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Public Policy Update rNAi BIOTEChNOLOGY DISCuSSIONS ENCOurAGED Jan Leach, Public Policy Board Chair, [email protected], and Elizabeth Grabau, Public Policy Board Member, [email protected] Biotechnology for improved management of plant diseases has the potential to reduce dependency of commercial and noncommercial growers on pesticides and to enhance food security for an increasing global population . APS has long opposed regulating food, feed, and fiber products based solely on the particular technology that was used to create the varieties/cultivars (www .apsnet .org/members/outreach/ppb/ positionstatements/Pages/BiotechnologyPositionStatement .aspx) . However, regulation of newly emerging approaches to crop modification, such as RNAi technology to silence expression of plant, pathogen, or insect genes, is now being discussed . The APS Public Policy Board (PPB) will hold a hot topics discussion session on RNAi use for biotechnology at the upcoming APS-CPS Joint Meeting . As a prelude to the meeting, and to encourage discussions on the topic among APS members now, we asked Vicki Vance, a virologist in the Biological Sciences Department of the University of South Carolina, to provide the following article as an introduction to RNAi and to summarize the pros and cons of using the approach for biotechnology purposes . Please feel free to add to the scientific discussion of the process by e-mailing any PPB member (www . apsnet .org/members/directories/Pages/PPB .aspx) with your comments or plan to attend the session during the joint meeting . rNAi Biotechnology: Pros and Cons for Crop Improvement Vicki Vance, University of South Carolina, [email protected] risk Assessment Genetically modified (GM) crops are subject to an environmental risk-assessment process designed to evaluate their safety, and approval by the relevant regulatory agency is required before commercial release of GM crops in all countries where these crops are currently grown, including the United States . The majority of approved GM crops have been transformed to produce one or more novel proteins that confer useful agronomic traits such as pathogen or insect resistance . However, a number of genetic engineering approaches use RNAi technology to confer useful traits to plants, and the measures needed to assess the safety of these approaches are currently under investigation . What exactly is RNAi? It stands for “RNA interference” and it refers to a set of related processes in which small regulatory RNAs direct sequence-specific repression of gene expression . RNAi pathways are evolutionarily ancient and various versions of these processes are found in virtually all eukaryotic organisms . All RNAi processes begin with an RNA precursor that is completely or partially double-stranded (dsRNA) . This precursor is then processed to produce the active small RNA by an enzyme called Dicer (or Dicer-like) . There are two major kinds of small RNAs that are currently used in biotechnological applications in plants: 1) short interfering RNAs (siRNAs), and 2) 40 March 2014 microRNAs (miRNAs) . The siRNA pathway is induced by invasive nucleic acids, such as viruses, transposons, or transgenes, and serves to defend the host plant against such invaders (Alvarado and Scholthof, 2009) . In contrast, miRNAs are produced by an endogenous pathway that is used to control an organism’s own gene expression (Vazquez et al ., 2010) . Once produced, the small RNA (either siRNA or miRNA) incorporates into a protein complex called RISC, where it serves as a guide to find target messenger RNAs using the base-pairing rules . The targeted messages are either degraded or their translation is blocked—either way, the encoded protein is not produced and the gene is said to be silenced . engineered to produce a transgene in which the endogenous miRNA in the precursor is replaced with one that is complementary to the targeted messenger RNA . The modified precursor is then processed to produce a novel miRNA called an artificial miRNA (Ossowski, et al ., 2008) . This approach produces a single small RNA rather than a population of small RNAs . The miRNA approach may be less effective in silencing the target gene (because only one small RNA is produced rather than a population), but it is more selective and less likely to result in off-target silencing . Artificial miRNAs thus provide an elegant method to silence genes in a very specific manner (Sablok et al ., 2011) sirNAs versus mirNAs Applications of rNAi Biotechnology (PrOs) RNAi technology differs depending on whether the approach relies on siRNAs or miRNAs . In a siRNA-based approach, a transgene is designed to produce dsRNA directly—these are called hairpin transgenes or RNAi constructs (Waterhouse et al ., 1998) . They produce a population of siRNAs that represents the entire dsRNA region and has complementarity to a chosen target messenger RNA . The siRNA population is generally very effective at silencing the target gene because there are many different small RNAs all targeting the same transcript . A miRNAbased approach is more precise . A natural endogenous miRNA gene is genetically RNAi has proven to be a powerful approach for silencing genes to improve agronomic traits in crop plants . The approach has been used to target specific plant genes for improved traits such as modified oil content in soybeans, increased lysine content in corn, and reduced caffeine content in coffee and to provide resistance to numerous viral diseases by targeting viral transcripts produced in planta (reviewed in Frizzi and Huang, 2010) . Remarkably, this same technology can be used to target genes in pest organisms that feed on plants, such as root-knot nematodes, corn rootworm, and bollworm (Baum et al ., 2007; Huang et al., 2006; Mao et al., 2007; Nowara et al., 2010). Thus, this technology provides a novel and potent new kind of pesticide. Limitations of RNAi Biotechnology (CONs) Because siRNA-based RNAi is an antiviral defense mechanism, many plant viruses encode proteins that suppress silencing (Burgyan, 2008; Alvarado and Scholthof, 2009). Furthermore, plant viral infections are very common. As a result, it is possible that viral infection in the field might suppress transgene-induced RNAi. In addition, suppressors of silencing from unrelated viruses are structurally unrelated and employ a variety of mechanisms to block silencing, making it difficult to engineer broad-spectrum protection against this problem. A second potential limitation arises from the fact that siRNAs comprise a population of molecules representing the entire sequence of the dsRNA trigger. Although this sequence heterogeneity could make it easy to silence a family of related genes with only one construct, it also opens the door to off-target effects, in which genes with regions of homology to the intended target get silenced unintentionally. A third potential limitation stems from the fact that post-transcriptional silencing in plants is mobile. It can be induced locally and will then spread throughout the plant. Thus, siRNA-based RNAi strategies might not be suitable for some applications requiring tissue-specific silencing of genes. The use of artificial miRNAs may alleviate some of these concerns, but they also bring their own set of limitations. On one hand, artificial miRNAs provide a much more specific way to silence genes than do siRNAs because miRNAs use only a single 21- or 22-nt sequence to identify the target, whereas siRNAs comprise a population of sequences. Thus, there is a reduced chance of off-target effects with miRNA-based RNAi, making it easier to target individual genes, even in a closely related gene family. Artificial miRNAs also provide a better option for tissue-specific silencing because miRNA-directed silencing tends not to move throughout the plant. However, a potential limitation of artificial miRNA-based RNAi is that the silencing might not be very durable because only a single 21- or 22-nt specificity determinant is involved. Escape from miRNA-directed silencing via mutation of the target, therefore, would be easier than in siRNA-directed silencing, in which a much larger sequence is targeted. Using two (or more) different GM Crop Strategies GM Crop Strategies A. PROTEIN PRODUCT Sense Transgene B. RNA INTERFERENCE Hairpin or miRNA Transgene transcription transcription mRNA dsRNA translation dicer Protein siRNA or miRNA Protein function confers desired trait Silence plant genes Silence genes in organism that eats plant Figure 1. Strategies for genetically modified (GM) crops. A) Most approved GM crops are based on producing a protein product. The plant is engineered with a sense transgene that is transcribed to produce a messenger RNA (mRNA) and then translated to produce the encoded protein. The protein is the functional element in this approach as well as the novel element from a risk assessment standpoint. B) In RNA interference (RNAi) strategies, the plant is engineered with either a hairpin transgene or with a transgene that that encodes a microRNA (miRNA) precursor. These transgenes produce RNA that is either completely double stranded (hairpin transgene) or partially double stranded (miRNA precursor). The small RNAs (either siRNAs or miRNAs) are the functional elements in this approach and the novel elements from the standpoint of safety assessment. artificial miRNAs against the target is a strategy that has been used to overcome this problem. Safety concerns for RNAi biotechnology (CONs). The current risk assessment procedures were designed to address potential risks posed by genetically engineered plants that encode and produce novel proteins. In the case of RNAi strategies, however, the genetically engineered plants encode and produce novel siRNAs or miRNAs, as well as their dsRNA precursors, rather than proteins. A new safety concern that is unique to RNAi approaches has recently been raised by a report that miRNAs made in plants are taken up by humans and other mammals when they eat plants (Zhang et al., 2012). The paper presented evidence that endogenous plant miRNAs from plant-derived foods are absorbed by cells of the mammalian GI tract and packaged into microvesicles, which protects them from degradation. The miRNAs are then trafficked via the bloodstream to a variety of tissues, where they are capable of regulating mammalian genes. The report has generated a good deal of excitement because of the implications with regard to uptake of therapeutic small RNAs to treat human disease. However, it also has implications for the safety of RNAi crop plants for human consumption. Many of these GM crops will harbor populations of small RNAs that don’t exist in nature and may have potential to suppress mammalian gene expression in unexpected ways. This is perhaps more of a concern for RNAi-based pesticides because that approach produces small RNAs designed to kill the targeted organism when it feeds on the plant. Subsequent publications have challenged whether uptake of ingested plant small RNAs occurs to any significant extent (Snow et al., 2013; Witwer et al., 2013). However, in view of the novel potential risks of RNAi-based GM crops, it seems that a careful re-evaluation of current risk assessment strategies is warranted in order to ensure the safety of RNAi crop plants. Biotechnology continued on page 42 Phytopathology News 41