Antikörper vermittelte Fusarien-Resistenz in transgenen Pflanzen

Abstract

Pathogenic fungi lead to large yield losses in agriculture and in ornamental plant production. They attack plants as well as seeds and contaminate them with mycotoxins and other metabolites. Resistant wild type species are often not available, so the only possibility to combat fungal pathogen is the use of fungicides which include risks for environment and the consumers as well as costs. The use of recombinant antibodies in transgenic plant to generate fungal pathogen resistant plants is a promising way to solve these problems in a consumer-friendly way. The goal of this work was to generate scFv antibody fragments against surface structures of Fusarium. These scFvs were be fused to antifungal peptides and proteins (AFP) and expressed in plants and should mediate resistance against the pathogenic fungi in planta. To generate scFv antibodies chicken were immunized with different fungal antigen-preparations. The total-RNA and the mRNA was isolated from spleen cells, the cDNS was synthesized, the VH- and VL-domains were amplified and assembled into a phagemid vector via restriction sites to generate scFv libraries. The diversity of the libraries was determined and the most promising libraries were combined and panned via Phage Display against fungal antigens. Soluble scFvs were tested for antigen binding activity after the third round of panning and the best binding scFvs were expressed in bacteria. The purified scFvs CWPD2, FPCWPA5 and SGB3 were characterised by ELISA, immunoblot and immunofluorescence. The scFvs were fused to different AFP and the scFvs as well as the AFP-scFv-fusions were cloned into different vector- and expression systems and their inhibitory effect was demonstrated in vitro. Resistent tests on stable transformed F2-plants of Arabidopsis demonstrated that the AFP-scFv constructs AG-scFv CWPD2, RS-scFv CWPD2, Lacto-scFv CWPD2 and Chi-scFv CWPD2 mediated antibody-resistance against F. oxysporum f.sp. matthiolae in A. thaliana cv. Columbia plants