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1

Genome Sequence of the Octopine-Type Agrobacterium tumefaciens Strain Ach5  

PubMed Central

We have sequenced the complete genome of the plant pathogen Agrobacterium tumefaciens strain LBA4213, a derivative of the wild-type strain A. tumefaciens Ach5 and the ancestor of A. tumefaciens strain LBA4404 used in genetic engineering. The genome consists of a circular chromosome and a linear chromosome, as well as a megaplasmid and a tumor-inducing plasmid. PMID:24675863

Henkel, Christiaan V.; den Dulk-Ras, Amke; Zhang, Xiaorong

2014-01-01

2

Genome Sequence of the Octopine-Type Agrobacterium tumefaciens Strain Ach5.  

PubMed

We have sequenced the complete genome of the plant pathogen Agrobacterium tumefaciens strain LBA4213, a derivative of the wild-type strain A. tumefaciens Ach5 and the ancestor of A. tumefaciens strain LBA4404 used in genetic engineering. The genome consists of a circular chromosome and a linear chromosome, as well as a megaplasmid and a tumor-inducing plasmid. PMID:24675863

Henkel, Christiaan V; den Dulk-Ras, Amke; Zhang, Xiaorong; Hooykaas, Paul J J

2014-01-01

3

Factors affecting transformation efficiency of embryogenic callus of Upland cotton ( Gossypium hirsutum ) with Agrobacterium tumefaciens  

Microsoft Academic Search

A reliable and high-efficiency system of transforming embryogenic callus (EC) mediated by Agrobacterium tumefaciens was developed in cotton. Various aspects of transformation were examined in efforts to improve the efficiency of producing transformants. LBA4404 and C58C3, harboring the p?gusBin19 plasmid containing neomycin phosphortransferase II (npt-II) gene as a selection marker, were used for transformation. The effects of Agrobacterium strains, acetosyringone

Shuangxia Jin; Xianlong Zhang; Shaoguang liang; Yichun Nie; Xiaoping Guo; Chao Huang

2005-01-01

4

[Agrobacterium-mediated sunflower transformation (Helianthus annuus L.) in vitro and in Planta using strain of LBA4404 harboring binary vector pBi2E with dsRNA-suppressor proline dehydrogenase gene].  

PubMed

To estimate the efficiency of proline dehydrogenase gene suppression towards increasing of sunflower (Helianthus annuus L.) tolerance level to water deficit and salinity, we employed strain LBA4404 harboring pBi2E with double-stranded RNA-suppressor, which were prepared on basis arabidopsis ProDH1 gene. The techniques of Agrobacterium-mediated transformation in vitro and in planta during fertilization sunflower have been proposed. There was shown the genotype-depended integration of T-DNA in sunflower genome. PCR-analysis showed that ProDH1 presents in genome of inbred lines transformed in planta, as well as in T1- and T2-generations. In trans-genic regenerants the essential accumulation of free L-proline during early stages of in vitro cultivation under normal conditions was shown. There was established the essential accumulation of free proline in transgenic regenerants during cultivation under lethal stress pressure (0.4 M mannitol and 2.0% sea water salts) and its decline upon the recovery period. These data are declared about effectiveness of suppression of sunflower ProDH and gene participation in processes connected with osmotolerance. PMID:25184200

Tishchenko, E N; Komisarenko, A G; Mikhal'skaia, S I; Sergeeva, L E; Adamenko, N I; Morgun, B V; Kochetov, A V

2014-01-01

5

A reliable protocol for transformation of Catharanthus roseus through Agrobacterium tumefaciens.  

PubMed

Proliferation of axillary shoot buds and multiple shoot formation in Catharanthus roseus was obtained in 96 % explants on MS medium (3 % sucrose) containing NAA + BA. 2,4-D induced callusing in both, the nodal as well as in leaf segments. Leaf-derived callus was used for transformation with Agrobacterium tumefaciens LBA4404/pBI-S1. Bacterial cell concentration, duration of co-cultivation and acetosyringone concentration influenced transformation efficiency. Under optimal co-cultivation conditions, 98 % of the explants showed GUS expression. PCR based amplification of the transformed and subsequently selected callus tissue indicated the presence of uidA, Gly I and nptII genes. PMID:23572917

Srivastava, Toolika; Das, Sandip; Sopory, Sudhir Kumar; Srivastava, P S

2009-01-01

6

Agrobacterium tumefaciens-mediated genetic transformation of haptophytes (Isochrysis species).  

PubMed

Isochrysis galbana and Isochrysis sp. are economically important microalgae from the division of haptophytes. Here, we report Agrobacterium-mediated stable DNA transfer into their nuclear genomes. Initial studies were performed to standardize co-cultivation media and determine the sensitivity of the microalgae to selective agents. Up to 1 mg/ml of the antibiotic hygromycin did not inhibit growth, whereas both the haptophytes bleached in artificial seawater (ASW) medium containing micromolar concentrations of the herbicide norflurazon. Co-cultivation of Isochrysis sp. and I. galbana with Agrobacterium tumefaciens strain LBA 4404 harboring the binary vector pCAMBIA 1380-pds-L504R yielded norflurazon-resistant (NR) colonies visible on selective plates after 20-30 days. pCAMBIA 1380-pds-L540R was constructed by cloning a mutated genomic phytoene desaturase (pds) gene from Haematococcus pluvialis as a selectable marker gene into the binary vector system pCAMBIA 1380. Co-cultivation of Isochrysis sp. with A. tumefaciens in ASW medium containing 200 ?M of acetosyringone for 72 h produced the highest number of NR cells. For I. galbana, 100 ?M of acetosyringone, ASW medium, and 48 h co-cultivation period appeared to be optimum co-cultivation parameters. The NR colonies kept their resistance phenotype for at least 24 months, even in the absence of selective pressure. The transfer of the pds gene in NR cells was shown by PCR amplification of the T-DNA sequences from the genomic DNA of NR cells and Southern blot analysis using T-DNA sequences as probes. The genetic manipulation described here will allow metabolic engineering and a better understanding of several biochemical pathways in the future. PMID:24993358

Prasad, Binod; Vadakedath, Nithya; Jeong, Hyun-Jeong; General, Thiyam; Cho, Man-Gi; Lein, Wolfgang

2014-10-01

7

Agrobacterium-mediated genetic transformation of a phalaenopsis orchid  

Microsoft Academic Search

Genetically transformed plants of a phalaenopsis orchid [Doritaenopsis Coral FantasyPhalaenopsis (Baby HatAnn Jessica)] were regenerated after cocultivation of cell clumps with Agrobacterium tumefaciens strains LBA4404 (pTOK233) and EHA101 (pIG121Hm) that harbored genes for ?-glucuronidase (GUS) and hygromycin resistance.\\u000a The efficiency of transformation was markedly increased by 10?h cocultivation of cell clumps with A. tumefaciens that had been induced with 200??m

M. M. Belarmino; M. Mii

2000-01-01

8

In vitro organogenesis and genetic transformation in popular Cucumis sativus L. through Agrobacterium tumefaciens.  

PubMed

The effect of growth regulators and culture conditions on the morphogenetic response of cotyledonary leaf discs was studied in popular cucumber variety (Cucumis sativus cv. Sheetal). Organogenesis was induced directly without any intervening callus phase on Murashige and Skoog medium supplemented with different concentrations of benzyladenine and indole propionic acid. Best results (93%) were obtained in the presence of the 4 mg/L benzyladenine and 1 mg/L IPA. The elongated shoots were rooted in basal medium with 1 mg/L indole butyric acid, hardened and transferred to the field conditions. Genetic transformation system has been established for Cucumis sativus cv. Sheetal, plants by infecting cotyledonary explants with Agrobacterium tumefaciens strain LBA4404 carrying binary plasmid pBI121, which contains scorable marker, beta-glucuronidase and selectable marker nptII under the CaMV 35S promoter. Infection was most effective when explants were infected with Agrobacterium for 15 min and co-cultivated for 2 days in the co-cultivation medium. Shoots were regenerated directly from cotyledonary leaf explants in the presence of kanamycin (50 microg/ml) and analysed. Southern blot analysis confirmed that transformation had occurred. This method will allow genetic improvement of this crop by the introduction of agronomically important genes. PMID:12635705

Soniya, E V; Das, M R

2002-03-01

9

GLYCINE RESISTANCE IN AGROBACTERIUM TUMEFACIENS  

PubMed Central

Beardsley, Robert E. (Manhattan College, New York, N. Y.). Glycine resistance in Agrobacterium tumefaciens. J. Bacteriol. 83:6–13. 1962.—The application of the fluctuation test of Luria and Delbrück to the distribution of glycine-resistant bacteria among cultures of Agrobacterium tumefaciens strain B6 indicates that resistance arises by mutation in the absence of glycine. On glycine-supplemented medium, additional resistant colonies arise during prolonged periods of incubation. Their appearance is proceded by L-form growth. In general, the number of generations over which glycine resistance is inherited in the absence of glycine is increased by serial transfers on the selection medium. In liquid medium containing glycine, sensitive bacteria form spheroplasts. Resistant bacteria continue to grow as rod forms. In the medium employed, spheroplasts are unstable. Images PMID:13866159

Beardsley, Robert E.

1962-01-01

10

Agrobacterium tumefaciens-mediated transformation of Campanula carpatica: factors affecting transformation and regeneration of transgenic shoots.  

PubMed

An efficient transformation system for Campanula carpatica was developed using Agrobacterium tumefaciens strains LBA4404 (harbouring the plasmid pBI121), and AGL0 (harbouring the plasmid pBEO210). This is the first report on the transformation of C. carpatica. Various factors affecting the transformation efficiency and subsequent regeneration were identified. The age of seedlings from which the explants for transformation studies were taken, and the growth conditions under which the seedlings were grown had a significant influence on the production of transformed shoots. Hypocotyls taken from 12-day-old seedlings grown in the dark were the most productive, with up to 25% of hypocotyls producing transformed shoots. Explants taken from 5-week-old seedlings produced only transformed callus. The medium used for co-cultivation and incubation also had a significant influence on transformation frequency and shoot regeneration. The cultivar "Blue Uniform" was more responsive than "White Uniform". Both bacterial strains and plasmids were equally effective in producing transformed tissue. Transformed shoots were selected on kanamycin medium, and the presence of the uidA and nptII genes in those selected shoots was confirmed by beta-glucuronidase and ELISA analyses, respectively. PMID:15114492

Sriskandarajah, Sridevy; Frello, Stefan; Jørgensen, Kirsten; Serek, Margrethe

2004-08-01

11

Ferrisiderophore reductase activity in Agrobacterium tumefaciens.  

PubMed Central

Reduction of the iron in ferriagrobactin by the cytoplasmic fraction of Agrobacterium tumefaciens strictly required NaDH as the reductant. Addition of flavin mononucleotide and anaerobic conditions were necessary for the reaction; when added with flavin mononucleotide, magnesium was stimulatory. This ferrisiderophore reductase activity may be a part of the iron assimilation process in A. tumefaciens. PMID:7056702

Lodge, J S; Gaines, C G; Arceneaux, J E; Byers, B R

1982-01-01

12

Ferrisiderophore reductase activity in Agrobacterium tumefaciens.  

PubMed

Reduction of the iron in ferriagrobactin by the cytoplasmic fraction of Agrobacterium tumefaciens strictly required NaDH as the reductant. Addition of flavin mononucleotide and anaerobic conditions were necessary for the reaction; when added with flavin mononucleotide, magnesium was stimulatory. This ferrisiderophore reductase activity may be a part of the iron assimilation process in A. tumefaciens. PMID:7056702

Lodge, J S; Gaines, C G; Arceneaux, J E; Byers, B R

1982-02-01

13

Exogenous phytohormone-independent growth and regeneration of tobacco plants transgenic for the 6b gene of Agrobacterium tumefaciens AKE10.  

PubMed Central

The 6b gene of Agrobacterium tumefaciens AKE10 (AK-6b) induces crown gall tumors on certain plants but so far there have been no reports of the gene being able to induce tumors on culture medium. We cloned T-DNA segments containing the 6b gene but lacking the auxin and cytokinin biosynthesis genes from A. tumefaciens AKE10. Tobacco (Nicotiana tabacum) leaf discs infected with A. tumefaciens LBA4404 carrying the clones produced shooty calli on hormone-free Murashige-Skoog medium. The relevant T-DNA segment was integrated into plant DNA as determined by Southern hybridization. Some of these immature shoots spontaneously developed into mature shoots, of which several leaves displayed morphological abnormalities. When leaf discs of these mature plants were placed onto the same medium numerous shoots developed from the wounding sites, indicating that the transgenic plants possessed a high regenerative potential. Northern blot and reverse transcriptase-polymerase chain reaction analyses showed a large accumulation of the AK-6b transcripts in the shooty calli, but only a limited degree in mature plants, demonstrating that AK-6b expression is regulated in plants and essential for the early stages of regeneration. Cytokinin levels in the shooty calli were comparable to those in normal shoots, suggesting that shoot regeneration is not mediated by the modulation of cytokinin content. PMID:8938404

Wabiko, H; Minemura, M

1996-01-01

14

Stimulation of Agrobacterium tumefaciens Growth by Azotobacter vinelandii Ferrisiderophores.  

PubMed

Azotobacter vinelandii stimulated the growth of Agrobacterium tumefaciens H2, H23, H24, H27, and ATCC 15955 on media containing insoluble iron sources. The Azotobacter vinelandii siderophores appeared to promote Agrobacterium tumefaciens growth by solubilizing mineral iron, and the ferrisiderophores so formed then acted as iron sources for Agrobacterium tumefaciens. Agrobactin, the Agrobacterium siderophore, appeared to be inefficient in solubilizing mineral iron directly. PMID:16347002

Page, W J; Dale, P L

1986-02-01

15

Stimulation of Agrobacterium tumefaciens Growth by Azotobacter vinelandii Ferrisiderophores  

PubMed Central

Azotobacter vinelandii stimulated the growth of Agrobacterium tumefaciens H2, H23, H24, H27, and ATCC 15955 on media containing insoluble iron sources. The Azotobacter vinelandii siderophores appeared to promote Agrobacterium tumefaciens growth by solubilizing mineral iron, and the ferrisiderophores so formed then acted as iron sources for Agrobacterium tumefaciens. Agrobactin, the Agrobacterium siderophore, appeared to be inefficient in solubilizing mineral iron directly. Images PMID:16347002

Page, William J.; Dale, Phyllis L.

1986-01-01

16

Agrobacterium-mediated engineering for sheath blight resistance of indica rice cultivars from different ecosystems  

Microsoft Academic Search

A concise T-DNA element was engineered containing the rice class-I chitinase gene expressed under the control of CaMV35S and the hygromycin phosphotransferase gene (hph) as a selectable marker. The binary plasmid vector pNO1 with the T-DNA element containing these genes of interest was mobilized\\u000a to Agrobacterium\\u000a \\u000a tumefaciens strain LBA4404 to act as an efficient donor of T-DNA in the transformation

K. Datta; Z. Koukolíková-Nicola; N. Baisakh; N. Oliva; S. K. Datta

2000-01-01

17

[Improvement of transformation frequency of rice mediated by Agrobacterium].  

PubMed

The factors influencing the frequency of rice transformation mediated by Agrobacterium have been investigated by using 16 commercially important indica and japonica rice cultivars or lines. The main results were as following: For most rice CC medium was the best for both callus initiation and subculture. With supplement of 2.5-5 mg/L ABA the quality of calli can be improved. The concentration of selective agent for Indica rice callus was lower than that for japonica rice callus. Agrobacterium tumefaciens strain EHA105 was more efficient than LBA4404 and AGL1 for rice transformation. The inhibitive effect of cefotaxime to Agrobacterium was better than that of carbenicillin. The partial desiccation treatment after co-cultivation was beneficial to inhibit the growth of Agrobacterium and increase transformation efficiency. A stable and efficient Agrobacterium-mediated transformation system has been established in ten different rice cultivars and fertile transgenic plants have been obtained. PMID:11329877

Yi, Z L; Cao, S Y; Wang, L; Chu, C C; Li, X; He, S J; Tang, Z S; Zhou, P H; Tian, W Z

2001-01-01

18

Agrobacterium tumefaciens -mediated transformation of Robinia pseudoacacia  

Microsoft Academic Search

Robinia pseudoacacia   (black locust) plants were regenerated after co-cultivation of stem and leaf segments with Agrobacterium tumefaciens strain GV3101 (pMP90) that harbored a binary vector that included genes for ?-glucuronidase (GUS) and hygromycin phosphotransferase. Successful transformation was confirmed by the ability of stem and\\u000a leaf segments to produce calli in the presence of hygromycin, by histochemical and fluorometric assays of

T. Igasaki; T. Mohri; H. Ichikawa; K. Shinohara

2000-01-01

19

Cellulose Synthesis in Agrobacterium tumefaciens  

SciTech Connect

We have cloned the celC gene and its homologue from E. coli, yhjM, in an expression vector and expressed the both genes in E. coli; we have determined that the YhjM protein is able to complement in vitro cellulose synthesis by extracts of A. tumefaciens celC mutants, we have purified the YhjM protein product and are currently examining its enzymatic activity; we have examined whole cell extracts of CelC and various other cellulose mutants and wild type bacteria for the presence of cellulose oligomers and cellulose; we have examined the ability of extracts of wild type and cellulose mutants including CelC to incorporate UDP-14C-glucose into cellulose and into water-soluble, ethanol-insoluble oligosaccharides; we have made mutants which synthesize greater amounts of cellulose than the wild type; and we have examined the role of cellulose in the formation of biofilms by A. tumefaciens. In addition we have examined the ability of a putative cellulose synthase gene from the tunicate Ciona savignyi to complement an A. tumefaciens celA mutant. The greatest difference between our knowledge of bacterial cellulose synthesis when we started this project and current knowledge is that in 1999 when we wrote the original grant very few bacteria were known to synthesize cellulose and genes involved in this synthesis were sequenced only from Acetobacter species, A. tumefaciens and Rhizobium leguminosarum. Currently many bacteria are known to synthesize cellulose and genes that may be involved have been sequenced from more than 10 species of bacteria. This additional information has raised the possibility of attempting to use genes from one bacterium to complement mutants in another bacterium. This will enable us to examine the question of which genes are responsible for the three dimensional structure of cellulose (since this differs among bacterial species) and also to examine the interactions between the various proteins required for cellulose synthesis. We have carried out one preliminary experiment of this type and have successfully complemented an A. tumefaciens CelC mutant with the homologous gene (yhjM) from E. coli.

Alan R. White; Ann G. Matthysse

2004-07-31

20

Agrobacterium tumefaciens is a diazotrophic bacterium  

SciTech Connect

This is the first report that Agrobacterium tumefaciens can fix nitrogen in a free-living condition as shown by its abilities to grown on nitrogen-free medium, reduce acetylene to ethylene, and incorporate {sup 15}N supplied as {sup 15}N{sub 2}. As with most other well-characterized diazotrophic bacteria, the presence of NH{sub 4}{sup +} in the medium and aerobic conditions repress nitrogen fixation by A. tumefaciens. The system requires molybdenum. No evidence for nodulation was found with pea, peanut, or soybean plants. Further understanding of the nitrogen-fixing ability of this bacterium, which has always been considered a pathogen, should cast new light on the evolution of a pathogenic versus symbiotic relationship.

Kanvinde, L.; Sastry, G.R.K. (Univ. of Leeds (England))

1990-07-01

21

Agrobactin, a siderophore from Agrobacterium tumefaciens.  

PubMed

A siderophore (microbial iron transport compound) was isolated from low iron cultures of Agrobacterium tumefaciens B6. The substance was characterized as a threonyl peptide of spermidine acylated with 3 residues of 2,3-dihydroxybenzoic acid, the carbonyl group of 1 residue of the latter participating in an oxazoline ring with the beta-hydroxyl of the threonine moiety. The compound, N-[3-(2,3-dihydroxybenzamido)propyl]-N-[4-(2,3-dihydroxybenzamido)butyl]-2-(2,3-dihydroxyphenyl)-trans-5-methyl-oxazoline-4-carboxamide, was given the trivial name agrobactin. Exposure to acid opened the oxazoline ring to afford agrobactin A. Ferric agrobactin A and agrobactin A itself, but not agrobactin or its ferric complex, had some capacity to feed iron to enterobactin-deficient strains of Escherichia coli and Salmonella typhimurium. Agrobactin was produced by A. tumefaciens in response to iron deficiency and was able to reverse the iron starvation in this organism precipitated by the presence of a ferric complexing agent not utilized by the cells. PMID:33987

Ong, S A; Peterson, T; Neilands, J B

1979-03-25

22

Agrobacterium tumefaciens-mediated transformation of Indian mulberry, Morus indica cv. K2: a time-phased screening strategy.  

PubMed

An efficient and reproducible protocol for the production of transgenic plants was developed for Morus indica cv. K2 by Agrobacterium tumefaciens-mediated transformation. The hypocotyls, cotyledon, leaf and leaf callus explants precultured for 5 days on regeneration medium were co-cultivated with a bacterial suspension at 10(9) cells/ml for 3 days in the dark. Infectivity of A. tumefaciens strain LBA4404 was more than that of strains GV2260 and A281, and among the various plasmids tried, pBI121 and pBI101:Act1 transformed nearly 100% of the explants followed closely by p35SGUSINT. About 90-100% of the explants tested positive in the beta-glucuronidase (GUS) histochemical assay performed after 3 days of co-cultivation. This high level of transient expression, however, decreased to 20-25% after 15 days. Gus activity was most stable in the callus explants, which emerged as the explant of choice for transformation. The transformed explants were selected on 50-75 mg/l kanamycin for 1 month, and 25-50% of the explants developed adventitious buds. On the basis of kanamycin-resistant shoots produced from the total number of explants inoculated, the transformation efficiency was 44%. After 1 month, 40% of these shoots displayed high gus activity as assessed by the GUS fluorometric assay. On a selection-free root induction medium, 80% of the shoots developed roots and 90% of the potted plantlets acclimatized to the growth room conditions. The 3-month-old regenerates showed gus and nptII(neomycin phosphotransferase II) gene activity as assayed by the GUS fluorometric assay and nptII enzyme assay, followed by PCR polymerase chain reaction (54.5%) analysis after 6-months. Transgene integration into the nuclear genome of 1-year-old regenerates was confirmed in 10 of the 18 transformants tested by Southern analysis. The transformation efficiency as defined by the number of transgenic plants produced from the total number of explants co-cultivated was 6%. PMID:12789417

Bhatnagar, S; Khurana, P

2003-03-01

23

Transformation of the plant Kalanchoë daigremontiana using Agrobacterium tumefaciens.  

PubMed

Kalanchoë daigremontiana can be stably transformed using the Agrobacterium tumefaciens-mediated T-DNA transfer method, as described here. Sterilized plant tissue is cocultivated with an A. tumefaciens suspension, transformants are selected and the shoots are grown in rooting medium and then in soil. Plant phenotypes can be examined approximately 3 mo after transfer of plants to soil. PMID:20147048

Garcês, Helena; Sinha, Neelima

2009-10-01

24

Transmission of an Agrobacterium tumefaciens Phage by Pristionchus Iheriteiri  

PubMed Central

Pristionchus lheriteiri (Maupas) Paramonov, a saprozoic nematode, served as a carrier of an unnamed phage of Agrobacterium tumefaciens (Smith and Townsend) Conn. Viable phage particles passed through the nematode, caused lysis and formed typical plaques on agar plates seeded to A. tumefaciens. Phage retention by carrier nematodes was extended several hr by restricting food intake. Female nematodes accumulated phage in greater quantities and more rapidly than male nematodes. PMID:19325671

Chantanao, A.; Jensen, H. J.

1969-01-01

25

Transgenic Pinus radiata from Agrobacterium tumefaciens –mediated transformation of cotyledons  

Microsoft Academic Search

A method for Agrobacterium tumefaciens-mediated transformation of Pinus radiata cotyledon explants was developed using commercially available open-pollinated seed. Pinus radiata is the most widely planted commercial conifer species in the Southern Hemisphere. Reports on transformation of this species have relied on particle bombardment of embryogenic callus derived from immature embryos. The main drawback to the method is the small number

J. E. Grant; P. A. Cooper; T. M. Dale

2004-01-01

26

Expression and Physiological Relevance of Agrobacterium tumefaciens Phosphatidylcholine Biosynthesis Genes?  

PubMed Central

Phosphatidylcholine (PC), or lecithin, is the major phospholipid in eukaryotic membranes, whereas only 10% of all bacteria are predicted to synthesize PC. In Rhizobiaceae, including the phytopathogenic bacterium Agrobacterium tumefaciens, PC is essential for the establishment of a successful host-microbe interaction. A. tumefaciens produces PC via two alternative pathways, the methylation pathway and the Pcs pathway. The responsible genes, pmtA (coding for a phospholipid N-methyltransferase) and pcs (coding for a PC synthase), are located on the circular chromosome of A. tumefaciens C58. Recombinant expression of pmtA and pcs in Escherichia coli revealed that the individual proteins carry out the annotated enzyme functions. Both genes and a putative ABC transporter operon downstream of PC are constitutively expressed in A. tumefaciens. The amount of PC in A. tumefaciens membranes reaches around 23% of total membrane lipids. We show that PC is distributed in both the inner and outer membranes. Loss of PC results in reduced motility and increased biofilm formation, two processes known to be involved in virulence. Our work documents the critical importance of membrane lipid homeostasis for diverse cellular processes in A. tumefaciens. PMID:18978052

Klüsener, Sonja; Aktas, Meriyem; Thormann, Kai M.; Wessel, Mirja; Narberhaus, Franz

2009-01-01

27

Transformation and regeneration of Brassica rapa using Agrobacterium tumefaciens  

Microsoft Academic Search

Transformation and regeneration procedures for obtaining transgenic Brassica rapa ssp. oleifera plants are described. Regeneration frequencies were increasedby using silver nitrate and by adjusting the duration of exposure to 2,4-D. For transformation, Agrobacterium tumefaciens strain EHA101 containing a binary plasmid with the neomycin phosphotransferase gene (NPT II) and the b-glucuronidase gene (GUS) was cocultivated with hypocotyl explants from the oilseed

Sharon E. Radke; Joann C. Turner; Daniel Facciotti

1992-01-01

28

Functions and regulation of quorum-sensing in Agrobacterium tumefaciens  

PubMed Central

In Agrobacterium tumefaciens, horizontal transfer and vegetative replication of oncogenic Ti plasmids involve a cell-to-cell communication process called quorum-sensing (QS). The determinants of the QS-system belong to the LuxR/LuxI class. The LuxI-like protein TraI synthesizes N-acyl-homoserine lactone molecules which act as diffusible QS-signals. Beyond a threshold concentration, these molecules bind and activate the LuxR-like transcriptional regulator TraR, thereby initiating the QS-regulatory pathway. For the last 20 years, A. tumefaciens has stood as a prominent model in the understanding of the LuxR/LuxI type of QS systems. A number of studies also unveiled features which are unique to A. tumefaciens QS, some of them being directly related to the phytopathogenic lifestyle of the bacteria. In this review, we will present the current knowledge of QS in A. tumefaciens at both the genetic and molecular levels. We will also describe how interactions with plant host modulate the QS pathway of A. tumefaciens, and discuss what could be the advantages for the agrobacteria to use such a tightly regulated QS-system to disseminate the Ti plasmids. PMID:24550924

Lang, Julien; Faure, Denis

2014-01-01

29

Transformation of Brassica napus L. using Agrobacterium tumefaciens : developmentally regulated expression of a reintroduced napin gene  

Microsoft Academic Search

Genetically transformed plants of Brassica napus L. (oilseed rape) were obtained from hypocotyl expiants using Agrobacterium tumefaciens vectors. Hypocotyl explants were inoculated with disarmed or oncogenic A. tumefaciens strains, EHA101 and A281, and then cultured on media containing kanamycin. The A. tumefaciens strains harbored a binary vector, which contained a neomycin phosphotransferase II (NPTII) gene driven by the 35S promoter

S. E. Radke; B. M. Andrews; M. M. Moloney; M. L. Crouch; J. C. Kridl; V. C. Knauf

1988-01-01

30

Plant responses to Agrobacterium tumefaciens and crown gall development  

PubMed Central

Agrobacterium tumefaciens causes crown gall disease on various plant species by introducing its T-DNA into the genome. Therefore, Agrobacterium has been extensively studied both as a pathogen and an important biotechnological tool. The infection process involves the transfer of T-DNA and virulence proteins into the plant cell. At that time the gene expression patterns of host plants differ depending on the Agrobacterium strain, plant species and cell-type used. Later on, integration of the T-DNA into the plant host genome, expression of the encoded oncogenes, and increase in phytohormone levels induce a fundamental reprogramming of the transformed cells. This results in their proliferation and finally formation of plant tumors. The process of reprogramming is accompanied by altered gene expression, morphology and metabolism. In addition to changes in the transcriptome and metabolome, further genome-wide (“omic”) approaches have recently deepened our understanding of the genetic and epigenetic basis of crown gall tumor formation. This review summarizes the current knowledge about plant responses in the course of tumor development. Special emphasis is placed on the connection between epigenetic, transcriptomic, metabolomic, and morphological changes in the developing tumor. These changes not only result in abnormally proliferating host cells with a heterotrophic and transport-dependent metabolism, but also cause differentiation and serve as mechanisms to balance pathogen defense and adapt to abiotic stress conditions, thereby allowing the coexistence of the crown gall and host plant. PMID:24795740

Gohlke, Jochen; Deeken, Rosalia

2014-01-01

31

Identification and analysis of a siderophore biosynthetic gene cluster from Agrobacterium tumefaciens C58  

Microsoft Academic Search

Using the complete genome sequence from Agrobacterium tumefaciens C58, the authors identified a secondary metabolite gene cluster that encodes the biosynthesis of a metabolite with siderophore activity. Support for this conclusion came from genetic and regulatory analysis of the gene cluster, along with the purification of a metabolite from A. tumefaciens C58 with iron-chelating activity. Genetic analysis of mutant strains

Michelle R. Rondon; Katie S. Ballering; Michael G. Thomas

2004-01-01

32

The Genome of the Natural Genetic Engineer Agrobacterium tumefaciens C58  

Microsoft Academic Search

The 5.67-megabase genome of the plant pathogen Agrobacterium tumefaciens C58 consists of a circular chromosome, a linear chromosome, and two plasmids. Extensive orthology and nucleotide colinearity between the genomes of A. tumefaciens and the plant symbiont Sinorhizobium meliloti suggest a recent evolutionary divergence. Their similarities include metabolic, transport, and regulatory systems that promote survival in the highly competitive rhizosphere; differences

Derek W. Wood; Joao C. Setubal; Rajinder Kaul; Dave E. Monks; Joao P. Kitajima; Vagner K. Okura; Yang Zhou; Lishan Chen; Gwendolyn E. Wood; Nalvo F. Almeida; Lisa Woo; Yuching Chen; Ian T. Paulsen; Peter D. Karp; Donald Bovee Sr; Peter Chapman; James Clendenning; Glenda Deatherage; Will Gillet; Charles Grant; Tatyana Kutyavin; Ruth Levy; Meng-Jin Li; Erin McClelland; Anthony Palmieri; Christopher Raymond; Gregory Rouse; Channakhone Saenphimmachak; Zaining Wu; Pedro Romero; David Gordon; Shiping Zhang; Heayun Yoo; Yumin Tao; Phyllis Biddle; Mark Jung; William Krespan; Michael Perry; Bill Gordon-Kamm; Li Liao; Sun Kim; Carol Hendrick; Zuo-Yu Zhao; Maureen Dolan; Forrest Chumley; Scott V. Tingey; Jean-Francois Tomb; Milton P. Gordon; Maynard V. Olson; Eugene W. Nester

2001-01-01

33

Genome Sequence of the Plant Pathogen and Biotechnology Agent Agrobacterium tumefaciens C58  

Microsoft Academic Search

Agrobacterium tumefaciens is a plant pathogen capable of transferring a defined segment of DNA to a host plant, generating a gall tumor. Replacing the transferred tumor-inducing genes with exogenous DNA allows the introduction of any desired gene into the plant. Thus, A. tumefaciens has been critical for the development of modern plant genetics and agricultural biotechnology. Here we describe the

Brad Goodner; Gregory Hinkle; Stacie Gattung; Nancy Miller; Mary Blanchard; Barbara Qurollo; Barry S. Goldman; Yongwei Cao; Manor Askenazi; Conrad Halling; Lori Mullin; Kathryn Houmiel; Jeffrey Gordon; Mark Vaudin; Oleg Iartchouk; Andrew Epp; Fang Liu; Clifford Wollam; Mike Allinger; Dahlia Doughty; Charlaine Scott; Courtney Lappas; Brian Markelz; Casey Flanagan; Chris Crowell; Jordan Gurson; Caroline Lomo; Carolyn Sear; Graham Strub; Chris Cielo; Steven Slater

2001-01-01

34

Genetic analysis of an Agrobacterium tumefaciens strain producing an agrocin active against biotype 3 pathogens  

Microsoft Academic Search

The successful biocontrol agent for crown gall disease, Agrobacterium radiobacter strain K84, is unable to protect grapevines from infection. We have identified a strain of Agrobacterium tumefaciens, J73, which produces an agrocin active both in vitro and in vivo against grapevine pathogens (Webster et al. 1986). We now report on the curing of this strain of its nopaline-type Ti plasmid

Jocelyn Webster; Jennifer Thomson

1988-01-01

35

Optimization of the uidA gene transfer into somatic embryos of rose via Agrobacterium tumefaciens  

E-print Network

; Somatic embryogenesis; Transformation; uidA gene 1. Introduction Roses are among the leading floriculturalOptimization of the uidA gene transfer into somatic embryos of rose via Agrobacterium tumefaciens somatic embryos via Agrobacterium-mediated transformation, but no transgenic plants were recovered

Korban, Schuyler S.

36

Salicylic Acid and Systemic Acquired Resistance Play a Role in Attenuating Crown Gall Disease Caused by Agrobacterium tumefaciens  

Microsoft Academic Search

We investigated the effects of salicylic acid (SA) and systemic acquired resistance (SAR) on crown gall disease caused by Agrobacterium tumefaciens. Nicotiana benthamiana plants treated with SA showed decreased susceptibility to Agrobacterium infection. Exogenous application of SA to Agrobacterium cultures decreased its growth, virulence, and attachment to plant cells. Using Agrobacterium whole-genome microarrays, we characterized the direct effects of SA

Ajith Anand; Srinivasa Rao Uppalapati; Choong-Min Ryu; Stacy N. Allen; Li Kang; Yuhong Tang; Kirankumar S. Mysore

2007-01-01

37

Growth of Agrobacterium tumefaciens under octopine limitation in chemostats.  

PubMed Central

Agrobacterium tumefaciens B6 and ATCC 15955 were grown under octopine or glutamate limitation in chemostats. Examination of the maximum specific growth rate (mu max) and substrate affinity (KS) for each strain indicated that strain B6 was highly inefficient in its use of octopine as either a nitrogen or carbon source compared with strain ATCC 15955. Examination of the yield coefficients showed that in both strains octopine was used more efficiently as a nitrogen source than as a carbon source. The data permitted predictions to be made concerning the outcome of competition for a single limiting substrate. Under octopine limitation, strain ATCC 15955 should dominate; under glutamate limitation, strain B6 should dominate. The results of an observed competition with glutamate as the limiting substrate confirmed the latter prediction, although B6 did dominate at a rate faster than was predicted from simple competition theory. B6 displayed higher growth rates and substrate affinities than ATCC 15955 on all concentrations of glutamate. The yield of B6 on octopine was also considerably higher. This latter attribute could provide an ecological advantage to B6 because of the importance of yield in the fate of competitions under multisubstrate regimens. These will be the most prevalent regimens in the area around the tumor (tumorosphere) or the rhizosphere. The increased performance on glutamate could provide an advantage in an opine-free environment when B6 is growing as a saprophyte. PMID:2383013

Bell, C R

1990-01-01

38

Plant cell transformation with Agrobacterium tumefaciens under simulated microgravity  

NASA Astrophysics Data System (ADS)

To investigate simulated microgravity (clinorotation) effect on plant cell transformation with Agrobacterium tumefaciens and crown gall formation, the culture of primary explants of potato and Jerusalem artichoke tubers was used. It is found that the efficiency of tumor formation and development in clinorotated explants are considerably reduced. When using the explants isolated from potato tubers clinorotated for 3, 5 and 19 days, drastic reduction of formation and development of crown gall tumors was observed. Conversely, the tumor number and their development increased when potato tubers were clinorotated for one day. As was estimated by us previously, cells of Jerusalem artichoke explants are the most sensitive to agrobacteria on 4-5 h of in vitro culturing and this time corresponds to the certain period of G1-stage of the cell cycle. We have also estimated that this period is characterized by the increase of binding of acridine orange by nuclear chromatin and increase in activity of RNA-polymerase I and II. Inoculation of explants with agrobacteria in this period was the most optimal for transformation and crown gall induction. We estimated that at four - hour clinorotation of explants the intensity of acridine orange binding to nuclei was considerably lower than on 4h in the control. At one-day clinorotation of potato tubers, a considerable increase in template accessibility of chromatin and in activity of RNA-polymerase I and II occurred. These results may serve as an evidence for the ability of plant dormant tissues to respond to microgravity. Another demonstration of dormant tissue response to changed gravity we obtained when investigating pathogenesis-related proteins (PR-proteins). PR-proteins were subjected to nondenaturing PAGE.and we have not found any effect of microgravity on PR-proteins of potato explants with normal or tumorous growth. We may suggest that such response derives from the common effects of two stress factors - wounding and changed gravity. Investigation of the effect of microgravity on PR-proteins of dormant potato tubers showed that an intensity of several electrophoretic fractions of these proteins with middle electrophoretic mobility increased and appeared two new minor fractions with high electrophoretic mobility under clinorotation of tubers. We discuss the possibility to use short term clinorotation of plant organs, from which the explants for the transformation with A. tumefaciens will be isolated, for an increase in the transformation efficiency of recalcitrant plants.

Sarnatska, Veresa; Gladun, Hanna; Padalko, Svetlana

39

Additional virulence genes and sonication enhance Agrobacterium tumefaciens -mediated loblolly pine transformation  

Microsoft Academic Search

Additional virulence (vir) genes in Agrobacterium tumefaciens and sonication were investigated for their impact on transformation efficiency in loblolly pine (Pinus taeda L.). Mature zygotic embryos of loblolly pine were co-cultivated with disarmed A. tumefaciens strain EHA105 containing either plasmid vector pCAMBIA1301 or vector pCAMBIA1301 with an additional 15.8-kb fragment carrying extra copies of the Vir B, Vir C, and

W. Tang

2003-01-01

40

Genetic transformation of Agave salmiana by Agrobacterium tumefaciens and particle bombardment  

Microsoft Academic Search

Agave salmiana was transformed using two different protocols: co-cultivation with Agrobacterium tumefaciens and particle bombardment. The uidA (?-glucuronidase) gene was used as a reporter gene for both methods whereas the nptII and bar genes were used as selectable markers for A. tumefaciens and biolistic transformation respectively. Previous reports for in vitro regeneration of A. salmiana have not been published; therefore the

Silvia Flores-Benítez; Juan F. Jiménez-Bremont; Sergio Rosales-Mendoza; Gerardo R. Argüello-Astorga; Rosalba Castillo-Collazo; Ángel Gabriel Alpuche-Solís

2007-01-01

41

Agrobacterium tumefaciens DNA and PS8 Bacteriophage DNA not Detected in Crown Gall Tumors  

Microsoft Academic Search

Renaturation kinetics of labeled Agrobacterium tumefaciens DNA are not influenced by addition of 104-fold excess of crown gall tumor DNA. Reconstruction experiments demonstrated that 0.01% added bacterial DNA produces a detectable increase in rate of renaturation of labeled DNA. Crown gall tumor DNA therefore cannot contain as much as 0.01% A. tumefaciens DNA (one entire bacterial genome per three diploid

Mary-Dell Chilton; Thomas C. Currier; Stephen K. Farrand; Arnold J. Bendich; Milton P. Gordon; Eugene W. Nester

1974-01-01

42

T-DNA transfer from Agrobacterium tumefaciens to the ectomycorrhizal fungus Pisolithus microcarpus.  

PubMed

The model ectomycorrhizal fungus Pisolithus microcarpus isolate 441 was transformed by using Agrobacterium tumefaciens LBA1100 and AGL-1. The selection marker was the Shble gene of Streptoallotecius hidustanus, conferring resistance to phleomycin, under the control of the gpd gene promoter and terminator of Schizophyllum commune. Transformation resulted in phleomycin resistant clones which were confirmed by PCR to contain the resistance cassette. A. tumefaciens-mediated gene transfer would allow the development of RNA interference technology in P. microcarpus. PMID:16178458

Pardo, A G; Kemppainen, M; Valdemoros, D; Duplessis, S; Martin, F; Tagu, D

2005-01-01

43

Crystal Structure of Uronate Dehydrogenase from Agrobacterium tumefaciens*  

PubMed Central

Uronate dehydrogenase from Agrobacterium tumefaciens (AtUdh) belongs to the short-chain dehydrogenase/reductase superfamily and catalyzes the oxidation of d-galacturonic acid and d-glucuronic acid with NAD+ as a cofactor. We have determined the crystal structures of an apo-form of AtUdh, a ternary form in complex with NADH and product (substrate-soaked structure), and an inactive Y136A mutant in complex with NAD+. The crystal structures suggest AtUdh to be a homohexamer, which has also been observed to be the major form in solution. The monomer contains a Rossmann fold, essential for nucleotide binding and a common feature of the short-chain dehydrogenase/reductase family enzymes. The ternary complex structure reveals a product, d-galactaro-1,5-lactone, which is bound above the nicotinamide ring. This product rearranges in solution to d-galactaro-1,4-lactone as verified by mass spectrometry analysis, which agrees with our previous NMR study. The crystal structure of the mutant with the catalytic residue Tyr-136 substituted with alanine shows changes in the position of Ile-74 and Ser-75. This probably altered the binding of the nicotinamide end of NAD+, which was not visible in the electron density map. The structures presented provide novel insights into cofactor and substrate binding and the reaction mechanism of AtUdh. This information can be applied to the design of efficient microbial conversion of d-galacturonic acid-based waste materials. PMID:21676870

Parkkinen, Tarja; Boer, Harry; Jänis, Janne; Andberg, Martina; Penttilä, Merja; Koivula, Anu; Rouvinen, Juha

2011-01-01

44

Effect of pre-plant soil fumigants on Agrobacterium tumefaciens, pythiaceous species, and subsequent soil recolonization by A. tumefaciens  

Microsoft Academic Search

Paradox (Juglans hindsii × J. regia), the dominant rootstock used in the California walnut industry, is susceptible to crown gall caused by Agrobacterium tumefaciens. In practice, soil fumigation has been a common pre-plant management strategy for crown gall, but even the industry standard, methyl bromide (MeBr), results in inconsistent disease control. To examine MeBr efficacy and identify potential alternatives, combinations of 1,3-dichloropropene

L. E. Yakabe; S. R. Parker; D. A. Kluepfel

2010-01-01

45

d-Glucaric Acid and Galactaric Acid Catabolism by Agrobacterium tumefaciens  

PubMed Central

Cell-free extract (crude extract) of Agrobacterium tumefaciens grown on d-glucuronate or d-glucarate converts d-glucarate and galactarate to a mixture of 2-keto-3-deoxy- and 4-deoxy-5-keto-d-glucarate. These compounds are then converted by partially purified crude extract to an intermediate tentatively identified as 2,5-diketoadipate. The same enzyme preparation further decarboxylates this intermediate to ?-ketoglutarate semialdehyde, which is subsequently oxidized in a nicotinamide adenine dinucleotide-dependent reaction to ?-ketoglutaric acid. Since A. tumefaciens converts d-glucuronic acid to d-glucarate, a pathway from d-glucuronate to ?-ketoglutarate in A. tumefaciens was determined. PMID:4314480

Chang, Yung Feng; Feingold, David Sidney

1970-01-01

46

Genome sequence of the plant pathogen and biotechnology agent Agrobacterium tumefaciens C58.  

PubMed

Agrobacterium tumefaciens is a plant pathogen capable of transferring a defined segment of DNA to a host plant, generating a gall tumor. Replacing the transferred tumor-inducing genes with exogenous DNA allows the introduction of any desired gene into the plant. Thus, A. tumefaciens has been critical for the development of modern plant genetics and agricultural biotechnology. Here we describe the genome of A. tumefaciens strain C58, which has an unusual structure consisting of one circular and one linear chromosome. We discuss genome architecture and evolution and additional genes potentially involved in virulence and metabolic parasitism of host plants. PMID:11743194

Goodner, B; Hinkle, G; Gattung, S; Miller, N; Blanchard, M; Qurollo, B; Goldman, B S; Cao, Y; Askenazi, M; Halling, C; Mullin, L; Houmiel, K; Gordon, J; Vaudin, M; Iartchouk, O; Epp, A; Liu, F; Wollam, C; Allinger, M; Doughty, D; Scott, C; Lappas, C; Markelz, B; Flanagan, C; Crowell, C; Gurson, J; Lomo, C; Sear, C; Strub, G; Cielo, C; Slater, S

2001-12-14

47

The genome of the natural genetic engineer Agrobacterium tumefaciens C58.  

PubMed

The 5.67-megabase genome of the plant pathogen Agrobacterium tumefaciens C58 consists of a circular chromosome, a linear chromosome, and two plasmids. Extensive orthology and nucleotide colinearity between the genomes of A. tumefaciens and the plant symbiont Sinorhizobium meliloti suggest a recent evolutionary divergence. Their similarities include metabolic, transport, and regulatory systems that promote survival in the highly competitive rhizosphere; differences are apparent in their genome structure and virulence gene complement. Availability of the A. tumefaciens sequence will facilitate investigations into the molecular basis of pathogenesis and the evolutionary divergence of pathogenic and symbiotic lifestyles. PMID:11743193

Wood, D W; Setubal, J C; Kaul, R; Monks, D E; Kitajima, J P; Okura, V K; Zhou, Y; Chen, L; Wood, G E; Almeida, N F; Woo, L; Chen, Y; Paulsen, I T; Eisen, J A; Karp, P D; Bovee, D; Chapman, P; Clendenning, J; Deatherage, G; Gillet, W; Grant, C; Kutyavin, T; Levy, R; Li, M J; McClelland, E; Palmieri, A; Raymond, C; Rouse, G; Saenphimmachak, C; Wu, Z; Romero, P; Gordon, D; Zhang, S; Yoo, H; Tao, Y; Biddle, P; Jung, M; Krespan, W; Perry, M; Gordon-Kamm, B; Liao, L; Kim, S; Hendrick, C; Zhao, Z Y; Dolan, M; Chumley, F; Tingey, S V; Tomb, J F; Gordon, M P; Olson, M V; Nester, E W

2001-12-14

48

Agrobacterium tumefaciens-mediated transformation of Mentha spicata and Mentha arvensis  

Microsoft Academic Search

The stable integration of GUS and NPTII genes in Mentha arvensis and M. spicata has been achieved by Agrobacterium tumefaciens-mediated\\u000a gene transfer. Transformation assays were performed by cocultivating plant leaf disks with either GV2260\\/GI or EHA105\\/MOG\\u000a Agrobacterium strains. Transgenic plants were selected on medium containing 150 mg l?1 kanamycin. Transgene presence and structure was studied by the use of PCR

F. Diemer; J. C. Caissard; S. Moja; F. Jullien

1999-01-01

49

Selection system and co-cultivation medium are important determinants of Agrobacterium-mediated transformation of sugarcane.  

PubMed

A reproducible method for transformation of sugarcane using various strains of Agrobacterium tumefaciens (A. tumefaciens) (AGL0, AGL1, EHA105 and LBA4404) has been developed. The selection system and co-cultivation medium were the most important factors determining the success of transformation and transgenic plant regeneration. Plant regeneration at a frequency of 0.8-4.8% occurred only when callus was transformed with A. tumefaciens carrying a newly constructed superbinary plasmid containing neomycin phosphotransferase (nptII) and beta-glucuronidase (gusA) genes, both driven by the maize ubiquitin (ubi-1) promoter. Regeneration was successful in plants carrying the nptII gene but not the hygromycin phosphotransferase (hph) gene. NptII gene selection was imposed at a concentration of 150 mg/l paromomycin sulphate and applied either immediately or 4 days after the co-cultivation period. Co-cultivation on Murashige and Skoog (MS)-based medium for a period of 4 days produced the highest number of transgenic plants. Over 200 independent transgenic lines were created using this protocol. Regenerated plants appeared phenotypically normal and contained both gusA and nptII genes. Southern blot analysis revealed 1-3 transgene insertion events that were randomly integrated in the majority of the plants produced. PMID:20041254

Joyce, Priya; Kuwahata, Melissa; Turner, Nicole; Lakshmanan, Prakash

2010-02-01

50

Unexpected phytostimulatory behavior for Escherichia coli and Agrobacterium tumefaciens model strains.  

PubMed

Plant-beneficial effects of bacteria are often underestimated, especially for well-studied strains associated with pathogenicity or originating from other environments. We assessed the impact of seed inoculation with the emblematic bacterial models Agrobacterium tumefaciens C58 (plasmid-cured) or Escherichia coli K-12 on maize seedlings in nonsterile soil. Compared with the noninoculated control, root biomass (with A. tumefaciens or E. coli) and shoot biomass (with A. tumefaciens) were enhanced at 10 days for 'PR37Y15' but not 'DK315', as found with the phytostimulator Azospirillum brasilense UAP-154 (positive control). In roots as well as in shoots, Agrobacterium tumefaciens and E. coli triggered similar (in PR37Y15) or different (in DK315) changes in the high-performance liquid chromatography profiles of secondary metabolites (especially benzoxazinoids), distinct from those of Azospirillum brasilense UAP-154. Genome sequence analysis revealed homologs of nitrite reductase genes nirK and nirBD and siderophore synthesis genes for Agrobacterium tumefaciens, as well as homologs of nitrite reductase genes nirBD and phosphatase genes phoA and appA in E. coli, whose contribution to phytostimulation will require experimental assessment. In conclusion, the two emblematic bacterial models had a systemic impact on maize secondary metabolism and resulted in unexpected phytostimulation of seedlings in the Azospirillum sp.-responsive cultivar. PMID:23360460

Walker, Vincent; Bruto, Maxime; Bellvert, Floriant; Bally, René; Muller, Daniel; Prigent-Combaret, Claire; Moënne-Loccoz, Yvan; Comte, Gilles

2013-05-01

51

Cloning and Characterization of Uronate Dehydrogenases from Two Pseudomonads and Agrobacterium tumefaciens Strain C58  

Microsoft Academic Search

Uronate dehydrogenase has been cloned from Pseudomonas syringae pv. tomato strain DC3000, Pseudomonas putida KT2440, and Agrobacterium tumefaciens strain C58. The genes were identified by using a novel comple- mentation assay employing an Escherichia coli mutant incapable of consuming glucuronate as the sole carbon source but capable of growth on glucarate. A shotgun library of P. syringae was screened in

Sang-Hwal Yoon; Tae Seok Moon; Pooya Iranpour; Amanda M. Lanza; Kristala Jones Prather

2009-01-01

52

Agrobacterium tumefaciens-mediated transformation of Allium cepa L.: the production of transgenic onions and shallots  

Microsoft Academic Search

This paper describes the development of a reliable transformation protocol for onion and shallot (Allium cepa L.) which can be used year-round. It is based on Agrobacterium tumefaciens as a vector, with three-week old callus, induced from mature zygotic embryos, as target tissue. For the development of the protocol a large number of parameters were studied. The expression of the

Si-Jun Zheng; Ludmila Khrustaleva; Betty Henken; Eri Sofiari; Evert Jacobsen; Chris Kik; Frans A. Krens

2001-01-01

53

Novel primers for detection of genetically diverse virulent Agrobacterium tumefaciens bv1 strains  

Technology Transfer Automated Retrieval System (TEKTRAN)

Novel primers were developed to amplify a 243 bp fragment of an intergenic region between gene5 and tms2 on the T-DNA of Agrobacterium tumefaciens. These primers exhibit 100% positive correlation with strain virulence, 100% negative correlation with avirulence and did not generate extraneous bands,...

54

Agrobacterium tumefaciens-mediated transformation of the soybean pathogen Phomopsis longicolla.  

PubMed

To facilitate functional genomics in the soybean pathogen Phomopsis longicolla, we developed a robust Agrobacterium tumefaciens-mediated transformation system that yielded 150-250 transformants per 1×10(6) conidia of P. longicolla. This first report of P. longicolla transformation provides a useful tool for insertional mutagenesis in an increasingly important pathogen of soybean. PMID:23305924

Li, Shuxian; Ridenour, John B; Kim, Hun; Hirsch, Robert L; Rupe, John C; Bluhm, Burton H

2013-03-01

55

An enrichment technique for auxotrophs of Agrobacterium tumefaciens using a combination of carbenicillin and lysozyme.  

PubMed

A procedure to enrich for auxotrophic and fermentation mutants of Agrobacterium tumefaciens is described. The method is based on the amplification of the killing power of carbenicillin by the addition of lysozyme. Isolation frequencies of some types of mutants are presented, with and without the application of the proposed procedure. The yield of mutants is usually enhanced a hundredfold per enrichment treatment. PMID:1104767

Klapwijk, P M; de Jonge, A J; Schilperoort, R A; Rörsch, A

1975-11-01

56

Genetic transformation of 9 in vitro clones of Alnus and Betula by Agrobacterium tumefaciens  

Microsoft Academic Search

Crown gall tumorigenesis, integration and expression of T-DNA encoded genes from Agrobacterium tumefaciens were investigated in 9 clones of Alnus glutinosa, A. incana and Betula papyrifera. Tumor formation on in vitro shoots was frequent in all clones with strain Ach5 and present in 8 clones with strain C58. Tumors excised from shoots were selected for autotrophic growth in vitro and

John Mackay; Armand Séguin; Maurice Lalonde

1988-01-01

57

Genome sequence of the arsenite-oxidizing strain Agrobacterium tumefaciens 5A.  

PubMed

Microbial transformations of arsenic influence its mobility and toxicity. We report the draft genome sequence of the arsenite-oxidizing strain Agrobacterium tumefaciens 5A isolated from an As-contaminated soil in the Madison River Valley, MT. A large number of metal (or metalloid) resistance genes, especially contributing to arsenite oxidation, were identified. PMID:22275101

Hao, Xiuli; Lin, Yanbing; Johnstone, Laurel; Liu, Guanghui; Wang, Gejiao; Wei, Gehong; McDermott, Timothy; Rensing, Christopher

2012-02-01

58

Evaluations and modifications of semi-selective media for improved isolation of Agrobacterium tumefaciens biovar 1 from cultivated walnut  

Technology Transfer Automated Retrieval System (TEKTRAN)

Agrobacterium tumefaciens, the causal agent of crown gall of walnut, is an aerobic, Gram negative bacterium belonging to the family Rhizobiaceae. Like many in this group, A. tumefaciens is a common inhabitant of soil and plant host tissue. Isolation from these complex environments is difficult even ...

59

SCREENING OF TRANSGENIC ANTHURIUMS FOR BACTERIAL BLIGHT AND NEMATODE RESISTANCE  

Technology Transfer Automated Retrieval System (TEKTRAN)

Anthuriums exhibit limited resistance to bacterial blight caused by Xanthomonas axonopodis pv. dieffenbachiae and to the nematodes Radopholus simile and Meloidogyne javanica. Agrobacterium tumefaciens transformation of embryogenic calli with strains LBA4404, EHA105, and AGLO resulted in transgenic p...

60

Replicon-Specific Regulation of Small Heat Shock Genes in Agrobacterium tumefaciens  

Microsoft Academic Search

Four genes coding for small heat shock proteins (sHsps) were identified in the genome sequence of Agrobacterium tumefaciens, one on the circular chromosome (hspC), one on the linear chromosome (hspL), and two on the pAT plasmid (hspAT1 and hspAT2). Induction of sHsps at elevated temperatures was revealed by immunoblot analyses. Primer extension experiments and translational lacZ fusions demonstrated that expres-

Sylvia Balsiger; Curdin Ragaz; Christian Baron; Franz Narberhaus

2004-01-01

61

Transformation of the cultivated mushroom Agaricus bisporus (Lange) using T-DNA from Agrobacterium tumefaciens  

Microsoft Academic Search

Agrobacterium tumefaciens is known to transfer parts of its tumor-inducing plasmid, the T-DNA, to plants, yeasts and filamentous fungi. We have used\\u000a this system to transform germinating basidiospores and vegetative mycelium of a commercial strain of the cultivated basidiomycete\\u000a Agaricus bisporus. Analysis of transformants shows that the T-DNA integrates at random sites into the host genome and that the selection

Thomas S. P. Mikosch; Brigitte Lavrijssen; Anton S. M. Sonnenberg

2001-01-01

62

Roles of Agrobacterium tumefaciens RirA in Iron Regulation, Oxidative Stress Response, and Virulence  

Microsoft Academic Search

The analysis of genetics and physiological functions of Agrobacterium tumefaciens RirA (rhizobial iron regulator) has shown that it is a transcription regulator and a repressor of iron uptake systems. The rirA mutant strain (NTLrirA) overproduced siderophores and exhibited a highly constitutive expression of genes involved in iron uptake (fhuA, irp6A, and fbpA) compared to that of the wild-type strain (NTL4).

Patchara Ngok-Ngam; Nantaporn Ruangkiattikul; A. Mahavihakanont; S. S. Virgem; R. Sukchawalit; S. Mongkolsuk

2009-01-01

63

Cell–cell signaling and the Agrobacterium tumefaciens Ti plasmid copy number fluctuations  

Microsoft Academic Search

The Agrobacterium tumefaciens oncogenic Ti plasmids replicate and segregate to daughter cells via repABC cassettes, in which repA and repB are plasmid partitioning genes and repC encodes the replication initiator protein. repABC cassettes are encountered in a growing number of plasmids and chromosomes of the ?-proteobacteria, and findings from particular representatives of agrobacteria, rhizobia and Paracoccus have began to shed

Katherine M. Pappas

2008-01-01

64

Root induction in Pinus ayacahuite by co-culture with Agrobacterium tumefaciens strains.  

PubMed

Transformation of in-vitro-derived shoots of Pinus ayacahuite Ehrenb. was achieved by co-culture with an oncogenic strain (A281 x 200) of Agrobacterium tumefaciens. During co-culture rooting also occurred; however, this rooting was not induced by genetic transformation of host cells, because a "disarmed" strain of A. tumefaciens (EHA101) also induced rooting. Furthermore, direct contact between shoots and bacterial cells was not required. Rooting occurred in agar-solidified medium and in a soilless substrate (9:1 vermiculite:peat mix). We conclude that A. tumefaciens strains induced rooting in P. ayacahuite through a change in the rhizosphere, probably by producing some root-inducing compound(s), and not through transformation of host cells. PMID:12651560

Saborio, Francisco; Moloney, Maurice M.; Tung, Pariana; Thorpe, Trevor A.

1999-05-01

65

Choline uptake in Agrobacterium tumefaciens by the high-affinity ChoXWV transporter.  

PubMed

Agrobacterium tumefaciens is a facultative phytopathogen that causes crown gall disease. For successful plant transformation A. tumefaciens requires the membrane lipid phosphatidylcholine (PC), which is produced via the methylation and the PC synthase (Pcs) pathways. The latter route is dependent on choline. Although choline uptake has been demonstrated in A. tumefaciens, the responsible transporter(s) remained elusive. In this study, we identified the first choline transport system in A. tumefaciens. The ABC-type choline transporter is encoded by the chromosomally located choXWV operon (ChoX, binding protein; ChoW, permease; and ChoV, ATPase). The Cho system is not critical for growth and PC synthesis. However, [14C]choline uptake is severely reduced in A. tumefaciens choX mutants. Recombinant ChoX is able to bind choline with high affinity (equilibrium dissociation constant [KD] of ?2 ?M). Since other quaternary amines are bound by ChoX with much lower affinities (acetylcholine, KD of ?80 ?M; betaine, KD of ?470 ?M), the ChoXWV system functions as a high-affinity transporter with a preference for choline. Two tryptophan residues (W40 and W87) located in the predicted ligand-binding pocket are essential for choline binding. The structural model of ChoX built on Sinorhizobium meliloti ChoX resembles the typical structure of substrate binding proteins with a so-called "Venus flytrap mechanism" of substrate binding. PMID:21803998

Aktas, Meriyem; Jost, Kathinka A; Fritz, Christiane; Narberhaus, Franz

2011-10-01

66

Overexpression of the HspL Promotes Agrobacterium tumefaciens Virulence in Arabidopsis Under Heat Shock Conditions.  

PubMed

Agrobacterium tumefaciens transfers a specific DNA fragment from the resident tumor-inducing (Ti) plasmid and effector virulence (Vir) proteins to plant cells during infection. A. tumefaciens VirB1-11 and VirD4 proteins assemble as the type IV secretion system (T4SS), which mediates transfer of the T-DNA and effector Vir protein into plant cells, thus resulting in crown gall disease in plants. Previous studies revealed that an ?-crystallin-type, small heat-shock protein (HspL) is a more effective VirB8 chaperone than three other small heat-shock proteins (HspC, HspAT1, and HspAT2). Additionally, HspL contributes to efficient T4SS-mediated DNA transfer and tumorigenesis under room-temperature growth. In this study, we aimed to characterize the impact of HspL on Agrobacterium-mediated transformation efficiency under heat-shock treatment. During heat shock, transient transformation efficiency and VirB8 protein accumulation were lower in the hspL deletion mutant than in the wild type. Overexpression of HspL in A. tumefaciens enhanced the transient transformation efficiency in root explants of both susceptible and recalcitrant Arabidopsis ecotypes. In addition, the reduced transient transformation efficiency during heat stress was recovered by overexpression of HspL in A. tumefaciens. HspL may help maintain VirB8 homeostasis and elevate Agrobacterium-mediated transformation efficiency under both heat-shock and nonheat-shock growth. PMID:25163013

Hwang, Hau-Hsuan; Liu, Yin-Tzu; Huang, Si-Chi; Tung, Chin-Yi; Huang, Fan-Chen; Tsai, Yun-Long; Cheng, Tun-Fang; Lai, Erh-Min

2015-02-01

67

Rapid, in situ detection of Agrobacterium tumefaciens attachment to leaf tissue.  

PubMed

Attachment of the plant pathogen Agrobacterium tumefaciens to host plant cells is an early and necessary step in plant transformation and agroinfiltration processes. However, bacterial attachment behavior is not well understood in complex plant tissues. Here we developed an imaging-based method to observe and quantify A. tumefaciens attached to leaf tissue in situ. Fluorescent labeling of bacteria with nucleic acid, protein, and vital dyes was investigated as a rapid alternative to generating recombinant strains expressing fluorescent proteins. Syto 16 green fluorescent nucleic acid stain was found to yield the greatest signal intensity in stained bacteria without affecting viability or infectivity. Stained bacteria retained the stain and were detectable over 72 h. To demonstrate in situ detection of attached bacteria, confocal fluorescent microscopy was used to image A. tumefaciens in sections of lettuce leaf tissue following vacuum-infiltration with labeled bacteria. Bacterial signals were associated with plant cell surfaces, suggesting detection of bacteria attached to plant cells. Bacterial attachment to specific leaf tissues was in agreement with known leaf tissue competencies for transformation with Agrobacterium. Levels of bacteria attached to leaf cells were quantified over time post-infiltration. Signals from stained bacteria were stable over the first 24 h following infiltration but decreased in intensity as bacteria multiplied in planta. Nucleic acid staining of A. tumefaciens followed by confocal microscopy of infected leaf tissue offers a rapid, in situ method for evaluating attachment of A. tumefaciens' to plant expression hosts and a tool to facilitate management of transient expression processes via agroinfiltration. PMID:22848046

Simmons, Christopher W; Nitin, N; Vandergheynst, Jean S

2012-01-01

68

Enhancers of Agrobacterium-mediated Transformation of Tibouchina semidecandra Selected on the Basis of GFP Expression.  

PubMed

Genetic engineering is a powerful tool for the improvement of plant traits. Despite reported successes in the plant kingdom, this technology has barely scratched the surface of the Melastomataceae family. Limited studies have led to some optimisation of parameters known to affect the transformation efficiency of these plants. The major finding of this study was to optimise the presence of selected enhancers [e.g., monosaccharides (D-glucose, D-galactose and D-fructose), tyrosine, aluminium chloride (AICI3) and ascorbic acid] to improve the transformation efficiency of Tibouchina semidecandra. Agrobacterium tumefaciens strain LBA4404 harbouring the disarmed plasmid pCAMBIA1304 was used to transform shoots and nodes of T. semidecandra. Different concentrations of the transformation enhancers were tested by using green fluorescent protein (GFP) as a reporter. The results obtained were based on the percentage of GFP expression, which was observed 14 days post-transformation. A combination of 120 ?M galactose and 100 ?M tyrosine supplemented with 600 ?M AICI3 in the presence of 15 mg/l ascorbic acid gave the highest percentage of positive transformants for T. semidecandra shoots. Whereas 60 ?M galactose and 50 ?M tyrosine with 200 ?M AICI3 in the presence of 15 mg/l ascorbic acid was optimum for T. semidecandra nodes. The presence of the hygromycin phosphotransferase II (hptII) transgene in the genomic DNA of putative T. semidecandra transformants was verified by PCR amplification with specific primers. PMID:24575204

Yong, Wilson Thau Lym; Henry, Erle Stanley; Abdullah, Janna Ong

2010-12-01

69

Enhancers of Agrobacterium-mediated Transformation of Tibouchina semidecandra Selected on the Basis of GFP Expression  

PubMed Central

Genetic engineering is a powerful tool for the improvement of plant traits. Despite reported successes in the plant kingdom, this technology has barely scratched the surface of the Melastomataceae family. Limited studies have led to some optimisation of parameters known to affect the transformation efficiency of these plants. The major finding of this study was to optimise the presence of selected enhancers [e.g., monosaccharides (D-glucose, D-galactose and D-fructose), tyrosine, aluminium chloride (AICI3) and ascorbic acid] to improve the transformation efficiency of Tibouchina semidecandra. Agrobacterium tumefaciens strain LBA4404 harbouring the disarmed plasmid pCAMBIA1304 was used to transform shoots and nodes of T. semidecandra. Different concentrations of the transformation enhancers were tested by using green fluorescent protein (GFP) as a reporter. The results obtained were based on the percentage of GFP expression, which was observed 14 days post-transformation. A combination of 120 ?M galactose and 100 ?M tyrosine supplemented with 600 ?M AICI3 in the presence of 15 mg/l ascorbic acid gave the highest percentage of positive transformants for T. semidecandra shoots. Whereas 60 ?M galactose and 50 ?M tyrosine with 200 ?M AICI3 in the presence of 15 mg/l ascorbic acid was optimum for T. semidecandra nodes. The presence of the hygromycin phosphotransferase II (hptII) transgene in the genomic DNA of putative T. semidecandra transformants was verified by PCR amplification with specific primers. PMID:24575204

Yong, Wilson Thau Lym; Henry, Erle Stanley; Abdullah, Janna Ong

2010-01-01

70

Gene cluster for ferric iron uptake in Agrobacterium tumefaciens MAFF301001.  

PubMed

Agrobacterium tumefaciens harboring a Ti plasmid causes crown gall disease in dicot plants by transferring its T-DNA into plant chromosomes. Iron acquisition plays an important role for pathogenicity in animal pathogens and several phytopathogens and for growth in the rhizosphere and on plant surfaces. Under iron-limiting condition, bacteria produce various iron-chelating agents called siderophores. Agrobacterium strains have the diversity in producing siderophores and a certain strain produces a typical catechol-type siderophore, called agrobactin, although its biosynthesis genes have not been analyzed yet. Here we describe the cloning and characterization of a functional gene cluster involved in ferric iron uptake in A. tumefaciens strain MAFF301001. Four complete open reading frames (ORFs) were found in 5-kb region of a genomic library clone 1A3. We named these genes agb, after agrobactin. agbC, agbE, agbB and agbA genes were identified in this order, and narrow intergenic spaces suggested that these genes constitute an operon. Predicted agb gene products and their phylogenetic analysis showed sequence similarity with enzymes which are involved in ferric iron uptake in other bacteria. Southern hybridization analysis clearly indicated the location of agb genes on the linear chromosome in strain MAFF301001 but the complete lack in another A. tumefaciens strain C58. Mutation analysis of agbB revealed that it is essential for growth and production of catechol compounds in iron-limiting medium. PMID:12207035

Sonoda, Hiroyuki; Suzuki, Katsunori; Yoshida, Kazuo

2002-06-01

71

Agrobacterium -mediated transformation of American ginseng with a rice chitinase gene  

Microsoft Academic Search

Transformation of American ginseng (Panax quinquefolius L.) with Agrobacterium strain LBA4404 containing a rice chitinase gene under control of the maize ubiquitin1 promoter and the phosphinothricin acetyltransferase (bar) and hygromycin phosphotransferase (hpt) genes as selectable markers is described. Epicotyl explants from 2- to 3-week-old ginseng seedlings were pre-cultured for 5-7 days on MS medium supplemented with 10 µM !-naphthaleneacetic acid

W. P. Chen; Z. K. Punja

2002-01-01

72

Transformation of Zea mays L. Using Agrobacterium tumefaciens and the Shoot Apex.  

PubMed

Agrobacterium tumefaciens is established as a vector for gene transfer in many dicotyledonous plants but is not accepted as a vector in monocotyledonous plants, especially in the important Gramineae. The use of Agrobacterium to transfer genes into monocot species could simplify the transformation and improvement of important crop plants. In this report we describe the use of Agrobacterium to transfer a gene into corn, the regeneration of plants, and detection of the transferred genes in the F(1) progeny. Shoot apices of Zea mays L. variety Funk's G90 were cocultivated with A. tumefaciens EHA 1, which harbored the plasmid pGUS3 containing genes for kanamycin resistance (NPT II) and beta-glucuronidase (GUS). Plants developed from these explants within 4 to 6 weeks. Fluorometric GUS assays of leaves and immature seeds from the plants exhibited low GUS activity. Both NOS and GUS gene fragments were amplified by polymerase chain reaction in the DNA isolated from the F(1) generations of one of the original transformed plants. Southern analysis showed both GUS and NPT probes hybridized to DNA in several of the F(1) progeny, demonstrating the incorporation of GUS and NPT II genes into high molecular weight DNA. These data establish successful gene transfer and sexual inheritance of the genes. PMID:16668001

Gould, J; Devey, M; Hasegawa, O; Ulian, E C; Peterson, G; Smith, R H

1991-02-01

73

Proteomic changes in grape embryogenic callus in response to Agrobacterium tumefaciens-mediated transformation.  

PubMed

Agrobacterium tumefaciens-mediated transformation is highly required for studies of grapevine gene function and of huge potential for tailored variety improvements. However, grape is recalcitrant to transformation, and the underlying mechanism is largely unknown. To better understand the overall response of grapevine to A. tumefaciens-mediated transformation, the proteomic profile of cv. Prime embryogenic callus (EC) after co-cultivation with A. tumefaciens was investigated by two-dimensional electrophoresis and MALDI-TOF-MS analysis. Over 1100 protein spots were detected in both inoculated and control EC, 69 of which showed significantly differential expression; 38 of these were successfully identified. The proteins significantly up-regulated 3 d after inoculation were PR10, resistance protein Pto, secretory peroxidase, cinnamoyl-CoA reductase and different expression regulators; down-regulated proteins were ascorbate peroxidase, tocopherol cyclase, Hsp 70 and proteins involved in the ubiquitin-associated protein-degradation pathway. A. tumefaciens transformation-induced oxidative burst and modified protein-degradation pathways were further validated with biochemical measurements. Our results reveal that agrobacterial transformation markedly inhibits the cellular ROS-removal system, mitochondrial energy metabolism and the protein-degradation machinery for misfolded proteins, while the apoptosis signaling pathway and hypersensitive response are strengthened, which might partially explain the low efficiency and severe EC necrosis in grape transformation. PMID:21889056

Zhao, Fengxia; Chen, Lihua; Perl, Avihai; Chen, Shangwu; Ma, Huiqin

2011-10-01

74

Generation of transgenic Lolium temulentum plants by Agrobacterium tumefaciens-mediated transformation.  

PubMed

Lolium temulentum L. (Darnel ryegrass) has been proposed to be used as a model species for functional genomics studies in forage and turf grasses, because it is a self-fertile, diploid species with a short life cycle and is closely related to other grasses. Embryogenic calluses were induced from mature embryos of a double haploid line developed through anther culture. The calluses were broken up into small pieces and used for Agrobacterium tumefaciens-mediated transformation. A. tumefaciens strain EHA105 harboring pCAMBIA1301 and pCAMBIA1305.2 vectors were used to infect embryogenic callus pieces. Hygromycin was used as a selection agent in stable transformation experiments. Hygromycin resistant calluses were obtained after 4-6 weeks of selection and transgenic plants were produced in 10-13 weeks after Agrobacterium-mediated transformation. Fertile plants were readily obtained after transferring the transgenics to the greenhouse. Transgenic nature of the regenerated plants was demonstrated by Polymerase chain reaction (PCR), Southern hybridization analysis, and GUS staining. Progeny analysis showed Mendelian inheritance of the transgenes. The transformation system provides a valuable tool for functionality tests of candidate genes in forage and turf grasses. PMID:17221228

Ge, Yaxin; Cheng, Xiaofei; Hopkins, Andrew; Wang, Zeng-Yu

2007-06-01

75

Agrobacterium tumefaciens-Mediated Transformation of the Lichen Fungus, Umbilicaria muehlenbergii  

PubMed Central

Transformation-mediated mutagenesis in both targeted and random manners has been widely applied to decipher gene function in diverse fungi. However, a transformation system has not yet been established for lichen fungi, severely limiting our ability to study their biology and mechanism underpinning symbiosis via gene manipulation. Here, we report the first successful transformation of the lichen fungus, Umbilicaria muehlenbergii, via the use of Agrobacterium tumefaciens. We generated a total of 918 transformants employing a binary vector that carries the hygromycin B phosphotransferase gene as a selection marker and the enhanced green fluorescent protein gene for labeling transformants. Randomly selected transformants appeared mitotically stable, based on their maintenance of hygromycin B resistance after five generations of growth without selection. Genomic Southern blot showed that 88% of 784 transformants contained a single T-DNA insert in their genome. A number of putative mutants affected in colony color, size, and/or morphology were found among these transformants, supporting the utility of Agrobacterium tumefaciens-mediated transformation (ATMT) for random insertional mutagenesis of U. muehlenbergii. This ATMT approach potentially offers a systematic gene functional study with genome sequences of U. muehlenbergii that is currently underway. PMID:24386304

Wang, Hai-Ying; Kim, Jung A.; Yu, Nan-Hee; Kim, Sungbeom; Cheong, Yong Hwa; Kang, Seogchan; Lee, Yong-Hwan; Hur, Jae-Seoun

2013-01-01

76

Genes responsible for the supervirulence phenotype of Agrobacterium tumefaciens A281.  

PubMed Central

Agrobacterium tumefaciens A281 induces large, rapidly appearing tumors on a variety of plants and has a wider host range than other strains of A. tumefaciens. By using Tn3HoHo1 transposon mutagenesis and complementation analysis, a 2.5-kilobase DNA fragment which is responsible for the supervirulence phenotype was identified in the virulence (vir) region of the Ti plasmid. This fragment contains the virG locus, as well as the 3' end of the virB operon. A clone of this fragment conferred the supervirulence phenotype on A348, a nonsupervirulent strain. The increased virulence was correlated with an increased expression of vir genes, which could be achieved by introducing an extra copy of the transcriptional activator virG or the supervirulence region for maximum virulence. The virulence of the supervirulent strain A281 could be increased even further if the entire virB operon was added in addition to the virG operon. A plasmid, pToK47, containing virB and virG increased the virulence of all A. tumefaciens strains into which the plasmid was introduced. These data suggest that a highly virulent binary vector system can be constructed which might prove especially useful in the transformation of certain higher plants. Images PMID:2443480

Jin, S G; Komari, T; Gordon, M P; Nester, E W

1987-01-01

77

Hfq Influences Multiple Transport Systems and Virulence in the Plant Pathogen Agrobacterium tumefaciens  

PubMed Central

The Hfq protein mediates gene regulation by small RNAs (sRNAs) in about 50% of all bacteria. Depending on the species, phenotypic defects of an hfq mutant range from mild to severe. Here, we document that the purified Hfq protein of the plant pathogen and natural genetic engineer Agrobacterium tumefaciens binds to the previously described sRNA AbcR1 and its target mRNA atu2422, which codes for the substrate binding protein of an ABC transporter taking up proline and ?-aminobutyric acid (GABA). Several other ABC transporter components were overproduced in an hfq mutant compared to their levels in the parental strain, suggesting that Hfq plays a major role in controlling the uptake systems and metabolic versatility of A. tumefaciens. The hfq mutant showed delayed growth, altered cell morphology, and reduced motility. Although the DNA-transferring type IV secretion system was produced, tumor formation by the mutant strain was attenuated, demonstrating an important contribution of Hfq to plant transformation by A. tumefaciens. PMID:22821981

Wilms, Ina; Möller, Philip; Stock, Anna-Maria; Gurski, Rosemarie; Lai, Erh-Min

2012-01-01

78

Increased 1-aminocyclopropane-1-carboxylate deaminase activity enhances Agrobacterium tumefaciens-mediated gene delivery into plant cells  

PubMed Central

Agrobacterium-mediated transformation is a useful tool for the genetic modification in plants, although its efficiency is low for several plant species. Agrobacterium-mediated transformation has three major steps in laboratory-controlled experiments: the delivery of T-DNA into plant cells, the selection of transformed plant cells, and the regeneration of whole plants from the selected cells. Each of these steps must be optimized to improve the efficiency of Agrobacterium-mediated plant transformation. It has been reported that increasing the number of cells transformed by T-DNA delivery can improve the frequency of stable transformation. Previously, we demonstrated that a reduction in ethylene production by plant cells during cocultivation with A. tumefaciens-expressing 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase resulted in increased T-DNA delivery into the plant cells. In this study, to further improve T-DNA delivery by A. tumefaciens, we modified the expression cassette of the ACC deaminase gene using vir gene promoter sequences. The ACC deaminase gene driven by the virD1 promoter was expressed at a higher level, resulting in a higher ACC deaminase activity in this A. tumefaciens strain than in the strain with the lac promoter used in a previous study. The newly developed A. tumefaciens strain improves the delivery of T-DNA into Solanum lycopersicum (tomato) and Erianthus ravennae plants and thus may be a powerful tool for the Agrobacterium-mediated genetic engineering of plants. PMID:24000136

Someya, Tatsuhiko; Nonaka, Satoko; Nakamura, Kouji; Ezura, Hiroshi

2013-01-01

79

EFFECT OF TEMPERATURE AND DETERGENTS ON AGROBACTERIUM TUMEFACIENS, THE CAUSAL PATHOGEN OF CROWN GALL DISEASE OF WALNUT  

Technology Transfer Automated Retrieval System (TEKTRAN)

Crown gall disease caused by the bacterium Agrobacterium tumefaciens causes significant economic losses in commercial walnut orchards and nursery operations in California. In an effort to develop integrated control strategies to ensure pathogen and disease free plant material at nurseries, the effe...

80

The regulatory VirA protein of Agrobacterium tumefaciens does not function at elevated temperatures.  

PubMed

Previous studies have shown that Agrobacterium tumefaciens causes tumors on plants only at temperatures below 32 degrees C, and virulence gene expression is specifically inhibited at temperatures above 32 degrees C. We show here that this effect persists even when the virA and virG loci are expressed under the control of a lac promoter whose activity is temperature independent. This finding suggests that one or more steps in the signal transduction process mediated by the VirA and VirG proteins are temperature sensitive. Both the autophosphorylation of VirA and the subsequent transfer of phosphate to VirG are shown to be sensitive to high temperatures (> 32 degrees C), and this correlates with the reduced vir gene expression observed at these temperatures. At temperatures of 32 degrees C and higher, the VirA molecule undergoes a reversible inactivation while the VirG molecule is not affected. vir gene induction is temperature sensitive in an acetosyringone-independent virA mutant background but not in a virG constitutive mutant which is virA and acetosyringone independent. These observations all support the notion that the VirA protein is responsible for the thermosensitivity of vir gene expression. However, an Agrobacterium strain containing a constitutive virG locus still cannot cause tumors on Kalanchoe plants at 32 degrees C. This strain induces normal-size tumors at temperatures up to 30 degrees C, whereas the wild-type Agrobacterium strain produces almost no tumors at 30 degrees C. These results suggest that at temperatures above 32 degrees C, the plant becomes more resistant to infection by A. tumefaciens and/or functions of some other vir gene products are lost in spite of their normal levels of expression. PMID:8226624

Jin, S; Song, Y N; Deng, W Y; Gordon, M P; Nester, E W

1993-11-01

81

Linear Chromosome-generating System of Agrobacterium tumefaciens C58: Protelomerase Generates and Protects Hairpin Ends  

SciTech Connect

Agrobacterium tumefaciens C58, the pathogenic bacteria that causes crown gall disease in plants, harbors one circular and one linear chromosome and two circular plasmids. The telomeres of its unusual linear chromosome are covalently closed hairpins. The circular and linear chromosomes co-segregate and are stably maintained in the organism. We have determined the sequence of the two ends of the linear chromosome thus completing the previously published genome sequence of A. tumefaciens C58. We found that the telomeres carry nearly identical 25-bp sequences at the hairpin ends that are related by dyad symmetry. We further showed that its Atu2523 gene encodes a protelomerase (resolvase) and that the purified enzyme can generate the linear chromosomal closed hairpin ends in a sequence-specific manner. Agrobacterium protelomerase, whose presence is apparently limited to biovar 1 strains, acts via a cleavage-and-religation mechanism by making a pair of transient staggered nicks invariably at 6-bp spacing as the reaction intermediate. The enzyme can be significantly shortened at both the N and C termini and still maintain its enzymatic activity. Although the full-length enzyme can uniquely bind to its product telomeres, the N-terminal truncations cannot. The target site can also be shortened from the native 50-bp inverted repeat to 26 bp; thus, the Agrobacterium hairpin-generating system represents the most compact activity of all hairpin linear chromosome- and plasmid-generating systems to date. The biochemical analyses of the protelomerase reactions further revealed that the tip of the hairpin telomere may be unusually polymorphically capable of accommodating any nucleotide.

Huang, Wai Mun; DaGloria, Jeanne; Fox, Heather; Ruan, Qiurong; Tillou, John; Shi, Ke; Aihara, Hideki; Aron, John; Casjens, Sherwood (Utah); (UMM)

2012-09-05

82

The regulatory VirA protein of Agrobacterium tumefaciens does not function at elevated temperatures.  

PubMed Central

Previous studies have shown that Agrobacterium tumefaciens causes tumors on plants only at temperatures below 32 degrees C, and virulence gene expression is specifically inhibited at temperatures above 32 degrees C. We show here that this effect persists even when the virA and virG loci are expressed under the control of a lac promoter whose activity is temperature independent. This finding suggests that one or more steps in the signal transduction process mediated by the VirA and VirG proteins are temperature sensitive. Both the autophosphorylation of VirA and the subsequent transfer of phosphate to VirG are shown to be sensitive to high temperatures (> 32 degrees C), and this correlates with the reduced vir gene expression observed at these temperatures. At temperatures of 32 degrees C and higher, the VirA molecule undergoes a reversible inactivation while the VirG molecule is not affected. vir gene induction is temperature sensitive in an acetosyringone-independent virA mutant background but not in a virG constitutive mutant which is virA and acetosyringone independent. These observations all support the notion that the VirA protein is responsible for the thermosensitivity of vir gene expression. However, an Agrobacterium strain containing a constitutive virG locus still cannot cause tumors on Kalanchoe plants at 32 degrees C. This strain induces normal-size tumors at temperatures up to 30 degrees C, whereas the wild-type Agrobacterium strain produces almost no tumors at 30 degrees C. These results suggest that at temperatures above 32 degrees C, the plant becomes more resistant to infection by A. tumefaciens and/or functions of some other vir gene products are lost in spite of their normal levels of expression. Images PMID:8226624

Jin, S; Song, Y N; Deng, W Y; Gordon, M P; Nester, E W

1993-01-01

83

Light strongly promotes gene transfer from Agrobacterium tumefaciens to plant cells.  

PubMed

Light conditions during Agrobacterium-based plant transformation, the most routinely used method in plant genetic engineering, differ widely and, to our knowledge, have not been studied systematically in relation to transformation efficiency. Here, light effects were examined in two already optimized transformation procedures: coculture of Agrobacterium tumefaciens with callus from two genotypes of the crop plant Phaseolus acutifolius (tepary bean) and coculture of root segments from two ecotypes of Arabidopsis thaliana. Except for the light conditions during coculture, all steps followed established procedures. Coculture was done either under continuous darkness, under a commonly used photoperiod of 16 h light/8 h darkness or under continuous light. beta-glucuronidase (GUS) production due to the transient expression of an intron-containing uidA gene in the binary vector was used to evaluate T-DNA transfer. In all situations, uidA expression correlated highly and positively with the light period used during coculture; it was inhibited severely by darkness and enhanced more under continuous light than under a 16 h light/8 h dark photoperiod. The promotive effect of light was observed with Agrobacterium strains harboring either a nopaline-, an octopine- or an agropine/succinamopine-type non-oncogenic helper Ti plasmid. The observed positive effect of light has obvious implications for developing and improving transient and stable transformation protocols, specifically those involving dark coculture conditions. PMID:12569399

Zambre, Mukund; Terryn, Nancy; De Clercq, Janniek; De Buck, Sylvie; Dillen, Willy; Van Montagu, Marc; Van Der Straeten, Dominique; Angenon, Geert

2003-02-01

84

Sequence and distribution of IS1312: evidence for horizontal DNA transfer from Rhizobium meliloti to Agrobacterium tumefaciens.  

PubMed Central

Two novel insertion sequences, IS1312 and IS1313, were found in pTiBo542, the Ti plasmid of Agrobacterium tumefaciens strains Bo542 and A281. Nucleotide sequencing and Southern hybridization revealed that IS1312 and IS1313 are homologous to Rhizobium meliloti ISRm1 and ISRm2, respectively. IS1312, ISRm1, and another Agrobacterium insertion sequence, IS426, belong to the same IS3 family of insertion sequences; however, IS1312 is more closely related to the Rhizobium ISRm1 than it is to the Agrobacterium IS426. The distribution patterns of these insertion elements and their sequence similarities suggest that IS1312 and IS1313 were horizontally transferred from R. meliloti to A. tumefaciens. PMID:7730290

Deng, W; Gordon, M P; Nester, E W

1995-01-01

85

Genetic transformation of 9 in vitro clones of Alnus and Betula by Agrobacterium tumefaciens.  

PubMed

Crown gall tumorigenesis, integration and expression of T-DNA encoded genes from Agrobacterium tumefaciens were investigated in 9 clones of Alnus glutinosa, A. incana and Betula papyrifera. Tumor formation on in vitro shoots was frequent in all clones with strain Ach5 and present in 8 clones with strain C58. Tumors excised from shoots were selected for autotrophic growth in vitro and axenic cultures were established. Octopine or nopaline, respective of the strain type used for inoculation, was detected in tumorous cultures. Southern blot analyses demonstrated T-DNA integration by hybridization of DNA from tumors with tmr and nos gene probes. One clone of B. papyrifera produced tumors with a morphogenic character, unusual in calli of this species, generating viable shoots which did not synthesize opine. PMID:24241754

Mackay, J; Séguin, A; Lalonde, M

1988-06-01

86

Cloning, expression, purification and crystallization of dihydrodipicolinate synthase from Agrobacterium tumefaciens  

PubMed Central

Dihydrodipicolinate synthase (DHDPS) catalyzes the first committed step of the lysine-biosynthesis pathway in bacteria, plants and some fungi. This study describes the cloning, expression, purification and crystallization of DHDPS (NP_354047.1) from the plant pathogen Agrobacterium tumefaciens (AgT-DHDPS). Enzyme-kinetics studies demonstrate that AgT-DHDPS possesses DHDPS activity in vitro. Crystals of AgT-DHDPS were grown in the unliganded form and in forms with substrate bound and with substrate plus allosteric inhibitor (lysine) bound. X-ray diffraction data sets were subsequently collected to a maximum resolution of 1.40?Å. Determination of the structure with and without substrate and inhibitor will offer insight into the design of novel pesticide agents. PMID:22949190

Atkinson, Sarah C.; Dogovski, Con; Dobson, Renwick C. J.; Perugini, Matthew A.

2012-01-01

87

Crystal Structure of AGR_C_4470p from Agrobacterium tumefaciens  

SciTech Connect

We report here the crystal structure at 2.0 {angstrom} resolution of the AGR{_}C{_}4470p protein from the Gram-negative bacterium Agrobacterium tumefaciens. The protein is a tightly associated dimer, each subunit of which bears strong structural homology with the two domains of the heme utilization protein ChuS from Escherichia coli and HemS from Yersinia enterocolitica. Remarkably, the organization of the AGR{_}C{_}4470p dimer is the same as that of the two domains in ChuS and HemS, providing structural evidence that these two proteins evolved by gene duplication. However, the binding site for heme, while conserved in HemS and ChuS, is not conserved in AGR{_}C{_}4470p, suggesting that it probably has a different function. This is supported by the presence of two homologs of AGR{_}C{_}4470p in E. coli, in addition to the ChuS protein.

Vorobiev,S.; Neely, H.; Seetharaman, J.; Ma, L.; Xiao, R.; Acton, T.; Montelione, G.; Tong, L.

2007-01-01

88

Analysis of the complete nucleotide sequence of the Agrobacterium tumefaciens virB operon.  

PubMed Central

The complete nucleotide sequence of the virB locus, from the octopine Ti plasmid of Agrobacterium tumefaciens strain 15955, has been determined. In the large virB-operon (9600 nucleotides) we have identified eleven open reading frames, designated virB1 to virB11. From DNA sequence analysis it is proposed that nearly all VirB products, i.e. VirB1 to VirB9, are secreted or membrane associated proteins. Interestingly, both a membrane protein (VirB4) and a potential cytoplasmic protein (VirB11) contain the consensus amino acid sequence of ATP-binding proteins. In view of the conjugative T-DNA transfer model, the VirB proteins are suggested to act at the bacterial surface and there play an important role in directing T-DNA transfer to plant cells. PMID:2837739

Thompson, D V; Melchers, L S; Idler, K B; Schilperoort, R A; Hooykaas, P J

1988-01-01

89

Isolation of a strain of Agrobacterium tumefaciens (Rhizobium radiobacter) utilizing methylene urea (ureaformaldehyde) as nitrogen source.  

PubMed

Methylene ureas (MU) are slow-release nitrogen fertilizers degraded in soil by microbial enzymatic activity. Improved utilization of MU in agricultural production requires more knowledge about the organisms and enzymes responsible for its degradation. A Gram-negative, MU-degrading organism was isolated from a soil in Sacramento Valley, California. The bacterium was identified as Agrobacterium tumefaciens (recently also known as Rhizobium radiobacter) using both genotypic and phenotypic characterization. The pathogenic nature of the organism was confirmed by a bioassay on carrot disks. The MU-hydrolyzing enzyme (MUase) was intracellular and was induced by using MU as a sole source of nitrogen. The bacterial growth was optimized in NH4Cl, urea, or peptone, whereas the production and specific activity of MUase were maximized with either NH4Cl or urea as a nitrogen source. The result has a practical significance, demonstrating a potential to select for this plant pathogen in soils fertilized with MU. PMID:15105883

Koivunen, Marja E; Morisseau, Christophe; Horwath, William R; Hammock, Bruce D

2004-03-01

90

Genomic Species Are Ecological Species as Revealed by Comparative Genomics in Agrobacterium tumefaciens  

PubMed Central

The definition of bacterial species is based on genomic similarities, giving rise to the operational concept of genomic species, but the reasons of the occurrence of differentiated genomic species remain largely unknown. We used the Agrobacterium tumefaciens species complex and particularly the genomic species presently called genomovar G8, which includes the sequenced strain C58, to test the hypothesis of genomic species having specific ecological adaptations possibly involved in the speciation process. We analyzed the gene repertoire specific to G8 to identify potential adaptive genes. By hybridizing 25 strains of A. tumefaciens on DNA microarrays spanning the C58 genome, we highlighted the presence and absence of genes homologous to C58 in the taxon. We found 196 genes specific to genomovar G8 that were mostly clustered into seven genomic islands on the C58 genome—one on the circular chromosome and six on the linear chromosome—suggesting higher plasticity and a major adaptive role of the latter. Clusters encoded putative functional units, four of which had been verified experimentally. The combination of G8-specific functions defines a hypothetical species primary niche for G8 related to commensal interaction with a host plant. This supports that the G8 ancestor was able to exploit a new ecological niche, maybe initiating ecological isolation and thus speciation. Searching genomic data for synapomorphic traits is a powerful way to describe bacterial species. This procedure allowed us to find such phenotypic traits specific to genomovar G8 and thus propose a Latin binomial, Agrobacterium fabrum, for this bona fide genomic species. PMID:21795751

Lassalle, Florent; Campillo, Tony; Vial, Ludovic; Baude, Jessica; Costechareyre, Denis; Chapulliot, David; Shams, Malek; Abrouk, Danis; Lavire, Céline; Oger-Desfeux, Christine; Hommais, Florence; Guéguen, Laurent; Daubin, Vincent; Muller, Daniel; Nesme, Xavier

2011-01-01

91

Proline antagonizes GABA-induced quenching of quorum-sensing in Agrobacterium tumefaciens.  

PubMed

Plants accumulate free L-proline (Pro) in response to abiotic stresses (drought and salinity) and presence of bacterial pathogens, including the tumor-inducing bacterium Agrobacterium tumefaciens. However, the function of Pro accumulation in host-pathogen interaction is still unclear. Here, we demonstrated that Pro antagonizes plant GABA-defense in the A. tumefaciens C58-induced tumor by interfering with the import of GABA and consequently the GABA-induced degradation of the bacterial quorum-sensing signal, 3-oxo-octanoylhomoserine lactone. We identified a bacterial receptor Atu2422, which is implicated in the uptake of GABA and Pro, suggesting that Pro acts as a natural antagonist of GABA-signaling. The Atu2422 amino acid sequence contains a Venus flytrap domain that is required for trapping GABA in human GABA(B) receptors. A constructed atu2422 mutant was more virulent than the wild type bacterium; moreover, transgenic plants with a low level of Pro exhibited less severe tumor symptoms than did their wild-type parents, revealing a crucial role for Venus flytrap GABA-receptor and relative abundance of GABA and Pro in host-pathogen interaction. PMID:19706545

Haudecoeur, E; Planamente, S; Cirou, A; Tannières, M; Shelp, B J; Moréra, S; Faure, D

2009-08-25

92

Integrative gene transfer in the truffle Tuber borchii by Agrobacterium tumefaciens-mediated transformation.  

PubMed

Agrobacterium tumefaciens-mediated transformation is a powerful tool for reverse genetics and functional genomic analysis in a wide variety of plants and fungi. Tuber spp. are ecologically important and gastronomically prized fungi ("truffles") with a cryptic life cycle, a subterranean habitat and a symbiotic, but also facultative saprophytic lifestyle. The genome of a representative member of this group of fungi has recently been sequenced. However, because of their poor genetic tractability, including transformation, truffles have so far eluded in-depth functional genomic investigations. Here we report that A. tumefaciens can infect Tuber borchii mycelia, thereby conveying its transfer DNA with the production of stably integrated transformants. We constructed two new binary plasmids (pABr1 and pABr3) and tested them as improved transformation vectors using the green fluorescent protein as reporter gene and hygromycin phosphotransferase as selection marker. Transformants were stable for at least 12 months of in vitro culture propagation and, as revealed by TAIL- PCR analysis, integration sites appear to be heterogeneous, with a preference for repeat element-containing genome sites. PMID:24949275

Brenna, Andrea; Montanini, Barbara; Muggiano, Eleonora; Proietto, Marco; Filetici, Patrizia; Ottonello, Simone; Ballario, Paola

2014-01-01

93

Integrative gene transfer in the truffle Tuber borchii by Agrobacterium tumefaciens-mediated transformation  

PubMed Central

Agrobacterium tumefaciens-mediated transformation is a powerful tool for reverse genetics and functional genomic analysis in a wide variety of plants and fungi. Tuber spp. are ecologically important and gastronomically prized fungi (“truffles”) with a cryptic life cycle, a subterranean habitat and a symbiotic, but also facultative saprophytic lifestyle. The genome of a representative member of this group of fungi has recently been sequenced. However, because of their poor genetic tractability, including transformation, truffles have so far eluded in-depth functional genomic investigations. Here we report that A. tumefaciens can infect Tuber borchii mycelia, thereby conveying its transfer DNA with the production of stably integrated transformants. We constructed two new binary plasmids (pABr1 and pABr3) and tested them as improved transformation vectors using the green fluorescent protein as reporter gene and hygromycin phosphotransferase as selection marker. Transformants were stable for at least 12 months of in vitro culture propagation and, as revealed by TAIL- PCR analysis, integration sites appear to be heterogeneous, with a preference for repeat element-containing genome sites. PMID:24949275

2014-01-01

94

Translation start sequences affect the efficiency of silencing of Agrobacterium tumefaciens T-DNA oncogenes.  

PubMed

Agrobacterium tumefaciens oncogenes cause transformed plant cells to overproduce auxin and cytokinin. Two oncogenes encode enzymes that convert tryptophan to indole-3-acetic acid (auxin): iaaM (tryptophan mono-oxygenase) and iaaH (indole-3-acetamide hydrolase). A third oncogene (ipt) encodes AMP isopentenyl transferase, which produces cytokinin (isopentenyl-AMP). Inactivation of ipt and iaaM (or iaaH) abolishes tumorigenesis. Because adequate means do not exist to control crown gall, we created resistant plants by introducing transgenes designed to elicit posttranscriptional gene silencing (PTGS) of iaaM and ipt. Transgenes that elicit silencing trigger sequence-specific destruction of the inducing RNA and messenger RNAs with related sequences. Although PTGS has proven effective against a variety of target genes, we found that a much higher percentage of transgenic lines silenced iaaM than ipt, suggesting that transgene sequences influenced the effectiveness of PTGS. Sequences required for oncogene silencing included a translation start site. A transgene encoding a translatable sense-strand RNA from the 5' end of iaaM silenced the iaaM oncogene, but deletion of the translation start site abolished the ability of the transgene to silence iaaM. Silencing A. tumefaciens T-DNA oncogenes is a new and effective method to produce plants resistant to crown gall disease. PMID:12972655

Lee, Hyewon; Humann, Jodi L; Pitrak, Jennifer S; Cuperus, Josh T; Parks, T Dawn; Whistler, Cheryl A; Mok, Machteld C; Ream, L Walt

2003-11-01

95

Host range conferred by the virulence-specifying plasmid of Agrobacterium tumefaciens.  

PubMed Central

The host range of Agrobacterium tumefaciens 1D1109, known to induce crown gall only on grapevine (Vitis spp.), was extended to include many plant species by transferring a tumor-inducing plasmid (pTi) from strain 1D1, a broad-host-range pathogen. The pTi plasmid was mobilized by the conjugative plasmid pRK2, which was inserted into 1D1 by mating with Escherichia coli J53(pRK2). The resulting transconjugants were screened for their ability to induce crown gall tumors on hosts other than grapevine by inoculation into sunflower. Transconjugants that were virulent on sunflower were then tested on 36 different host plants and compared with host-limited strain 1D1109 and the donor strain. Two transconjugants induced tumors on the same 28 plant species as those of the original plasmid donor 1D1(pRK2) (pTi). These results show that pRK2 promoted transfer of the pTi plasmid and suggest that the pTi plasmid rather than the A. tumefaciens chromosome determined the host range of the pathogen. Insertion of pRK2 alone did not extend the host range of strain 1D1109. Insertion of pS-a into A. tumefaciens 1D1 by mating with E. coli J53-1 (pS-a) resulted in the concomitant loss of pTi and virulence. There appears to be incompatibility between pTi and pS-a. Images PMID:457613

Loper, J E; Kado, C I

1979-01-01

96

Viral and chloroplastic signals essential for initiation and efficiency of translation in Agrobacterium tumefaciens.  

PubMed

The construction of high-level protein expression vectors using the CaMV 35S promoter in concert with highly efficient translation initiation signals for Agrobacterium tumefaciens is a relatively less explored field compared to that of Escherichia coli. In the current study, we experimentally investigated the capacity of the CaMV 35S promoter to direct GFP gene expression in A. tumefaciens in the context of different viral and chloroplastic translation initiation signals. GFP expression and concomitant translational efficiency was monitored by confocal microscopy and Western blot analysis. Among all of the constructs, the highest level of translation was observed for the construct containing the phage T7 translation initiation region followed by the chloroplastic Rubisco Large Subunit (rbcL) 58-nucleotide 5' leader region including its SD-like sequence (GGGAGGG). Replacing the SD-like (GGGAGGG) with non SD-like (TTTATTT) or replacing the remaining 52 nucleotides of rbcL with nonspecific sequence completely abolished translation. In addition, this 58 nucleotide region of rbcL serves as a translational enhancer in plants when located within the 5' UTR of mRNA corresponding to GFP. Other constructs, including those containing sequences upstream of the coat proteins of Alfalfa Mosaic Virus, or the GAGG sequence of T4 phage or the chloroplastic atpI and/or PsbA 5' UTR sequence, supported low levels of GFP expression or none at all. From these studies, we propose that we have created high expression vectors in A. tumefaciens and/or plants which contain the CaMV 35S promoter, followed by the translationally strong T7 SD plus RBS translation initiation region or the rbcL 58-nucleotide 5' leader region upstream of the gene of interest. PMID:25117444

Ahmad, Tauqeer; Venkataraman, Srividhya; Hefferon, Kathleen; AbouHaidar, Mounir G

2014-09-12

97

Identification and Characterization of a Second Quorum-Sensing System in Agrobacterium tumefaciens A6  

PubMed Central

Quorum sensing (QS) is a widespread mechanism of bacterial communication in which individual cells produce and respond to small chemical signals. In Agrobacterium tumefaciens, an acylhomoserine lactone-dependent QS mechanism is known to regulate the replication and conjugation of the tumor-inducing (Ti) plasmid. Most of the QS regulatory proteins are encoded within the Ti plasmid. Among them, TraI is the LuxI-type enzyme synthesizing the QS signal N-3-oxooctanoyl-l-homoserine lactone (3OC8HSL), TraR is the LuxR-type transcriptional factor that recognizes 3OC8HSL, and TraM is an antiactivator that antagonizes TraR. Recently, we identified a TraM homolog encoded by the traM2 gene in the chromosomal background of A. tumefaciens A6. In this study, we further identified additional homologs (TraI2 and TraR2) of TraI and TraR in this strain. We showed that similar to TraI, TraI2 could predominantly synthesize the QS signal 3OC8HSL. We also showed that TraR2 could recognize 3OC8HSL and activate the tra box-containing promoters as efficiently as TraR. Further analysis showed that traM2, traI2, and traR2 are physically linked on a mobile genetic element that is not related to the Ti plasmid. These findings indicate that A. tumefaciens A6 carries a second QS system that may play a redundant role in the regulation of the replication and conjugation of the Ti plasmid. PMID:24464459

Wang, Chao; Yan, Chunlan; Fuqua, Clay

2014-01-01

98

Analysis of transfer of tumor-inducing plasmids from Agrobacterium tumefaciens to Petunia protoplasts.  

PubMed Central

Petunia protoplasts were infected with the virulent Agrobacterium tumefaciens strain A348 or the avirulent strain A136 (lacking a Ti plasmid). The infection process was stopped at various time intervals up to 24 h after inoculation, and the DNA from the plant cells was isolated. Southern blot analysis indicated that the DNA isolated from infected Petunia cells was not detectably contaminated by bacterial DNA from lysed Agrobacterium cells. Analysis of the DNA from the virulent infections suggested that the transferred DNA (T-DNA) may be transferred to the plant cell rapidly (within 2 to 6 h) after the bacteria bind to the cell wall and that the T-DNA may exist in a rearranged state which is stable over the time period investigated. Dot blot analysis indicated that regions far outside the T-DNA may be transferred to the plant cell. Most of the DNA transferred to the plant cell during the initial hours of infection is rapidly degraded. Images PMID:3997773

Virts, E L; Gelvin, S B

1985-01-01

99

Agrobacterium tumefaciens mutants affected in crown gall tumorigenesis and octopine catabolism.  

PubMed Central

Mutants of Agrobacterium tumefaciens which affect virulence or the ability to catabolize octopine were isolated after Tn5-induced mutagenesis. Of 8,900 colonies tested, 7 mutants with Tn5 insertions in a specific region of other Ti plasmid unable to catabolize octopine were isolated. Thirty-seven mutants affected in tumorigenesis resulted from insertions in the Ti plasmid and the Agrobacterium chromosome. Of these mutations, 12 were chromosomal and 25 mapped on the plasmid. Twenty-three mapped within a 20-megadalton region, which is distinct from the Ti plasmid sequences found stably integrated into the plant cell genome T-deoxyribonucleic acid). Included in these were mutants that were either a virulent or produced tumors with unusual morphologies. Three mutants contained insertions in the T-deoxyribonucleic acid. These three mutants incited tumors which synthesized octopine but had an altered morphology due to either extensive proliferation of shoots or roots from the tumor callus. Three additional mutants not caused by Tn5 contained mutations in the Ti plasmid. Images PMID:6253441

Garfinkel, D J; Nester, E W

1980-01-01

100

Methylation of the T-DNA in Agrobacterium tumefaciens and in several crown gall tumors.  

PubMed Central

Methylation of the T-DNA in Agrobacterium tumefaciens and in four octopine-type (A6S/2, E9, 15955/1, 15955/01) and one nopaline-type (HT37#15) crown gall tumors was investigated using the isoschizomeric restriction endonucleases Msp I and Hpa II. T-DNA in the octopine-type Ti-plasmid pTiB6(806) was not methylated at the sequence 5'CCGG3' in Agrobacterium. With two possible exceptions, neither was the T-DNA of the nopaline-type Ti-plasmid pTiT37 methylated in the bacterium. In all tumor lines investigated, at least one copy of the T-DNA was not methylated. DNA methylation was not detected in the lines A6S/2, 15955/1, HT37#15, and the TL region of E9. DNA methylation of some copies of TR in the E9 tumor line, and possibly in the 15955/01 line, was detected. The methylation of some copies of TR in the E9 line may indicate that not all copies of TR are transcribed in this tumor. Images PMID:6306562

Gelvin, S B; Karcher, S J; DiRita, V J

1983-01-01

101

Historical account on gaining insights on the mechanism of crown gall tumorigenesis induced by Agrobacterium tumefaciens  

PubMed Central

The plant tumor disease known as crown gall was not called by that name until more recent times. Galls on plants were described by Malpighi (1679) who believed that these extraordinary growth are spontaneously produced. Agrobacterium was first isolated from tumors in 1897 by Fridiano Cavara in Napoli, Italy. After this bacterium was recognized to be the cause of crown gall disease, questions were raised on the mechanism by which it caused tumors on a variety of plants. Numerous very detailed studies led to the identification of Agrobacterium tumefaciens as the causal bacterium that cleverly transferred a genetic principle to plant host cells and integrated it into their chromosomes. Such studies have led to a variety of sophisticated mechanisms used by this organism to aid in its survival against competing microorganisms. Knowledge gained from these fundamental discoveries has opened many avenues for researchers to examine their primary organisms of study for similar mechanisms of pathogenesis in both plants and animals. These discoveries also advanced the genetic engineering of domesticated plants for improved food and fiber. PMID:25147542

Kado, Clarence I.

2014-01-01

102

Incidence of Agrobacterium tumefaciens biovar 1 in and on ‘Paradox’ (Juglans hindsii x Juglans regia) walnut seed collected from commercial nurseries  

Technology Transfer Automated Retrieval System (TEKTRAN)

The walnut rootstock Paradox (Juglans hindsii (Jeps) Rehder x J. regia L.) is susceptible to Agrobacterium tumefaciens (7) which often results in a high incidence of crown gall in nursery or walnut production orchards. Though A. tumefaciens is susceptible to the commonly used preplant soil fumigant...

103

Interaction of the Agrobacterium tumefaciens virulence protein VirD2 with histones.  

PubMed

Agrobacterium tumefaciens is a Gram-negative soil bacterium that genetically transforms plants and, under laboratory conditions, also transforms non-plant organisms, such as fungi and yeasts. During the transformation process a piece of ssDNA (T-strand) is transferred into the host cells via a type IV secretion system. The VirD2 relaxase protein, which is covalently attached at the 5' end of the T-strand through Tyr29, mediates nuclear entry as it contains a nuclear localization sequence. How the T-strand reaches the chromatin and becomes integrated in the chromosomal DNA is still far from clear. Here, we investigated whether VirD2 binds to histone proteins in the yeast Saccharomyces cerevisiae. Using immobilized GFP-VirD2 and in vitro synthesized His6-tagged S. cerevisiae proteins, interactions between VirD2 and the histones H2A, H2B, H3 and H4 were revealed. In vivo, these interactions were confirmed by bimolecular fluorescence complementation experiments. After co-cultivation of Agrobacterium strains expressing VirD2 tagged with a fragment of the yellow fluorescent protein analogue Venus with yeast strains expressing histone H2A or H2B tagged with the complementary part of Venus, fluorescence was detected in dot-shaped structures in the recipient yeast cells. The results indicated that VirD2 was transferred from Agrobacterium to yeast cells and that it interacted with histones in the host cell, and thus may help direct the T-DNA (transferred DNA) to the chromatin as a prelude to integration into the host chromosomal DNA. PMID:25505187

Wolterink-van Loo, Suzanne; Ayala, Abril A Escamilla; Hooykaas, Paul J J; van Heusden, G Paul H

2015-02-01

104

Agrobacterium tumefaciens -mediated transformation of embryogenic tissue and transgenic plant regeneration in Chamaecyparis obtusa Sieb. et Zucc  

Microsoft Academic Search

A genetic transformation procedure for Chamaecyparis obtusa was developed after co-cultivation of embryogenic tissues with disarmed Agrobacterium tumefaciens strain C58\\/pMP90, which harbours the sgfp (synthetic green fluorescent protein) visual reporter and nptII (neomycin phoshotransferase II) selectable marker genes. The highest transformation frequency was 22.5 independent transformed lines per dish (250 mg embryogenic tissue) following selection on kanamycin medium. Transgenic plantlets were regenerated

T. Taniguchi; M. Kurita; Y. Ohmiya; T. Kondo

2005-01-01

105

Regeneration of transgenic Picea glauca, P. Mariana , and P. abies after cocultivation of embryogenic tissue with Agrobacterium tumefaciens  

Microsoft Academic Search

Summary  Transgenic plants of three Picea species were produced after coculture of embryogenic tissue with the disarmed strain of Agrobacterium tumefaciens C58\\/pMP90\\/pBIV10 and selection on medium containing kanamycin. In addition to the nptII selectable gene (conferring resistance to kanamycin), the vector carried the uidA (?-glucuronidase) marker gene. Transformation frequencies were dependent on the species, genotype, and post-cocultivation\\u000a procedure. Of the three

Krystyna Klimaszewska; Denis Lachance; Gervais Pelletier; Marie-Anne Lelu; Armand Séguin

2001-01-01

106

Conversion of BAC clones into binary BAC (BIBAC) vectors and their delivery into basidiomycete fungal cells using Agrobacterium tumefaciens.  

PubMed

The genetic transformation of certain organisms, required for gene function analysis or complementation, is often not very efficient, especially when dealing with large gene constructs or genomic fragments. We have adapted the natural DNA transfer mechanism from the soil pathogenic bacterium Agrobacterium tumefaciens, to deliver intact large DNA constructs to basidiomycete fungi of the genus Ustilago where they stably integrated into their genome. To this end, Bacterial Artificial Chromosome (BAC) clones containing large fungal genomic DNA fragments were converted via a Lambda phage-based recombineering step to Agrobacterium transfer-competent binary vectors (BIBACs) with a Ustilago-specific selection marker. The fungal genomic DNA fragment was subsequently successfully delivered as T-DNA through Agrobacterium-mediated transformation into Ustilago species where an intact copy stably integrated into the genome. By modifying the recombineering vector, this method can theoretically be adapted for many different fungi. PMID:25239747

Ali, Shawkat; Bakkeren, Guus

2015-01-01

107

High-efficiency Agrobacterium-mediated transformation of chickpea (Cicer arietinum L.) and regeneration of insect-resistant transgenic plants.  

PubMed

To develop an efficient genetic transformation system of chickpea (Cicer arietinum L.), callus derived from mature embryonic axes of variety P-362 was transformed with Agrobacterium tumefaciens strain LBA4404 harboring p35SGUS-INT plasmid containing the uidA gene encoding ?-glucuronidase (GUS) and the nptII gene for kanamycin selection. Various factors affecting transformation efficiency were optimized; as Agrobacterium suspension at OD(600) 0.3 with 48 h of co-cultivation period at 20°C was found optimal for transforming 10-day-old MEA-derived callus. Inclusion of 200 ?M acetosyringone, sonication for 4 s with vacuum infiltration for 6 min improved the number of GUS foci per responding explant from 1.0 to 38.6, as determined by histochemical GUS assay. For introducing the insect-resistant trait into chickpea, binary vector pRD400-cry1Ac was also transformed under optimized conditions and 18 T(0) transgenic plants were generated, representing 3.6% transformation frequency. T(0) transgenic plants reflected Mendelian inheritance pattern of transgene segregation in T(1) progeny. PCR, RT-PCR, and Southern hybridization analysis of T(0) and T(1) transgenic plants confirmed stable integration of transgenes into the chickpea genome. The expression level of Bt-Cry protein in T(0) and T(1) transgenic chickpea plants was achieved maximum up to 116 ng mg(-1) of soluble protein, which efficiently causes 100% mortality to second instar larvae of Helicoverpa armigera as analyzed by an insect mortality bioassay. Our results demonstrate an efficient and rapid transformation system of chickpea for producing non-chimeric transgenic plants with high frequency. These findings will certainly accelerate the development of chickpea plants with novel traits. PMID:21516347

Mehrotra, Meenakshi; Sanyal, Indraneel; Amla, D V

2011-09-01

108

Replicon-Specific Regulation of Small Heat Shock Genes in Agrobacterium tumefaciens  

PubMed Central

Four genes coding for small heat shock proteins (sHsps) were identified in the genome sequence of Agrobacterium tumefaciens, one on the circular chromosome (hspC), one on the linear chromosome (hspL), and two on the pAT plasmid (hspAT1 and hspAT2). Induction of sHsps at elevated temperatures was revealed by immunoblot analyses. Primer extension experiments and translational lacZ fusions demonstrated that expression of the pAT-derived genes and hspL is controlled by temperature in a regulon-specific manner. While the sHsp gene on the linear chromosome turned out to be regulated by RpoH (?32), both copies on pAT were under the control of highly conserved ROSE (named for repression of heat shock gene expression) sequences in their 5? untranslated region. Secondary structure predictions of the corresponding mRNA strongly suggest that it represses translation at low temperatures by masking the Shine-Dalgarno sequence. The hspC gene was barely expressed (if at all) and not temperature responsive. PMID:15466035

Balsiger, Sylvia; Ragaz, Curdin; Baron, Christian; Narberhaus, Franz

2004-01-01

109

Agrobacterium tumefaciens mediated transient expression of plant cell wall-degrading enzymes in detached sunflower leaves.  

PubMed

For biofuel applications, synthetic endoglucanase E1 and xylanase (Xyn10A) derived from Acidothermus cellulolyticus were transiently expressed in detached whole sunflower (Helianthus annuus L.) leaves using vacuum infiltration. Three different expression systems were tested, including the constitutive CaMV 35S-driven, CMVar (Cucumber mosaic virus advanced replicating), and TRBO (Tobacco mosaic virus RNA-Based Overexpression Vector) systems. For 6-day leaf incubations, codon-optimized E1 and xylanase driven by the CaMV 35S promoter were successfully expressed in sunflower leaves. The two viral expression vectors, CMVar and TRBO, were not successful although we found high expression in Nicotiana benthamiana leaves previously for other recombinant proteins. To further enhance transient expression, we demonstrated two novel methods: using the plant hormone methyl jasmonic acid in the agroinfiltration buffer and two-phase optimization of the leaf incubation temperature. When methyl jasmonic acid was added to Agrobacterium tumefaciens cell suspensions and infiltrated into plant leaves, the functional enzyme production increased 4.6-fold. Production also increased up to 4.2-fold when the leaf incubation temperature was elevated above the typical temperature, 20C, to 30C in the late incubation phase, presumably due to enhanced rate of protein synthesis in plant cells. Finally, we demonstrated co-expression of E1 and xylanase in detached sunflower leaves. To our knowledge, this is the first report of (co)expression of heterologous plant cell wall-degrading enzymes in sunflower. PMID:25180328

Jung, Sang-Kyu; Lindenmuth, Benjamin E; McDonald, Karen A; Hwang, Hwang; Bui, Mai Q Nguyen; Falk, Bryce W; Uratsu, Sandra L; Phu, My L; Dandekar, Abhaya M

2014-01-01

110

The Agrobacterium tumefaciens Transcription Factor BlcR Is Regulated via Oligomerization  

SciTech Connect

The Agrobacterium tumefaciens BlcR is a member of the emerging isocitrate lyase transcription regulators that negatively regulates metabolism of {gamma}-butyrolactone, and its repressing function is relieved by succinate semialdehyde (SSA). Our crystal structure showed that BlcR folded into the DNA- and SSA-binding domains and dimerized via the DNA-binding domains. Mutational analysis identified residues, including Phe{sup 147}, that are important for SSA association; BlcR{sup F147A} existed as tetramer. Two BlcR dimers bound to target DNA and in a cooperative manner, and the distance between the two BlcR-binding sequences in DNA was critical for BlcR-DNA association. Tetrameric BlcR{sup F147A} retained DNA binding activity, and importantly, this activity was not affected by the distance separating the BlcR-binding sequences in DNA. SSA did not dissociate tetrameric BlcR{sup F147A} or BlcR{sup F147A}-DNA. As well as in the SSA-binding site, Phe{sup 147} is located in a structurally flexible loop that may be involved in BlcR oligomerization. We propose that SSA regulates BlcR DNA-binding function via oligomerization.

Pan, Yi; Fiscus, Valena; Meng, Wuyi; Zheng, Zhida; Zhang, Lian-Hui; Fuqua, Clay; Chen, Lingling (IMCB-Singapore); (Indiana)

2012-02-08

111

Nucleotide sequence and analysis of the plant-inducible locus pinF from Agrobacterium tumefaciens.  

PubMed Central

Several loci on the tumor-inducing plasmid from Agrobacterium tumefaciens were transcriptionally activated in the presence of wounded plant tissue or extracts. The inducible virulence loci were required for efficient tumor formation. In contrast, the plant-inducible locus pinF was not observed to be absolutely essential for virulence. Mutants in pinF showed an attenuated virulence on a variety of dicotyledonous hosts, and this attenuation became more pronounced with decreasing numbers of bacterial cells in the inoculum. The DNA sequence of a 5.5-kilobase region which included the pinF locus from the octopine-type tumor-inducing plasmid A6 was determined. Four open reading frames consistent with the observed transcription of pinF were observed. Two of the open reading frames, pinF1 and pinF2, coded for polypeptides with relative molecular weights of 47,519 (pinF1) and 46,740 (pinF2). A comparison of the amino acid sequences of pinF1 and pinF2 indicated that they were similar to each other and to known polypeptide sequences for cytochrome P-450 enzymes. PMID:2708311

Kanemoto, R H; Powell, A T; Akiyoshi, D E; Regier, D A; Kerstetter, R A; Nester, E W; Hawes, M C; Gordon, M P

1989-01-01

112

Fate of arsenate following arsenite oxidation in Agrobacterium tumefaciens?GW4.  

PubMed

The fate of arsenate (As(V) ) generated by microbial arsenite (As(III) ) oxidation is poorly understood. Agrobacterium tumefaciens wild-type strain (GW4) was studied to determine how the cell copes with As(V) generated in batch culture. GW4 grown heterotrophically with mannitol used As(III) as a supplemental energy supply as reflected by enhanced growth and increased cellular levels of NADH and ATP. Under low phosphate (Pi) conditions and presence of As(III) oxidation, up to ??50% of the resulting As(V) was taken up and found associated with the periplasm, membrane or cytoplasm fractions of the cells. Arsenic was found associated with proteins and polar lipids, but not in nucleic acids or sugars. Thin-layer chromatography and gas chromatography-mass spectrometry analysis suggested the presence of arsenolipids in membranes, presumably as part of the bilayer structure of the cell membrane and replacing Pi under Pi-limiting conditions. The potential role of a Pi-binding protein (PstS) for As(V) uptake was assessed with the His-tag purified protein. Intrinsic tryptophan fluorescence spectra analysis suggests that PstS can bind As(V) , but with lower affinity as compared with Pi. In early stationary phase cells, the As(V) ?:?Pi ratio was approximately 4.3 and accompanied by an altered cell ultrastructure. PMID:24673976

Wang, Qian; Qin, Dong; Zhang, Shengzhe; Wang, Lu; Li, Jingxin; Rensing, Christopher; McDermott, Timothy R; Wang, Gejiao

2014-03-27

113

The Agrobacterium tumefaciens Transcription Factor BlcR Is Regulated via Oligomerization*  

PubMed Central

The Agrobacterium tumefaciens BlcR is a member of the emerging isocitrate lyase transcription regulators that negatively regulates metabolism of ?-butyrolactone, and its repressing function is relieved by succinate semialdehyde (SSA). Our crystal structure showed that BlcR folded into the DNA- and SSA-binding domains and dimerized via the DNA-binding domains. Mutational analysis identified residues, including Phe147, that are important for SSA association; BlcRF147A existed as tetramer. Two BlcR dimers bound to target DNA and in a cooperative manner, and the distance between the two BlcR-binding sequences in DNA was critical for BlcR-DNA association. Tetrameric BlcRF147A retained DNA binding activity, and importantly, this activity was not affected by the distance separating the BlcR-binding sequences in DNA. SSA did not dissociate tetrameric BlcRF147A or BlcRF147A-DNA. As well as in the SSA-binding site, Phe147 is located in a structurally flexible loop that may be involved in BlcR oligomerization. We propose that SSA regulates BlcR DNA-binding function via oligomerization. PMID:21467043

Pan, Yi; Fiscus, Valena; Meng, Wuyi; Zheng, Zhida; Zhang, Lian-Hui; Fuqua, Clay; Chen, Lingling

2011-01-01

114

Transformation of Montmorency sour cherry ( Prunus cerasus L.) and Gisela 6 ( P. cerasus × P. canescens ) cherry rootstock mediated by Agrobacterium tumefaciens  

Microsoft Academic Search

Sour cherry (Prunus cerasus L.) scion cv. Montmorency and rootstock cv. Gisela 6 (P. cerasus × P. canescens) were transformed using Agrobacterium tumefaciens strain EHA105:pBISN1 carrying the neomycin phosphotransferase gene (nptII) and an intron interrupted ß-glucuronidase (GUS) reporter gene (gusA). Whole leaf explants were co-cultivated with A. tumefaciens, and selection and regeneration of transformed cells and shoots of both cultivars

Guo-Qing Song; K. C. Sink

2006-01-01

115

Agrobacterium tumefaciens-Mediated Transformation for Investigation of Somatic Recombination in the Fungal Pathogen Armillaria mellea?  

PubMed Central

Armillaria root disease is one of the most damaging timber and fruit tree diseases in the world. Despite its economic importance, many basic questions about the biology of the causal fungi, Armillaria spp., are unanswered. For example, Armillaria undergoes matings between diploid and haploid mycelia, which can result in a recombinant diploid without meiosis. Evidence of such somatic recombination in natural populations suggests that this reproductive mode may affect the pathogen's ecology. Investigations of the mechanisms and adaptive consequences of somatic recombination are, however, hampered by the lack of a method to reliably synthesize somatic recombinants. Here we report the first genetic transformation system for the genus Armillaria. We transformed A. mellea with selective markers for use in diploid-haploid matings to reliably synthesize somatic recombinants. This was accomplished with Agrobacterium tumefaciens carrying pBGgHg, which carries the hygromycin phosphotransferase gene (hph). hph was integrated into transformants, as evidenced by serial transfer to selective media, PCR, reverse transcription-PCR (RT-PCR), and Southern hybridization. Nuclear and mitochondrial markers were developed to genotype synthesized mycelia. In matings between a wild-type diploid and hygromycin-resistant haploids (transgenic), we identified recombinant, hygromycin-resistant diploids and, additionally, hygromycin-resistant triploids, all with the mitochondrial haplotype of the haploid partner. Our approach created no mycelium in which the haploid nucleus was replaced by the diploid nucleus, the typical outcome of diploid-haploid matings in Armillaria. This genetic transformation system, in combination with new markers to track chromosomal and cytoplasmic inheritance in A. mellea, will advance research aimed at characterizing the significance of somatic recombination in the ecology of this important fungus. PMID:20952653

Baumgartner, Kendra; Fujiyoshi, Phillip; Foster, Gary D.; Bailey, Andy M.

2010-01-01

116

Genetic transformation of Indian isolate of Lemna minor mediated by Agrobacterium tumefaciens and recovery of transgenic plants  

Microsoft Academic Search

Transgenic plants of an Indian isolate of Lemna minor have been developed for the first time using Agrobacterium tumefaciens and hard nodular cell masses ‘nodular calli’ developed on the BAP - pretreated daughter frond explants in B5 medium containing sucrose (1.0 %) with 2,4-D (5.0 ?M) and 2-iP (50.0 ?M) or 2,4-D (50.0 ?M) and TDZ (5.0 ?M) under light\\u000a conditions. These calli were co-cultured

Gulshan Chhabra; Darshna Chaudhary; Manish Sainger; Pawan K. Jaiwal

2011-01-01

117

A stable and efficient Agrobacterium tumefaciens-mediated genetic transformation of the medicinal plant Digitalis purpurea L.  

PubMed

In this study, we developed a rapid and efficient method for in vitro propagation and Agrobacterium tumefaciens-mediated transformation of Digitalis purpurea L. (syn. foxglove), an important medicinal plant. Mature leaf explants of D. purpurea were used for 100 % adventitious shoot regeneration on Murashige and Skoog (MS) medium supplemented with 1 mg L(-1) thidiazuron (TDZ) (a cytokine) and 0.1 mg L(-1) 1-naphthaleneacetic acid (NAA) (an auxin). Transformation was achieved by inoculating leaf explants with the A. tumefaciens strains GV2260/pBI121 or GV3101/pBI121. The binary vector pBI121 contained the reporter ?-glucuronidase gene (GUS) and kanamycin selection marker nptII. Kanamycin-resistant shoots were regenerated directly on the selection medium 4-6 weeks after co-cultivation. Approximately, 52.2 and 60 % of kanamycin-resistant shoots transformed with Agrobacterium strains GV2260 and GV3101, respectively, showed strong GUS staining by histochemical assay. Furthermore, PCR and Southern blot analysis confirmed the presence of nptII and GUS on the chromosome of the transformed D. purpurea plants, and stable GUS expression was detected in the transformants by RT-PCR analysis. This efficient method of shoot regeneration and genetic transformation of D. purpurea will provide a powerful tool to increase and produce valuable components such as digitoxin, digoxin, and digoxigenin in D. purpurea through improved secondary metabolic pathways via a biotechnological approach. PMID:24272685

Li, Ying; Gao, Zhenrui; Piao, Chunlan; Lu, Kaiwen; Wang, Zhiping; Cui, Min-Long

2014-02-01

118

Agrobacterium tumefaciens Deploys a Superfamily of Type VI Secretion DNase Effectors as Weapons for Interbacterial Competition In Planta  

PubMed Central

Summary The type VI secretion system (T6SS) is a widespread molecular weapon deployed by many Proteobacteria to target effectors/toxins into both eukaryotic and prokaryotic cells. We report that Agrobacterium tumefaciens, a soil bacterium that triggers tumorigenesis in plants, produces a family of type VI DNase effectors (Tde) that are distinct from previously known polymorphic toxins and nucleases. Tde exhibits an antibacterial DNase activity that relies on a conserved HxxD motif and can be counteracted by a cognate immunity protein, Tdi. In vitro, A. tumefaciens T6SS could kill Escherichia coli but triggered a lethal counterattack by Pseudomonas aeruginosa upon injection of the Tde toxins. However, in an in planta coinfection assay, A. tumefaciens used Tde effectors to attack both siblings cells and P. aeruginosa to ultimately gain a competitive advantage. Such acquired T6SS-dependent fitness in vivo and conservation of Tde-Tdi couples in bacteria highlights a widespread antibacterial weapon beneficial for niche colonization. PMID:24981331

Ma, Lay-Sun; Hachani, Abderrahman; Lin, Jer-Sheng; Filloux, Alain; Lai, Erh-Min

2014-01-01

119

Genetic Analysis of Agrobacterium tumefaciens Unipolar Polysaccharide Production Reveals Complex Integrated Control of the Motile-to-Sessile Switch  

PubMed Central

Summary Many bacteria colonize surfaces and transition to a sessile mode of growth. The plant pathogen Agrobacterium tumefaciens produces a unipolar polysaccharide (UPP) adhesin at single cell poles that contact surfaces. Here we report that elevated levels of the intracellular signal cyclic diguanosine monophosphate (c-di-GMP) lead to surface-contact-independent UPP production and a red colony phenotype due to production of UPP and the exopolysaccharide cellulose, when A. tumefaciens is incubated with the polysaccharide stain Congo Red. Transposon mutations with elevated Congo Red staining identified presumptive UPP negative regulators, mutants for which were hyperadherent, producing UPP irrespective of surface contact. Multiple independent mutations were obtained in visN and visR, activators of flagellar motility in A. tumefaciens, now found to inhibit UPP and cellulose production. Expression analysis in a visR mutant and isolation of suppressor mutations, identified three diguanylate cyclases inhibited by VisR. Null mutations for two of these genes decrease attachment and UPP production, but do not alter cellular c-di-GMP levels. However, analysis of catalytic site mutants revealed their GGDEF motifs are required to increase UPP production and surface attachment. Mutations in a specific presumptive cyclic diguanosine monophosphate phosphodiesterase also elevate UPP production and attachment, consistent with c-di-GMP activation of surface-dependent adhesin deployment. PMID:23829710

Xu, Jing; Kim, Jinwoo; Koestler, Benjamin J.; Choi, Jeong-Hyeon; Waters, Christopher M.; Fuqua, Clay

2013-01-01

120

CelR, an Ortholog of the Diguanylate Cyclase PleD of Caulobacter, Regulates Cellulose Synthesis in Agrobacterium tumefaciens  

PubMed Central

Cellulose fibrils play a role in attachment of Agrobacterium tumefaciens to its plant host. While the genes for cellulose biosynthesis in the bacterium have been identified, little is known concerning the regulation of the process. The signal molecule cyclic di-GMP (c-di-GMP) has been linked to the regulation of exopolysaccharide biosynthesis in many bacterial species, including A. tumefaciens. In this study, we identified two putative diguanylate cyclase genes, celR (atu1297) and atu1060, that influence production of cellulose in A. tumefaciens. Overexpression of either gene resulted in increased cellulose production, while deletion of celR, but not atu1060, resulted in decreased cellulose biosynthesis. celR overexpression also affected other phenotypes, including biofilm formation, formation of a polar adhesion structure, plant surface attachment, and virulence, suggesting that the gene plays a role in regulating these processes. Analysis of celR and ?cel mutants allowed differentiation between phenotypes associated with cellulose production, such as biofilm formation, and phenotypes probably resulting from c-di-GMP signaling, which include polar adhesion, attachment to plant tissue, and virulence. Phylogenetic comparisons suggest that species containing both celR and celA, which encodes the catalytic subunit of cellulose synthase, adapted the CelR protein to regulate cellulose production while those that lack celA use CelR, called PleD, to regulate specific processes associated with polar localization and cell division. PMID:24038703

Barnhart, D. Michael; Su, Shengchang; Baccaro, Brenna E.; Banta, Lois M.

2013-01-01

121

Response surface studies that elucidate the role of infiltration conditions on Agrobacterium tumefaciens-mediated transient transgene expression in harvested switchgrass ( Panicum virgatum)  

Microsoft Academic Search

Agrobacterium tumefaciens-mediated transient expression (agroinfiltration) experiments were performed in harvested switchgrass (Panicum virgatum) leaves to identify the effects of wounding by bead beating, surfactant concentration and vacuum application on in planta ?-glucuronidase expression and leaf decay. Expression was scored based on a consistent pattern of visual observations of histochemical staining over the leaf surface as might be observed in stable

J. S. VanderGheynst; H.-Y. Guo; C. W. Simmons

2008-01-01

122

Secretome Analysis Uncovers an Hcp-Family Protein Secreted via a Type VI Secretion System in Agrobacterium tumefaciens? †  

PubMed Central

Agrobacterium tumefaciens is a plant-pathogenic bacterium capable of secreting several virulence factors into extracellular space or the host cell. In this study, we used shotgun proteomics analysis to investigate the secretome of A. tumefaciens, which resulted in identification of 12 proteins, including 1 known secretory protein (VirB1*) and 11 potential secretory proteins. Interestingly, one unknown protein, which we designated hemolysin-coregulated protein (Hcp), is a predicted soluble protein without a recognizable N-terminal signal peptide. Western blot analysis revealed that A. tumefaciens Hcp is expressed and secreted when cells are grown in both minimal and rich media. Further biochemical and immunoelectron microscopy analysis demonstrated that intracellular Hcp is localized mainly in the cytosol, with a small portion in the membrane system. To investigate the mechanism of secretion of Hcp in A. tumefaciens, we generated mutants with deletions of a conserved gene, icmF, or the entire putative operon encoding a recently identified type VI secretion system (T6SS). Western blot analysis indicated that Hcp was expressed but not secreted into the culture medium in mutants with deletions of icmF or the t6ss operon. The secretion deficiency of Hcp in the icmF mutant was complemented by heterologous trans expression of icmF, suggesting that icmF is required for Hcp secretion. In tumor assays with potato tuber disks, deletion of hcp resulted in approximately 20 to 30% reductions in tumorigenesis efficiency, while no consistent difference was observed when icmF or the t6ss operon was deleted. These results increase our understanding of the conserved T6SS used by both plant- and animal-pathogenic bacteria. PMID:18263727

Wu, Hung-Yi; Chung, Pei-Che; Shih, Hsiao-Wei; Wen, Sy-Ray; Lai, Erh-Min

2008-01-01

123

An improved procedure for production of white spruce (Picea glauca) transgenic plants using Agrobacterium tumefaciens  

Microsoft Academic Search

An efficient and reproducible procedure for the transformation of white spruce (Picea glauca (Moench) Voss) embryogenic tissues was developed using A. tumefaciens-mediated gene transfer. Rapidly dividing white spruce embryogenic tissues were co-cultivated with disarmed A. tumefaciens strains containing additional copies of the virulence regions from plasmid PToK47. The plasmid pBi121, con- taining the neomycin phosphotransferase II (nptII) gene providing kanamycin

Julie Belles-Isles; Mathieu Dusabenyagasani; Francine M. Tremblay

2001-01-01

124

Agrobacterium tumefaciens-mediated transformation of embryogenic tissue and transgenic plant regeneration in Chamaecyparis obtusa Sieb. et Zucc.  

PubMed

A genetic transformation procedure for Chamaecyparis obtusa was developed after co-cultivation of embryogenic tissues with disarmed Agrobacterium tumefaciens strain C58/pMP90, which harbours the sgfp (synthetic green fluorescent protein) visual reporter and nptII (neomycin phoshotransferase II) selectable marker genes. The highest transformation frequency was 22.5 independent transformed lines per dish (250 mg embryogenic tissue) following selection on kanamycin medium. Transgenic plantlets were regenerated through the maturation and germination of somatic embryos. The intensity of GFP fluorescence, observed under a fluorescence microscope, varied from very faint to relatively strong, depending on the transgenic line or part of the transgenic plant. The integration of the genes into the genome of regenerated plantlets was confirmed by Southern blot analysis. PMID:15761663

Taniguchi, T; Kurita, M; Ohmiya, Y; Kondo, T

2005-03-01

125

Development of Protoporphyrinogen Oxidase as an Efficient Selection Marker for Agrobacterium tumefaciens-Mediated Transformation of Maize  

PubMed Central

In this article, we report the isolation of plant protoporphyrinogen oxidase (PPO) genes and the isolation of herbicide-tolerant mutants. Subsequently, an Arabidopsis double mutant (Y426M + S305L) was used to develop a selectable marker system for Agrobacterium tumefaciens-mediated transformation of maize (Zea mays) and to obtain multiple events tolerant to the PPO family of herbicides. Maize transformants were produced via butafenacil selection using a flexible light regime to increase selection pressure. Butafenacil selection per se did not change transgene copy number distribution relative to other selectable marker systems, but the most tolerant events identified in the greenhouse were more likely to contain multiple copies of the introduced mutant PPO gene. To date, more than 2,500 independent transgenic maize events have been produced using butafenacil selection. The high frequency of A. tumefaciens-mediated transformation via PPO selection enabled us to obtain single-copy transgenic maize lines tolerant to field levels of butafenacil. PMID:12972658

Li, Xianggan; Volrath, Sandy L.; Nicholl, David B.G.; Chilcott, Charles E.; Johnson, Marie A.; Ward, Eric R.; Law, Marcus D.

2003-01-01

126

Genetic transformation of Indian isolate of Lemna minor mediated by Agrobacterium tumefaciens and recovery of transgenic plants.  

PubMed

Transgenic plants of an Indian isolate of Lemna minor have been developed for the first time using Agrobacterium tumefaciens and hard nodular cell masses 'nodular calli' developed on the BAP - pretreated daughter frond explants in B5 medium containing sucrose (1.0 %) with 2,4-D (5.0 ?M) and 2-iP (50.0 ?M) or 2,4-D (50.0 ?M) and TDZ (5.0 ?M) under light conditions. These calli were co-cultured with A. tumefaciens strain EHA105 harboring a binary vector that contained genes for ?-glucuronidase with intron and neomycin phosphortransferase. Transformed cells selected on kanamycin selection medium were regenerated into fronds whose transgenic nature was confirmed by histochemical assay for GUS activity, PCR analysis and Southern hybridization. The frequency of transformation obtained was 3.8 % and a period of 11-13 weeks was required from initiation of cultures from explants to fully grown transgenic fronds. The pretreatment of daughter fronds with BAP, use of non-ionic surfactant, presence of acetosyringone in co-cultivation medium, co-culture duration of 3 d and 16 h photoperiod during culture were found crucial for callus induction, frond regeneration and transformation of L. minor. This transformation system can be used for the production of pharmaceutically important protein and in bioremediation. PMID:23573002

Chhabra, Gulshan; Chaudhary, Darshna; Sainger, Manish; Jaiwal, Pawan K

2011-04-01

127

Presence of unintended Agrobacterium tumefaciens cloning vector sequences in genetically modified plants.  

PubMed

Agrobacterium transformation was used in the production of genetically modified plants from oilseed rape (Brassica napus) and tobacco (Nicotiana tabacum). After inoculation stop with the antibiotic timentin, a subsequent one-week treatment eliminated the vector bacterium from the oilseed rape plate explant cultures. From the tobacco, however, we recorded vector-derived signals one week after potting the regenerants in the greenhouse and still 10 weeks later. Genetically modified plants produced through Agrobacterium-transformation therefore cannot be guaranteed to be completely free of unintended vector sequences after antibiotic treatment. PMID:16614922

Björklöf, Katarina; Färdig, Michael; Jørgensen, Kirsten S

2006-03-01

128

Agrobacterium tumefaciens-Mediated Transformation of Arabidopsis thaliana Root Explants by Using Kanamycin Selection  

Microsoft Academic Search

Culture conditions were developed that induce Arabidopsis thaliana (L.) Heynh. root cuttings to regenerate shoots rapidly and at 100% efficiency. The shoots produce viable seeds in vitro or after rooting in soil. A transformation procedure for Arabidopsis root explants based on kanamycin selection was established. By using this regeneration procedure and an Agrobacterium tumor-inducing Ti plasmid carrying a chimeric neomycin

Dirk Valvekens; Marc van Montagu; Mieke van Lijsebettens

1988-01-01

129

6-Hydroxy-3-Succinoylpyridine Hydroxylase Catalyzes a Central Step of Nicotine Degradation in Agrobacterium tumefaciens S33  

PubMed Central

Nicotine is a main alkaloid in tobacco and is also the primary toxic compound in tobacco wastes. It can be degraded by bacteria via either pyridine pathway or pyrrolidine pathway. Previously, a fused pathway of the pyridine pathway and the pyrrolidine pathway was proposed for nicotine degradation by Agrobacterium tumefaciens S33, in which 6-hydroxy-3-succinoylpyridine (HSP) is a key intermediate connecting the two pathways. We report here the purification and properties of an NADH-dependent HSP hydroxylase from A. tumefaciens S33. The 90-kDa homodimeric flavoprotein catalyzed the oxidative decarboxylation of HSP to 2,5-dihydroxypyridine (2,5-DHP) in the presence of NADH and FAD at pH 8.0 at a specific rate of about 18.8±1.85 µmol min?1 mg protein?1. Its gene was identified by searching the N-terminal amino acid residues of the purified protein against the genome draft of the bacterium. It encodes a protein composed of 391 amino acids with 62% identity to HSP hydroxylase (HspB) from Pseudomonas putida S16, which degrades nicotine via the pyrrolidine pathway. Considering the application potential of 2,5-DHP in agriculture and medicine, we developed a route to transform HSP into 2,5-DHP with recombinant HSP hydroxylase and an NADH-regenerating system (formate, NAD+ and formate dehydrogenase), via which around 0.53±0.03 mM 2,5-DHP was produced from 0.76±0.01 mM HSP with a molar conversion as 69.7%. This study presents the biochemical properties of the key enzyme HSP hydroxylase which is involved in the fused nicotine degradation pathway of the pyridine and pyrrolidine pathways and a new green route to biochemically synthesize functionalized 2,5-DHP. PMID:25054198

Huang, Haiyan; Wang, Shuning

2014-01-01

130

Agrobacterium tumefaciens-mediated transformation of Cleome gynandra L., a C4 dicotyledon that is closely related to Arabidopsis thaliana  

PubMed Central

In leaves of most C4 plants, the biochemistry of photosynthesis is partitioned between mesophyll and bundle sheath cells. In addition, their cell biology and development also differs from that in C3 plants. We have a poor understanding of the mechanisms that generate the cell-specific accumulation of proteins used in the C4 pathway, and there are few genes that have been shown to be important for the cell biology and development of C4 leaves. To facilitate functional analysis of C4 photosynthesis, and to enable knowledge from Arabidopsis thaliana to be translated to C4 species, an Agrobacterium tumefaciens-mediated transformation protocol was developed for the C4 species Cleome gynandra. A. tumefaciens, harbouring the binary vector SLJ1006, was used to transfer the uidA gene under the control of the CaMV 35S promoter into C. gynandra. Co-incubation of hypocotyls or cotyledons with SLJ1006 allowed efficient transfer of DNA into C. gynandra, and media that allowed callus production and then shoot regeneration were identified. Stable transformants of C. gynandra with detectable amounts of ?-glucuronidase (GUS) were produced at an efficiency of 14%. When driven by the CaMV 35S promoter, GUS was visible in all leaf cells, whereas uidA translationally fused to a CgRbcS gene generated GUS accumulation specifically in bundle sheath cells. This transformation procedure is the first for an NAD-ME type C4 plant and should significantly accelerate the analysis of mechanisms underlying C4 photosynthesis. PMID:20150516

Newell, Christine A.; Brown, Naomi J.; Liu, Zheng; Pflug, Alexander; Gowik, Udo; Westhoff, Peter; Hibberd, Julian M.

2010-01-01

131

The VirA protein of Agrobacterium tumefaciens is autophosphorylated and is essential for vir gene regulation.  

PubMed Central

The virA and virG gene products are required for the regulation of the vir regulon on the tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens. VirA is a membrane-associated protein which is homologous to the sensor molecules of other two-component regulatory systems. We overproduced truncated VirA proteins in Escherichia coli by deleting different lengths of the 5'-coding region of the virA gene and placing these genes under lacZ control. These proteins were purified from polyacrylamide gels and renatured. The renatured proteins became radiolabeled when they were incubated with [gamma-32P]ATP but not with [gamma-32P]GTP or [alpha-32P]ATP, which suggests an ATP gamma-phosphate-specific autophosphorylation. The smallest VirA protein, which retained only the C-terminal half of the protein, gave the strongest autophosphorylation signal, which demonstrates that the C-terminal domain has the autophosphorylation site. The phosphorylated amino acid was identified as phosphohistidine, and a highly conserved histidine was found in all of the VirA homologs. When this histidine was changed to glutamine, which cannot be phosphorylated, the resulting VirA protein lost both its ability to autophosphorylate and its biological function as a vir gene regulator. Results of this study indicate that VirA autophosphorylation is required for the induction of the vir regulon and subsequent tumor induction on plants by A. tumefaciens. Images FIG. 1 FIG. 2 FIG. 3 FIG. 4 FIG. 5 FIG. 6 PMID:2404940

Jin, S; Roitsch, T; Ankenbauer, R G; Gordon, M P; Nester, E W

1990-01-01

132

Agrobacterium tumefaciens-mediated transformation of Cleome gynandra L., a C(4) dicotyledon that is closely related to Arabidopsis thaliana.  

PubMed

In leaves of most C(4) plants, the biochemistry of photosynthesis is partitioned between mesophyll and bundle sheath cells. In addition, their cell biology and development also differs from that in C(3) plants. We have a poor understanding of the mechanisms that generate the cell-specific accumulation of proteins used in the C(4) pathway, and there are few genes that have been shown to be important for the cell biology and development of C(4) leaves. To facilitate functional analysis of C(4) photosynthesis, and to enable knowledge from Arabidopsis thaliana to be translated to C(4) species, an Agrobacterium tumefaciens-mediated transformation protocol was developed for the C(4) species Cleome gynandra. A. tumefaciens, harbouring the binary vector SLJ1006, was used to transfer the uidA gene under the control of the CaMV 35S promoter into C. gynandra. Co-incubation of hypocotyls or cotyledons with SLJ1006 allowed efficient transfer of DNA into C. gynandra, and media that allowed callus production and then shoot regeneration were identified. Stable transformants of C. gynandra with detectable amounts of beta-glucuronidase (GUS) were produced at an efficiency of 14%. When driven by the CaMV 35S promoter, GUS was visible in all leaf cells, whereas uidA translationally fused to a CgRbcS gene generated GUS accumulation specifically in bundle sheath cells. This transformation procedure is the first for an NAD-ME type C(4) plant and should significantly accelerate the analysis of mechanisms underlying C(4) photosynthesis. PMID:20150516

Newell, Christine A; Brown, Naomi J; Liu, Zheng; Pflug, Alexander; Gowik, Udo; Westhoff, Peter; Hibberd, Julian M

2010-03-01

133

Agrobacterium tumefaciens-mediated transformation in the entomopathogenic fungus Lecanicillium lecanii and development of benzimidazole fungicide resistant strains.  

PubMed

Lecanicillium lecanii has been used in the biological control of several insects in agricultural practice. Since the gene manipulation tools for this entomopathogenic fungus have not been sufficiently developed, Agrobacterium tumefaciens-mediated transformation (ATMT) in L. lecanii was investigated in this study, using the wild-type isolate FZ9906 as a progenitor strain and the hygromycin B resistance (hph) gene as a selection marker. Furthermore, a field carbendazim-resistant (mrt) gene from Botrytis cinerea was expressed in L. lecanii FZ9906 via the ATMT system. The results revealed that the frequency of transformation surpassed 25transformants/10(6) conidia, most of the putative transformants contained a single copy of T-DNA, and the T-DNA inserts were stably inherited after five generations. All putative transformants had indistinguishable biological characteristics relative to the wild-type strain, excepting two transformants with altered growth habits or virulence. Moreover, the resistance of the putative transformants to carbendazim (MBC) was improved, and the highest one was 380-fold higher than the wild-type strain. In conclusion, ATMT is an effective and suitable system for L. lecanii transformation, and will be a useful tool for the basic and application research of gene functions and gene modifications of this strain. PMID:25107375

Zhang, Yan-Jun; Zhao, Jin-Jin; Xie, Ming; Peng, De-Liang

2014-10-01

134

The complete nucleotide sequence of the TL-DNA of the Agrobacterium tumefaciens plasmid pTiAch5.  

PubMed Central

We have determined the complete primary structure (13 637 bp) of the TL-region of Agrobacterium tumefaciens octopine plasmid pTiAch5 . This sequence comprises two small direct repeats which flank the TL-region at each extremity and are involved in the transfer and/or integration of this DNA segment in plants. TL-DNA specifies eight open-reading frames corresponding to experimentally identified transcripts in crown gall tumor tissue. The eight coding regions are not interrupted by intervening sequences and are separated from each other by AT-rich regions. Potential transcriptional control signals upstream of the 5' and 3' ends of all the transcribed regions resemble typical eukaryotic signals: (i) transcriptional initiation signals ('TATA' or Goldberg- Hogness box) are present upstream to the presumed translational start codons; (ii) ' CCAAT ' sequences are present upstream of the proposed 'TATA' box; (iii) polyadenylation signals are present in the 3'-untranslated regions. Furthermore, no Shine-Dalgarno sequences are present upstream of the presumed translational start codons. PMID:6327292

Gielen, J; De Beuckeleer, M; Seurinck, J; Deboeck, F; De Greve, H; Lemmers, M; Van Montagu, M; Schell, J

1984-01-01

135

Characterization of the photolyase-like iron sulfur protein PhrB from Agrobacterium tumefaciens by Mössbauer spectroscopy  

NASA Astrophysics Data System (ADS)

High field Mössbauer spectroscopy has been used to characterize the [4Fe-4S] 2 +cluster of the protein PhrB from Agrobacterium tumefaciens which belongs to the cryptochrome/photolyase family (CPF) and which biological function has previously been shown to be DNA repair. Mössbauer spectra taken of the as prepared protein reveal ? = 0. 42 mms - 1, and ? E Q = 1. 26 mms - 1as well as an asymmetry parameter of ? = 0. 8. These parameters are characteristic for a ferredoxin-type [4Fe-4S] 2 +cluster. In order to investigate whether this cluster is involved in DNA-repair the protein has also been studied in its photoactivated state during DNA binding. The so obtained data sets exhibit essentially the same Mössbauer parameters as those of the non-activated PhrB. This indicates that during DNA repair the [4Fe-4S] 2 +cluster of PhrB has no significant amounts of transition states which have conformational changes compared to the resting state of the protein and which have life times of several seconds or longer.

Bauer, T. O.; Graf, D.; Lamparter, T.; Schünemann, V.

2014-04-01

136

Protein encoded by oncogene 6b from Agrobacterium tumefaciens has a reprogramming potential and histone chaperone-like activity  

PubMed Central

Crown gall tumors are formed mainly by actions of a group of genes in the T-DNA that is transferred from Agrobacterium tumefaciens and integrated into the nuclear DNA of host plants. These genes encode enzymes for biosynthesis of auxin and cytokinin in plant cells. Gene 6b in the T-DNA affects tumor morphology and this gene alone is able to induce small tumors on certain plant species. In addition, unorganized calli are induced from leaf disks of tobacco that are incubated on phytohormone-free media; shooty teratomas, and morphologically abnormal plants, which might be due to enhanced competence of cell division and meristematic states, are regenerated from the calli. Thus, the 6b gene appears to stimulate a reprogramming process in plants. To uncover mechanisms behind this process, various approaches including the yeast-two-hybrid system have been exploited and histone H3 was identified as one of the proteins that interact with 6b. It has been also demonstrated that 6b acts as a histone H3 chaperon in vitro and affects the expression of various genes related to cell division competence and the maintenance of meristematic states. We discuss current views on a role of 6b protein in tumorigenesis and reprogramming in plants. PMID:25389429

Ishibashi, Nanako; Kitakura, Saeko; Terakura, Shinji; Machida, Chiyoko; Machida, Yasunori

2014-01-01

137

GxySBA ABC transporter of Agrobacterium tumefaciens and its role in sugar utilization and vir gene expression.  

PubMed

Monosaccharides available in the extracellular milieu of Agrobacterium tumefaciens can be transported into the cytoplasm, or via the periplasmic sugar binding protein, ChvE, play a critical role in controlling virulence gene expression. The ChvE-MmsAB ABC transporter is involved in the utilization of a wide range of monosaccharide substrates but redundant transporters are likely given the ability of a chvE-mmsAB deletion strain to grow, albeit more slowly, in the presence of particular monosaccharides. In this study, a putative ABC transporter encoded by the gxySBA operon is identified and shown to be involved in the utilization of glucose, xylose, fucose, and arabinose, which are also substrates for the ChvE-MmsAB ABC transporter. Significantly, GxySBA is also shown to be the first characterized glucosamine ABC transporter. The divergently transcribed gene gxyR encodes a repressor of the gxySBA operon, the function of which can be relieved by a subset of the transported sugars, including glucose, xylose, and glucosamine, and this substrate-induced expression can be repressed by glycerol. Furthermore, deletion of the transporter can increase the sensitivity of the virulence gene expression system to certain sugars that regulate it. Collectively, the results reveal a remarkably diverse set of substrates for the GxySBA transporter and its contribution to the repression of sugar sensitivity by the virulence-controlling system, thereby facilitating the capacity of the bacterium to distinguish between the soil and plant environments. PMID:24957625

Zhao, Jinlei; Binns, Andrew N

2014-09-01

138

Agrobacterium tumefaciens recognizes its host environment using ChvE to bind diverse plant sugars as virulence signals  

PubMed Central

Agrobacterium tumefaciens is a broad host range plant pathogen that combinatorially recognizes diverse host molecules including phenolics, low pH, and aldose monosaccharides to activate its pathogenic pathways. Chromosomal virulence gene E (chvE) encodes a periplasmic-binding protein that binds several neutral sugars and sugar acids, and subsequently interacts with the VirA/VirG regulatory system to stimulate virulence (vir) gene expression. Here, a combination of genetics, X-ray crystallography, and isothermal calorimetry reveals how ChvE binds the different monosaccharides and also shows that binding of sugar acids is pH dependent. Moreover, the potency of a sugar for vir gene expression is modulated by a transport system that also relies on ChvE. These two circuits tune the overall system to respond to sugar concentrations encountered in vivo. Finally, using chvE mutants with restricted sugar specificities, we show that there is host variation in regard to the types of sugars that are limiting for vir induction. PMID:23267119

Hu, Xiaozhen; Zhao, Jinlei; DeGrado, William F.; Binns, Andrew N.

2013-01-01

139

Improvement in the Thermostability of d-Psicose 3-Epimerase from Agrobacterium tumefaciens by Random and Site-Directed Mutagenesis ?  

PubMed Central

The S213C, I33L, and I33L S213C variants of d-psicose 3-epimerase from Agrobacterium tumefaciens, which were obtained by random and site-directed mutagenesis, displayed increases of 2.5, 5, and 7.5°C in the temperature for maximal enzyme activity, increases of 3.3-, 7.2-, and 29.9-fold in the half-life at 50°C, and increases of 3.1, 4.3, and 7.6°C in apparent melting temperature, respectively, compared with the wild-type enzyme. Molecular modeling suggests that the improvement in thermostability in these variants may have resulted from increased putative hydrogen bonds and formation of new aromatic stacking interactions. The immobilized wild-type enzyme with and without borate maintained activity for 8 days at a conversion yield of 70% (350 g/liter psicose) and for 16 days at a conversion yield of 30% (150 g/liter psicose), respectively. After 8 or 16 days, the enzyme activity gradually decreased, and the conversion yields with and without borate were reduced to 22 and 9.6%, respectively, at 30 days. In contrast, the activities of the immobilized I33L S213C variant with and without borate did not decrease during the operation time of 30 days. These results suggest that the I33L S213C variant may be useful as an industrial producer of d-psicose. PMID:21873475

Choi, Jin-Geun; Ju, Yo-Han; Yeom, Soo-Jin; Oh, Deok-Kun

2011-01-01

140

Cell-autonomous cytokinin-independent growth of tobacco cells transformed by Agrobacterium tumefaciens strains lacking the cytokinin biosynthesis gene.  

PubMed Central

Mutations at the cytokinin biosynthesis locus (tmr) of Agrobacterium tumefaciens usually result in strains that induce tumors exhibiting the rooty phenotype associated with high auxin-to-cytokinin ratios. However, tobacco (Nicotiana tabacum cv Havana 425) leaf disc explants responded to tmr- mutant strain A356 by producing rapidly growing, unorganized tumors, indicating that these lines can grow in a cytokinin-independent fashion despite the absence of a functional tmr gene. Several methods have been used to characterize the physiological and cellular basis of this phenotype. The results indicate that tmr- tumors have a physiologically distinct mechanism for cytokinin-independent growth in comparison to tumors induced by wild-type bacteria. The cytokinin-independent phenotype of the tmr- transformants appears to be cell autonomous in nature: only the transformed cells and their progeny were capable of cytokinin-independent growth. Specifically, the tmr- tumors did not accumulate cytokinin, and clonal analysis indicated the tmr- transformed cells were not capable of stimulating the growth of neighboring nontransformed cells. Finally, the cytokinin-independent phenotype of the tmr- transformants was shown to be cold sensitive, whereas the wild-type tumors exhibited a cold-resistant cytokinin-independent phenotype. Potential mechanisms for this novel form of cytokinin-independent growth, including the role of the dehydrodiconiferyl alcohol glucosides found in both tumor types, are discussed. PMID:8058843

Black, R C; Binns, A N; Chang, C F; Lynn, D G

1994-01-01

141

Pleiotropic phenotypes caused by genetic ablation of the receiver module of the Agrobacterium tumefaciens VirA protein.  

PubMed Central

The VirA protein of Agrobacterium tumefaciens is a transmembrane sensory kinase that phosphorylates the VirG response regulator in response to chemical signals released from plant wound sites. VirA contains both a two-component kinase module and, at its carboxyl terminus, a receiver module. We previously provided evidence that this receiver module inhibited the activity of the kinase module and that inhibition might be neutralized by phosphorylation. In this report, we provide additional evidence for this model by showing that overexpressing the receiver module in trans can restore low-level basal activity to a VirA mutant protein lacking the receiver module. We also show that ablation of the receiver module restores activity to the inactive VirA (delta324-413) mutant, which has a deletion within a region designated the linker module. This indicates that deletion of the linker module does not denature the kinase module, but rather locks the kinase into a phenotypically inactive conformation, and that this inactivity requires the receiver module. These data provide genetic evidence that the kinase and receiver modules of VirA attain their native conformations autonomously. The receiver module also restricts the variety of phenolic compounds that have stimulatory activity, since removal of this module causes otherwise nonstimulatory phenolic compounds such as 4-hydroxyacetophenone to stimulate vir gene expression. PMID:8755904

Chang, C H; Zhu, J; Winans, S C

1996-01-01

142

Optimization of factors influencing microinjection method for Agrobacterium tumefaciens-mediated transformation of tomato.  

PubMed

A simple and efficient protocol for Agrobacterium-mediated genetic transformation of tomato was developed using combination of non-tissue culture and micropropagation systems. Initially, ESAM region of 1-day-old germinated tomato seeds were microinjected for one to five times with Agrobacterium inoculums (OD(600)?=?0.2-1.0). The germinated seeds were cocultivated in the MS medium fortified with (0-200 mM) acetosyringone and minimal concentrations of (0-20 mg?L(-1)) kanamycin, and the antibiotic concentration was doubled during the second round of selection. Bacterial concentration of OD(600)?=?0.6 served as an optimal concentration for infection and the transformation efficiency was significantly higher of about 46.28 %. In another set of experiment, an improved and stable regeneration system was adapted for the explants from the selection medium. Four-day-old double cotyledonary nodal explants were excised from the microinjected seedlings and cultured onto the MS medium supplemented with 1.5 mg?L(-1) thidiazuron, 1.5 mg?L(-1) indole-3-butyric acid, 30 mg?L(-1) kanamycin, and 0-1.5 mg?L(-1) adenine sulphate. Maximum of 9 out of 13 micropropagated shoots were shown positive to GUS assay. By this technique, the transformation efficiency was increased from 46.28 to 65.90 %. Thus, this paper reports the successful protocol for the mass production of transformants using microinjection and micropropagation techniques. PMID:23306888

Vinoth, S; Gurusaravanan, P; Jayabalan, N

2013-02-01

143

Analysis of Vir protein translocation from Agrobacterium tumefaciens using Saccharomyces cerevisiae as a model: evidence for transport of a novel effector protein VirE3  

PubMed Central

Agrobacterium tumefaciens causes crown gall disease on a variety of plants. During the infection process Agrobacterium transfers a nucleoprotein complex, the VirD2 T-complex, and at least two Vir proteins, VirE2 and VirF, into the plant cell via the VirB/VirD4 type IV secretion system. Recently, we found that T-DNA could also be transferred from Agrobacterium to Saccharomyces cerevisiae. Here, we describe a novel method to also detect trans-kingdom Vir protein transfer from Agrobacterium to yeast, using the Cre/lox system. Protein fusions between Cre and VirE2 or VirF were expressed in Agrobacterium. Transfer of the Cre-Vir fusion proteins from Agrobacterium to yeast was monitored by a selectable excision event resulting from site-specific recombination mediated by Cre on a lox-flanked transgene in yeast. The VirE2 and VirF proteins were transported to yeast via the virB-encoded transfer system in the presence of coupling factor VirD4, analogous to translocation into plant cells. The yeast system therefore provides a suitable and fast model system to study basic aspects of trans-kingdom protein transport from Agrobacterium into host cells. Using this method we showed that VirE2 and VirF protein transfer was inhibited by the presence of the Osa protein. Besides, we found evidence for a novel third effector protein, VirE3, which has a similar C-terminal signature to VirE2 and VirF. PMID:12560481

Schrammeijer, Barbara; den Dulk-Ras, Amke; Vergunst, Annette C.; Jurado Jácome, Esmeralda; Hooykaas, Paul J. J.

2003-01-01

144

Nodules are induced on alfalfa roots by Agrobacterium tumefaciens and Rhizobium trifolii containing small segments of the Rhizobium meliloti nodulation region  

SciTech Connect

Regions of the Rhizobium meliloti nodulation genes from the symbiotic plasmid were transferred to Agrobacterium tumefaciens and Rhizobium trifolii by conjugation. The A. tumefaciens and R. trifolii trans-conjugants were unable to elicit curling of alfalfa root hairs, but were able to induce nodule development at a low frequency. These were judged to be genuine nodules on the basis of cytological and developmental criteria. Like genuine alfalfa nodules, the nodules were initiated from divisions of the inner root cortical cells. They developed a distally positioned meristem and several peripheral vascular bundles. An endodermis separated the inner tissues of the nodule from the surrounding cortex. No infection threads were found to penetrate either root hairs or the nodule cells. Bacteria were found only in intercellular spaces. Thus, alfalfa nodules induced by A. tumefaciens and R. trifolii transconjugants carrying small nodulation clones of R. meliloti were completely devoid of intracellular bacteria. When these strains were inoculated onto white clover roots, small nodule-like protrusions developed that, when examined cytologically, were found to more closely resemble roots than nodules. Although the meristem was broadened and lacked a root cap, the protrusions had a central vascular bundle and other rootlike features. The results suggest that morphogenesis of alfalfa root nodules can be uncoupled from infection thread formation. The genes encoded in the 8.7-kilobase nodulation fragment are sufficient in A. tumefaciens or R. trifolii backgrounds for nodule morphogenesis.

Hirsch, A.M.; Drake, D.; Jacobs, T.W.; Long, S.R.

1985-01-01

145

Genome sequence and mutational analysis of plant-growth-promoting bacterium Agrobacterium tumefaciens CCNWGS0286 Isolated from a zinc-lead mine tailing.  

PubMed

The plant-growth-promoting bacterium Agrobacterium tumefaciens CCNWGS0286, isolated from the nodules of Robinia pseudoacacia growing in zinc-lead mine tailings, both displayed high metal resistance and enhanced the growth of Robinia plants in a metal-contaminated environment. Our goal was to determine whether bacterial metal resistance or the capacity to produce phytohormones had a larger impact on the growth of host plants under zinc stress. Eight zinc-sensitive mutants and one zinc-sensitive mutant with reduced indole-3-acetic acid (IAA) production were obtained by transposon mutagenesis. Analysis of the genome sequence and of transcription via reverse transcriptase PCR (RT-PCR) combined with transposon gene disruptions revealed that ZntA-4200 and the transcriptional regulator ZntR1 played important roles in the zinc homeostasis of A. tumefaciens CCNWGS0286. In addition, interruption of a putative oligoketide cyclase/lipid transport protein reduced IAA synthesis and also showed reduced zinc and cadmium resistance but had no influence on copper resistance. In greenhouse studies, R. pseudoacacia inoculated with A. tumefaciens CCNWGS0286 displayed a significant increase in biomass production over that without inoculation, even in a zinc-contaminated environment. Interestingly, the differences in plant biomass improvement among A. tumefaciens CCNWGS0286, A. tumefaciens C58, and zinc-sensitive mutants 12-2 (zntA::Tn5) and 15-6 (low IAA production) revealed that phytohormones, rather than genes encoding zinc resistance determinants, were the dominant factor in enhancing plant growth in contaminated soil. PMID:22636006

Hao, Xiuli; Xie, Pin; Johnstone, Laurel; Miller, Susan J; Rensing, Christopher; Wei, Gehong

2012-08-01

146

Genome Sequence and Mutational Analysis of Plant-Growth-Promoting Bacterium Agrobacterium tumefaciens CCNWGS0286 Isolated from a Zinc-Lead Mine Tailing  

PubMed Central

The plant-growth-promoting bacterium Agrobacterium tumefaciens CCNWGS0286, isolated from the nodules of Robinia pseudoacacia growing in zinc-lead mine tailings, both displayed high metal resistance and enhanced the growth of Robinia plants in a metal-contaminated environment. Our goal was to determine whether bacterial metal resistance or the capacity to produce phytohormones had a larger impact on the growth of host plants under zinc stress. Eight zinc-sensitive mutants and one zinc-sensitive mutant with reduced indole-3-acetic acid (IAA) production were obtained by transposon mutagenesis. Analysis of the genome sequence and of transcription via reverse transcriptase PCR (RT-PCR) combined with transposon gene disruptions revealed that ZntA-4200 and the transcriptional regulator ZntR1 played important roles in the zinc homeostasis of A. tumefaciens CCNWGS0286. In addition, interruption of a putative oligoketide cyclase/lipid transport protein reduced IAA synthesis and also showed reduced zinc and cadmium resistance but had no influence on copper resistance. In greenhouse studies, R. pseudoacacia inoculated with A. tumefaciens CCNWGS0286 displayed a significant increase in biomass production over that without inoculation, even in a zinc-contaminated environment. Interestingly, the differences in plant biomass improvement among A. tumefaciens CCNWGS0286, A. tumefaciens C58, and zinc-sensitive mutants 12-2 (zntA::Tn5) and 15-6 (low IAA production) revealed that phytohormones, rather than genes encoding zinc resistance determinants, were the dominant factor in enhancing plant growth in contaminated soil. PMID:22636006

Hao, Xiuli; Xie, Pin; Johnstone, Laurel; Miller, Susan J.

2012-01-01

147

A conserved mechanism of GABA binding and antagonism is revealed by structure-function analysis of the periplasmic binding protein Atu2422 in Agrobacterium tumefaciens.  

PubMed

Bacterial periplasmic binding proteins (PBPs) and eukaryotic PBP-like domains (also called as Venus flytrap modules) of G-protein-coupled receptors are involved in extracellular GABA perception. We investigated the structural and functional basis of ligand specificity of the PBP Atu2422, which is implicated in virulence and transport of GABA in the plant pathogen Agrobacterium tumefaciens. Five high-resolution x-ray structures of Atu2422 liganded to GABA, Pro, Ala, and Val and of point mutant Atu2422-F77A liganded to Leu were determined. Structural analysis of the ligand-binding site revealed two essential residues, Phe(77) and Tyr(275), the implication of which in GABA signaling and virulence was confirmed using A. tumefaciens cells expressing corresponding Atu2422 mutants. Phe(77) restricts ligand specificity to ?-amino acids with a short lateral chain, which act as antagonists of GABA signaling in A. tumefaciens. Tyr(275) specifically interacts with the GABA ?-amino group. Conservation of these two key residues in proteins phylogenetically related to Atu2422 brought to light a subfamily of PBPs in which all members could bind GABA and short ?-amino acids. This work led to the identification of a fingerprint sequence and structural features for defining PBPs that bind GABA and its competitors and revealed their occurrence among host-interacting proteobacteria. PMID:20630861

Planamente, Sara; Vigouroux, Armelle; Mondy, Samuel; Nicaise, Magali; Faure, Denis; Moréra, Solange

2010-09-24

148

Control of zinc homeostasis in Agrobacterium tumefaciens via zur and the zinc uptake genes znuABC and zinT.  

PubMed

The Agrobacterium tumefaciens zinc uptake regulator (Zur) was shown to negatively regulate the zinc uptake genes znuABC, encoding a zinc transport system belonging to the ATP-binding cassette (ABC) transporter family, and zinT, which encodes a periplasmic zinc-binding protein. The expression of znuABC and zinT was inducible when cells were grown in medium containing a metal chelator (EDTA), and this induction was shown to be specific for zinc depletion. The expression of znuABC was reduced in response to increased zinc in a dose-dependent manner, and zinT had a less pronounced but similar pattern of zinc-regulated expression. The inactivation of zur led to constitutively high expression of znuABC and zinT. In addition, a zur mutant had an increased total zinc content compared to the WT NTL4 strain, whereas the inactivation of zinT caused a reduction in the total zinc content. The zinT gene is shown to play a dominant role and to be more important than znuA and znuB for A. tumefaciens survival under zinc deprivation. ZinT can function even when ZnuABC is inactivated. However, mutations in zur, znuA, znuB or zinT did not affect the virulence of A. tumefaciens. PMID:25227896

Bhubhanil, Sakkarin; Sittipo, Panida; Chaoprasid, Paweena; Nookabkaew, Sumontha; Sukchawalit, Rojana; Mongkolsuk, Skorn

2014-11-01

149

Agrobacterium tumefaciens-mediated transformation of taro (Colocasia esculenta (L.) Schott) with a rice chitinase gene for improved tolerance to a fungal pathogen Sclerotium rolfsii.  

PubMed

Taro (Colocasia esculenta) is one of the most important crops in the Pacific Islands, however, taro yields have been declining in Hawaii over the past 30 years partly due to diseases caused by oomycete and fungal pathogens. In this study, an efficient Agrobacterium tumefaciens-mediated transformation method for taro is first reported. In total, approximately 200 pieces (8 g) of embryogenic calluses were infected with the super-virulent A. tumefaciens strain EHA105 harboring the plant transformation plasmid pBI121/ricchi11 that contains the rice chitinase gene ricchi11. The presence and expression of the transgene ricchi11 in six independent transgenic lines was confirmed using polymerase chain reaction (PCR) and reverse transcription-PCR (RT-PCR). Southern blot analysis of the six independent lines indicated that three out of six (50%) had integrated a single copy of the transgene, and the other three lines had two or three copies of the transgene. Compared to the particle bombardment transformation of taro method, which was used in the previous studies, the Agrobacterium-mediated transformation method obtained 43-fold higher transformation efficiency. In addition, these six transgenic lines via Agrobacterium may be more effective for transgene expression as a result of single-copy or low-copy insertion of the transgene than the single line with multiple copies of the transgene via particle bombardment. In a laboratory bioassay, all six transgenic lines exhibited increased tolerance to the fungal pathogen Sclerotium rolfsii, ranging from 42 to 63% reduction in lesion expansion. PMID:18301900

He, Xiaoling; Miyasaka, Susan C; Fitch, Maureen M M; Moore, Paul H; Zhu, Yun J

2008-05-01

150

A Genome-Wide Survey of Highly Expressed Non-Coding RNAs and Biological Validation of Selected Candidates in Agrobacterium tumefaciens  

PubMed Central

Agrobacterium tumefaciens is a plant pathogen that has the natural ability of delivering and integrating a piece of its own DNA into plant genome. Although bacterial non-coding RNAs (ncRNAs) have been shown to regulate various biological processes including virulence, we have limited knowledge of how Agrobacterium ncRNAs regulate this unique inter-Kingdom gene transfer. Using whole transcriptome sequencing and an ncRNA search algorithm developed for this work, we identified 475 highly expressed candidate ncRNAs from A. tumefaciens C58, including 101 trans-encoded small RNAs (sRNAs), 354 antisense RNAs (asRNAs), 20 5? untranslated region (UTR) leaders including a RNA thermosensor and 6 riboswitches. Moreover, transcription start site (TSS) mapping analysis revealed that about 51% of the mapped mRNAs have 5? UTRs longer than 60 nt, suggesting that numerous cis-acting regulatory elements might be encoded in the A. tumefaciens genome. Eighteen asRNAs were found on the complementary strands of virA, virB, virC, virD, and virE operons. Fifteen ncRNAs were induced and 7 were suppressed by the Agrobacterium virulence (vir) gene inducer acetosyringone (AS), a phenolic compound secreted by the plants. Interestingly, fourteen of the AS-induced ncRNAs have putative vir box sequences in the upstream regions. We experimentally validated expression of 36 ncRNAs using Northern blot and Rapid Amplification of cDNA Ends analyses. We show functional relevance of two 5? UTR elements: a RNA thermonsensor (C1_109596F) that may regulate translation of the major cold shock protein cspA, and a thi-box riboswitch (C1_2541934R) that may transcriptionally regulate a thiamine biosynthesis operon, thiCOGG. Further studies on ncRNAs functions in this bacterium may provide insights and strategies that can be used to better manage pathogenic bacteria for plants and to improve Agrobacterum-mediated plant transformation. PMID:23950988

Lee, Keunsub; Huang, Xiaoqiu; Yang, Chichun; Lee, Danny; Ho, Vincent; Nobuta, Kan; Fan, Jian-Bing; Wang, Kan

2013-01-01

151

Structural and functional analysis of a putative gene cluster for palatinose transport on the linear chromosome of Agrobacterium tumefaciens MAFF301001.  

PubMed

We identified a putative pal gene cluster (palR, palE, palF, palG, palK, palA, and palB) in the plant-tumorigenic bacterium Agrobacterium tumefaciens MAFF301001; by sequencing analyses, this cluster was found to be involved in palatinose transport, and its functional importance was revealed by mutational analyses. The pal gene products were highly homologous to those of putative trehalose/maltose ABC-type transport systems but were not essential to bacterial growth on trehalose. Insertion mutations in the palK and palE genes showed the necessity of these genes for bacterial growth and chemotaxis with palatinose as the carbon source, but no inhibition of tumorigenesis was observed. Growth on trehalose and maltose was not influenced by the mutations. PMID:12644509

De Costa, Devika M; Suzuki, Katsunori; Yoshida, Kazuo

2003-04-01

152

Structural and Functional Analysis of a Putative Gene Cluster for Palatinose Transport on the Linear Chromosome of Agrobacterium tumefaciens MAFF301001  

PubMed Central

We identified a putative pal gene cluster (palR, palE, palF, palG, palK, palA, and palB) in the plant-tumorigenic bacterium Agrobacterium tumefaciens MAFF301001; by sequencing analyses, this cluster was found to be involved in palatinose transport, and its functional importance was revealed by mutational analyses. The pal gene products were highly homologous to those of putative trehalose/maltose ABC-type transport systems but were not essential to bacterial growth on trehalose. Insertion mutations in the palK and palE genes showed the necessity of these genes for bacterial growth and chemotaxis with palatinose as the carbon source, but no inhibition of tumorigenesis was observed. Growth on trehalose and maltose was not influenced by the mutations. PMID:12644509

De Costa, Devika M.; Suzuki, Katsunori; Yoshida, Kazuo

2003-01-01

153

Acid-Induced Type VI Secretion System Is Regulated by ExoR-ChvG/ChvI Signaling Cascade in Agrobacterium tumefaciens  

PubMed Central

The type VI secretion system (T6SS) is a widespread, versatile protein secretion system in pathogenic Proteobacteria. Several T6SSs are tightly regulated by various regulatory systems at multiple levels. However, the signals and/or regulatory mechanisms of many T6SSs remain unexplored. Here, we report on an acid-induced regulatory mechanism activating T6SS in Agrobacterium tumefaciens, a plant pathogenic bacterium causing crown gall disease in a wide range of plants. We monitored the secretion of the T6SS hallmark protein hemolysin-coregulated protein (Hcp) from A. tumefaciens and found that acidity is a T6SS-inducible signal. Expression analysis of the T6SS gene cluster comprising the imp and hcp operons revealed that imp expression and Hcp secretion are barely detected in A. tumefaciens grown in neutral minimal medium but are highly induced with acidic medium. Loss- and gain-of-function analysis revealed that the A. tumefaciens T6SS is positively regulated by a chvG/chvI two-component system and negatively regulated by exoR. Further epistasis analysis revealed that exoR functions upstream of the chvG sensor kinase in regulating T6SS. ChvG protein levels are greatly increased in the exoR deletion mutant and the periplasmic form of overexpressed ExoR is rapidly degraded under acidic conditions. Importantly, ExoR represses ChvG by direct physical interaction, but disruption of the physical interaction allows ChvG to activate T6SS. The phospho-mimic but not wild-type ChvI response regulator can bind to the T6SS promoter region in vitro and activate T6SS with growth in neutral minimal medium. We present the first evidence of T6SS activation by an ExoR-ChvG/ChvI cascade and propose that acidity triggers ExoR degradation, thereby derepressing ChvG/ChvI to activate T6SS in A. tumefaciens. PMID:23028331

Shaw, Gwo-Chyuan; Lai, Erh-Min

2012-01-01

154

A Signaling Pathway Involving the Diguanylate Cyclase CelR and the Response Regulator DivK Controls Cellulose Synthesis in Agrobacterium tumefaciens  

PubMed Central

The production of cellulose fibrils is involved in the attachment of Agrobacterium tumefaciens to its plant host. Consistent with previous studies, we reported recently that a putative diguanylate cyclase, celR, is required for synthesis of this polymer in A. tumefaciens. In this study, the effects of celR and other components of the regulatory pathway of cellulose production were explored. Mutational analysis of celR demonstrated that the cyclase requires the catalytic GGEEF motif, as well as the conserved aspartate residue of a CheY-like receiver domain, for stimulating cellulose production. Moreover, a site-directed mutation within the PilZ domain of CelA, the catalytic subunit of the cellulose synthase complex, greatly reduced cellulose production. In addition, deletion of divK, the first gene of the divK-celR operon, also reduced cellulose production. This requirement for divK was alleviated by expression of a constitutively active form of CelR, suggesting that DivK acts upstream of CelR activation. Based on bacterial two-hybrid assays, CelR homodimerizes but does not interact with DivK. The mutation in divK additionally affected cell morphology, and this effect was complementable by a wild-type copy of the gene, but not by the constitutively active allele of celR. These results support the hypothesis that CelR is a bona fide c-di-GMP synthase and that the nucleotide signal produced by this enzyme activates CelA via the PilZ domain. Our studies also suggest that the DivK/CelR signaling pathway in Agrobacterium regulates cellulose production independent of cell cycle checkpoint systems that are controlled by divK. PMID:24443526

Barnhart, D. Michael; Su, Shengchang

2014-01-01

155

Characterization of a novel Agrobacterium tumefaciens Galactarolactone Cycloisomerase Enzyme for Direct Conversion of d-Galactarolactone to 3-Deoxy-2-keto-l-threo-hexarate*  

PubMed Central

Microorganisms use different pathways for d-galacturonate catabolism. In the known microbial oxidative pathway, d-galacturonate is oxidized to d-galactarolactone, the lactone hydrolyzed to galactarate, which is further converted to 3-deoxy-2-keto-hexarate and ?-ketoglutarate. We have shown recently that Agrobacterium tumefaciens strain C58 contains an uronate dehydrogenase (At Udh) that oxidizes d-galacturonic acid to d-galactarolactone. Here we report identification of a novel enzyme from the same A. tumefaciens strain, which we named Galactarolactone cycloisomerase (At Gci) (E.C. 5.5.1.-), for the direct conversion of the d-galactarolactone to 3-deoxy-2-keto-hexarate. The At Gci enzyme is 378 amino acids long and belongs to the mandelate racemase subgroup in the enolase superfamily. At Gci was heterologously expressed in Escherichia coli, and the purified enzyme was found to exist as an octameric form. It is active both on d-galactarolactone and d-glucarolactone, but does not work on the corresponding linear hexaric acid forms. The details of the reaction mechanism were further studied by NMR and optical rotation demonstrating that the reaction product of At Gci from d-galactaro-1,4-lactone and d-glucaro-1,4-lactone conversion is in both cases the l-threo form of 3-deoxy-2-keto-hexarate. PMID:22493433

Andberg, Martina; Maaheimo, Hannu; Boer, Harry; Penttilä, Merja; Koivula, Anu; Richard, Peter

2012-01-01

156

Agrobacterium tumefaciens VirC2 enhances T-DNA transfer and virulence through its C-terminal ribbon-helix-helix DNA-binding fold.  

PubMed

Agrobacterium tumefaciens VirC2 stimulates processing of single-stranded T-DNA that is translocated into plants to induce tumor formation, but how VirC2 functions is unclear. Here, we report the 1.7-A X-ray crystal structure of its trypsin-resistant C-terminal domain, VirC2(82-202), which reveals a form of the ribbon-helix-helix (RHH) DNA-binding fold contained within a single polypeptide chain. DNA-binding assays and mutagenesis indicate that VirC2 uses this RHH fold to bind double-stranded DNA but not single-stranded DNA. Mutations that severely affect VirC2 DNA binding are highly deleterious for both T-DNA transfer into yeast and the virulence of A. tumefaciens in different plants including Nicotiana glauca and Kalanchoe daigremontiana. These data suggest that VirC2 enhances T-DNA transfer and virulence through DNA binding with its RHH fold. The RHH fold of VirC2 is the first crystal structure representing a group of predicted RHH proteins that facilitate endonucleolytic processing of DNA for horizontal gene transfer. PMID:19482939

Lu, Jun; den Dulk-Ras, Amke; Hooykaas, Paul J J; Glover, J N Mark

2009-06-16

157

The Ctp Type IVb Pilus Locus of Agrobacterium tumefaciens Directs Formation of the Common Pili and Contributes to Reversible Surface Attachment  

PubMed Central

Agrobacterium tumefaciens can adhere to plant tissues and abiotic surfaces and forms biofilms. Cell surface appendages called pili play an important role in adhesion and biofilm formation in diverse bacterial systems. The A. tumefaciens C58 genome sequence revealed the presence of the ctpABCDEFGHI genes (cluster of type IV pili; Atu0216 to Atu0224), homologous to tad-type pilus systems from several bacteria, including Aggregatibacter actinomycetemcomitans and Caulobacter crescentus. These systems fall into the type IVb pilus group, which can function in bacterial adhesion. Transmission electron microscopy of A. tumefaciens revealed the presence of filaments, significantly thinner than flagella and often bundled, associated with cell surfaces and shed into the external milieu. In-frame deletion mutations of all of the ctp genes, with the exception of ctpF, resulted in nonpiliated derivatives. Mutations in ctpA (a pilin homologue), ctpB, and ctpG decreased early attachment and biofilm formation. The adherence of the ctpA mutant could be restored by ectopic expression of the paralogous pilA gene. The ?ctpA ?pilA double pilin mutant displayed a diminished biovolume and lower biofilm height than the wild type under flowing conditions. Surprisingly, however, the ctpCD, ctpE, ctpF, ctpH, and ctpI mutants formed normal biofilms and showed enhanced reversible attachment. In-frame deletion of the ctpA pilin gene in the ctpCD, ctpE, ctpF, ctpH, and ctpI mutants caused the same attachment-deficient phenotype as the ctpA single mutant. Collectively, these findings indicate that the ctp locus is involved in pilus assembly and that nonpiliated mutants, which retain the CtpA pilin, are proficient in attachment and adherence. PMID:24914181

Wang, Yi; Haitjema, Charles H.

2014-01-01

158

An IcmF Family Protein, ImpLM, Is an Integral Inner Membrane Protein Interacting with ImpKL, and Its Walker A Motif Is Required for Type VI Secretion System-Mediated Hcp Secretion in Agrobacterium tumefaciens  

Microsoft Academic Search

An intracellular multiplication F (IcmF) family protein is a conserved component of a newly identified type VI secretion system (T6SS) encoded in many animal and plant-associated Proteobacteria. We have previously identified ImpLM, an IcmF family protein that is required for the secretion of the T6SS substrate hemolysin- coregulated protein (Hcp) from the plant-pathogenic bacterium Agrobacterium tumefaciens. In this study, we

Lay-Sun Ma; Jer-Sheng Lin; Erh-Min Lai

2009-01-01

159

Optimizing shoot regeneration and transient expression factors for Agrobacterium tumefaciens transformation of sour cherry ( Prunus cerasus L.) cultivar Montmorency  

Microsoft Academic Search

An efficient, adventitious shoot regeneration protocol was devised, and transient expression studies were carried out to enable Agrobacterium-mediated stable transformation of sour cherry (Prunus cerasus L.) cultivar Montmorency. Leaves, from in vitro stock cultures, with the petiole removed and four partial cuts made transversely and equidistant through the midrib area were found to be the optimum explant type. A 24h

Guo-Qing Song; Kenneth C. Sink

2005-01-01

160

Transgenic ramie [Boehmeria nivea (L.) Gaud.]: factors affecting the efficiency of Agrobacterium tumefaciens-mediated transformation and regeneration.  

PubMed

In the present study, an efficient Agrobacterium-mediated gene transformation system was developed for ramie [Boehmeria nivea (L.) Gaud.] based on the examinations of several factors affecting plant transformation efficiency. The effects of Agrobacterium cell density, acetosyringone, co-cultivation temperature, co-cultivation duration, co-cultivation photoperiod and pH on stable transformation were evaluated. Agrobacterium at a concentration of OD = 0.5-0.8 improved the efficiency of transformation. Concentration of acetosyringone at 50 mg/L during co-cultivation significantly increased transformation efficiency. Co-cultivation at 20 degrees C, in comparison to 15, 25 and 28 degrees C, consistently resulted in higher transformation frequencies. A relatively short co-cultivation duration (3 days) was optimal for ramie transformation. Co-cultivation medium at pH 5.9 and co-cultivation in darkness both improved the transformation efficiencies of ramie. An overall scheme for producing transgenic ramie is presented, through which an average transformation rate from 10.5 to 24.7% in five ramie varieties was obtained. Stable expression and integration of the transgenes were confirmed by histochemical GUS assay, kanamycin painting assay, PCR and Southern blotting. This optimized transformation system should be employed for efficient Agrobacterium-mediated transformation of ramie. PMID:19533144

Wang, Bo; Liu, Lijun; Wang, Xuxia; Yang, Jinyu; Sun, Zhenxia; Zhang, Na; Gao, Shimei; Xing, Xiulong; Peng, Dingxiang

2009-09-01

161

In vivo analysis of DNA binding and ligand interaction of BlcR, an IclR-type repressor from Agrobacterium tumefaciens.  

PubMed

Agrobacterium tumefaciens BlcR represses transcription of the blcABC operon, which is involved in metabolism of ?-butyrolactone, and this repression is alleviated by succinate semialdehyde (SSA). BlcR exists as a homodimer, and the blcABC promoter DNA contains two BlcR-binding sites (IR1 and IR2) that correspond to two BlcR dimers. In this study, we established an in vivo system to examine the SSA-responsive control of BlcR transcriptional regulation. The endogenous blcR, encoded in the pAtC58 plasmid of A. tumefaciens C58, was not optimal for investigating the effect of SSA on BlcR repression, probably due to the SSA degradation mediated by the pAt-encoded blcABC. We therefore introduced blcR (and the blcABC promoter DNA, separately) exogenously into a strain of C58 cured of pAtC58 (and pTiC58). We applied this system to interrogate BlcR-DNA interactions and to test predictions from our prior structural and biochemical studies. This in vivo analysis confirmed the previously mapped SSA-binding site and supported a model by which DNA coordinates formation of a BlcR tetramer. In addition, we identified a specific lysine residue (K59) as an important determinant for DNA binding. Moreover, based on isothermal titration calorimetry analysis, we found IR1 to play the dominant role in binding to BlcR, relative to IR2. Together, these in vivo results expand the biochemical findings and provide new mechanistic insights into BlcR-DNA interactions. PMID:23449918

Pan, Yi; Wang, Yi; Fuqua, Clay; Chen, Lingling

2013-04-01

162

In vivo analysis of DNA binding and ligand interaction of BlcR, an IclR-type repressor from Agrobacterium tumefaciens  

PubMed Central

Agrobacterium tumefaciens BlcR represses transcription of the blcABC operon, which is involved in metabolism of ?-butyrolactone, and this repression is alleviated by succinate semialdehyde (SSA). BlcR exists as a homodimer, and the blcABC promoter DNA contains two BlcR-binding sites (IR1 and IR2) that correspond to two BlcR dimers. In this study, we established an in vivo system to examine the SSA-responsive control of BlcR transcriptional regulation. The endogenous blcR, encoded in the pAtC58 plasmid of A. tumefaciens C58, was not optimal for investigating the effect of SSA on BlcR repression, probably due to the SSA degradation mediated by the pAt-encoded blcABC. We therefore introduced blcR (and the blcABC promoter DNA, separately) exogenously into a strain of C58 cured of pAtC58 (and pTiC58). We applied this system to interrogate BlcR–DNA interactions and to test predictions from our prior structural and biochemical studies. This in vivo analysis confirmed the previously mapped SSA-binding site and supported a model by which DNA coordinates formation of a BlcR tetramer. In addition, we identified a specific lysine residue (K59) as an important determinant for DNA binding. Moreover, based on isothermal titration calorimetry analysis, we found IR1 to play the dominant role in binding to BlcR, relative to IR2. Together, these in vivo results expand the biochemical findings and provide new mechanistic insights into BlcR–DNA interactions. PMID:23449918

Pan, Yi; Wang, Yi; Fuqua, Clay

2013-01-01

163

Transformation of Montmorency sour cherry (Prunus cerasus L.) and Gisela 6 (P. cerasus x P. canescens) cherry rootstock mediated by Agrobacterium tumefaciens.  

PubMed

Sour cherry (Prunus cerasus L.) scion cv. Montmorency and rootstock cv. Gisela 6 (P. cerasus x P. canescens) were transformed using Agrobacterium tumefaciens strain EHA105:pBISN1 carrying the neomycin phosphotransferase gene (nptII) and an intron interrupted ss-glucuronidase (GUS) reporter gene (gusA). Whole leaf explants were co-cultivated with A. tumefaciens, and selection and regeneration of transformed cells and shoots of both cultivars was carried out for 12 weeks on selection medium containing 50 mg l(-1) kanamycin (Km) and 250 mg l(-1) timentin. These media were [Quoirin and Lepoivre (Acta Hortic 78:437-442, 1977)] supplemented with 0.5 mg l(-1) benzylaminopurine (BA) + 0.05 mg l(-1) indole-3-butyric acid (IBA), and woody plant medium [Lloyd and McCown (Proc Int Plant Prop Soc 30:421-427, 1980)] containing 2.0 mg l(-1) BA + 1.0 mg l(-1) IBA for cv. Montmorency and cv. Gisela 6, respectively. Seven out of 226 (3.1%) explants of cv. Montmorency and five out of 152 (3.9%) explants of cv. Gisela 6 produced 30/39 GUS- and PCR-positive shoots from the cut midribs via an intermediate callus. Southern analysis of the GUS- and PCR-positive transformants confirmed stable integration of the transgenes with 1-3 copy numbers in the genomes of seven lines of cv. Montmorency and five of cv. Gisela 6. The selected transformants have a normal phenotype in vitro. PMID:16369768

Song, Guo-Qing; Sink, K C

2006-03-01

164

Agrobacterium-mediated transformation of finger millet (Eleusine coracana (L.) Gaertn.) using shoot apex explants.  

PubMed

A new Agrobacterium-mediated transformation system was developed for finger millet using shoot apex explants. The Agrobacterium strain LBA4404 harboring binary vector pCAMBIA1301, which contained hygromycin phosphotransferase (hptII) as selectable marker gene and ?-glucuronidase (GUS) as reporter gene, was used for optimization of transformation conditions. Two finger millet genotypes, GPU 45 and CO 14, were used in this study. The optimal conditions for the Agrobacterium-mediated transformation of finger millet were found to be the co-cultivation of explants obtained on the 16th day after callus induction (DACI), exposure of explants for 30 min to agrobacterial inoculum and 3 days of co-cultivation on filter paper placed on medium supplemented with 100 ?M acetosyringone (AS). Addition of 100 ?M L: -cysteine in the selection medium enhanced the frequency of transformation and transgenic plant recovery. Both finger millet genotypes were transformed by Agrobacterium. A frequency of 19% transient expression with 3.8% stable transformation was achieved in genotype GPU 45 using optimal conditions. Five stably transformed plants were fully characterized by Southern blot analysis. A segregation analysis was also performed in four R(1) progenies, which showed normal Mendelian pattern of transgene segregation. The inheritance of transgenes in R(1) progenies was also confirmed by Southern blot analysis. This is the first report on Agrobacterium-mediated transformation of finger millet. This study underpins the introduction of numerous agronomically important genes into the genome of finger millet in the future. PMID:21584677

Ceasar, S Antony; Ignacimuthu, S

2011-09-01

165

Development of Efficient Plant Regeneration and Transformation System for Impatiens Using Agrobacterium tumefaciens and Multiple Bud Cultures as Explants  

PubMed Central

Background Impatiens (Impatiens walleriana) is a top selling floriculture crop. The potential for genetic transformation of Impatiens to introduce novel flower colors or virus resistance has been limited by its general recalcitrance to tissue culture and transformation manipulations. We have established a regeneration and transformation system for Impatiens that provides new alternatives to genetic improvement of this crop. Results In a first step towards the development of transgenic INSV-resistant Impatiens, we developed an efficient plant regeneration system using hypocotyl segments containing cotyledonary nodes as explants. With this regeneration system, 80% of explants produced an average of 32.3 elongated shoots per initial explant plated, with up to 167 elongated shoots produced per explant. Rooting efficiency was high, and 100% of shoots produced roots within 12 days under optimal conditions, allowing plant regeneration within approximately 8 weeks. Using this regeneration system, we developed an efficient Agrobacterium-mediated Impatiens transformation method using in vitro multiple bud cultures as explants and a binary plasmid (pHB2892) bearing gfp and nptII genes. Transgenic Impatiens plants, with a frequency up to 58.9%, were obtained within 12 to 16 weeks from inoculation to transfer of transgenic plants to soil. Transgenic plants were confirmed by Southern blot, phenotypic assays and T1 segregation analysis. Transgene expression was observed in leaves, stems, roots, flowers, and fruit. The transgenic plants were fertile and phenotypically normal. Conclusion We report the development of a simple and efficient Agrobacterium-mediated transformation system for Impatiens. To the best of our knowledge, there have been no reports of Agrobacterium-mediated transformation of Impatiens with experimental evidence of stable integration of T-DNA and of Agrobacterium-mediated transformation method for plants using in vitro maintained multiple bud cultures as explants. This transformation system has the advantages of 1) efficient, simple and rapid regeneration and transformation (with no need for sterilization or a greenhouse to grow stock plants), 2) flexibility (available all the time) for in vitro manipulation, 3) uniform and desirable green tissue explants for both nuclear and plastid transformation using Agrobacterium-mediated and biolistics methods, 4) no somaclonal variation and 5) resolution of necrosis of Agrobacterium-inoculated tissues. PMID:20696066

2010-01-01

166

Genetic complementation analysis of the Agrobacterium tumefaciens virB operon: virB2 through virB11 are essential virulence genes.  

PubMed Central

The Agrobacterium tumefaciens virB gene products are proposed to assemble into a transport system capable of exporting complexes of DNA and protein across the bacterial envelope en route to plant cells. Nonpolar null mutations were constructed in each of the 11 virB genes of the A. tumefaciens pTiA6NC plasmid. In tumorigenicity assays, delta virB1 mutants exhibited severely attenuated virulence and delta virB2 through delta virB11 mutants exhibited avirulence. NdeI restriction sites introduced at the predicted translational start sites of the virB genes were used to subclone each of the virB genes downstream of the lacZ or virB promoter on broad-host-range plasmids. virB gene expression plasmids were used to define promoter and general sequence requirements for genetic complementation of the deletion mutations. Whereas virB1 and virB2 complemented delta virB1 and delta virB2, respectively, only when expressed in trans from the virB promoter, virB3 through virB11 complemented the corresponding deletion mutations when expressed in trans from either the lacZ or virB promoter. Several virB genes required additional upstream or downstream sequences for complementation: (i) virB2 complemented the delta virB2 mutation only when the complementing plasmid coexpressed virB1 and virB2, (ii) virB6 and virB9 complemented the delta virB6 and delta virB9 mutations only when the complementing plasmids carried at most 55 and 230 bp of sequences residing 5' of these genes, respectively, and (iii) virB7 and virB8 complemented the delta virB7 and delta virB8 mutations only when the complementing plasmid coexpressed virB7 and virB8. These studies established that virB1 is an accessory virulence determinant and virB2 through virB11 are absolutely essential for the A. tumefaciens infection process. Images PMID:8206843

Berger, B R; Christie, P J

1994-01-01

167

Synthesis of methylerythritol phosphate analogues and their evaluation as alternate substrates for IspDF and IspE from Agrobacterium tumefaciens.  

PubMed

The methylerythritol phosphate biosynthetic pathway, found in most Bacteria, some parasitic protists, and plant chloroplasts, converts D-glyceraldehyde phosphate and pyruvate to isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), where it intersects with the mevalonate pathway found in some Bacteria, Archaea, and Eukarya, including the cytosol of plants. D-3-Methylerythritol-4-phosphate (MEP), the first pathway-specific intermediate in the pathway, is converted to IPP and DMAPP by the consecutive action of the IspD-H proteins. We synthesized five D-MEP analogues-D-erythritol-4-phosphate (EP), D-3-methylthrietol-4-phosphate (MTP), D-3-ethylerythritol-4-phosphate (EEP), D-1-amino-3-methylerythritol-4-phosphate (NMEP), and D-3-methylerythritol-4-thiolophosphate (MESP)-and studied their ability to function as alternative substrates for the reactions catalyzed by the IspDF fusion and IspE proteins from Agrobacterium tumefaciens, which covert MEP to the corresponding eight-membered cyclic diphosphate. All of the analogues, except MTP, and their products were substrates for the three consecutive enzymes. PMID:25184438

Krasutsky, Sergiy G; Urbansky, Marek; Davis, Chad E; Lherbet, Christian; Coates, Robert M; Poulter, C Dale

2014-10-01

168

Delineation of polar localization domains of Agrobacterium tumefaciens type IV secretion apparatus proteins VirB4 and VirB11  

PubMed Central

Agrobacterium tumefaciens transfers DNA and proteins to a plant cell through a type IV secretion apparatus assembled by the VirB proteins. All VirB proteins localized to a cell pole, although these conclusions are in dispute. To study subcellular location of the VirB proteins and to identify determinants of their subcellular location, we tagged two proteins, VirB4 and VirB11, with the visual marker green fluorescent protein (GFP) and studied localization of the fusion proteins by epifluorescence microscopy. Both GFP-VirB4 and GFP-VirB11 fusions localized to a single cell pole. GFP-VirB11 was also functional in DNA transfer. To identify the polar localization domains (PLDs) of VirB4 and VirB11, we analyzed fusions of GFP with smaller segments of the two proteins. Two noncontiguous regions in VirB4, residues 236–470 and 592–789, contain PLDs. The VirB11 PLD mapped to a 69 amino acid segment, residues 149–217, in the central region of the protein. These domains are probably involved in interactions that target the two proteins to a cell pole. PMID:25220247

Das, Aditi; Das, Anath

2014-01-01

169

Hypervirulence of Agrobacterium tumefaciens A281 is encoded in a region of pTiBo542 outside of T-DNA  

SciTech Connect

A binary-vectory strategy was used to study the hypervirulence of Agrobacterium tumefaciens A281, an L,L-succinamopine strain. Strain A281 is hypervirulent on several solanaceous plants. Plasmids were constructed (pCS65 and pCS277) carrying either the transferred DNA (T-DNA) or the remainder of the tumor-inducing (Ti) plasmid (pEHA101) from this strain and tested each of these constructs were tested in trans with complementary each of regions from heterologous Ti plasmids. Hypervirulence on tobacco could be reconstructed in a bipartite strain with the L,L-succinamopine T-DNA and the vir region on separate plasmids. pEHA101 was able to complement octopine T-DNA to hypervirulence on tobacco and tomato plants. Nopaline T-DNA was complemented better on tomato plants by pEHA101 than it was by its own nopaline vir region, but not to hypervirulence. L,L-Succinamopine T-DNA could not be complemented to hypervirulence on tobacco and tomato plants with either heterologous vir region. From these results the authors suggest that the hypervirulence of strain A281 is due to non-T-DNA sequences on the Ti plasmid.

Hood, E.E.; Helmer, G.L.; Fraley, R.T.; Chilton, M.D.

1986-12-01

170

The prokaryotic Cys2His2 zinc-finger adopts a novel fold as revealed by the NMR structure of Agrobacterium tumefaciens Ros DNA-binding domain  

PubMed Central

The first putative prokaryotic Cys2His2 zinc-finger domain has been identified in the transcriptional regulator Ros from Agrobacterium tumefaciens, indicating that the Cys2His2 zinc-finger domain, originally thought to be confined to the eukaryotic kingdom, could be widespread throughout the living kingdom from eukaryotic, both animal and plant, to prokaryotic. In this article we report the NMR solution structure of Ros DNA-binding domain (Ros87), providing 79 structural characterization of a prokaryotic Cys2His2 zinc-finger domain. The NMR structure of Ros87 shows that the putative prokaryotic Cys2His2 zinc-finger sequence is indeed part of a significantly larger zinc-binding globular domain that possesses a novel protein fold very different from the classical fold reported for the eukaryotic classical zinc-finger. The Ros87 globular domain consists of 58 aa (residues 9–66), is arranged in a ????? topology, and is stabilized by an extensive 15-residue hydrophobic core. A backbone dynamics study of Ros87, based on 15N R1, 15N R2, and heteronuclear 15N-{1H}-NOE measurements, has further confirmed that the globular domain is uniformly rigid and flanked by two flexible tails. Mapping of the amino acids necessary for the DNA binding onto Ros87 structure reveals the protein surface involved in the DNA recognition mechanism of this new zinc-binding protein domain. PMID:17956987

Malgieri, Gaetano; Russo, Luigi; Esposito, Sabrina; Baglivo, Ilaria; Zaccaro, Laura; Pedone, Emilia M.; Di Blasio, Benedetto; Isernia, Carla; Pedone, Paolo V.; Fattorusso, Roberto

2007-01-01

171

The thuEFGKAB Operon of Rhizobia and Agrobacterium tumefaciens Codes for Transport of Trehalose, Maltitol, and Isomers of Sucrose and Their Assimilation through the Formation of Their 3-Keto Derivatives  

PubMed Central

The thu operon (thuEFGKAB) in Sinorhizobium meliloti codes for transport and utilization functions of the disaccharide trehalose. Sequenced genomes of members of the Rhizobiaceae reveal that some rhizobia and Agrobacterium possess the entire thu operon in similar organizations and that Mesorhizobium loti MAFF303099 lacks the transport (thuEFGK) genes. In this study, we show that this operon is dedicated to the transport and assimilation of maltitol and isomers of sucrose (leucrose, palatinose, and trehalulose) in addition to trehalulose, not only in S. meliloti but also in Agrobacterium tumefaciens. By using genetic complementation, we show that the thuAB genes of S. meliloti, M. loti, and A. tumefaciens are functionally equivalent. Further, we provide both genetic and biochemical evidence to show that these bacteria assimilate these disaccharides by converting them to their respective 3-keto derivatives and that the thuAB genes code for this ketodisaccharide-forming enzyme(s). Formation of 3-ketotrehalose in real time in live S. meliloti is shown through Raman spectroscopy. The presence of an additional ketodisaccharide-forming pathway(s) in A. tumefaciens is also indicated. To our knowledge, this is the first report to identify the genes that code for the conversion of disaccharides to their 3-ketodisaccharide derivatives in any organism. PMID:23772075

Ampomah, Osei Yaw; Avetisyan, Anna; Hansen, Espen; Svenson, Johan; Huser, Thomas; Bhuvaneswari, T. V.

2013-01-01

172

A homolog of the Rhizobium meliloti nitrogen fixation gene fixN is involved in the production of a microaerobically induced oxidase activity in the phytopathogenic bacterium Agrobacterium tumefaciens.  

PubMed

Hybridization analysis using the Rhizobium meliloti nitrogen fixation gene fixN as a probe revealed the presence of a homologous DNA region in the phytopathogenic bacterium Agrobacterium tumefaciens. Hybridization signals were also detected with total DNAs of Rhizobium leguminosarum bv. phaseoli, Rhodobacter capsulatus and Escherichia coli, but not those of Xanthomonas campestris pv. campestris and Pseudomonas putida. The hybridizing fragment from A. tumefaciens was cloned and sequenced. The predicted gene product of one of the two open reading frames identified on the sequenced fragment shows homology to FixN of different Rhizobiaceae as well as a low but significant similarity to subunit I of heme copper oxidases from various bacteria. The presence of five strictly conserved histidine residues previously implicated in forming ligands to heme and CuB in oxidases and the predicted membrane topology provide evidence that the A. tumefaciens fixN-like gene product is a component of the heme copper oxidase superfamily. The incomplete open reading frame starting only 8 nucleotides downstream of the fixN-like gene exhibits homology to Rhizobium fixO. Using an uidA (GUS) gene fusion it could be shown that the A. tumefaciens fixN-like gene is preferentially expressed under microaerobic conditions. Expression of the uidA fusion is abolished in R. meliloti fixJ and fixK mutants, indicating that an Fnr-like protein is involved in transcriptional regulation of the fixN-like gene in A. tumefaciens. The presence of an upstream DNA sequence motif identical to the Fnr-consensus binding site (anaerobox) further supports this hypothesis. A. tumefaciens mutated in the fixN-like gene shows decreased TMPD-specific oxidase activity under microaerobic conditions, indicating that the fixN-like gene or operon codes for proteins involved in respiration under reduced oxygen availability. PMID:7753030

Schlüter, A; Rüberg, S; Krämer, M; Weidner, S; Priefer, U B

1995-04-20

173

Agrobacterium-mediated genetic transformation and development of herbicide-resistant sugarcane (Saccharum species hybrids) using axillary buds.  

PubMed

Direct regeneration from explants without an intervening callus phase has several advantages, including production of true type progenies. Axillary bud explants from 6-month-old sugarcane cultivars Co92061 and Co671 were co-cultivated with Agrobacterium strains LBA4404 and EHA105 that harboured a binary vector pGA492 carrying neomycin phosphotransferase II, phosphinothricin acetyltransferase (bar) and an intron containing beta-glucuronidase (gus-intron) genes in the T-DNA region. A comparison of kanamycin, geneticin and phosphinothricin (PPT) selection showed that PPT (5.0 mg l(-1)) was the most effective selection agent for axillary bud transformation. Repeated proliferation of shoots in the selection medium eliminated chimeric transformants. Transgenic plants were generated in three different steps: (1) production of putative primary transgenic shoots in Murashige-Skoog (MS) liquid medium with 3.0 mg l(-1) 6-benzyladenine (BA) and 5.0 mg l(-1) PPT, (2) production of secondary transgenic shoots from the primary transgenic shoots by growing them in MS liquid medium with 2.0 mg l(-1) BA, 1.0 mg l(-1) kinetin (Kin), 0.5 mg l(-1) alpha-napthaleneacetic acid (NAA) and 5.0 mg l(-1) PPT for 3 weeks, followed by five more cycles of shoot proliferation and selection under same conditions, and (3) rooting of transgenic shoots on half-strength MS liquid medium with 0.5 mg l(-1) NAA and 5.0 mg l(-1) PPT. About 90% of the regenerated shoots rooted and 80% of them survived during acclimatisation in greenhouse. Transformation was confirmed by a histochemical beta-glucuronidase (GUS) assay and PCR amplification of the bar gene. Southern blot analysis indicated integration of the bar gene in two genomic locations in the majority of transformants. Transformation efficiency was influenced by the co-cultivation period, addition of the phenolic compound acetosyringone and the Agrobacterium strain. A 3-day co-cultivation with 50 micro M acetosyringone considerably increased the transformation efficiency. Agrobacterium strain EHA105 was more effective, producing twice the number of transgenic shoots than strain LBA4404 in both Co92061 and Co671 cultivars. Depending on the variety, 50-60% of the transgenic plants sprayed with BASTA (60 g l(-1) glufosinate) grew without any herbicide damage under greenhouse conditions. These results show that, with this protocol, generation and multiplication of transgenic shoots can be achieved in about 5 months with transformation efficiencies as high as 50%. PMID:15133712

Manickavasagam, M; Ganapathi, A; Anbazhagan, V R; Sudhakar, B; Selvaraj, N; Vasudevan, A; Kasthurirengan, S

2004-09-01

174

Optimization and Utilization of Agrobacterium-mediated Transient Protein Production in Nicotiana  

PubMed Central

Agrobacterium-mediated transient protein production in plants is a promising approach to produce vaccine antigens and therapeutic proteins within a short period of time. However, this technology is only just beginning to be applied to large-scale production as many technological obstacles to scale up are now being overcome. Here, we demonstrate a simple and reproducible method for industrial-scale transient protein production based on vacuum infiltration of Nicotiana plants with Agrobacteria carrying launch vectors. Optimization of Agrobacterium cultivation in AB medium allows direct dilution of the bacterial culture in Milli-Q water, simplifying the infiltration process. Among three tested species of Nicotiana, N. excelsiana (N. benthamiana × N. excelsior) was selected as the most promising host due to the ease of infiltration, high level of reporter protein production, and about two-fold higher biomass production under controlled environmental conditions. Induction of Agrobacterium harboring pBID4-GFP (Tobacco mosaic virus-based) using chemicals such as acetosyringone and monosaccharide had no effect on the protein production level. Infiltrating plant under 50 to 100 mbar for 30 or 60 sec resulted in about 95% infiltration of plant leaf tissues. Infiltration with Agrobacterium laboratory strain GV3101 showed the highest protein production compared to Agrobacteria laboratory strains LBA4404 and C58C1 and wild-type Agrobacteria strains at6, at10, at77 and A4. Co-expression of a viral RNA silencing suppressor, p23 or p19, in N. benthamiana resulted in earlier accumulation and increased production (15-25%) of target protein (influenza virus hemagglutinin). PMID:24796351

Shamloul, Moneim; Trusa, Jason; Mett, Vadim; Yusibov, Vidadi

2014-01-01

175

Optimization and utilization of Agrobacterium-mediated transient protein production in Nicotiana.  

PubMed

Agrobacterium-mediated transient protein production in plants is a promising approach to produce vaccine antigens and therapeutic proteins within a short period of time. However, this technology is only just beginning to be applied to large-scale production as many technological obstacles to scale up are now being overcome. Here, we demonstrate a simple and reproducible method for industrial-scale transient protein production based on vacuum infiltration of Nicotiana plants with Agrobacteria carrying launch vectors. Optimization of Agrobacterium cultivation in AB medium allows direct dilution of the bacterial culture in Milli-Q water, simplifying the infiltration process. Among three tested species of Nicotiana, N. excelsiana (N. benthamiana × N. excelsior) was selected as the most promising host due to the ease of infiltration, high level of reporter protein production, and about two-fold higher biomass production under controlled environmental conditions. Induction of Agrobacterium harboring pBID4-GFP (Tobacco mosaic virus-based) using chemicals such as acetosyringone and monosaccharide had no effect on the protein production level. Infiltrating plant under 50 to 100 mbar for 30 or 60 sec resulted in about 95% infiltration of plant leaf tissues. Infiltration with Agrobacterium laboratory strain GV3101 showed the highest protein production compared to Agrobacteria laboratory strains LBA4404 and C58C1 and wild-type Agrobacteria strains at6, at10, at77 and A4. Co-expression of a viral RNA silencing suppressor, p23 or p19, in N. benthamiana resulted in earlier accumulation and increased production (15-25%) of target protein (influenza virus hemagglutinin). PMID:24796351

Shamloul, Moneim; Trusa, Jason; Mett, Vadim; Yusibov, Vidadi

2014-01-01

176

Cytokinin production by Agrobacterium and Pseudomonas spp.  

PubMed Central

The production of cytokinins by plant-associated bacteria was examined by radioimmunoassay. Strains producing trans-zeatin were identified in the genera Agrobacterium and Pseudomonas. Agrobacterium tumefaciens strains containing nopaline tumor-inducing plasmids, A. tumefaciens Lippia isolates, and Agrobacterium rhizogenes strains produced trans-zeatin in culture at 0.5 to 44 micrograms/liter. Pseudomonas solanacearum and Pseudomonas syringae pv. savastanoi produced trans-zeatin at levels of up to 1 mg/liter. In vitro cytokinin biosynthetic activity was measured for representative strains and was found to correlate with trans-zeatin production. The genetic locus for trans-zeatin secretion (tzs) was cloned from four strains: A. tumefaciens T37, A. rhizogenes A4, P. solanacearum K60, and P. syringae pv. savastanoi 1006. Southern blot analysis showed substantial homology of the Agrobacterium tzs genes to each other but not to the two Pseudomonas genes. Images PMID:3624204

Akiyoshi, D E; Regier, D A; Gordon, M P

1987-01-01

177

High frequency regeneration via direct somatic embryogenesis and efficient Agrobacterium-mediated genetic transformation of tobacco.  

PubMed

A direct somatic embryogenesis protocol was developed for four cultivars of Nicotiana species, by using leaf disc as an explant. Direct somatic embryogenesis of Nicotiana by using BAP and IAA has not been investigated so far. This method does not require formation of callus tissues which leads to somaclonal variations. The frequency of somatic embryogenesis was strongly influenced by the plant growth hormones. The somatic embryos developing directly from explant tissue were noticed after 6 d of culture. Somatic embryogenesis of a high frequency (87-96%) was observed in cultures of the all four genotypes (Nicotiana tabacum, N. benthamiyana, N. xanthi, N. t cv petihavana). The results showed that the best medium for direct somatic embryogenesis was MS supplemented with 2.5 mg/l, 0.2 mg/l IAA and 2% sucrose. Subculture of somatic embryos onto hormone free MS medium resulted in their conversion into plants for all genotypes. About 95% of the regenerated somatic embryos germinated into complete plantlets. The plants showed morphological and growth characteristics similar to those of seed-derived plants. Explants were transformed using Agrobacterium tumifacious LBA4404 plasmid pCAMBIA1301 harboring the GUS gene. The regenerated transgenic plants were confirmed by PCR analysis and histochemical GUS assay. The transformation efficiency obtained by using the Agrobacterium- mediated transformation was more than 95%. This method takes 6 wk to accomplish complete transgenic plants through direct somatic embryogenesis. The transgenic plantlets were acclimatized successfully with 98% survival in greenhouse and they showed normal morphological characteristics and were fertile. The regeneration and transformation method described herein is very simple, highly efficient and fast for the introduction of any foreign gene directly in tobacco through direct somatic embryogenesis. PMID:23518589

Pathi, Krishna Mohan; Tula, Suresh; Tuteja, Narendra

2013-06-01

178

Transformation of Brassica napus and Brassica oleracea Using Agrobacterium tumefaciens and the Expression of the bar and neo Genes in the Transgenic Plants  

PubMed Central

An efficient and largely genotype-independent transformation method for Brassica napus and Brassica oleracea was established based on neo or bar as selectable marker genes. Hypocotyl explants of Brassica napus and Brassica oleracea cultivars were infected with Agrobacterium strains containing chimeric neo and bar genes. The use of AgNO3 was a prerequisite for efficient shoot regeneration under selective conditions. Vitrification was avoided by decreasing the water potential of the medium, by decreasing the relative humidity in the tissue culture vessel, and by lowering the cytokinin concentration. In this way, rooted transformed shoots were obtained with a 30% efficiency in 9 to 12 weeks. Southern blottings and genetic analysis of S1-progeny showed that the transformants contained on average between one and three copies of the chimeric genes. A wide range of expression levels of the chimeric genes was observed among independent transformants. Up to 25% of the transformants showed no detectable phosphinotricin acetyltransferase or neomycin phosphotransferase II enzyme activities although Southern blottings demonstrated that these plants were indeed transformed. Images Figure 1 Figure 2 PMID:16667089

De Block, Marc; De Brouwer, Dirk; Tenning, Paul

1989-01-01

179

Plant–Agrobacterium interaction mediated by ethylene and super-Agrobacterium conferring efficient gene transfer  

PubMed Central

Agrobacterium tumefaciens has a unique ability to transfer genes into plant genomes. This ability has been utilized for plant genetic engineering. However, the efficiency is not sufficient for all plant species. Several studies have shown that ethylene decreased the Agrobacterium-mediated transformation frequency. Thus, A. tumefaciens with an ability to suppress ethylene evolution would increase the efficiency of Agrobacterium-mediated transformation. Some studies showed that plant growth-promoting rhizobacteria (PGPR) can reduce ethylene levels in plants through 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase, which cleaves the ethylene precursor ACC into ?-ketobutyrate and ammonia, resulting in reduced ethylene production. The whole genome sequence data showed that A. tumefaciens does not possess an ACC deaminase gene in its genome. Therefore, providing ACC deaminase activity to the bacteria would improve gene transfer. As expected, A. tumefaciens with ACC deaminase activity, designated as super-Agrobacterium, could suppress ethylene evolution and increase the gene transfer efficiency in several plant species. In this review, we summarize plant–Agrobacterium interactions and their applications for improving Agrobacterium-mediated genetic engineering techniques via super-Agrobacterium. PMID:25520733

Nonaka, Satoko; Ezura, Hiroshi

2014-01-01

180

Agrobacterium tumefaciens type IV secretion protein VirB3 is an inner membrane protein and requires VirB4, VirB7, and VirB8 for stabilization.  

PubMed

Agrobacterium tumefaciens VirB proteins assemble a type IV secretion apparatus and a T-pilus for secretion of DNA and proteins into plant cells. The pilin-like protein VirB3, a membrane protein of unknown topology, is required for the assembly of the T-pilus and for T-DNA secretion. Using PhoA and green fluorescent protein (GFP) as periplasmic and cytoplasmic reporters, respectively, we demonstrate that VirB3 contains two membrane-spanning domains and that both the N and C termini of the protein reside in the cytoplasm. Fusion proteins with GFP at the N or C terminus of VirB3 were fluorescent and, like VirB3, localized to a cell pole. Biochemical fractionation studies demonstrated that VirB3 proteins encoded by three Ti plasmids, the octopine Ti plasmid pTiA6NC, the supervirulent plasmid pTiBo542, and the nopaline Ti plasmid pTiC58, are inner membrane proteins and that VirB4 has no effect on membrane localization of pTiA6NC-encoded VirB3 (pTiA6NC VirB3). The pTiA6NC and pTiBo542 VirB2 pilins, like VirB3, localized to the inner membrane. The pTiC58 VirB4 protein was earlier found to be essential for stabilization of VirB3. Stabilization of pTiA6NC VirB3 requires not only VirB4 but also two additional VirB proteins, VirB7 and VirB8. A binary interaction between VirB3 and VirB4/VirB7/VirB8 is not sufficient for VirB3 stabilization. We hypothesize that bacteria use selective proteolysis as a mechanism to prevent assembly of unproductive precursor complexes under conditions that do not favor assembly of large macromolecular structures. PMID:20348257

Mossey, Pamela; Hudacek, Andrew; Das, Anath

2010-06-01

181

Cloning and Expression of TNF Related Apoptosis Inducing Ligand in Nicotiana tabacum  

PubMed Central

Molecular farming has been considered as a secure and economical approach for production of biopharmaceuticals. Human TNF Related Apoptosis Inducing Ligand (TRAIL) as a promising biopharmaceutical candidate has been produced in different expression hosts. However, little attention has been paid to molecular farming of the TRAIL in spite of numerous advantages of plant expression systems. Therefore, in this study the cytoplasmic production of the TRAIL was tackled in Nicotiana tabacum using Agrobacterium tumefaciens LBA 4404. Initially, the desired coding sequence was obtained using PCR technique on the constructed human cDNA library. Afterward, the necessary requirements for expression of the TRAIL in plant cell system were provided through sub-cloning into 35S-CaMV (Cauliflower Mosaic Virus) helper and final 0179-pGreen expression vectors. Then, the final TRAIL-pGreen expression vector was cloned into A. tumefaciens LBA 4404. Subsequently, the N. tabacum cells were transformed through co-culture method and expression of the TRAIL was confirmed by western blot analysis. Finally, the recombinant TRAIL was extracted through chromatographic technique and biological activity was evaluated through MTT assay (Methylthiazol Tetrazolium Assay). The result of western blot analysis indicated that only monomer and oxidized dimer forms of the TRAIL can be extracted from the N. tabacum cells. Moreover, the lack of trimeric assembly of the extracted TRAIL diminished its biological activity in sensitive A549 cell line. In conclusion, although N. tabacum cells can successfully produce the TRAIL, proper assembly and functionality of the TRAIL were unfavorable. PMID:25561925

Heidari, Hamid Reza; Bandehpour, Mojgan; Vahidi, Hossein; Barar, Jaleh; Kazemi, Bahram; Naderi-Manesh, Hossein

2015-01-01

182

Agrobacterium-mediated infectivity of cloned digitaria streak virus DNA.  

PubMed

A monomeric clone of double-stranded DNA synthesized in vitro DNA of the geminivirus Digitaria streak (DSV) was subcloned as a tandem dimeric unit into a binary vector of Agrobacterium tumefaciens, creating a plasmid pDS2. Inoculation of digitaria sanguinalis with A. tumefaciens carrying pDS2 resulted in viral infection. The symptoms, virus particles, and DNA forms obtained were indistinguishable from those of a natural DSV infection of D. sanguinalis. Inoculations have also induced infections in Zea mays and Avena sativa. The sequence of the Agrobacterium-mediated infectious clone of DSV has been determined. PMID:3341112

Donson, J; Gunn, H V; Woolston, C J; Pinner, M S; Boulton, M I; Mullineaux, P M; Davies, J W

1988-01-01

183

Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana  

Microsoft Academic Search

Summary The Agrobacterium vacuum infiltration method has made it possible to transform Arabidopsis thaliana without plant tissue culture or regeneration. In the present study, this method was evaluated and a substantially modified trans- formation method was developed. The labor-intensive vacuum infiltration process was eliminated in favor of simple dipping of developing floral tissues into a solution containing Agrobacterium tumefaciens, 5%

Steven J. Clough; Andrew F. Bent

1999-01-01

184

Transgenic plant production mediated by Agrobacterium in Indica rice  

Microsoft Academic Search

A reproducible system has been developed for the production of transgenic plants in indica rice using Agrobacterium-mediated gene transfer. Three-week-old scutella calli served as an excellent starting material. These were infected with an Agrobacterium tumefaciens strain EHA101 carrying a plasmid pIG121Hm containing genes for ß-glucuronidase (GUS) and hygromycin resistnace (HygR). Hygromycin (50 mg\\/l) was used as a selectable agent. Inclusion

Hamid Rashid; Shuuji Yokoi; Kinya Toriyama; Kokichi Hinata

1996-01-01

185

Agrobacterium-mediated genetic transformation of Prunus salicina  

Technology Transfer Automated Retrieval System (TEKTRAN)

We report Agrobacterium tumefaciens-mediated transformation from hypocotyls slices of two Prunus salicina varieties, 'Angeleno' and 'Larry Anne', using a modification of the technique previously described for P. domestica. Regeneration rates on thidiazuron (TDZ) and indole-3-butyric acid (IBA) supp...

186

Characterization of oncogene-silenced transgenic plants: implications for Agrobacterium biology and post-transcriptional gene silencing  

Microsoft Academic Search

SUMMARY Agrobacterium tumefaciens tumorigenesis is initiated by the horizontal transfer of a suite of oncogenes that alter hormone synthesis and sensitivity in infected plant cells. Transgenic plants silenced for the iaaM and ipt oncogenes are highly recalcitrant to tumorigenesis, and present a unique resource to elucidate funda- mental questions related to Agrobacterium biology and post- transcriptional gene silencing (PTGS). The

M. A. Escobar; E. L. Civerolo; V. S. Polito; K. A. Pinney; A. M. Dandekar

2003-01-01

187

UNIT 3D.3: PHENOTYPIC ANALYSES OF AGROBACTERIUM  

PubMed Central

Agrobacterium species are plant-associated relatives of the rhizobia. Several species cause plant diseases such as crown gall and hairy root, although there are also avirulent species. A. tumefaciens is the most intensively studied species and causes crown gall, a neoplastic disease that occurs on a variety of plants. Virulence is specified by large plasmids, and in the case of A. tumefaciens this is called the Ti (tumor-inducing) plasmid. During pathogenesis virulent agrobacteria copy a segment of the Ti plasmid and transfer it to the plant, where it subsequently integrates into the plant genome, and expresses genes that result in the disease symptoms. A. tumefaciens has been used extensively as a plant genetic engineering tool and is also a model microorganism that has been well studied for host-microbe associations, horizontal gene transfer, cell-cell communication, and biofilm formation. This unit describes standard protocols for simple phenotypic characterizations of A. tumefaciens. PMID:22549164

Morton, Elise R.; Fuqua, Clay

2012-01-01

188

Agrobacterium -mediated transformation of quaking aspen ( Populus tremuloides ) and regeneration of transgenic plants  

Microsoft Academic Search

Agrobacterium-mediated gene transformation of Populus tremuloides Michx was accomplished by co-cultivation of leaf disks excised from greenhouse plants with Agrobacterium tumefaciens containing a binary Ti-plasmid vector harboring chimeric neomycin phosphotransferase (NPT II) and ß-glucuronidase (GUS) genes. Shoot regeneration in the presence of kanamycin was achieved when thidiazuron (TDZ) was used as a plant growth regulator. Transformation was verified by amplification

Chung-Jui Tsai; Gopi K. Podila; Vincent L. Chiang

1994-01-01

189

Agrobacterium -mediated transformation of Fraxinus pennsylvanica hypocotyls and plant regeneration  

Microsoft Academic Search

A genetic transformation protocol for green ash (Fraxinus pennsylvanica) hypocotyl explants was developed. Green ash hypocotyls were transformed using Agrobacterium tumefaciens strain EHA105 harboring binary vector pq35GR containing the neomycin phosphotransferase (nptII) and ?-glucuronidase (GUS) fusion gene, and an enhanced green fluorescent protein gene. Pre-cultured hypocotyl explants were\\u000a transformed in the presence of 100 ?M acetosyringone using 90 s sonication plus 10 min

Ningxia Du; Paula M. Pijut

2009-01-01

190

Horticultural characteristics of transgenic tobacco expressing the rolC gene from Agrobacterium rhizogenes  

Microsoft Academic Search

Wisconsin 38 tobacco (Nicotiana tabacum L.) leaf discs were transformed with the disarmed Agrobacterium tumefaciens strain EHA 101 carrying the rolC gene from A. rhizogenes and NPT II and GUS genes. Shoots that regenerated on kanamycin-containing medium were confirmed as transgenic through GUS assays, polymerase chain reaction (PCR), Southern blot analyses, and transmission of the foreign genes through the sexual

R. Scorza; T. W. Zimmerman; J. M. Cordts; K. J. Footen; M. Ravelonandro

1994-01-01

191

Transformation of Pakchoi (Brassica rapa L. ssp. chinensis) by Agrobacterium infiltration  

Microsoft Academic Search

Transgenic pakchoi (Brassica rapa L. ssp. chinensis) plants were obtained in the progeny of plants infiltrated by an Agrobacterium tumefaciens strain carrying a gene for resistance to the herbicide phosphinotricin (Basta). Genetic analysis demonstrates the transmission of the herbicide resistant trait to the progeny. Molecular analyses show that the transgene was inserted in the plant genome and expressed. This work

Cao Ming Qing; Liu Fan; Yao Lei; David Bouchez; Colette Tourneur; Li Yan; Christophe Robaglia

2000-01-01

192

Agrobacterium-mediated maize transformation: immature embryos versus callus.  

PubMed

Transformation with Agrobacterium tumefaciens is the preferred method for delivery of transgenes into a wide range of plant species including maize. Optimized protocols for the Agrobacterium-mediated transformation of freshly isolated immature embryos and embryogenic Type I callus derived from plant seedlings are described. These protocols are suitable for the transformation of a wide variety of corn genotypes including commercial inbred lines. Agrobacterium harboring a binary vector containing the neomycin phosphotransferase (nptII) or the glyphosate resistant 5-enolpyruvylshikimate-3-phosphate (EPSPS) as selectable marker genes and also the green fluorescence protein gene (gfp) have been used. GFP is a visual screening marker which allows tracking of transformation during different selection and regeneration steps. The described protocols provide double digit transformation frequencies and can be routinely used for the production of a large numbers of transgenic plants. PMID:19378003

Sidorov, Vladimir; Duncan, David

2009-01-01

193

Meristem transformation of sunflower via Agrobacterium.  

PubMed

For transformation of sunflower (Helianthus annuus L. cv. Zebulon), shoot apical meristems were dissected from seeds and cocultivated with a disarmed Agrobacterium tumefaciens strain harboring a binary vector carrying genes encoding GUS- and NPTII-activity. The influence of the media conditions, the time of cocultivation and the stage of the developing seed on shoot development and meristem transformation was analysed. Transformants were selected by their ability to grow on kanamycin. Transformation was confirmed by assays for GUS and NPTII. GUS-positive shoots were rooted on rockwool and transferred to soil. Transformation of shoot meristem cells occurred at low frequencies. Chimaeric expression of the two genes was observed in transformed plants. Integration of the foreign DNA in the sunflower genome was confirmed with the polymerase chain reaction. PMID:24226429

Schrammeijer, B; Sijmons, P C; van den Elzen, P J; Hoekema, A

1990-07-01

194

Agrobacterium-mediated transformation of Ruta graveolens L.  

PubMed

Agrobacterium tumefaciens is used to develop a genetic transformation method for a medicinal plant Ruta graveolens. The direct plant regeneration strategy is preferred to callus line establishment. In vitro seedlings, 2- -to 3-wk-old, are used to excise hypocotyls and co-cultivated for 3 d with A. tumefaciens strain C58C1Rif containing plasmid pTDE4 harbouring neomycin phosphotransferase (npt II, kanamycin resistance) and beta-glucuronidase encoding genes. The Southern blot analysis has shown that 78% kanamycin resistant plants contain gene encoding beta-glucuronidase. The GUS histochemical assay shows that 67% transgenic plants exhibit the corresponding enzymatic activity. Routine transformation efficiency of R. graveolens L. is 11% and could reach up to 22%. Transgenic plants are grown in the greenhouse within 4 months after the initial seedlings. PMID:19521849

Lièvre, Karine; Tran, Thi Lê Minh; Doerper, Sébastien; Hehn, Alain; Lacoste, Paul; Thomasset, Brigitte; Bourgaud, Frédéric; Gontier, Eric

2009-01-01

195

Transformation of Vicia narbonensis via Agrobacterium-mediated gene transfer.  

PubMed

Shoot tips and epicotyl-segments of Vicia narbonensis were co-cultivated with Agrobacterium tumefaciens strain C58C1 pGV 3850 HPT, carrying a plasmid coding for hygromycin-phosphotransferase. On callus-induction medium containing 60 mg/l hygromycin for selection, approximately 18% of the explants produced hygromycin-resistant callus. After transfer to regeneration-medium these calluses produced hygromycin-resistant and nopaline-positive somatic embryos which could be regenerated to plantlets. The integration of the T-DNA into the plant genome was confirmed by Southern analysis. PMID:24220706

Pickardt, T; Meixner, M; Schade, V; Schieder, O

1991-02-01

196

Agrobacterium?-mediated transformation of embryogenic cell suspensions of the banana cultivar Rasthali (AAB)  

Microsoft Academic Search

A protocol was developed for establishing embryogenic suspension cultures from in vitro-grown, thin shoot-tip sections of\\u000a the banana cultivar Rasthali. The best medium for callus induction was an MS-based medium supplemented with 2?mg\\/l 2,4-D and\\u000a 0.2?mg\\/l zeatin. The callus was transferred to liquid medium to establish embryogenic cell suspensions. These cultures were\\u000a subsequently used for Agrobacterium-mediated transformation. The Agrobacterium tumefaciens

T. R. Ganapathi; N. S. Higgs; P. J. Balint-Kurti; C. J. Arntzen; G. D. May; J. M. Van Eck

2001-01-01

197

Nucleotide sequence of the T-DNA region from the A grobacterium tumefaciens octopine Ti plasmid pTi15955  

Microsoft Academic Search

The complete nucleotide sequence of the transferred region (T-DNA) of an octopine tumor inducing (Ti) plasmid fromAgrobacterium tumefaciens (pTi15955) has been determined. A total of 24 595 nucleotides extending approximately 900 bases to either side of the outermost, T-DNA boundaries was sequenced. Computer analysis of the sequenced portion of the Ti plasmid revealed that recognition sites for 72 restriction endonucleases

R. F. Barker; K. B. Idler; D. V. Thompson; J. D. Kemp

1983-01-01

198

Sri Lankan cassava mosaic virus replication associated protein (Rep) triggers transposition of IS426 in Agrobacterium.  

PubMed

We report a high rate of IS426 transposition in Agrobacterium tumefaciens in the presence of the Sri Lankan cassava mosaic virus (SLCMV) replication associated protein gene (Rep). Upon conjugal transfer of the binary plasmid pCam-SLCMV-Rep with the SLCMV Rep gene in the sense orientation under the transcriptional control of the Cauliflower mosaic virus (CaMV) 35S promoter into the A. tumefaciens vir helper strain EHA105, the binary plasmid size increased in all 15 transconjugants studied. Southern blot analysis of the transconjugants with the binary plasmid probe revealed that the 35S promoter and its proximal sequences in the T-DNA were rearranged. The rearranged sequences harboured the 1.3-kb IS426 element of A. tumefaciens. Conjugal mobilisation of the binary plasmid pCam-SLCMV-asRep, with the SLCMV Rep gene in antisense orientation, did not cause DNA rearrangement in EHA105. A mutated SLCMV Rep, in which a frame shift mutation caused retention of only 27 of the 351 amino acids, did not cause IS426 transposition in A. tumefaciens. These findings show that the multifunctional begomoviral Rep protein of SLCMV triggers transposition of IS426 in Agrobacterium. PMID:25135797

Resmi, Thulasi R; Nivedhitha, Sivarajan; Karthikeyan, Chockalingam; Veluthambi, Karuppannan

2014-11-01

199

Transformation of Morinda citrifolia via simple mature seed imbibition method.  

PubMed

Morinda citrifolia, is a valuable medicinal plant with a wide range of therapeutic properties and extensive transformation study on this plant has yet been known. Present study was conducted to establish a simple and reliable transformation protocol for M. citrifolia utilising Agrobacterium tumefaciens via direct seed exposure. In this study, the seeds were processed by tips clipping and dried and subsequently incubated in inoculation medium. Four different parameters during the incubation such as incubation period, bacterial density, temperature and binary vectors harbouring beta-glucuronidase (GUS) gene (pBI121 and pGSA1131), were tested to examine its effect on transformation efficiency. The leaves from the treated and germinated seedlings were analysed via Polymerase Chain Reaction (PCR), histochemical assay of the GUS gene and reverse transcription-PCR (RT-PCR). Results of the study showed that Agrobacterium strain LBA4404 with optical density of 1.0 and 2 h incubation period were optimum for M. citrifolia transformation. It was found that various co-cultivation temperatures tested and type of vector used did not affect the transformation efficiency. The highest transformation efficiency for M. citrifolia direct seed transformation harbouring pBI121 and pGSA1131 was determined to be 96.8% with 2 h co-cultivation treatment and 80.4% when using bacterial density of 1.0, respectively. The transformation method can be applied for future characterization study of M. citrifolia. PMID:24517006

Lee, J J; Ahmad, S; Roslan, H A

2013-12-15

200

The promoter of T L DNA gene 5 controls the tissue-specific expression of chimaeric genes carried by a novel type of Agrobacterium binary vector  

Microsoft Academic Search

A “plant gene vector cassette” to be used in combination with various Escherichia coli gene-cloning vectors was constructed. This cassette contains a replication and mobilization unit which allows it to be maintained and to be transferred back and forth between E. coli and Agrobacterium tumefaciens hosts provided these hosts contain plasmid RK2 replication and mobilization helper functions. The cassette also

Csaba Koncz; Jeff Schell

1986-01-01

201

Diversity of the limited-host range iaaH gene of Agrobacterium vitis strain Ag162.  

PubMed

The indole-3-acetamide hydrolase gene (iaaH) of the limited-host range strain AG162, a biotype III strain of Agrobacterium tumefaciens has been the subject of several studies and reviews, but its primary structure has not been previously reported. In the course of our own work we found that this gene hybridizes only weakly to a nucleic acid probe corresponding to the iaaH gene from a biotype I strain of A. tumefaciens. Analysis of the primary structure of the Ag162 iaaH gene revealed that it is diverse from biotype I iaaH genes and, surprisingly, also from the iaaH genes of previously characterized biotype III Agrobacterium strains. PMID:10520742

Oetiker, J H; Kato, A

1998-01-01

202

In planta transformation method for T-DNA transfer in orchids  

NASA Astrophysics Data System (ADS)

Transgenic plant technology is an efficient tool to study the function of gene(s) in plant. The most popular and widely used technique is Agrobacterium-mediated transformation in which cocultivation was done by immersing the plant tissues/organ in overnight bacterial cultured for about 30 minutes to one hour under in vitro condition. In this experiment, we developed more easier technique that omitted the in vitro step during cocultivation with Agrobacterium, namely in planta transformation method. Pollinaria (compact pollen mass of orchid) of Phalaenopsis amabilis and Spathoglottis plicata orchids were used as target explants that were immersed into bacterial culture for 30 minutes, then dried up the pollinaria, the transformed pollinaria was used to pollinate orchid flowers. The T-DNA used for this experiments were Ubipro?PaFT/A. tumefaciens GV3101 for P. amabilis and MeEF1?2 pro?GUS/ A. tumefaciens LBA 4404 for S.plicata. Seeds that were produced from pollinated flowers were grown onto 10 mg/l hygromicin containing NP (New Phalaenopsis) medium. The existance of transgene in putative transformant protocorm (developing orchid embryo) genome was confirmed using PCR with specific primers of either PaFT or GUS genes. Histochemical GUS assay was also performed to the putative transformants. The result showed that transformation frequencies were 2.1 % in P. amabilis, and 0,53% in S. plicata. These results indicates that in planta transformation method could be used for Agrobacterium-mediated genetic transformation, with advantage easier and more secure work from contaminants than that of the in vitro method.

Semiarti, Endang; Purwantoro, Aziz; Mercuriani, Ixora S.; Anggriasari, Anida M.; Jang, Seonghoe; Suhandono, Sony; Machida, Yasunori; Machida, Chiyoko

2014-03-01

203

Development of transgenic sweet potato with multiple virus resistance in South Africa (SA).  

PubMed

Multiple infections of Sweet potato feathery mottle virus (SPFMV), Sweet potato chlorotic stunt virus (SPCSV), Sweet potato virus G (SPVG) and Sweet potato mild mottle virus (SPMMV) cause a devastating synergistic disease complex of sweet potato (Ipomoea batatas Lam.) in KwaZulu-Natal, South Africa. In order to address the problem of multiple virus infections and synergism, this study aimed to develop transgenic sweet potato (cv. Blesbok) plants with broad virus resistance. Coat protein gene segments of SPFMV, SPCSV, SPVG and SPMMV were used to induce gene silencing in transgenic sweet potato. Transformation of apical tips of sweet potato cv. Blesbok was achieved by using Agrobacterium tumefaciens strain LBA4404 harboring the expression cassette. Polymerase chain reaction and Southern blot analyses showed integration of the transgenes occurred in six of the 24 putative transgenic plants and that all plants seemed to correspond to the same transformation event. The six transgenic plants were challenged by graft inoculation with SPFMV, SPCSV, SPVG and SPMMV-infected Ipomoea setosa Ker. Although virus presence was detected using nitrocellulose enzyme-linked immunosorbent assay, all transgenic plants displayed delayed and milder symptoms of chlorosis and mottling of lower leaves when compared to the untransformed control plants. These results warrant further investigation on resistance to virus infection under field conditions. PMID:24158330

Sivparsad, B J; Gubba, A

2014-04-01

204

Carrot (Daucus carota L.).  

PubMed

Plants are susceptible to infection by a broad range of fungal pathogens. A range of proteins have been evaluated that can enhance tolerance to these pathogens by heterologous expression in transgenic carrot tissues. The protocols for carrot transformation with Arabidopsis NPR1 (Non-Expressor of Pathogenesis-Related Proteins 1) are described in this chapter, using the herbicide resistance gene bar, which encodes phosphinothricin acetyltransferase, as a selectable marker. In this protocol, petiole segments (0.5-1.0 cm long) from aseptically grown carrot seedlings are exposed to Agrobacterium tumefaciens strain LBA4404 for 10-30 min and cocultivated for 2-3 days. Herbicide selection is then imposed for 8-12 weeks on a series of different tissue culture media until embryogenic calli are produced. The transfer of the embryogenic calli to hormone-free medium results in embryo development which eventually gives rise to transgenic plantlets. Embryogenic calli can also be propagated in suspension cultures. This protocol has yielded transgenic carrot plants with defined T-DNA inserts at the rate of between 1 and 3 Southern-positive independent events out of 100. PMID:25416249

Wally, Owen S D; Punja, Zamir K

2015-01-01

205

[Subcellular localization and resistance to Gibberella fujikuroi of AtELHYPRP2 in transgenic tobacco].  

PubMed

The subcellular localization and the resistance to fungal pathogen Gibberella fujikuroi of the protein encoded by Arabidopsis AtELHYPRP2 (EARLI1-LIKE HYBRID PROLINE-RICH PROTEIN 2, AT4G12500) were investigated using transgenic tobacco plants. The coding sequence of AtELHYPRP2 was amplified from genomic DNA of Col-0 ecotype. After restriction digestion, the PCR fragment was ligated into pCAMBIA1302 to produce a fusion expression vector, pCAMBIA1302-AtELHYPRP2-GFP. Then the recombinant plasmid was introduced into Agrobacterium tumefaciens strain LBA4404 and transgenic tobacco plants were regenerated and selected via leaf disc transformation method. RT-PCR and Western blotting analyses showed that AtELHYPRP2 expressed effectively in transgenic tobacco plants. Observation under laser confocal microscopy revealed that the green fluorescence of AtELHYPRP2-GFP fusion protein could overlap with the red fluorescence came from propidium iodide staining, indicating AtELHYPRP2 is localized to cell surface. Antimicrobial experiments exhibited that the constitutive expression of AtELHYPRP2 could enhance the resistance of tobacco to fungal pathogen G. fujikuroi and the infection sites could accumulate H2O2 obviously. The basal expression levels of PR1 and the systemic expression levels of PR1 and PR5 in transgenic tobacco plants were higher than that of the wild-type plants, suggesting AtELHYPRP2 may play a role in systemic acquired resistance. PMID:25007583

Chai, Qiuxia; Li, Benchang; Xu, Ziqin

2014-03-01

206

Molecular analysis of a tryptophan-2-monooxygenase gene (IaaM) of Agrobacterium vitis.  

PubMed

Tryptophan-2-monooxygenase genes occur in a number of bacteria and encode the conversion of tryptophan to the plant hormone precursor indole-3-acetamide. The role of these genes in the plant-bacteria interaction is often unclear. However, their function as a virulence determinant is established for Pseudomonas savastanoi and Agrobacterium tumefaciens. Some members of the Agrobacteria, such as Agrobacterium vitis have a limited host range. We have characterized the tryptophan-2-monooxygenase (iaaM) gene of A. vitis strain AG162 and show it is different from other A. vitis strains and related to iaaM of A. rhizogenes. The sequence of AG162 iaaM was deposited in the Genbank database under the accession number AF142716. PMID:10727091

Oetiker, J H; Lee, D H; Kato, A

1999-01-01

207

Fate of Agrobacterium radiobacter K84 in the environment.  

PubMed Central

Agrobacterium radiobacter K84 is an effective, commercially applied, biological control agent for the plant disease crown gall, yet little is known about the survival and dissemination of K84. To trace K84 in the environment, spontaneous antibiotic-resistant mutants were used. Growth rates and phenotypes of streptomycin- or rifampin-resistant K84 were similar to those of the parental K84, except the rifampin-resistant mutant produced less agrocin 84 as determined by bioassay. K84 and a strain of Agrobacterium tumefaciens established populations averaging 10(5) CFU/g in the rhizosphere of cherry and persisted on roots for 2 years. K84 established rhizosphere populations between 10(4) and 10(6) CFU/g on cherry, ryegrass, and 11 other herbaceous plants. Populations of K84 declined substantially in fallow soil or water over a 16-week period. K84 was detected in the rhizosphere of ryegrass located up to 40 cm from an inoculum source, indicating lateral dissemination of K84 in soil. In gall tissue on cherry, K84 established populations of 10(5) CFU/g, about 10- to 100-fold less than that of the pathogen. These data demonstrate that K84 persists for up to 2 years in a field environment as a rhizosphere inhabitant or in association with crown gall tissue. PMID:8357247

Stockwell, V O; Moore, L W; Loper, J E

1993-01-01

208

Arginine catabolism in Agrobacterium strains: role of the Ti plasmid.  

PubMed Central

We present a study of the enzymatic activities involved in the pathway for arginine catabolism by Agrobacterium tumefaciens. Nitrogen from arginine is recovered through the arginase-urease pathway; the genes for these two activities are probably chromosomally born. Arginase was found to be inducible during growth in the presence of arginine or ornithine. Urease was constitutively expressed. Ornithine, resulting from the action of arginase on arginine, could be used as a nitrogen source via transamination to delta 1-pyrroline-5-carboxylate and reduction of the latter compound to proline by a reductase (both enzymatic activities are probably chromosomally encoded). Ornithine could also be used as a carbon source. Thus, we identified an ornithine cyclase activity that was responsible for direct conversion of ornithine to proline. This activity was found to be Ti plasmid encoded and inducible by growth in medium containing octopine or nopaline. The same activity was also chromosomally encoded in some Agrobacterium strains. In such strains, this activity was inducible during growth in arginine-containing medium. PMID:3957872

Dessaux, Y; Petit, A; Tempé, J; Demarez, M; Legrain, C; Wiame, J M

1986-01-01

209

Efficient method for Agrobacterium mediated transformation of Artemisia annua L.  

PubMed

Artemisinin, a potent antimalarial natural products isolated from aerial parts of Artemisia annua L. Many patents have been reported that the demand for artemisinin is exponentially increasing year after year due to increased incidences of drug resistant malaria throughout the world. Leaf explants were used frequently as target tissue to generate transgenic of Artemisia. annua L. However, obtaining a large number of transgenic lines through out the year is a laborious and delicate process. To circumvent this, we have developed a highly efficient leaf explant based Agrobacterium mediated transformation of A. annua L. plant. The gus gene was used as screenable marker to assess and optimize the performance of T-DNA delivery. The age of explant, kind of bacterial inoculation, suspension duration, infection times and co-culture conditions were optimized. The co-culture was carried out with Agrobacterium tumefaciens strain EHA105 under desiccation condition in the dark at 25-28 0C for 2-4 days. Complete analysis of transgene insertion demonstrated that the optimized method of transformation from leaf explants of A. annua L. was efficient and highly reproducible. PMID:22642822

Alam, Pravej; Mohammad, Anis; Ahmad, M M; Khan, Mather Ali; Nadeem, Mohd; Khan, Riyazuddeen; Akmal, Mohd; Ahlawat, Seema; Abdin, M Z

2014-01-01

210

In vitro regeneration and Agrobacterium-mediated genetic transformation of Euonymus alatus.  

PubMed

An in vitro plant regeneration method and an Agrobacterium tumefaciens-mediated genetic transformation protocol were developed for Euonymus alatus. More than 60% of cotyledon and 70% of hypocotyl sections from 10-day-old seedlings of E. alatus produced 2-4 shoots on woody plant medium (WPM) supplemented with 5.0 mg/l 6-benzylaminopurine (BA) plus 0.2 mg/l alpha-naphthalene acetic acid (NAA), and 77% of shoots produced roots on WPM medium with 0.3 mg/l NAA and 0.5 mg/l Indole-3-butyricacid (IBA). On infection with Agrobacterium tumefaciens strain EHA105 harboring a gusplus gene that contained a plant recognizable intron from the castor bean catalase gene to ensure plant-specific beta-glucuronidase (GUS) expression, 16% of cotyledon and 15% of hypocotyl explants produced transgenic shoots using kanamycin as a selection agent, and 67% of these shoots rooted. Stable insertion of T-DNA into the host genome was determined with organ- and tissue-specific expression of the gusplus gene and further confirmed with a PCR-based molecular analysis. PMID:16733742

Chen, Yongqin; Lu, Litang; Deng, Wei; Yang, Xingyu; McAvoy, Richard; Zhao, Degang; Pei, Yan; Luo, Keming; Duan, Hui; Smith, William; Thammina, Chandra; Zheng, Xuelian; Ellis, Donna; Li, Yi

2006-10-01

211

Factors influencing Agrobacterium-mediated embryogenic callus transformation of Valencia sweet orange (Citrus sinensis) containing the pTA29-barnase gene.  

PubMed

Valencia sweet orange (Citrus sinensis (L.) Osbeck) calluses were used as explants to develop a new transformation system for citrus mediated by Agrobacterium tumefaciens. Factors affecting Agrobacterium-mediated transformation efficiency included mode of pre-cultivation, temperature of cocultivation and presence of acetosyringone (AS). The highest transformation efficiency was obtained with a 4-day pre-cultivation period in liquid medium. Transformation efficiency was higher when cocultivation was performed for 3 days at 19 degrees C than at 23 or 28 degrees C. Almost no resistant callus was obtained if the cocultivation medium lacked AS. The transformation procedure yielded transgenic Valencia plants containing the pTA29-barnase gene, as verified by PCR amplification and confirmed by Southern blotting. Because male sterility is a common factor leading to seedlessness in citrus cultivars with parthenocarpic characteristics, production of seedless citrus genotypes by Agrobacterium-mediated genetic transformation is a promising alternative to conventional breeding methods. PMID:14597430

Li, D D; Shi, W; Deng, X X

2003-12-01

212

Expression of Shigella flexneri ipaB Gene in Tobacco  

PubMed Central

Background Shigellosis is a leading cause of diarrhea in many developing countries and although the disease can be controlled and managed with antibiotics, the constant emergence of resistant species requiring ever newer antibacterial drugs make development of an effective vaccine necessary. The bacteria are highly contagious and since immunity to Shigella is serotype-specific a multi-serotype vaccine is required for adequate protection. Proteins encoded by Shigella invasion plasmid, which are part of the Type Three Secretion System (TTSS) of this bacteria, are good candidate as vaccine targets since they are both immunogenic and conserved between different Shigella species. The advent of molecular farming, which is a low cost system, has opened up new venues for production of recombinant proteins. In view of the difficulties encountered in expressing IpaB in Escherichia coli (E. coli), the feasibility of the expression of this protein in tobacco has been investigated. Methods The ipaB gene was cloned in place of the Hygromycin gene in pCambia1304 containing GFP as a reporter gene. The vector was then transferred into competent Agrobacterium tumefaciens (A. tumefaciens) strain LBA4404 which was used for agro-infiltration of Nicotiana tobaccum (N. tobaccum) leaves. Transformation was confirmed by expression of GFP. The gene was also cloned in pBAD/geneIII A and transformed E. coli host containing the construct was induced using different amounts of L-arabinose as inducer. Expression of IpaB gene by both hosts was determined by Western blotting using anti-IpaB monoclonal antibody. Results The data obtained showed that IpaB was expressed in plant leaves but expression in E. coli was not detectable. Conclusion This study showed that N. tobaccum is capable of expressing this protein without its specific chaperon and in levels detectable by Western blotting. PMID:23799180

Ohadi, Mandana; Rasouli, Rahimeh; Darzi-Eslam, Elham; Jafari, Anis; Ehsani, Parastoo

2013-01-01

213

Transgenic Pearl Millet Male Fertility Restorer Line (ICMP451) and Hybrid (ICMH451) Expressing Brassica juncea Nonexpressor of Pathogenesis Related Genes 1 (BjNPR1) Exhibit Resistance to Downy Mildew Disease  

PubMed Central

Brassica juncea Nonexpressor of pathogenesis-related genes 1 (BjNPR1) has been introduced into pearl millet male fertility restorer line ICMP451 by Agrobacterium tumefaciens-mediated genetic transformation. Transgenic pearl millet plants were regenerated from the phosphinothricin-resistant calli obtained after co-cultivation with A. tumefaciens strain LBA4404 harbouring Ti plasmid pSB111-bar-BjNPR1. Molecular analyses confirmed the stable integration and expression of BjNPR1 in transgenic pearl millet lines. Transgenes BjNPR1 and bar were stably inherited and disclosed co-segregation in subsequent generations in a Mendelian fashion. Transgenic pearl millet hybrid ICMH451-BjNPR1 was developed by crossing male-sterile line 81A X homozygous transgenic line ICMP451-BjNPR1. T3 and T4 homozygous lines of ICMP451-BjNPR1 and hybrid ICMH451-BjNPR1 exhibited resistance to three strains of downy mildew pathogen, while the untransformed ICMP451 and the isogenic hybrid ICMH451 plants were found susceptible. Following infection with S. graminicola, differential expression of systemic acquired resistance pathway genes, UDP-glucose salicylic acid glucosyl transferase and pathogenesis related gene 1 was observed in transgenic ICMP451-BjNPR1 and untransformed plants indicating the activation of systemic acquired resistance pathway contributing to the transgene-mediated resistance against downy mildew. The transgenic pearl millet expressing BjNPR1 showed resistance to multiple strains of S. graminicola and, as such, seems promising for the development of durable downy mildew resistant hybrids. PMID:24603762

Khareedu, Venkateswara Rao; Vudem, Dashavantha Reddy

2014-01-01

214

Deletion analysis of the mannopine synthase gene promoter in sunflower crown gall tumors and Agrobacterium tumefaciens  

Microsoft Academic Search

We have used deletion mutagenesis to analyze a TR-DNA promoter from the octopine-type Ti plasmid pTiB6806. The promoter for the gene encoding mannopine synthase (mas) was cloned upstream of the bacterial kanamycin-resistance gene neomycin phosphotransferase II (NPT II). Bal31 deletion mutagenesis was used to generate deletion derivatives of the mas\\/NPTII gene beginning 1353 bp upstream of the initiation of transcription

Victor J. DiRita; Stanton B. Gelvin

1987-01-01

215

TECHNICAL ADVANCE Genetic transformation of the actinorhizal tree Allocasuarina verticillata by Agrobacterium tumefaciens  

E-print Network

C. Franche1, * , D. Diouf1, Q.V. Le1, D. Bogusz1,A. This capacity for rapid growth comes from a symbiotic N’Diaye 1, H. Gherbi1, C. Gobé1 and E. Duhoux1,2 association with an actinomycete of the genus Frankia, 1Physiologie Cellulaire et Moléculaire des Arbres which allows A. verticillata to fix dinitrogen in root nodules, (ORSTOM GeneTrop), 911 avenue Agropolis, BP 5045, so-called actinorhizae (Benson and Silvester, 1993). 34032 Montpellier cédex 1, France Actinorhizae are found in at least 20 other angiosperm 2Université Paris VII-Denis Diderot, 2 Place Jussieu, genera that belong to eight different families. Little is

unknown authors

216

Generation of transgenic Lolium temulentum plants by Agrobacterium tumefaciens -mediated transformation  

Microsoft Academic Search

Lolium temulentum L. (Darnel ryegrass) has been proposed to be used as a model species for functional genomics studies in forage and turf grasses,\\u000a because it is a self-fertile, diploid species with a short life cycle and is closely related to other grasses. Embryogenic\\u000a calluses were induced from mature embryos of a double haploid line developed through anther culture. The

Yaxin Ge; Xiaofei Cheng; Andrew Hopkins; Zeng-Yu Wang

2007-01-01

217

Leaf disc transformation of cultivated tomato ( L. esculentum ) using Agrobacterium tumefaciens  

Microsoft Academic Search

The leaf disc transformation\\/regeneration system was modified for tomato (L. esculentum). Both leaf explants and cotyledon\\/hypocotyl sections can be used to regenerate transformed plants. We have obtained over 300 transgenic plants from eight tomato cultivars. We have evidence for both single and multi-copy insertions of the T-DNA, and have demonstrated inheritance of the T-DNA insert in the expected Mendelian ratios.

Sheila McCormick; Jeanne Niedermeyer; Joyce Fry; Arlene Barnason; Robert Horsch; Robert Fraley

1986-01-01

218

Agrobacterium tumefaciens-Induced Bacteraemia Does Not Lead to Reporter Gene Expression in Mouse Organs  

E-print Network

. Agrobacteria carrying Cauliflower mosaic virus (CaMV) 35S promoter-based constructs and isolated from the injected mice retained their capacity to promote green fluorescent protein (GFP) synthesis in Nicotiana often represent a preferred source of recombinant proteins and biopharmaceuticals for human consump

Citovsky, Vitaly

219

Improved Agrobacterium-mediated transformation of three maize inbred lines using MS salts.  

PubMed

Transformation technology as a research or breeding tool to improve maize is routinely used in most industrial and some specialized public laboratories. However, transformation of many inbred lines remains a challenging task, especially when using Agrobacterium tumefaciens as the delivery method. Here we report success in generating transgenic plants and progeny from three maize inbred lines using an Agrobacterium-mediated standard binary vector system to target maize immature embryos. Eleven maize inbred lines were pre-screened for transformation frequency using N6 salts. A subset of three maize inbred lines was then systematically evaluated for frequency of post-infection embryogenic callus induction and transformation on four media regimes: N6 or MS salts in each of two distinct media backgrounds. Transgenic plants recovered from inbred lines B104, B114, and Ky21 were analyzed for transgene integration, expression, and transmission. Average transformation frequencies of 6.4% (for B104), 2.8% (for B114), and 8% (for Ky21) were achieved using MS salts. Availability of Agrobacterium-mediated maize inbred line transformation will improve future opportunities for maize genetic and functional genomic studies. PMID:16710703

Frame, Bronwyn R; McMurray, Jennifer M; Fonger, Tina M; Main, Marcy L; Taylor, Kyle W; Torney, François J; Paz, Margie M; Wang, Kan

2006-10-01

220

Cytokinins secreted by Agrobacterium promote transformation by repressing a plant myb transcription factor.  

PubMed

Agrobacterium-mediated transformation is the most widely used technique for generating transgenic plants. However, many crops remain recalcitrant. We found that an Arabidopsis myb family transcription factor (MTF1) inhibited plant transformation susceptibility. Mutating MTF1 increased attachment of several Agrobacterium strains to roots and increased both stable and transient transformation in both susceptible and transformation-resistant Arabidopsis ecotypes. Cytokinins from Agrobacterium tumefaciens decreased the expression of MTF1 through activation of the cytokinin response regulator ARR3. Mutating AHK3 and AHK4, genes that encode cytokinin-responsive kinases, increased the expression of MTF1 and impaired plant transformation. Mutant mtf1 plants also had increased expression of AT14A, which encodes a putative transmembrane receptor for cell adhesion molecules. Plants overexpressing AT14A exhibited increased susceptibility to transformation, whereas at14a mutant plants exhibited decreased attachment of bacteria to roots and decreased transformation, suggesting that AT14A may serve as an anchor point for Agrobacteria. Thus, by promoting bacterial attachment and transformation of resistant plants and increasing such processes in susceptible plants, treating roots with cytokinins may help engineer crops with improved features or yield. PMID:24255177

Sardesai, Nagesh; Lee, Lan-Ying; Chen, Huabang; Yi, Hochul; Olbricht, Gayla R; Stirnberg, Alexandra; Jeffries, Jacob; Xiong, Kia; Doerge, R W; Gelvin, Stanton B

2013-11-19

221

Agrobacterium-Mediated Transformation of Subterranean Clover (Trifolium subterraneum L.).  

PubMed Central

We have developed a rapid and reproducible transformation system for subterranean clover (Trifolium subterraneum L.) using Agrobacterium tumefaciens-mediated gene delivery. Hypocotyl segments from seeds that had been allowed to imbibe were used as explants, and regeneration was achieved via organogenesis. Glucose and acetosyringone were required in the co-cultivation medium for efficient gene transfer. DNA constructs containing four genes encoding the enzymes phosphinothricin acetyl transferase, [beta]-glucuronidase (GUS), neomycin phosphotransferase, and an [alpha]-amylase inhibitor were used to transform subterranean clover. Transgenic shoots were selected on a medium containing 50 mg/L of phosphinothricin. Four commercial cultivars of subterranean clover (representing all three subspecies) have been successfully transformed. Southern analysis revealed the integration of T-DNA into the subterranean clover genome. The expression of the introduced genes has been confirmed by enzyme assays and northern blot analyses. Transformed plants grown in the glasshouse showed resistance to the herbicide Basta at applications equal to or higher than rates recommended for killing subterranean clover in field conditions. In plants grown from the selfed seeds of the primary transformants, the newly acquired gene encoding GUS segregated as a dominant Mendelian trait. PMID:12232188

Khan, MRI.; Tabe, L. M.; Heath, L. C.; Spencer, D.; Higgins, TJV.

1994-01-01

222

Isozyme gene expression in potato tumors incited by Agrobacterium.  

PubMed

Two plant tumors (crown galls and hairy roots) were experimentally provoked on potato cv. 'Désirée' by oncogenic strains of Agrobacterium tumefaciens and A. rhizogenes. A marked shift in the expression of some organ-specific genes occurred in crown galls derived from the central zone of tubers: two novel isozyme genes (Est-B and Pox-E) were expressed, two others (Est-C and Pox-F) were suppressed and the remaining ones were maintained in the original state. When the starting tissue was the stem segment, a smaller shift occurred, namely the activation of Adh-A and the suppression of Pox-F. In all cases, the isozyme profiles characterizing all crown galls, whatever their origin, were identical. Under normal aeration conditions, Adh-A was not expressed in either tumoral or non-tumoral roots. However, under the relative anaerobic conditions of in vitro cultures, a difference existed between both types of roots: Adh-A was expressed in normal but not in tumoral roots. This means that hairy roots can tolerate higher levels of anaerobiosis without giving rise to an anaerobic response. For the remaining isozymes, no alteration occurred in either organized (hairy root) or unorganized (crown gall) tumors, as compared to the corresponding non-tumoral tissues (normal root and callus, respectively). PMID:24247945

Oliver, J L

1986-06-01

223

Development of an Agrobacterium-Mediated Stable Transformation Method for the Sensitive Plant Mimosa pudica  

PubMed Central

The sensitive plant Mimosa pudica has long attracted the interest of researchers due to its spectacular leaf movements in response to touch or other external stimuli. Although various aspects of this seismonastic movement have been elucidated by histological, physiological, biochemical, and behavioral approaches, the lack of reverse genetic tools has hampered the investigation of molecular mechanisms involved in these processes. To overcome this obstacle, we developed an efficient genetic transformation method for M. pudica mediated by Agrobacterium tumefaciens (Agrobacterium). We found that the cotyledonary node explant is suitable for Agrobacterium-mediated transformation because of its high frequency of shoot formation, which was most efficiently induced on medium containing 0.5 µg/ml of a synthetic cytokinin, 6-benzylaminopurine (BAP). Transformation efficiency of cotyledonary node cells was improved from almost 0 to 30.8 positive signals arising from the intron-sGFP reporter gene by using Agrobacterium carrying a super-binary vector pSB111 and stabilizing the pH of the co-cultivation medium with 2-(N-morpholino)ethanesulfonic acid (MES) buffer. Furthermore, treatment of the explants with the detergent Silwet L-77 prior to co-cultivation led to a two-fold increase in the number of transformed shoot buds. Rooting of the regenerated shoots was efficiently induced by cultivation on irrigated vermiculite. The entire procedure for generating transgenic plants achieved a transformation frequency of 18.8%, which is comparable to frequencies obtained for other recalcitrant legumes, such as soybean (Glycine max) and pea (Pisum sativum). The transgene was stably integrated into the host genome and was inherited across generations, without affecting the seismonastic or nyctinastic movements of the plants. This transformation method thus provides an effective genetic tool for studying genes involved in M. pudica movements. PMID:24533121

Mano, Hiroaki; Fujii, Tomomi; Sumikawa, Naomi; Hiwatashi, Yuji; Hasebe, Mitsuyasu

2014-01-01

224

Agrobacterium-mediated genetic transformation and plant regeneration of the hardwood tree species Fraxinus profunda.  

PubMed

This transformation and regeneration protocol provides an integral framework for the genetic improvement of Fraxinus profunda (pumpkin ash) for future development of plants resistant to the emerald ash borer. Using mature hypocotyls as the initial explants, an Agrobacterium tumefaciens-mediated genetic transformation system was successfully developed for pumpkin ash (Fraxinus profunda). This transformation protocol is an invaluable tool to combat the highly aggressive, non-native emerald ash borer (EAB), which has the potential to eliminate native Fraxinus spp. from the natural landscape. Hypocotyls were successfully transformed with Agrobacterium strain EHA105 harboring the pq35GR vector, containing an enhanced green fluorescent protein (EGFP) as well as a fusion gene between neomycin phosphotransferase (nptII) and gusA. Hypocotyls were cultured for 7 days on Murashige and Skoog (MS) medium with 22.2 ?M 6-benzyladenine (BA), 4.5 ?M thidiazuron (TDZ), 50 mg L(-1) adenine hemisulfate (AS), and 10 % coconut water (CW) prior to transformation. Hypocotyls were transformed using 90 s sonication plus 10 min vacuum infiltration after Agrobacterium was exposed to 100 ?M acetosyringone for 1 h. Adventitious shoots were regenerated on MS medium with 22.2 ?M BA, 4.5 ?M TDZ, 50 mg L(-1) AS, 10 % CW, 400 mg L(-1) timentin, and 20 mg L(-1) kanamycin. Timentin at 400 and 20 mg L(-1) kanamycin were most effective at controlling Agrobacterium growth and selecting for transformed cells, respectively. The presence of nptII, GUS (?-glucuronidase), and EGFP in transformed plants was confirmed using polymerase chain reaction (PCR), while the expression of EGFP was also confirmed through fluorescent microscopy and reverse transcription-PCR. This transformation protocol provides an integral foundation for future genetic modifications of F. profunda to provide resistance to EAB. PMID:24493252

Stevens, Micah E; Pijut, Paula M

2014-06-01

225

Development of an Agrobacterium-mediated stable transformation method for the sensitive plant Mimosa pudica.  

PubMed

The sensitive plant Mimosa pudica has long attracted the interest of researchers due to its spectacular leaf movements in response to touch or other external stimuli. Although various aspects of this seismonastic movement have been elucidated by histological, physiological, biochemical, and behavioral approaches, the lack of reverse genetic tools has hampered the investigation of molecular mechanisms involved in these processes. To overcome this obstacle, we developed an efficient genetic transformation method for M. pudica mediated by Agrobacterium tumefaciens (Agrobacterium). We found that the cotyledonary node explant is suitable for Agrobacterium-mediated transformation because of its high frequency of shoot formation, which was most efficiently induced on medium containing 0.5 µg/ml of a synthetic cytokinin, 6-benzylaminopurine (BAP). Transformation efficiency of cotyledonary node cells was improved from almost 0 to 30.8 positive signals arising from the intron-sGFP reporter gene by using Agrobacterium carrying a super-binary vector pSB111 and stabilizing the pH of the co-cultivation medium with 2-(N-morpholino)ethanesulfonic acid (MES) buffer. Furthermore, treatment of the explants with the detergent Silwet L-77 prior to co-cultivation led to a two-fold increase in the number of transformed shoot buds. Rooting of the regenerated shoots was efficiently induced by cultivation on irrigated vermiculite. The entire procedure for generating transgenic plants achieved a transformation frequency of 18.8%, which is comparable to frequencies obtained for other recalcitrant legumes, such as soybean (Glycine max) and pea (Pisum sativum). The transgene was stably integrated into the host genome and was inherited across generations, without affecting the seismonastic or nyctinastic movements of the plants. This transformation method thus provides an effective genetic tool for studying genes involved in M. pudica movements. PMID:24533121

Mano, Hiroaki; Fujii, Tomomi; Sumikawa, Naomi; Hiwatashi, Yuji; Hasebe, Mitsuyasu

2014-01-01

226

Direct visualization of Agrobacterium-delivered VirE2 in recipient cells  

PubMed Central

Agrobacterium tumefaciens is a natural genetic engineer widely used to deliver DNA into various recipients, including plant, yeast and fungal cells. The bacterium can transfer single-stranded DNA molecules (T–DNAs) and bacterial virulence proteins, including VirE2. However, neither the DNA nor the protein molecules have ever been directly visualized after the delivery. In this report, we adopted a split-GFP approach: the small GFP fragment (GFP11) was inserted into VirE2 at a permissive site to create the VirE2-GFP11 fusion, which was expressed in A. tumefaciens; and the large fragment (GFP1–10) was expressed in recipient cells. Upon delivery of VirE2-GFP11 into the recipient cells, GFP fluorescence signals were visualized. VirE2-GFP11 was functional like VirE2; the GFP fusion movement could indicate the trafficking of Agrobacterium-delivered VirE2. As the natural host, all plant cells seen under a microscope received the VirE2 protein in a leaf-infiltration assay; most of VirE2 moved at a speed of 1.3–3.1 ?m sec?1 in a nearly linear direction, suggesting an active trafficking process. Inside plant cells, VirE2-GFP formed filamentous structures of different lengths, even in the absence of T-DNA. As a non-natural host recipient, 51% of yeast cells received VirE2, which did not move inside yeast. All plant cells seen under a microscope transiently expressed the Agrobacterium-delivered transgene, but only 0.2% yeast cells expressed the transgene. This indicates that Agrobacterium is a more efficient vector for protein delivery than T-DNA transformation for a non-natural host recipient: VirE2 trafficking is a limiting factor for the genetic transformation of a non-natural host recipient. The split-GFP approach could enable the real-time visualization of VirE2 trafficking inside recipient cells. PMID:24299048

Li, Xiaoyang; Yang, Qinghua; Tu, Haitao; Lim, Zijie; Pan, Shen Q

2014-01-01

227

Morphogenetic and chemical stability of long-term maintained Agrobacterium-mediated transgenic Catharanthus roseus plants.  

PubMed

Transgenic Catharanthus roseus plants (transgenic Dhawal [DT] and transgenic Nirmal [NT]) obtained from the Agrobacterium tumefaciens and Agrobacteriumrhizognenes-mediated transformations, respectively, have been maintained in vitro for 5 years. Plants were studied at regular intervals for various parameters such as plant height, leaf size, multiplication rate, alkaloid profile and presence of marker genes. DT plant gradually lost the GUS gene expression and it was not detected in the fifth year while NT plant demonstrated the presence of genes rolA, rolB and rolC even in the fifth year, indicating the more stable nature of Ri transgene. Vindoline content in the DT was two times more than in non-transformed control plants. Alkaloid and tryptophan profiles were almost constant during the 5 years. The cluster analysis revealed that the DT plant is more close to the control Nirmal plant followed by NT plant. PMID:25102992

Verma, Priyanka; Sharma, Abhishek; Khan, Shamshad Ahmad; Mathur, Ajay Kumar; Shanker, Karuna

2015-02-01

228

Agrobacterium-mediated transformation of Solanum tuberosum L. cv. 'Russet Burbank'.  

PubMed

Stem sections from shoot cultures maintained in vitro were used to produce transgenic plants of the potato, Solanum tuberosum L. cv. 'Russet Burbank'. Stem internode pieces inoculated with Agrobacterium tumefaciens containing coat protein genes from potato virus X and potato virus Y, produced shoots with a frequency of 60% in the absence of selection and 10% on medium containing 100 mg/l kanamycin monosulfate. Regenerated shoots were assayed for kanamycin resistance by placing stem segments on callus induction medium containing an increased level of kanamycin. Of a total 255 regenerated shoots, 47 (18%) were kanamycin resistant. Of the kanamycin resistant shoots, 25 (53%) expressed the PVX or PVY coat protein genes as assayed by enzyme-linked immunosorbent assay or Western immunoblot analysis. PMID:24226160

Newell, C A; Rozman, R; Hinchee, M A; Lawson, E C; Haley, L; Sanders, P; Kaniewski, W; Tumer, N E; Horsch, R B; Fraley, R T

1991-05-01

229

Occurrence of enzymes involved in biosynthesis of indole-3-acetic acid from indole-3-acetonitrile in plant-associated bacteria, Agrobacterium and Rhizobium.  

PubMed Central

The occurrence of a hitherto unknown pathway involving the action of two enzymes, a nitrile hydratase and an amidase for the biosynthesis of indole-3-acetic acid was discovered in phytopathogenic bacteria Agrobacterium tumefaciens and in leguminous bacteria Rhizobium. The nitrile hydratase acting on indole-3-acetonitrile was purified to homogeneity through only two steps from the cell-free extract of A. tumefaciens. The molecular mass of the purified enzyme estimated by HPLC was about 102 kDa, and the enzyme consisted of four subunits identical in molecular mass. The enzyme exhibited a broad absorption spectrum in the visible range with absorption maxima at 408 nm and 705 nm, and it contained cobalt and iron. The enzyme stoichiometrically catalyzed the hydration of indole-3-acetonitrile into indole-3-acetamide with a specific activity of 13.7 mol per min per mg and a Km of 7.9 microM. Images Fig. 1 PMID:11607511

Kobayashi, M; Suzuki, T; Fujita, T; Masuda, M; Shimizu, S

1995-01-01

230

Occurrence of enzymes involved in biosynthesis of indole-3-acetic acid from indole-3-acetonitrile in plant-associated bacteria, Agrobacterium and Rhizobium.  

PubMed

The occurrence of a hitherto unknown pathway involving the action of two enzymes, a nitrile hydratase and an amidase for the biosynthesis of indole-3-acetic acid was discovered in phytopathogenic bacteria Agrobacterium tumefaciens and in leguminous bacteria Rhizobium. The nitrile hydratase acting on indole-3-acetonitrile was purified to homogeneity through only two steps from the cell-free extract of A. tumefaciens. The molecular mass of the purified enzyme estimated by HPLC was about 102 kDa, and the enzyme consisted of four subunits identical in molecular mass. The enzyme exhibited a broad absorption spectrum in the visible range with absorption maxima at 408 nm and 705 nm, and it contained cobalt and iron. The enzyme stoichiometrically catalyzed the hydration of indole-3-acetonitrile into indole-3-acetamide with a specific activity of 13.7 mol per min per mg and a Km of 7.9 microM. PMID:11607511

Kobayashi, M; Suzuki, T; Fujita, T; Masuda, M; Shimizu, S

1995-01-31

231

Expression of a fungal ferulic acid esterase in alfalfa modifies cell wall digestibility  

PubMed Central

Background Alfalfa (Medicago sativa) is an important forage crop in North America owing to its high biomass production, perennial nature and ability to fix nitrogen. Feruloyl esterase (EC 3.1.1.73) hydrolyzes ester linkages in plant cell walls and has the potential to further improve alfalfa as biomass for biofuel production. Results In this study, faeB [GenBank:AJ309807] was synthesized at GenScript and sub-cloned into a novel pEACH vector containing different signaling peptides to target type B ferulic acid esterase (FAEB) proteins to the apoplast, chloroplast, endoplasmic reticulum and vacuole. Four constructs harboring faeB were transiently expressed in Nicotiana leaves, with FAEB accumulating at high levels in all target sites, except chloroplast. Stable transformed lines of alfalfa were subsequently obtained using Agrobacterium tumefaciens (LBA4404). Out of 136 transgenic plants regenerated, 18 independent lines exhibited FAEB activity. Subsequent in vitro digestibility and Fourier transformed infrared spectroscopy (FTIR) analysis of FAEB-expressing lines showed that they possessed modified cell wall morphology and composition with a reduction in ester linkages and elevated lignin content. Consequently, they were more recalcitrant to digestion by mixed ruminal microorganisms. Interestingly, delignification by alkaline peroxide treatment followed by exposure to a commercial cellulase mixture resulted in higher glucose release from transgenic lines as compared to the control line. Conclusion Modifying cell wall crosslinking has the potential to lower recalcitrance of holocellulose, but also exhibited unintended consequences on alfalfa cell wall digestibility due to elevated lignin content. The combination of efficient delignification treatment (alkaline peroxide) and transgenic esterase activity complement each other towards efficient and effective digestion of transgenic lines. PMID:24650274

2014-01-01

232

Agrobacterium-mediated genetic transformation of yam (Dioscorea rotundata): an important tool for functional study of genes and crop improvement  

PubMed Central

Although genetic transformation of clonally propagated crops has been widely studied as a tool for crop improvement and as a vital part of the development of functional genomics resources, there has been no report of any existing Agrobacterium-mediated transformation of yam (Dioscorea spp.) with evidence of stable integration of T-DNA. Yam is an important crop in the tropics and subtropics providing food security and income to over 300 million people. However, yam production remains constrained by increasing levels of field and storage pests and diseases. A major constraint to the development of biotechnological approaches for yam improvement has been the lack of an efficient and robust transformation and regeneration system. In this study, we developed an Agrobacterium-mediated transformation of Dioscorea rotundata using axillary buds as explants. Two cultivars of D. rotundata were transformed using Agrobacterium tumefaciens harboring the binary vectors containing selectable marker and reporter genes. After selection with appropriate concentrations of antibiotic, shoots were developed on shoot induction and elongation medium. The elongated antibiotic-resistant shoots were subsequently rooted on medium supplemented with selection agent. Successful transformation was confirmed by polymerase chain reaction, Southern blot analysis, and reporter genes assay. Expression of gusA gene in transgenic plants was also verified by reverse transcription polymerase chain reaction analysis. Transformation efficiency varied from 9.4 to 18.2% depending on the cultivars, selectable marker genes, and the Agrobacterium strain used for transformation. It took 3–4 months from Agro-infection to regeneration of complete transgenic plant. Here we report an efficient, fast and reproducible protocol for Agrobacterium-mediated transformation of D. rotundata using axillary buds as explants, which provides a useful platform for future genetic engineering studies in this economically important crop. PMID:25309562

Nyaboga, Evans; Tripathi, Jaindra N.; Manoharan, Rajesh; Tripathi, Leena

2014-01-01

233

Resistance to crown gall disease in transgenic grapevine rootstocks containing truncated virE2 of Agrobacterium.  

PubMed

A truncated form of the Ti-plasmid virE2 gene from Agrobacterium tumefaciens strains C58 and A6, and A. vitis strain CG450 was transferred and expressed in somatic embryos of grapevine rootstocks 110 Richter (Vitis rupestris × V. berlandieri), 3309 Couderc (V. rupestris × V. riparia) and Teleki 5C (V. berlandieri × V. riparia) via Agrobacterium-mediated transformation to confer resistance to crown gall disease. Transformation was confirmed in 98% of the 322 lines by enzyme-linked immunosorbent assay for the neomycin phosphotransferase II protein and 97% of 295 lines by polymerase chain reaction for the truncated virE2 transgene. Southern blot analysis revealed the insertion of truncated virE2 at one to three loci in a subset of seven transgenic 110 Richter lines. In vitro resistance screening assays based on inoculations of shoot internode sections showed reduced tumorigenicity and very small galls in 23 of 154 transgenic lines. Non-transformed controls had a 100% tumorigenicity rate with very large galls. Disease resistance assay at the whole plant level in the greenhouse revealed seven transgenic lines (3 lines of 110 Richter, 2 lines of 3309 Couderc and 2 lines of Teleki 5C) were resistant to A. tumefaciens strain C58 and A. vitis strains TM4 and CG450 with a substantially reduced percentage of inoculation sites showing gall as compared to controls. No association was found between the level of resistance to crown gall disease and the source Agrobacterium strain of virE2. Taken together, our data showed that resistance to crown gall disease can be achieved by expressing a truncated form of virE2 in grapevines. PMID:20182792

Krastanova, Stoyanka V; Balaji, Vasudevan; Holden, Michele R; Sekiya, Mary; Xue, Baodi; Momol, Esengul A; Burr, Thomas J

2010-12-01

234

Assessment of factors influencing the Agrobacterium-mediated in planta seed transformation of brinjal (Solanum melongena L.).  

PubMed

An efficient and reproducible in planta transformation method was developed for brinjal using seed as an explant. The brinjal seeds were infected with Agrobacterium tumefaciens EHA 105 harbouring pCAMBIA 1301-bar plasmid, and the transformants were selected against BASTA®. Several parameters influencing the in planta seed transformation such as pre-culture duration, acetosyringone concentration, surfactants, duration of sonication, vacuum pressure and vacuum duration have been evaluated. The putatively transformed (T 0) brinjal plants were screened by GUS histochemical analysis. Among the different combinations and concentrations tested, when the 18-h pre-cultured brinjal seeds were sonicated for 20 min and vacuum infiltered for 3 min at 500 mm of Hg in Agrobacterium suspension containing 100 ?M acetosyringone, 0.2 % Silwett L-77 favoured the Agrobacterium infection and showed maximum transformation efficiency. Among the five brinjal varieties evaluated, Arka Samhitha showed maximum transformation efficiency at 45.66 %. The transgene was successfully transmitted to progeny plants (T 1) which was evidenced by GUS histochemical analysis, polymerase chain reaction and Southern hybridisation. The in planta protocol developed in the present study would be beneficial to transfer the economically and nutritionally important genes into different varieties of brinjal, and the transgenic brinjal plants can be produced in less time (approximately 27 days). PMID:23852797

Subramanyam, Kondeti; Rajesh, Manoharan; Jaganath, Balusamy; Vasuki, Amirthalingam; Theboral, Jeevaraj; Elayaraja, Dhandapani; Karthik, Sivabalan; Manickavasagam, Markandan; Ganapathi, Andy

2013-09-01

235

Agrobacterium VirD2 protein interacts with plant host cyclophilins  

PubMed Central

Agrobacterium tumefaciens induces crown gall tumors on plants by transferring a nucleoprotein complex, the T-complex, from the bacterium to the plant cell. The T-complex consists of T-DNA, a single-stranded DNA segment of the tumor-inducing plasmid, VirD2, an endonuclease covalently bound to the 5? end of the T-DNA, and perhaps VirE2, a single-stranded DNA binding protein. The yeast two-hybrid system was used to screen for proteins interacting with VirD2 and VirE2 to identify components in Arabidopsis thaliana that interact with the T-complex. Three VirD2- and two VirE2-interacting proteins were identified. Here we characterize the interactions of VirD2 with two isoforms of Arabidopsis cyclophilins identified by using this analysis. The VirD2 domain interacting with the cyclophilins is distinct from the endonuclease, omega, and the nuclear localization signal domains. The VirD2–cyclophilin interaction is disrupted in vitro by cyclosporin A, which also inhibits Agrobacterium-mediated transformation of Arabidopsis and tobacco. These data strongly suggest that host cyclophilins play a role in T-DNA transfer. PMID:9618535

Deng, Wanyin; Chen, Lishan; Wood, Derek W.; Metcalfe, Tracee; Liang, Xiaoyou; Gordon, Milton P.; Comai, Luca; Nester, Eugene W.

1998-01-01

236

Genetic Transformation of Metroxylon sagu (Rottb.) Cultures via Agrobacterium-Mediated and Particle Bombardment  

PubMed Central

Sago palm (Metroxylon sagu) is a perennial plant native to Southeast Asia and exploited mainly for the starch content in its trunk. Genetic improvement of sago palm is extremely slow when compared to other annual starch crops. Urgent attention is needed to improve the sago palm planting material and can be achieved through nonconventional methods. We have previously developed a tissue culture method for sago palm, which is used to provide the planting materials and to develop a genetic transformation procedure. Here, we report the genetic transformation of sago embryonic callus derived from suspension culture using Agrobacterium tumefaciens and gene gun systems. The transformed embryoids cells were selected against Basta (concentration 10 to 30?mg/L). Evidence of foreign genes integration and function of the bar and gus genes were verified via gene specific PCR amplification, gus staining, and dot blot analysis. This study showed that the embryogenic callus was the most suitable material for transformation as compared to the fine callus, embryoid stage, and initiated shoots. The gene gun transformation showed higher transformation efficiency than the ones transformed using Agrobacterium when targets were bombarded once or twice using 280?psi of helium pressure at 6 to 8?cm distance. PMID:25295258

Ibrahim, Evra Raunie

2014-01-01

237

Genetic transformation of Metroxylon sagu (Rottb.) cultures via Agrobacterium-mediated and particle bombardment.  

PubMed

Sago palm (Metroxylon sagu) is a perennial plant native to Southeast Asia and exploited mainly for the starch content in its trunk. Genetic improvement of sago palm is extremely slow when compared to other annual starch crops. Urgent attention is needed to improve the sago palm planting material and can be achieved through nonconventional methods. We have previously developed a tissue culture method for sago palm, which is used to provide the planting materials and to develop a genetic transformation procedure. Here, we report the genetic transformation of sago embryonic callus derived from suspension culture using Agrobacterium tumefaciens and gene gun systems. The transformed embryoids cells were selected against Basta (concentration 10 to 30?mg/L). Evidence of foreign genes integration and function of the bar and gus genes were verified via gene specific PCR amplification, gus staining, and dot blot analysis. This study showed that the embryogenic callus was the most suitable material for transformation as compared to the fine callus, embryoid stage, and initiated shoots. The gene gun transformation showed higher transformation efficiency than the ones transformed using Agrobacterium when targets were bombarded once or twice using 280?psi of helium pressure at 6 to 8?cm distance. PMID:25295258

Ibrahim, Evra Raunie; Hossain, Md Anowar; Roslan, Hairul Azman

2014-01-01

238

An efficient Agrobacterium-mediated transformation method for the edible mushroom Hypsizygus marmoreus.  

PubMed

Hypsizygus marmoreus is one of the major edible mushrooms in East Asia. As no efficient transformation method, the molecular and genetics studies were hindered. The glyceraldehyde-3-phosphate dehydrogenase (GPD) gene of H. marmoreus was isolated and its promoter was used to drive the hygromycin B phosphotransferase (HPH) and enhanced green fluorescent protein (EGFP) in H. marmoreus. Agrobacterium tumefaciens-mediated transformation (ATMT) was successfully applied in H. marmoreus. The transformation parameters were optimized, and it was found that co-cultivation of bacteria with protoplast at a ratio of 1000:1 at a temperature of 26 °C in medium containing 0.3 mM acetosyringone resulted in the highest transformation efficiency for Agrobacterium strain. Besides, three plasmids, each carrying a different promoter (from H. marmoreus, Ganoderma lucidum and Lentinula edodes) driving the expression of an antibiotic resistance marker, were also tested. The construct carrying the H. marmoreus gpd promoter produced more transformants than other constructs. Our analysis showed that over 85% of the transformants tested remained mitotically stable even after five successive rounds of subculturing. Putative transformants were analyzed for the presence of hph gene by PCR and Southern blot. Meanwhile, the expression of EGFP in H. marmoreus transformants was detected by fluorescence imaging. This ATMT system increases the transformation efficiency of H. marmoreus and may represent a useful tool for molecular genetic studies in this mushroom species. PMID:24612605

Zhang, Jin jing; Shi, Liang; Chen, Hui; Sun, Yun qi; Zhao, Ming wen; Ren, Ang; Chen, Ming jie; Wang, Hong; Feng, Zhi yong

2014-01-01

239

Agrobacterium-mediated transformation of Fraxinus pennsylvanica hypocotyls and plant regeneration.  

PubMed

A genetic transformation protocol for green ash (Fraxinus pennsylvanica) hypocotyl explants was developed. Green ash hypocotyls were transformed using Agrobacterium tumefaciens strain EHA105 harboring binary vector pq35GR containing the neomycin phosphotransferase (nptII) and beta-glucuronidase (GUS) fusion gene, and an enhanced green fluorescent protein gene. Pre-cultured hypocotyl explants were transformed in the presence of 100 microM acetosyringone using 90 s sonication plus 10 min vacuum-infiltration. Kanamycin at 20 mg l(-1) was used for selecting transformed cells. Adventitious shoots regenerated on Murashige and Skoog medium supplemented with 13.3 microM 6-benzylaminopurine, 4.5 microM thidiazuron, 50 mg l(-1) adenine sulfate, and 10% coconut water. GUS- and polymerase chain reaction (PCR)-positive shoots from the cut ends of hypocotyls were produced via an intermediate callus stage. Presence of the GUS and nptII genes in GUS-positive shoots were confirmed by PCR and copy number of the nptII gene in PCR-positive shoots was determined by Southern blotting. Three transgenic plantlets were acclimatized to the greenhouse. This transformation and regeneration system using hypocotyls provides a foundation for Agrobacterium-mediated transformation of green ash. Studies are underway using a construct containing the Cry8Da protein of Bacillus thuringiensis for genetic transformation of green ash. PMID:19343350

Du, Ningxia; Pijut, Paula M

2009-06-01

240

Improved Agrobacterium-mediated co-transformation and selectable marker elimination in transgenic rice by using a high copy number pBin19-derived binary vector.  

PubMed

A high copy number, selectable marker gene (SMG)-free Agrobacterium binary vector pBin19?nptII was constructed by deleting the nptII gene from pBin19. The binary vectors with the RK2 and pVS replication origins exist in 12 and 3 copies, respectively, in Agrobacterium. The tobacco osmotin gene (ap24) was cloned in pBin19?nptII and the resultant plasmid pBin19?nptII-ap24 was mobilized into the Agrobacterium tumefaciens strain C58C1 Rif(r) harbouring the single-copy cointegrate vector pGV2260::pSSJ1. The T-DNA of the cointegrate vector harboured the hph (SMG) and gus genes. Transformation of Oryza sativa L. var. Pusa Basmati1 with Agrobacterium tumefaciens (pGV2260::pSSJ1, pBin19?nptII-ap24) yielded 14 independent hyg+/GUS+ transgenic plants. Southern blot analysis with hph and ap24 probes revealed that 12 out of the 14 transgenic plants were co-transformed and harboured hph, gus and ap24 genes. The new multi-copy binary vector yielded 86% co-transformation efficiency. SMG elimination by genetic separation of the cointegrate T-DNA with the hph/gus genes and binary vector T-DNA with the ap24 gene was accomplished in four out of ten primary co-transformants that were forwarded to the T? generation. PMID:21497712

Sripriya, Rajasekaran; Sangeetha, Manoharan; Parameswari, Chidambaram; Veluthambi, Balamani; Veluthambi, Karuppannan

2011-06-01

241

Identification of two glutamine synthetases in Agrobacterium.  

PubMed Central

Two distinct glutamine synthetases have been identified in Agrobacterium and in the fast-growing rhizobia. A limited survey indicates that GSII may be found only in the Rhizobiaceae family. PMID:6102556

Fuchs, R L; Keister, D L

1980-01-01

242

Applications of optical manipulation in plant biology  

NASA Astrophysics Data System (ADS)

Measuring small forces in biology is important for determining basic physiological parameters of a cell. The plant cell wall provides a primary defense and presents a barrier to research. Magnitudes of small forces are impossible to measure with mechanical transducers, glass needles, atomic force microscopy, or micropipet-based force transduction due to the cell wall. Therefore, a noninvasive method of breaching the plant cell wall to access the symplastic region of the cell is required. Laser light provides sub-micrometer positioning, particle manipulation without mechanical contact, and piconewton force determination. Consequently, the extension of laser microsurgery to expand an experimental tool for plant biology encompassed the overall objective. A protocol was developed for precisely inserting microscopic objects into the periplasmic region of plant callus cells using laser microsurgery. Ginkgo biloba and Agrobacterium rhizogenes were used as the model system for developing the optical tweezers and scalpel techniques. Better than 95% survival was achieved after plasmolyzing G. biloba cells, ablating a 2-4 ?m hole through the cell wall using a pulsed UV laser beam, trapping and manipulating bacteria into the periplasmic region, and deplasmolyzing the cells. Optical trapping experiments implied a difference existed between the bacteria models. Determining the optical trapping efficiency of Agrobacterium rhizogenes and A. tumefaciens strains indicated the A. rhizogenes strain, ATCC 11325, was significantly less efficiently trapped than strains A4 and ATCC 15834 and the A. tumefaciens strain LBA4404. Differences were also found in capsule generation, growth media viscosity, and transmission electron microscopy negative staining implying that a difference in surface structure exists. Calcofluor fluorescence suggests the difference involves an exopolysaccharide. Callus cell plasmolysis revealed Hechtian strands interconnecting the plasma membrane and the cell wall. The spring tension of these strands was measured in normal and cold-hardened G. biloba and N. tabacum callus cells. There was little change in flexibility between the groups of cultured cells in either species studied. Microspheres were attached to Hechtian strands in normal cultured Nicotiana tabacum and the cells were deplasmolyzed and replasmolyzed to determine the fate of Hechtian strands. The microspheres either moved to the plasma membrane and adhered or moved to the cell wall and adhered. The attached microspheres occasionally moved independently on the same strand. Inserted microspheres provided a visual probe to follow physiological events within a plant cell.

Buer, Charles S.

243

Agrobacterium-derived cytokinin influences plastid morphology and starch accumulation in Nicotiana benthamiana during transient assays  

PubMed Central

Background Agrobacterium tumefaciens-based transient assays have become a common tool for answering questions related to protein localization and gene expression in a cellular context. The use of these assays assumes that the transiently transformed cells are observed under relatively authentic physiological conditions and maintain ‘normal’ sub-cellular behaviour. Although this premise is widely accepted, the question of whether cellular organization and organelle morphology is altered in Agrobacterium-infiltrated cells has not been examined in detail. The first indications of an altered sub-cellular environment came from our observation that a common laboratory strain, GV3101(pMP90), caused a drastic increase in stromule frequency. Stromules, or ‘stroma-filled-tubules’ emanate from the surface of plastids and are sensitive to a variety of biotic and abiotic stresses. Starting from this observation, the goal of our experiments was to further characterize the changes to the cell resulting from short-term bacterial infestation, and to identify the factor responsible for eliciting these changes. Results Using a protocol typical of transient assays we evaluated the impact of GV3101(pMP90) infiltration on chloroplast behaviour and morphology in Nicotiana benthamiana. Our experiments confirmed that GV3101(pMP90) consistently induces stromules and alters plastid position relative to the nucleus. These effects were found to be the result of strain-dependant secretion of cytokinin and its accumulation in the plant tissue. Bacterial production of the hormone was found to be dependant on the presence of a trans-zeatin synthase gene (tzs) located on the Ti plasmid of GV3101(pMP90). Bacteria-derived cytokinins were also correlated with changes to both soluble sugar level and starch accumulation. Conclusion Although we have chosen to focus on how transient Agrobacterium infestation alters plastid based parameters, these changes to the morphology and position of a single organelle, combined with the measured increases in sugar and starch content, suggest global changes to cell physiology. This indicates that cells visualized during transient assays may not be as ‘normal’ as was previously assumed. Our results suggest that the impact of the bacteria can be minimized by choosing Agrobacterium strains devoid of the tzs gene, but that the alterations to sub-cellular organization and cell carbohydrate status cannot be completely avoided using this strategy. PMID:24886417

2014-01-01

244

Elevated Temperature Differentially Affects Virulence, VirB Protein Accumulation, and T-Pilus Formation in Different Agrobacterium tumefaciens and Agrobacterium vitis Strains  

Microsoft Academic Search

That gene transfer to plant cells is a temperature-sensitive process has been known for more than 50 years. Previous work indicated that this sensitivity results from the inability to assemble a functional T pilus required for T-DNA and protein transfer to recipient cells. The studies reported here extend these observations and more clearly define the molecular basis of this assembly

CHRISTIAN BARON; NATALIE DOMKE; MICHAEL BEINHOFER; SIEGFRIED HAPFELMEIER

2001-01-01

245

Finger millet [Eleusine coracana (L.) Gaertn].  

PubMed

Millets are the primary food source for millions of people in tropical regions of the world supplying mineral nutrition and protein. In this chapter, we describe an optimized protocol for the Agrobacterium-mediated transformation of finger millet variety GPU 45. Agrobacterium strain LBA4404 harboring plasmid pCAMBIA1301 which contains hygromycin phosphotransferase (hph) as selectable marker gene and ?-glucuronidase (GUS) as reporter gene has been used. This protocol utilizes the shoot apex explants for the somatic embryogenesis and regeneration of finger millet after the transformation by Agrobacterium. Desiccation of explants during cocultivation helps for the better recovery of transgenic plants. This protocol is very useful for the efficient production of transgenic plants in finger millet through Agrobacterium-mediated transformation. PMID:25300836

Ceasar, Stanislaus Antony; Ignacimuthu, Savarimuthu

2015-01-01

246

Use of Agrobacterium expressing green fluorescent protein to evaluate colonization of sonication-assisted Agrobacterium-  

E-print Network

Agrobacterium for plant transformation is the organism's host speci¢city, resulting in low levels spectinomycin (Sigma, St Louis, MO, USA) were screened for GFPexpression with a Leica M8 stereo dissecting micro

Finer, John J.

247

Biological activity of the rolB-like 5' end of the A4-orf8 gene from the Agrobacterium rhizogenes TL-DNA.  

PubMed

The iaaM gene from different plant-associated bacteria encodes a tryptophan monooxygenase (IaaM) that catalyzes the synthesis of indole-3-acetamide (IAM), a precursor of indole-3-acetic acid (IAA). Unlike the IaaM proteins from other bacteria, Agrobacterium spp. T-DNA-encoded IaaM proteins carry a 200 amino acid N-terminal extension with low homology to various members of the RolB protein family. This family is composed of 18 highly divergent T-DNA-encoded proteins, the basic functions of which are still largely undetermined. Deletion of the 5' rolB-like extension of the iaaM gene from Agrobacterium tumefaciens strain Ach5 did not lead to a reduction in IAM synthesis in plants. When expressed in tobacco, the rolB-like fragment did not affect growth or morphology. An iaaM homolog (A4-orf8) from the TL-DNA of Agrobacterium rhizogenes strain A4 also was investigated. Neither the full-size A4-orf8 gene nor the 5'-truncated form induced detectable IAM synthesis. Plants expressing the rolB-like part of the A4-orf8 gene, however, were dwarfed and mottled to various extents and synthesized abnormally high amounts of glucose, fructose, sucrose, and starch. PMID:11277438

Otten, L; Helfer, A

2001-03-01

248

Nucleotide sequence analysis of TL-DNA of Agrobacterium rhizogenes agropine type plasmid. Identification of open reading frames.  

PubMed

We have determined the nucleotide sequence of the Ri TL-DNA region from an Agrobacterium rhizogenes agropine-type plasmid using subcloned regions from the essentially identical Ri TL-DNAs from strains A4 and HRI. This sequenced region of 21,126 base pairs (bp) contains the complete TL-DNA region of the Ri plasmid as determined by analysis of TL-DNA borders in the genome of infected, clonal, Convolvulus arvensis plants. The left and right borders of the TL-DNA are flanked by 25-bp sequences which match the 25-bp terminal sequences found near the borders of T-DNA regions of Agrobacterium tumefaciens Ti plasmids. Other DNA sequences similar to these 25-bp terminal sequences are found within the TL region, and some of these sequences appear to be associated with Ri TL-DNA structures found in transformed tobacco plants. The TL-DNA region contains 18 open reading frames, many of which have 5' and 3' regulatory elements similar to those found in eukaryotic genes. In many cases, CCAAT and TATA elements were found upstream from putative transcriptional initiation codons, and poly(A) addition (AATAAA) elements were observed in presumed 3'-noncoding regions. Comparison of Ri TL-DNA coding and noncoding sequence regions with T-DNA sequence regions from octopine type Ti plasmid pTi15955 reveals no extensive sequence homologies. PMID:3001043

Slightom, J L; Durand-Tardif, M; Jouanin, L; Tepfer, D

1986-01-01

249

Variable internal flexibility characterizes the helical capsid formed by agrobacterium VirE2 protein on single-stranded DNA.  

PubMed

Agrobacterium is known for gene transfer to plants. In addition to a linear ssDNA oligonucleotide, Agrobacterium tumefaciens secretes an abundant ssDNA-binding effector, VirE2. In many ways VirE2 adapts the conjugation mechanism to transform the eukaryotic host. The crystal structure of VirE2 shows two compact domains joined by a flexible linker. Bound to ssDNA, VirE2 forms an ordered solenoidal shell, or capsid known as the T-complex. Here, we present a three-dimensional reconstruction of the VirE2-ssDNA complex using cryo-electron microscopy and iterative helical real-space reconstruction. High-resolution refinement was not possible due to inherent heterogeneity in the protein structure. By a combination of computational modeling, chemical modifications, mass spectroscopy, and electron paramagnetic resonance, we found that the N-terminal domain is tightly constrained by both tangential and longitudinal links, while the C terminus is weakly constrained. The quaternary structure is thus rigidly assembled while remaining locally flexible. This flexibility may be important in accommodating substrates without sequence specificity. PMID:23769668

Bharat, Tanmay A M; Zbaida, David; Eisenstein, Miriam; Frankenstein, Ziv; Mehlman, Tevie; Weiner, Lev; Sorzano, Carlos Oscar S; Barak, Yoav; Albeck, Shira; Briggs, John A G; Wolf, Sharon G; Elbaum, Michael

2013-07-01

250

Analysis of Hydroxycinnamic Acid Degradation in Agrobacterium fabrum Reveals a Coenzyme A-Dependent, Beta-Oxidative Deacetylation Pathway  

PubMed Central

The soil- and rhizosphere-inhabiting bacterium Agrobacterium fabrum (genomospecies G8 of the Agrobacterium tumefaciens species complex) is known to have species-specific genes involved in ferulic acid degradation. Here, we characterized, by genetic and analytical means, intermediates of degradation as feruloyl coenzyme A (feruloyl-CoA), 4-hydroxy-3-methoxyphenyl-?-hydroxypropionyl–CoA, 4-hydroxy-3-methoxyphenyl-?-ketopropionyl–CoA, vanillic acid, and protocatechuic acid. The genes atu1416, atu1417, and atu1420 have been experimentally shown to be necessary for the degradation of ferulic acid. Moreover, the genes atu1415 and atu1421 have been experimentally demonstrated to be essential for this degradation and are proposed to encode a phenylhydroxypropionyl-CoA dehydrogenase and a 4-hydroxy-3-methoxyphenyl-?-ketopropionic acid (HMPKP)–CoA ?-keto-thiolase, respectively. We thus demonstrated that the A. fabrum hydroxycinnamic degradation pathway is an original coenzyme A-dependent ?-oxidative deacetylation that could also transform p-coumaric and caffeic acids. Finally, we showed that this pathway enables the metabolism of toxic compounds from plants and their use for growth, likely providing the species an ecological advantage in hydroxycinnamic-rich environments, such as plant roots or decaying plant materials. PMID:24657856

Campillo, Tony; Renoud, Sébastien; Kerzaon, Isabelle; Vial, Ludovic; Baude, Jessica; Gaillard, Vincent; Bellvert, Floriant; Chamignon, Cécile; Comte, Gilles; Lavire, Céline; Hommais, Florence

2014-01-01

251

Potassium chloride and rare earth elements improve plant growth and increase the frequency of the Agrobacterium tumefaciens -mediated plant transformation  

Microsoft Academic Search

Plant transformation efficiency depends on the ability of the transgene to successfully interact with plant host factors.\\u000a Our previous work and the work of others showed that manipulation of the activity of host factors allows for increased frequency\\u000a of transformation. Recently we reported that exposure of tobacco plants to increased concentrations of ammonium nitrate increases\\u000a the frequency of both homologous

Alex Boyko; Aki Matsuoka; Igor Kovalchuk

2011-01-01

252

Transformation of five grape rootstocks with plant virus genes and a virE2 gene from Agrobacterium tumefaciens  

Microsoft Academic Search

Summary  To facilitate the development of transgenic grapevines that are resistant to grapevine fanleaf virus (GFLV), grapevine leafroll-associated\\u000a closterovirus (GLRaV-3) and crown gall diseases, we developed a rapid system for regenerating root-stocks: Couderc 3309, Vitis riparia ‘Gloire de Montpellier’, Teleki 5C, Millardet et De Grasset 101-14, and 110 Richter via somatic embryogenesis. Embryo culture\\u000a and grape regeneration were accomplished with four

B. Xue; K. S. Ling; C. L. Reid; S. Krastanova; M. Sekiya; E. A. Momol; S. Süle; J. Mozsar; D. Gonsalves; T. J. Burr

1999-01-01

253

Relatedness Among Rhizobium and Agrobacterium Species Determined by Three Methods of Nucleic Acid Hybridization  

PubMed Central

Deoxyribonucleic acid (DNA) was isolated from 20 strains of Rhizobium and Agrobacterium and from one strain of Serratia marcescens; the guanine plus cytosine content of each DNA sample was determined by thermal denaturation. Radioactive DNA was isolated from three reference strains following the uptake of [2-14C]thymidine in the presence of deoxyadenosine. Ribonucleic acid (RNA) polymerase was used to synthesize radioactive RNA on DNA templates from the three reference strains. Radioactive DNA and RNA from the three reference strains were each hybridized with filter-bound DNA from all of the 21 test strains in 6 × SSC (standard saline citrate) and 50% formamide at 43 C for 40 hr. DNA/DNA relatedness was also determined by spectrophotometric measurement of the rates of association of single-stranded DNA. The order of relatedness between strains was similar by each method. Overall standard deviations for the DNA/DNA and DNA/RNA membrane filter techniques were ±0.87 and ±1.03%, respectively; that for the spectrophotometric technique was ±4.11%. The DNA/DNA membrane technique gave higher absolute values of hybridization than did the DNA/RNA technique. R. leguminosarum and R. trifolii could not be distinguished from each other by these techniques. These results also indicated close relationships between R. lupini and R. japonicum, and (with less certainty) between R. meliloti and R. phaseoli. Of all the rhizobia tested against the A. tumefaciens 371 reference strain, the R. japonicum strains were the most unrelated. The three Agrobacterium strains used were as related to the R. lupini and R. leguminosarum references as were several rhizobium strains. PMID:4591471

Gibbins, Ann M.; Gregory, K. F.

1972-01-01

254

Agrobacterium: nature’s genetic engineer  

PubMed Central

Agrobacterium was identified as the agent causing the plant tumor, crown gall over 100 years ago. Since then, studies have resulted in many surprising observations. Armin Braun demonstrated that Agrobacterium infected cells had unusual nutritional properties, and that the bacterium was necessary to start the infection but not for continued tumor development. He developed the concept of a tumor inducing principle (TIP), the factor that actually caused the disease. Thirty years later the TIP was shown to be a piece of a tumor inducing (Ti) plasmid excised by an endonuclease. In the next 20 years, most of the key features of the disease were described. The single-strand DNA (T-DNA) with the endonuclease attached is transferred through a type IV secretion system into the host cell where it is likely coated and protected from nucleases by a bacterial secreted protein to form the T-complex. A nuclear localization signal in the endonuclease guides the transferred strand (T-strand), into the nucleus where it is integrated randomly into the host chromosome. Other secreted proteins likely aid in uncoating the T-complex. The T-DNA encodes enzymes of auxin, cytokinin, and opine synthesis, the latter a food source for Agrobacterium. The genes associated with T-strand formation and transfer (vir) map to the Ti plasmid and are only expressed when the bacteria are in close association with a plant. Plant signals are recognized by a two-component regulatory system which activates vir genes. Chromosomal genes with pleiotropic functions also play important roles in plant transformation. The data now explain Braun’s old observations and also explain why Agrobacterium is nature’s genetic engineer. Any DNA inserted between the border sequences which define the T-DNA will be transferred and integrated into host cells. Thus, Agrobacterium has become the major vector in plant genetic engineering. PMID:25610442

Nester, Eugene W.

2015-01-01

255

A high-throughput Agrobacterium-mediated transformation system for the grass model species Brachypodium distachyon L.  

PubMed

In the ongoing process of developing Brachypodium distachyon as a model plant for temperate cereals and forage grasses, we have developed a high-throughput Agrobacterium-mediated transformation system for a diploid accession. Embryogenic callus, derived from immature embryos of the accession BDR018, were transformed with Agrobacterium tumefaciens strain AGL1 carrying two T-DNA plasmids, pDM805 and pWBV-Ds-Ubi-bar-Ds. Transient and stable transformation efficiencies were optimised by varying the pre-cultivation period, which had a strong effect on stable transformation efficiency. On average 55% of 17-day-old calli co-inoculated with Agrobacterium regenerated stable transgenic plants. Stable transformation frequencies of up to 80%, which to our knowledge is the highest transformation efficiency reported in graminaceous species, were observed. In a study of 177 transgenic lines transformed with pDM805, all of the regenerated transgenic lines were resistant to BASTA, while the gusA gene was expressed in 88% of the transgenic lines. Southern blot analysis revealed that 35% of the tested plants had a single T-DNA integration. Segregation analysis performed on progenies of ten selected T(0) plants indicated simple Mendelian inheritance of the two transgenes. Furthermore, the presence of two selection marker genes, bar and hpt, on the T-DNA of pWBV-Ds-Ubi-bar-Ds allowed us to characterize the developed transformation protocol with respect to full-length integration rate. Even when not selected for, full-length integration occurred in 97% of the transformants when using bialaphos as selection agent. PMID:18064538

P?curar, Daniel Ioan; Thordal-Christensen, Hans; Nielsen, Klaus Kristian; Lenk, Ingo

2008-10-01

256

Iron-Binding Compounds from Agrobacterium spp.: Biological Control Strain Agrobacterium rhizogenes K84 Produces a Hydroxamate Siderophore  

PubMed Central

Iron-binding compounds were produced in various amounts in response to iron starvation by a collection of Agrobacterium strains belonging to the species A. tumefaciens, A. rhizogenes, and A. vitis. The crown gall biocontrol agent A. rhizogenes strain K84 produced a hydroxamate iron chelator in large amounts. Production of this compound, and also of a previously described antibiotic-like substance called ALS84, occurred only in cultures of strain K84 grown in iron-deficient medium. Similarly, sensitivity to ALS84 was expressed only when susceptible cells were tested in low-iron media. Five independent Tn5-induced mutants of strain K84 affected in the production of the hydroxamate iron chelator showed a similar reduction in the production of ALS84. One of these mutants, M8-10, was completely deficient in the production of both agents and grew poorly compared to the wild type under iron-limiting conditions. Thus, the hydroxamate compound has siderophore activity. A 9.1-kb fragment of chromosomal DNA containing the Tn5 insertion from this mutant was cloned and marker exchanged into wild-type strain K84. The homogenote lost the ability to produce the hydroxamate siderophore and also ALS84. A cosmid clone was isolated from a genomic library of strain K84 that restored to strain M8-10 the ability to produce of the siderophore and ALS84, as well as growth in iron-deficient medium. This cosmid clone contained the region in which Tn5 was located in the mutant. Sequence analysis showed that the Tn5 insert in this mutant was located in an open reading frame coding for a protein that has similarity to those of the gramicidin S synthetase repeat superfamily. Some such proteins are required for synthesis of hydroxamate siderophores by other bacteria. Southern analysis revealed that the biosynthetic gene from strain K84 is present only in isolates of A. rhizogenes that produce hydroxamate-type compounds under low-iron conditions. Based on physiological and genetic analyses showing a correlation between production of a hydroxamate siderophore and ALS84 by strain K84, we conclude that the two activities share a biosynthetic route and may be the same compound. PMID:11157228

Penyalver, Ramón; Oger, Philippe; López, María M.; Farrand, Stephen K.

2001-01-01

257

Agrobacterium Uses a Unique Ligand-Binding Mode for Trapping Opines and Acquiring A Competitive Advantage in the Niche Construction on Plant Host  

PubMed Central

By modifying the nuclear genome of its host, the plant pathogen Agrobacterium tumefaciens induces the development of plant tumours in which it proliferates. The transformed plant tissues accumulate uncommon low molecular weight compounds called opines that are growth substrates for A. tumefaciens. In the pathogen-induced niche (the plant tumour), a selective advantage conferred by opine assimilation has been hypothesized, but not experimentally demonstrated. Here, using genetics and structural biology, we deciphered how the pathogen is able to bind opines and use them to efficiently compete in the plant tumour. We report high resolution X-ray structures of the periplasmic binding protein (PBP) NocT unliganded and liganded with the opine nopaline (a condensation product of arginine and ?-ketoglurate) and its lactam derivative pyronopaline. NocT exhibited an affinity for pyronopaline (KD of 0.6 µM) greater than that for nopaline (KD of 3.7 µM). Although the binding-mode of the arginine part of nopaline/pyronopaline in NocT resembled that of arginine in other PBPs, affinity measurement by two different techniques showed that NocT did not bind arginine. In contrast, NocT presented specific residues such as M117 to stabilize the bound opines. NocT relatives that exhibit the nopaline/pyronopaline-binding mode were only found in genomes of the genus Agrobacterium. Transcriptomics and reverse genetics revealed that A. tumefaciens uses the same pathway for assimilating nopaline and pyronopaline. Fitness measurements showed that NocT is required for a competitive colonization of the plant tumour by A. tumefaciens. Moreover, even though the Ti-plasmid conjugal transfer was not regulated by nopaline, the competitive advantage gained by the nopaline-assimilating Ti-plasmid donors led to a preferential horizontal propagation of this Ti-plasmid amongst the agrobacteria colonizing the plant-tumour niche. This work provided structural and genetic evidences to support the niche construction paradigm in bacterial pathogens. PMID:25299655

Planamente, Sara; El Sahili, Abbas; Blin, Pauline; Aumont-Nicaise, Magali; Dessaux, Yves; Moréra, Solange; Faure, Denis

2014-01-01

258

Horticultural characteristics of transgenic tobacco expressing the rolC gene from Agrobacterium rhizogenes  

SciTech Connect

Wisconsin 38 tobacco (Nicotiana tabacum L.) leaf discs were transformed with the disarmed Agrobacterium tumefaciens strain EHA 101 carrying the rolC gene from A. rhizogenes and NPT II and GUS genes. Shoots that regenerated on kanamycin-containing medium were confirmed as transgenic through GUS assays, polymerase chain reaction (PCR), Southern blot analyses, and transmission of the foreign genes through the sexual cycle. Transgenic plants were as short as half the height of control plants; were earlier flowering by up to 35 days; and had smaller leaves, shorter internodes, smaller seed capsules, fewer seeds, smaller flowers, and reduced pollen viability. The number of seed capsules, leaf number, and specific root length were similar between transgenic and control plants. Transgenic clones varied in the expression of the rolC-induced growth alterations as did the first generation of seedlings from these clones. Such differences suggested the potential for selecting for different levels of expression. Transformation with the rolC gene presents a potentially useful method of genetically modifying horticultural crops, particularly for flowering date, height, and leaf and flower size. Chemical names used: neomycin phosphotransferase (NPTII), [beta]-glucuronidase (GUS).

Scorza, R.; Zimmerman, T.W.; Cordts, J.M.; Footen, K.J. (Dept. of Agriculture, Kearneysville, WV (United States)); Ravelonandro, M. (Inst. National Recherche Agronomique, Villenave d'Ornon (France). Station de Pathologie Vegetale)

1994-09-01

259

Physical, Chemical, Developmental, and Genetic Factors that Modulate the Agrobacterium-Vitis Interaction 1  

PubMed Central

Tumor formation in Vitis species and hybrids, incited by Agrobacterium tumefaciens, was altered by chemical, physical, developmental, and genetic variables. Knowledge of the effect of these variables was used to develop a stringent in vitro assay system to select parents for a study of genetic factors that modulate tumor formation. Tumor formation was reduced by short day preconditioning of assay plants and by inoculation of the morphological apex of isolated stem segments. Pretreatment of plants with auxin or cytokinin altered specificity in various combinations of strains and host genotypes. All Vitis species and hybrids formed tumors in response to strains designated as limited host range, but some displayed a necrotic reaction (cell death at and below site of inoculation) or a null response (same as the response to inoculation with an avirulent strain) to strains designated as wide host range (VC Knauf, CG Panagopoulos, EW Nester [1982] Phytopathology 72: 1545-1549). Screens of F1 progeny, derived from crosses of null, necrotic, and tumor-producing phenotypes, demonstrated that the null and the necrotic phenotypes were modulated by dominant and recessive host genes. The extent of cellular necrosis in the necrotic phenotype was modified by the morphological location of the inoculation site, by the presence of buds on the host stem, and by deletion of the tryptophane monooxygenase locus gene of the Ti-plasmid. ImagesFigure 2Figure 3 PMID:16668140

Lowe, Brenda Ann; Krul, William Robert

1991-01-01

260

Efficient soybean regeneration and Agrobacterium-mediated transformation using a whole cotyledonary node as an explant.  

PubMed

An optimized regeneration and Agrobacterium-mediated transformation protocol based on whole cotyledonary node explants was developed in soybean (Glycine max) cultivar Zhong Huang 13. Adding 6-benzylaminopurine (BAP) in a germinating medium could significantly increase regeneration efficiency; the optimal BAP concentration for shoot formation was 0.5 mg/L. The concentrations of plant growth regulators in a shoot induction medium were optimized by the orthogonal test [L9 (3(3))]. The best combination for shoot regeneration was a medium of Murashige & Skoog salts with B5 vitamins (MSB) supplemented with 3.5 mg/L BAP, 0.2 mg/L indole-3-butyric acid (IBA), and 0.2 mg/L kinetin (KT). Under this favorable condition, one node could regenerate 28-30 shoots. Soybean whole cotyledonary nodes were transformed by inoculation with A. tumefaciens strain EHA105 harboring a vector pBI121 containing a ?-glucuronidase gene (gus). GUS assay, polymerase chain reaction, and Southern blot analysis indicated that the gus gene was transformed into soybean plants with 23.1% transformation efficiency. Transgenic plants could be obtained within 5-6 weeks, which was about 4 weeks less than that of a traditional single cotyledonary node method. PMID:24974933

Zhang, Fuli; Chen, Can; Ge, Honglian; Liu, Jinmei; Luo, Yunling; Liu, Kun; Chen, Long; Xu, Kedong; Zhang, Yi; Tan, Guangxuan; Li, Chengwei

2014-01-01

261

Gene manipulation of a heavy metal hyperaccumulator species Thlaspi caerulescens L. via Agrobacterium-mediated transformation.  

PubMed

Thlaspi caerulescens L. is well known as a Zn/Cd hyperaccumulator. The genetic manipulation of T. caerulescens through transgenic technology can modify plant features for use in phytoremediation. Here, we describe the efficient transformation of T. caerulescens using Agrobacterium tumefaciens strain EHA105 harboring a binary vector pBI121 with the nptII gene as a selectable marker, the gus gene as a reporter and a foreign catalase gene. Based on the optimal concentration of growth regulators, the shoot cluster regeneration system via callus phase provided the basis of the genetic transformation in T. caerulescens. The key variables in transformation were examined, such as co-cultivation period and bacterial suspension density. Optimizing factors for T-DNA delivery resulted in kanamycin-resistant transgenic shoots with transformation efficiency more than 20%, proven by histochemical GUS assay and PCR analysis. Southern analysis of nptII and RT-PCR of catalase gene demonstrated that the foreign genes were integrated in the genome of transformed plantlets. Moreover, the activity of catalase enzyme in transgenic plants was obviously higher than in wild-type plants. This method offers new prospects for the genetic engineering of this important hyperaccumulator species. PMID:18427996

Guan, Zi Qiu; Chai, Tuan Yao; Zhang, Yu Xiu; Xu, Jin; Wei, Wei; Han, Lu; Cong, Lin

2008-09-01

262

Agrobacterium-mediated genetic transformation of the desiccation tolerant resurrection plant Ramonda myconi (L.) Rchb.  

PubMed

In this paper we describe the first procedure for Agrobacterium tumefaciens-mediated genetic transformation of the desiccation tolerant plant Ramonda myconi (L.) Rchb. Previously, we reported the establishment of a reliable and effective tissue culture system based on the integrated optimisation of antioxidant and growth regulator composition and the stabilisation of the pH of the culture media by means of a potassium phosphate buffer. This efficient plant regeneration via callus phase provided a basis for the optimisation of the genetic transformation in R. myconi. For gene delivery, both a standard (method A) and a modified protocol (method B) have been applied. Since the latter has previously resulted in successful transformation of another resurrection plant, Craterostigma plantagineum, an identical protocol was utilized in transformation of R. myconi, as this method may prove general for dicotyledonous resurrection plants. On this basis, physical and biochemical key variables in transformation were evaluated such as mechanical microwounding of plant explants and in vitro preinduction of vir genes. While the physical enhancement of bacterial penetration was proved to be essential for successful genetic transformation of R. myconi, an additional two-fold increase in the transformation frequency was obtained when the above physical and biochemical treatments were applied in combination. All R0 and R1 transgenic plants were fertile, and no morphological abnormalities were observed on the whole-plant level. PMID:16362301

Tóth, Sándor; Kiss, Csaba; Scott, Peter; Kovács, Gabriella; Sorvari, Seppo; Toldi, Ottó

2006-05-01

263

Systematic Dissection of the Agrobacterium Type VI Secretion System Reveals Machinery and Secreted Components for Subcomplex Formation  

PubMed Central

The type VI secretion system (T6SS) is widely distributed in pathogenic Proteobacteria. Sequence and structural analysis of T6SS reveals a resemblance to the T4 bacteriophage tail, in which an outer sheath structure contracts an internal tube for injecting nucleic acid into bacterial cells. However, the molecular details of how this phage tail-like T6SS structure is assembled in vivo and executed for exoprotein or effector secretion remain largely unknown. Here, we used a systematic approach to identify T6SS machinery and secreted components and investigate the interaction among the putative sheath and tube components of Agrobacterium tumefaciens. We showed that 14 T6SS components play essential roles in the secretion of the T6SS hallmark exoprotein Hcp. In addition, we discovered a novel T6SS exoprotein, Atu4347, that is dispensable for Hcp secretion. Interestingly, Atu4347 and the putative tube components, Hcp and VgrG, are mainly localized in the cytoplasm but also detected on the bacterial surface. Atu4342 (TssB) and Atu4341 (TssC41) interact with and stabilize each other, which suggests that they are functional orthologs of the sheath components TssB (VipA) and TssC (VipB), respectively. Importantly, TssB interacts directly with the three exoproteins (Hcp, VgrG, and Atu4347), in which Hcp also interacts directly with VgrG-1 on co-purification from Escherichia coli. Further co-immunoprecipitation and pulldown assays revealed these subcomplex(es) in A. tumefaciens and thereby support T6SS functioning as a contractile phage tail-like structure. PMID:23861778

Lin, Jer-Sheng; Ma, Lay-Sun; Lai, Erh-Min

2013-01-01

264

Dimerization of VirD2 binding protein is essential for Agrobacterium induced tumor formation in plants.  

PubMed

The Type IV Secretion System (T4SS) is the only bacterial secretion system known to translocate both DNA and protein substrates. The VirB/D4 system from Agrobacterium tumefaciens is a typical T4SS. It facilitates the bacteria to translocate the VirD2-T-DNA complex to the host cell cytoplasm. In addition to protein-DNA complexes, the VirB/D4 system is also involved in the translocation of several effector proteins, including VirE2, VirE3 and VirF into the host cell cytoplasm. These effector proteins aid in the proper integration of the translocated DNA into the host genome. The VirD2-binding protein (VBP) is a key cytoplasmic protein that recruits the VirD2-T-DNA complex to the VirD4-coupling protein (VirD4 CP) of the VirB/D4 T4SS apparatus. Here, we report the crystal structure and associated functional studies of the C-terminal domain of VBP. This domain mainly consists of ?-helices, and the two monomers of the asymmetric unit form a tight dimer. The structural analysis of this domain confirms the presence of a HEPN (higher eukaryotes and prokaryotes nucleotide-binding) fold. Biophysical studies show that VBP is a dimer in solution and that the HEPN domain is the dimerization domain. Based on structural and mutagenesis analyses, we show that substitution of key residues at the interface disrupts the dimerization of both the HEPN domain and full-length VBP. In addition, pull-down analyses show that only dimeric VBP can interact with VirD2 and VirD4 CP. Finally, we show that only Agrobacterium harboring dimeric full-length VBP can induce tumors in plants. This study sheds light on the structural basis of the substrate recruiting function of VBP in the T4SS pathway of A. tumefaciens and in other pathogenic bacteria employing similar systems. PMID:24626239

Padavannil, Abhilash; Jobichen, Chacko; Qinghua, Yang; Seetharaman, Jayaraman; Velazquez-Campoy, Adrian; Yang, Liu; Pan, Shen Q; Sivaraman, J

2014-03-01

265

Dimerization of VirD2 Binding Protein Is Essential for Agrobacterium Induced Tumor Formation in Plants  

PubMed Central

The Type IV Secretion System (T4SS) is the only bacterial secretion system known to translocate both DNA and protein substrates. The VirB/D4 system from Agrobacterium tumefaciens is a typical T4SS. It facilitates the bacteria to translocate the VirD2-T-DNA complex to the host cell cytoplasm. In addition to protein-DNA complexes, the VirB/D4 system is also involved in the translocation of several effector proteins, including VirE2, VirE3 and VirF into the host cell cytoplasm. These effector proteins aid in the proper integration of the translocated DNA into the host genome. The VirD2-binding protein (VBP) is a key cytoplasmic protein that recruits the VirD2–T-DNA complex to the VirD4-coupling protein (VirD4 CP) of the VirB/D4 T4SS apparatus. Here, we report the crystal structure and associated functional studies of the C-terminal domain of VBP. This domain mainly consists of ?-helices, and the two monomers of the asymmetric unit form a tight dimer. The structural analysis of this domain confirms the presence of a HEPN (higher eukaryotes and prokaryotes nucleotide-binding) fold. Biophysical studies show that VBP is a dimer in solution and that the HEPN domain is the dimerization domain. Based on structural and mutagenesis analyses, we show that substitution of key residues at the interface disrupts the dimerization of both the HEPN domain and full-length VBP. In addition, pull-down analyses show that only dimeric VBP can interact with VirD2 and VirD4 CP. Finally, we show that only Agrobacterium harboring dimeric full-length VBP can induce tumors in plants. This study sheds light on the structural basis of the substrate recruiting function of VBP in the T4SS pathway of A. tumefaciens and in other pathogenic bacteria employing similar systems. PMID:24626239

Padavannil, Abhilash; Jobichen, Chacko; Qinghua, Yang; Seetharaman, Jayaraman; Velazquez-Campoy, Adrian; Yang, Liu; Pan, Shen Q.; Sivaraman, J.

2014-01-01

266

Agrobacterium-Mediated Transformation of Tomato with rolB Gene Results in Enhancement of Fruit Quality and Foliar Resistance against Fungal Pathogens  

PubMed Central

Tomato (Solanum lycopersicum L.) is the second most important cultivated crop next to potato, worldwide. Tomato serves as an important source of antioxidants in human diet. Alternaria solani and Fusarium oxysporum cause early blight and vascular wilt of tomato, respectively, resulting in severe crop losses. The foremost objective of the present study was to generate transgenic tomato plants with rolB gene and evaluate its effect on plant morphology, nutritional contents, yield and resistance against fungal infection. Tomato cv. Rio Grande was transformed via Agrobacterium tumefaciens harbouring rolB gene of Agrobacterium rhizogenes. rolB. Biochemical analyses showed considerable improvement in nutritional quality of transgenic tomato fruits as indicated by 62% increase in lycopene content, 225% in ascorbic acid content, 58% in total phenolics and 26% in free radical scavenging activity. Furthermore, rolB gene significantly improved the defence response of leaves of transgenic plants against two pathogenic fungal strains A. solani and F. oxysporum. Contrarily, transformed plants exhibited altered morphology and reduced fruit yield. In conclusion, rolB gene from A. rhizogenes can be used to generate transgenic tomato with increased nutritional contents of fruits as well as improved foliar tolerance against fungal pathogens. PMID:24817272

Arshad, Waheed; Haq, Ihsan-ul-; Waheed, Mohammad Tahir; Mysore, Kirankumar S.; Mirza, Bushra

2014-01-01

267

Agrobacterium-mediated transformation of tomato with rolB gene results in enhancement of fruit quality and foliar resistance against fungal pathogens.  

PubMed

Tomato (Solanum lycopersicum L.) is the second most important cultivated crop next to potato, worldwide. Tomato serves as an important source of antioxidants in human diet. Alternaria solani and Fusarium oxysporum cause early blight and vascular wilt of tomato, respectively, resulting in severe crop losses. The foremost objective of the present study was to generate transgenic tomato plants with rolB gene and evaluate its effect on plant morphology, nutritional contents, yield and resistance against fungal infection. Tomato cv. Rio Grande was transformed via Agrobacterium tumefaciens harbouring rolB gene of Agrobacterium rhizogenes. rolB. Biochemical analyses showed considerable improvement in nutritional quality of transgenic tomato fruits as indicated by 62% increase in lycopene content, 225% in ascorbic acid content, 58% in total phenolics and 26% in free radical scavenging activity. Furthermore, rolB gene significantly improved the defence response of leaves of transgenic plants against two pathogenic fungal strains A. solani and F. oxysporum. Contrarily, transformed plants exhibited altered morphology and reduced fruit yield. In conclusion, rolB gene from A. rhizogenes can be used to generate transgenic tomato with increased nutritional contents of fruits as well as improved foliar tolerance against fungal pathogens. PMID:24817272

Arshad, Waheed; Haq, Ihsan-ul-; Waheed, Mohammad Tahir; Mysore, Kirankumar S; Mirza, Bushra

2014-01-01

268

Agrobacterium-mediated genetic transformation of Coffea arabica (L.) is greatly enhanced by using established embryogenic callus cultures  

PubMed Central

Background Following genome sequencing of crop plants, one of the main challenges today is determining the function of all the predicted genes. When gene validation approaches are used for woody species, the main obstacle is the low recovery rate of transgenic plants from elite or commercial cultivars. Embryogenic calli have frequently been the target tissue for transformation, but the difficulty in producing or maintaining embryogenic tissues is one of the main problems encountered in genetic transformation of many woody plants, including Coffea arabica. Results We identified the conditions required for successful long-term proliferation of embryogenic cultures in C. arabica and designed a highly efficient and reliable Agrobacterium tumefaciens-mediated transformation method based on these conditions. The transformation protocol with LBA1119 harboring pBin 35S GFP was established by evaluating the effect of different parameters on transformation efficiency by GFP detection. Using embryogenic callus cultures, co-cultivation with LBA1119 OD600 = 0.6 for five days at 20 °C enabled reproducible transformation. The maintenance conditions for the embryogenic callus cultures, particularly a high auxin to cytokinin ratio, the age of the culture (optimum for 7-10 months of proliferation) and the use of a yellow callus phenotype, were the most important factors for achieving highly efficient transformation (> 90%). At the histological level, successful transformation was related to the number of proembryogenic masses present. All the selected plants were proved to be transformed by PCR and Southern blot hybridization. Conclusion Most progress in increasing transformation efficiency in coffee has been achieved by optimizing the production conditions of embryogenic cultures used as target tissues for transformation. This is the first time that a strong positive effect of the age of the culture on transformation efficiency was demonstrated. Our results make Agrobacterium-mediated transformation of embryogenic cultures a viable and useful tool both for coffee breeding and for the functional analysis of agronomically important genes. PMID:21595964

2011-01-01

269

Expression and Functional Characterization of the Agrobacterium VirB2 Amino Acid Substitution Variants in T-pilus Biogenesis, Virulence, and Transient Transformation Efficiency  

PubMed Central

Agrobacterium tumefaciens is a phytopathogenic bacterium that causes crown gall disease by transferring transferred DNA (T-DNA) into the plant genome. The translocation process is mediated by the type IV secretion system (T4SS) consisting of the VirD4 coupling protein and 11 VirB proteins (VirB1 to VirB11). All VirB proteins are required for the production of T-pilus, which consists of processed VirB2 (T-pilin) and VirB5 as major and minor subunits, respectively. VirB2 is an essential component of T4SS, but the roles of VirB2 and the assembled T-pilus in Agrobacterium virulence and the T-DNA transfer process remain unknown. Here, we generated 34 VirB2 amino acid substitution variants to study the functions of VirB2 involved in VirB2 stability, extracellular VirB2/T-pilus production and virulence of A. tumefaciens. From the capacity for extracellular VirB2 production (ExB2+ or ExB2?) and tumorigenesis on tomato stems (Vir+ or Vir?), the mutants could be classified into three groups: ExB2?/Vir?, ExB2?/Vir+, and ExB2+/Vir+. We also confirmed by electron microscopy that five ExB2?/Vir+ mutants exhibited a wild-type level of virulence with their deficiency in T-pilus formation. Interestingly, although the five T-pilus?/Vir+ uncoupling mutants retained a wild-type level of tumorigenesis efficiency on tomato stems and/or potato tuber discs, their transient transformation efficiency in Arabidopsis seedlings was highly attenuated. In conclusion, we have provided evidence for a role of T-pilus in Agrobacterium transformation process and have identified the domains and amino acid residues critical for VirB2 stability, T-pilus biogenesis, tumorigenesis, and transient transformation efficiency. PMID:24971727

Wu, Hung-Yi; Chen, Chao-Ying; Lai, Erh-Min

2014-01-01

270

Plant viral vectors for delivery by Agrobacterium.  

PubMed

Plant viral vectors delivered by Agrobacterium are the basis of several manufacturing processes that are currently in use for producing a wide range of proteins for multiple applications, including vaccine antigens, antibodies, protein nanoparticles such as virus-like particles (VLPs), and other protein and protein-RNA scaffolds. Viral vectors delivered by agrobacterial T-DNA transfer (magnifection) have also become important tools in research. In recent years, essential advances have been made both in the development of second-generation vectors designed using the 'deconstructed virus' approach, as well as in the development of upstream manufacturing processes that are robust and fully scalable. The strategy relies on Agrobacterium as a vector to deliver DNA copies of one or more viral RNA/DNA replicons; the bacteria are delivered into leaves by vacuum infiltration, and the viral machinery takes over from the point of T-DNA transfer to the plant cell nucleus, driving massive RNA and protein production and, if required, cell-to-cell spread of the replicons. Among the most often used viral backbones are those of the RNA viruses Tobacco mosaic virus (TMV), Potato virus X (PVX) and Cowpea mosaic virus (CPMV), and the DNA geminivirus Bean yellow dwarf virus. Prototypes of industrial processes that provide for high yield, rapid scale up and fast manufacturing cycles have been designed, and several GMP-compliant and GMP-certified manufacturing facilities are in place. These efforts have been successful as evidenced by the fact that several antibodies and vaccine antigens produced by magnifection are currently in clinical development. PMID:23949286

Gleba, Yuri Y; Tusé, Daniel; Giritch, Anatoli

2014-01-01

271

Isolation and identification of TL-DNA/plant junctions in Convolvulus arvensis transformed by Agrobacterium rhizogenes strain A4.  

PubMed

We have constructed a Charon 4A phage library containing insert DNA isolated from a morning glory (Convolvulus arvensis) plant (clone 7) regenerated from a root organ culture incited by Agrobacterium rhizogenes, strain A4. Using a subcloned region of the Ri plasmid as P-labeled probe, two lambda clones containing most of the 'left' T-DNA (TL) region were isolated. One of these lambda clones contains the left TL-DNA/plant junction, which was located by comparing nucleotide sequences from the appropriate regions of the Ri plasmid and this lambda clone. A 25-bp sequence found near this left TL-DNA/plant junction matches the 25-bp terminal sequence found at or near T-DNA/plant junctions of both nopaline- and octopine-type A. tumefaciens Ti plasmids. A possible location for the right Ri TL-DNA/plant junction in C. arvensis clone 7 was found by obtaining the nucleotide sequence surrounding its mapped location. Hybridization of plant DNA found adjacent to the left TL-DNA/plant junction against total C. arvensis DNA shows that this T-DNA integration occurred in a plant DNA region that does not contain highly repetitive DNA sequences. Nucleotide sequence analysis of 1004 bp of this plant DNA revealed no complete or partial open reading frames, but this plant DNA does have the potential to form various secondary structures which might play a role in the T-DNA integration event. PMID:16453649

Slightom, J L; Jouanin, L; Leach, F; Drong, R F; Tepfer, D

1985-12-01

272

Vitreoscilla hemoglobin promotes Salecan production by Agrobacterium sp. ZX09*  

PubMed Central

Salecan is a novel exopolysaccharide produced by the strain Agrobacterium sp. ZX09, and it is composed of only glucose monomers. The unique chemical composition and excellent physicochemical properties make Salecan a promising material for applications in coagulation, lubrication, protection against acute liver injury, and alleviating constipation. In this study, we cloned the Vitreoscilla hemoglobin gene into a broad-host-range plasmid pCM158. Without antibiotic selection, there was negligible loss of the plasmid in the host Agrobacterium sp. ZX09 after one passage of cultivation. The expression of Vitreoscilla hemoglobin was demonstrated by carbon monoxide (CO) difference spectrum. The engineered strain Agrobacterium sp. ZX09 increased Salecan yield by 30%. The other physiological changes included its elevated respiration rate and cellular invertase activity. PMID:25367790

Chen, Yun-mei; Xu, Hai-yang; Wang, Yang; Zhang, Jian-fa; Wang, Shi-ming

2014-01-01

273

A Putative Transmembrane Leucine Zipper of Agrobacterium VirB10 Is Essential for T-Pilus Biogenesis but Not Type IV Secretion  

PubMed Central

The Agrobacterium tumefaciens VirB/VirD4 type IV secretion system is composed of a translocation channel and an extracellular T pilus. Bitopic VirB10, the VirB7 lipoprotein, and VirB9 interact to form a cell envelope-spanning structural scaffold termed the “core complex” that is required for the assembly of both structures. The related pKM101-encoded core complex is composed of 14 copies each of these VirB homologs, and the transmembrane (TM) ? helices of VirB10-like TraF form a 55-Å-diameter ring at the inner membrane. Here, we report that the VirB10 TM helix possesses two types of putative dimerization motifs, a GxxxA (GA4) motif and two leucine (Leu1, Leu2) zippers. Mutations in the Leu1 motif disrupted T-pilus biogenesis, but these or other mutations in the GA4 or Leu2 motif did not abolish substrate transfer. Replacement of the VirB10 TM domain with a nondimerizing poly-Leu/Ala TM domain sequence also blocked pilus production but not substrate transfer or formation of immunoprecipitable complexes with the core subunits VirB7 and VirB9 and the substrate receptor VirD4. The VirB10 TM helix formed weak homodimers in Escherichia coli, as determined with the TOXCAT assay, whereas replacement of the VirB10 TM helix with the strongly dimerizing TM helix from glycophorin A blocked T-pilus biogenesis in A. tumefaciens. Our findings support a model in which VirB10's TM helix contributes to the assembly or activity of the translocation channel as a weakly self-interacting membrane anchor but establishes a heteromeric TM-TM helix interaction via its Leu1 motif that is critical for T-pilus biogenesis. PMID:23625845

Garza, Isaac

2013-01-01

274

A protocol for Agrobacterium-mediated transformation in rice  

Microsoft Academic Search

Agrobacterium-mediated transformation of rice is an important method that has been widely adopted by many laboratories. However, because current approaches rely on culture systems, routine protocols have been established only in japonica rice, especially those varieties with higher regeneration potential. Some very efficient methods have been developed for japonica varieties that enable high-throughput functional analysis in rice; however, many elite

Ikuko Aichi; Makoto Matsuoka; Asuka Nishimura

2007-01-01

275

[Agrobacterium rubi strains from blueberry plants are highly diverse].  

PubMed

The diversity of a collection of Agrobacterium rubi strains isolated from blueberries from different regions of Argentina was studied by conventional microbiological tests and molecular techniques. Results from biochemical and physiological reactions, as well as from rep-PCR and RFLP analysis of PCR-amplified 23S rDNA showed high phenotypic and genotypic intraspecific variation. PMID:25444133

Abrahamovich, Eliana; López, Ana C; Alippi, Adriana M

2014-01-01

276

Cell Host & Microbe Agrobacterium Induces Expression of a Host  

E-print Network

pathogens, suggesting that Agrobacterium has co-opted a plant defense response and that bacterial Vir of the best-studied strategies employed by diverse plant pathogens is defense suppression. For example, Pseudo- monas syringae has evolved avirulence proteins that suppress the plant basal defense mediated by the RIN

Citovsky, Vitaly

277

ORIGINAL PAPER Agrobacterium-mediated transformation of black cherry  

E-print Network

ORIGINAL PAPER Agrobacterium-mediated transformation of black cherry for flowering control cherry is one of the most valuable hardwood species for cabinetry, furniture, and veneer. The goal of this study was to develop transgenic black cherry plants with reproductive sterility and enhanced insect

278

Opine utilization by Agrobacterium spp.: octopine-type Ti plasmids encode two pathways for mannopinic acid degradation.  

PubMed Central

Octopine-type strains of Agrobacterium tumefaciens degrade the opine mannopinic acid through a specific pathway which involves cleavage of the molecule at the C--N bond between the amino acid and the sugar moieties. Mannose was identified as a product of the reaction. This pathway was inducible by mannopinic and agropinic acids, but not by mannopine or agropine, the two other mannityl opines. The transport system for this pathway appeared to be specific for mannopinic acid. A second, nonspecific pathway for mannopinic acid degradation was also identified. This involved some of the catabolic functions associated with the metabolism of mannopine and agropine. This second pathway was inducible by mannopine and agropine but not by mannopinic or agropinic acids. The transport system for this pathway appeared to have a broad specificity. Transposon Tn5 insertion mutants affected in the specific catabolic pathway were isolated and analyzed. These mutants continued to catabolize mannopine and agropine. Both mapped to a region of the Ti plasmid previously shown to be associated with the catabolism of mannopinic acid. Restriction enzyme analysis of the Ti plasmid from strain 89.10, an octopine strain that is naturally unable to utilize mannopinic acid, showed a deletion in this same region encoding the specific mannopinic acid degradation pathway. Analysis of recombinant clones showed that the second, nonspecific pathway was encoded in a region of the Ti plasmid associated with mannopine and agropine catabolism. This region shared no structural overlap with the segment of the plasmid encoding the specific mannopinic acid degradative pathway. Images PMID:2838452

Dessaux, Y; Guyon, P; Petit, A; Tempé, J; Demarez, M; Legrain, C; Tate, M E; Farrand, S K

1988-01-01

279

Agrobacterium mediated transformation of gypsophila ( Gypsophila paniculata L.)  

Microsoft Academic Search

As a major contributor to the flower market, Gypsophila paniculata is an important target for the breeding of new varieties. However, gypsophila breeding is strongly hampered by the sterility\\u000a of this species’ genotypes and the lack of a genetic-transformation procedure for this genus. Here we describe the establishment\\u000a of a transformation procedure for gypsophila (Gypsophila paniculata L.) based on Agrobacterium

Michal Moyal Ben Zvi; Amir Zuker; Marianna Ovadis; Elena Shklarman; Hagit Ben-Meir; Shamir Zenvirt; Alexander Vainstein

2008-01-01

280

Development of transformation system of rice based on binary bacterial artificial chromosome (BIBAC) vector.  

PubMed

An Agrobacterium-mediated transformation protocol using binary bacterial artificial chromosome (BIBAC) vector system in rice (Oryza sativa L.) was developed. Calli derived from mature embryos of japonica rice cv. H1493 were used as target tissues. Various aspects in transformation and regeneration processes including callus induction and culture, Agrobacterium concentration and duration of co-cultivation, bacterial elimination and transformant selection were examined in order to improve the transformation efficiency. An optimized transformation conditions was established including: using an Agrobacterium strain, LBA4404(HP4404), which carries a super-virulent helper plasmid pCH32, for the infection; a modified N6 medium system for callus induction and culture; pH 5.6 for media in pre-cultivation and co-cultivation; Agrobacterium concentration at OD600 = 1.0 for 3 days co-cultivation and 7 days for a resting period of the infected calli. Based on PCR and Southern blot analysis, it was demonstrated that insert DNA and marker genes carried by BIBAC2 were integrated into the rice genome. PMID:16553216

He, Rui-Feng; Wang, Yuan-Yuan; Du, Bo; Tang, Ming; You, Ai-Qing; Zhu, Li-Li; He, Guang-Cun

2006-03-01

281

ENHANCING THE FREQUENCY OF SOMATIC EMBRYOGENESIS FOLLOWING AGROBACTERIUM-MEDIATED TRANSFORMATION OF  

E-print Network

ENHANCING THE FREQUENCY OF SOMATIC EMBRYOGENESIS FOLLOWING AGROBACTERIUM-MEDIATED TRANSFORMATION media containing 25 mg l21 hygromycin in subsequent selection periods. However, somatic embryogenesis embryogenic explants along with the location of embryogenesis- and transformation-competent cells

Korban, Schuyler S.

282

Genetically engineering plants using agrobacterium, Robert HorschSite: DNA Interactive (www.dnai.org)  

NSDL National Science Digital Library

Interviewee: Robert Horsch DNAi Location:Manipulation>Techniques>transferring & storing>interviews Bacterial transfer Robert Horsch talks about agrobacterium as a ready-made delivery system for getting foreign DNA into plants.

2008-10-06

283

The use of transient GUS expression to develop an Agrobacterium -mediated gene transfer system for kiwifruit  

Microsoft Academic Search

Summary  We have monitored transient GUS expression 4–5 days after cocultivation of leaf explants with Agrobacterium, in order to optimise parameters of cocultivation and so develop an efficient, reproducible gene transfer system in kiwifruit. Factors that were important included the health of the explant, the strain of Agrobacterium, and the binary vector used. Pre-culture of the leaf explants before cocultivation inhibited

Bart-Jan Janssen; Richard C. Gardner

1993-01-01

284

Phylogeny of the Rhizobium-Allorhizobium-Agrobacterium clade supports the delineation of Neorhizobium gen. nov.  

PubMed

The genera Agrobacterium, Allorhizobium, and Rhizobium belong to the family Rhizobiaceae. However, the placement of a phytopathogenic group of bacteria, the genus Agrobacterium, among the nitrogen-fixing bacteria and the unclear position of Rhizobium galegae have caused controversy in previous taxonomic studies. To resolve uncertainties in the taxonomy and nomenclature within this family, the phylogenetic relationships of generic members of Rhizobiaceae were studied, but with particular emphasis on the taxa included in Agrobacterium and the "R. galegae complex" (R. galegae and related taxa), using multilocus sequence analysis (MLSA) of six protein-coding housekeeping genes among 114 rhizobial and agrobacterial taxa. The results showed that R. galegae, R. vignae, R. huautlense, and R. alkalisoli formed a separate clade that clearly represented a new genus, for which the name Neorhizobium is proposed. Agrobacterium was shown to represent a separate cluster of mainly pathogenic taxa of the family Rhizobiaceae. A. vitis grouped with Allorhizobium, distinct from Agrobacterium, and should be reclassified as Allorhizobium vitis, whereas Rhizobium rhizogenes was considered to be the proper name for former Agrobacterium rhizogenes. This phylogenetic study further indicated that the taxonomic status of several taxa could be resolved by the creation of more novel genera. PMID:24581678

Mousavi, Seyed Abdollah; Österman, Janina; Wahlberg, Niklas; Nesme, Xavier; Lavire, Céline; Vial, Ludovic; Paulin, Lars; de Lajudie, Philippe; Lindström, Kristina

2014-05-01

285

Biodegradation of Glycerol Trinitrate and Pentaerythritol Tetranitrate by Agrobacterium radiobacter  

PubMed Central

Bacteria capable of metabolizing highly explosive and vasodilatory glycerol trinitrate (GTN) were isolated under aerobic and nitrogen-limiting conditions from soil, river water, and activated sewage sludge. One of these strains (from sewage sludge) chosen for further study was identified as Agrobacterium radiobacter subgroup B. A combination of high-pressure liquid chromatography and nuclear magnetic resonance analyses of the culture medium during the growth of A. radiobacter on basal salts-glycerol-GTN medium showed the sequential conversion of GTN to glycerol dinitrates and glycerol mononitrates. Isomeric glycerol 1,2-dinitrate and glycerol 1,3-dinitrate were produced simultaneously and concomitantly with the disappearance of GTN, with significant regioselectivity for the production of the 1,3-dinitrate. Dinitrates were further degraded to glycerol 1- and 2-mononitrates, but mononitrates were not biodegraded. Cells were also capable of metabolizing pentaerythritol tetranitrate, probably to its trinitrate and dinitrate analogs. Extracts of broth-grown cells contained an enzyme which in the presence of added NADH converted GTN stoichiometrically to nitrite and the mixture of glycerol dinitrates. The specific activity of this enzyme was increased 160-fold by growth on GTN as the sole source of nitrogen. PMID:16535244

White, G. F.; Snape, J. R.; Nicklin, S.

1996-01-01

286

Efficient production of transgenic melon via Agrobacterium-mediated transformation.  

PubMed

Oriental melon (Cucumis melo L. var. makuwa) is an important fruit for human consumption. However, this plant species is one of the most recalcitrant to genetic transformation. The lack of an efficient in vitro system limits the development of a reproducible genetic transformation protocol for Oriental melon. In this study, an efficient transgenic production method for Agrobacterium-mediated transformation using cotyledon explants of Oriental melon was developed. Cotyledon explants were pre-cultivated for two days in the dark, and the optimal conditions for transformation of melon were determined to be a bacteria concentration of OD600 0.6, inoculation for 30 min, and two days of co-cultivation. Transgenic melon plants were produced from kanamycin-resistant shoots. A total of 11 independent transgenic plants were regenerated with a transformation efficiency of 0.8% of the inoculated explants. The transgenic plants were phenotypically normal and fully fertile, which might be a consequence of the co-cultivation time. PMID:24841654

Bezirganoglu, I; Hwang, S Y; Shaw, J F; Fang, T J

2014-01-01

287

Transgenic cowpea (Vigna unguiculata) seeds expressing a bean alpha-amylase inhibitor 1 confer resistance to storage pests, bruchid beetles.  

PubMed

Cowpea is one of the important grain legumes. Storage pests, Callosobruchus maculatus and C. chinensis cause severe damage to the cowpea seeds during storage. We employ a highly efficient Agrobacterium-mediated cowpea transformation method for introduction of the bean (Phaseolus vulgaris) alpha-amylase inhibitor-1 (alphaAI-1) gene into a commercially important Indian cowpea cultivar, Pusa Komal and generated fertile transgenic plants. The use of constitutive expression of additional vir genes in resident pSB1 vector in Agrobacterium strain LBA4404, thiol compounds during cocultivation and a geneticin based selection system resulted in twofold increase in stable transformation frequency. Expression of alphaAI-1 gene under bean phytohemagglutinin promoter results in accumulation of alphaAI-1 in transgenic seeds. The transgenic protein was active as an inhibitor of porcine alpha-amylase in vitro. Transgenic cowpeas expressing alphaAI-1 strongly inhibited the development of C. maculatus and C. chinensis in insect bioassays. PMID:18784925

Solleti, Siva Kumar; Bakshi, Souvika; Purkayastha, Jubilee; Panda, Sanjib Kumar; Sahoo, Lingaraj

2008-12-01

288

Agrobacterium-Mediated Transformation of Bread and Durum Wheat Using Freshly Isolated Immature Embryos  

NASA Astrophysics Data System (ADS)

Agrobacterium-mediated transformation of wheat is becoming a viable alternative to the more established biolistic protocols. It offers advantages in terms of simple, low-copy-number integrations and can be applied with similar efficiencies to specific durum wheat and spring and winter bread wheat types varieties.

Huixia, Wu; Angela, Doherty; Jones, Huw D.

289

Agrobacterium-Mediated Transformation of the Recalcitrant Vanda Kasem's Delight Orchid with Higher Efficiency  

PubMed Central

The presented study established Agrobacterium-mediated genetic transformation using protocorm-like bodies (PLBs) for the production of transgenic Vanda Kasem's Delight Tom Boykin (VKD) orchid. Several parameters such as PLB size, immersion period, level of wounding, Agrobacterium density, cocultivation period, and concentration of acetosyringone were tested and quantified using gusA gene expression to optimize the efficiency of Agrobacterium-mediated genetic transformation of VKD's PLBs. Based on the results, 3-4?mm PLBs wounded by scalpel and immersed for 30 minutes in Agrobacterium suspension of 0.8 unit at A600nm produced the highest GUS expression. Furthermore, cocultivating infected PLBs for 4 days in the dark on Vacin and Went cocultivation medium containing 200?𝜇M acetosyringone enhanced the GUS expression. PCR analysis of the putative transformants selected in the presence of 250?mg/L cefotaxime and 30?mg/L geneticin proved the presence of wheatwin1, wheatwin2, and nptII genes. PMID:24977213

Gnasekaran, Pavallekoodi; James Antony, Jessica Jeyanthi; Uddain, Jasim; Subramaniam, Sreeramanan

2014-01-01

290

Assessment of conditions affecting Agrobacterium-mediated soybean transformation using the cotyledonary node explant  

Microsoft Academic Search

Summary Conditions affecting Agrobacterium-mediated transformation of soybean (Glycine max (L.) Merr.), including seed vigor of explant source, selection system, and cocultivation conditions, were investigated. A negative correlation between seed sterilization duration and seed vigor, and a positive correlation between seed vigor and regenerability of explants were observed in the study, suggesting that use of high vigor seed and minimum seed

Margie M. Paz; Huixia Shou; Zibiao Guo; Zhanyuan Zhang; Anjan K. Banerjee; Kan Wang

2004-01-01

291

Saponin production by cultures of Panax ginseng transformed with Agrobacterium rhizogenes  

Microsoft Academic Search

Hairy root culture of Ginseng (Panax ginseng) was established after roots were induced on callus following infection with Agrobacterium rhizogenes. The transformed cultures of ginseng could be subcultured as an axenic root culture in the absence of phytohormones, and grew with extensive lateral branches more rapidly than the ordinary cultured roots induced by hormonal control from ginseng callus. The hairy

Takafumi Yoshikawa; Tsutomu Furuya

1987-01-01

292

Growth pattern and ginsenoside production of Agrobacterium -transformed Panax ginseng roots  

Microsoft Academic Search

Panax ginseng roots transformed by Agrobacterium rhizogenes grew rapidly in a hormone-free medium. The transformed roots showed biphasic growth: rapid during the first two weeks and slower thereafter. Sucrose in the medium was almost all converted to glucose and fructose during the first two weeks, and the root growth slowed down after the depletion of sucrose in the medium. Periodic

Shinji Inomata; Mineyuki Yokoyama; Yoko Gozu; Toshiaki Shimizu; Mitsuo Yanagi

1993-01-01

293

Author's personal copy Epigenetic control of Agrobacterium T-DNA integration  

E-print Network

Author's personal copy Review Epigenetic control of Agrobacterium T-DNA integration Shimpei Magori the phosphorylation of histone H2AX, play an important role in DNA repair. Thus, by implication, such epigenetic codes and the epigenetic information in the host chromatin. This article is part of a Special Issue entitled: Epigenetic

Citovsky, Vitaly

294

A Fruiting Body Tissue Method for Efficient Agrobacterium-Mediated Transformation of Agaricus bisporus  

Microsoft Academic Search

We have devised a highly efficient, convenient, and expedi- tious genetic transformation system for the button mushroom Agaricus bisporus. Our method is based on the Agrobacterium- mediated fungal transformation (agro-transformation) system originally described for the yeast Saccharomyces cerevisiae (1, 2). The unavailability of a practical gene transfer system is the single largest obstacle precluding the use of molecular ap- proaches

XI CHEN; MICHELLE STONE; CARL SCHLAGNHAUFER; C. PETER ROMAINE

2000-01-01

295

A transgenic tropical maize line generated by the direct transformation of the embryo-scutellum by A. tumefaciens  

Microsoft Academic Search

An efficient Agrobacterium-mediated transformation system, from which transgenic tropical maize plants were directly generated without previous crosses\\u000a with laboratory or temperate lines, was established. Experimental evaluations were focused on two main issues: (i) establishment\\u000a of appropriate tissue culture conditions, which induced somatic embryogenesis from the scutellum-cells, and (ii) the delivery\\u000a of T-DNA toward these cells. High rates of embryogenic-calli, mainly

Angel Valdez-Ortiz; Sergio Medina-Godoy; M. Elena Valverde; Octavio Paredes-López

2007-01-01

296

BIBAC and TAC clones containing potato genomic DNA fragments larger than 100 kb are not stable in Agrobacterium  

Microsoft Academic Search

Development of efficient methods to transfer large DNA fragments into plants will greatly facilitate the map-based cloning of genes. The recently developed BIBAC and TAC vectors have shown potential to deliver large DNA fragments into plants via Agrobacterium-mediated transformation. Here we report that BIBAC and TAC clones containing potato genomic DNA fragments larger than 100 kb are not stable in Agrobacterium.

J. Song; J. M. Bradeen; S. K. Naess; J. P. Helgeson; J. Jiang

2003-01-01

297

Introduction of pathogen defense genes and a cytokinin biosynthesis gene into sugarbeet (Beta vulgaris L.) by Agrobacterium or particle bombardment  

Microsoft Academic Search

Two different methods for sugarbeet (Beta vulgaris L.) transformation were developed, one using Agrobacterium with excised cotyledons, the other, particle bombardment of embryogenic hypocotyl callus. Transformation efficiencies averaged\\u000a 0.7% for the Agrobacterium method (number of transgenic plants obtained per treated cotyledon) and about 8% for the bombardment method (number of transgenic\\u000a plants obtained per plate of embryogenic callus treated). Transgenic

G. W. Snyder; J. C. Ingersoll; A. C. Smigocki; L. D. Owens

1999-01-01

298

Efficient transformation of Arabidopsis thaliana : comparison of the efficiencies with various organs, plant ecotypes and Agrobacterium strains  

Microsoft Academic Search

Summary  The efficiency of Agrobacterium-mediated transformation of Arabidopsis thaliana was compared with different organs, Arabidopsis ecotypes, and Agrobacterium strains. Efficiency of shoot regeneration was examined using hypocotyl, cotyledon and root explants prepared from young seedlings. Hypocotyl expiants had the highest regeneration efficiency in all of the four Arabidopsis ecotypes tested, when based on a tissue culture system of callus-inducing medium (CIM:

Kazuhito Akama; Hideaki Shiraishi; Shozo Ohta; Kenzo Nakamura; Kiyotaka Okada; Yoshiro Shimura

1992-01-01

299

Novel high- and low-copy stable cosmids for use in Agrobacterium and Rhizobium.  

PubMed

Presented are a set of cosmids based on the unit copy Agrobacterium plasmid, pTAR, and the high-copy-number mutant plasmid, pUCD500, of pTiC58. The addition of a par function derived from pTAR to the vectors allowed them to be stably maintained throughout the cell population in the absence of selective pressure. These vectors, designed for Agrobacterium and Rhizobium, also work in Escherichia coli. The vectors can be cotransferred to Rhizobiaceae from E. coli with the helper plasmid, pRK2013. The pTiC58 origin containing vectors, pUCD1000 and pUCD1001 were found to be incompatible with a 250-kb plasmid harbored by R. meliloti RM102Z1. RM102Z1(pUCD1000) was still capable of nodulating roots in alfalfa. PMID:3906714

Gallie, D R; Novak, S; Kado, C I

1985-09-01

300

Agrobacterium rhizogenes-mediated transformation and regeneration of the legume Astragalus sinicus (Chinese milk vetch)  

Microsoft Academic Search

A system for the Agrobacterium rhizogenes-mediated transformation and root and then shoot regeneration of the legume species Astragalus sinicus (Chinese milk vetch) has been developed.A. rhizogenes DC-AR2 strain harboring the binary vector pBI121, carrying the uidA gene encoding GUS activity, was used to transform seedlings in wound sites. Transformation was monitored by detection of mikimopine and histochemical ?-glucuronidase (GUS) activity.

Hyeon-Je Cho; Jack M. Widholm; Nobukazu Tanaka; Yasuaki Nakanishi; Yoshikatsu Murooka

1998-01-01

301

Assessment of conditions affecting Agrobacterium -mediated soybean transformation using the cotyledonary node explant  

Microsoft Academic Search

Conditions affecting Agrobacterium-mediated transformation of soybean [Glycine max (L.) Merr.], including seed vigor of explant source, selection system, and cocultivation conditions, were investigated. A\\u000a negative correlation between seed sterilization duration and seed vigor, and a positive correlation between seed vigor and\\u000a regenerability of explants were observed in the study, suggesting that use of high vigor seed and minimum seed sterilization

Margie M. Paz; Huixia Shou; Zibiao Guo; Zhanyuan Zhang; Anjan K. Banerjee; Kan Wang

2004-01-01

302

Selection of Maize Inbred Lines with High Regeneration and Susceptibility to Agrobacterium tumifacien  

Microsoft Academic Search

Ten-maize inbred lines of maize (Zea mays L.) with high-induction rate and proliferation ability of embryonic calli were selected from 70-maize inbred lines by immature embryo culturing. Some of the embryonic calli were transferred onto regeneration medium to examine the ability of regeneration, some were transformed via Agrobacterium tumifaciens C58 carrying intron-?-glucuronidase (gus) gene, and GV3301 carrying the green fluorescent

Yu Wang; Shaohong Fu; Ying Wen; Zhiming Zhang; Yanli Xia; Yuzhen Liu; Tingzhao Rong; Guangtang Pan

2007-01-01

303

Thidiazuron: an efficient plant growth regulator for enhancing Agrobacterium -mediated transformation in Petunia hybrida  

Microsoft Academic Search

Efficient shoot regeneration and Agrobacterium-mediated genetic transformation systems were developed for Petunia hybrida cv. Mitchell. Leaf explants of petunia were cultured on Murashige and Skoog (MS) medium with different concentrations of\\u000a thidiazuron (TDZ) without auxin. The highest frequency of shoot regeneration (52.1%) and mean number of shoots per explant\\u000a (4.1) were obtained on medium containing 2 mg l?1 TDZ. Leaf explants inoculated

Gunaratnam Thirukkumaran; Valentine Otang Ntui; Raham Sher Khan; Masahiro Mii

2009-01-01

304

pGreen: a versatile and flexible binary Ti vector for Agrobacterium-mediated plant transformation  

Microsoft Academic Search

Binary Ti vectors are the plasmid vectors of choice in Agrobacterium-mediated plant transformation protocols. The pGreen series of binary Ti vectors are configured for ease-of-use and to meet the demands of a wide range of transformation procedures for many plant species. This plasmid system allows any arrangement of selectable marker and reporter gene at the right and left T-DNA borders

Roger P. Hellens; E. Anne Edwards; Nicola R. Leyland; Samantha Bean; Philip M. Mullineaux

2000-01-01

305

Transformation of opium poppy ( Papaver somniferum L.) with Agrobacterium rhizogenes MAFF 03-01724  

Microsoft Academic Search

Summary  Transformed cultures of opium poppy (Papaver somniferum L.) were established by infecting hypocotyl segments with Agrobacterium rhizogenes MAFF 03-01724. Undifferentiated calli formed on the infected site grew satisfactorily on phytohormone-free solid medium in the dark and produced opine, mikimopine, which could not be detected in a normal culture. Numerous adventitious shoots developed from transformed calli during subculture. The transformed shoots

Kayo Yoshimatsu; Koichiro Shimomura

1992-01-01

306

Agrobacterium–mediated transformation and stable expression of the green fluorescent protein in Brassica rapa  

Microsoft Academic Search

An efficient Agrobacterium–mediated method for transformation, regeneration and screening of Brassica rapa subsp. oleifera (synonym to B. campestris) was developed. For transformation of B. rapa subsp. oleifera, 5-d-old cotyledons were co-cultivated for 2 d with Agrobacteria (strain AGL1) harbouring a binary vector carrying a gene for green fluorescent protein (GFP). For regeneration, cultivation of explants in Murashige–Skoog-based media supplemented with 2 mg

Tony Wahlroos; Petri Susi; Lidia Tylkina; Svetlana Malyshenko; Svetlana Zvereva; Timo Korpela

2003-01-01

307

Effect of culture medium ions on chromate reduction by resting cells of Agrobacterium radiobacter  

Microsoft Academic Search

The influence of some ions in pre-growth culture medium on chromate reduction by resting cells of Agrobacterium radiobacter strain EPS-916 was investigated. The reduction was dependent on the Fe2+ content of the culture medium: the higher the iron content, the lower the reduction rate. The cells showed maximum chromate reduction when pre-grown in the presence of 0.243 µm Mg2+, 20

Santiago LLovera; Ramon Bonet; Maria Dolores Simon-Pujol; Francisco Congregado

1993-01-01

308

Mixture of endophytic Agrobacterium and Sinorhizobium meliloti strains could induce nonspecific nodulation on some woody legumes  

Microsoft Academic Search

Agrobacterium sp. II CCBAU 21244 isolated from root nodules of Wisteria sinensis was verified as an endophytic bacterium by inoculation and reisolation tests. However, inoculation with a mixture of this\\u000a strain and a Sinorhizobium meliloti strain could induce root nodules on W. sinensis and two other woody legumes, which do not form a symbiosis with S. meliloti alone. Rod-shaped and

Jie LiuEn; En Tao Wang; Da Wei Ren; Wen Xin Chen

2010-01-01

309

Identification of an opd (Organophosphate Degradation) Gene in an Agrobacterium Isolate  

Microsoft Academic Search

We isolated a bacterial strain, Agrobacterium radiobacter P230, which can hydrolyze a wide range of organo- phosphate (OP) insecticides. A gene encoding a protein involved in OP hydrolysis was cloned from A. radiobacter P230 and sequenced. This gene (called opdA) had sequence similarity to opd, a gene previously shown to encode an OP-hydrolyzing enzyme in Flavobacterium sp. strain ATCC 27551

Irene Horne; Tara D. Sutherland; Rebecca L. Harcourt; Robyn J. Russell; John G. Oakeshott

2002-01-01

310

Phosphomannose-isomerase ( pmi ) gene as a selectable marker for Agrobacterium -mediated transformation of rapeseed  

Microsoft Academic Search

A transformation method using the phosphomannose-isomerase (pmi) gene as a selectable marker was developed for Brassica napus. The pmi-gene, which converts mannose-6-phosphate to fructose-6-phosphate allowing for selection of transgenic plants on mannose-selective\\u000a medium, was transferred to B. napus hypocotyl explants by Agrobacterium-mediated transformation. More than 350 transgenic plants from three rapeseed varieties were obtained with transformation\\u000a frequencies up to 24.2%

M. Wallbraun; K. Sonntag; C. Eisenhauer; G. Krzcal; Y. P. Wang

2009-01-01

311

[A toxicological evaluation of an Agrobacterium radiobacter-based biological fertilizer].  

PubMed

Pathogenic properties (for warm-blooded organisms) of the industrial strain Agrobacterium radiobacter (strain 204) and toxicity of biofertilizer on its base--rhizoagrin--have been studied. It is established that the studied microorganisms are avirulent, nontoxic, nontoxicogenic and may be recommended for making biopreparations. The preparation rhizoagrin is not toxic for warm-blooded animals and may be used as an alternative of chemical mineral fertilizers. PMID:1331718

Omel'ianets, T G; Guloian, T E; Filatova, I N

1992-01-01

312

Plant regeneration from transformed embryogenic callus of an elite indica rice via Agrobacterium  

Microsoft Academic Search

The present study describes a simple and efficient protocol for plant regeneration from scutellar-derived embryogenic calli of an elite basmati indica rice (Oryza sativa L., cv Pusa Basmati 1) transformed with Agrobacterium. A supervirulent plasmid pTOK233 as well as a non-supervirulent plasmid pJB90GI containing ß-glucuronidase (gus) and hygromycin phosphotransferase (hpt) chimeric genes were used to assess transformation and regeneration efficiency.

R. Kumria; B. Waie; M. V. Rajam

2001-01-01

313

Lox-dependent gene expression in transgenic plants obtained via Agrobacterium-mediated transformation.  

PubMed

Lox sites of the Cre/lox recombination system from bacteriophage P1 were analyzed for their ability to affect on transgene expression when inserted upstream from a gene coding sequence adjacent to the right border (RB) of T-DNA. Wild and mutated types of lox sites were tested for their effect upon bar gene expression in plants obtained via Agrobacterium-mediated and biolistic transformation methods. Lox-mediated expression of bar gene, recognized by resistance of transgenic plants to PPT, occurred only in plants obtained via Agrobacterium-mediated transformation. RT-PCR analysis confirms that PPT-resistant phenotype of transgenic plants obtained via Agrobacterium-mediated transformation was caused by activation of bar gene. The plasmid with promoterless gus gene together with the lox site adjacent to the RB was constructed and transferred to Nicotiana tabacum as well. Transgenic plants exhibited GUS activity and expression of gus gene was detected in plant leaves. Expression of bar gene from the vectors containing lox site near RB allowed recovery of numerous PPT-resistant transformants of such important crops as Beta vulgaris, Brassica napus, Lactuca sativa and Solanum tuberosum. Our results demonstrate that the lox site sequence adjacent to the RB can be used to control bar gene expression in transgenic plants. PMID:23821951

Shcherbak, N; Kishchenko, O; Sakhno, L; Komarnytsky, I; Kuchuk, M

2013-01-01

314

[Methods for the detection of Agrobacterium from plant, soil and water samples].  

PubMed

The genus Agrobacterium includes phytopathogenic bacteria that induce the development of root crown galls and/or aerial galls at the base of the stem or hairy roots on more than 600 species of plants belonging to 90 dicotyledonous families and non-pathogenic species. These bacteria being natural soil inhabitants are particularly difficult to eradicate, which is a problem in nurseries where more than 80% of infections occur. Since early detection is crucial to avoid the inadvertent spread of the disease, the aim of this work was to develop sensitive and precise identification techniques by using a set of semi-selective and differential culture media in combination with a specific PCR to amplify a partial sequence derived from the virC operon, as well as a multiplex PCR on the basis of 23SrDNA sequences, and biological assays to identify and differentiate species and biovars of Agrobacterium obtained either from soil, water or plant samples. The combination of the different assays allowed us to reduce the number of false positive and negative results from bacteria isolated from any of the three types of samples. Therefore, the combination of multiplex PCR, specific PCR, isolations in semi-selective D1, D1-M and YEM-RCT media combined with bioassays on cut leaves of Kalanchoe and seedlings of California Wonder pepper cultivar constitute an accurate tool to detect species and biovars of Agrobacterium for diagnostic purposes. PMID:22274826

Alippi, Adriana M; López, Ana C; Balatti, Pedro A

2011-01-01

315

An alternative approach for gene transfer in trees using wild-type Agrobacterium strains.  

PubMed

Micropropagated shoots of three forest tree species, poplar (Populus tremula x P. alba), wild cherry (Prunus avium L.) and walnut (Juglans nigra x J. regia), were inoculated each with six different wild-type Agrobacterium strains. Poplar and wild cherry developed tumors that grew hormone-independently, whereas on walnut, gall formation was weak. On poplar and wild cherry, tumors induced by nopaline strains developed spontaneously shoots that had a normal phenotype and did not carry oncogenic T-DNA. From these observations, we have established a co-inoculation method to transform plants, using poplar as an experimental model. The method is based on inoculation of stem internodes with an Agrobacterium suspension containing both an oncogenic strain that induces shoot differentiation and a disarmed strain that provides the suitable genes in a binary vector. We used the vector pBI121 carrying neo (kanamycin resistance) and uidA (beta-glucuronidase) genes to facilitate early selection and screening. Poplar plants derived from kanamycin-resistant shoots that did not carry oncogenic T-DNA, were shown to contain and to express neo and uidA genes. These results suggest that wild-type Agrobacterium strains that induce shoot formation directly from tumors can be used as a general tool for gene transfer, avoiding difficult regeneration procedures. PMID:1653060

Brasileiro, A C; Leplé, J C; Muzzin, J; Ounnoughi, D; Michel, M F; Jouanin, L

1991-09-01

316

An efficient regeneration protocol for Agrobacterium-mediated transformation of melon (Cucumis melo L.).  

PubMed

An efficient selection and plant regeneration protocol for Agrobacterium-mediated transformation, using cotyledon node zone-stem connection region of melon, has been developed. The new Agrobacterium-mediated transformation methodology, independent of organ culture, used the entire germinated seed as explants. The transformation system was maximized to maintain the integrity of melon itself, thus avoiding the limitations of traditional tissue culture methods. The transformation was carried out under a non-sterile environment. The incorporation of a selectable marker (neomycin phosphotransferase II) into the genome of transgenic plants was confirmed by PCR and Southern blot analyses. The transformation frequency based on the PCR was 13%. Transgenic melon plants were usually detected by PCR in less than 1 month after Agrobacterium inoculation, and seeds could be harvested in 3 months. The growth characteristics and morphology of the transgenic plants were identical to the untransformed wild-type plants. This method would be beneficial for facilitating the characteristics of gene functions and for boosting the manipulation of melon transformation for commercial purposes. PMID:24446287

Zhang, H J; Gao, P; Wang, X Z; Luan, F S

2014-01-01

317

RNA interference of 1-aminocyclopropane-1-carboxylic acid oxidase (ACO1 and ACO2) genes expression prolongs the shelf life of Eksotika (Carica papaya L.) papaya fruit.  

PubMed

The purpose of this study was to evaluate the effectiveness of using RNA interference in down regulating the expression of 1-aminocyclopropane-1-carboxylic acid oxidase gene in Eksotika papaya. One-month old embryogenic calli were separately transformed with Agrobacterium strain LBA 4404 harbouring the three different RNAi pOpOff2 constructs bearing the 1-aminocyclopropane-1-carboxylic acid oxidase gene. A total of 176 putative transformed lines were produced from 15,000 calli transformed, selected, then regenerated on medium supplemented with kanamycin. Integration and expression of the targeted gene in putatively transformed lines were verified by PCR and real-time RT-PCR. Confined field evaluation of a total of 31 putative transgenic lines planted showed a knockdown expression of the targeted ACO1 and ACO2 genes in 13 lines, which required more than 8 days to achieve the full yellow colour (Index 6). Fruits harvested from lines pRNAiACO2 L2-9 and pRNAiACO1 L2 exhibited about 20 and 14 days extended post-harvest shelf life to reach Index 6, respectively. The total soluble solids contents of the fruits ranged from 11 to 14° Brix, a range similar to fruits from non-transformed, wild type seed-derived plants. PMID:24950439

Sekeli, Rogayah; Abdullah, Janna Ong; Namasivayam, Parameswari; Muda, Pauziah; Abu Bakar, Umi Kalsom; Yeong, Wee Chien; Pillai, Vilasini

2014-01-01

318

Production of recombinant single chain antibodies (scFv) in vegetatively reproductive Kalanchoe pinnata by in planta transformation.  

PubMed

We developed an asexual reproductive plant, Kalanchoe pinnata, as a new bioreactor for plant-based molecular farming using a newly developed transformation method. Leaf crenate margins were pin-pricked to infect the plant with the Agrobacterium strain LBA4404 and vacuum infiltration was also applied to introduce the target gene into the plants. Subsequently, the young mother leaf produced new clones at the leaf crenate margins without the need for time- and labor-consuming tissue culture procedures. The average transformation rates were approximately 77 and 84% for pin-prickling and vacuum-infiltration methods, respectively. To functionally characterize an introduced target protein, a nucleic acid hydrolyzing recombinant 3D8 scFv was selected and the plant based 3D8 scFv proteins were purified and analyzed. Based on abzyme analysis, the purified protein expressed with this system had catalytic activity and exhibited all of properties of the protein produced in an E. coli system. This result suggested that vegetatively reproductive K. pinnata can be a novel and potent bioreactor for bio-pharmaceutical proteins. PMID:19688214

Jung, Yuchul; Rhee, Yong; Auh, Chung-Kyoon; Shim, Hyekyung; Choi, Jung-Jin; Kwon, Suk-Tae; Yang, Joo-Sung; Kim, Donggiun; Kwon, Myung-Hee; Kim, Yong-Sung; Lee, Sukchan

2009-10-01

319

AGROBEST: an efficient Agrobacterium-mediated transient expression method for versatile gene function analyses in Arabidopsis seedlings  

PubMed Central

Background Transient gene expression via Agrobacterium-mediated DNA transfer offers a simple and fast method to analyze transgene functions. Although Arabidopsis is the most-studied model plant with powerful genetic and genomic resources, achieving highly efficient and consistent transient expression for gene function analysis in Arabidopsis remains challenging. Results We developed a highly efficient and robust Agrobacterium-mediated transient expression system, named AGROBEST (Agrobacterium-mediated enhanced seedling transformation), which achieves versatile analysis of diverse gene functions in intact Arabidopsis seedlings. Using ?-glucuronidase (GUS) as a reporter for Agrobacterium-mediated transformation assay, we show that the use of a specific disarmed Agrobacterium strain with vir gene pre-induction resulted in homogenous GUS staining in cotyledons of young Arabidopsis seedlings. Optimization with AB salts in plant culture medium buffered with acidic pH 5.5 during Agrobacterium infection greatly enhanced the transient expression levels, which were significantly higher than with two existing methods. Importantly, the optimized method conferred 100% infected seedlings with highly increased transient expression in shoots and also transformation events in roots of ~70% infected seedlings in both the immune receptor mutant efr-1 and wild-type Col-0 seedlings. Finally, we demonstrated the versatile applicability of the method for examining transcription factor action and circadian reporter-gene regulation as well as protein subcellular localization and protein–protein interactions in physiological contexts. Conclusions AGROBEST is a simple, fast, reliable, and robust transient expression system enabling high transient expression and transformation efficiency in Arabidopsis seedlings. Demonstration of the proof-of-concept experiments elevates the transient expression technology to the level of functional studies in Arabidopsis seedlings in addition to previous applications in fluorescent protein localization and protein–protein interaction studies. In addition, AGROBEST offers a new way to dissect the molecular mechanisms involved in Agrobacterium-mediated DNA transfer. PMID:24987449

2014-01-01

320

Characterization of Plasmid-Borne and Chromosome-Encoded Traits of Agrobacterium Biovar 1, 2, and 3 Strains from France  

PubMed Central

We collected 111 Agrobacterium isolates from galls of various origins (most of them from France) and analyzed both their plasmid-borne and chromosome-encoded traits. Phenotypic analysis of these strains allowed their classification in three phena which exactly matched the delineation of biovars 1, 2, and 3. A fourth phenon was identified which comprises three atypical strains. The phenotypic analysis has also allowed us to identify 12 additional characteristics which could be used to identify the three biovars of Agrobacterium. Our results also suggest that biovar 1 and 2 represent distinct species. Analysis of plasmid-borne traits confirmed that tartrate utilization is a common feature of biovar 3 strains (now named Agrobacterium vitis) and of Agrobacterium grapevine strains in general. Among pathogenic strains of Agrobacterium, several exhibited unusual opine synthesis and degradation patterns, and one strain of biovar 3 induced tumors containing vitopine and a novel opine-like molecule derived from putrescine. We have named this compound ridéopine. PMID:10788345

Ridé, Michel; Ridé, Suzanne; Petit, Annik; Bollet, Claude; Dessaux, Yves; Gardan, Louis

2000-01-01

321

Novel Tellurite-Amended Media and Specific Chromosomal and Ti Plasmid Probes for Direct Analysis of Soil Populations of Agrobacterium Biovars 1 and 2  

PubMed Central

Ecology and biodiversity studies of Agrobacterium spp. require tools such as selective media and DNA probes. Tellurite was tested as a selective agent and a supplement of previously described media for agrobacteria. The known biodiversity within the genus was taken into account when the selectivity of K2TeO3 was analyzed and its potential for isolating Agrobacterium spp. directly from soil was evaluated. A K2TeO3 concentration of 60 ppm was found to favor the growth of agrobacteria and restrict the development of other bacteria. Morphotypic analyses were used to define agrobacterial colony types, which were readily distinguished from other colonies. The typical agrobacterial morphotype allowed direct determination of the densities of agrobacterial populations from various environments on K2TeO3-amended medium. The bona fide agrobacterium colonies growing on media amended with K2TeO3 were confirmed to be Agrobacterium colonies by using 16S ribosomal DNA (rDNA) probes. Specific 16S rDNA probes were designed for Agrobacterium biovar 1 and related species (Agrobacterium rubi and Agrobacterium fici) and for Agrobacterium biovar 2. Specific pathogenic probes from different Ti plasmid regions were used to determine the pathogenic status of agrobacterial colonies. Various morphotype colonies from bulk soil suspensions were characterized by colony blot hybridization with 16S rDNA and pathogenic probes. All the Agrobacterium-like colonies obtained from soil suspensions on amended media were found to be bona fide agrobacteria. Direct colony counting of agrobacterial populations could be done. We found 103 to 104 agrobacteria · g of dry soil?1 in a silt loam bulk soil cultivated with maize. All of the strains isolated were nonpathogenic bona fide Agrobacterium biovar 1 strains. PMID:11133429

Mougel, Christophe; Cournoyer, Benoit; Nesme, Xavier

2001-01-01

322

REVIEW ARTICLE published: 22 November 2011  

E-print Network

on plant pathogen-encoded F-box effectors, such asVirF of Agrobacterium tumefaciens, GALAs of Ralstonia, protein degradation, Agrobacterium, Ralstonia, Polerovirus INTRODUCTION Diverse pathogens have evolved

Citovsky, Vitaly

323

Stable Agrobacterium-Mediated Transformation of Maritime Pine Based on Kanamycin Selection  

PubMed Central

An efficient transformation protocol based on kanamycin selection was developed for Agrobacterium-mediated transformation of maritime pine embryonal masses. The binary vector pBINUbiGUSint, which contained neomycin phosphotransferase II (nptII) as a selectable marker gene and ?-glucuronidase (uidA) as a reporter gene, was used for transformation studies. Different factors, such as embryogenic line, bacterial strain, bacterial concentration, and coculture duration, were examined and optimized. For selection of transformants, 15?mgL?1 kanamycin was used. The highest transformation efficiency (11.4 events per gram of fresh mass) was achieved when a vigorously growing embryonal mass (embryogenic line L01) was cocultivated with Agrobacterium strain AGL1 at the optical density (OD600?nm) of 0.3 for 72?h. Evidence of the stable transgene integration was obtained by polymerase chain reaction for the nptII and uidA genes and expression of the uidA gene. Maturation capacity of the transgenic lines was negatively affected by the transformation process. Induction of axillary shoots by preculturing the embryos with benzyladenine allowed overcoming the low maturation rates of some transformed lines. The transgenic embryos were germinated and the axillar shoots were rooted. Transgenic plants were transferred to potting substrate showing normal growth. PMID:24376383

Alvarez, José M.; Ordás, Ricardo J.

2013-01-01

324

Genetic transformation of Centaurium erythraea Rafn by Agrobacterium rhizogenes and the production of secoiridoids.  

PubMed

Hairy roots of Centaurium erythraea were obtained by infection with Agrobacterium rhizogenes strain LBA 9402. They spontaneously regenerated adventitious shoots in Woody Plant liquid medium without growth regulators. The shoots were grown continuously in Murashige and Skoog (MS) liquid or agar solidified media supplemented with 0.1 mg l(-1) indole-3-acetic acid and 1.0 mg l(-1) 6-benzylaminopurine. These shoots produced roots 4 weeks after transfer into agar-solidified MS medium without phytohormones. Regenerated plants grown and flowered under greenhouse conditions. The transgenic value of the regenerated plants was confirmed by the polymerase chain reaction amplification. Transformation by Agrobacterium rhizogenes alters plant morphology and production of secoiridoid glucosides. The level of secoiridoids was also modified by development stage of transformed plants. The total content of the compounds (expressed as the sum of gentiopicroside, sweroside and swertiamarin) in 10-week old pRi-transformed regenerants was 280 mg g(-1) dry weight and was 8-times the content in the sample of commercially available C. erythraea herb. PMID:16841219

Piatczak, Ewelina; Krolicka, Aleksandra; Wysokinska, Halina

2006-12-01

325

Screening Chinese soybean genotypes for Agrobacterium-mediated genetic transformation suitability*  

PubMed Central

The Agrobacterium-mediated transformation system is the most commonly used method in soybean transformation. Screening of soybean genotypes favorable for Agrobacterium-infection and tissue regeneration is the most important step to establish an efficient genetic transformation system. In this study, twenty soybean genotypes that originated from different soybean production regions in China were screened for transient infection, regeneration capacity, and stable transgenic efficiency. Three genotypes, Yuechun 04-5, Yuechun 03-3, and Tianlong 1, showed comparable stable transgenic efficiencies with that of the previously reported American genotypes Williams 82 and Jack in our experimental system. For the Tianlong 1, the average stable transformation efficiency is 4.59%, higher than that of control genotypes (Jack and Williams 82), which is enough for further genomic research and genetic engineering. While polymerase chain reaction (PCR), LibertyLink strips, and ?-glucuronidase (GUS) staining assays were used to detect the insertion and expression of the transgene, leaves painted with 135 mg/L Basta could efficiently identify the transformants. PMID:23549846

Song, Zhang-yue; Tian, Jing-luan; Fu, Wei-zhe; Li, Lin; Lu, Ling-hong; Zhou, Lian; Shan, Zhi-hui; Tang, Gui-xiang; Shou, Hui-xia

2013-01-01

326

Transient transformation of sunflower leaf discs via an Agrobacterium-mediated method: applications for gene expression and silencing studies  

Microsoft Academic Search

The sunflower belongs to the Compositae family and is an economically important crop because of the quality of its oil. Unfortunately, molecular analyses are limited due to the lack of genomic information, mutant libraries and efficient and rapid transformation protocols. In a wide variety of species, Agrobacterium-mediated transient transformation is a useful tool that can provide valuable insight into many

Pablo A Manavella; Raquel L Chan

2009-01-01

327

Influences of Agrobacterium rhizogenes strains, plant genotypes, and tissue types on the induction of transgenic hairy roots in Vitis species  

Technology Transfer Automated Retrieval System (TEKTRAN)

Agrobacterium rhizogenes-mediated induction of transgenic hairy roots was previously demonstrated in Vitis vinifera L. and a few other Vitis species. In this study, 13 Vitis species, including V. aestivalis, V. afghanistan, V. champinii, V. doaniana, V. flexuosa, V. labrusca, V. nesbittiana, V. pal...

328

Whole-genome analysis of herbicide-tolerant mutant rice generated by agrobacterium-mediated gene targeting.  

PubMed

Gene targeting (GT) is a technique used to modify endogenous genes in target genomes precisely via homologous recombination (HR). Although GT plants are produced using genetic transformation techniques, if the difference between the endogenous and the modified gene is limited to point mutations, GT crops can be considered equivalent to non-genetically modified mutant crops generated by conventional mutagenesis techniques. However, it is difficult to guarantee the non-incorporation of DNA fragments from Agrobacterium in GT plants created by Agrobacterium-mediated GT despite screening with conventional Southern blot and/or PCR techniques. Here, we report a comprehensive analysis of herbicide-tolerant rice plants generated by inducing point mutations in the rice ALS gene via Agrobacterium-mediated GT. We performed genome comparative genomic hybridization (CGH) array analysis and whole-genome sequencing to evaluate the molecular composition of GT rice plants. Thus far, no integration of Agrobacterium-derived DNA fragments has been detected in GT rice plants. However, >1,000 single nucleotide polymorphisms (SNPs) and insertion/deletion (InDels) were found in GT plants. Among these mutations, 20-100 variants might have some effect on expression levels and/or protein function. Information about additive mutations should be useful in clearing out unwanted mutations by backcrossing. PMID:25378689

Endo, Masaki; Kumagai, Masahiko; Motoyama, Ritsuko; Sasaki-Yamagata, Harumi; Mori-Hosokawa, Satomi; Hamada, Masao; Kanamori, Hiroyuki; Nagamura, Yoshiaki; Katayose, Yuichi; Itoh, Takeshi; Toki, Seiichi

2015-01-01

329

Whole-Genome Analysis of Herbicide-Tolerant Mutant Rice Generated by Agrobacterium-Mediated Gene Targeting  

PubMed Central

Gene targeting (GT) is a technique used to modify endogenous genes in target genomes precisely via homologous recombination (HR). Although GT plants are produced using genetic transformation techniques, if the difference between the endogenous and the modified gene is limited to point mutations, GT crops can be considered equivalent to non-genetically modified mutant crops generated by conventional mutagenesis techniques. However, it is difficult to guarantee the non-incorporation of DNA fragments from Agrobacterium in GT plants created by Agrobacterium-mediated GT despite screening with conventional Southern blot and/or PCR techniques. Here, we report a comprehensive analysis of herbicide-tolerant rice plants generated by inducing point mutations in the rice ALS gene via Agrobacterium-mediated GT. We performed genome comparative genomic hybridization (CGH) array analysis and whole-genome sequencing to evaluate the molecular composition of GT rice plants. Thus far, no integration of Agrobacterium-derived DNA fragments has been detected in GT rice plants. However, >1,000 single nucleotide polymorphisms (SNPs) and insertion/deletion (InDels) were found in GT plants. Among these mutations, 20–100 variants might have some effect on expression levels and/or protein function. Information about additive mutations should be useful in clearing out unwanted mutations by backcrossing. PMID:25378689

Endo, Masaki; Kumagai, Masahiko; Motoyama, Ritsuko; Sasaki-Yamagata, Harumi; Mori-Hosokawa, Satomi; Hamada, Masao; Kanamori, Hiroyuki; Nagamura, Yoshiaki; Katayose, Yuichi; Itoh, Takeshi; Toki, Seiichi

2015-01-01

330

[Transformation of embryogenic Calli of Siberian wildrye grass (Elymus sibiricus L. cv. Chuancao No.2) mediated by agrobacterium].  

PubMed

Formation of embryogenic calli of Siberian wildrye grass (Elymus sibiricus L. cv. Chuancao No.2) was induced from mature seeds as explants, and proliferated on MS medium containing 2,4-D 5.0 mg/L and KT 0.05 mg/L. An effective and stable callus regeneration system was established by optimizing the culture conditions (Tables 1, 2 and Fig.2). After the calli were subcultured 8 weeks, selected the whitish-yellow-coloured compact nodular calli that transformed with plasmid pCAMBIA1304 carrying hygromycin resistance gene (hptII) and Pseudomonas pseudoalcaligenes insecticidal protein gene (ppIP), which was mediated by an Agrobacterium strain EHA105. Resistant plants were obtained after hygromycin selection (Figs.3, 4). Some important factors that affect the transformation efficiency were studied, which included selection pressure, time of embryogenic calli proliferation, OD value of Agrobacterium suspension, temperature, medium and time of co-cultivation, and concentration of antibiotics used for suppressing the overgrowth of Agrobacterium in the course of transformation plant regeneration. This research is the first successful genetic transformation of Elymus sibiricus L. cv. Chuancao No.2 mediated by Agrobacterium. PMID:16477130

Li, Da-Xu; Zhang, Jie; Zhao, Jian; Zhang, Yi; Li, Li; Liu, Su-Jun; Chen, Fei; Yang, Zhi-Rong

2006-02-01

331

Enhanced anthocyanin synthesis in foliage plant Caladium bicolor.  

PubMed

A protocol was developed for Agrobacterium-mediated genetic transformation of monocotyledon foliage plant Caladium bicolor cv. Jackie Suthers using leaf disc and petiole as the explants. The explants were inoculated with Agrobacterium strain LBA4404 harboring a binary vector with the maize anthocyanin regulatory gene Lc under the control of the cauliflower mosaic virus promoter. Callus formation was induced in MS medium supplemented with 0.5 mg/l 6-benzylaminopurine (6-BA), 0.1 mg/1 2,4-dichlorophenoxyacetic acid (2,4-D), 30 g/l sucrose and kanamycin 50 mg/l for selection. Resistant calli were induced for shoot generation in MS medium with 2 mg/l 6-BA and 0.2 mg/l alpha-naphthaleneacetic acid. As much as 10% of the explants gave rise to kanamycin-resistant shoots with our procedure. Transformed plants had enhanced anthocyanin accumulation in the roots, leaves and stems (epidermis and vascular bundles). Integration of the transgene into the host genome was confirmed by genomic Southern blot hybridization, and RNA blot hybridization analysis indicated that the expression of the transgene correlated with anthocyanin accumulation. This investigation illustrates the utility of anthocyanin regulatory genes in the genetic manipulation of the color of foliage plants. It also supports the premise that the Lc gene can be used as a powerful non-destructive cell autonomous visual marker in a wide variety of plants, as exemplified by the perfect symmetrical half-green/half-red plant presumably derived from the symmetrical division of one transgenic and one non-transgenic precursor meristematic cell. PMID:15372198

Li, S J; Deng, X M; Mao, H Z; Hong, Y

2005-03-01

332

Characterization of a new pathovar of Agrobacterium vitis causing banana leaf blight in China.  

PubMed

A new banana leaf blight was found in Nanning city, China, during a 7-year survey (2003-2009) of the bacterial diseases on banana plants. Eight bacterial strains were isolated from affected banana leaves, and identified as an intraspecific taxon of Agrobacterium vitis based on their 16S rDNA sequence similarities with those of 37 randomly selected bacterial strains registered in GenBank database. The representative strain Ag-1 was virulent on banana leaves and shared similar growth and biochemical reactions with the reference strain IAM14140 of A. vitis. The strains causing banana leaf blight were denominated as A. vitis pv. musae. The traditional A. vitis strains virulent to grapevines were proposed to be revised as A. vitis pv. vitis. This is the first record of a new type of A. vitis causing banana leaf blight in China. PMID:23828501

Huang, Siliang; Long, Mengling; Fu, Gang; Lin, Shanhai; Qin, Liping; Hu, Chunjin; Cen, Zhenlu; Lu, Jie; Li, Qiqin

2015-01-01

333

Catapol production in Chinese foxglove (Rehmannia glutinosa Libos.) hairy roots transformed with Agrobacterium rhizogenes ATCC15834.  

PubMed

Hairy root clones of Rehmannia glutinosa were established via transformation with Agrobacterium rhizogenes ATCC15834. To optimize the culturing conditions for both root growth and catalpol production, the effects of various combinations of seven basal media, pH, and carbon sources were examined in the dark. The fastest root growth was obtained in an SH medium containing 4% sucrose, pH 5.8. The highest catalpol content (0.54% dry weight) was achieved in a WPM medium supplemented with 4% sucrose, pH 5.8. Effects of plant growth regulators and chitosan were also investigated. Auxin 2 mg/L IAA significantly increased both root length and the frequency of lateral roots. Both 50 mg/L chitosan and 0.5 mg/L GA3 induced catalpol production, with contents calculated at 0.7% dry weight and 0.65% dry weight, respectively. PMID:19521851

Hwang, Sung Jin

2009-01-01

334

Agrobacterium-mediated transformation of Malus robusta with tomato iron transporter gene.  

PubMed

The tomato iron transporter gene (LeIRT2) was introduced to Malus robusta Rehd. via Agrobacterium-mediated transformation to produce iron-deficiency tolerant apple rootstock. A total of 19 putative transformants were obtained, 11 of which were verified by PCR amplification to carry a fragment of the transgene. Among them, nine were confirmed to carry the transgene by Southern blot analysis with one to three copies of the transgene integrated into the plant genome. Two transgenic plants, one carrying one copy and the other three copies of the transgene, were hydroponically cultured to test their tolerance to iron-deficiency, which was found only in the transgenic plant with a single copy, which weighted 21%-4% greater than those of the control plants. PMID:15961896

Qu, Shen-Chun; Huang, Xiao-De; Zhang, Zhen; Yao, Quan-Hong; Tao, Jian-Min; Qiao, Yu-Shan; Zhang, Jun-Yi

2005-06-01

335

The ORF8 gene product of Agrobacterium rhizogenes TL-DNA has tryptophan 2-monooxygenase activity.  

PubMed

The open reading frame 8 (ORF8) is located on the TL-DNA of the phytopathogenic soil bacterium Agrobacterium rhizogenes strain A4. The predicted ORF8 protein has a particular structure and is possibly a natural fusion protein. The N-terminal domain shows homology to the A. rhizogenes rolB protein and may modulate the auxin responsiveness of host cells. The C terminus has up to 38% homology to tryptophan 2-monooxygenases (t2m). We show that ORF8 overexpressing plants contain a fivefold higher concentration of indole-3-acetamide (IAM) than untransformed plants. Protein extracts from seedlings and Escherichia coli overexpressing ORF8 show significantly higher turnover rates of tryptophan to IAM than negative controls. We conclude that the ORF8 gene product has tryptophan 2-monooxygenase activity. PMID:10875340

Lemcke, K; Prinsen, E; van Onckelen, H; Schmülling, T

2000-07-01

336

Correlative Association between Resident Plasmids and the Host Chromosome in a Diverse Agrobacterium Soil Population  

PubMed Central

Soil samples collected from a fallow field which had not been cultivated for 5 years harbored a population of Agrobacterium spp. estimated at 3 × 107 CFU/g. Characterization of 72 strains selected from four different isolation media showed the presence of biovar 1 (56%) and bv. 2 (44%) strains. Pathogenicity assays on five different test plants revealed a high proportion (33%) of tumorigenic strains in the resident population. All tumorigenic strains belonged to bv. 1. Differentiation of the strains by restriction fragment length polymorphism analysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cellular proteins, and utilization patterns of 95 carbon substrates (Biolog GN microplate) revealed a diversified bv. 1 population, composed of five distinct chromosomal backgrounds (chr A, C, D, E, and F), and a homogeneous bv. 2 population (chr B). chr A, B, C, and D were detected at similar levels throughout the study site. According to opine metabolism, pathogenicity, and agrocin sensitivity, chr A strains carried a nopaline Ti plasmid (pTi), whereas chr C strains had an octopine pTi. In addition, four of six nontumorigenic bv. 1 strains (two chr D, one chr E, and one chr F) had distinct and unusual opine catabolism patterns. chr B (bv. 2) strains were nonpathogenic and catabolized nopaline. Although agrocin sensitivity is a pTi-borne trait, 14 chr B strains were sensitive to agrocin 84, apparently harboring a defective nopaline pTi similar to pAtK84b. The other two chr B strains were agrocin resistant. The present analysis of chromosomal and plasmid phenotypes suggests that in this Agrobacterium soil population, there is a preferential association between the resident plasmids and their bacterial host. Images PMID:16348927

Bouzar, Hacène; Ouadah, Djaouida; Krimi, Zoulikha; Jones, Jeffrey B.; Trovato, Maurizio; Petit, Annik; Dessaux, Yves

1993-01-01

337

HvCKX2 gene silencing by biolistic or Agrobacterium-mediated transformation in barley leads to different phenotypes  

PubMed Central

Background CKX genes encode cytokinin dehydrogenase enzymes (CKX), which metabolize cytokinins in plants and influence developmental processes. The genes are expressed in different tissues and organs during development; however, their exact role in barley is poorly understood. It has already been proven that RNA interference (RNAi)-based silencing of HvCKX1 decreased the CKX level, especially in those organs which showed the highest expression, i.e. developing kernels and roots, leading to higher plant productivity and higher mass of the roots [1]. The same type of RNAi construct was applied to silence HvCKX2 and analyze the function of the gene. Two cultivars of barley were transformed with the same silencing and selection cassettes by two different methods: biolistic and via Agrobacterium. Results The mean Agrobacterium-mediated transformation efficiency of Golden Promise was 3.47% (±2.82). The transcript level of HvCKX2 in segregating progeny of T1 lines was decreased to 34%. The reduction of the transcript in Agrobacterium-derived plants resulted in decreased CKX activity in the developing and developed leaves as well as in 7 DAP (days after pollination) spikes. The final phenotypic effect was increased productivity of T0 plants and T1 lines. Higher productivity was the result of the higher number of seeds and higher grain yield. It was also correlated with the higher 1000 grain weight, increased (by 7.5%) height of the plants and higher (from 0.5 to 2) numbers of spikes. The transformation efficiency of Golden Promise after biolistic transformation was more than twice as low compared to Agrobacterium. The transcript level in segregating progeny of T1 lines was decreased to 24%. Otherwise, the enzyme activity found in the leaves of the lines after biolistic transformation, especially in cv. Golden Promise, was very high, exceeding the relative level of the control lines. These unbalanced ratios of the transcript level and the activity of the CKX enzyme negatively affected kernel germination or anther development and as a consequence setting the seeds. The final phenotypic effect was the decreased productivity of T0 plants and T1 lines obtained via the biolistic silencing of HvCKX2. Conclusion The phenotypic result, which was higher productivity of silenced lines obtained via Agrobacterium, confirms the hypothesis that spatial and temporal differences in expression contributed to functional differentiation. The applicability of Agrobacterium-mediated transformation for gene silencing of developmentally regulated genes, like HvCKX2, was proven. Otherwise low productivity and disturbances in plant development of biolistic-silenced lines documented the unsuitability of the method. The possible reasons are discussed. PMID:23134638

2012-01-01

338

Acquisition of an Agrobacterium Ri Plasmid and Pathogenicity by Other  -Proteobacteria in Cucumber and Tomato Crops Affected by Root Mat  

Microsoft Academic Search

Root mat of cucumbers and tomatoes has previously been shown to be caused by Agrobacterium radiobacter strains harboring a root-inducing Ri plasmid (pRi). Nine other pRi-harboring -Proteobacteria have subse- quently been isolated from root mat-infected crops. Fatty acid profiling and partial 16S rRNA sequence analysis identified three of these strains as being in the genus Ochrobactrum, five as being in

S. A. Weller; D. E. Stead; J. P. W. Young

2004-01-01

339

Enhanced targeted integration mediated by translocated I-SceI during the Agrobacterium mediated transformation of yeast.  

PubMed

Agrobacterium mediated transformation (AMT) has been embraced by biotechnologists as the technology of choice to introduce or alter genetic traits of plants. However, in plants it is virtually impossible to predetermine the integration site of the transferred T-strand unless one is able to generate a double stranded break (DSB) in the DNA at the site of interest. In this study, we used the model organism Saccharomyces cerevisiae to investigate whether the Agrobacterium mediated translocation of site-specific endonucleases via the type IV secretion system (T4SS), concomitantly with T-DNA transfer is possible and whether this can improve the gene targeting efficiency. In addition to that, the effect of different chromatin states on targeted integration, was investigated. It was found that Agrobacterium mediated translocation of the homing endonuclease I-SceI has a positive effect on the integration of T-DNA via the homologous repair (HR) pathway. Furthermore, we obtained evidence that nucleosome removal has a positive effect on I-SceI facilitated T-DNA integration by HR. Reversely; inducing nucleosome formation at the site of integration removes the positive effect of translocated I-SceI on T-DNA integration. PMID:25662162

Rolloos, Martijn; Hooykaas, Paul J J; van der Zaal, Bert J

2015-01-01

340

Survival of Agrobacterium radiobacter K84 on various carriers for crown gall control.  

PubMed Central

Screening was performed on nine carriers to find an improved formulation for Agrobacterium radiobacter K84 cells. The survival data showed that it is possible to preserve A. radiobacter cells on dry solid supports for a long time provided that the storage temperature is 4 degrees C and that the inoculation volume for 4 x 10(9) CFU g-1 is not less than 0.15 ml g of carrier-1. On the other hand, a substantial carrier water content was necessary for room temperature storage. Many materials proved to be suitable as microbial carriers; in some cases, vermiculite allowed long storage times comparable to those reported for peat or carboxymethyl cellulose, which are already employed in some commercial A. radiobacter K84 products. Furthermore, vermiculite assured full and immediate biological activity in the prevention of crown gall, showing that it is suitable for a new formulation of strain K84. A hypothesis to explain the different survival abilities in wet and dry conditions is presented. PMID:1892394

Pesenti-Barili, B; Ferdani, E; Mosti, M; Degli-Innocenti, F

1991-01-01

341

In vitro regeneration and Agrobacterium mediated genetic transformation of Artemisia aucheri Boiss.  

PubMed

In the present study, we developed an efficient protocol for in vitro plant regeneration and genetically transformed root induction in medicinal plant Artemisia aucheri Boiss. Leaf explants were cultivated in MS medium supplemented by combination of plant growth regulators including ?-naphthalene-acetic acid, 6-benzyl-aminopurine, indole-3-acetic acid and 2, 4-dichlorophenoxyaceticacid. The highest frequency of shoot organogenesis occurred on MS medium supplemented with 0.05 mg/l NAA plus 2 mg/l BA (96.3 %) and MS medium supplemented with 0.5 mg/l IAA plus 2 mg/l BA (88.3 %). Root induction was obtained on MS medium supplemented with 0.5 mg/l IBA. This is a simple, reliable, rapid and high efficient regeneration system for A. aucheri Boiss in short period via adventitious shoot induction approach. Also, an efficient genetically transformed root induction for A. aucheri was developed through Agrobacterium rhizogenes-mediated transformation by four bacterial strains, A4, ATCC15834, MSU440, and A13 (MAFF-02-10266). The maximum frequency of hairy root induction was obtained using MSU440 (93 %) and ATCC15834 (89 %) bacterial strains. Hairy root lines were confirmed by PCR using the rolB gene specific primers and Southern blot analysis. PMID:25320471

Sharafi, Ali; Sohi, Haleh Hashemi; Mirzaee, Hooman; Azadi, Pejman

2014-10-01

342

Production of herbicide-tolerant zoysiagrass by Agrobacterium-mediated transformation.  

PubMed

Herbicide-resistant zoysiagrass (Zoysia japonica Steud.) has been developed by Agrobacterium-mediated transformation. A callus-type transformation system was established by optimizing several factors that affect the rate of transformation, including co-cultivation period and concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D), CaCl2 and acetosyringone. Maximal GUS expression was observed when a Type 3 callus was co-cultivated on 2,4-D-free co-cultivation medium for 9 d. In addition, removal of calcium and addition of 50-100 mg/L acetosyringone during co-cultivation enhanced GUS expression. When this optimized protocol was applied to the transformation of the bialaphos resistance gene (bar), four plants per 700 mg of infected calluses survived on the selective medium. DNA gel-blot analysis showed that two copies of the transgene had been integrated. After application of 2 g/L bialaphos for a week the transgenic plants survived herbicide spraying, while untransformed zoysiagrasses and invading weeds died. The herbicide-tolerant zoysiagrass will permit more efficient weed control in this widely cultivated turf grass. PMID:14503840

Toyama, Koichi; Bae, Chang-Hyu; Kang, Joeng-Gu; Lim, Yong-Pyo; Adachi, Taiji; Riu, Key-Zung; Song, Pill-Soon; Lee, Hyo-Yeon

2003-08-31

343

Purification, properties, and sequence of glycerol trinitrate reductase from Agrobacterium radiobacter.  

PubMed Central

Glycerol trinitrate (GTN) reductase, which enables Agrobacterium radiobacter to utilize GTN and related explosives as sources of nitrogen for growth, was purified and characterized, and its gene was cloned and sequenced. The enzyme was a 39-kDa monomeric protein which catalyzed the NADH-dependent reductive scission of GTN (Km = 23 microM) to glycerol dinitrates (mainly the 1,3-isomer) with a pH optimum of 6.5, a temperature optimum of 35 degrees C, and no dependence on metal ions for activity. It was also active on pentaerythritol tetranitrate (PETN), on isosorbide dinitrate, and, very weakly, on ethyleneglycol dinitrate, but it was inactive on isopropyl nitrate, hexahydro-1,3,5-trinitro-1,3,5-triazine, 2,4,6-trinitrotoluene, ammonium ions, nitrate, or nitrite. The amino acid sequence deduced from the DNA sequence was homologous (42 to 51% identity and 61 to 69% similarity) to those of PETN reductase from Enterobacter cloacae, N-ethylmaleimide reductase from Escherichia coli, morphinone reductase from Pseudomonas putida, and old yellow enzyme from Saccharomyces cerevisiae, placing the GTN reductase in the alpha/beta barrel flavoprotein group of proteins. GTN reductase and PETN reductase were very similar in many respects except in their distinct preferences for NADH and NADPH cofactors, respectively. PMID:9401040

Snape, J R; Walkley, N A; Morby, A P; Nicklin, S; White, G F

1997-01-01

344

An efficient method for Agrobacterium-mediated genetic transformation and plant regeneration in cumin (Cuminum cyminum L.).  

PubMed

Cumin is an annual herbaceous medicinally important plant having diverse applications. An efficient and reproducible method of Agrobacterium-mediated genetic transformation was herein established for the first time. A direct regeneration method without callus induction was optimised using embryos as explant material in Gamborg's B5 medium supplemented with 0.5-?M 6-benzyladenine and 2.0-?M ?-naphthalene acetic acid. About 1,020 embryos (a mean of 255 embryos per batch) were used for the optimisation of transformation conditions. These conditions were an Agrobacterium cell suspension of 0.6 OD600, a co-cultivation time of 72 h, 300-?M acetosyringone and wounding of explants using a razor blade. Pre-cultured elongated embryos were treated using optimised conditions. About 720 embryos (a mean of 180 embryos per batch) were used for transformation and 95 % embryos showed transient ?-glucuronidase expression after co-cultivation. Putative transformed embryos were cultured on B5 medium for shoot proliferation and 21 regenerated plants were obtained after selection and allowed to root. T0 plantlets showed ?-glucuronidase expression and gene integration was confirmed via PCR amplification of 0.96 and 1.28 kb fragments of the hygromycin-phosphotransferase II and ?-glucuronidase genes, respectively. In this study, a transformation efficiency of 1.5 % was demonstrated and a total of 11 transgenic plants were obtained at the hardening stage, however, only four plants acclimatised during hardening. Gene copy number was analysed by Southern blot analysis of hardened plants and single-copy gene integration was observed. This is the first successful attempt of Agrobacterium-mediated genetic transformation of cumin. PMID:23813408

Pandey, Sonika; Mishra, Avinash; Patel, Manish Kumar; Jha, Bhavanath

2013-09-01

345

Salt tolerance and activity of antioxidative enzymes of transgenic finger millet overexpressing a vacuolar H(+)-pyrophosphatase gene (SbVPPase) from Sorghum bicolor.  

PubMed

A vacuolar proton pyrophosphatase cDNA clone was isolated from Sorghum bicolor (SbVPPase) using end-to-end gene-specific primer amplification. It showed 80-90% homology at the nucleotide and 85-95% homology at the amino acid level with other VPPases. The gene was introduced into expression vector pCAMBIA1301 under the control of the cauliflower mosaic virus 35S (CaMV35S) promoter and transformed into Agrobacterium tumifaciens strain LBA4404 to infect embryogenic calli of finger millet (Eleusine coracana). Successful transfer of SbVPPase was confirmed by a GUS histochemical assay and PCR analysis. Both, controls and transgenic plants were subjected to 100 and 200mM NaCl and certain biochemical and physiological parameters were studied. Relative water content (RWC), plant height, leaf expansion, finger length and width and grain weight were severely reduced (50-70%), and the flowering period was delayed by 20% in control plants compared to transgenic plants under salinity stress. With increasing salt stress, the proline and chlorophyll contents as well as the enzyme activities of superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), guaiacol peroxidase (GPX) and glutathione reductase (GR) increased by 25-100% in transgenics, while malondialdehyde (MDA) showed a 2-4-fold decrease. The increased activities of antioxidant enzymes and the reduction in the MDA content suggest efficient scavenging of reactive oxygen species (ROS) in transgenics and, as a consequence, probably alleviation of salt stress. Also, the leaf tissues of the transgenics accumulated 1.5-2.5-fold higher Na(+) and 0.4-0.8-fold higher K(+) levels. Together, these results clearly demonstrate that overexpression of SbVPPase in transgenic finger millet enhances the plant's performance under salt stress. PMID:24877670

Anjaneyulu, Ediga; Reddy, Palle Surender; Sunita, Merla Srilakshmi; Kishor, Polavarapu B Kavi; Meriga, Balaji

2014-06-15

346

Composite Medicago truncatula plants harbouring Agrobacterium rhizogenes-transformed roots reveal normal mycorrhization by Glomus intraradices  

PubMed Central

Composite plants consisting of a wild-type shoot and a transgenic root are frequently used for functional genomics in legume research. Although transformation of roots using Agrobacterium rhizogenes leads to morphologically normal roots, the question arises as to whether such roots interact with arbuscular mycorrhizal (AM) fungi in the same way as wild-type roots. To address this question, roots transformed with a vector containing the fluorescence marker DsRed were used to analyse AM in terms of mycorrhization rate, morphology of fungal and plant subcellular structures, as well as transcript and secondary metabolite accumulations. Mycorrhization rate, appearance, and developmental stages of arbuscules were identical in both types of roots. Using Mt16kOLI1Plus microarrays, transcript profiling of mycorrhizal roots showed that 222 and 73 genes exhibited at least a 2-fold induction and less than half of the expression, respectively, most of them described as AM regulated in the same direction in wild-type roots. To verify this, typical AM marker genes were analysed by quantitative reverse transcription-PCR and revealed equal transcript accumulation in transgenic and wild-type roots. Regarding secondary metabolites, several isoflavonoids and apocarotenoids, all known to accumulate in mycorrhizal wild-type roots, have been found to be up-regulated in mycorrhizal in comparison with non-mycorrhizal transgenic roots. This set of data revealed a substantial similarity in mycorrhization of transgenic and wild-type roots of Medicago truncatula, validating the use of composite plants for studying AM-related effects. PMID:19574251

Mrosk, Cornelia; Forner, Susanne; Hause, Gerd; Küster, Helge; Kopka, Joachim; Hause, Bettina

2009-01-01

347

Functional genomic analysis of cotton genes with agrobacterium-mediated virus-induced gene silencing.  

PubMed

Cotton (Gossypium spp.) is one of the most agronomically important crops worldwide for its unique textile fiber production and serving as food and feed stock. Molecular breeding and genetic engineering of useful genes into cotton have emerged as advanced approaches to improve cotton yield, fiber quality, and resistance to various stresses. However, the understanding of gene functions and regulations in cotton is largely hindered by the limited molecular and biochemical tools. Here, we describe the method of an Agrobacterium infiltration-based virus-induced gene silencing (VIGS) assay to transiently silence endogenous genes in cotton at 2-week-old seedling stage. The genes of interest could be readily silenced with a consistently high efficiency. To monitor gene silencing efficiency, we have cloned cotton GrCla1 from G. raimondii, a homolog gene of Arabidopsis Cloroplastos alterados 1 (AtCla1) involved in chloroplast development, and inserted into a tobacco rattle virus (TRV) binary vector pYL156. Silencing of GrCla1 results in albino phenotype on the newly emerging leaves, serving as a visual marker for silencing efficiency. To further explore the possibility of using VIGS assay to reveal the essential genes mediating disease resistance to Verticillium dahliae, a fungal pathogen causing severe Verticillium wilt in cotton, we developed a seedling infection assay to inoculate cotton seedlings when the genes of interest are silenced by VIGS. The method we describe here could be further explored for functional genomic analysis of cotton genes involved in development and various biotic and abiotic stresses. PMID:23386302

Gao, Xiquan; Shan, Libo

2013-01-01

348

Agrobacterium Mediated Transient Gene Silencing (AMTS) in Stevia rebaudiana: Insights into Steviol Glycoside Biosynthesis Pathway  

PubMed Central

Background Steviol glycoside biosynthesis pathway has emerged as bifurcation from ent-kaurenoic acid, substrate of methyl erythritol phosphate pathway that also leads to gibberellin biosynthesis. However, the genetic regulation of steviol glycoside biosynthesis has not been studied. So, in present study RNA interference (RNAi) based Agrobacterium mediated transient gene silencing (AMTS) approach was followed. SrKA13H and three SrUGTs (SrUGT85C2, SrUGT74G1 and SrUGT76G1) genes encoding ent-kaurenoic acid-13 hydroxylase and three UDP glycosyltransferases of steviol glycoside biosynthesis pathway were silenced in Stevia rebaudiana to understand its molecular mechanism and association with gibberellins. Methodology/Principal Findings RNAi mediated AMTS of SrKA13H and three SrUGTs has significantly reduced the expression of targeted endogenous genes as well as total steviol glycoside accumulation. While gibberellins (GA3) content was significantly enhanced on AMTS of SrUGT85C2 and SrKA13H. Silencing of SrKA13H and SrUGT85C2 was found to block the metabolite flux of steviol glycoside pathway and shifted it towards GA3 biosynthesis. Further, molecular docking of three SrUGT proteins has documented highest affinity of SrUGT76G1 for the substrates of alternate pathways synthesizing steviol glycosides. This could be a plausible reason for maximum reduction in steviol glycoside content on silencing of SrUGT76G1 than other genes. Conclusions SrKA13H and SrUGT85C2 were identified as regulatory genes influencing carbon flux between steviol glycoside and gibberellin biosynthesis. This study has also documented the existence of alternate steviol glycoside biosynthesis route. PMID:24023961

Guleria, Praveen; Yadav, Sudesh Kumar

2013-01-01

349

Exploration of new perspectives and limitations in Agrobacterium-mediated gene transfer technology. Final report, June 1, 1992--May 31, 1995  

SciTech Connect

Genetic manipulation of plants often involves the introduction of homologous or partly homologous genes. Ectropic introduction of homologous sequences into plant genomes may trigger epigenetic changes, making expression of the genes unpredictable. The main project objective was to examine the feasibility of using Agrobacterium-mediated gene transfer for homologous gene targeting in plants.

Marton, L.

1996-02-01

350

Agrobacterium-mediated genetic transformation and regeneration of transgenic plants using leaf midribs as explants in ramie [Boehmeria nivea (L.) Gaud].  

PubMed

In this study, leaf midribs, the elite explants, were used for the first time to develop an efficient regeneration and transformation protocol for ramie [Boehmeria nivea (L.) Gaud.] via Agrobacterium-mediated genetic transformation. Sensitivity of leaf midribs regeneration to kanamycin was evaluated, which showed that 40 mg l(-1) was the optimal concentration needed to create the necessary selection pressure. Factors affecting the ramie transformation efficiency were evaluated, including leaf age, Agrobacterium concentration, length of infection time for the Agrobacterium solution, acetosyringone concentration in the co-cultivation medium, and the co-cultivation period. The midrib explants from 40-day-old in vitro shoots, an Agrobacterium concentration at OD600 of 0.6, 10-min immersion in the bacteria solution, an acetosyringone concentration of 50 mg l(-1) in the co-cultivation medium and a 3-day co-cultivation period produced the highest efficiencies of regeneration and transformation. In this study, the average transformation rate was 23.25%. Polymerase chain reactions using GUS and NPTII gene-specific primers, Southern blot and histochemical GUS staining analyses further confirmed that the transgene was integrated into the ramie genome and expressed in the transgenic ramie. The establishment of this system of Agrobacterium-mediated genetic transformation and regeneration of transgenic plants will be used not only to introduce genes of interest into the ramie genome for the purpose of trait improvement, but also as a common means of testing gene function by enhancing or inhibiting the expression of target genes. PMID:24488319

An, Xia; Wang, Bo; Liu, Lijun; Jiang, Hui; Chen, Jie; Ye, Shengtuo; Chen, Leiyu; Guo, Pingan; Huang, Xing; Peng, Dingxiang

2014-05-01

351

Transformation of several species of higher plants by Agrobacterium rhizogenes: sexual transmission of the transformed genotype and phenotype.  

PubMed

The T-DNA of the Ri plasmid from Agrobacterium rhizogenes is compatible with the regeneration of whole plants from genetically transformed roots and is transmitted through meiosis to the progeny of genetically transformed plants in carrot, tobacco, and morning glory (Convolvulus arvensis). The presence of Ri T-DNA is correlated with a phenotype that in some respects is invariable from species to species and in other respects varies as a function of species, organ clone within species, or individual. The transformed phenotype concerns a variety of morphological and physiological traits, is dominantly inherited in tobacco, but does not in general appear to be deleterious. The Ri T-DNA may provide a molecular starting point for studying a number of basic phenomena in plant morphology and physiology. PMID:6744417

Tepfer, D

1984-07-01

352

Production of human interferon alfa 2b in plants of Nicotiana excelsior by Agrobacterium-mediated transient expression.  

PubMed

Human interferon alpha2b gene was transiently expressed in Nicotiana excelsior plants. Fusion with N. plumbaginifolia calreticulin signal peptide for improved apoplast targeting and carrying out the expression under optimized conditions resulted in maximal interferon activity of 3.2 x 10(3) IU/g fresh weight (FW) with an average of 2.1 +/- 0.8 x 10(3) IU/g FW. It proves that N. excelsior is a suitable host for Agrobacterium-mediated transient expression of genes encoding physiologically active human proteins. The transient expression conditions optimized for GFP marker protein were confirmed to be preferable for hIFN alpha2b. PMID:21058531

Sindarovska, Y R; Gerasymenko, I M; Sheludko, Y V; Olevinskaya, Z M; Spivak, N Y; Kuchuk, N V

2010-01-01

353

Agrobacterium-mediated transformation of cotton (Gossypium hirsutum) shoot apex with a fungal phytase gene improves phosphorus acquisition.  

PubMed

Cotton is an important world economic crop plant. It is considered that cotton is recalcitrant to in vitro proliferation. Somatic embryogenesis and plant regeneration has been successful by using hypocotyl, whereas it is highly genotype dependent. Here, a genotype-independent cotton regeneration protocol from shoot apices is presented. Shoot apices from 3- to 5-day-old seedlings of cotton are infected with an Agrobacterium strain, EHA105, carrying the binary vector pC-KSA contained phytase gene (phyA) and the marker gene neomycin phosphotransferase (NPTII), and directly regenerated as shoots in vitro. Rooted shoots can be obtained within 6-8 weeks. Plants that survived by leaf painting kanamycin (kan) were -further analyzed by DNA and RNA blottings. The transgenic plants with increased the phosphorus (P) acquisition efficiency were obtained following the transformation method. PMID:23143496

Ma, Zhiying; Liu, Jianfeng; Wang, Xingfen

2013-01-01

354

Agrobacterium-mediated transformation of durum wheat (Triticum turgidum L. var. durum cv Stewart) with improved efficiency.  

PubMed

An efficient Agrobacterium-mediated durum wheat transformation system has been developed for the production of 121 independent transgenic lines. This improved system used Agrobacterium strain AGL1 containing the superbinary pGreen/pSoup vector system and durum wheat cv Stewart as the recipient plant. Acetosyringone at 400 microM was added to both the inoculation and cultivation medium, and picloram at 10 mg l(-1) and 2 mg l(-1) was used in the cultivation and induction medium, respectively. Compared with 200 microM in the inoculation and cultivation media, the increased acetosyringone concentration led to significantly higher GUS (beta-glucuronidase) transient expression and T-DNA delivery efficiency. However, no evident effects of acetosyringone concentration on regeneration frequency were observed. The higher acetosyringone concentration led to an improvement in average final transformation efficiency from 4.7% to 6.3%. Furthermore, the concentration of picloram in the co-cultivation medium had significant effects on callus induction and regeneration. Compared with 2 mg l(-1) picloram in the co-cultivation medium, increasing the concentration to 10 mg l(-1) picloram resulted in improved final transformation frequency from 2.8% to 6.3%, with the highest frequency of 12.3% reached in one particular experiment, although statistical analysis showed that this difference in final transformation efficiency had a low level of significance. Stable integration of foreign genes, their expression, and inheritance were confirmed by Southern blot analyses, GUS assay, and genetic analysis. Analysis of T(1) progeny showed that, of the 31 transgenic lines randomly selected, nearly one-third had a segregation ratio of 3:1, while the remainder had ratios typical of two or three independently segregating loci. PMID:20202997

He, Y; Jones, H D; Chen, S; Chen, X M; Wang, D W; Li, K X; Wang, D S; Xia, L Q

2010-06-01

355

Agrobacterium-mediated transformation of durum wheat (Triticum turgidum L. var. durum cv Stewart) with improved efficiency  

PubMed Central

An efficient Agrobacterium-mediated durum wheat transformation system has been developed for the production of 121 independent transgenic lines. This improved system used Agrobacterium strain AGL1 containing the superbinary pGreen/pSoup vector system and durum wheat cv Stewart as the recipient plant. Acetosyringone at 400??M was added to both the inoculation and cultivation medium, and picloram at 10?mg l?1 and 2?mg l?1 was used in the cultivation and induction medium, respectively. Compared with 200??M in the inoculation and cultivation media, the increased acetosyringone concentration led to significantly higher GUS (?-glucuronidase) transient expression and T-DNA delivery efficiency. However, no evident effects of acetosyringone concentration on regeneration frequency were observed. The higher acetosyringone concentration led to an improvement in average final transformation efficiency from 4.7% to 6.3%. Furthermore, the concentration of picloram in the co-cultivation medium had significant effects on callus induction and regeneration. Compared with 2?mg l?1 picloram in the co-cultivation medium, increasing the concentration to 10?mg l?1 picloram resulted in improved final transformation frequency from 2.8% to 6.3%, with the highest frequency of 12.3% reached in one particular experiment, although statistical analysis showed that this difference in final transformation efficiency had a low level of significance. Stable integration of foreign genes, their expression, and inheritance were confirmed by Southern blot analyses, GUS assay, and genetic analysis. Analysis of T1 progeny showed that, of the 31 transgenic lines randomly selected, nearly one-third had a segregation ratio of 3:1, while the remainder had ratios typical of two or three independently segregating loci. PMID:20202997

He, Y.; Jones, H. D.; Chen, S.; Chen, X. M.; Wang, D. W.; Li, K. X.; Wang, D. S.; Xia, L. Q.

2010-01-01

356

Analysis of TR-DNA\\/plant junctions in the genome of a Convolvulus arvensis clone transformed by Agrobacterium rhizogenes strain A4  

Microsoft Academic Search

A Charon 4A phage library, containing insert DNA isolated from a morning glory (Convolvulus arvensis) plant genetically transformed by Ri T-DNA from Agrobacterium rhizogenes strain A4, was used to isolate a lambda clone that contains part of the Ri TL-DNA and the complete TR-DNA. The two Ri T-DNAs were recovered adjacent to each other in a tail-to-tail configuration (i.e. with

Lise Jouanin; David Bouchez; Roger F. Drong; David Tepfer; Jerry L. Slightom

1989-01-01

357

Influence of Agrobacterium rhizogenes on induction of hairy roots and benzylisoquinoline alkaloids production in Persian poppy ( Papaver bracteatum Lindl.): preliminary report  

Microsoft Academic Search

Persian poppy (Papaver bracteatum Lindl.) is an important medicinal plant and source of the opium alkaloids codeine, morphine and thebaine. Transgenic root\\u000a cultures of P. bracteatum Lindl. are well-defined model systems to investigate the molecular and metabolic regulation of benzylisoquinoline alkaloid\\u000a biosynthesis. Agrobacterium rhizogenes was able to produce hairy roots on wounded Persian poppy seedlings. Excised shoots from 7-day-old Persian

Sara Rostampour; Haleh Hashemi Sohi; Esmat Jourabchi; Ehsan Ansari

2009-01-01

358

Expression of a crown gall biological control phenotype in an avirulent strain of Agrobacterium vitis by addition of the trifolitoxin production and resistance genes  

PubMed Central

Background Agrobacterium vitis is a causal agent of crown-gall disease. Trifolitoxin (TFX) is a peptide antibiotic active only against members of a specific group of ?-proteobacteria that includes Agrobacterium and its close relatives. The ability of TFX production by an avirulent strain of Agrobacterium to reduce crown gall disease is examined here. Results TFX was shown to be inhibitory in vitro against several A. vitis strains. TFX production, expressed from the stable plasmid pT2TFXK, conferred biological control activity to an avirulent strain of A. vitis. F2/5, against three virulent, TFX-sensitive strains of A. vitis tested on Nicotiana glauca. F2/5(pT2TFXK) is significantly reduces number and size of galls when co-inoculated with tumorigenic strain CG78 at a 10:1 ratio, but is ineffective at 1:1 or 1:10 ratios. F2/5(pT2TFXK) is effective when co-inoculated with tumorigenic strain CG435 at 10:1 and 1:1 ratios, but not at a 1:10 ratio. When F2/5(pT2TFXK) is co-inoculated with CG49 at a 10:1 ratio, the incidence of gall formation does not decline but gall size decreases by more than 70%. A 24 h pre-inoculation with F2/5(pT2TFXK) does not improve biological control at the 1:10 ratio. Conclusions TFX production by an avirulent strain of Agrobacterium does confer in that strain the ability to control crown gall disease on Nicotiana glauca. This is the first demonstration that the production of a ribosomally synthesized, post-translationally modified peptide antibiotic can confer reduction in plant disease incidence from a bacterial pathogen. PMID:11882255

Herlache, Thomas C; Triplett, Eric W

2002-01-01

359

A novel and fully scalable Agrobacterium spray-based process for manufacturing cellulases and other cost-sensitive proteins in plants.  

PubMed

Transient transfection of plants by vacuum infiltration of Agrobacterium vectors represents the state of the art in plant-based protein manufacturing; however, the complexity and cost of this approach restrict it to pharmaceutical proteins. We demonstrated that simple spraying of Nicotiana plants with Agrobacterium vectors in the presence of a surfactant can substitute for vacuum inoculation. When the T-DNA of Agrobacterium encodes viral replicons capable of cell-to-cell movement, up to 90% of the leaf cells can be transfected and express a recombinant protein at levels up to 50% of total soluble protein. This simple, fast and indefinitely scalable process was successfully applied to produce cellulases, one of the most volume- and cost-sensitive biotechnology products. We demonstrate here for the first time that representatives of all hydrolase classes necessary for cellulosic biomass decomposition can be expressed at high levels, stored as silage without significant loss of activity and then used directly as enzyme additives. This process enables production of cellulases, and other potential high-volume products such as noncaloric sweetener thaumatin and antiviral protein griffithsin, at commodity agricultural prices and could find broad applicability in the large-scale production of many other cost-sensitive proteins. PMID:25470212

Hahn, Simone; Giritch, Anatoli; Bartels, Doreen; Bortesi, Luisa; Gleba, Yuri

2014-12-01

360

Agrobacterium-mediated transformation of leaf base derived callus tissues of popular indica rice (Oryza sativa L. sub sp. indica cv. ADT 43).  

PubMed

A simple and efficient protocol for the Agrobacterium-mediated transformation of an agronomically useful abiotic sensitive popular indica rice cv. ADT 43 has been developed. Initiation of calli were best achieved from the leaf bases of 4 days old rice seedlings on LS medium supplemented with 2.5mg/L 2,4-D and 1.0mg/L thiamine-HCl. Rice calli immersed in Agrobacterium suspension (strain EHA 105, OD(600)=0.8) were co-cultured on LS30-AsPC medium for 2 days at 25±2°C in the dark. Based on GUS expression analysis, 10min co-cultivation time with 100?M acetosyringone was found optimum for the delivery of gus gene. Calli were proved to be very sensitive to Agrobacterium infection and we found that the level of necrotic response can be minimized after co-cultivation with 30% LS, 10g/L PVP, 10% coconut water and 250mg/L timentin which improved the final transformation efficiency to 9.33%. Molecular and genetic analysis of transgenic plants reveals the integration, expression and inheritance of transgene in the progeny (T(1)) of these plants. The copy number of transgenes has been found to vary from 1 to 2 in transgenic plants (T(0) and T(1)). PMID:21763536

Karthikeyan, Alagarsamy; Pandian, Shunmugiah Karutha; Ramesh, Manikandan

2011-09-01

361

The T-DNA oncogene A4-orf8 from Agrobacterium rhizogenes A4 induces abnormal growth in tobacco.  

PubMed

The related orf8 and iaaM T-DNA genes from Agrobacterium are each composed of two distinct parts. The 5' parts (called Norf8 or NiaaM) encode a 200-amino-acid (aa) sequence with homology to various T-DNA oncoproteins such as RolB, RolC, and 6b. The 3' parts (Corf8 or CiaaM) encode a 550-aa sequence with homology to IaaM proteins from Pseudomonas and Pantoea spp. Whereas iaaM genes encode flavin adenine dinucleotide (FAD)-dependent tryptophan 2-monooxygenases that catalyze the synthesis of indole-3-acetamide (IAM), A4-orf8 from Agrobacterium rhizogenes A4 does not. Plants expressing a 2x35S-A4-Norf8 construct accumulate soluble sugars and starch. We now have regenerated plants that express the full-size 2x35S-A4-orf8 and the truncated 2x35S-A4-Corf8 gene. 2x35S-A4-Corf8 plants accumulate starch and show reduced growth like 2x35S-A4-Norf8 plants but, in addition, display a novel set of characteristic growth modifications. These consist of leaf hypertrophy and hyperplasia (blisters); thick, dark-green leaves; thick stems; and swollen midveins. Mutations in the putative FAD-binding site of A4-Orf8 did not affect the blister syndrome. Plants expressing 2x35S-A4-Corf8 had a normal phenotype but contained less starch and soluble sugars than did wild-type plants. When 2x35S-A4-Corf8 plants were crossed to starch-accumulating 2x35S-A4-Norf8 plants with reduced growth, A4-Corf8 partially restored growth and reduced starch accumulation. A4-Corf8xA4-Norf8 crosses did not lead to the blister syndrome, suggesting that this requires physical linkage of the A4-NOrf8 and A4-COrf8 sequences. PMID:15782634

Umber, Marie; Clément, Bernadette; Otten, Léon

2005-03-01

362

PAPAYA (CARICA PAPAYA L.)  

Technology Transfer Automated Retrieval System (TEKTRAN)

Transgenic papaya plants were initially obtained using particle bombardment, a method having a poor efficiency in producing intact, single copy insertion of transgenes. Subsequently, transformation has been improved using Agrobacterium tumefaciens. With rapid progress being made in genome sequencing...

363

Comparison of the 'Ca Liberibacter asiaticus' genome adapted for an intracellular lifestyle with other members of the rhizobiales  

Technology Transfer Automated Retrieval System (TEKTRAN)

An intracellular plant pathogen ‘Ca. Liberibacter asiaticus,’ a member of the Rhizobiales, is related to Sinorhizobium meliloti, Bradyrhizobium japonicum, Agrobacterium tumefaciens and Bartonella henselae, an intracellular mammalian pathogen. Whole chromosome comparisons identified at least 52 clust...

364

RECENT ADVANCES IN BARLEY TRANSFORMATION  

Technology Transfer Automated Retrieval System (TEKTRAN)

Barley, an important member of the cereals, has been successfully transformed through various methods such as particle bombardment, Agrobacterium-tumefaciens, DNA uptake, and electroporation. Initially, the transformation in barley concentrated on developing protocols using marker genes such as gus,...

365

Effect of clove oil on plant pathogenic bacteria and bacterial wilt of tomato and geranium  

Technology Transfer Automated Retrieval System (TEKTRAN)

We determined the antibacterial activity of clove oil against seven different genera of plant pathogenic bacteria including Gram-negative Agrobacterium tumefaciens, Erwinia carotovora pv. carotovora, Pseudomonas syringae pv. syringae, Ralstonia solanacearum, and Xanthomonas campestris pv. pelargonii...

366

Robert Horsch, still imageSite: DNA Interactive (www.dnai.org)  

NSDL National Science Digital Library

Image of Robert Horsch. As a plant biologist for Monsanto, Robert Horsch and two other colleagues published a paper about a soil bacterium, Agrobacterium tumefaciens, that detailed its natural genetic engineering abilities.

2008-10-06

367

Robert Horsch, still imageSite: DNA Interactive (www.dnai.org)  

NSDL National Science Digital Library

Image of Dr. Robert Horsch As a plant biologist for Monsanto, Robert Horsch and two other colleagues published a paper about a soil bacterium, Agrobacterium tumefaciens, that detailed its natural genetic engineering abilities.

2008-10-06

368

Effect of Nitrogen Source Concentration on Curdlan Production by Agrobacterium sp. ATCC 31749 Grown on Prairie Cordgrass Hydrolysates.  

PubMed

The effect of nitrogen source concentration on the production of the polysaccharide curdlan by the bacterium Agrobacterium sp. ATCC 31749 from hydrolysates of prairie cordgrass was examined. The highest curdlan concentrations were produced by ATCC 31749 when grown on a medium containing a solids-only hydrolysate and the nitrogen source ammonium phosphate (2.2 mM) or a medium containing a complete hydrolysate and 3.3 mM ammonium phosphate. The latter medium sustained a higher level of bacterial curdlan production than the former medium after 144 h. Biomass production by ATCC 31749 was highest after 144 h when grown on a medium containing a solids-only hydrolysate and 2.2 or 8.7 mM ammonium phosphate. On the medium containing the complete hydrolysate, biomass production by ATCC 31749 was highest after 144 h when 3.3 mM ammonium phosphate was present. Bacterial biomass production after 144 h was greater on the complete hydrolysate medium compared to the solids-only hydrolysate medium. Curdlan yield produced by ATCC 31749 after 144 h from the complete hydrolysate medium containing 3.3 mM ammonium phosphate was higher than from the solids-only hydrolysate medium containing 2.2 mM ammonium phosphate. PMID:25397813

West, Thomas P

2014-11-14

369

Biotin production under limiting growth conditions by Agrobacterium/Rhizobium HK4 transformed with a modified Escherichia coli bio operon.  

PubMed

The E. coli biotin (bio) operon was modified to improve biotin production by host cells: (a) the divergently transcribed wild-type bio operon was re-organized into one transcriptional unit; (b) the wild-type bio promoter was replaced with a strong artificial (tac) promoter; (c) a potential stem loop structure between bioD and bioA was removed; and (d) the wild-type bioB ribosomal binding site (RBS) was replaced with an artificial RBS that resulted in improved bioB expression. The effects of the modifications on the bio operon were studied in E. coli by measuring biotin and dethiobiotin production, and bio gene expression with mini-cells and two-dimensional polyacrylamide gel electrophoresis. The modified E. coli bio operon was introduced into a broad host-range plasmid and used to transform Agrobacterium/Rhizobium HK4, which then produced 110 mg L-1 of biotin in a 2-L fermenter, growing on a defined medium with diaminononanoic acid as the starting material. Biotin production was not growth-phase dependent in this strain, and the rate of production remained high under limiting (maintenance) and zero growth conditions. PMID:10455485

Shaw; Lehner; Fuhrmann; Kulla; Brass; Birch; Tinschert; Venetz; Venetz; Sanchez; Tonella; Hochstrasser

1999-06-01

370

Investigating plasmodesmata genetics with virus-induced gene silencing and an agrobacterium-mediated GFP movement assay.  

PubMed

Plasmodesmata (PD) are channels that connect the cytoplasm of adjacent plant cells, permitting intercellular transport and communication. PD function and formation are essential to plant growth and development, but we still know very little about the genetic pathways regulating PD transport. Here, we present a method for assaying changes in the rate of PD transport following genetic manipulation. Gene expression in leaves is modified by virus-induced gene silencing. Seven to ten days after infection with Tobacco rattle virus carrying a silencing trigger, the gene(s) of interest is silenced in newly arising leaves. In these new leaves, individual cells are then transformed with Agrobacterium to express GFP, and the rate of GFP diffusion via PD is measured. By measuring GFP diffusion both within the epidermis and between the epidermis and mesophyll, the assay can be used to study the effects of silencing a gene(s) on PD transport in general, or transport through secondary PD specifically. Plant biologists working in several fields will find this assay useful, since PD transport impacts plant physiology, development, and defense. PMID:25287205

Brunkard, Jacob O; Burch-Smith, Tessa M; Runkel, Anne M; Zambryski, Patricia

2015-01-01

371

Characterisation and microstructure of reduced-fat chicken patties made with a novel polymer from Agrobacterium radiobacter k84.  

PubMed

Chicken patties elaborated with a novel polymer from Agrobacterium radiobacter k84 (ARB) were characterised during 60days of frozen storage. After cooking, formulations without ARB (F0), with ARB 5g/100g (F5) and ARB 10g/100g (F10) presented 4.23%, 2.83% and 0.11% fat, respectively. No differences were observed to water holding capacity, cooking yield and shear force among formulations. Microstructural analysis showed formation of meat emulsion for F5 and gel for F10. Colour and chicken flavour decreased with increase of ARB; no difference was found for tenderness among the formulations. Overall acceptance showed higher scores for F0 when compared to F5 and F10. Lipid oxidation was not a limiting factor for stability of patties; all formulations presented suitable microbiological quality over the assessed period. These results suggest ARB as a promising fat substitute, capable of maintain the quality aspects of chicken patties, although a negative impact in colour has been found. PMID:25466137

Calliari, Caroline Maria; de Souza, Evandro Leite; Castro-Goméz, Raúl Jorge Hernan; Honório, Vanessa Gonçalves; Magnani, Marciane

2015-04-15

372

T-DNA gene 5 of Agrobacterium modulates auxin response by autoregulated synthesis of a growth hormone antagonist in plants.  

PubMed Central

Oncogenes carried by the transferred DNA (T-DNA) of Agrobacterium Ti plasmids encode the synthesis of plant growth factors, auxin and cytokinin, and induce tumour development in plants. Other T-DNA genes regulate the tumorous growth in ways that are not yet understood. To determine the function of T-DNA gene 5, its coding region was expressed in Escherichia coli. Synthesis of the gene 5 encoded protein (26 kDa) correlated with a 28-fold increase in conversion of tryptophan to indole-3-lactate (ILA), an auxin analogue. Expression of chimeric gene 5 constructs in transgenic tobacco resulted in overproduction of ILA that enhanced shoot formation in undifferentiated tissues and increased the tolerance of germinating seedlings to the inhibitory effect of externally supplied auxin. Promoter analysis of gene 5 in plants revealed that its expression was inducible by auxin and confined to the vascular phloem cells. cis-regulatory elements required for auxin regulation and phloem specific expression of gene 5 were mapped to a 90 bp promoter region that carried DNA sequence motifs common to several auxin induced plant promoters, as well as a binding site for a nuclear factor, Ax-1. ILA was found to inhibit the auxin induction of the gene 5 promoter and to compete with indole-3-acetic acid (IAA) for in vitro binding to purified cellular auxin binding proteins. It is suggested therefore that ILA autoregulates its own synthesis and thereby modulates a number of auxin responses in plants. Images PMID:1756712

Körber, H; Strizhov, N; Staiger, D; Feldwisch, J; Olsson, O; Sandberg, G; Palme, K; Schell, J; Koncz, C

1991-01-01

373

Transformation of sunflower ( Helianthus annuus L.) following wounding with glass beads  

Microsoft Academic Search

A procedure was developed for transformation of Helianthus annuus (sunflower) using Agrobacterium tumefaciens. Cotyledons were removed from young seedlings, and the remaining tissue was uniformly wounded by shaking with glass beads. The wounded tissue was then co-cultivated with a hypervirulent strain of Agrobacterium tumefaciens harboring the binary plasmid pCNL56. Minimal use of defined medium was required, and no callus was

W. Scott Grayburn; Brady A. Vick

1995-01-01

374

[Transformation of bar gene to tetraploid of Isatis indigotica].  

PubMed

The transgenic tetraploid of Isatis indigotica mediated by Agrobacterium tumefaciens was obtained. To transfer the plant binary expression vector pCAMBIA 3300 carrying bar gene, the Agrobacterium tumefaciens strain EHA 105 was used as engineering bacterium. The results of PCR indicated that the bar gene had been transferred into and merged with the genome of Isatis indigotica. This study will make foundation for improvement of other characters of this species with genetic engineering. PMID:14535009

Xu, Tiefeng; Tang, Kexuan; Zhang, Hanming; Guo, Meili; Zhang, Lei; Li, Fupeng

2003-05-01

375

Brucella abortus efp gene is required for an efficient internalization in HeLa cells  

Microsoft Academic Search

Numerous chromosomal virulence genes (chv) have been shown to play an important role in the ability of Agrobacterium tumefaciens to transform plants. The A. tumefaciens chvH gene encodes a protein similar in sequence to the Escherichia coli elongation factor P (EF-P). In A. tumefaciens this factor is required for tumor formation and for full expression of the vir genes, exerting its activity

Florencia Iannino; Juan E. Ugalde; Nora Iñón de Iannino

376

Visual marker and Agrobacterium-delivered recombinase enable the manipulation of the plastid genome in greenhouse-grown tobacco plants.  

PubMed

Successful manipulation of the plastid genome (ptDNA) has been carried out so far only in tissue-culture cells, a limitation that prevents plastid transformation being applied in major agronomic crops. Our objective is to develop a tissue-culture independent protocol that enables manipulation of plastid genomes directly in plants to yield genetically stable seed progeny. We report that in planta excision of a plastid aurea bar gene (bar(au) ) is detectable in greenhouse-grown plants by restoration of the green pigmentation in tobacco leaves. The P1 phage Cre or PhiC31 phage Int site-specific recombinase was delivered on the Agrobacterium T-DNA injected at the axillary bud site, resulting in the excision of the target-site flanked marker gene. Differentiation of new apical meristems was forced by decapitating the plants above the injection site. The new shoot apex that differentiated at the injection site contained bar(au)-free plastids in 30-40% of the injected plants, of which 7% transmitted the bar(au)-free plastids to the seed progeny. The success of obtaining seed with bar(au)-free plastids depended on repeatedly forcing shoot development from axillary buds, a process that was guided by the size and position of green sectors in the leaves. The success of in planta plastid marker excision proved that manipulation of the plastid genomes is feasible within an intact plant. Extension of the protocol to in planta plastid transformation depends on the development of new protocols for the delivery of transforming DNA encoding visual markers. PMID:22268515

Tungsuchat-Huang, Tarinee; Maliga, Pal

2012-05-01

377

Induction of hairy roots by various strains of Agrobacterium rhizogenes in different types of Capsicum species explants  

PubMed Central

Background Capsicum annuum and Capsicum frutescens, also known as “chilies”, belong to the Solanaceae family and have tremendous beneficial properties. The application of hairy root culture may become an alternative method for future development of these species by adding value, such as by increasing secondary metabolites and improving genetic and biochemical stability compared with normal Capsicum plants. Therefore, in this research, different types of explants of both species were infected with various Agrobacterium rhizogenes strains to provide more information about the morphology and induction efficiency of hairy roots. After 2 weeks of in vitro seed germination, young seedling explants were cut into three segments; the cotyledon, hypocotyl, and radical. Then, the explants were co-cultured with four isolated A. rhizogenes strains in Murashige & Skoog culture media (MS) containing decreasing carbenicillin disodium concentrations for one month. Results In this experiment, thick and short hairy roots were induced at all induction sites of C. annuum while thin, elongated hairy roots appeared mostly at wound sites of C. frutescens. Overall, the hairy root induction percentages of C. frutescens were higher compared with C. annuum. Hairy root initiation was observed earliest using radicles (1st week), followed by cotyledons (2nd week), and hypocotyls (3rd week). Cotyledon explants of both species had the highest induction frequency with all strains compared with the other explants types. Strains ATCC 13333 and ATCC 15834 were the most favourable for C. frutescens while ATCC 43056 and ATCC 43057 were the most favourable for C. annuum. The interactions between the different explants and strains showed significant differences with p-values?

2014-01-01

378

The rolB-like part of the Agrobacterium rhizogenes orf8 gene inhibits sucrose export in tobacco.  

PubMed

Many Agrobacterium T-DNA genes belong to the highly diverse rolB family. The mode of action of most of these genes is still unknown. rolB-like sequences also are present at the 5' ends of the T-DNA-located iaaM genes and the iaaM homolog orf8, whereas iaaM genes from Pseudomonas and Erwinia spp. lack such sequences. iaaM genes encode tryptophan monooxygenases; these enzymes convert tryptophan into indole-3-acetamide, a precursor of indole-3-acetic acid. Tobacco plants expressing the rolB-like part of the A4 orf8 gene (2x35S-A4-Norf8 plants) accumulate glucose, fructose, sucrose, and starch and resemble sucrose transporter (NtSUT1) antisense plants. Different lines of evidence indicate that 2x35S-A4-Norf8 plants export less sucrose from source leaves. Glucose, fructose, sucrose, and starch accumulate in source leaves during sink-source transition, whereas sink tissues like petioles and midveins contain lower levels than normal. Petiole exudation experiments demonstrate a significant decrease in export of label after 14C-sucrose infiltration and after 14CO2 labeling. Grafting of stunted homozygous 2x35S-A4-Norf8 plants onto wild-type rootstocks restores growth, indicating that unloading is not affected. Growth of 2x35S-A4-Norf8 seedlings is inhibited on naphthalene acetic acid-containing media, suggesting a link between sucrose transport and auxin sensitivity. PMID:12236602

Umber, Marie; Voll, Lars; Weber, Andreas; Michler, Pierre; Otten, Léon

2002-09-01

379

Development of Agrobacterium-mediated virus-induced gene silencing and performance evaluation of four marker genes in Gossypium barbadense.  

PubMed

Gossypiumbarbadense is a cultivated cotton species and possesses many desirable traits, including high fiber quality and resistance to pathogens, especially Verticilliumdahliae (a devastating pathogen of Gossypium hirsutum, the main cultivated species). These elite traits are difficult to be introduced into G. hirsutum through classical breeding methods. In addition, genetic transformation of G. barbadense has not been successfully performed. It is therefore important to develop methods for evaluating the function and molecular mechanism of genes in G. barbadense. In this study, we had successfully introduced a virus-induced gene silencing (VIGS) system into three cultivars of G. barbadense by inserting marker genes into the tobacco rattle virus (TRV) vector. After we optimized the VIGS conditions, including light intensity, photoperiod, seedling age and Agrobacterium strain, 100% of plants agroinfiltrated with the GaPDS silencing vector showed white colored leaves. Three other marker genes, GaCLA1, GaANS and GaANR, were employed to further test this VIGS system in G. barbadense. The transcript levels of the endogenous genes in the silenced plants were reduced by more than 99% compared to control plants; these plants presented phenotypic symptoms 2 weeks after inoculation. We introduced a fusing sequence fragment of GaPDS and GaANR gene silencing vectors into a single plant, which resulted in both photobleaching and brownish coloration. The extent of silencing in plants agroinfiltrated with fusing two-gene-silencing vector was consistent with plants harboring a single gene silencing vector. The development of this VIGS system should promote analysis of gene function in G. barbadense, and help to contribute desirable traits for breeding of G. barbadense and G. hirsutum. PMID:24023833

Pang, Jinhuan; Zhu, Yue; Li, Qing; Liu, Jinzhi; Tian, Yingchuan; Liu, Yule; Wu, Jiahe

2013-01-01

380

Formation of Se (0) Nanoparticles by Duganella sp. andAgrobacterium sp. isolated from Se-laden soil of North-East Punjab, India  

PubMed Central

Background Selenium (Se) is an essential trace element, but is toxic at high concentrations. Depending upon the geological background, the land use or on anthropogenic pollution, different amounts of Se may be present in soil. Its toxicity is related to the oxyanions selenate and selenite as they are water soluble and bioavailable. Microorganisms play an important role in Se transformations in soil and its cycling in the environment by transforming water-soluble oxyanions into water insoluble, non-toxic elemental Se (0). For this study, soil samples were collected from selenium-contaminated agricultural soils of Punjab/India to enrich and isolate microbes that interacted with the Se cycle. Results A mixed microbial culture enriched from the arable soil of Punjab could reduce 230?mg/l of water soluble selenite to spherical Se (0) nanoparticles during aerobic growth as confirmed by SEM-EDX. Four pure cultures (C 1, C 4, C 6, C 7) of Gram negative, oxidase and catalase positive, aerobic bacteria were isolated from this mixed microbial consortium and identified by 16?S rDNA gene sequence alignment as two strains of Duganella sp. (C 1, C 4) and two strains of Agrobacterium sp.(C 6, C 7). SEM/TEM-EDX analyses of the culture broth of the four strains revealed excretion of uniformly round sharply contoured Se (0) nanoparticles by all cultures. Their size ranged from 140–200?nm in cultures of strains C 1 and C 4, and from 185–190?nm in cultures of strains C 6 and C 7. Both Duganella sp. revealed better selenite reduction efficiencies than the two Agrobacterium sp. Conclusions This is the first study reporting the capability of newly isolated, aerobically growing Duganella sp. and Agrobacterium sp. from soils of Punjab/India to form spherical, regularly formed Se (0) nanoparticles from water soluble selenite. Among others, the four strains may significantly contribute to the biogeochemical cycling of Se in soil. Bioconversion of toxic selenite to non-toxic Se (0) nanoparticles under aerobic conditions in general may be useful for detoxification of agricultural soil, since elemental Se may not be taken up by the roots of plants and thus allow non-dangerous fodder and food production on Se-containing soil. PMID:22607265

2012-01-01

381

Analysis of TR-DNA/plant junctions in the genome of a Convolvulus arvensis clone transformed by Agrobacterium rhizogenes strain A4.  

PubMed

A Charon 4A phage library, containing insert DNA isolated from a morning glory (Convolvulus arvensis) plant genetically transformed by Ri T-DNA from Agrobacterium rhizogenes strain A4, was used to isolate a lambda clone that contains part of the Ri TL-DNA and the complete TR-DNA. The two Ri T-DNAs were recovered adjacent to each other in a tail-to-tail configuration (i.e. with the TR-DNA inverted with respect to the TL-DNA). Comparison of nucleotide sequences from this lambda clone with the corresponding sequences from the Ri plasmid allowed us to determine the location of the T-DNA/plant junction for the right end of the TL-DNA and the left and right ends of the TR-DNA. We located, near each of these borders, a 24 bp sequence that is similar to the 24 bp consensus sequence found near the pTi T-DNA extremities. In addition, sequences similar to the "core" overdrive sequence from pTi are located near each right border. Hybridization and nucleotide sequence analysis of the DNA adjacent to the TL/TR junction shows that no plant DNA is located between the TL and TR-DNAs and suggests that the plant DNA adjacent to the end of the TR-DNA may have been rearranged during the integration into the plant genome. PMID:24272719

Jouanin, L; Bouchez, D; Drong, R F; Tepfer, D; Slightom, J L

1989-01-01

382

The quorum-sensing system AvsR-AvsI regulates both long-chain and short-chain acyl-homoserine lactones in Agrobacterium vitis E26.  

PubMed

Agrobacterium vitis strain E26 is a nonpathogenic bacterium isolated from grape crown gall. In this study, the identification of a luxR-luxI type quorum-sensing system in strain E26 is reported. This system is involved in the induction of hypersensitive response on tobacco, but not its biocontrol activity. The deduced components AvsI(E26) and AvsR(E26) show the greatest similarity to AvsI(F2/5) and AvsR(F2/5), respectively from A. vitis strain F2/5. The mutant in AvsI(E26) abolished the production of both long-chain and short-chain acyl-homoserine lactones signals as well as the ability to cause hypersensitive response on tobacco. Complementaion of avsI (E26) and avsR (E26) genes restored the lost phenotypes to the level of wild type E26. In pot trial, no significant difference on biocontrol efficiency against grapevine crown gall was found between the wide type E26 and its quorum sensing negative mutants. PMID:17906938

Wang, Yuan-Hong; Zhang, Li-Qun; Li, Jin-Yun; Wang, Jian-Hui; Wang, Hui-Min

2008-03-01

383

Utilization of Trihalogenated Propanes by Agrobacterium radiobacter AD1 through Heterologous Expression of the Haloalkane Dehalogenase from Rhodococcus sp. Strain m15-3  

PubMed Central

Trihalogenated propanes are toxic and recalcitrant organic compounds. Attempts to obtain pure bacterial cultures able to use these compounds as sole carbon and energy sources were unsuccessful. Both the haloalkane dehalogenase from Xanthobacter autotrophicus GJ10 (DhlA) and that from Rhodococcus sp. strain m15-3 (DhaA) were found to dehalogenate trihalopropanes to 2,3-dihalogenated propanols, but the kinetic properties of the latter enzyme are much better. Broad-host-range dehalogenase expression plasmids, based on RSF1010 derivatives, were constructed with the haloalkane dehalogenase from Rhodococcus sp. strain m15-3 under the control of the heterologous promoters Plac, PdhlA, and Ptrc. The resulting plasmids yielded functional expression in several gram-negative bacteria. A catabolic pathway for trihalopropanes was designed by introducing these broad-host-range dehalogenase expression plasmids into Agrobacterium radiobacter AD1, which has the ability to utilize dihalogenated propanols for growth. The recombinant strain AD1(pTB3), expressing the haloalkane dehalogenase gene under the control of the dhlA promoter, was able to utilize both 1,2,3-tribromopropane and 1,2-dibromo-3-chloropropane as sole carbon sources. Moreover, increased expression of the haloalkane dehalogenase resulted in elevated resistance to trihalopropanes. PMID:10508091

Bosma, Tjibbe; Kruizinga, Edwin; de Bruin, Erik J.; Poelarends, Gerrit J.; Janssen, Dick B.

1999-01-01

384

Utilization of trihalogenated propanes by Agrobacterium radiobacter AD1 through heterologous expression of the haloalkane dehalogenase from Rhodococcus sp. strain M15-3.  

PubMed

Trihalogenated propanes are toxic and recalcitrant organic compounds. Attempts to obtain pure bacterial cultures able to use these compounds as sole carbon and energy sources were unsuccessful. Both the haloalkane dehalogenase from Xanthobacter autotrophicus GJ10 (DhlA) and that from Rhodococcus sp. strain m15-3 (DhaA) were found to dehalogenate trihalopropanes to 2,3-dihalogenated propanols, but the kinetic properties of the latter enzyme are much better. Broad-host-range dehalogenase expression plasmids, based on RSF1010 derivatives, were constructed with the haloalkane dehalogenase from Rhodococcus sp. strain m15-3 under the control of the heterologous promoters P(lac), P(dhlA), and P(trc). The resulting plasmids yielded functional expression in several gram-negative bacteria. A catabolic pathway for trihalopropanes was designed by introducing these broad-host-range dehalogenase expression plasmids into Agrobacterium radiobacter AD1, which has the ability to utilize dihalogenated propanols for growth. The recombinant strain AD1(pTB3), expressing the haloalkane dehalogenase gene under the control of the dhlA promoter, was able to utilize both 1,2,3-tribromopropane and 1,2-dibromo-3-chloropropane as sole carbon sources. Moreover, increased expression of the haloalkane dehalogenase resulted in elevated resistance to trihalopropanes. PMID:10508091

Bosma, T; Kruizinga, E; de Bruin, E J; Poelarends, G J; Janssen, D B

1999-10-01

385

Agrobacterium-transformed rice plants expressing synthetic cryIA(b) and cryIA(c) genes are highly toxic to striped stem borer and yellow stem borer.  

PubMed

Over 2,600 transgenic rice plants in nine strains were regenerated from >500 independently selected hygromycin-resistant calli after Agrobacterium-mediated transformation. The plants were transformed with fully modified (plant codon optimized) versions of two synthetic cryIA(b) and cryIA(c) coding sequences from Bacillus thuringiensis as well as the hph and gus genes, coding for hygromycin phosphotransferase and beta-glucuronidase, respectively. These sequences were placed under control of the maize ubiquitin promoter, the CaMV35S promoter, and the Brassica Bp10 gene promoter to achieve high and tissue-specific expression of the lepidopteran-specific delta-endotoxins. The integration, expression, and inheritance of these genes were demonstrated in R0 and R1 generations by Southern, Northern, and Western analyses and by other techniques. Accumulation of high levels (up to 3% of soluble proteins) of CryIA(b) and CryIA(c) proteins was detected in R0 plants. Bioassays with R1 transgenic plants indicated that the transgenic plants were highly toxic to two major rice insect pests, striped stem borer (Chilo suppressalis) and yellow stem borer (Scirpophaga incertulas), with mortalities of 97-100% within 5 days after infestation, thus offering a potential for effective insect resistance in transgenic rice plants. PMID:9501164

Cheng, X; Sardana, R; Kaplan, H; Altosaar, I

1998-03-17

386

Agrobacterium-transformed rice plants expressing synthetic cryIA(b) and cryIA(c) genes are highly toxic to striped stem borer and yellow stem borer  

PubMed Central

Over 2,600 transgenic rice plants in nine strains were regenerated from >500 independently selected hygromycin-resistant calli after Agrobacterium-mediated transformation. The plants were transformed with fully modified (plant codon optimized) versions of two synthetic cryIA(b) and cryIA(c) coding sequences from Bacillus thuringiensis as well as the hph and gus genes, coding for hygromycin phosphotransferase and ?-glucuronidase, respectively. These sequences were placed under control of the maize ubiquitin promoter, the CaMV35S promoter, and the Brassica Bp10 gene promoter to achieve high and tissue-specific expression of the lepidopteran-specific ?-endotoxins. The integration, expression, and inheritance of these genes were demonstrated in R0 and R1 generations by Southern, Northern, and Western analyses and by other techniques. Accumulation of high levels (up to 3% of soluble proteins) of CryIA(b) and CryIA(c) proteins was detected in R0 plants. Bioassays with R1 transgenic plants indicated that the transgenic plants were highly toxic to two major rice insect pests, striped stem borer (Chilo suppressalis) and yellow stem borer (Scirpophaga incertulas), with mortalities of 97–100% within 5 days after infestation, thus offering a potential for effective insect resistance in transgenic rice plants. PMID:9501164

Cheng, Xiongying; Sardana, Ravinder; Kaplan, Harvey; Altosaar, Illimar

1998-01-01

387

Comparative investigation of the reaction mechanisms of the organophosphate-degrading phosphotriesterases from Agrobacterium radiobacter (OpdA) and Pseudomonas diminuta (OPH).  

PubMed

Metal ion-dependent, organophosphate-degrading enzymes have acquired increasing attention due to their ability to degrade and thus detoxify commonly used pesticides and nerve agents such as sarin. The best characterized of these enzymes are from Pseudomonas diminuta (OPH) and Agrobacterium radiobacter (OpdA). Despite high sequence homology (>90 % identity) and conserved metal ion coordination these enzymes display considerable variations in substrate specificity, metal ion affinity/preference and reaction mechanism. In this study, we highlight the significance of the presence (OpdA) or absence (OPH) of an extended hydrogen bond network in the active site of these enzymes for the modulation of their catalytic properties. In particular, the second coordination sphere residue in position 254 (Arg in OpdA, His in OPH) is identified as a crucial factor in modulating the substrate preference and binding of these enzymes. Inhibition studies with fluoride also support a mechanism for OpdA whereby the identity of the hydrolysis-initiating nucleophile changes as the pH is altered. The same is not observed for OPH. PMID:25104333

Pedroso, Marcelo M; Ely, Fernanda; Miti?, Nataša; Carpenter, Margaret C; Gahan, Lawrence R; Wilcox, Dean E; Larrabee, James L; Ollis, David L; Schenk, Gerhard

2014-12-01

388

Tall fescue (Festuca arundinacea Schreb.).  

PubMed

Tall fescue (Festuca arundinacea Schreb.) is the predominant cool-season perennial grass in the United States. It is widely used for both forage and turf purposes. This chapter describes a protocol that allows for the generation of a large number of transgenic tall fescue plants by Agrobacterium tumefaciens-mediated transformation. Embryogenic calli induced from caryopsis are used as explants for inoculation with A. tumefaciens. The Agrobacterium strain used is EHA105. Hygromycin phosphotransferase gene (hph) is used as the selectable marker, and hygromycin is used as the selection agent. Calli resistant to hygromycin are obtained after 4-6 weeks of selection. Soil-grown tall fescue plants can be regenerated 4-5 months after Agrobacterium tumefaciens-mediated transformation. PMID:25416272

Ge, Yaxin; Wang, Zeng-Yu

2015-01-01

389

An Efficient Method of Agrobacterium-Mediated Genetic Transformation and Regeneration in Local Indian Cultivar of Groundnut (Arachis hypogaea) Using Grafting.  

PubMed

Groundnut (Arachis hypogaea L.) is an industrial crop used as a source of edible oil and nutrients. In this study, an efficient method of regeneration and Agrobacterium-mediated genetic transformation is reported for a local cultivar GG-20 using de-embryonated cotyledon explant. A high regeneration 52.69?±?2.32 % was achieved by this method with 66.6 ?M 6-benzylaminopurine (BAP), while the highest number of shoot buds per explant, 17.67?±?3.51, was found with 20 ?M BAP and 10 ?M 2,4-dichlorophenoxyacetic acid (2,4-D). The bacterial culture OD, acetosyringone and L-cysteine concentration were optimized as 1.8, 200 ?M and 50 mg L(-1), respectively, in co-cultivation media. It was observed that the addition of 2,4-D in co-cultivation media induced accumulation of endogenous indole-3-acetic acid (IAA). The optimized protocol exhibited 85 % transformation efficiency followed by 14.65?±?1.06 % regeneration, of which 3.82?±?0.6 % explants were survived on hygromycin after selection. Finally, 14.58?±?2.95 % shoots (regenerated on survived explants) were rooted on rooting media (RM3). In grafting method, regenerated shoots (after hygromycin selection) were grafted on the non-transformed stocks with 100 % survival and new leaves emerged in 3 weeks. The putative transgenic plants were then confirmed by PCR, Southern hybridization, reverse transcriptase PCR (RT-PCR) and ?-glucuronidase (GUS) histochemical assay. The reported method is efficient and rapid and can also be applied to other crops which are recalcitrant and difficult in rooting. PMID:25308617

Tiwari, Vivekanand; Chaturvedi, Amit Kumar; Mishra, Avinash; Jha, Bhavanath

2014-10-12

390

Degradation of 4-aminobenzenesulfonate by a two-species bacterial coculture. Physiological interactions between Hydrogenophaga palleronii S1 and Agrobacterium radiobacter S2.  

PubMed

The mutualistic interactions in a 4-aminobenzenesulfonate (sulfanilate) degrading mixed bacterial culture were studied. This coculture consisted of Hydrogenophaga palleronii strain S1 and Agrobacterium radiobacter strain S2. In this coculture only strain S1 desaminated sulfanilate to catechol-4-sulfonate, which did not accumulate in the medium but served as growth substrate for strain S2. During growth in batch culture with sulfanilate as sole source of carbon, energy, nitrogen and sulfur, the relative cell numbers (colony forming units) of both strains were almost constant. None of the strains reached a cell number which was more than threefold higher than the cell number of the second strain. A mineral medium with sulfanilate was inoculated with different relative cell numbers of both strains (relative number of colony forming units S1:S2 2200:1 to 1:500). In all cases, growth was found and the proportion of both strains moved towards an about equal value of about 3:1 (strain S1:strain S2). In contrast to the coculture, strain S1 did not grow in a mineral medium in axenic culture with 4-aminobenzenesulfonate or any other simple organic compound tested. A sterile culture supernatant from strain S2 enabled strain S1 to grow with 4-aminobenzenesulfonate. The same growth promoting effect was found after the addition of a combination of 4-aminobenzoate, biotin and vitamin B12. Strain S1 grew with 4-aminobenzenesulfonate plus the three vitamins with about the same growth rate as the mixed culture in a mineral medium. When (resting) cells of strain S1 were incubated in a pure mineral medium with sulfanilate, up to 30% of the oxidized sulfanilate accumulated as catechol-4-sulfonate in the culture medium. In contrast, only minor amounts of catechol-4-sulfonate accumulated when strain S1 was grown with 4ABS in the presence of the vitamins. PMID:8782393

Dangmann, E; Stolz, A; Kuhm, A E; Hammer, A; Feigel, B; Noisommit-Rizzi, N; Rizzi, M; Reuss, M; Knackmuss, H J

1996-06-01

391

Optimal inductive and cultural conditions of Polygonum multiflorum transgenic hairy roots mediated with Agrobacterium rhizogenes R1601 and an analysis of their anthraquinone constituents  

PubMed Central

Background: Polygonum multiflorum is an important medicinal plant. Hairy roots systems obtained by transforming plant tissues with the natural genetic engineer Agrobacterium rhizogenes can produce valuable biological active substances, which have immense potential in the pharmaceutical industry. Objective: To optimize the inductive and cultural conditions of P. multiflorum hairy roots and to identify the major active secondary metabolites in hairy roots. Materials and Methods: P. multiflorum hairy root were mediated with A. rhizogenes R1601 to induce hairy roots. Four combinations, including Murashige–Skoog (MS), 1/2 MS, B5, and White, were investigated to optimize the culture medium. MS medium was selected for the growth measurement. The qualitative and quantitative determinations of free anthraquinone in hairy roots were compared with the calli and aseptic plantlets using high-performance liquid chromatography. Results: The inductive rates of hairy roots by leaves were higher than for any other explants. The presence of agropine in the P. multiflorum hairy roots confirmed that they were indeed transgenic. MS medium was the most suitable of the four media for hairy root growth. Meanwhile, the growth kinetics and nutrient consumption results showed that the hairy roots displayed a sigmoidal growth curve and that their optimal inoculation time was 18-21 days. The determination of the anthraquinone constituents indicated that the rhein content of the hairy roots reached 2.495 ?g g?1 and was 2.55-fold higher than that of natural plants. Conclusion: Transgenic hairy roots of P. multiflorum could be one of the most potent materials for industrial-scale production of bioactive anthraquinone constituents. PMID:24696550

Huang, Bing; Lin, Huanjie; Yan, Chuanyan; Qiu, Hongyan; Qiu, Lipeng; Yu, Rongmin

2014-01-01

392

Immobilization of organophosphohydrolase OpdA from Agrobacterium radiobacter by overproduction at the surface of polyester inclusions inside engineered Escherichia coli.  

PubMed

Organophosphorus pesticides (OP) are highly toxic and are widely used as insecticides. Bacterial organophosphohydrolases which hydrolyze a variety of OPs have been considered for the clean-up of polluted environments. This study describes the engineering of Escherichia coli towards the overproduction of the organophosphohydrolase (OpdA) from Agrobacterium radiobacter at the surface of polyester inclusions. The OpdA was N-terminally fused via a designed linker region to the C-terminus of polyester inclusion-forming enzyme PhaC of Ralstonia eutropha. The PhaC-L-OpdA fusion protein was overproduced by using the strong T7 promoter and when coexpressed with genes phaA (encoding ?-ketothiolase) and phaB (encoding acetoacetyl-CoA reductase) from R. eutropha this led to formation of polyester inclusions abundantly displaying OpdA. These OpdA beads showed organophosphohydrolase activity of 1,840 U/g wet polyester beads or 4,412 U/g protein. Steady state kinetics revealed that when compared with free OpdA the k(cat) (s(-1)) of 139 of immobilized OpdA was reduced by about 16.5-fold while the K(M) (M) of 2.5 × 10(-4) was increased by 1.6-fold. The immobilized OpdA showed increased temperature stability. Moreover, the stability of OpdA immobilized to polyester beads was assessed by incubating OpdA beads at 25°C for up to 11 days and no significant loss in enzyme activity was detected. The application performance of the OpdA beads with respect to hydrolysis of OPs in contaminated environments was demonstrated in wool scour spiked with fluorescent coumaphos. This study demonstrated a new strategy toward the efficient recombinant production of immobilized organophosphohydrolase, the OpdA, suitable for bioremediation applications. PMID:22170266

Blatchford, Paul A; Scott, Colin; French, Nigel; Rehm, Bernd H A

2012-05-01

393

Potential of a 16S rRNA-Based Taxonomic Microarray for Analyzing the Rhizosphere Effects of Maize on Agrobacterium spp. and Bacterial Communities†  

PubMed Central

Bacterial diversity is central to ecosystem sustainability and soil biological function, for which the role of roots is important. The high-throughput analysis potential of taxonomic microarray should match the breadth of bacterial diversity. Here, the power of this technology was evidenced through methodological verifications and analysis of maize rhizosphere effect based on a 16S rRNA-based microarray developed from the prototype of H. Sanguin et al. (Environ. Microbiol. 8:289-307, 2006). The current probe set was composed of 170 probes (41 new probes in this work) that targeted essentially the Proteobacteria. Cloning and sequencing of 16S rRNA amplicons were carried out on maize rhizosphere and bulk soil DNA. All tested clones that had a perfect match with corresponding probes were positive in the hybridization experiment. The hierarchically nested probes were reliable, but the level of taxonomic identification was variable, depending on the probe set specificity. The comparison of experimental and theoretical hybridizations revealed 0.91% false positives and 0.81% false negatives. The microarray detection threshold was estimated at 0.03% of a given DNA type based on DNA spiking experiments. A comparison of the maize rhizosphere and bulk soil hybridization results showed a significant rhizosphere effect, with a higher predominance of Agrobacterium spp. in the rhizosphere, as well as a lower prevalence of Acidobacteria, Bacteroidetes, Verrucomicrobia, and Planctomycetes, a new taxon of interest in soil. In addition, well-known taxonomic groups such as Sphingomonas spp., Rhizobiaceae, and Actinobacteria were identified in both microbial habitats with strong hybridization signals. The taxonomic microarray developed in the present study was able to discriminate and characterize bacterial community composition in related biological samples, offering extensive possibilities for systematic exploration of bacterial diversity in ecosystems. PMID:16751545

Sanguin, Hervé; Remenant, Benoît; Dechesne, Arnaud; Thioulouse, Jean; Vogel, Timothy M.; Nesme, Xavier; Moënne-Loccoz, Yvan; Grundmann, Geneviève L.

2006-01-01

394

Genetic Transformation of Switchgrass  

NASA Astrophysics Data System (ADS)

Switchgrass (Panicum virgatum L.) is a highly productive warm-season C4 species that is being developed into a dedicated biofuel crop. This chapter describes a protocol that allows the generation of transgenic switchgrass plants by Agrobacterium tumefaciens-mediated transformation. Embryogenic calluses induced from caryopses or inflorescences were used as explants for inoculation with A. tumefaciens strain EHA105. Hygromycin phosphotransferase gene (hph) was used as the selectable marker and hygromycin was used as the selection agent. Calluses resistant to hygromycin were obtained after 5-6 weeks of selection. Soil-grown switchgrass plants were regenerated about 6 months after callus induction and Agrobacterium-mediated transformation.

Xi, Yajun; Ge, Yaxin; Wang, Zeng-Yu

395

Hairy root induction of Papaver somniferum var. album , a difficult-to-transform plant, by A. rhizogenes LBA 9402  

Microsoft Academic Search

Two strains of Agrobacterium rhizogenes (15834, LBA 9402) and one Agrobacterium tumefaciens strain [GV 3101 (PMP90RK, p35SGUS-2)] and four culture media were tested and compared for their ability to induce hairy root formation on wounded Papaver somniferum L. hypocotyls. Five weeks after the infection with A. rhizogenes LBA 9402, hairy roots appeared on 80% of the hypocotyls maintained in the

V. Le Flem-Bonhomme; D. Laurain-Mattar

2004-01-01

396

Maize (Zea mays L.).  

PubMed

Agrobacterium tumefaciens-mediated transformation is an effective method for introducing genes into maize. In this chapter, we describe a detailed protocol for genetic transformation of the maize genotype Hi II. Our starting plant material is immature embryos cocultivated with an Agrobacterium strain carrying a standard binary vector. In addition to step-by-step laboratory transformation procedures, we include extensive details in growing donor plants and caring for transgenic plants in the greenhouse. PMID:25300834

Frame, Bronwyn; Warnberg, Katey; Main, Marcy; Wang, Kan

2015-01-01

397

Comparative anatomy of gall development on Gypsophila paniculata induced by bacteria with different mechanisms of pathogenicity  

Microsoft Academic Search

Galls induced on Gypsophila paniculata by Pantoea agglomerans pv. gypsophilae (Pag) and Agrobacterium tumefaciens (At), bacteria with different mechanisms of pathogenicity, were compared morphologically and anatomically. The pathogenicity of Pag is dependent on the presence of an indigenous plasmid that harbors hrp gene cluster, genes encoding Hop virulence proteins and biosynthetic genes for auxin (IAA) and cytokinins (CKs), whereas that

L. Chalupowicz; I. Barash; M. Schwartz; R. Aloni; S. Manulis

2006-01-01

398

Clonal Propagation of walnut rootstock genotypes for genetic improvement 2010  

Technology Transfer Automated Retrieval System (TEKTRAN)

The soilborne bacterium Agrobacterium tumefaciens is the causal agent of crown gall disease of walnut. Large tumors located near the crown of the tree are hallmark symptoms induced by the bacterial pathogen. Untreated tumors can have an adverse effect on tree health resulting in reduced nut yield an...

399

Abstract Transgenic orchid (Dendrobium Madame Thong-In) plants were regenerated by inoculating thin-  

E-print Network

Abstract Transgenic orchid (Dendrobium Madame Thong-In) plants were regenerated by inoculating thin a binary vector that carried the orchid DOH1 antisense gene. The transformation was performed through two, indicating a role for DOH1 in the basic plant architecture in orchid. Keywords Agrobacterium tumefaciens

Yu, Hao

400

The Canon of Potato Science: 6. Genetic Modification and Cis and Transgenesis  

Microsoft Academic Search

What is it? Genetic modification is the act of inserting one or more agriculturally important genes into the genome of a potato plant by in vitro techniques and by using modified Agrobacterium tumefaciens as a natural gene transfer tool. The end product is a genetically modified (GM) plant. Important preconditions for transformation are in vitro regeneration and transformation ability of

E. Jacobsen

2007-01-01

401

Genetic transformation and gene silencing mediated by multiple copies of a transgene in eastern white pine  

Microsoft Academic Search

An efficient transgenic eastern white pine (Pinus strobus L.) plant regeneration system has been estab- lished using Agrobacterium tumefaciens strain GV3850- mediated transformation and the green fluorescent protein (gfp) gene as a reporter in this investigation. Stable integration of transgenes in the plant genome of pine was confirmed by polymerase chain reaction (PCR), Southern blot, and northern blot analyses. Transgene

Wei Tang; Ronald J. Newton; Douglas A. Weidner

2007-01-01

402

Transformation of sunflower ( Helianthus annuus L.): a reliable protocol  

Microsoft Academic Search

A reliable protocol for the transformation of cultivated sunflower (Helianthus annuus L.) has been established, based on microprojectile bombardment of half shoot apices in combination with Agrobacterium tumefaciens coculture. Transgenic shoots have been obtained from 5 inbred lines, although transformation efficiencies varied with the genotype. Plants expressing the transgenes could be recovered from up to 7% of the explants. A

Nathalie Knittel; Véronique Gruber; Günther Hahne; Philippe Lénée

1994-01-01

403

Relative strengths of the 35S califlower mosaic virus, 1?, 2?, and nopaline synthase promoters in transformed tobacco sugarbeet and oilseed rape callus tissue  

Microsoft Academic Search

The 35S promoter of cauliflower mosaic virus and promoters from the nopaline synthase, 1' and 2' genes of Agrobacterium tumefaciens T-DNA were fused to the bacterial octopine synthase and chitinase gene coding regions. These chimaeric gene constructions were introduced into tobacco, sugarbeet and oilseed rape cells and their relative levels of expression measured by primer extension analysis of RNA isolated

Mark H. Harpster; Jeffrey A. Townsend; Jonathan D. G. Jones; John Bedbrook; Pamela Dunsmuir

1988-01-01

404

Evaluaton of Wild Juglans Species for Crown Gall Resistance  

Technology Transfer Automated Retrieval System (TEKTRAN)

Crown Gall disease of walnut is caused by the ubiquitous soil-borne bacterium, Agrobacterium tumefaciens, which is able to transfer a specific piece of its own DNA into the genome of the plant host cell. The result of this genetic transformation is the autonomous undifferentiated massive growth of ...

405

[Genetic transformation of sugar beet: evolution of theoretical and experimental approaches].  

PubMed

The review is dedicated to several aspects of sugar beet (Beta vulgaris L.) biotechnology: in vitro cultivation, callus induction, plant regeneration and genetic transformation. Media composition, methods of plant regeneration via somatic embryogenesis and protoplast culture are analysed. The use of Agrobacterium tumefaciens and gold particle bombardment is the base for modern genetic transformation methods. PMID:16250243

Golovko, A E; Dovzhenko, A A; Gleba, Iu Iu

2005-01-01

406

GhSEM-1 marker potentially associated with regeneration ability in cotton.  

Technology Transfer Automated Retrieval System (TEKTRAN)

A marker protein for embryogenic potential could be useful in determining if target tissue for Agrobacterium tumefaciens or microprojectile bombardment has the ability to regenerate plants. Certain varieties of cotton, especially Coker 312, are known to form somatic embryos readily, while others are...

407

AN IN VIVO LUCIFERASE-BASED TRANSIENT ASSAY SYSTEM USING AGROBACTERIA-INFILTRATION: IMPLICATIONS FOR POST-TRANSCRIPTIONAL GENE SILENCING  

Technology Transfer Automated Retrieval System (TEKTRAN)

An in vivo assay system for analyzing transient luciferase expression in Agrobactera-infused tobacco leaves is described. The system makes use of Agrobacterium tumefaciens harboring T-DNA vectors containing either an intron-containing firefly (Photinus pyralis) luciferase (EC 1.13.12.7) gene or an ...

408

Particle bombardment and the genetic enhancement of crops: myths and realities  

Microsoft Academic Search

DNA transfer by particle bombardment makes use of physical processes to achieve the transformation of crop plants. There is no dependence on bacteria, so the limitations inherent in organisms such as Agrobacterium tumefaciens do not apply. The absence of biological constraints, at least until DNA has entered the plant cell, means that particle bombardment is a versatile and effective transformation

Fredy Altpeter; Niranjan Baisakh; Roger Beachy; Ralph Bock; Teresa Capell; Paul Christou; Henry Daniell; Karabi Datta; Swapan Datta; Philip J. Dix; Claude Fauquet; Ning Huang; Ajay Kohli; Hans Mooibroek; Liz Nicholson; Thi Thanh Nguyen; Gregory Nugent; Krit Raemakers; Andrea Romano; David A. Somers; Eva Stoger; Nigel Taylor; Richard Visser

2005-01-01

409

Evaluation of wild Juglans species for crown gall resistance  

Technology Transfer Automated Retrieval System (TEKTRAN)

Paradox, the most widely used rootstock in CA walnut production, is highly susceptible to the causal agent of crown gall (CG) Agrobacterium tumefaciens. The bacterial pathogen induces the formation of large tumors around the crown of the tree resulting in a reduction in both vigor and yield. If left...

410

In planta transformation of Arabidopsis thaliana  

Microsoft Academic Search

Transformants of Arabidopsis thaliana can be generated without using tissue culture techniques by cutting primary and secondary inflorescence shoots at their bases and inoculating the wound sites with Agrobacterium tumefaciens suspensions. After three successive inoculations, treated plants are grown to maturity, harvested and the progeny screened for transformants on a selective medium. We have investigated the reproducibility and the overall

Vesna Katavic; George W. Haughn; Darwin Reed; Marilyn Martin; Ljerka Kunst

1994-01-01

411

Methoxybifurcarenone: an antifungal and antibacterial meroditerpenoid from the brown alga Cystoseira tamariscifolia  

Microsoft Academic Search

A meroditerpenoid metabolite has been isolated from the brown alga Cystoseira tamariscifolia and characterized as methoxybifurcarenone, by spectral analysis. Methoxybifurcarenone possesses antifungal activity against three tomato pathogenic fungi: Botrytis cinerea, Fusarium oxysporum sp. mycopersici and Verticillium alboatrum and antibacterial activity against Agrobacterium tumefaciens and Escherichia coli.

Ahmed Bennamara; Abdelmjid Abourriche; Mohamed Berrada; M'hamed Charrouf; Nezha Chaib; Mohammed Boudouma; François Xavier Garneau

1999-01-01

412

Evidence for VirB4-mediated dislocation of membrane-integrated VirB2 pilin during biogenesis of the Agrobacterium VirB/VirD4 type IV secretion system.  

PubMed

Agrobacterium VirB2 pilin is required for assembly of the VirB/VirD4 type IV secretion system (T4SS). The propilin is processed by signal sequence cleavage and covalent linkage of the N and C termini, and the cyclized pilin integrates into the inner membrane (IM) as a pool for assembly of the secretion channel and T pilus. Here, by use of the substituted cysteine accessibility method (SCAM), we defined the VirB2 IM topology and then identified distinct contributions of the T4SS ATPase subunits to the pilin structural organization. Labeling patterns of Cys-substituted pilins exposed to the membrane-impermeative, thiol-reactive reagent 3-(N-maleimidopropionyl)biocytin (MPB) supported a topology model in which two hydrophobic stretches comprise transmembrane domains, an intervening hydrophilic loop (residues 90 to 94) is cytoplasmic, and the hydrophilic N and C termini joined at residues 48 and 121 form a periplasmic loop. Interestingly, the VirB4 ATPase, but not a Walker A nucleoside triphosphate (NTP) binding motif mutant, induced (i) MPB labeling of Cys94, a residue that in the absence of the ATPase is located in the cytoplasmic loop, and (ii) release of pilin from the IM upon osmotic shock. These findings, coupled with evidence for VirB2-VirB4 complex formation by coimmunoprecipitation, support a model in which VirB4 functions as a dislocation motor to extract pilins from the IM during T4SS biogenesis. The VirB11 ATPase functioned together with VirB4 to induce a structural change in the pilin that was detectable by MPB labeling, suggestive of a role for VirB11 as a modulator of VirB4 dislocase activity. PMID:20656905

Kerr, Jennifer E; Christie, Peter J

2010-10-01

413

Cherry.  

PubMed

Agrobacterium tumefaciens-mediated transformation of sour chgahtvy (Prunus cerasus L.) "Montmorency" and sweet cherry rootstocks "Gisela 6" and "Gisela 7" (P. cerasus × P. canescens) is described. Briefly, leaf explants from in vitro shoots are cocultivated with A. tumefaciens either directly (for "Gisela 6" and "Gisela 7") or after pretreatment (for "Montmorency") on cocultivation medium; selection and regeneration of transformed shoots are carried out on selection medium containing 50 mg/L kanamycin (Km) and 250 mg/L timentin (or cefotaxime) for 3-5 months. In this protocol, the optimal media for shoot proliferation and shoot regeneration from leaf explants are genotype dependent. PMID:25416255

Song, Guo-Qing

2015-01-01

414

Transgenic broccoli expressing a Bacillus thuringiensis insecticidal crystal protein: Implications for pest resistance management strategies  

Microsoft Academic Search

We usedAgrobacterium tumefaciens to transform flowering stalk explants of five genotypes of broccoli with a construct containing the neomycin phosphotransferase gene and aBacillus thuringiensis (Bt) gene [CryIA(c) type] optimized for plant expression. Overall transformation efficiency was 6.4%; 181 kanamycin-resistant plants were recovered. Of the 162 kanamycin-resistant plants tested, 112 (69%) caused 100% morality of 1st-instar larvae of aBt-susceptible diamondback moth

Timothy D. Metz; Richard T. Roush; Juliet D. Tang; Anthony M. Shelton; Elizabeth D. Earle

1995-01-01

415

Expression of Chlamydophila psittaci MOMP heat-labile toxin B subunit fusion gene in transgenic rice  

Microsoft Academic Search

A DNA fragment encoding the MOMP gene of Chlamydophila psittaci was fused to the heat-labile toxin B subunit gene (LTB-MOMP) and transferred into rice callus by Agrobacterium tumefaciens-mediated transformation. The LTB-MOMP fusion gene was detected in genomic DNA from transformed rice leaves by Southern blot and RT-PCR amplification. Synthesis and assembly of the LTB-MOMP fusion protein into pentamers was detected

Xiuxiang Zhang; Ziguo Yuan; Xuejun Guo; Jingwen Li; Zhaonan Li; Qingyu Wang

2008-01-01

416

Biofilms on Indwelling Urethral Catheters Produce Quorum Sensing Signal Molecules In Situ and In Vitro  

Microsoft Academic Search

Acylated homoserine lactones (AHLs) are chemical signals that mediate population density-dependent (quorum-sensing) gene expression in numerous gram-negative bacteria. In this study, gram-negative bacilli isolated from catheters were screened for AHL production by a cross-feeding assay utilizing an AHL-responsive Agrobacterium tumefaciens reporter strain. Positive reactions were obtained from 14 isolates of Pseudomonas aeruginosa; negative or weakly positive reactions were recorded for

DAVID J. STICKLER; NICOLA S. MORRIS; ROBERT J. C. MCLEAN; CLAY FUQUA

417

The nucleotide sequence and genome structure of the geminivirus miscanthus streak virus  

Microsoft Academic Search

A tandem dimer of miscanthus streak virus (MiSV) DNA was inserted into the T-DNA of the binary plasmid vector pBIN19 and agroinoculated into several monocotyledonous plants (monocots) using Agrobacterium tumefaciens or A. rhizogenes. Disease symptoms and geminate particles were produced in maize and Panicum milaceum plants, and MiSV- specific double-stranded and single-stranded DNAs were found in these plants. The nucleotide

Masaaki Chatani; Yoshinori Matsumoto; Haruyoshi Mizuta; Masato Ikegami; Margaret I. Boulton; Jeffrey W. Davies

1991-01-01

418

Transferring cucumber mosaic virus-white leaf strain coat protein gene into Cucumis melo L. and evaluating transgenic plants for protection against infections  

Microsoft Academic Search

A single regeneration procedure using cotyledon examples effectively regenerated five commercially grown muskmelon cultivars. This regeneration scheme was used to facilitate gene transfers using either Agrobacterium tumefaciens or microprojectile bombardment methods. In both cases, the transferred genes were from the T-DNA region of the binary vector plasmid pGA482GG\\/cp cucumber mosaic virus-white leaf strain (CMV-WL), which contains genes that encode neomycin

C. Gonsalves; B. Xue; M. Yepes; M. Fuchs; K. Ling; S. Namba

1994-01-01

419

Rapid and efficient transformation of diploid Medicago truncatula and Medicago sativa ssp. falcata lines improved in somatic embryogenesis  

Microsoft Academic Search

We describe a simple and efficient protocol for regeneration-transformation of two diploid Medicago lines: the annual M. truncatula R108-1(c3) and the perennial M. sativa ssp. falcata (L.) Arcangeli PI.564263 selected previously as highly embryogenic genotypes. Here, embryo regeneration of R108-1 to complete\\u000a plants was further improved by three successive in vitro regeneration cycles resulting in the line R108-1(c3). Agrobacterium tumefaciens-mediated

T. H. Trinh; P. Ratet; E. Kondorosi; P. Durand; K. Kamaté; P. Bauer; A. Kondorosi

1998-01-01

420

Rapid transformation and regeneration of alfalfa (Medicago falcata L.) via direct somatic embryogenesis  

Microsoft Academic Search

Two simple, rapid and efficient protocols for theregeneration of transformed tetraploid lines ofalfalfa (Medicago falcata L.) have beendeveloped and compared. Leaf explants fromembryogenic lines 47\\/1-150 and 47\\/1-5 were inoculatedwith Agrobacterium tumefaciens containingconstructs carrying the nptII selectable markergene and promoter:gusA gene fusions under thecontrol of the CaMV 35S or Arabidopsis cdc2a,CycB1 and CycA2 promoters. In the firstregeneration system (the MSH system),

C. Y. Shao; E. Russinova; A. Iantcheva; A. Atanassov; A. McCormac; D. F. Chen; M. C. Elliott; A. Slater

2000-01-01

421

Replication of tomato yellow leaf curl virus (TYLCV) DNA in agroinoculated leaf discs from selected tomato genotypes  

Microsoft Academic Search

The leaf disc agroinoculation system was applied to study tomato yellow leaf curl virus (TYLCV) replication in explants from susceptible and resistant tomato genotypes. This system was also evaluated as a potential selection tool in breeding programmes for TYLCV resistance. Leaf discs were incubated with a head-to-tail dimer of the TYLCV genome cloned into the Ti plasmid ofAgrobacterium tumefaciens. In

H. Czosnek; A. Kheyr-Pour; B. Gronenborn; E. Remetz; M. Zeidan; A. Altman; H. D. Rabinowitch; S. Vidavsky; N. Kedar; Y. Gafni; D. Zamir

1993-01-01

422

Efficient transformation of Actinidia arguta by reducing the strength of basal salts in the medium to alleviate callus browning  

Microsoft Academic Search

An efficient transformation system for high-throughput functional genomic studies of kiwifruit has been developed to overcome\\u000a the problem of necrosis in Actinidia arguta explants. The system uses Agrobacterium tumefaciens strain EHA105 harbouring the binary vector pART27-10 to inoculate leaf strips. The vector contains neomycin phosphotransferase\\u000a (nptII) and ?-glucuronidase (GUS) (uidA) genes. A range of light intensities and different strengths of

Meili Han; Andrew P. Gleave; Tianchi Wang

2010-01-01

423

Transgenic caraway, Carum carvi L.: a model species for metabolic engineering  

Microsoft Academic Search

Regeneration in caraway was obtained via two different routes. Hypocotyls showed delayed shoot formation after a callus phase\\u000a and at relatively low frequencies. In contrast, high-frequency, direct regeneration occurred when cotyledonary node explants\\u000a were used. Transient expression of ?-glucuronidase was monitored after inoculation of both explant types with Agrobacterium tumefaciens AGL0(pMOG410). Gene transfer was more efficient when using cotyledonary node

F. A. Krens; L. C. P. Keizer; I. E. M. Capel

1997-01-01

424

Nodulin gene expression and ENOD2 localization in effective, nitrogen fixing and ineffective, bacteria-free nodules of alfalfa  

Microsoft Academic Search

Alfalfa plants form bacteria-free nodules in response to a number of agents, including Rhizobium meliloti exo mutants, Agrobacterium tumefaciens transconjugants carrying cloned R. meliloti nodulation genes, and compounds that function as auxin transport inhibitors, N-( 1-naphthyl)phthalamic acid or 2,3,5-triiodobenzoic acid. These bacteria-free nodules contain transcripts for the nodulins Nms30 and MsENOD2; transcripts for late nodulins like leghemoglobin are not detected.

Wiel van de C. C. M; J. H. Nurris; B. Bocheneck; Rebecca Dickstein; Ton Bisseling; Ann M. Hirsch

1990-01-01

425

Transgenic tomato cv. Pusa Uphar expressing a bacterial mannitol-1-phosphate dehydrogenase gene confers abiotic stress tolerance  

Microsoft Academic Search

A bacterial mannitol-1-phosphate dehydrogenase (mtlD) gene driven by the constitutive cauliflower mosaic virus (CaMV) 35S promoter was transferred into tomato plants using an Agrobacterium tumefaciens-mediated transformation protocol in an attempt to improve abiotic stress tolerance in the transformed plants. Transgene integration\\u000a was confirmed by PCR analysis and Southern blot analysis, and transgene expression was confirmed by reverse transcription\\u000a (RT)-PCR and

Neeraj KhareDanswrang; Danswrang Goyary; Narendra Kumar Singh; Pramila Shah; Meenal Rathore; Sivalingam Anandhan; Dinesh Sharma; Mohomad Arif; Zakwan Ahmed

2010-01-01

426

A Simple and General Method for Transferring Genes into Plants  

Microsoft Academic Search

Transformed petunia, tobacco, and tomato plants have been produced by means of a novel leaf disk transformation-regeneration method. Surface-sterilized leaf disks were inoculated with an Agrobacterium tumefaciens strain containing a modified tumor-inducing plasmid (in which the phytohormone biosynthetic genes from transferred DNA had been deleted and replaced with a chimeric gene for kanamycin resistance) and cultured for 2 days. The

R. B. Horsch; J. E. Fry; N. L. Hoffmann; D. Eichholtz; S. G. Rogers; R. T. Fraley

1985-01-01

427

Transgenic plants of rutabaga ( Brassica napobrassica ) tolerant to pest insects  

Microsoft Academic Search

Cotyledons cut from axenic seedlings were immersed inAgrobacterium tumefaciens suspension which was treated with acetosyringone and nopaline at low pH overnight. The infected cotyledon explants were cultured on MSB medium (MS salts + B5 Vitamins) containing 6-BA 3mg\\/1 for 2–3 days, and transferred onto selective medium (MSB with kanamycin 50–100 mg\\/l). Kanamycin-resistant shoots were selected. More than 60 regenerated plants

Xue-bao Li; Hui-Zhu Mao; Yong-Yan Bai

1995-01-01

428

Soybean [Glycine max (L.) Merr].  

PubMed

In this chapter we describe an Agrobacterium tumefaciens transformation method of soybean that utilizes mature half seeds and regeneration from the cotyledonary node region. This method results in fertile transformed soybean plants and transgenic seed in approximately 9 months. Using mature half seeds as starting material has proven to be a reliable method that does not require additional wounding for infection to occur. We have continued to make improvements in the protocol, resulting in an efficient plant regeneration system. PMID:25300848

Luth, Diane; Warnberg, Katey; Wang, Kan

2015-01-01

429

Nonbioluminescent Strains of Photobacterium phosphoreum Produce the Cell-to-Cell Communication Signal N-(3-Hydroxyoctanoyl)homoserine Lactone  

Microsoft Academic Search

Bioluminescence is a common phenotype in marine bacteria, such as Vibrio and Photobacterium species, and can be quorum regulated by N-acylated homoserine lactones (AHLs). We extracted a molecule that induced a bacterial AHL monitor (Agrobacterium tumefaciens</