Isolation and characterization of mobile sRNA/target pairs in plants by using a viral protein as a probe

Projektbeschreibung

Verfolgung mobiler RNA-Moleküle in Pflanzen und Entdeckung ihrer Targets für die Gen-Stilllegung

Regulative kleine RNA (sRNA) gibt es überall in Pflanzen. Sie spielen eine entscheidende Rolle bei der Ausbreitung von RNA-Interferenz (RNAi) in einer Pflanze, welche ein genetisches Regulationssystem ist, das die Aktivität bestimmter Gene stilllegt. Angesichts der Vielfalt der sRNA und ihrer Targets sowie aufgrund ihrer Mobilität war es schwierig, Paare mobiler sRNA und ihrer Targets zu identifizieren und vollständig zu charakterisieren. Das EU-finanzierte Projekt GeminiDECODER verwendet dazu eine neuartige Sonde mit beispielloser Auflösung. Diese Sonde ist ein Protein in einem Pflanzenvirus, das die Ausbreitung von RNAi von Zelle zu Zelle beeinflusst, wie kürzlich gezeigt wurde. Sie soll Forschenden dabei helfen, mobile sRNA und ihre Targets unter unterschiedlichen Bedingungen in verschiedenen Pflanzen zu isolieren., mit potenzieller Anwendung bei der Regulierung der Nahrungspflanzenproduktivität.

Ziel

In plants, RNA interference (RNAi) is involved in processes ranging from development to stress responses. The core element of this regulatory mechanism, the small RNA (sRNA), has been characterized as a mobile molecule able to spread the RNAi beyond the initiation sites, acting as a cell non-autonomous process. Examples of mobile sRNAs are known: mobile small interfering RNAs (siRNAs) deriving from viruses spread throughout the plant, and are responsible for the activation of the viral silencing in uninfected tissues; other siRNAs, together with microRNAs (miRNAs), have been described to move through different cell layers, acting as morphogens; finally, some miRNAs have been shown to move through the vasculature under stress conditions, activating protective responses. Mobile sRNAs are therefore of capital importance for plant biology, and many approaches have been followed to analyze them; however, the number of mobile sRNA/target pairs fully characterized is scarce. Recently, the C4 protein from Tomato yellow leaf curl virus (TYLCV) has been shown to interfere specifically with the cell-to-cell movement of RNAi, affecting neither accumulation nor activity of the sRNAs. In this project, C4 will be used as a novel and powerful tool to isolate mobile sRNAs and their targets, under different conditions (basal vs viral infection) and in different plants (Arabidopsis and tomato), in a large scale and with unprecedented resolution. By using C4-expressing plants, out-of-location mobile sRNAs as well as increased levels of their unprocessed targets are expected to be easily detected. Thanks to this viral probe, a whole picture of the different populations of mobile sRNAs and their targets will be obtained, and the biological relevance of the most prominent candidates will be assessed. As a result, relevant new knowledge about the regulation of key biological processes in plants will be obtained, which may open new avenues for the design of more productive crops.