Jacques Tempé

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.

Conjugal Transfer of Nopaline and Agropine Ti-Plasmids- The Role of Agrocinopines

SummaryThe conjugative behaviour of nopaline and agropine Ti-plasmids has been investigated. Using a technique which avoids enrichment of transconjugants on a mating medium we have shown that preculture in the presence of agrocinopines A or B of donor strains harbouring nopaline Ti plasmids promotes plasmid transfer whereas preculture of the same strains in the presence of nopaline has no such effect. Similarly, preculture in the presence of agrocinopines C or D promotes Ti-plasmid transfer from strains harbouring agropine Ti-plasmids.

Multiple transformation of plant cells by Agrobacterium may be responsible for the complex organization of T-DNA in crown gall and hairy root

SummaryInoculation of carrot discs and Lotus corniculatus plantlets with mixtures of different Agrobacterium rhizogenes or of A. rhizogenes and A. tumefaciens or with Agrobacterium strains harboring both an Ri and a modified Ti plasmid resulted in frequent multiple (pluribacterial) transformation of cells, as revealed by the mixed opine-type of hairy roots arising from them. Multiple transformation may account for the presence of dispersed T-DNA inserts in crown gall and hairy root lines. A plant genetic engineering strategy based on segregation of T-DNA inserts in the progeny of multiple transformants is proposed.

Production of indole alkaloids by in vitro root cultures from Catharanthus trichophyllus

Abstract Following in vitro infection of an aseptic Catharanthus trichophyllus plant with Agrobacterium rhizogenes (15834), six hairy root cultures were established. These were compared with normal root cultures, derived from the same plant, with respect to production of indole alkaloids. A rapidly growing hairy root line was used for fermenter (201) cultures. The alkaloid content of the roots obtained was examined. Seventeen monomeric indole alkaloids were purified and characterized, including five hitherto undescribed substances. Normal root and hairy root line cultures showed similar alkaloid composition. Analyses, performed at five-week intervals on five-week-old cultures showed variable alkaloid yields.

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.

Silver nitrate-positive opines in crown gall tumors

Abstract Agrobacterium tumefaciens strains harboring octopine- or agropine-type Ti-plasmids induce crown gall tumors on Kalanchoe tubiflora , sunflower or carrot, that contain agropine, mannopine, mannopinic acid, and agropinic acid. Since oxidation of these compounds on electrophoretograms by an alkaline silver nitrate reagent yields darkly stained spots due to reduction of the silver ions, they are called ‘silver nitrate-positive’. These silver nitrate-positive compounds are not present in normal plant tissues nor in tumors induced by A. tumefaciens strains that contain a nopaline-type Ti-plasmid. All four compounds are opines since octopine- and agropine-type Ti-plasmids, but not nopaline-type Ti-plasmids, confer on the bacterial host the ability to catabolize them for growth. Silver nitrate-positive opines were always present in primary octopine- or agropine-type crown gall tumors. However, cultured crown gall tumors frequently did not contain detectable silver nitrate-positive opines even though other types of opines, such as octopine, were usually present.

Induction of Cell Proliferation by Agrobacterium tumefaciens and A. Rhizogenes: A Parasite’s Point of View

The crown gall and the hairy root diseases which affect dicotyledonous plants are caused by the pathogenic soil bacteria Agrobacterium tumefaciens and A. rhizogenes. In these organisms genes responsible for pathogenicity are borne on large plasmids (200–400 kb) called respectively Ti plasmids (Tumor inducing) and Ri plasmids (Root inducing) (Zaenen et al., 1974; Van Larebeke et al., 1974, 1975; Watson et al., 1975; White and Nester, 1980 a; Costantino et al., 1981; Chilton et al., 1982; Petit et al., 1983). Both diseases afflict crop plants and produce tumorous or rooty overgrowths which usually develop on the roots or at the crown of the plant, or more rarely on stems. In the laboratory, inoculation of a supsension of virulent bacteria produces typical symptoms (Fig. 1). The molecular basis for pathogenicity is the transfer, integration, and expression of a segment of Ti or Ri plasmid DNA into the nuclear genome of the host cells (Chilton et al., 1977, 1980, 1982; Schell et al., 1979; Lemmers et al., 1980; Thomashow et al., 1980; Willmitzer et al., 1980, 1982; Spano et al., 1982; White et al., 1982). This segment, called T-DNA (Transferred DNA) carries several genes which confer upon the plant cell a specific phenotype (Garfinkel et al., 1981; Willmitzer et al., 1982; Leemans et al., 1982, see also chapter 11).

Genes for the Catabolism and Synthesis of a Nodule-Specific, Opine-Like Compound are Closely Linked and on the Sym Plasmid of Rhizobium Meliloti

The biological rationale for the Agrobacterium-crowngall interaction is the production of opines (1,2). The redirection of plant metabolites into a form which the inducing bacteria, but few others, can utilize ensures a competitive advantage to the bacteria.