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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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Characterization of TMV-Cg-derived small RNA populations.(A) Size distribution of viral small RNA populations. Proportions of viral siRNAs derived from the genomic sense (B) and antisense (C) strands in each size class are also shown as the percentage of the total viral small RNA reads. Data from different source libraries are represented in black (Col-0; TMV-Cg 3dpi), white (rdr1-1; TMV-Cg 3dpi), and grey (rdr6-15; TMV-Cg 3dpi) histograms, respectively.
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pone-0004971-g003: Characterization of TMV-Cg-derived small RNA populations.(A) Size distribution of viral small RNA populations. Proportions of viral siRNAs derived from the genomic sense (B) and antisense (C) strands in each size class are also shown as the percentage of the total viral small RNA reads. Data from different source libraries are represented in black (Col-0; TMV-Cg 3dpi), white (rdr1-1; TMV-Cg 3dpi), and grey (rdr6-15; TMV-Cg 3dpi) histograms, respectively.

Mentions: The abundance of TMV-Cg-derived small RNAs recovered by deep sequencing allowed detailed characterization of the viral small populations captured from different genetic backgrounds. We found that the TMV-Cg-derived small RNAs from infected wild type Arabidopsis was predominated by the 21- (78.4%) and 22-nt species (12.9%), with the 21-nt species being, by far, the most abundant (Fig. 3A), indicating that the Arabidopsis DCL4 and DCL2 were the major dicing activities involved in biogenesis of TMV-Cg-derived siRNAs. This observation is consistent with previous reports from small RNA blot-based studies on several other RNA viruses, including TCV, a modified TRV, and CMV [12], [19]. When compared with the infected wild type library, it is obvious that the diminished viral small RNA reads in both rdr1 and rdr6 libraries resulted from a partial loss of both the 21- and 22-nt species in the mutant plants, suggesting that both RDR1 and RDR6 function in viral small RNA biogenesis through the DCL4- and DCL2-dependent pathways (Fig. 3A). When the polarity of TMV-Cg-derived small RNAs was examined with respect to the viral genome, nearly 80% of the TMV-Cg-derived small RNA reads in the infected wild type library were found to be “sense”, indicating a strong strand bias of the viral small RNA population. More specifically, the 21- and 22-nt sense viral small RNA reads accounted for 62.0% and 10.3% of the total viral small RNA reads in the library, respectively (Fig. 3B and 3C). The strand-biased feature of the viral small RNA population is consistent with previous observations made in low-throughput sequencing-based studies on CymRSV-infected Nicotiana benthamiana and TuMV-infected Brassica juncea [15], [16]. Interestingly, the infected rdr libraries not only had a reduction in TMV-Cg-derived small RNA reads, but also exhibited a substantially reduced strand bias in the viral small RNA population, with the portion of “antisense” small RNA reads increased to 35.1% and 34.0% in rdr1 and rdr6 libraries, respectively. These changes were most prominent in the 21- and 22-nt viral small RNA species (Fig. 3B and C). The ratio of sense/antisense 21-nt viral siRNA reads dropped from 3.77 in the wild type library to 1.52 and 1.78 in rdr1 and rdr6 libraries, respectively. Similarly, the ratio of sense/antisense 22-nt viral siRNA reads dropped from 4.02 in the wild type library to 1.71 and 2.06 in rdr1 and rdr6 libraries, respectively.


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)

Characterization of TMV-Cg-derived small RNA populations.(A) Size distribution of viral small RNA populations. Proportions of viral siRNAs derived from the genomic sense (B) and antisense (C) strands in each size class are also shown as the percentage of the total viral small RNA reads. Data from different source libraries are represented in black (Col-0; TMV-Cg 3dpi), white (rdr1-1; TMV-Cg 3dpi), and grey (rdr6-15; TMV-Cg 3dpi) histograms, respectively.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2654919&req=5

pone-0004971-g003: Characterization of TMV-Cg-derived small RNA populations.(A) Size distribution of viral small RNA populations. Proportions of viral siRNAs derived from the genomic sense (B) and antisense (C) strands in each size class are also shown as the percentage of the total viral small RNA reads. Data from different source libraries are represented in black (Col-0; TMV-Cg 3dpi), white (rdr1-1; TMV-Cg 3dpi), and grey (rdr6-15; TMV-Cg 3dpi) histograms, respectively.
Mentions: The abundance of TMV-Cg-derived small RNAs recovered by deep sequencing allowed detailed characterization of the viral small populations captured from different genetic backgrounds. We found that the TMV-Cg-derived small RNAs from infected wild type Arabidopsis was predominated by the 21- (78.4%) and 22-nt species (12.9%), with the 21-nt species being, by far, the most abundant (Fig. 3A), indicating that the Arabidopsis DCL4 and DCL2 were the major dicing activities involved in biogenesis of TMV-Cg-derived siRNAs. This observation is consistent with previous reports from small RNA blot-based studies on several other RNA viruses, including TCV, a modified TRV, and CMV [12], [19]. When compared with the infected wild type library, it is obvious that the diminished viral small RNA reads in both rdr1 and rdr6 libraries resulted from a partial loss of both the 21- and 22-nt species in the mutant plants, suggesting that both RDR1 and RDR6 function in viral small RNA biogenesis through the DCL4- and DCL2-dependent pathways (Fig. 3A). When the polarity of TMV-Cg-derived small RNAs was examined with respect to the viral genome, nearly 80% of the TMV-Cg-derived small RNA reads in the infected wild type library were found to be “sense”, indicating a strong strand bias of the viral small RNA population. More specifically, the 21- and 22-nt sense viral small RNA reads accounted for 62.0% and 10.3% of the total viral small RNA reads in the library, respectively (Fig. 3B and 3C). The strand-biased feature of the viral small RNA population is consistent with previous observations made in low-throughput sequencing-based studies on CymRSV-infected Nicotiana benthamiana and TuMV-infected Brassica juncea [15], [16]. Interestingly, the infected rdr libraries not only had a reduction in TMV-Cg-derived small RNA reads, but also exhibited a substantially reduced strand bias in the viral small RNA population, with the portion of “antisense” small RNA reads increased to 35.1% and 34.0% in rdr1 and rdr6 libraries, respectively. These changes were most prominent in the 21- and 22-nt viral small RNA species (Fig. 3B and C). The ratio of sense/antisense 21-nt viral siRNA reads dropped from 3.77 in the wild type library to 1.52 and 1.78 in rdr1 and rdr6 libraries, respectively. Similarly, the ratio of sense/antisense 22-nt viral siRNA reads dropped from 4.02 in the wild type library to 1.71 and 2.06 in rdr1 and rdr6 libraries, respectively.

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