Agrobacterium Tumefaciens Plasmid Research Articles

THE TENDENCY OF TRANSGENIC PLANTS SPREAD IN 2019-2021

The achievement of modern biotechnology, in particular genetic engineering in crop production, provides not only the possibility of increasing production, but also more efficient use of natural resources and reduces the negative impact on the environment. Recent publications indicate the widespread distribution of genetically modified (GM) agricultural crops in the world that are currently in commercial use, research and development. All over the world, the most cultivated GM crops are cotton, soybeans, corn, and rapeseed, and the area under these biotechnological crops continues to grow every year. However, the analysis of publicly available sources of information revealed the risks of negative effects of GMOs on human and animal bodies, as well as on the environment and biodiversity.
 The article analyzes the results of research on the presence of GM ingredients in plant raw materials, processing products, and feed for productive and non-productive animals for the period 2019-2021. The study was conducted by the polymerase chain reaction method in real time (PCR-RF), according to DSTU ISO 21569:2008 (ISO 21569:2005, IDT), and DSTU ISO 21571:2008 (ISO 21571:2005, IDT). In the studied samples were detected target sequences of the promoter p35S CaMV (cauliflower mosaic virus), promoter p34S FMV, terminator NOS (T-NOS) T1 of the Agrobacterium tumefaciens plasmid, genes Pat, EPSPS, Cry 3A.
 Transgenic plants are present in circulation in the agricultural market of Ukraine, and the area under their crops is increasing.
 The largest number of GM plants was found in samples of rapeseed, soybeans, and feed for productive and non-productive animals. There was a tendency for an increase in the number of transgenic plants in tested samples. During rapeseed research in 2019, the number of positive samples was 6.5%, in 2020 – 7.4%, and in 2021 – 14.3%. When studying soybeans in 2019, the number of positive samples was 6.7%, in 2020 – 16.7%, and in 2021 – 18.2%.
 GM ingredients were also found in feed for productive and non-productive animals. In 2020 16.7% of compound feed for poultry were positive, while in 2021 there were no samples for research.
 As for feeds for non-productive animals, during 2019–2020 there were no detected positive samples, while in 2021 the number of positive samples was 12%.

The Composite 259-kb Plasmid of Martelella mediterranea DSM 17316T–A Natural Replicon with Functional RepABC Modules from Rhodobacteraceae and Rhizobiaceae

A multipartite genome organization with a chromosome and many extrachromosomal replicons (ECRs) is characteristic for Alphaproteobacteria. The best investigated ECRs of terrestrial rhizobia are the symbiotic plasmids for legume root nodulation and the tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens. RepABC plasmids represent the most abundant alphaproteobacterial replicon type. The currently known homologous replication modules of rhizobia and Rhodobacteraceae are phylogenetically distinct. In this study, we surveyed type-strain genomes from the One Thousand Microbial Genomes (KMG-I) project and identified a roseobacter-specific RepABC-type operon in the draft genome of the marine rhizobium Martelella mediterranea DSM 17316T. PacBio genome sequencing demonstrated the presence of three circular ECRs with sizes of 593, 259, and 170-kb. The rhodobacteral RepABC module is located together with a rhizobial equivalent on the intermediate sized plasmid pMM259, which likely originated in the fusion of a pre-existing rhizobial ECR with a conjugated roseobacter plasmid. Further evidence for horizontal gene transfer (HGT) is given by the presence of a roseobacter-specific type IV secretion system on the 259-kb plasmid and the rhodobacteracean origin of 62% of the genes on this plasmid. Functionality tests documented that the genuine rhizobial RepABC module from the Martelella 259-kb plasmid is only maintained in A. tumefaciens C58 (Rhizobiaceae) but not in Phaeobacter inhibens DSM 17395 (Rhodobacteraceae). Unexpectedly, the roseobacter-like replication system is functional and stably maintained in both host strains, thus providing evidence for a broader host range than previously proposed. In conclusion, pMM259 is the first example of a natural plasmid that likely mediates genetic exchange between roseobacters and rhizobia.

КОНТРОЛЬ ГЕНЕТИЧНО МОДИФІКОВАНИХ РОСЛИН – ГАРАНТІЯ ПРОДОВОЛЬЧОЇ БЕЗПЕКИ

One of the problems in formation of mechanisms of development of environmentally safe environment is the proliferation of genetically modified organisms (GMOs). The use of genetic engineering in agriculture, food and agricultural industry have created the new opportunities for manufacturing the food and feed for meet the ever growing needs of the world's population. However, the opinions of scientists about the benefits and risks of genetic engineering is different. So today is particularly acute issue unpredictable consequences and danger for human health and the environment which are connected with GMO.The article present the results of determination of genetically modified organisms in plant material for the period 2013 – 2015 years, from the farms of different ownership forms. Analysis of the conducted research indicates on the circulation in farms of Lviv region transgenic plants.Found that the most common genetically modified plants were maize, soya and rape. The research of plant material on the presence of GMOs was performed by polymerase chain reaction in real time (PCR RF) by setting in them target sequences of promoter 35S virus of cauliflower mosaic (CaMV), promoter of FMV and (or) terminator NOS (T–NOS) T1 plasmid of Agrobacterium tumefaciens, Pat, EPSPS, Cry 3A, and determined species identification of positive samples. The use of PCR–RF allows quickly and qualitatively detect the GMO in the research samples. In 2013, the percentage of positive samples was 3.9% of the total, in 2014 – 2.3% and in 2015 – 3.3%. It is therefore necessary to monitor the vegetable raw and seed material on the presence of genetically modified organisms, to monitor the situation on GMO in Ukraine, because the problem of biosafety and the evaluation of the potential risks from their use is not studied.

Application of Vascular Puncture for Evaluation of Curtovirus Resistance in Chile Pepper and Tomato

AbstractCurtoviruses cause severe damage to tomatoes and peppers. Functional field resistance to curtoviruses in these plants is desirable but difficult to produce and difficult to screen for because it is time‐consuming and resistance could be achieved by developing resistance either to the virus or to insect feeding. To improve and speed curtovirus resistance testing in tomato (Solanum lycopersicum) and pepper (Capsicum annuum) plants, two puncture methods were developed and compared to leafhopper inoculation and feeding preference assays. The two puncture methods were adapted to introduce a modified Agrobacterium tumefaciens plasmid carrying a recombinant curtovirus into the meristem tissue of tomato plants and into newly germinated chile pepper seedlings. The puncture techniques were used to screen for resistance to curtoviruses in chile pepper and tomato breeding lines and varieties. Similarly, the peppers and tomatoes were assayed for curtovirus resistance using leafhopper inoculation and feeding preference, which was assessed by stylet sheath staining. Virus infection by puncture and leafhopper feeding was monitored using PCR and ELISA. ELISA was performed using an antibody to bacterially expressed coat protein. While pepper cvs Tabasco, NuMex Las Cruces cayenne and New Mexico 6‐4 were infected using both puncture and leafhopper inoculation methods, New Mexico 6‐4 had higher infection rates than the other two cultivars. Stylet sheath staining results suggest that leafhoppers prefer to feed on New Mexico 6‐4 rather than Tabasco and NuMex Las Cruces cayenne. Eight tomato cultivars were infected using meristem removal injection inoculation. Three tomatoes cultivars (CVF‐11, Saladmaster and Supersteak) were infected using leafhopper inoculation, although stylet sheath staining results suggested that the first two cultivars were not preferred by the insect vector. Our results suggest that puncture methods and leafhopper inoculation are successful in resistance screening, and both methods should be used as part of screening, because they assess different types of resistance.

Insect peptide metchnikowin confers on barley a selective capacity for resistance to fungal ascomycetes pathogens

The potential of metchnikowin, a 26-amino acid residue proline-rich antimicrobial peptide synthesized in the fat body of Drosophila melanogaster was explored to engineer disease resistance in barley against devastating fungal plant pathogens. The synthetic peptide caused strong in vitro growth inhibition (IC50 value ∼1 μM) of the pathogenic fungus Fusarium graminearum. Transgenic barley expressing the metchnikowin gene in its 52-amino acid pre-pro-peptide form under the control of the inducible mannopine synthase (mas) gene promoter from the Ti plasmid of Agrobacterium tumefaciens displayed enhanced resistance to powdery mildew as well as Fusarium head blight and root rot. In response to these pathogens, metchnikowin accumulated in plant apoplastic space, specifying that the insect signal peptide is functional in monocotyledons. In vitro and in vivo tests revealed that the peptide is markedly effective against fungal pathogens of the phylum Ascomycota but, clearly, less active against Basidiomycota fungi. Importantly, germination of the mutualistic basidiomycete mycorrhizal fungus Piriformospora indica was affected only at concentrations beyond 50 μM. These results suggest that antifungal peptides from insects are a valuable source for crop plant improvements and their differential activities toward different phyla of fungi denote a capacity for insect peptides to be used as selective measures on specific plant diseases.

Dimerization of the quorum‐sensing transcription factor TraR enhances resistance to cytoplasmic proteolysis

TraR is a LuxR-type quorum-sensing protein encoded by the tumour-inducing plasmid of Agrobacterium tumefaciens. TraR requires the pheromone N-3-oxooctanoyl-L-homoserine lactone (OOHL) for biological activity, and is dimeric both in solution and when bound to DNA. Dimerization is mediated primarily by two alpha-helices, one in the N-terminal OOHL binding domain, and the other in the C-terminal DNA binding domain. Each of these helices forms a parallel coiled coil with the identical helix of the opposite subunit. We have previously shown that OOHL is essential for resistance to proteolysis, and here we asked whether dimerization is also required for protease resistance. We constructed a series of site-directed mutations at the dimer interface, and tested these mutants for activity in vivo. Alteration of residues A149, A150, A153, A222 and I229 completely abolished activity, while alteration of three other residues also caused significant defects. All mutants were tested for dimerization as well as for specific DNA binding. The cellular abundance of these proteins in A. tumefaciens was measured using Western immunoblots and OOHL sequestration, while the half-life was measured by pulse-chase radiolabelling. We found a correlation between defects in in vivo activity, in vitro dimerization, DNA binding and protein half-life. We conclude that dimerization of TraR enhances resistance to cellular proteases.

Revaluating the evolution of the Toc and Tic protein translocons

The origin of the plastid from a cyanobacterial endosymbiont necessitated the establishment of specialized molecular machines (translocons) to facilitate the import of nuclear-encoded proteins into the organelle. To improve our understanding of the evolution of the translocons at the outer and inner envelope membrane of chloroplasts (Toc and Tic, respectively), we critically reassess the prevalent notion that their subunits have a function exclusive to protein import. We propose that many translocon components are multifunctional, conserving ancestral pre-endosymbiotic properties that predate their recruitment into the primitive translocon (putatively composed of subunits Toc34, Toc75 and Tic110 and associated chaperones). Multifunctionality seems to be a hallmark of the Tic complex, in which protein import is integrated with a broad array of plastid processes.

Construction of an infectious full-length cDNA clone of potato virus M

An infectious full-length cDNA clone of potato virus M (PVM) was produced. Total RNA was extracted from PVM-infected Nicotiana hesperis plants and used for cDNA synthesis. Subsequent RT-PCR produced two DNA fragments of about 5.5 and 3.2 kbp, which were ligated downstream of an enhanced 35S cauliflower mosaic virus promoter. After cloning of the enhanced 35S promoter with the PVM sequence into a modified pBIN19 plasmid and electroporation of Agrobacterium tumefaciens, the agroinoculated PVM full-length clone (pPVM-flc) led to systemic PVM infections in different host plants, causing symptoms indistinguishable from those caused by wild-type PVM.

AttG and attC mutations of Agrobacterium tumefaciens are dominant negative mutations that block attachment and virulence

The cryptic plasmid (pAT) of Agrobacterium tumefaciens was not required for virulence or attachment to plant surfaces. However, mutations in the attC and attG genes located on pAT caused the bacteria to become avirulent and non-attaching on tomato, carrot, and Bryophyllum daigremontiana. This was the case whether the mutation was in the copy of the genes located on pAT or whether it was carried in a second copy of the attA-G operon located on a plasmid in cells that contained a wild-type copy of pAT. Thus attC and attG mutations are dominant negative mutations. The mechanism by which these mutations block attachment and virulence is unknown.

Cloning of Salt Stress Responsive cDNA from Wheat and Resistant Analysis of Differential Fragment SR07 in Transgenic Tobacco

Analysis of the gene expression differentiation in leaves of wheat ( Triticum aestivum L.) cultivar Baofeng 7228, under salt stress, was carried out by Differential-Display Reverse Transcription-polymerase Chain Reaction (DDRT-PCR.) Twenty-seven differential cDNA fragments were obtained. The expression of the SR07 fragment was induced noticeably by salt treatment, and the nucleotide sequence homology of 87% between the SR07 fragment and PIPs (water channel proteins) was observed. Further research showed that a 561 bp open read frame was present in the SR07 fragment. Plant expression vector of pCAMBIA-SR07 was constructed and three transformants of tobacco ( Nicotiana tobacum) mediated by Agrobacterium tumefaciens plasmid were obtained. Resistance to salt, PEG, and mannitol stresses of the three transformants were examined. No significant difference ( P > 0.05) was observed between the control and the transformants in resistance to salt stress, but there was significant difference ( P < 0.05) between the control and the transformants in resistance to PEG and mannitol stresses. Therefore, the expression of the SR07 fragment may play an important role in the water regulation of the plant.

Identification of a megaplasmid centromere reveals genetic structural diversity within the repABC family of basic replicons

The basic replication unit of many plasmids and second chromosomes in the alpha-proteobacteria consists of a repABC locus that encodes the trans- and cis-acting components required for both semiautonomous replication and replicon maintenance in a cell population. In terms of physical genetic organization and at the nucleotide sequence level, repABC loci are well conserved across various genera. As with all repABC-type replicons that have been genetically characterized, the 1.4 Mb pSymA and 1.7 Mb pSymB megaplasmids from the plant endosymbiont Sinorhizobium meliloti encode strong incompatibility (inc) determinants. We have identified a novel inc sequence upstream of the repA2 gene in pSymA that is not present on pSymB and not reported in other repABC plasmids that have been characterized. This region, in concert with the repA and repB genes, stabilizes a test plasmid indicating that it constitutes a partitioning (par) system for the megaplasmid. Purified RepB binds to this sequence and binding may be enhanced by RepA. We have isolated 19 point mutations that eliminate incompatibility, reduce RepB binding or the stabilization phenotype associated with this sequence and all of these map to a 16-nucleotide palindromic sequence centred 330 bp upstream of the repA2 gene. An additional five near-perfect repeats of this palindrome are located further upstream of the repA2 gene and we show that they share some conservation with known RepB binding sites in different locations on other repABC plasmids and to two sequences found on the tumour inducing plasmid of Agrobacterium tumefaciens. These additional palindromes also bind RepB but one of them does not display obvious incompatibility effects. A heterogenic distribution of par sequences demonstrates unexpected diversity in the structural genetic organization of repABC loci, despite their obvious levels of similarity.

Agroinfection as a rapid method for propagating Cowpea mosaic virus-based constructs

To increase the efficiency of infections with Cowpea mosaic virus (CPMV)-based constructs, clones suitable for agroinfection were constructed. Full-length copies of RNA-1 and RNA-2 were inserted between the sequence of a Cauliflower mosaic virus (CaMV) 35S promoter and a nos terminator and were introduced into the Agrobacterium tumefaciens plasmid, pBINPLUS. Infiltration of leaves of either Nicotiana benthamiana or cowpea ( Vigna unguiculata) with a bacterial suspension containing a mixture of the RNA-1- and RNA-2-based plasmids resulted in the plants developing typical CPMV symptoms. To confirm the utility of this approach for use with CPMV-based vectors, a GFP construct based on RNA-2 was adapted for agroinfection. Infiltration of N. benthamiana leaves with a mixture of Agrobacteria containing this construct and the RNA-1 plasmid resulted in high levels of GFP expression. The results demonstrate that agroinfection is a suitable method for the propagation of CPMV-based derivatives.

Identification of a chromosomal tra-like region in Agrobacterium tumefaciens.

The virD4 gene is one of the virulence genes present on the pTiC58 plasmid of Agrobacterium tumefaciens. Unexpectedly, we found that a pTi-free A. tumefaciens strain carried a protein of similar size to the plasmid-encoded VirD4 protein which reacted with VirD4-specific antibodies. This suggested that this strain may contain a homologue of the VirD4 protein. A chromosomal fragment encoding a protein of similar sequence to VirD4 was isolated and a 7.8 kilobase region surrounding the gene encoding this putative homologue was sequenced. This region contained four open reading frames, encoding putative proteins similar to proteins of known bacterial transfer and conjugation systems, viz., orf1 encoded a putative homologue of the TraA protein of the Rhizobium symbiosis plasmid pNGR234 and the TraA protein encoded by pTiC58 from A. tumefaciens plasmid pTiC58, orf3 encoded a protein very similar to the MobC protein encoded by the IncQ plasmid RSF1010 of E. coli and to MobS encoded by pTF1 from Thiobacillus ferrooxidans, whereas the predicted product of orf4 displayed similarity to the TraG protein encoded by the IncPalpha plasmid RP4 of E. coli, TraG and VirD4 encoded by A. tumefaciens plasmid pTiC58. The product of orf2 showed no significant similarity to any known protein. Preliminary assays with two orf4 mutants suggested that the product of this orf is involved in DNA transfer. The 7.8 kb chromosomal fragment seems to be closely related to the tra region of different conjugative plasmids and appears to be confined to Agrobacterium species, raising the question of the role of a chromosomal tra-like region during evolution.

Xenognosin sensing in virulence: is there a phenol receptor in Agrobacterium tumefaciens?

Background: The mechanisms of signal perception and transmission in the ‘two-component’ autokinase transmitters/response regulators are poorly understood, especially considering the vast number of such systems now known. Virulence induction from the tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens represents one of the best understood systems with regard to the chemistry of the activating signal, and yet the existing data does not support a receptor-mediated perception event for the xenognostic phenols. Results: Here we provide the first conclusive evidence that a specific receptor must be involved in xenognostic phenol perception, detail structural requirements of the xenognosins necessary for perception by this receptor, and develop a genetic strategy that demonstrates critical components of the phenol recognition system are not encoded on the Ti plasmid. Conclusions: Although the basic elements of the two-component system required for phenol-mediated induction of virulence gene expression are encoded on the Ti plasmid, they are dependent on the chromosomal background for even the very first stage of signal perception. This discovery suggests a curious evolutionary history, and also provides functional insight into the mechanisms of two-component signal detection and transmission in general.

Natural competence for DNA transformation in Helicobacter pylori: identification and genetic characterization of the comB locus.

The gram-negative bacterial pathogen Helicobacter pylori, an important aetiological agent of gastroduodenal disease in humans, belongs to a group of bacterial species displaying competence for genetic transformation. Here, we describe the comB gene locus of H. pylori involved in DNA transformation competence. It consists of a cluster of four tandemly arranged genes with partially overlapping open reading frames, orf2, comB1, comB2 and comB3, constituting a single transcriptional unit. Orf2 encodes a 37-amino-acid peptide carrying a signal sequence, whereas comB1, comB2 and comB3 produce 29 kDa, 38 kDa and 42 kDa proteins, respectively, as demonstrated by immunoblotting with specific antisera. For Orf2 and ComB1, no homologous proteins were identified in the database. For ComB3, the best homologies were found with TraS/TraB from the Pseudomonas aeruginosa conjugative plasmid RP1 and TrbI of plasmid RP4, VirB10 from the Ti plasmid of Agrobacterium tumefaciens and PtlG, a protein involved in secretion of pertussis toxin of Bordetella pertussis. Defined transposon knock-out mutants in individual comB genes resulted in transformation-defective phenotypes ranging from a 90% reduction to a complete loss of the natural transformation efficiency. The comB2 and comB3 genes show homology to HP0528 and HP0527, respectively, located on the cagII pathogenicity island of H. pylori strain 26695.

Reassessing T-DNA insertions

Agrobacterium is a marvellous organism for plant genetic engineering, but we would do well to remember that it has evolved to serve its own ends and not ours. In last month's issue of Trends in Plant Science, Natasha Smith[1xMore T-DNA than meets the eye. Smith, N. Trends Plant Sci. 1998; 3: 85Abstract | Full Text PDFSee all References][1]made clear that the processing of T-DNA plays a key role in defining the type of T-DNA molecule being integrated, and is of critical importance in plant biotechnology.A practical definition of the left border is that it is the point beyond which there is no T-DNA transfer, because it is the point where most of the T-DNA ends on the Ti plasmid — at least in simple systems such as the nopaline Ti plasmid pTi C58 and its derivatives. Nevertheless, it has been known for some time that several species of Agrobacterium carry more than one T-DNA on their Ti plasmid. For example, the octopine type of Ti plasmid, pTiA6, contains four T-DNA borders delimiting, in theory, three T-DNAs: TR (right), TC (central) and TL (left). However, the system is even more complex, because this type of plasmid is able to generate six different types of T-strands (composed of TR, TR+TC, TR+TC+TL, TC, TC+TL and TL (Refs [2xActivation of Agrobacterium tumefaciens vir gene expression generates multiple single-stranded T-strand molecules from the pTiA6 T-region: requirement for 5′ virD gene products. Stachel, S., Timmerman, B., and Zambryski, P. EMBO J. 1987; 6: 857–863PubMedSee all References][2]and [3xVirulence genes, borders, and overdrive generate single-stranded T-DNA molecules from the A6 Ti plasmid of Agrobacterium tumefaciens. Veluthambi, K., Ream, W., and Gelvin, S.B. J. Bacteriol. 1988; 170: 1523–1532PubMedSee all References][3]). The `left border' of TR can, alternatively, act as a left border; not be recognized and allow the transfer of TR+TC; or even play the role of a right border to initiate the transfer of TC+TL. Similar observations have been made with limited-host-range strains of Agrobacterium, where it has been postulated that the different lengths of T-DNA (encoding different numbers of oncogenes) could play a role in the host-range specificity[4xLimited-host-range plasmid of Agrobacterium tumefaciens: molecular and genetic analysis of transferred DNA. Yanofsky, M. et al. J. Bacteriol. 1985; 163: 341–348PubMedSee all References, 5xRole of T-region border in Agrobacterium host range. Paulus, F. et al. Mol. Plant–Microbe Interact. 1991; 4: 163–172Crossref | PubMedSee all References]. Thus, unprocessed left borders are not merely an invention of binary plasmid designers.Are there ways in which to improve processing at the left border and to transfer T-DNA in a more precise manner? In order to answer this question, it would be interesting to investigate the DNA region surrounding the left border, because an `attenuation activity' of sequences flanking the left border has been reported[6xSequence context of the T-DNA border repeat element determines its relative activity during T-DNA transfer to plant cells. Wang, K. et al. Mol. Gen. Genet. 1987; 210: 338–346Crossref | Scopus (27)See all References][6]. Termination of T-strand production probably involves nicking of the left border, and although this has not been fully investigated, the nicking probably has to be performed at a high rate, without initiation of a new T-strand to the left of the left border. Some as yet undescribed activity of virulence proteins might be responsible for this process; these proteins could recognize the region surrounding the left borders (e.g. the `attenuation domain') and block T-strand initiation. Certainly, a better understanding of the role of the left border during T-strand formation would allow the design of better vectors for Agrobacterium-mediated transformation: a combination of attenuation regions in the binary plasmid and of the corresponding virulence region on the helper plasmid might lead to the transfer of well-delimited T-DNA.In assessing the efficacy of T-DNA transfer for biotechnology, it is important to consider the small number of Ti plasmids that naturally occurs in Agrobacterium cells. Processing of the borders from a Ti plasmid involves a stoichiometric reaction between the endonuclease VirD2 and the border sequences. As a consequence, there might be a risk in using binary plasmids present in high copy number (especially in suboptimal induction of the virulence region), because the excess of borders could titrate-out VirD2, resulting in incomplete processing.Despite these considerations, in most cases correct processing is still observed. Although it is possible to improve on our present tools, it might be more convenient simply to eliminate those transgenic plants that contain DNA from outside the T-DNA integrated in their genome. As far as I am aware, colleagues who have undertaken field trials with plants transformed using Agrobacterium had to prove that the integrated foreign DNA was restricted to the region between the left and the right border.

An Agrobacterium virulence factor encoded by a Ti plasmid gene or a chromosomal gene is required for T-DNA transfer into plants.

Mutagenesis of the vir region on the Ti plasmid of Agrobacterium tumefaciens revealed a new locus, virJ, that is induced by the plant-wound signal molecule, acetosyringone (AS). virJ lies between virA and virB, and is transcribed in the same direction. The amino acid sequence of virJ is similar to a region of a previously characterized chromosomal gene, acvB, required for virulence. virJ can complement the avirulent phenotype of an acvB mutant, indicating that virJ and acvB encode the same factor required for tumorigenesis. Southern analysis revealed that virJ is present on the Ti plasmid of an octopine but not a nopaline strain whereas acvB is present on the chromosomes of both octopine and nopaline strains. While virJ is regulated by AS under the control of the virA/virG two-component regulatory system, acvB is not induced by AS. VirJ possesses a putative signal peptide and was found predominantly in the periplasmic fraction. The strain lacking both acvB and virJ had an impaired ability to transfer T-DNA into plant cells, suggesting that the factor encoded by virJ or acvB is required for T-DNA transfer from A. tumefaciens to plant cells. acvB is the first chromosomal gene implicated in T-DNA transfer, but whether it functions specifically for this process is not clear. We hypothesize that virJ evolved from acvB, presumably for a more specialized role in tumorigenesis.

Nonspecific transmission of the NPTII gene from E. coli helper plasmid to a plasmid of Agrobacterium tumefaciens.

Nonspecific transmission of the NPTII gene from E. coli helper plasmid to a plasmid of Agrobacterium tumefaciens.

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

During the inception of crown gall tumorigenesis, the transferred DNA (T-DNA) is processed from the Ti (tumor inducing) plasmid of Agrobacterium tumefaciens and is transferred to plant cells. T-DNA processing and transfer require the induction of vir (virulence) genes by phenolic compounds secreted by wounded plant cells. After vir gene induction, both single-stranded (T-strands) and double-stranded forms of processed T-DNA accumulate in the bacteria. Although current models favor the transfer of T-strands to plants, there has yet been no experimental evidence to show this. In this paper, we show that T-strands disappear from acetosyringone-induced A. tumefaciens within 30 min of bacterial cocultivation with tobacco protoplasts. PCR analysis of T-DNA associated with protoplasts indicates that single-stranded, but not double-stranded, T-DNA can be detected in the plant cells within 30 min of bacterial cocultivation. Control experiments show that this T-DNA does not originate from lysed contaminating bacterial cells. T-DNA transfer depends on a functional bacterial virB operon. Protoplast infections using an A. tumefaciens virE mutant result in a low level of accumulation of T-strands in the plant cells.

Fruit‐specific expression of the A. tumefaciens isopentenyl transferase gene in tomato: effects on fruit ripening and defense‐related gene expression in leaves

SummaryThis paper describes the analysis of tomato plants transformed with a chimeric gene consisting of the promoter region of a fruit specifically expressed tomato gene linked to the ipt gene coding sequences from the Ti plasmid of Agrobacterium tumefaciens. The pattern of expression of this chimeric gene was found to be consistent with the expression of the endogenous fruit‐specific gene and consequently, plants expressing the chimeric gene were phenotypically normal until fruit maturation and ripening. A dramatically altered fruit phenotype, islands of green pericarp tissue remaining on otherwise deep red ripe fruit, was then evident in many of the transformed plants. Cytokinin levels in transformed plant fruit tissues were 10 to 100‐fold higher than in control fruit. In the leaves of a fruit‐bearing transformant, despite a lack of detectable ipt mRNA accumulation, approximately fourfold higher than control leaf levels of cytokinin were detected. It is suggested that cytokinin produced in fruit is being transported to the leaves since accumulation in leaves of PR‐1 and chitinase mRNAs, which encode defense‐related proteins known to be induced by cytokinin, occurred only when the transformant was reproductively active. Effects of elevated cytokinin levels on tomato fruit gene expression and cellular differentiation processes are also described.