A tobacco homologue of the Ri-plasmid orf13 gene causes cell proliferation in carrot root discs.

Abstract

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 (torf13-1 and torf13-2) of the Ri-plasmid orf13 gene from Nicotiana tabacum L. cv. Havana 425. The clone torf13-1 has a 594-bp open reading frame (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, mikimopine, and mannopine Ri-plasmids and the N. glauca homologue Ngorf13. Southern analyses showed that there are at least two torf13 genes derived from the N. tomentosiformis ancestor of tobacco, strongly suggesting that torf13 resulted from an ancient transfer between ancestors of 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 microM alpha-naphthaleneacetic acid) and cytokinin (1.4 microM kinetin) resulted in a marked down-regulation of torf13 mRNA accumulation. Therefore, 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 orf13 and torf13-1 regulated by the strong cauliflower mosaic virus 35S RNA promoter induced the formation of dense green callus on the disc surface. These findings indicate that at least one function of the orf13 ORF is conserved in the tobacco homologue, and provide direct evidence that a c-rol gene can influence cell proliferation.