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Post-transcriptional Gene Silencing (post-transcriptional + gene_silencing)
Selected AbstractsLoss of sense transgene-induced post-transcriptional gene silencing by sequential introduction of the same transgene sequences in tobaccoFEBS JOURNAL, Issue 7 2010Sayaka Hirai RNA silencing is an epigenetic inhibition of gene expression and is guided by small interfering RNAs. Sense transgene-induced post-transcriptional gene silencing (S-PTGS) occurs in a portion of a transgenic plant population. When a sense transgene encoding a tobacco endoplasmic reticulum ,-3 fatty acid desaturase (NtFAD3) was introduced into tobacco plants, an S-PTGS line, S44, was obtained. Introduction of another copy of the NtFAD3 transgene into S44 plants caused a phenotypic change from S-PTGS to overexpression. Because this change was associated with the methylation of the promoter sequences of the transgene, reduced transcriptional activity may abolish S-PTGS and residual transcription of the sense transgene may account for the overexpression. To clarify whether RNA-directed DNA methylation (RdDM) can repress the transcriptional activity of the S44 transgene locus, we introduced several RdDM constructs targeting the transgene promoter. An RdDM construct harboring a 200-bp-long fragment of promoter sequences efficiently abrogated the generation of NtFAD3 small interfering RNAs in S44 plants. Transcription of the transgene was partially repressed, but the resulting NtFAD3 mRNAs successfully accumulated and an overexpressed phenotype was established. Our results indicate an example in which overexpression of the transgene is established by complex epigenetic interactions among the transgenic loci. [source] Highly efficient targeting and accumulation of a Fab fragment within the secretory pathway and apoplast of Arabidopsis thalianaFEBS JOURNAL, Issue 15 2001Koen Peeters To further improve antibody production in plants, constructs were designed to minimize transgene silencing and to retain a Fab fragment within the secretory pathway of transgenic Arabidopsis thaliana plants. The levels of antibody accumulation suggest that placing the sequences that encode Fd and light chain under the control of nonidentical 3, regions reduces susceptibility to post-transcriptional gene silencing compared with when the individual polypeptide-encoding sequences are placed under the control of identical 3, regions. High levels of accumulation (up to 6% of total soluble protein) were found for both secreted and intracellularly targeted antibody fragments. Immunofluorescence microscopic analysis showed that Fab fragments devoid of any additional C-terminal sequence were efficiently secreted, whereas retention of Fab fragments within the endomembrane system of the secretory pathway was achieved by C-terminal fusion of the DIKDEL sequence to the antibody light chain. Furthermore, analysis by immunoprecipitation and ELISA showed that intracellular retention of antibody fragments did not affect antigen-binding activity, and more than 80% of the isolated antibody fragments were found to bind antigen. Taken together, our results provide improvements to the technology of recombinant antibody production in transgenic plants. [source] Structural and biochemical advances in mammalian RNAiJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 5 2006Robert E. Collins Abstract RNAi is a collection of processes mediated by small RNAs that silence gene expression in a sequence-specific manner. Studies of processes as divergent as post-transcriptional gene silencing, transcriptional silencing through RNA-directed DNA methylation, or heterochromatin formation, and even RNA-guided DNA elimination have converged on a core pathway. This review will highlight recent structural and mechanistic studies illustrating siRNA and miRNA processing, RISC formation, the execution of RNAi by RISC, and the regulation of these pathways, with a specific focus on vertebrate systems. J. Cell. Biochem. 99: 1251,1266, 2006. © 2006 Wiley-Liss, Inc. [source] Resistance to cassava mosaic disease in transgenic cassava expressing antisense RNAs targeting virus replication genesPLANT BIOTECHNOLOGY JOURNAL, Issue 4 2005Peng Zhang Summary African cassava mosaic virus (ACMV) is a major contributor to cassava mosaic disease (CMD), the economically most important and devastating disease of cassava in Africa. We have developed transgenic cassava plants with increased ACMV resistance using improved antisense RNA technology by targeting the viral mRNAs of Rep (AC1), TrAP (AC2) and REn (AC3). Viral DNA replication assays in detached leaves demonstrated that replication of two ACMV isolates was strongly reduced or inhibited in most transgenic lines. After ACMV infection of plants using biolistic inoculation, several lines remained symptomless at lower infection pressure (100 ng viral DNA/plant). Symptom development was reduced and attenuated even at higher DNA doses. Transgenic ACMV-resistant plants had significantly reduced viral DNA accumulation in their infected leaves. Short sense and antisense RNAs specific to AC1 were identified in transgenic lines expressing AC1 antisense RNA, suggesting that the short RNAs mediate interference by post-transcriptional gene silencing. Our results demonstrate that resistance to ACMV infection of cassava can be achieved with high efficacy by expressing antisense RNAs against viral mRNAs encoding essential non-structural proteins, providing a new tool to combat CMD in Africa. [source] The plasma membrane Na+/H+ antiporter SOS1 is essential for salt tolerance in tomato and affects the partitioning of Na+ between plant organsPLANT CELL & ENVIRONMENT, Issue 7 2009RAQUEL OLÍAS ABSTRACT We have identified a plasma membrane Na+/H+ antiporter gene from tomato (Solanum lycopersicum), SlSOS1, and used heterologous expression in yeast to confirm that SlSOS1 was the functional homolog of AtSOS1. Using post-transcriptional gene silencing, we evaluated the role played by SlSOS1 in long-distance Na+ transport and salt tolerance of tomato. Tomato was used because of its anatomical structure, more complex than that of Arabidopsis, and its agricultural significance. Transgenic tomato plants with reduced expression of SlSOS1 exhibited reduced growth rate compared to wild-type (WT) plants in saline conditions. This sensitivity correlated with higher accumulation of Na+ in leaves and roots, but lower contents in stems of silenced plants under salt stress. Differential distribution of Na+ and lower net Na+ flux were observed in the xylem sap in the suppressed plants. In addition, K+ concentration was lower in roots of silenced plants than in WT. Our results demonstrate that SlSOS1 antiporter is not only essential in maintaining ion homeostasis under salinity, but also critical for the partitioning of Na+ between plant organs. The ability of tomato plants to retain Na+ in the stems, thus preventing Na+ from reaching the photosynthetic tissues, is largely dependent on the function of SlSOS1. [source] SERRATE is a novel nuclear regulator in primary microRNA processing in ArabidopsisTHE PLANT JOURNAL, Issue 6 2006Li Yang Summary The Arabidopsis gene SERRATE (SE) controls leaf development, meristem activity, inflorescence architecture and developmental phase transition. It has been suggested that SE, which encodes a C2H2 zinc finger protein, may change gene expression via chromatin modification. Recently, SE has also been shown to regulate specific microRNAs (miRNAs), miR165/166, and thus control shoot meristem function and leaf polarity. However, it remains unclear whether and how SE modulates specific miRNA processing. Here we show that the se mutant exhibits some similar developmental abnormalities as the hyponastic leaves1 (hyl1) mutant. Since HYL1 is a nuclear double-stranded RNA-binding protein acting in the DICER-LIKE1 (DCL1) complex to regulate the first step of primary miRNA transcript (pri-miRNA) processing, we hypothesized that SE could play a previously unrecognized and general role in miRNA processing. Genetic analysis supports that SE and HYL1 act in the same pathway to regulate plant development. Consistently, SE is critical for the accumulation of multiple miRNAs and the trans -acting small interfering RNA (ta-siRNA), but is not required for sense post-transcriptional gene silencing. We further demonstrate that SE is localized in the nucleus and interacts physically with HYL1. Finally, we provide evidence that SE and HYL1 probably act with DCL1 in processing pri-miRNAs before HEN1 in miRNA biogenesis. In plants and animals, miRNAs are known to be processed in a stepwise manner from pri-miRNA. Our data strongly suggest that SE plays an important and general role in pri-miRNA processing, and it would be interesting to determine whether animal SE homologues may play similar roles in vivo. [source] Manipulation of DET1 expression in tomato results in photomorphogenic phenotypes caused by post-transcriptional gene silencingTHE PLANT JOURNAL, Issue 3 2004Ganga Rao Davuluri Summary The tomato HIGH PIGMENT-2 gene encodes an orthologue of the Arabidopsis nuclear protein DE-ETIOLATED 1 (DET1). From genetic analyses it has been proposed that DET1 is a negative regulator of light signal transduction, and recent results indicate that it may control light-regulated gene expression at the level of chromatin remodelling. To gain further understanding about the function of DET1 during plant development, we generated a range of overexpression constructs and introduced them into tomato. Unexpectedly, we only observed phenotypes characteristic of DET1 inactivation, i.e. hyper-responsiveness to light. Molecular analysis indicated in all cases that these phenotypes were a result of suppression of endogenous DET1 expression, due to post-transcriptional gene silencing. DET1 silencing was often lethal when it occurred at relatively early stages of plant development, whereas light hyper-responsive phenotypes were obtained when silencing occurred later on. The appearance of phenotypes correlated with the generation of siRNAs but not DNA hypermethylation, and was most efficient when using constructs with mutations in the DET1 coding sequence or with constructs containing only the 3,-terminal portion of the gene. These results indicate an important function for DET1 throughout plant development and demonstrate that silencing of DET1 in fruits results in increased carotenoids, which may have biotechnological potential. [source] Viroid-induced RNA silencing of GFP-viroid fusion transgenes does not induce extensive spreading of methylation or transitive silencingTHE PLANT JOURNAL, Issue 1 2004Ulrike Vogt Summary Viroid infection is associated with the production of short interfering RNAs (siRNAs), a hallmark of post-transcriptional gene silencing (PTGS). However, viroid RNAs autonomously replicating in the nucleus have not been shown to trigger the degradation of homologous RNA in the cytoplasm. To investigate the potential of viroids for the induction of gene silencing, non-infectious fragments of potato spindle tuber viroid (PSTVd) cDNA were transcriptionally fused to the 3, end of the green fluorescent protein (GFP)-coding region. Introduction of such constructs into tobacco plants resulted in stable transgene expression. Upon PSTVd infection, transgene expression was suppressed and partial de novo methylation of the transgene was observed. PSTVd-specific siRNA was detected but none was found corresponding to the gfp gene. Methylation was restricted almost entirely to the PSTVd-specific part of the transgene. Neither a gfp transgene construct lacking viroid-specific elements was silenced nor was de novo methylation detected, when it was introduced into the genetic background of the PSTVd-infected plant lines containing silenced GFP:PSTVd transgenes. The absence of gfp -specific siRNAs and of significant methylation within the gfp -coding region demonstrated that neither silencing nor DNA methylation spread from the initiator region into adjacent 5, regions. [source] Graft transmission of induced and spontaneous post-transcriptional silencing of chitinase genesTHE PLANT JOURNAL, Issue 5 2001Patrice Crété Summary Sense and antisense tobacco chitinase (CHN) transgenes, Luciferase-CHN transcriptional fusions, and promoterless CHN cDNAs were introduced biolistically into CHN transformants of tobacco that never exhibit spontaneous gene silencing. All of the constructs tested induced systemic silencing of the resident CHN transgene and endogenes. Nuclear run-on transcription assays showed that local introduction of additional gene copies triggers systemic post-transcriptional gene silencing (PTGS). Together, this provides evidence that additional transgene copies need not be either highly transcribed or produce sense transcripts to evoke production of systemic PTGS signals. CHN PTGS was transmitted by top grafting, but not by reciprocal grafting of mature stems or the exchange of tissue plugs. Thus, the commonly encountered difficulties in achieving graft-transmission could reflect the method used. Silencing in sense but not antisense transformants was transmitted by grafting to a high-expressing sense CHN scion suggesting that the elaboration of mobile signals may not be an essential feature of antisense-mediated gene silencing. [source] RNA commutes to work: regulation of plant gene expression by systemically transported RNA moleculesBIOESSAYS, Issue 12 2001Shoko Ueki Although long-distance movement of endogenous mRNAs in plants is well established, the functional contributions of these transported RNA molecules has remained unclear. In a recent report, Kim et al.2001 showed that systemically transported mRNA is capable of causing phenotypic change in developing tissue. Here, this finding and its significance are reviewed and discussed in detail. In addition, in order to give proper perspective, long-distance transport of other types of RNAs, e.g., RNA elicitors of post-transcriptional gene silencing and RNA genomes of plant viruses, and its possible regulation are discussed. BioEssays 23:1087,1090, 2001. © 2001 John Wiley & Sons, Inc. [source] | ||||||||||