Plant WRKY Transcription Factors Research Articles

The role of WRKY transcription factors in plants

The WRKY proteins are one of the largest families of transcriptional regulators in plants. Their biosynthesis is induced during certain stages of plant development and upon pathogen infection. A single WRKY transcription factor may be involved in the regulation of several apparently disparate processes. Their hallmark is strong conservation of the DNA binding domain which contains an invariant WRKYGQK amino acid sequence and a zinc binding motif. However, the overall sequences of individual representatives are highly divergent. Little is known about the 3D-structure of the WRKY proteins. Up to date there have only been results of structural strudies of DNA binding domain available. In this review, the biological function as well the structural studies of the WRKY proteins have been recapitulated.

Expression and Functional Analysis of WRKY Transcription Factors in Chinese Wild Hazel, Corylus heterophylla Fisch.

Plant WRKY transcription factors are known to regulate various biotic and abiotic stress responses. In this study we identified a total of 30 putative WRKY unigenes in a transcriptome dataset of the Chinese wild Hazel, Corylus heterophylla, a species that is noted for its cold tolerance. Thirteen full-length of these ChWRKY genes were cloned and found to encode complete protein sequences, and they were divided into three groups, based on the number of WRKY domains and the pattern of zinc finger structures. Representatives of each of the groups, Unigene25835 (group I), Unigene37641 (group II) and Unigene20441 (group III), were transiently expressed as fusion proteins with yellow fluorescent fusion protein in Nicotiana benthamiana, where they were observed to accumulate in the nucleus, in accordance with their predicted roles as transcriptional activators. An analysis of the expression patterns of all 30 WRKY genes revealed differences in transcript abundance profiles following exposure to cold, drought and high salinity conditions. Among the stress-inducible genes, 23 were up-regulated by all three abiotic stresses and the WRKY genes collectively exhibited four different patterns of expression in flower buds during the overwintering period from November to April. The organ/tissue related expression analysis showed that 18 WRKY genes were highly expressed in stem but only 2 (Unigene9262 and Unigene43101) were greatest in male anthotaxies. The expression of Unigene37641, a member of the group II WRKY genes, was substantially up-regulated by cold, drought and salinity treatments, and its overexpression in Arabidopsis thaliana resulted in better seedling growth, compared with wild type plants, under cold treatment conditions. The transgenic lines also had exhibited higher soluble protein content, superoxide dismutase and peroxidase activiety and lower levels of malondialdehyde, which collectively suggets that Unigene37641 expression promotes cold tolerance.

WRKY transcription factors down-regulate the expression of H2S-generating genes, LCD and DES in Arabidopsis thaliana

Hydrogen sulfide (H2S) is a gasotransmitter playing a vital role in response to biotic and abiotic stress for plants. In order to understand the transcriptional regulation of the genes that are responsible for endogenous H2S generation, the promoter sequences of L-cysteine desulfhydrase (LCD), D-cysteine desulfhydrase (DCD1, DCD2), desulfhydrase (DES) and nitrogen fixation synthetase (NFS1, NFS2) were analyzed. They are all found to contain a W-box, a characteristic core binding site for the plant WRKY transcription factors, which have important roles in the plant’s responses to numerous stresses by modifying the expression patterns of their target genes. An electrophoretic mobility shift assay indicated that WRKY18 and WRKY60 interacted with the W-box in the promoters of the LCD, DCD1, DCD2, DES and NFS2 genes, whereas WRKY40 bound to the W-box of the NFS1 promoter. The expression levels of the LCD, DES and DCD1 genes were up-regulated, but the DCD2 was down-regulated in the plants with WRKY18, WRKY40 or WRKY60 mutations. The plants with double and triple mutations of WRKY18, WRKY40 and WRKY60 had a higher rate of H2S production during cadmium stress and were more resistant to the cadmium stress than the wild type or single mutants. These results suggest that WRKY transcription factors regulate the H2S signaling pathway in plants, allowing them to cope with cadmium stress.

Overexpression of BnWRKY33 in oilseed rape enhances resistance to Sclerotinia sclerotiorum

Sclerotinia sclerotiorum causes a devastating disease in oilseed rape (Brassica napus) resulting in a tremendous yield loss worldwide. Studies on various host-pathogen interactions have shown that plant WRKY transcription factors are essential for defence. For the B. napus-S. sclerotiorum interaction, little direct evidence has been found with regard to the biological roles of specific WRKY genes in host resistance. In this study, we isolated a B. napus WRKY gene, BnWRKY33, and found that the gene is highly responsive to S. sclerotiorum infection. Transgenic B. napus plants overexpressing BnWRKY33 showed markedly enhanced resistance to S. sclerotiorum, constitutive activation of the expression of BnPR1 and BnPDF1.2, and inhibition of H2 O2 accumulation in response to pathogen infection. Further, we isolated a mitogen-activated protein (MAP) kinase substrate gene, BnMKS1, and found that not only can BnWRKY33 interact with BnMKS1, which can also interact with BnMPK4, using the yeast two-hybrid assay, consistent with their collective nuclear localization, but also BnWRKY33, BnMKS1 and BnMPK4 are substantially and synergistically expressed in response to S. sclerotiorum infection. In contrast, the three genes showed differential expression in response to phytohormone treatments. Together, these results suggest that BnWRKY33 plays an important role in B. napus defence to S. sclerotiorum, which is most probably associated with the activation of the salicylic acid (SA)- and jasmonic acid (JA)-mediated defence response and inhibition of H2 O2 accumulation, and we propose a potential mechanism in which BnMPK4-BnMKS1-BnWRKY33 exist in a nuclear localized complex to regulate resistance to S. sclerotiorum in oilseed rape.

A salicylic acid‐induced rice (Oryza sativa L.) transcription factor OsWRKY77 is involved in disease resistance of Arabidopsis thaliana

Plant WRKY transcription factors act as either positive or negative regulators of plant basal disease resistance. To comprehensively characterise the complicated functional network, we isolated OsWRKY77 from rice seedlings treated with salicylic acid. OsWRKY77 is a typical WRKY transcription factor, based on in its protein structure analysis, nuclear localisation of the fused OsWRKY77-GFP protein and gel electrophoretic mobility shift assay binding, which demonstrated that OsWRKY77 was able to bind a W-box. Transgenic Arabidopsis lines overexpressing OsWRKY77 repressed growth of Pseudomonas syringae pv. tomato DC3000 (PstDC300), and the reduced susceptibility was associated with enhanced expression of defence-related PR1, PR2 and PR5 genes. These results show that OsWRKY77 is a positive regulator of PR gene expression and basal resistance to the bacterial pathogen PstDC3000.

ABO3, a WRKY transcription factor, mediates plant responses to abscisic acid and drought tolerance in Arabidopsis

The biological functions of WRKY transcription factors in plants have been widely studied, but their roles in abiotic stress are still not well understood. We isolated an ABA overly sensitive mutant, abo3, which is disrupted by a T-DNA insertion in At1g66600 encoding a WRKY transcription factor AtWRKY63. The mutant was hypersensitive to ABA in both seedling establishment and seedling growth. However, stomatal closure was less sensitive to ABA, and the abo3 mutant was less drought tolerant than the wild type. Northern blot analysis indicated that the expression of the ABA-responsive transcription factor ABF2/AREB1 was markedly lower in the abo3 mutant than in the wild type. The abo3 mutation also reduced the expression of stress-inducible genes RD29A and COR47, especially early during ABA treatment. ABO3 is able to bind the W-box in the promoter of ABF2in vitro. These results uncover an important role for a WRKY transcription factor in plant responses to ABA and drought stress.

Stress- and Pathogen-Induced Arabidopsis WRKY48 is a Transcriptional Activator that Represses Plant Basal Defense

Plant WRKY transcription factors can function as either positive or negative regulators of plant basal disease resistance. Arabidopsis WRKY48 is induced by mechanical and/or osmotic stress due to infiltration and pathogen infection and, therefore, may play a role in plant defense responses. WRKY48 is localized to the nucleus, recognizes the TTGACC W-box sequence with a high affinity in vitro and functions in plant cells as a strong transcriptional activator. To determine the biological functions directly, we have isolated loss-of-function T-DNA insertion mutants and generated gain-of-function transgenic overexpression plants for WRKY48 in Arabidopsis. Growth of a virulent strain of the bacterial pathogen Pseudomonas syringae was decreased in the wrky48 T-DNA insertion mutants. The enhanced resistance of the loss-of-function mutants was associated with increased induction of salicylic acid-regulated PR1 by the bacterial pathogen. By contrast, transgenic WRKY48-overexpressing plants support enhanced growth of P. syringae and the enhanced susceptibility was associated with reduced expression of defense-related PR genes. These results suggest that WRKY48 is a negative regulator of PR gene expression and basal resistance to the bacterial pathogen P. syringae.

Functional analysis of Arabidopsis WRKY25 transcription factor in plant defense against Pseudomonas syringae

BackgroundA 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.ResultsWe 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.ConclusionThe 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.

Arabidopsis WRKY33 transcription factor is required for resistance to necrotrophic fungal pathogens

Plant WRKY transcription factors are key regulatory components of plant responses to microbial infection. In addition to regulating the expression of defense-related genes, WRKY transcription factors have also been shown to regulate cross-talk between jasmonate- and salicylate-regulated disease response pathways. The two pathways mediate resistance against different types of microbial pathogens, and there are numerous reports of antagonistic interactions between them. Here we show that mutations of the Arabidopsis WRKY33 gene encoding a WRKY transcription factor cause enhanced susceptibility to the necrotrophic fungal pathogens Botrytis cinerea and Alternaria brassicicola concomitant with reduced expression of the jasmonate-regulated plant defensin PDF1.2 gene. Ectopic over-expression of WRKY33, on the other hand, increases resistance to the two necrotrophic fungal pathogens. The wrky33 mutants do not show altered responses to a virulent strain of the bacterial pathogen Pseudomonas syringae, although the ectopic expression of WRKY33 results in enhanced susceptibility to this pathogen. The susceptibility of WRKY33-over-expressing plants to P. syringae is associated with reduced expression of the salicylate-regulated PR-1 gene. The WRKY33 transcript is induced in response to pathogen infection, or treatment with salicylate or the paraquat herbicide that generates activated oxygen species in exposed cells. WRKY33 is localized to the nucleus of plant cells and recognizes DNA molecules containing the TTGACC W-box sequence. Together, these results indicate that pathogen-induced WRKY33 is an important transcription factor that regulates the antagonistic relationship between defense pathways mediating responses to P. syringae and necrotrophic pathogens.

The Structure and Function of Plant WRKY Transcription Factors

WRKY transcription factors that are unique to plants are the new type transcriptional regulatory factors in which N-terminal ends contain a conserved WRKYGQR amino acids sequences. WRKY transcription factors regulate the target genes expression that contain the W-box elements in the promoter regions by specifically binding to (T)(T)TGAC(C/T) sequence. Therefore, the WRKY transcription factors participate in the plant various kinds defense responses and regulate the plant growth and development. This article reviews the progress of the basic structure and biological function of plant WRKY transcription factors.

A hot pepper gene encoding WRKY transcription factor is induced during hypersensitive response to Tobacco mosaic virus and Xanthomonas campestris

Plant WRKY transcription factors were previously implicated in the alteration of gene expression in response to various pathogens. The WRKY proteins constitute a large family of plant transcription factors, whose precise functions have yet to be elucidated. Using a domain-specific differential display procedure, we isolated a WRKY gene, which is rapidly induced during an incompatible interaction between hot pepper and Tobacco mosaic virus (TMV) or Xanthomonas campestris pv . vesicatoria (Xcv). The full-length cDNA of CaWRKY-a (Capsicum annuum WRKY-a) encodes a putative polypeptide of 546 amino acids, containing two WRKY domains with a zinc finger motif. The expression of CaWRKY-a could be rapidly induced by not only chemical elicitor such as salicylic acid (SA) or ethephon but also wounding treatments. The nuclear localization of CaWRKY-a was determined in transient expression system using tobacco BY-2 cells by polyethylene glycol (PEG)-mediated transformation experiment. With oligonucleotide molecules containing the putative W-box sequences as a probe, we confirmed that CaWRKY-a protein had W-box-binding activity. These results suggest that CaWRKY-a might be involved as a transcription factor in plant defense-related signal transduction pathways.