A TOBACCO HOMOLOG OF THE RI-PLASMID ORF13 GENE CAUSES CELL-PROLIFERATION IN CARROT ROOT DISCS

The tobacco genome contains genes, called cellular rol (c-rol) genes, that are very similar in sequence to genes present in the T-DNA of the Agrobacterium rhizogenes Ri-plasmid. We have cloned two homologues (t orf13-1 and torf13-2) of the Ri-plasmid orf13 gene from Nicotiana taba cum L. cv. Havana 425. The clone torf13-1 has a 594-bp open reading fr ame (ORF) which is similar in sequence (77-82% for DNA and 67-77% for the deduced amino acid sequence) to orf13 genes of the agropine, mikim opine, and mannopine Ri -plasmids and the N. glauca homologue Ngorf13. Southern ana lyses showed that there are at least two torf13 genes de rived from the N. tomentosiformis ancestor of tobacco, strongly sugges ting that torf13 resulted from an ancient transfer between ancestors o f modern A. rhizogenes and tobacco. Steady-state expression of torf13 mRNA is high in sepals, petals, shoot tips and in younger leaves, but considerably lower in stem tissues, lower leaves and roots. Treatment of cultured leaf discs for 5-20 days on medium containing auxin (10.7 mu M alpha-naphthaleneacetic acid) and cytokinin (1.41 mu M kinetin) r esulted in a marked down-regulation of torf13 mRNA accumulation. There fore, torf13 is transcriptionally active in normal tobacco tissues and the steady-state mRNA level is regulated. Inoculation of carrot-root discs with A. tumefaciens strains carrying the mannopine Ri-plasmid or f13 and torf13-1 regulated by the strong cauliflower mosaic virus 35S RNA promoter induced the formation of dense green callus on the disc s urface. These findings indicate that at least one function of the orf1 3 ORF is conserved in the tobacco homologue, and provide direct eviden ce that a c-rol gene can influence cell proliferation.