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Editorial on the Research Topic: Advances and Challenges of RNAi Based Technologies for Plants—Volume 2

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.

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.

Facing current climate challenges and drastically reduced chemical options for plant protection, the exploitation of RNA interference (RNAi) as an agricultural biotechnology tool has unveiled possible new solutions to the global problems of agricultural losses caused by pests and other biotic and abiotic stresses. While the use of RNAi as a tool in agriculture is still limited to a few transgenic crops, and only adopted in restricted parts of the world, scientists and industry are already seeking innovations in leveraging and exploiting the potential of RNAi in the form of RNA-based biocontrol compounds for external applications. Here, we highlight the expanding research and development pipeline, commercial landscape and regulatory environment surrounding the pursuit of RNA-based biocontrol compounds with improved environmental profiles. The commitments of well-established agrochemical companies to invest in research endeavours and the role of start-up companies are crucial for the successful development of practical applications for these compounds. Additionally, the availability of standardized guidelines to tackle regulatory ambiguities surrounding RNA-based biocontrol compounds will help to facilitate the entire commercialization process. Finally, communication to create awareness and public acceptance will be key to the deployment of these compounds. © 2019 Society of Chemical Industry.

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.

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.

Aufgrund von verschiedenen Herausforderungen und Restriktionen sind im Bereich Pflanzenschutz neue Lösungsansätze gefordert, um zukünftigen Schädlingsproblemen zu begegnen. Eine mögliche Alternative zu konventionellen chemischen Pflanzenschutzmitteln sind Produkte, die auf dem natürlicherweise in Tieren und Pflanzen vorhandenen Kontroll- und Abwehrmechanismus der RNA-Interferenz (RNAi) beruhen. Praktische Verwendung fand RNAi bisher unter anderem bei der Erzeu­gung gentechnisch veränderter Pflanzen mit Virus­resistenz und mit eingebautem Schutz gegen Insekten. In den letzten Jahren hat aber auch der Anwendungs­bereich von extern applizierten RNA-basierten Pflanzenschutzmitteln zunehmend an Bedeutung gewonnen, und einige derartige Produkte sind schon weit in der Entwicklung. Um den sicheren und nachhaltigen Einsatz dieses neuen, durch hohe Spezifität gekennzeichneten Wirkprinzips im Pflanzenschutz zu ermöglichen, sind geeignete Regulierungsstandards und angepasste Testmethoden zur Risikoanalyse notwendig., Journal für Kulturpflanzen, Bd. 73 Nr. 1-2 (2021)

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.

A joint assessment of two separate approaches investigated the occurrence of volunteer oilseed rape (Brassica napus L.; OSR) as affected by cultivar, field history and environment. Approach I comprised surveys for volunteers on >100 farmers’ fields in Germany in the years 2009 and 2010. Volunteers were assigned to the cultivars grown in previous years by inter-simple sequence repeat-PCR and cluster analysis. High-dormancy cultivars resulted in 0–7 and low-dormancy cultivars in 0–1.3 volunteers m−2. Highest numbers originated from the most recent harvests 2007 and 2006. Approach II was a meta-analysis based on 116 data sets from field trials and farmers’ fields in Germany to evaluate and to rank the impact of management factors on the soil seedbank and volunteers in following crops. Varietal disposition to seed dormancy turned out to be the significantly most relevant factor. The contribution of varietal dormancy to variation in the soil seedbank and of volunteers in the 1st and 2nd following crop was 1.2–2.3 times as great as the contribution of post-harvest tillage. Up to 45% of the variation in the observations originated from factors that can be controlled by human actions on a given location in a given year. The overall analysis confirmed the results from independent short-term trials and showed that both agronomists and breeders need to contribute to reducing OSR volunteers.

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.

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.

Pod dehiscence of oilseed rape (OSR) occurs before and during harvest, resulting in seed losses and in seed persistence in the soil for several years. Persisting seeds can emerge as volunteers in subsequent crops. Volunteers with deviating traits and especially genetically modified (GM) oilseed rape volunteers can impair the product quality of an oilseed rape crop. Under experimental field conditions seed persistence and volunteer abundance were found to be efficiently controlled by delayed post-harvest tillage and selection of cultivars with low secondary seed dormancy. To investigate the long-term factors affecting volunteer abundance in farmer-managed fields, an on-farm survey was carried out in winter oilseed rape fields at different locations in Germany, using artificial sowing gaps as one method to detect putative volunteers. Molecular marker analysis (ISSR-PCR), which was performed for 11 out of 31 fields, confirmed that most of the plants identified within sowing gaps were indeed volunteers. In fields with low OSR cropping frequency high percentages of volunteers could be assigned to previously cultivated varieties. Results indicate that seeds of varieties with high secondary dormancy (HD) persist in the fields for longer times and in larger numbers than seeds of varieties with low secondary dormancy. However, cultivation of HD varieties did not necessarily result in high volunteer abundance, as location and management factors have further impact on long-term seed survival. Nevertheless, with respect to GM oilseed rape cultivation proper variety selection will be an important means to assure co-existence with conventional oilseed rape.

Genetically modified oilseed rape is currently grown on about 23 % of the global oilseed rape acreage. In order to separate transgenic and non-transgenic oilseed rape production and to ensure co-existence of different agricultural cultivation schemes, as is specified by the European legislation, confinement measures have to be defined. Pollen-mediated gene flow is the most important means by which transgenes are dispersed between fields. In contrast to the majority of the previous investigations the objective of this study was to assess the extent of gene flow in the case of multiple pollen-donor fields. A high erucic acid rape genotype was used as biochemical marker for the quantification of outcrossing into a low erucic acid oilseed rape variety. Outcrossing data were obtained from two experimental locations. As expected, multiple pollen sources in a fragmented landscape can result in high gene transfer frequencies, thus requiring larger isolation distances than a field design with a single pollen donor source. The results of the study are transferable to homozygous transgenic oilseed rape varieties.

The major genetic factors affecting the expression of newly introduced genes in transgenic plants including epigenetic effects are summarized. Examples of (trans)gene silencing and the genetic signals involved are given. Based on current knowledge, several strategies to generate stable transgenic lines can be followed. Although initial laboratory and field tests over few generations allow good predictions about the long-term expression stability of single transgenic lines, the combination of different events by genetic crossing raises new questions. As different transgenic lines often carry the same or similar promoter elements and as coding regions may have sequence homologies, new epigenetic interactions can arise, thus justifying thorough expression testing of stacked transformation events obtained through cross-breeding.

The genomic structure surrounding a T-DNA integration site in a transgenic petunia plant, which shows deregulation of a root-specific promoter, was investigated. We have already demonstrated that T-DNA integration in this transformant (P13) had occurred close to a scaffold/matrix attachment region (S/MAR). A major question regarding the observed promoter leakiness was whether the T-DNA had integrated into the centre or at the border of the Petun-SAR and whether other regulatory elements are located within this genomic region. While small rearrangements were shown to occur during T-DNA integration in agreement with other reports, we find indications of the presence of a SINE retroposon – an apparent landmark for recombinogenic targets – at the integration site. Binding assays to both plant and animal nuclear scaffolds, supported by biomathematical analyses, reveal that the T-DNA is definitely located at the border of a strong S/MAR, which is in agreement with current models on the structure of integration sites. These results, together with a developmentally regulated leaf-specific enhancer effect of the Petun-SAR on gene expression in transgenic tobacco plants, indicate that the Petun-SAR demarcates the right border of a chromatin domain with genes predominantly active in leaves.

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.

International audience; Canola ([i]Brassica napus[/i]) varieties containing inherent restrictions on gene flow may address problems of seed- and pollen-mediated gene flow. The aim of this study was to explore the effectiveness of 1) low-dormancy varieties in avoiding GM soil seedbanks and volunteers, and 2) cleistogamous (clg) genotypes to avoid outcrossing.GM and non-GM varieties with altered seed ingredients had a wide dormancy range that turned out to be genetically determined. Quantitative trait locus (QTL) mapping identified four QTLs, which together explained 35% of the phenotypic variance. The fiber content of the seeds appeared to additionally affect seed dormancy. A qualitative polymerase chain reaction (PCR) assay was developed for clg canola by cloning and sequencing of clg1-specific DNA fragments. Detection of characteristic bands in 0.1% clg spiked samples illustrated the sensitivity of this method.The inherent traits of low dormancy and cleistogamy may be feasible tools to minimize gene flow and may be considered for coexistence of GM and non-GM canola.

When growing different transgenic herbicide-resistant oilseed rape cultivars side by side, seeds with multiple herbicide resistance can arise, possibly causing problems for the management of volunteer plants. Large-scale field experiments were performed in the years 1999/2000 and 2000/2001 in order to investigate the frequencies and the consequences of the transfer of herbicide resistance genes from transgenic oilseed rape to cultivars grown on neighboring agricultural fields. Transgenic oilseed rape with resistance to glufosinate-ammonium (LibertyLink, LL) and with glyphosate resistance (RoundupReady, RR), respectively, was sown in adjacent 0.5 ha plots, surrounded by about 8 ha non-transgenic oilseed rape. The plots and the field were either in direct contact (0.5 m gap width) or they were separated by 10 m of fallow land. Seed samples taken during harvest in the transgenic plots at different distances were investigated for progeny with resistance to the respective other herbicide. It was found that outcrossing frequencies were reduced to different extents by a 10 m isolation distance. In addition to pollen-mediated transgene flow as a result of outcrossing, we found considerable seed-mediated gene flow by adventitious dispersal of transgenic seeds through the harvesting machine. Volunteer plants with double herbicide resistance emerging in the transgenic plots after harvest were selected by suitable applications of the complementary herbicides Basta and Roundup Ultra. In both years, double-resistant volunteers were largely restricted to the inner edges of the plots. Expression analysis under controlled laboratory conditions of double-resistant plants generated by manual crosses revealed stability of transgene expression even at elevated temperatures. Greenhouse tests with double-resistant oilseed rape plants gave no indication that the sensitivity to a range of different herbicides is changed as compared to non-transgenic oilseed rape.

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.

35 ref.; International audience