Progress 08/01/06 to 01/31/09
Outputs
OUTPUTS: Using a crucifer-infecting strain of Tobacco Mosaic Virus (TMV-Cg) and Arabidopsis thaliana as a model system, we analyzed the viral small RNA profiles in wild-type plants as well as in the rdr1 and rdr6 mutants by small RNA deep sequencing. Small RNA libraries were constructed using RNAs prepared from the virus infected wild-type and rdr mutants as well as from mock-infected wild-type control plants. Deep sequencing of the small RNA libraries using the Solexa/Illumina's platform yielded sufficient abundance of viral siRNA sequences that allow meaningful comparison between viral small RNA profiles captured from the wild type plants and those from the rdr mutants. A high-resolution genomic map for TMV-Cg-derived small RNAs was generated. We analyze the genomic hot spots for viral siRNA generation as well as strand bias of the viral siRNA population at different genetic backgrounds. The effects of loss-of-function mutations in RDR1 and RDR6 on biogenesis of TMV-Cg-derived siRNAs were assessed. Finally, we examined the sequence homology between viral siRNA and the host gene transcripts. Based on the hypothesis that some of the viral siRNAs may target host transcripts for posttranscriptional regulation, we computationally predicted the host genes that could be potentially targeted by the TMV-Cg-derived siRNAs. A selected small subset of these predicted targets were subjected to experimental validation. The PD presented our findings at the "Small RNA Workshop" on the 17th International Plant and Animal Genome Conference, which was held in January 10-14, 2009 at San Diego, CA. In addition, the complete dataset of TMV-Cg-derived small RNAs resulted from this work has been submitted to the NCBI's Gene Expression Omnibus (GEO) for public access. The computational tools that were developed in this work for analyzing viral siRNAs have also been made freely accessible to the community (see publications below) PARTICIPANTS: This project supported a full-time postdoctoral research associate to gain training in the areas of virus-host interaction and small RNA biology. It also stimulated interdisciplinary collaboration between biologists and computer scientists--it provided excellent research opportunity for a part-time computer science majored graduate research assistant to develop computational tools for analyzing large sequence data set. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
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 α), respectively, yielded a positive result in cleavage validation by 5'RACE assays. Our work provided evidence for direct involvement of RDR1 and RDR6 in viral siRNA biogenesis and revealed potentially widespread functions for viral siRNAs in regulating host gene expression. These results raised the interesting possibility for viral siRNA-mediated virus-host interactions that may contribute to viral pathogenicity and host specificity.
Publications
- Qi X, Bao FS, and Xie Z. (2009) Small RNA deep sequencing reveals role for Arabidopsis thaliana RNA-dependent RNA polymerases in viral siRNA biogenesis. PLoS ONE 4: e4971
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Progress 08/01/07 to 07/31/08
Outputs
OUTPUTS: Using a crucifer-infecting strain of Tobacco Mosaic Virus (TMV-Cg) and Arabidopsis thaliana as a model virus-host system, we analyzed the viral small RNA profiles in wild type Arabidopsis as well as rdr mutant plants by applying small RNA deep sequencing technology. Large sets TMV-Cg-specific small RNA reads, mostly of 21- and 22-nucleotide in size and originating predominately from the genomic sense RNA strand, were captured at an early infection stage, generating the first near-saturated, high-resolution small RNA map for the genome of a plant virus. The TMV-Cg genome was found to harbore 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. Additionally, an informatics analysis showed that a large set of host genes could be potentially targeted by TMV-Cg-derived siRNAs for posttranscriptional silencing, raising the interesting possibility for a novel layer of potentially widespread virus-host interactions that may have contributed to viral pathogenicity and host specificity. PARTICIPANTS: Xiaopeng Qi, Postdoctoral Research Associate; Forrest S. Bao, Graduate student majored in Computer Science. This project provided funding support for a full-time postdoctoral research associate to gain training in the areas of virus-host interaction and small RNA biology. It also stimulated interdisciplinary collaboration between biologists and computer scientists--it provided excellent research opportunity for a part-time computer science majored graduate research assistant to develop computational tools for analyzing large sequencing datasets. TARGET AUDIENCES: This project provided learning experience to a high school student through the Clark Scholar Program at Texas Tech University (Summer 2007) PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Our work revealed several important features of viral small RNA population that provide mechanistic insights on their biogenesis and potential biological functions. Given the highly conserved nature of the RNA silencing machinery, we anticipate that our findings should be highly applicable to a wide range of virus-host systems involving many economically important plant species.
Publications
- Xie Z., and Qi X. (2008) Diverse Small RNA-directed Silencing Pathways in Plants. Biochim Biophys Acta doi:10.1016/j.bbagrm.2008.02.009 [Epub ahead of print on March 4, 2008]
- Qi X., Bao F. S., and Xie Z.(2008). Small RNA Deep Sequencing Reveals Role for Arabidopsis thaliana RNA-dependent RNA Polymerases in Viral siRNA Biogenesis. (Submitted for review)
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Progress 08/01/06 to 07/31/07
Outputs
Progress has been made in the following three areas: (1) To genetically examine the role of Arabidopsis RNA-dependent RNA polymerases (RDR) in the biogenesis of virus-derived siRNAs, we have constructed rdr double and triple mutant lines using the previously characterized rdr1-1, rdr2-1, and rdr6-15 alleles. Homozygous mutant plants were infected with Tobacco mosaic virus (TMV-Cg) and early systemic tissues were collected for small RNA analysis. Small RNA libraries have been made using total RNAs prepared from systemically infected tissues and are currently being sequenced using the Solexa deep sequencing technology. Computational tools have been developed for analyzing the sequencing data, which including sequence sorting and mapping along the plant and viral genomes. (2) Green fluorescent protein (GFP)- and epitope-tagged RDR1 constructs have been made for characterizing the cellular expression pattern of RDR1. Transgenic Arabidopsis plants expressing the tagged RDR1
in both wild-type and rdr1 background are currently being characterized. (3) Out of the six identifiable RDR genes in the rice genome, we have cloned the cDNAs for three of the putative rice RDRs. The function of these RDR homologs will be examined using a genetic complementation approach in Arabidopsis.
Impacts
This work will advance our current understanding on the contribution of specific host core RNA silencing factors in antiviral defense. Given the highly conserved nature of the RNA silencing machinery, results from this work should be highly applicable to a wide range of plant species including economically important crop plants.
Publications
- No publications reported this period
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