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Pathogenesis-related-10 (PR10) proteins play significant roles in plant defence against biotic and abiotic stresses. Recently, two banana PR10 proteins (MaPR10-BeB5 and MaPR10-GNA5) were characterised and shown to exhibit antifungal properties against Aspergillus fumigatus in vitro . In rice, transgenic overexpression of PR10 proteins conferred resistance to pathogen infection and drought tolerance without affecting productivity, highlighting their potential for agricultural applications. However, PR10 proteins also include the Bet v 1-like family of allergens implicated in pollen food allergy syndromes, raising concerns about potential adverse effects on human health. In this study, we evaluated the allergenic potential of the recently isolated banana PR10 proteins. We first predicted the presence of IgE epitopes of the Bet v 1 allergen family in the deduced PR10 peptide sequences in silico . We then predicted the structures of four human IgE scFv protein sequences and three plant PR10 protein sequences. Based on the quality of the predicted structures, one IgE scFv protein structure was selected for docking with the three plant PR10 proteins. We confirmed the docking results with immunoblot analysis performed using recombinant MaPR10-BeB5 and MaPR10-GNA5 proteins against the sera of banana-allergic patients. Our experimental results substantiated the notion that both protein variants are potentially allergenic since these proteins were recognised by 26.6% of banana-allergic patients with broad PR10 protein recognition. We caution that the allergenic potential of MaPR10 proteins should be carefully considered before implementing transgenic overexpression strategies to improve crops, with a suggestion to limit their expression to non-edible plant tissues.

Soybean is an important plant source of protein worldwide. Increasing demands for soybean can be met by improving the quality of its seed protein. In this study, GmCG-1, which encodes the β-conglycinin α' subunit, was identified via combined genome-wide association study and transcriptome analysis. We subsequently knocked down GmCG-1 and its paralogues GmCG-2 and GmCG-3 with CRISPR-Cas9 technology and generated two stable multigene knockdown mutants. As a result, the β-conglycinin content decreased, whereas the 11S/7S ratio, total protein content and sulfur-containing amino acid content significantly increased. Surprisingly, the globulin mutant exhibited salt tolerance in both the germination and seedling stages. Little is known about the relationship between seed protein composition and the salt stress response in soybean. Metabonomics and RNA-seq analysis indicated that compared with the WT, the mutant was formed through a pathway that was more similar to that of active salicylic acid biosynthesis; however, the synthesis of cytokinin exhibited greater defects, which could lead to increased expression of plant dehydrin-related salt tolerance proteins and cell membrane ion transporters. Population evolution analysis suggested that GmCG-1, GmCG-2, and GmCG-3 were selected during soybean domestication. The soybean accessions harboring GmCG-1 Hap1 presented relatively high 11S/7S ratios and relatively high salt tolerance. In conclusion, knockdown of the β-conglycinin α and α' subunits can improve the nutritional quality of soybean seeds and increase the salt tolerance of soybean plants, providing a strategy for designing soybean varieties with high nutritional value and high salt tolerance., (© 2024 Society for Experimental Biology and John Wiley & Sons Ltd.)

Pru p 7 (also named Peamaclein) is a member of the gibberellin-regulated protein family, which is the latest foodborne allergenic protein identified in peach. In this paper, the prokaryotic expression and identification of Pru p 7 were performed, and the protein properties, structure, and homology were analyzed. In addition, a preliminary screening of B-cell linear epitopes of Pru p 7 was performed by the bioinformatics software prediction method, and three epitopes were identified using slot-blot immune microarray assay combined with an immune score matrix (P-1, AA16-21, AGYQER; P-2, AA40-46, TYGNKDE; P-3, AA52-59, DLKNSKGN). Moreover, the electrostatic potential of these epitopes was analyzed, and the stability after ultrahigh pressure treatment was also verified. Finally, the amino acids that play key immune roles in the epitopes were obtained by amino acid mutations. These results may contribute to the further understanding of Pru p 7 and the prevention of peach allergy.

Expanded agriculture production is required to support the world's population but can impose substantial environmental and climate change costs, particularly with intensifying animal production and protein demand. Shifting from an animal- to a plant-based protein diet has numerous health benefits. Soybean (Glycine max [L.] Merr.) is a major source of protein for human food and animal feed; improved soybean protein content and amino acid composition could provide high-quality soymeal for animal feed, healthier human foods, and a reduced carbon footprint. Nonetheless, during the soybean genome evolution, a balance was established between the amount of seed protein, oil, and carbohydrate content, burdening the development of soybean cultivars with high proteins (HPs). We isolated 2 high-seed protein soybean mutants, HP1 and HP2, with improved seed amino acid composition and stachyose content, pointing to their involvement in controlling seed rebalancing phenomenon. HP1 encodes β-conglycinin (GmCG-1) and HP2 encodes sucrose-binding protein (GmSBP-1), which are both highly expressed in soybean seeds. Mutations in GmSBP-1, GmCG-1, and the paralog GmCG-2 resulted in increased protein levels, confirming their role as general regulators of seed protein content, amino acid seed composition, and seed vigor. Biodiversity analysis of GmCG and GmSBP across 108 soybean accessions revealed haplotypes correlated with protein and seed carbohydrate content. Furthermore, our data revealed an unprecedented role of GmCG and GmSBP proteins in improving seed vigor, crude protein, and amino acid digestibility. Since GmSBP and GmCG are present in most seed plants analyzed, these genes could be targeted to improve multiple seed traits., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

This study investigated the effects of genetic diversity in the allergenicity of peanut and assessed the allergenic capacity of six Arachis hypogaea accessions using a Balb/c mouse model. It also explored potential cross-reactivities between Ara h 3 (peanut allergen) and Gly m (soybean allergen) using computational tools. Female Balb/c mice were injected with peanut protein extracts and alum. Serum-specific antibodies (IgE, IgGt, IgG1, IgG2a) were measured using ELISA, and allergic protein profiles were examined via western blot. Structural homology, B cell epitopes, and molecular interactions between Ara h 3 and Gly m with human IgE were also investigated. The mice developed high sIgE and sIgG1 responses, with antibodies recognizing 19 bands on western blot. Notably, Saharan accessions showed unique features such as no bands on western blot profiles, reduced anaphylactic symptoms, lower IgE titers, and less intestinal tissue damage. Molecular docking results suggest significant cross-allergenicity, supported by allergenicity predictions and structural homology analysis. This comprehensive analysis provides insights into shared epitopes, potential competition for binding sites, and molecular dynamics of cross-reactive responses, enhancing understanding of food allergen interactions. The study recommends using Algerian Sahara peanut accessions in breeding, genomics studies, and industry for safer peanut options for individuals with allergies. SIGNIFICANCE: The significance of this study lies in its contribution to addressing a major public health issue: peanut allergy, which represents a significant cause of anaphylaxis affecting numerous individuals and families worldwide. By exploring the genetic diversity of peanut proteins and identifying hypoallergenic accessions through experimental and computational approaches, this research offers valuable insights for mitigating allergic reactions. The findings highlight that certain accessions from the Saharan region exhibit reduced allergenicity, resulting in attenuated anaphylactic symptoms, lower IgE levels, and reduced intestinal damage in murine models. Furthermore, the study's in silico analysis sheds light on the issue of cross-reactivity between peanut and soybean allergens, providing crucial information for understanding allergen interactions at the molecular level. Overall, this research contributes to advancing knowledge in the field of food allergen research and has practical implications for improving the quality of life for individuals allergic to peanuts, particularly through the selection of safer peanut varieties and their cultivation., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Allergen detection methods support food labeling and quality assessment at the allergen component level of allergen preparations used for allergy diagnosis and immunotherapy (AIT). Commonly applied enzyme-linked immunosorbent assay (ELISA) requires animal antibodies but potentially shows batch variations. We developed synthetic aptamers as alternative binders in allergen detection to meet the replacement, reduction, and refinement (3R) principle on animal protection in science. ssDNA aptamers were specifically selected against the major peanut allergen Ara h 1 and identified by next-generation sequencing. Application in various detection systems (ELISA-like assays, western blot, and surface plasmon resonance) was demonstrated. The ELISA-like assay comprised a sensitivity of 10 ng/mL Ara h 1, comparable to published antibody-based ELISA, and allowed Ara h 1 detection in various peanut flours, similar to those used in peanut AIT as well as in processed food. This ELISA-like aptamer-based assay proofs antibody-free allergen detection for food labeling or quality assessment of diagnostic and therapeutic allergen products.

Soybean β-conglycinin is a major allergen that adversely affects the nutritional properties of soybean. Soybean deficient in β-conglycinin is associated with low allergenicity and high nutritional value. Long intergenic noncoding RNAs (lincRNAs) regulate gene expression and are considered important regulators of essential biological processes. Despite increasing knowledge of the functions of lincRNAs, relatively little is known about the effects of lincRNAs on the accumulation of soybean β-conglycinin. The current study presents the identification of a lincRNA lincCG1 that was mapped to the intergenic noncoding region of the β-conglycinin α-subunit locus. The full-length lincCG1 sequence was cloned and found to regulate the expression of soybean seed storage protein (SSP) genes via both cis- and trans- acting regulatory mechanisms. Loss-of-function lincCG1 mutations generated using the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system led to the deficiency of the allergenic α'-, α-, and β-subunits of soybean β-conglycinin as well as higher content of proteins, sulfur-containing amino acids, and free arginine. The dominant null allele LincCG1 , and consequently, the β-conglycinin-deficient phenotype associated with the lincCG1 -gene-edited line was stably inherited by the progenies in a Mendelian fashion. The dominant null allele LincCG1 may therefore be exploited for engineering/developing novel hypoallergenic soybean varieties. Furthermore, Cas9-free and β-conglycinin-deficient homozygous mutant lines were obtained in the T 1 generation. This study is the first to employ the CRISPR/Cas9 technology for editing a lincRNA gene associated with the soybean allergenic protein β-conglycinin. Moreover, this study reveals that lincCG1 plays a crucial role in regulating the expression of the β-conglycinin subunit gene cluster, besides highlighting the efficiency of employing the CRISPR/Cas9 system for modulating lincRNAs, and thereby regulating soybean seed components.

Pollen from Salsola kali , i.e., saltwort, Russian thistle, is a major allergen source in the coastal regions of southern Europe, in Turkey, Central Asia, and Iran. S. kali- allergic patients mainly suffer from hay-fever (i.e., rhinitis and conjunctivitis), asthma, and allergic skin symptoms. The aim of this study was to investigate the importance of individual S. kali allergen molecules. Sal k 1, Sal k 2, Sal k 3, Sal k 4, Sal k 5, and Sal k 6 were expressed in Escherichia coli as recombinant proteins containing a C-terminal hexahistidine tag and purified by nickel affinity chromatography. The purity of the recombinant allergens was analyzed by SDS-PAGE. Their molecular weight was determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and their fold and secondary structure were studied by circular dichroism (CD) spectroscopy. Sera from clinically well-characterized S. kali -allergic patients were used for IgE reactivity and basophil activation experiments. S. kali allergen-specific IgE levels and IgE levels specific for the highly IgE cross-reactive profilin and the calcium-binding allergen from timothy grass pollen, Phl p 12 and Phl p 7, respectively, were measured by ImmunoCAP. The allergenic activity of natural S. kali pollen allergens was studied in basophil activation experiments. Recombinant S. kali allergens were folded when studied by CD analysis. The sum of recombinant allergen-specific IgE levels and allergen-extract-specific IgE levels was highly correlated. Sal k 1 and profilin, reactive with IgE from 64% and 49% of patients, respectively, were the most important allergens, whereas the other S. kali allergens were less frequently recognized. Specific IgE levels were highest for profilin. Of note, 37% of patients who were negative for Sal k 1 showed IgE reactivity to Phl p 12, emphasizing the importance of the ubiquitous cytoskeletal actin-binding protein, profilin, for the diagnosis of IgE sensitization in S. kali -allergic patients. rPhl p 12 and rSal k 4 showed equivalent IgE reactivity, and the clinical importance of profilin was underlined by the fact that profilin-monosensitized patients suffered from symptoms of respiratory allergy to saltwort. Accordingly, profilin should be included in the panel of allergen molecules for diagnosis and in molecular allergy vaccines for the treatment and prevention of S. kali allergy., Competing Interests: RV received research grants from Worg Pharmaceuticals, Hangzhou, China; HVD Biotech, Vienna, Austria; and Viravaxx, Vienna, Austria and serves as a consultant for Worg and Viravaxx. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Peterkova, Trifonova, Gattinger, Focke-Tejkl, Garib, Magbulova, Djambekova, Zakhidova, Ismatova, Sekerel, Tuten Dal, Tulaev, Kundi, Keller, Karaulov and Valenta.)

Food allergies mediated by specific IgE (sIgE) have a significant socioeconomic impact on society. Evaluating the IgE cross-reactivity between allergens from different allergen sources can enable the better management of these potentially life-threatening adverse reactions to food proteins and enhance food safety. A novel banana fruit allergen, S-adenosyl-L-homocysteine hydrolase (SAHH), has been recently identified and its recombinant homolog was heterologously overproduced in E. coli . In this study, we performed a search in the NCBI (National Center for Biotechnology Information) for SAHH homologs in ryegrass, latex, and kiwifruit, all of which are commonly associated with pollen-latex-fruit syndrome. In addition, Western immunoblot analysis was utilized to identify the cross-reactive IgE to banana SAHH in the sera of patients with a latex allergy, kiwifruit allergy, and ryegrass allergy. ClustalOmega analysis showed more than 92% amino acid sequence identity among the banana SAHH homologs in ryegrass, latex, and kiwifruit. In addition to five B-cell epitopes, in silico analysis predicted eleven T-cell epitopes in banana SAHH, seventeen in kiwifruit SAHH, twelve in ryegrass SAHH, and eight in latex SAHH, which were related to the seven-allele HLA reference set (HLA-DRB1*03:01, HLA-DRB1*07:01, HLA-DRB1*15:01, HLA-DRB3*01:01, HLA-DRB3*02:02, HLA-DRB4*01:01, HLA-DRB5*01:01). Four T-cell epitopes were identical in banana and kiwifruit SAHH (positions 328, 278, 142, 341), as well as banana and ryegrass SAHH (positions 278, 142, 96, and 341). All four SAHHs shared two T-cell epitopes (positions 278 and 341). In line with the high amino acid sequence identity and B-cell epitope homology among the analyzed proteins, the cross-reactive IgE to banana SAHH was detected in three of three latex-allergic patients, five of six ryegrass-allergic patients, and two of three kiwifruit-allergic patients. Although banana SAHH has only been studied in a small group of allergic individuals, it is a novel cross-reactive food allergen that should be considered when testing for pollen-latex-fruit syndrome.

Common ragweed pollen allergy has become a health burden worldwide. One of the major allergens in ragweed allergy is Amb a 1, which is responsible for over 90% of the IgE response in ragweed-allergic patients. The major allergen isoform Amb a 1.01 is the most allergenic isoform in ragweed pollen. So far, no recombinant Amb a 1.01 with similar allergenic properties to its natural counterpart (nAmb a 1.01) has been produced. Hence, this study aimed to produce a recombinant Amb a 1.01 with similar properties to the natural isoform for improved ragweed allergy management. Amb a 1.01 was expressed in insect cells using a codon-optimized DNA construct with a removable N -terminal His-Tag (rAmb a 1.01). The recombinant protein was purified by affinity chromatography and physicochemically characterized. The rAmb a 1.01 was compared to nAmb a 1.01 in terms of the IgE binding (enzyme-linked immunosorbent assay (ELISA), immunoblot) and allergenic activity (mediator release assay) in well-characterized ragweed-allergic patients. The rAmb a 1.01 exhibited similar IgE reactivity to nAmb a 1.01 in different IgE-binding assays (i.e., IgE immunoblot, ELISA, quantitative ImmunoCAP inhibition measurements). Furthermore, the rAmb a 1.01 showed comparable dose-dependent allergenic activity to nAmb a 1.01 regarding basophil activation. Overall, the results showed the successful expression of an rAmb a 1.01 with comparable characteristics to the corresponding natural isoform. Our findings provide the basis for an improvement in ragweed allergy research, diagnosis, and immunotherapy.

Glycinin is an important allergenic protein. A1a is the acidic chain of the G1 subunit in glycinin (G1A1a), and it has strong allergenicity. In this study, we used phage display technology to express the protein of G1A1a and its overlapping fragments and an indirect enzyme-linked immunosorbent assay (iELISA) to determine the antigenicity and allergenicity of the expressed protein. After three rounds of screening, it was determined that fragment A1a-2-B-I ( 151 SLENQLDQMPRRFYLAGNQEQEFLKYQQEQG 181 ) is the allergenic domain of G1A1a destroyed by thermal processing. In addition, three overlapping peptides were synthesized from fragments A1a-2-B-I, and a linear epitope was found in this domain through methods including dot blot and iELISA. Peptide 2 ( 157 DQMPRRFYLANGNQE 170 ) showed allergenicity, and after replacing it with alanine, it was found that amino acids D 157 , Q 158 , M 159 , and Y 164 were the key amino acids that affected its antigenicity, while Q 158 , M 159 , R 162 , and N 168 affected allergenicity.

Novel allergen immunotherapy (AIT) approaches necessitate the use of more effective and safe therapeutics, which can be accomplished by employing novel adjuvants for improved innate immune cell activation, as well as hypoallergenic allergen forms. In this study, we investigate the immunomodulatory effects of a chimera rBet v 1a-BanLec wt (rBv1a-BL wt ; C wt ) composed of the major birch pollen allergen Bet v 1a and banana lectin (BanLec wt ; BLwt) and two novel chimeras, rBv1l-BL H84T (rBet v 1l-BanLec H84T ; C1) and rBL H84T -Bv1l (rBanLec H84T -Bet v 1l; C2), both composed of BL H84T and hypoallergenic birch pollen allergen Bv1l in the co-culture model Caco-2/THP-1, and PBMCs from donors with birch pollen allergy. The chimeric molecules rBv1l-BL H84T (C1) and rBL H84T -Bv1l (C2) were created in silico and then produced in E. coli using recombinant DNA technology. Real-time PCR analysis of gene expression following compound treatment in the co-culture model revealed that all three chimeras have the potential to induce the anti-inflammatory cytokine IL-10 gene expression in Caco-2 cells and IFN-γ gene expression in THP-1 cells. Sandwich ELISA revealed that Cwt increased IL-10 secretion and IFN-/IL-4 levels in PBMCs from birch pollen allergic donors, whereas C1 and C2 were less effective. The findings suggest that Cwt should be analyzed further due to its potential benefit in AIT., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The hazel allergen Cor a 1 is a PR-10 protein, closely related to the major birch pollen allergen Bet v 1. Hazel allergies are caused by cross-reactive IgE antibodies originally directed against Bet v 1. Despite the importance of PR-10 proteins in allergy development, their function and localization in the plant remain largely elusive. Therefore, the presence of Cor a 1 mRNA and proteins was investigated in different tissues, i.e., the female flower, immature and mature nuts, catkins, and pollen. Four yet unknown Cor a 1 isoallergens, i.e., Cor a 1.0501-1.0801, and one new Cor a 1.03 variant were discovered and characterized. Depending on the isoallergen, the occurrence and level of mRNA expression varied in different tissues, suggesting different functions. Interestingly, Cor a 1.04 previously thought to be only present in nuts, was also detected in catkins and pollen. The corresponding Cor a 1 genes were expressed in Escherichia coli. The purified proteins were analysed by CD and NMR spectroscopy. Immunoblots and ELISAs to determine their allergenic potential showed that the new proteins reacted positively with sera from patients allergic to birch, hazel and elder pollen and were recognized as novel isoallergens/variants by the WHO/IUIS Allergen Nomenclature Sub-Committee., (© 2024. The Author(s).)

Ambrosia trifida (giant ragweed) is an invasive plant that can cause serious damage to natural ecosystems and severe respiratory allergies. However, the genomic basis of invasive adaptation and pollen allergens in Ambrosia species remain largely unknown. Here, we present a 1.66 Gb chromosome-scale reference genome for giant ragweed and identified multiple types of genome duplications, which are responsible for its rapid environmental adaptation and pollen development. The largest copies number and species-specific expansions of resistance-related gene families compared to Heliantheae alliance might contribute to resist stresses, pathogens and rapid adaptation. To extend the knowledge of evolutionary process of allergic pollen proteins, we predicted 26 and 168 potential pollen allergen candidates for giant ragweed and other Asteraceae plant species by combining machine learning and identity screening. Interestingly, we observed a specific tandemly repeated array for potential allergenic pectate lyases among Ambrosia species. Rapid evolutionary rates on putative pectate lyase allergens may imply a crucial role of nonsynonymous mutations on amino acid residues for plant biological function and allergenicity. Altogether, this study provides insight into the molecular ecological adaptation and putative pollen allergens prediction that will be helpful in promoting invasion genomic research and evolution of putative pollen allergy in giant ragweed., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024. Published by Elsevier B.V.)

Introduction: Artemisia species are widely spread in north hemisphere. Artemisia sieversiana pollen is one of the common pollen allergens in the north of China. At present, seven allergens were identified and had been listed officially from A. sieversiana pollen, but the remaining allergens are still insufficiently studied, which need to be found., Methods: Pectate lyase was purified from the extracts of A. sieversiana pollen by anion exchange, size exclusion, and HPLC-hydrophobic interaction chromatography. The gene of A. sieversiana pectate lyase (Art si pectate lyase) was cloned and expressed in Escherichia coli. The enzyme activity and circular dichroism (CD) spectrum of natural and recombinant proteins were analyzed. The allergenicity of Art si pectate lyase was characterized by enzyme-linked immunosorbent assay (ELISA), Western blot, inhibition ELISA, and basophil activation test. The allergen's physicochemical properties, three-dimensional structure, sequence profiles with homologous allergens and phylogenetic tree were analyzed by in silico methods., Results: Natural Art si pectate lyase (nArt si pectate lyase) was purified from A. sieversiana pollen extracts by three chromatographic strategies. The cDNA sequence of Art si pectate lyase had a 1191-bp open reading frame encoding 396 amino acids. Both natural and recombinant pectate lyase (rArt si pectate lyase) exhibited similar CD spectrum, and nArt si pectate lyase had higher enzymatic activity. Moreover, the specific immunoglobulin E (IgE) binding rate against nArt si pectate lyase and rArt si pectate lyase was determined as 40% (6/15) in patients' serum with Artemisia species pollen allergy by ELISA. The nArt si pectate lyase and rArt si pectate lyase could inhibit 76.11% and 47.26% of IgE binding activities to the pollen extracts, respectively. Art si pectate lyase was also confirmed to activate patients' basophils. Its structure contains a predominant motif of classic parallel helical core, consisting of three parallel β-sheets, and two highly conserved features (vWiDH, RxPxxR) which may contribute to pectate lyase activity. Moreover, Art si pectate lyase shared the highest sequence identity of 73.0% with Art v 6 among currently recognized pectate lyase allergen, both were clustered into the same branch in the phylogenetic tree., Conclusion: In this study, pectate lyase was identified and comprehensively characterized as a novel allergen in A. sieversiana pollen. The findings enriched the allergen information for this pollen and promoted the development of component-resolved diagnosis and molecular therapy of A. sieversiana pollen allergy., (© 2024 S. Karger AG, Basel.)

Raw strawberries contain allergens that cause oral allergic syndrome. Fra a 1 is one of the major allergens in strawberries and might decrease their allergenicity by heating, likely due to structural changes in the allergen leading to decreased recognition of the allergens in the oral cavity. In the present study, to understand the relationship between allergen structure and allergenicity, the expression and purification of 15N-labeled Fra a 1 were examined and the sample was used for NMR analysis. Two isoforms, Fra a 1.01 and Fra a 1.02, were used and expressed in E. coli BL21(DE3) in M9 minimal medium. Fra a 1.02 was purified as a single protein by using the GST tag approach, whereas histidine × 6-tag (his6-tag) Fra a 1.02 was obtained both as the full-length (∼20 kDa) and a truncated (∼18 kDa) form. On the other hand, his6-tag Fra a 1.01 was purified as a homogeneous protein. 1 ⁵N-labeled HSQC NMR spectra suggested that Fra a 1.02 was thermally denatured at lower temperatures than Fra a 1.01, despite the high amino acid sequence homology (79.4%) of these isoforms. Furthermore, the samples in the present study allowed us to analyze ligand binding that probably affects structural stability. In conclusion, GST tag was effective for obtaining a homogeneous protein when his6-tag failed to give a single form, and the present study provided a sample that could be used for NMR studies of the details of the allergenicity and structure of Fra a 1., (Copyright © 2023 Elsevier Inc. All rights reserved.)

The term allergy was coined in 1906 by the Austrian scientist and pediatrician Clemens Freiherr von Pirquet. In 1976, Dietrich Kraft became the head of the Allergy and Immunology Research Group at the Department of General and Experimental Pathology of the University of Vienna. In 1983, Kraft proposed to replace natural extracts used in allergy diagnostic tests and vaccines with recombinant allergen molecules and persuaded Michael Breitenbach to contribute his expertise in molecular cloning as one of the mentors of this project. Thus, the foundation for the Vienna School of Molecular Allergology was laid. With the recruitment of Heimo Breiteneder as a young molecular biology researcher, the work began in earnest, resulting in the publication of the cloning of the first plant allergen Bet v 1 in 1989. Bet v 1 has become the subject of a very large number of basic scientific as well as clinical studies. Bet v 1 is also the founding member of the large Bet v 1-like superfamily of proteins with members-based on the ancient conserved Bet v 1 fold-being present in all three domains of life, i.e., archaea, bacteria and eukaryotes. This suggests that the Bet v 1 fold most likely already existed in the last universal common ancestor. The biological function of this protein was probably related to lipid binding. However, during evolution, a functional diversity within the Bet v 1-like superfamily was established. The superfamily comprises 25 families, one of which is the Bet v 1 family, which in turn is composed of 11 subfamilies. One of these, the PR-10-like subfamily of proteins, contains almost all of the Bet v 1 homologous allergens from pollen and plant foods. Structural and functional comparisons of Bet v 1 and its non-allergenic homologs of the superfamily will pave the way for a deeper understanding of the allergic sensitization process.

Structural and functional information about food allergens is essential for understanding the allergenicity of food proteins. All allergens belong to a small number of protein families. Various allergens from different families have been successfully produced recombinantly in E. coli for their characterization and applications in allergy diagnosis and treatment. However, recombinant hexameric 11S seed storage protein has not been reported, although numerous 11S legumins are known to be food allergens, including the recently identified macadamia nut allergen Mac i 2. Here we report the production of a macadamia nut legumin by expressing it in E. coli with a substrate site of HRV 3C protease and cleaving the purified protein with HRV 3C protease. The protease divided the protein into two chains and left a native terminus for the C-terminal chain, resulting in a recombinant hexameric 11S allergen for the first time after the residues upstream to the cleavage site flipped out of the way of the trimer-trimer interaction. The 11S allergens are known to have multiple isoforms in many species. The present study removed an obstacle in obtaining homogeneous allergens needed for studying allergens and mitigating allergenicity. Immunoreactivity of the protein with serum IgE confirmed it to be a new isoform of Mac i 2., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest associated with this publication. Mention of trade names or commercial products in this publication is solely to provide specific information and does not imply recommendation or endorsement by USDA. USDA is an equal opportunity provider and employer., (Published by Elsevier Inc.)

Linear IgE epitopes play essential roles in persistent allergies, including peanut and tree nut allergies. Using chemically synthesized peptides attached to membranes and microarray experiments is one approach for determining predominant epitopes that has seen success. However, the overall expense of this approach and the inherent challenges in scaling up the production and purification of synthetic peptides precludes the general application of this approach. To overcome this problem, we have constructed a plasmid vector for expressing peptides sandwiched between an N-terminal His-tag and a trimeric protein. The vector was used to make overlapping peptides derived from peanut allergens Ara h 2. All the peptides were successfully expressed and purified. The resulting peptides were applied to identify IgE binding epitopes of Ara h 2 using four sera samples from individuals with known peanut allergies. New and previously defined dominant IgE binding epitopes of Ara h 2 were identified. This system may be readily applied to produce agents for component- and epitope-resolved food allergy diagnosis., Competing Interests: Conflicts of interest The authors have no conflict of interest to declare. Mention of trade names or commercial products in this publication is solely to provide specific information and does not imply recommendation or endorsement by USDA. USDA is an equal opportunity provider and employer., (Published by Elsevier Ltd.)

In this work, three allergen-encoding genes (Ana o 1, Ana o 2, Ana o 3) were investigated for the detection of cashew nut as an allergenic food. Normalised and single-tube nested real-time PCR approaches targeting the Ana o 2 or Ana o 3 genes are proposed and compared. Normalised real-time PCR detected 10 pg, while single-tube nested real-time PCR achieved 1 pg of cashew nut DNA. Single-tube nested real-time PCR targeting Ana o 3 allowed the best relative sensitivities (10 mg/kg cashew nut in dough/biscuit), being successfully validated regarding precision/accuracy. The normalised real-time PCR did not show acceptable accuracy for both targets. Sensitivity of single-tube nested real-time PCR was affected by the matrix (pasta), but not by thermal processing (dough/biscuit). Herein, two highly sensitive and specific single-tube nested real-time PCR targeting allergen-encoding genes are proposed for the first time as quantitative/validated tools for cashew nut analysis as an allergenic food., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Gibberellin-regulated protein (GRP) is a fruit severe allergen. The amounts of GRP expression normalized against actin in peach were determined by reverse transcription-quantitative PCR (RT-qPCR). The results were consistent with those determined by enzyme-linked immunosorbent assay (ELISA). The GRP expression was more evident in flesh than peel and increased rapidly in the maturing period. This approach is applicable to estimate the amount of GRP in other plants., (© The Author(s) 2022. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

Giant ragweed (Ambrosia trifida) pollen is closely associated with respiratory allergy in late summer and autumn, and the prevalence of giant ragweed pollen allergy progressively increases. Compared with short ragweed (Ambrosia artemisiifolia), allergenic components from giant ragweed pollen are poorly investigated. To promote component-resolved diagnosis and treatment for giant ragweed pollen allergy, it becomes necessary to identify and characterize unknown allergens from giant ragweed pollen. In the present study, we identified and characterized a new cysteine-protease (CP) allergen from giant ragweed pollen, named as Amb t CP. The cloned Amb t CP gene encoded 387 amino acids. Recombinant Amb t CP (rAmb t CP) and natural Amb t CP (nAmb t CP) were purified by high-affinity Ni 2+ resin and immunoaffinity chromatography respectively. During refolding, purified rAmb t CP could autocatalytically converted to its mature forms displaying a higher enzymatic activity. Moreover, the autocatalytic conversion of proforms to mature forms of nAmb t CP could cause their amount to change in giant ragweed pollen extracts. Then, the allergenicity of Amb t CP was characterized: 23 (33.8%) of 68 Chinese patients with ragweed pollen allergy showed positive IgE binding to nAmb t CP by enzyme-linked immunosorbent assay (ELISA); the result of subsequent ELISA showed that IgE-binding activity of proforms and mature forms of rAmb t CP was different, with positive rate of 39.1% (9/23) and 47.8% (11/23) respectively; Amb t CP showed IgE cross-reactivity with the CP components from short ragweed, Artemisia annua and Artemisia sieversiana pollen. Our findings will help to promote component-resolved diagnosis and treatment for giant ragweed pollen allergy, standardize allergen products and individualize allergen-specific immunotherapy., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

The cultivated peanut ( Arachis hypogaea L.) is a legume consumed worldwide in the form of oil, nuts, peanut butter, and candy. Improving peanut production and nutrition will require new technologies to enable novel trait development. Clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9 (CRISPR-Cas9) is a powerful and versatile genome-editing tool for introducing genetic changes for studying gene expression and improving crops, including peanuts. An efficient in vivo transient CRISPR-Cas9- editing system using protoplasts as a testbed could be a versatile platform to optimize this technology. In this study, multiplex CRISPR-Cas9 genome editing was performed in peanut protoplasts to disrupt a major allergen gene with the help of an endogenous tRNA-processing system. In this process, we successfully optimized protoplast isolation and transformation with green fluorescent protein (GFP) plasmid, designed two sgRNAs for an allergen gene, Ara h 2 , and tested their efficiency by in vitro digestion with Cas9. Finally, through deep-sequencing analysis, several edits were identified in our target gene after PEG-mediated transformation in protoplasts with a Cas9 and sgRNA-containing vector. These findings demonstrated that a polyethylene glycol (PEG)-mediated protoplast transformation system can serve as a rapid and effective tool for transient expression assays and sgRNA validation in peanut.

Birch belongs to the order Fagales and the family Betulaceae. Birch pollen is one of the most important airborne inhaled allergens in the north temperate zone, leading to allergic rhinitis, asthma and pollen-related food allergy. The sensitization rate to birch pollen is about 8-16% in the general population and 7-57% in patients seen at various allergy centers. Seven birch pollen allergens have been recognized by the International Allergen Nomenclature Sub-committee, with Bet v 1 as the sole major allergen. Component-resolved diagnostics can help to discriminate broad cross-reactivity and false-positive diagnoses of pollen allergy caused by specific IgE to pan-allergens such as Bet v 2, 4 or Bet v 7 from true birch allergy represented by the major allergen Bet v 1-specific IgE. Patients with allergic symptoms to birch pollen showed significantly higher serum anti-Bet v 1 IgE concentrations than asymptomatic individuals with birch sensitization. A higher level of IgE to Bet v 1 also predicted oral allergy syndrome after the ingestion of Rosaceae fruits, nuts, or Apiaceae vegetables, which have cross-reactive homologous allergens with birch allergens. Bet v 1 is one of the first allergens developed using recombinant technology. Many forms of genetically modified Bet v 1 hypo-allergens have been developed and have shown benefit in animal models or even clinical trials of allergen immunotherapy., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Background Quantitative detection of allergens is of great significance for clarifying the cause, treatment, and prevention of allergy disease. Birch pollen is one of the most common inhalational allergens and Bet v1 is the major component allergen of birch allergen. This study aims to develop a stable and sensitive chemiluminescence immunoassay (CLIA) for the detection of birch pollen allergic specific IgE (sIgE) based on recombinant Bet v1 (rBet v1) protein. Methods rBet v1 protein was expressed in Escherichia coli and purified. Then rBet v1 was applied to detect sIgE in human serum. The performance of the established CLIA was evaluated and compared with Phadia rBet v1 fluorescence enzyme immunoassay (FEIA) system. Results The developed CLIA for sIgE to rBet v1 detection shows excellent performance. The assay showed a linear range from 0.1 to 100 IU/mL, with a low detection limit of 0.06 IU/mL. A total of 164 samples were evaluated by CLIA and compared with the results of FEIA. The positive, negative, and total coincidence rate was 90.6% (87/96), 91.2% (62/68), and 90.9% (149/164), respectively. The r-value of Spearman's rank correlation analysis was 0.935 (P < 0.001). The use of high levels of bilirubin (50 mg/dL), hemoglobin (400 mg/dL) and lipid (2000 mg/dL) didn't interfere with the results. Conclusions The proposed CLIA exhibits excellent performance for the detection of rBet v1 specific IgE. It can be a reliable tool for the early diagnosis of hypersensitivity., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Ber e 1, a major Brazil nut allergen, has been successfully produced in the yeast Pichia pastoris expression system as homogenous recombinant Ber e 1 (rBer e 1) with similar physicochemical properties and identical immunoreactivity to its native counterpart, nBer e 1. However, O-linked glycans was detected on the P.pastoris-derived rBer e 1, which is not naturally present in nBer e 1, and may contribute to the allergic sensitisation. In this study, we addressed the glycosylation differences between P. pastoris-derived recombinant Ber e 1 and its native counterparts. We also determined whether this fungal glycosylation could affect the antigenicity and immunogenicity of the rBer e 1 by using dendritic cells (DC) as an immune cell model due to their role in modulating the immune response. We identified that the glycosylation occurs at Ser96, Ser101 and Ser110 on the large chain and Ser19 on the small polypeptide chain of rBer e 1 only. The glycosylation on rBer e 1 was shown to elicit varying degree of antigenicity by binding to different combination of human leukocyte antigens (HLA) at different frequencies compared to nBer e 1 when tested using human DC-T cell assay. However, both forms of Ber e 1 are weak immunogens based from their low response indexes (RI). Glycans present on rBer e 1 were shown to increase the efficiency of the protein recognition and internalization by murine bone marrow-derived dendritic cells (bmDC) via C-type lectin receptors, particularly the mannose receptor (MR), compared to the non-glycosylated nBer e 1 and SFA8, a weak allergenic 2S albumin protein from sunflower seed. Binding of glycosylated rBer e 1 to MR alone was found to not induce the production of IL-10 that modulates bmDC to polarise Th2 cell response by suppressing IL-12 production and DC maturation. Our findings suggest that the O-linked glycosylation by P. pastoris has a small but measurable effect on the in vitro antigenicity of the rBer e 1 compared to its non-glycosylated counterpart, nBer e 1, and thus may influence its applications in diagnostics and immunotherapy., Competing Interests: The authors have declared that no competing interests exist.

Scope: Around 25% of food allergic persons in Central Europe suffer from carrot allergy caused by the major carrot allergen Dau c 1. Three different isoallergens, Dau c 1.01, Dau c 1.02 and Dau c 1.03 are identified. However, information about the qualitative and quantitative composition of natural (n)Dau c 1 is scarce., Methods and Results: The new carrot allergen Dau c 1.0401 is identified on the mRNA and protein level by RT-PCR and mass spectrometry. It displays only around 60% sequence identity to the other known Dau c 1 isoallergens. NMR and CD-spectra are typical for a well-folded protein containing both α-helices and β-strands. It showed a poor refolding capacity after incubation at 95 °C. IgE-binding is impaired in immunoblots, whereas in inhibition assays IgE binding to soluble Dau c 1.0401 is detected and it clearly provoked a response in mediator release assays., Conclusion: Dau c 1.0401 is a new isoallergen which contributes to the allergenicity of carrots. The absence of immunoreactivity in immobilized assays indicates that IgE binding is impaired when the protein is blotted on a solid phase. Altogether, the results point out that its allergenicity can be reduced upon carrot processing., (© 2021 The Authors. Molecular Nutrition & Food Research published by Wiley-VCH GmbH.)

Key Message: Linkage mapping and GWAS identified 67 QTLs related to soybean glycinin, β-conglycinin and relevant traits. Polymorphisms of the candidate gene Gy1 promoter were associated with the glycinin content in soybean. The major components of storage proteins in soybean seeds are glycinin and β-conglycinin, which play important roles in determining protein nutrition and soy food processing properties. Increasing the protein content while improving the ratio of glycinin to β-conglycinin is substantially important for soybean protein improvement. To investigate the genetic mechanism of storage protein subunits, 184 recombinant inbred lines (RILs) derived from a cross of Kefeng No. 1 and Nannong 1138-2 and 211 diverse soybean cultivars were used to detect loci related to glycinin (11S), β-conglycinin (7S), the sum of glycinin and β-conglycinin (SGC), and the ratio of glycinin to β-conglycinin (RGC). Sixty-seven QTLs and 11 hot genomic regions were identified as affecting the four traits. One genetic region (q10-1) on chromosome 10 was associated with multiple traits by both linkage and association analysis. Eight genes in 11 hot genomic regions might be related to soybean protein subunit. The candidate gene analysis showed that polymorphisms in Gy1 promoters were significantly correlated with the 11S content. The QTLs and candidate genes identified in the present study allow for further understanding the genetic basis of 11S and 7S regulation and provide useful information for marker-assisted selection (MAS) in soybean quality improvement.

About 50-70% of patients allergic to birch pollen suffer from sensitization after apple ingestion. Apple allergenicity was established in only few varieties. Studies were performed on apple fruits of 21 traditional and nine modern varieties organically, intensively, or integratively produced. The aim of the study was to assess whether the factors like cultivation method, maturity stage, genotype, or type of tissue place an impact on the allergenic potential of apples. To answer these questions, we used semiquantitative real-time PCR, ELISA, and immunoblotting. Apple allergen genes present divergent expression across apple cultivars. Expression of the Mal d 1.06A correlates with the Mal d 1 level and is affected by the cultivation method and maturity of the fruit. The content of the main allergen Mal d 1 varied widely across cultivars. Interestingly, in our study, the Gala variety presented a low Mal d 1 concentration regardless of the cultivation method. Based on the Mal d 1.06A expression, the Mal d 1 protein content, and the immunoreactivity assay, the Kandil Sinap, Kosztela, Rumianka from Alma-Ata, Kantówka Gdańska, Reinette Coulon, and Gala cultivars emerged as potentially hypoallergenic apple cultivars. Our study allowed distinguishing between potentially low, medium, and highly allergenic varieties.

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated endonuclease 9 (Cas9) system is being rapidly developed for mutagenesis in higher plants. Ideally, foreign DNA introduced by this system is removed in the breeding of edible crops and vegetables. Here, we report an efficient generation of Cas9-free mutants lacking an allergenic gene, Gly m Bd 30K, using biolistic transformation and the CRISPR/Cas9 system. Five transgenic embryo lines were selected on the basis of hygromycin resistance. Cleaved amplified polymorphic sequence analysis detected only two different mutations in e all of the lines. These results indicate that mutations were induced in the target gene immediately after the delivery of the exogenous gene into the embryo cells. Soybean plantlets (T 0 plants) were regenerated from two of the transgenic embryo lines. The segregation pattern of the Cas9 gene in the T 1 generation, which included Cas9-free plants, revealed that a single copy number of transgene was integrated in both lines. Immunoblot analysis demonstrated that no Gly m Bd 30K protein accumulated in the Cas9-free plants. Gene expression analysis indicated that nonsense mRNA decay might have occurred in mature mutant seeds. Due to the efficient induction of inheritable mutations and the low integrated transgene copy number in the T 0 plants, we could remove foreign DNA easily by genetic segregation in the T 1 generation. Our results demonstrate that biolistic transformation of soybean embryos is useful for CRISPR/Cas9-mediated site-directed mutagenesis of soybean for human consumption.

Identifying causative genes for a target trait in conifer reproduction is challenging for species lacking whole-genome sequences. In this study, we searched for the male-sterility gene (MS1) in Cryptomeria japonica, aiming to promote marker-assisted selection (MAS) of male-sterile C. japonica to reduce the pollinosis caused by pollen dispersal from artificial C. japonica forests in Japan. We searched for mRNA sequences expressed in male strobili and found the gene CJt020762, coding for a lipid transfer protein containing a 4-bp deletion specific to male-sterile individuals. We also found a 30-bp deletion by sequencing the entire gene of another individual with the ms1. All nine breeding materials with the allele ms1 had either a 4-bp or 30-bp deletion in gene CJt020762, both of which are expected to result in faulty gene transcription and function. Furthermore, the 30-bp deletion was detected from three of five individuals in the Ishinomaki natural forest. From our findings, CJt020762 was considered to be the causative gene of MS1. Thus, by performing MAS using two deletion mutations as a DNA marker, it will be possible to find novel breeding materials of C. japonica with the allele ms1 adapted to the unique environment of each region of the Japanese archipelago.

Reducing allergenicity and increasing oleic content are important goals in groundnut breeding studies. Ara h 1 is a major allergen gene and Delta(12)-fatty-acid desaturase (FAD2) is responsible for converting oleic into linoleic acid. These genes have homoeologues with one copy in each subgenome, identified as Ara h 1.01, Ara h 1.02, ahFAD2A and ahFAD2B in tetraploid groundnut. To alter functional properties of these genes we have generated an Ethyl Methane Sulfonate (EMS) induced mutant population to be used in Targeting Induced Local Lesions in Genomes (TILLING) approach. Seeds were exposed to two EMS concentrations and the germination rates were calculated as 90.1% (1353 plants) for 0.4% and 60.4% (906 plants) for 1.2% EMS concentrations in the M 1 generation. Among the 1541 M 2 mutants, 768 were analyzed by TILLING using four homoeologous genes. Two heterozygous mutations were identified in the ahFAD2B and ahFAD2A gene regions from 1.2% and 0.4% EMS-treated populations, respectively. The mutation in ahFAD2B resulted in an amino acid change, which was serine to threonine predicted to be tolerated according to SIFT analysis. The other mutation causing amino acid change, glycine to aspartic acid was predicted to affect protein function in ahFAD2A. No mutations were detected in Ara h 1.01 and Ara h 1.02 for both EMS-treatments after sequencing. We estimated the overall mutation rate to be 1 mutation every 2139 kb. The mutation frequencies were also 1/317 kb for ahFAD2A in 0.4% EMS and 1/466 kb for ahFAD2B in 1.2% EMS treatments. The results demonstrated that TILLING is a powerful tool to interfere with gene function in crops and the mutagenized population developed in this study can be used as an efficient reverse genetics tool for groundnut improvement and functional genomics.

The highly variable and species-specific pollen surface patterns are formed by sporopollenin accumulation. The template for sporopollenin deposition and polymerization is the primexine that appears on the tetrad surface, but the mechanism(s) by which primexine guides exine patterning remain elusive. Here, we report that the Poaceae-specific EXINE PATTERN DESIGNER 1 ( EPAD1 ), which encodes a nonspecific lipid transfer protein, is required for primexine integrity and pollen exine patterning in rice ( Oryza sativa ). Disruption of EPAD1 leads to abnormal exine pattern and complete male sterility, although sporopollenin biosynthesis is unaffected. EPAD1 is specifically expressed in male meiocytes, indicating that reproductive cells exert genetic control over exine patterning. EPAD1 possesses an N-terminal signal peptide and three redundant glycosylphosphatidylinositol (GPI)-anchor sites at its C terminus, segments required for its function and localization to the microspore plasma membrane. In vitro assays indicate that EPAD1 can bind phospholipids. We propose that plasma membrane lipids bound by EPAD1 may be involved in recruiting and arranging regulatory proteins in the primexine to drive correct exine deposition. Our results demonstrate that EPAD1 is a meiocyte-derived determinant that controls primexine patterning in rice, and its orthologs may play a conserved role in the formation of grass-specific exine pattern elements., (© 2020 American Society of Plant Biologists. All rights reserved.)

Almond (Prunus dulcis) is not only widely used as a human food as a result of its flavor, nutrients, and health benefits, but it is also one of the most likely tree nuts to trigger allergies. Almond allergens, however, have not been studied as extensively as those of peanuts and other selected tree nuts. This review provides an update of the molecular properties of almond allergens to clarify some confusion about the identities of almond allergens and our perspective on characterizing putative almond allergens. At present, the following almond allergens have been designated by the World Health Organization/International Union of Immunological Societies Allergen Nomenclature Sub-Committee: Pru du 3 (a non-specific lipid transfer protein 1, nsLTP1), Pru du 4 (a profilin), Pru du 5 (60S acidic ribosomal protein 2), Pru du 6 (an 11S legumin known as prunin) and Pru du 8 (an antimicrobial protein with cC3C repeats). Besides, almond vicilin and almond γ-conglutin have been identified as food allergens, although further characterization of these allergens is still of interest. In addition, almond 2S albumin was reported as a food allergen as a result of the misidentification of Pru du 8. Two more almond proteins have been called allergens based on their sequence homology with known food allergens and their 'membership' in relevant protein families that contain allergens in many species. These include the pathogenesis related-10 protein (referred to as Pru du 1) and the thaumatin-like protein (referred to as Pru du 2). Almonds thus have five known food allergens and five more likely ones that need to be investigated further. Published 2020. This article is a U.S. Government work and is in the public domain in the USA., (Published 2020. This article is a U.S. Government work and is in the public domain in the USA.)

Transcription infidelity (TI) is a mechanism that increases RNA and protein diversity. We found that single-base omissions (i.e., gaps) occurred at significantly higher rates in the RNA of highly allergenic legumes. Transcripts from peanut, soybean, sesame, and mite allergens contained a higher density of gaps than those of nonallergens. Allergen transcripts translate into proteins with a cationic carboxy terminus depleted in hydrophobic residues. In mice, recombinant TI variants of the peanut allergen Ara h 2, but not the canonical allergen itself, induced, without adjuvant, the production of anaphylactogenic specific IgE (sIgE), binding to linear epitopes on both canonical and TI segments of the TI variants. The removal of cationic proteins from bovine lactoserum markedly reduced its capacity to induce sIgE. In peanut-allergic children, the sIgE reactivity was directed toward both canonical and TI segments of Ara h 2 variants. We discovered 2 peanut allergens, which we believe to be previously unreported, because of their RNA-DNA divergence gap patterns and TI peptide amino acid composition. Finally, we showed that the sIgE of children with IgE-negative milk allergy targeted cationic proteins in lactoserum. We propose that it is not the canonical allergens, but their TI variants, that initiate sIgE isotype switching, while both canonical and TI variants elicit clinical allergic reactions.

Immunoglobulin E (IgE) is pivotal for manifestation and persistence of most immediate-type allergies and some asthma phenotypes. Consequently, IgE represents a crucial target for both, diagnostic purposes as well as therapeutic approaches. In fact, allergen-specific immunotherapy - aiming to re-route an IgE-based inflammatory response into an innocuous immune reaction against the allergen - is the only curative approach for IgE-mediated allergic diseases known so far. However, this requires the cognate allergen to be known. Unfortunately, even in well-characterized allergics or asthmatics, often just a small fraction of total IgE can be assigned to specific target allergens. To overcome this knowledge gap, we have devised an analytical platform for unbiased IgE target epitope detection. The system relies on chemically produced random peptide libraries immobilized on polystyrene beads ("one-bead-one-compound (OBOC) libraries") capable to present millions of different peptide motifs simultaneously to immunoglobulins from biological samples. Beads binding IgE are highlighted with a fluorophore-labeled anti-IgE antibody allowing fluorescence-based detection and isolation of positives, which then can be characterized by peptide sequencing. Setting-up this platform required an elaborate optimization process including proper choice of background suppressants, secondary antibody and fluorophore label as well as incubation conditions. For optimal performance our procedure involves a sophisticated pre-adsorption step to eliminate beads that react nonspecifically with anti-IgE secondary antibodies. This step turned out to be important for minimizing detection of "false positive" motifs that otherwise would erroneously be classified as IgE epitopes. In validation studies we were able to retrieve artificial test-peptide beads spiked into our library by using IgE directed against those test-peptides at physiological concentrations (≤20 IU/ml of specific IgE), and disease-relevant bead-bound epitopes of the major peanut allergen Ara h 2 by screening with sera from peanut allergics. Thus, we established a platform with which one can find and validate new immunoglobulin targets using patient material which displays a largely unknown immunoglobulin repertoire., (Copyright © 2020 Krause, Röckendorf, Meckelein, Sinnecker, Schwager, Möckel, Jappe and Frey.)

Although strawberries are highly appreciated fruits, their intake can induce allergic reactions in atopic patients. These reactions can be due to the patient's previous sensitization to the major birch pollen allergen Bet v 1, by which IgE generated in response to Bet v 1 cross-reacts with the structurally related strawberry Fra a 1 protein family. Fra a 1.02 is the most expressed paralog in ripe strawberries and is highly allergenic. To better understand the molecular mechanisms regulating this allergic response, we have determined the three-dimensional structure of Fra a 1.02 and four site-directed mutants that were designed based on their positions in potential epitopes. Fra a 1.02 and mutants conform to the START fold. We show that the cross-reactivity of all the mutant variants to IgE from patients allergic to Bet v 1 was significantly reduced without altering the conserved structural fold, so that they could potentially be used as hypoallergenic Fra a 1 variants for the generation of vaccines against strawberry allergy in atopic patients.

This study was conducted to evaluate the effects of recombinant probiotic bacteria as a candidate for oral vaccine with the potential of treating allergy to Amaranthus retroflexus pollens. The main gene of this allergen, Ama r 2, was cloned into the food grade plasmid pNZ7025 and then was electrotransformed into the food grade Lactococcus lactis NZ1330. No expression was observed in the primary structure due to the distance between the ribosome binding site and the start codon. Therefore, the vector structure was corrected using the site-directed mutagenesis (SDM) technique. The cell extract of this strain was used for assessing the expression of the recombinant allergen in western blot analysis, and the existence of this protein with a molecular weight of 14.2 kDa was confirmed. To evaluate the efficacy of this strain in the treatment of allergies as an oral vaccine, probiotic ice cream was prepared. After the sensitization of mice, the treatment was performed by oral immunotherapy for 4 weeks, 4 to 5 times per week. 20  μ l of functional ice cream with 10 12  CFU/ml of r- L. lactis NZ1330 significantly reduced the serum IgE level. The levels of IFN- γ and TGF- β cytokines increased in the 20  μ l ice cream treatment group as well as 40  μ g/ml pure allergen compared with the PBS-treated group, and IL-4 cytokine levels decreased compared with the PBS-treated group. Overall, 20  μ l ice cream with 10 12  CFU/ml of the recombinant bacteria resulted in the best performance in terms of improving allergies to Th1 and Treg responses., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2020 Alireza Vasiee et al.)

ATP sulfurylase, an enzyme which catalyzes the conversion of sulfate to adenosine 5'-phosphosulfate (APS), plays a significant role in controlling sulfur metabolism in plants. In this study, we have expressed soybean plastid ATP sulfurylase isoform 1 in transgenic soybean without its transit peptide under the control of the 35S CaMV promoter. Subcellular fractionation and immunoblot analysis revealed that ATP sulfurylase isoform 1 was predominantly expressed in the cell cytoplasm. Compared with that of untransformed plants, the ATP sulfurylase activity was about 2.5-fold higher in developing seeds. High-resolution 2-D gel electrophoresis and immunoblot analyses revealed that transgenic soybean seeds overexpressing ATP sulfurylase accumulated very low levels of the β-subunit of β-conglycinin. In contrast, the accumulation of the cysteine-rich Bowman-Birk protease inhibitor was several fold higher in transgenic soybean plants when compared to the non-transgenic wild-type seeds. The overall protein content of the transgenic seeds was lowered by about 3% when compared to the wild-type seeds. Metabolite profiling by LC-MS and GC-MS quantified 124 seed metabolites out of which 84 were present in higher amounts and 40 were present in lower amounts in ATP sulfurylase overexpressing seeds compared to the wild-type seeds. Sulfate, cysteine, and some sulfur-containing secondary metabolites accumulated in higher amounts in ATP sulfurylase transgenic seeds. Additionally, ATP sulfurylase overexpressing seeds contained significantly higher amounts of phospholipids, lysophospholipids, diacylglycerols, sterols, and sulfolipids. Importantly, over expression of ATP sulfurylase resulted in 37-52% and 15-19% increases in the protein-bound cysteine and methionine content of transgenic seeds, respectively. Our results demonstrate that manipulating the expression levels of key sulfur assimilatory enzymes could be exploited to improve the nutritive value of soybean seeds.

Allergic sensitization to the major allergen Bet v 1 represents the dominating factor inducing a vast variety of allergic symptoms in birch pollen allergic patients worldwide, including the pollen food allergy syndrome. In order to overcome the huge socio-economic burden associated with allergic diseases, allergen-specific immunotherapy (AIT) as a curative strategy to manage the disease was introduced. Still, many hurdles related to this treatment exist making AIT not the patients' first choice. To improve the current situation, the development of hypoallergen-based drug products has raised attention in the last decade. Herein, we investigated the efficacy of the novel AIT candidate BM4, a hypoallergenic variant of Bet v 1, to induce treatment-relevant cross-reactive Bet v 1-specific IgG antibodies in two different mammals, Wistar rats and New Zealand White rabbits. We further analyzed the cross-reactivity of BM4-induced Wistar rat antibodies with the birch pollen-associated food allergens Mal d 1 and Cor a 1, and the functional capability of the induced antibodies to act as IgE-blocking IgG antibodies. Enzyme-linked immunosorbent assay (ELISA) was used to determine the titers of rat IgG1, IgG2a, IgG2b, and IgE, as well as rabbit IgG and IgE antibodies. To address the functional relevance of the induced IgG antibodies, the capacity of rat sera to suppress binding of human IgE to Bet v 1 was investigated by using an inhibition ELISA and an IgE-facilitated allergen-binding inhibition assay. We found that the treatment with BM4 induced elevated Bet v 1-specific IgG antibody titers in both mammalian species. In Wistar rats, high BM4-specific IgG1, IgG2a, and IgG2b titers (10 4 to 10 6 ) were induced, which cross-reacted with wild-type Bet v 1, and the homologous allergens Mal d 1 and Cor a 1. Rat allergen-specific IgG antibodies sustained upon treatment discontinuation. Sera of rats immunized with BM4 were able to significantly suppress binding of human IgE to the wild-type allergens and CD23-mediated human IgE-facilitated Bet v 1 binding on B cells. By contrast, treatment-induced IgE antibody levels were low or undetectable. In summary, BM4 induced a robust IgG immune response that efficiently blocked human IgE-binding to wild-type allergens, underscoring its potential therapeutic value in AIT., (Copyright © 2020 Aglas, Bethanis, Chrusciel, Stolz, Gruen, Jaakkola, Jongejan, Yatkin and Van Ree.)

Scope: The major carrot allergen Dau c 1 belongs to the group of pathogenesis related class 10 (PR-10) proteins and is homologous to the birch pollen allergen Bet v 1. In contrast to most other PR-10 allergens, Dau c 1 can elicit Bet v 1 independent sensitization. Although Dau c 1 is considered heat labile, allergic reactions against cooked carrots are possible., Methods and Results: The pH and temperature stability as well as the allergenic potential before and after treatment of purified natural (n) Dau c 1 and different recombinant (r) isoallergens is investigated: rDau c 1.0104, rDau c 1.0105, rDau c 1.0201, rDau c 1.0301. All proteins except rDau c 1.0201 are able to refold at physiological pH. pH conditions around the pI (4.4-5.5) or the presence of the carrot matrix reduce the refolding capacity. Below the pI, most isoallergens are heat resistant and still able to cause mediator release, indicating allergenicity. Moreover, cooked carrot extract is still able to provoke mediator release due to remaining soluble Dau c 1., Conclusion: Patients allergic to carrots should avoid processed carrot containing foodstuff because heating or pH treatment do not completely abolish the allergenicity of Dau c 1., (© 2020 The Authors. Published by Wiley-VCH GmbH.)

Thaumatin-like proteins (TLPs), which are defined as pathogenesis-related protein family 5 (PR5) members, are common plant proteins involved in defense responses and confer antifungal activity against many plant pathogens. Our earlier studies have reported that the TaTLP1 gene was isolated from wheat and proved to be involved in wheat defense in response to leaf rust attack. The present study aims to identify the interacting proteins of TaTLP1 and characterize the role of the interaction between wheat and Puccinia triticina (Pt). Pull-down experiments designed to isolate the molecular target of TaTLP1 in tobacco resulted in the identification of TaPR1, a pathogenesis-related protein of family 1, and the interaction between TaTLP1 and TaPR1 was confirmed by yeast two-hybrid experiments (Y2H), bimolecular fluorescence complementation (BiFC), and co-immunoprecipitation (Co-IP). In vitro, TaTLP1 and TaPR1 together increased antifungal activity against Pt. In vivo, the disease resistance phenotype, histological observations of fungal growth and host responses, and accumulation of H2O2 in TaTLP1-TaPR1 in co-silenced plants indicated that co-silencing significantly enhanced wheat susceptibility compared to single knockdown TaTLP1 or TaPR1 plants. The accumulation of reactive oxygen species (ROS) was significantly reduced in co-silenced plants compared to controls during Pt infection, which suggested that the TaTLP1-TaPR1 interaction positively modulates wheat resistance to Pt in an ROS-dependent manner. Our findings provide new insights for understanding the roles of two different PRs, TaTLP1 and TaPR1, in wheat resistance to leaf rust., Competing Interests: The authors have declared that no competing interests exist.

The tradeoff between protein and oil storage in oilseed crops has been tested here in oilseed rape ( Brassica napus ) by analyzing the effect of suppressing key genes encoding protein storage products (napin and cruciferin). The phenotypic outcomes were assessed using NMR and mass spectrometry imaging, microscopy, transcriptomics, proteomics, metabolomics, lipidomics, immunological assays, and flux balance analysis. Surprisingly, the profile of storage products was only moderately changed in RNA interference transgenics. However, embryonic cells had undergone remarkable architectural rearrangements. The suppression of storage proteins led to the elaboration of membrane stacks enriched with oleosin (sixfold higher protein abundance) and novel endoplasmic reticulum morphology. Protein rebalancing and amino acid metabolism were focal points of the metabolic adjustments to maintain embryonic carbon/nitrogen homeostasis. Flux balance analysis indicated a rather minor additional demand for cofactors (ATP and NADPH). Thus, cellular plasticity in seeds protects against perturbations to its storage capabilities and, hence, contributes materially to homeostasis. This study provides mechanistic insights into the intriguing link between lipid and protein storage, which have implications for biotechnological strategies directed at improving oilseed crops., (© 2020 American Society of Plant Biologists. All rights reserved.)

Natural rubber (NR), principally comprising cis -1,4-polyisoprene, is an industrially important natural hydrocarbon polymer because of its unique physical properties, which render it suitable for manufacturing items such as tires. Presently, industrial NR production depends solely on latex obtained from the Pará rubber tree, Hevea brasiliensis . In latex, NR is enclosed in rubber particles, which are specialized organelles comprising a hydrophobic NR core surrounded by a lipid monolayer and membrane-bound proteins. The similarity of the basic carbon skeleton structure between NR and dolichols and polyprenols, which are found in most organisms, suggests that the NR biosynthetic pathway is related to the polyisoprenoid biosynthetic pathway and that rubber transferase, which is the key enzyme in NR biosynthesis, belongs to the cis -prenyltransferase family. Here, we review recent progress in the elucidation of molecular mechanisms underlying NR biosynthesis through the identification of the enzymes that are responsible for the formation of the NR backbone structure.

Hordeins are the major barley seed storage proteins and are elicitors of celiac disease. Attempts to reduce the hordein level in barley have been made; however, the resultant pleiotropic effects are less understood. Here, data-independent acquisition mass spectrometry was used to measure proteome-wide abundance differences between wild-type and single hordein-null barley lines. Using comparative quantitative proteomics, we detected proteome-wide changes (∼59%) as a result of the specific reduction in hordein proteins. The comparative analysis and functional annotation revealed an increase in non-gluten storage proteins, such as globulins and lipid transfer proteins, and proteins rich in essential amino acids in the null lines. This study yields an informative molecular portrait of the hordein-null lines and the underlying mechanisms of storage protein biosynthesis. This study indicates the extent to which protein content can be manipulated without biological consequence, and we envision its wide-scale application for studying modified crops.

Background: Peanut allergy is a severe and increasingly frequent disease with high medical, psychosocial, and economic burden for affected patients and wider society. A causal, safe, and effective therapy is not yet available., Objective: We sought to develop an immunogenic, protective, and nonreactogenic vaccine candidate against peanut allergy based on virus-like particles (VLPs) coupled to single peanut allergens., Methods: To generate vaccine candidates, extracts of roasted peanut (Ara R) or the single allergens Ara h 1 or Ara h 2 were coupled to immunologically optimized Cucumber Mosaic Virus-derived VLPs (CuMVtt). BALB/c mice were sensitized intraperitoneally with peanut extract absorbed to alum. Immunotherapy consisted of a single subcutaneous injection of CuMVtt coupled to Ara R, Ara h 1, or Ara h 2., Results: The vaccines CuMVtt-Ara R, CuMVtt-Ara h 1, and CuMVtt-Ara h 2 protected peanut-sensitized mice against anaphylaxis after intravenous challenge with the whole peanut extract. Vaccines did not cause allergic reactions in sensitized mice. CuMVtt-Ara h 1 was able to induce specific IgG antibodies, diminished local reactions after skin prick tests, and reduced the infiltration of the gastrointestinal tract by eosinophils and mast cells after oral challenge with peanut. The ability of CuMVtt-Ara h 1 to protect against challenge with the whole extract was mediated by IgG, as shown via passive IgG transfer. FcγRIIb was required for protection, indicating that immune complexes with single allergens were able to block the allergic response against the whole extract, consisting of a complex allergen mixture., Conclusions: Our data suggest that vaccination using single peanut allergens displayed on CuMVtt may represent a novel therapy against peanut allergy with a favorable safety profile., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Plant lipid transfer proteins (LTPs) are small basic proteins that play important roles in the regulation of various plant biological processes as well as the response to biotic and abiotic stresses. However, knowledge is limited on how this family of proteins is regulated in response to nematode infection in cucumber. In the present study, a total of 39 CsLTP&#95;2 genes were identified by querying databases for cucumber-specific LTP&#95;2 using a Hidden Markov Model approach and manual curation. The family has a five-cysteine motif (5CM) with the basic form CC-Xn-CXC-Xn-C, which differentiates it from typical nsLTPs. The members of CsLTP&#95;2 were grouped into six families according to their structure and their phylogenetic relationships. Expression data of CsLTP&#95;2 genes in 10 cucumber tissues indicated that they were tissue-specific genes. Two genes showed significant expression change in roots of resistant and susceptible lines during nematode infection, indicating their involvement in response to Meloidogyne incognita . This systematic analysis provides a foundation of knowledge for future studies of the biological roles of CsLTP&#95;2 genes in cucumber in response to nematode infection and may help in the efforts to improve M. incognita -resistance breeding in cucumber.

Pleiotropy, the control of multiple phenotypes by a single locus, is expected to slow the rate of adaptation by increasing the chance that beneficial alleles also have deleterious effects. However, a prediction arising from classical theory of quantitative trait evolution states that pleiotropic alleles may have a selective advantage when phenotypes are distant from their selective optima. We examine the role of pleiotropy in regulating adaptive differentiation among populations of common ragweed (Ambrosia artemisiifolia); a species that has recently expanded its North American range due to human-mediated habitat change. We employ a phenotype-free approach by using connectivity in gene networks as a proxy for pleiotropy. First, we identify loci bearing footprints of local adaptation, and then use genotype-expression mapping and co-expression networks to infer the connectivity of the genes. Our results indicate that the putatively adaptive loci are highly pleiotropic, as they are more likely than expected to affect the expression of other genes, and they reside in central positions within the gene networks. We propose that the conditionally advantageous alleles at these loci avoid the cost of pleiotropy by having large phenotypic effects that are beneficial when populations are far from their selective optima. We further use evolutionary simulations to show that these patterns are in agreement with a model where populations face novel selective pressures, as expected during a range expansion. Overall, our results suggest that highly connected genes may be targets of positive selection during environmental change, even though they likely experience strong purifying selection in stable selective environments., Competing Interests: The authors have declared that no competing interests exist.

Aim: To study the prophylactic effect of recombinant Lactococcus lactis (rLl) harbouring Ara h 2.02 peanut allergen, in sensitized and challenged mice., Methods and Results: Ara h 2.02 cDNA was cloned into pNZ8048 for heterologous expression in L. lactis. The purified recombinant allergen showed IgE binding comparable with native Ara h 2. Balb/c mice were fed with either recombinant (rLl), nonrecombinant L. lactis (Ll) or NaHCO 3 (Sham) prior to sensitization and challenged with rAra h 2.02, whereas the baseline group was only fed with Ll. Allergen-specific immunoglobulin and splenocyte cytokines responses were determined for each mouse. Mice fed with either Ll or rLl showed significant alleviation of IgE and IgG 1 compared to the Sham group. Despite no significant decrease in Th2 (IL-4, IL-13, IL-6) or increase in Th1 (IFN-γ) cytokines, both groups showed lower IL-10 level, while the IL-4 : IFN-γ ratio was significantly lower for rLl compared to Ll group., Conclusions: Oral administration of rLl harbouring Ara h 2.02 demonstrated alleviation of Th2-associated responses in allergen-challenged mice and a possible added allergen-specific prophylactic effect., Significance and Impact of the Study: Ara h 2.02 coupled with the intrinsic properties of probiotic L. lactis as a delivery vehicle can be explored for the development of a commercially scalable vaccine., (© 2019 The Society for Applied Microbiology.)

Background: Small, basic peanut proteins are often poorly extracted in pH-neutral buffers that are optimal for the extraction of peanut storage proteins such as Ara h 1. As a result, such proteins are easily missed as potential allergens., Objective: To analyse the allergenic composition of the basic peanut protein (BPP) fraction., Methods: A peanut extract prepared at pH 4 was fractionated by physicochemical procedures. Chemical analysis was performed by SDS-PAGE and mass spectrometry. Because immunoblotting was found to be inefficient for most of these small basic proteins, IgE-binding activity was measured by coupling the fractions to CNBr-activated Sepharose, followed by incubation with sera from 55 Dutch peanut-allergic children and 125 I-labelled anti-IgE., Results: Most IgE reactivity of the BPP fraction was due to the 5-7 kDa amino-terminal fragment of Ara h 1. This finding was confirmed by the use of the fragment in recombinant form, to which 25/55 of the sera was IgE-positive., Conclusion: The amino-terminal fragment of Ara h 1, a member of a family of small anti-microbial proteins, is an allergen independent of the carboxy-terminal fragment of Ara h 1., (© 2019 John Wiley & Sons Ltd.)