Induced resistance in nectarine fruit by Bacillus licheniformis W10 for the control of brown rot caused by Monilinia fructicola.

Brown rot caused by Monilinia fructicola has led to considerable preharvest and postharvest losses in all major nectarine fruit-growing areas. In our previous study, we successfully identified a biocontrol strain of bacteria, Bacillus licheniformis W10, that can be used to control brown rot. However, the possible mechanism of the control of brown rot by B. licheniformis W10 is still unclear. Therefore, the objectives of this study were to determine whether B. licheniformis W10 induces resistance by activating defense-related enzymes including antioxidant enzymes in nectarine. Treatment of nectarine fruit with B. licheniformis W10 reduced both M. fructicola-induced oxidative damage and reactive oxygen species (ROS) production. Furthermore, application of B. licheniformis to nectarine fruit resulted in a significant increase in the activity of antioxidant and defense-related enzymes and increase in the expression of the corresponding genes. Overall, our results verified the proposed mechanism of B. licheniformis W10 in controlling M. fructicola via regulation of ROS levels and activation of antioxidant and defense-related enzymes.

Silencing of one copy of the translation initiation factor eIFiso4G in Japanese plum (Prunus salicina) impacts susceptibility to Plum pox virus (PPV) and small RNA production.

BACKGROUND: In plants, host factors encoded by susceptibility (S) genes are indispensable for viral infection. Resistance is achieved through the impairment or the absence of those susceptibility factors. Many S genes have been cloned from model and crop species and a majority of them are coding for members of the eukaryotic translation initiation complex, mainly eIF4E, eIF4G and their isoforms. The aim of this study was to investigate the role of those translation initiation factors in susceptibility of stone fruit species to sharka, a viral disease due to Plum pox virus (PPV). RESULTS: For this purpose, hairpin-inducing silencing constructs based on Prunus persica orthologs were used to generate Prunus salicina (Japanese plum) 4E and 4G silenced plants by Agrobacterium tumefaciens-mediated transformation and challenged with PPV. While down-regulated eIFiso4E transgenic Japanese plums were not regenerated in our conditions, eIFiso4G11-, but not the eIFiso4G10-, silenced plants displayed durable and stable resistance to PPV. We also investigated the alteration of the si- and mi-RNA profiles in transgenic and wild-type Japanese plums upon PPV infection and confirmed that the newly generated small interfering (si) RNAs, which are derived from the engineered inverted repeat construct, are the major contributor of resistance to sharka. CONCLUSIONS: Our results indicate that S gene function of the translation initiation complex isoform is conserved in Prunus species. We discuss the possibilities of using RNAi silencing or loss-of-function mutations of the different isoforms of proteins involved in this complex to breed for resistance to sharka in fruit trees.

ENEA, a peach and apricot IgE-binding protein cross-reacting with the latex major allergen Hev b 5.

Peach and apricot can cause allergic reactions with symptoms ranging from mild to very severe, including anaphylaxis. Sometimes subjects allergic to fruits of the Prunus genus have been reported to be also allergic to rubber latex products. The objective of this study is the characterization of a newly identified peach and apricot protein showing similarities with the allergens Hev b 5 from rubber latex and Man e 5 from manioc. This protein has been named ENEA on the basis of the single letter amino acid code of the first four N-terminal residues of the isolated molecule. It has been found in very variable amounts in different peach cultivars and batches. ENEA was isolated from peach pulp extracts by chromatographic separations and identified by direct protein sequencing. At that time, the full length sequence was available only for the homologous protein of the taxonomically closely related apricot, which was produced as a recombinant molecule in Escherichia coli. The following availability of the full length sequence of peach ENEA revealed a very high identity (97%) with the apricot homolog. Similarly to Hev b 5 and to Man e 5, the structural characterization indicated that ENEA is an intrinsically disordered protein. The immunological properties, investigated by dot blotting, the ABA system and the FABER test, showed that ENEA is recognized by specific IgE of allergic patients. In a selected population of 31 patients reporting allergic reactions to peach fruit and/or IgE positive to Hev b 5, 28 and 27 subjects resulted co-sensitized to rENEA and Hev b 5 in the ABA and ISAC test, respectively. In a random population of 3305 suspected allergic patients, analyzed with the FABER test, 17 of them were sensitized to rENEA and 10 of them were also positive to Hev b 5. In addition, both the natural molecule from peach and the recombinant protein of apricot partially inhibited the IgE binding to Hev b 5. In conclusion, a new peach and apricot IgE-binding protein, cross-reacting with the major latex allergen Hev b 5, has been identified. Its variable concentration in the fruit might explain some occasionally occurring allergic reactions. The apricot molecule has recently been registered by the WHO/IUIS Allergen Nomenclature Sub-Committee with the allergen name Pru ar 5. The recombinant form of apricot ENEA, now available, will contribute to allergy diagnosis.

New Data Completing the Spectrum of the Ma, RMia, and RMja Genes for Resistance to Root-Knot Nematodes (Meloidogyne spp.) in Prunus.

Root-knot nematodes (RKN) (Meloidogyne spp.) are worldwide pests that affect a considerable number of plants, among which stone fruit (Prunus spp.) are severely attacked. Prevalent RKN species are Meloidogyne arenaria, M. incognita, and M. javanica in stone fruit but the emergent M. ethiopica and M. enterolobii are also reported to challenge perennial crops. In Prunus spp., the complete-spectrum resistance (R) gene Ma from plum and the more restricted-spectrum R genes RMia from peach and RMja from almond completely inhibit nematode multiplication and gall formation of the RKN species that they control. This study aimed to update the resistance spectra of these three major genes by evaluating their activity toward one isolate of the yet-untested RKN species mentioned above. To state whether a given gene controls a particular species, the principle of our experiment was to genotype with appropriate markers a number of individuals segregating for this gene and then to phenotype these individuals. A perfect matching of the genotype and the phenotype of individuals indicates that the gene of interest is active against and, thus, controls the corresponding isolate of this RKN species. Segregating materials used were an Ma F1 plum progeny, an RMia F2 peach progeny, and an RMja F2 almond progeny. In addition to previous data, our results establish a clear spectrum for each of the three genes toward isolates from both the three prevalent species and the two emerging species. Ultimately, our results reveal that (i) Ma controls all of them, (ii) RMja controls all species except M. incognita and M. floridensis, and (iii) RMia controls M. arenaria, M. incognita, and M. ethiopica but not M. javanica or M. enterolobii. Our data should have wide implications for RKN resistance management and breeding and for deciphering the molecular mechanisms of the spectrum of RKN R genes.

Development of a LC-MS method for the discrimination between trace level Prunus contaminants of spices.

The need for an analytical procedure for the identification of allergens present at trace levels in foods was highlighted by conflicting results in a case of contamination of the spice cumin. The application of a bottom-up proteomics experiment was investigated to identify marker peptides for potential contaminant nuts which could then be monitored with high specificity and sensitivity by selective reaction monitoring experiments. The method developed allowed for the distinction between two closely related Prunus species, almond and mahaleb, in two different spices, cumin and paprika. The paprika sample was found to be contaminated with almond and the cumin sample, contaminated at a much lower level, was found to be contaminated with mahaleb. The method could be applied to any protein-dense food matrix allergen so long as suitable control and reference samples can be acquired.

PPV susceptibility of commonly used peach rootstock-scion combinations.

Sharka disease is one of the most devastating plant epidemics of Prunus species, caused by plum pox virus (PPV). The viral infection affects the fruits by weight-loss and degradation of quality properties. Breeding of resistant rootstocks and cultivars is one of the most effective disease control methods. PPV determines the peach production all over the world. On the world's fruit production list peach is in the sixth, in the Mediterranean region in the fourth place. In this study new data were shown about PPV susceptibility of commonly used rootstock-scion combinations from Hungary. Reverse transcription PCR (RT-PCR) analysis was conducted on the samples from a commercial orchard; the results were evaluated by chi-square test and binary logistic regression. Four rootstock ('GF677', 'PeMa', 'Cadaman' and almond seedlings) and three scion cultivars (Prunus persicae 'Michelini', 'Babygold 6' and 'Cresthaven') were included in this experiment. The rootstocks did not show any significant differences in regard to the resistance of the virus infection (40-50%), but in case of scions, strong significant relations were observed. In case of the combinations there were results in both directions; tolerant and susceptible combinations were observed as well.

Do Skin Prick Test and In Vitro Techniques Diagnose Sensitization to Peach Lipid Transfer Protein and Profilin Equally Well in Allergy to Plant Food and Pollen?

OBJECTIVE: To compare the skin prick test (SPT) with in vitro techniques (single and multiplex fluorescence enzyme-immunoassay [FEIA]) for detecting sensitization to profilin and lipid transfer protein (LTP). METHODS: We retrospectively studied 181 patients with pollen and/or plant food allergy and 61 controls. SPT was performed with date palm profilin (Pho d 2) and peach LTP (Pru p 3), and specific IgE (sIgE) to Phl p 12 and Pru p 3 was analyzed using single FEIA and microarray. RESULTS: Fifteen of 201 patients with negative results for LTP in the SPT were sensitized to this allergen in the in vitro tests, and 18 of 41 patients with positive results for LTP in the SPT were not sensitized according to the in vitro tests. Seventeen of 186 patients with negative results for profilin in the SPT were sensitized to Phl p 12 by serum sIgE, and 30 out of 56 patients with positive results for profilin in SPT were not sensitized to Phl p 12 according to the other tests. Moderate agreement was observed between the 3 techniques studied. CONCLUSIONS: SPT is a sensitive technique for detecting sensitization to LTP and profilin. Its results are similar to those of in vitro techniques, especially in patients with negative SPT results for peach LTP and palm tree profilin.

Hypersensitivity to Tomato (Lycopersicon esculentum) in Peach-Allergic Patients: rPrup 3 and rPrup 1 Are Predictive of Symptom Severity.

UNLABELLED: Background: The role of allergens in the severity of tomato allergy symptoms has not yet been studied. OBJECTIVES: To evaluate the relationship between severe allergic reactions to peach and tomato and between tomato allergy symptoms and the pattern of IgE positivity for rPru p 1, rPru p 3, rPru p 4, rBetv 1, rBetv 2, rBetv4, rPhl p 1, and rPhl p 12 in order to identify the role of recombinant allergens in the severity of reactions to tomato. METHODS: We studied peach-allergic patients with clinical reactions to tomato by performing an open food challenge, skin prick test, and determination of serum specific IgE to tomato and to recombinant peach, birch, and grass allergens. Statistical analysis was carried out to evaluate the relationship between the severity of tomato symptoms and IgE positivity to the different allergens and to peach-induced symptoms. RESULTS: We found a significant association between severe reactions to tomato and severe reactions to peach (P = .01 7) and levels of IgE to rPru p3 (P = .029) and between mild tomato allergy symptoms and levels of IgE to rPru p1 (P = .047), anti-rBetv 1 (P = .0414), anti-rBetv 2 (P = .0457), and Phleum pratense (P = .0022). CONCLUSION: We observed a significant relationship between peach and symptoms of tomato allergy. IgE positivity for rPru p3 seems to be a surrogate biochemical marker for severe tomato allergy, whereas the presence of anti-rPru p 1 IgE may be an indicator of mild tomato allergy.

Tomato nsLTP as an "In Vivo" Diagnostic Tool: Sensitization in a Mediterranean Population.

BACKGROUND: Tomato allergies have been extensively studied but component-resolved in vivo diagnosis with purified allergens has yet to be performed. OBJECTIVES: To evaluate the prevalence of sensitization to Sola l 3 in a Mediterranean population, and to compare the resulting sensitization profile with that of individuals sensitized to tomato, peach, and/or purified lipid transfer protein (LTP). METHODS: Sola l 3 was purified, characterized, and used to prepare skin prick tests (SPTs). Two groups of patients were selected. Group 1 consisted of patients with at least 1 positive SPT to tomato, peach, or LTP mixture (marker extracts) who were subsequently tested with Sola l 3 (n = 280). Group 2 (prevalence study) consisted of patients who underwent simultaneous SPT with the 3 marker extracts and Sola l 3 (n = 658). Patients from either group who were positive to any of the 4 extracts were studied in detail (study group, n = 1 23). ELISA and immunoblot assays were performed in individuals with a positive SPT to Sola l 3 to detect the presence of specific IgE antibodies to this allergen. RESULTS: Prevalence of sensitization to Sola l 3 was 3.2% overall and 54.7% in tomato-positive patients. Most tomato-sensitized patients were asymptomatic. Symptoms were more common in Sola l 3-positive individuals. Sensitization to peach and the LTP mixture did not discriminate between Sola l 3-positive and Sola l 3-negative patients. CONCLUSIONS: This study confirms that LTP, not only from peach but also from other fruit and vegetables, including tomato, is an important allergen in the Mediterranean area. Sensitization to Sola l 3 is associated with more symptoms in tomato-sensitized patients.

Impact of glutathione on the allergenicity of the peach lipid transfer protein Pru p 3.

BACKGROUND AND OBJECTIVE: The allergenic potential of proteins can be altered under various physicochemical conditions. Glutathione (GSH) is a reducing agent that is used as an antioxidant in food products. We aimed to characterize the natural folding of peach proteins and test the allergenicity of reduced and natural Pru p 3, the major peach allergen. METHODS: Pru p 3 was purified from peach, and its conformation was analyzed by means of circular dichroism. Using a thiol fluorescent probe, reduced proteins were detected in fresh peach. GSH-reduced Pru p 3 was tested in vitro for T-cell proliferation and in vivo using skin prick testing. RESULTS: GSH-reduced Pru p 3 produced variable skin prick reactions in peach-allergic patients. The proliferative response of peripheral blood mononuclear cells from allergic patients to reduced Pru p 3 tended to be less intense, whereas secretion of the cytokines IFN-γ, IL-5, and IL-10 was comparable. In a pool of sera from peach-allergic patients, reduction hardly impaired IgE-binding. Moreover, the stability of reduced Pru p 3 to gastrointestinal digestion was similar to that of the natural form. CONCLUSIONS: GSH can at least transiently reduce Pru p 3. We found that the effect of reduction on the allergenicity of Pru p 3 varied. Therefore, as an additive, GSH does not seem to eliminate the risk of reactions for peach-allergic patients.

Structural features, IgE binding and preliminary clinical findings of the 7kDa Lipid Transfer Protein from tomato seeds.

Allergic reactions caused by 9kDa Lipid Transfer Proteins (9k-LTP), such as Pru p 3, have been widely investigated, whereas a possible contribution of components of 7kDa LTP (7k-LTP) sub-family in triggering allergic symptoms has been overlooked so far. With the aim to investigate the contribution of 7k-LTPs to the food allergies, we have identified, isolated and characterised a tomato seed 7k-LTP (Sola l 7k-LTP). The protein was purified by chromatographic separations, identified by direct protein sequencing and mass spectrometry and a molecular model was built. Functional evaluation of the allergen has been performed by skin testing. Sola l 7k-LTP consists of 68 amino acids producing a molecular mass of 7045Da and displays 41% sequence identity with Pru p 3, the allergenic 9k-LTP from peach. IgE antibodies specifically recognising Sola l 7k-LTP were found within the population claiming tomato ingestion-related symptoms, but also in subjects tolerant on tomato exposure. A few subjects were mono-sensitised to Sola l 7k-LTP, which is biologically active as shown by the positive skin test. In line with the immunological results, the molecular model shows structural similarities between the IgE binding regions of the two sub-families. Therefore, Sola l 7k-LTP shares some structural and immunological features with Pru p 3, but it also displays individual features that could be responsible for mono-specific IgE binding. In conclusion, Sola l 7k-LTP is a new identified allergenic LTP, the description of which may contribute to the improvement of allergy diagnosis and to the formulation of a safe and personalised diet. In addition, to avoid current confusing classifications, a new nomenclature policy for LTP sub-families is proposed in this paper. We now suggest that 7-kDa LTP (so far named LTP2) be renamed 7k-LTP and 9-kDa LTP (so far named LTP1) be renamed 9k-LTP.

Occupational Allergy to Peach (Prunus persica) Tree Pollen and Potential Cross-Reactivity between Rosaceae Family Pollens.

Orchard workers in north China are highly exposed to orchard pollens, especially peach and other Rosaceae family pollens during pollination season. The aim of this study was to investigate whether occupational allergy to peach tree pollen as a member of Rosaceae family is IgE-mediated and to evaluate the cross-reactivity among Rosaceae family pollens. Allergen skin test and conjunctival challenge test were performed; enzyme linked immune-sorbent assay (ELISA), inhibiting ELISA, western immunoblotting and inhibiting western immunoblotting were done with Rosaceae family orchard pollens, including peach, apricot, cherry, apple and pear tree pollens. Mass spectrometry was also performed to probe the main allergen component and cross-reactive protein. Sensitizations to peach pollen were found in both skin test and conjunctival challenge in the patients. Serum specific IgE to three pollens (peach, apricot and cherry) were detected through ELISA. When peach pollen used as solid phase, ELISA inhibition revealed other four kinds of pollens capable of inducing partial to strong inhibitions (45% to 87%), with the strongest inhibition belonging to apricot pollen (87%). Western blotting showed predominant IgE binding to a 20 KD protein among these pollens, which appeared to be a cross-reactive allergen component through western blotting inhibition. It was recognized as a protein homologous to glutathione s-transferase 16 from Arabidopsis thaliana. Peach and other Rosaceae family tree pollen may serve as a potential cause of IgE mediated occupational respiratory disease in orchard workers in north China.

Allergen component specific ige measurement with the Immulite™ 2000 system: diagnostic accuracy and intermethod comparison.

BACKGROUND: The identification of the allergenic molecules, associated to the advances in the field of recombinant allergens, led to the development of a new concept in allergy diagnosis called component-resolved diagnosis. The aim of our study was to evaluate the diagnostic accuracy of different allergen components using the full automatic singleplex quantitative platform Immulite™ 2000. METHODS: One hundred ninety-five allergic outpatients (35 to olive pollen, 35 to birch pollen, 35 to profilin, 35 to house dust mites, 35 to peach, and 20 to shrimp) and 20 negative controls were enrolled for the study. Bet v 1, Bet v 2, Ole e 1, Der p 1, Der p 2, Der f 1, Der f 2, Pru p 3, tropomyosin were tested both with Immulite™ 2000 and ImmunoCAP™ (Thermo Fisher Scientific, Uppsala, Sweden). RESULTS: Sensitivity of allergen-specific Immunoglobulin E (sIgE) to Ole e 1, Bet v 1, Der p 1, Der p 2, Der f 1, Der f 2, Pen m 1, and Pru p 3 with Immulite™ 2000 was 100%, 100%, 77.1%, 94.3%, 71.4%, 94%, 75%, and 97.1%, respectively, and the specificity was 100% for all the allergens. The overall agreement between Immulite™ 2000 and ImmunoCAP™ (Thermo Fisher Scientific) platforms was 98.6% (Cohen's kappa = 0.979; confidence interval [CI] 95%: 0.960-0.997). From moderate to strong, positive linear correlations between the assays (r(2) from 0.322 to 0.860, and Spearman's rho from 0.824 to 0.971) were showed. CONCLUSIONS: A high diagnostic accuracy of the sIgE to allergen components measurement with Immulite™ 2000 and a high agreement with ImmunoCAP™ platforms were shown in this study.

Value of microarray allergen assay in the management of eosinophilic oesophagitis.

BACKGROUND: Eosinophilic oesophagitis (EoE) is a disorder characterised by oesophageal dysfunction and, histologically, by eosinophilic inflammation. Although treatment, which includes dilatations, oral corticosteroids and restrictive diets, is often effective, choosing the foods to be eliminated from the diet is difficult. OBJECTIVE: Component resolved diagnostic by microarray allergen assay may be useful in detecting allergens that might be involved in the inflammatory process. METHODS: We studied 67 patients with EoE, diagnosed clinically and histologically by endoscopic biopsy. CRD analysis with microarray technology was carried out in the 67 EoE patients, 50 patients with pollen allergy without digestive symptoms, and 50 healthy controls. RESULTS: Allergies were not detected by microarray in only seven of the 67 patients with EoE. Controls with pollen allergy showed sensitisation to different groups of pollen proteins without significant differences. In EoE patients with response to some allergens, the predominant allergens were grasses group 1 and, in particular, nCyn d 1 (Cynodon dactylon) or Bermuda grass pollen in 59.5%, followed by lipid transfer proteins (LTP) of peach (19.40%), hazelnut (17.91%) and Artemisia (19.40%). CONCLUSIONS: In patients with EoE, sensitisation to plant foods and pollen is important. The proteins most frequently involved are nCyn d 1 and lipid transfer proteins, hazelnuts and walnuts. After one year of an array-guided exclusion diet and pollen-specific immunotherapy in the case of high levels of response, patients with EoE showed preliminary significant improvements.

Expression, purification, and characterization of almond (Prunus dulcis) allergen Pru du 4.

Biochemical characterizations of food allergens are required for understanding the allergenicity of food allergens. Such studies require a relatively large amount of highly purified allergens. The level of Pru du 4 in almond is low, and its expression in a soluble form in Escherichia coli required an expression tag. An MBP tag was used to enhance its expression and solubility. Sumo was used for the first time as a peptidase recognition site. The expression tag was removed with a sumo protease, and the resulting wild-type Pru du 4 was purified chromatographically. The stability of the allergen was investigated with chemical denaturation. The Gibbs free energy of Pru du 4 folding-unfolding transition was determined to be 5.4 ± 0.7 kcal/mol.

Rootstock-to-scion transfer of transgene-derived small interfering RNAs and their effect on virus resistance in nontransgenic sweet cherry.

Small interfering RNAs (siRNAs) are silencing signals in plants. Virus-resistant transgenic rootstocks developed through siRNA-mediated gene silencing may enhance virus resistance of nontransgenic scions via siRNAs transported from the transgenic rootstocks. However, convincing evidence of rootstock-to-scion movement of siRNAs of exogenous genes in woody plants is still lacking. To determine whether exogenous siRNAs can be transferred, nontransgenic sweet cherry (scions) was grafted on transgenic cherry rootstocks (TRs), which was transformed with an RNA interference (RNAi) vector expressing short hairpin RNAs of the genomic RNA3 of Prunus necrotic ringspot virus (PNRSV-hpRNA). Small RNA sequencing was conducted using bud tissues of TRs and those of grafted (rootstock/scion) trees, locating at about 1.2 m above the graft unions. Comparison of the siRNA profiles revealed that the PNRSV-hpRNA was efficient in producing siRNAs and eliminating PNRSV in the TRs. Furthermore, our study confirmed, for the first time, the long-distance (1.2 m) transfer of PNRSV-hpRNA-derived siRNAs from the transgenic rootstock to the nontransgenic scion in woody plants. Inoculation of nontransgenic scions with PNRSV revealed that the transferred siRNAs enhanced PNRSV resistance of the scions grafted on the TRs. Collectively, these findings provide the foundation for 'using transgenic rootstocks to produce products of nontransgenic scions in fruit trees'.

In patients with LTP syndrome food-specific IgE show a predictable hierarchical order.

BACKGROUND: Lipid transfer protein (LTP) is a widely cross-reacting allergen in plant foods. OBJECTIVE: To assess whether IgE to vegetable foods show predictable trends in LTP allergic patients. METHODS: Clinical allergy to plant foods other than peach was sought in 15 consecutive peach-allergic adults monosensitized to LTP. IgE specific for peach, apple, hazelnut, walnut, peanut, lentil, maize, soybean, tomato, sesame, mustard melon, kiwi, and celery as well as to mugwort pollen was measured. RESULTS: Peach-specific IgE levels exceeded IgE to all other study foods. The higher were peach-specific IgE levels, the higher was the probability that other plant-derived foods scored positive. Mean IgE levels specific for all study foods were strongly correlated to peach specific IgE. Food-specific IgE followed a rather precise hierarchy, both in terms of number of positive in-vitro tests and of IgE levels, with apple at the second place after peach, followed by walnut, hazelnut, peanut, lentil, maize, soybean, tomato, kiwi, sesame, mustard, melon, and celery. Such hierarchy was not necessarily paralleled by clinical allergy as lentil, maize, and soybean scored positive in the majority of patients, but induced allergy in 0, 1, and 0 patients, respectively. IgE levels were not necessarily correlated with the severity of clinical allergy. Little or no IgE reactivity to mugwort pollen was found. CONCLUSIONS: In LTP syndrome, IgE reactivity to foods other than peach is in most cases predictable and follows a regular sequence that probably depends on the degree of homology with Pru p3. The reasons why some foods are tolerated by most patients despite elevated IgE reactivity remains to be elucidated.

Pomegranate ( Punica granatum L.) expresses several nsLTP isoforms characterized by different immunoglobulin E-binding properties.

BACKGROUND: Pomegranate allergy is associated with sensitization to non-specific lipid transfer proteins (nsLTPs). Our aim was to identify and characterize the non-specific nsLTPs expressed in pomegranate at the molecular level and to study their allergenic properties in terms of immunoglobulin E (IgE)-binding and cross-reactivity with peach nsLTP (Pru p 3). METHODS: A non-equilibrium two-dimensional (2-D) electrophoretic approach based on acid-urea PAGE and sodium dodecyl sulfate PAGE was set up to separate pomegranate nsLTPs. Their immunoreactivity was tested by immunoblotting carried out with anti-Pru p 3 polyclonal antibodies and sera from pomegranate-allergic patients. For final identification, pomegranate nsLTPs were purified by chromatography and subjected to trypsin digestion and mass spectrometry (MS) analysis. For this purpose, the sequences obtained by cDNA cloning of three pomegranate nsLTPs were integrated in the database that was subsequently searched for MS data interpretation. RESULTS: Four nsLTPs were identified by 2-D immunoblotting. The detected proteins showed different IgE-binding capacity and partial cross-reactivity with Pru p 3. cDNA cloning and MS analyses led to the identification of three nsLTP isoforms with 66-68% amino acid sequence identity named Pun g 1.0101, Pun g 1.0201 and Pun g 1.0301. CONCLUSIONS: By 2-D electrophoresis, we could separate different nsLTP isoforms possessing different IgE-binding properties, which might reflect peculiar allergenic potencies. The contribution of Pru p 3 to prime sensitization is not central as in other plant nsLTPs.