Salmonella Pathogenicity Islands (SPIs) in pathogenesis of Salmonella enterica Typhimurium infection in the chicken is poorly studied, while many studies have been completed in murine models. The Type VI Secretion System (T6SS) is a recently described protein secretion system in Gram-negative bacteria. The genus Salmonella contains five phylogenetically distinct T6SS encoded in differentially distributed genomic islands. S. Typhimurium harbors a T6SS encoded in SPI-6 (T6SSSPI-6), which contributes to the ability of Salmonella to colonize mice. On the other hand, serotype Gallinarum harbors a T6SS encoded in SPI-19 (T6SSSPI-19) that is required for colonization of chicks. In this work, we investigated the role of T6SSSPI-6 in infection of chicks by S. Typhimurium. Oral infection of White Leghorn chicks showed that a ΔT6SSSPI-6 mutant had reduced colonization of the gut and internal organs, compared with the wild-type strain. Transfer of the intact T6SSSPI-6 gene cluster into the T6SS mutant restored bacterial colonization. In addition, our results showed that transfer of T6SSSPI-19 from S. Gallinarum to the ΔT6SSSPI-6 mutant of S. Typhimurium not only complemented the colonization defect but also resulted in a transient increase in the colonization of the cecum and ileum of chicks at days 1 and 3 post-infection. Our data indicates that T6SSSPI-6 contributes to chicken colonization and suggests that both T6SSSPI-6 and T6SSSPI-19 perform similar functions in vivo despite belonging to different phylogenetic families.The role of the  Salmonella gastroenteritis has an estimated global burden of 93.8 million cases per year, of which 80.3 million cases are likely to be food-borne S. enterica serovar Enteritidis and S. enterica serovar Typhimurium Salmonella enterica serovar Typhimurium (S. Typhimurium) is a broad host-range pathogen able to infect humans, mice and birds. In mice, this serovar causes a systemic infection similar to human typhoid fever that results from infection with serovar Typhi  Salmonella infection. In humans however, S. Typhimurium causes self-limiting gastroenteritis characterized by abdominal pain, vomiting and inflammatory diarrhea Nontyphoidal in vitro cell culture systems have identified numerous genes required to establish a successful infection by S. Typhimurium. Most genes are clustered in genomic islands known as Salmonella Pathogenicity Islands (SPIs) Salmonella enterica, the SPI-1 and SPI-2 are the two major virulence determinants of Salmonella. Each of these SPIs encodes two different type III secretion systems (T3SS) that deliver effector proteins directly into the cytoplasm of eukaryotic cells SPI-1 is mainly involved in invasion of intestinal epithelial cells Salmonella containing vacuole inside infected cells SPI-2 is essential for survival within phagocytic cells and systemic infection Studies conducted using murine models of infection and S. Typhimurium infection in the chicken are few and are sometimes contradictory. While some authors reported that both T3SSSPI-1 and T3SSSPI-2 are required for the infection process SPI-1 nor T3SSSPI-2 is critical for colonization of chickens Salmonella-resistant mice and one-week-old chickens by S. Typhimurium Salmonella infection of the chicken, and that genes in addition to the highly conserved SPIs are required for chicken colonization and systemic spread.Studies on the role of the SPIs in the pathogenesis of Salmonella contains five phylogenetically distinct T6SS loci; four of them are differentially distributed among serovars of S. enterica, while the fifth T6SS is present in S. bongoriSPI-6 and T6SSSPI-19, have been linked to Salmonella pathogenesis. T6SSSPI-6 is required for intracellular replication in macrophages and systemic dissemination in mice by S. Typhimurium S. Typhi SPI-19 contributes to colonization of the gastrointestinal tract and internal organs of chickens by S. Gallinarum strain 287/1 Type VI secretion systems participate in a variety of different processes, ranging from inter-bacterial relationships to pathogenesis SPI-6 to S. Typhimurium ability to colonize the gastrointestinal tract and internal organs of White Leghorn chicks. We have also addressed whether T6SSSPI-19 of S. Gallinarum can rescue the colonization defect of a S. Typhimurium mutant lacking T6SSSPI-6. Through competitive index experiments we demonstrate that T6SSSPI-6 is crucial to gastrointestinal colonization and systemic spread of S. Typhimurium in chicks. In addition, we show that transfer of T6SSSPI-19 restores the colonization defect of a mutant lacking T6SSSPI-6, indicating that both T6SS perform similar functions in vivo despite belonging to different phylogenetic families.In this study we have investigated the contribution of T6SSThe bacterial strains used in this work are listed in XbaI restriction enzyme (Fermentas) and T4 DNA ligase (New England Biolabs) were used as per manufacturer instructions. DNA samples were routinely analyzed by electrophoresis in 1% agarose gels (1X Tris-acetate-EDTA buffer) and visualized under UV light after ethidium bromide staining.DNA manipulations were performed using standard protocols. Plasmid DNA was isolated from overnight cultures using the QIAprep Spin Miniprep Kit (QIAGEN), according to the manufactureŕs instructions. Genomic DNA was isolated from overnight cultures utilizing the GenElute Bacterial Genomic DNA kit (Sigma) according to the manufactureŕs instructions. PCR products were purified using the QIAquick PCR Purification Kit (QIAGEN). Primers were designed using the Vector NTI Advance 10.0 software (Invitrogen) and are listed in S. Typhimurium carrying deletions of the T6SSSPI-6 gene cluster and the clpV (STM0272) or phoN genes were constructed using the Lambda-Red System int-201. To be able to identify wild type versus mutant colonies in the mixed competition experiments, the S. Typhimurium ΔphoN mutant was used as the wild type strain. phoN+ and phoN- strains can be distinguished by blue-white selection on 5-bromo-4-chloro-3-indolyl phosphate (XP) containing media, phoN- strains form white colonies while phoN+ strains appear blue. Mutations in phoN do not affect the ability of S. Typhimurium to colonize and persist in the chick Mutants of SPI-6 gene cluster from S. Typhimurium 14028s onto plasmid R995 was performed by the VEX-Capture technique for the targeted excision and cloning of large DNA fragments loxP sites were inserted at each side of the targeted genomic region by homologous recombination of PCR products by the Lambda-Red system, using as templates the plasmids pVEX1212 and pVEX2212 that encode Sp and Cam resistance cassettes, respectively. Correct insertion of loxP sites was confirmed by PCR using primers SPI-6_OUT5 and STM0266_VEX_H2_U2 for loxP insertion located in the upstream region of the T6SS cluster, and primers SPI-6_OUT_DOWN and STM0298_VEX_H2_D2 for the downstream loxP insertion. This cluster was excised from the chromosome as a non-replicating circular DNA molecule by specific recombination of loxP sites mediated by the action of Cre recombinase encoded in plasmid pEKA30. This intermediate was captured into the R995-VC6 vector by a homologous recombination event, producing the R995+SPI-6 plasmid. The R995-VC6 plasmid contains a 1,209 bp internal region of homology to the T6SSSPI-6 cluster, cloned by PCR amplification with primers STM_VC_OUT5 and STM_VC_OUT3 . For competitive infections in chickens, the in vivo stability of plasmids R995 and R995+SPI-6 was assessed in each organ at each time point studied. No differences were observed on colony forming units (CFU) indicating that R995 and its derivatives are highly stable in vivo.Plasmid R995+SPI-6 was transferred to S. Typhimurium strains were grown aerobically at 42°C for 16 hours in LB broth. This temperature of incubation was used because it corresponds to the body temperature of chicks. For single and competitive infections, fifteen 4-day old unsexed White Leghorn chicks were orally inoculated with 109 CFU of a single strain or with an equal mixture of the strains to be tested in a volume of 100 µl of sterile PBS. The inoculum was serially diluted and plated to determine the titer and input ratio. Five birds from the infected group were sacrificed by asphyxiation with CO2 on days 1, 3 and 9 post-infection. Ileum, cecum (including contents), liver and spleen were collected. These organs were homogenized in sterile PBS and serial ten-fold dilutions spread on LB agar plates containing the appropriate antibiotics for determination of CFU. For histopathological analysis, the cecum and liver of experimental animals were fixed in 10% formalin for 24 h followed by incubation in 70% ethanol and then embedded in paraffin. The samples were stained with hematoxylin and eosin and 10 fields per sample were examined and scored by a trained veterinary pathologist to determine histopathological changes.t-test. P values of <0.05 were considered statistically significant .Data obtained from competitive infection experiments were calculated as a mean ratio of logarithmically converted CFU of mutant to wild type normalized to the input ratio. Error bars indicate standard error. Statistical significance was determined using a two-tailed Students All animal experiments in this study were approved by the Texas A&M University Institutional Animal Care and Use Committee (TAMU AUP# 2010-38) and were carried out in accordance with the Guide to the Care and Use of Laboratory Animals, the Public Health Service Policy on the Human Care and Use of Laboratory Animals.S. Typhimurium.Single infections and competitive index experiments were performed to determine the contribution of the SPI-6 T6SS to intestinal and systemic colonization of chicks by SPI-6 mutant (MTM35) or a ΔclpV deletion mutant (MTM2640) and colonization of the cecum, ileum, liver and spleen was evaluated over 9 days of infection. ClpV, a conserved structural component of the T6SS that belongs to Clp/Hsp100 AAA+ of ATPase superfamily, is required for the activity of the secretion system SPI-6 and ΔclpV mutant strains showed an overall lower degree of colonization of the cecum and ileum from day 3 post-infection and of the liver and spleen from day one post-infection, suggesting a role for the SPI-6 T6SS in chick colonization.For single infections, White Leghorn chicks were orally-infected with either the wild-type strain, a ΔT6SSSPI-6 and ΔclpV mutants during intestinal and systemic colonization from day 1 post-infection. This markedly attenuated phenotype was more pronounced at the third day post-infection and it was maintained throughout day 9 in each organ analyzed. These results indicate that S. Typhimurium requires a functional T6SS to efficiently colonize the avian host.In order to determine the competitive fitness within the host, of each mutant strain, competitive index experiments were performed. White leghorn chicks were orally infected with a mixture of each mutant with the wild-type strain at a 1∶1 ratio and colonization of each organ was evaluated over 9 days of infection. As shown in S. Typhimurium 14028s .To directly link the absence of the T6SSSPI-6 mutant and the wild type, each bearing the empty vector  and colonization was determined at days 1, 3, and 9 post infection. As shown in SPI-6 gene cluster to the ΔT6SSSPI-6 mutant restored its ability to colonize the cecum and the ileum at all time points. On the other hand, in the spleen and liver, the results were not conclusive due to a very low and heterogeneous colonization of these deeper tissues by S. Typhimurium harbouring the R995 plasmid (data not shown). Nevertheless, complementation of the defective phenotype of the ΔT6SSSPI-6 mutant in the gastrointestinal tract supports the contribution of T6SSSPI-6 in chicken colonization.The complemented strain (MTM35R6) was tested in a competition experiment against the ΔT6SSSPI-19 contributes to efficient colonization of infected chicks by S. Gallinarum 287/91 SPI-6 and T6SSSPI-19 have different evolutionary histories, and were probably acquired at different times during Salmonella evolution Salmonella colonization of infected chicks, we examined the possibility that both T6SS could contribute to chicken colonization in a similar extent. To test whether T6SSSPI-19 can restore the ability of the S. Typhimurium ΔT6SSSPI-6 mutant to efficiently colonize the avian host, the complete T6SSSPI-19 gene cluster captured from S. Gallinarum 287/91 in the R995 plasmid was transferred to S. Typhimurium ΔT6SSSPI-6 by conjugation. The resulting strain (MTM35R19) was tested in a competition experiment with the wild-type S. Typhimurium strain bearing the empty R995 vector (WT/R995). The results showed that introduction of the T6SSSPI-19 complemented the colonization defect of the ΔT6SSSPI-6 mutant in both the cecum and ileum  Scheme of the VEX-Capture procedure: loxP sites were inserted in the chromosome of S. Typhimurium 14028s at each side of the T6SSSPI-6 gene cluster through homologous recombination of PCR products using the Lambda-Red system. In presence of pEKA30, a plasmid that constitutively expresses the Cre recombinase, the T6SS cluster was excised from the chromosome as a non-replicative, circular DNA intermediate that was captured through homologous recombination in R995-VC6, a derivative of R995 plasmid harboring an internal region of homology to T6SSSPI-6. (B) Tiling-PCR analysis of the T6SSSPI-6 gene cluster cloned onto the R995 plasmid. Specific primers were designed to amplify ten fragments that cover the entire T6SSSPI-6 region and whose lengths vary between 3,298 and 4,274 bp.(TIF)Click here for additional data file.


StBEL5 RNA, its role in controlling tuber formation, and the function of the 503-nt 3′ untranslated region (UTR) of the RNA in mediating transport. Because the 3′ UTR of StBEL5 is a key element in regulating several aspects of RNA metabolism, a potato leaf cDNA library was screened using the 3′ UTR of StBEL5 as bait in the yeast three-hybrid (Y3H) system to identify putative partner RNA-binding proteins (RBPs). From this screen, 116 positive cDNA clones were isolated based on nutrient selection, HIS3 activation, and lacZ induction and were sequenced and classified. Thirty-five proteins that were predicted to function in either RNA- or DNA-binding were selected from this pool. Seven were monitored for their expression profiles and further evaluated for their capacity to bind to the 3′ UTR of StBEL5 using β-galactosidase assays in the Y3H system and RNA gel-shift assays. Among the final selections were two RBPs, a zinc finger protein, and one protein, StLSH10, from a family involved in light signaling. In this study, the Y3H system is presented as a valuable tool to screen and verify interactions between target RNAs and putative RBPs. These results can shed light on the dynamics and composition of plant RNA-protein complexes that function to regulate RNA metabolism.Heterografting and RNA transport experiments have demonstrated the long-distance mobility of  StBEL5  has also been clearly established in both cucumber and pumpkin  in the cell, and a number of RNA-protein interactions have been established. RBPs function in splicing, nuclear export, RNA transport and localization, translation, and stability  system  similar in design to the stolon library described by Chen et al.  of the 3′ UTR of n et al.  was usedStBEL5 full-length 3′ UTR hybrid RNA was transformed with 60 μg of the potato cDNA library. The entire transformation mixture (about 10 mL) was spread onto plates containing SD/-his/-leu/-ura and 1.0 mM 3-aminotrizole (3-AT), a competitive inhibitor of the HIS3 gene product. To calculate the transformation efficiency, the transformation mixture was serially diluted  and grown on small SD/-leu/-ura plates. After 1st and 2nd rounds of screening on different concentration  of 3-AT-containing plates, positive colonies were applied to β-galactosidase assays in order to measure the induction of the lacZ reporter gene using a Yeast β-galactosidase Assay Kit (Pierce Biotechnology), according to the manufacturer’s protocol. From the assays, 116 colonies were selected. The 3′ UTR of StBEL5 with StPTB6-pAD  with slight modifications. Positive yeast colonies were picked from the plate and inoculated in 3.0 ml of SD/-leu. Overnight grown cells were pelleted and incubated at 30°C for at least 30 min after resuspension in 480 μl Buffer SE/β-mercaptoethanol and 40 μl lyticase solutions. After incubation, yeast plasmid DNA was isolated by following the kit protocols. The rescued yeast plasmids were transformed into E. coli BL21-Codon (DE3) cells (Stratagene). The recombinant proteins were induced with 0.4 mM IPTG, and purified using HisPur Cobalt Purification Kit (Pierce Biotechnology). For in vitro transcription to generate RNA probes, T3 promoter-containing sense primers were created by adding T3 sequences on the 5′ end of the sense primers of the target sequences (Table TM Gel RNA Recovery kit (ZymoResearch). Five femtomoles of biotin-labeled RNA probes were incubated with indicated amounts of purified recombinant proteins in the binding buffer provided by the Light Shift Chemiluminescent RNA EMSA kit (Pierce Biotechnology) on ice for 45 min. The RNA-protein complexes were separated in 2.5% agarose (for the full-length probe) or 5% polyacrylamide gel (for the IRE probe) and transferred onto BrightStar-Plus (Ambion) nylon membranes. The signal was detected using the EMSA kit according to the manufacturer’s manual.The PCR-amplified fragments with gene-specific primer sets  were used for testing HIS3 expression. The 281 colonies were streaked on these plates, and 194 colonies were grown on SD/-his/-leu/-ura plates containing at least 5.0 mM 3-AT. Finally, 116 colonies were selected based on β-galactosidase and HIS3 activation and were sequenced and analyzed  exhibited significant matches to previously characterized or known genes , and their putative RNA-binding function, seven cDNA clones were selected for verification as protein partners of StBEL5, and designated as StBEL5 RNA-binding protein (B5RBP) one to seven , containing a sucrose synthase motif for the sucrose metabolic pathway and a glycosyltransferase motif for biosynthetic processes  protein with a Domain of Unknown Function  in the C-terminal region. It is involved in light regulation of seedling development  containing both KOW  protein family member containing a coiled-coil motif (CC) and a RRM  tail and control its length  that is involved in photomorphogenesis was shown to mediate the expression of At5G65260  family protein containing two ankyrin repeats for protein-protein interactions, and two zinc finger-C3H1 domains (ZF-CCCH) for zinc ion binding, and nucleic acid binding  functions in mRNA stability  family protein that shares sequence similarity with Arabidopsis AtRNP1 (AT4G14300) containing two RRM domains  that is an ortholog of the human nuclear import receptor transportin1 protein  appeared 10 times from the Y3H screening . B5RBP1 and -2 exhibited the strongest interaction with sequence located toward the 3′ end of the UTR (T2 and UA baits). B5RBP4, the potato ortholog of eIF5A, exhibited equivalent strength of activity with all three truncated baits suggesting a degree of non-specific binding. These results with the potato eIF5A are consistent with previous work showing that a pumpkin form of eIF5A exhibited RNA-binding that was non-sequence-specific in nature  appear to function in the intracellular transport of RNAs. Two RBPs identified in the current study, eIF5A and HSP70, were isolated from a phloem-mobile RNP complex of pumpkin  in stolons and young tubers  also suggest a possible role in tuber development. Screening a library of proteins from a sink organ like the tuberizing stolon would further expand our understanding of the RNP complex responsible for delivery of the mobile StBEL5 transcript to its functional site. Overall, these results demonstrate the utility of the Y3H system in screening and verifying interactions between target RNAs and putative RBPs.Factors known to affect The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Functional RNA molecules participate in numerous biological processes, ranging from gene regulation to protein synthesis. Analysis of functional RNA motifs and elements in RNA sequences can obtain useful information for deciphering RNA regulatory mechanisms. Our previous work, RegRNA, is widely used in the identification of regulatory motifs, and this work extends it by incorporating more comprehensive and updated data sources and analytical approaches into a new platform.An integrated web-based system, RegRNA 2.0, has been developed for comprehensively identifying the functional RNA motifs and sites in an input RNA sequence. Numerous data sources and analytical approaches are integrated, and several types of functional RNA motifs and sites can be identified by RegRNA 2.0: (i) splicing donor/acceptor sites; (ii) splicing regulatory motifs; (iii) polyadenylation sites; (iv) ribosome binding sites; (v) rho-independent terminator; (vi) motifs in mRNA 5'-untranslated region (5'UTR) and 3'UTR; (vii) AU-rich elements; (viii) C-to-U editing sites; (ix) riboswitches; (x) RNA cis-regulatory elements; (xi) transcriptional regulatory motifs; (xii) user-defined motifs; (xiii) similar functional RNA sequences; (xiv) microRNA target sites; (xv) non-coding RNA hybridization sites; (xvi) long stems; (xvii) open reading frames; (xviii) related information of an RNA sequence. User can submit an RNA sequence and obtain the predictive results through RegRNA 2.0 web page.http://regrna2.mbc.nctu.edu.tw.RegRNA 2.0 is an easy to use web server for identifying regulatory RNA motifs and functional sites. Through its integrated user-friendly interface, user is capable of using various analytical approaches and observing results with graphical visualization conveniently. RegRNA 2.0 is now available at  Numerous functional RNA motifs have been identified as playing significant roles in many essential biological processes, including transcriptional and post-transcriptional regulation of gene expression, control of mRNA stability, alternative splicing, and transcription termination. The biological activities of functional RNA motifs usually rely on a combination of their primary sequences and specific secondary structures, which act as target sites of RNA-binding factors or directly interact with translation machinery . For inscis-regulatory RNA elements. Each family is represented by a multiple sequence alignment of known and predicted representative members, and annotated with a consensus base-paired secondary structure. It facilitates the identification and classification of new members of known RNA families, and provides the glimpses of conservation of multiple ncRNA families across a wide taxonomic range. fRNAdb [Several databases were established for collecting functional RNA molecules ,4-14. UT. fRNAdb ,7 is a d. fRNAdb . The dat. fRNAdb ,10 is a . fRNAdb ,12 is an. fRNAdb  is the ccis-regulatory elements in various regions surrounding. RBSfinder [Various approaches were developed for identifying functional RNA motifs or elements -26. GeneBSfinder  is a proBSfinder  can rapiBSfinder  was deveBSfinder  was deveBSfinder  is a sysBSfinder  was deveBSfinder  is a praBSfinder  is an imBSfinder  is an apAnalysis of functional RNA motifs and sites in RNA sequences can obtain useful information for deciphering RNA regulatory mechanisms. Our previous work, RegRNA , is wideThis work presents an integrated web server, RegRNA 2.0, for identifying functional RNA motifs and sites in an input RNA sequence. Numerous data sources, such as Rfam , fRNAdb The functional RNA motifs and sites supported in RegRNA 2.0 are categorized into several types: (i) splicing donor/acceptor sites; (ii) splicing regulatory motifs; (iii) polyadenylation sites; (iv) ribosome binding sites; (v) rho-independent terminator; (vi) motifs in mRNA 5'-untranslated region (5'UTR) and 3'-UTR; (vii) AU-rich elements; (viii) C-to-U editing sites; (ix) riboswitches; (x) RNA cis-regulatory elements; (xi) transcriptional regulatory motifs; (xii) user-defined motifs; (xiii) similar functional RNA sequences; (xiv) microRNA target sites; (xv) non-coding RNA hybridization sites; (xvi) long stems; (xvii) open reading frames; (xviii) related information of an RNA sequence.cis-regulatory families, 11 profiles of ERPIN RNA elements, 12 descriptors of RiboSW riboswitches, 48 models of UTRsite motifs and 2,171 transcription factor binding matrices of TRNASFAC, 294 sequence patterns of AEdb splicing regulatory motifs, 5 sequence patterns of ARED AU-rich elements, 475,318 fRNAdb sequences whose length are less than 500 nt, 21,643 miRNA sequences of miRBase and 170,581 ncRNA sequences of NONCODE collected for identifying different functional RNA motifs and sites.The process flow of RegRNA 2.0 is depicted in Figure cis-regulatory families. miRanda [Numerous analytical approaches and data sources were integrated in RegRNA 2.0 Table . GeneSpl miRanda  and miRN miRanda  and sequ miRanda  is utili miRanda -33. For  miRanda  is used  miRanda  is integ miRanda  are usedAn integrated web-based system with user-friendly interface Figure  was deveRegRNA 2.0 provides an intuitive graphical visualization  Joint Research Program under Grant Number: VGHUST101-G5-1-1. This publishing charge is also partially supported by MOE ATU.The authors approved the submission of this paper to BMC Bioinformatics Volume 14 Supplement 2, 2013: Selected articles from the Eleventh Asia Pacific Bioinformatics Conference (APBC 2013): Bioinformatics. The full contents of the supplement are available online at http://www.biomedcentral.com/bmcbioinformatics/supplements/14/S2.This article has been published as part of A cartoon representation of the mechanism of genetic regulation by the guanine riboswitch [Click here for file


Arabidopsis thaliana, they are silenced by the siRNA–mediated transcriptional gene silencing pathway where long non-coding RNAs (lncRNAs) produced by RNA Polymerase V (Pol V) guide ARGONAUTE4 (AGO4) to chromatin and attract enzymes that establish repressive chromatin modifications. It is unknown how chromatin modifying enzymes are recruited to chromatin. We show through chromatin immunoprecipitation (ChIP) that SPT5L/KTF1, a silencing factor and a homolog of SPT5 elongation factors, binds chromatin at loci subject to transcriptional silencing. Chromatin binding of SPT5L/KTF1 occurs downstream of RNA Polymerase V, but independently from the presence of 24-nt siRNA. We also show that SPT5L/KTF1 and AGO4 are recruited to chromatin in parallel and independently of each other. As shown using methylation-sensitive restriction enzymes, binding of both AGO4 and SPT5L/KTF1 is required for DNA methylation and repressive histone modifications of several loci. We propose that the coordinate binding of SPT5L and AGO4 creates a platform for direct or indirect recruitment of chromatin modifying enzymes.Eukaryotic genomes contain significant amounts of transposons and repetitive DNA elements, which, if transcribed, can be detrimental to the organism. Expression of these elements is suppressed by establishment of repressive chromatin modifications. In  Arabidopsis thaliana, however for many of them their position in the silencing pathway is unknown. One of those proteins is SPT5L/KTF1, a homolog of an elongation factor associated with RNA Polymerase II. Here we establish the position of SPT5L in the silencing pathway and propose the molecular mechanism of its function. This gives further knowledge of the mechanism of transcriptional gene silencing and is important to understand how transposons are controlled.Transposons and other repetitive elements occupy vast areas of the eukaryotic genomes. They pose a threat to genome integrity but at the same time regulate expression of many genes and have been proposed to be a major factor contributing to genome evolution. One of the processes responsible for controlling activity of transposons and other repetitive elements is transcriptional gene silencing. This process uses small interfering RNA and long non-coding RNA to recruit enzymes that establish repressive chromatin modifications. Several proteins have been identified to be needed for siRNA–mediated transcriptional silencing in  Eukaryotic genomes contain significant amounts of transposons and other repetitive DNA elements, which usually remain transcriptionally inactive. Efficient silencing of transposon transcription is essential for preventing their mobility and for maintaining genome integrity Arabidopsis thaliana, single-stranded RNA precursors for siRNA biogenesis are produced by RNA Polymerase II (Pol II) or RNA Polymerase IV (Pol IV), while the second strand is synthesized by RDR2 (RNA-Dependent RNA Polymerase 2). DCL3 (Dicer-like 3) cleaves double-stranded RNA into siRNAs that are then incorporated into ARGONAUTE4 (AGO4) Transposons are silenced at both transcriptional and post-transcriptional levels by mechanisms that involve small interfering RNAs (siRNAs) Schizosaccharomyces pombe where loci subject to siRNA-mediated transcriptional silencing are actively transcribed by RNA Polymerase II S. pombe was observed by the ability of Argonaute proteins to cleave RNA. This ability is required for the establishment of repressive chromatin modifications Recognition of target loci by AGO4-siRNA complexes requires sequence identity between siRNAs and the genomic loci. These loci, however, are often actively transcribed, and it is not clear if siRNAs base-pair interact with DNA or nascent RNA transcripts Arabidopsis where transcriptional silencing requires a specialized RNA Polymerase complex known as RNA Polymerase V (Pol V) This mechanism may be similar in RNA Polymerases and AGO4 are assisted in their functions by several other known protein components of the plant silencing system, all of which are required for efficient establishment and maintenance of transcriptional silencing Despite the recent progress in understanding the mechanisms of transcriptional gene silencing, it is not known how siRNAs work with Pol V transcripts, AGO4 and other proteins to recruit chromatin modifying enzymes to their target loci in chromatin. It is unknown how chromatin-bound AGO4 recruits enzymes that establish repressive chromatin modifications. It is unknown if other protein components of the silencing system help AGO4 recruit chromatin modifying enzymes. It is also unknown in what order proteins involved in silencing are recruited to chromatin. Here we try to resolve the mechanism of siRNA-mediated recruitment of chromatin modifying enzymes to chromatin and the function of SPT5L in this process. We show that SPT5L physically interacts with chromatin and that SPT5L works downstream of Pol V but does not require 24-nt siRNA. SPT5L and AGO4 are recruited to chromatin in parallel and at least partially independently of each other and both are needed for DNA methylation and repressive histone modifications at several loci. We propose that the coordinate binding of SPT5L and AGO4 creates a platform for direct or indirect recruitment of chromatin modifying enzymes.IGN5 and solo LTR DNA from Col-0 wild type at much higher levels than from spt5l mutant which represents the background level  loci (The interaction of SPT5L with AGO4 nd level . This sh 8) loci , which aIGN20, IGN22, IGN23, IGN25 and IGN26  methylation at tested loci. Consistently with previous reports, DNA methylation was strongly reduced at AtSN1 locus in both ago4spt5l mutants IGN5 locus in ago4 mutant IGN5 locus in spt5l mutant and at IGN23, IGN25 and IGN26 loci in both ago4 and spt5l mutants . At IGN5 and IGN26 loci, H3K9me2 was reduced in all three mutants , dms3-4, and ago4-1 introgressed into Col-0 background were described previously rdr2-1 mutant was obtained from J. Carrington. spt5l-1  mutant line, affinity-purified anti-SPT5L (anti-KTF1), affinity-purified anti-Pol V (anti-NRPE1) and affinity-purified anti-AGO4 antibodies were described previously T method relative to inputs ChIP was performed essentially as described HaeIII, AluI or AvaII restriction enzymes (NEB) for 20 min. After heat-inactivation of the enzyme DNA was amplified using 0.75u Platinum Taq (Invitrogen).For DNA methylation analysis genomic DNA was extracted from above-ground tissue of 2-week old plants using DNeasy Plant Mini Kit (Qiagen). 100 ng of genomic DNA was digested with 10u of Total RNA was extracted from 2-week old plants using RNeasy Plant Mini Kit (Qiagen) and amplified using SuperScript III Platinum SYBR Green One-Step qRT-PCR Kit (Invitrogen) in Applied Biosystems 7500 real time PCR machine.Oligonucleotide primers used in this study are shown in Table S1Loci assayed in this study, their accession numbers and oligonucleotide primers.(PDF)Click here for additional data file.Text S1Detailed Chromatin Immunoprecipitation (ChIP) protocol.(PDF)Click here for additional data file.


Patterns of herbivory can alter the spatial structure of ecosystems, with important consequences for ecosystem functions and biodiversity. While the factors that drive spatial patterns in herbivory in terrestrial systems are well established, comparatively less is known about what influences the distribution of herbivory in coral reefs.We quantified spatial patterns of macroalgal consumption in a cross-section of Ningaloo Reef . We used a combination of descriptive and experimental approaches to assess the influence of multiple macroalgal traits and structural complexity in establishing the observed spatial patterns in macroalgal herbivory, and to identify potential feedback mechanisms between herbivory and macroalgal nutritional quality. Spatial patterns in macroalgal consumption were best explained by differences in structural complexity among habitats. The biomass of herbivorous fish, and rates of herbivory were always greater in the structurally-complex coral-dominated outer reef and reef flat habitats, which were also characterised by high biomass of herbivorous fish, low cover and biomass of macroalgae and the presence of unpalatable algae species. Macroalgal consumption decreased to undetectable levels within 75 m of structurally-complex reef habitat, and algae were most abundant in the structurally-simple lagoon habitats, which were also characterised by the presence of the most palatable algae species. In contrast to terrestrial ecosystems, herbivory patterns were not influenced by the distribution, productivity or nutritional quality of resources , and we found no evidence of a positive feedback between macroalgal consumption and the nitrogen content of algae.This study highlights the importance of seascape-scale patterns in structural complexity in determining spatial patterns of macroalgal consumption by fish. Given the importance of herbivory in maintaining the ability of coral reefs to reorganise and retain ecosystem functions following disturbance, structural complexity emerges as a critical feature that is essential for the healthy functioning of these ecosystems. Spatial heterogeneity in ecosystems can strongly influence population structure, community composition and ecosystem processes This study focuses on the processes that control the spatial distribution of herbivory in coral reefs, ecosystems that are characterised by some of the highest rates of herbivory Early studies dealing with spatial patterns in coral reefs focused on the distribution of marine plants, and highlighted the importance of herbivory for maintaining differences among habitats Roving herbivorous fishes have been clearly identified as the key herbivores in undisturbed coral reefs In this study, we quantified spatial patterns of macroalgal herbivory by fishes across a coral reef, tested for similar spatial patterns in potential explanatory variables, and then used manipulative and mensurative experiments to test some hypotheses arising from the patterns observed. We tested for the presence of spatial variation in herbivory by quantifying consumption of erect macroalgae and measuring the biomass and composition of herbivorous fishes among a cross section of a coral reef . We then related patterns in herbivory to the spatial distribution of algal cover, algal biomass and structural complexity in these three habitats. In the manipulative and mensurative experiments, we selected two habitats with contrasting levels of herbivory (reef flat and lagoon) and used herbivore exclusion and feeding experiments to test hypotheses about the mechanisms that might cause the observed consumption patterns. In particular, we asked: (1) Does consumption of macroalgae relate to spatial patterns in macroalgal productivity, nutritional quality, community composition and/or palatability? (2) Does herbivory influence macroalgal nutritional quality? (3) Does benthic structural complexity and proximity to reef influence the distribution of macroalgal herbivory?Sargassum) among habitats, a pattern that was constant at all sites . Since Sargassum was much more abundant in the lagoon than in other habitats (see below), and consumption may depend on local availability of this resource, we incorporated Sargassum availability into our analysis of consumption by comparing a relative consumption index (RCI) for this taxa across habitats, where RCI  =  proportion of Sargassum consumed x proportion of Sargassum present  as for rates of herbivory.There was a significant difference in the rates of herbivory  of all roving herbivorous fish yielded a high classification success of 93.1% across all habitat types (i.e. only 6.9% misclassification error). Correlations with CAP axis scores indicate that a high biomass of eef flat , where tof 59.7% . Correlachlegeli .Lobophora variegata, Turbinaria ornata and an unidentified filamentous green alga, whereas the outer reef habitat was characterised by red algae belonging to the genera Amphiroa and Amansia  but not between macroalgal herbivory and algal biomass . In addition, there were no significant relationships between measurements of herbivory rates and total herbivorous fish biomass , or browser fish biomass . Rates of herbivory were higher in the sites and habitats that were more structurally complex, as reflected by a strong linear relationship between rates of consumption of tethered Sargassum and rugosity , where rugosity explained 90% of the variance in consumption.We found a near-significant logarithmic relationship between site averages for measurements of macroalgal herbivory and algal cover  showed that changes in algae biomass were strongly influenced by the habitat in which thalli were deployed and whether thalli were caged, as shown by a Habitat x Herbivory interaction , i.e. the presence of cages did not confound the interpretation of the effects of herbivory on algal biomass. In the absence of herbivory, there were no differences in L. variegata biomass accumulation between habitats  , but theLobophora variegata after 5 days of deployment (mean consumption (± SE) reef flat morphotype  = 2.57±1.14 cm2, lagoon morphotype  = 2.13±0.59 cm2; t = −0.6359, df = 12, p = 0.537, 95% CI  =  −4.006, 1.416). The two morphotypes did not differ in their nitrogen content (mean nitrogen content (± SE) reef flat morphotype  = 1.22±0.05%, lagoon morphotype  = 1.21±0.02%; Welch's t = 0.170, df = 4.657, p = 0.873; 95% CI  =  −0.134, 0.153). The C:N ratio of the reef flat morphotype tended to be lower than the lagoon morphotype, although statistical differences between the two only approached significance (mean carbon:nitrogen ratio (± SE) reef flat morphotype  = 21.87±0.3, lagoon morphotype  = 23.65±0.81; t = −2.057, df = 8, p = 0.074). The 95% confidence intervals of this near-significant result were relatively wide and non-symmetrical around zero , suggesting that a difference in C:N ratio may exist but was not detected by our test. We found no differences among morphotypes in their phenolic content (mean phenolic content (± SE) reef flat morphotype  = 1.56±0.1%, lagoon morphotype  = 1.60±0.24%; t = −0.151, df = 8, p = 0.883; 95% CI = −0.643, 0.564).We detected no significant difference in area loss between lagoon and reef flat morphotypes of Sargassum varied with increasing distance from the boundary between the reef flat and lagoon, and the nature of the differences among distances varied among sites . Macroalgal cover in the lagoon at Ningaloo ranged between 10–80%, values that are similar to inner shelf systems in the GBR (36–66%), while the outer reef at Ningaloo (located about 1 km offshore) hosted <10% algal cover, values that are more similar to mid-shelf or outer-shelf reefs located 50–100 km offshore in the GBR (0–15%) The distinct spatial patterns in consumption of algae described in this study are also similar to the GBR We found a near-significant negative logarithmic relationship between algal cover and algal consumption that suggests that the cover of macroalgae is reduced under high herbivory conditions, but it also depends on other factors under low herbivory conditions. The conclusion that this relationship is causal, rather than just correlative, is supported by the growth in macroalgae with experimental exclusion of herbivores. We did not detect a similar relationship between macroalgal biomass and herbivory, probably because much of the algal biomass collected in our quadrats was present under coral plates and in small crevices that were effectively inaccessible to consumers, whereas algae surveyed as percentage cover better reflect algae that is available to herbivores.Lobophora variegata transplanted to the high herbivory reef flat and low herbivory lagoon. Similarly, we did not find any among-habitat differences in the nitrogen content of algae. We did, however, detect some differences between habitats in the availability of nitrogen per unit carbon, which tended to be higher for thalli placed in the reef flat (i.e. lower C:N ratio). Lower C:N ratios are generally associated with higher palatability, and many marine herbivores are known to preferentially consume and grow faster on diets with low C:N ratios L. variegata, the two morphotypes were equally consumed, despite near-significant differences in C:N ratio. This contrasts with the findings of Coen and Tanner Spatial patterns of macroalgal consumption were not related to among-habitat differences in macroalgal production (measured as biomass change) or nutrient content, since these algal traits were similar in habitats with contrasting levels of herbivory. Experimental exclusion of herbivores resulted in very similar growth of Sargassum species, which are readily eaten by macroalgal browsers worldwide when accessible  was characterised by high abundance of palatable Lobophora variegata specimens from habitats with contrasting levels of herbivory (lagoon and reef flat morphotypes) indicates that potential differences in nitrogen fixation or other nitrogen uptake mechanisms between habitats are not having an effect on macroalgal nitrogen content in Ningaloo Reef.In many terrestrial systems, herbivores have a positive effect on the plants they consume through enhancing nutrient recycling and availability, a feedback mechanism known as grazing optimisation In this study, structural complexity was identified as the key feature mediating spatial patterns of macroalgal consumption by fish. This conclusion is supported by three lines of evidence: (1) different outcomes from experimental exclusion of herbivorous fish in structurally-complex reef habitat and structurally-simple lagoon habitat; (2) a strong linear relationship between macroalgal consumption and structural complexity; and, (3) a decrease in herbivory with increasing distance from structurally-complex reef habitat. These results are consistent with other studies that have shown an increase in herbivore density and grazing rates with topographical complexity Understanding the mechanisms that drive spatial patterns of ecological processes in coral reefs is particularly important for the management of these systems, because the ability of individual coral reefs to reorganise and maintain ecosystem function following disturbance is considered to strongly depend on the matrix of adjacent reefs and habitats in the surrounding seascape The Department of the Environment and Conservation of Western Australia provided a permit to the authors during 2008–2009 to perform this study within the Sanctuary Zones of the Ningaloo Marine Park (Permit Number CE002084) and to collect flora samples (Permit Number SF006457).This study was conducted at Ningaloo Reef , a fringing reef approximately 290 km in length. Ningaloo Reef is an arid-zone reef where extensive coral reefs occurs in close proximity to the mainland. The study was conducted between April 2008 and September 2009 in the Mandu  and Maud  sanctuary zones of the Ningaloo Marine Park. At Ningaloo, the reef crest is narrow and mostly devoid of coral growth, the reef flat landward of the reef crest hosts coral communities across a width of approximately 150 m, and the outer reef slopes seaward of the reef crest presenting a well-developed spur and groove morphology to depths of 30 m Most of this study took place in the Mandu sanctuary zone, where we haphazardly selected three study sites in each of three habitats that characterise the area: lagoon, reef flat and outer reef . We perfSargassum myriocystum. This brown alga is rapidly eaten (within hours) and has a relatively simple morphology, which allowed us to estimate biomass loss from differences in length before and after deployment. S. myriocystum plants bear 3–6 main lateral branches per individual, and each of these has a consistent length-weight relationship (148.01±9.241 mg/cm). Patterns of herbivory were determined by placing lateral branches of S. myriocystum of 15 cm in length and similar weight  in three sites at each of the lagoon, reef flat and outer reef habitats. In each site, S. myriocystum lateral branches (n = 25) were distributed haphazardly and tethered to the available substrata using cable ties. In the lagoon habitat, branches were either tethered to other macroalgae or to lose pieces of dead coral on the sand at around 1.5 m depth. In the reef flat habitat, branches were mostly tethered to pieces of dead coral covered in epilithic algal matrix adjacent to live corals, at 1–2 m depth. In the outer reef habitat, branches were mostly tethered to coral pieces and crustose coralline rocky surfaces at about 6 m depth – the shallowest depth that we could easily access in regular swell conditions. Tethered algae were collected 4–7 hours after deployment, and mass consumed was estimated from the total length consumed, and converted to mass consumed per hour. Some tethered algae became detached and lost, leading to an unbalanced data set . Replicates where algae became wholly detached were not included in the analysis because we could not be sure that detachment was due to herbivory.Relative differences in consumption of erect macroalgae between lagoon, reef flat and outer reef habitats at all Mandu sites were measured in April 2008 using tethered pieces of b, where W is weight in grams, TL is total length and parameters a and b are constants obtained from the literature Kyphosus vaigiensis, Naso lituratus, Naso spp., Naso unicornis, Scarus ghobban, S. schlegeli, initial-phase Scarus sp., Siganus argenteus, S. doliatus, and S. trispilos.Censuses of the herbivorous fish assemblage were carried out during a two-week period in November 2008 at three sites in each of the three habitats in the Mandu Sanctuary zone . Fishes To determine whether spatial patterns in macroalgal herbivory were related to macroalgal distribution, we measured algal cover, algal biomass and community composition at each site at Mandu in November 2008. Algal cover was quantified using the line intercept benthic survey method described by Fox and Bellwood 2 haphazardly placed quadrats of all macroalgae  at each site. Algal samples were bagged and returned to the laboratory, where they were sorted to genus level (where possible) and weighed. Algal taxa that we were unable to identify were classified according to broad functional groups .Macroalgal biomass and community composition were measured by clearing three 0.25 mTo determine whether patterns in consumption of macroalgae were related to topographic complexity, we measured coral cover  and estimated a rugosity ratio (n = 3) at each site. Live coral cover was quantified using the line intercept benthic survey method described above for algal cover. To measure rugosity, a 10 m light chain was placed along the substrate contour, and the equivalent straight line horizontal distance encompassed by the 10 m of chain was measured (n = 3). The rugosity ratio (R) was calculated as the straight line horizontal distance along the reef divided by the total chain length, with values close to unity indicating a flat substratum and lower values indicating a structured habitat Lobophora variegata . Open (uncaged) plots were marked with steel reinforcing bar along the corners. In caged plots, fences and roofs were made with plastic-coated metal mesh (2.5 cm mesh size), thereby excluding large herbivorous fish. Partial cages consisted of steel reinforcing bars along the corners with one fenced side and a roof, and were used to control for cage artefacts. All plots had a base of plastic coated mesh to which thalli were attached. The experiment ran for 6 weeks, and the cages were cleaned of fouling organisms once after 2 weeks, although these were not abundant. We recorded blotted wet-weight of algae (n = 3) at the beginning and at the end of the experiment and calculated biomass change.We used triangular cages of 1082 cmL. variegata sections of intermediate age  were used in the chemical analyses, and care was taken to gently remove any epibiota. Some thalli were so heavily grazed that we used the entire thallus in the analysis, and in some instances we did not have enough mass to conduct chemical analyses, leading to an unbalanced data set.At the end of the experiment, algal thalli were freeze-dried and ground. Nitrogen and carbon content of individual thalli were analysed using a Europa Scientific ANCA-NT 20-20 mass spectrometer. Where possible, Lobophora variegata from high and low herbivory habitats  with an experiment performed in April 2008 in the Mandu sanctuary zone. Lagoon and reef flat habitats are characterised by hosting different morphotypes of L. variegata. As in other coral-reef ecosystems, an erect ruffled form (hereafter referred to as ‘lagoon’ morphotype) is usually found on sandy substrata where herbivores are less abundant, whereas the flat decumbent form (hereafter referred to as ‘reef flat’ morphotype) is usually found underneath coral plates in coral-dominated habitat where herbivores are often more common L. variegata were offered in pairs of similar initial area in the reef-flat habitat, where we recorded highest herbivore activity. Replicate pairs (n = 15) were at least 3 metres apart from each other. An equal number of controls (n = 15) were individually protected from herbivores with plastic window-screen mesh cages (3 mm mesh size). L. variegata pairs were left in the field for five days. Replicates with one or two wholly detached algae were not included in the analysis because we could not guarantee that detachment was due to herbivory. Photographs of each algal specimen were taken at the beginning and at the end of the experiment and consumption was measured as changes in area determined using ImageJ analysis software. Five additional thalli of each morphotype were collected at the beginning of the experiment for carbon, nitrogen and phenolic chemical analyses to further identify potential differences in nutritional traits between habitats. Total phenolic content was quantified spectrophotometrically using a modified Folin-Ciocalteu assay We compared the palatability and chemical composition of Sargassum myriocystum lateral branches at a range of distances from the reef flat/lagoon boundary and measured the amount of algae consumed after 48 hours. We predicted that if structural complexity positively influences herbivory, the rates of macroalgal removal would be higher near the structurally complex reef flat habitat than in the flat lagoon habitat. This experiment was performed in the Maud sanctuary zone. We were unable to perform this experiment in the Mandu sanctuary zone because the reef flat/lagoon boundary in that part of the Ningaloo reef is diffuse, with isolated coral heads scattered irregularly near the boundary. In contrast, the reef flat and lagoon habitats are clearly defined at Maud. These two sanctuary zones have similar rates of macroalgal removal and a similar herbivorous fish assemblage . Sargassum myriocystum (n = 3) lateral branches of about 40 cm in length were tethered at 9 distances relative to the reef flat/lagoon boundary  at each of three separate sites. Replicates within each distance per site were approximately 15 m apart and parallel to the reef flat/lagoon boundary, and sites were approximately 250 m apart. The length of each individual S. myriocystum lateral branch was measured at the beginning and at the end of the experimental period. Since sand was the most common substrate away from the reef, S. myriocystum lateral branches were tethered to lose pieces of dead coral that were buried in the sand.To test the effects of proximity to reef on macroalgal removal, we tethered All data were checked for normality and equality of variances by visual inspection of scatterplots and distribution of residuals Patterns in consumption of tethered algae, total fish biomass, coral cover and in the species composition of herbivorous fish and algae communities were analysed using PERMANOVA testing for differences between sites  and habitats . Patterns in the cover and biomass of macroalgae and in rugosity were analysed using analysis of variance (ANOVA) with the same design. When significant differences were detected between main effects in ANOVA, Student-Newman-Keuls (SNK) tests were used to resolve the differences among means.Bray-Curtis distance was our metric in all multivariate analyses and data were fourth-root transformed prior to analyses to reduce the effects of numerically large values (i.e. abundant schooling species) We used regressions to determine whether rates of herbivory matched patterns in algal cover, algal biomass and rugosity, and to assess the relationship between herbivory rates and roving herbivorous and browser fish biomass. All data were averaged at the site level. Linear, polynomial and logarithmic regressions were fitted to the data, and the significant regression that best-fit the data was selected using Akaike's Information Criterion.L. variegata after 6 weeks were analysed using a four-way ANOVA, testing for differences among sites , habitats , large herbivore-exclusion treatments  and plots . Differences in feeding between lagoon and reef flat L. variegata were analysed using a t-test as outlined by Peterson and Renaud In the herbivore exclusion x habitat experiment, changes in biomass and chemical composition in All PERMANOVA and multivariate analyses were performed using Primer-E v6 software Figure S1Study area: Mandu and Maud sanctuary zones in Ningaloo Reef, Western Australia. In Mandu, there were three sites within each habitat: lagoon (diamond icons), reef flat (circle icons) and outer reef (square icons).(EPS)Click here for additional data file.Table S1PERMANOVA results on removal rates of Sargassum myriocystum lateral branches between sites and habitats.(DOCX)Click here for additional data file.Table S2Results of the two factor univariate analyses assessing differences in (a) all roving herbivore biomass, (b) macroalgal browser biomass, (c) algal cover, (d) total algal biomass, (e) coral cover and (f) rugosity between sites and habitats . Pooling procedure was used in accordance to Underwood (DOCX)Click here for additional data file.Table S3Results of the two factor PERMANOVAs assessing multivariate differences between sites and habitats in assemblages of (a) all roving herbivorous fish, (b) browser herbivorous fish and (c) algae.(DOCX)Click here for additional data file.Table S4ANOVA results on (a) biomass change and PERMANOVA results on changes in (b) nitrogen and (c) C/N of Lobophora variegata transplanted specimens in lagoon and reef flat habitats over the three sites.(DOCX)Click here for additional data file.Table S5ANOVA results on removal of Sargassum myriocystum lateral branches at three sites and nine distances from the reef flat/lagoon boundary.(DOCX)Click here for additional data file.


Oncotarget, Morachis et al. describe identification of three distinct drugs that inhibit mRNA synthesis [Transcriptional regulation by RNA polymerase II (RNA Pol II) is a highly regulated process involving the action of multiple transcription factors that collectively regulate synthesis of messenger RNA (mRNA). These complexes sit atop core promoter regions of DNA composed of sequence motifs that mark the starting point of transcription . Studiesynthesis . SurprisThe p53 tumor suppressor protein promotes longevity by reducing somatic mutations or the survival and proliferation of mutant cells . Almost The same group has recently demonstrated that p53 target promoters are structurally diverse and display pronounced differences in Poll II occupancy . Their s


Due to its small size and limited protein coding capacity, HDV is exquisitely reliant upon host cellular proteins to facilitate its transcription and replication. Remarkably, HDV does not encode an RNA-dependent RNA polymerase which is traditionally required to catalyze RNA-templated RNA synthesis. Furthermore, HDV lacks enzymes responsible for post-transcriptional and -translational modification, processes which are integral to the HDV life cycle. This review summarizes the known HDV-interacting proteins and discusses their significance in HDV biology.The hepatitis  The HDV genome is comprised of a small, G/C-rich, single-stranded, circular RNA molecule of ∼ 1,700 nt and adopts a highly base-paired, rod-like secondary structure , which  195 aa; ,12. Post2.All viruses must subvert host cellular machinery for genome expression and virion production. HDV is almost completely reliant upon the host cell for these processes due to its extremely limited protein coding capacity. Several host proteins interact with HDV during the course of infection, including those in the nucleus that facilitate transcription and replication and those in the cytoplasm that are principally associated with production of HDAgs. Host proteins that interact with components of HDV can be divided into two categories – those that interact with HDAg  and thos2.1.Co-immunoprecipitation studies of cells expressing HDAg-S have recently reported that over 100 cellular proteins might interact with HDAg-S alone; over 25 percent of these proteins were shown to affect HDV RNA accumulation . These HCellular proteins involved in post-translational modification have been shown to play a critical role in HDV biology. HDAgs undergo much post-translational modification including serine and threonine phosphorylation –48, argitrans-repression of HDV transcription in the nucleus and facilitates viral packaging with HBV envelope proteins [Two phosphorylated isoforms of HDAg-S and one phosphorylated isoform of HDAg-L are found in infected cells . While Hproteins ,28. HDAgCellular proteins involved in post-transcriptional modification thus play a crucial role in progression of the HDV life cycle through modulation of HDAg function. Despite the propensity for extensive post-translational modification of HDAg, cellular HDAg exists in several states of post-translational modification in infected cells and it is unclear which proportion of HDAg is modified, and the nature and extent of these modifications, during infection.In addition to several sites for post-translational modification, HDAg contains a nuclear localization signal (NLS) composed of ten amino acids located at position 66–75 that is necessary for nuclear entry of HDV RNPs . This seNuclear proteins provide attractive candidates for HDAg-interacting proteins that regulate transcription and replication. Nucleolin, also known as C23, is a multifunctional nuclear phosphoprotein that has been demonstrated to interact with HDAg . NucleolNucleophosmin, also known as B23, is another nuclear phosphoprotein that has been shown to interact with both HDAg-S and HDAg-L . Like nuYin Yang 1 (YY1) is a transcription factor that binds both nucleophosmin and nucleolin and has recently been shown to interact with both HDAg-S and HDAg-L . ThroughHistone H1e is another nuclear protein that was identified as an HDAg-S interacting partner . HistoneThe protein MOV10 was recently shown to interact with HDAg . Interesdelta interacting protein A (DIPA) has also been identified [in vivo [A potential human homologue of HDAg-L known as entified . DIPA sh[in vivo ,81. Over[in vivo . Further[in vivo . FurtherWhile many proteins have been shown to interact with HDAgs, the role of most of these proteins in the HDV life cycle and their relation to HDV-mediated pathogenesis remains unclear. One possible mechanism by which HDV coerces host cell replication and virion assembly is through host cell gene regulation. The following section will discuss HDAg interactions with host cell components that results in alteration in gene expression.2.2.Genome-wide proteomic analyses of Huh7 hepatocellular carcinoma cells transfected with HDV RNA, HDAg-S, or HDAg-L have shown that at least 32 proteins demonstrate differential expression in the presence of HDV components . The affin vitro and in vivo, presumably by an indirect method such as association with or sequestration of host transcription factors [HDAg, particularly HDAg-L, has been shown to regulate gene expression through a number of cellular pathways. RNAP II transcription from both active and basal promoters has been shown to be inhibited by both isoforms of HDAg  factors . However factors  and euka factors  in varioMore recently, HDAg-L has been demonstrated to stimulate transcription from the serum response element (SRE) pathway in a manner that does not change the DNA-binding activity of the serum response factor (SRF) protein, the endogenous transcription factor associated with SRE-responsive gene regulation ,87. It hin vitro and in vivo [To date, there are few documented instances of direct HDAg interaction with cellular transcription factors resulting in altered host gene expression. In one case, HDAg-S and HDAg-L have been shown to interact with the Smad3 and c-Jun transcription factors both  in vivo . Smad3 i in vivo . c-Jun i in vivo . While i in vivo . Both HD in vivo . Althoug in vivo . From th in vivo  and comp in vivo .Clustrin, a secreted protein with chaperone activity that is involved in the regulation of cell death (reviewed by ), has betrans-activation of host genes, providing a temporal regulation of host gene expression dependent on the stage of HDV infection. Further studies will be required to fully elucidate HDAg interacting proteins and determine their involvement in host cell gene regulation.In the absence of direct interaction with host transcription factors, little is known about the additional ways in which HDV infection might regulate host gene expression. It has been shown that the RNA binding domain of HDAg bears considerable homology to canonical DNA-binding domains (DBDs) such as the one found in the eukaryotic SRY gene . This ob2.3.in vitro transcription assays [One of the most important aspects of HDV biology is that HDV must interact with and utilize host DNA-dependent RNA polymerases (RNAPs) for transcription and replication since it does not encode an RNA-dependent RNA polymerase. Since HDAg-S was shown to be necessary for the accumulation of HDV RNA in infected cells , it was n assays ,100,101 in vitro [HDAg bears weak homology to the A polypeptide chain of negative elongation factor (NELF), a transcriptional regulatory factor that interacts directly with RNAP II to repress transcription . NELF anin vitro . HDAgs, in vivo co-immunoprecipitation experiments [i.e., RNA versus DNA) is also affected, providing a mechanism by which RNAP II is capable of utilizing this HDV RNA template.Subsequent studies would indeed demonstrate the direct interaction of HDAg with RNAP II during RNAP II transcription ,39. HDAgeriments . The HDAeriments ,39. It heriments . HDAg-S eriments . Since bin vitro [HDV RNA has also been shown to interact directly with RNAP II and its components. Ribonucleoprotein immunoprecipitation assays have shown interaction between both genomic and antigenomic HDV RNA with RNAP II in cells replicating HDV ,64. Intein vitro . This RNin vitro . The strin vitro , has alsin vitro . Togethein vitro synthesis of the antigenome is largely diminished upon immunodepletion with α-SL1 antibody, implicating RNAP I in antigenome synthesis [The observation that replication of HDV, under some circumstances, is resistant to high levels of α-amanitin has prompted investigation into the involvement of RNAP I in HDV replication ,104. Metynthesis . Howeverynthesis . Interesynthesis . These sBoth genomic and antigenomic HDV RNA were recently shown to associate with RNAP I in cells replicating HDV . Interes2.4.in vivo since RNA accumulation is impaired by deletion of these motifs [delta ribozyme [HDAg itself is an RNA binding protein; HDAg interacts with HDV RNA and is proposed to shuttle it to the nucleus for replication, to the cytosol for packaging and export, and to modulate its transcription and replication . HDAge motifs . Furthere motifs . This RBribozyme . The binribozyme . Multimeribozyme . The cenribozyme , possiblThe unbranched, rod-like secondary structure of HDV RNA is necessary for HDAg binding ,115. Thidelta ribozyme. It was reported that glyceraldehyde 3-phosphate dehydrogenase (GAPDH) interacts with the antigenome of HDV to enhance delta ribozyme activity [delta ribozyme activity almost two-fold in agreement with a previous study, which showed that GAPDH increases the rate of cis-cleavage of hammerhead ribozymes [Another critical step in the replication of HDV is the autocatalytic cleavage of its RNA genome and antigenome by the activity . Interacactivity . Althougactivity . GAPDH pactivity . The GAPibozymes ,120. Thein vitro [As aforementioned, PKR is a protein that interacts with and phosphorylates HDAg . It has in vitro ,121. Binin vitro . It is pin vitro ,61. Furtnrb, were identified as HDV RNA-interacting partners both in vitro and in cells replicating HDV [nrb shares much homology with the C-terminal portion of PSF [nrb have been shown to interact with the CTD of RNAP II during both transcription initiation and elongation, and PSF can likely interact with RNA and the CTD of RNAP II simultaneously [nrb, might provide a direct physical link between HDV RNA and RNAP II, promoting RNAP II-mediated transcription from a non-canonical RNA template. Further characterization of the role of PSF in the HDV life cycle might thus provide novel insight into the mechanism of HDV replication by a host DNA-dependent RNA polymerase. Furthermore, exploration of the role of PSF in HDV biology could provide clues to the mechanism of HDV pathogenesis since mutations in the PSF gene have been identified in both cervical cancer and papillary renal carcinoma cell lineages [Recently, five additional proteins, eEF1A1, ASF/SF2, hnRNP-L, PSF, and p54ting HDV ,67. Due ting HDV  and has ting HDV –126. It ting HDV . ASF/SF2ting HDV ). Furtheting HDV . The polting HDV ). p54nrbn of PSF  and has n of PSF . Both PSaneously . Interesaneously ,67, inclaneously ,100. It aneously . These olineages ,134 and lineages –138. Due3.The rapidly increasing number of host proteins that interact with HDAgs and HDV RNA is testament to the complexity of the host-pathogen interaction upon HDV infection. Such interactions might enable HDV propagation, exert pathogenic effects on the host, or be completely benign; it is thus imperative to determine which host cellular proteins interact with HDV following infection and to establish their roles in the HDV life cycle and pathogenesis. Through identification of HDV-interacting factors and determination of their roles in HDV biology it will become possible to understand the molecular mechanisms by which HDV causes disease and to develop novel strategies to combat HDV infection. More generally, characterization of the manner in which HDV RNA is capable of replication and transcription by using host proteins, specifically DNA-dependent RNA polymerases, will provide global insight into polymerase function and potentially reveal novel pathways of RNA synthesis, metabolism, and regulation.


Spontaneous neoplasm metastasis, a fatalist pathological feature of cancer, is a long-evolving, multi-steps process that can now only be treated or controlled by drugs or immuno-modulators. As we have previously hypothesized, each drug or immuno-modulator might act differently within various stages of a metastasis. Therefore any researches helping to determine these differences will be beneficial for updating therapeutics for metastasis. In this work, we have testified this hypothesis by using a series of well-known anti-metastatic agents – Bisdioxopiperazine compounds. There have been two most difficult problems in cancer biology and therapeutics, neoplasm metastasis and multi-drug-resistances (MDR). Among these two thorny problems, treatments of neoplasm metastasis are especially difficulty and should be placed on higher agenda of the highest for its fatalist pathogenesis features and unpredictability of therapeutic outcome at the stage of drug initiation. Also, metastasized tumors often concomitantly manifest the characters of MDR. Therapeutic reports of metastasis treatment nowadays are overwhelmed with researches and applications of antivascular (angiogenesis) and MMPs inhibitors and more than 300 agents of different chemical formulae have been mentioned in the literatures –3. Curretumor detachment from primary location;tumor cells flow in the blood or lymphatic vessels;tumor cell attachment and penetration through blood vessels of distant organs and angiogenesis .Tumor metastases involve a fixed course of pathophysiological processes, and are responsible for more than 60% of cancer deaths worldwide –7. SpontWe have previously hypothesized that many anti-cancer or anti-metastatic drugs might act differently in the different courses of these substages . In ordeRaz), ICRF-186 and ICRF-187 (two stereo-isomers of Raz) and ICRF-193, developed in the UK, have been a series of serendipitous agents found to be effective against a model of spontaneous metastasis  [Bisdioxopiperazine compounds (Biz), including ICRF-154, Razoxane ICRF-159, , Raz, ICma, 3LL) . Ever sima, 3LL)  and Bimoma, 3LL) –16 were The researches of anti-metastatic drugs, though at the odds nowadays facing, will be the hottest spot and discipline in the future. This work is among this very phase of great transitions of understanding and knowledge about metastasis treatments from drug discovery only into clinical drug options in combinations with.Drugs and Reagents: Pro, Bim and Raz were prepared by Prof Yun-Feng Ren at the Department of Medicinal Chemistry, Shanghai Institute of Materia Medica, Chinese Academy of Science and suspended in 5 g/L carboxymethylcelluose (CMC) solution before use.Animals and Tumor Models: C57BL/6j strain mice were purchased from Shanghai Center of Laboratory Animal Breeding, Chinese Academy of Sciences and were conducted experimentations in compliance with the Guidelines for the Care and Use of Research Animals, NIH, established by Washington University’s Animal Studies Committee.50) to basically offset a possible lethal or mutational toxicity of conventional therapies involved in such experimental study. Biological inoculation with pulmonary nodules of 3LL was a method used by Yue et al [6 cells) from donor mice. The experiment was undergone in two different sets of schedules for day2 and day8 initiation of therapy. Either from day2 or day8 after tumor inoculation, the mice were injected ip with drugs daily in 10 injections. On day 18, all mice were sacrificed, and locally grown tumors were separated from skin and muscles and weighed. The 10 lungs of 5 host mice of each group were sterilely homogenized with sterilized normal saline (2 ml). These homogenized lungs were separately injected sc into 10 healthy C57BL/6j mice. The numbers of mice with newly locally grown tumors were counted after 1 month later.Neoplasm Metastases Determination: Since anti-metastatic therapeutic would be rather preventive than indiscrimination cytotoxic against normal and neoplasm tissues, we used a low dosage of Biz compounds  in the following therapeutic experiment.We used the doses of 1/10 and 1/5 LDThe Effects of Biz on Pulmonary Metastasis of 3LL: 14C-probimane tracing and autoradiography [++-Mg++-ATPase [Since tumor metastasis is responsible for more than 60% cancer death worldwide, metastasis therapy, as a result, remains to be a biggest challenge to all cancer therapeutics. Biz compounds are well-known for their anti-metastasis action. However, their modes of action and therapeutic profile remain to be investigated for their potential clinical availability. The research work for data has been mostly focusing on anti-angiogenesis action –20 and oiography , 25, an iography , 14. The+-ATPase .This experimental evidence further supports our hypothesis that each drug or immuno-modulators might act differently within various stages of a metastatic course. In general, we propose that the MMPs inhibitors might be more active in preventing tumor cells from detaching from primary locations. Immuno-modulators might promote the activity of macrophages in killing of tumor cells in vascular and lymphatic circulation . However


Hibiscus cannabilis L.) leaves, respectively. The p23 and HCRSV coat protein (CP) coding regions were both amplified from total RNA extracted from isolated nuclei. Viral RNA in the nucleus may be used to generate viral microRNAs (vir-miRNAs), as five putative vir-miRNAs were predicted from HCRSV using the vir-miRNAs prediction database. The vir-miRNA (hcrsv-miR-H1-5p) was detected using TaqMan® stem-loop real-time PCR, and by northern blot using DIG-end labeled probe in HCRSV-infected kenaf leaves. Finally, a novel nuclear localization signal (NLS) was discovered in p23 of HCRSV. The NLS interacts with importin α and facilitates viral RNA genome to enter nucleus. We demonstrate the presence of a (+)-sense single-stranded viral RNA within nucleus.Viruses contain either DNA or RNA as genomes. DNA viruses replicate within nucleus, while most RNA viruses, especially (+)-sense single-stranded RNA, replicate and are present within cytoplasm. We proposed a new thought that is contrary to the common notion that (+)-sense single-stranded RNA viruses are present only in the cytoplasm. In this study, we question whether the genome of a plant RNA virus  is present in the nucleus of infected cells? Hibiscus chlorotic ringspot virus (HCRSV) RNA was detected in the nucleus of infected cells, as shown by fluorescent in situ hybridization. Western blot using anti-histone 3 and anti-phosphoenolpyruvate carboxylase showed that nuclei were highly purified from mock and HCRSV-infected kenaf ( A virus is a small infectious agent that can replicate inside the living cells of organisms. Viruses infect all types of organisms, from animals and plants to bacteria and archaea http://psort.hgc.jp/), the p23 was shown to be able to enter nucleus. It also contains a DNA binding motif. A nuclear localization signal (NLS) is a short stretch of amino acids (aa) that mediates the import of a protein into nucleus Hibiscus chlorotic ringspot virus (HCRSV) belongs to the genus Carmovirus MicroRNAs (miRNAs) are small RNAs (∼22 nucleotides) that have regulatory function on gene expression Hibiscus cannabilis L.) plants, it is plausible that the p23 coding region is able to generate both the p23 protein and vir-miRNAs. A total of five vir-miRNAs have been predicted from the HCRSV genome . In this regard, we investigated if HCRSV RNA is found in the nucleus, where vir-miRNAs are generated.Previously it has been shown that a single RNA transcript containing both the protein-coding region and the miRNA coding sequence can be translated to produce a protein and to generate miRNAs V genome  using ViStrong Cy3 signals (red dots) were detected in the cytoplasm and nucleus of HCRSV-infected cells hybridized with Cy3-labeled probe (corresponding to the HCRSV p23 coding region at nt 350-301). The specific Cy3 red dots present in the nucleus were highlighted with white arrows . The results showed that as early as 4 hpt, HCRSV RNA was detected in the nucleus of the transfected protoplasts. Time points at 0, 4, 48 and 72 hpt were selected to show the final merged images. The intensity of the red dots observed represent an increase in the amount of HCRSV RNA at 48 and 72 hpt, indicating that replication of HCRSV RNA increased gradually  can facilitate RNA to enter nucleus Arabidopsis was generated. Putative p23 transgenic line 1 (L1) and line 5 (L5) were determined by PCR using vector forward primer  and gene reverse primer   , as wellCCTGAAA)  using DICCTGAAA)  to detecEven if the p23 can be detected in the nucleus, it is not essential for it to contain a NLS since it can bind to other carrier protein to enter nucleus. Hence, the p23 was divided into different fragments, according to its predicted cleavage sites, corresponding constructs were shown . Firstly2R→AX2A, RX5K→AX5A, HX8K→AX8A and KX2RX5H→AX2AX5A). Six representatives were shown  and localization of fusion protein was investigated. From the mutant clones, three basic aa mutations  located in positions (20–41 aa) in the p23 of HCRSV. Any one of the basic aa mutation abolished nuclear localization of p23-GFP fusion protein. A total of seven mutants were generated by replacing each basic aa with an alanine residue . Eluted protein was probed with anti-importin α antibody. Compared with the wild type plant, the importin α was only detected in the protein extract from p23-GFP transgenic Arabidopsis plants  experiment was carried out. The results showed that importin α was detected in both sonicated mock and HCRSV-infected kenaf leaves, suggesting that importin α was present in the cross-linked protein-RNA complex , five candidates , namely In order to prove that there is vir-miRNAs existed in the HCRSV, two approaches were carried out. Firstly, mature miRNAs were quantified using TaqMan® MicroRNA Assays, in which the stem-loop RT primers are more specific than the conventional ones. The results showed that vir-miRNAs were detected in total RNA extracted from HCRSV-infected kenaf leaves. In order to exclude the involvement of siRNAs, total RNAs isolated from highly purified kenaf nuclei were also used for the vir-miRNA detection, using TaqMan real-time PCR. A plant conserved miRNA mi395a (as a positive control) showed high expression level . Since tAgrobacterium-infiltrated p23-GFP gene was performed. Highly purified nuclei were extracted from corresponding genes  infiltrated into kenaf leaves after 72 h. Then total RNAs were extracted from these purified nuclei. RT-PCR results showed that the sequence encoding p23 was only detected from p23-GFP Agrobacterium-infiltrated kenaf leaves, but not from empty vector (GFP) or negative control (p27-GFP). Similarly, vir-miRNA hcrsv-miR-H1-5p was also detected only in total RNA from the nuclei of p23-GFP agro-infiltrated kenaf leaves, using TaqMan real time PCR and northern blot.In order to further prove that the small RNA signals detected were miRNAs, but not siRNAs that were generated from HCRSV infection. Experiments using In this study, we have identified the presence of a viral RNA genome in nucleus. Using FISH, we demonstrated the presence of the Cy3-labelled probe targeting viral RNA in the nucleus of single cells isolated from paraformaldehyde-fixed HCRSV-infected kenaf leaves and protoplasts. Western blot analysis verified that the isolated nuclei were highly purified. Using total RNA extracted from the purified nuclei, both of the p23 coding region  and the CP gene  were amplified using RT-PCR. Finally, using the customized stem loop primers, vir-miRNA was detected using Taqman® real-time PCR. In addition, small RNAs were detected using both 5′ and 3′ miRNA probes, suggesting that both of the mature miRNA strands may have higher expression level in the cells LMP1 gene HCRSV RNA entering nucleus may be used to synthesize vir-miRNAs. Since the first vir-miRNA was discovered, its functions have been studied. It represses both host cellular genes and viral genes to aid viral replication and to function as orthologs of cellular miRNAs. While vir-miRNAs have been discoverred progressively from DNA viruses, there is none reported in RNA viruses. The current knowledge shows that RNA virus replication is associated with the cytoplasmic membranes in the host cells. As the dicer-like processing complex is located in the nucleus and viral RNA is not known to enter nucleus where vir-miRNA is generated, it is not apparent how RNA viruses can be processed to yield mature miRNAs in the cytoplasm Using constructed vir-miRNA hcrsv-miR-H1-5p (amip23) for co-infiltration experiments, we tested if hcrsv-miR-H1-5p can target the p23 to regulate its expression. Our results showed that amip23 can target and downregulate p23 gene in co-infiltrated pGreen-p23-GFP/amip23, as compared with p23-GFP infiltrated alone, or pGreen-p23-GFP/amip27 . As expeKKKRRVP, is from the SV40 large T antigen KRPAATKKAGQAKKKK, is from the nucleoplasmin LSPQLLKLSRTPVSLHEILASL) which is leucine rich and highly hydrophobic. The NLS of p23 overlaps with the predicted DNA binding domain. It agrees with a previous report in which the authors found that there is an overlap in the NLS and DNA binding region of 90% of the proteins for which both the NLS motif and DNA-binding regions were found Our results showed that the p23 of HCRSV contains a novel NLS, which is different from the classical NLS (cNLS) for nuclear protein import. The best characterized transport cNLS consists of either one or two clusters of basic aa. One type of the cNLSs, as represented by Arabidopsis as co-IP materials is due to low expression level of p23 in vivo. The p23 is predicted to be a transcription factor. In addition, since the p23 is highly hydrophobic, it is difficult to express it. Attempts to produce a monoclonal antibody to p23 was unsuccessful. Using the p23 with a GFP tag (p23-GFP) which was driven by a strong Cauliflower mosic virus (CaMV) 35S promoter, it is possible to obtain sufficient quantity of p23 protein for immunoprecipitation. Furthermore, RNA-CHIP experiments also showed that importin α can bind to HCRSV RNA, forming a p23-importin α-HCRSV RNA complex, and facilitates HCRSV RNA to enter nucleus.For a protein with an NLS entering nucleus, the molecular basis for recognition of a cNLS by importin α has been defined using x-ray crystallography Viral genetic material enterring nucleus can affect nucleus, for example, the target of rapamycin (TOR) protein, which is a conserved regulator of ribosome biogenesis, can modulate nucleolar structure Our results showed that similar to DNA viruses, viral RNA genome can also enter nucleus. This will open up many studies to unravel more functions of RNA viruses. For example, do all RNA viruses enter nucleus of host cells? Does viral RNA interact with host components in nucleus to regulate gene expression? Better understanding on how viral replication is regulated will allow researchers to control viral diseases by designing specific molecules to target viral pathogens in future.In conclusion, we have identified the presence of a (+)-sense ss viral RNA in nucleus, where vir-miRNAs are generated. In addition, a novel NLS in p23 facilitates viral RNA to enter nucleus through its binding to importin α.CCGGAATTCATGCTTTCTCAATTGCTTTC and p23-R: CGCGGATCCCGGGCGAGTACCCCTGAAA primers. A construct 35SpGreen-p23-GFP was transferred into Agrobacterium. Cloning of p23 with predicted cleavage fragments were constructed as p23 (1–49 aa), p23 (50–209 aa), p23 (1–19 aa), p23 (20–49 aa) and p23 (20–41 aa) were made to identify the NLS. Within the p23 (20–41 aa) fragment, mutations at three basic aa  were constructed using routine high-fidelity PCR (KAPA) by point mutations. In addition, three leucine residues (as negative controls) were mutated to confirm the NLS in the p23. Arabidopsis seedlings were grown in long-day conditions . The p23-GFP gene was transformed into wild type Arabidopsis using floral dip method following published protocols Kenaf seedlings were grown under 16 h light 8 h dark cycles at 25°C. The HCRSV p23 coding region was PCR amplified using p23-F: Southern blot was performed strictly following a protocol . Briefly, Ten µg genomic DNA was digested with EcoRI and separated on an agarose gel overnight. After denaturing with buffer , the DNA was transferred to nylon membrane using capillary transfer method. The transferred DNA was cross linked by UV and hybridized with DIG-labeled p23 DNA probe at 42°C, 16 h. Finally, the membrane was probed with anti-DIG antibody before it was developed with CPSD substrate.Vir-miRNA hcrsv-miR-H1-5p was engineered into the endogenous miR319a precursor (plasmid pRS300) by site-directed mutagenesis, following the protocol by Rebecca Scheab Max-Plank Institute for Developmental Biology, Tuebingen, Germany (2005) . The artificial miRNAs amip23 and amip27  and p27 (2622-2614 nt), respectively) were generated.Agrobacteria culture grown to OD600 nm = 1.0–1.5 was harvested, containing the pGreen-p23-GFP, truncated p23 fragments or artificial vir-miRNA plasmids. The cell pellet was resuspended in buffer (pH = 7) containing 10 mM each of MgCl2 and MES, and 100 µM acetosyringone, infiltration was performed after 4 h. Negative controls include original plasmid (35S pGreen+GFP) and 35S pGreen+p27+GFP. All the infiltration transient expression experiments were repeated twice. For co-infiltration experiments, agroinfiltration mixtures of pGreen-GFP/amip23, pGreen-p23-GFP/amip23, pGreen-CP-GFP/amip23, pGreen-GFP/amip27, pGreen-p23-GFP/amip27 were prepared.In vivo co-IP was carried out in accordance with the manufacturer's protocol . Briefly, 7 days later, wild type and p23-GFP transgenic Arabidopsis seedlings (0.1 g) were homogenized on ice in 0.2 ml of protein extraction buffer . The extract was centrifuged at 12,000× g for 10 min to remove cellular debris, and the supernatant  was mixed with equal volumes of coupling buffer and incubated for 2 h with amine-reactive gel previously coupled to 750 µg rabbit anti-GFP antibody. The amine-reactive gel was washed three times with coupling buffer and the immunoprecipitated proteins were subsequently eluted from the gel with elution buffer. The immunoprecipitates were probed with anti-importin α antibody.CHIP experiment was performed following previous protocol 2O before digested with filter-sterile cellulase (0.8%) and macerase (0.25%) for 16 h. The cells were isolated accordingly in vitro transcribed RNA from full-length HCRSV cDNA . After 0, 4, 48 and 72 h transfection, protoplasts were collected and fixed with fixation buffer described above. The FISH procedures 5′-GTTGGAGTGCCCCCAAAATGTAGCTTTGCTGCGTTGCCGCATGGAGAGC-3′) for each reaction was used to localize HCRSV RNA. Images were obtained using a confocal laser scanning microscope .Mock and HCRSV-infected kenaf leaves were cut into 1 mm×1 mm and fixed with 4% (w/v) paraformaldehyde, 2.5% (w/v) sucrose in 0.1 M phosphate buffer (pH 7.2), 2 h at room temperature. The cells were washed twice with sterile Hhttp://www.le.ac.uk/bl/phh4/agnor.htm) described by Heslop-Harrison and Schwarzacher of the Molecular Cytogenetics Research Group at University of Leicaster, U.K.Lower epidermal layer of kenaf leaves was peeled off using a pair of forceps and stained with DAPI or silver according to the protocol ((Instructions of the CelLytic PN Plant Nuclei Isolation/Extraction Kit (Sigma-Aldrich) were strictly followed. Blotted proteins were probed with anti-phosphoenolpyruvate carboxylase (PEPC) and anti-histone 3 (H3), followed by alkaline phosphatase-conjugated protein A or anti-rabbit IgG peroxidase conjugate. Finally, membranes were visualized using nitroblue tetrazolium/5-bromo-4-chloroindol-3-yl phosphate staining or Thermo Scientific SuperSignal West Pico Chemiluminescent substrate. RT-PCR primers designed to amplify the p23 and the CP coding regions of HCRSV were carried out, using total RNA extracted from the highly purified kenaf nuclei.Total RNAs were extracted using TRIzol reagent (Invitrogen). Reverse transcription was carried out using reverse transcriptase . HCRSV CP gene was amplified with H-CP-F and H-CP-R .http://alk.ibms.sinica.edu.tw) Candidate sequences for vir-miRNA in HCRSV were predicted using the Vir-Mir database (https://products.appliedbiosystems.com). Since the probe sequence is derived from proprietary bioinformatics analysis by the company, an assay ID CSKAJSI (Applied Biosystems), instead of the actual sequence, was assigned to a predicted HCRSV vir-miRNA for future ordering. The hcrsv-miR-H1-5p was detected subsequently using TaqMan real-time PCR Standard TaqMan assays for vir-miRNA were obtained from the custom TaqMan small RNA assay design tool (Two DIG-end-labeled probes (26 nt each) labeled with DIG oligonucleotide tailing kit (Roche Applied Science) were used to detect miRNA. The probes used were:5′-GTGCCCCCAAAATGTAGCTTTGCTGC-3′) andHcmi305pro-5p (5′-GCCGCACAGCAAGCTCATTCGAGCGC-3′).Hcmi305pro-3p  were superimposed onto the differential interference contrast (DIC) image. No Cy3 signal (red color) was detected in the nuclei of the mock transfected kenaf protoplast.(MPG)Click here for additional data file.Movie S2Three dimensional rotating image of a HCRSV-transfected protoplast 72 hpi using FISH method. DAPI-stained nuclei (blue-color foci) were superimposed onto the differential interference contrast (DIC) image. Single RNA molecules (red dots within the blue-color foci) were detected in nuclei of the protoplast transfected with in vitro transcript of full-length cDNA clone of HCRSV.(MPG)Click here for additional data file.Movie S3Three dimensional animation images of a mock-transfected protoplast 72 hpi using FISH method. DAPI-stained nuclei (blue-color foci) were superimposed onto the differential interference contrast (DIC) image. No Cy3 signal (red color) was detected in the nuclei of the mock transfected kenaf protoplast.(MPG)Click here for additional data file.Movie S4Three dimensional animation images of a HCRSV-transfected protoplast 72 hpi using FISH method. DAPI-stained nuclei (blue-color foci) were superimposed onto the differential interference contrast (DIC) image. Single RNA molecules (red dots within the blue-color foci) were detected in nuclei of the protoplast transfected with in vitro transcript of full-length cDNA clone of HCRSV.(MPG)Click here for additional data file.Table S1Primers used in this study.(DOC)Click here for additional data file.