Recombination analysis of Maize dwarf mosaic virus (MDMV) in the Sugarcane mosaic virus (SCMV) subgroup of potyviruses.

Recombination among RNA viruses is a natural phenomenon that appears to have played a significant role in the species development and the evolution of many strains. It also has particular significance for the risk assessment of plants which have been genetically modified for disease resistance by incorporating viral sequences into their genomes. However, the exact recombination events taking place in viral genomes are not investigated in detail for many virus groups. In this analysis, different single-stranded positive-sense RNA potyviruses were compared using various in silico recombination detection methods and new recombination events in the Sugarcane mosaic virus (SCMV) subgroup were detected. For an extended in silico recombination analysis, two of the analyzed Maize dwarf mosaic virus full-length genomes were sequenced additionally during this work. These results strengthen the evidence that recombination is a major driving force in virus evolution, and the emergence of new virus variants in the SCMV subgroup, paired with mutations, could generate viruses with altered biological properties. The intra- and interspecific homolog recombinations seem to be a general trait in this virus group, causing little or no changes to the amino acid of the progenies. However, we found a few breakpoints between the members of SCMV subgroup and the weed-infecting distant relatives, but only a few methods of the RDP3 package predicted these events with low significance level.

The epitopes in wheat proteins for defining toxic units relevant to human health.

Wheat-related disorders are well-studied health problems. Knowledge of the composition and amounts of epitopes present in a single wheat sample represents a significant gap, and the detailed wheat proteome datasets now available can provide the necessary information to carry out an estimation of allergen prediction for a single cultivar. The combined use of genome sequence and allergen databases, prediction methodology, and cereal chemistry results in better understanding of the level of toxicity present in the end-products produced from wheat flour. The workflow presented in this review provides information about the number and distribution of epitopes at single protein, or protein fraction, levels. In addition, epitopes present in the highest frequency and harmful proteins expressed in the highest amount can be identified. The "epitope toxicity" value obtained in this way is a significant research output from the analysis of large datasets that can be applied to the food industry.

Immunoanalytic investigation of grain proteins antigenic for celiac disease patients in an einkorn collection.

Our study focuses on the complex characterization of a wild and cultivated einkorn collection of the Cereal Gene Bank of Agriculture Research Institute in Hungary, using proteomics, immune analytics and bioinformatics analyses. In a serological ELISA pre-screen of 208 different Triticum monococcum L. ssp. monococcum and Triticum monococcum L. ssp. aegilopoides genotypes with celiac disease samples high diversity was observed in the immune response. Based on the immune analytic results, four genotypes with significantly reduced immune reactivity were selected for detailed proteomics characterization. Our results confirm the benefits of high-throughput/large-scale pre-screening and the use of a complex examination platform to get relevant information about the genetic diversity of celiac disease-relevant proteins in the analyzed einkorn genotypes. These genotypes cannot be incorporated into the daily diet of celiac patients; however, they may represent candidates - especially in combination with enzymatic treatments - to improve the lifestyle of individuals suffering from other clinical conditions like non-celiac wheat sensitivity.

Effect of additional water supply during grain filling on protein composition and epitope characteristics of winter oats.

Pure oats in gluten-free diets (GFD) represent important nutritional benefits for people suffering from celiac disease (CD). However, oat cultivars do not contain the typical CD-related wheat gliadin analog polypeptides. Emerging evidence suggests that oat cultivars containing gluten-like epitopes in avenin sequences may pose potential health risks for celiac patients in rare cases, depending on the individual's susceptibility. Consequently, it is necessary to screen oats in terms of protein and epitope composition, to be able to select safe varieties for gluten-free applications. The overall aim of our study is to investigate the variation of oat protein composition directly related to health-related and techno-functional properties and to examine how the protein compositional parameters change due to irrigation during the grain-filling period as compared to the natural rain-fed grown, in a large winter oat population of different geographic origin. Elements of an oat sample population representing 164 winter oat varieties from 8 countries and the protein composition of resulting samples have been characterized. Size distribution of the total protein extracts has been analyzed by SE-HPLC, while the 70% ethanol extracted proteins were analyzed by RP-HPLC. Protein extracts are separated into 3 main groups of fractions on the SE-HPLC column; polymeric, avenin, and non-avenin monomeric protein groups, representing 59.17-80.87%, 12.89-31.03%, and 3.40-9.41% of total protein content, respectively. The ratio of polymeric to monomeric proteins varied between 1.71 and 6.07. 91 RP-HPLC-separated peaks have been differentiated from the ethanol extractable proteins of the entire population. The various parameters identified a lot of variation, confirming the significance of genotypic variation. In addition, it was also established that the additional water supply during grain filling significantly affected the various quantitative parameters of protein content, but not its qualitative structure. This environmental effect, however, was strongly genotype-dependent. Winter oat genotypes with low levels of epitope content were identified and it was proven that these characteristics were independent of the environmental factor of water availability. These genotypes are appropriate for initiating a specific breeding program to yield oat cultivars suitable for CD patients.

The Effect of Abiotic Stresses on the Protein Composition of Four Hungarian Wheat Varieties.

Global climate change in recent years has resulted in extreme heat and drought events that significantly influence crop production and endanger food security. Such abiotic stress during the growing season has a negative effect on yield as well as on the functional properties of wheat grain protein content and composition. This reduces the value of grain, as these factors significantly reduce end-use quality. In this study, four Hungarian bread wheat cultivars (Triticum aestivum ssp. aestivum) with different drought and heat tolerance were examined. Changes in the size- and hydrophobicity-based distribution of the total proteins of the samples have been monitored by SE- and RP-HPLC, respectively, together with parallel investigations of changes in the amounts of the R5 and G12 antibodies related to celiac disease immunoreactive peptides. Significant difference in yield, protein content and composition have been observed in each cultivar, altering the amounts of CD-related gliadin, as well as the protein parameters directly related to techno-functional properties (Glu/Gli ratio, UPP%). The extent of changes largely depended on the timing of the abiotic stress. The severity of the negative effect depended on the growth stage in which abiotic stress occurred.

Investigation of Protein and Epitope Characteristics of Oats and Its Implications for Celiac Disease.

The use of pure oats (oats cultivated with special care to avoid gluten contamination from wheat, rye, and barley) in the gluten-free diet (GFD) represents important nutritional benefits for the celiac consumer. However, emerging evidence suggests that some oat cultivars may contain wheat gliadin analog polypeptides. Consequently, it is necessary to screen oats in terms of protein and epitope composition to be able to select safe varieties for gluten-free applications. The overall aim of our study is to investigate the variability of oat protein composition directly related to health-related and techno-functional properties. Elements of an oat sample population representing 162 cultivated varieties from 20 countries and the protein composition of resulting samples have been characterized. Size distribution of the total protein extracts has been analyzed by size exclusion-high performance liquid chromatography (SE-HPLC) while the 70% ethanol-extracted proteins were analyzed by RP-HPLC. Protein extracts separated into three main groups of fractions on the SE-HPLC column: polymeric proteins, avenins (both containing three subgroups based on their size), and soluble proteins, representing respectively 68.79-86.60, 8.86-27.72, and 2.89-11.85% of the total protein content. The ratio of polymeric to monomeric proteins varied between 1.37 and 3.73. Seventy-six reversed phase-HPLC-separated peaks have been differentiated from the ethanol extractable proteins of the entire population. Their distribution among the cultivars varied significantly, 6-23 peaks per cultivar. The number of appearances of peaks also showed large variation: one peak has been found in 107 samples, while 15 peaks have been identified, which appeared in less than five cultivars. An estimation method for ranking the avenin-epitope content of the samples has been developed by using MS spectrometric data of collected RP-HPLC peaks and bioinformatics methods. Using ELISA methodology with the R5 antibody, a high number of the investigated samples were found to be contaminated with wheat, barley, or rye.

Natural insertions within the N-terminal region of the coat protein of Maize dwarf mosaic potyvirus (MDMV) have an effect on the RNA stability.

A 13 amino acid residue insertion was found in the N-terminal region of the coat protein of several Maize dwarf mosaic virus isolates (MDMV). These insertions seem to be the result of a direct duplication event, but differ in some positions. In order to evaluate the influence of the insertion on the RNA secondary structure and stability, the RNA secondary structures and minimum free energies (MFE) of all existing MDMV coat protein sequences were estimated using three different softwares, the Vienna RNA Package, NUPACK, and UNAFold, and compared to the secondary structure and MFE of various random sequence collections preserving the nucleotide distribution of MDMV. The bioinformatic analysis showed that the insertion stabilizes the RNA structure of the coat protein gene.

Genetic diversity of Hungarian Maize dwarf mosaic virus isolates.

The genetic diversity of the coat-protein (CP) region and the untranslated C-terminal region (3'UTR) of Maize dwarf mosaic virus (MDMV) was analyzed to evaluate the variability between isolates (inter-isolate sequence diversity). The results of inter-isolate sequence diversity analysis showed that the diversity of the MDMV CP gene is fairly high (p-distance: up to 0.136). During sequence analysis, a 13 amino-acid residue insertion and an 8 amino-acid residue deletion were found within the N-terminal region of the CP gene. The phylogenetic analysis showed that-unlike other potyvirus species in this subgroup-the MDMV isolates could not be distinguished on the basis of their host plants or geographic origins.

Genome mapping of seed-borne allergens and immunoresponsive proteins in wheat.

Wheat is an important staple grain for humankind globally because of its end-use quality and nutritional properties and its adaptability to diverse climates. For a small proportion of the population, specific wheat proteins can trigger adverse immune responses and clinical manifestations such as celiac disease, wheat allergy, baker's asthma, and wheat-dependent exercise-induced anaphylaxis (WDEIA). Establishing the content and distribution of the immunostimulatory regions in wheat has been hampered by the complexity of the wheat genome and the lack of complete genome sequence information. We provide novel insights into the wheat grain proteins based on a comprehensive analysis and annotation of the wheat prolamin Pfam clan grain proteins and other non-prolamin allergens implicated in these disorders using the new International Wheat Genome Sequencing Consortium bread wheat reference genome sequence, RefSeq v1.0. Celiac disease and WDEIA genes are primarily expressed in the starchy endosperm and show wide variation in protein- and transcript-level expression in response to temperature stress. Nonspecific lipid transfer proteins and α-amylase trypsin inhibitor gene families, implicated in baker's asthma, are primarily expressed in the aleurone layer and transfer cells of grains and are more sensitive to cold temperature. The study establishes a new reference map for immunostimulatory wheat proteins and provides a fresh basis for selecting wheat lines and developing diagnostics for products with more favorable consumer attributes.

Characterization of globulin storage proteins of a low prolamin cereal species in relation to celiac disease.

Brachypodium distachyon, a small annual grass with seed storage globulins as primary protein reserves was used in our study to analyse the toxic nature of non-prolamin seed storage proteins related to celiac disease. The main storage proteins of B. distachyon are the 7S globulin type proteins and the 11S, 12S seed storage globulins similar to oat and rice. Immunoblot analyses using serum samples from celiac disease patients were carried out followed by the identification of immune-responsive proteins using mass spectrometry. Serum samples from celiac patients on a gluten-free diet, from patients with Crohn's disease and healthy subjects, were used as controls. The identified proteins with intense serum-IgA reactivity belong to the 7S and 11-12S seed globulin family. Structure prediction and epitope predictions analyses confirmed the presence of celiac disease-related linear B cell epitope homologs and the presence of peptide regions with strong HLA-DQ8 and DQ2 binding capabilities. These results highlight that both MHC-II presentation and B cell response may be developed not only to prolamins but also to seed storage globulins. This is the first study of the non-prolamin type seed storage proteins of Brachypodium from the aspect of the celiac disease.

Polyamines may influence phytochelatin synthesis during Cd stress in rice.

Although the metabolism of phytochelatins and higher polyamines are linked with each other, the direct relationship between them under heavy metal stress has not yet been clarified. Two approaches were used to reveal the influence of polyamine content on cadmium stress responses, particularly with regard to phytochelatin synthesis: putrescine pre-treatment of rice plants followed by cadmium stress, and treatment with the putrescine synthesis inhibitor, 2-(difluoromethyl)ornithine combined with cadmium treatment. The results indicated that putrescine pre-treatment enhanced the adverse effect of cadmium, while the application of 2-(difluoromethyl)ornithine reduced it to a certain extent. These differences were associated with increased polyamine content, more intensive polyamine metabolism, but decreased thiol and phytochelatin contents. The gene expression level and enzyme activity of phytochelatin synthase also decreased in rice treated with putrescine prior to cadmium stress, compared to cadmium treatment alone. In contrast, the inhibition of putrescine synthesis during cadmium treatment resulted in higher gene expression level of phytochelatin synthase. The results suggest that polyamines may have a substantial influence on phytochelatin synthesis at several levels under cadmium stress in rice.