In eukaryotes, RNA silencing is a conserved mechanism of gene regulation and has essential antiviral roles in in plants, nematodes, and insects. Antiviral RNA silencing is triggered by viral dsRNA and is characterized by the accumulation of small interfering RNAs (siRNAs) derived from viral RNA (primary siRNAs) formed by Dicer- like proteins (DCL). Features of viral RNA that trigger silencing have not been determined. Potential triggers include viral dsRNA in replication intermediates and self-complementary sequences in viral genomic RNA. Additionally, during an amplification phase, cellular RNA-dependent-RNA polymerases (RDR) synthesize dsRNA that is processed into secondary virus-derived siRNA by DCL. In plants, RDR1 and RDR6 are the genetic determinants of silencing amplification. However, the mechanisms that bring plant cellular RDRs in contact with their viral RNA substrates are not known. This project is focused in two poorly characterized phases of the plant antiviral RNA silencing: Initiation and amplification. The goals are to 1) identify the viral triggers of RNA silencing and 2) to determine the mechanisms that route viral RNA into cellular RDR- dependent amplification. Our hypothesis is that primary virus-derived siRNAs in association with ARGONAUTE (AGO) proteins flag viral RNA as a substrate for cellular RDRs. The approach consists on genetically blocking cellular RDR-dependent amplification to identify viral RNA triggers based on the profiles of primary virus- derived, and by co-precipitation of DCL and AGO proteins with their viral RNA targets. To determine the mechanisms of RNA silencing amplification, RDR1 and RDR6 interaction partners will be determined by co- precipitation and mass spectrometry, and a synthetic system will be developed to induce and measure antiviral RNA silencing amplification. This system is designed to determine the functionality of structurally different primary virus-derived siRNAs, in association with AGO proteins, in flagging viral RNA as a substrate for cellular RDR1 and RDR6. This work will help define how plant viral RNAs are recognized as distinct from cellular (non- targeted) RNA, and determine the mechanisms of antiviral RNA silencing initiation and amplification. Expected findings will contribute to our understanding and manipulation of an antiviral immunity system that determines the outcome, disease or no disease, in plant-virus interactions. Some viruses cause important human diseases, others cause devastating diseases in primary staple crops. In plants, RNA silencing is an essential part of antiviral immunity. During their replication, all viruses go through and RNA phase that potentially exposes them to antiviral responses. This project uses model plants and RNA viruses to determine the mechanisms of initiation and amplification of antiviral RNA silencing.