T-DNA Oncogenes Research Articles

Prospects for the study of natural GMOs

Naturally transgenic plants are plants that have been subjected to “Agrobacterium” mediated transformation in natural conditions without any human impact. They contain T-DNA-like sequences, called cellular T-DNA (cT-DNA) in their genomes and transfer them from generation to generation [1].
 At the moment, several dozen species of natural GMOs are known, and this list is constantly updated. Based on the available data on the diversity of natural GMOs, it can be concluded that in each case, plants have their own set of functionally active transgenes. Accordingly, each cT-DNA performs its own functions. This set of active transgenes will define promising areas for nGMO research, such as:
 
 description of the structures and functions of opine synthesis genes and the biological activity of their products in the regulation of plant-microbial interactions [2];
 description of the effect of oncogenes on plant morphogenesis, their primary and secondary metabolism [1].
 
 In addition, sequences, newly acquired by plants, can be successfully used in phylogenetic studies [3].
 These topics will be the subject of a report at the conference.
 The work was supported by a grant from the Russian Science Foundation using the equipment of the resource centers of St. Petersburg State University “Biobank” “Chemical Analysis and Materials” and “Development of molecular and cellular technologies”.
 Keywords: natural GMO; cellular T-DNA; phylogenetic studies; opines; T-DNA oncogenes.

In the interkingdom horizontal gene transfer, the small rolA gene is a big mystery.

The biological function of the agrobacterial oncogene rolA is very poorly understood compared to other components of the mechanism of horizontal gene transfer during agrobacterial colonization of plants. Research groups around the world have worked on this problem, and available information is reviewed in this review, but other rol oncogenes have been studied much more thoroughly. Having one unexplored element makes it impossible to form a complete picture. However, the limited data suggest that the rolA oncogene and its regulatory apparatus have great potential in plant biotechnology and genetic engineering. Here, we collect and discuss available experimental data about the function and structure of rolA. There is still no clear understanding of the mechanism of RolA and its structure and localization. We believe this is because of the nucleotide structure of a frameshift in the most well-studied rolA gene of the agropine type pRi. In fact, interest in the genes of agrobacteria as natural tools for the phenotypic or biochemical engineering of plants increased. We believe that a detailed understanding of the molecular mechanisms will be forthcoming. KEY POINTS: • Among pRi T-DNA oncogenes, rolA is the least understood in spite of many studies. • Frameshift may be the reason for the failure to elucidate the role of agropine rolA. • Understanding of rolA is promising for the phenotypic and biochemical engineering of plants.

Evolutionary classification of tumor- and root-inducing plasmids based on T-DNAs and virulence regions

Tumor-inducing (Ti) and root-inducing (Ri) plasmids of Agrobacterium that display a large diversity are involved in crown gall and hairy root plant diseases. Their phylogenetic relationships were inferred from an exhaustive set of Ti and Ri plasmids (including 36 new complete Ti plasmids) by focusing on T-DNA and virulence regions. The opine synthase gene content of T-DNAs revealed 13 opine types corresponding to former classifications based on opines detected in diseased plants, while the T-DNA gene content more finely separate opine types in 18 T-DNA organizations. This classification was supported by the phylogeny of T-DNA oncogenes of Ti plasmids. The five gene organizations found in Ti/Ri vir regions was supported by the phylogeny of common vir genes. The vir organization was found to be likely an ancestral plasmid trait separating “classic” Ti plasmids (with one or two T-DNAs) and “Ri and vine-Ti” plasmids. A scenario generally supported by the repABC phylogeny. T-DNAs likely evolved later with the acquisition of opine characteristics as last steps in the Ti/Ri plasmid evolution. This novel evolutionary classification of Ti/Ri plasmids was found to be relevant for accurate crown gall and hairy root epidemiology.

A molecular system to investigate the effects of compound heterozygous mutations on multimeric pathogenic proteins

The RolA protein belongs to the RolB class of plant T-DNA oncogenes, and shares structural similarity with the papilloma virus E2 DNA-binding domain. It has potentially as an inducer of plant secondary metabolism, although its role in biotechnology has yet to be realised. In this investigation, a Rubia cordifolia callus culture transformed with the rolA plant oncogene for more than 10 years was analysed. Expression of the rolA gene in the callus line was stable during long-term cultivation, and growth parameters were both elevated and stable, exceeding those of the non-transformed control culture. The rolA-transformed calli not only demonstrated remarkably stable growth, but also the ability to increase the yield of anthraquinones (AQs) in long-term cultivation. After ten years of cultivating rolA callus lines, we observed an activation of AQ biosynthesis from 200 mg/l to 874 mg/l. The increase was mainly due to activation of ruberitrinic acid biosynthesis. The expression of key AQ biosynthesis genes was strongly activated in rolA-transgenic calli. We compared the effects of the rolA gene with those of the rolB gene, which was previously considered the most potent inducer of secondary metabolism, and showed that rolA was more productive under conditions of long-term cultivation.

Resveratrol supported on magnesium DiHydroxide (Resv@MDH, REVIFAST) represents an improved formulation of resveratrol with fast absorption and bioavailability in humans

The RolA protein belongs to the RolB class of plant T-DNA oncogenes, and shares structural similarity with the papilloma virus E2 DNA-binding domain. It has potentially as an inducer of plant secondary metabolism, although its role in biotechnology has yet to be realised. In this investigation, a Rubia cordifolia callus culture transformed with the rolA plant oncogene for more than 10 years was analysed. Expression of the rolA gene in the callus line was stable during long-term cultivation, and growth parameters were both elevated and stable, exceeding those of the non-transformed control culture. The rolA-transformed calli not only demonstrated remarkably stable growth, but also the ability to increase the yield of anthraquinones (AQs) in long-term cultivation. After ten years of cultivating rolA callus lines, we observed an activation of AQ biosynthesis from 200 mg/l to 874 mg/l. The increase was mainly due to activation of ruberitrinic acid biosynthesis. The expression of key AQ biosynthesis genes was strongly activated in rolA-transgenic calli. We compared the effects of the rolA gene with those of the rolB gene, which was previously considered the most potent inducer of secondary metabolism, and showed that rolA was more productive under conditions of long-term cultivation.

Activation of anthraquinone biosynthesis in long-cultured callus culture of Rubia cordifolia transformed with the rolA plant oncogene

The RolA protein belongs to the RolB class of plant T-DNA oncogenes, and shares structural similarity with the papilloma virus E2 DNA-binding domain. It has potentially as an inducer of plant secondary metabolism, although its role in biotechnology has yet to be realised. In this investigation, a Rubia cordifolia callus culture transformed with the rolA plant oncogene for more than 10 years was analysed. Expression of the rolA gene in the callus line was stable during long-term cultivation, and growth parameters were both elevated and stable, exceeding those of the non-transformed control culture. The rolA-transformed calli not only demonstrated remarkably stable growth, but also the ability to increase the yield of anthraquinones (AQs) in long-term cultivation. After ten years of cultivating rolA callus lines, we observed an activation of AQ biosynthesis from 200 mg/l to 874 mg/l. The increase was mainly due to activation of ruberitrinic acid biosynthesis. The expression of key AQ biosynthesis genes was strongly activated in rolA-transgenic calli. We compared the effects of the rolA gene with those of the rolB gene, which was previously considered the most potent inducer of secondary metabolism, and showed that rolA was more productive under conditions of long-term cultivation.

Agrobacterium-Mediated Transformation in the Evolution of Plants.

In most cases, the genetic engineering of plants uses Agrobacterium-mediated transformation to introduce novel genes. In nature, insertion of T-DNA into the plant genome and its subsequent transfer via sexual reproduction have been shown for several species in the genera Nicotiana, Ipomoea , and Linaria . A sequence homologous to T-DNA of the Ri plasmid of Agrobacterium rhizogenes was found in the genome of wild-type Nicotiana glauca (section Noctiflorae) more than 30years ago and was named "cellular T-DNA" (cT-DNA). It comprises an imperfect inverted repeat and contains homologs of several T-DNA oncogenes (NgrolB, NgrolC, Ngorf13, Ngorf14) and an opine synthesis gene (Ngmis). Multiple cT-DNAs have also been found in species of the sections Tomentosae and Nicotiana of the genus Nicotiana. These ancient cT-DNA genes are still expressed, indicating that they may play a role in the evolution of these plants. In 2012-2013, cT-DNA was detected and characterized in Linaria vulgaris and L. genistifolia ssp. dalmatica. Their cT-DNA is present in two copies and organized as an imperfect direct tandem repeat, containing LvORF2, LvORF3, LvORF8, LvrolA, LvrolB, LvrolC, LvORF13, LvORF14, and the Lvmis genes. In 2015, cT-DNA was found in Ipomoea. Two types of T-DNA-like sequences were described within this genera, and their distribution varied among cultured hexaploid, tetraploid, and wild diploid forms. Thus, several independent T-DNA integration events occurred in the genomes of these three plant genera. We propose that the events of T-DNA insertion in the plant genome might have affected their evolution, resulting in the creation of new plant species. In this chapter, we focus on the structure and functions of cT-DNA in Linaria, Nicotiana, and Ipomoea and discuss their possible evolutionary role.

T-6b allocates more assimilation product for oil synthesis and less for polysaccharide synthesis during the seed development of Arabidopsis thaliana

BackgroundAs an Agrobacterium tumefaciens T-DNA oncogene, T-6b induces the development of tumors and the enation syndrome in vegetative tissues of transgenic plants. Most of these effects are related to increases in soluble sugar contents. To verify the potential roles of T-6b in the distribution of carbon in developing seeds, not in vegetative tissues, we fused an endosperm-specific promoter to the T-6b gene for expression in transgenic Arabidopsis thaliana plants.ResultsThe expression of T-6b in reproductive organs did not induce the development of the enation syndrome, and moreover, promoted endosperm expansion, which increased the total seed biomass by more than 10%. Additionally, T-6b also increased oil content in mature seeds by more than 10% accompanied with the decrease of starch and mucilage content at the same time.ConclusionsT-6b enhances seed biomass and helps oil biosynthesis but not polysaccharides in reproductive organs without disturbing vegetative growth and development. Our findings suggest T-6b may be very useful for increasing oil production in biodiesel plants.

Morphological analysis of the 6b oncogene-induced enation syndrome.

The T-DNA 6b oncogene induces complex and partly unprecedented phenotypic changes in tobacco stems and leaves, which result from hypertrophy and hyperplasia with ectopic spot-like, ridge-like and sheet-like meristems. The Agrobacterium T-DNA oncogene 6b causes complex growth changes in tobacco including enations; this unusual phenotype has been called "6b enation syndrome". A detailed morphological and anatomical analysis of the aerial part of Nicotiana tabacum plants transformed with a dexamethasone-inducible dex-T-6b gene revealed several striking growth phenomena. Among these were: uniform growth of ectopic photosynthetic cells on the abaxial leaf side, gutter-like petioles with multiple parallel secondary veins, ectopic leaf primordia emerging behind large glandular trichomes, corniculate structures emerging from distal ends of secondary veins, pin-like structures with remarkable branching patterns, ectopic vascular strands in midveins and petioles extending down along the stem, epiascidia and hypoascidia, double enations and complete inhibition of leaf outgrowth. Ectopic stipule-like leaves and inverted leaves were found at the base of the petioles. Epinastic and hyponastic growth of petioles and midveins yielded complex but predictable leaf folding patterns. Detailed anatomical analysis of over sixty different 6b-induced morphological changes showed that the different modifications are derived from hypertrophy and abaxial hyperplasia, with ectopic photosynthetic cells forming spot-like, ridge-like and sheet-like meristems and ectopic vascular strands forming regular patterns in midveins, petioles and stems. Part of the enation syndrome is due to an unknown phloem-mobile enation factor. Graft experiments showed that the 6b mRNA is mobile and could be the enation factor. Our work provides a better insight in the basic effects of the 6b oncogene.

Horizontal gene transfer from Agrobacterium to plants.

Most genetic engineering of plants uses Agrobacterium mediated transformation to introduce novel gene content. In nature, insertion of T-DNA in the plant genome and its subsequent transfer via sexual reproduction has been shown in several species in the genera Nicotiana and Linaria. In these natural examples of horizontal gene transfer from Agrobacterium to plants, the T-DNA donor is assumed to be a mikimopine strain of A. rhizogenes. A sequence homologous to the T-DNA of the Ri plasmid of Agrobacterium rhizogenes was found in the genome of untransformed Nicotiana glauca about 30 years ago, and was named “cellular T-DNA” (cT-DNA). It represents an imperfect inverted repeat and contains homologs of several T-DNA oncogenes (NgrolB, NgrolC, NgORF13, NgORF14) and an opine synthesis gene (Ngmis). A similar cT-DNA has also been found in other species of the genus Nicotiana. These presumably ancient homologs of T-DNA genes are still expressed, indicating that they may play a role in the evolution of these plants. Recently T-DNA has been detected and characterized in Linaria vulgaris and L. dalmatica. In Linaria vulgaris the cT-DNA is present in two copies and organized as a tandem imperfect direct repeat, containing LvORF2, LvORF3, LvORF8, LvrolA, LvrolB, LvrolC, LvORF13, LvORF14, and the Lvmis genes. All L. vulgaris and L. dalmatica plants screened contained the same T-DNA oncogenes and the mis gene. Evidence suggests that there were several independent T-DNA integration events into the genomes of these plant genera. We speculate that ancient plants transformed by A. rhizogenes might have acquired a selective advantage in competition with the parental species. Thus, the events of T-DNA insertion in the plant genome might have affected their evolution, resulting in the creation of new plant species. In this review we focus on the structure and functions of cT-DNA in Linaria and Nicotiana and discuss their possible evolutionary role.

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.

The rolC gene induces expression of a pathogenesis-related β-1,3-glucanase in transformed ginseng cells

The Agrobacterium rhizogenes rolC oncogene is capable of stimulating production of secondary metabolites in transformed plant cells that suggest its possible involvement in plant defense reactions. We tested whether the gene could also affect production of pathogenesis-related proteins. Using a well-known group of PR-proteins, such as β-1,3-glucanases, we observed a 10-fold increase in total β-1,3-glucanase activity in rolC-transformed Panax ginseng cells compared with normal cells. The increase was due to the production of a salicylic acid-activated β-1,3-glucanase isoform. We isolated cDNA of the corresponding β-1,3-glucanase gene ( Pg-glu1), which shared 38–60% sequence identity with previously reported sequences of plant β-1,3-glucanases at the protein level. Levels of Pg-glu1 mRNA transcripts were tightly correlated with expression of the rolC gene. Our data, together with previously reported information, indicate that A. rhizogenes can activate plant defense reactions via expression of T-DNA oncogenes.

Translation start sequences affect the efficiency of silencing of Agrobacterium tumefaciens T-DNA oncogenes.

Agrobacterium tumefaciens oncogenes cause transformed plant cells to overproduce auxin and cytokinin. Two oncogenes encode enzymes that convert tryptophan to indole-3-acetic acid (auxin): iaaM (tryptophan mono-oxygenase) and iaaH (indole-3-acetamide hydrolase). A third oncogene (ipt) encodes AMP isopentenyl transferase, which produces cytokinin (isopentenyl-AMP). Inactivation of ipt and iaaM (or iaaH) abolishes tumorigenesis. Because adequate means do not exist to control crown gall, we created resistant plants by introducing transgenes designed to elicit posttranscriptional gene silencing (PTGS) of iaaM and ipt. Transgenes that elicit silencing trigger sequence-specific destruction of the inducing RNA and messenger RNAs with related sequences. Although PTGS has proven effective against a variety of target genes, we found that a much higher percentage of transgenic lines silenced iaaM than ipt, suggesting that transgene sequences influenced the effectiveness of PTGS. Sequences required for oncogene silencing included a translation start site. A transgene encoding a translatable sense-strand RNA from the 5' end of iaaM silenced the iaaM oncogene, but deletion of the translation start site abolished the ability of the transgene to silence iaaM. Silencing A. tumefaciens T-DNA oncogenes is a new and effective method to produce plants resistant to crown gall disease.

Agrobacterium tumefaciens transfers extremely long T-DNAs by a unidirectional mechanism.

During crown gall tumorigenesis, part of the Agrobacterium tumefaciens tumor-inducing (Ti) plasmid, the T-DNA, integrates into plant DNA. Direct repeats define the left and right ends of the T-DNA, but tumorigenesis requires only the right-hand repeat. Virulence (vir) genes act in trans to mobilize the T-DNA into plant cells. Transfer of T-DNA begins when the VirD endonuclease cleaves within the right-hand border repeat. Although the T-DNA right-border repeat promotes T-DNA transmission best in its normal orientation, an inverted right border exhibits reduced but significant activity. Two models may account for this diminished tumorigenesis. The right border may function bidirectionally, with strong activity only in its wild-type orientation, or it may promote T-DNA transfer in a unidirectional manner such that, with an inverted right border, transfer proceeds around the entire Ti plasmid before reaching the T-DNA. To determine whether a substantial portion of the Ti plasmid is transferred to plant cells, as predicted by the unidirectional-transfer hypothesis, we examined T-DNAs in tumors induced by strains containing a Ti plasmid with a right border inverted with respect to the T-DNA oncogenes. These tumors contained extremely long T-DNAs corresponding to most or all of the Ti plasmid. To test whether the right border can function bidirectionally, we inserted T-DNAs with either a properly oriented or an inverted right border into a specific site in the A. tumefaciens chromosome. A border situated to transfer the oncogenes first directed T-DNA transfer even from the bacterial chromosome, whereas a border in the opposite (inverted) orientation did not transfer the oncogenes to plant cells. Our results indicate that the right-border repeat functions in a unidirectional manner.

Introduction and expression of the octopine T-DNA oncogenes in tobacco plants and their progeny

The tumour-inducing T-DNA genes 1, 2 and 4 of the octopine Ti-plasmid pTiAch5 were cloned and introduced into tobacco cells by cocultivation or leaf disk transformation using pTi derived vectors. When a selectable marker was needed, we used a aminoglycoside phosphotransferase II (nos-APH(3′)II) chimeric gene conferring kanamycin resistance to plant cells. The expression of gene 4 in transformed tissue cultures precluded the regeneration of normal transformed plants. Normal transformed plants were obtained with the construction carrying genes 1 or 2. We report in vivo complementation of genes 1 and 2 after crosses of transformed plants. Strategies are described for the use of genes 1 and 2 as selection or screening markers in plant cells or regenerated plants.

Agrobacterium T-DNA oncogenes encode plant growth regulators: are there cellular homologues in plants?

Agrobacterium T-DNA oncogenes encode plant growth regulators: are there cellular homologues in plants?

Comparison of T-DNA oncogene complements of Agrobacterium tumefaciens tumor-inducing plasmids with limited and wide host ranges.

The T-DNA oncogene complements of the limited-host-range tumor-inducing plasmid pTiAg63 and the wide-host-range plasmid pTiA6 were compared. The resulting data indicate that pTiAg63 has DNA sequences related to most of the genes encoded by the oncogene region, the TL-DNA, of pTiA6 and that these sequences are divided between two T-DNA regions, the TA-DNA, which encoded sequences related to pTiA6 genes 4 (the cytokinin independence gene) and 6a, as well as to a pTiA6 TL-DNA fragment that encoded gene 6b and a portion of gene 3, and the TB-DNA, which encoded sequences related to genes 1 and 2 (the auxin independence genes). Tumor tissues of Nicotiana rustica incited by Agrobacterium tumefaciens harboring either pTiA6 or pTiAg63 grew axenically in vitro on phytohormone-free medium. The morphologies of the tissues, however, differed; whereas those incited with pTiA6 grew as loose, friable, unorganized callus, the tumors incited by pTiAg63 grew as clumps of rootlike structures. Thus, the T-DNA oncogene complements of these plasmids were not equivalent. The results are discussed in relation to the A. tumefaciens host range.

Host range encoded by the Agrobacterium tumefaciens tumor-inducing plasmid pTiAg63 can be expanded by modification of its T-DNA oncogene complement.

Agrobacterium tumefaciens harboring pTiA6 incite unorganized tumors on Nicotiana rustica, sunflowers, carrots, and tomatoes, whereas isogenic strains of agrobacteria harboring pTiAg63 form "rooty" tumors on N. rustica and are essentially avirulent on sunflowers, carrots, and tomatoes. In this report we show that the different host range characteristics of these two plasmids were due, in part, to differences in the T-DNA oncogene complements of the plasmids. Specifically, we constructed derivatives of pTiAg63 that contained pTiA6 oncogenes 4, 6a, and 6b inserted into the TB-DNA region and found that agrobacteria harboring these plasmids could incite unorganized tumors on N. rustica, tomatoes, carrots, and the inbred sunflower line HA202R. Undefined host factors, however, also appeared to be involved in determining A. tumefaciens host range since three inbred sunflower lines, HA303B, HA89B, and HA290B, were susceptible to tumor formation by agrobacteria harboring pTiA6 but not by strains harboring pTiAg63 or the modified pTiAg63 plasmids.