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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 rhizobiaceae1

https://doi.org/10.1006/jmbi.2001.4488Get rights and content

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.

Introduction

It is well known that some bacteria of the Rhizobiaceae contain giant plasmids essential for their parasitism or symbiosis to their host plants. The parasitism or infection of Agrobacterium rhizogenes and Agrobacterium tumefaciens into target dicotyledonous plants, is caused by the transfer of the oncogenic T-DNA region of the root-inducing (Ri) and tumor-inducing (Ti) plasmids, respectively (Zambryski et al., 1989). The virulence (vir) genes on those plasmids are involved in the T-DNA transfer. Through the action of T-DNA genes, the infected plant cells proliferate and grow abnormal hairy roots in the case of the Ri plasmid, or crown gall tumors in the case of the Ti plasmid. T-DNA also encodes synthesis genes for unusual amino acid derivatives or sugar-phosphodiesters, which are generally termed opines. Typical opines, octopine and nopaline for example, are utilized as nitrogen sources by Agrobacterium sp. The T-DNA transfer system has been the fundamental basis for the creation of plant vectors and transgenic plants (Chilton, 1993). On the other hand, the symbiosis between Rhizobiacease endosymbiotic bacteria and Leguminosae plants is caused by the symbiotic (Sym) plasmids of the bacteria, the infected legume cells grow nodules, which contain nitrogen-fixing bacteria (Schultze & Kondorosi, 1998). Sym’s nod and nif genes are required to establish a symbiotic relationship between bacteria and plants (Fischer, 1994). The complete sequence of a Sym plasmid (pNGR234a) has been determined (Freiberg et al., 1997). Recently, the complete sequence of a nopaline-type Ti plasmid (pTi-SAKURA) has been determined Hattori et al 2000, Suzuki et al 2000, reflecting the interest in the relationship between T-DNA transfer and trans-kingdom conjugation Inomata et al 1994, Nishikawa et al 1990, Nishikawa et al 1992, Nishikawa and Yoshida 1998, Sawasaki et al 1996, Yoshida et al 1997.

To understand the evolutionary relationship of the parasitic/symbiotic system and cross-talk system between Agrobacterium spp/Rhizobium spp and target plants through the comparison of giant plasmids, their entire genome sequence is required. Unfortunately, the genome structure of the Ri plasmid has been poorly studied except for its T-DNA and vir genes Tanaka and Oka 1994a, Liang et al 1998. Therefore, a project to sequence an Ri plasmid was undertaken. As a target plasmid, a mikimopine-type Ri plasmid, pRi1724 of A. rhizogenes MAFF301724 was selected (Shiomi et al., 1987) because of the following advantages: (1) pRi1724 is one of the most studied Ri plasmids, and its physical map has been designed with the cosmid linking library (Tanaka & Oka, 1994b) and lambda linking library (Moriguchi et al, 2000). (2) It has high capability of T-DNA transfer and root induction, in spite of being smaller than a typical agropine-type Ri plasmid, e.g. pRiA4b (Huffman et al., 1984). This guarantees quick nucleotide sequencing and an easy prediction of the minimum gene organization of the Ri plasmid in nature. (3) Because there is no mikimopine-type in Ti plasmids, it is considered to represent the specific Ri plasmid opine type. Thus, when predicting its evolution, we can exclude the possibility of horizontal gene transfer between Ri and Ti plasmids. (4) The genome analyses are expected to elucidate the mikimopine metabolism pathway, which has not been analyzed. Here, we describe the first complete sequencing and analysis of a typical Ri plasmid, pRi1724 and its evolutionary relationship with Ti and Sym plasmids.

Section snippets

Sequence features of pRi1724

As a result of complete sequencing, a total of 173 open reading frames (ORFs) were estimated for pRi1724, as shown in Table 1 and Figure 1. These ORFs are asymmetrically distributed: 115 ORFs clockwise and 58 ORFs counter-clockwise. As shown in Figure 2, the average G+C content of pRi1724 (57.3 %) is between those of Ti (pTi-SAKURA, 56.0 %) and Sym (pNGR234a, 58.5 %) plasmids. Figure 2 shows the distribution of G+C content of the entire pRi1724 DNA. T-DNA (46.1 %), rep (53.0 %) and vir (54.1 %)

Discussion

Here, we describe the first complete sequence of a Ri plasmid (pRi1724). Based on the sequence data, it is possible to estimate the evolutionary process of the infection/symbiotic and the cross-talk system between Agrobacterium/Rhizobium and plants by comparing the Ti and Sym plasmids. The minimum essential genes and elements for the Agrobacterium parasitic process seem to be attributed to the vir and opine catabolism (opc) genes and the T-DNA because they are normally included in the

Ri plasmid DNA

Plasmid pRi1724::kan in A. tumefaciens strain DC-AT2 (Tanaka et al., 1993b) was used as a DNA source throughout this study. The field strain A. rhizogenes MAFF301724 harbors extra plasmids as well as the original pRi1724, causing difficulty in isolating an ample amount of pRi1724 DNA. Therefore, we used the DC-AT2 strain, which is a pRi1724::kan-induced transformant of Ti-less A. tumefaciens C58C1. The Ri plasmid prepared was as described Hattori et al 1997, Suzuki et al 2000.

Culture of bacteria and phages

The culture of

Acknowledgements

We thank Dr Fang-Sik Che, Nara Institute of Science and Technology, for kindly providing us with mikimopine; Dr Takahiko Tsudzuki, Aichi Gakuin University, for his help in constructing the circular gene map; Dr Katsunori Suzuki for his constant advice and help in our laboratory; and Dr Takakazu Kaneko, Kazusa DNA Research Institute, for his helpful discussions and suggestions. We thank the Cryogenic Center of Hiroshima University for supplying us with cryogen (liquid nitrogen).

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    Edited by N.-H. Chua

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    Present address: K. Moriguchi, Plant Genetics, National Institute of Genetics, 1111 Yata, Mishima 411-8540, Japan.

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