Arabidopsis H3K9me2 histone methyltransferase, Su(var)3-9 homolog 4/Kryptonite (SUVH4/KYP), as a bona fide cellular target of TrAP. TrAP interacts with the catalytic domain of KYP and inhibits its activity in vitro. TrAP elicits developmental anomalies phenocopying several TGS mutants, reduces the repressive H3K9me2 mark and CHH DNA methylation, and reactivates numerous endogenous KYP-repressed loci in vivo. Moreover, KYP binds to the viral chromatin and controls its methylation to combat virus infection. Notably, kyp mutants support systemic infection of TrAP-deficient Geminivirus. We conclude that TrAP attenuates the TGS of the viral chromatin by inhibiting KYP activity to evade host surveillance. These findings provide new insight on the molecular arms race between host antiviral defense and virus counter defense at an epigenetic level.Transcriptional gene silencing (TGS) can serve as an innate immunity against invading DNA viruses throughout Eukaryotes. Geminivirus code for TrAP protein to suppress the TGS pathway. Here, we identified an DOI:http://dx.doi.org/10.7554/eLife.06671.001 Many viruses can infect plants and cause diseases that can reduce crop yields. The Geminiviruses are a family of plant viruses that are transmitted by insects and infect tomato, cabbage, and many other crop plants. These viruses hijack the plant cells that they infect and force the plant cells to make viral proteins using instructions provided by the genes in the virus' own DNA. To make proteins, DNA is first copied into molecules of messenger ribonucleic acid (or mRNA) in a process called transcription. However, plants can defend themselves by blocking the transcription of viral DNA through ‘transcriptional gene silencing’.In plant cells, DNA is packaged around proteins called histones to form a structure called chromatin. Small chemical tags attached to the histones can alter the structure of chromatin to regulate the activity of the genes encoded within it. For example, ‘methyl’ tags added to certain histones can block transcription and lower the activity of a gene. DNA from viruses can also associate with histones inside plant cells meaning that transcriptional gene silencing can take place by the addition of these methyl tags. Many Geminiviruses produce a protein called TrAP, which can activate transcription, but it is not clear how this works.Arabidopsis thaliana, was genetically engineered to produce high levels of these TrAP proteins. These ‘transgenic’ plants did not develop properly: they grew more slowly, had abnormal leaves, and flowered earlier. Furthermore, hundreds of plant genes were more active than usual in the transgenic plants, which suggests that TrAP inhibits transcriptional gene silencing. Further experiments showed that TrAP directly binds to a plant enzyme called KYP—which normally deposits methyl groups on chromatin and prevents it from working. This reduces the number of methyl groups that are attached to histones associated with both viral and plant chromatin, which results in the activation of genes that would normally be switched off.Castillo-González et al. studied the TrAP proteins from two different Geminiviruses that can infect crop plants. The commonly used model plant, Castillo-González et al.'s findings show how Geminiviruses can stop transcriptional gene silencing of chromatin that contains virus DNA to evade the host plant's defenses. The next challenge is to understand how TrAP inhibits KYP, which may present new ways to genetically engineer plants to become resistant to infection by viruses.DOI:http://dx.doi.org/10.7554/eLife.06671.002 RNA silencing is a host defense mechanism to combat invading nucleic acids. One type of RNA silencing is referred to as post-transcriptional gene silencing (PTGS). In PTGS, double-stranded RNAs (dsRNAs) are processed by Dicer-like ribonucleases into small-interfering RNAs (siRNAs). Mature siRNAs are incorporated into an Argonaute (AGO)-centered RNA-induced silencing complex (RISC) to regulate expression of target genes through RNA cleavage or translational repression. PTGS has evolved as a universal defense response toward all viruses because dsRNAs can result from intermediates in RNA virus replication, highly structured RNA virus genomes, or from viral transcripts. To evade this surveillance mechanism, virtually all plant viruses are known to encode suppressor proteins that are able to block different key steps of the PTGS pathway .Arabidopsis Su(var)3-9 homolog 4, Kryptonite (KYP)   level is poorly understood. In eukaryotes, the nuclear DNA is wrapped onto histone octamers to constitute chromatin. The chromatin undergoes various DNA and histone methylations, and these modifications have variable effects on gene expression depending on the precise residues, contexts, and modification complexity. Histone methylation takes place on lysine and arginine residues of the amino-terminal tails . The prete (KYP) , and itsaf (CLF) . Local Haf (CLF) , 2014a.Arabidopsis mutants are hypersusceptiple to Geminivirus infection and show exacerbated disease symptoms  and Cabbage Leaf Curl Virus (CaLCuV). We found that constitutive expression of TGMV-TrAP in Arabidopsis thaliana caused morphological abnormalities that mimic loss-of-function mutants of numerous TGS components including lhp1 (like-heterochromatin1) and clf. Microarray analyses of TrAP transgenic plants and lhp1 mutants revealed a substantial overlap in reprogrammed host genes at a genome-wide level. Through biochemical screening, we identified KYP as the bona fide target of TrAP. We demonstrated in vitro that TrAP binds to the catalytic domain of KYP and inhibits its enzymatic activity; while in vivo, TrAP decreases the repressive H3K9me2 marks and H3K9me2-dependent CHH methylation in gene-rich regions. We also found that KYP directly associates with the Geminivirus minichromosome and deposits H3K9me2 marks on viral chromatin. In addition, kyp mutants but not wild-type plants sustain low systemic infection of CaLCuV lacking TrAP protein. Taken together, we propose that KYP-catalyzed H3K9me2 is a line of the innate immunity against invading DNA pathogens, and Geminivirus TrAP functions to inactivate KYP to counter host defense. Thus, this study provides new insight into the host–virus interaction at the TGS level.Here, we investigated the suppression mechanism of TrAP proteins, encoded by two Geminivirus members, Arabidopsis transgenic lines overexpressing full-length TGMV TrAP, with or without Flag-Myc4, 3HA, or CFP epitopes. These transgenic plants were confirmed by northern (data not shown) or western blot assays  and ago1-27, a hypomorphic allele of ago1 . Of these, 295 transcripts were elevated whereas 291 were reduced  were also upregulated in lhp1 mutant. This is significantly greater than 1.28% expected by chance . Interestingly, among the co-upregulated genes are a group of flowering-stimulated transcriptional factors including the key flowering-time integrator, FT, and 12 other genes such as TFL1, AGL5, and AGL9  were also repressed in lhp1 mutant. This is significantly greater than 1.25% expected by chance  . These results suggested a possible hyposensitivity to auxin in lhp1 mutants and TrAP transgenic plants, which could explain the smaller statues of both genotypes. Consistent with this hypothesis, numerous auxin-repressed loci including PS2 and aging genes like TET9, SAG13, and SRG1 were upregulated in both lines. Concomitantly, six genes related to cell growth and five genes engaged in cell wall loosening were also repressed. Significant genome-wide overlap of 35S-TrAP and lhp1 loss-of-function-responsive genes suggested that TrAP might be genetically involved in the LHP1-related TGS pathway.Similarly, out of 291 genes significantly downregulated in the ed test) . Genes rTrAP also deregulates expression of heterochromatic loci. To this end, we selected numerous transposable elements (TEs) that were not recovered from the microarray assays and assessed them directly by qRT-PCR. Excitingly, most of the tested transposons were transcriptionally active , indirectly leading to deregulation of the epigenetic marks. Analysis of the microarray data challenged this possibility as no significant changes in the transcripts of any canonical TGS components were revealed (Nicotiana benthamiana (N. benthamiana). When NLuc and CLuc are brought together through interaction of the test proteins, catalytic activity is restored and recorded through CCD camera. In our LCI screening, we recovered LHP1 and KYP, a SUVH-type H3K9me2 methyltransferase, suggesting that TrAP is physically close to LHP1 and/or KYP proteins in vivo    followeder (XVE) . TogetheWe performed in vitro pull-down assays to examine whether TrAP interacts directly with KYP. We found that maltose-binding protein (MBP)-KYP, but not MBP and other MBP-tagged control proteins, was able to pull down GST-tagged TGMV- and CaLCuV-encoded TrAP proteins . This inSUVH2 display early-flowering phenotype, suggesting that TrAP might target this protein in vivo as well.Since the SET domain is well conserved among histone methyltransferases (HMTase) , we nextEscherichia coli as the enzyme source, recombinant histone 3 as the substrate, and methyl-14C-SAM as the methyl donor (50])  . Thus, oddm1, kyp, and lhp1 control plants. Remarkably, all tested loci in TrAP transgenic lines displayed consistent reduction of H3K9me2 and H3K27me3, whereas changes of H3K4me3 were variable. This molecular phenotype mimicked kyp mutant at all tested loci, but not lhp1 mutant. This result indicated inhibitory effect of TrAP on KYP activity on the TEs in vivo.Given that in vivo TrAP genetically interferes with the TGS pathway, and in vitro it physically interacts with HMTases  and inhibits the activity of KYP, we wondered whether TrAP alters KYP function in vivo. To address this question, we conducted chromatin immunoprecipitation (ChIP) analyses of H3K4me3, H3K9me2, and K3K27me3 marks  on numerTrAP-responsive protein-coding genes is associated with changes in the histone methylation status. First, we screened numerous loci that showed transcriptional deregulation in the TrAP overexpression lines  and CHH (∼21.7%), but not in CG (∼2%) contexts when compared to Col-0. To our surprise, TrAP transgenic plants only exhibited decrease in methylation of CHH (∼11%) but not CHG  methylation in bidopsis . We pred not CHG .10.7554/kyp and TrAP overexpression plants with Col-0    by scanning the genomes in 200 bp tiles and comparing the level of methylation among ethods’) . Given tethods’) , we sepacontexts . Remarkactively) . Notablyctively) . This su regions . Togethekyp mutants are hypersusceptible to Geminivirus infection . These observations suggest a possible homoeostatic regulation of this critical TGS component.In animals, H3K9 methylation promotes TGS and latency of integrated viruses. In plants, Geminivirus constitutes into a minichromosome that also undergoes epigenetic regulation. The specific TrAP-KYP interaction, inhibition of KYP activity in vitro, and reduction of H3K9me2 and CHH methylation in vivo suggest that KYP is a major factor in combating viruses. Previous studies showed that nfection ; these enfection . Moreoveromoters ; furtherkyp mutant, and the methylation marks were further restored by the KYP transgene, suggesting that viral chromatin H3K9me2 is catalyzed by KYP. Notably, the H3K9me2 amount was substantially enhanced in the locus defined by Primer #9 in the transgenic KYP overexpression plants  by changing a single nucleotide (T to A) that produces an amber mutation and introduces an XbaI restriction site after the sixth codon in the TrAP gene  DNA . TrAP isrAP gene . Then, wp plants . Consisto CaLCuV , these po CaLCuV . Consisto CaLCuV  or syste and PCR . Particuion site . Excitins in vitro  TrAin vitro ; (4) TrAin vitro , and corin vitro ; (5) TrAin vitro ; (6) metin vitro ; (7) KYPin vitro ; and (8) protein . To our kyp mutants displayed hypersusceptibility to Geminivirus infection, which could be rescued by exogenous wild-type KYP gene. KYP directly acts on viral chromatin and deposits the repressive H3K9me2 mark on the viral chromatin. Moreover, viruses lacking the essential TrAP protein can, although inefficiently, cause systemic infection in KYP-deficient hosts. All these facts point out the unambiguous role of KYP-mediated TGS in defense against viral infection. On the other hand, TrAP functions to inhibit KYP catalytic activity, reducing the repressive H3K9me2 mark, to activate transcription of viral genes. Our KYP reconstitution assays show that TrAP:KYP molar ratio of around 2 is enough to cause ∼50% inhibition of KYP activity, indicating that TrAP is a potent inhibitor of HMTase activity. Thus, direct inhibition of KYP represents a novel counter-defense mechanism for virus survival in the hosts. This mechanism could account for the long-documented essential role of TrAP in expression of viral genes including the coat protein and the nuclear shuttle protein , and an early flowering phenotype similar to loss-of-function mutant of the kyp paralog, SUVH2, the transgenic plants are phenotypically different from kyp single mutant. This could be due to functional redundancy between KYP and its paralogs in vivo; alas, the morphological phenotype of the higher-order mutants has not been fully documented.Our in vitro assays clearly demonstrated that TrAP predominantly binds to the catalytic domain of KYP and inhibited its enzymatic activity. Whether TrAP might alter KYP conformation or block the accessibility of substrates to the active sites upon interaction awaits future structural analysis. We note, however, that activity . ArisingTrAP-overexpression plants are morphologically similar to lhp1, which is characteristic of the H3K27me3 pathway. Moreover, the substantial overlapping of TrAP-responsive genes with lhp1-regulated genes strongly suggested that TrAP might target the LHP1-related H3K27me3 pathway  . The despromoter  was modipromoter  plasmid -DC-nLUC  for tranKYP start codon with the primers PKYP EcoRV for and PKYP BamHI rev. The binary vector pBA002a Flag-4Myc-KYP was obtained by the Gateway system using LR clonase. The resultant plasmid and PCR product harboring KYP promoter were digested with EcoRV/BamHI and ligated with T4 DNA ligase. The plasmid was confirmed by sequencing using the primer PKYP seq For.The cDNA or DNA fragments were cloned into pENTR/D vectors, confirmed by sequencing, and then transferred to the appropriate destination vectors by recombination using the LR Clonase (Invitrogen). The primers for the cloning are listed in the E. coli DH5α. The plasmids were further confirmed by sequencing and transformed into E. coli BL21 Rossetta DE3 for expression.For expression of recombinant proteins, the cDNA or DNA fragments were cloned into pMCSG9 or pMCSG10 vectors to produce His-MBP- or His-GST-tagged proteins respectively, by ligation independent cloning  using prtrap DNA A infective clone the pNSB1090 plasmid was subjected to site directed mutagenesis by amplification with the primers CaLCuV_AL2_null_XbaI_for and CaLCuV_AL2_null_XbaI_rev  plants were transformed with binary vectors by the floral-dip method (kyp mutant (SALK_130630C) was obtained from the Arabidopsis Biological Resource Center (ABRC) and confirmed by genotyping and qRT-PCR.p method . The trap method  containi2HPO4, 1.8 mM KH2HPO4, pH 7.3, 10 mM 2-mercaptoethanol) incubated with the sepharose glutathione 1 hr at 4°C, eluted with elution buffer . The elute was further purified through the Ni-NTA column and finally through SEC in column buffer ; the fractions containing the target protein were pulled together, concentrated to 25 μM and aliquoted for usage.For in vitro pull-down or HMTase reconstitution assays, the recombinant proteins were purified following either one or two-step affinity purification procedure. His-MBP-tagged proteins were first purified through Immobilized Metal Affinity Chromatography (IMAC) using the Ni-NTA resin (Qiagen), followed by amylose resin (NEB) according to manufacturers' protocols. His-GST-tagged proteins were initially purified through sepharose glutathione column  followed by IMAC. Specifically for HMTase assays, His-MBP-CaLCuV_TrAP, His-MBP-TGMV_TrAP, and His-MBP were prepared in lysis buffer , incubated with the Ni-NTA resin at 4°C for 1 hr, eluted with 300 mM imidazole and immediately incubated with the amylose resin (NEB) at 4°C for 1 hr. The proteins were eluted with 10 mM maltose and the elute was further separated by size exclusion chromatography (SEC) in column buffer ; the fractions containing the target protein were pulled together, concentrated to 100 µM, aliquoted and stored at −80°C until usage. His-GST-KYP was prepared in PBS buffer  and the bait (His-MBP-Bait) proteins were purified by IMAC using the lysis buffer , incubated with the Ni-NTA resin at 4°C for 1 hr, eluted with 300 mM imidazole and immediately dialyzed in storage buffer  at 4°C overnight.g for 2 min, transferred to a second tube containing 2.5 µg of the MBP-tagged bait protein, and incubated at room temperature for 2 hr. The protein complexes were harvested by adding 50 µl amylose resin beads, followed by 2 hr incubation at room temperature, and cleaned with six vigorous washes with buffer. The pulled-down proteins were resolved by SDS-PAGE and the preys were detected by western blot using anti-GST antibody.In vitro pull-down assays and in vivo Co-IP were done as described . BrieflyN. benthamiana leaves were collected 2 days after agroinfiltration, ground in liquid nitrogen and stored at −80°C until use. For the assay, total proteins were extracted from 0.4 g of ground powder in 1.2 ml (3 vol) of IP buffer ; then, the soluble proteins were cleared twice by ultracentrifugation at 20,000 × rcf for 15 min at 4°C. The protein complexes were immunoprecipitated with 15 µl Anti-c-Myc-agarose affinity gel (Sigma–Aldrich #A7470) at 4°C for 2 hr, the unspecific-bound proteins were removed by four consecutive washes with the IP buffer with 10 min incubation each at 4°C. The protein complexes were eluted in 200 μl of elution buffer  for 20 min. The supernatant was collected, frozen in liquid nitrogen and dried using the Savant SpeedVac concentrator; finally, the sample was solubilized in 50 μl of 2× SDS-loading buffer for western blot analyses.For Co-IP experiments, Arabidopsis transgenic plants expressing XVE-Flag-Myc4-TrAP were induced for 16 hr with 25 µM ß-estradiol in liquid MS media, ground in liquid nitrogen and stored at −80°C until use. For the assay, total proteins were extracted from 10 g of ground powder in 40 ml (4 vol) of IP buffer ; then, the soluble proteins were cleared twice by ultracentrifugation at 20,000 × rcf for 15 min at 4°C. The protein complexes were first immunoprecipitated using 500 μl of Anti-FLAG M2 magnetic beads  and incubated in slow rotation for 2 hr at 4°C, the nonspecific-bound proteins were removed by three consecutive washes with 15 ml of IP buffer for 10 min incubation each at 4°C. The protein complexes were then eluted by competition with 100 mg/ml FLAG peptide and subsequently immunoprecipitated with 100 µl Anti-c-Myc-agarose affinity gel (Sigma–Aldrich #A7470) at 4°C for 1.5 hr, the nonspecific-bound proteins were removed by five consecutive washes with the IP buffer with 5 min incubation each at 4°C. The protein complexes were eluted in 200 μl of elution buffer  for 20 min. The supernatant was collected, frozen in liquid nitrogen and dried using the Savant SpeedVac concentrator; finally, the sample was solubilized in 30 μl of 2× SDS-loading buffer and run in 10% SDS-PAGE. The samples were run to one-third of the gel, stained with Coomassie blue and collected by excising the whole lane for mass spectrometry analysis in the Taplin Mass Spectrometry Facility at Harvard Medical School.9-day-old wild-type control and 32P-labeled probe targeting the CR region of CaLCuV DNA A. The probe was obtained by PCR using the primers CR_F and CR_R ; then total DNA was extracted with phenol:chlorophorm:isoamyl alcohol (25:24:1) and precipitated with 2-propanol. The DNA was treated with RNase A and further purified with phenol:chlorophorm:isoamyl alcohol (25:24:1) and precipitated with ethanol, then dissolved in ultrapure water. The specified amount of DNA was separated by electrophoresis in 0.8% agarose, transferred overnight by capillarity to a Hybond-N membrane , and probed with and CR_R  and labeTotal RNA was extracted using Trizol reagent from either adult rosette leaves or 2-week-old seedlings of independent transgenic lines, the RNA blots were then performed as previously described .Immunoprecipitation of Flag-AGO1-associated small RNAs were performed as described . RNA wasN. benthamiana leaves infiltrated with various combinations of A. tumefaciens GV3101 harboring pCambia Myc-TrAP-nLUC or pCambia Myc-nLUC and A. tumefaciens ABI carrying pER10cLUC-3HA or pER10cLUC-3HA-candidate proteins. The agrobacteria containing the pER10 plasmids were incubated with 25 µM beta-estradiol for 3 hr prior infiltration, and all the cultures were adjusted to OD600 = 0.8. The transfected leaves were assayed 2 days after agroinfiltration by adding the substrate (10 mM luciferin). The sprayed leaves were incubated in total darkness for 5 min and photographed using an electronmultiplying charge-coupled device (EMCCD) camera, Cascade II 512, from Photomerics . The images were processed with WinView32 Ver 2.5.19.7 (Roper Scientific).The LCI was performed on 4-week-old N. benthamiana) were agroinfiltrated with syringe without needle as previously described . The co-localization was evaluated using a Nikon inverted microscope Eclipse Ti-E . CFP signal was measured by excitation with Shutter 10-3 filter 3 (CFPHQ [Ex]), and emission was detected at 485 nm; YFP used Shutter 10-3 filter 4 (YFPHQ [Ex]) and emission was detected at 540 nm. The images were processed using NIS-Elements-AR 4.30.01 (Nikon) and Adobe Photoshop software.Leaves of 4-week-old tobacco plants . FRET was determined by the acceptor photobleaching method  was incubated for 0–10 min at 37°C. The reaction products were separated by 18% SDS-polyacrylamide gel electrophoresis and visualized by Coomassie Brilliant Blue R-250 staining; then, the gels were fixed 1 hr in fixing solution  and scintillated for 30 min in 1 M sodium salicylate. Gels were dried 2 hr at 80°C. The 14C signal was detected by fluorography using in a preflashed Classic autoradiography film blue sensitive; Filters Kodak Wratten No. 22 and No. 96 were used together for preflashing. Preflashed film was exposed 5–7 days at −80°C. The film was developed using a Kodak M35A X-OMAT Processor, and the results were digitalized in a Chemi-Doc XRS System and analyzed with the Image Lab Software .In vitro HMTase reactions were modified from  as folloTo assess the impact of TrAP on KYP activity, His-MBP-TrAP or His-MBP were pre-incubated with His-GST-KYP in different molar ratios, ranging from 0 to 10, for 1 hr at room temperature, then the assays were proceeded as described above. The experiments were performed 3–5 times for statistical analysis.2, 60 mM KCl, 15 mM NaCl, 1 mM CaCl2, 1 mM PMSF, 1 pellet/50 ml Complete EDTA-free Protease inhibitor [Roche]), filtered through two layers of Miracloth and centrifuged at 11,000 × rcf for 10 min in 4°C. After discarding the supernatant, the pellet was resuspended in 1 ml of Nuclei lysis buffer ; the samples were sonicated in 10 cycles 30 s ON and 90 s OFF, using the Bioruptor  at the highest power in 4°Cs. The sonicated samples were centrifuged for 10 min at 21,000 × rcf in 4°C. 100 μl of the clarified chromatin was diluted 10-fold with Nuclei lysis buffer without SDS for each assay. The immunoprecipitation was accomplished by the addition of 40 µl Protein A Dynabeads (Invitrogen) and 3 μl of the pertinent antibody, followed by 6 hr incubation at 4°C on mild rotation. The beads-conjugated complexes were washed with 1 ml of Low salt buffer , followed by 1 ml of high salt buffer , then with 1 ml of LiCl buffer , and finally twice with 1 ml of TE  by incubating 5 min at 4°C in between washes. The samples were eluted twice at room temperature with 250 μl of elution buffer , for 15 and 30 min, respectively. The samples were decrosslinked with 100 mM NaCl at 65°C overnight, followed by Proteinase K treatment for 90 min at 45°C. The DNA was purified by Phenol:Chloroform:IsoamylAlcohol 25:24:1, and precipitated in 100% Ethanol at −80°C.The analysis of histone modifications was performed as described . Two graThe antibodies used are mono-clonal anti-H3K9me2 ; monoclonal anti-H3K4me3 ; mono-clonal anti-H3K27me3 .2, 0.5% Triton X-100, 5 mM DTT, 2 mM PMSF, 1 pellet/25 ml Complete EDTA-free Protease inhibitor [Roche]), filtered through two layers of Miracloth and centrifuged at 2000 × rcf for 15 min in 4°C. After discarding the supernatant, the nuclear precipitates were washed three times with 1 ml of Honda buffer; subsequently, the pellet was suspended in 300 μl of Nuclei lysis buffer  and sonicated in ten cycles 30 s ON and 90 s OFF, using the Bioruptor (Diagenode) at the highest power in 4°Cs. The sonicated samples were centrifuged for 10 min at 21000 × rcf in 4°C. 100 μl of the clarified chromatin was diluted 10-fold with ChIP dilution buffer  per ChIP. The immunoprecipitation was accomplished by the addition of 40 µl of Anti-FLAG M2 magnetic beads , followed by 2 hr incubation at 4°C on mild rotation. The beads were washed five times with 1 ml of Washing buffer  incubating 5 min at 4°C in between; then, two more washes with 1 ml TE buffer incubating 5 min at 4°C. Finally, the samples were eluted twice at room temperature with 125 μl of Elution buffer , for 15 and 30 min, respectively. The samples were decrosslinked and the DNA extracted as above.The immunoprecipitation of Flag-4Myc-KYP-Chromatin complexes was done as in , using A35S-TrAP transgenic plants, and lhp1 mutants. Seedlings were grown on MS medium with 1% sucrose for 7 days. One mg of total RNA was used for reverse transcription using MessageAmp II aRNA kits (Ambion) and 15 mg of labeled cRNA for hybridization. GeneChip hybridization and scanning were performed at the Genomics Resource Center, Rockefeller University, New York.Microarray analyses using the Affymetrix ATH1 platform were performed with two biological replicates using wild-type plants, Statistical analysis of microarray data was performed using R software. Initially the microarray plates were tested for quality by an M plot and the data normalized by the RMA method from the Affy package. Subsequently, the distribution of the samples was assessed with scatter plots and the normalized data sets were evaluated with the Moderate t-test from R package limma for p-value computation. Then, the eBays method was used to compute moderated t-statistics and log-odds of differential expression by empirical Bayes shrinkage of the standard errors towards a common value. The moderated t-statistic (t) is the logFC to its standard error. In our DEG results our thresholds are p-value < 0.05 and logFC >1 (up-regulated) or logFC < −1 (down-regulated). The False Discovery Rate was approximated from the eBayes adjusted p-value.The significance of the overlapping data sets was calculated through Pearson's Chi-squared test with 1° of freedom.Expression levels of the tested genes were examined by quantitative RT-PCR. Total RNAs were prepared from 9 days-old seedlings and treated with DNase before being subjected to cDNA synthesis using Superscript III reverse transcriptase (Invitrogen) primed by random primers. The EF1a gene  was inclArabidopsis genome (Version: TAIR10) using Bismark v0.14.3  at eight true leaves developmental stage, then we harvested nine to eleven newly emerged rosette leaves of each individual 18 days after inoculation . Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.eLife posts the editorial decision letter and author response on a selection of the published articles . An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent  and three reviewers. All four discussed their comments before we reached this decision, and the editor has assembled the following comments to help you prepare a revised submission.Thank you for sending your work entitled “Geminivirus-encoded TrAP suppressor inhibits SUVH4/KYP activity to counter host defense” for consideration at In your manuscript, you propose that a geminivirus encoded protein, TrAP, acts by interfering with the action of the SET domain histone methyltransferase SUVH4, a component of the plant host's transcriptional gene silencing machinery, providing a means that promotes virus survival in the arms race between virus and host. The topic is of broad interest given the conservation of HMTases in multicellular eukaryotes and the existence of other DNA viruses that affect animals, including humans. Your findings expand upon the previously proposed pleiotropic functions of TrAP proteins in suppression of TGS, such as adenosine kinase inhibition and SAMDC1 stabilisation. As well as illustrating a new role for TrAP during viral pathogenesis, your work could provide a useful tool for scientists, who could employ TrAP as a dominant inhibitor of H3K9me modification.TrAP overexpression causing developmental defects reminiscent of certain epigenetic mutants, on physical interaction of TrAP with SUVH4 and inhibition of SUVH4 histone methyltransferase activity in vitro, and on SUVH4 effects on the viral genome. While there was in principle broad enthusiasm for the work among the reviewers, there were several substantial concerns that preclude acceptance of the work in its current form. However, we would be very interested in a substantially revised version, in which you provide:Your conclusions regarding the TrAP/SUNVH4 interaction are based on suvh4 mutants and TrAP overexpressers.1) Whole-genome comparisons of DNA and H3K9 methylation in 2) More evidence of direct interaction of SUVH4 and TrAP using co-IPs from complemented mutants.Arabidopsis SET domain proteins or any of the other previously documented TrAP targets. The differences in phenotypes between TrAP overexpressers and suvh4 mutants would suggest that the SUVH4 interaction is not the entire story. To further support the conclusion that TrAP specifically interacts with SUVH4 in vivo, you should conduct co-IP experiments using constructs expressed at endogenous levels. You already have a tagged version of SUVH4 expressed under its endogenous promoter and you have many plant lines expressing varying levels of TrAP, and these lines could be easily crossed. Even better, but not mandatory, would be affinity purification of the epitope tagged TrAP followed by mass spectrometry.The first major point of your manuscript concerns the interaction of TrAP with SUVH4. While we were convinced that TrAP can interact with SUVH4 in vitro, the in vivo data with overexpression constructs were less convincing. This is especially important considering there might be competition for binding to other SET domain proteins. As is, it is difficult to determine whether TrAP is indeed exerting its effect largely through SUVH4 rather than through any of the other 31+ suvh4 single mutant, nor the suvh456 triple mutant, in which H3K9me levels are drastically reduced. In fact, you start out by comparing TrAP overexpressers with lhp1 mutants. In any case, the phenotypic discrepancies suggests that expression of TrAP disrupts many biological processes not connected with SUVH4 or its closest homologs, and it raises the possibility that TrAP generally targets SET domain HMTases, a possibility that you acknowledge in the Discussion. In and of itself, such potential promiscuity of TrAP is not an issue, as it would be just as interesting if a single viral protein could inhibit many SET domain HMTases. However, to make the argument that part of the effects of TrAP are through inhibition of SUVH4, you should compare the effects of TrAP overexpression to the effects of suvh4 mutants rather than the effects of other chromatin regulatory factors like LHP1. As there are very few genes mis-regulated in suvh4 single mutants, you should compare global levels of DNA and H3K9 methylation in TrAP overexpressers to those observed in a suvh4 mutant (or suv456 triple mutants). We also note that LHP1 has been shown to bind H3K27me3 and that it is in the CURLY LEAF/Polycomb pathway, and that there is no evidence that LHP1 is related to SUVH4 function. It is not clear how these distinct effects can be easily reconciled, and it tends to make the current presentation appear disjointed. On the other hand, in TrAP overexpression tends to phenocopy methylation patterns of suvh4 mutants at several endogenous loci, more so than the lhp1 knockout. These issues would most easily be reconciled with whole-genome data, as suggested above.A second main point is that TrAP inhibits SUVH4. Again, the in vitro data are very good, but the in vivo data are less conclusive. For example, you demonstrate that constitutive expression of TrAP causes developmental defects reminiscent of mutations affecting various epigenetic modifications. However, such defects are not observed in the suvh4 mutants are more susceptible to geminivirus infection. You also show that SUVH4 associates with the geminivirus genome by ChIP. However H3K9me modification of the viral genome is largely unaffected by loss of SUVH4, and there is no evidence that the observed changes influence expression of viral genes. You should compare viral gene expression in wild type and suvh4 mutants, to determine if transcriptional gene silencing by SUVH4 does indeed affect viral gene expression. If no change in expression is observed in the suvh4 mutant, this would argue against the presented model and suggest TrAP is functioning via an alternative mechanism.A final main point concerns the hypothesis that TrAP-mediated inhibition of SUVH4 is a means of suppressing host defense. In support of this notion, you cite (and reproduce) previous data showing that eLife. Your revised article has been favorably evaluated by Detlef Weigel (Senior Editor) and three reviewers. The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below.Thank you for resubmitting your work entitled “Geminivirus-encoded TrAP suppressor inhibits the histone methyltransferase SUVH4/KYP to counter host defense” for further consideration at Specifically, we would like you to follow up as much as possible the suggestions of Reviewer 2, and also to provide BS-seq data.Reviewer #1:The authors have addressed all my concerns. I understand the difficulties the authors had to address the major concern #2 and I think that, even when the requested experiment would improve the manuscript its absence doesn't make the manuscript any weaker.Reviewer #2:TrAP overexpression affects the activity of KYP in vivo. As outlined below, I remain concerned about the interpretation of this data. Regarding the final point, the role of KYP in regulating the expression of virus genes, the authors provide additional genetic evidence supporting a role of KYP in virus infection in a manner that is specifically connected to the TrAP gene. While this is not a direct assessment of the role of KYP in transcriptional regulation of viral genes, it does further support a link between TrAP, KYP, and viral defense. Taken together, the new experiments presented significantly strengthen the connection between KYP and TrAP, but the extent to which the TrAP protein modulates KYP HMTase activity at endogenous loci remains less clear.In the revised version of their manuscript, Castillo-Gonzalez et al. addressed several, but not all, of my main concerns. Regarding the in vivo relevance of the KYP-TrAP interaction, the authors conducted TrAP IP mass spectrometry experiments and found many peptides corresponding to KYP, confirming an interaction under biologically relevant conditions. A second concern was in regards to the H3K9me2 ChIP data supporting the conclusion that ChIP data:TrAP overexpression lines which is in support of their hypothesis that TrAP inhibits KYP activity in vivo. However, as detailed below, it is not clear which, if any of the 10 genes tested in TrAP overexpression lines and the kyp mutants. Thus, while this data is in support of their conclusion that TrAP affects KYP activity in vivo, it is unclear how broadly TrAP is affecting KYP function. Inclusion of more loci known to be targets of KYP would significantly strengthen the conclusion that TrAP affects KYP function in vivo.Castillo-Gonzalez present H3K9me ChIP data at a total of 14 endogenous loci in TrAP overexpression plants compared to WT plants…”. However, in looking at available H3K9me2 data on the UCSC browser, these genes are largely devoid of H3K9me signal. To determine if there is H3K9me2 signal at the sites tested in TrAP overexpression lines, the WT signal should not be normalized to one and instead should be presented as a % input  alongside the enrichment level at a gene without H3K9me (like actin). Such analysis should not require repeating the ChIP assays and thus can be done in the absence of a good lot of Abcam 1220 antibody. However, if these sites do not have sufficient H3K9me2 signal in Wt plants then testing additional loci will become necessary. In this case, looking at DNA methylation levels (even if they have been shown to be affected by other TrAP proteins the Bisaro lab) in Wt plants, TrAP overexpressor lines, and kyp mutants can serve in support of the authors’ claims without relying on the Abcam H3K9me2 antibody.The ChIP data presented in Mass spectrometry data:The text referring to the TrAP mass spectrometry data is quite brief and ambiguous as written. For example it is stated that 31 peptides uniquely match KYP, but it is unclear if these were only recovered in induced samples. Additional details should be provided in the text. For example in the response to the reviewers it is clearly stated that no peptides to KYP were identified in Col controls and that KYP was the most abundant hit from the mass spectrometry. It is not clear why these important details would be omitted from the text.Reviewer #3:TrAP overexpression mimics loss of SUHV4 activity. The papers that the authors mention “Dr. David Bisaro's lab . We also note that LHP1 has been shown to bind H3K27me3 and that it is in the CURLY LEAF/Polycomb pathway, and that there is no evidence that LHP1 is related to SUVH4 function. It is not clear how these distinct effects can be easily reconciled, and it tends to make the current presentation appear disjointed. On the other hand, inyou show that TrAP overexpression tends to phenocopy methylation patterns of suvh4 mutants at several endogenous loci, more so than the lhp1 knockout. These issues would most easily be reconciled with whole-genome data, as suggested above.suvh4. We do not have to reconcile the relevance of LHP1 with SUVH4  since TrAP could target both pathways as stated in the discussion. We agree that our manuscript would be further improved if we could conduct whole-genome comparisons of H3K9 methylation in suvh4 mutants and TrAP transgenic lines. With the advice as a guide, we have been actively pursuing this experiment. We prepared all materials and were ready for the ChIP-seq. However, we experienced an unexpected problem with the antibodies: as you know, the Abcam anti-H3K9me2 antibody (Ab1220) is the preferred antibody for ChIP-seq, and it is also the one we have successfully used before. Unfortunately, we ran out of our previous antibody (lot number 138716-4), and the newly purchased lots have not worked. We have screened the antibody from three lots already, with only one replacement barely immunoprecipitating H3K9me2 . To date, we have spent many weeks doing all kinds of tests in all possible conditions. The conclusion is that we need to wait for a new lot of antibody from Abcam to be tested, and to perform the ChIP-seq if it is good.This is an insightful point. It is true that TrAP transgenic plants phenocopy the morphology of mutants in the lhp1/leafy/polycomb pathway, but they also recapture the molecular phenotype of http://compbio.med.harvard.edu/antibodies/targets/37 andI thought to mention that the reason for us to choose the Abcam antibody was that Abcam is known to produce good anti-H3K9m2 antibody. In a big collaborative project funded by NIH to screen varieties of antibodies from many vendors worldwide, to filter out noise and to find the best ones in the epigenetics field , Abcam’s ab1220 was reported as the best antibody for H3K9me2 ChIP. A website was provided for evaluation of the performance of each antibody (i.e. http://compbio.med.harvard.edu/antibodies). There are several other companies producing anti-H3K9me2 . Data from this project suggested that the antibodies from these vendors could not be used for ChIP-seq.kyp mutant and Col-0 , inoculated kyp mutants and Col-0 control. We found that control plants did not allow replication of CaLCuV ∆TrAP at a detectable level. In contrast, the kyp mutant plants displayed systemic virus accumulation to a certain extent  previous data showing that suvh4 mutants are more susceptible to geminivirus infection. You also show that SUVH4 associates with the geminivirus genome by ChIP. However H3K9me modification of the viral genome is largely unaffected by loss of SUVH4, and there is no evidence that the observed changes influence expression of viral genes. You should compare viral gene expression in wild type and suvh4 mutants, to determine if transcriptional gene silencing by SUVH4 does indeed affect viral gene expression. If no change in expression is observed in the suvh4 mutant, this would argue against the presented model and suggest TrAP is functioning via an alternative mechanism.Arabidopsis, as the kyp mutants are hypersensitive, the complemented lines are as sensitive as the wild type, and the over expression lines are more resistant to infection. Moreover, we have shown that KYP expression affects the methylation status of the viral chromatin. Together, these data point to a direct effect of KYP in the pathogenesis of CaLCuV.Another insightful point. So far, we have provided evidence of the effect of KYP levels on the progression of CaLCuV infection in kyp mutant plants not only show more severe symptoms of infection, but also they accumulate the virus titers at more than 20-fold higher level than that in the wild type plants. The virus accumulation and systemic infection entails TrAP protein, as it is required for the expression of the late genes (coat protein and nuclear shuttle protein) that are necessary for the accumulation of the infective ssDNA particle. Most importantly, we have engineered a TrAP null mutant virus and we have performed infection studies in both the wild type and kyp mutant plants. As previously shown, begomoviruses deficient in TrAP are unable to cause systemic infection in wild type hosts; however, kyp mutant hosts can sustain systemic infection of this virus, although at low levels (Please refer to our response above).With the comment as a guidance, we have performed Southern blots to determine the populations of the viral DNA in the infected plants, and we have now shown that Specifically, we would like you to follow up as much as possible the suggestions of Reviewer 2, and also to provide BS-seq data.Reviewer #1:The authors have addressed all my concerns. I understand the difficulties the authors had to address the major concern #2 and I think that, even when the requested experiment would improve the manuscript its absence doesn't make the manuscript any weaker.Reviewer #2:In the revised version of their manuscript, Castillo-Gonzalez et al. addressed several, but not all, of my main concerns. Regarding the in vivo relevance of the KYP-TrAP interaction, the authors conducted TrAP IP mass spectrometry experiments and found many peptides corresponding to KYP, confirming an interaction under biologically relevant conditions. A second concern was in regards to the H3K9me2 ChIP data supporting the conclusion that TrAP overexpression affects the activity of KYP in vivo. As outlined below, I remain concerned about the interpretation of this data. Regarding the final point, the role of KYP in regulating the expression of virus genes, the authors provide additional genetic evidence supporting a role of KYP in virus infection in a manner that is specifically connected to the TrAP gene. While this is not a direct assessment of the role of KYP in transcriptional regulation of viral genes, it does further support a link between TrAP, KYP, and viral defense. Taken together, the new experiments presented significantly strengthen the connection between KYP and TrAP, but the extent to which the TrAP protein modulates KYP HMTase activity at endogenous loci remains less clear.ChIP data:Castillo-Gonzalez present H3K9me ChIP data at a total of 14 endogenous loci inand conclude that ∼11/14 show reduced H3K9me levels in the TrAP overexpression lines which is in support of their hypothesis that TrAP inhibits KYP activity in vivo. However, as detailed below, it is not clear which, if any of the 10 genes tested inare normally marked H3K9me2, as would be expected for bona fide KYP targets. Without further clarification on these loci, that leaves the 4 transposons tested in, of which only two show a >2x reduction in signal in the TrAP overexpression lines and the kyp mutants. Thus, while this data is in support of their conclusion that TrAP affects KYP activity in vivo, it is unclear how broadly TrAP is affecting KYP function. Inclusion of more loci known to be targets of KYP would significantly strengthen the conclusion that TrAP affects KYP function in vivo.This is a very important point. Obviously, we missed the opportunity of presenting our point clearly in the previous versions. Here in this version, we emphasized this critical point.Arabidopsis thaliana, Genome Biology, 2009; Bernatavichute et al., Genome-Wide Association of Histone H3 Lysine Nine Methylation with CHG DNA Methylation in Arabidopsis thaliana, PLoS ONE, 2008; Zhang et al., PLoS Biology, 2007) allowed us to pinpoint the specific histone modifications  on the genome and to provide a guide for selection of the regions for ChIP-qPCR  their transcription was significantly affected by the expression of the TrAP transgene ; and 2) We should also mention that there might be few cases in which our data was not fully consistent with the published work. The reason is that plant materials in our experiments differ from the materials used in previous studies. We used 11 days-old seedlings grown in short day conditions (8h light/ 16h dark), while some of the data reported was obtained from 3 weeks old adult plants grown under constant light (i.e. H3K9me2 and H3K4me3), or from 10-14 days-old seedlings grown in long day condition (16h light/ 8h dark) (i.e. H3K27me3). These differences imply a huge disparity in the developmental stage of the samples, as H3K9me2 and H3K4me3 mark were measured from flowered adult plants. Taking into consideration that we focused on flowering genes, it is not surprising that some differences between our data and others’ exist.The ChIP data presented in/B should be separated out to indicate which genes are marked by each chromatin modification in WT plants. Each gene will be predominantly marked by only one modification . Without knowing the levels of each modification in WT plants, the data cannot be fully interpreted as presented because the WT sample is artificially set to one. For example if a gene (like RDR1) is expressed and marked by H3K4me in Wt plants, but is not marked by H3K9me or H3K27me then a ChIP signal showing reduced H3K9me or H3K27me is likely noise rather than a real signal. This is a critical point in the paper, as they use this data make the statement that TrAP causes decreased H3K9me levels. In the subsection “TrAP reduced H3K9me2 repressive marks in vivo” they state: “specifically, eight out of 10 TrAP deregulated loci, including the flowering-promoting genes, displayed reduced H3K9me in the TrAP overexpression plants compared to WT plants…”. However, in looking at available H3K9me2 data on the UCSC browser, these genes are largely devoid of H3K9me signal. To determine if there is H3K9me2 signal at the sites tested inin WT plants, and to determine if this signal is meaningfully reduced in the TrAP overexpression lines, the WT signal should not be normalized to one and instead should be presented as a % input  alongside the enrichment level at a gene without H3K9me (like actin). Such analysis should not require repeating the ChIP assays and thus can be done in the absence of a good lot of Abcam 1220 antibody. However, if these sites do not have sufficient H3K9me2 signal in Wt plants then testing additional loci will become necessary. In this case, looking at DNA methylation levels (even if they have been shown to be affected by other TrAP proteins the Bisaro lab) in Wt plants, TrAP overexpressor lines, and kyp mutants can serve in support of the authors’ claims without relying on the Abcam H3K9me2 antibody.This is another insightful point; and thanks to this reviewer for guiding us on how to present our ChIP data in a more accurate way.First, based on the advice from this reviewer, we recalculated our ChIP-qPCR and completely reformatted the data taking into account the background signal, by redoing qPCR using the remaining ChIP DNA from the original experiments. In the new figures, each histone modification is shown separately. Also, the data is presented as enrichment relative to tubulin (TUB8) or relative to input alongside to TUB8 . We would like to emphasize that our remaining ChIP-DNA from previous experiments only allowed us to repeat 7 of 12 protein-coding genes in our original manuscript, and the 5 transposable elements presented in the current version.We would like to reiterate that the previously published H3K9me2 ChIP-seq data are obtained using plants at a developmental stage different from ours as stated above, and this fact could cause differences at the chromatin level that may render the data not readily comparable. For instance, the reported H3K9me2 marks at some of our target genes are low in the 3-week old plants, but one could not imply that they do not exist at the seedling stage. As an additional case, the published H3K27me3 (obtained from seedlings) and H3K4me3 (obtained from adult plants) available datasets, if directly compared, might mislead us to conclude that both modifications coexist at the flowering genes.Mass spectrometry data:The text referring to the TrAP mass spectrometry data is quite brief and ambiguous as written. For example it is stated that 31 peptides uniquely match KYP, but it is unclear if these were only recovered in induced samples. Additional details should be provided in the text. For example in the response to the reviewers it is clearly stated that no peptides to KYP were identified in Col controls and that KYP was the most abundant hit from the mass spectrometry. It is not clear why these important details would be omitted from the text.We stated this point in the previous point-to-point response letter; and now we have moved this critical information in the main text.Reviewer #3:The manuscript has improved, and I can understand the difficulties with the H3K9me2 ChiP-seq, but I dont really agree with the reasoning to not do the BSseq. This is easy enough and while changes in DNA methylation might represent a downstream effect, the DNA methylation changes are well characterised in in suvh4 so they could still get a good idea of whether or not TrAP overexpression mimics loss of SUHV4 activity. The papers that the authors mention “Dr. David Bisaro's lab ” only perform BSseq at specific loci, not genome wide.suvh4/kyp, and 35S-TrAP plants. The new result is now included in the new subsection “TrAP decreases CHH DNA methylation” and new As requested, we have performed whole genome bisulfite sequencing in our Col-0 wild type, TrAP overexpression did not affect CHG methylation; however, it reduced CHH methylation in gene rich regions that were almost completely overlapped with the suvh4 mutant. These regions included FT, CUC1, and AT4G17810, in accordance with our reported loss of H3K9me2 enrichment in TrAP transgenic lines and suvh4 mutants. Our new result is a bit surprising, but more exciting, that is to say, CHH and CHG could be regulated separately – it is known that CMT2 – the main CHH DNA methyltransferase – has greater affinity for H3K9me2 over H3K9me1. Other non-CG methyltransferases, such as CMT3  do not show binding preference among different methylation levels in the histone tail. We therefore envision methylation at CHH sites to be more sensitive to changes in H3K9me2 methylation than CHG. In all, our data is consistent with decrease in H3K9me2, which could be explained by partial inhibition of SUVH4 activity.We found that


The nucleosome positioning regulates the gene expression and many other DNA-related processes in eukaryotes. Genome-wide mapping of nucleosome positions and correlation of genome-wide nucleosomal remodeling with the changes in the gene expression can help us understanding gene regulation on genome level.A. thaliana. We have mapped genome-wide nucleosomes by performing tiling microarray using 146 bp mononucleosomal template DNA. The average nucleosomal coverage is approximately 346 bp per nucleosome both under the control and the SA-treated conditions. The nucleosomal coverage is more in the coding region than in the 5′ regulatory regions. We observe approximately 50% nucleosomal remodeling on SA treatment where significant nucleosomal depletion and nucleosomal enrichment around the transcription start site (TSS) occur in SA induced genes and SA repressed genes respectively in response to SA treatment. Especially in the case of the SA-induced group, the nucleosomal remodeling over the minimal promoter in response to SA is especially significant in the Non-expresser of PR1 (NPR1)-dependent genes. A detailed investigation of npr1-1 mutant confirms a distinct role of NPR1 in the nucleosome remodeling over the core promoter. We have also identified several motifs for various hormonal responses; including ABRE elements in the remodeled nucleosomal regions around the promoter region in the SA regulated genes. We have further identified that the W-box and TGACG/C motif, reported to play an important role in SA-mediated induction, are enriched in nucleosome free regions (NFRs) of the promoter region of the SA induced genes.In the present study, we correlate the gene expression and the genomic nucleosomal remodeling in response to salicylic acid (SA) treatment in A. thaliana. It also reports significant role of NPR1 in genome-wide nucleosomal remodeling in response to SA.This is the first study reporting genome-wide effects of SA treatment on the chromatin architecture of The online version of this article (doi:10.1186/s12870-014-0404-2) contains supplementary material, which is available to authorized users. PR-1a) of tobacco is remodeled on induction with salicylic acid (SA) [PR-1a core promoter. Several reports suggest that the position of the nucleosome is critical in globally regulating the in vivo binding of transcription factors by either allowing or blocking their binding to the nucleosome [in vivo packing mechanisms of nucleosome organization have also been proposed for the nucleosomes in yeast [It is now well known that the position of the nucleosome along a particular sequence of DNA has profound effects on its accessibility for all DNA processes such as transcription regulation, recombination, and repair -3. For ecid (SA) . This sucleosome -9. A nuccleosome -16. In ain yeast .Saccharomyces cerevisiae, Drosophila melanogaster, Homo sapiens, Caenorhabditis elegans and A. thaliana [Genome-wide mapping of nucleosome positions of eukaryotic genomes is a prerequisite to understand the basic mechanism of chromosomal organization. Previously genome-wide nucleosome positioning has been mapped and analyzed in thaliana ,18-27. Dthaliana , Microcothaliana -25,27, wA. thaliana has concluded for the first time that the nucleosomes are more densely packed in pericentromeric regions containing heavily methylated DNA than in the euchromatin region [A. thaliana.Previously a study of global nucleosome positioning in n region . Taking n region ,30. Thisn region . NPR1 han region -34. The A. thaliana chromosomes under both control and SA-treated conditions. We have also correlated the changes in nucleosome positioning to the global transcriptional response to SA in A. thaliana. Our results report distinct chromatin remodeling in core promoter and 5′ upstream promoter regulatory regions of SA-induced genes. We also find a distinct role of non-expresser of PR1 (NPR1) in the nucleosomal remodeling in SA-regulated genes. For this, we identify SA- regulated NPR1-dependent (ND) and NPR1-independent (NI) genes by performing the whole genome expression analysis under control and SA-treated conditions both in the wild-type and the npr1 mutant plants. The nucleosomal occupancy study of these groups reveals a correlation between SA-regulated ND genes and the dependence of NPR1 for nucleosomal remodeling at their core promoter region. We also find many defense related motifs both in the core promoter region and in the upstream regulatory region of SA regulated genes. While some of these are present in the nucleosome occupied regions (NORs), rest others are present in the nucleosome free region (NFRs).Briefly, we have mapped global nucleosome positioning in A. thaliana Col-0 (used as the wild-type) and npr1-1 mutant. All seeds were grown in vitro in MS (Murashige and Skoog) medium by keeping them on a mesh for stratification at 4°C for 2 days before placing them in the growth chamber. A. thaliana plants were grown under controlled environmental conditions . After two weeks of growth, the medium was exchanged either with water or with 2 mM SA, followed by 24 h incubation in light at 22 ± 1°C.Seed sterilization and plant growth conditions were followed as reported earlier  for bothnpr1-1) were treated either with water or SA, rinsed with water, blotted dry, and ground to powder in liquid nitrogen. Nuclei isolation buffer NIB1 , 0.5 mM EDTA, 0.1% Triton X-100, and 7 mM 2-mercaptoethanol) was added to it. The extract was first filtered through a 4-layered muslin cloth and then sequentially through 80, 60, 40, and 20 μm mesh sieves. The filtrate was centrifuged at 3000 g at 4°C for 10 min. The pellet was suspended in NIB2 (NIB1 without Triton X-100) and centrifuged again. The pellet was suspended in 5% percoll , loaded on a 20–80% percoll step gradient, and centrifuged. The nuclei were removed from the 20–80% percoll interface, washed in NIB2, and resuspended in NIB1 buffer. The nuclear preparation was incubated with micrococcal nuclease  in a buffer containing 25 mM KCl, 4 mM MgCl2, 1 mM CaCl2, 50 mM Tris-Cl (pH 7.4), and 12.5% glycerol at 37°C for 10 min at the concentration of 1unit/μg of genomic DNA from untreated sample and 0.6unit/μg of genomic DNA from treated sample  and 25 mM dNTP (with dTTP and dUTP in 4:1 ratio) in 20 μl aliquots for a total volume of 100 μl that was sufficient to obtain approximately 10 μg of purified PCR products for each sample. The PCR products were purified with the PCR cleanup kit . Fragmentation and labeling was done as per Affymetrix ChIP Assay Protocol (P/N 702238) using GeneChip® WT Double-Stranded DNA Terminal Labeling Kit (P/N 900812). Hybridization, washing, and staining were done according to the Affymetrix Chromatin Immunoprecipitation Assay Protocol onto GeneChip® A. thaliana Tiling 1.0R array (Reverse) using GeneChip® Hybridization, Wash, and Stain Kit (P/N 900720) on Affymetrix Fluidics station 450 according to the manufacturer’s protocol. Arrays were scanned on an Affymetrix GeneChip® 3000 7G scanner, and the signals were quantified with the Affymetrix GeneChip® Operating Software (GCOS) to generate cel files. The data can be found in NCBI public repository as GSE25553 [Isolated mononucleosomal DNA fragments (SA-treated and untreated) and genomic control DNA were amplified with random primers (N6), Klenow fragment (3′ → 5′ exoGSE25553 . The expcel files), which included the use of quantile normalization, probe analysis, and two-sample analysis to generate normalized log2 transformed values. The two-sample comparison analysis included datasets, either control versus genomic or SA-treated versus genomic, that could help in identifying consistently differentially enriched regions along the genome in control and SA-treated conditions respectively as compared to background region. Since genomic DNA was randomly sheared DNA, the comparison also yielded negative log2 transformed signal intensity values for certain probe sets in both cases. Throughout this article, this log2 transformed signal intensity value has been directly referred to as enrichment value. These enrichment values were then used for further analysis in a comparative fashion in order to determine the nucleosomal remodeling across the genome.In the subsequent analysis, raw data from all probes were mapped onto TAIR10. Tiling Analysis software (TAS) from Affymetrix technology was used to analyze tiling array data  have been used as seven stringency levels. For example, stringency level ≥0.0 means that if a probe has enrichment value ≥0.0, then only it may serve as a nucleosomal signal, not otherwise.C script to develop the dataset of distinct and fuzzy nucleosomes: Tab separated file containing the genomic positions and the log2 transformed signal intensity values from all the three replicates was used as input for both control and SA treated datasets to get the nucleosomal regions (distinct and fuzzy) at different stringency levels. For a given stringency level, the presence of a nucleosome is inferred, if the enrichment value is ≥ the value of the stringency level continuously at 4 probes (for distinct nucleosomes) or at >4 probes (for fuzzy nucleosomes) in all the three replicates in the dataset under study. For example, for inferring the presence of a distinct nucleosome at stringency level ≥0.0, four continuous probes should have enrichment value ≥0.0.A. thaliana from TAIR10 tabular files.Perl script to develop a database of global positions of TSS, start codon, stop codon, and mRNA end for all the genes along all the five chromosomes of C script to find out the remodeled nucleosomal regions and common nucleosomal regions: The nucleosomal regions present either only in control conditions or only in SA treated conditions were considered as remodeled nucleosomal regions and the nucleosomal regions present in both conditions were considered as the common nucleosomal regions for the purpose of input data in this script.The strand position (either forward or reverse) and the TSS (Transcription start site) of gene.The nucleosomal dataset both for distinct and fuzzy nucleosomes (coming from first script).C script to search every 50 bp window size for the nucleosomal coverage of 1000 bp upstream and downstream from TSS site of the genes in different groups: Two different input files were used –Four different custom scripts were developed to predict nucleosomal occupied regions from the signal intensity data of TAS  in different steps –If a continuous stretch of ≥25 bp was predicted to be having nucleosome occupancy in 50 bp window, then it was marked as count 1 (considered as having a nucleosome). On the other hand, if the continuous nucleosome coverage was <25 bp in 50 bp window, then it was marked as count 0 (considered as having no nucleosome).http://ncgd.nbri.res.in/nucleosome_prediction.aspx.All of these scripts are available at A total of 140 datasets were developed over seven stringency levels, two conditions and two types (distinct and fuzzy) of nucleosomes for the five chromosomes using first custom script. Nucleosome regions covering an area of 131–161 bp represented distinct nucleosomes and the nucleosome regions covering an area ≥161 bp represented fuzzy nucleosomes.A. thaliana  divided by the total region covered by nucleosomes (distinct + fuzzy) for all the five chromosomes of Regions having nucleosomes shifted/removed under SA-treated condition as compared to control condition.Regions having nucleosomes added under SA-treated condition as compared to control condition.Regions retaining nucleosomes under both conditions.Nucleosomal regions were identified for the following conditions using third custom scriptThe first two regions taken together (i.e. a + b) formed a remodeled nucleosomal region, whereas all regions taken together formed total nucleosomal coverage (i.e. a + b + c).npr1-1) in two independent biological replicates using Spectrum plant total RNA Kit (Sigma-Aldrich) according to the manufacturer’s instructions and eluted with nuclease-free water. After DNaseI (Ambion) treatment, the RNA was quantified and checked for integrity using a Bioanalyzer .Total RNA was isolated from two week old seedlings (both Col-0 and in vitro transcription and labeling using a GeneChip® 3′ IVT Express kit (Affymetrix) to generate cRNA as per manufacturer’s protocol.Direct labeling procedure was used i.e., double-stranded cDNA was synthesized using 250 ng total RNA, either with an oligo-dT primer (for QPCR purpose) or a T7 promoter-containing oligo (dT) primer, followed by The cRNA was fragmented to approx. <100 bases, hybridized to ATH1 Genome chip and were incubated at 45°C temperature for 16 hrs at 60 rpm in hybridization chamber. Washing and staining was performed by using an Affymetrix Fluidics Station 450 according to the manufacturer’s instructions . The arrays were scanned using Gene Chip Scanner 3000 7G (Affymetrix). Scanned images were processed and analyzed using GCOS software (Affymetrix). The data has been deposited in NCBI (GSE 51626).Arrays Images were captured and then raw cel files were analyzed using affylmGUI biocondcutor R package for Robust Multi-array Average (RMA) algorithm  by perfoGenes with log2 fold change ≥2 were considered as up-regulated, hence were called as SI genes,Genes with fold change ≤−2 were considered as down-regulated, hence were called as SR group,Uninduced genes with an equal and consistent expression profile in all the replicates of SA and control array files were grouped into silent (SIL) genes  and constitutive (CON) genes .The fold change criteria along with P-values ≤0.05 has been used to classify the genes in different groups.npr1-1 mutants under untreated and treated conditions (GSE51626) for the identification of ND and NI genes. Thereafter, two-way ANOVA was used under R script developed by Pavlidis et al. [npr1-1 mutant array files were processed for ANOVA analysis. Genes with adjusted P-value ≤0.05 were considered as SA regulated genes . The Ct (threshold cycle) value for each gene was determined and QPCR data analysis was done by comparing Ct values [For validating the ≥−1.0 stringency level, primers were designed both from the regions showing the presence of the nucleosome in both control and SA-treated conditions (exonic regions) and from the regions showing the absence of the nucleosome in both control and SA-treated conditions , as depicted in our tiling array analysis . For the internal control set, five exonic regions  were selected; the selected exons did not show nucleosomal remodeling on SA treatment. Moreover, they did not belong to any groups such as SI, SR, ND, or NI. The average Ct values of each of the three genes was normalized to the average Ct values of a complete set of internal control genes to obtain ΔCt values. Similar treatment was followed for all the sets; that is, wild-type Control (Wt-Ct), wild-type SA treated (Wt-SA), npr1-1 Control (NPR1-Ct), and npr1-1 SA treated (NPR1-SA). Thereafter, the average ΔCt value for the set of three genes was determined and average fold change was calculated and plotted along with standard error (SE). Student’s t-test was applied to test the significance of the changes under both conditions in wild-type versus mutant plants in a pair-wise manner.To assess the effect of NPR1 on nucleosomal occupancy on the promoter region in all the six groups, mononucleosomes were prepared as described earlier from both treated and untreated wild-type and Analysis of the relative transcript abundance of each group  was done using total RNA for each group prepared under similar conditions and using the same analyses strategy as done for studying nucleosomal occupancy described earlier.t-test  for each window in all the groups. Only the adjusted P-values ≤0.05  were considered as significant.Only the regions consisting of −1000 bp to +1000 bp with respect to TSS of a particular gene were considered for this purpose. Overlapping coordinates or genomic regions were filtered from −1000 to +1000 bp region with respect to TSS in all genes in the constitutive (CON), silent (SIL), SA induced (SI), SA repressed (SR), SA induced-ND (SI-ND), SA repressed-ND (SR-ND), SA induced-NI (SI-NI), and SA repressed-NI (SR-NI) groups. These regions were then removed from each group. The regions were mapped for the nucleosomal coverage  using fourth custom script. Statistical significance analysis at a P-value ≤0.05 was performed using Student’s t-test  was applied along with the Q-value package for multiple hypothesis testing correction. Only the adjusted P-values ≤0.05 were considered as significant.The presence or absence of a nucleosome in −1000 to +1000 bp-long region was calculated on per gene basis for all genes for both control and SA-treated conditions in different groups . Based on the remodeling at each position, percentage of the genes showing remodeling was calculated for each position for all groups. A graph representing the percentage of genes having nucleosome at each position was drawn along with the statistical significance data for comparison between different groups. Statistical analysis was done for −1000 to +1000 bp region with respect to TSS over a window of 150 bp (so as to cover one nucleosome long region) to find differences in the form of nucleosomal enrichment and nucleosomal depletion at the proximal promoter region among SI versus unaffected groups (i.e. CON and SIL groups together) and SR versus unaffected groups, SI-ND versus SI-NI and SR-ND versus SR-NI. For this, Student’s All the groups were analyzed for their enriched gene ontologies using agriGO software  under SEThird custom script was used to count the number of nucleosomal occupied, nucleosomal free and remodeled nucleosomal regions in the dataset of different groups. Nucleosomal bases were calculated in control as well as SA treated condition. Common nucleosomal bases present in the both the conditions were also calculated. Remodeled nucleosomal bases were calculated by subtracting the common nucleosomal bases from total nucleosomal bases (Control and SA treated).a) For SI genes: Remodeled nucleosomal regions (199)  of 1000 bp upstream regions from SI genes (111) were extracted from TAIR10 Database. Motif analysis was done for these regions. MEME (ver. 4.2)  was deplA. Thaliana (W) were used to identify novel cis-regulatory elements specifically enriched in SR genes (with respect to SI genes) present in the remodeled regions. A database was constructed consisting of all possible combinations of 6 bases length. Thereafter, the motif analysis was done using following two methods:i)Lift methodLift  was calcii)Motif enrichment (OE) methodThe expected occurrence of a particular hexameric motif was counted by the method used for counting W-Box and TGA motifs. Thereafter, the ratios between observed and expected occurrence for each motif found in the SI and W remodeled regions were calculated. Similarly, the ratios between observed and expected occurrence for each motif found in the SR and W remodeled regions was also calculated. Finally, ratio of motifs enriched in SR as compared to W and the motif enriched in SI as compared to W was calculated to obtain enriched motif in SR genes with respect to SI genes.B) For SR genes: The promoter regions −600 to −800 upstream TSS (200 bp) of SI and SR genes were extracted from TAIR10 database. Additionally, as control dataset, the remodeled nucleosomal regions  from whole genome of cis-regulatory W-box motif (TTGAC) and TGA motif (TGACG/C) was independently calculated for the following four regions:total promoter region,region having the nucleosome occupied under both conditions (NOR),region lacking the nucleosome under any of the two conditions ( NFR), andregions with remodeled nucleosomes.Nucleosomal regions from the 1000 bp upstream TSS (promoter regions) were extracted for all the groups. In these regions, the enrichment of First of all, the local nucleotide frequency (F) of a base was calculated in the above mentioned regions as per the formula –Say, T = total no of all bases in a particular regionThen F = (count of the a particular base in a region)/Tcis-regulatory motif was calculated based on the local nucleotide frequency of each constituent base of the motif under study using the following formula:Thereafter, the expected frequency of occurrence of a particular The occurrence of these motifs has been further corrected for their expected frequency of occurrences and for the differences due to database sizes for total number of promoters, NORs, remodeled nucleosomes, and NFRs. The corrected frequency of occurrence of a particular motif was calculated by normalizing the observed frequency with the expected frequency of their occurrence. Next, graphs showing the significance of each motif in all groups were plotted at 95% binomial confidence interval with respect to the total promoter region.A. thaliana on SA treatment. To determine the changes in the nucleosome positions that eventually lead to changes in the gene expression, 2-week old control or SA-treated A. thaliana seedlings were used for mononucleosomal DNA preparation . PR1 (pathogenesis-related gene1) is a marker gene of the SA pathway. Earlier, it was demonstrated by us in case of Nicotiana tabacum that a distinct nucleosome spans the TATA-box and TSS of PR-1a minimal promoter at the resting state, which gets remodeled and removed on activation with SA [PR1 in A. thaliana, similar to tobacco PR-1a, is also an SA-induced NPR1-dependent gene; thus, PR1 was selected to validate the results obtained by tiling microarray on SA-induced NPR1-dependent genes. The A. thaliana PR1 gene (At2g14610) is located on the reverse strand of chromosome 2; its genic region spans from position 6,241,704 to 6,243,463 (1760 bp) encompassing the promoter region which spans from 6,242,463 to 6,243,463 with distinct TATA-box at position 6,242,491 ; and extending further into both the promoter region and the region downstream TSS, indicating the presence of many nucleosomes in that region. After SA induction, distinct MNase1 sensitivity was observed in this region and, thus, lack of hybridization signals around the region −221 to +78 bp, indicating the nucleosomal remodeling and removal of the nucleosome from the region  to −2.67  for extracting mononucleosomes, thus interpreting only an average position of a nucleosome rather than the distinct nucleosome present in a specific cell lineage, as also observed in the case of Caenorhabditis elegans [The chosen stringency level was subjected to further verification using QPCR analysis over four loci having a nucleosome under control conditions and four other loci lacking any nucleosome under control conditions. The results are concurrent with the interpretation made for each gene locus based on the tilling microarray experiments  than the regulatory region  in conjunction to our nucleosomal mapping experiment. SI, SR, CON and SIL groups were identified  at the TSS was ascertained in all these groups on SA treatment. For this, the total numbers of genes, showing nucleosomal occupancy under any condition, were retrieved and the percentage of the genes showing either nucleosomal depletion or enrichment on SA treatment around −1000 to +1000 bp region with respect to TSS was calculated. Mapping of the cumulative data showed interesting patterns. It was found that the region downstream of TSS show comparatively less remodeling in all the four groups Figure . The CONTo understand the reason behind distinct nucleosomal remodeling profiles of different groups, collective expression profile of these groups was obtained by annotating these groups on the basis of their functions. It is found that most of the CON genes were significantly expressed in different metabolic pathways, while most of the SIL genes were significantly expressed in reproductive and floral development pathways  gene during transcription activation in yeast [et al. have also shown the same for the beta-phaseolin gene [The data analysis at the minimal promoter region of the ve genes ,50,52. Tve genes . In the in yeast ,52,54,55in yeast  where itin yeast . Li et alin gene . Till daPR1 locus using a mononucleosomal template  over the entire region on SA treatment. The data  in the SI genes is affected by the presence of NPR1. Thus, SA-regulated -ND and -NI genes were identified  and t Figure . First, 5 Figure . SI-ND a5 Figure  indicatecis-regulatory motifs were identified in the remodeled nucleosomal region in the promoters (1 kb upstream of TSS) of 111 SI genes  respectthaliana , in the thaliana , as a cothaliana ) respectthaliana . The prePR1 in the SAR pathway have been reported to be highly enriched in W-box motif TTGAC [PR1 regulon genes. However, both TGA factors and WRKY transcription factors are reported to regulate SA response by binding TGACG motif and W-box, respectively, in the PR1 promoter [PR-1a gene regulation [Promoters of the genes co-regulated with if TTGAC , providipromoter ,74 and gpromoter ,76. Therpromoter ,78, and gulation . Hence, A. thaliana [extA gene in B. napa in response to wounding and tensile stress [B. napa, rice, barley and wheat in relation to abscissic acid regulated transcription [cis-regulatory elements that are involved in both the SA-mediated response of SA-induced genes and the JA- and ABA-mediated response of SA-repressed genes.The SR group shows characteristic nucleosomal remodeling by enrichment on SA treatment at an upstream region, that is, −600 to –800 bp upstream of TSS Figure D. Using thaliana ,80. TGA1thaliana ,82 respethaliana  and an ethaliana  respectie stress ,86. Manycription ,68,87,88cription ,89,90. Tcription . Motifs cription -101 werecription ,102-106.Perturbations change the transcriptional profile of an organism. These changes require both silencing and activation of certain genes and, thus, demand chromatin remodeling of such gene loci. In our study, conspicuous nucleosomal remodeling is found in the promoter region of salicylic acid regulated genes, where, under any of the two conditions, 40% of the chromatin is occupied by nucleosomes. The nucleosomal coverage is higher in 3′ coding regions as compared to the non-coding regions. Likewise, the nucleosome coverage is higher over the entire chromosome except the centromeric region with marginally higher coverage in the pericentromeric regions. The nucleosomal remodeling at the promoter region in constitutive and silent groups remains insensitive to the salicylic acid treatment, whereas both salicylic acid induced and salicylic acid repressed groups show nucleosomal depletion and enrichment, respectively, on salicylic acid treatment. The nucleosomal depletion in the promoter region of the salicylic acid induced genes is a general feature of the group. A distinct role of Non-expresser of PR1 (NPR1) in salicylic acid regulated remodeling is observed. The salicylic acid induced-NPR1dependent genes operate through nucleosomal depletion, and salicylic acid repressed-NPR1 dependent genes operate through nucleosomal enrichment in response to salicylic acid induction in an NPR1-dependent manner. All the four groups, that is, salicylic acid induced-NPR1 dependent, salicylic acid repressed-NPR1 dependent, salicylic acid induced-NPR1 independent, and salicylic acid repressed-NPR1 independent, show significant nucleosomal enrichment in the absence of NPR1 on salicylic acid treatment.The remodeled nucleosomal regions in salicylic acid induced groups harbour many important motifs that are known to be enriched in the genes involved in defense mechanisms such as ABRE elements, MYB box etc. Nevertheless, prominent motifs in the salicylic acid pathway, that is, W-box and TGACG/C, are enriched in nucleosome free regions of the promoter region. Even the upstream region of salicylic acid repressed groups harbours many motifs that are involved in defense pathways.A. thaliana. It also interprets the enrichment of relevant motifs with respect to overall nucleosomal positioning in the promoter regions of salicylic acid regulated genes.Overall, this study reports, for the first time, the genome-wide effects of salicylic acid treatment on the chromatin architecture of http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE25553 and http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE51626.The datasets supporting the results of this article are available in the NCBI GEO repository as,