These are representative sample records from Science.gov related to your search topic.
For comprehensive and current results, perform a real-time search at Science.gov.
1

Plant hormone effect of antibiotics on the transformation efficiency of plant tissues by Agrobacterium tumefaciens cells  

Microsoft Academic Search

The inhibition of bacterial growth by carbenicillin and cefotaxime was demonstrated using three different Agrobacterium tumefaciens strains, LBA4404, C58 and EHA101. LBA4404 was the most sensitive strain to carbenicillin and cefotaxime. Significantly toxic effects were observed when tobacco leaf explants were grown on MS medium containing 250 ?g\\/ml carbenicillin and 1 ?g\\/ml 2,4-dichlorophenoxyacetic acid (2,4-D). The regeneration of tobacco shoots

Jhy-Jhu Lin; Nacyra Assad-Garcia; Jonathan Kuo

1995-01-01

2

Agrobacterium tumefaciens -mediated transformation of japonica and indica rice varieties  

Microsoft Academic Search

Genetic transformation of rice (Oryza sativa L.) mediated by Agrobacterium ttumefaciens has been confirmed for japonica varieties and extended to include the more recalcitrant indica varieties. Immature embryos were inoculated with either A. tumefaciens At656 (pCNL56) or LBA4404 (pTOK233). Experimental conditions were developed initially for immature embryos treated with strain At656, based upon both transient and stable ß-glucuromdase (GUS) activities.

Rhodora R. Aldemita; Thomas K. Hodges

1996-01-01

3

Agrobacterium tumefaciens mediated genetic transformation of selected elite clone(s) of Eucalyptus tereticornis  

Microsoft Academic Search

Procedure for the Agrobacterium\\u000a tumefaciens mediated T-DNA delivery into the elite clone(s) of Eucalyptus tereticornis using leaf explants from microshoots has been developed. Amongst two strains of A. tumefaciens namely, EHA105 and LBA4404 (harbouring pBI121 plasmid), strain EHA105 was found to be more efficient. Pre-culturing of tissue\\u000a (2 days) on medium supplemented with 100 ?M acetosyringone, before bacterial infection significantly increased transient

Diwakar Aggarwal; Anil Kumar; M. Sudhakara Reddy

4

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

5

High efficiency transformation protocol for three Indian cotton varieties via Agrobacterium tumefaciens  

Microsoft Academic Search

A protocol for consistent production of transgenic cotton plants in three Indian varieties was established utilizing Agrobacterium-mediated transformation. Shoot tip explants were transformed by cocultivation with Agrobacterium tumefaciens strain LBA 4404. The strain harbors a binary vector pBAL2 carrying the reporter gene ?-glucuronidase intron (GUS-INT) and the marker gene neomycin phosphotransferase (NPTII). Regeneration potential of explants or different hormones was

V. V Satyavathi; V Prasad; B Gita Lakshmi; G Lakshmi Sita

2002-01-01

6

The persistence of engineered Agrobacterium tumefaciens in agroinfected plants.  

PubMed

Several plant species, including tomato (Lycopersicon esculentum), Gynura aurantiaca, avocado (Persea americana), and grapefruit (Citrus paradisi) grafted on Troyer citrange (Poncirus trifoliata x C. sinensis) were "agro-infected" with Agrobacterium tumefaciens strain LBA-4404, carrying a mini-Ti plasmid with a dimeric cDNA of citrus exocortis viroid (CEVd). Extracts prepared from tissues of the agroinfected plants 38-90 days after inoculation were plated on selective media and found to contain large amounts of the engineered bacteria. These observations suggest the need for more stringent quarantine measures when handling A. tumefaciens cells harboring constructs for "agroinoculation" with plant viruses or viroids. PMID:8274776

Mogilner, N; Zutra, D; Gafny, R; Bar-Joseph, M

1993-01-01

7

Transformation of oil palm using Agrobacterium tumefaciens.  

PubMed

Transgenic oil palm (Elaeis guineensis Jacq.) plantlets are regenerated after Agrobacterium tumefaciens-mediated transformation of embryogenic calli derived from young leaves of oil palm. The calli are transformed with an Agrobacterium strain, LBA4404, harboring the plasmid pUBA, which carries a selectable marker gene (bar) for resistance to the herbicide Basta and is driven by a maize ubiquitin promoter. Modifications of the transformation method, treatment of the target tissues using acetosyringone, exposure to a plasmolysis medium, and physical injury via biolistics are applied. The main reasons for such modifications are to activate the bacterial virulence system and, subsequently, to increase the transformation efficiency. Transgenic oil palm cells are selected and regenerated on a medium containing herbicide Basta. Molecular analyses revealed the presence and integration of the introduced bar gene into the genome of the transformants. PMID:22351008

Izawati, Abang Masli Dayang; Parveez, Ghulam Kadir Ahmad; Masani, Mat Yunus Abdul

2012-01-01

8

Developing an Agrobacterium tumefaciens -mediated genetic transformation for a selenium-hyperaccumulator Astragalus racemosus  

Microsoft Academic Search

Agrobacterium\\u000a tumefaciens strain LBA4404 containing the plasmid pBI121, carrying the reporter gene uidA and the kanamycin resistance gene nptII, was used for gene transfer experiments in selenium (Se)-hyperaccumulator Astragalus racemosus. The effects of kanamycin on cell growth and division and acetosyringone on transformation efficiency were evaluated. The\\u000a optimal concentration of kanamycin that could effectively inhibit cell growth and division in

Diane E. Darlington; Chiu-Yueh Hung; Jiahua Xie

2009-01-01

9

[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

10

Salttolerant transgenic perennial ryegrass ( Lolium perenne L.) obtained by Agrobacterium tumefaciens-mediated transformation of the vacuolar Na +\\/H + antiporter gene  

Microsoft Academic Search

The objective of this study was to obtain a salt-tolerant perennial ryegrass (Lolium perenne L.) by transforming it with a rice vacuolar membrane Na+\\/H+ antiporter gene via the Agrobacterium-mediated method. To optimize the transformation conditions, two Agrobacterium tumefaciens strains, LBA4404 and EHA105, carrying plasmid pCAMBIA3301, were used to transform embryogenic calli of perennial ryegrass; two factors affecting transformation efficiency, acetosyringone

Yu-Ye Wu; Qi-Jun Chen; Min Chen; Jia Chen; Xue-Chen Wang

2005-01-01

11

Agrobacterium tumefaciens -mediated transformation of Withania somnifera (L.) Dunal: an important medicinal plant  

Microsoft Academic Search

This report describes Agrobacterium tumefaciens-mediated transformation of Withania somnifera—an important Indian medicinal plant. A. tumefaciens strain LBA4404, containing the binary vector pIG121Hm was used for transformation, along with the gusA reporter gene with intron under the transcriptional control of the Cauliflower Mosaic Virus (CaMV) 35S promoter. The leaf\\u000a segments from two-and-a-half-month-old green house-grown seedlings were more efficient in transformation, as

Vibha Pandey; Pratibha Misra; Pankaj Chaturvedi; Manoj K. Mishra; Prabodh K. Trivedi; Rakesh Tuli

2010-01-01

12

A stable and reproducible transformation system for the wetland monocot Juncus accuminatus (bulrush) mediated by Agrobacterium tumefaciens  

Microsoft Academic Search

A highly reproducible Agrobacterium-mediated transformation system was developed for the wetland monocot Juncus accuminatus. Three Agrobacterium tumefaciens binary plasmid vectors, LBA4404\\/pTOK233, EHA105\\/pCAMBIA1201, and EHA105\\/pCAMBIA1301 were used. All vectors contained the\\u000a 35SCaMV promoter driven, intron containing, ?-glucuronidase (gus), and hygromycin phosphotransferase (hptII) genes within their T-DNA. After 48 h of cocultivation, 21-d-old seedling derived calli were placed on medium containing\\u000a timentin at

Rangaraj Nandakumar; Li Chen; Suzanne M. D. Rogers

2007-01-01

13

Successful Agrobacterium -Mediated Genetic Transformation of Maize Elite Inbred lines  

Microsoft Academic Search

An efficient transformation system was developed for maize (Zea mays L.) elite inbred lines using Agrobacterium-mediated gene transfer by identifying important factors that affected transformation efficiency. The hypervirulent Agrobacterium tumefaciens strain EHA105 proved to be better than octopine LBA4404 and nopaline GV3101. Improved transformation efficiencies were obtained when immature embryos were inocubated with Agrobacterium suspension cells (A600 = 0.8) for 20 min in

Xueqing Huang; Zhiming Wei

2005-01-01

14

Construction of an engineering strain producing high yields of ?-transglucosidase via Agrobacterium tumefaciens-mediated transformation of Asperillus niger.  

PubMed

In this study, Agrobacterium tumefaciens-mediated transformation (ATMT) was used in breeding industrial strains for the purpose of improving ?-transglucosidase production. Firstly, an efficient ATMT system for Asperillus niger was established by optimization of several influencing factors, in which transformation efficiency was improved up to 14-fold compared with the initial conditions. Furthermore, binary vector pBI-Glu containing an ?-transglucosidase expression cassette was constructed and transferred into Agrobacterium tumefaciens LBA4404 in order to infect A. niger. By the efficient ATMT method, the gene for ?-transglucosidase, driven by strong promoter PglaA (the glucoamylase gene promoter), had a high expression level in A. niger A-8 (25.02 U/mL). The optimized ATMT system was found to be effective and suitable for A. niger, and should be a useful tool for studying the function of A. niger genes and for industrial breeding of this strain. PMID:24018680

Li, Ming; Zhou, Liying; Liu, Meng; Huang, Yunyan; Sun, Xin; Lu, Fuping

2013-01-01

15

Preincubation of cut tobacco leaf explants promotes Agrobacterium-mediated transformation by increasing vir gene induction  

Microsoft Academic Search

The effects of preincubation of cut tobacco leaf explants on Agrobacterium transformation efficiency and induction of Agrobacterium virE-lacZ fusion were evaluated. Transformation efficiency was evaluated by histochemical and fluorometric analysis of ?-glucuronidase in leaf rings transformed with Agrobacterium tumefaciens strain LBA4404(pKIWI105). The transformation efficiency increased by 2-fold, 5-fold, and 4.3-fold upon preincubation for 24, 48, and 72 h, respectively. Preincubation

G. Sunilkumar; K. Vijayachandra; K. Veluthambi

1999-01-01

16

Agrobacterium -mediated genetic transformation of safflower ( Carthamus tinctorius  L.)  

Microsoft Academic Search

Reproducible and highly efficient protocols for shoot regeneration and genetic transformation mediated by Agrobacterium have been established for safflower (Carthamus tinctorius L.). Agrobacterium tumefaciens strain LBA 4404 with gus reporter gene and hygromycin (hpt gene) as plant selection marker was used as the plant transformation vector. Genetic transformation experiments were carried\\u000a out to evaluate the efficacy of various parameters such as genotype,

K. Sri Shilpa; V. Dinesh Kumar; M. Sujatha

2010-01-01

17

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

18

Use of the GUS gene as a selectable marker for Agrobacterium-mediated transformation of Rubus  

Microsoft Academic Search

A transformation system was established for red raspberry, blackberry and blackberry x raspberry hybrids, utilizing the binary vector system of Agrobacterium tumefaciens. Leaf discs or internodal stem segments were inoculated with Agrobacterium strain LBA4404 containing the binary vectors PBI121.X, which has the ß-glucuronidase (GUS) marker gene, or Bin 19, which has the neomycin phosphotransferase II (NPT II) gene. Regenerants were

Julie Graham; R. J. McNicol; A. Kumar

1990-01-01

19

Agrobacterium tumefaciens-mediated transformation of eggplant (Solanum melongena L.) using root explants.  

PubMed

An efficient variety-independent method for producing transgenic eggplant (Solanum melongena L.) via Agrobacterium tumefaciens-mediated genetic transformation was developed. Root explants were transformed by co-cultivation with Agrobacterium tumefaciens strain LBA4404 harbouring a binary vector pBAL2 carrying the reporter gene beta-glucuronidase intron (GUS-INT) and the marker gene neomycin phosphotransferase (NPTII). Transgenic calli were induced in media containing 0.1 mg l(-1) thidiazuron (TDZ), 3.0 mg l(-1) N(6)-benzylaminopurine, 100 mg l(-1) kanamycin and 500 mg l(-1) cefotaxime. The putative transgenic shoot buds elongated on basal selection medium and rooted efficiently on Soilrite irrigated with water containing 100 mg l(-1) kanamycin sulphate. Transgenic plants were raised in pots and seeds subsequently collected from mature fruits. Histochemical GUS assay and polymerase chain reaction analysis of field-established transgenic plants and their offsprings confirmed the presence of the GUS and NPTII genes, respectively. Integration of T-DNA into the genome of putative transgenics was further confirmed by Southern blot analysis. Progeny analysis of these plants showed a pattern of classical Mendelian inheritance for both the NPTII and GUS genes. PMID:12789429

Franklin, G; Lakshmi Sita, G

2003-02-01

20

Transgenic Medicago truncatula plants obtained from Agrobacterium tumefaciens -transformed roots and Agrobacterium rhizogenes-transformed hairy roots.  

PubMed

Medicago truncatula, barrel medic, is a forage crop that has been developed into a model legume. The development of new transformation methods is important for functional genomic studies in this species. Based on Agrobacterium tumefaciens-mediated transformation of root explants, we developed an effective system for producing M. truncatula (genotype R108) transgenic plants. Among the four A. tumefaciens strains (AGL1, C58C1, EHA105 and LBA4404) tested, EHA105 and AGL1 were most effective in regenerating transgenics. Callus induction frequency from root explants was 69.8%, and plantlet/shoot regeneration frequency was 41.3% when EHA105 was used. Transgenic nature of the regenerated plants was confirmed by PCR and Southern hybridization analyses. Progeny analysis revealed stable Mendelian meiotic transmission of transgenes. Because M. truncatula is particularly useful for the study of root endosymbiotic associations, we further developed a plant regeneration system from A. rhizogenes-transformed hairy roots of M. truncatula. Fertile true transgenic plants were regenerated from the hairy roots, thus allowing the assessment of gene functions at the whole plant level. Segregation analysis revealed that the hairy root genes could be segregated out in the progenies. By coupling A. rhizogenes-mediated hairy root transformation and the regeneration system reported here, once potential genes of interest are identified, the transformed hairy roots carrying such genes could be directly regenerated into plants for more detailed characterization of the genes. PMID:16575594

Crane, Cynthia; Wright, Elane; Dixon, Richard A; Wang, Zeng-Yu

2006-05-01

21

Transformation of a Texas cotton cultivar by using Agrobacterium and the shoot apex  

Microsoft Academic Search

Transgenic cotton (Gossypium hirsutum L.) plants of a Texas cultivar CUBQHRPIS were obtained using Agrobacterium-mediated transformation coupled with the use of shoot-apex explants. After inoculation with A. tumefaciens strain LBA 4404 containing the pBI121 plasmid, regeneration of primary plants was carried out in a medium containing kanamycin\\u000a (100? mg?l-1). Progeny obtained by selfing were germinated in the greenhouse and selected

C. Zapata; S. H. Park; K. M. El-Zik; R. H. Smith

1999-01-01

22

Agrobacterium -mediated transformation of cotton ( Gossypium hirsutum ) using a heterologous bean chitinase gene  

Microsoft Academic Search

Cotton (Gossypium hirsutum L., var. Coker 312) hypocotyl explants were transformed with three strains of Agrobacterium tumefaciens, LBA4404, EHA101 and C58, each harboring the recombinant binary vector pBI121 containing the chi gene insert and neomycin phosphotransferase (nptII) gene, as selectable marker. Inoculated tissue sections were placed onto cotton co-cultivation medium. Transformed calli were selected on MS medium containing 50 mg l?1

Masoud Tohidfar; Mojtaba Mohammadi; Behzad Ghareyazie

2005-01-01

23

Agrobacterium -mediated co-transformation of multiple genes in upland cotton  

Microsoft Academic Search

Two cotton genotypes, Simian 3 (SM 3) and WC, were co-transformed using a mixture of four Agrobacterium tumefaciens cultures of strain LBA4404, each carrying a plasmid harboring the following genes, Bt + sck (for Bacillus thuringenesis protein and modified Cowpea trypsin inhibitor), bar (for glufosinate), keratin, and fibroin. The frequency of callus induction, embryogenesis, and plant regeneration were notably different between the

Fei-Fei Li; Shen-Jie Wu; Tian-Zi Chen; Jie Zhang; Hai-Hai Wang; Wang-Zhen Guo; Tian-Zhen Zhang

2009-01-01

24

Agrobacterium tumefaciens-mediated genetic transformation of Salix matsudana Koidz. using mature seeds.  

PubMed

An Agrobacterium tumefaciens-mediated transformation method was developed for Salix matsudana Koidz. using mature seeds as starting material. Multiple shoots were induced directly from embryonic shoot apices of germinating seeds. Although thidiazuron, 6-benzylaminopurine and zeatin induced multiple shoot induction with high frequency, zeatin (4.5 ?M) was more effective for elongation of shoots and roots. The binary vector pCAMBIA1303, which contained neomycin phosphotransferase as a selectable marker gene and ?-glucuronidase as a reporter gene, was used for transformation. Factors affecting transformation efficiency were examined for optimization of the procedure. Up to 35 of 180 seeds regenerated kanamycin-resistant shoots under optimal transformation conditions as follows: seeds were precultured for 4 days, apices of embryonic shoots were removed and infected with A. tumefaciens strain LBA4404 grown to a cell density equivalent (OD600) of 0.6, and then the infected explants were cultivated at 21 °C for 4 days. Storage of seeds at -20 °C for as long as 3 years had no significant effect on the induction of kanamycin-resistant shoots. Using this method, transgenic plants were obtained within ?5 months with a transformation frequency of 7.2%. Analysis by polymerase chain reaction (PCR) showed that 36.4-93.8% of plants from all 13 tested kanamycin-resistant lines were PCR positive. Several 'escapes' were eliminated by a second round of selection. PCR, Southern blot and reverse transcriptase-PCR analyses of selected transgenic individuals 2 years after cutting propagation confirmed the successful generation of stable transformants. Our method, which minimizes the duration of axenic culture, may provide an alternative procedure for transformation of other recalcitrant Salix species. PMID:23771952

Yang, Jingli; Yi, Jaeseon; Yang, Chuanping; Li, Chenghao

2013-06-01

25

Transformation of rice mediated by Agrobacterium tumefaciens  

Microsoft Academic Search

Agrobacterium tumefaciens has been routinely utilized in gene transfer to dicotyledonous plants, but monocotyledonous plants including important cereals were thought to be recalcitrant to this technology as they were outside the host range of crown gall. Various challenges to infect monocotyledons including rice with Agrobacterium had been made in many laboratories, but the results were not conclusive until recently. Efficient

Yukoh Hiei; Toshihiko Komari; Tomoaki Kubo

1997-01-01

26

Tissue culture-independent In Planta Transformation s trategy: an Agrobacterium tumefaciens-Mediated gene Transfer Method to o vercome recalcitrance in cotton (Gossypium hirsutum L.)  

Microsoft Academic Search

The major constraint in cotton improvement has been the recalcitrance of cotton varieties to tissue culture. alternate methods that avoid\\/ minimize tissue culture would be beneficial for the improvement of cotton. in this report, trans- genic cotton plants have been produced by a tissue-culture independent Agrobacterium tume- faciens - mediated transformation procedure. Agrobacterium strain lBa4404 harboring the binary vector pKiWi105

E. Keshamma; S. Rohini; K. S. Rao; B. Madhusudhan; M. Udaya Kumar

27

Agroinfiltration by Cytokinin-Producing Agrobacterium sp. Strain GV3101 Primes Defense Responses in Nicotiana tabacum.  

PubMed

Transient infiltrations in tobacco are commonly used in plant studies, but the host response to different disarmed Agrobacterium strains is not fully understood. The present study shows that pretreatment with disarmed Agrobacterium tumefaciens GV3101 primes the defense response to subsequent infection by Pseudomonas syringae in Nicotiana tabacum. The presence of a trans-zeatin synthase (tzs) gene in strain GV3101 may be partly responsible for the priming response, as the tzs-deficient Agrobacterium sp. strain LBA4404 only weakly imparts such responses. Besides inducing the expression of defense-related genes like PR-1 and NHL10, GV3101 pretreatment increased the expression of tobacco mitogen-activated protein kinase (MAPK) pathway genes like MEK2, WIPK (wound-induced protein kinase), and SIPK (salicylic acid-induced protein kinase). Furthermore, the GV3101 strain showed a stronger effect than the LBA4404 strain in activating phosphorylation of the tobacco MAPK, WIPK and SIPK, which presumably prime the plant immune machinery. Lower doses of exogenously applied cytokinins increased the activation of MAPK, while higher doses decreased the activation, suggesting a balanced level of cytokinins is required to generate defense response in planta. The current study serves as a cautionary warning for plant researchers over the choice of Agrobacterium strains and their possible consequences on subsequent pathogen-related studies. PMID:25054409

Sheikh, Arsheed Hussain; Raghuram, Badmi; Eschen-Lippold, Lennart; Scheel, Dierk; Lee, Justin; Sinha, Alok Krishna

2014-11-01

28

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

29

Agrobacterium tumefaciens-mediated transformation of poinsettia, Euphorbia pulcherrima, with virus-derived hairpin RNA constructs confers resistance to Poinsettia mosaic virus  

PubMed Central

Agrobacterium-mediated transformation for poinsettia (Euphorbia pulcherrima Willd. Ex Klotzsch) is reported here for the first time. Internode stem explants of poinsettia cv. Millenium were transformed by Agrobacterium tumefaciens, strain LBA 4404, harbouring virus-derived hairpin (hp) RNA gene constructs to induce RNA silencing-mediated resistance to Poinsettia mosaic virus (PnMV). Prior to transformation, an efficient somatic embryogenesis system was developed for poinsettia cv. Millenium in which about 75% of the explants produced somatic embryos. In 5 experiments utilizing 868 explants, 18 independent transgenic lines were generated. An average transformation frequency of 2.1% (range 1.2–3.5%) was revealed. Stable integration of transgenes into the poinsettia nuclear genome was confirmed by PCR and Southern blot analysis. Both single- and multiple-copy transgene integration into the poinsettia genome were found among transformants. Transgenic poinsettia plants showing resistance to mechanical inoculation of PnMV were detected by double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). Northern blot analysis of low molecular weight RNA revealed that transgene-derived small interfering (si) RNA molecules were detected among the poinsettia transformants prior to inoculation. The Agrobacterium-mediated transformation methodology developed in the current study should facilitate improvement of this ornamental plant with enhanced disease resistance, quality improvement and desirable colour alteration. Because poinsettia is a non-food, non-feed plant and is not propagated through sexual reproduction, this is likely to be more acceptable even in areas where genetically modified crops are currently not cultivated. PMID:18327592

Spetz, Carl; Haugslien, Sissel; Xing, Shaochen; Dees, Merete W.; Moe, Roar; Blystad, Dag-Ragnar

2008-01-01

30

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

31

Transport of nonmetabolizable opines by Agrobacterium tumefaciens  

SciTech Connect

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

Krishnan, M.; Burgner, J.W.; Chilton, W.S.; Gelvin, S.B. (Purdue Univ., West Lafayette, IN (USA))

1991-01-01

32

SEASONAL FLUCTUATION OF AGROBACTERIUM TUMEFACIENS POPULATIONS IN WALNUT ORCHARD SOIL  

Technology Transfer Automated Retrieval System (TEKTRAN)

Crown gall disease caused by the bacterium Agrobacterium tumefaciens causes significant economic loss in commercial walnut orchards and nursery operations in California. To aid in development of disease control strategies, it is important to understand the structure and dynamics of A. tumefaciens p...

33

Transformation of arctic bramble (Rubus arcticus L.) by Agrobacterium tumefaciens  

Microsoft Academic Search

Genetic transformation of arctic bramble (Rubus arcticus L.) was achieved utilizing a Ti-plasmid vector system of Agrobacterium tumefaciens. Internodal stem segments were inoculated with Agrobacterium strain EHA101 carrying a T-DNA with the CaMV 35 S promoter-gus-int marker gene from which ?-glucuronidase (GUS) is expressed only in plants. Regenerants were produced on Murashige and Skoog medium. Growth of Agrobacterium was inhibited

H. I. Kokko; S. O. Kärenlampi

1998-01-01

34

Isolation of a strain of Agrobacterium tumefaciens (Rhizobium radiobacter) utilizing methylene urea  

E-print Network

Isolation of a strain of Agrobacterium tumefaciens (Rhizobium radiobacter) utilizing methylene urea (recently also known as Rhizobium radiobacter) using both genotypic and phenotypic characterization, ureaformaldehyde, slow-release fertilizer, soil, nitrogen, isolation, Agrobacterium tumefaciens, Rhizobium

Hammock, Bruce D.

35

Agrobacterium-mediated sorghum transformation.  

PubMed

Agrobacterium tumefaciens was used to genetically transform sorghum. Immature embryos of a public (P898012) and a commercial line (PHI391) of sorghum were used as the target explants. The Agrobacterium strain used was LBA4404 carrying a 'Super-binary' vector with a bar gene as a selectable marker for herbicide resistance in the plant cells. A series of parameter tests was used to establish a baseline for conditions to be used in stable transformation experiments. A number of different transformation conditions were tested and a total of 131 stably transformed events were produced from 6175 embryos in these two sorghum lines. Statistical analysis showed that the source of the embryos had a very significant impact on transformation efficiency, with field-grown embryos producing a higher transformation frequency than greenhouse-grown embryos. Southern blot analysis of DNA from leaf tissues of T0 plants confirmed the integration of the T-DNA into the sorghum genome. Mendelian segregation in the T1 generation was confirmed by herbicide resistance screening. This is the first report of successful use of Agrobacterium for production of stably transformed sorghum plants. The Agrobacterium method we used yields a higher frequency of stable transformation that other methods reported previously. PMID:11202440

Zhao, Z Y; Cai, T; Tagliani, L; Miller, M; Wang, N; Pang, H; Rudert, M; Schroeder, S; Hondred, D; Seltzer, J; Pierce, D

2000-12-01

36

Agrobacterium tumefaciens responses to plant-derived signaling molecules  

PubMed Central

As a special phytopathogen, Agrobacterium tumefaciens infects a wide range of plant hosts and causes plant tumors also known as crown galls. The complexity of Agrobacterium–plant interaction has been studied for several decades. Agrobacterium pathogenicity is largely attributed to its evolved capabilities of precise recognition and response to plant-derived chemical signals. Agrobacterium perceives plant-derived signals to activate its virulence genes, which are responsible for transferring and integrating its Transferred DNA (T-DNA) from its Tumor-inducing (Ti) plasmid into the plant nucleus. The expression of T-DNA in plant hosts leads to the production of a large amount of indole-3-acetic acid (IAA), cytokinin (CK), and opines. IAA and CK stimulate plant growth, resulting in tumor formation. Agrobacterium utilizes opines as nutrient sources as well as signals in order to activate its quorum sensing (QS) to further promote virulence and opine metabolism. Intriguingly, Agrobacterium also recognizes plant-derived signals including ?-amino butyric acid and salicylic acid (SA) to activate quorum quenching that reduces the level of QS signals, thereby avoiding the elicitation of plant defense and preserving energy. In addition, Agrobacterium hijacks plant-derived signals including SA, IAA, and ethylene to down-regulate its virulence genes located on the Ti plasmid. Moreover, certain metabolites from corn (Zea mays) also inhibit the expression of Agrobacterium virulence genes. Here we outline the responses of Agrobacterium to major plant-derived signals that impact Agrobacterium–plant interactions. PMID:25071805

Subramoni, Sujatha; Nathoo, Naeem; Klimov, Eugene; Yuan, Ze-Chun

2014-01-01

37

Efficient production of transgenic Alstroemeria plants by using Agrobacterium tumefaciens  

Microsoft Academic Search

A highly efficient and reproducible protocol was developed to obtain transgenic Alstroemeria plants by combining Agrobacterium tumefaciens with friable embryogenic callus (FEC). To develop this transformation method, factors such as infection time, cocultivation period, effect of acetosyringone (AS), different dilution concentrations of the bacterium and temperature during cocultivation were evaluated. A protocol was developed in which transient GUS expression activity

J. B. Kim; C. J. J. M. Raemakers; E. Jacobsen; R. G. F. Visser

2007-01-01

38

The transformation of Zea mays seedlings with Agrobacterium tumefaciens  

Microsoft Academic Search

Virulent strains of the soil bacterium Agrobacterium tumefaciens infect dicotyledonous plants and elicit a profound neoplastic response which results in crown gall formation (18). The inciting agent has been shown to be a high molecular weight plasmid (Ti) a section of which, the T-DNA, integrates into the host plant's genome (4, 28, 30). Although transformation of this kind was presumed

Anne C. F. Graves; S. L. Goldman

1986-01-01

39

Virulence of Agrobacterium tumefaciens strain A281 on legumes  

SciTech Connect

This study addresses the basis of host range on legumes of Agrobacterium tumefaciens strain A281, an L,L-succinamopine strain. The authors tested virulence of T-DNA and vir region constructs from this tumor-inducing (Ti) plasmid with complementary Ti plasmid regions from heterologous nopaline and octopine strains.

Hood, E.E.; Fraley, R.T.; Chilton, M.D.

1987-03-01

40

Mechanism of cellulose synthesis in Agrobacterium tumefaciens.  

PubMed Central

Extracts of Agrobacterium tumefaciens incorporated UDP-[14C]glucose into cellulose. When the extracts were fractionated into membrane and soluble components, neither fraction was able to synthesize cellulose. A combination of the membrane and soluble fractions restored the activity found in the original extracts. Extracts of cellulose-minus mutants showed no significant incorporation of UDP-glucose into cellulose. When mixtures of the extracts were made, the mutants were found to fall into two groups: extracts of mutants from the first group could be combined with extracts of the second group to obtain cellulose synthesis. No synthesis was observed when extracts of mutants from the same group were mixed. The groups of mutants corresponded to the two operons identified in sequencing the cel genes (A. G. Matthysse, S. White, and R. Lightfoot. J. Bacteriol. 177:1069-1075, 1995). Extracts of mutants were fractionated into membrane and soluble components, and the fractions were mixed and assayed for the ability to synthesize cellulose. When the membrane fraction from mutants in the celDE operon was combined with the soluble fraction from mutants in the celABC operon, incorporation of UDP-glucose into cellulose was observed. In order to determine whether lipid-linked intermediates were involved in cellulose synthesis, permeablized cells were examined for the incorporation of UDP-[14C]glucose into material extractable with organic solvents. No radioactivity was found in the chloroform-methanol extract of mutants in the celDE operon, but radioactive material was recovered in the chloroform-methanol extract of mutants in the celABC operon. The saccharide component of these compounds was released after mild acid hydrolysis and was found to be mainly glucose for the celA insertion mutant and a mixture of cellobiose, cellotriose, and cellotetrose for the celB and celC insertion mutants. The radioactive compound extracted with chloroform-methanol form the celC insertion mutant was incorporated into cellulose by membrane preparations from celE mutants, which suggests that this compound is a lipid-linked intermediate in cellulose synthesis. PMID:7860586

Matthysse, A G; Thomas, D L; White, A R

1995-01-01

41

Characterization of nonattaching mutants of Agrobacterium tumefaciens.  

PubMed Central

The first step in tumor formation by Agrobacterium tumefaciens is the site-specific binding of the bacteria to plant host cells. Transposon mutants of the bacteria which fail to attach to carrot suspension culture cells were isolated. These mutants showed no significant attachment to carrot cells with either microscopic or viable cell count assays of bacterial binding. The nonattaching mutants were all avirulent. When revertants of the mutants were obtained by enriching for bacteria which do bind to carrot cells, the bacteria were found to have regained the ability to bind to carrot cells and virulence simultaneously. These results suggest that the ability of the bacteria to bind to plant cells is required for virulence. Like the parent strain, all of the nonattaching mutants synthesized cellulose, but unlike the parent strain, they failed to aggregate carrot suspension culture cells. The transposon Tn5, which was used to obtain the mutants, was located on a 12-kilobase EcoRI fragment of the bacterial chromosomal DNA in all of the nonattaching mutants from strain C58. That the mutant phenotype was due to the Tn5 insertion was shown by cloning the Tn5-containing DNA fragment from the mutant bacteria and using it to replace the wild-type fragment in the parent strain by marker exchange. The resulting bacteria had the same mutant phenotype as the original Tn5 mutants; they did not attach to carrot cells, they did not cause the aggregation of carrot cells, and they were avirulent. No difference was seen between the parent strain and the nonattaching mutants in hydrophobicity, motility, flagella, fimbriae, beta-2-glucan content, size of lipopolysaccharide, or ability of the lipopolysaccharide to inhibit bacterial attachment to tissue culture cells. Differences were seen between the parent strain and the nonattaching mutants in the polypeptides removed from the bacteria during the preparation of spheroplasts. Three of the mutants were lacking a polypeptide of about 34 kilodaltons (kDa). One mutant was lacking the 34-kDa polypeptide and another polypeptide of about 38 kDa. The fifth mutant was lacking a polypeptide slightly smaller than the 34-kDa polypeptide missing in the other four mutants. These missing polypeptides all reappeared in the revertants of the mutants. Thus, bacterial binding to plant cells appears to require the presence of these polypeptides. Images PMID:3025176

Matthysse, A G

1987-01-01

42

The T-pilus of Agrobacterium tumefaciens  

Microsoft Academic Search

T-pilus biogenesis uses a conserved transmembrane nucleoprotein- and protein-transport apparatus for the transport of cyclic T-pilin subunits to the Agrobacterium cell surface. T-pilin subunits are processed from full-length VirB2 pro-pilin into a cyclized peptide, a rapid reaction that is Agrobacterium specific and can occur in the absence of Ti-plasmid genes.

Erh-Min Lai; Clarence I Kado

2000-01-01

43

Agrobacterium tumefaciens -mediated genetic transformation of sesame ( Sesamum indicum L.)  

Microsoft Academic Search

Sesame (Sesamum indicum) is an important oil seed crop that has not yet been transformed genetically. We report herein for the first time successful\\u000a recovery of fertile transgenic plants of sesame from cotyledon explants inoculated with Agrobacterium tumefaciens carrying a binary vector pCAMBIA2301 that contains a neomycin phosphotransferase gene (nptII) and a ?-glucuronidase (GUS) gene (uidA) interrupted with an intron.

Manju Yadav; Darshna Chaudhary; Manish Sainger; Pawan K. Jaiwal

2010-01-01

44

Transformation of Brassica napus with Agrobacterium tumefaciens based vectors  

Microsoft Academic Search

A reproducible system to produce transgenic Brassica napus plants has been developed using stem segments. Stem segments from 6–7 week old plants were inoculated with an Agrobacterium tumefaciens strain containing a disarmed tumor-inducing plasmid pTiT37-SE carrying a chimeric bacterial gene encoding kanamycin resistance (pMON200). Stem explants were cocultured for 2 days before transfer to kanamycin selection medium. Shoots regenerated directly

Joyce Fry; Arlene Barnason; Robert B. Horsch

1987-01-01

45

Agrocin 84 sensitivity: A plasmid determined property in Agrobacterium tumefaciens  

Microsoft Academic Search

It was shown for some oncogenic Agrobacterium tumefaciens strains that agrocin 84 sensitivity is determined by the presence of a large closed circular DNA plasmid, called the Ti-plasmid. Whereas wild-type strain C58 is agrocin 84 sensitive, all Ti-plasmid cured derivatives were found to be fully resistant. Moreover all independently isolated agrocin 84 resistant colonies were stably non-oncogenic and plasmid negative.

G. Engler; M. Holsters; M. Van Montagu; J. Schell; J. P. Hernalsteens; R. Schilperoort

1975-01-01

46

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

47

Transfection and transformation of Agrobacterium tumefaciens  

Microsoft Academic Search

The freeze thaw transfection procedure of Dityatkin et al. (1972) was adapted for the transfection and transformation of A. tumefaciens. Transfection of the strains B6S3 and B6-6 with DNA of the temperate phage PS8cc186 yielded a maximum frequency of 2 10-7 transfectants per total recipient population. In transformation of the strain GV3100 with the P type plasmid RP4 a maximum

M. Holsters; D. de Waele; A. Depicker; E. Messens; M. van Montagu; J. Schell

1978-01-01

48

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

49

Transformation of forage legumes using Agrobacterium tumefaciens.  

PubMed

Galls were induced in six species of forage legumes following inoculation with wild-type strains of A. tumefaciens. The plant species was more influential than the bacterial strain in determining the type of tumour produced. Inoculation of Medicago sativa resulted in small, disorganised tumours. The three Trifolium species, T. repens, T. hybridum and T. pratense, formed galls which tended to produce roots and both Onobrychis viciifolia and Lotus corniculatus produced teratomatous galls. The shoots elongated in the latter species only. In L. corniculatus, tissues that were infected by five bacterial strains were capable of shoot regeneration when cultured on a hormone-free medium. The transformed nature of these shoots was confirmed by their failure to root, the production of callus from leaves cultured on hormone-free medium and the presence of opines. PMID:24247771

Webb, K J

1986-04-01

50

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

51

Progress of cereal transformation technology mediated by Agrobacterium tumefaciens  

PubMed Central

Monocotyledonous plants were believed to be not transformable by the soil bacterium Agrobacterium tumefaciens until two decades ago, although convenient protocols for infection of leaf disks and subsequent regeneration of transgenic plants had been well established in a number of dicotyledonous species by then. This belief was reinforced by the fact that monocotyledons are mostly outside the host range of crown gall disease caused by the bacterium and by the failures in trials in monocotyledons to mimic the transformation protocols for dicotyledons. However, a key reason for the failure could have been the lack of active cell divisions at the wound sites in monocotyledons. The complexity and narrow optimal windows of critical factors, such as genotypes of plants, conditions of the plants from which explants are prepared, tissue culture methods and culture media, pre-treatments of explants, strains of A. tumefaciens, inducers of virulence genes, transformation vectors, selection marker genes and selective agents, kept technical hurdles high. Eventually it was demonstrated that rice and maize could be transformed by co-cultivating cells of callus cultures or immature embryos, which are actively dividing or about to divide, with A. tumefaciens. Subsequently, these initial difficulties were resolved one by one by many research groups, and the major cereals are now transformed quite efficiently. As many as 15 independent transgenic events may be regenerated from a single piece of immature embryo of rice. Maize transformation protocols are well established, and almost all transgenic events deregulated for commercialization after 2003 were generated by Agrobacterium-mediated transformation. Wheat, barley, and sorghum are also among those plants that can be efficiently transformed by A. tumefaciens. PMID:25426132

Hiei, Yukoh; Ishida, Yuji; Komari, Toshihiko

2014-01-01

52

Interactions and DNA Transfer between Agrobacterium tumefaciens, the Ti-Plasmid and the Plant Host  

Microsoft Academic Search

Agrobacterium tumefaciens is a gram-negative bacterium with the unique capacity to induce neoplasmic transformations in dicotyledonous plants. Recently, both the mechanism and the biological significance of this transformation have been elucidated. Agrobacterium tumefaciens strains contain a large extrachromosomal DNA plasmid (the Ti-plasmid). This Ti-plasmid is responsible for the oncogenic properties of Agrobacterium strains. A particular segment of the Ti-plasmid, containing

J. Schell; M. van Montagu; M. de Beuckeleer; M. de Block; A. Depicker; M. de Wilde; G. Engler; C. Genetello; J. P. Hernalsteens; M. Holsters; J. Seurinck; B. Silva; F. van Vliet; R. Villarroel

1979-01-01

53

Rice scutellum induces Agrobacterium tumefaciens vir genes and T-strand generation  

Microsoft Academic Search

For successful transformation of a plant by Agrobacterium tumefaciens it is essential that the explant used in cocultivation has the ability to induce Agrobacterium tumour-inducing (Ti) plasmid virulence (vir) genes. Here we report a significant variation in different tissues of Indica rice (Oryza sativa L. cv. Co43) in their ability to induce Agrobacterium tumefaciens vir genes and T-strand generation, using

K. Vijayachandra; K. Palanichelvam; K. Veluthambi

1995-01-01

54

Agrobacterium tumefaciens-mediated transient transformation of Arabidopsis thaliana leaves.  

PubMed

Transient assays provide a convenient alternative to stable transformation. Compared to the generation of stably transformed plants, agroinfiltration is more rapid, and samples can be analyzed a few days after inoculation. Nevertheless, at difference of tobacco and other plant species, Arabidopsis thaliana remains recalcitrant to routine transient assays. In this chapter, we describe a transient expression assay using simple infiltration of intact Arabidopsis leaves with Agrobacterium tumefaciens carrying a plasmid expressing a reporter fluorescent protein. In this protocol, Agrobacterium aggressiveness was increased by a prolonged treatment in an induction medium deficient in nutrients and containing acetosyringone. Besides, Arabidopsis plants were cultivated in intermediate photoperiod (12 h light-12 h dark) to promote leaf growth. PMID:24057365

Mangano, Silvina; Gonzalez, Cintia Daniela; Petruccelli, Silvana

2014-01-01

55

Development of Transgenic Papaya through Agrobacterium-Mediated Transformation.  

PubMed

Transgenic papaya plants were regenerated from hypocotyls and immature zygotic embryo after cocultivation with Agrobacterium tumefaciens LBA-4404 carrying a binary plasmid vector system containing neomycin phosphotransferase (nptII) gene as the selectable marker and ?-glucuronidase (GUS) as the reporter gene. The explants were co-cultivated with Agrobacterium tumefaciens on regeneration medium containing 500?mg/L carbenicillin?+?200?mg/L cefotaxime for one week. The cocultivated explants were transferred into the final selection medium containing 500?mg/L carbenicillin?+?200?mg/L cefotaxime?+?50?mg/L kanamycin for callus induction as well as plant regeneration. The callus derived from the hypocotyls of Carica papaya cv. Shahi showed the highest positive GUS activities compared to Carica papaya cv. Ranchi. The transformed callus grew vigorously and formed embryos followed by transgenic plantlets successfully. The result of this study showed that the hypocotyls of C. papaya cv. Shahi and C. papaya cv. Ranchi are better explants for genetic transformation compared to immature embryos. The transformed C. papaya cv. Shahi also showed the maximum number of plant regeneration compared to that of C. papaya cv. Ranchi. PMID:24066284

Azad, Md Abul Kalam; Rabbani, Md Golam; Amin, Latifah; Sidik, Nik Marzuki

2013-01-01

56

Development of Transgenic Papaya through Agrobacterium-Mediated Transformation  

PubMed Central

Transgenic papaya plants were regenerated from hypocotyls and immature zygotic embryo after cocultivation with Agrobacterium tumefaciens LBA-4404 carrying a binary plasmid vector system containing neomycin phosphotransferase (nptII) gene as the selectable marker and ?-glucuronidase (GUS) as the reporter gene. The explants were co-cultivated with Agrobacterium tumefaciens on regeneration medium containing 500?mg/L carbenicillin?+?200?mg/L cefotaxime for one week. The cocultivated explants were transferred into the final selection medium containing 500?mg/L carbenicillin?+?200?mg/L cefotaxime?+?50?mg/L kanamycin for callus induction as well as plant regeneration. The callus derived from the hypocotyls of Carica papaya cv. Shahi showed the highest positive GUS activities compared to Carica papaya cv. Ranchi. The transformed callus grew vigorously and formed embryos followed by transgenic plantlets successfully. The result of this study showed that the hypocotyls of C. papaya cv. Shahi and C. papaya cv. Ranchi are better explants for genetic transformation compared to immature embryos. The transformed C. papaya cv. Shahi also showed the maximum number of plant regeneration compared to that of C. papaya cv. Ranchi. PMID:24066284

Azad, Md. Abul Kalam; Rabbani, Md. Golam; Amin, Latifah; Sidik, Nik Marzuki

2013-01-01

57

Agrobacterium rhizogenes GALLS Protein Substitutes for Agrobacterium tumefaciens Single-Stranded DNA-Binding Protein VirE2  

Microsoft Academic Search

Agrobacterium tumefaciens and Agrobacterium rhizogenes transfer plasmid-encoded genes and virulence (Vir) proteins into plant cells. The transferred DNA (T-DNA) is stably inherited and expressed in plant cells, causing crown gall or hairy root disease. DNA transfer from A. tumefaciens into plant cells resembles plasmid conjugation; single-stranded DNA (ssDNA) is exported from the bacteria via a type IV secretion system comprised

Larry D. Hodges; Josh Cuperus; Walt Ream

2004-01-01

58

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

59

Microprojectile bombardment of plant tissues increases transformation frequency by Agrobacterium tumefaciens  

Microsoft Academic Search

Bombardment of plant tissues with microprojectiles in an effective method of wounding to promote Agrobacterium-mediated transformation. Tobacco cv. Xanthi leaves and sunflower apical meristems were wounded by microprojectile bombardment prior to application of Agrobacterium tumefaciens strains containing genes within the T-DNA encoding GUS or NPTII. Stable kanamycin-resistant tobacco transformants were obtained using an NPTII construct from particle\\/plasmid, particle-wounded\\/Agrobacterium-treated or scalpel-wounded\\/Agrobacterium-treated

Dennis Bidney; Chris Scelonge; Joanie Martich; Monique Burrus; Lynn Sims; Gary Huffman

1992-01-01

60

Large plasmid in Agrobacterium tumefaciens essential for crown gall-inducing ability  

Microsoft Academic Search

THE gram-negative bacterium Agrobacterium tumefaciens induces crown gall tumours in many, mostly dicotyledonous, plants. Zaenen et al.1 demonstrated the presence of one or more large plasmids in a number of crown gall-inducing Agrobacterium strains belonging to seven different Agrobacterium groups. They were not able to find such plasmids in eight non-pathogenic Agrobacterium strains belonging to four of the same groups2,3.

N. Van Larebeke; G. Engler; M. Holsters; S. Van den Elsacker; I. Zaenen; R. A. Schilperoort; J. Schell

1974-01-01

61

Application of sonication in combination with vacuum infiltration enhances the agrobacterium-mediated genetic transformation in Indian soybean cultivars.  

PubMed

Soybean is a recalcitrant crop to Agrobacterium-mediated genetic transformation. Development of highly efficient, reproducible, and genotype-independent transformation protocol is highly desirable for soybean genetic improvement. Hence, an improved Agrobacterium-mediated genetic transformation protocol has been developed for cultivar PK 416 by evaluating various parameters including Agrobacterium tumefaciens strains (LBA4404, EHA101, and EHA105 harboring pCAMBIA1304 plasmid), sonication duration, vacuum infiltration pressure, and vacuum duration using cotyledonary node explants of soybean prepared from 7-day-old seedlings. The transformed plants were successfully developed through direct organogenesis system. Transgene expression was assessed by GUS histochemical and gfp visual assays, and integration was analyzed by PCR and Southern blot hybridization. Among the different combinations and durations evaluated, a maximum transformation efficiency of 18.6 % was achieved when the cotyledonary node explants of cv. PK 416 were sonicated for 20 s and vacuum infiltered for 2 min at 250 mmHg in A. tumefaciens EHA105 suspension. The amenability of the standardized protocol was tested on four more soybean cultivars JS 90-41, Hara Soy, Co 1, and Co 2 in which all the cultivars responded favorably with transformation efficiency ranging from 13.3 to 16.6 %. The transformation protocol developed in the present study would be useful to transform diverse soybean cultivars with desirable traits. PMID:25480345

Arun, Muthukrishnan; Subramanyam, Kondeti; Mariashibu, Thankaraj Salammal; Theboral, Jeevaraj; Shivanandhan, Ganeshan; Manickavasagam, Markandan; Ganapathi, Andy

2015-02-01

62

Transformation of white spruce and other conifer species by Agrobacterium tumefaciens  

Microsoft Academic Search

Studies of the ability ofAgrobacterium to transform white spruce (Picea glauca), Engelmann spruce (P. engelmanni), Sitka spruce (P. sitchensis) and Douglas-fir (Pseudotsuga menziesii) showed frequencies of gall formation from 0–80% depending upon the strain ofAgrobacterium, and the conifer species. Thirty sixA. tumefaciens strains and oneA. rhizogenes strain were tested on 6 month old white spruce seedlings. NineA. tumefaciens strains induced

David Ellis; Dane Roberts; Ben Sutton; Wayne Lazaroff; David Webb; Barry Flinn

1989-01-01

63

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

64

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

65

Mutants of Agrobacterium tumefaciens with elevated vir gene expression  

SciTech Connect

Expression of Agrobacterium tumefaciens virulence (vir) genes requires virA, virG, and a plant-derived inducing compound such as acetosyringone. To identify the critical functional domains of virA and virG, a mutational approach was used. Agrobacterium A136 harboring plasmid pGP159, which contains virA, virG, and a reporter virB:lacZ gene fusion, was mutagenized with UV light or nitrosoguanidine. Survivors that formed blue colonies on a plate containing 5-bromo-4-chloro-3-indolyl beta-D-galactoside were isolated and analyzed. Quantification of beta-galactosidase activity in liquid assays identified nine mutant strains. By plasmid reconstruction and other procedures, all mutations mapped to the virA locus. These mutations caused an 11- to 560-fold increase in the vegetative level of virB:lacZ reporter gene expression. DNA sequence analysis showed that the mutations are located in four regions of VirA: transmembrane domain one, the active site, a glycine-rich region with homology to ATP-binding sites, and a region at the C terminus that has homology to the N terminus of VirG.

Pazour, G.J.; Ta, C.N.; Das, A. (University of Minnesota, St. Paul (USA))

1991-08-15

66

Improved Agrobacterium -mediated genetic transformation of GNA transgenic sugarcane  

Microsoft Academic Search

Six plasmids carrying a snowdrop lectin (Galanthus nivalis agglutinin, GNA) and one of three selection markers were successfully transferred into two sugarcane cultivars (FN81–745\\u000a and Badila) via Agrobacterium-mediated transformation. Agrobacterium strains LBA4404, EHA105 and A281 that harboured a super-binary vector were used for sugarcane transformation. The use of\\u000a the hygromycin (Hyg) resistance gene (hpt II), phosphinothrincin (PPT) resistance gene (bar)

Dongting Zhangsun; Sulan Luo; Rukai Chen; Kexuan Tang

2007-01-01

67

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

Technology Transfer Automated Retrieval System (TEKTRAN)

Paradox (Juglans hindsii x 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 preplant management strategy for crown gall, but even an industry standard, methyl b...

68

Optimization of Agrobacterium mediated genetic transformation of cotyledonary node explants of Vigna radiata.  

PubMed

A reproducible and highly efficient protocol for genetic transformation mediated by Agrobacterium has been established for greengram (Vigna radiata L. Wilczek). Double cotyledonary node (DCN) explants were inoculated with Agrobacterium tumefaciens strain LBA 4404 harboring a binary vector pCAMBIA 2301 containing neomycin phosphotransferase (npt II) gene as selectable marker, ?-glucuronidase (GUS) as a reporter (uidA) gene and annexin 1 bj gene. Important parameters like optical density of Agrobacterium culture, culture quantity, infection medium, infection and co-cultivation time and acetosyringone concentration were standardized to optimize the transformation frequency. Kanamycin at a concentration of 100 mg/l was used to select transformed cells. Transient and stable GUS expressions were studied in transformed explants and regenerated putative plants, respectively. Transformed shoot were produced on regeneration medium containing 100 mg/l kanamycin and 250 mg/l cefotaxime and rooted on ˝ MS medium. Transient and constitutive GUS expression was observed in DCN explants and different tissues of T(0) and T(1) plants. Rooted T(0) and T(1) shoots confirming Polymerase Chain Reaction (PCR) positive for npt II and annexin 1bj genes were taken to maturity to collect the seeds. Integration of annexin gene into the greengram genome was confirmed by Southern blotting. PMID:23420384

Yadav, Sushil Kumar; Katikala, Sweety; Yellisetty, Varalaxmi; Kannepalle, Annapurna; Narayana, Jyothi Lakshmi; Maddi, Vanaja; Mandapaka, Maheswari; Shanker, Arun Kumar; Bandi, Venkateswarlu; Bharadwaja, Kirti Pulugurtha

2012-12-01

69

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

70

Cyclic diguanylic acid and cellulose synthesis in Agrobacterium tumefaciens  

SciTech Connect

The occurrence of the novel regulatory nucleotide bis(3',5')-cyclic diguanylic acid (c-di-GMP) and its relation to cellulose biogenesis in the plant pathogen Agrobacterium tumefaciens was studied. c-di-GMP was detected in acid extracts of {sup 32}P-labeled cells grown in various media, and an enzyme responsible for its formation from GTP was found to be present in cell-free preparations. Cellulose synthesis in vivo was quantitatively assessed with ({sup 14}C)glucose as a tracer. The organism produced cellulose during growth in the absence of plant cells, and this capacity was retained in resting cells. Synthesis of a cellulosic product from UDP-glucose in vitro with membrane preparations was markedly stimulated by c-di-GMP and its precursor GTP and was further enhanced by Ca2+. The calcium effect was attributed to inhibition of a c-di-GMP-degrading enzyme shown to be present in the cellulose synthase-containing membranes.

Amikam, D.; Benziman, M. (Hebrew Univ. of Jerusalem (Israel))

1989-12-01

71

Extended Host Range of Agrobacterium tumefaciens in the Genus Pinus.  

PubMed

Two-to 4-month-old seedlings of nine pine species (Pinus eldarica Medw., Pinus elliottii Engelm., Pinus jeffreyi Grev. & Balf., Pinus lambertiana Dougl., Pinus ponderosa Laws., Pinus radiata D. Don, Pinus sylvestris L., Pinus taeda L., Pinus virginiana Mill), Douglas fir (Pseudotsuaa menziesii (Mirb.) Franco) and incense cedar (Libocedrus decurrens Torr.) were inoculated with five strains of Agrobacterium tumefaciens. Transformation occurred in all conifer species tested as determined by gall formation and opine production. The frequency of gall formation varied by host species, by bacterial strain, and was related to the age of the stem when inoculated. Galls were visible 8 to 12 weeks after inoculation and were small (often less than 2.5 millimeters in diameter). Fewer than half (230 of 502) of the galls originally formed on the trees were present after 1 year, and 26 of these grew to diameters greater than 2 centimeters. The majority of these larger galls (18 of 26) were found in P. radiata. Bacterial strain-specific opines were found in 67 of the 81 gall tissues sampled. PMID:16667394

Stomp, A M; Loopstra, C; Chilton, W S; Sederoff, R R; Moore, L W

1990-04-01

72

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

73

Genome engineering of Agrobacterium tumefaciens using the lambda Red recombination system.  

PubMed

Agrobacterium tumefaciens has been widely used as a tool for transgenesis in plants. The availability of its genome sequence should facilitate the directed engineering of improved properties; however, the current genome engineering options are laborious. Here, we investigated whether the lambda Red operon can be applied for recombineering of the A. tumefaciens genome. First, we built an expression plasmid for A. tumefaciens employing a tetracycline-inducible promoter to regulate the Red operon. This multicopy plasmid was then itself modified in A. tumefaciens to verify that the Red operon was functional. Then, we modified the endogenous A. tumefaciens tumor-inducing plasmid and the linear chromosome. These results extend recombineering technology to a new host and indicate a fast and convenient way to engineer the A. tumefaciens genome for functional genomics and strain improvements. PMID:24297480

Hu, Shengbiao; Fu, Jun; Huang, Fan; Ding, Xuezhi; Stewart, A Francis; Xia, Liqiu; Zhang, Youming

2014-03-01

74

Common loci for Agrobacterium tumefaciens and Rhizobium meliloti exopolysaccharide synthesis and their roles in plant interactions  

SciTech Connect

The authors isolated approximately 100 analogous EPS-deficient (Exo) mutants of the closely related plant pathogen Agrobacterium tumefaciens, including strains whose EPS deficiencies were specifically complemented by each of five cloned, R. meliloti exo loci. They also cloned A. tumefaciens genes which complemented EPS defects in three of the R. meliloti Exo mutants. In two of these cases, symbiotic defects were also complemented. All of the A. tumefaciens Exo mutants formed normal crown gall tumors on four different plant hosts, except ExoC mutants, which were nontumorigenic and unable to attach to plant cells in vitro. Like their R. meliloti counterparts, A. tumefaciens Exo mutants were deficient in production of succinoglycan, the major acidic EPS species produced by both genera. A. tumefaciens ExoC mutants also produced extremely low levels of another major EPS, cyclic 1,2-..beta..-D-glucan. This deficiency has been noted previously in a different set of nontumorigenic, attachment-defective A. tumefaciens mutants.

Cangelosi, G.A.; Hung, L.; Puvanesarajah, V.; Stacey, G.; Ozga, D.A.; Leigh, J.A.; Nester, E.W.

1987-05-01

75

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

76

High-efficiency gene transfer to recalcitrant plants by Agrobacterium tumefaciens  

Microsoft Academic Search

Agrobacterium tumefaciens efficiently transforms most plants. A few dicotyledonous plants and most monocotyledonous plants are, however, recalcitrant\\u000a to A. tumefaciens infection. We investigated whether the constitutive synthesis of a high level of the T-strand DNA intermediate can improve\\u000a the transformation efficiency of plants. We previously described a mutation in the vir gene regulator virG, virGN54D, that allows constitutive expression of

J. Ke; R. Khan; T. Johnson; D. A. Somers; A. Das

2001-01-01

77

Genetic control of quorum-sensing signal turnover in Agrobacterium tumefaciens  

Microsoft Academic Search

A signal turnover system is an essential component of many genetic regulatory mechanisms. The best-known example is the ubiquitin-dependent protein degradation system that exists in many organisms. We found that Agrobacterium tumefaciens adopts a unique signal turnover system to control exiting from a quorum-sensing mode. A. tumefaciens regulates Ti plasmid conjugal transfer by a quorum-sensing signal, N-3-oxo-octanoyl homoserine lactone (3OC8HSL),

Hai-Bao Zhang; Lian-Hui Wang; Lian-Hui Zhang

2002-01-01

78

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

79

Agrobacterium tumefaciens-mediated transformation as a tool for insertional mutagenesis in medicinal fungus Cordyceps militaris  

Microsoft Academic Search

Cordyceps militaris is an insect-born fungus with various biological and pharmacological activities. The mutant library of C. militaris was constructed by improved Agrobacterium tumefaciens-mediated transformation (ATMT), for the ultimate identification of genes involved in isolate degeneration during fruiting body production. Successful transformation of C. militaris JM4 by A. tumefaciens AGL-1 carrying vector pATMT1 was performed, with efficiency in the range of

Zhuangli Zheng; Chuanhua Huang; Li Cao; Cuihong Xie; Richou Han

2011-01-01

80

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

81

Developmental Effects of Zeatin, Ribosyl-Zeatin, and Agrobacterium tumefaciens B6 on Certain Mosses  

PubMed Central

Eight species of mosses studied were divided into two groups on the basis of their developmental responses to ribosyl-trans-zeatin and Agro-bacterium tumefaciens B6. All eight produced either gametophores or callus on the protonema in response to 6-(?,?-dimethylallylamino) purine and trans-zeatin. Three which produced normal gametophores with A. tumefaciens yielded callus or abnormal gametophores with ribosyl-trans-zeatin. Ribosyl-trans-zeatin and A. tumefaciens were relatively ineffective on five other mosses. Characteristics of protonemal growth common to each of these two groups are described. PMID:16659608

Spiess, Luretta D.

1976-01-01

82

[Influence of Pseudomonas aureofaciens exopolysaccharides on tumour formation caused by Agrobacterium tumefaciens].  

PubMed

The ability of exopolysaccharides of Pseudomonas aureofaciens strains UKM B-111 and UKM B-306, components of insectofungicidal preparation Gaupsin, to influence the process of tumour formation caused by Agrobacterium tumefaciens. Bacterial polysaccharides, obtained on Kozer medium with glucose (possible alginates) blocked agrobacterium cell adhesion by 29-49%. Those obtained on the medium with sucrose polysaccharides (possible levans) by 30-75% inhibit later stages of tumour formation. The paper is presented in Ukrainian. PMID:19938600

Balko, O I; Avdieieva, L V

2009-01-01

83

Production of herbicide-resistant transgenic sweet potato plants through Agrobacterium tumefaciens method  

Microsoft Academic Search

Transgenic herbicide-resistant sweet potato plants [Ipomoea batatas (L.) Lam.] were produced through Agrobacterium-mediated transformation system. Embryogenic calli derived from shoot apical meristems were infected with Agrobacterium tumefaciens strain EHA105 harboring the pCAMBIA3301 vector containing the bar gene encoding phosphinothricin N-acetyltransferase (PAT) and the gusA gene encoding ?-glucuronidase (GUS). The PPT-resistant calli and plants were selected with 5 and 2.5 mg l?1 PPT,

Hye Jin Choi; Thummala Chandrasekhar; Hyo-Yeon Lee; Kyung-Moon Kim

2007-01-01

84

Factors Affecting Transformation Efficiency of Poplar Hybrid Line NC5331 by Agrobacterium tumefaciens  

Microsoft Academic Search

Acetosyringone, pH, and glucose, whichmay affectAgrobacterium-mediated gene transformation to poplar hybrid line rC5331, were investigated in an attempt to raise thegene transfer efficiency.The Agrobacterium tumefaciens strain used harbored disarmed vector (pMON9749) carrying a Pglucuronidase gene and a kanamycin resistant marker. With the addition of ace- tosyringone at 25 to 75 (iM, the transformationefficiency was significantlyenhanced,but dependent on pH. Acetosyringone required

Xin Y. Li; Feng H. Huang; Edward E. Gbur

1997-01-01

85

Agrobacterium tumefaciens-mediated genetic transformation of rye (Secale cereale L.)  

Microsoft Academic Search

A system for the genetic transformation of rye by co-cultivation with Agrobacterium tumefaciens is described. A total of 45 independent transgenic plants were regenerated with a transformation efficiency of 1 to % of the inoculated explants. The co-cultivation of Agrobacterium-strain AGL0, harboring plasmid pJFnptII and rye im-mature embryos in liquid medium allowed a high throughput and facilitated washing of the

Juan Carlos Popelka; Fredy Altpeter

2003-01-01

86

Rhizobium meliloti Genes Required for Nodule Development are Related to Chromosomal Virulence Genes in Agrobacterium tumefaciens  

Microsoft Academic Search

Symbiotically essential genes have been identified in Rhizobium meliloti that are structurally and functionally related to chromosomal virulence (chv) genes of Agrobacterium tumefaciens. Homologous sequences also exist in the genomes of other fast-growing rhizobia including Rhizobium trifolii, Rhizobium leguminosarum, and Rhizobium phaseoli. In Agrobacterium, the chvA and chvB loci are known to be essential for oncogenic transformation of dicotyledonous plants

T. Dylan; L. Ielpi; S. Stanfield; L. Kashyap; C. Douglas; M. Yanofsky; E. Nester; D. R. Helinski; G. Ditta

1986-01-01

87

Timentin as an alternative antibiotic for suppression of Agrobacterium tumefaciens in genetic transformation  

Microsoft Academic Search

The effects of timentin on shoot regeneration of tobacco (Nicotiana tabaccum) and Siberian elm (Ulmus pumila L.) and its use for the suppression of Agrobacterium tumefaciens in Agrobacterium-mediated genetic transformation were determined. Timentin is a mixture of ticarcillin and clavulanic acid, and at concentrations\\u000a of 200–500 mg\\/l with ratios of ticarcillin:clavulanic acid of 50:1 and 100:1, it had little effect

Z.-M. Cheng; J. A. Schnurr; J. A. Kapaun

1998-01-01

88

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

89

Agrobacterium-mediated high efficiency transformation of tall fescue (Festuca arundinacea).  

PubMed

Tall fescue (Festuca arundinacea) is the predominant cool-season pasture grass in the USA. Embryogenic calluses were induced from seeds/caryopsis of elite tall fescue cultivars Jesup and Kentucky-31, and were broken up into small pieces and used for Agrobacterium tumefaciens-mediated transformation. Agrobacterium strains LBA4404 and EHA105 harboring pCAMBIA vectors or the super-binary vector pTOK233 were used to infect the embryogenic callus pieces. The number of hygromycin resistant calluses obtained per dish of infected callus pieces was in the range of 2.0-5.8, and the number of transgenic plants recovered per dish of infected callus pieces was in the range of 0.4-1.7. When transformation efficiency was calculated based on the number of transgenic plants recovered and the number of original intact calluses used, the transformation frequency was in the range of 1.9-8.7%. The use of the easily available pCAMBIA vectors could produce equivalent results as the superbinary vector pTOK233. The transgenic nature of the regenerated plants was demonstrated by Southern hybridization analysis using undigested and digested genomic DNA samples. Expression of the transgenes was confirmed by northern hybridization analysis, GUS staining, and detection of GFP signals. Fertile transgenic plants were obtained after vernalization in the greenhouse. Progeny analysis revealed Mendelian inheritance of the transgenes. PMID:15700425

Wang, Zeng-Yu; Ge, Yaxin

2005-01-01

90

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...

91

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

92

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

93

Genetic transformation of the apple scion cultivar ‘Delicious’ viaAgrobacterium tumefaciens  

Microsoft Academic Search

Transformed shoots of the major apple scion cultivar ‘Delicious’ (Malus × domestica Borkh.) were obtained by cocultivation withAgrobacterium tumefaciens carrying disarmed plasmids. The transformation efficiency was influenced by the type of plasmid and by the inoculation temperature. Initial selection involved a callus stage followed by shoot regeneration. Shoot regeneration occurred only in the dark. Shoots grew in the light and

Sridevy Sriskandarajah; Peter B. Goodwin; Jim Speirs

1994-01-01

94

In vitro transformation of cultured cells from Nicotiana tabacum by Agrobacterium tumefaciens  

Microsoft Academic Search

CROWN GALL, the plant tumour of many dicotyledonous and some monocotyledonous species, is caused by Agrobacterium tumefaciens infection after wounding1. Crown gall cells, unlike cells derived from healthy plants, have the ability to proliferate indefinitely in culture without the growth hormones auxin and cytokinin2. The properties of the tumours, such as morphology (rough or smooth type). and the synthesis of

L. Márton; G. J. Wullems; L. Molendijk; R. A. Schilperoort

1979-01-01

95

Acetosyringone promotes high efficiency transformation of Arabidopsis thaliana explants by Agrobacterium tumefaciens  

Microsoft Academic Search

High frequency transformation of Arabidopsis thaliana leaf explants has been obtained using a disarmed Ti plasmid containing the coding region of a neomycin phosphotransferase gene (NPT II) as a selectable marker. The rate of transformation ranged from 55 to 63 percent when acetosyringone (AS), a natural wound response molecule, was added to an Agrobacterium tumefaciens culture prior to incubation with

Shahla N. Sheikholeslam

1987-01-01

96

X-ray Structure of Imidazolonepropionase from Agrobacterium tumefaciens at 1.87 angstrom Resolution  

SciTech Connect

Histidine degradation in agrobacterium tumefaciens involves four enzymes, including histidase, urocanase, imidazolonepropionase, and N-formylglutamate amido hydrolase. The third enzyme of the pathway, imidazolone-propionase, a 45.6 kDa protein, catalyzes conversion of imidazolone-5-propanoate to N-forminio-t-glutamate.

Tyagi,R.; Kumaran, D.; Burley, S.; Swaminathan, S.

2007-01-01

97

from E. coli and the 3 terminator re-gion from Agrobacterium tumefaciens  

E-print Network

from E. coli and the 3 terminator re- gion from Agrobacterium tumefaciens cloned in pBluescript® KS. Direct clone characterization from plaques and colonies by the polymerase chain reaction. Nucleic Acids.F. Fritsch and T. Maniatis. 1989. Molecular Cloning: A Laboratory Man- ual. CSH Laboratory Press, Cold Spring

Bardwell, James

98

CONDITIONS FOR EFFICIENT AGROBACTERIUM TUMEFACIENS-MEDIATED TRANSFORMATION OF ASCOCHYTA RABIEI.  

Technology Transfer Automated Retrieval System (TEKTRAN)

In order to develop an insertional mutagenesis transformation system to study pathogenicity factors of Ascochyta rabie, the causal agent of Ascochyta blight of chickpea, the conditions for efficient transformation of A. rabiei using Agrobacterium tumefaciens-Mediated Transformation have been determi...

99

DETECTION AND IMPLICATIONS OF EARLY AGROBACTERIUM TUMEFACIENS INFECTION OF PARADOX SEEDS AND SEEDLINGS  

Technology Transfer Automated Retrieval System (TEKTRAN)

Paradox (Juglans hindsii x J. regia), the dominant rootstock used in California, USA walnut production, has many desirable horticultural characteristics, but is highly susceptible to crown gall. Crown gall, caused by the soil-borne bacterium Agrobacterium tumefaciens, can not be consistently control...

100

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,...

101

Multilocus Sequence-Based Analysis Delineates a Clonal Population of Agrobacterium (Rhizobium) radiobacter (Agrobacterium tumefaciens) of Human Origin ?  

PubMed Central

The genus Agrobacterium includes plant-associated bacteria and opportunistic human pathogens. Taxonomy and nomenclature within the genus remain controversial. In particular, isolates of human origin were all affiliated with the species Agrobacterium (Rhizobium) radiobacter, while phytopathogenic strains were designated under the synonym denomination Agrobacterium tumefaciens. In order to study the relative distribution of Agrobacterium strains according to their origins, we performed a multilocus sequence-based analysis (MLSA) on a large collection of 89 clinical and environmental strains from various origins. We proposed an MLSA scheme based on the partial sequence of 7 housekeeping genes (atpD, zwf, trpE, groEL, dnaK, glnA, and rpoB) present on the circular chromosome of A. tumefaciens C58. Multilocus phylogeny revealed that 88% of the clinical strains belong to genovar A7, which formed a homogeneous population with linkage disequilibrium, suggesting a low rate of recombination. Comparison of genomic fingerprints obtained by pulsed-field gel electrophoresis (PFGE) showed that the strains of genovar A7 were epidemiologically unrelated. We present genetic evidence that genovar A7 may constitute a human-associated population distinct from the environmental population. Also, phenotypic characteristics, such as culture at 42°C, agree with this statement. This human-associated population might represent a potential novel species in the genus Agrobacterium. PMID:21398532

Aujoulat, Fabien; Jumas-Bilak, Estelle; Masnou, Agnčs; Sallé, Fanny; Faure, Denis; Segonds, Christine; Marchandin, Hélčne; Teyssier, Corinne

2011-01-01

102

Membrane lipids in Agrobacterium tumefaciens: biosynthetic pathways and importance for pathogenesis  

PubMed Central

Many cellular processes critically depend on the membrane composition. In this review, we focus on the biosynthesis and physiological roles of membrane lipids in the plant pathogen Agrobacterium tumefaciens. The major components of A. tumefaciens membranes are the phospholipids (PLs), phosphatidylethanolamine (PE), phosphatidylglycerol, phosphatidylcholine (PC) and cardiolipin, and ornithine lipids (OLs). Under phosphate-limited conditions, the membrane composition shifts to phosphate-free lipids like glycolipids, OLs and a betaine lipid. Remarkably, PC and OLs have opposing effects on virulence of A. tumefaciens. OL-lacking A. tumefaciens mutants form tumors on the host plant earlier than the wild type suggesting a reduced host defense response in the absence of OLs. In contrast, A. tumefaciens is compromised in tumor formation in the absence of PC. In general, PC is a rare component of bacterial membranes but amount to ~22% of all PLs in A. tumefaciens. PC biosynthesis occurs via two pathways. The phospholipid N-methyltransferase PmtA methylates PE via the intermediates monomethyl-PE and dimethyl-PE to PC. In the second pathway, the membrane-integral enzyme PC synthase (Pcs) condenses choline with CDP-diacylglycerol to PC. Apart from the virulence defect, PC-deficient A. tumefaciens pmtA and pcs double mutants show reduced motility, enhanced biofilm formation and increased sensitivity towards detergent and thermal stress. In summary, there is cumulative evidence that the membrane lipid composition of A. tumefaciens is critical for agrobacterial physiology and tumor formation. PMID:24723930

Aktas, Meriyem; Danne, Linna; Möller, Philip; Narberhaus, Franz

2014-01-01

103

Quantitative Estimation of Agrobacterium tumefaciens DNA in Crown Gall Tumor Cells  

PubMed Central

Several reports suggest that Agrobacterium tumefaciens nucleic acids can induce transformation of the cells of susceptible host plants and that bacteria-free tissue cultures of transformed cells contain A. tumefaciens DNA, RNA, antigens, or bacteriophages. We assayed Vinca rosea tumor DNA for base sequence homologies with A. tumefaciens DNA by DNA·DNA solution enrichment and DNA·DNA filter saturation hybridization techniques. No homologies were found by either method. The filter saturation hybridization technique included model filters containing known percentages of bacterial DNA mixed with V. rosea leaf DNA. Using this sensitive technique, we found that no more than 0.02% of the crown gall tumor genome could be homologous to A. tumefaciens DNA. This upper estimate of homology corresponds to 0.2 bacterial genome equivalent per diploid tumor cell. PMID:4530329

Drlica, Karl A.; Kado, C. I.

1974-01-01

104

Endophytic occupation of root nodules and roots of Melilotus dentatus by Agrobacterium tumefaciens.  

PubMed

Agrobacterium strains have been frequently isolated from the root nodules of different legumes. Various possible mechanisms have been proposed to explain the existence of these bacteria in nodules, but there is no sufficient experimental evidence to support the estimations. In this work, we proved that the Agrobacterium strain CCBAU 81181, which was originally isolated from the root nodules of Onobrychis viciaefolia, and a symbiotic strain of Sinorhizobium meliloti CCBAU 10062 could coinhabit the root nodules of Melilotus dentatus. Analyses were performed by using a fluorescence marker, reisolation of bacteria from nodules, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of whole cellular proteins, and polymerase chain reaction amplification of symbiotic genes. The inoculation of A. tumefaciens CCBAU 81181 did not affect the growth and nodulation of plants. CCBAU 81181 and 24 other Agrobacterium strains isolated from nodules were incapable of nodulating on their original or alternative host and 22 strains of these strains were endophytes in the roots and stems of their hosts. Also, the tumor-inducing A. tumefaciens strains IAM 13129(T) and C58 were found capable of entering the roots of Glycyrrhiza pallidiflora, but did not cause pathogenic symptoms. With these results, we conclude that A. tumefaciens strains could be endophytic bacteria in the roots, stems, and root nodules. This finding partially explains why Agrobacterium strains were frequently isolated from the surface-sterilized nodules. PMID:16897296

Wang, Ling Ling; Wang, En Tao; Liu, Jie; Li, Ying; Chen, Wen Xin

2006-10-01

105

The multifaceted roles of the interspecies signalling molecule indole in Agrobacterium tumefaciens.  

PubMed

Bacteria utilize signal molecules to ensure their survival in environmental niches, and indole is an interspecies and interkingdom signalling molecule, which is widespread in the natural environment. In this study, we sought to identify novel roles of indole in soil-borne bacterium Agrobacterium tumefaciens. Agrobacterium tumefaciens was found not to synthesize indole and to degrade it rapidly. The addition of exogenous indole dose-dependently inhibited A.?tumefaciens growth and decreased its motility. Surprisingly, indole markedly increased A.?tumefaciens biofilm formation on polystyrene, glass and nylon membrane surfaces and enhanced its antibiotic tolerance. Transcriptional analysis showed that indole markedly up-regulated several biofilm-related (celA, cheA, exoR, phoB, flgE, fliR and motA), stress-related genes (clpB, dnaK, gsp, gyrB, marR and soxR) and efflux genes (emrA, norM, and Atu2551) in A.?tumefaciens, which partially explained the increased biofilm formation and antibiotic tolerance. In contrast, the plant auxin indole-3-acetic acid did not affect biofilm formation, antibiotic tolerance or gene expression. Interestingly, indole was found to exhibit several similarities with antibiotics, as it inhibited the growth of non-indole-producing bacteria, whereas these bacteria countered its effects by rapidly degrading indole, and by enhancing biofilm formation and antibiotic tolerance. PMID:25040348

Lee, Jin-Hyung; Kim, Yong-Guy; Baek, Kwang-Hyun; Cho, Moo Hwan; Lee, Jintae

2015-04-01

106

Crown gall transformation of tobacco callus cells by cocultivation with Agrobacterium tumefaciens  

SciTech Connect

Incubation of cells from squashed tobacco callus tissue with virulent Agrobacterium tumefaciens leads to the production of cells displaying a crown gall phenotype. In vitro crown gall transformation of dicotyledonous plant cells has been demonstrated after cocultivation of cell-wall regenerating mesophyll protoplasts with Agrobacterium tumefaciens cells. In addition, it has been shown that protoplasts freshly isolated from suspension cultures, when treated with A. tumefaciens spheroplasts and a fusogen, also generated cells displaying a typical crown gall phenotype, i.e., phytohormone-independent growth and opine synthesis. Subsequently, both techniques were used to transfer and express foreign genes in plant cells via A. tumefaciens T-DNA integration. For practical purposes, it would be advantageous to be able to perform crown gall transformation of plant cells in tissue culture. The authors report here for the first time the production of Nicotiana tabacum crown gall cells after cocultivation of callus tissue with A. tumefaciens A136 cells. 11 references, 1 figure, 1 table.

Muller, A.; Manzara, T.; Lurquin, P.F.

1984-09-17

107

Reconciliation of sequence data and updated annotation of the genome of Agrobacterium tumefaciens C58, and distribution of a linear chromosome in the genus Agrobacterium.  

PubMed

Two groups independently sequenced the Agrobacterium tumefaciens C58 genome in 2001. We report here consolidation of these sequences, updated annotation, and additional analysis of the evolutionary history of the linear chromosome, which is apparently limited to the biovar I group of Agrobacterium. PMID:23241979

Slater, Steven; Setubal, Joăo C; Goodner, Brad; Houmiel, Kathryn; Sun, Jian; Kaul, Rajinder; Goldman, Barry S; Farrand, Stephen K; Almeida, Nalvo; Burr, Thomas; Nester, Eugene; Rhoads, David M; Kadoi, Ryosuke; Ostheimer, Trucian; Pride, Nicole; Sabo, Allison; Henry, Erin; Telepak, Erin; Cromes, Lindsey; Harkleroad, Alana; Oliphant, Louis; Pratt-Szegila, Phil; Welch, Roy; Wood, Derek

2013-02-01

108

Reconciliation of Sequence Data and Updated Annotation of the Genome of Agrobacterium tumefaciens C58, and Distribution of a Linear Chromosome in the Genus Agrobacterium  

PubMed Central

Two groups independently sequenced the Agrobacterium tumefaciens C58 genome in 2001. We report here consolidation of these sequences, updated annotation, and additional analysis of the evolutionary history of the linear chromosome, which is apparently limited to the biovar I group of Agrobacterium. PMID:23241979

Slater, Steven; Setubal, Joăo C.; Houmiel, Kathryn; Sun, Jian; Kaul, Rajinder; Goldman, Barry S.; Farrand, Stephen K.; Almeida, Nalvo; Burr, Thomas; Nester, Eugene; Rhoads, David M.; Kadoi, Ryosuke; Ostheimer, Trucian; Pride, Nicole; Sabo, Allison; Henry, Erin; Telepak, Erin; Cromes, Lindsey; Harkleroad, Alana; Oliphant, Louis; Pratt-Szegila, Phil; Welch, Roy; Wood, Derek

2013-01-01

109

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 ...

110

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

111

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

112

Enhanced soybean infection by the legume "super-virulent" Agrobacterium tumefaciens strain KAT23.  

PubMed

Agrobacterium tumefaciens KAT23 harbors a nopaline-type Ti plasmid and is "super-virulent" to soybean (Glycine max) and other leguminous plants. The right and left border sequences of the essential cis-element for T-DNA transfer were removed in order to utilize the high infectivity of this strain in an Agrobacterium-mediated soybean transformation system. The resulting strain, named Soy2, showed no oncogenic activity. After inoculation with disarmed Soy2 harboring binary vector pIG121-Hm and pCAMBIA-WR, soybean epicotyls exhibited high beta-glucuronidase activities, with efficiencies higher than EHA105, an A. tumefaciens strain widely used in making transgenic plants. PMID:18603788

Yukawa, Kiyoshi; Kaku, Hisatoshi; Tanaka, Hiroshi; Koga-Ban, Yasunori; Fukuda, Masao

2008-07-01

113

Transformation of cotton (Gossypium hirsutum L.) by Agrobacterium tumefaciens and regeneration of transgenic plants  

Microsoft Academic Search

Cotton (Gossypium hirsutum L.) cotyledon tissues have been efficiently transformed and plants have been regenerated. Cotyledon pieces from 12-day-old aseptically germinated seedlings were inoculated with Agrobacterium tumefaciens strains containing avirulent Ti (tumor-inducing) plasmids with a chimeric gene encoding kanamycin resistance. After three days cocultivation, the cotyledon pieces were placed on a callus initiation medium containing kanamycin for selection. High frequencies

Ebrahim Firoozabady; David L. DeBoer; Donald J. Merlo; Edward L. Halk; Lorraine N. Amerson; Kay E. Rashka; Elizabeth E. Murray

1987-01-01

114

Integration of Agrobacterium tumefaciens T-DNA in the Saccharomyces cerevisiae Genome by Illegitimate Recombination  

Microsoft Academic Search

Agrobacterium tumefaciens can transfer part of its Ti plasmid, the T-DNA, to plant cells where it integrates into the nuclear genome via illegitimate recombination. Integration of the T-DNA results in small deletions of the plant target DNA, and may lead to truncation of the T-DNA borders and the production of filler DNA. We showed previously that T-DNA can also be

Paul Bundock; Paul J. J. Hooykaas

1996-01-01

115

An Agrobacterium tumefaciens -mediated transformation system using callus of Zoysia tenuifolia Willd. ex Trin  

Microsoft Academic Search

Zoysia tenuifolia Willd. ex Trin. is one of the most popularly cultivated turfgrass. This is the first report of successful plant regeneration\\u000a and genetic transformation protocols for Z. tenuifolia using Agrobacterium tumefaciens. Initial calli was induced from stem nodes incubated on a Murashige and Skoog (1962) (MS) medium supplemented with 2 mg l?1 2,4-dichlorophenoxyacetic acid (2,4-D) and 1 mg l?1 6-benzyladenine (BA), with a

Meiru Li; Hongqing Li; Xiaoying Hu; Xiaoping Pan; Guojiang Wu

2010-01-01

116

Transfer of the Agrobacterium tumefaciens TI Plasmid to Avirulent Agrobacteria and to Rhizobium ex planta  

Microsoft Academic Search

SUMMARY A mutant of A. tumefaciens strain ~6~3, carrying the R factor RP4, was able to transfer its TI plasmid to various avirulent Agrobacterium strains and to a strain of Rhizobium. Strains carrying the TI(~6s3) plasmid were selected by their ability to utilize octopine. The isolates were able to induce tumours and exclude phage API . The tumours induced on

P. J. J. Hooykaas; P. M. Klapwijk; M. P. Nuti; R. A. Schiliperoort

1977-01-01

117

Transformation of pollen embryo-derived explants by Agrobacterium tumefaciens in Hyoscyamus niger  

Microsoft Academic Search

Leaf, root, stem, petiole, hypocotyl, and zygotic embryo explants, as well as pollen embryoids, and redifferentiated tissues from pollen embryoid-derived plantlets of Hyoscyamus niger L. (black henbane) were inoculated with Agrobacterium tumefaciens, harboring binary vectors (pGS Gluc1) and then cultured on media containing kanamycin. Transient ß-glucuronidase activity and kanamycin resistant callus formation were influenced by explant origin. Transgenic calluses were

Shanjun Tu; R. S. Sangwan; V. Raghavan; D. P. S. Verma; B. S. Sangwan-Norreel

2005-01-01

118

Promotion of Monacolin K production by Agrobacterium tumefaciens-mediated transformation in Monascus albidus 9901.  

PubMed

The binary vector pCAMBIA3300-gpdA-hph-trpC with hygromycin B phosphotransferase (hph) was constructed and transformed into Monascus albidus 9901 by Agrobacterium tumefaciens-mediated transformation, with gene hph as the selective marker. In order to improve the efficiency of A. tumefaciens-mediated transformation in M. albidus 9901, we optimized various factors including concentration of M. albidus 9901 spores, cell density of A. tumefaciens, co-cultivation time, temperature, and acetosyringone concentration. Most transformants of M. albidus 9901 could grow stably on media containing 50 ?g ml?ą hygromycin B up to five generations. The presence of hph was identified by PCR. Two transformants H1 and H2 which produced more Monacolin K than M. albidus 9901 were screened, and the concentration of Monacolin K in the fermented millet by H1 and H2 increased by 42.15% and 40.34% respectively compared with that produced by M. albidus 9901. PMID:20717674

Wang, Lu; Wang, Wu; Xu, Ganrong

2011-02-01

119

A simple method for the transformation of Agrobacterium tumefaciens by foreign DNA.  

PubMed

A method for successful introduction of Ti plasmid vector ( > 10 kb) into Agrobacterium tumefaciens is described. Competent A.tumefaciens cells were prepared with 50 mmol/L CaC1(2) at room temperature and introduction was done on ice followed by a 28 degrees C heat pulse. The efficiency of transformation was 10(-4)-10(-5) transformants per total recipient population or 10(6) transformants per microgram DNA. The effects of growth phase of A. tumefaciens cells, concentration of CaC1(2) solution, temperature, liquid N2, heat pulse, recovery time, and storage time of competent cells at 4 degrees C or -20 degrees C (in 15% glycerin) on transformation were studied. PMID:8739105

Cui, W; Liu, W; Wu, G

1995-01-01

120

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

Microsoft Academic Search

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 ?-glucuronidase ( gus-intron) genes in the

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

2004-01-01

121

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

122

Restoration of virulence of Vir region mutants of Agrobacterium tumefaciens strain B6S3 by coinfection with normal and mutant Agrobacterium strains  

Microsoft Academic Search

Three avirulent Tn7 insertion mutants mapping in the vir E region of the Agrobacterium tumefaciens plasmid pTiB6S3 regain virulence by co-infection with several wildtype strains and with a number of strains carrying mutations in other regions of the Ti plasmid. This finding indicates that during tumour induction normal Agrobacterium strains produce a diffusable factor required for transformation and might allow

L. Otten; H. De Greve; J. Leemans; R. Hain; P. Hooykaas; J. Schell

1984-01-01

123

Profound impact of Hfq on nutrient acquisition, metabolism and motility in the plant pathogen Agrobacterium tumefaciens.  

PubMed

As matchmaker between mRNA and sRNA interactions, the RNA chaperone Hfq plays a key role in riboregulation of many bacteria. Often, the global influence of Hfq on the transcriptome is reflected by substantially altered proteomes and pleiotropic phenotypes in hfq mutants. Using quantitative proteomics and co-immunoprecipitation combined with RNA-sequencing (RIP-seq) of Hfq-bound RNAs, we demonstrate the pervasive role of Hfq in nutrient acquisition, metabolism and motility of the plant pathogen Agrobacterium tumefaciens. 136 of 2544 proteins identified by iTRAQ (isobaric tags for relative and absolute quantitation) were affected in the absence of Hfq. Most of them were associated with ABC transporters, general metabolism and motility. RIP-seq of chromosomally encoded Hfq3xFlag revealed 1697 mRNAs and 209 non-coding RNAs (ncRNAs) associated with Hfq. 56 ncRNAs were previously undescribed. Interestingly, 55% of the Hfq-bound ncRNAs were encoded antisense (as) to a protein-coding sequence suggesting that A. tumefaciens Hfq plays an important role in asRNA-target interactions. The exclusive enrichment of 296 mRNAs and 31 ncRNAs under virulence conditions further indicates a role for post-transcriptional regulation in A. tumefaciens-mediated plant infection. On the basis of the iTRAQ and RIP-seq data, we assembled a comprehensive model of the Hfq core regulon in A. tumefaciens. PMID:25330313

Möller, Philip; Overlöper, Aaron; Förstner, Konrad U; Wen, Tuan-Nan; Sharma, Cynthia M; Lai, Erh-Min; Narberhaus, Franz

2014-01-01

124

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

125

Profound Impact of Hfq on Nutrient Acquisition, Metabolism and Motility in the Plant Pathogen Agrobacterium tumefaciens  

PubMed Central

As matchmaker between mRNA and sRNA interactions, the RNA chaperone Hfq plays a key role in riboregulation of many bacteria. Often, the global influence of Hfq on the transcriptome is reflected by substantially altered proteomes and pleiotropic phenotypes in hfq mutants. Using quantitative proteomics and co-immunoprecipitation combined with RNA-sequencing (RIP-seq) of Hfq-bound RNAs, we demonstrate the pervasive role of Hfq in nutrient acquisition, metabolism and motility of the plant pathogen Agrobacterium tumefaciens. 136 of 2544 proteins identified by iTRAQ (isobaric tags for relative and absolute quantitation) were affected in the absence of Hfq. Most of them were associated with ABC transporters, general metabolism and motility. RIP-seq of chromosomally encoded Hfq3xFlag revealed 1697 mRNAs and 209 non-coding RNAs (ncRNAs) associated with Hfq. 56 ncRNAs were previously undescribed. Interestingly, 55% of the Hfq-bound ncRNAs were encoded antisense (as) to a protein-coding sequence suggesting that A. tumefaciens Hfq plays an important role in asRNA-target interactions. The exclusive enrichment of 296 mRNAs and 31 ncRNAs under virulence conditions further indicates a role for post-transcriptional regulation in A. tumefaciens-mediated plant infection. On the basis of the iTRAQ and RIP-seq data, we assembled a comprehensive model of the Hfq core regulon in A. tumefaciens. PMID:25330313

Möller, Philip; Overlöper, Aaron; Förstner, Konrad U.; Wen, Tuan-Nan; Sharma, Cynthia M.; Lai, Erh-Min; Narberhaus, Franz

2014-01-01

126

Multiple copies of virG enhance the transient transformation of celery, carrot and rice tissues by Agrobacterium tumefaciens  

Microsoft Academic Search

In an effort to improve the T-DNA-mediated transformation frequency of economically important crops, we investigated the possible enhancement effect of multiple copies of virG genes contained in Agrobacterium tumefaciens strains upon the transient transformation of celery, carrot and rice tissues. Four days after A. tumefaciens infection, we performed histochemical ß-glucuronidase (GUS) assays to determine the frequency of transient transformation of

Chang-Nong Liu; Xiu-Qing Li; Stanton B. Gelvin

1992-01-01

127

Involvement of circular intermediates in the transfer of T-DNA from Agrobacterium tumefaciens to plant cells  

Microsoft Academic Search

Co-cultivation of Agrobacterium tumefaciens with plant cells leads to the induction of circular copies of the T-DNA segment of the large tumour-inducing (Ti) plasmid in the bacterial cells. These circular molecules are presumably intermediates in DNA transfer from the A. tumefaciens genome to the plant cells. In support of this suggestion, the junction of the T-DNA circles occurs precisely in

Zdena Koukolíková-Nicola; Raymond D. Shillito; Barbara Hohn; Kan Wang; Marc Van Montagu; Patricia Zembryski

1985-01-01

128

Agrobacterium tumefaciens -mediated transgenic plant production via direct shoot bud organogenesis from pre-plasmolyzed leaf explants of Catharanthus roseus  

Microsoft Academic Search

Production of Agrobacterium tumefaciens-mediated transgenic plants, via direct shoot bud organogenesis from leaves of Catharanthus roseus, is reported. A. tumefaciens harbouring the plasmid pBI121 with GUS gene uidA and kanamycin resistance gene nptII was used. Highest transformation efficiency of 1.4 transgenic shoots\\/responded explant was obtained when pre-plasmolysed\\u000a leaves, pre-incubated on shoot bud induction medium for 10 days, were subjected to sonication

Priyanka Verma; Ajay Kumar Mathur

2011-01-01

129

Transgenic plants of coffee Coffea canephora from embryogenic callus via Agrobacterium tumefaciens-mediated transformation  

Microsoft Academic Search

Embryogenic calli were induced from leaf explants of coffee (Coffea canephora) on McCown's woody plant medium (WPM) supplemented with 5??M N6–(2-isopentenyl)-adenosine (2-iP). These calli were co-cultured with Agrobacterium tumefaciens EHA101 harboring pIG121-Hm, containing ?-glucuronidase (GUS), hygromycin phosphotransferase (HPT), and neomycin phosphotransferase\\u000a II genes. Selection of putative transgenic callus was performed by gradual increase in hygromycin concentration (5, 50, 100?mg\\/l).\\u000a The

T. Hatanaka; Y. E. Choi; T. Kusano; H. Sano

1999-01-01

130

Introduction of foreign genes into potato cultivars Bintje and Désirée using an Agrobacterium tumefaciens binary vector.  

PubMed

Tuber discs of Solanum tuberosum cv Bintje and Désirée were cocultivated with an Agrobacterium tumefaciens binary vector, carrying both the neomycine phosphotransferase and the E. coli ?-glucuronidase gene fused to resp. the nopaline synthase and Cauliflower mosaic virus 35S promotor.Inoculated tuber discs produce transgenic shoots in selective media containing kanamycin. The transgenic plants are phenotypically normal and contain the euploid number of chromosomes. Both the neomycin phosphotransferase as well as the ?-glucuronidase gene are expressed conferring resp. kanamycin resistance and ?-glucuronidase activity to the plants. PMID:24241414

Stiekema, W J; Heidekamp, F; Louwerse, J D; Verhoeven, H A; Dijkhuis, P

1988-01-01

131

A combination of overgrowth-control antibiotics improves Agrobacterium tumefaciens -mediated transformation efficiency for cultivated tomato ( L. esculentum )  

Microsoft Academic Search

Summary  The transformation efficiency of cultivated tomato (Lycopersicon esculentum cv. UC82) using Agrobacterium tumefaciens was improved from 14% in a previous report to 25% in the present study. Several variables potentially involved in the improvement\\u000a of transformation efficiency were evaluated, including enhancements in the regeneration system, antibiotics used for Agrobacterium-overgrowth control, and method of applying kanamycin for selection. The most important

Wei Hu; Gregory C. Phillips

2001-01-01

132

Localization and orientation of the VirD4 protein of Agrobacterium tumefaciens in the cell membrane  

Microsoft Academic Search

The virD4 gene of Agrobacterium tumefaciens is essential for the formation of crown galls. Analysis of the nucleotide sequence of virD4 has suggested that the N-terminal region of the encoded protein acts as a signal peptide for the transport of the VirD4 protein to the cell membrane of Agrobacterium. We have examined the localization and orientation of this protein in

Shigehisa Okamoto; Akiko Toyoda-Yamamoto; Kenji Ito; Itaru Takebe; Yasunori Machida

1991-01-01

133

X-ray structure of imidazolonepropionase from Agrobacterium tumefaciens at 1.87 Ĺ resolution  

SciTech Connect

Histidine degradation in Agrobacterium tumefaciens involves four enzymes, including histidase (EC 4.3.1.3), urocanase (EC 4.2.1.49), imidazolonepropionase (EC 3.5.2.7), and N-formylglutamate amidohydrolase (EC 3.5.3.8). The third enzyme of the pathway, imidazolone-propionase, a 45.6 kDa protein, catalyzes conversion of imidazolone-5-propanoate to N-forminio-L-glutamate. Initial studies of the role of imidazolonepropionase in histidine degradation were published in 1953. Subsequent publications have been limited to enzyme kinetics, crystallization, and a recently reported structure determination. The imidazolonepropionases are members of metallodepenent-hydrolases (or amidohydroase) superfamily, which includs ureases, adenosine deaminases, phosphotriesterases, dihydroorotases, allantoinases, hydantoinases, adenine and cytosine deaminases, imidazolonepropionases, aryldial-kylphosphatases, chlorohydrolases, and formylmethanofuran dehydroases. Proteins belonging to this large group share a common three-dimensional structural motif (an eightfold {alpha}/{beta} or TIM barrel) with similar active sites. Most superfamily members also share a conserved metal binding site, involving four histidine residues and one aspartic acid. Imidazolonepropionase is one of the targets selected for X-ray crystallpgrahpic structure determination by the New York Structural GenomiX Research Consortium (NYSGXRC) Target ID: 9252b to correlate the structure function relationship of poorly studied by important enzyme. Here they report the crystal structure of imidazolonepropionase from Agrobacterium tumefaciens determined at 1.87 {angstrom} resolution.

Tyagi, Rajiv; Kumaran, Desigan; Burley, Stephen K.; Swaminathan, Subramanyam (SGX); (BNL)

2010-01-12

134

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

135

Transformation of white spruce and other conifer species byAgrobacterium tumefaciens.  

PubMed

Studies of the ability ofAgrobacterium to transform white spruce (Picea glauca), Engelmann spruce (P. engelmanni), Sitka spruce (P. sitchensis) and Douglas-fir (Pseudotsuga menziesii) showed frequencies of gall formation from 0-80% depending upon the strain ofAgrobacterium, and the conifer species. Thirty sixA. tumefaciens strains and oneA. rhizogenes strain were tested on 6 month old white spruce seedlings. NineA. tumefaciens strains induced gall formation on more than 50% of the inoculated trees and at greater than 10% of the inoculated sites. One strain, B2/74 gave rise to galls at 28% of the inoculated sites on white spruce and induced the highest overall frequency of gall formation on all the conifer species tested. Relative frequency of gall formation was consistent among species, although the overall frequency was much higher on Douglas-fir. Of the well characterized strains for which disarmed derivatives are available only A281 (carrying the supervirulent tumor inducing plasmid, pTiBo542) gave efficient transformation. Stable integration of T-DNA encoded genes has been confirmed by the expression of opine synthesis and hormone autonomous growth. The transfer and long-term stable expression of kanamycin resistance and firefly luciferase activity using binary vector systems was also achieved. PMID:24232587

Ellis, D; Roberts, D; Sutton, B; Lazaroff, W; Webb, D; Flinn, B

1989-05-01

136

Agrobacterium tumefaciens ExoR represses succinoglycan biosynthesis and is required for biofilm formation and motility  

PubMed Central

The ubiquitous plant pathogen Agrobacterium tumefaciens attaches efficiently to plant tissues and abiotic surfaces and can form complex biofilms. A genetic screen for mutants unable to form biofilms on PVC identified disruptions in a homologue of the exoR gene. ExoR is a predicted periplasmic protein, originally identified in Sinorhizobium meliloti, but widely conserved among alphaproteobacteria. Disruptions in the A. tumefaciens exoR gene result in severely compromised attachment to abiotic surfaces under static and flow conditions, and to plant tissues. These mutants are hypermucoid due to elevated production of the exopolysaccharide succinoglycan, via derepression of the exo genes that direct succinoglycan synthesis. In addition, exoR mutants have lost flagellar motility, do not synthesize detectable flagellin and are diminished in flagellar gene expression. The attachment deficiency is, however, complex and not solely attributable to succinoglycan overproduction or motility disruption. A. tumefaciens ExoR can function independently of the ChvG–ChvI two component system, implicated in ExoR-dependent regulation in S. meliloti. Mutations that suppress the exoR motility defect suggest a branched regulatory pathway controlling succinoglycan synthesis, motility and biofilm formation. PMID:20576688

Tomlinson, Amelia D.; Ramey-Hartung, Bronwyn; Day, Travis W.; Merritt, Peter M.; Fuqua, Clay

2010-01-01

137

Genetic analysis of nonpathogenic Agrobacterium tumefaciens mutants arising in crown gall tumors.  

PubMed Central

Little is known about the effect of the host on the genetic stability of bacterial plant pathogens. Crown gall, a plant disease caused by Agrobacterium tumefaciens, may represent a useful model to study this effect. Indeed, our previous observations on the natural occurrence and origin of nonpathogenic agrobacteria suggest that the host plant might induce loss of pathogenicity in populations of A. tumefaciens. Here we report that five different A. tumefaciens strains initially isolated from apple tumors produced up to 99% nonpathogenic mutants following their reintroduction into axenic apple plants. Two of these five strains were also found to produce mutants on pear and/or blackberry plants. Generally, the mutants of the apple isolate D10B/87 were altered in the tumor-inducing plasmid, harboring either deletions in this plasmid or point mutations in the regulatory virulence gene virG. Most of the mutants originating from the same tumor appeared to be of clonal origin, implying that the host plants influenced agrobacterial populations by favoring growth of nonpathogenic mutants over that of wild-type cells. This hypothesis was confirmed by coinoculation of apple rootstocks with strain D10B/87 and a nonpathogenic mutant. PMID:7601840

Bélanger, C; Canfield, M L; Moore, L W; Dion, P

1995-01-01

138

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

Microsoft Academic Search

The detailed structural organization of DNA sequences transferred to the plant genome via Agrobacterium tumefaciens has been determined in 11 transgenic tomato plants that carry the transferred DNA (T-DNA) at a single genetic locus. The majority (seven) of these plants were found to carry multiple copies of T-DNA arranged in inverted repeat structures. Such a high frequency of inverted repeats

Richard Jorgensen; Christine Snyder; Jonathan D. G. Jones

1987-01-01

139

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...

140

Stimulation of oleandrin production by combined Agrobacterium tumefaciens mediated transformation and fungal elicitation in Nerium oleander cell cultures  

Microsoft Academic Search

Suspension cultures derived from Agrobacterium tumefaciens-transformed calli were established in Nerium oleander L. The presence of the bacterial T-DNA in the transformed calli was detected by the polymerase chain reaction as well as plant hormone autotrophy. The ability of the cultures to accumulate oleandrin was confirmed using high performance liquid chromatography. The effect of fungal elicitors prepared from Aspergillus niger

Amany K. Ibrahim; Sherief Khalifa; Ishrak Khafagi; Diaa Youssef; Ikhlas Khan; Mostafa Mesbah

2007-01-01

141

Genetic modification of the commercial apple cultivars Gala, Golden Delicious and Elstar via an Agrobacterium tumefaciens-mediated transformation method  

Microsoft Academic Search

Modern biotechnological techniques, including genetic modification, offer important tools to plant breeders for the introduction of resistance genes into a fruit crop such as apple. However, the transformation methods necessary to generate transgenic apple plants from commercial cultivars are often tedious or unavailable. In this article we report on an Agrobacterium tumefaciens-mediated transformation method for the commerical apple cultivars Gala,

Klaas J. Puite; Jan G. Schaart

1996-01-01

142

DEVELOPMENT OF A CULTURE-INDEPENDENT REAL-TIME PCR ASSAY FOR DETECTION OF AGROBACTERIUM TUMEFACIENS IN SOIL  

Technology Transfer Automated Retrieval System (TEKTRAN)

Crown gall disease caused by the bacterium Agrobacterium tumefaciens can cause significant economic loss in both commercial walnut orchards and in nursery operations in California. This results from the fact that, Paradox, one of the most popular walnut rootstocks in California, is extremely suscep...

143

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

144

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

145

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

146

Factors enhancing Agrobacterium tumefaciens-mediated gene transfer in peanut (Arachis hypogaea L.)  

NASA Technical Reports Server (NTRS)

Parameters enhancing Agrobacterium-mediated transfer of foreign genes to peanut (Arachis hypogaea L.) cells were investigated. An intron-containing beta-glucuronidase uidA (gusA) gene under the transcriptional control of CaMV 35S promoter served as a reporter. Transformation frequency was evaluated by scoring the number of sectors expressing GUS activity on leaf and epicotyl explants. The 'Valencia Select' market type cv. New Mexico was more amenable to Agrobacterium transformation than the 'runner' market type cultivars tested (Florunner, Georgia Runner, Sunrunner, or South Runner). The disarmed Agrobacterium tumefaciens strain EHA101 was superior in facilitating the transfer of uidA gene to peanut cells compared to the disarmed strain C58. Rinsing of explants in half-strength Murashige-Skoog (MS) media prior to infection by Agrobacterium significantly increased the transformation efficiency. The use of cocultivation media containing high auxin [1.0 or 2.5 mg/l (4.53 micromolar or 11.31 micromolar) 2,4-D] and low cytokinin [0.25 or 0.5 mg/l (1.0 micromolar or 2.0 micromolar) BA] promoted higher transformation than either hormone-free or thidiazuron-containing medium. The polarity of the epicotyl during cocultivation was important; explants incubated in an inverted (vertically) manner followed by a vertically upright position resulted in improved transformation and shoot regeneration frequencies. Preculture of explants in MS basal medium or with 2.5 mg thidiazuron per l prior to infection drastically decreased the number of transformed zones. The optimized protocol was used to obtain transient transformation frequencies ranging from 12% to 36% for leaf explants, 15% to 42% for epicotyls. Initial evidence of transformation was obtained by polymerase chain reaction and subsequently confirmed by Southern analysis of regenerated plants.

Egnin, M.; Mora, A.; Prakash, C. S.; Mortley, D. G. (Principal Investigator)

1998-01-01

147

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

148

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

PubMed

The key amino acid residues that influence the function of the Agrobacterium tumefaciens iron response regulator protein (Irr(At) ) were investigated. Several Irr(At) mutant proteins containing substitutions in amino acids corresponding to candidate metal- and haem-binding sites were constructed. The ability of the mutant proteins to repress the promoter of the membrane bound ferritin (mbfA) gene was investigated using a promoter-lacZ fusion assay. A single mutation at residue H94 significantly decreased the repressive activity of Irr(At) . Multiple mutation analysis revealed the importance of H45, H65, the HHH motif (H92, H93 and H94) and H127 for the repressor function of Irr(At) . H94 is essential for the iron responsiveness of Irr(At) . Furthermore, the Irr(At) mutant proteins showed differential abilities to complement the H(2) O(2) -hyper-resistant phenotype of an irr mutant. PMID:22817265

Bhubhanil, Sakkarin; Ruangkiattikul, Nantaporn; Niamyim, Phettree; Chamsing, Jareeya; Ngok-Ngam, Patchara; Sukchawalit, Rojana; Mongkolsuk, Skorn

2012-10-01

149

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?Ĺ. 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

150

Agrobacterium tumefaciens-mediated genetic transformation of the phytopathogenic fungus Penicillium digitatum * §  

PubMed Central

Agrobacterium tumefaciens-mediated transformation (ATMT) system was assessed for conducting insertional mutagenesis in Penicillium digitatum, a major fungal pathogen infecting post-harvest citrus fruits. A transformation efficiency of up to 60 transformants per 106 conidia was achieved by this system. The integration of the hph gene into the fungal genome was verified by polymerase chain reaction (PCR) amplification and sequencing. These transformants tested were also shown to be mitotically stable. Southern blot analysis of 14 randomly selected transformants showed that the hph gene was randomly integrated as single copy into the fungal genome of P. digitatum. Thus, we conclude that ATMT of P. digitatum could be used as an alternatively practical genetic tool for conducting insertional mutagenesis in P. digitatum to study functional genomics. PMID:18837111

Wang, Ji-ye; Li, Hong-ye

2008-01-01

151

Population Heterogeneity of Agrobacterium tumefaciens in Galls of Populus L. from a Single Nursery  

PubMed Central

This study focused on the natural crown gall infections occurring in a Leuce poplar nursery. Soil effects on crown gall frequency were detected, indicating that contamination was due to a resident Agrobacterium tumefaciens population, which was present before seedling plantation. The crown gall frequency on poplar progenies varied from 3 to 67%, indicating the feasibility of improvement in crown gall resistance. Of 129 tumor isolates, 128 were pathogenic. These isolates were of biotype 1 or 2. Biochemical, serological, and antibiotic resistance typing results concurred, indicating the presence of four biotype 1 and two biotype 2 resident subpopulations. No significant change was noticed in the relative proportions of subpopulations from one year to another. Pathogenic subpopulations both in vitro and in planta were susceptible to Kerr K84 (P. B. New and A. Kerr, J. Appl. Bacteriol. 90:172-179, 1972). In addition, no serological cross-reactions were found to occur between K84 and the pathogenic subpopulations. PMID:16347314

Nesme, Xavier; Michel, Marie-France; Digat, Bernard

1987-01-01

152

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

153

Peptidoglycan Synthesis Machinery in Agrobacterium tumefaciens During Unipolar Growth and Cell Division  

PubMed Central

ABSTRACT The synthesis of peptidoglycan (PG) in bacteria is a crucial process controlling cell shape and vitality. In contrast to bacteria such as Escherichia coli that grow by dispersed lateral insertion of PG, little is known of the processes that direct polar PG synthesis in other bacteria such as the Rhizobiales. To better understand polar growth in the Rhizobiales Agrobacterium tumefaciens, we first surveyed its genome to identify homologs of (~70) well-known PG synthesis components. Since most of the canonical cell elongation components are absent from A. tumefaciens, we made fluorescent protein fusions to other putative PG synthesis components to assay their subcellular localization patterns. The cell division scaffolds FtsZ and FtsA, PBP1a, and a Rhizobiales- and Rhodobacterales-specific l,d-transpeptidase (LDT) all associate with the elongating cell pole. All four proteins also localize to the septum during cell division. Examination of the dimensions of growing cells revealed that new cell compartments gradually increase in width as they grow in length. This increase in cell width is coincident with an expanded region of LDT-mediated PG synthesis activity, as measured directly through incorporation of exogenous d-amino acids. Thus, unipolar growth in the Rhizobiales is surprisingly dynamic and represents a significant departure from the canonical growth mechanism of E. coli and other well-studied bacilli. PMID:24865559

Cameron, Todd A.; Anderson-Furgeson, James; Zupan, John R.; Zik, Justin J.

2014-01-01

154

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

155

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

156

Structural Analysis of ADP-Glucose Pyrophosphorylase From the Bacterium Agrobacterium Tumefaciens  

SciTech Connect

ADP-glucose pyrophosphorylase (ADPGlc PPase) catalyzes the conversion of glucose 1-phosphate and ATP to ADP-glucose and pyrophosphate. As a key step in glucan synthesis, the ADPGlc PPases are highly regulated by allosteric activators and inhibitors in accord with the carbon metabolism pathways of the organism. Crystals of Agrobacterium tumefaciens ADPGlc PPase were obtained using lithium sulfate as a precipitant. A complete anomalous selenomethionyl derivative X-ray diffraction data set was collected with unit cell dimensions a = 85.38 {angstrom}, b = 93.79 {angstrom}, and c = 140.29 {angstrom} ({alpha} = {beta} = {gamma} = 90{sup o}) and space group I{sub 222}. The A. tumefaciens ADPGlc PPase model was refined to 2.1 {angstrom} with an R{sub factor} = 22% and R{sub free} = 26.6%. The model consists of two domains: an N-terminal {alpha}{beta}{alpha} sandwich and a C-terminal parallel {beta}-helix. ATP and glucose 1-phosphate were successfully modeled in the proposed active site, and site-directed mutagenesis of conserved glycines in this region (G20, G21, and G23) resulted in substantial loss of activity. The interface between the N- and the C-terminal domains harbors a strong sulfate-binding site, and kinetic studies revealed that sulfate is a competitive inhibitor for the allosteric activator fructose 6-phosphate. These results suggest that the interface between the N- and C-terminal domains binds the allosteric regulator, and fructose 6-phosphate was modeled into this region. The A. tumefaciens ADPGlc PPase/fructose 6-phosphate structural model along with sequence alignment analysis was used to design mutagenesis experiments to expand the activator specificity to include fructose 1,6-bisphosphate. The H379R and H379K enzymes were found to be activated by fructose 1,6-bisphosphate.

Cupp-Vickery, J.R.; Igarashi, R.Y.; Perez, M.; Poland, M.; Meyer, C.R.

2009-05-14

157

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...

158

Assessing the Genetic Diversity of Agrobacterium Tumefaciens in CA Walnut Growing Regions and Resistance to the Biocontrol Agent, A. Rhizogenes K84  

Technology Transfer Automated Retrieval System (TEKTRAN)

Crown gall of walnut (Juglans sp.), caused by the bacterium Agrobacterium tumefaciens, greatly impacts the CA walnut industry. To determine the genetic diversity of A. tumefaciens throughout the Central Valley of CA, we collected isolates from ten walnut growing counties. A total of 340 A. tumefac...

159

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

160

Agrobacterium tumefaciens-mediated transformation of Phellodendron amurense Rupr. using mature-seed explants.  

PubMed

An efficient transformation protocol was developed for Agrobacterium-mediated transformation of Phellodendron amurense Rupr. for using explants from mature seeds. The binary vector pCAMBIA1303, which contained hygromycin phosphotransferase (hptII) as a selectable marker gene and ?-glucuronidase (GUS) as a reporter gene, was used for transformation studies. Different factors that affect survival of transformed buds, namely Agrobacterium infection method, bacterial strain, pre-culture duration, acetosyringone concentration, co-culture duration, and co-culture temperature were examined and optimized for transformation efficiency on the basis of GUS staining of hygromycin-resistant buds. Polymerase chain reaction (PCR), Southern blot and reverse transcription PCR confirmed the presence of the GUS gene. A transformation frequency of 13.1 % was achieved under optimized conditions for transformation (A. tumefaciens strain EHA105, 4 days co-cultivation at 4 °C, and infection of the pre-cultured mature-seed explants for 2 days). This is the first report of a successful genetic transformation protocol for P. amurense. PMID:23065217

Yang, Jingli; Zhao, Bo; Kim, Yeon Bok; Zhou, Chenguang; Li, Chunyan; Chen, Yunlin; Zhang, Haizhen; Li, Cheng Hao

2013-01-01

161

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

162

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

163

Plant Transformation by Coinoculation with a Disarmed Agrobacterium tumefaciens Strain and an Escherichia coli Strain Carrying Mobilizable Transgenes  

Microsoft Academic Search

Transformation of Nicotiana tabacum leaf explants was attempted with Escherichia coli as a DNA donor either alone or in combination with Agrobacterium tumefaciens. We constructed E. coli donor strains harboring either the promiscuous IncP-type or IncN-type conjugal transfer system and second plasmids containing the respec- tive origins of transfer and plant-selectable markers. Neither of these conjugation systems was able to

Katherine M. Pappas; Stephen C. Winans

2003-01-01

164

Shoot regeneration in stem expiants and its amenability to Agrobacterium tumefaciens mediated gene transfer in Brassica carinata  

Microsoft Academic Search

Immature stem segments of seven different genotypes of Brassica carinata produced shoots with variable frequencies when cultured in MS medium with BAP and picloram at 0.2 mg\\/l each. Line 171, which produced shoots with 100% efficiency from both cut ends of the expiant, was selected for testing the amenability of this regeneration protocol for genetic transformation. A non-oncogenic Agrobacterium tumefaciens

S. B. Narasimhulu; P. B. Kirti; T. Mohapatra; Shyam Prakash; V. L. Chopra

1992-01-01

165

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

166

Integration of Complete Transferred DNA Units is Dependent on the Activity of Virulence E2 Protein of Agrobacterium tumefaciens  

Microsoft Academic Search

Agrobacterium tumefaciens transfers transferred DNA (T-DNA), a single-stranded segment of its tumor-inducing (Ti) plasmid, to the plant cell nucleus. The Ti-plasmid-encoded virulence E2 (VirE2) protein expressed in the bacterium has single-stranded DNA (ssDNA)-binding properties and has been reported to act in the plant cell. This protein is thought to exert its influence on transfer efficiency by coating and accompanying the

L. Rossi; B. Hohn; B. Tinland

1996-01-01

167

Covalently Bound VirD2 Protein of Agrobacterium tumefaciens Protects the T-DNA from Exonucleolytic Degradation  

Microsoft Academic Search

We show that upon induction of Agrobacterium tumefaciens, free linear double-stranded T-DNA molecules as well as the previously described T-strands are generated from the Ti plasmid. A majority of these molecules are bound to a protein. We show that this protein is the product of the virulence gene virD2. This protein was found to be attached to the 5' terminus

Franz Durrenberger; Andreas Crameri; Barbara Hohn; Zdena Koukolikova-Nicola

1989-01-01

168

Cotransformation with one Agrobacterium tumefaciens strain containing two binary plasmids as a method for producing marker-free transgenic plants  

Microsoft Academic Search

Co-transformation was investigated as a method that would allow the use of a selectable marker during plant regeneration\\u000a followed by recovery of progeny which contain the desired gene(s) but lack a marker gene. Rapeseed (Brassica napus cv `212\\/86') and tobacco (Nicotiana tabacum cv `Xanthi NC') were co-cultivated with a single Agrobacterium tumefaciens strain containing two binary plasmids. Genes from both

M. Daley; V. C. Knauf; K. R. Summerfelt; J. C. Turner

1998-01-01

169

An analysis of the boundaries of the octopine TL-DNA in tumors induced by Agrobacterium tumefaciens  

Microsoft Academic Search

The octopine Ti plasmid of Agrobacterium tumefaciens strain Ach5 contains a 13.5 kb TL-region and a 6 kb TR-region which can independently be transferred to plant nuclear DNA. A direct repeat of 25 bp flanks the TL-region, and is related to the direct repeat flanking the nopaline T-region (Zambryski et al. 1982; Yadav et al. 1982). Two right TL-DNA borders,

M. Holsters; R. Villarroel; J. Gielen; J. Seurinck; H. De Greve; M. Van Montagu; J. Schell

1983-01-01

170

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

Microsoft Academic Search

Agrobacterium tumefaciens genetically transforms plant cells by transferring a copy of its T-DNA to the plant where it is integrated and stably maintained. In the presence of wounded plant cells this process is activated and mediated by the products of the vir genes which are grouped into six distinct loci. The largest is the virB locus spanning 9.5 kb. Transposon

Gretchen A. Kuldau; Guido Vos; John Owen; Gretchen McCaffrey; Patricia Zambryski

1990-01-01

171

ThednaKJOperon ofAgrobacterium tumefaciens: Transcriptional Analysis and Evidence for a New Heat Shock Promoter  

Microsoft Academic Search

The dnaKJ operon of Agrobacterium tumefaciens was cloned and sequenced and was found to be highly homologous to previously analyzeddnaKJoperons. Transcription of this operon inA. tumefacienswas stimu- lated by heat shock as well as by exposure to ethanol and hydrogen peroxide. There were two transcripts representing the dnaKJ operon: one containing the dnaK and dnaJ genes and the second containing

GIL SEGAL; ANDELIORA Z. RON; S. Wise

1995-01-01

172

High-efficiency transformation of Lycium barbarum mediated by Agrobacterium tumefaciens and transgenic plant regeneration via somatic embryogenesis  

Microsoft Academic Search

We have developed a reliable and high-frequency system of transformation and regeneration via somatic embryogenesis (SE) of Lycium barbarum. Leaf segments were co-cultivated with Agrobacterium tumefaciens EHA101 (pIG121Hm) carrying the neomycin phosphotransferase II gene as a selectable marker and an intron-#-glucuronidase (GUS) gene as a reporter marker. On the medium for callus-induction, which contained 50 mg l-1 kanamycin (Km), approximately

Z. Hu; J. Yang; G. Guo; G. Zheng

2002-01-01

173

Evaluation of four Agrobacterium tumefaciens strains for the genetic transformation of tomato (Solanum lycopersicum L.) cultivar Micro-Tom.  

PubMed

KEY MESSAGE : Agrobacterium tumefaciens strains differ not only in their ability to transform tomato Micro-Tom, but also in the number of transgene copies that the strains integrate in the genome. The transformation efficiency of tomato (Solanum lycopersicum L.) cv. Micro-Tom with Agrobacterium tumefaciens strains AGL1, EHA105, GV3101, and MP90, harboring the plasmid pBI121 was compared. The presence of the nptII and/or uidA transgenes in regenerated T(0) plants was determined by PCR, Southern blotting, and/or GUS histochemical analyses. In addition, a rapid and reliable duplex, qPCR TaqMan assay was standardized to estimate transgene copy number. The highest transformation rate (65 %) was obtained with the Agrobacterium strain GV3101, followed by EHA105 (40 %), AGL1 (35 %), and MP90 (15 %). The mortality rate of cotyledons due to Agrobacterium overgrowth was the lowest with the strain GV3101. The Agrobacterium strain EHA105 was more efficient than GV3101 in the transfer of single T-DNA insertions of nptII and uidA transgenes into the tomato genome. Even though Agrobacterium strain MP90 had the lowest transformation rate of 15 %, the qPCR analysis showed that the strain MP90 was the most efficient in the transfer of single transgene insertions, and none of the transgenic plants produced with this strain had more than two insertion events in their genome. The combination of higher transformation efficiency and fewer transgene insertions in plants transformed using EHA105 makes this Agrobacterium strain optimal for functional genomics and biotechnological applications in tomato. PMID:23099543

Chetty, V J; Ceballos, N; Garcia, D; Narváez-Vásquez, J; Lopez, W; Orozco-Cárdenas, M L

2013-02-01

174

A Photolyase-Like Protein from Agrobacterium tumefaciens with an Iron-Sulfur Cluster  

PubMed Central

Photolyases and cryptochromes are evolutionarily related flavoproteins with distinct functions. While photolyases can repair UV-induced DNA lesions in a light-dependent manner, cryptochromes regulate growth, development and the circadian clock in plants and animals. Here we report about two photolyase-related proteins, named PhrA and PhrB, found in the phytopathogen Agrobacterium tumefaciens. PhrA belongs to the class III cyclobutane pyrimidine dimer (CPD) photolyases, the sister class of plant cryptochromes, while PhrB belongs to a new class represented in at least 350 bacterial organisms. Both proteins contain flavin adenine dinucleotide (FAD) as a primary catalytic cofactor, which is photoreduceable by blue light. Spectral analysis of PhrA confirmed the presence of 5,10-methenyltetrahydrofolate (MTHF) as antenna cofactor. PhrB comprises also an additional chromophore, absorbing in the short wavelength region but its spectrum is distinct from known antenna cofactors in other photolyases. Homology modeling suggests that PhrB contains an Fe-S cluster as cofactor which was confirmed by elemental analysis and EPR spectroscopy. According to protein sequence alignments the classical tryptophan photoreduction pathway is present in PhrA but absent in PhrB. Although PhrB is clearly distinguished from other photolyases including PhrA it is, like PhrA, required for in vivo photoreactivation. Moreover, PhrA can repair UV-induced DNA lesions in vitro. Thus, A. tumefaciens contains two photolyase homologs of which PhrB represents the first member of the cryptochrome/photolyase family (CPF) that contains an iron-sulfur cluster. PMID:22066008

Oberpichler, Inga; Pierik, Antonio J.; Wesslowski, Janine; Pokorny, Richard; Rosen, Ran; Vugman, Michal; Zhang, Fan; Neubauer, Olivia; Ron, Eliora Z.; Batschauer, Alfred; Lamparter, Tilman

2011-01-01

175

Role a Agrobacterium tumefaciens ChvA protein in export of. beta. -1,2-glucan  

SciTech Connect

Functional chvA and chvB genes are required for attachment of Agrobacterium tumefaciens to plant cells, an early step in crown gall tumor formation. Strains defective in these loci do not secrete normal amounts of cyclic {beta}-1,2-glucan. Whereas chvB is required for {beta}-1,2-glucan synthesis, the role of chvA in glucan synthesis or export has not been clearly defined. We found that cultures of chvA mutants contained as much neutral {beta}-1,2-glucan in the cell pellets as did the wild type, with no detectable accumulation of glucan in the culture supernatant. The cytoplasm of chvA mutant cells contained over three times more soluble {beta}-1,2-glucan than did the cytoplasm of the wild-type parent. Unlike the wild type, chvA mutants contained no detectable periplasmic glucan. The amino acid sequence of chvA is highly homologous to the sequences of bacterial and eucaryotic export proteins, as observed previously in the case of ndvA, a rhizobial homolog of chvA. Strong sequence homology within this family of export proteins is concentrated in the carboxy-terminal portions of the proteins, but placement of consensus ATP-binding sites, internal signal sequences, and hydrophobic domains are conserved over their entire lengths. These data suggest a model for {beta}-1,2-glucan synthesis in A. tumefaciens in which glucan is synthesized inside the inner membrane with the participation of ChvB and transported across the inner membrane with the participation of ChvA.

Cangelosi, G.A.; Martinetti, G.; Leigh, J.A.; Lee, Chi Chang; Theines, C. (Univ. of Washington, Seattle (USA))

1989-03-01

176

Fha Interaction with Phosphothreonine of TssL Activates Type VI Secretion in Agrobacterium tumefaciens  

PubMed Central

The type VI secretion system (T6SS) is a widespread protein secretion system found in many Gram-negative bacteria. T6SSs are highly regulated by various regulatory systems at multiple levels, including post-translational regulation via threonine (Thr) phosphorylation. The Ser/Thr protein kinase PpkA is responsible for this Thr phosphorylation regulation, and the forkhead-associated (FHA) domain-containing Fha-family protein is the sole T6SS phosphorylation substrate identified to date. Here we discovered that TssL, the T6SS inner-membrane core component, is phosphorylated and the phosphorylated TssL (p-TssL) activates type VI subassembly and secretion in a plant pathogenic bacterium, Agrobacterium tumefaciens. Combining genetic and biochemical approaches, we demonstrate that TssL is phosphorylated at Thr 14 in a PpkA-dependent manner. Further analysis revealed that the PpkA kinase activity is responsible for the Thr 14 phosphorylation, which is critical for the secretion of the T6SS hallmark protein Hcp and the putative toxin effector Atu4347. TssL phosphorylation is not required for the formation of the TssM-TssL inner-membrane complex but is critical for TssM conformational change and binding to Hcp and Atu4347. Importantly, Fha specifically interacts with phosphothreonine of TssL via its pThr-binding motif in vivo and in vitro and this interaction is crucial for TssL interaction with Hcp and Atu4347 and activation of type VI secretion. In contrast, pThr-binding ability of Fha is dispensable for TssM structural transition. In conclusion, we discover a novel Thr phosphorylation event, in which PpkA phosphorylates TssL to activate type VI secretion via its direct binding to Fha in A. tumefaciens. A model depicting an ordered TssL phosphorylation-induced T6SS assembly pathway is proposed. PMID:24626341

Lin, Jer-Sheng; Wu, Hsin-Hui; Hsu, Pang-Hung; Ma, Lay-Sun; Pang, Yin-Yuin; Tsai, Ming-Daw; Lai, Erh-Min

2014-01-01

177

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

178

Evidence of Biological Control of Agrobacterium tumefaciens Strains Sensitive and Resistant to Agrocin 84 by Different Agrobacterium radiobacter Strains on Stone Fruit Trees  

PubMed Central

The effectiveness of Agrobacterium radiobacter K84, 0341, and a K84 non-agrocin-producing mutant (K84 Agr-) in biological control of crown gall on rootstocks of stone fruit trees was determined in three experiments. In experiment 1, K84 and 0341 controlled crown gall on plum plants in soil inoculated with two strains of Agrobacterium tumefaciens resistant to agrocin 84. In experiment 2, K84 controlled crown gall on peach plants in soils inoculated with strains of A. tumefaciens sensitive or resistant to agrocin 84 or with a mixture of both. However, the effectiveness of K84 was higher against the sensitive strain than against the resistant strain. There was a residual effect of K84 from one year to another in soil inoculated with the sensitive strains. In experiment 3, K84 and K84 Agr- controlled crown gall on plum and peach plants in soils inoculated with strains of A. tumefaciens sensitive or resistant to agrocin 84. The control afforded by K84 was higher than that provided by K84 Agr- against the sensitive strain but was similar against the resistant strain. PMID:16347881

López, María M.; Gorris, María Teresa; Salcedo, Carmina I.; Montojo, Ana M.; Miró, Marcela

1989-01-01

179

Attempts to Detect Agrobacterium tumefaciens DNA in Crown-Gall Tumor Tissue 1  

PubMed Central

Primary and secondary crown gall tissue cultures were established from sunflower plants (Helianthus annuus, variety Mammoth Russian) wound-inoculated with Agrobacterium tumefaciens (Smith and Townsend) Conn strain B6. Growth rates of tumor tissues and habituated healthy sunflower stem section tissues on basal medium lacking auxin and cytokinin were compared to those of healthy sunflower stem section tissue grown on the same medium with added phytohormones. No difference was detected in the thermal denaturation midpoints (74.8 C) and melting profiles in 25 mm sodium phosphate (pH 6.8), or the buoyant densities in cesium chloride equilibrium centrifugation (1.687 g cm?3), between deoxyribonucleic acids (DNAs) isolated from crude nuclear preparations of the four tissue types. No satellite DNA was observed in equilibrium centrifugation of unsheared plant DNAs. Heterologous DNA renaturation kinetic analyses were performed in 0.14 m sodium phosphate (pH 6.8) at 70 C. Thermal stability measurements of reassociated DNA revealed less than 1% of mismatched base pairs. Reannealing of sheared, denatured, radioactive A. tumefaciens B6 DNA (molecular weight, 325,000 daltons) in the presence of a 5400-fold excess of sheared calf thymus, healthy tissue, or secondary sunflower crown gall DNA obeyed second order kinetics, with a Cot˝ of 2.8, identical to that observed when B6 DNA was reannealed in the absence of foreign DNA. Reannealing rates of B6 DNA in the presence of 5400-fold excesses of DNA from two lines of primary sunflower crown gall were increased 2.24- or 1.47-fold. Digestion of the tumor DNA preparations with pancreatic deoxyribonuclease I until no detectable DNA remained, followed by restoration of solution viscosity by added calf thymus DNA, failed to remove the acceleration effect of the tumor DNA preparations. Reisolation of the reannealed nucleic acid formed in this experiment, and digestion with ribonuclease A or deoxyribonuclease I revealed that the double-stranded fraction was composed entirely of DNA-DNA duplexes, with no detectable DNA-RNA hybrids. The data indicate that tumor, but not healthy tissue DNA preparations contain some factor or factors (not DNA) which accelerate the reannealing of bacterial DNA. Sunflower tumor tissue DNAs, therefore, do not contain integrated A. tumefaciens DNA sequences in amounts greater than a random ? of the bacterial genome per diploid amount of plant DNA, or a complete bacterial genome per five diploid plant cell DNA equivalents. Further, the possibility of the presence of many copies of a specific portion greater than 5% of the bacterial genome is excluded. Images PMID:16659607

Merlo, Donald J.; Kemp, John D.

1976-01-01

180

[Establishment of high efficiency genetic transformation system of maize mediated by Agrobacterium tumefaciens].  

PubMed

In order to establish high-frequency regeneration and high-efficiency genetic transformation system in maize, the significance of the 11 factors influencing maize embryonic callus induction and 9 factors affecting embryonic callus differentiation was researched by orthogonal experiment. The results showed that genotype had highly significant impact on induction of embryonic callus. The concentration of 6-BA, AgNO3, 2,4-D, ABA, and medium are the significant factors. The Multi-comparison showed that ABA 2 mg/L has a significant influence. Among the callus differentiation factors, the genotype and 6-BA concentration showed a strong main effect, the concentrations of NAA, medium, KT and 2,4-D had significant impacts on callus differentiation. Southern blotting analysis demonstrated that the resistant callus rate under the selection pressure of 25 mg/L hygromycin was a reliable indicator for system optimization in resistance screening. The concentration of acetosyringone (AS) showed sensitive differences among genotypes. The highest transformation rate was found with the optimized combination of 24-25 degrees C for co-culture temperature, 0.7 ODx15 min for Agrobacterium tumefa-ciens concentration and incubation-time, and pH 5.5-6.2. By this optimized combination, the survival rate of resistant calli as an index for the stable transformation rates of inbred lines Huangzao 4 and Zong 31 by introducing GUS gene into maize inbred lines was as high as 48.6% and 46.2%, respectively. PMID:19933098

WEI, Kai-Fa

2009-11-01

181

Extended Host Range of Agrobacterium tumefaciens in the Genus Pinus1  

PubMed Central

Two-to 4-month-old seedlings of nine pine species (Pinus eldarica Medw., Pinus elliottii Engelm., Pinus jeffreyi Grev. & Balf., Pinus lambertiana Dougl., Pinus ponderosa Laws., Pinus radiata D. Don, Pinus sylvestris L., Pinus taeda L., Pinus virginiana Mill), Douglas fir (Pseudotsuaa menziesii (Mirb.) Franco) and incense cedar (Libocedrus decurrens Torr.) were inoculated with five strains of Agrobacterium tumefaciens. Transformation occurred in all conifer species tested as determined by gall formation and opine production. The frequency of gall formation varied by host species, by bacterial strain, and was related to the age of the stem when inoculated. Galls were visible 8 to 12 weeks after inoculation and were small (often less than 2.5 millimeters in diameter). Fewer than half (230 of 502) of the galls originally formed on the trees were present after 1 year, and 26 of these grew to diameters greater than 2 centimeters. The majority of these larger galls (18 of 26) were found in P. radiata. Bacterial strain-specific opines were found in 67 of the 81 gall tissues sampled. Images Figure 1 PMID:16667394

Stomp, Anne-Marie; Loopstra, Carol; Chilton, W. Scott; Sederoff, Ronald R.; Moore, Larry W.

1990-01-01

182

Structure and function of a decarboxylating Agrobacterium tumefaciens keto-deoxy-d-galactarate dehydratase.  

PubMed

Agrobacterium tumefaciens (At) strain C58 contains an oxidative enzyme pathway that can function on both d-glucuronic and d-galacturonic acid. The corresponding gene coding for At keto-deoxy-d-galactarate (KDG) dehydratase is located in the same gene cluster as those coding for uronate dehydrogenase (At Udh) and galactarolactone cycloisomerase (At Gci) which we have previously characterized. Here, we present the kinetic characterization and crystal structure of At KDG dehydratase, which catalyzes the next step, the decarboxylating hydrolyase reaction of KDG to produce ?-ketoglutaric semialdehyde (?-KGSA) and carbon dioxide. The crystal structures of At KDG dehydratase and its complexes with pyruvate and 2-oxoadipic acid, two substrate analogues, were determined to 1.7 Ĺ, 1.5 Ĺ, and 2.1 Ĺ resolution, respectively. Furthermore, mass spectrometry was used to confirm reaction end-products. The results lead us to propose a structure-based mechanism for At KDG dehydratase, suggesting that while the enzyme belongs to the Class I aldolase protein family, it does not follow a typical retro-aldol condensation mechanism. PMID:25454257

Taberman, Helena; Andberg, Martina; Parkkinen, Tarja; Jänis, Janne; Penttilä, Merja; Hakulinen, Nina; Koivula, Anu; Rouvinen, Juha

2014-12-30

183

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

184

Biogenesis of T Pili in Agrobacterium tumefaciens Requires Precise VirB2 Propilin Cleavage and Cyclization  

PubMed Central

VirB2 propilin is processed by the removal of a 47-amino-acid signal peptide to generate a 74-amino-acid peptide product in both Escherichia coli and Agrobacterium tumefaciens. The cleaved VirB2 protein is further cyclized to form the T pilin in A. tumefaciens but not in E. coli. Mutations in the signal peptidase cleavage sequence of VirB2 propilin cause the formation of aberrant T pilin and also severely attenuate virulence. No T pilus was observed in these mutants. The potential role of the exact VirB2 propilin cleavage and cyclization in T pilus biogenesis and virulence is discussed. PMID:11741876

Lai, Erh-Min; Eisenbrandt, Ralf; Kalkum, Markus; Lanka, Erich; Kado, Clarence I.

2002-01-01

185

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

186

The Agrobacterium tumefaciens rnd Homolog Is Required for TraR-Mediated Quorum-Dependent Activation of Ti Plasmid tra Gene Expression  

Microsoft Academic Search

Conjugal transfer of Agrobacterium tumefaciens Ti plasmids is regulated by quorum sensing via TraR and its cognate autoinducer, N-(3-oxo-octanoyl)-L-homoserine lactone. We isolated four Tn5-induced mutants of A. tumefaciens C58 deficient in TraR-mediated activation of tra genes on pTiC58DaccR. These mutations also affected the growth of the bacterium but had no detectable influence on the expression of two tester gene systems

ZHAO-QING LUO; STEPHEN K. FARRAND

2001-01-01

187

Agrobacterium tumefaciens-mediated genetic transformation of the Taxol-producing endophytic fungus Ozonium sp EFY21.  

PubMed

An efficient Agrobacterium tumefaciens-mediated genetic transformation method was successfully established for a newly isolated Taxol-producing fungus, Ozonium sp EFY21. A specific hygromycin B resistance expression vector, pCAMBIA1304'AN7-1, was constructed for fungal transformation. Key factors affecting transformation efficiency were thoroughly investigated and optimized. PCR amplification and Southern hybridization were used to verify the transformation events. This study should pave the way for future genetic modification studies of Ozonium sp EFY21. PMID:24065647

Liu, L; Wei, Y M; Zhou, X W; Lin, J; Sun, X F; Tang, K X

2013-01-01

188

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

189

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

190

Characterization of the mmsAB-araD1 (gguABC) Genes of Agrobacterium tumefaciens?  

PubMed Central

The chvE-gguABC operon plays a critical role in both virulence and sugar utilization through the activities of the periplasmic ChvE protein, which binds to a variety of sugars. The roles of the GguA, GguB, and GguC are not known. While GguA and GguB are homologous to bacterial ABC transporters, earlier genetic analysis indicated that they were not necessary for utilization of sugars as the sole carbon source. To further examine this issue, in-frame deletions were constructed separately for each of the three genes. Our growth analysis clearly indicated that GguA and GguB play a role in sugar utilization and strongly suggests that GguAB constitute an ABC transporter with a wide range of substrates, including l-arabinose, d-fucose, d-galactose, d-glucose, and d-xylose. Site-directed mutagenesis showed that a Walker A motif was vital to the function of GguA. We therefore propose renaming gguAB as mmsAB, for multiple monosaccharide transport. A gguC deletion affected growth only on l-arabinose medium, suggesting that gguC encodes an enzyme specific to l-arabinose metabolism, and this gene was renamed araD1. Results from bioinformatics and experimental analyses indicate that Agrobacterium tumefaciens uses a pathway involving nonphosphorylated intermediates to catabolize l-arabinose via an l-arabinose dehydrogenase, AraAAt, encoded at the Atu1113 locus. PMID:21984786

Zhao, Jinlei; Binns, Andrew N.

2011-01-01

191

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

192

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

193

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

194

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

195

The Agrobacterium tumefaciens virulence gene chvE is part of a putative ABC-type sugar transport operon.  

PubMed Central

The Agrobacterium tumefaciens virulence determinant ChvE is a periplasmic binding protein which participates in chemotaxis and virulence gene induction in response to monosaccharides which occur in the plant wound environment. The region downstream of the A. tumefaciens chvE gene was cloned and sequenced for nucleotide and expression analysis. Three open reading frames transcribed in the same direction as chvE were revealed. The first two, together with chvE, encode putative proteins of a periplasmic binding protein-dependent sugar uptake system, or ABC-type (ATP binding cassette) transporter. The third open reading frame encodes a protein of unknown function. The deduced transporter gene products are related on the amino acid level to bacterial sugar transporters and probably function in glucose and galactose uptake. We have named these genes gguA, -B, and -C, for glucose galactose uptake. Mutations in gguA, gguB, or gguC do not affect virulence of A. tumefaciens on Kalanchoe diagremontiana; growth on 1 mM galactose, glucose, xylose, ribose, arabinose, fucose, or sucrose; or chemotaxis toward glucose, galactose, xylose, or arabinose. PMID:9079938

Kemner, J M; Liang, X; Nester, E W

1997-01-01

196

A novel antibacterial peptide active against peach crown gall (Agrobacterium tumefaciens) isolated from cyanide-tolerant actinomycetes G19.  

PubMed

An antimicrobial peptide was extracted from the antagonistic actinomycetes G19. It was designated as G19-F. By using MALDI-TOF mass spectrometry, the molecular weight of G19-F was determined. The primary structure of the antimicrobial peptide was determined using N-terminal sequencing and mass spectrometry. Results showed that the peptide had eleven amino acids, with the sequence D-V-C-D-G-G-D-G-D-E-D, and a calculated molecular mass of 1,096 Da. G19-F showed antimicrobial activity against peach crown gall caused by Agrobacterium tumefaciens. The antimicrobial peptide maintained its activity after being heated to 100 °C and exhibited stability from pH 4 to 10. Its activity has also remained after ultraviolet irradiation. The mechanism by which G19-F inhibits A. tumefaciens was to increase permeability of the cell membrane and destroy the cell wall structure. Furthermore, as a novel peptide, it has a potential for cure A. tumefaciens infection. PMID:25358422

Wang, Shufang; Ji, Jinglin; Ma, Huanpu; Liu, Zhimin

2015-01-01

197

A high throughput Agrobacterium tumefaciens-mediated transformation method for functional genomics of perennial ryegrass (Lolium perenne L.).  

PubMed

A robust and high throughput Agrobacterium genetic transformation procedure has been developed for perennial ryegrass (Lolium perenne L.). Embryogenic callus lines were selected and maintained as plants in vitro. Embryogenic calli derived from meristematic regions of the vegetative tillers were co-cultivated with Agrobacterium tumefaciens strain EHA101 carrying the plasmid pCAMBIA 1305.1 in the presence of acetosyringone for 3-4 days. The calli were grown under 94.8 and 151.6 microM hygromycin selection, respectively for two cycles of 2-weeks each, followed by transfer to regeneration medium with 47.4 microM hygromycin. Regenerated plants were rooted and successfully transferred to soil. The transgenic nature of the regenerated plants was confirmed by DNA gel blot analysis and gene expression demonstrated by GUS histochemical assay and/or reverse transcription PCR. After development of the transformation procedure, we used Agrobacterium strain EHA101 carrying a modified binary plasmid pMH bearing genes of interest. In the past 2 years, we have produced more than 1,000 plants with constructs encoding different genes of interest from perennial ryegrass. PMID:16518636

Bajaj, Shivendra; Ran, Yidong; Phillips, Jonathan; Kularajathevan, Gunaseelan; Pal, Sunil; Cohen, Dan; Elborough, Kieran; Puthigae, Sathish

2006-07-01

198

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

199

Shoot regeneration in stem expiants and its amenability to Agrobacterium tumefaciens mediated gene transfer in Brassica carinata.  

PubMed

Immature stem segments of seven different genotypes of Brassica carinata produced shoots with variable frequencies when cultured in MS medium with BAP and picloram at 0.2 mg/l each. Line 171, which produced shoots with 100% efficiency from both cut ends of the expiant, was selected for testing the amenability of this regeneration protocol for genetic transformation. A non-oncogenic Agrobacterium tumefaciens containing plasmid PCV 730, a binary vector carrying resistance genes for kanamycin and hygromycin, was used. A cocultivation period of 4 d with a bacterial concentration of approximately 2.5×10 cells/ml, followed by a recovery period of 2 d, produced transformed shoots that could be selected and rooted in the presence of kanamycin at 15 mg/l. Transformation was confirmed by neomycin phospho-transferase assay and Southern blot analysis. Seed analysis of transformed plants indicated that kanamycin resistance was inherited in the progeny. PMID:24201439

Narasimhulu, S B; Kirti, P B; Mohapatra, T; Prakash, S; Chopra, V L

1992-07-01

200

Improved dominant selection markers and co-culturing conditions for efficient Agrobacterium tumefaciens-mediated transformation of Ustilago scitaminea.  

PubMed

Ustilago scitaminea is the causal agent of sugar-cane smut disease. There is, however, no genetic transformation method for it. Here we report the development of an efficient mutagenesis method based on Agrobacterium tumefaciens-mediated transformation. To improve transformation efficiency, a range of conditions, including the codon-usage preference of the selection marker gene, promoters and the culture conditions for transformation were optimized. A strong promoter to drive marker gene expression, optimized codon usage of selection marker gene, controlled water content and pH of co-culture medium were critical factors affecting transformation efficiency. Our findings provide a useful tool for genetic analysis of this important plant pathogen. PMID:24563317

Sun, Longhua; Yan, Meixin; Ding, Zhaojian; Liu, Yanbin; Du, Minge; Xi, Pinggen; Liao, Jinling; Ji, Lianghui; Jiang, Zide

2014-06-01

201

Variation in hormone autonomy and regenerative potential of cells transformed by strain A66 of Agrobacterium tumefaciens  

SciTech Connect

Mutant Agrobacterium tumefaciens strain A66 is shown to differ from its wild-type progenitor (strain A6) by a spontaneous 2.7 kb DNA insert into the T-DNA region of its Ti plasmid. Tobacco stems transformed by A66 exhibit an attenuated response characterized by slow growth and shoot proliferation. Clonal analysis demonstrates that this response is due to an alteration in the growth and regenerative potential of transformed cells, rather than to variation in the frequency of fully autonomous cells within the primary tumor. Cloned A66 transformed tobacco cells exhibit an auxin requirement for growth that can be overcome by shoot proliferation. Other host species, however, may complement the A66 mutation yielding fully auxin-independent tumors when transformed by this bacterium.

Binns, A.N. (Univ. of Pennsylvania, Philadelphia); Sciaky, D.; Wood, H.N.

1982-12-01

202

The Agrobacterium tumefaciens virC1 gene product binds to overdrive, a T-DNA transfer enhancer.  

PubMed Central

In Agrobacterium tumefaciens, a cis-active 24-base-pair sequence adjacent to the right border of the T-DNA, called overdrive, stimulates tumor formation by increasing the level of T-DNA processing. Recent results from our laboratory have suggested that the virC operon which enhances T-DNA processing probably does so because the VirC1 protein interacts with overdrive (N. Toro, A. Datta, M. Yanofsky, and E. W. Nester, Proc. Natl. Acad. Sci. USA 85:8558-8562, 1988). We report here the purification of the VirC1 protein from cells of Escherichia coli harboring a plasmid containing the coding sequences of the virC locus of the octopine Ti plasmid. By gel mobility shift and DNase I footprinting assays, we showed that this purified virC1 gene product binds to overdrive but not to the right border of T-DNA. Images PMID:2592351

Toro, N; Datta, A; Carmi, O A; Young, C; Prusti, R K; Nester, E W

1989-01-01

203

Agrobacterium tumefaciens ExoR Controls Acid Response Genes and Impacts Exopolysaccharide Synthesis, Horizontal Gene Transfer, and Virulence Gene Expression  

PubMed Central

Agrobacterium tumefaciens is a facultative plant pathogen and the causative agent of crown gall disease. The initial stage of infection involves attachment to plant tissues, and subsequently, biofilms may form at these sites. This study focuses on the periplasmic ExoR regulator, which was identified based on the severe biofilm deficiency of A. tumefaciens exoR mutants. Genome-wide expression analysis was performed to elucidate the complete ExoR regulon. Overproduction of the exopolysaccharide succinoglycan is a dramatic phenotype of exoR mutants. Comparative expression analyses revealed that the core ExoR regulon is unaffected by succinoglycan synthesis. Several findings are consistent with previous observations: genes involved in succinoglycan biosynthesis, motility, and type VI secretion are differentially expressed in the ?exoR mutant. In addition, these studies revealed new functional categories regulated by ExoR, including genes related to virulence, conjugation of the pAtC58 megaplasmid, ABC transporters, and cell envelope architecture. To address how ExoR exerts a broad impact on gene expression from its periplasmic location, a genetic screen was performed to isolate suppressor mutants that mitigate the exoR motility phenotype and identify downstream components of the ExoR regulatory pathway. This suppression analysis identified the acid-sensing two-component system ChvG-ChvI, and the suppressor mutant phenotypes suggest that all or most of the characteristic exoR properties are mediated through ChvG-ChvI. Subsequent analysis indicates that exoR mutants are simulating a response to acidic conditions, even in neutral media. This work expands the model for ExoR regulation in A. tumefaciens and underscores the global role that this regulator plays on gene expression. PMID:24982308

Heckel, Brynn C.; Tomlinson, Amelia D.; Morton, Elise R.; Choi, Jeong-Hyeon

2014-01-01

204

GENETIC TRANSFORMATION OF SCLEROTINIA SCLEROTIORUM THROUGH AGROBACTERIUM TUMEFACIENS-MEDIATED TRANSFORMATION.  

Technology Transfer Automated Retrieval System (TEKTRAN)

In order to study genetic factors of pathogenicity, insertional mutants of Sclerotinia sclerotiorum were generated using Agrobacterium tumefacience-mediated transformation with both mycelial fragments and ascospores. Transformants were tested for number of insertions by Southern hybridization and f...

205

Global Analysis of Differentially Expressed Genes and Proteins in the Wheat Callus Infected by Agrobacterium tumefaciens  

PubMed Central

Agrobacterium-mediated plant transformation is an extremely complex and evolved process involving genetic determinants of both the bacteria and the host plant cells. However, the mechanism of the determinants remains obscure, especially in some cereal crops such as wheat, which is recalcitrant for Agrobacterium-mediated transformation. In this study, differentially expressed genes (DEGs) and differentially expressed proteins (DEPs) were analyzed in wheat callus cells co-cultured with Agrobacterium by using RNA sequencing (RNA-seq) and two-dimensional electrophoresis (2-DE) in conjunction with mass spectrometry (MS). A set of 4,889 DEGs and 90 DEPs were identified, respectively. Most of them are related to metabolism, chromatin assembly or disassembly and immune defense. After comparative analysis, 24 of the 90 DEPs were detected in RNA-seq and proteomics datasets simultaneously. In addition, real-time RT-PCR experiments were performed to check the differential expression of the 24 genes, and the results were consistent with the RNA-seq data. According to gene ontology (GO) analysis, we found that a big part of these differentially expressed genes were related to the process of stress or immunity response. Several putative determinants and candidate effectors responsive to Agrobacterium mediated transformation of wheat cells were discussed. We speculate that some of these genes are possibly related to Agrobacterium infection. Our results will help to understand the interaction between Agrobacterium and host cells, and may facilitate developing efficient transformation strategies in cereal crops. PMID:24278131

Zhou, Xiaohong; Wang, Ke; Lv, Dongwen; Wu, Chengjun; Li, Jiarui; Zhao, Pei; Lin, Zhishan; Du, Lipu; Yan, Yueming; Ye, Xingguo

2013-01-01

206

Maize (Zea mays L.) transformation by Agrobacterium tumefaciens infection of pollinated ovules.  

PubMed

A novel transformation system was established for maize using Agrobacterium infection of in vitro cultured ovules. The maize ovules were isolated 24h after pollination and infected with Agrobacterium. The embryos were isolated from the pollinated ovules 2-3 weeks after Agrobacterium infection, regenerated to plantlets and investigated for transgene expression and inheritance. Experimental evaluations were focused on the four main aspects. Firstly, through the introduction of gus gene for monitoring transformation and development of embryo, it was confirmed that transgenic plants can be generated from in vitro cultured maize ovules infected with Agrobacterium. Secondly, in order to standardize the transformation protocol, several important factors that affected transformation efficiency were optimized. They included Agrobacterium delivery approach, surfactant, AS concentration, and cocultivation duration. Thirdly, stable expression and Mendelian inheritance of the introduced genes were analyzed in independent lines over two generations. Fourthly, the pollinated ovule culture-regeneration potential and transformation efficiency of five maize inbred lines were investigated to confirm the genotype independence of this transformation system. We conclude that the transformation system established in this study can be used to generate high-quality transgenic maize plants rapidly and directly. PMID:24333124

Chen, Liang; Cong, Yuanyuan; He, Hongxia; Yu, Ying

2014-02-10

207

Optimization of the uid A gene transfer of Rosa hybrida via Agrobacterium tumefaciens : an assessment of factors influencing the efficiency of gene transfer  

Microsoft Academic Search

To develop a transformation protocol of Rosa hybrida ‘Samantha’ via Agrobacterium tumefaciens, the authors examined the effect of different factors on T-DNA transfer by measuring transient expression levels of an intron-containing\\u000a ?-glucuronidase gene. The results indicate that explant, light condition, salt concentration and acetosyringone (AS) concentration\\u000a in co-culture medium are the most important factors, and factors like co-culture temperature, co-culture

Liping Gao; Manzhu Bao

2004-01-01

208

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

Microsoft Academic Search

The 11 gene products of the Agrobacterium tumefaciens virB operon, together with the VirD4 protein, are proposed to form a membrane complex which mediates the transfer of T-DNA to plant cells. This study examined one putative component of that complex, VirB4. A deletion of the virB4 gene on the Ti plasmid pTiA6NC was constructed by replacing the virB4 gene with

Karla Jean Fullner; Kathryn M. Stephens; Eugene W. Nester

1994-01-01

209

Clonal analysis of heterogeneous crown gall tumor tissues induced by wild-type and shooter mutant strains of Agrobacterium tumefaciens -expression of T-DNA genes  

Microsoft Academic Search

Summary Tumors were induced by anAgrobacterium tumefaciens strain with a wild-type octopine Ti plasmid and by shooter mutants with a transposon insertion in the auxin-locus of the T-region. Cloning of isolated axenic tumor tissues revealed that in all cases they consisted of tumor cells (10–26%) next to a majority of normal cells. The tumor clones that had been induced by

G. M. S. van Slogteren; J. H. C. Hoge; P. J. J. Hooykaas; R. A. Schilperoort

1983-01-01

210

Genetic analysis of the individual T-DNA genes of Agrobacterium tumefaciens ; further evidence that two genes are involved in indole-3-acetic acid synthesis  

Microsoft Academic Search

The T-DNA genes of Ti plasmids of Agrobacterium tumefaciens can induce tumorous growth on a wide range of dicotyledonous plants. We subcloned the individual onc genes of the pTiC58 T-DNA and reintroduced them in the T-region of the Ti plasmid gene vector pGV3850 (from which the onc genes had been removed (Zambryski et al. 1983)). These experiments were designed to

D. Inzé; A. Follin; M. Van Lijsebettens; C. Simoens; C. Genetello; M. Van Montagu; J. Schell

1984-01-01

211

Study of the organization of the genomes of Escherichia coli, Brucella melitensis and Agrobacterium tumefaciens by insertion of a unique restriction site  

Microsoft Academic Search

TnSMap, a Tn5 derivative containing the 18 bp I-Scel site, was delivered from a RP4mobilizable, RK6-derived suicide vector to Escherichia coli HB101, Brucella melitensis and Agrobacterium tumefaciens C58, which all lack natural I-Scel sites in their genomes. Digestion of the DNA from TnZMap-containing strains and analysis by pulsed-field gel electrophoresis (PFGE) revealed that these derivatives contained a single transposon insertion.

Estelle Jumas-Bilak; Claude Maugard; Sylvie Michaux-Charachon; Annick Allardet-Servent; Arnaud Perrin; D. O'Callaghan; M. Ramuz

1995-01-01

212

Genetic and Environmental Factors Affecting T-Pilin Export and T-Pilus Biogenesis in Relation to Flagellation of Agrobacterium tumefaciens  

Microsoft Academic Search

The T pilus, primarily composed of cyclic T-pilin subunits, is essential for the transmission of the Ti-plasmid T-DNA from Agrobacterium tumefaciens to plant cells. Although the virB2 gene of the 11-gene virB operon was previously demonstrated to encode the full-length propilin, and other genes of this operon have been implicated as members of a conserved transmembrane transport apparatus, the role

ERH-MIN LAI; OLGA CHESNOKOVA; LOIS M. BANTA; CLARENCE I. KADO

2000-01-01

213

Agrobacterium rhizogenes and A. tumefaciens co-transformation to obtain grapevine hairy roots producing the coat protein of grapevine chrome mosaic nepovirus  

Microsoft Academic Search

Hairy root cultures of grapevine were obtained from plantlets co-inoculated by virulent Agrobacterium rhizogenes strains and\\u000a disarmed A. tumefaciens strains harbouring the binary vectors pKHG4 and pKVHG 2+. These plasmids contain the nptII, hpt and\\u000a gus genes and differ for the presence of the gene encoding for the grapevine chrome mosaic virus coat protein. For the cultivar\\u000a ‘Gravesac’, 72% of

L. Torregrosa; A. Bouquet

1997-01-01

214

High efficiency Agrobacterium tumefaciens -mediated transformation of Arabidopsis thaliana leaf and cotyledon explants  

Microsoft Academic Search

A highly efficient and fast Agrobacterium-mediated leaf disc transformation system for the Arabidopsis thaliana L. genotype C24 was developed. This protocol is also amenable to other ecotypes - as could be shown for Landsberg erecta and Wassllewskija. Besides the hygromycin selection also the G418 and kanamycin selection were established. Furthermore the described procedure is appliable not only to leaf explants

Renate Schmidt; Lothar Willmitzer

1988-01-01

215

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

216

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

217

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

SciTech Connect

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. The authors overproduced truncated VirA proteins in Escherichia coli be deleting different lengths of the 5{prime}-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}-{sup 32}P)ATP but not with ({gamma}-{sup 32}P)GTP or ({alpha}-{sup 32}P)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.

Jin, S.; Roitsch, T.; Ankenbauer, R.G.; Gordon, M.P.; Nester, E.W. (Univ. of Washington, Seattle (USA))

1990-02-01

218

Antiparallel and Interlinked Control of Cellular Iron Levels by the Irr and RirA Regulators of Agrobacterium tumefaciens?†  

PubMed Central

The plant pathogen Agrobacterium tumefaciens encodes predicted iron-responsive regulators, Irr and RirA, that function in several other bacteria to control the response to environmental iron levels. Deletion mutations of irr and rirA, alone and in combination, were evaluated for their impact on cellular iron response. Growth was severely diminished in the ?irr mutant under iron-limiting conditions, but reversed to wild-type levels in an ?irr ?rirA mutant. The level of uncomplexed iron in the ?irr mutant was decreased, whereas the ?rirA mutant exhibited elevated iron levels. Sensitivity of the ?irr and ?rirA mutants to iron-activated antimicrobial compounds generally reflected their uncomplexed-iron levels. Expression of genes that encode iron uptake systems was decreased in the ?irr mutant, whereas that of iron utilization genes was increased. Irr function required a trihistidine repeat likely to mediate interactions with heme. Iron uptake genes were derepressed in the ?rirA mutant. In the ?irr ?rirA mutant, iron uptake and utilization genes were derepressed, roughly combining the phenotypes of the single mutants. Siderophore production was elevated in the rirA mutant, but most strongly regulated by an RirA-controlled sigma factor. Expression of rirA itself was regulated by Irr, RirA, and iron availability, in contrast to irr expression, which was relatively stable in the different mutants. These studies suggest that in A. tumefaciens, the Irr protein is most active under low-iron conditions, inhibiting iron utilization and activating iron acquisition, while the RirA protein is active under high-iron conditions, repressing iron uptake. PMID:21602352

Hibbing, Michael E.; Fuqua, Clay

2011-01-01

219

Temperature Affects the T-DNA Transfer Machinery of Agrobacterium tumefaciens  

Microsoft Academic Search

Early studies onAgrobacterium tumefaciensshowed that development of tumors on plants following infection byA. tumefacienswas optimal at temperatures around 22&C and did not occur at temperatures above 29&C. To assesswhetherthisinabilitytoinducetumorsisduetoadefectintheT-DNAtransfermachinery,mobilization of an incompatibility group Q (IncQ) plasmid by the T-DNA transfer machinery ofA. tumefacienswas tested at various temperatures. Optimal transfer occurred when matings were performed at 19&C, and transfer was not

KARLA JEAN FULLNER; ANDEUGENE W. NESTER

1996-01-01

220

Genotype-independent leaf disc transformation of potato ( Solanum tuberosum ) using Agrobacterium tumefaciens  

Microsoft Academic Search

Leaves of the in vitro grown potato cultivars ‘Bintje’, ‘Berolina’, ‘Desiree’, and ‘Russet Burbank’ were wounded and co-cultivated with Agrobacterium strains having chimeric bar and nptII genes on a disarmed T-DNA. Each leaf from these cultivars formed numerous calli on kanamycin-containing medium, and almost all calli regenerated shoots. For ‘Russet Burbank’, it was necessary to include AgNO3 in the medium

M. Block

1988-01-01

221

Factors affecting Agrobacterium tumefaciens -mediated genetic transformation of Lycium barbarum L  

Microsoft Academic Search

Summary  Using the system for genetic transformation and transgenic plant regeneration via somatic embryogenesis (SE) of Lycium barbarum established in this laboratory, this study reports the optimization of the factors affecting the efficiency of transformation,\\u000a including pre-culture period, leaf explant source, use of acetosyringone, strains and density of Agrobacterium, and temperature of co-cultivation. The optimized transformation protocol for L. barbarum included

Zhong Hu; Yi-Rui Wu; Wei Li; Huan-Huan Gao

2006-01-01

222

Altered Growth and Wood Characteristics in Transgenic Hybrid Aspen Expressing Agrobacterium tumefaciens T-DNA Indoleacetic Acid-Biosynthetic Genes.  

PubMed Central

A key regulator of cambial growth is the plant hormone indoleacetic acid (IAA). Here we report on altered wood characteristics and growth patterns in transgenic hybrid aspen (Populus tremula L. x Populus tremuloides Michx.) expressing Agrobacterium tumefaciens T-DNA IAA-biosynthetic iaaM and iaaH genes. Eighteen lines simultaneously expressing both genes were regenerated. Of these, four lines, verified to be transgenic by northern blot analysis, were selected and raised under controlled growth conditions. All four lines were affected in their growth patterns, including alterations in height and stem diameter growth, internode elongation, leaf enlargement, and degree of apical dominance. Two transgenic lines, showing the most distinct phenotypic deviation from the wild type, were characterized in more detail for free and conjugated IAA levels and for wood characteristics. Both lines showed an altered IAA balance, particularly in mature leaves and roots where IAA levels were elevated. They also exhibited changes in wood anatomy, most notably a reduction in vessel size, an increase in vessel density, and changes in ray development. Thus, the recent development of techniques for gene transfer to forest trees enabled us to investigate the influence of an altered IAA balance on xylem development in an intact experimental system. In addition, the results demonstrate the possibility of manipulating wood properties in a forest tree through controlled changes of IAA concentration and distribution. PMID:12228661

Tuominen, H.; Sitbon, F.; Jacobsson, C.; Sandberg, G.; Olsson, O.; Sundberg, B.

1995-01-01

223

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

224

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

225

T-DNA and opine synthetic loci in tumors incited by Agrobacterium tumefaciens A281 on soybean and alfalfa plants  

SciTech Connect

A report is given of the molecular characterization of transferred DNA (T-DNA) in leguminous tumors incited by Agrobacterium tumefaciens A281 harboring the tumor-inducing plasmid pTiBo542. The T-DNA is composed of two regions named T/sub L/ (left portion)-DNA and T/sub R/ (right portion)-DNA, in accordance with the nomenclature for the octopine strains. T/sub L/-DNA is defined by several internal HindIII restriction fragments totaling 10.8 kilobase pairs (kbp) in uncloned soybean and alfalfa tumors. Alfalfa tumor DNA may contain one more HindIII fragment at the left end of T/sub L/-DNA that does soybean tumor DNA. T/sub R/-DNA has a 5.8-kbp BamHI-EcoRi internal fragment. All borders other than the left border of T/sub L/-DNA appear to be the same within the detection limits of Southern blot hybridization experiments. The two T-DNA regions are separated by 16 to 19 kbp of DNA not stably maintained in tumors. The distance from the left border of T/sub L/-DNA to the right border of T/sub R/-DNA is approximately 40 kbp. Loci for the mannityl opines are situated in T/sub R/-DNA, based on genetic and biochemical criteria.

Hood, E.E.; Chilton, W.S.; Chilton, M.D.; Fraley, R.T.

1986-12-01

226

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

227

Expression, characterization, and improvement of a newly cloned halohydrin dehalogenase from Agrobacterium tumefaciens and its application in production of epichlorohydrin.  

PubMed

A gene encoding halohydrin dehalogenase (HHDH) from Agrobacterium tumefaciens CCTCC M 87071 was cloned and expressed in Escherichia coli. To increase activity and stability of HHDH, 14 amino acid residues around the active site and substrate-binding pocket based on the structural analysis and molecular docking were selected as targets for site-directed mutagenesis. The studies showed that the mutant HHDH (Mut-HHDH) enzyme had a more accessible substrate-binding pocket than the wild-type HHDH (Wt-HHDH). Molecular docking revealed that the distance between the substrate and active site was closer in mutant which improved the catalytic activity. The expressed Wt-HHDH and Mut-HHDH were purified and characterized using 1,3-dichloro-2-propanol (1,3-DCP) as substrates. The specific activity of the mutant was enhanced 26-fold and the value of k cat was 18.4-fold as compared to the Wt-HHDH, respectively. The Mut-HHDH showed threefold extension of half-life at 45 °C than that of Wt-HHDH. Therefore it is possible to add 1,3-DCP concentration up to 100 mM and epichlorohydrin (ECH) was produced at a relatively high conversion and yield (59.6 %) using Mut-HHDH as catalyst. This Mut-HHDH could be a potential candidate for the upscale production of ECH. PMID:24777710

Liu, Zhi-Qiang; Gao, Ai-Cun; Wang, Ya-Jun; Zheng, Yu-Guo; Shen, Yin-Chu

2014-07-01

228

Covalently bound VirD2 protein of Agrobacterium tumefaciens protects the T-DNA from exonucleolytic degradation.  

PubMed Central

We show that upon induction of Agrobacterium tumefaciens, free linear double-stranded T-DNA molecules as well as the previously described T-strands are generated from the Ti plasmid. A majority of these molecules are bound to a protein. We show that this protein is the product of the virulence gene virD2. This protein was found to be attached to the 5' terminus of processed T-DNA at the right border and to the rest of the Ti plasmid at the left border. The protein remnant after Pronase digestion rendered the right end of the double-stranded T-DNA resistant to 5'----3' exonucleolytic attack in vitro. The protein-DNA association was resistant to SDS, mercaptoethanol, mild alkali, piperidine, and hydroxylamine, indicating that it involves a covalent linkage. The possible involvement of this T-DNA-protein complex in replication, transduction to the plant, nuclear targeting, and integration into the plant nuclear DNA is discussed. Images PMID:2556703

Dürrenberger, F; Crameri, A; Hohn, B; Koukolíková-Nicola, Z

1989-01-01

229

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

230

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

231

Potential Application of N-Carbamoyl-?-Alanine Amidohydrolase from Agrobacterium tumefaciens C58 for ?-Amino Acid Production?  

PubMed Central

An N-carbamoyl-?-alanine amidohydrolase of industrial interest from Agrobacterium tumefaciens C58 (?carAt) has been characterized. ?carAt is most active at 30°C and pH 8.0 with N-carbamoyl-?-alanine as a substrate. The purified enzyme is completely inactivated by the metal-chelating agent 8-hydroxyquinoline-5-sulfonic acid (HQSA), and activity is restored by the addition of divalent metal ions, such as Mn2+, Ni2+, and Co2+. The native enzyme is a homodimer with a molecular mass of 90 kDa from pH 5.5 to 9.0. The enzyme has a broad substrate spectrum and hydrolyzes nonsubstituted N-carbamoyl-?-, -?-, -?-, and -?-amino acids, with the greatest catalytic efficiency for N-carbamoyl-?-alanine. ?carAt also recognizes substrate analogues substituted with sulfonic and phosphonic acid groups to produce the ?-amino acids taurine and ciliatine, respectively. ?carAt is able to produce monosubstituted ?2- and ?3-amino acids, showing better catalytic efficiency (kcat/Km) for the production of the former. For both types of monosubstituted substrates, the enzyme hydrolyzes N-carbamoyl-?-amino acids with a short aliphatic side chain better than those with aromatic rings. These properties make ?carAt an outstanding candidate for application in the biotechnology industry. PMID:19011069

Martínez-Gómez, Ana Isabel; Martínez-Rodríguez, Sergio; Pozo-Dengra, Joaquín; Tessaro, Davide; Servi, Stefano; Clemente-Jiménez, Josefa María; Rodríguez-Vico, Felipe; Las Heras-Vázquez, Francisco Javier

2009-01-01

232

Potential application of N-carbamoyl-beta-alanine amidohydrolase from Agrobacterium tumefaciens C58 for beta-amino acid production.  

PubMed

An N-carbamoyl-beta-alanine amidohydrolase of industrial interest from Agrobacterium tumefaciens C58 (beta car(At)) has been characterized. Beta car(At) is most active at 30 degrees C and pH 8.0 with N-carbamoyl-beta-alanine as a substrate. The purified enzyme is completely inactivated by the metal-chelating agent 8-hydroxyquinoline-5-sulfonic acid (HQSA), and activity is restored by the addition of divalent metal ions, such as Mn(2+), Ni(2+), and Co(2+). The native enzyme is a homodimer with a molecular mass of 90 kDa from pH 5.5 to 9.0. The enzyme has a broad substrate spectrum and hydrolyzes nonsubstituted N-carbamoyl-alpha-, -beta-, -gamma-, and -delta-amino acids, with the greatest catalytic efficiency for N-carbamoyl-beta-alanine. Beta car(At) also recognizes substrate analogues substituted with sulfonic and phosphonic acid groups to produce the beta-amino acids taurine and ciliatine, respectively. Beta car(At) is able to produce monosubstituted beta(2)- and beta(3)-amino acids, showing better catalytic efficiency (k(cat)/K(m)) for the production of the former. For both types of monosubstituted substrates, the enzyme hydrolyzes N-carbamoyl-beta-amino acids with a short aliphatic side chain better than those with aromatic rings. These properties make beta car(At) an outstanding candidate for application in the biotechnology industry. PMID:19011069

Martínez-Gómez, Ana Isabel; Martínez-Rodríguez, Sergio; Pozo-Dengra, Joaquín; Tessaro, Davide; Servi, Stefano; Clemente-Jiménez, Josefa María; Rodríguez-Vico, Felipe; Las Heras-Vázquez, Francisco Javier

2009-01-01

233

GxySBA ABC Transporter of Agrobacterium tumefaciens and Its Role in Sugar Utilization and vir Gene Expression  

PubMed Central

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

2014-01-01

234

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

235

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

236

Ti plasmid-specified chemotaxis of Agrobacterium tumefaciens C58C1 toward vir-inducing phenolic compounds and soluble factors from monocotyledonous and dicotyledonous plants.  

PubMed Central

Twelve phenolic compounds with related structures were analyzed for their ability to act as chemoattractants for Agrobacterium tumefaciens C58C1 and as inducers of the Ti plasmid virulence operons. The results divided the phenolic compounds into three groups: compounds that act as strong vir inducers and are chemoattractants for A. tumefaciens C58C1 harboring the nopaline Ti plasmid pDUB1003 delta 31, but not the isogenic cured strain; compounds that are at best weak vir inducers and are weak chemoattractants for Ti plasmid-harboring and cured A. tumefaciens C58C1; and compounds that are vir noninducers and are also nonattractants. A strong correlation between vir-inducing ability and Ti plasmid requirement for chemotaxis is thus established. In addition, chemical structure rules for vir induction and chemotaxis are outlined. Positive chemotaxis toward root and shoot homogenates from monocotyledonous and dicotyledonous plants was observed. At low extract concentrations, chemotaxis was enhanced by the presence of Ti plasmid. The chemoattractants do not derive from intact cell walls. Lack of attraction is not responsible for the apparent block to monocot transformation by A. tumefaciens. PMID:3410827

Ashby, A M; Watson, M D; Loake, G J; Shaw, C H

1988-01-01

237

DNA modifying enzymes of Agrobacterium tumefaciens: effect of DNA topoisomerase, restriction endonuclease, and unique DNA endonuclease on plasmid and plant DNA.  

PubMed Central

Extracts from Agrobacterium tumefaciens strain ID135 contain three enzymes that have been characterized and partially purified. The first enzyme, a DNA topoisomerase, appeared to relax only negatively twisted DNA. The second enzyme, Atu I, a type II restriction endonuclease, generated the identical DNA digestion pattern as EcoRII when several DNAs were used. The third enzyme, endonuclease A, showed a preference for superhelical DNAs as substrates. When plasmid pCK135DNA, obtained from the virulent strain IDI135 of A. tumefaciens, or plant DNA was exposed to the three enzymes, changes in DNA patterns were observed due to either conformational changes or digestion of the DNAs. These enzymes may function in vivo in the processing and incorporation of bacterial DNA in plant cells. Images PMID:212732

LeBon, J M; Kado, C I; Rosenthal, L J; Chirikjian, J G

1978-01-01

238

Transformation of passionfruit (Passiflora edulis fv flavicarpa Degener.) using Agrobacterium tumefaciens.  

PubMed

Leaf and stem explants of passionfruit (Passiflora eadulis fv flavicarpa) were co-cultivated with a disarmed strain of Agrobacterium tunefaciens harbouring the co-integrate vector pMON200. Four plants of passionfruit were regenerated from leaf explants on agar-solidified Murashige and Skoog (1962) based medium containing 4.43 ?M 6-benzyl-aminopurine and supplemented with 86 ?M kanamycin sulphate. The four plants were rooted by transfer to MS based medium with 14.7 ?M 3-indolebutyric acid and 2.68 ?M ?-naphthyleneacetic acid for 7 d, followed by MS based medium lacking growth regulators. Both media used for rooting contained 172 ?M kanamycin sulphate. Rooted plants were potted and grown to maturity. Three of the plants synthesised nopaline and expressed neomycin phosphotransferase activity; DNA dot blot and polymerase chain reaction analyses confirmed the presence of the neomycin phosphotransferase gene in three plants. PMID:24193523

Manders, G; Otoni, W C; d'Utra Vaz, F B; Blackball, N W; Power, J B; Davey, M R

1994-09-01

239

Genotype-independent leaf disc transformation of potato (Solanum tuberosum) using Agrobacterium tumefaciens.  

PubMed

Leaves of the in vitro grown potato cultivars 'Bintje', 'Berolina', 'Desiree', and 'Russet Burbank' were wounded and co-cultivated with Agrobacterium strains having chimeric bar and nptII genes on a disarmed T-DNA. Each leaf from these cultivars formed numerous calli on kanamycin-containing medium, and almost all calli regenerated shoots. For 'Russet Burbank', it was necessary to include AgNO3 in the medium to obtain efficient shoot regeneration. The transformed plants have one to a few copies of the T-DNA, show NPT-II and PAT activities, and are resistant to high doses of the commercial preparation of phospinotricin (glufosinate). Almost no somaclonal variation was detected in trans-genic plants. PMID:24232356

De Block, M

1988-11-01

240

Agrobacterium tumefaciens-Mediated Transformation of Valsa mali: An Efficient Tool for Random Insertion Mutagenesis  

PubMed Central

Valsa mali is a causal agent of apple and pear trees canker disease, which is a destructive disease that causes serious economic losses in eastern Asia, especially in China. The lack of an efficient transformation system for Valsa mali retards its investigation, which poses difficulties to control the disease. In this research, a transformation system for this pathogen was established for the first time using A. tumefaciens-mediated transformation (ATMT), with the optimal transformation conditions as follows: 106/mL conidia suspension, cocultivation temperature 22°C, cocultivation time 72 hours, and 200??M acetosyringone (AS) in the inductive medium. The average transformation efficiency was 1015.00 ± 37.35 transformants per 106 recipient conidia. Thirty transformants were randomly selected for further confirmation and the results showed the presence of T-DNA in all hygromycin B resistant transformants and also revealed random and single gene integration with genetic stability. Compared with wild-type strain, those transformants exhibited various differences in morphology, conidia production, and conidia germination ability. In addition, pathogenicity assays revealed that 14 transformants had mitigated pathogenicity, while one had enhanced infection ability. The results suggest that ATMT of V. mali is a useful tool to gain novel insight into this economically important pathogen at molecular levels. PMID:24381526

Wang, Caixia; Guan, Xiangnan; Wang, Hanyan; Li, Guifang; Dong, Xiangli; Wang, Guoping

2013-01-01

241

Analogs of the Autoinducer 3-Oxooctanoyl-Homoserine Lactone Strongly Inhibit Activity of the TraR Protein of Agrobacterium tumefaciens  

Microsoft Academic Search

The TraR and TraI proteins of Agrobacterium tumefaciens mediate cell-density-dependent expression of the Ti plasmid tra regulon. TraI synthesizes the autoinducer pheromone N-(3-oxooctanoyl)-L-homoserine lactone (3-oxo-C8-HSL), while TraR is an 3-oxo-C8-HSL-responsive transcriptional activator. We have compared the abilities of 3-oxo-C8-HSL and 32 related compounds to activate expression of a TraR-regulated promoter. In a strain that expresses wild-type levels of TraR, only

JUN ZHU; JOHN W. BEABER; MARGRET I. MORE; CLAY FUQUA; ANATOL EBERHARD; STEPHEN C. WINANS

1998-01-01

242

Molecular basis of ChvE function in sugar binding, sugar utilization, and virulence in Agrobacterium tumefaciens.  

PubMed

ChvE is a chromosomally encoded protein in Agrobacterium tumefaciens that mediates a sugar-induced increase in virulence (vir) gene expression through the activities of the VirA/VirG two-component system and has also been suggested to be involved in sugar utilization. The ChvE protein has homology to several bacterial periplasmic sugar-binding proteins, such as the ribose-binding protein and the galactose/glucose-binding protein of Escherichia coli. In this study, we provide direct evidence that ChvE specifically binds the vir gene-inducing sugar d-glucose with high affinity. Furthermore, ChvE mutations resulting in altered vir gene expression phenotypes have been isolated and characterized. Three distinct categories of mutants have been identified. Strains expressing the first class are defective in both virulence and d-glucose utilization as a result of mutations to residues lining the sugar-binding cleft. Strains expressing a second class of mutants are not adversely affected in sugar binding but are defective in virulence, presumably due to impaired interactions with the sensor kinase VirA. A subset of this second class of mutants includes variants of ChvE that also result in defective sugar utilization. We propose that these mutations affect not only interactions with VirA but also interactions with a sugar transport system. Examination of a homology model of ChvE shows that the mutated residues associated with the latter two phenotypes lie in two overlapping solvent-exposed sites adjacent to the sugar-binding cleft where conformational changes associated with the binding of sugar might have a maximal effect on ChvE's interactions with its distinct protein partners. PMID:19633083

He, Fanglian; Nair, Gauri R; Soto, Cinque S; Chang, Yehchung; Hsu, Lillian; Ronzone, Erik; DeGrado, William F; Binns, Andrew N

2009-09-01

243

Genetic transformation of selected mature cork oak ( Quercus suber L.) trees  

Microsoft Academic Search

A transformation system for selected mature cork oak ( Quercus suber L.) trees using Agrobacterium tumefaciens has been established. Embryos obtained from recurrent proliferating embryogenic masses were inoculated with A. tumefaciens strains EHA105, LBA4404 or AGL1 harbouring the plasmid pBINUbiGUSint [carrying the neomycin phosphotransferase II ( nptII) and ?-glucuronidase ( uidA) genes]. The highest transformation efficiency (4%) was obtained when freshly

R. Álvarez; P. Alonso; M. Cortizo; C. Celestino; I. Hernández; M. Toribio; R. J. Ordás

2004-01-01

244

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

245

Small high-yielding binary Ti vectors pLSU with co-directional replicons for Agrobacterium tumefaciens-mediated transformation of higher plants.  

PubMed

Small high-yielding binary Ti vectors of Agrobacterium tumefaciens were constructed to increase the cloning efficiency and plasmid yield in Escherichia coli and A. tumefaciens for transformation of higher plants. We reduced the size of the binary vector backbone to 4566bp with ColE1 replicon (715bp) for E. coli and VS1 replicon (2654bp) for A. tumefaciens, a bacterial kanamycin resistance gene (999bp), and the T-DNA region (152bp). The binary Ti vectors with the truncated VS1 replicon were stably maintained with more than 98% efficiency in A. tumefaciens without antibiotic selection for 4 days of successive transfers. The transcriptional direction of VS1 replicon can be the same as that of ColE1 replicon (co-directional transcription), or opposite (head-on transcription) as in the case of widely used vectors (pPZP or pCambia). New binary vectors with co-directional transcription yielded in E. coli up to four-fold higher transformation frequency than those with the head-on transcription. In A. tumefaciens the effect of co-directional transcription is still positive in up to 1.8-fold higher transformation frequency than that of head-on transcription. Transformation frequencies of new vectors are over six-fold higher than those of pCambia vector in A. tumefaciens. DNA yields of new vectors were three to five-fold greater than pCambia in E. coli. The proper functions of the new T-DNA borders and new plant selection marker genes were confirmed after A. tumefaciens-mediated transformation of tobacco leaf discs, resulting in virtually all treated leaf discs transformed and induced calli. Genetic analysis of kanamycin resistance trait among the progeny showed that the kanamycin resistance and sensitivity traits were segregated into the 3:1 ratio, indicating that the kanamycin resistance genes were integrated stably into a locus or closely linked loci of the nuclear chromosomal DNA of the primary transgenic tobacco plants and inherited to the second generation. PMID:22404832

Lee, Seokhyun; Su, Guiying; Lasserre, Eric; Aghazadeh, Monty Arta; Murai, Norimoto

2012-05-01

246

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

247

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

248

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

Microsoft Academic Search

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\\u000a transformation were evaluated. Agrobacterium at a concentration of OD = 0.5–0.8 improved the efficiency of transformation.

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

2009-01-01

249

Large Deletions in the pAtC58 Megaplasmid of Agrobacterium tumefaciens Can Confer Reduced Carriage Cost and Increased Expression of Virulence Genes  

PubMed Central

The accessory plasmid pAtC58 of the common laboratory strain of Agrobacterium tumefaciens confers numerous catabolic functions and has been proposed to play a role in virulence. Genomic sequencing of evolved laboratory strains of A. tumefaciens revealed the presence of multiple deletion events in the At plasmid, with reductions in plasmid size ranging from 25% to 30% (115–194 kb). Flanking both ends of the sites of these deletions is a short-nucleotide repeat sequence that is in a single copy in the deleted plasmids, characteristic of a phage- or transposon-mediated deletion event. This repeat sequence is widespread throughout the C58 genome, but concentrated on the At plasmid, suggesting its frequency to be nonrandom. In this study, we assess the prevalence of the larger of these deletions in multiple C58 derivatives and characterize its functional significance. We find that in addition to elevating virulence gene expression, this deletion is associated with a significantly reduced carriage cost to the cell. These observations are a clear demonstration of the dynamic nature of the bacterial genome and suggest a mechanism for genetic plasticity of these costly but otherwise stable plasmids. Additionally, this phenomenon could be the basis for some of the dramatic recombination events so ubiquitous within and among megaplasmids. PMID:23783172

Morton, Elise R.; Merritt, Peter M.; Bever, James D.; Fuqua, Clay

2013-01-01

250

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

251

Production of pineapple transgenic plants assisted by temporary immersion bioreactors  

Microsoft Academic Search

A procedure for producing pineapple [Ananas comosus (L.) Merr.] transgenic plants was developed that involved selection by micropropagation in temporary immersion bioreactors (TIBs). Pineapple calluses ranging in size from 1.5 mm to 2.0 mm that were co-cultivated with Agrobacterium tumefaciens strains AT2260 (pIG121Hm) and LBA4404 (pTOK233) for 24 h produced the highest percentage (40%) of GUS+ calluses. Phosphinothricin and hygromycin,

P. Espinosa; J. Lorenzo; A. Iglesias; L. Yabor; E. Menéndez; J. Borroto; L. Hernández; A. Arencibia

2002-01-01

252

Effect of over-expression of Linum usitatissimum PINORESINOL LARICIRESINOL REDUCTASE ( LuPLR ) gene in transgenic Phyllanthus amarus  

Microsoft Academic Search

Shoot tip explants of Phyllanthus amarus were cocultivated with Agrobacterium tumefaciens strain LBA 4404 carrying plasmid pCAMBIA 2301 harbouring genes coding for betaglucuronidase (gus), kanamycin (kan), and neomycin\\u000a phosphotransferase II (nptII) along with a gene coding for Linum usitatissimum PINORESINOL LARICIRESINOL REDUCTASE (Lu-PLR). Transformed shoot tip explants were maintained in a Murashige and Skoog (MS) medium containing TDZ 1.54 mg l?1, kan

Anindita Banerjee; Sharmila Chattopadhyay

2010-01-01

253

Morphological changes in transgenic poplar induced by expression of the rice homeobox gene OSH1  

Microsoft Academic Search

Genetically transformed lombardy poplar (Po-pulus nigra L. var. italica Koehne) plants were regenerated after co-cultivation of stem segments with Agrobacterium tumefaciens strain LBA4404 that harbored a binary vector which included the rice gene for a homeodomain protein (OSH1) and a gene for\\u000a neomycin phosphotransferase. The expression of the OSH1 gene under control of the cauliflower mosaic virus 35S promoter induced

T. Mohri; T. Igasaki; N. Futamura; K. Shinohara

1999-01-01

254

Transgenic indica rice cv. ADT 43 expressing a ? 1 -pyrroline-5-carboxylate synthetase ( P5CS ) gene from Vigna aconitifolia demonstrates salt tolerance  

Microsoft Academic Search

To develop salt tolerant rice, the P5CS gene of Vigna aconitifolia, encoding for proline synthesis, was introduced into the popular indica rice cultivar ADT 43. Agrobacterium tumefaciens strain LBA 4404 harboring the binary vector pCAMBIA 1301\\/P5CS, carrying the proline synthesis encoding gene P5CS, was co-cultivated with embryogenic callus of rice. Adding 100 ?M acetosyringone to the Linsmaier and Skoog (LS) liquid

Alagarsamy Karthikeyan; Shumugiah Karutha Pandian; Manikandan Ramesh

255

Cadmium resistance in transgenic tobacco plants enhanced by expressing bean heavy metal-responsive gene PvSR2  

Microsoft Academic Search

PvSR2 (Phaseolus vulgaris stress-related gene) has been cloned from French bean and shown to be expressed specifically upon heavy metal treatment.\\u000a In order to investigate the role of PvSR2 in plant, PvSR2 gene under the control of cauliflower mosaic virus 35S promoter was introduced into tobacco mediated with Agrobacterium tumefaciens LBA4404. The regenerated plantlets were selected on medium with 100

Tuanyao Chai; Qiong Chen; Yuxiu Zhang; Juan Dong; Chengcai An

2003-01-01

256

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

257

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

258

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

259

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

260

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

261

Development of an efficient transformation method by Agrobacterium tumefaciens and high throughput spray assay to identify transgenic plants for woodland strawberry (Fragaria vesca) using NPTII selection.  

PubMed

KEY MESSAGE : We developed an efficient Agrobacterium -mediated transformation method using an Ac/Ds transposon tagging construct for F. vesca and high throughput paromomycin spray assay to identify its transformants for strawberry functional genomics. Genomic resources for Rosaceae species are now readily available, including the Fragaria vesca genome, EST sequences, markers, linkage maps, and physical maps. The Rosaceae Genomic Executive Committee has promoted strawberry as a translational genomics model due to its unique biological features and transformability for fruit trait improvement. Our overall research goal is to use functional genomic and metabolic approaches to pursue high throughput gene discovery in the diploid woodland strawberry. F. vesca offers several advantages of a fleshy fruit typical of most fruit crops, short life cycle (seed to seed in 12-16 weeks), small genome size (206 Mbb/C), small plant size, self-compatibility, and many seeds per plant. We have developed an efficient Agrobacterium tumefaciens-mediated strawberry transformation method using kanamycin selection, and high throughput paromomycin spray assay to efficiently identify transgenic strawberry plants. Using our kanamycin transformation method, we were able to produce up to 98 independent kanamycin resistant insertional mutant lines using a T-DNA construct carrying an Ac/Ds transposon Launchpad system from a single transformation experiment involving inoculation of 22 leaf explants of F. vesca accession 551572 within approx. 11 weeks (from inoculation to soil). Transgenic plants with 1-2 copies of a transgene were confirmed by Southern blot analysis. Using our paromomycin spray assay, transgenic F. vesca plants were rapidly identified within 10 days after spraying. PMID:23160638

Pantazis, Christopher J; Fisk, Sarah; Mills, Kerri; Flinn, Barry S; Shulaev, Vladimir; Veilleux, Richard E; Dan, Yinghui

2013-03-01

262

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

263

Genetic analysis of the agrocinopine catabolic region of Agrobacterium tumefaciens Ti plasmid pTiC58, which encodes genes required for opine and agrocin 84 transport.  

PubMed Central

The acc region, subcloned from pTiC58 of classical nopaline and agrocinopine A and B Agrobacterium tumefaciens C58, allowed agrobacteria to grow using agrocinopine B as the sole source of carbon and energy. acc is approximately 6 kb in size. It consists of at least five genes, accA through accE, as defined by complementation analysis using subcloned fragments and transposon insertion mutations of acc carried on different plasmids within the same cell. All five regions are required for agrocin 84 sensitivity, and at least four are required for agrocinopine and agrocin 84 uptake. The complementation results are consistent with the hypothesis that each of the five regions is separately transcribed. Maxicell experiments showed that the first of these genes, accA, encodes a 60-kDa protein. Analysis of osmotic shock fractions showed this protein to be located in the periplasm. The DNA sequence of the accA region revealed an open reading frame encoding a predicted polypeptide of 59,147 Da. The amino acid sequence encoded by this open reading frame is similar to the periplasmic binding proteins OppA and DppA of Escherichia coli and Salmonella typhimurium and OppA of Bacillus subtilis. Images PMID:8366042

Hayman, G T; Beck von Bodman, S; Kim, H; Jiang, P; Farrand, S K

1993-01-01

264

Agrobacterium tumefaciens-mediated transgenic plant and somaclone production through direct and indirect regeneration from leaves in Stevia rebaudiana with their glycoside profile.  

PubMed

Agrobacterium tumefaciens (EHA-105 harboring pCAMBIA 1304)-mediated transgenic plant production via direct regeneration from leaf and elite somaclones generation through indirect regeneration in Stevia rebaudiana is reported. Optimum direct regeneration frequency along with highest transformation frequency was found on MS?+?1 mg/l BAP?+?1 mg/l NAA, while indirect regeneration from callus was obtained on MS?+?1 mg/l BAP?+?2 mg/l NAA. Successful transfer of GUS-positive (GUS assay and PCR-based confirmation) transgenic as well as four somaclones up to glasshouse acclimatization has been achieved. Inter-simple sequence repeat (ISSR) profiling of transgenic and somaclonal plants showed a total of 113 bands, out of which 49 were monomorphic (43.36 %) and 64 were polymorphic (56.64 %). Transgenic plant was found to be closer to mother plant, while on the basis of steviol, stevioside, and rebaudioside A profile, somaclone S2 was found to be the best and showed maximum variability in ISSR profiling. PMID:24154495

Khan, Shamshad Ahmad; Ur Rahman, Laiq; Shanker, Karuna; Singh, Manju

2014-05-01

265

Evidence for Host Genome Involvement in Cytokinin Metabolism by Male and Female Cells of Mercurialis annua Transformed by Strain 15,955 of Agrobacterium tumefaciens  

PubMed Central

When male and female individuals of a dioecious species Mercurialis annua L. were inoculated with the same strain of Agrobacterium tumefaciens (15,955), the corresponding tumor tissues of each sex clearly differed in their endogenous cytokinin content; only the male tumors had a morphogenetic feminizing effect on male flowers. In male tumor tissues, zeatin (Z) in higher quantity than ribosyl-zeatin (RZ) became the major metabolite in contrast with the general situation for crown-galls; the female tumor tissues were characterized by an increase of total endogenous cytokinins and by the appearance of some specific metabolites such as a methyl-thio-Z and several glycosylated Z derivatives that had not been detected in healthy apices. In both male and female tumor tissues, the cis form of RZ, present in healthy apices as 30% of trans-RZ form, was no longer detectable. Quantitative and qualitative differences characterize male and female tumor tissues (host genes expression) but since differences also appeared between healthy male and female apices and their corresponding tumor tissues (TDNA gene expression), it can be tentatively concluded that a complex interaction between host cytokinin genes and those of TDNA control the endogenous metabolism of tumor tissues. Images Fig. 1 PMID:16663368

Guerin, B.; Kahlem, G.; Teller, G.; Durand, Bernard

1984-01-01

266

Genetic analysis of the agrocinopine catabolic region of Agrobacterium tumefaciens Ti plasmid pTiC58, which encodes genes required for opine and agrocin 84 transport.  

PubMed

The acc region, subcloned from pTiC58 of classical nopaline and agrocinopine A and B Agrobacterium tumefaciens C58, allowed agrobacteria to grow using agrocinopine B as the sole source of carbon and energy. acc is approximately 6 kb in size. It consists of at least five genes, accA through accE, as defined by complementation analysis using subcloned fragments and transposon insertion mutations of acc carried on different plasmids within the same cell. All five regions are required for agrocin 84 sensitivity, and at least four are required for agrocinopine and agrocin 84 uptake. The complementation results are consistent with the hypothesis that each of the five regions is separately transcribed. Maxicell experiments showed that the first of these genes, accA, encodes a 60-kDa protein. Analysis of osmotic shock fractions showed this protein to be located in the periplasm. The DNA sequence of the accA region revealed an open reading frame encoding a predicted polypeptide of 59,147 Da. The amino acid sequence encoded by this open reading frame is similar to the periplasmic binding proteins OppA and DppA of Escherichia coli and Salmonella typhimurium and OppA of Bacillus subtilis. PMID:8366042

Hayman, G T; Beck von Bodman, S; Kim, H; Jiang, P; Farrand, S K

1993-09-01

267

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

268

Genetic transformation of Fusarium avenaceum by Agrobacterium tumefaciens mediated transformation and the development of a USER-Brick vector construction system  

PubMed Central

Background The plant pathogenic and saprophytic fungus Fusarium avenaceum causes considerable in-field and post-field losses worldwide due to its infections of a wide range of different crops. Despite its significant impact on the profitability of agriculture production and a desire to characterize the infection process at the molecular biological level, no genetic transformation protocol has yet been established for F. avenaceum. In the current study, it is shown that F. avenaceum can be efficiently transformed by Agrobacterium tumefaciens mediated transformation. In addition, an efficient and versatile single step vector construction strategy relying on Uracil Specific Excision Reagent (USER) Fusion cloning, is developed. Results The new vector construction system, termed USER-Brick, is based on a limited number of PCR amplified vector fragments (core USER-Bricks) which are combined with PCR generated fragments from the gene of interest. The system was found to have an assembly efficiency of 97% with up to six DNA fragments, based on the construction of 55 vectors targeting different polyketide synthase (PKS) and PKS associated transcription factor encoding genes in F. avenaceum. Subsequently, the ?FaPKS3 vector was used for optimizing A. tumefaciens mediated transformation (ATMT) of F. avenaceum with respect to six variables. Acetosyringone concentration, co-culturing time, co-culturing temperature and fungal inoculum were found to significantly impact the transformation frequency. Following optimization, an average of 140 transformants per 106 macroconidia was obtained in experiments aimed at introducing targeted genome modifications. Targeted deletion of FaPKS6 (FA08709.2) in F. avenaceum showed that this gene is essential for biosynthesis of the polyketide/nonribosomal compound fusaristatin A. Conclusion The new USER-Brick system is highly versatile by allowing for the reuse of a common set of building blocks to accommodate seven different types of genome modifications. New USER-Bricks with additional functionality can easily be added to the system by future users. The optimized protocol for ATMT of F. avenaceum represents the first reported targeted genome modification by double homologous recombination of this plant pathogen and will allow for future characterization of this fungus. Functional linkage of FaPKS6 to the production of the mycotoxin fusaristatin A serves as a first testimony to this. PMID:25048842

2014-01-01

269

In vitro regeneration and Agrobacterium tumefaciens-mediated genetic transformation in asakura-sanshoo (Zanthoxylum piperitum (L.) DC. F. inerme Makino) an important medicinal plant  

PubMed Central

Context: Asakura-sanshoo (Zanthoxylum piperitum [L.] DC. f. inerme Makino) is an important medicinal plant in East Asia. Transgenic technique could be applied to improve plant traits and analyze gene function. However, there is no report on regeneration and genetic transformation in Asakura-sanshoo. Aims: To establish a regeneration and Agrobacterium tumefaciens-mediated genetic transformation system in Asakura-sanshoo, which could be used for cultivar improvement and gene function analysis. Settings and Design: The various combinations of indole-3-butyric acid (IBA), 6-benzylaminopurine (BA) and naphthalene acetic acid (NAA) were explored for the optimal plant regeneration from petiole and stem of Asakura-sanshoo. The half-strength woody plant medium (WPM) with different concentrations of NAA and IBA was used to induce root. For genetic transformation, A. tumefaciens strain EHA-105 harboring the plasmid pBin-Ex-H-ipt which carries the isopentenyl transferase (ipt) gene, ?-glucuronidase (GUS) gene and kanamycin resistance gene neomycin phosphotransferase II (NPTII) were used. The transformation efficiency was detected by the kanamycin resistant frequency. Materials and Methods: Petioles and stems were obtained from the in vitro cultured Asakura-sanshoo. The petiole and stem segments were precultured for 3 days, and then inflected using the bacterium at the concentration of OD600 0.5–0.8 for 10 min, followed by 3 days co-cultivation. Selection of the transgenic plants was carried out after 7 days the regeneration using gradient kanamycin at 30 mg/L and 50 mg/L, respectively. Successful transformed plants were confirmed by GUS histochemical assays, polymerase chain reaction (PCR), reverse transcription-PCR (RT-PCR), and Southern blotting analysis. Results: The highest shoots regeneration was obtained on WPM supplement with 0.5 mg/L BA and 0.2 mg/L NAA. The optimal rooting medium was half strength macro-element WPM. The kanamycin resistant frequency of petiole and stem was 24.66% and 25.93%, respectively. Thirty-five shoots in thousands adventitious buds were confirmed through GUS histochemical assays, PCR, RT-PCR, and Southern blotting. The regeneration shoot per explants elevated 5.85 fold compared with the wild-type plants. Conclusions: Individual transgenic Asakura-sanshoo lines were obtained. In this paper, it first revealed the expression of ipt gene significantly promoted the adventitious buds induction in Asakura-sanshoo as the same action as in other plants.

Zeng, Xiaofang; Zhao, Degang

2015-01-01

270

An enhancer-like element present in the promoter of a T-DNA gene from the Ti plasmid of Agrobacterium tumefaciens  

PubMed Central

The promoter of the 780 gene of T-right [Thomashow, M., Nutter, R., Montoya, A., Gordon, M. & Nester, E. (1980) Cell 19, 729-739] from Agrobacterium tumefaciens Ti plasmid (pTi15955) was shown to contain an upstream cis-acting element (activator) having enhancer-like properties. To characterize the properties of this promoter element, it was placed in both polarities, upstream and downstream of a ?-37 deletion mutant of the 780 gene. The ?-37 deletion contains the entire 780 gene with the 5? flanking sequences deleted upstream of TATA to -37. The effect of the activator on in vivo transcriptional activity was assessed by S1 nuclease mapping utilizing a homologous reference gene as an internal standard. Transcript levels from sunflower crown gall tumors were between 127% and 90% of the wild-type 780 gene depending on the polarity of the activator element when placed directly upstream to the 780 gene ?-37 promoter. Repositioning the activator element 613 base pairs further upstream increased transcription by 2-fold regardless of polarity. However, the activator element did not promote transcription when placed in either polarity ?200 base pairs downstream of the poly(A) addition site. Upstream promoter fragments (TATA-distal) from the octopine synthase (ocs) and agropine synthase (ags) genes were also tested for activation of the ?-37 construction. The ocs sequences activated transcription of the ?-37 deletion to 14% of wild-type levels when placed upstream in the B (reverse) orientation. All other constructions with the ocs and ags sequences showed no enhancement of promoter activity. Images PMID:16593943

Bruce, Wesley B.; Bandyopadhyay, Ram; Gurley, William B.

1988-01-01

271

Dynamic FtsA and FtsZ localization and outer membrane alterations during polar growth and cell division in Agrobacterium tumefaciens.  

PubMed

Growth and cell division in rod-shaped bacteria have been primarily studied in species that grow predominantly by peptidoglycan (PG) synthesis along the length of the cell. Rhizobiales species, however, predominantly grow by PG synthesis at a single pole. Here we characterize the dynamic localization of several Agrobacterium tumefaciens components during the cell cycle. First, the lipophilic dye FM 4-64 predominantly stains the outer membranes of old poles versus growing poles. In cells about to divide, however, both poles are equally labeled with FM 4-64, but the constriction site is not. Second, the cell-division protein FtsA alternates from unipolar foci in the shortest cells to unipolar and midcell localization in cells of intermediate length, to strictly midcell localization in the longest cells undergoing septation. Third, the cell division protein FtsZ localizes in a cell-cycle pattern similar to, but more complex than, FtsA. Finally, because PG synthesis is spatially and temporally regulated during the cell cycle, we treated cells with sublethal concentrations of carbenicillin (Cb) to assess the role of penicillin-binding proteins in growth and cell division. Cb-treated cells formed midcell circumferential bulges, suggesting that interrupted PG synthesis destabilizes the septum. Midcell bulges contained bands or foci of FtsA-GFP and FtsZ-GFP and no FM 4-64 label, as in untreated cells. There were no abnormal morphologies at the growth poles in Cb-treated cells, suggesting unipolar growth uses Cb-insensitive PG synthesis enzymes. PMID:23674672

Zupan, John R; Cameron, Todd A; Anderson-Furgeson, James; Zambryski, Patricia C

2013-05-28

272

The Integrity of the Periplasmic Domain of the VirA Sensor Kinase Is Critical for Optimal Coordination of the Virulence Signal Response in Agrobacterium tumefaciens?‡  

PubMed Central

The plant pathogen Agrobacterium tumefaciens responds to three main signals at the plant-bacterium interface: phenolics, such as acetosyringone (AS), monosaccharides, and acidic pH (?5.5). These signals are transduced via the chromosomally encoded sugar binding protein ChvE and the Ti plasmid-encoded VirA/VirG two-component regulatory system, resulting in the transcriptional activation of the Ti plasmid virulence genes. Here, we present genetic and physical evidence that the periplasmic domain of VirA dimerizes independently of other parts of the protein, and we examine the effects of several engineered mutations in the periplasmic and transmembrane regions of VirA on vir-inducing capacity as indicated by AS sensitivity and maximal level of vir-inducing activity at saturating AS levels. The data indicate that helix-breaking mutations throughout the periplasmic domain of VirA or mutations that reposition the second transmembrane domain (TM2) of VirA relieve the periplasmic domain's repressive effects on the maximal activity of this kinase in response to phenolics, effects normally relieved only when ChvE, sugars, and low pH are also present. Such relief, however, does not sensitize VirA to low concentrations of phenolics, the other major effect of the ChvE-sugar and low pH signals. We further demonstrate that amino acid residues in a small Trg-like motif in the periplasmic domain of VirA are crucial for transmission of the ChvE-sugar signal to the cytoplasmic domain. These experiments provide evidence that small perturbations in the periplasmic domain of VirA can uncouple sugar-mediated changes in AS sensitivity from the sugar-mediated effects on maximal activity. PMID:21216996

Nair, Gauri R.; Lai, Xiaoqin; Wise, Arlene A.; Rhee, Benjamin Wonjae; Jacobs, Mark; Binns, Andrew N.

2011-01-01

273

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

274

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

275

Transfer of non-T-DNA portions of the Agrobacterium tumefaciens Ti plasmid pTiA6 from the left terminus of TL-DNA  

Microsoft Academic Search

We introduced a plant selection marker, nptII, to the left of border A in the Agrobacterium Ti plasmid pTiA6. Infection of tobacco leaf discs with the modified Agrobacterium strain gave rise to kanamycin-resistant calli which grew in a hormone-dependent manner. Southern hybridization analysis of DNA isolated from four transformants indicated initiation of DNA transfer at or near border A and

Vai. Ramanathan; K. Veluthambi

1995-01-01

276

Regeneration from mature and immature embryos and transient gene expression via Agrobacterium-mediated transformation in emmer wheat (Triticum dicoccum Schuble).  

PubMed

The present study establishes a regeneration protocol and optimizes conditions for Agrobacterium-mediated transformation of the tetraploid emmer wheat, Triticum dicoccum. Regeneration from mature and immature embryos was accomplished as a two-step process involving callus induction in the presence of 2,4-D followed by regeneration on a 2,4-D free, cytokinin-containing medium (RM1). Higher concentrations of 2,4-D (4 mg/l) though conducive for callusing (89.39% in mature embryos and 96% in immature embryos) proved detrimental for further regeneration. At lower 2,4-D (1 mg/ml) although callusing was suboptimal, (56.8% and 84% from mature and immature embryos, respectively) the regeneration response was the highest on RM1 medium (64.4% and 56.6% from mature and immature embryos, respectively). Overall, the regeneration response of immature embryos was lower than the mature embryos by 10-12%. Due to the ease of availability of mature embryos the mature embryo-derived calli were chosen as the target tissue for Agrobacterium-mediated transformation in the two Indian varieties DDK1001 and DDK1009. Histochemical GUS expression revealed the suitability of the mature embryo-derived calli for such investigations. Of the CaMV35S and Act1 promoters employed, the monocot promoter Act1 displayed higher GUS gene activity in the mature embryo derived calli when co-cultivated with LBA4404 (pBI101::Act1). PMID:13677634

Khurana, Jigyasa; Chugh, Archana; Khurana, Paramjit

2002-11-01

277

Engineering cotton ( Gossypium hirsutum L.) for resistance to cotton leaf curl disease using viral truncated AC1 DNA sequences  

Microsoft Academic Search

Several important biological processes are performed by distinct functional domains found on replication-associated protein\\u000a (Rep) encoded by AC1 of geminiviruses. Two truncated forms of replicase (tAC1) gene, capable of expressing only the N-terminal\\u000a 669 bp (5?AC1) and C-terminal 783 bp (3?AC1) nucleotides cloned under transcriptional control of the CaMV35S were introduced\\u000a into cotton (Gossypium hirsutum L.) using LBA4404 strain of Agrobacterium tumefaciens

Jamil A. HashmiYusuf; Yusuf Zafar; Muhammad Arshad; Shahid Mansoor; Shaheen Asad

2011-01-01

278

Agrobacterium and Tumor Induction: A Model System.  

ERIC Educational Resources Information Center

The author offers laboratory procedures for experiments using the bacterium, Agrobacterium tumefaciens, which causes crown gall disease in a large number of plants. Three different approaches to growing a culture are given. (SA)

Lennox, John E.

1980-01-01

279

Molecular Cloning and Characterization of cgs, the Brucella abortus Cyclic ?(1-2) Glucan Synthetase Gene: Genetic Complementation of Rhizobium meliloti ndvB and Agrobacterium tumefaciens chvB Mutants  

PubMed Central

The animal pathogen Brucella abortus contains a gene, cgs, that complemented a Rhizobium meliloti nodule development (ndvB) mutant and an Agrobacterium tumefaciens chromosomal virulence (chvB) mutant. The complemented strains recovered the synthesis of cyclic ?(1-2) glucan, motility, virulence in A. tumefaciens, and nitrogen fixation in R. meliloti; all traits were strictly associated with the presence of an active cyclic ?(1-2) glucan synthetase protein in the membranes. Nucleotide sequencing revealed the presence in B. abortus of an 8.49-kb open reading frame coding for a predicted membrane protein of 2,831 amino acids (316.2 kDa) and with 51% identity to R. meliloti NdvB. Four regions of the B. abortus protein spanning amino acids 520 to 800, 1025 to 1124, 1284 to 1526, and 2400 to 2660 displayed similarities of higher than 80% with R. meliloti NdvB. Tn3-HoHo1 mutagenesis showed that the C-terminal 825 amino acids of the Brucella protein, although highly conserved in Rhizobium, are not necessary for cyclic ?(1-2) glucan synthesis. Confirmation of the identity of this protein as B. abortus cyclic ?(1-2) glucan synthetase was done by the construction of a B. abortus Tn3-HoHo1 insertion mutant that does not form cyclic ?(1-2) glucan and lacks the 316.2-kDa membrane protein. The recovery of this mutant from the spleens of inoculated mice was decreased by 3 orders of magnitude compared with that of the parental strain; this result suggests that cyclic ?(1-2) glucan may be a virulence factor in Brucella infection. PMID:9721274

Ińón de Iannino, Nora; Briones, Gabriel; Tolmasky, Marcelo; Ugalde, Rodolfo A.

1998-01-01

280

The Agrobacterium tumefaciens virB7 gene product, a proposed component of the T-complex transport apparatus, is a membrane-associated lipoprotein exposed at the periplasmic surface.  

PubMed Central

The Agrobacterium tumefaciens virB7 gene product contains a typical signal sequence ending with a consensus signal peptidase II cleavage site characteristic of bacterial lipoproteins. VirB7 was shown to be processed as a lipoprotein by (i) in vivo labeling of native VirB7 and a VirB7::PhoA fusion with [3H]palmitic acid and (ii) inhibition of VirB7 processing by globomycin, a known inhibitor of signal peptidase II. A VirB7 derivative sustaining a Ser substitution for the invariant Cys-15 residue within the signal peptidase II cleavage site could not be visualized immunologically and failed to complement a delta virB7 mutation, establishing the importance of this putative lipid attachment site for VirB7 maturation and function. VirB7 partitioned predominantly with outer membrane fractions from wild-type A348 cells as well as a delta virB operon derivative transformed with a virB7 expression plasmid. Expression of virB7 fused to phoA, the alkaline phosphatase gene of Escherichia coli, gave rise to high alkaline phosphatase activities in E. coli and A. tumefaciens cells, providing genetic evidence for the export of VirB7 in these hosts. VirB7 was shown to be intrinsically resistant to proteinase K; by contrast, a VirB7::PhoA derivative was degraded by proteinase K treatment of A. tumefaciens spheroplasts and remained intact upon treatment of whole cells. Together, the results of these studies favor a model in which VirB7 is topologically configured as a monotopic protein with its amino terminus anchored predominantly to the outer membrane and with its hydrophilic carboxyl domain located in the periplasmic space. Parallel studies of VirB5, VirB8, VirB9, and VirB10 established that each of these membrane-associated proteins also contains a large periplasmic domain whereas VirB11 resides predominantly or exclusively within the interior of the cell. PMID:8655494

Fernandez, D; Dang, T A; Spudich, G M; Zhou, X R; Berger, B R; Christie, P J

1996-01-01

281

Generation of single-stranded T-DNA molecules during the initial stages of T-DNA transfer from Agrobacterium tumefaciens to plant cells  

Microsoft Academic Search

Activation of the T-DNA transfer process of Agrobacterium by the plant signal molecule acetosyringone generates a single-stranded, unipolar, linear T-DNA molecule (T-strand)-a potential conjugative intermediate in the transfer of the T-DNA to plant cells. Acetosyringone induction also leads to S1 nuclease-sensitive sites at the Ti plasmid T-DNA borders, and other molecular changes associated with the Ti plasmid T-DNA sequences, which

Scott E. Stachel; Benedikt Timmerman; Patricia Zambryski

1986-01-01

282

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

PubMed Central

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

2013-01-01

283

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

284

Horizontal gene transfer of atrazine-degrading genes (atz) from Agrobacterium tumefaciens St96-4 pADP1::Tn5 to bacteria of maize-cultivated soil.  

PubMed

The plasmid pADP1::Tn5 derived from pADP1[Atr+] carrying a Tn5 transposon conferring kanamycin and streptomycin resistances was constructed and introduced in Agrobacterium tumefaciens St96-4. This genetically modified strain was inoculated (approximately 10(8) cfu g(-1)) in potted soils planted with maize and treated or not with atrazine (1.5 mg kg(-1)). Bulk and maize rhizosphere soils were sampled 39 days after planting to look for soil indigenous bacteria that had acquired pADP1::Tn5. Four transconjugants were isolated from four different soil samples. The estimated transfer frequency of pADP1::Tn5 was 10(-4) per donor. Maize rhizosphere and atrazine treatment had no obvious effect on pADP1::Tn5 transfer frequency. The sequencing of the 16S rDNA sequences of the transconjugants revealed that they were almost identical and highly similar to that of Variovorax spp (97%). In addition, their characterization suggested that the atzA and atzB genes had been transferred from pADP1::Tn5 to the bacterial chromosome in two of the four transconjugants. These data suggest that the atz degrading genes are horizontally transferred in soil and possibly subjected to gene rearrangement. PMID:16032656

Devers, Marion; Henry, Sonia; Hartmann, Alain; Martin-Laurent, Fabrice

2005-09-01

285

Development of Agrobacterium-mediated transformation technology for mature seed-derived callus tissues of indica rice cultivar IR64.  

PubMed

Indica rice cultivar IR64 is most recalcitrant to regenerate, which affects the transformation efficiency especially when mature seed-derived callus tissues are used as explants. Therefore, a simple, rapid and improved genetic transformation protocol has been developed for the indica rice cultivar IR64 using Agrobacterium-mediated genetic transformation. With different hormonal combination tested, the maximum callus induction was observed on MS medium supplemented with 2.5 mg/l 2,4-D and 0.15 mg/l BAP from the scutellum explants. Three weeks old scutellum derived callus explants were immersed in Agrobacterium suspension (strain LBA4404, OD600=1.0) and co-cultured at 26±2°C in dark for 2 d. The maximum transformation efficiency (12%) was achieved with infection of callus explants for 20 min along with use of 150 ?m acetosyringone. The maximum plant regeneration was observed on MS medium supplemented with 3 mg/l BAP, 1 mg/l Kinetin and 0.5 mg/l NAA. The maximum root induction was observed on MS medium along with 10 g/l glucose and 20 g/l sucrose. The integration of the transgene in T1 transgenic plants was confirmed by polymerase chain reaction and Southern blot analyses. The copy number of transgenes has been found to vary from 1 to 2 in transgenic plants. By using this improved method we have successfully raised transgenic rice plants within 3 mo from seed inoculation to plant regeneration. PMID:22538224

Sahoo, Ranjan Kumar; Tuteja, Narendra

2012-01-01

286

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

PubMed

Plant transformation efficiency depends on the ability of the transgene to successfully interact with plant host factors. Our previous work and the work of others showed that manipulation of the activity of host factors allows for increased frequency of transformation. Recently we reported that exposure of tobacco plants to increased concentrations of ammonium nitrate increases the frequency of both homologous recombination and plant transgenesis. Here we tested the influence of KCl and salts of rare earth elements, Ce and La on the efficiency of Agrobacterium-mediated plant transformation. We found that exposure to KCl, CeCl(3) and LaCl(3) leads to an increase in recombination frequency in Arabidopsis and tobacco. Plants grown in the presence of CeCl(3) and LaCl(3) had higher biomass, longer roots and greater root number. Analysis of transformation efficiency showed that exposure of tobacco plants to 50 mM KCl resulted in ~6.0-fold increase in the number of regenerated calli and transgenic plants as compared to control plants. Exposure to various concentrations of CeCl(3) showed a maximum increase of ~3.0-fold in both the number of calli and transgenic plants. Segregation analysis showed that exposure to KCl and cerium (III) chloride leads to more frequent integrations of the transgene at a single locus. Analysis of transgene intactness showed better preservation of right T-DNA border during transgene integration. Our data suggest that KCl and CeCl(3) can be effectively used to improve quantity and quality of transgene integrations. PMID:21132499

Boyko, Alex; Matsuoka, Aki; Kovalchuk, Igor

2011-04-01

287

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

288

Genetic transformation of selected mature cork oak (Quercus suber L.) trees.  

PubMed

A transformation system for selected mature cork oak (Quercus suber L.) trees using Agrobacterium tumefaciens has been established. Embryos obtained from recurrent proliferating embryogenic masses were inoculated with A. tumefaciens strains EHA105, LBA4404 or AGL1 harbouring the plasmid pBINUbiGUSint [carrying the neomycin phosphotransferase II (nptII) and beta-glucuronidase (uidA) genes]. The highest transformation efficiency (4%) was obtained when freshly isolated explants were inoculated with A. tumefaciens strain AGL1. Evidence of stable transgene integration was obtained by PCR for the nptII and uidA genes, Southern blotting and expression of the uidA gene. The transgenic embryos were germinated and successfully transferred to soil. PMID:15185122

Alvarez, R; Alonso, P; Cortizo, M; Celestino, C; Hernández, I; Toribio, M; Ordás, R J

2004-10-01

289

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

PubMed

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

290

Agrobacterium-mediated transformation of Arabidopsis thaliana using the floral dip method  

E-print Network

the transformation efficiency5. This version of transformation approach was often referred to as the ``AgrobacteriumAgrobacterium-mediated transformation of Arabidopsis thaliana using the floral dip method Xiuren of various methods for Agrobacterium tumefaciens­mediated transformation of Arabidopsis thaliana. Among these

Franks, Robert

291

Status Perkembangan Perbaikan Sifat Genetik Padi Menggunakan Transformasi Agrobacterium  

Microsoft Academic Search

Development Status of Rice Genetic Improvement using Agrobacterium Transformation. Syamsidah Rahmawati. Genetic transformation of rice becomes an important re- search area in recent years. Rice is staple food for almost half of world population and has been extensively used as a plant model system for monocotyledonous plant. Compare to direct DNA transfer techniques (PEG, electroporation, and DNA bombardment), Agrobacterium tumefaciens-mediated

Syamsidah Rahmawati

292

Stability analysis of chickpea large genomic DNA inserts in Agrobacterium.  

Technology Transfer Automated Retrieval System (TEKTRAN)

Agrobacterium tumefaciens-mediated transformation of large DNA inserts directly into plants facilitates the transfer of gene clusters and flanking regulatory elements. It is recommended that the integrity of large genomic fragments in Agrobacterium be verified prior to plant transformation. In this ...

293

Barley Transformation Using Agrobacterium-Mediated Techniques  

NASA Astrophysics Data System (ADS)

Methods for the transformation of barley using Agrobacterium-mediated techniques have been available for the past 10 years. Agrobacterium offers a number of advantages over biolistic-mediated techniques in terms of efficiency and the quality of the transformed plants produced. This chapter describes a simple system for the transformation of barley based on the infection of immature embryos with Agrobacterium tumefaciens followed by the selection of transgenic tissue on media containing the antibiotic hygromycin. The method can lead to the production of large numbers of fertile, independent transgenic lines. It is therefore ideal for studies of gene function in a cereal crop system.

Harwood, Wendy A.; Bartlett, Joanne G.; Alves, Silvia C.; Perry, Matthew; Smedley, Mark A.; Leyland, Nicola; Snape, John W.

294

[Expression of a partially modified delta-endotoxin gene from Bacillus thuringiensis var. tenebrionis in transgenic potato plants].  

PubMed

A modified gene (Bt77) of delta-endotoxin from Bacillus thuringiensis var. tenebrionis was constructed and cloned into pMON505. This binary transformation vector was introduced into Agrobacterium tumefaciens strains containing different helper disarmed Ti-plasmids, LBA4404, A281, and CBE21. These Agrobacterium strains were used to transform potato stem segments (S. tuberosum, cv Desiree, Resy, Temp, Granat). Regenerants were selected on kanamycin-containing media. The presence of the Bt77 sequence in plant genomic DNA was confirmed by PCR analysis. Bt gene expression was studied in regenerated plants. Western blot analysis revealed that transgenic plants produced the Bt protein in the range of 0.005-0.02% of total protein. Total protection against insect damage of leaf tissue from these plants was observed in laboratory bioassays with of Colorado beetle larvae. Transgenic plants showed incomplete protection from CB larvae. PMID:7990839

Gulina, I V; Shul'ga, O A; Mironov, M V; Revenkova, E V; Kraev, A S; Pozmogova, G E; Iakovleva, G A; Skriabin, K G

1994-01-01

295

Improved Agrobacterium -mediated transformation of cowpea via sonication and vacuum infiltration  

Microsoft Academic Search

An improved method of Agrobacterium-mediated transformation of cowpea was developed employing both sonication and vacuum infiltration treatments. 4 day-old cotyledonary\\u000a nodes were used as explants for co-cultivation with Agrobacterium tumefaciens strain EHA105 harbouring the binary vector pSouv-cry1Ac. Among the different injury treatments, vacuum infiltration and their\\u000a combination treatments tested, sonication for 20 s followed by vacuum infiltration for 5 min with A. tumefaciens

Souvika Bakshi; Ayan Sadhukhan; Sagarika Mishra; Lingaraj Sahoo

296

Agrobacterium-mediated transformation of Easter lily (Lilium longiflorum cv. Nellie White)  

Technology Transfer Automated Retrieval System (TEKTRAN)

Conditions were optimized for transient transformation of Lilium longiflorum cv. Nellie White using Agrobacterium tumefaciens. Bulb scale and basal meristem explants were inoculated with A. tumefaciens strain AGL1 containing the binary vector pCAMBIA 2301 which has the uidA gene that codes for ß-gl...

297

AGROBACTERIUM -MEDIATED TRANSFORMATION OF INDICA RICE CULTIVARS GROWN IN VIETNAM  

Microsoft Academic Search

Genetic transformation of rice (Oryza sativa L.) mediated by Agrobacterium tumefaciens has been performed on 5 indica cultivars grown in Vietnam. Embryogenic calli derived from mature seeds were inoculated with A. tumefaciens LBA4044 in which the T-DNA contained plasmid pSBbarB-UbiCre or plasmid pSB35L-Hyg-L-Gus. Transformation efficiency recorded as the number of independent transgenic plants showing Southern blot + per the number

TRAN THI; CUC HOA; AMIRHUSIN BAHAGIAWATI; THOMAS K. HODGES

298

Agrobacterium -mediated transformation and regeneration of kiwi fruit  

Microsoft Academic Search

Genetically transformed kiwi fruit (Actinidia deliciosa) plants were obtained from hypocotyl and stem segments co-cultured with Agrobacterium tumefaciens strain EHA101 harboring a binary vector, pLAN411 or pLAN421, which contained the neomycin phosphotransferase II (nptII) gene and the ß-glucuronidase (GUS) gene. After co-culturing with the A. tumefaciens, the hypocotyl or stem segments were cultured on a selection medium containing 25µg\\/ml kanamycin

Chiyomi Uematsu; Makoto Murase; Hiroaki Ichikawa; Jun Imamura

1991-01-01

299

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

300

An optimized Agrobacterium-mediated transformation for soybean for expression of binary insect resistance genes  

Microsoft Academic Search

Here we described a method that used the soybean [Glycine max (L.) Merr] embryonic tip for Agrobacterium-mediated transformation. To improve transformation efficiencies, the effect of several factors were examined by measuring transient expression levels of ?-glucuronidase and the number of resistant explants with PPT selection. The hypervirulent Agrobacterium tumefaciens strain KYRT1 proved to be a better transformer than EHA105 and

Wei Dang; Zhi-ming Wei

2007-01-01

301

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

302

An in vivo, luciferase-based, Agrobacterium -infiltration assay system: implications for post-transcriptional gene silencing  

Microsoft Academic Search

An in vivo assay system for analyzing transient luciferase expression in tobacco leaves infused with Agrobacterium\\u000a tumefaciens is described. The system makes use of A. tumefaciens harboring T-DNA vectors containing either an intron-containing firefly (Photinus pyralis) luciferase (EC 1.13.12.7) gene or an intron-containing sea pansy (Renilla reniformis) luciferase (EC 1.13.12.5) gene. Single or mixed Agrobacterium lines were infiltrated into leaf

Christopher Ian Cazzonelli; Jeff Velten

2006-01-01

303

Appropriate choice of antibiotic and Agrobacterium strain improves transformation of antibiotic-sensitive Fragaria vesca and F. v. semperflorens  

Microsoft Academic Search

By identifying antibiotics that eliminated Agrobacterium tumefaciens with the least phytotoxic effects, we were able to select appropriate A. tumefaciens strains for a more efficient transformation of seasonal Fragaria vesca and everbearing F. v. semperflorens. An efficient and reproducible method of shoot regeneration from leaf discs was developed with optimal shoot regeneration obtained on medium supplemented with 0.25 mg l-1

M. Alsheikh; H.-P. Suso; M. Robson; N. Battey; A. Wetten

2002-01-01

304

High efficiency Agrobacterium-mediated transformation and regeneration of citrus  

Microsoft Academic Search

A procedure for increased efficiency of production of transgenic citrus plants was developed by extending the exposure of the explants to the selection agent and by grafting in vitro the regenerated shoot apices onto seedling rootstocks. Carrizo citrange (Citrus sinensis L. Osbeck × Poncirus trifoliata L. Raf.) stem segments from in vitro grown seedlings were cocultivated with Agrobacterium tumefaciens EHA

Leandro Pena; Magdalena Cervera; José Juárez; Carmen Ortega; José A. Pina; Nuria Durán-Vila; Luis Navarro

1995-01-01

305

GENETIC TRANSFORMATION OF ASCOCHYTA RABIEI USING AGROBACTERIUM-MEDIATED TRANSFORMATION  

Technology Transfer Automated Retrieval System (TEKTRAN)

The conditions for efficient transformation of Ascochyta rabiei using Agrobacterium-mediated transformation have been determined. A. tumefaciens cells were co-cultivated with conidia of A. rabiei strain AR628, highly virulent on chickpea. Hygromycin B resistance (hph) was found to be superior to g...

306

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...

307

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

308

Agrobacterium-mediated transformation of round leaved sundew ( Drosera rotundifolia L.)  

Microsoft Academic Search

Agrobacterium tumefaciens-mediated genetic transformation method of the carnivorous medicinal plant round leaved sundew (Drosera rotundifolia L.) was developed. The micropropagation conditions of sundew aseptically germinated seeds were defined and the internal kanamycin resistance of sundew was tested. Transformation was made by cocultivation of micropropagated sundew leaves with A. tumefaciens strain C58C1 containing a cointegrate plasmid vector with neomycin phosphotransferase and

Merja Hirsikorpi; Terttu Kämäräinen; Teemu Teeri; Anja Hohtola

2002-01-01

309

Agrobacterium mediated transformation of Vigna sesquipedalis Koern (asparagus bean).  

PubMed

Agrobacterium mediated transformation of Vigna sesquipedalis was achieved using cotyledonary node explants prepared from 5 days old seedlings germinated on B5 basal medium, and transformed using Agrobacterium tumefaciens strain EHA101, carrying the phosphinothricin-N-acetyltransferase gene and neomycin-3-phosphotransferase-II gene as selectable markers and GUS gene as a screenable marker. Gene transfer was achieved by inoculation of cotyledonary node explants with a bacterial suspension and a further cocultivation with Agrobacterium suspension for 3 days on B5 basal medium. Only 10% of the explants were transformed with EHA101 and exhibited transient expression of GUS genes, while 2% of shoots exhibited stable integration of genes and developed into plants. Transgenic character of tissues was confirmed by GUS assay and Southern analysis. Histological analysis of GUS gene expression directly after cocultivation revealed a high competence of subepidermal cell layers of cotyledonary node and associated cotyledons for transformation with Agrobacterium. PMID:11272416

Ignacimuthu, S

2000-05-01

310

Agrobacterium -mediated transformation of mature Prunus serotina (black cherry) and regeneration of transgenic shoots  

Microsoft Academic Search

A protocol for Agrobacterium-mediated transformation was developed for in vitro leaf explants of an elite, mature Prunus serotina tree. Agrobacterium tumefaciens strain EHA105 harboring an RNAi plasmid with the black cherry AGAMOUS (AG) gene was used. Bacteria were induced for 12 h with 200 ?M acetosyringone for vir gene induction before leaf explant inoculation. Explants were co-cultured for 3 days, and then cultured

Xiaomei Liu; Paula M. Pijut

2010-01-01

311

Agrobacterium -mediated transformation of germinating seeds of Arabidopsis thaliana : A non-tissue culture approach  

Microsoft Academic Search

Germinating seeds of Arabidopsis thaliana were cocultivated with an Agrobacterium tumefaciens strain (C58Clrif) carrying the pGV3850:pAK1003 Ti plasmid. This Ti plasmid contains the neomycin phosphotransferase II gene (NPT II) which confers resistance to kanamycin and G418. Seeds (T1 generation) imbibed for 12 h before a 24 h exposure to Agrobacterium gave rise to the highest number of transformed progeny (T2

Kenneth A. Feldmann; M. David Marks

1987-01-01

312

T-DNA integration and expression in a monocot crop plant after induction of Agrobacterium  

Microsoft Academic Search

Agrobacterium tumefaciens induces tumorous proliferations on dicotyledonous plants by transferring a specific region (T-DNA) of its tumour-inducing (Ti) plasmid into wound-activated plant cells, where it becomes integrated into nuclear DNA1 and expressed2. The vir region of the Ti plasmid is essential for T-DNA transfer3,4 and is induced by specific wound substances from plants5-9. Agrobacterium is therefore a powerful tool for

Willi Schäfer; Andrea Görz; Günter Kahl

1987-01-01

313

Shoot regeneration and Agrobacterium -mediated transformation of Fragaria vesca L  

Microsoft Academic Search

An efficient and reliable method for shoot regeneration from leaf disks of Fragaria vesca L. has been developed. This protocol has been successfully employed to obtain transformed plants using Agrobacterium tumefaciens as gene vector. Murashige and Skoog basal medium supplemented with benzyladenine (4 mg\\/l) and indole-3-butyric acid (0.25 mg\\/l) induced the maximum percentage of shoot regeneration (98%) and the highest

Iman Mansouri; José A. Mercado; Victoriano Valpuesta; José M. López-Aranda; Fernando Pliego-Alfaro; Miguel A. Quesada

1996-01-01

314

At the maize\\/ Agrobacterium interface: natural factors limiting host transformation  

Microsoft Academic Search

Background: Agrobacterium tumefaciens has been successfully harnessed as the only natural vector for the incorporation of foreign genes into higher plants, but its use in the grain crops is often limited. Low transformation efficiency has been partly attributed to a failure in the initial events in the transformation process, specifically in the capacity of the VirA\\/VirG two-component system to induce

Jin Zhang; Laural Boone; Remigiusz Kocz; Chuhan Zhang; Andrew N Binns; David G Lynn

2000-01-01

315

Gene targeting in plants using the Agrobacterium vector system  

Microsoft Academic Search

In the past decade several methods have been developed for the introduction of foreign DNA into plant cells to obtain transgenic\\u000a plants. In some of these methods, purified DNA is directly introduced into protoplasts that for some species can be regenerated\\u000a into mature plants. The more commonly used protocols, however, employ the natural capacity ofAgrobacterium tumefaciens to transfer a defined

Remko Offringa; Paul J. J. Hooykaas

1992-01-01

316

Agrobacterium -mediated transformation and regeneration of cotton plants  

Microsoft Academic Search

Cotton (Gossypium hirsutum L.) was transformed by the EHA101 strain of Agrobacterium tumefaciens harboring a binary vector pGA482GG plasmid carrying the marker genes for neomycin phosphotransferase II (nptII) determining resistance to kanamycin and ?-glucuronidase (GUS). The cotyledons, hypocotyls, shoot meristem tissue, and its segments taken from in vitro growing seedlings were used as\\u000a explants. Explants were cultured in a Murashige

S. Unlu Yuceer; N. K. Koc

2006-01-01

317

Revised cocultivation conditions produce effective Agrobacterium-mediated genetic transformation of carnation ( Dianthus caryophyllus L.)  

Microsoft Academic Search

The cocultivation medium is one of the most important factors for the successful genetic transformation of carnation node explants. High genetic transformation efficiency of node explants cocultured by Agrobacterium tumefaciens strain AGL0 with pKT3 plasmid (containing ?-glucuronidase (GUS) and neomycin phosphotransferase (NPTII)) was achieved when the explants were cocultivated on filter paper soaked with water or water and acetosyringone (AS).

Chalermsri Nontaswatsri; Seiichi Fukai; Masanori Goi

2004-01-01

318

Agrobacterium?mediated transformation of lavandin (Lavandula x intermedia Emeric ex Loiseleur)  

Microsoft Academic Search

Lavandin (Lavandula x Emeric ex Loiseleur) is an aromatic plant, the essential oil of which is widely used in the perfume, cosmetic, flavouring and pharmaceutical industries. The qualitative or quantitative modification of its terpenes-containing essential oil by genetic engineering could have important scientific and commercial applications. In this study, we report the first Agrobacterium tumefaciens-mediated gene transfer into lavandin. The

Sandrine Dronne; Sandrine Moja; Frédéric Jullien; Françoise Berger

1999-01-01

319

Agrobacterium rhizogenes-induced cotton hairy root culture as an alternative tool for cotton functional genomics  

Technology Transfer Automated Retrieval System (TEKTRAN)

Although well-accepted as the ultimate method for cotton functional genomics, Agrobacterium tumefaciens-mediated cotton transformation is not widely used for functional analyses of cotton genes and their promoters since regeneration of cotton in tissue culture is lengthy and labor intensive. In cer...

320

Nodulation of Sesbania Species by Rhizobium (Agrobacterium) Strain IRBG74 and Other Rhizobia  

Technology Transfer Automated Retrieval System (TEKTRAN)

Concatenated sequence analysis with 16S rRNA, rpoB and fusA genes identified a strain (IRBG74) isolated from root nodules of the aquatic legume Sesbania cannabina as a close relative of the plant pathogen Rhizobium radiobacter (syn. Agrobacterium tumefaciens). However, DNA:DNA hybridisation with R. ...

321

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

322

Genetic transformation of Fusarium oxysporum f.sp. gladioli with Agrobacterium to study pathogenesis in Gladiolus  

Technology Transfer Automated Retrieval System (TEKTRAN)

Fusarium rot caused by Fusarium oxysporum f.sp. gladioli (Fog) is one of the most serious diseases of Gladiolus, both in the field and in stored bulbs. In order to study the pathogenesis of this fungus, we have transformed Fog with Agrobacterium tumefaciens binary vectors containing the hygromycin B...

323

DNA transfer from Agrobacterium to Zea mays or Brassica by agroinfection is dependent on bacterial virulence functions  

Microsoft Academic Search

Summary  DNA transfer fromAgrobacterium tumefaciens, a soil bacterium, to the non-host graminaceous monocotyledonous plantZea mays, was analysed using the recently developed technique of agroinfection. Agroinfection ofZ. mays with maize streak virus using strains ofA. tumefaciens carrying mutations in the pTiC58 virulence region showed an almost absolute dependence on the products of the bacterialvirC genes. In contrast, agroinfection of the control hostBrassica

Nigel Grimsley; Barbara Hohn; Cynthia Ramos; Clarence Kado; Peter Rogowsky

1989-01-01

324

High-frequency transformation of Arabidopsis thaliana by Agrobacterium tumefaciens  

Microsoft Academic Search

Incorporation of 5 mg\\/L silver thiosulphate into media for seed germination and callus induction, as used in the transformation\\u000a protocol originally described by Valvekens et al. (1988), was found to increase the frequency of regeneration of transformants\\u000a ofArabidopsis thaliana ecotypes C24 and Landsbergerecta by at least 10- to 100-fold. Other factors, such as density of the bacterial inoculation culture, density

Michael C. Clarke; Wenbin Wei; Keith Lindsey

1992-01-01

325

Genetic Transformation of Wheat Mediated by Agrobacterium tumefaciens  

Microsoft Academic Search

A rapid Agrobacferium fumefaciens-mediated transformation system for wheat was developed using freshly isolated immature embryos, precultured immature embryos, and embryogenic calli as explants. lhe explants were inoculated with a disarmed A. tumefa- ciens strain C58 (ABI) harboring the binary vector pMON18365 containing the p-glucuronidase gene with an intron, and a select- able marker, the neomycin phosphotransferase II gene. Various factors

Ming Cheng; Joyce E. Fry; Shengzhi Pang; Huaping Zhou; Catherine M. Hironaka; David R. Duncan; Timothy W. Conner; Yuechun Wan

1997-01-01

326

Dose Response of Agrobacterium Tumefaciens to Soil Fumigants  

Technology Transfer Automated Retrieval System (TEKTRAN)

Cut flower growers in California have routinely used methyl bromide with and without chloropicrin for pre-plant soil fumigation for the control of soilborne pathogens and weeds. Recent research to identify alternatives to methyl bromide for flower growers has involved combinations of 1,3-dichlorop...

327

An efficient Agrobacterium-mediated transformation system for poplar.  

PubMed

Poplar is a model system for the regeneration and genetic transformation of woody plants. To shorten the time required for studies of transgenic poplar, efforts have been made to optimize transformation methods that use Agrobacterium tumefaciens. In this study, an Agrobacterium infective suspension was treated at 4 °C for at least 10 h before infecting explants. By transforming the Populus hybrid clone "Nanlin895" (Populus deltoides×P. euramericana) with Agrobacterium harboring the PBI121:CarNAC6 binary vector, we showed that the transformation efficiency was improved significantly by multiple independent factors, including an Agrobacterium infective suspension with an OD600 of 0.7, an Agrobacterium infection for 120 min, an Agrobacterium infective suspension at a pH of 5.0, an acetosyringone concentration of 200 µM, a cocultivation at 28 °C, a cocultivation for 72 h and a sucrose concentration of 30 g/L in the cocultivation medium. We also showed that preculture of wounded leaf explants for two days increased the regeneration rate. The integration of the desired gene into transgenic poplars was detected using selective medium containing kanamycin, followed by southern blot analysis. The expression of the transgene in the transgenic lines was confirmed by northern blot analysis. PMID:24933641

Movahedi, Ali; Zhang, Jiaxin; Amirian, Rasoul; Zhuge, Qiang

2014-01-01

328

An Efficient Agrobacterium-Mediated Transformation System for Poplar  

PubMed Central

Poplar is a model system for the regeneration and genetic transformation of woody plants. To shorten the time required for studies of transgenic poplar, efforts have been made to optimize transformation methods that use Agrobacterium tumefaciens. In this study, an Agrobacterium infective suspension was treated at 4 °C for at least 10 h before infecting explants. By transforming the Populus hybrid clone “Nanlin895” (Populus deltoides × P. euramericana) with Agrobacterium harboring the PBI121:CarNAC6 binary vector, we showed that the transformation efficiency was improved significantly by multiple independent factors, including an Agrobacterium infective suspension with an OD600 of 0.7, an Agrobacterium infection for 120 min, an Agrobacterium infective suspension at a pH of 5.0, an acetosyringone concentration of 200 µM, a cocultivation at 28 °C, a cocultivation for 72 h and a sucrose concentration of 30 g/L in the cocultivation medium. We also showed that preculture of wounded leaf explants for two days increased the regeneration rate. The integration of the desired gene into transgenic poplars was detected using selective medium containing kanamycin, followed by southern blot analysis. The expression of the transgene in the transgenic lines was confirmed by northern blot analysis. PMID:24933641

Movahedi, Ali; Zhang, Jiaxin; Amirian, Rasoul; Zhuge, Qiang

2014-01-01

329

Development of highly efficient genetic transformation protocols for table grape Sugraone and Crimson Seedless at low Agrobacterium density  

Microsoft Academic Search

Highly efficient genetic transformation protocols and the regeneration of transgenic plants of Sugraone and Crimson Seedless\\u000a grapevines (Vitis vinifera L.) were achieved from embryogenic calli co-cultured with low Agrobacterium tumefaciens densities. The sensitivity of embryogenic cultures to kanamycin, as well as the effect of Agrobacterium strains, C58(pMP90) or EHA105, and the bacterial concentration (0.06 or 0.2 at Optical Density OD600)

Antonio-José López-Pérez; Leonardo Velasco; María Pazos-Navarro; Mercedes Dabauza

2008-01-01

330

Arabidopsis VIRE2 INTERACTING PROTEIN2 Is Required for Agrobacterium T-DNA Integration in Plants W  

E-print Network

, Michigan 48109 Agrobacterium tumefaciens­mediated genetic transformation is an efficient tool for genetic Brook, New York 11794 c Institute of Genetics, Martin Luther University, D-06099 Halle (Saale), Germany domain/leucine zipper motif­containing protein required for nuclear import and integration of T

Citovsky, Vitaly

331

Attachment of Agrobacterium to plant surfaces  

PubMed Central

Agrobacterium tumefaciens binds to the surfaces of inanimate objects, plants, and fungi. These bacteria are excellent colonizers of root surfaces. In addition, they also bind to soil particles and to the surface of artificial or man-made substances, such as polyesters and plastics. The mechanisms of attachment to these different surfaces have not been completely elucidated. At least two types of binding have been described unipolarpolysaccharide-dependent polar attachment and unipolar polysaccharide-independent attachment (both polar and lateral). The genes encoding the enzymes for the production of the former are located on the circular chromosome, while the genes involved in the latter have not been identified. The expression of both of these types of attachment is regulated in response to environmental signals. However, the signals to which they respond differ so that the two types of attachment are not necessarily expressed coordinately. PMID:24926300

Matthysse, Ann G.

2014-01-01

332

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

333

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

334

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

Microsoft Academic Search

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\\u000a 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\\u000a were

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

2008-01-01

335

Agrobacterium -mediated transformation of cotton ( Gossypium hirsutum L.) via vacuum infiltration  

Microsoft Academic Search

AnAgrobacterium-mediated gene transfer system with recovery of putative transformants was developed for cotton (Gossypium hirsutum L.) cv. Cocker-312. Two-month-old hypocotyl-derived embryogenic calli were infected through agroinfiltration for 10 min at\\u000a 27 psi in a suspension ofAgrobacterium tumefaciens strain GV3101 carrying tDNA with theGUS gene, encoding ?-glucuronidase (GUS), and the neomycin phosphotransferase II (nptII) gene as a kanamycin-resistant plant-selectable marker. Six

2004-01-01

336

Agrobacterium mediated transformation of chickpea ( Cicer arietinum L.) embryo axes  

Microsoft Academic Search

Embryo axes of four accessions of chickpea (Cicer arietinum L.) were treated with Agrobacterium tumefaciens strains C58C1\\/GV2260 carrying the plasmid p35SGUSINT and EHA101 harbouring the plasmid pIBGUS. In both vectors the GUS gene\\u000a is interrupted by an intron. After inoculation shoot formation was promoted on MS medium containing 0.5?mg\\/l BAP under a selection\\u000a pressure of 100?mg\\/l kanamycin or 10?mg\\/l phosphinothricin,

K. V. Krishnamurthy; K. Suhasini; A. P. Sagare; M. Meixner; A. de Kathen; T. Pickardt; O. Schieder

2000-01-01

337

Agrobacterium-mediated transformation of maize (Zea mays) immature embryos.  

PubMed

Agrobacterium tumefaciens-mediated transformation is one of the most efficient and simple gene delivery systems for genetic improvement and biology studies in maize. This system has become more widely used by both public and private laboratories. However, transformation efficiencies vary greatly from laboratory to laboratory for the same genotype. Here, we illustrate our advanced Agrobacterium-mediated transformation method in Hi-II maize using simple binary vectors. The protocol utilizes immature embryos as starting explants and the bar gene as a selectable marker coupled with bialaphos as a selective agent. The protocol offers efficient transformation results with high reproducibility, provided that some experimental conditions are well controlled. This transformation method, with minor modifications, can be also employed to transform certain maize inbreds. PMID:24243211

Lee, Hyeyoung; Zhang, Zhanyuan J

2014-01-01

338

Agrobacterium-mediated transformation of the commercially important grapefruit cultivar Rio Red (Citrus paradisi Macf.)  

Microsoft Academic Search

Transgenic plants of grapefruit cv. Rio Red (Citrus paradisi Macf.) have been obtained by Agrobacterium tumefaciens-mediated gene transfer using seedling-derived epicotyl segments as explants and kanamycin as the selective agent. The transformation\\u000a procedure includes a shoot elongation phase with a liquid medium overlay, which provides additional selection against non-transgenic\\u000a shoots. Transformed shoots are invigorated and multiplied on a non-selective medium

Z. N. Yang; I. L. Ingelbrecht; E. Louzada; M. Skaria; T. E. Mirkov

2000-01-01

339

Increased Agrobacterium -mediated transformation and rooting efficiencies in canola ( Brassica napus L.) from hypocotyl segment explants  

Microsoft Academic Search

An efficient protocol for the production of transgenic Brassica napus cv. Westar plants was developed by optimizing two important parameters: preconditioning time and co-cultivation time. Agrobacterium tumefaciens-mediated transformation was performed using hypocotyls as explant tissue. Two variants of a green fluorescent protein (GFP)-encoding gene--mGFP5-ER and eGFP--both under the constitutive expression of the cauliflower mosaic virus 35S promoter, were used for

V. Cardoza; C. N. Stewart

2003-01-01

340

Agrobacterium-mediated transformation of citrange: factors affecting transformation and regeneration  

Microsoft Academic Search

The effects of cocultivation with Agrobacterium tumefaciens, regeneration and selection conditions on the transformation efficiency of citrange (Citrus sinensis L. Osbeck×Poncirus trifoliata L. Raf.) have been investigated. Factors such as cocultivation period, preculture of explants, use of acetosyringone or feeder\\u000a plates during cocultivation, cocultivation on a medium rich in auxins, postcultivation in darkness, and different kanamycin\\u000a concentrations for selection were

M. Cervera; J. A. Pina; J. Juárez; L. Navarro; L. Peńa

1998-01-01

341

Adaptation of Cotton Shoot Apex Culture to Agrobacterium-Mediated Transformation  

Microsoft Academic Search

A protocol is presented for rapid genotype-independent transformation and regeneration of cotton (Gossypium spp.) from shoots isolated from germinating seedlings. Isolated shoots are inoculated with a super-virulent strain of Agrobacterium tumefaciens, subjected to a mild antibiotic selection, and directly regenerated as shoots in vitro. Shoots do not dedifferentiate and mutation rates are low. Rooted shoots can be obtained within 6–10 weeks

Jean H. Gould; Maria Magallanes-Cedeno

1998-01-01

342

Agrobacterium -mediated transformation of Hyacinthus orientalis with thaumatin II gene to control fungal diseases  

Microsoft Academic Search

Transgenic plants of hyacinth (Hyacinthus orientalis L.) cvs. Edisson and Chine Pink have been obtained by Agrobacterium-mediated transformation. Leaf explants of the both hyacinth cultivars regenerated shoots on MS medium containing 2.2 ?M BAP\\u000a and 0.3 ?M NAA at a frequency of 95%. A. tumefaciens strain CBE21 carrying binary vector pBIThau35 was used for transformation. Plasmid pBIThau35 has been produced by cloning

E. A. Popowich; A. P. Firsov; T. Y. Mitiouchkina; V. L. Filipenya; S. V. Dolgov; V. N. Reshetnikov

2007-01-01

343

Genetic transformation of wheat via Agrobacterium-mediated DNA delivery.  

PubMed

The method described involves an initial incubation of wheat immature embryos in a liquid culture of Agrobacterium tumefaciens. The Agrobacterium strain is engineered to contain a binary vector with a gene of interest and a selectable marker gene placed between the T-DNA borders; the T-DNA is the region transferred to the plant cells, thus harnessing the bacterium's natural ability to deliver specific DNA into host cells. Following the initial inoculation with the Agrobacterium, the embryos are co-cultivated for several days after which the Agrobacterium is selectively destroyed using an antibiotic. Tissue culture of the embryos on plant media with a correct balance of hormones allows embryogenic callus formation followed by regeneration of plantlets, and in the later stages of tissue culture a selectable marker (herbicide) is included to minimize the incidence of non-transformed plants. This protocol has been used successfully to generate transformed plants of a wide range of wheat varieties, both spring and winter bread wheats (T. aestivum L.) and durum wheats (T. turgidum L.). PMID:24243208

Sparks, Caroline A; Doherty, Angela; Jones, Huw D

2014-01-01

344

Reiterated DNA sequences in Rhizobium and Agrobacterium spp.  

PubMed

Repeated DNA sequences are a general characteristic of eucaryotic genomes. Although several examples of DNA reiteration have been found in procaryotic organisms, only in the case of the archaebacteria Halobacterium halobium and Halobacterium volcanii [C. Sapienza and W. F. Doolittle, Nature (London) 295:384-389, 1982], has DNA reiteration been reported as a common genomic feature. The genomes of two Rhizobium phaseoli strains, one Rhizobium meliloti strain, and one Agrobacterium tumefaciens strain were analyzed for the presence of repetitive DNA. Rhizobium and Agrobacterium spp. are closely related soil bacteria that interact with plants and that belong to the taxonomical family Rhizobiaceae. Rhizobium species establish a nitrogen-fixing symbiosis in the roots of legumes, whereas Agrobacterium species is a pathogen in different plants. The four strains revealed a large number of repeated DNA sequences. The family size was usually small, from 2 to 5 elements, but some presented more than 10 elements. Rhizobium and Agrobacterium spp. contain large plasmids in addition to the chromosomes. Analysis of the two Rhizobium strains indicated that DNA reiteration is not confined to the chromosome or to some plasmids but is a property of the whole genome. PMID:3450286

Flores, M; González, V; Brom, S; Martínez, E; Pińero, D; Romero, D; Dávila, G; Palacios, R

1987-12-01

345

Reiterated DNA sequences in Rhizobium and Agrobacterium spp.  

PubMed Central

Repeated DNA sequences are a general characteristic of eucaryotic genomes. Although several examples of DNA reiteration have been found in procaryotic organisms, only in the case of the archaebacteria Halobacterium halobium and Halobacterium volcanii [C. Sapienza and W. F. Doolittle, Nature (London) 295:384-389, 1982], has DNA reiteration been reported as a common genomic feature. The genomes of two Rhizobium phaseoli strains, one Rhizobium meliloti strain, and one Agrobacterium tumefaciens strain were analyzed for the presence of repetitive DNA. Rhizobium and Agrobacterium spp. are closely related soil bacteria that interact with plants and that belong to the taxonomical family Rhizobiaceae. Rhizobium species establish a nitrogen-fixing symbiosis in the roots of legumes, whereas Agrobacterium species is a pathogen in different plants. The four strains revealed a large number of repeated DNA sequences. The family size was usually small, from 2 to 5 elements, but some presented more than 10 elements. Rhizobium and Agrobacterium spp. contain large plasmids in addition to the chromosomes. Analysis of the two Rhizobium strains indicated that DNA reiteration is not confined to the chromosome or to some plasmids but is a property of the whole genome. Images PMID:3450286

Flores, M; González, V; Brom, S; Martínez, E; Pińero, D; Romero, D; Dávila, G; Palacios, R

1987-01-01

346

Agrobacterium rhizogenes-induced cotton hairy root culture as an alternative tool for cotton functional genomics.  

PubMed

Although well-accepted as the ultimate method for cotton functional genomics, Agrobacterium tumefaciens-mediated cotton transformation is not widely used for functional analyses of cotton genes and their promoters since regeneration of cotton in tissue culture is lengthy and labor intensive. In certain cases, A. rhizogenes-induced hairy root culture has been a suitable molecular tool for functional analyses of genes and promoters for plants that are difficult to regenerate by A. tumefaciens-mediated transformation. Similarly, A. rhizogenes-induced hairy root cultures are an alternative tool for cotton functional genomics. In this chapter, the advantages and disadvantages of using A. rhizogenes-induced cotton hairy root culture over A. tumefaciens-mediated cotton transformation are discussed. The procedures for transformation, generation, selection, and molecular analyses of transgenic cotton hairy roots are introduced by describing the functional analysis of a cotton promoter in cotton hairy roots generated by A. rhizogenes-mediated transformation. PMID:23143493

Kim, Hee Jin

2013-01-01

347

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

348

Agrobacterium-mediated disruption of a nonribosomal peptide synthetase gene in the invertebrate pathogen Metarhizium anisopliae reveals a peptide spore factor  

Technology Transfer Automated Retrieval System (TEKTRAN)

Numerous secondary metabolites have been isolated from the insect pathogenic fungus Metarhizium anisopliae, but the roles of these compounds as virulence factors in disease development are poorly understood. We targeted for disruption by Agrobacterium tumefaciens-mediated transformation a putative n...

349

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

350

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

351

Macro elements in inoculation and co-cultivation medium strongly affect the efficiency of Agrobacterium -mediated transformation in Lilium  

Microsoft Academic Search

A highly efficient Agrobacterium-mediated transformation system for Lilium × formolongi was established by modifying the medium used for inoculation and co-cultivation. Meristematic nodular calli of Lilium were inoculated with an overnight culture of A. tumefaciens strain EHA101 containing the plasmid pIG121-Hm harboring an intron-containing ?-glucuronidase (GUS), hygromycin phosphotransferase,\\u000a and neomycin phosphotransferase II genes. The effects of ten different types of media and

Pejman Azadi; Dong Poh Chin; Kiyo Kuroda; Raham Sher Khan; Masahiro Mii

2010-01-01

352

Regeneration of transgenic plants by Agrobacterium -mediated transformation of somatic embryos of juvenile and mature Quercus robur  

Microsoft Academic Search

A protocol was developed for genetic transformation of somatic embryos derived from juvenile and mature Quercus robur trees. Optimal transformation conditions were evaluated on the basis of the results of transient GUS expression assays with\\u000a five oak embryogenic lines and a strain of Agrobacterium tumefaciens (EHA105) harbouring a p35SGUSINT plasmid containing a nptII and a uidA (GUS) genes. For stable

N. Vidal; R. Mallón; S. Valladares; A. M. Meijomín; A. M. Vieitez

2010-01-01

353

Localization of target cells and improvement of Agrobacterium -mediated transformation efficiency by direct acetosyringone pretreatment of carrot root discs  

Microsoft Academic Search

Summary Localization of target cells forAgrobacterium-mediated transformation in the carrot root disc model has been achieved after inoculation with a disarmedA. tumefaciens strain harbouring a GUS-intron construct. The first GUS positive cells could be detected on both sides of the discs 48 h after inoculation. The transformed cells were always more numerous on the apical side, mainly localized in the

Anne Guivarc'h; J.-C. Caissard; S. Brown; Dominique Marie; W. Dewitte; H. Van Onckelen; Dominique Chriqui

1993-01-01

354

Agrobacterium -mediated transformation as a useful tool for the molecular genetic study of the phytopathogen Curvularia lunata  

Microsoft Academic Search

In order to explore the molecular mechanisms of virulence and genetic variance of Curvularia lunata in maize, an ATMT (Agrobacterium tumefaciens-mediated transformation) system was established in order to create a wide range of insertional transformants of C. lunata. Our results showed that the germinating conidia were the ideal starting material for transformation. Based on our optimised\\u000a transformation condition, the transformation

Tong Liu; Lixing Liu; Xue Jiang; Jumei Hou; Kehe Fu; Feihong Zhou; Jie Chen

2010-01-01

355

Enhanced Agrobacterium -mediated Transformation of Embryogenic Calli of Upland Cotton via Efficient Selection and Timely Subculture of Somatic Embryos  

Microsoft Academic Search

Agrobacterium-tumefaciens-mediated transformation of cotton embryogenic calli (EC) was enhanced by choosing appropriate EC and improving efficiency\\u000a of coculture, selection cultivation, and plant regeneration. After 48-h cocultivation, the number of ?-glucuronidase (GUS)-positive\\u000a calli characterized by yellow, loose, and fine-grained EC was twofold greater than that of gray, brown, and coarse-granule\\u000a EC. It indicated that efficiency of transient transformation was affected by

Shen-Jie Wu; Hai-Hai Wang; Fei-Fei Li; Tian-Zi Chen; Jie Zhang; Yan-Jie Jiang; Yezhang Ding; Wang-Zhen Guo; Tian-Zhen Zhang

2008-01-01

356

Agrobacterium -mediated transformation of cotton ( Gossypium hirsutum L. cv. Zhongmian 35 ) using glyphosate as a selectable marker  

Microsoft Academic Search

The most economically significant Chinese cotton cultivar (Gossypium hirsutum L. cv. Zhongmian 35) was transformed via Agrobacterium tumefaciens-mediated DNA transfer. The aroA-M1 gene that confers resistance to the glyphosate was fused with a chloroplast-transit peptide of Arabidopsis thaliana 5-enolpyruvyl-3-phosphoshikimate synthase (ASP) and expressed in cotton plants under the control of a CaMV35S promoter. Transgenic plants were directly selected on medium

Fu-Yong Zhao; Yun-Feng Li; Peilin Xu

2006-01-01

357

T-DNA from Agrobacterium Ti Plasmid is in the Nuclear DNA Fraction of Crown Gall Tumor Cells  

Microsoft Academic Search

The crown gall teratoma tumor line BT37, incited by Agrobacterium tumefaciens strain T37, has been found to contain part of the tumor-inducing plasmid, pTi T37, of the inciting strain. This foreign DNA segment, called T-DNA, is maintained at several copies per diploid tumor cell. We have examined subcellular DNA fractions from this tumor line in an effort to determine whether

Mary-Dell Chilton; Randall K. Saiki; Narendra Yadav; Milton P. Gordon; Francis Quetier

1980-01-01

358

Prediction of candidate small non-coding RNAs in Agrobacterium by computational analysis.  

PubMed

Small non-coding RNAs with important regulatory roles are not confined to eukaryotes. Recent work has uncovered a growing number of bacterial small RNAs (sRNAs), some of which have been shown to regulate critical cellular processes. Computational approaches, in combination with molecular experiments, have played an important role in the identification of these sRNAs. At present, there is no information on the presence of small non-coding RNAs and their genes in the Agrobacterium tumefaciens genome. To identify potential sRNAs in this important bacterium, deep sequencing of the short RNA populations isolated from Agrobacterium tumefaciens C58 was carried out. From a data set of more than 10,000 short sequences, 16 candidate sRNAs have been tentatively identified based on computational analysis. All of these candidates can form stem-loop structures by RNA folding predictions and the majority of the secondary structures are rich in GC base pairs. Some are followed by a short stretch of U residues, indicative of a rho-independent transcription terminator, whereas some of the short RNAs are found in the stem region of the hairpin, indicative of eukaryotic-like sRNAs. Experimental strategies will need to be used to verify these candidates. The study of an expanded list of candidate sRNAs in Agrobacterium will allow a more complete understanding of the range of roles played by regulatory RNAs in prokaryotes. PMID:23554609

Zhao, Tingting; Zhang, Ren; Wang, Mingbo

2010-01-01

359

Mechanisms and regulation of surface interactions and biofilm formation in Agrobacterium  

PubMed Central

For many pathogenic bacteria surface attachment is a required first step during host interactions. Attachment can proceed to invasion of host tissue or cells or to establishment of a multicellular bacterial community known as a biofilm. The transition from a unicellular, often motile, state to a sessile, multicellular, biofilm-associated state is one of the most important developmental decisions for bacteria. Agrobacterium tumefaciens genetically transforms plant cells by transfer and integration of a segment of plasmid-encoded transferred DNA (T-DNA) into the host genome, and has also been a valuable tool for plant geneticists. A. tumefaciens attaches to and forms a complex biofilm on a variety of biotic and abiotic substrates in vitro. Although rarely studied in situ, it is hypothesized that the biofilm state plays an important functional role in the ecology of this organism. Surface attachment, motility, and cell division are coordinated through a complex regulatory network that imparts an unexpected asymmetry to the A. tumefaciens life cycle. In this review, we describe the mechanisms by which A. tumefaciens associates with surfaces, and regulation of this process. We focus on the transition between flagellar-based motility and surface attachment, and on the composition, production, and secretion of multiple extracellular components that contribute to the biofilm matrix. Biofilm formation by A. tumefaciens is linked with virulence both mechanistically and through shared regulatory molecules. We detail our current understanding of these and other regulatory schemes, as well as the internal and external (environmental) cues mediating development of the biofilm state, including the second messenger cyclic-di-GMP, nutrient levels, and the role of the plant host in influencing attachment and biofilm formation. A. tumefaciens is an important model system contributing to our understanding of developmental transitions, bacterial cell biology, and biofilm formation. PMID:24834068

Heindl, Jason E.; Wang, Yi; Heckel, Brynn C.; Mohari, Bitan; Feirer, Nathan; Fuqua, Clay

2014-01-01

360

Highly Efficient Agrobacterium-Mediated Transformation of Wheat Via In Planta Inoculation  

NASA Astrophysics Data System (ADS)

This chapter details a reproducible method for the transformation of spring wheat using Agrobacterium tumefaciens via the direct inoculation of bacteria into immature seeds in planta as described in patent WO 00/63398(1. Transformation efficiencies from 1 to 30% have been obtained and average efficiencies of at least 5% are routinely achieved. Regenerated plants are phenotypically normal with 30-50% of transformation events carrying introduced genes at single insertion sites, a higher rate than is typically reported for transgenic plants produced using biolistic transformation methods.

Risacher, Thierry; Craze, Melanie; Bowden, Sarah; Paul, Wyatt; Barsby, Tina

361

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 Agrobacterium rhizognenes-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

2015-01-01

362

Characterization of copper-resistant agrobacterium isolated from legume nodule in mining tailings.  

PubMed

A copper-resistant bacteria CCNWSX2332 was isolated from root nodules of Lespedeza cuneata growing in a gold mining tailing region in northwest of China. The specific growth rate of the strain was 0.62 microh(-1) in the presence of 2.0 mM Cu(2+) in TY liquid media, and the maximum copper accumulation of whole cell reached 147.03 microM Cu(2+) per gram (dry weight) after 4 h incubation. A partial sequence of the copper resistance gene copA was amplified from the strain, and the phylogenetic analysis based on 16S rDNA sequence showed that CCNWSX2332 belonged to Agrobacterium, and it had 100% similarity with Agrobacterium tumefaciens type strain IAM13129(T). PMID:18979058

Yu, Jianfu; Fan, Lianmei; Yang, Shushen; Tang, Ming; Yang, Wenquan; Li, Huifen; Wei, Gehong

2009-03-01

363

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

364

Agrobacterium-Mediated Disruption of a Nonribosomal Peptide Synthetase Gene in the Invertebrate Pathogen Metarhizium anisopliae Reveals a Peptide Spore Factor  

Microsoft Academic Search

Numerous secondary metabolites have been isolated from the insect pathogenic fungus Metarhizium aniso- pliae, but the roles of these compounds as virulence factors in disease development are poorly understood. We targeted for disruption by Agrobacterium tumefaciens-mediated transformation a putative nonribosomal peptide synthetase (NPS) gene, MaNPS1. Four of six gene disruption mutants identified were examined further. Chemical analyses showed the presence

Yong-Sun Moon; Bruno G. G. Donzelli; Stuart B. Krasnoff; Heather McLane; Mike H. Griggs; Peter Cooke; John D. Vandenberg; Donna M. Gibson; Alice C. L. Churchill

2008-01-01

365

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

366

Root and shoot parts of strawberry: factories for production of functional human pro-insulin.  

PubMed

Diabetes, a disease caused by excessive blood sugar, is caused by the lack of insulin. For commercial production, insulin is made in bacteria or yeast by protein recombinant technology. The focus of this research is evaluating another resource and producing of recombinant insulin protein in as strawberry as this plant has high potential in production of pharmaceutical proteins. Strawberry is a suitable bioreactor for production of recombinant proteins especially edible vaccines. In this research, human pro-insulin gene was cloned in pCAMBIA1304 vector under CaMV35S promoter and NOS terminator. Agrobacterium tumefaciens LBA4404, AGL1, EHA105, EHA101, C58, C58 (pGV2260) and C58 (pGV3101) strains were used for transformation of pro-insulin gene into strawberry cv. Camarosa, Selva, Sarian Hybrid, Pajaro, Paros, Gaviota, Alpine. Additionally, Agrobacterium rhizogenes K599, R1000, A4 and MSU440 strains were utilized for gene transformation into hairy roots. PCR analysis indicated the presence of transformed human pro-insulin gene in the strawberry and hairy roots. Also, its transcription was confirmed using RT-PCR. Furthermore, the analysis of plants, fruits and hairy roots at the level of proteins using dot blot, ELISA, SDS-PAGE and ECL tests re-confirmed the expression of this protein in the transgenic plants as well as hairy roots. Protein purification of human pro-insulin from transgenic tissues was performed using affinity chromatography. Finally, the bioassay of recombinant pro-insulin was performed. The analysis of second generations of transgenic plants (T1) at DNA and protein levels was also performed as a complementary experiment. This study opens a new avenue in molecular farming of human pro-insulin through its mass production in roots and shoots of strawberry. PMID:25403333

Tavizi, Ashkan; Javaran, Mokhtar Jalali; Moieni, Ahmad; Mohammadi-Dehcheshmeh, Manijeh; Mohebodini, Mehdi; Ebrahimie, Esmaeil

2014-11-18

367

Cloning, Transformation and Expression of Proinsulin Gene in Tomato (Lycopersicum esculentum Mill.)  

PubMed Central

Background: Plants are among promising and suitable platform systems for production of recombinant biopharmaceutical proteins due to several features such as safety, no need for fermentation, inexpensive investment, and fast and easy scale-up. Human insulin is one of the most widely used medicines in the world. Up to now different expression systems including Escherichia coli, yeast and CHO have been exploited for producing recombinant human insulin and a variety of different recombinant insulin are extensively used. Objectives: This study reports on the transformation and expression of proinsulin gene in tomato plants for the first time in Iran. Materials and Methods: This study reports the cloning, transformation and expression of proinsulin gene in tomato plants. Specific primers were designed and used for PCR amplification and cloning of the proinsulin gene in the plant expression vector pCAMBIA1304. The recombinant construct was transferred into Agrobacterium tumefaciens strain LBA4404, and used for Agrobacterium mediated stable transformation of tomato plants. Presence of the desired gene in transgenic lines was confirmed through colony PCR and sequencing. The expression of the protein in transgenic lines was confirmed by immunodot blot assay. Results: The presence of the proinsulin gene in the genomic DNA of transgenic tomato was confirmed by PCR. Also total protein of transgenic tomato was extracted and the expression of proinsulin was detected using dotblot assay. Conclusions: This survey addresses the possibility of proinsulin gene transfer and expression in tomato transgenic lines. This study can be used as a basis for future researches to produce human proinsulin in tomato and other candidate plants. PMID:24644433

Soltanmohammadi, Behnoush; Jalali-Javaran, Mokhtar; Rajabi-Memari, Hamid; Mohebodini, Mehdi

2014-01-01

368

DNA transfer from Agrobacterium to Zea mays or Brassica by agroinfection is dependent on bacterial virulence functions.  

PubMed

DNA transfer from Agrobacterium tumefaciens, a soil bacterium, to the non-host graminaceous monocotyle-donous plant Zea mays, was analysed using the recently developed technique of agroinfection. Agroinfection of Z. mays with maize streak virus using strains of A. tumefaciens carrying mutations in the pTiC58 virulence region showed an almost absolute dependence on the products of the bacterial virC genes. In contrast, agroinfection of the control host Brassica rapa with cauliflower mosaic virus was less dependent on the virC gene products. In other respects, the basic mechanism of the plant-bacterium interaction was found to be similar. While intact virA, B, D and G functions were absolutely necessary, mutants in virE were attenuated. Agroinfection of maize was effective in the absence of an exogenously supplied vir gene inducer, and indeed wounded Z. mays tissues were found to produce substance(s) which induced the expression of A. tumefaciens vir genes. These findings are discussed in the light of current knowledge about the function of Agrobacterium vir genes. PMID:2770696

Grimsley, N; Hohn, B; Ramos, C; Kado, C; Rogowsky, P

1989-06-01

369

Agrobacterium infection and plant defense—transformation success hangs by a thread  

PubMed Central

The value of Agrobacterium tumefaciens for plant molecular biologists cannot be appreciated enough. This soil-borne pathogen has the unique capability to transfer DNA (T-DNA) into plant systems. Gene transfer involves both bacterial and host factors, and it is the orchestration of these factors that determines the success of transformation. Some plant species readily accept integration of foreign DNA, while others are recalcitrant. The timing and intensity of the microbially activated host defense repertoire sets the switch to “yes” or “no.” This repertoire is comprised of the specific induction of mitogen-activated protein kinases (MAPKs), defense gene expression, production of reactive oxygen species (ROS) and hormonal adjustments. Agrobacterium tumefaciens abuses components of the host immunity system it mimics plant protein functions and manipulates hormone levels to bypass or override plant defenses. A better understanding of the ongoing molecular battle between agrobacteria and attacked hosts paves the way toward developing transformation protocols for recalcitrant plant species. This review highlights recent findings in agrobacterial transformation research conducted in diverse plant species. Efficiency-limiting factors, both of plant and bacterial origin, are summarized and discussed in a thought-provoking manner. PMID:24391655

Pitzschke, Andrea

2013-01-01

370

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

371

Transfer of nitrogen fixation genes from a bacterium with the characteristics of both Rhizobium and Agrobacterium.  

PubMed Central

Strain T1K, reported to be Rhizobium trifolii strain T1 carrying the drug resistance plasmid RU-1drd, was able to transfer a cluster of nif+ genes to Escherichia coli K-12. Additional genetic material, resembling the gal-chlA region of E. coli, was also transferred from strain T1K. The segregation pattern of these transferred genes suggested that they were on a plasmid. Although strain TIK was able to nodulate red and white clover, it also formed very slow-growing galls on tomato stems and shared many physiological properties with Agrobacterium tumefaciens, to which it seemed more closely related than to R. trifolii. The R. trifolii hybrid T1 (R1-19drd), constructed by conjugation, did not share any of these properties of both A. tumefaciens. Thus, strain T1K appears to be a bacterium with properties of both A. tumefaciens and R. trifolii and with the capacity to transfer nif+ genes and other functions which it may have "cloned" from another bacterium such as Klebsiella. Images PMID:342496

Skotnicki, M L; Rolfe, B G

1978-01-01

372

Rapid and accurate species and genomic species identification and exhaustive population diversity assessment of Agrobacterium spp. using recA-based PCR.  

PubMed

Agrobacteria are common soil bacteria that interact with plants as commensals, plant growth promoting rhizobacteria or alternatively as pathogens. Indigenous agrobacterial populations are composites, generally with several species and/or genomic species and several strains per species. We thus developed a recA-based PCR approach to accurately identify and specifically detect agrobacteria at various taxonomic levels. Specific primers were designed for all species and/or genomic species of Agrobacterium presently known, including 11 genomic species of the Agrobacterium tumefaciens complex (G1-G9, G13 and G14, among which only G2, G4, G8 and G14 still received a Latin epithet: pusense, radiobacter, fabrum and nepotum, respectively), A. larrymoorei, A. rubi, R. skierniewicense, A. sp. 1650, and A. vitis, and for the close relative Allorhizobium undicola. Specific primers were also designed for superior taxa, Agrobacterium spp. and Rhizobiaceace. Primer specificities were assessed with target and non-target pure culture DNAs as well as with DNAs extracted from composite agrobacterial communities. In addition, we showed that the amplicon cloning-sequencing approach used with Agrobacterium-specific or Rhizobiaceae-specific primers is a way to assess the agrobacterial diversity of an indigenous agrobacterial population. Hence, the agrobacterium-specific primers designed in the present study enabled the first accurate and rapid identification of all species and/or genomic species of Agrobacterium, as well as their direct detection in environmental samples. PMID:23578959

Shams, M; Vial, L; Chapulliot, D; Nesme, X; Lavire, C

2013-07-01

373

Ecological dynamics and complex interactions of Agrobacterium megaplasmids  

PubMed Central

As with many pathogenic bacteria, agrobacterial plant pathogens carry most of their virulence functions on a horizontally transmissible genetic element. The tumor-inducing (Ti) plasmid encodes the majority of virulence functions for the crown gall agent Agrobacterium tumefaciens. This includes the vir genes which drive genetic transformation of host cells and the catabolic genes needed to utilize the opines produced by infected plants. The Ti plasmid also encodes, an opine-dependent quorum sensing system that tightly regulates Ti plasmid copy number and its conjugal transfer to other agrobacteria. Many natural agrobacteria are avirulent, lacking the Ti plasmid. The burden of harboring the Ti plasmid depends on the environmental context. Away from diseased hosts, plasmid costs are low but the benefit of the plasmid is also absent. Consequently, plasmidless genotypes are favored. On infected plants the costs of the Ti plasmid can be very high, but balanced by the opine benefits, locally favoring plasmid bearing cells. Cheating derivatives which do not incur virulence costs but can benefit from opines are favored on infected plants and in most other environments, and these are frequently isolated from nature. Many agrobacteria also harbor an At plasmid which can stably coexist with a Ti plasmid. At plasmid genes are less well characterized but in general facilitate metabolic activities in the rhizosphere and bulk soil, such as the ability to breakdown plant exudates. Examination of A. tumefaciens C58, revealed that harboring its At plasmid is much more costly than harboring it’s Ti plasmid, but conversely the At plasmid is extremely difficult to cure. The interactions between these co-resident plasmids are complex, and depend on environmental context. However, the presence of a Ti plasmid appears to mitigate At plasmid costs, consistent with the high frequency with which they are found together. PMID:25452760

Platt, Thomas G.; Morton, Elise R.; Barton, Ian S.; Bever, James D.; Fuqua, Clay

2014-01-01

374

Use of Ti plasmid DNA probes for determining tumorigenicity of agrobacterium strains  

SciTech Connect

Probes consisting of T-DNA genes from the Ti plasmid of Agrobacterium tumefaciens were used for determining tumorigenicity of strains. Two {sup 32}P-labeled probes hybridized with 28 of 28 tumorigenic strains of the pathogen but not with 20 of 22 nontumorigenic strains. One probe, pTHE17, consists of all but the far left portion of the T-DNA of strain C58. Probe SmaI7 consists of SmaI fragment 7 of pTiC58, including onc genes 1, 4, and 6a and most of 2. Another probe, pAL4044, consisting of the vir region of strain Ach-5, hybridized with several nontumorigenic as well as tumorigenic strains. Colony hybridizations were done with 28 tumorigenic and 22 nontumorigenic Agrobacterium strains. About 10{sup 6} CFU of the different tumorigenic strains were detectable with this method. Southern analyses confirmed the presence or absence of Ti plasmids in strains for which tumorigenicity was questioned. Colony hybridization with the T-DNA probes provides a rapid and sensitive means for determining the tumorigenic nature of Agrobacterium strains.

Burr, T.J.; Norelli, J.L.; Katz, B.H.; Bishop, A.L. (Cornell Univ., Geneva, NY (USA))

1990-06-01

375

Okadaic acid and trifluoperazine enhance Agrobacterium-mediated transformation in eastern white pine.  

PubMed

Mature zygotic embryos of recalcitrant Christmas tree species eastern white pine (Pinus strobus L.) were used as explants for Agrobacterium tumefaciens strain GV3101-mediated transformation using the uidA (beta-Glucuronidase) gene as a reporter. Influence of the time of sonication and the concentrations of protein phosphatase inhibitor (okadaic acid) and kinase inhibitor (trifluoperazine) on Agrobacterium-mediated transformation have been evaluated. A high transformation frequency was obtained after embryos were sonicated for 45-50 s, or treated with 1.5-2.0 microM okadaic acid or treated with 100-200 microM trifluoperazine, respectively. Protein phosphatase and kinase inhibitors enhance Agrobacterium-mediated transformation in eastern white pine. A 2-3.5-fold higher rate of hygromycin-resistant callus was obtained with an addition of 2 microM okadaic acid or 150 microM trifluoperazine or sonicated embryos for 45 s. Stable integration of the uidA gene in the plant genome of eastern white pine was confirmed by polymerase chain reaction (PCR), Southern and northern blot analyses. These results demonstrated that a stable and enhanced transformation system has been established in eastern white pine and this system would provide an opportunity to transfer economically important genes into this Christmas tree species. PMID:17242943

Tang, Wei; Lin, Jinxing; Newton, Ronald J

2007-05-01

376

Agrobacterium-mediated transformation of protocorm-like bodies in Cymbidium.  

PubMed

Genetically transformed plants of Cymbidium were regenerated after cocultivating protocorm-like bodies (PLB) with Agrobacterium tumefaciens strain EHA101 (pIG121Hm) that harbored genes for beta-glucuronidase (gus), hygromycin phosphotransferase (hpt) and neomycin phosphotransferase II (nptII). PLB of three genotypes maintained in liquid new Dogashima medium (NDM), were subjected to transformation experiments. The PLB inoculated with Agrobacterium produced secondary PLB, 4 weeks after transfer onto 2.5 g L(-1) gellan gum-solidified NDM containing 10 g L(-1) sucrose, 20 mg L(-1) hygromycin and 40 mg L(-1) meropenem. Transformation efficiency was affected by genotype and the presence of acetosyringone during cocultivation. The highest transformation efficiency was obtained when PLB from the genotype L4 were infected and cocultivated with Agrobacterium on medium containing 100 muM acetosyringone. Transformation of the hygromycin-resistant plantlets regenerated from different sites of inoculated PLB was confirmed by histochemical GUS assay, PCR analysis and Southern blot hybridization. PMID:17205333

Chin, Dong Poh; Mishiba, Kei-ichiro; Mii, Masahiro

2007-06-01

377

Opine catabolic loci from Agrobacterium plasmids confer chemotaxis to their cognate substrates.  

PubMed

Opines are carbon compounds produced by crown galls and hairy roots induced by Agrobacterium tumefaciens and A. rhizogenes, respectively. These novel condensation products of plant metabolic intermediates are utilized as nutritional sources by the Agrobacterium strains that induced the growths. Thus, opines are thought to favor the propagation of agrobacteria in the tumorsphere. Certain Agrobacterium strains were chemoattracted to opines. The chemotactic activities to octopine, to nopaline, to mannopine, and to agrocinopines A + B were dependent on the type of the Ti plasmid present in the bacterium. The determinants for chemotaxis to these opines were localized to the regions of the octopine- and nopaline-type Ti plasmids coding for transport and catabolism of that opine. An insertion in accA, which encodes the periplasmic binding protein for agrocinopines A + B, abolished chemotaxis while an insertion in accC, which encodes a component of the transport system, and an insertion in accF, which encodes a function required for agrocinopine catabolism, did not affect chemotaxis to this opine. Thus, transport and catabolism of these opines are not required for the chemotactic activity. Analyses of subclones of the acc region confirmed that accA is the only gene required from the Ti plasmid for chemotaxis to agrocinopines A + B. PMID:9450336

Kim, H; Farrand, S K

1998-02-01

378

High frequency Agrobacterium tumefaciens -mediated plant transformation induced by ammonium nitrate  

Microsoft Academic Search

Success in plant genetic transformation depends on the efficiency of explant regeneration and transgene integration. Whereas\\u000a the former one depends on explant totipotency, the latter depends on the activity of host DNA repair and chromatin organisation\\u000a factors. We analyzed whether factors that result in an increase in recombination frequency can also increase transformation\\u000a efficiency. Here, we report that a threefold

Alex Boyko; Aki Matsuoka; Igor Kovalchuk

2009-01-01

379

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

E-print Network

, transgenic plants were only induced from transformed primary embryogenic callus. Stable integration crops in the world. Breeding of roses using sexual hybridization is rather cumbersome due to a limited breeding methods [1]. Availability of both efficient regeneration and transfor- mation systems

Korban, Schuyler S.

380

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

Microsoft Academic Search

Several nopaline degrading strains and one octopine degrading strain are shown to loose oncogenicity as well as the ability to utilize these guanidine compounds when they are cured of their TI plasmid. To investigate whether the specific genes involved in the utilization of one or the other compound are located on the plasmid, plasmid-transfer experiments have been performed.

Gerrit Bomhoff; Pieter M. Klapwijk; Harry C. M. Kester; Robbert A. Schilperoort; Jean Pierre Hernalsteens; Jef Schell

1976-01-01

381

A rapid transformation method for Solanum tuberosum using binary Agrobacterium tumefaciens vectors  

Microsoft Academic Search

A tuber disc transformation and regeneration system was devised for potato (Solanum tuberosum). Tuber discs were found to be the most morphogenetic organ on a medium previously optimised for tomato regeneration. Shoot regeneration from tuber discs was rapid and transformed as shown by nopaline assays and Southern blot analysis. The ease and speed of the tuber disc method will allow

S. Sheerlnan; M. W. Bevan

1988-01-01

382

Agrobacterium tumefaciens-mediated transformation of the soybean pathogen Phomopsis longicolla  

Technology Transfer Automated Retrieval System (TEKTRAN)

Phomopsis seed decay (PSD) of soybean is caused primarily by the fungal pathogen Phomopsis longicolla. PSD impairs seed germination, reduces seedling vigor, and can substantially reduce stand establishment. In hot and humid conditions, PSD can cause significant yield losses. Few studies have explore...

383

Systemic Agrobacterium tumefaciens–mediated transfection of viral replicons for efficient transient expression in plants  

Microsoft Academic Search

Plant biotechnology relies on two approaches for delivery and expression of heterologous genes in plants: stable genetic transformation and transient expression using viral vectors. Although much faster, the transient route is limited by low infectivity of viral vectors carrying average-sized or large genes. We have developed constructs for the efficient delivery of RNA viral vectors as DNA precursors and show

Sylvestre Marillonnet; Carola Thoeringer; Romy Kandzia; Victor Klimyuk; Yuri Gleba

2005-01-01

384

BREEDING & GENETICS Linkage Analysis of Transgenes Inserted into Cotton via Agrobacterium tumefaciens Transformation  

Microsoft Academic Search

The location of transgenes inserted into a genome are important in genetic studies and breeding programs. We conducted linkage analysis between 2,4-D resistant transgenes and 14 morphological marker genes in upland cotton (Gossypium hirsutum L.). Two separate germlines that exhibited monogenic dominance for resistance to 2,4-D were selected for linkage analysis. Multiple marker lines T582 and T586 were crossed with

Russell J. Kohel; Jerry E. Quisenberry; Greg Cartwright; John Yu

2000-01-01

385

Purification and characterization of a methylene urea-hydrolyzing enzyme from Rhizobium radiobacter (Agrobacterium tumefaciens)  

E-print Network

Purification and characterization of a methylene urea-hydrolyzing enzyme from Rhizobium radiobacter , Bruce D. Hammockb a Department of Land, Air, and Water Resources, University of California, One Shields) to homogeneity using a four-step purification procedure with an overall yield of 3%. The active enzyme has

Hammock, Bruce D.

386

Dual Promoter of Agrobacterium tumefaciens Mannopine Synthase Genes is Regulated by Plant Growth Hormones  

Microsoft Academic Search

Temporal and spacial distribution of mannopine synthase (mas) promoter activity was determined throughout the development of transgenic tobacco plants using bacterial luciferase luxA and luxB as reporter genes. Luciferase activity was determined by luminometry in vitro and visualized by computer-enhanced single-photon video imaging in vivo. The activity of the mas dual promoters increased basipetally in developing plants and was wound-inducible

W. H. R. Langridge; K. J. Fitzgerald; C. Koncz; J. Schell; A. A. Szalay

1989-01-01

387

Dual promoter of Agrobacterium tumefaciens mannopine synthase genes is regulated by plant growth hormones  

Microsoft Academic Search

Temporal and spacial distribution of man- nopine synthase (mar) promoter activity was determined throughout the development of transgenic tobacco plants using bacterial luciferase luxA and luxB as reporter genes. Luciferase activity was determined by luminometry in vifm and visualized by computer-enhanced single-photon video imaging in vivo. The activity of the mas dual promoters increased basipetally in developing plants and was

388

Evidence for stable transformation of wheat by floraldip in Agrobacterium tumefaciens  

Technology Transfer Automated Retrieval System (TEKTRAN)

Hexaploid wheat is one of the world’s most important staple crops but genetic transformation is still challenging. We have developed a floral transformation protocol that does not utilize tissue culture. Three T-DNA wheat transformants have been produced in the germplasm line, Crocus, using this p...

389

Efficient regeneration and Agrobacterium tumefaciens mediated transformation of recalcitrant sweet potato (Ipomoea batatas L.) cultivars  

Microsoft Academic Search

Sweet potato is a major world crop and its behavior under in vitro culture is genotype dependent. We studied several factors influencing the regeneration and transformation efficiency of two recalcitrant cultivars: Jewel and CEMSA 78354. Growth regulators, explant preparation and removal of apical dominance were evaluated in order to optimize the regeneration steps. At the same time, the influence of

Rolando García González; Danalay Somonte Sánchez; Zurima Zaldúa Guerra; Jesús Mena Campos; Alina López Quesada; Rolando Morán Valdivia; Ariel D. Arencibia; Karla Quiroz Bravo; Peter DS

390

INSERTIONAL MUTAGENESIS OF SCLEROTINIA SCLEROTIORUM THROUGH AGROBACTERIUM TUMEFACIENS-MEDIATED TRANSFORMATION.  

Technology Transfer Automated Retrieval System (TEKTRAN)

Stem rot caused by Sclerotinia sclerotiorum is one of the most serious diseases of many economically important crops under conducive conditions. Despite extensive investigations on the disease, the genetic factors that control the pathogenesis of S. sclerotiorum are incompletely understood, except ...

391

First report of crown gall caused by Agrobacterium tumefaciens on Euphorbia esula/virgata in Europe  

Technology Transfer Automated Retrieval System (TEKTRAN)

Hypertrophy and hyperplasia resembling crown galls were found on roots of Euphorbia esula virgata occurring at a single site (47°34’32.52”N, 21° 27’ 38.31”E) in east-central Hungary in 2005. Leafy spurge (E. esula/virgata) is an invasive species causing substantial economic losses to the value of gr...

392

Organogenesis and Agrobacterium tumefaciens -mediated transformation of Eucalyptus saligna with P5CS gene  

Microsoft Academic Search

The purpose of this research was Eucalyptus saligna in vitro regeneration and transformation with P5CSF129A gene, which encodes ?1-pyrroline-5-carboxylate synthetase (P5CS), the key enzyme in proline biosynthesis. After selection of the most responsive genotype, shoot organogenesis was induced\\u000a on leaf explants cultured on a callus induction medium (CI) followed by subculture on a shoot induction medium (SI). Shoots\\u000a were subsequently

R. Dibax; C. Deschamps; J. C. Bespalhok Filho; L. G. E. Vieira; H. B. C. Molinari; M. K. F. De Campos; M. Quoirin

2010-01-01

393

Characterization of a putative RND-type efflux system in Agrobacterium tumefaciens  

Microsoft Academic Search

Sequencing of a 7277 bp fragment adjacent to the chvH locus of Agrobacteriumtumefaciens revealed four open reading frames (ORFs), designated ameR, ameA, ameB and ameC, respectively. These ORFs exhibit amino acid similarities to components of Resistance-Nodulation-Cell Division (RND) type efflux systems. AmeA and AmeB show high homology to membrane fusion proteins (MFP) and RND-type transporters, whereas AmeC shows similarity to

Wen-Tao Peng; Eugene W Nester

2001-01-01

394

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

395

Efficient gene targeting in Penicillium chrysogenum using novel Agrobacterium-mediated transformation approaches.  

PubMed

The industrial production of ?-lactam antibiotics by Penicillium chrysogenum has increased tremendously over the last decades, however, further optimization via classical strain and process improvement has reached its limits. The availability of the genome sequence provides new opportunities for directed strain improvement, but this requires the establishment of an efficient gene targeting (GT) system. Recently, mutations affecting the non-homologous end joining (NHEJ) pathway were shown to increase GT efficiencies following PEG-mediated DNA transfer in P. chrysogenum from 1% to 50%. Apart from direct DNA transfer many fungi can efficiently be transformed using the T-DNA transfer system of the soil bacterium Agrobacterium tumefaciens, however, for P. chrysogenum no robust system for Agrobacterium-mediated transformation was available. We obtained efficient AMT of P. chrysogenum spores with the nourseothricin acetyltransferase gene as selection marker, and using this system we investigated if AMT in a NHEJ mutant background could further enhance GT efficiencies. In general, AMT resulted in higher GT efficiencies than direct DNA transfer, although the final frequencies depended on the Agrobacterium strain and plasmid backbone used. Providing overlapping and complementing fragments on two different plasmid backbones via the same Agrobacterium host was shown to be most effective. This so-called split-marker or bi-partite method resulted in highly efficient GT (>97%) almost exclusively without additional ectopic T-DNA insertions. As this method provides for an efficient GT method independent of protoplasts, it can be applied to other fungi for which no protoplasts can be generated or for which protoplast transformation leads to varying results. PMID:23994321

de Boer, Paulo; Bronkhof, Jurian; Duki?, Karolina; Kerkman, Richard; Touw, Hesselien; van den Berg, Marco; Offringa, Remko

2013-12-01

396

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

397

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

398

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

399

Biological control of crown gall of grapevine, rose, and tomato by nonpathogenic Agrobacterium vitis strain VAR03-1.  

PubMed

A nonpathogenic strain of Agrobacterium vitis VAR03-1 was tested as a biological control agent for crown gall of grapevine (Vitis vinifera). When roots of grapevine, rose (Rose multiflora), and tomato (Lycopersicon esculentum) were soaked in a cell suspension of antagonists before planting in soil infested with tumorigenic A. vitis, A. rhizogenes, and A. tumefaciens, respectively, treatment with VAR03-1 significantly reduced the number of plants with tumors and disease severity in the three plant species. The inhibitory effects of treatment with VAR03-1 and the nonpathogenic A. rhizogenes strain K84 on crown gall of rose and tomato were almost identical, and the inhibitory effect of VAR03-1 on grapevine was superior to that of K84. Moreover, VAR03-1 greatly controlled crown gall of grapevine due to tumorigenic A. vitis in the field. VAR03-1 established populations averaging 10(6) colony forming units (CFU)/g of root in the rhizosphere of grapevine and persisted on roots for 2 years. VAR03-1 was bacteriocinogenic, producing a halo of inhibition against those three species of Agrobacterium. This is the first report that a nonpathogenic strain, VAR03-1, can effectively control crown gall caused by tumorigenic A. vitis, A. rhizogenes, and A. tumefaciens. PMID:18943411

Kawaguchi, A; Inoue, K; Ichinose, Y

2008-11-01

400

[A simple and highly efficient Agrobacterium-mediated rice transformation system].  

PubMed

A simple and highly efficient rice transformation system was established based on the studying of factors influencing the Agrobacterium-mediated rice transformation. Embryogenic calli derived from mature embryos were infected and cocultivated with A. tumefaciens EHA101 harboring binary vectors: pHQ9, pHQ10, pHQT3. The highest transformation frequency was about 100 hygromycin resistance calli per gram of calli explants, and above 85% of these calli could be regenerated into plants. The putative transgenic plants were confirmed by GUS assay and southern-blot analysis. Hygromycin resistance tests indicated that the segregation of transgene in T1 progeny corresponded to the Mendelian ratio, 3:1. This system will benefit the functional genomic study of rice by using T-DNA insertion mutagenesis and gene targeting. PMID:14574993

Li, Mei Ru; Li, Hong Qing

2003-08-01

401

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

402

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

PubMed

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

403

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

404

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.

405

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

406

Visualization of VirE2 protein translocation by the Agrobacterium type IV secretion system into host cells.  

PubMed

Type IV secretion systems (T4SS) can mediate the translocation of bacterial virulence proteins into host cells. The plant pathogen Agrobacterium tumefaciens uses a T4SS to deliver a VirD2-single stranded DNA complex as well as the virulence proteins VirD5, VirE2, VirE3, and VirF into host cells so that these become genetically transformed. Besides plant cells, yeast and fungi can efficiently be transformed by Agrobacterium. Translocation of virulence proteins by the T4SS has so far only been shown indirectly by genetic approaches. Here we report the direct visualization of VirE2 protein translocation by using bimolecular fluorescence complementation (BiFC) and Split GFP visualization strategies. To this end, we cocultivated Agrobacterium strains expressing VirE2 tagged with one part of a fluorescent protein with host cells expressing the complementary part, either fused to VirE2 (for BiFC) or not (Split GFP). Fluorescent filaments became visible in recipient cells 20-25 h after the start of the cocultivation indicative of VirE2 protein translocation. Evidence was obtained that filament formation was due to the association of VirE2 with the microtubuli. PMID:24376037

Sakalis, Philippe A; van Heusden, G Paul H; Hooykaas, Paul J J

2014-02-01

407

A Rapid and Stable Agrobacterium-Mediated Transformation Method of a Medicinal Plant Chelone glabra L.  

PubMed

Transformation approach is a useful tool for the study of gene function, the mechanism of molecular regulation, and increase usefulness of components by reverse genetic approach in plants. In this study, we developed a stable and rapid method for Agrobacterium-mediated transformation of a medicinal plant Chelone glabra L. using leaf explants. Stable transformants were obtained using Agrobacterium tumefaciens strains GV2260 and GV3101 that harbored the binary vector pBI121 and contained the neomycin phosphotransferase gene (NPT II) as a selectable marker and a reporter gene ?-glucuronidase (GUS). Putative transformants were identified by kanamycin selection and a histochemical assay. PCR and Southern blot analysis confirmed the integration of the GUS gene into transformed genomes as well as detected stable expression of the ?-glucuronidase gene (GUS) by RT-PCR. Resulting transformed plants had morphologically normal phenotypes. This method requires two changes of medium and few leaf explants as well as the transformation efficiency of 2-8 % after 2-3 months of inoculation. This method can provide a quick and economical transformation method for reverse genetic approach to change the secondary metabolic pathway to increase useful components in C. glabra. PMID:25492686

Gao, Zhenrui; Li, Ying; Chen, Jinhua; Chen, Zhixing; Cui, Min-Long

2015-03-01

408

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

409

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

410

Agrobacterium-mediated genetic transformation of Pogostemon cablin (Blanco) Benth. Using leaf explants: bactericidal effect of leaf extracts and counteracting strategies.  

PubMed

An optimized protocol for Agrobacterium tumefaciens-mediated transformation of patchouli using leaf disk explants is reported. In vitro antibacterial activity of leaf extracts of the plants revealed Agrobacterium sensitivity to the extracts. Fluorometric assay of bacterial cell viability indicated dose-dependent cytotoxic activity of callus extract against Agrobacterium cells. Addition of 0.1% Tween 20 and 2 g/l L-glutamine to Agrobacterium infection medium counteracted the bactericidal effect and significantly increased the T-DNA delivery to explants. A short preculture of explants for 2 days followed by infection with Agrobacterium in medium containing 150 ?M of acetosyringone were found essential for efficient T-DNA delivery. Cocultivation for 3 days at 22 °C in conjunction with other optimized factors resulted in maximum T-DNA delivery. The Agrobacterium-mediated transformation of leaf disk explants were found significantly related to physiological age of the explants, age and origin of the of the donor plant. Leaf explants from second node of the 3-month-old in vivo plants showed highest transformation efficiency (94.3%) revealed by transient GUS expression assay. Plants selected on medium containing 20 mg/l kanamycin showed stable GUS expression in leaves and stem. The elongated shoots readily developed roots on kanamycin-free rooting medium and on transfer to soil, plants were successfully established. Polymerase chain reaction (PCR) and reverse-transcriptase PCR analysis in putative plants confirmed their transgenic nature. The established transformation method should provide new opportunities for the genetic improvement of patchouli for desirable trait. PMID:22434351

Paul, Anamika; Bakshi, Souvika; Sahoo, Debee Prasad; Kalita, Mohan Chandra; Sahoo, Lingaraj

2012-04-01

411

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

412

Agrobacterium-mediated transformation of cotton.  

PubMed

There are many methods and techniques that can be used to transfer foreign genes into cells. In plant biotechnology, Agrobacterium-mediated transformation is a widely used traditional method for inserting foreign genes into plant genome and obtaining transgenic plants, particularly for dicot plant species. Agrobacterium-mediated transformation of cotton involves several important and also critical steps, which includes coculture of cotton explants with Agrobacterium, induction and selection of stable transgenic cell lines, recovery of plants from transgenic cells majorly through somatic embryogenesis, and detection and expression analysis of transgenic plants. In this chapter, we describe a detailed step-by-step protocol for obtaining transgenic cotton plants via Agrobacterium-mediated transformation. PMID:23143481

Zhang, Baohong

2013-01-01

413

Agrobacterium-Mediated Gene Transfer Results Mainly in Transgenic Plants Transmitting T-DNA as a Single Mendelian Factor  

PubMed Central

Forty-four independent transformed tobacco plants were obtained from a cocultivation experiment with Agrobacterium tumefaciens strains carrying modified Ti-plasmids. The transformed plants were either self-fertilized or crossed with nontransformed plants or with other transformed plants. The segregation of a phenotypic marker (kanamycin resistance) in the progenies of these plants was determined. In 40 cases out of 44, the segregation of the kanamycin resistance marker is consistent with Mendelian genetics. Among these 40 clones, 35 contain a single kanamycin resistance locus. The five others segregate two independent resistance loci. In two of the single insert clones, the segregation ratio after selfing indicates that the T-DNA insertion may have caused a recessive lethal mutation. PMID:17246346

Budar, F.; Thia-Toong, L.; Van Montagu, M.; Hernalsteens, J.-P.

1986-01-01

414

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

415

Agrobacterium-mediated genetic transformation of plants: biology and biotechnology  

Microsoft Academic Search

Agrobacterium-mediated genetic transformation is the dominant technology used for the production of genetically modified transgenic plants. Extensive research aimed at understanding and improving the molecular machinery of Agrobacterium responsible for the generation and transport of the bacterial DNA into the host cell has resulted in the establishment of many recombinant Agrobacterium strains, plasmids and technologies currently used for the successful

Tzvi Tzfira; Vitaly Citovsky

2006-01-01

416

Biological systems of the host cell involved in Agrobacterium infection  

Microsoft Academic Search

Summary Genetic transformation of plants by Agrobacterium, which in nature causes neoplastic growths, repre- sents the only known case of trans-kingdom DNA transfer. Furthermore, under laboratory conditions, Agrobacterium can also transform a wide range of other eukaryotic species, from fungi to sea urchins to human cells. How can the Agrobacterium virulence machinery function in such a variety of evolutionarily distant

Vitaly Citovsky; Stanislav V. Kozlovsky; Benoît Lacroix; Adi Zaltsman; Mery Dafny-Yelin; Shachi Vyas; Andriy Tovkach; Tzvi Tzfira

2007-01-01

417

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

418

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

419

Increased Agrobacterium-mediated transformation and rooting efficiencies in canola (Brassica napus L.) from hypocotyl segment explants  

NASA Technical Reports Server (NTRS)

An efficient protocol for the production of transgenic Brassica napus cv. Westar plants was developed by optimizing two important parameters: preconditioning time and co-cultivation time. Agrobacterium tumefaciens-mediated transformation was performed using hypocotyls as explant tissue. Two variants of a green fluorescent protein (GFP)-encoding gene--mGFP5-ER and eGFP--both under the constitutive expression of the cauliflower mosaic virus 35S promoter, were used for the experiments. Optimizing the preconditioning time to 72 h and co-cultivation time with Agrobacterium to 48 h provided the increase in the transformation efficiency from a baseline of 4% to 25%. With mGFP5-ER, the transformation rate was 17% and with eGFP it was 25%. Transgenic shoots were selected on 200 mg/l kanamycin. Rooting efficiency was 100% on half-strength Murashige and Skoog medium with 10 g/l sucrose and 0.5 mg/l indole butyric acid in the presence of kanamycin.

Cardoza, V.; Stewart, C. N.

2003-01-01

420

Molecular and genetic analysis of the transferred DNA regions of the root-inducing plasmid of Agrobacterium rhizogenes.  

PubMed Central

The T-DNA regions of the root-inducing (Ri) plasmid pRiA4b of Agrobacterium rhizogenes were characterized. Two regions, designated TL-DNA and TR-DNA, were found to be integrated and stably maintained in the plant genome. The TL-DNA spanned a 15- to 20-kilobase region of pRiA4b and was separated from the TR-DNA region by at least 15 kilobases of nonintegrated plasmid DNA. The TR-DNA region also spanned a 15- to 20-kilobase region of pRiA4b and included a region of homology to the tms morphogenic loci of the tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens. Eighteen deletions and 95 transposon insertions were generated in the T-DNA regions and tested for alterations in virulence. Insertions into four loci in the TL-DNA affected the morphology of root formation of Kalanchoë diagremontiana leaves and stems, but had no visible effects on other host plants. Insertions into two loci (tms-1 and tms-2) in the TR-DNA eliminated virulence symptoms on all plants tested, with the exception of K. diagremontiana stems, where sparse root formation occurred. Complementation experiments with Ri and Ti plasmid T-DNA mutations indicate that the tms genes of the two plasmids serve similar functions and suggest a functional relationship between one or more genes of the TL-DNA and the cytokinin synthesis locus tmr of the Ti plasmid. Images PMID:4044524

White, F F; Taylor, B H; Huffman, G A; Gordon, M P; Nester, E W

1985-01-01

421

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

422

Genome Sequences of Three Agrobacterium Biovars Help Elucidate the Evolution of Multichromosome Genomes in Bacteria? †  

PubMed Central

The family Rhizobiaceae contains plant-associated bacteria with critical roles in ecology and agriculture. Within this family, many Rhizobium and Sinorhizobium strains are nitrogen-fixing plant mutualists, while many strains designated as Agrobacterium are plant pathogens. These contrasting lifestyles are primarily dependent on the transmissible plasmids each strain harbors. Members of the Rhizobiaceae also have diverse genome architectures that include single chromosomes, multiple chromosomes, and plasmids of various sizes. Agrobacterium strains have been divided into three biovars, based on physiological and biochemical properties. The genome of a biovar I strain, A. tumefaciens C58, has been previously sequenced. In this study, the genomes of the biovar II strain A. radiobacter K84, a commercially available biological control strain that inhibits certain pathogenic agrobacteria, and the biovar III strain A. vitis S4, a narrow-host-range strain that infects grapes and invokes a hypersensitive response on nonhost plants, were fully sequenced and annotated. Comparison with other sequenced members of the Alphaproteobacteria provides new data on the evolution of multipartite bacterial genomes. Primary chromosomes show extensive conservation of both gene content and order. In contrast, secondary chromosomes share smaller percentages of genes, and conserved gene order is restricted to short blocks. We propose that secondary chromosomes originated from an ancestral plasmid to which genes have been transferred from a progenitor primary chromosome. Similar patterns are observed in select Beta- and Gammaproteobacteria species. Together, these results define the evolution of chromosome architecture and gene content among the Rhizobiaceae and support a generalized mechanism for second-chromosome formation among bacteria. PMID:19251847

Slater, Steven C.; Goldman, Barry S.; Goodner, Brad; Setubal, Joăo C.; Farrand, Stephen K.; Nester, Eugene W.; Burr, Thomas J.; Banta, Lois; Dickerman, Allan W.; Paulsen, Ian; Otten, Leon; Suen, Garret; Welch, Roy; Almeida, Nalvo F.; Arnold, Frank; Burton, Oliver T.; Du, Zijin; Ewing, Adam; Godsy, Eric; Heisel, Sara; Houmiel, Kathryn L.; Jhaveri, Jinal; Lu, Jing; Miller, Nancy M.; Norton, Stacie; Chen, Qiang; Phoolcharoen, Waranyoo; Ohlin, Victoria; Ondrusek, Dan; Pride, Nicole; Stricklin, Shawn L.; Sun, Jian; Wheeler, Cathy; Wilson, Lindsey; Zhu, Huijun; Wood, Derek W.

2009-01-01

423

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

424

Nucleotide Sequences of the Pseudomonas savastanoi Indoleacetic Acid Genes Show Homology with Agrobacterium tumefaciens T-DNA  

Microsoft Academic Search

We report the nucleotide sequences of iaaM and iaaH, the genetic determinants for, respectively, tryptophan 2-monooxygenase and indoleacetamide hydrolase, the enzymes that catalyze the conversion of L-tryptophan to indoleacetic acid in the tumor-forming bacterium Pseudomonas syringae pv. savastanoi. The sequence analysis indicates that the iaaM locus contains an open reading frame encoding 557 amino acids that would comprise a protein

Tetsuji Yamada; Curtis J. Palm; Bob Brooks; Tsune Kosuge

1985-01-01

425

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

426

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

427

The roles of bacterial and host plant factors in Agrobacterium-mediated genetic transformation  

E-print Network

transfer tool for biotechnology. KEY WORDS: Agrobacterium, genetic transformation, macromolecular transportThe roles of bacterial and host plant factors in Agrobacterium-mediated genetic transformation York, Stony Brook, NY, USA ABSTRACT The genetic transformation of plants mediated by Agrobacterium

Citovsky, Vitaly

428

77 FR 40880 - Agrobacterium radiobacter; Registration Review Proposed Decision; Notice of Availability  

Federal Register 2010, 2011, 2012, 2013, 2014

...EPA-HQ-OPP-2009-0878; FRL-9354-5] Agrobacterium radiobacter; Registration Review Proposed...registration review decision for the pesticide Agrobacterium radiobacter and opens a public comment...registration review decision for the pesticide Agrobacterium radiobacter strains...

2012-07-11

429

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

430

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

431

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

432

Agrobacterium vitis rezisztencia kialaktsa az iaaM szekvencia segtsgvel  

E-print Network

Agrobacterium vitis rezisztencia kialakítása az iaaM szekvencia segítségével DIPLOMAMUNKA iaaM(S4) génben 17 4. EREDMÉNYEK és MEGVITATÁSUK 18 4.1. Az A. vitis S4 iaaM szekvencia izolálása 18 4MS4 szekvenciát tartalmazó Agrobacterium vektor létrehozása 20 4.5. A. vitis S4 iaaM mutáns baktérium

433

Agrobacterium T-DNA integration: molecules and models  

E-print Network

, NY 11794-5215, USA Genetic transformation mediated by Agrobacterium involves the transfer of a DNA-kingdom DNA transfer [1]. Although used mainly for plant genetic engineering [2], Agrobacterium can transform unexplored, although several different T-DNA integration mechanisms have been suggested. Recent genetic

Citovsky, Vitaly

434

A highly efficient Agrobacterium mediated transformation system for chickpea wilt pathogen Fusarium oxysporum f. sp. ciceri using DsRed-Express to follow root colonisation.  

PubMed

The soil-borne fungus Fusarium oxysporum f. sp. ciceri (Foc) causes vascular wilt of chickpea (Cicer arietinum L.), resulting in substantial yield losses worldwide. Agrobacterium tumefaciens mediated transformation (ATMT) has served as a resourceful tool for plant-pathogen interaction studies and offers a number of advantages over conventional transformation systems. Here, we developed a highly efficient A. tumefaciens mediated transformation system for Foc. In addition, a binary vector for constitutive expression of red fluorescent protein (DsRed-Express) was used to study developmental stages and host-pathogen interactions. Southern hybridisation was performed to confirm the transformation event and the presence of T-DNA in selected hygromycin resistant transformants. Most of the transformants showed single copy integrations at random positions. Microscopic studies revealed significant levels of fluorescent protein, both in conidia and mycelia. Confocal microscopy of chickpea roots infected with the transformed Foc showed rapid colonisation. These studies will allow us to develop strategies to determine the mechanisms of Foc-chickpea interaction in greater detail and to apply functional genomics for the characterisation of involved genes at the molecular level either by insertional mutagenesis or gene knock-out. PMID:22397973

Islam, Md Nazrul; Nizam, Shadab; Verma, Praveen K

2012-06-20

435

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

436

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

437

The virC and virD operons of the Agrobacterium Ti plasmid are regulated by the ros chromosomal gene: analysis of the cloned ros gene.  

PubMed Central

The ros chromosomal gene is present in octopine and nopaline strains of Agrobacterium tumefaciens as well as in Rhizobium meliloti. This gene encodes a 15.5-kDa protein that specifically represses the virC and virD operons in the virulence region of the Ti plasmid. The ros gene was cloned from a genomic bank by electroporation and complementation in Agrobacterium cells. Reporter fusion to the ros gene indicates that the level of transcription is controlled in part by autoregulation. A consensus inverted repeat sequence present in the ros promoter and in the virC and virD promoters of pTiC58, pTiA6, and pRiA4b suggests that a specific Ros binding site exists in these promoters. In the virC and virD promoter region, this binding site is within a cluster of vir box consensus sequences in which the VirG protein binds. This suggests possible binding competition between Ros and VirG at the virC and virD promoters. That the Ros protein binds DNA is suggested by the presence of a 'zinc finger' consensus sequence in the protein. Images PMID:2013576

Cooley, M B; D'Souza, M R; Kado, C I

1991-01-01

438

A new high-frequency Agrobacterium-mediated transformation technique for Sesamum indicum L. using de-embryonated cotyledon as explant.  

PubMed

In spite of the economic importance of sesame (Sesamum indicum L.) and the recent availability of its genome sequence, a high-frequency transformation protocol is still not available. The only two existing Agrobacterium-mediated transformation protocols that are available have poor transformation efficiencies of less than 2%. In the present study, we report a high-frequency, simple, and reproducible transformation protocol for sesame. Transformation was done using de-embryonated cotyledons via somatic embryogenic stages. All the critical parameters of transformation, like incubation period of explants in pre-regeneration medium prior to infection by Agrobacterium tumefaciens, cocultivation period, concentrations of acetosyringone in cocultivation medium, kanamycin concentration, and concentration of plant hormones, including 6-benzylaminopurine, have been optimized. This protocol is superior to the two existing protocols in its high regeneration and transformation efficiencies. The transformed sesame lines have been tested by PCR, RT-PCR for neomycin phosphotransferase II gene expression, and ?-glucuronidase (GUS) assay. The regeneration frequency and transformation efficiency are 57.33 and 42.66%, respectively. T0 and T1 generation transgenic plants were analyzed, and several T1 plants homozygous for the transgenes were obtained. PMID:24590594

Chowdhury, Supriyo; Basu, Arpita; Kundu, Surekha

2014-09-01

439

Hairy Root Transformation Using Agrobacterium rhizogenes as a Tool for Exploring Cell Type-Specific Gene Expression and Function Using Tomato as a Model1[W][OPEN  

PubMed Central

Agrobacterium rhizogenes (or Rhizobium rhizogenes) is able to transform plant genomes and induce the production of hairy roots. We describe the use of A. rhizogenes in tomato (Solanum spp.) to rapidly assess gene expression and function. Gene expression of reporters is indistinguishable in plants transformed by Agrobacterium tumefaciens as compared with A. rhizogenes. A root cell type- and tissue-specific promoter resource has been generated for domesticated and wild tomato (Solanum lycopersicum and Solanum pennellii, respectively) using these approaches. Imaging of tomato roots using A. rhizogenes coupled with laser scanning confocal microscopy is facilitated by the use of a membrane-tagged protein fused to a red fluorescent protein marker present in binary vectors. Tomato-optimized isolation of nuclei tagged in specific cell types and translating ribosome affinity purification binary vectors were generated and used to monitor associated messenger RNA abundance or chromatin modification. Finally, transcriptional reporters, translational reporters, and clustered regularly interspaced short palindromic repeats-associated nuclease9 genome editing demonstrate that SHORT-ROOT and SCARECROW gene function is conserved between Arabidopsis (Arabidopsis thaliana) and tomato. PMID:24868032

Ron, Mily; Kajala, Kaisa; Pauluzzi, Germain; Wang, Dongxue; Reynoso, Mauricio A.; Zumstein, Kristina; Garcha, Jasmine; Winte, Sonja; Masson, Helen; Inagaki, Soichi; Federici, Fernán; Sinha, Neelima; Deal, Roger B.; Bailey-Serres, Julia; Brady, Siobhan M.

2014-01-01

440

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

441

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

442

Stable transformation and direct regeneration in Coffea canephora P ex. Fr. by Agrobacterium rhizogenes mediated transformation without hairy-root phenotype.  

PubMed

A system for genetic transformation of Coffea canephora by co-cultivation with Agrobacterium rhizogenes harbouring a binary vector has been developed. The objective of the present study was the genetic transformation and direct regeneration of transformants through secondary embryos bypassing an intervening hairy root stage. Transformants were obtained with a transformation efficiency up to 3% depending on the medium adjuv