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Small RNA deep sequencing reveals role for Arabidopsis thaliana RNA-dependent RNA polymerases in viral siRNA biogenesis.

Qi X, Bao FS, Xie Z - PLoS ONE (2009)

Bottom Line: Significantly, both the rdr1 and rdr6 mutants exhibited globally reduced levels of viral small RNA production as well as reduced strand bias in viral small RNA population, revealing an important role for these host RDRs in viral siRNA biogenesis.Two of such predicted host targets, which encode a cleavage and polyadenylation specificity factor (CPSF30) and an unknown protein similar to translocon-associated protein alpha (TRAP alpha), respectively, yielded a positive result in cleavage validation by 5'RACE assays.Our data raised the interesting possibility for viral siRNA-mediated virus-host interactions that may contribute to viral pathogenicity and host specificity.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Texas Tech University, Lubbock, Texas, United States of America.

ABSTRACT
RNA silencing functions as an important antiviral defense mechanism in a broad range of eukaryotes. In plants, biogenesis of several classes of endogenous small interfering RNAs (siRNAs) requires RNA-dependent RNA Polymerase (RDR) activities. Members of the RDR family proteins, including RDR1and RDR6, have also been implicated in antiviral defense, although a direct role for RDRs in viral siRNA biogenesis has yet to be demonstrated. Using a crucifer-infecting strain of Tobacco Mosaic Virus (TMV-Cg) and Arabidopsis thaliana as a model system, we analyzed the viral small RNA profile in wild-type plants as well as rdr mutants by applying small RNA deep sequencing technology. Over 100,000 TMV-Cg-specific small RNA reads, mostly of 21- (78.4%) and 22-nucleotide (12.9%) in size and originating predominately (79.9%) from the genomic sense RNA strand, were captured at an early infection stage, yielding the first high-resolution small RNA map for a plant virus. The TMV-Cg genome harbored multiple, highly reproducible small RNA-generating hot spots that corresponded to regions with no apparent local hairpin-forming capacity. Significantly, both the rdr1 and rdr6 mutants exhibited globally reduced levels of viral small RNA production as well as reduced strand bias in viral small RNA population, revealing an important role for these host RDRs in viral siRNA biogenesis. In addition, an informatics analysis showed that a large set of host genes could be potentially targeted by TMV-Cg-derived siRNAs for posttranscriptional silencing. Two of such predicted host targets, which encode a cleavage and polyadenylation specificity factor (CPSF30) and an unknown protein similar to translocon-associated protein alpha (TRAP alpha), respectively, yielded a positive result in cleavage validation by 5'RACE assays. Our data raised the interesting possibility for viral siRNA-mediated virus-host interactions that may contribute to viral pathogenicity and host specificity.

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TMV-Cg-derived small RNAs captured from virus-infected wild type Arabidopsis and rdr mutants by deep sequencing.(A) Detection of TMV-Cg genomic RNA from infected (3 dpi) leaves by Northern blot assay using a CP-specific probe. The relative abundance (in parentheses) of viral RNA was normalized to signal from the actin probe. (B) A flowchart showing stepwise computational extraction of TMV-Cg-specific small RNA reads from a multiplex small RNA library sequencing sample. (C) Virus- and host-specific small RNA reads recovered from each of the four source libraries.
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pone-0004971-g001: TMV-Cg-derived small RNAs captured from virus-infected wild type Arabidopsis and rdr mutants by deep sequencing.(A) Detection of TMV-Cg genomic RNA from infected (3 dpi) leaves by Northern blot assay using a CP-specific probe. The relative abundance (in parentheses) of viral RNA was normalized to signal from the actin probe. (B) A flowchart showing stepwise computational extraction of TMV-Cg-specific small RNA reads from a multiplex small RNA library sequencing sample. (C) Virus- and host-specific small RNA reads recovered from each of the four source libraries.

Mentions: Profiling of virus-derived small RNAs can help decipher the mechanism for their biogenesis. Recently developed next-generation DNA sequencing technologies offer a cost-effective approach for small RNA profiling [21], [22]. A small RNA deep sequencing approach was adopted that allows analysis of multiplexed small RNA libraries in parallel, to characterize the TMV-Cg-derived small RNA populations in wild type Arabidopsis and rdr mutant plants. TMV-Cg infects Arabidopsis systemically and causes mild disease symptoms [23], [24]. TMV-Cg was selected because it induces the expression of RDR1 in Arabidopsis [24] and provides an opportunity to assess the role of RDR1 and RDR6 in viral small RNA biogenesis. Systemically infected (3 dpi) tissues were used as the source materials for small RNA library construction. By Northern blot using a capsid protein (CP) gene-specific probe, TMV-Cg full-length genomic RNA (gRNA) was readily detectable at 3 dpi in the local inoculated leaves of wild type Arabidopsis (Fig. 1A, lane 2). Slightly elevated levels of TMV-Cg gRNA were detected in rdr1 and rdr6 mutants, respectively (Fig. 1A, lanes 3 and 4). These data confirmed successful and uniform infection of TMV-Cg in wild type Arabidopsis and rdr mutant plants.


Small RNA deep sequencing reveals role for Arabidopsis thaliana RNA-dependent RNA polymerases in viral siRNA biogenesis.

Qi X, Bao FS, Xie Z - PLoS ONE (2009)

TMV-Cg-derived small RNAs captured from virus-infected wild type Arabidopsis and rdr mutants by deep sequencing.(A) Detection of TMV-Cg genomic RNA from infected (3 dpi) leaves by Northern blot assay using a CP-specific probe. The relative abundance (in parentheses) of viral RNA was normalized to signal from the actin probe. (B) A flowchart showing stepwise computational extraction of TMV-Cg-specific small RNA reads from a multiplex small RNA library sequencing sample. (C) Virus- and host-specific small RNA reads recovered from each of the four source libraries.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2654919&req=5

pone-0004971-g001: TMV-Cg-derived small RNAs captured from virus-infected wild type Arabidopsis and rdr mutants by deep sequencing.(A) Detection of TMV-Cg genomic RNA from infected (3 dpi) leaves by Northern blot assay using a CP-specific probe. The relative abundance (in parentheses) of viral RNA was normalized to signal from the actin probe. (B) A flowchart showing stepwise computational extraction of TMV-Cg-specific small RNA reads from a multiplex small RNA library sequencing sample. (C) Virus- and host-specific small RNA reads recovered from each of the four source libraries.
Mentions: Profiling of virus-derived small RNAs can help decipher the mechanism for their biogenesis. Recently developed next-generation DNA sequencing technologies offer a cost-effective approach for small RNA profiling [21], [22]. A small RNA deep sequencing approach was adopted that allows analysis of multiplexed small RNA libraries in parallel, to characterize the TMV-Cg-derived small RNA populations in wild type Arabidopsis and rdr mutant plants. TMV-Cg infects Arabidopsis systemically and causes mild disease symptoms [23], [24]. TMV-Cg was selected because it induces the expression of RDR1 in Arabidopsis [24] and provides an opportunity to assess the role of RDR1 and RDR6 in viral small RNA biogenesis. Systemically infected (3 dpi) tissues were used as the source materials for small RNA library construction. By Northern blot using a capsid protein (CP) gene-specific probe, TMV-Cg full-length genomic RNA (gRNA) was readily detectable at 3 dpi in the local inoculated leaves of wild type Arabidopsis (Fig. 1A, lane 2). Slightly elevated levels of TMV-Cg gRNA were detected in rdr1 and rdr6 mutants, respectively (Fig. 1A, lanes 3 and 4). These data confirmed successful and uniform infection of TMV-Cg in wild type Arabidopsis and rdr mutant plants.

Bottom Line: Significantly, both the rdr1 and rdr6 mutants exhibited globally reduced levels of viral small RNA production as well as reduced strand bias in viral small RNA population, revealing an important role for these host RDRs in viral siRNA biogenesis.Two of such predicted host targets, which encode a cleavage and polyadenylation specificity factor (CPSF30) and an unknown protein similar to translocon-associated protein alpha (TRAP alpha), respectively, yielded a positive result in cleavage validation by 5'RACE assays.Our data raised the interesting possibility for viral siRNA-mediated virus-host interactions that may contribute to viral pathogenicity and host specificity.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Texas Tech University, Lubbock, Texas, United States of America.

ABSTRACT
RNA silencing functions as an important antiviral defense mechanism in a broad range of eukaryotes. In plants, biogenesis of several classes of endogenous small interfering RNAs (siRNAs) requires RNA-dependent RNA Polymerase (RDR) activities. Members of the RDR family proteins, including RDR1and RDR6, have also been implicated in antiviral defense, although a direct role for RDRs in viral siRNA biogenesis has yet to be demonstrated. Using a crucifer-infecting strain of Tobacco Mosaic Virus (TMV-Cg) and Arabidopsis thaliana as a model system, we analyzed the viral small RNA profile in wild-type plants as well as rdr mutants by applying small RNA deep sequencing technology. Over 100,000 TMV-Cg-specific small RNA reads, mostly of 21- (78.4%) and 22-nucleotide (12.9%) in size and originating predominately (79.9%) from the genomic sense RNA strand, were captured at an early infection stage, yielding the first high-resolution small RNA map for a plant virus. The TMV-Cg genome harbored multiple, highly reproducible small RNA-generating hot spots that corresponded to regions with no apparent local hairpin-forming capacity. Significantly, both the rdr1 and rdr6 mutants exhibited globally reduced levels of viral small RNA production as well as reduced strand bias in viral small RNA population, revealing an important role for these host RDRs in viral siRNA biogenesis. In addition, an informatics analysis showed that a large set of host genes could be potentially targeted by TMV-Cg-derived siRNAs for posttranscriptional silencing. Two of such predicted host targets, which encode a cleavage and polyadenylation specificity factor (CPSF30) and an unknown protein similar to translocon-associated protein alpha (TRAP alpha), respectively, yielded a positive result in cleavage validation by 5'RACE assays. Our data raised the interesting possibility for viral siRNA-mediated virus-host interactions that may contribute to viral pathogenicity and host specificity.

Show MeSH
Related in: MedlinePlus