The virB operon of Agrobacterium tumefaciens pTiC58 encodes 11 open reading frames

Summary Agrobacterium tumefaciens genetically transforms plant cells by transferring a copy of its T-DNA to the plant where it is integrated and stably maintained. In the presence of wounded plant cells this process is activated and mediated by the products of the vir genes which are grouped into six distinct loci. The largest is the virB locus spanning 9.5 kb. Transposon mutagenesis studies have shown that virB gene products are required for virulence but their functions remain largely unknown. To provide information relevant to understanding the function of VirB polypeptides, the nucleotide sequence of the virB operon from a nopaline plasmid, pTiC58, is presented here. Eleven open reading frames (ORFs) are predicted from this sequence. The predicted sizes of 10 of the 11 VirB polypeptides are verified by specific expression in Escherichia coli. Only the product of the smallest ORF potentially encoding a 5.8 kDa polypeptide has not been detected. The initiation of translation of five virB ORFs occurs at codons that overlap the termination codons of the ORF immediately upstream; thus, translational coupling may be an important mechanism for efficient translation of the large virB polycistronic mRNA. Based on hydropathy plot analysis nine of the virB ORFs encode proteins that may interact with membranes; these data support the earlier hypothesis (Engstromm et al. 1987) that virB gene products may form a membrane pore or channel to mediate exit of the T-DNA copy (T-strands) from Agrobacterium into the plant cell. A comparison of the two published octopine virB sequences with the nopaline sequence presented here is made.     

Klebsiella pneumoniae nif-lac fusions are expressed in Agrobacterium tumefaciens C58

Summary Plasmids containing hybrid genes, in which different Klebsiella pneumoniae nif (nitrogen-fixation) promoters were fused with the structural part of the Escherichia coli lac operon, were introduced into a double auxotrophic derivative of Agrobacterium tumefaciens C58. A study of their expression in the new host was made simple by the inherent inability of A. tumefaciens C58 to produce -galactosidase unless provided with the wild-type lac operon of E. coli. As shown by quantitative measurements of the enzyme, all K. pneumoniae promoters were expressed well in A. tumefaciens C58, even under conditions known to repress them. It also has been shown that the activity of K. pneumoniae nif A is essential for the expression of nifHDK even when introduced into A. tumefaciens. After entering the new host the plasmids, the nif genes and the fusion alleles contained in them, remained stable. Possible mechanisms responsible for the constitutive behaviour of nif promoters in A. tumefaciens are discussed.     

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     

A functional map of the nopaline catabolism genes on the Ti plasmid of Agrobacterium tumefaciens C58

Summary The nopaline catabolism (noc) genes are located in a 14.4 kb region on the pTiC58 plasmid of A. tumefaciens C58. These genes permit the bacterium to grow on nopaline N²-(1,3-dicarboxylpropyl) arginine, a substrate produced in plant tumors initiated by strain C58. The functions of the noc genes include the use of nopaline and L-ornithine as sole carbon and nitrogen sources. Using Tn5 insertional mutants, we have identified and mapped the positions of the genes that are responsible for nopaline catabolism (NopC), ornithine catabolism (OrnC) and nopaline uptake (NopU). A polar relationship was found between these phenotypes, which extended leftward over the noc region to the T-region. The NopC mutants were also deficient in nopaline oxidase, an enzyme that liberates free arginine from nopaline.The noc region also encodes the synthesis of a periplasmic protein, n1 that was induced by nopaline. Tn5 insertional mutations and molecular cloning were used to map the n1 production locus. The recombinant plasmids, pSa4480 and pSa4481, containing the 8.9 kb right-hand end of the noc region, conferred n1 production when introduced into a pTi-free strain of A. tumefaciens. Production of n1 by the strains carrying these plasmids required nopaline induction.We have identified in toto three noc loci: nocB, nocC, and nocA, which confer n1 production, nopaline oxidase production and ornithine catabolism respectively. A model is proposed whereby the noc genes of pTiC58 are contained on a leftward reading operon in the order nocB, nocC, and nocA.     

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.     

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.     

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     

T-DNA is organized predominantly in inverted repeat structures in plants transformed with Agrobacterium tumefaciens C58 derivatives

Summary The detailed structural organization of DNA sequences transferred to the plant genome via Agrobacterium tumefaciens has been determined in 11 transgenic tomato plants that carry the transferred DNA (T-DNA) at a single genetic locus. The majority (seven) of these plants were found to carry multiple copies of T-DNA arranged in inverted repeat structures. Such a high frequency of inverted repeats among transgenotes has not been previously reported and appears to be characteristic of transformation events caused by C58/pGV3850 strains of Agrobacterium. The inverted repeats were found to be centered on either the left or the right T-DNA boundary and both types were observed at similar frequency. In several plants both types of inverted repeat were found to coexist in the same linear array of elements. Direct repeats were observed in two plants, each time at the end of an array of inverted repeat elements, and at a lower frequency than inverted repeats. The junctions between T-DNA elements and plant DNA sequences and the junctions between adjacent T-DNA elements were mapped in the same 11 plants, allowing the determination of the distribution of junction points at each end for both types of junction. Based on a total of 17 distinct junctions at the right end of T-DNA and 19 at the left end, the distribution of junction points was found to be much more homogeneous at the right end than at the left end. Left end junctions were found to be distributed over a 3 kb region of T-DNA with two thirds of the junctions within 217 bp of the left repeat. Two thirds of the right end junctions were found to lie within 11 bp of the right repeat with the rest more than 39 bp from the right repeat. T-DNA::plant DNA junctions and T-DNA::T-DNA inverted repeat junctions showed similar distributions of junction points at both right and left ends. The possibilities that T-DNA inverted repeats are unstable in plants and refractory to cloning in wild type Escherichia coli is discussed. Two distinct types of mechanisms for inverted repeat formation are contrasted, replication and ligation mechanisms.     

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.     

T-DNA structure and gene expression in petunia plants transformed by Agrobacterium tumefaciens C58 derivatives

Summary We have previously described substantial variation in the level of expression of two linked genes which were introduced into transgenic petunia plants using Agrobacterium tumefaciens. These genes were (i) nopaline synthase (nos) and (ii) a chimeric chlorophyll a/b binding protein/octopine synthase (cab/ocs) gene. In this report we analyze the relationship between the level of expression of the introduced genes and T-DNA structure and copy number in 40 transgenic petunia plants derived from 26 transformed calli. Multiple shoots were regenerated from 8 of these calli and in only 6 cases were multiple regenerated shoots from each callus genotypically identical to each other. Many genotypes showed no nos gene expression (22/28). Most of the plants (16/22) which lacked nos gene expression did contain nos-encoding DNA with the expected restriction enzyme map. Similarly, amongst the genotypes showing no cab/ocs gene expression, the majority (11/28) did not show any alterations in restriction fragments corresponding to the expected cab/ocs coding sequences (10/11). Approximately half of the plants carried multiple copies of T-DNA in inverted repeats about the left or right T-DNA boundaries. No positive correlation was observed between the copy number of the introduced DNA and the level of expression of the introduced genes. However, plants with high copy number complex insertions composed of multiple inverted repeats in linear arrays usually showed low levels of expression of the introduced genes.     

Octopine and nopaline synthesis and breakdown genetically controlled by a plasmid of Agrobacterium tumefaciens

Summary Several nopaline degrading strains and one octopine degrading strain are shown to loose oncogenicity as well as the ability to utilize these guanidine compounds when they are cured of their TI plasmid. To investigate whether the specific genes involved in the utilization of one or the other compound are located on the plasmid, plasmid-transfer experiments have been performed.The plasmid from a nopaline degrading strain has been transferred to a naturally non oncogenic Agrobacterium namely A. radiobacter. Furthermore, the plasmid from an octopine degrading strain has been transferred to a plasmid-cured strain which originally had the capacity to utilize nopaline. Both kinds of experiments prove that the TI plasmid determines the strain specificity with regard to the utilization of either octopine or nopaline.They also demonstrate that the synthesis of either octopine or nopaline in crown gall cells is also determined by genes located on the TI plasmid harboured by the transforming A. tumefaciens strains.     

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.     

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.     

A spontaneous insertion in the agrocin sensitivity region of the Ti-plasmid of Agrobacterium tumefaciens C58

A spontaneous agrocin-resistant mutant of Agrobacterium tumefaciens strain C58 was shown to have an insertion of 1.2 kb in the agrocin-sensitivity region of pTiC58. The insertion was cloned from the Ti-plasmid, and a subclone containing only DNA internal to the insertion was used to probe the Ti-plasmid and chromosomal DNA of the wild-type strain C58. The probe showed homology to chromosomal sequences but showed no homology to wild-type pTiC58. Homology was also detected with chromosomal sequences of A. tumefaciens strains, B6, K24, and T37. These results suggest that an indigenous insertion sequence of 1.2 kb transposed from the chromosome to the agrocin-sensitivity region of the Ti-plasmid in this spontaneous mutant of C58.     

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。     

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.