Summary DNA methylation is a conserved epigenetic mark in plants and mammals. In Arabidopsis, DNA methylation can be triggered by small interfering RNAs (siRNAs) through an RNA-directed DNA methylation (RdDM) pathway. Here we report the identification of a new RdDM effector, RDM3/KTF1. Loss-of-function mutations in RDM3/KTF1 reduce DNA methylation and release the silencing of RdDM target loci without abolishing the siRNA triggers. KTF1 has similarity to the transcription elongation factor SPT5 and contains a C-terminal extension rich in GW/WG repeats. KTF1 colocalizes with ARGONAUTE 4 (AGO4) in punctate nuclear foci, and binds AGO4 and RNA transcripts. Our results suggest KTF1 as an adaptor protein that binds scaffold transcripts generated by Pol V and recruits AGO4 and AGO4-bound siRNAs to form an RdDM effector complex. The dual interaction of an effector protein with AGO and small RNA target transcripts may be a general feature of RNA silencing effector complexes.

RNA-directed DNA methylation (RdDM) is an RNAi-based mechanism for establishing transcriptional gene silencing in plants. The plant-specific RNA polymerases IV and V are required for the generation of 24-nucleotide (nt) siRNAs and for guiding sequence-specific DNA methylation by the siRNAs, respectively. However, unlike the extensively studied multisubunit Pol II, our current knowledge about Pol IV and Pol V is restricted to only the two largest subunits NRPD1a/NRPD1 and NRPD1b/NRPE1 and the one second-largest subunit NRPD2a. It is unclear whether other subunits may be required for the functioning of Pol IV and Pol V in RdDM. From a genetic screen for second-site suppressors of the DNA demethylase mutant ros1, we identified a new component (referred to as RDM2) as well as seven known components (NRPD1, NRPE1, NRPD2a, AGO4, HEN1, DRD1, and HDA6) of the RdDM pathway. The differential effects of the mutations on two mechanistically distinct transcriptional silencing reporters suggest that RDM2, NRPD1, NRPE1, NRPD2a, HEN1, and DRD1 function only in the siRNA-dependent pathway of transcriptional silencing, whereas HDA6 and AGO4 have roles in both siRNA-dependent and -independent pathways of transcriptional silencing. In the rdm2 mutants, DNA methylation and siRNA accumulation were reduced substantially at loci previously identified as endogenous targets of Pol IV and Pol V, including 5S rDNA, MEA-ISR, AtSN1, AtGP1, and AtMU1. The amino acid sequence of RDM2 is similar to that of RPB4 subunit of Pol II, but we show evidence that RDM2 has diverged significantly from RPB4 and cannot function in Pol II. An association of RDM2 with both NRPD1 and NRPE1 was observed by coimmunoprecipitation and coimmunolocalization assays. Our results show that RDM2/NRPD4/NRPE4 is a new component of the RdDM pathway in Arabidopsis and that it functions as part of Pol IV and Pol V.[Keywords: DNA methylation; transcriptional gene silencing; siRNAs; Pol IV; Pol V] Supplemental material is available at http://www.genesdev.org.

RNA-directed DNA methylation (RdDM) is a conserved mechanism for epigenetic silencing of transposons and other repetitive elements. We report that the rdm4 (RNA-directed DNA Methylation4) mutation not only impairs RdDM, but also causes pleiotropic developmental defects in Arabidopsis. Both RNA polymerase II (Pol II)-and Pol V-dependent transcripts are affected in the rdm4 mutant. RDM4 encodes a novel protein that is conserved from yeast to humans and interacts with Pol II and Pol V in plants. Our results suggest that RDM4 functions in epigenetic regulation and plant development by serving as a transcriptional regulator for RNA Pol V and Pol II, respectively. DNA methylation and histone modifications are important epigenetic silencing mechanisms in eukaryotic cells (Chan et al. 2005;Matzke and Birchler 2005). In plants, DNA methylation is catalyzed by DNA methytransferase MET1, CMT3, and DRM2 (Chan et al. 2005). MET1 and CMT3 mainly function in maintaining DNA hypermethylation at CG and CHG sites (H is A, T, or C) during DNA duplication, while the function of DRM2 is involved in de novo DNA methylation at CHH sites directed by RNA (Chan et al. 2005). RNA-directed DNA methylation (RdDM) was first discovered in plants (Wassenegger et al. 1994), and plays important roles in transgene silencing, genome integrity, and transposon stability (Matzke et al. 2009).In the RdDM pathway, both 24-nucleotide (nt) siRNAs and long noncoding RNA transcripts are essential for de novo DNA methylation (Wierzbicki et al. 2008). The 24-nt siRNAs are generated in a pathway involving the putative DNA-directed RNA polymerase IV (Pol IV), RDR2 (RNA-dependent RNA polymerase 2), and DCL3 (Dicer-like RNA Pol IV and Pol V have distinct largest subunits-NRPD1 and NRPE1, respectively-but share some common subunits such as NRPD/E2 and NRPD/E4 (He et al. 2009b). Some subunits of Pol IV and Pol V are also shared with Pol II (Huang et al. 2009;Ream et al. 2009). It appears that the plant-specific RNA Pol IV and Pol V evolved from an ancestral RNA polymerase to function specifically in RdDM. The transcription activity of Pol II is tightly regulated with the help of a series of general transcription factors (Kornberg 2007). Although many general transcription factors of Pol II have been studied extensively, nothing is known about the regulation of transcription by Pol IV and Pol V.In the present study, we carried out a forward genetic screen for second site suppressors of the DNA demethylase mutant ros1, and identified a transcription factor, RDM4 (RNA-directed DNA Methylation4), that is required for RdDM in Arabidopsis. Interestingly, unlike other RdDM mutants, the rdm4 mutant plants display pleiotropic developmental phenotypes. RDM4 encodes a novel protein that is conserved in eukaryotic organisms. We found that RDM4 interacts with Pol II and Pol V in plants. Our results suggest that RDM4 is a regulator of Pol II and Pol V transcription, and thereby contributes to both development and RdDM. Results and DiscussionThe rdm4 mutation suppresses transcr...

The Arabidopsis thalianaT-DNA insertion mutant rh57-1 exhibited hypersensitivity to glucose (Glc) and abscisic acid (ABA). The other two rh57 mutants also showed Glc hypersensitivity similar to rh57-1, strongly suggesting that the Glc-hypersensitive feature of these mutants results from mutation of AtRH57. rh57-1 and rh57-3 displayed severely impaired seedling growth when grown in Glc concentrations higher than 3%. The gene, AtRH57 (At3g09720), was expressed in all Arabidopsis organs and its transcript was significantly induced by ABA, high Glc and salt. The new AtRH57 belongs to class II DEAD-box RNA helicase gene family. Transient expression of AtRH57-EGFP (enhanced green fluorescent protein) in onion cells indicated that AtRH57 was localized in the nucleus and nucleolus. Purified AtRH57-His protein was shown to unwind double-stranded RNA independent of ATP in vitro. The ABA biosynthesis inhibitor fluridone profoundly redeemed seedling growth arrest mediated by sugar. rh57-1 showed increased ABA levels when exposed to high Glc. Quantitative real time polymerase chain reaction analysis showed that AtRH57 acts in a signaling network downstream of HXK1. A feedback inhibition of ABA accumulation mediated by AtRH57 exists within the sugar-mediated ABA signaling. AtRH57 mutation and high Glc conditions additively caused a severe defect in small ribosomal subunit formation. The accumulation of abnormal pre-rRNA and resistance to protein synthesis-related antibiotics were observed in rh57 mutants and in the wild-type Col-0 under high Glc conditions. These results suggested that AtRH57 plays an important role in rRNA biogenesis in Arabidopsis and participates in response to sugar involving Glc- and ABA signaling during germination and seedling growth.