Inhibition of in vivo Slicer activity of Argonaute protein 1 by the viral 2b protein independent of its dsRNA-binding function

The 2b protein of Cucumber mosaic virus (CMV) has several unique properties, such as targeting to the nucleolus and interaction with both Argonautes (AGOs) and short and long double-stranded RNA (dsRNA). We have recently uncoupled the domain requirements for dsRNA binding and nucleolar targeting from the physical interactions with AGO proteins, and have found that the direct 2b-AGO interaction is sufficient to inhibit the in vitro AGO1 Slicer function independent of the other biochemical properties of 2b. Because the AGO binding activity of 2b is not required for its suppressor function in vivo, this raises the question of whether in vivo 2b-AGO interaction is possible to inhibit the in vivo AGO Slicer function. In this study, by taking advantage of a technology for the production of artificial trans-acting small interfering RNA (tasiRNA), a process uniquely associated with AGO1-mediated in vivo Slicer activity, we demonstrated that the expression of the 2b protein in planta interfered with the production of tasiRNA. Through further detailed analysis with deletion mutants of 2b proteins, we found that the inhibition of in vivo AGO1 Slicer function required the nucleolar localization signal (NoLS), in addition to the AGO-binding domain, of the 2b protein. Our finding demonstrates that in vivo 2b-AGO1 interaction is sufficient to inhibit AGO1 Slicer function independent of the dsRNA-binding activity of the 2b protein.