Genetically modified α-amylase inhibitor peas are not specifically allergenic in mice.

Weevils can devastate food legumes in developing countries, but genetically modified peas (Pisum sativum), chickpeas and cowpeas expressing the gene for alpha-amylase inhibitor-1 (αAI) from the common bean (Phaseolus vulgaris) are completely protected from weevil destruction. αAI is seed-specific, accumulated at high levels and undergoes post-translational modification as it traverses the seed endomembrane system. This modification was thought to be responsible for the reported allergenicity in mice of the transgenic pea but not the bean. Here, we observed that transgenic αAI peas, chickpeas and cowpeas as well as non-transgenic beans were all allergenic in BALB/c mice. Even consuming non-transgenic peas lacking αAI led to an anti-αAI response due to a cross-reactive response to pea lectin. Our data demonstrate that αAI transgenic peas are not more allergenic than beans or non-transgenic peas in mice. This study illustrates the importance of repeat experiments in independent laboratories and the potential for unexpected cross-reactive allergic responses upon consumption of plant products in mice.

Comparison of the α-amylase inhibitor-1 from common bean (Phaseolus vulgaris) varieties and transgenic expression in other legumes--post-translational modifications and immunogenicity.

The seeds of peas (Pisum sativum) and chickpeas (Cicer arietinum) expressing a gene for α-amylase inhibitor-1 (αAI) from the common bean (Phaseolus vulgaris) are protected from damage by old world bruchids (pea and cowpea weevils). Here, we used electrospray ionization time-of-flight mass spectrometry to compare the post-translational modifications of αAI from transgenic sources with the processed forms of the protein from several bean varieties. All sources showed microheterogeneity with differences in the relative abundance of particular variants due to differences in the frequency of addition of glycans, variable processing of glycans, and differences of C-terminal exopeptidase activity. The structural variation among the transgenics was generally within the range of the bean varieties. Previously, mice showed allergic reactions following ingestion of transgenic pea αAI but not bean αAI. Here, only minor differences were observed following intraperitoneal sensitization. Both of the transgenic pea and bean forms of αAI elicited Th1 and Th2 antibody isotype responses, suggesting that both proteins are immunogenic and could potentially be allergenic.

Cowpea bruchid midgut transcriptome response to a soybean cystatin--costs and benefits of counter-defence.

The insect digestive system is the first line of defence protecting cells and tissues of the body from a broad spectrum of toxins and antinutritional factors in its food. To gain insight into the nature and breadth of genes involved in adaptation to dietary challenge, a collection of 20 352 cDNAs was prepared from the midgut tissue of cowpea bruchid larvae (Callosobruchus maculatus) fed on regular diet and diets containing antinutritional compounds. Transcript responses of the larvae to dietary soybean cystatin (scN) were analysed using cDNA microarrays, followed by quantitative real-time PCR (RT-PCR) confirmation with selected genes. The midgut transcript profile of insects fed a sustained sublethal scN dose over the larval life was compared with that of insects treated with an acute high dose of scN for 24 h. A total of 1756 scN-responsive cDNAs was sequenced; these clustered into 967 contigs, of which 653 were singletons. Many contigs (451) did not show homology with known genes, or had homology only with genes of unknown function in a Blast search. The identified differentially regulated sequences encoded proteins presumptively involved in metabolism, structure, development, signalling, defence and stress response. Expression patterns of some scN-responsive genes were consistent in each larval stage, whereas others exhibited developmental stage-specificity. Acute (24 h), high level exposure to dietary scN caused altered expression of a set of genes partially overlapping with the transcript profile seen under chronic lower level exposure. Protein and carbohydrate hydrolases were generally up-regulated by scN whereas structural, defence and stress-related genes were largely down-regulated. These results show that insects actively mobilize genomic resources in the alimentary tract to mitigate the impact of a digestive protease inhibitor. The enhanced or restored digestibility that may result is possibly crucial for insect survival, yet may be bought at the cost of weakened response to other stresses.

Effectiveness of Bacillus thuringiensis-transgenic chickpeas and the entomopathogenic fungus Metarhizium anisopliae in controlling Helicoverpa armigera (Lepidoptera: Noctuidae).

The use of genetically modified (Bt) crops expressing lepidopteran-specific Cry proteins derived from the soil bacterium Bacillus thuringiensis is an effective method to control the polyphagous pest Helicoverpa armigera. As H. armigera potentially develops resistance to Cry proteins, Bt crops should be regarded as one tool in integrated pest management. Therefore, they should be compatible with biological control. Bioassays were conducted to understand the interactions between a Cry2Aa-expressing chickpea line, either a susceptible or a Cry2A-resistant H. armigera strain, and the entomopathogenic fungus Metarhizium anisopliae. In a first concentration-response assay, Cry2A-resistant larvae were more tolerant of M. anisopliae than susceptible larvae, while in a second bioassay, the fungus caused similar mortalities in the two strains fed control chickpea leaves. Thus, resistance to Cry2A did not cause any fitness costs that became visible as increased susceptibility to the fungus. On Bt chickpea leaves, susceptible H. armigera larvae were more sensitive to M. anisopliae than on control leaves. It appeared that sublethal damage induced by the B. thuringiensis toxin enhanced the effectiveness of M. anisopliae. For Cry2A-resistant larvae, the mortalities caused by the fungus were similar when they were fed either food source. To examine which strain would be more likely to be exposed to the fungus, their movements on control and Bt chickpea plants were compared. Movement did not appear to differ among larvae on Bt or conventional chickpeas, as indicated by the number of leaflets damaged per leaf. The findings suggest that Bt chickpeas and M. anisopliae are compatible to control H. armigera.

Transgenic expression of bean alpha-amylase inhibitor in peas results in altered structure and immunogenicity.

The development of modern gene technologies allows for the expression of recombinant proteins in non-native hosts. Diversity in translational and post-translational modification pathways between species could potentially lead to discrete changes in the molecular architecture of the expressed protein and subsequent cellular function and antigenicity. Here, we show that transgenic expression of a plant protein (alpha-amylase inhibitor-1 from the common bean (Phaseolus vulgaris L. cv. Tendergreen)) in a non-native host (transgenic pea (Pisum sativum L.)) led to the synthesis of a structurally modified form of this inhibitor. Employing models of inflammation, we demonstrated in mice that consumption of the modified alphaAI and not the native form predisposed to antigen-specific CD4+ Th2-type inflammation. Furthermore, consumption of the modified alphaAI concurrently with other heterogeneous proteins promoted immunological cross priming, which then elicited specific immunoreactivity of these proteins. Thus, transgenic expression of non-native proteins in plants may lead to the synthesis of structural variants possessing altered immunogenicity.

Pest and disease protection conferred by expression of barley ß-hordothionin and Nicotiana alata proteinase inhibitor genes in transgenic tobacco.

Proteinase inhibitors and thionins are among the many proteins thought to have a role in plant defence against pests and pathogens. Complementary DNA clones encoding the precursors of a multi-domain proteinase inhibitor from Nicotiana alata Link et Otto (NA-PI) (Mr approximately 43 000) and a ß-hordothionin (ß-HTH) (Mr approximately 13 000) from barley, were linked to constitutive promoters and subsequently transferred by Agrobacterium-mediated transformation into tobacco. The NA-PI and ß-HTH precursor proteins were synthesised and post-translationally processed in transgenic tobacco and accumulated as polypeptides of apparent size Mr approximately 6000 and Mr approximately 8500, respectively. The na-pi and ß-hth genes were stably inherited for at least two generations. Transgenic tobacco plants containing the highest amounts of NA-PI and ß-HTH were crossed to produce plants containing both genes. Helicoverpa armigera (tobacco budworm) larvae that ingested transgenic tobacco leaves expressing both NA-PI and ß-HTH, exhibited higher mortality and slower development relative to larvae fed on non-transgenic tobacco. NA-PI and ß-HTH, either alone, or in combination, also conferred protection against the fungal pathogen, Botrytis cinerea (grey mould) and the bacterial pathogen, Pseudomonas solanacearum (bacterial wilt). The effect of the two proteins depended upon the organism tested and the contribution of each gene to the protective effects was not necessarily equal. The genetic engineering of plants with proteinase inhibitors or thionins, therefore, has potential for improving crop productivity by simultaneously increasing resistance to both pests and pathogens.

Sulphur and nitrogen nutrition influence the response of chickpea seeds to an added, transgenic sink for organic sulphur.

In order to increase the concentration of the nutritionally essential sulphur amino acids in seed protein, a transgene encoding a methionine- and cysteine-rich protein, sunflower seed albumin (SSA), was transferred to chickpeas (Cicer arietinum L). Transgenic seeds that accumulated SSA contained more methionine and less oxidized sulphur than the controls, suggesting that additional demand for sulphur amino acids from the expression of the transgene stimulated sulphur assimilation. In addition, the activity of trypsin inhibitors, a known family of endogenous, sulphur-rich chickpea seed proteins, was diminished in transgenic, SSA-containing seeds compared with the non-transgenic controls. Together, these results indicate that the reduced sulphur sequestered into SSA was supplied partly by additional sulphur assimilation in the developing transgenic seeds, and partly by some diversion of sulphur amino acids from endogenous seed proteins. Growth of chickpeas on nutrient with a high sulphur-to-nitrogen ratio increased the total seed sulphur content and the accumulation of sulphur amino acids in the seeds, and partly mitigated the effect of SSA accumulation on the trypsin inhibitor amount. The results suggest that free methionine and O-acetylserine (OAS) acted as signals that modulated chickpea seed protein composition in response to the variation in sulphur demand, as well as in response to variation in the nitrogen and sulphur status of the plant.

Growth, fecundity and competitive ability of transgenic Trifolium subterraneum subsp. subterraneum cv. Leura expressing a sunflower seed albumin gene.

Ecological risk assessment is an important step in the production and commercialisation of transgenic plants. To date, however, most risk assessment studies have been performed on crop plants, and few have considered the ecological consequences associated with genetic modification of pasture species. In this study we compared the growth, yield, population dynamics and competitive ability of transgenic Trifolium subterraneum subsp. subterraneum cv. Leura (subclover) expressing a nutritive sunflower seed albumin (ssa) gene with the equivalent non-transgenic commercial line in a glasshouse competition trial. Plants were grown in low-fertility soil typical of unimproved native southeastern Australian grasslands. We measured survivorship, seed production rate, seed germination rate, seed weight, dry weight yield and the intrinsic rate of population increase (lambda) of plants grown in mixtures and monocultures over a range of densities (250 to 2000 plants m(-2)), and also determined intragenotypic and intergenotypic competition coefficients for each line. There were no significant differences between transgenic and non-transgenic plants in any of the measured variables except survivorship; transgenic plants had a significantly lower survival rate than non-transgenic plants when grown at high densities (p<0.01). However, density-dependent effects were observed for all measured variables, and in all models plant density affected the response variables more than the presence of the transgene. Based on these results, we conclude that the ssa gene construct appears to confer no advantage to transgenic T. s. subterraneum cv. Leura growing in mixed or pure swards under the fertility and density regimes examined in the trial. Our data also suggest that transgenic subterranean clover expressing the ssa gene is unlikely to exhibit a competitive advantage over associated non-transgenic commercial cultivars when grown in dense swards in low-fertility pastures.

A plant-based allergy vaccine suppresses experimental asthma via an IFN-gamma and CD4+CD45RBlow T cell-dependent mechanism.

Allergic asthma is currently considered a chronic airway inflammatory disorder associated with the presence of activated CD4(+) Th2-type lymphocytes, eosinophils, and mast cells. Interestingly, therapeutic strategies based on immune deviation and suppression have been shown to successfully attenuate the development of the asthma phenotype. In this investigation, we have for the first time used a genetically modified (GM) plant, narrow leaf lupin (Lupinus angustifolius L.), expressing a gene for a potential allergen (sunflower seed albumin) (SSA-lupin) to examine whether a GM plant/food-based vaccine strategy can be used to suppress the development of experimental asthma. We show that oral consumption of SSA-lupin promoted the induction of an Ag-specific IgG2a Ab response. Furthermore, we demonstrate that the plant-based vaccine attenuated the induction of delayed-type hypersensitivity responses and pathological features of experimental asthma (mucus hypersecretion, eosinophilic inflammation, and enhanced bronchial reactivity (airways hyperreactivity). The suppression of experimental asthma by SSA-lupin was associated with the production of CD4(+) T cell-derived IFN-gamma and IL-10. Furthermore, we show that the specific inhibition of experimental asthma was mediated via CD4(+)CD45RB(low) regulatory T cells and IFN-gamma. Thus, our data demonstrate that a GM plant-based vaccine can promote a protective immune response and attenuate experimental asthma, suggesting that plant-based vaccines may be potentially therapeutic for the protection against allergic diseases.

The redistribution of protein sulfur in transgenic rice expressing a gene for a foreign, sulfur-rich protein.

Sulfur amino acid composition is an important determinant of seed protein quality. A chimeric gene encoding sunflower seed albumin (SSA), one of the most sulfur-rich seed storage proteins identified so far, was introduced into rice (Oryza sativa) in order to modify cysteine and methionine content of the seed. Analysis of a transgenic line expressing SSA at approximately 7% of total seed protein revealed that the mature grain showed little change in the total sulfur amino acid content compared to the parental genotype. This result indicated that the transgenic rice grain was unable to respond to the added demand for cysteine and methionine imposed by the production of SSA. Analysis of the protein composition of the transgenic grain showed changes in the relative levels of the major seed storage proteins, as well as some non-storage proteins, compared to non-transgenic controls. Changes observed at the protein level were concomitant with differences in mRNA accumulation but not always with the level of transcription. The limited sulfur reserves appeared to be re-allocated from endogenous proteins to the new sulfur sink in the transgenic grain. We hypothesize that this response is mediated by a signal transduction pathway that normally modulates seed storage protein composition in response to environmental fluctuations in sulfur availability, via both transcriptional and post-transcriptional control of gene expression.

Plasticity of seed protein composition in response to nitrogen and sulfur availability.

Seed composition is genetically programmed, but the implementation of that program is affected by many factors including the nutrition of the parent plant. In particular, seeds demonstrate a remarkable capacity to maintain nitrogen homeostasis in conditions of varying sulfur supply. They do this by altering the expression of individual genes encoding abundant storage proteins. The signal transduction pathways that modulate gene expression in seeds in response to N and S availability involve both transcriptional and post-transcriptional mechanisms.