Association Between the Seed Storage Proteins 2S Albumin and 11S Globulin and Severe Allergic Reaction After Flaxseed Intake

Background: Given the increased popularity of flaxseed in meals, several cases of allergy to these seeds have been reported. Little is known about the allergens implicated in hypersensitivity reactions to flaxseed. The present study aimed to identify the allergens involved in IgE-mediated reactions in 5 patients with a clinical history of severe systemic symptoms after flaxseed consumption. Methods: Proteins that were potential allergens with IgE-binding capacity were purified from flaxseed extract using chromatography and identified via MALDI-TOF mass spectrometry. Immunoassays were performed using the 5 allergic patients’ sera tested individually and as a pool. Results: Immunoblotting of the flaxseed extract revealed a low-molecular-mass protein (around 13 kDa) in 4 of the 5 patients, while a protein of approximately 55 kDa was detected in 2 patients. The proteins were identified by mass spectrometry as flaxseed 2S albumin, which is included in the WHO/IUIS allergen nomenclature as Lin u 1, and 11S globulin. Inhibition assays revealed in vitro IgE-mediated cross-reactivity between Lin u 1 and peanut and cashew nut proteins, while IgE-mediated recognition of 11S globulin by patients’ sera was partially inhibited by several plant-derived sources. Conclusions: Seed storage proteins from flaxseed were involved in the development of severe symptoms in the 5 patients studied and exhibited cross-reactivity with other allergenic sources. Besides the severity of flaxseed allergy in patients sensitized to 2S albumin, this is the first time that 11S globulin has been identified as a potential allergen. Taking these data into account should ensure a more accurate diagnosis. (AU)

Antecedentes: Dada la creciente popularidad de la linaza en las comidas, se han notificado varios casos de alergia a estas semillas. La información acerca de los alérgenos implicados en las reacciones de hipersensibilidad a estas semillas es escasa. El presente trabajo pretende identificar los alérgenos implicados en las reacciones mediadas por IgE en cinco pacientes con una historia clínica de síntomas sistémicos graves tras el consumo de linaza. Métodos: Las proteínas susceptibles de ser alérgenos con capacidad de unir IgE se purificaron a partir del extracto de linaza mediante técnicas cromatográficas. Su identificación se realizó mediante espectrometría de masas MALDI-TOF. Se realizaron inmunoensayos con los sueros de los cinco pacientes alérgicos, utilizados de forma individual o como mezclas. Resultados: Cuatro de los cinco pacientes reconocieron una proteína de baja masa molecular (alrededor de 13 kDa) en inmunoensayos con extracto de linaza, mientras que dos pacientes reconocieron una proteína de aproximadamente 55 kDa. Se identificaron por espectrometría de masas como albúmina 2S de linaza, incluida en la nomenclatura de alérgenos de la OMS/IUIS como Lin u 1, y globulina 11S, respectivamente. Los ensayos de inhibición in vitro revelaron la existencia de reactividad cruzada de la Lin u 1 con las proteínas del cacahuete y del anacardo, mientras que el reconocimiento por parte de la IgE de la globulina 11S por parte de los sueros de los pacientes fue parcialmente inhibido por varias fuentes vegetales. Conclusiones: Las proteínas de almacenamiento de las semillas de lino estaban implicadas en el desarrollo de síntomas graves en cinco individuos y mostraron una reactividad cruzada con otras fuentes alergénicas. Además de la gravedad de la alergia a la linaza en los pacientes sensibilizados a la albúmina 2S, es la primera vez que se identifica la globulina 11S como un alérgeno potencial.

Effect of a Bacillus subtilis strain on flax protection against Fusarium oxysporum and its impact on the root and stem cell walls.

In Normandy, flax is a plant of important economic interest because of its fibres. Fusarium oxysporum, a telluric fungus, is responsible for the major losses in crop yield and fibre quality. Several methods are currently used to limit the use of phytochemicals on crops. One of them is the use of plant growth promoting rhizobacteria (PGPR) occurring naturally in the rhizosphere. PGPR are known to act as local antagonists to soil-borne pathogens and to enhance plant resistance by eliciting the induced systemic resistance (ISR). In this study, we first investigated the cell wall modifications occurring in roots and stems after inoculation with the fungus in two flax varieties. First, we showed that both varieties displayed different cell wall organization and that rapid modifications occurred in roots and stems after inoculation. Then, we demonstrated the efficiency of a Bacillus subtilis strain to limit Fusarium wilt on both varieties with a better efficiency for one of them. Finally, thermo-gravimetry was used to highlight that B. subtilis induced modifications of the stem properties, supporting a reinforcement of the cell walls. Our findings suggest that the efficiency and the mode of action of the PGPR B. subtilis is likely to be flax variety dependent.

Multiple functional self-association interfaces in plant TIR domains.

The self-association of Toll/interleukin-1 receptor/resistance protein (TIR) domains has been implicated in signaling in plant and animal immunity receptors. Structure-based studies identified different TIR-domain dimerization interfaces required for signaling of the plant nucleotide-binding oligomerization domain-like receptors (NLRs) L6 from flax and disease resistance protein RPS4 from Arabidopsis Here we show that the crystal structure of the TIR domain from the Arabidopsis NLR suppressor of npr1-1, constitutive 1 (SNC1) contains both an L6-like interface involving helices αD and αE (DE interface) and an RPS4-like interface involving helices αA and αE (AE interface). Mutations in either the AE- or DE-interface region disrupt cell-death signaling activity of SNC1, L6, and RPS4 TIR domains and full-length L6 and RPS4. Self-association of L6 and RPS4 TIR domains is affected by mutations in either region, whereas only AE-interface mutations affect SNC1 TIR-domain self-association. We further show two similar interfaces in the crystal structure of the TIR domain from the Arabidopsis NLR recognition of Peronospora parasitica 1 (RPP1). These data demonstrate that both the AE and DE self-association interfaces are simultaneously required for self-association and cell-death signaling in diverse plant NLRs.

Flaxseed extract exhibits mucosal protective effect in acetic acid induced colitis in mice by modulating cytokines, antioxidant and antiinflammatory mechanisms.

New treatments for inflammatory bowel disease are of interest due to high rate of remission failure. Natural products have been effective in IBD therapeutics as they have multiple constituents. The aim of the present study was to evaluate the effect of Flaxseed extract (Fs.Cr) on ulcerative colitis and identify the possible mechanisms involved. Colitis was induced by intrarectal administration of 6% AA in BALB/c mice. Colonic mucosal damage was assessed after 24h by calculating disease activity index (DAI), macroscopic and histological damage scores, biochemical measurement of myeloperoxidase (MPO), malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT), and total glutathione activities. Since cytokines are involved in exacerbating inflammatory cascade with emerging role of innate immune cytokines in IBD therapeutics, we hence assessed the effect on the levels of TNF-α, IFN-γ and IL-17, at 6, 12 and 24h by ELISA. Fs.Cr ameliorated the severity of AA colitis as evident by improved DAI, macroscopic damage and the histopathological scores along with restoration of goblet cells. Fs.Cr decreased MDA and MPO activities and enhanced antioxidant activity compared to the AA group. Finally, Fs.Cr in doses (300 and 500mg/kg) decreased TNF-α and IFN-γ levels at all time points with simultaneous increase in IL-17 levels at 24h as compared to the AA group. These results suggest that Fs.Cr ameliorates the severity of AA colitis by reducing goblet cell depletion, scavenging oxygen-derived free radicals, reduce neutrophil infiltration that may be attributed due to decreasing IFN-γ and TNF-α and increasing IL-17 levels.

[Case report: anaphylaxis caused by linseed included in baked bread].

We recently experienced a 29-year-old female with anaphylaxis caused by linseed included in homemade bread. This is the first report of linseed-induced allergy in Japan. She obtained the linseed-containing ingredients of bread by mail order. We performed skin-prick tests and basophil degranulation tests using extracts of the ingredients and commercially available linseeds; both tests showed positive results for linseed. The patient's serum was also positive for linseed-specific IgE. Since linseeds are included in various kinds of foods and exposure to them is increasing, linseeds may need to be recognized as a potential trigger of immediate-type allergy in Japan.

Structures of the flax-rust effector AvrM reveal insights into the molecular basis of plant-cell entry and effector-triggered immunity.

Fungal and oomycete pathogens cause some of the most devastating diseases in crop plants, and facilitate infection by delivering a large number of effector molecules into the plant cell. AvrM is a secreted effector protein from flax rust (Melampsora lini) that can internalize into plant cells in the absence of the pathogen, binds to phosphoinositides (PIPs), and is recognized directly by the resistance protein M in flax (Linum usitatissimum), resulting in effector-triggered immunity. We determined the crystal structures of two naturally occurring variants of AvrM, AvrM-A and avrM, and both reveal an L-shaped fold consisting of a tandem duplicated four-helix motif, which displays similarity to the WY domain core in oomycete effectors. In the crystals, both AvrM variants form a dimer with an unusual nonglobular shape. Our functional analysis of AvrM reveals that a hydrophobic surface patch conserved between both variants is required for internalization into plant cells, whereas the C-terminal coiled-coil domain mediates interaction with M. AvrM binding to PIPs is dependent on positive surface charges, and mutations that abrogate PIP binding have no significant effect on internalization, suggesting that AvrM binding to PIPs is not essential for transport of AvrM across the plant membrane. The structure of AvrM and the identification of functionally important surface regions advance our understanding of the molecular mechanisms underlying how effectors enter plant cells and how they are detected by the plant immune system.

Deciphering the responses of root border-like cells of Arabidopsis and flax to pathogen-derived elicitors.

Plant pathogens including fungi and bacteria cause many of the most serious crop diseases. The plant innate immune response is triggered upon recognition of microbe-associated molecular patterns (MAMPs) such as flagellin22 and peptidoglycan. To date, very little is known of MAMP-mediated responses in roots. Root border cells are cells that originate from root caps and are released individually into the rhizosphere. Root tips of Arabidopsis (Arabidopsis thaliana) and flax (Linum usitatissimum) release cells known as "border-like cells." Whereas root border cells of pea (Pisum sativum) are clearly involved in defense against fungal pathogens, the function of border-like cells remains to be established. In this study, we have investigated the responses of root border-like cells of Arabidopsis and flax to flagellin22 and peptidoglycan. We found that both MAMPs triggered a rapid oxidative burst in root border-like cells of both species. The production of reactive oxygen species was accompanied by modifications in the cell wall distribution of extensin epitopes. Extensins are hydroxyproline-rich glycoproteins that can be cross linked by hydrogen peroxide to enhance the mechanical strength of the cell wall. In addition, both MAMPs also caused deposition of callose, a well-known marker of MAMP-elicited defense. Furthermore, flagellin22 induced the overexpression of genes involved in the plant immune response in root border-like cells of Arabidopsis. Our findings demonstrate that root border-like cells of flax and Arabidopsis are able to perceive an elicitation and activate defense responses. We also show that cell wall extensin is involved in the innate immunity response of root border-like cells.

Population processes at multiple spatial scales maintain diversity and adaptation in the Linum marginale--Melampsora lini association.

Host-pathogen coevolution is a major driver of species diversity, with an essential role in the generation and maintenance of genetic variation in host resistance and pathogen infectivity. Little is known about how resistance and infectivity are structured across multiple geographic scales and what eco-evolutionary processes drive these patterns. Across southern Australia, the wild flax Linum marginale is frequently attacked by its rust fungus Melampsora lini. Here, we compare the genetic and phenotypic structure of resistance and infectivity among population pairs from two regions where environmental differences associate with specific life histories and mating systems. We find that both host and pathogen populations are genetically distinct between these regions. The region with outcrossing hosts and pathogens that go through asexual cycles followed by sexual reproduction showed greater diversity of resistance and infectivity phenotypes, higher levels of resistance and less clumped within-population spatial distribution of resistance. However, in the region where asexual pathogens infect selfing hosts, pathogens were more infective and better adapted to sympatric hosts. Our findings largely agree with expectations based on the distinctly different host mating systems in the two regions, with a likely advantage for hosts undergoing recombination. For the pathogen in this system, sexual reproduction may primarily be a survival mechanism in the region where it is observed. While it appears to potentially have adverse effects on local adaptation in the short term, it may be necessary for longer-term coevolution with outcrossing hosts.

Structural and functional analysis of a plant resistance protein TIR domain reveals interfaces for self-association, signaling, and autoregulation.

The Toll/interleukin-1 receptor (TIR) domain occurs in animal and plant immune receptors. In the animal Toll-like receptors, homodimerization of the intracellular TIR domain is required for initiation of signaling cascades leading to innate immunity. By contrast, the role of the TIR domain in cytoplasmic nucleotide-binding/leucine-rich repeat (NB-LRR) plant immune resistance proteins is poorly understood. L6 is a TIR-NB-LRR resistance protein from flax (Linum usitatissimum) that confers resistance to the flax rust phytopathogenic fungus (Melampsora lini). We determine the crystal structure of the L6 TIR domain and show that, although dispensable for pathogenic effector protein recognition, the TIR domain alone is both necessary and sufficient for L6 immune signaling. We demonstrate that the L6 TIR domain self-associates, most likely forming a homodimer. Analysis of the structure combined with site-directed mutagenesis suggests that self-association is a requirement for immune signaling and reveals distinct surface regions involved in self-association, signaling, and autoregulation.

Development of antibodies against secoisolariciresinol--application to the immunolocalization of lignans in Linum usitatissimum seeds.

Lignans are widely distributed plant metabolites associated with a large range of biological activities. In order to gain insight into their biosynthesis and their spatio-temporal accumulation an immunological probe was developed. Secondary metabolites generally have too small molecular weight to be antigenic and have to be associated with a carrier protein. Secoisolariciresinol was chosen as the hapten and was linked to bovine serum albumin via a spacer arm, the p-aminohippuric acid. The artificial antigen was injected to New Zealand rabbits. The successful production of polyclonal antibodies against secoisolariciresinol was assessed with indirect enzyme immunosorbent assay (ELISA) by comparison with pre-immune serum and by competitive assays using dilutions of secoisolariciresinol standards. The antibodies had an IC(50) value of 94 µg/ml and showed moderate cross-reactivities with structurally related compounds. They were thus used to immunolocalize lignans in flaxseed (Linum usitatissimum), one of the richest sources of lignans. The immunohistochemical labeling allowed us to localize for the first time lignans in planta. They are mainly localized in the secondary wall of the sclerite cells of the outer integument of the seed. A very light labeling is also observed in cytoplasmic inclusions of the endosperm. The results were correlated with HPLC analytical results which enabled to evaluate the relative lignan quantities: in flaxseed about 90% of the metabolites are localized in the outer integument.

Prospective study of sensitization and food allergy to flaxseed in 1317 subjects.

BACKGROUND: Foods containing flaxseed proteins rich inpolyunsaturatedfatty acids are new on the market. OBJECTIVES: In a population of patients attending the allergology department, we evaluated the frequency of sensitization to flaxseed, characterized allergens and looked for modifications related to industrial processing. METHODS: Natural, heated and extruded flaxseeds were tested using prick-in-prick tests (PIP using the fresh seed), SDS PAGE, immunoblots, immunoblot inhibition and Fourier Transform Infrared (FTIR) spectroscopy. RESULTS: PIP tests to natural flaxseed were positive in 5.8% of the 1317 patients. 73 of 77 PIP-positive patients were atopic. There was cross-reactivity with five seeds. peanut, soybean, rapeseed, lupine and wheat, and with rape pollen. Immunoblot inhibition by bromelain confirmed the presence of specific IgE to cross-reactive carbohydrate determinants (CCD). 0.15% of this population presented with food allergy to flaxseed and positive PIP to heated and extruded flaxseed. Two sera showed that clinically relevant allergens in industrial products had MW between 25 and 38 kDa. Sensitization to processed flaxseed characterized only the allergic subjects. FTIR spectroscopy showed major modifications in 3 and alpha structures following industrial processing. CONCLUSION: Positive prick tests to natural flaxseed were mainly due to cross-reactions. Flaxseed allergy is rare and could be detected by PIP to heated extruded flaxseed. Increasing consumption callsfor monitoring of clinical risk.

Internalization of flax rust avirulence proteins into flax and tobacco cells can occur in the absence of the pathogen.

Translocation of pathogen effector proteins into the host cell cytoplasm is a key determinant for the pathogenicity of many bacterial and oomycete plant pathogens. A number of secreted fungal avirulence (Avr) proteins are also inferred to be delivered into host cells, based on their intracellular recognition by host resistance proteins, including those of flax rust (Melampsora lini). Here, we show by immunolocalization that the flax rust AvrM protein is secreted from haustoria during infection and accumulates in the haustorial wall. Five days after inoculation, the AvrM protein was also detected within the cytoplasm of a proportion of plant cells containing haustoria, confirming its delivery into host cells during infection. Transient expression of secreted AvrL567 and AvrM proteins fused to cerulean fluorescent protein in tobacco (Nicotiana tabacum) and flax cells resulted in intracellular accumulation of the fusion proteins. The rust Avr protein signal peptides were functional in plants and efficiently directed fused cerulean into the secretory pathway. Thus, these secreted effectors are internalized into the plant cell cytosol in the absence of the pathogen, suggesting that they do not require a pathogen-encoded transport mechanism. Uptake of these proteins is dependent on signals in their N-terminal regions, but the primary sequence features of these uptake regions are not conserved between different rust effectors.

Engineering Flax with the GT Family 1 Solanum sogarandinum Glycosyltransferase SsGT1 Confers Increased Resistance to Fusarium Infection.

The aim of this study was to engineer a flax with increased resistance to pathogens. The approach was based on the recent analysis of the Solanum sogarandinum -derived glycosyltransferase (UGT) protein, designated SsGT1 (previously called 5UGT). On the basis of enzyme studies, the recombinant SsGT1 is a 7-O-glycosyltransferase, the natural substrates of which include both anthocyanidins and flavonols such as kaempferol and quercetin. Because flavonoids act as antioxidants and glycosylation increases the stability of flavonoids, it has been suggested that the accumulation of a higher quantity of flavonoid glycosides in transgenic plants might improve their resistance to pathogen infection. Flax overproducing SsGT1 showed higher resistance to Fusarium infection than wild-type plants, and this was correlated with a significant increase in the flavonoid glycoside content in the transgenic plants. Overproduction of glycosyltransferase in transgenic flax also resulted in proanthocyanin, lignan, phenolic acid, and unsaturated fatty acid accumulation in the seeds. The last is meaningful from a biotechnological point of view and might suggest the involvement of polyphenol glycosides in the protection of unsaturated fatty acids against oxidation and thus improve oil storage. It is thus suggested that introduction of SsGT1 is sufficient for engineering altered pathogen resistance in flax.

Crystal structures of flax rust avirulence proteins AvrL567-A and -D reveal details of the structural basis for flax disease resistance specificity.

The gene-for-gene mechanism of plant disease resistance involves direct or indirect recognition of pathogen avirulence (Avr) proteins by plant resistance (R) proteins. Flax rust (Melampsora lini) AvrL567 avirulence proteins and the corresponding flax (Linum usitatissimum) L5, L6, and L7 resistance proteins interact directly. We determined the three-dimensional structures of two members of the AvrL567 family, AvrL567-A and AvrL567-D, at 1.4- and 2.3-A resolution, respectively. The structures of both proteins are very similar and reveal a beta-sandwich fold with no close known structural homologs. The polymorphic residues in the AvrL567 family map to the surface of the protein, and polymorphisms in residues associated with recognition differences for the R proteins lead to significant changes in surface chemical properties. Analysis of single amino acid substitutions in AvrL567 proteins confirm the role of individual residues in conferring differences in recognition and suggest that the specificity results from the cumulative effects of multiple amino acid contacts. The structures also provide insights into possible pathogen-associated functions of AvrL567 proteins, with nucleic acid binding activity demonstrated in vitro. Our studies provide some of the first structural information on avirulence proteins that bind directly to the corresponding resistance proteins, allowing an examination of the molecular basis of the interaction with the resistance proteins as a step toward designing new resistance specificities.

Flax rust resistance gene specificity is based on direct resistance-avirulence protein interactions.

Genetic studies of the flax-flax rust interaction led to the formulation of the gene-for-gene hypothesis and identified resistance genes (R) in the host plant and pathogenicity genes, including avirulence (Avr) and inhibitor of avirulence genes (I), in the rust pathogen. R genes have now been cloned from four of the five loci in flax and all encode proteins of the Toll, Interleukin-1 receptor, R gene-nucleotide binding site-leucine-rich repeat (TIR-NBS-LRR) class. Avr genes have been cloned from four loci in flax rust and encode small secreted proteins with no between locus similarity and no close homologs in current data bases. It is postulated that Avr proteins enter the host cell, have virulence effector functions, and in resistant host genotypes, are recognized by direct and specific interaction with host R proteins, leading to activation of rust resistance defense responses. Direct interaction between R and Avr proteins is the basis of gene-for-gene specificity in the flax-flax rust system and both R and Avr genes have the signatures of diversifying selection, suggesting the existence of a coevolutionary arms race between the host plant and its obligate rust pathogen.

Antibodies with specificities for D-xylose and for D-galacturonic acid residues of flaxseed polysaccharides.

Two sets of anti-carbohydrate antibodies, one with specificity for D-xylose residues of flaxseed polysaccharides and the other with specificity for D-galacturonic acid residues, have been isolated by affinity chromatography from the immune serum of rabbits immunized with a vaccine of the polysaccharides and Freunds complete adjuvant. A number of properties of the antibodies are described. Of special note is the finding that, like other anti-carbohydrate antibodies, the new antibodies are biosynthesized in multi-molecular forms.

Effects of exposure to flax dust in Polish farmers: work-related symptoms and immunologic response to microbial antigens associated with dust.

Medical examinations were performed in a group of 51 Polish farmers heavily exposed to flax dust during harvesting and scutching (threshing) and in a group of 50 healthy urban dwellers not exposed to organic dusts (controls). The examinations included: interview concerning the occurrence of respiratory disorders and work-related symptoms, physical examination, X-ray examination of chest, lung function tests, oxymetric examinations, determination of the concentration of cytokines (IL-1alpha IL-6, TNFalpha) in blood serum and allergological tests with microbial antigens associated with organic dust, comprising: skin prick test with 4 antigens, agar-gel precipitation test with 12 antigens and test for specific inhibition of leukocyte migration with 4 antigens. As many as 32 farmers (62.7%) reported the occurrence of work-related symptoms during harvesting, transporting and scutching of flax. The most common complaint was general weakness reported by 15 farmers (29.4%), followed by headache reported by 14 (27.5%), blocking of the nose - by 11 (21.6%), dry cough, shivering, and eyes itching - each by 10 (19.8%), chest tightness and hoarseness - each by 9 (17.6%). No control subjects reported these work-related symptoms. The mean spirometric values in the examined group of farmers were within a normal range and did not show a significant post-shift decline. In contrast, a significant post-shift decline of oxymetric values was found among flax farmers. The farmers showed a frequency of the positive early skin reactions to environmental allergens in the range of 0-19.6%, a frequency of positive precipitin reactions in a range of 0-56.9%, and frequency of positive reactions of leukocyte migration inhibition in a range of 7.8-21.6%. The members of the control group responded to the majority of allergens with a significantly lower frequency of positive results compared to the farmers. Elevated concentrations of IL-1alpha and IL-6, but not TNFalpha, were found in blood sera of flax farmers. In conclusion, farmers engaged in harvesting and scutching of flax represent a group of elevated professional risk because of high incidence of work-related symptoms and high frequency of allergic reactions to bacteria and fungi associated with organic dust.