Polygalacturonase-inhibiting proteins (PGIPs) are plant proteins believed to playa role in the defence against pathogenic fungi. In this study. it was hypothesized that apple PGIPI could be used to confer enhanced resistance against Verticillium-wilt. a major disease of potato caused by the fungus Verticillillm dahliae. Transgenic lines containing the apple pgip1 gene under control of the enhanced CaMV 35S (e35S) promoter had been generated previously. Stable integration of the transgene into the potato genome was shown by the polymerase chain reaction (PCR) and Southern blot with a DIG¬labelled apple pgip1 fragment as probe. Polygalacturonase (PG)-inhibiting assays (the agarose diffusion assay and reducing sugar assays) were employed to investigate the inhibiting activity of apple PGIP I extracts, prepared from the transgenic potato lines. on the PGs secreted by V. dahliae grown on pectin medium. Inhibition was successful for all but one of the transgenic lines. Active PGIPI was expressed in the leaves of in vitro- and glasshouse grown plants, as well as in roots of in vitro-grown plants. Due to the success of the in vitro inhibition results. it was anticipated that the apple pgip1 transgene would protect the transgenic lines against Verticillium-wilt in a subsequent glasshouse trial. The transgenic lines and untransformed BP I potato control were planted in soil inoculated with V. dahliae microsclerotia and control soil. Assessments of the visual symptoms of yellowing and wilt were made on a scale of 1-5. Colonisation of stem sections was determined by plating onto potato dextrose agar plates. Disease index values were calculated from the symptom and colonisation data. Analysis of variance indicated six lines to be significantly different from the rest when grown in the inoculated soil, but five of them also showed significantly slower senescence symptoms when grown in the control soil. It is proposed that the physiological effect of an extended juvenile phase resulted in the apparent increased disease resistance. This could be caused by transformation or tissue culture¬-induced somaclonal variation of the potato plants. The hypothesis that transformation of the apple pgip1 gene into potato would confer enhanced resistance against Verticillium-wilt was not supported by the data that was obtained. Expression of antifungal genes by pathogen-inducible promoters is a valuable strategy in the development of disease resistant crops of importance. A construct containing the apple pgipl gene under control of the pathogen-inducible gst1 promoter from Arabidopsis thaliana (L.) Heynh was generated. Agrobacterium tumefaciens GV31OI(pMP90RK) was transfonned with the plant transformation vector pCAMBIA2300 containing the gst1 and e35S promoter-pgip1 inserts. A. thaliana was transformed using the floral-dip method, and putative transgenic progeny were selected by kanamycin selection of the seeds. PCR verified the insertion of the transgene into the genomes of T2 and T3 lines. Gene expression from the two promoters was compared by performing PGIP extractions and the agarose diffusion assay. The gst1 promoter was active even without induction by methyl-salicylate. Both constructs led to the expression of active apple PGIP1 against V. dahliae PG in the heterologous plant A. thaliana.
Dissertation (MSc (Plant Biotechnology))--University of Pretoria, 2006.