The Arabidopsis PHD-finger protein EDM2 has multiple roles in balancing NLR immune receptor gene expression.

Plant NLR-type receptors serve as sensitive triggers of host immunity. Their expression has to be well-balanced, due to their interference with various cellular processes and dose-dependency of their defense-inducing activity. A genetic "arms race" with fast-evolving pathogenic microbes requires plants to constantly innovate their NLR repertoires. We previously showed that insertion of the COPIA-R7 retrotransposon into RPP7 co-opted the epigenetic transposon silencing signal H3K9me2 to a new function promoting expression of this Arabidopsis thaliana NLR gene. Recruitment of the histone binding protein EDM2 to COPIA-R7-associated H3K9me2 is required for optimal expression of RPP7. By profiling of genome-wide effects of EDM2, we now uncovered additional examples illustrating effects of transposons on NLR gene expression, strongly suggesting that these mobile elements can play critical roles in the rapid evolution of plant NLR genes by providing the "raw material" for gene expression mechanisms. We further found EDM2 to have a global role in NLR expression control. Besides serving as a positive regulator of RPP7 and a small number of other NLR genes, EDM2 acts as a suppressor of a multitude of additional NLR genes. We speculate that the dual functionality of EDM2 in NLR expression control arose from the need to compensate for fitness penalties caused by high expression of some NLR genes by suppression of others. Moreover, we are providing new insights into functional relationships of EDM2 with its interaction partner, the RNA binding protein EDM3/AIPP1, and its target gene IBM1, encoding an H3K9-demethylase.

The Arabidopsis RRM domain protein EDM3 mediates race-specific disease resistance by controlling H3K9me2-dependent alternative polyadenylation of RPP7 immune receptor transcripts.

The NLR-receptor RPP7 mediates race-specific immunity in Arabidopsis. Previous screens for enhanced downy mildew (edm) mutants identified the co-chaperone SGT1b (EDM1) and the PHD-finger protein EDM2 as critical regulators of RPP7. Here, we describe a third edm mutant compromised in RPP7 immunity, edm3. EDM3 encodes a nuclear-localized protein featuring an RNA-recognition motif. Like EDM2, EDM3 promotes histone H3 lysine 9 dimethylation (H3K9me2) at RPP7. Global profiling of H3K9me2 showed EDM3 to affect this silencing mark at a large set of loci. Importantly, both EDM3 and EDM2 co-associate in vivo with H3K9me2-marked chromatin and transcripts at a critical proximal polyadenylation site of RPP7, where they suppress proximal transcript polyadeylation/termination. Our results highlight the complexity of plant NLR gene regulation, and establish a functional and physical link between a histone mark and NLR-transcript processing.

Aphid effector Me10 interacts with tomato TFT7, a 14-3-3 isoform involved in aphid resistance.

We demonstrated previously that expression of Macrosiphum euphorbiae salivary protein Me10 enhanced aphid reproduction on its host tomato (Solanum lycopersicum). However, the mechanism of action of Me10 remained elusive. To confirm the secretion of Me10 by the aphid into plant tissues, we produced Me10 polyclonal antibodies. To identify the plant targets of Me10, we developed a tomato immune induced complementary DNA yeast two-hybrid library and screened it with Me10 as bait. Immunoprecipitation and bimolecular fluorescence complementation (BiFC) assays were performed to validate one of the interactions in planta, and virus-induced gene silencing was used for functional characterization in tomato. We demonstrated that Me10 is secreted into the plant tissues and interacts with tomato 14-3-3 isoform 7 (TFT7) in yeast. Immunoprecipitation assays confirmed that Me10 and its homologue in Aphis gossypii, Ag10k, interact with TFT7 in planta. Further, BiFC revealed that Me10 interaction with TFT7 occurs in the plant cell cytoplasm. While silencing of TFT7 in tomato leaves did not affect tomato susceptibility to M. euphorbiae, it enhanced longevity and fecundity of A. gossypii, the non-host aphid. Our results suggest the model whereby TFT7 plays a role in aphid resistance in tomato and effectors of the Me10/Ag10k family interfere with TFT7 function during aphid infestation.

An alternative polyadenylation mechanism coopted to the Arabidopsis RPP7 gene through intronic retrotransposon domestication.

Transposable elements (TEs) can drive evolution by creating genetic and epigenetic variation. Although examples of adaptive TE insertions are accumulating, proof that epigenetic information carried by such "domesticated" TEs has been coopted to control host gene function is still limited. We show that COPIA-R7, a TE inserted into the Arabidopsis thaliana disease resistance gene RPP7 recruited the histone mark H3K9me2 to this locus. H3K9me2 levels at COPIA-R7 affect the choice between two alternative RPP7 polyadenylation sites in the pre-mRNA and, thereby, influence the critical balance between RPP7-coding and non-RPP7-coding transcript isoforms. Function of RPP7 is fully dependent on high levels of H3K9me2 at COPIA-R7. We present a direct in vivo demonstration for cooption of a TE-associated histone mark to the epigenetic control of pre-mRNA processing and establish a unique mechanism for regulation of plant immune surveillance gene expression. Our results functionally link a histone mark to alternative polyadenylation and the balance between distinct transcript isoforms from a single gene.

The Arabidopsis defense component EDM2 affects the floral transition in an FLC-dependent manner.

Arabidopsis thaliana EDM2 was previously shown to be specifically required for disease resistance mediated by the R protein RPP7. Here we provide additional data showing that the role of EDM2 in plant immunity is limited and does not include a function in basal defense. In addition, we found that EDM2 has a promoting effect on the floral transition. We further found that the protein kinase WNK8 physically interacts with EDM2 in the nucleus. Unlike EDM2, which serves as a substrate of this kinase, WNK8 appears not to be required for RPP7-mediated defense. As reported previously, however, WNK8 does affect flowering time. Epistasis analyses suggested that EDM2 acts upstream of the floral repressor FLC (AT5G10140) and downstream of WNK8 (AT5G41990) in a regulatory module that resembles the autonomous floral promotion pathway, comprising a set of mechanisms that are known to affect the floral transition by regulating FLC transcript levels.

EMSY-like genes are required for full RPP7-mediated race-specific immunity and basal defense in Arabidopsis.

The Arabidopsis thaliana gene enhanced downy mildew 2 (EDM2) encodes a nuclear protein required for RPP7-mediated race-specific disease resistance against Hyaloperonospora arabidopsidis, proper floral transition and additional developmental processes. Transcript levels of the disease-resistance gene RPP7 are enhanced by EDM2 while those of the floral suppressor FLC are repressed by EDM2. By yeast two-hybrid screening for EDM2-interacting proteins, we identified AtEML1, a member of a small group of four Arabidopsis proteins containing an EMSY N-terminal domain, a central Agenet domain, and a C-terminal coiled-coil motif. Using T-DNA mutants combined with silencing by artificial microRNAs, we found AtEML1, AtEML2, and, likely, AtEML4 to contribute to RPP7-mediated immunity. Besides this, AtEML1 and AtEML2 participate in a second EDM2-dependent function and affect floral transition. Unlike EDM2, whose role in immunity appears to be limited to RPP7-mediated disease resistance, some AtEML members contribute to basal defense, an unspecific general defense mechanism. Domain architectures of EDM2 as well as AtEML proteins suggest roles of these proteins in the regulation of chromatin states. Thus, possible cooperation of AtEML members with EDM2 at the level of chromatin dynamics may link race-specific pathogen recognition to general defense mechanisms.

Co-option of EDM2 to distinct regulatory modules in Arabidopsis thaliana development.

BACKGROUND: Strong immunity of plants to pathogenic microorganisms is often mediated by highly specific mechanisms of non-self recognition that are dependent on disease resistance (R) genes. The Arabidopsis thaliana protein EDM2 is required for immunity mediated by the R gene RPP7. EDM2 is nuclear localized and contains typical features of transcriptional and epigenetic regulators. In addition, to its role in immunity, EDM2 plays also a role in promoting floral transition. This developmental function of EDM2, but not its role in RPP7-mediated disease resistance, seems to involve the protein kinase WNK8, which physically interacts with EDM2 in nuclei. RESULTS: Here we report that EDM2 affects additional developmental processes which include the formation of leaf pavement cells and leaf expansion as well as the development of morphological features related to vegetative phase change. EDM2 has a promoting effect of each of these processes. While WNK8 seems not to exhibit any vegetative phase change-related function, it has a promoting effect on the development of leaf pavement cells and leaf expansion. Microarray data further support regulatory interactions between WNK8 and EDM2. The fact that the effects of EDM2 and WNK8 on leaf pavement cell formation and leaf expansion are co-directional, while WNK8 counteracts the promoting effect of EDM2 on floral transition, is surprising and suggests that WNK8 can modulate the activity of EDM2. CONCLUSION: We propose that EDM2 has been co-opted to distinct regulatory modules controlling a set of different processes in plant immunity and development. WNK8 appears to modulate some functions of EDM2.

Straight walk: a modified method of ligation-mediated genome walking for plant species with large genomes.

Polymerase chain reaction (PCR)-based genome walking techniques are commonly used to clone unknown genomic regions flanking known sequences. However, these methods are typically problematic when applied to highly complex DNA templates isolated from plants with large genomes. Here we describe a reliable and efficient genome walking method that is particularly effective for plants with large genomes. Our ligation-mediated PCR method, Straight Walk, has improved sensitivity and specificity due to optimization of sequences of adaptors and adaptor primers. Successful genome walking in lily, which has one of the largest genomes in plants, indicates that Straight Walk is applicable for most plant species.

A novel Arabidopsis pathosystem reveals cooperation of multiple hormonal response-pathways in host resistance against the global crop destroyer Macrophomina phaseolina.

Dubbed as a "global destroyer of crops", the soil-borne fungus Macrophomina phaseolina (Mp) infects more than 500 plant species including many economically important cash crops. Host defenses against infection by this pathogen are poorly understood. We established interactions between Mp and Arabidopsis thaliana (Arabidopsis) as a model system to quantitatively assess host factors affecting the outcome of Mp infections. Using agar plate-based infection assays with different Arabidopsis genotypes, we found signaling mechanisms dependent on the plant hormones ethylene, jasmonic acid and salicylic acid to control host defense against this pathogen. By profiling host transcripts in Mp-infected roots of the wild-type Arabidopsis accession Col-0 and ein2/jar1, an ethylene/jasmonic acid-signaling deficient mutant that exhibits enhanced susceptibility to this pathogen, we identified hundreds of genes potentially contributing to a diverse array of defense responses, which seem coordinated by complex interplay between multiple hormonal response-pathways. Our results establish Mp/Arabidopsis interactions as a useful model pathosystem, allowing for application of the vast genomics-related resources of this versatile model plant to the systematic investigation of previously understudied host defenses against a major crop plant pathogen.

Use of enhancer trapping to identify pathogen-induced regulatory events spatially restricted to plant-microbe interaction sites.

Plant genes differentially expressed during plant-pathogen interactions can be important for host immunity or can contribute to pathogen virulence. Large-scale transcript profiling studies, such as microarray- or mRNA-seq-based analyses, have revealed hundreds of genes that are differentially expressed during plant-pathogen interactions. However, transcriptional responses limited to a small number of cells at infection sites can be difficult to detect using these approaches, as they are under-represented in the whole-tissue datasets typically generated by such methods. This study examines the interactions between Arabidopsis thaliana (Arabidopsis) and the pathogenic oomycete Hyaloperonospora arabidopsidis (Hpa) by enhancer trapping to uncover novel plant genes involved in local infection responses. We screened a ß-glucuronidase (GUS) reporter-based enhancer-trap population for expression patterns related to Hpa infection. Several independent lines exhibited GUS expression in leaf mesophyll cells surrounding Hpa structures, indicating a regulatory response to pathogen infection. One of these lines contained a single enhancer-trap insertion in an exon of At1g08800 (MyoB1, Myosin Binding Protein 1) and was subsequently found to exhibit reduced susceptibility to Hpa. Two additional Arabidopsis lines with T-DNA insertions in exons of MyoB1 also exhibited approximately 30% fewer spores than wild-type plants. This study demonstrates that our enhancer-trapping strategy can result in the identification of functionally relevant pathogen-responsive genes. Our results further suggest that MyoB1 either positively contributes to Hpa virulence or negatively affects host immunity against this pathogen.

Genomic structure and differential expression of two tandem-arranged GSTZ genes in rice.

Glutathione S-transferases (GSTs) are scavenging enzymes that detoxify cellular xenobiotics and toxins by catalyzing the conjugation of these substrates with a tripeptide glutathione. GSTs are classified depending on gene organization and sequence similarity. The sequence analysis of genomic DNA for zeta class GST (GSTZ) locus in rice indicated that two homologous GSTZ genes lay in a tandem orientation with a short (0.4 kb) intergenic spacer. The upstream OsGSTZ1 and downstream OsGSTZ2 spanned 3.5 and 3.2 kb with nine coding exons, respectively. The transcript of OsGSTZ1 had a long 3' untranslated region (3' UTR) that was mostly encoded by a 10th noncoding exon, whereas OsGSTZ2 mRNA contained a long 5' UTR. Northern blot analysis showed that OsGSTZ1/2 messages were strongly expressed in leaf blades, while transcripts from roots were low level. Because OsGSTZ1/2 messages in leaf tissues were strongly induced only by water treatment, it was difficult to assay for the induction of OsGSTZ1/2 transcripts by various stress treatments. Thus, using rice culture cells, we analyzed the respective responses of OsGSTZ1 and OsGSTZ2 genes against various treatments by real-time reverse transcriptase-polymerase chain reaction (RT-PCR). The results showed that OsGSTZ1 was expressed at a level ca. 1000-fold higher than OsGSTZ2 in suspension cells without stress treatment. OsGSTZ1 was expressed constitutively under various stress conditions. In contrast, the expression of OsGSTZ2 gene was strongly enhanced to 30-fold by treatment with jasmonic acid. These observations suggested that the expression of OsGSTZ1 and OsGSTZ2 genes are differentially regulated in the culture cell of rice.

The PHD-finger module of the Arabidopsis thaliana defense regulator EDM2 can recognize triply modified histone H3 peptides.

Recently we reported that the Arabidopsis thaliana PHD-finger protein EDM2 (enhanced downy mildew 2) impacts disease resistance by affecting levels of di-methylated lysine 9 of histone H3 (H3K9me2) at an alternative polyadenylation site in the immune receptor gene RPP7. EDM2-dependent modulation of this post-translational histone modification (PHM) shifts the balance between full-length RPP7 transcripts and prematurely polyadenylated transcripts, which do not encode the RPP7 protein. Our previous work genetically linked, for the first time, PHMs to alternative polyadenylation and established EDM2 as a critical component mediating PHM-dependent polyadenylation control. However, how EDM2 is recruited to its genomic target sites and how it affects H3K9me2 levels is unknown. Here we show the PHD-finger module of EDM2 to recognize histone H3 bearing certain combinations of 3 distinct PHMs. Our results suggest that targeting of EDM2 to specific genomic regions is mediated by the histone-binding selectivity of its PHD-finger domain.

Mutations in EDM2 selectively affect silencing states of transposons and induce plant developmental plasticity.

We previously reported on the A. thaliana gene EDM2, which is required for several developmental processes and race-specific immunity. Although EDM2 encodes a nuclear protein with features commonly observed in epigenetic factors, its role in chromatin silencing remains unknown. Here we demonstrate that silencing states of several transposons in edm2 mutants are altered. Levels of their transcripts anti-correlate with those of the repressive epigenetic marks H3K27me1, H3K9me2, and DNA-methylation at CHG sites. In addition, double mutant analysis revealed epistasis between EDM2 and the major histone H3K9-methyltransferase gene KRYPTONITE/SUVH4 in the control of H3K9me2 and CHG methylation. Moreover, we found that the expressivity of several mutant edm2 phenotypes exhibits stochastic variation reminiscent of mutants of known epigenetic modifiers. We propose that EDM2 affects the expression of transposons and developmentally important genes by modulating levels of repressive chromatin marks in a locus dependent manner.

NSD1 mitigates caspase-1 activation by listeriolysin O in macrophages.

Mammals and plants share pathogen-sensing systems named nod-like receptors (NLRs). Some NLRs form the inflammasome, a protein scaffold that regulates the secretion of interleukin (IL)-1ß and IL-18 by cleaving catalytically inactive substrates into mature cytokines. Here, we show an immune conservation between plant and mammalian NLRs and demonstrate that the murine nuclear receptor binding SET domain protein 1 (NSD1), a protein that bears similarity to the NLR regulator enhanced downy mildew 2 (EDM2) in Arabidopsis, diminishes caspase-1 activity during extracellular stimulation with Listeria monocytogenes listeriolysin O (LLO). EDM2 is known to regulate plant developmental processes, whereas NSD1 is associated with developmental disorders. We observed that NSD1 neither affects nuclear factor (NF)-κB signaling nor regulates NLRP3 inflammasome gene expression at the chromatin, transcriptional or translational level during LLO stimulation of macrophages. Silencing of Nsd1 followed by LLO stimulation led to increased caspase-1 activation, enhanced post-translational maturation of IL-1ß and IL-18 and elevated pyroptosis, a form of cell death associated with inflammation. Furthermore, treatment of macrophages with LLO(W492A), which lacks hemolytic activity due to a tryptophan to alanine substitution in the undecapeptide motif, indicates the importance of functional LLO for NSD1 regulation of the NLRP3 inflammasome. Taken together, our results indicate that NLR signaling in plants may be used for gene discovery in mammals.

EDM2 is required for RPP7-dependent disease resistance in Arabidopsis and affects RPP7 transcript levels.

Specific disease resistance of Arabidopsis thaliana against the Hyaloperonospora parasitica isolate Hiks1 (HpHiks1) is mediated by RPP7. Although this disease resistance gene encodes a typical nucleotide binding site leucine-rich repeat (NB-LRR) disease resistance protein, its function is independent of the defense hormone salicylic acid and most known genes required for plant immune responses. We identified EDM2 (enhanced downy mildew 2) in a genetic screen for RPP7 suppressors. Mutations of EDM2 phenocopy RPP7 mutations, but do not affect other tested disease resistance genes. We isolated EDM2 by map-based cloning. The predicted EDM2 protein is structurally unrelated to previously identified components of the plant immune system, bears typical features of transcriptional regulators, including plant homeodomain (PHD)-finger-like domains, and defines a plant-specific protein family. In edm2 mutants both constitutive and HpHiks1-induced RPP7 transcript levels are reduced, suggesting that EDM2 is either a direct or an indirect regulator of RPP7 expression. Microarray analyses defined a set of defense-associated genes, the expression of which is suppressed during successful HpHiks1 colonization of either rpp7 or edm2 plants. This transcriptional phenotype is counteracted by an EDM2/RPP7-dependent mechanism.