Unknown,Transcriptomics,Genomics,Proteomics

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High-throughput pyrosequencing of plant virus small RNAs

ABSTRACT: Plant virus infection involves the production of viral small RNAs (vsRNAs) with the potential to associate with distinct Argonaute (AGO)-containing silencing complexes and mediate diverse silencing effects on RNA and chromatin. We used multiplexed, high-throughput pyrosequencing to profile populations of vsRNAs from plants infected with viruses from different genera. Sense and antisense vsRNAs of 20 to 24 nucleotides (nts) spread throughout the entire viral genomes in an overlapping configuration; virtually all genomic nucleotide positions were represented in the dataset. We present evidence to suggest that every genomic position could be a putative cleavage site for vsRNA formation, although viral genomes contain specific regions that serve as preferential sources of vsRNA production. Hotspots for vsRNAs of 21-, 22-, and 24-nt usually coincide in the same genomic regions, indicating similar target affinities among Dicer-like (DCL) enzymes. In the light of our results, the overall contribution of perfectly base paired double-stranded RNA and imperfectly base paired structures within single-stranded RNA to vsRNA formation is discussed. Our census of vsRNAs extends the current view of the distribution and composition of vsRNAs in virus-infected plants, and contributes to define a more comprehensive scenario of vsRNA biogenesis and their regulatory functions in plants Raw data files are available on our FTP site: ftp://ftp.ncbi.nlm.nih.gov/pub/geosup/Series/GSE16996 10 samples examined: Arabidopsis plants infected with TRV, TuMV or CMV; Nicothiana benthamiana plants infected with CymRSV, PVX or PMMoV; Cucumis melo plants infected with MNSV, quimeric MNSV or WMV and Solanum lycopersicum plants infected with TYLCV.

ORGANISM(S): Melon necrotic spot virus

SUBMITTER: Yu Wang

PROVIDER: E-GEOD-16996 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Deep-sequencing of plant viral small RNAs reveals effective and widespread targeting of viral genomes.

Donaire Livia L Wang Yu Y Gonzalez-Ibeas Daniel D Mayer Klaus F KF Aranda Miguel A MA Llave César C

Virology 20090807 2

Plant virus infection involves the production of viral small RNAs (vsRNAs) with the potential to associate with distinct Argonaute (AGO)-containing silencing complexes and mediate diverse silencing effects on RNA and chromatin. We used multiplexed, high-throughput pyrosequencing to profile populations of vsRNAs from plants infected with viruses from different genera. Sense and antisense vsRNAs of 20 to 24 nucleotides (nts) spread throughout the entire viral genomes in an overlapping configuratio ...[more]

PMID: 19665162

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Project description:Melon (Cucumis melo L.) is a commercially important fruit crop that is cultivated worldwide. The melon research community has recently benefited from the determination of a complete draft genome sequence and the development of associated genomic tools, which have allowed us to focus on small RNAs (sRNAs). These are short, non-coding RNAs 21â24 nucleotides in length with diverse physiological roles. In plants, they regulate gene expression and heterochromatin assembly, and control protection against virus infection. Much remains to be learned about the role of sRNAs in melon. We constructed 10 sRNA libraries from two stages of developing ovaries, fruits and photosynthetic cotyledons infected with viruses, and carried out high-throughput pyrosequencing. We catalogued and analyzed the melon sRNAs, resulting in the identification of 26 known miRNA families (many conserved with other species), the prediction of 84 melon-specific miRNA candidates, the identification of trans-acting siRNAs, and the identification of chloroplast, mitochondrion and transposon-derived sRNAs. In silico analysis revealed more than 400 potential targets for the conserved and novel miRNAs. This analysis provides insight into the composition and function of the melon small RNAome, and paves the way towards an understanding of sRNA-mediated processes that regulate melon fruit development and melonâvirus interactions. 11 small RNA libraries from several tissues of melon are included en the raw data. 2 samples from ovary, 2 samples from fruit, 1 sample from healthy cotyledons (Cultivar Tendral), 1 samples from healthy cotyledons (genotype TGR-1551), 1 sample from cotyledons (cultivar Tendral) infected with Watermelon mosaic virus (WMV), 1 sample from cotyledons (cultivar TGR-1551) infected with WMV, 1 sample from cotyledons (cultivar Tendral) infected with Melon necrotic spot virus (MNSV, Malfa5 isolate), 1 sample from cotyledons (cultivar Tendral) infected with MNSV (chimeric virus with Malfa5-264 isolates), 1 library from synthetic RNA oligos. Raw reads were obtained from two independent 454 runs, ~22,000 reads each one, to a total of 447,180 reads

Project description:In response to infection, viral genomes are processed by Dicer-like (DCL) ribonuclease proteins into viral small RNAs (vsRNAs) of discrete sizes. vsRNAs are then used as guides for silencing the viral genome. The profile of vsRNAs produced during the infection process has been extensively studied for some group of viruses. However, nothing is known for members of the economically important family Luteoviridae, a group of phloem-restricted viruses. Here, the population of vsRNAs from cotton plants infected with Cotton leafroll dwarf virus (CLRDV), a member of the genus Polerovirus, family Luteoviridae, is characterized.Deep sequencing of small RNAs (sRNAs) from CLRDV-infected cotton leaves was performed. Results showed 21-nt to 24-nt long vsRNAs matching all the viral genome, with a higher frequency of matches in the 3M-CM-"M-BM-^@M-BM-^Y region. Equivalent amounts of sense and antisense vsRNAs were found, and the 22-nt long small RNA class was the most prominent one. Looking for cotton Dcl transcripts levels during infection, we could observe that Dcl4 seems to be up-regulated, while Dcl2 seems to be down-regulated.This is the first report on the profile of sRNAs coming from a plant infected with a member of the family Luteoviridae. Our sequence data strongly suggest that virus-derived double-stranded RNA functions as one of the main precursors of vsRNAsOur results indicate that secondary structures of the viral RNAs are not the main source of the viRNAs observed, as suggested for other viruses. Judging by the profiled size classes, all cotton DCLs might be working to silence the virus. The possible causes for the unexpected high accumulation of 22-nt viRNAvsRNAs are discussed. CLRDV is the causal agent of worldwide cotton pathology named Cotton blue disease. Our results are an important contribution for understanding the molecular mechanisms involved in this and related diseases. Total RNA obtained from leaves of the cotton plant 5 days post-infection with Cotton leafrol dawrf virus (CLRDV) compared to not-infected control.

Dataset Information

High-throughput pyrosequencing of plant virus small RNAs

Publications

Deep-sequencing of plant viral small RNAs reveals effective and widespread targeting of viral genomes.

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