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.     

A Bayesian approach to efficient differential allocation for resampling-based significance testing

Background  

Large-scale statistical analyses have become hallmarks of post-genomic era biological research due to advances in high-throughput assays and the integration of large biological databases. One accompanying issue is the simultaneous estimation of p-values for a large number of hypothesis tests. In many applications, a parametric assumption in the null distribution such as normality may be unreasonable, and resampling-based p-values are the preferred procedure for establishing statistical significance. Using resampling-based procedures for multiple testing is computationally intensive and typically requires large numbers of resamples.     

Flow regimes in a two phase reversed flow jet loop bioreactor

Reversed flow jet loop bioreactors (RFJLB) have been used extensively for 2 or 3 phase biochemical reactions. From visual observations and gas holdup data, 3 distinct flow regimes are identified in RFJLB, namely: (1) Bubble free regime (BFR), where bubbles are observed in the draft tube only; (2) Transition regime (TR), where bubbles are observed in both the draft tube and the annulus, but without circulation; and (3) Complete bubble circulation regime (CBCR), where bubbles circulate in both the draft tube and annulus. CBCR is the most desirable regime, since the reactor operation in this regime gives a higher gas holdup and mass transfer rate than in the other two regimes. In the present study, the hydrodynamic behavior of RFJLB was investigated under various operational and geometrical conditions, such as gas and liquid velocity and nozzle configuration. Factors affecting the critical liquid circulation velocity (CLCV) above which the CBCR is established were identified and evaluated quantitatively.     

Recombination is suppressed in an alien introgression in peanut harboring Rma, a dominant root-knot nematode resistance gene

Rma, a dominant root-knot nematode resistance gene introduced into tetraploid peanut (Arachis hypogaea) from a synthetic allotetraploid donor (TxAG-6), has been widely deployed in modern cultivars. The genomic location and borders of the alien chromosome segment introgressed from TxAG-6 into NemaTAM (a BC₇-derived introgression line) and other modern cultivars carrying Rma have not been genetically mapped, and resistance gene candidates (RGCs) have not been identified for Rma. Our study focused on densely populating the alien introgression with codominant DNA markers, identifying and mapping the borders of the alien introgression carried by NemaTAM, and identifying RGCs for Rma. Altogether, 2,847 simple sequence repeat (SSR) and 380 single strand conformational polymorphism (SSCP) markers were screened for linkage to Rma-247 of the SSCP markers targeted 202 nucleotide binding site (NBS) leucine-rich repeat (LRR) and other resistance (R) gene homologs (75 were identified by mining a peanut EST database). SSR, NBS-LRR, and Ser/Thr receptor-like protein loci within the alien introgression co-segregated with Rma in an F₄ population (Gregory × Tifguard) and were tightly linked and spanned 3.4 cM in an F₅ population (NemaTAM × GP-NC-WS-14). By comparative mapping in the A-genome progenitor of peanut (A. duranensis), Rma was discovered to have been introduced on an interstitial alien chromosome segment spanning one-third to one-half of chromosome 9A. Numerous codominant DNA markers were identified for finer mapping of Rma, shortening the alien introgression harboring Rma by marker-assisted selection, and introducing novel root-knot nematode R-genes into peanut by targeting syntenic segments on chromosomes 9A and 9B in wild diploid donors.     

Current applications of bicelles in NMR studies of membrane-associated amphiphiles and proteins

This review covers current trends in studies of membrane amphiphiles and membrane proteins using both fast tumbling bicelles and magnetically aligned bicelle media for both solution state and solid state NMR. The fast tumbling bicelles provide a versatile biologically mimetic membrane model, which in many cases is preferable to micelles, both because of the range of lipids and amphiphiles that may be combined and because radius of curvature effects and strain effects common with micelles may be avoided. Drug and small molecule binding and partitioning studies should benefit from their application in fast tumbling bicelles, tailored to mimic specific membranes. A wide range of topology and immersion depth studies have been shown to be effective in fast tumbling bicelles, while residual dipolar couplings add another dimension to structure refinement possibilities, particularly for situations in which the peptide is uniformly labeled with 15N and 13C. Solid state NMR studies of polytopic transmembrane proteins demonstrate that it is possible to express, purify, and reconstitute membrane proteins, ranging in size from single transmembrane domains to seven-transmembrane GPCRs, into bicelles. The line widths and quality of the resulting 15NH dipole-15N chemical shift spectra demonstrate that there are no insurmountable obstacles to the study of large membrane proteins in magnetically aligned media.     

CpG-induced IFNgamma expands TLR4-specific IL-18 responses in vivo

Serum IL-18 responses to LPS increase after pretreatment with CpG-containing DNA. Compared to saline-pretreated controls, mice pretreated with CpG for two days produced 20-fold more serum IL-18 when challenged with lipopolysaccharide (LPS). In contrast, IFNgamma-deficiency or anti-IFNgamma pretreatment reduced CpG-expanded IL-18 responses to LPS by 67 and 83%, respectively. Mice pretreated with either IFNgamma or CpG comparably increased LPS-inducible serum IL-18 responses. LPS, compared to challenge with other TLR agonists, was best able to trigger high serum IL-18 levels in CpG-pretreated mice and this response was TLR4-dependent. CpG, compared to pretreatment with other TLR agonists, optimally expanded LPS-induced IL-18 responses that correlated with higher levels of circulating IFNgamma levels prior to LPS challenge. High-level serum IL-18 responses were caspase-1-dependent and P2X7 receptor-independent. We conclude that CpG promotes high-level IL-18 synthesis by an IFNgamma-dependent and IFNgamma-sufficient mechanism in vivo that is optimally triggered by LPS.     

Tetracycline treatment targeting Wolbachia affects expression of an array of proteins in Brugia malayi parasite

Wolbachia is an intracellular endosymbiont of Brugia malayi parasite whose presence is essential for the survival of the parasite. Treatment of B. malayi‐infected jirds with tetracycline eliminates Wolbachia, which affects parasite survival and fitness. In the present study we have tried to identify parasite proteins that are affected when Wolbachia is targeted by tetracycline. For this Wolbachia depleted parasites (B. malayi) were obtained by tetracycline treatment of infected Mongolian jirds (Meriones unguiculatus) and their protein profile after 2‐DE separation was compared with that of untreated parasites harboring Wolbachia. Approximately 100 protein spots could be visualized followed by CBB staining of 2‐D gel and included for comparative analysis. Of these, 54 showed differential expressions, while two new protein spots emerged (of 90.3 and 64.4 kDa). These proteins were subjected to further analysis by MALDI‐TOF for their identification using Brugia coding sequence database composed of both genomic and EST sequences. Our study unravels two crucial findings: (i) the parasite or Wolbachia proteins, which disappeared/down‐regulated appear be essential for parasite survival and may be used as drug targets and (ii) tetracycline treatment interferes with the regulatory machinery vital for parasites cellular integrity and defense and thus could possibly be a molecular mechanism for the killing of filarial parasite. This is the first proteomic study substantiating the wolbachial genome integrity with its nematode host and providing functional genomic data of human lymphatic filarial parasite B. malayi.     

Extrusion of electroformed giant unilamellar vesicles through track-etched membranes

Unilamellar vesicle populations having a narrow size distribution and mean radius below 100 nm are preferred for drug delivery applications. In the present work, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) was used to prepare giant unilamellar vesicles (GUVs) by electroformation and multilamellar vesicles (MLVs) by thin film hydration. Our experiments show that in contrast to MLVs, a single-pass extrusion of GUVs through track-etched polycarbonate membranes at moderate pressure differences is sufficient to produce small liposomes having low polydispersity index. Moreover, we observe that the drug encapsulating potential of extruded liposomes obtained from GUVs is significantly higher compared to liposomes prepared by extrusion of MLVs. Furthermore, our experiments carried out for varying membrane pore diameters and extrusion pressures suggest that the size of extruded liposomes is a function of the velocity of GUV suspensions in the membrane pore.