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C2: In vitro characterization of viral suppressors of plant RNA silencing

Supervisor: Prof. Dr. Sven-Erik Behrens

 

Specific aim/topic

(1)     Exploring the roles of TBSV P19 and TCV P38 in affecting antiviral and cellular RNA silencing

 

Background and significance

Small RNA-mediated RNA silencing is the primary adaptive immune response against (+)RNA viruses in higher plants. Inductors are structured elements of the viral genome or double-stranded RNAs that are generated by cellular RNA polymerases or during viral replication via (-)RNA intermediates. The RNA triggers are processed by the Dicers DCL2, 3 and 4 into 21-24 nt long vsiRNAs, which move ahead of the infection in the plant and may establish antiviral RNA silencing and immunity. As a key-feature of silencing, vsiRNAs are incorporated into RNA-induced silencing complexes (RISC) that contain ARGONAUTE (AGO) nucleases and other, yet uncharacterized components. Ten AGO proteins were identified in A. thaliana, from which AGO1, 2, 3, 5 and 7 were indicated to contribute to antiviral protection (see previous studies). In antiviral RISC the vsiRNA guide strand directs the AGO/RISC to the cognate viral RNA that is inactivated by cleavage in the siRNA–RNA duplex. Another RNA silencing pathway in plants is mediated by micro RNAs (miRs) that are processed by DCL1 from genome-encoded RNA precursors. MiRs are also incorporated into RISC and may mediate endonucleolytic cleavage or translational repression of mRNAs. During plant and virus co-evolution, most viruses developed viral suppressor proteins of RNA silencing (VSRs). VSRs are often pathogenic itself by targeting dicing, RNA amplification or RISC assembly in anti-viral but also cellular RNA silencing. Though VSRs of different viruses show little homology, they often affect similar processes. For example, the Tombusvirus P19 and the Carmovirus P38 were both reported to locally increase the amount of miR168 in the plant . MiR168 represses the expression of AGO1 mRNA , and P19 was demonstrated to inhibit antiviral silencing by decreasing the AGO1 level via mir168 induction. P19 was shown to inhibit silencing also by sequestering vsiRNAs. P38 was indicated to associate via the GW/WG ‘AGO hook motif’ with AGO1 and to impair sRNA loading and de novo formation of AGO1/RISC. Infections with P38-expressing virus correlate with an increase in the amount of DCL1 in the plant, and it was proposed that AGO1/P38 interactions impact the level of miR162, which is a negative regulator of the DCL1 mRNA. The unequivocal characterization of the anti-silencing activities of VSRs is hampered by their multifunctional nature. For example, P38 also acts as a virus coat protein; accordingly mutations in the protein frequently interfere with virus viability in planta. Here, we propose to apply a newly developed in vitro system that enables studies on P19 and P38 independently from the viral infection and assembly processes.