Root-Inducing Region of Mikimopine Type Ri Plasmid pRi1724

Abstract

The soil bacterium Agrobacterium rhizogenes MAFF 03-O1 724 was isolated in Japan from melon plants showing hairy root disease (Shiomi et al., 1987). This strain induces hairy roots on the inoculated sites of dicotyledonous plants like other A. rhizogenes strains. These hairy roots produce a new type of opine named mikomopine (Isogai et al., 1988(Isogai et al., , 1990, which is a stereoisomer of cucumopine (Davvioud et al., 1987). A mikimopine type Ri plasmid, pRi1724, was found in the strain and a 7.4-kb fragment encoding rol genes conferring root-inducing activity was cloned (Table I). This 7.4-kb fragment was introduced into a multicloning site of plant binary vector pEIlOl (Jefferson et al., 1987), and the recombinant plasmid was transferred to Agrobacterium tumefaciens (LBA4404 by tri-parenta1 mating from Escherichia coli HB101. Transgenic tobacco plants were obtained on Murashige and Skoog medium containing phytohormones and kanamycin by co-cultivation of leaf discs with Agrobacterium. At the same time, a cloned 4.3-kb fragment encoding rol genes of agropine type Ri plasmid pRiA4b (Jouanin, 1984) was used for transformation of tobacco.The transgenic plants transformed with 7.4-or 4.3-kb fragments showed similar morphological changes, such as active root growth, dwarfness, and increase of shoot branching; leaf wrinking was observed only in the transformants with the 4.3-kb fragment of pRiA4b. To clarify the similarities or differences on the morphology of the transgenic plants depending on the type of Ri plasmids, the nucleotide sequences of TO[ genes from pRi1724 were determined. Four ORFs corresponding to rolA, B, and C and ORF13 were observed by a computer analysis. A comparison of the coding regions of pRi1724 with those of agropine type pRiA4b (Slightom et al., 1986) and mannopine type pRi8196 (Hansen et al., 1991) showed very strong homology throughout the sequences (Table 11). The percentage of sequence homology increased at the 3' end. The cDNA was synthesized from transgenic or nontransgenic tobacco as the template for reverse treanscription of RNA followed by the PCR. Using primers designed to amplify rol genes, the expression of rolA, B, and C from pRiA4b was detected, whereas the expression of the rolA gene from pRi1724 was not. It was predicted that Ri plasmid pRil724, Cosmid library in pHC79 was constructed from partially digested Ri plasmid. The fragment screened with the radiolabeled hybrid-cosmid pLJ1 (Jouanin, 1984), which contains TL-DNA of pRiHRI, was digested and inserted into a plant binary vector. Both strands were sequenced using the dideoxy method. Function:Root-inducing activity.the morphological differences resulted from the specific expression of the rolA gene from pRiA4b. The enzymic functions of rolB and rolC proteins had been reported to be responsible for auxin-glucosidase (Estruch et al., 1991b) and cytokinin-P-glucosidase (Estruch et al., 1991a) activities, respectively. However, functions of rolA and ORF13 proteins had not been elucidated at that time. Here we su...

A maize cDNA encoding a member of the retinoblastoma protein family: involvement in endoreduplication.

“…3A). Among them are the Arabidopsis thaliana cyclin A1-3 proteins (11), Agrobacterium tumefaciens VirD4 (26), Agrobacterium rhizogenes RolD (27), and several replication-associated proteins encoded by single-stranded DNA monopartite geminiviruses (13). The RepA protein encoded by WDV was of particular interest, since it has been shown to interact with the human Rb-related protein, p130, and its LxCxE motif was found to be required for efficient viral DNA replication in plant cells (13).…”

Horizontal gene transfer and mutation: Ng rol genes in the genome of Nicotiana glauca

“…11). rolB homologues of strains of A. rhizogenes other than the agropinetype strain also have coding regions longer than that of the NgrolB gene (28)(29)(30). Although the ORF of NgrolB starts at the corresponding base of RirolB, differences in bases at sites 633 and 723 bp from the initiation codon generate early-termination codons.…”

Quorum-Dependent Mannopine-Inducible Conjugative Transfer of an Agrobacterium Opine-Catabolic Plasmid

“…The conservation of this large region is particularly interesting considering the varied nature of the plasmids and the geographical locations from which the parental strains were isolated. MAFF 301724, a biovar 1 strain from which pRi1724 was identified, was isolated in Japan from a melon plant with hairy root disease (92,93). The parental strain of pRi2659, also a biovar 1 strain, was isolated in the United Kingdom from a similarly diseased cucumber plant (91).…”