Promoter sequences from Hevea brasiliensis hevein genes

The present invention relates to the isolation of DNA sequences from the Hevea brasiliensis containing the promoter and regulatory region of hevein genes. The promoter sequences of the hevein genes also act as an inducible promoter regulated by wounds and pathogen infection. (Résumé d'auteur

Qualitative comparison of cassiicolin in four strains of Corynespora cassiicola

Corynespora cassiicola is a necrotrophic ascomycete fungus affecting a wide range of plants. In rubber tree, C. cassiicola causes the Corynespora Leaf Fall (CLF) disease, responsible for sporadic but often severe epidemics in rubber plantations in most Asian and African producing countries. Divergence in the susceptibility of rubber clones to CLFD in various locations revealed the existence of different physiological races. A toxin secreted by the fungus (cassiicolin) has been identified as the primary determinant of C. cassiicola pathogenicity. An optimized purification protocol allowed the preparation of highly purified toxin in sufficient amount to fulfil its molecular characterization. Cassiicolin was shown to be a 27 amino acids glycosylated protein with 3 disulfide bounds. To test whether qualitative differences in the toxin may influence the pathogenicity, four strains of various geographic origins and with different virulence profiles were analysed comparatively. Biochemical purification of the toxin followed by mass spectrometry was attempted from all four strains, as well as cloning and expression analysis of the full cassiicolin-encoding gene, when detected. The results are discussed with respect to the pathogenicity of the selected strains. (Résumé d'auteur

Rubber genetics and breeding at Cirad-France Country report of activities from 2007 to 2011 (AGAP research unit)

In Cirad and the research unit " AGAP ", the two groups EGV (Marc Seguin et al.) and BURST (Pascal Montoro et al.) work on rubber genetics and molecular physiology respectively. Whereas BURST group is related with the IRRDB biotechnology group, EGV is more closely associated with the IRRDB breeding group. The main rubber research activities of EGV group are the development of molecular genetic markers for application to rubber breeding including clonal identification, genetic diversity analysis, and QTL-mapping. QTL-mapping was notably applied to the analysis of genetic determinism of SALB resistance sources in Wickham x Amazonian families (CMB = Cirad-Michelin-Brazil project), and extended to a Wickham x Wickham family for the study of growth, latex production and molar mass distribution of rubber chains in native rubber. In the CMB project, SSH banks were created for the generation of ESTs and candidate gene identification, from which a new series of EST-SSR markers were produced. In Brazil, the CMB project also develops a conventional programme of creation and selection of clones, either productive and tolerant to SALB, or adapted to supoptimal areas. Hevea x Corynespora interactions are studied with view to develop resistance breeding. In partnership with IFC (French Rubber Institute), a collaborative network of Large Scale Clonal Trials (LSCT) was developed for characterizing rubber clones, including the IRCA clones issued from a CNRA-Cirad cooperation in Côte d'Ivoire, thus allowing the up-dating of clonal recommendations in Africa. (Résumé d'auteur

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

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