Enhancement of post-transcriptional gene silencing by grafting.

We showed previously that grafting transmitted silencing occurred when transgenic ACC oxidase 1 (ACO1) overexpressing tomato plants that also produced siRNAs were grafted onto transgenic stocks that already showed strong silencing. The presence of siRNAs in these overexpressing scions may indicate that silencing, though inefficient, may already occur at a low level before grafting. To test if a silencing state with a relatively high level of target mRNA can be shifted towards further more effective silencing, we grafted an ACO1 antisense (AS) line with a high level of antisense ACO1 transgene mRNA and low level of siRNAs to the ACO1 strong silencer stock. The AS mRNA level was reduced dramatically two weeks after grafting. More interestingly, self-grafting of ACO1 overexpressers and AS lines also induced strong silencing in the scions. We suggest that grafting transmitted silencing may involve the switching from an inefficient or weak silencing state to a stronger silencing by a systemic silencing signal, similar to the change of silencing states that sometimes occurs during development. Control experiments using non-transgenic stocks designed to test whether wounding alone is responsible for generating a signal that enhances silencing in transgenic scions gave negative results. We propose that the build-up of silencing signal and/or molecules at both sides of the grafting junction and their sudden release when the phloem is reconnected may be critical to grafting transmitted silencing.