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Despite the existence of numerous links between WRKY transcription factors and plant defence mechanisms, direct evidence for the involvement of WRKY proteins in this process remains limited. In most cases, their expression is up-regulated in pathogen-infected or SA-treated plants. This up-regulation is often correlated with pathogen resistance as they are expressed specifically during incompatible reactions (Liu et al., 2004; Park et al., 2006). Functional studies have only been performed in A. thaliana for a few WRKY transcription factors. To date, two Arabidopsis WRKY factors (AtWRKY22 and AtWRKY29) have been identified as important downstream compounds of the MAPK pathway that confer resistance to both bacterial and fungal pathogens (Asai et al., 2002). Overexpression of AtWRKY70 increased resistance to virulent pathogens (Li et al., 2004, 2006). Recently, a WRKY transcription factor (OsWRKY71) was overexpressed in rice, and transgenic plants showed an enhanced resistance to virulent bacteria (Liu et al., 2006). Similar results have been shown with Arabidopsis plants expressing AtWRKY18, albeit in a development-dependent manner (Chen and Chen, 2002). However, nearly all such lines showed altered leaf morphologies and changes in flowering time (Chen and Chen, 2002; Robatzek and Somssich, 2002; Li et al., 2004).
In the present study, a cDNA, designated VvWRKY1, encoding a polypeptide of 151 amino acids, was identified. Phylogenetic analysis showed that VvWRKY1 belongs to the subgroup IIc (Dong et al., 2003), with StWRKY1 and AtWRKY75 (corresponding to AtWRKY64 in Dong et al., 2003) as its closest homologues. In grapevine, numerous partial sequences from EST databases showing significant homologies with WRKY genes have been identified from libraries constructed after abiotic stresses or from different stages of flower and fruit development. However, no functional analyses of genes encoding these sequences have been published to date.
Consistent with its putative role as a transcription factor, the VvWRKY1 protein contains a nuclear targeting sequence. Moreover, since phosphorylation of WRKY proteins is thought to play a role in their activation (Yang et al., 1999; Yamamoto et al., 2004), the presence of phosphorylation sites in the VvWRKY1 deduced protein sequence was sought. Indeed, numerous potential phosphorylation sites were found using the PSORT program (Nakai and Kanehisa, 1992; http://psort.nibb.ac.jp) (data not shown). This suggests a possible regulation of VvWRKY1 activity by phosphorylation via different protein kinases.
WRKY proteins were described as transcription factors capable of binding W-box-containing sequences (Eulgem et al., 2000) that are present in the promoter regions of a large number of defence genes including PR genes. The present data show that the VvWRKY1 protein also binds specifically to W-box cis-elements in different nucleotidic environments. Thus, one must assume that VvWRKY1 might regulate the expression of genes containing the W-box within their promoters.
WRKY genes have been duplicated many times over during plant evolution, resulting in a large gene family involved in regulating a large set of genes and thereby ensuring proper cellular responses to physiological processes, internal and external stimuli. For example, the expression of several Arabidopsis WRKY genes is strongly up-regulated during plant senescence (Hindehofer and Zentgraf, 2001; Robatzek and Somssich, 2001; Chen et al., 2002; Guo et al., 2004). In the present study, VvWRKY1 appeared to be developmentally regulated in leaves and in berries. VvWRKY1 expression was very weak in berries before veraison but there was a significant increase at veraison that continued through ripening (Fig. 4A). This finding is in agreement with previous results obtained with the CaWRKY1 gene in pepper. CaWRKY1 expression is strongly up-regulated in red fruit and may play an important role in pepper fruit maturation (Ulker and Somssich, 2004). Furthermore, VvWRKY1 expression in grape berries appears to be co-regulated with some PR-like class genes, such as class IV chitinase, thaumatin-like protein, lipid transfer protein, and metallothionein, that are differentially expressed during ripening, some peaking after veraison (Robinson et al., 1997; Tattersall et al., 1997; Salzman et al., 1998; Davies and Robinson, 2000). Even if some of these genes are induced by pathogen infection in a susceptible cultivar (Jacobs et al., 1999), up-regulation of PR genes seems to be correlated with resistance to powdery mildew (Chellemi and Marois, 1992).
The response to pathogens is regulated by multiple signal transduction pathways in which SA, jasmonic acid, and ethylene function as key signalling molecules (Glazebrook, 2001). SA is a key endogenous secondary signal involved in activation and/or potentiation of plant defence responses (Dempsey et al., 1999). It is required for the establishment of SAR. A second signalling pathway, which is generally antagonistic with the SA-dependent pathway, involves methyl jasmonate/ethylene as key intermediates. Ethylene plays a role in controlling symptom development due to virulent bacteria or fungi (Lund et al., 1998). However, simultaneous activation of these two pathways is fully compatible in Arabidopsis (Van Wees et al., 2000). H2O2 is also an important component of plant defence mechanisms even if its signalling role is not completely defined (Tenhaken et al., 1995). All these key molecules act in a very complex signalling network and share several steps or enzymes with each other (Reymond and Farmer, 1998). In the present assays, all of these molecules affected VvWRKY1 expression, with transcription of VvWRKY1 being up-regulated in response to SA, H2O2, and ethephon. The closest homologues of VvWRKY1, StWRKY1 and AtWRKY75, have also been shown to be induced by elicitors, SA treatment, and pathogen infection (Dellagi et al., 2000; Dong et al., 2003). Moreover, numerous studies showed induction of WRKY gene expression in response to SA (Yang et al., 1999; Chen and Chen, 2000, 2002; Dong et al., 2003; Li et al., 2004), but few authors analysed the effect of other signal molecules, such as H2O2 (Miao et al., 2004; Rhizhsky et al., 2004) or ethylene (Li et al., 2004). Only AtWRKY70 expression can be activated by SA and ACC (a natural precursor of ethylene; Li et al., 2004). On the other hand, no variation in expression of GaWRKY1 was noted by Xu et al. (2004) in response to SA and H2O2. VvWRKY1 activation of transcription was also previously described in grapevine cell suspension after treatment by ergosterol, a non-specific fungal elicitor (Laquitaine et al., 2006). Taken together, these results are consistent with the finding that expression of many WRKY genes in various plant species is rapidly activated upon pathogen challenge and/or elicitor treatments. The induction of VvWRKY1 transcription by wounding, fungal elicitors, or signalling molecules strongly suggests the involvement of this transcription factor in grapevine stress responses.
In this work, an attempt was also made to analyse the biological impact of the constitutive expression of VvWRKY1 in tobacco. All independent transgenic tobacco lines showed no visible phenotypic changes, which is consistent with the present finding that these lines do not have constitutive high elevated levels of PR gene transcripts. This last finding was quite surprising because overexpression of WRKY genes often induced constitutive PR gene expression (Chen and Chen, 2002; Li et al., 2004; Liu et al., 2006). However, the present analysis of PR gene expression was not exhaustive and the possibility cannot be ruled out that the expression of other PR genes can be induced in the transgenic lines. Nevertheless, evidence presented here shows that overexpression of VvWRKY1 in tobacco results in a slight but significant decrease in susceptibility towards three different fungi, namely Pythium, and the downy and the powdery mildew agents. These findings indicate that constitutive ectopic expression of VvWRKY1 alone is insufficient to activate defence responses but requires additional unknown pathogen-induced plant components to exert its functions. Similar results have been obtained in Arabidopsis for AtWRKY18, which alone is not able to activate PR gene expression in transgenic plants during the early stages of development (Chen and Chen, 2002). Like many other complex biological processes, plant defence responses to pathogen infection involve transcriptional regulation of a large number of plant host genes (Rushton and Somssich, 1998). Taken together, these results suggest that the VvWRKY1 transcription factor requires co-ordination with developmentally regulated components and/or other transcription factors induced by plant–pathogen interaction in order to activate plant defence responses in tobacco.
In conclusion, a new grape WRKY gene, designated VvWRKY1, has been identified whose expression is strongly regulated during berry and leaf development, and under stress conditions. This transcription factor appears to be induced by abiotic and biotic stresses in grapevine. Its overexpression in tobacco induces a decrease in susceptibility towards some fungi, suggesting a role in the plant defence response to fungal pathogens. However, the decrease in susceptibility is more significant against a necrotrophic fungus (Pythium) and a biotrophic fungus (powdery mildew) than against the two other biotrophic pathogens tested (downy mildew and PVY). Mechanisms of plant defence against biotrophic and necrotrophic pathogens use different signalling pathways that activate various responses allowing resistance (Glazebrook, 2005). In the case of biotrophs, the HR, often mediated by SA signalling, generally results in resistance, while resistance to necrotrophs involves a different set of defence responses activated by jasmonic acid and/or ethylene signalling. The data presented here suggest that VvWRKY1 may be involved in both signalling pathways. Consistent with this finding, AtWRKY70 has also been shown to be involved in defence against biotrophic and necrotrophic pathogens (AbuQamar et al., 2006; Li et al., 2006). However, the difference in susceptibility observed between VvWRKY1 tobacco transgenic lines and control plants towards all the selected fungi is relatively slight and may be due to the use of a heterologous plant system. Further investigations using a homologous expression system in which VvWRKY1 will find its endogenous target genes and/or partners are in progress and might provide new insights into the biological function of WRKY genes in grapevine and their role in enhancing protection against various fungi.
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