A Genetic Network for Systemic RNA Silencing in Plants

Non-cell autonomous RNA silencing can spread from cell to cell and over long-distance in animals and plants. However, the genetic requirements and signals involved in plant mobile silencing remain poorly understood. Here, we identified a DICER-LIKE2 (DCL2)-dependent mechanism for systemic spread of post-transcriptional RNA silencing (also known as post-transcriptional gene silencing, PTGS) in Nicotiana benthamiana. Using a suite of transgenic DCL RNAi lines coupled with a GFP reporter, we demonstrated that N. benthamiana DCL1, DCL2, DCL3 or DCL4 is required to produce microRNAs, 22, 24 or 21nt small interfering RNAs (siRNAs), respectively. All sized siRNAs produced in local incipient cells were present at low levels in distal tissues. Inhibition of DCL2 expression reduced the long-distance spread of silencing, as opposed to suppression of DCL3 or DCL4 which enhanced systemic PTGS. DCL2-DCL4 double-RNAi developed systemic PTGS like DCL2 RNAi lines, but in contrast to DCL4 RNAi lines. We further show that 21 or 24nt local siRNAs processed by DCL4 or DCL3 were not correlated with long-distance spread of systemic silencing. Grafting experiments demonstrated that DCL2 was required in the scion to respond to the signal, but not in the rootstock to produce/send the signal. These results suggest a coordinated DCL genetic pathway in which DCL2 plays an essential role in systemic PTGS, whilst both DCL4 and DCL3 may act as attenuators of systemic PTGS in N. benthamiana. The potential role of 21, 22 and 24nt siRNAs in long-distance spread of PTGS is discussed.