Roles of Dicer-Like Proteins 2 and 4 in Intra- and Intercellular Antiviral Silencing.

RNA silencing is an innate antiviral mechanism conserved in organisms across kingdoms. Such a cellular defense involves DICER or DICER-LIKEs (DCLs) that process plant virus RNAs into viral small interfering RNAs (vsiRNAs). Plants encode four DCLs that play diverse roles in cell-autonomous intracellular virus-induced RNA silencing (known as VIGS) against viral invasion. VIGS can spread between cells. However, the genetic basis and involvement of vsiRNAs in non-cell-autonomous intercellular VIGS remains poorly understood. Using GFP as a reporter gene together with a suite of DCL RNAi transgenic lines, here we show that despite the well-established activities of DCLs in intracellular VIGS and vsiRNA biogenesis, DCL4 acts to inhibit intercellular VIGS whereas DCL2 is required (likely along with DCL2-processed/dependent vsiRNAs and their precursor RNAs) for efficient intercellular VIGS trafficking from epidermal to adjacent cells. DCL4 imposed an epistatic effect on DCL2 to impede cell-to-cell spread of VIGS. Our results reveal previously unknown functions for DCL2 and DCL4 that may form a dual defensive frontline for intra- and intercellular silencing to double-protect cells from virus infection in Nicotiana benthamiana.