Assembly of the VirB transport complex for DNA transfer from Agrobacterium tumefaciens to plant cells

The VirB transporter is a type IV secretion system that mediates the genetic transformation of plant cells by Agrobacterium tumefaciens. Assembly of this transporter depends on, first, formation of a VirB7/B9 complex that stabilizes many of the VirB proteins, second, formation of a virulence-specific pilus composed primarily of VirB2 and VirB5, and, third, post-translational processing of VirB1 and VirB2.     

A chemotaxis cluster from Agrobacterium tumefaciens

We report the DNA sequence of a 9.6-kb region of the Agrobacterium tumefaciens chromosome containing a putative 8-kb chemotaxis operon. The putative operon begins with orf1, whose predicted protein product shows strong sequence identity to methyl-accepting chemotaxis proteins (MCPs), followed by orf2, cheY1, cheA, cheR, cheB, cheY2, orf9, orf10. All of the identified homologues show a high degree of sequence conservation with their counterparts in the che operons from Sinorhizobium meliloti and Rhodobacter sphaeroides, and are arranged in a similar order. Mutations in orf1 and cheA result in impaired chemotaxis, whereas deletion of orf10, appears to have no effect on chemotaxis or motility. Although the putative operon does not contain a cheW homologue, heterologous probing and PCR using consensus primers indicates that cheW maps elsewhere in the Agrobacterium genome.     

An essential virulence protein of Agrobacterium tumefaciens, VirB4, requires an intact mononucleotide binding domain to function in transfer of T-DNA

The 11 gene products of the Agrobacterium tumefaciens virB operon, together with the VirD4 protein, are proposed to form a membrane complex which mediates the transfer of T-DNA to plant cells. This study examined one putative component of that complex, VirB4. A deletion of the virB4 gene on the Ti plasmid pTiA6NC was constructed by replacing the virB4 gene with the kanamycin resistance-conferring nptII gene. The virB4 gene was found to be necessary for virulence on plants and for the transfer of IncQ plasmids to recipient cells of A. tumefaciens. Genetic complementation of the deletion strain by the virB4 gene under control of the virB promoter confirmed that the deletion was nonpolar on downstream virB genes. Genetic complementation was also achieved with the virB4 gene placed under control of the lac promoter, even though synthesis of the VirB4 protein from this promoter is far below wild-type levels. Having shown a role for the VirB4 protein in DNA transfer, lysine-439, found within the conserved mononucleotide binding domain of VirB4, was changed to a glutamic acid, methionine, or arginine by oligonucleotide-directed mutagenesis. virB4 genes bearing these mutations were unable to complement the virB4 deletion for either virulence or for IncQ transfer, showing that an intact mononucleotide binding site is necessary for the function of VirB4 in DNA transfer. The necessity of the VirB4 protein with an intact mononucleotide binding site for extracellular complementation of virE2 mutants was also shown. In merodiploid studies, lysine-439 mutations present in trans decreased IncQ plasmid transfer frequencies, suggesting that VirB4 functions within a complex to facilitate DNA transfer.     

DNA sequences homologous to the T DNA region of Agrobacterium tumefaciens are present in diverse Rhizobium species

Summary DNA sequences homologous to the T DNA region of the octopine-type Ti plasmid from Agrobacterium tumefaciens are present in different Rhizobium species. Plasmid DNA from each of two R. leguminosarum, two R. meliloti, and four slow-growing Rhizobium strains examined contain restriction endonuclease fragments that hybridize with the T DNA region, or with DNA sequences at or near the adjacent Ti plasmid transfer (ra) region. Four different BamHI fragments that contain homology to the T DNA region were cloned from R. leguminosarum 300 plasmid DNA. Cloned fragments of 5.9 kb and 10.3 kb hybridize to each other and are homologous to sequences which map at the right boundary region (EcoRI fragment 24) of the core T DNA. Ti plasmid sequences homologous to those present in cloned fragments of 10.9 kb and 2.0 kb map in adjacent fragments near the tra genes, approximately 10 kb to the right of the core T DNA.     

Behavior of Inc-Q plasmids in Agrobacterium tumefaciens

Inc-Q plasmids were introduced into Agrobacterium tumefaciens, by mobilization from Escherichia coli with an Inc-P plasmid, or by transformation with purified plasmid DNA. It was found that they were stably maintained. The presence of an Inc-Q plasmid did not influence tumorigenicity. These results suggest that these plasmids may be used in genetic complementation studies of Ti plasmid mutants in A. tumefaciens.     

Transfer of RP4::Mu plasmids to Agrobacterium tumefaciens

Transfers of RP4::Mu plasmids from Escherichia coli to Agrobacterium tumefaciens are very inefficient in contrast to the very efficient transfer of RP4. Apparently, one or more Mu functions prevent RPR::Mu plasmids from establishing in some Gram-negatives other than E. coli. This problem was eliminated by the use of a mutant Mu prophage, Mu cts62r23, in RP4. Moreover, the transfer of RP4::Mu cts62r23 to the Agrobacterium strain C58 was found to be affected by a restriction modification system. The target for this restriction was located on Mu DNA and not on RP4 DNA. The plaque-forming phage production of Mu cts62r23 in Agrobacterium was found to be 10⁶ times lower than in E. coli.     

农杆菌介导的棉花枯萎病菌转化体系的优化

【背景】海岛棉相对陆地棉更易感枯萎病,一旦发生很难根治,使得枯萎病逐渐成为威胁新疆海岛棉产业发展的主要病害,但其致病机理目前还不是十分明确。【目的】揭示棉花枯萎病菌的遗传变异和致病机理,同时获得带有绿色荧光蛋白(Green Fluorescent Protein,GFP)标记的棉花枯萎病菌转化子用于观察其侵染海岛棉的途径。【方法】采用农杆菌介导的遗传转化(Agrobacterium tumefaciens-Mediated Transformation,ATMT)方法,对棉花枯萎病菌7号生理小种st89进行了遗传转化并对转化条件进行优化。【结果】农杆菌介导的遗传转化法转化棉花枯萎病菌的最佳条件为:150 mg/L的潮霉素浓度能完全抑制棉花枯萎病菌的生长,浓度为200 mg/L的头孢噻肟钠能完全抑制农杆菌LBA4404生长,农杆菌起始浓度OD₆₀₀为0.2,农杆菌预培养时间为8 h,棉花枯萎病菌分生孢子浓度为10⁵个/mL,枯萎病菌孢子悬液和农杆菌LBA4404比例为1:1,乙酰丁香酮浓度为200μmol/mL,共培养时间为4 d,转化后培养温...     

Agrobacterium tumefaciens TR-DNA encodes a pathway for agropine biosynthesis

Summary A pathway for biosynthesis of the crown-gall opine, agropine is proposed and three potential new precursors characterised. The location of genes involved in the three steps of this pathway was determined by site directed insertions and deletions in the T_R region of the octopine Ti plasmid, pTiB6Tra^c. The proposed biosynthetic pathway for agropine which involves three T-DNA genes is in contrast to the biosynthesis of octopine and nopaline where single T-DNA genes are involved.     

Plasmid DNA of Agrobacterium tumefaciens detected in a presumed habituated tobacco cell line

Summary Tabac anergié tissue has been used by many investigators as a habituated tobacco cell line. In this study, we have found sequences which are homologous to approximately 9 megadaltons of an Agrobacterium octopine-type plasmid in DNA isolated from Tabac anergié tissue. These T-DNA sequences were shown to be the same core DNA sequences found to be present in several other unorganized tumor lines which have been studied in our laboratory. The T-DNA was also shown to be integrated into plant DNA. Thus, the Tabac anergié tissue is a crown gall tumor induced by a strain of Agrobacterium tumefaciens containing an octopinetype plasmid. These results indicated that the plants regenerated from 40 clones of the Tabac anergié line by Sacristán and Melchers (1977) were actually reverted plants obtained from an unorganized crown gall tumor, kept in culture for more than 25 years. It is necessary to re-evaluate observations and conclusions which have been based upon Tabac anergié being a habituated tissue.     

Detection of T-DNA transfer to plant cells by A. tumefaciens virulence mutants using agroinfection

Summary To test whether virulence mutants of Agrobacterium tumefaciens are capable of promoting T-DNA transfer into plant cells, a tandem array of Cauliflower Mosaic Virus (CaMV) DNA was cloned between T-region border sequences on a wide host range plasmid and introduced into various virulence mutants. The resulting strains were used to infect Brassica rapa cv. Just Right. This assay, recently referred to as agroinfection, is based on the appearance of viral symptoms following transfer of T-DNA to plant cells, and is shown to be at least 100 times more sensitive in detecting T-DNA transfer than tumour formation. Mutants in the loci vir A, B and G, which were avirulent on turnip, failed to induce virus symptoms. Of the two vir D mutants tested, neither induced tumours, but one was capable of inducing virus symptoms. Mutants in vir E, C and F, which induced respectively no, small and normal tumours on turnip, all induced virus symptoms.     

Cloning and characterisation of the recA gene of Agrobacterium tumefaciens C58

Summary A gene library of Agrobacterium tumefaciens C58 has been constructed in the plasmid vector pACYC184. A recombinant plasmid was isolated from the library by interspecific complementation in E. coli, which contained the A. tumefaciens recA gene. Heterologous Southern blotting and DNA sequence analysis have demonstrated the existence of considerable homology between the recA genes of A. tumefaciens, E. coli and R. meliloti.Abbreviations MMS methyl methanesulfonate - UV ultraviolet light - bp base pairs - kbp kilo base pairs - dATP deoxyadenosine 5-triphosphate - dNTP deoxynucleoside triphosphate - Ap ampicillin - Cm chloramphenicol - Km kanamycin - Tet tetracycline     

根癌农杆菌介导的玫瑰茄愈伤组织的遗传转化

以根癌农杆菌LBA4404和EHA105为供体菌株,对玫瑰茄愈伤组织进行了转化条件的研究,建立了一套玫瑰茄愈伤组织遗传转化体系。利用该转化体系获得了2个稳定表达新霉素磷酸转移酶活性的玫瑰茄转化细胞系。GUS活性组织化学检测和PCR扩增鉴定的结果表明,愈伤组织的转化率为4%。说明采用农杆菌介导法将外源基因经愈伤组织导入玫瑰茄细胞是可行的。     

Expression of various genes to enhance ubiquinone metabolic pathway in Agrobacterium tumefaciens

Ubiquinone (UQ), a lipid-soluble component, acts as a mobile component of the respiratory chain by playing an essential role in the electron transport system in many organisms, and has been widely used in pharmaceuticals due to its antioxidant property. The biosynthesis of UQ involves 10 sequential reactions brought about by various enzymes. In this study, dps gene, which encodes decaprenyl diphosphate synthase, involved in ubiquinone biosynthesis from Agrobacterium tumefaciens, and coq2 gene of Saccharomyces cerevisiae, ppt1 gene of Schizosaccahromyces pombe and ubiA gene of Escherichia coli, all of them encoding 4-hydroxybenzoate:polyprenyl diphosphate (4-HB:PPP) transferase, were reconfigured into an operon under the control of a single promoter to yield various plasmids including pBIV-dps, pBIV-dpsq, pBIV-dpsp and pBIV-dpsca. The recombinant A. tumefaciens containing dps-ubiC-ubiA gene showed the highest level ubiquinone production than that of the other recombinants and the nonrecombinant bacterium. In an aerobic fed-batch fermentation, A. tumefaciens containing the pBIV-dpsca plasmid produced 25.2 mg of ubiquinone-10 per liter which was 1.68 times higher than that of nonrecombinant type. While in microaerobic fed-batch fermentation, recombinant cell pBIV-dpsca produced 30.8 mg L⁻¹ of ubiquinone-10. Compared to the original A. tumefaciens, the ubiquinone-10 yield and productivities of the recombinant bacterium pBIV-dpsca increased 88.9% and 77.7%, respectively, under microaerobic fed-batch conditions.     

Suppression of tumorigenicity by the IncW R plasmid pSa in Agrobacterium tumefaciens

Summary The effect of the IncW R plasmid, pSa, on tumorigenicity and on the expression and maintenance of the Ti plasmid in tumorigenic strains of A. tumefaciens was determined. Plasmid pSa could be transferred into and stably maintained by both octopine-and nopaline-utilizing A. tumefaciens strains. The R plasmid had no effect on Ti plasmid maintenance or on Ti plasmid functions, such as octopine utilization or conjugal bacterial transfer. However, A. tumefaciens strains harboring both the R plasmid and the Ti plasmid in most instances failed to induce tumors on a number of plant species. This effect on tumorigenicity is specific to pSa. When pSa is cured from the A. tumefaciens transconjugants or when their Ti plasmids are genetically transferred to an appropriate recipient, the resultant strains lacking the R plasmid regain tumorigenicity. Restriction endonuclease analysis of plasmid DNA isolated from transconjugants harboring pSa showed no difference in Ti plasmid cleavage patterns when compared to plasmid DNA isolated from the tumorigenic parent strain. These results indicate that pSa does not induce detectable permanent genetic alteration of the Ti plasmid. Rather, it appears that the R plasmid suppresses some Ti plasmid function(s) necessary for tumorigenicity.     

Cloning of the Agrobacterium tumefaciens C58 trpE gene by complementation in Escherichia coli

Summary The trpE gene of Agrobacterium tumefaciens C58 was cloned from a gene library by complementation in Escherichia coli. It was shown to be unlinked to trpD gene in this organism. It was also shown that the nontumorigenic phenotype of tryptophan auxotrophs of A. tumefaciens could be complemented by addition of exogenous tryptophan. The role of bacterially synthesised tryptophan in the process of tumour formation is discussed.Abbreviations Ap ampicillin - Cm chloramphenicol - Gent gentamycin - Km kanamycin - dATP deoxyadenosine 5-triphosphate - IAA indole acetic acid - NB nutrient broth - MinAB minimal Agrobacterium medium     

Tzs, a nopaline Ti plasmid gene from Agrobacterium tumefaciens associated with trans-zeatin biosynthesis

Summary The tzs gene, present in nopaline Ti plasmids, confers on Agrobacterium tumefaciens the ability to produce the phytohormone, trans-zeatin (Regier and Morris (1982) Biochem Biophys Res Comm 104:1560–1566). This gene has now been cloned from the nopaline Ti plasmid pTiC58. It occurs outside the T-DNA in a region close to that associated with virulence functions. Sequence studies indicate that tzs has substantial homology with the T-region gene, ipt, which is known to encode a dimethylallylpyrophosphate transferase, the first enzyme of the cytokinin biosynthetic pathway. As expected from its homology with ipt, tzs possesses significant DMA transferase activity but when expressed in Escherichia coli it causes secretion of trans-zeatin.     

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.     

Regions of DNA sequence homology between an octopine and a nopaline Ti plasmid of Agrobacterium tumefaciens

Summary Despite the fact that pTiC58 and pTiB6S3 functionally, have been shown to date to have only tumorigenicity and phage AP1 exclusion in common, many restriction fragments of the plasmids contain DNA sequences common to both. The bulk of this homologous DNA is concentrated in a few restriction endonuclease fragments and the remainder is organized in short discontinuous regions spread over many fragments. In pTiB6S3 the bulk of the homology is distributed throughout a 29x10⁶ dalton segment comprising 8 Sma I fragments. This region includes those sequences which are transferred to and transcribed in tumorigenic plant cells induced by B6-806 or closely related strains. The pattern of homology within this portion of the plasmid shows a region of low sequence homology (Sma I Fragment 3 b) apparently corresponding to the gene or genes coding for octopine synthesis in the plant tumor cells, surrounded by regions of high sequence homology. The extent of inter-plasmid homology then decreases with increasing distance from fragment 3b. The remainder of the homology is distributed throughout a segment of maximum size 21.5x10⁶ daltons comprising two Sma I fragments and cannot yet be definitely linked with any specific plasmid function.