Fungal pathogens of cereal crops: Proteomic insights into fungal pathogenesis, host defense, and resistance.

Fungal infections of cereal crops pose a significant risk to global food security through reduced grain production and quality, as well as contamination of animal feed and human products for consumption. To combat fungal disease, we need to understand how the pathogen adapts and survives within the hostile environment of the host and how the host's defense response can be modulated for protection from disease. Such investigations offer insight into fungal pathogenesis, host immunity, the development of resistance, and mechanisms of action for currently-used control strategies. Mass spectrometry-based proteomics provides a technologically-advanced platform to define differences among fungal pathogens and their hosts at the protein level, supporting the discovery of proteins critical for disease, and uncovering novel host responses driving susceptibly or resistance of the host. In this Review, we explore the role of mass spectrometry-based proteomics in defining the intricate relationship between a pathogen and host during fungal disease of cereal crops with a focus on recent discoveries derived from the globally-devastating diseases of Fusarium head blight, Rice blast, and Powdery mildew. We highlight advances made for each of these diseases and discuss opportunities to extrapolate findings to further our fight against fungal pathogens on a global scale.

GWAS provides biological insights into mechanisms of the parasitic plant (Striga) resistance in sorghum.

BACKGROUND: Sorghum yields in sub-Saharan Africa (SSA) are greatly reduced by parasitic plants of the genus Striga (witchweed). Vast global sorghum genetic diversity collections, as well as the availability of modern sequencing technologies, can be potentially harnessed to effectively manage the parasite. RESULTS: We used laboratory assays - rhizotrons to screen a global sorghum diversity panel to identify new sources of resistance to Striga; determine mechanisms of resistance, and elucidate genetic loci underlying the resistance using genome-wide association studies (GWAS). New Striga resistant sorghum determined by the number, size and biomass of parasite attachments were identified. Resistance was by; i) mechanical barriers that blocked parasite entry, ii) elicitation of a hypersensitive reaction that interfered with parasite development, and iii) the inability of the parasite to develop vascular connections with hosts. Resistance genes underpinning the resistance corresponded with the resistance mechanisms and included pleiotropic drug resistance proteins that transport resistance molecules; xylanase inhibitors involved in cell wall fortification and hormonal regulators of resistance response, Ethylene Response Factors. CONCLUSIONS: Our findings are of fundamental importance to developing durable and broad-spectrum resistance against Striga and have far-reaching applications in many SSA countries where Striga threatens the livelihoods of millions of smallholder farmers that rely on sorghum as a food staple.

Protective responses of tolerant and sensitive wheat seedlings to systemic and local zearalenone application - Electron paramagnetic resonance studies.

BACKGROUND: Mycotoxins are among the environmental stressors whose oxidative action is currently widely studied. The aim of this paper was to investigate the response of seedling leaves to zearalenone (ZEA) applied to the leaves (directly) and to the grains (indirectly) in tolerant and sensitive wheat cultivars. RESULTS: Biochemical analyses of antioxidant activity were performed for chloroplasts and showed a similar decrease in this activity irrespective of plant sensitivity and the way of ZEA application. On the other hand, higher amounts of superoxide radical (microscopic observations) were generated in the leaves of plants grown from the grains incubated in ZEA solution and in the sensitive cultivar. Electron paramagnetic resonance (EPR) studies showed that upon ZEA treatment greater numbers of Mn - aqua complexes were formed in the leaves of the tolerant wheat cultivar than in those of the sensitive one, whereas the degradation of Fe-protein complexes occurred independently of the cultivar sensitivity. CONCLUSION: The changes in the quantity of stable, organic radicals formed by stabilizing reactive oxygen species on biochemical macromolecules, indicated greater potential for their generation in leaf tissues subjected to foliar ZEA treatment. This suggested an important role of these radical species in protective mechanisms mainly against direct toxin action. The way the defense mechanisms were activated depended on the method of the toxin application.

Extraction of total wheat (Triticum aestivum) protein fractions and cross-reactivity of wheat allergens with other cereals.

A one-step mild extraction of total wheat protein fractions was developed in this study, and the allergic cross-reactivity among dietary cereals were assessed by SDS-PAGE, western blotting, indirect ELISA, and inhibition ELISA using sera from 12 wheat allergic patients. The fractions of albumin, globulin, gliadin and glutenins in wheat flour can be obtained by a one-step extraction with Na2CO3-NaHCO3 (20 mM, pH 9.6, 0.5 M NaCl, 40% ethanol, 1 mM PMSF) in comparison to sequential extractions. Results showed high cross-reactivity in wheat, barley and rye due to close resemblance and high sequence identity (>50%), whereas nearly negligible cross-reactivity among rice, buckwheat, and quinoa was observed. Our research findings suggest that people with wheat allergy should rely primarily on the use of rice, quinoa and non-grain buckwheat, which is an effective substitute for wheat, while those with hypersensitivity should avoid the use of barley and rye in their diet.

Snow mold of winter cereals: a complex disease and a challenge for resistance breeding.

KEY MESSAGE: Snow mold resistance is a complex quantitative trait highly affected by environmental conditions during winter that must be addressed by resistance breeding. Snow mold resistance in winter cereals is an important trait for many countries in the Northern Hemisphere. The disease is caused by at least four complexes of soilborne fungi and oomycetes of which Microdochium nivale and M. majus are among the most common pathogens. They have a broad host range covering all winter and spring cereals and can basically affect all plant growth stages and organs. Their attack leads to a low germination rate, and/or pre- and post-emergence death of seedlings after winter and, depending on largely unknown environmental conditions, also to foot rot, leaf blight, and head blight. Resistance in winter wheat and triticale is governed by a multitude of quantitative trait loci (QTL) with mainly additive effects highly affected by genotype × environment interaction. Snow mold resistance interacts with winter hardiness in a complex way leading to a co-localization of resistance QTLs with QTLs/genes for freezing tolerance. In practical breeding, a multistep procedure is necessary with (1) freezing tolerance tests, (2) climate chamber tests for snow mold resistance, and (3) field tests in locations with and without regularly occurring snow cover. In the future, resistance sources should be genetically characterized also in rye by QTL mapping or genome-wide association studies. The development of genomic selection procedures should be prioritized in breeding research.

Clinical and in vitro cross-reactivity of cereal grains in children with IgE-mediated wheat allergy

INTRODUCTION AND OBJECTIVES: Wheat and cereal grains have a broad range of cross-reactivity, but the clinical relevance of this cross-reactivity is uncertain. This study aimed to evaluate clinical and in vitro cross-reactivity with barley, oat, and Job's tears among wheat-allergic patients. MATERIALS AND METHODS: Patients aged 5 to 15 years with IgE-mediated wheat allergy were enrolled. Skin prick test (SPT) and specific IgE (sIgE) to wheat, barley, and oat, and SPT to Job's tears were performed. Oral food challenge (OFC) was conducted if the SPT was ≤5 mm in size and there was no history of anaphylaxis to each grain. Profiles of sIgE bound allergens of wheat, barley, and oat, and inhibition ELISA of IgE binding to barley and oat with wheat were performed. RESULTS: Ten patients with a median age of 8 years were enrolled. Nine of those patients had a history of wheat anaphylaxis. The median SPT size and sIgE level to wheat was 7.3 mm and 146.5 kUA/l, respectively. The cross-reactivity rate for barley, oat, and Job's tears was 60.0%, 33.3%, and 20.0%, respectively. Significantly larger SPT size and higher sIgE level were observed in patients with positive cross-reactivity to barley and oat when compared to patients without cross-reactivity. Barley and oat extracts inhibited 59% and 16% of sIgE bound to wheat gliadins and glutenins, respectively. CONCLUSION: The cross-reactivity rate was quite low for oat and Job's tears compared to that of barley; therefore, avoidance of all cereal grains may be unnecessary in patients with severe wheat allergy

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X6: A Novel Antibody for Potential Use in Gluten Quantification.

Gliadin is a fraction of gluten, known to trigger celiac disease in susceptible people. To date, the life-long gluten-free diet is used for the prevention of this disease. Hence, methods for gluten control in foods are of significant importance. Being one of the most-used methods used for this purpose, ELISA should use high-affinity antibodies to gliadin peptides involved into celiac process. This study investigates the characteristics of a novel anti-gliadin antibody X6. We found the QXQPFPXP site to be a recognized epitope that provides specific binding of the antibody to cereal prolamins involved in celiac disease manifestation. A specificity study using immunoblotting shows the recognition of wheat, barley and rye proteins-as well as α-gliadin homologs from non-edible cereals (Dasypyrum villosum). Reactivity to avenin was less pronounced, as this protein does not contain the PFP motif most critical for antibody recognition. The proteins of Zea mays and Setaria italica were not recognized by X6. X6-based ELISA highly correlated with R5 and G12, which are Codex Alimentarius standards in the quantitative assessment of gluten content (Pearson's R = 0.86 and 0.87, respectively). Qualitative assessment revealed no significant differences between R5 and G12 and X6.

Clinical and in vitro cross-reactivity of cereal grains in children with IgE-mediated wheat allergy.

INTRODUCTION AND OBJECTIVES: Wheat and cereal grains have a broad range of cross-reactivity, but the clinical relevance of this cross-reactivity is uncertain. This study aimed to evaluate clinical and in vitro cross-reactivity with barley, oat, and Job's tears among wheat-allergic patients. MATERIALS AND METHODS: Patients aged 5 to 15 years with IgE-mediated wheat allergy were enrolled. Skin prick test (SPT) and specific IgE (sIgE) to wheat, barley, and oat, and SPT to Job's tears were performed. Oral food challenge (OFC) was conducted if the SPT was ≤5 mm in size and there was no history of anaphylaxis to each grain. Profiles of sIgE bound allergens of wheat, barley, and oat, and inhibition ELISA of IgE binding to barley and oat with wheat were performed. RESULTS: Ten patients with a median age of 8 years were enrolled. Nine of those patients had a history of wheat anaphylaxis. The median SPT size and sIgE level to wheat was 7.3 mm and 146.5 kUA/l, respectively. The cross-reactivity rate for barley, oat, and Job's tears was 60.0%, 33.3%, and 20.0%, respectively. Significantly larger SPT size and higher sIgE level were observed in patients with positive cross-reactivity to barley and oat when compared to patients without cross-reactivity. Barley and oat extracts inhibited 59% and 16% of sIgE bound to wheat gliadins and glutenins, respectively. CONCLUSION: The cross-reactivity rate was quite low for oat and Job's tears compared to that of barley; therefore, avoidance of all cereal grains may be unnecessary in patients with severe wheat allergy.

Assessing the health impact of interventions for baker's allergy and asthma in supermarket bakeries: a group randomised trial.

PURPOSE: To assess the impact of an intervention for baker's allergy and asthma in supermarket bakeries. METHODS: A group randomised trial conducted in 31 bakeries (n = 337 bakers) that were randomly assigned to one of two intervention groups (n = 244 bakers) and a control group (n = 93 bakers). Health data collected prior to and 1-year after the intervention included information obtained from an ECRHS questionnaire; tests for atopy and serum-specific IgE to cereal flours; fractional exhaled nitric oxide (FeNO). Data from the two intervention groups were combined to form one intervention group for purposes of the statistical analysis. RESULTS: At 1 year of follow-up, the incidence and level of decline of work-related ocular-nasal and chest symptoms, sensitisation status and elevated FeNO (FeNO > 25 ppb) was similar in both intervention and control groups. The mean FeNO difference was also similar across both groups (2.2 ppb vs 1.7 ppb, p = 0.86). In those with FeNO > 25 ppb at baseline, the decline was greater in the intervention compared to control group (16.9 ppb vs 7.7 ppb, p = 0.24). Multivariate logistic regression models (adjusting for smoking, baseline sensitisation to cereal flour, baseline FeNO > 25 ppb) did not demonstrate an appreciable FeNO decline (≥ 10%) in the intervention compared to control group. However, stratification by the presence of work-related ocular-nasal symptoms in bakers at baseline demonstrated a significant FeNO decline (≥ 10%) in the intervention compared to the control group (OR 3.73, CI 1.22-11.42). CONCLUSION: This study demonstrates some evidence of an intervention effect on FeNO 1 year after an intervention, particularly in bakers with work-related ocular-nasal symptoms.

The Pros and Cons of Using Oat in a Gluten-Free Diet for Celiac Patients.

A therapeutic gluten-free diet often has nutritional limitations. Nutritional qualities such as high protein content, the presence of biologically active and beneficial substances (fiber, beta-glucans, polyunsaturated fatty acids, essential amino acids, antioxidants, vitamins, and minerals), and tolerance by the majority of celiac patients make oat popular for use in gluten-free diet. The health risk of long-time consumption of oat by celiac patients is a matter of debate. The introduction of oat into the diet is only recommended for celiac patients in remission. Furthermore, not every variety of oat is also appropriate for a gluten-free diet. The risk of sensitization and an adverse immunologically mediated reaction is a real threat in some celiac patients. Several unsolved issues still exist which include the following: (1) determination of the susceptibility markers for the subgroup of celiac patients who are at risk because they do not tolerate dietary oat, (2) identification of suitable varieties of oat and estimating the safe dose of oat for the diet, and (3) optimization of methods for detecting the gliadin contamination in raw oat used in a gluten-free diet.

Prevention of Food Allergy: The Significance of Early Introduction.

Over the last two decades, the prevalence of food allergies has registered a significant increase in Westernized societies, potentially due to changes in environmental exposure and lifestyle. The pathogenesis of food allergies is complex and includes genetic, epigenetic and environmental factors. New evidence has highlighted the role of the intestinal microbiome in the maintenance of the immune tolerance to foods and the potential pathogenic role of early percutaneous exposure to allergens. The recent increase in food allergy rates has led to a reconsideration of prevention strategies for atopic diseases, mainly targeting the timing of the introduction of solid foods into infants' diet. Early recommendation for high atopy risk infants to delay the introduction of potential food allergens, such as cow's milk, egg, and peanut, until after the first year of life, has been rescinded, as emerging evidence has shown that these approaches are not effective in preventing food allergies. More recently, high-quality clinical trials have suggested an opposite approach, which promotes early introduction of potential food allergens into infants' diet as a means to prevent food allergies. This evidence has led to the production of new guidelines recommending early introduction of peanut as a preventive strategy for peanut allergy. However, clinical trials investigating whether this preventive dietary approach could also apply to other types of food allergens have reported ambiguous results. This review focuses on the latest high-quality evidence from randomized controlled clinical trials examining the timing of solid food introduction as a strategy to prevent food allergies and also discusses the possible implications of early complementary feeding on both the benefits and the total duration of breastfeeding.

The AvrPm3-Pm3 effector-NLR interactions control both race-specific resistance and host-specificity of cereal mildews on wheat.

The wheat Pm3 resistance gene against the powdery mildew pathogen occurs as an allelic series encoding functionally different immune receptors which induce resistance upon recognition of isolate-specific avirulence (AVR) effectors from the pathogen. Here, we describe the identification of five effector proteins from the mildew pathogens of wheat, rye, and the wild grass Dactylis glomerata, specifically recognized by the PM3B, PM3C and PM3D receptors. Together with the earlier identified AVRPM3A2/F2, the recognized AVRs of PM3B/C, (AVRPM3B2/C2), and PM3D (AVRPM3D3) belong to a large group of proteins with low sequence homology but predicted structural similarities. AvrPm3b2/c2 and AvrPm3d3 are conserved in all tested isolates of wheat and rye mildew, and non-host infection assays demonstrate that Pm3b, Pm3c, and Pm3d are also restricting the growth of rye mildew on wheat. Furthermore, divergent AVR homologues from non-adapted rye and Dactylis mildews are recognized by PM3B, PM3C, or PM3D, demonstrating their involvement in host specificity.

Performance of specific immunoglobulin E tests for diagnosing occupational asthma: a systematic review and meta-analysis.

OBJECTIVES: To determine the test performance parameters for the retrievable range of high-molecular-weight (HMW) and low-molecular-weight (LMW) occupational allergens and to evaluate the impact of allergenic components and the implementation of measures for test validation. METHODS: A protocol with predefined objectives and inclusion criteria was the basis of an electronic literature search of MEDLINE and EMBASE (time period 1967-2016). The specific inhalation challenge and serial peak flow measurements were the reference standards for the specific IgE (sIgE) test parameters. All of the review procedures were reported according to Preferred Reporting Items for Systematic Reviews and Meta-Analyses. RESULTS: Seventy-one studies were selected, and 62 entered meta-analysis. Pooled pairs analysis indicated a sensitivity of 0.74(95% CI 0.66 to 0.80) and specificity of 0.71(95% CI 0.63 to 0.77) for HMW allergens and a sensitivity of 0.28(95% CI 0.18 to 0.40) and specificity of 0.89(95% CI 0.77 to 0.95) for LMW allergens. Component-specific analysis improved the test parameters for some allergens. Test validation was handled heterogeneously among studies. CONCLUSION: sIgE test performance is rather satisfactory for a wide range of HMW allergens with the potential for component-specific approaches, whereas sensitivity for LMW allergens is considerably lower, indicating methodological complications and/or divergent pathomechanisms. A common standard for test validation is needed.

Targeted Metabolite Profiling-Based Identification of Antifungal 5-n-Alkylresorcinols Occurring in Different Cereals against Fusarium oxysporum.

A rapid and convenient biochemometrics-based analysis of several cereal-derived extracts was used to identify n-alkyl(enyl)resorcinols (AR) as antifungals against Fusarium oxysporum. Total AR content and liquid chromatography/mass spectrometry (LC-MS)-based profiles were recorded for each extract, in addition to their antifungal activity, to help integrate these chemical and biological datasets by orthogonal partial least squares regression. In this study, we developed and used a micro-scale amended medium (MSAM) assay to evaluate the in vitro mycelial growth inhibition at low amounts of extracts. Triticale husk-derived extracts had the highest AR content (662.1 µg olivetol equivalent/g dry extract), exhibiting >79% inhibition at the highest doses (10.0⁻1.0 µg/µL). Correlation of the chemical and antifungal datasets using supervised metabolite profiling revealed that 5-n-nonadecanylresorcinol, 5-n-heneicosylresorcinol, and 5-n-tricosyl-resorcinol were the most active ARs occurring in cereal products from Colombia. Hence, we propose the biochemometrics-based approach as a useful tool for identifying AR-like antifungals against F. oxysporum.

Antibiotic producing endophytic Streptomyces spp. colonize above-ground plant parts and promote shoot growth in multiple healthy and pathogen-challenged cereal crops.

The Streptomyces spp. used in this work were previously isolated as diazotrophic endophytes from sorghum stems. Here, we characterized the Streptomyces spp. for their colonization ability, plant growth promotion and protection against fungal disease in three cereals. In vitro analysis by dual culture study showed inhibitory effect on the rice pathogen Magnaporthe oryzae B157 along with inhibition of the ubiquitous phytopathogen Rhizoctonia solani by the Streptomyces spp. used in this study. The active compounds responsible for phytopathogen inhibition were extracted with ethyl acetate and tested positive against the fungal pathogens. GC-MS based identification of the active compounds responsible for fungal pathogen inhibition showed them to be 2-(chloromethyl)-2-cyclopropyloxirane, 2, 4- ditert-butylphenol and 1-ethylthio-3-methyl-1, 3-butadiene in extracts of culture supernatants from the three different strains respectively. EGFP tagged Streptomyces strains showed profuse colonization in roots as well as aerial parts of cereal plants. Direct inhibitory action against M. oryzae B157 and R. solani correlated with the observation that upon fungal pathogen challenge, the bacterized rice, sorghum and wheat plants showed significantly good plant growth, particularly in aerial parts as compared to unbacterized controls. In addition, benefit was seen in inoculated healthy plants in terms of increase in wet weight of roots and shoots as compared to the uninoculated controls. The mechanism of biocontrol also involved induction of plant defense response as evidenced by the upregulation of PR10a, NPR1, PAL and LOX2 in Streptomyces colonized plants.

Development of a Nanobody-AviTag Fusion Protein and Its Application in a Streptavidin-Biotin-Amplified Enzyme-Linked Immunosorbent Assay for Ochratoxin A in Cereal.

Ochratoxin A (OTA) is a common food contaminant that threatens consumers' safety and health. A sensitive and selective biotin-streptavidin-amplified enzyme-linked immunosorbent assay (BA-ELISA) for OTA using a nanobody-AviTag fusion protein (Nb-AviTag) was developed in this study. The prokaryotic expression vector Nb28-AviTag-pAC6 for Nb-AviTag was constructed, followed by transformation to the AVB101 cells for antibody expression and in vivo biotinylation. The purified Nb28-AviTag was used to establish the BA-ELISA and the procedures for this Nb-AviTag-based BA-ELISA were optimized. The Nb-AviTag-based BA-ELISA exhibited the half maximal inhibitory concentration (IC50) of 0.14 ng mL-1 and the limit of detection (LOD = IC10) of 0.028 ng mL-1 for OTA basing on the optimized experiment parameters. The assay sensitivity was improved 4.6 times and 4.3 times compared to Nb-based ELISA, respectively. This method had LODs of 1.4 µg kg-1 in barley, 0.56 µg kg-1 in oats, and 0.84 µg kg-1 in rice for OTA. The average recovery percent was in a range of 84-137%, and the relative standard derivation percent ranged from 0.64% to 7.8%. The content of OTA in contaminated cereal samples was determined by both the developed Nb-AviTag-based method and liquid chromatography-tandem mass spectrometry (LC-MS/MS). The results demonstrated that the Nb-AviTag was a robust and promising bioreceptor in highly sensitive detection of OTA and other low molecular weight compounds using BA system.

Specific IgE for Fag e 3 Predicts Oral Buckwheat Food Challenge Test Results and Anaphylaxis: A Pilot Study.

BACKGROUND: Buckwheat (BW) is the source of a life-threatening allergen. Fag e 3-specific serum IgE (sIgE) is more useful than BW-sIgE for diagnosis; however, it is unknown whether Fag e 3-sIgE can predict oral food challenge (OFC) results and anaphylaxis. This study aimed to clarify the efficacy of Fag e 3-sIgE in predicting OFC results and anaphylaxis. METHODS: We conducted a retrospective review of BW- and Fag e 3-sIgE data obtained using the ImmunoCAP® assay system and fluorescent enzyme-linked immunosorbent assay from children who underwent OFC using 3,072 mg of BW protein between July 2006 and March 2014 at Sagamihara National Hospital, Kanagawa, Japan. RESULTS: We analyzed 60 patients aged 1.9-13.4 years (median 6.0 years); 20 (33%) showed objective symptoms upon BW OFC. The patients without symptoms had significantly lower Fag e 3-sIgE than those with non-anaphylactic (p < 0.001) and anaphylactic reactions to BW (p = 0.004). Fag e 3-sIgE was the only tested factor that significantly predicted positive OFC results (odds ratio 8.93, 95% confidence interval 3.10-25.73, p < 0.001) and OFC-induced anaphylaxis (2.67, 1.12-6.35, p = 0.027). We suggest that a threshold Fag e 3-sIgE level of 18.0 kUE/L has 95% probability of provoking a positive reaction to BW. CONCLUSIONS: Fag e 3-sIgE predicted OFC results and OFC-induced anaphylaxis. We further emphasize paying careful attention to the risk of BW OFC-induced anaphylaxis.