Identification and Characterization of the Grape WRKY Family

WRKY transcription factors have functions in plant growth and development and in response to biotic and abiotic stresses. Many studies have focused on functional identification of WRKY transcription factors, but little is known about the molecular phylogeny or global expression patterns of the complete WRKY family. In this study, we identified 80 WRKY proteins encoded in the grape genome. Based on the structural features of these proteins, the grape WRKY genes were classified into three groups (groups 1–3). Analysis of WRKY genes expression profiles indicated that 28 WRKY genes were differentially expressed in response to biotic stress caused by grape whiterot and/or salicylic acid (SA). In that 16 WRKY genes upregulated both by whiterot pathogenic bacteria and SA. The results indicated that 16 WRKY proteins participated in SA-dependent defense signal pathway. This study provides a basis for cloning genes with specific functions from grape.


Introduction
Various defense mechanisms have evolved in plants to combat microbial infection. Transcription factors are central to this process, and transcription factor families have expanded and evolved in plants to coordinate gene expression. Expression of a large number of defense-related plant genes is regulated at the transcriptional level in response to pathogen infection [1]. Timely transcriptional regulation of defenserelated genes is crucial for effective responses to pathogens [2]. Proteins of the WRKY family are the most important transcription factors for the regulation of plant defense response pathways [3][4][5]. The WRKY name is derived from the conserved DNA binding domain sequence WRKYGQK; the conservative domain is approximately 60 residues, followed by a Cys2His2 or Cys2HisCys zinc-binding motif [6,7], and WRKY proteins were divided into Group I-III based on the number of WRKY domains and the structure of zinc fingers [8,9]. The Group II WRKY proteins are classified into a, b, c, d, and e subgroups based on their zinc finger motifs [5].
In higher plants, the WRKY gene family members play a variety of roles. Accumulating evidence indicates that WRKY transcription factors are involved in responses to biotic stresses as well as in plant development [7,10,11]. Salicylic acid (SA) is an response endogenous phytohormone and an important signal substances in the deployment of systemic acquired resistance (SAR) [12]. SAR is characterized by an increase in endogenous salicylic acid (SA) and enhanced resistance to a broad spectrum of virulent pathogens. SA is necessary for SAR, and a series of studies demonstrated that SA triggers host defence mechanisms against pathogen infections [13,14]. Many WRKY genes are key factors controlling plant response to disease resistance especially pathogen infections that can trigger SA-dependent defense signaling. In Arabidopsis, WRKY70 was identified as an important node of SA signaling during plant defense responses [15]. In addition, treatment of Arabidopsis with a bacterial pathogen or salicylic acid (SA) resulted in differential expression of WRKY genes [15,16]. This pattern of expression was also observed in other plant species [10,[17][18][19].
Cultivated grapevines are susceptible to many pathogens including phytoplasmas, viruses, bacteria, and fungi [22]. Among these, grape white rot (Coniothyrium diplodiella) is the most important agriculturally because it causes extensive losses in quantity and quality of harvested berries. As a consequence, table and wine grape cultivation requires extensive use of phytochemicals. In China, grape white rot is the main fungal disease of grapes causing heavy losses in grape production [23]. Vitis vinifera L. ("European" grape) is the most economically important and widespread species of Vitis spp. producing more than 90% of world's production of table, wine, and raisin grapes. More than 80% of cultivated grape varieties are derived from this species. All V. vinifera L. varieties are susceptible to grape white rot [24]. The disease is found in most grape-growing regions of the world [25] and results in poor berry quality and weakened vines in warm and humid climates. Multiple research strategies are currently being pursued so that wine growers may produce healthy fruits right up to maturity with minimum use of chemical treatments.
The complete V. vinifera PN40024 genomic sequence [26,27] has been determined. The V. vinifera genome was the first fruit tree species genome to be sequenced, making V. vinifera an ideal model system for fruit trees [26]. The release of the latest V. vinifera genome sequence, 12X (http:// www.genoscope.cns.fr/externe/GenomeBrowser/Vitis/), and the many WRKY gene and protein sequences from other Vitis spp. present in the NCBI (http://www.ncbi.nlm.nih .gov/) and PlantTFDB (http://planttfdb.cbi.pku.edu.cn/index .php?sp=Vvi) databases provided an opportunity to analyze and further understand the grape WRKY gene family. In this study, we identified a comprehensive and nonredundant set of eighty WRKY genes in the grape genome. Phylogenetic and motif analysis and characterization of WRKY expression induced by pathogens and salicylic acid (SA) were also performed to lay a solid foundation for further comparative genomics studies.

Characterization of Putative WRKY Proteins in Grape.
The latest 12X V. vinifera genomic and protein sequences were downloaded from the NCBI database. The procedure used to survey grape WRKY proteins was similar to identification methods described for other species. The hidden Markov model (HMM) profile for the WRKY domain from the Pfam database was used as a query to survey all potential proteins. The Pfam database was then used to decide if the candidate proteins contained features typical of WRKY proteins. Identical and defective sequences were eliminated using manual inspection in the MEGA ver4.0 software. Nonoverlapping WRKY protein sequences were used for further analysis.

Phylogenetic Analysis Based on Conserved WRKY
Domains. WRKY genes of was retrieved by TBlastN software from the publicly available information in the database (http://www.ncbi.nlm.nih.gov, http://www.genoscope.cns .fr/externe/GenomeBrowser/Vitis/). Conserved WRKY domains of the VvWRKY proteins were identified by manual inspection using the Pfam software and used to generate a multiple sequence alignment of the WRKY domains. Phylogenetic trees based on 58 representative domains from poplar, Arabidopsis, rice, and grape were constructed using Clustal X ver1.83 and Mega ver4.0 [28] by the neighborjoining (NJ) method to produce improved classifications of the different clades. Bootstrap values were calculated from 1,000 iterations.

Analysis of Conserved Motif Distribution and Structure and Gene Duplication in Grape.
To assess the structural divergence of VvWRKY genes, conserved motifs in the encoded proteins were identified using the multiple expectation maximization for motif elicitation (MEME) online software (http://meme.sdsc.edu/meme/intro.html). Parameters employed in the analysis were as follows: maximum number of motifs, 20; minimum motif width, 6; and maximum motif width, 50. The online software 2ZIP (http://2zip.molgen.mpg.de/index.html) was used to predict the conserved Leu zipper motif. HARF, LXXLL, and LXLXLX motifs were identified manually.

Expression Analysis.
Chinese wild grape V. vinifera "Pinor" leaves (numbered by the National Repository for Grapevine (Zhengzhou grape germplasm repository)) were used for all experiments. When shoots of vines were 25-35 cm in length, the third through fifth fully expanded young leaves beneath the apex were selected for biotic and abiotic treatments. The plants were subjected to abiotic stress and SA treatments according to Ramamoorthy et al. [29]. For SA treatment, plants were sprayed with a 0.1 mM SA solution. Treatment with deionized water served as a control and was repeated three times on three independent plants. Leaves were collected 0, 9, 12, 24, and 48 h after treatment and immediately frozen in liquid nitrogen for further study.
For pathogen treatment, leaves were inoculated with Coniothyrium diplodiella mycelium gelose discs from a 3day-old culture at six sites and placed on PDA medium at 28 ∘ C. Leaves sprayed with PDA medium were used as negative control. Leaves were collected 0, 9, 12, 24, and 48 h after inoculation (hpi). Treatment with deionized water was performed as a control. After harvest, the materials were immediately frozen in liquid nitrogen and stored at −80 ∘ C for further analysis.
Grape total RNA was extracted as previously described [30]. Grapevine total RNA was extracted from V. vinifera "Pinor" leaves using an improved SDS/phenol method [31] at 0, 9, 12, 24, and 48 h after infection with Coniothyrium diplodiella. The following primers were used for RT-PCR amplification: 5 -GCGGGCAAGAGATACCTCAA-3 and 5 -TCAATCTGTCTAGGAAAGGAAG-3 for EF1 (AF176496). Three independent PCR reactions were carried out for each gene and similar results were obtained. Amplification products were quantified using a Roche 480 II realtime PCR instrument.

Identification of WRKY Proteins from Grape.
To identify WRKY proteins encoded in the grape genome, publicly available genome sequences were searched using the BlastP software based on an HMM (PF03106). A total of 80 putative grape WRKY protein sequences were initially identified.

Phylogenetic Analysis and Classification of WRKY Groups in Grape.
A phylogenetic tree was constructed based on the conserved WRKY domains to examine the phylogenetic relationships among all 80 members. VvWRKY domains of the Group I include two domains (the C-terminal and Nterminal domain). A multiple sequence alignment of the 80 WRKY domains was performed ( Figure 1). Three major groups were identified as described by Wu et al. [20]. In addition, several subgroups were revealed by the phylogenetic analysis.
The positions of the C-and N-terminal WRKY domains in the WRKY proteins were relatively consistent. Group I contained 19 WRKY proteins all of which contained two WRKY domains. Three Group I-CTWD (C-terminal     domain) members, VvWRKY32-1C, VvWRKY32-2C, and VpWRKY2C, were more closely related to the N-terminal members of Group I than to the other C-terminal members (Figures 2, and 3). While Group I VvWRKY44C was far away from both I-CTWD and N-terminal, and unique, which suggesting a special origin of the domain. The largest number of WRKY proteins of Group II was divided to five among 23 major subgroups: IIa, IIb, IIc, IId, and IIe. Group IIa (seven members) and Group IIb (eight members) were two subgroups from the same branch, while Group IId (nine members) and Group IIe (eight members) were close in genetic tree. Five members of Group IIc were more similar to Groups IIa and IIb from the same branch based on the phylogenetic analysis. Four members of Group IIc, VvWRKY13-1, 13-2, 12-1, and 12-2, were more closely related to the C-terminal WRKY domains of Group I than those of other groups. This is consistent with a recent analysis supporting the hypothesis that loss of the Group I N-terminal WRKY domain led to the origin of the Group II WRKY proteins [5].   Seven WRKY domains belonged to Group III, which is generally considered to be the most evolutionarily advanced group and the most adaptable [5]. Phylogenetic relationships between the Group III WRKY domains of 15 AtWRKY, 28 OsWRKY, 10 PtWRKY, and 7 VvWRKY proteins were examined ( Figure 4). The poplar WRKY domains shared higher sequence homology with the other dicot plants (Arabidopsis and grape) than with monocot rice. Similarly, the six AtWRKY domains clustered in two groups ( Figure 4). However, this diversity was not found in Arabidopsis, poplar, or grape, suggesting that VvWRKY genes respond to different environmental signals.

Conserved Motifs and Domains in Grape WRKY Proteins.
With the exception of the conserved 60 amino acid residues, no functional or structural homologies were present in the WRKY protein sequences [7]. Few WRKY proteins contain a conserved leucine zipper motif, a hypothetical structure common to a class of DNA-binding proteins [7,32]. Using the online 2ZIP software, we found that none of the grape WRKY proteins contained this structure with the exception of VvWRKY18. The conserved HARF sequence motif (RTGHARFRR(A/G)P) was found in six members (VvWRKY7-1, -7-2, -11-1, -11-2, -11-3, and VtWRKY11) of the Group IId WRKY proteins in Arabidopsis, although no putative function has been clearly identified for this motif [7,33]. WRKY proteins are transcription factors associated with activation and repression of plant immune responses [7,10,34]. The coactivator motif, LXXLL (L, leucine; X, any amino acid), and the active repressor motif, LXLXLX [35,36], were searched for in the VvWRKY protein sequences by manual inspection. Seven VvWRKY proteins, VpWRKY3 and VvWRKY20-3, -42, -9, -18, -3-2, and -40-2, contained the LXLXLX motif and three VvWRKY proteins (VvWRKY7-2, -11-2, and 11-3) contained the LXXLL motif. The Multiple expectation maximization for motif elicitation online software was used as a secondary method to analyze motif distribution and confirm the results of domain prediction ( Figure 5; Table 2). LZ indicates potential leucine zipper structures that were also predicted by the COILSCAN and COIL programs but none was found. The conserved motifs 1, 2, 3, 4, 5, and 6 in Figure 5 were characterized as WRKY domains and were broadly distributed among the VxWRKY protein sequences. Motif 10 and motif 13, a conserved NLS motif, were found mainly among the Groups I and IId proteins, while motif 12 was found only among Groups I and IId proteins, although its function is unknown.

Discussion
The WRKY transcription factor gene family appears to be involved in the regulation of a variety of processes [7,21,37]. The complex features and functions of this family have been studied extensively in the model herbaceous plants Arabidopsis and rice and in the woody plant poplar. Characterization of the WRKY genes in grape (Vitis spp.) would facilitate a broader understanding of this gene superfamily. In this study, 80 WRKY genes were characterized. The lengths of these sequences were highly varied implying a high degree of complexity among the VvWRKY genes.
The WRKY genes can be divided into three main groups based on their structural features. PtWRKY27 OsWRKY79 OsWRKY38 OsWRKY53 OsWRKY70 numbers of Arabidopsis, rice, poplar, and grape WRKY genes in Groups IIa and IIb suggest that all VvWRKY genes belonging to these subgroups have been identified, but there were only Group Ia in grape (Table 3). Fewer Group III WRKY genes have been identified in grape compared to in Arabidopsis and rice, close to the poplar quantitatively, implying that the number of VvWRKY genes in this group has either declined over evolutionary time or was underestimated in our analysis. Of the VvWRKY genes, 67.9% belong to Group II and 9.7% to Group III. This distribution is more  Figure 5: Phylogenetic analysis of 80 members of the VvWRKY family. Amino acid sequences from the single WRKY domain of Groups II and III proteins or the C-terminal WRKY domain of Group I proteins were aligned. Conserved primary structure features of the VvWRKY family located outside the WRKY domains were identified using MEME software (http://www.sdsc.edu/MEME) and are shown below the tree. Schematic representations of typical proteins of each (sub) group are shown on the bottom; motifs of amino acid sequences were shown in Table 2; LZ indicates potential leucine zipper structures that were also predicted by the COILSCAN and COIL (Wisconsin Package Version 10.0) programs.         I   32  34  50  16  3  Ia  14  14  23  16  3  Ib  18  20  27   II   26  30  44  IIa  3  4  5  5  2  IIb  8  8  9  7  IIc  7  7  13  22  IId  8  11  13  7  2  IIe  4  8  III  14  36  10  7  1  Total  72  100  104  72  8 a According to Wu et al. [20] and He et al. [21]. b Vitis thunbergii, Vitis thunbergii, Vitis pseudoreticulata, and Vitis hybrid cultivar. similar to that of the PtWRKY family than the AtWRKY or OsWRKY family, indicating a similar evolutionary history between grape and poplar (Table 3). A phylogenetic tree was constructed based on 58 Group III genes from Arabidopsis, rice, poplar, and grape.
The WRKY genes may act as a regulatory node that plays a crucial role in responses to abiotic stresses or in stressinduced defense signaling pathways [17]. Plant response to pathogens is regulated by multiple signal transduction pathways, in which SA functions as key signaling molecules [15,38]. Considerable effort has been directed toward elucidating the regulatory network controlling expression of SA-inducible genes. AtWRKY70 of Arabidopsis thaliana was in the SA-signal transduction pathway leading to PR gene expression [15]. Many of the WRKY genes were responsive to fungal infection and SA treatment, leading us to suspect that they may also play a regulatory role in the establishment of disease tolerance and in the SA signal transduction pathway in grape [34,39,40]. Expression analysis of AtWRKY genes in Arabidopsis showed that almost 70% are differentially regulated in response to pathogen infection and SA treatment, suggesting that the major role of WRKY genes in flowering plants is to mediate defense responses [41]. Details of the roles of the WRKY genes in defending dicotyledonous plants against pathogens can be found in three excellent reviews [4,7,42]. In our results, WRKY genes in grape showed that almost 57% (16 genes) were differentially regulated in response to both pathogen infection and SA treatment. It was lower than Arabidopsis. We speculated that the reason was that WRKY genes belong to V. vinifera PN40024 genomic sequence, and the test material was V. vinifera "Pinor, " which was susceptible cultivars Europe grape.

Disclosure
The English in this document has been checked by at least two professional editors, both native speakers of English. For a certificate, please see http://www.textcheck.com/certificate/oPFplV.