Investigation of transitive and systemic RNA silencing in plants

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With our proposed project we aim to generate new important insights into the mechanism of transitive and systemic silencing. In plants, small RNAs (sRNAs), including micro RNAs (miRNAs) and small interfering RNAs (siRNAs), are loaded onto ARGONAUTE proteins (AGOs) to form the RNA induced silencing complex (RISC). RISC targets homologous transcripts usually for cleavage, in a process called RNA silencing or RNA interference (RNAi). Upon cleavage, target transcripts are generally exonucleolytically degraded. However, some targeted transcripts are copied by RNA-DIRECTED RNA POLYMERASEs (RDRs) into double stranded RNA (dsRNA). The generated dsRNA is processed by DICER-LIKE enzymes (DCLs) into secondary siRNAs. The secondary siRNAs are again loaded onto AGOs initiating a self-reinforcing RNAi process. This 5´ and 3´ spreading of RNA silencing beyond the primary target site is termed transitivity or transitive silencing. The mechanistic details of transitivity remain largely unknown. The size and structure of sRNAs seem to effect the initiation of the process. For example, 22-nt sRNAs are assumed to recruit RDRs to their targets. In addition to transitive silencing, plants display systemic silencing. Long distance-moving silencing signals can be produced in tissue in which local silencing is triggered. In the apical leaves receiving these signals, silencing, including transitive silencing, of homologous transcripts can be induced. However, it is not clear if systemic silencing requires the activation of local silencing or if systemic silencing signals are produced simply upon introduction of a putative silencing trigger. We therefore aim to study the initiation process of systemic silencing. In general, transgenes are more prone to transitive and systemic silencing than endogenes are. The nature of the target is likely to affect the initiation of both processes. In the current proposal, we aim to investigate the prerequisites for the initiation of transitive and systemic silencing of a selection of transgenic and endogenous targets in Nicotiana benthamiana using synthetic siRNAs as silencing triggers. The introduction of synthetic siRNAs allows for the first time to study the effect of silencing triggers' nature on RNA silencing. In addition, targets that are highly prone to the production of secondary siRNAs, e.g. transcripts derived from intronless transgenes, will be compared with targets that appear to be resistant, e.g. transcripts derived from intron-containing endogenes and transgenes. The novelty of our combinatorial approach and subsequent analysis will provide new important insights into the mechanism of transitive and systemic silencing. Such knowledge will be important for the development of strategies that will be based on the application of sRNAs as a tool to combat viruses, to control weeds and fungi and to improve plant traits.