Search

We have constructed an Agrobacterium‐mediated plant vector,pHTT88,which upon integration into the plant chromosomes can produce transcriptional fusions between upstream control elements in the plant genome and the gene for neomycin phosphotransferase II (npt‐II). The vector is based on a juxtaposition of a T‐DNA right border to the coding sequence of npt‐II. Using this system it is possible to isolate kanamycin‐resistant plant calli where the site of integration is suitably positioned downstream of plant transcription signals. We have demonstrated that the NPT‐II activity of such tobacco transformants shows different tissue‐specific patterns of regulation in the regenerated plants. Among eight transformants analyzed, we have isolated one gene fusion expressed only in roots and others expressed only in stem. The results demonstrate that a gene fusion vector can be used effectively in plants to identify and characterize regulatory elements.

The lacZ gene of Escherichia coli, coding for beta‐galactosidase, is a widely used reporter gene for gene expression studies in microbial and animal systems. To demonstrate that it is also a powerful reporter gene in plants, lacZ was fused to 5′ regulatory elements of several genes known to be functional in plant cells. By measuring LacZ activities in transgenic plants containing these gene constructs, we showed that the reporter is correctly monitoring the regulatory properties of the well‐characterized promoters fused to lacZ. beta‐Galactosidase was assayed directly in plant extracts when they contained high levels of LacZ or, when LacZ was expressed at low level, by separating the endogenous and LacZ activities electrophoretically and detecting the enzymes with a fluorogenic substrate. The most outstanding property of the marker is its amenability to histochemical detection. Due to its stability, LacZ can be fixed in the tissue with glutaraldehyde without loss of activity and detected with high resolution by using XGal. We could reveal expression patterns unnoticed earlier for many of the regulatory elements studied. The chlorophyll a/b binding protein gene, expressed at very high levels in green tissues, is also expressed at a low level in the vascular cylinder of the root. The Agrobacterium T‐DNA gene encoding octopine synthase is especially active in the epidermis of the root tip and the TR2′ gene was shown to be root specific in the intact plant and stimulated by wounding in the leaf tissue. The TR1′ gene, fused to nptII, shows similar characteristics suggesting co‐regulation of this tightly linked dual promoter.

The T‐DNA transfer process of Agrobacterium tumefaciens is activated by the induction of the Ti plasmid virulence (vir) loci by plant signal molecules such as acetosyringone. Upon initiation of the T‐DNA transfer process, site‐specific nicks occur at the 25‐bp border sequences. This cleavage leads to the generation of a free, linear ssT‐DNA molecule which is bound by sequence non‐specific VirE proteins. Here we present evidence for the involvement of other acetosyringone‐induced proteins in the formation of a covalent complex between the T‐strand and protein, designated the T‐complex. Alkaline gel‐electrophoretic analysis showed that proteins specifically bind to the 5′ termini of nicked T‐DNA molecules. The T‐complex can be formed in Escherichia coli when the VirD1 and VirD2 proteins are expressed.

The TL‐DNA in octopine crown galls encodes seven transcripts most, if not all, expressed from individual promoters. Site‐specific deletions and substitutions in the T‐region of the octopine plasmid pTiB6S3 indicate some of the functions of the TL‐DNA transcripts. Two of the seven genes are sufficient to allow tumorous growth. T‐DNA transfer and oncogenicity are controlled by different and independently acting functions. None of the transcripts of TL‐DNA appear to be essential for T‐DNA transfer. Four, possibly five, of the TL‐DNA transcripts act by suppressing organ development. Shoot and root formation are suppressed by the action of different transcripts.

Superoxide dismutases (SODs) are metalloproteins that catalyse the dismutation of superoxide radicals to oxygen and hydrogen peroxide. The enzyme has been found in all aerobic organisms examined, where it plays a major role in the defence against toxic reduced oxygen species which are generated in many biological oxidations. Here we report the complete primary structure of a plant manganese superoxide dismutase (MnSOD), deduced from a cDNA clone of Nicotiana plumbaginifolia. The plant protein is highly homologous to MnSODs from other organisms and also contains an N‐terminal leader sequence resembling a transit peptide for mitochondrial targeting. The location of the mature protein within the mitochondria has been demonstrated by subcellular fractionation experiments. We have analysed the expression profile of this MnSOD and found that it is dramatically induced during stress conditions, most notably in tissue culture as a result of sugar metabolism and also as part of the pathogenesis response of the plant, being induced by ethylene, salicylic acid, and Pseudomonas syringae infection. This induction is always accompanied by an increase in cytochrome oxidase activity, which suggests a specific protective role for MnSOD during conditions of increased mitochondrial respiration.

We showed that phosphorus‐containing metabolites of crown gall tissues were all taken up by appropriate pTi+agrobacteria. All but one were also taken up by pTi‐bacteria. This one compound, produced by nopaline‐, but not by octopine‐type tumours, was the only phosphorylated organic compound actively secreted by healthy crown gall cells, and it appears to be agrocinopine A. Testing crown gall cell exudates may be a general procedure for the identification of opines by transformed plant cells.

We have determined the complete primary structure (13 637 bp) of the TL‐region of Agrobacterium tumefaciens octopine plasmid pTiAch5 . This sequence comprises two small direct repeats which flank the TL‐region at each extremity and are involved in the transfer and/or integration of this DNA segment in plants. TL‐DNA specifies eight open‐reading frames corresponding to experimentally identified transcripts in crown gall tumor tissue. The eight coding regions are not interrupted by intervening sequences and are separated from each other by AT‐rich regions. Potential transcriptional control signals upstream of the 5′ and 3′ ends of all the transcribed regions resemble typical eukaryotic signals: (i) transcriptional initiation signals (‘TATA’ or Goldberg‐ Hogness box) are present upstream to the presumed translational start codons; (ii) ‘ CCAAT ‘ sequences are present upstream of the proposed ‘TATA’ box; (iii) polyadenylation signals are present in the 3′‐untranslated regions. Furthermore, no Shine‐Dalgarno sequences are present upstream of the presumed translational start codons.

In an attempt to elucidate the transfer and integration mechanism of AgrobacteriumDNA upon crown gall induction, we translocated a borderless T‐DNA to different sites of the C58 Ti plasmid. As a result of the physical linkage of the T‐DNA oncgenes with other Ti plasmid functions, the concerned strain retained tumor‐inducing capacity. However, when the borderless T‐DNA is separated on an independent replicon while all other pTi functions are provided in trans, the strain can no longer induce tumors on plants. We provide evidence that the right T‐DNA border region harbors one or more in cisactive functions essential in the transfer and/or stabilization of the T‐DNA into plant cells. The strains used in these experiments allowed us to conclude that some function(s) of the Ti plasmid can induce plant cell proliferations independently of the T‐DNA transformation event. The results described here indicate that other Ti plasmid sequences than solely the T‐region can be transferred to plant cells.

A Ti plasmid mutant was constructed in which all the on‐cogenic functions of the T‐DNA have been deleted and replaced by pBR322. This Ti plasmid, pGV3850, still mediates efficient transfer and stabilization of its truncated T‐DNA into infected plant cells. Moreover, integration and expression of this minimal T‐DNA in plant cells does not interfere with normal plant cell differentiation. A DNA fragment cloned in a pBR vector can be inserted in the pGV3850 T‐region upon a single recombination event through the pBR322 region of pGV3850 producing a co‐integrate useful for the transformation of plant cells. Based upon these properties, pGV3850 is proposed as an extremely versatile vector for the introduction of any DNA of interest into plant cells.

Seventeen in vivoand in vitroinsertion mutations in the TR‐region of the octopine Ti plasmid B6S3 of Agrobacterium tumefacienswere constructed. They include mutations in each of the five TR transcripts. All mutants retained their oncogenic properties and induced tumors with wild‐type morphology. Determination of opines in tumors induced by mutant Agrobacteriumstrains showed that production of mannopine and agropine are TR‐linked traits. Two transcripts are necessary for the synthesis of mannopine and a third one for the conversion of mannopine into agropine. None of the mutations prevents the excretion of opines. In addition, none of the five transcripts is essential for transfer and integration of the TR‐region.

Signals necessary for in vivo expression of Ti plasmid T‐DNA‐encoded octopine and nopaline synthase genes were studied in crown gall tumors by constructing mutated genes carrying various lengths of sequences upstream of the 5′ initiation site of their mRNAs. Deletions upstream of position ‐294 did not interfere with expression of the octopine synthase gene while those extending upstream of position ‐170 greatly reduced the gene expression. The estimated size of the octopine synthase promoter is therefore 295 bp. The maximal length of 5′ upstream sequences involved in the in vivo expression of the nopaline synthase gene is 261 bp. Our results also demonstrated that Ti plasmid‐derived sequences contain all signals essential for expression of opine synthase genes in plants. Expression of these genes, therefore, is independent of the direct vicinity of the plant DNA sequences and is not activated by formation of plant DNA and T‐DNA border junction.

Opine synthases are enzymes produced in dicotyledonous plants as the result of a natural gene transfer phenomenon. Agrobacteria contain Ti plasmids that direct the transfer, stable integration and expression of a number of genes in plants, including the genes coding for octopine or nopaline synthase. This fact was used as the basis for the construction of a number of chimeric genes combining the 5′ upstream promoter sequences and most of the untranslated leader sequence of the nopaline synthase (nos) gene with the coding sequence of two bacterial genes: the aminoglycoside phosphotransferase (APH(3′)II) gene of Tn5 and the methotrexate‐insensitive dihydrofolate reductase (DHFR MtxR) of the R67 plasmid. The APH(3′)II enzyme inactivates a number of aminoglycoside antibiotics such as kanamycin, neomycin and G418. Kanamycin, G418 and methotrexate are very toxic to plants. The chimeric NOS‐APH(3′)II gene, when transferred to tobacco cells using the Ti plasmid as a gene vector, was expressed and conferred resistance to kanamycin to the plant cells. Kanamycin‐resistant tobacco cells were shown to contain a typical APH(3′)II phosphorylase activity. This chimeric gene can be used as a potent dominant selectable marker in plants. Similar results were also obtained with a NOS‐DHFR MtxRgene. Our results demonstrate that foreign genes are not only transferred but are also functionally expressed when the appropriate constructions are made using promoters known to be active in plant cells.

Sequences in the 3′‐untranslated region of two different octopine T‐DNA genes were analyzed with regard to their significance in polyadenylation. Poly(A) addition sites were localized precisely by S1 nuclease mapping with T‐DNA‐derived mRNAs isolated from tobacco. The gene encoding transcript 7’ contains two AATAAA hexanucleotides, respectively 119 bp and 170 bp downstream of the TAA stop codon. A single poly(A) site was mapped 24‐25 bp downstream of the first AATAAA. Further, we show that a mutant octopine synthase gene, which has lost part of its 3′‐untranslated region by deletion, is still active. This mutant gene terminates 19 bp upstream from the major wild‐type polyadenylation site. The deletion also removes the AATAAT signal preceding this site. The mutant octopine synthase gene contains a minimum of four different poly(A) sites. The most prominent of these sites is identical to the minor poly(A) site of the wild‐type gene, and is preceded by a sequence AATGAATATA. Three other sites are located within the adjacent plant DNA, giving rise to hybrid T‐DNA/plant DNA transcripts. The two most distal sites are probably dependent on a motif AATAAATAAA, found 29 bp away from the T‐DNA/plant DNA junction.

Transmission of ColE1/pMB1‐derived plasmids, such as pBR322, from Escherichia coli donor strains was shown to be an efficient way to introduce these plasmids into Agrobacterium. This was accomplished by using E. coli carrying the helper plasmids pGJ28 and R64drd11 which provide the ColE1 mob functions and tra functions, respectively. For example, the broad host‐range replication plasmid, pGV1150, a co‐integrate plasmid between pBR322 and the W‐type mini‐Sa plasmid, pGV1106, was transmitted from E. coli to A. tumefaciens with a transfer frequency of 4.5 x 10(‐3). As pBR322 clones containing pTiC58 fragments were unable to replicate in Agrobacterium, these clones were found in Agrobacterium only if the acceptor carried a Ti plasmid, thus allowing a co‐integration of the pBR322 clones with the Ti plasmid by homology recombination. These observations were used to develop an efficient method for site‐specific mutagenesis of the Ti plasmids. pTiC58 fragnents, cloned in pBR322, were mutagenized in vitro and transformed into E. coli. The mutant clones were transmitted from an E. coli donor strain containing pGJ28 and R64drd11 to an Agrobacterium containing a target Ti plasmid. Selecting for stable transfer of the mutant clone utilizing its antibiotic resistance marker(s) gave exconjugants that already contained a co‐integrate plasmid between the mutant clone and the Ti plasmid. A second recombination can dissociate the co‐integrate plasmid into the desired mutant Ti plasmid and a non‐replicating plasmid formed by the vector plasmid pBR322 and the target Ti fragment. These second recombinants lose the second plasmid and they are identified by screening for the appropriate marker combination.

Phosphinothricin (PPT) is a potent inhibitor of glutamine synthetase in plants and is used as a non‐selective herbicide. The bargene which confers resistance in Streptomyces hygroscopicusto bialaphos, a tripeptide containing PPT, encodes a phosphinothricin acetyltransferase (PAT) (see accompanying paper). The bargene was placed under control of the 35S promoter of the cauliflower mosaic virus and transferred to plant cells using Agrobacterium‐mediated transformation. PAT was used as a selectable marker in protoplast co‐cultivation. The chimeric bargene was expressed in tobacco, potato and tomato plants. Transgenic plants showed complete resistance towards high doses of the commercial formulations of phosphinothricin and bialaphos. These data present a successful approach to obtain herbicide‐resistant plants by detoxification of the herbicide.

The Bacillus subtilis phage phi 105 repressor specifically recognizes a 14‐bp operator sequence which does not exhibit 2‐fold rotational symmetry. To facilitate a genetic analysis of this sequence‐dependent DNA binding a B. subtilis strain was constructed in which mutations affecting the phi 105 repressor‐operator interaction cause a selectable phenotype, chloramphenicol resistance. After in vivo mutagenesis, we isolated and mapped 22 different mutations in the repressor coding sequence, 15 of which are missense substitutions. These are exclusively located in the N‐terminal part (positions 1‐43) of the 144 residue long polypeptide. Two nonsense mutants, at positions 70 and 89, respectively, still show partial repressor activity. These data suggest that the phi 105 repressor consists of at least two independently folding structural domains, of which the N‐terminal is involved in operator binding. Twelve missense mutations are clustered in a region extending from Gln‐18 to Arg‐37, which we propose to be the DNA‐binding alpha‐helix–beta‐turn–alpha‐helix motif, common to all lambda Cro‐like repressors. The second (‘recognition’) helix shows significant homology with the corresponding sequence in Tn3 resolvase, and there is also a striking similarity between the phi 105 operator and the consensus sequence for a Tn3 res half‐site. Based on these observations, and on the previously isolated phi 105 0c mutants, we tentatively assign some specific contacts between base pairs from the first half of a phi 105 operator site and amino acids from the repressor's ‘recognition helix’.