The mechanism of graft transmission of sense and antisense gene silencing in tomato plants

We investigated the effect of target mRNA level on grafting-transmitted gene silencing in tomato plants by using a strong ACC oxidase 1 (ACO1) silencer as the stock and transgenic ACO1 overexpressers as scions. Manifestation of graft transmission of sense gene silencing required a high initial level of target mRNA in the scion. A relatively high level of siRNA, similar to that in the strong ACO1 silencer, was also detected in the silencing-susceptible strong ACO1 overexpressers prior to grafting. After grafting the silencing signal from the stock enhanced the level of the siRNAs in the scion and the ACO1 mRNA level was reduced dramatically. Using stock and scions producing different siRNAs we provided evidence that the transmissible silencing signal does not correspond to the bulk siRNAs in the stock. We also showed, contrary to a previous report, that antisense silencing was graft-transmissible but it took longer to manifest itself. The delay in graft transmission from antisense-silenced plants could be attributed to the difference in the nature or strength of the signal or the mechanism of its amplification, but is further evidence of mechanistic similarities between sense and antisense silencing.     

Scion on a Stock Producing siRNAs of Potato Spindle Tuber Viroid (PSTVd) Attenuates Accumulation of the Viroid

Plants can attenuate the replication of plant viruses and viroids by RNA silencing induced by virus and viroid infection. In higher plants, silencing signals such as small interfering RNAs (siRNAs) produced by RNA silencing can be transported systemically through phloem, so it is anticipated that antiviral siRNA signals produced in a stock would have the potential to attenuate propagation of viruses or viroids in the scion. To test whether this is indeed the case, we prepared transgenic tobacco (Nicotiana benthamiana) expressing a hairpin RNA (hpRNA) of Potato spindle tuber viroid (PSTVd) in companion cells by using a strong companion cell-specific promoter. A grafting experiment of the wild type tobacco scion on the top of the transgenic tobacco stock revealed that accumulation of PSTVd challenge-inoculated into the scion was apparently attenuated compared to the control grafted plants. These results indicate that genetically modified rootstock expressing viroid-specific siRNAs can attenuate viroid accumulation in a non-genetically modified scion grafted on the stock.     

Phloem flow strongly influences the systemic spread of silencing in GFP Nicotiana benthamiana plants

The term 'RNA silencing' describes a process that results in the specific degradation of an RNA target. In plants, silenced tissues can initiate the spreading of the process into non-silenced regions by a mobile signal that can be transmitted over long distances. In the present work, we made use of a modified grafting approach to elucidate the driving force behind long-distance transport of the silencing signal. We made reciprocal grafts of two GFP-transgenic Nicotiana benthamiana lines, the non-silenced line 16c (sensor) and the silenced line 6.4 (inducer). We show that the direction of systemic spread of silencing from inducer to sensor can be manipulated by altering sink/source relations in the plant. Using radioactive phosphate as a phloem tracer, we demonstrated that plants that transmitted silencing from silenced scion to non-silenced rootstock had developed a persisting phloem flow from scion to rootstock. These data provide experimental proof of what has been hypothesized so far, that the silencing signal travels via phloem from source to sink. We present here evidence that the appearance of systemic silencing is not an accidental stochastic process, but can be predicted on the basis of the direction of phloem flow.