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Binding of siRNA molecules is crucial for RNAi suppressor activity of Rice hoja blanca virus NS3 in plants

¹ Rockefeller University, Laboratory of Plant Molecular Biology, NY, USA;
² Hubrecht Institute, Utrecht, The Netherlands;
³ Laboratory of Virology, Wageningen University, The Netherlands;
⁴ Keygene N.V., Wageningen, The Netherlands

⁵ E-mail: rob.goldbach{at}wur.nl

The NS3 protein of rice hoja blanca tenuivirus represents a viral suppressor of RNAi that sequesters small interfering (si)RNAs in vitro. To determine whether this siRNA binding property is the critical determinant for the suppressor activity of NS3, an alanine point mutational analysis was performed and the resulting mutant proteins were tested for both siRNA binding ability and RNAi suppressor activity in plants. Alanine substitutions of lysine residues at position 173-175 resulted in mutant proteins that lost both their affinity for siRNAs and their RNAi suppressor activity in planta. This indicates that siRNA binding of NS3 is indeed essential for the suppressor function of NS3 and that residues at position 173-175 are involved in the siRNA binding and suppressor activity.

Received 22 January 2009; accepted 4 March 2009.