Conserved WRKY Domain Research Articles

Identification of WRKY Gene Family from Dimocarpus longan and Its Expression Analysis during Flower Induction and Abiotic Stress Responses

Longan is an important fruit tree in the subtropical region of Southeast Asia and Australia. However, its blooming and its yield are susceptible to stresses such as droughts, high salinity, and high and low temperature. To date, the molecular mechanisms of abiotic stress tolerance and flower induction in longan have not been elucidated. WRKY transcription factors (TFs), which have been studied in various plant species, play important regulatory roles in plant growth, development, and responses to stresses. However, there is no report about WRKYs in longan. In this study, we identified 55 WRKY genes with the conserved WRKY domain and zinc finger motif in the longan genome. Based on the structural features of WRKY proteins and topology of the phylogenetic tree, the longan WRKY (DlWRKY) family was classified into three major groups (I–III) and five subgroups (IIa–IIe) in group II. Tissue expression analysis showed that 25 DlWRKYs were highly expressed in almost all organs, suggesting that these genes may be important for plant growth and organ development in longan. Comparative RNA-seq and qRT-PCR-based gene expression analysis revealed that 18 DlWRKY genes showed a specific expression during three stages of flower induction in “Sijimi” (“SJ”), which exhibited the “perpetual flowering” (PF) habit, indicating that these 18 DlWRKY genes may be involved in the flower induction and the genetic control of the perpetual flowering trait in longan. Furthermore, the RT-qPCR analysis illustrated the significant variation of 27, 18, 15, 17, 27, and 23 DlWRKY genes under SA (Salicylic acid), MeJA (Methyl Jasmonate), heat, cold, drought, or high salinity treatment, respectively, implicating that they might be stress- or hormone-responsive genes. In summary, we systematically and comprehensively analyzed the structure, evolution, and expression pattern of the DlWRKY genes. The results presented here increase our understanding of the WRKY family in fruit trees and provide a basis for the further elucidation of the biological function of DlWRKY genes in longan.

GmWRKY49, a Salt-Responsive Nuclear Protein, Improved Root Length and Governed Better Salinity Tolerance in Transgenic Arabidopsis.

Plant WRKY transcription factors (TFs) are active guardians against pathogens’ insurgency, key components in developmental processes, contributors in signal transduction pathways, and regulators of diverse biotic and abiotic stress responses. In this research, we isolated, cloned, and functionally characterized a new WRKY TF GmWRKY49 from soybean. GmWRKY49 is a nuclear protein which contains two highly conserved WRKY domains and a C2H2-type zinc-finger structure. The normalized expression (log2 ratio) of GmWRKY49 was 2.75- and 1.90-fold in salt-tolerant and salt-susceptible soybean genotypes, respectively. The transcripts of GmWRKY49 could be detected in roots, stems, leaves, flowers, and almost no expression in pod tissues. The salinity-tolerance response of this gene was studied through overexpression in soybean composite seedlings and transgenic Arabidopsis. The effect of GmWRKY49 overexpression on root length of transgenic Arabidopsis was also investigated. Under salt stress, several parameters including germination rate, survival rate, root length, rosette diameter, relative electrolyte leakage, and proline content were significantly higher in composite seedlings and transgenic Arabidopsis than those in wild-type. Moreover, GmWRKY49 enhanced salinity tolerance in soybean mosaic seedlings and transgenic Arabidopsis. These results suggest that GmWRKY49 is a positive regulator of salinity tolerance in soybean and has high potential utilization for crop improvement.

Transcriptome wide identification and characterization of NO-responsive WRKY transcription factors in Arabidopsis thaliana L.

WRKY transcription factors are important plant-specific regulatory genes characterized by one or two conserved WRKY domain(s) usually followed by a zinc-finger motif. In this study using Arabidopsis thaliana, the RNA-Seq based transcriptomic analysis showed differential expression of 33 genes encoding WRKY TFs in response to the nitric oxide (NO) donor S-Nitrosocysteine (CySNO). Interestingly, 93.9% of these TFs were up-regulated with at least 2-fold change, suggesting their putative involvement in NO mediated gene regulation. GO- analysis of all the 33 transcriptomic elements showed their putative involvement in biological processes such as abiotic stress tolerance and defense against fungal pathogens (89.39 fold enrichment). Analysis of the NO-responsive AtWRKY TFs promoter region revealed the presence of the cis-acting elements such as ABRE, EIRE, ERE, and MBS involved in osmotic stress response, maximal elicitor-mediated activation, and drought-stress regulation. The analysis of NO-responsive AtWRKY TF motifs and their comparison with rice, soybean, and tomato orthologs suggested that members of the WRKY family belonging to the same group shared similar motifs and phylogenetic tree suggested that these TFs were highly conserved. Validation of transcriptomic data through quantitative real time-PCR showed a high correlation coefficient (0.85) indicating the high reliability and similarity of both types of analysis. Comparison of the NO-responsive and non-responsive WRKYs showed the presence of tyrosine (T) and cysteine (C) residues at a distance of 7 residues from the WRKYGQK motif which may serve as potential targets for modification by NO via tyrosine nitration and S-nitrosylation. We also validated the response of WRKYs through in vivo analysis using atwrky62 loss of function mutant and the results indicated a negative role of AtWRKY62 in plant growth. Furthermore, atwrky62 showed significantly less SNO contents compared to wild type plants indicating putative role of AtWRKY62 in NO metabolism.

Genome-wide identification of WRKY transcription factors in kiwifruit (Actinidia spp.) and analysis of WRKY expression in responses to biotic and abiotic stresses.

As one of the largest transcriptional factor families in plants, WRKY transcription factors play important roles in various biotic and abiotic stress responses. To date, WRKY genes in kiwifruit (Actinidia spp.) remain poorly understood. In our study, o total of 97 AcWRKY genes have been identified in the kiwifruit genome. An overview of these AcWRKY genes is analyzed, including the phylogenetic relationships, exon-intron structures, synteny and expression profiles. The 97 AcWRKY genes were divided into three groups based on the conserved WRKY domain. Synteny analysis indicated that segmental duplication events contributed to the expansion of the kiwifruit AcWRKY family. In addition, the synteny analysis between kiwifruit and Arabidopsis suggested that some of the AcWRKY genes were derived from common ancestors before the divergence of these two species. Conserved motifs outside the AcWRKY domain may reflect their functional conservation. Genome-wide segmental and tandem duplication were found, which may contribute to the expansion of AcWRKY genes. Furthermore, the analysis of selected AcWRKY genes showed a variety of expression patterns in five different organs as well as during biotic and abiotic stresses. The genome-wide identification and characterization of kiwifruit WRKY transcription factors provides insight into the evolutionary history and is a useful resource for further functional analyses of kiwifruit.

Genome-wide analysis of WRKY transcription factors and their response to abiotic stress in celery (Apium graveolens L.)

ABSTRACTCelery (Apium graveolens L.) is rich in nutrient substances and is cultivated worldwide. WRKY protein family is one of the largest transcription factor families in plants and is involved in growth and development, signal transduction, senescence and stress resistance. In this study, we identified 69 celery WRKY family transcription factors based on conserved WRKY domain sequence from the celery genomic and transcriptomic data. Phylogenetic analysis of celery and Arabidopsis WRKY proteins divided these celery WRKY proteins into three groups, seven subgroups. The numbers of WRKY proteins in celery and other 16 species were compared to illustrate the evolution of WRKY transcription factors in plants. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to analyse the expression profiles of five selected AgWRKY genes under abiotic stress (abscisic acid, cold, drought and salt) treatments. The qRT-PCR results indicated that those selected AgWRKY family genes were upregulated under abiotic stress treatments. This study provides a basic research for further investigation of the structure and function of AgWRKY genes.

Overexpression of DgWRKY4 Enhances Salt Tolerance in Chrysanthemum Seedlings.

High salinity seriously affects the production of chrysanthemum, so improving the salt tolerance of chrysanthemum becomes the focus and purpose of our research. The WRKY transcription factor (TF) family is highly associated with a number of processes of abiotic stress responses. We isolated DgWRKY4 from Dendranthema grandiflorum, and a protein encoded by this new gene contains two highly conserved WRKY domains and two C2H2 zinc-finger motifs. Then, we functionally characterized that DgWRKY4 was induced by salt, and DgWRKY4 overexpression in chrysanthemum resulted in increased tolerance to high salt stress compared to wild-type (WT). Under salt stress, the transgenic chrysanthemum accumulated less malondialdehyde, hydrogen peroxide (H2O2), and superoxide anion () than WT, accompanied by more proline, soluble sugar, and activities of antioxidant enzymes than WT; in addition, a stronger photosynthetic capacity and a series of up-regulated stress-related genes were also found in transgenic chrysanthemum. All results demonstrated that DgWRKY4 is a positive regulatory gene responding to salt stress, via advancing photosynthetic capacity, promoting the operation of reactive oxygen species-scavenging system, maintaining membrane stability, enhancing the osmotic adjustment, and up-regulating transcript levels of stress-related genes. So, DgWRKY4 can serve as a new candidate gene for salt-tolerant plant breeding.

Genome-Wide Identification and Expression Analysis of the WRKY Gene Family in Cassava.

The WRKY family, a large family of transcription factors (TFs) found in higher plants, plays central roles in many aspects of physiological processes and adaption to environment. However, little information is available regarding the WRKY family in cassava (Manihot esculenta). In the present study, 85 WRKY genes were identified from the cassava genome and classified into three groups according to conserved WRKY domains and zinc-finger structure. Conserved motif analysis showed that all of the identified MeWRKYs had the conserved WRKY domain. Gene structure analysis suggested that the number of introns in MeWRKY genes varied from 1 to 5, with the majority of MeWRKY genes containing three exons. Expression profiles of MeWRKY genes in different tissues and in response to drought stress were analyzed using the RNA-seq technique. The results showed that 72 MeWRKY genes had differential expression in their transcript abundance and 78 MeWRKY genes were differentially expressed in response to drought stresses in different accessions, indicating their contribution to plant developmental processes and drought stress resistance in cassava. Finally, the expression of 9 WRKY genes was analyzed by qRT-PCR under osmotic, salt, ABA, H2O2, and cold treatments, indicating that MeWRKYs may be involved in different signaling pathways. Taken together, this systematic analysis identifies some tissue-specific and abiotic stress-responsive candidate MeWRKY genes for further functional assays in planta, and provides a solid foundation for understanding of abiotic stress responses and signal transduction mediated by WRKYs in cassava.

Cloning and Functional Analysis of a WRKY28-like Gene in Soybean

WRKY transcription factors play important roles in regulating plant growth and development, and participate in plant response to abiotic and biotic stresses. Arabidopsis WRKY28 is an essential transcription factor for plant response to necrotrophic pathogen and abiotic stress. To explore the function of the homologues gene of AtWRKY28 in soybean, we cloned GmWRKY28- like (Glyma.14G028900) and performed bioinformatics analysis. Its sub-cellular localization and tissue expression patterns were further investigated. We also analyzed the expression levels of GmWRKY28-like under ABA, PEG, and NaCl stress treatments. Our results showed that the coding DNA sequence (CDS) of GmWRKY28-like gene is 1008 bp in length, encoding 335 amino acids. GmWRKY28-like contains a conserved WRKY domain with 22 serine, one threonine and two tyrosine, without any trans- membrane domain or signal peptide. Phylogenetic analysis showed that the WRKY28 from Phaseolus vulgaris was most similar to GmWRKY28-like. GmWRKY28-like was verified to be located in the nucleus. This gene expressed at low levels in root and seed while high levels in true leaf, flower and shoot apical meristem. The 1500 bp upstream region of GmWRKY28-like contains a

Genome-wide analysis of WRKY transcription factors in white pear (Pyrus bretschneideri) reveals evolution and patterns under drought stress.

BackgroundWRKY transcription factors (TFs) constitute one of the largest protein families in higher plants, and its members contain one or two conserved WRKY domains, about 60 amino acid residues with the WRKYGQK sequence followed by a C2H2 or C2HC zinc finger motif. WRKY proteins play significant roles in plant development, and in responses to biotic and abiotic stresses. Pear (Pyrus bretschneideri) is one of the most important fruit crops in the world and is frequently threatened by abiotic stress, such as drought, affecting growth, development and productivity. Although the pear genome sequence has been released, little is known about the WRKY TFs in pear, especially in respond to drought stress at the genome-wide level.ResultsWe identified a total of 103 WRKY TFs in the pear genome. Based on the structural features of WRKY proteins and topology of the phylogenetic tree, the pear WRKY (PbWRKY) family was classified into seven groups (Groups 1, 2a–e, and 3). The microsyteny analysis indicated that 33 (32 %) PbWRKY genes were tandemly duplicated and 57 genes (55.3 %) were segmentally duplicated. RNA-seq experiment data and quantitative real-time reverse transcription PCR revealed that PbWRKY genes in different groups were induced by drought stress, and Group 2a and 3 were mainly involved in the biological pathways in response to drought stress. Furthermore, adaptive evolution analysis detected a significant positive selection for Pbr001425 in Group 3, and its expression pattern differed from that of other members in this group. The present study provides a solid foundation for further functional dissection and molecular evolution of WRKY TFs in pear, especially for improving the water-deficient resistance of pear through manipulation of the PbWRKYs.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-2233-6) contains supplementary material, which is available to authorized users.

Cloning and expression analysis of transcription factor gene DoWRKY1 in Dendrobium officinale

WRKY transcription factors are novel transcriptional regulatory factors, which play an important role in regulating plant development, metabolism and other physiological processes. In this study, a new Dendrobium officinale WRKY transcription factor, designated as DoWRKY1 was cloned by using RT-PCR and RACE (GenBank Accession No. KF953910). Bioinformatic analysis demonstrated that, the full-length cDNA of DoWRKY1 was 1,704 bp. And DoWRKY1 contained a 1,629 bp open reading frame (ORF) that encoding a peptide of 542 amino acid residues. The putative DoWRKY1 protein contained two conserved WRKY domains and it belonged to the group I WRKY family protein. Yeast one-hybrid experiment showed that DoWRKY1 had transcriptional activation ability in yeast, and it could activate the expression of downstream report genes (His3 and Ade2). Semi-quantitative RT-PCR experiment showed that DoWRKY1 expressed in roots, stems, leaves and protocorm-like bodies. Real-time qRT-PCR proved that DoWRKY1 could be induced by methyl jasmonate (MeJA) and chitosan (Chitosan), and the expression level of this gene can reach the expression peak at 2 h and 1 h, respectively. These results are useful for further determination of the regulation function of this gene in secondary metabolism of D. officinale.

Expression patterns of WRKY genes in di-haploid Populus simonii × P. nigra in response to osmotic stress and ABA treatment

In this study, 108 WRKY genes with complete conserved WRKY domain and zinc-finger motif were identified from poplar in silico, and they were classified into three subfamilies named I, II, and III. The WRKY members in group II were further clustered into five subgroups (IIa–e), where members in group I and III formed a single cluster. Thirteen highly expressed genes were employed to profile their spatio-temporal expression patterns in response to osmotic stress and ABA treatment by qRT-PCR. Under both osmotic stress and ABA treatment, overall gene expression displayed a dynamic pattern of “induced amplification—recover” in leaves, but not in roots and stems. Regarding individual gene expression across the seven time points and the three tissues, five genes (PthWRKY28, 56, 62, 88, and 106) were involved in regulation of both osmotic stress tolerance and ABA signaling transduction pathways. Meanwhile, four genes (PthWRKY41, 44, 51, and 116) played distinct roles in either osmotic stress or ABA treatment condition. These suggested that WRKY genes and corresponding pathways may play a critical role in regulating poplar responses to osmotic stress and ABA treatment in a dynamic and tissue-specific manner.

Isolation, Expression, and Promoter Analysis of GbWRKY2: A Novel Transcription Factor Gene from Ginkgo biloba.

WRKY transcription factor is involved in multiple life activities including plant growth and development as well as biotic and abiotic responses. We identified 28 WRKY genes from transcriptome data of Ginkgo biloba according to conserved WRKY domains and zinc finger structure and selected three WRKY genes, which are GbWRKY2, GbWRKY16, and GbWRKY21, for expression pattern analysis. GbWRKY2 was preferentially expressed in flowers and strongly induced by methyl jasmonate. Here, we cloned the full-length cDNA and genomic DNA of GbWRKY2. The full-length cDNA of GbWRKY2 was 1,713 bp containing a 1,014 bp open reading frame encoding a polypeptide of 337 amino acids. The GbWRKY2 genomic DNA had one intron and two exons. The deduced GbWRKY2 contained one WRKY domain and one zinc finger motif. GbWRKY2 was classified into Group II WRKYs. Southern blot analysis revealed that GbWRKY2 was a single copy gene in G. biloba. Many cis-acting elements related to hormone and stress responses were identified in the 1,363 bp-length 5′-flanking sequence of GbWRKY2, including W-box, ABRE-motif, MYBCOREs, and PYRIMIDINE-boxes, revealing the molecular mechanism of upregulated expression of GbWRKY2 by hormone and stress treatments. Further functional characterizations in transiently transformed tobacco leaves allowed us to identify the region that can be considered as the minimal promoter.

Molecular characterization of 26 cotton WRKY genes that are expressed differentially in tissues and are induced in seedlings under high salinity and osmotic stress

WRKY proteins that contain highly conserved WRKYGQK sequence and zinc-finger-like motif belong to a large transcription factor family in plants. In this study, 26 genes encoding putative WRKY proteins were identified in cotton (Gossypium hirsutum). On the basis of their conserved WRKY domain sequences, all of the GhWRKY proteins can be assigned to the previously defined groups I–III. Among 26 members of cotton WRKY family, five with two WRKY domains belong to group I, sixteen contribute to group II, and five containing C2HC zinc-finger pattern fall into group III. Phylogenetic analysis of GhWRKY proteins with Arabidopsis WRKYs revealed that they were indeed classified into three groups, and also their relative distance can be used as clues to confer their putative functions. Quantitative RT-PCR analysis showed that the isolated GhWRKY genes were expressed differentially in different tissues (such as leaves, roots, cotyledons, petals, anthers, or/and fibers), suggesting they may have an impact on cotton development. Under NaCl, mannitol and abscisic acid treatments, expressions of the GhWRKY genes are up-regulated in roots, cotyledons and/or hypocotyls of cotton seedlings, suggesting that the isolated GhWRKY genes may take part in plant response to abiotic stress during cotton development. Thus, our results may provide a valuable reference data set as the basis for further studying the roles of these WRKY genes in cotton.

Genome-wide identification of WRKY family genes and their response to cold stress in Vitis vinifera.

BackgroundWRKY transcription factors are one of the largest families of transcriptional regulators in plants. WRKY genes are not only found to play significant roles in biotic and abiotic stress response, but also regulate growth and development. Grapevine (Vitis vinifera) production is largely limited by stressful climate conditions such as cold stress and the role of WRKY genes in the survival of grapevine under these conditions remains unknown.ResultsWe identified a total of 59 VvWRKYs from the V. vinifera genome, belonging to four subgroups according to conserved WRKY domains and zinc-finger structure. The majority of VvWRKYs were expressed in more than one tissue among the 7 tissues examined which included young leaves, mature leaves, tendril, stem apex, root, young fruits and ripe fruits. Publicly available microarray data suggested that a subset of VvWRKYs was activated in response to diverse stresses. Quantitative real-time PCR (qRT-PCR) results demonstrated that the expression levels of 36 VvWRKYs are changed following cold exposure. Comparative analysis was performed on data from publicly available microarray experiments, previous global transcriptome analysis studies, and qRT-PCR. We identified 15 VvWRKYs in at least two of these databases which may relate to cold stress. Among them, the transcription of three genes can be induced by exogenous ABA application, suggesting that they can be involved in an ABA-dependent signaling pathway in response to cold stress.ConclusionsWe identified 59 VvWRKYs from the V. vinifera genome and 15 of them showed cold stress-induced expression patterns. These genes represented candidate genes for future functional analysis of VvWRKYs involved in the low temperature-related signal pathways in grape.

Optimization of expression and purification of recombinant Salvia miltiorrhiza WRKY1 protein in Escherichia coli

WRKY transcription factor is one of the Zinc finger proteins which contains a highly conserved WRKY domain and is a family of the plant-specific transcription factor. The plasmid pET28a-SmWRKY1 harboring Salvia miltiorrhiza WRKY1 (SmWRKY1) gene was successfully transformed and expressed in Escherichia coli BL21 (DE3). The conditions on protein expression of SmWRKY1 in E. coli, including induction duration, temperature, IPTG concentration and the E. coli concentration were optimized. The results showed that the highest protein expression of SmWRKY1 was obtained at 24 hours after the E. coli was cultured in the presence of 0.2 mol x L(-1) IPTG at 20 degrees C with A600 values of 1.0-1.5. This recombinant histidine-tagged protein was expressed at 2.454 g x L(-1) as inclusion body, which was first extracted using urea, and then purified by Ni2+ affinity chromatography and identified by SDS-PAGE. The expression of SmWRKY1 in E. coli was further confirmed by western blotting analysis.

Characterization of HbWRKY1, a WRKY transcription factor from Hevea brasiliensis that negatively regulates HbSRPP

Small rubber particle protein (SRPP) is a major component of Hevea brasiliensis (H. brasiliensis) latex, which is involved in natural rubber (NR) biosynthesis. However, little information is available on the regulation of SRPP gene (HbSRPP) expression. To study the transcriptional regulation of HbSRPP, the yeast one-hybrid experiment was performed to screen the latex cDNA library using the HbSRPP promoter as bait. One cDNA that encodes the WRKY transcription factor, designated as HbWRKY1, was isolated from H. brasiliensis. HbWRKY1 contains a 1437 bp open reading frame that encodes 478 amino acids. The deduced HbWRKY1 protein was predicted to possess two conserved WRKY domains and a C2H2 zinc-finger motif. HbWRKY1 was expressed at different levels, with the highest transcription in the flower, followed by the bark, latex, and leaf. Furthermore, the co-expression of pHbSRP::GUS with CaMV35S::HbWRKY1 significantly decreased the GUS activity in transgenic tobacco, indicating that HbWRKY1 significantly suppressed the HbSRPP promoter. These results suggested that HbWRKY1 maybe a negative transcription regulator of HbSRPP involved in NR biosynthesis in H. brasiliensis.

Genome-wide analysis of the WRKY gene family in physic nut (Jatropha curcas L.)

The WRKY proteins, which contain highly conserved WRKYGQK amino acid sequences and zinc-finger-like motifs, constitute a large family of transcription factors in plants. They participate in diverse physiological and developmental processes. WRKY genes have been identified and characterized in a number of plant species. We identified a total of 58 WRKY genes (JcWRKY) in the genome of the physic nut (Jatropha curcas L.). On the basis of their conserved WRKY domain sequences, all of the JcWRKY proteins could be assigned to one of the previously defined groups, I–III. Phylogenetic analysis of JcWRKY genes with Arabidopsis and rice WRKY genes, and separately with castor bean WRKY genes, revealed no evidence of recent gene duplication in JcWRKY gene family. Analysis of transcript abundance of JcWRKY gene products were tested in different tissues under normal growth condition. In addition, 47 WRKY genes responded to at least one abiotic stress (drought, salinity, phosphate starvation and nitrogen starvation) in individual tissues (leaf, root and/or shoot cortex). Our study provides a useful reference data set as the basis for cloning and functional analysis of physic nut WRKY genes.

WRKY Transcription Factors in Wheat and Their Induction by Biotic and Abiotic Stress

WRKY proteins constitute a large family of transcription factors in higher plants, and are involved in the regulation of development, senescence and stress resistance. However, little is known with respect to the constitution and function of WRKY genes in bread wheat (Triticum aestivum). We identified a set of 92 wheat WRKY genes from publicly available sequence databases, and constructed a phylogeny map based on the conserved WRKY domain peptide sequences. The wheat sequences were classified into the standard set of WRKY groups and sub-groups established from plant model genomes. Our results show the WRKY orthologues of wheat and rice are more similar compared to their Arabidopsis counterparts. Therefore, we argue some expansion of the WRKY family occurred following the divergence of these three species. The induction of sequence variation in a subset of 18 TaWRKY genes via somatic hybridization was studied by comparing the sequences from cv. JN177 and its derivative cv. SR3. We also analyzed the responses of these 18 genes to imposed salinity, PEG, ABA and SA using quantitative real time PCR. Nine of the 18 TaWRKY genes were up-regulated by exogenous SA. Six genes were up-regulated by salinity or PEG treatment, and their expression was also induced by exogenous ABA. Interestingly, induction by salinity or PEG was impaired when ABA biosynthesis was inhibited. The indication was that most of the TaWRKY genes involved in the abiotic stress response acted in an ABA-dependent manner.

Characterization of a wheat transcription factor, TaWRKY45, and its effect on Fusarium head blight resistance in transgenic wheat plants

The WRKY transcription factors belong to a large protein family characterized by the conserved WRKY domain. These factors have been identified to play biological functions in various plant developmental processes. WRKY proteins are also known to be involved in regulating plant responses to pathogen attack and stress-related hormones. In this study, we report the isolation and characterization of the gene (TaWRKY45) for the wheat WRKY45 transcription factor. Amino acid sequence alignment and phylogenetic analyses demonstrated that the TaWRKY45 protein is orthologous to rice OsWRKY45. Our analysis of its expression in wheat indicated that TaWRKY45 was constitutively expressed in various organs and throughout the lifetime of the plant. We observed that TaWRKY45 was upregulated in response to benzothiadiazole (BTH), a plant immune system strengthner, and Fusarium graminearum, which is a causal fungus for Fusarium head blight (FHB). The constitutive overexpression of the TaWRKY45 transgene conferred an enhanced resistance against F. graminearum to transgenic wheat plants grown under greenhouse conditions. These results indicate that TaWRKY45 is involved in the defense systems for the biotic stressors in wheat and that it may be potentially utilized to improve the disease resistance of wheat.

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.