that infectious hyphae were present in the leaves infiltrated with
F. sporotrichioides at 2 dpi (Figures 1I-N). Thus, theseresults indicate that Arabidopsis is a host plant ofF. sporotrichioides.
We next examined whether T-2 toxin accumulated
in F. sporotrichioides–inoculated leaves. The type B
trichothecenes, such as DON, were detected by matrix-

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Figure 1 Inoculation of Arabidopsis leaves and flower buds with Fusarium sporotrichioides. Photographs of mock-treated (A, E, I) andconidial suspension–treated Arabidopsis leaves (B–D, F–H, J–L) at 2 dpi. (E–H) represents magnification of the inner box of panels A–D,
respectively. Trypan blue staining of mock-treated leaves (I) and leaves inoculated with F. sporotrichioides (J–L). (M, N) Cross-sections of Trypan
blue staining of leaves inoculated with F. sporotrichioides. Arrowheads show the hyphae. Photographs of mock-treated (O) and conidial
suspension–treated flower buds (P) at 2 dpi. The scale bars indicate 1 cm (A–D), 1 mm (E–H, O, P), and 100 μm (I–N).

assisted laser desorption/ionization time-of-flight mass
spectrometry (MALDI-TOF MS) [25]. Thus, we thenestablished a method for quantification of T-2 toxin
using MALDI-TOF MS. We tested sodium azide as amatrix for detecting T-2 toxin. Additional file 1 showsthat T-2 toxin was successfully ionized by sodium azide
at 489.2 Da, corresponding to the sodium adduct of thetoxin. In addition, the detection limit for T-2 toxin wasAsano et al. Proteome Science 2012, 10:61
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approximately 50 fmol (data not shown). Anothertype A trichothecene, diacetoxyscirpenol (DAS), was also
detected by the same method (Additional file 1). Because
DAS was not detected in any of the samples inoculatedwith F. sporotrichioides, we used DAS as an internal
control to quantify T-2 toxin in F. sporotrichioides–
inoculated tissues. We then measured the concentrationof T-2 toxin in rosette leaves inoculated with conidial
suspensions of F. sporotrichioides by the infiltration
method. T-2 toxin appeared to have accumulated in theF. sporotrichioides–inoculated leaves to an average levelof 17.6 ± 6.84 ng per leaf (n = 6), whereas T-2 toxin was
not detected in the mock-treated leaves. The accumulation of this volume of T-2 toxin in Arabidopsis leaves
caused the cell death and defense response [9]. Thus,
the accumulation of T-2 toxin in the F. sporotrichioides–
inoculated leaves was sufficient to contribute to theirvirulence.
After infiltration inoculation with highly concentrated
microconidial suspensions, infectious F. sporotrichioides
hyphae were observed in Arabidopsis leaf cells (Figures 1M,
N; arrowheads). Arabidopsis has previously been
demonstrated to show interaction with F. sporotrichioides in that the expression of PR1 and PDF1.2a
mRNAs in Arabidopsis were induced by inoculation
with fumonisins-producing F. moniliforme [13], F.
oxysporum [26], F. graminearum [9]. We therefore
investigated the amount of PR1 and PDF1.2a mRNAsof F. sporotrichioides-inoculated Arabidopsis leaves byquantitative real-time RT-PCR (qRT-PCR) analysis. The
PR1 mRNAs were induced by inoculation with F. sporotrichioides at 24 and 48 h post inoculation (Figure 2A).
The PDF1.2a were induced by inoculation with F. sporotrichioides at 48 h post inoculation (Figure 2A).
Moreover, the activities of p47 (MPK6) and p44(MPK3) have been shown to be activated by bacterial andfungal pathogen–associated molecular patterns (PAMPs)
during plant–pathogen interactions [27]. MPK6 andMPK3 have also been shown to be activated by T-2 toxin
and DON [9]. To investigate the defense response of
Arabidopsis to F. sporotrichioides, we performed anin-gel kinase assay using myelin basic protein. The
activities of MPK6 and MPK3 were not different between
F. sporotrichioides–inoculated and mock-treated leaves
by 6 h post inoculation (Figure 2B). However, theactivities of MPK6 was increased by inoculation of
F. sporotrichioides compared to mock treatment by
24 h post inoculation (Figure 2B). On the other hand,the activation of MPK3 was weak compared with MPK6
(Figure 2B). These results indicate that the defense response of Arabidopsis is induced by F. sporotrichioides
infection.
To profile the defense response of Arabidopsis leaves
against F. sporotrichioides, we performed a proteomic

Page 4 of 10

Figure 2 The expression pattern of PDF1.2a and PR1 mRNA in
Arabidopsis by inoculation of F. sporotrichioides. (A) The
expression analysis of PR1 and PDF1.2a mRNA in Arabidopsis after
inoculation. ACT2 and ACT8 were used as reference genes. Theseexperiments were repeated 3 times. (B) Activity of MPK3 and MPK6
in response to inoculation with F. sporotrichioides. Upper panel:
results of an in-gel kinase assay. Lower panel: CBB staining of theloading control. The amplification efficiency for qRT-PCR of PR-1,
PDF1.2 and ACT2, 8 were 91.6, 86.7 and 98.2%, respectively. Theseexperiments were repeated 3 times.

analysis in mature Arabidopsis leaves inoculated with
F. sporotrichioides by the infiltration method. For thispurpose, we performed two-dimensional (2D) difference gel electrophoresis. Total protein was extracted
from mock-treated and F. sporotrichioides–inoculated
Arabidopsis leaves, and fluorescently labeled with Cy3
and Cy5, respectively. The resulting fluorescently labeledproteins were mixed and subjected to 2D electrophoresison the same gel. As shown in Figures 3A–E, 24 protein
spots exhibited significantly different expression patterns (upregulated or down regulated) in Arabidopsis
leaves inoculated with F. sporotrichioides. These protein spots were digested with trypsin, and the resulting
peptides were identified by MALDI-TOF/TOF analyzer.
Many proteins belonging to GSTs were increased
in F. sporotrichioides–inoculated Arabidopsis leaves(Figure 3F). Table 1 shows that the levels of 5 GSTs
from spots A3, A5, A7, A8, and A9 were upregulated
to 2.66, 3.28, 1.82, 204, and 1.35-fold by inoculation,
respectively (Table 1). The spots A5 (GSTF4a) and A7

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Figure 3 Proteomic analysis of Arabidopsis leaves inoculated with a conidial suspension of F. sporotrichioides. (A) Protein spots ofmock-treated samples (Cy3-labeled total protein). (B) Protein spots of F. sporotrichioides–inoculated leaves (Cy5-labeled total protein). (C) Merge of(A) and (B) images. Spots of downregulated (D) and upregulated (E) proteins in F. sporotrichioides–inoculated leaves are shown. The spot
numbers correspond to the results of protein identification (Table 1). This experiment was repeated 3 times. Functional classification of theidentified proteins is shown. (F) The proteins increased and (G) decreased in response to inoculation with F. sporotrichioides.

(GSTF4b) were identified as same GSTF4. Some posttranslational modifications of GSTs have been identified in Arabidopsis [28]. This different position of
GSTF4 might be the post-translational modification.
GSTs in Arabidopsis were used as marker protein of
pathogen defense response [11]. GSTs are known tofunction in the maintenance of the redox state and thedetoxification of toxins in many species [29]. The formation of DON-glutathione (DON-GSH) conjugates islikely to play a role in the detoxification of DON [16].
Therefore, these enhanced GSTs might be involved in
the detoxification of T-2 toxin.
Moreover, superoxide dismutase (SOD) from spot A4
was upregulated to 1.3-fold by inoculation (Table 1).
The ascorbate peroxidase (APX) protein from spot A12
was enhanced to 2.0-fold by inoculation (Table 1). It
is known that T-2 toxin causes the accumulation ofhydrogen peroxide in Arabidopsis leaves [9]. Accumulated

reactive oxygen species (ROS) are detoxified by SOD and
APX [30]. Thus, the induction of SOD and APX by inoculation of F. sporotrichioides might also be associatedwith the detoxification of superoxide in Arabidopsis
leaves [29].
To compare the defense response in Arabidopsis toF. sporotrichioides and other Fusarium species, we carried out the proteomic analysis with inoculation of two
known plant-fungal F. graminearum. F. graminearum
H3 and ZEA-1 were used in this study. Total proteinsof mock, F. graminearum H3 and F. graminearum
ZEA-1-treated Arabidopsis leaves were fluorescently
labeled with Cy2, Cy3 and Cy5, respectively. The
expression of GSTF7 was upregulated to 1.20-fold by
inoculation of F. graminearum H3 compared with mocktreatment (Figure 4A, B, Table 2). GSTF4a, GSTF4b,
GSTF3, GSTF9, APX and SOD were not increased withinoculation of F. graminearum H3 (Figure 4A, B, D, E,

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Table 1 Identification of protein spots that were differentially expressed after the inoculation of Arabidopsis leaves
with F. sporotrichioides
Spot No.

Cov. (%)aTotalbDescription

SpeciesProtein IDc

Fusarium/Mockd

A1

38.2

14.0

LHCA1

A. thaliana

gi|15233115

6.15

A1

35.2

10.4

Putative H+-transporting ATP synthase

A. thaliana

gi|18491181

6.15

A2

8.10

1.06

Transketolase-like protein

A. thaliana

gi|7329685

62.4

A3

30.8

6.00

Glutathione S-transferase 6 (GSTF3)

A. thaliana

gi|15218640

2.66

A4

29.5

12.0

Superoxide dismutase

A. thaliana

gi|4455253

1.34

A5

59.9

18.8

Glutathione S-transferase (GSTF4a)

A. thaliana

sp|P46422|GSTF4_ARATH

3.28

A6

9.00

2.06

Alpha-xylosidase

A. thaliana

sp|Q9S7Y7|XYL1_ARATH

22.6

A7

57.5

15.9

Glutathione S-transferase (GSTF4b)

A. thaliana

sp|P46422|GSTF4_ARATH

1.82

A8

42.1

12.5

Glutathione S-transferase 11 (GSTF9)

A. thaliana

sp|Q9SRY5|GSTF9_ARATH

204

A9

54.4

24.0

Glutathione S-transferase 9 (GSTF7)

A. thaliana

gi|15224581

1.35

A10

34.1

11.5

ATP synthase gamma chain

A. thaliana

gi|5708095

1.15

A11

34.6

15.7

Formate dehydrogenase

A. thaliana

gi|15241492

1.38

A12

12.5

3.20

putative ascorbate Peroxidase APX4

A. thaliana

gi|31980500

1.95

B1

44.4

18.4

Rubisco activase

A. thaliana

gi|18405145

0.65

B2

44.3

24.4

Rubisco activase

A. thaliana

gi|18405145

0.44

B3

10.4

11.9

Embryo defective 2726

A. thaliana

gi|18417320

0.57

B4

43.0

22.2

Rubisco activase

A. thaliana

gi|18405145

0.76

B5

26.7

10.0

Glutamine synthetase 2

A. thaliana

gi|15238559

0.67

B6

38.5

20.0

Rubisco activase

A. thaliana

gi|30687999

0.70

B7

17.1

13.1

Chaperonin, putative

A. thaliana

gi|15231255

0.88

B7

18.3

6.01

ATP synthase CF1 alpha subunit

A. thaliana

gi|7525018

0.88

B8

19.5

6.15

ATP synthase gamma chain,chloroplast precursor

A. thaliana

gi|5708095

0.54

B8

7.80

4.00

Indole-3-acetonitrile nitrilase

A. thaliana

gi|30692067

0.54

B9

12.9

6.09

Glyceraldehyde-3-phosphate
dehydrogenase B subunit

A. thaliana

gi|336390

0.96

B10

21.7

26.3

Arabidopsis thaliana glycine
Decarboxylase P-protein 2

A. thaliana

gi|15225249

0.78

B11

24.9

24.1

Arabidopsis thaliana glycine
Decarboxylase P-protein 1

A. thaliana

gi|15234036

0.65

B12

28.7

10.7

Ferredoxin-NADPH(+)-Oxidoreductase 2

A. thaliana

gi|15223753

0.89

a

Cov. (%); Coverage: the percent ratio of all amino acids from valid peptide matches to the total number of amino acids in the protein. bTotal ProtScore wascalculated from confidence for all peptides detected for a given identified protein. cProtein ID indicates the accession number of the protein. dThe ratio of
Fusarium/Mock shows the fold-change in expression levels between Fusarium-inoculated leaves and mock-treated leaves for each protein. Data are representativeof 3 independent experiments.

Table 2). On the other hand, GSTF4a, GSTF4b, GSTF7and APX were upregulated to 1.48, 1.19, 1.36 and2.17-fold by inoculation of F. graminearum ZEA-1,
respectively (Figure 4A, C, D, F, Table 2). The extracellular matrix from F. moniliforme contains various
elicitors to Arabidopsis, such as chitosan [11]. The
expression of GSTF7 was enhanced by inoculation of
F. sporotrichioides, F. graminearum H3 and F. graminearum ZEA-1 (Figure 4G). These results indicatethat GSTF7 might be enhanced by common elicitorsof Fusarium species. On the other hand, the expressionof GSTF4a, GSTF4b and APX were increased with F.
sporotrichioides and F. graminearum ZEA-1 (Figure 4G).
We suggest that GSTF4a, GSTF4b, GSTF7 and APX
were enhanced by common elicitors of F. sporotrichioides
and F. graminearum H3.
Also, the expression of GSTF3, GSTF9 and SOD wereuniquely enhanced by inoculation with F. sporotrichioides(Figure 4G). F. sporotrichioides produces trichothecenes,
which is T-2 toxin [6]. T-2 toxin from F. sporotrichioides
induces the defense response in Arabidopsis [9]. The expression of GSTF3, GSTF9 and SOD might be enhanced

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Figure 4 The expression pattern of GSTs, APX and SOD in Arabidopsis leaves inoculated with a conidial suspension of F. graminearum
H3 and ZEA-1. (A, D) Protein spots of mock-treated samples (Cy2-labeled total protein). (B, E) Protein spots of F. graminearum H3-inoculated
leaves (Cy3-labeled total protein). (C, F) Protein spots of F. graminearum ZEA-1-inoculated leaves (Cy5-labeled total protein). This experiment was
repeated 3 times. (G) Venn diagram showing of expression pattern of GSTs, APX and SOD proteins in Arabidopsis leaves inoculated with aconidial suspension of F. sporotrichioides, F. graminearum H3 and ZEA-1.

by T-2 toxin. We suggest that the defense response in
Arabidopsis against F. sporotrichioides causes by thevarious elicitors.
Conversely, the expression of photosynthesis- andmetabolism-related proteins such as Rubisco activase,
ATP synthase, and ferredoxin-NADP+ oxidoreductase
was downregulated in Arabidopsis leaves by inoculation
with F. sporotrichioides (Figure 3G). Rubsico activase
from spots B1, B2, B4, and B6 were downregulated
0.65, 0.44, 0.76, and 0.70-fold by inoculation (Table 1).
Rubisco activase is required to allow the regeneration of

the critical carbamate in the active site of Rubisco [31].
Moreover, glutamine synthetase 2 was decreased to 0.67fold by inoculation (Table 1). Glutamine synthetase is
one of the essential enzymes for glutamine production[32]. We propose that the activation of the defensesignaling pathway was enhanced, while the expression ofphotosynthesis- and metabolism-related proteins was
suppressed by inoculation with F. sporotrichioides.
Five days after inoculation with F. graminearum, GSTs
and SOD were upregulated or induced, and Rubisco
activase were downregulated [14,21]. These results

Table 2 The protein spots that were differentially expressed after the inoculation of Arabidopsis leaves with two
F. graminearum H3 and ZEA-1 strains
Spot No.

Description

Protein IDa

F. graminearum
H3/Mockb

F. graminearum
ZEA-1/Mockb

A3

Glutathione S-transferase 6 (GSTF3)

gi|15218640

1.03

1.13

A4

Superoxide Dismutase

gi|4455253

1.10

1.10

A5

Glutathione S-transferase (GSTF4a)

sp|P46422|GSTF4_ARATH

1.13

1.48

A7

Glutathione S-transferase (GSTF4b)

sp|P46422|GSTF4_ARATH

1.03

1.19

A8

Glutathione S-transferase 11 (GSTF9)

sp|Q9SRY5|GSTF9_ARATH

N. D.

N. D.

A9

Glutathione S-transferase 9 (GSTF7)

gi|15224581

1.20

1.36

A12

Putative Ascorbate Peroxidase APX4

gi|31980500

0.80

2.17

a
Protein ID indicates the accession number of the protein. bThe ratio of Fusarium/Mock shows the fold-change in expression levels between Fusarium-inoculated
leaves and mock-treated leaves for each protein. N. D. shows the not detected the spot. Data are representative of 3 independent experiments.

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indicate that the interaction between Arabidopsis andF. sporotrichioides is similar to the interaction between
wheat and F. graminearum at the proteomic level.

Page 8 of 10

plants were incubated in a chamber under about 100%
relative humidity, at 22°C, and a 16/8-h light/dark cycle.
Trypan blue staining

Conclusions
Some phytopathogenic Fusarium species, including
F. sporotrichioides, are known to produce type A trichothecenes, such as T-2 toxin. However, the interactions between type A trichothecene–producing Fusarium
species and plants have not been well studied. In thisstudy, virulence of F. sporotrichioides was observed in
Arabidopsis leaves after inoculation. Even when the
Arabidopsis leaves were infiltrated with conidial suspensions of F. sporotrichioides, invasive hyphae wereobserved in the leaves. PR1 and PDF1.2 mRNA were
induced by inoculation of F. sporotrichioides. MPK3
and MPK6, which are MAP kinases in Arabidopsis,
were also activated in 24 h by F. sporotrichioides.
Proteomic analysis revealed that some defense-related
proteins, including 5 GSTs, SOD, and APX, wereincreased in the F. sporotrichioides–infiltrated leaves.
Also, GSTF3, GSTF9 and SOD were uniquely enhanced
by inoculation of F. sporotrichioides. These results indicate that a defense response is caused in Arabidopsis
leaves by infection with F. sporotrichioides.
Methods
Plant and fungal growth

The Columbia (Col-0) ecotype of A. thaliana (L.) Heynh.
was used in this study. Arabidopsis seeds were sownin soil, placed at 4°C in the dark for 2 days, andsubsequently grown at 22°C under a 16/8-h light/darkcycle. F. sporotrichioides strain IFO 9955 (previously
misidentified as Fusarium solani) was used in this study[33]. F. graminearum H3 (MAFF101551) and ZEA-1
strains were used in this study. The fungi was grown at
22°C under weak light on Synthetic Low Nutrient (SN)agar medium. The production of microconidia was
induced by SN liquid medium (0.1% KH2PO4, 0.1%
KNO3, 0.1% MgSO4·7H2O, 0.05% KCl, 0.02% glucose,
0.02% sucrose) [34].
Fungal inoculation

For the preparation of conidia, F. sporotrichioides wascultured in SN liquid medium with shaking for 2 days at
22°C, in constant darkness. The conidia were collectedby centrifugation (14,000 g at room temperature for5 min) and were washed with phosphate-buffered saline(PBS) at least 3 times. The collected conidia were suspended in PBS and the number of conidia counted
using a hemocytometer. For the infiltration inoculation, aconidial suspension (1 × 106 conidia/mL) or PBS (mock)without detergent was injected into the abaxial sides ofthe leaves with a needleless syringe [35]. The inoculated

The hyphae of inoculated leaves were stained by previously
described solution [36]. After staining, leaves were washedin chloral hydrate [36].
The quantitative real-time RT-PCR

Total RNA was isolated using the Plant RNA Isolation
Mini Kit (Agilent Technologies, CA, USA). First-strand
cDNA was synthesized using the PrimeScript RT Reagent
Kit (Takara Bio, Shiga, Japan). The qRT-PCR was carriedout with SYBRW Premix Ex Taq™ II (Perfect Real Time)(Takara Bio, Shiga, Japan), gene specific primer pairs for
PR1, PDF1.2 or ACT2/8, respectively, and cDNA as
template. Arabidopsis ACT2/8 was used as reference
genes. The primers used for qRT-PCR were as previously
described (PR1 [37], PDF1.2a [38], and ACT2/8 [39]).
The qRT-PCR analysis was performed using the Mx3000P
QPCR System (Agilent Technologies, CA, USA). The
following PCR program was used: initial denaturation,
95°C, 10 s; 40 cycles of 95°C for 5 s, 60°C for 20 s
and 72°C for 30 s with a temperature transition rateof 20°C/s; and a melting curve analysis, at 95°C for 0s and 65°C for 15 s, and an increase to 95°C with atemperature transition rate of 0.1°C/s. To generate astandard curve, homologous standards were used in all
experiments. The cDNA quantities of target genes were
calculated using Mxpro QPCR software (Agilent Technologies, CA, USA). The q-PCR analysis was carried out
3 times.
In-gel kinase assay

Crude extracts were prepared from F. sporotrichioides–
inoculated or mock-treated leaves. An in-gel kinase assay
was performed as previously described [9].
Quantification of T-2 toxin

The plant and fungal materials were ground to a fine
powder in liquid nitrogen with a mortar and pestle. For
T-2 toxin extraction, the fine powder was added to
10 mL of an acetonitrile–water (84:16) solution containing
1 μg/mL diacetoxyscirpenol (DAS) as an internal standard.
The solution was then incubated on a rotator for 60 min
at room temperature. The extraction mixture was centrifuged (2,000 g, at room temperature, for 5 min), andthe supernatant collected.
To purify T-2 toxin, we used MycoSep 227 Trichcolumns (Romer Labs, Inc., MO, USA). The extraction
mixture was added to a test tube, and a column was
slowly inserted into the test tube. The purified solutions(fraction 1) were collected using the columns andtransferred to new tubes. An acetonitrile–water (84:16)

Asano et al. Proteome Science 2012, 10:61
http://www.proteomesci.com/content/10/1/61

solution (3 mL) containing 1 μg/mL DAS was added tothe test tube, a column was slowly inserted into the test
tube again, and these purified solutions were added to
fraction 1. Aliquots of these extraction mixtures were
evaporated using a SpeedVac concentrator.
To quantify T-2 toxin, the extracts were mixed with
1 mg/mL sodium azide as a matrix and analyzed using
a 4800 Plus MALDI TOF/TOFTM analyzer (AB Sciex,
CA, USA).
Proteomic analysis of Arabidopsis rosette leaves
inoculated with F. sporotrichioides

The plant materials were ground to a fine powder inliquid nitrogen using a mortar and pestle. For protein
extraction, approximately 2 g of this fine powder wasadded to 5 mL of PBS buffer containing 1% Triton-X
100, 1 mM phenylmethanesulfonyl fluoride (PMSF)
and 1/1000 protease inhibitor cocktail (Sigma-Aldrich,
MO, USA) and the solution thoroughly mixed by vortexing. The extract was centrifuged (14,000 g at 4°C
for 15 min) and the supernatant collected. The concentration of protein in each sample was measured
with an RC DC Protein Assay Kit (Bio-Rad Japan,
Tokyo, Japan). Next, the protein was precipitated with
5% trichloroacetic acid (TCA), and the resulting pellet
was washed with 100% acetone at least twice. The
acetone was briefly evaporated from the protein by
aspiration for 5 min. The protein was subsequently
dissolved in lysis buffer (8 M urea, 2% CHAPS, and30 mM Tris–HCl, pH 8.5). The insoluble material was
removed by centrifugation (15,000 g at 4°C for 15 min),and the pH of the supernatant (ranging from 8 to 9)
confirmed with litmus paper.
The proteins were labeled using CyDye DIGE Fluors
developed for fluorescence 2-D technology (GE Healthcare, Tokyo, Japan) according to the manufacturer’s
recommendations. Each sample was covalently labeledwith a different fluorescent dye: Cy3 (mock) or Cy5(Arabidopsis inoculated with F. sporotrichioides). CyDyelabeled proteins (each 30 μg) were loaded onto an 18-cm
rehydration strip with an immobilized pH gradient of
3–10 and separated on a Multiphor electrophoresis
unit (GE Healthcare, Tokyo, Japan) using the following
setting: a 1.5-h gradient from 300–3,500 V, and 5 h at
3,500 V. After the isoelectric focusing (IEF), the rehydration strip was mounted onto the top of a 10%
SDS-polyacrylamide gel with a stacking gel, in a Hoefer
SE 600 Ruby system (GE Healthcare, Tokyo, Japan).
Electrophoresis was performed at a constant voltage of
10 mA for 7 h. After the 2D electrophoresis, the acrylamide gels were directly scanned using a TyphoonTM
9400 imager (GE Healthcare, Tokyo, Japan). The scanned
images were applied to ImageQuant V5.2 software (GEHealthcare, Tokyo, Japan). PDQuest Advanced software

Page 9 of 10(Bio-Rad Japan, Tokyo, Japan) was used to compareindividual images and to localize some of the spots.
Differentially expressed proteins were identified byStudent’s t-test of spot intensity (P < 0.05, n = 3). The
protein spots were collected from the gels using an Ettan
Spot Picker (GE Healthcare, Tokyo, Japan).
Identification of proteins by MALDI-TOF/TOF analysis

MALDI-TOF/TOF analysis was performed, as previously
described [40].
Functional classification of proteins

The identified proteins were classified according to functional categories using The Plant Proteome Database(http://ppdb.tc.cornell.edu/default.aspx).

Additional file
Additional file 1: Mass spectra of T-2 toxin and diacetoxyscirpenolusing MALDI-TOF analysis. (A) T-2 toxin peaks were detected at m/z
489.2. (B) Peaks of the matrix (sodium azide) only. (C) Diacetoxyscirpenol
peaks were detected at m/z 389.1. (D) Peaks of the matrix (sodium azide)
only.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
TA carried out the experiments. MK isolated F. graminearum H3 and ZEA-1
strains. TA and TN designed experiments and wrote the manuscript. TN
designed this project. All authors read and approved the final manuscript.
Acknowledgements
We are grateful to Prof. Kim Hammond-Kosack (Rothamsted Research, UK)for her very valuable suggestions and to Dr. Takayuki Aoki (RIKEN, Japan) for
helpful discussions. This work was supported by Grants-in-Aid for Young
Scientists (no. 21770038) and Scientific Research (no. 23580060) from the
Ministry of Education, Culture, Sports, Science and Technology of Japan. The
work was also supported in part by Research for Promoting TechnologicalSeeds (no. 07–070) from the Japan Science and Technology Agency (JST).
Author details
1
Division of Functional Genomics, Advanced Science Research Center,
Kanazawa University, 13-1 Takaramachi, Kanazawa 920-0934, Japan.
2
Equipment Support Promotion office, Advanced Science Research Center,
Kanazawa University, Kanazawa 920-1192, Japan. 3Plant & Microbial MetabolicEngineering Research Unit, Discovery Research Institute (DRI), RIKEN, 2–1
Hirosawa, Wako, Saitama 351-0198, Japan. 4Division of Life Science, Graduate
School of Natural Science and Technology, Kanazawa University, Kanazawa
920-1192, Japan.
Received: 29 July 2012 Accepted: 5 October 2012
Published: 30 October 2012
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