The interaction of Agrobacterium Ti-plasmid DNA and plant cells

The tumour-inducing plasmids of Agrobacterium tumefaciens (Ti-plasmids) reveal several interesting properties. They are catabolic plasmids, which, instead of rendering Agrobacterium strains capable of catabolizing compounds found in Nature, force a plant to synthesize these catabolites (denoted 'opines'). This situation is obtained by insertion of a segment of the Ti-plasmid (the T-DNA) into the plant nucleus, where T-DNA genes become expressed and intervene in the biosynthesis of these opines. Cells containing the T-DNA behave as neoplasms (crown gall cells). Southern blotting shows that the insertion process responsible for T-DNA transfer probably recognizes special sequences on the T-DNA since the length of the T-DNA segment observed in different, independently isolated tumour lines was found to be similar. For the nopaline Ti-plasmids both left-hand and right-hand borders were found to be constant. For the octopine plasmid the left border was constant and at least two classes of right-hand borders were found. Upon redifferentiation of the transformed plant cells, the T-DNA was found to be conserved in all somatic cells examined. However, small deletions at the border fragments of the T-DNA have been observed. The exact arrangement and copy number of the T-DNA in a nucleus is still under study, but genomic cloning has already revealed that an interspersed tandem arrangement is dominant in nopaline tumours. Clones containing both the right border of one T-DNA and the left border of the neighbouring tandem T-DNA were isolated. In order to identify the different T-plasmid encoded functions an extensive use was made of transposon insertion mutagenesis. When an antibiotic resistance transposon was inserted into the non-essential regions of the T-DNA, a linked transfer to the plant DNA of the transposon together with the T-DNA was observed. This indicates that Ti-plasmids are possible vectors for genetic engineering in plants. A strategy is described for insertion of any cloned DNA segment into the T-DNA.     

转移DNA

农杆菌能够将其环状质粒上的一段转移ＤＮＡ(ｔｒａｎｓｆｅｒＤＮＡ ,Ｔ ＤＮＡ)转移并整合到受体细胞的基因组中 ,并使之携带的基因在受体细胞中表达。利用这种天然载体转化系统 ,除了可以在细菌间进行接合转移外 ,还可以向真菌、放线菌等低等真核生物和许多高等植物基因组转移[1] 。利用人工构建的转移复合物可以将ＤＮＡ转运到哺乳动物的细胞核中[2 ] 。虽然不同农杆菌质粒ＤＮＡ同源性有较大差异 ,但仍有一些共有的保守区段 ,如Ｔ ＤＮＡ区、Ｖｉｒ区、质粒复制起始区、质粒接合转移毒性区等。其中Ｔ ＤＮＡ区和Ｖｉｒ区是与Ｔ ＤＮＡ…     

Transfer of T-DNA from Agrobacterium to the plant cell.

Agrobacterium tumefaciens is the causative agent of crown gall, a disease of dicotyledonous plants characterized by a tumorous phenotype. Earlier in this century, scientific interest in A. tumefaciens was based on the possibility that the study of plant tumors might reveal mechanisms that were also operating in animal neoplasia. In the recent past, the tumorous growth was shown to result from the expression of genes coded for by a DNA segment of bacterial origin that was transferred and became stably integrated into the plant genome. This initial molecular characterization of the infection process suggested that Agrobacterium might be used to deliver genetic material into plants. The potential to genetically engineer plants generated renewed interest in the study of A. tumefaciens. In this review, we concentrate on the most recent advances in the study of Agrobacterium-mediated gene transfer, its relationship to conjugation, DNA processing and transport, and nuclear targeting. In the following discussion, references for earlier work can be found in more comprehensive reviews (Hooykaas and Schilperoort, 1992; Zambryski, 1992; Hooykaas and Beijersbergen, 1994).     

Agrobacterium rhizogenes-mediated DNA transfer inPinus halepensis Mill.

Agrobacterium rhizogenes strain LBA9402 was used to transformPinus halepensis embryos, seedlings and shoots. Mature embryos exhibited susceptibility to the agrobacterium as monitored by -glucurortidase (GUS) expression, with more than 85% showing considerable transient GUS expression in the radicle. GUS expression was also observed in cotyledons, but at a lower rate of about 24% of the embryos (1–5 spots/embryo). Stable transformation was evidenced by the regeneration of GUS-expressing roots and calli from infectedP. halepensis seedlings. Inoculum injections into intact seedling hypocotyls induced callus and root formation at the wound sites in 64% of the seedlings. Dipping seedling cuttings in a bacterial suspension resulted in adventitious root formation in 7I% of the seedling cuttings, all of which expressed GUS activity. Adventitious shoots, that were induced on 2.5-year-old seedlings by pruning and spraying with 6-benzylaminopurine, were infected by injecting of bacterial suspension into their basal side. Two months later, adventitious roots and root primordia regenerated in 74% and 40% of 2- and 5-month-old shoots, respectively. Non-transformed shoots, either without or with auxin application, failed to form roots. Polymerase chain reaction and Southern blot analyses confirmed theuidA-transgenic nature of the root and callus, as well as the presence ofrolC androlB genes in roots from infectedP. halepensis seedlings.Abbreviations BA 6-benzylaminopurine - NOS nopaline synthase - PCR polymerise chain reaction - EtOH ethanol - GUS -glucuronidase - NPTII neomycin phosphotransferase II - CaMV cauliflower mosaic virus - X-gluc 5-bromo-4-chloro-3-indolyl -D-glucuronic acid     

Formation of Complex Extrachromosomal T-DNA Structures in Agrobacterium tumefaciens-Infected Plants

Agrobacterium tumefaciens is a unique plant pathogenic bacterium renowned for its ability to transform plants. The integration of transferred DNA (T-DNA) and the formation of complex insertions in the genome of transgenic plants during A. tumefaciens-mediated transformation are still poorly understood. Here, we show that complex extrachromosomal T-DNA structures form in A. tumefaciens-infected plants immediately after infection. Furthermore, these extrachromosomal complex DNA molecules can circularize in planta. We recovered circular T-DNA molecules (T-circles) using a novel plasmid-rescue method. Sequencing analysis of the T-circles revealed patterns similar to the insertion patterns commonly found in transgenic plants. The patterns include illegitimate DNA end joining, T-DNA truncations, T-DNA repeats, binary vector sequences, and other unknown "filler" sequences. Our data suggest that prior to T-DNA integration, a transferred single-stranded T-DNA is converted into a double-stranded form. We propose that termini of linear double-stranded T-DNAs are recognized and repaired by the plant's DNA double-strand break-repair machinery. This can lead to circularization, integration, or the formation of extrachromosomal complex T-DNA structures that subsequently may integrate.     

Protamine Sulfate Provides Enhanced and Reproducible Intravenous Gene Transfer by Cationic Liposome/DNA Complex

Abstract

A novel lipid/polycation/DNA (LPD) formulation has been developed for in vivo gene transfer. It involves the condensation of plasmid DNA with protamine sulfate, a cationic polypeptide, followed by the addition of DOTAP cationic liposomes. Compared with DOTAP/DNA complex, LPD offers greater protection of plasmid DNA against enzymatic digestion and gives consistently higher gene expression in mice via tail vein injection. The in vivo efficiency of LPD was dependent upon charge ratio and was also affected by the lipid used. Increasing the amount of DNA delivered induced an increase in gene expression. The optimal dose was approximately 50 μg per mouse, at which concentration approximately 10 ng luciferase protein per mg extracted tissue protein could be detected in the lung. Gene expression in the lung was detected as early as 1 h after injection, peaked at 6 h, and declined thereafter. Using LacZ as a reporter gene, it was shown that endothelial cells were the primary locus of transgene expression in both lung and spleen. No sign of inflammation in these organs was noticed. Since protamine sulfate has been proven to be non-toxic and only weakly immunogenic in humans, this novel vector may be useful for the clinical use of gene therapy.     

苜蓿高含硫氨基酸蛋白转基因植株再生

通过农杆菌介导法将高含硫氨基酸蛋白基因转入苜蓿,成功地诱导转基因植株再生,转化植株生长和发育良好,苜蓿子叶外植体是较理想的转化受体。冷凉湿润的环境条件是苜蓿移栽成活率高所必需的。     

DNA 的电荷转移

在双链DNA分子中,电荷常出现远距离的迁移现象,这与很多生物学功能有密切的关系,本文说明了DNA电荷转移的形成机制,分析了误配、绞联、三股螺旋及DNA结合蛋白对DNA电荷转移效率的影响,同时论述了DNA电荷转移的生物学意义。     

T-DNA-mediated transfer of Agrobacterium tumefaciens chromosomal DNA into plants

Besides the well-documented integration of DNA flanked by the transfer DNA borders, occasional insertion of fragments from the tumor-inducing plasmid into plant genomes has also been reported during Agrobacterium tumefaciens-mediated transformation. We demonstrate that large (up to approximately 18 kb) gene-bearing fragments of Agrobacterium chromosomal DNA (AchrDNA) can be integrated into Arabidopsis thaliana genomic DNA during transformation. One in every 250 transgenic plants may carry AchrDNA fragments. This has implications for horizontal gene transfer and indicates a need for greater scrutiny of transgenic plants for undesired bacterial DNA.     

Reiterated DNA sequences in Rhizobium and Agrobacterium spp.

Repeated DNA sequences are a general characteristic of eucaryotic genomes. Although several examples of DNA reiteration have been found in procaryotic organisms, only in the case of the archaebacteria Halobacterium halobium and Halobacterium volcanii [C. Sapienza and W. F. Doolittle, Nature (London) 295:384-389, 1982], has DNA reiteration been reported as a common genomic feature. The genomes of two Rhizobium phaseoli strains, one Rhizobium meliloti strain, and one Agrobacterium tumefaciens strain were analyzed for the presence of repetitive DNA. Rhizobium and Agrobacterium spp. are closely related soil bacteria that interact with plants and that belong to the taxonomical family Rhizobiaceae. Rhizobium species establish a nitrogen-fixing symbiosis in the roots of legumes, whereas Agrobacterium species is a pathogen in different plants. The four strains revealed a large number of repeated DNA sequences. The family size was usually small, from 2 to 5 elements, but some presented more than 10 elements. Rhizobium and Agrobacterium spp. contain large plasmids in addition to the chromosomes. Analysis of the two Rhizobium strains indicated that DNA reiteration is not confined to the chromosome or to some plasmids but is a property of the whole genome.     

Agrobacterium tumefaciens supports DNA replication of diverse geminivirus types

We have previously shown that the soil-borne plant pathogen Agrobacterium tumefaciens supports the replication of tomato leaf curl geminivirus (Australian isolate) (TLCV) DNA. However, the reproducibility of this observation with other geminiviruses has been questioned. Here, we show that replicative DNA forms of three other geminiviruses also accumulate at varying levels in Agrobacterium. Geminiviral DNA constructs that lacked the ability to replicate in Agrobacterium were rendered replication-competent by changing their configuration so that two copies of the viral ori were present. Furthermore, we report that low-level replication of TLCV DNA can occur in Escherichia coli containing a dimeric TLCV construct in a high copy number plasmid. These findings were reinforced by expression studies using beta-glucuronidase which revealed that all six TLCV promoters are active in Agrobacterium, and two are functional in E. coli.     

Azospirillum DNA shows homology with Agrobacterium chromosomal virulence genes

DNA from Azospirillum brasilense Sp7 (ATCC 29145) was hybridized with probes containing the T-region and the vir-region of a nopaline Ti-plasmid, and the chromosomal virulence region (chv) of Agrobacterium tumefaciens. Homology to chv was found. Hybridization signals with the chv probe were detected in 7 A. brasilense strains and 1 A. lipoferum strain tested.     

Characterization of Agrobacterium tumefaciens DNA ligases C and D

Agrobacterium tumefaciens encodes a single NAD⁺-dependent DNA ligase and six putative ATP-dependent ligases. Two of the ligases are homologs of LigD, a bacterial enzyme that catalyzes end-healing and end-sealing steps during nonhomologous end joining (NHEJ). Agrobacterium LigD1 and AtuLigD2 are composed of a central ligase domain fused to a C-terminal polymerase-like (POL) domain and an N-terminal 3′-phosphoesterase (PE) module. Both LigD proteins seal DNA nicks, albeit inefficiently. The LigD2 POL domain adds ribonucleotides or deoxyribonucleotides to a DNA primer-template, with rNTPs being the preferred substrates. The LigD1 POL domain has no detectable polymerase activity. The PE domains catalyze metal-dependent phosphodiesterase and phosphomonoesterase reactions at a primer-template with a 3′-terminal diribonucleotide to yield a primer-template with a monoribonucleotide 3′-OH end. The PE domains also have a 3′-phosphatase activity on an all-DNA primer-template that yields a 3′-OH DNA end. Agrobacterium ligases C2 and C3 are composed of a minimal ligase core domain, analogous to Mycobacterium LigC (another NHEJ ligase), and they display feeble nick-sealing activity. Ligation at DNA double-strand breaks in vitro by LigD2, LigC2 and LigC3 is stimulated by bacterial Ku, consistent with their proposed function in NHEJ.     

The Infidelity of Conjugal DNA Transfer in ESCHERICHIA COLI

The accuracy of replication and transfer of a lacI gene on an F' plasmid was measured. Following conjugal transfer of the F', a small but reproducible increase (1.8-fold) in the frequency of lacI- mutations was detected. Among these, however, the frequency of nonsense mutations was 15-fold higher than in the absence of transfer. This corresponds to a 300-fold increase in the rate of base substitutions per round of replication compared with normal vegetative DNA replication. The amber mutational spectra revealed that, following conjugal transfer, mutation frequencies were increased markedly at all sites detected. In addition, an increase in G:C leads to A:T transitions was noted and was due almost entirely to an enhanced proportion of mutants recovered at the spontaneous hotspots (amber sites 6, 15 and 34). recA-dependent processes were not responsible for the increase in mutation, since similar results were observed with various recA- donor and recipient combinations. These results demonstrate that the fidelity of conjugal DNA replication is considerably lower than that of vegetative DNA replication.     

Transfer of the Ti plasmid from Agrobacterium tumefaciens into Escherichia coli cells

We have screened strains of Agrobacterium tumefaciens for spontaneous mutants showing constitutive transfer of the nopaline Ti plasmid pTiC58 during conjugation. The Ti plasmid derivatives obtained could be transferred not only to A. tumefaciens but also to E. coli cells. The Ti plasmid cannot survive as a freely replicating plasmid in E. coli, but it can occasionally integrate into the E. coli chromosome. However, insertion in tandem of plasmids carrying fd replication origins (pfd plasmids) into the T-DNA provides an indicator for all transfer events into E. coli cells, providing fd gene 2 protein is present in these cells. This viral protein causes the excision of one copy of the pfd plasmid and allows its propagation in the host cell. By using this specially designed Ti plasmid, which was also made constitutive in transfer functions, we found plasmid exchange among A. tumefaciens strains and between A. tumefaciens and E. coli cells to be equally efficient. A Ti plasmid with repressed transfer functions was transferred to E. coli with a rate similar to the low frequency at which it was transferred to A. tumefaciens. The expression of transfer functions of plasmid RP4 either in A. tumefaciens or in E. coli did not increase the transfer of the Ti plasmid into E. coli cells, nor did the addition of acetosyringone, an inducer of T-DNA transfer to plant cells. The results show that A. tumefaciens can transfer the Ti plasmid to E. coli with the same efficiency as within its own species. Conjugational transmission of extrachromosomal DNA like the narrow-host-range Ti plasmid may often not only occur among partners allowing propagation of the plasmid, but also on a 'try-all' basis including hosts which do not replicate the transferred DNA.