The proposed renewal project focuses on mechanistic aspects of antiviral RNA silencing using the Arabidopsis model. The genetic, genomic and technical aspects of this model have proven exceptionally useful in revealing RNA silencing functions that limit virus infection at the cell-autonomous and cellnonautonomous levels, as well as silencing functions that govern stress responses, development, and repressive chromatin. In the current project period, Arabidopsis was used to identify the roles of cellular and viral factors, such as virus-encoded suppressors of RNA silencing, during antiviral defense and counterdefensive processes. This, and work from several other groups, led to conceptualization of a threephase model for antiviral silencing in Arabidopsis. This model describes events occurring during the initial targeting phase, the siRNA amplification phase and the systemic silencing phase. This proposed research seeks to test key elements of each phase of the model. The three Aims are summarized as follows: ? Aim 1 - Understand silencing events during the initial targeting phase. New high-throughput sequencing technology will be used to analyze the genetic requirements for initial targeting of viral genomes by DCL factors during early stages of infection, and test the hypothesis that initial targeting yields primarily (+)-sense siRNA that seed the subsequent amplification step. ? Aim 2 - Understand silencing events during the amplification phase. The genetic requirements and detailed accumulation patterns of siRNA formed during the RDR6-dependent amplification phase will be determined, and the small RNA populations that interact with a silencing suppressor during antiviral silencing will be identified. ? Aim 3 - Understand silencing events during the systemic phase. The hypothesis that viral silencing suppressors function in vascular cells to inhibit cell-nonautonomous, DCL4-dependent systemic signals that limit virus invasiveness will be tested.
Recent discoveries have revealed the central importance of small RNA-mediated processes during virushost interactions. Small RNA from viruses and from host cells govern susceptibility and defense responses, and can affect virulence, latency and immunity. This project uses a genetically tractable model organism to understand principles governing small RNA-directed silencing of viruses as a defense response.
|Minoia, Sofia; Carbonell, Alberto; Di Serio, Francesco et al. (2014) Specific argonautes selectively bind small RNAs derived from potato spindle tuber viroid and attenuate viroid accumulation in vivo. J Virol 88:11933-45|
|Carbonell, Alberto; Takeda, Atsushi; Fahlgren, Noah et al. (2014) New generation of artificial MicroRNA and synthetic trans-acting small interfering RNA vectors for efficient gene silencing in Arabidopsis. Plant Physiol 165:15-29|
|Cumbie, Jason S; Kimbrel, Jeffrey A; Di, Yanming et al. (2011) GENE-counter: a computational pipeline for the analysis of RNA-Seq data for gene expression differences. PLoS One 6:e25279|
|Fischer, Sylvia E J; Montgomery, Taiowa A; Zhang, Chi et al. (2011) The ERI-6/7 helicase acts at the first stage of an siRNA amplification pathway that targets recent gene duplications. PLoS Genet 7:e1002369|
|Garcia-Ruiz, Hernan; Takeda, Atsushi; Chapman, Elisabeth J et al. (2010) Arabidopsis RNA-dependent RNA polymerases and dicer-like proteins in antiviral defense and small interfering RNA biogenesis during Turnip Mosaic Virus infection. Plant Cell 22:481-96|
|Dunoyer, Patrice; Schott, Gregory; Himber, Christophe et al. (2010) Small RNA duplexes function as mobile silencing signals between plant cells. Science 328:912-6|
|Wei, Taiyun; Zhang, Changwei; Hong, Jian et al. (2010) Formation of complexes at plasmodesmata for potyvirus intercellular movement is mediated by the viral protein P3N-PIPO. PLoS Pathog 6:e1000962|
|Cosson, Patrick; Sofer, Luc; Le, Quang Hien et al. (2010) RTM3, which controls long-distance movement of potyviruses, is a member of a new plant gene family encoding a meprin and TRAF homology domain-containing protein. Plant Physiol 154:222-32|