A NOVEL TRANSIENT ASSAY SYSTEM DEMONSTRATES THAT DT-A(TSM) IS A TEMPERATURE-SENSITIVE TOXIN IN PLANT-TISSUES

We describe a rapid transient assay system for analysing the efficacy of diphtheria toxin A chain (DT-A) derivatives as agents of genetic ce ll ablation in plant tissues. Because of the extreme cytotoxic effects of DT-A in eukaryotic cells we are investigating the use of temperatu re sensitive (DT-A(tsm)) and attenuated (tox 176) derivatives of DT-A to generate more intermediate phenotypes. The relative toxicities of D T-A, DT-A(tsm) and tox 176 were measured by determining their effects on the expression of a co-transfected luciferase reporter gene constru ct, using microprojectile bombardment into plant tissues. In pollen an d leaves incubated at 16 degrees C, the expression of DT-A or DT-A A(t sm) resulted in a significant reduction in luciferase (LUG) activity. However, at 30 degrees C the expression of DT-A A(tsm) had no effect o n the level of LUC activity, in contrast to the DT-A gene, which aboli shed 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 syst ems, the toxicity of tox 176 was not attenuated relative to DT-A.