# Identification and Characterization of the Grape WRKY Family

Authors: Ying Zhang; Jian can Feng
Journal: BioMed Research International
 (2014)

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## Abstract

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 grapeWRKY 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.


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## Body

## 1. 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 defense-related 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 [3–5]. 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, theWRKY 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. InArabidopsis, WRKY70 was identified as an important node of SA signaling during plant defense responses [15]. In addition, treatment ofArabidopsis 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, 17–19].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 viniferaL. (“European” grape) is the most economically important and widespread species ofVitis 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. AllV. viniferaL. 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 completeV. vinifera PN40024 genomic sequence [26, 27] has been determined. TheV. vinifera genome was the first fruit tree species genome to be sequenced, makingV. vinifera an ideal model system for fruit trees [26]. The release of the latestV. viniferagenome sequence, 12X (http://www.genoscope.cns.fr/externe/GenomeBrowser/Vitis/), and the many WRKY gene and protein sequences from otherVitisspp. 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 grapeWRKY gene family. In this study, we identified a comprehensive and nonredundant set of eightyWRKY 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.


## 2. Materials and Methods

### 2.1. Characterization of Putative WRKY Proteins in Grape

The latest 12XV. 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.


### 2.2. 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 neighbor-joining (NJ) method to produce improved classifications of the different clades. Bootstrap values were calculated from 1,000 iterations.


### 2.3. Analysis of Conserved Motif Distribution and Structure and Gene Duplication in Grape

To assess the structural divergence ofVvWRKY 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.


### 2.4. Expression Analysis

Chinese wild grapeV. 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 withConiothyrium diplodiella mycelium gelose discs from a 3-day-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 fromV. vinifera“Pinor” leaves using an improved SDS/phenol method [31] at 0, 9, 12, 24, and 48 h after infection withConiothyrium diplodiella. The following primers were used for RT-PCR amplification: 5′-GCGGGCAAGAGATACCTCAA-3′ and 5′-TCAATCTGTCTAGGAAAGGAAG-3′ forEF1γ (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 real-time PCR instrument.


## 2.1. Characterization of Putative WRKY Proteins in Grape

The latest 12XV. 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.


## 2.2. 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 neighbor-joining (NJ) method to produce improved classifications of the different clades. Bootstrap values were calculated from 1,000 iterations.


## 2.3. Analysis of Conserved Motif Distribution and Structure and Gene Duplication in Grape

To assess the structural divergence ofVvWRKY 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.


## 2.4. Expression Analysis

Chinese wild grapeV. 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 withConiothyrium diplodiella mycelium gelose discs from a 3-day-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 fromV. vinifera“Pinor” leaves using an improved SDS/phenol method [31] at 0, 9, 12, 24, and 48 h after infection withConiothyrium diplodiella. The following primers were used for RT-PCR amplification: 5′-GCGGGCAAGAGATACCTCAA-3′ and 5′-TCAATCTGTCTAGGAAAGGAAG-3′ forEF1γ (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 real-time PCR instrument.


## 3. Results

### 3.1. 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. With the exception of VvWRKY1, VvWRKY2, VpWRKY1, and VpWRKY2, none of the proteins were described previously. Based on manual inspection using the MEGA ver4.0 software, seven sequences were discarded due to redundancy or insufficient conservation of WRKY sequences. The remaining WRKY proteins were reviewed using the Pfam program to confirm that all candidates contained the conserved WRKY motif. Properties of the proteins including numbers of amino acids, molecular weights, and isoelectric points (PI) are listed in Table1. The average VvWRKY sequence length was 382 amino acids and lengths ranged from 151 (VvWRKY1-1) to 798 (VvWRKY9) residues, while the isoelectric points (PI) ranged from 4.7 (VvWRKY22-4) to 9.84 (VvWRKY21).Table 1
Members of the grape WRKY superfamily of transcription factors.
Name
Proteina
Chr
Deduced polypeptide
Organism
Group
Domain
Length (aa)
PI
MW (kDa)
Family name
Pattern
VvWRKY 1-1
GI:50953501
*
151
9.61
17.7
Vitis vinifera
IIc
1-1
C-X4-C-X23-HXH
VvWRKY 1-2
GI:50953502
*
297
9.67
17.7
Vitis vinifera
IIc
1-2
C-X4-C-X23-HXH
VvWRKY 2-1
GI:359476618
4
700
6.52
75.9
Vitis vinifera
I-NTWD
2-1N
C-X4-C-X22-HXH
I-CTWD
2-1C
C-X4-C-X23-HXH
VvWRKY 2-2
GI:225463536
19
734
5.7
80.3
Vitis vinifera
I-NTWD
2-1N
C-X4-C-X22-HXH
I-CTWD
2-2C
C-X4-C-X23-HXH
VvWRKY 2-3
GI:225469228
*
536
7.21
58.44
Vitis vinifera
I-NTWD
2-3N
C-X4-C-X22-HXH
I-CTWD
2-3C
C-X4-C-X23-HXH
VvWRKY 2-4
GI:359478811
6
336
6.31
21.7
Vitis vinifera
IIc
2-4
C-X4-C-X23-HXH
VvWRKY 3-1
GI:48686707
1
317
7.64
58.3
Vitis vinifera
I-NTWD
3-1N
C-X4-C-X22-HXH
I-CTWD
3-1C
C-X4-C-X23-HXH
VvWRKY 3-2
GI:315272006
*
534
8.22
35.3
Vitis vinifera
IIa
3-2
C-X5-C-X23-HXH
VvWRKY 4
GI:315272008
*
625
8.71
34.4
Vitis vinifera
IIa
4
C-X5-C-X25-HXH
VvWRKY 6-1
GI:359485613
12
753
6.48
64.1
Vitis vinifera
IIb
6-1
C-X5-C-X23-HXH
VvWRKY 6-2
GI:225444291
10
535
6.04
57.5
Vitis vinifera
IIb
6-2
C-X5-C-X23-HXH
VvWRKY 7-1
GI:225458699
18
347
9.36
38.1
Vitis vinifera
IId
7-1
C-X5-C-X23-HXH
VvWRKY 7-2
GI:225438803
7
535
9.57
36.6
Vitis vinifera
IId
7-2
C-X5-C-X23-HXH
VvWRKY 9
GI:225447777
12
798
5.16
52.4
Vitis vinifera
IIb
9
C-X5-C-X23-HXH
VvWRKY 11-1
GI:225466161
4
338
9.49
36.6
Vitis vinifera
IId
11-1
C-X5-C-X23-HXH
VvWRKY 11-2
GI:225445976
11
297
9.73
32.4
Vitis vinifera
IId
11-2
C-X5-C-X23-HXH
VvWRKY 11-3
GI:262091438
*
297
9.73
32.4
Vitis vinifera
IId
1-3
C-X5-C-X23-HXH
VvWRKY 12-1
GI:225453346
15
228
7.68
26.1
Vitis vinifera
IIc
12-1
C-X4-C-X23-HXH
VvWRKY 12-2
GI:225453345
15
228
7.8
26.1
Vitis vinifera
IIc
12-2
C-X4-C-X23-HXH
VvWRKY 13-1
GI:359472522
1
305
5.8
33.4
Vitis vinifera
IIc
13-1
C-X4-C-X23-HXH
VvWRKY 13-1
GI:359472523
1
305
5.81
33.4
Vitis vinifera
IIc
13-1
C-X4-C-X23-HXH
VvWRKY 14
GI:225444177
10
438
5.16
47.5
Vitis vinifera
IIe
14
C-X5-C-X23-HXH
VvWRKY 18
GI:359476150
4
261
9.1
28.9
Vitis vinifera
IIa
18
C-X5-C-X23-HXH
VvWRKY 20-1
GI:359496861
*
604
6.01
65.7
Vitis vinifera
I-NTWD
20-1N
C-X4-C-X22-HXH
I-CTWD
20-1C
C-X4-C-X23-HXH
VvWRKY 20-2
GI:359494165
19
580
6.27
63.4
Vitis vinifera
I-NTWD
20-2N
C-X4-C-X23-HXH
I-CTWD
20-2C
C-X4-C-X23-HXH
VvWRKY 20-3
GI:225447598
12
407
5.02
44.8
Vitis vinifera
I-NTWD
20-3N
C-X4-C-X22-HXH
I-CTWD
20-3C
C-X4-C-X23-HXH
VvWRKY 20-4
GI:359496860
12
514
6.6
56.2
Vitis vinifera
I-NTWD
20-4N
C-X4-C-X23-HXH
I-CTWD
20-4C
C-X4-C-X23-HXH
VvWRKY 20-5
GI:359494164
19
595
6.8
65
Vitis vinifera
I-NTWD
20-5N
C-X4-C-X23-HXH
I-CTWD
20-5C
C-X4-C-X22-HXH
VvWRKY 20-6
GI:359485885
12
407
4.8
44.8
Vitis vinifera
I-NTWD
20-6N
C-X4-C-X23-HXH
I-CTWD
20-6C
C-X4-C-X23-HXH
VvWRKY 21
GI:225437249
7
340
9.84
38
Vitis vinifera
IId
21
C-X5-C-X23-HXH
VvWRKY 22-1
GI:359480165
7
233
6.06
26.8
Vitis vinifera
IIe
22-1
C-X5-C-X23-HXH
VvWRKY 22-2
GI:225454298
15
348
5.73
38
Vitis vinifera
IIe
22-2
C-X5-C-X23-HXH
VvWRKY 22-3
GI:225426142
2
331
5.71
36.6
Vitis vinifera
IIe
22-3
C-X5-C-X23-HXH
VvWRKY 22-4
GI:225464629
*
166
4.7
18.4
Vitis vinifera
IIe
22-4
C-X5-C-X23-HXH
VvWRKY 23
GI:225437606
7
302
6.74
33.8
Vitis vinifera
IIc
23
C-X4-C-X23-HXH
VvWRKY 24
GI:359489647
15
165
9.61
19
Vitis vinifera
IIc
24
C-X4-C-X23-HXH
VvWRKY 28-1
GI:225463412
10
319
6.76
35
Vitis vinifera
IIc
28-1
C-X4-C-X23-HXH
VvWRKY 28-2
GI:225446835
12
311
6.92
34.8
Vitis vinifera
IIc
28-2
C-X4-C-X23-HXH
VvWRKY 30
GI:40846374
*
349
5.02
39.3
Vitis aestivalis
III
30
C-X7-C-X23-HTC
VvWRKY 32-1
GI:225445873
11
499
6.12
53.8
Vitis vinifera
I-NTWD
32-1N
C-X4-C-X22-HXH
I-CTWD
32-1C
C-X4-C-X23-HXH
VvWRKY 32-2
GI:225430477
4
475
8.47
52.6
Vitis vinifera
I-NTWD
32-2N
C-X4-C-X22-HXH
I-CTWD
32-2C
C-X4-C-X23-HXH
VvWRKY 33-1
GI:225439574
8
552
7.29
61
Vitis vinifera
I-NTWD
33-1N
C-X4-C-X22-HXH
I-CTWD
33-1C
C-X4-C-X23-HXH
VvWRKY 33-2
GI:225434421
6
603
6.42
66.3
Vitis vinifera
I-NTWD
33-2N
C-X4-C-X22-HXH
I-CTWD
33-2C
C-X4-C-X23-HXH
VvWRKY 40-1
GI:225443178
9
311
5.16
52.4
Vitis vinifera
IIa
40-1
C-X5-C-X25-HXH
VvWRKY 40-2
GI:225430340
4
317
8.22
35.3
Vitis vinifera
IIa
40-2
C-X5-C-X23-HXH
VvWRKY 41
GI:225426000
2
342
6.05
38.6
Vitis vinifera
III
41
C-X7-C-X23-HTC
VvWRKY 42
GI:359494147
19
511
9.2
55.1
Vitis vinifera
IIb
42
C-X5-C-X23-HXH
VvWRKY 44
GI:225439779
8
477
8.84
52.3
Vitis vinifera
I-NTWD
44C
C-X4-C-X22-HXH
I-CTWD
44N
C-X4-C-X23-HXH
VvWRKY 45
GI:225451489
14
182
9.41
20.8
Vitis vinifera
IIc
45
C-X4-C-X23-HXH
VvWRKY 46
GI:225454483
15
349
5.01
39.2
Vitis vinifera
III
46
C-X7-C-X23-HTC
VvWRKY 47
GI:225437767
7
505
8.03
54.7
Vitis vinifera
IIb
47
C-X5-C-X23-HXH
VvWRKY 48
GI:225432004
5
309
5.72
34.4
Vitis vinifera
IIc
48
C-X4-C-X23-HXH
VvWRKY 49
GI:225440394
8
299
5.16
52.4
Vitis vinifera
IIc
49
C-X4-C-X23-HXH
VvWRKY 50
GI:225429590
4
166
5.2
18.9
Vitis vinifera
IIc
50
C-X4-C-X23-HXH
VvWRKY 51-1
GI:359476460
4
136
9.39
15.8
Vitis vinifera
IIc
51-1
C-X4-C-X23-HXH
VvWRKY 51-2
GI:359480857
7
149
9.07
17.2
Vitis vinifera
IIc
51-2
C-X4-C-X23-HXH
VvWRKY 51-3
GI:225466167
4
191
5.58
21.5
Vitis vinifera
IIc
51-3
C-X4-C-X23-HXH
VvWRKY 51-4
GI:359480856
7
193
7.1
21.5
Vitis vinifera
IIc
51-4
C-X4-C-X23-HXH
VvWRKY 53
GI:359490533
16
364
5.45
40
Vitis vinifera
III
53
C-X7-C-X23-HTC
VvWRKY 57-1
GI:225423515
1
305
5.62
33.4
Vitis vinifera
IIc
57-1
C-X4-C-X23-HXH
VvWRKY 57-2
GI:225425363
1
189
9.4
21.3
Vitis vinifera
IIc
57-2
C-X4-C-X23-HXH
VvWRKY 65-1
GI:225443744
10
278
5.14
3.9
Vitis vinifera
IIe
65-1
C-X5-C-X23-HXH
VvWRKY 65-2
GI:225446682
12
244
5.41
26.4
Vitis vinifera
IIe
65-2
C-X5-C-X23-HXH
VvWRKY 65-3
GI:359485307
12
244
5.4
26.4
Vitis vinifera
IIe
65-3
C-X5-C-X23-HXH
VvWRKY 55
GI:225448719
13
364
5.97
40.3
Vitis vinifera
III
55
C-X7-C-X23-HTC
VvWRKY 70-1
GI:225448721
13
313
5.45
35.3
Vitis vinifera
III
70-1
C-X7-C-X23-HTC
VvWRKY 70-2
GI:225439707
8
322
5.49
36.6
Vitis vinifera
III
70-2
C-X7-C-X23-HTC
VvWRKY 72-1
GI:359491334
17
611
7.9
65.7
Vitis vinifera
IIb
72-2
C-X5-C-X23-HXH
VvWRKY 72-2
GI:359488978
14
755
5.85
59.7
Vitis vinifera
IIb
72-2
C-X5-C-X23-HXH
VvWRKY 72-3
GI:359473376
1
547
5.92
59.7
Vitis vinifera
IIb
72-3
C-X5-C-X23-HXH
VvWRKY 74
GI:225463956
14
362
9.68
41.3
Vitis vinifera
IId
74
C-X5-C-X23-HXH
VvWRKY 75
GI:225456341
17
151
9.67
17.7
Vitis vinifera
IIc
75
C-X4-C-X23-HXH
VtWRKY5
GI:183979104
*
529
7.72
57.7
Vitis thunbergii
I-NTWD
Vt5N
C-X4-C-X22-HXH
I-CTWD
Vt5C
C-X4-C-X23-HXH
VtWRKY7
GI:183979108
*
603
6.42
66.3
Vitis thunbergii
I-NTWD
Vt7N
C-X4-C-X22-HXH
I-CTWD
Vt7C
C-X4-C-X23-HXH
VtWRKY11
GI:183979106
*
338
9.49
36.6
Vitis thunbergii
IId
Vt11
C-X5-C-X23-HXH
VaWRKY4
GI:40060529
*
311
8.71
34.4
Vitis aestivalis
IIa
Va4
C-X5-C-X23-HXH
VpWRKY1
GI:263199372
*
322
5.58
36.5
Vitis pseudoreticulata
III
Vp1
C-X7-C-X23-HTC
VpWRKY2
GI:290894627
*
499
6.23
53.9
Vitis pseudoreticulata
I-NTWD
Vp2N
C-X4-C-X22-HXH
I-CTWD
Vp2C
C-X4-C-X23-HXH
VpWRKY3
GI:345104746
*
319
7.67
35.5
Vitis pseudoreticulata
IIa
Vp3
C-X5-C-X23-HXH
VbcWRKY50
GI:163914201
*
127
9.4
14.3
Vitis hybrid cultivar
IId
Vbc50
C-X5-C-X23-HXH
aGenBank protein number
Chr.: chromosome; ORF: open reading frame; *chromosome unknown.The WRKY domain is approximately 60 amino acid residues in length and is considered to be a crucial element for interaction with the W-box (C/T)TGAC(T/C) to activate many defense-related genes. In our study, 80 WRKY domains contained highly conserved WRKYGQK sequences, while the other WRKY domains had one amino acid mismatch in the conserved WRKY sequence (Figure1). In VvWRKY51-2, 51-3, and 51-3, the WRKY domain sequence was WRKYGKK. As described by Eulgem et al. [7], the metal-chelating zinc finger motif (C-X
4
-
5-X
22
-
23-H-X-H or C-X
5
–
8-C-X
25
–
28-H-X
1
-
2-C) is another important characteristic of WRKY proteins. Zinc-finger-like motifs were identified in all of the grape WRKY proteins.The WRKY conservative domain. (a) Comparison of WRKY domain sequences from VvWRKY proteins. Sequences encoding the peptide stretch WRKYGQK were found by the BLAST programs tblastn and blastp programs in genomic and EST databases. Gaps (dots) have been inserted for optimal alignment. Residues that are highly conserved within each of the major groups are in black and potential zinc ligands are highlighted in red boxes, and the different amino acid residues are highlighted in green boxes. (b) The fourβ-strands are shown in red. I CT and I NT denote the N- and C-terminal WRKY domains from Group I WRKY proteins. As in (b), the β
1, β
2, β
3, and β
4 motifs are shown in green and the Zn finger is indicated by a green line.


### 3.2. 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 N-terminal domain). A multiple sequence alignment of the 80 WRKY domains was performed (Figure1). 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 (Figures2 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].Figure 2
Phylogenetic tree based on amino acid sequences as determined by the MEGA ver4.0 software using the neighbor-joining method. Bootstrap values (≥500) based on 1,000 replications are exhibited beside the nodes.Figure 3
Histogram of the number and distribution of three groups of WRKY genes on 19 chromosomes.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 inArabidopsis, poplar, or grape, suggesting that VvWRKY genes respond to different environmental signals.Figure 4
Phylogram of Group III WRKY domains fromArabidopsis (AtWRKY), rice (OsWRKY), poplar (PtWRKY), and grape (VvWRKY and VpWRKY). The alignment of amino acid sequences was produced using the MEGA ver4.0 program with the neighbor-joining method.


### 3.3. 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 inArabidopsis, 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.Table 2
Motif sequences.
Motif
Width
Best possible match
1
31
GCPVRKHVERCSEDPSMVITTYEGEHNHPVP
2
21
ILDDGYRWRKYGQKVIKGNPY
3
41
EKPSEDGYNWRKYGQKQVKGSEYPRSYYKCTHPNCPVKKKV
4
8
PRSYYRCT
5
29
EEINKKDKKKGHKKIREPRFCFQTRSEVD
6
31
ERSHDGQITEIIYKGTHNHPKPQPNRRYAVG
7
50
YRQMRPAKLPIARSPCFTIPPGLSPTCLLDSPVFLSNMKVEPSPTTGTFP
8
41
ETGVLVEELNRMNEENKKLREMLEIMCENYNALQMHLMELM
9
29
FLVEQMTAAITKDPNFTAALAAAISGIIL
10
29
SGRCHCSKRRKMRVKRTIRVPAISSKIAD
11
29
QMASMMCPISMSTPFPTITLDLTKPTSFS
12
29
INHFDCREITDYTVSKFKRVISILNRTGH
13
41
NRNNIHGSVGNNTYSTSMDDIFRKKREETDKIKFRRVYYIT
14
29
PAATAMASTTSAAASMLLSGSMTSQDGLM
15
15
GDEDDEDEPDSKRWK
16
29
GFSKMDEQIAIQEAASAGLKSMEHLIRLL
17
50
DPNGHANFQENPELGSQGQMGNLNKPNEGLPAYSLPGMDQETTQAMPLHL
18
24
WEHKTLINELTQGREMAKQLKIHL
19
41
TSMESVPIEVDYDKLQQRQHFNIGVQASQSEQKETNPIIVV
20
41
NWMAASLDLNANPLRLFDDTPKKEVQDDFTGLGLKVVSLKEFigure 5
Phylogenetic analysis of 80 members of theVvWRKY 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 theVvWRKY 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.


### 3.4. Quantitative RT-PCR Analysis of Expression of 28 WRKY Genes

To analyze expression patterns ofWRKY genes during plant defense responses, we analyzed the expression profiles of 28WRKY genes under disease conditions and in response to SA treatment using quantitative RT-PCR (Figure 6). Many of theWRKY genes exhibited significant changes in their expression levels in response to disease or SA. We isolated total RNA from leaves at various time points after infection with the fungal pathogenConiothyrium diplodiella or after treatment with SA. The expression patterns of the 28WRKY genes (VvWRKY1-1,VvWRKY2-1,VvWRKY3,VvWRKY6-1,VvWRKY7-1,VvWRKY11-4,VvWRKY14,VvWRKY18,VvWRKY22-1,VvWRKY28,VvWRKY30,VvWRKY32,VvWRKY40,VvWRKY41,VvWRKY42,VvWRKY45,VvWRVvKY46,VvWRKY48,VvWRKY51,VvWRKY53,VvWRKY55,VvWRKY65,VvWRKY70-1,VvWRKY70-2,WRKY72,andVvWRKY74) were determined. After pathogen infection, threeWRKY genes (VvWRKY48,VvWRKY51, andVvWRKY45) showed little or no change in transcript levels, but the other 25 genes showed altered expression patterns. Among the 25 differentially regulated genes, the expression of two (VvWRKY3 andVvWRKY41) was repressed and that of 23 was induced. After SA treatment, 5 genes (VvWRKY41, VvWRKY30, VvWRKY42, VvWRKY46,andVvWRKY70-1) showed little or no change in transcript levels, but expression of the other 23 genes was induced and upregulated. 16 of 23 (VvWRKY1-1, VvWRKY2-1, VvWRKY3, VvWRKY6-1, VvWRKY7-1, VvWRKY11-4, VvWRKY14, VvWRKY22-1, VvWRKY28, VvWRKY32, VvWRKY40, VvWRKY53, VvWRKY55, VvWRKY65, VvWRKY70-1, VvWRKY70-2, WRKY72,andVvWRKY74) upregulatedVvWRKY genes were induced and upregulated by both pathogen infection and SA treatment (Figure 6).Quantitative RT-PCR analysis of VdWRKY subgroup gene expression in response to pathogenic fungal infection and SA treatment. 0 h (control) indicates treatment with deionized water. Grape EFγ gene (accession number AF176496) expression served as an internal control.
(a)
(b)
(c)
(d)


## 3.1. 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. With the exception of VvWRKY1, VvWRKY2, VpWRKY1, and VpWRKY2, none of the proteins were described previously. Based on manual inspection using the MEGA ver4.0 software, seven sequences were discarded due to redundancy or insufficient conservation of WRKY sequences. The remaining WRKY proteins were reviewed using the Pfam program to confirm that all candidates contained the conserved WRKY motif. Properties of the proteins including numbers of amino acids, molecular weights, and isoelectric points (PI) are listed in Table1. The average VvWRKY sequence length was 382 amino acids and lengths ranged from 151 (VvWRKY1-1) to 798 (VvWRKY9) residues, while the isoelectric points (PI) ranged from 4.7 (VvWRKY22-4) to 9.84 (VvWRKY21).Table 1
Members of the grape WRKY superfamily of transcription factors.
Name
Proteina
Chr
Deduced polypeptide
Organism
Group
Domain
Length (aa)
PI
MW (kDa)
Family name
Pattern
VvWRKY 1-1
GI:50953501
*
151
9.61
17.7
Vitis vinifera
IIc
1-1
C-X4-C-X23-HXH
VvWRKY 1-2
GI:50953502
*
297
9.67
17.7
Vitis vinifera
IIc
1-2
C-X4-C-X23-HXH
VvWRKY 2-1
GI:359476618
4
700
6.52
75.9
Vitis vinifera
I-NTWD
2-1N
C-X4-C-X22-HXH
I-CTWD
2-1C
C-X4-C-X23-HXH
VvWRKY 2-2
GI:225463536
19
734
5.7
80.3
Vitis vinifera
I-NTWD
2-1N
C-X4-C-X22-HXH
I-CTWD
2-2C
C-X4-C-X23-HXH
VvWRKY 2-3
GI:225469228
*
536
7.21
58.44
Vitis vinifera
I-NTWD
2-3N
C-X4-C-X22-HXH
I-CTWD
2-3C
C-X4-C-X23-HXH
VvWRKY 2-4
GI:359478811
6
336
6.31
21.7
Vitis vinifera
IIc
2-4
C-X4-C-X23-HXH
VvWRKY 3-1
GI:48686707
1
317
7.64
58.3
Vitis vinifera
I-NTWD
3-1N
C-X4-C-X22-HXH
I-CTWD
3-1C
C-X4-C-X23-HXH
VvWRKY 3-2
GI:315272006
*
534
8.22
35.3
Vitis vinifera
IIa
3-2
C-X5-C-X23-HXH
VvWRKY 4
GI:315272008
*
625
8.71
34.4
Vitis vinifera
IIa
4
C-X5-C-X25-HXH
VvWRKY 6-1
GI:359485613
12
753
6.48
64.1
Vitis vinifera
IIb
6-1
C-X5-C-X23-HXH
VvWRKY 6-2
GI:225444291
10
535
6.04
57.5
Vitis vinifera
IIb
6-2
C-X5-C-X23-HXH
VvWRKY 7-1
GI:225458699
18
347
9.36
38.1
Vitis vinifera
IId
7-1
C-X5-C-X23-HXH
VvWRKY 7-2
GI:225438803
7
535
9.57
36.6
Vitis vinifera
IId
7-2
C-X5-C-X23-HXH
VvWRKY 9
GI:225447777
12
798
5.16
52.4
Vitis vinifera
IIb
9
C-X5-C-X23-HXH
VvWRKY 11-1
GI:225466161
4
338
9.49
36.6
Vitis vinifera
IId
11-1
C-X5-C-X23-HXH
VvWRKY 11-2
GI:225445976
11
297
9.73
32.4
Vitis vinifera
IId
11-2
C-X5-C-X23-HXH
VvWRKY 11-3
GI:262091438
*
297
9.73
32.4
Vitis vinifera
IId
1-3
C-X5-C-X23-HXH
VvWRKY 12-1
GI:225453346
15
228
7.68
26.1
Vitis vinifera
IIc
12-1
C-X4-C-X23-HXH
VvWRKY 12-2
GI:225453345
15
228
7.8
26.1
Vitis vinifera
IIc
12-2
C-X4-C-X23-HXH
VvWRKY 13-1
GI:359472522
1
305
5.8
33.4
Vitis vinifera
IIc
13-1
C-X4-C-X23-HXH
VvWRKY 13-1
GI:359472523
1
305
5.81
33.4
Vitis vinifera
IIc
13-1
C-X4-C-X23-HXH
VvWRKY 14
GI:225444177
10
438
5.16
47.5
Vitis vinifera
IIe
14
C-X5-C-X23-HXH
VvWRKY 18
GI:359476150
4
261
9.1
28.9
Vitis vinifera
IIa
18
C-X5-C-X23-HXH
VvWRKY 20-1
GI:359496861
*
604
6.01
65.7
Vitis vinifera
I-NTWD
20-1N
C-X4-C-X22-HXH
I-CTWD
20-1C
C-X4-C-X23-HXH
VvWRKY 20-2
GI:359494165
19
580
6.27
63.4
Vitis vinifera
I-NTWD
20-2N
C-X4-C-X23-HXH
I-CTWD
20-2C
C-X4-C-X23-HXH
VvWRKY 20-3
GI:225447598
12
407
5.02
44.8
Vitis vinifera
I-NTWD
20-3N
C-X4-C-X22-HXH
I-CTWD
20-3C
C-X4-C-X23-HXH
VvWRKY 20-4
GI:359496860
12
514
6.6
56.2
Vitis vinifera
I-NTWD
20-4N
C-X4-C-X23-HXH
I-CTWD
20-4C
C-X4-C-X23-HXH
VvWRKY 20-5
GI:359494164
19
595
6.8
65
Vitis vinifera
I-NTWD
20-5N
C-X4-C-X23-HXH
I-CTWD
20-5C
C-X4-C-X22-HXH
VvWRKY 20-6
GI:359485885
12
407
4.8
44.8
Vitis vinifera
I-NTWD
20-6N
C-X4-C-X23-HXH
I-CTWD
20-6C
C-X4-C-X23-HXH
VvWRKY 21
GI:225437249
7
340
9.84
38
Vitis vinifera
IId
21
C-X5-C-X23-HXH
VvWRKY 22-1
GI:359480165
7
233
6.06
26.8
Vitis vinifera
IIe
22-1
C-X5-C-X23-HXH
VvWRKY 22-2
GI:225454298
15
348
5.73
38
Vitis vinifera
IIe
22-2
C-X5-C-X23-HXH
VvWRKY 22-3
GI:225426142
2
331
5.71
36.6
Vitis vinifera
IIe
22-3
C-X5-C-X23-HXH
VvWRKY 22-4
GI:225464629
*
166
4.7
18.4
Vitis vinifera
IIe
22-4
C-X5-C-X23-HXH
VvWRKY 23
GI:225437606
7
302
6.74
33.8
Vitis vinifera
IIc
23
C-X4-C-X23-HXH
VvWRKY 24
GI:359489647
15
165
9.61
19
Vitis vinifera
IIc
24
C-X4-C-X23-HXH
VvWRKY 28-1
GI:225463412
10
319
6.76
35
Vitis vinifera
IIc
28-1
C-X4-C-X23-HXH
VvWRKY 28-2
GI:225446835
12
311
6.92
34.8
Vitis vinifera
IIc
28-2
C-X4-C-X23-HXH
VvWRKY 30
GI:40846374
*
349
5.02
39.3
Vitis aestivalis
III
30
C-X7-C-X23-HTC
VvWRKY 32-1
GI:225445873
11
499
6.12
53.8
Vitis vinifera
I-NTWD
32-1N
C-X4-C-X22-HXH
I-CTWD
32-1C
C-X4-C-X23-HXH
VvWRKY 32-2
GI:225430477
4
475
8.47
52.6
Vitis vinifera
I-NTWD
32-2N
C-X4-C-X22-HXH
I-CTWD
32-2C
C-X4-C-X23-HXH
VvWRKY 33-1
GI:225439574
8
552
7.29
61
Vitis vinifera
I-NTWD
33-1N
C-X4-C-X22-HXH
I-CTWD
33-1C
C-X4-C-X23-HXH
VvWRKY 33-2
GI:225434421
6
603
6.42
66.3
Vitis vinifera
I-NTWD
33-2N
C-X4-C-X22-HXH
I-CTWD
33-2C
C-X4-C-X23-HXH
VvWRKY 40-1
GI:225443178
9
311
5.16
52.4
Vitis vinifera
IIa
40-1
C-X5-C-X25-HXH
VvWRKY 40-2
GI:225430340
4
317
8.22
35.3
Vitis vinifera
IIa
40-2
C-X5-C-X23-HXH
VvWRKY 41
GI:225426000
2
342
6.05
38.6
Vitis vinifera
III
41
C-X7-C-X23-HTC
VvWRKY 42
GI:359494147
19
511
9.2
55.1
Vitis vinifera
IIb
42
C-X5-C-X23-HXH
VvWRKY 44
GI:225439779
8
477
8.84
52.3
Vitis vinifera
I-NTWD
44C
C-X4-C-X22-HXH
I-CTWD
44N
C-X4-C-X23-HXH
VvWRKY 45
GI:225451489
14
182
9.41
20.8
Vitis vinifera
IIc
45
C-X4-C-X23-HXH
VvWRKY 46
GI:225454483
15
349
5.01
39.2
Vitis vinifera
III
46
C-X7-C-X23-HTC
VvWRKY 47
GI:225437767
7
505
8.03
54.7
Vitis vinifera
IIb
47
C-X5-C-X23-HXH
VvWRKY 48
GI:225432004
5
309
5.72
34.4
Vitis vinifera
IIc
48
C-X4-C-X23-HXH
VvWRKY 49
GI:225440394
8
299
5.16
52.4
Vitis vinifera
IIc
49
C-X4-C-X23-HXH
VvWRKY 50
GI:225429590
4
166
5.2
18.9
Vitis vinifera
IIc
50
C-X4-C-X23-HXH
VvWRKY 51-1
GI:359476460
4
136
9.39
15.8
Vitis vinifera
IIc
51-1
C-X4-C-X23-HXH
VvWRKY 51-2
GI:359480857
7
149
9.07
17.2
Vitis vinifera
IIc
51-2
C-X4-C-X23-HXH
VvWRKY 51-3
GI:225466167
4
191
5.58
21.5
Vitis vinifera
IIc
51-3
C-X4-C-X23-HXH
VvWRKY 51-4
GI:359480856
7
193
7.1
21.5
Vitis vinifera
IIc
51-4
C-X4-C-X23-HXH
VvWRKY 53
GI:359490533
16
364
5.45
40
Vitis vinifera
III
53
C-X7-C-X23-HTC
VvWRKY 57-1
GI:225423515
1
305
5.62
33.4
Vitis vinifera
IIc
57-1
C-X4-C-X23-HXH
VvWRKY 57-2
GI:225425363
1
189
9.4
21.3
Vitis vinifera
IIc
57-2
C-X4-C-X23-HXH
VvWRKY 65-1
GI:225443744
10
278
5.14
3.9
Vitis vinifera
IIe
65-1
C-X5-C-X23-HXH
VvWRKY 65-2
GI:225446682
12
244
5.41
26.4
Vitis vinifera
IIe
65-2
C-X5-C-X23-HXH
VvWRKY 65-3
GI:359485307
12
244
5.4
26.4
Vitis vinifera
IIe
65-3
C-X5-C-X23-HXH
VvWRKY 55
GI:225448719
13
364
5.97
40.3
Vitis vinifera
III
55
C-X7-C-X23-HTC
VvWRKY 70-1
GI:225448721
13
313
5.45
35.3
Vitis vinifera
III
70-1
C-X7-C-X23-HTC
VvWRKY 70-2
GI:225439707
8
322
5.49
36.6
Vitis vinifera
III
70-2
C-X7-C-X23-HTC
VvWRKY 72-1
GI:359491334
17
611
7.9
65.7
Vitis vinifera
IIb
72-2
C-X5-C-X23-HXH
VvWRKY 72-2
GI:359488978
14
755
5.85
59.7
Vitis vinifera
IIb
72-2
C-X5-C-X23-HXH
VvWRKY 72-3
GI:359473376
1
547
5.92
59.7
Vitis vinifera
IIb
72-3
C-X5-C-X23-HXH
VvWRKY 74
GI:225463956
14
362
9.68
41.3
Vitis vinifera
IId
74
C-X5-C-X23-HXH
VvWRKY 75
GI:225456341
17
151
9.67
17.7
Vitis vinifera
IIc
75
C-X4-C-X23-HXH
VtWRKY5
GI:183979104
*
529
7.72
57.7
Vitis thunbergii
I-NTWD
Vt5N
C-X4-C-X22-HXH
I-CTWD
Vt5C
C-X4-C-X23-HXH
VtWRKY7
GI:183979108
*
603
6.42
66.3
Vitis thunbergii
I-NTWD
Vt7N
C-X4-C-X22-HXH
I-CTWD
Vt7C
C-X4-C-X23-HXH
VtWRKY11
GI:183979106
*
338
9.49
36.6
Vitis thunbergii
IId
Vt11
C-X5-C-X23-HXH
VaWRKY4
GI:40060529
*
311
8.71
34.4
Vitis aestivalis
IIa
Va4
C-X5-C-X23-HXH
VpWRKY1
GI:263199372
*
322
5.58
36.5
Vitis pseudoreticulata
III
Vp1
C-X7-C-X23-HTC
VpWRKY2
GI:290894627
*
499
6.23
53.9
Vitis pseudoreticulata
I-NTWD
Vp2N
C-X4-C-X22-HXH
I-CTWD
Vp2C
C-X4-C-X23-HXH
VpWRKY3
GI:345104746
*
319
7.67
35.5
Vitis pseudoreticulata
IIa
Vp3
C-X5-C-X23-HXH
VbcWRKY50
GI:163914201
*
127
9.4
14.3
Vitis hybrid cultivar
IId
Vbc50
C-X5-C-X23-HXH
aGenBank protein number
Chr.: chromosome; ORF: open reading frame; *chromosome unknown.The WRKY domain is approximately 60 amino acid residues in length and is considered to be a crucial element for interaction with the W-box (C/T)TGAC(T/C) to activate many defense-related genes. In our study, 80 WRKY domains contained highly conserved WRKYGQK sequences, while the other WRKY domains had one amino acid mismatch in the conserved WRKY sequence (Figure1). In VvWRKY51-2, 51-3, and 51-3, the WRKY domain sequence was WRKYGKK. As described by Eulgem et al. [7], the metal-chelating zinc finger motif (C-X
4
-
5-X
22
-
23-H-X-H or C-X
5
–
8-C-X
25
–
28-H-X
1
-
2-C) is another important characteristic of WRKY proteins. Zinc-finger-like motifs were identified in all of the grape WRKY proteins.The WRKY conservative domain. (a) Comparison of WRKY domain sequences from VvWRKY proteins. Sequences encoding the peptide stretch WRKYGQK were found by the BLAST programs tblastn and blastp programs in genomic and EST databases. Gaps (dots) have been inserted for optimal alignment. Residues that are highly conserved within each of the major groups are in black and potential zinc ligands are highlighted in red boxes, and the different amino acid residues are highlighted in green boxes. (b) The fourβ-strands are shown in red. I CT and I NT denote the N- and C-terminal WRKY domains from Group I WRKY proteins. As in (b), the β
1, β
2, β
3, and β
4 motifs are shown in green and the Zn finger is indicated by a green line.


## 3.2. 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 N-terminal domain). A multiple sequence alignment of the 80 WRKY domains was performed (Figure1). 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 (Figures2 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].Figure 2
Phylogenetic tree based on amino acid sequences as determined by the MEGA ver4.0 software using the neighbor-joining method. Bootstrap values (≥500) based on 1,000 replications are exhibited beside the nodes.Figure 3
Histogram of the number and distribution of three groups of WRKY genes on 19 chromosomes.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 inArabidopsis, poplar, or grape, suggesting that VvWRKY genes respond to different environmental signals.Figure 4
Phylogram of Group III WRKY domains fromArabidopsis (AtWRKY), rice (OsWRKY), poplar (PtWRKY), and grape (VvWRKY and VpWRKY). The alignment of amino acid sequences was produced using the MEGA ver4.0 program with the neighbor-joining method.


## 3.3. 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 inArabidopsis, 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.Table 2
Motif sequences.
Motif
Width
Best possible match
1
31
GCPVRKHVERCSEDPSMVITTYEGEHNHPVP
2
21
ILDDGYRWRKYGQKVIKGNPY
3
41
EKPSEDGYNWRKYGQKQVKGSEYPRSYYKCTHPNCPVKKKV
4
8
PRSYYRCT
5
29
EEINKKDKKKGHKKIREPRFCFQTRSEVD
6
31
ERSHDGQITEIIYKGTHNHPKPQPNRRYAVG
7
50
YRQMRPAKLPIARSPCFTIPPGLSPTCLLDSPVFLSNMKVEPSPTTGTFP
8
41
ETGVLVEELNRMNEENKKLREMLEIMCENYNALQMHLMELM
9
29
FLVEQMTAAITKDPNFTAALAAAISGIIL
10
29
SGRCHCSKRRKMRVKRTIRVPAISSKIAD
11
29
QMASMMCPISMSTPFPTITLDLTKPTSFS
12
29
INHFDCREITDYTVSKFKRVISILNRTGH
13
41
NRNNIHGSVGNNTYSTSMDDIFRKKREETDKIKFRRVYYIT
14
29
PAATAMASTTSAAASMLLSGSMTSQDGLM
15
15
GDEDDEDEPDSKRWK
16
29
GFSKMDEQIAIQEAASAGLKSMEHLIRLL
17
50
DPNGHANFQENPELGSQGQMGNLNKPNEGLPAYSLPGMDQETTQAMPLHL
18
24
WEHKTLINELTQGREMAKQLKIHL
19
41
TSMESVPIEVDYDKLQQRQHFNIGVQASQSEQKETNPIIVV
20
41
NWMAASLDLNANPLRLFDDTPKKEVQDDFTGLGLKVVSLKEFigure 5
Phylogenetic analysis of 80 members of theVvWRKY 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 theVvWRKY 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.


## 3.4. Quantitative RT-PCR Analysis of Expression of 28 WRKY Genes

To analyze expression patterns ofWRKY genes during plant defense responses, we analyzed the expression profiles of 28WRKY genes under disease conditions and in response to SA treatment using quantitative RT-PCR (Figure 6). Many of theWRKY genes exhibited significant changes in their expression levels in response to disease or SA. We isolated total RNA from leaves at various time points after infection with the fungal pathogenConiothyrium diplodiella or after treatment with SA. The expression patterns of the 28WRKY genes (VvWRKY1-1,VvWRKY2-1,VvWRKY3,VvWRKY6-1,VvWRKY7-1,VvWRKY11-4,VvWRKY14,VvWRKY18,VvWRKY22-1,VvWRKY28,VvWRKY30,VvWRKY32,VvWRKY40,VvWRKY41,VvWRKY42,VvWRKY45,VvWRVvKY46,VvWRKY48,VvWRKY51,VvWRKY53,VvWRKY55,VvWRKY65,VvWRKY70-1,VvWRKY70-2,WRKY72,andVvWRKY74) were determined. After pathogen infection, threeWRKY genes (VvWRKY48,VvWRKY51, andVvWRKY45) showed little or no change in transcript levels, but the other 25 genes showed altered expression patterns. Among the 25 differentially regulated genes, the expression of two (VvWRKY3 andVvWRKY41) was repressed and that of 23 was induced. After SA treatment, 5 genes (VvWRKY41, VvWRKY30, VvWRKY42, VvWRKY46,andVvWRKY70-1) showed little or no change in transcript levels, but expression of the other 23 genes was induced and upregulated. 16 of 23 (VvWRKY1-1, VvWRKY2-1, VvWRKY3, VvWRKY6-1, VvWRKY7-1, VvWRKY11-4, VvWRKY14, VvWRKY22-1, VvWRKY28, VvWRKY32, VvWRKY40, VvWRKY53, VvWRKY55, VvWRKY65, VvWRKY70-1, VvWRKY70-2, WRKY72,andVvWRKY74) upregulatedVvWRKY genes were induced and upregulated by both pathogen infection and SA treatment (Figure 6).Quantitative RT-PCR analysis of VdWRKY subgroup gene expression in response to pathogenic fungal infection and SA treatment. 0 h (control) indicates treatment with deionized water. Grape EFγ gene (accession number AF176496) expression served as an internal control.
(a)
(b)
(c)
(d)


## 4. 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 plantsArabidopsis and rice and in the woody plant poplar. Characterization of theWRKY genes in grape (Vitisspp.) would facilitate a broader understanding of this gene superfamily. In this study, 80WRKY genes were characterized. The lengths of these sequences were highly varied implying a high degree of complexity among theVvWRKY genes.TheWRKY genes can be divided into three main groups based on their structural features. The Groups Ib and IIWRKY genes are likely to have evolved from Group IaWRKY genes through loss of the N-terminal WRKY domain. Replacement of the conserved His residue in the metal-chelating zinc finger motif with a Cys residue may have led to the evolution of Group IIIWRKY genes [33]. The similar numbers ofArabidopsis, rice, poplar, and grapeWRKY genes in Groups IIa and IIb suggest that allVvWRKY genes belonging to these subgroups have been identified, but there were only Group Ia in grape (Table 3). Fewer Group IIIWRKYgenes have been identified in grape compared to inArabidopsis and rice, close to the poplar quantitatively, implying that the number ofVvWRKY genes in this group has either declined over evolutionary time or was underestimated in our analysis. Of theVvWRKY genes, 67.9% belong to Group II and 9.7% to Group III. This distribution is more 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 fromArabidopsis, rice, poplar, and grape.Table 3
Numbers of the various types ofWRKY genes in Arabidopsis, rice, poplar, and grape.
Group
Subgroup
Gene number
AtWRKYa
OsWRKYa
PtWRKYa
VvWRKYa
VxWRKYb
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
aAccording to Wu et al. [20] and He et al. [21].
b
Vitis thunbergii, Vitis thunbergii, Vitis pseudoreticulata, and Vitis hybrid cultivar.TheWRKY genes may act as a regulatory node that plays a crucial role in responses to abiotic stresses or in stress-induced 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 ofArabidopsis thaliana was in the SA-signal transduction pathway leading to PR gene expression [15]. Many of theWRKY 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 ofAtWRKYgenes inArabidopsisshowed that almost 70% are differentially regulated in response to pathogen infection and SA treatment, suggesting that the major role ofWRKYgenes in flowering plants is to mediate defense responses [41]. Details of the roles of theWRKYgenes in defending dicotyledonous plants against pathogens can be found in three excellent reviews [4, 7, 42]. In our results,WRKYgenes in grape showed that almost 57% (16 genes) were differentially regulated in response to both pathogen infection and SA treatment. It was lower thanArabidopsis. We speculated that the reason was thatWRKY genes belong toV. vinifera PN40024 genomic sequence, and the test material wasV. vinifera“Pinor,” which was susceptible cultivars Europe grape.



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*Source: 787680-2014-04-27.xml*