Marijn Knip; Maria E. Constantin; Hans Thordal-Christensen

“ The biggest revelations may come in the form of factors that are involved in RNA export from the producing cell, its physical extracellular transport and its import into target cells. These mechanisms are very enigmatic at this point, and we can only speculate by comparison to analogous phenomena within organisms. So far, HIGS has focused on the function of target genes, but we foresee that it could be used to dissect the process of trans-kingdom RNA-silencing transfer by setting up carefully designed screens. ”

“ RNA secretion is believed to generate the extracellular RNA that is transported between hosts and parasites. For instance, T. cruzi–produced vesicles are loaded with an Argonaute protein and sRNAs, which potentially influence host gene expression [23] . Extracellular vesicular transport of sRNA is a candidate mechanism to facilitate trans-kingdom RNA transfer between other species as well. Plant leaves attacked by the powdery mildew fungus deliver both shedding vesicles and exosomes at the fungal attack site, and interference of the latter hampers plant defense [53] – [54] . ”

“ The examples in Table 1 and Figure 1 suggest that there is a framework, widely conserved in eukaryotes, that allows production, transfer, and perception of RNA signals between very distantly related organisms across the branches of different kingdoms of the tree of life. In the HIGS examples, sRNA-producing constructs are designed to target genes in the interacting organisms, often of different kingdoms. However, evidence is available that natural, endogenous sRNA also target genes in a trans-kingdom manner. ”

“ Mobility of sRNA molecules within organisms is a well-known phenomenon, facilitating gene silencing between cells and tissues. sRNA signals are also transmitted between organisms of the same species and of different species. Remarkably, in recent years many examples of RNA-signal exchange have been described to occur between organisms of different kingdoms. These examples are predominantly found in interactions between hosts and their pathogens, parasites, and symbionts. ”

“ On the right, transfer of sRNA in shedding vesicles (SV) , which are generated directly from the PM, is depicted. How RNA is loaded into SV is unknown. The recipient cell takes up the sRNA after fusion of the SV with the PM in a process that requires fusogenic proteins. SVs might be taken up in an endocytosis-dependent manner and exosomes might be taken up in a membrane fusion event. In the cytosol of the recipient cell, the sRNA is recognized by the RNAi machinery and triggers gene silencing, either through post-transcriptional gene silencing (PTGS) or transcriptional gene silencing (TGS) . ”

“ Ideas of how free sRNA might be taken up by target cells come from studies in C. elegans, which revealed key components for uptake of free extracellular dsRNA. These are the transmembrane protein channel, Systemic RNAi-Deficient (SID) -1 [40] , and the single-pass transmembrane receptor, SID-2 [37] . It is thought that free dsRNA is internalized from the intestinal lumen by a SID-2–receptor mediated endocytosis, after which dsRNA can escape from the endosome into the cytoplasm by SID-1 (Figure 2) [37] . Unlike SID-1, which is conserved in animals, SID-2 is poorly conserved [41] . ”

“ Here, the B. cinerea fungus produces 73 sRNAs with potential targets in Arabidopsis and tomato. Of these, three 21-nt retrotransposon-derived siRNAs target four plant transcripts important for pathogen defense, despite three to five mismatches. This increases the chance of sRNAs to be functional in an interacting organism and leads to speculation on whether such mechanisms have arisen fortuitously. Since the presence of matching sRNAs can provide a clear selective advantage, it is likely not to be a random occurrence. ”

“ Trans-kingdom RNA signaling is now a documented phenomenon with intriguing implications for our understanding of biological interactions. Similar to ideas proposed by Sarkies and Miska on “social RNA” [41] , the presented evidence for trans-kingdom RNAi suggests that genetic interaction between organisms at the RNA level is common. Organisms become genetically programmed according to endogenous and environmental input. Hitherto, we have known these to include physical and chemical stimuli from other organisms. ”

“ It is plausible that in trans-kingdom transfer, sRNA signals will also be amplified in their target cells to be able to induce gene silencing. It has been suggested that organisms ingesting plant material that contains sRNAs amplify these signals in the cells lining the digestive gut [26] . ”

“ We believe that RNA signals that travel between organisms rely on similar mechanisms as those observed for extracellular transport within an organism (Figure 2) . In humans, sRNAs have been found to be present in extracellular fluids. This is a hostile environment for RNA, which needs to be protected from degradation. Exported sRNA has been found inside extracellular vesicles and in association with High-Density Lipoprotein (HDL) cholesterol particles (Figure 2) [33] – [34] . ”

“ Even though convincing, the data available for trans-kingdom RNAi are fragmented and mostly based on input sRNA sequences and phenotypic effects in the receiving organisms. No example provides information for the whole RNA signaling chain, which conceptually should involve sRNA production, secretion, uptake, perception, amplification and manifestation. ”

“ In vitro cultivation of one of the two organisms following the interaction overcomes the obvious contamination problem when determining presence of transferred sRNA. Weiberg et al. [19] study the plant RNAi machinery to support the hypothesis that the fungal-induced plant gene suppression indeed is caused by the fungus sRNA functioning in the plant. Plant RNAi in general is required for fungal resistance, and by knocking out DCL1, Weiberg et al. ”

“ Through evolution, hosts and their invaders have undergone amazing arms races involving appearances of receptors and downstream response mechanisms for detection and defense on the host side, and, e.g., defense suppressing effectors on the side of the invaders. Hitherto, the interactions are described to be based on transfer of proteins and low-molecular-weight molecules between the organisms. ”

“ However, now we see that genetic programming also is influenced by genetic stimuli in the form of environmental RNA. Obvious biological niches where such RNA communication could be prolific would be in the soil, where evidence for this already has been seen between plants and symbiotic mycorrhizal fungi [16] , and on the skin and in the gut of animals. ”

“ When first discovered, extracellular vesicles were merely considered to result from stressed cells shedding waste products [44] . However, after the discovery of nucleic acid sequences in these vesicles, they were considered much more interesting, as this suggested that they might facilitate genetic signaling [33] . sRNA-containing vesicles in human plasma are either shedding vesicles, formed by outward-budding at the plasma membrane, or exosomes, formed by inward budding of intraluminal vesicles (ILV) at endosomal membranes of multi-vesicular bodies (MVBs) . ”

“ Another way of obtaining host resistance may be based on the fact that pathogens and perhaps parasites also make use of sRNAs in the interaction with hosts. Therefore, the host genes targeted by them could be re-coded to make them insensitive. ”

“ Kogel, K-H & van Bel, AJE, “Multivesicular bodies participate in a cell wall-associated defence response in barley leaves attacked by the pathogenic powdery mildew fungus”, Cell Microbiol 8: 1009–1019, [ [pmid:16681841 AnQHückelhovenRKogelK-Hvan BelAJE] ] ↑ Zhang, W; Pedersen, C; Kwaaitaal, M; Gregersen, PERL & Mørch, SM, “Interaction of barley powdery mildew effector candidate CSEP0055 with the defence protein PR17c”, Mol Plant Pathol 13: 1110–1119, [ [pmid:22863200 ZhangWPedersenCKwaaitaalMGregersenPERLMørchSMet al] ] ↑ Molnar, A ”