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RNA silencing is a part of the plant innate immune system that could effectively cope with intruders, like viruses. However, viruses evolved proteins that can suppress RNA silencing thus supporting virus spreading in the host. To counteract RNA silencing, suppressor proteins attack different players of RNA silencing pathway. The P1 protein of the Sweet potato mild mottle virus binds and inactivates small RNA loaded RISC complexes. Using a deletion series in the P1 protein we aimed to identify the possible smallest working version of P1. Our results revealed that the minimal RNA silencing suppressor domain of P1 is as small as 210 amino acids in size. Acta Biol Szeged 58(2): (2014) Key WorDS RNA silencing plant Sweet potato mild mottle virus RNA silencing suppressor Accepted Dec 18, 2014 *Corresponding author. E-mail: lakatos.lorant@med.u-szeged.hu 1 RNA silencing is a post-transcriptional gene regulation mechanism conserved in almost all eukaryotes and involved in many essential biological processes, from development, physiological activity to the regulation of abiotic and biotic stress responses by micro RNAs (miRNA), genome defense by 24 nucleotide (nt) long small interfering RNAs (siRNAs) and in particular antiviral defense by 21 nt siRNAs. RNA silencing negatively regulates genes expression (Herr and Baulcombe 2004). The trigger of RNA silencing can be pri-miRNAs for miRNAs or double stranded (ds) RNAs as replicative forms of plant viruses. Trigger RNAs are processed into siand miRNAs by the RNAse III type enzymes Dicers, then small RNAs are loaded into the Argonaute (AGO) protein containing protein complexes called RNA induced silencing complex (RISC). This process is referred to as RISC assembly. In RISC, one strand of the small RNA is eliminated resulting in the single stranded (ss) small RNA containing RISC complex (active RISC). Active RISC complexes are able to hamper gene expression either by cleaving the target RNA or by inhibiting the translation of the target RNA (Hutvagner and Simard 2008). Thus, Argonaute (AGO) proteins play a key role in RNA silencing. In the last few years, several AGO binding proteins were identified. Most of them contain GW/WG (Gly-Trp/Trp-Gly) domains, which mediate the interaction with AGO. This group of proteins was named GW/WG proteins after the founding member GW182 protein of human. GW182 in animals bind AGO and mediate the interaction with the polyA binding proteins to repress the translation of the target RNA (Pfaff et al. 2013). In Schizosaccharomyces pombe, transcriptional silencing involves AGO and TAS3 that shows high similarity to animal GW182 proteins (Pfaff et al. 2013). In plants, heterochromatin silencing exploits a couple of GW/WG proteins, such as the second largest subunit of the RNA polymerase V complex and SPT5 protein (Bies-Etheve et al. 2009; Herr et al. 2005). Moreover, the GW/WG protein SUO was identified in Arabidopsis thaliana to be involved in miRNA driven RNA silencing (Yang et al. 2012). Thus, these GW/ WG proteins have a positive effect on RNA silencing and are absolutely required for the efficient carrying RNA silencing into execution. Two viral proteins bearing WG/GW domains were found to have a negative effect on RNA silencing. The p38 protein of the Turnip crinkle virus (TCV) sequesters small RNA unloaded AGO1 (Azevedo et al. 2010). However, the P1 RNA silencing suppressor protein of the Sweet potato mild mottle virus (SPMMV) is markedly different from that of p38. SPMMV P1 binds AGO1 loaded with sior miRNAs, thus inhibiting target cleavage of active RISC complexes. Mutational analysis revealed three WG/GW motifs, resembling the AGO binding platform conserved in plants and metazoans, at the N-terminal part of P1, which are required for AGO binding and silencing suppressor activity (Giner et al. 2010). Recently, we found that the first 383 amino acid (aa) region of the SPMMV P1 renders RNA silencing suppressor activity (Giner et al. 2010). Thus, our aim was to find the smallest region of the P1 protein bearing fully functional suppressor activity. Our results based on a series of truncation of

Characterization of AGO-binding silencing suppressors and creating the first artificial RNA silencing suppressor