Since the recent sequencing of the rice genome, the functional identification of rice genes has become increasingly important. Various tagged lines have been generated; however, the number of tagged genes available is not sufficient for extensive study of gene function. To help identify the functions of genes in rice, we developed a Gateway vector, pANDA, for RNA interference of rice genes. This vector can be used for Agrobacterium transformation of rice and allows easy and fast construction of efficient RNAi vectors. In the construct, hairpin RNA derived from a given gene is transcribed from a strong maize ubiquitin promoter, and an intron is placed 5' upstream of inverted repeats to enhance RNA expression. Analysis of rice genes using this vector showed that suppression of mRNA expression was observed in more than 90% of transgenic plants examined, and short interfering RNA indicative of RNA silencing was detected in each silenced plant. A similar vector, pANDA-mini, was also developed for direct transfer into leaf cells or protoplasts. This vector can be used for transient suppression of gene function in rice. These vectors should help identify the functions of rice genes whose tagged mutants are not available at present and complement existing methods for functional genomics of rice.

Active DNA demethylation is an important part of epigenetic regulation in plants and animals. How active DNA demethylation is regulated and its relationship with histone modification patterns are unclear. Here, we report the discovery of IDM1, a regulator of DNA demethylation in Arabidopsis. IDM1 is required for preventing DNA hypermethylation of highly homologous multicopy genes and other repetitive sequences that are normally targeted for active DNA demethylation by Repressor of Silencing 1 and related 5-methylcytosine DNA glycosylases. IDM1 binds methylated DNA at chromatin sites lacking histone H3K4 di- or trimethylation and acetylates H3 to create a chromatin environment permissible for 5-methylcytosine DNA glycosylases to function. Our study reveals how some genes are indicated by multiple epigenetic marks for active DNA demethylation and protection from silencing.

RNA silencing with inverted repeat (IR) constructs has been used to suppress gene expression in various organisms. However, the transitive RNA-silencing effect described in plants may preclude the use of RNA silencing for a gene family. Here, we show that, in rice (Oryza sativa), transitive RNA silencing (spreading of double-stranded RNA along the target mRNA) occurred with the green fluorescent protein transgene but not with the endogenous phytoene desaturase gene. We fused IR copies of unique 3# untranslated regions derived from the rice OsRac gene family to a strong promoter and stably introduced them into rice. Each of the seven members of the OsRac gene family was specifically suppressed by its respective IR construct. We also examined IR constructs in which multiple 3# untranslated regions were fused and showed that three members of the OsRac gene family were effectively suppressed by a single construct. Using highly conserved regions of the two members of the OsRac gene family, we also suppressed the expression of all members of the gene family with variable efficiencies. These results suggest that RNA silencing is a useful method for the functional analysis of gene families in rice and other plants.

DNA methylation is an important epigenetic mark in many eukaryotes1-5. In plants, 24-nt small interfering RNAs (siRNAs) bound to the effector protein, Argonaute 4 (AGO4) can direct de novo DNA methylation by the methyltransferase DRM22,4-6. Here we report a new regulator of RNA-directed DNA methylation (RdDM) in Arabidopsis: RDM1. Loss-of-function mutations in the RDM1 gene impair the accumulation of 24-nt siRNAs, reduce DNA methylation, and release transcriptional gene silencing at RdDM target loci. RDM1 encodes a small protein that appears to bind single-stranded methyl DNA, and associates and co-localizes with RNA polymerase II, AGO4 and DRM2 in the nucleus. Our results suggest that RDM1 is a component of the RdDM effector complex and may play a role in linking siRNA production with pre-existing or de novo cytosine methylation. Our results also suggest that although RDM1 and Pol V may function together at some RdDM target sites in the peri-nucleolar siRNA processing center, Pol II rather than Pol V is associated with the RdDM effector complex at target sites in the nucleoplasm.

SUMMARY DNA methylation is a conserved epigenetic mark that plays important roles in plant and vertebrate development, genome stability, and gene regulation. Canonical Methyl-CpG-Binding Domain (MBD) proteins are important interpreters of DNA methylation that recognize methylated CG sites and recruit chromatin remodelers, histone deacetylases and histone methyltransferases to repress transcription. Here, we show that Arabidopsis MBD7 and Increased DNA Methylation 3 (IDM3) are anti-silencing factors that prevent gene repression and DNA hypermethylation. MBD7 preferentially binds to highly methylated, CG-dense regions and physically associates with other anti-silencing factors, including the histone acetyltransferase IDM1 and the alpha-crystallin domain proteins IDM2 and IDM3. IDM1 and IDM2 were previously shown to facilitate active DNA demethylation by the 5-methylcytosine DNA glycosylase/lyase ROS1. Thus, MBD7 tethers the IDM proteins to methylated DNA, which enables the function of DNA demethylases that in turn limit DNA methylation and prevent transcriptional gene silencing.

Homologous recombination-based gene targeting is a powerful tool for precise genome modification and has been widely used in organisms ranging from yeast to higher organisms such as Drosophila and mouse. However, gene targeting in higher plants, including the most widely used model plant Arabidopsis thaliana, remains challenging. Here we report a sequential transformation method for gene targeting in Arabidopsis. We find that parental lines expressing the bacterial endonuclease Cas9 from the egg cell- and early embryo-specific DD45 gene promoter can improve the frequency of single-guide RNA-targeted gene knock-ins and sequence replacements via homologous recombination at several endogenous sites in the Arabidopsis genome. These heritable gene targeting can be identified by regular PCR. Our approach enables routine and fine manipulation of the Arabidopsis genome.

DNA methylation is important for the silencing of transposons and other repetitive elements in many higher eukaryotes. However, plant and mammalian genomes have evolved to contain repetitive elements near or inside their genes. How these genes are kept from being silenced by DNA methylation is not well understood. A forward genetics screen led to the identification of the putative chromatin regulator Enhanced Downy Mildew 2 (EDM2) as a cellular antisilencing factor and regulator of genome DNA methylation patterns. EDM2 contains a composite Plant Homeo Domain that recognizes both active and repressive histone methylation marks at the intronic repeat elements in genes such as the Histone 3 lysine 9 demethylase gene Increase in BONSAI Methylation 1 (IBM1) and is necessary for maintaining the expression of these genes by promoting mRNA distal polyadenylation. Because of its role in maintaining IBM1 expression, EDM2 is required for preventing CHG methylation in the bodies of thousands of genes. Our results thus increase the understanding of antisilencing, genome methylation patterns, and regulation of alternative RNA processing by intronic heterochromatin.

DNA methylation is an important epigenetic mark for transcriptional gene silencing (TGS) in diverse organisms [1][2][3][4][5][6] . Recent studies suggest that the methylation status of a number of genes is dynamically regulated by methylation and demethylation [7][8][9][10] . In Arabidopsis, active DNA demethylation is mediated by the ROS1 (repressor of silencing 1) subfamily of 5-methylcytosine DNA glycosylases through a base excision repair pathway 8,[10][11][12][13] . These demethylases play critical roles in erasing DNA methylation and preventing TGS of target genes 7,8,10 . However, it is not known how the demethylases are targeted to specific sequences. We report here the identification of ROS3, an essential regulator of DNA demethylation that contains an RNA recognition motif. Analysis of ros3 mutant and ros1ros3 double mutant suggests that ROS3 acts in the same genetic pathway as ROS1 to prevent DNA hypermethylation and TGS. Gel mobility shift assays and analysis of ROS3 immunoprecipitate from plant extracts showed that ROS3 binds to small RNAs in vitro and in vivo. Immunostaining shows that ROS3 and ROS1 proteins colocalize in discrete foci dispersed throughout the nucleus. These results demonstrate a critical role for ROS3 in preventing DNA hypermethylation and suggest that DNA demethylation by ROS1 may be guided by RNAs bound to ROS3.We developed a sensitive assay system in Arabidopsis to genetically dissect active DNA demethylation 10,14 . The system consists of the RD29A-LUC transgene (firefly luciferase reporter driven by the stress-responsive RD29A promoter) and the non-allelic endogenous RD29A gene. The RD29A promoter is subjected to continuous siRNA-directed DNA methylation such that active DNA demethylation is required to keep the RD29A and RD29A-LUC genes transcriptionally active. In ros1 mutants, the RD29A promoter for both the transgene and endogenous gene becomes hypermethylated and both genes are silenced 10 . In addition, the 35S-NPTII transgene linked to RD29A-LUC is also silenced such that ros1 mutant plants are sensitive to kanamycin. We isolated the ros3 mutant from a T-DNA mutagenized population 15 Fig. 1a and Supplementary Fig. 1a and 1b) as well as sensitivity to kanamycin (Fig. 1b). Genetic analysis indicated that the ros3 mutation is recessive and affects a nuclear gene (data not shown).Northern blot ( Fig. 1c) and nuclear run-on ( Fig. 1d) Fig. 2a and 2b). Treatment with the cytosine methylation inhibitor 5-aza-2'-deoxycytidine increased RD29A-LUC expression in the ros3 mutant to the wild type level ( Supplementary Fig. 3). These results suggest that DNA hypermethylation is responsible for the TGS in ros3 mutant plants.The nrpd1a-1 mutation in the largest subunit of RNA polymerase IVa blocks the accumulation of 24-nt siRNAs corresponding to the RD29A promoter (data not shown). Analysis of nrpd1aros3 double mutant showed that the nrpd1a mutation causes a significant increase in RD29A-LUC expression ( Fig. 2c and 2d) and substantial decrease in CpG, CpNpG and CpNpN methyl...