Characterizing the Role of TaWRKY13 in Salt Tolerance

The WRKY transcription factor superfamily is known to participate in plant growth and stress response. However, the role of this family in wheat (Triticum aestivum L.) is largely unknown. Here, a salt-induced gene TaWRKY13 was identified in an RNA-Seq data set from salt-treated wheat. The results of RT-qPCR analysis showed that TaWRKY13 was significantly induced in NaCl-treated wheat and reached an expression level of about 22-fold of the untreated wheat. Then, a further functional identification was performed in both Arabidopsis thaliana and Oryza sativa L. Subcellular localization analysis indicated that TaWRKY13 is a nuclear-localized protein. Moreover, various stress-related regulatory elements were predicted in the promoter. Expression pattern analysis revealed that TaWRKY13 can also be induced by polyethylene glycol (PEG), exogenous abscisic acid (ABA), and cold stress. After NaCl treatment, overexpressed Arabidopsis lines of TaWRKY13 have a longer root and a larger root surface area than the control (Columbia-0). Furthermore, TaWRKY13 overexpression rice lines exhibited salt tolerance compared with the control, as evidenced by increased proline (Pro) and decreased malondialdehyde (MDA) contents under salt treatment. The roots of overexpression lines were also more developed. These results demonstrate that TaWRKY13 plays a positive role in salt stress.


Introduction
Unlike animals, plants cannot move when exposed to stress. However, complex signaling network have been established to cope with stress [1]. Under stress, a series of responses are induced to prevent or minimize damage. These are accompanied by many physiological, biochemical and developmental changes [2]. Current research on plant stress response has reached the level of cells and molecules, and combined with genetics, we can explore the stress responsive mechanisms in order to improve plant growth under conditions of stress [3][4][5][6][7].

Identification and Genome Structure Analysis of WRKYs in Triticum aestivum
According to the Plant Transcription Factor Database website (http://planttfdb.cbi.pku.edu.cn/ index.php), wheat has 171 TaWRKYs, which are distributed across all chromosomes (1AL, 1BL, 1DL, 2AL, 2AS, 2BS, 2DL, 2DS, 3AL, 3B, 3DL, 4AL, 4AS, 4DS, 5AL, 5BS, 5BL, 5DL, 6AL, 6AS, 6BS, 6DS, 7AL, 7DL). Here, PF03106 was used as a key word to blast WRKYs in wheat on the Phytozome website (https://phytozome.jgi.doe.gov/pz/portal.html). Nucleic acid and amino acid sequences of 100 TaWRKYs that harbor at least one WRKY domain are shown in Supplementary Table S2. Based on the rule that the CDS of TaWRKYs were more than 300 base pairs [30], some TaWRKYs were removed, and then combined with the NCBI database (https://www.ncbi.nlm.nih.gov/pubmed), meaning that 57 TaWRKYs were identified with the annotation gene's name, ID, transcript name and location ( Table 1). The location of 57 TaWRKYs on chromosomes was analyzed by using the online website http://mg2c.iask.in/mg2c_v2.0/. From the map, we can see that the locations of TaWRKYs were different on each chromosome; for example, TaWRKY6, 38,50,27,48, and 57 were located at the end of chromosome 3B (forward or reverse), while TaWRKY70 and TaWRKY 71 were located near the centromere of chromosome 1D. Moreover, the distribution of TaWRKYs on 4A was the combination of both distributions described above (Figure 1). To further explore gene structure differences, a gene structure figure of 56 TaWRKYs is displayed in Figure 2. The TaWRKYs are all different in structure. Most TaWRKYs contain 1 to 5 different exons, which may contain different functional structures, such as zinc finger, leucine, kinase structure, exerting different biological functions. TaWRKY7, 22,23,24,33,56, and 90 do not harbor introns, only containing exons and/or an upstream structure.

Identification and Biological Analysis of TaWRKY13
To find wheat stress-responsive genes under salt stress, the roots of three-leaf wheat seedlings were immersed in 150 mM NaCl solution for 1 h. Control_Leaf represents the leaf tissue without NaCl treatment, NaCl_Leaf represents the leaf tissue treated as per the above description; each treatment involved two independent replicates which were then sampled for RNA-seq (Supplementary Table S1). Twelve TaWRKYs (TaWRKY4, 9, 12, 13, 15, 22, 29, 33, 34, 44, 53, and 70) were selected based on the rule log 2 (NaCl_Leaf /Control_Leaf) > 2. As shown in Figure 3, TaWRKY13 gave the highest relative expression in response to salt stress, peaking at more than 20-fold at 1 h. TaWRKY13 (ID: 31962353, Traes_2AS_6269D889E.1) was selected for further investigation. TaWRKY13 contained a 975 bp open reading frame (ORF) encoding 324 amino acids; the molecular weight of the protein was 81.02 kDa with pI 4.99 (https://web.expasy.org/protparam/). The predicted amino acid sequence showed that TaWRKY13 only harbored one WRKY domain with a highly conserved WRKYGQK motif and a CX4-5CX22-23HXH zinc-finger motif.

Phylogenetic Analysis of AtWRKYs, OsWRKYs and TaWRKYs
Phylogenetic analysis is a useful method that can provide some clues to the possible functions of predicted or analyzed target genes. It would be useful to know the homologs of Triticum aestivum WRKYs (TaWRKYs), especially TaWRKY13, with WRKYs of Arabidopsis thaliana (AtWRKYs) and WRKYs of Oryza sativa (OsWRKYs) with reference to previous results. A phylogenic tree was constructed by the neighbor-joining method [39] to investigate the evolutionary relationships between AtWRKYs, OsWRKYs and TaWRKYs. There are 398 WRKYs for phylogenetic analysis (90 AtWRKYs, 128 OsWRKYs and 171 TaWRKYs) ( Figure 4). According to Figure 4, AtWRKYs, OsWRKYs and TaWRKYs were scattered across different branches of the phylogenic tree, and all WRKYs were divided into three broad categories; among them, there were more WRKYs in groups I and II than in group III. TaWRKY13 (ID: Traes_2AS_6269D889E.1) and AtWRKY13 (ID: AT4G39410) were in group II, and OsWRKY13 (ID: LOC-Os01g546600) belonged to group I. The results of phylogenetic analysis preliminarily indicated that TaWRKY13 has a closer homology with AtWRKY13 than OsWRKY13.

TaWRKY13 was Localized in the Nucleus
To investigate the biological activity of TaWRKY13, the coding sequence fused to the N-terminus of the green fluorescent protein (GFP) was inserted into wheat mesophyll protoplasts by the PEG-mediated method. As the control, 35S::GFP was transformed [40]. The fluorescence of the control GFP was distributed throughout the cells, whereas the fluorescence of 35S::TaWRKY13-GFP was specifically localized in the nucleus ( Figure 5). Thus, TaWRKY13 is a nuclear-located protein.

Tissue-Specific Expression of TaWRKY13
Studies of genes with a specific expression in different tissues are necessary to understand the regulatory mechanisms of plant growth and development and the relationship between cell type and function. Here, the promoter sequence of TaWRKY13 was fused to the pCAMBIA1305 vector, which contains a β-glucuronidase (GUS) reporter gene in the N-terminus ( Figure 6). The GUS reporter gene can preliminarily determine the tissue specificity of the gene by observing the tissue location with a blue color after staining [41]. qRT-PCR was used to further verify the relative expression level at the molecular level. TaWRKY13 was expressed in the roots, stems and leaves of T 3 generation transgenic Arabidopsis plants under normal and salt-stress conditions, with the relative expression in roots being higher than in leaves and stems. After NaCl treatment, the expression levels in roots, stems and leaves were significantly increased, indicating that TaWRKY13 might be responsive to salt stress. All data are means ± SDs of three independent biological replicates. The ANOVA demonstrated significant differences (* p < 0.05, ** p < 0.01).

TaWRKY13 Is Involved in Various Stress Responses
WRKY proteins are reported to be involved in various biotic and abiotic stresses [25]. Expression pattern analyses were conducted to determine whether TaWRKY13 was responsive to abiotic stresses. The results indicated that TaWRKY13 participated in salt PEG, ABA and cold-stress responses (Figure 7). For PEG treatment, the relative expression level of TaWRKY13 was rapidly induced at 1 h after the imposition of PEG stress ( Figure 7A). After NaCl treatment for 1 h, TaWRKY13 was highly induced at a maximum level of about 22-fold ( Figure 7B). Exogenous ABA and cold stress also significantly affected the expression of TaWRKY13 ( Figure 7C,D). The rapid increase in relative expression levels of TaWRKY13 following different stress treatments indicated an important role at the initial stages of stress response. Error bars represent standard deviations (SDs). All data are means ± SDs of three independent biological replicates. The ANOVA demonstrated significant differences (** p < 0.01).

Stress-Related Regulatory Elements in the Promoter of TaWRKY13
The 1.856 kb promoter region upstream of the TaWRKY13 ATG start codon was isolated to gain an insight into the regulatory mechanism. We searched for putative cis-acting elements in the promoter regions using the database PLACE (http://www.dna.affrc.go.jp/PLACE/). The results are shown in Table 2. Numerous stress-related regulatory elements were present, including a W-BOX, MYB element and TATA-BOX, which take part in the response to both drought and high-salt stress, as well as low-temperature responsive (LTR), ABA-responsive element (ABRE) and GT1, which mainly participate in salt-stress response. Moreover, there were various light, gibberellin, SA (salicylic acid) and high-temperature responsive elements, indicating that TaWRKY13 is involved in abiotic stress response and plant hormone-related signal transduction.

Root System Analysis Indicates That Overexpression Lines Respond to Salt Stress in Arabidopsis
To explore the mechanism of TaWRKY13 under salt stress, a pCAMBIA1302-TaWRKY13 (35S::TaWRKY13) vector was constructed and transformed into Arabidopsis for root length assay [40]. The results of the identification of homozygotes by agarose gel electrophoresis (AGE) and the selection of three transgenic lines (35S::TaWRKY13#1, #2, #3) by RT-qPCR are available in Supplementary Figure S1. Seedlings of control (Columbia-0) and three T 3 generation overexpression lines were first grown on MS (Murashige & Skoog) medium for one week and then transplanted to MS medium supplemented with various NaCl concentrations (0, 100, 120 mM) for salt treatment. As shown in Figure 8, the overexpression lines have an advantage in terms of the main root length and total surface area compared to Col-0 under NaCl treatment.

TaWRKY13 Overexpression Response to Salt Stress in Oryza sativa
Two-week-old T 3 rice lines seedlings of the control (Nipponbare) and three overexpression lines (35S::TaWRKY13#1, #2, #3) were grown hydroponically in untreated control solution or in the same solution supplemented with 150 mM NaCl to explore the physiological tolerance of TaWRKY13 overexpression rice lines to salt stress [42]. The verification of homozygotes and the selection of three transgenic lines were conducted by AGE and RT-qPCR, respectively ( Figure 9A,B). As shown in Figure 9C, before NaCl treatment, both Nipponbare and the three transgenic lines showed similar growth patterns, with no or little difference in plant height, root length, and proline (Pro) and malondialdehyde (MDA) contents. After 7 days of NaCl treatment, both Nipponbare and the overexpression lines showed leaf shedding ( Figure 9D). Compared with the transgenic lines, Nipponbare plants showed evidence of wilting, water loss and yellowing, whereas the transgenics lines showed less severe symptoms. Meanwhile, the overexpression of TaWRKY13 increased the proline content and decreased MDA content under NaCl treatment ( Figure 9E,F). The root length of Nipponbare was significantly lower than for transgenic plants; the surface areas of transgenic plants were higher than for Nipponbare ( Figure 9G,H). These results indicated that the overexpression of TaWRKY13 enhanced salt tolerance in rice.

Discussion
Regarded as one group among many important transcription factors in plants, WRKY TFs are represented by 90 members in Arabidopsis and more than 100 in rice [43]. The functions of WRKY TFs have been studied in detail in various plant species since their first discovery.
Since the application of transcriptome sequencing technology, researchers have sequenced the genome of wheat [44,45]. However, owing to the large and complex genome of heterohexaploid wheat, the task has posed many challenges [46]. Recently, transgenic Arabidopsis plants of TaWRKY2 and TaWRKY19 have shown improved stress tolerance, and the overexpression of TaWRKY2 and TaWRKY19 has exhibited salt, osmotic/dehydration and freezing stress tolerance [47]. More than 160 TaWRKYs were characterized according to their sequence alignment, motif type and phylogenetic relationship analysis by Sezer et al. [48]. Although the WRKY genes associated with stress can be identified by transcriptome sequencing and family analysis, functional identification and mechanism analysis in wheat is limited. Salt stress is one of the most serious stresses that cannot be reversed after damage [49].
Here, on the basis of the previous research, combining RNA-Seq, real-time quantitative PCR (RT-qPCR), and the latest wheat database, TaWRKY13 was isolated from the wheat genome for further study. RNA-Seq was conducted first (Supplementary Table S1); meanwhile, using the wheat database, 57 TaWRKY genes were annotated ( Table 1). The results showed that TaWRKYs were differently distributed (number and location) on wheat chromosomes (Figure 1). Studies of the genome structure and the phylogenetic analysis of TaWRKY genes were initially difficult, because the wheat genome was too complex for statistical analysis; there were 171 TaWRKY genes according to the database (https://phytozome.jgi.doe.gov/pz/portal.html). Based on the rule that the CDS of TaWRKYs were more than 300 base pairs, we removed redundant TaWRKY genes and, combined with the NCBI database (https://www.ncbi.nlm.nih.gov/pubmed), 56 TaWRKY genes were selected for the analysis of the gene structure ( Figure 2). Major TaWRKY genes harbored different CDS and binding motifs responsible for special function; for example, TaWRKY1 contained an N-terminal CUT domain and a C-terminal NL domain [30]. To further explore TaWRKY genes that respond to salt stress, 12 TaWRKY genes were chosen for verification by qRT-PCR ( Figure 3). All 12 genes were up-regulated under salt stress, and TaWRKY13 was chosen for further study due to its higher expression level under salt treatment. Phylogenetic analysis demonstrated that TaWRKY genes have different evolutionary relationships and homologies to WRKYs in Arabidopsis and rice ( Figure 4); compared to OsWRKY13, AtWRKY13 was closer to TaWRKY13, possibly indicating similar biological functions [50]. For OsWRKY13, the non-conservation of evolution may provide a basis for the subsequent functional identification of TaWRKY13 in rice, in that the influence of rice itself in OsWRKY13 was eliminated. Subcellular localization showed that TaWRKY13 is a nuclear protein ( Figure 5) which may mainly be involved in nuclear signal transduction [51,52]. Although many cotton (Gossypium hirsutum) WRKY genes were expressed at low levels during development, a few GhWRKYs expressed highly in specific tissues such as roots, stems, leaves and embryos [53]. Our results showed that TaWRKY13 was expressed in roots, stems and leaves in transgenic lines, the relative expression level of roots was higher than stems and leaves in transgenic lines, and under salt-stress conditions, the relative expression level was double that of the normal condition ( Figure 6).
An increasing number of studies have shown that WRKY TFs play important roles in abiotic stress response; for instance, the overexpression of GmWRKY21 improved cold tolerance in Arabidopsis, because of the regulation of DREB2A and STZ/Zat10. GmWRKY54 conferred salt and drought tolerance; GmWRKY13, which was insensitive to ABA (abscisic acid) but markedly sensitive to salt and mannitol, may function in both lateral root development and the abiotic stress response [54]. Expression pattern analyses revealed that TaWRKY13 was induced significantly by PEG, salt, low-temperature and ABA (Figure 7). Compared with PEG, low-temperature, and ABA stress, TaWRKY13 achieved the highest relative expression level under salt treatment, which was in accordance with the following root length assay in Arabidopsis and the rice resistance assay. Products of WRKY TFs bind to specific cis-regulatory sequences such as the W-BOX in the promoter to induce the expression of downstream target genes [55]. Many regulatory cis-elements that are responsive to drought (W-BOX, MYB and TATA-BOX), high salt (LTR, ABRE and GT1), SA (salicylic acid, WRKY) and cold were recognized in the TaWRKY13 promoter, showing that TaWRKY13 is capable of responding to stress ( Table 2). WRKY13 participated in various physiological processes; for example, a weaker stem phenotype, reduced sclerenchyma development, and altered lignin synthesis were observed in an AtWRKY13 mutant, showing that it functioned in stem development [56]. When AtWRKY13 was disturbed under short-day conditions, AtWRKY13 promoted flowering [57]. Furthermore, WRKY13 was also involved in the cross talk between abiotic and biotic stress signaling pathways, and OsWRKY13 displayed selective binding to different cis-elements to regulate various stress [58]. In this study, a root length assay of overexpression lines was conducted in Arabidopsis for an analysis of the stress tolerance of TaWRKY13; overexpression lines had longer root lengths and a higher total root area than Col-0 ( Figure 8A-C). Additionally, the overexpression of TaWRKY13 enhanced salt tolerance in transgenic rice (Figure 9). Under NaCl treatment, the transgenic lines of TaWRKY13 grew vigorously, whereas Nipponbare seedlings were more wilted and yellow ( Figure 9D); the transgenic lines also had higher proline (Pro) and reduced malondialdehyde (MDA) contents ( Figures 8F and 9E ) under NaCl treatment. In addition, the roots of transgenic lines were longer and more developed than Nipponbare ( Figure 9G,H). These results all showed that TaWRKY13 was responsive to salt stress, in agreement with data from other species [54,56,58]. In accordance with the present study, the results suggested that TaWRKY13 has a potential role in improving salt tolerance in wheat. These results are only preliminary in exploring the putative role of TaWRKY13 in salt tolerance; more researches about the role and regulation mechanism of TaWRKY13 are still needed in wheat. For instance, based on the above findings, TaWRKY13 was transformed into wheat for functional verification and mechanism analysis to further improve the role of TaWRKY13 in wheat stress tolerance pathways.

De Novo Transcriptome Sequencing of Salt-Treated Wheat
Wheat (Triticum aestivum L. cultivar Jinhe 9123, from the Institute of Genetics and Physiology, Hebei Academy of Agriculture and Forestry Sciences, Shijiazhuang, China) was cultivated in a 10 cm × 10 cm pot (vermiculite:soil, 1:3) supplemented with Hoagland's liquid medium at 22 • C under a 16 h light/8 h darkness photoperiod for 10 days. When the wheat seedlings were at the three-leaf stage, the pots were immersed in 150 mM NaCl solution and water (control) for 1 h, respectively [30], prior to the sampling of 0.1 g fresh leaf tissue. Samples were submerged immediately in liquid nitrogen and stored at −80 • C for RNA-Seq. The experiment was performed in three independent replications. In Supplementary  Table S1, Control_Leaf means a sample without NaCl treatment, and NaCl_Leaf means a sample with salt treatment; each treatment involved three independent replicates, which were then sampled for RNA-Seq. Data are shown in Supplementary Table S1.

Structure Analysis and Phylogenetic Analysis of TaWRKYs
According to the information listed in Table 1, the chromosome location of TaWRKYs was analyzed by using the online website http://mg2c.iask.in/mg2c_v2.0/. For gene structure analysis, the data of TaWRKYs that were identified in Section 4.2 were uploaded to GSDS (http://gsds.cbi.pku.edu.cn/) to obtain the map of the TaWRKYs' structure. For phylogenetic analysis, a tree of WRKYs from wheat, rice and Arabidopsis was constructed using the neighbor-joining method in MEGA 6.0 with 1000 bootstrap replications [39]. Data for gene structure and the phylogenetic tree analysis were downloaded from PlantTFDB (http://planttfdb.cbi.pku.edu.cn/) and are shown in Supplementary  Tables S2 and S3.

RNA Extraction of Stress Treatments and RT-qPCR Analyses
Wheat seeds were sown as previously described; vermiculite and soil were removed by water after being grown for 10 days, and the fresh leaf tissue of three-leaf-stage wheat seedlings were used for the RNA extraction of different stress treatments. For the identification of TaWRKY responses to salt stress, the seedlings roots were immersed in 150 mM NaCl solution, and 0.1 g of fresh leaf tissue was sampled at different times (0, 0.5, 1, 2, 4, 8, 12 and 24 h). For the expression pattern analyses, the roots of wheat seedlings were immersed in 10% PEG6000, 150 mM NaCl and 100 µmol·L −1 ABA solutions. Wheat seedlings for cold treatment were placed in a 10 h light/14 h darkness, 4/2 • C chamber and sampled at different periods (0, 1, 6 and 24 h) [30,59,60]. For specific tissue expression assays, T 3 generation transgenic Arabidopsis (35S::pTaWRKY13) plants were surface-sterilized with 10% Chloros and washed three times with sterile water. Sterilized seeds were sown on MS (Murashige & Skoog) medium, vernalized in darkness for 3-4 days at 4 • C, then grown in a chamber at 22 • C and 75% humidity under a 16 h light/8 h darkness photoperiod for one week. The seedings were transplanted to soil (vermiculite:soil, 1:3), 0.1 g fresh roots, stems and leaves tissue of 10-day-old transgenic Arabidopsis seedlings with or without 150 mM NaCl treatment were sampled for RNA the extraction of different tissues [56].
All samples after collection were submerged immediately in liquid nitrogen and stored at −80 • C for RNA extraction using an RNA prep plant kit (TIANGEN, Beijing, China); cDNA was synthesized using a Prime Script First-Strand cDNA Synthesis Kit (TransGen, Beijing, China) following the manufacturer's instructions. RT-qPCR was performed with Super Real PreMix Plus (TransGen, Beijing, China) on an ABI Prism 7500 system (Applied Biosystems, Foster city, CA, USA). Specific primers for TaActin, AtActin and TaWRKY4, 9,12,13,15,22,19,33,34,44,53 and 70 for RT-qPCR are listed in Supplementary Table S4. Three biological replicates were used for RT-qPCR analysis, and the 2 −∆∆Ct method was used for quantification.

Gene Isolation and Subcellular Localization
The ORF (open reading frame) of TaWRKY13 was amplified by PCR with specific primers from wheat cDNA (cultivar Jinhe 9123). The PCR product was fused into pZeroBack vector (TIANGEN, Beijing, China) and sequenced for further study. The correct sequencing plasmids were treated as templates, the segment with restriction sites was amplified by specific primers, and the PCR product was inserted into the N-terminus of the green fluorescent protein (GFP) containing the CaMV35S promoter for subcellular localization; the 35S::GFP vector was used as the control. Both 35S::GFP and 35S::TaWRKY13-GFP were transferred into wheat mesophyll protoplasts by the PEG-mediated method [29]. A confocal laser scanning microscope (LSM700; CarlZeiss, Oberkochen, Germany) was used to observe the fluorescence after incubation in darkness at 22 • C for 18-20 h. All primers are listed in Supplementary Table S4.

Tissue-Specific Expression of TaWRKY13 and GUS Staining
Tissue-specific expression analysis of TaWRKY13 was conducted by two methods. In the first one, the CDS of TaWRKY13 was amplified as described in Section 4.5, then cloned into the pCAMBIA1302 vector; then, the infected inflorescence of Arabidopsis was determined by the Agrobacterium-mediated method [61], grown as described in Section 4.4, until T 3 generation transgenic Arabidopsis seeds were obtained. The identification of homozygotes and selection of three transgenic lines were conducted by agarose gel electrophoresis and RT-qPCR, respectively [59]. The transgenic Arabidopsis seedlings with or without NaCl (150 mM) treatment were used for RT-qPCR as described in Section 4.4. In the second method, promoter fragments of TaWRKY13 (pTaWRKY13) were obtained from Ensemble Plants (plants.ensembl.org/index.html); the pTaWRKY13 was amplified by PCR with specific primers from wheat cDNA (Jinhe 9123), and the PCR product was fused into pLB vector (TIANGEN, Beijing, China) and sequenced. The fragment of TaWRKY13 promoter was cloned to the pCAMBIA1305 vector harboring a β-glucuronidase (GUS) tag, obtaining the T 3 generation transgenic Arabidopsis seeds as per the previous description. T 3 generation transgenic Arabidopsis (35S::pTaWRKY13) seeds were surface-sterilized, sown on MS medium, vernalized, and grown in a chamber at 22 • C and 75% humidity under a 16 h light/8 h darkness photoperiod for one week as described in Section 4.4. Ten-day-old transgenic Arabidopsis seedlings were submerged to 150 mM NaCl solution for 1 h. After salt treatment, the liquid was drained with filter paper, and the plant material was subjected to GUS staining solution supplemented with 5-bromo-4-chloro-3-indolylb-d-glucuronic acid (X-gluc) for 3 h; 70% (vol/vol) ethanol was used to remove the chlorophyll following the manufacturer's protocol (Real-Times, Beijing, China) [56]. GUS staining was observed by a Leica microscope (Wetzlar, Germany). Primers are listed in Supplementary Table S4. 4.7. Cis-Acting Elements in the TaWRKY13 Promoter A 1.856 kb promoter fragment upstream of the ATG start codon of TaWRKY13 was obtained from the Ensemble Plants website (http://plants.ensembl.org/index.html). Cis-acting elements that respond to various stresses in the promoter region were analyzed by PLACE (http://www.dna.affrc.go. jp/PLACE/) [29].

Root Growth Assays of TaWRKY13 under Salt Stress in Arabidopsis
T 3 generation transgenic Arabidopsis lines were obtained as previously described (Section 4.6). Seeds of Col-0 and transgenic lines (35S::TaWRKY13#1, #2, #3) were surface-sterilized, sown on MS medium, vernalized, grown in a chamber at 22 • C and 75% humidity under a 16 h light/8 h darkness photoperiod for one week as described above (Section 4.4). Three ten-day-old Arabidopsis seedlings (Col-0 and transgenic lines) were transferred to MS medium containing different concentrations of NaCl (0, 100, 120 mM) for one week [40].

Generation of Transgenic Rice and Stress Identification of TaWRKY13 to Salt Tolerance
Plant expression vector pCAMBIA1305-TaWRKY13 was constructed and transformed to competent cells of EHA105 as previously described [30]. Genetic transformation was conducted by Dr Chuan-Yin Wu and colleagues at the Institute of Crop Science, Chinese Academy of Agricultural Sciences using the agrobacterium-mediated method, and Nipponbare was used as the control [62]. The selection of three transgenic lines was made by agarose gel electrophoresis and RT-qPCR, respectively, as previously described ( Figure S1). T 3 generation transgenics (35S::TaWRKY13#1, #2, #3) and Nipponbare were used for further study. Rice seeds were treated with 0.7% hydrogen peroxide for one day for surface sterilization, breaking dormancy and promoting germination, then replaced with 0.7% hydrogen peroxide with water and germinated at 37 • C for 3 days (changing the water once a day). When seeds showed white buds, bare seeds were transplanted to 96-well plates (24 seeds of Nipponbare and 35S::TaWRKY13#1,#2,#3, respectively) and placed in a growth chamber at 28 • C and a 16 h light/8 h darkness photoperiod and 70% relative humidity for the hydroponic culture. Seedings were cultured in water for one week, then cultured in water supplemented with Hoagland's hydroponic culture solution. The culture solution was replaced every 5 days, and the pH was set at 5.5 [63]. Three-leaf seedlings were treated. For salt treatment, the 96-well plates growing three-leaf stage seedlings were transferred to YS hydroponic culture solution and a YS hydroponic culture solution supplemented with 150 mM NaCl for several days until phenotypes appeared [62]. For each salt treatment, there were three independent replicates. Primers are listed in Supplementary Table S4.

Measurements of Proline (Pro) and Malondialdehyde (MDA) Contents
To better understand the function of TaWRKY13 under salt treatment, proline and MDA contents were measured with Pro and MDA assay kits (Comin, Beijing, China) based on the manufacturer's protocols. Main root lengths and total surface areas of Arabidopsis and rice roots were measured by the WinRHIZO system (Hang xin, Guangzhou, China). Measurements were made on all three biological replicates; means ± SD and statistically significant differences were based on the ANOVA (* p < 0.05, ** p < 0.01).

Conclusions
We identified the salt-induced WRKY gene TaWRKY13 (ID: 31962353) from a wheat RNA-Seq database (https://phytozome.jgi.doe.gov/pz/portal.html) and real-time quantitative PCR (RT-qPCR). TaWRKY13 is a nuclear protein that was expressed in the roots, stems and leaves of transgenic Arabidopsis. TaWRKY13 was responsive to PEG, salt, cold, and exogenous abscisic acid (ABA) treatment. The overexpression of TaWRKY13 was responsive to salt stress in both Arabidopsis and rice, as evidenced by the promotion of root length and the total root surface area. These results provide a basis for further understanding the functions of TaWRKY13 in wheat when subjected to salt stress.   Acknowledgments: We thank Li-Na Ning for critically reading the manuscript.

Conflicts of Interest:
The authors declare no conflict of interest.

ROS
Reactive oxygen species SnRK2 Sucrose