protein–protein interactions in the regulation of wrky...

Molecular Plant • Volume 6 • Number 2 • Pages 287–300 • March 2013 REVIEW ARTICLE

Protein–Protein Interactions in the Regulation of WRKY Transcription FactorsYingjun Chia, Yan Yanga, Yuan Zhoua, Jie Zhoua,b, Baofang Fanb, Jing-Quan Yua and Zhixiang Chena,b,1

a Department of Horticulture, Zhejiang University, Hangzhou, Chinab Department of Botany and Plant Pathology, Purdue University, West Lafayette, IN 47907, USA

ABSTRACT It has been almost 20 years since the first report of a WRKY transcription factor, SPF1, from sweet potato. Great progress has been made since then in establishing the diverse biological roles of WRKY transcription factors in plant growth, development, and responses to biotic and abiotic stress. Despite the functional diversity, almost all ana-lyzed WRKY proteins recognize the TTGACC/T W-box sequences and, therefore, mechanisms other than mere recognition of the core W-box promoter elements are necessary to achieve the regulatory specificity of WRKY transcription factors. Research over the past several years has revealed that WRKY transcription factors physically interact with a wide range of proteins with roles in signaling, transcription, and chromatin remodeling. Studies of WRKY-interacting proteins have provided important insights into the regulation and mode of action of members of the important family of transcrip-tion factors. It has also emerged that the slightly varied WRKY domains and other protein motifs conserved within each of the seven WRKY subfamilies participate in protein–protein interactions and mediate complex functional interactions between WRKY proteins and between WRKY and other regulatory proteins in the modulation of important biologi-cal processes. In this review, we summarize studies of protein–protein interactions for WRKY transcription factors and discuss how the interacting partners contribute, at different levels, to the establishment of the complex regulatory and functional network of WRKY transcription factors.

Key words: WRKY transcription factors; protein–protein interactions; VQ proteins; protein phosphorylation; chromatin remodeling; HDA19.

InTRoDuCTIonWRKY proteins are a relatively recently discovered class of sequence-specific DNA-binding transcription factors (Rushton et al., 2010). WRKY proteins are so named because of the highly conserved WRKY domain, which contains the almost invariant WRKYGQK sequence at the N-terminus followed by a Cx4–5Cx22–23HxH or Cx7Cx23HxC zinc-finger motif (Rushton et al., 2010). Genes encoding WRKY proteins are found in some non-photosynthetic eukaryotes, indicating their origin before the emergence of photosynthetic eukaryotes. In higher plants, WRKY proteins are encoded by large families with more than 70 members in Arabidopsis thaliana and more than 100 members in rice (Wu et al., 2005; Zhang and Wang, 2005). WRKY proteins were initially classified into three groups based on the number and structure of the conserved WRKY zinc-finger motifs (Eulgem et al., 2000). The first group contains two Cx4Cx22–23HxH zinc-finger motifs, the second group contains one Cx4–5Cx23HxH zinc-finger motif, and the third group contains one Cx7Cx23HxC zinc-finger motif. More recent analyses have shown that Group II WRKY proteins can be further divided into five subgroups (IIa, IIb, IIc, IId, and IIe) (Zhang and Wang, 2005; Rushton et al., 2010). The green

algae Chlamydomonas reinhardtii, non-photosynthetic slime mold Dictyostelium discoideum, and unicellular protest Giardi alamblia all contain a single Group I WRKY gene, suggesting that Group I WRKY proteins with two WRKY domains are the ancestors to the other groups of WRKY proteins (Zhang and Wang, 2005).

Extensive studies have established that plant WRKY tran-scription factors play important roles in the two branches of the plant innate immune system, which are triggered by pathogen-associated molecular patterns (PAMP-triggered immunity or PTI) and pathogen virulent effectors (effec-tor-triggered immunity or ETI) (Jones and Dangl, 2006). A large number of studies have reported WRKY genes from a wide range of plant species are responsive to pathogens

1 To whom correspondence should be addressed. E-mail [email protected].

© The Author 2013. Published by the Molecular Plant Shanghai Editorial Office in association with Oxford University Press on behalf of CSPB and IPPE, SIBS, CAS.

doi:10.1093/mp/sst026, Advance Access publication 2 March 2013

Received 30 December 2012; accepted 30 January 2013

10.1093/mp/sst026

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288 Chi et al. • WRKY Transcription Factors

or pathogen elicitors. In Arabidopsis, more than 70% of the WRKY gene family members belonging to different groups are responsive to pathogen infection and salicylic acid (SA) treatment (Dong et al., 2003), suggesting a major role of the gene family in plant defense. Studies using knockout or knockdown mutants or overexpression lines of WRKY genes have shown that WRKY transcription factors can positively or negatively regulate various aspects of plant PTI and ETI (Chen and Chen, 2000, 2002; Li et al., 2004; Eulgem, 2006; Kim et al., 2006; Li et al., 2006; Xu et al., 2006; Zheng et al., 2006; Eulgem and Somssich, 2007; Zheng et al., 2007; Kim et al., 2008; Lai et al., 2008; Pandey and Somssich, 2009; Lai et al., 2011a; Mao et al., 2011). WRKY proteins also regulate plant hormone signaling (Chen et al., 2010; Shang et al., 2010), secondary metabolism (Wang et al., 2010b; Suttipanta et al., 2011), and plant responses to abiotic stresses (Jiang and Deyholos, 2009; Li et al., 2009, 2011). In addition, several WRKY proteins are involved in the regulation of plant growth and developmen-tal processes including trichome (Johnson et al., 2002) and seed development (Luo et al., 2005), germination (Jiang and Yu, 2009), and leaf senescence (Robatzek and Somssich, 2002; Miao et al., 2004). Thus, WRKY proteins have important roles in diverse biological processes in plants.

While great progress has been made over the last decade in establishing the wide range of biological functions of plant WRKY genes, our understanding of the regulation and mode of action of the family of transcription factors is relatively limited. As critical regulators of diverse biological processes, WRKY transcription factors are subject to regulation by a wide range of developmental and environmental signals. Our current knowledge about the regulation of WRKY tran-scription factors has been restricted mostly to the dynamic changes of the levels of WRKY gene transcripts under vari-ous biotic and abiotic stress conditions. For many well-stud-ied transcription factors, however, regulation is often highly complex, involving changes not only in transcript and protein levels, but also in DNA binding, subcellular localization, and other properties through posttranslational mechanisms. In addition, despite the diversity in biological functions, almost all analyzed WRKY proteins recognize the TTGACC/T. W-box sequences (de Pater et al., 1996; Rushton et al., 1996; Wang et al., 1998; Chen and Chen, 2000; Cormack et al., 2002) and it is unclear how a WRKY protein recognizes specific target genes to achieve functional specificity.

It has been well established that proteins, particularly regulatory proteins, rarely act alone. Very often, they physi-cally interact either transiently or permanently with each other to undertake biological functions in living systems. Therefore, mapping of dynamic protein–protein interac-tions is a critical step towards understanding a complex molecular process. Over the last several years, a substan-tial number of interacting partners of plant WRKY proteins with roles in signaling, transcription, chromatin remod-eling, and other cellular processes have been identified. Interaction of transcription factors with signaling proteins

is a common mechanism of signal transduction into the nucleus. WRKY-interacting proteins with roles in transcrip-tion and chromatin remodeling could affect the DNA-binding and transcription-regulatory activities of WRKY transcription factors, thereby controlling or modulating WRKY-regulated gene expression. In this review, we focus on WRKY-interacting proteins in an attempt to provide a mechanistic view of the regulation and mode of action of WRKY transcription factors.

WRKY–WRKY InTERACTIonSThere is a growing body of evidence for extensive WRKY–WRKY protein interactions (Figure 1A). In Arabidopsis, the three Group IIa WRKY proteins (AtWRKY18, AtWRKY40, and AtWRKY60) interact with themselves and with each other through the leucine zipper motifs present at the N-termini of the subclass of WRKY proteins (Xu et al., 2006). In addition, two Group IIb Arabidopsis WRKY proteins (AtWRKY6 and AtWRKY42) interact with each other (Chen et al., 2009). More recently, the 13 Arabidopsis Group III WRKY proteins were tested for self- and mutual interactions with a split-ubiquitin yeast two-hybrid system and prominent interaction of AtWRKY30 with AtWRKY53, AtWRKY54, and AtWRKY70 was found (Besseau et al., 2012). While Group IIa WRKY proteins contain canonical leucine zipper sequences, many of the WRKY proteins belonging to other Group II and Group III subclasses also have multiple leucine/isoleucine/valine residues at approximate seven-residue intervals at their N-termini that are predicted to form amphipathic alpha helixes similar to the secondary structure of a leucine zipper (Z. Chen, unpublished results). The extensive protein–protein interactions within members of a subclass of WRKY proteins might be mediated by these potential leucine zipper sequences. Consistently with this possibility, interactions between WRKY proteins of two different subclasses have also been identified. In rice, OsWRKY71, a Group IIa WRKY protein, interacted not only with itself, but also with a Group IId WRKY protein, OsWRKY51, in the nuclei of aleurone cells based on bimolecular fluorescence complementation (BiFC) assays (Xie et al., 2006). Yeast two-hybrid assays also revealed that two Arabidopsis Group IIa WRKY proteins (AtWRKY40 and AtWRKY60) interact with AtWRKY36 (a Group IId WRKY protein) and AtWRKY38 (a Group III WRKY protein) (Arabidopsis Interactome Mapping Consortium, 2011). Thus, interactions between WRKY proteins from at least four of the seven WRKY subclasses have been experimentally demonstrated (Figure 1A).

Extensive WRKY–WRKY protein interactions will have important implications in our understanding of the complex modes of functional interactions among WRKY transcrip-tion factors. So far, however, there have only been a lim-ited number of studies that aim to address the functional interactions among the interacting WRKY partners through analysis of DNA binding, target gene expression, and



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Chi et al. • WRKY Transcription Factors 289

mutant phenotypes. Analysis of interacting OsWRKY51 and OsWRKY71 from rice showed cooperative functional interac-tion between the two WRKY proteins in the regulation of rice abscisic acid (ABA)-repressible and gibberellic acid (GA)-inducible Amy32b α-amylase gene during seed germination (Xie et al., 2006). In vitro, OsWRKY51 did not bind to the two tandem-repeat W-boxes in the Amy32b gene promoter. However, the presence of OsWRKY51 in the assay mixture strongly enhanced the binding of OsWRKY71 to the Amy32b gene promoter. Based on promoter reporter assays in aleu-rone cells, overexpression of the two interacting rice WRKY genes repressed the rice Amy32b gene in a specific and syner-gistic manner (Xie et al., 2006). Thus, the physical interaction of OsWRKY51 and OsWRKY71 acts as an important mecha-nism for synergistic functional interaction in regulating gene expression involved in ABA and GA signaling crosstalk in aleurone cells (Xie et al., 2006).

The functional interactions among Arabidopsis interacting AtWRKY18, AtWRKY40, and AtWRKY60 are very complex with additive, cooperative, and antagonistic effects. Mutant analysis of the three WRKY genes indicates that they are negative regulators of plant basal defense (Xu et al., 2006). They also act as negative regulators of ABA signaling in seed germination and post-germination growth through transcriptional repression of ABA-responsive genes (Chen et al., 2010; Shang et al., 2010). High levels of ABA recruit AtWRKY40, a key negative regulator, and probably AtWRKY18 and AtWRKY60 as well, from the nucleus to the cytosol by promoting their interaction with the chloroplast envelope ABA receptor, the magnesium-protophorphyrin IX chelatase H subunit (Shang et al., 2010). The additive nature of the functional interactions among the three interacting WRKY proteins is evident from their partially redundant roles as negative regulators in plant responses to the hemibiotrophic

Figure 1. WRKY–WRKY Protein Interactions.(A) WRKY proteins belonging to various subclasses (IIa, IIb, IId, and III) can interact with themselves and wither each other to form homo- and hetero-complexes with unknown stoichiometry. Pairs of WRKY proteins whose interactions have been experimentally demonstrated are listed.(B) Closely spaced W-boxes (orange boxes) in a target gene promoter can be recognized by specific interacting WRKY proteins (W) for cooperative or antagonistic regulation of gene transcription.(C) Distantly separated W-boxes (orange boxes) in a gene promoter can be recognized by interacting WRKY proteins (W) from the same protein complex possibly through formation of a DNA loop. WRKY-induced looping could regulate the expression of a target gene by affecting the binding of other DNA-binding transcription factors and chromatin organization in the looped region. WRKY-induced looping could also provide long-distance communications between W-box-containing cis-regulatory modules in promoter and enhancers or other regulatory sequences of a target gene.



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bacterial pathogen Pseudomonas syringae based on the phenotypes of single, double, and triple mutants (Xu et al., 2006). AtWRKY18 and AtWRKY40 also act redundantly as negative regulators in PAMP-triggered basal defense against powdery mildew fungus, Golovinomyces orontii, based on the strong resistance phenotype of the atwrky18 atwrky40 double mutants and the negative effects of the two WRKY genes on induction of basal defense genes (Shen et al., 2007). The redundant roles of AtWRKY18 and AtWRKY40 in PAMP-triggered basal defense suggest that the homo-complex of the two WRKY proteins have similar regulatory activities. The partially redundant roles of AtWRKY18 and AtWRKY40 observed in other aspects of plant basal defense could be attributed to the dosage effects of the two WRKY homo-complexes. Alternatively, the AtWRKY18/AtWRKY40 hetero-complexes may have enhanced regulatory activities compared with the AtWRKY18 and AtWRKY40 homo-complexes. In vitro assays have indeed shown that interaction between AtWRKY18 and AtWRKY40 substantially enhanced their binding to a series of oligo DNA molecules containing two W-box sequences separated by various numbers of nucleotides (Xu et al., 2006).

The cooperative and antagonistic functional interactions among AtWRKY18, AtWRKY40, and AtWRKY60 have also been reported. In spite of its role as a negative regulator in basal defense based on mutant analysis, overexpression of AtWRKY18 enhanced resistance to P. syringae and caused constitutive PATHOGENESIS-RELATED (PR) gene expression (Chen and Chen, 2002), consistently with one reported study showing an enhanced disease susceptibility phenotype of the atwrky18 mutant plants (Wang et al., 2006). However, co-expression of AtWRKY18 with AtWRKY40 or AtWRKY60 abolished constitutive PR gene expression and made plants susceptible to the bacterial pathogen (Xu et al., 2006). These results indicated antagonistic interactions among the interacting proteins in the regulation of plant defense and defense-regulated genes in these overexpression plants. On the other hand, during the analysis of ABA regulation of the three WRKY genes, it was found that ABA-induced expres-sion of AtWRKY60 was almost completely abolished in the atwrky18 and atwrky40 single mutants, suggesting coopera-tion between AtWRKY18 and AtWRKY40 in the activation of ABA-induced expression of AtWRKY60 (Chen et al., 2010). The three WRKY proteins also recognize the W-boxes in the promoters of the ABA-responsive ABI4 and ABI5 genes and repress their expression in a cooperative and complex manner (Liu et al., 2012). The complex patterns of functional interac-tions can be, at least in part, attributed to their physical inter-actions in plant cells.

In vitro assays showed that interaction between AtWRKY18 and AtWRKY40 substantially enhanced their binding to a series of oligo DNA molecules containing two W-box sequences separated by zero to four nucleotides (Xu et al., 2006). AtWRKY60 alone has little DNA-binding activity for these W-box sequences but could enhance the binding

of AtWRKY18 for a DNA molecule with two direct repeats of W-boxes spaced by three nucleotides (Xu et al., 2006). By contrast, the DNA-binding activity of AtWRKY40 was reduced when AtWRKY60 was included. Thus, AtWRKY60 differen-tially affects the DNA-binding activity of AtWRKY18 and AtWRKY40 (Xu et al., 2006). When the W-box-containing pro-moter sequences from ABA-regulated ABI4 and ABI5 genes were used as probes, the three WRKY proteins displayed differential binding activities when assayed separately (Liu et al., 2012). Effects, mostly antagonistic, of one WRKY pro-tein on the DNA-binding activity of another WRKY protein to some of the tested promoter elements were also observed (Liu et al., 2012). Interestingly, inclusion of all three WRKY proteins in the binding reaction abolished binding to three of the five tested promoter probes, suggesting possible physi-cal interactions not only between two, but also among three, WRKY proteins in plant cells (Liu et al., 2012).

Many WRKY-regulated genes, particularly defense-related genes, are enriched in W-box elements in their promoters (Maleck et al., 2000; Dong et al., 2003). Some of the W-boxes in the promoter sequences are often clustered and organized in closely spaced direct or inverted repeats (Maleck et al., 2000; Dong et al., 2003). In light of the extensive WRKY–WRKY interactions, these W-box repeats or clusters could well be the target sequences of WRKY complexes with multi-ple WRKY protein subunits (Figure 1B). Depending on the ori-entation and the number of intervening nucleotides, a W-box repeat may be recognized or discriminated by WRKY protein complexes with specific arrangements and conformations of WRKY subunits. As a result, interacting WRKY proteins may bind to a closely spaced W-box repeat and regulate the cor-responding target gene cooperatively or antagonistically (Figure 1B).

W-boxes in gene promoters can also be separated by a substantial number of nucleotides and may be recognized by interacting WRKY proteins from the same protein complex through formation of a DNA loop (Figure 1C), which are involved in many genetic processes including transcription regulation (Chen et al., 2012; Kulaeva et al., 2012; Sanyal et al., 2012). The ability of a WRKY protein complex to bridge two W-box sequences depends on the ability of elastic energy necessary to form a loop in the promoter. Depending on the organization of WRKY DNA-binding domains in the WRKY complex and the distance between the two W-box sequences, formation of a DNA loop may require only the bending or bending plus twisting energy of the DNA molecule. Among the five W-box-containing promoter fragments from ABA-regulated ABI4 and ABI5 genes tested for binding by interacting Arabidopsis ATWRKY18, AtWRKY40, and AtWRKY60, two contain only a single W-box while the other three contain two W-box elements separated by 85, 111, 130 nucleotides (Liu et al., 2012). Significant effects of one WRKY protein on the DNA-binding activity of another WRKY protein to these five promoter elements were observed but the most dramatic effects were the completely abolished binding to



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the three promoter probes containing two distantly spaced W-boxes when all three WRKY proteins were included in the binding reactions, indicating the high sensitivity of binding to altered compositions of the WRKY complexes (Liu et al., 2012). Therefore, WRKY-induced looping, if it exists, could provide a highly dynamic and selective mechanism for recognition of promoter W-box elements by WRKY proteins. In addition, WRKY-induced looping could affect the binding of other DNA-binding transcription factors and chromatin organization in the looped regions and provide long-distance communications between W-box-containing cis-regulatory modules in promoter and other regulatory regions such as enhancers of the target genes.

The cis-regulatory modules of plant gene promoters are often very complex, consisting of distinct types of cis-acting elements recognized by different classes of DNA-binding tran-scription factors. Therefore, direct and indirect physical and functional interactions of WRKY proteins with other DNA-binding transcription factors are likely to be as prevalent and as important as WRKY–WRKY interactions in the regulation of many WRKY target genes. For example, the barley Amy32b α-amylase gene promoter contains at least five cis-acting ele-ments critical for its highly regulated expression pattern and its repression involves physical interaction of a WRKY protein, HvWRKY38, and a DOF protein, BPBF, in barley aleurone cells (Zou et al., 2008).

WRKY–VQ InTERACTIonSOver the past several years, several groups have reported that WRKY proteins interact with proteins containing a con-served FxxxVQxLTG or VQ motif (Figure 2). In Arabidopsis, AtWRKY25 and AtWRKY33 form complexes with a VQ pro-tein, MKS1 (MAP KINASE SUBSTRATE1), which also interacts with and acts as a substrate of MIOTGEN-ACTIVATED PROTEIN KINASE (MAPK) 4 (MPK4) (Andreasson et al., 2005; Qiu et al., 2008). Both AtWRKY25 and AtWRKY33 are Group I WRKY transcription factors and AtWRKY33 is important for plant resistance to necrotrophic pathogens (Zheng et al., 2006). The critical role of AtWRKY33 in plant resistance to necro-trophic pathogens is associated with its positive roles in the regulation of defense genes including biosynthetic genes for antimicrobial phytoalexin camalexin. Based on the dynamic association among MPK4, MKS1, and AtWRKY33, it was pro-posed that the physical interaction allows for sequestration of AtWRKY33 in the absence of pathogen infection (Qiu et al., 2008). Upon pathogen infection or elicitor treatment, acti-vated MPK4 phosphorylates MKS1 and releases AtWRKY33, which then targets the expression of camalexin biosynthetic gene PAD3 (Qiu et al., 2008). Another Arabidopsis VQ pro-tein, HAIKU1 (IKU1), important for endosperm growth and seed size (Wang et al., 2010a), interacts with AtWRKY10, which also regulates Arabidopsis seed size (Luo et al., 2005).

More recently, we have shown that AtWRKY33 also interacts with two other VQ proteins in the nucleus, SIGMA

FACTOR-INTERACTING PROTEIN1 (SIB1) and SIB2 (Lai et al., 2011a). Interestingly, both SIB1 and SIB2 are also targeted to chloroplasts and interact with plastid-encoded plastid RNA polymerase SIGMA FACTOR 1 (SIG1) (Morikawa et al., 2002). Group I WRKY proteins contain two WRKY domains and the C-terminal WRKY domain is primarily responsible for their sequence-specific DNA-binding activity (Ishiguro and Nakamura, 1994; de Pater et al., 1996). Through tests of a number of truncated AtWRKY33, it was revealed that MKS1, SIB1, and SIB2 bind to the C-terminal WRKY domain and stimulate the DNA-binding activity of AtWRKY33 (Lai et al., 2011a). The conserved V and Q residues in the VQ motif of

Figure 2. Interactions of WRKY Transcription Factors with Other Proteins.A variety of environmental and developmental signals can trigger acti-vation of MAPK signaling cascade and increase in cellular Ca2+ levels. Activated MAPK cascade leads to phosphorylation of specific WRKY proteins (W), which can alter their DNA-binding or other transcrip-tion-regulatory activities directly or indirectly through affecting their interaction with additional proteins such as 14–3–3 proteins. Increased cellular Ca2+ levels would promote CaM binding to specific WRKY proteins and alter their important transcription-regulatory activities. Activated MAPK cascade and increased cellular Ca2+ levels also affect phosphorylation and CaM binding of VQ proteins, thereby influencing their interactions with Group I and IIc WRKY proteins. Altered DNA-binding and other regulatory properties of WRKY proteins due to phosphorylation or interactions with signaling proteins will ultimately affect the transcription of their target genes. Interactions of WRKY proteins with histone deacetylases (HDA) can result in the removal of acetyl groups from histone tails and more tight wrapping of DNA by histones, leading to repression of target gene transcription.



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SIB1 are important for interaction with AtWRKY33, indicating that the short VQ motif is the core of a WRKY-interacting domain (Lai et al., 2011a). Both SIB1 and SIB2 are induced by the necrotrophic pathogen Botrytis cinerea. Mutation of SIB1 and SIB2 compromises while overexpression of SIB1 enhances resistance to B. cinerea. These results indicate that dual-targeted SIB1 and SIB2 function as activators of WRKY33 in plant defense against necrotrophic pathogens (Lai et al., 2011a).

There are 34 genes in Arabidopsis that encode VQ motif-containing proteins (Cheng et al., 2012). A majority of Arabidopsis VQ genes contain no intron and encode rela-tively small proteins of 100–200-amino acid residues with very diverse sequences beyond the short VQ motif. Despite of the structural diversity, all 34 Arabidopsis VQ proteins are capable of interacting with WRKY proteins in yeast (Cheng et al., 2012). Survey of these VQ-interacting WRKY proteins revealed that they all belong to Group I and Group IIc sub-families (Cheng et al., 2012). Since the N-terminal WRKY domain of AtWRKY33 fails to interact with VQ proteins, the interactions of Group I WRKY proteins with VQ proteins are through their C-terminal WRKY domains. The amino acid sequences of the C-terminal WRKY domain of Group I WRKY proteins and the single WRKY domain of Group IIc WRKY proteins are highly similar and share several critical struc-tural features including four amino acid residues intervening with the two conserved zinc-finger cysteine residues and two aspartate residues in the region immediately preceding the WRKYGQK signature sequence (Rushton et al., 2010; Cheng et al., 2012). Site-directed mutagenesis indicated that these structural features shared by the Group I and Group IIc WRKY domains are important for interactions with VQ proteins (Cheng et al., 2012). The C-terminal WRKY domain of Group I WRKY proteins is an anti-parallel β-sheet forming from four β-strands (Yamasaki et al., 2005). The two conserved aspar-tate residues and the four intervening residues between the two zinc-finger cysteine residues are in close proximity at one end of the β-sheet and may be part of the interface for inter-action with the small VQ motif. As the β-sheet of the WRKY domain is in direct contact with DNA bases (Yamasaki et al., 2005, 2012), VQ proteins could affect the DNA binding of interacting WRKY proteins by directly altering the conforma-tion of the WRKY DNA-binding domains.

In Arabidopsis, there are 32 Group I and IIc WRKY genes, compared to 34 VQ genes. From yeast two-hybrid assays, the C-terminal WRKY domains of AtWRKY25 and AtWRKY33 interact with a majority of VQ proteins with varying degrees (Cheng et al., 2012). About 50% of the VQ proteins tested also interact with the WRKY domain of Group IIc AtWRKY51 (Cheng et al., 2012). Thus, despite their similar numbers, the interactions between the two groups of interacting proteins are not on the one-to-one partnership. Therefore, a single Group I or Group IIc WRKY protein can potentially interact with multiple VQ proteins, which may differentially affect DNA-binding or other properties of the interacting WRKY

protein. Some VQ proteins may stimulate while others may inhibit the DNA-binding activity of their interacting WRKY proteins. VQ proteins may also affect the DNA-binding speci-ficity of their interacting WRKY proteins, for example, by altering the preference for the nucleotides neighboring the conserved W-boxes. Regulation of the DNA-binding activity of a WRKY protein by interacting VQ proteins may function to fine-tune the levels of target gene expression. Altering DNA-binding specificity of a WRKY protein by interacting VQ partners, on the other hand, will result in change of target gene specificity and, consequently, the biological functions of the WRKY transcription factor. Interaction of a single WRKY protein with multiple VQ proteins may, therefore, confer broad biological functions to the WRKY protein. For exam-ple, Arabidopsis AtWRKY33, which interacts with multiple VQ proteins, is critical not only for resistance to necrotrophic pathogens, but also for tolerance to important abiotic stresses (Zheng et al., 2006; Jiang and Deyholos, 2009; Li et al., 2011).

The amino acid sequences flanking the short FxxxVQxxLTG motifs are highly diverse among VQ proteins. Besides the direct effects on DNA binding, the variable regions of VQ proteins could regulate the interacting WRKY proteins through other mechanisms including interactions with additional proteins. Indeed, some Arabidopsis VQ proteins also interact with signaling proteins such as MPK3, MPK4, and CaM1 (Perruc et al., 2004; Qiu et al., 2008; Cheng et al., 2012). Interactions of VQ proteins with these signaling proteins may lead to conformational changes or posttranslation modifications that can, in turn, have activating or inhibitory effects on their WRKY-interacting partners. Thus, some VQ proteins may act as effectors of signaling proteins for activating or repressing WRKY-mediated transcription in response to specific environmental and developmental signals. A substantial number of VQ proteins such as SIB1 and SIB2 are also targeted to chloroplasts or mitochondria and may be involved in the coordination of transcription or retrograde signaling from the organelles to the nucleus. Other VQ proteins may provide as a scaffold for recruitment of additional proteins including transcription machinery proteins and those involved in chromatin remolding that are required for WRKY-regulated gene transcription. A comprehensive analysis of VQ proteins should, therefore, provide a wealth of information about the regulation and mode of action of the two most ancient subclasses of WRKY transcription factors.

WRKY–MAPK InTERACTIonSSignaling through MAPKs is evolutionarily conserved among eukaryotes and plays critical roles in plant response to patho-gens and stress conditions. In tobacco, Arabidopsis, rice, and other plants, stress/pathogen-responsive MAPKs have been identified and extensively studied. In tobacco, WOUND-INDUCED PROTEIN KINASE (WIPK) and SALICYLIC ACID-INDUCED PROTEIN KINASES (SIPK) are activated in resistant tobacco plants upon infection of TMV and are involved



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in pathogen- or pathogen-elicitor-induced hypersensitive responses (HR) (Zhang and Klessig, 2000). In Arabidopsis, MPK3, MPK6 (orthologs to tobacco WIPK and SIPK), and MPK4 are also activated by biotic and abiotic stresses and functional analyses using both loss- and gain-of-function approaches indicate their critical roles in plant disease resistance and stress tolerance (Pitzschke et al., 2009). Among the increas-ing number of identified substrates of the stress/pathogen-responsive MAPKs are Group I WRKY proteins (Figure 2), which function as critical components of the MPAK signaling pathways in regulation of plant defense and stress responses.

In tobacco, a Group I WRKY protein, NtWRKY1, has been identified as a SIPK-interacting protein through a targeted yeast two-hybrid assay (Menke et al., 2005). NtWRKY1 is phos-phorylated by SIPK, which leads to increased DNA-binding activity to a W-box-containing DNA molecule. Co-expression of SIPK and NtWRKY1 in tobacco promotes cell death. This result suggests that NtWRKY1 might function as a component in the SIPK signaling pathway involved in pathogen-induced hypersensitive cell death (Menke et al., 2005). In Nicotiana benthamiana, another Group I WRKY protein, NbWRKY8, is a substrate of SIPK, WIPK, and the paralog NTF4 (Ishihama et al., 2011). Phosphorylation of NbWRKY8 enhances its DNA-binding and transcription-activating activities. Overexpression of a phospo-mimicking NbWRKY8 mutant results in induced expression of defense genes, including a gene encoding 3-hydroxy-3-methylglutuaryl CoA reductase critical for bio-synthesis of isoprenoid phytoalexins in solanaceous plants (Ishihama et al., 2011). This result supports that phosphoryla-tion of NbWRKY8 by stress/pathogen-responsive MAPKs has a critical role in tobacco defense responses, including path-ogen-induced phytoalexin formation (Ishihama et al., 2011).

In Arabidopsis, AtWRKY33 is the closest Arabidopsis homolog of NbWRKY8 and is also required for pathogen-induced production of the indolic phytoalexin camalexin. As discussed earlier, AtWRKY33 and closely related AtWRKY25 interact with MKS1, a substrate of MPK4. Further studies have shown that, in the absence of pathogens, MPK4 is present as a complex with AtWRKY33 in the nucleus through mutual interactions with MKS1 (Qiu et al., 2008). Upon infection by the bacterial pathogen P. syringae or flagellin treatment, acti-vated MPK4 phosphorylates MKS1 and releases AtWRKY33. The released AtWRKY33 then targets the promoter of PAD3, which encodes a biosynthetic enzyme for camalexin produc-tion (Qiu et al., 2008). Intriguingly, while AtWRKY33 has a critical role in resistance to necrotrophic pathogens, MKS1 affects only SA-mediated defense responses (Andreasson et al., 2005). MPK4, on the other hand, plays a positive role in defense against necrotrophic pathogens despite its sequestering of AtWRKY33 (Qiu et al., 2008). Furthermore, camalexin can be induced regardless of MPK4 activation (Ren et al., 2008). Therefore, the functional relevance of the dynamic MPK4–MKS1–AtWRKY33 complexes in pathogen-induced PAD3 expression and camalexin production requires further analysis.

More recent research has indicated that the critical role of AtWRKY33 in Botrytis-induced camalexin biosynthesis is regulated through phosphorylation by functionally redun-dant MPK3 and MPK6 (Mao et al., 2011). Activation of the MPK3/MPK6 cascade results in coordinated up-regulation of multiple camalexin biosynthetic genes including CYP71A3 and PAD3. AtWRKY33 expression is also up-regulated by the MPK3/MPK6 cascade. In the atwrky33 mutant plants, induc-tion of camalexin production and expression of camalexin biosynthetic genes by gain-of-function MPK3/MPK6 and path-ogen infection is compromised. AtWRKY33 is phosphorylated by MPK3/MPK6 both in vitro and in vivo and the mutant AtWRKY33 gene for the MPK3/MPK6 phosphorylation sites is unable to fully complement the deficiency of camalexin production in the atwrky33 mutant. ChIP–PCR assays indicate that AtWRKY33 binds to its own promoter and the promoter of PAD3 in vivo. These results indicate that AtWRKY33 acts downstream of MPK3/MPK6 cascade in regulation of path-ogen-induced camalexin biosynthesis. Induction of its own expression by activated AtWRKY33 upon phosphorylation by MPK3/MPK6 constitutes a potential positive feedback mecha-nism for rapid and strong induction of AtWRKY33-mediated defense responses, including camalexin biosynthesis (Mao et al., 2011).

In rice, yeast two-hybrid screening identified a number of proteins that interact with OsBWMK1, a MAPK involved in SA-dependent defense responses. One of these interacting proteins is OsWRKY33, whose gene is induced by several defense signals including a fungal elicitor, SA, jasmonic acid (JA), and hydrogen peroxide (Koo et al., 2009). OsWRKY33 is phosphorylated by OsBWMK1 and in vitro assays indicate that the phosphorylation enhances the DNA-binding activity of OsWRKY33. Co-expression of OsWRKY33 and OsBWMK1 in Arabidopsis protoplasts enhances SA-induced expression of a reporter gene driven by the W-box sequences or the defense-related PR1 gene promoter (Koo et al., 2009). In another reported study, it was found that rice OsWRKY30 interacts with several rice MAPKs and can be phosphorylated by some of the interacting MAPKs (Shen et al., 2012). Overexpression of OsWRKY30 enhances drought tolerance. Mutation of all the SP sites in OsWRKY30 compromises its function of transcriptional activation and abolishes its ability to enhance drought tolerance in transgenic plants (Shen et al., 2012). These results support that phosphorylation of WRKY proteins by stress/pathogen-responsive MAPKs is important for their biological functions in plant defense and stress responses, although the possibility of the SP mutations affecting aspects of the protein other than phosphorylation cannot be completely ruled out.

A majority of the WRKY proteins from tobacco, Arabidopsis, and rice characterized as substrates of stress/pathogen-responsive MAPKs are Group I WRKY proteins with two WRKY domains. These WRKY proteins contain clus-tered proline-directed serines (SP clusters) as potential phos-phorylation sites of MAPKs (Ishihama and Yoshioka, 2012).



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Interestingly, other Group I WRKY proteins from plants also have the conserved SP clusters and even the WRKY protein of the unicellular green algae Chlamydomonas has partially conserved SP clusters, underscoring the highly conserved nature of MAPK-dependent regulation of Group I WRKY pro-teins (Ishihama and Yoshioka, 2012). In addition, some of the Group I WRKY proteins contain a MAPK-docking site named the D domain with the general feature of a cluster of basic residues upstream of LxL motif (Ishihama and Yoshioka, 2012). Other Group I WRKY proteins, however, contain no D-domain sequences and, therefore, may rely on other domains for interactions with MAPKs. The variation in sequences among the MAPK-docking domains and other MAPK-interacting domains may dictate the selectivity of interactions between WRKY and MAPK proteins (Ishihama and Yoshioka, 2012).

The target WRKY proteins of MAPK cascades are appar-ently not restricted to Group I subclass members. By probing high-density protein microarrays using 10 different activated MPKs from Arabidopsis, several hundreds of phosphoryla-tion substrates of MPKs were isolated, which are enriched in transcription factors including 25 WRKY proteins (Popescu et al., 2009). These 25 WRKY proteins include five Group I (AtWRKY3, 20, 23, 44, and 58), one Group IIa (AtWRKY40), five Group IIb (AtWRKY6, 36, 47, 61, and 72), two Group IIc (AtWRKY8 and 75), three Group IId (AtWRKY7, 21, and 39), four Group IIe (AtWRKY22, 27, 65, and 69), and five Group III (AWRKY46, 53, 55, 62, and 66) WRKY proteins. Survey of the amino acid sequences revealed SP or TP sites in 23 of the 25 WRKY proteins but not in AtWRKY55 or AtWRKY77, suggesting other phosphorylation sites recognized by the Arabidopsis MAPKs. Characterization of some of these WRKY proteins also provided new information about their phos-phorylation-dependent regulation by interacting MAPKs. For example, phosphorylation of AtWRKY6 when expressed alone in N. benthamiana is very low but is increased if co-expressed with an MAPK kinase (MKK)/MPK-activating mod-ule (Popescu et al., 2009). If the tissue expressing AtWRKY6 is treated with phosphatase, rapid degradation of AtWRKY6 was observed (Popescu et al., 2009). Thus, the stability of AtWRKY6 appears to be regulated by its phosphorylation state. Indeed, the stability of AtWRKY6, a regulator of plant responses to low-Pi stress, is decreased under high Pi levels, most likely resulting from 26S proteasome-mediated prote-olysis (Chen et al., 2009). It will be of interest to determine whether Pi-induced degradation of AtWRKY6 is dependent on its phosphorylation by a MAPK cascade.

MAPKs are catalytically inactive in their base state and require phosphorylation for activation by MAP kinase kinases (MKKs), which in turn are activated through phosphoryla-tion by upstream MAP kinase kinase kinases (MEKKs). In one reported case, regulation of a WRKY protein by the MAPK cascade uses a short cut by an upstream MEKK. Arabidopsis MEKK1 acts as a DNA-binding protein that recognizes a cis-acting element in the promoter of AtWRKY53, which partici-pates in regulation of leaf senescence (Miao et al., 2007). The

binding motif of MEKK1 in the AtWRKY53 promoter is neces-sary for the age-regulated expression of AtWRKY55. In addi-tion, MEKK1 interacts with AtWRKY53 and phosphorylates the transcription factor (Miao et al., 2007). Phosphorylation of AtWRKY53 increases its DNA-binding activity in vitro and stimulates the AtWRKY53 promoter activity in vivo (Miao et al., 2007). Thus, MEKK1 acts as both a DNA-binding protein and a protein kinase that regulates both the expression of AtWRKY55 and the activity of the gene product.

InTERACTInG WITH CHRoMATIn REMoDELInG PRoTEInSIn eukaryotic cells, nuclear DNA is packaged with proteins into chromatin (Glatt et al., 2011). The basic unit of chromatin is the nucleosome, consisting of a segment of DNA (~147 bp) wrapped around a histone octamer of two copies each of four core histones: H2A, H2B, H3, and H4. The linker histone H1 binds to the linker DNA region between nucleosomes and also locks the DNA around a nucleosome by binding the core histone octamer and the entry and exit sites of the DNA. Transcriptional regulation of a gene requires access of its promoter sequence by the transcriptional machinery proteins including RNA polymerase, transcription factors, activators, and repressors (Glatt et al., 2011). Tightly packaged and condensed chromatin structure occludes the regulatory DNA sequence, thereby preventing interaction with transcriptional machinery proteins for regulated gene expression. Remodeling the structure, composition, and positing of nucleosomes alters chromatin architecture to allow dynamic access of condensed DNA for transcriptional regulation (Glatt et al., 2011). Histone-modifying enzyme complexes can catalyze modifications such as acetylation, methylation, phosphorylation, and ubiquitination of histones. Such modifications may affect gene expression by directly loosening or tightening the condensed DNA wrapped around histones (Glatt et al., 2011). For example, histone acetylation loses chromatin condensation, leading to increased gene transcription (Santos-Rosa and Caldas, 2005). Histone modification can also affect gene expression by generating a so-called ‘histone code’ recognized by other proteins for such purposes (Rando, 2012). The recognition leads to recruitment of these proteins to the promoter sequence to activate or repress transcription.

Arabidopsis AtWRKY38 and AtWRKY62 genes encode two structurally similar Group III WRKY proteins and are respon-sive to SA in an NPR1-dependent manner (Kim et al., 2008). Functional analysis using both knockout mutants and overex-pression lines indicate that AtWRKY38 and AtWRKY62 func-tion partially redundantly as negative regulators in plant basal resistance to virulent P. syringae and in pathogen-induced PR1 expression. Yeast two-hybrid screens identified Arabidopsis HISTONE DEACETYLASE 19 (HDA19) as an interacting partner of both AtWRKY38 and AtWRKY62 (Kim et al., 2008) (Figure 2). Both BiFC and co-immunoprecipitation indicated that the



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interaction occurs in the nucleus and is highly specific. Histone deacetylases catalyze the removal of acetyl groups from his-tone tails, which allows the histones to wrap the DNA more tightly, leading to repression of the transcription of genes. In a reporter gene assay in plant cells, overexpression of HDA19 indeed results in repression of transcription-activating activity of AtWRKY38 and AtWRKY62 (Kim et al., 2008).

Arabidopsis HDA19 is a global modulator of plant gene expression. HDA19 is associated with transcriptional core-pressor TOPLESS (TP) or TOPLESS-RELATED (TPR) proteins and the transcriptional repressor complexes are recruited by AP2/EREBP-type transcriptional repressors involved in plant devel-opment and stress responses (Song et al., 2005; Krogan et al., 2012). In addition, Arabidopsis SNC1, a Toll-like/interleukin-1 receptor (TIR)–NB–LRR resistance protein, activates immune response through its association with TPR1, which forms com-plexes with HDA19 (Zhu et al., 2010). Mutations of TPR1 and its close homologs including TP compromise while its overex-pression activates SNC1-mediated immune responses against P. syringae (Zhu et al., 2010). These results suggest that the TPL and TPR/HDA19 transcription repressor complexes func-tion positively to regulate plant immune responses against the hemibiotrophic bacterial pathogen. Consistently with the positive role, we have previously shown that the phenotypes of hda9 mutants and its overexpression lines are similar to those for TPR1 in response to P. syringae (Kim et al., 2008). Intriguingly, a recent study has shown that HDA19 functions in repression of SA biosynthesis and SA-mediated defense responses (Choi et al., 2012). The discrepancy might arise from the difference in growth conditions and assay methods, as HDA19 has critical roles in a variety of stress-associated signal-ing pathways. In addition to its role in SA-mediated defense, HDA19 is involved in JA and ethylene signaling as well as ABA and drought stress responses (Zhou et al., 2005).

Very recently, a WRKY protein from banana, MaWRKY1, interacts with a banana linker histone H1, MaHIS1 (Wang et al., 2012). Expression of MaHIS1 is induced by ethylene during fruit postharvest ripening as well as other exogenous hormones including JA, ABA, and hydrogen peroxide, and chilling and pathogen infection (Wang et al., 2012). Drought-induced histone genes encoding linker H1 or H1 variant pro-teins have also been isolated in plants (Wei and O’Connell, 1996; Ascenzi and Gantt, 1997; Przewloka et al., 2002; Kim et al., 2010; Trivedi et al., 2012). The responsive expression of linker histone genes to a variety of stresses and the interac-tion with a WRKY transcription factor suggest that these chro-matin components may act together with their interacting transcription factors in the regulation of gene expression. In other eukaryotic organisms, a number of transcription regula-tors repress or activate transcription by direct interaction with histones. In yeast, Sir3p, Sir4p, and Tup1p repress transcrip-tion of genes through interaction with histones (Moretti and Shore, 2001). The domains of these transcription regulators required for interaction with histone H3 and H4 are the same as required for transcriptional repression (Moretti and Shore,

2001). The proline-rich transcriptional activation domain of mammalian nuclear factor 1 (NF-1) also interacts with histone H3 (Cirillo et al., 2002). NF-1 is also required for efficient ini-tiation of SV40 DNA replicates by binding auxiliary sequences and acting on the chromatin structure of the replication ori-gin. Evaluation of a series of deletion and point mutants of NF-1 indicates that the H3-binding domain and the replication activity match perfectly (Muller and Mermod, 2000).

InTERACTIonS WITH CALMoDuLIn AnD 14–3–3 PRoTEInSCalmodulin (CaM) is a Ca2+-binding signaling protein found in all eukaryotic organisms. The primary role of CaM is transducing Ca2+ signals by binding the ions and then altering its interactions with various target proteins. Through screening of an Arabidopsis cDNA expression library using CaM as a probe, positive clones encoding AtWRKY7, a Group IId WRKY protein, were isolated (Park et al., 2005) (Figure 2). The binding was verified by other procedures including gel mobility shift assay, split-ubiquitin yeast two-hybrid assay, and a competition assaying with the Ca2+/CaM-dependent phosphodiesterase. AtWRKY7 contains a short amino acid region (VAVNSFKKVISLLGRSR) called C-motif, which is the CaM binding domain. Similar C-motifs (DxxVxKFKxVISLLxxxR) are also present in the other 10 Arabidopsis Group IId WRKY proteins, which are also bound by CaM (Park et al., 2005). Among the functionally characterized Group IId WRKY proteins are AtWRKY7, AtWRKY11, and AtWRKY17 from Arabidopsis, which functions as negative regulators of plant basal defense (Journot-Catalino et al., 2006; Kim et al., 2006). In plant cells, AtWRKY7 acts as a transcriptional repressor (Kim et al., 2006). OsWRKY51, also a Group IId WRKY protein from rice, binds to OsWRKY71 and the formed complex represses the rice Amy32b gene in a specific and synergistic manner (Xie et al., 2006). Interactions between OsWRKY71 and OsWRKY51 are probably mediated by the potential amphipathic alpha helixes present at their N-terminus not only in Group IIa WRKY proteins like OsWRKY71, but also in OsWRKY51 and other Group IId, IIb, and III WRKY proteins. C-motifs of Group IId WRKY proteins also form amphipathic alpha helixes at the N-terminus of Group IId WRKY proteins (Park et al., 2005). A variety of environmental and developmental signals can trigger increase in cellular Ca2+ levels (Knight, 2000), which would promote CaM binding to the Goup II WRKY proteins. If the WRKY–WRKY and WRKY–CaM interaction domains on the WRKY proteins are in close proximity or even overlap, binding of a CaM to a WRKY protein will prevent WRKY–WRKY interactions, thereby providing a possible mechanism for potential regulation of WRKY–WRKY interaction by cellular Ca2+ levels.

14–3–3 proteins regulate a variety of cellular processes through interactions with target proteins typically in a phos-phorylation-dependent manner (Shen et al., 2003). Proteomic profiling of tandem affinity-purified 14–3–3 protein com-plexes in Arabidopsis have identified seven WRKY proteins



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(AtWRKY6, 16, 18, 19, 27, 32, and 40) that form complexes with 14–3–3 proteins (Chang et al., 2009) (Figure 2). Among the 14–3–3 WRKY clients are two TIR–NBS–LRR–WRKY pro-teins (AtWRKY16 and AtWRKY19). It has been shown that insertion of a single amino acid residue in the WRKY domain of AtWRKY19 causes activation of defense response and hypersensitive cell death (Noutoshi et al., 2005), suggesting a negative role of the WRKY domain in plant disease resist-ance. AtWRKY6 is involved in the regulation of plant senes-cence and low-Pi stress response (Robatzek and Somssich, 2002; Chen et al., 2009). AtWRKY18 and AtWRKY40 function in a complex manner in plant defense and ABA signaling (Xu et al., 2006; Chen et al., 2010; Shang et al., 2010; Liu et al., 2012). The mutant plants for AtWRKY27 exhibit delayed symptom development after infection by the bacterial wilt pathogen Ralstonia solanacearum (Mukhtar et al., 2008).

Interactions of 14–3–3 proteins with many client proteins are promoted by phosphorylation of target-binding site of the clients (Shen et al., 2003). It is, therefore, possible that these WRKY clients of the 14–3–3 proteins are subject to regulation by phosphorylation by stress/pathogen-responsive kinase cascades. Because 14–3–3 proteins dimerize and each 14–3–3 dimer can bind two clients (Shen et al., 2003), those WRKY proteins with phosphorylated binding sites could form complexes with other protein indirectly through mutual interactions with a 14–3–3 dimer (Figure 2). Since the purified 14–3–3/WRKY complexes may contain additional proteins, the association of a WRKY protein with 14–3–3 proteins may be indirect and, therefore, regulation of the WRKY protein by phosphorylation could also be through a true 14–3–3 cli-ent in the complex. Thus, the 14–3–3 proteins could act as dynamic interaction nodes in the complex WRKY protein interaction map. Indeed, 14–3–3 proteins often function as adaptor proteins that bind a multitude of regulatory and signaling proteins (Fukuhara et al., 2005; Schoonheim et al., 2007; Xu et al., 2010; Arulpragasam et al., 2012).

oTHER WRKY-InTERACTInG PARTnERSIn ETI, recognition of strain-specific pathogen effectors encoded by avirulent (Avr) genes by resistance (R) proteins are linked with activation of defense genes in the nucleus. In a number of characterized cases, effector-triggered acti-vation of defense genes apparently result from inactivation of specific WRKY repressors by associated R proteins. In bar-ley, the avirulent A10 effector from powder mildew fungal pathogen Blumeria graminis is recognized by MLA10, a CC–NBR–LRR-type R protein (Shen et al., 2007). The recognition promotes association of MLA10 through its N-terminal CC domain with two closely related Group IIa WRKY proteins, HvWRKY1 and HvWRKY2 (Shen et al., 2007). Experiments using both virus-induced gene silencing and overexpression indicated that HvWRKY1 and HvWRKY2 act as repressors of PAMP-triggered basal defense (Shen et al., 2007). Apparently, effector-induced association of MLA10 with HvWRKY1 and

HvWRKY2 acts as a molecular switch for derepression of basal defense genes through inactivation of the repressor func-tions of the WRKY proteins. In Arabidopsis and other plants, there are unusual R proteins that contain not only the TIR/CC–NB–LRR structures but also a WRKY domain. As discussed earlier, a single amino acid insertion into the WRKY domain of the TIR–NBS–LRR–WRKY-type AtWRKY19 protein results in activation of defense responses and hypersensitive cell death, indicating that the WRKY domain also has a repressor function (Noutoshi et al., 2005). For this type of R protein, inactivation of the WRKY repressor function may be achieved through intra-molecular interactions between the WRKY and other domains of the same protein upon recognition, directly or indirectly, of an avirulent effector.

Protein–protein interactions can also provide a regulatory bridge linking a cellular process with WRKY-mediated transcription. In Arabidopsis, AtWRKY33 also interacts with ATG18a, a critical component of autophagy (Lai et al., 2011b). In plants, autophagy is involved in nutrient recycling and in responses to a range of abiotic stresses (Bassham, 2007). Autophagy also regulates pathogen-induced programmed cell death in plant defense against biotrophic pathogens (Liu et al., 2005). Interaction of a critical component of autophagy with AtWRKY33 suggests a possible role of autophagy in plant defense against necrotrophic pathogens. Indeed, expression of autophagy genes and formation of autophagosomes are both induced by infection of necrotrophic pathogens. Arabidopsis mutants deficient in autophagy are compromised in resistance to necrotrophic fungal pathogens B. cinerea and Alternaria brassicicola (Lai et al., 2011b). Thus, autophagy plays a critical role in plant resistance to necrotrophic fungal pathogens. The physical interactions between AtWRKY33 and ATG18 may provide a mechanism for cooperation between autophagy and AtWRKY33-mediated gene transcription in the regulation of plant defense. Indeed, AtWRKY33 appears to be required for sustained induction of autophagy gene expression and autophagosome formation, indicating that ATWRKY33 is a positive regulator of pathogen-induced autophagy (Lai et al., 2011b). Through ATG18, autophagy may also regulate the transcription-regulatory activity of AtWRKY33. In yeast, ATG18 and ATG21 interact with the transcriptional activator RTG3 and are required for RTG3-regulated gene expression (Georgakopoulos et al., 2001).

The ubiquitin–proteasome system (UPS) plays an important role in almost every aspect of plant growth, development, and responses to environmental conditions. In UPS, ubiquitin is covalently attached to target proteins through the action of E1, E2, and E3 enzymes, which results in the degradation of the target genes in the 26S proteasome. Common target proteins of UPS in plant signaling are transcriptional repressors, whose degradation leads to derepression of signaling pathways (Santner and Estelle, 2010). There are also reports that some WRKY transcription factors are subject to regulation by UPS. In Arabidopsis, the protein level of AtWRKY6, a repressor of PHO1, is decreased under low-Pi stress (Chen et al., 2009). MG132, a



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26S proteasome inhibitor, inhibits low-Pi-induced degradation of AtWRKY6, suggesting that PHO1 derepression may result from degradation of AtWRKY6 repressor by UPS (Chen et al., 2009). In addition, a HECT domain E3 ubiquitin ligase UPL5 interacts with AtWRKY53, a positive regulator of plant senes-cence (Miao and Zentgraf, 2010). In vitro, AtWRKY52 acts as a substrate of UPL5 for polyubquitination and induced overex-pression of UPL5 caused increased degradation of AtWRKY53 in vivo (Miao and Zentgraf, 2010). Mutation of UPL5 results in increased senescence, particularly in the transgenic plants overexpressing AtWRKY53 (Miao and Zentgraf, 2010). These results indicate that AtWRKY53 is subject to negative regula-tion of UPS to prevent premature senescence.

ConCLuDInG REMARKSOver the past several years, a substantial number of WRKY-interacting proteins have been identified. Some of the identi-fied WRKY-interacting proteins such as MAPKs and CaM act upstream of WRKY transcription factors in signal transduc-tion from specific cellular or environmental signals to altered transcription in the nucleus. The extensive WRKY–WRKY interactions provide a mechanism for functional cooperation and antagonism among WRKY proteins for dynamic regula-tion of target genes (Figure 1). Other WRKY-interacting pro-teins such as VQ proteins act as cofactors that positively or negatively regulate the regulatory properties of WRKY tran-scription factors (Figure 2). With the identification of HDAC and histone proteins as WRKY-interacting proteins (Figure 2), we are also beginning to reveal chromatin remodeling as a mechanism for regulation of target gene transcription by members of the important family of transcription factors. Therefore, identified WRKY-interacting proteins encompass components that are critical for the regulation and mode of action of WRKY transcription factors (Figure 2). Direct target genes of a number of WRKY transcription factors have also been identified and analyzed using chromatin immunopre-cipitation and other molecular and genomic tools. This pro-gress is starting to make it feasible to construct a framework for signaling pathways of a few well-studied WRKY transcrip-tion factors from their regulation by signaling proteins to their action of activating or repressing target genes in the nucleus. Over the next few years, it is expected that additional WRKY-interacting proteins will be identified by both tradi-tional procedures such as yeast two-hybrid screens and more recently developed methods such as high-density protein microarrays. The roles of these WRKY-interacting proteins in the regulation and mode of action of WRKY transcription fac-tors can be analyzed in association with specific target genes and biological processes. A particularly important effort will be the integration of these identified dynamic and complex protein–protein and protein–DNA interactions in WRKY-mediated transcription of important target genes to develop a comprehensive understanding of the WRKY signaling and

transcriptional regulatory network. To this end, it will be cen-tral to utilize the approach of systems biology, by which the dynamic behaviors of WRKY proteins and associated interact-ing components are quantitatively and systematically meas-ured using genomic, bioinformatics, and proteomic tools and the dynamic outcomes of WRKY-regulated gene expression and associated biological processes can be described and pre-dicted using mathematical and computational models. These studies can lead to a better understanding of not only WRKY transcription factors, but also the important biological pro-cesses that they regulate.

FUNDING

Research on plant WRKY transcription factors is supported by grants from the US National Science Foundation, the Natural Science Foundation of China, and the Ministry of Agriculture of China. No conflict of interest declared.

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