This Article Abstract Full Text (PDF) PPT slides of all figures Supplemental Data All Versions of this Article: 20/9/2357    most recent tpc.107.055566v1 Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Related articles in Plant Cell Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Kim, K.-C. Articles by Chen, Z. Search for Related Content PubMed PubMed Citation Articles by Kim, K.-C. Articles by Chen, Z. Agricola Articles by Kim, K.-C. Articles by Chen, Z.

Arabidopsis WRKY38 and WRKY62 Transcription Factors Interact with Histone Deacetylase 19 in Basal Defense^[W]

Department of Botany and Plant Pathology, Purdue University, West Lafayette, Indiana 47907-2054

¹ Address correspondence to zhixiang{at}purdue.edu.

	ABSTRACT

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION METHODS REFERENCES

 
Arabidopsis thaliana WRKY38 and WRKY62, encoding two structurally similar type III WRKY transcription factors, are induced in a Nonexpressor of PR Gene1 (NPR1)–dependent manner by salicylic acid (SA) or by virulent Pseudomonas syringae. Disease resistance and SA-regulated Pathogenesis-Related1 (PR1) gene expression are enhanced in the wrky38 and wrky62 single mutants and, to a greater extent, in the double mutants. Overexpression of WRKY38 or WRKY62 reduces disease resistance and PR1 expression. Thus, WRKY38 and WRKY62 function additively as negative regulators of plant basal defense. WRKY38 and WRKY62 interact with Histone Deacetylase 19 (HDA19). Expression of HDA19 is also induced by P. syringae, and the stability of its induced transcripts depends on SA and NPR1 in infected plants. Disruption of HDA19 leads to compromised resistance, whereas its overexpression results in enhanced resistance to P. syringae. Thus, HDA19 has a role opposite from those of WRKY38 and WRKY62 in basal resistance to the bacterial pathogen. Both WRKY38 and WRKY62 are transcriptional activators in plant cells, but their activation activities are abolished by overexpressed HDA19. Interaction of WRKY38 and WRKY62 with HDA19 may act to fine-tune plant basal defense responses.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION METHODS REFERENCES

 
Detection of an invading pathogen by a plant triggers a complex set of signal transduction pathways and a battery of defense mechanisms. Upon perception of pathogen/microbe-associated molecular patterns (PAMPs), plants can activate distinct mitogen-activated protein kinase cascades, leading to PAMP-triggered immunity (PTI) (Jones and Dangl, 2006). Successful pathogens suppress PTI through secreted effector proteins and, as a result, cause diseases. Coevolution of plant hosts with the virulent pathogens can give rise to specific plant disease resistance (R) proteins that recognize pathogen effectors and activate highly efficient effector-triggered immunity (ETI) (Jones and Dangl, 2006).

Both PTI and ETI are associated with the accumulation of defense signal molecules such as salicylic acid (SA), ethylene (ET), and jasmonic acid (JA). In Arabidopsis thaliana, SA-regulated defense responses including Pathogenesis-Related (PR) gene expression require the function of the Nonexpressor of PR Gene1 (NPR1) gene, which encodes a 66-kD protein with ankyrin repeats (Cao et al., 1997). Arabidopsis mutants deficient in SA biosynthesis or signaling are compromised in resistance to biotrophic pathogens that feed on living host tissue during the whole or part of their infection cycle. ET- and JA-mediated signaling pathways, on the other hand, often mediate plant defense against necrotrophic pathogens that promote host cell death at early stages of infection (Glazebrook, 2004). A number of studies have shown that SA and ET/JA signaling pathways are mutually antagonistic (Kunkel and Brooks, 2002). For example, mutations of JA signaling regulators such as COI1 can enhance SA accumulation and signaling in pathogen-infected plants (Kloek et al., 2001), and blocking SA accumulation can promote JA signaling (Spoel et al., 2003). Other studies have shown that SA and ET/JA signaling pathways can interact positively or even synergistically in plant defense responses (Kloek et al., 2001; Mur et al., 2006). It has been suggested that the apparent discrepancy of the relationship between SA and JA signaling may arise from the concentration-specific outcomes of their interactions (Mur et al., 2006). When both signals were applied at low levels, a transient synergistic enhancement was observed in the expression of genes associated with JA or SA. When applied at high levels or at prolonged times, the two signals become antagonistic to each other.

WRKY DNA binding transcription factors play important roles in the regulation of genes associated with plant defense responses (Eulgem and Somssich, 2007). Recent mutant analyses in Arabidopsis have revealed direct links between specific WRKY proteins and complex defense responses. Arabidopsis WRKY70 has been shown to regulate the crosstalk between SA- and JA-mediated signaling by promoting SA-dependent and suppressing JA-dependent responses (Li et al., 2004, 2006). Mutations of WRKY70 have been shown to enhance plant susceptibility to both biotrophic and necrotrophic pathogens, including the bacterial pathogen Erwinia carotovora, as well as the fungal pathogens Erysiphe cichoracearum and Botrytis cinerea (Li et al., 2004, 2006; AbuQamar et al., 2006). In addition, wrky70 mutants are compromised in both basal defense and R gene (RPP4)–mediated disease resistance to the oomycete Hyaloperonospora parasitica (Knoth et al., 2007). By contrast, a number of Arabidopsis WRKY proteins, including WRKY7, WRKY11, and WRKY17, function as negative regulators of plant basal defense (Park et al., 2005; Journot-Catalino et al., 2006; Kim et al., 2006). Mutations of these genes enhance basal plant resistance to virulent Pseudomonas syringae strains. The structurally related WRKY18, WRKY40, and WRKY60 have partially redundant roles as negative regulators in plant resistance against the biotrophic bacterial pathogen P. syringae and the fungal pathogen E. cichoracearum (Xu et al., 2006; Shen et al., 2007). The wrky18 wrky40 double mutant and the wrky18 wrky40 wrky60 triple mutant, however, are more susceptible to the necrotrophic fungal pathogen B. cinerea (Xu et al., 2006). Thus, these three WRKY proteins appear to be involved in the antagonistic crosstalk of defense mechanisms against different types of microbial pathogens. In a previously reported study, Arabidopsis mitogen-activated protein kinase4 (MPK4), an activator of JA/ET-mediated defense and a repressor of SA-dependent resistance (Petersen et al., 2000), was found to interact with the MPK4 substrate MKS1, which, in turn, interacts with Arabidopsis WRKY25 and WRKY33 (Andreasson et al., 2005). In addition, WRKY25 and WRKY33 are phosphorylated by MPK4 in vitro, and a wrky33 knockout mutant expresses elevated levels of PR1 under short-day growth conditions (Andreasson et al., 2005). Disruption of WRKY33 results in enhanced susceptibility to necrotrophic fungal pathogens and impaired expression of JA/ET-regulated defense genes (Zheng et al., 2006). These results indicate that WRKY33 functions as a positive regulator of JA/ET-mediated pathways and plays an important role in disease resistance to necrotrophic fungal pathogens. Although the wrky33 mutants respond normally to P. syringae, mutations of WRKY25 enhance tolerance to the bacterial pathogen (Zheng et al., 2007). In addition, overexpression of either WRKY25 or WRKY33 enhances susceptibility to the bacterial pathogen and suppresses SA-regulated PR1 gene expression (Zheng et al., 2006, 2007). These results suggest that WRKY25 and WRKY33 function as downstream components of the MPK4-mediated SA-repressing and JA/ET-activating signaling pathways. Thus, WRKY transcription factors play diverse roles in plant defense responses. These diverse roles of WRKY proteins may reflect the complex signaling and transcriptional networks of plant defense through the actions of a wide range of interactive positive and negative regulators.

Arabidopsis WRKY38 and WRKY62, encoding two structurally related type III WRKY transcription factors, are induced by both pathogen infection and SA treatment (Yu et al., 2001; Dong et al., 2003; Kalde et al., 2003; Mao et al., 2007). In this study, we report that WRKY38 and WRKY62 function additively as negative regulators of plant basal defense. WRKY38 and WRKY62 interact in the nucleus with Histone Deacetylase 19 (HDA19), which functions as a positive regulator of plant basal disease resistance. Both WRKY38 and WRKY62 activate transcription in plant cells, but this activity can be abolished by overexpressed HDA19. Thus, the physical interactions provide a specific mechanism for the functional antagonism of the defense-repressing WRKY transcriptional activators for the defense-activating HDA19 transcriptional repressor that may function in the tight regulation and fine-tuning of plant defense responses.

	RESULTS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION METHODS REFERENCES

 
Structures, DNA Binding, and Subcellular Localization of WRKY38 and WRKY62
WRKY proteins can be classified into three groups (Eulgem et al., 2000). The first group contains two Cys₂His₂ zinc-finger motifs, and the second group contains only one Cys₂His₂ zinc-finger motif. The third group of WRKY proteins contains a single Cys₂HisCys motif. Based on this classification, WRKY38 and WRKY62 belong to group III WRKY proteins, each with a single Cys₂HisCys motif (see Supplemental Figure 1A online). Besides the conserved WRKY domains, the two proteins share substantial levels of homology at their N termini, but the sequence similarity at their C termini is relatively low (see Supplemental Figure 1A online).

WRKY transcription factors are thought to function by binding their cognate TTGACC/T W-box cis elements in the promoter regions of target genes and activating or repressing their expression (Ulker and Somssich, 2004). A number of isolated WRKY proteins have been shown to bind W-box sequences (Rushton et al., 1996; Chen and Chen, 2000; Yu et al., 2001). To examine the DNA binding activity of WRKY38 and WRKY62, we expressed the genes in Escherichia coli, purified the recombinant protein, and assayed their binding to an oligonucleotide that contains a TTGACC W-box sequence (P12a; see Supplemental Figure 1B online) using electrophoretic mobility shift assays. A protein/DNA complex with reduced mobility was detected when purified recombinant WRKY38 or WRKY62 protein was incubated with the P12a probe (see Supplemental Figure 1B online). Binding of the WRKY proteins to a mutant probe (mP12a) in which the TTGACC sequence was changed to TTGAAC was not detectable (see Supplemental Figure 1B online). Thus, binding of WRKY38 and WRKY62 to the TTGACC W-box sequence is highly specific.

To determine the subcellular location of WRKY38 and WRKY62, we constructed green fluorescent protein (GFP) fusions to the C termini of the two WRKY proteins. The fusion constructs, driven by the cauliflower mosaic virus (CaMV) 35S promoter, were directly bombarded into onion (Allium cepa) epidermal cells. The transiently expressed WRKY38-GFP and WRKY62-GFP fusion proteins were localized exclusively to the nucleus (see Supplemental Figure 1C online). By contrast, GFP was found in both the nucleus and cytoplasm (see Supplemental Figure 1C online).

SA- and NPR1-Dependent Expression of WRKY38 and WRKY62
To analyze the involvement of WRKY38 and WRKY62 in plant basal defense, we analyzed their expression in response to the virulent P. syringae pv tomato strain DC3000 (PstDC3000). As shown in Figure 1A , WRKY38 and WRKY62 transcripts were undetectable in healthy uninfected plants or in plants infiltrated with 50 mM MgCl₂ (mock inoculation). In plants infiltrated with PstDC3000, transcripts for WRKY38 or WRKY62 remained undetectable at 4 h after inoculation (HAI) but were elevated at 8 and 12 HAI before declining to almost basal levels by 24 HAI (Figure 1A). To determine the role of SA in pathogen-induced WRKY38 and WRKY62 expression, we compared their pathogen-induced expression in wild-type plants with those in the SA signaling–defective npr1-3 mutant and the sid2-3 mutant (SALK_133146), which contains a T-DNA insertion in the SID2 gene important for SA biosynthesis (Cao et al., 1997; Wildermuth et al., 2001). As shown in Figure 1B, transcripts for both WRKY38 and WRKY62 were elevated at 8 and 12 HAI in pathogen-infected wild-type plants but not in the pathogen-infected sid2 and npr1 mutants. Quantitative RT-PCR showed that the levels of WRKY38 and WRKY62 transcripts were elevated 8- to 10-fold at 8 and 12 HAI in wild-type plants but not in the sid2 and npr1 mutants (see Supplemental Figure 2 online). Thus, induction of WRKY38 and WRKY62 by the virulent bacterial pathogen is dependent on SA and NPR1.

View larger version (60K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 1. Pathogen- and SA-Induced Expression of WRKY38 and WRKY62. (A) Time course of pathogen-induced expression of WRKY38 and WRKY62. Five-week-old Arabidopsis plants (Col-0) were infiltrated with 10 mM MgCl2 or PstDC3000 (OD600 = 0.0001 in 10 mM MgCl2). The infiltrated leaves were collected at the indicated times after inoculation for RNA isolation. RNA gel blot analysis was performed with a 32P-labeled WRKY38 probe. The blot was stripped and reprobed with the WRKY62 probe. Ethidium bromide staining of rRNA is shown for the assessment of equal loading. (B) SA and NPR1 dependence of pathogen-induced expression of WRKY38 and WRKY62. Five-week-old wild-type (Col-0), npr1-3, and sid2-3 mutant plants were infiltrated with PstDC3000. Leaf collection, RNA isolation, and RNA gel blot analysis of WRKY38 and WRKY62 expression were performed as in (A). (C) Time course of SA-induced expression of WRKY38 and WRKY62. Five-week-old Arabidopsis plants (Col-0) were sprayed with water or SA (1 mM). Leaf collection, RNA isolation, and RNA gel blot analysis of WRKY38 and WRKY62 expression were performed as in (A). (D) NPR1 dependence of SA-induced expression of WRKY38 and WRKY62. Five-week-old wild-type (Col-0) and npr1-3 mutant plants were sprayed with 1 mM SA. Leaf collection, RNA isolation, and RNA gel blot analysis of WRKY38 and WRKY62 expression were performed as in (A). These experiments were performed three times with similar results.

Disruption of WRKY38 and WRKY62 Enhances Plant Basal Defense
To analyze the role of WRKY38 and WRKY62 directly, we identified T-DNA insertion or transposon-tagged mutants for both WRKY38 and WRKY62. The wrky38-1 mutant (WiscDsLox489-492C21; Columbia [Col-0] ecotype) contains a T-DNA insertion in the second intron, while wrky38-2 (RATM11-6950-1_H; Nossen-0 [No-0] ecotype) contains a Ds transposon insertion in the last exon of the WRKY38 gene (see Supplemental Figure 3A online). The wrky62-1 (GABI_016H10; Col-0 ecotype) and wrky62-2 (RATM11-6212-1_G; No-0 ecotype) mutants contain a T-DNA and a Ds transposon insertion, respectively, in the second exon of the WRKY62 gene (see Supplemental Figure 3A online). Homozygous mutant plants were identified by PCR with WRKY38- or WRKY62-specific primers. RNA gel blot analysis failed to detect WRKY38 or WRKY62 transcripts of the expected sizes in the respective homozygous mutants after SA treatment (see Supplemental Figures 3B and 3C online). To determine possible functional redundancy, we also generated the wrky38-1 wrky62-1 (Col-0 ecotype) and wrky38-2 wrky62-2 (No-0 ecotype) double mutants through genetic crossing. The wrky38 and wrky62 single and double mutants showed no differences in growth, development, or morphology from wild-type plants.

To determine possible changes of the mutants in plant basal disease resistance, we inoculated them with PstDC3000 and monitored both bacterial growth and disease symptom development. As shown in Figures 2A and 2B , the wrky38 and wrky62 single mutants had 2.5- to 4-fold reductions in the growth of the bacterial pathogen. The single mutants also developed significantly less severe disease symptoms than wild-type plants after infection (Figures 2C and 2D). A greater reduction (8- to 11-fold) of bacterial growth was observed in the wrky38 wrky62 double mutants relative to that in the wild-type plants (Figures 2A and 2B). The marked reduction in bacterial growth in the double mutants was accompanied by a substantially reduced development of disease symptoms (Figures 2C and 2D). These results suggest that WRKY38 and WRKY62 function additively with a negative role in basal resistance to the virulent bacterial pathogen.

View larger version (45K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 2. Altered Responses of the WRKY Mutants to PstDC3000. (A) and (B) Altered bacterial growth in the WRKY mutants. Wild type, single mutants, and double mutants for WRKY38 and WRKY62 were infiltrated with a suspension of PstDC3000 (OD600 = 0.0001 in 10 mM MgCl2). Samples were taken at 0 (open bars) or 3 (closed bars) DAI to determine bacterial growth. Means and SE were calculated from 10 plants for each treatment. According to Duncan's multiple range test (P = 0.05), means of colony-forming units do not differ at 0 DAI significantly if they are indicated with the same lowercase letter and do not differ significantly at 3 DAI if they are indicated with the same uppercase letter. (C) and (D) Altered disease symptom development in the WRKY mutants. Pathogen inoculation of wild-type and mutant plants was performed as in (A) and (B). Photographs of pairs of representative inoculated leaves were taken at 4 DAI (C) and 5 DAI (D). (E) and (F) Pathogen-induced PR1 expression. Wild type, single mutants, and double mutants for WRKY38 and WRKY62 were infiltrated with a suspension of PstDC3000 (OD600 = 0.0001 in 10 mM MgCl2). Inoculated leaves were collected at the indicated DAI for RNA isolation. RNA gel blot analysis was performed with 32P-labeled PR1. These experiments were performed four times with similar results.

Constitutive overexpression of a number of Arabidopsis WRKY genes, such as WRKY7 and WRKY18, resulted in reduced growth, altered flowering time, and changed leaf morphology (Chen and Chen, 2002; Kim et al., 2006). Analysis of F3 homozygous plants from WRKY38- or WRKY62-overexpressing transgenic plants revealed no differences in growth, development, or morphology from wild-type plants. Following inoculation with PstDC3000, the transgenic WRKY38 and WRKY62 overexpression lines displayed greater bacterial growth (4- to 6-fold) than wild-type plants (Figure 3A ). The inoculated leaves of WRKY38- and WRKY62-overexpressing plants also developed more severe disease symptoms than those of wild-type plants after infection (Figure 3B). For comparison, the npr1-3 mutant had 10-fold higher bacterial growth than wild-type plants and developed even more severe disease symptoms than the WRKY38- or WRKY62-overexpressing plants (Figures 3A and 3B). These results support the hypothesis that WRKY38 and WRKY62 have negative roles in basal resistance to the bacterial pathogen.

View larger version (43K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 3. Characterization of WRKY38 and WRKY62 Overexpression Lines. (A) Altered bacterial growth. Wild-type, overexpression line, and npr1 mutant plants were infiltrated with a suspension of PstDC3000 (OD600 = 0.0001 in 10 mM MgCl2). Samples were taken at 0 (open bars) or 3 (closed bars) DAI to determine the growth of the bacterial pathogen. Means and SE were calculated from 10 plants for each treatment. According to Duncan's multiple range test (P = 0.05), means of colony-forming units do not differ significantly at 0 DAI if they are indicated with the same lowercase letter and do not differ significantly at 3 DAI if they are indicated with the same uppercase letter. (B) Altered disease symptom development. Pathogen inoculation was performed as in (A). Photographs of representative inoculated leaves were taken at 3 DAI. (C) Pathogen-induced PR1 expression. Wild-type, overexpression line, and npr1 mutant plants were infiltrated with a suspension of PstDC3000 (OD600 = 0.0001 in 10 mM MgCl2). Inoculated leaves were collected at the indicated DAI for RNA isolation. RNA gel blot analysis was performed with 32P-labeled PR1. These experiments were performed four times with similar results.

To further investigate the role of WRKY38 and WRKY62 in SA-regulated defense gene expression, we examined the sensitivity of the mutants and overexpression lines to SA for PR1 expression. The mutant and overexpression plants were sprayed with various concentrations of SA and examined for PR1 expression 24 h later. As shown in Figure 4 , the wrky38-1 and wrky62-1 single mutants had slightly higher levels of PR1 transcripts than wild-type plants, particularly after treatment with relatively higher concentrations (0.5 and 1.0 mM) of SA. The wrky38-1 wrky62-1 double mutant had substantially higher levels of PR1 transcripts than wild-type plants at all of the SA concentrations tested (Figure 4). By contrast, the WRKY38- and WRKY62-overexpressing lines accumulated lower levels of PR1 transcripts than wild-type plants after treatment of different concentrations of SA (Figure 4). Thus, WRKY38 and WRKY63 negatively regulate plant responsiveness to SA for PR1 expression.

View larger version (78K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 4. Roles of WRKY38 and WRKY62 in Plant SA Sensitivity for PR1 Induction. Five-week-old wild-type (Col-0), knockout mutant, and overexpression plants for WRKY38 and WRKY62 were sprayed with SA at the indicated concentrations. Leaf samples were collected at the indicated times after spraying for RNA isolation. RNA gel blot analysis was performed with 32P-labeled PR1. These experiments were performed twice with similar results.

We subsequently tested their interactions in plant cells using the bimolecular fluorescence complementation (BiFC) assay in Agrobacterium tumefaciens–infiltrated tobacco (Nicotiana benthamiana) leaves (Cui et al., 2007). WRKY62 was fused to the N-terminal yellow fluorescent protein (YFP) fragment and HDA19 was fused to the C-terminal YFP fragment. When WRKY62-N-YFP was coexpressed with HDA19-C-YFP, a strong BiFC signal was observed predominantly in the nuclear compartment, based on staining with 4,6-diamidino-2-phenylindole (DAPI) (Figure 5 ). Since WRKY38 is structurally and functionally related to WRKY62, we also examined its interaction with HDA19 in plant cells using the BiFC assay. Indeed, when WRKY38 was fused to the terminal YFP fragment and coexpressed with HDA19-C-YFP in tobacco leaves, a BiFC signal was also observed predominantly in the nuclear compartment (Figure 5). Control experiments in which WRKY38-N-YFP or WRKY62-N-YFP was coexpressed with unfused C-YFP protein or unfused N-YFP was coexpressed with HDA19-C-YFP did not show any fluorescence (Figure 5). These experiments provide strong evidence that both WRKY38 and WRKY62 form complexes with HDA19 in the nuclear compartment of plant cells. Interactions of WRKY38 and WRKY62 with HDA19 in the nucleus are consistent with the subcellular localization of WRKY38, WRKY62, and HDA19 in the nuclear compartment (see Supplemental Figure 1C online) (Long et al., 2006).

View larger version (87K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 5. BiFC Analysis of WRKY Protein Interactions with HDA19. Fluorescence was observed from complementation of the N-terminal part of the YFP fused with WRKY38 (WRKY38-N-YFP) or WRKY62 (WRKY62-N-YFP) with the C-terminal part of the YFP fused with HDA19 (HDA19-C-YFP) and colocalized with DAPI stains in the nuclear compartment of tobacco leaf epidermal cells. No fluorescence was observed when WRKY38-N-YFP or WRKY62-N-YFP was coexpressed with unfused C-YFP or when unfused N-YFP was coexpressed with HDA19-C-YFP. These experiments were performed three times with similar results.

Regulated Expression of HDA19
As a first step to characterize the role of HDA19 in plant resistance to PstDC3000, we analyzed its expression in wild-type plants after infection of the bacterial pathogen. The HDA19 transcript levels were very low before inoculation and at 12 HAI (Figure 6A ). However, HDA19 transcripts were readily detected by RNA gel blot analysis in pathogen-inoculated plants at 24 HAI and continued to increase gradually during the next 24 h. No significant induction of HDA19 was observed in plants infiltrated with MgCl₂ (Figure 6A). Thus, expression of HDA19 is induced by PstDC3000. In addition, transcripts for HDA19 were elevated in SA- or methyl jasmonate (MeJA)–treated plants (Figure 6B), although the induction was delayed when compared with those of WRKY38 and WRKY62 in SA-treated plants (Figure 1A). Pathogen-, SA-, and MeJA-induced expression of HDA19 was also detected by quantitative RT-PCR (see Supplemental Figure 6 online).

View larger version (90K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 6. Expression of HDA19. (A) Time course of pathogen-induced expression of HDA19. Five-week-old Arabidopsis plants (Col-0) were infiltrated with 10 mM MgCl2 or PstDC3000 (OD600 = 0.0001 in 10 mM MgCl2). The infiltrated leaves were collected at the indicated times after inoculation for RNA isolation. RNA gel blot analysis was performed with a 32P-labeled HDA19 fragment. (B) Time course of induced expression of HDA19 by SA, 1-aminocyclopropane-1-carboxylic acid (ACC), and MeJA. Five-week-old Arabidopsis plants (Col-0) were sprayed with SA (1 mM), ACC (0.1 mM), and MeJA (0.1 mM). Leaf collection, RNA isolation, and RNA gel blot analysis of HDA19 expression were performed as in (A). (C) Pathogen-induced expression of HDA19 in defense signaling mutants. Five-week-old wild-type (Col-0), sid2-3, npr1-3, coi1-1, and ein2-1 mutant plants were infiltrated with a suspension of PstDC3000 (OD600 = 0.0001 in 10 mM MgCl2). Leaf collection, RNA isolation, and RNA gel blot analysis of HDA19 expression were performed as in (A). The experiments were performed three times with similar results.

Knockout Mutant and Overexpression Plants for HDA19
To determine its role in plant basal disease resistance, we identified two T-DNA insertion mutants for HDA19 (hda19-3 and hda19-4) that both contain a T-DNA insertion in the first exon of the gene (see Supplemental Figure 7A online). We inoculated the hda19 knockout mutant plants with PstDC3000 and monitored both bacterial growth and disease symptom development. As shown in Figure 7A , the hda19 mutants had an approximately fivefold increase in the growth of the bacterial pathogen. The mutants also developed more severe disease symptoms than wild-type plants after the infection (Figure 7C). Thus, unlike WRKY38 and WRKY62, HDA19 functions as a positive regulator in basal resistance to PstDC3000.

View larger version (49K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 7. Altered Responses to P. syringae by the Mutant and Overexpression Plants for HDA19. (A) and (B) Altered bacterial growth. The wild type (Col-0), hda19 mutants (A), and the overexpression lines (B) were infiltrated with a suspension of PstDC3000 (OD600 = 0.0001 in 10 mM MgCl2). Samples were taken at 0 (open bars) or 3 (closed bars) DAI to determine the growth of the bacterial pathogen. Means and SE were calculated from 10 plants for each treatment. According to Duncan's multiple range test (P = 0.05), means of colony-forming units do not differ significantly at 0 DAI if they are indicated with the same lowercase letter and do not differ significantly at 3 DAI if they are indicated with the same uppercase letter. (C) and (D) Altered disease symptom development. Pathogen inoculation was performed as in (A) and (B). Photographs of representative inoculated leaves to determine altered disease responses of the hda19 mutants (C) were taken at 3 DAI. Photographs for the overexpression line (D) were taken at 4 DAI. (E) and (F) Pathogen-induced PR1 expression. Wild-type, hda19, and HDA19-overexpressing plants were infiltrated with a suspension of PstDC3000 (OD600 = 0.0001 in 10 mM MgCl2). Inoculated leaves were collected at the indicated times after inoculation for RNA isolation. RNA gel blot analysis was performed with 32P-labeled PR1. These experiments were performed four times with similar results.

To investigate the molecular basis for the altered plant basal resistance against PstDC3000, we analyzed PR1 expression in the hda19-3 mutant and HDA19-overexpressing transgenic plants after infection with the bacterial pathogen. After PstDC3000 inoculation, the levels of PR1 transcripts were reduced in the hda19 mutant relative to those in the wild-type plants (Figure 7E). On the other hand, the levels of PR1 transcripts in the HDA19-overexpressing plants were higher than those in wild-type plants, particularly at later stages of infection (i.e., at 48 and 72 HAI) (Figure 7F). These results suggest that HDA19 is a positive regulator of SA-regulated PR1 gene expression.

Repression of Transcriptional Activation Activity of WRKY38 and WRKY62 by HDA19
Histone deacetylases catalyze the removal of acetyl groups from histone tails and often repress the transcription of genes by reducing the access of DNA by transcription factors (Zhou et al., 2005). Since HDA19 has a role in plant basal defense opposite to those of WRKY38 and WRKY62, it is possible that HDA19, through physical interaction, can reduce the transcriptional regulatory activity of the two transcription factors in plant cells. To test this possibility, we analyzed the transcriptional activation or repression activity of WRKY38 and WRKY62 using a transgenic system previously used for the analysis of WRKY7 (Kim et al., 2006). In this system, the transcriptional regulatory activity of a protein is determined through assays of a β-glucuronidase (GUS) reporter gene in stably transformed plants. The GUS reporter gene is driven by a synthetic promoter consisting of the –100 minimal CaMV 35S promoter and eight copies of the LexA operator sequence (see Supplemental Figure 8A online). Due to the minimal 35S promoter used, these transgenic plants constitutively expressed low levels of the GUS reporter gene, thereby making them suitable for assays of transcription activation or repression by determining increases or decreases in GUS activities following coexpression of an effector protein.

To generate the WRKY38 and WRKY62 effectors, we fused their coding sequences with that of the DNA binding domain (DBD) of LexA. The fusion construct was subcloned behind the steroid-inducible Gal4 promoter in pTA7002 (Aoyama and Chua, 1997) (see Supplemental Figure 8A online) and transformed into transgenic plants that already contain the GUS reporter construct. Unfused WRKY38, WRKY62, and LexA DBD genes were also subcloned into pTA7002 and transformed into transgenic GUS reporter plants as controls (see Supplemental Figure 8A online). Transgenic plants containing both the reporter and an effector construct were identified through antibiotic resistance screens. To determine how the effectors influence GUS reporter gene expression, we determined the changes of GUS activity in these transgenic plants following induction of the effector gene expression by spraying 20 µM dexamethasone (DEX), a steroid. In the transgenic plants that expressed the unfused WRKY38, WRKY62, or LexA DBD effector gene, the ratios of GUS activities measured before DEX treatment to those measured after DEX treatment were close to 1 (see Supplemental Figure 8B online). These results indicated that induced expression of WRKY38, WRKY62, or LexA DBD alone had no significant effect on expression of the GUS reporter gene. In the transgenic plants harboring the LexA DBD-WRKY38 or LexA DBD-WRKY62 effector gene, induction of the fusion effector after DEX treatment resulted in an approximately fourfold to sixfold increase in GUS activity (see Supplemental Figure 8B online). These results indicate that WRKY38 and WRKY62 are transcriptional activators in plant cells.

To determine how HDA19 affects the transcriptional activation activity of WRKY38 and WRKY62, we crossed the transgenic 35S:HDA19-L1 line with transgenic plants harboring both the GUS reporter and an effector gene. A transgenic line harboring an empty vector was also crossed with the transgenic reporter/effector double transformants as controls. As shown in Figure 8 , in the progeny plants from the control cross with no constitutive HDA19 overexpression, induced expression of the fused LexA-WRKY38 or LexA-WRKY62 effector gene after DEX treatment resulted in 3.5- to 6-fold induction in GUS activity, which was similar to that observed in their respective parental reporter/effector lines (see Supplemental Figure 8B online). On the other hand, in the progeny plants that constitutively overexpressed HDA19, induced expression of the fused LexA-WRKY38 or LexA-WRKY62 effector after DEX treatment resulted in no significant change in GUS activity (Figure 8). Thus, overexpressed HDA19 effectively abolished the transcriptional activation activity of WRKY38 and WRKY62.

View larger version (13K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 8. Antagonism of the Transcriptional Activation Activity of WRKY38 and WRKY62 by HDA19. Effects of overexpressed HDA19 and HDA19m on the transcriptional activation activity of WRKY38 and WRKY62. The HDA19- or HDA19m-overexpressing line was crossed to lines harboring both the GUS reporter and one of the five tested effectors. A transgenic line containing an empty vector was also crossed to the same GUS/effector double transformants as controls. The ratios of GUS activities were calculated from the GUS activities determined in the leaves harvested 18 h after DEX treatment (+) over those determined prior to DEX treatment (–).

To determine whether the ability of overexpressed HDA19 to abolish the transcription activation activity of a transcription factor is correlated with physical interaction, we examined the effect of overexpressed HDA19 on the transcription activation or repression activity of WRKY18 and WRKY48, which do not interact with HDA19 based on BiFC (see Supplemental Figure 5 online). However, induced expression of the LexA DBD-WRKY18 effector gene had no significant effect on the GUS activity of the transgenic reporter/effector plants, indicating that WRKY18 has little transcription activation or repression activity in plant cells (data not shown). On the other hand, in the transgenic plants harboring the LexA DBD-WRKY48 effector gene, induction of the fusion effector after DEX treatment resulted in an 20-fold increase in GUS activity (see Supplemental Figure 10 online). This result indicated that WRKY48 is a strong transcription activator. The transgenic plants harboring both the GUS reporter and the LexA DBD-WRKY48 effector gene was then crossed with the transgenic 35S:HDA19-L1 line. In the progeny plants that constitutively overexpressed HDA19, induced expression of the fused LexA-WRKY48 after DEX treatment resulted in an 20-fold induction in GUS activity, which was similar to that observed in the progeny derived from a cross with a control line transformed with an empty vector (see Supplemental Figure 10 online). Thus, overexpressed HDA19 did not significantly affect the transcription activation activity of WRKY48.

Functional Interaction of WRKY62 with HDA19 in Plant Disease Resistance
To analyze the functional interaction of the SA-regulated WRKY genes with HDA19 in plant disease resistance, we examined the effects of overexpression of WRKY62 on plant responses to PstDC3000 in both the wild-type and hda19 mutant backgrounds. The transgenic 35S:WRKY62-L1 line was crossed with the hda19-3 mutant, and the hda19-3/35S:WRKY62 plants in the F2 generation were identified by PCR and RNA gel blotting and compared with wild-type, hda19-3 mutant, and 35S:WRKY62-L1 plants for responses to PstDC3000. Following inoculation with the virulent bacterial pathogen, overexpression of WRKY62 in the wild-type background (35S:WRKY62-L1) caused an approximately fivefold increase in bacterial growth (Figure 9A ). Overexpression of WRKY62 in the hda19-3 mutant background (hda19-3/35S:WRKY62-L1), on the other hand, led to an 15-fold increase in bacterial growth (Figure 9A). The inoculated leaves of the hda19-3/35S:WRKY62-L1 plants also developed more severe disease symptoms than those of hda19-3 and 35S:WRKY62-L1 plants after infection (Figure 9B). These results support the functional interaction of WRKY62 with HDA19 in plant disease resistance.

View larger version (49K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 9. Altered Responses to P. syringae by Overexpression of WRKY62 in the Wild-Type and hda19 Mutant Backgrounds. (A) Altered bacterial growth. Wild-type, 35S:WRKY62-L1, hda19-3, and hda19-3/35S:WRKY62 plants were infiltrated with a suspension of PstDC3000 (OD600 = 0.0001 in 10 mM MgCl2). Samples were taken at 0 (open bars) or 3 (closed bars) DAI to determine the growth of the bacterial pathogen. Means and SE were calculated from 10 plants for each treatment. According to Duncan's multiple range test (P = 0.05), means of colony-forming units at 0 DAI do not differ significantly if they are indicated with the same lowercase letter, and means of colony-forming units at 3 DAI do not differ significantly if they are indicated with the same uppercase letter. (B) Altered disease symptom development. Pathogen inoculation was performed as in (A). Photographs of representative inoculated leaves were taken at 3 DAI.

	DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION METHODS REFERENCES

A large number of Arabidopsis WRKY genes are induced by infection with PstDC3000, and a number of these pathogen-responsive WRKY genes have been analyzed for roles in plant basal disease resistance to the bacterial pathogen. Intriguingly, a majority of these functionally characterized pathogen-responsive WRKY genes function to repress plant basal resistance to the bacterial pathogen (Kim et al., 2006; Wang et al., 2006; Xu et al., 2006; Zheng et al., 2006, 2007; Shen et al., 2007). Some of the PAMP-induced WRKY negative regulators of plant defense have been proposed to provide a functional interface between PTI and ETI. For example, barley (Hordeum vulgare) WRKY1 and WRKY2 function as PAMP-inducible suppressors of basal defense (Shen et al., 2007). Upon avirulent effector recognition, barley MLA resistance protein can translocate to the nucleus and physically interact with the two WRKY proteins. The interactions apparently can interfere with the suppressor activity of the WRKY proteins, thereby derepressing PAMP-dependent basal defense during the activation of ETI (Shen et al., 2007). Possible inactivation of defense-suppressing WRKY proteins during ETI has also been proposed for Arabidopsis WRKY52/RRS1 R protein, which confers resistance toward the bacterial pathogen Ralstonia solanacearum (Eulgem and Somssich, 2007). A previous study has shown that the WRKY domain in WRKY52/RRS1 may play a negative role in defense signaling (Noutoshi et al., 2005). The interaction of RRS1 with its cognate effector PopP2 may inactivate the WRKY domain and activate high-amplitude defense mechanisms by derepression. Thus, during PTI, these defense-repressing WRKY proteins are induced by PAMP and may act to downregulate PAMP-induced plant defense responses so that they are not too deleterious to the host. During ETI, these defense-repressing WRKY proteins are inactivated upon functional recognition of avirulent factors by their cognate R proteins for derepression of defense mechanisms.

Both PstDC3000 and SA induced WRKY38 and WRKY62 relatively quickly but transiently (Figure 1). The induction of the two negative regulators during the early stages of infection might serve as a mechanism to prevent unnecessary or even harmful overactivation of pathogen-induced defense mechanisms when the population of the pathogen is still at relatively low levels. As pathogen growth increases, enhanced defense mechanisms would be necessary, and this could be achieved at least partially by suppressed expression and inactivation of negative regulators such as WRKY38 and WRKY62. Indeed, the transcript levels of both WRKY38 and WRKY62 started to decline and eventually reached nearly basal levels between 12 and 24 h after pathogen infection (Figure 1). In addition, as the expression of WRKY38 and WRKY62 started to decrease, the transcript levels of HDA19 started to increase concomitantly (Figure 6). The elevated HDA19 proteins from its induced expression would help to inactivate the remaining WRKY38 and WRKY62 transcriptional activators for a stronger defense response.

Pathogen-induced expression of negative defense regulators during plant defense responses could also be explained by their possible involvement in the antagonistic crosstalk of distinct signaling pathways against different types of microbial pathogens. SA-mediated signaling activates defense mechanisms effective against biotrophic pathogens but can suppress ET/JA-mediated signaling in defense against necrotrophic pathogens (Glazebrook, 2004). A WRKY protein that represses defense against one type of pathogen may function as an activator of defense against another type of microbial pathogen. For example, while its overexpression enhances susceptibility to biotrophic PstDC3000, WRKY33 is an important positive regulator of plant resistance to necrotrophic fungal pathogens (Zheng et al., 2006). Although relatively resistant to PstDC3000, the wrky38 and wrky62 mutants respond normally to the necrotrophic pathogens (data not shown). These results suggest that WRKY38 and WRKY62 do not play a major role in the antagonistic crosstalk of defense signaling pathways against these two types of pathogens. However, it is still possible that the two WRKY proteins play a positive role in plant responses to certain unknown abiotic or biotic stresses that may or may not be antagonized by SA- and NPR1-mediated defense.

Functional Antagonisms through Physical Interactions
Both defense-activating and defense-repressing WRKY proteins have been identified (Journot-Catalino et al., 2006; Kim et al., 2006; Li et al., 2006; Zheng et al., 2007), indicating that they are critical regulators of differential and graded plant defense responses to distinct types of microbial pathogens. Understanding how these WRKY proteins interact functionally with each other and with other defense regulators will provide important insights into the molecular basis of the tight regulation and fine-tuning of plant defense responses. It has been proposed that the defense-repressing barley WRKY1 and WRKY2 can be inactivated by the interacting MLA R protein (Shen et al., 2007). Likewise, the interaction of Arabidopsis WRKY52/RRS1 with its cognate effector PopP2 has been suggested to inactivate the WRKY domain of RRS1 to activate high-amplitude defense mechanisms by derepression (Eulgem and Somssich, 2007). How the physical interaction leads to the inactivation of these WRKY proteins is unknown. In this study, we have demonstrated a simple mechanism by which the defense-repressing WRKY38 and WRKY62 can be inactivated by the interacting HDA19. Although WRKY38 and WRKY62 suppress disease resistance and repress defense gene expression, they act as transcriptional activators in plant cells (Figure 8). Thus, WRKY38 and WRKY62 do not appear to repress defense genes directly; more likely, they first activate certain unknown negative regulators that, in turn, repress defense genes (Figure 10 ). HDA19, on the other hand, is a positive regulator of plant basal resistance to PstDC3000 (Figure 7). Histone deacetylases are often associated with transcriptional corepressor complexes by reducing histone acetylation levels to create repressed chromatin regions. Thus, one mode of action by HDA19 as a positive regulator of plant basal disease resistance is to counteract the WRKY38 and WRKY62 negative regulators of plant defense by binding directly to them and inactivating their transcriptional activation activity (Figure 10).

View larger version (5K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 10. A Model for the Functional Interactions of WRKY38 and WRKY62 with HDA19 during Plant Defense Responses. Infection by P. syringae leads to the accumulation of SA, which induces the expression of WRKY38 and WRKY62 in an NPR1-dependent manner. WRKY38 and WRKY62, as transcriptional activators, activate the expression of unknown regulatory genes that, in turn, repress the expression of defense genes (e.g., PR1) and basal disease resistance. Infection by P. syringae also induces HDA19, whose transcripts are stabilized by SA- and NPR1-mediated signaling. HDA19 represses the transcriptional activation activity of WRKY38 and WRKY62 and, as a result, reduces the activation of negative regulatory genes of plant basal defense by the two WRKY transcription factors.

The host-selective toxin HC-toxin produced by the filamentous fungus Cochliobolus carbonum is a critical determinant of virulence in the interaction with the host, maize (Zea mays) (Walton, 2006). HC-toxin inhibits maize histone deacetylases both in vitro and in vivo (Brosch et al., 1995; Ransom and Walton, 1997). It has been suggested that by inhibiting histone deacetylases, HC-toxin may interfere with the proper expression of a subset of plant host genes necessary for the host to mount an effective defense against the fungal pathogen (Ransom and Walton, 1997). Therefore, plant histone deacetylases might play a general role in maintaining the appropriate acetylation state of histones for proper induction of plant defense genes.

	METHODS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION METHODS REFERENCES

 
Plant Growth Conditions
The Arabidopsis thaliana wild-type, mutant, and transgenic plants used in the study were all grown in growth chambers at 22°C and 120 µE·m^–2·s^–1 light on a 12-h-light/12-h-dark photoperiod.

Production of Recombinant Protein, and Electrophoretic Mobility Shift Assays
To generate the WRKY38 and WRKY62 recombinant proteins, their full-length cDNAs were cloned into pET32a (Novagen) and transformed into Escherichia coli strain BL21 (DE3). Induction of expression and purification of recombinant His-tagged proteins were performed according to the protocol provided by Novagen. The purified proteins were dialyzed overnight against a nuclear extraction buffer (25 mM HEPES/KOH, pH 7.5, 40 mM KCl, 0.1 mM EDTA, 10% glycerol, 1 mM DTT, and 30 µg/L phenylmethylsulfonyl fluoride) at 4°C. Double-stranded synthetic oligonucleotides were labeled to specific activities of 10⁵ cpm/ng using the Klenow fragment of DNA polymerase I. DNA and protein complexes were allowed to form at room temperature for 30 min and resolved on a 10% polyacrylamide gel in 0.5x Tris-borate-EDTA at 4°C.

Subcellular Localization
The WRKY38 and WRKY62 cDNAs were amplified and fused with the GFP gene in-frame in a pBluescript II SK vector. The empty GFP plasmid was used as a control. The plasmid was isolated using Qiagen kits, concentrated to 1 µg/µL, and used to coat the gold particles for bombardment experiments. Transient expression of the GFP fusion genes in onion (Allium cepa) epidermal cells through particle bombardment and subsequent localization of the proteins were performed as described previously (Xu et al., 2006).

RNA Gel Blotting
For RNA gel blot analysis, total RNA (5 µg) was separated on agarose–formaldehyde gels and blotted to nylon membranes. Blots were hybridized with [-³²P]dATP-labeled gene-specific probes. Hybridization was performed in PerfectHyb plus hybridization buffer (Sigma-Aldrich) overnight at 68°C. The membrane was then washed for 10 min twice with 2x SSC (1x SSC is 0.15 M NaCl and 0.015 M sodium citrate) and 1% SDS and for 10 min with 0.1x SSC and 1% SDS at 68°C. The probes used in RNA gel blotting were as follows: WRKY38, an 450-nucleotide 3' fragment obtained by XbaI/XhoI digestion of a full-length cDNA clone isolated from a cDNA library (in ZAP Express vector from Stratagene) prepared from Arabidopsis plants harvested 4 h after spraying with 2 mM SA (Xu et al., 2006); WRKY62, an 400-nucleotide 3' fragment obtained by HindIII/XhoI digestion of a full-length cDNA clone; HDA19, a 747-nucleotide internal HindIII fragment from its full-length cDNA clone; and PR1, a 410-nucleotide PCR fragment amplified from the Arabidopsis genomic DNA using two PR1-specific primers (5'-TTCTTCCCTCGAAAGCTCAA-3' and 5'-CGTTCACATAATTCCCACGA-3').

Quantitative RT-PCR
Total RNA was isolated from treated leaves and treated with DNA-free (Ambion) to remove contaminated genomic DNA. First-strand cDNA was synthesized with SuperScript II reverse transcriptase (Invitrogen) following the instructions of the manufacturer. Diluted first-strand cDNA was used as template, and real-time PCR was conducted with both ubiquitin primers (5'-GAAGGCGAAGATCCAAGACAAG-3' and 5'-TCCCGGCGAAAATCAATC-3') and gene-specific primers (WRKY38, 5'-CGCCATGCGGTTGAAGAG-3' and 5'-TAACTTGAAAGCGGTCCACCAT-3'; WRKY62, 5'-CCAACCAGCTGCTCATCATG-3' and 5'-GGCCAAATCCTCCCTTTCC-3'; HDA19, 5'-GACTGTGATTACAACACACCGT-3' and 5'-AATTGCCGCCAGTATCCAT-3'). The PCR was set up using SYBR Green PCR Master Mix (Applied Biosystems) and run on the ABI Prism 7000 system. The relative specific mRNA abundance was calculated using ubiquitin as an internal control.

Isolation of Knockout Mutants
The wrky38-1 (WiscDsLox489-492C21; in Col-0 ecotype) and wrky38-2 (RATM11-6950-1_H; in No-0 ecotype) mutants each contain a Ds transposon in the second intron and the third exon of the WRKY38 gene, respectively. Homozygous wrky38 mutant plants were identified by PCR using a pair of primers corresponding to sequences flanking the Ds tagging sites (pW38F, 5'-ATGAACTCCCCACACGAAAAG-3'; pW38R, 5'-AAAGTAAAACTGATCATAACGATCCCA-3'). The wrky62-1 (GABI_016H10; in Col-0 ecotype) and wrky62-2 (RATM11-6212-1_G; in No-0 ecotype) mutants each contain a T-DNA insertion and a Ds transposon in the second exon of WRKY62. Homozygous wrky62 mutant plants were identified by PCR using a pair of primers corresponding to sequences flanking the insertion sites (pW62F, 5'-ATGAACTCTTGCCAACAAAAGGCT-3'; pW62R, 5'-TGATGATAAGTCGTGAGATGTCCA-3'). The hda19-3 (SALK_139445) and hda19-4 (SALK_139443) mutants each contain a T-DNA insertion in the first exon of the HDA19 gene. Plants homozygous for the T-DNA insertions were identified by PCR using a pair of primers corresponding to sequence flanking the T-DNA insertion site (pHDA19F, 5'-CGCTCACTACGGTCTCCTTC-3'; pHDA19R, 5'-TAAAGAACACGCTGCAAACG-3'). The sid2-3 mutant (SALK_042603) contains a T-DNA insertion in the fourth intron of the SID2 gene. Homozygous sid2-3 mutant plants were identified by PCR using a pair of primers flanking the insertion site (pSID2F, 5'-TAGTTAGTGTGGCCATGCTAAG-3'; pSID2R, 5'-CCTAATTCCACGAGCCAAAA-3').

Construction of WRKY38, WRKY62, and HDA19 Overexpression Plants
An EcoRI/HindIII fragment that contains the CaMV 35S promoter with double enhancers, multiple cloning sites, and 35S terminator was excised from pFF19 and cloned into the same sites of the transformation vector pOCA28 to generate pOCA30 (Chen and Chen, 2002). To generate the 35S:WRKY38 construct, the cDNA fragment that contains the full coding sequence and 3' untranslated region of WRKY38 was excised with SacI and KpnI from a cloning plasmid and subcloned into the same restriction sites of pOCA30 in the sense orientation behind the 35S promoter. The 35S:WRKY62 construct was generated in a similar way by subcloning the full-length cDNA for WRKY62 into the SmaI and XbaI sites of pOCA30. To generate the 35S:HDA19 construct, the cDNA fragment that contains the full coding sequence and 3' untranslated region of HDA19 was excised with SpeI and XhoI from a cloning plasmid and subcloned into the XbaI and SalI sites of pOCA30 in the sense orientation behind the 35S promoter. HDA19m with Ala substitutions for the catalytic His-148 and His-149 residues was generated by overlapping PCR and confirmed by sequencing. Briefly, two pairs of primers (pA408, 5'-ATCGAGCTCGTCGACGTAATGGATACTGGCGGCAA-3'/pA409, 5'-TCGCACTTCTTAGCGGCAGCGAGACCACCA-3' and pA410, 5'-TGGTGGTCTCGCTGCCGCTAAGAAGTGCGA-3'/pA411, 5'-AGCATAAAATGCCTCCTCCA-3') were first used to amplify two DNA fragments from the full-length HDA19 cDNA clone. The amplified two fragments were then used as templates for overlapping PCR using pA408 and pA411 as primers to amplify a DNA fragment of 530 bp corresponding to the 5' region of the HDA19 coding sequence with introduced mutations. The fragment was digested with SacI and used to replace the corresponding wild-type SacI fragment of the full-length HDA19 cDNA clone.

Arabidopsis transformation was performed by the floral dip procedure (Clough and Bent, 1998). The seeds were collected from the infiltrated plants and selected in Murashige and Skoog medium containing 50 µg/mL kanamycin. Kanamycin-resistant plants were transferred to soil 9 d later and grown in a growth chamber for further analysis.

Pathogen Inoculation
Pathogen inoculations were performed by infiltration of leaves of at least six plants for each treatment with the Pseudomonas syringae pv tomato DC3000 strain (OD₆₀₀ = 0.0001 in 10 mM MgCl₂). Inoculated leaves were harvested 3 d after infiltration and homogenized in 10 mM MgCl₂. Diluted leaf extracts were plated on King's B medium supplemented with rifampicin (100 µg/mL) and kanamycin (25 µg/mL) and incubated at 25°C for 2 d before counting the colony-forming units.

CytoTrap Two-Hybrid Screening
WRKY62-interacting proteins were identified using the CytoTrap two-hybrid system as described by the manufacturer (Stratagene). The Arabidopsis pMyr two-hybrid cDNA library was prepared from Arabidopsis plants harvested 4 h after spraying with 2 mM SA. The WRKY62 cDNA was inserted into the pSOS plasmid to generate bait plasmids. The pMyr cDNA library and the corresponding bait plasmid were used to transform yeast strain cdc25H. Yeast transformants were plated onto the synthetic glucose minimal medium lacking uracil and Leu [SD/glucose(–UL)]. After growth at 25°C for 2 to 4 d, the colonies were replica-plated on the SD/galactose(–UL) plates and kept at 37°C. Those positive clones that grew on the SD/galactose(–UL) plates but not on the SD/glucose(–UL) plates were saved and analyzed further. Plasmid DNA was recovered from positive yeast colonies, transformed into E. coli strain DH5, and isolated for DNA sequencing.

BiFC Assays
DNA sequences for the N-terminal 173–amino acid EYFP (N-YFP) and C-terminal 64–amino acid (C-YFP) fragments were PCR-amplified and cloned into the plant expression vectors pOCA30 (Chen and Chen, 2002) and pFGC5941 to generate pOCA-N-YFP and pFGC-C-YFP, respectively. The WRKY18, WRKY38, WRKY62, WRKY48, and WRKY70 coding sequences were inserted into pOCA-N-YFP to generate the N-terminal in-frame fusions with N-YFP, whereas HDA19 and WRKY40 were introduced into pFGC-C-YFP to form C-terminal in-frame fusions with C-YFP. The resulting clones were verified through sequencing. The plasmids were introduced into Agrobacterium tumefaciens (strain GV3101), and infiltration of tobacco (Nicotiana benthamiana) was performed as described previously (Cui et al., 2007). Infected tissues were analyzed at 16 to 24 h after infiltration. Fluorescence and DAPI staining were visualized by confocal microscopy using a Bio-Rad MRC-1024 laser scanning confocal imaging system.

Immunoprecipitation
To generate the FLAG- or MYC-tagged proteins, cDNA fragments for WRKY38, WRKY62, and HDA19 were generated by PCR amplification and subsequently subcloned into a tagging plasmid behind the FLAG or MYC tag sequence as described previously (Xu et al., 2006). The tagged genes were subcloned into the plant transformation vector pOCA30, introduced into A. tumefaciens (strain GV3101), and infiltrated into tobacco as described previously (Cui et al., 2007). Preparation of protein extracts, immunoprecipitation, and detection of interacting proteins with protein gel blot analysis were performed as described previously (Xu et al., 2006).

Assays of Transcriptional Regulatory Activity of WRKY38 and WRKY62
Transgenic Arabidopsis plants containing a GUS reporter gene driven by a synthetic promoter consisting of the –100 minimal CaMV 35S promoter and eight copies of the LexA operator sequence were described previously (Kim et al., 2006). To generate effector genes, the DNA fragment for the LexA DBD was digested from the plasmid pEG202 (Clontech) using HindIII and EcoRI and cloned into the same sites in pBluescript. The full-length WRKY18, WRKY38, WRKY48, and WRKY62 cDNA fragments were subsequently subcloned behind the LexA DBD to generate translational fusions. The LexA DBD-WRKY fusion genes were cloned into the XhoI and SpeI sites of pTA2002 behind the steroid-inducible promoter (Aoyama and Chua, 1997). As controls, the unfused LexA DBD, WRKY38, and WRKY62 genes were also cloned into the same sites of pTA7002. These effector constructs were directly transformed into the transgenic GUS reporter plants, and double transformants were identified through screening for antibiotic (hygromycin) resistance. Determination of the activation or repression of GUS reporter gene expression by the effector proteins was performed as described previously (Kim et al., 2006).

Accession Numbers
Arabidopsis Genome Initiative numbers for the genes discussed in this article are as follows: WRKY18, At4g31800; WRKY38, At5g22570; WRKY48, At5g49520; WRKY62, At5g01900; WRKY70, At3g56400; HDA19, At4g38130; PR1, At2g14610; NPR1, At1g64280; SID2, At1g74710; COI1, At2g39940; EIN2, At5g03280.

Supplemental Data
The following materials are available in the online version of this article.

Supplemental Figure 1. Sequences, DNA Binding, and Subcellular Localization of WRKY38 and WRKY62.

Supplemental Figure 2. Analysis of the Expression of WRKY38 (A) and WRKY62 (B) by Quantitative RT-PCR.

Supplemental Figure 3. Loss-of-Function Mutants and Overexpression Lines for WRKY38 and WRKY62.

Supplemental Figure 4. Determination of Protein–Protein Interactions by Yeast Two-Hybrid Assays and Coimmunoprecipitation.

Supplemental Figure 5. BiFC Analysis of Protein–Protein Interactions.

Supplemental Figure 6. Analysis of the Expression of HDA19 by Quantitative RT-PCR.

Supplemental Figure 7. Generation and Characterization of Mutants and Overexpression Lines for HDA19.

Supplemental Figure 8. WRKY38 and WRKY62 Are Transcription Activators.

Supplemental Figure 9. Evolutionarily Invariant His Residues in a Highly Conserved Deacetylase Motif from Arabidopsis HDA19, Yeast Rpd3, and Hos2.

Supplemental Figure 10. Effect of the Transcription Activation Activity of WRKY48 by Overexpressed HDA19.

	Acknowledgments

 
We thank the ABRC at Ohio State University and the RIKEN Tsukuba Institute for the Arabidopsis mutants. We are grateful to Jixin Dong for his help with this work. This work was supported by National Science Foundation Grant MCB-0209819. This is journal paper 2007-18187 of the Purdue University Agricultural Research Program.

	Footnotes

 
The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: Zhixiang Chen (zhixiang{at}purdue.edu).

^[W] Online version contains Web-only data.

www.plantcell.org/cgi/doi/10.1105/tpc.107.055566

Received September 6, 2007; Revision received August 11, 2008. accepted August 18, 2008.

	REFERENCES

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION METHODS REFERENCES

 
AbuQamar, S., Chen, X., Dhawan, R., Bluhm, B., Salmeron, J., Lam, S., Dietrich, R.A., and Mengiste, T. (2006). Expression profiling and mutant analysis reveals complex regulatory networks involved in Arabidopsis response to Botrytis infection. Plant J. 48: 28–44.[CrossRef][Web of Science][Medline]

Andreasson, E., et al. (2005). The MAP kinase substrate MKS1 is a regulator of plant defense responses. EMBO J. 24: 2579–2589.[CrossRef][Web of Science][Medline]

Aoyama, T., and Chua, N.-H. (1997). A glucocorticoid-mediated transcriptional induction system in transgenic plants. Plant J. 11: 605–612.[CrossRef][Web of Science][Medline]

Broder, Y.C., Katz, S., and Aronheim, A. (1998). The ras recruitment system, a novel approach to the study of protein-protein interactions. Curr. Biol. 8: 1121–1124.[CrossRef][Web of Science][Medline]

Brosch, G., Ransom, R., Lechner, T., Walton, J.D., and Loidl, P. (1995). Inhibition of maize histone deacetylases by HC toxin, the host-selective toxin of Cochliobolus carbonum. Plant Cell 7: 1941–1950.[Abstract]

Cao, H., Glazebrook, J., Clarke, J.D., Volko, S., and Dong, X. (1997). The Arabidopsis NPR1 gene that controls systemic acquired resistance encodes a novel protein containing ankyrin repeats. Cell 88: 57–63.[CrossRef][Web of Science][Medline]

Chen, C., and Chen, Z. (2000). Isolation and characterization of two pathogen- and salicylic acid-induced genes encoding WRKY DNA-binding proteins from tobacco. Plant Mol. Biol. 42: 387–396.[CrossRef][Web of Science][Medline]

Chen, C., and Chen, Z. (2002). Potentiation of developmentally regulated plant defense response by AtWRKY18, a pathogen-induced Arabidopsis transcription factor. Plant Physiol. 129: 706–716.[Abstract/Free Full Text]

Clough, S.J., and Bent, A.F. (1998). Floral dip: A simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16: 735–743.[CrossRef][Web of Science][Medline]

Cui, X., Fan, B., Scholz, J., and Chen, Z. (2007). Roles of Arabidopsis cyclin-dependent kinase C complexes in cauliflower mosaic virus infection, plant growth, and development. Plant Cell 19: 1388–1402.[Abstract/Free Full Text]

Dong, J., Chen, C., and Chen, Z. (2003). Expression profile of the Arabidopsis WRKY gene superfamily during plant defense response. Plant Mol. Biol. 51: 21–37.[CrossRef][Web of Science][Medline]

Eulgem, T., Rushton, P.J., Robatzek, S., and Somssich, I.E. (2000). The WRKY superfamily of plant transcription factors. Trends Plant Sci. 5: 199–206.[CrossRef][Web of Science][Medline]

Eulgem, T., and Somssich, I.E. (2007). Networks of WRKY transcription factors in defense signaling. Curr. Opin. Plant Biol. 10: 366–371.[CrossRef][Web of Science][Medline]

Glazebrook, J. (2004). Contrasting mechanisms of defense against biotrophic and necrotrophic pathogens. Annu. Rev. Phytopathol. 43: 205–227.[CrossRef][Web of Science]

Glazebrook, J., Chen, W., Estes, B., Chang, H.S., Nawrath, C., Metraux, J.P., Zhu, T., and Katagiri, F. (2003). Topology of the network integrating salicylate and jasmonate signal transduction derived from global expression phenotyping. Plant J. 34: 217–228.[CrossRef][Web of Science][Medline]

Jones, J.D., and Dangl, J.L. (2006). The plant immune system. Nature 444: 323–329.[CrossRef][Web of Science][Medline]

Journot-Catalino, N., Somssich, I.E., Roby, D., and Kroj, T. (2006). The transcription factors WRKY11 and WRKY17 act as negative regulators of basal resistance in Arabidopsis thaliana. Plant Cell 18: 3289–3302.[Abstract/Free Full Text]

Kadosh, D., and Struhl, K. (1998). Histone deacetylase activity of Rpd3 is important for transcriptional repression in vivo. Genes Dev. 12: 797–805.[Abstract/Free Full Text]

Kalde, M., Barth, M., Somssich, I.E., and Lippok, B. (2003). Members of the Arabidopsis WRKY group III transcription factors are part of different plant defense signaling pathways. Mol. Plant Microbe Interact. 16: 295–305.[Web of Science][Medline]

Kim, K.C., Fan, B., and Chen, Z. (2006). Pathogen-induced Arabidopsis WRKY7 is a transcriptional repressor and enhances plant susceptibility to Pseudomonas syringae. Plant Physiol. 142: 1180–1192.[Abstract/Free Full Text]

Kloek, A.P., Verbsky, M.L., Sharma, S.B., Schoelz, J.E., Vogel, J., Klessig, D.F., and Kunkel, B.N. (2001). Resistance to Pseudomonas syringae conferred by an Arabidopsis thaliana coronatine-insensitive (coi1) mutation occurs through two distinct mechanisms. Plant J. 26: 509–522.[CrossRef][Web of Science][Medline]

Knoth, C., Ringler, J., Dangl, J.L., and Eulgem, T. (2007). Arabidopsis WRKY70 is required for full RPP4-mediated disease resistance and basal defense against Hyaloperonospora parasitica. Mol. Plant Microbe Interact. 20: 120–128.[CrossRef][Web of Science][Medline]

Kunkel, B.N., and Brooks, D.M. (2002). Cross talk between signaling pathways in pathogen defense. Curr. Opin. Plant Biol. 5: 325–331.[CrossRef][Web of Science][Medline]

Li, J., Brader, G., Kariola, T., and Palva, E.T. (2006). WRKY70 modulates the selection of signaling pathways in plant defense. Plant J. 46: 477–491.[CrossRef][Web of Science][Medline]

Li, J., Brader, G., and Palva, E.T. (2004). The WRKY70 transcription factor: A node of convergence for jasmonate-mediated and salicylate-mediated signals in plant defense. Plant Cell 16: 319–331.[Abstract/Free Full Text]

Long, J.A., Ohno, C., Smith, Z.R., and Meyerowitz, E.M. (2006). TOPLESS regulates apical embryonic fate in Arabidopsis. Science 312: 1520–1523.[Abstract/Free Full Text]

Mao, P., Duan, M., Wei, C., and Li, Y. (2007). WRKY62 transcription factor acts downstream of cytosolic NPR1 and negatively regulates jasmonate-responsive gene expression. Plant Cell Physiol. 48: 833–842.[Abstract/Free Full Text]

Mur, L.A., Kenton, P., Atzorn, R., Miersch, O., and Wasternack, C. (2006). The outcomes of concentration-specific interactions between salicylate and jasmonate signaling include synergy, antagonism, and oxidative stress leading to cell death. Plant Physiol. 140: 249–262.[Abstract/Free Full Text]

Noutoshi, Y., Ito, T., Seki, M., Nakashita, H., Yoshida, S., Marco, Y., Shirasu, K., and Shinozaki, K. (2005). A single amino acid insertion in the WRKY domain of the Arabidopsis TIR-NBS-LRR-WRKY-type disease resistance protein SLH1 (sensitive to low humidity 1) causes activation of defense responses and hypersensitive cell death. Plant J. 43: 873–888.[CrossRef][Web of Science][Medline]

Park, C.Y., Lee, J.H., Yoo, J.H., Moon, B.C., Choi, M.S., Kang, Y.H., Lee, S.M., Kim, H.S., Kang, K.Y., Chung, W.S., Lim, C.O., and Cho, M.J. (2005). WRKY group IId transcription factors interact with calmodulin. FEBS Lett. 579: 1545–1550.[CrossRef][Web of Science][Medline]

Petersen, M., et al. (2000). Arabidopsis MAP kinase 4 negatively regulates systemic acquired resistance. Cell 103: 1111–1120.[CrossRef][Web of Science][Medline]

Ransom, R.F., and Walton, J.D. (1997). Histone hyperacetylation in maize in response to treatment with HC-toxin or infection by the filamentous fungus Cochliobolus carbonum. Plant Physiol. 115: 1021–1027.[Abstract]

Rushton, P.J., Torres, J.T., Parniske, M., Wernert, P., Hahlbrock, K., and Somssich, I.E. (1996). Interaction of elicitor-induced DNA-binding proteins with elicitor response elements in the promoters of parsley PR1 genes. EMBO J. 15: 5690–5700.[Web of Science][Medline]

Sharma, V.M., Tomar, R.S., Dempsey, A.E., and Reese, J.C. (2007). Histone deacetylases RPD3 and HOS2 regulate the transcriptional activation of DNA damage-inducible genes. Mol. Cell. Biol. 27: 3199–3210.[Abstract/Free Full Text]

Shen, Q.H., Saijo, Y., Mauch, S., Biskup, C., Bieri, S., Keller, B., Seki, H., Ulker, B., Somssich, I.E., and Schulze-Lefert, P. (2007). Nuclear activity of MLA immune receptors links isolate-specific and basal disease-resistance responses. Science 315: 1098–1103.[Abstract/Free Full Text]

Spoel, S.H., et al. (2003). NPR1 modulates cross-talk between salicylate- and jasmonate-dependent defense pathways through a novel function in the cytosol. Plant Cell 15: 760–770.[Abstract/Free Full Text]

Tian, L., and Chen, Z.J. (2001). Blocking histone deacetylation in Arabidopsis induces pleiotropic effects on plant gene regulation and development. Proc. Natl. Acad. Sci. USA 98: 200–205.[Abstract/Free Full Text]

Tian, L., Wang, J., Fong, M.P., Chen, M., Cao, H., Gelvin, S.B., and Chen, Z.J. (2003). Genetic control of developmental changes induced by disruption of Arabidopsis histone deacetylase 1 (AtHD1) expression. Genetics 165: 399–409.[Abstract/Free Full Text]

Ulker, B., and Somssich, I.E. (2004). WRKY transcription factors: From DNA binding towards biological function. Curr. Opin. Plant Biol. 7: 491–498.[CrossRef][Web of Science][Medline]

Walton, J.D. (2006). HC-toxin. Phytochemistry 67: 1406–1413.[CrossRef][Web of Science][Medline]

Wang, D., Amornsiripanitch, N., and Dong, X. (2006). A genomic approach to identify regulatory nodes in the transcriptional network of systemic acquired resistance in plants. PLoS Pathog. 2: e123.[CrossRef][Medline]

Wildermuth, M.C., Dewdney, J., Wu, G., and Ausubel, F.M. (2001). Isochorismate synthase is required to synthesize salicylic acid for plant defence. Nature 414: 562–565.[CrossRef][Web of Science][Medline]

Xu, X., Chen, C., Fan, B., and Chen, Z. (2006). Physical and functional interactions between pathogen-induced Arabidopsis WRKY18, WRKY40, and WRKY60 transcription factors. Plant Cell 18: 1310–1326.[Abstract/Free Full Text]

Yu, D., Chen, C., and Chen, Z. (2001). Evidence for an important role of WRKY DNA binding proteins in the regulation of NPR1 gene expression. Plant Cell 13: 1527–1540.[Abstract/Free Full Text]

Zheng, Z., Mosher, S.L., Fan, B., Klessig, D.F., and Chen, Z. (2007). Functional analysis of Arabidopsis WRKY25 transcription factors in plant defense against Pseudomonas syringae. BMC Plant Biol. 7: 2.[CrossRef][Medline]

Zheng, Z., Qamar, S.A., Chen, Z., and Mengiste, T. (2006). Arabidopsis WRKY33 transcription factor is required for resistance to necrotrophic fungal pathogens. Plant J. 48: 592–605.[CrossRef][Web of Science][Medline]

Zhou, C., Zhang, L., Duan, J., Miki, B., and Wu, K. (2005). HISTONE DEACETYLASE19 is involved in jasmonic acid and ethylene signaling of pathogen response in Arabidopsis. Plant Cell 17: 1196–1204.[Abstract/Free Full Text]

Related articles in Plant Cell:

Basal Defense in Arabidopsis: WRKYs Interact with Histone Deacetylase HDA19

Jennifer Mach
Plant Cell 2008 20: 2282. [Full Text]  

This article has been cited by other articles:

				S. P. Pandey and I. E. Somssich The Role of WRKY Transcription Factors in Plant Immunity Plant Physiology, August 1, 2009; 150(4): 1648 - 1655. [Full Text] [PDF]

				J. E. Parker The Quest for Long-Distance Signals in Plant Systemic Immunity Sci. Signal., May 12, 2009; 2(70): pe31 - pe31. [Abstract] [Full Text] [PDF]

				J. Mach Basal Defense in Arabidopsis: WRKYs Interact with Histone Deacetylase HDA19 PLANT CELL, September 1, 2008; 20(9): 2282 - 2282. [Full Text] [PDF]

This Article Abstract Full Text (PDF) PPT slides of all figures Supplemental Data All Versions of this Article: 20/9/2357    most recent tpc.107.055566v1 Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Related articles in Plant Cell Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Kim, K.-C. Articles by Chen, Z. Search for Related Content PubMed PubMed Citation Articles by Kim, K.-C. Articles by Chen, Z. Agricola Articles by Kim, K.-C. Articles by Chen, Z.