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Design of tomato fruits with reduced allergenicity by dsRNAi-mediated inhibition of ns-LTP (Lyc e 3) expression.
Plant Biotechnol J. 2006 Mar; 4(2):231-42.PB

Abstract

Plant genetic engineering has the potential to introduce new allergenic proteins into foods but, at the same time, it can be used to remove established allergens. Here, we report the molecular characterization of Lyc e 3, a new tomato (Lycopersicon esculentum) allergen, and the efficient down-regulation of its expression in transgenic tomato plants. Following the identification of an immunoglobulin E (IgE)-binding 9-kDa polypeptide in tomato peel, designated Lyc e 3, its partial amino acid sequence was determined by N-terminal protein sequencing. Sequence comparison revealed that Lyc e 3 encodes a nonspecific lipid transfer protein (ns-LTP). In plants, ns-LTPs are encoded by large gene families which differ in primary amino acid sequence, expression and proposed cellular function. To identify Lyc e 3 encoding complementary DNAs (cDNAs), public tomato expressed sequence tag (EST) databases were screened for ns-LTP sequences. Following this strategy, two cDNAs, LTPG1 and LTPG2, with high homology to the N-terminal sequence of Lyc e 3, were identified. Ectopic expression of LTPG1 and LTPG2 in Escherichia coli, followed by immunoblotting, verified their IgE reactivity. Subsequently, transgenic tomato plants constitutively expressing LTPG1- or LTPG2-specific double-stranded RNA interference (dsRNAi) constructs were created and tested for the suppression of Lyc e 3 accumulation. Efficient silencing of Lyc e 3 was documented by Northern and Western blotting. In both cases, Lyc e 3 accumulation was decreased to levels below the detection limit (less than 0.5% of the wild-type protein). The allergenic potential of Lyc e 3-deficient tomato fruits was tested by measuring histamine release from sensitized human basophils stimulated with transgenic and parental lines. These assays revealed a strong (10- to 100-fold) decrease in histamine release of human basophils challenged with transgenic fruit extracts when compared with control extracts. These results demonstrate the feasibility of creating low allergenic tomato fruits by means of dsRNAi inhibition.

Authors+Show Affiliations

Institut für Pflanzengenetik and Kulturpflanzenforschung Gatersleben, Corrensstrasse 3, 06466 Gatersleben, Germany.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

17177799

Citation

Le, Lien Quynh, et al. "Design of Tomato Fruits With Reduced Allergenicity By dsRNAi-mediated Inhibition of ns-LTP (Lyc E 3) Expression." Plant Biotechnology Journal, vol. 4, no. 2, 2006, pp. 231-42.
Le LQ, Lorenz Y, Scheurer S, et al. Design of tomato fruits with reduced allergenicity by dsRNAi-mediated inhibition of ns-LTP (Lyc e 3) expression. Plant Biotechnol J. 2006;4(2):231-42.
Le, L. Q., Lorenz, Y., Scheurer, S., Fötisch, K., Enrique, E., Bartra, J., Biemelt, S., Vieths, S., & Sonnewald, U. (2006). Design of tomato fruits with reduced allergenicity by dsRNAi-mediated inhibition of ns-LTP (Lyc e 3) expression. Plant Biotechnology Journal, 4(2), 231-42.
Le LQ, et al. Design of Tomato Fruits With Reduced Allergenicity By dsRNAi-mediated Inhibition of ns-LTP (Lyc E 3) Expression. Plant Biotechnol J. 2006;4(2):231-42. PubMed PMID: 17177799.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Design of tomato fruits with reduced allergenicity by dsRNAi-mediated inhibition of ns-LTP (Lyc e 3) expression. AU - Le,Lien Quynh, AU - Lorenz,Yvonne, AU - Scheurer,Stephan, AU - Fötisch,Kay, AU - Enrique,Ernesto, AU - Bartra,Joan, AU - Biemelt,Sophia, AU - Vieths,Stefan, AU - Sonnewald,Uwe, PY - 2006/12/21/pubmed PY - 2007/2/3/medline PY - 2006/12/21/entrez SP - 231 EP - 42 JF - Plant biotechnology journal JO - Plant Biotechnol J VL - 4 IS - 2 N2 - Plant genetic engineering has the potential to introduce new allergenic proteins into foods but, at the same time, it can be used to remove established allergens. Here, we report the molecular characterization of Lyc e 3, a new tomato (Lycopersicon esculentum) allergen, and the efficient down-regulation of its expression in transgenic tomato plants. Following the identification of an immunoglobulin E (IgE)-binding 9-kDa polypeptide in tomato peel, designated Lyc e 3, its partial amino acid sequence was determined by N-terminal protein sequencing. Sequence comparison revealed that Lyc e 3 encodes a nonspecific lipid transfer protein (ns-LTP). In plants, ns-LTPs are encoded by large gene families which differ in primary amino acid sequence, expression and proposed cellular function. To identify Lyc e 3 encoding complementary DNAs (cDNAs), public tomato expressed sequence tag (EST) databases were screened for ns-LTP sequences. Following this strategy, two cDNAs, LTPG1 and LTPG2, with high homology to the N-terminal sequence of Lyc e 3, were identified. Ectopic expression of LTPG1 and LTPG2 in Escherichia coli, followed by immunoblotting, verified their IgE reactivity. Subsequently, transgenic tomato plants constitutively expressing LTPG1- or LTPG2-specific double-stranded RNA interference (dsRNAi) constructs were created and tested for the suppression of Lyc e 3 accumulation. Efficient silencing of Lyc e 3 was documented by Northern and Western blotting. In both cases, Lyc e 3 accumulation was decreased to levels below the detection limit (less than 0.5% of the wild-type protein). The allergenic potential of Lyc e 3-deficient tomato fruits was tested by measuring histamine release from sensitized human basophils stimulated with transgenic and parental lines. These assays revealed a strong (10- to 100-fold) decrease in histamine release of human basophils challenged with transgenic fruit extracts when compared with control extracts. These results demonstrate the feasibility of creating low allergenic tomato fruits by means of dsRNAi inhibition. SN - 1467-7652 UR - https://www.unboundmedicine.com/medline/citation/17177799/Design_of_tomato_fruits_with_reduced_allergenicity_by_dsRNAi_mediated_inhibition_of_ns_LTP__Lyc_e_3__expression_ L2 - https://doi.org/10.1111/j.1467-7652.2005.00175.x DB - PRIME DP - Unbound Medicine ER -