Overexpression of SpWRKY1 promotes resistance to Phytophthora nicotianae and tolerance to salt and drought stress in transgenic tobacco

Jing-bin Li, Yu-shi Luan* and Zhen Liu

School of Life Science and Biotechnology, Dalian University of Technology, No. 2 Linggong Road, Ganjingzi District, Dalian 116024, Liaoning Province, China

*Corresponding author, e-mail: [email protected] WRKY transcription factors are key regulatory components of plant responses to biotic and abiotic stresses. SpWRKY1, a pathogen-induced WRKY gene, was isolated from tomato (Solanum pimpinellifolium L3708) using in silico cloning and RT-PCR methods. SpWRKY1 expression was significantly induced following oomycete pathogen infection and treatment with salt, drought, salicylic acid (SA), methyl jasmonate (MeJA) and abscisic acid (ABA). Overexpression of SpWRKY1 in tobacco conferred greater resistance to Phytophthora nicotianae infection, as evidenced by lower malonaldehyde (MDA) content and relative electrolyte leakage (REL), and higher chlorophyll content, peroxidase (POD, EC 1.11.1.7), superoxide dismutase (SOD, EC 1.15.1.1) and phenylalanine ammonia-lyase (PAL, EC 4.3.1.24) activities. This resistance was also coupled with enhanced expression of SA- and JA-associated genes (NtPR1, NtPR2, NtPR4, NtPR5 and NtPDF1.2), as well as various defense-related genes (NtPOD, NtSOD and NtPAL). In addition, transgenic tobacco plants also displayed an enhanced tolerance to salt and drought stresses, mainly demonstrated by the transgenic lines exhibiting lower accumulation of MDA content, and higher POD (EC 1.11.1.7), SOD (EC 1.15.1.1), chlorophyll content, photosynthetic rate and stomatal conductance, accompanied by enhanced expression of defense-related genes (NtPOD, NtSOD, NtLEA5, NtP5CS and NtNCED1) under salt and drought stresses. Overall, these findings suggest that SpWRKY1 acts as a positive regulator involved in tobacco defense responses to biotic and abiotic stresses.

This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/ppl.12315 This article is protected by copyright. All rights reserved

Abbreviations – ABA, abscisic acid; CaMV, cauliflower mosaic virus; EST, expression sequence tag; MDA, malondialdehyde; MeJA, methyl jasmonate; ORF, open reading frame; PAL, phenylalanine ammonia-lyase; PDF1.2, plant defensin1.2; PEG, polyethyleneglycol; POD, peroxidase; PR gene, pathogenesis-related gene; qRT-PCR, quantitative real-time polymerase chain reaction; REL, relative electrolyte leakage; ROS, reactive oxygen species; RT-PCR, reverse transcriptase polymerase chain reaction; SA, salicylic acid; SOD, superoxide dismutase; WT, wild type.

Introduction Plants are constantly challenged by various harsh environmental stresses, such as pathogens infection, high salinity and drought, which can limit their growth and productivity. In order to cope with these challenges, plants have evolved sophisticated molecular mechanisms to sense and react to these diverse external signals resulting in adaptive responses through physiological and morphological changes (Agarwal et al. 2011, Wang et al. 2013). Transcription factors are essential components in the signaling network, which have been implicated in the regulation of plant stress response via inducing and/or repressing the expressions of a battery of downstream defense-related genes (Nakashima et al. 2009, Ma et al. 2013). Recently, a range of transcription factors in plants have been identified (Xiong et al. 2005). Among the different transcription factors, WRKY has received increasing attention for its roles in plant defense (Rushton et al. 2010). Each WRKY transcription factor has at least one WRKY domain of approximately 60 amino acids containing the conserved amino acid sequence WRKYGQK at its N-terminus (from which the factors take their name) and either a C2H2 (C-X4-5-C-X22-23-H-X-H) or a C2HC (C-X7-C-X23-H-X-C) zinc finger motif at its C terminus (Rushton et al. 2012). Proteins from group I contain two WRKY domains and a C2H2 zinc-finger motif, while those from groups II and III only contain one WRKY domain and a C2H2 or C2HC zinc-finger motif, respectively (Rushton et al. 2010). The WRKY domain can bind to the TTGAC(C/T) of W-box found in the promoter sequence of numerous plant defense or defense-related genes and WRKY genes themselves (Ciolkowski et al. 2008). Since the first WRKY protein was cloned from sweet potato, more and more WRKY genes have been experimentally identified in various plant species, such as 74 members in Arabidopsis thaliana (Eulgem and Somssich 2007), 102 in rice (Oryza sativa) (Ross et al. 2007), 119 in maize This article is protected by copyright. All rights reserved

(Zea mays) (Wei et al. 2012), and 81 in tomato (Solanum lycopersicum) (Huang et al. 2012). Salicylic acid (SA) and jasmonic acid (JA) are two well-studied key mediators involved in regulating defense responses in plants (Miljkovic et al. 2012). SA-dependent signaling pathway is believed to mediate the resistance to biotrophic pathogens. By contrast, JA-dependent signaling pathway is thought to be necessary for resistance to necrotrophic pathogens (Qiu et al. 2007). Moreover, two signaling pathways communicate with one another, either synergistically or antagonistically (Miljkovic et al. 2012). Thus, it is conceivable that transcription activation of SAand/or JA-responsive defense genes is essential for plant disease resistance conferred by the two pathways. Recent investigations indicated that WRKY transcription factors may play a key role involved in plant disease resistance and modulation of a subset of SA- and/or JA-responsive gene expression (Qiu et al. 2007, Yu et al. 2012). In Arabidopsis, overexpression of AtWRKY18, AtWRKY46 and AtWRKY70 results in increased resistance to Pseudomonas syringae and enhanced expression of SA-responsive marker genes, such as pathogenesis-related (PR) gene (Chen and Chen 2002, Li et al. 2004, Hu et al. 2012). Furthermore, transgenic Arabidopsis overexpressing AtWRKY28 and AtWRK75 showed increased resistance to Sclerotinia sclerotiorum and enhanced the expression of JA-responsive marker genes, such as plant defensin1.2 (PDF1.2) gene which encoding an antimicrobial defensin (Chen et al. 2013a). In rice, overexpression of OsWRKY45 and OsWRKY53 led to enhanced resistance to Magnaporthe grisea (Chujo et al. 2007, Shimono et al. 2007). Meanwhile, overexpression of rice OsWRKY6 and OsWRKY23 in Arabidopsis also exhibited increased disease resistance to Xanthomonas oryzae or P. syringae, and enhanced expression of the PR gene (Jing et al. 2009, Hwang et al. 2011). In addition to pathogen defense responses, WRKY transcription factors also play important roles in responses to abiotic stress. Overexpression of AtWRKY25, AtWRKY33, ZmWRKY33 and ThWRKY4 in Arabidopsis results in enhanced tolerance to salt (Jiang and Deyholos 2009, Li et al. 2013, Zheng et al. 2013). As well, transgenic Arabidopsis overexpressing TaWRKY2 and TaWRKY19 from wheat displayed enhanced salt and drought tolerance (Niu et al. 2012). Moreover, overexpression of OsWRKY11 and OsWRKY30 in rice also dramatically increased drought tolerance (Wu et al. 2009, Shen et al. 2012). The functions of WRKY genes were extensively explored in various plants, especially in rice and Arabidopsis. Only a few members of the WRKY transcription factors from tomato have hitherto been functionally characterized, including SlWRKY70, SlWRKY72, SlWRKY and SlDRW1 (Bhattarai et al. This article is protected by copyright. All rights reserved

2010, Atamian et al. 2012, Li et al. 2012, Liu et al. 2014), the function of the majority tomato WRKY members remains unknown. In the present study, we isolated a pathogens-inducible tomato WRKY gene, designated as SpWRKY1, and investigated its expression patterns in response to pathogen infection, salt, drought and phytohormones. To further characterize the function of SpWRKY1, transgenic tobacco plants were generated and assayed for resistance to pathogen attack and tolerance to salt and drought stresses. We also performed a comparison of the physiological changes and expression level of defense-related genes in wild-type (WT) and transgenic tobaccos plants before and after treatment. This study provides key clues in understanding the roles of SpWRKY1 in plant defense response to biotic and abiotic stresses.

Materials and methods Plant materials and treatments Tomato (Solanum pimpinellifolium L3708) seeds were sown into pots filled with perlite: vermiculite (1:3, v/v) until root emergence and the seedlings were then grown in hydroponic culture at 25 ± 3°C under a 16 h light and 8 h dark photoperiod, as previously described in Li and Luan (2014). For the tissue-specific expression analysis, the root, stem and leaf were harvested separately from five-leaf stage tomato seedlings. Five-leaf stage tomato seedlings were also used for the various treatments. For pathogen inoculation, 2 ml aliquots of Phytophthora infestans zoospores (1.0×106 zoospores ml–1) were sprayed onto seedlings. Salt and drought treatments were applied to the seedlings in accordance with Li and Luan (2014). Phytohormones treatments were performed by spraying the seedlings with 2 mM SA, 0.1 mM methyl jasmonate (MeJA) or 0.1 mM abscisic acid (ABA). Leaves harvested at various time points after treatment were quickly frozen in liquid nitrogen and stored at –80°C for RNA extraction. Three independent biological replications were performed for each experiment.

Cloning and sequence analyses In our previous research, several EST fragments, which putatively encodes a WRKY transcription factor, were isolated by reverse transcriptase-PCR (RT-PCR) using degenerate primers during the incompatible interaction between the late blight resistant tomato varieties S. pimpinellifolium L3708 and the oomycete pathogen P. infestans (Li and Luan 2014). To obtain a tomato WRKY gene, in silico This article is protected by copyright. All rights reserved

cloning was performed as previously described by Li and Luan (2014). To verify the assembled sequence, gene-specific primers (forward primer (FP): 5’-ATGGCTGCTTCAAGTTTCTCT-3’ and reverse primer (RP): 5’-TCAGCAAAGCAATGACTCCAT-3’) were used to amplify the open reading frame (ORF) regions. Template was a mixture of the first strand cDNA reversed transcribed from total RNA extracted from samples in the incompatible combination. The PCR product was purified, cloned into pMD18-T vector (TaKaRa, Dalian, China) and sequenced (Sangon, Shanghai, China). The deduced protein sequence analyses were performed using different software in online SIB resources (http://www.expasy.org/proteomics). Transmembrane domain and signal peptide prediction were analyzed using the TMHMM procedure (http://www.cbs.dtu.dk/services/TMHMM-2.0/) and SignalP procedure (http://www.cbs.dtu.dk/services/SignalP/). Subcellular localization and nuclear localization signals

(NLS)

were

predicted

using

(http://www.csbio.sjtu.edu.cn/bioinf/euk-multi-2/)

and

Euk-mPLoc NLS

mapper

program program

(http://nls-mapper.iab.keio.ac.jp). The alignment of the deduced protein sequences and phylogenetic tree analyses were, respectively, done by ClustalX 1.81, GeneDoc and MEGA 4.0 software using the standard parameters.

Gene expression analysis by quantitative real-time PCR Tissue-specific expression and expression patterns of SpWRKY1 after different treatments were determined using quantitative real-time PCR (qRT-PCR) analyses with a pair of primers (FP: 5'-CAGTGGCAACCACTCAATTAAC-3'

and

RP:

5’-TCAGACTGTTGTGGCCTTATAC-3’)

following the methods described in Li and Luan (2014). The expression of tomato actin gene (accession number: FJ532351) was used as an internal reference to normalize all data with the primers FP: 5’-ACCTTCAACGTTCCAGCTATG-3’ and RP: 5’-TCACCAGAGTCCAACACAATAC-3’. The relative expression of SpWRKY1 was calculated by the 2–ΔΔCT method (Livak and Schmittgen 2001). Data acquisition and analysis were performed by using Rotor-gene 6 software (Corbett Research Pty Ltd). All reactions were carried out in three times with three independent biological replicates.

Overexpression vector construction and generation of transgenic tobacco An overexpression vector was constructed on the base of the pBI121 vector. The ORF of the SpWRKY1 gene was amplified with specific primers modified to include BamHI and SacI restriction This article is protected by copyright. All rights reserved

sites

(underlined),

FP:

5’-CGGGATCCATGGCTGCTTCAAGTTTCTCT-3’

and

RP:

5’-CGAGCTCTCAGCAAAGCAATGACTCCAT-3’. The amplification product was cloned into Bam HI and Sac I restriction sites of the vector pBI121, replacing the GUS gene, under the control of Cauliflower mosaic virus (CaMV) 35S promoter. The recombinant plasmid was mobilized into Agrobacterium tumefaciens strain GV3101 by the freeze-thaw method, and from there into leaves of four-week-old tobacco (Nicotiana tabacum cv 89) by means of the Agrobacterium-mediated leaf disc method (Oh et al. 2005). Putative transgenic plants were selected on Murashige and Skoog (MS) agar medium containing 50 mg l–1 kanamycin. The integration of the transgene into different transgenic lines was confirmed by PCR and RT-PCR with gene-specific

primers

(FP:

5’-ATGGCTGCTTCAAGTTTCTCT-3’

and

RP:

5’-TCAGCAAAGCAATGACTCCAT-3’). The expression level of SpWRKY1 in these selected positive transgenic lines was further examined by qRT-PCR using the primers (FP: 5'-CAGTGGCAACCACTCAATTAAC-3' and RP: 5’-TCAGACTGTTGTGGCCTTATAC-3’). The tobacco actin gene (accession number: AB158612) (FP: 5′-TCAAGCTGTGTTGTCCCTATAC-3′ and RP: 5′-GGAAGGGCGTAACCTTCATAA-3′) was used for the normalization of the qRT-PCR analysis.

Disease resistance analysis of transgenic plants Four-week-old tobacco seedlings (WT and transgenic lines) were transferred into triangular flask containing aerated quarter-strength Hoagland nutrient solution and cultured for one week in the greenhouse at 25 ± 3°C with a 16 h light and 8 h dark photoperiod cycle. Phytophthora nicotianae was cultivated on CA medium (carrot, 200 g l–1; sucrose 20 g l–1; agar 15 g l–1; pH 6.5) at 28°C for 5– 7 days. Preparation of mycelia and zoospore production of P. nicotianae followed the method described by Shan et al. (2004) and the concentration was adjusted to 1.0×105 zoospores ml–1 for inoculation. For detached-leaf inoculation assay, healthy leaves from WT and transgenic tobacco plants were detached, and 20 μl of zoospores suspension were dropped onto the leaf surface at sites subject to toothpick wounding (four sites per leaf). The inoculated leaves were then placed on filter paper saturated with sterilized water in a porcelain dish and covered with plastic film. The inoculated leaves were maintained in the dark at high humidity for 24 h, and then moved to the greenhouse at 28°C with This article is protected by copyright. All rights reserved

a 16 h light and 8 h dark photoperiod cycle. The areas of necrosis surrounding inoculation sites and disease indices were measured at two weeks days after inoculation. Disease grades (g) were categorized from 0 to 3 based on the lesion area: g0 = no symptoms, g1 = lesion diameter smaller than 0.5 cm, g2 = lesion diameter between 0.5 and 1 cm, g3 = lesion diameter larger than 1 cm and lesions merged. The resistance of a plant was indicated by the disease index (DI) defined as DI (%) =∑ (gi×ni)×100/(n×gimax), where gi is the value of disease grades, ni is the number of leaves with each disease grade, and n is the total number of leaves inoculated in the plant. A high-DI value means a low resistance. For whole plant inoculation assay, 2 ml aliquots of zoospores suspension was applied to tobacco leaves with a hand held sprayer until run off. The inoculated plants were maintained in the dark at high humidity for 24 h, and then moved to the greenhouse at 25 ± 3°C with a 16 h light and 8 h dark photoperiod cycle. After two weeks, the leaf symptoms of tobacco plants were photographed and the key physiological parameters were analyzed. The leaf surface area and lesion area in each leaf was quantified upon image acquisition using ImageJ software (Larroque et al. 2013). Each experiment was carried out at least three times.

Analysis of transgenic plants exposed to salt and drought treatment The 7-day-old aseptic tube seedlings of WT and transgenic tobacco plants were transferred to glass bottles containing half-strength MS agar medium. The root length and fresh weight of each plant were measured under normal conditions and after two weeks treatment with 200 mM NaCl and 15% PEG 6000. For the future salt and drought tolerance assay, three-week-old tobacco plants (WT and transgenic lines) were transferred into pots filled with a mixture of soil, perlite and vermiculite (1:1:1, v/v/v) cultured for two weeks in the greenhouse and irrigated with quarter-strength Hoagland nutrient solution (once every three days). For the salt stress treatment, plants were irrigated with quarter-strength Hoagland nutrient solution supplemented with 200 mM NaCl for two weeks (once every two days). For drought stress treatment, plants were withheld from watering for two weeks, and were then rewatered for three days. After stress treatments, the phenotypes were photographed and the leaves of WT and transgenic lines were harvested for physiological parameter measurements. Each salt and drought tolerance experiment was carried out at least three times.

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Measurements of MDA content, REL, enzyme activities, chlorophyll content and photosynthesis parameters Malondialdehyde (MDA) content was determined using the thiobarbituric acid (TBA) reaction, as described by Ma et al. (2013). Relative electrolyte leakage (REL) was examined as described by Cao et al. (2007). Peroxidase (POD, EC 1.11.1.7), superoxide dismutase (SOD, EC 1.15.1.1) and phenylalanine ammonia-lyase (PAL, EC 4.3.1.24) activities were measured according to the method as described previously (Chen et al. 2013b). All enzyme extraction procedures were conducted at 4°C. The total chlorophyll content was determined in 80% (v/v) acetone extract according to the method of Talaat and Shawky (2014). The Portable Photosynthesis System CIRAS-2 was used to measure photosynthesis parameters, such as photosynthetic rate and stomatal conductance (Li et al. 2014).

Expression analysis of downstream genes regulated by SpWRKY1 Expressions

of

the

SA-associated

genes,

JA-associated

genes,

ROS-related

genes

and

stress-responsive genes in tobacco (WT and transgenic lines) were analyzed by qRT-PCR. The primer sequences

were

as

follows:

for

NtPR1

(accession

number:

X06361),

FP:

5′-

GTTGAGATGTGGGTCGATGAG-3′ and RP: 5′-CGCCAAACCACCTGAGTATAG-3′; for NtPR2 (accession

number:

M60460),

FP:

5′-CAACCCGCCCAAAGATAGTA-3′

and

RP:

5′-TGGCTAAGAGTGGAAGGTTATG-3′; for NtPR4 (accession number: X60281), FP: 5′CGAACACAGGAACAGGAACT-3′ and RP: 5′-GCTGATAGCCCACTCCATTT-3′; for NtPR5 (accession

number:

AF154636),

FP:

5′-CTCTAGCATGGTGGATTGACTT-3′;

5′-GCTCGATTACGTCTTGTCTCTC-3′ for

NtPDF1.2

(accession

number:

and

X99403),

RP: FP:

5′-CTGTACCAGCCCTTGCTAAT-3′ and RP: 5′-TAGCTAGCACGTCCATCTTTG-3′; for NtPOD (accession

number:

AB178953),

FP:

5′-CTCCATTTCCATGACTGCTTTG-3′

5′-GTTGGGTGGTGAGGTCTTT-3′;

for

NtSOD

(accession

number:

and

RP:

AB093097),

FP:

5′-CGGCAATTAGCGGTGACATA-3′ and RP: 5′-ATGGCGTCATGTAGCTGTTC-3′; for NtPAL (accession

number:

AB289452),

5′-CCGTTCTTGGTTCTCCTATGT-3′;

FP:

and

RP:

AF053076),

FP:

5′-AGCTAGTAGTGATTGGGTTATGG-3′

for

NtLEA5

(accession

number:

5’-GTTACCATACCACGTCCCATAG-3’ and RP: 5’-GAGCTAGGACGCTCCATATTT-3’; for NtP5CS (accession number: HM854026), FP: 5’-GACACGGACTGATGGAAGATTAG-3’ and RP: 5’-GCACCTGAAGTCACCAGAATAA-3’; for NtNCED1 (accession number: HM068892), FP: This article is protected by copyright. All rights reserved

5’-ACGAACTCCAACACCCTTTAC-3’ and RP: 5’-AGGGAGTGAGAGACTGGATTT-3’. The tobacco actin gene (accession number: AB158612) was used as an internal reference to normalize all data

with

the

primers

FP:

5′-TCAAGCTGTGTTGTCCCTATAC-3′

and

RP:

5′-GGAAGGGCGTAACCTTCATAA-3′.

Statistical analysis All of the above numerical data expressed as means ± standard deviation (SD) of three independent experiments performed. Duncan multiple range tests were performed by using one-way analysis of variance (ANOVA) on SPSS version 17.0 software and a P value 40 was instability). The protein grand average of hydropathicity was –1.062, when grand average of hydropathicity was less than 0; the protein was a hydrophilic, indicating that the SpWRKY1 was a hydrophilic protein. Transmembrane prediction with TMHMM showed that the inferred amino acid sequence from SpWRKY1 had no transmembrane regions. SignalP analysis showed that SpWRKY1 without the signal peptide. A subcellular localization analysis predicted that SpWRKY1 may exist in the nucleus. A subcellular localization analysis This article is protected by copyright. All rights reserved

predicted that SpWRKY1 may exist in the nucleus and its nuclear targeting is due to the presence of a nuclear localization signal (NLS) at amino acid 338–348 of SpWRKY1. Multiple alignments showed that SpWRKY1 contained two typical WRKY domains followed by two C-X4-5-C-X22-23-H-X-H type zinc finger motifs and shares high similarity with group I WRKY transcription factor of other plant species (Fig. 1A). Thus, it was classified as a group I WRKY transcription factor according to Rushton's classification method (Rushton et al. 2010). The relationships among various members of group I WRKY from different plants were further analyzed by means of the neighbor-joining method using their full length amino acid sequences, and phylogenetic tree pointed out the existence of two main clusters (Fig. 1B). SpWRKY1 clusters with CaWRKY2 (89% identity) reported as rapidly induced by pathogens (Oh et al. 2006), with NtWRKY1 (53% identity) which was induced by fungal elicitors (Yamamoto et al. 2004). It also clusters with other WRKY transcription factor implicated in pathogens response (BnWRKY33, OsWRKY53, AtWRKY25 and AtWRKY33) and/or abiotic stress response (SlWRKY, AtWRKY25, AtWRKY33 and TaWRKY2) which were differently modulated by SA, JA or ABA (Zheng et al. 2006, Chujo et al. 2007, Zheng et al. 2007, Jiang and Deyholos 2009, Yang et al. 2009, Li et al. 2011, Li et al. 2012, Niu et al. 2012). The second group comprises WRKY transcription factor were only involved in pathogens response (VvWRKY2, AtWRKY3 and AtWRKY4) (Mzid et al. 2007, Lai et al. 2008). Thus, we propose that SpWRKY1 may be involved in the response to biotic and abiotic stresses.

Tissue-specific expression and expression patterns of SpWRKY1 under various conditions To explore the tissue-specific expression of SpWRKY1, we first examined various tomato organs by qRT-PCR. As shown in Fig. 2A, SpWRKY1 showed low expression in root and stem, whereas relatively high expression was observed in leaf. To test if SpWRKY1 is involved in the plant response to biotic and abiotic stresses, the expression patterns of SpWRKY1 in tomato leaves were measured by qRT-PCR after treatment with P. infestans, salt and drought. After inoculation with P. infestans, the transcript of SpWRKY1 was up-regulated at 6 h, and then reached its highest level at 12 h with 5.09-fold (Fig. 2B). When treated with salt stress, the transcript of SpWRKY1 increased transiently and reached the highest peaks at 12 h with 8.57-fold (Fig. 2C). When treated with drought stress, the transcript of SpWRKY1 was significantly induced and reached the highest peaks at 2 h with 5.23-fold (Fig. 2D). This article is protected by copyright. All rights reserved

Phytohormones, including SA, MeJA (an analogue of JA) and ABA, are implicated in complex signaling pathways and play vital roles in regulating plant responses to various biotic and abiotic stresses (Rajendra and Jones 2009). To assess the possible involvement of SpWRKY1 in signaling pathways regulated by these phytohormones, the expressions of SpWRKY1 in tomato leaves were examined by qRT-PCR following treatment with exogenously applied SA, MeJA and ABA. For the SA treatment, the transcript of SpWRKY1 was more rapidly accumulated within 2 h and reached a maximum at 4 h with 4.27-fold (Fig. 2E). For the MeJA treatment, the transcript of SpWRKY1 was significantly induced and reached the highest peaks at 2 h with 6.32-fold (Fig. 2F). For the ABA treatment, SpWRKY1 transcripts began to increase at 2 h and reached its peak at 4 h with 4.11-fold and then decreased (Fig. 2G).

Overexpression of SpWRKY1 in transgenic tobacco promotes resistance to P. nicotianae To gain further insight into SpWRKY1 function in response to multiple stresses, the full-length SpWRKY1 sequence was cloned into the plant binary vector pBI121 under the control of CaMV 35S promoter and then was transform tobacco via the leaf disc method. Kanamycin tolerant plants were checked by PCR and RT-PCR, transgenic lines exhibited the expected transgene-specific 1608-bp band, but the corresponding band was not found in the WT plants (Fig. 3A, B). Interestingly, it was not possible to detect any expression of the SpWRKY1 gene in line 5 by RT-PCR (data not shown), although this transgenic line was verified as positive for the SpWRKY1 gene in its genomes by PCR detection. This is most likely to be a result of gene silencing due to multiple copies of the transgene (Luo et al. 2005). Subsequently, qRT-PCR was employed to estimate the levels of SpWRKY1 expression in leaf tissue of the three selected lines (Fig. 3C). SpWRKY1 overexpression was found to be 1.43 times in Line 1 and 1.26 times in Line 2 (relative to the expression of SpWRKY1 in Line 4). Hence, Line 1, Line 2 and Line 4 were selected for further study. Disease resistance tests were performed on the transgenic tobacco plants using oomycete pathogen P. nicotianae. Visually, the detached leaves of the transgenic plants exhibited more resistance to P. nicotianae. We observed the WT plants exhibited browning in the injection zone and the brown necrotic areas had expanded to cover over one half of the leaf areas, whereas symptoms on transgenic lines were much less (Fig. 4A). Moreover, the degree of resistance was calculated by the DI. As show in Fig. 4B, transgenic lines had lower DI than WT tobacco plants and the data was This article is protected by copyright. All rights reserved

consistent with what was observed visually. In a further study, ratio of lesion area to leaf area was compared in the whole plant of WT and transgenic lines inoculated with P. nicotianae. After inoculation, transgenic plants exhibited lower ratio of lesion area to leaf area compared to the WT plants (Fig. 4C). These results indicated to some extent that overexpression of SpWRKY1 in tobacco resulted in increased resistance to P. nicotianae. To investigate the physiological differences between WT and transgenic plants, some important physiological parameters were measured. MDA as an indicator of lipid peroxidation (Li et al. 2012) and REL as an indicator of membrane damage (Huang et al. 2011). Before inoculation with P. nicotianae, WT and transgenic lines showed no significant difference in MDA content and REL (Fig. 5A, B). After inoculation, there were a marked increase in MDA content and REL between WT and transgenic lines. However, the accumulations of MDA content and REL were significantly lower in the transgenic lines relative to WT plants, indicating that the WT suffered from severe membrane damage (Fig. 5A, B). Chlorophyll content is a reliable marker of photosynthetic damage (Liu et al. 2011). It was no significant difference between the WT and transgenic lines before inoculation (Fig. 5C). After inoculation, there was a marked decrease in chlorophyll content for both WT and transgenic lines. However, the accumulation of chlorophyll content was significantly higher in the transgenic lines than WT plants (Fig. 5C). Pathogen invasion could result in damage to plants via oxidative stress involving the generation of excess reactive oxygen species (ROS) (Tian et al. 2013). Plants have evolved antioxidant defense systems to scavenge excess ROS and maintain cellular ROS homeostasis (Mittler 2002). POD and SOD is well-known ROS scavenger enzyme in the antioxidant defense system (Shi et al. 2014). PAL, the first enzyme of phenylpropanoid pathway plays an important role in the regulation of antimicrobial compound biosynthesis in plants as a response to pathogen infection (Liu et al. 2011). Before inoculation, the activities of POD, SOD and PAL were no significant difference (Fig. 5D–F). After inoculation, the activities of these enzymes were all increased in both WT and transgenic lines. However, the increase degree was much more obvious in the transgenic lines than those in WT plants (Fig. 5D–F). Taken together, these results indicate that overexpression of SpWRKY1 indeed decreased lipid peroxidation and photosynthetic damage, and increased defense-related enzymes activities in transgenic tobacco, in turn, which may contributed to transgenic tobacco plants increased resistance to P. nicotianae. To gain further insight into the molecular mechanism underlying the increased disease resistance This article is protected by copyright. All rights reserved

of the transgenic plants, the transcript levels of known defense-related genes were investigated by qRT-PCR in the WT and transgenic lines before and after inoculation with P. nicotianae. The known genes are involved in response to SA (NtPR1, NtPR2 and NtPR5), response to JA (NtPR4 and NtPDF1.2), ROS detoxification (NtPOD and NtSOD) and pathogen challenge (NtPAL) (Li et al. 2004, Shi et al. 2014, Kim and Hwang 2014). Before inoculation, the expression levels of NtPR1, NtPR2, NtPR4 and NtPR5 in the transgenic lines were higher than those in WT plants (Fig. 6A–D). However, the expression levels of NtPDF1.2, NtPOD, NtSOD and NtPAL were no significant difference between the WT and transgenic lines (Fig. 6E–H). Following P. nicotianae infection, each of these eight genes was significantly upregulated in the transgenic lines as compared to the WT plants (Fig. 6). Taken together, these data show that SpWRKY1 overexpression in transgenic tobacco plants affected the transcriptional expression (directly or indirectly) of each of the eight tested defense-related genes in response to P. nicotianae infection.

Overexpression of SpWRKY1 enhances salt and drought tolerance in transgenic tobacco To detect the capacity of SpWRKY1 in abiotic stress tolerance, aseptic seedlings of WT and transgenic tobacco plants were transferred to half-strength MS agar medium with and without stress treatment. Under normal conditions, no significant difference in the root length and fresh weight were observed between the WT and transgenic lines (Fig. 7A, B). Upon exposure to salt stress, the root length and fresh weight in both WT and transgenic lines showed decrease (Fig. 7A, B). However, the transgenic lines exhibited much improved growth features (Fig. 7C), as well as longer root and higher fresh weight, than the WT plants (Fig. 7A, B). In addition, the aseptic seedlings were also transferred to half-strength MS agar medium containing 15% PEG6000 for the drought stress treatment. No significant difference was observed between the WT and transgenic plants under normal conditions (Fig. 7D, E). After treatment with drought stress, there was a marked decrease in root length and fresh weight between WT and the transgenic lines (Fig. 7D, E). However, the transgenic lines showed longer root and higher fresh weight than WT plants in response to drought stress (Fig. 7D–F). These results suggest that the overexpression of SpWRKY1 confers an enhanced tolerance to salt and drought stresses during early seedling development. Seedlings were also transferred from half-strength MS agar medium to soil for two weeks, and then also subjected to salt and drought stress treatments. For salt tolerance assay, both WT and This article is protected by copyright. All rights reserved

transgenic tobacco plants were irrigated with 200 mM NaCl for two weeks. It was observed that the growth of the WT plant was completely inhibited and leaf growth was retarded. However, the transgenic lines showed normal growth with expanded leaves (Fig. 8A). After drought treatment for two weeks, compared with transgenic lines, the WT plants exhibited smaller and more withered. With subsequent watering, the WT plants remained in the wilted stage, whereas transgenic lines restored normal growth (Fig. 10A). These phenotypic characterizations suggested that overexpression of SpWRKY1 also enhanced salt and drought stress tolerance after transplanting. To investigate the physiological differences between WT and transgenic tobacco plants before and after salt and drought stress treatment, some important physiological indices were measured. MDA content was reported as indicator for the membrane damage. Before treatment, WT and transgenic lines showed no significant difference in MDA content (Fig. 8B and Fig. 10B). After treatment with salt and drought stress, there was a marked increase in MDA content between WT and the transgenic lines (Fig. 8B and Fig. 10B). However, the accumulation of MDA was much lower in the transgenic lines relative to WT (Fig. 8B and Fig. 10B). Salt and drought stresses could result in damage to plants via oxidative stress involving the generation of ROS (Xiong et al. 2002). We monitored changes in the activities of POD and SOD between WT and transgenic lines in response to salt and drought stress. Before stress treatment, WT and transgenic lines showed no significant difference in the activities of POD and SOD (Fig. 8C, D and Fig. 10C, D). After salt and drought stress treatment, there were a marked increase in POD and SOD activities between WT and transgenic lines. However, the activities of POD and SOD were significantly higher in the transgenic lines relative to WT plants (Fig. 8C, D and Fig. 10C, D). Moreover, some important photosynthetic parameters, including chlorophyll content, photosynthetic rate and stomatal conductance were also assayed. As shown in Fig. 8E–G and Fig. 10E–G, chlorophyll content, photosynthetic rate and stomatal conductance were no significant difference between the WT and transgenic lines before treatment. After treatment, there were dramatically decreased in both WT and transgenic lines (Fig. 8E–G and Fig. 10E–G). However, chlorophyll content, photosynthetic rate and stomatal conductance were significantly higher in the transgenic lines than WT following salt and drought treatment. The physiological characterization results suggested that overexpression of SpWRKY1 in tobacco resulted in enhanced salt and drought stresses tolerance may via reduced lipid peroxidation, enhanced antioxidant enzyme activity and maintained photosynthesis. This article is protected by copyright. All rights reserved

To further investigate the mechanisms of SpWRKY1 improving tolerance to salt and drought stresses, the expression levels of several stress-responsive genes were examined in all lines before and after stress treatment. These genes encode enzymes involved in ROS detoxification (NtPOD and NtSOD), stress defensive (NtLEA5), biosynthesis of proline (NtP5CS) and ABA (NtNCED1). Before stress treatment, there was no significant difference in the expression of these stress-response genes (Fig. 9 and Fig. 11). After salt and drought treatment, the expression levels of these genes were significantly up-regulated in both WT and the transgenic lines (Fig. 9 and Fig. 11). However, these genes were induced at much higher levels in the transgenic lines as compared to WT plants (Fig. 9 and Fig. 11). Therefore, we infer that the underlying mechanism of SpWRKY1 transgenic lines enhanced salt and drought tolerance resulted from enhancing the expression level of some stress-responsive genes.

Discussion WRKY proteins are a class of zinc finger-containing transcription factors that are encoded by large gene family in higher plants (Rushton et al. 2010). Recently, increased attention has been focused on WRKY transcription factors and their participation in the regulation of plant responses to various biotic and abiotic stresses (Zheng et al. 2006, Zheng et al. 2007, Jiang and Deyholos 2009, Zheng et al. 2013). Moreover, WRKY transcription factors act as transcriptional regulators by recognizing and binding the W-box in the promoter regions of various stress-related genes, thereby enhancing plant tolerance to multiple stresses (Wang et al. 2014a). To date, a series of stress-responsive WRKY transcription factors have been identified and characterized from different plant species. However, most functional analyses of WRKY genes have been restricted to model plants like A. thaliana and O. sativa, and data associated with the WRKY family members from tomato are notably limited. In the present study, we isolated a pathogen-induced WRKY gene from S. pimpinellifolium L3708, which is a particular late blight resistant tomato variety. It has two WRKY DNA-binding domains and belonged to group I WRKY family. We designated this gene SpWRKY1 and actually corresponds to SlWRKY31 (Solyc06g066370.2.1) of Huang et al. (2012) classification. However, the expression and functional identification of SpWRKY1 under biotic and abiotic stresses has not been published, thus we continued our investigations. WRKY transcription factors have rapid and strong induction in response to certain pathogens, This article is protected by copyright. All rights reserved

high salinity, drought and phytohormones (Dang et al. 2014, Li and Luan 2014). The expression pattern of a gene is usually an indicator of its function (Shi et al. 2014). The results of qRT-PCR analyses indicated that the transcription level of SpWRKY1 was increased by not only pathogen attack, salt and drought stress, but also signal molecules such as SA, MeJA and ABA (Fig. 2). These results encouraged us to probe the function of SpWRKY1 in response to biotic and abiotic stresses. To investigate the effects of overexpression of SpWRKY1 on the response to oomycete pathogen resistance, transgenic tobacco plants were inoculated with P. nicotianae. Both assays，the detached lead assay and inoculation on the whole plant, indicated tobacco plants overexpressing SpWRKY1 was more resistant to P. nicotianae infection than WT plants (Fig. 4). Pathogen invasion is often followed by the production of ROS, which are important determinants of hypersensitive response (HR) in incompatible pathogen–plant interactions (Kotchoni and Gachomo 2006, Wi et al. 2012). Low levels of ROS can act as signaling molecules in response to pathogen infection. However, late massive ROS can induce lipid peroxidation and damage cellular structure, lead to oxidative stress and disease susceptibility (Sathiyaraj et al. 2011, Wi et al. 2012). The control of ROS accumulation is quite important for disease resistance. MDA is one of the most frequently used indicators of lipid peroxidation (Li et al. 2012). REL is an indicator of membrane damage (Huang et al. 2011). In the present study, we found that transgenic tobacco plants generated lower MDA and experienced less REL than in the WT after two weeks inoculation with P. nicotianae (Fig. 5A, B). Chlorophyll contents are extremely important biomolecules and serve as good indicators of photosynthetic function (Liu et al. 2011). POD and SOD are important enzymes in the ROS scavenging systems, which can scavenge excess ROS and protect cells from oxidative damage (Shi et al. 2014). After infection with P. nicotianae, transgenic lines contain higher chlorophyll content and activities of POD and SOD in comparison with WT (Fig. 5C–E). In addition, the greater expression levels of NtPOD and NtSOD that encode ROS-scavenging enzymes in transgenic lines may produce an increase in the activities of these enzymes, thus conferring more resistance against to P. nicotianae (Fig. 6F, G). It is suggested that overexpression of SpWRKY1 may regulate these genes and confer a more efficient antioxidant system to decreased production of ROS or more effectively scavenging of excess ROS (Fig. 12). However, the regulatory roles of SpWRKY1 in the ROS scavenging pathway should be explored further. Phenylalanine ammonia-lyase (PAL) catalyzes the first step of the

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phenylpropanoid pathway and is involved in the biosynthesis of phenolic compounds and phytoalexins (Liu et al. 2011). Compared with its activity in WT plants, the activity of PAL was significantly increased in the transgenic lines following pathogen infection (Fig. 5F). Similarly, the expression of NtPAL in the transgenic lines was higher than the WT (Fig. 6H). The increased activity of PAL may result in the accumulation of antifungal secondary metabolites which contributed to increased resistance to P. nicotianae (Fig. 12). Previous studies have demonstrated that WRKY transcription factors can induce the expression of a number of defense-related genes, including the well-studied PR genes, via binding to the W-box in the promoter of these genes (Yu et al. 2012). PR genes are widely used as molecular markers in pathogen resistance assays. PR gene induction is correlated with enhanced disease resistance in many cases (Shi et al. 2014). In the present study, we found that the PR genes, including NtPR1, NtPR2, NtPR4 and NtPR5, were greater in the transgenic lines than the expression in the WT plants and disease resistance in transgenic plants has been improved (Fig. 6A–D). Notably, NtPR1, NtPR2 and NtPR5 are marker genes for SA signaling (Seo et al. 2008), and PR4 is the marker gene for JA signaling (Shi et al. 2014). Moreover, the expression of the JA-responsive marker genes NtPDF1.2 (Li et al. 2004) was also enhanced (Fig. 6E). Most PR genes, if not all, are induced through the coordinate action of SA and JA signaling pathways and underlie the systemic acquired resistance (SAR) that develops in resistant plants upon infection with pathogens (Seo et al. 2008). SA-dependent defenses are often triggered by biotrophic pathogens, whereas JA-dependent plant defenses are generally activated by necrotrophic pathogens (Shi et al. 2014). SA and JA signaling usually act antagonistically; however, synergism between these two phytohormones has also been observed (Mur et al. 2006, Miljkovic et al. 2012). Therefore, we speculate that SpWRKY1-dependent activation of downstream PR genes and plant defensin gene plays a pivotal role in the resistance of transgenic lines to pathogens, and that this resistance might be related to SA-dependent and JA-dependent defense pathways (Fig. 12). Similar observations have also been reported for the Gossypium hirsutum WRKY transcription factor GhWRKY15 and GhWRKY39-1 (Yu et al. 2012, Shi et al. 2014). However, reports of tomato WRKY transcription factors that function in both SA- and JA-dependent pathways are relatively limited. Taken together, the molecular mechanisms that underlie the activities of SpWRKY1 in pathogen defense are fairly complex and multiple signaling pathways may function together that need to be explored through further research. A proposed functional model of SpWRKY1 in defense This article is protected by copyright. All rights reserved

responses to pathogen was shown in Fig. 12. In addition to the vital roles of WRKY transcription factors in disease resistance, their role in abiotic stress tolerance, such as salt and drought has been also well documented (Li et al. 2012, Niu et al. 2012). Our results showed that overexpression of SpWRKY1 in tobacco remarkably improved plants’ tolerance to salt and drought stress. It was observed that transgenic lines showed better growth than WT tobacco when plants were grown in the half-strength MS medium (Fig. 7) and planted on soils (Fig. 8A and 10A). Exposure of plants to high salinity and drought stresses will lead to the rapid generation and high accumulation of ROS (Li et al. 2012, Shu et al. 2012). Increasing evidences show that low levels of ROS can act as signaling molecules in response to abiotic stress, however excessive ROS can damage cellular membranes and mediate lipid peroxidation, leading to oxidative stress, indicating that the control of ROS to suitable levels is important for abiotic stress tolerance (Wang et al. 2014b). Previous reports have shown that WRKY transcription factor confer salt and drought stresses tolerance in transgenic plant through accumulation of ROS-scavenging enzymes, such as POD and SOD (Li et al. 2012, Liu et al. 2013). After salt and drought stress treatment, a lower accumulation of MDA and a higher activity of antioxidant enzymes, including POD and SOD, were detected in the transgenic lines relative to the WT plants (Fig. 8B–D and Fig. 10B–D). In addition, the expression levels of NtPOD and NtSOD were also up-regulated in the transgenic lines after salt (Fig. 9A, B) and drought stress treatment (Fig. 11A, B), consistent with the greater activity of these antioxidant enzymes. This may presumably explain the activation of the antioxidant enzymes and the lower ROS levels in the transgenic lines. We reasoned that overexpression of SpWRKY1 improved the activities of the antioxidant enzymes POD and SOD and results in the suppression of ROS accumulation in order to suffer less from oxidative damage under salt and drought conditions. Chlorophyll content can absorb light energy and convert it into chemical energy in the process of photosynthesis (Zhao et al. 2012). Salt and drought can greatly decrease chlorophyll content and influence photosynthesis (Shu et al. 2012). Thus, protecting chlorophyll from degradation is important for maintaining plant photosynthesis and enhancing plant stress tolerance (Wang et al. 2014b). To test whether the difference in tolerance to salt and drought stress between WT and the transgenic lines was related to photosynthesis, chlorophyll content and photosynthetic rate were investigated. Before treatment, no dramatic differences were observed (Fig. 8E, F and Fig. 10E, F). After salt and drought stress treatment, there were marked decreases in both WT and the transgenic lines. However, the This article is protected by copyright. All rights reserved

content of chlorophyll and photosynthetic rate were significantly higher in the transgenic lines than WT in response to salt and drought stress (Fig. 8E, F and Fig. 10E, F). In addition, reduction in photosynthetic rate may be due to limitation of CO2 diffusion through stomata (Chaves et al. 2009). The present study found that stomatal conductance result followed the same pattern as photosynthetic rate after salt and drought treatment (Fig. 8G and Fig. 10G). Based on the physiological and biochemical analysis, it can be determined that the enhanced salt and drought tolerance in the SpWRKY1-overexpression tobacco plants is correlated with the maintenance of higher activities of ROS-scavenging enzymes and better photosynthesis. To reveal the possible underlying molecular mechanisms of enhanced salt and drought stress tolerance in the transgenic lines, the expression of several abiotic stress-response genes were determined. LEA5 genes encode group 5 late embryogenesis abundant (LEA) proteins. Circumstantial evidence has demonstrated that LEA5 proteins function in osmotic stress tolerance, stabilizing labile enzymes, and protecting macromolecule and membranes structures (Shao et al. 2005, Hundertmark and Hincha 2008). The present observations show that NtLEA5 expression was unregulated in transgenic tobacco plants after salt and drought stress treatment (Fig. 9C and Fig. 11C). Greater induction of this gene suggests that more LEA proteins might be synthesized in the transgenic lines, and therefore contributes to facilitating osmotic adjustment and protection of membrane structure under adverse stress conditions. Additionally, we found that the expression of NtP5CS was also increased in transgenic lines compared with the WT plant after salt and drought treatment (Fig. 9D and Fig. 11D). The delta1-pyrroline-5-carboxylate synthetase (P5CS) gene encodes an enzyme which was involved in catalysis of the first two steps in proline biosynthesis (Yue et al. 2012). The increased expression of NtP5CS may lead to further increased proline production in transgenic tobacco. Proline has been reported to play roles in protecting enzymes from denaturation, regulating the cytosolic acidity and increasing water retention capacity (Liu et al. 2007). Proline accumulates in many plant species in response to environmental stress, such as salt and drought, and its accumulation frequently correlates with tolerance to salt and drought stress in plants (Ben et al. 2008, Huang et al. 2011). ABA is an important phytohormone and plays a critical role in plant responses to abiotic stress (Huang et al. 2013). Biochemical and genetic evidence reveals that many enzymes are involved in the ABA biosynthetic pathway, but 9-cis-epoxycarotenoid dioxygenase (NCED) is the rate-limiting enzyme that regulates ABA biosynthesis in plants and ABA levels under the stresses are closely correlated This article is protected by copyright. All rights reserved

with the transcript abundance of NCED genes, suggesting that NCEDs play a pivotal role in ABA accumulation (Xian et al. 2014, Zhang et al. 2014). The results in this study showed that the transcript level of NtNCED1 was enhanced in transgenic lines as compared with the WT after salt and drought treatment (Fig. 9E and Fig. 11E). Thus, we hypothesized that SpWRKY1 may be involved in salt and drought stress response via an ABA-dependent signaling pathway, and further research is needed. Some WRKY transcription factors can positively regulate abiotic and biotic stress responses. GhWRKY39 (from cotton) and VpWRKY3 (from grapevine) were implicated in dual resistance to biotic and abiotic stresses when they were overexpressed in tobacco (Zhu et al. 2012, Shi et al. 2014). In our present work, overexpression of SpWRKY1 resulted in enhanced resistance to pathogen infection and tolerance to salt and drought stress. This was partially correlated with the activation of ROS-related antioxidant genes/enzymes, leading to less accumulation of ROS under stress. Meanwhile, some defense-related genes were also found to be up-regulated by SpWRKY overexpression. On the basis of our study, it can be inferred that SpWRKY1 may facilitate crosstalk between the complicated biotic and abiotic stress response pathways. Although the influence of overexpression of SpWRKY1 in tobacco was explored, the mechanism underlying the function of SpWRKY1 should be elucidated in transgenic tomato. Construction and characterization of transgenic tomato lines with overexpression and virus-induced gene silencing (VIGS)-mediated suppression of SpWRKY1 gene is underway. As we learn more about SpWRKY1 and its regulation, the design of efficient strategies for crop improvement should become possible.

Author contributions Conceived and designed the experiments: Jing-bin Li and Yu-shi Luan; performed the experiments: Jing-bin Li and Zhen Liu; analyzed the data: Jing-bin Li and Zhen Liu; contributed reagents/materials/analysis tools: Jing-bin Li and Yu-shi Luan; wrote the paper: Jing-bin Li.

Acknowledgements – This work was supported by grants from the National Natural Science Foundation of China (31272167 and 30972001).

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Edited by M. Uemura

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Figure legends

Fig. 1. Characterization and sequence analysis of SpWRKY1. (A) Comparison of SpWRKY1 sequence with NtWRKY1, CaWRKY2, TaWRKY2, AtWRKY33 and OsWRKY53. Identical residues were colored in black and conserved residues in dark gray. Two WRKY domains were underlined with solid lines. The Cys and His residues forming the zinc-finger motif were indicated by arrows. (B) Phylogenetic analysis of SpWRKY1 in relation to other plant group I WRKY transcription factors. Phylogenetic tree constructed by neighbor-joining algorithms of MEGA software version 4.0 after the multiple WRKY protein sequences alignment using the ClustalX 1.81 program. Bootstrapping was performed 1000 times to obtain support values for each branch. The WRKY transcription factors used are as follows: AtWRKY33 (NP_181381.2), BnWRKY33 (ACI14397.1), TaWRKY2 (ACD80357.1), This article is protected by copyright. All rights reserved

OsWRK KY53 (BAF775367.1), NttWRKY1 (B BAA82107.11), CaWRKY Y2 (ABD65 5255.1), AtW WRKY25 (NP_1800584.1), SlW WRKY (XP_0004251909.11), VvWRKY Y2 (AAT460067.1), AtWR RKY3 (AAK K28311.1) and AtW WRKY4 (AA AL13048.1).

Quantitative real-time PCR analysis of expression patterns of SpWRKY Y1 in differennt tissues Fig. 2. Q and in response r to various v treattments. (A) Tissue-speciific expression of SpWR RKY1 in tom mato root, stem and leaf. (B–G G) Expressioon patterns of o SpWRKY11 in tomato leaves at the indicated time t and with Phytophhthora infestaans, NaCl, PEG P 6000, SA A, MeJA and d ABA. The data are pressented as treated w the meaan ± standard d deviation (SD) of threee independennt experimen nts. The vallues indicateed by the differentt letters are significantlyy different att P < 0.05, as a determinedd using Dunncan’s multipple range tests.

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Fig. 3. Molecular analysis of putatively transgenic tobacco plants. (A) Genomic DNA-PCR analysis of WT plant and transgenic lines. (B) RT-PCR analysis of SpWRKY1 transcript levels in WT and transgenic lines under normal conditions. (C) QRT-PCR analysis of the selected transgenic lines (Line 1, Line 2 and Line 4) for determination of the expression level of SpWRKY1. The data are presented as the mean ± SD of three independent experiments. M, DL2000 DNA Marker; P, positive control by using pBI121-SpWRKY1 plasmid DNA as PCR template; WT, untransformed wild-type tobacco; L 1– 6, independent transgenic tobacco lines overexpressing SpWRKY1.

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Fig. 4. Overexpression of SpWRKY1 in transgenic tobacco increased Phytophthora nicotianae resistance. (A) Disease signs on the detached leaves from WT and transgenic lines at two weeks after inoculation with P. nicotianae. (B) Disease index was measured at two weeks after inoculation. (C) Measurement of the ratio of lesion area to leaf area in P. nicotianae-inoculation leaves at two weeks after inoculation. The data are presented as the mean ± SD of three independent experiments. The values indicated by the different letters are significantly different at P < 0.05, as determined using Duncan’s multiple range tests.

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Fig. 5. Physiological changes in WT and transgenic tobacco plants before and after inoculation with P. nicotianae. (A) MDA content. (B) Relative electrolyte leakage. (C) Chlorophyll content. (D) POD activity. (E) SOD activity. (F) PAL activity. The data are presented as the mean ± SD of three independent experiments. The values indicated by the different letters are significantly different at P < 0.05, as determined using Duncan’s multiple range tests.

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Fig. 6. Relative expression levels of some defense-related genes in the leaves from the WT and transgenic tobacco plants before and after inoculation with P. nicotianae. (A-H) Relative transcript levels of NtPR1, NtPR2, NtPR4, NtPR5, NtPDF1.2, NtPOD, NtSOD and NtPAL. The data are presented as the mean ± SD of three independent experiments. The values indicated by the different letters are significantly different at P < 0.05, as determined using Duncan’s multiple range tests.

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Fig. 7. Analysis of root length and fresh weight between WT and transgenic tobacco plants before and after salt and drought treatment. (A–B) The root length and fresh weight of WT and transgenic lines before and after salt treatment. (C) Phenotypic comparison of WT and transgenic lines after salt treatment. (D–E) The root length and fresh weight of WT and transgenic lines before and after drought treatment. (F) Phenotypic comparison of WT and transgenic lines after drought treatment. The data are presented as the mean ± SD of three independent experiments. The values indicated by the different letters are significantly different at P < 0.05, as determined using Duncan’s multiple range tests.

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Fig. 8. Analysis of the enhanced salt tolerance in transgenic tobacco plants. (A) Phenotype comparison of WT and transgenic lines before and after salt treatment. (B) MDA content. (C) POD activity. (D) SOD activity. (E) Chlorophyll content. (F) Photosynthetic rate. (G) Stomatal conductance. The data are presented as the mean ± SD of three independent experiments. The values indicated by the different letters are significantly different at P < 0.05, as determined using Duncan’s multiple range tests.

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Fig. 9. Relative expression levels of some defense-related genes in the leaves from the WT and transgenic tobacco plants before and after salt treatment. (A–E) Relative transcript levels of NtPOD, NtSOD, NtLEA5, NtP5CS and NtNCED1. The data are presented as the mean ± SD of three independent experiments. The values indicated by the different letters are significantly different at P < 0.05, as determined using Duncan’s multiple range tests.

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Fig. 10. Analysis of the enhanced drought tolerance in transgenic tobacco plants. (A) Phenotype comparison of WT and transgenic lines before and after drought treatment. (B) MDA content. (C) POD activity. (D) SOD activity. (E) Chlorophyll content. (F) Photosynthetic rate. (G) Stomatal conductance. The data are presented as the mean ± SD of three independent experiments. The values indicated by the different letters are significantly different at P < 0.05, as determined using Duncan’s multiple range tests.

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Fig. 11. Relative expression levels of some defense-related genes in the leaves from the WT and transgenic tobacco plants before and after drought treatment. (A–E) Relative transcript levels of NtPOD, NtSOD, NtLEA5, NtP5CS and NtNCED1. The data are presented as the mean ± SD of three independent experiments. The values indicated by the different letters are significantly different at P < 0.05, as determined using Duncan’s multiple range tests.

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Fig. 12. A proposed model for the putative role of SpWRKY1 in the regulation of pathogen defense responses. Solid lines, regulation may be executed by direct or indirect action. Dotted lines, regulation has been confirmed in other plant species.

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