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Hevea genetic transformation using a single-chain variable fragment (scFv) specific to cassiicolin, the causal agent of Corynespora leaf disease

Sunderasan E., Kadir R.A., Pujade-Renaud V., Malik A., Ezam Badarudin B., Yeang H.Y., Nathan S.. 2011. In : Sunderasan E. (ed.), Arija Mad Arif Siti (ed.). Report on the International Rubber Research and Development Board Biotechnology Workshop : Malaysian Rubber Board Headquarters, Kuala Lumpur, Malaysia, 30 May - 1 June 2011. s.l. : s.n., p. 28-28. IRRBD Biotechnology Workshop, 2011-05-30/2011-06-01, Kuala Lumpur (Malaisie).

The pathogenic fungus Corynespora cassiicola a serious threat to rubber trees (Hevea brasiliensis); the infected leaves develop necrotic lesions and abscise, leaving the tree unproductive. The destructiveness of C. cassiicola has been largely attributed to cassiicolin, a secreted protein toxin of the fungus. Recombinant antibody technology coupled to genetic transformation offer hope to curtail the disease in Hevea. Single chain variable fragment (scFv) specific to cassiicolin could bind and deactivate the toxin in genetically modified rubber trees that harbour the functional antibody gene. A scFv phage library was constructed from cassiicolin immunized Balb/C mice spleen IgG heavy and light variable chains. Biopanning of the phage library yielded a scFv clone with high specificity to cassiicolin. Nucleotide sequence and deduced amino acid sequence information of the scFv were obtained. The hemagglutinin (HA) tagged scFv expressed in Escherichia coli is discerned as a band at circa 30 kDa on SDS-PAGE. The corresponding band was detected by anti-HA IgG on a Western immunoblot of the gel. Homology-based modelling was employed to create three-dimensional structures of the scFv antibody and cassiicolin, and binding (docking) of the antibody to the toxin antigen was simulated. Furthermore, deactivation of cassiicolin by the scFv was demonstrated by detached leaf bio-assay on selected susceptible Hevea clones. Efforts are underway to genetically transform Hevea clones with the cassiicolin-specific scFv gene to enhance resistance to Corynespora leaf disease. (Texte intégral)

Mots-clés : hévéa; hevea brasiliensis; glycoprotéine; transformation génétique; corynespora cassiicola; inde; malaisie; cassiicoline

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