The complete nucleotide sequence of a plant root-inducing (Ri) plasmid indicates its chimeric structure and evolutionary relationship between tumor-inducing (Ti) and symbiotic (Sym) plasmids in rhizobiaceae

Abstract

The Ri (root-inducing) plasmid in Agrobacterium rhizogenes and Ti (tumor-inducing) plasmid in Agrobacterium tumefaciens have provided the fundamental basis for the construction of plant vectors and transgenic plants. Recently, the determination of the first complete nucleotide sequence of the Ti plasmid (pTi-SAKURA) has been successful. To understand the general structure of these oncogenic T-DNA transfer plasmids, the whole nucleotide sequence of a mikimopine-type Ri plasmid, pRi1724, was analyzed. The plasmid is 217,594 bp in size, and has 173 open reading frames (ORFs) in total, which are asymmetrically distributed. Except for 27 ORFs, which are unknown, 173 ORFs were classified into 12 groups as follows: three for DNA replication, nine for plasmid modification, 22 for conjugation, 26 for virulence, 11 for T-DNA gene, 19 for mikimopine/mikimopine-lactam transport, ten for an unknown opine metabolism, seven for transcriptional regulator, five for sugar transport, five for glycerol metabolism, four for chemoreceptor and 32 for others. The elucidated chimeric structure of pRi1724 interestingly indicates that the evolution of Rhizobiaceae plasmids seems to have kept interactions among the plasmids; especially, the genes and elements for a conjugal transfer of pRi1724 had clearly closer kinship to those of a Sym (symbiotic) plasmid, pNGR234a in Rhizobium sp. than those of Ti plasmids. By using sequencing and Northern analysis, we examined the metabolic pathway and gene expression of mikimopine, which is probably an Ri-specific opine.