A transcriptomic approach to food safety - Fruit allergens and mycotoxins

Chapter 1 - Food safety has become a public health priority, since food-related diseases affect up to one third of the population of the rich countries, and are spreading also in the developing ones. During the last decades, the integration of agricultural and food industries, and the food globalization have changed the patterns of food production and distribution, creating the ideal environment for the diffusion of new food-related disorders. Among the most important are the food allergies and the contamination of food and feed with mycotoxins such as aflatoxins or ochratoxins. The international community is aware of the problem and is trying to focus on a "farm to fork" approach operating in every step of the food chain with new regulations and preventive methods. In this context, nutritional genomics may represent a key scientific area, but its most important tool, which is transgenesis, is not well accepted by the consumers. Therefore, the biotechnologists are exploring alternative ways of using innovative genomic technologies to deliver crop improvements in a reasonable time. Marker assisted selection and functional genomics studies may allow an easier exploitation of natural variation, and to study the effective interactions existing between environment and genotype to set up innovative agricultural practices aimed at specifically modifying the expression of the genes of interest. In the present research, a transcriptomic approach was adopted for studying fruit allergens and mycotoxins, by using high-throughput techniques such as real-time PCR and cDNA-AFLP. The major findings of this study concern the factors affecting the allergenic potential in apple and peach, and the regulation of ochratoxin A biosynthesis in Aspergillus carbonarius, a filamentous fungus contaminating grapes and wines. Chapter 2 - Apple consumption is highly recommended for a healthy diet because of its nutritional properties. However, freshly consumed apples can cause allergic reactions in the worldwide population because of the presence of four classes of allergens, namely, Mal d 1, Mal d 2, Mal d 3 and Mal d 4, and their cross reactivity with sensitizing allergens of other species. Knowledge of the environmental and endogenous factors affecting the allergenic potential of apples would give important information to apple breeders, growers and consumers for the selection of hypoallergenic genotypes, the adoption of agronomical practices decreasing the allergenic potential, and the consumption of fruits with no or reduced amount of allergens. In the present research, expression studies were performed for all the known genes encoding allergen isoforms belonging to the four classes. Fruit specimens collected from fifteen apple varieties were investigated by means of Real Time PCR, and Principal Component Analysis (PCA) was performed for the ordination and classification of varieties according to their total allergenic potentials. Three different trials were carried out to assess the effects of shadowing, elevation and storage, and water stress on the expression of allergen-related genes. Shadowing was shown to be effective in reducing and increasing the allergenic potential in cortex and epidermis, respectively, whereas elevation was found to increase the global allergenic potential, especially in the cortex. Storage of fruits harvested at low elevations enhanced the global allergen-related transcription in the cortex and decreased it in the epidermis. An opposite trend was determined by storage of fruits harvested at high elevations. In spite of the dramatic effect on both fruit size and vegetative growth of the trees, water stress was shown to slightly affect the expression of only two genes. The possible implications of the results for breeders, growers and consumers are critically discussed. Chapter 3 - Section 1 - The full length cDNAs of Pp-LTP1 and Pp-LTP2, two members of the Lipid Transfer Protein multigene family in peach, have been isolated along with the proximal promoter and the complete genomic sequence of Pp-LTP1. As observed in other plant species, Pp-LTP1 is organized in two exons and, according to the deduced amino acid sequence of the secreted protein, it encodes the 9 kDa allergenic protein Pru p 3. Pp-LTP1 transcripts and Pru p 3 protein markedly accumulate in epicarp of ripe fruit in all of the peach and nectarine varieties examined (including "Royal Gem") with the exception of "Rita Star" that shows weak specific mRNA hybridization signal and no Pru p 3 accumulation. SDS-PAGE clearly displays a different banding pattern in correspondence of 9 kDa between "Rita Star" and "Royal Gem". In immunoblotting analysis, performed using sera of eight LTP positive patients, a strong band is present when using "Royal Gem" epicarp extract but not in the case of "Rita Star". According to these data, "Rita Star" appears an interesting model to better elucidate the role of LTP in fruit development and allergenic reactions. Section 2 - Recombinant allergenic proteins represent an important tool in the diagnosis of allergic reactions to foods of plant origin, relying upon a deep and precise knowledge of the gene sequences encoding allergens. In the present study, the list of the known allergens of peach was implemented with candidate allergenic proteins identified by means of a bioinformatics approach. Possible implications for the development of new diagnostic tools are discussed. Section 3 - In the Mediterranean area, the fruits of the Rosaceae species are most frequently involved in allergic reactions in the adult population and in patients older than 3-years of age. In peach, both true allergens and cross-reactive proteins cause hypersensitive reactions involving a wide variety of symptoms. Three known classes of allergenic proteins, namely Pru p 1, Pru p 3 and Pru p 4, have been reported to be mostly involved, but an exhaustive understanding concerning the proteins determining the overall allergenic potential, their biological function and the factors affecting the expression of the related genes is missing. In the present study, the expression profiles of some selected known and newly identified genes encoding allergen isoforms are characterized during fruit growth and development, and upon different fruit load and light radiation regimes. According to the results herein presented, enhancing light radiation and decreasing the fruit load achieved a reduction of the transcription rate of the majority of allergen-encoding genes. Chapter 4 - Aspergillus carbonarius is responsible for the majority of mycotoxin contaminations in grapes and its derivatives, infecting berries from the early developmental stages throughout the whole winemaking process. Most of A. carbonarius strains are ochratoxin A (OTA) producers, even though at very different levels. This broad variability was used to identify genes whose expression is linked with the ability of producing OTA. A cDNA-AFLP differential display screening was performed in two strains of Aspergillus carbonarius, antagonists for the ability of producing OTA, allowing the identification of 119 differentially expressed sequences putatively involved in the biosyntesis of OTA and its regulation. A likely connection was pointed out between the biosynthesis of the toxin, vegetative growth and sexual/asexual developmental progression, along with common signalling pathways involving G protein and Ca2+/calmodulin dependent phosphorylation and dephoshorylation cascades. Chapter 5 - The transcriptomic approach adopted in the present research proved to be efficient, and allowed to achieve the following results: 1. The identification of putative hypoallergenic apple varieties 2. The identification of several factors affecting allergen-related gene expression in apple and peach 3. The identification of a peach variety with low content of Pru p 3 allergen 4. The classification of new candidate peach allergens 5. A model for OTA biosynthesis regulation in Aspergillus carbonarius The present research put the basis for the future development of: 1. Breeding programmes for the constitution of hypoallergenic apple varieties 2. Agricultural practices reducing the allergenic potential of apple and peach fruits 3. Recombinant allergens of peach for diagnostic purpose 4. Biocompetition strategies to avoid the contamination of grapes and wines with ochratoxin A