INVESTIGATION OF THE MECHANISM UNDERLYING THE INHIBITORY EFFECT OF HETEROLOGOUS RAS GENES IN PLANT-CELLS

The ras genes from yeast and mammalian cells were fused to plant expre ssion promoters, and introduced into plant cells via Agrobacterium, to study their effect on cell growth and development. All introduced ras genes had a strong inhibitory effect on callus and shoot regeneration from plant tissues. This is consistent with earlier findings that het erologous ras genes were highly lethal to protoplasts following direct DNA uptake. These effects could not be reversed by increasing exogeno us or endogenous cytokinin levels. These effects were also independent of the v-Ha-ras mutations in functionally important regions of Ras pr oteins such as effector-binding and membrane-binding sites. Similarly, co-transformation with the genes encoding the Ras-negative regulators , GTPase-activating protein and neurofibromin did not affect the ras i nhibitory effect, indicating that the mechanism of ras inhibition of p lant cells is not related to normal ras cellular functions. This concl usion was supported by further studies in which ras gene expression wa s modified using various promoters and antisense constructs. The intro duced ras sequences remained fully inhibitory regardless of which prom oters (inducible or tissue-specific) or which orientations (sense or a ntisense) were tested. This strongly suggests that the ras DNA sequenc e itself, rather than the Ras protein or ras mRNA, is directly involve d in the inhibitory effect. The mechanism underlying this novel phenom enon remains unknown. Introduced ras genes may inhibit plant cell grow th by inducing co-suppression of unknown endogenous ras or ras-related genes, thereby leading to the arrest of cell growth.