Genome-wide identification, phylogeny and expression analyses of group III WRKY genes in cotton (Gossypium hirsutum)

Abstract Cotton (Gossypium hirsutum) is an important economic crop, so it is of great significance to analyze its molecular mechanisms of resistance to Verticillium Wilt. The WRKY gene family encodes transcription factors (TFs) involved in plant resistance to biotic and abiotic stress. Here, we identified 17 group III WRKY genes in cotton based on the cotton genome project. The phylogenetic relationship and evolutionary of WRKY Group III TFs in Arabidopsis thaliana and G. hirsutum showed that all the WRKY Group III TFs were divided into two clades. Overall, Group III WRKYs with similar motif patterns showed a tendency to cluster into the same group in the phylogenetic tree. Elements related to jasmonic acid (JA) and salicylic acid (SA) regulation were identified in the promoter region of most WRKY Group III genes. The expression levels of five Group III genes were up-regulated more than two-fold and three genes were down-regulated after Verticillium dahliae Kleb. infection. Moreover, three group III GhWRKY genes with JA or SA regulatory element in the promoter region could be significantly induced by JA or SA, respectively. Subcellular localization and transcriptional activation analysis in yeast showed that Cot_14615 protein localized to the nucleus of mesophyll cells in Nicotiana tabacum and had transcription activation function. Our data on cotton group III WRKY genes can serve as a basis for further studies into their potential function in cotton resistant to Verticillium Wilt.


Introduction
Cotton (Gossypium spp.), the world's leading natural fiber crop and the second largest oil crop, is affected by a series of biotic and abiotic stresses in the process of growth. This results in decreased yield and fiber quality. Among the various stresses, Verticillium wilt, caused by the soil-borne fungal pathogen Verticillium dahliae Kleb., is one of the most serious soil-born diseases in cotton. Verticillium wilt causes severe cotton yield reductions and losses [1]. V. dahliae first adheres to the cotton root surface, and its hyphae penetrate the roots and colonize the cortex. It is usually considered that Verticillium wilt is a vascular disease transmitted through the cotton vascular tissue of roots only. According to new research, the vascular tissue is not the sole transmission route of V. dahliae in cotton plants. V. dahliae can infect any organ of upland cotton plants and then spread to the entire plant from the infected organ through the surface and interior of the organ [2]. Therefore, V. dahliae infection-induced damages have also been exacerbated because of very broad host range and diversity of means of spread.
Plants are frequently stimulated in adverse environmental conditions. Being sessile in nature, plants have evolved specific defensive mechanisms to deal with biotic and abiotic stresses. In this process, transcription factors (TFs) play an important role by regulating gene expression, which is a major part of the complex signal transduction networks of defensive mechanisms [3]. In the plant TFs family, several families are especially bound with biotic and abiotic stresses, including the WRKY, NAC and MYB gene families named according to their domain structures [4][5][6][7]. The WRKY gene family is the second largest family after the NAC gene family. WRKYs play a central role not only in plant growth and development but also in plant stress responses [4]. WRKY TFs have conserved WRKY domains composed of approximately 60 amino acids with 'WRKYGQK' , and can be classified into three main categories: group I, II and III according to the number and characteristics of the conserved WRKY domains [8,9]. In addition, group II are divided into five distinct subgroups (IIa, IIb, IIc, IId and IIe) [10]. Noteworthy, Group III WRKY TFs have undergone a change in the zinc-finger motif from C 2 H 2 of Group I and II to C 2 HC (C-X 7 -C-X 23 -H-X 1 -C), which has been considered the most advanced in plant evolution to acquire the adaptability to different environmental pressure [8,11].
Studies have demonstrated that most Group III TFs of Arabidopsis thaliana are involved in different plant defense signaling pathways, indicating that they may have evolved as a result of increasing biological requirements [8,11]. For example, AtWRKY38 (III) and AtWRKY62 (III), as negative regulators of plant basal defense, reduce disease resistance by reducing SA-regulated PR1 gene expression [12]. Overexpression of AtWRKY41 (III) negatively affects the response to Erwinia carotovora infection, but not to Pseudomonas syringae infection [13]. AtWRKY70 (III) as a node of convergence for salicylic acid (SA) and jasmonic acid (JA)-mediated defense signal pathways, together with WRKY54 (III), enhanced the resistance to the hemibiotroph Pseudomonas syringae pv tomato (Pst) DC3000, but increased the susceptibility of Arabidopsis to the necrotrophic pathogens Pectobacterium carotovorum and Botrytis cinerea [14]. Additionally, certain WRKY III genes from rice and tomato have an effect on different sorts of stresses. OsWRKY74 (III), for instance, is involved in the tolerance to phosphate (Pi) starvation [15]. Similarly, WRKY Group III genes in tomato could also respond to TYLCV infection. Taken together, WRKY III genes play significant and complex roles in the defense against biotic and abiotic stresses.
To date, the functional analysis of group III WRKYs of cotton has been reported less and mainly with a focus on fiber development, leaf senescence and abiotic stress, with limited analysis of the possible roles in resisting Verticillium wilt [16]. In this study, we analyzed the symptom characteristics and the phylogenetic relationships of group III WRKY proteins in G. hirsutum and A. thaliana, and the expression levels of group III GhWRKY genes of cotton were confirmed after infection with V. dahliae. In addition, three group III GhWRKY genes of cotton with SA and JA regulatory elements in their promoter sequence were selected to explore the expression levels after SA and JA treatment. We aimed to obtain novel insights into the interaction of WRKY Group III TFs with V. dahliae infection in cotton.

Identification of group III WRKY genes in G. hirsutum and A. thaliana
Group III WRKY members and their domain proteins in Arabidopsis and G. hirsutum were downloaded from the Arabidopsis genome, TAIR 9.0 release (http://www. Arabidopsis.org/) and the cotton genome project (http://grand.cricaas.com.cn/page/species/detail?id=2), respectively.

Phylogenetic analysis and characterization of group III WRKY proteins
Multiple sequence alignment was conducted using Clustal X2, and MEGA 7 was used to conduct the phylogenetic tree based on the neighbor-joining method with 1000 bootstraps [17,18]. The conserved motifs of Group III WRKY protein sequences of cotton and Arabidopsis were analyzed on MEME server (http:// meme-suite. org/tools/meme) [19].

Exon/intron structure and promoter region analysis of WRKY
The exon and intron structures of each WRKY Group III TF in cotton were analyzed by Gene Structure Display Server (GSDS v2.0, http://gsds.gao-lab.org/) [20]. Approximately 1.5 kb of DNA sequence upstream from the codons of WRKY Group III TFs was downloaded from the cotton genome project (https://cottonfgd.org/analyze/). PlantCARE (http://bioinformatics. psb.ugent.be/webtools/plantcare/html/) was used to analyze the promoter region of WRKY Group III TFs [21].

Culturing of Verticillium dahliae
The highly aggressive, defoliating V. dahliae strain Linxi2-1, obtained from Key Laboratory for Crop Germplasm Resources of Hebei, was cultured for 7 d at 25 °C on 25% potato dextrose agar (PDA) medium. It was then inoculated into a Czapek medium, and was cultured for 10 d at 25 °C. This suspension was adjusted to a density of 10 7 conidia per milliliter prior to use [22].

Plant materials, growth conditions and treatments
Seeds of the resistant cotton cultivar 'CCRI12' and the susceptible cotton cultivar 'CCRI 8' , obtained from Cotton Research Institute (Anyang City of Henan Province, China) were surface-sterilized with 0.1% HgCl 2 for 5 min and rinsed in sterile water five times for 3 min each time. The seeds were then soaked in sterile water for 48 h at 28 °C. Germinated seeds were transferred to tissue culture pots containing Murashige and Skoog (MS) medium (pH 5.8) supplemented with 10 g/L of sucrose and 2 g/L of phytagel, and then incubated for another 5 d at 28 °C, under a 16-h photoperiod (2000 Lux).
One part of the seedlings were removed from their pots and inoculated by root-dipping for 30 s in conidia suspension. Afterwards, they were transferred to fresh pots containing the original MS medium and incubated at 25 °C in a growth room. After incubating for 48 h, hypocotyl tissues were cut from randomly selected seedlings for each treatment. Hypocotyl tissues were first surface-sterilized for 15 s in 70% ethanol followed by five washes in sterile water to ensure that the mycelia were removed from the tissue, and then cultured on PDA medium at 25 °C [22]. After incubating for 10 d, the stem of cotton seedlings was cut with a blade to observe the browning of vascular bundles. To analyze the gene expression patterns of group III WRKYs, cotton roots were collected at 0, 2, 6, 12, 24 and 48 h after infection with V. dahliae by root-dipping for 30 s in conidia suspension, then frozen in liquid nitrogen and stored at −80 °C.
The other part of the 'CCRI12' seedlings were sprayed with 100 μmol/L SA or 100 μmol/L JA, and cotton cotyledons were collected at 0, 2, 6, 12, 24 and 48 h after treatment, then frozen in liquid nitrogen and stored at −80 °C.

Quantitative real-time PCR analysis
Total RNA was extracted from cotton roots and cotyledons using a RN09-EASYspin RNA Plant Mini Kit (Aidlab Biotechnologies Co. Ltd., Beijing, China), and treated with DNase I (TaKaRa Biotechnology (Dalian) Co., Ltd., Dalian, China) to remove DNA contamination. Approximately 2 μg of total RNA was reversely transcribed into first-strand cDNA by uEIris II RT-PCR system. The synthesized cDNA samples were diluted 10-fold, and then 1 μL was taken as an RT-qPCR template. The GhUbiquitin7 (GhUB7, as internal control) and WRKY Group III genes were evaluated by qRT-PCR with SYBR Green Mix (Takara) and the Roche LightCycler 2.0 (Roche, Germany). All primers were designed according to the complete CDS sequences of all genes using primer5; the primers, annealing temp and PCR product length are shown in Supplemental Table S1. Thermal-cycling conditions included an initial 95 °C for 10 s; followed by 45 cycles of 95 °C for 10 s, annealing temperature for 15 s and 72 °C for 15 s; Then a final melting curve step from annealing temperature to 95 °C, ramp rate of 0.05 °C/s. The data of qRT-PCR from three replicate experiments were analyzed by the 2 −ΔΔCt method [23].

Subcellular localization of CotAD_14615
The complete ORF sequences of CotAD_14615 without a stop codon was amplified by RT-PCR containing HindIII and SalI restriction sites. The purified PCR products were digested with HindIII and SalI and then fused to the pSuper1300-GFP vector to get a fusion protein CotAD_14615::GFP under the control of the CaMV 35S promoter. For construction of 35S::nls::mKate::RFP vector, the nuclear signal peptide (MDPKKKRKV) was fused to the far-RFP mKate of pBWA(V) HS vector driven by CaMV 35S promoter. The construct 35S::GFP was used as a control. CotAD_14615::GFP and 35S::GFP were agro-infiltrated into leaves of Nicotiana tabacum, respectively. The fluorescence signal was observed with a confocal microscope (Leica TCS SP5) after transformation for 12-16 h. GFP signal was excited using a wavelength of 488 nm. The RFP emission signal was collected between 587 and 610 nm.

Analysis of transcriptional activation in yeast
The full-length coding sequence of CotAD_14615 was fused to the pGBKT7 vector with EcoRI and SalI restriction sites. The construction was named pGBKT7-CotAD_14615. Empty vector pGBKT7 and pGBKT7-GAL4 were taken as negative and positive control, respectively. The construct and controls were introduced into Saccharomyces cerevisiae strain AH109 by the lithium acetate method. The yeast cells were grown on selective medium (SD) medium without tryptophan (SD-T) and screened by PCR. The positive clones were cultured on the SD-T, SD without tryptophan, histidine (SD-T/H) and SD without tryptophan, histidine, and adenine (SD-T/H/A with X-D-Galactosidase (X-gal)) medium at 30 °C. The images were photographed after 3 d to detect the transcriptional activation [24].

Phylogenetic analysis of WRKY group III TFs
To investigate the phylogenetic relationship and evolution of Arabidopsis and cotton based on WRKY Group III TFs peptide sequences, multiple full-length peptide sequence alignment was performed by Clustal X2. Then a phylogenetic tree was conducted using MEGA7 ( Figure 1). Based on the phylogenetic relationship, all the WRKY Group III TFs were divided into two clades. Both Clade 1 and Clade 2 consist of 15 members. Clade 1 contained 5 Arabidopsis members and 10 cotton members. Clade 2 is made up of 8 members of Arabidopsis and 7 cotton members.
In addition, the conserved motifs for GhWRKY and AtWRKY Group III genes were identified using MEME; six motifs were observed in the 30 protein construct domains ( Figure 2). The lengths of the six conserved motifs were 29, 21, 21, 29, 32 and 15 aa, respectively (Supplemental Table S2). All WRKYs sequences in Group III had motif 1 motif 2. The members that are closely related in the phylogenetic tree share common motif compositions, as expected. For example, the CotAD_36424, CotAD_47724, CotAD_28446, AtWRKY53, AtWRKY41, AtWRKY30, CotAD_02678, CotAD_35535, CotAD_17081, CotAD_21710, CotAD_29630 have the same motif patterns and share the motifs 1-6. Other Group III WRKYs, however, contain 4, 3 or 2 motif patterns. All Group III WRKYs genes of Arabidopsis contain motifs 1-4, indicating that these motifs are conserved during evolution.

Exon/intron structure analysis of GhWRKY group III TFs
To gain deeper insights into the structures of WRKY Group III TFs, their exon/intron distribution in cotton was analyzed on the website of GSDS (Figure 3). Except for CotAD_64005 with one intron, all the WRKY Group III TFs members contain two introns. The CotAD_64005 lost the second exon and the first intron compared with Group III TFs members containing two introns. For cotton WRKY Group III TFs with two introns, the intron length varied, and seven Group III TFs members contained long introns.

Promoter region analysis of GhWRKY group III TFs
To determine the cis-acting elements, promoter regions, about 1.5-kb DNA sequences upstream from the codons of WRKY Group III TFs in cotton, were identified and analyzed by plant CARE (Figure 4). We found some basic elements, including TATA elements (core promoter element at around −30 bp of the transcription start) and CAAT-box, in all promoters. In addition, there were elements related to hormone regulation, such as TCA-element (cis-acting element involved in salicylic acid responsiveness), CGTCA-motif and TGACG element (cis-acting elements involved in the MeJA responsiveness), ABRE (cis-acting element involved in the abscisic acid responsiveness) and P-box (gibberellin-responsive element) in most Group III TF promoter regions. Moreover, cis-acting elements related to abiotic stress were found in several GhWRKY TFs promoter regions, for example, LTR (cis-acting element involved in low-temperature responsiveness), MBS (MYB binding site involved in drought-inducibility), and TC-rich repeats (cis-acting element involved in defense and stress responsiveness). Remarkably, a series of W or W-like boxes were found in several WRKY TF promoters, which indicated complex interaction of WRKY TFs with each other by binding with W-box elements [25,26].

Detection of Verticillium dahliae accumulation in resistant and susceptible cotton cultivars
To control the contamination of undesired microbes, sterile cotton seedlings from 'CCRI12' and 'CCRI8' were used for disease resistance identification ( Figure 5A). Sterile seedlings of cotton cultivars ('CCRI12' and 'CCRI8') showed different symptoms after V. dahliae infection. After V. dahliae infection for 48 h, more mycelia were isolated from the hypocotyl of 'CCRI8' than 'CCRI12' (Figure 5B, C). 'CCRI8' showed more obvious symptoms than 'CCRI12' (Figure 5D), and the vascular bundle of 'CCRI8' was browning more than that of 'CCRI12' after infection with V. dahliae for 10 d ( Figure 5E). These results show that 'CCRI8' was more susceptible to V. dahliae infection than 'CCRI12' . Seedlings grown in soil-filled pots are usually used to identify the resistance of variety by dipping the roots into conidia suspension of V. dahliae strain [27]. However, the method of sterile seedlings inoculation  with V. dahliae via root-dipping could provide better and more reliable basis for resistance assays under controlled environmental conditions.

Expression profiles of part GhWRKY group III genes in response to Verticillium dahliae infection
Gene expression analysis can provide essential clues for gene function. Therefore, we carried out qRT-PCR to elucidate the expression patterns of nine GhWRKY Group III genes, which are homologous with AtWRKY70, AtWRKY54 and early drought response factor AtWRKY53 in the evolutionary tree, after V. dahliae infection at different times in resistant cotton cultivars ( Figure 6). More than two-fold difference in transcript levels was considered to be the true difference for the genes under treatments. The expression levels of 5 Group III genes were up-regulated at least at one or two-time points after V. dahliae infection, except for CotAD_16771, CotAD_28446, CotAD_32464 and CotAD_47724. After V. dahliae induction, the maximum expression was observed with 4.5-fold in CotAD_42959 at 24 h, 3.8-fold in CotAD_57168, 11-fold in CotAD_14615 at 2 h, 3.7-fold in CotAD_12532 and 2.8-fold in CotAD_24423. Among these results, it is worth noting that the expression of CotAD_14615 reached the maximum at 2 h in the resistant varieties after inoculation with V. dahliae. Two hours after the infection of V. dahliae is probably the key period for the signal recognition of pathogen in cotton root. In addition, it is generally believed that closely related members in the phylogenetic tree have similar sequences and common functions. The sequence of CotAD_14615 is most similar to AtWRKY70 and AtWRKY54. Therefore, we speculate that this gene may play an important role in the resistance to V. dahliae invasion in cotton.

Group III WRKY genes participate in cotton abiotic stresses
SA and JA are two essential plant hormones that promote immunity against biotrophic and semibiotrophic pathogens [28,29]. Many WRKY genes play important roles in SA and JA mediated biological processes, which is involved in pathogen-associated molecular pattern triggered immunity. Three GhWRKY Group III genes with a SA or JA cis-regulatory element in there promoter regions were selected to investigate the expression patterns after SA and JA treatment ( Figure  7). The expression level of CotAD_57168 reached a high level 6 h after treatment with SA and then declined, however, showed a strong increase in expression at 6 and 12 h after treatment with JA. CotAD_14615 was not clearly induced after spraying with SA, but it was strongly induced by JA and reached the maximum expression at 6 h. Moreover, CotAD_02678 was induced by SA and JA and reached a high level expression at 24 h. Homologous genes usually share similar functions. CotAD_57168 is close to AtWRKY54 and 70 in the phylogenetic tree, and the expression pattern was also similar to AtWRKY54 and AtWRKY70 after treatment with SA, which plays crucial roles in basal defense and the establishment of systemic acquired resistance [30,31]. In addition, the expression pattern of three genes was also consistent with the regulatory  elements of their promoter. This result indicates that three members of WRKY group III may have a related, SA or JA-dependent function in signal transduction.

Subcellular localization of CotAD_14615
Because the expression of CotAD_14615 was significantly up-regulated after treatment with V. dahliae and JA, we studied the subcellular localization of CotAD_14615. The full-length ORF of CotAD_14615 without a stop codon was fused to the N-terminal of GFP reporter protein of pSuper1300-GFP vector driven by CaMV 35S promoter, generating a fusion construct CotAD_14615::GFP ( Figure 8). A nuclear signal peptide fused to a RFP protein mKate [32] was used as a positive control, and the pSuper1300-GFP vector was used as a negative control. The pSuper1300-GFP vector, CotAD_14615::GFP and red fluorescent protein (RFP)-mKATE were co-transformed into Nicotiana tabacum mesophyll cells. Microscopic visualization demonstrated that the CotAD_14615-GFP fusion protein fluorescence perfectly overlapped with RFP fluorescence ( Figure  9A-D), indicating that CotAD_14615 was localized in the nucleus. However, green fluorescence was exclusively detected in the entire cell region when the pSuper1300-GFP vector was transformed into Nicotiana tabacum mesophyll cells ( Figure 9E-H). The CotAD_14615-GFP fusion protein was exclusively localized to the nucleus of Nicotiana tabacum mesophyll cells in a transient expression assay. The result is similar to a previous study [33].

Transcriptional activation assay of CotAD_14615 in yeast
Transcriptional activation of CotAD_14615 was detected with the GAL4 yeast expression system. Yeast strain AH109 was transformed with the construct pGBKT7-CotAD_14615, PGBKT7-GAL4 and pGBKT7. PGBKT7-GAL4 and pGBKT7 were taken as a positive and negative control, respectively. The yeast cells transformed with pGBKT7-CotAD_14615 and PGBKT7-GAL4 grew well on the SD-THA/X medium, and turned blue in the presence of X-α-gal. Meanwhile, the cells with the empty vector pGBKT7 could only survive on the SD/-T medium ( Figure 10). The results indicated that CotAD_14615 functioned as a transcriptional activator.

Discussion
WRKY genes are one of the largest gene families, and can be classified into three main groups: groups I, II and III [9]. WRKY Group III is expressed only in higher plants, and most of these proteins are related to plant responses to biotic stress, whereas group I is expressed in ferns and some eukaryotic cells besides higher plants [34][35][36]. Therefore, Group III TFs may have evolved to acquire the adaptability to the pressure of different environments [8,11]. Many members of WRKY Group III in Arabidopsis have been proved to play an important role in stress resistance [8,11]. In the present study, 17 group III WRKY genes in cotton were identified and analyzed. Elements related to JA and SA regulation were identified in the promoter region of most of these Group III genes. The expression levels of part of the Group III genes were up-regulated or down-regulated after V. dahliae infection. Moreover, group III GhWRKY genes with a JA or SA regulatory element in the promoter region could be significantly induced by JA or SA, respectively. These results indicate that members of Group III TFs play different roles in cotton biotic and abiotic stress.
Multiple sequence alignment showed that Group III GhWRKYs of cotton and Arabidopsis were divided into two clades, and those with similar motif patterns tend to cluster into the same group. It has been previously demonstrated that most Group III TFs of plants were involved in different plant defense signaling pathways [11,30,37]. This distribution may be related to the split of different families in dicotyledon. Therefore, we speculated that members of this subfamily may have a conserved function in plant defense. Compared with Arabidopsis, the number of motifs of Group III WRKYs genes in cotton changed greatly, suggesting these WRKY genes may have more functions. Changes in the number of exons and introns can alter the gene structure, which plays an important role in the evolution of multigene families [38][39][40][41][42]. As well as the number of introns, the length of the exons/introns is also different among Group III GhWRKYs. In the process of multigene family evolution, exon/intron acquisition or loss have always occurred, which may be related to the functional diversification of gene family members. The difference in the size and number of introns suggested that WRKY Group III TFs reflect the diversity of functions during the growth, development or resistance to adversity in cotton.
After infection with V. dahliae, some group III genes were significantly induced or repressed ( Figure 6), indicating that group III may play an important role in the ability of plants to resist V. dahliae infection. The expression of CotAD_14615 was dramatically induced by V. dahliae invasion or JA treatment. This result indicates that the gene may have a cross-regulation function under different stresses. Furthermore, AtWRKY53 is an early factor in drought response and overexpression lines were hypersensitive to drought stress compared with Col-0 plants [43]. CotAD_28446, CotAD_32464 and CotAD_47724, which are homologous with AtWRKY53 in the phylogenetic tree, were downregulated after infection with V. dahliae. The study found that the increase of AtWRKY53 expression can significantly reduce the H 2 O 2 content [43]. In the process of SA-induced system acquired disease resistance (SAR), the marked event was the ROS burst [44]. It has been Figure 10. transcriptional activity assay of cotaD_14615 protein in yeast. the transformants were incubated on the SD-t, SD-t/h, and SD-t/h/a with X-α-gal medium. the pgBt7-gal4 (positive control) and pgBt7-cotaD_14615 could survive on three different medium. the negative control could only survive on the SD/-t medium.
reported for Arabidopsis that H 2 O 2 activates the SA pathway, mediated by PAD4, which acts to amplify the SA defenses that eventually lead to programmed cell death [45]. We speculated that the reduced expression level of 3 Group III genes homologous with AtWRKY53 after infection with V. dahliae probably prevented the decline in the H 2 O 2 level, and activated the SA signaling pathway. So, the resistance of plants to biotic stress and abiotic stress is not isolated, and one gene may be involved in integrated resistance of plants.
Subcellular localization experiments showed that most WRKY transcription factors were mainly localized in the nucleus. For example, in wheat (Triticum aestivum L.), the TaWRKY46 protein was expressed in the nucleus [24]. In this work, the subcellular localization and transcriptional activation assay experiments showed that CotAD_14615 was located in the nucleus ( Figure 8) and had transcriptional activation activity ( Figure 9) possibly by binding the promoter sequence of target genes.
Overall, our work suggested that the Group III GhWRKYs of cotton, especially the CotAD_14615, may play an important role in the resistance to V. dahliae. The findings may help identify candidate resistance-related genes to study the molecular mechanism, which is the foundation of biotechnology for genetic improvement of cotton resistance to adverse conditions.

Conclusions
In the present study, 17 group III WRKY genes in cotton were identified through searching the G. hirsutum genome. They could be classified into two clades with group III WRKY genes from Arabidopsis thaliana. The conserved WRKY DNA-binding domain was found in all group III WRKY proteins according to conserved motif analysis. Gene structure analysis showed that 16 of the 17 group III WRKY genes in G. hirsutum contain two introns. Elements related to biotic and abiotic stresses were identified in the promoter region of most Group III TFs. Furthermore, some Group III WRKY genes were up-regulated or down-regulated after V. dahliae and JA or SA treatment. The Cot_14615 protein was confirmed to be localized in the nucleus and has transcriptional activation activity. Our data on cotton group III WRKY genes can serve as a basis for further studies into their potential function in cotton resistant to Verticillium Wilt.