Both of these examples used sense transgenes, therefore this type of silencing effect commonly is referred to as co-suppression. Several other groups working with similar systems have reported analogous results . In contrast, antisense silencing was shown in tobacco to be not graft-transmissible regardless of whether the signal originated in the scion or root stock . In tomato grafting experiments with the ACC oxidase gene, antisense silencing of scion ACC oxidase was not seen early after graft establishment, however after several weeks a graft-transmissable silencing was observed . This time lag may account for why the earlier experiments concluded that there was no silencing in grafted antisense lines. A high level of expression of the target gene in the scion was necessary for the detection of silencing by Northern hybridization, as a result of expression of the antisense construct in the root stock, a situation similar to the nitrate reductase experiments discussed earlier . Thus, experimental time lines, the levels of target gene expression, and the model organisms used may be important determinants of the efficacy of antisense silencing in grafted systems. It has also been shown that even when target gene are not present in the recipient graft, transgenic siRNAs can accumulate from donor grafts . Arabidopsis containing a GFP inverted-repeat silencing construct as the donor was grafted withWT or GFP-expressing scions as recipients. The sRNAs identified in scion tissues included siRNAs generated as a result of the GFP construct and a substantial population of endogenous sRNAs from the root stock donor as well. Size classes ranging from 21 to 25 nt were most abundant, and the 24-nt class directed epigenetic modification of the GFP signal in the scion. The massively parallel deep sequencing methods used by this group showed that if a silencing target was not present in the recipient ,plastic planters bulk then siRNAs generated from hairpin-GFP in the root stock were still present in the scion, albeit at levels several of orders of magnitude lower.

This could be why previous experiments using less sensitive detection techniques, such as Northern blots, did not detect mobility of the signal. A recent report has shown that beyond the 24-nt siRNAs mentioned above, all size classes of siRNAs can trigger homologous sequence-specific methylation of targets at long-distances, at least in Arabidopsis . What facilitates the movement of sRNAs? sRNAs and associated RNPs are small enough to be translocated based on their size, since experiments have shown that a 27-kDa GFP is able to diffuse into the vascular system . Results of experiments where movement proteins are included indicate that spreading of the silencing signal is at least partially dependent on the size of the plasmodesmatal apertures . Alternatively, movement of the silencing signal might be selective, perhaps requiring protein– protein, or protein–nucleic acid interactions in order to obviate the apparent plasmodesmatal aperture size exclusion limit. This view is supported by experiments involving mutants deffective or deficient in the ability to move signals . Regardless of uncertainties related to the mechanism of sRNA movement, the evidence demonstrates that movement does indeed occur through the phloem component of the vascular system and is mediated by plasmodesmata, at least to some degree. Many experiments have been performed regarding the mobility of RNAs, both large and small, but whether the same pathways that are used for the movement of mRNA are used for miRNA or siRNA movement has not been determined. The emerging idea that sRNAs are involved in physiology, defense, and development, both cell autonomously and for long-distance signaling, is becoming more widely accepted . Given the variability in mobility detected across several studies, it seems that plasmodesmata-based transport of sRNAs is a regulated process. However, the molecular mechanisms that mediate sRNA mobility and whether they are cis or trans-acting are unknown.

Researchers have successfully employed strategies that utilize the expression of siRNAs in order to protect the plant root zone from pests and pathogens . For example, in soybean, resistance strategies that target soybean cyst nematode genes, including those associated with stimulating root growth in infected plants, sperm production, and female development have been tested . By grafting these plants to WT scions, systemic protection may be achieved in a manner similar to the virus resistance reported in tobacco and more recently in cassava in experiments demonstrating control of the devastating Cassava brown streak Uganda virus . Aside from pathogen resistance, down–regulation, and/or epigenetic modification of transcripts and genetic networks in the scion or the root stock also appear to be possible through the use of siRNAs and could influence scion-specific characteristics, such as flowering time, fruit production or quality, or root characteristics, such as tuberization in potatoes .In addition to RNAs, proteins may be transported over long distances in a regulated fashion. Certain motifs, reminiscent of nuclear localization signals, allow protein entry into CC and subsequently into the phloem for long-distance movement. Despite the evidence for selective and regulated processes for protein long-distance translocation, there is also evidence that shows nonspecific “leakage” of supposedly cell-autonomous proteins into sieve tubes and subsequently into sink tissues. Xylem vessels,which mainly transport water and low molecular weight inorganic and organic solutes, have been shown to contain proteins, although at lower concentrations than in phloem sap . Proteins targeted to the apoplast may inadvertently enter xylem or phloem vasculature and subsequently be transported to and unloaded in sink tissues. Examples of movement of proteins include exogenous viral movement proteins, endogenous transcription factors and xylem/phloem proteins . Some of the first studies of xylem protein transport involved viral movement proteins , but as knowledge has progressed, more researchers have been able to demonstrate mobility of endogenous plant proteins. For many years, proteins had been observed in the phloem, but the idea of a coordinated, selective, and regulated process of trafficking, influencing not only development, but plant responses to environmental cues is a more recent idea that has gained support .

Mobile proteins or non-cell-autonomous proteins may be encoded by as many as 20% of the genes in Arabidopsis . A comprehensive analysis of phloem sap proteins in pumpkin and cucumber using high resolution 2-D gel electrophoresis and partial sequencing by mass spectrometry identified several hundred proteins in the phloem, and the majority of these proteins may have roles in stress and defense reactions . Models of the mechanics underlying protein mobility in the vasculature include the structures associated with the vascular tissue. Within the phloem, SE, which lack a nucleus, ribosomes, and a vacuole, depend on neighboring CC for maintenance of their metabolic tasks . Because mature SE cells cannot synthesize proteins, the likely origins of proteins in the phloem are immature SE or CC. Structurally different from the plasmodesmata that connect mesophyll cells, specialized plasmodesmata between CC and SE are branched with all of the branches on the CC side funneling to a single opening on the SE membrane side. The requirements for specificity of transport between CC and SE are not completely known but accumulating evidence points to the importance of these branched plasmodesmata. Reviews from two research groups establish plasmodesmata as the “gatekeepers” of macromolecular transport into the SE . The specific mechanisms governing the regulation of plasmodesmatal apertures are still a mystery, but fluorescently labeled dextrans and GFP expression have been used to study plasmodesmatal size exclusion limits and their function under differing conditions. Through grafting,collection pot the vascular networks of both root stock and scion become connected and what is mobile in the root stock vascular networks is likely to become mobile in the vascular networks of the scion. In a thorough heterografting experiment involving 11 interspecific and intergeneric Cucurbit graft combinations, several structural P-proteins appeared in the recipient phloem exudate, as shown by SDS-PAGE and Coomassie staining. The results effectively demonstrated the direction of transmission was dependent on the combination of heterograft used, with some graft partners taking the role of donor or acceptor, and some able to perform both roles . This has clear implications for choosing of graft partners for GE-modifified root stocks. Fluorescence microscopy of graft junctions has shown sieve tube bridges connecting scion external bundle phloem to internal bundle root stock phloem when mobility was demonstrated. This observation identified physical continuity within the phloem as a prerequisite for mobility of proteins, but did not resolve the selective directionality observed .When two Cucurbit structural P-proteins, PP1 and PP2 were examined in intergeneric grafts, RT-PCR and Northern blots demonstrated that protein products rather than mRNA transcripts were translocated across the graft junctions. In addition to structural proteins, RNA-binding proteins appear to be abundant in the phloem translocation stream. Phloem sap collected and analyzed from four different sources all contained sRNAs of 18–25 nt sizes with various abundance profiles for each species. Fractionation of the phloem sap from pumpkin, cucumber, and lupine also identified a small∼27 kDa protein that bound strongly to 18–24 nt ssRNA. After cloning the pumpkin PSPR1 gene, microinjection studies demonstrated that PSPR1 specifically shuttled a high percentage of the ssRNAs across cell boundaries. In these studies, co-injection and subsequent movement of a 20-kDa fluorescent dextran showed that plasmodesmatal aperture was at least 20 kDa. Apparently, dilated plasmodesmata alone were not sufficient to allow the movement of ssRNAs between cells, since use of another protein shown to increase plasmodesmatal apertures was not sufficient to allow the movement of the ssRNAs .

Given that the ssRNAs were approximately 8 kDa, their lack of movement when KN1 was provided suggested a sequestration mechanism or a more complex ssRNA-binding protein interaction than is currently presumed. In an informative experiment, rice thioredoxin a major phloem sieve tube protein with basic antioxidant functions, was expressed in E. coli and fluorescently labeled with FITC . In tobacco, the labeled, heterologously expressed RPP13-1 protein was observed to migrate beyond the site of injection. However, the similarly purified and labeled E. coli homolog of RPP13-1 was not phloem-mobile under duplicate conditions, suggesting significant sequence or structure requirements for movement. Co-injection of rice RPP13-1 and FITC-labeled dextrans established that RPP13-1 increased the plasmodesmatal size exclusion limit to 9–20 kDa, from ∼1 kDa. Furthermore, two mutants of RPP13-1 that were deficient for mobility were identified and crystal structure prediction studies suggested that charged clusters of residues on the outer surface were responsible for binding and/or transport of RPP13-1 through the companion cell-plasmodesmata complex . Aoki et al. demonstrated the importance of protein structure for mobility using two heat shock proteins , CmHsc70-1 and CmHsc70-2, that had been isolated from pumpkin phloem sap. In microinjection experiments, CmHsc70-1 and CmHsc70-2, interacted with plasmodesmata, increasing the size exclusion limit and thereby, enhanced their own cell-to-cell transport. The C-terminal region of these HSPs potentiated their noncell-autonomous mobility through the plasmodesmata. A gainof-function experiment in which the C-terminal cucumber HSP motif was fused to a human Hsp70 protein established that the fusion protein, but not WT human Hsp70, could move from cellto-cell following micro-injection into pumpkin cotyledons, much like the movement of injected intact CmHsc70-1 and CmHsc70- 2. Interestingly, fusing the HSP C-terminal motif to GFP did notresult in cell-to-cell migration, suggesting that at least in this case, the targeting motif was only active in the context of highly conserved HSPs . Unlike nuclear localization signals or ER-targeting peptides, vascular system targeting peptides may have several different motifs, perhaps suggesting specialized interactions with different families of proteins, and/or selective import/export mechanisms. While targeting motifs appear to be important in regulating mobility, a non-regulated diffusion-based mechanism in the symplast from one cell to another is supported by the observation that protein size influences non-targeted movement of GFP but differences appear to be species- and developmental stage-dependent. Earlier studies indicated that non-regulated diffusion is limited to ∼50 kDa proteins in mature leaves and 60 kDa proteins in developing leaves . Unregulated diffusion-based movement across the sieve tube element–companion cell complex has been observed when CC specific promoters regulate 27 kDa GFP expression.While it was perhaps not surprising to detect the GFP in the vascular system due to the porous end plates of the SE, unloading of the GFP into the mesophyll sink cells was unexpected. Using the same promoter, GFP-fusions as large as 67 kDa subsequently were shown to traffic from CC to SE in root tips, although larger variants were restricted to a zone of cells adjacent to the mature protophloem.