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.

Public Health Relevance

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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
4R37AI043288-17
Application #
8307600
Study Section
Special Emphasis Panel (NSS)
Program Officer
Park, Eun-Chung
Project Start
1997-09-30
Project End
2018-02-28
Budget Start
2013-03-01
Budget End
2014-02-28
Support Year
17
Fiscal Year
2013
Total Cost
$310,200
Indirect Cost
$122,200
Name
Donald Danforth Plant Science Center
Department
Type
DUNS #
044193006
City
St. Louis
State
MO
Country
United States
Zip Code
63132
Carbonell, Alberto; Carrington, James C; Daròs, José-Antonio (2016) Fast-forward generation of effective artificial small RNAs for enhanced antiviral defense in plants. RNA Dis 3:
Fahlgren, Noah; Hill, Steven T; Carrington, James C et al. (2016) P-SAMS: a web site for plant artificial microRNA and synthetic trans-acting small interfering RNA design. Bioinformatics 32:157-8
Wang, Haifeng; Beyene, Getu; Zhai, Jixian et al. (2015) CG gene body DNA methylation changes and evolution of duplicated genes in cassava. Proc Natl Acad Sci U S A 112:13729-34
Fahlgren, Noah; Feldman, Maximilian; Gehan, Malia A et al. (2015) A Versatile Phenotyping System and Analytics Platform Reveals Diverse Temporal Responses to Water Availability in Setaria. Mol Plant 8:1520-35
Garcia-Ruiz, Hernan; Carbonell, Alberto; Hoyer, J Steen et al. (2015) Roles and programming of Arabidopsis ARGONAUTE proteins during Turnip mosaic virus infection. PLoS Pathog 11:e1004755
Carbonell, Alberto; Carrington, James C (2015) Antiviral roles of plant ARGONAUTES. Curr Opin Plant Biol 27:111-7
Carbonell, Alberto; Fahlgren, Noah; Mitchell, Skyler et al. (2015) Highly specific gene silencing in a monocot species by artificial microRNAs derived from chimeric miRNA precursors. Plant J 82:1061-75
Gilbert, Kerrigan B; Fahlgren, Noah; Kasschau, Kristin D et al. (2014) Preparation of Multiplexed Small RNA Libraries From Plants. Bio Protoc 4:
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
Zhang, Xiaoming; Niu, DongDong; Carbonell, Alberto et al. (2014) ARGONAUTE PIWI domain and microRNA duplex structure regulate small RNA sorting in Arabidopsis. Nat Commun 5:5468

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