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J Tempé

Publications and source records attributed to J Tempé.

At least 19 recordsLinked to original sources
PubMed1994

A new open reading frame, encoding a putative regulatory protein, in Agrobacterium rhizogenes T-DNA.

We report the presence of an open reading frame, named ORF13a, encoding a putative regulatory protein on the T-DNA of Agrobacterium rhizogenes 8196 Ri plasmid. Homologous ORFs are present at the same location in two other types of Ri plasmids. We present evidence that ORF13a is transcriptionally active. Expression of ORF13a was investigated by analysis of glucuronidase (GUS) activity in transgenic tobacco containing an ORF13aGUS fusion. The gene fusion was expressed at higher level in roots than in leaves. The putative protein encoded by ORF13a has an isoelectric point of 11.55 and carries SPXX repeated motifs suggesting a possible regulatory function for this gene.

Amino Acid Sequence↗
PubMed1993

Characterization of genes for synthesis and catabolism of a new rhizopine induced in nodules by Rhizobium meliloti Rm220-3: extension of the rhizopine concept.

Rhizopines are selective growth substrates synthesized in nodules only by strains of rhizobia capable of their catabolism. We report the isolation and study of genes for the synthesis and catabolism of a new rhizopine, scyllo-inosamine (sIa), from alfalfa nodules induced by Rhizobium meliloti Rm220-3. This compound is similar in structure to the previously described rhizopine 3-O-methyl-scyllo-inosamine from R. meliloti L5-30 (P.J. Murphy, N. Heycke, Z. Banfalvi, M.E. Tate, F.J. de Bruijn, A. Kondorosi, J. Tempé, and J. Schell, Proc. Natl. Acad. Sci. USA 84:493-497, 1987). The synthesis (mos) and catabolism (moc) genes for the Rm220-3 rhizopine are closely linked and located on the nod-nif Sym plasmid. The mos genes are directly controlled by the NifA/NtrA regulatory system. A comparison of the sequence of the 5' regions of the two mos loci shows very extensive conservation of sequence as well as strong homology to the nifH coding region. Restriction mapping and hybridization to DNA from the four open reading frames (ORFs) of the L5-30 mos locus indicate the absence of mosA and presence of the other three ORFs (ORF1 and mosB and -C) in Rm220-3. We suggest that the L5-30 mosA gene product is involved in the conversion of scyllo-inosamine to 3-O-methyl-scyllo-inosamine. Restriction fragment length polymorphism analysis of the moc regions of both strains shows that they are very similar. Regulation studies indicate that the moc region is not controlled by the common regulatory gene nifA, ntrA, and ntrC. We discuss the striking similarities in gene structure, location, and regulation between these two rhizopine loci in relation to the rhizopine concept.

Base Sequence↗
PubMed1992

A T-DNA transfer stimulator sequence in the vicinity of the right border of pRi8196.

An 8 bp sequence repeated 6 times is present to the right of the mannopine type pRi8196 T-DNA right-border sequence. Experiments were designed to test whether these repeats have a role in T-DNA transfer. Several constructs in which different lengths of pRi8196 right-border region were linked to the cucumopine synthesis gene on an Agrobacterium-Escherichia coli shuttle vector were made. The recombinant plasmids were tested for their efficiency to act as a source of T-DNA in a binary system in which a wild-type Ri plasmid provided virulence and root-inducing functions. The T-DNA transfer efficiency of the constructs was assessed by computing the relative frequency of roots containing cucumopine. Depending on the Ri plasmid used as source of virulence functions, a high level of T-DNA transfer was observed only if 6 (pRi8196) or 5 (pRiA4) repeats were present. These results were confirmed by looking for single-stranded T-DNA molecules (T-strands) in bacteria induced for virulence. The repetition of the 8 bp unit was named 'T-DNA transfer stimulator sequence' (TSS).

Base Sequence↗
PubMed1991

Agrobacterium rhizogenes pRi8196 T-DNA: mapping and DNA sequence of functions involved in mannopine synthesis and hairy root differentiation.

This paper presents the map and DNA sequence analysis of pRi8196 transferred DNA (T-DNA) genes encoding root-inducing and mannopine synthesis functions. A canonical 24-base-pair border repeat as well as two "pseudoborders" are present at the functional right T-DNA border. To the left of this border are homologs of the mas1' and mas2' genes of TR pRiA4. Next to these are five open reading frames (ORFs) homologous to ORFs 10-14 of TL of pRiA4. ORFs 10-12 (rolA, rolB, and rolC) are less related to their pRiA4 homologs than are the other large ORFs analyzed here. In contrast to T-DNA genes of pRiA4, pRi8196 T-DNA ORFs 11 and 12 (rolB and rolC) are sufficient to induce hairy roots on carrot disks.

Base Sequence↗
PubMed1991

Single rol Genes from the Agrobacterium rhizogenes T(L)-DNA Alter Some of the Cellular Responses to Auxin in Nicotiana tabacum.

Two kinds of cellular responses to auxin, the hyperpolarization of protoplasts and the division of protoplast-derived cells, were compared in Nicotiana tabacum plants transformed by different T-DNA fragments of Agrobacterium rhizogenes strain A4. Using transmembrane potential difference measurements to characterize hormonal sensitivity of mesophyll protoplasts, we found a 30-fold increase in sensitivity to auxin in protoplasts transformed by the whole Ri A4 T-DNA. Furthermore, the rol genes of the Ri A4 T(L)-DNA, together or as single genes, were able to increase the sensitivity to auxin by factors up to 10(4). The different effects of the single rol genes on the sensitivity of mesophyll protoplasts to auxin, rolB being the most powerful, were consistent with their respective rhizogenic effects on leaf fragments (A Spena, T Schmülling, C Koncz, J Schell [1987] EMBO J 6: 3891-3899). No difference was seen concerning the effects of auxin on division of cells derived from normal or transformed protoplasts. These results suggest that only some cellular responses to auxin could be selectively altered by rol genes. They also show that rol-transformed tobaccos can be a model system to study auxin action in plants.

Journal Article↗
PubMed1990

Auxin regulates the promoter of the root-inducing rolB gene of Agrobacterium rhizogenes in transgenic tobacco.

The regulation in tobacco of the rolB and rolC promoters of Agrobacterium rhizogenes pRi 1855 TL-DNA was studied by using the beta-glucuronidase (GUS) reporter system in transgenic plants. A 20- to 100-fold increase of GUS activity was selectively induced by auxin in rolB-GUS transformed mesophyll protoplasts, whereas this auxin-dependent increase was only 5-fold in rolC-GUS protoplasts. Moreover, both gene fusions exhibited similar tissue-specific expression in aerial parts but different patterns in roots. The spatial pattern of rolB-GUS expression could be strongly modified by the addition of exogenous auxin, further suggesting that auxin plays a central role in the regulation of the rolB promoter in tobacco. The tissue-specific and auxin-dependent regulation of the rolB promoter is discussed in relation to the effects of the rolB gene on rhizogenesis and on cellular responses to auxin.

Cloning, Molecular↗
PubMed1990

High Sensitivity to Auxin is a Common Feature of Hairy Root.

The responses to auxin of Lycopersicon esculentum roots transformed by (T(l)+T(r))-DNA of the Ri plasmid of agropine-type Agrobacterium rhizogenes strain 15834 and Catharanthus trichophyllus roots transformed by the (T(l)+T(r))-DNA, and by T(l)- or T(r)- DNA alone of the same bacterial strain were compared to that of their normal counterparts. The transmembrane electrical potential difference of root protoplasts was measured as a function of the concentration of exogenous naphthalene acetic acid. The sensitivity to auxin expressed by this response was shown to be independent of the measurement conditions and of the basal polarization of isolated protoplasts. According to this electrical response, as well as to the modulation by auxin of proton excretion by root tips and root tip elongation, roots transformed by (T(l)+T(r)) DNA are 100 to 1000 times more sensitive to exogenous auxin than normal roots, as is the case with normal and transformed roots from Lotus corniculatus (WH Shen, A Petit, J Guern, J Tempé [1988] Proc Natl Acad Sci USA 85: 3417-3421). Further-more, transformed roots of C. trichophyllus are not modified in their sensitivity to fusicoccin, illustrating the specificity of the modification of the auxin sensitivity. Roots transformed by the T(r)-DNA alone showed the same sensitivity to auxin as normal roots, whereas the roots transformed by the T(l)-DNA alone exhibited an auxin sensitivity as high as the roots transformed by (T(l)+T(r))-DNA. It was concluded that the high sensitivity to auxin is controlled by the T(l)-DNA in agropine type Ri plasmids.

Journal Article↗
PubMed1988

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 (TL-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 TL-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.

Base Sequence↗
PubMed1988

Hairy roots are more sensitive to auxin than normal roots.

Responses to auxin of Lotus corniculatus root tips or protoplasts transformed by Agrobacterium rhizogenes strains 15834 and 8196 were compared to those of their normal counterparts. Three different types of experiments were performed, involving long-term, medium-term, or short-term responses to a synthetic auxin, 1-naphthaleneacetic acid. Root tip elongation, proton excretion by root tips, and transmembrane electrical potential difference of root protoplasts were measured as a function of exogenous auxin concentration. The sensitivity of hairy root tips or protoplasts to exogenous auxin was found to be 100-1000 times higher than that of untransformed material.

Journal Article↗
PubMed1988

Opine utilization by Agrobacterium spp.: octopine-type Ti plasmids encode two pathways for mannopinic acid degradation.

Octopine-type strains of Agrobacterium tumefaciens degrade the opine mannopinic acid through a specific pathway which involves cleavage of the molecule at the C--N bond between the amino acid and the sugar moieties. Mannose was identified as a product of the reaction. This pathway was inducible by mannopinic and agropinic acids, but not by mannopine or agropine, the two other mannityl opines. The transport system for this pathway appeared to be specific for mannopinic acid. A second, nonspecific pathway for mannopinic acid degradation was also identified. This involved some of the catabolic functions associated with the metabolism of mannopine and agropine. This second pathway was inducible by mannopine and agropine but not by mannopinic or agropinic acids. The transport system for this pathway appeared to have a broad specificity. Transposon Tn5 insertion mutants affected in the specific catabolic pathway were isolated and analyzed. These mutants continued to catabolize mannopine and agropine. Both mapped to a region of the Ti plasmid previously shown to be associated with the catabolism of mannopinic acid. Restriction enzyme analysis of the Ti plasmid from strain 89.10, an octopine strain that is naturally unable to utilize mannopinic acid, showed a deletion in this same region encoding the specific mannopinic acid degradation pathway. Analysis of recombinant clones showed that the second, nonspecific pathway was encoded in a region of the Ti plasmid associated with mannopine and agropine catabolism. This region shared no structural overlap with the segment of the plasmid encoding the specific mannopinic acid degradative pathway.

Arginine↗
PubMed1987

Studies on the introduction and mobility of the maize Activator element in Arabidopsis thaliana and Daucus carota.

We have co-transformed carrot (Daucus carota) and Arabidopsis thaliana with an Agrobacterium tumefaciens non-tumorigenic T-DNA carrying the maize transposable element Activator (Ac) and an Agrobacterium rhizogenes Ri T-DNA. We present evidence that the Ac element transposes in transformed root or root-derived callus cultures of both species. We show that fertile plants can be regenerated from transformed, root-derived callus cultures of Arabidopsis, demonstrating the utility of the Ri plasmid for introducing the maize Ac element into plants. We also present evidence that Ac elements that excise from the transforming T-DNA early after transformation continue to be mobile in carrot root cultures.

Cells, Cultured↗
PubMed1987

Genetic analysis of mannityl opine catabolism in octopine-type Agrobacterium tumefaciens strain 15955.

The genetic organization of functions responsible for mannityl opine catabolism of the Ti plasmid of Agrobacterium tumefaciens strain 1,5955 was investigated. A partial HindIII digest of pTi1,5955 was cloned into a broad host range cosmid and the clones obtained were tested for ability to confer mannityl opine degradation upon Agrobacterium. Inserts containing genes for catabolism of mannopinic acid, mannopine, agropine, and agropinic acid were obtained, spanning a segment of 43 kb on the Ti plasmid. Two clones conferring upon Agrobacterium the ability to catabolize the mannityl opines were mobilized to several Rhizobium sp., to Pseudomonas putida and P. fluorescens and to Escherichia coli. The catabolic functions were phenotypically expressed in all Rhizobium sp. tested, and in P. fluorescens, but not in P. putida or in E. coli.

Arginine↗
PubMed1987

Genes for the catabolism and synthesis of an opine-like compound in Rhizobium meliloti are closely linked and on the Sym plasmid.

In alfalfa nodules induced by Rhizobium meliloti strain L5-30 the compound L-3-O-methyl-scyllo-inosamine (3-O-MSI) is synthesized. This compound is also catabolized specifically by this strain. Its biological properties are therefore similar to the Agrobacterium opines. To answer the question whether opine-like compounds ("Rhizopines") play a role in a plant symbiotic interaction, we isolated the genes for the catabolism of 3-O-MSI (moc genes) and for the induction of its synthesis in the nodule [mos gene(s)]. moc and mos genes were shown to be closely linked and located on the Sym plasmid of L5-30, suggesting that they have co-evolved and may be important in symbiosis. These genes have been cloned into a broad host-range vector that can be mobilized into other R. meliloti strains where they are expressed. The location of the mos genes in the bacteria extends the opine concept, initially developed for a plant pathological interaction, to a symbiotic one.

Journal Article↗
PubMed1986

Agrobacterium Ti and Ri plasmids specify enzymic lactonization of mannopine to agropine.

A novel enzymic activity, responsible for the conversion of mannopine to agropine by lactonization, has been identified in Agrobacterium strains. This activity is encoded by octopine-type and agropine-type Ti or Ri plasmids, and is inducible by mannopine and agropine. In crude extracts it is stable for long periods and can be used for preparative synthesis of agropine from mannopine. The physiological role of this activity is not understood. However, it is probably involved in degradation of opines of the agropine family since it is always associated with agropine utilization in wild-type strains.

Chemical Phenomena↗
PubMed1986

Arginine catabolism in Agrobacterium strains: role of the Ti plasmid.

We present a study of the enzymatic activities involved in the pathway for arginine catabolism by Agrobacterium tumefaciens. Nitrogen from arginine is recovered through the arginase-urease pathway; the genes for these two activities are probably chromosomally born. Arginase was found to be inducible during growth in the presence of arginine or ornithine. Urease was constitutively expressed. Ornithine, resulting from the action of arginase on arginine, could be used as a nitrogen source via transamination to delta 1-pyrroline-5-carboxylate and reduction of the latter compound to proline by a reductase (both enzymatic activities are probably chromosomally encoded). Ornithine could also be used as a carbon source. Thus, we identified an ornithine cyclase activity that was responsible for direct conversion of ornithine to proline. This activity was found to be Ti plasmid encoded and inducible by growth in medium containing octopine or nopaline. The same activity was also chromosomally encoded in some Agrobacterium strains. In such strains, this activity was inducible during growth in arginine-containing medium.

Ammonia-Lyases↗
PubMed1984

Physical map of the Agrobacterium rhizogenes strain 8196 virulence plasmid.

Virulence of Agrobacterium rhizogenes, agent of hairy root disease, is conferred by large plasmids called Ri (root-inducing) plasmids. We have determined the BamHI fragment map of pRi8196, MW 143 Mda, principally by analysis of recombinant plasmids containing overlapping BamHI partial-digest fragments. Clones containing solitary BamHI inserts of remaining unmapped fragments were used to probe a series of Southern-blotted, pRi8196-derived EcoRI, PstI, HindIII, SalI, or SmaI digests. Continguous hybridized bands represented complements of EcoRI, PstI, HindIII, SalI, or SmaI fragments which bridged the unmapped BamHI fragments. We present, in addition, a detailed map of the core T-DNA region with respect to the restriction endonucleases SalI, EcoRI, HpaI, and HindIII.

Chromosome Mapping↗
PubMed1984

Succinamopine: a new crown gall opine.

Agrobacterium tumefaciens strains can incite plant tumors consisting of transformed cells that synthesize novel metabolites called opines. The pattern of opine synthesis is dictated by plasmid-borne genes in the pathogen; additional plasmid genes confer on the pathogen the ability to catabolize the same pattern of opines synthesized. One group of A. tumefaciens strains, AT181, EU6, and T10/73, contains closely related tumor-inducing (Ti) plasmids that encode the ability to degrade the opine nopaline; but tumors incited by these strains do not synthesize nopaline. We demonstrated by Southern blot hybridization that AT181(pTi) has no DNA homologous to the nopaline synthase gene of pTi T37, a nopaline Ti plasmid that appears to be most closely related to this group based on fingerprint analysis. Tumors incited by these seemingly anomalous strains contain a new opine that we designate succinamopine. Its structure is analogous to that of nopaline, with asparagine replacing arginine. Evidence for the structure of succinamopine, as well as those of two related metabolites, succinamopine lactam and succinopine lactam, will be published elsewhere. Ability to catabolize succinamopine, succinamopine lactam, and succinopine lactam is encoded by pTi AT181, pTi EU6, and pTi T10/73, but not by any of 15 other Ti and root-inducing plasmids tested. Three avirulent strains tested did not catabolize succinamopine, succinamopine lactam, or succinopine lactam. We propose that pTi AT181, pTi EU6, and pTi T10/73 be designated the succinamopine Ti plasmids.

Amino Acids↗