A novel transient assay system demonstrates that DT-A tsm is a temperature-sensitive toxin in plant tissues

Abstract

We describe a rapid transient assay system for analysing the efficacy of diphtheria toxin A chain (DT-A) derivatives as agents of genetic cell ablation in plant tissues. Because of the extreme cytotoxic effects of DT-A in eukaryotic cells we are investigating the use of temperature sensitive (DT-A tsm) and attenuated (tox 176) derivatives of DT-A to generate more intermediate phenotypes. The relative toxicities of DT-A, DT-A tsm and tox 176 were measured by determining their effects on the expression of a co-transfected luciferase reporter gene construct, using microprojectile bombardment into plant tissues. In pollen and leaves incubated at 16°C, the expression of DT-A or DT-A tsm resulted in a significant reduction in luciferase (LUC) activity. However, at 30°C the expression of DT-A tsm had no effect on the level of LUC activity, in contrast to the DT-A gene, which abolished LUC activity. Furthermore, at the lower permissive temperature DT-A tsm was between 50- and 200-fold less toxic than DT-A. Similar co-bombardment experiments showed that, in contrast to the mammalian systems, the toxicity of tox 176 was not attenuated relative to DT-A.