Identification of amino acid residues important for the function of Agrobacterium tumefaciens Irr protein

The key amino acid residues that influence the function of the Agrobacterium tumefaciens iron response regulator protein (Irr(At) ) were investigated. Several Irr(At) mutant proteins containing substitutions in amino acids corresponding to candidate metal- and haem-binding sites were constructed. The ability of the mutant proteins to repress the promoter of the membrane bound ferritin (mbfA) gene was investigated using a promoter-lacZ fusion assay. A single mutation at residue H94 significantly decreased the repressive activity of Irr(At) . Multiple mutation analysis revealed the importance of H45, H65, the HHH motif (H92, H93 and H94) and H127 for the repressor function of Irr(At) . H94 is essential for the iron responsiveness of Irr(At) . Furthermore, the Irr(At) mutant proteins showed differential abilities to complement the H(2) O(2) -hyper-resistant phenotype of an irr mutant.     

Biological control of crown gall, Agrobacterium tumefaciens

Biological control of crown gall caused by Agrobacteriurn turnefaciens (Smith & Townsend) Conn, pioneered by Dr A. Kerr in South Australia, is effected through the establishment of a high population of the related non-pathogen A. radiobacter (Beijerinck & van Delden) Conn, strain 84 in the rhizosphere of susceptible plants. Strain 84 produces a bacteriocin to which many strains of the pathogen in Australasia, North America and Britain are sensitive. The disease is present in Britain on a variety of hosts including cherry. At East Malling cherry leaf scars, invaluable as an avenue of infection for bacterial canker infectivity titrations, have been used successfully in crown gall studies. Live cells of strain 84, but neither an avirulent strain of the pathogen nor a soil bacterium highly antagonistic to A. tumefaciens in vitro, inhibited gall formation in cherry leaf scars. Heat-killed cells had no effect. In a field experiment at East Malling hardwood cuttings of the new rootstock Colt have been dipped in strain 84 and total inhibition of crown gall is expected to ensue. The results of other experiments where the disease is already established on cherry-rootstock layer-beds and in blackberry plantations are less predictable. In time we hope to solve this problem. Only time will show whether this method of biological control is long lasting or will eventually break down.     

Efficient transformation of Agrobacterium tumefaciens by electroporation

High-voltage electroporation was used to transform Agrobacterium tumefaciens strains A136 and A348, reaching the efficiency of 1-3 x 10(8) transformants/micrograms DNA. Transformation frequency was dependent on the electrical field strength and the pulse length. No significant reduction in transformation efficiency was observed when the transforming DNA contained sites sensitive to endonuclease AtuCI of A. tumefaciens.     

Transformation of rice mediated by Agrobacterium tumefaciens

Agrobacterium tumefaciens has been routinely utilized in gene transfer to dicotyledonous plants, but monocotyledonous plants including important cereals were thought to be recalcitrant to this technology as they were outside the host range of crown gall. Various challenges to infect monocotyledons including rice with Agrobacterium had been made in many laboratories, but the results were not conclusive until recently. Efficient transformation protocols mediated by Agrobacterium were reported for rice in 1994 and 1996. A key point in the protocols was the fact that tissues consisting of actively dividing, embryonic cells, such as immature embryos and calli induced from scutella, were co-cultivated with Agrobacterium in the presence of acetosyringonc, which is a potent inducer of the virulence genes. It is now clear that Agrobacterium is capable of transferring DNA to monocotyledons if tissues containing competent cells are infected. The studies of transformation of rice suggested that numerous factors including genotype of plants, types and ages of tissues inoculated, kind of vectors, strains of Agrobacterium, selection marker genes and selective agents, and various conditions of tissue culture, are of critical importance. Advantages of the Agrobacterium-mediated transformation in rice, like on dicotyledons, include the transfer of pieces of DNA with defined ends with minimal rearrangements, the transfer of relatively large segments of DNA, the integration of small numbers of copies of genes into plant chromosomes, and high quality and fertility of transgenic plants. Delivery of foreign DNA to rice plants via A. tumefaciens is a routine technique in a growing number of laboratories. This technique will allow the genetic improvement of diverse varieties of rice, as well as studies of many aspects of the molecular biology of rice.     

Constitutive activation of two-component response regulators: characterization of VirG activation in Agrobacterium tumefaciens

Response regulators are the ultimate modulators in two-component signal transduction pathways. The N-terminal receiver domains generally accept phosphates from cognate histidine kinases to control output. VirG for example, the response regulator of the VirA/VirG two-component system in Agrobacterium tumefaciens, mediates the expression of virulence genes in response to plant host signals. Response regulators have a highly conserved structure and share a similar conformational activation upon phosphorylation, yet the sequence and structural features that determine or perturb the cooperative activation events are ill defined. Here we use VirG and the unique features of the Agrobacterium system to extend our understanding of the response regulator activation. Two previously isolated constitutive VirG mutants, VirGN54D and VirGI77V/D52E, provide the foundation for our studies. In vivo phosphorylation patterns establish that VirGN54D is able to accumulate phosphates from small-molecule phosphate donors, such as acetyl phosphate, while the VirGI77V/D52E allele carries conformational changes mimicking the active conformation. Further structural alterations on these two alleles begin to reveal the changes necessary for response regulator activation.     

Transformation of azuki bean by Agrobacterium tumefaciens

Stable transformation and regeneration was developed for a grain legume, azuki bean (Vigna angularis Willd. Ohwi & Ohashi). Two constructs containing the neomycin phosphotransferase II gene (nptII) and either the -glucuronidase (GUS) gene or the modified green fluorescent protein [sGFP(S65T)] gene were introduced independently via Agrobacterium tumefaciens-mediated transformation. After 2 days of co-cultivation on MS medium supplemented with 100 M acetosyringone and 10 mg l^–1 6-benzyladenine, seedling epicotyl explants were placed on regeneration medium containing 100 mg l^–1 kanamycin. Adventitious shoots developing from explant calli were excised onto rooting medium containing 100 mg l^–1 kanamycin. Rooted shoots were excised and repeatedly selected on the same medium containing kanamycin. Surviving plants were transferred to soil and grown in a green house to produce viable seeds. This process took 5 to 7 months after co-cultivation. Molecular analysis confirmed the stable integration and expression of foreign genes.     

Transformation of Corynespora cassiicola by Agrobacterium tumefaciens

The fungus causing target spot disease, Corynespora cassiicola (Berk. & M. A. Curtis) C. T. Wei, poses an increasing threat to watermelon (Citrullus lanatus), muskmelon (Cucumis melo), and cucumber (Cucumis sativus); the most economically important cucurbit crops grown in China. An understanding of the molecular mechanisms underlying the pathogenicity of C. cassiicola is essential for the development of new strategies to control this disease-causing fungus. Agrobacterium tumefaciens-mediated transformation (ATMT) might be useful to obtain transformants of C. cassiicola, for the ultimate identification of genes involved in pathogenicity. In the present work, we established and optimized an ATMT protocol using A. tumefaciens strain AGL-1 carrying the vector pATMT1 for C. cassiicola. Efficiency of ATMT was 102–148 transformants per 10⁶ conidia and successive subculturing of transformants on non-selective and selective media demonstrated that the integrated transfer (T)-DNA was stably inherited in C. cassiicola transformants. The integration of the hygromycin B phosphotransferase (hph) gene into C. cassiicola was validated by PCR and Southern blot analyses, which revealed that nearly 90 % of the transformants contained single-copy T-DNA. The transformants with altered phenotypes were characterized. Three of these transformants completely lost pathogenicity and other three showed strongly impaired pathogenicity relative to the Cc-GX strain on muskmelon leaves. These results strongly suggest that ATMT may be used as a molecular tool for identifying genes relevant to pathogenicity in the fungus C. cassiicola, an emerging threat to several agronomically important plants in China.     

Dual control of Agrobacterium tumefaciens Ti plasmid virulence genes.

The virulence genes of nopaline (pTiC58) and octopine (pTiA6NC) Ti plasmids are similarly affected by the Agrobacterium tumefaciens ros mutation. Of six vir region complementation groups (virA, virB, virG, virC, virD, and virE) examined by using fusions to reporter genes, the promoters of only two (virC and virD) responded to the ros mutation. For each promoter that was affected by ros, the level of expression of its associated genes was substantially elevated in the mutant. This increase was not influenced by Ti plasmid-encoded factors, and the mutation did not interfere with the induction of pTiC58 vir genes by phenolic compounds via the VirA/VirG regulatory control mechanism. The effects of the ros mutation and acetosyringone were cumulative for all vir promoters examined. The pleiotropic characteristics of the ros mutant include the complete absence of the major acidic capsular polysaccharide.     

Genetic Transformation of Wheat Mediated by Agrobacterium tumefaciens

A rapid Agrobacterium tumefaciens-mediated transformation system for wheat was developed using freshly isolated immature embryos, precultured immature embryos, and embryogenic calli as explants. The explants were inoculated with a disarmed A. tumefaciens strain C58 (ABI) harboring the binary vector pMON18365 containing the [beta]-glucuronidase gene with an intron, and a selectable marker, the neomycin phosphotransferase II gene. Various factors were found to influence the transfer-DNA delivery efficiency, such as explant tissue and surfactants present in the inoculation medium. The inoculated immature embryos or embryogenic calli were selected on G418-containing media. Transgenic plants were regenerated from all three types of explants. The total time required from inoculation to the establishment of plants in soil was 2.5 to 3 months. So far, more than 100 transgenic events have been produced. Almost all transformants were morphologically normal. Stable integration, expression, and inheritance of the transgenes were confirmed by molecular and genetic analysis. One to five copies of the transgene were integrated into the wheat genome without rearrangement. Approximately 35% of the transgenic plants received a single copy of the transgenes based on Southern analysis of 26 events. Transgenes in T1 progeny segregated in a Mendelian fashion in most of the transgenic plants.     

Genetic transformation of Robinia pseudoacacia by Agrobacterium tumefaciens

Transgenic Robinia pseudoacacia plants were obtained by Agrobacterium tumefaciens mediated gene transfer. Agrobacterium strain LBA4404 harbouring a binary vector that contained the chimeric neomycin phosphotransferase II (NPTII) and beta-glucuronidase (GUS) genes was co-cultivated with hypocotyl segments of in vitro raised seedlings of Robinia. Parameters important for high efficiency regeneration and transformation rates included type of explant, pre-conditioning of explants and appropriate length of co-cultivation period with Agrobacterium. A transformation frequency 16.67% was obtained by 48 hr of pre-conditioning followed by 48 hr of co-cultivation. Transformed tissue was selected by the ability to grow on kanamycin containing medium. Successful regeneration was followed after histochemical GUS assay for the detection of transgenic tissue. This transformation procedure has the potential to expand the range of genetic variation in Robinia.     

Transformation of Metarhizium anisopliae mediated by Agrobacterium tumefaciens

A simple, highly efficient, and reliable Agrobacterium tumefaciens-mediated transformation method was developed for the insect pathogenic fungus Metarhizium anisopliae. Expression of the green fluorescent protein gene, egfp, and the benomyl resistance gene, benA3, were used as markers in transformed M. anisopliae. Transformation efficiencies were dependent on the strain of A. tumefaciens used. With strain AGL-1, 17.0 +/- 1.4 transformants per plate could be obtained using conidial concentrations of 10(6) conidia/mL and a 2 day co-cultivation in the presence of 200 micromol/L acetosyringone. On the other hand, transformations using strain LBA4404 were unsuccessful. Ten transformants were tested by Southern analysis and found to contain a single copy T-DNA. Twenty transformants were subcultured for five generations on nonselective media, and 95% of the transformants were mitotically stable. Agrobacterium tumefaciens-mediated transformation of M. anisopliae can serve as a useful tool to investigate genes involved in insect pathogenicity.     

根癌农杆菌介导的大豆遗传转化

农杆菌介导法是大豆遗传转化的重要方法之一 ,许多实验室应用该方法得到了转基因大豆 ,但目前使用该方法进行转化的效率还比较低 ,尚需深入研究。农杆菌菌株、大豆基因型、组织培养条件、T-DNA的转移效率和转化后的筛选模式都会影响大豆转化的效率。概述了近年来根癌农杆菌介导的大豆遗传转化的一些重要成果 ,以及转化过程中大豆的易感性与农杆菌的转化能力、乙酰丁香酮促进vir基因活化、转化的受体系统和巯基混合物减轻受体材料的褐化、提高T DNA的转移效率等几个重要因素的研究进展 ,并介绍了转化中常用的几个筛选标记基因 (nptⅡ、hpt、bar基因和突变的ahas基因 )及通过共转化法去除标记基因的方法 ,同时对今后研究的重点进行了讨论.     

Genetic transformation of Populus nigra by Agrobacterium tumefaciens

Two clones of Populus nigra L. were tested in vivo and in vitro for their susceptibility to three different Agrobacterium tumefaciens wild-type strains evaluating number and size of resulting calluses. Strain C58 proved to be the most virulent.Various parameters affecting Agrobacterium-mediated transformation of P. nigra clones were further analyzed using ß-glucuronidase gene transient expression. The clone Jean Pourtet proved to be more susceptible than the clone San Giorgio. A. tumefaciens strain A281 pKIWI105 proved to be the most virulent. The optimal procedure involved dipping of leaf discs into a bacterial suspension (7×10⁸ cells/ml) for 20 min, followed by a 48 h co-cultivation period on semi-solid regeneration medium.Leaf explants were co-cultivated with two disarmed A. tumefaciens strains. Plantlets of San Giorgio were regenerated, tested for ß-glucuronidase activity and rooted on selective medium containing kanamycin. Polymerase chain reaction analysis and Southern blot hybridization confirmed the integration of the neomycin phosphotransferase II gene into the poplar genome.Abbreviations BAP 6-benzyl-aminopurine - CaMV Cauliflower Mosaic Virus - 2,4-D 2,4-dichlorophenoxyacetic acid - GUS and gus ß-glucuronidase - hpt hygromycin phosphotransferase - IBA indole-3-butyric acid - KIN kinetin - LB Luria Bertani - MS Murashige and Skoog - NAA ßnaphthaleneacetic acid - NOS Nopaline synthase - NPTII and nptII neomycin phosphotransferase II - PCR Polymerase chain reaction - PVC poly-vinyl-cloride - SDS sodium dodecyl sulfate - SSC sodium cloride-sodium citrate - Tris tris(hydroxymethyl)amino-methane - WPM Woody Plant Medium     

Enhancement of Agrobacterium tumefaciens Infectivity by Mitomycin C

The ability of Agrobacterium tumefaciens to induce pinto leaf tumors may be enhanced two- to threefold after treatment with mitomycin C. The enhancement may be obtained with either lethal or nonlethal concentrations. With 10-min treatments, an optimal response was obtained with 0.005 mug of mitomycin C per ml in the absence of any change in the number of viable cells. Both the tumor induction process and the tumors induced by treated cultures appear qualitatively the same as controls. To account for these results, the antibiotic must increase the proportion of viable cells that will subsequently initiate tumors. One, or at most a few, random lesions in the bacterial chromosome seem to be the necessary requirement for this promotion. At mitomycin concentrations of 1 and 5 mug/ml, the ability of A. tumefaciens to initiate tumors is rapidly lost, indicating that a fairly intact bacterial chromosome is one of the essentials for the tumor induction process.     

Transport of nonmetabolizable opines by Agrobacterium tumefaciens.

We have examined the uptake of [14C]octopine and [14C]nopaline by Agrobacterium tumefaciens strains containing the C58 chromosomal background in medium suitable for the induction of vir genes. All strains tested could transport both of these opines, regardless of the presence or type of Ti plasmid (octopine or nopaline) present in the bacterium. The transport of these opines required active cellular metabolism. Nonradioactive octopine, nopaline, and arginine competed effectively with [14C]octopine and [14C]nopaline for transport into A. tumefaciens A136, suggesting that the transport of these opines occurs via an arginine transport pathway not encoded by the Ti plasmid.