Leon Otten

Mendelian Transmission of Genes Introduced into Plants by the Ti Plasmids of Agrobacterium tumefaciens

SummaryInsertion of the bacterial transposon Tn7 was used to obtain mutants of an octopine Ti plasmid. Crown gall tumours induced on tobacco by an Agrobacterium tumefaciens strain carrying a particular mutant Ti plasmid (pGV2100) were found to give rise to shoots. These shoots were grown in vitro and one of them (rGV-1) was found to contain the T-DNA specific enzyme lysopine dehydrogenase (LpDH) and to form roots. After transfer to soil, rGV-1 developed into a morphologically and functionally normal tobacco plant. All cells of the regenerant and of vegetatively produced offspring were shown, by cloning of leaf protoplasts, to contain T-DNA and LpDH activity. rGV-1 and vegetatively produced offspring flowered normally. Plantlets obtained from haploid anther cultures were tested for LpDH activity Forty-one percent of these plantlets were LpDH positive. Moreover, both self-pollination of rGV-1 and crosses between rGV-1 and normal tobacco plants showed that the LpDH character was transmitted both through the pollen and through the eggs of rGV-1 as a single dominant factor with Mendelian segregation ratios typical for monohybrid crosses. By repeated selfing, homozygous plants were obtained which bred true with respect to LpDH. The importance of these findings with respect to the use of Agrobacterium tumefaciens and Ti plasmids for genetic engineering in plants is discussed.

Restoration of virulence of Vir region mutants of Agrobacterium tumefaciens strain B6S3 by coinfection with normal and mutant Agrobacterium strains

SummaryThree avirulent Tn7 insertion mutants mapping in the vir E region of the Agrobacterium tumefaciens plasmid pTiB6S3 regain virulence by co-infection with several wildtype strains and with a number of strains carrying mutations in other regions of the Ti plasmid. This finding indicates that during tumour induction normal Agrobacterium strains produce a diffusable factor required for transformation and might allow the isolation of such a factor.

Nuclear and Polysomal Transcripts of T-DNA in Octopine Crown Gall Suspension and Callus-Cultures

SummaryTo establish a detailed map of the transcribed parts of the T-DNA in two octopine crown gall lines grown in suspension culture, T-DNA-derived steady-state nuclear and polysomal RNA as well as RNA synthesized in isolated nuclei purified from the crown gall tissues, was analyzed by Southern blot hybridization to specific fragments of the T-region of the octopine plasmid pTi ACH5. In addition total RNA isolated from the same lines grown as callus tissue on solid agar, was analyzed for T-DNA specific transcripts. The results show that all of the T-DNA is trancribed although different segments are transcribed to significantly different extents. Roughly the same hybridization patterns was found for nuclear and polysomal poly-A+ and poly-A− RNA. The transcription pattern was found to be different for cells in the stationary phase of growth compared with actively growing cells.

Identification of an Agrobacterium tumefaciens pTiB6S3 vir region fragment that enhances the virulence of pTiC58

SummaryThe Agrobacterium tumefaciens octopine strain B6S3 and the nopaline strain C58 were compared for their ability to induce opine synthesis on Kalanchoe daigremontiana stem fragments. Whereas B6S3 induced high levels of octopine synthesis, C58 induced only low levels of nopaline synthesis. However, C58-induced nopaline synthesis was greatly increased by mixed infection with B6S3. This effect (called “helper-effect”) was shown to be due to the activity of a 5 kb fragment from the virulence region of the B6S3 Ti plasmid, since incorporation of this fragment into the C58 plasmid enabled C58 to induce high levels of nopaline synthesis in the absence of a helper strain. The 5kb region contains the vir F locus, as defined earlier (Hooykaas et al. 1984b). A possible correlation between the helper function and vir F is discussed. Our results show that large differences in virulence on particular host plants exist between natural Agrobacterium strains and can be overcome by mixed infections.

Agrobacterium vitis nopaline Ti plasmid pTiAB4: relationship to other Ti plasmids and T-DNA structure

The Ti plasmid of the Agrobacterium vitis nopaline-type strain AB4 was subcloned and mapped. Several regions of the 157 kb Ti plasmid are similar or identical to parts of the A. vitis octopine/cucumopine (o/c)-type Ti plasmids, and other regions are homologous to the nopaline-type Ti plasmid pTiC58. The T-DNA of pTiAB4 is a chimaeric structure of recent origin: the left part is 99.2% homologous to the left part of the TA-DNA of the o/c-type Ti plasmids, while the right part is 97.1 % homologous to the right part of an unusual nopaline T-DNA recently identified in strain 82.139, a biotype Il strain from wild cherry. The 3′ non-coding regions of the ipt genes from pTiAB4 and pTi82.139 are different from those of other ipt genes and contain a 62 by fragment derived from the coding sequence of an ipt gene of unknown origin. A comparison of different ipt gene sequences indicates that the corresponding 62 by sequence within the coding region of the AB4 ipt gene has been modified during the course of its evolution, apparently by sequence transfer from the 62 by sequence in the 3′ non-coding region. In pTi82.139 the original coding region of the ipt gene has remained largely unmodified. The pTiAB4 6b gene differs from its pTi82.139 counterpart by the lack of a 12 by repeat in the 3′ part of the coding sequence. This leads to the loss of four glutamic acid residues from a series of ten. In spite of these differences, the ipt and 6b genes of pTiAB4 are functional. Our results provide new insight into the evolution of Agrobacterium Ti plasmids and confirm the remarkable plasticity of these genetic elements. Possible implications for the study of bacterial phylogeny are discussed.

Plant-Cells Transformed by Modified Ti-Plasmids - a Model System to Study Plant Development

Ti plasmids are harbored by a group of soil bacteria (Agrobacteria) and are responsible for the capacity of these bacteria to induce so-called “crown gall” tumors on most dicotyledonous plants. (For recent reviews see Gordon 1979, Schell et al. 1979, Schilperoort et al. 1979, Van Montagu and Schell 1979, Schell and Van Montagu 1980, Schilperoort et al. 1980, Van Montagu et al. 1980). The plant tumor cells, unlike nontrans-formed plant tissues, can be cultured under axenic conditions on synthetic media in the absence of growth hormones, i.e. cytokinins and auxins (Braun 1956); the tumor cells also produce low molecular weight compounds called opines not found in untransformed plant tissues. The type of opine produced defines crown galls as octopine, nopaline, or agropine type tumors (Guyon et al. 1980).

Rapid divergence of Agrobacterium vitis octopine-cucumopine Ti plasmids from a recent common ancestor

The octopine/cucumopine (o/c) Ti plasmids of the grapevine-associated Agrobacterium vitis strains constitute a family of related DNA molecules. Restriction maps were established of two limited-host-range o/c Ti plasmids, pTiAg57 and pTiAB3, and of the wide-host-range o/c Ti plasmid pTiHml. Together with the previously obtained map of the wide-host-range o/c Ti plasmid pTiTm4, about 1000 kb were mapped with a resolution of 0.2 kb, allowing a detailed comparison of the various structures. One region of the o/c Ti plasmids is highly conserved and differs mainly by the presence or absence of relatively small DNA fragments (0.9–2.7 kb); the other region has been modified more extensively and carries large sequences specific for each Ti plasmid type. The sequence similarity within large conserved regions shows that these plasmids have diverged recently and that their evolution was driven by large-scale genetic events rather than single nucleotide changes. These results have important implications for studies on bacterial evolution.

Ti Plasmids and Directed Genetic Engineering

Publisher Summary This chapter discusses Ti plasmids and directed genetic engineering. Intermediate vectors with which a specific DNA sequence can be introduced at a predetermined site in the T region of a complete Ti plasmid have been applied successfully. Their use is somewhat cumbersome, because it is difficult to work with DNA sequences that are not linked to a genetic marker with which the necessary recombinational events can be monitored. A vector would be able to function after it is introduced directly into plant protoplasts by liposome or protoplast fusion, by PEG treatment, or by Agrobacterium strains containing a resident Ti plasmid providing in trans the missing Onc functions. Introduction of plasmid DNA in Agrobacteria can be achieved by transformation. The T-region vector plasmid can be constructed such that it will replicate both in E. Coli and in Agrobacterium . An alternative approach was to introduce genes at specified sites in the T region of a functional Ti plasmid. The principle is to make an intermediate vector consisting of a common E. coli cloning vehicle. This intermediate vector is introduced subsequently, via mobilization, into an A. tumefaciens strain carrying a Ti plasmid that has been made constitutive for transfer and that already carries antibiotic resistance markers.

Ti-Plasmids: Genetic Engineering of Plants

Crown gall is a neoplastic disease of most dicotyledonous plants and is caused by the soil bacterium Agrobacterium tumefaciens. A large extra-chromosomal plasmid in these bacteria was found to be responsible for its tumor-inducing capacity and was, therefore, called Ti-plasmid (1). Bacteria-free crown gall cells can be cultured in the absence of phytohormones and this hormone-independent growth defines tumor cells in plants (2). Sterile tumor tissues have been shown to contain a DNA segment (called T-DNA) which is homologous and colinear with a defined fragment of the Ti-plasmid, and it is covalently linked to plant DNA (3–9). The T-DNA has been localized in the nucleus (10, 11) and is directly responsible for the hormone-independent growth of the tumor cells. It is also responsible for the synthesis of low molecular weight compounds, called opines, which are not found in normal plant tissue. The opine produced defines crown galls as octopine, nopaline or agropine type tumors (12). Opines can be utilized by A. tumefaciens selectively as sources for carbon, nitrogen and energy, and, thus, the interaction between these bacteria and plants can be seen as a special parasitic relationship which benefits the bacteria (4).

Regeneration of Plants from Crown Gall Cells A step in the T-DNA mediated genetic engineering of plants

The mechanism whereby the plant pathogenic soil bacterium Ag-robacterium tumefaciens induces Crown Gall plant tumors is the result of a remarkable example of naturally occurring genetic engineering. A fragment of about 20 kb (called the T-region) of a 120 kb plasmid (called Ti plasmid for tumor-inducing plasmid) is transferred to the plant cell where it becomes covalently linked to the nuclear DNA (1–7). This integrated DNA has been named the T-DNA. Crown Gall cells have two distinctive properties: They grow without hormones (8,9) and they produce so-called opines: compounds not found in normal plant cells and used specifically by Agrobacterium tumefaciens as a carbon, nitrogen and energy source with the help of a Ti plasmid-encoded uptake and degradation system (10–13). Opines moreover have the capacity to induce a plasmid transfer system which can spread the opine degradation system through an Agrobacterium population (14,15). For recent reviews see 3, 16–20.