Paul J. Rushton

The WRKY superfamily of plant transcription factors.

The WRKY proteins are a superfamily of transcription factors with up to 100 representatives in Arabidopsis. Family members appear to be involved in the regulation of various physio-logical programs that are unique to plants, including pathogen defense, senescence and trichome development. In spite of the strong conservation of their DNA-binding domain, the overall structures of WRKY proteins are highly divergent and can be categorized into distinct groups, which might reflect their different functions.

WRKY transcription factors

WRKY transcription factors are one of the largest families of transcriptional regulators in plants and form integral parts of signalling webs that modulate many plant processes. Here, we review recent significant progress in WRKY transcription factor research. New findings illustrate that WRKY proteins often act as repressors as well as activators, and that members of the family play roles in both the repression and de-repression of important plant processes. Furthermore, it is becoming clear that a single WRKY transcription factor might be involved in regulating several seemingly disparate processes. Mechanisms of signalling and transcriptional regulation are being dissected, uncovering WRKY protein functions via interactions with a diverse array of protein partners, including MAP kinases, MAP kinase kinases, 14-3-3 proteins, calmodulin, histone deacetylases, resistance proteins and other WRKY transcription factors. WRKY genes exhibit extensive autoregulation and cross-regulation that facilitates transcriptional reprogramming in a dynamic web with built-in redundancy.

Interaction of elicitor-induced DNA-binding proteins with elicitor response elements in the promoters of parsley PR1 genes.

PR1 is a pathogenesis‐related protein encoded in the parsley genome by a family of three genes (PR1–1, PR1–2 and PR1–3). Loss‐ and gain‐of‐function experiments in a transient expression system demonstrated the presence of two fungal elicitor responsive elements in each of the PR1–1 and PR1–2 promoters. These elements, W1, W2 and W3, contain the sequence (T)TGAC(C) and mutations that disrupt this sequence abolish function. Gel shift experiments demonstrated that W1, W2 and W3 are bound specifically by similar nuclear proteins. Three cDNA clones encoding sequence‐specific DNA‐binding proteins were isolated by South‐Western screening and these proteins, designated WRKY1, 2 and 3, also bind specifically to W1, W2 and W3. WRKY1, 2 and 3 are members of the family of sequence‐specific DNA‐binding proteins, which we call the WRKY family. Treatment of parsley cells with the specific oligopeptide elicitor Pep25 induced a transient and extremely rapid increase in mRNA levels of WRKY1 and 3. WRKY2 mRNA levels in contrast showed a concomitant transient decrease. These rapid changes in WRKY mRNA levels in response to a defined signal molecule suggest that WRKY1, 2 and 3 play a key role in a signal transduction pathway that leads from elicitor perception to PR1 gene activation.

EARLY NUCLEAR EVENTS IN PLANT DEFENCE SIGNALLING : RAPID GENE ACTIVATION BY WRKY TRANSCRIPTION FACTORS

Parsley WRKY proteins comprise a family of plant‐specific zinc‐finger‐type factors implicated in the regulation of genes associated with pathogen defence. In vitro, these proteins bind specifically to functionally defined TGAC‐containing W box promoter elements within the Pathogenesis‐Related Class10 (PR‐10) genes. Here we present in vivo data demonstrating that WRKY1 is a transcriptional activator mediating fungal elicitor‐induced gene expression by binding to W box elements. In situ RNA hybridization revealed that the WRKY1 gene is rapidly and locally activated in parsley leaf tissue around fungal infection sites. Transient expression studies in parsley protoplasts showed that a specific arrangement of W box elements in the WRKY1 promoter itself is necessary and sufficient for early activation and that WRKY1 binds to such elements. Our results demonstrate that WRKY transcription factors play an important role in the regulation of early defence‐response genes including regulation of WRKY1.

Synthetic plant promoters containing defined regulatory elements provide novel insights into pathogen- and wound- induced signaling

Pathogen-inducible plant promoters contain multiple cis-acting elements, only some of which may contribute to pathogen inducibility. Therefore, we made defined synthetic promoters containing tetramers of only a single type of element and present evidence that a range of cis-acting elements (boxes W1, W2, GCC, JERE, S, Gst1, and D) can mediate local gene expression in planta after pathogen attack. The expression patterns of the promoters were monitored during interactions with a number of pathogens, including compatible, incompatible, and nonhost interactions. Interestingly, there were major differences in the inducibilities of the various promoters with the pathogens tested as well as differences in the speed of induction and in the basal expression levels. We also show that defense signaling is largely conserved across species boundaries at the cis-acting element level. Many of these promoters also direct local wound-induced expression, and this provides evidence for the convergence of resistance gene, nonhost, and wound responses at the level of the promoter elements. We have used these cis-acting elements to construct improved synthetic promoters and show the effects of varying the number, order, and spacing of such elements. These promoters are valuable additions to the study of signaling and transcriptional activation during plant–pathogen interactions.

Transcriptional control of plant genes responsive to pathogens

Transcriptional activation of genes is a vital part of the plants defence system against pathogens. Cis-acting elements within the promoters of many of these genes have recently been defined and investigators have started to isolate their cognate trans-acting factors. Some of these factors have counterparts in animals, whereas others are present only in plants, reflecting the fact that plants have developed a unique defence system.

WRKY transcription factors: key components in abscisic acid signalling

WRKY transcription factors (TFs) are key regulators of many plant processes, including the responses to biotic and abiotic stresses, senescence, seed dormancy and seed germination. For over 15 years, limited evidence has been available suggesting that WRKY TFs may play roles in regulating plant responses to the phytohormone abscisic acid (ABA), notably some WRKY TFs are ABA-inducible repressors of seed germination. However, the roles of WRKY TFs in other aspects of ABA signalling, and the mechanisms involved, have remained unclear. Recent significant progress in ABA research has now placed specific WRKY TFs firmly in ABA-responsive signalling pathways, where they act at multiple levels. In Arabidopsis, WRKY TFs appear to act downstream of at least two ABA receptors: the cytoplasmic PYR/PYL/RCAR-protein phosphatase 2C-ABA complex and the chloroplast envelope-located ABAR-ABA complex. In vivo and in vitro promoter-binding studies show that the target genes for WRKY TFs that are involved in ABA signalling include well-known ABA-responsive genes such as ABF2, ABF4, ABI4, ABI5, MYB2, DREB1a, DREB2a and RAB18. Additional well-characterized stress-inducible genes such as RD29A and COR47 are also found in signalling pathways downstream of WRKY TFs. These new insights also reveal that some WRKY TFs are positive regulators of ABA-mediated stomatal closure and hence drought responses. Conversely, many WRKY TFs are negative regulators of seed germination, and controlling seed germination appears a common function of a subset of WRKY TFs in flowering plants. Taken together, these new data demonstrate that WRKY TFs are key nodes in ABA-responsive signalling networks.

Tobacco Transcription Factors: Novel Insights into Transcriptional Regulation in the Solanaceae

Tobacco (Nicotiana tabacum) is a member of the Solanaceae, one of the agronomically most important groups of flowering plants. We have performed an in silico analysis of 1.15 million gene-space sequence reads from the tobacco nuclear genome and report the detailed analysis of more than 2,500 tobacco transcription factors (TFs). The tobacco genome contains at least one member of each of the 64 well-characterized TF families identified in sequenced vascular plant genomes, indicating that evolution of the Solanaceae was not associated with the gain or loss of TF families. However, we found notable differences between tobacco and non-Solanaceae species in TF family size and evidence for both tobacco- and Solanaceae-specific subfamily expansions. Compared with TF families from sequenced plant genomes, tobacco has a higher proportion of ERF/AP2, C2H2 zinc finger, homeodomain, GRF, TCP, zinc finger homeodomain, BES, and STERILE APETALA (SAP) genes and novel subfamilies of BES, C2H2 zinc finger, SAP, and NAC genes. The novel NAC subfamily, termed TNACS, appears restricted to the Solanaceae, as they are absent from currently sequenced plant genomes but present in tomato (Solanum lycopersicum), pepper (Capsicum annuum), and potato (Solanum tuberosum). They constitute approximately 25% of NAC genes in tobacco. Based on our phylogenetic studies, we predict that many of the more than 50 tobacco group IX ERF genes are involved in jasmonate responses. Consistent with this, over two-thirds of group IX ERF genes tested showed increased mRNA levels following jasmonate treatment. Our data are a major resource for the Solanaceae and fill a void in studies of TF families across the plant kingdom.

Leucine zipper-containing WRKY proteins widen the spectrum of immediate early elicitor-induced WRKY transcription factors in parsley

Two new WRKY transcription factors from parsley (Petroselinum crispum), WRKY4 and WRKY5, were isolated using the yeast one-hybrid system. In yeast, both proteins interacted sequence-specifically with W boxes (TTGACC) and activated transcription. They appear to contain functional leucine zippers, which increase their affinities for W boxes. Co-transfection experiments in parsley protoplasts confirmed their in vivo-binding specificity for W boxes. Elicitor-mediated expression of the WRKY5 gene, the first parsley member of the group III family of WRKY proteins, is extremely transient, with high mRNA levels occurring within a time window of less than 1 h. WRKY4 and -5, as well as the previously identified parsley transcription factors WRKY1 and -3, are encoded by immediate early elicitor-activated genes that differ in their sensitivity to cycloheximide (CHX) and their activation kinetics. We propose that a number of the pathways activated during the plant defense response require the induction of several distinct WRKY transcription factors with different DNA binding-site preferences to fine-tune the activation of a wide spectrum of target genes.

Tobacco Transcription Factors NtMYC2a and NtMYC2b Form Nuclear Complexes with the NtJAZ1 Repressor and Regulate Multiple Jasmonate-Inducible Steps in Nicotine Biosynthesis

Biotic and abiotic stress lead to elevated levels of jasmonic acid (JA) and its derivatives and activation of the biosynthesis of nicotine and related pyridine alkaloids in cultivated tobacco (Nicotiana tabacum L.). Among the JA-responsive genes is NtPMT1a, encoding putrescine N-methyl transferase, a key regulatory enzyme in nicotine formation. We have characterized three genes (NtMYC2a, b, c) encoding basic helix-loop-helix (bHLH) transcription factors (TFs) whose expression is rapidly induced by JA and that specifically activate JA-inducible NtPMT1a expression by binding a G-box motif within the NtPMT1a promoter in in vivo and in vitro assays. Using split-YFP assays, we further show that, in the absence of JA, NtMYC2a and NtMYC2b are present as nuclear complexes with the NtJAZ1 repressor. RNA interference (RNAi)-mediated knockdown of NtMYC2a and NtMYC2b expression results in significant decreases in JA-inducible NtPMT1a transcript levels, as well as reduced levels of transcripts encoding other enzymes involved in nicotine and minor alkaloid biosynthesis, including an 80-90% reduction in the level of transcripts encoding the putative nicotine synthase gene NtA662. In contrast, ectopic overexpression of NtMYC2a and NtMYC2b had no effect on NtPMT1a expression in the presence or absence of exogenously added JA. These data suggest that NtMYC2a, b, c are required components of JA-inducible expression of multiple genes in the nicotine biosynthetic pathway and may act additively in the activation of JA responses.