Tokuji Tsuchiya

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

Significance We show the histone mark H3K9me2, which is known to mediate transposon silencing, to determine the choice between alternative polyadenylation sites within the Arabidopsis thaliana disease resistance gene RPP7. High H3K9me2 levels recruited to the first RPP7 intron by the COPIA-R7 retrotransposon suppress use of a promoter-proximal polyadenylation site. Modulating H3K9me2 levels at this site shifts the balance between full-length and incomplete RPP7 transcripts. By recruiting H3K9me2-dependent polyadenylation control to RPP7, the COPIA-R7 insertion provided a new switch to fine-tune RPP7 expression. Selective advantages resulting from this mechanism likely contributed to the domestication of COPIA-R7 at RPP7. 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.

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.

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

BackgroundStrong 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.ResultsHere 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.ConclusionWe 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.

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)-1β 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-1β and IL-18 and elevated pyroptosis, a form of cell death associated with inflammation. Furthermore, treatment of macrophages with LLOW492A, 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.

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 β-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.