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Although the very first WRKY was isolated by Ishiguro and Nakamura in 1994 (Ishiguro and Nakamura, 1994), it was only 2 years later that this family received its name, based on its conserved amino acid sequence ‘WRKYGQK’

(Rushton et al., 1996). In this latter study, WRKY proteins were identified through a search for factors binding a particular response element of the PR-1 gene promoter called the W box. By doing so, the group of Imre E. Somssich was looking for the factor responsible for the induction of the PR-1 gene in elicitor­treated parsley cells (Petroselinum crispum). A bacteriophage cDNA library was made from total RNA extracted from parsley cells treated with a fungal oligopeptide elicitor and screened with various W­box oligonucleotides.

Out of the four clones isolated, three coding sequences were recovered and named PcWRKY1, 2 and 3.

As more and more of the Arabidopsis genome was revealed, the WRKY members were divided into three groups depending on the number and precise sequence of their DNA­binding WRKY domain (Eulgem et al., 2000). With 74 genes in Arabidopsis and 90 in rice the WRKY family is recognized as an important family of plant TF (Ulker and Somssich, 2004). One clue that links the WRKY to the plant defence response is the induction of their expression during infection or treatment with an elicitor. Indeed, 49 WRKY genes out of 72 tested in Arabidopsis respond to infection by P. syringae or treatment with SA (Dong et al., 2003). Furthermore, using an inducible version of NPR1, Wang et al. identified eight WRKY factors as direct targets of this defence response regulator (Wang et al., 2006). However, it is only in recent years that a direct link with defence was definitely established when loss­of­function mutations of WRKY genes were shown to affect defence responses (Eulgem and Somssich, 2007). Using overexpression and antisense lines of Arabidopsis

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WRKY70, it was shown that the expression of this gene is directly correlated to resistance to both Erwinia carotovora and P. syringae (Li et al., 2004).

Later, two insertion KO lines for this same gene were isolated and they both showed an increased susceptibility to H. parasitica (Knoth et al., 2007). Also, an insertion KO mutant in AtWRKY18 was reported to be defective in SAR, whereas KO plants of AtWRKY58 were more resistant to P. syringae after treatment with suboptimal levels of benzothiadiazole (BTH), an analogue of SA that is used to protect plants against diseases in the fields (Wang et al., 2006).

This indicates that AtWRKY18 and AtWRKY58 act as positive and negative regulators, respectively, of plant defence responses. More recently, a KO of gene AtWRKY27 was shown to have delayed symptoms when infected by the pathogen Ralstonia solanacearum (Mukhtar et al., 2008). On the other hand, an insertion KO line for the gene AtWRKY25 did not reveal differences in susceptibility to P. syringae, although disease symptoms were reduced (Zheng et al., 2007).

WRKY factors have also been implicated in resistance to necrotrophic pathogens, as a KO line for Arabidopsis WRKY33 has an increased susceptibility to B. cinerea as well as to A. brassicicola (Zheng et al., 2006).

In rice, WRKY45 was shown to be induced by BTH (Shimono et al., 2007).

Overexpression of this gene also conferred strong resistance to the blast disease (Magnaporthe grisea) while silencing of the gene had the opposite effect. In barley, HvWRKY1 and HvWRKY2 proteins were shown to interact directly with the mildew A (MLA) R protein (Shen et al., 2007). When these two genes are silenced using a viral vector, infection by the virulent Blumeria graminis is significantly reduced. They were therefore identified as repressor of the basal defence response in barley.

Studies of multiple loss­of­function lines suggest that a complex interaction network exists between the different WRKY proteins. For instance, WRKY70 function was shown to be partially redundant with the function of WRKY53 (Wang et al., 2006). As expected, the double mutant wrky53 wrky70 showed enhanced susceptibility to P. syringae as compared to the single KO plants. In another study, it was shown that the Arabidopsis proteins WRKY18, WRKY40 and WRKY60 physically interacted with each other in yeast (Xu et al., 2006).

Gel retardation was then used to show that these proteins can form hetero­

complexes in vitro with changes in their binding affinity and/or specificity.

Mutant lines for these genes were obtained and revealed that, while only the single KO wrky18 showed increased resistance to P. syringae, the multiple KOs wrky18/40, wrky18/60 and wrky18/40/60 showed even more resistance.

The same trend was observed in regard to infection by B. cinerea, illustrating the possible negative effect of these WRKY proteins on resistance as well as the partial redundancy that exists inside this family (Xu et al., 2006). These results concerning the resistance of the wrky18 line contrast with those mentioned earlier by Wang et al. (2006). However, this last group tested the SAR, a specific component of plant defence, while Xu et al. tested the basal defence of the plant (Xu et al., 2006). Taken together, these two studies indicate that WRKY18 is a negative regulator of basal resistance and a positive regulator of acquired resistance which is truly interesting. In yet another study,

Transcription Factor Families and Plant Defence 153 it was shown that WRKY11 and WRKY17 also have partially redundant functions as negative regulators of defence (Journot­Catalino et al., 2006). It was shown that mutation of WRKY11 alone resulted in an increased resistance to P. syringae. Mutation of WRKY17 alone did not show altered resistance, but the wrky11/wrky17 double mutant line showed even more resistance than the wrky11 line. These results constitute convincing evidence regarding a role for WRKYs as both negative and positive regulators of resistance. It will be interesting in the future to learn about the complex interactions controlling the balance between these two roles

The WRKY genes are usually activated during the response to pathogens so they can modulate the transcriptome of the plant (Eulgem et al., 2000).

Overexpression of an activator WRKY will lead to constitutive expression of SA­inducible genes and will usually be accompanied by a decrease in expression of JA/ET­inducible genes (reviewed in Fobert, 2006). As indicated above, WRKY factors are known to bind a specific sequence known as the W box (Rushton et al., 1996). This was shown by a DNA–ligand binding screen as well as cotransfection assays in parsley cells. The specificity of this binding was further tested by random binding site selection (Du and Chen, 2000) and the consensus binding site TGAC­C/T was established. The direct interaction of WRKY proteins with DNA in vivo was shown by ChIP assays in parsley cells (Turck et al., 2004). After elicitation, PcWRKY1 was shown to bind prefer­

entially to fragments containing W boxes inside the promoters of PcWRKY1 and PcPR1-1. These results were later confirmed by another study showing that the AtWRKY33 promoter region is occupied by WRKY proteins before treatment with an elicitor and that it is occupied even more afterwards (Lippok et al., 2007). While these studies answered an important question, they also raised some more as they showed that some unidentified WRKY proteins were constitutively bound to the W box inside the genes even before elicitation.

We can therefore imagine that the WRKY binding activity is regulated by the formation of different homo and heterocomplexes of WRKY proteins. The study by Xu et al. clearly indicated that association of different WRKYs resulted in different binding strengths and specificities in vitro (Xu et al., 2006). The activity of a defence­related gene would then be the result of the ratio of different WRKY proteins present on the promoter. More precise ChIP studies could eventually shed some light on this matter. It is also possible that the WRKY activity is regulated by post­translational modifications. Indeed, two­

dimensional Western blotting revealed that a single WRKY protein can be present in different forms and that some of these forms become more abundant after elicitation (Turck et al., 2004). Another study showed that WRKY22 and WRKY29 were downstream components of a MAPK signalling cascade in Arabidopsis (Asai et al., 2002). This shows that, just like the ERF factors, WRKYs could also be activated by specific MAPK cascades.

The WRKY domain is defined as the DNA­binding domain of the WRKY proteins (reviewed in Eulgem et al., 2000). It is composed of approximately 60 amino acids that are the most conserved residues in the WRKY proteins.

Importantly, the WRKY domain is sufficient for mediating sequence­specific DNA binding. A putative zinc­binding motif C₂H₂ or C₂HC was originally

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found in the sequence coding for the WRKY domain. Complete loss of DNA­

binding activity upon treatment of these proteins with the divalent metal chelator 1,10­o­phenanthroline provided evidence that zinc binding was important for proper domain folding and/or DNA binding of the WRKY domain (Rushton et al., 1995; de Pater et al., 1996).

Recently, the three­dimensional structures of the DNA­binding domain of AtWRKY4 and AtWRKY1 were obtained by NMR spectroscopy (Yamasaki et al., 2005) and by X­ray crystallography (Duan et al., 2007), respectively. The structures can nearly be superimposed, suggesting that the WRKY proteins share a common DNA­binding mechanism. The structures consist of a four­

stranded (AtWRKY4) or a five­stranded (AtWRKY1) antiparallel β­sheet with a zinc­binding pocket. The WRKY domain is structurally related to the Glial Cells Missing (GCM) family of transcription factors for which a structure bound to DNA exists (Cohen et al., 2003). NMR­titration experiments and DNA docking enabled elaboration of a WRKY/DNA model (Yamasaki et al., 2005) (see Fig.

6.1). This model is in good agreement with a model obtained by comparative modelling using the structure of GCM/DNA as a canvas. According to these models, the β­sheet of the WRKY domain lies perpendicular to the DNA axis so that the β­strand containing the invariant WRKYGQK motif enters deeply into the major groove of the DNA making contacts with a 6­bp region.

However, the structural determinants of the sequence­specific binding of the WRKY domain are still unknown.