Rootstock‐to‐scion transfer of transgene‐derived small interfering RNAs and their effect on virus resistance in nontransgenic sweet cherry

Abstract

SummarySmall interfering RNAs (siRNAs) are silencing signals in plants. Virus-resistant transgenic rootstocks developed through siRNA-mediated gene silencing may enhance virus resistance of nontransgenic scions via siRNAs transported from the transgenic rootstocks. However, convincing evidence of rootstock-to-scion movement of siRNAs of exogenous genes in woody plants is still lacking. To determine whether exogenous siRNAs can be transferred, nontransgenic sweet cherry (scions) was grafted on transgenic cherry rootstocks (TRs), which was transformed with an RNA interference (RNAi) vector expressing short hairpin RNAs of the genomic RNA3 of Prunus necrotic ringspot virus (PNRSV-hpRNA). Small RNA sequencing was conducted using bud tissues of TRs and those of grafted (rootstock/scion) trees, locating at about 1.2 m above the graft unions. Comparison of the siRNA profiles revealed that the PNRSV-hpRNA was efficient in producing siRNAs and eliminating PNRSV in the TRs. Furthermore, our study confirmed, for the first time, the long-distance (1.2 m) transfer of PNRSV-hpRNA-derived siRNAs from the transgenic rootstock to the nontransgenic scion in woody plants. Inoculation of nontransgenic scions with PNRSV revealed that the transferred siRNAs enhanced PNRSV resistance of the scions grafted on the TRs. Collectively, these findings provide the foundation for 'using transgenic rootstocks to produce products of nontransgenic scions in fruit trees'.

Immunity to tomato yellow leaf curl virus in transgenic tomato is associated with accumulation of transgene small RNA

“…5 and 6). The number of mobile t-siRNAs was larger than that shown by Molnar et al [26] in Arabidopsis plants (moving from shoot to root) and from a transgenic sweet cherry root to scion [24]. The size distribution and the profile of the mobile t-siRNA in our experiment was similar to that characterized in the transgenic rootstock, dominated by the 21-to 22-nt sizes, as shown previously in tobacco [29].…”

“…This was in turn processed to siRNA molecules of about 21-24 nt length that directed the RNA silencing pathway [22]. It has been shown that siRNAs produced from hairpin loci can move through the vasculature or a graft union [23,24] and are functional in mediating post-transcriptional gene silencing of endogenous and exogenous targets, [25][26][27]. This trait was exploited for the development of viral resistance of a non-transgenic scion grafted onto transgenic rootstock expressing siRNA to target an invading virus [28][29][30].…”

Literature review of baseline information to support the risk assessment of RNAi‐based GM plants

“…For example, post-transcriptional silencing of an endogenous gene in the shoot was observed in Nicotiana when a wild-type scion was grafted on a rootstock synthesizing a siRNA signal (Kasai et al, 2011). The signal can spread from a rootstock can travel quite far as demonstrated in cherry, where siRNAs were detected 1.2 m from the graft union (Zhao and Song, 2014).…”

Generation of Transgenic Rootstock Plum ((Prunus pumila L. × P. salicina Lindl.) × (P. cerasifera Ehrh.)) Using Hairpin-RNA Construct for Resistance to the Plum pox virus

“…The success of this approach, however, has largely depended on the species, the level of expression and the interactions of the small interfering ribonucleic acid (siRNA) with the target gene [15]. For example, the transfer of transgene-derived siRNAs from the transgenic cherry rootstocks to the non-transgenic scions in grafted trees was effective in inducing resistance to the Prunus necrotic ringspot virus [16]. In apples, however, the transmission of siRNAs from transgenic rootstock to non-transgenic scion was not observed [17].…”