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E W Nester

Publications and source records attributed to E W Nester.

At least 91 records · Page 5Linked to original sources
PubMed1986

Plant phenolic compounds induce expression of the Agrobacterium tumefaciens loci needed for virulence.

The virulence loci of Agrobacterium tumefaciens are a set of linked transcriptional units that play an essential role in the early stages of plant tumorigenesis. These loci are induced upon cocultivation of the bacteria with plant cells. Seven phenolic compounds that are widely distributed among the angiosperm plants--catechol, gallic acid, pyrogallic acid, p-hydroxybenzoic acid, protocatechuic acid, beta-resorcylic acid, and vanillin--are able to induce the expression of the virulence loci. These phenolics in combination induce each transcriptional locus of the vir loci. Furthermore, this induction displays similar kinetics and genetic control to that observed during cocultivation of the bacteria with plant cells.

Cells, Cultured↗
PubMed1986

The genetic and transcriptional organization of the vir region of the A6 Ti plasmid of Agrobacterium tumefaciens.

The genetic transformation of plant cells by Agrobacterium tumefaciens is mediated by the genes of the Ti plasmid vir region. To determine the genetic and transcriptional organization of the vir region of pTiA6, vir plasmid clones were saturated with insertion mutations of a Tn3-lacZ transposon. This element is both an insertion mutagen and a reporter for the expression of the sequences into which it has inserted. One hundred and twenty-four vir::Tn3-lac insertions were analyzed for their mutagenic effect on Agrobacterium virulence, and for their expression of beta-galactosidase activity, the lacZ gene product, in vegetative bacteria and in bacteria cocultivated with plant cells. These data in conjunction with genetic complementation results show that the pTiA6 vir region contains six distinct vir complementation groups: virA, virB, virC, virD, virE and virG. Mutations in these loci eliminate (virA, virB, virD and virG) or significantly restrict (virC and virE) the ability of Agrobacterium to transform plant cells. Each of the vir loci corresponds to a single vir transcription unit: virA is constitutively expressed and non-inducible; virB, virC, virD and virE are expressed only upon activation by plant cells; and virG is both constitutively expressed and plant-inducible. The two largest vir operons, virB and virD, are probably polycistronic. The pTiA6 vir region also contains plant-inducible loci (pin) which are non-essential for virulence.

Cloning, Molecular↗
PubMed1986

A plant cell factor induces Agrobacterium tumefaciens vir gene expression.

The virulence genes of Agrobacterium are required for this organism to genetically transform plant cells. We show that vir gene expression is specifically induced by a small (<1000 Da) diffusible plant cell metabolite present in limiting quantities in the exudates of a variety of plant cell cultures. Active plant cell metabolism is required for the synthesis of the vir-inducing factor, and the presence of bacteria does not stimulate this production. vir-inducing factor is (i) heat and cold stable; (ii) pH stable, although vir induction with the factor is sensitive above pH 6.0; and (iii) partially hydrophobic. Induction of vir gene expression was assayed by monitoring beta-galactosidase activity in Agrobacterium strains that carry gene fusions between each of the vir loci and the lacZ gene of Escherichia coli. vir-inducing factor (partially purified on a C-18 column) induces both the expression in Agrobacterium of six distinct loci and the production of T-DNA circular molecules, which are thought to be involved in the transformation process. vir-inducing factor potentially represents the signal that Agrobacterium recognizes in nature as a plant cell susceptible to transformation.

Journal Article↗
PubMed1986

A gene essential for Agrobacterium virulence is homologous to a family of positive regulatory loci.

The vir region of Agrobacterium tumefaciens spans at least six transcriptional loci required for crown gall tumorigenesis. The transcriptional induction of two of these vir loci in response to cocultivation with tobacco suspension cells was measured by using bacteria containing mutations in each of the six vir loci located on the Ti plasmid. Induction of these vir genes occurred only in bacteria that had functional copies of virA and virG. The nucleic acid sequence of a 1.25-kilobase clone encompassing virG contains one open reading frame capable of coding for a protein of about 30,000 daltons. The amino acid sequence of the predicted virG product is homologous to that of eight bacterial proteins, including that of the ompR gene of Escherichia coli. Most, although not all, of these proteins, like VirG, are positive regulatory elements.

Amino Acid Sequence↗
PubMed1986

Analysis of Agrobacterium tumefaciens virulence mutants in leaf discs.

The leaf disc transformation system provides a simple means to score expression of various T-DNA markers within days of infection by Agrobacterium tumefaciens as well as long-term selection for growth of transformed callus and shoots. In this report, we describe the application of this system to evaluation of marker transfer and integration in a comprehensive set of defined avirulent mutants of A. tumefaciens. We conclude that virC is not essential when the T-DNA is present on a binary vector. The most likely explanation for this is that the virC gene product is involved in generation of a T-DNA intermediate.

Arginine↗
PubMed1986

Physical and functional map of supervirulent Agrobacterium tumefaciens tumor-inducing plasmid pTiBo542.

Agrobacterium tumefaciens strains carrying pTiBo542 induce large, fast-appearing tumors and have an unusually wide host range. A clone bank was made from this 250-kilobase plasmid in a wide-host-range vector, and restriction maps were determined for BamHI and SalI. The virulence genes, transferred DNA genes, plasmid incompatibility region, and a region that inhibits growth of certain A. tumefaciens strains were localized. The six virulence genes and two tms genes were highly homologous to the genes of pTiA6, but the tmr gene was not. Mutations in each of the six vir loci of pTiA6 were complemented by clones from the vir region of pTiBo542.

Chromosome Mapping↗
PubMed1986

Molecular characterization of a host-range-determining locus from Agrobacterium tumefaciens.

The virulence loci play an essential role in tumor formation by Agrobacterium tumefaciens. This study focused on the virC locus, which affects the host range Agrobacterium species. virC mutants display an attenuated or avirulent phenotype on certain host plants, but remain fully virulent on other plant hosts. The nucleotide sequence revealed that the virC locus of pTiA6NC is an operon consisting of two open reading frames. These two open reading frames, designated virC1 and virC2, encode protein products of 25,713 and 22,710 daltons, respectively, which were visualized by polyacrylamide gel electrophoresis. Only two nucleotides separated the stop codon for virC1 from the start codon for virC2, indicating that these genes may be translationally coupled.

Arginine↗
PubMed1985

Unusual plasmid DNA organization in an octopine crown gall tumor.

A cloned tobacco crown gall tumor, 1595501, incited by A. tumefaciens strain 15955 was studied. Molecular analysis of the organization of the T-DNA by means of Southern transfer and hybridization techniques indicated that the 1595501 tumor has about 10 copies of TL DNA, five of which are complete TL DNA, whereas most octopine tumors have only one to two copies of complete TL DNA. Hybridization studies and genomic cloning indicated that some segments of the T-DNA have undergone deletions. One of the clones contained two copies of T-DNA which were inverted in orientation with respect to each other. Two left ends of TL DNA from the 1595501 tumor line and the corresponding region of the octopine plasmid were sequenced. Comparison of the various cloned T-DNA sequences with Ti-plasmid sequence indicated that while there is an association with a 25 base pair direct repeat, there is no specific set of base pairs in the T-DNA at which divergence from Ti-plasmid sequences occurred.

Arginine↗
PubMed1985

New cloning vehicles for transformation of higher plants.

We have constructed a set of small vectors based on the tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens which allow the transfer of exogenous DNA into plant chromosomes. These vectors contain: (i) a chimeric gene containing the transcriptional control signals from the nopaline synthase gene and the coding sequence for neomycin phosphotransferase; (ii) the ColE1 replicon; (iii) the cos site of bacteriophage lambda; (iv) the border sequences from the ends of the T-DNA region of the Ti plasmid; and (v) a wide host range replicon. Due to the small size of these cosmid vectors, DNA fragments up to 35 kbp can be inserted by an in vitro packaging method in Escherichia coli. The ability of these vectors to be stably replicated in both E. coli and A. tumefaciens allows their subsequent transfer to and maintenance in Agrobacterium without intermediate genetic manipulations. We demonstrate that DNA cloned into these vectors in A. tumefaciens can efficiently transform plants when in trans with a wild-type Ti plasmid which donates the functions necessary for DNA transfer and integration. We also show that only the right border of the T-DNA is necessary for DNA transformation.

Journal Article↗
PubMed1985

A Tn3 lacZ transposon for the random generation of beta-galactosidase gene fusions: application to the analysis of gene expression in Agrobacterium.

The construction and use of a Tn3-lac transposon, Tn3-HoHo1, is described. Tn3-HoHo1 can serve as a transposon mutagen and provides a new and useful system for the random generation of both transcriptional and translational lacZ gene fusions. In these fusions the production of beta-galactosidase, the lacZ gene product, is placed under the control of the gene into which Tn3-HoHo1 has inserted. The expression of the gene can thus be analyzed by monitoring beta-galactosidase activity. Tn3-HoHo1 carries a non-functional transposase gene; consequently, it can transpose only if transposase activity is supplied in trans, and is stable in the absence of this activity. A system for the insertion of Tn3-HoHo1 into sequences specifically contained within plasmids is described. The applicability of Tn3-HoHo1 was demonstrated studying three functional regions of the Agrobacterium tumefaciens A6 Ti plasmid. These regions code for octopine catabolism, virulence and plant tumor phenotype. The regulated expression of genes contained within each of these regions was analyzed in Agrobacterium employing Tn3-HoHo1 generated lac fusions.

Amino Acid Sequence↗
PubMed1985

Identification and genetic analysis of an Agrobacterium tumefaciens chromosomal virulence region.

A genetic analysis of Agrobacterium tumefaciens chromosomal functions required for virulence was undertaken. Large Tn5-containing cosmid clones were isolated from DNA of avirulent A. tumefaciens mutants having chromosomal Tn5 insertions and exhibiting defective attachment to plant cells. The clones from several different mutants each contained overlapping segments of a 30-kilobase A. tumefaciens chromosomal region, which were physically mapped. All chromosomal Tn5 insertions leading to the avirulent, attachment-defective phenotype were localized within an 11-kilobase portion of this chromosomal virulence region. Transposon Tn3::HoHo1 (Tn3 containing lacZ) was used to simultaneously mutagenize and create lac fusions within the virulence region. This analysis demonstrated the presence of two distinct chromosomal virulence loci, which were 1.5 and 5 kilobases long; transposon insertions into these loci led to avirulence and defective attachment. The beta-galactosidase activity associated with various Tn3::HoHo1-created lac fusions indicated that the loci are transcribed in opposite directions, and complementation studies suggested that each locus consists of a single transcriptional unit. A cosmid clone of the chromosomal virulence region containing a lac fusion in the extreme 3' portion of the 5-kilobase locus was used to demonstrate that expression of this region is dependent on the presence of sequences in the 5' portion of the locus, confirming its operon-like nature.

Chromosomes, Bacterial↗
PubMed1985

Role for 2-linked-beta-D-glucan in the virulence of Agrobacterium tumefaciens.

Phenol-water cell extracts of virulent Agrobacterium tumefaciens A348 and several avirulent mutants with a reduced ability to attach to plant surfaces were examined. A low-molecular-weight 2-linked-beta-D-glucan was identified in the cell wall extracts of the virulent wild-type strain. Analyses of phenol-water extracts and culture filtrates of four mutant strains showed that the mutants did not produce any 2-linked-beta-D-glucan. When these mutants were complemented, the ability to produce the glucan described above was restored. These results suggest that there is a role for 2-linked-beta-D-glucans in the attachment of A. tumefaciens to plant cells. One avirulent, attachment-defective mutant retained its ability to produce the low-molecular-weight glucan. This mutation, however, mapped to a different transcriptional unit than the mutants deficient in the glucan described above. Thus, it appears that 2-linked-beta-D-glucan is only one component that may be necessary for attachment of A. tumefaciens to plant cell surfaces.

Adhesiveness↗
PubMed1985

Molecular and genetic analysis of the transferred DNA regions of the root-inducing plasmid of Agrobacterium rhizogenes.

The T-DNA regions of the root-inducing (Ri) plasmid pRiA4b of Agrobacterium rhizogenes were characterized. Two regions, designated TL-DNA and TR-DNA, were found to be integrated and stably maintained in the plant genome. The TL-DNA spanned a 15- to 20-kilobase region of pRiA4b and was separated from the TR-DNA region by at least 15 kilobases of nonintegrated plasmid DNA. The TR-DNA region also spanned a 15- to 20-kilobase region of pRiA4b and included a region of homology to the tms morphogenic loci of the tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens. Eighteen deletions and 95 transposon insertions were generated in the T-DNA regions and tested for alterations in virulence. Insertions into four loci in the TL-DNA affected the morphology of root formation of Kalanchoë diagremontiana leaves and stems, but had no visible effects on other host plants. Insertions into two loci (tms-1 and tms-2) in the TR-DNA eliminated virulence symptoms on all plants tested, with the exception of K. diagremontiana stems, where sparse root formation occurred. Complementation experiments with Ri and Ti plasmid T-DNA mutations indicate that the tms genes of the two plasmids serve similar functions and suggest a functional relationship between one or more genes of the TL-DNA and the cytokinin synthesis locus tmr of the Ti plasmid.

Chromosome Deletion↗
PubMed1985

New class of limited-host-range Agrobacterium mega-tumor-inducing plasmids lacking homology to the transferred DNA of a wide-host-range, tumor-inducing plasmid.

Biotype 1 and 2 strains of Agrobacterium tumefaciens were isolated from crown gall tumors of Lippia canescens plants growing as ground cover in Arizona. The isolates were agrocin 84 sensitive, did not catabolize octopine, nopaline, agropine, or mannopine, and were limited in their tumorigenic host range. One biotype 2 strain, AB2/73, showed the most limited host range; it incited tumors only on Lippia strains, the cucurbit family of plants, and Nicotiana glauca. Megaplasmids were detected in the isolates by vertical agarose gel electrophoresis. The unusual host range, as well as sensitivity to agrocin 84, were plasmid specified since they were conjugally cotransferred with plasmids from donor strain AB2/73. Correlation of deletions with concomitant loss of virulence and agrocin 84 sensitivity identified the megaplasmid pAtAB2/73d as the virulence element in strain AB2/73. The estimated size of this tumor-inducing plasmid was 500 kilobases. Axenic growth of tumor tissue incited by strains carrying pAtAB2/73d was phytohormone independent. Although the limited-host-range megaplasmid pAtAB2/73d lacked any detectable homology to the phytohormone-biosynthetic genes in wide-host-range transferred DNA (tms-1, tms-2, tmr), it showed homology to the wide-host-range virB, virC, virD, and virG loci. Therefore, pAtAB2/73d represents a new class of tumor-inducing plasmids distinguished by its large size, the absence of determinants for the catabolism of several known opines, the presence of agrocin 84 sensitivity, and its lack of homology to wide-host-range transferred DNA contrasted with its conservation of sequences from the wise-host-range vir region.

Adenine Nucleotides↗
PubMed1984

T-DNA of Agrobacterium tumefaciens encodes an enzyme of cytokinin biosynthesis.

Phytohormone overproduction in crown gall tumors is due to the expression of several T-DNA genes. The data strongly suggest that the tmr gene (transcript 4) is responsible for cytokinin overproduction by encoding dimethylallyl-pyrophosphate:AMP dimethylallyltransferase (DMA transferase), an enzyme directly involved in cytokinin biosynthesis. Cell-free extracts of Escherichia coli strains containing the tmr gene from pTiA6NC had DMA transferase activity. No activity was present in the control strain containing only the plasmid vector. The cytokinins synthesized were isopentenyladenine, isopentenyladenosine, and isopentenyladenosine 5'-monophosphate. DMA transferase activity was also detected in cloned crown gall tumors incited by Agrobacterium tumefaciens wild-type A6NC and a tms mutant. Enzymatic activity in cell-free extracts of E. coli and tumors could be abolished by transposon insertion within the tmr gene.

Base Sequence↗
PubMed1984

Hairy-root-inducing plasmid: physical map and homology to tumor-inducing plasmids.

A physical map was constructed for the 250-kilobase plasmid pRiA4b, which confers the virulence properties of a strain of Agrobacterium rhizogenes for hairy root disease in plants. The complete HindIII and KpnI restriction map was determined from a collection of overlapping HindIII partial digest clones. Homologous regions with two well-characterized plasmids that confer virulence for crown gall disease, plasmids pTiA6 and pTiT37, were mapped on pRiA4b. As much as 160 kilobases of pRiA4b had detectable homology to one or both of these crown-gall-tumor-inducing plasmids. About 33 kilobases of pRiA4b hybridized to the vir region of pTiA6, a segment of DNA required for virulence of Agrobacterium tumefaciens. Portions of pTiA6 and pTiT37 transferred into plant cells in crown gall disease (T-DNA), shared limited homology with scattered regions of pRiA4b. The tumor morphology loci tms-1 and tms-2 from the T-DNA of pTiA6 hybridized to pRiA4b. A T-DNA fragment containing the tml and tmr tumor morphology loci also hybridized to pRiA4b, but the homology has not been defined to a locus and is probably not specific to tmr. A segment of pRiA4b T-DNA which was transferred into plant cells in hairy root disease lacked detectable homology to pTiA6 and had limited homology at one end to the T-DNA of pTiT37.

Chromosome Mapping↗
PubMed1983

Plant tumors.

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Photography↗