As previously described for other species [12, 17, 18], including N. benthamiana [19, 20], here we describe a simple and highly efficient micrografting protocol for N. attenuata with transgenic lines. Our data support the employment of N. attenuata grafts that combine WT genotype either to ov- or ir-transgenic lines, this latter only when used as rootstocks, given the altered transcript accumulation of WT roots promoted by silenced shoots. The motivation to adopt this well-known grafting method for N. attenuata is grounded in the several layers of inter-related plant defenses that have described in N. attenuata. Systemic signals produced upon herbivore attack enhance the indigestibility of N. attenuata leaves by augmented TPI activity  and increase its toxicity by producing nicotine  and diterpene glucosides . Systemic defense signals are also involved in recruiting predators to attacking herbivores by producing volatiles that betray the location of the herbivores on attacked plants [6, 22], as well as in reallocation of energy to roots that are later remobilized for reproduction and thereby enhances a plant's tolerance of herbivore attack . In addition, it is known that N. attenuata can change its flower opening time in order to recruit new pollinators that do not have larval stages that are herbivores of the plant and thereby reduces future herbivore loads . Taken together, the establishment of micrografting method for N. attenuata will allow us to examine the systemic signals and gene function in above- versus below-ground parts of a plant in an ecological context.
Meristematic activity in the graft junction is an important determinant of attaining high grafting success rates . The reason why previous attempts of cleft grafting with adult N. attenuata plants resulted in low success rate may be due to poor callus formation in stem tissues. The hypocotyls of germinated seeds have proved to be the most reliable explant to induce meristematic activity for tissue culture and regeneration of N. attenuata  and this tissue's particularly high meristematic activity appears also to be the reason behind the success of the of the grafting procedure described here (Figure 1C).
Other than its simplicity and high efficiency, a major advantage of this method for N. attenuata is that its impact on a plant's adult life is expected to be minor because the grafting takes place in an early phase of plant development. Only five to six days after grafting, completely healed and healthy grafted seedlings are obtained (Figure 1B), which do not show morphological or fitness compromises when compared to intact WT N. attenuata plants at later stages in development (Figure 2). Moreover, the wounding inflicted by the grafting procedure itself could potentially lead to activation of defense related traits such as augmented TPI expression or nicotine accumulation [27, 28]. However TPI and PMT transcript accumulation levels of five-week-old WT/WT resembled those of intact WT N. attenuata plants (Figure 3A, B). In addition, given that the graft junction is established at the shoot-root interface, this protocol allows for the manipulation of a larger long-distance signaling system in plants, rather than only within shoots .
Recent molecular studies suggest the graft hybridization can occur by the exchange of genetic material among neighboring cells and across the graft junction [30–32]. However, as reported for N. benthamiana, micrografted roots harboring 35S-derived RNAi constructs were unable to promote silencing of its target in nonsilenced shoots . To validate the micrografting method for N. attenuata with transgenic lines, transmission of the silencing effect in rootstocks to scions or vice versa should be examined. Our data are consistent with the lack, or very weak upward transmission of the silencing signal (Figure 3A). On the other hand, when irPMT shoots were grafted onto WT roots of one-week-old seedlings, the roots' ability to accumulate PMT transcripts in later developmental stages was reduced (Figure 3B), consistent with the concept of source-to-sink facilitated movement of sRNA silencing signals . Regardless of the molecular mechanism underlying the spread of the silencing from shoot to root observed in irPMT/WT grafts, these data suggest a limitation to the use of grafts consisting of ir construct-derived transgenic lines scions and WT rootstocks for addressing ecological questions.
Roots are thought to play a role in the control of developmental processes of the aboveground parts of plants, such as shoot branching and flowering [12, 33]. As for plant defenses, it has been shown that roots of N. attenuata account for both plant resistance (e.g. nicotine production, ) and tolerance (e.g. changes in within-plant carbon allocation, ) to herbivore attack. However, micrografting can further extend our understanding of a plant's below-ground interactions and molecular mechanisms and their final contribution to the whole-plant performance. For instance, the signaling underlying the JA-induced nicotine synthesis in the roots of N. attenuata after leaf damage can be investigated by analyzing grafted plants that have WT shoots and roots deficient in JA perception (irCOI1, ), synthesis (irLOX3, ) or activation (i.e. conversion of JA to its active JA-Ile form, ) (Additional file 1). In addition, ethylene is known to attenuate MeJA-induced accumulation of PMT transcripts as well as the production of nicotine . Given the readily available ethylene-related transgenic lines (ovETR1 and irACO, ) of N. attenuata (Additional file 1), it would be interesting to determine whether ethylene biosynthesis or perception in the roots is the limiting step in the regulation of nicotine synthesis upon herbivore attack. The movement of small RNA is also important in long-distance signaling. Silenced lines of RNA-directed RNA polymerase genes (irRdR1, irRdR2 and irRdR3, [38–40]) enable us to find small RNAs that move from WT shoot to small RNA-deficient root after herbivore attack. In addition, silenced lines of Dicer-like (DCL) proteins, which will be available soon, are also useful in manipulating the role of small RNA in shoot-root signaling. Finally, scrutiny of field-grown micrografted N. attenuata plants displaying markedly differences in performance could lead to the identification of novel root-derived traits that account for plants' Darwinian fitness as well as serve as a complementary approach to the molecular characterization of genes . Therefore we predict that the protocol reported here will be valuable for unraveling potential root-based traits that profoundly affect plant development and fitness.