Transfer of T-DNA from Agrobacterium to the Plant Cell' (original) (raw)

Delivery of T-DNA from the Agrobacterium tumefaciens chromosome into plant cells

The EMBO journal, 1984

The intact T-region of the B6Ti plasmid of Agrobacterium tumefaciens was stepwise cloned into a site in transposon Tn3. In this way a suitable vehicle (Tn1882) was obtained for translocating the T-region to different replicons, i.e., to other plasmids or the chromosome. The IncP plasmid R772::Tn1882 conferred tumorigenicity on Agrobacterium if the virulence genes were provided in trans in the same cell. This result showed that the T-region present on Tn1882 was transferred efficiently to plant cells. Normal tumor development also occurred if the T-region was placed in the chromosome of A. tumefaciens and an R' plasmid was present carrying virA-E or virA-F. We conclude that the plasmid location of the T-region is not a prerequisite for transfer to the plant cell. The apparently normal delivery of the T-DNA from a bacterial chromosomal location supports a model involving a processing step within Agrobacterium effecting transfer of the T-region as a separate entity.

Interactions and DNA transfer between Agrobacterium tumefaciens , the Ti-plasmid and the plant host

Proceedings of the Royal Society of London. Series B. Biological Sciences, 1979

Agrobacterium tumefaciens is a gram-negative bacterium with the unique capacity to induce neoplasmic transformations in dicotyledonous plants. Recently, both the mechanism and the biological significance of this transformation have been elucidated. Agrobacterium tumefaciens strains contain a large extrachromosomal DNA plasmid (the Ti-plasmid). This Ti-plasmid is responsible for the oncogenic properties of Agrobacterium strains. A particular segment of the Ti-plasmid, containing information determining the tumorous growth pattern and the synthesis of so-called ‘opines’, e. g. octopine ( N-α -(D-l-carboxyethyl)-L-arginine) and nopaline ( N-α -(l, 3-dicarboxypropyl)-L-arginine), is transferred and stably main­tained and expressed in the transformed plant cells. This phenomenon can be understood as a ‘genetic colonization’ of the plant cells by bacterial plasmid DNA so that the transformed plant cells will produce and secrete into the medium amino acid derivatives (the opines) that Ti-p...

Deviating T-DNA transfer fromAgrobacterium tumefaciens to plants

Plant Molecular Biology, 1996

We analyzed 29 T-DNA inserts in transgenicArabidopsis thaliana plants for the junction of the right border sequences and the flanking plant DNA. DNA sequencing showed that in most lines the right border sequences transferred had been preserved during integration, corroborating literature data. Surprisingly, in four independent transgenic lines a complete right border repeat was present followed by binary vector sequences. Cloning of two of these T-DNA inserts by plasmid rescue showed that in these lines the transferred DNA consisted of the complete binary vector sequences in addition to the T-region. On the basis of the structure of the transferred DNA we propose that in these lines T-DNA transfer started at the left-border repeat, continued through the vector part, passed the right border repeat, and ended only after reaching again this left-border repeat.

Association of single-stranded transferred DNA from Agrobacterium tumefaciens with tobacco cells

Proceedings of the National Academy of Sciences, 1994

During the inception of crown gall tumorigenesis, the traferred DNA (T-DNA) is processed from the Ti (tumor Indc n)pl d of Agrobacterium twfaciens and is transferred to plant cells. T-DNA processing and tr er require the induction of vir (virulence) genes by phenolic compounds se by wounded plant cells. After vir gene induction, both single-stranded (T-strands) and doublestranded forms of proessd T-DNA ac Late in the bacteria. Although current models favor the trnser of T-strands to plants, there has yet been no experinental evidence to show this. In this paper, we show that T-strands disappear from acetosyringone-induced A. twmefaciens within 30 min of bacterial cocultivation with tobacco protoplasts. PCR analysis of T-DNA associated with protoplasts indicates that singlestranded, but not double-stranded, T-DNA can be detected in the plant cells within 30 min of bacterial cocultivation. Control

Mechanisms of crown gall formation: T-DNA transfer fromAgrobacterium tumefaciens to plant cells

The Botanical Magazine Tokyo, 1989

Agrobacterium tumefaciens harbouring the Ti plasmid incites crown gall tumor on dicotyledonous species. Upon infection of these plants, T-DNA in the Ti plasmid is transferred by unknown mechanisms to plant cells to be integrated into nuclear DNA. When Agrobacterium is incubated with protoplasts or seedlings of dicotyledonous plants, circularization of T-DNA and expression of vir (virulence) genes on the Ti plasmid are induced. The circularization event is efficiently induced by mesophyll protoplasts of tobacco which are highly competent for transformation by the T-DNA, and is also induced by diffusible phenolic compounds excreted from the protoplasts. The circularization and formation of crown gall both require the expression of the v/rD locus, one of the inducible vir genes. These results suggest that the circularization of T-DNA reflects one of steps of the T-DNA transfer during formation of crown gall. In contrast to dicotyledonous plants, monocotyledonous plants are thought to be unresponsive to infection by Agrobacterium. We showed that monocotyledonous plants do not excrete diffusible inducers for the expression of v/r genes, while the); contain a novel type of a signal substance(s). This inducer is not detected in the exudates of seedlings of monocotyledonous plants, but is found in the extracts from the seedlings, and also those from the seeds, bran and germ of wheat and oats. This finding suggests that T-DNA processing, and possibly its transfer, should take place when Agrobacterium invades seedlings and seeds of monocotyledonous plants.

New Approaches to Agrobacterium tumefaciens-Mediated Gene Transfer to Plants

Genetic Engineering - An Insight into the Strategies and Applications, 2016

Agrobacterium tumefaciens, a plant pathogen, is commonly used as a vector for the introduction of foreign genes into plants and consequent regeneration of transgenic plants. A. tumefaciens naturally infects the wound sites in dicotyledonous plants and induces diseases known as crown gall. The bacterium has a large plasmid that induces tumor induction, and for this reason, it was named tumor-inducing (Ti) plasmid. The expression of T-DNA genes of Ti-plasmid in plant cells causes the formation of tumors at the infection site. The molecular basis of Agrobacterium-mediated transformation is the stable integration of a DNA sequence (T-DNA) from Ti (tumor-inducing) plasmid of A. tumefaciens into the plant genome. A. tumefaciens-mediated transformation has some advantages compared with direct gene transfer methods such as integration of low copy number of T-DNA into plant genome, stable gene expression, and transformation of large size DNA segments. That is why manipulations of the plant, bacteria and physical conditions have been applied to increase the virulence of bacteria and to increase the transformation efficiency. Preculturing explants before inoculation, modification of temperature and medium pH, addition chemicals to inoculation medium such as acetosyringone, changing bacterial density, and cocultivation period, and vacuum infiltration have been reported to increase transformation. In this chapter, four new transformation protocols that can be used to increase the transformation efficiency via A. tumefaciens in most plant species are described.

Formation of complex extrachromosomal T-DNA structures in Agrobacterium tumefaciens-infected plants

Plant physiology, 2012

Agrobacterium tumefaciens is a unique plant pathogenic bacterium renowned for its ability to transform plants. The integration of transferred DNA (T-DNA) and the formation of complex insertions in the genome of transgenic plants during A. tumefaciensmediated transformation are still poorly understood. Here, we show that complex extrachromosomal T-DNA structures form in A. tumefaciens-infected plants immediately after infection. Furthermore, these extrachromosomal complex DNA molecules can circularize in planta. We recovered circular T-DNA molecules (T-circles) using a novel plasmid-rescue method. Sequencing analysis of the T-circles revealed patterns similar to the insertion patterns commonly found in transgenic plants. The patterns include illegitimate DNA end joining, T-DNA truncations, T-DNA repeats, binary vector sequences, and other unknown "filler" sequences. Our data suggest that prior to T-DNA integration, a transferred single-stranded T-DNA is converted into a doublestranded form. We propose that termini of linear double-stranded T-DNAs are recognized and repaired by the plant's DNA double-strand break-repair machinery. This can lead to circularization, integration, or the formation of extrachromosomal complex T-DNA structures that subsequently may integrate.

Intracellular Agrobacterium can Transfer DNA to the Cell Nucleus of the Host Plant

Proceedings of The National Academy of Sciences, 1995

Agrobacterium tumefaciens is a Gramnegative, soil-borne bacterium responsible for the crown gall disease ofplants. The galls result from genetic transformation of plant cells by the bacteria. Genes located on the transferred DNA (T-DNA), which is part of the large tumor-inducing (Ti) plasmid of Agrobacterium, are integrated into host plant chromosomes and expressed. This transfer requires virulence