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SUMMARY: Cold stress restricts the metabolic and physiological activities of plants, thereby affecting their growth and development. Although broad‐complex, tramtrack, and bric‐à‐brac (BTB) proteins are essential for diverse biological processes and stress responses, the mechanisms underlying BTB‐mediated cold responses remain not fully understood. Here, we characterize the function of the cold‐induced SlBTB19 protein in tomato (Solanum lycopersicum). Overexpression of SlBTB19 resulted in increased plant sensitivity to cold stress, whereas SlBTB19 knockout mutants exhibited a cold‐tolerance phenotype. Further analyses, including protein–protein interaction studies and cell‐free degradation assays, revealed that SlBTB19 interacts with and destabilizes the transcription factor SlWRKY2. Using virus‐induced gene silencing (VIGS) to silence SlWRKY2 in both wild‐type and slbtb19 mutants, we provided genetic evidence that SlWRKY2 acts downstream of SlBTB19 in regulating cold tolerance. Importantly, we demonstrated that SlWRKY2 positively regulates cold tolerance in a CRT/DRE binding factor (CBF)‐dependent manner. Under cold stress, SlWRKY2 binds to the W‐box in the CBF1 and CBF3 promoters, directly activating their expression. In summary, our findings identify a SlBTB19‐SlWRKY2 module that negatively regulates the CBF‐dependent cold tolerance pathway in tomato. Significance Statement: Our findings not only contribute to the broader understanding of the molecular basis of plant responses to cold stress but also highlight the importance of post‐transcriptional mechanisms in regulating these responses. In summary, we proposed an updated model: A cold‐induced BTB family protein SlBTB19 negatively regulates cold tolerance. SlBTB19 interacts with and facilitates the proteolysis of the SlWRKY2 protein. SlWRKY2 is a positive regulator of cold tolerance, which activates cold‐responsive genes SlCBFs. [ABSTRACT FROM AUTHOR]

Chilling stress is a major environmental factor that significantly reduces crop production. To adapt to chilling stress, plants activate a series of cellular responses and accumulate an array of metabolites, particularly proline. Here, we report that the transcription factor SlWRKY51 increases proline contents in tomato (Solanum lycopersicum) under chilling stress. SlWRKY51 expression is induced under chilling stress. Knockdown or knockout of SlWRKY51 led to chilling‐sensitive phenotypes, with lower photosynthetic capacity and more reactive oxygen species (ROS) accumulation than the wild type (WT). The proline contents were significantly reduced in SlWRKY51 knockdown and knockout lines under chilling stress, perhaps explaining the phenotypes of these lines. D‐1‐pyrroline‐5‐carboxylate synthetase (P5CS), which catalyses the rate‐limiting step of proline biosynthesis, is encoded by two closely related P5CS genes (P5CS1 and P5CS2). We demonstrate that SlWRKY51 directly activates the expression of P5CS1 under chilling stress. In addition, the VQ (a class of plant‐specific proteins containing the conserved motif FxxhVQxhTG) family member SlVQ10 physically interacts with SlWRKY51 to enhance its activation of P5CS1. Our study reveals that the chilling‐induced transcription factor SlWRKY51 enhances chilling tolerance in tomato by promoting proline accumulation. Summary statement: The transcription factor WRKY51 enhances proline production in tomato by regulating the expression of the D‐1‐pyrroline‐5‐carboxylate synthetase gene SlP5CS1 under chilling stress. [ABSTRACT FROM AUTHOR]

MYB transcription factors have been linked to anthocyanin synthesis and various color phenotypes in plants. In apple, MYB10 confers a red‐flesh phenotype due to a minisatellite insertion in its R6 promoter, but R6:MYB10 genotypes exhibit various degrees of red pigmentation in the flesh, suggesting the involvement of other genetic factors. Here, it is shown that MdWRKY10, a transcription factor identified via DNA pull‐down trapping, binds to the promoter of MdMYB10 and activates its transcription. MdWRKY10 specifically interacts with the WDR protein MdTTG1 to join the apple MYB‐bHLH‐WDR (MBW) complex, which significantly enhances its transcriptional activation activity. A 163‐bp InDel detected in the promoter region of the alleles of MdWRKY10 in a hybrid population of identical heterozygous genotypes regarding R6 by structural variation analysis, contains a typical W‐box element that MdWRKY10 binds to for transactivation. This leads to increased transcript levels of MdWRKY10 and MdMYB10 and enhanced anthocyanin synthesis in the flesh, largely accounting for the various degrees of flesh red pigmentation in the R6 background. These findings reveal a novel regulatory role of the WRKY‐containing protein complex in the formation of red flesh apple phenotypes and provide broader insights into the molecular mechanism governing anthocyanin synthesis in plants. [ABSTRACT FROM AUTHOR]

Summary: Glomerella leaf spot (GLS), caused by the fungus Colletotrichum fructicola, is considered one of the most destructive diseases affecting apples. The VQ‐WRKY complex plays a crucial role in the response of plants to biotic stresses. However, our understanding of the defensive role of the VQ‐WRKY complex on woody plants, particularly apples, under biotic stress, remains limited. In this study, we elucidated the molecular mechanisms underlying the defensive role of the apple MdVQ37‐MdWRKY100 module in response to GLS infection. The overexpression of MdWRKY100 enhanced resistance to C. fructicola, whereas MdWRKY100 RNA interference in apple plants reduced resistance to C. fructicola by affecting salicylic acid (SA) content and the expression level of the CC‐NBS‐LRR resistance gene MdRPM1. DAP‐seq, Y1H, EMSA, and RT‐qPCR assays indicated that MdWRKY100 inhibited the expression of MdWRKY17, a positive regulatory factor gene of SA degradation, upregulated the expression of MdPAL1, a key enzyme gene of SA biosynthesis, and promoted MdRPM1 expression by directly binding to their promotors. Transient overexpression and silencing experiments showed that MdPAL1 and MdRPM1 positively regulated GLS resistance in apples. Furthermore, the overexpression of MdVQ37 increased the susceptibility to C. fructicola by reducing the SA content and expression level of MdRPM1. Additionally, MdVQ37 interacted with MdWRKY100, which repressed the transcriptional activity of MdWRKY100. In summary, these results revealed the molecular mechanism through which the apple MdVQ37‐MdWRKY100 module responds to GLS infection by regulating SA content and MdRPM1 expression, providing novel insights into the involvement of the VQ‐WRKY complex in plant pathogen defence responses. [ABSTRACT FROM AUTHOR]

Pathogens generate and secrete effector proteins to the host plant cells during pathogenesis to promote virulence and colonization. If the plant carries resistance (R) proteins that recognize pathogen effectors, effector‐triggered immunity (ETI) is activated, resulting in a robust immune response and hypersensitive response (HR). The bipartite effector AvrRps4 from Pseudomonas syringae pv. pisi has been well studied in terms of avirulence function. In planta, AvrRps4 is processed into two parts. The C‐terminal fragment of AvrRps4 (AvrRps4C) induces HR in turnip and is recognized by the paired resistance proteins AtRRS1/AtRPS4 in Arabidopsis. Here, we show that AvrRps4C targets a group of Arabidopsis WRKY, including WRKY46, WRKY53, WRKY54, and WRKY70, to induce its virulence function. Indeed, AvrRps4C suppresses the general binding and transcriptional activities of immune‐positive regulator WRKY54 and WRKY54‐mediated resistance. AvrRps4C interferes with WRKY54's binding activity to target gene SARD1 in vitro, suggesting WRKY54 is sequestered from the SARD1 promoter by AvrRps4C. Through the interaction of AvrRps4C with four WRKYs, AvrRps4 enhances the formation of homo‐/heterotypic complexes of four WRKYs and sequesters them in the cytoplasm, thus inhibiting their function in plant immunity. Together, our results provide a detailed virulence mechanism of AvrRps4 through its C‐terminus. [ABSTRACT FROM AUTHOR]

Maize (Zea mays L.) is an important food crop with a wide range of uses in both industry and agriculture. Drought stress during its growth cycle can greatly reduce maize crop yield and quality. However, the molecular mechanisms underlying maize responses to drought stress remain unclear. In this work, a WRKY transcription factor‐encoding gene, ZmWRKY30, from drought‐treated maize leaves was screened out and characterized. ZmWRKY30 gene expression was induced by dehydration treatments. The ZmWRKY30 protein localized to the nucleus and displayed transactivation activity in yeast. Compared with wild‐type (WT) plants, Arabidopsis lines overexpressing ZmWRKY30 exhibited a significantly enhanced drought stress tolerance, as evidenced by the improved survival rate, increased antioxidant enzyme activity by superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT), elevated proline content, and reduced lipid peroxidation recorded after drought stress treatment. In contrast, the mutator (Mu)‐interrupted ZmWRKY30 homozygous mutant (zmwrky30) was more sensitive to drought stress than its null segregant (NS), characterized by the decreased survival rate, reduced antioxidant enzyme activity (SOD, POD, and CAT) and proline content, as well as increased malondialdehyde accumulation. RNA‐Seq analysis further revealed that, under drought conditions, the knockout of the ZmWRKY30 gene in maize affected the expression of genes involved in reactive oxygen species (ROS), proline, and myo‐inositol metabolism. Meanwhile, the zmwrky30 mutant exhibited significant downregulation of myo‐inositol content in leaves under drought stress. Combined, our results suggest that ZmWRKY30 positively regulates maize responses to water scarcity. This work provides potential target genes for the breeding of drought‐tolerant maize. [ABSTRACT FROM AUTHOR]

Summary: Jasmonate (JA) and abscisic acid (ABA) are two major phytohormones involved in pathogen resistance. However, how their biosynthesis is regulated is not well understood.We silenced NaWRKY70 in wild tobacco Nicotiana attenuata and determined its role in regulating genes involved in the production of JA, ABA and the phytoalexin capsidiol in response to the fungal pathogen Alternaria alternata using techniques including electrophoretic mobility shift, chromatin immunoprecipitation, transient overexpression and virus‐induced gene silencing.Silencing NaWRKY70 dramatically reduced both basal and A. alternata‐induced jasmonoyl‐isoleucine (JA‐Ile) and ABA. Further evidence showed that NaWRKY70 directly binds to the W‐boxes of the promoters of NaAOS and NaJAR4 (JA biosynthesis), NaNCED1 and NaXD1‐like (ABA biosynthesis), and NaMPK4 (ABA signaling) to activate their expression, while binding but repressing the expression of NaCYP707A4‐like3 (ABA degradation). Additionally, NaWRKY70 regulates capsidiol production through its key enzyme genes NaEASs and NaEAHs, and interacts with its regulator NaERF2‐like to enhance their expression, whereas ABA negatively regulates capsidiol biosynthesis.Our results highlight the key role of NaWRKY70 in controlling both JA‐Ile and ABA production, as well as capsidiol production, thus providing new insight into the defense mechanism of plant resistance to A. alternata. [ABSTRACT FROM AUTHOR]

Plant cells undergo extensive transcriptional reprogramming following pathogen infection, with these reprogramming patterns becoming more complex when pathogens, such as hemibiotrophs, exhibit different lifestyles. These transcriptional changes are often orchestrated by MYB, WRKY and AP2/ERF transcription factors (TFs), which modulate both growth and defence‐related gene expression. Transcriptional analysis of defence‐related genes in avocado (Persea americana) infected with Phytophthora cinnamomi indicated differential immune response activation when comparing a partially resistant and susceptible rootstock. This study identified 226 MYB, 82 WRKY, and 174 AP2/ERF TF‐encoding genes in avocado, using a genome‐wide approach. Phylogenetic analysis revealed substantial sequence conservation within TF groups underscoring their functional significance. RNA‐sequencing analysis in a partially resistant and susceptible avocado rootstock infected with P. cinnamomi was indicative of an immune response switch occurring in either rootstock after 24 and 6 h post‐inoculation, respectively. Different clusters of co‐expressed TF genes were observed at these times, suggesting the activation of necrotroph‐related immune responses at varying intervals between the two rootstocks. This study aids our understanding of avocado immune response activation following P. cinnamomi infection, and the role of the TFs therein, elucidating the transcriptional reprogramming disparities between partially resistant and susceptible rootstocks. [ABSTRACT FROM AUTHOR]

Cold stress is a major meteorological threat to crop growth and yield. Abscisic acid (ABA) plays important roles in plant cold tolerance by activating the expression of cold‐responsive genes; however, the underlying transcriptional regulatory module remains unknown. Here, we demonstrated that the cold‐ and ABA‐responsive transcription factor ETHYLENE RESPONSE FACTOR 15 (ERF15) positively regulates ABA‐mediated cold tolerance in tomato. Exogenous ABA treatment significantly enhanced cold tolerance in wild‐type tomato plants but failed to rescue erf15 mutants from cold stress. Transcriptome analysis showed that ERF15 was associated with the expression of cold‐responsive transcription factors such as CBF1 and WRKY6. Further RT‐qPCR assays confirmed that the ABA‐induced increased in CBF1 and WRKY6 transcripts was suppressed in erf15 mutants when the plants were subjected to cold treatment. Moreover, yeast one‐hybrid assays, dual‐luciferase assays and electrophoretic mobility shift assays demonstrated that ERF15 activated the transcription of CBF1 and WRKY6 by binding their promoters. Silencing CBF1 or WRKY6 significantly decreased cold tolerance. Overall, our study identified the role of ERF15 in conferring ABA‐mediated cold tolerance in tomato plants by activating CBF1 and WRKY6 expression. This study not only broadens our knowledge of the mechanism of ABA‐mediated cold tolerance in plants but also highlights ERF15 as an ideal target gene for cold‐tolerant crop breeding. Summary statement: This study demonstrated that the cold‐ and abscisic acid (ABA)‐responsive transcription factor ETHYLENE RESPONSE FACTOR 15 positively regulates ABA‐mediated cold tolerance in tomato plants by activating CBF1 and WRKY6 expression. [ABSTRACT FROM AUTHOR]

SUMMARY: Plants growing under natural conditions experience high light (HL) intensities that are often accompanied by elevated temperatures. These conditions could affect photosynthesis, reduce yield, and negatively impact agricultural productivity. The combination of different abiotic challenges creates a new type of stress for plants by generating complex environmental conditions that often exceed the impact of their individual parts. Transcription factors (TFs) play a key role in integrating the different molecular signals generated by multiple stress conditions, orchestrating the acclimation response of plants to stress. In this study, we show that the TF WRKY48 negatively controls the acclimation of Arabidopsis thaliana plants to a combination of HL and heat stress (HL + HS), and its expression is attenuated by jasmonic acid under HL + HS conditions. Using comparative physiological and transcriptomic analyses between wild‐type and wrky48 mutants, we further demonstrate that under control conditions, WRKY48 represses the expression of a set of transcripts that are specifically required for the acclimation of plants to HL + HS, hence its suppression during the HL + HS stress combination contributes to plant survival under these conditions. Accordingly, mutants that lack WRKY48 are more resistant to HL + HS, and transgenic plants that overexpress WRKY48 are more sensitive to it. Taken together, our findings reveal that WRKY48 is a negative regulator of the transcriptomic response of Arabidopsis to HL + HS and provide new insights into the complex regulatory networks of plant acclimation to stress combination. Significance Statement: Our study reveals that the transcription factor WRKY48 plays a key role as a suppressor of Arabidopsis thaliana tolerance to the combination of high light and heat stress. We further propose that jasmonic acid negatively regulates WRKY48 expression under stress combination. [ABSTRACT FROM AUTHOR]

SUMMARY: Plant immune receptors, known as NOD‐like receptors (NLRs), possess unique integrated decoy domains that enable plants to attract pathogen effectors and initiate a specific immune response. The present study aimed to create a library of these integrated domains (IDs) and screen them with pathogen effectors to identify targets for effector virulence and NLR–effector interactions. This works compiles IDs found in NLRs from seven different plant species and produced a library of 78 plasmid clones containing a total of 104 IDs, representing 43 distinct InterPro domains. A yeast two‐hybrid assay was conducted, followed by an in planta interaction test, using 32 conserved effectors from Ralstonia pseudosolanacearum type III. Through these screenings, three interactions involving different IDs (kinase, DUF3542, WRKY) were discovered interacting with two unrelated type III effectors (RipAE and PopP2). Of particular interest was the interaction between PopP2 and ID#85, an atypical WRKY domain integrated into a soybean NLR gene (GmNLR‐ID#85). Using a Förster resonance energy transfer‐fluorescence lifetime imaging microscopy technique to detect protein–protein interactions in living plant cells, PopP2 was demonstrated to physically associate with ID#85 in the nucleus. However, unlike the known WRKY‐containing Arabidopsis RRS1‐R NLR receptor, GmNLR‐ID#85 could not be acetylated by PopP2 and failed to activate RPS4‐dependent immunity when introduced into the RRS1‐R immune receptor. The generated library of 78 plasmid clones, encompassing these screenable IDs, is publicly available through Addgene. This resource is expected to be valuable for the scientific community with respect to discovering targets for effectors and potentially engineering plant immune receptors. Significance Statement: This work provides a library of 104 unique integrated domains cloned from seven crop and model plants. It allows the exploition of NLR gene evolutionary diversity for the detection of pathogen effector virulence targets and putative effector–NOD‐like receptor (NLR) pairs. When screening for interaction with conserved Ralstonia pseudosolanacearum type III effectors, we uncovered an atypical WRKY domain integrated in a soybean NLR as a target of PopP2. [ABSTRACT FROM AUTHOR]

SUMMARY: 'Candidatus Phytoplasma tritici' ('Ca. P. tritici') is an insect‐borne obligate pathogen that infects wheat (Triticum aestivum) causing wheat blue dwarf disease, and leads to yield losses. SWP12 is a potential effector secreted by 'Ca. P. tritici' that manipulates host processes to create an environment conducive to phytoplasma colonization, but the detailed mechanism of action remains to be investigated. In this study, the expression of SWP12 weakened the basal immunity of Nicotiana benthamiana and promoted leaf colonization by Phytophthora parasitica, Sclerotinia sclerotiorum, and tobacco mild green mosaic virus. Moreover, the expression of SWP12 in wheat plants promoted phytoplasma colonization. Triticum aestivum WRKY74 and N. benthamiana WRKY17 were identified as host targets of SWP12. The expression of TaWRKY74 triggered reactive oxygen species bursts, upregulated defense‐related genes, and decreased TaCRR6 transcription, leading to reductions in NADH dehydrogenase complex (NDH) activity. Expression of TaWRKY74 in wheat increased plant resistance to 'Ca. P. tritici', and silencing of TaWRKY74 enhanced plant susceptibility, which indicates that TaWRKY74 is a positive regulator of wheat resistance to 'Ca. P. tritici'. We showed that SWP12 weakens plant resistance and promotes 'Ca. P. tritici' colonization by destabilizing TaWRKY74. Significance Statement: Phytoplasma is an important group of plant pathogens, and infection of wheat (Triticum aestivum) can lead to yield reduction. Understanding the pathogenic mechanism of wheat blue dwarf phytoplasma effectors will help to improve the prevention and control strategies of wheat phytoplasma diseases. [ABSTRACT FROM AUTHOR]

Summary: CaWRKY40 in pepper (Capsicum annuum) promotes immune responses to Ralstonia solanacearum infection (RSI) and to high‐temperature, high‐humidity (HTHH) stress, but how it interacts with upstream signalling components remains poorly understood.Here, using approaches of reverse genetics, biochemical and molecular biology we functionally characterised the relationships among the WRKYGMK‐containing WRKY protein CaWRKY27b, the calcium‐dependent protein kinase CaCDPK29, and CaWRKY40 during pepper response to RSI or HTHH.Our data indicate that CaWRKY27b is upregulated and translocated from the cytoplasm to the nucleus upon phosphorylation of Ser137 in the nuclear localisation signal by CaCDPK29. Using electrophoretic mobility shift assays and microscale thermophoresis, we observed that, due to the replacement of Q by M in the conserved WRKYGQK, CaWRKY27b in the nucleus failed to bind to W‐boxes in the promoters of immunity‐ and thermotolerance‐related marker genes. Instead, CaWRKY27b interacted with CaWRKY40 and promoted its binding and positive regulation of the tested marker genes including CaNPR1, CaDEF1 and CaHSP24. Notably, mutation of the WRKYGMK motif in CaWRKY27b to WRKYGQK restored the W‐box binding ability.Our data therefore suggest that CaWRKY27b is phosphorylated by CaCDPK29 and acts as a transcriptional activator of CaWRKY40 during the pepper response to RSI and HTHH. [ABSTRACT FROM AUTHOR]

Geminiviruses constitute the largest group of known plant viruses and cause devastating losses to a wide range of crops and woody plants globally. Mulberry mosaic dwarf‐associated virus (MMDaV), identified from Chinese mulberry trees via small RNA‐based deep sequencing, is a divergent monopartite geminivirus belonging to the genus Mulcrilevirus of the family Geminiviridae. Previous studies have shown that plants employ multiple layers of defence to protect themselves from geminivirus infection. The interplay between plant and MMDaV is nevertheless less studied. This study presents evidence that MMDaV triggers hypersensitive response (HR)‐mediated antiviral defence in Nicotiana benthamiana plants. We show that the RepA protein of MMDaV is engaged in HR‐type cell death induction. We find that the RepA mutants with compromised nuclear localization ability impair their capabilities of cell death induction. Virus‐induced gene silencing of the key components of the R protein‐mediated signalling pathway reveals that down‐regulation of the nucleus‐targeting NbWRKY1 alleviates the cell death induction activity of RepA. We further demonstrate that RepA up‐regulates the transcript level of NbWRKY1. Furthermore, expression of RepA in N. benthamiana confers plant resistance against two begomoviruses. We propose that plant resistance against RepA can be potentially used to improve plant defence against geminiviruses in crops. [ABSTRACT FROM AUTHOR]

Septoria tritici blotch (STB), caused by the fungus Zymoseptoria tritici, is currently the main threat to worldwide wheat production in temperate climates. Understanding the genetic mechanisms that underpin the Z. tritici–wheat interaction will be crucial for generating new control strategies against STB. Plant hormones are essential regulators of growth and immunity. In particular, jasmonic acid (JA) plays a central role in defence against necrotrophic plant pathogens, but its role in mediating immunity against Z. tritici is largely unknown. Here we identify the transcription factor TaWRKY10 that is taxonomically restricted to the grass subfamily Pooideae as a novel regulator of JA responses in wheat. We demonstrate by using virus‐induced gene silencing that silencing TaWRKY10 leads to increased resistance against Z. tritici with an earlier onset of necrotic symptoms, but with reduced pathogen sporulation. Wheat plants silenced for TaWRKY10 show an up‐regulated JA response. Transcriptional profiling of TaWRKY10 knock‐down plants indicates that it is a negative regulator of the JA receptor TaCOI1 gene expression. Our data indicate that TaWRKY10 down‐regulates JA responses, contributing to the susceptibility of wheat to Z. tritici. We postulate that manipulating TaWRKY10 may provide a strategy to boost STB resistance in wheat. [ABSTRACT FROM AUTHOR]

Summary: Plant Phosphate Transporter 1 (PHT1) proteins, probably the only influx transporters for phosphate (Pi) uptake, are partially degraded on sufficient Pi levels to prevent excessive Pi accumulation. Therefore, the basal/constitutive expression level of PHT1 genes is vital for maintaining Pi uptake under Pi‐replete conditions.Rice (Oryza sativa) OsPHT1;1 is a unique gene as it is highly expressed and not responsive to Pi, however the mechanism for maintaining its basal/constitutive expression remains unknown. Using biochemical and genetic approaches, we identified and functionally characterised the transcription factors maintaining the basal/constitutive expression of OsPHT1;1.OsWRKY21 and OsWRKY108 interact within the nucleus and both bind to the W‐box in the OsPHT1;1 promoter. Overexpression of OsWRKY21 or OsWRKY108 led to increased Pi accumulation, resulting from elevated expression of OsPHT1;1. By contrast, oswrky21 oswrky108 double mutants showed decreased Pi accumulation and OsPHT1;1 expression in a Pi‐dependent manner. Moreover, similar to ospht1;1 mutants, plants expressing the OsWRKY21–SRDX fusion protein (a chimeric dominant suppressor) were impaired in Pi accumulation in Pi‐replete roots, accompanied by downregulation of OsPHT1;1 expression.Our findings demonstrated that rice WRKY transcription factors function redundantly to promote Pi uptake by activating OsPHT1;1 expression under Pi‐replete conditions, and represent a novel pathway independent of the central Pi signalling system. [ABSTRACT FROM AUTHOR]

CaWRKY40 was previously found to be transcriptionally up‐regulated by Ralstonia solanacearum inoculation (RSI) or heat stress (HS), but the underlying mechanism remains unknown. Herein, we report that a double‐W box‐element (DWE) in the promoter of CaWRKY40 is critical for these responses. The upstream W box unit WI of this composite element is crucial for preferential binding by CaWRKY40 and responsiveness to RSI or HS. DWE‐driven CaWRKY40 can be transcriptionally and nonspecifically regulated by itself and by CaWRKY58 and CaWRKY27. The DWE was also found in the promoters of CaWRKY40 orthologs, including AtWRKY40, VvWRKY40, GmWRKY40, CplWRKY40, SaWRKY40, SpWRKY40, NtWRKY40, and NaWRKY40. DWEAtWRKY40 was analogous to DWECaWRKY40 by responding to RSI or HS and AtWRKY40 expression. These data suggest that a conserved response of plants to pathogen infection or HS is probably mediated by binding of the DWE by WRKY40. [ABSTRACT FROM AUTHOR]

SUMMARY: Rhizoctonia solani is a soil‐borne necrotrophic fungus that causes sheath blight in grasses. The basal resistance of compatible interactions between R. solani and rice is known to be modulated by some WRKY transcription factors (TFs). However, genes and defense responses involved in incompatible interaction with R. solani remain unexplored, because no such interactions are known in any host plants. Recently, we demonstrated that Bd3‐1, an accession of the model grass Brachypodium distachyon, is resistant to R. solani and, upon inoculation with the fungus, undergoes rapid induction of genes responsive to the phytohormone salicylic acid (SA) that encode the WRKY TFs BdWRKY38 and BdWRKY44. Here, we show that endogenous SA and these WRKY TFs positively regulate this accession‐specific R. solani resistance. In contrast to a susceptible accession (Bd21), the infection process in the resistant accessions Bd3‐1 and Tek‐3 was suppressed at early stages before the development of fungal biomass and infection machinery. A comparative transcriptome analysis during pathogen infection revealed that putative WRKY‐dependent defense genes were induced faster in the resistant accessions than in Bd21. A gene regulatory network (GRN) analysis based on the transcriptome dataset demonstrated that BdWRKY38 was a GRN hub connected to many target genes specifically in resistant accessions, whereas BdWRKY44 was shared in the GRNs of all three accessions. Moreover, overexpression of BdWRKY38 increased R. solani resistance in Bd21. Our findings demonstrate that these resistant accessions can activate an incompatible host response to R. solani, and BdWRKY38 regulates this response by mediating SA signaling. Significance Statement: Rhizoctonia solani is a plant pathogenic fungus that causes sheath blight in grasses. In accordance with its necrotrophic lifestyle with a broad host range, the incompatible plant–R. solani interactions are not known in any host plants. Here, we demonstrate that such interactions could occur in the small grass Brachypodium distachyon, and BdWRKY38 is a key transcription factor that positively regulates this interaction by mediating the salicylic acid signaling pathway. [ABSTRACT FROM AUTHOR]

Leaf senescence is a complex and precise regulatory process that is correlated with numerous internal and environmental factors. Leaf senescence is tightly related to the redistribution of nutrients, which significantly affects productivity and quality, especially in crops. Evidence shows that the mediation of transcriptional regulation by WRKY transcription factors is vital for the fine‐tuning of leaf senescence. However, the underlying mechanisms of the involvement of WRKY in leaf senescence are still unclear in wheat.Using RNA sequencing data, we isolated a novel WRKY transcription factor, TaWRKY40‐D, which localizes in the nucleus and is basically induced by the progression of leaf senescence.TaWRKY40‐D is a promoter of natural and dark‐induced leaf senescence in transgenic Arabidopsis thaliana and wheat. We also demonstrated a positive response of TaWRKY40‐D in wheat upon jasmonic acid (JA) and abscisic acid (ABA) treatment. Consistent with this, the detached leaves of TaWRKY40‐D VIGS (virus‐induced gene silencing) wheat plants showed a stay‐green phenotype, while TaWRKY40‐D overexpressing Arabidopsis plants showed premature leaf senescence after JA and ABA treatment. Moreover, our results revealed that TaWRKY40‐D positively regulates leaf senescence, possibly by altering the biosynthesis and signalling of JA and ABA pathway genes.Together, our results suggest a new regulator of JA‐ and ABA‐related leaf senescence, as well as a new candidate gene that can be used for molecular breeding in wheat. [ABSTRACT FROM AUTHOR]

Phosphate (Pi) deficiency severely restricts plant growth and development, as Pi is an essential macronutrient. Calcium (Ca2+) is a ubiquitous second messenger in plants; calcineurin B-like proteins (CBL) and CBL-interacting protein kinases (CIPK) are signalling pathways that act as an important Ca2+ signalling network which integrates plants to fine tune the response to stress; however, whether CIPK are involved in Pi deficiency stress remains largely unknown. • In this study, we carried out a reverse genetic strategy to screen T-DNA insertion mutants of CIPK isoforms under Pi deficiency in Arabidopsis thaliana. Then Pi content, transcription of phosphate starvation-induced (PSI) genes, acid phosphatase activity and hydrogen peroxide were determined in the wild-type (WT) and cipk1 mutant, respectively. The phenotype of CIPK1 complementation lines was analysed. • The cipk1 mutant had a more sensitive phenotype, with lower root elongation and root length, and decreased Pi content compared with the WT under Pi deficiency. Moreover, CIPK1 mutation caused phosphate starvation-induced (PSI) genes to be significantly induced under Pi deficiency. Histological staining demonstrated that the cipk1 mutant had increased acid phosphatase activity and hydrogen peroxide concentration under Pi deficiency. By using the yeast two-hybrid system, we further demonstrated the interaction between CIPK1 and the WRKY transcription factors, WRKY6 and WRKY42. • Overall, we demonstrate that CIPK1 is involved in the Pi deficiency signalling pathway in A. thaliana, revealing the important role of Ca2+ in the Pi nutrition signalling pathway, and potentially providing a theoretical foundation for molecular breeding of crops with better Pi utilization efficiency. [ABSTRACT FROM AUTHOR]

Summary: Boron (B) deficiency is one of the major causes of growth inhibition and yield reduction in Brassica napus (B. napus). However, the molecular mechanisms of low B adaptation in B. napus are largely unknown. Here, fifty‐one BnaWRKY transcription factors were identified as responsive to B deficiency in B. napus, in which BnaAn.WRKY26, BnaA9.WRKY47, BnaA1.WKRY53 and BnaCn.WRKY57 were tested in yeast one‐hybrid assays and showed strong binding activity with conserved sequences containing a W box in the promoters of the B transport‐related genes BnaNIP5;1s and BnaBOR1s. Green fluorescent protein fused to the target protein demonstrated the nuclear localization of BnaA9.WRKY47. CRISPR/Cas9‐mediated knockout lines of BnaA9.WRKY47 in B. napus had increased sensitivity to low B and lower contents of B than wild‐type plants. In contrast, overexpression of BnaA9.WRKY47 enhanced the adaptation to low B with higher B contents in tissues than in wild‐type plants. Consistent with the phenotypic response and B accumulation in these transgenic lines, the transcription activity of BnaA3.NIP5;1, a B efficiency candidate gene, was decreased in the knockout lines but was significantly increased in the overexpressing lines under low B conditions. Electrophoretic mobility shift assays, transient expression experiments in tobacco and in situ hybridizations showed that BnaA9.WRKY47 directly activated BnaA3.NIP5;1 expression through binding to the specific cis‐element. Taken together, our findings support BnaWRKYs as new participants in response to low B, and BnaA9.WRKY47 contributes to the adaptation of B. napus to B deficiency through up‐regulating BnaA3.NIP5;1 expression to facilitate efficient B uptake. [ABSTRACT FROM AUTHOR]

Summary: The accumulation of secondary metabolites and the regulation of tissue acidity contribute to the important traits of grape berry and influence plant performance in response to abiotic and biotic factors. In several plant species a highly conserved MYB–bHLH–WD (MBW) transcriptional regulatory complex controls flavonoid pigment synthesis and transport, and vacuolar acidification in epidermal cells. An additional component, represented by a WRKY‐type transcription factor, physically interacts with the complex increasing the expression of some target genes and adding specificity for other targets. Here we investigated the function of MBW(W) complexes involving two MYBs (VvMYB5a and VvMYB5b) and the WRKY factor VvWRKY26 in grapevine (Vitis vinifera L.). Using transgenic grapevine plants we showed that these complexes affected different aspects of morphology, plant development, pH regulation, and pigment accumulation. Transcriptomic analysis identified a core set of putative target genes controlled by VvMYB5a, VvMYB5b, and VvWRKY26 in different tissues. Our data indicated that VvWRKY26 enhances the expression of selected target genes induced by VvMYB5a/b. Among these targets are genes involved in vacuolar hyper‐acidification, such as the P‐type ATPases VvPH5 and VvPH1, and trafficking, and genes involved in the biosynthesis of flavonoids. In addition, VvWRKY26 is recruited specifically by VvMYB5a, reflecting the functional diversification of VvMYB5a and VvMYB5b. The expression of MBWW complexes in vegetative organs, such as leaves, indicates a possible function of vacuolar hyper‐acidification in the repulsion of herbivores and/or in developmental processes, as shown by defects in transgenic grape plants where the complex is inactivated. Significance Statement: The two R2R3‐MYB paralogs VvMYB5a and VvMYB5b and the WRKY‐type transcription factor VvWRKY26 are involved in the regulation of vacuolar hyper‐acidification during early development of grapevine organs and the alteration of their expression affects morphology, plant development, and pigment accumulation. VvWRKY26 is part of the MYB5‐driven MBWW complexes, enhances the expression of VvMYB5a and VvMYB5b target genes, and is apparently recruited specifically by VvMYB5a, therefore contributing to the functional diversification of VvMYB5a and VvMYB5b. [ABSTRACT FROM AUTHOR]

Summary: Systemic acquired acclimation (SAA) plays a key role in optimizing growth and preventing damage associated with fluctuating or abrupt changes in the plant environment. To be effective, SAA has to occur at a rapid rate and depend on rapid signaling pathways that transmit signals from affected tissues to all parts of the plant. Although recent studies have identified several different rapid systemic signaling pathways that could mediate SAA, very little information is known about the extent of their involvement in mediating transcriptomic responses. Here we reveal that the systemic transcriptomic response of plants to excess light stress is extensive in its context and involves an early (2 min) and transient stage of transcript expression that includes thousands of genes. This early response is dependent on the respiratory burst oxidase homolog D protein, and the function of the reactive oxygen species (ROS) wave. We further identify a core set of transcripts associated with the ROS wave and suggest that some of these transcripts are involved in linking ROS with calcium signaling. Priming of a systemic leaf to become acclimated to a particular stress during SAA involves thousands of transcripts that display a rapid and transient expression pattern driven by the ROS wave. Significance Statement: The reactive oxygen species (ROS) wave is required for a rapid, early and transient systemic transcriptomic response to light stress that includes thousands of transcripts, many of them important for light stress acclimation. A core set of transcripts that includes several transcriptional regulators involved in H2O2 signaling is directly associated with the ROS wave and could be associated with systemic responses to many other stresses. [ABSTRACT FROM AUTHOR]

Summary: Our previous studies indicated that tomato WRKY1 transcription factor acts as a positive regulator during tomato resistance to Phytophthora infestans. However, the molecular mechanism of WRKY1‐mediated resistance regulation remains unclear. Here, we used a comparative transcriptome analysis between wild‐type and WRKY1‐overexpressing tomato plants to identify differentially expressed genes (DEGs) and long non‐coding RNAs (DELs), and we examined long non‐coding RNA (lncRNA)‐gene networks. The promoter sequences of the upregulated DEGs and DELs were analyzed. Among 1073 DEGs and 199 DELs, 1 kb 5′‐upstream regions of 59 DEGs and 22 DELs contain the W‐box, the target sequence of the WRKY1. The results of promoter−β‐glucuronidase (GUS) fusion and yeast one‐hybrid assay showed that lncRNA33732 was activated by WRKY1 through sequence‐specific interactions with the W‐box element in its promoter. The overexpression and silencing analysis of lncRNA33732 in tomato showed that lncRNA33732 acts as a positive regulator and enhanced tomato resistance to P. infestans by induction of the expression of respiratory burst oxidase (RBOH) and increase in the accumulation of H2O2. When the expression of RBOH gene was inhibited in tomato plants, H2O2 accumulation decreased and resistance were impaired. These findings suggest that lncRNA33732 activated by WRKY1 induces RBOH expression to increase H2O2 accumulation in early defense reaction of tomato to P. infestans attack. Our results provide insights into the WRKY1−lncRNA33732−RBOH module involved in the regulation of H2O2 accumulation and resistance to P. infestans, as well as provide candidates to enhance broad‐spectrum resistance to pathogens in tomato. Significance Statement: This study describes that WRKY1 can activate the expression of lncRNA33732 through sequence‐specific interactions with the W‐box element in its promoter, and that lncRNA33732 can induce RBOH expression to increase H2O2 accumulation during early defense reactions. Our results provide insights into the WRKY1−lncRNA33732−RBOH module involved in the regulation of H2O2 accumulation and resistance to P. infestans. [ABSTRACT FROM AUTHOR]

Summary: WRKY transcription factors (TFs) belong to a large family of regulatory proteins in plants that modulate many plant processes. Extensive studies have been conducted on WRKY‐mediated defense response in Arabidopsis thaliana and several crop species. Here, we aimed to investigate the potential roles and contributions of WRKY TFs in improving the defense response in the resynthesized Arabidopsis allotetraploids (Arabidopsis suecica) derived from two related autotetraploid progenitors, Arabidopsis thaliana (At4) and Arabidopsis arenosa (Aa). Rapid and differential induction of WRKY18 and WRKY40 expression was evident in response to Pseudomonas syringae and salicylic acid (SA) treatments in the allotetraploids. Selected direct targets of the WRKYs and PR1 also showed altered induction kinetics in the allotetraploids. Cleaved amplified polymorphic sequence analysis further revealed the accumulation of preferential homoeologous alleles (AtWRKY18, AaWRKY40, and AtWRKY60) in the allotetraploids, suggesting the potential for altered protein–protein interaction networks in the hybrids. Indeed, results showed that the cis‐interacting AtWRKY18/AtWRKY18 homodimer or trans‐interacting AtWRKY18/AaWRKY40 heterodimer exists as the preferred dimer interaction. Moreover, differential affinities of WRKY18 and WRKY40 homo‐ and heterodimers toward the W‐boxes in the WRKY60 promoter were observed. Transient and stable expression of the selected WRKYs in transgenic Arabidopsis further supported the idea that differential interactions lead to changes in PR1 induction and direct target expression under stress, respectively. Our data suggest that differential expression as well as differences in the strength of protein–protein and/or protein–DNA interactions among the WRKY homoeologs could lead to altered regulatory networks of defense genes, contributing to improved defense in allotetraploids. Significance statement: Differential expression kinetics and preferential homoeologous WRKY interactions contribute to altered biotic stress responses in Arabidopsis allotetraploids. [ABSTRACT FROM AUTHOR]

The emission of volatiles is a common, but mostly neglected, ability of bacteria that is important for inter‐ and intraspecific interactions. Currently, limited information is available on how the bacterial volatile (mVOC) signal is integrated into a plant's life at the physiological, transcriptional and metabolic level. Previous results provided evidence for volatile‐dependent regulation of WRKY18, a pathogen‐responsive transcription factor of Arabidopsis thaliana in co‐culture with two rhizobacteria, Serratia plymuthica HRO‐C48 and Stenotrophomonas maltophilia R3089.Dual cultures of these bacteria and A. thaliana; application of the common mVOC 2‐phenyl‐ethanol; extraction of metabolites of A. thaliana after exposure to bacterial volatiles; and analysis of the metabolomes (GC‐TOF/MS) were carried out.The prominent microbial aromatic compound 2‐phenyl‐ethanol, emitted by both bacteria, negatively affects growth of A. thaliana wild type, whereas WRKY18 T‐DNA insertion mutants were significantly more tolerant than wild‐type seedlings. This paper also demonstrates for the first time the impact of the rhizobacterial volatiles on the metabolome of A. thaliana. Upon mVOC exposure the plants rearrange their metabolism by accumulation of e.g. amino acids and TCA intermediates that potentially allow plants to cope with and survive this stress.Our findings illustrate the high degree of complexity of metabolic rearrangements underlying the interactions of bacterial volatile elicitors and resulting plant responses. Furthermore, the impact of the volatile 2‐phenyl‐ethanol as a signal in the WRKY18‐dependent pathway highlights this compound as an important molecular player. [ABSTRACT FROM AUTHOR]

Summary: 3‐O‐caffeoylquinic acid, also known as chlorogenic acid (CGA), functions as an intermediate in lignin biosynthesis in the phenylpropanoid pathway. It is widely distributed among numerous plant species and acts as an antioxidant in both plants and animals. Using GC‐MS, we discovered consistent and extreme variation in CGA content across a population of 739 4‐yr‐old Populus trichocarpa accessions. We performed genome‐wide association studies (GWAS) from 917 P. trichocarpa accessions and expression‐based quantitative trait loci (eQTL) analyses to identify key regulators. The GWAS and eQTL analyses resolved an overlapped interval encompassing a hydroxycinnamoyl‐CoA:shikimate hydroxycinnamoyl transferase 2 (PtHCT2) that was significantly associated with CGA and partially characterized metabolite abundances. PtHCT2 leaf expression was significantly correlated with CGA abundance and it was regulated by cis‐eQTLs containing W‐box for WRKY binding. Among all nine PtHCT homologs, PtHCT2 is the only one that responds to infection by the fungal pathogen Sphaerulina musiva (a Populus pathogen). Validation using protoplast‐based transient expression system suggests that PtHCT2 is regulated by the defense‐responsive WRKY. These results are consistent with reports of CGA functioning as an antioxidant in response to biotic stress. This study provides insights into data‐driven and omics‐based inference of gene function in woody species. [ABSTRACT FROM AUTHOR]

We previously reported that one of the ethylene response factors ( ERFs), Nt ERF3, and other members of the subgroup VIII-a ERFs of the AP2/ ERF family exhibit cell death-inducing ability in tobacco leaves. In this study, we focused on the involvement of Nt ERF3 in a cell death signalling pathway in tobacco plants, particularly downstream of Nt SIPK/Nt WIPK and Nt WRKY1, which are mitogen-activated protein kinases and a phosphorylation substrate of Nt SIPK, respectively. An ERF-associated amphiphilic repression ( EAR) motif-deficient Nt ERF3b mutant ( Nt ERF3bΔ EAR) that lacked cell death-inducing ability suppressed the induction of cell death caused by Nt ERF3a. The transient co-expression of Nt ERF3bΔ EAR suppressed the hypersensitive reaction ( HR)-like cell death induced by Nt SIPK and Nt WRKY1. The induction of cell death by Nt SIPK and Nt WRKY1 was also inhibited in transgenic plants expressing Nt ERF3bΔ EAR. Analysis of gene expression, ethylene production and cell death symptoms in salicylic acid-deficient tobacco plants suggested the existence of some feedback regulation in the HR cell death signalling pathway mediated by SIPK/ WIPK and WRKY1. Overall, these results suggest that Nt ERF3 functions downstream of Nt SIPK/ Nt WIPK and Nt WRKY1 in a cell death signalling pathway, with some feedback regulation. [ABSTRACT FROM AUTHOR]

Although seed dormancy is an important agronomic trait, its molecular basis is poorly understood. ABSCISIC ACID INSENSITIVE 3 (ABI3) plays an essential role in the establishment of seed dormancy. Here, we show that the lack of a seed-expressed WRKY transcription factor, WRKY41, confers reduced primary seed dormancy and thermoinhibition, phenotypes resembling those for a lack of ABI3. Loss-of-function abi3-17 and wrky41 alleles also both confer reduced sensitivity to ABA during germination and early seedling growth. Absence of WRKY41 decreases ABI3 transcript abundance in maturing and imbibed seeds, whereas transgenically overexpressing WRKY41 increases ABI3 expression. Moreover, transgenic overexpression of ABI3 completely restores seed dormancy phenotypes on wrky41. ChIP- qPCR and EMSA reveal that WRKY41 binds directly to the ABI3 promoter through three adjacent W-boxes, and a transactivation assay indicates that these W-boxes are essential for ABI3 expression. Whilst RT-qPCR analysis shows that the regulation of ABI3 by WRKY41 is not through ABA and other factors known to promote ABI3 transcription during seed maturation and germination, we also show that high concentrations of ABA might promote negative feedback regulation of WRKY41 expression. Finally, analysis of the wrky41 aba2 double mutant confirms that WRKY41 and ABA collaboratively regulate ABI3 expression and seed dormancy. In summary, our results demonstrate that WRKY41 is an important regulator of ABI3 expression, and hence of seed dormancy. [ABSTRACT FROM AUTHOR]

Foliar spray with BABA led to a significant reduction of lesion development in Brassica carinata caused by Alternaria brassicae. To get better insight into molecular mechanisms underlying priming of defence responses by BABA, expression pattern of Bc WRKY genes and marker genes for the SA and JA pathway namely PR-1 and PDF 1.2 was examined. Q- RT- PCR analysis revealed priming of Bc WRKY70, Bc WRKY11 and Bc WRKY53 gene expression in BABA-pretreated Brassica plants challenged with pathogen. However, the expression of Bc WRKY72 and Bc WRKY18 remained unchanged. Furthermore, Bc WRKY7 gene was found to be upregulated in water-treated plants in response to pathogen indicating its role in susceptibility. In addition, BABA application potentiated expression of defence genes PR-1, PDF1.2 and PAL in response to the pathogen. In conclusion, BABA-primed expression of Bc WRKY70, Bc WRKY11 and Bc WRKY53 genes is strongly correlated with enhanced expression of PR-1, PDF1.2 and PAL hence suggesting their role in BABA-induced resistance. [ABSTRACT FROM AUTHOR]

The development of a multicellular embryo from a single zygote is a complex and highly organized process that is far from understood. In higher plants, apical-basal patterning mechanisms are crucial to correctly specify root and shoot stem cell niches that will sustain and drive post-embryonic plant growth and development. The auxin-responsive AtWRKY23 transcription factor is expressed from early embryogenesis onwards and the timing and localization of its expression overlaps with the root stem cell niche. Knocking down WRKY23 transcript levels or expression of a dominant-negative WRKY23 version via a translational fusion with the SRDX repressor domain affected both apical-basal axis formation as well as installation of the root stem cell niche. WRKY23 expression is affected by two well-known root stem cell specification mechanisms, that is, SHORTROOT and MONOPTEROS-BODENLOS signalling and can partially rescue the root-forming inability of mp embryos. On the basis of these results, we postulate that a tightly controlled WRKY23 expression is involved in the regulation of both auxin-dependent and auxin-independent signalling pathways towards stem cell specification. [ABSTRACT FROM AUTHOR]

The transcriptional activator WRKY45 plays a major role in the salicylic acid/benzothiadiazole-induced defense program in rice. Here, we show that the nuclear ubiquitin-proteasome system (UPS) plays a role in regulating the function of WRKY45. Proteasome inhibitors induced accumulation of polyubiquitinated WRKY45 and transient up-regulation of WRKY45 target genes in rice cells, suggesting that WRKY45 is constantly degraded by the UPS to suppress defense responses in the absence of defense signals. Mutational analysis of the nuclear localization signal indicated that UPS-dependent WRKY45 degradation occurs in the nuclei. Interestingly, the transcriptional activity of WRKY45 after salicylic acid treatment was impaired by proteasome inhibition. The same C-terminal region in WRKY45 was essential for both transcriptional activity and UPS-dependent degradation. These results suggest that UPS regulation also plays a role in the transcriptional activity of WRKY45. It has been reported that AtNPR1, the central regulator of the salicylic acid pathway in Arabidopsis, is regulated by the UPS. We found that OsNPR1/NH1, the rice counterpart of NPR1, was not stabilized by proteasome inhibition under uninfected conditions. We discuss the differences in post-translational regulation of salicylic acid pathway components between rice and Arabidopsis. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) are small 21-nucleotide RNAs that post-transcriptionally regulate gene expression. MiR396 controls leaf development by targeting GRF and bHLH transcription factors in Arabidopsis. WRKY transcription factors, unique to plants, have been identified as mediating varied stress responses. The sunflower ( Helianthus annuus) HaWRKY6 is a particularly divergent WRKY gene exhibiting a putative target site for the miR396. A possible post-transcriptional regulation of HaWRKY6 by miR396 was investigated., Here, we used expression analyses, performed by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and northern blots together with computational approaches to establish the regulatory interaction between HaWRKY6 and the identified sunflower miR396. Arabidopsis plants expressing a mi396-resistant version of HaWRKY6 confirmed the miRNA-dependency of the HaWRKY6 silencing., Sunflower plants exposed to high temperatures or salicylic acid presented opposite expression of HaWRKY6 and miR396. Experiments using the wildtype and miRNA-resistant versions of HaWRKY6 showed altered stress responses. Our results showed a role of the recently evolved miR396 regulation of HaWRKY6 during early responses to high temperature., Our study reveals how a miRNA that normally regulates development has been recruited for high-temperature protection in sunflower, a plant particularly well adapted to this type of stress. [ABSTRACT FROM AUTHOR]

Interactions with the (a)biotic environment play key roles in a plant's fitness and vitality. In addition to direct surface-to-surface contact, volatile chemicals can also affect the physiology of organism. Volatiles of Serratia plymuthica and Stenotrophomonas maltophilia significantly inhibited growth and induced H2O2 production in Arabidopsis in dual culture. Within 1 day, transcriptional changes were observed by promoter-GUS assays using a stress-inducible W-box-containing 4x GST1 construct. Expression studies performed at 6, 12 and 24 h revealed altered transcript levels for 889 genes and 655 genes in response to Se. plymuthica or St. maltophilia volatiles, respectively. Expression of 162 genes was altered in both treatments. Meta-analysis revealed that specifically volatile-responsive genes were significantly overlapping with those affected by abiotic stress. We use the term mVAMP (microbial volatile-associated molecular pattern) to describe these volatile-specific responses. Genes responsive to both treatments were enriched for W-box motifs in their promoters, and were significantly enriched for transcription factors ( ERF2, ZAT10, MYB73 and WRKY18). The susceptibility of wrky18 mutant lines to volatiles was significantly delayed, suggesting an indispensable role for WRKY18 in bacterial volatile responses. [ABSTRACT FROM AUTHOR]

Arabidopsis seed size is regulated by the IKU pathway that includes IKU2 (a leucine-rich repeat kinase) and MINI3 (a WRKY transcription factor). We report the cloning of the IKU1 ( At2g35230) gene. iku1 mutants cause reduced endosperm growth and the production of small seeds. IKU1 encodes a protein containing a VQ motif, which is a motif specific to plants. IKU1 is expressed in the early endosperm and its progenitor, the central cell. Restoration of IKU1 function in the endosperm is sufficient to rescue seed size. A genomic construct carrying mutations in the VQ motif failed to complement the iku1 mutation, suggesting an essential role for the VQ motif. IKU1 interacts with MINI3 in the yeast two-hybrid system, consistent with an IKU1 function in the IKU-MINI pathway. Our data support the proposition that endosperm development is an important determinant of seed size. [ABSTRACT FROM AUTHOR]

• WRKY transcription factors are key regulators that activate and fine-tune stress responses, including defense responses against pathogens. We isolated a poplar ( Populus tremula× Populus alba) cDNA sequence, PtWRKY23, that encodes the ortholog of Arabidopsis WRKY23 and present the functional analysis of WRKY23, with emphasis on its potential role in resistance to rust infection. • To investigate the function of PtWRKY23, we examined PtWRKY23 expression after stress treatments by qRT-PCR and generated PtWRKY23-misexpressing plants. Transgenic plants were assessed for resistance to Melampsora rust and were analyzed using the poplar Affymetrix GeneChip® and histological techniques to study the consequences of PtWRKY23 misexpression. • PtWRKY23 is rapidly induced by Melampsora infection and elicitor treatments and poplars overexpressing and underexpressing PtWRKY23 were both more susceptible to Melampsora infection than wild type. Transcriptome analysis of PtWRKY23 overexpressors revealed a significant overlap with the Melampsora-infection response. Transcriptome analysis also suggests that PtWRKY23 affects redox homeostasis and cell wall-related metabolism, which was confirmed by analyses that showed that PtWRKY23-misexpressing plants have altered peroxidase activity, apparent H2O2 accumulation and lignin deposition. • Our results show that PtWRKY23 affects resistance to Melampsora infection and that this may be caused by deregulation of genes that disrupt redox homeostasis and cell wall metabolism. [ABSTRACT FROM AUTHOR]

• Pseudomonas fluorescens WCS417r bacteria and β-aminobutyric acid can induce disease resistance in Arabidopsis, which is based on priming of defence. • In this study, we examined the differences and similarities of WCS417r- and β-aminobutyric acid-induced priming. • Both WCS417r and β-aminobutyric acid prime for enhanced deposition of callose-rich papillae after infection by the oomycete Hyaloperonospora arabidopsis. This priming is regulated by convergent pathways, which depend on phosphoinositide- and ABA-dependent signalling components. Conversely, induced resistance by WCS417r and β-aminobutyric acid against the bacterial pathogen Pseudomonas syringae are controlled by distinct NPR1-dependent signalling pathways. As WCS417r and β-aminobutyric acid prime jasmonate- and salicylate-inducible genes, respectively, we subsequently investigated the role of transcription factors. A quantitative PCR-based genome-wide screen for putative WCS417r- and β-aminobutyric acid-responsive transcription factor genes revealed distinct sets of priming-responsive genes. Transcriptional analysis of a selection of these genes showed that they can serve as specific markers for priming. Promoter analysis of WRKY genes identified a putative cis-element that is strongly over-represented in promoters of 21 NPR1-dependent, β-aminobutyric acid-inducible WRKY genes. • Our study shows that priming of defence is regulated by different pathways, depending on the inducing agent and the challenging pathogen. Furthermore, we demon-strated that priming is associated with the enhanced expression of transcription factors. [ABSTRACT FROM AUTHOR]

Insect feeding on plants causes a complex series of coordinated defence responses. Little is known, however, about the time-dependent aspect of induced changes. Here we present a time series-based investigation of Arabidopsis thaliana L er subjected to attack by a specialist pest of Brassicaceae species, Brevicoryne brassicae. Transcriptome and metabolome changes were studied at 6, 12, 24 and 48 h after infestation to monitor the progress of early induced responses. The use of full-genome oligonucleotide microarrays revealed the initiation of extensive gene expression changes already during the first 6 h of infestation. Data indicated the involvement of reactive oxygen species (ROS) and calcium in early signalling, and salicylic acid (SA) and jasmonic acid (JA) in the regulation of defence responses. Transcripts related to senescence, biosynthesis of anti-insect proteins, indolyl glucosinolates (GS) and camalexin, as well as several uncharacterized to date WRKY transcription factors, were induced. Follow-up studies of defence-involved secondary metabolites revealed depositions of callose at the insects’ feeding sites, a decrease in the total level of aliphatic GS, particularly 3-hydroxypropyl glucosinolate, and accumulation of 4-methoxyindol-3-ylmethyl glucosinolate 48 h after the attack. The novel role of camalexin, induced as a part of defence against aphids, was verified in fitness experiments. Fecundity of B. brassicae was reduced on camalexin-accumulating wild-type (WT) plants as compared with camalexin-deficient pad3-1 mutants. Based on experimental data, a model of plant–aphid interactions at the early phase of infestation was proposed. [ABSTRACT FROM AUTHOR]

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

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

Abstract: Calmodulin (CaM) is a ubiquitous Ca2+-binding protein known to regulate diverse cellular functions by modulating the activity of various target proteins. We isolated a cDNA encoding AtWRKY7, a novel CaM-binding transcription factor, from an Arabidopsis expression library with horseradish peroxidase-conjugated CaM. CaM binds specifically to the Ca2+-dependent CaM-binding domain (CaMBD) of AtWRKY7, as shown by site-directed mutagenesis, a gel mobility shift assay, a split-ubiquitin assay, and a competition assay using a Ca2+/CaM-dependent enzyme. Furthermore, we show that the CaMBD of AtWRKY7 is a conserved structural motif (C-motif) found in group IId of the WRKY protein family. [Copyright &y& Elsevier]

Mitogen-activated protein kinase (MAPK) cascades are important signaling modules in eukaryotic cells that convert signals generated from the receptors/sensors to cellular responses. Upon activation, MAPKs can be translocated into nuclei where they phosphorylate transcription factors, which in turn activate gene expression. We recently identified NtMEK2, a tobacco MAPK kinase, as the upstream kinase of SIPK and WIPK, two well-characterized tobacco stress-responsive MAPKs. In the conditional gain-of-function NtMEK2 DD transgenic tobacco plants, the activation of endogenous SIPK and WIPK by NtMEK2DD induces several groups of defense genes, including 3-hydroxy-3-methlyglutaryl CoA reductase ( HMGR), basic pathogenesis related ( PR) genes, systemic acquired resistance gene 8.2 ( Sar 8.2), and harpin-induced gene1 ( Hin1). To identify the transcription factor(s) involved in the activation of these defense genes, we performed gel-mobility shift assays using nuclear extracts from NtMEK2 DD plants. Among the common cis-acting elements present in the promoters of defense-related genes, we observed a strong increase in the binding activity to the W box in nuclear extracts from the NtMEK2 DD plants but not the control NtMEK2 KR plants. The elevated W-box-binding activity in the nuclear extracts cannot be reversed by phosphatase treatment, excluding the possibility of a direct phosphorylation regulation of WRKY transcription factors by SIPK/WIPK. Instead, we observed a rapid increase in the expression of several WRKY genes in the NtMEK2 DD plants. These results suggest that the increase in W-box-binding activity after SIPK/WIPK activation is a result of WRKY gene activation, and the NtMEK2–SIPK/WIPK cascade is involved in regulating the expression of genes ranging from transcription factors to defense genes further downstream during plant defense responses. [ABSTRACT FROM AUTHOR]