Development of a luciferase-based reporter of transcriptional gene silencing that enables bidirectional mutant screening in Arabidopsis thaliana
© Won et al.; licensee BioMed Central Ltd. 2012
Received: 1 March 2012
Accepted: 18 April 2012
Published: 7 June 2012
Cytosine methylation is an important chromatin modification that maintains genome integrity and regulates gene expression through transcriptional gene silencing. Major players in de novo methylation guided by siRNAs (known as RNA-directed DNA methylation, or RdDM), maintenance methylation, and active demethylation have been identified in Arabidopsis. However, active demethylation only occurs at a subset of RdDM loci, raising the question of how the homeostasis of DNA methylation is achieved at most RdDM loci. To identify factors that regulate the levels of cytosine methylation, we aimed to establish a transgenic reporter system that allows for forward genetic screens in Arabidopsis.
We introduced a dual 35 S promoter (d35S) driven luciferase reporter, LUCH, into Arabidopsis and isolated a line with a moderate level of luciferase activity. LUCH produced transgene-specific 24 nucleotide siRNAs and its d35S contained methylated cytosine in CG, CHG and CHH contexts. Treatment of the transgenic line with an inhibitor of cytosine methylation de-repressed luciferase activity. Mutations in several components of the RdDM pathway but not the maintenance methylation genes resulted in reduced d35S methylation, especially CHH methylation, and de-repression of luciferase activity. A mutation in MOM1, which is known to cooperate with RdDM to silence transposons, reduced d35S DNA methylation and de-repressed LUCH expression. A mutation in ROS1, a cytosine demethylation enzyme, increased d35S methylation and reduced LUCH expression.
We developed a luciferase-based reporter, LUCH, which reports both DNA methylation directed by small RNAs and active demethylation by ROS1 in Arabidopsis. The moderate basal level of LUCH expression allows for bi-directional genetic screens that dissect the mechanisms of DNA methylation as well as demethylation.
KeywordsCytosine methylation Demethylation RdDM MOM1 Transcriptional gene silencing siRNA
Cytosine methylation is a major epigenetic mechanism that establishes transcriptional gene silencing (TGS) to maintain genome integrity and regulate gene expression in plants and mammals (reviewed in ). Well-known biological phenomena involving DNA methylation as an underlying mechanism include imprinting, paramutation and X chromosome inactivation. In plants, transposons and repetitive elements are methylated, thereby keeping transposons silenced and immobilized and consequently protecting the genome from damage by these mobile elements. Also, when transposons or repeats are located in the regulatory regions of genes, DNA methylation at the transposons or repeats may influence the transcription of the nearby genes through TGS.
The enzymes that initiate, maintain, and erase DNA methylation in Arabidopsis have been identified and characterized (reviewed in ). De novo DNA methylation, also known as RNA-directed DNA methylation (RdDM), requires DOMAIN REARRANGED METHYLTRANSFERASE2 (DRM2), which is guided to specific genomic loci by 24 nucleotide small interfering RNAs (siRNAs). siRNAs are synthesized from repeats and transposons in an RNA polymerase IV (Pol IV)-, RNA DEPENDENT RNA POLYMERASE2 (RDR2)-, and DICERLIKE3 (DCL3)-dependent manner. Pol IV is thought to transcribe these loci into single-stranded RNAs, which are then rendered double-stranded by RDR2. DCL3 dices the double-stranded RNAs into 24 nucleotide siRNAs, which are loaded into the ARGONAUTE4 (AGO4)-clade of AGO proteins (reviewed in ). Base-pairing between the AGO4-loaded siRNAs and nascent transcripts produced by Pol V is thought to recruit AGO4/siRNAs and DRM2 to the RdDM targets, resulting in de novo methylation in a sequence-specific manner (reviewed in ). After the initial establishment of DNA methylation, hemimethylated cytosines in CG and CHG contexts resulting from DNA replication are fully methylated by METHYLTRANSFERASE1 (MET1) and CHROMOMETHYLTRANSFERASE3 (CMT3), respectively (reviewed in ). The positive feedback loop in which DNA methylation promotes siRNA biogenesis, which guides de novo DNA methylation, needs to be kept in check to prevent the expansion of heterochromatin and the sporadic silencing of genic regions. One such mechanism is DNA demethylation. Four DNA glycosylase/lyase enzymes remove methyl cytosine through a base excision repair mechanism (reviewed in ). DEMETER establishes imprinting during female gametogenesis and REPRESSOR OF SILENCING1 (ROS1), DEMETER-LIKE2 (DML2) and DML3 prevent hypermethylation in vegetative tissues.
Although the enzymes that deposit or erase DNA methylation are known, how these enzymes are regulated to achieve the proper homeostasis of DNA methylation is still nebulous. Although demethylation can keep DNA methylation in check, whole genome bisulfite sequencing in the ros1 dml2 dml3 triple mutant revealed that only a few hundred loci are hypermethylated  and are thus targets of demethylation. Since thousands of loci harbor DNA methylation, generate siRNAs and are targets of RdDM, it remains to be determined how most RdDM loci achieve homeostasis of DNA methylation. It is likely that other, as yet unknown, mechanisms prevent the hypermethylation of RdDM loci.
In addition to the RdDM pathway, MORPHEUS’ MOLECULE1 (MOM1) impacts TGS in a complex manner usually without affecting the levels of cytosine methylation at target loci [5–7]. It encodes a protein with similarities to chromatin remodeling ATPases and silences endogenous loci and transgenes by an unknown mechanism . MOM1 exhibits a complex relationship with RdDM depending on the target loci . It functions either in the same pathway as RdDM or in a parallel pathway, or it could even antagonize the silencing by RdDM. Some loci are transcriptionally suppressed by MOM1 independently of RdDM.
Forward genetic screens in Arabidopsis can help reveal mechanisms that regulate DNA methylation. In fact, most of the currently known genes involved in DNA methylation or demethylation were uncovered through genetic screens. However, most prior genetic screens were based on the isolation of mutations that release RdDM to result in de-repressed reporter gene expression, thus precluding the identification of negative regulators of DNA methylation. So far, the only known negative factors in DNA methylation, ROS1 and ROS3 (a protein required for ROS1-mediated demethylation), were isolated from genetic screens using the RD29A::LUC transgene system [8, 9]. Therefore, RD29A::LUC happens to be a target of ROS1-mediated demethylation. As mentioned above, the relatively lower number of ROS1/DML2/DML3 target loci in the genome as compared to the number of RdDM loci suggests the presence of unknown negative factors for methylation acting independently of, or in combination with, active demethylation by ROS1/DML2/DML3. Consequently, it is valuable to develop additional RdDM reporter transgenes inserted into different genomic locations to allow for the identification of these negative players.
Here, we report the establishment of a firefly LUCIFERASE (LUC)-based reporter transgene driven by a dual 35S promoter that harbors DNA methylation in CG, CHG, and CHH contexts in Arabidopsis. We show that LUC expression is repressed mainly through CHH methylation in an RdDM-dependent manner. MOM1 also plays a role in DNA methylation and TGS of the reporter. More importantly, the moderate level of basal LUC expression in wild-type plants allows for genetic screens that aim at the isolation of mutants with not only defective but also enhanced DNA methylation. In fact, a ros1 allele with reduced transgene expression was isolated using this system. The reporter line will prove to be an effective tool in dissecting the mechanisms that regulate DNA methylation.
Results and discussion
Generation of the luciferase reporter line, LUCH
LUCHdoes not report miRNA activity
Since LUCH contained a miR172 binding site, we first investigated whether it was able to report miRNA activity. If it were repressed by miR172, we would expect mutations in miRNA biosynthesis genes (reviewed in ), such as DICERLIKE1 (DCL1), HYPONASTIC LEAVES1 (HYL1), and SERRATE (SE) to de-repress LUCH expression. In the F2 population of LUCH crossed to dcl1–7, LUC luminescence was moderately increased in 12 out of 216 segregating seedlings (Additional file 1: Figure S2A). Since LUCH and DCL1 are not linked, the small proportion of seedlings with the moderately high LUC luminescence was not consistent with dcl1–7 being able to de-repress LUCH expression. Indeed, genotyping confirmed that only one of the 12 was homozygous for dcl1–7, and three of the 12 were homozygous for the wild-type DCL1 allele. Therefore, the moderate increase was likely due to inherent variations in LUCH expression or other background mutations. hyl1 and se-1 mutations also failed to increase LUC luminescence [see Additional file 1: Figure S2B and S2C. These results demonstrate that LUCH was unable to report miRNA activities even though the LUC transcript contains a miRNA-binding site in the 3′ UTR.
LUCHis regulated by RdDM-mediated TGS
Next, since RdDM target loci produce siRNAs, we determined the accumulation of siRNAs from the LUCH and d35S::NPTII transgenes. Even though we did not artificially introduce any hairpin source of d35S-specific siRNAs, siRNAs were detected in the LUCH line by northern blotting using a d35S-specific probe (Figure 2C). High throughput sequencing was conducted to examine the small RNAs from the transgenes in more detail. siRNAs mapping to both DNA strands of the two transgenes were found; and 22 nucleotide siRNAs were the most abundant small RNA species [see Additional file 1: Figure S4A and S4B]. Even though LUCH was introduced into rdr6–11 to prevent S-PTGS by blocking the biogenesis of secondary siRNAs, 21 nucleotide and 22 nucleotide siRNAs mapping to the transgene were present, which suggests that PTGS was still occurring. Perhaps the siRNAs were primary siRNAs resulting from sense and antisense transcription from the locus or secondary siRNAs from the activities of RDR2. Twenty four nucleotide siRNAs, which are associated with RdDM, were also present. Among 18 to 27 nucleotide small RNAs that mapped to d35S in LUCH, 24 nucleotide siRNAs accounted for approximately 19% of the total [see Additional file 1: Figure S4A]. The d35S promoters driving LUC and NPTII were 96% identical in sequences. We took advantage of the sequence differences to determine whether both regions generated siRNAs. Indeed, siRNAs specific to each d35S were found [see Additional file 1: Figure S4C; Additional file 2], indicating that each d35S gave rise to siRNAs. The reverse strand 24 nucleotide siRNAs were similar in quantity between the two transgenes (123 and 106 reads for LUCH and d35S::NPTII, respectively). Interestingly, forward strand 24 nucleotide siRNAs were different in quantity between the two transgenes: 509 and 120 reads were from d35S::NPTII and LUCH, respectively. The abundance of d35S::NPTII-specific siRNAs was attributed to both higher diversity of siRNA species and higher levels of a subset of species [see Additional file 2]. The basis for the differential siRNA levels is unknown but may be due to differences in read-through transcription at the two d35S. Taken together, LUCH exhibits the molecular characteristics associated with RdDM, such as CHH methylation and 24 nucleotide siRNA production.
LUCH is regulated by MOM1
LUCH is regulated by ROS1-mediated DNA demethylation
We developed a transgenic LUC reporter system that reports both TGS by RdDM and MOM1, and ROS1-mediated demethylation. Moderate expression of the reporter enables genetic screens in two directions to isolate mutants with decreased as well as increased DNA methylation. Considering that existing TGS reporter systems, such as the NOSpro, α’pro, and clk-sk lines, are mainly suitable for the isolation of positive players in RdDM, LUCH is a useful genetic resource for the identification of negative players in RdDM, for which nothing is known. Moreover, LUCH will potentially contribute to the better understanding of MOM1-mediated TGS or the mechanisms of active demethylation. For the latter, although RD29::LUC reports ROS1-mediated DNA demethylation, as a second reporter of ROS1-mediated demethylation residing at a different genomic location, LUCH will enrich our resources to tackle the mechanisms of demethylation.
Arabidopsis mutants used in this study were rdr6–11 , dcl1–7 , se-1 , hyl1 , met1–3 , cmt3–7  and drd3–1  and newly isolated drm2–6, ago4–6, drd1–12, hen1–9, ros1–5 and mom1–5. For map-based cloning of newly isolated mutants, LUCH rdr6–11 in the Columbia-0 (Col-0) accession was introgressed into Landsberg erecta (Ler) by backcrossing to Ler five times and one line with a similar level of LUC activity as LUCH in Col-0 was isolated. The isolated mutants from LUCH rdr6–11 in Col were each crossed to LUCH rdr6–11 in Ler, and in the F2 population, seedlings with high (for drm2–6, ago4–6, drd1–12, hen1–9, and mom1–5) or low (ros1–5) luciferase activities were identified and served as the mapping population. Polymorphisms between Col-0 and Ler were utilized to map and clone the genes.
Growth conditions and luciferase live imaging
Arabidopsis thaliana seeds were surface-sterilized, planted on MS-agar plates containing 1% sucrose, and stratified at 4 °C for three days. Seedlings were grown at 23 °C under continuous lights for ten days. All experiments were performed with ten-day old seedlings unless otherwise specified. For luciferase live imaging, 1 mM luciferin (a substrate of luciferase; Promega, Madison, Wisconsin, USA) in 0.01% Triton X-100 was sprayed onto the seedlings, which were then transferred to a Stanford Photonics Onyx Luminescence Dark Box. Luciferase images were taken with a Roper Pixis 1024B camera controlled by the WinView32 software at a two minute exposure time. Identical exposure conditions were used to capture all images in this study. The images were displayed and analyzed with WinView32 such that image contrast was adjusted to effectively distinguish the difference in intensities between different lines within a plate as previously described .
Construction of transgene, southern blot analysis and TAIL-PCR
The LUC coding region was amplified using the Rlucp1 and Rlucp2 primers and pRL-SV40 (Promega) as the template. d35S::LUC was constructed by replacing GFP in pAVA321  with the LUC coding region using NcoI and BamHI restriction sites. The d35S::LUC cassette was cloned into the pPZP211  at the SalI and BamHI restriction sites. An AP2 fragment including the miR172 binding site was amplified from Col-0 genomic DNA with the primers AP2p26 and AP2p28 and inserted downstream of d35S::LUC in pPZP211 using BamHI and EcoRI to generate d35S::LUC-AP2, which will be referred to as LUCH. The construct was introduced into rdr6–11 plants by Agrobacterium tumefaciens-mediated transformation. Southern blot analysis was performed according to the standard protocol  to evaluate the copy number of LUCH using the full-length LUC coding region as the probe. The probe was amplified with the primers lucp6 and lucp7, and radiolabeled with the RPN1633 Rediprime II Random Prime Labeling System (GE Healthcare Biosciences, Pittsburgh, Pennsylvania, USA). TAIL-PCR was performed as described . Primers used are listed in Additional file 3: Table S1.
Analysis of DNA cytosine methylation
For the McrBC-PCR assay, two reactions were set up for each genomic DNA sample: McrBC-treated and untreated reactions. A total of 300 ng genomic DNA was digested with 3 units of McrBC (New England Biolabs, Ipswich, Massachusetts, USA) for 25 minutes at 37 °C in a 20 μl reaction. Using 1 μl (15 ng) of restricted genomic DNA as the template, genomic regions corresponding to d35S or full length LUC in the LUCH transgene were amplified using the 35Sf and LUC 0.13 k R primers or the lucp6 and lucp7 primers, respectively. ACT1 was amplified with the Actin1-F and Actin1-R primers and used as a loading control. PCR products were analyzed on a 2% agarose gel stained with ethidium bromide. For Southern blot analysis, 15 μg of genomic DNA was digested with AluI (NEB) and hybridization was performed following standard methods . The d35S promoter was PCR-amplified with the 35Sf and 35Sr primers and radiolabeled using the RPN1633 Rediprime II random prime labeling system (GE Healthcare). For bisulfite sequencing, 1 μg of genomic DNA was subjected to bisulfite conversion using the EpiTect Bisulfite Kit according to the manufacturer’s instructions (Qiagen, Hilden, Germany). Converted DNA was subjected to PCR reactions with primers YZ 35 S Bis F and YZ LUC Bis R and the PCR products were cloned into the pGEM-T Easy vector (Promega). At least 26 colonies were sequenced for each sample. Unique clones were obtained and analyzed for DNA methylation with Kithmeth (http://katahdin.mssm.edu/kismeth/revpage.pl) . For 5-aza-2′-deoxycytidine (Sigma, St. Louis, Missouri, USA) treatment, seeds were germinated and grown on MS-agar medium containing 7 μg/ml of the chemical for two weeks and luciferase images were taken. Primers used are listed in Additional file 3: Table S1.
Analysis of small RNA accumulation
RNA isolation and hybridization to detect small RNAs were performed as described previously . To detect siRNAs from the d35S promoter, a DNA fragment was amplified from the d35S promoter using the 35Sf and 35Sr primers and cloned into the pGEM-T Easy vector (Promega). The plasmid was linearized by SpeI (NEB) and used as a template for in vitro transcription with T7 RNA polymerase (Promega) in the presence of [α-32P] UTP. The labeled in vitro transcripts were used as the probe in northern blotting. Radioactive signals were detected with a Phosphorimager. For small RNA deep sequencing, a small RNA library was constructed using the TruSeq Small RNA Sample Prep Kit (Illumina, San Diego, California, USA) according to the manufacturer’s instructions with some modifications. Instead of total RNA, 15 to 40 nucleotide long RNAs were used as the starting material. The small RNA library was sequenced by Illumina Hiseq2000 at the genomics core facility at the University of California Riverside. After the raw reads were filtered by the Illumina quality control pipeline and the adaptor sequences were trimmed, 14,363,865 reads between 18 nucleotides and 28 nucleotides were matched to the Arabidopsis genome (TAIRv10) as well as the transgenes with SOAP2 . A total of 8,710,699 and 22,245 reads were mapped to the Arabidopsis genome and the transgenes, respectively, with no mismatches.
cDNA was synthesized from 5 μg of DNaseI (Roche, Basel, Switzerland)-treated total RNA using Reverse Transcriptase (Fermentas, Burlington, Ontario, Canada) and oligo-dT (Fermentas) as the primer. Using cDNA and gene-specific primers, PCR was performed and RT-PCR products were analyzed on a 2% agarose gel stained with ethidium bromide. The sequences of primers are listed in Additional file 3: Table S1.
dual 35 S promoter from Cauliflower mosaic virus
- LUCH LUC :
repressed by CHH methylation
RNA-directed DNA methylation
reverse transcriptase-polymerase chain reaction
small interfering RNA
thermal asymmetric interlaced PCR
transcriptional gene silencing
The work was supported by grants from the National Science Foundation (MCB-1021465) and the National Institutes of Health (GM061146) to X.C. The work was also supported by the Howard Hughes Medical Institute and the Gordon and Betty Moore Foundation. TD was supported by an NSF ChemGen IGERT program (DGE0504249). XZ was the recipient of a Chinese Academy of Sciences visiting scholar fund. HY was supported by funds from the China Scholarship Council.
- Law JA, Jacobsen SE: Establishing, maintaining and modifying DNA methylation patterns in plants and animals. Nat Rev Genet. 2010, 11: 204-220. 10.1038/nrg2719.PubMed CentralView ArticlePubMedGoogle Scholar
- Haag JR, Pikaard CS: Multisubunit RNA polymerases IV and V: purveyors of non-coding RNA for plant gene silencing. Nat Rev Mol Cell Biol. 2011, 12: 483-492. 10.1038/nrm3152.View ArticlePubMedGoogle Scholar
- Furner IJ, Matzke M: Methylation and demethylation of the Arabidopsis genome. Curr Opin Plant Biol. 2011, 14: 137-141. 10.1016/j.pbi.2010.11.004.View ArticlePubMedGoogle Scholar
- Lister R, O’Malley RC, Tonti-Filippini J, Gregory BD, Berry CC, Millar AH, Ecker JR: Highly integrated single-base resolution maps of the epigenome in Arabidopsis. Cell. 2008, 133: 523-536. 10.1016/j.cell.2008.03.029.PubMed CentralView ArticlePubMedGoogle Scholar
- Yokthongwattana C, Bucher E, Caikovski M, Vaillant I, Nicolet J, Mittelsten Scheid O, Paszkowski J: MOM1 and Pol-IV/V interactions regulate the intensity and specificity of transcriptional gene silencing. EMBO J. 2010, 29: 340-351. 10.1038/emboj.2009.328.PubMed CentralView ArticlePubMedGoogle Scholar
- Vaillant I, Schubert I, Tourmente S, Mathieu O: MOM1 mediates DNA-methylation-independent silencing of repetitive sequences in Arabidopsis. EMBO Rep. 2006, 7: 1273-1278. 10.1038/sj.embor.7400791.PubMed CentralView ArticlePubMedGoogle Scholar
- Amedeo P, Habu Y, Afsar K, Mittelsten Scheid O, Paszkowski J: Disruption of the plant gene MOM releases transcriptional silencing of methylated genes. Nature. 2000, 405: 203-206. 10.1038/35012108.View ArticlePubMedGoogle Scholar
- Zheng X, Pontes O, Zhu J, Miki D, Zhang F, Li WX, Iida K, Kapoor A, Pikaard CS, Zhu JK: ROS3 is an RNA-binding protein required for DNA demethylation in Arabidopsis. Nature. 2008, 455: 1259-1262. 10.1038/nature07305.PubMed CentralView ArticlePubMedGoogle Scholar
- Gong Z, Morales-Ruiz T, Ariza RR, Roldan-Arjona T, David L, Zhu JK: ROS1, a repressor of transcriptional gene silencing in Arabidopsis, encodes a DNA glycosylase/lyase. Cell. 2002, 111: 803-814. 10.1016/S0092-8674(02)01133-9.View ArticlePubMedGoogle Scholar
- Chen X: A microRNA as a translational repressor of APETALA2 in Arabidopsis flower development. Science. 2004, 303: 2022-2025. 10.1126/science.1088060.View ArticlePubMedGoogle Scholar
- Peragine A, Yoshikawa M, Wu G, Albrecht HL, Poethig RS: SGS3 and SGS2/SDE1/RDR6 are required for juvenile development and the production of trans-acting siRNAs in Arabidopsis. Genes Dev. 2004, 18: 2368-2379. 10.1101/gad.1231804.PubMed CentralView ArticlePubMedGoogle Scholar
- Mourrain P, Beclin C, Elmayan T, Feuerbach F, Godon C, Morel JB, Jouette D, Lacombe AM, Nikic S, Picault N, Rémoué K, Sanial M, Vo TA, Vaucheret H: Arabidopsis SGS2 and SGS3 genes are required for posttranscriptional gene silencing and natural virus resistance. Cell. 2000, 101: 533-542. 10.1016/S0092-8674(00)80863-6.View ArticlePubMedGoogle Scholar
- Dalmay T, Hamilton A, Rudd S, Angell S, Baulcombe DC: An RNA-dependent RNA polymerase gene in Arabidopsis is required for posttranscriptional gene silencing mediated by a transgene but not by a virus. Cell. 2000, 101: 543-553. 10.1016/S0092-8674(00)80864-8.View ArticlePubMedGoogle Scholar
- Chen X: Small RNAs and their roles in plant development. Annu Rev Cell Dev Biol. 2009, 25: 21-44. 10.1146/annurev.cellbio.042308.113417.View ArticlePubMedGoogle Scholar
- Matzke M, Aufsatz W, Kanno T, Daxinger L, Papp I, Mette MF, Matzke AJ: Genetic analysis of RNA-mediated transcriptional gene silencing. Biochim Biophys Acta. 2004, 1677: 129-141. 10.1016/j.bbaexp.2003.10.015.View ArticlePubMedGoogle Scholar
- Aufsatz W, Mette MF, Matzke AJ, Matzke M: The role of MET1 in RNA-directed de novo and maintenance methylation of CG dinucleotides. Plant Mol Biol. 2004, 54: 793-804. 10.1007/s11103-004-0179-1.View ArticlePubMedGoogle Scholar
- Lindroth AM, Cao X, Jackson JP, Zilberman D, McCallum CM, Henikoff S, Jacobsen SE: Requirement of CHROMOMETHYLASE3 for maintenance of CpXpG methylation. Science. 2001, 292: 2077-2080. 10.1126/science.1059745.View ArticlePubMedGoogle Scholar
- Robinson-Beers K, Pruitt RE, Gasser CS: Ovule development in wild-type Arabidopsis and two female-sterile mutants. Plant Cell. 1992, 4: 1237-1249.PubMed CentralView ArticlePubMedGoogle Scholar
- Prigge MJ, Wagner DR: The Arabidopsis SERRATE gene encodes a zinc-finger protein required for normal shoot development. Plant Cell. 2001, 13: 1263-1280.PubMed CentralView ArticlePubMedGoogle Scholar
- Lu C, Fedoroff N: A mutation in the Arabidopsis HYL1 gene encoding a dsRNA binding protein affects responses to abscisic acid, auxin, and cytokinin. Plant Cell. 2000, 12: 2351-2366.PubMed CentralView ArticlePubMedGoogle Scholar
- Saze H, Scheid OM, Paszkowski J: Maintenance of CpG methylation is essential for epigenetic inheritance during plant gametogenesis. Nat Genet. 2003, 34: 65-69. 10.1038/ng1138.View ArticlePubMedGoogle Scholar
- Kanno T, Huettel B, Mette MF, Aufsatz W, Jaligot E, Daxinger L, Kreil DP, Matzke M, Matzke AJM: Atypical RNA polymerase subunits required for RNA-directed DNA methylation. Nat Genet. 2005, 37: 761-765. 10.1038/ng1580.View ArticlePubMedGoogle Scholar
- Chinnusamy V, Stevenson B, Lee BH, Zhu JK: Screening for gene regulation mutants by bioluminescence imaging. Sci STKE. 2002, 2002: pl10-PubMedGoogle Scholar
- von Arnim AG, Deng XW, Stacey MG: Cloning vectors for the expression of green fluorescent protein fusion proteins in transgenic plants. Gene. 1998, 221: 35-43. 10.1016/S0378-1119(98)00433-8.View ArticlePubMedGoogle Scholar
- Hajdukiewicz P, Svab Z, Maliga P: The small, versatile pPZP family of Agrobacterium binary vectors for plant transformation. Plant Mol Biol. 1994, 25: 989-994. 10.1007/BF00014672.View ArticlePubMedGoogle Scholar
- Sambrook J, Fritsch EF, Maniatis T: Molecular Cloning: A Laboratory Manual. 1989, Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 2Google Scholar
- Liu Y-G, Chen Y: High-efficiency thermal asymmetric interlaced PCR for amplification of unknown flanking sequences. Biotechniques. 2007, 43: 649-656. 10.2144/000112601.View ArticlePubMedGoogle Scholar
- Gruntman E, Qi Y, Slotkin RK, Roeder T, Martienssen R, Sachidanandam R: Kismeth: analyzer of plant methylation states through bisulfite sequencing. BMC Bioinforma. 2008, 9: 371-10.1186/1471-2105-9-371.View ArticleGoogle Scholar
- Park W, Li J, Song R, Messing J, Chen X: CARPEL FACTORY, a dicer homolog, and HEN1, a novel protein, act in microRNA metabolism in Arabidopsis thaliana. Curr Biol. 2002, 12: 1484-1495. 10.1016/S0960-9822(02)01017-5.View ArticlePubMedGoogle Scholar
- Li R, Yu C, Li Y, Lam T-W, Yiu S-M, Kristiansen K, Wang J: SOAP2: an improved ultrafast tool for short read alignment. Bioinformatics. 2009, 25: 1966-1967. 10.1093/bioinformatics/btp336.View ArticlePubMedGoogle Scholar
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