Root-Directed Expression of Alien Genes in Transgenic Potato

Abstract

Bacterial pathogens of potato, e.g. Pseudomonas solanacearum, are known to infect potato roots causing severe losses in relatively warm climates. Our intention was to express, in potato roots, a bactericidal peptide that was identified in the larvae of the flesh fly (Sarcophaga peregrina) by Natori and associates in 1977. The cDNA coding for this peptide was subsequently isolated and it was termed sarcotoxin IA (sarco) by these investigators. The respective protein was also characterized and the mature proteins mass is about 5kDa. We used the Tob promoter that is root specific, to direct sarco expression in roots. In parallel we used the Gus gene as a reporter gene for this (Tob) promoter activity. Thus, two constructions of fusion genes were made. In one we inserted Tob up-stream of sarco and in the second Tob was inserted up-stream of Gus. In both cases the coding region was followed by a terminator. Both transformation cassettes contained also a kanamycin (hand) resistance gene (nptll) that was inserted as a selective marker under the 35S (CaMV) promoter. Agrobacterium mediated genetic-transformation was performed with potato tuber discs. Five potato cultivars and breeding lines were used: Desiree, Achi- rana INTA, LT-9, TS-10, TS-15. Potato plants that regenerated from Agrobacterium infected tuber-discs and rooted on selective medium were regarded putative transformants and were further analyzed. We found that putative transformants that resulted from transformation of a vector that contained Gus driven by Tob, indeed expressed the reporter gene in their roots. This verified the potency and specificity of the chimeric genes in the transformation vector. Polyclonal anti-sarco antibodies were produced and used to evaluate the expression of sarco in the putative transgenic potato plants. Preliminary western- blot assays indicated that indeed the roots of some of the plants that were transformed with the chimeric-gene that contained the Tob promoter and the sarco cDNA, showed bands that reacted with the antisarco antibodies.