DNA demethylases target promoter transposable elements to positively regulate stress responsive genes in Arabidopsis

Background

DNA demethylases regulate DNA methylation levels in eukaryotes. Arabidopsis encodes four DNA demethylases, DEMETER (DME), REPRESSOR OF SILENCING 1 (ROS1), DEMETER-LIKE 2 (DML2), and DML3. While DME is involved in maternal specific gene expression during seed development, the biological function of the remaining DNA demethylases remains unclear.

Results

We show that ROS1, DML2, and DML3 play a role in fungal disease resistance in Arabidopsis. A triple DNA demethylase mutant, rdd (ros1 dml2 dml3), shows increased susceptibility to the fungal pathogen Fusarium oxysporum. We identify 348 genes differentially expressed in rdd relative to wild type, and a significant proportion of these genes are downregulated in rdd and have functions in stress response, suggesting that DNA demethylases maintain or positively regulate the expression of stress response genes required for F. oxysporum resistance. The rdd-downregulated stress response genes are enriched for short transposable element sequences in their promoters. Many of these transposable elements and their surrounding sequences show localized DNA methylation changes in rdd, and a general reduction in CHH methylation, suggesting that RNA-directed DNA methylation (RdDM), responsible for CHH methylation, may participate in DNA demethylase-mediated regulation of stress response genes. Many of the rdd-downregulated stress response genes are downregulated in the RdDM mutants nrpd1 and nrpe1, and the RdDM mutants nrpe1 and ago4 show enhanced susceptibility to F. oxysporum infection.

Conclusions

Our results suggest that a primary function of DNA demethylases in plants is to regulate the expression of stress response genes by targeting promoter transposable element sequences.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-014-0458-3) contains supplementary material, which is available to authorized users.     

Spatial indicators for nature conservation from European to local scale

The paper presents an overview of the objectives and exemplary results of the FP 5 project “Spatial Indicators for European Nature Conservation” (SPIN). The SPIN project is focused on the development and testing of advanced classification methods and spatial indicators based on multisensor satellite data and GIS to accomplish monitoring and management tasks in the context of Natura 2000 and nature conservation. A representative selection of eight regional test areas covers a pan-European network and allows comparative investigations to provide accepted recommendations for regional and European nature conservation. The selected results of four case studies are presented and discussed. The range of work covers the production of regional and local habitat maps by object-oriented classification, a case-based reasoning method for change detection as a management support tool for planning and regulating local land use, the selection and application of structural indicators for the monitoring of Natura 2000 habitats and the downscaling and disaggregation of soil information. Results and the further implementation of presented methods are discussed in the conclusions.     

Angiogenin levels and ANG genotypes: dysregulation in amyotrophic lateral sclerosis

Objective

To determine whether 5 single nucleotide polymorphisms (SNPs) associate with ALS in 3 different populations. We also assessed the contribution of genotype to angiogenin levels in plasma and CSF.

Methods

Allelic association statistics were calculated for polymorphisms in the ANG gene in 859 patients and 1047 controls from Sweden, Ireland and Poland. Plasma, serum and CSF angiogenin levels were quantified and stratified according to genotypes across the ANG gene. The contribution of SNP genotypes to variance in circulating angiogenin levels was estimated in patients and controls.

Results

All SNPs showed association with ALS in the Irish group. The SNP rs17114699 replicated in the Swedish cohort. No SNP associated in the Polish cohort. Age- and sex-corrected circulating angiogenin levels were significantly lower in patients than in controls (p<0.001). An allele dose-dependent regulation of angiogenin levels was observed in controls. This regulation was attenuated in the ALS cohort. A significant positive correlation between CSF plasma angiogenin levels was present in controls and abolished in ALS.

Conclusions

ANG variants associate with ALS in the Irish and Swedish populations, but not in the Polish. There is evidence of dysregulation of angiogenin expression in plasma and CSF in sporadic ALS. Angiogenin expression is likely to be important in the pathogenesis of ALS.     

Molecular cloning and characterization of <Emphasis Type="Italic">clyA</Emphasis> genes in various serotypes of <Emphasis Type="Italic">Salmonella enterica</Emphasis>

Cytolysin A (ClyA) is a pore-forming hemolytic protein encoded by the clyA gene. It has been identified in Salmonella enterica serovars Typhi and Paratyphi A. To identify and characterize the clyA genes in various Salmonella enterica strains, 21 different serotypes of strains isolated from clinical specimens were presently examined. Full-length clyA genes were found in S. enterica serovar Brandenburg, Indiana, Panama, and Schwarzengrund strains by polymerase chain reaction amplification. The ClyA proteins from these four strains showed >97% amino acid identity to that of S. enterica serovar Typhi. Although all four serovars expressed detectable levels of ClyA as determined by Western blot analysis, they did not show a strong hemolytic effect on blood agar, indicating that ClyA may not be efficiently expressed or secreted. Escherichia coli transformed with clyA genes from the four serovars enhanced production of ClyA proteins and hemolytic activities to a level similar to S. enterica serovar Typhi ClyA. The present results suggest that ClyA may play a role in the pathogenesis of S. enterica serovar Brandenburg, Indiana, Panama and Schwarzengrund.     

Immunological responses induced by <Emphasis Type="Italic">asd</Emphasis> and <Emphasis Type="Italic">wzy/asd</Emphasis> mutant strains of <Emphasis Type="Italic">Salmonella enterica</Emphasis> serovar Typhimurium in BALB/c mice

Attenuated bacteria have long been developed as vaccine candidates but can have some disadvantages, such as the potential for damage to immune organs due to insufficient clearance. To minimize these disadvantages, we generated Salmonella enterica serovar Typhimurium mutants SHJ2104 (asd::cm) and HTSaYA (wzy::km, asd::cm). The wzy gene codes for the O-antigen polymerase, which is involved in lipopolysaccharide (LPS) biosynthesis, and asd codes for aspartate β-semialdehyde dehydrogenase, which participates in cell wall formation. The strains synthesized LPS with a short-chain length, and showed lower cytotoxicity and reduced intracellular proliferation in animal cells compared to wild-type bacteria. After oral infection, the mutants were cleared in immune tissues, including the Peyer’s patch, mesenteric lymph node, and spleen, within 5 days. The LD50 of the mutants in Balb/c mice was estimated to be 10⁶ higher than wild-type bacteria when administered either via an oral or i.p. route, indicating that the two strains are highly attenuated. To compare the immune response to and protective effects of the mutants against wild-type bacterial infection, we inoculated the mutants into mice via an oral (1×10¹⁰CFU) or i.p. (1×10⁷ CFU) route once or twice at a two week interval. All immune responses, such as serum IgG and secretory IgA levels, cytokine production, and delayed hypersensitivity, were highly induced by two rounds of immunization. HTSaYA and SHJ2104 induced similar immune responses, and mice immunized with HTSaYA or SHJ2104 via an i.p. route were protected against wild-type Salmonella infection even at 100-fold of the LD₅₀ (5×10⁶ CFU). Taken together, these data indicate that HTSaYA and SHJ2104 could be developed as live attenuated Salmonella vaccine candidates.     

Native plant/herbivore interactions as determinants of the ecological and evolutionary effects of invasive mammalian herbivores: the case of the common brushtail possum

In their native range, mammalian herbivores exist in a suite of direct and indirect ecological and evolutionary relationships with plant populations and communities. Outside their native range these herbivores become embedded in a multitude of new ecological and evolutionary interactions with native plant species in the new range. Sound knowledge of the plant/herbivore interactions in the herbivores’ native range provides an ideal framework to better understand their effects in the introduced range. The example of the common brushtail possum (Trichosurus vulpecula) and its introduction to New Zealand from Australia provides an excellent case study. In Australia, the common brushtail possum is a widespread generalist herbivore and it is thought that this generalist lifestyle has equipped the species well for successful colonisation of New Zealand. In Australia the brushtail possum has co-existed with highly chemically defended foliage since the Oligocene and recent papers have supported the role of possums as agents of selection on eucalypt defences. While the chemical profile of New Zealand foliage is comparatively poorly understood, possums do show clear selectivity between and within populations and some of these interactions may be mediated by the animals ability to ‘cope’ with PSMs, coupled with maintaining its generalist diet. While possums have had less time to effect evolutionary change in New Zealand species, their impacts on plant fitness have been well documented. However, further knowledge on variation and heritability of foliage traits driving possum preferences is needed to elucidate the ecological and evolutionary plant/possum interactions in the invasive range.     

Novel protein interactors of urokinase-type plasminogen activator receptor

The urokinase-type plasminogen activator receptor (uPAR) has been implicated in tumor growth and metastasis. The crystal structure of uPAR revealed that the external surface is largely free to interact with a number of proteins. Additionally, due to absence of an intracellular cytoplasmic protein domain, many of the biological functions of uPAR necessitate interactions with other proteins. Here, we used yeast two-hybrid screening of breast cancer cDNA library to identify hSpry1 and HAX1 proteins as putative candidate proteins that interact with uPAR bait constructs. Interaction between these two candidates and uPAR was confirmed by GST-pull down, co-immunoprecipitation assays and confocal microscopy. These novel interactions that have been identified may also provide further evidence that uPAR can interact with a number of other proteins which may influence a range of biological functions.     

Hypercapnia induced shifts in gill energy budgets of Antarctic notothenioids

Mechanisms responsive to hypercapnia (elevated CO₂ concentrations) and shaping branchial energy turnover were investigated in isolated perfused gills of two Antarctic Notothenioids (Gobionotothen gibberifrons, Notothenia coriiceps). Branchial oxygen consumption was measured under normo- versus hypercapnic conditions (10,000 ppm CO₂) at high extracellular pH values. The fractional costs of ion regulation, protein and RNA synthesis in the energy budgets were determined using specific inhibitors. Overall gill energy turnover was maintained under pH compensated hypercapnia in both Antarctic species as well as in a temperate zoarcid (Zoarces viviparus). However, fractional energy consumption by the examined processes rose drastically in G. gibberifrons (100–180%), and to a lesser extent in N. coriiceps gills (7–56%). In conclusion, high CO₂ concentrations under conditions of compensated acidosis induce cost increments in epithelial processes, however, at maintained overall rates of branchial energy turnover.     

Generalized Voronoi Tessellation as a Model of Two-dimensional Cell Tissue Dynamics

Voronoi tessellations have been used to model the geometric arrangement of cells in morphogenetic or cancerous tissues, however, so far only with flat hyper-surfaces as cell-cell contact borders. In order to reproduce the experimentally observed piecewise spherical boundary shapes, we develop a consistent theoretical framework of multiplicatively weighted distance functions, defining generalized finite Voronoi neighborhoods around cell bodies of varying radius, which serve as heterogeneous generators of the resulting model tissue. The interactions between cells are represented by adhesive and repelling force densities on the cell contact borders. In addition, protrusive locomotion forces are implemented along the cell boundaries at the tissue margin, and stochastic perturbations allow for non-deterministic motility effects. Simulations of the emerging system of stochastic differential equations for position and velocity of cell centers show the feasibility of this Voronoi method generating realistic cell shapes. In the limiting case of a single cell pair in brief contact, the dynamical nonlinear Ornstein–Uhlenbeck process is analytically investigated. In general, topologically distinct tissue conformations are observed, exhibiting stability on different time scales, and tissue coherence is quantified by suitable characteristics. Finally, an argument is derived pointing to a tradeoff in natural tissues between cell size heterogeneity and the extension of cellular lamellae.     

A Case of Probable Mixed-Infection with Clonorchis sinensis and Fasciola sp.: CT and Parasitological Findings

We report here a human case probably mixed-infected with Clonorchis sinensis and Fasciola sp. who was diagnosed by computed tomography (CT) scan, serological findings, and/or fecal examination. The patient was a 43-year-old Korean female and was admitted to Kyung Hee University Hospital with the complaints of fever and abdominal pain. On admission, marked eosinophilia was noted in her peripheral blood. CT scan showed specific lesions for clonorchiasis and fascioliasis in the liver, along with lesions suggestive of amebic abscess. Micro-ELISA revealed positive results for the 2 helminthic infections. Eggs of C. sinensis and trophozoites of Entamoeba histolytica were observed in the stool. Treatment with praziquantel followed by metronidazole and tinidazole reduced abnormalities in the liver and eosinophilia. This is the first case report of a possible co-infection with 2 kinds of liver flukes in the Republic of Korea.