Plant Science 227 (2014) 145–156

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Tomato WRKY transcriptional factor SlDRW1 is required for disease resistance against Botrytis cinerea and tolerance to oxidative stress Bo Liu a,b , Yong-Bo Hong a , Ya-Fen Zhang a , Xiao-Hui Li a , Lei Huang a , Hui-Juan Zhang a , Da-Yong Li a , Feng-Ming Song a,∗ a b

National Key Laboratory for Rice Biology, Institute of Biotechnology, Zhejiang University, Hangzhou, Zhejiang, 310058, China Weinan Vocational and Technical College, Weinan, Shanxi, China

a r t i c l e

i n f o

Article history: Received 15 May 2014 Received in revised form 1 August 2014 Accepted 3 August 2014 Available online 10 August 2014 Keywords: Tomato (Solanum lycopersicum) WRKY proteins SlDRW1 Botrytis cinerea oxidative stress defense response

a b s t r a c t WRKY proteins comprise a large family of transcription factors that play important roles in plant responses to biotic and abiotic stresses; however, only a few of tomato WRKYs have been studied for their biological functions. In the present study, we identified a Botrytis cinerea-responsive WRKY gene SlDRW1 (Solanum lycopersicum defense-related WRKY1) from tomato. SlDRW1 is a nucleus localized protein with transactivation activity in yeast. Expression of SlDRW1 was significantly induced by B. cinerea, leading to 10–13 folds of increase than that in the mock-inoculated plants but not by Pseudomonas syringae pv. tomato (Pst) DC3000. Silencing of SlDRW1 resulted in increased severity of disease caused by B. cinerea, but did not affect the phenotype of disease caused by Pst DC3000. In addition, silencing of SlDRW1 also resulted in decreased tolerance against oxidative stress but did not affect drought stress tolerance. Furthermore, silencing of SlDRW1 attenuated defense response such as expression of defense-related genes after infection by B. cinerea. Our results demonstrate that SlDRW1 is a positive regulator of defense response in tomato against B. cinerea and oxidative stress. © 2014 Elsevier Ireland Ltd. All rights reserved.

1. Introduction During their lifetime, plants have to confront different kinds of biotic and abiotic stresses around their growth environment. To cope with these stresses to survive, plants have developed a series of defense mechanisms regulated by a complicated signaling network, which is often initiated upon perceiving environmental cues [1–5]. Activation of defense responses against biotic and abiotic stress is always accompanied with significant alterations in expression of a large set of genes, which are regulated by different types of transcription factors (TFs). Thus, TFs are critical regulatory factors that determine the outcome of plant interactions with biotic and abiotic stress through modulating the temporal and spatial expression of the genes involved in defense response. In recent years, many TFs belonging to the NAC, ERF, MYB, WRKY and bZIP families have been identified to play important roles in plant responses to biotic and abiotic stress [6–10]. The WRKY proteins comprise one of the largest families and contain either one or two copies of the conserved WRKY domain, followed by a C2 H2 or C2 HC zinc finger motif [11]. Based on

∗ Corresponding author. Tel.: +86 571 88982481. E-mail address: [email protected] (F.-M. Song). http://dx.doi.org/10.1016/j.plantsci.2014.08.001 0168-9452/© 2014 Elsevier Ireland Ltd. All rights reserved.

the number of WRKY domains and the type of the zinc finger motifs, the WRKY proteins can be classified into three groups [11,12]. Group I WRKYs contain two WRKY domains whereas Group II and III WRKYs contain only one WRKY domain. Another, WRKYs belonging to Group I and Group II have a C2 H2 zinc finger motif whereas the Group III WRKYs have C2 HC zinc finger motif [11,12]. Although WRKYs have recently been implicated in the regulation of plant growth and development [12,13], the most important functions for WRKYs seem to act as regulators of defense response against biotic and abiotic stresses. Expression of WRKY genes can be induced strongly and rapidly by different biotic and abiotic stresses in numerous plant species [11,14,15]. For example, 49 out of 72 Arabidopsis WRKY genes can respond to bacterial infection or salicylic acid (SA) and most of the Group III WRKY genes can be induced by both SA and pathogens [16,17]. A large body of studies with functional genomic approaches by analyzing phenotypes of the knockout/knockdown or overexpression lines has demonstrated that the WRKY proteins play critical roles in regulating disease resistance responses in plants. In Arabidopsis thaliana, WKRY3 and WRKY4 [18], WRKY8 [19], WRKY11 and WRKY17 [20], WRKY18/WRKY40/WRKY60 [21], WRKY22 [22], WRKY27 [23], WRKY25 [24], WRKY33 [25], WRK46 [26], WRKY70 [27,28] and WRKY72 [29] have been shown to

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be either positive or negative regulators of defense response against different types of pathogens. Similarly, at least 10 rice WRKYs, i.e. OsWRKY03 [30], OsWRKY71 [31], OsWRKY13 [32], OsWRKY45 [33,34], OsWRK89 [35], OsWRKY31 [36], OsWRKY22 [37], OsWRKY30 [38], OsWRKY28 [39], OsWRKY76 [40] and OsWRKY62 [41], have been implicated in immune responses against fungal and bacterial pathogens. Alternatively, some of the WRKY proteins have also been shown to play important roles in regulating abiotic stress tolerance (for review see [42]), such as Arabidopsis WRKY6 and WRKY75 in low phosphorus stress [43,44], WRKY25 and WRKY39 in heat stress [45,46], WRKY63 (ABO3) and WRKY57 in drought stress [47,48], WRKY34 in cold stress [49]), WRKY18, WRKY40, WRKY70 and WRKY54 in osmotic stress [50,51], WRKY30 in oxidative stress [52] and WRKY46 in aluminum toxicity [53], and rice OsWRKY30 in drought stress [54]. These observations suggest that, compared with other TF families, a relatively larger proportion of the WRKY family members in a given plant species play roles in regulating defense response against diverse biotic and abiotic stresses, demonstrating the importance of the WRKY proteins in plant stress responses. Recent genome-wide bioinformatics analysis identified a total of 81 WRKY genes in tomato genome [55]. However, only a few of tomato WRKYs have been characterized at molecular level for their biological functions. It was recently reported that SlWRKY70 and SlWRKY72 are required for R gene Mi-1-mediated resistance to aphids and nematodes [29,56] and SlWRKY72 also contributes to basal immunity against Pseudomonas syringae [29]. Overexpression of a tomato WRKY gene in transgenic tobacco plants resulted in increased expression of defense genes and improved abiotic stress tolerance [57]. Furthermore, some tomato WRKY genes were found to show differential expression patterns after infection with viral, bacterial and fungal pathogens [55,58–60] or treatment with pathogen-derived elicitors [61]. However, the biological functions for the majority of the tomato WRKYs are not clear yet. In this study, we performed VIGS-based assays to identify putative WRKYs that are involved in defense response in tomato against B. cinerea, the causal agent of grey mold disease on a number of economically important crops. We found that silencing of one tomato WRKY gene led to increased severity of disease caused by B. cinerea and designated this putative WRKY gene as SlDRW1 (Solanum lycopersicum defense-related WRKY1). Results from further experiments demonstrate that SlDRW1 plays important roles in defense response against B. cinerea and oxidative stress tolerance in tomato.

2. Materials and methods 2.1. Plant growth and treatments Tomato (S. lycopersicum) cv. Suhong 2003 was used in this study. Tomato and Nicotiana benthamiana plants were grown in a mixture of perlite:vermiculite:plant ash (1:6:2) in a growth room at 22 ◦ C under a 16 h light (350 ␮mol s−1 m−2 photons m−2 s−1 ) and 8 h dark regime. For analysis of gene expression in response to defense signaling hormones, 4-week-old tomato plants were treated by foliar spraying with 100 ␮M methyl jasmonate (MeJA), 100 ␮M 1amino cyclopropane-1-carboxylic acid (ACC), 1 mM salicylic acid (SA) or water as a control. For analysis of gene expression in response to pathogen infection, 4-week-old plants were inoculated with spore suspension of B. cinerea, bacterial suspension of Pst DC3000 or with same volume of buffer as a mock-inoculation control (see below “Disease assays” for details). Leaf samples were collected at indicated time points after treatment or inoculation and stored at −80 ◦ C until use.

2.2. Extraction of total RNA Total RNA was extracted from leaf tissues using Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions and treated with PrimeScript RT reagent Kit With gDNA Eraser (Takara, Dalian, China) to eliminate DNA according to the manufacturer’s protocols. The total RNA samples were stored at −80 ◦ C until use. 2.3. Cloning of SlDRW3 and bioinformatics analysis First-strand cDNA was synthesized using the AMV reverse transcriptase (Takara, Dalian, China) with oligo d(T) primer according to the manufacturer’s instructions. Full-length cDNA of SlDRW1 was amplified using a pair of SlDRW1-specific primers SlDRW1orf-1F (5 -ATG GCT GCT TCA AGT TTC TCT TTT CC-3 ) and SlDRW1-orf-1R (5 -TTC ATA AAC TTC AAT TCG TGC TCC T-3 ). The PCR product was purified and cloned into pMD-19T vector (Takara, Dalian, China), followed by sequencing for confirmation. Similarity analyses of nucleotide and amino acid sequences were carried out using BLAST program at the NCBI GenBank database (http://www.ncbi.nlm.nih.gov/BLAST/). Plant WRKY protein sequences were retrieved from NCBI GenBank. Sequence alignment was performed using ClustalX (version 2.0.8) and phylogenetic tree was generated by neighbor-joining algorithm with p-distance method using MEGA version 6.05. A bootstrap statistical analysis was performed with 1000 replicates to test the phylogeny. 2.4. Construction of VIGS vector and agroinfiltration A 437 bp fragments of SlDRW1 was amplified with a pair of SlDRW1-specific primers SlDRW1-vigs-1F (5 -GCG TCT AGA TGA CGA CTT CTT TCA CCG ACC TT-3 , a XbaI site underlined) and SlDRW1-vigs-1R (5 -ATA GGA TCC TGT GGG CTC TTG ACA ATT CCA T-3 , a BamHI site underlined) using plasmid pMD19T-SlDRW1 as templates. The resulting products were digested and cloned into pYL156, yielding TRV-SlDRW1 construct. For construction of TRV-GUS, a 396 bp fragments of the GUS gene was amplified with primers GUS-vigs-1F (5 -CGG TCT AGAACC TGG GTG GAC GAT ATC AC-3 , a XbaI site underlined) and GUS-vigs-1R (5 -CGG GGA TCC GTG CAC CATC AGC ACG TTA T -3 , a BamHI site underlined), and cloned into pYL156, yielding TRV-GUS construct. The recombinant plasmids TRV-SlDRW1 and TRV-GUS were transformed into Agrobacterium tumefaciens strain GV3101 by electroporation. Agrobacteria carrying TRV-SlDRW1 or TRV-GUS construct were cultivated in YEP liquid medium with 50 ␮g/mL kanamycin, 50 ␮g/mL rifampicin and 25 ␮g/mL gentamicin to OD600 = 0.8∼1.0. Cells were centrifuged and resuspended in infiltration buffer containing 10 mM MgCl2 , MES (pH 5.7) and 150 ␮M acetosyringone. The agrobacteria carrying TRV-GUS or TRV-SlDRW1 were mixed with agrobacteria carrying pTRV1 in a ratio of 1:1 and maintained at OD600 = 1.5 for 3 h at room temperature. The mixed agrobacteria suspension was infiltrated into the abaxial surface of the 2-weekold seedlings using a 1 mL needleless syringe. Efficiency of the VIGS protocol was evaluated using phytoene desaturase (PDS) gene as a marker of silencing in tomato plants according to Liu et al. [62]. The VIGS-infiltrated plants were allowed to grow for 3 weeks under the same conditions as mentioned above and then used for all experiments. 2.5. Transient expression in N. benthamiana The coding sequence of SlDRW1 was amplified using a pair of primers SlDRW1-oe-1F (5 -ATA GGA TCC ATG GAA TTT ACC AGT TTG GT-3 , a BamHI site underlined) and SlDRW1-oe-1R

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(5 -GCT GAA TTC TTA CCA TCT CCC TGT CTG AT-3 , an EcoRI site underlined) and cloned into pCAMBIA991 vector, yielding pCAMBIA991-SlDRW1. The recombinant plasmid pCAMBIA991SlDRW1 was transformed into A. tumefaciens strain GV3101 by electroporation. Agrobacteria carrying pCAMBIA991-SlDRW1 or pCAMBIA991 empty vector were infiltrated into leaves of 4-weekold plants using a 1 mL syringe without a needle. Leaf samples were collected at 2 days after agroinfiltration for analyzing the expression level of SlDRW1 and were used for disease assays. 2.6. Subcellular localization analysis The open reading frame of SlDRW1 was obtained by PCR amplification using a pair of primers SlDRW1-gfp-1F (5 -AGT GGA TCC ATG GCT GCT TCA AGT TTC TCT T-3 , a BamHI site underlined) and SlDRW1-gfp-1R (5 -GCG TCT AGA TCA GCA A AGC AAT GAC TCC ATA-3 , an XbaI site underlined). The PCR product was cloned into pFGC-eGFP vector to yield pFGC-SlDRW1 construct and confirmed by enzymatic digestion with BamHI and XbaI. The recombinant plasmid pFGC-SlDRW1 and pFGC-eGFP (as a control) were transformed into A. tumefaciens strain GV3101 by electroporation. Agrobacteria carrying pFGC-SlDRW1 or pFGC-eGFP were grown at 28 ◦ C in YEP medium (50 ␮g/ml kanamycin, 50 ␮g/ml rifampicin and 25 ␮g/ml gentamicin), pelleted by centrifugation and then resuspended in infiltration buffer (10 mM MgCl2 , 10 mM MES and 150 ␮M acetosyringone) to OD600 = 0.6. Agrobacterial suspensions were infiltrated using 1 mL syringes without needles into leaves of N. benthamiana for subcellular localization analysis or into leaves of 4-week-old tomato plants for transient expression assays. N. benthamiana leaves infiltrated with agrobacteria were collected at 24 h after infiltration and GFP was detected with a confocal laser scanning microscope (Zeiss LSM 510 META; argon laser excitation wavelength, 488 nm). Tomato plants infiltrated with agrobacteria were used for disease assays at 3 days after agroinfiltration. 2.7. Transactivation activity assay The coding sequence of SlDRW1 was PCR amplified using a pair of primers SlDRW1-TA-1F (5 -ATG GTC GAC ATG GCT GCT TCA AGT TTC TCT T-3 , a SalI site underlined) and SlDRW1-TA1R (5 -CGA CTG CAG TCA GCA AAG CAA TGA CTC CAT A-3 , a PstI site underlined) and cloned into pBD-GAL4Cam vector, yielding plasmid pBD-SlDRW1. The plasmid pBD-SlDRW1 and pBD empty vector (as a negative control) were transformed into yeast strain AH109. The transformed yeast was cultivated on the SD/Trp− and SD/Trp− His− medium for 3 days at 28 ◦ C, followed by addition of x-␣-gal. The transactivation activity of the fusion protein was evaluated according to the growth situation and production of blue pigments after the addition of x-␣-gal of the transformed yeast cells on SD/Trp− His− medium. 2.8. Disease assays Inoculation of tomato plants with B. cinerea was carried out as described previously [63]. Briefly, spores were collected in 1% maltose buffer from 10-day-old B. cinerea cultures grown on 2 × V8 agar (36% V8 juice, 0.2% CaCO3 , 2% agar) by passing through two layers of chesscloth and spore density was adjusted to 1 × 105 spores/mL. Detached fully expanded leaves were inoculated by drop inoculation method according to a previously reported procedure [63]. The inoculated leaves were covered with a transparent plastic film and kept in a growth chamber with similar condition as for plant growth. Diameters of each lesion were recorded 4 days post inoculation (dpi). Leaves from at least 10 individual plants were used in each independent experiment.

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P. syringae pv. tomato (Pst) DC3000 grown overnight in King’s B liquid medium containing 25 ␮g/mL rifampicin were diluted and re-grown to OD600 = ∼1.0. Bacteria were collected and resuspended in 10 mM MgCl2 to OD600 = 0.002 (∼106 CFU/mL). Four-week-old plants were inoculated by infiltrating 20 ␮L Pst DC3000 suspension at two sites of each leaf with 1-mL syringes without needles. The inoculated plants were covered with a transparent plastic film and disease symptoms were observed daily. For measurement of bacterial growth curve, leaf punches from six individual plants were surface sterilized in 70% ethanol for 10 s, homogenized in 200 ␮L of 10 mM MgCl2 , diluted in 10 mM MgCl2 , and plated on KB agar plates containing 100 ␮g/mL rifampicin. 2.9. Quantitative RT-PCR analysis of gene expression Silencing efficiency of SlDRW1 as reduction of mRNA transcript level in TRV-SlDRW1-infiltrated plants, expression of SlDRW1 and defense genes was analyzed by qRT-PCR. Tomato SlActin was used as a reference gene with primers of SlActin-1F (5 -GAA ATA GCA TAA GAT GGC AGA CG-3 ) and SlActin-1R (5 -ATA CCC ACC ATC ACA CCA GTA T-3 ). Primers for SlDRW1 and other defense genes are as follows: SlDRW1-q-1F, 5 -CAA CCA AAG GAC TTG CTG ATA GA-3 ; SlDRW1-q-1F, 5 -TTG GAC TTA AAC CAG GAG GAA TAG-3 ; SlPR1a-q-1F, 5 -TCT TGT GAG GCC CAA AAT TC-3 ; SlPR1a-q-1R, 5 ATA GTC TGG CCT CTC GGA CA-3 ; SlPR1b-q-1F, 5 -CCA AGA CTA TCT TGC GGT TC-3 ; SlPR1b-q-1R, 5 -GAA CCT AAG CCA CGA TAC CA-3 ; SlPR5-q-1F, 5 -AAT TGC AAT TTTA ATG GTG C-3 ; SlPR5-q-1R, 5 TAG CAG ACC GTT TAA GAT GC-3 ; SlPR7-q-1F, 5 -AAC TGC AGA ACA AGT GAA GG-3 ; SlPR7-q-1R, 5 -AAC GTG ATT GTA GCA ACA GG-3 ; SlTPK1b-q-1F, 5 -CTG TTA GCA TAG ATG GTG GTG AT-3 ; SlTPK1bq-1R, 5 -CGA AAG TTC CTA GTG GCT GTT TT-3 ; SlMAPKKKε-qR-1F, 5 -GGA GTG TTA AAT GCT AGA CCA GGA A-3 ; SlMAPKKKε-q-1R, 5 -CAC ATG AAG GAT TTG ACG GTT GT-3 . Relative expression was calculated using 2−CT method as described previously [64]. The experiments were repeated independently with three biological replicates using SYBR Green PCR master mix kit (Takara, Dalian, China) in a CFX96 real-time PCR detection system (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s instructions. 2.10. Detection of reactive oxygen species (ROS) Detection of H2 O2 and superoxide anion (O2 − ) in leaf tissues was performed by 3,3-diaminobenzidine (DAB) and nitroblue tetrazolium (NBT) staining, respectively, according to the methods described previously [65,66]. Leaf samples were collected from inoculated plants at 0 and 24 h after inoculation with B. cinerea and dipped into DAB solution (1 mg/ml, pH 3.8) for staining of H2 O2 , or in NBT solution (1 mg/mL nitroblue tetrazolium in 10 mM NaN3 and 10 mM phosphate buffer, pH 7.8) for staining of H2 O2 and superoxide anion, respectively. Photos showing accumulation of H2 O2 and superoxide anion in leaves was taken using a digital camera. 2.11. Oxidative and drought stress assays and chlorophyll content measurement For oxidative stress assays, fully expanded leaves from TRVSlDRW1- or TRV-GUS-infiltrated plants were collected 2 weeks after VIGS infiltration and rinsed with sterile distilled water. Leaf discs (13 mm in diameter) were made by a hole puncher from at least six individual plants for each experiment and were incubated in 1/2 MS buffer supplemented with 20 mM H2 O2 or with H2 O (as a control) for 3 days under illumination condition (200 ␮mol m−2 s−1 ). Measurement of chlorophyll content was performed as described before [67] and the chlorophyll content was calculated according to the formula Chl (A + B) = 5.24A664 + 22.24A648 . For drought stress assays, the

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TRV-SlDRW1- or TRV-GUS-infiltrated plants were allowed for further growth with normal watering regime for 2 weeks after VIGS infiltration and then were subjected to drought stress by stopping watering for a certain period of time until the wilting symptoms were obvious. Measurement of the relative water content (RWC) in leaves was performed as described before [68]. Fully expanded leaves from six individual plants were detached to measure the leaf fresh weight (WF ), turgid leaf weight (WT ) and dry weights (WD ) and RWC were calculated from the equation RWC (%) = (WF − WD )/(WT − WD ) × 100% [68]. All experiments were repeated independently for 3 times. 3. Results 3.1. Characterization of SlDRW1 To identify putative WRKYs that regulate defense response in tomato against necrotrophic fungal pathogens, we examined by VIGS-based assays the possible involvement of six WRKY genes in

defense response against B. cincrea and found that silencing of one WRKY gene (Solyc06g066370) led to increased severity of disease caused by B. cinerea. By contrast, silencing of each of the other five WRKY genes (Solyc03g116890, Solyc08g067340, Solyc06g066370, Solyc09g015770 and Solyc03g095770) did not affect the phenotype of disease caused by B. cinerea (data not shown). Thus, this WRKY gene Solyc06g066370, designated as SlDRW1 for S. lycopersicum defense-related WRKY1, was chosen for further study. The fulllength cDNA of SlDRW1 is 2016 bp with an open reading frame of 1608 bp, which encodes a protein of 535 residues with a calculated molecular mass of 59.6 kDa and a theoretical pI of 7.2. The SlDRW1 protein contains two WRKY domains, a deduced D domain and a SP cluster that is rich in potential MAPK phosphorylation sites (Fig. 1A). In addition, two putative zinc finger motifs located in the WRKY domains and two putative nuclear localization sequences with one in WRKY domain I and another one in the region between two WRKY domains were also identified (Fig. 1A). Phylogenetic tree analysis revealed that SlDRW1 shows 71–93% of identity to potato StWRKY8, pepper CaWRKY2 [69], tobacco NtWRKY8, NbWRKY8

Fig. 1. Sequence alignment and phylogenetic tree analysis of SlDRW1 with other plant WRKY proteins. (A) Alignment of SlDRW1 with NbWRKY8, CaWRKY2 and AtWRKY33. Conserved D domain, SP cluster and two WRKY domains are indicated. Asterisks indicate two putative nuclear localization sequences and arrows indicated cysteines and histidines that may form a zinc finger motif. (B) Phylogenetic tree analysis of SlDRW1 with other plant WRKY proteins. Phylogenetic tree was constructed by neighbour-joining method using MEGA program version 6.05. SlDRW1 in the tree is indicated by an arrow. Plant WRKY proteins used and their GenBank accessions are as follows: Arabidopsis AtWRKY33 (AAM34736), pepper CaWRKY2 (ABD65255), sweet potato SPF1 (BAA06278), Nicotiana benthamiana NbWRKY7 (BAI63295) and NbWRKY82 (AB44539), tobacco NtWRKY1 (BAA82107), NtWRKY6 (BAB61053) and NtWRKY8 (AEX49954) and StWRKY8 (BAI63294).

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[70] and sweet potato IbSPF4 [71]) and also exhibits 50.5% of identity to Arabidopsis AtWRKY33, which is the closest homolog of SlDRW1 in Arabidopsis (Fig. 1B).

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result indicates that the SlDRW1 is localized to nucleus of cells, as expected for a TF. 3.3. SlDRW1 has transactivation activity in yeast

3.2. SlDRW1 is a nuclear protein To determine the subcellular localization of SlDRW1, a SlDRW1GFP fusion construct was generated and infiltrated into leaves of N. benthamiana plants. When transiently expressed, the SlDRW1-GFP fusion protein was localized exclusively in nucleus, while the GFP fluorescence was observed throughout the entire cytoplasm and the nucleus without specific compartment localization (Fig. 2). This

Transactivation activity of SlDRW1 was examined using a yeast assay system. As shown in Fig. 2B, both yeast transformants carrying pBD-SlDRW1 and pBD vector grew well on SD/Trp− medium. However, only yeast transformants containing pBD-SlDRW1 was able to grow on the SD/Trp− His− medium and produced a blue pigment after the addition of x-␣-gal, showing a ␤-galactosidase activity, as expected; whereas transformants containing the pBD

Fig. 2. Subcellular localization and transactivation activity of SlDRW1. (A) Subcellular localization of SlDRW1 when transiently expressed in N. benthamiana leaves. The SlDRW1::GFP and GFP alone constructs were transiently expressed through agroinfiltration in N. benthamiana leaves and green fluorescence of the GFP was viewed under a confocal laser microscopy. The cells were examined under the fluorescence (left), bright field (middle), and as a merged image (right) showing either the diffused (control plasmid) or the nuclear localization of the SlDRW1-GFP fusion protein. Bar = 50 ␮M. (B) Transactivation activity of SlDRW1 in yeast. Yeasts carrying pBD-SlDRW1 or pBD empty vector (as a negative control) were streaked on the SD/Trp− plates (left) or SD/Trp− His− plates (middle) for 3 days at 28 ◦ C. The x-␣-gal was added to the SD/Trp− His− plates and kept at 28 ◦ C for 6 h (right).

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empty vector did not. These results indicate that SlDRW1 has transactivation activity in yeast cells. 3.4. Expression of SlDRW1 in response to B. cinerea and defense signaling hormones To explore the possible involvement of SlDRW1 in tomato disease resistance response, we analyzed the expression dynamics of SlDRW1 in response to infection by different types of pathogens and treatment with defense signaling hormones. As shown in Fig. 3A, the expression of SlDRW1 in mock-inoculated plants maintained unchanged during the experimental period; however, the expression level of SlDRW1 increased dramatically at 24 h post inoculation (hpi), showing an increase of 13 folds over that in mock-inoculated plants. The expression level of SlDRW1 maintained at a very high level until 72 hpi, having approximately 10 folds higher than that in mock-inoculated plants (Fig. 3A). However, similar expression patterns of SlDRW1 in Pst DC3000-inoculated and mock-inoculated plants was observed (Fig. 3B), indicating that Pst DC3000 did not induce expression of SlDRW1. In defense signaling hormonetreated plants, expression of SlDRW1 was induced by JA, showing an increase of approximately one fold over that in control plants at 12 and 24 h after treatment (Fig. 3C). However, SA and ACC did not induce expression of SlDRW1 (Fig. 3C). These results indicate that expression of SlDRW1 can be induced by infection of B. cinerea and treatment with JA. 3.5. SlDRW1 is required for resistance against B. cinerea To explore the possible function of SlDRW1 in disease resistance, we used the TRV-based gene silencing system [62] to knockdown the expression level of SlDRW1 in tomato plants and compared the disease phenotype between the silenced and the control plants. For this purpose, we made TRV-mediated VIGS constructs for SlDRW1 genes and performed standard VIGS procedure on 2-week-old tomato seedlings. In each independent experiment, when >90% of the TRV-PDS-infiltrated plants showed bleacing phenotype, indicating a high level of silencing for the endogenous PDS gene, the TRV-SlDRW1-infiltrated plants in the same batch were used for various experiments 3 weeks after VIGS infiltration. The silencing efficiency for SlDRW1 under our experimental conditions was ∼70% (Fig. 4A), as examined by qRT-PCR analysis of the transcript level in the TRV-SlDRW1-infiltrated plants and compared with that in the TRV-GUS-infiltrated negative control plants. We first examined the disease phenotype of the TRV-SlDRW1infiltrated plants after inoculation with B. cinerea using a detached leaf inoculation assay. Under our disease assay conditions, typical disease symptom, e.g. necrotic lesions, was observed in the leaves from the TRV-SlDRW1- and TRV-GUS-infiltrated plants at 2 dpi but the lesions in the leaves from the TRV-SlDRW1-infiltrated plants expanded much rapidly and were larger than those in the TRV-GUSinfiltrated plants (Fig. 4B). At 4 dpi, the lesion size in the leaves from the TRV-SlDRW1-infiltrated plants showed an average of 6.9 mm, giving an increase of 33% over that in the TRV-GUS-infiltrated plants (average of 5.2 mm for lesion size) (Fig. 4C). Meanwhile, we also explored whether transient expression of SlDRW1 in tomato leaves can confer an increased resistance against B. cinerea. As shown in Fig. 4D, the expression level of SlDRW1 in leaves of plants infiltrated with agrobacteria carrying pFGC-SlDRW1 construct increase significantly at 2 days after infiltration, leading to 3 times higher over that in the control plants infiltrated with agrobacteria carrying pFGCeGFP only. Disease assays revealed that the lesions on leaves of the pFGC-SlDRW1-infiltrated plants were smaller than those on leaves of the pFGC-eGFP-infiltrated plants (Fig. 4E), resulting in a reduction of 30% in size (Fig. 4F). These data indicate that silencing of

Fig. 3. Expression of SlDRW1 in response to pathogen infection and treatment with defense signaling hormones. Four-week-old tomato seedlings were inoculated by spore suspension of B. cinerea (A), bacterial suspension of Pst DC3000 (B) or similar volume of buffer as mock-inoculation control or treated by foliar spraying with 1 mM SA, 100 ␮M MeJA, 100 ␮M ACC solutions or sterilized distill water as a control (C). Leaf samples were collected at different time points after inoculation or treatment as indicated. Total RNA was extracted and used for qRT-qPCR analysis. Data presented are the means ± SD from three independent experiments and * above the error bars or different letters above the columns indicate significant differences at p < 0.05 level.

SlDRW1 resulted in increased disease while transient overexpression led to reduced disease caused by B. cinerea, demonstrating that SlDRW1 plays an important role in resistance against B. cinerea in tomato. We further examined whether SlDRW1 is also involved in resistance against Pst DC3000. In out experiments, no significant difference in disease progress and severity as well as in

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Fig. 4. SlDRW1 is a positive regulator of defense response against B. cinerea. (A–C) Silencing of SlDRW1 led to ehnahced susceptibility to B. cinerea. (A) Silencing efficiency in TRV-SlDRW1-infiltrated tomato plants. Two-week-old seedlings were infiltrated with agrobacteria carrying TRV-SlDRW1 or TRV-GUS and leaf samples were collected 4 weeks after VIGS treatment. The transcript of SlDRW1 was analysed by qRT-PCR. (B and C) Disease phenotype and lesion size on detached leaves of TRV-SlDRW1- or TRV-GUS-infiltrated plants after inoculation with B. cinerea, respectively. (D and E) Transient overexpression of SlDRW1 resulted in increased resistance to B. cinerea. (D) Expression of SlDRW1 in pFGC-SlDRW1- or pFGC-eGFP-infiltrated plants. Three-week-old seedlings were infiltrated with agrobacteria carrying pFGC-SlDRW1 or pFGC-eGFP vector and leaf samples were collected 3 days after infiltration. (E and F), Disease phenotype and lesion size on detached leaves of pFGC-SlDRW1- or pFGC-eGFP-infiltrated plants after inoculation with B. cinerea, respectively. Data presented are the means ± SD from three independent experiments and different letters above the columns indicate significant differences at p < 0.05 level.

bacterial growth in planta between the TRV-SlDRW1- and TRVGUS-infiltrated plants at 4 dpi was observed (Fig. 5), indicating that SlDRW1 does not have a function in resistance against Pst DC3000 in tomato. 3.6. Silencing of SlDRW1 affects defense response against B. cinerea Reactive oxyen species (ROS) has been implicated in plant responses against B. cinerea [72]. We examined whether silencing of SlDRW1 affects accumulation of ROS upon infection of B.cinera. No significant accumulation of H2 O2 and superoxide anion was

detected in leaves of the TRV-SlDRW1- and TRV-GUS-infiltrated plants at 0 h after inoculation (Fig. 6A and B), indicating that silencing of SlDRW1 did not affect accumulation of ROS in tomato plants. At 24 h after inoculation with B. cinerea, similar accumulation of H2 O2 and superoxide anion was observed in leaves of the TRVSlDRW1- and TRV-GUS-infiltrated plants, indicating that silencing of SlDRW1 did not affect the generation of ROS upon infection of B. cinerea (Fig. 6B). We further examined the effect of SlDRW1 silencing on the expression of defense- and signaling-related genes in tomato plants after infection by B. cinerea. For this purpose, we analyzed and compared the expression levels of SlPR1a, SlPR1b, SlPR5, STPK1a and

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observed in leaf discs from the TRV-SlDRW1- and TRV-GUSinfiltrated plants; however, bleaching and chlorosis symptoms in leaf discs from the TRV-SlDRW1-infiltrated plants was much severe than those of the TRV-GUS-infiltrated plants (Fig. 7A). This observation was further confirmed by measuring chlorophyll contents in leaf discs from the TRV-SlDRW1- and TRV-GUS-infiltrated plants after H2 O2 treatments (Fig. 7B). Without H2 O2 treatment, no significant difference in relative chlorophyll contents in leaf discs from the TRV-SlDRW1- and TRV-GUS-infiltrated plants was observed. However, relative chlorophyll contents in leaf discs from the TRV-SlDRW1- and TRV-GUS-infiltrated plants were dramatically decreased after treatments with H2 O2 (Fig. 7B). Notably, relative chlorophyll contents, measuring approximately 5.5% at 5 days after treatment, in leaf discs from the TRV-SlDRW1-infiltrated plants were significantly lower than that, measuring about 35.4%, from the TRV-GUS-infiltrated plants (Fig. 7B). These results indicate that silencing of SlDRW1 attenuates oxidative stress tolerance in tomato plants. In drought stress assays, no difference in appearance and severity of wilting phenotype in the TRV-SlDRW1- and TRV-GUSinfiltrated plants was observed over a 10 days of experimental period (Fig. 8A). Similarly, no difference in relative water contents in leaves of the TRV-SlDRW1- and TRV-GUS-infiltrated plants was observed before and after drought stress treatment (Fig. 8B). These data indicate that SlDRW1 may not participate in drought stress response in tomato plants.

4. Discussion

Fig. 5. SlDRW1 is not required for resistance against P. syringae pv. tomato DC3000. Two-week-old seedlings were infiltrated with agrobacteria carrying TRV-SlDRW1 or TRV-GUS and were inoculated with Pst DC3000 at 2 weeks after VIGS infiltration. Disease phenotype (A) and bacterial population (B) on leaves of TRV-SlDRW1or TRV-GUS-infiltrated plants were recorded or measured. Data presented are the means ± SD from three independent experiments and different letters above the columns indicate significant differences at p < 0.05 level.

SlMAPKKKε in the TRV-SlDRW1- and TRV-GUS-infiltrated plants after infection by B. cinerea. As shown in Fig. 6C, expression levels of SlPR1a and SlPR1b in the TRV-SlDRW1-infiltrated plants were significantly increased, showing 8–10 folds higher than those in the TRV-GUS-infiltrated plants after infection with B. cinerea. However, the expression levels of SlPR7, SlTPK1b and SlMAPKKKε in the TRV-SlDRW1-infiltrated plants were markedly reduced, leading to reduction of 50–73% as compared with those in the TRV-GUSinfiltrated plants after infection with B. cinerea (Fig. 6C). These data indicate that silencing of SlDRW1 affects the expression of a set of genes upon infection with B. cinerea. 3.7. SlDRW1 is required for oxidative stress tolerance To explore whether SlDRW1 has a function in abiotic stress response, we analyzed and compared the oxidative stress tolerance and drought tolerance of the TRV-SlDRW1- and TRV-GUSinfiltrated plants. In oxidative stress assays, leaf discs from leaves of the TRV-SlDRW1- and TRV-GUS-infiltrated plants were treated in H2 O2 solution as an artificial oxidative stress condition. During an experimental period of 5 days, no significant phenotype appeared on the leaf discs from the TRV-SlDRW1- and TRVGUS-infiltrated plants without H2 O2 treatment (Fig. 7A). With the treatment of H2 O2 , bleaching and chlorosis symptom were

Transcriptional regulation of genes in host plants is a critical step that activates inducible defense responses upon pathogen infection. Compared with the facts that extensive studies have been performed and a large number of WRKYs has been implicated in biotic and abiotic stress response in model plant species such as Arabidopsis and rice [6,11–13,42,73–77], little is known about the biological function of the tomato WRKYs. To date, only a few of tomato WRKYs have been studied functionally by overexpression in transgenic tobacco lines or VIGS-based knockdown in tomato [29,56,57]. In this study, we demonstrated that SlDRW1 functions as a positive regulator in defense response against B. cienrea and tolerance against oxidative stress. As a large TF family, members of the WRKY family probably participate in diverse biotic and abiotic stress responses. Our findings presented in this study provide a previously uncharacterized member of the tomato WRKY family with clear functions in biotic and abiotic stress response. The WRKY family members can be classified into three groups according to the number of WRKY domains and the type of the zinc finger motifs [11]. The SlDRW1 protein contains two WRKY domains and two putative zinc finger motifs (Fig. 1A) and shows the highest level of identity to AtWRKY33 among all 72 Arabidopsis WRKYs (Fig. 1B). These structure characteristics and phylogenetic similarity suggest that SlDRW1 belongs to Group I of plant WRKYs [11]. In yeast, SlDRW1 was shown to have transactivation activity (Fig. 2B), which is similar to NbWRKY8 and CaWRKY2, two closest homologs of SlDRW1, having transactivation activity in yeast and in plants, respectively [69,70]. Thus, it is likely that SlDRW1 is a transcriptional activator, like NbWRKY8 and CaWRKY2 [69,70]. Furthermore, phosphorylation of WRKY proteins by MPKs seems to be a critical event for their activity [70,78]. Putative MPK phosphorylation sites were also identified in SlDRW1 protein, raising the possibility that activity of SlDRW1 may be regulated at both transcription and post-transcription levels. Three lines of experimental evidence presented in this study suggest that SlDRW1 is a positive regulator of defense response against B. cinerea. Expression of SlDRW1 was dramatically induced

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Fig. 6. Generation of reactiv oxygen species and expression of defense gene in SlDRW1-silenced plants after infection with B. cinerea. Two-week-old seedlings were infiltrated with agrobacteria carrying TRV-SlDRW1 or TRV-GUS and were inoculated by spraying with spore suspension of B. cinerea at 2 weeks after VIGS infiltration. Leaf samples were collected at 0 (as controls) and 24 h after inoculation for detection of ROS and analyses of defense gene expression. (A and B) Detection of H2 O2 and superoxide anion by DAB and NBT staining, respectively. Representative stained leaves are shown and the experiments were repeated twice with similar results. (C), Expression of defense genes after infection with B. cinerea. Relative expression levels were calculated by comparing with the corresponding values at 0 h after treatment (as a control). At least six leaves from six individual silenced or control plants were used for each experiment. Data presented are the means ± SD from three independent experiments and different letters above the columns indicate significant differences at p < 0.05 level.

by B. cinerea, giving 10–13 folds increase over the mock-inoculated control during 24–72 h after inoculation (Fig. 3A). This is similar to the expression pattern of AtWRKY33, the closest homolog of SlDRW1 in Arabidopsis, in response to B. cinerea [25,79] and the induced expression patterns of CaWRKY2 by different types of pathogens in pepper [69]. Surprisingly, expression of SlDRW1 was not induced by Pst DC3000 (Fig. 3B), differing from that of AtWRKY33, which was significantly upregulated by both virulent and avirulent strains of Pst DC3000 [16,25]. Expression of SlDRW1 in response to signaling hormones also showed different patterns as of AtWRKY33 and CaWRKY2. SlDRW1 only responded to JA but not to SA and ACC (Fig. 3C), while AtWRKY33 was induced by SA and ACC but not by MeJA [25] and CaWRKY2 was induced by all these hormones [69]. The B. cinerea- and JA-inducible expression features imply that SlDRW1 is positively involved in defense response against B. cinerea. In our study, we found that silencing of SlDRW1 resulted in increased severity of disease whereas transient expression of SlDRW1 led to decrease severity of disease cused by B. cinerea (Fig. 4). This is in line with the observations that knockout of AtWRKY33 or knockdown of NbWRKY8, showing high level of identity to SlDRW1 (Fig. 1B), markedly increased severities of diseases

caused by B. cinerea in Arabidopsis [25] and by Phytophthora infestans in N. benthamiana [70]. Interestingly, silencing of SlDRW1 did not affect the phenotype of disease caused by Pst DC3000, as revealed by the disease symptom and bacterial growth in planta (Fig. 5). Similar observation was also obtained for AtWRKY33 [25]. However, overexpression of AtWRKY33 led to enhanced susceptibility to Pst DC3000 [25]. Together, these data suggest that SlDRW1, like its closest Arabidopsis homolog AtWRKY33, is required for disease resistance against B. cinerea. Silencing of SlDRW1 did not alter the accumulation of superoxide anion and H2 O2 in tomato leaves after infection with B. cinerea (Fig. 6A and B), although it was found that infection of B. cinerea could affect the activity of superoxide dismutase and catalase induced by B. cinerea [80,81]. ROS accumulated in late stage of infection has been implicated in susceptible response against necrotrophic fungi like B. cinerea [72]. Thus, it is possible that the function of SlDRW1 in defense response against B. cinerea does not link to ROS generation. In SlDRW1-silenced plants, expression of SlPR1a and SlPR1b was induced significantly but expression of SlPR7, SlTPK1b and SlMKKKε was reduced markedly after infection of B. cinerea (Fig. 6C). The PR1 gene is mainly regulated through

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Fig. 7. Silencing of SlDRW1 reduced tolerance to oxidative stress. Two-week-old seedlings were infiltrated with agrobacteria carrying TRV-SlDRW1 or TRV-GUS and leaf discs were taken from leaves of TRV-SlDRW1- or TRV-GUS-infiltrated plants at 2 weeks after VIGS infiltration. Leaf discs were immerged in 1/2 MS buffer supplemented with 20 mM H2 O2 or H2 O as a control. Phenotype (A) and relative chlorophyll contents (B) in leaf discs from TRV-SlDRW1- or TRV-GUS-infiltrated plants under oxidatve stress. Photos were taken and samples were collected for analysis of chlorohpyll contents at 5 days after treatment. Data presented in (B) are the means ± SD from three independent experiments and different letters above the columns indicate significant differences at p < 0.05 level.

SA-mediated signaling pathway against biotrophic pathogens [82]. The upregulated expression of SlPR1a and SlPR1b might indicate an activated SA-mediated signaling pathway in the SlDRW1-silenced plants after infection of B. cinerea. This is in agreement with the observation that expression of PR1 in atwrky33 mutant plants was induced significantly by infection of B. cinerea [25]. However, expression of SlTPK1b, a regulator of the ethylene (ET)-dependent signaling pathways [63], was downregulated in SlDRW1-silenced plants after infection of B. cinerea (Fig. 6C), indicating that silencing of SlDRW1 attenuates the ET-dependent signaling pathway, which is important for resistance to necrotrophic pathogens [72]. Generally, defense response against necrotrophic fungi is activated through the JA/ET-dependent signaling pathway [72,83]. The downregulated expression of SlTPK1b, together with the JA-induced

expression pattern, may indicate that SlDRW1 is involved in the JA/ET-dependent signaling pathway leading to defense response against B. cinerea. Furthermore, antagonistic effect between the SA- and JA/ET-dependent signaling pathways may exist in the SlDRW1-silenced plants after infection of B. cinerea, as revealed by the expression patterns of SlPR1s and SlTPK1b genes (Fig. 6C). Several Arabidopsis WRKYs including AtWRKY33, the closest Arabidopsis WRKY of SlDRW1, have been shown to mediate the cross-talk between the SA- and JA/ET-dependent signaling pathways [25,79,84]. In addition to the role in biotic stress response, the WRKYs have also been implicated in abiotic stress response [42,74]. We found in this study that silencing of SlDRW1 resulted in reduced tolerance to oxidative stress (Fig. 7), indicating a role for SlDRW1

Fig. 8. SlDRW1 is not involved in drought stress response. Two-week-old seedlings were infiltrated with agrobacteria carrying TRV-SlDRW1 or TRV-GUS and allowed for further growth for another 2 weeks. The TRV-SlDRW1- or TRV-GUS-infiltrated plants were treated for drought stress by stoping watering for a period until wilting symptom was appeared. Phenotype (A) and relative water contents (B) in leaves from the TRV-SlDRW1- or TRV-GUS-infiltrated plants were taken at 5 days after drought treatment. Data presented in (B) are the means ± SD from three independent experiments and different letters above the columns indicate significant differences at p < 0.05 level.

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in regulating oxidative stress response in tomato. It was recently found that overexpression of Arabidopsis WKRY30 and WRKY28 increased oxidative stress tolerance [52,85] whereas overexpression of WKRY25 attenuated oxidative stress tolerance [86]. Thus, it becomes clear that the plant WRKY proteins also participate in oxidative stress response, probably having association with the ROS production during abiotic stress response. However, no difference in accumulation of ROS in SlDRW1-silenced plants grown under normal condition was observed (Fig. 6A and B). The mechanism for SlDRW1 in oxidative stress response needs to be further investigated. Alternatively, although silencing of SlDRW1 did not result in any change in drought stress phenotype (Fig. 8), the involvement of SlDRW1 in drought and other abiotic stress response cannot be simply ruled out before abiotic stress phenotype of stable SlDRW1-overexpressing transgenic plants is carefully examined. For example, mutations in Arabidopsis WRKY25 or WKRY33 did not show clear phenotype in salt stress response whereas overexpression of either gene conferred significant tolerance to salt stress [86]. Acknowledgements This work was supported by the National Basic Research Program of China (2009CB119005), the National Key Technology R & D Program of China (2011BAD12B04), the National High-Tech R & D Program (No. 2012AA101504) and the Research Fund for the Doctoral Program of Higher Education of China (20120101110070). References [1] T. Boller, S.Y. He, Innate immunity in plants: an arms race between pattern recognition receptors in plants and effectors in microbial pathogens, Science 324 (2009) 742–744. [2] P.N. Dodds, J.P. Rathjen, Plant immunity: towards an integrated view of plant–pathogen interactions, Nat. Rev. Genet. 11 (2010) 539–548. [3] J. Zhang, J.M. Zhou, Plant immunity triggered by microbial molecular signatures, Mol. Plant 3 (2010) 783–793. [4] V. Chinnusamy, K. Schumaker, J.-K. Zhu, Molecular genetic perspectives on cross-talk and specificity in abiotic stress signalling in plants, J. Exp. Bot. 55 (2004) 225–236. [5] K. Shinozaki, K. Yamaguchi-Shinozaki, Gene networks involved in drought stress response and tolerance, J. Exp. Bot. 58 (2007) 221–227. [6] T. Eulgem, I.E. Somssich, Networks of WRKY transcription factors in defense signaling, Curr. Opin. Plant Biol. 10 (2007) 366–371. [7] N. Gutterson, T.L. Reuber, Regulation of disease resistance pathways by AP2/ERF transcription factors, Curr. Opin. Plant Biol. 7 (2004) 465–471. [8] F. Licausi, M. Ohme-Takagi, P. Perata, APETALA2/ethylene responsive factor (AP2/ERF) transcription factors: mediators of stress responses and developmental programs, New Phytol. 199 (2013) 639–649. [9] M. Nuruzzaman, A.M. Sharoni, S. Kikuchi, Roles of NAC transcription factors in the regulation of biotic and abiotic stress responses in plants, Front Microbiol. 4 (2013) 248. [10] M.S. Alves, S.P. Dadalto, A.B. Gonc¸alves, G.B. De Souza, V.A. Barros, L.G. Fietto, Plant bZIP transcription factors responsive to pathogens: a review, Int. J. Mol. Sci. 14 (2013) 7815–7828. [11] T. Eulgem, P.J. Rushton, S. Robatzek, I.E. Somssich, The WRKY superfamily of plant transcription factors, Trends Plant Sci. 5 (2000) 199–206. [12] P.J. Rushton, I.E. Somssich, P. Ringler, Q.J. Shen, WRKY transcription factors, Trends Plant Sci. 15 (2010) 247–258. [13] B. Ulker, I.E. Somssich, WRKY transcription factors: from DNA binding towards biological function, Curr. Opin. Plant Biol. 7 (2004) 491–498. [14] R. Ramamoorthy, S.Y. Jiang, N. Kumar, P.N. Venkatesh, S. Ramachandran, A comprehensive transcriptional profiling of the WRKY gene family in rice under various abiotic and phytohormone treatments, Plant Cell Physiol. 49 (2008) 865–879. [15] S. Berri, P. Abbruscato, O. Faivre-Rampant, A.C. Brasileiro, I. Fumasoni, K. Satoh, S. Kikuchi, L. Mizzi, P. Morandini, M.E. Pè, P. Piffanelli, Characterization of WRKY co-regulatory networks in rice and Arabidopsis, BMC Plant Biol. 9 (2009) 120. [16] J. Dong, C. Chen, Z. Chen, Expression profiles of the Arabidopsis WRKY gene superfamily during plant defense response, Plant Mol. Biol. 51 (2003) 21–37. [17] M. Kalde, M. Barth, I.E. Somssich, B. Lippok, Members of the Arabidopsis WRKY group III transcription factors are part of different plant defense signaling pathways, Mol. Plant Microbe Interact. 16 (2003) 295–305. [18] Z. Lai, K. Vinod, Z. Zheng, B. Fan, Z. Chen, Roles of Arabidopsis WRKY3 and WRKY4 transcription factors in plant responses to pathogens, BMC Plant Biol. 8 (2008) 68.

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