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      Genome-wide analysis of tomato long non-coding RNAs and identification as endogenous target mimic for microRNA in response to TYLCV infection

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          Abstract

          Recently, a large number of long noncoding RNAs (lncRNAs) have emerged as important regulators of many biological processes in animals and plants. However, how lncRNAs function during plant DNA virus infection is largely unknown. We performed strand-specific paired-end RNA sequencing of tomato samples infected with Tomato yellow leaf curl virus (TYLCV) with three biological replicates. Overall, we predicted 1565 lncRNAs including long intergenic ncRNAs (lincRNAs) and natural antisense transcripts (lncNATs) and definitively identified lnRNAs that are involved in TYLCV infection by virus-induced gene silencing (VIGS). We also verified the functions of a set of lncRNAs that were differentially expressed between 0 and 7 days post inoculation (dpi). More importantly, we found that several lncRNAs acted as competing endogenous target mimics (eTMs) for tomato microRNAs involved in the TYLCV infection. These results provide new insight into lncRNAs involved in the response to TYLCV infection that are important components of the TYLCV network in tomatoes.

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          Most cited references35

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          Virus-induced gene silencing in tomato.

          We have previously demonstrated that a tobacco rattle virus (TRV)-based vector can be used in virus-induced gene silencing (VIGS) to study gene function in Nicotiana benthamiana. Here we show that recombinant TRV infects tomato plants and induces efficient gene silencing. Using this system, we suppressed the PDS, CTR1 and CTR2 genes in tomato. Suppression of CTR1 led to a constitutive ethylene response phenotype and up-regulation of an ethylene response gene, CHITINASE B. This phenotype is similar to Arabidopsis ctr1 mutant plants. We have constructed a modified TRV vector based on the GATEWAY recombination system, allowing restriction- and ligation-free cloning. Our results show that tomato expressed sequence tags (ESTs) can easily be cloned into this modified vector using a single set of primers. Using this vector, we have silenced RbcS and an endogenous gene homologous to the tomato EST cLED3L14. In the future, this modified vector system will facilitate large-scale functional analysis of tomato ESTs.
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            Protocol: a highly sensitive RT-PCR method for detection and quantification of microRNAs

            MicroRNAs (miRNAs) are a class of small non-coding RNAs with a critical role in development and environmental responses. Efficient and reliable detection of miRNAs is an essential step towards understanding their roles in specific cells and tissues. However, gel-based assays currently used to detect miRNAs are very limited in terms of throughput, sensitivity and specificity. Here we provide protocols for detection and quantification of miRNAs by RT-PCR. We describe an end-point and real-time looped RT-PCR procedure and demonstrate detection of miRNAs from as little as 20 pg of plant tissue total RNA and from total RNA isolated from as little as 0.1 μl of phloem sap. In addition, we have developed an alternative real-time PCR assay that can further improve specificity when detecting low abundant miRNAs. Using this assay, we have demonstrated that miRNAs are differentially expressed in the phloem sap and the surrounding vascular tissue. This method enables fast, sensitive and specific miRNA expression profiling and is suitable for facilitation of high-throughput detection and quantification of miRNA expression.
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              Systematic identification of long noncoding RNAs expressed during zebrafish embryogenesis.

              Long noncoding RNAs (lncRNAs) comprise a diverse class of transcripts that structurally resemble mRNAs but do not encode proteins. Recent genome-wide studies in humans and the mouse have annotated lncRNAs expressed in cell lines and adult tissues, but a systematic analysis of lncRNAs expressed during vertebrate embryogenesis has been elusive. To identify lncRNAs with potential functions in vertebrate embryogenesis, we performed a time-series of RNA-seq experiments at eight stages during early zebrafish development. We reconstructed 56,535 high-confidence transcripts in 28,912 loci, recovering the vast majority of expressed RefSeq transcripts while identifying thousands of novel isoforms and expressed loci. We defined a stringent set of 1133 noncoding multi-exonic transcripts expressed during embryogenesis. These include long intergenic ncRNAs (lincRNAs), intronic overlapping lncRNAs, exonic antisense overlapping lncRNAs, and precursors for small RNAs (sRNAs). Zebrafish lncRNAs share many of the characteristics of their mammalian counterparts: relatively short length, low exon number, low expression, and conservation levels comparable to that of introns. Subsets of lncRNAs carry chromatin signatures characteristic of genes with developmental functions. The temporal expression profile of lncRNAs revealed two novel properties: lncRNAs are expressed in narrower time windows than are protein-coding genes and are specifically enriched in early-stage embryos. In addition, several lncRNAs show tissue-specific expression and distinct subcellular localization patterns. Integrative computational analyses associated individual lncRNAs with specific pathways and functions, ranging from cell cycle regulation to morphogenesis. Our study provides the first systematic identification of lncRNAs in a vertebrate embryo and forms the foundation for future genetic, genomic, and evolutionary studies.
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                Author and article information

                Journal
                Sci Rep
                Sci Rep
                Scientific Reports
                Nature Publishing Group
                2045-2322
                18 December 2015
                2015
                : 5
                : 16946
                Affiliations
                [1 ]Jiangsu Key Laboratory for Bioresources of Saline Soils, Provincial Key Laboratory of Agrobiology, Jiangsu Academy of Agricultural Sciences , Nanjing 210014, China
                [2 ]Shanghai Center for Plant Stress Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences , Shanghai 201602, China
                [3 ]College of Horticulture and Plant Protection, Yangzhou University , Yangzhou 225009, China
                Author notes
                Article
                srep16946
                10.1038/srep16946
                4683531
                26679690
                ec0a6c38-eb5f-4521-a160-beb885d74072
                Copyright © 2015, Macmillan Publishers Limited

                This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

                History
                : 14 June 2015
                : 22 October 2015
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