WRKY transcription factors

Review

Feature Review

WRKY transcription factors Paul J. Rushton1, Imre E. Somssich2, Patricia Ringler3 and Qingxi J. Shen3 1

Department of Biology and Microbiology, South Dakota State University, Brookings, SD 57007, USA Max-Planck-Institut fu¨r Pflanzenzu¨chtungsforschung, Abteilung Molekulare Phytopathologie, Carl-von-Linne´-Weg 10, D-50829 Ko¨ln, Germany 3 School of Life Sciences, University of Nevada, Las Vegas, NV 89154, USA 2

WRKY transcription factors are one of the largest families of transcriptional regulators in plants and form integral parts of signalling webs that modulate many plant processes. Here, we review recent significant progress in WRKY transcription factor research. New findings illustrate that WRKY proteins often act as repressors as well as activators, and that members of the family play roles in both the repression and derepression of important plant processes. Furthermore, it is becoming clear that a single WRKY transcription factor might be involved in regulating several seemingly disparate processes. Mechanisms of signalling and transcriptional regulation are being dissected, uncovering WRKY protein functions via interactions with a diverse array of protein partners, including MAP kinases, MAP kinase kinases, 14-3-3 proteins, calmodulin, histone deacetylases, resistance proteins and other WRKY transcription factors. WRKY genes exhibit extensive autoregulation and cross-regulation that facilitates transcriptional reprogramming in a dynamic web with builtin redundancy. WRKY transcription factors – a historical perspective Fifteen years have passed since the first reports of WRKY transcription factors [1–3] and substantial progress has been achieved since then. The first two reports were of illdefined DNA binding proteins that played potential roles in the regulation of gene expression by sucrose (SPF1) [1] or during germination (ABF1 and ABF2) [2]. A third report identified WRKY1, WRKY2 and WRKY3 from parsley (Petroselinum crispum) and coined the name WRKY (pronounced ‘worky’) [3]. This paper also provided the first evidence that WRKY proteins play roles in regulating plant responses to pathogens, and many reports have since shown this to be a major role of WRKY transcription factors. In particular, WRKY factors are key regulators, both positive and negative, of the two partly interconnected branches of plant innate immunity, microbe- or pathogen-associated molecular pattern-triggered immunity (MTI or PTI) and effector-triggered immunity (ETI) (see Glossary) [4]. The WRKY family is among the ten largest families of transcription factors in higher plants and is found throughout the green lineage (green algae and land plants) [5]. The family has expanded during the evolution of plants. This expansion is likely to be associated with the ongoing development of highly sophisticated Corresponding author: Shen, Q.J. ([email protected])

defence mechanisms co-evolving in land plants together with their adapted pathogens. Significant advances regarding our understanding of WRKY proteins have occurred in the past ten years since the publication of the first review on WRKY transcription factors in 2000 [6]. However, more recently, research has focussed on additional roles of WRKY factors in plant processes such as germination, senescence and responses to abiotic stresses such as drought and cold. Moreover, such studies are no longer restricted to model plants such as Arabidopsis (Arabidopsis thaliana) but are rapidly expanding to include in particular, crop species. In this review we will examine some of the recent advances in our knowledge in this rapidly moving area of plant research. This includes new information on the roles of WRKY transcription factors in plants, the mechanisms of WRKY protein function, autoregulation and cross-regulation in signalling involving WRKY transcription factors and the latest information about the evolution of WRKY genes based on recently sequenced plant genomes. Previous reviews have mainly concentrated on the roles of WRKY transcription factors in defence responses. Although this aspect will be considered, we have placed a major emphasis on all other known roles of WRKY transcription factors in order to produce a more comprehensive review of their roles in plants. The WRKY domain and the W box The defining feature of WRKY transcription factors is their DNA binding domain. This is called the WRKY domain after the almost invariant WRKY amino acid sequence at the N-terminus (Figure 1a) [3,6]. In a few WRKY proteins, the WRKY amino acid sequences have been replaced by WRRY, WSKY, WKRY, WVKY or WKKY [7]. The WRKY

Glossary ETI: Effector-triggered immunity. This is race- or cultivar-specific resistance based on the perception of effector proteins by resistance (R) proteins. This leads to a quick and strong induction of defence responses. MAMP or PAMP: Microbe- or pathogen-associated molecular pattern. This is a conserved structure of microbes or pathogens that are recognized by the host. MTI or PTI: MAMP or PAMP-triggered immunity. This is a basal defence mechanism that is induced by MAMP or PAMP perception and is effective against a broad range of pathogens. W box: The minimal consensus sequence (TTGACC/T) required for specific DNA binding of most WRKY transcription factors. WRKY domain: The DNA binding domain that is the defining feature of WRKY transcription factors. The WRKY domain contains the WRKY amino acid signature near the N-terminus and a novel zinc-finger structure at the Cterminus.

1360-1385/$ – see front matter ß 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.tplants.2010.02.006 Available online 19 March 2010

247

Review

Trends in Plant Science Vol.15 No.5

Figure 1. The WRKY domain. (a) The WRKY domain consensus for each WRKY subfamily in higher plants. MEME [91] was used to produce each consensus sequence using WRKY domains from Arabidopsis thaliana, rice (Oryza sativa), poplar (Populus trichocarpa) and soybean (Glycine max). The WRKY motif is highlighted in yellow and the cysteines and histidines that form the zinc finger are shown in blue. The four b-strands are shown in red. I CT and I NT denote the N-terminal and C-terminal WRKY domains from Group I WRKY proteins. (b) Two views of a spacefill structural model of the C-terminal WRKY domain from AtWRKY4. The model was produced using Venn [92] and was based on the solution structure by Yamasaki et al. [10]. As in Figure 1(a), the WRKYGQK motif is shown in yellow and the cysteines and histidines that form the zincbinding pocket are shown in blue.

domain is about 60 residues in length, and as well as containing the WRKY signature it also has an atypical zinc-finger structure at the C-terminus. The zinc-finger structure is either Cx4–5Cx22–23HxH or Cx7Cx23HxC. Initially, in the absence of a complete gene family from any plant species, the WRKY transcription factors were divided into three groups based on the number of WRKY domains (two domains in Group I proteins and one in the others) and the structure of their zinc fingers (C2HC in Group III proteins) [6]. Group II genes were further divided into IIa, IIb, IIc, IId and IIe based on the primary amino acid sequence (Figure 1a). Later analysis based purely on phylogenetic data showed that the WRKY family in higher plants is more accurately divided into Groups I, IIa + IIb, IIc, IId + IIe, and III. The Group II genes are not monophyletic (Figure 2) [8,9]. 248

Structure of the WRKY domain The first accurate description of the WRKY domain also presented the first evidence that it might contain a novel zinc-finger structure [2]. Binding of wild oat (Avena fatua) ABF1 and ABF2 to their W box (TTGACC/T) binding sites was abolished by the addition of the divalent metal chelator 1,10-o-phenanthroline, thus supporting a zinc-finger structure. In 2005, Yamasaki et al. reported the first solution structure of a WRKY domain [10]. The WRKY domain consists of a four-stranded b-sheet, with the zinc coordinating Cys/His residues forming a zinc-binding pocket (Figure 1b). The WRKYGQK residues correspond to the most N-terminal b-strand, which partly protrude from one surface of the protein, thereby enabling access to the major DNA groove and contacts with the DNA. It was proposed that the b-strand containing the WRKYGQK motif

Review

Figure 2. Phylogenetic tree of the WRKY family in higher plants. The WRKY domains from the complete WRKY gene families in soybean (Glycine max, red dots), rice (Oryza sativa, yellow), Arabidopsis thaliana (blue) and poplar (Populus trichocarpa, green) were used and the evolutionary history of the WRKY gene family was inferred using the Neighbor-Joining method. The WRKY subfamilies are indicated; I NT and I CT indicate the N-terminal and C-terminal domains from Group I WRKY proteins, respectively. The tree supports the I, IIa + IIb, IIc, IId + IIe, and III division of the WRKY family and the observation that Group II genes are not monophyletic. The optimal tree is shown. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method and are in the units of the number of amino acid substitutions per site. Phylogenetic analyses were conducted in MEGA4 [93].

makes contact with an approximately 6-bp region, and this is consistent with the length of the W box [10]. A crystal structure of the AtWRKY1 C-terminal WRKY domain showed a similar structure with an additional b-strand N-terminal to the other four that became apparent because the domain used was longer [11]. The structure of the WRKY domain shares some similarity with both the NAC and GCM DNA-binding domains, and this led to the proposition that the WRKY and GCM domains are evolutionarily related [10–12]. The WRKY and GCM zinc-fingers do have similarities in their zinc-binding motifs, but the hallmark WRKYGQK signature is missing from GCM proteins. How meaningful these similarities are is questionable because the WRKY domain seems to be more ancestral than the GCM domain, and any relatedness between the two lies way back in evolutionary history before the plant kingdom diverged from the other eukaryotic kingdoms [10]. W boxes and clustered W boxes The conservation of the WRKY domain is mirrored by a remarkable conservation of its cognate binding site, the W

Trends in Plant Science

Vol.15 No.5

box (TTGACC/T) [3,6]. Gel shift experiments, random binding site selection, yeast one-hybrid screens and cotransfection assays performed with many different WRKY proteins have shown that the W box is the minimal consensus required for specific DNA binding [3,13]. Almost all WRKY transcription factors bind preferentially to the same core sequence, and this poses the question: how do WRKY factors show specificity for their target promoters? A detailed study of the binding selectivity of five Arabidopsis WRKY proteins using gel shift experiments showed that although the W box core is required, adjacent sequences also partly determine binding site preference [13]. AtWRKY6 and AtWRKY11 show high affinity towards W boxes that have a G residue directly 50 to the element, whereas AtWRKY26, AtWRKY38 and AtWRKY43 bind better to the same motif if this residue is a T, C or A. Another important observation concerned the identity of ‘W box-like sequences’ that do not function as W boxes; neither CTGACC nor TTGACA were bound by any of the five WRKY representatives tested, suggesting that a minimal W box element might be defined as TTGACC/T [13]. Both bioinformatic-based and functional studies of plant promoters have found clusters of W boxes in stress-inducible promoters [14]. In the case of PcWRKY1, the multiple W boxes appear to have a synergistic effect on transcription [15]; in barley (Hordeum vulgare), HvWRKY38 requires two adjacent W boxes for efficient binding [16]. There are a few reports of WRKY proteins binding to non-W box sequences. OsWRKY13 can bind to the PRE4 element (TGCGCTT) as well as to W boxes [17], and SUSIBA2 (a sucrose-regulated barley WRKY transcription factor which is also called HvWRKY46 [18]) can bind to both W boxes and a sugar-responsive (SURE) element (TAAAGATTACTAATAGGAA [19,20]) whereas tobacco (Nicotiana tabacum) NtWRKY12 appears to bind a SURE-like element but not the W box [20]. NtWRKY12 has a GKK amino acid sequence following WRKY rather than the more common GQK, and binds specifically to the WK box (TTTTCCAC). Mutation of the GKK motif into GQK or GEK abolished interaction with the WK box, suggesting that these amino acids may be important for binding site recognition [21]. Roles played by WRKY transcription factors From the beginning of research into WRKY transcription factors, it was evident that they play roles in regulating several different plant processes; however, for convenience, some of these are dealt with individually below. The recent data presented here lead us to a new insight, namely that it is common for a single WRKY transcription factor to regulate transcriptional reprogramming associated with multiple plant programmes. The dynamic web of signalling in which WRKY factors operate has multiple inputs and outputs. Biotic stress The majority of reports concerning WRKY transcription factors have indicated that numerous members of the multigene family play roles in the transcriptional reprogramming associated with the plant immune response. This is an active research area that has been extensively reviewed and therefore will only be briefly touched upon 249

Review

Trends in Plant Science Vol.15 No.5

Figure 3. WRKY transcription factors mediate immune responses. Two pathways are shown. The pathway in barley (Hordeum vulgare) involves the resistance protein MLA and WRKY transcription factors [94]. The other pathway in Arabidopsis involves a MAP kinase (MAPK) pathway consisting of a MAP kinase kinase kinase (MEKK), a MAP kinase kinase (MKK), and a MAP kinase (MPK). In barley (right), MAMP or PAMP are perceived by receptors that initiate signalling via intracellular MAPK cascades. This stimulates the induction of unknown WRKY transcriptional activators (red) and HvWRKY1/2 repressors (blue). The WRKY repressors may prevent chronic defence gene activation. Autorepressed MLA receptors are folded by RAR1, SGT1 and cytosolic HSP90 and the result is basal defence. Upon coactivation of one or several MAMP or PAMP receptors and MLA by cognate Blumeria graminis f. sp. hordei (powdery mildew) effectors (in this case AVRA), an integrated MAMP or PAMP and MLA-triggered immune response is triggered. Activated MLA stimulates nuclear association with the WRKY1/2 repressors, thereby de-repressing MAMP-triggered immunity. Derepression of basal defence responses is thought to amplify the expression of defence-related genes. In Arabidopsis, the MEKK1–MKK1/2–MPK4 module is activated by MAMP or PAMP [75,76]. This leads to nuclear dissociation of the MPK4–MKS1–WRKY33 complex and release of WRKY33 and MKS1. WRKY33 enhances the expression of PAD3. PAD3 is required for the synthesis of the antimicrobial camalexin [76]. Abbreviations: ETI, effector-triggered immunity; HSP90, heat shock protein 90; MAMP, microbe-associated molecular pattern; MLA, mildew-resistance locus A; MTI, microbe-associated molecular pattern-triggered immunity; PAD4, phytoalexin deficient 4; PAMP, pathogen-associated molecular pattern; PTI, pathogen-associated molecular pattern-triggered immunity; RAR1, required for Mla12 resistance 1; and SGT1, suppressor of G-two allele of skp1.

here [4,22]. What we have learned is that WRKY factors are central components of many aspects of the innate immune system of the plant, including MTI or PTI, ETI, basal defence and systemic acquired resistance [4]. There are now many examples where overexpression or knockdown of WRKY gene expression has effects on plant defence, and this has allowed the unravelling of some components of the web of signalling (Figures 3–5; see Supplementary material Table S1) [22–25]. Within these networks, several WRKY proteins are targets of MAP kinases [26]. In barley, ETI to barley powdery mildew (Blumeria graminis f. sp. hordei) requires the recognition of the fungal avirulence AVR10 effector by the resistance protein MLA (mildew-resistance locus A) in the cytoplasm and the subsequent association of MLA with 250

HvWRKY1 and HvWRKY2 within the nucleus (Figure 3) [27]. HvWRKY1 and HvWRKY2 were also shown to act as negative regulators of PTI and, therefore, this study provided a mechanistic link between these two major defence signalling pathways. Despite intense studies, few additional components involved in linking the terminal signalling stages of these defence pathways with WRKY factors, or with any other transcription factors for that matter, have been identified. A recent study in rice has revealed that the pair of allelic genes OsWRKY45-1 and OsWRKY45-2, which encode proteins differing in ten amino acids, play opposite roles in rice–bacteria interactions [28]. The OsWRKY451 allele is found in the japonica subspecies of Oryza sativa, and the OsWRKY45-2 allele in the indica subspecies.

Review

Trends in Plant Science

Vol.15 No.5

Figure 4. The roles of WRKY transcription factors in signalling pathways in response to herbivore attack and dehydration. (a) A signalling cascade in native tobacco (Nicotiana attenuata) in response to wounding by herbivores [30]. Two WRKY genes, NaWRKY3 and NaWRKY6, coordinate responses to herbivory. NaWRKY3 is required for NaWRKY6 elicitation by fatty acid–amino conjugates (FACs) in Manduca sexta larval oral secretions. The two WRKY transcription factors regulate expression of jasmonic acid (JA) biosynthesis genes (LOX, AOS, AOC and OPR), thereby increasing the levels of JA and JA-isoleucine. This is turn regulates direct and indirect defences against herbivores. (b) A dehydration-induced signalling pathway in the resurrection plant Boea hygrometrica [40]. Galactinol synthase plays an important role in drought tolerance and the BhGolS1 gene is inducible by both dehydration and ABA. The BhGolS1 promoter contains four W boxes that are bound in vivo by the early dehydration and ABAinducible BhWRKY1. This interaction is associated with activation of transcription and drought tolerance [40]. Abbreviations: ABA, abscisic acid; AOC, allene oxide cyclase; AOS, allene oxide synthase; LOX, 13-lipoxygenase; and OPR, 12-oxo-phytodienoic acid reductase.

Overexpression studies of both alleles show that they positively regulate resistance to the rice fungal pathogen Magnaporthe grisea [28,29], but they differentially regulate resistance to Xanthomonas oryzae (bacterial rice leaf blight). OsWRKY45-1 appears to be a negative regulator and OsWRKY45-2 appears to be a positive regulator of plant response to X. oryzae. In addition, it appears that these genes work through different pathways because differential expression of OsWRKY45-1 modulates salicylic acid and jasmonic acid (JA) levels, whereas differential expression of OsWRKY45-2 appears to only significantly modulate JA levels. An elegant set of experiments in the native tobacco Nicotiana attenuata showed that two WRKY genes, NaWRKY3 and NaWRKY6, coordinate responses to herbivory [30]. NaWRKY3 is required for NaWRKY6 elicitation by fatty acid–amino conjugates in Manduca sexta larval oral secretions, and silencing either or both genes made plants highly vulnerable to herbivores. It appears that the two WRKY genes help plants differentiate mechanical wounding from herbivore attack (Figure 4a) [30]. WRKY transcription factors are also involved in responses to nematodes. The Arabidopsis gene AtWRKY23 is upregulated almost immediately upon nematode infection and knockdown lines show increased resistance to the cyst nematode Heterodera schachtii [31]. Finally, an Arabidopsis WRKY gene previously shown to have a role in pathogen defence was reported to negatively regulate susceptibility

of the plant to Agrobacterium-mediated transformation [32]. Abiotic stress WRKY transcription factors play pivotal roles in regulating many stress reactions in plants; however, unravelling their roles in abiotic stress responses has lagged behind that of biotic stresses. This may be a reflection of crosstalk and redundancy intrinsic to such responses and also a lack of suitable mutant lines. In one of the earliest studies, a WRKY gene isolated from a xerophytic evergreen C3 shrub, the creosote bush (Larrea tridentata), was shown to be an activator of abscisic acid (ABA) signalling [33]. ABA mediates plant responses to abiotic stresses, and, hence is called a ‘stress hormone’. In another transient expression study using aleurone cells, OsWRKY24 and OsWRKY45 were found to act as repressors of an ABA-inducible promoter, and OsWRKY72 and OsWRKY77 were shown to be activators of the same promoter [34]. Some of the other earliest evidence of roles related to abiotic stress responses came from transcription profiling; however, recent functional analyses have provided more direct evidence. For example, in rice, heat shock inducible HSP101 promoter-driven overexpression of OsWRKY11 led to enhanced heat and drought tolerance [35]. Likewise, overexpression of OsWRKY45 resulted in enhanced salt and drought tolerance, in addition to increased disease resistance [25]. In Arabidopsis, overexpression of either AtWRKY25 or AtWRKY33 increases salt 251

Review

Trends in Plant Science Vol.15 No.5

Figure 5. The roles of WRKY transcription factors in signalling pathways during germination and senescence. (a) Amy32b a-amylase gene expression in barley (Hordeum vulgare) aleurone cells during germination [52]. Abscisic acid (ABA) promotes onset and maintenance of seed dormancy. By contrast, gibberellins (GAs) break seed dormancy and promote seed germination. Negative regulators HvWRKY38, BPBF (a DOF transcription factor), HRT and HvMCB1 (a MYB transcription factor) bind to their corresponding cis-acting elements in the absence of GA and form a ‘repressosome’ [52,95]. Positive regulators RAMY (a zinc-finger transcription factor), SAD (a DOF transcription factor), HvGAMYB, and HvMYBS3 bind to the same cis-acting elements in the presence of GA and form an ‘enhanceosome’ [52,96]. This results in transcription of the Amy32b gene and the production of a-amylase enzyme. How the ‘repressosome’ is replaced by the ‘enhanceosome’ during the transition of Amy32b from the repressed to the activated state is unknown. (b) A senescence-induced signalling pathway in Arabidopsis. MEKK1 was found to bind to the AtWRKY53 promoter. AtWRKY53 itself is phosphorylated by MEKK1 and this may increase binding of AtWRKY53 to its own promoter [59]. Epigenetic regulation has also been linked to the regulation of AtWRKY53. Upon activation of the locus during senescence, H3K4me2 and H3K4me3 methylation significantly increases in its promoter and coding regions [61]. Abbreviations: H3K4me2, di-methylated histone H3 at lysine 4; H3K4me3, tri-methylated histone H3 at lysine 4; HRT, hordeum repressor of transcription; and MEKK1, MAP kinase kinase kinase.

tolerance [36]. Additional work on AtWRKY25 using both null mutants and overexpression lines showed that it is also involved in the response to heat stress [37]. These examples illustrate that WRKY factors form part of the signalling processes associated with transcriptional reprogramming when plants encounter high salt, heat, osmotic stress, high CO2 levels, high ozone concentrations, cold or drought (see Supplementary material Table S1). Further evidence has come from altered plant responses to different abiotic stresses following the overexpression of three stress-inducible soybean (Glycine max) WRKY genes in Arabidopsis. GmWRKY21-overexpressing Arabidopsis plants were more tolerant to cold stress than wild-type plants, whereas GmWRKY54-overexpressing plants were more salt and drought tolerant, and GmWRKY13 overexpression resulted in increased sensitivity to salt and mannitol stress [38]. This recent progress shows that many WRKY genes play roles in multiple stress-induced signalling pathways (see Supplementary material Table S1) and that the ‘WRKY web’ of signalling includes both biotic stress and abiotic stress components [39]. There is currently little information about other components of WRKY transcription factor-regulated abiotic stress-induced signalling pathways. Recently, however, 252

advances have been made in a dehydration tolerance signalling pathway in the resurrection plant Boea hygrometrica (Figure 4b) [40]. Galactinol synthase plays an important role in drought and cold tolerance [41] and the BhGolS1 gene is inducible by both dehydration and ABA. The BhGolS1 promoter contains four W boxes and chromatin immunoprecipitation showed that it is bound in vivo by the early dehydration and ABA-inducible BhWRKY1 [40]. These data provide direct evidence linking a dehydration-inducible WRKY factor with a downstream target gene that plays a vital role in drought responses. Seed development The role of WRKY genes in seed development is implicated in several gene expression studies. The WRKY transcription factor DGE1 of orchardgrass (Dactylis glomerata) is expressed during somatic embryogenesis [42]. A Group Ia WRKY transcription factor in wild potato (Solanum chacoense), ScWRKY1, was found to be expressed strongly and transiently in fertilized ovules at the late torpedo stage [43]. SUSIBA2 is expressed in the endosperm and regulates starch production [20]. Arabidopsis AtWRKY10, also known as MINISEED3, is expressed in pollen and the globular embryo as well as in

Review developing endosperm from the two-nuclei stage through the cellularization stage. Plants homozygous for a knockout mutation in this gene produce small seeds. The development of these seeds showed reduced growth of the embryo and early cellularization of the endosperm, highlighting the key role that WRKY genes play during this process [44]. Seed dormancy and germination In cereals, a-amylase enzymes are involved in hydrolysis of starch, an important step in germination and postgermination growth of cereals. These genes are gibberellin (GA)-inducible and ABA-repressible and, hence, are ideal for GA and ABA crosstalk studies. In an early study of the WRKY family, two wild oat WRKY transcription factors, ABF1 and ABF2, were found to bind to W boxes in the promoters of the a-amylase gene a-Amy2 [2]. Several studies have demonstrated that rice and barley homologues of these two wild oat WRKY genes are ABA-inducible and GA-repressible in aleurone cells and embryos. Via bombardment-mediated transient expression in rice and barley, rice OsWRKY51 and OsWRKY71 were found to encode repressors of the rice RAmy1A a-amylase and the barley Amy32b a-amylase genes. By forming a heterotetramer, OsWRKY51/71 antagonises GAMYB, a well-documented transcriptional activator of GA signalling. Exogenous GA treatment destabilizes GFP:OsWRKY71 whereas the proteasome inhibitor MG132 blocks the degradation of this fusion protein [7,45,46]. These lines of evidence suggest that OsWRKY51 and OsWRKY71 are key regulators mediating the crosstalk of GA and ABA in aleurone cells and embryos. A more detailed study addressed how WRKY proteins suppress GA signalling, using the putative barley orthologue of OsWRKY71, HvWRKY38 [16,47]. Each of the four cis-acting elements essential for GA induction of the barley Amy32b a-amylase gene can be bound by both transcriptional repressors and activators in barley aleurone cells [48–51]. This implies that both GA induction and ABA antagonistic suppression of GA effects are mediated by the same set of cis-acting DNA elements. Based on these studies [48–51], a model has been proposed for the antagonistic regulation of Amy32b expression by GA and ABA, which involves a ‘repressosome’ and an ‘enhanceosome’ (Figure 5a) [52]. Switching from the ‘repressosome’ to the ‘enhanceosome’ during the transition of Amy32b from the repressed to the activated state is likely to be mediated by the GA-promoted degradation of repressors [45] and GA-induced expression of activators such as GAMYB [53]. By contrast, ABA induces the expression of the repressor genes, blocks the expression of the activator genes, and hence promotes switching of the promoter binding complex from ‘enhanceosome’ to ‘repressosome’. As a result, GA induction is antagonized by ABA. Although genetic evidence is required to validate that OsWRKY51, OsWRKY71 and HvWRKY38 really mediate cereal seed germination, studies in Arabidopsis also argue for the importance of WRKY genes in controlling plant responses to GA. RGA, encoding a DELLA protein, is a negative regulator of GA signalling. Analysis of RGA mutants indicated that AtWRKY27 is a direct target of

Trends in Plant Science

Vol.15 No.5

RGA, possibly through interactions with other DNA binding proteins. AtWRKY27 was also found to be negatively regulated in an immediate/early manner by GA, supporting a potential role of this transcription factor as a negative regulator of GA signalling [54]. Furthermore, studies of TDNA insertion mutants have indicated a possible role of Arabidopsis AtWRKY2 as a negative feedback regulator of ABA-mediated arrest of seed germination and post-germination growth [36]. Senescence WRKY transcription factors are involved in the regulation of leaf senescence. Expression profiling in Arabidopsis revealed that WRKY transcription factors are the second largest family of transcription factors in the senescence transcriptome [55]. The first evidence of a role in senescence came from studies of AtWRKY6 [56,57]. AtWRKY6 is strongly upregulated during senescence, and analysis of AtWRKY6 target genes identified the SENESCENCEINDUCED RECEPTOR KINASE/FLG22-INDUCED RECEPTOR-LIKE KINASE (SIRK/FRK1). SIRK/FRK1 encodes a receptor-like protein kinase whose expression is strongly and specifically induced during leaf senescence [57]. Other WRKY genes that regulate senescence include AtWRKY53, AtWRKY70 and OsWRKY23. Overexpression or RNAi knockdown of AtWRKY53 led to senescenceassociated phenotypes [58], and a role for MEKK1 in this process has been determined [59] (Figure 5b). Knockout lines for AtWRKY70 showed that it acts as a negative regulator of senescence [60], and overexpression of OsWRKY23 accelerated leaf senescence [24]. This illustrates that members of the WRKY transcription factor family both positively and negatively regulate this process. Recently, epigenetic programming has also been implicated in the mechanism whereby AtWRKY53 regulates senescence (see below) [61]. Development WRKY transcription factors such as SUSIBA2 and MINISEED3 play roles in the regulation of seed development and many WRKY proteins regulate senescence, but there are few reports of WRKY factor involvement in other developmental processes. One exception is Transparent Testa Glabra 2 (TTG2)/AtWRKY44, which plays a role in trichome development and also effects mucilage and tannin synthesis in the seed coat [62]. TTG2 is expressed in young leaves, trichomes, seed coats and root hairless cells. A mechanism for TTG2 function is suggested by the observation that the expression of TTG2 is regulated by bHLH (basic helix–loop–helix) and R2R3MYB transcription factors such as WEREWOLF, GLABRA1 and TRANSPARENT TESTA, and that TTG2 in turn appears to regulate the expression of GLABRA2 [63]. Furthermore, maternally expressed TTG2 controls lethality in interploidy crosses of Arabidopsis and maps to the Doctor Strangelove 1 quantitative trait locus (QTL) that affects seed survival [64]. Roles in multiple processes and additional roles WRKY transcription factors demonstrate that a single transcription factor may be involved in regulating several seemingly disparate processes. HvWRKY38 and HvWRKY1 253

Review appear to be allelic because their sequences are >99% identical at the DNA level (1239 of 1241 bp). Interestingly, HvWRKY38 has been shown to be involved in cold and drought responses [16], whereas HvWRKY1 is a repressor of basal defence and directly interacts with the MLA resistance protein (Figure 3) [27]. In addition, HvWRKY1/38 and other similar genes from rice and wild oat have also been shown to act as repressors of seed germination [47,52]. The conclusion from these studies is that HvWRKY1/38 is a regulator of at least three different processes: biotic stress responses, abiotic stress responses and germination. Additional roles for members of the WRKY family have been suggested. AtWRKY75 appears to act as a modulator of phosphate starvation [65] and CjWRKY1 regulates steps in alkaloid biosynthesis [66]. Recently, the role of a WRKY gene that is found outside of the plant kingdom was demonstrated. The sole WRKY gene found in the intestinal protozoan parasite Giardia lamblia is involved in the transcriptional regulation of cyst wall protein genes [67]. Interestingly, the non-plant WRKY transcription factor binds specifically to W boxes, including several in its own promoter, suggesting an autoregulation mechanism similar to that found with some plant WRKY genes. Mechanisms of WRKY function: activation, repression and de-repression of transcription WRKY proteins can activate or repress transcription and are generally rich in potential transcriptional activation and repression domains. Some WRKY factors possess both functions. For example, in yeast, AtWRKY53 was found to activate or repress transcription of a reporter gene depending on the promoter context [68]. AtWRKY53 cotransformed with reporter constructs into Arabidopsis protoplasts negatively regulated its own promoter while acting on other promoters as an activator, which is consistent with the results in yeast [68]. Similarly, Arabidopsis AtWRKY6 negatively autoregulates its own promoter while transcriptionally activating SIRK/FRK1 [57]. Via transient expression studies, OsWRKY72 and OsWRKY77 have been shown to be activators of ABA signalling, but repressors of GA signalling in aleurone cells [7]. Substantial evidence now indicates that many genes are repressed by WRKY factors bound to their promoters. The extent of this role of WRKY proteins in repression is one of the insights into WRKY functions. Observations made on PcWRKY1 led to the proposal of a model to explain PcPR10 gene activation [5]. In this model, W box elements are generally occupied by WRKY factors. Following recognition of an elicitor molecule by a receptor, a mitogenactivated protein kinase (MAPK) cascade is activated, with a protein kinase being translocated into the nucleus of the cell where it can modify bound WRKY factors directly. This allosteric interaction causes their release and possible replacement by other WRKY transcription factors from their cognate W box elements, thereby derepressing PcPR10 and PcWRKY1. Other examples of WRKY factors that act as repressors have been mentioned earlier, such as OsWRKY71, which is a repressor of GA-inducible a-amylase gene activation, and HvWRKY1 and HvWRKY2, which bind to and repress basal defence genes (Figure 3) [27]. 254

Trends in Plant Science Vol.15 No.5

MAP kinase pathways are involved in controlling WRKY transcription factor activity [59,69–75]. In Arabidopsis, a recent report has shown that the WRKY transcription factor AtWRKY33 exists in nuclear complexes with the MAP kinase MPK4. MAMP or PAMP perception activates a MEKK1–MKK1/2–MPK4 module consisting of a MAP kinase (MPK), a MAP kinase kinase (MKK) and a MAP kinase kinase kinase (MEKK) [76]. This leads to nuclear dissociation of an MPK4–MKS1–WRKY33 complex and release of AtWRKY33 and MKS1. AtWRKY33 then activates the expression of PAD3 ( phytoalexin deficient 3). PAD3 is required for the synthesis of the antimicrobial camalexin [76]. Another insight concerns a senescence-induced signalling pathway in Arabidopsis. Interestingly, MEKK1 was found to bind directly to the AtWRKY53 promoter at a site (WP1) upstream of a W box where AtWRKY53 can bind to its own promoter. AtWRKY53 itself is phosphorylated by MEKK1 and this may increase binding of AtWRKY53 to its own promoter (Figure 5b) [59]. MEKK1 appears to direct a shortcut in signalling by both directly phosphorylating AtWRKY53 and also binding to the promoter of its encoding gene [59]. Histone modifications may also play essential roles in these repression–de-repression processes. The Arabidopsis WRKY genes AtWRKY38, AtWRKY53, AtWRKY62 and AtWRKY70 have all been implicated in processes involving covalent histone modifications [61,77,78]. AtWRKY70 is a primary target for the Arabidopsis homologue of Trithorax (ATX1). ATX1 activates the expression of AtWRKY70 by establishing nucleosomal histone H3K4 trimethylation marks, and it has been suggested that the PR-1 and THI2.1 defence genes are controlled via this epigenetic regulation of AtWRKY70 [77]. Epigenetic regulation has also been linked to the regulation of AtWRKY53, a key modulator of leaf senescence. Upon activation of the locus during senescence, H3K4me2 and H3K4me3 methylation significantly increases in its promoter and coding regions [61]. Another study of histone modifications centred on the AtWRKY38 and AtWRKY62 genes [78]. These WRKY factors are negative regulators of basal defence and interact with Histone Deacetylase 19 (HDA19). HDA19 represses transcription by removing acetyl groups from histone tails, and this suggests a mechanism whereby WRKY transcription factors can repress defence gene activation. Another mechanism of WRKY function probably operates through small RNAs (smRNAs). These are broadly classified into micro RNAs (miRNAs) and small interfering RNAs (siRNAs), and have emerged as a fundamental layer of the regulation of gene expression. This is an area that promises rapid advances in the near future because the predicted targets for several miRNAs encode WRKY transcription factors [22]. It is likely that future studies will not only show that WRKY factors regulate smRNA populations but also that WRKY transcription factors themselves are targeted by smRNAs. Autoregulation and cross-regulation: negative feedback loops and feed forward modules One feature of the WRKY web of signalling is autoregulation by interaction of WRKY transcription factors with their own promoters and cross-regulation by other WRKY

Review

Trends in Plant Science

transcription factors [4,15,57]. The first example was PcWRKY1, which has a conserved arrangement of three synergistically acting W boxes (WABC) in its promoter [15]. In response to PAMP treatment, PcWRKY1 transcripts accumulate rapidly and transiently [3]. Surprisingly, temporal chromatin immunoprecipitation (ChIP) studies showed that the three W boxes are constitutively occupied by WRKY transcription factors [79] and recruitment of PcWRKY1 to its own promoter coincides with the downregulation of PcWRKY1. It seems that the transient activation of PcWRKY1 [3] is achieved through crossregulation by other WRKY factors that activate transcription, followed by repression caused by a negative feedback loop [79]. Many WRKY gene promoters are statistically enriched for W boxes and this suggests that autoregulation and cross-regulation are common features of WRKY action [4,80]. Interacting partners Only a few components of signalling pathways interacting with WRKY transcription factors have been identified. In addition to histone deacetylases, and MAP kinases mentioned above, other interacting partners have been reported. Arabidopsis Group IId WRKY proteins contain a calmodulin (CaM)-binding domain, designated the Cmotif (DxxVxKFKxVISLLxxxR), which is bound by CaM in vitro, suggesting possible regulation by CaM and Ca2+ fluxes [81]. Tandem affinity purification tag experiments have also shown that at least seven Arabidopsis WRKY factors form complexes with 14-3-3 proteins [82]. The 14-33 proteins are known to regulate diverse cellular functions through hundreds of different protein–protein interactions. Many DNA binding experiments suggest that WRKY proteins can bind as monomers to W boxes. The extent to which WRKY proteins form functional dimers is unknown, but there is clear evidence for homo- or heterodimer formation among some WRKY proteins. This is a feature of the leucine zipper-containing Group IIa WRKY proteins [2,83], and the Arabidopsis Group IIa proteins AtWRKY18, AtWRKY40 and AtWRKY60 form homo- and hetero-complexes [84]. The rice proteins OsWRKY51 and OsWRKY71 interact in the nucleus of aleurone cells and this enhances the binding of OsWRKY71 to the Amy32b promoter, even though OsWRKY51 itself does not bind [46].

Vol.15 No.5

NBS–LRR–WRKY proteins One unexpected discovery was the existence of chimeric proteins comprising domains typical for both intracellular type-R proteins (NBS–LRR proteins) and WRKY transcription factors [85]. AtWRKY52/RRS1 contains a Group III WRKY domain C-terminal to a TIR–NBS–LRR (Toll/ interleukin-1 receptor–nucleotide-binding site–leucine-rich repeat) domain and confers immunity towards the bacterial pathogen Ralstonia solanacearum by nuclear interaction with the bacterial effector PopP2 [85,86]. A missense mutation within the WRKY domain results in conditional activation of defence responses and a loss of binding to W boxes [87]. Moreover, AtWRKY52/RRS1 acts together with the R protein RPS4 to provide dual resistance towards fungal and bacterial pathogens [88]. This suggests that the TIR–NBS–LRR–WRKY may allow a shortcut of the ETI pathway, leading to defence gene activation. In addition to AtWRKY52/RRS1, AtWRKY16/TTR1 and AtWRKY19, NBS–LRR–WRKY proteins can be found in several other dicots. The presence of WRKY domains from different subfamilies suggests that this mechanistic shortcut has evolved on several occasions. Interestingly, AtWRKY19 also contains a domain in its C-terminus that is highly sequence-related to the previously mentioned MEKK1. WRKY transcription factors across the plant kingdom There has been a lineage-specific expansion of the WRKY gene family during the course of plant evolution and the number of genes range from a single WRKY transcription factor in the unicellular green alga Chlamydomonas reinhardtii to 37 in the moss Physcomitrella patens, 74 in Arabidopsis and almost 200 in soybean [5] (Table 1). In higher plants, genes from all seven WRKY subfamilies (Groups I, IIa, IIb, IIc, IId, IIe and III) are found (Figure 2). The appearance of the subfamilies can be traced back through the green plant lineage, with Group I being the apparent ancestral type of WRKY gene because it is the only one present in Chlamydomonas reinhardtii [5]. Five Group III genes have been found in the moss Physcomitrella patens, although they are a slightly variant type with the amino acid sequence WKKYGNK instead of WRKYGQK [89]. The ancient origin of Group III genes was a surprise because it was previously assumed to be the last group to evolve due to its expansion in monocots [5,9]. In fact, Group IIa and

Table 1. WRKY gene number in various organisms. Species Arabidopsis lyrata (lyrate rock cress) Arabidopsis thaliana (mouse ear cress) Glycine max (soybean) Medicago truncatula (barrel clover) Populus trichocarpa (black cottonwood) Oryza sativa ssp. indica (rice) Oryza sativa ssp. japonica (rice) Sorghum bicolor (sorghum) Selaginella moellendorffii Physcomitrella patens Chlamydomonas reinhardtii Ostreococcus sp. RCC809 Dictyostelium discoideum Giardia lamblia

Type of organism Dicot plant Dicot plant Dicot plant Dicot plant Dicot plant Monocot plant Monocot plant Monocot plant Spike moss Moss Single-celled green alga Single-celled green alga Slime mould Protozoan

Number of genes 79 74 197 75 103 102 97 93 35 37 1 3 1 1

Refs http://supfam.mrc-lmb.cam.ac.uk/SUPERFAMILY [5] [97] http://supfam.mrc-lmb.cam.ac.uk/SUPERFAMILY http://supfam.mrc-lmb.cam.ac.uk/SUPERFAMILY [98] Table S2 in the supplementary material online http://supfam.mrc-lmb.cam.ac.uk/SUPERFAMILY http://supfam.mrc-lmb.cam.ac.uk/SUPERFAMILY [89] [5] http://supfam.mrc-lmb.cam.ac.uk/SUPERFAMILY [5] [5]

255

Review Group IIe WRKY genes appear to have evolved last as they are the only two groups absent from the moss P. patens [89]. Interestingly, there are a few examples of Group I WRKY genes in non-photosynthetic eukaryotes; one in the slime mould Dictyostelium discoideum and another in the unicellular protist G. lamblia [5]. Both of these non-photosynthetic eukaryotic WRKY genes are classified as Group I, again lending additional support that they are the ancestral type. As these genes contain two WRKY domains, one possibility is that a ‘proto-WRKY’ with a single domain was the true ancestor of plant WRKY genes [9]. Alternatively, deletion of one of the two WRKY domains may have occurred [7]. A comparison between tomato, Arabidopsis and Capsella revealed extensive colinearity of a genomic region encompassing the WRKY orthologues AtWRKY10, tomato Le-D and the Capsella Cr-D, and provides possible evidence in support of this alternative theory [90]. The tomato WRKY gene harbours two WRKY domains whereas Arabidopsis and Capsella harbour only one. One major unanswered question is why so few non-plant species have WRKY genes. The sporadic distribution of the WRKY family in phylogenetically distant eukaryotes is suggestive of their possible spread through intra-eukaryotic lateral transfers [12]. It has been suggested that the WRKY family may represent a lineage-specific expansion following the emergence of an ancestral WRKY gene from a transposon [12], and it seems more likely that a few nonplant species may have gained WRKY genes rather than the wholesale loss of WRKY genes in multiple lineages. Conclusions and future prospects Major advances in WRKY transcription factor research have occurred over the past 15 years. Genetic, molecular and computational biology analyses have provided valuable insights into their regulatory roles in diverse plant stress and hormone responses, in development, as well as in the evolution of the WRKY gene family itself. Individual proteins interacting with a few selected WRKY factors have been identified along with a limited number of WRKY target genes. Moreover, we are beginning to grasp the complexity of the signalling web in which WRKY transcription factors operate and the plant processes that they regulate. Thanks to recent technological breakthroughs we can move forward to address and to unravel several important unresolved questions. For example, use of chromatin immunoprecipitation in combination with massively parallel sequencing (ChIP-Seq) should enable us to detect the specific DNA target sites of WRKY transcription factors in response to diverse signals on a global scale. Such studies should not only enable us to identify WRKYregulated genes but should provide vital information regarding the possible existence of additional minor or major in vivo WRKY binding sites, and also help to resolve how promoter discrimination can be achieved despite the stereotypic binding preference of WRKY transcription factors to the W box. It is also becoming feasible to capture, purify and characterize the highly dynamic, WRKY-associated transcriptional complexes at distinct promoter sites following signalling through defined pathways. One can foresee that such research should provide the biochemical 256

Trends in Plant Science Vol.15 No.5

basis for exploring how the convergence of synergistic and antagonistic signals at a given promoter is resolved and, thereby, help to disentangle how WRKY-dependent crosstalk is achieved. Acknowledgements We thank Senthil Subramanian for helpful comments on the manuscript, and Prateek Tripathi, Ashley Boken and Tanner Langum for help with Table 1. This project was supported by the National Research Initiative Competitive Grant No. 2008-35100-04519 from the USDA National Institute of Food and Agriculture to P.J.R. and Q.J.S., Grant No. 200735304-18297 to Q.J.S, and the DFG Support AFGN Grant SO235/5-4 to I.E.S.

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.tplants. 2010.02.006. References 1 Ishiguro, S. and Nakamura, K. (1994) Characterization of a cDNA encoding a novel DNA-binding protein, SPF1, that recognizes SP8 sequences in the 50 upstream regions of genes coding for sporamin and beta-amylase from sweet potato. Mol. Gen. Genet. 244, 563–571 2 Rushton, P.J. et al. (1995) Members of a new family of DNA-binding proteins bind to a conserved cis-element in the promoters of alphaAmy2 genes. Plant Mol. Biol. 29, 691–702 3 Rushton, P.J. et al. (1996) Interaction of elicitor-induced DNA-binding proteins with elicitor response elements in the promoters of parsley PR1 genes. EMBO J. 15, 5690–5700 4 Eulgem, T. and Somssich, I.E. (2007) Networks of WRKY transcription factors in defense signaling. Curr. Opin. Plant Biol. 10, 366–371 ¨ lker, B. and Somssich, I.E. (2004) WRKY transcription factors: from 5 U DNA binding towards biological function. Curr. Opin. Plant Biol. 7, 491–498 6 Eulgem, T. et al. (2000) The WRKY superfamily of plant transcription factors. Trends Plant Sci. 5, 199–206 7 Xie, Z. et al. (2005) Annotations and functional analyses of the rice WRKY gene superfamily reveal positive and negative regulators of abscisic acid signaling in aleurone cells. Plant Physiol. 137, 176–189 8 Rushton, P.J. et al. (2008) Tobacco transcription factors: novel insights into transcriptional regulation in the solanaceae. Plant Physiol. 147, 280–295 9 Zhang, Y. and Wang, L. (2005) The WRKY transcription factor superfamily: its origin in eukaryotes and expansion in plants. BMC Evol. Biol. 5, 1 10 Yamasaki, K. et al. (2005) Solution structure of an Arabidopsis WRKY DNA binding domain. Plant Cell 17, 944–956 11 Duan, M.R. et al. (2007) DNA binding mechanism revealed by high resolution crystal structure of Arabidopsis thaliana WRKY1 protein. Nucleic Acids Res. 35, 1145–1154 12 Babu, M.M. et al. (2006) The natural history of the WRKY-GCM1 zinc fingers and the relationship between transcription factors and transposons. Nucleic Acids Res. 34, 6505–6520 13 Ciolkowski, I. et al. (2008) Studies on DNA-binding selectivity of WRKY transcription factors lend structural clues into WRKYdomain function. Plant Mol. Biol. 68, 81–92 14 Maleck, K. et al. (2000) The transcriptome of Arabidopsis thaliana during systemic acquired resistance. Nat. Genet. 26, 403–410 15 Eulgem, T. et al. (1999) Early nuclear events in plant defence signalling: rapid gene activation by WRKY transcription factors. EMBO J. 18, 4689–4699 16 Mare`, C. et al. (2004) Hv-WRKY38: a new transcription factor involved in cold- and drought-response in barley. Plant Mol. Biol. 55, 399–416 17 Cai, M. et al. (2008) Identification of novel pathogen-responsive ciselements and their binding proteins in the promoter of OsWRKY13, a gene regulating rice disease resistance. Plant Cell Environ. 31, 86–96 18 Mangelsen, E. et al. (2008) Phylogenetic and comparative gene expression analysis of barley (Hordeum vulgare) WRKY transcription factor family reveals putatively retained functions between monocots and dicots. BMC Genomics 9, 194

Review 19 Grierson, C. et al. (1994) Separate cis sequences and trans factors direct metabolic and developmental regulation of a potato tuber storage protein gene. Plant J. 5, 815–826 20 Sun, C. et al. (2003) A novel WRKY transcription factor, SUSIBA2, participates in sugar signaling in barley by binding to the sugarresponsive elements of the iso1 promoter. Plant Cell 15, 2076–2092 21 van Verk, M.C. et al. (2008) A novel WRKY transcription factor is required for induction of PR-1a gene expression by salicylic acid and bacterial elicitors. Plant Physiol. 146, 1983–1995 22 Pandey, S.P. and Somssich, I.E. (2009) The role of WRKY transcription factors in plant immunity. Plant Physiol. 150, 1648–1655 23 Encinas-Villarejo, S. et al. (2009) Evidence for a positive regulatory role of strawberry (Fragaria  ananassa) Fa WRKY1 and Arabidopsis At WRKY75 proteins in resistance. J. Exp. Bot. 60, 3043–3065 24 Jing, S. et al. (2009) Heterologous expression of OsWRKY23 gene enhances pathogen defense and dark-induced leaf senescence in Arabidopsis. Plant Growth Regul. 58, 181–190 25 Qiu, Y.P. and Yu, D.Q. (2009) Over-expression of the stress-induced OsWRKY45 enhances disease resistance and drought tolerance in Arabidopsis. Environ. Exp. Bot. 65, 35–47 26 Popescu, S.C. et al. (2009) MAPK target networks in Arabidopsis thaliana revealed using functional protein microarrays. Genes Dev. 23, 80–92 27 Shen, Q.H. et al. (2007) Nuclear activity of MLA immune receptors links isolate-specific and basal disease-resistance responses. Science 315, 1098–1103 28 Tao, Z. et al. (2009) A pair of allelic WRKY genes play opposite roles in rice-bacteria interactions. Plant Physiol. 151, 936–948 29 Shimono, M. et al. (2007) Rice WRKY45 plays a crucial role in benzothiadiazole-inducible blast resistance. Plant Cell 19, 2064–2076 30 Skibbe, M. et al. (2008) Induced plant defenses in the natural environment: Nicotiana attenuata WRKY3 and WRKY6 coordinate responses to herbivory. Plant Cell 20, 1984–2000 31 Grunewald, W. et al. (2008) A role for AtWRKY23 in feeding site establishment of plant-parasitic nematodes. Plant Physiol. 148, 358–368 32 Gelvin, S.B. (2009) Agrobacterium in the genomics age. Plant Physiol. 150, 1665–1676 33 Zou, X. et al. (2004) A WRKY gene from creosote bush encodes an activator of the abscisic acid signaling pathway. J. Biol. Chem. 279, 55770–55779 34 Xie, Z. et al. (2005) Regulatory networks of the phytohormone abscisic acid. Vitam. Horm. 72, 235–269 35 Wu, X. et al. (2009) Enhanced heat and drought tolerance in transgenic rice seedlings overexpressing OsWRKY11 under the control of HSP101 promoter. Plant Cell Rep. 28, 21–30 36 Jiang, Y. and Deyholos, M. (2009) Functional characterization of Arabidopsis NaCl-inducible WRKY25 and WRKY33 transcription factors in abiotic stresses. Plant Mol. Biol. 69, 91–105 37 Li, S. et al. (2009) Functional analysis of an Arabidopsis transcription factor WRKY25 in heat stress. Plant Cell Rep. 28, 683–693 38 Zhou, Q.Y. et al. (2008) Soybean WRKY-type transcription factor genes, GmWRKY13, GmWRKY21, and GmWRKY54, confer differential tolerance to abiotic stresses in transgenic Arabidopsis plants. Plant Biotechnol. J. 6, 486–503 39 Eulgem, T. (2006) Dissecting the WRKY web of plant defense regulators. PLoS Pathog. 2, e126 40 Wang, Z. et al. (2009) A WRKY transcription factor participates in dehydration tolerance in Boea hygrometrica by binding to the W-box elements of the galactinol synthase (BhGolS1) promoter. Planta 230, 1155–1166 41 Teruaki, T. et al. (2002) Important roles of drought- and cold-inducible genes for galactinol synthase in stress tolerance in Arabidopsis thaliana. Plant J. 29, 417–426 42 Alexandrova, K.S. and Conger, B.V. (2002) Isolation of two somatic embryogenesis-related genes from orchardgrass (Dactylis glomerata). Plant Sci. 162, 301–307 43 Lagace, M. and Matton, D.P. (2004) Characterization of a WRKY transcription factor expressed in late torpedo-stage embryos of Solanum chacoense. Planta 219, 185–189 44 Luo, M. et al. (2005) MINISEED3 (MINI3), a WRKY family gene, and HAIKU2 (IKU2), a leucine-rich repeat (LRR) KINASE gene, are regulators of seed size in Arabidopsis. Proc. Natl. Acad. Sci. U. S. A. 102, 17531–17536

Trends in Plant Science

Vol.15 No.5

45 Zhang, Z.L. et al. (2004) A rice WRKY gene encodes a transcriptional repressor of the gibberellin signaling pathway in aleurone cells. Plant Physiol. 134, 1500–1513 46 Xie, Z. et al. (2006) Interactions of two abscisic-acid induced WRKY genes in repressing gibberellin signaling in aleurone cells. Plant J. 46, 231–242 47 Xie, Z. et al. (2007) Salicylic acid inhibits gibberellin-induced alphaamylase expression and seed germination via a pathway involving an abscisic-acid-inducible WRKY gene. Plant Mol. Biol. 64, 293–303 48 Diaz, I. et al. (2005) The DOF protein, SAD, interacts with GAMYB in plant nuclei and activates transcription of endosperm-specific genes during barley seed development. Plant J. 42, 652–662 49 Moreno-Risueno, M.A. et al. (2007) The HvDOF19 transcription factor mediates the abscisic acid dependent repression of hydrolase genes in germinating barley aleurone. Plant J. 51, 352–365 50 Peng, R.H. et al. (2004) A new rice zinc-finger protein binds to the O2S box of the alpha-amylase gene promoter. Eur. J. Biochem. 271, 2949– 2955 51 Raventos, D. et al. (1998) HRT, a novel zinc finger, transcriptional repressor from barley. J. Biol. Chem. 273, 23313–23320 52 Zou, X. et al. (2008) Interactions of two transcriptional repressors and two transcriptional activators in modulating gibberellin signaling in aleurone cells. Plant Physiol. 148, 176–186 53 Gubler, F. et al. (1995) Gibberellin-regulated expression of a Myb gene in barley aleurone cells - evidence for Myb transactivation of a high-pl alpha-amylase gene promoter. Plant Cell 7, 1879–1891 54 Zentella, R. et al. (2007) Global analysis of DELLA direct targets in early gibberellin signaling in Arabidopsis. Plant Cell 19, 3037–3057 55 Guo, Y. et al. (2004) Transcriptome of Arabidopsis leaf senescence. Plant Cell Environ. 27, 521–549 56 Robatzek, S. and Somssich, I.E. (2001) A new member of the Arabidopsis WRKY transcription factor family, AtWRKY6, is associated with both senescence- and defence-related processes. Plant J. 28, 123–133 57 Robatzek, S. and Somssich, I.E. (2002) Targets of AtWRKY6 regulation during plant senescence and pathogen defense. Genes Dev. 16, 1139– 1149 58 Miao, Y. et al. (2004) Targets of the WRKY53 transcription factor and its role during leaf senescence in Arabidopsis. Plant Mol. Biol. 55, 853–867 59 Miao, Y. et al. (2007) Arabidopsis MEKK1 can take a short cut: it can directly interact with senescence-related WRKY53 transcription factor on the protein level and can bind to its promoter. Plant Mol. Biol. 65, 63–76 ¨ lker, B. et al. (2007) The WRKY70 transcription factor of Arabidopsis 60 U influences both the plant senescence and defense signaling pathways. Planta 226, 125–137 61 Ay, N. et al. (2009) Epigenetic programming via histone methylation at WRKY53 controls leaf senescence in Arabidopsis thaliana. Plant J. 58, 333–346 62 Johnson, C.S. et al. (2002) TRANSPARENT TESTA GLABRA2, a trichome and seed coat development gene of Arabidopsis, encodes a WRKY transcription factor. Plant Cell 14, 1359–1375 63 Ishida, T. et al. (2007) Arabidopsis TRANSPARENT TESTA GLABRA2 is directly regulated by R2R3 MYB transcription factors and is involved in regulation of GLABRA2 transcription in epidermal differentiation. Plant Cell 19, 2531–2543 64 Dilkes, B.P. et al. (2008) The maternally expressed WRKY transcription factor TTG2 controls lethality in interploidy crosses of Arabidopsis. PLoS Biol. 6, 2707–2720 65 Devaiah, B.N. et al. (2007) WRKY75 transcription factor is a modulator of phosphate acquisition and root development in Arabidopsis. Plant Physiol. 143, 1789–1801 66 Kato, N. et al. (2007) Identification of a WRKY protein as a transcriptional regulator of benzylisoquinoline alkaloid biosynthesis in Coptis japonica. Plant Cell Physiol. 48, 8–18 67 Pan, Y.J. et al. (2009) A novel WRKY-like protein involved in transcriptional activation of cyst wall protein genes in Giardia lamblia. J. Biol. Chem. 284, 17975–17988 68 Miao, Y. et al. (2008) A novel upstream regulator of WRKY53 transcription during leaf senescence in Arabidopsis thaliana. Plant Biol. (Stuttg.) 10 (Suppl 1), 110–120 69 Asai, T. et al. (2002) MAP kinase signaling cascade in Arabidopsis innate immunity. Nature 415, 977–983

257

Review 70 Kim, C.Y. and Zhang, S. (2004) Activation of a mitogen-activated protein kinase cascade induces WRKY family of transcription factors and defense genes in tobacco. Plant J. 38, 142–151 71 Liu, Y. et al. (2004) Involvement of MEK1 MAPKK, NTF6 MAPK, WRKY/MYB transcription factors, COI1 and CTR1 in N-mediated resistance to tobacco mosaic virus. Plant J. 38, 800–809 72 Hofmann, M.G. et al. (2008) Cloning and characterization of a novel LpWRKY1 transcription factor in tomato. Plant Physiol. Biochem. 46, 533–540 73 Andreasson, E. et al. (2005) The MAP kinase substrate MKS1 is a regulator of plant defense responses. EMBO J. 24, 2579–2589 74 Koo, S.C. et al. (2009) OsBWMK1 mediates SA-dependent defense responses by activating the transcription factor OsWRKY33. Biochem. Biophys. Res. Commun. 387, 365–370 75 Fiil, B.K. et al. (2009) Gene regulation by MAP kinase cascades. Curr. Opin. Plant Biol. 12, 615–621 76 Qiu, J-L. et al. (2008) Arabidopsis MAP kinase 4 regulates gene expression through transcription factor release in the nucleus. EMBO J. 27, 2214–2221 77 Alvarez-Venegas, R. et al. (2007) Epigenetic control of a transcription factor at the cross section of two antagonistic pathways. Epigenetics 2, 106–113 78 Kim, K.C. et al. (2008) Arabidopsis WRKY38 and WRKY62 transcription factors interact with histone deacetylase 19 in basal defense. Plant Cell 20, 2357–2371 79 Turck, F. et al. (2004) Stimulus-dependent, promoter-specific binding of transcription factor WRKY1 to its native promoter and the defenserelated gene PcPR1-1 in parsley. Plant Cell 16, 2573–2585 80 Dong, J. et al. (2003) Expression profiles of the Arabidopsis WRKY gene superfamily during plant defense response. Plant Mol. Biol. 51, 21–37 81 Park, C.Y. et al. (2005) WRKY group IId transcription factors interact with calmodulin. FEBS Lett. 579, 1545–1550 82 Chang, I.F. et al. (2009) Proteomic profiling of tandem affinity purified 14-3-3 protein complexes in Arabidopsis thaliana. Proteomics 9, 2967– 2985 83 Cormack, R.S. et al. (2002) Leucine zipper-containing WRKY proteins widen the spectrum of immediate early elicitor-induced WRKY transcription factors in parsley. Biochim. Biophys. Acta. 1576, 92–100 84 Xu, X.P. et al. (2006) Physical and functional interactions between pathogen-induced Arabidopsis WRKY18, WRKY40, and WRKY60 transcription factors. Plant Cell 18, 1310–1326

258

Trends in Plant Science Vol.15 No.5 85 Deslandes, L. et al. (2002) Resistance to Ralstonia solanacearum in Arabidopsis thaliana is conferred by the recessive RRS1-R gene, a member of a novel family of resistance genes. Proc. Natl. Acad. Sci. U. S. A. 99, 2404–2409 86 Deslandes, L. et al. (2003) Physical interaction between RRS1-R, a protein conferring resistance to bacterial wilt, and PopP2, a type III effector targeted to the plant nucleus. Proc. Natl. Acad. Sci. U. S. A. 100, 8024–8029 87 Noutoshi, Y. et al. (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 88 Narusaka, M., Shirasu, K., Noutoshi, Y., Kubo, Y., Shiraishi, T., Iwabuchi, M. and Narusaka, Y. (2009) RRS1 and RPS4 provide a dual resistance-gene system against fungal and bacterial pathogens. Plant J. 60, 218–226 89 Rensing, S.A. et al. (2008) The physcomitrella genome reveals evolutionary insights into the conquest of land by plants. Science 319, 64–69 90 Rossberg, M. et al. (2001) Comparative sequence analysis reveals extensive microcolinearity in the lateral suppressor regions of the tomato, Arabidopsis, and Capsella genomes. Plant Cell 13, 979–988 91 Bailey, T.L. et al. (2009) MEME SUITE: tools for motif discovery and searching. Nucl. Acids Res. 37 (Suppl 2), W202–W208 92 Vyas, J. et al. (2009) VENN, a tool for titrating sequence conservation onto protein structures. Nucl. Acids Res. 37, e124 93 Tamura, K. et al. (2007) MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol. Biol. Evol. 24, 1596–1599 94 Shen, Q-H. and Schulze-Lefert, P. (2007) Rumble in the nuclear jungle: compartmentalization, trafficking, and nuclear action of plant immune receptors. EMBO J. 26, 4293–4301 95 Rubio-Somoza, I. et al. (2006) HvMCB1, a R1MYB transcription factor from barley with antagonistic regulatory functions during seed development and germination. Plant J. 45, 17–30 96 Rubio-Somoza, I. et al. (2006) Ternary complex formation between HvMYBS3 and other factors involved in transcriptional control in barley seeds. Plant J. 47, 269–281 97 Schmutz, J. et al. (2010) Genome sequence of the palaeopolyploid soybean. Nature 463, 178–183 98 Ross, C.A. et al. (2007) The WRKY gene family in rice (Oryza sativa). J. Integr. Plant Biol. 49, 827–842