The unique carnation stunt-associated pararetroviroid.

The discovery and description of a pararetroviroid associated with carnation stunt was one of the most outstanding achievements gained by the research team directed by Ricardo Flores, as it opened up a whole new world in plant virology and quickly led to the identification of further viroid diseases. The carnation stunt-associated viroid-like RNA has been proved to exist in both RNA and DNA form, a discovery that pointed to new ways of studying the co-evolution of plants and viroid-like RNAs. This paper aims to summarise the scientific work of Ricardo Flores, a source of inspiration to both present and future generations of scientists.

Bioeconomy: Biomass and biomass-based energy supply and demand.

This paper addresses the challenges of the transition from a fossil fuel-dependent to a bio-based economy and implications related to the production of food, feed, bioenergy and other bio-based materials. The objective is to provide a comprehensive review of global biomass and biomass-based energy supplies and demand, with particular attention to the EU. Furthermore, factors related to setting priorities in the use of non-food biomass are discussed, as food security will remain the top priority. Finally, the changes in the bioenergy balance indicators in the Member States of the EU and new plant breeding technologies are analyzed. Overall, this study describes the complexity of the bio-based value chains in making decisions on how best to use biomass. The article presents a comprehensive review on global biomass and biomass based energy supplies and demand, discusses the European chemical industry perspective, analyzes the changes in the biomass based energy balance indicators in the Member States of the EU, and considers the challenges of the new plant breeding technologies.

Safety evaluation of the food enzyme α-amylase from a genetically modified strain of Bacillus licheniformis (DP-Dzb54).

The food enzyme α-amylase (4-α-d-glucan glucanohydrolase; EC 3.2.1.1) is produced with the genetically modified Bacillus licheniformis strain DP-Dzb54 by Danisco. The α-amylase is intended to be used in starch processing for the production of glucose syrups. Residual amounts of total organic solids are removed by the purification steps applied during the production of glucose syrups; consequently, dietary exposure was not calculated. The parental strain meets all the requirements for the Qualified Presumption of Safety approach for risk assessment, except the absence of acquired antimicrobial resistance genes. However, this has no practical consequence for the food enzyme as it has been shown not to contain viable cells and DNA from the production strain. As no other concerns arising from the microbial source and its subsequent genetic modification or from the manufacturing process have been identified, the Panel considers that toxicological tests are not needed for the assessment of this food enzyme. Similarity of the amino acid sequence to those of known allergens was searched and no match was found. The Panel considered that, under the intended conditions of use, the risk of allergic sensitisation and elicitation reactions by dietary exposure cannot be excluded, but the likelihood to occur is considered to be low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

Molecular Modeling for Better Understanding of Cucumovirus Pathology.

Cucumber mosaic virus (CMV) is a small RNA virus capable of infecting a wide variety of plant species. The high economic losses due to the CMV infection made this virus a relevant subject of scientific studies, which were further facilitated by the small size of the viral genome. Hence, CMV also became a model organism to investigate the molecular mechanism of pathogenesis. All viral functions are dependent on intra- and intermolecular interactions between nucleic acids and proteins of the virus and the host. This review summarizes the recent data on molecular determinants of such interactions. A particular emphasis is given to the results obtained by utilizing molecular-based planning and modeling techniques.

Safety evaluation of the food enzyme ß-amylase obtained from soybean (Glycine max).

The food enzyme considered in this opinion is a ß-amylase (EC 3.2.1.2) from soybean submitted by Nagase (Europa) GmbH. This ß-amylase is intended to be used in the starch processing for maltose syrup production and the manufacture of a Japanese rice cake type. Based on the maximum use levels recommended for the respective food processes, dietary exposure to the food enzyme-total organic solids (TOS) was estimated on the basis of Japanese consumption data. Conservative average infant formula consumption, as reported in the EFSA Draft Guidance on risk assessment of substances present in food intended for infants below 16 weeks of age, was used to estimate the exposure to a fraction of soybean comparable to the food enzyme-TOS, resulting from the consumption of soybean-derived foods. The exposure estimate to the food enzyme-TOS was found to be lower than the comparable fraction from the source material. Potential allergenicity of the ß-amylase was evaluated by searching for similarity of the amino acid sequence to those of known allergens, and no match was found. The ß-amylase is produced from soybean, which is a known allergenic food. Japanese rice cake, consequently, may contain traces of soybean allergens, which may give rise to safety concerns in soybean-allergic consumers. Based on the origin of the food enzyme from edible parts of soybean, the manufacturing process, the compositional and biochemical data provided and the dietary intake estimates, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use, except that Japanese rice cake produced with this food enzyme may contain traces of soybean allergens.

Safety evaluation of the food enzyme ß-amylase from genetically modified Bacillus licheniformis strain NZYM-JA.

The food enzyme considered in this opinion is a ß-amylase (4-α-d-glucan maltohydrolase; EC 3.2.1.2), produced with genetically modified Bacillus licheniformis strain NZYM-JA by Novozymes A/S (Denmark). The ß-amylase food enzyme is intended to be used in starch processing for the production of glucose syrups. Since the residual amounts of total organic solids (TOS) in glucose syrups after filtration and purification during starch processing were considered negligible, no dietary exposure was calculated. Toxicological tests made with the food enzyme under application indicated that there was no concern with respect to genotoxicity, mutagenicity or systemic toxicity. The allergenicity was evaluated by searching for similarity of the amino acid sequence to those of known allergens; no match was found. The Panel considers that there are no indications for allergic reactions. Based on the genetic modifications performed, the manufacturing process, the compositional and biochemical data provided, the findings in the toxicological studies and allergenicity assessment, the Panel concludes that this food enzyme does not give rise to safety concerns under the intended conditions of use.

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.

Engineering resistance to PVY in different potato cultivars in a marker-free transformation system using a 'shooter mutant' A. tumefaciens.

In this work, Potato virus Y (PVY) resistant potatoes were generated using an environmentally safe construct. For this purpose, a 'shooter' mutant Agrobacterium-based transformation system was used. The isopentenyl transferase gene (ipt) present on the Ti plasmid of 'shooter' strains enhances shoot regeneration and can be used as a phenotypic selection marker. The introduced marker-free binary vector carried a hairpin construct derived from the coat protein gene of PVY-NTN strain in order to induce gene silencing. Transformation resulted in high regeneration rates (1.4-5.7 shoots per explant). With pre-selection for the ipt (+) phenotype the transformation frequency was 24-53%, while without selection 12-28% of the shoots were PCR positive. The presence of the transgene was verified by Southern hybridization. In 16 of 31 challenged transformant lines PVY could be detected neither by RT-PCR nor by back inoculation. A 62.5% of these resistant lines proved to be also ipt-free. This transformation system was reproducible in four potato cultivars, suggesting that it could easily be adapted for other species.

A novel strategy for creating recombinant infectious RNA virus genomes.

Reverse transcriptases with RNase H activity are particularly apt to switch templates and generate recombinant molecules in vitro. This property has been exploited for the first time to create a library of recombinant RNAs 3 between two strains of Cucumber mosaic virus (CMV) or between CMV and Tomato aspermy virus (TAV), which share 75 and 63% sequence identity, respectively. The recombination events were almost entirely of the precise homologous type, and occurred at the same sites as those previously identified in co-infected plants, making it possible to use this strategy to create numerous cDNA fragments with crossovers similar to those occurring in vivo. Sub-cloning of recombinant fragments into an infectious full-length clone was accomplished by homologous recombination in yeast, alleviating the need for in vitro ligation at common restriction sites. Most of the recombinant genomes were infectious. Association of these two methods constitutes an efficient and practical means for generating numerous infectious viral genomes equivalent to ones that might arise by precise homologous recombination between two parental viral genomes in nature.

The necrotic pathotype of the cucumber mosaic virus (CMV) ns strain is solely determined by amino acid 461 of the 1a protein.

The unique Ns isolate of Cucumber mosaic virus (CMV) induces necrotic lesions on several Nicotiana spp. in contrast to other strains that cause systemic mosaic on these plants. By using biologically active RNA transcripts from cDNAs of Ns-CMV and a reference subgroup I strain Rs-CMV, we confined the genetic determinant solely responsible for necrosis induction to amino acid 461 of the la protein translated from genomic RNA1. An Arg to Cys change at this position (R461C) rendered Rs-CMV necrotic, whereas the reciprocal C461R mutation reverted the necrotic phenotype of Ns-CMV. Necrotic (Ns-CMV, R461C) and non-necrotic (Rs-CMV and C461R) viruses accumulated to similar levels in Nicotiana clevelandii protoplasts. Deletion of the residue at position 461 abolished replicase activity of the Ns-CMV 1a protein. The R461C mutation also was introduced into the 1a protein of Trk7-CMV, a subgroup II isolate. Symptoms induced by the Trk7/R461C mutant were identical to those caused by wild-type Trk7-CMV, even when the mutant Trk7 RNA1 was co-inoculated with RNA2 and 3 of the necrotic Ns strain.

Limited utility of blue fluorescent protein (BFP) in monitoring plant virus movement.

While the green fluorescent protein (GFP) is a routinely used marker gene in higher plants, there are only a few data concerning the use of blue fluorescent protein (BFP). These proteins together are used for dual colour tagging experiments in various biological systems; however, the benefits of this technique in plant virology have not been exploited yet. In this work, our aim was to determine whether the BFP is a suitable second marker in conjunction with GFP for following the progress of virus infection. Nicotiana clevelandii, N. benthamiana and N. tabacum cv. Xanthi-nc plants were infected with potato virus X vector carrying the GFP or the Y66H type BFP gene. While GFP was brightly fluorescent in all species, the fluorescence intensity of BFP varied widely, from the bright fluorescence observed in N. clevelandii to the absence of fluorescence in N. tabacum cv. Xanthi-nc. Since at even mild acidic pH BFP rapidly fades, the more acidic cytosol of N. tabacum could be responsible for impaired in vivo fluorescence. After infiltration of the infected leaves of N. clevelandii with pH 5 phosphate buffer, the fluorescence faded thus confirming this situation.

Heterologous movement protein strongly modifies the infection phenotype of cucumber mosaic virus.

A hybrid virus (CMVcymMP) constructed by replacing the movement protein (MP) of cucumber mosaic cucumovirus (CMV) with that of cymbidium ringspot tombusvirus (CymRSV) was viable and could efficiently spread both cell to cell and long distance in host plants. The hybrid virus was able to move cell to cell in the absence of functional CP, whereas CP-deficient CMV was restricted to single inoculated cells. In several Chenopodium and Nicotiana species, the symptom phenotype of the hybrid virus infection was clearly determined by the foreign MP gene. In Nicotiana debneyi and Nicotiana tabacum cv. Xanthi, the hybrid virus could move systemically, contrary to CymRSV.