Eugene W. Nester

Stable incorporation of plasmid DNA into higher plant cells: the molecular basis of crown gall tumorigenesis

Abstract Evidence is presented that crown gall tumors are caused by the incorporation of part of a virulence plasmid carried by the inciting bacterium, Agrobacterium tumefaciens. The rate of reassociation of labeled plasmid DNA was slightly accelerated in the presence of tobacco crown gall tumor DNA, but not normal tobacco DNA. Treatment of tumor DNA with DNAase abolished the acceleration. To determine whether all plasmid sequences are represented in tumor DNA, the labeled plasmid DNA was separated into specific fragments after digestion with restriction endonuclease Sma I. Renaturation rates for DNA from bands 1, 2, 7, 8, 9, 12 and 14 were not affected by tumor DNA. DNA from band 3 showed a slight rate increase in the presence of tumor DNA, indicating 21–27 copies of 14–18% of the DNA sequences in this (doublet) band. The band 3 doublet was separated by electrophoresis into bands 3a and 3b. Tumor DNA had little effect on the rate of reassociation of labeled band 3a DNA. Band 3b DNA renatured rapidly in the presence of tumor DNA, and its rate increase indicated that approximately 18 copies of 40% of band 3b DNA sequences are present per diploid tumor cell. This amounts to 3.7 × 10 6 daltons of foreign genetic information and represents a contribution of 0.0011% to the DNA content of the tumor cell. The relationship between this plant tumor and virally induced animal tumor systems is discussed.

Genetic Analysis of Crown Gall: Fine Structure Map of the T-DNA by Site-Directed Mutagenesis

Abstract Seventy-five Tn5 and three Tn3 insertions were generated, characterized and mapped in the pTiA6NC plasmid sequences which are stably integrated in crown gall tumors (T-DNA). Four mutants containing Tn5 insertions in a specific region of the T-DNA incited tumors that no longer synthesized octopine. No single insertion resulted in complete loss of oncogenicity. Twenty-five transposon insertions defined three distinct loci affecting tumor morphology. The first group ( tml ), of seven mutants, contained Tn5 insertions within a 1.25 kilobase (kb) region and incited tumors two to three times larger than normal. The second group ( tmr ), of nine mutants, incited tumors with a massive amount of roots proliferating from the tumor callus and contained Tn5 insertions in a 1 kb cluster. The third group ( tms ), of nine mutants, incited tumors with shoots growing from the tumor callus and contained three Tn3 insertions and six Tn5 insertions distributed over a 3.1 kb region. Each of these loci was separated by Tn5 insertions that did not noticeably change tumor formation. We identified two more regions of the T-DNA where transposon insertions did not appear to affect tumor morphology: one contained 28 Tn5 insertions distributed over 3.2 kb; the other contained 17 Tn5 insertions distributed over 7 kb and the octopine synthesis locus. A detailed functional map of the T-DNA of pTiA6NC resulted from the characterization of these insertions. The incorporation of Tn5 sequences into the plant genome was also demonstrated. We discuss these results in relation to the map location of tumor-derived RNA transcribed from the T-DNA, the role of phytohormones in crown gall tumorigenesis and the eventual use of the Ti plasmid as a vehicle for introducing genes of choice into the genomes of higher plants.

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 λ; (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.

Wide host range cloning vectors: A cosmid clone bank of an Agrobacterium Ti plasmid

Abstract The plasmids pVK100, pVK101, and pVK102 are described. Their properties include RK2 replication properties, P1 incompatibility, non-self-transmissibility, mobilization by the helper plasmid pRK2013, and single restriction sites wherein cloned inserts can inactivate either kanamycin or tetracycline resistance markers. The vectors pVK100 and pVK102 are cosmids. Single Eco R1 sites are present in pVK100 and pVK101; pVK101 also contains a Bgl II site suitable for cloning. The cosmid pVK102 was used to generate a clone bank of Agrobacterium tumefaciens plasmid pTiA6. By identification of restriction fragments shared by clones with inserts of pTiA6 DNA that overlapped, restriction fragment maps of pTiA6 were established for the enzymes Sal 1, Eco R1, Xho 1, and Sst 1. Various members of the clone bank representing all regions of pTiA6 were mated from E. coli strain HB101 into A. tumefaciens strains A136 and A856. The region of pTiA6 associated with octopine catabolism was localized by identifying which clones conferred octopine utilization upon strain A136. The inability of certain strain A856 transconjugants to utilize octopine identified the clones which eliminated the octopine-type Ti plasmid of strain A856 by incompatibility. The use of such inc clones to cure plasmids and distinguish incompatibility groups of plasmids is discussed.

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.

Isolation of covalently closed circular DNA of high molecular weight from bacteria

Abstract A simple method is described in detail for the efficient isolation of high molecular weight covalently closed circular DNA (ccc-DNA) from Agrobacterium . Although this method was developed for isolating ccc-DNA of molecular weights greater than 10 8 daltons in Agrobacterium , the technique also proves to be useful in isolating ccc-DNA of varying sizes from a variety of other bacteria. The technique involves the shearing and alkali denaturation of the chromosomal DNA, followed by the preferential removal of the single-stranded DNA by phenol extraction. The DNA which remains is largely ccc-DNA. The DNA is then concentrated, and the ccc-DNA is separated from the chromosomal DNA by centrifugation in a cesium chloride-ethidium bromide density gradient. By this technique, ccc-DNA of varying sizes has also been isolated from species of Escherichia, Rhizobium, Citrobacter , and Lactobacillus .

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.

Pilus assembly by agrobacterium T-DNA transfer genes

Agrobacterium tumefaciens can genetically transform eukaryotic cells. In many bacteria, pili are required for interbacterial DNA transfer. The formation of pili by Agrobacterium required induction of tumor-inducing (Ti) plasmid-encoded virulence genes and growth at low temperature. A genetic analysis demonstrated that virA, virG, virB1 through virB11, and virD4 are the only Ti plasmid genes necessary for pilus assembly. The loss and gain of pili in various mutants correlated with the loss and gain of transferred DNA (T-DNA) transfer functions, which is consistent with the view that Agrobacterium pili are required for transfer of DNA to plant cells in a process similar to that of conjugation.

Plant gene expression response to Agrobacterium tumefaciens

To elucidate the nature of plant response to infection and transformation by Agrobacterium tumefaciens, we compared the cDNA-amplified fragment length polymorphism (AFLP) pattern of Agrobacterium- and mock-inoculated Ageratum conyzoides plant cell cultures. From 16,000 cDNA fragments analyzed, 251 (1.6%) were differentially regulated (0.5% down-regulated) 48 h after cocultivation with Agrobacterium. From 75 strongly regulated fragments, 56 were already regulated 24 h after cocultivation. Sequence similarities were obtained for 20 of these fragments, and reverse transcription–PCR analysis was carried out with seven to confirm their cDNA-AFLP differential pattern. Their sequence similarities suggest a role for these genes in signal perception, transduction, and plant defense. Reverse transcription–PCR analysis indicated that four genes involved in defense response are regulated in a similar manner by nonpathogenic bacteria, whereas one gene putatively involved in signal transduction appeared to respond more strongly to Agrobacterium. A nodulin-like gene was regulated only by Agrobacterium. These results demonstrate a rapid plant cell response to Agrobacterium infection, which overlaps a general response to bacteria but also has Agrobacterium-specific features.

Foreign DNA of bacterial plasmid origin is transcribed in crown gall tumours

Agrobacterium tumefaciens incites cancerous growths in dicotyledonous plants called crown gall tumours. Large plasmids in oncogenic Agrobacterium strains1 carry genetic information which is essential for tumour induction2,3. We recently reported that axenic crown gall tumour tissue contains multiple copies of a small part of the oncogenic (Ti) plasmid of the inciting bacterial strain4. The mechanism of induction of crown gall tumours thus seems to resemble that of some virally induced animal tumours: the eukaryotic cell is transformed by addition of new genetic information which is stably maintained through subsequent cell divisions5–7. The means by which foreign DNA brings about the tumorous growth pattern of crown gall cells is unknown. The first step in elucidating its mode of action is reported here: we have detected RNA transcripts of the foreign genetic information in the tumour cell.