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      RNA-Seq reveals virus–virus and virus–plant interactions in nature

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          Abstract

          As research on plant viruses has focused mainly on crop diseases, little is known about these viruses in natural environments. To understand the ecology of viruses in natural systems, comprehensive information on virus–virus and virus–host interactions is required. We applied RNA-Seq to plants from a natural population of Arabidopsis halleri subsp. gemmifera to simultaneously determine the presence/absence of all sequence-reported viruses, identify novel viruses and quantify the host transcriptome. By introducing the criteria of read number and genome coverage, we detected infections by Turnip mosaic virus (TuMV), Cucumber mosaic virus and Brassica yellows virus. Active TuMV replication was observed by ultramicroscopy. De novo assembly further identified a novel partitivirus, Arabidopsis halleri partitivirus 1. Interestingly, virus reads reached a maximum level that was equivalent to that of the host's total mRNA, although asymptomatic infection was common. AhgAGO2, a key gene in host defence systems, was upregulated in TuMV-infected plants. Multiple infection was frequent in TuMV-infected leaves, suggesting that TuMV facilitates multiple infection, probably by suppressing host RNA silencing. Revealing hidden plant–virus interactions in nature can enhance our understanding of biological interactions and may have agricultural applications.

          Abstract

          RNA-Seq, coupled with de novo assembly, enables the comprehensive and solid determination of virus infection, and revealed the virus–virus and virus–host interactions in naturally growing plants.

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          Most cited references54

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          Controlling the False Discovery Rate: A Practical and Powerful Approach to Multiple Testing

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            Double indexing overcomes inaccuracies in multiplex sequencing on the Illumina platform

            Due to the increasing throughput of current DNA sequencing instruments, sample multiplexing is necessary for making economical use of available sequencing capacities. A widely used multiplexing strategy for the Illumina Genome Analyzer utilizes sample-specific indexes, which are embedded in one of the library adapters. However, this and similar multiplex approaches come with a risk of sample misidentification. By introducing indexes into both library adapters (double indexing), we have developed a method that reveals the rate of sample misidentification within current multiplex sequencing experiments. With ~0.3% these rates are orders of magnitude higher than expected and may severely confound applications in cancer genomics and other fields requiring accurate detection of rare variants. We identified the occurrence of mixed clusters on the flow as the predominant source of error. The accuracy of sample identification is further impaired if indexed oligonucleotides are cross-contaminated or if indexed libraries are amplified in bulk. Double-indexing eliminates these problems and increases both the scope and accuracy of multiplex sequencing on the Illumina platform.
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              Dual RNA-seq unveils noncoding RNA functions in host-pathogen interactions.

              Bacteria express many small RNAs for which the regulatory roles in pathogenesis have remained poorly understood due to a paucity of robust phenotypes in standard virulence assays. Here we use a generic 'dual RNA-seq' approach to profile RNA expression simultaneously in pathogen and host during Salmonella enterica serovar Typhimurium infection and reveal the molecular impact of bacterial riboregulators. We identify a PhoP-activated small RNA, PinT, which upon bacterial internalization temporally controls the expression of both invasion-associated effectors and virulence genes required for intracellular survival. This riboregulatory activity causes pervasive changes in coding and noncoding transcripts of the host. Interspecies correlation analysis links PinT to host cell JAK-STAT signalling, and we identify infection-specific alterations in multiple long noncoding RNAs. Our study provides a paradigm for a sensitive RNA-based analysis of intracellular bacterial pathogens and their hosts without physical separation, as well as a new discovery route for hidden functions of pathogen genes.
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                Author and article information

                Journal
                FEMS Microbiol Ecol
                FEMS Microbiol. Ecol
                femsec
                FEMS Microbiology Ecology
                Oxford University Press
                0168-6496
                1574-6941
                21 August 2016
                November 2016
                21 August 2016
                : 92
                : 11
                : fiw176
                Affiliations
                [1 ]Center for Ecological Research, Kyoto University, Hirano 2-509-3, Otsu, Shiga 520-2113, Japan
                [2 ]Faculty of Agriculture, Ryukoku University, Yokotani 1-5, Seta Oe-cho, Otsu, Shiga 520-2914, Japan
                [3 ]JST PRESTO, Japan Science and Technology Agency, Honcho, Kawaguchi, Saitama 332-0012, Japan
                Author notes
                [* ] Corresponding author: Center for Ecological Research, Kyoto University, Hirano 2-509-3, Otsu 520-2113, Japan. Tel: +81-77-549-8200, Fax: +81-77-549-8201, E-mail: kamitani@ 123456ecology.kyoto-u.ac.jp
                Article
                fiw176
                10.1093/femsec/fiw176
                5854034
                27549115
                2a138a1c-9fdb-4066-8fe3-f93480a911e5
                © FEMS 2016.

                This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

                History
                : 16 August 2016
                : 10 April 2016
                Page count
                Pages: 11
                Funding
                Funded by: Aid for Scientific Research
                Award ID: 26221106
                Funded by: Aid for Japan Society for the Promotion of Science Fellows
                Award ID: 15J00628
                Categories
                Research Article

                Microbiology & Virology
                argonaute2,arabidopsis halleri,arabidopsis halleri partitivirus 1,multiple infection,rna-seq,turnip mosaic virus

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