WRKY Domain Research Articles

Phenotypic characterization and genetic mapping of a new gene required for male and female gametophyte development in rice

Gametophyte development is an important process in flowering plants. Seeds are the primary organs for harvesting, and therefore spikelet fertility is especially vital for rice yield. Here, we report the characterization and genetic mapping of a novel rice male and female sterile mutant, mfs1, which was generated from a transgenic line of japonica rice variety Zhonghua 9. Anther transverse sections indicate that the microspores in these plants were abnormally degenerated at the meiotic leptotene stage, while the tapetum layer was not degenerated and remained present; therefore, no functional pollen grains formed in the mutant anther locules. In addition, the majority of mutant embryo sacs did not undergo differentiation or the differentiation was incomplete, resulting in the absence of functional embryo sac formation. Genetic analysis of three F2 populations indicated that the mutant phenotype was controlled by a single recessive gene. The mutant phenotype was confirmed by linkage analysis to be not due to a foreign T-DNA insertion. A map-based cloning strategy was employed to map the mutant gene, and, finally, the mfs1 locus was mapped to a 99-kb physical interval between simple sequence repeat markers RM12585 and D051 on rice chromosome 2. Sequence analysis indicates that 11 rice candidate genes are located in this region, including three encoded putative proteins and eight encoded known proteins, such as transcription factors having WRKY and zinc finger domains, a ubiquitin fusion degradation protein, chaperone protein clpB 1, a two-component response regulator, the ZOS2-04-C2H2 zinc finger protein, a peptidase of the T1 family and a protein kinase.

Identification of coffee WRKY transcription factor genes and expression profiling in resistance responses to pathogens

In plants, WRKY proteins are a group of transcription factors existing as a gene superfamily that play important roles in regulation of defense response pathways. To assess the diversity of this protein family in coffee (Coffea spp.), data mining methods were used on a set of around 200,000 coffee expressed sequence tags. A total of 53 different putative WRKY genes were obtained, but only 22 unigenes encoding a protein with a WRKY domain were identified, eight of which are supported by full-length cDNA sequences. Alignment of WRKY domain sequences of the coffee unigenes and 72 Arabidopsis thaliana WRKY genes showed that the 22 coffee WRKY members were distributed among the main A. thaliana WRKY subgroups and shared conserved peptide domains. To assess the involvement of WRKY genes in coffee defense response pathways, their expression was analyzed under biotic (nematode and rust fungus infection), hormonal (salicylic acid, methyl-jasmonate), and wounding treatments, leaf senescence, and fruit development. Five members of WRKY groups IId and III were regulated only by pathogens and hormone treatments. Although a significant correlation of WRKY genes expression after MeJA and rust treatments was observed, expression of coffee genes involved in JA biosynthesis and lipoxygenase (EC 1.13.11.12) activity assays did not support the involvement of JA in the early coffee resistance responses to the rust pathogen. The five WRKY transcription factor members identified might play important roles as regulators of pathogen resistance responses and could be useful for improving coffee tolerance to various biotic stresses.

GhWRKY3, a novel cotton (Gossypium hirsutum L.) WRKY gene, is involved in diverse stress responses

WRKY proteins play important roles in plant defense responses. In the present study, a novel WRKY gene, nominated as GhWRKY3, was isolated from cotton (Gossypium hirsutum L.). The full-length cDNA of GhWRKY3 is 1,705bp in length and encodes a protein with 507 amino acids containing two typical WRKY domains and two zinc finger motifs. Amino acid sequence alignment revealed that GhWRKY3 shares a high degree of identity with other higher plant WRKY proteins. The subcellular localization assay indicated that GhWRKY3 is localized to the nucleus. Analysis of 5'-flanking region of GhWRKY3 revealed a group of putative cis-acting elements. The results of expression analysis indicated that GhWRKY3 is constitutively expressed in roots, stems and leaves. Semi-quantitative RT-PCR showed that GhWRKY3 is up-regulated by application of various phytohormones including salicylic acid (SA), methyl jasmonate (MeJA), abscisic acid (ABA), gibberellins (GAs) and ethylene (ET). Furthermore, the transcripts of GhWRKY3 are enhanced after infection with Rhizoctonia solani, Colletotrichum gossypii and Fusarium oxysporum f. sp. vasinfectum, respectively. Also, GhWRKY3 can be induced by wounding treatment, but not by cytokinin (6-benzylaminopurine, 6-BA), auxin analogue, drought, NaCl, and cold (4°C). These data suggested that GhWRKY3 might play an important role in plant defense responses and fulfill a pivotal role in plant development.

Expression analyses indicate the involvement of sunflower WRKY transcription factors in stress responses, and phylogenetic reconstructions reveal the existence of a novel clade in the Asteraceae

The sunflower belongs to the Asteraceae family, whose members represent ten percent of the flowering plants. The genome sequence of this species is unknown and the information regarding the huge quantity of expressed sequence tags (ESTs) from Helianthus spp., available in public databases, has not yet been well explored. Transcription factors (TFs) are key proteins regulating many signal transduction pathways. Within TFs, the WRKY proteins constitute a family involved in the plant abiotic and biotic stress responses. To identify and characterise sunflower WRKY TFs, we utilised in silico approaches and gene expression surveys. Through a bioinformatics analysis, the existence of 97 sunflower WRKY members derived from EST databases was estimated. A comparative in silico analysis among Arabidopsis, rice, lettuce and sunflower WRKY proteins allowed us to assign the putative orthologues. Phylogenetic trees constructed with WRKY domains resolved the same seven groups assigned to Arabidopsis and additionally, a novel clade with traits apparently specific to Asteraceae. Expression patterns of one selected member of each group indicated that these genes are regulated by biotic and abiotic stresses as well as by stress-associated hormones. Moreover, the analysis performed in Sclerotinia-susceptible and resistant lines have shown that some of the sunflower WRKYs are related to such tolerance. This study indicates that the Asteraceae WRKY family has undergone a particular diversification, which could be the source of specific new functions. On the other hand, the sunflower WRKYs could play a role in the biotic stress response and could be markers of tolerance to necrotrophic pathogens.

Isolation and molecular characterization of a SoWRKY1 transcription factor from spinach (Spinacia oleracea)

WRKY genes encode proteins which belong to a large family of transcription factors that are involved in various developmental and physiological processes, response to pathogen infections and wound stress in plants. The molecular characteristics of WRKY genes involved in these are becoming clear in model plants, like Arabidopsis. However, knowledge of WRKY genes in other plants, for instance vegetable, is still not enough. In the present studies, using a yeast one-hybrid system we isolated a cDNA from a spinach cDNA library and characterized its function. The cDNA, designated SoWRKY1, encodes a putative polypeptide of 362 amino acids which is highly homologous to Arabidopsis WRKY40 (AtWRKY40). The putative primary structure of SoWRKY1 contains a single WRKY domain of Cys2His2 zinc finger motif and a potential nuclear localization signal (NLS), whose structure is characteristic of group IIa type WRKY protein. SoWRKY1-sGFP fusion protein was localized to the nucleus when the protein was expressed in onion epidermal cells. SoWRKY1 showed high binding affinity to DNA molecules containing TTGAC(C/T) W-box sequences and transcriptional activation activity in yeast. SoWRKY1 transcripts in spinach leaves were transiently induced by wounding treatment and salicylic acid (SA). The transcripts accumulated following treatment with cycloheximide (CHX), a protein biosynthesis inhibitor. Functional analysis of SoWRKY1 in vivo was performed by overexpression in Arabidopsis, and pathogenesis-related PR1 and PR2 gene expression level increased in the transgenic plants. These results suggest that SoWRKY1 might be involved as a transcription factor in defense-related signaling transduction pathways of spinach.

Isolation and Characterization of AaWRKY1, an Artemisia annua Transcription Factor that Regulates the Amorpha-4,11-diene Synthase Gene, a Key Gene of Artemisinin Biosynthesis

Amorpha-4,11-diene synthase (ADS) of Artemisia annua catalyzes the conversion of farnesyl diphosphate into amorpha-4,11-diene, the first committed step in the biosynthesis of the antimalarial drug artemisinin. The promoters of ADS contain two reverse-oriented TTGACC W-box cis-acting elements, which are the proposed binding sites of WRKY transcription factors. A full-length cDNA (AaWRKY1) was isolated from a cDNA library of the glandular secretory trichomes (GSTs) in which artemisinin is synthesized and sequestered. AaWRKY1 encodes a 311 amino acid protein containing a single WRKY domain. AaWRKY1 and ADS genes were highly expressed in GSTs and both were strongly induced by methyl jasmonate and chitosan. Transient expression analysis of the AaWRKY1-GFP (green fluorescent protein) reporter revealed that AaWRKY1 was targeted to nuclei. Biochemical analysis demonstrated that the AaWRKY1 protein was capable of binding to the W-box cis-acting elements of the ADS promoters, and it demonstrated transactivation activity in yeast. Co-expression of the effector construct 35S::AaWRKY1 with a reporter construct ADSpro1::GUS greatly activated expression of the GUS (beta-glucuronidase) gene in stably transformed tobacco. Furthermore, transient expression experiments in agroinfiltrated Nicotiana benthamiana and A. annua leaves showed that AaWRKY1 protein transactivated the ADSpro2 promoter activity by binding to the W-box of the promoter; disruption of the W-box abolished the activation. Transient expression of AaWRKY1 cDNA in A. annua leaves clearly activated the expression of the majority of artemisinin biosynthetic genes. These results strongly suggest the involvement of the AaWRKY1 transcription factor in the regulation of artemisinin biosynthesis, and indicate that ADS is a target gene of AaWRKY1 in A. annua.

Identification and expression analysis of WRKY transcription factor genes in canola (Brassica napus L.) in response to fungal pathogens and hormone treatments.

BackgroundMembers of plant WRKY transcription factor families are widely implicated in defense responses and various other physiological processes. For canola (Brassica napus L.), no WRKY genes have been described in detail. Because of the economic importance of this crop, and its evolutionary relationship to Arabidopsis thaliana, we sought to characterize a subset of canola WRKY genes in the context of pathogen and hormone responses.ResultsIn this study, we identified 46 WRKY genes from canola by mining the expressed sequence tag (EST) database and cloned cDNA sequences of 38 BnWRKYs. A phylogenetic tree was constructed using the conserved WRKY domain amino acid sequences, which demonstrated that BnWRKYs can be divided into three major groups. We further compared BnWRKYs to the 72 WRKY genes from Arabidopsis and 91 WRKY from rice, and we identified 46 presumptive orthologs of AtWRKY genes. We examined the subcellular localization of four BnWRKY proteins using green fluorescent protein (GFP) and we observed the fluorescent green signals in the nucleus only.The responses of 16 selected BnWRKY genes to two fungal pathogens, Sclerotinia sclerotiorum and Alternaria brassicae, were analyzed by quantitative real time-PCR (qRT-PCR). Transcript abundance of 13 BnWRKY genes changed significantly following pathogen challenge: transcripts of 10 WRKYs increased in abundance, two WRKY transcripts decreased after infection, and one decreased at 12 h post-infection but increased later on (72 h). We also observed that transcript abundance of 13/16 BnWRKY genes was responsive to one or more hormones, including abscisic acid (ABA), and cytokinin (6-benzylaminopurine, BAP) and the defense signaling molecules jasmonic acid (JA), salicylic acid (SA), and ethylene (ET). We compared these transcript expression patterns to those previously described for presumptive orthologs of these genes in Arabidopsis and rice, and observed both similarities and differences in expression patterns.ConclusionWe identified a set of 13 BnWRKY genes from among 16 BnWRKY genes assayed, that are responsive to both fungal pathogens and hormone treatments, suggesting shared signaling mechanisms for these responses. This study suggests that a large number of BnWRKY proteins are involved in the transcriptional regulation of defense-related genes in response to fungal pathogens and hormone stimuli.

A negative regulator encoded by a rice WRKY gene represses both abscisic acid and gibberellins signaling in aleurone cells

Abscisic acid (ABA) and gibberellins (GAs) control several developmental processes including seed maturation, dormancy, and germination. The antagonism of these two hormones is well-documented. However, recent data from transcription profiling studies indicate that they can function as agonists in regulating the expression of many genes although the underlying mechanism is unclear. Here we report a rice WRKY gene, OsWRKY24, which encodes a protein that functions as a negative regulator of both GA and ABA signaling. Overexpression of OsWRKY24 via particle bombardment-mediated transient expression in aleurone cells represses the expression of two reporter constructs: the beta-glucuronidase gene driven by the GA-inducible Amy32b alpha-amylase promoter (Amy32b-GUS) and the ABA-inducible HVA22 promoter (HVA22-GUS). OsWRKY24 is unlikely a general repressor because it has little effect on the expression of the luciferase reporter gene driven by a constitutive ubiquitin promoter (UBI-Luciferase). As to the GA signaling, OsWRKY24 differs from OsWRKY51 and -71, two negative regulators specifically function in the GA signaling pathway, in several ways. First, OsWRKY24 contains two WRKY domains while OsWRKY51 and -71 have only one; both WRKY domains are essential for the full repressing activity of OsWRKY24. Second, binding of OsWRKY24 to the Amy32b promoter appears to involve sequences in addition to the TGAC cores of the W-boxes. Third, unlike OsWRKY71, OsWRKY24 is stable upon GA treatment. Together, these data demonstrate that OsWRKY24 is a novel type of transcriptional repressor that inhibits both GA and ABA signaling.

Identification and characterization of the WRKY transcription factor family in Pinus monticola

The WRKY gene family represents an ancient and highly complex group of transcription factors involved in signal transduction pathways of numerous plant developmental processes and host defense response. Up to now, most WRKY proteins have been identified in a few angiosperm species. Identification of WRKY genes in a conifer species would facilitate a comprehensive understanding of the evolutionary and function-adaptive process of this superfamily in plants. We performed PCR on genomic DNA to clone WRKY sequences from western white pine (Pinus monticola), one of the most valuable conifer species endangered by white pine blister rust (Cronartium ribicola). In total, 83 P. monticola WRKY (PmWRKY) sequences were identified using degenerate primers targeted to the WRKY domain. A phylogenetic analysis revealed that PmWRKY members fell into four major groups (1, 2a+2b, 2c, and 2d+2e) described in Arabidopsis and rice. Because of high genetic diversity of the PmWRKY family, a modified AFLP method was used to detect DNA polymorphism of this gene family. Polymorphic fragments accounted for 17%-35% of total PCR products in the AFLP profiles. Among them, one WRKY AFLP marker was linked to the major resistance gene (Cr2) against C. ribicola. The results of this study provide basic genomic information for a conifer WRKY gene family, which will pave the way for elucidating gene evolutionary mechanisms in plants and unveiling the precise roles of PmWRKY in conifer development and defense response.

Enhanced heat and drought tolerance in transgenic rice seedlings overexpressing OsWRKY11 under the control of HSP101 promoter

An OsWRKY11 gene, which encodes a transcription factor with the WRKY domain, was identified as one of the genes that was induced by both heat shock and drought stresses in seedlings of rice (Oryza sativa L.). To determine if overexpression of OsWRKY11 confers heat and drought tolerance, OsWRKY11 cDNA was fused to the promoter of HSP101 of rice and introduced into a rice cultivar Sasanishiki. Overexpression of OsWRKY11 was induced by heat treatment. After heat pretreatment, the transgenic lines showed significant heat and drought tolerance, as indicated by the slower leaf-wilting and less-impaired survival rate of green parts of plants. They also showed significant desiccation tolerance, as indicated by the slower water loss in detached leaves. Our results indicate that the OsWRKY11 gene plays a role in heat and drought stress response and tolerance, and might be useful for improvement of stress tolerance.

Submergence-responsive MicroRNAs are Potentially Involved in the Regulation of Morphological and Metabolic Adaptations in Maize Root Cells

Anaerobic or low oxygen conditions occur when maize plants are submerged or subjected to flooding of the soil. Maize survival under low oxygen conditions is largely dependent on metabolic, physiological and morphological adaptation strategies; the regulation mechanisms of which remain unknown. MicroRNAs (miRNAs) play critical roles in the response to adverse biotic or abiotic stresses at the post-transcriptional level. The aim of this study was to understand submergence-responsive miRNAs and their potential roles in submerged maize roots. A custom muParaflo microfluidic array containing plant miRNA (miRBase: http://microrna.sanger.ac.uk) probes was used to explore differentially expressed miRNAs. Small RNAs from treated roots were hybridized with the microarray. The targets and their cis-acting elements of small RNA were predicted and analysed by RT-PCR. Microarray data revealed that the expression levels of 39 miRNAs from nine maize and some other plant miRNA families were significantly altered (P < 0.01). Four expression profiles were identified across different submergence time-points. The zma-miRNA166, zma-miRNA167, zma-miRNA171 and osa-miRNA396-like were induced in the early phase, and their target genes were predicted to encode important transcription factors, including; HD-ZIP, auxin response factor, SCL and the WRKY domain protein. zma-miR159, ath-miR395-like, ptc-miR474-like and osa-miR528-like were reduced at the early submergence phase and induced after 24 h of submergence. The predicted targets for these miRNAs were involved in carbohydrate and energy metabolism, including starch synthase, invertase, malic enzyme and ATPase. In addition, many of the predicted targets were involved in the elimination of reactive oxygen species and acetaldehyde. Overall, most of the targets of induced miRNAs contained the cis-acting element, which is essential for the anaerobic response or hormone induction. Submergence-responsive miRNAs are involved in the regulation of metabolic, physiological and morphological adaptations of maize roots at the post-transcriptional level.

Identification of genes encoding receptor-like protein kinases as possible targets of pathogen- and salicylic acid-induced WRKY DNA-binding proteins in Arabidopsis

To understand how plant host genes are regulated during the activation of plant defence responses, we are studying a group of pathogen- and salicylic acid (SA)-induced DNA-binding proteins containing the novel WRKY domain. To identify downstream target genes of these WRKY proteins, we have searched the Arabidopsis genome and identified four closely linked genes on chromosome IV that contain an unusually large number of the W-box sequences [(T)TGAC(C/T)] recognized by WRKY proteins within a few hundred base pairs upstream of their coding regions. All four genes encode proteins characteristic of receptor-like protein kinases (RLK), each consisting of an N-terminal signal sequence, an extracellular receptor domain, a single transmembrane domain and a C-terminal cytoplasmic serine/threonine protein kinase domain. All four RLK genes were induced by treatment with SA or infection by a bacterial pathogen. Studies with one of the RLK genes (RLK4) indicated that a cluster of W-box elements in its promoter region were recognized by both purified WRKY proteins and SA-induced W-box binding activities from SA-treated Arabidopsis plants. Further analysis using the RLK4 gene promoter fused to a reporter gene in transgenic Arabidopsis indicated that the consensus WRKY protein-binding sites in the RLK4 gene promoter were important for the inducible expression of the reporter gene. These results indicate that pathogen- and SA-induced W-box binding proteins regulate not only genes encoding defence proteins with direct or indirect anti-microbial activities, but also genes encoding proteins with regulatory functions.

Genetic variants of miRNA sequences and non small cell lung cancer survival

The Arabidopsis immune receptors RPS4 and RRS1 interact to co-confer responsiveness to bacterial effectors. The RRS1-R allele, with RPS4, responds to AvrRps4 and PopP2, whereas RRS1-S responds only to AvrRps4. Here, we show that the C terminus of RRS1-R but not RRS1-S is phosphorylated. Phosphorylation at Thr1214 in the WRKY domain maintains RRS1-R in its inactive state and also inhibits acetylation of RRS1-R by PopP2. PopP2 in turn catalyzes O-acetylation at the same site, thereby preventing its phosphorylation. Phosphorylation at other sites is required for PopP2 but not AvrRps4 responsiveness and facilitates the interaction of RRS1’s C terminus with its TIR domain. Derepression of RRS1-R or RRS1-S involves effector-triggered proximity between their TIR domain and C termini. This effector-promoted interaction between these domains relieves inhibition of TIRRPS4 by TIRRRS1. Our data reveal effector-triggered and phosphorylation-regulated conformational changes within RRS1 that results in distinct modes of derepression of the complex by PopP2 and AvrRps4.

Identification of 118 Arabidopsis Transcription Factor and 30 Ubiquitin-Ligase Genes Responding to Chitin, a Plant-Defense Elicitor

Chitin, found in the cell walls of true fungi and the exoskeleton of insects and nematodes, is a well-established elicitor of plant defense responses. In this study, we analyzed the expression patterns of Arabidopsis thaliana transcription factor (TF) and ubiquitin-ligase genes in response to purified chitooctaose at different treatment times (15, 30, 60, 90, and 120 min after treatment), using both quantitative polymerase chain reaction and the Affymetrix Arabidopsis whole-genome array. A total of 118 TF genes and 30 ubiquitin-ligase genes were responsive to the chitin treatment. Among these genes, members from the following four TF families were overrepresented: APETALA2/ethylene-reponsive element binding proteins (27), C2H2 zinc finger proteins (14), MYB domain-containing proteins (11), and WRKY domain transcription factors (14). Transcript variants from a few of these genes were found to respond differentially to chitin, suggesting transcript-specific regulation of these TF genes.

The potential of the WRKY gene family for phylogenetic reconstruction: An example from the Malvaceae

The WRKY gene family of transcription factors is involved in several diverse pathways and includes components of plant-specific, ancient regulatory networks. WRKY genes contain one or two highly conserved DNA binding domains interrupted by an intron. We used partial sequences of five independent WRKY loci to assess their potential for phylogeny reconstruction. Loci were originally isolated from Theobroma cacao L. by PCR with a single pair of degenerate primers; loci-specific primers were subsequently designed. We tested those loci across the sister genera Herrania Goudot and Theobroma L., with Guazuma ulmifolia Lam. as the outgroup. Overall, the combined WRKY matrices performed as well or better than other genes in resolving the intrageneric phylogeny of Herrania and Theobroma. The ease of isolating numerous, independent WRKY loci from diverse plant species with a single pair of degenerate primers designed to the highly conserved WRKY domain, renders them extremely useful tools for generating multiple, single or low copy nuclear loci for molecular phylogenetic studies at lower taxonomic levels. This is the first demonstration of the potential for members of the WRKY gene family for phylogenetic reconstruction.

The WRKY Gene Family in Rice (Oryza sativa)

Abstract WRKY genes encode transcription factors that are involved in the regulation of various biological processes. These zinc‐finger proteins, especially those members mediating stress responses, are uniquely expanded in plants. To facilitate the study of the evolutionary history and functions of this supergene family, we performed an exhaustive search for WRKY genes using HMMER and a Hidden Markov Model that was specifically trained for rice. This work resulted in a comprehensive list of WRKY gene models in Oryza sativa L. ssp. indica and L. ssp. japonica. Mapping of these genes to individual chromosomes facilitated elimination of the redundant, leading to the identification of 98 WRKY genes in japonica and 102 in indica rice. These genes were further categorized according to the number and structure of their zinc‐finger domains. Based on a phylogenetic tree of the conserved WRKY domains and the graphic display of WRKY loci on corresponding indica and japonica chromosomes, we identified possible WRKY gene duplications within, and losses between the two closely related rice subspecies. Also reviewed are the roles of WRKY genes in disease resistance and responses to salicylic acid and jasmonic acid, seed development and germination mediated by gibberellins, other developmental processes including senescence, and responses to abiotic stresses and abscisic acid in rice and other plants. The signaling pathways mediating WRKY gene expression are also discussed.

DNA binding mechanism revealed by high resolution crystal structure of Arabidopsis thaliana WRKY1 protein.

WRKY proteins, defined by the conserved WRKYGQK sequence, are comprised of a large superfamily of transcription factors identified specifically from the plant kingdom. This superfamily plays important roles in plant disease resistance, abiotic stress, senescence as well as in some developmental processes. In this study, the Arabidopsis WRKY1 was shown to be involved in the salicylic acid signaling pathway and partially dependent on NPR1; a C-terminal domain of WRKY1, AtWRKY1-C, was constructed for structural studies. Previous investigations showed that DNA binding of the WRKY proteins was localized at the WRKY domains and these domains may define novel zinc-binding motifs. The crystal structure of the AtWRKY1-C determined at 1.6 Å resolution has revealed that this domain is composed of a globular structure with five β strands, forming an antiparallel β-sheet. A novel zinc-binding site is situated at one end of the β-sheet, between strands β4 and β5. Based on this high-resolution crystal structure and site-directed mutagenesis, we have defined and confirmed that the DNA-binding residues of AtWRKY1-C are located at β2 and β3 strands. These results provided us with structural information to understand the mechanism of transcriptional control and signal transduction events of the WRKY proteins.

Identification of a new member of the WRKY family in tobacco. Involved in ozone-induced gene regulation?

Recent studies showed, that ozone-induced gene expression occurs via at least two different signalling mechanisms that are ethylene-dependent (β-1,3-glucanases) and ethylene-independent independent (stilbene synthase). To identify trans-acting factors involved in ozone-induced gene expression we analyzed a 150 bp PCR fragment of an ozone-responsive promoter segment of the grape vine resveratrol synthase gene (Vst1) in combination of a cDNA library prepared from ozone-treated tobacco plants, using the yeast one-hybrid screening system. Two cDNA clones that encode WRKY binding proteins were isolated by this screening technique. The open reading frame of NtWRKY10 and NtWRKY11 showed an identity of 93.5% and the deduced amino acid sequence an identity of 89.3%. According to the WRKY domain classification in Arabidopsis, both proteins belong to subgroup II. Comparison with known tobacco WRKY proteins indicate that WRKY10 and WRKY11 belong to a new class of tobacco WRKY transcription factors. Electrophoretic mobility shift assays (EMSA) of yeast extracts, containing the WRKY fusion protein showed a weak binding to the radioactively labelled 150 bp ozone-responsive Vst1 fragment. These results are consistent with an involvement of WRKY proteins in ozone-induced phytoalexin gene expression.

Interactions of two abscisic‐acid induced WRKY genes in repressing gibberellin signaling in aleurone cells

Gibberellins (GA) promote while abscisic acid (ABA) inhibits seed germination and post-germination growth. To address the cross-talk of GA and ABA signaling, we studied two rice WRKY genes (OsWRKY51 and OsWRKY71) that are ABA-inducible and GA-repressible in embryos and aleurone cells. Over-expression of these two genes in aleurone cells specifically and synergistically represses induction of the ABA-repressible and GA-inducible Amy32b alpha-amylase promoter reporter construct (Amy32b-GUS) by GA or the GA-inducible transcriptional activator, GAMYB. The physical interactions of OsWRKY71 proteins themselves and that of OsWRKY71 and OsWRKY51 are revealed in the nuclei of aleurone cells using bimolecular fluorescence complementation (BiFC) assays. Although OsWRKY51 itself does not bind to the Amy32b promoter in vitro, it interacts with OsWRKY71 and enhances the binding affinity of OsWRKY71 to W boxes in the Amy32b promoter. The binding activity of OsWRKY71 is abolished by deleting the C-terminus containing the WRKY domain or substituting the key amino acids in the WRKY motif and the zinc finger region. However, two of these non-DNA-binding mutants are still able to repress GA induction by enhancing the binding affinity of the wild-type DNA-binding OsWRKY71 repressors. In contrast, the third non-DNA-binding mutant enhances GA induction of Amy32b-GUS, by interfering with the binding of the wild-type OsWRKY71 or the OsWRKY71/OsWRKY51 repressing complex. These data demonstrate the synergistic interaction of ABA-inducible WRKY genes in regulating GAMYB-mediated GA signaling in aleurone cells, thereby establishing a novel mechanism for ABA and GA signaling cross-talk.

Dissecting the WRKY Web of Plant Defense Regulators

During the past ten years, a large body of circumstantial evidence has accumulated, implicating WRKY factors in transcriptional reprogramming during plant immune responses [1–3]. Encoded by complex gene families in higher plants [4], these transcription factors share a DNA-binding domain (WRKY domain) comprising approximately 60 amino acids [5]. Additional conserved features of WRKYs are limited to separate subgroups of this family and include putative leucine zippers, nuclear localization signals, calmodulin binding sites, and several domains of unknown function [5–7]. Multiple studies have demonstrated the ability of WRKYs to bind to promoters of defense-associated genes via specific interactions of their WRKY domains with pathogen response elements termed W boxes (TTGACC/T) [8–11]. Both activating and repressing effects of WRKYs and W boxes on transcription have been observed [12–14]. It has also been shown that stable and transient overexpression of several WRKYs in the model plant Arabidopsis thaliana (Arabidopsis) conveys enhanced resistance to various bacterial or fungal pathogens [15–17].