Condensate Formation by Structured dsRNA Binding Domains Drives RNA Sequestration in Plants

A key antiviral strategy in plants involves the sequestration of viral genomic RNA by dsRNA-binding proteins DRB2, DRB3, and DRB5 within phase-separated viral replication complexes (VRCs). While these proteins colocalize in VRC condensates to suppress viral replication, the molecular mechanisms underlying viral RNA sequestration and replication arrest in the antiviral defense pathway remain enigmatic. Here, we show that the oligomerization-prone dsRBD2 domain of DRB2/3/5 adopts a modified dsRBD fold and undergoes phase separation upon interaction with dsRNA. We find that the structural modifications lead to a unique surface charge distribution that promotes multivalent, surface-exposed interaction patches on dsRBD. These features enable concentration-dependent transient self-association in DRB2/3/5 and guide the formation of gel-like condensates in the presence of individual triggers such as dsRNA, molecular crowding, or ATP. Our study identifies a plausible mechanism of dsRNA-binding protein induced phase separation and suggests that RNA sequestration via condensate formation contributes to plant antiviral immunity.