Inheritance of faba bean resistance to Broomrape, genetic diversity and QTL mapping analysis.

Six faba bean parents and their F1 and F2 generations were used in this investigation to study the genetic system controlling resistance of faba bean (Vicia faba L.) to broomrape (Orobanche crenata). Most of the F1 hybrids were tolerant to broomrape. In the F2 generation, the population P5 × P6 (Assiut 125 × Romy 12) gave the highest value of relative yield and tolerance index. Heterosis and inbreeding depression were only positive in number of tillers/plant and seed yield/plant characters. The results indicated that the additive effect was more important than the dominance one (D > H1) only for No. of pods/plant in the F1 generation. Moreover, the narrow-sense heritability was low for most of the studied traits. Three molecular marker systems, namely RAPD, ISSR and SRAP were used for identification and estimation of the genetic diversity among the six faba bean genotypes. The three molecular markers generated DNA unique bands for all genotypes. Only, eight DNA fragments were related to Orobanche tolerance. Clearly and reproducible polymorphic markers were subjected to QTL analysis. The linkage analysis showed that, out of 34 marker loci segregated in the F2 population, 29 (85.29%) were mapped on three linkage groups. QTL analysis using SIM method performed for the 29 markers assigned to LG-1, LG-2 and LG-3 with the eight traits, number of tillers/plant, plant height, number of pods/plant, seed yield/plant, number of broomrape spikes per plant, height of broomrape spikes, relative yield and tolerance index, showing 12 putative QTLs for all traits except number of tillers/plant. From this study, it is clear that P5 × P6 (Assiut 125 × Romy12) population could be considered promising for selection for resistance to broomrape infestation.

Intestinal transcriptional profiling reveals fava bean-induced immune response in DBA/1 mice.

BACKGROUND: Fava beans (FBs) have long been used as food, and their principal disadvantage is derived from their haemotoxicity. We hypothesized that FB ingestion alters the intestinal gene expression pattern, thereby inducing an immune response. RESULTS: In-depth sequence analysis identified 769 differentially expressed genes (DEGs) associated with the intestine in FB-treated DBA/1 mouse intestines. The identified genes were shown to be associated with biological processes (such as response to stimulus and immune system processes), human disease pathways (such as infectious diseases, endocrine and metabolic diseases, and immune diseases), and organismal system pathways (such as the digestive system, endocrine system, environmental adaptation, and immune system). Moreover, plasma total immunoglobulin E (IgE), histamine, interleukin (IL)-4 and IL-13 levels were significantly increased when the mice were treated with FBs. CONCLUSIONS: These results demonstrated that FBs affect the intestinal immune response and IgE and cytokine secretion in DBA/1 mice.

Intestinal transcriptional profiling reveals fava bean-induced immune response in DBA/1 mice

BACKGROUND: Fava beans (FBs) have long been used as food, and their principal disadvantage is derived from their haemotoxicity. We hypothesized that FB ingestion alters the intestinal gene expression pattern, thereby inducing an immune response. RESULTS: In-depth sequence analysis identified 769 differentially expressed genes (DEGs) associated with the intestine in FB-treated DBA/1 mouse intestines. The identified genes were shown to be associated with biological processes (such as response to stimulus and immune system processes), human disease pathways (such as infectious diseases, endocrine and metabolic diseases, and immune diseases), and organismal system pathways (such as the digestive system, endocrine system, environmental adaptation, and immune system). Moreover, plasma total immunoglobulin E (IgE), histamine, interleukin (IL)-4 and IL-13 levels were significantly increased when the mice were treated with FBs. CONCLUSIONS: These results demonstrated that FBs affect the intestinal immune response and IgE and cytokine secretion in DBA/1 mice.

Broad bean-induced anaphylaxis: a case report

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Large-Scale Transcriptome Analysis in Faba Bean (Vicia faba L.) under Ascochyta fabae Infection.

Faba bean is an important food crop worldwide. However, progress in faba bean genomics lags far behind that of model systems due to limited availability of genetic and genomic information. Using the Illumina platform the faba bean transcriptome from leaves of two lines (29H and Vf136) subjected to Ascochyta fabae infection have been characterized. De novo transcriptome assembly provided a total of 39,185 different transcripts that were functionally annotated, and among these, 13,266 were assigned to gene ontology against Arabidopsis. Quality of the assembly was validated by RT-qPCR amplification of selected transcripts differentially expressed. Comparison of faba bean transcripts with those of better-characterized plant genomes such as Arabidopsis thaliana, Medicago truncatula and Cicer arietinum revealed a sequence similarity of 68.3%, 72.8% and 81.27%, respectively. Moreover, 39,060 single nucleotide polymorphism (SNP) and 3,669 InDels were identified for genotyping applications. Mapping of the sequence reads generated onto the assembled transcripts showed that 393 and 457 transcripts were overexpressed in the resistant (29H) and susceptible genotype (Vf136), respectively. Transcripts involved in plant-pathogen interactions such as leucine rich proteins (LRR) or plant growth regulators involved in plant adaptation to abiotic and biotic stresses were found to be differently expressed in the resistant line. The results reported here represent the most comprehensive transcript database developed so far in faba bean, providing valuable information that could be used to gain insight into the pathways involved in the resistance mechanism against A. fabae and to identify potential resistance genes to be further used in marker assisted selection.

Plant Immune Responses: Aphids Strike Back.

To survive and complete their life cycle, herbivorous insects face the difficult challenge of coping with the arsenal of plant defences. A new study reports that aphids secrete evolutionarily conserved cytokines in their saliva to suppress host immune responses.

A Secreted MIF Cytokine Enables Aphid Feeding and Represses Plant Immune Responses.

Aphids attack virtually all plant species and cause serious crop damages in agriculture. Despite their dramatic impact on food production, little is known about the molecular processes that allow aphids to exploit their host plants. To date, few aphid salivary proteins have been identified that are essential for aphid feeding, and their nature and function remain largely unknown. Here, we show that a macrophage migration inhibitory factor (MIF) is secreted in aphid saliva. In vertebrates, MIFs are important pro-inflammatory cytokines regulating immune responses. MIF proteins are also secreted by parasites of vertebrates, including nematodes, ticks, and protozoa, and participate in the modulation of host immune responses. The finding that a plant parasite secretes a MIF protein prompted us to question the role of the cytokine in the plant-aphid interaction. We show here that expression of MIF genes is crucial for aphid survival, fecundity, and feeding on a host plant. The ectopic expression of aphid MIFs in leaf tissues inhibits major plant immune responses, such as the expression of defense-related genes, callose deposition, and hypersensitive cell death. Functional complementation analyses in vivo allowed demonstrating that MIF1 is the member of the MIF protein family that allows aphids to exploit their host plants. To our knowledge, this is the first report of a cytokine that is secreted by a parasite to modulate plant immune responses. Our findings suggest a so-far unsuspected conservation of infection strategies among parasites of animal and plant species.

Hypersensitivity linked to exposure of broad bean protein(s) in allergic patients and BALB/c mice.

OBJECTIVE: Broad bean (Vicia faba L.), a common vegetable, belongs to the family Fabaceae and is consumed worldwide. Limited studies have been done on allergenicity of broad beans. The aim of this study was to determine if broad bean proteins have the ability to elicit allergic responses due to the presence of clinically relevant allergenic proteins. METHODS: Simulated gastric fluid (SGF) assay and immunoglobulin E (IgE) immunoblotting were carried out to identify pepsin-resistant and IgE-binding proteins. The allergenicity of broad beans was assessed in allergic patients, BALB/c mice, splenocytes, and RBL-2H3 cells. RESULTS: Eight broad bean proteins of approximate molecular weight 70, 60, 48, 32, 23, 19, 15, and 10 kDa that remained undigested in SGF, showed IgE-binding capacity as well. Of 127 allergic patients studied, broad bean allergy was evident in 16 (12%). Mice sensitized with broad bean showed increased levels of histamine, total and specific IgE, and severe signs of systemic anaphylaxis compared with controls. Enhanced levels of histamine, prostaglandin D2, cysteinyl leukotriene, and ß-hexosaminidase release were observed in the primed RBL-2H3 cells following broad bean exposure. The levels of interleukin IL-4, IL-5, IL-13 and regulated on activation, normal T-cell expressed and secreted were found enhanced in broad bean-treated splenocytes culture supernatant compared with controls. CONCLUSION: This study inferred that broad bean proteins have the ability to elicit allergic responses due to the presence of clinically relevant allergenic proteins.

SNP discovery and high-density genetic mapping in faba bean (Vicia faba L.) permits identification of QTLs for ascochyta blight resistance.

Ascochyta blight, caused by the fungus Ascochyta fabae Speg., is a common and destructive disease of faba bean (Vicia faba L.) on a global basis. Yield losses vary from typical values of 35-40% to 90% under specific environmental conditions. Several sources of resistance have been identified and used in breeding programs. However, introgression of the resistance gene determinants into commercial cultivars as a gene pyramiding approach is reliant on selection of closely linked genetic markers. A total of 14,552 base variants were identified from a faba bean expressed sequence tag (EST) database, and were further quality assessed to obtain a set of 822 high-quality single nucleotide polymorphisms (SNPs). Sub-sets of 336 EST-derived simple sequence repeats (SSRs) and 768 SNPs were further used for high-density genetic mapping of a biparental faba bean mapping population (Icarus×Ascot) that segregates for resistance to ascochyta blight. The linkage map spanned a total length of 1216.8 cM with 12 linkage groups (LGs) and an average marker interval distance of 2.3 cM. Comparison of map structure to the genomes of closely related legume species revealed a high degree of conserved macrosynteny, as well as some rearrangements. Based on glasshouse evaluation of ascochyta blight resistance performed over two years, four genomic regions controlling resistance were identified on Chr-II, Chr-VI and two regions on Chr-I.A. Of these, one (QTL-3) may be identical with quantitative trait loci (QTLs) identified in prior studies, while the others (QTL-1, QTL-2 and QTL-4) may be novel. Markers in close linkage to ascochyta blight resistance genes identified in this study can be further validated and effectively implemented in faba bean breeding programs.

Modulation of IgE immunoreactivity to broad bean proteins after food processing in a Moroccan population.

BACKGROUND: The aim of this study was to assess the sensitivity profile of the population of Fez and Casablanca in Morocco to dry broad bean (Vicia faba), and to investigate the effect of food processing (heat and/or enzymatic hydrolysis by pepsin) on the human IgE binding capacity to broad bean proteins (BBP). METHODS: Sera samples from 146 patients with atopic hypersensitivity were recruited in order to evaluate specific IgE levels to native and processed broad bean proteins by ELISA. Under the same conditions, we assessed the immunoreactivity of rabbit IgG obtained by immunisation with native BBP. RESULTS: High IgE levels to BBP were found; in fact, 79.3% of children and 80.4% of adults had positive values. The heat treatment (70°C during 60 min) of dry beans proteins showed slight reduction in recognition of these antigens by rabbit IgG (22%) and by human IgE (12%). Pepsin hydrolysis decreased rabbit-IgG recognition by 55% in the first 30 min of treatment. In contrast, and under the same conditions, pepsin increased human-IgE recognition with an average of 143% for all patients. However, the combination of the two treatments (heating and pepsin digestion) showed a decrease of 16% in BBP recognition for all patients. CONCLUSIONS: This study demonstrates a high sensitivity of a Moroccan population to broad bean proteins which was resistant to heat and digestion by pepsin.

Characterization of non-host resistance in broad bean to the wheat stripe rust pathogen.

BACKGROUND: Non-host resistance (NHR) confers plant species immunity against the majority of microbial pathogens and represents the most robust and durable form of plant resistance in nature. As one of the main genera of rust fungi with economic and biological importance, Puccinia infects almost all cereals but is unable to cause diseases on legumes. Little is known about the mechanism of this kind of effective defense in legumes to these non-host pathogens. RESULTS: In this study, the basis of NHR in broad bean (Vicia faba L.) against the wheat stripe rust pathogen, Puccinia striiformis f. sp. tritici (Pst), was characterized. No visible symptoms were observed on broad bean leaves inoculated with Pst. Microscopic observations showed that successful location of stomata and haustoria formation were significantly reduced in Pst infection of broad bean. Attempted infection induced the formation of papillae, cell wall thickening, production of reactive oxygen species, callose deposition and accumulation of phenolic compounds in plant cell walls. The few Pst haustoria that did form in broad bean cells were encased in reactive oxygen and callose materials and those cells elicited cell death. Furthermore, a total of seven defense-related genes were identified and found to be up-regulated during the Pst infection. CONCLUSIONS: The results indicate that NHR in broad bean against Pst results from a continuum of layered defenses, including basic incompatibility, structural and chemical strengthening of cell wall, posthaustorial hypersensitive response and induction of several defense-related genes, demonstrating the multi-layered feature of NHR. This work also provides useful information for further determination of resistance mechanisms in broad bean to rust fungi, especially the adapted important broad bean rust pathogen, Uromyces viciae-fabae, because of strong similarity and association between NHR of plants to unadapted pathogens and basal resistance of plants to adapted pathogens.

Screening and analysis of genes expressed upon infection of broad bean with Clover yellow vein virus causing lethal necrosis.

Clover yellow vein virus (ClYVV) causes lethal systemic necrosis in legumes, including broad bean (Vicia faba) and pea (Pisum sativum). To identify host genes involved in necrotic symptom expression after ClYVV infection, we screened cDNA fragments in which expression was changed in advance of necrotic symptom expression in broad bean (V. faba cv. Wase) using the differential display technique and secondarily with Northern blot analysis. Expression changes were confirmed in 20 genes, and the six that exhibited the most change were analyzed further. These six genes included a gene that encodes a putative nitrate-induced NOI protein (VfNOI), and another was homologous to an Arabidopsis gene that encodes a glycine- and proline-rich protein GPRP (VfGPRP). We recently reported that necrotic symptom development in ClYVV-infected pea is associated with expression of salicylic acid (SA)-dependent pathogenesis-related (PR) proteins and requires SA-dependent host responses. Interestingly, VfNOI and VfGPRP expression was correlated with that of the putative SA-dependent PR proteins in ClYVV-infected broad bean. However, broad bean infected with a recombinant ClYVV expressing the VfGPRP protein showed weaker symptoms and less viral multiplication than that infected with ClYVV expressing the GFP protein. These results imply that VfGPRP plays a role in defense against ClYVV rather than in necrotic symptom expression.

Expression of human cytomegalovirus pp150 gene in transgenic Vicia faba L. and immunogenicity of pp150 protein in mice.

The pp150 gene of human cytomegalovirus (HCMV) was transferred into Vicia faba plants by Agrobacterium tumefaciens-mediated transformation. Three of five hygromycin resistant V. faba plants were identified as positive by PCR and dot-blot hybridization. The ELISA results indicated that pp150 protein from three plants of transformed V. faba leaves and seeds made up 0.005-0.015% of the total soluble protein. The results of detection by immunoblot and inhibition of immunofluorescent assay (IFA) showed that pp150 soluble protein had immunity activity. HCMV pp150-specific antibody (IgG, IgA) and IFN-gamma producing T cells were detected in 100% of the mice immunized with pp150 transgenic V. faba seeds by ELISA and intracellular staining and flow cytometry analysis, respectively. The transgenic V. faba plants will provide new material for the development of edible vaccination against HCMV infection.

Validation of QTLs for Orobanche crenata resistance in faba bean (Vicia faba L.) across environments and generations.

Broomrape (Orobanche crenata Forsk.) is a major root-parasite of faba bean (Vicia faba L.), that seriously limits crop cultivation in the whole Mediterranean area. This parasitic weed is difficult to control, difficult to evaluate and the resistance identified so far is of polygenic nature. This study was conducted to identify genetic regions associated with broomrape resistance in recombinant inbred lines (RILs) and to validate their previous location in the original F(2) population derived from the cross between lines Vf6 and Vf136. A progeny consisting of 165 F(6) RILs was evaluated in three environments across two locations in 2003 and 2004. Two hundred seventy seven molecular markers were assigned to 21 linkage groups (9 of them assigned to specific chromosomes) that covered 2,856.7 cM of the V. faba genome. The composite interval mapping on the F(6) map detected more quantitative trait loci (QTL) than in the F(2) analysis. In this sense, four QTLs controlling O. crenata resistance (Oc2-Oc5) were identified in the RI segregant population in three different environments. Only Oc1, previously reported in the F(2) population, was not significant in the advanced lines. Oc2 and Oc3 were found to be associated with O. crenata resistance in at least two of the three environments, while the remaining two, Oc4 and Oc5, were only detected in Córdoba-04 and Mengíbar-04 and seemed to be environment dependent.