5.3.26
EXPRESSION OF AN ERWINIA CAROTOVORA PEKTATE LYASE IN TRANSGENIC POTATOES, A WAY TO ENHANCE SOFT-ROT RESISTANCE OF TUBER TISSUE

CB WEGENER1 and JHP WEBER2

1Federal Centre for Breeding Research on Cultivated Plants, 181 90 Groll Losewitz, Germany; 2Carlsberg Laboratory, 2500 Copenhagen, Denmark

Background and objectives
Pectate lyase (PL) secreted by Erwinia carotovora bacteria depolymerizes plant cell-wall pectins into unsaturated oligogalacturonates (OG), predominantly dimers and trimers. These have been shown to induce tissue responses against potato soft rot [1]. This led to the idea to construct transgenic potato plants that express the isoenzyme PL3 of E. carotovora subsp. atroseptica. Liberated by tissue wounding, the PL3 would release OG from the cell wall pectins that trigger plant defence reactions. The advanced defence could reduce the danger of a progressive soft rot. Greenhouse-grown transgenic potatoes were analysed for their PL enzyme production and its effect against Erwinia soft-rot infection. The first field experiments were carried out in 1997 at Groll Lijsewitz.

Material and methods
Transformation: the gene encoding the Erwinia PL3 was introduced into potatoes by Agrobacterium tumefaciens. Integration of the gene was examined by PCR. Gene expression driven by the patatin B33 or the CaMV 35S promoter was analysed by Western immunoblots. PL enzyme activity was assayed in the purified cell extract of tuber tissue. The release of OG was determined by monitoring the A235.
Decrease in cell viability: tissue slices (10 mm diameter, 2 mm thick) were cut from tubers and incubated between filter papers soaked in a suspension of E. carotovora subsp. atroseptica bacteria at 1x108 c.f.u./ml for 16 h at 20C. Controls were incubated with water. Decrease in cell viability was determined by the neutral-red vital staining method [1].
Tissue maceration: tissue cylinders (8 mm diameter, 4 cm long) were cut from tubers, weighed and placed in holes containing 200 pLI of the Erwinia bacteria at 1x 08 c.f.u./ml. After incubation for 16 h at 20C, the cell debris was removed and the remaining tissue was weighed. Maceration is presented as percentage weight reduction.

Results and conclusions
The PL3 protein was faithfully translated in the plants, retained in the cytoplasm. Assay of PL activity in cell extracts of tuber tissue revealed differences among the transgenic lines ranging from 30 to 344 mU/ml extract. The decrease in cell viability caused by the bacteria was remarkably inhibited in tissue slices of PL-active transgenic lines. Tuber tissue of the untransformed, PL-inactive control showed a decrease in cell viability of 52.1%, while that of the PL-active line was reduced by 22.7%. Similarly, the application of Erwinia bacteria to cylinders cut from transgenic PL-active tubers revealed less maceration compared to PL-inactive plants. Cylinders of the 13 PL-inactive lines showed a maceration of 257.2%, while that of the 15 lines expressing PL3 had a weight loss amounting to 156.5%. Corresponding to these results, the growth of Erwinia bacteria was reduced on tuber tissue expressing PL3. An inoculum at 1x104 c.f.u./ml was completely inhibited, while the propagation of bacteria succeeded in tissue of the PL-inactive control. The results obtained for greenhouse plants showed that the expression of a PL patho-enzyme in transgenic potatoes enhanced the resistance of tuber tissue against invading Erwinia bacteria.

The first field experiments with transgenic potatoes were carried out during the 1997 growing season. Phenotypic differences between transgenic and untransformed plant lines were not observed. All transgenic lines grown in the field produced the PL enzyme. This is a good starting point for the enhancement of soft-rot resistance of potato tuber tissue. Conclusive results will be obtained after a further year of field experiments.

References
1. Weber J, Oisen O, Wegener C, von Wettstein D, 1996. Physiological and Molecular Plant Pathology 48, 389-401.