Sequence context of the T-DNA border repeat element determines its relative activity during T-DNA transfer to plant cells

Summary We present a detailed analysis of the function of the right and left T-DNA border regions of the nopaline Ti plasmid of Agrobacterium tumefaciens. An avirulent deletion of the right border of the nopaline Ti plasmid (pGV3852) was used as an acceptor for 14 different T-DNA border constructs. The functional activities of these constructs were assayed by their ability to restore virulence, i.e. transformation on inoculated plants. Tumorigenicities were measured in several independent experiments over a 2 year period and the statistical significance of their relative levels was evaluated. The data indicate: (i) the entire sequence of the 25 bp direct repeat of the T-DNA is required to provide an efficient substrate for mediating T-DNA transfer events; deletion derivatives of either the conserved or the vaiable domain of the repeat are defective in T-DNA transfer; (ii) while the 25 bp direct repeat alone can promote the T-DNA transfer, the flanking sequences of the repeats enhance (on the right) or attenuate (on the left) their activity; and (iii) tumorigenicity measurements vary depending on the plant assay system: potato discs are more sensitive than wounded tobacco leaves in detecting differences in T-DNA border activity.     

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

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. WhenAgrobacterium is incubated with protoplasts or seedlings of dicotyledonous plants, circulation of T-DNA and expression ofvir (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 thevirD locus, one of the induciblevir 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 byAgrobacterium. We showed that monocotyledonous plants do not excrete diffusible inducers for the expression ofvir genes, while they 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 whenAgrobacterium invades seedlings and seeds of monocotyledonous plants. Recipient of the Botanical Society Award for Young Scientists, 1987.     

Stable expression of the GUS reporter gene in chrysanthemum depends on binary plasmid T-DNA

Three chrysanthemum (Dendranthema grandiflora) cultivars were cocultivated with 2 Agrobacterium tumefaciens strains in combination with 4 pBIN19 derived binary plasmids, all carrying the Nosnptll selection gene and 35Sgus(intron) reporter gene. All binary plasmids transferred DNA to chrysanthemum explants but only pMOG410 gave good stable expression of GUS. This plasmid differs from the other plasmids in 2 aspects: 1) It carries a restored nptll gene and 2) the selection gene is positioned at the left border side of the reporter gene. Cocultivation with AGLO(pMOG410) yielded up to 13 GUS positive shoots per 100 explants. The presence of the gus and nptll gene in recovered shoots was confirmed by PCR and Southern blot analysis.     

Multiple transformation of plant cells by Agrobacterium may be responsible for the complex organization of T-DNA in crown gall and hairy root

Summary Inoculation of carrot discs and Lotus corniculatus plantlets with mixtures of different Agrobacterium rhizogenes or of A. rhizogenes and A. tumefaciens or with Agrobacterium strains harboring both an Ri and a modified Ti plasmid resulted in frequent multiple (pluribacterial) transformation of cells, as revealed by the mixed opine-type of hairy roots arising from them. Multiple transformation may account for the presence of dispersed T-DNA inserts in crown gall and hairy root lines. A plant genetic engineering strategy based on segregation of T-DNA inserts in the progeny of multiple transformants is proposed.     

Phytohormones in the formation of crown gall tumors

Crown gall tumors were initiated in a variety of plant species by infection with Agrobacterium tumefaciens strain B6 and the concomitant changes in the tissue levels of phytohormones, mainly indole-3-acetic acid (IAA) and cytokinins, were analyzed. A comparison was made of these hormones with those produced by virulent and avirulent strains of the bacterium in liquid culture and with those of bacteria-free crown gall callus cultures. Specific radioimmunoassays were employed for hormone determinations. An assay for the quantitation of femto-mol amounts of isopentenyladenosine and related cytokinins was newly developed and is described in detail. The results can be summarized as follows: Virulence in strain B 6 is associated with the ability to release trans-zeatin and increased amounts of IAA into the surrounding environment. In many, but not all plants analyzed, the development of crown gall tumors is also associated with a sharp rise in the levels of trans-zeatin-type zytokinins and IAA (e.g., Euphorbia lathyris, Catharanthus roseus). Crown gall calli growing on hormone-free media varied greatly in their cytokinin levels. In a culture of Nicotiana tabacum, both trans-zeatin and isopentenyladenine or related cytokinins were not detected. Thus, tumor growth cannot be explained on the basis of elevated levels of IAA and/or cytokinins alone.Abbreviations ABA abscisic acid - GC-MS gas chromatography-mass spectroscopy - HPLC high pressure liquid chromatography - IAA indole-3-acetic acid - RIA radioimmunoassay - TLC thin layer chromatography Part 19 in the series Use of immunoassay in plant science     

Clones from a shooty tobacco crown gall tumor II: irregular T-DNA structures and organization,T-DNA methylation and conditional expression of opine genes

Summary Transformed clones from a shooty tobacco crown gall tumor, induced byAgrobacterium tumefaciens strain LBA1501, having the auxin locus of the TL-region inactivated by a Tn1831 insertion, were investigated for their T-DNA structure and expression. It has been described previously (28) that in addition to clones with an expected phenotype (phytohormone independent growth in tissue culture (Aut⁺), shoot regeneration (Reg⁺) and octopine synthesis (Ocs⁺)), clones were obtained with an aberrant phenotype. One of these clones, TSO38, is Aut⁺Reg⁺ but shows little or no octopine synthesis activity (Ocs^-). Subclones of TSO38, however, are either Ocs^- or Ocs⁺. Ocs^- shoots become Ocs⁺ under certain states of differentiation, indicating that the octopine synthase gene is present. The fact that in the Ocs^- subclones the octopine synthase gene is not expressed, is probably due to DNA methylation (29). The present paper describes that shoots derived from both an Ocs⁺ and an Ocs^- subclone of TSO38, which were negative for the presence of mannopine (Mas^-) and agropine (Ags^-), became Mas⁺Ags⁺ after culturing on medium containing the hypomethylating agent 5-azacytidine. This means that both in the Ocs^- line and in the Ocs⁺ line expression of TR-DNA opine genes most likely was hampered by DNA methylation. The T-DNA structures of an Ocs^- and an Ocs⁺ TSO38 subclone proved to be identical and surprisingly complex. No intact copy of Tn1831 was present. TL-DNA and TR-DNA segments, present in high copy numbers, were truncated; several T-DNA segments existed in tandem arrangements. When DNA from an Ocs⁺ and an Ocs^- subclone of TSO38 were compared for cleavability by the methylation sensitive restriction enzymes HpaII and MspII, differences were detected, but it became also clear that both lines contained methylated T-DNA segments. This indicates that the Ocs^- and the Ocs⁺ TSO38 subclones differ only quantitatively in respect to degree of T-DNA methylation.     

T-DNA rearrangements due to tissue culture: somaclonal variation in crown gall tissues

After three years of apparent stability in tissue culture, the single cell derived shooty crown gall line sNT1.013 produced a revertant shoot which had switched from non-rooting (Rod⁺) and octopine synthesizing (Ocs⁺) to Rod^- Ocs^-, indicating that in this revertant TL-DNA genes 4 (causing the Rod⁺ trait) and gene 3 (causing the Ocs⁺ trait) had been inactivated. Southern blots revealed that the inactivation of these T-DNA genes was the result of a considerable rearrangement of DNA sequences, accompanied by deletions and possibly also by DNA amplifications. This study for the first time unambiguously proves that foreign genes which have been introduced via Agrobacterium tumefaciens can, at a low frequency, be inactivated after T-DNA integration because of reorganization of T-DNA sequences during tissue culture. This can be considered as an event of somaclonal variation.     

Plant-inducible recombination between the 25 bp border sequences of T-DNA in Agrobacterium tumefaciens

Summary We developed a model system for detecting and assaying the circular forms of T-DNA which may be generated in Agrobacterium by intramolecular recombination between the 25 bp border repeats of T-DNA. We demonstrated using this system that the DNA region flanked by the 25 bp direct repeats is in fact circularized by recombination between these repeats in cells of Agrobacterium cocultured with tobacco protoplasts. Furthermore, quantitative analysis of the recombination revealed the following: (1) the recombination is also induced when the agrobacterial cells are incubated in protoplast-free conditioned medium prepared by filtering the protoplast culture. The conditioned medium is effective, even after it has been heated at 100°C. (2) The DNA region encompassing the virulence region of the Ti-plasmid is required for recombination. (3) The recombination takes place only between 25 bp repeats with the same orientation. On the basis of these results, we conclude that the circular form of T-DNA is generated by homologous recombination between the border repeats which is mediated by gene product(s) encoded by the virulence region of the Ti-plasmid. Either the recombination itself, or the expression of the virulence gene(s) responsible for the recombination, is induced by diffusible and heatstable factor(s) secreted by plant cells.     

A T-DNA transfer stimulator sequence in the vicinity of the right border of pRi8196

An 8 bp sequence repeated 6 times is present to the right of the mannopine type pRi8196 T-DNA righ-border sequence. Experiments were designed to test whether these repeats have a role in T-DNA transfer. Several constructs in which different lengths of pRi8196 right-border region were linked to the cucumopine synthesis gene on anAgrobacterium-Escherichia coli shuttle vector were made. The recombinant plasmids were tested for their efficiency to act as a source of T-DNA in a binary system in which a wild-type Ri plasmid provided virulence and root-inducing functions. The T-DNA transfer efficiency of the constructs was assessed by computing the relative frequency of roots containing cucumopine. Depending on the Ri plasmid used as source of virulence functions, a high level of T-DNA transfer was observed only if 6 (pRi8196) or 5 (pRiA4) repeats were present. These results were confirmed by looking for single-stranded T-DNA molecules (T-strands) in bacteria induced for virulence. The repetition of the 8 bp unit was named T-DNA transfer stimulator sequence (TSS).     

Insights into recognition of the T-DNA border repeats as termination sites for T-strand synthesis by Agrobacterium tumefaciens

The recognition of the T-DNA left border (LB) repeat is affected by its surrounding sequences. Here, the LB regions were further characterized by molecular analysis of transgenic plants, obtained after Agrobacterium tumefaciens-mediated transformation with T-DNA vectors that had been modified in this LB region. At least the 24-bp LB repeat by itself was insufficient to terminate the T-strand synthesis. Addition of the natural inner and/or outer border regions to at least the LB repeat, even when present at a distance, enhanced the correct recognition of the LB repeat, reducing the number of plants containing vector backbone sequences. In tandem occurrence of both the octopine and nopaline LB regions with their repeats terminated the T-strand synthesis most efficiently at the LB, yielding a reproducibly high number of plants containing only the T-DNA. Furthermore, T-strand synthesis did not terminate efficiently at the right border (RB) repeat, which might indicate that signals in the outer RB region inhibit the termination of T-strand synthesis at the RB repeat.     

Conserved regions in the T-DNA of different Agrobacterium rhizogenes root-inducing plasmids

The T-regions of the three so far identified types of Ri plasmids-corresponding to the synthesis of three different hairy root opines, agropine, mannopine and cucumopine-have been compared in detail by Southern blot cross hybridizations. Two distinct zones of very strong sequence homology, approximately 4 and 3 kilobases in length respectively, have been identified in all three T-regions. The highly conserved sequences, not present in Ti plasmid T-DNA, may encode essential rhizogenic functions common to all Agrobacterium rhizogenes T-DNAs.     

Absence in monocotyledonous plants of the diffusible plant factors inducing T-DNA circularization and vir gene expression in Agrobacterium

Summary T-DNA circularization is one of the molecular events specifically induced in agrobacterial cells upon their infection of dicotyledonous plant cells. We developed a seedling co-cultivation procedure to determine whether or not monocotyledonous plants have the ability to induce T-DNA circularization and vir gene expression. Co-cultivation of Agrobacterium tumefaciens with seedlings of dicotyledonous plants showed that the circularization event takes place efficiently. The exudates and extracts of the seedlings also effectively induced T-DNA circularization and vir gene expression, indicating that dicotyledonous seedlings contain diffusible factors capable of inducing these molecular events. In contrast, neither T-DNA circularization nor vir gene expression was detectable when Agrobacterium was incubated with seedlings of monocotyledonous plants. Supplementing with acetosyringone, a known inducer of vir gene expression and T-DNA circularization, resulted in the induction of circularization during co-cultivation with monocotyledonous seedlings. These results indicate that the seedlings of monocotyledonous plants have no detectable amounts of diffusible inducers, unlike dicotyledonous seedlings. Therefore, it is unlikely that the vir genes are expressed in Agrobacterium inoculated in monocotyledonous plants. This may be one of the blocks in tumorigenesis of monocotyledonous plants by Agrobacterium.     

New plant binary vectors with selectable markers located proximal to the left T-DNA border

Five new binary vectors have been constructed which have the following features: (1) different plant selectable markers including neomycin phosphotransferase (nptII), hygromycin phosphotransferase (hpt), dihydrofolate reductase (dhfr), phosphinothricin acetyl transferase (bar), and bleomycin resistance (ble); (2) selectable markers are located near the T-DNA left border and; (3) selectable marker and -glucuronidase (uidA) reporter genes are divergently organized for efficient expression, and can easily be removed or replaced as needed.     

Neoplastic progression in crown gall in tobacco without elevated auxin levels

We have isolated two stable variants from a crown-gall teratoma tissue of tobacco (Nicotiana tabacum L.) transformed by Agrobacterium tumefaciens strain A66, a mutant of the virulent A6 strain containing an insertion sequence in the tumor-inducing (Ti) plasmid at the locus coding for auxin biosynthesis. Normally tobacco cells transformed by strain A66 spontaneously form shoots in culture and will not grow on hormone-free medium unless shoots develop. The variant tissue lines, isolated from the teratoma tissue after prolonged culture in the dark, grew as friable and unorganized tissues on hormone-free growth medium. Growth of the variants was more sensitive to auxin feeding than growth of the parental teratoma line, and the auxin dose-response curves of the variant lines were similar to those obtained with A6-transformed tobacco cells. Southern blot analysis of DNA from the parental teratoma line and one of the variants showed no differences in copy number or organization of the oncogenic DNA sequence (T-DNA) transferred from the bacterium, indicating that the variant phenotype did not result from reversion of the A66 mutation. Radio-immunoassay analysis showed similar levels of indole-3-acetic acid (IAA) in the variants and parental teratoma line (3–50 and 38–42 pmol·(gFW)^-1, respectively), whereas an A6-transformed cell line contained much higher IAA levels (150–1200 pmol·(g FW)^-1). Low levels of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid in the variants and the parental teratoma line (<5 nmol·(g FW)^-1) as compared with that found in the A6-transformed line (>100 nmol· (g FW)^-1) provided additional, indirect evidence for low auxin levels in the variant lines. These results indicate that crown-gall teratoma tissues of tobacco may switch to the unorganized, auxin-sensitive phenotype without an increase in auxin content.Abbreviations IAA indole-3-acetic acid - kb kilobase - NAA -naphthalene acetic acid - NAM -naphthaleneacetamide - T-DNA DNA transferred from the Ti plasmid to the plant - T_L-DNA the left transferred region of pTiA6 containing the T-DNA oncogenes     

Deviating T-DNA transfer fromAgrobacterium tumefaciens to plants

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.