Ri Plasmid Research Articles

Cell-Specific Expression of the rolC Gene of the TL-DNA of Ri Plasmid in Transgenic Tobacco Plants

Transgenic tobacco plants possessing a chimeric gene of the promoter region of the Ri plasmid ORF12 gene (rolC) and a structural gene of bacterial β-glycuronidase were obtained

Expression of a chimaeric kanamycin resistance gene introduced into the wild soybeanGlycine canescens using a cointegrate Ri plasmid vector

Seedling hypocotyl explants ofGlycine canescens were inoculated withAgrobacterium rhizogenes carrying a chimaeric NPTII gene cointegrated into the TL-DNA of pRiA4. Transformed roots produced shoots on B5 based medium with 10.0 mgl(-1) BAP, 0.05 mgl(-1) IBA and 50 μgml(-1) kanamycin. Cultured roots and regenerated plants expressed NPTII enzyme activity which was correlated with the presence of Ri TL-DNA and the structural sequence of the NPTII gene.

Upstream non-coding region which confers polar expression to Ri plasmid root inducing gene rolB

Root differentiation could be elicited on carrot discs by transformation with the agropine Ri plasmid rolB gene cloned in the binary vector Bin19, provided two conditions were met. Firstly, an adequate auxin supply had to be provided. This was achieved by co-inoculation with a strain carrying only the auxin synthetic genes of the TR-DNA. Most of the resulting roots were then shown to harbour only rolB and no aux genes. Secondly, an extended non-coding region (∼1200 bp) at the 5′ end of rolB had to be included in the construction. A shorter (∼300 bp) 5′ region, including TATA and CCAAT boxes, was not sufficient to trigger root differentiation. Both the extended (B1185) and reduced (B310) 5′ regions of rolB were then cloned upstream of the β-glucuronidase (GUS) reporter gene and infections carried out both on the apical and on the basal side of carrot discs. Strong expression of GUS, visualized histochemically as an intense blue colouring of transformed cells was observed with B1185-GUS constructions on the apical side of the discs. Only occasionally could coloured cells be observed on the basal side of the discs with B1185-GUS and on both apical and basal sides with B310-GUS constructions. Strong GUS expression was, on the contrary, achieved on cells of both auxin-rich (apical) and auxin-depleted (basal) sides of the discs with the strong constitutive viral promoter, CaMV35S. These results indicate the presence of an upstream regulatory region which confers polar expression to the rolB gene and suggest a role for auxin in its activation.

Expression of a mouse metallothionein gene in transgenic plant tissues

Three gene constructions based on a mouse metallothionein I gene ( mMT-I) were introduced into tobacco using an Ri plasmid vector system to test the effectiveness of animal gene regulatory signals in plant cells. No transcription from the native mouse gene was observed. In plant cells bearing chimeric mMT-I genes in which transcription was driven by the nopaline synthase promoter, neither polyadenylation nor splicing of mMT-I pre-mRNA was observed. Detailed comparisons of mMT-I sequences with those of known plant genes were carried out; slight differences in regions of known consensus sequences may be at least partly responsible for the non-recognition of mMT-I gene regulatory signals in plant cells, though other as yet unidentified, potentially necessary sequences may also be involved.

Expression of a synthetic gene for improved protein quality in transformed potato plants

The essential amino acid composition of plant foodstuffs is a very important nutritional property, especially in the developing countries where people often depend heavily on plant proteins from a single source. In these situations, it would be highly beneficial to ‘engineer’ the plant to produce proteins with a balanced essential amino acid content. Recombinant DNA technology and Agrobacterium-based vector systems offer a novel approach to modify plant proteins and raise the possibility of increasing their nutritive value. Methods are described for the inoculation of in vitro potato plants with Agrobacterium rhizogenes containing, within the Ri plasmid, a synthetic gene sequence coding for a protein high in essential amino acids. This synthetic gene fragment (HEAAE-DNA), 292 basepairs in length, codes for a protein composed of about 80% essential amino acids. Plants were regenerated from the Agrobacterium-induced hairy roots and we present data that these transformed plants contain the synthetic gene insert. Furthermore, our evidence shows that this novel gene was transcribed and translated within the transformed plant. Tubers from regenerated potato plants were subjected to analysis for introduction and expression of this gene. Integration of the gene into the plant genome and its expression into mRNAs and HEAAE-proteins have been demonstrated using Southern, northern and western analysis.

Oligoside production by hairy root cultures transformed by Ri plasmids.

Convenient methods have been developed to transform Panax gineseng with Ri plasmids of Agrobacterium rhizogenes. Crown galls were formed on the stems of ginseng plants by infecting them wirh A__-. rhizogenes. Hairy roots were generated from the crown galls when the stem segments bearing the crown galls were incubated on solid Murashige-Skoog's (MS) medium. The hairy roots were also generated on excised ginseng root segments which had been inoculated with A. rhizogenes. The ginseng hairy root cultures, established as liquid cultures in MS medium, produced 0.34-0.82% ginsenosides on a dry weight basis. The ginseng hairy roots were found to contain nucleosides, adenosine and guanosine, at signifivantly higher levels than normal ginseng plants. The transformed hairy roots of Glycyrrhiza uralensis were obtained by the infection of A__-. rhizogenes either in asceptically grown plantlets or in excised plant stems. One of the established liquid culture lines produced glycyrrhizin at an yield of 4.7%.

Analysis of TR-DNA/plant junctions in the genome of a Convolvulus arvensis clone transformed by Agrobacterium rhizogenes strain A4

A Charon 4A phage library, containing insert DNA isolated from a morning glory (Convolvulus arvensis) plant genetically transformed by Ri T-DNA from Agrobacterium rhizogenes strain A4, was used to isolate a lambda clone that contains part of the Ri TL-DNA and the complete TR-DNA. The two Ri T-DNAs were recovered adjacent to each other in a tail-to-tail configuration (i.e. with the TR-DNA inverted with respect to the TL-DNA). Comparison of nucleotide sequences from this lambda clone with the corresponding sequences from the Ri plasmid allowed us to determine the location of the T-DNA/plant junction for the right end of the TL-DNA and the left and right ends of the TR-DNA. We located, near each of these borders, a 24 bp sequence that is similar to the 24 bp consensus sequence found near the pTi T-DNA extremities. In addition, sequences similar to the "core" overdrive sequence from pTi are located near each right border. Hybridization and nucleotide sequence analysis of the DNA adjacent to the TL/TR junction shows that no plant DNA is located between the TL and TR-DNAs and suggests that the plant DNA adjacent to the end of the TR-DNA may have been rearranged during the integration into the plant genome.

Use of a Disarmed Ri Plasmid Vector in Analysis of Transformed Root Induction

Use of a Disarmed Ri Plasmid Vector in Analysis of Transformed Root Induction

Improvements in the growth and yield of olive trees by transformation with the Ri plasmid of Agrobacterium rhizogenes

Results of experiments to determine the beneficial effects of Agrobacterium rhizogenes inoculation on olive seedlings are reported herein. Young, bare root stock, olive trees treated with Agrobacterium rhizogenes 232 possessed a larger ratio of new root to older root mass, a larger total root mass, and a greater total top dry weight after 60 days than those treated with autoclaved A. rhizogenes 232 or a strain of the bacterium lacking the Ri plasmid. Over a course of 3 years, transformed trees grew more quickly and produced significantly more flowers, fruits, and oil than control trees, in the field. New secondary roots initiated by A. rhizogenes were well integrated into the existing vascular system of the older roots. The treatment of olive seedlings with A. rhizogenes should be seriously considered as an agronomic practice deserving further testing.

Function of heterologous and pseudo border repeats in T region transfer via the octopine virulence system of Agrobacterium tumefaciens

The successful transfer of the Ti plasmid T region to the plant cell is mediated by its 24 bp border repeats. Processing of the T-region prior to transfer to the plant cell is started at the right border repeat and is stimulated by a transfer enhancer sequence called "overdrive". Left and right border repeats differ somewhat in nucleotide sequence; moreover, the repeats of different Ti and Ri plasmids are slightly different. Our data indicate that these differences do not have a significant influence on border activity. However, the overdrive sequence is essential for the efficient transfer of a T region via an octopine transfer system. Our data suggest that an overdrive sequence must also be present next to the right border repeats of the nopaline Ti plasmid and the agropine of octopine and nopaline Ti plasmids express some differences in T-DNA processing activities. of cotopine and nopaline Ti plasmids express some differences in T-DNA processing activities.Furthermore, we demonstrate that certain pseudo border repeats, sequences that resemble the native 24 bp border repeat and naturally occur within the octopine Ti plasmid T-region, are able to mediate T region transfer to the plant cell, albeit with much reduced efficiency as compared to wild-type border repeats.

RolA locus of the Ri plasmid directs developmental abnormalities in transgenic tobacco plants.

Plants containing the left T-DNA (TL) of Agrobacterium rhizogenes show a variety of developmental abnormalities that include severely wrinkled leaves, loss of apical dominance, reduced geotropism of roots, reduced internode distances, and floral hyperstyly. The TL-DNA also affects the morphology of tumor tissue at the site of inoculation on Kalanchoe diagremontiana leaves. Single mutations at four loci of the TL-DNA (rolA, rolB, rolC, and rolD) are known to affect tumor morphology on K. diagremontiana leaves. We regenerated plants from tissues transformed with TL-DNA containing mutations in each of the rol loci in order to determine which of the rol loci, if any, control the abnormal plant phenotype. Only plants regenerated after infection with bacteria containing a mutation in rolA locus showed loss of the wrinkled leaf phenotype. The rolA locus was cloned into the plant transformation vector pGA472 and introduced alone into plants. Transgenic plants containing rolA displayed the abnormal phenotype. These results indicate that rolA is the primary determinant of the severely wrinkled phenotype of Ri plasmid transgenic plants. Other rol loci may influence the degree of developmental abnormalities.

Detection of Ri T-DNA in transformed oilseed rape regenerated from hairy roots

A fertile plant with altered phenotype of oilseed rape (Brassica napus) spring cultivar HM-81 was regenerated from hairy root tumors incited by Agrobacterium rhizogenes 15834. Southern blot hybridization analysis revealed several copies of Tl and Tr-DNA of Ri plasmid.

Homology mapping of T-DNA regions on three Agrobacterium rhizogenes Ri plasmids by electron microscope heteroduplex studies

Recombinant plasmids carrying segments of the Agrobacterium rhizogenes T-DNA regions of the three Ri plasmids 1855 (T L-DNA only), 8196, and 2659 were used for establishing homology maps by electron microscope examination of heteroduplexes. Plasmid DNA was linearized by digestion with suitable restriction endonucleases in order to generate large T-DNA segments. Heteroduplexes were prepared in 50% formamide and spread under standard conditions. Measurements of double and single strands allowed the drawing of homology maps. The three T-DNAs share mainly two homologous sequences of respectively about 2.5 and 1.5 kb, bracketing a largely nonhomologous central part which is about 5.5 kb long. The T-DNAs from pRi1855 and pRi2659 appear to be more related to each other than to that of pRi8196. With reference to the published nucleotide sequence of the T L-DNA of pRiA4 (probably identical to that of pRi1855), ORFs 8 and 14 seem to be the most conserved sequences of the three T-DNAs. The significance of these conserved sequences is unclear since the genetic loci involved in rhizogenicity of agropine strains identified previously are located in nonhomologous regions.

T-DNA length variability in mannopine hairy root: more than 50 kilobasepairs of pRi T-DNA can integrate in plant cells

Independent carrot (Daucus carota) hairy root lines were established by inoculation of discs taken from the same carrot with Agrobacterium rhizogenes 8196 and A. tumefaciens C58C1(pRi8196) carrying pRi8196. Several lines were compared with respect to T-DNA length. One of them was found to have integrated sequences covering more than 50 kbp of the Ri plasmid.

Mini-Mu lac transposons with broad-host-range origins of conjugal transfer and replication designed for gene regulation studies in Rhizobiaceae

Novel mini-Mu derivatives were constructed, carrying a truncated lacZYA operon fused to the terminal 117 bp of the Mu S-end, for the isolation of translational lac fusions by mini-Mu-mediated insertion mutagenesis. Different selectable markers (chloramphenicol resistance; gentamycin resistance) were introduced to allow selection for mini-Mu insertions in different replicons and bacterial strains. A mini-Mu lac derivative carrying the site for conjugal transfer of plasmid RP4 ( oriT) and the origin of replication of the Agrobacterium rhizogenes Ri plasmid ( oriRiHRI) was constructed to enable one-step lac-fusion mutagenesis of cloned (plasmid-borne) regions in Escherichia coli and efficient conjugal transfer of gene fusions to a variety of Gram-negative bacteria. The conjugation frequency, stability and copy number of replicons carrying mini-Mu lac derivatives with oriT and ori'RiHRI in members of the Rhizobiaceae such as Rhizobium meliloti, Azorhizobium caulinodans ORS571 and Agrobacterium tumefaciens C58 was examined.

Production of secondary metabolites by hairy roots and regenerated plants transformed with Ri plasmids.

Transformation of plants belonging to several families was investigated with Agrobacterium rhizogenes which harbours Ri plasmids and transfers T-DNA into the plant genome. Transformed Nicotiana tabacum had typical hairy roots, whereas Cassia torosa, C. occidentalisand C. obtusifolia had rather thick hairy roots lacking finelateral branching. The hairy roots of N. tabacum regenerated plants with typical phenotypes of Ri plasmid transformants. The nicotine content in the leaves of the regenerants were the highest in the plant that showed a heavily transformed phenotype. The hairy roots of Cassia plants produced anthraquinones and a xanthone, whose production profiles basically reflected those of the parent plant roots.

Organ-specific mRNA in transgenic tobacco plants possessing T-DNA of Ri plasmids

The structure and mRNA accumulation of T-DNA of Ri (A4) plasmids integrated into the genome of Nicotiana tabacum var. Samsun were investigated. Two inserts of TL-DNA were found in the transformed plant (R1) and one (R2) of the progenies obtained by self-crossing of the R1 plant. The expression level of T-DNA-derived transgenic plants was high in roots and stems, but very low in leaves. The gene encoding the major transcript of integrated Ri TL-DNA coincided with the ORF12 (Slightom et al., J. Biol. Chem., 261 (1986) 108).

Ｒｉプラスミドのわい化遺伝子

Ｒｉプラスミドのわい化遺伝子

Regeneration of flax plants transformed by Agrobacterium rhizogenes

Regeneration of flax (Linum usitatissimum) following transformation by either Agrobacterium tumefaciens carrying a disarmed Ti-plasmid vector, or Agrobacterium rhizogenes carrying an unmodified Ri plasmid, was examined. Hypocotyl and cotyledon explants inoculated with A. tumefaciens formed transformed callus, but did not regenerate transformed shoots either directly or via callus. However, cotyledon explants inoculated with A. rhizogenes formed transformed roots which did regenerate transformed shoots. Ri T-DNA encoded opines were detected in the transformed plantlets and Southern hybridization analysis confirmed the presence of T-DNA from the Ri plasmid in their DNA. Transformed plantlets had curled leaves, short internodes and some had a more developed root system characterized by plagiotropic behaviour.

Identification of the Region Encoding Opine Synthesis and of a Region Involved in Hairy Root Induction on the T-DNA of Cucumber-Type Ri Plasmid

Identification of the Region Encoding Opine Synthesis and of a Region Involved in Hairy Root Induction on the T-DNA of Cucumber-Type Ri Plasmid