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1.  A Wheat WRKY Transcription Factor TaWRKY10 Confers Tolerance to Multiple Abiotic Stresses in Transgenic Tobacco 
PLoS ONE  2013;8(6):e65120.
WRKY transcription factors are reported to be involved in defense regulation, stress response and plant growth and development. However, the precise role of WRKY transcription factors in abiotic stress tolerance is not completely understood, especially in crops. In this study, we identified and cloned 10 WRKY genes from genome of wheat (Triticum aestivum L.). TaWRKY10, a gene induced by multiple stresses, was selected for further investigation. TaWRKY10 was upregulated by treatment with polyethylene glycol, NaCl, cold and H2O2. Result of Southern blot indicates that the wheat genome contains three copies of TaWRKY10. The TaWRKY10 protein is localized in the nucleus and functions as a transcriptional activator. Overexpression of TaWRKY10 in tobacco (Nicotiana tabacum L.) resulted in enhanced drought and salt stress tolerance, mainly demonstrated by the transgenic plants exhibiting of increased germination rate, root length, survival rate, and relative water content under these stress conditions. Further investigation showed that transgenic plants also retained higher proline and soluble sugar contents, and lower reactive oxygen species and malonaldehyde contents. Moreover, overexpression of the TaWRKY10 regulated the expression of a series of stress related genes. Taken together, our results indicate that TaWRKY10 functions as a positive factor under drought and salt stresses by regulating the osmotic balance, ROS scavenging and transcription of stress related genes.
doi:10.1371/journal.pone.0065120
PMCID: PMC3677898  PMID: 23762295
2.  Roles of arabidopsis WRKY18, WRKY40 and WRKY60 transcription factors in plant responses to abscisic acid and abiotic stress 
BMC Plant Biology  2010;10:281.
Background
WRKY transcription factors are involved in plant responses to both biotic and abiotic stresses. Arabidopsis WRKY18, WRKY40, and WRKY60 transcription factors interact both physically and functionally in plant defense responses. However, their role in plant abiotic stress response has not been directly analyzed.
Results
We report that the three WRKYs are involved in plant responses to abscisic acid (ABA) and abiotic stress. Through analysis of single, double, and triple mutants and overexpression lines for the WRKY genes, we have shown that WRKY18 and WRKY60 have a positive effect on plant ABA sensitivity for inhibition of seed germination and root growth. The same two WRKY genes also enhance plant sensitivity to salt and osmotic stress. WRKY40, on the other hand, antagonizes WRKY18 and WRKY60 in the effect on plant sensitivity to ABA and abiotic stress in germination and growth assays. Both WRKY18 and WRKY40 are rapidly induced by ABA, while induction of WRKY60 by ABA is delayed. ABA-inducible expression of WRKY60 is almost completely abolished in the wrky18 and wrky40 mutants. WRKY18 and WRKY40 recognize a cluster of W-box sequences in the WRKY60 promoter and activate WRKY60 expression in protoplasts. Thus, WRKY60 might be a direct target gene of WRKY18 and WRKY40 in ABA signaling. Using a stable transgenic reporter/effector system, we have shown that both WRKY18 and WRKY60 act as weak transcriptional activators while WRKY40 is a transcriptional repressor in plant cells.
Conclusions
We propose that the three related WRKY transcription factors form a highly interacting regulatory network that modulates gene expression in both plant defense and stress responses by acting as either transcription activator or repressor.
doi:10.1186/1471-2229-10-281
PMCID: PMC3023790  PMID: 21167067
3.  The WRKY transcription factor superfamily: its origin in eukaryotes and expansion in plants 
Background
WRKY proteins are newly identified transcription factors involved in many plant processes including plant responses to biotic and abiotic stresses. To date, genes encoding WRKY proteins have been identified only from plants. Comprehensive search for WRKY genes in non-plant organisms and phylogenetic analysis would provide invaluable information about the origin and expansion of the WRKY family.
Results
We searched all publicly available sequence data for WRKY genes. A single copy of the WRKY gene encoding two WRKY domains was identified from Giardia lamblia, a primitive eukaryote, Dictyostelium discoideum, a slime mold closely related to the lineage of animals and fungi, and the green alga Chlamydomonas reinhardtii, an early branching of plants. This ancestral WRKY gene seems to have duplicated many times during the evolution of plants, resulting in a large family in evolutionarily advanced flowering plants. In rice, the WRKY gene family consists of over 100 members. Analyses suggest that the C-terminal domain of the two-WRKY-domain encoding gene appears to be the ancestor of the single-WRKY-domain encoding genes, and that the WRKY domains may be phylogenetically classified into five groups. We propose a model to explain the WRKY family's origin in eukaryotes and expansion in plants.
Conclusions
WRKY genes seem to have originated in early eukaryotes and greatly expanded in plants. The elucidation of the evolution and duplicative expansion of the WRKY genes should provide valuable information on their functions.
doi:10.1186/1471-2148-5-1
PMCID: PMC544883  PMID: 15629062
4.  Genome-wide analysis of WRKY gene family in Cucumis sativus 
BMC Genomics  2011;12:471.
Background
WRKY proteins are a large family of transcriptional regulators in higher plant. They are involved in many biological processes, such as plant development, metabolism, and responses to biotic and abiotic stresses. Prior to the present study, only one full-length cucumber WRKY protein had been reported. The recent publication of the draft genome sequence of cucumber allowed us to conduct a genome-wide search for cucumber WRKY proteins, and to compare these positively identified proteins with their homologs in model plants, such as Arabidopsis.
Results
We identified a total of 55 WRKY genes in the cucumber genome. According to structural features of their encoded proteins, the cucumber WRKY (CsWRKY) genes were classified into three groups (group 1-3). Analysis of expression profiles of CsWRKY genes indicated that 48 WRKY genes display differential expression either in their transcript abundance or in their expression patterns under normal growth conditions, and 23 WRKY genes were differentially expressed in response to at least one abiotic stresses (cold, drought or salinity). The expression profile of stress-inducible CsWRKY genes were correlated with those of their putative Arabidopsis WRKY (AtWRKY) orthologs, except for the group 3 WRKY genes. Interestingly, duplicated group 3 AtWRKY genes appear to have been under positive selection pressure during evolution. In contrast, there was no evidence of recent gene duplication or positive selection pressure among CsWRKY group 3 genes, which may have led to the expressional divergence of group 3 orthologs.
Conclusions
Fifty-five WRKY genes were identified in cucumber and the structure of their encoded proteins, their expression, and their evolution were examined. Considering that there has been extensive expansion of group 3 WRKY genes in angiosperms, the occurrence of different evolutionary events could explain the functional divergence of these genes.
doi:10.1186/1471-2164-12-471
PMCID: PMC3191544  PMID: 21955985
5.  WRKY54 and WRKY70 co-operate as negative regulators of leaf senescence in Arabidopsis thaliana 
Journal of Experimental Botany  2012;63(7):2667-2679.
The plant-specific WRKY transcription factor (TF) family with 74 members in Arabidopsis thaliana appears to be involved in the regulation of various physiological processes including plant defence and senescence. WRKY53 and WRKY70 were previously implicated as positive and negative regulators of senescence, respectively. Here the putative function of other WRKY group III proteins in Arabidopsis leaf senescence has been explored and the results suggest the involvement of two additional WRKY TFs, WRKY 54 and WRKY30, in this process. The structurally related WRKY54 and WRKY70 exhibit a similar expression pattern during leaf development and appear to have co-operative and partly redundant functions in senescence, as revealed by single and double mutant studies. These two negative senescence regulators and the positive regulator WRKY53 were shown by yeast two-hydrid analysis to interact independently with WRKY30. WRKY30 was expressed during developmental leaf senescence and consequently it is hypothesized that the corresponding protein could participate in a senescence regulatory network with the other WRKYs. Expression in wild-type and salicylic acid-deficient mutants suggests a common but not exclusive role for SA in induction of WRKY30, 53, 54, and 70 during senescence. WRKY30 and WRKY53 but not WRKY54 and WRKY70 are also responsive to additional signals such as reactive oxygen species. The results suggest that WRKY53, WRKY54, and WRKY70 may participate in a regulatory network that integrates internal and environmental cues to modulate the onset and the progression of leaf senescence, possibly through an interaction with WRKY30.
doi:10.1093/jxb/err450
PMCID: PMC3346227  PMID: 22268143
Arabidopsis thaliana; ROS; SA; senescence; WRKY transcription factors
6.  Characterization of WRKY co-regulatory networks in rice and Arabidopsis 
BMC Plant Biology  2009;9:120.
Background
The WRKY transcription factor gene family has a very ancient origin and has undergone extensive duplications in the plant kingdom. Several studies have pointed out their involvement in a range of biological processes, revealing that a large number of WRKY genes are transcriptionally regulated under conditions of biotic and/or abiotic stress. To investigate the existence of WRKY co-regulatory networks in plants, a whole gene family WRKYs expression study was carried out in rice (Oryza sativa). This analysis was extended to Arabidopsis thaliana taking advantage of an extensive repository of gene expression data.
Results
The presented results suggested that 24 members of the rice WRKY gene family (22% of the total) were differentially-regulated in response to at least one of the stress conditions tested. We defined the existence of nine OsWRKY gene clusters comprising both phylogenetically related and unrelated genes that were significantly co-expressed, suggesting that specific sets of WRKY genes might act in co-regulatory networks. This hypothesis was tested by Pearson Correlation Coefficient analysis of the Arabidopsis WRKY gene family in a large set of Affymetrix microarray experiments. AtWRKYs were found to belong to two main co-regulatory networks (COR-A, COR-B) and two smaller ones (COR-C and COR-D), all including genes belonging to distinct phylogenetic groups. The COR-A network contained several AtWRKY genes known to be involved mostly in response to pathogens, whose physical and/or genetic interaction was experimentally proven. We also showed that specific co-regulatory networks were conserved between the two model species by identifying Arabidopsis orthologs of the co-expressed OsWRKY genes.
Conclusion
In this work we identified sets of co-expressed WRKY genes in both rice and Arabidopsis that are functionally likely to cooperate in the same signal transduction pathways. We propose that, making use of data from co-regulatory networks, it is possible to highlight novel clusters of plant genes contributing to the same biological processes or signal transduction pathways. Our approach will contribute to unveil gene cooperation pathways not yet identified by classical genetic analyses. This information will open new routes contributing to the dissection of WRKY signal transduction pathways in plants.
doi:10.1186/1471-2229-9-120
PMCID: PMC2761919  PMID: 19772648
7.  OsWRKY IIa Transcription Factors Modulate Rice Innate Immunity 
Rice (New York, N.y.)  2010;3(1):36-42.
WRKY transcription factors regulate diverse plant processes including responses to biotic stresses. Our previous studies indicate that OsWRKY62, an OsWRKY IIa subfamily member, functions as a negative regulator of the rice defense against Xanthomonas oryzae pv. oryzae. Here, we report that a large inverted repeat construct designed to knock down the expression of the four OsWRKY IIa subfamily members (OsWRKY62, OsWRKY28, OsWRKY71, and OsWRKY76) leads to overexpression of all four genes and disease resistance in some transgenic plants. These phenotypes are stably inherited as reflected by progeny analysis. A pathogenesis-related gene, PR10, is up-regulated in plants overexpressing the OsWRKY IIa genes. These results suggest that OsWRKY IIa proteins interact functionally to modulate plant innate immunity.
doi:10.1007/s12284-010-9039-6
PMCID: PMC3175632  PMID: 21961049
Rice; OsWRKY IIa; Xoo; Innate immunity
8.  Identification and expression analysis of WRKY transcription factor genes in canola (Brassica napus L.) in response to fungal pathogens and hormone treatments 
BMC Plant Biology  2009;9:68.
Background
Members 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.
Results
In 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.
Conclusion
We 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.
doi:10.1186/1471-2229-9-68
PMCID: PMC2698848  PMID: 19493335
9.  Functional analysis of Arabidopsis WRKY25 transcription factor in plant defense against Pseudomonas syringae 
BMC Plant Biology  2007;7:2.
Background
A common feature of plant defense responses is the transcriptional regulation of a large number of genes upon pathogen infection or treatment with pathogen elicitors. A large body of evidence suggests that plant WRKY transcription factors are involved in plant defense including transcriptional regulation of plant host genes in response to pathogen infection. However, there is only limited information about the roles of specific WRKY DNA-binding transcription factors in plant defense.
Results
We analyzed the role of the WRKY25 transcription factor from Arabidopsis in plant defense against the bacterial pathogen Pseudomonas syringae. WRKY25 protein recognizes the TTGACC W-box sequences and its translational fusion with green fluorescent protein is localized to the nucleus. WRKY25 expression is responsive to general environmental stress. Analysis of stress-induced WRKY25 in the defense signaling mutants npr1, sid2, ein2 and coi1 further indicated that this gene is positively regulated by the salicylic acid (SA) signaling pathway and negatively regulated by the jasmonic acid signaling pathway. Two independent T-DNA insertion mutants for WRKY25 supported normal growth of a virulent strain of P. syringae but developed reduced disease symptoms after infection. By contrast, Arabidopsis constitutively overexpressing WRKY25 supported enhanced growth of P. syringae and displayed increased disease symptom severity as compared to wild-type plants. These WRKY25-overexpressing plants also displayed reduced expression of the SA-regulated PR1 gene after the pathogen infection, despite normal levels of free SA.
Conclusion
The nuclear localization and sequence-specific DNA-binding activity support that WRKY25 functions as a transcription factor. Based on analysis of both T-DNA insertion mutants and transgenic overexpression lines, stress-induced WRKY25 functions as a negative regulator of SA-mediated defense responses to P. syringae. This proposed role is consistent with the recent finding that WRKY25 is a substrate of Arabidopsis MAP kinase 4, a repressor of SA-dependent defense responses.
doi:10.1186/1471-2229-7-2
PMCID: PMC1780049  PMID: 17214894
10.  Ectopic Expression of a WRKY Homolog from Glycine soja Alters Flowering Time in Arabidopsis 
PLoS ONE  2013;8(8):e73295.
Flowering is a critical event in the life cycle of plants; the WRKY-type transcription factors are reported to be involved in many developmental processes sunch as trichome development and epicuticular wax loading, but whether they are involved in flowering time regulation is still unknown. Within this study, we provide clear evidence that GsWRKY20, a member of WRKY gene family from wild soybean, is involved in controlling plant flowering time. Expression of GsWRKY20 was abundant in the shoot tips and inflorescence meristems of wild soybean. Phenotypic analysis showed that GsWRKY20 over-expression lines flowered earlier than the wild-type plants under all conditions: long-day and short-day photoperiods, vernalization, or exogenous GA3 application, indicating that GsWRKY20 may mainly be involved in an autonomous flowering pathway. Further analyses by qRT-PCR and microarray suggests that GsWRKY20 accelerating plant flowering might primarily be through the regulation of flowering-related genes (i.e., FLC, FT, SOC1 and CO) and floral meristem identity genes (i.e., AP1, SEP3, AP3, PI and AG). Our results provide the evidence demonstrating the effectiveness of manipulating GsWRKY20 for altering plant flowering time.
doi:10.1371/journal.pone.0073295
PMCID: PMC3753250  PMID: 23991184
11.  Molecular Phylogenetic and Expression Analysis of the Complete WRKY Transcription Factor Family in Maize 
The WRKY transcription factors function in plant growth and development, and response to the biotic and abiotic stresses. Although many studies have focused on the functional identification of the WRKY transcription factors, much less is known about molecular phylogenetic and global expression analysis of the complete WRKY family in maize. In this study, we identified 136 WRKY proteins coded by 119 genes in the B73 inbred line from the complete genome and named them in an orderly manner. Then, a comprehensive phylogenetic analysis of five species was performed to explore the origin and evolutionary patterns of these WRKY genes, and the result showed that gene duplication is the major driving force for the origin of new groups and subgroups and functional divergence during evolution. Chromosomal location analysis of maize WRKY genes indicated that 20 gene clusters are distributed unevenly in the genome. Microarray-based expression analysis has revealed that 131 WRKY transcripts encoded by 116 genes may participate in the regulation of maize growth and development. Among them, 102 transcripts are stably expressed with a coefficient of variation (CV) value of <15%. The remaining 29 transcripts produced by 25 WRKY genes with the CV value of >15% are further analysed to discover new organ- or tissue-specific genes. In addition, microarray analyses of transcriptional responses to drought stress and fungal infection showed that maize WRKY proteins are involved in stress responses. All these results contribute to a deep probing into the roles of WRKY transcription factors in maize growth and development and stress tolerance.
doi:10.1093/dnares/dsr048
PMCID: PMC3325079  PMID: 22279089
WRKY transcription factor; maize; phylogenetic analysis; expression profile; development
12.  The large soybean (Glycine max) WRKY TF family expanded by segmental duplication events and subsequent divergent selection among subgroups 
BMC Plant Biology  2013;13:148.
Background
WRKY genes encode one of the most abundant groups of transcription factors in higher plants, and its members regulate important biological process such as growth, development, and responses to biotic and abiotic stresses. Although the soybean genome sequence has been published, functional studies on soybean genes still lag behind those of other species.
Results
We identified a total of 133 WRKY members in the soybean genome. According to structural features of their encoded proteins and to the phylogenetic tree, the soybean WRKY family could be classified into three groups (groups I, II, and III). A majority of WRKY genes (76.7%; 102 of 133) were segmentally duplicated and 13.5% (18 of 133) of the genes were tandemly duplicated. This pattern was not apparent in Arabidopsis or rice. The transcriptome atlas revealed notable differential expression in either transcript abundance or in expression patterns under normal growth conditions, which indicated wide functional divergence in this family. Furthermore, some critical amino acids were detected using DIVERGE v2.0 in specific comparisons, suggesting that these sites have contributed to functional divergence among groups or subgroups. In addition, site model and branch-site model analyses of positive Darwinian selection (PDS) showed that different selection regimes could have affected the evolution of these groups. Sites with high probabilities of having been under PDS were found in groups I, II c, II e, and III. Together, these results contribute to a detailed understanding of the molecular evolution of the WRKY gene family in soybean.
Conclusions
In this work, all the WRKY genes, which were generated mainly through segmental duplication, were identified in the soybean genome. Moreover, differential expression and functional divergence of the duplicated WRKY genes were two major features of this family throughout their evolutionary history. Positive selection analysis revealed that the different groups have different evolutionary rates. Together, these results contribute to a detailed understanding of the molecular evolution of the WRKY gene family in soybean.
doi:10.1186/1471-2229-13-148
PMCID: PMC3850935  PMID: 24088323
13.  Male gametophyte-specific WRKY34 transcription factor mediates cold sensitivity of mature pollen in Arabidopsis 
Journal of Experimental Botany  2010;61(14):3901-3914.
Mature pollen is very sensitive to cold stress in chilling-sensitive plants. Plant WRKY DNA-binding transcription factors are key regulators in plant responses to abiotic and biotic stresses. Previous studies have suggested that WRKY34 (At4g26440) gene might be involved in pollen viability, although the mechanism involved is unclear. In this study, it is shown that cold treatment increased WRKY34 expression in the wild type, and promoter-GUS analysis revealed that WRKY34 expression is pollen-specific. Enhanced green fluorescent protein-tagged WRKY34 was localized in the nuclei. Pollen harbouring the wrky34 allele showed higher viability than pollen with the WRKY34 allele after cold treatment. Further functional analysis indicated that the WRKY34 transcription factor was involved in pollen development regulated by the pollen-specific MIKC* class of MADS-domain transcription factors under cold stress, and cold-insensitivity of mature wrky34 pollen might be partly attributable to the enhanced expression of transcriptional activator CBFs in the mutants. Thus, the WRKY34 transcription factor negatively mediated cold sensitivity of mature Arabidopsis pollen and might be involved in the CBF signal cascade in mature pollen.
doi:10.1093/jxb/erq204
PMCID: PMC2935866  PMID: 20643804
Arabidopsis; cold stress; pollen; transcription factor; WRKY34
14.  Exploring transcriptional signalling mediated by OsWRKY13, a potential regulator of multiple physiological processes in rice 
BMC Plant Biology  2009;9:74.
Background
Rice transcription regulator OsWRKY13 influences the functioning of more than 500 genes in multiple signalling pathways, with roles in disease resistance, redox homeostasis, abiotic stress responses, and development.
Results
To determine the putative transcriptional regulation mechanism of OsWRKY13, the putative cis-acting elements of OsWRKY13-influenced genes were analyzed using the whole genome expression profiling of OsWRKY13-activated plants generated with the Affymetrix GeneChip Rice Genome Array. At least 39 transcription factor genes were influenced by OsWRKY13, and 30 of them were downregulated. The promoters of OsWRKY13-upregulated genes were overrepresented with W-boxes for WRKY protein binding, whereas the promoters of OsWRKY13-downregulated genes were enriched with cis-elements putatively for binding of MYB and AP2/EREBP types of transcription factors. Consistent with the distinctive distribution of these cis-elements in up- and downregulated genes, nine WRKY genes were influenced by OsWRKY13 and the promoters of five of them were bound by OsWRKY13 in vitro; all seven differentially expressed AP2/EREBP genes and six of the seven differentially expressed MYB genes were suppressed by in OsWRKY13-activated plants. A subset of OsWRKY13-influenced WRKY genes were involved in host-pathogen interactions.
Conclusion
These results suggest that OsWRKY13-mediated signalling pathways are partitioned by different transcription factors. WRKY proteins may play important roles in the monitoring of OsWRKY13-upregulated genes and genes involved in pathogen-induced defence responses, whereas MYB and AP2/EREBP proteins may contribute most to the control of OsWRKY13-downregulated genes.
doi:10.1186/1471-2229-9-74
PMCID: PMC3224702  PMID: 19534828
15.  Ectopic Expression of a Wheat WRKY Transcription Factor Gene TaWRKY71-1 Results in Hyponastic Leaves in Arabidopsis thaliana 
PLoS ONE  2013;8(5):e63033.
Leaf type is an important trait that closely associates with crop yield. WRKY transcription factors exert diverse regulatory effects in plants, but their roles in the determination of leaf type have not been reported so far. In this work, we isolated a WRKY transcription factor gene TaWRKY71-1 from a wheat introgression line SR3, which has larger leaves, superior growth capacity and higher yield than its parent common wheat JN177. TaWRKY71-1 specifically expressed in leaves, and produced more mRNA in SR3 than in JN177. TaWRKY71-1 localized in the nucleus and had no transcriptional activation activity. TaWRKY71-1 overexpression in Arabidopsis resulted in hyponastic rosette leaves, and the hyponastic strength was closely correlative with the transcription level of the transgene. The spongy mesophyll cells at abaxial side of leaves were drastically compacted by TaWRKY71-1 overexpression. In TaWRKY71-1 overexpression Arabidopsis, the expression of IAMT1 that encodes a methyltransferase converting free indole-3-acetic acid (IAA) to methyl-IAA ester (MeIAA) to alter auxin homeostatic level was induced, and the induction level was dependent on the abundance of TaWRKY71-1 transcripts. Besides, several TCP genes that had found to be restricted by IAMT1 had lower expression levels as well. Our results suggest that TaWRKY71-1 causes hyponastic leaves through altering auxin homeostatic level by promoting the conversion of IAA to MeIAA.
doi:10.1371/journal.pone.0063033
PMCID: PMC3650005  PMID: 23671653
16.  MusaWRKY71 Overexpression in Banana Plants Leads to Altered Abiotic and Biotic Stress Responses 
PLoS ONE  2013;8(10):e75506.
WRKY transcription factors are specifically involved in the transcriptional reprogramming following incidence of abiotic or biotic stress on plants. We have previously documented a novel WRKY gene from banana, MusaWRKY71, which was inducible in response to a wide array of abiotic or biotic stress stimuli. The present work details the effects of MusaWRKY71 overexpression in transgenic banana plants. Stable integration and overexpression of MusaWRKY71 in transgenic banana plants was proved by Southern blot analysis and quantitative real time PCR. Transgenic banana plants overexpressing MusaWRKY71 displayed enhanced tolerance towards oxidative and salt stress as indicated by better photosynthesis efficiency (Fv/Fm) and lower membrane damage of the assayed leaves. Further, differential regulation of putative downstream genes of MusaWRKY71 was investigated using real-time RT-PCR expression analysis. Out of a total of 122 genes belonging to WRKY, pathogenesis-related (PR) protein genes, non-expressor of pathogenesis-related genes 1 (NPR1) and chitinase families analyzed, 10 genes (six belonging to WRKY family, three belonging to PR proteins family and one belonging to chitinase family) showed significant differential regulation in MusaWRKY71 overexpressing lines. These results indicate that MusaWRKY71 is an important constituent in the transcriptional reprogramming involved in diverse stress responses in banana.
doi:10.1371/journal.pone.0075506
PMCID: PMC3792942  PMID: 24116051
17.  Cooperation of three WRKY-domain transcription factors WRKY18, WRKY40, and WRKY60 in repressing two ABA-responsive genes ABI4 and ABI5 in Arabidopsis  
Journal of Experimental Botany  2012;63(18):6371-6392.
Three evolutionarily closely related WRKY-domain transcription factors WRKY18, WRKY40, and WRKY60 in Arabidopsis were previously identified as negative abscisic acid (ABA) signalling regulators, of which WRKY40 regulates ABI4 and ABI5 expression, but it remains unclear whether and how the three transcription factors cooperate to regulate expression of ABI4 and ABI5. In the present experiments, it was shown that WRKY18 and WRKY60, like WRKY40, interact with the W-box in the promoters of ABI4 and ABI5 genes, though the three WRKYs have their own preferential binding domains in the two promoters. WRKY18 and WRKY60, together with WRKY40, inhibit expression of the ABI5 and/or ABI4 genes, which is consistent with their negative roles in ABA signalling. Further, genetic evidence is provided that mutations of ABI4 and ABI5 genes suppress ABA-hypersensitive phenotypes of the null mutant alleles of WRKY18 and WRKY60 genes, demonstrating that ABI4 and ABI5 function downstream of these two WRKY transcription factors in ABA signalling. A working model of cooperation of the three WRKYs in repressing ABI4 and ABI5 expression is proposed, in which the three WRKYs antagonize or aid each other in a highly complex manner. These findings help to understand the complex mechanisms of WRKY-mediated ABA signal transduction.
doi:10.1093/jxb/ers293
PMCID: PMC3504491  PMID: 23095997
ABA-responsive gene; ABA signalling; ABI4; ABI5; Arabidopsis thaliana; WRKY transcription factor; WRKY18; WRKY40; WRKY60
18.  Elucidating the evolutionary conserved DNA-binding specificities of WRKY transcription factors by molecular dynamics and in vitro binding assays 
Nucleic Acids Research  2013;41(21):9764-9778.
WRKY transcription factors constitute a large protein family in plants that is involved in the regulation of developmental processes and responses to biotic or abiotic stimuli. The question arises how stimulus-specific responses are mediated given that the highly conserved WRKY DNA-binding domain (DBD) exclusively recognizes the ‘TTGACY’ W-box consensus. We speculated that the W-box consensus might be more degenerate and yet undetected differences in the W-box consensus of WRKYs of different evolutionary descent exist. The phylogenetic analysis of WRKY DBDs suggests that they evolved from an ancestral group IIc-like WRKY early in the eukaryote lineage. A direct descent of group IIc WRKYs supports a monophyletic origin of all other group II and III WRKYs from group I by loss of an N-terminal DBD. Group I WRKYs are of paraphyletic descent and evolved multiple times independently. By homology modeling, molecular dynamics simulations and in vitro DNA–protein interaction-enzyme-linked immunosorbent assay with AtWRKY50 (IIc), AtWRKY33 (I) and AtWRKY11 (IId) DBDs, we revealed differences in DNA-binding specificities. Our data imply that other components are essentially required besides the W-box-specific binding to DNA to facilitate a stimulus-specific WRKY function.
doi:10.1093/nar/gkt732
PMCID: PMC3834811  PMID: 23975197
19.  Genome-wide identification of WRKY45-regulated genes that mediate benzothiadiazole-induced defense responses in rice 
BMC Plant Biology  2013;13:150.
Background
The rice transcription factor WRKY45 plays a crucial role in salicylic acid (SA)/benzothiadiazole (BTH)-induced disease resistance. Its knockdown severely reduces BTH-induced resistance to the fungal pathogen Magnaporthe oryzae and the bacterial pathogen Xanthomonas oryzae pv. oryzae (Xoo). Conversely, overexpression of WRKY45 induces extremely strong resistance to both of these pathogens. To elucidate the molecular basis of WRKY45-dependent disease resistance, we analyzed WRKY45-regulated gene expression using rice transformants and a transient gene expression system.
Results
We conducted a microarray analysis using WRKY45-knockdown (WRKY45-kd) rice plants, and identified WRKY45-dependent genes among the BTH-responsive genes. The BTH-responsiveness of 260 genes was dependent on WRKY45. Among these, 220 genes (85%), many of which encoded PR proteins and proteins associated with secondary metabolism, were upregulated by BTH. Only a small portion of these genes overlapped with those regulated by OsNPR1/NH1, supporting the idea that the rice SA pathway branches into WRKY45- regulated and OsNPR1/NH1-regulated subpathways. Dexamethazone-induced expression of myc-tagged WRKY45 in rice immediately upregulated transcription of endogenous WRKY45 and genes encoding the transcription factors WRKY62, OsNAC4, and HSF1, all of which have been reported to have defense-related functions. This was followed by upregulation of defense genes encoding PR proteins and secondary metabolic enzymes. Many of these genes were also induced after M. oryzae infection. Their temporal transcription patterns were consistent with those after dexamethazone-induced WRKY45 expression. In a transient expression system consisting of particle bombardment of rice coleoptiles, WRKY45 acted as an effector to trans-activate reporter genes in which the luciferase coding sequence was fused to upstream and intragenic sequences of WRKY62 and OsNAC4. Trans-activation of transcription occurred through a W-box-containing sequence upstream of OsNAC4 and mutations in the W-boxes abolished the trans-activation.
Conclusions
These data suggest a role of WRKY45 in BTH-induced disease resistance as a master regulator of the transcriptional cascade regulating defense responses in one of two branches in the rice SA pathway.
doi:10.1186/1471-2229-13-150
PMCID: PMC3850545  PMID: 24093634
WRKY; Salicylic acid; Benzothiadiazole; Magnaporthe oryzae; OsNPR1
20.  Cross activity of orthologous WRKY transcription factors in wheat and Arabidopsis 
Journal of Experimental Botany  2010;62(6):1975-1990.
WRKY proteins are transcription factors involved in many plant processes including plant responses to pathogens. Here, the cross activity of TaWRKY78 from the monocot wheat and AtWRKY20 from the dicot Arabidopsis on the cognate promoters of the orthologous PR4-type genes wPR4e and AtHEL of wheat and Arabidopsis, respectively, was investigated. In vitro analysis showed the ability of TaWRKY78 to bind a –17/+80 region of the wPR4e promoter, containing one cis-acting W-box. Moreover, transient expression analysis performed on both TaWRKY78 and AtWRKY20 showed their ability to recognize the cognate cis-acting elements present in the wPR4e and AtHEL promoters, respectively. Finally, this paper provides evidence that both transcription factors are able to cross-regulate the orthologous PR4 genes with an efficiency slightly lower than that exerted on the cognate promoters. The observation that orthologous genes are subjected to similar transcriptional control by orthologous transcription factors demonstrates that the terminal stages of signal transduction pathways leading to defence are conserved and suggests a fundamental role of PR4 genes in plant defence. Moreover, these results corroborate the hypothesis that gene orthology imply similar gene function and that diversification between monocot and dicot has most likely occurred after the specialization of WRKY function.
doi:10.1093/jxb/erq396
PMCID: PMC3060686  PMID: 21193575
Arabidopsis thaliana; gene expression; gene orthology; PR4 genes; transient expression analysis; transcription; regulation; Triticum aestivum
21.  Roles of Arabidopsis WRKY3 and WRKY4 Transcription Factors in Plant Responses to Pathogens 
BMC Plant Biology  2008;8:68.
Background
Plant WRKY DNA-binding transcription factors are involved in plant responses to biotic and abiotic responses. It has been previously shown that Arabidopsis WRKY3 and WRKY4, which encode two structurally similar WRKY transcription factors, are induced by pathogen infection and salicylic acid (SA). However, the role of the two WRKY transcription factors in plant disease resistance has not been directly analyzed.
Results
Both WRKY3 and WRKY4 are nuclear-localized and specifically recognize the TTGACC W-box sequences in vitro. Expression of WRKY3 and WRKY4 was induced rapidly by stress conditions generated by liquid infiltration or spraying. Stress-induced expression of WRKY4 was further elevated by pathogen infection and SA treatment. To determine directly their role in plant disease resistance, we have isolated T-DNA insertion mutants and generated transgenic overexpression lines for WRKY3 and WRKY4. Both the loss-of-function mutants and transgenic overexpression lines were examined for responses to the biotrophic bacterial pathogen Pseudomonas syringae and the necrotrophic fungal pathogen Botrytis cinerea. The wrky3 and wrky4 single and double mutants exhibited more severe disease symptoms and support higher fungal growth than wild-type plants after Botrytis infection. Although disruption of WRKY3 and WRKY4 did not have a major effect on plant response to P. syringae, overexpression of WRKY4 greatly enhanced plant susceptibility to the bacterial pathogen and suppressed pathogen-induced PR1 gene expression.
Conclusion
The nuclear localization and sequence-specific DNA-binding activity support that WRKY3 and WRKY4 function as transcription factors. Functional analysis based on T-DNA insertion mutants and transgenic overexpression lines indicates that WRKY3 and WRKY4 have a positive role in plant resistance to necrotrophic pathogens and WRKY4 has a negative effect on plant resistance to biotrophic pathogens.
doi:10.1186/1471-2229-8-68
PMCID: PMC2464603  PMID: 18570649
22.  The WRKY transcription factor family in Brachypodium distachyon 
BMC Genomics  2012;13:270.
Background
A complete assembled genome sequence of wheat is not yet available. Therefore, model plant systems for wheat are very valuable. Brachypodium distachyon (Brachypodium) is such a system. The WRKY family of transcription factors is one of the most important families of plant transcriptional regulators with members regulating important agronomic traits. Studies of WRKY transcription factors in Brachypodium and wheat therefore promise to lead to new strategies for wheat improvement.
Results
We have identified and manually curated the WRKY transcription factor family from Brachypodium using a pipeline designed to identify all potential WRKY genes. 86 WRKY transcription factors were found, a total higher than all other current databases. We therefore propose that our numbering system (BdWRKY1-BdWRKY86) becomes the standard nomenclature. In the JGI v1.0 assembly of Brachypodium with the MIPS/JGI v1.0 annotation, nine of the transcription factors have no gene model and eleven gene models are probably incorrectly predicted. In total, twenty WRKY transcription factors (23.3%) do not appear to have accurate gene models. To facilitate use of our data, we have produced The Database of Brachypodium distachyon WRKY Transcription Factors. Each WRKY transcription factor has a gene page that includes predicted protein domains from MEME analyses. These conserved protein domains reflect possible input and output domains in signaling. The database also contains a BLAST search function where a large dataset of WRKY transcription factors, published genes, and an extensive set of wheat ESTs can be searched. We also produced a phylogram containing the WRKY transcription factor families from Brachypodium, rice, Arabidopsis, soybean, and Physcomitrella patens, together with published WRKY transcription factors from wheat. This phylogenetic tree provides evidence for orthologues, co-orthologues, and paralogues of Brachypodium WRKY transcription factors.
Conclusions
The description of the WRKY transcription factor family in Brachypodium that we report here provides a framework for functional genomics studies in an important model system. Our database is a resource for both Brachypodium and wheat studies and ultimately projects aimed at improving wheat through manipulation of WRKY transcription factors.
doi:10.1186/1471-2164-13-270
PMCID: PMC3583182  PMID: 22726208
WRKY transcription factor; Brachypodium distachyon; Wheat; Comparative genomics; Database
23.  Arabidopsis WRKY2 transcription factor mediates seed germination and postgermination arrest of development by abscisic acid 
BMC Plant Biology  2009;9:96.
Background
Plant WRKY DNA-binding transcription factors are key regulators in certain developmental programs. A number of studies have suggested that WRKY genes may mediate seed germination and postgermination growth. However, it is unclear whether WRKY genes mediate ABA-dependent seed germination and postgermination growth arrest.
Results
To determine directly the role of Arabidopsis WRKY2 transcription factor during ABA-dependent seed germination and postgermination growth arrest, we isolated T-DNA insertion mutants. Two independent T-DNA insertion mutants for WRKY2 were hypersensitive to ABA responses only during seed germination and postgermination early growth. wrky2 mutants displayed delayed or decreased expression of ABI5 and ABI3, but increased or prolonged expression of Em1 and Em6. wrky2 mutants and wild type showed similar levels of expression for miR159 and its target genes MYB33 and MYB101. Analysis of WRKY2 expression level in ABA-insensitive and ABA-deficient mutants abi5-1, abi3-1, aba2-3 and aba3-1 further indicated that ABA-induced WRKY2 accumulation during germination and postgermination early growth requires ABI5, ABI3, ABA2 and ABA3.
Conclusion
ABA hypersensitivity of the wrky2 mutants during seed germination and postgermination early seedling establishment is attributable to elevated mRNA levels of ABI5, ABI3 and ABI5-induced Em1 and Em6 in the mutants. WRKY2-mediated ABA responses are independent of miR159 and its target genes MYB33 and MYB101. ABI5, ABI3, ABA2 and ABA3 are important regulators of the transcripts of WRKY2 by ABA treatment. Our results suggest that WRKY2 transcription factor mediates seed germination and postgermination developmental arrest by ABA.
doi:10.1186/1471-2229-9-96
PMCID: PMC2719644  PMID: 19622176
24.  DNA binding mechanism revealed by high resolution crystal structure of Arabidopsis thaliana WRKY1 protein 
Nucleic Acids Research  2007;35(4):1145-1154.
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.
doi:10.1093/nar/gkm001
PMCID: PMC1851648  PMID: 17264121
25.  WRKY76 is a rice transcriptional repressor playing opposite roles in blast disease resistance and cold stress tolerance 
Journal of Experimental Botany  2013;64(16):5085-5097.
OsWRKY76 encodes a group IIa WRKY transcription factor of rice. The expression of OsWRKY76 was induced within 48h after inoculation with rice blast fungus (Magnaporthe oryzae), and by wounding, low temperature, benzothiadiazole, and abscisic acid. Green fluorescent protein-fused OsWRKY76 localized to the nuclei in rice epidermal cells. OsWRKY76 showed sequence-specific DNA binding to the W-box element in vitro and exhibited W-box-mediated transcriptional repressor activity in cultured rice cells. Overexpression of OsWRKY76 in rice plants resulted in drastically increased susceptibility to M. oryzae, but improved tolerance to cold stress. Microarray analysis revealed that overexpression of OsWRKY76 suppresses the induction of a specific set of PR genes and of genes involved in phytoalexin synthesis after inoculation with blast fungus, consistent with the observation that the levels of phytoalexins in the transgenic rice plants remained significantly lower than those in non-transformed control plants. Furthermore, overexpression of OsWRKY76 led to the increased expression of abiotic stress-associated genes such as peroxidase and lipid metabolism genes. These results strongly suggest that OsWRKY76 plays dual and opposing roles in blast disease resistance and cold tolerance.
doi:10.1093/jxb/ert298
PMCID: PMC3830488  PMID: 24043853
Blast disease resistance; cold stress; phytoalexin; rice; transcriptional repressor; WRKY.

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