RNA-basierte antivirale Immunität in einem pflanzlichen in vitro-System

First observed in plants, suppression of viral infections by small RNAs is yet established as an ancient immune response that is mechanistically related to RNA interference (RNAi). The immune response initiates with the recognition of viral double-stranded and/or structured RNA by DICER-LIKE (DCL) RNAse III enzymes that generate 21–25 nucleotide (nt) RNA fragments. Some of these small interfering RNAs (siRNAs) direct specific silencing of the homologous viral genomic and/or messenger RNAs by RNaseH-like Argonaute protein(s). The Argonaute proteins are believed to be components of yet incompletely characterized effector complexes, such as RNA-induced silencing complexes (RISC). In Arabidopsis Thaliana, four DCLs and ten Argonaute proteins are reported. So far, it was shown that DCL2 and DCL4 that generate 22 nt and 21 nt siRNAs act redundantly, and either was found sufficient to initiate small RNA-directed immunity against various positive-strand RNA viruses. Concerning the Argonaute proteins, controversial data exist on the role of Ago1 mediating antiviral activity. Ago2 and Ago5 bind viral siRNAs but their role in viral immunity is tentative. Importantly, many plant viruses have evolved proteins that suppress at different stages of the silencing pathway. The best-characterized silencing suppressor is the p19 protein of Tombusviruses, which specifically binds 21-nt siRNAs in vitro and in vivo preventing siRNA incorporation into effector complexes such as RISC. Taking advantage of an in vitro translation/replication system of the Tombusvirus Tomato bushy stunt virus (TBSV), this project aims at performing an in depth characterization of the function of TBSV RNA generated siRNAs and of individual cellular proteins in RNA-based antiviral immunity.