Transformation of vitis tissue by different strains of Agrobacterium tumefaciens containing the T-6b gene

Summary Stem pieces and leaf disks of Vitis spp. were cocultured with Agrobacterium tumefaciens strains carrying the UidA (ß-glucuronidase = GUS) gene. The transformation efficiency was highly increased by using a modified T-6b gene (a gene from pTiTm4) which interferes with normal growth and allows regeneration of normal Nicotiana rustica plants (Tinland 1990). The strains first tested on stem segments were subsequently tested in a leaf explant system. On leaves the transformation efficiency of the strains was much lower than with stems. Both the T-6b gene and the hsp 70-T-6b gene (a modified T-6b gene under the control of a heat shock promoter) allowed the initiation of GUS-positive buds.Abbreviations GUS ß-glucuronidase - BAP benzylaminopurine - X-gluc 5-bromo-4-chloro-3-indolyl glucuronide     

The Agrobacterium oncogene AB-6b causes a graft-transmissible enation syndrome in tobacco

Agrobacterium 6b oncogenes induce tumours and modify plant growth in various ways. Here we show that the AB-6b gene from strain AB4 placed under 2x35S promoter control (2x35S-AB-6b) induces a complex enation syndrome in transgenic Nicotiana tabacum plants, that also occurs in a few rare cases of genetic enations. In Arabidopsis thaliana, 2x35S-AB-6b induced radially symmetrical tubes on the abaxial side of the leaves, which must therefore be considered as the Arabidopsis equivalents of enations on other plant species. Tobacco and Arabidopsis 2x35S-AB-6b leaves contained small, supernumerary densely packed cells between the spongy mesophyll and the abaxial epidermis, close to vascular strands arising at an early stage of leaf development. On tobacco, the 2x35S-AB-6b enation syndrome could be transmitted across graft junctions to growing tissues of untransformed plants, both acropetally and basipetally. We propose that the AB-6b gene encodes the synthesis of one or more enation factor(s) that are transported by the phloem and modify the growth of developing tissues.     

New plant growth-modifying properties of the Agrobacterium T-6b oncogene revealed by the use of a dexamethasone-inducible promoter

Agrobacterium 6b oncogenes induce tumours on Nicotiana glauca and enations and associated modifications in transgenic N. tabacum plants. 2x35S-AB-6b tobacco rootstocks produced a graft-transmissible factor that induced enations in wild-type scions; the nature of this enation factor remains to be identified. Here, we report on the properties of tobacco plants carrying a dexamethasone-inducible T-6b gene (dex-T-6b). Induction with dex led to complex growth modifications, many of which have not been reported previously. Modifications were only found in growing tissues; mature tissues remained unaffected. Growth could be either stimulated or inhibited. Dex induction of young plants led to morphogenetic gradients that included enations, tubular leaves and fragmented leaf primordia. Root elongation was increased or slowed down, while radial root growth was strongly enhanced. Additional cell divisions were found in the root pericycle and vasculature. Enation factor import from mature tissues did not have the same effects on growing tissues as local T-6b synthesis: normal scions grafted on induced dex-T-6b rootstocks formed enations, whereas local dex-T-6b induction at the shoot apex led to numerous dark-green spots on the abaxial side of the leaves. In leaf patch assays, the 23-kDa T-6b protein was found to move through leaves and to enter the vascular system. This and the fact that rootstocks of spontaneous tobacco enation mutants did not modify wild-type scions contrary to 6b plants indicate that the 6b protein might be the enation factor.     

The Agrobacterium tumefaciens T-DNA gene 6b is an onc gene

In this article it is shown that the T-DNA of Agrobacterium tumefaciens contains besides the well-known cyt and aux genes another gene with an oncogenic effect in plants. The gene in question is called 6^b and causes the formation of small tumors in plant species such as Nicotiana glauca and Kalanchoe tubiflora.     

Expression of a chimaeric heat-shock-inducible Agrobacterium 6b oncogene in Nicotiana rustica

The T-6b gene of Agrobacterium tumefaciens strain Tm4 induces tumours on Nicotiana rustica by an as yet unknown mechanism. These tumours cannot be regenerated into normal plants. To study the effect of the T-6b gene product on normal plant cells, the T-6b gene was placed under control of the Drosophila melanogaster hsp70 heat-shock promoter and introduced into N. rustica. Progeny of an hsp70-T-6b transformant developed into normal plants. The inducibility of the hsp70-T-6b construct was shown by northern analysis and by heat-shock-dependent growth alterations on the level of whole seedlings. Upon wounding at normal temperature conditions hsp70-T-6b plants formed small tumours on leaves and stems. Grafts between transformed plants and normal plants led to a wound callus which remained limited to transformed tissues, indicating that the T-6b gene product does not diffuse. Protoplasts of hsp70-T-6b plants divided in the same way as control protoplasts under standard culture conditions. However, when protoplast cultures were started in the absence of hormones, normal cells rapidly lost their sensitivity towards hormones, whereas hsp70-T-6b cells remained sensitive for a significantly longer period. Thus, the T-6b gene product alters hormone sensitivity during the initial phases of protoplast culture.     

Abnormal accumulation of sugars and phenolics in tobacco roots expressing the Agrobacterium T-6b oncogene and the role of these compounds in 6b-induced growth

The Agrobacterium T-DNA oncogene 6b induces tumors and modifies the growth of transgenic plants by an unknown mechanism. We have investigated changes in roots of tobacco seedlings that express a dexamethasone-inducible T-6b (dex-T-6b) gene. On induction medium with sucrose, intact or isolated dex-T-6b roots accumulated sucrose, glucose, and fructose and changed their growth, contrary to noninduced roots. Root fragments bridging agar blocks with or without sucrose accumulated sugars at the site of sucrose uptake, resulting in local growth. Induced root fragments showed enhanced uptake of 14C-labeled sucrose, glucose, and fructose. When seedlings were placed on sucrose-free induction medium, sugar levels strongly decreased in roots and increased in cotyledons. Collectively, these results demonstrate that 6b stimulates sugar uptake and retention with drastic effects on growth. Apart from sugars, phenolic compounds also have been found to accumulate in 6b tissues and have been proposed earlier to play a role in 6b-induced growth. Induced dex-T-6b roots accumulated high levels of 5-caffeoylquinic acid (or chlorogenic acid [CGA]), but only under conditions where endogenous sugars increased. Inhibition of phenylalanine ammonia-lyase with the competitive inhibitor 2-aminoindan-2-phosphonic acid (AIP) abolished CGA accumulation without modifying sugar accumulation or affecting the 6b phenotype. We conclude that the absorption, retention, and abnormal accumulation of sugars are essential factors in 6b-induced growth changes, whereas phenylpropanoids only marginally contribute to the 6b seedling phenotype.     

Transfer of T-DNA and Vir proteins to plant cells by Agrobacterium tumefaciens induces expression of host genes involved in mediating transformation and suppresses host defense gene expression

Agrobacterium tumefaciens is a plant pathogen that incites crown gall tumors by transferring to and expressing a portion of a resident plasmid in plant cells. Currently, little is known about the host response to Agrobacterium infection. Using suppressive subtractive hybridization and DNA macroarrays, we identified numerous plant genes that are differentially expressed during early stages of Agrobacterium-mediated transformation. Expression profiling indicates that Agrobacterium infection induces plant genes necessary for the transformation process while simultaneously repressing host defense response genes, thus indicating successful utilization of existing host cellular machinery for genetic transformation purposes. A comparison of plant responses to different strains of Agrobacterium indicates that transfer of both T-DNA and Vir proteins modulates the expression of host genes during the transformation process.     

Reduction of polar auxin transport in tobacco by the tumorigenic Agrobacterium tumefaciens AK-6b gene

The plant-tumorigenic 6b (AK-6b) gene of Agrobacterium tumefaciens strain AKE10 induces morphological alterations to tobacco plants, Nicotiana tabacum. To investigate the molecular mechanisms underlying these processes, we generated transgenic tobacco harboring the AK-6b gene under the control of a dexamethazone-inducible promoter. Upon induction, transgenic tobacco seedlings exhibited distinct classes of aberrant morphologies, most notably adventitious outgrowths and stunted epicotyls. Histological analysis revealed massive proliferation and altered venation in the newly established outgrowths. Prominent vascular development suggested that auxin metabolism or signaling had been altered. Indeed, basipetal auxin transport in the hypocotyls of the transgenic seedlings was reduced by 50–80%, whereas intracellular auxin contents were only slightly reduced. Analysis of cell extracts by HPLC revealed a large accumulation of phenolic compounds, including the flavonoid kaempferol-3-rutinoside, in transgenic plants compared with wild-type seedlings. As some naturally occurring flavonoids have been shown to affect auxin transport, we suggest that the AK-6b gene expression impairs auxin transport via modulation of phenylpropanoid metabolism, and ultimately results in the observed morphological alterations. Electronic Supplementary Material Supplementary material is available for this article at     

Interaction between <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis> oncoprotein 6b and a tobacco nucleolar protein that is homologous to TNP1 encoded by a transposable element of <Emphasis Type="Italic">Antirrhinum majus</Emphasis>

When gene 6b on the T-DNA of Agrobacterium tumefaciens is transferred to plant cells, its expression causes plant hormone-independent division of cells in in vitro culture and abnormal cell growth, which induces various morphological defects in 6b-expressing transgenic Arabidopsis thaliana and Nicotiana tabacum plants. Protein 6b localizes to the nuclei, a requirement for the abnormal cell growth, and binds to a tobacco nuclear protein called NtSIP1 and histone H3. In addition, 6b has histone chaperone-like activity in vitro and affects the expression of various plant genes, including cell division-related genes and meristem-related class 1 KNOX homeobox genes, in transgenic Arabidopsis. Here, we report that 6b binds to a newly identified protein NtSIP2, whose amino acid sequence is predicted to be 30% identical and 51% similar to that of the TNP1 protein encoded by the transposon Tam1 of Antirrhinum majus. Immunolocalization analysis using anti-T7 antibodies showed nucleolar localization of most of the T7 epitope-tagged NtSIP2 proteins. A similar analysis with the T7-tagged 6b protein also showed subnucleolar as well as nuclear localization of the 6b protein. These results suggest the involvement of 6b along with NtSIP2 in certain molecular processes in the nucleolus as well as the nucleoplasm.     

The natural compound trans-chalcone induces programmed cell death in Arabidopsis thaliana roots

Chalcone (1,3-diphenyl-2-propen-1-one) is an aromatic ketone precursor of important molecules in plants such as flavonoids or anthocyanins. Its phytotoxicity has been demonstrated on different plant species, but to date little is known about the mechanisms of action of this secondary metabolite at plant cellular level. Detailed analysis by light and transmission electron microscopy (TEM) was conducted to examine the root meristems' ultrastructure of control and chalcone-treated Arabidopsis seedlings. Mitochondrial dysfunction was analysed by measuring mitochondrial membrane potential with JC-1 fluorochrome. Finally, acridine orange/ethidium bromide staining was used for the detection of programmed cell death. Microscopy revealed tissue alterations, inhibition of root hair formation and important changes after 7 and 14 d at the chalcone IC(50) value. Chalcone-treated cells showed signs of programmed cell death such as mitochondrial condensation, disruption of organelles and chromatin fragmentation. Acridine orange/ethidium bromide staining confirmed the programmed cell death, which could be induced by the reduction of mitochondrial transmembrane potential (ΔΨ(m)) that was detected after chalcone treatment. These results confirm the phytotoxic activity of chalcone on Arabidopsis seedlings, the alteration of mitochondrial membrane potential and the induction of programmed cell death.     

Organization of the TC and TE cellular T‐DNA regions in Nicotiana otophora and functional analysis of three diverged TE‐6b genes

Nicotiana otophora contains Agrobacterium‐derived T‐DNA sequences introduced by horizontal gene transfer (Chen et al., 2014). Sixty‐nine contigs were assembled into four different cellular T‐DNAs (cT‐DNAs) totalling 83 kb. TC and TE result from two successive transformation events, each followed by duplication, yielding two TC and two TE inserts. TC is also found in other Nicotiana species, whereas TE is unique to N. otophora. Both cT‐DNA regions are partially duplicated inverted repeats. Analysis of the cT‐DNA divergence patterns allowed reconstruction of the evolution of the TC and TE regions. TC and TE carry 10 intact open reading frames. Three of these are TE‐6b genes, derived from a single 6b gene carried by the Agrobacterium strain which inserted TE in the N. otophora ancestor. 6b genes have so far only been found in Agrobacterium tumefaciens or Agrobacterium vitis T‐DNAs and strongly modify plant growth (Chen and Otten, 2016). The TE‐6b genes were expressed in Nicotiana tabacum under the constitutive 2 × 35S promoter. TE‐1‐6b‐R and TE‐2‐6b led to shorter plants, dark‐green leaves, a strong increase in leaf vein development and modified petiole wings. TE‐1‐6b‐L expression led to a similar phenotype, but in addition leaves show outgrowths at the margins, flowers were modified and plants became viviparous, i.e. embryos germinated in the capsules at an early stage of their development. Embryos could be rescued by culture in vitro. The TE‐6b phenotypes are very different from the earlier described 6b phenotypes and could provide new insight into the mode of action of the 6b genes.     

The effect of decitabine on the expression and methylation of the PPP1CA,BTG2, and PTEN in association with changes in miR-125b,miR-17, and miR-181b in NALM6 cell line

Precursor B-cell acute lymphoblastic leukemia (B-ALL) is the most prevalent pediatric cancer. DNA methylation and changes in the microRNAs (miRNAs) expression are known to be important causes of B-ALL. Decitabine as a DNA methyltransferase inhibitor agent is able to induce hypomethylation in several tumor suppressor genes. Much evidence has proven BTG2, PPP1CA, and PTEN act as tumor suppressor genes in many malignancies. In this case control study, the messenger RNA (mRNA) expression of PPP1CA, BTG2, and PTEN genes using quantitative real-time polymerase chain reaction (rRT-PCR) in Nalm6 cell line and five patients suffer from ALL with mean age 5.6 years were determined in compare with seven normal healthy donors age and sex matched. qRT-PCR analysis revealed that the expression levels of PPP1CA, BTG2, and PTEN genes were significantly decreased in Nalm6 ([FC] = 0.46, [FC] = 0.046, [FC] = 0.54) and according to the Methylation-specific PCR (MSP) analysis, these genes were hypermethylated in Nalm6. In next step, the effects of decitabine treatment on the methylation and expression of these genes in association with changes in miR-125b, miR-17, and miR-181b expression levels were evaluated in optimal concentration 2.5 µM of decitabine. Our data showed that decitabine is able to restore the expression levels of aforementioned genes and downregulate expression levels of oncomiRs; including miR-125b, miR-17, and miR-181b in Nalm6 cell line. Therefore, it seems that decitabine can be used as a potential drug for the first line treatment of patients with B-ALL, but further in vivo investigation is necessary.     

The novel ruthenium—γ‐linolenic complex [Ru2(aGLA)4Cl] inhibits C6 rat glioma cell proliferation and induces changes in mitochondrial membrane potential,increased reactive oxygen species generation and apoptosis in vitro

The present study reports the synthesis of a novel compound with the formula [Ru₂(aGLA)4Cl] according to elemental analyses data, referred to as Ru₂GLA. The electronic spectra of Ru₂GLA is typical of a mixed valent diruthenium(II,III) carboxylate. Ru₂GLA was synthesized with the aim of combining and possibly improving the anti‐tumour properties of the two active components ruthenium and γ‐linolenic acid (GLA). The properties of Ru₂GLA were tested in C6 rat glioma cells by analysing cell number, viability, lipid droplet formation, apoptosis, cell cycle distribution, mitochondrial membrane potential and reactive oxygen species. Ru₂GLA inhibited cell proliferation in a time and concentration dependent manner. Nile Red staining suggested that Ru₂GLA enters the cells and ICP‐AES elemental analysis found an increase in ruthenium from <0.02 to 425 mg/Kg in treated cells. The sub‐G1 apoptotic cell population was increased by Ru₂GLA (22 ± 5.2%) when analysed by FACS and this was confirmed by Hoechst staining of nuclei. Mitochondrial membrane potential was decreased in the presence of Ru₂GLA (44 ± 2.3%). In contrast, the cells which maintained a high mitochondrial membrane potential had an increase (18 ± 1.5%) in reactive oxygen species generation. Both decreased mitochondrial membrane potential and increased reactive oxygen species generation may be involved in triggering apoptosis in Ru₂GLA exposed cells. The EC₅₀ for Ru₂GLA decreased with increasing time of exposure from 285 µM at 24 h, 211 µM at 48 h to 81 µM at 72 h. In conclusion, Ru₂GLA is a novel drug with antiproliferative properties in C6 glioma cells and is a potential candidate for novel therapies in gliomas. Copyright © 2009 John Wiley & Sons, Ltd.