Exploiting breakdown in nonhost effector-target interactions to boost host disease resistance.

Plants are resistant to most microbial species due to nonhost resistance (NHR), providing broad-spectrum and durable immunity. However, the molecular components contributing to NHR are poorly characterised. We address the question of whether failure of pathogen effectors to manipulate nonhost plants plays a critical role in NHR. RxLR (Arg-any amino acid-Leu-Arg) effectors from two oomycete pathogens, Phytophthora infestans and Hyaloperonospora arabidopsidis, enhanced pathogen infection when expressed in host plants (Nicotiana benthamiana and Arabidopsis, respectively) but the same effectors performed poorly in distantly related nonhost pathosystems. Putative target proteins in the host plant potato were identified for 64 P. infestans RxLR effectors using yeast 2-hybrid (Y2H) screens. Candidate orthologues of these target proteins in the distantly related non-host plant Arabidopsis were identified and screened using matrix Y2H for interaction with RxLR effectors from both P. infestans and H. arabidopsidis. Few P. infestans effector-target protein interactions were conserved from potato to candidate Arabidopsis target orthologues (cAtOrths). However, there was an enrichment of H. arabidopsidis RxLR effectors interacting with cAtOrths. We expressed the cAtOrth AtPUB33, which unlike its potato orthologue did not interact with P. infestans effector PiSFI3, in potato and Nicotiana benthamiana. Expression of AtPUB33 significantly reduced P. infestans colonization in both host plants. Our results provide evidence that failure of pathogen effectors to interact with and/or correctly manipulate target proteins in distantly related non-host plants contributes to NHR. Moreover, exploiting this breakdown in effector-nonhost target interaction, transferring effector target orthologues from non-host to host plants is a strategy to reduce disease.

Time-series transcriptomics reveals a BBX32-directed control of acclimation to high light in mature Arabidopsis leaves.

The photosynthetic capacity of mature leaves increases after several days' exposure to constant or intermittent episodes of high light (HL) and is manifested primarily as changes in chloroplast physiology. How this chloroplast-level acclimation to HL is initiated and controlled is unknown. From expanded Arabidopsis leaves, we determined HL-dependent changes in transcript abundance of 3844 genes in a 0-6 h time-series transcriptomics experiment. It was hypothesized that among such genes were those that contribute to the initiation of HL acclimation. By focusing on differentially expressed transcription (co-)factor genes and applying dynamic statistical modelling to the temporal transcriptomics data, a regulatory network of 47 predominantly photoreceptor-regulated transcription (co-)factor genes was inferred. The most connected gene in this network was B-BOX DOMAIN CONTAINING PROTEIN32 (BBX32). Plants overexpressing BBX32 were strongly impaired in acclimation to HL and displayed perturbed expression of photosynthesis-associated genes under LL and after exposure to HL. These observations led to demonstrating that as well as regulation of chloroplast-level acclimation by BBX32, CRYPTOCHROME1, LONG HYPOCOTYL5, CONSTITUTIVELY PHOTOMORPHOGENIC1 and SUPPRESSOR OF PHYA-105 are important. In addition, the BBX32-centric gene regulatory network provides a view of the transcriptional control of acclimation in mature leaves distinct from other photoreceptor-regulated processes, such as seedling photomorphogenesis.

Downy Mildew effector HaRxL21 interacts with the transcriptional repressor TOPLESS to promote pathogen susceptibility.

Hyaloperonospora arabidopsidis (Hpa) is an oomycete pathogen causing Arabidopsis downy mildew. Effector proteins secreted from the pathogen into the plant play key roles in promoting infection by suppressing plant immunity and manipulating the host to the pathogen's advantage. One class of oomycete effectors share a conserved 'RxLR' motif critical for their translocation into the host cell. Here we characterize the interaction between an RxLR effector, HaRxL21 (RxL21), and the Arabidopsis transcriptional co-repressor Topless (TPL). We establish that RxL21 and TPL interact via an EAR motif at the C-terminus of the effector, mimicking the host plant mechanism for recruiting TPL to sites of transcriptional repression. We show that this motif, and hence interaction with TPL, is necessary for the virulence function of the effector. Furthermore, we provide evidence that RxL21 uses the interaction with TPL, and its close relative TPL-related 1, to repress plant immunity and enhance host susceptibility to both biotrophic and necrotrophic pathogens.

Nitric oxide sensing in plants is mediated by proteolytic control of group VII ERF transcription factors.

Nitric oxide (NO) is an important signaling compound in prokaryotes and eukaryotes. In plants, NO regulates critical developmental transitions and stress responses. Here, we identify a mechanism for NO sensing that coordinates responses throughout development based on targeted degradation of plant-specific transcriptional regulators, the group VII ethylene response factors (ERFs). We show that the N-end rule pathway of targeted proteolysis targets these proteins for destruction in the presence of NO, and we establish them as critical regulators of diverse NO-regulated processes, including seed germination, stomatal closure, and hypocotyl elongation. Furthermore, we define the molecular mechanism for NO control of germination and crosstalk with abscisic acid (ABA) signaling through ERF-regulated expression of ABSCISIC ACID INSENSITIVE5 (ABI5). Our work demonstrates how NO sensing is integrated across multiple physiological processes by direct modulation of transcription factor stability and identifies group VII ERFs as central hubs for the perception of gaseous signals in plants.

Time-Series Transcriptomics Reveals That AGAMOUS-LIKE22 Affects Primary Metabolism and Developmental Processes in Drought-Stressed Arabidopsis.

In Arabidopsis thaliana, changes in metabolism and gene expression drive increased drought tolerance and initiate diverse drought avoidance and escape responses. To address regulatory processes that link these responses, we set out to identify genes that govern early responses to drought. To do this, a high-resolution time series transcriptomics data set was produced, coupled with detailed physiological and metabolic analyses of plants subjected to a slow transition from well-watered to drought conditions. A total of 1815 drought-responsive differentially expressed genes were identified. The early changes in gene expression coincided with a drop in carbon assimilation, and only in the late stages with an increase in foliar abscisic acid content. To identify gene regulatory networks (GRNs) mediating the transition between the early and late stages of drought, we used Bayesian network modeling of differentially expressed transcription factor (TF) genes. This approach identified AGAMOUS-LIKE22 (AGL22), as key hub gene in a TF GRN. It has previously been shown that AGL22 is involved in the transition from vegetative state to flowering but here we show that AGL22 expression influences steady state photosynthetic rates and lifetime water use. This suggests that AGL22 uniquely regulates a transcriptional network during drought stress, linking changes in primary metabolism and the initiation of stress responses.

The Transcription Factor ATHB5 Affects GA-Mediated Plasticity in Hypocotyl Cell Growth during Seed Germination.

Gibberellic acid (GA)-mediated cell expansion initiates the seed-to-seedling transition in plants and is repressed by DELLA proteins. Using digital single-cell analysis, we identified a cellular subdomain within the midhypocotyl, whose expansion drives the final step of this developmental transition under optimal conditions. Using network inference, the transcription factor ATHB5 was identified as a genetic factor whose localized expression promotes GA-mediated expansion specifically within these cells. Both this protein and its putative growth-promoting target EXPANSIN3 are repressed by DELLA, and coregulated at single-cell resolution during seed germination. The cellular domains of hormone sensitivity were explored within the Arabidopsis (Arabidopsis thaliana) embryo by putting seeds under GA-limiting conditions and quantifying cellular growth responses. The middle and upper hypocotyl have a greater requirement for GA to promote cell expansion than the lower embryo axis. Under these conditions, germination was still completed following enhanced growth within the radicle and lower axis. Under GA-limiting conditions, the athb5 mutant did not show a phenotype at the level of seed germination, but it did at a cellular level with reduced cell expansion in the hypocotyl relative to the wild type. These data reveal that the spatiotemporal cell expansion events driving this transition are not determinate, and the conditional use of GA-ATHB5-mediated hypocotyl growth under optimal conditions may be used to optionally support rapid seedling growth. This study demonstrates that multiple genetic and spatiotemporal cell expansion mechanisms underlie the seed to seedling transition in Arabidopsis.

Transcriptional Dynamics Driving MAMP-Triggered Immunity and Pathogen Effector-Mediated Immunosuppression in Arabidopsis Leaves Following Infection with Pseudomonas syringae pv tomato DC3000.

Transcriptional reprogramming is integral to effective plant defense. Pathogen effectors act transcriptionally and posttranscriptionally to suppress defense responses. A major challenge to understanding disease and defense responses is discriminating between transcriptional reprogramming associated with microbial-associated molecular pattern (MAMP)-triggered immunity (MTI) and that orchestrated by effectors. A high-resolution time course of genome-wide expression changes following challenge with Pseudomonas syringae pv tomato DC3000 and the nonpathogenic mutant strain DC3000hrpA- allowed us to establish causal links between the activities of pathogen effectors and suppression of MTI and infer with high confidence a range of processes specifically targeted by effectors. Analysis of this information-rich data set with a range of computational tools provided insights into the earliest transcriptional events triggered by effector delivery, regulatory mechanisms recruited, and biological processes targeted. We show that the majority of genes contributing to disease or defense are induced within 6 h postinfection, significantly before pathogen multiplication. Suppression of chloroplast-associated genes is a rapid MAMP-triggered defense response, and suppression of genes involved in chromatin assembly and induction of ubiquitin-related genes coincide with pathogen-induced abscisic acid accumulation. Specific combinations of promoter motifs are engaged in fine-tuning the MTI response and active transcriptional suppression at specific promoter configurations by P. syringae.

Wigwams: identifying gene modules co-regulated across multiple biological conditions.

MOTIVATION: Identification of modules of co-regulated genes is a crucial first step towards dissecting the regulatory circuitry underlying biological processes. Co-regulated genes are likely to reveal themselves by showing tight co-expression, e.g. high correlation of expression profiles across multiple time series datasets. However, numbers of up- or downregulated genes are often large, making it difficult to discriminate between dependent co-expression resulting from co-regulation and independent co-expression. Furthermore, modules of co-regulated genes may only show tight co-expression across a subset of the time series, i.e. show condition-dependent regulation. RESULTS: Wigwams is a simple and efficient method to identify gene modules showing evidence for co-regulation in multiple time series of gene expression data. Wigwams analyzes similarities of gene expression patterns within each time series (condition) and directly tests the dependence or independence of these across different conditions. The expression pattern of each gene in each subset of conditions is tested statistically as a potential signature of a condition-dependent regulatory mechanism regulating multiple genes. Wigwams does not require particular time points and can process datasets that are on different time scales. Differential expression relative to control conditions can be taken into account. The output is succinct and non-redundant, enabling gene network reconstruction to be focused on those gene modules and combinations of conditions that show evidence for shared regulatory mechanisms. Wigwams was run using six Arabidopsis time series expression datasets, producing a set of biologically significant modules spanning different combinations of conditions. AVAILABILITY AND IMPLEMENTATION: A Matlab implementation of Wigwams, complete with graphical user interfaces and documentation, is available at: warwick.ac.uk/wigwams. .

An RxLR effector from Phytophthora infestans prevents re-localisation of two plant NAC transcription factors from the endoplasmic reticulum to the nucleus.

The potato late blight pathogen Phytophthora infestans secretes an array of effector proteins thought to act in its hosts by disarming defences and promoting pathogen colonisation. However, little is known about the host targets of these effectors and how they are manipulated by the pathogen. This work describes the identification of two putative membrane-associated NAC transcription factors (TF) as the host targets of the RxLR effector PITG_03192 (Pi03192). The effector interacts with NAC Targeted by Phytophthora (NTP) 1 and NTP2 at the endoplasmic reticulum (ER) membrane, where these proteins are localised. Transcripts of NTP1 and NTP2 rapidly accumulate following treatment with culture filtrate (CF) from in vitro grown P. infestans, which acts as a mixture of Phytophthora PAMPs and elicitors, but significantly decrease during P. infestans infection, indicating that pathogen activity may prevent their up-regulation. Silencing of NTP1 or NTP2 in the model host plant Nicotiana benthamiana increases susceptibility to P. infestans, whereas silencing of Pi03192 in P. infestans reduces pathogenicity. Transient expression of Pi03192 in planta restores pathogenicity of the Pi03192-silenced line. Moreover, colonisation by the Pi03192-silenced line is significantly enhanced on N. benthamiana plants in which either NTP1 or NTP2 have been silenced. StNTP1 and StNTP2 proteins are released from the ER membrane following treatment with P. infestans CF and accumulate in the nucleus, after which they are rapidly turned over by the 26S proteasome. In contrast, treatment with the defined PAMP flg22 fails to up-regulate NTP1 and NTP2, or promote re-localisation of their protein products to the nucleus, indicating that these events follow perception of a component of CF that appears to be independent of the FLS2/flg22 pathway. Importantly, Pi03192 prevents CF-triggered re-localisation of StNTP1 and StNTP2 from the ER into the nucleus, revealing a novel effector mode-of-action to promote disease progression.

From bench to bountiful harvests: a road map for the next decade of Arabidopsis research.

In the face of an increasing world population and climate instability, the demands for food and fuel will continue to rise. Plant science will be crucial to help meet these exponentially increasing requirements for food and fuel supplies. Fundamental plant research will play a major role in providing key advances in our understanding of basic plant processes that can then flow into practical advances through knowledge sharing and collaborations. The model plant Arabidopsis thaliana has played a major role in our understanding of plant biology, and the Arabidopsis community has developed many tools and resources to continue building on this knowledge. Drawing from previous experience of internationally coordinated projects, The international Arabidopsis community, represented by the Multinational Arabidopsis Steering Committee (MASC), has drawn up a road map for the next decade of Arabidopsis research to inform scientists and decision makers on the future foci of Arabidopsis research within the wider plant science landscape. This article provides a summary of the MASC road map.

Conserved noncoding sequences highlight shared components of regulatory networks in dicotyledonous plants.

Conserved noncoding sequences (CNSs) in DNA are reliable pointers to regulatory elements controlling gene expression. Using a comparative genomics approach with four dicotyledonous plant species (Arabidopsis thaliana, papaya [Carica papaya], poplar [Populus trichocarpa], and grape [Vitis vinifera]), we detected hundreds of CNSs upstream of Arabidopsis genes. Distinct positioning, length, and enrichment for transcription factor binding sites suggest these CNSs play a functional role in transcriptional regulation. The enrichment of transcription factors within the set of genes associated with CNS is consistent with the hypothesis that together they form part of a conserved transcriptional network whose function is to regulate other transcription factors and control development. We identified a set of promoters where regulatory mechanisms are likely to be shared between the model organism Arabidopsis and other dicots, providing areas of focus for further research.

Arabidopsis defense against Botrytis cinerea: chronology and regulation deciphered by high-resolution temporal transcriptomic analysis.

Transcriptional reprogramming forms a major part of a plant's response to pathogen infection. Many individual components and pathways operating during plant defense have been identified, but our knowledge of how these different components interact is still rudimentary. We generated a high-resolution time series of gene expression profiles from a single Arabidopsis thaliana leaf during infection by the necrotrophic fungal pathogen Botrytis cinerea. Approximately one-third of the Arabidopsis genome is differentially expressed during the first 48 h after infection, with the majority of changes in gene expression occurring before significant lesion development. We used computational tools to obtain a detailed chronology of the defense response against B. cinerea, highlighting the times at which signaling and metabolic processes change, and identify transcription factor families operating at different times after infection. Motif enrichment and network inference predicted regulatory interactions, and testing of one such prediction identified a role for TGA3 in defense against necrotrophic pathogens. These data provide an unprecedented level of detail about transcriptional changes during a defense response and are suited to systems biology analyses to generate predictive models of the gene regulatory networks mediating the Arabidopsis response to B. cinerea.

Genome sequence and analysis of the Irish potato famine pathogen Phytophthora infestans.

Phytophthora infestans is the most destructive pathogen of potato and a model organism for the oomycetes, a distinct lineage of fungus-like eukaryotes that are related to organisms such as brown algae and diatoms. As the agent of the Irish potato famine in the mid-nineteenth century, P. infestans has had a tremendous effect on human history, resulting in famine and population displacement. To this day, it affects world agriculture by causing the most destructive disease of potato, the fourth largest food crop and a critical alternative to the major cereal crops for feeding the world's population. Current annual worldwide potato crop losses due to late blight are conservatively estimated at $6.7 billion. Management of this devastating pathogen is challenged by its remarkable speed of adaptation to control strategies such as genetically resistant cultivars. Here we report the sequence of the P. infestans genome, which at approximately 240 megabases (Mb) is by far the largest and most complex genome sequenced so far in the chromalveolates. Its expansion results from a proliferation of repetitive DNA accounting for approximately 74% of the genome. Comparison with two other Phytophthora genomes showed rapid turnover and extensive expansion of specific families of secreted disease effector proteins, including many genes that are induced during infection or are predicted to have activities that alter host physiology. These fast-evolving effector genes are localized to highly dynamic and expanded regions of the P. infestans genome. This probably plays a crucial part in the rapid adaptability of the pathogen to host plants and underpins its evolutionary potential.

A local regulatory network around three NAC transcription factors in stress responses and senescence in Arabidopsis leaves.

A model is presented describing the gene regulatory network surrounding three similar NAC transcription factors that have roles in Arabidopsis leaf senescence and stress responses. ANAC019, ANAC055 and ANAC072 belong to the same clade of NAC domain genes and have overlapping expression patterns. A combination of promoter DNA/protein interactions identified using yeast 1-hybrid analysis and modelling using gene expression time course data has been applied to predict the regulatory network upstream of these genes. Similarities and divergence in regulation during a variety of stress responses are predicted by different combinations of upstream transcription factors binding and also by the modelling. Mutant analysis with potential upstream genes was used to test and confirm some of the predicted interactions. Gene expression analysis in mutants of ANAC019 and ANAC055 at different times during leaf senescence has revealed a distinctly different role for each of these genes. Yeast 1-hybrid analysis is shown to be a valuable tool that can distinguish clades of binding proteins and be used to test and quantify protein binding to predicted promoter motifs.

MEME-LaB: motif analysis in clusters.

SUMMARY: Genome-wide expression analysis can result in large numbers of clusters of co-expressed genes. Although there are tools for ab initio discovery of transcription factor-binding sites, most do not provide a quick and easy way to study large numbers of clusters. To address this, we introduce a web tool called MEME-LaB. The tool wraps MEME (an ab initio motif finder), providing an interface for users to input multiple gene clusters, retrieve promoter sequences, run motif finding and then easily browse and condense the results, facilitating better interpretation of the results from large-scale datasets. AVAILABILITY: MEME-LaB is freely accessible at: http://wsbc.warwick.ac.uk/wsbcToolsWebpage/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

The Pseudomonas syringae type III effector HopD1 suppresses effector-triggered immunity, localizes to the endoplasmic reticulum, and targets the Arabidopsis transcription factor NTL9.

• Pseudomonas syringae type III effectors are known to suppress plant immunity to promote bacterial virulence. However, the activities and targets of these effectors are not well understood. • We used genetic, molecular, and cell biology methods to characterize the activities, localization, and target of the HopD1 type III effector in Arabidopsis. • HopD1 contributes to P. syringae virulence in Arabidopsis and reduces effector-triggered immunity (ETI) responses but not pathogen-associated molecular pattern-triggered immunity (PTI) responses. Plants expressing HopD1 supported increased growth of ETI-inducing P. syringae strains compared with wild-type Arabidopsis. We show that HopD1 interacts with the membrane-tethered Arabidopsis transcription factor NTL9 and demonstrate that this interaction occurs at the endoplasmic reticulum (ER). A P. syringae hopD1 mutant and ETI-inducing P. syringae strains exhibited enhanced growth on Arabidopsis ntl9 mutant plants. Conversely, growth of P. syringae strains was reduced in plants expressing a constitutively active NTL9 derivative, indicating that NTL9 is a positive regulator of plant immunity. Furthermore, HopD1 inhibited the induction of NTL9-regulated genes during ETI but not PTI. • HopD1 contributes to P. syringae virulence in part by targeting NTL9, resulting in the suppression of ETI responses but not PTI responses and the promotion of plant pathogenicity.

High-resolution temporal profiling of transcripts during Arabidopsis leaf senescence reveals a distinct chronology of processes and regulation.

Leaf senescence is an essential developmental process that impacts dramatically on crop yields and involves altered regulation of thousands of genes and many metabolic and signaling pathways, resulting in major changes in the leaf. The regulation of senescence is complex, and although senescence regulatory genes have been characterized, there is little information on how these function in the global control of the process. We used microarray analysis to obtain a high-resolution time-course profile of gene expression during development of a single leaf over a 3-week period to senescence. A complex experimental design approach and a combination of methods were used to extract high-quality replicated data and to identify differentially expressed genes. The multiple time points enable the use of highly informative clustering to reveal distinct time points at which signaling and metabolic pathways change. Analysis of motif enrichment, as well as comparison of transcription factor (TF) families showing altered expression over the time course, identify clear groups of TFs active at different stages of leaf development and senescence. These data enable connection of metabolic processes, signaling pathways, and specific TF activity, which will underpin the development of network models to elucidate the process of senescence.

Subcellular localization of the Hpa RxLR effector repertoire identifies a tonoplast-associated protein HaRxL17 that confers enhanced plant susceptibility.

Filamentous phytopathogens form sophisticated intracellular feeding structures called haustoria in plant cells. Pathogen effectors are likely to play a role in the establishment and maintenance of haustoria in addition to their better-characterized role in suppressing plant defence. However, the specific mechanisms by which these effectors promote virulence remain unclear. To address this question, we examined changes in subcellular architecture using live-cell imaging during the compatible interaction between the oomycete Hyaloperonospora arabidopsidis (Hpa) and its host Arabidopsis. We monitored host-cell restructuring of subcellular compartments within plant mesophyll cells during haustoria ontogenesis. Live-cell imaging highlighted rearrangements in plant cell membranes upon infection, in particular to the tonoplast, which was located close to the extra-haustorial membrane surrounding the haustorium. We also investigated the subcellular localization patterns of Hpa RxLR effector candidates (HaRxLs) in planta. We identified two major classes of HaRxL effector based on localization: nuclear-localized effectors and membrane-localized effectors. Further, we identified a single effector, HaRxL17, that associated with the tonoplast in uninfected cells and with membranes around haustoria, probably the extra-haustorial membrane, in infected cells. Functional analysis of selected effector candidates in planta revealed that HaRxL17 enhances plant susceptibility. The roles of subcellular changes and effector localization, with specific reference to the potential role of HaRxL17 in plant cell membrane trafficking, are discussed with respect to Hpa virulence.

Multiple candidate effectors from the oomycete pathogen Hyaloperonospora arabidopsidis suppress host plant immunity.

Oomycete pathogens cause diverse plant diseases. To successfully colonize their hosts, they deliver a suite of effector proteins that can attenuate plant defenses. In the oomycete downy mildews, effectors carry a signal peptide and an RxLR motif. Hyaloperonospora arabidopsidis (Hpa) causes downy mildew on the model plant Arabidopsis thaliana (Arabidopsis). We investigated if candidate effectors predicted in the genome sequence of Hpa isolate Emoy2 (HaRxLs) were able to manipulate host defenses in different Arabidopsis accessions. We developed a rapid and sensitive screening method to test HaRxLs by delivering them via the bacterial type-three secretion system (TTSS) of Pseudomonas syringae pv tomato DC3000-LUX (Pst-LUX) and assessing changes in Pst-LUX growth in planta on 12 Arabidopsis accessions. The majority (~70%) of the 64 candidates tested positively contributed to Pst-LUX growth on more than one accession indicating that Hpa virulence likely involves multiple effectors with weak accession-specific effects. Further screening with a Pst mutant (ΔCEL) showed that HaRxLs that allow enhanced Pst-LUX growth usually suppress callose deposition, a hallmark of pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI). We found that HaRxLs are rarely strong avirulence determinants. Although some decreased Pst-LUX growth in particular accessions, none activated macroscopic cell death. Fewer HaRxLs conferred enhanced Pst growth on turnip, a non-host for Hpa, while several reduced it, consistent with the idea that turnip's non-host resistance against Hpa could involve a combination of recognized HaRxLs and ineffective HaRxLs. We verified our results by constitutively expressing in Arabidopsis a sub-set of HaRxLs. Several transgenic lines showed increased susceptibility to Hpa and attenuation of Arabidopsis PTI responses, confirming the HaRxLs' role in Hpa virulence. This study shows TTSS screening system provides a useful tool to test whether candidate effectors from eukaryotic pathogens can suppress/trigger plant defense mechanisms and to rank their effectiveness prior to subsequent mechanistic investigation.

MEDIATOR25 acts as an integrative hub for the regulation of jasmonate-responsive gene expression in Arabidopsis.

The PHYTOCHROME AND FLOWERING TIME1 gene encoding the MEDIATOR25 (MED25) subunit of the eukaryotic Mediator complex is a positive regulator of jasmonate (JA)-responsive gene expression in Arabidopsis (Arabidopsis thaliana). Based on the function of the Mediator complex as a bridge between DNA-bound transcriptional activators and the RNA polymerase II complex, MED25 has been hypothesized to function in association with transcriptional regulators of the JA pathway. However, it is currently not known mechanistically how MED25 functions to regulate JA-responsive gene expression. In this study, we show that MED25 physically interacts with several key transcriptional regulators of the JA signaling pathway, including the APETALA2 (AP2)/ETHYLENE RESPONSE FACTOR (ERF) transcription factors OCTADECANOID-RESPONSIVE ARABIDOPSIS AP2/ERF59 and ERF1 as well as the master regulator MYC2. Physical interaction detected between MED25 and four group IX AP2/ERF transcription factors was shown to require the activator interaction domain of MED25 as well as the recently discovered Conserved Motif IX-1/EDLL transcription activation motif of MED25-interacting AP2/ERFs. Using transcriptional activation experiments, we also show that OCTADECANOID-RESPONSIVE ARABIDOPSIS AP2/ERF59- and ERF1-dependent activation of PLANT DEFENSIN1.2 as well as MYC2-dependent activation of VEGETATIVE STORAGE PROTEIN1 requires a functional MED25. In addition, MED25 is required for MYC2-dependent repression of pathogen defense genes. These results suggest an important role for MED25 as an integrative hub within the Mediator complex during the regulation of JA-associated gene expression.