Mass spectrometric analysis of digesta does not improve the allergenicity assessment of GM crops.

An investigation of the potential allergenicity of newly expressed proteins in genetically modified (GM) crops comprises part of the assessment of GM crop safety. However, allergenicity is not completely predictable from a definitive assay result or set of protein characteristics, and scientific opinions regarding the data that should be used to assess allergenicity are continuously evolving. Early studies supported a correlation between the stability of a protein exposed to digestive enzymes such as pepsin and the protein's status as a potential allergen, but over time the conclusions of these earlier studies were not confirmed. Nonetheless, many regulatory authorities, including the European Food Safety Authority (EFSA), continue to require digestibility analyses as a component of GM crop risk assessments. Moreover, EFSA has recently investigated the use of mass spectrometry (MS), to make digestion assays more predictive of allergy risk, because it can detect and identify small undigested peptides. However, the utility of MS is questionable in this context, since known allergenic peptides are unlikely to exist in protein candidates intended for commercial development. These protein candidates are pre-screened by the same bioinformatics processes that are normally used to identify MS targets. Therefore, MS is not a standalone allergen identification method and also cannot be used to predict previously unknown allergenic epitopes. Thus, the suggested application of MS for analysis of digesta does not improve the poor predictive power of digestion assays in identifying allergenic risk.

Molecular characterization for food safety assessment of a genetically modified late blight resistant potato: an unusual case.

Standard food safety assessments of genetically modified crops require a thorough molecular characterization of the novel DNA as inserted into the plant that is intended for commercialization, as well as a comparison of agronomic and nutritional characteristics of the genetically modified to the non-modified counterpart. These characterization data are used to identify any unintended changes in the inserted DNA or in the modified plant that would require assessment for safety in addition to the assessment of the intended modification. An unusual case of an unintended effect discovered from the molecular characterization of a genetically modified late blight resistant potato developed for growing in Bangladesh and Indonesia is presented here. Not only was a significant portion of the plasmid vector backbone DNA inserted into the plant along with the intended insertion of an R-gene for late blight resistance, but the inserted DNA was split into two separate fragments and inserted into two separate chromosomes. One fragment carries the R-gene and the other fragment carries the NPTII selectable marker gene and the plasmid backbone DNA. The implications of this for the food safety assessment of this late blight resistant potato are considered.

Assessment of the potential allergenicity of genetically-engineered food crops.

An extensive safety assessment process exists for genetically-engineered (GE) crops. The assessment includes an evaluation of the introduced protein as well as the crop containing the protein with the goal of demonstrating the GE crop is "as-safe-as" non-GE crops in the food supply. One of the evaluations for GE crops is to assess the expressed protein for allergenic potential. Currently, no single factor is recognized as a predictor for protein allergenicity. Therefore, a weight-of-the-evidence approach, which accounts for a variety of factors and approaches for an overall assessment of allergenic potential, is conducted. This assessment includes an evaluation of the history of exposure and safety of the gene(s) source; protein structure (e.g. amino acid sequence identity to human allergens); stability of the protein to pepsin digestion in vitro; heat stability of the protein; glycosylation status; and when appropriate, specific IgE binding studies with sera from relevant clinically allergic subjects. Since GE crops were first commercialized over 20 years ago, there is no proof that the introduced novel protein(s) in any commercialized GE food crop has caused food allergy.

uORF-mediated translation allows engineered plant disease resistance without fitness costs.

Controlling plant disease has been a struggle for humankind since the advent of agriculture. Studies of plant immune mechanisms have led to strategies of engineering resistant crops through ectopic transcription of plants' own defence genes, such as the master immune regulatory gene NPR1 (ref. 1). However, enhanced resistance obtained through such strategies is often associated with substantial penalties to fitness, making the resulting products undesirable for agricultural applications. To remedy this problem, we sought more stringent mechanisms of expressing defence proteins. On the basis of our latest finding that translation of key immune regulators, such as TBF1 (ref. 3), is rapidly and transiently induced upon pathogen challenge (see accompanying paper), we developed a 'TBF1-cassette' consisting of not only the immune-inducible promoter but also two pathogen-responsive upstream open reading frames (uORFsTBF1) of the TBF1 gene. Here we demonstrate that inclusion of uORFsTBF1-mediated translational control over the production of snc1-1 (an autoactivated immune receptor) in Arabidopsis thaliana and AtNPR1 in rice enables us to engineer broad-spectrum disease resistance without compromising plant fitness in the laboratory or in the field. This broadly applicable strategy may lead to decreased pesticide use and reduce the selective pressure for resistant pathogens.

Fungal and Oomycete Diseases of Tropical Tree Fruit Crops.

The tropics produce a range of fruit from tree crops that cannot be grown in colder climates. Bananas, mangos, several nuts, spices, coffee, and cacao are widely traded and much sought after around the world. However, the sustainable production of these tropical tree fruit crops faces significant challenges. Among these, losses due to pests and diseases play a large part in reducing yields, quality, and profitability. Using bananas and cacao as key examples, we outline some of the reasons fungal and oomycete diseases cause such significant losses to tropical tree crops. Cultivation of monocultures derived from limited genetic diversity, environmental conditions conducive for disease development, high levels of disease incidence and severity, a lack of disease resistance in planting materials, shortages of labor, and inadequate infrastructure and investment pose significant challenges, especially for smallholder producers. The expansion of travel and trade has given rise to emerging infectious plant diseases that add further insecurity and pressure. We conclude that holistic actions are needed on multiple fronts to address the growing problem of disease in tropical fruit tree crops.

Glycolipid biosurfactants: main properties and potential applications in agriculture and food industry.

Glycolipids, consisting of a carbohydrate moiety linked to fatty acids, are microbial surface active compounds produced by various microorganisms. They are characterized by high structural diversity and have the ability to decrease the surface and interfacial tension at the surface and interface, respectively. Rhamnolipids, trehalolipids, mannosylerythritol lipids and cellobiose lipids are among the most popular glycolipids. They have received much practical attention as biopesticides for controlling plant diseases and protecting stored products. As a result of their antifungal activity towards phytopathogenic fungi and larvicidal and mosquitocidal potencies, glycolipid biosurfactants permit the preservation of plants and plant crops from pest invasion. Also, as a result of their emulsifying and antibacterial activities, glycolipids have great potential as food additives and food preservatives. Furthermore, the valorization of food byproducts via the production of glycolipid biosurfactant has received much attention because it permits the bioconversion of byproducts on valuable compounds and decreases the cost of production. Generally, the use of glycolipids in many fields requires their retention from fermentation media. Accordingly, different strategies have been developed to extract and purify glycolipids. © 2016 Society of Chemical Industry.

Assessment of potential adjuvanticity of Cry proteins.

Genetically modified (GM) crops have achieved success in the marketplace and their benefits extend beyond the overall increase in harvest yields to include lowered use of insecticides and decreased carbon dioxide emissions. The most widely grown GM crops contain gene/s for targeted insect protection, herbicide tolerance, or both. Plant expression of Bacillus thuringiensis (Bt) crystal (Cry) insecticidal proteins have been the primary way to impart insect resistance in GM crops. Although deemed safe by regulatory agencies globally, previous studies have been the basis for discussions around the potential immuno-adjuvant effects of Cry proteins. These studies had limitations in study design. The studies used animal models with extremely high doses of Cry proteins, which when given using the ig route were co-administered with an adjuvant. Although the presumption exists that Cry proteins may have immunostimulatory activity and therefore an adjuvanticity risk, the evidence shows that Cry proteins are expressed at very low levels in GM crops and are unlikely to function as adjuvants. This conclusion is based on critical review of the published literature on the effects of immunomodulation by Cry proteins, the history of safe use of Cry proteins in foods, safety of the Bt donor organisms, and pre-market weight-of-evidence-based safety assessments for GM crops.

Allergenicity assessment of genetically-modified tobacco expressing salt tolerance cbl gene.

It is mandatory to assess the allergenic potential of genetically modified (GM) crops before their commercialization. Recently, a transgene [Calcineurin B-like (CBL) protein] has been introduced into tobacco plant to make the crop salt resistance. Therefore, it was felt necessary to assess the allergenic potential of the cbl gene product, which was introduced and expressed in Nicotiana tabacum (tobacco) plant and compared the allergenic effects with the wild-type (WT) counterpart. Bioinformatic analysis revealed that there was no significant sequence homology with known allergens. Also, no difference between the protein digestibility profiles of GM and WT tobacco was found. Rapid digestion of CBL protein (Mol Wt 35 kDa) by simulated gastric fluid (SGF) indicated reduced chances of this protein to induce allergenicity. In addition, BALB/c mice sensitized by intraperitoneal administration of WT and GM tobacco protein showed comparable levels of clinical score, specific IgE, IgG1, histamine level, similar effect on different organs as well as IgE binding proteins. These findings indicate that insertion of cbl gene in tobacco did not cause any additional allergic risk to consumer and the GM and native tobacco proteins behave similarly in both in vitro and in vivo situations even after genetic modification.

Evaluation of global sequence comparison and one-to-one FASTA local alignment in regulatory allergenicity assessment of transgenic proteins in food crops.

To address the high false positive rate using >35% identity over 80 amino acids in the regulatory assessment of transgenic proteins for potential allergenicity and the change of E-value with database size, the Needleman-Wunsch global sequence alignment and a one-to-one (1:1) local FASTA search (one protein in the target database at a time) using FASTA were evaluated by comparing proteins randomly selected from Arabidopsis, rice, corn, and soybean with known allergens in a peer-reviewed allergen database (http://www.allergenonline.org/). Compared with the approach of searching >35%/80aa+, the false positive rate measured by specificity rate for identification of true allergens was reduced by a 1:1 global sequence alignment with a cut-off threshold of ≧30% identity and a 1:1 FASTA local alignment with a cut-off E-value of ≦1.0E-09 while maintaining the same sensitivity. Hence, a 1:1 sequence comparison, especially using the FASTA local alignment tool with a biological relevant E-value of 1.0E-09 as a threshold, is recommended for the regulatory assessment of sequence identities between transgenic proteins in food crops and known allergens.

Assessment of possible allergenicity of hypothetical ORFs in common food crops using current bioinformatic guidelines and its implications for the safety assessment of GM crops.

Before a genetically modified (GM) crop can be commercialized it must pass through a rigorous regulatory process to verify that it is safe for human and animal consumption, and to the environment. One particular area of focus is the potential introduction of a known or cross-reactive allergen not previously present within the crop. The assessment of possible allergenicity uses the guidelines outlined by the Food and Agriculture Organization (FAO) and World Health Organization's (WHO) Codex Alimentarius Commission (Codex) to evaluate all newly expressed proteins. Some regulatory authorities have broadened the scope of the assessment to include all DNA reading frames between stop codons across the insert and spanning the insert/genomic DNA junctions. To investigate the utility of this bioinformatic assessment, all naturally occurring stop-to-stop frames in the non-transgenic genomes of maize, rice, and soybean, as well as the human genome, were compared against the AllergenOnline (www.allergenonline.org) database using the Codex criteria. We discovered thousands of frames that exceeded the Codex defined threshold for potential cross-reactivity suggesting that evaluating hypothetical ORFs (stop-to-stop frames) has questionable value for making decisions on the safety of GM crops.

In silico assessment of the potential allergenicity of transgenes used for the development of GM food crops.

Genetically modified (GM) crops require allergenicity and toxicity assessment of the novel protein(s) to ensure complete safety to the consumers. These assessments are performed in accordance with the guidelines proposed by Codex (2003) and ICMR (2008). The guidelines recommend sequence homology analysis as a preliminary step towards allergenicity prediction, later in vitro experiments may be performed to confirm allergenicity. In the present study, an in silico approach is employed to evaluate the allergenic potential of six transgenes routinely used for the development of GM food crops. Among the genes studied, manganese superoxide dismutase (MnSOD) and osmotin shares greater than 90% identity with Hev b 10 and Cap a 1w, respectively. Chitinase shares greater than 70% identity with allergens namely Pers a 1 and Hev b 11, and fungal chitinase showed significant IgE binding with 7 of 75 patients' sera positive to different food extracts. Glucanases (alfalfa, wheat) and glycine betaine aldehyde dehydrogenase gene share 50% homology with allergens like - Ole e 9, Cla h 10 and Alt a 10. The results demonstrate the allergenic potential of six genes and can serve as a guide for selection of transgenes to develop GM crops.

Field testing, gene flow assessment and pre-commercial studies on transgenic Solanum tuberosum spp. tuberosum (cv. Spunta) selected for PVY resistance in Argentina.

Solanum tuberosum ssp. tuberosum (cv. Spunta) was transformed with a chimeric transgene containing the Potato virus Y (PVY) coat protein (CP) sequence. Screening for PVY resistance under greenhouse conditions yielded over 100 independent candidate lines. Successive field testing of selected lines allowed the identification of two genetically stable PVY-resistant lines, SY230 and SY233, which were further evaluated in field trials at different potato-producing regions in Argentina. In total, more than 2,000 individuals from each line were tested along a 6-year period. While no or negligible PVY infection was observed in the transgenic lines, infection rates of control plants were consistently high and reached levels of up to 70-80%. Parallel field studies were performed in virus-free environments to assess the agronomical performance of the selected lines. Tubers collected from these assays exhibited agronomical traits and biochemical compositions indistinguishable from those of the non-transformed Spunta cultivar. In addition, an interspecific out-crossing trial to determine the magnitude of possible natural gene flow between transgenic line SY233 and its wild relative Solanum chacoense was performed. This trial yielded negative results, suggesting an extremely low probability for such an event to occur.

Influence of cultivated landscape composition on variety resistance: an assessment based on wheat leaf rust epidemics.

In plant pathology, the idea of designing variety management strategies at the scale of cultivated landscapes is gaining more and more attention. This requires the identification of effects that take place at large scales on host and pathogen populations. Here, we show how the landscape varietal composition influences the resistance level (as measured in the field) of the most grown wheat varieties by altering the structure of the pathogen populations. For this purpose, we jointly analysed three large datasets describing the wheat leaf rust pathosystem (Puccinia triticina/Triticum aestivum) at the country scale of France with a Bayesian hierarchical model. We showed that among all compatible pathotypes, some were preferentially associated with a variety, that the pathotype frequencies on a variety were affected by the landscape varietal composition, and that the observed resistance level of a variety was linked to the frequency of the most aggressive pathotypes among all compatible pathotypes. This data exploration establishes a link between the observed resistance level of a variety and landscape composition at the national scale. It illustrates that the quantitative aspects of the host-pathogen relationship have to be considered in addition to the major resistance/virulence factors in landscape epidemiology approaches.

Engineering pathogen resistance in crop plants.

As the world population continues to increase, food supplies must also grow to meet nutritional requirements. One means of ensuring the stability and plentitude of the food supply is to mitigate crop loss caused by plant pathogens. Strategies for combating disease include traditional technologies such as plant breeding and chemical applications; current technologies such as generating transgenic plants that express components of known defense signaling pathways; and the adaptation of newer technologies such as RNA silencing of pathogen and plant transcripts. Breeding has been used to pyramid resistance (R) genes into many different plants including rice. Chemical strategies include application of salicylic acid (SA) analogs to stimulate systemic acquired resistance (SAR) responses. Genetic screens in Arabidopsis have identified genes controlling SAR and these genes have been manipulated and used to engineer crop plants. The diseases caused by plant viruses are being thwarted through the initiation of endogenous RNA silencing mechanisms. Many of these strategies show great promise, some limitations, and exciting opportunities to develop many new tools for combating plant pests.