Final Report Summary - MEVISP (Genetic, biochemical and cell biological mechanisms of virus silencing in plants) RNA silencing is an ancient mechanism used by plants, animals and fungi to protect against invasive nucleic acids, including viruses and transposable elements, and to control endogenous gene expression. Previous work has established the central pathway responsible for RNA silencing against viruses: plant Dicer-like proteins (DCLs) cleave long double-stranded RNA (dsRNA) into populations of 21-24 nucleotide long small-interfering RNAs (siRNAs) which are loaded into Argonaute proteins (AGOs), the central effectors of the RNA-induced silencing complex (RISC) that repress RNAs with complementary sequences. dsRNA is created when viruses replicate within cells, by secondary foldback structures, and also following the action of host RNA-dependent RNA polymerases (RDRs) aided by the RNA-binding protein SGS3. RNA silencing effectively kills viruses, and consequently viruses have evolved to negate the pathway by expressing Viral Suppressors of RNA silencing (VSRs). Our experimental plan makes use of viral strains with impaired VSR function, including Turnip Crinkle Virus (TCV) and Turnip Mosaic Virus (TuMV), as described in (Pumplin & Voinnet, Nature Reviews Microbiology 11, 745–760 (2013)).Experimental work carried out under the Marie Curie project MeViSP focused heavily on poorly understood questions surrounding subcellular organization of RNA silencing. A large collection of stable plant lines that express fluorescent protein-tagged reporters was generated and analyzed by live-cell microscopy. Together with complementary experiments including immunolocalization and biochemical approaches, a comprehensive and detailed assessment of silencing pathways from the cellular perspective could be gained. These experiments revealed first, that the major anti-viral protein DCL4 is in fact localized in the cytoplasm, and governed by a complex homeostatic mechanism involving alternative mRNA isoforms under epigenetic regulation. This discovery was enabled by the painstaking effort to create reporter lines that accurately reflect endogenous gene regulation. In addition, we observed that silencing components change localization during virus infection, an idea which was merely a hypothesis during the creation of this project. These discoveries were further expanded through the establishment of international collaborations. The techniques and material generated can now be used to ask additional detailed questions of RNA silencing organization within cells. Furthermore, we will release the lines to the greater scientific community to enable diverse researchers to build on this knowledge.The MeViSP project also outlined a mutagenesis screen to discover novel genes that mediate virus defense using a novel method. At the start of the support period, material was created to express a mutant virus under an inducible promoter system and tested as outlined in the plan. Subsequently, technical limitations arose due to the virus chosen for the screen, as the inducible system showed 1) virus infection in the absence of induction and 2) silencing of the inducible virus transgene; These unforeseen difficulties precluded a reliable genetic screen. In a collaboration with a fellow member of the group, we overcame this limitation by creating a new set of material with a second virus that in preliminary experiments is not prone to the problems of transgene instability. This material is now in the final stages of control experiments, and will be used for a screen as described in the proposal.In total, the MeViSP project has already resulted in 1) creation of novel and valuable plant resources that will shed light on the cell biogy and genetics of RNA silencing and anti-viral defense in plants; 2) crucial basic discoveries into the regulation and localization of DCL proteins in plants; 3) international collaborations and dissemination of discoveries at international meetings and invited seminars. In the long term, this research project will greatly improve our understanding of both RNA silencing and anti-viral defense in plants, as well as gene regulation during seed development, a crucial agronomic topic. Knowledge generated will improve researchers’ ability to use RNA silencing for plant and agricultural improvement, with the specific possibility to alleviate viral diseases of plants.
