Abstract
BackgroundDNA 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.ResultsWe 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.ConclusionsOur 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.
Highlights
DNA demethylases regulate DNA methylation levels in eukaryotes
The rdd mutant shows enhanced susceptibility to Fusarium oxysporum Three-week-old wild-type (WT) Col-0 plants and rdd mutant plants were inoculated with F. oxysporum f. sp. conglutinans (Fo) by root dipping and grown on either sucrose-free Murashige and Skoog (MS) agar (MS[S-]) or in soil
Only 25% of the Col-0 plants were found severely diseased compared to more than 85% of the rdd plants (Figure 1C, right). These results indicated that plant disease resistance is compromised in rdd
Summary
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. De novo cytosine methylation is mediated by RNA-directed DNA methylation (RdDM), a plant-specific pathway that can generate 5-methylcytosines at all sequence contexts (CG, CHG, and CHH where H stands for A, C, or T) [2]. RdDM is directed by 24-nt small interfering RNAs (siRNAs) produced by the combined function of RNA POLYMERASE IV (Pol IV), RNA-DEPENDENT RNA POLYMERASE 2 (RDR2), and DICER-LIKE 3 (DCL3) These siRNAs bind to ARGONAUTE 4 (AGO4) to form and guide the RNA-induced silencing complex to target DNA through interaction with long non-coding RNA transcribed by Pol V. This AGO4-siRNA-long non-coding RNA complex recruits the de novo methyltransferase DRM2 (and DRM1) via an unknown mechanism, resulting in sequence-specific cytosine methylation. In plants DNA methylation occurs mainly in transposons and repetitive DNA sequences [1]
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