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