Grapevine-mould Plasmopara viticola infection (downy mildew) elicits the expression of allergenic PR-proteins

Background: grapevine-related occupational respiratory symptoms can be induced by either pollens (in spring time) or exogenous factors like infecting moulds, i.e. Plasmopara viticola (PV). In both cases no allergens have been identified so far. Although PV IgE-binding proteins have been previously detected we cannot exclude that the mould might also induce the expression of vine allergenic proteins as a response to infection. Indeed, pathogenesis-related proteins (PRs) represent a group of stress-induced proteins frequently described as allergens in many plants. The object of the present work is to verify by a proteomic approach whether the allergenic reactions experienced by a farmer working in a vineyard infected by PV are elicited by PR-proteins expressed upon infection or by allergens present in PV. Method: A 54 year old male suffering of asthma in august working in a vineyard was evaluated by SPT with a panel of commercial allergens (birch positive) and by Prick-to-Prick using PV-infected (positive) and non-infected vine leaves (negative). Proteins were extracted from the leaves with a cocktail of 7 M urea, 2 M thiourea, 80 mM citric acid, 3% CHAPS, 5% PVPP, precipitated with cold acetone and finally solubilized in 7 M urea, 2 M thiourea, 3% CHAPS, 40mM Tris, 1% pH 3-10 ampholyte, 1% DTT. A sample of PV was isolated after several artificial infection cycles on surface-sterilized leaves and extracted as described above. Proteins were separated by 1 (1-D) and 2-dimensional (2-D) electrophoreses. Gels were either stained with Coomassie dye or subjected to immunoblotting with the patient serum (1:10 dilution). Spots giving positive signal by immunoblotting were cut out from 2-D Coomassie-stained gels and subjected to trypsin digestion. Peptides were analyzed by nanoHPLC Chip MS/MS mass spectrometry. Results: 1-D IgE-immunoblotting indicates that the allergens are intensely expressed by the plant upon infection since no signals were detected in PV extract and only traces were found in non-infected leaves. Two IgE-binding bands were detected at 42 and 35 kDa. The 2-D analysis allowed to resolve the 35 kDa band into two spot trains at 35.6 and 34 kDa identified by MS as β-glucanases (PR-2) and Harpin Binding Proteins (HBP1). Conclusion: We could confirm that PV induces the expression of IgE-binding PR-2 and HBP1, the latter never described before as an allergen, strongly suggesting that these molecules contribute to eliciting the symptoms in the patient.