Keywords antifungal activity; Kunitz trypsin inhibitor; prokaryotic expression; Rhizoctonia solani; transgenic tobacco Correspondence C.-C.. Taken together, these studies suggest that Nt
Trang 1from 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: cczheng@sdau.edu.cn
*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.
Trang 2They 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 solaniinoculation, 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 P1 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
Trang 3members of Arabidopsis and rice blast analysis of the
Arabidopsisprotein 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
75 NtKTI1
71 CAN81015
71 AAC49969
71 LeMIR
69 At1g17860
79 MIR
72 TC-21
69 AtKTI1
66 RASI
66 KTI3
M
K
E
L
T M
M M M M M T
N K K K L M K
T T T I M S K T M M
L T N N S L T T V K
L S Q Q S S A K S S
L F L L L F T T L T
L L F F L F A M R I
L F L F Y F V N L F
S S P P I V V P P F
L L F F F S L K L L
S L L L L A L F I F
V L I I L L L Y L L
I I F L L L F L L F
P A T A A A A V S C
I L I I V A F L L A
A A S S F A T A L F
L V F F I A S L A T
C K N N S N K T I T
V P S S H P S A S S
P F F L R L Y V F Y
N P L L G L F L S L
P V S S V S F A C P
S A S S T A G S S S
R
F
L V
A A S A T A A
G E A A E D N
S S E E A S A N A A
S S A S A A A A A I
P P P P V P N Y P A
S D P P E N S G P D
P P A E P P P A P F
V V V V V V V V V V
L L V V K L L V Y L
D D D D D D D D D D
I T I I I I T I T N
N E A D N D D D E E
G G G G G G G G G G
D K K K K E D N H N
K Q K I S K E A E P
V L L L L L L M L L
K R R R L R Q F S E
V S T T T T T H A N
G G G G G G G D G
L V I V V T V E G G
N D D D N N Q S S T
Y Y Y Y Y Y Y Y Y Y
F Y Y Y Y Y Y Y Y Y
V I I I I I V V V I
L L L L L V L L L L
P P P P P P S P P S
V V V V V V S V A D
I I V V I L I I S I
R R R R R R S R P T
G G G G G D G G G A
R R R R R H A R H F
G G G G G G G G G G
G G G G G G G G G G
G G G G G G G G G I
G R
L L L L L L L L L A
L T T T T T A T T A
P L L M M V L L M P
S A D D S S G A A T
N S S S N A R G P G
V T T I L T A R R N
K G G G K T T G V E
Q P
N
G
N N N D T T G G
N E E K E F Q Q L
T N S M T V S P P R
C C C C C C C C C C
P P P P P P P P P P
R L L L T P E Y L L
D D D D S R I D L T
149 NtKTI1
144 CAN81015
146 AAC49969
146 LeMIR
136 At1g17860
155 MIR
148 TC-21
144 AtKTI1
142 RASI
136 KTI3
A A
I V V V V V V I V V
I V V V I V V V A V
Q Q Q Q Q Q Q Q Q Q
N E E E D T R E E S
S Q Q H Q R R S T R
D H Q N F K S S D N
E E E E E E D E E E
V V I I V V L V R L
Q S K D S D D D R D
E N N Q Q H N E K K
G G G G G D G G G G
L L L L L R T I F I
P P P P P P P P P G
V L L L V L V V V T
V T T T K A I K R I
F F F F F F F F F I
A T T T S F S S T S
P P P P P P N N P S
F V V V Y E A W W P
N N N D D N D R G Y
T P P P P S L G
A
A
A
P
K K K K K K K K E R
K K K K S E D V D I
G G G G R D D A R R
V V V V T V V F T F
V I I I I V V V I I
R R R R P R R P R A
L V E E V V V E V E
S S S S S S S S S G
I T T T T T T Q T H
D D D D D D D N D P
L H L L V L V L V L
N N N N N N N N R S
V I I I I I I I I L
R K K I K N E E R K
F F F F F F F T F F
F S S S S S V D N D
T A A A P A P V A S
P S A N F I G A F
T T S S M R T A
P D V
C R A I
R T M
I I I I W L I I L
C C C C T C C C C
A V V V S S I V V
R Q Q Q T T Q Q G
E S S S S S S I
T T P
T T T T T T T T T
I L L Q I V V Y E E
W W W W W W W W W W
K K K K E R R R H S
L L L L L L L V V V
G D D A D D G G V
T E D D N K N E D E
Y Y F F F Y Y F E D
D D D D D D D D P L
D E E E E E N H L P
K S T T T S S E T E
L S T T T T A R G G
K G G G K G G K A P
Q Q K Q Q Q K Q R A
Y R Y Y W Y W Y V
F F F F F F W F R K
I V I I I V V V V I
V T T T S T T V V G
T T I L T I T A T E
G G G G C G D G G N
G G G G G G G P P K
V V N D V V V K L D
E E E Q E K K P I
G G G G G G G E G
N N N N N N E G
P P P P P P P F P
G G G G G G G G S
P X R V Q P P Q P
Q E E E K E N D S
209 NtKTI1
203 CAN81015
210 AAC49969
205 LeMIR
196 At1g17860
220 MIR
221 TC-21
215 AtKTI1
200 RASI
215 KTI3
T T T T T T T S G A
L L I I V I L L R M
S D S S D S C K E D
S N N N N S S S N G
W W W W W W W F A W
F F F F F F F F F F
K K K K K K K K R R
I I I I I I I I V L
E E E E D E E E E E
K K K K K E K K K R
L Y F Y F F A S Y V
G E E D E C G G G S
T D R R K G V E G D
D D D D D S L D G D
G G E
F A F
N
N
Y Y Y Y Y Y Y Y Y Y
K K K K K K K K K K
F L L L I L F F L L
V V V L R V R V V V
F F Y Y F F F F S F
C C C C C C C C C C
P P P P P P P P R P
S T T T T T S R Q
V V V V V V V T Q
C C C C C C C C A
K D N D N G D D D E
I F F F F S S S S
C C C C C C C G
K K K K K K T N D
V P V V V V T P D
I V I I I K L K K
C C C C C C C C C C
K G K R R G S S Q G
D D D D D D D D D D
V I V I V V I V L I
G G G G G G G G G G
I I I I V I R I V I
Y Y F F F Y H F S S
T I I I V I S I R I
K Q Q Q Q D D D D D
D N D D D Q D E G H
K D L D
D
G G G G G G G G A G
V Y I V K R Q V R T
R R R R R R I R A R
F R R R R R R R W R
L L L L L L L L L L
A A A A A A A A G V
L L L L L L L L A V
S S S S S S S S S S
K
D D D D D D D D Q N
T V V V V K N K P K
P P P P P P E P P P
L F F F L F W F H L
R K K K K A A L V V
V V V V V F W V V V
M M M M M E M M V Q
F F F F F F F F F F
K K K K K N K K K Q
K K K K R K K K K K
T A A A A T A A A L
F Q Y V S N R D
V Y K V P K
V F T T S E
K I E P S
D K V P L
Q S E A
V S K
V K K
N T N
A M H
N G
D L
S
R
S
**
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 P1 and P1¢ 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.
Trang 4genome 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) as an
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 m M NaCl
NtKTI1
Nt C7 EF1- a
100 µ M MeJA
NtKTI1 PR1c EF1- a
Wounding
NtKTI1 PR1c EF1-a
100 µ M ABA
NtKTI1
Nt din EF1-a
5 m M SA
NtKTI1 PR1c EF1- a
R solani NtKTI1
PR1c EF1-a
Stem
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.
Trang 5gene 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 gradgrad-ually 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 paPhytophtho-rasitica 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
Inhibitor protein (µg)
100 80 60 40 20 0
NtKTI1
(kDa) 97.4
66.2
43.0
29.0
20.1
2
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.
Trang 6plants 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 NtKTI1enhanced 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
2
Fig 4 Inhibition of fungal growth by NtKTI1 in vitro (A) Inhibition of Rhizoctonia solani growth by NtKTI1 after 24 h: 1, 20 lL of 5 mgÆmL)1 heat-inactivated NtKTI1 protein in 20 m M phosphate buffer (pH 6.5); 2, 3 and 4, 20 lL of 1, 2 and 5 mgÆmL)1NtKTI1 in the same buffer (B) Antibacterial activity assay of NtKTI1 against E coli: 1, 20 lL of 5 mgÆmL)1 heat-inactivated NtKTI1 protein in 20 m M phosphate buffer (pH 6.5); 2, 20 lL of 5 mgÆmL)1ampicillin in the same buffer (pH 6.5); 3 and 4, 20 lL of 2 and 5 mgÆmL)1NtKTI1 (C) Inhibition of Rhizo-pus nigricans growth by NtKTI1 after 72 h: 1, 20 lL of 10 mgÆmL)1heat-inactivated NtKTI1 protein in 20 m M phosphate buffer (pH 6.5); 2,
20 lL of 10 mgÆmL)1 NtKTI1 in the same buffer (D) Inhibition of Phytophthora parasitica growth by NtKTI1 after 48 h: 1, 20 lL of
10 mgÆmL)1heat-inactivated NtKTI1 protein in 20 m M phosphate buffer (pH 6.5); 2, 20 lL of 10 mgÆmL)1NtKTI1 in the same buffer Scale bars represent 1 cm.
Trang 7pest 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 NtKTI1and 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
Days after infection
NtKTI1
rRNA
50
40
30
20
10
0
5 DAI
3 DAI
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.
Trang 8AtDr4 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 26–28C
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 (SMART 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 104 (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-32P] 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 SuperScript II reverse transcriptase (Invitrogen, Carlsbad,
CA, USA), and the cDNA product served as template for
Trang 9RT-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) (94C for 3 min; 25
cycles of 94C for 1 min, 57 C for 45 s and 72 C for
2 min; 72C 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 28C, 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 A253caused 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 NaH2PO4, 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 30C for
48 h on PDA plates and maintained at 23C for 2 weeks before use in the experiment After 3 days of incubation in the dark at 30C, a colonized disk of agar (2 mm2
) 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 28C 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 28C 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 30C 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.
Trang 10downstream 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 seedlcorrespond-ings, 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
30C 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 30C, a colonized disk of agar (2 mm2)
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 mm2) 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 28C 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 (http://www.compbio dundee.ac.uk/www-jpred/index.html) [58] Predicted second-ary and tertisecond-ary structures were compared and used to help align variable sites and indels
Arabidopsis KTIs were obtained by searching The Ara-bidopsis Information Resource (TAIR, http://www.arabid-opsis.org/) and GenBank (http://www.ncbi.nlm.nih.gov) 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) (http://rgp.dna.affrc.go.jp), The Rice Genome Annota-tion Project Database and Resource (http://rice plantbiology.msu.edu/) and TIGR Rice Genome Annota-tion Database and Resource (http://www.tigr.org/tdb/e2k1/ 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 (http://multalin.toulouse.inra.fr/multalin/multalin.html) [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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