EFSA guidelines on environmental risk assessment of GM animals, including insects

Future applications for the marketing of genetically modified organisms (GMOs) in the EU may include food/feed products derived from genetically modified (GM) animals, and the release of GM animals, including insects, into the environment. Efforts towards the development of GM insects to control insect vectors of human diseases and manage agricultural pests have progressed substantially with various GM insect × trait combinations in the development pipeline. As a proactive measure, the scientific GMO Panel of the European Food Safety Authority (EFSA) has developed guidelines on: (1) the risk assessment of food/feed derived from GM animals including animal health and welfare aspects; and (2) the environmental risk assessment (ERA) of living GM animals, including insects, released into the environment for commercial purposes. The latter assists applicants in the preparation and presentation of their applications by describing the elements and data requirements for a structured ERA of GM insects consistent with the current Directive 2001/18/EC. A dedicated Working Group (WG) was involved in the elaboration of the ERA guidelines on GM insects, which underwent a public consultation before their finalisation. Relevant comments received were considered by the WG. The WG also took into account the external scientific report on GM insects commissioned by EFSA (Benedict et al., 2010). This report provided background information by mapping relevant fields of expertise and identified essential elements to be considered when performing an ERA of GM insects. Content and stakeholder involvement for the EFSA guidelines are presented

RNAi-based GM plants: Food for thought for risk assessors

SummaryRNA 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

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

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Results</p> <p>Here, we studied the nuclear import pathway of IN by using multiple <it>in vivo </it>and <it>in vitro </it>systems. Nuclear import was not observed in an importin α temperature-sensitive yeast mutant, indicating an importin α-mediated process. Direct interaction between the full-length IN and importin α was demonstrated <it>in vivo </it>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.</p> <p>Conclusion</p> <p>Our present findings support the view that nuclear import of IN occurs via the importin α 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.</p

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) No1829/2003 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 AnnexII of Regulation (EU) No503/2013 are considered necessary for the risk assessment of the presence at low levels of genetically modified plant material in food and feed. (C) 2017 European Food Safety Authority. EFSA Journal published by John Wiley and Sons Ltd on behalf of European Food Safety Authority

Phosphorylation of bamboo mosaic virus satellite RNA (satBaMV)-encoded protein P20 downregulates the formation of satBaMV-P20 ribonucleoprotein complex

Bamboo mosaic virus (BaMV) satellite RNA (satBaMV) depends on BaMV for its replication and encapsidation. SatBaMV-encoded P20 protein is an RNA-binding protein that facilitates satBaMV systemic movement in co-infected plants. Here, we examined phosphorylation of P20 and its regulatory functions. Recombinant P20 (rP20) was phosphorylated by host cellular kinase(s) in vitro, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and mutational analyses revealed Ser-11 as the phosphorylation site. The phosphor-mimic rP20 protein interactions with satBaMV-translated mutant P20 were affected. In overlay assay, the Asp mutation at S11 (S11D) completely abolished the self-interaction of rP20 and significantly inhibited the interaction with both the WT and S11A rP20. In chemical cross-linking assays, S11D failed to oligomerize. Electrophoretic mobility shift assay and subsequent Hill transformation analysis revealed a low affinity of the phospho-mimicking rP20 for satBaMV RNA. Substantial modulation of satBaMV RNA conformation upon interaction with nonphospho-mimic rP20 in circular dichroism analysis indicated formation of stable satBaMV ribonucleoprotein complexes. The dissimilar satBaMV translation regulation of the nonphospho- and phospho-mimic rP20 suggests that phosphorylation of P20 in the ribonucleoprotein complex converts the translation-incompetent satBaMV RNA to messenger RNA. The phospho-deficient or phospho-mimicking P20 mutant of satBaMV delayed the systemic spread of satBaMV in co-infected Nicotiana benthamiana with BaMV. Thus, satBaMV likely regulates the formation of satBaMV RNP complex during co-infection in planta

Bean dwarf mosaic virus : a model system for the study of viral movement

Bean dwarf mosaic virus -[Colombia:1987] (BDMV-[CO:87]) is a single-stranded plant DNA virus, a member of the genus Begomovirus of the family Geminiviridae .BDMV virions are twinned incomplete isosahedra measuring 18 × 30 nm. The viral particle is composed of 110 subunits of coat protein, organized as 22 pentameric capsomers. Each subunit has a molecular mass of ∼29 kDa. BDMV possesses two DNA components (designated DNA-A and DNA-B), each ∼2.6 kb in size.The natural and most important host of BDMV is the common bean ( Phaseolus vulgaris ). Nicotiana benthamiana is often used as an experimental host. Common bean germplasm can be divided into two major gene pools: Andean materials, which are mostly susceptible to BDMV, and Middle American materials, which are mostly resistant to BDMV.The symptom intensity in common bean plants depends on the stage of infection. Early infection of susceptible bean seedlings will result in severe stunting and dwarfing, leaf distortion and mottling or mosaic, as well as chlorotic or yellow spots or blotches. BDMV-infected plants usually abort their flowers or produce severely distorted pods. Late infection of susceptible plants or early infection of moderately resistant genotypes may show a mild mosaic, mottle and crumpling or an irregular distribution of variegated patches.As a member of the Begomovirus group, BDMV is transmitted from plant to plant by the whitefly Bemisia tabaci . BDMV is a nonphloem-limited virus and can replicate and move in the epidermal, cortical and phloem cells. As a nonphloem-limited virus, it is sap-transmissible.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/79128/1/j.1364-3703.2010.00619.x.pd

Viral protein targeting to the cortical endoplasmic reticulum is required for cell–cell spreading in plants

Sorting signal-mediated oligomerization and localization of the viral protein TGBp3 to curved ER tubules is essential for viral movement between cells in plants

Intracellular Transport of Plant Viruses: Finding the Door out of the Cell

Plant viruses are a class of plant pathogens that specialize in movement from cell to cell. As part of their arsenal for infection of plants, every virus encodes a movement protein (MP), a protein dedicated to enlarging the pore size of plasmodesmata (PD) and actively transporting the viral nucleic acid into the adjacent cell. As our knowledge of intercellular transport has increased, it has become apparent that viruses must also use an active mechanism to target the virus from their site of replication within the cell to the PD. Just as viruses are too large to fit through an unmodified plasmodesma, they are also too large to be freely diffused through the cytoplasm of the cell. Evidence has accumulated now for the involvement of other categories of viral proteins in intracellular movement in addition to the MP, including viral proteins originally associated with replication or gene expression. In this review, we will discuss the strategies that viruses use for intracellular movement from the replication site to the PD, in particular focusing on the role of host membranes for intracellular transport and the coordinated interactions between virus proteins within cells that are necessary for successful virus spread

ANK, a Host Cytoplasmic Receptor for the Tobacco mosaic virus Cell-to-Cell Movement Protein, Facilitates Intercellular Transport through Plasmodesmata

Plasmodesma (PD) is a channel structure that spans the cell wall and provides symplastic connection between adjacent cells. Various macromolecules are known to be transported through PD in a highly regulated manner, and plant viruses utilize their movement proteins (MPs) to gate the PD to spread cell-to-cell. The mechanism by which MP modifies PD to enable intercelluar traffic remains obscure, due to the lack of knowledge about the host factors that mediate the process. Here, we describe the functional interaction between Tobacco mosaic virus (TMV) MP and a plant factor, an ankyrin repeat containing protein (ANK), during the viral cell-to-cell movement. We utilized a reverse genetics approach to gain insight into the possible involvement of ANK in viral movement. To this end, ANK overexpressor and suppressor lines were generated, and the movement of MP was tested. MP movement was facilitated in the ANK-overexpressing plants, and reduced in the ANK-suppressing plants, demonstrating that ANK is a host factor that facilitates MP cell-to-cell movement. Also, the TMV local infection was largely delayed in the ANK-suppressing lines, while enhanced in the ANK-overexpressing lines, showing that ANK is crucially involved in the infection process. Importantly, MP interacted with ANK at PD. Finally, simultaneous expression of MP and ANK markedly decreased the PD levels of callose, β-1,3-glucan, which is known to act as a molecular sphincter for PD. Thus, the MP-ANK interaction results in the downregulation of callose and increased cell-to-cell movement of the viral protein. These findings suggest that ANK represents a host cellular receptor exploited by MP to aid viral movement by gating PD through relaxation of their callose sphincters