Investigation of transitive and systemic RNA silencing in plants

通过我们提出的项目，我们的目标是对传递性和系统性沉默的机制产生新的重要见解。在植物中，小RNA (sRNAs)，包括微RNA （miRNAs）和小干扰RNA (sirna)，被装载到ARGONAUTE蛋白（AGOs）上，形成RNA诱导沉默复合体（RISC）。RISC通常靶向同源转录物进行切割，这一过程称为RNA沉默或RNA干扰（RNAi）。在切割过程中，目标转录物通常被外核降解。然而，一些靶向转录本被RNA定向RNA聚合酶（RDRs）复制成双链RNA （dsRNA）。生成的dsRNA被DICER-LIKE酶（DCLs）加工成次级sirna。次级sirna再次装载到AGOs上，启动一个自我强化的RNAi过程。RNA沉默在主要靶点之外的这种5′和3′扩散被称为传递性或传递性沉默。及物性的机制细节在很大程度上仍然未知。srna的大小和结构似乎影响了这一过程的启动。例如，假设22-nt srna将rdr招募到它们的目标。除了传递性沉默，植物还表现出系统性沉默。在触发局部沉默的组织中可以产生远距离移动的沉默信号。在接收这些信号的顶端叶片中，同源转录物的沉默（包括传递性沉默）可以被诱导。然而，目前尚不清楚系统性沉默是否需要激活局部沉默，或者系统性沉默信号是否仅仅在引入假定的沉默触发器后产生。因此，我们旨在研究系统性沉默的启动过程。一般来说，转基因基因比内源基因更容易发生传递性和系统性沉默。目标的性质可能会影响这两个过程的启动。在目前的提案中，我们的目的是研究利用合成sirna作为沉默触发器，对烟草中选择的转基因和内源性靶点进行传递性和系统性沉默的先决条件。合成sirna的引入使得首次研究沉默触发器的性质对RNA沉默的影响成为可能。此外，将高度容易产生次级sirna的靶标（例如，来自无内含子的转基因的转录本）与似乎具有抗性的靶标（例如，来自含内含子的内源和转基因的转录本）进行比较。我们的组合方法和后续分析的新颖性将为传递性和系统性沉默的机制提供新的重要见解。这些知识对于制定基于应用srna作为对抗病毒、控制杂草和真菌以及改善植物性状的工具的战略将是重要的。

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.