Abstract
Grafting of commercial varieties onto transgenic stress-tolerant rootstocks is attractive approach, because fruit from the non-transgenic plant body does not contain foreign genes. RNA silencing can modulate gene expression and protect host plants from viruses and insects, and small RNAs (sRNAs), key molecules of RNA silencing, can move systemically. Here, to evaluate the safety of foods obtained from sRNA-recipient plant bodies, we investigated the effects of rootstock-derived sRNAs involved in mediating RNA-directed DNA methylation (RdDM) on non-transgenic scions. We used tobacco rootstocks showing RdDM against the cauliflower mosaic virus (CaMV) 35S promoter. When scions harboring CaMV 35S promoter sequence were grafted onto RdDM-inducing rootstocks, we found that RdDM-inducing sRNAs were only weakly transported from the rootstocks to the scion, and we observed a low level of DNA methylation of the CaMV 35S promoter in the scion. Next, wild-type (WT) tobacco scions were grafted onto RdDM-inducing rootstocks (designated NT) or WT rootstocks (designated NN), and scion leaves were subjected to multi-omics analyses. Our transcriptomic analysis detected 55 differentially expressed genes between the NT and NN samples. A principal component analysis of proteome profiles showed no significant differences. In the positive and negative modes of LC-ESI-MS and GC-EI-MS analyses, we found a large overlap between the metabolomic clusters of the NT and NN samples. In contrast, the negative mode of a LC-ESI-MS analysis showed separation of clusters of NT and NN metabolites, and we detected 6 peak groups that significantly differed. In conclusion, we found that grafting onto RdDM-inducing rootstocks caused a low-level transmission of sRNAs, resulting in limited DNA methylation in the scion. However, the causal relationships between sRNA transmission and the very slight changes in the transcriptomic and metabolomic profiles of the scions remains unclear. The safety assessment points for grafting with RdDM rootstocks are discussed.