Genome structure of Ri plasmid (2). Sequencing analysis of T-DNA and its flanking regions of pRi1724 in Japanese Agrobacterium rhizogenes.

We sequenced 42.6 kb including T-DNA and its flanking regions which corresponds to about 1/5 of entire length of a mikimopine-type Ri plasmid, pRi1724 in A. rhizogenes. We identified 37 ORFs (Open Reading Frames) including genes in total. Among them, 20 ORFs are probably new genes. Those ORFs have similarity with those in Agrobacterium and 9 ORFs of them was newly found on Ri plasmids.     

Organization of the agropine synthesis region of the T-DNA of the Ri plasmid from Agrobacterium rhizogenes.

The agropine/mannopine synthesis region of the TR region of the Ri plasmid of Agrobacterium rhizogenes strain A4 was localized on the basis of sequence similarity with probes from Ti plasmids of Agrobacterium tumefaciens and analysis of transposon insertions. The nucleotide sequence of the right part of the TR-DNA of pRiA4, encompassing the three genes involved in mannityl-opine synthesis, was determined and compared to the sequence of the corresponding region of the octopine-type Ti plasmid pTi15955. The organization of this region is strongly conserved between Ri and Ti plasmids, but the similarity is restricted to the coding sequences: no homology was detected in the 5' and 3' flanking sequences. The mas1' and ags proteins are the most conserved, showing more than 68% amino acid conservation, whereas the mas2' proteins are only 59% identical. Significant G/C content and codon usage differences are observed between pTi15955 and pRiA4. An open reading frame strongly similar to that of bacterial repressors is situated immediately to the right of the TR region.     

Disarming and sequencing of Agrobacterium rhizogenes strain K599 (NCPPB2659) plasmid pRi2659

Agrobacterium rhizogenes strain K599 (pRi2659), a causative agent of hairy root disease, effectively induces hairy root formation in a variety of plant species, including numerous soybean (Glycine max) cultivars. Because Agrobacterium-mediated transformation of soybean remains challenging and labor intensive, K599 appeared a suitable progenitor for new agrobacteria strains for plant transformation. In this paper, we report the disarming and sequencing of pRi2659 and the usefulness of the resulting disarmed strain in plant transformation studies of Arabidopsis thaliana, maize (Zea mays), tomato (Lycopersicon esculentum), and soybean (G. max). Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

lacZ gene fusions and insertion mutagenesis in the TL-region of Agrobacterium rhizogenes Ri plasmid

Agrobacterium rhizogenes induces root formation and inserts a fragment of its plasmid into the genome of infected plants. A part of the transferred region (TL-region) of the Ri plasmid of A. rhizogenes strain A4 was cloned in pBR322. Insertions of the Escherichia coli lacZ coding region into the hybrid plasmids were made in vivo using mini-Mu-duction. Two mini-Mus were used, one with the Mu A and B transposase genes (MudII1681) and the other without (MudII1734). Two inserts which result in E. coli lacZ expression where shown to be located in the T-DNA region. This indicates that portions of the T-DNA are capable of expression in bacteria. When these two hybrid plasmids were transformed into Agrobacterium only the one harboring MudII1734 insert gave transformants which correspond to homologous recombination. These results indicate that gene fusion and insertion directed mutagenesis can be simultaneously obtained with this mini-Mu and could be used to study Agrobacterium gene expression.     

TL-DNA from Agrobacterium rhizogenes plasmid pRi1855 reduces the osmotic pressure in transformed plants grown in vitro

Growth, water content, osmotic pressure and solute content were examined for normal potato (Solanum tuberosum L. cv. Desiree) and a derivative (line D9X8a), which was genetically transformed with T_L-DNA from Agrobacterium rhizogenes. Plants were grown (i) in vitro, (ii) in a growth chamber and (iii) in the field. In vitro, the transformed potato plants produced more biomass than the untransformed plants, partly because they had a higher water content. Potassium concentration and osmotic pressure were lower in cell sap extracted from the transformed potato shoots. In some cases the difference was as much as 50%. These differences were less clear, absent or reversed in plants from a growth chamber or from the field. In the field, however, transformed potato senesced early. It is suggested that a cellular basis for these observations may be changes induced by Ri T_L-DNA expression products in plant membrane properties.Abbreviations Ri root inducing - Ti tumour inducing - T-DNA transferred DNA     

发根农杆菌Ri质粒rolB基因研究进展（综述）

RiT-DNArolB基因是发根农杆菌转化植物的决定因子,rolB基因表达引起转基因植物形成大量毛状根（hairy root)。本文介绍近年来rolB基因的位点,表达与调控及RolB蛋白结构与功能方面的研究进展。     

Regulation of the vir genes of Agrobacterium tumefaciens plasmid pTiC58.

The virulence (vir) region of pTiC58 was screened for promoter activities by using gene fusions to a promoterless lux operon in the broad-host-range vector pUCD615. Active vir fragments contained the strongly acetosyringone-inducible promoters of virB, virC, virD, and virE and the weakly inducible promoters of virA and virG. Identical induction patterns were obtained with freshly sliced carrot disks, suggesting that an inducer is released after plant tissue is wounded. Optimal conditions for vir gene induction were pH 5.7 for 50 microM acetosyringone or sinapic acid. The induction of virB and virE by acetosyringone was strictly dependent on intact virA and virG loci. An increase in the copy number of virG resulted in a proportional, acetosyringone-independent increase in vir gene expression, and a further increase occurred only if an inducing compound and virA were present.     

Identification of hairy root loci in the T-regions of Agrobacterium rhizogenes Ri plasmids

Summary Agrobacterium rhizogenes induces root formation at the wound site of inoculation in plants and inserts a fragment of its plasmid (Ri) into the plant nuclear DNA. Parts of the transferred region (T-region) of the Ri plasmid of A. rhizogenes strain A4 or 8196 are cloned in Escherichia coli. Insertions of the E. coli lacZ coding region into the hybrid plasmids were made in vivo using transduction by miniMu. Twenty insertions localized in the T_L-DNA of pRiA4 (or pRi1855) and 2 inserts in the T-DNA of pRi8196 were obtained in E. coli. One of the T_L-DNA insertions is saved up because it is linked to an internal T-DNA deletion; the others because they confer a lactose plus phenotype on E. coli; this indicates that the T-DNA harbours sequences that are expressed in E. coli. Fifteen of these T-DNA insertions were transfered to Agrobacterium where they substitute the corresponding wild-type T-DNA of the Ri plasmid by homologous recombination. These strains corresponding to insertion-directed mutagenesis were used to inoculate Daucus carota slices and stems and leaves of Kalanchoe daigremontiana. The two insertions strains obtained in the T-DNA of pRi8196 are avirulent on K. daigremontiana; but their phenotypes differ on D. carota slices, suggesting that insertions affect distinct loci on the T-DNA involved in hairy root formation. Only one insertion out of the twenty obtained in the T_L-DNA of pRiA4 (or 1855) induces a loss of virulence on leaves of K. daigremontiana. However the T_L-DNA deletion harbouring strain induces a loss of virulence on D. carota and K. daigremontiana (stems and leaves), confirming the importance of the T_L-DNA for hairy root induction. re]19850711 rv]19851230 ac]19860114     

Design and development of amplifiable broad-host-range cloning vectors: analysis of the vir region of Agrobacterium tumefaciens plasmid pTiC58

The construction of a set of new plasmids that are suitable as general cloning vectors in Escherichia coli and Agrobacterium tumefaciens is described. Plasmid pUCD2 is amplifiable in E. coli, replicates in a wide range of gram-negative hosts and contains a number of useful restriction endonuclear cleavage sites and antibiotic resistance genes. This includes unique sites for KpnI, SacI, SacII, PstI, ClaI, SalI, EcoRV, and PvuII and the genes for resistance to kanamycin, tetracycline, ampicillin, and spectinomycin/streptomycin. Derivatives of pUCD2 include pUCD4, which has a unique XbaI site and the cosmid pUCD5, which also contains a unique EcoRI site. Two smaller plasmids pUCD9P and pUCD9X, contain many of the same unique sites as pUCD2 and pUCD4, but carry only the pBR322 replication origin and therefore do not display the extensive host-range of pSa. These plasmids were used to isolate and manipulate fragments of the A. tumefaciens pTiC58 plasmid in both E. coli and A. tumefaciens. Fragments from the virulence (vir) region of pTiC58 inserted immediately upstream of the spectinomycin resistance gene of pUCD2 resulted in spectinomycin resistance levels that varied greatly depending on the particular fragment and its orientation of insertion. Using this property we find that a major portion of the vir region of pTiC58 is transcribed in A. tumefaciens and E. coli from left to right toward the T region.     

vir genes influence conjugal transfer of the Ti plasmid of Agrobacterium tumefaciens.

Mutation of the genes virA, virB, virC, and virG of the Agrobacterium tumefaciens octopine-type Ti plasmid pTiR10 was found to cause a 100- to 10,000-fold decrease in the frequency of conjugal transfer of this plasmid between Agrobacterium cells. This effect was not absolute, however, in that it occurred only during early times (18 to 24 h) of induction of the conjugal transfer apparatus by octopine. Induction of these mutant Agrobacterium strains by octopine for longer periods (48 to 72 h) resulted in a normal conjugal transfer frequency. The effect of these vir gene mutations upon conjugation could be restored by the introduction of cosmids harboring wild-type copies of the corresponding disrupted vir genes into the mutant Agrobacterium strains. In addition, transfer of the self-mobilizable plasmid pPH1JI was not impaired in any of the mutant Agrobacterium strains tested. The effect of vir gene function on the conjugal transfer of the Ti plasmid suggests that a relationship may exist between the processes that control the transfer of the T-DNA from Agrobacterium to plant cells and the conjugal transfer of the Ti plasmid between bacterial cells.     

Opines stimulate induction of the vir genes of the Agrobacterium tumefaciens Ti plasmid.

Upon incubation of Agrobacterium tumefaciens A348 with acetosyringone, the vir genes encoded by the Ti (tumor-inducing) plasmid are induced. The addition of certain opines, including octopine, nopaline, leucinopine, and succinamopine, enhanced this induction 2- to 10-fold. The compounds mannopine, acetopine, arginine, pyruvate, and leucine did not stimulate the induction of the vir genes to such an extent. The enhancement of vir gene induction by opines depended on acetosyringone and the genes virA and virG. Opines stimulated the activity of the vir genes, the double-stranded cleavage of the T (transferred)-DNA at the border repeat sequences, and the production of T-strands by the bacterium. The transformation efficiency of cotton shoot tips was markedly increased by the addition of acetosyringone and nopaline at the time of infection.     

Analysis of unique variable region of a plant root inducing plasmid, pRi1724, by the construction of its physical map and library.

Ri plasmids in Agrobacterium rhizogenes specifically induce the hairy root syndrome on various dicotyledonous plants. Its T-DNA transfer system as well as those of Ti plasmids have successfully provided the fundamental technique to introduce exogenous genes into plants. To study the Ri genome structure, we constructed a complete BamHI physical map and a lambda library of pRi1724 of A. rhizogenes strain 1724. By using these, we carried out the complete sequence of the 74-kb region between the right border of T-DNA and tra operon, which is the highly variable region (VAR) among Ri and Ti plasmids. As a result, we found three kinds of putative ABC-type transport operons, histidine utilization operon, glycerol utilization operon and two chemoreceptor genes. In addition, a virulence-related gene, tzs was located independently of the vir region.     

Molecular and genetic analysis of the transferred DNA regions of the root-inducing plasmid of Agrobacterium rhizogenes.

The T-DNA regions of the root-inducing (Ri) plasmid pRiA4b of Agrobacterium rhizogenes were characterized. Two regions, designated TL-DNA and TR-DNA, were found to be integrated and stably maintained in the plant genome. The TL-DNA spanned a 15- to 20-kilobase region of pRiA4b and was separated from the TR-DNA region by at least 15 kilobases of nonintegrated plasmid DNA. The TR-DNA region also spanned a 15- to 20-kilobase region of pRiA4b and included a region of homology to the tms morphogenic loci of the tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens. Eighteen deletions and 95 transposon insertions were generated in the T-DNA regions and tested for alterations in virulence. Insertions into four loci in the TL-DNA affected the morphology of root formation of Kalancho? diagremontiana leaves and stems, but had no visible effects on other host plants. Insertions into two loci (tms-1 and tms-2) in the TR-DNA eliminated virulence symptoms on all plants tested, with the exception of K. diagremontiana stems, where sparse root formation occurred. Complementation experiments with Ri and Ti plasmid T-DNA mutations indicate that the tms genes of the two plasmids serve similar functions and suggest a functional relationship between one or more genes of the TL-DNA and the cytokinin synthesis locus tmr of the Ti plasmid.