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The use of RNA interference (RNAi) enables the silencing of target genes in plants or plant-dwelling organisms, through the production of double stranded RNA (dsRNA) resulting in altered plant characteristics. Expression of properly synthesized dsRNAs in plants can lead to improved crop quality characteristics or exploit new mechanisms with activity against plant pests and pathogens. Genetically modified (GM) crops exhibiting resistance to viruses or insects via expression of dsRNA have received authorization for cultivation outside Europe. Some products derived from RNAi plants have received a favourable opinion from the European Food Safety Authority (EFSA) for import and processing in the European Union (EU). The authorization process in the EU requires applicants to produce a risk assessment considering food/feed and environmental safety aspects of living organisms or their derived food and feed products. The present paper discusses the main aspects of the safety assessment (comparative assessment, molecular characterization, toxicological assessment, nutritional assessment, gene transfer, interaction with target and non-target organisms) for GM plants expressing dsRNA, according to the guidelines of EFSA. Food/feed safety assessment of products from RNAi plants is expected to be simplified, in the light of the consideration that no novel proteins are produced. Therefore, some of the data requirements for risk assessment do not apply to these cases, and the comparative compositional analysis becomes the main source of evidence for food/feed safety of RNAi plants. During environmental risk assessment, the analysis of dsRNA expression levels of the GM trait, and the data concerning the observable effects on non-target organisms (NTO) will provide the necessary evidence for ensuring safety of species exposed to RNAi plants. Bioinformatics may provide support to risk assessment by selecting target gene sequences with low similarity to the genome of NTOs possibly exposed to dsRNA. The analysis of these topics in risk assessment indicates that the science-based regulatory process in Europe is considered to be applicable to GM RNAi plants, therefore the evaluation of their safety can be effectively conducted without further modifications. Outcomes from the present paper offer suggestions for consideration in future updates of the EFSA Guidance documents on risk assessment of GM organisms.

BackgroundRNA interference (RNAi), a natural regulatory mechanism involved in gene regulation, transgene silencing and virus defense in plants, has been widely used for improving the quality of crops and for conferring resistance to plant pests and pathogens. For the sustained application of RNAi-based genetically modified (GM) crops in agriculture, stable expression of the silencing-inducing hairpin transgene is important. As RNA interference may be accompanied by RNA-directed methylation of homologous DNA, including the RNAi transgene, gene body methylation may spread into the promoter and eventually lead to progressive transcriptional silencing of the RNAi inducing transgene. This would be detrimental for the effective use of RNAi crops and may have safety implications if food allergens or antinutrients are the target. Methods and ResultsIn order to gain knowledge about the molecular structure of transgenes sensitive to gene silencing as well as about the early detection of unstable plant lines, an unstable GM tobacco line was analyzed with respect to the integration structure and methylation patterns. This plant line was selected because it lost reporter gene expression in progeny plants due to post-transcriptional gene silencing (PTGS) and was subject to transcriptional gene silencing (TGS) during prolonged tissue culture propagation of the primary transformant. It was found to contain an inverted repeat integration with transgene constructs separated by the bacterial transposon Tn5393 and to exist in two epigenetic states with respect to transgene methylation.ConclusionsInverted repeat integration containing bacterial transposon sequences may be a possible molecular trigger for the observed silencing of transgene expression in this unstable GM line.

Increasing pest and pathogen challenges as well as having fewer conventional pesticides to employ require innovative and sustainable solutions for plant protection. One group of pesticides that is in the pipeline and is expected to be subject to regulation and risk assessment procedures in the near future, is based on the natural gene silencing mechanism RNA interference (RNAi). These dsRNA-based products can be highly specific for a target organism due to the sequence-specific interaction between effective small interfering RNAs (siRNAs) and a complementary target RNA. General regulatory frameworks for pesticide authorization in the U.S. and in the EU are presented. In addition, production and application procedures and specific characteristics of dsRNA-based pesticides relevant for risk assessment and regulation are considered.

This paper evaluates the potential hazards of food and feed derived from RNAi plants including: adverse changes of plant metabolism; mechanisms and potential for non-target gene silencing in humans and livestock, including gut microbiome; bioinformatics tools for predictionof off-target sequences of interfering RNA; the possible non-specific effects of dsRNA and siRNA in mammals; and the comparison of data requirements for safety assessment of food and feed from RNAi plants and from plants expressing recombinant proteins. It also discusses exposure and RNAi-specific risk assessment.

RNA interference (RNAi) is being developed and exploited to improve plants by modifying endogenous gene expression as well as to target pest and pathogen genes both within plants (i.e. host-induced gene silencing) and/or as topical applications (e.g. spray-induced gene silencing). RNAi is a natural mechanism which can be exploited to make a major contribution towards integrated pest management and sustainable agricultural strategies needed worldwide to secure current and future food production. RNAi plants are being assessed and regulated using existing regulatory frameworks for GMO. However, there is an urgent need to develop appropriate science-based risk assessment procedures for topical RNAi applications within existing plant protection products legislation.

RNA interference (RNAi) occurs naturally in plants and is involved in developmental gene regulation as well as in transposon silencing and defence against viruses. RNAi is initiated by small double-stranded RNAs (dsRNAs) of different origins, eventually leading to the silencing of genes with complementary sequences. Besides its use as a basic tool to study gene function, RNAi has widely been exploited for the generation of pathogen resistant crops and for modifying plant metabolism. For the elucidation of gene functions, silencing-inducing dsRNA is often delivered transiently by means of agroinfiltration. This method is independent of plant regeneration and allows rapid testing of multiple silencing constructs. To get more insight into the applicability of transient gene silencing, we compared a variety of different transient and stable approaches to induce silencing of the β-glucuronidase (gus) transgene by the expression of gus hairpin (hpGus) transcripts. While stable expression of the hairpin always resulted in specific gene silencing, transiently expressed hpGus transcripts could only trigger silencing when the hairpin construct was introduced simultaneously with or prior to the target gene.

RNA interference (RNAi) is a biological process in which double-stranded ribonucleic acid (dsRNA) molecules inhibit protein expression. In recent years, the application of dsRNA has been used in the development of agricultural products for pest control. The 2019 Organisation for Economic Cooperation and Development (OECD) Conference on RNAi Based Pesticides (“the Conference”) brought together academic, industry, and government experts in various aspects of RNAi to discuss the current state of knowledge and topics to help in developing considerations for risk assessment. The Conference focused on environment, with some discussion of human health. Along with presentations on the use of dsRNA-based products in agriculture, government regulation, risk assessment, and a background on the Draft OECD Working Paper on “Considerations for the Environmental Risk Assessment of the Application of Sprayed or Externally Applied dsRNA-Based Pesticides” (“OECD Working Paper”), the Conference included panel discussions from presenters at the end of each session and a larger discussion session with Conference participants on the environmental fate of dsRNA, non-target organism (NTO) risk assessment, and human health risk assessment. This paper summarizes input from presenters and Conference participants during these discussions. Key considerations from these discussions have already been incorporated into the OECD Working Paper, that once finalized and published, will facilitate regulators in evaluating externally applied dsRNA-based products for potential environmental risks.

Transgenes in genetically modified plants are often not reliably expressed during development or in subsequent generations. Transcriptional gene silencing (TGS) as well as post-transcriptional gene silencing (PTGS) have been shown to occur in transgenic plants depending on integration pattern, copy number and integration site. In an effort to reduce position effects, to prevent read-through transcription and to provide a more accessible chromatin structure, a P35S-ß-glucuronidase (P35S-gus) transgene flanked by a scaffold/matrix attachment region from petunia (Petun-SAR), was introduced in Nicotiana tabacum plants by Agrobacterium tumefaciens mediated transformation. It was found that Petun-SAR mediates enhanced expression and copy number dependency up to 2 gene copies, but did not prevent gene silencing in transformants with multiple and rearranged gene copies. However, in contrast to the non-SAR transformants where silencing was irreversible and proceeded during long-term vegetative propagation and in progeny plants, gus expression in Petun-SAR plants was re-established in the course of development. Gene silencing was not necessarily accompanied by DNA methylation, while the gus transgene could still be expressed despite considerable CG methylation within the coding region.

Reciprocal gene exchange between cultivated sugar beet and wild beets in seed production areas is probably the reason for the occurence of weed beets in sugar beet production fields. Therefore, when releasing transgenic sugar beet plants into the environment, gene transfer to wild beets (Beta vulgaris ssp. maritima) has to be considered. In this study the transfer of BNYVV- (beet necrotic yellow vein virus) resistance and herbicide-tolerance genes from two transgenic sugar beet lines that were released in field experiments in 1993 and 1994 in Germany to different wild beet accessions was investigated. In order to evaluate the consequences of outcrossing, manual pollinations of emasculated wild beet plants with homozygous transgenic sugar beet plants were performed. In the resulting hybrids the transgenes were stably inherited according to Mendelian law. Gene expression in leaves and roots of the hybrids was in the same range as in the original transgenic sugar beet plants. Moreover, it was found that in one of the wild beet accessions, transfer and expression of the BNYVV resistance gene did considerably increase the level of virus resistance.

The cultivation of genetically modified (GM) herbicide resistant oilseed rape (Brassica napus) has increased over the past few years. The transfer of herbicide resistance genes via pollen (gene flow) from GM crops to non-GM crops is of relevance for the realisation of co-existence of different agricultural cultivation forms as well as for weed management. Therefore the likelihood of pollen-mediated gene flow has been investigated in numerous studies. Despite the difficulty to compare different experiments with varying levels of outcrossing, we performed a literature search for world-wide studies on cross-fertilisation in fully fertile oilseed rape. The occurrence and frequency of pollen-mediated intraspecific gene flow (outcrossing rate) can vary according to cultivar, experimental design, local topography and environmental conditions. The outcrossing rate from one field to another depends also on the size and arrangement of donor and recipient populations and on the ratio between donor and recipient plot size. The outcrossing levels specified in the presented studies are derived mostly from experiments where the recipient field is either surrounding the donor field (continuous design) or is located as a patch at different distances from the donor field (discontinuous design). Reports of gene flow in Brassica napus generally show that the amount of cross-fertilisation decreases as the distance from the pollen source increases. The evidence given in various studies reveals that the bulk of GM cross-fertilisation occurs within the first 10 m of the recipient field. The removal of the first 10 m of a non-transgenic field facing a GM crop might therefore be more efficient for reducing the total level of cross-fertilisation in a recipient sink population than to recommend separation distances. Future experiments should investigate cross-fertilisation with multiple adjacent donor fields at the landscape level under different spatial distributions of rapeseed cultivars and different cropping systems. The level of cross-fertilisation occurring over the whole field is mainly important for co-existence and has not been investigated in agricultural scale experiments until now. Potential problems with herbicide resistant oilseed rape volunteers arising from intraspecific gene flow can be largely solved by the choice of suitable cultivars and herbicides as well as by soil management.

Integration of foreign genes into plant genomes by the Agrobacterium T-DNA transfer system has been considered to occur at random. Also, the expression level of genes introduced into the plant genome is not predictable and can vary substantially between individual plants transformed with the same gene construct (Odell et al. 1987; Rogers et al. 1985), in some cases to the extent of loss of organ specificity (van der Hoeven et al. 1994). This variability of expression has been referred to as “position effect”. It may partly be due to endogenous regulatory elements like promoters or enhancers close to the integration site and to the chromosomal structure/transcriptional state of the integration site.