Báo cáo khoa học: NtKTI1, a Kunitz trypsin inhibitor with antifungal activity from Nicotiana tabacum, plays an important role in tobacco’s defense response pot

NtKTI1, a Kunitz trypsin inhibitor with antifungal activity
from Nicotiana tabacum, plays an important role in
tobacco’s defense response
Hao Huang*, Sheng-Dong Qi*, Fang Qi, Chang-Ai Wu, Guo-Dong Yang and Cheng-Chao Zheng
State Key Laboratory of Crop Biology, College of Life Sciences, Shandong Agricultural University, China
Introduction
Phytopathogen attack represents a major problem for
agriculture in general, and has caused devastating
famines throughout human history [1,2]. Fungal
pathogens are responsible for significant crop losses
worldwide, resulting from both the infection of grow-
ing plants and the destruction of harvested crops [3].
To counter attacks by various fungi with different
infection strategies, plants have evolved multiple and
complex defense mechanisms throughout their life
cycles [4].
Proteinase inhibitors (PIs) are one of the most
important classes of defense proteins, and have been
identified from a broad range of plant species [5,6]. PIs
possess an enormous diversity of function by regulat-
ing the proteolytic activity of their target proteinases,
resulting in the formation of a stable PI complex [7].
Keywords
antifungal activity; Kunitz trypsin inhibitor;
prokaryotic expression; Rhizoctonia solani;
transgenic tobacco
Correspondence
C C. Zheng, State Key Laboratory of Crop
Biology, College of Life Sciences, Shandong
Agricultural University, Taian, Shandong
271018, China

Fax: 86 538 8226399
Tel: 86 538 8242894
E-mail:
*These authors contributed equally to this
work
Database
The nucleotide sequence of NtKTI1 is
available in the GenBank database under
accession number FJ494920
(Received 20 May 2010, revised 21 July
2010, accepted 30 July 2010)
doi:10.1111/j.1742-4658.2010.07803.x
A cDNA library from tobacco inoculated with Rhizoctonia solani was con-
structed, and several cDNA fragments were identified by differential
hybridization screening. One cDNA clone that was dramatically repressed,
NtKTI1, was confirmed as a member of the Kunitz plant proteinase inhibi-
tor family. RT-PCR analysis revealed that NtKTI1 was constitutively
expressed throughout the whole plant and preferentially expressed in the
roots and stems. Furthermore, RT-PCR analysis showed that NtKTI1
expression was repressed after R. solani inoculation, mechanical wounding
and salicylic acid treatment, but was unaffected by methyl jasmonate,
abscisic acid and NaCl treatment. In vitro assays showed that NtKTI1 exerted
prominent antifungal activity towards R. solani and moderate antifungal
activity against Rhizopus nigricans and Phytophthora parasitica var. nicoti-
anae. Bioassays of transgenic tobacco demonstrated that overexpression of
NtKTI1 enhanced significantly the resistance of tobacco against R. solani,
and the antisense lines exhibited higher susceptibility than control lines
towards the phytopathogen. Taken together, these studies suggest that
NtKTI1 may be a functional Kunitz trypsin inhibitor with antifungal activ-
ity against several important phytopathogens in the tobacco defense

response.
Abbreviations
ABA, abscisic acid; BAEE, N-a-benzoyl-
L-arginine ethyl ester; KTI, Kunitz trypsin inhibitor; MeJA, methyl jasmonate; PCD, programmed cell
death; PDA, potato dextrose agar; PI, proteinase inhibitor; PR, pathogenesis-related; SA, salicylic acid; SQRT-PCR, semiquantitative RT-PCR.
4076 FEBS Journal 277 (2010) 4076–4088 ª 2010 The Authors Journal compilation ª 2010 FEBS
They are generally present at high concentration in
storage tissues (up to 10% of protein content), but can
also be induced in response to attacks by insects and
pathogenic microorganisms [8]. Their defense mecha-
nism relies on the inhibition of proteinases produced
by microorganisms, causing a reduction in the avail-
ability of the amino acids necessary for their growth
and development [8,9].
Currently, 59 distinct PI families have been recog-
nized [10]. PIs were initially classified into nonspecific
and class-specific superfamilies, and the latter was sub-
categorized into several families, including serine, cys-
teine, aspartic and metalloproteinase inhibitors [11].
Serine proteinases appears to be the largest family of
proteinases, and plant serine PIs have been classified
into several subfamilies, including soybean (Kunitz),
Bowman–Birk, potato I, potato II, squash, barley,
cereal, Ragi A1 and Thaumatin-like inhibitors [12].
Kunitz PIs are single-chain polypeptides of around
20 kDa with low cysteine content, generally with four
cysteine residues arranged into two intra-chain disul-
fide bridges [7]. The members of this family have one
reactive site and are mostly active against serine pro-
teinases, but may also inhibit other proteinases [13].

They are widespread in plants and have been reported
to respond to various forms of abiotic stress, such as a
radish PI containing the Kunitz motif induced by
NaCl treatment, and BnD22 and AtDr4 responding to
drought stress in rape [14] and Arabidopsis [15], respec-
tively. In potato tubers, Kunitz PIs are induced under
multiple treatments and water-deficient conditions
[16–19]. The rapid synthesis of Kunitz PIs is one of
the most common inducible herbivore defenses in
plants. Several Kunitz trypsin inhibitors (KTIs) have
been reported to be rapidly induced by wounding and
herbivore attack in trembling aspen (Populus tremuloides
Michx.) and poplar (Populus trichocarpa · Populus
deltoides) [6,20]. A chickpea KTI, CaTPI-2, is induced
by mechanical wounding in epicotyls and leaves [21].
To date, only limited plant Kunitz PIs that respond to
pathogen attack have been characterized. Arabidopsis
KTI, AtKTI1, was found to be an antagonist of cell
death triggered by phytopathogens and fumonisin B1,
which modulates programmed cell death (PCD) in
plant–pathogen interactions [22]. Several KTIs are
repressed by the infection of Melampsora medusae in
hybrid poplar [23]. However, all the Kunitz PIs from
different species in these studies were induced by both
biotic and abiotic stress; previously, no tobacco PIs
whose expression is repressed in biotic stress have been
identified.
In this study, we isolated and characterized a KTI
gene (NtKTI1) encoding a functional PI protein in
tobacco. NtKTI1 was preferentially expressed in

tobacco roots and stems, and was repressed in Rhizoc-
tonia solani inoculation, mechanical wounding and sali-
cylic acid (SA) treatment. In vitro antimicrobial assay
and in planta studies demonstrated that NtKTI1 is an
antifungal protein that increases the resistance of
tobacco to fungal attack.
Results
Isolation and characterization of a cDNA encod-
ing NtKTI1
A cDNA clone, NtKTI1, was isolated from tobacco by
differential hybridization screening to identify genes
responding to the infection of the fungus R. solani.It
consisted of 840 nucleotides and contained a 627-bp
open reading frame encoding a polypeptide of 209 resi-
dues weighing approximately 23.1 kDa. By comparing
the genome DNA sequence, we determined NtKTI1 to
be an intronless gene.
A search of the National Center for Biotechnology
Information database revealed that the deduced
NtKTI1 showed similarity to a number of putative
proteins from other plant species, including tomato
Lemir [24], miracle fruit MIR [25], Tc-21, a member of
the Kunitz PI family [26], RASI, an a-amylase ⁄ subtili-
sin inhibitor precursor from rice [27], and Arabidopsis
At1g17860 and At1g73260 (Fig. 1A). To improve the
quality of the alignment, secondary and tertiary struc-
ture predictions were made by JPred and SWISS-
MODEL, which were used to manually edit and refine
the alignment. We included the extensively studied soy-
bean (Glycine max) KTI3 with confirmed inhibitor

activity [28] for comparison. These KTIs typically con-
tain the Kunitz motif (Fig. 1A, conserved residues
denoted by inverted triangles) and four cysteine resi-
dues that form two conserved intramolecular disulfide
bonds. The variability of the second conserved cysteine
residue (Fig. 1A, boxed area outlined by broken line)
does not influence the formation of the disulfide bond.
Most of these KTIs also have two additional free cys-
teine residues located in a loop (Fig. 1A, plus signs).
However, it is interesting that the most conserved
regions correspond to predicted b-sheets. Furthermore,
although some conserved residues are found within the
reactive loop of these KTIs, this loop is highly vari-
able, including the P
1
residue of the reactive site
(Fig. 1A, boxed area and starred residues). The reac-
tive loop of RASI has atypical residues compared with
that of KTI3 and other proteins.
To better characterize NtKTI1, we analyzed the evo-
lutionary relationships of NtKTI1 with the KTI family
H. Huang et al. NtKTI1 participates in tobacco’s fungal defense
FEBS Journal 277 (2010) 4076–4088 ª 2010 The Authors Journal compilation ª 2010 FEBS 4077
members of Arabidopsis and rice. blast analysis of the
Arabidopsis protein database (TAIR8 proteins) resulted
in the identification of seven genes that encode potential
orthologs of NtKTI1. After multiple blast searches
of several databases (see Materials and methods), only
one putative OsKTI (Os04g0526600) in the rice
75NtKTI1

71CAN81015
71AAC49969
71LeMIR
69At1g17860
79MIR
72TC-21
69AtKTI1
66RASI
66KTI3
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144CAN81015
146AAC49969
146LeMIR
136At1g17860
155MIR
148TC-21
144AtKTI1
142RASI
136KTI3
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209NtKTI1

203CAN81015
210AAC49969
205LeMIR
196At1g17860
220MIR
221TC-21
215AtKTI1
200RASI
215KTI3
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**
At1g72290
At1g73330
At1g73325
At1g73260
At1g17860
At3g04320
At3g04330
Os04g05266 00
NtKTI1
10 PAM
A2
A1
B
A3
A
B
Fig. 1. Characterization of NtKTI1 . (A) Sequence alignment of the deduced amino acid sequence of NtKTI1 with other homologous proteins.
Sequences were retrieved from the National Center for Biotechnology Information with the following accession numbers: CAN81015, a
hypothetical protein from grapes (Vitis vinifera), which gained the highest scores, is the closest homolog to NtKTI1; AAC49969, from com-
mon tobacco (Nicotiana tabacum), is a tumor-related protein; Lemir from tomato (Lycopersicon esculentum) (AAC63057); At1g17860 and

AtKTI1 from Arabidopsis; MIR, a miraculin precursor, from Synsepalum dulcificum (BAA07603); Tc-21, a member of the Kunitz proteinase
inhibitor family, from cacao (Theobroma cacao) (1802409A). RASI, an a-amylase ⁄ subtilisin inhibitor precursor (Os04g0526600) from rice
(Oryza sativa) (P29421) and soybean (Glycine max) Kunitz trypsin inhibitor KTI3 (AAB23464) are shown for comparison. The GenBank acces-
sion number of NtKTI1 is FJ494920. Shading shows conserved (black) and similar (gray) amino acid residues, and dots represent sequence
gaps. A full line above the alignment marks the signal peptides, and inverted triangles denote the Kunitz motif. Below the alignment, two
disulfide bridges formed by four conserved cysteine residues are shown in brackets; plus signs (+) denote free cysteine residues. The
known structural features of KTI3 are indicated as follows: arrows above the alignment (M) delineate b-sheets, a boxed region (full line) indi-
cates the reactive loop, and asterisks (*) denote the P
1
and P
1
¢ reactive site residues of KTI3. (B) Phylogenetic analysis of NtKTI1 with homo-
logs of rice and Arabidopsis. The phylogenetic tree was constructed using the default settings of the web-based alignment tool
MULTALIN.
A triangle (m) denotes the tree root.
NtKTI1 participates in tobacco’s fungal defense H. Huang et al.
4078 FEBS Journal 277 (2010) 4076–4088 ª 2010 The Authors Journal compilation ª 2010 FEBS
genome was obtained. Genome sequence analysis and
online prediction revealed that all KTI genes are
intronless and encode proteins with a putative signal
peptide for cell secretion.
Using the default settings of the web-based align-
ment tool multalin, a phylogenetic tree, including
full-length NtKTI1, OsKTI and AtKTIs, was con-
structed (Fig. 1B). Inspection of the phylogenetic tree
reveals that the members of the KTI family are divided
into two clades. Clade A can be further divided into
three groups. At1g72290, a Kunitz-type cysteine PI
[29], forms the single-member group A1. At1g73325, a
Dr4-related protein [22], and At1g73330, a protein

encoded by the drought-repressed Dr4 gene [15], form
group A2. At1g73260, an antagonist of cell death trig-
gered by phytopathogens [22], At1g17860, the closest
homolog of NtKTI1, Os04g0526600, rice RASI, and
NtKTI1 form group A3. Clade B includes At3g04320
and At3g04330, which both contain an incomplete
C-terminal (only two cysteine residues that form one
disulfide bond) compared with the other KTIs.
Expression of NtKTI1 is spatially regulated and
repressed by multiple stimuli
To determine the accumulation pattern of NtKTI1
transcripts in tobacco, semiquantitative RT-PCR
(SQRT-PCR) analysis was performed using the total
RNA isolated from roots, stems, leaves, flowers, young
seeds and mature seeds. The same cDNA was also
used to amplify elongation factor-1a (EF1a)asan
internal control. As shown in Fig. 2A, NtKTI1 was
constitutively expressed throughout the whole plant.
The expression of NtKTI1 was higher in roots and
stems than in other organs (Fig. 2A). In young and
mature seeds, the transcript of NtKTI1 was difficult to
detect. These results suggest that NtKTI1 is preferen-
tially expressed in roots and is not a storage protein.
In addition, the expression level of NtKTI1 increased
in stems and roots at the later developmental stage
(Fig. 2B), indicating that NtKTI1 might be temporally
regulated.
To elucidate the potential involvement of NtKTI1 in
plant defense, we characterized the expression of the
NtKTI1

Young
seed
Mature
seed
Root Stem Leaf Flower
EF1-a
Root
Water
NtKTI1
PR1c
EF1-a
300 mM NaCl
NtKTI1
Nt C7
EF1-a
100 µM MeJA
NtKTI1
PR1c
EF1-a
Wounding
NtKTI1
PR1c
EF1-a
100 µM ABA
NtKTI1
Nt din
EF1-a
5 mM SA
NtKTI1
PR1c

EF1-a
R. solani
NtKTI1
PR1c
EF1-a
Stem
0 1 3 6 12 24 h
0 1 2 3 4 6 d
1 2 3 1
2 3
NtKTI1
EF1-a
A
B
C
Fig. 2. NtKTI1 gene expression patterns determined by SQRT-PCR
analysis. (A) NtKTI1 transcript accumulation in roots, stems, leaves,
flowers, young seeds and mature seeds. (B) NtKTI1 transcript accu-
mulation in stems and roots at different developmental stages: 1,
1-month-old tobacco; 2, 3-month-old tobacco; 3, 5-month-old
tobacco. (C) Time course of NtKTI1 expression following treatment
with Rhizoctonia solani, mechanical wounding, SA, MeJA, ABA,
NaCl and water. Water treatment served as a control. To confirm
the efficacy of treatment, PR1c [60] was used as a positive control
for R. solani, mechanical wounding and SA treatment [61]. Primers
specific for Ntdin, which is induced in response to ABA [61,62],
and NtC7, which is induced in response to NaCl [63], were also
used. EF1a (AF120093) was used as an internal control. Experi-
ments were repeated at least three times. There are three biologi-
cal replications for each independent experiment. The photographs

represent one of three independent experiments that gave similar
results.
H. Huang et al. NtKTI1 participates in tobacco’s fungal defense
FEBS Journal 277 (2010) 4076–4088 ª 2010 The Authors Journal compilation ª 2010 FEBS 4079
gene in plants as a function of exposure to R. solani,
mechanical wounding, SA and methyl jasmonate
(MeJA). As shown in Fig. 2C, when 4-week-old
tobacco seedlings were exposed to R. solani, PR1c was
induced 3 days after inoculation and enhanced at
4 days. Although the transcriptional level of NtKTI1
was not affected significantly during the first 3 days, it
was strongly repressed at 4 days and was barely detect-
able at 6 days. During the 24 h period of mechanical
wounding treatment, the level of NtKTI1 mRNA grad-
ually decreased, whereas PR1c gradually increased.
During SA treatment, PR1c was induced after 12 h
and accumulated to a high level at the 24 h point,
whereas NtKTI1 was clearly repressed after 3 h. In
addition, the expression of NtKTI1 was not affected
by MeJA, abscisic acid (ABA) and NaCl treatments
and water control (Fig. 2C).
NtKTI1 displays in vitro antifungal activity as a
trypsin inhibitor
To elucidate the functional identity of the NtKTI1
gene, we produced an N-terminally His-tagged protein
with and without the predicted signal peptide in
Escherichia coli BL21 (DE3 pLysS), and determined
its biological activity. As shown in Fig. 3A, the puri-
fied recombinant NtKTI1 protein without the signal
peptide had the expected size of about 31.0 kDa and

exhibited a similar inhibitory effect on bovine trypsin
activity to soybean TI (Fig. 3B), strongly suggesting
that NtKTI1 encodes a functional KTI in tobacco.
However, the full-length NtKTI1 protein was not
detectable by SDS ⁄ PAGE (Fig. 3A, lane 2), sug-
gesting that it might form inclusion bodies that are
insoluble.
The antimicrobial activity of NtKTI1 was tested
against an array of fungi and several bacteria in vitro.
The results showed that NtKTI1 obviously inhibited
the hyphal growth of three important phytopathogenic
fungi: R. solani, Rhizopus nigricans and Phytophtho-
ra parasitica var. nicotianae. The antifungal activity
towards R. solani was prominent (Fig. 4A), with anti-
fungal action clearly observed 24 h after loading the
samples. Meanwhile, NtKTI1 also showed moderate
activity against Rh. nigricans (Fig. 4C) and P. parasiti-
ca var. nicotianae (Fig. 4D), but, although the protein
concentration of NtKTI1 was much higher than that
in the in vitro antifungal assay towards R. solani, the
antifungal action was still weak. The fungi grew more
slowly when there was a higher concentration of
NtKTI1 in the plates. However, we did not detect any
activity of NtKTI1 against bacteria, such as E. coli
DH5a (Fig. 4B).
Tobacco plants overexpressing NtKTI1 show
enhanced resistance to R. solani infection
To evaluate the in planta role of NtKTI1 in defense,
sense and antisense lines under the control of the cauli-
flower mosaic virus 35S promoter (35S) were generated.

Stable transgenic integration into plants regenerated on
a selective medium was confirmed by northern blot
analyses (Fig. 5A). Six T2 transgenic lines (three sense
lines and three antisense lines) were constructed and
employed to evaluate the disease resistance of trans-
genic tobacco using a standard detached leaf assay.
The leaves of 3-month-old sense, antisense and control
Trypsin activity (%)
Inhibitor protein (µ
g
)
100
80
60
40
20
0
NtKTI1
(kDa)
97.4
66.2
43.0
29.0
20.1
012345
21345
A
B
Fig. 3. Production of recombinant NtKTI1 protein and in vitro assay
of trypsin inhibitory activity. (A) Coomassie-stained SDS ⁄ PAGE gel

showed bacterial expression and purification of His-tagged NtKTI1
protein without a signal peptide. Lane 1, soluble sample from unin-
duced Escherichia coli extraction with full-length NtKTI1 construct;
lane 2, soluble sample from induced E. coli extraction with full-
length NtKTI1 construct by isopropyl thio-b-
D-galactoside; lane 3,
soluble sample from uninduced E. coli extraction with NtKTI1
construct without putative signal peptide; lane 4, soluble sample
protein from induced E. coli extraction with NtKTI1 construct with-
out putative signal peptide by isopropyl thio-b-
D-galactoside; lane 5,
purified NtKTI1 is indicated by an arrow in the right lane. (B) In vitro
bovine trypsin inhibition by the recombinant NtKTI1 protein (open
circles). Soybean trypsin inhibitor (triangles) was assayed in parallel
as a positive control. Experiments were repeated three times, with
similar results.
NtKTI1 participates in tobacco’s fungal defense H. Huang et al.
4080 FEBS Journal 277 (2010) 4076–4088 ª 2010 The Authors Journal compilation ª 2010 FEBS
plants were inoculated with the fungal pathogen
R. solani. The results showed that fungal hyphae grew
concentrically from the site of inoculation, resulting in
visible necrosis 3 days after infection in all three lines.
However, the detectable necrosis was substantially
smaller in sense plants than in antisense and control
plants: 5 days after infection, the diameter of the
lesions was about 44 mm in the leaves of antisense
plants, but only 17 mm in the leaves of the sense line
(Fig. 5A). Overall, the resistance levels were consistent
with the expression levels of NtKTI1 in different lines,
indicating that the overexpression of NtKTI1 reduced

susceptibility at the early stage of infection and affected
the development and extension of R. solani hyphae in
leaves.
In certain plants, susceptibility to infection by
R. solani decreases with increasing age of the plant;
young tobacco seedlings have been shown to be
severely affected [30]. As shown in Fig. 2B, NtKTI1
may play an important role in the susceptibility of
tobacco towards R. solani at different developmental
stages. To determine whether overexpression of
NtKTI1 enhanced the resistance to pathogens, we inoc-
ulated the seedlings of all three lines with R. solani,a
fungal pathogen. After transplantation into inoculated
soil, the sense lines showed more vigorous growth and
a decrease in seedling mortality relative to control and
antisense lines. Specifically, disease progressed rapidly
in both control and antisense plants: 74% and 85%,
respectively, had died after 30 days (Fig. 5B). By con-
trast, seedlings from sense lines were substantially less
susceptible, and disease progressed much more slowly
than in the other two lines. After 30 days, only 43%
had died. Taken together, these results indicate that
overexpression of NtKTI1 could significantly increase
the resistance against fungal pathogens in both
detached leaves and whole plants.
Discussion
KTIs have been studied in various plant species, often
with a focus on their potential for biotechnology-based
1
2

3
4
1
2
3
4
AB
2
1
1
2
CD
Fig. 4. Inhibition of fungal growth by NtKTI1 in vitro. (A) Inhibition of Rhizoctonia solani growth by NtKTI1 after 24 h: 1, 20 lLof5mgÆmL
)1
heat-inactivated NtKTI1 protein in 20 mM phosphate buffer (pH 6.5); 2, 3 and 4, 20 lL of 1, 2 and 5 mgÆmL
)1
NtKTI1 in the same buffer. (B)
Antibacterial activity assay of NtKTI1 against E. coli:1,20lLof5mgÆmL
)1
heat-inactivated NtKTI1 protein in 20 mM phosphate buffer
(pH 6.5); 2, 20 lLof5mgÆmL
)1
ampicillin in the same buffer (pH 6.5); 3 and 4, 20 lL of 2 and 5 mgÆmL
)1
NtKTI1. (C) Inhibition of Rhizo-
pus nigricans growth by NtKTI1 after 72 h: 1, 20 lLof10mgÆmL
)1
heat-inactivated NtKTI1 protein in 20 mM phosphate buffer (pH 6.5); 2,
20 lLof10mgÆmL
)1

NtKTI1 in the same buffer. (D) Inhibition of Phytophthora parasitica growth by NtKTI1 after 48 h: 1, 20 lLof
10 mgÆmL
)1
heat-inactivated NtKTI1 protein in 20 mM phosphate buffer (pH 6.5); 2, 20 lLof10mgÆmL
)1
NtKTI1 in the same buffer. Scale
bars represent 1 cm.
H. Huang et al. NtKTI1 participates in tobacco’s fungal defense
FEBS Journal 277 (2010) 4076–4088 ª 2010 The Authors Journal compilation ª 2010 FEBS 4081
pest control for agriculture [6] and their response to
abiotic stress [31]. However, very little is known about
the antifungal role of KTIs. In this study, we report
the cloning and characterization of a KTI from
tobacco. The deduced NtKTI1 displays the conserved
features of the Kunitz PI family, such as a conserved
region at the N-terminus corresponding to a signal
peptide [18,26] and the signature pattern [32]. How-
ever, it does not show the vacuolar targeting motif
present in the N- or C-terminus of other Kunitz family
members [33–36], suggesting that NtKTI1 is not a vac-
uolar protein. Indeed, the programs SignalP-3.0 [37]
and psort [38] predict that the propeptide forms a sig-
nal peptide and that the mature protein is secreted
extracellularly. Further immunolocalization studies
could help to confirm the subcellular localization of
this polypeptide.
Recently, a 20.5-kDa KTI from Pseudostellaria hete-
rophylla roots has demonstrated antifungal activity
against Fusarium oxysporum [39]. AFP-J, a serine PI
belonging to the Kunitz family purified from tubers of

potato, strongly inhibits the human pathogenic fungi
Candida albicans, Trichosporon beigelii and Saccharo-
myces cerevisiae, whereas it exhibits no activity against
crop fungal pathogens [40]. NtKTI1 displays obviously
antifungal activity against R. solani, Rh. nigricans and
P. parasitica var. nicotianae, but does not inhibit
F. oxysporum, Physalospora piricola, Alternaria alter-
nata, Magnaporthe grisea, Colletotrichum orbiculare,
Bipolaris sorokiniana or E. coli DH5a (Fig. 4). There-
fore, we suggest that, although these antifungal pro-
teins belong to the same family of plant PIs, they have
different antifungal spectra.
Rhizoctonia solani, a soil-borne pathogen responsible
for serious damage to many important crops, primarily
infects the roots and stems of plants [41,42]. Surpris-
ingly, NtKTI1 mRNA was detected mostly in the roots
and stems of tobacco seedlings (Fig. 2A). Unlike serine
PIs of other species, which frequently accumulate in
the plant organs most vulnerable to herbivore damage,
such as leaves and seeds [43], little NtKTI1 mRNA
was accumulated in these two organs (Fig. 2A). The
lack of NtKTI1 transcripts in seeds (Fig. 2A) suggests
that NtKTI1 is not a storage protein, which is in
agreement with previous reports [44]. However,
increasing expression of NtKTI1 in stems and roots at
later developmental stages (Fig. 2B) suggests that it
may be responsible for the susceptibility of tobacco to
R. solani. Thus, our results indicate that there is signif-
icant correlation between the expression pattern of
NtKTI1 and the location of R. solani infection. In gen-

eral, the expression of most Kunitz PIs can be induced
by both biotic and abiotic stress [20,31]. In our study,
however, when tobacco seedlings were treated with
R. solani, SA and mechanical wounding, the level
of NtKTI1 transcripts decreased. Similarly, in other
studies, the transcripts of two other KTIs, Arabidopsis
Sense
Control
Antisense
100
80
60
40
20
0
Seedling nortality (%)
0 6 9 12 15 18 2421 27 30
Days after infection
Sense Control Antisense
NtKTI1
rRNA
50
40
30
20
10
0
Diameter of lesion (mm)
5 DAI
3 DAI

Sense Control Antisense
A
B
Fig. 5. Disease evaluation of transgenic tobacco plants. (A)
Detached leaf assay on sense, antisense and control plants inocu-
lated with Rhizoctonia solani. The northern blots show the expres-
sion of NtKTI1 in each representative transgenic and control line.
Scale bars represent 5 cm. Photographs were taken 6 days after
infection (top panel) and data (size of lesion in millimeters) were
recorded 3 and 5 days after the infection of tobacco leaves (bottom
panel). (B) Rate of seedling mortality of sense (filled circles), anti-
sense (triangles) and control (open circles) lines. Data represent
four independent experiments with 60 plants used in each. Error
bars are standard errors of the determinations. The experiment
was repeated three times with similar results, and a representative
experiment is shown. DAI, days after infection.
NtKTI1 participates in tobacco’s fungal defense H. Huang et al.
4082 FEBS Journal 277 (2010) 4076–4088 ª 2010 The Authors Journal compilation ª 2010 FEBS
AtDr4 and chickpea CaTPI-1, were repressed by pro-
gressive drought and constant lighting, respectively
[15,45]. Furthermore, SA can reduce the mRNA level
of KTIs in tomato [46,47]. These studies indicate that
plant PIs rely on different regulation mechanisms when
responding to different forms of stress.
Many phytopathogenic fungi are known to produce
extracellular proteinases [48], which play an active role
in the pathogenicity, virulence and development of dis-
eases [49,50]. In response to the proteinases secreted by
phytopathogens, plants synthesize inhibitory proteins
that can suppress enzyme activity [11]. Based on our

results, we propose that, when tobacco is challenged
with phytopathogens, NtKTI1 inhibits the extracellular
proteinases produced by phytopathogens, thus leading
to the inhibition of hyphal growth of phytopathogens.
Serine proteinases of plants can be induced after
pathogen attack, which also triggers a series of bio-
chemical responses in plants, including the accumula-
tion of a characteristic group of proteins called
pathogenesis-related (PR) proteins [51,52]. As shown
in Fig. 2C, the reduced expression level of NtKTI1 cor-
relates with the increased expression level of PR1c,
suggesting that the SA, but not MeJA, defense signal-
ing pathway is activated. After the recognition of
tobacco and phytopathogen, the transcript of NtKTI1
is repressed and the signal transduction pathway of
plant defense, such as the SA signaling pathway, is
activated, together with the expression of PR proteins.
In summary, our data strongly suggest that NtKTI1
may function as an antifungal protein to several phy-
topathogens during the plant defense response.
Materials and methods
Plant materials and treatments
Tobacco plants (Nicotiana tabacum L. cv. NC89, supplied
by Professor Xingqi Guo, Shandong Agricultural Univer-
sity, China) were grown aseptically on Murashige and Sko-
og medium containing 2% sucrose (pH 5.8) at 26–28 °C
under natural and additional artificial light (16 h ⁄ 8 h pho-
toperiod). One-, three- and five-month-old tobacco plants
were used for NtKTI1 expression detection. Four-week-old
tobacco seedlings in a growth room were used for treat-

ments. For wounding experiments, four fully developed
leaves were cut on four sites with scissors and pooled for
each time point. For chemical treatments, uniformly devel-
oped plants were sprayed with 5 mm SA, 100 lm MeJA or
100 lm ABA for the given time periods. For NaCl treat-
ment, uniformly developed seedlings were cultured in solu-
tions containing 300 mm NaCl for the given time periods.
Mock treatments were performed by spraying plants with
water. Leaves from three plants were pooled for each time
point, frozen in liquid nitrogen and stored at )80 °C for
later use. All experiments were conducted at least twice.
cDNA library construction and screening
Poly(A)+ RNA (0.5 lg), isolated from NC89 seedlings
treated with R. solani for 24 h, was used to synthesize first-
strand cDNA, and then amplified by long-distance PCR
according to the manufacturer’s protocol (SMARTÔ
cDNA Library Construction Kit; Clontech, Mountain
View, CA, USA). The double-stranded cDNA was digested
by SfiI enzyme, and then fractionated by Chroma Spin-400.
Fragments longer than 500 bp were cloned into
SfiI-digested dephosphorylated kTripIEx2 arms with T4
DNA ligase. The recombinants were packaged in vitro with
Packagene (Promega, Madison, WI, USA).
The cDNA library was screened by differential hybridiza-
tion (one with untreated seedling cDNA probe, one with
R. solani-treated plant cDNA probe). Plaques at a density
of 10
4
(plate diameter, 15 cm) were transferred onto the
membrane. Prehybridization, hybridization and washing

were performed as described previously [53]. Positive clones
were plaque purified by two additional rounds of plaque
hybridization with the same probes. Clones exclusively or
preferentially hybridized by the R. solani-treated plant
cDNA probe were selected. Of these, one cDNA clone,
NtKTI1, is described in this paper.
Gene cloning and northern blot analysis
Total RNA was extracted using the RNeasy Plant Mini kit
(Qiagen, Fremont, CA, USA) according to the manufac-
turer’s instructions. RNA samples for each experiment were
analyzed in at least two independent blots. The procedure
of hybridization was performed in the same manner as
cDNA library screening. The specific NtKTI1 cDNA frag-
ment was labeled with [a-
32
P] dCTP by priming a gene
labeling system from Promega, and used as the hybridiza-
tion probe. The blots were autoradiographed at )80 °C for
up to 7 days. The ethidium bromide-stained rRNA band in
the agarose gel is shown as a loading control.
SQRT-PCR analysis
Total RNA was extracted from tobacco seedlings using the
RNeasy Plant Mini kit and treated with RNase-free
DNase-I (Takara, Dalian, China) to remove genomic
DNA. RNA was stored in RNase-free water and diluted in
10 mm Tris (pH 7.5), and quantified via UV spectropho-
tometry (GeneQuant II; Pharmacia Biotech, Piscataway,
NJ, USA). Then, first-strand cDNA was synthesized using
SuperScriptÔ II reverse transcriptase (Invitrogen, Carlsbad,
CA, USA), and the cDNA product served as template for

H. Huang et al. NtKTI1 participates in tobacco’s fungal defense
FEBS Journal 277 (2010) 4076–4088 ª 2010 The Authors Journal compilation ª 2010 FEBS 4083
RT-PCR. The constitutively expressed gene in tobacco,
EF1a, was also subjected to RT-PCR at the same time as
an internal standard control. Twenty-five cycles of PCR
using Taq DNA polymerase (Takara) (94 °C for 3 min; 25
cycles of 94 °C for 1 min, 57 °C for 45 s and 72 °C for
2 min; 72 °C for 7 min) were performed to amplify
NtKTI1, PR1c (X17681), NtC7 (AB087235), Ntdin
(AB026439) and EF1a (AF120093). The primers used in
RT-PCR are described in Table 1. Twenty-five microliters
of the RT-PCR products were run on a 1.2% agarose gel
and visualized on ethidium bromide-stained gels using the
GelDoc-It TS Imaging System (Ultra Violet Products,
Upland, CA, USA). Each experiment was repeated at least
three times. Figure 2 represents one of these independent
experiments.
Prokaryotic expression, purification and trypsin
activity assay
The full-length NtKTI1 gene was amplified from the
tobacco genome and subsequently cloned into pMD18-T
simple vector (Takara). After sequence confirmation, the
coding regions with and without the putative N-terminal
signal sequence were subcloned into the EcoRI and HindIII
restriction sites of pET30a (Novagen, Madison, WI, USA).
Expression was induced with 0.5 mm isopropyl thio-b-d-
galactoside for 3 h at 28 °C, and the collected cells were
solubilized in native binding buffer. Recombinant NtKTI1
proteins were affinity purified under native conditions, as
described in the manufacturer’s protocol for nickel nitrilo-

triacetic acid agarose (Invitrogen). The activity of recombi-
nant NtKTI1 protein was determined by measuring the
change in A
253
caused by cleavage of the trypsin substrate
N-a-benzoyl-l-arginine ethyl ester (BAEE; Sigma, St Louis,
MO, USA), as described previously [22], with some modifi-
cations. Briefly, reaction mixtures containing 400 lL bovine
pancreas trypsin solution (0.5 lgÆlL
)1
; Sigma), 80 lL
sodium phosphate buffer (0.5 m, pH 6.5) and recombinant
NtKTI1 in elution buffer, or an equal volume of elution
buffer (50 mm NaH
2
PO
4
, 300 mm NaCl, 250 mm imidazole,
pH 8.0) as control, were adjusted to 530 lL and incubated
at room temperature for 30 min. Incubation mixtures (50 lL)
were added to a cuvette containing 3 mL BAEE substrate
(0.25 mm BAEE, 67 mm phosphate buffer, pH 7.0).
Antimicrobial assays of purified NtKTI1
All bacterial and fungal strains used in this study were
identified and kindly provided by Professor Guangmin
Zhang, Shandong Agricultural University, China. Physalos-
pora piricola, AIternaria alternata, Magnaporthe grisea, Col-
letotrichum orbiculare, Bipolaris sorokiniana, Rh. nigricans,
P. parasitica var. nicotianae, F. oxysporum and R. solani
were employed for the assay of antifungal activity. All

fungi were grown in potato dextrose agar (PDA). In vitro
antifungal activity assay was performed as described previ-
ously [39,54] with minor modifications. Cultures of
R. solani AG-4 were incubated in the dark at 30 °C for
48 h on PDA plates and maintained at 23 °C for 2 weeks
before use in the experiment. After 3 days of incubation in
the dark at 30 °C, a colonized disk of agar (2 mm
2
) was
transferred to another PDA plate. This plate was subcul-
tured for another 3 days under the same conditions. In
brief, the assay was executed using sterile Petri plates
(100 · 15 mm) containing 20 mL of PDA. The mycelia
were initially grown on the plates at 28 °C to obtain colo-
nies with a size of 30–40 mm in diameter. The potential
antifungal samples dissolved in 20 mm phosphate buffer
(pH 6.5) were then loaded onto sterile filter paper disks
(0.5 cm in diameter) which rested at a distance of 10 mm
away from the rim of the fungal colonies. The plates were
incubated in the dark at 28 °C and the zones of fungal inhi-
bition around the disks were checked daily. The plates
produced crescents of inhibition around disks containing
samples with antifungal activity.
The assay for antibacterial activity was conducted using
sterile Petri plates (100 · 15 mm) containing 10 mL Luria–
Bertani medium (1.5% agar). Warm nutrient agar (10 mL,
0.7%) containing E. coli DH5a was poured into each plate.
Sterile filter paper disks (0.5 cm in diameter) were placed
on the agar. Then, a sample solution (20 lL) in 20 mm
phosphate buffer (pH 6.5) was added to one of the disks.

Only the buffer was added to the control disk. The plate
was incubated at 30 °C for 20–24 h. A transparent ring
around the paper disks signified antibacterial activity.
Ampicillin (5 mgÆmL
)1
) served as a positive control. All
antimicrobial assays, including antifungal and antibacterial
assays, were performed in triplicate.
Generation of sense and antisense transgenic
tobacco lines
The vector pBI121, which contains the TM2 fragment
(GenBank accession number AF373415) [55] isolated
from the tobacco line (Nicotiana tabacum L cv. Nc89)
inserted into the HindIII site upstream and the EcoRI site
Table 1. Sequences of primers used in SQRT-PCR.
Name Primer sequence
KTI-RT5 5¢-GATTCTTAGCAGGTTCATCGCCATCT-3¢
KTI-RT3 5¢-TGCACACACTTGGACAGAACAC-3¢
PR1c-5 5¢-GCGAAAACCTAGCTTGGGGAAG-3¢
PR1c-3 5¢-TATATAACGTGAAATGGACGC-3¢
EF1a5 5¢-GAAGCTCTTCAGGAGGCACTTCCT-3¢
EF1a3 5¢-CAATGGTGGGTACGCAGAGAGGAT-3¢
NtC7-5 5¢-GAAGCTTACGTTCCGATGCAAAGTC-3¢
NtC7-3 5¢-AGAAAGTACAAATATCCATTC-3¢
Ntdin5 5¢-GAATTTAGTGATGGGCATGCTCCTG-3¢
Ntdin3 5¢-AGTAATCTTATCAGATTCACCAC-3¢
NtKTI1 participates in tobacco’s fungal defense H. Huang et al.
4084 FEBS Journal 277 (2010) 4076–4088 ª 2010 The Authors Journal compilation ª 2010 FEBS
downstream of the 35S::gusA cassette, was used for tobacco
transformation. The b-glucuronidase reporter gene of

pBI121 was eliminated and the tagged NtKTI1 were
inserted into the corresponding sites of pBI121 in the sense
and antisense orientations.
Wild-type Nicotiana tobacum L. cv. Nc89 was grown on
soil until the six-leaf stage. The fusion gene constructs were
transferred to Agrobacterium tumefaciens strain LBA4404
by the freeze–thaw method and the leaf pieces were trans-
formed as described previously [56]. The pBI121-TM2
empty vector was transformed as a control. For each plas-
mid, 50 leaf disks were treated at one time, and the series
was repeated three times. T0 transgenic tobacco plants were
identified by PCR to amplify the nptII gene with specific
primers (5¢-CGCATGATTGAACAAGATGG-3¢ and
5¢-TCCCGCTCAGAAGAACTCGTC-3¢). The correspond-
ing T1 transgenic tobacco seedlings, segregated at a ratio of
3 : 1 (resistant : sensitive), were selected to propagate the
T2 generation, which was used for further analysis. PCR-
screened positive transgenic plants were subjected to north-
ern blot analysis.
R. solani resistance analysis on transgenic plants
Cultures of R. solani AG-4 were incubated in the dark at
30 °C for 48 h on PDA plates, and maintained at 23 °C for
2 weeks before use in the experiment. After a 3-day incuba-
tion in the dark at 30 °C, a colonized disk of agar (2 mm
2
)
was transferred to another PDA plate, where it was subcul-
tured for another 3 days under the same conditions.
Leaves of 3-month-old plants were used as hosts for
R. solani infection. A colonized piece of 3-day-old agar

(2 mm
2
) was placed at the center of the adaxial surface of
each leaf, on two layers of moist filter paper saturated with
1 ⁄ 2 Murashige and Skoog solution (pH 5.8), in a Petri dish,
and kept under a 16 h photoperiod at 28 °C. The length
(mm) of the lesions on infected leaves was measured, and
photographs were taken 6 days after infection. Each treat-
ment consisted of 18 plants from three different lines of
transgenic or controls, and was replicated three times.
Fungal cultures grown on PDA plates were homogenized
and suspended in sterile water and mixed with sterile soil
(five plates for 3 L of soil) [57]. Transgenic tobacco lines
were transplanted into soil inoculated with R. solani AG-4.
Plants were maintained under the same conditions as prior
to inoculation, except that the relative humidity was
increased to 99%. The development of disease symptoms
was observed for 30 days and the seedling mortality was
calculated. Each condition was tested in triplicate.
Sequence alignments, database search and
phylogenetic constructions
Multiple alignments of amino acid sequences were per-
formed using the informatics application DNAMAN
(Lynnon BioSoft, Montreal, QC, Canada), and were manu-
ally adjusted. To improve alignments, secondary structure
predictions were made using Jpred (pbio.
dundee.ac.uk/www-jpred/index.html) [58]. Predicted second-
ary and tertiary structures were compared and used to help
align variable sites and indels.
Arabidopsis KTIs were obtained by searching The Ara-

bidopsis Information Resource (TAIR, bid-
opsis.org/) and GenBank ().
Rice KTI genes were obtained by multiple blast searches
of databases using the Kunitz motif sequences including
GenBank, the Rice Genome Research Program (RGP) and
The International Rice Genome Sequencing Project (IRS-
GP) (), The Rice Genome Annota-
tion Project Database and Resource (http://rice.
plantbiology.msu.edu/) and TIGR Rice Genome Annota-
tion Database and Resource ( />osa1/). Gene predictions were performed with the Rice
Genome Automated Annotation System (http://rice-
gaas.dna.affrc.go.jp/). The predicted genes were compared
with their expressed sequence tags and cDNAs obtained
from the Internet.
A phylogenetic tree was constructed online using the
default settings of the web-based alignment tool multa-
lin ( />[59].
Acknowledgements
This work was supported by the National Natural Sci-
ence Foundation (Grant No. 30970230), the Program
for Changjiang Scholars and Innovative Research
Team in University (Grant No. IRT0635) and the
Genetically Modified Organisms Breeding Major Pro-
jects (Grant No. 2009ZX08009-092B) in China.
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