Risk Assessment Considerations for Genetically Modified RNAi Plants: EFSA's Activities and Perspective.

Genetically modified plants (GMPs) intended for market release can be designed to induce "gene silencing" through RNA interference (RNAi). The European Food Safety Authority (EFSA) and other international risk assessment bodies/regulatory agencies have taken several actions to determine whether the existing risk assessment approaches for GMPs are appropriate for the risk assessment of RNAi-based GMPs or require complementary or alternative approaches. To our knowledge, at the international level, no dedicated guidelines have been developed for the risk assessment and regulation of RNAi-based GMPs, confirming that existing science-based risk assessment approaches for GMPs are generally considered suitable for RNAi-based GMPs. However, some specificities have been identified for the risk assessment of RNAi-based GMPs. Here, we report on some of these specificities as identified and addressed by the EFSA GMO Panel for the molecular characterisation, food/feed safety assessment and environmental risk assessment of RNAi-based GMPs, using the DvSnf7 dsRNA-expressing maize MON87411 as a case study.

Managing evidence in food safety and nutrition.

Evidence ('data') is at the heart of EFSA's 2020 Strategy and is addressed in three of its operational objectives: (1) adopt an open data approach, (2) improve data interoperability to facilitate data exchange, and (3) migrate towards structured scientific data. As the generation and availability of data have increased exponentially in the last decade, potentially providing a much larger evidence base for risk assessments, it is envisaged that the acquisition and management of evidence to support future food safety risk assessments will be a dominant feature of EFSA's future strategy. During the breakout session on 'Managing evidence' of EFSA's third Scientific Conference 'Science, Food, Society', current challenges and future developments were discussed in evidence management applied to food safety risk assessment, accounting for the increased volume of evidence available as well as the increased IT capabilities to access and analyse it. This paper reports on presentations given and discussions held during the session, which were centred around the following three main topics: (1) (big) data availability and (big) data connection, (2) problem formulation and (3) evidence integration.

Annual post-market environmental monitoring report on the cultivation of genetically modified maize MON 810 in 2016.

Following a request from the European Commission, EFSA assessed the annual post-market environmental monitoring (PMEM) report for the 2016 growing season of the Cry1Ab-expressing maize event MON 810 provided by Monsanto Europe S.A. Partial compliance with refuge requirements was reported in Spain, as observed in previous years. EFSA reiterates the need to achieve full compliance in areas of high maize MON 810 adoption to delay resistance evolution, and therefore advocates increasing the level of compliance in such areas. Resistance monitoring data do not indicate a decrease in susceptibility to the Cry1Ab protein in the field corn borer populations tested in the 2016 season. However, EFSA identified some methodological and reporting limitations pertaining to resistance monitoring that need improvement in future PMEM reports. No complaints related to corn borer infestation of maize MON 810 were received via the farmer alert system during the 2016 cultivation season. EFSA encourages the consent holder to provide more information on this complementary resistance monitoring tool. The data on general surveillance do not indicate any unanticipated adverse effects on human and animal health or the environment arising from the cultivation of maize MON 810. EFSA reiterates its recommendations on the methodology and analysis of farmer questionnaires, and considers that future literature searches on maize MON 810 performed in the context of annual PMEM reports should follow the guidelines given in the 2017 EFSA explanatory note on literature searching. Moreover, EFSA encourages relevant stakeholders to implement a methodological framework that enables the use of existing networks in the broader context of environmental monitoring. EFSA concludes that no new evidence has been reported in the 2016 PMEM report that would invalidate previous EFSA evaluations on the safety of maize MON 810.

Predictive Protein Toxicity and Its Use in Risk Assessment.

In the EU novel proteins used in food or feed are assessed for their potential toxic effects in humans and livestock animals. The discovery of clear molecular features linked to the toxicity of a protein may be an important step towards the use of predictive protein toxicity in risk assessment.

Assessment of the potential integration of the DNA plasmid vaccine CLYNAV into the salmon genome.

The European Commission mandated EFSA to review a new data package provided by the company Elanco, for the possible integration/non-integration of the DNA plasmid vaccine CLYNAV into the genome of Atlantic salmon (Salmo salar) and to indicate whether EFSA agrees with the conclusions drawn by Elanco. The vaccine is injected into fish to confer protection against pancreas disease caused by the salmonid alphavirus. The majority of the experimental data provided by the company was for muscle tissue close to the injection site and for gonadal tissue. EFSA considers that the long persistence of DNA plasmid in muscle tissue close to the injection site and the potential heritability of an integration event in gonad cells support the focus of the assessment on both these tissues. The experimental data did not provide scientifically robust evidence for a true integration event. The company overall concluded that the likelihood of integration is negligible, based on considerations in the context of the company's environmental risk assessment, but did not provide a quantitative value for the rate of integration linked to the term 'negligible'. It is therefore not possible to evaluate this statement specifically with regard to integration rates. EFSA notes that knowledge about homologous and non-homologous integration predicts that integration could occur with certain frequency. Therefore, EFSA has constructed worst-case scenarios leading to upper estimates for possible integration rates of the DNA plasmid vaccine into the Atlantic salmon genome. EFSA concludes that, based on the worst-case scenarios described here and taking into account additional factors decreasing the likelihood of integration, the actual integration rate is likely to be orders of magnitude lower than the upper estimated integration rate calculated in the context of the worst-case scenarios. With the available evidence, the actual integration rate cannot be estimated with more precision.

Guidance for the risk assessment of the presence at low level of genetically modified plant material in imported food and feed under Regulation (EC) No 1829/2003.

This document provides guidance for the risk assessment under Regulation (EC) No 1829/2003 of the unintended, adventitious or technically unavoidable presence in food and feed of low level of genetically modified plant material intended for markets other than in the European Union. In this context, the presence at low level is defined to be maximum 0.9% of genetically modified plant material per ingredient. This guidance is intended to assist applicants by indicating which scientific requirements of Annex II of Regulation (EU) No 503/2013 are considered necessary for the risk assessment of the presence at low levels of genetically modified plant material in food and feed.

Risk assessment of new sequencing information on genetically modified soybean event 305423.

The GMO Panel has previously assessed genetically modified (GM) soybean 305423 as a single event and as part of a two-event stack, 305423 × 40-3-2. These soybean events were found to be as safe as their conventional counterparts and other appropriate comparators with respect to potential effects on human and animal health and the environment. On 23 February 2017, European Commission requested EFSA to analyse new nucleic acid sequencing data and updated bioinformatics data for soybean event 305423 and to indicate whether the previous conclusions of the GMO Panel on the previously assessed GM soybeans remain valid. The new sequencing data indicated a four base pair (bp) difference compared to the sequencing data originally provided: one bp located in the genomic 3' flanking region, two bp located in a gene silencing cassette and one bp in a partial promoter. These bp reported as differences in the new nucleic acid sequencing data on soybean event 305423 were already present in the original plant material used for the risk assessment. Thus, with the exception of bioinformatics analyses, including an off-target search with the dsRNA expression cassette, the studies performed for the risk assessment of the single event soybean 305423 and the two-event stack soybean 305423 × 40-3-2 remain valid. The new sequencing data and the bioinformatic analyses performed on the new sequence including the RNAi off-target search, did not give rise to safety issues. Therefore, EFSA concludes that the original risk assessment of the single soybean event 305423 and the two-event stack soybean 305423 × 40-3-2 remains valid.

Risk assessment of new sequencing information on genetically modified soybean event 40-3-2.

The GMO Panel has previously assessed genetically modified (GM) soybean 40-3-2 as a single event and as part of a two-event stack, 305423 × 40-3-2. These soybean events were found to be as safe as their conventional counterparts and other appropriate comparators with respect to potential effects on human and animal health and the environment. On 4 April 2017, European Commission requested EFSA to analyse new nucleic acid sequencing and updated bioinformatics data for soybean event 40-3-2 and to indicate whether the conclusions of the GMO Panel on the previously assessed GM soybeans remain valid. The new sequencing data indicated that, the sequence of soybean event 40-3-2 as present in the stacked soybean 305423 × 40-3-2 contains an additional nucleotide in the 5' flanking region of a 72 bp additional insert of CP4 EPSPS present in soybean 40-3-2. Re-examination of the original sequencing data of the single soybean event 40-3-2 by the applicant, indicated that this additional nucleotide was already present in the original plant material used for the risk assessment of soybean event 40-3-2. Thus, with the exception of bioinformatics analyses, the studies performed for the risk assessment of the single event soybean 40-3-2 and the two-event stack soybean 305423 × 40-3-2 remain valid. The updated bioinformatic analyses performed on the corrected sequence did not give rise to safety issues. Therefore, EFSA concludes, based on the information provided, that the original risk assessment of the soybean event 40-3-2 as a single and the stacked soybean 305423 × 40-3-2 remains valid.

RNAi-based GM plants: food for thought for risk assessors.

RNA interference (RNAi) is an emerging technology that offers new opportunities for the generation of new traits in genetically modified (GM) plants. Potential risks associated with RNAi-based GM plants and issues specific to their risk assessment were discussed during an international scientific workshop (June 2014) organized by the European Food Safety Authority (EFSA). Selected key outcomes of the workshop are reported here.

EFSA's scientific activities and achievements on the risk assessment of genetically modified organisms (GMOs) during its first decade of existence: looking back and ahead.

Genetically modified organisms (GMOs) and derived food and feed products are subject to a risk analysis and regulatory approval before they can enter the market in the European Union (EU). In this risk analysis process, the role of the European Food Safety Authority (EFSA), which was created in 2002 in response to multiple food crises, is to independently assess and provide scientific advice to risk managers on any possible risks that the use of GMOs may pose to human and animal health and the environment. EFSA's scientific advice is elaborated by its GMO Panel with the scientific support of several working groups and EFSA's GMO Unit. This review presents EFSA's scientific activities and highlights its achievements on the risk assessment of GMOs for the first 10 years of its existence. Since 2002, EFSA has issued 69 scientific opinions on genetically modified (GM) plant market registration applications, of which 62 for import and processing for food and feed uses, six for cultivation and one for the use of pollen (as or in food), and 19 scientific opinions on applications for marketing products made with GM microorganisms. Several guidelines for the risk assessment of GM plants, GM microorganisms and GM animals, as well as on specific issues such as post-market environmental monitoring (PMEM) were elaborated. EFSA also provided scientific advice upon request of the European Commission on safeguard clause and emergency measures invoked by EU Member States, annual PMEM reports, the potential risks of new biotechnology-based plant breeding techniques, evaluations of previously assessed GMOs in the light of new scientific publications, and the use of antibiotic resistance marker genes in GM plants. Future challenges relevant to the risk assessment of GMOs are discussed. EFSA's risk assessments of GMO applications ensure that data are analysed and presented in a way that facilitates scientifically sound decisions that protect human and animal health and the environment.

Inhibition of HIV-1 integrase nuclear import and replication by a peptide bearing integrase putative nuclear localization signal.

BACKGROUND: The integrase (IN) of human immunodeficiency virus type 1 (HIV-1) has been implicated in different steps during viral replication, including nuclear import of the viral pre-integration complex. The exact mechanisms underlying the nuclear import of IN and especially the question of whether it bears a functional nuclear localization signal (NLS) remain controversial. RESULTS: Here, we studied the nuclear import pathway of IN by using multiple in vivo and in vitro systems. Nuclear import was not observed in an importin alpha temperature-sensitive yeast mutant, indicating an importin alpha-mediated process. Direct interaction between the full-length IN and importin alpha was demonstrated in vivo using bimolecular fluorescence complementation assay (BiFC). Nuclear import studies in yeast cells, with permeabilized mammalian cells, or microinjected cultured mammalian cells strongly suggest that the IN bears a NLS domain located between residues 161 and 173. A peptide bearing this sequence -NLS-IN peptide- inhibited nuclear accumulation of IN in transfected cell-cycle arrested cells. Integration of viral cDNA as well as HIV-1 replication in viral cell-cycle arrested infected cells were blocked by the NLS-IN peptide. CONCLUSION: Our present findings support the view that nuclear import of IN occurs via the importin alpha pathway and is promoted by a specific NLS domain. This import could be blocked by NLS-IN peptide, resulting in inhibition of viral infection, confirming the view that nuclear import of the viral pre-integration complex is mediated by viral IN.

Microtubule-associated protein AtMPB2C plays a role in organization of cortical microtubules, stomata patterning, and tobamovirus infectivity.

AtMPB2C is the Arabidopsis (Arabidopsis thaliana) homolog of MPB2C, a microtubule-associated host factor of tobacco mosaic virus movement protein that was been previously identified in Nicotiana tabacum. To analyze the endogenous function of AtMPB2C and its role in viral infections, transgenic Arabidopsis plant lines stably overexpressing green fluorescent protein (GFP)-AtMPB2C were established. The GFP-AtMPB2C fusion protein was detectable in various cell types and organs and localized at microtubules in a punctuate pattern or in filaments. To determine whether overexpression impacted on the cortical microtubular cytoskeleton, GFP-AtMPB2C-overexpressing plants were compared to known microtubular marker lines. In rapidly elongated cell types such as vein cells and root cells, GFP-AtMPB2C overexpression caused highly unordered assemblies of cortical microtubules, a disturbed, snake-like microtubular shape, and star-like crossing points of microtubules. Phenotypically, GFP-AtMPB2C transgenic plants showed retarded growth but were viable and fertile. Seedlings of GFP-AtMPB2C transgenic plants were characterized by clockwise twisted leaves, clustered stomata, and enhanced drought tolerance. GFP-AtMPB2C-overexpressing plants showed increased resistance against oilseed rape mosaic virus, a close relative of tobacco mosaic virus, but not against cucumber mosaic virus when compared to Arabidopsis wild-type plants. These results suggest that AtMPB2C is involved in the alignment of cortical microtubules, the patterning of stomata, and restricting tobamoviral infections.

Movement profiles: a tool for quantitative analysis of cell-to-cell movement of plant viral movement proteins.

Movement proteins (MPs) are virally encoded factors that mediate transport of viral nucleic acid between plant cells. Many MPs are able to move between cells themselves. This feature serves as the basis for evaluation of the transport activity of individual MPs. MPs are transiently expressed as a fusion to autofluorescent proteins such as green fluorescent protein (GFP) in individual epidermal cells of leaves by biolistic delivery. Expressing cells can be directly monitored for subcellular localization and cell-to-cell movement of the MP:GFP fusion protein into neighboring cells by confocal scanning microscopy. During the time frame of transient expression, numerous cells are evaluated at several time points, and the accumulated data are depicted in a graph termed "movement profile." Thus, a movement profile will provide information on the correlation between subcellular localization of the MP in the expressing cell and the efficiency of cell-to-cell transport, the time course and efficiency of targeting of the MP to plasmodesmata, and the translocation efficiency of the MP into neighboring cells.

MPB2C, a microtubule-associated plant factor, is required for microtubular accumulation of tobacco mosaic virus movement protein in plants.

Movement protein binding 2C (MPB2C) is a plant endogenous microtubule-associated protein previously identified as an interaction partner of tobacco (Nicotiana tabacum) mosaic virus movement protein (TMV-MP). In this work, the role of MPB2C in cell-to-cell transport of TMV-MP, viral spread of TMV, and subcellular localization of TMV-MP was examined. To this end, plants with reduced MPB2C levels were generated by a gene-silencing strategy. Local and systemic spread of TMV and cell-to-cell movement of TMV-MP were unimpaired in MPB2C-silenced plants as compared to nonsilenced plants, indicating that MPB2C is not required for intercellular transport of TMV-MP itself or spread of TMV. However, a clear change in subcellular distribution of TMV-MP characterized by a nearly complete loss of microtubular localization was observed in MPB2C-silenced plants. This result shows that the MPB2C is a central player in determining the complex subcellular localization of TMV-MP, in particular its microtubular accumulation, a phenomenon that has been frequently observed and whose role is still under discussion. Clearly, MPB2C mediated accumulation of TMV-MP at microtubules is not required for intercellular spread but may be a means to withdraw the TMV-MP from the cell-to-cell transport pathway.

RNA chaperone activity of protein components of human Ro RNPs.

Ro ribonucleoprotein (RNP) complexes are composed of one molecule of a small noncoding cytoplasmic RNA, termed Y RNA, and the two proteins Ro60 and La. Additional proteins such as hnRNP I, hnRNP K, or nucleolin have recently been shown to be associated with subpopulations of Y RNAs. Ro RNPs appear to be localized in the cytoplasm of all higher eukaryotic cells but their functions have remained elusive. To shed light on possible functions of Ro RNPs, we tested protein components of these complexes for RNA chaperone properties employing two in vitro chaperone assays and additionally an in vivo chaperone assay. In these assays the splicing activity of a group I intron is measured. La showed pronounced RNA chaperone activity in the cis-splicing assay in vitro and also in vivo, whereas no activity was seen in the trans-splicing assay in vitro. Both hnRNP I and hnRNP K exhibited strong chaperone activity in the two in vitro assays, however, proved to be cytotoxic in the in vivo assay. No chaperone activity was observed for Ro60 in vitro and a moderate activity was detected in vivo. In vitro chaperone activities of La and hnRNP I were completely inhibited upon binding of Y RNA. Taken together, these data suggest that the Ro RNP components La, hnRNP K, and hnRNP I possess RNA chaperone activity, while Ro60-Y RNA complexes might function as transporters, bringing other Y RNA binding proteins to their specific targets.

Detection of nanometer-sized particles in living cells using modern fluorescence fluctuation methods.

Nanosized materials are increasingly used in medicine and biotechnology but originate also from various aerosol sources. A detailed understanding of their interaction with cells is a prerequisite for specific applications and appraisal of hazardous effects. Fluorescence fluctuation methods are applied to follow the time-course of the translocation and distribution of fluorescent 20 nm polystyrene nanoparticles with negative surface charges in HeLa cells under almost physiological conditions. The experimental results demonstrate that singular particles enter the cell without significant contribution by endocytotic mechanisms and are distributed within the cytoplasm. Subsequently aggregation is observed, which can be blocked by cytotoxins, like Genistein and Cytochalasin B, interfering with cellular uptake processes. The observed non-active uptake is due to non-specific interactions with the cell surface and could be responsible for distribution of nanometer-sized materials in tissue.

Mimicking carboxyterminal phosphorylation differentially effects subcellular distribution and cell-to-cell movement of Tobacco mosaic virus movement protein.

Phosphorylation of Tobacco mosaic virus movement protein (TMV-MP) at three carboxyterminal Ser/Thr sites negatively regulates TMV-MP gating function and viral spread in Nicotiana tabacum but not in Nicotiana benthamiana, indicating a host dependant inactivation strategy. Here, we examine the effect of mimicking carboxyterminal phosphorylation on cell-to-cell transport of TMV-MP protein itself in host plants Nicotiana clevelandii, N. benthamiana, Nicotiana glutinosa and N. tabacum. Since TMV-MP transport function was inactivated only in N. tabacum, this host was chosen to explore the contribution of individual carboxyterminal phosphorylation sites. Selective mimicking of phosphorylation at one site enhances TMV-MP cell-to-cell transport, whereas a negative effect requires mimicking of phosphorylation at two or three sites. Potentially, during viral infection in N. tabacum, MP phosphorylation may occur sequentially: first, MP phosphorylation at a single site might ensure effective viral movement; only thereafter, further phosphorylation events may lead to inactivation of TMV-MP transport function.

Bioimprinted QCM sensors for virus detection-screening of plant sap.

Surface imprinting techniques on polymer-coated quartz-crystal microbalances (QCM) have been used to detect tobacco mosaic viruses (TMV) in aqueous media. Molecularly imprinted polymers (MIP), tailor-made by self organisation of monomers around a template (TMV), were generated directly on the gold electrodes. Imprinted trenches on the polymer surface mimicking the shape and surface functionality of the virus serve as recognition sites for re-adsorption after washing out of the template. The sensors are applicable to TMV detection ranging from 100 ng mL(-1) to 1 mg mL(-1) within minutes. Furthermore, direct measurements without time-consuming sample preparation are possible in complex matrices such as tobacco plant sap.