Isolation of antibody fragments recognising phytopathogen secreted enzymes and the expression of scFvs in transgenic tobacco

The expression of antibody fragments has been suggested as a possible mechanism for the introduction of disease resistance into transgenic plants. Important pathogenicity factors of certain phytopathogens are secreted cell wall degrading enzymes. Pectate lyases and polygalacturonase are important cell wall degrading enzymes secreted by Erwinia carotovora and Botrytis cinerea respectively. The blocking of activities of these enzymes could offer an opportunity for reducing the ingress of these pathogens. Pectate lyase C from E. carotovara was purified to near homogeneity by cation exchange chromatography following overexpression in E.coli. Hybridoma lines recognising this enzyme were obtained and an scFv (single chain variable fragment) isolated from one of these lines. This scFv did not recognise pectate lyase.;The purified enzyme was used to inoculate mice to construct libraries of scFvs derived from spleen mRNA. These libraries did not yield any scFvs recognising pectate lyase. A synthetic human scFv library was then screened with pectate lyase and Botrytis cinerea polygalacturonase (PG), and scFvs recognising PG isolated. These scFvs were tested against a range of antigens to determine specificity. One scFv, C1, was found to bind PG but did not bind to control antigens bovine serum antigen, hen egg lysozyme or pectate lyase. Neither did this scFv bind to PG isolated from another fungal pathogen, Cryphonectria parasistica. scFv C1 was found to bind to a carbohydrate epitope on the B. cinerea polygalacturonase.