Design of tomato fruits with reduced allergenicity by dsRNAi‐mediated inhibition of ns‐LTP (Lyc e 3) expression

Abstract

SummaryPlant 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. 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.

RNA Interference-Mediated Repression of Cell Wall Invertase Impairs Defense in Source Leaves of Tobacco

“…AB055500]). RNAi is homology dependent, and earlier studies revealed that 90% sequence identity between two nucleotide sequences is sufficient to trigger effective RNA degradation (Schweizer et al, 2000;Le et al, 2006;Chen et al, 2008).…”

“…AF506005), primers were designed from the highly homologous sequence region of both sequences (bp 755-1,162 according to LIN8 sequence). In this region, both sequences share 81.9% sequence identity, which is thought to be sufficient to silence both isoforms by a single construct (Le et al, 2006;Chen et al, 2008). A 400-bp cDNA fragment of LIN8 was amplified by PCR using the primers LIN8-5# 5#-caccggtactggaaattggg-3# and LIN8-3# 5#-acaggccattgaaccaattg-3# (nt 755-1,162 of LIN8), and tomato (Solanum lycopersicum) leaf cDNA as a template.…”

RNA interference: concept to reality in crop improvement

“…In peanut, the Ara h 2 is one of the most allergic protein present out of 7 allergenic proteins. To remove Lyc e3 from tomato, Le et al (2006) have demonstrated an efficient silencing of Lyc e3, a tomato allergen that encodes a nonspecific lipid transfer protein in the RNAi-silenced tomato plants. Siritungam and Sayre (2003) have reduced the linamarin content, a cyanogenic substance in cassava plants.…”

Plant lipid transfer proteins: Evolution, expression and function

“…Later nsLTPs have also been identified as allergens in e.g. oranges, tomato, hazelnut, olive tree pollen and cabbage (Tejera et al, 1999;Pastorello et al, 2002;Ahrazem et al, 2005;Le et al, 2006;Palacin et al, 2006). Due to the similarity in structure between nsLTPs from different plants, there is a high rate of cross-reactivity (Egger et al, 2010).…”