Chantal David

Further extension of the opine concept: Plasmids in Agrobacterium rhizogenes cooperate for opine degradation

SummaryPrimary hairy root tissues as well as aseptic hairy root culture lines contain specific compounds that have been biologically characterized as opines. These substances are agropine, mannopine, mannopinic acid, and agropinic acid; they have been synthesized and their electrophoretic behavior has been studied. Hairy root tissues also contain agrocinopines. According to the opine content of hairy root tissues, two types of Agrobacterium rhizogenes strains have been identified. Agropine-type strains (A4, 15834, HRI) elicit roots containing agropine, mannopine, mannopinic acid, and agropinic acid, whereas mannopine-type strains (8196, TR7, TR101) elicit roots containing only mannopine, mannopinic acid and agropinic acid. A. rhizogenes strains catabolize the opines whose synthesis they induce in the hairy root tissues. However, strain HRI only catabolizes agropine. Except for strain HRI, all A. rhizogenes strains studied contain three plasmids, of which the largest appears to be a cointegrate of the two others. Transconjugants of A. rhizogenes plasmids in A. tumefaciens have been obtained by selection on opines. Their properties have been studied and related to their plasmid content. In the mannopine strain C58C1(pRi8196), the virulence functions and the opine-related functions are located on the same plasmid (pRi8196). In agropine strains the catabolic functions are dissociated: agropine degradation is specified by the virulence plasmid, which also specifies opine synthesis in hairy root tissue, however, mannopine, mannopinic acid and agropinic acid degradation are specified by the smaller plasmid. Strain HRI contains only the virulence plasmid, which explains its inability to degrade mannopine, mannopinic acid, and agropinic acid.

Genetic transformation ofCatharanthus roseus G. Don byAgrobacterium rhizogenes.

Catharanthus roseus plantlets were inoculated with differentAgrobacterium rhizogenes strains. This plant species is known to produce secondary metabolites and axenic hairy-root cultures are an alternative to extraction of plant tissue for the compounds which are synthesized in roots. Hairy root lines were established from inoculations with the agropine strain 15834 and transformed plants were obtained after spontaneous regeneration. Phenotypic alterations of both the root system and the aerial parts were observed in transformed plants. All the tissues analyzed contained agropine and mannopine. T-DNA analysis confirmed the presence of TL- and TR-DNAs either as distinct inserts, or as a single and continuous insert including the region between TL and TR on pRi 15834.

Agrobacterium rhizogenes mediated transformation of grapevine (Vitis vinifera L.)

Genetically transformed grapevine (Vitis vinifera L.) roots were obtained after inocultation of in vitro grown whole plants (cv. Grenache) with Agrobacterium rhizogenes. The strain used contains two plasmids: the wild-type Ri plasmid pRi 15834 and a Ti-derived plasmid which carries a chimaeric neomycin phosphotrans-ferase gene (NPT II) and the nopaline synthase gene. Expression of the NPT II gene can confer kanamycin resistance to transformed plant cells. Slowly growing axenic root cultures derived from single root tips were obtained. Opine analysis indicated the presence of agropine and/or nopaline in established root cultures. For one culture, the presence of T-DNA was confirmed by dot-blot hybridization with pRi 15834 TL-DNA. Callogenesis was induced by subculturing root fragments on medium supplemented with benzylaminopurine and indoleacetic acid.Transformation of in vitro cultured grapevine cells has recently been reported (baribault T.J. et al., Plant Cell Rep (1989) 8: 137–140). In contrast with the results presented here, expession of the NPT II gene Conferred kanamycin resistance to Vitis vinifera calli that was sufficient for selection of trasformed cells.

The 3' promoter region involved in RNA synthesis directed by the turnip yellow mosaic virus genome in vitro

We have previously shown that the last 100 nucleotides from the 3′ end of turnip yellow mosaic virus (TYMV) RNA compete in vitro with genomic RNA for the TYMV‐specific RNA‐dependent RNA polymerase (RdRp). To further characterize the promoter on genomic RNA that produces complementary RNA strands, shorter fragments corresponding to the 3′ region of the viral RNA were generated and used in in vitro assays. Fragments as short as 38 nucleotides corresponding to the 3′ end of TYMV RNA compete with the viral RNA for the RdRp suggesting that the 3′ promoter on plus strand RNA is probably ≤ 38 nucleotides long. These transcripts are themselves used as templates in vitro.

Host-tissue differences in transformation of pumpkin (Cucurbita pepo L.) by Agrobacterium rhizogenes

Agrobacterium rhizogenes (wild-type strains 8196 and 15834) transformation of pumpkin (Cucurbita pepo L.) intact seedlings grown in vivo, and 6–8-day-old excised cotyledons cultured in axenic conditions was investigated. Transformed (hairy) roots were successfully induced only on the excised cotyledons with the strain 8196, while intact seedlings failed to form hairy roots with either of the two different bacterial strains. Axenic hairy-root cultures established on MS medium without hormones grew vigorously. Mannopine was detected in all transgenic root clones examined. The peroxidase activity in transformed roots was higher compared with normal roots. Electrophoretic analyses of soluble proteins and isoperoxidases showed substantial differences between transformed and normal pumpkin roots.

T-DNA of the Agrobacterium Ti and Ri Plasmids as Vectors

The study of T-DNA transmission from Agrobacterium Ti or Ri plasmid into the genomes of higher plant cells has revealed much about the consequences of transformation. It is now clear that the transformed phenotype is caused by hormonal changes produced directly or indirectly by T-DNA genes. The opine synthases are enzymes encoded in T-DNA that function in the plant cell. Our level of understanding of T-DNA-encoded functions is already sufficient to reveal clear and feasible ways to exploit T-DNA as a gene vector. What remains to challenge the crown gall investigator are many questions of fundamental importance: What is the mechanism of the seemingly illegitimate recombination between T-DNA and plant DNA, and is this process catalyzed by bacterial or host plant enzymes, or both? Do T-DNA genes encode enzymes that catalyze biosynthesis of auxin- and cytokinin-active substances? What gene in T-DNA confers immunity to A. tumefaciens, and what is its mode of action? Does T-DNA insert into random or specific sites in the host plant genome? Did T-DNA derive from plant genetic information or has prokaryotic DNA arrived at functional eukaryotic gene structure by convergent evolution? Although there is keen interest in T-DNA as a vector for genetic engineering, it holds equal interest as a unique interface between the biology of prokaryotes and eukaryotes.