Milton P. Gordon

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.

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.

Changes in T-DNA methylation and expression are associated with phenotypic variation and plant regeneration in a crown gall tumor line.

SummaryPhenotypic variation of an octopine-type crown gall tumor line resulting from changes in the pattern of T-DNA methylation and expression is described. Variants that grow as unorganized callus always express T-DNA transcripts 1 and 2. In shoot-forming variants (teratomas) only T-DNA transcript 4 is expressed. This line also regenerates normal-appearing, rooted plants in which all T-DNA expression is suppressed. Tissues from these plants require phytohormones for growth in vitro. These plants are selffertile and transmit T-DNA through meiosis, and T-DNA suppression is maintained in the next generation. After treatment of regenerated plant tissue with 5-azacytidine, an inhibitor of DNA methylation, T-DNA transcription and phytohormone-independent tumorous growth resume. The T-DNA of cell lines in which T-DNA is not expressed is highly methylated, whereas the level of T-DNA methylation is reduced in 5-azacytidine treated cells that resume T-DNA expression and phytohormone-independent growth. The correlation between the degree of T-DNA methylation and the level of T-DNA expression indicates that hypermethylation is responsible for the suppression of T-DNA transcription.

Systemically wound-responsive genes in poplar trees encode proteins similar to sweet potato sporamins and legume Kunitz trypsin inhibitors

When the lower leaves of hybrid poplar trees are mechanically wounded, several novel mRNAs accumulate in the unwounded upper leaves (Parsons TJ, Bradshaw HD, Gordon MP: Systemic accumulation of specific mRNAs in response to wounding in poplar trees, Proc Natl Acad Sci USA, in press). A partial cDNA clone corresponding to a transcript from the wound-responsive gene designated win 3 (wound-inducible) has been cloned by differential hybridization to 32P-labelled cDNA from the leaves of wounded trees. Northern blots show a large accumulation of win 3 transcripts in the unwounded leaves of wounded trees. Southern blot analysis of poplar DNA suggests that win 3 is a member of a multigene family. The nucleotide sequences of several win 3 cDNA clones have been determined, indicating that at least three win 3 gene family members are transcribed. A genomic clone of a win 3 gene family member has been isolated and a 1.5 kb Hind III fragment containing the predicted protein-coding and 5′ upstream regions has been sequenced. The putative win 3 gene product is similar to the major soluble proteins of sweet potato tubers, sporamin A and sporamin B. Both Win3 and the sporamins share significant amino acid sequence identity with Kunitz-type trypsin inhibitors from legume seeds. The Kunitz family of proteinase inhibitors thus joins three other proteinase inhibitor families which are systemically responsive to wounding.

Molecular and genetic analysis of factors controlling host range in Agrobacterium tumefaciens

SummaryWe have investigated the factors which contribute to the host specificity of a tumor inducing plasmid of Agrobacterium, pTiAg162, which confers a narrow host range. Determinants both within the T-DNA and virulence regions contribute to host specificity. Within the T-DNA a defective cytokinin biosynthetic gene limits host range. Nucleotide sequence analysis revealed a large deletion in the 5′ coding region of this gene when compared with the homologous gene from the wide host range tumor inducing plasmid, pTiA6. Introduction of the wide host range cytokinin biosynthesis gene into the T-DNA of the limited host range strain expanded the host range and suppressed the rooty morphology of tumors incited by the limited host range strain. Two genes from the virulence region of the wide host range plasmid, designated virA and virC, must also be introduced into the limited host range strain in order to restore a wide host range phenotype. The wide host range strain is avirulent on some cultivars of Vitis plants on which the limited host range strain induces tumors. This avirulence is apparently due to a hypersensitive response in which infected plant cells are killed at the site of inoculation. Mutations within the virC locus of the wide host range plasmid prevented the hypersensitive response and allowed the formation of tumors by the wide host range strain.

VirE1 is a specific molecular chaperone for the exported single‐stranded‐DNA‐binding protein VirE2 in Agrobacterium

Agrobacterium tumefaciens induces tumours on plants by transferring a nucleoprotein complex, the T‐complex, from the bacterium to the plant cell. The T‐complex consists of a single‐stranded DNA (ssDNA) segment, the T‐DNA, and VirD2, an endonuclease covalently attached to the 5′ end of the T‐DNA. A type IV secretion system encoded by the virB operon and virD4 is required for the entry of the T‐complex and VirE2, a ssDNA‐binding protein, into plant cells. The VirE1 protein is specifically required for the export of the VirE2 protein, as demonstrated by extracellular complementation and tumour formation. In this report, using a yeast two‐hybrid system, we demonstrated that the VirE1 and VirE2 proteins interact and confirmed this interaction by in vitro binding assays. Although VirE2 is a ssDNA‐binding protein, addition of ssDNA into the binding buffer did not interfere with the interaction of VirE1 and VirE2. VirE2 also interacts with itself, but the interaction between VirE1 and VirE2 is stronger than the VirE2 self‐interaction, as measured in a lacZ reporter gene assay. In addition, the interaction of VirE2 with itself is inhibited by VirE1, indicating that VirE2 binds VirE1 preferentially. Analysis of various virE2 deletions indicated that the VirE1 interaction domain of VirE2 overlaps the VirE2 self‐interaction domain. Incubation of extracts from Escherichia coli overexpressing His‐VirE1 with the extracts of E. coli overexpressing His‐VirE2 increased the yield of His‐VirE2 in the soluble fraction. In a similar purified protein solubility assay, His‐VirE1 increased the amount of His‐VirE2 partitioning into the soluble fraction. In Agrobacterium, VirE2 was undetectable in the soluble protein fraction unless VirE1 was co‐expressed. When urea was added to solubilize any large protein aggregates, a low level of VirE2 was detected. These results indicate that VirE1 prevents VirE2 from aggregating, enhances the stability of VirE2 and, perhaps, maintains VirE2 in an export‐competent state. Analysis of the deduced amino acid sequence of the VirE1 protein revealed that the VirE1 protein shares a number of properties with molecular chaperones that are involved in the transport of specific proteins into animal and plant cells using type III secretion systems. We suggest that VirE1 functions as a specific molecular chaperone for VirE2, the first such chaperone linked to the presumed type IV secretion system.

Foreign DNA sequences in crown gall teratomas and their fate during the loss of the tumorous traits

SummaryA cloned tobacco crown gall teratoma induced by A. tumefaciens strain T37 was found to contain part of the Ti plasmid (T-DNA) of the causative bacterium. The tumor line contained transcripts of the ends of the T-DNA; transcripts of the intervening T-DNA were not detected.When grafted to a healthy tobacco plant, the cloned tumor line produced a plant that flowered and set seed. Tissues from leaves and flower petals when placed in tissue culture resumed a malignant growth pattern and grew in the absence of phytohormones. Both leaf and flower petal tissues were found to contain T-DNA, but in smaller quantity than observed in the parental line. Haploid tissue derived from anther and tissue from F1 progeny plants, which morphologically appear to be completely normal and require phytohormones for growth in vitro, were found to be free from T-DNA. Thus, meiosis acts either to cause or select for loss of the foreign DNA. The correlation between the presence of foreign DNA and the tumor phenotype indicates that the continued presence of T-DNA is required for the maintenance of the tumorous state.

DNA from the A6S/2 crown gall tumor contains scrambled Ti-plasmid sequences near its junctions with plant DNA

The A6S/2 tumor incited on tobacco by Agrobacterium tumefaciens harboring the octopine-type A6 Ti plasmid contains one insert of Ti-plasmid sequences (the T DNA). This 13 kb insert is derived from a colinear sequence in the Ti plasmid (the T region) and becomes attached to plant DNA in the nucleus of the host cell. We have determined the DNA sequence encompassing the left end of the T region of the A6 Ti plasmid and the corresponding portion of the A6S/2 T DNA. The two sequences are identical for at least 806 bp. To the left of the divergence point, the tumor contains five partially overlapping sequences that are direct or inverted repeats of sequences to the right of the divergence point. The Ti plasmid contains only the right member of each of these repeats. We have also performed heteroduplex studies that indicate that this T DNA has a 520 bp inverted repeat of an internal sequence at the right end near its junction with plant DNA. The repeated sequences near the ends of the T DNA resemble the repeats of adenovirus type 12 sequences found near its junction with host DNA. We discuss data suggesting that the 23 bp to the immediate right of the divergence point of the A6 left junction form a site important in some step in the transfer of T-region DNA from the bacteria to the plant.

Regeneration of a transgenic woody legume (Robinia pseudoacacia L., black locust) and morphological alterations induced by Agrobacterium rhizogenes-mediated transformation

Abstract A repeatable and fast transformation-regeneration system has been developed for a woody legume, black locust ( Robinia pseudoacacia L.). Hairy roots were produced from hypocotyl segments inoculated with Agrobacterium rhizogenes R1601, a super-virulent strain with eukaryotic kanamycin resistance gene, in less than 1 week and resulted in subsequent shoot regeneration within 4 weeks. Transgenic black locust trees were established in soil 12 weeks after inoculation with the A. rhizogenes strain. The hairy root phenotype found in regenerated plants, which has been characterized as having wrinkled leaves and abundant root development, became evident after 3 months of growth. Southern blot analysis clearly showed multiple integration of T-DNA (transferred DNA) and the presence of neomycin phosphotransferase (NPTII) gene sequences in the transformed black locust genome.