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: 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. 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 . . . . . 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 149NtKTI1 144CAN81015 146AAC49969 146LeMIR 136At1g17860 155MIR 148TC-21 144AtKTI1 142RASI 136KTI3 . . 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 . 209NtKTI1 203CAN81015 210AAC49969 205LeMIR 196At1g17860 220MIR 221TC-21 215AtKTI1 200RASI 215KTI3 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 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 (http://www.compbio. 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, 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. References 1 Agrios GN (1998) Plant Pathology, 4th edn. Academic Press, San Diego, CA. 2 Strange RN & Scott PR (2005) Plant disease: a threat to global food security. Annu Rev Phytopathol 43, 83–116. 3 Whitby SM (2001) The potential use of plant pathogens against crops. 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