Posttranscriptional gene silencing (PTGS) is a form of small (s)RNA-mediated gene regulation with important roles in development, stress responses, and antiviral defense in plants. PTGS effectors, called RNA-induced silencing complexes (RISCs), have ARGONAUTE (AGO) proteins bound to single-stranded (ss) guide sRNAs as core components. RISCs recognize target RNA displaying complementarity to guide sRNAs, and inactivate them by endonucleolytic cleavage (or ‘slicing’) or by slicer-independent translational repression. A precise dissection of the underlying mechanisms has been, however, hampered by a lack of plant cell-free systems allowing reconstruction of key PTGS steps in vitro. Recently, a cell-free AGO1-RISC assembly system was developed in Japan, using extracts from evacuolated tobacco BY-2 protoplasts (BYL) and allowed to establish that AGO1-RISC assembly relies on molecular chaperoning by HSP90 and co-factor CYP40. The proposed project will further deepen the understanding of plant RISC assembly mechanisms and decipher the molecular underpinnings of other PTGS steps including Dicer processing and RISC-mediated translational repression. In parallel, the BYL system will be used to investigate, in a strict comparative manner, the direct targets and modes of action of several plant viral suppressors of RNA silencing (VSRs). These proteins have been studied so far mostly in transgenic plants, where feedback PTGS regulations, intrinsically variable VSR expression levels and indirect effects have led to an inaccurate- and sometimes contradictory- view of their action. BYL will bypass these caveats and improve our knowledge of VSRs on antiviral silencing, but also of PTGS, as VSRs will be used to stabilize transient, and thus far inaccessible forms of protein-protein and protein-RNA complexes. Knowledge on plant cell-free systems implemented via the proposed project will finally contribute to European excellence and competitiveness in the field.