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1

Stable inheritance of transgenes in rice plants transformed by Agrobacterium tumefaciens  

Microsoft Academic Search

Inheritance of foreign genes in rice transformed by Agrobacterium tumefaciens has been investigated up to the R4 progeny. Rice cultivar Tsukinohikari was transformed with A. tumefaciens strains LBA4404(pTOK233) and EHA101(pIG121Hm). Cultivar Koshihikari was transformed with LBA4404(pTOK233). pTOK233 is a \\

Y. Hiei; T. Komari

2

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

3

Transformation of the monocotyledonous Alstroemeria by Agrobacterium tumefaciens  

Microsoft Academic Search

An efficient procedure is described for the transformation of the monocotyledonous Alstroemeria by Agrobacterium tumefaciens via callus regeneration. Calli derived from ovules were co-cultivated with A. tumefaciens strains EHA101 and LBA4404, which harbored the binary vector plasmids pIG121Hm and pTOK233, respectively. These plasmids contain the ß-glucuronidase gene ( gusA) as a reporter gene and the hygromycin phosphotransferase and neomycin phosphotransferase II

M. Akutsu; T. Ishizaki; H. Sato

2004-01-01

4

Optimization of Agrobacterial ( Agrobacterium tumefaciens ) Transformation of Maize Embryogenic Callus  

Microsoft Academic Search

The method for genetic transformation of maize (Zea mays L.) via embryogenic callus infection with Agrobacterium tumefaciens was developed. Calli were co-cultivated with the overnight culture of A. tumefaciens strain LBA4404 harboring the pBI121 plasmid with the nptII and uidA genes. Thereafter, the sensitivity of calli and regenerated plantlets to kanamycin (Km) was determined. It was shown that kanamycin selection

S. A. Danilova; Yu. I. Dolgikh

2005-01-01

5

Carrot ( Daucus carota ) hypocotyl transformation using Agrobacterium tumefaciens  

Microsoft Academic Search

Daucus carota hypocotyl sections were transformed withAgrobacterium tumefaciens LBA4404 containing CaMV 35S promoter, ß-glucuronidase coding sequence and the nopaline synthase (Nos) poly adenylation sequences in Bin 19. Sliced sterile seedling hypocotyl segments were preincubated for 2 days, co-cultivated withAgrobacterium for an additional 2 days, and then transferred to medium containing 100ug\\/ml of kanamycin and 400ug\\/ml carbenicillin. In 6 weeks kanamycin

John C. Thomas; Mark J. Guiltinan; Silvia Bustos; Terry Thomas; Craig Nessler

1989-01-01

6

Effects of antibiotics on the elimination of Agrobacterium tumefaciens from loblolly pine ( Pinus taeda ) zygotic embryo explants and on transgenic plant regeneration  

Microsoft Academic Search

Three antibiotics were evaluated for their effects on the elimination of Agrobacterium tumefaciens during the genetic transformation of loblolly pine ( Pinus taeda L.) using mature zygotic embryos as targets. Agrobacterium tumefaciens strains, EHA105, GV3101, and LBA 4404, all harbouring the plasmid pCAMBIA1301, which carries the selectable marker gene, hygromycin phosphotransferase ( hpt) controlled by the cauliflower mosaic virus 35S

Wei Tang; Hongsong Luo; Ronald J. Newton

2004-01-01

7

Agrobacterium tumefaciens -mediated transformation of blueberry ( Vaccinium corymbosum L.)  

Microsoft Academic Search

Transient expression studies using blueberry leaf explants and monitored by ?-glucuronidase (GUS) assays indicated Agrobacterium tumefaciens strain EHA105 was more effective than LBA4404 or GV3101; and the use of acetosyringone (AS) at 100 ? M for inoculation and 6 days co-cultivation was optimum compared to 2, 4, 8, 10 or 12 days. Subsequently, explants of the cultivars Aurora, Bluecrop, Brigitta, and Legacy were

Guo-Qing Song; K. C. Sink

2004-01-01

8

Improved protocols for transformation of indica rice mediated by Agrobacterium tumefaciens  

Microsoft Academic Search

A highly efficient gene transfer method mediated by Agrobacterium tumefaciens was developed for Group I indica rice, which had been quite recalcitrant in tissue culture and transformation. Freshly isolated immature embryos from plants grown in a greenhouse were inoculated with A. tumefaciens LBA4404 that harbored super-binary vector pTOK233 or pSB134, which had a hygromycin-resistance gene and a GUS gene in

Yukoh Hiei; Toshihiko Komari

2006-01-01

9

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

10

A reliable protocol for transformation of Catharanthus roseus through Agrobacterium tumefaciens  

Microsoft Academic Search

Proliferation of axillary shoot buds and multiple shoot formation in Catharanthus roseus was obtained in 96 % explants on MS medium (3 % sucrose) containing NAA + BA. 2,4-D induced callusing in both, the nodal\\u000a as well as in leaf segments. Leaf-derived callus was used for transformation with Agrobacterium tumefaciens LBA4404\\/pBI-S1. Bacterial cell concentration, duration of co-cultivation and acetosyringone concentration

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

2009-01-01

11

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

12

Stable transformation of protocorm-like bodies in Phalaenopsis orchid mediated by Agrobacterium tumefaciens  

Microsoft Academic Search

Genetically transformed plantlets of Phalaenopsis were regenerated after co-cultivation of protocorm-like bodies (PLBs) with Agrobacterium tumefaciens strain LBA4404 containing the vector pTOK233 that harbors genes for ?-glucuronidase and hygromycin resistance. Four lines of Phalaenopsis orchid (T0, T5, T10 and Hikaru) were tested on three multiplication media for the production of PLBs. Two types of explants, intact and transversely bisected PLBs,

M. L Chai; C. J Xu; K. K Senthil; J. Y Kim; D. H Kim

2002-01-01

13

An efficient protocol for sugarcane (Saccharum spp. L.) transformation mediated by Agrobacterium tumefaciens  

Microsoft Academic Search

This is the first successful report of the recovery of morphologically normal transgenic sugarcane plants from co-cultivation of calluses with Agrobacterium tumefaciens. Transformation frequencies (total of transgenic plants\\/number of cell clusters) were between 9.4 × 10-3 and 1.15 × 10-2. In our experiments, both LBA4404 (pTOK233) and EHA101 (pMTCA3IG), carrying a super-binary vector or supervirulent strain, respectively, were successful for

Ariel D. Arencibia; Elva R. Carmona; Pilar Tellez; Ming-Tsair Chan; Su-May Yu; Luis E. Trujillo; Pedro Oramas

1998-01-01

14

Genetic transformation mediated by Agrobacterium tumefaciens of florists' chrysanthemum ( Dendranthema xgrandiflorum ) cultivar ‘Peach Margaret’  

Microsoft Academic Search

Summary  A genetic transformation method usingAgrobacterium tumefaciens strain LBA4404 and based on the neomycin phosphotransferase II (nptII) selectable marker gene is described for a cultivar of florists' chrysanthemum,Dendranthema Xgrandiflorum ‘Peach Margaret’. We used the flavonoid regulatory cDNA,Leaf color (Lc) from the monocotZea mays (maize), under control of the cauliflower mosaic virus 35S promoter, to test if it would serve as a

M. R. Boase; J. M. Bradley; N. K. Borst

1998-01-01

15

Haploid transformation in Brassica napus using an octopine-producing strain of Agrobacterium tumefaciens  

Microsoft Academic Search

Microspore-derived embryos of Brassica napus were transformed using the disarmed octopine-producing LBA4404 strain of Agrobacterium tumefaciens containing the binary vector pBin19. Octopine-producing strains have previously been reported to be ineffective in transforming Brassica. Four actively growing yellow\\/ green sectors were selected from the embryos on 50 mg\\/l kanamycin and plants regenerated. Analysis for NPT-II activity in these young plants initially

E. B. Swanson; L. R. Erickson

1989-01-01

16

Factors influencing Agrobacterium tumefaciens-mediated transformation and regeneration of the safflower cultivar ‘centennial’  

Microsoft Academic Search

The effects of co-cultivation conditions on transformation efficiency and direct shoot regeneration from seedling explants of safflower cv. ‘Centennial’ were examined. Agrobacterium tumefaciens strain EHA105\\/p35SGUSInt was more infective than LBA4404\\/pBI121 as determined by numbers of sectors expressing ß-glucuronidase activity. Compared to nontransformed controls, efficiency of direct shoot regeneration was markedly decreased by co-cultivation with EHA105 and the decrease exacerbated by

Teresa K. Orlikowska; Harwood J. Cranston; William E. Dyer

1995-01-01

17

Agrobacterium tumefaciens -mediated creeping bentgrass ( Agrostis stolonifera L.) transformation using phosphinothricin selection results in a high frequency of single-copy transgene integration  

Microsoft Academic Search

Genetic transformation of creeping bentgrass mediated by Agrobacterium tumefaciens has been achieved. Embryogenic callus initiated from seeds (cv. Penn-A-4) was infected with an A. tumefaciens strain (LBA4404) harboring a super-binary vector that contained an herbicide-resistant bar gene driven either by the CaMV 35S promoter or a rice ubiquitin promoter. Plants were regenerated from 219 independent transformation events. The overall stable transformation

H. Luo; Q. Hu; K. Nelson; C. Longo; A. P. Kausch; J. M. Chandlee; J. K. Wipff; C. R. Fricker

2004-01-01

18

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

Microsoft Academic Search

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\\u000a by Agrobacterium tumefaciens, strain LBA 4404, harbouring virus-derived hairpin (hp) RNA gene constructs to induce RNA silencing-mediated resistance to\\u000a Poinsettia mosaic virus (PnMV). Prior to transformation, an efficient somatic embryogenesis system was developed for poinsettia

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

2008-01-01

19

Agrobacterium tumefaciens-mediated transformation of Pinus pinea L. cotyledons: an assessment of factors influencing the efficiency of uidA gene transfer  

Microsoft Academic Search

This study is the first report of a protocol for transfer and expression of foreign chimeric genes into cotyledons excised\\u000a from Pinus pinea L. embryos. Agrobacterium tumefaciens EHA105 harbouring the plasmid p35SGUSint was more infective than LBA4404 or C58 GV3850, as determined by the percentage of\\u000a cotyledons showing uidA expression. Factors which significantly affected the T-DNA transfer included: (1) preinduction

J. M. Humara; M. López; R. J. Ordás

1999-01-01

20

Agrobacterium-mediated sorghum transformation  

Microsoft Academic Search

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

Zuo-yu Zhao; Tishu Cai; Laura Tagliani; Mike Miller; Ning Wang; Hong Pang; Marjorie Rudert; Sheryl Schroeder; Dave Hondred; Jon Seltzer; Dortie Pierce

2000-01-01

21

Obtaining of transgenic french bean plants ( Phaseolus vulgaris L.) resistant to the herbicide pursuit by agrobacterium-mediated transformation  

Microsoft Academic Search

The transgenic plants of French bean (Phaseolus vulgaris) resistant herbicide Pursuit and kanamycin have been obtained. The\\u000a genetic transformation was carried out with Agrobacterium tumefaciens strain LBA4404 containing binary vector carrying mutant ahas\\/als and selective nptII genes. Integration of the transgenes into plant genome was confirmed by polymerase chain reaction.

S. N. Nifantova; I. K. Komarnickiy; N. V. Kuchuk

2011-01-01

22

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

2013-09-07

23

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

24

Agrobacterium tumefaciens-mediated transformation of Narcissus tazzeta var. chinensis.  

PubMed

Phytoene synthase (PSY), as a key regulatory enzyme for carotene biosynthesis, plays an important role in regulating color formation in many species. In the present study, a protocol was developed for the transformation of Narcissus tazzeta var chinensis using Agrobacterium tumefaciens strain LBA4404 harboring a binary vector pCAMBIA1301 plasmid which contained an antisense phytoene synthase gene, a reporter beta-glucuronidase gene and a selectable marker hygromycin phosphotransferase gene. Effects of some factors on efficiency of transformation and regeneration were examined. Preculture of the explants for 6 days before inoculation enhanced the transient GUS expression. The addition of acetosyringone (AS) at 100 micromol l(-1) for inoculation and a period of 3 days co-cultivation yielded efficient transient GUS expression. Transformants were obtained through selection on MS medium containing 5 mg l(-1) 6-benzylaminopurine (BA), 0.1 mg l(-1)alpha-naphthalene acetic acid (NAA) and 40 mg l(-1) hygromycin. The transformation frequency was 1.24% based on PCR analysis of gus gene. One or two copies of transgene were demonstrated in different transformations by Southern blotting analyses. Northern blotting results confirmed that the transcription of the endogenous psy gene in transgenic plants was inhibited or silenced. The method reported here provides new opportunities for improvement of quality traits of Narcissus tazzeta via genetic transformation. PMID:17541598

Lu, Gang; Zou, Qingcheng; Guo, Deping; Zhuang, Xiaoying; Yu, Xiaolin; Xiang, Xun; Cao, Jiashu

2007-06-01

25

Agrobacterium-mediated genetic transformation of a phalaenopsis orchid  

Microsoft Academic Search

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

M. M. Belarmino; M. Mii

2000-01-01

26

Highly efficient Agrobacterium -mediated transformation of banana cv. Rasthali (AAB) via sonication and vacuum infiltration  

Microsoft Academic Search

A reproducible and efficient transformation method was developed for the banana cv. Rasthali (AAB) via Agrobacterium-mediated genetic transformation of suckers. Three-month-old banana suckers were used as explant and three Agrobacterium tumefaciens strains (EHA105, EHA101, and LBA4404) harboring the binary vector pCAMBIA1301 were used in the co-cultivation. The banana\\u000a suckers were sonicated and vacuum infiltered with each of the three A.

Kondeti Subramanyam; Koona Subramanyam; K. V. Sailaja; M. Srinivasulu; K. Lakshmidevi

2011-01-01

27

An effective method of sonication-assisted Agrobacterium -mediated transformation of chickpeas  

Microsoft Academic Search

An efficient and reproducible transformation method of sonication- assisted Agrobacterium-mediated transformation (SAAT) was developed for chickpea (Cicer arietinum L.). Agrobacterium tumefaciens (LBA4404) harboring pCAMBIA1305.2 was used to transform decapitated embryo explants of two cultivars of chickpeas. By using\\u000a a series of co-cultivation, callus induction, shoot initiation and root inducing media, a large number of transgenic plants\\u000a were recovered. Transient expressions

Malabika Roy Pathak; Riyad Yousif Hamzah

2008-01-01

28

Transgenic grasspea ( Lathyrus sativus L.): Factors influencing Agrobacterium -mediated transformation and regeneration  

Microsoft Academic Search

A reproducible procedure was developed for genetic transformation of grasspea using epicotyl segment co-cultivation with Agrobacterium. Two disarmed Agrobacterium tumefaciens strains, EHA 105 and LBA 4404, both carrying the binary plasmid p35SGUSINT with the neomycin phosphotransferase II (nptII) gene and the ?-glucuronidase (gus)-intron, were studied as vector systems. The latter was found to have a higher transforming ability. Several key factors modifying the

D. P. Barik; U. Mohapatra; P. K. Chand

2005-01-01

29

Transgenic regal pelargoniums that express the rol C gene from Agrobacterium rhizogenes exhibit a dwarf floral and vegetative phenotype  

Microsoft Academic Search

Summary  The regal pelargonium, ev. Dubonnet, was transformed using the disarmed Agrobacterium tumefaciens strains LBA4404 or EHA105 containing the binary vector pLN70. This plasmid carries on its T-DNA the rolC gene from Agrobacterium rhizogenes under control of the CaMV 35S promoter and the npt II selectable marker gene under a NOS promoter. Six independent transformants were produced and grouped according to

M. R. Boase; C. S. Winefield; T. A. Lill; M. J. Bendall

2004-01-01

30

Transgenic tea [ Camellia sinensis (L.) O. Kuntze cv. Kangra Jat] plants obtained by Agrobacterium -mediated transformation of somatic embryos  

Microsoft Academic Search

A protocol for the production of transgenic tea [Camellia sinensis (L.) O. Kuntze cv. Kangra Jat] was developed via Agrobacterium-mediated genetic transformation of somatic embryos. Two disarmed Agrobacterium tumefaciens strains, EHA 105 and LBA 4404, both carrying the binary plasmid p35SGUSINT with the nptII gene and gus-intron were evaluated as vector systems. A number of parameters were tested with respect

T. K. Mondal; A. Bhattacharya; P. S. Ahuja; P. K. Chand

2001-01-01

31

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

Microsoft Academic Search

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\\u000a 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 OD600 0.3 with 48 h

Meenakshi Mehrotra; Indraneel Sanyal; D. V. Amla

32

Stable transformation of suspension-cultured Glycyrrhiza inflata batalin cells with Agrobacterium tumefaciens.  

PubMed

A protocol for the efficient genetic transformation of licorice (Glycyrrhiza inflata Batalin) cells in suspension culture using Agrobacterium tumefaciens-mediated T-DNA delivery is described. G. inflata cells in suspension culture were infected with A. tumefaciens strain LBA4404 harbouring the binary vector pCAMBIA1303, which contains the beta-glucuronidase (GUS) reporter gene and a hygromycin resistance gene (hpt II), respectively, under the transcriptional control of the CaMV35S promoter. Optimal transformation efficiency was achieved with an A. tumefaciens suspension having an OD600 of 0.4 and a period of 24 h of co-cultivation with 3-day-old cells in a medium supplemented with 200 microM acetosyringone. The transgenic cell lines have been maintained in suspension subculture for 5 months. PCR and Southern blot analyses confirmed the stable integration of transgenes into the G. inflata genome. The introduced genes had no discernable effect on cell growth or accumulation of total licorice flavonoids in the transgenic cell lines. This study provides the basis for the development of transgenic G. inflata cells. PMID:23413755

Li, Yali; Li, Shutao; Dong, Yanshan; Zhang, Yu; Fu, Chunhua; Yu, Longjiang

33

Agrobacterium tumefaciens-mediated transformation of blueberry (Vaccinium corymbosum L.).  

PubMed

Transient expression studies using blueberry leaf explants and monitored by beta-glucuronidase (GUS) assays indicated Agrobacterium tumefaciens strain EHA105 was more effective than LBA4404 or GV3101; and the use of acetosyringone (AS) at 100 microM for inoculation and 6 days co-cultivation was optimum compared to 2, 4, 8, 10 or 12 days. Subsequently, explants of the cultivars Aurora, Bluecrop, Brigitta, and Legacy were inoculated with strain EHA105 containing the binary vector pBISN1 with the neomycin phosphotransferase gene (nptII) and an intron-interrupted GUS gene directed by the chimeric super promoter (Aocs)3AmasPmas. Co-cultivation was for 6 days on modified woody plant medium (WPM) plus 100 microM AS. Explants were then placed on modified WPM supplemented with 1.0 mg l(-1) thidiazuron, 0.5 mg l(-1) alpha-naphthaleneacetic, 10 mg l(-1) kanamycin (Km), and 250 mg l(-1) cefotaxime. Selection for Km-resistant shoots was carried out in the dark for 2 weeks followed by culture in the light at 30 microE m(-2) s(-1) at 25 degrees C. After 12 weeks, selected shoots that were both Km resistant and GUS positive were obtained from 15.3% of the inoculated leaf explants of cultivar Aurora. Sixty-eight independent clones derived from such shoots all tested positive by the polymerase chain reaction using a nptII primer. Eight of eight among these 68 clones tested positive by Southern hybridization using a gusA gene derived probe. The transformation protocol also yielded Km-resistant, GUS-positive shoots that were also PCR positive at frequencies of 5.0% for Bluecrop, 10.0% for Brigitta and 5.6% for Legacy. PMID:15300402

Song, Guo-Qing; Sink, K C

2004-08-05

34

Agrobacterium -mediated transformation of herbicide resistance in creeping bentgrass and colonial bentgrass  

Microsoft Academic Search

Embryogenic calli were induced from the seeds of creeping bentgrass (Agrostis palustris Huds.) cv. Regent and colonial bentgrass (Agrostis Tenuis Sibth. Fl. Oxen.) cv. Tiger. The embryogenic calli were precultured on fresh medium for 4–7 days and then co-cultivated withAgrobacterium tumefaciens, LBA4404. which contains plasmid vector-pSBGM harboring bar coding region, synthetic green fluorescent protein (sGFP) coding\\u000a region and matrix attachment

Ming-liang Chai; Bing-liang Wang; Jae-yeoul Kim; Jong-min Lee; Doo-hwan Kim

2003-01-01

35

Chrysanthemum cultivar– Agrobacterium interactions revealed by GUS expression time course experiments  

Microsoft Academic Search

Nine 24-day GUS expression time course experiments involving 2760 leaf explants revealed complex host–bacterium interactions between four cultivars of florists' chrysanthemum from two sources (in vitro and glasshouse) and four disarmed strains of Agrobacterium tumefaciens (EHA105, LBA4404, MOG101, MOG301) containing the binary vectors pMOG410 or pKIWI110. Cultivars Peach Margaret and Yellow Lucondra [DendranthemaGrandiflorum] were more easily transformed than cv. Korean

M. R Boase; R. C Butler; N. K Borst

1998-01-01

36

Agrobacterium -mediated transformation and regeneration of fertile transgenic plants of chinese cabbage ( brassica campestris ssp. pekinensis cv. ‘spring flavor’)  

Microsoft Academic Search

A procedure for the regeneration of fertile transgenic Chinese cabbage (Brassica campestris ssp. pekinensis cv. Spring Flavor) is presented in this report. The protocol is based on infection of cotyledon explants of 5-d-old seedlings with an Agrobacterium tumefaciens strain LBA4404 carrying a disarmed binary vector pTOK\\/BKS-1. The T-DNA region of this binary vector contains the nopaline synthase\\/neomycin phosphotransferase II (nptII)

Se Jun; Seok Yoon Kwon; Kee Yoeup Pack; Kyung-Hee Paek

1995-01-01

37

[Factors affecting Agrobacterium tumefaciens-mediated transformation of wheat (Triticum aestivum L.)].  

PubMed

Immature embryos and embryo-derived calli from two cultivars of winter wheat (Triticum aestivum L.), BAU146 and BAU170, were transformed with three strains of Agrobacterium tumefaciens, AGL-1, EHA105 and LBA4404 harboring expression vector p3301 or pBTAaB. Both vectors contained bar gene and p3301 contained also gus gene with an intron. The highest explant survival rate and transformation efficiency was obtained when the bacterial cell density was OD600 1.0 with 1 h of infection incubation. Higher osmotic treatment of the explants before inoculation had a positive effect on transformation, while addition of acetosyringone showed ambiguous one, depending on the explant types and bacterium strains. The efficiencies of transformation and transgenic plant regeneration were varied greatly with the bacterium strain, receptor genotype, explant type and its age and physiological state. After optimizing these factors, a large number of PPT-resistant calli and some of PPT-resistant plants were obtained. The resistant plantlet tested and 50% to 60% of the resistant calli were GUS-positive. The integration of foreign DNA into the genome of transgenic plants (3 out 6) was further confirmed by PCR and Southern Blot analysis. PMID:12182083

Wang, Yong-Qin; Xiao, Xing-Guo; Zhang, Ai-Min

2002-01-01

38

Genetic transformation in two potato cultivars with T-DNA from disarmed Agrobacterium  

Microsoft Academic Search

Derivatives of potato (Solanum tuberosum cv.'s ‘Maris Bard’ and ‘Desiree’) transformed with disarmed T-DNA from genetically engineered Agrobacterium tumefaciens strains were isolated. The transformed plants were recovered from shoot-forming tumours induced by infection of wounds with mixedcultures of shoot-inducing A. tumefaciens strains T37 and either Agrobacterium strain LBA1834(pRAL1834), (Hille et al. 1983) or LBA4404(pBIN6; pRAL4404), (Bevan 1984). Two small-scale feasibility

G. Ooms; M. M. Burrell; A. Karp; M. Bevan; J. Hille

1987-01-01

39

Transformation of Liquidambar styraciflua using Agrobacterium tumefaciens  

Microsoft Academic Search

We describe the molecular transformation of Liquidambar styraciflua using Agrobacterium tumefaciens. A binary TI-plasmid vector containing a chimeric neomycin phosphotransferagene which confers resistance to kanamycin and either a chimeric Bacillus thuringiensis toxin gene, a chimeric E. coli ß-glucuronida(GUS), or a chimeric tobacco anionic peroxidase gene was introduced into sweetgum by co-cultivation with Agrobacterium tumefaciens. Sweetgum shoots regenerated in the presence

Janet Sullivan; L. Mark Lagrimini

1993-01-01

40

Agrobacterium tumefaciens-mediated creeping bentgrass (Agrostis stolonifera L.) transformation using phosphinothricin selection results in a high frequency of single-copy transgene integration.  

PubMed

Genetic transformation of creeping bentgrass mediated by Agrobacterium tumefaciens has been achieved. Embryogenic callus initiated from seeds (cv. Penn-A-4) was infected with an A. tumefaciens strain (LBA4404) harboring a super-binary vector that contained an herbicide-resistant bar gene driven either by the CaMV 35S promoter or a rice ubiquitin promoter. Plants were regenerated from 219 independent transformation events. The overall stable transformation efficiency ranged from 18% to 45%. Southern blot and genetic analysis confirmed transgene integration in the creeping bentgrass genome and normal transmission and stable expression of the transgene in the T1 generation. All independent transformation events carried one to three copies of the transgene, and a majority (60-65%) contained only a single copy of the foreign gene with no apparent rearrangements. We report here the successful use of Agrobacterium for the large-scale production of transgenic creeping bentgrass plants with a high frequency of a single-copy transgene insertion that exhibit stable inheritance patterns. PMID:14615907

Luo, H; Hu, Q; Nelson, K; Longo, C; Kausch, A P; Chandlee, J M; Wipff, J K; Fricker, C R

2003-11-13

41

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

42

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

PubMed Central

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

Wabiko, H; Minemura, M

1996-01-01

43

Differenzialdiagnostische Wirtspflanzen für Agrobacterium tumefaciens und Agrobacterium rhizogenes  

Microsoft Academic Search

\\u000a Abstract  P?i naší práci s nádorotvornými bakteriemi se ukázalo, žeBryophyllum diagremontiana L.,Bryophyllum tubiflorum L. aSolanum laciniatum L. jsou vhodné diferen?ní rostliny proAgrobacterium tumefaciens, Agrobacterium rhizogenes a sm?s obou t?chto bakterií.\\u000a \\u000a \\u000a 1. \\u000a \\u000a NaAgrobacterium tumefaciens reagujeBryophyllum diagremontiana tvorbou nádor? za 32 dní po o?kování a stimulaci r?stu ko?ínk? z okraj? ná dor? (za 52 dn?). UBryophyllum tubiflorum je inkubacni doba nádor? i ko?ínk?

Jaroslav Limberk; Wissenschaften Praha

1962-01-01

44

Agrobacterium -mediated transformation of Codonopsis lanceolata using the ?-TMT gene  

Microsoft Academic Search

Efficient transformation of leaf disc-derived callus of Codonopsis lanceolata was obtained using Agrobacterium tumefaciens strain LBA4404 harboring a binary vector, pYBI121, that carries the neomycin phosphotransferase (npt II) gene as a selectable marker. The green shoots recovered from agroinfected explants on selection medium (containing 0.1 mg\\/l\\u000a ?-naphthaleneacetic acid (NAA), 1 mg\\/l 6-benzylaminopurine (BAP), 100 mg\\/l kanamycin, and 250 mg\\/l cefotaxime) were rooted\\u000a on Murashige

Bimal Kumar Ghimire; Eun Soo Seong; Jung Dae Lim; Kweon Heo; Myong Jo Kim; Ill-Min Chung; John A. Juvik; Chang Yeon Yu

2008-01-01

45

The Oncogenes of Agrobacterium Tumefaciens and Agrobacterium Rhizogenes  

Microsoft Academic Search

The common soil bacteria Agrobacterium tumefaciens and Agrobacterium rhizogenes are unique genetic pathogens capable of fundamentally redirecting plant metabolism in order to generate macroscopic tissue\\u000a masses (crown galls and hairy roots, respectively) which support the growth of large populations of Agrobacteria. Central to pathogenesis is the horizontal transfer of a suite of oncogenes from the tumor-inducing (Ti) plasmids of A.

Monica T. Britton; Matthew A. Escobar; Abhaya M. Dandekar

46

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

PubMed

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

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

2001-01-01

47

Plant gene expression response to Agrobacterium tumefaciens  

PubMed Central

To elucidate the nature of plant response to infection and transformation by Agrobacterium tumefaciens, we compared the cDNA-amplified fragment length polymorphism (AFLP) pattern of Agrobacterium- and mock-inoculated Ageratum conyzoides plant cell cultures. From 16,000 cDNA fragments analyzed, 251 (1.6%) were differentially regulated (0.5% down-regulated) 48 h after cocultivation with Agrobacterium. From 75 strongly regulated fragments, 56 were already regulated 24 h after cocultivation. Sequence similarities were obtained for 20 of these fragments, and reverse transcription–PCR analysis was carried out with seven to confirm their cDNA-AFLP differential pattern. Their sequence similarities suggest a role for these genes in signal perception, transduction, and plant defense. Reverse transcription–PCR analysis indicated that four genes involved in defense response are regulated in a similar manner by nonpathogenic bacteria, whereas one gene putatively involved in signal transduction appeared to respond more strongly to Agrobacterium. A nodulin-like gene was regulated only by Agrobacterium. These results demonstrate a rapid plant cell response to Agrobacterium infection, which overlaps a general response to bacteria but also has Agrobacterium-specific features.

Ditt, Renata F.; Nester, Eugene W.; Comai, Luca

2001-01-01

48

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

49

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

50

Control of Pantothenate Accumulation in Agrobacterium tumefaciens  

PubMed Central

Two pantothenate-requiring mutants of Agrobacterium tumefaciens have been isolated. One of them (strain WMP-1) is unusual in that growth levels equivalent to the parent strain are achieved only when the medium is additionally supplemented with aspartate or another compound related to the tricarboxylic acid cycle. Extracts of cells grown on limiting aspartate were found to contain four times more 14C-pantothenate than those grown at optimal aspartate concentrations. This difference was found in both the perchloric acid-soluble and -insoluble fractions, presumably the coenzyme A pool and acyl carrier protein, respectively. These findings are discussed in terms of membrane integrity and the control of fatty acid biosynthesis.

Kaneshiro, Tsuneo; Arthur, Larry O.; Nickerson, Kenneth W.

1973-01-01

51

Auxin synthesis in Agrobacterium tumefaciens and A. tumefaciens-transformed plant tissue  

Microsoft Academic Search

Problems of IAA biosynthesis, IAA precursors and IAA biosynthetic pathways in Agrobacterium tumefaciens-transformed plant tissue, especially in tobacco tissue culture are reviewed. The knowkedge about levels of IAA and the IAA biosynthetic pathways in Agrobacterium tumefaciens itself is also summarized.

M. Kutá?ek; J. Rovenská

1991-01-01

52

Bacterial transposons are co-transferred with T-DNA to rice chromosomes during Agrobacterium-mediated transformation.  

PubMed

Agrobacterium tumefaciens is widely utilized for delivering a foreign gene into a plant's genome. We found the bacterial transposon Tn5393 in transgenic rice plants. Analysis of the flanking sequences of the transferred-DNA (T-DNA) identified that a portion of the Tn5393 sequence was present immediately next to the end of the T-DNA. Because this transposon was present in A. tumefaciens strain LBA4404, but not in EHA105 and GV3101, our findings indicated that Tn5393 was transferred from LBA4404 into the rice genome during the transformation process. We also noted that another bacterial transposon, Tn5563, is present in transgenic plants. Analyses of 331 transgenic lines revealed that 26.0% carried Tn5393 and 2.1% contained Tn5563. In most of the lines, an intact transposon was integrated into the T-DNA and transferred to the rice chromosome. More than one copy of T-DNA was introduced into the plants, often at a single locus. This resulted in T-DNA repeats of normal and transposon-carrying TDNA that generated deletions of a portion of the T-DNA, joining the T-DNA end to the bacterial transposon. Based on these data, we suggest that one should carefully select the appropriate Agrobacterium strain to avoid undesirable transformation of such sequences. PMID:22570148

Kim, Sung-Ryul; An, Gynheung

2012-05-07

53

AMINO ACID CROSS RESISTANCE IN AGROBACTERIUM TUMEFACIENS  

PubMed Central

Beardsley, Robert E. (Manhattan College, New York, N. Y.). Amino acid cross resistance in Agrobacterium tumefaciens. J. Bacteriol. 84:1237–1240. 1962.—Resistant clones selected on medium supplemented with glycine were also resistant to d-methionine, d-valine, dl-norleucine, and dl-serine. Cross resistance was similarly exhibited by clones selected on d-methionine, d-valine, or dl-norleucine. Two types of resistant organisms were observed. One produced colonies containing normal rods on selection medium. The other produced translucent colonies containing L forms. Both grew as typical rods in unsupplemented medium. Some resistant clones did not produce a temperate phage carried by the parental strain, but these retained immunity to homologous phage. The toxicity of d-methionine and d-valine for nonresistant bacteria is not reversed by the l isomers. The lethal effects of toxic amino acids are additive.

Beardsley, Robert E.

1962-01-01

54

Untersuchungen zur genetischen Transformation zwischen Agrobacterium tumefaciens und Rhizobium spec  

Microsoft Academic Search

Mit DNA von virulenten Agrobacterium tumefaciens-Stämmen gelingt die Übertragung der Fähigkeit zur Induktion pflanzlicher Tumoren auf einige Rhizobium-Stämme. Unter Berücksichtigung kritischer Fehlerquellen machen die beschriebenen Untersuchungen das Vorliegen einer echten genetischen Transformation sehr wahrscheinlich.

Hartmut Kern

1965-01-01

55

High efficiency transformation of tall fescue with Agrobacterium tumefaciens  

Microsoft Academic Search

An efficient genetic transformation system for tall fescue (Festuca arundinacea Schreb.), using Agrobacterium tumefaciens-mediated T-DNA delivery, is described. Seed-derived embryogenic calli were infected with Agrobacterium tumefaciens strain EHA105 harboring plasmids pTOK47 and pCAMBIA1301. Infected calli were selected at 250mgL?1 hyg B and the regenerated plantlets at 50mgL?1. Using the protocol developed, 34% of the calli infected were hyg B resistant,

Shujie Dong; Rongda Qu

2005-01-01

56

High efficiency transformation of tall fescue with Agrobacterium tumefaciens  

Microsoft Academic Search

An efficient genetic transformation system for tall fescue ( Festuca arundinacea Schreb.), using Agrobacterium tumefaciens-mediated T- DNA delivery, is described. Seed-derived embryogenic calli were infected with Agrobacterium tumefaciens strain EHA105 harboring plasmids pTOK47 and pCAMBIA1301. Infected calli were selected at 250 mg L1 hyg B and the regenerated plantlets at 50 mg L1. Using the protocol developed, 34% of the

Shujie Dong; Rongda Qu

2005-01-01

57

ACC deaminase activity in avirulent Agrobacterium tumefaciens D3.  

PubMed

Some plant-growth-promoting bacteria encode the enzyme 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase, which breaks down ACC, the direct precursor of ethylene biosynthesis in all higher plants, into ammonia and ?-ketobutyrate and, as a result, reduces stress ethylene levels in plants caused by a wide range of biotic and abiotic stresses. It was previously shown that ACC deaminase can inhibit crown gall development induced by Agrobacterium tumefaciens and can partially protect plants from this disease. Agrobacterium tumefaciens D3 has been previously reported to contain a putative ACC deaminase structural gene (acdS) and a regulatory gene (acdR = lrpL). In the present study, it was found that A. tumefaciens D3 is an avirulent strain. ACC deaminase activity and its regulation were also characterized. Under gnotobiotic conditions, wild-type A. tumefaciens D3 was shown to be able to promote plant root elongation, while the acdS and lrpL double mutant strain A. tumefaciens D3-1 lost that ability. When co-inoculated with the virulent strain, A. tumefaciens C58, in wounded castor bean plants, both the wild-type A. tumefaciens D3 and the mutant A. tumefaciens D3-1 were found to be able to significantly inhibit crown gall development induced by A. tumefaciens C58. PMID:21491979

Hao, Youai; Charles, Trevor C; Glick, Bernard R

2011-04-01

58

Genetic transformation of tobacco NT1 cells with Agrobacterium tumefaciens  

Microsoft Academic Search

This protocol is used to produce stably transformed tobacco (Nicotiana tabacum) NT1 cell lines, using Agrobacterium tumefaciens–mediated DNA delivery of a binary vector containing a gene encoding hepatitis B surface antigen and a gene encoding the kanamycin selection marker. The NT1 cultures, at the appropriate stage of growth, are inoculated with A. tumefaciens containing the binary vector. A 3-day cocultivation

Kristin J Mayo; Barbara J Gonzales; Hugh S Mason

2006-01-01

59

High throughput genetic transformation mediated by Agrobacterium tumefaciens in maize  

Microsoft Academic Search

A high throughput genetic transformation system in maize has been developed with Agrobacterium tumefaciens mediated T-DNA delivery. With optimized conditions, stable callus transformation frequencies for Hi-II immature embryos averaged approximately 40%, with results in some experiments as high as 50%. The optimized conditions include N6 medium system for Agrobacterium inoculation, co-cultivation, resting and selection steps; no AgNo3 in the infection

Zuo-yu Zhao; Weining Gu; Tishu Cai; Laura Tagliani; David Hondred; Diane Bond; Sheryl Schroeder; Marjorie Rudert; Dottie Pierce

2002-01-01

60

Genetic transformation of Populus nigra by Agrobacterium tumefaciens  

Microsoft Academic Search

Two clones of Populus nigra L. were tested in vivo and in vitro for their susceptibility to three different Agrobacterium tumefaciens wild-type strains evaluating number and size of resulting calluses. Strain C58 proved to be the most virulent.Various parameters affecting Agrobacterium-mediated transformation of P. nigra clones were further analyzed using ß-glucuronidase gene transient expression. The clone Jean Pourtet proved to

M. Confalonieri; A. Balestrazzi; S. Bisoffi

1994-01-01

61

AGROBACTERIUM TUMEFACIENS-MEDIATED TRANSFORMATION OF TRICHODERMA HARZIANUM  

Microsoft Academic Search

The development of a transformation system is a significant technical hurdle in the study of many filamentous fungi. Agrobacterium tumefaciens has been shown to transform yeasts, fungi and even human cells. By using this transformation system, filamentous fungus Trichoderma harzianum was successfully transformed with an efficiency of 60-110 transformants per 10 7 spores. PCR and Southern analysis showed that the

Qian Yang; Li-ming Yang; Pi-Gang Liu; Sen Li; Jinzhu Song

62

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

63

Untersuchungen zur genetischen Transformation zwischen Agrobacterium tumefaciens und Rhizobium spec  

Microsoft Academic Search

In Ergänzung zu den Untersuchungen an den Partnern einer genetischen Transformation zwischen Agrobacterium tumefaciens (Donator) und Rhizobium leguminosarum (Acceptor) und einem hierdurch entstandenen Transformanten werden vergleichende Analysen an entsprechenden DNA-Präparaten durchgeführt. Bei gleichen physikalischen Eigenschaften und ähnlicher chemischer Zusammensetzung sind geringe Differenzen im Basengehalt der einzelnen Präparate festzustellen. Säulenchromatographische Fraktionierungen der basisch hydrolysierten Apyrimidinsäuren ergenben keine spezifischen Unterschiede in der

Hartmut Kern

1965-01-01

64

Untersuchungen zur genetischen Transformation zwischen Agrobacterium tumefaciens und Rhizobium spec  

Microsoft Academic Search

Vergleichende Untersuchungen an den Partnern einer genetischen Transformation zwischen Agrobacterium tumefaciens (Donator) und Rhizobium leguminosarum (Acceptor) sowie einem hieraus hervorgegangenen virulenten Transformanten ergeben klare serologische und morphologische Unterschiede. Es wird gezeigt, daß der transformierte Stamm nicht nur die Fähigkeit zur Tumorinduktion an Pflanzen erlangt hat, sondern auch in einigen physiologischen und biochemischen Eigenschaften so verändert wurde, daß er zwischen denen

Hartmut Kern

1965-01-01

65

Plant cell transformation with Agrobacterium tumefaciens under simulated microgravity  

Microsoft Academic Search

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

Veresa Sarnatska; Hanna Gladun; Svetlana Padalko

2008-01-01

66

Alkylsyringamides, new inducers of Agrobacterium tumefaciens virulence genes  

Microsoft Academic Search

The virulence genes of Agrobacterium tumefaciens are specifically activated by plant phenolic compounds and allow this organism to genetically transform plant cells. New types of phenolic compounds, three phenol amides derived from syringic acid, were synthesized. Introduction of an amide group in syringic acid strongly enhances its vir gene inducing activity.

F. Dyé; K. Berthelot; B. Griffon; D. Delay; F. M. Delmotte

1997-01-01

67

Alkylsyringamides, new inducers of Agrobacterium tumefaciens virulence genes.  

PubMed

The virulence genes of Agrobacterium tumefaciens are specifically activated by plant phenolic compounds and allow this organism to genetically transform plant cells. New types of phenolic compounds, three phenol amides derived from syringic acid, were synthesized. Introduction of an amide group in syringic acid strongly enhances its vir gene inducing activity. PMID:9195039

Dyé, F; Berthelot, K; Griffon, B; Delay, D; Delmotte, F M

1997-01-01

68

Genetic transformation of lignin degrading fungi facilitated by Agrobacterium tumefaciens  

Microsoft Academic Search

BACKGROUND: White-rot fungi are primarily the major degraders of lignin, a major obstacle for commercial exploitation of plant byproducts to produce bioethanol and other industrially important products. However, to improve their efficacy for lignin degradation, it has become necessary to genetically modify these organisms using appropriate vectors. Agrobacterium tumefaciens, a soil phytopathogenic bacterium, generally transforms plants by delivering a portion

Krishna K Sharma; Ramesh C Kuhad

2010-01-01

69

Agrobacterium tumefaciens-gene transfer into wheat tissues  

Microsoft Academic Search

DNA can be transferred by Agrobacterium tumefaciens to wheat, albeit at very low frequencies. Transfer of agrobacterial DNA occurred in cultures where the embryos had been subjected to partial enzymatic digestion prior to cocultivation with the bacteria. It is unclear whether this is by the normal process mediated by the Ti virulence genes and the border repeats of the T-DNA.

Pauline A. Mooney; Peter B. Goodwin; Elizabeth S. Dennis; Danny J. Llewellyn

1991-01-01

70

[Efficient Agrobacterium-mediated transformation of soybean].  

PubMed

To improve Agrobacterium tumefaciens mediated transformation of embryonic tips of soybean [Glycine max (L.) Merr], the effect of several factors on transformation efficiency were examined by measuring transient expression levels of beta-glucuronidase and the number of resistant explants. The hypervirulent Agrobacterium tumefaciens strain KYRT1 was proved to be a better transformer than EHA105 and LBA4404. Improved transformation efficiencies were obtained when embryonic tips were incubated with an Agrobacterium suspension (A600=0.5) for 20 h. Optimized co-cultivation was performed in acidic medium (pH 5.4) at 22 degrees C in the dark for 5 days. Resting culture and step-by-step selection culture were beneficial to the survial of resistant explants. By combining the best treatments, transgenic soybeans of seven cultivars were obtained that simultaneously express the cryIA (c) and Pinellia ternata agglutinins (pta) genes. Most of the transgenic plants (about 70%) are fertile. The transformation frequency [(the number of PCR-positive regenerated plants/the number of infected explants) x 100%] ranged from 4.29% to 18.0%. PCR and Southern analyses confirmed the stable integration of the binary insect resistance genes in the primary transgenic plants. PMID:17674770

Dang, Wei; Wei, Zhi Ming

2007-06-01

71

Differentiation of Petunia hybrida tissues transformed by Agrobacterium rhizogenes and Agrobacterium tumefaciens  

Microsoft Academic Search

Petunia hybrida plants were inoculated with differentAgrobacterium rhizogenes andA. tumefaciens strains and developed tumors were further cultivatedin vitro. Transformed flowering plants differentiated from tumors induced byA. rhizogenes strains 8196 and TRIOL Transformed but non-rooted plants developed also from tumors incited byA. tumefaciens T37. Cultures of roots transformed byA. rhizogenes strain 15834 did not show increased incidence of chromosomal aberrations in

M. Ond?ej; R?zena Bísková

1986-01-01

72

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

Microsoft Academic Search

Medicago\\u000a 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,

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

2006-01-01

73

Proteins on Escherichia coli andAgrobacterium tumefaciens Translation Systems  

Microsoft Academic Search

Theeffects of30type1andof2 (ricin andvolkensin) type2ribosome-inactivating proteins (RIPs) on Escherichia coli andAgrobacterium tumefaciens cell-free translation systems were compared withtheeffects on a rabbit reticulocyte translation system. Thedepurinating activity ofRIPson E.coli ribosomes was also evaluated. Onlysixtype1RIPsinhibited endogenous mRNA-directed translational activity ofE.coli lysates, withsubmicromolar 50% inhibitory concentrations. FourRIPshadsimilar activities on poly(U)-directed phenylalanine polymerization byE.coli ribosomes, andthree RIPsinhibited poly(U)-directed polyphenylala- ninesynthesis byA.tumefaciens ribosomes, withsubmicromolar 50%1inhibitory

TOMAS GIRBES; MIGUEL FERRERAS; F. JAVIER ARIAS; M. ANGELES ROJO; ROSARIO IGLESIAS; CARLOS ALEGRE; FIORENZO STIRPE

74

Agrobacterium tumefaciens and Agrobacterium rhizogenes transformed roots of the parasitic plant Triphysaria versicolor retain parasitic competence  

Microsoft Academic Search

Parasitic plants in the Orobanchaceae invade roots of neighboring plants to rob them of water and nutrients. Triphysaria is facultative parasite that parasitizes a broad range of plant species including maize and Arabidopsis. In this paper we describe transient and stable transformation systems for Triphysaria\\u000a versicolor Fischer and C. Meyer. Agrobacterium\\u000a tumefaciens and Agrobacterium\\u000a rhizogenes were both able to transiently

Alexey Tomilov; Natalya Tomilova; John I. Yoder

2007-01-01

75

Expression and physiological relevance of Agrobacterium tumefaciens phosphatidylcholine biosynthesis genes.  

PubMed

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

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

2008-10-31

76

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

77

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

78

Factors affecting Agrobacterium tumefaciens -mediated transformation of peppermint  

Microsoft Academic Search

Substantial improvement in peppermint (Mentha x piperita L. var. Black Mitcham) genetic transformation has been achieved so that the frequency of transgenic plants regenerated (percent\\u000a of leaf explants that produced transformed plants) was 20-fold greater than with the original protocol. Essential modifications\\u000a were made to conditions for Agrobacterium tumefaciens co-cultivation that enhanced infection, and for selection of transformed cells and

X. Niu; X. Li; P. Veronese; R. A. Bressan; S. C. Weller; P. M. Hasegawa

2000-01-01

79

The Cell-Cell Communication System of Agrobacterium Tumefaciens  

Microsoft Academic Search

The Ti plasmids of Agrobacterium tumefaciens carry almost all of the genes required for the formation of crown gall tumors and for the utilization of opines that are\\u000a produced by these tumors. These plasmids also encode a cell-cell signalling (quorum sensing) system that is homologous to\\u000a the LuxR-LuxI system of Vibrio fischeri. The LuxI orthologue TraI synthesizes a specific N-acylhomoserine

Catharine E. White; Stephen C. Winans

80

Mechanisms and Regulation of Polar Surface Attachment in Agrobacterium tumefaciens  

PubMed Central

Summary Agrobacterium tumefaciens is a plant pathogen that transfers a segment of its own DNA into host plants to cause Crown Gall disease. The infection process requires intimate contact between the infecting bacteria and the host tissue. A. tumefaciens attaches efficiently to plant tissues and to abiotic surfaces, and can establish complex biofilms at colonization sites. The dominant mode of attachment is via a single pole in contact with the surface. Several different appendages, adhesins and adhesives play roles during attachment, and foster the transition from free-swimming to sessile growth. This polar surface interaction reflects a more fundamental cellular asymmetry in A. tumefaciens that influences and is congruent with its attached lifestyle.

Tomlinson, Amelia D.; Fuqua, Clay

2009-01-01

81

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

82

Agrobacterium tumefaciens mutants affected in attachment to plant cells.  

PubMed Central

An analysis of Agrobacterium tumefaciens mutants with Tn5 insertions in chromosomal DNA showed that the chromosome of A. tumefaciens codes for a specific ability of this bacterium to attach to plant cells. This ability is associated with tumorigenesis by A. tumefaciens, the ability of avirulent A. tumefaciens to inhibit tumorigenesis, and the ability to adsorb certain phages. A second class of chromosomal mutations affects tumorigenesis without altering the ability to attach to plant cells. The attachment of A. tumefaciens to plant cells was assayed by mixing radiolabeled bacteria with suspensions of tobacco tissue culture cells or freshly isolated Zinnia leaf mesophyll cells. Under the conditions of this assay, an avirulent Ti plasmid-cured strain attached to the same extent as the same strain containing pTiB6806. Six of eight avirulent mutants with Tn5 insertions in chromosomal DNA showed defective attachment, whereas two retained wild-type attachment ability. In contrast to the strains showing wild-type attachment, the attachment-defective mutants failed to inhibit tumorigenesis when inoculated onto Jerusalem artichoke slices before inoculation of a virulent strain and also showed a loss of sensitivity to two Agrobacterium phages. The loss of phage sensitivity appeared to be due to a loss of ability to adsorb the phages. Staining with Calcofluor indicated that the mutants retained the ability to synthesize cellulose fibrils, which have been implicated in the attachment process. Southern filter hybridizations demonstrated that each mutant contained a single Tn5 insertion, and genetic linkage between the Tn5 insertion in one mutant and the attachment phenotype has also been demonstrated. Images

Douglas, C J; Halperin, W; Nester, E W

1982-01-01

83

Agrobacterium - tumefaciens -mediated transformation of Helminthosporium turcicum , the maize leaf-blight fungus  

Microsoft Academic Search

Agrobacterium tumefaciens has the ability to transfer its T-DNA to plants, yeast, filamentous fungi, and human cells and integrate it into their genome. Conidia of the maize pathogen Helminthosporium turcicum were transformed to hygromycin B resistance by a Agrobacterium- tumefaciens-mediated transformation system using a binary plasmid vector containing the hygromycin B phosphotransferase ( hph) and the enhanced green fluorescent protein

Yeshitila Degefu; Mubashir Hanif

2003-01-01

84

Production of terpenes by differentiated shoot cultures of Mentha citrata transformed with Agrobacterium tumefaciens T37  

Microsoft Academic Search

Crown gall initiation on Mentha × piperita var. citrata (Ehrh.) Briq. (mint) was investigated using a range of wild type and mutant strains of Agrobacterium tumefaciens. Axenic transformed shoot cultures of Mentha ‘citrata’ were established on plant stems inoculated with the nopaline strain T37 of Agrobacterium tumefaciens. The presence of T-DNA in the transformed tissues and the absence of bacterial

Andrew Spencer; John D. Hamill; Michael J. C. Rhodes

1990-01-01

85

Highly efficient Agrobacterium-mediated transformation of banana cv. Rasthali (AAB) via sonication and vacuum infiltration.  

PubMed

A reproducible and efficient transformation method was developed for the banana cv. Rasthali (AAB) via Agrobacterium-mediated genetic transformation of suckers. Three-month-old banana suckers were used as explant and three Agrobacterium tumefaciens strains (EHA105, EHA101, and LBA4404) harboring the binary vector pCAMBIA1301 were used in the co-cultivation. The banana suckers were sonicated and vacuum infiltered with each of the three A. tumefaciens strains and co-cultivated in the medium containing different concentrations of acetosyringone for 3 days. The transformed shoots were selected in 30 mg/l hygromycin-containing selection medium and rooted in rooting medium containing 1 mg/l IBA and 30 mg/l hygromycin. The presence and integration of the hpt II and gus genes into the banana genome were confirmed by GUS histochemical assay, polymerase chain reaction, and southern hybridization. Among the different combinations tested, high transformation efficiency (39.4 ± 0.5% GUS positive shoots) was obtained when suckers were sonicated and vacuum infiltered for 6 min with A. tumefaciens EHA105 in presence of 50 ?M acetosyringone followed by co-cultivation in 50 ?M acetosyringone-containing medium for 3 days. These results suggest that an efficient Agrobacterium-mediated transformation protocol for stable integration of foreign genes into banana has been developed and that this transformation system could be useful for future studies on transferring economically important genes into banana. PMID:21212957

Subramanyam, Kondeti; Subramanyam, Koona; Sailaja, K V; Srinivasulu, M; Lakshmidevi, K

2011-01-07

86

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

87

Methionine biosynthesis in Agrobacterium tumefaciens: study of the first enzyme.  

PubMed

Here we characterize the first step in methionine biosynthesis in Agrobacterium tumefaciens, an ?-proteobacterium. We explored the metA gene and its products and found several unique properties. Although the gene was annotated as a homoserine transsuccinylase, based upon sequence similarity to characterized homologs in other bacteria, including Escherichia coli, the enzyme uses acetyl-CoA as a substrate and therefore is functionally a transacetylase. Moreover, the protein is thermolabile and the gene is under regulation of heat shock transcriptional activator ?32. 3. The gene has a SAM-riboswitch, which shuts off transcription by ?-32 as well as by the vegetative ?-70. PMID:23085540

Rotem, Or; Biran, Dvora; Ron, Eliora Z

2012-10-17

88

Transgenic Acacia sinuata from Agrobacterium tumefaciens -mediated transformation of hypocotyls  

Microsoft Academic Search

Transgenic herbicide tolerant Acacia sinuata plants were produced by transformation with the bar gene conferring phosphinothricin resistance. Precultured hypocotyl explants were infected with Agrobacterium tumefaciens strain EHA105 in the presence of 100 ?M acetosyringone and shoots regenerated on MS (Murashige and Skoog, 1962, Physiol Plant 15:473–497) medium with 13.3 ?M benzylaminopurine, 2.6 ?M indole-3-acetic acid, 1 g l?1 activated charcoal, 1.5 mg l?1 phosphinothricin, and 300 mg l?1 cefotaxime.

G. Vengadesan; S. Amutha; M. Muruganantham; R. Prem Anand; A. Ganapathi

2006-01-01

89

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

90

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

91

Agrobacterium tumefaciens mediated transformation of marine microalgae Schizochytrium.  

PubMed

Schizochytrium was a known docosahexaenoic acid producing marine microalgae. In this study, we have developed a novel transformation approach of Schizochytrium using the Agrobacterium tumefaciens (A. tumefaciens) binary vector system. After co-cultivation of Schizochytrium protoplasts with A. tumefaciens harboring pCAMBIA2301 containing the neomycin phosphotransferase II (NPT II) gene as the selectable marker which confers resistance to G418, the Schizochytrium transformants were successfully obtained on the G418-containing plates. The integration and expression of the transgenes were confirmed by PCR analysis and GUS activity assay. To further validate the transformation system, pCAMBIA2301-EGFP containing the egfp gene was introduced into Schizochytrium. The following results demonstrated that the exogenous egfp gene has been successfully incorporated into the genome of Schizochytrium. In addition, the introduced egfp gene expressed efficiently according to the Western blot and fluorescence assay results. More importantly, the majority of the transformants displayed similar biomass and fatty acid production comparing with the wild type strain. Our results demonstrated that exogenous genes could be expressed efficiently in transgenic Schizochytrium, suggesting that genetically engineered Schizochytrium could be explored by this system. PMID:21641193

Cheng, Rubin; Ma, Ruijuan; Li, Ke; Rong, Hui; Lin, Xiangzhi; Wang, Zhaokai; Yang, Shanjun; Ma, Yong

2012-03-20

92

Inhibition of lipopolysaccharide synthesis in Agrobacterium tumefaciens and Aeromonas salmonicida.  

PubMed

Lipopolysaccharide (LPS) synthesis was inhibited, new lipid A metabolites accumulated, and growth ceased, when the plant pathogen Agrobacterium tumefaciens and the fish pathogen Aeromonas salmonicida were treated with an antibacterial agent which specifically inhibits CTP:CMP-3-deoxy-manno-octulosonate cytidylyltransferase (CMP-KDO synthase). The new lipid A metabolites were purified by chromatography on DEAE-cellulose and chemically analysed. Metabolites isolated from both bacterial species contained glucosamine and phosphate in a 1:1 molar ratio, and 3-OH-C14:0 was the major fatty acid present (1 mol and 1.4 mol per mol glucosamine for A. tumefaciens and A. salmonicida, respectively). Inhibition of LPS synthesis by CMP-KDO synthase inhibitor had no effect on the initial kinetics of A. tumefaciens attachment to cultured carrot cells, but did inhibit cell aggregation normally induced by bacterial cellulose synthesis. Bacteria treated with inhibitor remained viable and able to synthesize protein at 15% the rate of control cells, indicating that the lack of cellulose-induced aggregation was not due to the inability of bacteria to make protein, but rather the inability to respond normally to the bacterial-plant cell interaction. PMID:1324975

Goldman, R C; Capobianco, J O; Doran, C C; Matthysse, A G

1992-07-01

93

Linear Chromosome-generating System of Agrobacterium tumefaciens C58  

PubMed Central

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

2012-01-01

94

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

2013-08-28

95

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.

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

2013-01-01

96

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

97

Agrobacterium-mediated transformation of rough lemon (Citrus jambhiri Lush) with yeast HAL2 gene  

PubMed Central

Background Rough lemon (Citrus jambhiri Lush.) is the most commonly used Citrus rootstock in south Asia. It is extremely sensitive to salt stress that decreases the growth and yield of Citrus crops in many areas worldwide. Over expression of the yeast halotolerant gene (HAL2) results in increasing the level of salt tolerance in transgenic plants. Results Transformation of rough lemon was carried out by using Agrobacterium tumefaciens strains LBA4404 harboring plasmid pJRM17. Transgenic shoots were selected on kanamycin 100?mg?L-1 along with 250?mg?L-1 each of cefotaxime and vancomycin for effective inhibition of Agrobacterium growth. The Murashige and Skoog (MS) medium containing 200??M acetoseryngone (AS) proved to be the best inoculation and co-cultivation medium for transformation. MS medium supplemented with 3?mg?L-1 of 6-benzylaminopurine (BA) showed maximum regeneration efficiency of the transformed explants. The final selection of the transformed plants was made on the basis of PCR and Southern blot analysis. Conclusion Rough lemon has been successfully transformed via Agrobacterium tumefaciens with ?-glucuronidase (GUS) and HAL2. Various factors affecting gene transformation and regeneration efficiency were also investigated.

2012-01-01

98

Preliminary crystallographic studies of glycogen synthase from Agrobacterium tumefaciens.  

PubMed

Crystals of the glycogen synthase (GS) from Agrobacterium tumefaciens have been grown that diffract to 2.6 A resolution. The enzyme, which is homologous to the starch synthases of plants, catalyzes the last reaction step in the biosynthesis of glycogen. It is a alpha-retaining glucosyltransferase that uses ADP-glucose to incorporate additional glucose monomers onto the growing glycogen polymer. Its homology with mammalian GSs is marginal, but several regions shown to be important in catalysis are strictly conserved. Knowledge of the crystal structure of GS will be a major advance in the understanding of glycogen/starch metabolism and its regulation. A rational approach in enzyme engineering can subsequently be envisaged. The multiwavelength anomalous diffraction approach will be used to solve the phase problem. PMID:12595715

Guerin, Marcelo E; Buschiazzo, Alejandro; Ugalde, Juan E; Ugalde, Rodolfo A; Alzari, Pedro M

2003-02-21

99

Active Transport of Glucose-1-Phosphate in Agrobacterium tumefaciens  

PubMed Central

The presence of an active transport system for glucose-1-phosphate in Agrobacterium tumefaciens was demonstrated from the following observations. (i) The bacterium could grow on a medium containing glucose-1-phosphate as carbon source; (ii) the entry of glucose-1-phosphate into the resting cells occurred against concentration gradient obeying Michaelis-Menten kinetics; and (iii) the entry reaction was energy-dependent. The transport system for glucose-1-phosphate was formed inducibly by growing the organism on a glucose-1-phosphate or sucrose medium. From the inhibition and kinetics studies it was found that the transport system had a high specificity for glucose-1-phosphate with a high affinity, Km value of 4.5 × 10?6m at pH 8.2. The existence of glucose-1-phosphate binding factor was proved in the shock fluid which was prepared from the cells grown on both glucose-1-phosphate and sucrose media by osmotic shock.

Fukui, Sakuzo; Miyairi, Sachio

1970-01-01

100

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

101

The FNR-type transcriptional regulator SinR controls maturation of Agrobacterium tumefaciens biofilms  

Microsoft Academic Search

Summary Agrobacterium tumefaciens is a plant pathogen that persists as surface-associated populations on plants or soil particles. A genetic screen for A. tumefaciens mutants deficient for surface interactions identified a mutant that forms thin, sparsely populated biofilms, but is proficient for initial attachment. The mutant is disrupted in a gene designated sinR , encoding a member of the DNR subfamily

Bronwyn E. Ramey; Ann G. Matthysse; Clay Fuqua

102

Genome Sequence of the Quorum-Sensing-Signal-Producing Nonpathogen Agrobacterium tumefaciens Strain P4  

PubMed Central

Agrobacterium tumefaciens P4 is a quorum-sensing-signal-producing bacterium that has been isolated from the tobacco rhizosphere. This strain belongs to genomospecies 1 of the A. tumefaciens complex; it is avirulent on various putative host plants, devoid of the Ti plasmid, and contains a luxI homolog on the At plasmid.

Mondy, Samuel; Lalouche, Ouaghlis; Dessaux, Yves

2013-01-01

103

Genome Sequence of the Quorum-Quenching Agrobacterium tumefaciens Strain WRT31.  

PubMed

Agrobacterium tumefaciens strain WRT31 is a quorum-sensing signal-degrading bacterium that has been isolated from the rhizosphere of tobacco plants. This strain belongs to A. tumefaciens genomovar G1, is avirulent on various putative host plants, devoid of Ti plasmid, and contains the blcC gene encoding a gamma-butyrolactonase. PMID:23969055

Mondy, Samuel; Lalouche, Ouaghlis; Dessaux, Yves; Faure, Denis

2013-08-22

104

Genome Sequence of the Quorum-Sensing-Signal-Producing Nonpathogen Agrobacterium tumefaciens Strain P4.  

PubMed

Agrobacterium tumefaciens P4 is a quorum-sensing-signal-producing bacterium that has been isolated from the tobacco rhizosphere. This strain belongs to genomospecies 1 of the A. tumefaciens complex; it is avirulent on various putative host plants, devoid of the Ti plasmid, and contains a luxI homolog on the At plasmid. PMID:24092790

Mondy, Samuel; Lalouche, Ouaghlis; Dessaux, Yves; Faure, Denis

2013-10-03

105

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

106

Light strongly promotes gene transfer from Agrobacterium tumefaciens to plant cells  

Microsoft Academic Search

Light conditions during Agrobacterium-based plant transformation, the most routinely used method in plant genetic engineering, differ widely and, to our knowledge, have not been studied systematically in relation to transformation efficiency. Here, light effects were examined in two already optimized transformation procedures: coculture of Agrobacterium tumefaciens with callus from two genotypes of the crop plant Phaseolus acutifolius (tepary bean) and

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

2003-01-01

107

High efficiency transformation of peppermint ( Mentha × piperita L.) with Agrobacterium tumefaciens  

Microsoft Academic Search

Transgenic peppermint (Mentha × piperita L.) plants were obtained by using Agrobacterium tumefaciens-mediated gene transfer. The effects of the coculture period and of the Agrobacterium strain were tested. 10% transformed plants were regenerated by leaf disk culture after inoculation with strain EHA105MOG harbouring ?-glucuronidase and neomycin phosphotransferase II genes, with a coculture period of 5 days. Rooting of regenerated plants

F. Diemer; F. Jullien; O. Faure; S. Moja; M. Colson; E. Matthys-Rochon; J. C. Caissard

1998-01-01

108

Ethylene Production in Plants during Transformation Suppresses vir Gene Expression in Agrobacterium tumefaciens  

Microsoft Academic Search

Ethylene evolution from plants inhibits Agrobacterium-mediated genetic transformation, but the mechanism is little understood. In this study, the possible role of ethylene in Agrobacterium-mediated genetic transformation was clarified. It was tested whether or not plant ethylene sensitivity affected genetic transformation; the sensitivity might regulate bacterial growth during co-cultivation and vir gene expression in Agrobacterium tumefaciens. For these experiments, melon (Cucumis

Satoko Nonaka; Ken-Ichi Yuhashi; Keita Takada; Masayuki Sugaware; Kiwamu Minamisawa; Hiroshi Ezura

2008-01-01

109

ER disruption and GFP degradation during non-regenerable transformation of flax with Agrobacterium tumefaciens  

Microsoft Academic Search

Flax is considered as plant species susceptible to Agrobacterium-mediated genetic transformation. In this study, stability of flax transformation by Agrobacterium rhizogenes versus Agrobacterium tumefaciens was tested by using combined selection for antibiotic resistance and visual selection of green fluorescent protein (GFP)-fusion\\u000a reporter targeted to the endoplasmic reticulum (ER). Transformation with A. rhizogenes was stable for over 2 years, whereas transformation by

Juraj Bleho; Bohuš Obert; Tomáš Taká?; Beáta Petrovská; Claudia Heym; Diedrik Menzel; Jozef Šamaj

110

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

111

Genetic transformation of lignin degrading fungi facilitated by Agrobacterium tumefaciens  

PubMed Central

Background White-rot fungi are primarily the major degraders of lignin, a major obstacle for commercial exploitation of plant byproducts to produce bioethanol and other industrially important products. However, to improve their efficacy for lignin degradation, it has become necessary to genetically modify these organisms using appropriate vectors. Agrobacterium tumefaciens, a soil phytopathogenic bacterium, generally transforms plants by delivering a portion of the resident Ti- plasmid, the T-DNA (transfer DNA). The trans-Kingdom gene transfer is initiated by the activity of Ti-plasmid encoded vir (virulence) genes in response to low-molecular-mass phenolic compounds such as acetosyringone. A. tumefaciens played a major role in plant genetic engineering and basic research in molecular biology, accounting for nearly 80% of the transgenic plants produced so far. Initially, it was believed that only dicotyledons, gymnosperms and a few monocotyledonous species could be transformed by this bacterium; but recent reports have totally changed this scenario by demonstrating that many 'recalcitrant' species not included in its natural host range can also be transformed, especially filamentous fungi. Results This paper describes an efficient and convenient Agrobacterium-mediated gene transformation system for successful delivery of T-DNA, carrying the genes coding for ?-glucuronidase (uidA), green fluorescent protein (gfp) and hygromycin phosphotransferase (hpt) to the nuclear genome of lignin degrading white-rot fungi such as Phanerochaete chrysosporium, Ganoderma sp. RCKK-02, Pycnoporous cinnabarinus, Crinipellis sp. RCK-1, Pleurotus sajor-caju and fungal isolate BHR-UDSC without supplementation of acetosyringone. The fungal transformants were confirmed by PCR and Southern hybridization. The expression vector pCAMBIA 1304-RCKK was constructed by the addition of GPD promoter from plasmid p416 to the binary vector backbone pCAMBIA1304, which controls uidA and gfp gene. Transmission Electron Microscope (TEM) analysis revealed the attachment of bacterial cells to the fungal hyphae. Transformation frequency varied from 50 to 75% depending on the fungal species used in this study. The transformation efficiency was maximum at 20°C whereas no transfer was observed at temperature above 29°C. Conclusion These findings provide a rapid and reproducible transformation method without external addition of acetosyringone, which could be useful for improving white-rot fungi for their various biotechnological applications.

2010-01-01

112

Obtençăo de mutantes de Aspergillus carbonarius via transformaçăo genética mediada por Agrobacterium tumefaciens Generation of Aspergillus carbonarius mutants by Agrobacterium tumefaciens-mediated transformation method  

Microsoft Academic Search

Aspergillus carbonarius is a potent ochratoxin A producer , a mycotoxin that has nephrotoxic and carcinogenic effects. The knowledge of genes involved in biosynthesis of this toxin may be useful for the development of detection and control methods. The Agrobacterium tumefaciens-mediated transformation method has been demonstrated as a powerful tool to obtain insertional mutants for the characterization of new genes.

Lígia Uno Lunardi; Carla Beatriz Fier; Fernando Yuldi Ashikaga; Luiz Rodrigo Morioka; Maria Helena Pelegrinelli Fungaro

113

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

114

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

115

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

116

Atypical Adaptive and Cross-Protective Responses Against Peroxide Killing in a Bacterial Plant Pathogen, Agrobacterium tumefaciens  

Microsoft Academic Search

Physiological adaptive and cross-protection responses to oxidants were investigated in Agrobacterium tumefaciens. Exposure of A. tumefaciens to sublethal concentrations of H 2O 2 induced adaptive protection to lethal concentrations of H 2O 2. Similar treatments with organic peroxide and menadione did not produce adaptive protection to subsequent exposure to lethal concentrations of these oxidants. Pretreatment of A. tumefaciens with an

Paiboon Vattanaviboon; Warawan Eiamphungporn; Skorn Mongkolsuk

2003-01-01

117

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

118

Regeneration of transgenic loblolly pine ( Pinus taeda L.) from zygotic embryos transformed with Agrobacterium tumefaciens  

Microsoft Academic Search

Embryos of 24 open-pollinated families of loblolly pine (Pinus teade L.) were used as explants to conduct in vitro regeneration. Then, Agrobacterium tumefaciens strain GV3101 harboring the plasmid pPCV6NFHygGUSINT was used to transform mature zygotic embryos of seven families of loblolly pine. The frequency of transformation varied among families infected with A. tumefaciens. The highest frequency (100%) of transient #-glucuronidase

Wei Tang; Ron Sederoff; Ross Whetten

2001-01-01

119

Factors influencing Agrobacterium tumefaciens-mediated transformation of Artemisia annua L  

Microsoft Academic Search

Following our previously described Agrobacterium tumefaciens-mediated transformation procedure for Artemisia annua L., we have undertaken several additional experiments to establish the importance of some parameters such as explant type,\\u000a age of explant source, A. tumefaciens strain and type of binary vector. Several binary vectors were useful for the production of transgenic callus on explants\\u000a of different ages. In transformed calli,

A. Vergauwe; E. Van Geldre; D. Inzé; M. Van Montagu; E. Van den Eeckhout

1998-01-01

120

Agrobacterium tumefaciens -mediated transformation for random insertional mutagenesis in Colletotrichum lagenarium  

Microsoft Academic Search

Random insertional mutagenesis using a marker DNA fragment is an effective method for identifying fungal genes relevant to morphogenesis, metabolism, and so on. Agrobacterium tumefaciens-mediated transformation (AtMT) has long been used as a tool for the genetic modification of a wide range of plant species. Recent study has indicated that A. tumefaciens could transfer T-DNA not only to plant cells

Gento Tsuji; Satoshi Fujii; Naoki Fujihara; Chika Hirose; Seiji Tsuge; Tomonori Shiraishi; Yasuyuki Kubo

2003-01-01

121

Factors influencing the Agrobacterium tumefaciens-mediated transformation of carrot (Daucus carota L.)  

Microsoft Academic Search

To develop an efficient procedure for Agrobacterium tumefaciens-mediated genetic transformation of carrot (Daucus carota L.) the effects of several factors were studied. Parameters which significantly affected the transformation frequency were the variety, the explant type, and the co-cultivation period. Under optimal conditions, using the A. tumefaciens C58C1 containing either pGSTRN943 or pGSGluc1 and 3 days of co-cultivation, the frequency of

Nathalie Pawlicki; Rajbir S. Sangwan; Brigitte S. Sangwan-Norreel

1992-01-01

122

Efficient Agrobacterium tumefaciens -mediated transformation using embryogenic suspension cultures in sweetpotato, Ipomoea batatas (L.) Lam  

Microsoft Academic Search

Efficient Agrobacterium tumefaciens-mediated transformation was achieved using embryogenic suspension cultures of sweetpotato (Ipomoea batatas (L.) Lam.) cv. Lizixiang. Cell aggregates from embryogenic suspension cultures were cocultivated with the A. tumefaciens strain EHA105 harboring a binary vector pCAMBIA1301 with gusA and hygromycin phosphotransferase II gene (hpt II) genes. Selection culture was conducted using 25 mg l?1 hygromycin. A total of 2,218 plants were

Bo Yu; Hong Zhai; Yuping Wang; Ning Zang; Shaozhen He; Qingchang Liu

2007-01-01

123

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

124

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

125

Transformation of suspension cultures of bromegrass ( Bromus inermis ) by Agrobacterium tumefaciens  

Microsoft Academic Search

Smooth bromegrass (Bromus inermis Leyss) is an extremely cold hardy perennial grass and its cell culture is an excellent system for studying mechanisms of\\u000a cold hardiness induced by low temperature or abscisic acid (ABA). Agrobacterium tumefaciens-mediated transformation of non-embryogenic bromegrass cultures was attempted. Agrobacterium strain EHA105 carrying a binary vector that contained the neomycin phosphotransferase (NPT II), beta-glucuronidase (GUS)\\u000a and

Toshihide Nakamura; Masaya Ishikawa

2006-01-01

126

Agrobacterium tumefaciens -mediated genetic transformation of a recalcitrant grain legume, lentil ( Lens culinaris Medik)  

Microsoft Academic Search

A simple and reproducible Agrobacterium-mediated transformation protocol for a recalcitrant legume plant, lentil (Lens culinaris M.) is reported. Application of wounding treatments and efficiencies of three Agrobacterium tumefaciens strains, EHA105, C58C1, and KYRT1 were compared for T-DNA delivery into lentil cotyledonary node tissues. KYRT1 was found\\u000a to be on average 2.8-fold more efficient than both EHA105 and C58C1 for producing

Ufuk Celikkol Akcay; M. Mahmoudian; H. Kamci; M. Yucel; H. A. Oktem

2009-01-01

127

Factors influencing Agrobacterium tumefaciens -mediated genetic transformation of Eleusine coracana (L.) Gaertn  

Microsoft Academic Search

Agrobacterium-mediated transformation protocol has been developed for Eleusine coracana (var. PR-202) by varying several factors which influence T-DNA delivery. Green nodular regenerative calli with meristematic\\u000a nodules of seed origin were used as the target tissue for Agrobacterium\\u000a tumefaciens-mediated gene transfer. The highest frequency of transformation (44.4%) was observed when callus was infected, co-cultivated\\u000a and incubated at 22°C. Incorporation of higher

Manju Sharma; Aditi Kothari-Chajer; Swati Jagga-Chugh; S. L. Kothari

2011-01-01

128

Cloning and characterization of the dxs gene, encoding 1-deoxy- d-xylulose 5-phosphate synthase from Agrobacterium tumefaciens, and its overexpression in Agrobacterium tumefaciens  

Microsoft Academic Search

A newly isolated gene dxs11 from Agrobacterium tumefaciens (KCCM 10413), an organism with potential for the industrial production of ubiquinone-10 (UbiQ10), encoding a 1-deoxy-d-xylulose 5-phosphate synthase (Dxs), was cloned in Escherichia coli and its nucleotide sequence was determined. DNA sequence analysis revealed an open reading frame of 1920bp, capable of encoding a polypeptide of 640 amino acids residues with a

Jung-Kul Lee; Deok-Kun Oh; Sang-Yong Kim

2007-01-01

129

Transformation of chickpea: effect of genotype, explant, Agrobacterium -strain and composition of culture medium  

Microsoft Academic Search

Reproducible and high-frequency transgenic plant regeneration from callus and embryo axes of four different genotypes of chickpea\\u000a (Cicer arietinum) was achieved after Agrobacterium-mediated transformation. Three different strains of Agrobacterium (EHA105, AGL1 and LBA4404) harboring the binary vector pCAMBIA1301 containing ?-glucuronidase (GUS) and hygromycin phosphotransferase (hpt) genes under the control of a CaMV35S promoter were used. The highest number of transgenic

B. Bhattacharjee; M. Mohan; S. Nair

2010-01-01

130

Linear Chromosome-generating System of Agrobacterium tumefaciens C58 PROTELOMERASE GENERATES AND PROTECTS HAIRPIN ENDS  

Microsoft Academic Search

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

Wai Mun Huang; Jeanne DaGloria; Heather Fox; Qiurong Ruan; John Tillou; Ke Shi; Hideki Aihara; John Aron; Sherwood Casjens

2012-01-01

131

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

PubMed

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

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

2013-01-07

132

Genetic transformation of Artemisia annua by Agrobacterium tumefaciens and artemisinin synthesis in transformed cultures  

Microsoft Academic Search

Transformed organ cultures of Artemisia annua were established following infection with two wild type nopaline strains of Agrobacterium tumefaciens. Parameters like explant type, strain type, age of the plant source for explants, affected the tumorigenesis frequency significantly. Crown galls were formed both on in vivo and in vitro plants: 2–3% of the in vitro galls regenerated spontaneously to produce shooty

Biswajit Ghosh; Swapna Mukherjee; Sumita Jha

1997-01-01

133

Withanolide synthesis in cultures of Withania somnifera transformed with Agrobacterium tumefaciens  

Microsoft Academic Search

Transformed organ cultures of Withania somnifera were established following infection with wild type nopaline and octopine strains of Agrobacterium tumefaciens. The oncogenic strains had different levels of virulence on two genotypes studied, the main difference was found in the nature of the galls formed and in their subsequent morphological competence. Ten percent of the galls obtained following infection with nopaline

Swagata Ray; Sumita Jha

1999-01-01

134

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

135

Transformation of Agrobacterium tumefaciens into a NonOncogenic Species by an Escherichia coli RNA  

Microsoft Academic Search

Transforming RNA excreted by showdomycin-resistant Escherichia coli induces a persistent, heritable, and spectacular change in Agrobacterium tumefaciens B6, a bacterium that carries the oncogenic principle for tumor induction in plants. Transformants possessing new physiological and biochemical properties have completely or partially lost the capacity for tumor induction. They synthetize new ribosomes whose components are profoundly modified. On the basis of

Mirko Beljanski; Monique Beljanski; Pierre Manigault; Pierre Bourgarel

1972-01-01

136

Transformation of Rhododendron spp. using Agrobacterium tumefaciens with a GUS-intron chimeric gene  

Microsoft Academic Search

The five Rhododendron cultivars, ‘America’, ‘Catawbiense grandiflorum roseum’, ‘Madame Carvalho’, ‘Mars’ and ‘Nova Zembla’ were used for transformation by Agrobacterium tumefaciens carrying T-DNA with the gusA gene encoding ?-glucuronidase (GUS) gene and the neomycin phosphotransferase II gene as a selectable marker gene. The GUS reporter gene was successfully transferred into all five cultivars as indicated by fluorimetric staining, polymerase chain

Daniela Pavingerová; Jind?ich Bríza; Karel Kodýtek; Hana Niedermeierová

1997-01-01

137

Forskolin synthesis in in vitro cultures of Coleus forskohlii Briq transformed with Agrobacterium tumefaciens  

Microsoft Academic Search

Agrobacterium tumefaciens mediated tumor tissue and shooty teratomas of Coleus forskohlii were cultured in vitro. Forskolin was detected in tumorous callus (0.002%), rhizogenic callus (0.011%) and root cultures (0.014%), but not in shooty teratomas. Forskolin synthesis and accumulation in tumorous C. forskohlii cultures may permit the elucidation of diterpene metabolism in this species.

Swapna Mukherjee; Biswajit Ghosh; Sumita Jha

1996-01-01

138

Crown gall of tobacco caused by Agrobacterium tumefaciens biovar 1 in tobacco fields  

Microsoft Academic Search

Crown gall disease of tobacco was found in Iwate Prefecture, Japan in 1995. Ten bacterial isolates, obtained from the galls of tobacco, were identified as Agrobacterium tumefaciens (Smith and Townsend 1907) Conn 1942 biovar 1 based on their ability to induce galls on the 14 tested plants, including tobacco after needle-prick inoculation, and on 12 cultural, physiological, and biological characteristics.

Naruto Furuya; Fumika Shimokusuzono; Yutaka Nakamura; Kishiko Nishimura; Minoru Takeshita; Nobuaki Matsuyama; Kayo Manabe; Youichi Takanami

2004-01-01

139

Agrobacterium tumefaciens -mediated transformation to alter ethylene and cytokinin biosynthesis in broccoli  

Microsoft Academic Search

Broccoli (Brassica oleracea var. italica) deteriorates rapidly following harvest. Postharvest treatment of broccoli with 6-benzylaminopurine delays senescence, whilst exogenous ethylene has been shown to accelerate this process following harvest. To alter ethylene biosynthesis, broccoli was transformed, using Agrobacterium tumefaciens-mediated transformation, with an antisense ACC oxidase gene from broccoli driven by the asparagine synthetase promoter from asparagus. In addition, broccoli was

Nigel E. Gapper; Marian J. McKenzie; Mary C. Christey; Robert H. Braun; Simon A. Coupe; Ross E. Lill; Paula E. Jameson

2002-01-01

140

Effect of age and type of tissue on genetic transformation of Lotus corniculatus by Agrobacterium tumefaciens  

Microsoft Academic Search

Various experiments of Lotus corniculatus cv. Leo were infected with Agrobacterium tumefaciens strains C58 (wild-type) and GV3101 (control). A maximum of 83 per cent of cotyledons excised from 7 day old seedlings and 63 per cent of leaves excised from seedlings grown in vitro formed galls in culture. The genotype of the seedling had an effect on the response.

Ian P. Armstead; K. Judith Webb

1987-01-01

141

Agrobacterium tumefaciens -mediated transformation of the antitumor clavaric acid-producing basidiomycete Hypholoma sublateritium  

Microsoft Academic Search

The basidiomycete Hypholoma sublateritium produces clavaric acid, an antitumor isoprenoid compound. Arthrospores of this fungus were transformed by Agrobacterium tumefaciens-mediated conjugation. Five plasmids carrying different regulatory sequences to drive expression of the hph (hygromycin phosphotransferase) gene were tested. The promoter used was critically important in order to express heterologous genes in H. sublateritium. Constructions carrying the Agaricus bisporus glyceraldehyde-3-phosphate dehydrogenase

R. P. Godio; R. Fouces; E. J. Gudińa; J. F. Martín

2004-01-01

142

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

PubMed

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

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

2012-02-01

143

Direct Transfer and Expression of Human GM-CSF in Tobacco Suspension Cell using Agrobacterium -Mediated Transfer System  

Microsoft Academic Search

Direct gene transfer methods were established for cell suspension cultures of tobacco (Nicotiana tabacum cv. Havana) using Agrobacterium-mediated transformation system. Agrobacterium strain LBA4404 containing a binary vector carrying the hGM-CSF gene was introduced into tobacco suspension cells. Cell culture cycle affected transformation efficiency. The cells at 5 days after subculture showed the highest response growing 9.7 transformed cells per plate.

Young Sook Kim; Tae Ho Kwon; Yang Moon Sik

2004-01-01

144

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

145

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.

Slater, Steven; Setubal, Joao 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

146

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

2008-07-07

147

Optimizing the production of transformed pea ( Pisum sativum L.) callus using disarmed Agrobacterium tumefaciens strains  

Microsoft Academic Search

For optimization of the transformation procedure with Pisum sativum L. stern explant callus was used to test the effect of disarmed Agrobacterium tumefaciens strains, cocultivation procedures (preconditioning of explants; use of Nicotiana tabacum L. nurse cultures), duration of cocultivation (2, 3 or 4 days), and agents for selection (kanamycin or hygromycin). The succinamopine strain EHA101(pBI1042) produced the highest percentage of

Monika M. Lulsdorf; Hans Rempel; Jennie A. Jackson; David S. Baliski; Shaun L. A. Hobbs

1991-01-01

148

Shoot regeneration of mesophyll protoplasts transformed by Agrobacterium tumefaciens , not achievable with untransformed protoplasts  

Microsoft Academic Search

Alternative methods for shoot regeneration in protoplast derived cultures were developed in Nicotiana paniculata and Physalis minima. In both species protoplast derived callus is not regeneratable to shoots by conventional methods, e.g. hormone treatment. Leaf discs and stem segments of N. paniculata and P. minima were incubated with Agrobacterium tumefaciens “shooter” strains harbouring pGV 2215 or pGV 2298 or wildtype

A. Steffen; T. Eriksson; O. Schieder

1986-01-01

149

Host and T-DNA determinants of cytokinin autonomy in tobacco cells transformed by Agrobacterium tumefaciens  

Microsoft Academic Search

The hormone autonomy of tobacco (Nicotiana tabacum L.) cells transformed byAgrobacterium tumefaciens containing mutations attmr (the “rooty” locus) of the pTiT37 plasmid has been examined. These cells require cytokinin, but not auxin for continuous growth in culture, indicating that the function of thetmr locus is to specify or induce cytokinin autonomy. Examination of tissues from plants regenerated from cells transformed

Andrew N. Binns

1983-01-01

150

Ornamental traits modification by Rol genes in Osteospermum ecklonis transformed with Agrobacterium tumefaciens  

Microsoft Academic Search

Summary  Transgenic plants of Osteospermum ecklonis were produced by cocultivation of leaf fragments with Agrobacterium tumefaciens harboring rol genes from A. rhizogenes. The phenotypic alterations caused by the different transgenes were evaluated in field trials. The genetic manipulation produced\\u000a transgenic plants characterized by the following features: 1) increased number of flowers (e.g., 35SrolC and rolABC); 2) early flowering (e.g., 35SrolC); 3)

Annalisa Giovannini; Michela Zottini; Giacomo Morreale; Angelo Spena; Andrea Allavena

1999-01-01

151

Translation Start Sequences Affect the Efficiency of Silencing of Agrobacterium tumefaciens T-DNA Oncogenes  

Microsoft Academic Search

Agrobacterium tumefaciens oncogenes cause transformed plant cells to overproduce auxin and cytokinin. Two oncogenes encode enzymes that convert tryptophan to indole-3-acetic acid (auxin): iaaM (tryptophan mono-oxygenase) and iaaH (indole-3-acetamide hydrolase). A third oncogene (ipt) encodes AMP isopentenyl transferase, which produces cytokinin (isopentenyl-AMP). Inactivation of ipt and iaaM (or iaaH) abolishes tumorigenesis. Because adequate means do not exist to control crown

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

2003-01-01

152

Transformation of a CAM plant, the facultative halophyte Mesembryanthemum crystallinum by Agrobacterium tumefaciens  

Microsoft Academic Search

This study is the first to demonstrate that a foreign DNA can be delivered into cells of facultative halophyte crassulacean\\u000a acid metabolism (CAM) plant, Mesembryanthemum crystallinum L. with Agrobacterium tumefaciens. This plant can be induced to\\u000a change from C3 to CAM by drought and various stresses, and is a good model to study the environment stress on metabolism not\\u000a only

Ken Ishimaru

1999-01-01

153

Efficient transformation of Beauveria bassiana by Agrobacterium tumefaciens -mediated insertional mutagenesis  

Microsoft Academic Search

An efficient transformation system employing Agrobacterium tumefaciens-mediated transformation was developed for the fungal biocontrol agent Beauveria bassiana. The system was successfully applied to integrate the hygromycin resistance gene and green fluorescent protein gene into\\u000a B. bassiana with a transformation frequency of 13–22 transformants per 106 conidia. The majority of transformants harboured a randomly integrated single copy of T-DNA that was

J. Wu; H. Ridgway; M. Carpenter; T. Glare

2008-01-01

154

Pathogenesis-related proteins in plants and tissues of Nicotiana tabacum transformed by Agrobacterium tumefaciens  

Microsoft Academic Search

Large amounts of pathogenesis-related (PR) proteins were found inNicotiana tabacum crown gall tissue, following transformation of normal tobacco cells withAgrobacterium tumefaciens. In contrast, PR proteins were not detected in leaves of grafted plants that had been recovered from crown gall tissue even though these plants were still transformed as shown by their inability to form roots and ability to produce

J. F. Antoniw; G. Ooms; R. F. White; G. J. Wullems; L. v. Vloten-Doting

1983-01-01

155

Shoot regeneration and genetic transformation by Agrobacterium tumefaciens of Hydrangea macrophylla Ser. leaf discs  

Microsoft Academic Search

A reproducible procedure was developed for genetic transformation of Hydrangea macrophylla Ser. cv. Blaumeise by Agrobacterium tumefaciens following the development of an efficient regeneration system using leaf discs excised from 12 to 15 weeks old meristem-derived vitroplants. Explants were cultivated on solid B5 medium complemented with maltose 110mM, BAP 10?M and NAA 0.5?M. A low light regime of 17?molm?2s?1 improved

Latifa Hamama; Linda Voisine; Didier Peltier; Jacques Boccon-Gibod

2011-01-01

156

Genetic transformation of prickly-pear cactus ( Opuntia ficus-indica ) by Agrobacterium tumefaciens  

Microsoft Academic Search

A system for genetic transformation of an elite prickly pear cactus (Opuntia ficus-indica L., cultivar Villa Nueva) by Agrobacterium tumefaciens was developed. Beginning with direct bacterial infection by using a hypodermic syringe to the meristematic tissue termed areoles, transgenic plants were obtained by selection with 100 mg l?1 kanamycin. Transient and stable GUS activities were monitored on kanamycin-resistant shoots and regenerated plants,

H. Silos-Espino; A. Valdez-Ortiz; Q. Rascón-Cruz; E. Rodríguez-Salazar; O. Paredes-López

2006-01-01

157

Agrobacterium tumefaciens -mediated transformation of antifungal lipopeptide producing fungus Coleophoma empetri F-11899  

Microsoft Academic Search

The filamentous fungus Coleophoma empetri F-11899 produces an echinocandin-like compound FR901379, the original source for micafungin which is prescribed to treat\\u000a deep-seated mycoses. Despite its industrial importance, no genetic information on C. empetri F-11899 is currently available. To characterize FR901379 biosynthetic genes by insertional mutagenesis and to improve the\\u000a compound production genetically, Agrobacterium tumefaciens-mediated transformation (ATMT) was attempted to make

Masato Yamada; Kazunobu Yawata; Yohsuke Orino; Satoshi Ueda; Yasuhiro Isogai; Goro Taguchi; Makoto Shimosaka; Seiji Hashimoto

2009-01-01

158

Agrobacterium tumefaciens -mediated transformation of broccoli ( Brassica oleracea var. italica ) and cabbage ( B. oleracea var. capitata )  

Microsoft Academic Search

Transgenic broccoli (Brassica oleracea var. italica) was produced by two Agrobacterium tumefaciens-mediated transformation methods. One used flowering stalk explants from mature plants; the other used hypocotyl and petiole explants from in vitro-grown seedlings. Several hundred transformants containing a Bacillus thuringiensis ?-endotoxin gene (CryIA(c)-type) and the neomycin phosphotransferase gene were recovered. Rooted transformants were obtained in as little as 3 months

Timothy D. Metz; Ram Dixit; Elizabeth D. Earle

1995-01-01

159

Agrobacterium tumefaciens -mediated transformation of the plant pathogenic fungus Rosellinia necatrix  

Microsoft Academic Search

Rosellinia necatrix is a soil-borne root pathogen affecting a wide range of commercially important plant species. The mycelium of R. necatrix was transformed to hygromycin B resistance by an Agrobacterium tumefaciens-mediated transformation system using a binary plasmid vector containing the hygromycin B phosphotransferase (hph) gene controlled by the heterologous fungal Aspergillus nidulans P-gpd (glyceraldehyde 3-phosphate dehydrogenase) promoter and the trpC

Sanae Kano; Takuma Kurita; Satoko Kanematsu; Tsutomu Morinaga

2011-01-01

160

Agrobacterium tumefaciens -mediated transformation as a tool for random mutagenesis of Colletotrichum trifolii  

Microsoft Academic Search

We transformed Colletotrichum trifolii, the causal agent of alfalfa anthracnose, using Agrobacterium tumefaciens as a new tool for random insertional mutagenesis. Fungal spores of C. trifolii were transformed with T-DNA including the hygromycin phosphotransferase gene ( hph). Southern analysis showed that every randomly selected transformant had a unique hybridization pattern of T-DNA, suggesting that the T-DNA was randomly integrated into

Hiroyuki Takahara; Gento Tsuji; Yasuyuki Kubo; Mikihiro Yamamoto; Kazuhiro Toyoda; Yoshishige Inagaki; Yuki Ichinose; Tomonori Shiraishi

2004-01-01

161

Parameters affecting the efficiency of Agrobacterium tumefaciens -mediated transformation of Colletotrichum graminicola  

Microsoft Academic Search

We have developed an Agrobacterium\\u000a tumefaciens-mediated transformation (ATMT) protocol for the plant pathogenic fungus Colletotrichum graminicola, the cause of anthracnose leaf blight and stalk rot of corn. The ATMT results in higher transformation efficiencies than\\u000a previously available polyethylene glycol-mediated protocols, and falcate spores can be used instead of protoplasts for transformation.\\u000a Various experimental parameters were tested for their effects on

Jennifer L. Flowers; Lisa J. Vaillancourt

2005-01-01

162

An Agrobacterium tumefaciens transformation protocol effective on a variety of cottonwood hybrids (genus Populus )  

Microsoft Academic Search

We describe a protocol for Agrobacterium tumefaciens-mediated transformation of hybrid cottonwoods (Populus sections Tacamahaca Spach. and Aigeiros Duby). The protocol has allowed routine transformation of several economically important\\u000a cottonwood hybrids (Populus trichocarpa Torr. & Gray×P. deltoides Bartr. ex. Marsh. and P. deltoides×P. nigra L.) that were previously difficult to transform. The procedure was applied to 11 different hybrid cottonwood genotypes

K.-H. Han; R. Meilan; C. Ma; S. H. Strauss

2000-01-01

163

Trehalose synthase gene transfer mediated by Agrobacterium tumefaciens enhances resistance to osmotic stress in sugarcane  

Microsoft Academic Search

Trehalose synthase gene (TSase) fromGrifola frondosa was transferred into sugarcane (Saccharum officinarum L.) usingAgrobacterium-mediated method to improve sugarcane drought-tolerance. The results indicated that embryogenic callus of sugarcane was sensitive\\u000a toA. tumefaciens EHA105 strain in the transformation system employed. The high frequency of PPT-resistant plants were obtained from transformated\\u000a with 3 weeks callus after incubation, which reached 4.5% on average. The

Zi-Zhang Wang; Shu-Zhen Zhang; Ben-Peng Yang; Yang-Rui Li

2005-01-01

164

Production of Transgenic Tall Fescue Plants with Enhanced Stress Tolerances by Agrobacterium tumefaciens-Mediated Transformation  

Microsoft Academic Search

In order to improve stress tolerances of turf-type tall fescue (Festuca arundinacea Schreb.), Agrobacterium tumefaciens strain EHA105 carrying plasmid pCMD containing stress tolerance-related CBF1 gene from Arabidopsis thaliana was used to transform mature seeds-derived embryogenic calli of four cultivars. A total of 112 transgenic plants were regenerated from 32 independent lines and verified by histochemical detection of GUS activity, PCR

Guan-ting WU; Jin-qing CHEN; Zhang-hua HU; Chun-xiu LANG; Xiao-yun CHEN; Fu-lin WANG; Wei JIN; Ying-wu XIA

2006-01-01

165

Optimization of Agrobacterium tumefaciens mediated genetic transformation protocol for aromatic rice  

Microsoft Academic Search

A rapid, efficient and reproducible genetic transformation protocol was optimized for four aromatic rice varieties by using the established plant regeneration protocol. Mature embryos were inoculated with Agrobacterium tumefaciens strain EHA105 carrying a binary vector pIG121-Hm with GUS (reporter gene) and hpt (hygromycin resistance) gene and the transformation experiment was performed by optimizing two important parameters viz. infection times and

M. R. Hossain; L. Hassan; A. K. Patwary; M. J. Ferdous

2009-01-01

166

Cloning and sequencing of the serine dehydrogenase gene from Agrobacterium tumefaciens.  

PubMed

The structural gene for NADP+-dependent serine dehydrogenase [EC 1.1.1.-] from Agrobacterium tumefaciens ICR 1600 was cloned into Escherichia coli cells and its complete DNA sequence was analyzed. The gene encodes a polypeptide containing 249 amino acid residues. The enzyme had high sequence similarity to short-chain alcohol dehydrogenases from bacteria and unknown proteins of Haemophilus influenzae, Escherichia coli, and Saccharomyces cerevisiae. PMID:12092831

Fujisawa, Hisae; Nagata, Shinji; Chowdhury, Emran Kabir; Matsumoto, Miki; Misono, Haruo

2002-05-01

167

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

168

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

2010-08-18

169

Genetic Transformation of Aloe barbadensis Miller by Agrobacterium tumefaciens  

Microsoft Academic Search

Despite the importance of aloe in cosmetic and pharmaceutical industries, improvement of aloe (Aloe barbadensis Miller) by genetic engineering was seldom reported previously. In this study, regeneration and transformation conditions, including explant selection and surface sterilization, use of different Agrobacterium strains, and co-culture processing, are optimized. The use of 20.0% sodium hypochloride (25 min) for sterilization was less detrimental to

Congfen He; Jiaxing Zhang; Jie Chen; Xingguo Ye; Lipu Du; Yinmao Dong; Hua Zhao

2007-01-01

170

Agrobacterium-mediated transformation of herbicide resistance in creeping bentgrass and colonial bentgrass.  

PubMed

Embryogenic calli were induced from the seeds of creeping bentgrass (Agrostis palustris Huds.) cv. Regent and colonial bentgrass (Agrostis Tenuis Sibth. Fl. Oxen.) cv. Tiger. The embryogenic calli were precultured on fresh medium for 4-7 days and then co-cultivated with Agrobacterium tumefaciens, LBA4404, which contains plasmid vector-pSBGM harboring bar coding region, synthetic green fluorescent protein (sGFP) coding region and matrix attachment region (MAR). After 3 days of co-cultivation, the calli were washed thoroughly and transferred to MS medium containing 2 mg/L of 2, 4-D, 12-15 mg/L phosphinothricin (PPT) and 250 mg/L of cefotaxime. After 2-3 months of selection, the actively growing calli of 'Regent' and 'Tiger' were transferred to MS medium with 12-15 mg/L PPT and 250 mg/L cefotaxime for regeneration. The putative transformants were maintained on MS medium with 3 mg/L PPT for long period but control died within 1 month. After establishing in greenhouse, the transformants also showed strong resistance to 0.4% of herbicide Basta but control plants died within 2 weeks. Under confocal microscope, both young leaves and roots showed significant GFP expression. PCR analysis revealed the presence of a DNA fragment of GFP gene at the expected size (380 bp) in the transformants and its absence in a randomly selected control plant. PMID:12765291

Chai, Ming-Liang; Wang, Bing-Liang; Kim, Jae-Yeoul; Lee, Jong-Min; Kim, Doo-Hwan

171

A new PCR system for Agrobacterium tumefaciens detection based on amplification of T-DNA fragment.  

PubMed

The design of the PCR system presented in this work is based on the knowledge of the molecular character of the crown gall disease. The virulence of Agrobacterium tumefaciens requires the presence of a big (up to 235,000 bp) plasmid Ti (pTi-tumour inducing plasmid). This plasmid carries the so-called T-DNA fragment (T-DNA-transferred DNA), which integrates into cell chromosomes of the infected plants and subsequently changes the plant morphology nad metabolism. In cannot be excluded that after T-DNA integration the presence of Agrobacterium is not necessary for the development of pathological changes. Thus, T-DNA is the only sign that must be present both in virulent bacteria and in infected plants in any stadium of the disease and even before the infection. This is why T-DNA was chosen as the target region for PCR amplification. Primers flanking a 220 bp fragment of one of the conservative regions responsible for Agrobacterium pathogenicity, namely tms2 gene coding for indolacetamide amidohydrolase (the second step of auxin biosynthesis) were designed as the optimal for PCR amplification. The PCR amplification reactions were performed for matrixes isolated from cultures of reference strains giving one predicted product for each sample. First attempts of T-DNA detection in infected soils and plants were performed. We hope that the presented new PCR system for Agrobacterium tumefaciens detection will help to fight the crown gall disease in the nearest future. PMID:9429288

Sachadyn, P; Kur, J

1997-01-01

172

Choline Uptake in Agrobacterium tumefaciens by the High-Affinity ChoXWV Transporter?  

PubMed Central

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

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

2011-01-01

173

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

PubMed

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

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

2011-07-29

174

Interactions and DNA transfer between Agrobacterium tumefaciens, the Ti-plasmid and the plant host.  

PubMed

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 information determining the tumorous growth pattern and the synthesis of so-called 'opines', e.g. octopine (N-alpha-(D-1-carboxyethyl)-L-arginine) and nopaline (N-alpha-(1,3-dicarboxypropyl)-L-argine), is transferred and stably maintained and expressed in the transformed plant cells. This phenomenon can be understood as a 'genetic colonization' of the plant cells by bacterial plasmid DNA so that the transformed plant cells will produce and secrete into the medium amino acid derivatives (the opines) that Ti-plasmid carrying agrobacteria can selectively use as carbon and nitrogen sources. PMID:36626

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

1979-04-11

175

Tropane alkaloid production by hairy roots of Atropa belladonna obtained after transformation with Agrobacterium rhizogenes 15834 and Agrobacterium tumefaciens containing rol A, B, C genes only  

Microsoft Academic Search

Atropa belladonna leaf disks were infected by a wild strain Agrobacterium rhizogenes 15834 harboring the Ri-TL-DNA and by a disarmed Agrobacterium tumefaciens strain harboring a construction with only rolABC and npt II genes. Thirteen root lines were established and examined for their growth rate and alkaloid productivity to evaluate the possible role of rol genes in morphological differentiation and in

Valérie Bonhomme; Dominique Laurain-Mattar; Jérôme Lacoux; Marc-André Fliniaux; Annie Jacquin-Dubreuil

2000-01-01

176

Agrobacterium -mediated transformation in chickpea ( Cicer arietinum L.) with an insecticidal protein gene: optimisation of different factors  

Microsoft Academic Search

Agrobacterium-mediated transformation in chickpea was developed using strain LBA4404 carrying nptII, uidA and cryIAc genes and transformants selected on Murashige and Skoog’s basal medium supplemented with benzyladenine, kinetin and kanamycin.\\u000a Integration of transgenes was demonstrated using polymerase chain reaction and Southern blot hybridization of T0 plants. The expression of CryIAc delta endotoxin and GUS enzyme was shown by enzyme linked

Shivani Indurker; Hari S. Misra; Susan Eapen

2010-01-01

177

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

178

The Tzs protein and exogenous cytokinin affect virulence gene expression and bacterial growth of Agrobacterium tumefaciens.  

PubMed

The soil phytopathogen Agrobacterium tumefaciens causes crown gall disease in a wide range of plant species. The neoplastic growth at the infection sites is caused by transferring, integrating, and expressing transfer DNA (T-DNA) from A. tumefaciens into plant cells. A trans-zeatin synthesizing (tzs) gene is located in the nopaline-type tumor-inducing plasmid and causes trans-zeatin production in A. tumefaciens. Similar to known virulence (Vir) proteins that are induced by the vir gene inducer acetosyringone (AS) at acidic pH 5.5, Tzs protein is highly induced by AS under this growth condition but also constitutively expressed and moderately upregulated by AS at neutral pH 7.0. We found that the promoter activities and protein levels of several AS-induced vir genes increased in the tzs deletion mutant, a mutant with decreased tumorigenesis and transient transformation efficiencies, in Arabidopsis roots. During AS induction and infection of Arabidopsis roots, the tzs deletion mutant conferred impaired growth, which could be rescued by genetic complementation and supplementing exogenous cytokinin. Exogenous cytokinin also repressed vir promoter activities and Vir protein accumulation in both the wild-type and tzs mutant bacteria with AS induction. Thus, the tzs gene or its product, cytokinin, may be involved in regulating AS-induced vir gene expression and, therefore, affect bacterial growth and virulence during A. tumefaciens infection. PMID:23593941

Hwang, Hau-Hsuan; Yang, Fong-Jhih; Cheng, Tun-Fang; Chen, Yi-Chun; Lee, Ying-Ling; Tsai, Yun-Long; Lai, Erh-Min

2013-09-01

179

The efficiency of Arabidopsis thaliana floral dip transformation is determined not only by the Agrobacterium strain used but also by the physiology and the ecotype of the dipped plant.  

PubMed

To evaluate the chromosomal background of different Agrobacterium strains on floral dip transformation frequency, eight wild-type Agrobacterium strains, provided by Laboratorium voor Microbiologie Gent (LMG) and classified in different genomic groups, were compared with the commonly used Agrobacterium strains C58C1 Rif(r) (pMP90) and LBA4404 in Arabidopsis thaliana Columbia (Col-0) and C24 ecotypes. The C58C1 Rif(r) chromosomal background in combination with the pMP90 virulence plasmid showed high Col-0 floral dip transformation frequencies (0.76 to 1.57%). LMG201, which is genetically close to the Agrobacterium C58 strain, with the same virulence plasmid showed comparable or even higher transformation frequencies (1.22 to 2.28%), whereas the LBA4404 strain displayed reproducibly lower transformation frequencies (<0.2%). All other tested LMG Agrobacterium chromosomal backgrounds had transformation frequencies between those of the C58C1 Rif(r) (pMP90) and LBA4404 reference strains. None of the strains could transform the C24 ecotype with a frequency higher than 0.1%. Strikingly, all Arabidopsis Col-0 floral dip transformation experiments showed a high transformation variability from plant to plant (even more than 50-fold) within and across the performed biological repeats for all analyzed Agrobacterium strains. Therefore, the physiology of the plant and, probably, the availability of competent flowers to be transformed determine, to a large extent, floral dip transformation frequencies. PMID:23581821

Ghedira, Rim; De Buck, Sylvie; Nolf, Jonah; Depicker, Ann

2013-07-01

180

[Research on Festuca arundinacea transformation mediated by Agrobacterium tumefaciens].  

PubMed

The embryo-derived calli from four types of tall fescues (Festuca arundinacea Schreb) were transformed with two Agrobactrium tumefaciens strains AGL1 and GV3101. AGL1 harbors an intron-AtNHX1 expression vector pROK2U containing ubiqutin promoter and npt II marker gene. GV3101 harbors an intron-AtNHX1 expression vector pROK2 containing 35S promoter and npt II gene. After infection and co-culture with AGL1 or GV3101, the embyogenic calli were selected with 50-150 mg/L paromomycine and 1126 resistant plants regenerated from the resistant calli. All plants were selected further with 10-20 mg/L Kanamycin and 525 of them remained green. Genome DNA of the resistant plants was checked with specific primers and probe from AtNHX1 gene. The results of PCR assay and Southern blot analysis indicted that exogenous target gene (AtNHX1 gene) had been transferred into different cultivars of Festuca arundinacea. Different transformation frequencies among the four cultivars were obtained. PMID:16018184

Zhao, Jun-Sheng; Zhi, Da-Ying; Xue, Zhe-Yong; Xia, Guang-Min

2005-06-01

181

Production of transgenic creeping bentgrass Agrostis stolonifera var. palustris plants by Agrobacterium tumefaciens -mediated transformation using hygromycin selection  

Microsoft Academic Search

A protocol was developed for Agrobacterium tumefaciens-mediated transformation of creeping bentgrass [Agrostis stolonifera L. var. palustris (Huds) Farw]. The transformation was performed using the vector pCAMBIA 1301 which contains the reporter (uidA) gene and the selectable marker hygromycin phosphotransferase (hph) gene. Embryogenic calli initiated from mature seeds were infected with A. tumefaciens strain EHA105 followed by hygromycin selection. Effects of

Ning Han; Dong Chen; Hong-Wu Bian; Min-Juan Deng; Mu-Yuan Zhu

2005-01-01

182

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

183

The Chloramphenicol-Inducible catB Gene in Agrobacterium tumefaciens Is Regulated by Translation Attenuation†  

PubMed Central

Agrobacterium tumefaciens strains C58, A136, and BG53 are chloramphenicol resistant, and each contains the catB gene originally identified by Tennigkeit and Matzuran (Gene 99:113-116, 1991). The chloramphenicol acetyltransferase activity in all of the strains is chloramphenicol inducible. Examination of the catB gene in strain BG53 indicates that it is regulated by an attenuation mechanism similar to translation attenuation that regulates inducible catA genes resident in gram-positive bacteria and the inducible cmlA gene that confers chloramphenicol resistance in Pseudomonas spp.

Rogers, Elizabeth J.; Rahman, M. Sayeedur; Hill, Russell T.; Lovett, Paul S.

2002-01-01

184

Agrobacterium tumefaciens Mediated Transformation of ChiV Gene to Trichoderma harzianum  

Microsoft Academic Search

As a soil-borne filamentous fungus, Trichoderma harzianum exhibits biological control properties because it parasitizes a large variety of phytopathogenic fungi. In this study, the\\u000a vectors pBI121 and pCAMBIA1301 and cloning vector pUC18 were used to successfully construct expression vector pCA-GChiV for\\u000a filamentous fungi transformation mediated by Agrobacterium tumefaciens.The ChiV gene was successfully transferred into the biocontrol fungus T. harzianum with

Liming Yang; Qian Yang; Kening Sun; Ye Tian; Hulun Li

2011-01-01

185

Agrobacterium tumefaciens -mediated transformation of Cry I A ( b ) gene to Trichoderma harzianum  

Microsoft Academic Search

In this study,Cry IA(b) gene was successfully transferred into the biocontrol fungusTrichoderma harzianum with an efficiency of 60–180 transformants per 106 spores by usingAgrobacterium tumefaciens-mediated transformation. Putative transformants were analyzed to test the presence ofCry IA(b) gene by Southern blot. Most transformants contained a single T-DNA copy. RT-PCR analysis showed that theCry IA(b) gene was transcribed. Antifungal activities and insecticidal

Xingxi Gao; Qian Yang

2004-01-01

186

Identification and localization of transformed cells in Agrobacterium tumefaciens -induced plant tumors  

Microsoft Academic Search

.  \\u000a Agrobacterium tumefaciens-induced tumors of dicotyledonous plants consist of well-defined vascular bundle-like structures originating from transformed\\u000a cells. The current view that 25% of the tumor cells are transformed has been re-investigated by using ?-glucuronidase (gus)-gene-containing wild-type bacteria (A281 p35S gus-int). Regularly growing stem and leaf tumors showed irregular GUS-staining patterns in the different plant species, Ricinus communis L., Cucurbita maxima

Claudia Rezmer; Ralf Schlichting; Rebecca Wächter; Cornelia I. Ullrich

1999-01-01

187

Accumulation of copper in Trichoderma reesei transformants, constructed with the modified Agrobacterium tumefaciens -mediated transformation technique  

Microsoft Academic Search

A modified Agrobacterium tumefaciens-mediated transformation method was established for the construction of mutants with improved copper tolerance and accumulation\\u000a capability in Trichoderma reesei. One transformant, AT01, exhibited the highest copper accumulation capability. With copper at 0.7 mM, AT01 removed 13 mg\\u000a copper\\/g biomass (removal rate of 96%), whereas the wild-type strain removed only 6 mg copper\\/g biomass (removal rate of 50%).\\u000a Optimal conditions

Kehe Fu; Lixing Liu; Lili Fan; Tong Liu; Jie Chen

2010-01-01

188

Overexpression of the chitosanase gene in Fusarium solani via Agrobacterium tumefaciens-mediated transformation.  

PubMed

To overexpress the chitosanase gene (csn) in F. solani, a vector based on pCAMBIA 1300 was constructed. The csn gene, which is under control of the Aspergillus nidulans gpdA promoter and A. nidulans trpC terminator, was introduced back into the F. solani genome by Agrobacterium tumefaciens-mediated transformation, and the herbicide-resistance gene bar from Streptomyces hygroscopicus was used as the selection marker. Transformants which showed a significant increase in chitosanase production (approximately 2.1-fold than control) were obtained. Southern blot analysis indicated that most transformants had a single-copy T-DNA integration. PMID:19093150

Liu, Huaiwei; Bao, Xiaoming

2008-12-18

189

Phospholipids of Rhizobium meliloti and Agrobacterium tumefaciens : Lack of effect of Ti plasmid  

Microsoft Academic Search

We have studied the phospholipid composition ofRhizobium meliloti strains which do or do not contain the large, tumor-inducing (Ti) plasmid ofAgrobacterium tumefaciens. The major phospholipids of stationary phase cells were phosphatidylethanolamine (PE) (22%), phosphatidyl-N-methylethanolamine\\u000a (22%), phosphatidylcholine (PC) (27%), phosphatidylglycerol (11.4%), and cardiolipin (11%); as average percent of lipid phosphorus.\\u000a Phosphatidyl-N,N-dimethylethanolamine (3.7%) was also present. The proportions of PE were higher,

Emily A. Thompson; Allan E. Kaufman; Norah C. Johnston; Howard Goldfine

1983-01-01

190

STRUCTURE\\/FUNCTION ANALYSIS OF THE MINOR T-PILUS COMPONENT VIRB5 FROM AGROBACTERIUM TUMEFACIENS  

Microsoft Academic Search

Type IV Secretion Systems (T4SS) are machineries required for the virulence of many Gram-negative pathogens. They contribute to bacterial competence, conjugation and the translocation of toxins from bacteria into eukaryotic hosts. In the plant pathogen Agrobacterium tumefaciens, the T4SS complex is composed of 11 VirB proteins (VirB1-VirB11) and VirD4. The VirB\\/D4 complex spans the bacterial envelope and assembles filamentous T-pili,

Khaled Ahmed Aly

2010-01-01

191

Efficient gene knockout in the maize pathogen Setosphaeria turcica using Agrobacterium tumefaciens-mediated transformation.  

PubMed

Setosphaeria turcica, a hemibiotrophic pathogenic dothideomycete, is the causal agent of Northern Leaf Blight of maize, which periodically causes significant yield losses worldwide. To explore molecular mechanisms of fungal pathogenicity and virulence to the host, an efficient targeted gene knockout transformation system using Agrobacterium tumefaciens was established with field collected strains. The starting materials, incubation time, induction medium type, Agrobacterium cell density, and method of co-incubation were optimized for deletion of 1,3,8-trihydroxynaphthalene reductase, a gene in the melanin biosynthesis pathway, as a test case. Four additional genes were deleted in two different S. turcica field isolates to confirm robustness of the method. One of these mutant strains was reduced in virulence compared with the wild-type strain when inoculated on susceptible maize. Transformation efficiency was ?20 ± 3 transformants per 1× 10(6) germlings and homologous recombination efficiency was 33.3 to 100%. PMID:23384859

Xue, Chunsheng; Wu, Dongliang; Condon, Bradford J; Bi, Qing; Wang, Weiwei; Turgeon, B Gillian

2013-06-01

192

[Transgenic soybean obtained with Agrobacterium tumefaciens-mediated transformation of embryonic tip of soybean mature seeds].  

PubMed

Regenerated embryonic tips were inoculated with Agrobacterium tumefaciens strain EHA105, which contains binary vector pCAMBIA2301, and cultured for 20 h. Our results showed that the T-DNA transfer efficiency reached up to 63.3% (Table 1) and the transformation efficiency reached up to 6.4%-12.1% (Table 2). The effect of infection time on T-DNA delivery into soybean embryonic tips was determined (Table 1). We also discuss the effects of days of co-cultivation to transient expression and the effects of different AS concentrations to transient expression of gus gene (Figs. 1,2). These data indicate that the embryonic tip regeneration system can be used for efficient, effective Agrobacterium-mediated transformation. PMID:15643082

Liu, Hai-Kun; Wei, Zhi-Ming

2004-12-01

193

A New Type IV Secretion System Promotes Conjugal Transfer in Agrobacterium tumefaciens  

PubMed Central

Two DNA transfer systems encoded by the tumor-inducing (Ti) plasmid have been previously identified in Agrobacterium tumefaciens. The virB operon is required for the transfer of transferred DNA to the plant host, and the trb system encodes functions required for the conjugal transfer of the Ti plasmid between cells of Agrobacterium. Recent availability of the genome sequence of Agrobacterium allowed us to identify a third system that is most similar to the VirB type IV secretion system of Bartonella henselae. We have designated this system avhB for Agrobacterium virulence homologue virB. The avhB loci reside on pAtC58 and encode at least 10 proteins (AvhB2 through AvhB11), 7 of which display significant similarity to the corresponding virulence-associated VirB proteins of the Ti plasmid. However, the AvhB system is not required for tumor formation; rather, it mediates the conjugal transfer of the pAtC58 cryptic plasmid between cells of Agrobacterium. This transfer occurs in the absence of the Ti plasmid-encoded VirB and Trb systems. Like the VirB system, AvhB products promote the conjugal transfer of the IncQ plasmid RSF1010, suggesting that these products comprise a mating-pair formation system. The presence of plasmid TiC58 or plasmid RSF1010 reduces the conjugal transfer efficiency of pAtC58 10- or 1,000-fold, respectively. These data suggest that complex substrate interactions exist among the three DNA transfer systems of Agrobacterium.

Chen, Lishan; Chen, Yuching; Wood, Derek W.; Nester, Eugene W.

2002-01-01

194

A repression system for genes encoding proteins toxic to bacteria in Agrobacterium tumefaciens-mediated plant transformation  

Microsoft Academic Search

Transfer of a chimeric gene encoding secretory T4 lysozyme under the control of the mannopine synthase promoter (pMAS) into tobacco by Agrobacterium-mediated gene transfer has proven to be difficult. This can be due to unwanted expression of the lysozyme gene inserted into the T-DNA in Agrobacterium tumefaciens. Two different strategies have been successfully tested to inhibit expression of foreign genes

Astrid Jahnke; Horst Lörz

1998-01-01

195

Agrobacterium tumefaciens-mediated transformation of Swingle citrumelo ( Citrus paradisi Macf.× Poncirus trifoliata L. Raf.) using thin epicotyl sections  

Microsoft Academic Search

Transgenic plants of the important citrus rootstock Swingle citrumelo (Citrus paradisiMacf.×Poncirustrifoliata L. Raf.) were obtained using Agrobacterium tumefaciens-mediated transformation of seedling epicotyl tissue. Thin sections were co-cultured with Agrobacterium strain EHA105 carrying pBI121, a plasmid that contains the nptII (pnos) gene and the uidA (p35S) gene for scorable marker ?-glucuronidase (GUS). The use of thin sections and the application of

H. B. C Molinari; J. C Bespalhok; A. K Kobayashi; L. F. P Pereira; L. G. E Vieira

2004-01-01

196

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

Microsoft Academic Search

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

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

2006-01-01

197

Agrobacterium tumefaciens-mediated transformation of the plant pathogenic fungus Rosellinia necatrix.  

PubMed

Rosellinia necatrix is a soil-borne root pathogen affecting a wide range of commercially important plant species. The mycelium of R. necatrix was transformed to hygromycin B resistance by an Agrobacterium tumefaciens-mediated transformation system using a binary plasmid vector containing the hygromycin B phosphotransferase (hph) gene controlled by the heterologous fungal Aspergillus nidulans P-gpd (glyceraldehyde 3-phosphate dehydrogenase) promoter and the trpC terminator. Co-cultivation of R. necatrix strain W1015 and A. tumefaciens strain AGL-1 at 25 degrees C using the binary vector pAN26-CB1300, which contained the hygromycin B resistance cassette based on pAN26 and pCAMBIA1300, resulted in high frequencies of transformation. The presence of the hph gene in the transformants was detected by PCR, and single-copy integration of the marker gene was demonstrated by Southern blot analysis. This report of an Agrobacterium-mediated transformation method should allow the development of T-DNA tagging as a system f or insertional mutagenesis in R necatrix and provide a simple and reliable method for genetic manipulation. PMID:21513216

Kano, Sanae; Kurita, Takuma; Kanematsu, Satoko; Morinaga, Tsutomu

198

Rhizobitoxine production in Agrobacterium tumefaciens C58 by Bradyrhizobium elkanii rtxACDEFG genes.  

PubMed

We examined the genetic basis and transfer for production of rhizobitoxine, an inhibitor of ethylene biosynthesis in plants, directed by the rtx genes of Bradyrhizobium elkanii. Comparison with genome sequences of Bradyrhizobium japonicum and Xanthomonas oryzae suggests that the rtx genes extend from the previously identified rtxAC genes through four additional genes rtxDEFG. Reverse transcription-PCR analysis showed that the rtxACDEFG genes are expressed as an operon. Mutational analysis indicated that rtxDEG mutants reduced rhizobitoxine biosynthesis, while the rtxA gene is essential for its synthesis. Introduction of the rtxACDEFG into Agrobacterium tumefaciens resulted in strong expression of rtxACDEFG and production of RtxA protein, but no rhizobitoxine was detectable. Addition of O-acetylhomoserine, a precursor of rhizobitoxine, to the Agrobacterium derivative, however, fostered production of rhizobitoxine in culture. The diluted culture supernatant inhibited the activities of beta-cystathionase and 1-aminocyclopropane-1-carboxylate synthase, indicating that A. tumefaciens carrying rtxACDEFG genes excreted biologically active rhizobitoxine. PMID:17227467

Sugawara, Masayuki; Haramaki, Ryota; Nonaka, Satoko; Ezura, Hiroshi; Okazaki, Shin; Eda, Shima; Mitsui, Hisayuki; Minamisawa, Kiwamu

2007-01-15

199

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

200

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

PubMed

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

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

2007-01-13

201

Genes responsible for the supervirulence phenotype of Agrobacterium tumefaciens A281.  

PubMed Central

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

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

1987-01-01

202

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.

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

2010-01-01

203

Agrobacterium tumefaciens-mediated transformation of Aspergillus aculeatus for insertional mutagenesis.  

PubMed

Agrobacterium tumefaciens-mediated transformation (AMT) was applied to Aspergillus aculeatus. Transformants carrying the T-DNA from a binary vector pBIG2RHPH2 were sufficiently mitotically stable to allow functional genomic analyses. The AMT technique was optimized by altering the concentration of acetosyringone, the ratio and concentration of A. tumefaciens and A. aculeatus cells, the duration of co-cultivation, and the status of A. aculeatus cells when using conidia, protoplasts, or germlings. On average, 30 transformants per 104 conidia or 217 transformants per 107 conidia were obtained under the optimized conditions when A. tumefaciens co-cultured with fungi using solid or liquid induction media (IM). Although the transformation frequency in liquid IM was 100-fold lower than that on solid IM, the AMT method using liquid IM is better suited for high-throughput insertional mutagenesis because the transformants can be isolated on fewer selection media plates by concentrating the transformed germlings. The production of two albino A. aculeatus mutants by AMT confirmed that the inserted T-DNA disrupted the polyketide synthase gene AapksP, which is involved in pigment production. Considering the efficiency of AMT and the correlation between the phenotypes and genotypes of the transformants, the established AMT technique offers a highly efficient means for characterizing the gene function in A. aculeatus. PMID:22166586

Kunitake, Emi; Tani, Shuji; Sumitani, Jun-Ichi; Kawaguchi, Takashi

2011-12-14

204

Agrobacterium tumefaciens-mediated transformation of the plant pathogenic fungus, Magnaporthe grisea.  

PubMed

An effective way to study the infection mechanisms of fungal pathogens is to disrupt their genes via transformation in both targeted and random manners. This isolates the mutants that exhibit altered virulence. In this paper, we report the successful transformation of Magnaporthe grisea, the causal agent for rice blast, that is mediated by Agrobacterium tumefaciens. Employing the binary vector pBHt2, which carries the bacterial hygromycin B phosphotransferase gene under the control of the Aspergillus nidulans trpC promoter as a selectable marker, led to the production of 500 to > 1,000 hygromycin B-resistant transformants per 1 x 10(6) conidia of M. grisea. The transformation efficiency is correlated with the number of A. tumefaciens cells used, pre-treating bacterial cells with acetosyringone prior to co-cultivation with fungal spores, and the duration of co-cultivation. All of the transformants tested remained mitotically stable, maintaining their hygromycin B resistance after several generations of growth in the absence of hygromycin B. A genomic Southern blot analysis showed that over 60% of the transformants contained a single T-DNA insert on their genome. Considering the efficiency and flexibility of A. tumefaciens-mediated transformation (ATMT), this technique offers highly efficient means for characterizing the genes that are important for the pathogenicity of M. grisea. PMID:11804343

Rho, H S; Kang, S; Lee, Y H

2001-12-31

205

A fine control of quorum-sensing communication in Agrobacterium tumefaciens  

PubMed Central

The bacterial pathogen Agrobacterium tumefaciens produces the quorum-sensing (QS) signal 3-oxo-octanoylhomoserine lactone (OC8HSL) for controlling horizontal transfer of its tumor inducing (Ti) plasmid that carries both the T-DNA and the virulence genes. Over-accumulation of OC8HSL also increases severity of plant symptoms (number of emerging tumors at infection site) by an unknown mechanism. A. tumefaciens strain C58 expresses two lactonases, AttM (BlcC) and AiiB, that cleave OC8HSL and are potential modulators of QS. Recent data highlight the direct contribution of lactonases AttM and AiiB in the control of OC8HSL level and QS-regulated functions such as conjugation of Ti plasmid and seriousness of plant symptoms. Expression of the two lactonases is regulated by different plant signals. A working model of QS in the course of the A. tumefaciens-plant host interaction is proposed and discussed.

Haudecoeur, Elise

2010-01-01

206

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

PubMed

Additional virulence (vir) genes in Agrobacterium tumefaciens and sonication were investigated for their impact on transformation efficiency in loblolly pine (Pinus taeda L.). Mature zygotic embryos of loblolly pine were co-cultivated with disarmed A. tumefaciens strain EHA105 containing either plasmid vector pCAMBIA1301 or vector pCAMBIA1301 with an additional 15.8-kb fragment carrying extra copies of the Vir B, Vir C, and Vir G regions from the supervirulent plasmid pTOK47. pCAMBIA1301 contains hygromycin resistance and the beta-glucuronidase (GUS) reporter gene. Expression of GUS was observed after 3-6 days of co-cultivation, with peak expression at approximately 21 days. The highest numbers of GUS-expressing areas were visible up to 21 days after co-cultivation, declining rapidly thereafter. Both transient and stable transformation efficiencies increased when the explants were sonicated before co-cultivation and/or the additional virB, virC, and virG genes were included with the pCAMBIA1301 plasmid T-DNA. Use of the plasmid with additional vir genes and sonication dramatically enhanced the efficiency of Agrobacterium-mediated gene transfer not only in transient expression but also in the recovery of hygromycin-resistant lines. Stably transformed cultures and transgenic plants were produced from embryos transformed with A. tumefaciens EHA105 carrying pCAMBIA1301 or pCAMBIA1301+pTOK47 in the three families of loblolly pine. The presence of the introduced GUS and hygromycin phosphotransferase genes in the transgenic plants was confirmed by polymerase chain reaction and Southern hybridization analyses. PMID:12789430

Tang, W

2002-11-26

207

DNA sequences homologous to the T DNA region of Agrobacterium tumefaciens are present in diverse Rhizobium species  

Microsoft Academic Search

DNA sequences homologous to the T DNA region of the octopine-type Ti plasmid from Agrobacterium tumefaciens are present in different Rhizobium species. Plasmid DNA from each of two R. leguminosarum, two R. meliloti, and four slow-growing Rhizobium strains examined contain restriction endonuclease fragments that hybridize with the T DNA region, or with DNA sequences at or near the adjacent Ti

R. G. Hadley; A. A. Szalay

1982-01-01

208

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

209

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

PubMed

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

2013-09-02

210

Evaluation of a wide range of pepper genotypes for regeneration and transformation with an Agrobacterium tumefaciens shooter strain  

Microsoft Academic Search

A regeneration protocol developed for the Agrobacterium tumefaciens-mediated transformation of pepper (Capsicum annuum L.) was used to evaluate the potential for genetic transformation of 107 doubled haploid (DH) pepper genotypes belonging to 12 main cultivar groups. The genotypes were scored on the basis of the ratio of regenerated shoots compared to the commercial cultivar Fehérözön, which exhibited 30–70% regeneration from

E. Balázs; Á. Bukovinszki; M. Csányi; G. Csilléry; Z. Divéki; I. Nagy; J. Mitykó; K. Salánki; V. Mihálka

2008-01-01

211

Efficient production of transgenic potato ( S. tuberosum L. ssp. andigena) plants via Agrobacterium tumefaciens-mediated transformation  

Microsoft Academic Search

Potato is an important target crop for biotechnological applications and is a valuable model system for studying signaling processes. Efficient transformation is critical for rapid genetic analyses. The production of transgenic potato shoots within 4 weeks from the time of initial inoculation of leaf explants by Agrobacterium tumefaciens has been established with the Solanum tuberosum subspecies andigena. Vigorous stock plants,

Anjan K. Banerjee; Salomé Prat; David J. Hannapel

2006-01-01

212

Full structural characterization of the lipid A components from the Agrobacterium tumefaciens strain C58 lipopolysaccharide fraction  

Microsoft Academic Search

For the first time, the complete structure of the lipid A from the lipopolysaccharide of an Agrobacterium species is here reported. In particular, the structure of the lipid A from A. tumefaciens strain C58, a soil pathogen bacterium strictly related to Rhizobiaceae, was determined. The structural study, carried out by chemical analysis, mass spectrometry, and nuclear magnetic resonance spectroscopy, revealed

Alba Silipo; Cristina De Castro; Rosa Lanzetta; Antonio Molinaro; Michelangelo Parrilli

2004-01-01

213

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...

214

Agrobacterium tumefaciens-mediated transformation of hybrid larch (Larix kaempferi T L. decidua) and transgenic plant regenerationn  

Microsoft Academic Search

A transformation procedure was developed for hybrid larch embryogenic tissue using Agrobacterium tumefaciens. The cocultivation procedure yielded one to two transformation events per 100 cocultivated masses. The addition of 100 m coniferyl alcohol increased the yield. This improved procedure was successfully applied to three other genotypes. After 3\\u000a months on selective medium, the transgenic tissue remained embryogenic, which allowed production

V. Levée; M.-A. Lelu; L. Jouanin; D. Cornu; G. Pilate

1997-01-01

215

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

216

Linear chromosome-generating system of Agrobacterium tumefaciens C58: protelomerase generates and protects hairpin ends.  

PubMed

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. PMID:22582388

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

2012-05-10

217

Agrobacterium tumefaciens-mediated genetic transformation of a recalcitrant grain legume, lentil (Lens culinaris Medik).  

PubMed

A simple and reproducible Agrobacterium-mediated transformation protocol for a recalcitrant legume plant, lentil (Lens culinaris M.) is reported. Application of wounding treatments and efficiencies of three Agrobacterium tumefaciens strains, EHA105, C58C1, and KYRT1 were compared for T-DNA delivery into lentil cotyledonary node tissues. KYRT1 was found to be on average 2.8-fold more efficient than both EHA105 and C58C1 for producing transient beta-glucuronidase (GUS) gene (gus) expression on cotyledonary petioles. Wounding of the explants, use of an optimized transformation protocol with the application of acetosyringone and vacuum infiltration treatments in addition to the application of a gradually intensifying selection regime played significant roles in enhancing transformation frequency. Lentil explants were transformed by inoculation with Agrobacterium tumefaciens strain, KYRT1 harboring a binary vector pTJK136 that carried neomycin phosphotransferase gene (npt-II) and an intron containing gusA gene on its T-DNA region. GUS-positive shoots were micrografted on lentil rootstocks. Transgenic lentil plants were produced with an overall transformation frequency of 2.3%. The presence of the transgene in the lentil genome was confirmed by GUS assay, PCR, RT-PCR and Southern hybridization. The transgenic shoots grafted on rootstocks were successfully transferred to soil and grown to maturity in the greenhouse. GUS activity was detected in vegetative and reproductive organs of T(0), T(1), T(2) and T(3) plants. PCR assays of T(1), T(2) and T(3) progenies confirmed the stable transmission of the transgene to the next generations. PMID:19083242

Akcay, Ufuk Celikkol; Mahmoudian, M; Kamci, H; Yucel, M; Oktem, H A

2008-12-16

218

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

219

A disarmed binary vector from Agrobacterium tumefaciens functions in Agrobacterium rhizogenes  

Microsoft Academic Search

Binary Ti plasmid vector systems consist of two plasmids in Agrobacterium, where one plasmid contains the DNA that can be transferred to plant cells and the other contains the virulence (vir) genes which are necessary for the DNA transfer but are not themselves stably transferred. We have constructed two nononcogenic vectors (pARC4 and pARC8) based on the binary Ti plasmid

Robert B. Simpson; Albert Spielmann; Linda Margossian; Thomas D. McKnight

1986-01-01

220

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

PubMed

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

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

2002-10-16

221

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.

Wang, Ji-ye; Li, Hong-ye

2008-01-01

222

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

223

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.

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

1987-01-01

224

[Production of transgenic rape plants (Brassica napus L.) using Agrobacterium tumefaciens].  

PubMed

The procedure for genetic transformation of two spring and one winter rapeseed cultivars was developed. No-paline strains of Agrobacterium tumefaciens GV3101 and EHA105 were shown to be preferable for gene transfer, as compared to the octopine strain GV2260. With two types of plant explants, the segments of hypocotyls and cotyledons, transformation was successful; however, its efficiency was somewhat higher with the fragments of hypocotyls. Analysis of regenerated plants by PCR and Southern blotting confirmed the presence of the nptII and nisA genes in transformants. RNA analysis by Northern blotting showed expression of the nisA gene in transformed shoots. The transgenes were inherited in T2 as Mendelian traits. The effect of biotic and abiotic factors on the efficiency of genetic transformation in rapeseed is discussed. PMID:10994497

Radchuk, V V; Klocke, E; Radchuk, R I; Neumann, M; Blume YaB

2000-07-01

225

Improved production of transgenic Dioscorea zingiberensis (Dioscoreaceae) by Agrobacterium tumefaciens-mediated transformation.  

PubMed

The establishment of high-efficiency Agrobacterium-mediated transformation techniques could improve the production of Dioscorea zingiberensis, a medicinal species with a high diosgenin content. We co-cultivated embryogenic calli induced from mature seeds with A. tumefaciens strain EHA105. A binary vector, pCAMBIA1381, which contains the gfp and hpt genes under the control of the ubiquitin promoter and the CaMV 35S promoter, respectively, was used for transformation. Pre-culture, basic medium, acetosyringone, and bacterial density were evaluated to establish the most efficient protocol. The optimal conditions consisted of MS medium without CaCl(2) for pre- and co-cultivation, three days for pre-culture, addition of 200 ?M AS, and an OD(600) of 0.5. The transgenic plants grown under selection were confirmed by PCR analysis and Southern blot analysis. This protocol produced transgenic D. zingiberensis plants in seven months, with a transformation efficiency of 6%. PMID:22370891

Shi, L; Fan, J Q; Hu, C G; Luo, J; Yao, J L

2012-02-03

226

Production of fertile transgenic peanut (Arachis hypogaea L.) plants using Agrobacterium tumefaciens.  

PubMed

Fertile transgenic plants of peanut (Arachis hypogaea L. cv. New Mexico Valencia A) were produced using an Agrobacterium-mediated transformation system. Leaf section explants were inoculated with A. tumefaciens strain EHA105 harboring the binary vector pBI121 containing the genes for ?-glucuronidase (GUS) and neomycin phosphotransferase II (NPTII). Approximately 10% of the shoots regenerated on selection medium were GUS-positive. Five independent transformation events resulted in the production of 52 fertile transgenic peanut plants. On average, 240 d were required between seed germination for explant preparation and the production of mature t1 seed by T0 plants. Molecular analysis of transgenic plants confirmed the stable integration of the transgenes into the peanut genome. GUS expression segregated in a 3?1 Mendelian ratio in most T1 generation plants. PMID:24178604

Cheng, M; Jarret, R L; Li, Z; Xing, A; Demski, J W

1996-05-01

227

Transsexuality in the Rhizosphere: Quorum Sensing Reversibly Converts Agrobacterium tumefaciens from Phenotypically Female to Male?  

PubMed Central

Conjugative plasmids generally encode proteins that block the conjugative entry of identical or similar plasmids into the host cell, a phenomenon known as entry exclusion. Here, we demonstrate that two Ti plasmids of Agrobacterium tumefaciens encode robust entry exclusion functions. Two proteins, TrbJ and TrbK, can each mediate entry exclusion and act synergistically. The trbJ and trbK genes are included within the trb operon, which is tightly regulated by the quorum-sensing regulator TraR and the cognate acylhomoserine lactone. In the absence of quorum-sensing signals, these proteins are not significantly expressed, and cells lacking TrbJ and TrbK are efficient Ti plasmid recipients. In the presence of these signals, these strains block the entry of Ti plasmids and instead become efficient conjugal donors.

Cho, Hongbaek; Pinto, Uelinton M.; Winans, Stephen C.

2009-01-01

228

Genomic species are ecological species as revealed by comparative genomics in Agrobacterium tumefaciens.  

PubMed

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

2011-07-27

229

O -methyltransferase(s)-suppressed plants produce lower amounts of phenolic vir inducers and are less susceptible to Agrobacterium tumefaciens infection  

Microsoft Academic Search

The first step of Agrobacterium tumefaciens\\/plant interaction corresponds to the activation of a transduction pathway of the bacterium by plant exudate. Phenolic compounds\\u000a rapidly secreted by wounded plant cells induce the expression of bacterial virulence (vir) genes; however, little is known about their biosynthesis in plant. Here we show that inoculation of an Agrobacterium tumefaciens virulent strain on orthodiphenol-O-methyltransferases-suppressed tobacco

Stéphane Maury; A. Delaunay; F. Mesnard; D. Crônier; B. Chabbert; P. Geoffroy; M. Legrand

2010-01-01

230

Translation Start Sequences Affect the Efficiency of Silencing of Agrobacterium tumefaciens T-DNA Oncogenes1  

PubMed Central

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

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

2003-01-01

231

Agrobacterium tumefaciens mediated transformation of ChiV gene to Trichoderma harzianum.  

PubMed

As a soil-borne filamentous fungus, Trichoderma harzianum exhibits biological control properties because it parasitizes a large variety of phytopathogenic fungi. In this study, the vectors pBI121 and pCAMBIA1301 and cloning vector pUC18 were used to successfully construct expression vector pCA-GChiV for filamentous fungi transformation mediated by Agrobacterium tumefaciens.The ChiV gene was successfully transferred into the biocontrol fungus T. harzianum with an efficiency of 90-110 transformants per 10(7) spores using A. tumefaciens-mediated transformation. Putative transformants were analyzed to test the transformation by the southern blot, and the expression of ChiV was detected by reverse transcription PCR. The transformants were co-cultured to assay antifungal activities with Rhizoctonia solani. The inhibition rates of the transformants and no ChiV gene transferred T. harzianum were 98.56% and 82.42%, respectively, on the fourth day.The results showed that the ChiV transformants had significantly higher inhibition activity. PMID:20936373

Yang, Liming; Yang, Qian; Sun, Kening; Tian, Ye; Li, Hulun

2010-10-10

232

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

PubMed

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

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

2009-08-13

233

Bioassays of quorum sensing compounds using Agrobacterium tumefaciens and Chromobacterium violaceum.  

PubMed

In most bacteria, a global level of regulation exists involving intercellular communication via the production and response to cell density-dependent signal molecules. This cell density-dependent regulation has been termed quorum sensing (QS). QS is a global regulator, which has been associated with a number of important features in bacteria including virulence regulation and biofilm formation. Consequently, there is considerable interest in understanding, detecting, and inhibiting QS. Acyl homoserine lactones (acyl HSLs) are used as extracellular QS signals by a variety of Gram-negative bacteria. Chromobacterium violaceum, a Gram-negative bacterium commonly found in soil and water, produces the characteristic purple pigment violacein, the production of which is regulated by acyl HSL-mediated QS. Based on this readily observed pigmentation phenotype, C. violaceum strains can be used to detect various aspects of acyl HSL-mediated QS activity. In another commonly used bioassay organism, Agrobacterium tumefaciens, QS can be detected by the use of a reporter gene such as lacZ. Here, we describe several commonly used approaches incorporating C. violaceum and A. tumefaciens that can be used to detect acyl HSLs and QS inhibition. PMID:21031300

Chu, Weihua; Vattem, Dhiraj A; Maitin, Vatsala; Barnes, Mary B; McLean, Robert J C

2011-01-01

234

Establishment of an efficient Agrobacterium tumefaciens-mediated leaf disc transformation of Thellungiella halophila.  

PubMed

Thellungiella halophila is a salt-tolerant close relative of Arabidopsis, which is adopted as a halophytic model for stress tolerance research. We established an Agrobacterium tumefaciens-mediated transformation procedure for T. halophila. Leaf explants of T. halophila were incubated with A. tumefaciens strain EHA105 containing a binary vector pCAMBIA1301 with the hpt gene as a selectable marker for hygromycin resistance and an intron-containing beta-glucuronidase gene as a reporter gene. Following co-cultivation, leaf explants were cultured on selective medium containing 10 mg l(-1) hygromycin and 500 mg l(-1) cefotaxime. Hygromycin-resistant calluses were induced from the leaf explants after 3 weeks. Shoot regeneration was achieved after transferring the calluses onto fresh medium of the same composition. Finally, the shoots were rooted on half strength MS basal medium supplemented with 10 mg l(-1) hygromycin. Incorporation and expression of the transgenes were confirmed by PCR, Southern blot analysis and GUS histochemical assay. Using this protocol, transgenic T. halophila plants can be obtained in approximately 2 months with a high transformation frequency of 26%. PMID:17551729

Li, Hong-Qing; Xu, Jie; Chen, Lei; Li, Mei-Ru

2007-06-06

235

Agrobacterium tumefaciens-mediated genetic transformation of the cardenolide-producing plant Digitalis minor L.  

PubMed

A repeatable transformation system has been established for Digitalis minor using Agrobacterium tumefaciens. Leaf explants from 30-day-old seedlings were inoculated with either EHA105 (carrying the nptII and gusA genes) or AGL1 (with the bar and gusA genes) strains. Among the tested factors influencing T-DNA transfer to plants, the EHA105 strain and the addition of acetosyringone to the co-culture medium increased transformation. The highest transformation efficiency (8.4 %) was obtained when freshly isolated explants, soaked in a bacterial suspension with an OD550 of 0.9, were subcultured on selection medium after a 4-day co-culture with the bacteria. Evidence of stable transgene integration was obtained by PCR, growth on media selective for nptII or bar genes, and expression of the gusA gene. Southern hybridisation, performed in six plants, provided information about the number of inserts. More than 200 transgenic plants were recovered from 65 independent explants. Thirty of these plants were successfully established in soil. This is the first report on transgenic Digitalis spp plants using an A. tumefaciens-mediated leaf disc transformation procedure. PMID:12624819

Sales, Ester; Segura, Juan; Arrillaga, Isabel

2003-02-01

236

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

PubMed Central

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

Haudecoeur, E.; Planamente, S.; Cirou, A.; Tannieres, M.; Shelp, B. J.; Morera, S.; Faure, D.

2009-01-01

237

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

PubMed

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. PMID:457613

Loper, J E; Kado, C I

1979-08-01

238

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

2012-10-11

239

Efficient Agrobacterium tumefaciens-mediated transformation of soybeans using an embryonic tip regeneration system.  

PubMed

Here, we report the establishment of an efficient, in vitro, shoot organogenesis, regeneration system for soybeans [Glycine max (L.) Merr.]. Mature soybean seeds were soaked for 24 h, the embryonic tips were collected and cultured on MSB5 medium supplemented with 3.5 mg l(-1) N6-benzylaminopurine (BAP) for 24 h, and explants were transferred to MSB5 medium supplemented with 0.2 mg l(-1) BAP and 0.2 mg l(-1) indolebutyric acid. Use of embryonic tips yielded a higher regeneration frequency (87.7%) than regeneration systems using cotyledonary nodes (40.3%) and hypocotyl segments (56.4%) as starting materials. Regenerated embryonic tips were inoculated with Agrobacterium tumefaciens strain EHA105, which contains the binary vector pCAMBIA2301, and cultured for 20 h. Our results showed that the T-DNA transfer efficiency reached up to 78.2% and the transformation efficiency reached up to 15.8%. These data indicate that the embryonic tip regeneration system can be used for efficient, effective Agrobacterium-mediated transformation. PMID:15605177

Liu, Hai-Kun; Yang, Chao; Wei, Zhi-Ming

2004-07-16

240

Agrobacterium tumefaciens -mediated transformation of Lesquerella fendleri L., a potential new oil crop with rich lesquerolic acid  

Microsoft Academic Search

A protocol was developed for regeneration and Agrobacterium-mediated genetic transformation of Lesquerella fendleri. Calli were first induced from hypocotyls and cotyledons on MS plus 0.5 mg l?1 BA, 1 mg l?1 NAA and 1 mg l?1 2,4-D, then co-cultivated for 2–3 days in darkness on MS supplemented with 0.5 mg l?1 BA, 0.2 mg l?1 NAA and 100 ?mol l?1As together with Agrobacterium tumefaciens strain EHA105\\/pCAMBIA1301 that harbored genes for uidA (GUS) and

Wenyan Wang; Chenggang Wang; Bang-Lian Huang; Bangquan Huang

2008-01-01

241

Functional domains of Agrobacterium tumefaciens single-stranded DNA-binding protein VirE2.  

PubMed Central

The transferred DNA (T-DNA) portion of the Agrobacterium tumefaciens tumor-inducing (Ti) plasmid enters infected plant cells and integrates into plant nuclear DNA. Direct repeats define the T-DNA ends; transfer begins when the VirD2 endonuclease produces a site-specific nick in the right-hand border repeat and attaches to the 5' end of the nicked strand. Subsequent events liberate the lower strand of the T-DNA from the Ti plasmid, producing single-stranded DNA molecules (T strands) that are covalently linked to VirD2 at their 5' ends. A. tumefaciens appears to transfer T-DNA into plant cells as a T-strand-VirD2 complex. The bacterium also transports VirE2, a cooperative single-stranded DNA-binding protein, into plant cells during infection. Both VirD2 and VirE2 contain nuclear localization signals that may direct these proteins, and bound T strands, into plant nuclei. Here we report the locations of functional regions of VirE2 identified by eight insertions of XhoI linker oligonucleotides, and one deletion mutation, throughout virE2. We examined the effects of these mutations on virulence, single-stranded DNA (ssDNA) binding, and accumulation of VirE2 in A. tumefaciens. Two of the mutations in the C-terminal half of VirE2 eliminated ssDNA binding, whereas two insertions in the N-terminal half altered cooperativity. Four of the mutations, distributed throughout virE2, decreased the stability of VirE2 in A. tumefaciens. In addition, we isolated a mutation in the central region of VirE2 that decreased tumorigenicity but did not affect ssDNA binding or VirE2 accumulation. This mutation may affect export of VirE2 into plant cells or nuclear localization of VirE2, or it may affect an uncharacterized activity of VirE2.

Dombek, P; Ream, W

1997-01-01

242

Biological Activity of the tzs Gene of Nopaline Agrobacterium tumefaciens GV3101 in Plant Regeneration and Genetic Transformation.  

PubMed

Agrobacterium tumefaciens has been widely used in plant genetic transformation. Hormone-encoding genes residing in the T-DNA region have been removed, resulting in disarmed Agrobacterium strains that are used in various transformation experiments. Nopaline Agrobacterium strains, however, carry another hormone gene, trans-zeatin synthesizing (tzs), that codes for trans-zeatin in the virulence region of the tumor-inducing plasmids. We investigated the activity and function of the tzs gene of a nopaline Agrobacterium sp. strain GV3101 in plant in vitro regeneration. Leaf explants of tobacco and Nicotiana benthamiana co-cultured with strain GV3101 exhibited active shoot regeneration in media without added plant growth regulators. On medium without plant growth regulators, transgenic shoots were also induced from explants co-cultured with GV3101 containing a binary vector. Enzyme-linked immunosorbent assay showed that cell-free extracts of Agrobacterium sp. strain GV3101 culture contained the trans-zeatin at 860 ng/liter. Polymerase chain reaction using tzs-specific primers showed that the tzs gene was present in strain GV3101 but not in other Agrobacterium strains. The study showed that the tzs gene in GV3101 was actively expressed, and that trans-zeatin produced in the Agrobacterium strain can promote plant shoot regeneration. PMID:24088018

Han, Zhao-Fen; Hunter, David M; Sibbald, Susan; Zhang, Ji-Shu; Tian, Lining

2013-11-01

243

Consistent and stable expression of the npt II, uid A and bar genes in transgenic Pinus radiata after Agrobacterium tumefaciens -mediated transformation using nurse cultures  

Microsoft Academic Search

An Agrobacterium tumefaciens-mediated transformation protocol has been developed for embryogenic cell cultures of Pinus radiata. Transgenic lines were only produced when embryogenic tissue was placed on nurse tissue during the Agrobacterium co-cultivation and recovery stages of the procedure. Plantlets were regenerated via somatic embryogenesis from ten of the 11 transgenic lines tested and at least 20 of each line were

J. A. Charity; L. Holland; L. J. Grace; C. Walter

2005-01-01

244

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

245

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

Microsoft Academic Search

We have previously described substantial variation in the level of expression of two linked genes which were introduced into transgenic petunia plants using Agrobacterium tumefaciens. These genes were (i) nopaline synthase (nos) and (ii) a chimeric chlorophyll a\\/b binding protein\\/octopine synthase (cab\\/ocs) gene. In this report we analyze the relationship between the level of expression of the introduced genes and

Jonathan D. G. Jones; David E. Gilbert; Karen L. Grady; Richard A. Jorgensen

1987-01-01

246

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

Microsoft Academic Search

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

A. N. Binns; D. Sciaky; H. N. Wood

1982-01-01

247

High Efficiency Transgene Segregation in Co-Transformed Maize Plants using an Agrobacterium Tumefaciens 2 T-DNA Binary System  

Microsoft Academic Search

For regulatory issues and research purposes it would be desirable to have the ability to segregate transgenes in co-transformed maize. We have developed a highly efficient system to segregate transgenes in maize that was co-transformed using an Agrobacterium tumefaciens 2 T-DNA binary system. Three vector treatments were compared in this study; (1) a 2 T-DNA vector, where the selectable marker

Michael Miller; Laura Tagliani; Ning Wang; Benjamin Berka; Dennis Bidney; Zuo-Yu Zhao

2002-01-01

248

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

249

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

250

Factors involved in Agrobacterium tumefaciens-mediated gene transfer into Pinus nigra Arn. ssp. salzmannii (Dunal) Franco  

Microsoft Academic Search

Cotyledons from dissected sterile embryos of salgareńo pine (Pinus nigra Arn. ssp. salzmannii (Dunal) Franco) were inoculated with different disarmed Agrobacterium tumefaciens strains harbouring the binary vector p35SGUSint. The transient expression of a ?-glucuronidase gene (uidA) was studied, using a histochemical staining procedure. Nineteen days after inoculation, the activity of ?-glucuronidase\\u000a was detected in epidermal and subepidermal layers of cotyledonary

Marián López; Jaime M. Humara; Roberto Rodríguez; Ricardo J. Ordás

2000-01-01

251

Agrobacterium tumefaciens -mediated transformation of blackgram: An assessment of factors influencing the efficiency of uidA gene transfer  

Microsoft Academic Search

Agrobacterium tumefaciens strain EHA105 carrying a binary vector pCAMBIA2301, which contains a neomycin phosphotransferase gene (nptII) and a ?-glucuronidase (GUS) gene (uidA) interrupted with an intron, was used for transformation of Vigna mungo cotyledonary node explants. Various factors such as preculture and wounding of explants, manipulations in inoculation and\\u000a co-cultivation conditions were found to play a significant role in influencing

R. Saini; P. K. Jaiwal

2007-01-01

252

Agrobacterium tumefaciens -mediated transformation of the legume Astragalus sinicus using kanamycin resistance selection and green fluorescent protein expression  

Microsoft Academic Search

To develop an efficient protocol for the transformation of the legume Astragalus sinicus (Chinese milk vetch), cotyledon segments were infected with Agrobacterium tumefaciens strain EHA105 harboring the binary vector pBINm-gfp5-ER which carries the gfp5 gene encoding green fluorescent protein and the kanamycin (Km) resistance gene nptII. The infected explants were cultured on shoot regeneration (SR) medium containing 1.0 mg l-1

Hyeon-Je Cho; Jack M. Widholm

2002-01-01

253

Spatial location and requirements for the assembly of the Agrobacterium tumefaciens type IV secretion apparatus  

PubMed Central

Type IV secretion is used by pathogenic microorganisms to transfer effector macromolecules to eukaryotic target cells. The VirB/D4 apparatus of Agrobacterium tumefaciens transfers DNA and proteins to plant cells. We postulated that the cell pole is the site of assembly of the A. tumefaciens type IV apparatus. Using immunofluorescence microscopy, we now demonstrate that 10 of the VirB proteins localized primarily to one cell pole and a macromolecular VirB complex is assembled at the pole. Neither the assembly of the complex nor polar localization of a VirB protein requires ATP utilization by the VirB ATPases. The requirement of other VirB proteins for the polar localization of at least six VirB proteins indicates an essential role of protein–protein interaction in polar targeting. Four proteins (VirB3, VirB4, VirB8, and VirB11) could target themselves to a cell pole independent of a VirB protein. We provide evidence that VirB6–VirB10 are the structural components of the type IV apparatus. Using strains that express defined subsets of the virB genes, we demonstrate that VirB7–VirB10 are the minimum components sufficient for the assembly of a polar VirB complex. VirB6 associates with this complex to form the type IV secretion apparatus. VirB8 functions as the assembly factor and targets the apparatus to the cell pole.

Judd, Paul K.; Kumar, Renu B.; Das, Anath

2005-01-01

254

Recovery of Nonpathogenic Mutant Bacteria from Tumors Caused by Several Agrobacterium tumefaciens Strains: a Frequent Event??  

PubMed Central

We have evaluated the interaction that bacterial genotypes and plant hosts have with the loss of pathogenicity in tumors, using seven Agrobacterium tumefaciens strains inoculated on 12 herbaceous and woody hosts. We performed a screening of the agrobacteria present inside the tumors, looking for nonpathogenic strains, and found a high variability of those strains in this niche. To verify the origin of the putative nonpathogenic mutant bacteria, we applied an efficient, reproducible, and specific randomly amplified polymorphic DNA analysis method. In contrast with previous studies, we recovered a very small percentage (0.01%) of nonpathogenic strains that can be considered true mutants. Of 5,419 agrobacterial isolates examined, 662 were nonpathogenic in tomato, although only 7 (from pepper and tomato tumors induced by two A. tumefaciens strains) could be considered to derive from the inoculated strain. Six mutants were affected in the transferred DNA (T-DNA) region; one of them contained IS426 inserted into the iaaM gene, whereas the whole T-DNA region was apparently deleted in three other mutants, and the virulence of the remaining two mutants was fully restored with the T-DNA genes as well. The plasmid profile was altered in six of the mutants, with changes in the size of the Ti plasmid or other plasmids and/or the acquisition of new plasmids. Our results also suggest that the frequent occurrence of nonpathogenic clones in the tumors is probably due to the preferential growth of nonpathogenic agrobacteria, of either endophytic or environmental origin, but different from the bacterial strain inducing the tumor.

Llop, Pablo; Murillo, Jesus; Lastra, Beatriz; Lopez, Maria M.

2009-01-01

255

A Glutathione Transferase from Agrobacterium tumefaciens Reveals a Novel Class of Bacterial GST Superfamily  

PubMed Central

In the present work, we report a novel class of glutathione transferases (GSTs) originated from the pathogenic soil bacterium Agrobacterium tumefaciens C58, with structural and catalytic properties not observed previously in prokaryotic and eukaryotic GST isoenzymes. A GST-like sequence from A. tumefaciens C58 (Atu3701) with low similarity to other characterized GST family of enzymes was identified. Phylogenetic analysis showed that it belongs to a distinct GST class not previously described and restricted only in soil bacteria, called the Eta class (H). This enzyme (designated as AtuGSTH1-1) was cloned and expressed in E. coli and its structural and catalytic properties were investigated. Functional analysis showed that AtuGSTH1-1 exhibits significant transferase activity against the common substrates aryl halides, as well as very high peroxidase activity towards organic hydroperoxides. The crystal structure of AtuGSTH1-1 was determined at 1.4 Ĺ resolution in complex with S-(p-nitrobenzyl)-glutathione (Nb-GSH). Although AtuGSTH1-1 adopts the canonical GST fold, sequence and structural characteristics distinct from previously characterized GSTs were identified. The absence of the classic catalytic essential residues (Tyr, Ser, Cys) distinguishes AtuGSTH1-1 from all other cytosolic GSTs of known structure and function. Site-directed mutagenesis showed that instead of the classic catalytic residues, an Arg residue (Arg34), an electron-sharing network, and a bridge of a network of water molecules may form the basis of the catalytic mechanism. Comparative sequence analysis, structural information, and site-directed mutagenesis in combination with kinetic analysis showed that Phe22, Ser25, and Arg187 are additional important residues for the enzyme's catalytic efficiency and specificity.

Skopelitou, Katholiki; Dhavala, Prathusha; Papageorgiou, Anastassios C.; Labrou, Nikolaos E.

2012-01-01

256

T-DNA locus structure in a large population of soybean plants transformed using the Agrobacterium-mediated cotyledonary-node method.  

PubMed

Designing transformation experiments for either functional genomics or crop improvement requires knowledge of the transgene locus structure, number, transmission and expression resulting from a specific transformation method. We recently reported an improvement to the soybean [Glycine max (L.) Merrill] cotyledonary-node transformation method that resulted in the efficient production of transgenic plants. To characterize the transgene loci resulting from this method, we analysed 270 independent T0 plants and 95 randomly selected T1 progenies for T-DNA locus complexity using Southern analysis. The lines were transformed with Agrobacterium tumefaciens strains LBA4404 or EHA105 carrying the binary plasmids pGPTV, pTOK233, pCAMBIA1303 or pCAMBIA1309, and regenerated in medium supplemented with or without silver nitrate (AgNO3). Analysis in the T0 generation showed that the number of hpt-hybridizing fragments per plant ranged from 1-15, with 31.5% of the lines having a single hpt-hybridizing fragment. Each primary soybean transformant had, on average, 2.0 unlinked transgene loci and that half of the segregating loci in the T1 progenies were single, simple T-DNA insertions. Of the loci containing multiple T-DNA fragments, a low frequency had tandem and inverted repeat T-DNA structures. Integration of binary plasmid backbone sequences occurred in 37% of primary transformants. A. tumefaciens strain, binary plasmid and thiol treatment had no significant effect on transgene locus structure, numbers or expression. Interestingly, exposure of soybean explants to AgNO3 throughout shoot induction and elongation increased T-DNA locus complexity in the primary transformants and decreased silencing of gusA expression in the T1 generation. PMID:17134390

Olhoft, Paula M; Flagel, Lex E; Somers, David A

2004-07-01

257

Agrobacterium tumefaciens-mediated transformation of Penicillium expansum PE-12 and its application in molecular breeding.  

PubMed

Lipase produced by Penicillium expansum is widely used in laundry detergent and leather industry; however, the absence of an efficient transformation technology sets a major obstacle for further enhancement of its lipase productivity through advanced gene engineering. In this work, Agrobacterium tumefaciens-mediated transformation (ATMT) was investigated for P. expansum PE-12 transformation, using hygromycin phosphotransferase (hph) as a selectable marker gene. As a result, we revealed that the frequency of transformation surpassed 100 transformants/10(5)condida, most of the integrated T-DNA appeared as a single copy at a random position in chromosomal DNA, and all the transformants showed mitotic stability. Facilitated by this newly established method, for the first time, P. expansum PE-12 was genetically engineered to improve the lipase yield, through a homologous expression vector carrying the endogenous lipase gene (PEL) driven by the strong constitutive promoter of the glyceraldehydes-3-phosphate dehydrogenase gene (gpdA) from Aspergillus nidulans. The highest expression level of the engineered strain reached up to 1700 U/mL, nearly 2-fold of the original industrial strain (900 U/mL). Our reproducible ATMT system has not only revealed the great potential of homologous expression-directed genetic engineering, which is more efficient and specific compared to traditional mutagenesis, but also provided new possibilities and perspectives for any other practical applications of P. expansum-related genetic engineering in the future. PMID:23265791

Zhang, Tian; Qi, Zhen; Wang, Yueyue; Zhang, Fangyuan; Li, Renyong; Yu, Qingsheng; Chen, Xiangbin; Wang, Huojun; Xiong, Xin; Tang, Kexuan

2012-12-21

258

Stable transformation of Medicago truncatula cv. Jemalong for gene analysis using Agrobacterium tumefaciens.  

PubMed

Medicago truncatula is a model legume that has all the genomic resources to be suitable as a model for functional genomics. Transformation to produce transgenic plants is part of this toolkit, enabling a spectrum of approaches to study gene function: including knockdown, overexpression, reporter genes for gene expression, and proteins tagged with fluorescent proteins such as GFP. A special genetic line is necessary for transformation and Jemalong 2HA derived from cv. Jemalong is used in the methods described. Leaf explants can be used for the transformation of the embryonic stem cells to produce the transgenic somatic embryos for regeneration. An auxin and a cytokinin are the key hormone requirements for regeneration by somatic embryogenesis but other hormones such as abscisic acid can be used to augment the system. As the explants used in this system are from leaves, rather than immature embryos or meristematic tissues often used in other species, it is a quite straightforward system. Agrobacterium tumefaciens containing a binary vector suitable for the particular objectives is used to deliver the transgene of interest. PMID:23996317

Song, Youhong; Nolan, Kim E; Rose, Ray J

2013-01-01

259

Structural characterization of a group II 2/2 hemoglobin from the plant pathogen Agrobacterium tumefaciens.  

PubMed

Within the 2/2 hemoglobin sub-family, no group II 2/2Hbs from proteobacteria have been so far studied. Here we present the first structural characterization of a group II 2/2Hb from the soil and phytopathogenic bacterium Agrobacterium tumefaciens (At-2/2HbO). The crystal structure of ferric At-2/2HbO (reported at 2.1Ĺ resolution) shows the location of specific/unique heme distal site residues (e.g., His(42)CD1, a residue distinctive of proteobacteria group II 2/2Hbs) that surround a heme-liganded water molecule. A highly intertwined hydrogen-bonded network, involving residues Tyr(26)B10, His(42)CD1, Ser(49)E7, Trp(93)G8, and three distal site water molecules, stabilizes the heme-bound ligand. Such a structural organization suggests a path for diatomic ligand diffusion to/from the heme. Neither a similar distal site structuring effect nor the presence of distal site water molecules has been so far observed in group I and group III 2/2Hbs, thus adding new distinctive information to the complex picture of currently available 2/2Hb structural and functional data. This article is part of a Special Issue entitled: Protein Structure and Function in the Crystalline State. PMID:21070893

Pesce, Alessandra; Nardini, Marco; Labarre, Marie; Richard, Christian; Wittenberg, Jonathan B; Wittenberg, Beatrice A; Guertin, Michel; Bolognesi, Martino

2010-11-09

260

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

261

Transgenic Acacia sinuata from Agrobacterium tumefaciens-mediated transformation of hypocotyls.  

PubMed

Transgenic herbicide tolerant Acacia sinuata plants were produced by transformation with the bar gene conferring phosphinothricin resistance. Precultured hypocotyl explants were infected with Agrobacterium tumefaciens strain EHA105 in the presence of 100 microM acetosyringone and shoots regenerated on MS (Murashige and Skoog, 1962, Physiol Plant 15:473-497) medium with 13.3 microM benzylaminopurine, 2.6 microM indole-3-acetic acid, 1 g l(-1) activated charcoal, 1.5 mg l(-1) phosphinothricin, and 300 mg l(-1) cefotaxime. Phosphinothricin at 1.5 mg l(-1) was used for the selection. Shoots surviving selection on medium with phosphinothricin expressed GUS. Following Southern hybridization, eight independent shoots regenerated of 500 cocultivated explants were demonstrated to be transgenic, which represented transformation frequency of 1.6%. The transgenics carried one to four copies of the transgene. Transgenic shoots were propagated as microcuttings in MS medium with 6.6 microM 6-benzylaminopurine and 1.5 mg l(-1) phosphinothricin. Shoots elongated and rooted in MS medium with gibberellic acid and indole-3-butyric acid, respectively both supplemented with 1.5 mg l(-1) phosphinothricin. Micropropagation of transgenic plants by microcuttings proved to be a simple means to bulk up the material. Several transgenic plants were found to be resistant to leaf painting with the herbicide Basta. PMID:16807750

Vengadesan, G; Amutha, S; Muruganantham, M; Anand, R Prem; Ganapathi, A

2006-06-29

262

Crystal Structure of Exotype Alginate Lyase Atu3025 from Agrobacterium tumefaciens*  

PubMed Central

Alginate, a major component of the cell wall matrix in brown seaweeds, is degraded by alginate lyases through a ?-elimination reaction. Almost all alginate lyases act endolytically on substrate, thereby yielding unsaturated oligouronic acids having 4-deoxy-l-erythro-hex-4-enepyranosyluronic acid at the nonreducing end. In contrast, Agrobacterium tumefaciens alginate lyase Atu3025, a member of polysaccharide lyase family 15, acts on alginate polysaccharides and oligosaccharides exolytically and releases unsaturated monosaccharides from the substrate terminal. The crystal structures of Atu3025 and its inactive mutant in complex with alginate trisaccharide (H531A/?GGG) were determined at 2.10- and 2.99-? resolutions with final R-factors of 18.3 and 19.9%, respectively, by x-ray crystallography. The enzyme is comprised of an ?/?-barrel + anti-parallel ?-sheet as a basic scaffold, and its structural fold has not been seen in alginate lyases analyzed thus far. The structural analysis of H531A/?GGG and subsequent site-directed mutagenesis studies proposed the enzyme reaction mechanism, with His311 and Tyr365 as the catalytic base and acid, respectively. Two structural determinants, i.e. a short ?-helix in the central ?/?-barrel domain and a conformational change at the interface between the central and C-terminal domains, are essential for the exolytic mode of action. This is, to our knowledge, the first report on the structure of the family 15 enzyme.

Ochiai, Akihito; Yamasaki, Masayuki; Mikami, Bunzo; Hashimoto, Wataru; Murata, Kousaku

2010-01-01

263

Agrobacterium tumefaciens-Mediated Transformation of Aspergillus fumigatus: an Efficient Tool for Insertional Mutagenesis and Targeted Gene Disruption  

PubMed Central

Agrobacterium tumefaciens was used to transform Aspergillus fumigatus by either random or site-directed integration of transforming DNA (T-DNA). Random mutagenesis via Agrobacterium tumefaciens-mediated transformation (ATMT) was accomplished with T-DNA containing a hygromycin resistance cassette. Cocultivation of A. fumigatus conidia and Agrobacterium (1:10 ratio) for 48 h at 24°C resulted in high frequencies of transformation (>100 transformants/107 conidia). The majority of transformants harbored a randomly integrated single copy of T-DNA and were mitotically stable. We chose alb1, a polyketide synthase gene, as the target gene for homologous integration because of the clear phenotype difference between the white colonies of ?alb1 mutant strains and the bluish-green colonies of wild-type strains. ATMT with a T-DNA-containing alb1 disruption construct resulted in 66% albino transformants. Southern analysis revealed that 19 of the 20 randomly chosen albino transformants (95%) were disrupted by homologous recombination. These results suggest that ATMT is an efficient tool for transformation, random insertional mutagenesis, and gene disruption in A. fumigatus.

Sugui, Janyce A.; Chang, Yun C.; Kwon-Chung, K. J.

2005-01-01

264

Plant transformation by coinoculation with a disarmed Agrobacterium tumefaciens strain and an Escherichia coli strain carrying mobilizable transgenes.  

PubMed

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 respective origins of transfer and plant-selectable markers. Neither of these conjugation systems was able to stably transform plant cells at detectable levels, even when VirE2 was expressed in the donor cells. However, when an E. coli strain expressing the IncN-type conjugation system was coinoculated with a disarmed A. tumefaciens strain, plant tumors arose at high frequencies. This was caused by a two-step process in which the IncN transfer system mobilized the entire shuttle plasmid from E. coli to the disarmed A. tumefaciens strain, which in turn processed the T-DNA and transferred it to recipient plant cells. The mobilizable plasmid does not require a broad-host-range replication origin for this process to occur, thus reducing its size and genetic complexity. Tumorigenesis efficiency was further enhanced by incubation of the bacterial strains on medium optimized for bacterial conjugation prior to inoculation of leaf explants. These techniques circumvent the need to construct A. tumefaciens strains containing binary vectors and could simplify the creation of transgenic plants. PMID:14602634

Pappas, Katherine M; Winans, Stephen C

2003-11-01

265

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

PubMed Central

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 respective origins of transfer and plant-selectable markers. Neither of these conjugation systems was able to stably transform plant cells at detectable levels, even when VirE2 was expressed in the donor cells. However, when an E. coli strain expressing the IncN-type conjugation system was coinoculated with a disarmed A. tumefaciens strain, plant tumors arose at high frequencies. This was caused by a two-step process in which the IncN transfer system mobilized the entire shuttle plasmid from E. coli to the disarmed A. tumefaciens strain, which in turn processed the T-DNA and transferred it to recipient plant cells. The mobilizable plasmid does not require a broad-host-range replication origin for this process to occur, thus reducing its size and genetic complexity. Tumorigenesis efficiency was further enhanced by incubation of the bacterial strains on medium optimized for bacterial conjugation prior to inoculation of leaf explants. These techniques circumvent the need to construct A. tumefaciens strains containing binary vectors and could simplify the creation of transgenic plants.

Pappas, Katherine M.; Winans, Stephen C.

2003-01-01

266

Heat shock transcription of the groESL operon of Agrobacterium tumefaciens may involve a hairpin-loop structure.  

PubMed

The groESL operon of Agrobacterium tumefaciens was cloned and sequenced and found to be highly homologous to previously analyzed groE operons in nucleotides of the coding region and in amino acid sequence. Transcription of this operon in A. tumefaciens was considerably stimulated by heat shock. Primer extension analysis revealed that the groE transcripts from cells under heat shock were initiated from the same promoter (a sigma-70-like promoter) as transcripts from untreated cells, and no sequence homology with the Escherichia coli heat shock promoters was observed. The DNA sequence downstream of the transcription start site contains an inverted repeat that has a strong similarity to other groESL operons of both gram-positive and gram-negative bacteria (such as cyanobacteria and chlamydiae). This conserved region is thought to form a hairpin-loop structure and may play a role in gene regulation during heat shock. PMID:8098329

Segal, G; Ron, E Z

1993-05-01

267

Cloning, sequencing, and transcriptional analysis of the gene coding for the vegetative sigma factor of Agrobacterium tumefaciens.  

PubMed

The sigA gene of Agrobacterium tumefaciens was cloned and sequenced. Comparison with previously analyzed sigA genes revealed a high degree of similarity in nucleotide and amino acid sequences of regions two, three, and four of vegetative sigma factors. However, the upstream regulatory region shows no sequence homology with the Escherichia coli heat shock (sigma 32) promoters. It also does not contain the hairpin-loop structure (inverted repeat sequence) that was found in the upstream region of the groE operon in A. tumefaciens. The transcription initiation site of the gene was determined and found to be at the same position during normal growth and under heat shock conditions. Furthermore, no heat shock activation was observed at the transcriptional level. PMID:8491721

Segal, G; Ron, E Z

1993-05-01

268

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

PubMed Central

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

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

2003-01-01

269

Agrobacterium -mediated transformation of the wetland monocot Typha latifolia L. (Broadleaf cattail)  

Microsoft Academic Search

An Agrobacterium-mediated model transformation system was standardized for the wetland monocot Typha latifolia L. to achieve the long-term objective of introducing candidate genes for phytoremediation. Two binary plasmid vectors, pCAMBIA1301\\/EHA105 and pTOK233\\/LBA4404, both containing the gus (ß-glucuronidase) and hptII (hygromycin phosphotransferase II) genes, were used for transformation. Fifty-day-old 5 mg\\/l picloram-derived calli were cocultivated and selected on medium containing 20 mg\\/l or 40 mg\\/l

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

2005-01-01

270

Site-specific mutagenesis of the Ti plasmid by transformation of Agrobacterium tumefaciens with mutagenized T-DNA fragments cloned in E. coli plasmids  

Microsoft Academic Search

A DNA fragment covering the complete T-region of the Ti plasmid from Agrobacterium tumefaciens strain C58 was cloned in the Escherichia coli cosmid pHC79. This fragment was mutagenized by insertion of transposon Tn5. The isolated DNA from hybrid plasmids was used to transform cells of A. tumefaciens strain C58 applying the freeze-thaw method. Although the E. coli plasmids with the

Peter Zahm; Christine Hohmeyer; Klaus Geider

1984-01-01

271

Agrobacterium tumefaciens soxR Is Involved in Superoxide Stress Protection and Also Directly Regulates Superoxide-Inducible Expression of Itself and a Target Gene?  

PubMed Central

Inactivation of Agrobacterium tumefaciens soxR increases sensitivity to superoxide generators. soxR expression is highly induced by superoxide stress and is autoregulated. SoxR also directly regulates the superoxide-inducible expression of atu5152. Taken together, the physiological role of soxR and the mechanism by which it regulates expression of target genes make the A. tumefaciens SoxR system different from other bacterial systems.

Eiamphungporn, Warawan; Charoenlap, Nisanart; Vattanaviboon, Paiboon; Mongkolsuk, Skorn

2006-01-01

272

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

273

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.

Zhao, Jinlei; Binns, Andrew N.

2011-01-01

274

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.

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

2010-01-01

275

Agrobacterium tumefaciens-mediated transformation for investigation of somatic recombination in the fungal pathogen Armillaria mellea.  

PubMed

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

2010-10-15

276

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

2013-08-12

277

Stable genetic transformation of castor ( Ricinus communis L.) via Agrobacterium tumefaciens -mediated gene transfer using embryo axes from mature seeds  

Microsoft Academic Search

A protocol for the transformation of castor embryo axes using the pCAMBIA vector 1304 in disarmed Agrobacterium tumefaciens strain EHA105 is presented. Co-cultivated explants were initially subjected to expansion and proliferation on MS medium with 0.5 mg l-1 TDZ followed by three cycles of selection on medium with 0.5 mg l-1 BA and increasing concentrations of hygromycin (20–40–60 mg l-1). Selected shoot clusters were transferred to

M. Sujatha; M. Sailaja

2005-01-01

278

Evaluation of methods for celery ( Apium Graveolens L.) transformation using Agrobacterium tumefaciens and the bar gene as selectable marker  

Microsoft Academic Search

Callus selection (CS) and the flamingo-bill explant (FB) methods were evaluated for efficacy in transformation for celery.\\u000a Agrobacterium tumefaciens strains EHA105 and GV3101, each with the bar gene under the promoters NOS (pGPTV-BAR) or 35S (pDHB321.1), were used. Leaf explants were inoculated and co-cultivated for\\u000a 2 d in the dark. Calluses emerged on the explants on callus medium (C), Murashige and

A. V. Loskutov; G.-Q. Song; K. C. Sink

2008-01-01

279

Additional copies of virG from pTiBo542 provide a super-transformation ability to Agrobacterium tumefaciens in citrus  

Microsoft Academic Search

Agrobacterium tumefaciens strain A281 carrying pTiBo542 was described as able to induce large and early-appearing tumours on a wider range of plants than other Agrobacterium strains. Here we show a strict correlation between the supervirulence of A281 and super-transformation ability of its disarmed derivative EHA105\\/p35SGUSINT. This strain produced much higher transformation than the disarmed derivative of the common strain C58,

Riadh Ghorbel; Stefano La-Malfa; Maria M. López; Annik Petit; Luis Navarro; Leandro Peńa

2001-01-01

280

In vitro susceptibility of two tropical Acacia species to Agrobacterium tumefaciens Suscetibilidade ‡ infecÁŞo por Agrobacterium tumefaciens de duas espŘcies tropicais de Acacia in vitro  

Microsoft Academic Search

Acacia mangium and A. mearnsii are leguminous N-fixing trees which can be used for afforestation of degraded soils. The application of modern techniques of genetic transformation to such species should allow some traits, like insect resistance, to be improved, or to modify lignin metabolism. In order to establish an efficient transformation system for these species, ten wild strains of Agrobacterium

Marguerite Quoirin; Wilhelm E. Hagiwara; Flávio Zanette; Dulce E. de Oliveira

281

Genetic and molecular analyses of picA, a plant-inducible locus on the Agrobacterium tumefaciens chromosome.  

PubMed Central

picA is an Agrobacterium tumefaciens chromosomal locus, identified by Mu d11681 mutagenesis, that is inducible by certain acidic polysaccharides found in carrot root extract. Cloning and genetic analysis of a picA::lacZ fusion defined a region of the picA promoter that is responsible for the induction of this locus. Furthermore, we identified a possible negative regulator of picA expression upstream of the picA locus. This sequence, denoted pgl, has extensive homology to polygalacturonase genes from several organisms and inhibited the induction of the picA promoter when present in multiple copies in A. tumefaciens. DNA sequence analysis indicated at least two long open reading frames (ORFs) in the picA region. S1 nuclease mapping was used to identify the transcription initiation site of picA. Mutation of ORF1, but not ORF2, of the picA locus was responsible for an increased aggregation of A. tumefaciens, forming "ropes" in the presence of pea root cap cells. In addition, a potato tuber disk virulence assay indicated that a preinduced picA mutant was more virulent than was the wild-type control, a further indication that the picA locus regulates the surface properties of the bacterium in the presence of plant cells or plant cell extracts. Images

Rong, L J; Karcher, S J; Gelvin, S B

1991-01-01

282

Agrobacterium tumefaciens-Mediated Transformation of Maize Endosperm as a Tool to Study Endosperm Cell Biology1[OA  

PubMed Central

Developing maize (Zea mays) endosperms can be excised from the maternal tissues and undergo tissue/cell-type differentiation under in vitro conditions. We have developed a method to transform in vitro-grown endosperms using Agrobacterium tumefaciens and standard binary vectors. We show that both aleurone and starchy endosperm cells can be successfully transformed using a short cocultivation with A. tumefaciens cells. The highest transformation rates were obtained with the A. tumefaciens EHA101 strain and the pTF101.1 binary vector. The percentage of aleurone cells transformed following this method varied between 10% and 22% whereas up to the eighth layer of starchy endosperm cells underneath the aleurone layer showed transformed cells. Cultured endosperms undergo normal cell type (aleurone and starchy endosperm) differentiation and storage protein accumulation, making them suitable for cell biology and biochemical studies. In addition, transgenic cultured endosperms are able to express and accumulate epitope-tagged storage proteins that can be isolated for biochemical assays or used for immunolabeling techniques.

Reyes, Francisca C.; Sun, Beimeng; Guo, Hena; Gruis, Darren (Fred); Otegui, Marisa S.

2010-01-01

283

Genetic analysis of Agrobacterium tumefaciens unipolar polysaccharide production reveals complex integrated control of the motile-to-sessile switch.  

PubMed

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 c-di-GMP 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

2013-07-29

284

De novo synthesis of bacterial glycogen: Agrobacterium tumefaciens glycogen synthase is involved in glucan initiation and elongation.  

PubMed

Evidence is presented indicating that initiation of glycogen synthesis in Agrobacterium tumefaciens does not require the presence of alpha(1,4)-linked glucans. Crude cell extracts incubated with ADP-glucose (Glc) were able to form alpha(1,4)-linked glucans despite the fact that cells used for extract preparation displayed a genotype that prevented synthesis of Glc-containing sugar nucleotides and thus preformation of alpha(1,4)-linked glucans and that the defined growth medium used contained glycerol as carbon source. A. tumefaciens glycogen synthase (GS) purified to homogeneity from the above-mentioned cells was able to build its own primer by transferring Glc residues from ADP-Glc to an amino acid(s) in the same protein. Primed GS then became the substrate for further GS-catalyzed glucan elongation. It was concluded that, contrary to what happens in mammalian and yeast cells in which two different proteins are required for linear alpha(1,4)-linked glucan formation (glycogenin for initiation and GS for further elongation), in A. tumefaciens and probably in all other bacteria, the same protein is involved in both glycogen initiation and elongation. PMID:12960388

Ugalde, Juan E; Parodi, Armando J; Ugalde, Rodolfo A

2003-09-05

285

Metabolic modeling of denitrification in Agrobacterium tumefaciens: a tool to study inhibiting and activating compounds for the denitrification pathway.  

PubMed

A metabolic network model for facultative denitrification was developed based on experimental data obtained with Agrobacterium tumefaciens. The model includes kinetic regulation at the enzyme level and transcription regulation at the enzyme synthesis level. The objective of this work was to study the key factors regulating the metabolic response of the denitrification pathway to transition from oxic to anoxic respiration and to find parameter values for the biological processes that were modeled. The metabolic model was used to test hypotheses that were formulated based on the experimental results and offers a structured look on the processes that occur in the cell during transition in respiration. The main phenomena that were modeled are the inhibition of the cytochrome c oxidase by nitric oxide (NO) and the (indirect) inhibition of oxygen on the denitrification enzymes. The activation of transcription of nitrite reductase and NO reductase by their respective substrates were hypothesized. The general assumption that nitrite and NO reduction are controlled interdependently to prevent NO accumulation does not hold for A. tumefaciens. The metabolic network model was demonstrated to be a useful tool for unraveling the different factors involved in the complex response of A. tumefaciens to highly dynamic environmental conditions. PMID:23087683

Kampschreur, Marlies J; Kleerebezem, Robbert; Picioreanu, Cristian; Bakken, Lars; Bergaust, Linda; de Vries, Simon; Jetten, Mike S M; van Loosdrecht, Mark C M

2012-10-18

286

Metabolic modeling of denitrification in Agrobacterium tumefaciens: a tool to study inhibiting and activating compounds for the denitrification pathway  

PubMed Central

A metabolic network model for facultative denitrification was developed based on experimental data obtained with Agrobacterium tumefaciens. The model includes kinetic regulation at the enzyme level and transcription regulation at the enzyme synthesis level. The objective of this work was to study the key factors regulating the metabolic response of the denitrification pathway to transition from oxic to anoxic respiration and to find parameter values for the biological processes that were modeled. The metabolic model was used to test hypotheses that were formulated based on the experimental results and offers a structured look on the processes that occur in the cell during transition in respiration. The main phenomena that were modeled are the inhibition of the cytochrome c oxidase by nitric oxide (NO) and the (indirect) inhibition of oxygen on the denitrification enzymes. The activation of transcription of nitrite reductase and NO reductase by their respective substrates were hypothesized. The general assumption that nitrite and NO reduction are controlled interdependently to prevent NO accumulation does not hold for A. tumefaciens. The metabolic network model was demonstrated to be a useful tool for unraveling the different factors involved in the complex response of A. tumefaciens to highly dynamic environmental conditions.

Kampschreur, Marlies J.; Kleerebezem, Robbert; Picioreanu, Cristian; Bakken, Lars; Bergaust, Linda; de Vries, Simon; Jetten, Mike S. M.; van Loosdrecht, Mark C. M.

2012-01-01

287

The dnaKJ operon of Agrobacterium tumefaciens: transcriptional analysis and evidence for a new heat shock promoter.  

PubMed

The dnaKJ operon of Agrobacterium tumefaciens was cloned and sequenced and was found to be highly homologous to previously analyzed dnaKJ operons. Transcription of this operon in A. tumefaciens was stimulated 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 only the dnaK gene. Primer extension analysis indicated that transcription started from the same site in heat-shocked cells and in untreated cells. The upstream regulatory region of the dnaKJ operon of A. tumefaciens does not contain the highly conserved inverted repeat sequence previously found in the groESL operon of this bacterium, as well as in many other groE and dnaK operons. Sequence analysis of the promoter region of several groESL and dnaK operons from alpha-purple proteobacteria indicates the existence of a putative promoter sequence different from the known consensus promoter sequences recognized by the Escherichia coli vegetative or heat shock sigma factor. This promoter may constitute the heat shock promoter of these alpha-purple proteobacteria. PMID:7592349

Segal, G; Ron, E Z

1995-10-01

288

The use of amoxicillin and ticarcillin in combination with a ?-lactamase inhibitor as decontaminating agents in the Agrobacterium tumefaciens-mediated transformation of Artemisia annua L  

Microsoft Academic Search

Artemisinin is a new very promising antimalarial compound produced in the areal parts of the plant Artemisia annua L. (Asteraceae). There is a great interest in the overproduction of artemisinin by means of transgenic plants. The existing Agrobacterium tumefaciens-mediated transformation procedure uses the expensive decontaminating agent vancomycin. In addition vancomycin exhibits a low activity against the Agrobacteria. This paper describes

Annemieke Vergauwe; Els Van Geldre; Dirk Inzé; Marc Van Montagu; Elfride Van den Eeckhout

1996-01-01

289

Silent T-DNA genes in plant lines transformed by Agrobacterium tumefaciens are activated by grafting and by 5-azacytidine treatment  

Microsoft Academic Search

A shooty tumor induced by a shooter mutant of an octopine strain of Agrobacterium tumefaciens was cloned. One clone obtained (TS038) behaved aberrantly in that it grew as a shooty tumor tissue on phytohormone free medium, but did not contain octopine synthase activity. In line TS038 the genes for octopine synthase and for the enzymes involved in agropine and mannopine

G. M. S. Slogteren; P. J. J. Hooykaas; R. A. Schilperoort

1984-01-01

290

The development of a reproducible Agrobacterium tumefaciens transformation system for garlic ( Allium sativum L.) and the production of transgenic garlic resistant to beet armyworm ( Spodoptera exigua Hübner)  

Microsoft Academic Search

This paper describes the development of a reliable transformation system for garlic (Allium sativum L.) and its application in producing insect resistant GM garlic lines. The transformation system is based on Agrobacterium tumefaciens as a vector, using young callus derived from different callus sources: callus induced from both apical and non-apical root segments of in vitro plantlets, true garlic seeds

Si-Jun Zheng; Betty Henken; Yul Kyun Ahn; Frans A. Krens; Chris Kik

2004-01-01

291

The tomato UV-damaged DNA-binding protein-1 (DDB1) is implicated in pathogenesis-related (PR) gene expression and resistance to Agrobacterium tumefaciens.  

PubMed

Plants defend themselves against potential pathogens via the recognition of pathogen-associated molecular patterns (PAMPs). However, the molecular mechanisms underlying this PAMP-triggered immunity (PTI) are largely unknown. In this study, we show that tomato HP1/DDB1, coding for a key component of the CUL4-based ubiquitin E3 ligase complex, is required for resistance to Agrobacterium tumefaciens. We found that the DDB1-deficient mutant (high pigment-1, hp1) is susceptible to nontumorigenic A. tumefaciens. The efficiency of callus generation from the hp1 cotyledons was extremely low as a result of the necrosis caused by Agrobacterium infection. On infiltration of nontumorigenic A. tumefaciens into leaves, the hp1 mutant moderately supported Agrobacterium growth and developed disease symptoms, but the expression of the pathogenesis-related gene SlPR1a1 and several PTI marker genes was compromised at different levels. Moreover, exogenous application of salicylic acid (SA) triggered SlPR1a1 gene expression and enhanced resistance to A. tumefaciens in wild-type tomato plants, whereas these SA-regulated defence responses were abolished in hp1 mutant plants. Thus, HP1/DDB1 may function through interaction with the SA-regulated PTI pathway in resistance against Agrobacterium infection. PMID:21726402

Liu, Jikai; Li, Hanjing; Miao, Min; Tang, Xiaofeng; Giovannoni, Jim; Xiao, Fangming; Liu, Yongsheng

2011-07-04

292

Improved frequency of transformation in rice and maize by treatment of immature embryos with centrifugation and heat prior to infection with Agrobacterium tumefaciens  

Microsoft Academic Search

The efficiency of transformation was improved by treating immature embryos with heat and centrifugation before infection with Agrobacterium tumefaciens in rice and maize. Because the effects were detected both in the levels of transgene expression after co-cultivation and in the number of independent transgenic plants obtained per embryo, conditions were first optimized based on the transgene expression, and then transformants

Yukoh Hiei; Yuji Ishida; Keisuke Kasaoka; Toshihiko Komari

2006-01-01

293

The Agrobacterium tumefaciens virulence D2 protein is responsible for precise integration of T-DNA into the plant genome.  

PubMed Central

The VirD2 protein of Agrobacterium tumefaciens was shown to pilot T-DNA during its transfer to the plant cell nucleus. We analyze here its participation in the integration of T-DNA by using a virD2 mutant. This mutation reduces the efficiency of T-DNA transfer, but the efficiency of integration of T-DNA per se is unaffected. Southern and sequence analyses of integration events obtained with the mutated VirD2 protein revealed an aberrant pattern of integration. These results indicate that the wild-type VirD2 protein participates in ligation of the 5'-end of the T-strand to plant DNA and that this ligation step is not rate limiting for T-DNA integration. Images

Tinland, B; Schoumacher, F; Gloeckler, V; Bravo-Angel, A M; Hohn, B

1995-01-01

294

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

295

Nucleotide sequences of the Pseudomonas savastanoi indoleacetic acid genes show homology with Agrobacterium tumefaciens T-DNA  

PubMed Central

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 with a molecular weight of 61,783; the iaaH locus contains an open reading frame of 455 amino acids that would comprise a protein with a molecular weight of 48,515. Significant amino acid sequence homology was found between the predicted sequence of the tryptophan monooxygenase of P. savastanoi and the deduced product of the T-DNA tms-1 gene of the octopine-type plasmid pTiA6NC from Agrobacterium tumefaciens. Strong homology was found in the 25 amino acid sequence in the putative FAD-binding region of tryptophan monooxygenase. Homology was also found in the amino acid sequences representing the central regions of the putative products of iaaH and tms-2 T-DNA. The results suggest a strong similarity in the pathways for indoleacetic acid synthesis encoded by genes in P. savastanoi and in A. tumefaciens T-DNA. Images

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

1985-01-01

296

The groESL operon of Agrobacterium tumefaciens: evidence for heat shock-dependent mRNA cleavage.  

PubMed

The heat shock response of the groESL operon of Agrobacterium tumefaciens was studied at the RNA level. The operon was found to be activated under heat shock conditions and transcribed as a polycistronic mRNA that contains the groES and groEL genes. After activation, the polycistronic mRNA appeared to be cleaved between the groES and groEL genes and formed two monocistronic mRNAs. The groES cleavage product appeared to be unstable and subjected to degradation, while the groEL cleavage product appeared to be stable and became the major mRNA representing the groESL operon after long periods of growth at a high temperature. The polycistronic mRNA containing the groES and groEL genes was the major mRNA representing the groESL operon at a low temperature, and it reappeared when the cells were returned to the lower growth temperature after heat shock induction. These findings indicate that the cleavage event is part of the heat shock regulation of the groESL operon in A. tumefaciens. PMID:7530710

Segal, G; Ron, E Z

1995-02-01

297

Heat shock activation of the groESL operon of Agrobacterium tumefaciens and the regulatory roles of the inverted repeat.  

PubMed

Deletions were constructed in the conserved inverted repeat (IR) found in the groESL operon of Agrobacterium tumefaciens and in many other groE and dnaK operons and genes in eubacteria. These deletions affected the level of expression of the operon and the magnitude of its heat shock activation. The IR seems to operate at the DNA level, probably as an operator site that binds a repressor under non-heat shock conditions. The IR was also found to function at the mRNA level, since under non-heat shock conditions transcripts containing deletions of one side of the IR had longer half-lives than did transcripts containing the wild-type IR. Under heat shock conditions, the half-life of the mRNA was unaffected by this deletion because of heat shock-dependent cleavage. However, the groESL operon was found to be heat shock activated even after most of the IR was deleted. This observation, together with the fact that the groESL operon of A. tumefaciens was heat shock activated in Escherichia coli and vice versa, suggests that a heat shock promoter regulates the heat shock activation of this operon. The primary role of the IR appears to be in reducing the MRNA levels from this promoter under non-heat shock conditions. PMID:8655565

Segal, G; Ron, E Z

1996-06-01

298

In Vitro Characterization of the Enzyme Properties of the Phospholipid N-Methyltransferase PmtA from Agrobacterium tumefaciens?  

PubMed Central

Agrobacterium tumefaciens requires phosphatidylcholine (PC) in its membranes for plant infection. The phospholipid N-methyltransferase PmtA catalyzes all three transmethylation reactions of phosphatidylethanolamine (PE) to PC via the intermediates monomethylphosphatidylethanolamine (MMPE) and dimethylphosphatidylethanolamine (DMPE). The enzyme uses S-adenosylmethionine (SAM) as the methyl donor, converting it to S-adenosylhomocysteine (SAH). Little is known about the activity of bacterial Pmt enzymes, since PC biosynthesis in prokaryotes is rare. In this article, we present the purification and in vitro characterization of A. tumefaciens PmtA, which is a monomeric protein. It binds to PE, the intermediates MMPE and DMPE, the end product PC, and phosphatidylglycerol (PG) and phosphatidylinositol. Binding of the phospholipid substrates precedes binding of SAM. We used a coupled in vitro assay system to demonstrate the enzymatic activity of PmtA and to show that PmtA is inhibited by the end products PC and SAH and the antibiotic sinefungin. The presence of PG stimulates PmtA activity. Our study provides insights into the catalysis and control of a bacterial phospholipid N-methyltransferase.

Aktas, Meriyem; Narberhaus, Franz

2009-01-01

299

The lipoprotein VirB7 interacts with VirB9 in the membranes of Agrobacterium tumefaciens.  

PubMed Central

VirB9 and VirB7 are essential components of the putative VirB membrane channel required for transfer of the T-complex from Agrobacterium tumefaciens into plants. In this report, we present a biochemical analysis of their interaction and cellular localization. A comparison of relative electrophoretic mobilities under nonreducing and reducing conditions suggested that they form thiol-sensitive complexes with other proteins. Two-dimensional gel electrophoresis identified one complex as a heterodimer of VirB9 and VirB7 covalently linked by a disulfide bond, as well as VirB7 homodimers and monomers. Immunoprecipitation with VirB9-specific antiserum isolated the heterodimeric VirB9-VirB7 complex. Incubation with reducing agent split the complex into its constituent VirB9 and VirB7, which further confirmed linkage via cysteine residues. The interaction between VirB9 and VirB7 also was observed in the yeast two-hybrid system. Membrane attachment of VirB9-VirB7 may be conferred by lipoprotein modification, since labeling with [3H]palmitic acid in A. tumefaciens verified that VirB7 is a lipoprotein associated with VirB9. VirB9 and VirB7 showed equal distribution between inner and outer membranes, in accord with their proposed association with the transmembrane VirB complex.

Baron, C; Thorstenson, Y R; Zambryski, P C

1997-01-01

300

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

PubMed

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

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

2011-05-11

301

Heat shock activation of the groESL operon of Agrobacterium tumefaciens and the regulatory roles of the inverted repeat.  

PubMed Central

Deletions were constructed in the conserved inverted repeat (IR) found in the groESL operon of Agrobacterium tumefaciens and in many other groE and dnaK operons and genes in eubacteria. These deletions affected the level of expression of the operon and the magnitude of its heat shock activation. The IR seems to operate at the DNA level, probably as an operator site that binds a repressor under non-heat shock conditions. The IR was also found to function at the mRNA level, since under non-heat shock conditions transcripts containing deletions of one side of the IR had longer half-lives than did transcripts containing the wild-type IR. Under heat shock conditions, the half-life of the mRNA was unaffected by this deletion because of heat shock-dependent cleavage. However, the groESL operon was found to be heat shock activated even after most of the IR was deleted. This observation, together with the fact that the groESL operon of A. tumefaciens was heat shock activated in Escherichia coli and vice versa, suggests that a heat shock promoter regulates the heat shock activation of this operon. The primary role of the IR appears to be in reducing the MRNA levels from this promoter under non-heat shock conditions.

Segal, G; Ron, E Z

1996-01-01

302

Genetic transformation of strawberry by Agrobacterium tumefaciens using a leaf disk regeneration system  

Microsoft Academic Search

An efficient genetic transformation protocol has been developed for strawberry cv. Redcoat using Agrobacterium tumefadens. The protocol relies on a high frequency (84%) shoot regeneration system from leaf disks. The leaf disks were inoculated with a non-oncogenic Agrobacterium tumefadens strain MP90 carrying a binary vector plasmid pBI121 which contains a chimeric nopaline synthase (NOS) promoter driven neomycin phosphotransferase (NPT II)

Narender S. Nehra; Ravindra N. Chibbar; Kutty K. Kartha; Raju S. S. Datla; William L. Crosby; Cecil Stushnoff

1990-01-01

303

Transfer of Rhizobium meliloti pSym genes into Agrobacterium tumefaciens: host-specific nodulation by atypical infection.  

PubMed

The pSym megaplasmid of Rhizobium meliloti 2011 mobilized by plasmid RP4, or plasmid pGMI42, an RP4-prime derivative which carries a 290-kilobase pSym fragment including nitrogenase and nod genes, was introduced into Agrobacterium tumefaciens. The resulting transconjugants induced root deformations specifically on the homologous hosts Medicago sativa and Melilotus alba and not on the heterologous hosts Trifolium pratense and Trifolium repens. The root deformations were shown to be genuine nodules by physiological and cytological studies. Thus, host specificity nodulation genes are located on the pSym megaplasmid. Host nodulation specificity did not seem to require recognition at the root hair level since no infection threads could be detected in the root hairs. Cytological observations indicated that bacteria penetrated only the superficial layers of the host root tissue by an atypical infection process. The submeristematic zone and the central tissue of the nodules were bacteria free. Thus, nodule organogenesis was probably triggered from a distance by the bacteria. Agrobacterium transconjugants carrying pSym induced the formation of more numerous and larger nodules than those carrying the RP4-prime plasmid pGMI42, suggesting that some genes influencing nodule organogenesis are located in a pSym region(s) outside that which has been cloned into pGMI42. PMID:6690420

Truchet, G; Rosenberg, C; Vasse, J; Julliot, J S; Camut, S; Denarie, J

1984-01-01

304

Agrobacterium tumefaciens mediated fused egfp-hph gene expression under the control of gpd promoter in Pleurotus ostreatus.  

PubMed

A transformation system for the basidiomycete Pleurotus ostreatus was established using agrobacterium-mediated infection. Following P. ostreatus glyceraldehyde-3-phosphate dehydrogenase gene analysis, its promoter region including two introns was used as cis-regulatory element to drive expression of enhanced green fluorescent protein (eGFP). As a selection marker, the hygromycin phosphotransferase (hph) gene cassette was used in the binary vector pPEH. Mycelia without pretreatment were found to be the most efficient recipients in transformation experiments while fruiting body tissue or basidiospores showed lower transformation rates. A transformation efficiency of 75% was achieved. After subculturing, putative transformants were screened by PCR and Southern blot analysis showing the expected ectopic integration of the transforming DNA. At the same time, the promotor region was shown to drive expression of selection marker as well as eGFP that could be visualized, which will be helpful for future investigation using Agrobacterium tumefaciens mediated transformation for functional characterization of genes in the mushroom forming basidioymcete P. ostreatus. PMID:20869218

Ding, Yi; Liang, Shen; Lei, Jinghang; Chen, Liguo; Kothe, Erika; Ma, Aimin

2010-09-23

305

Genetic transformation and molecular analysis of polyhydroxybutyrate biosynthetic gene expression in oil palm (Elaeis guineensis Jacq. var Tenera) tissues  

Microsoft Academic Search

Bioplastics are an alternative substitute for petrochemical synthetic plastics. Polyhydroxybutyrate (PHB) genes are involved in bioplastic synthesis. In this study, bioplastic synthesis genes were incorporated into the genome of oil palm because this plant has a high concentration of the PHB precursor acetyl-CoA. Immature embryos (IEs) of Elaeis guineensis var Tenera were infected with Agrobacterium tumefaciens LBA4404 that contained the

Ismanizan Ismail; Nor Fakhrana Iskandar; Gor Mian Chee; Ruslan Abdullah

2010-01-01

306

Assessment of the efficiency of cotransformation of the T-DNA of disarmed binary vectors derived from Agrobacterium tumefaciens and the T-DNA of A. rhizogenes  

Microsoft Academic Search

Co-transfer of Agrobacterium rhizogenes T-DNA and T-DNA from the A. tumefaciens binary vector pBin19 (Bevan, 1984) was studied in detail using Nicotiana rustica. High frequencies of co-transfer of T-DNA's were observed, even when no selection pressure was exerted. Increased levels of pBin19 T-DNA were found in hairy root cultures with selection at higher levels of kanamycin sulphate (50–200 µg ml-1).

John D. Hamill; Andrea Prescott; Cathie Martin

1987-01-01

307

An efficient method for the production of transgenic plants of peanut ( Arachis hypogaea L .) through Agrobacterium tumefaciens-mediated genetic transformation  

Microsoft Academic Search

Cotyledon explants from mature peanut seeds (Arachis hypogaea L.) were optimized to obtain adventitious shoot buds with high frequencies (>90%). Efficient transformation of these cotyledons by using Agrobacterium tumefaciens strain C58 carrying neomycin phosphotransferase II (nptII) and ß-glucuronidase (GUS; uidA), or coat protein gene of the Indian peanut clump virus (IPCVcp) and nptII on binary vectors (pBI121; pROKII:IPCVcp) led to

Kiran K Sharma; Vanamala Anjaiah

2000-01-01

308

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

309

Development of an efficient gene targeting system in Colletotrichum higginsianum using a non-homologous end-joining mutant and Agrobacterium tumefaciens -mediated gene transfer  

Microsoft Academic Search

The hemibiotrophic ascomycete Colletotrichum higginsianum is the casual agent of anthracnose disease of cruciferous plants. High efficiency transformation by Agrobacterium tumefaciens-mediated gene transfer has been established for this fungus. However, targeted gene mutagenesis through homologous recombination\\u000a rarely occurs in C. higginsianum. We have identified and disrupted the C. higginsianum homologue of the human Ku70 gene, ChKU70, which encodes a protein

Takuma Ushimaru; Hiroshi Terada; Kie Tsuboi; Yuki Kogou; Ayumu Sakaguchi; Gento Tsuji; Yasuyuki Kubo

2010-01-01

310

Rapid Assay of Foreign Gene Expression in Leaf Discs Transformed by Agrobacterium tumefaciens: Role of T-DNA Borders in the Transfer Process  

Microsoft Academic Search

We have developed a sensitive leaf disc transformation procedure for studying early and\\/or transient T-DNA expression during Agrobacterium tumefaciens-mediated transformation of plant cells. Using this system, we have examined the function of T-DNA border sequences on the early expression of T-DNA genes and on the stable integration of those genes in infected cells. Deletion of the right border from the

R. B. Horsch; H. J. Klee

1986-01-01

311

Suppression of transfer of non-T-DNA ‘vector backbone’ sequences by multiple left border repeats in vectors for transformation of higher plants mediated by Agrobacterium tumefaciens  

Microsoft Academic Search

Vectors for transformation of higher plants mediated by Agrobacterium tumefaciens were modified so that one, two or three additional copies of the left border (LB) sequences were inserted close to the original LB of the T-DNA. A gene for ß-glucuronidase (gusA) was placed outside the T-DNA to monitor the transfer to plants of 'vector backbone' sequences. The expression of GUS

Yoshiki Kuraya; Shozo Ohta; Miyuki Fukuda; Yukoh Hiei; Nobuhiko Murai; Kazuyuki Hamada; Jun Ueki; Hidemasa Imaseki; Toshihiko Komari

2004-01-01

312

Enhanced transformation of tomato co-cultivated with Agrobacterium tumefaciens C58C1Rif r ::pGSFR1161 in the presence of acetosyringone  

Microsoft Academic Search

Explants of tomato (Lycopersicon esculentum Mill cv. Ailsa Craig) were co-cultivated with Agrobacterium tumefaciens C58C1Rifr::pGSFR1161 in the presence of 20 (µM acetosyringone). Transformed root clones were selected on kanamycin medium and the presence of the nptII gene in the plant DNA confirmed by the polymerase chain reaction. Root clones derived from acetosyringone treatment grew more vigorously in the presence of

Kátia H. Lipp Joăo; Terence A. Brown

1993-01-01

313

Multiple gene co-integration in Arabidopsis thaliana predominantly occurs in the same genetic locus after simultaneous in planta transformation with distinct Agrobacterium tumefaciens strains  

Microsoft Academic Search

A total of 243 transgenic T1-plant lines were produced by in planta transformation of Arabidopsis by a mixture of three distinct Agrobacterium tumefaciens strains carrying different plasmid constructs. PCR analysis indicated that about 30% of the transgenic plants were transformed with two different T-DNAs and additional 9.5% of the plants carried all genes from three different T-DNAs. Southern analysis revealed

Volodymyr V. Radchuk; Dang Thi Van; Evelyn Klocke

2005-01-01

314

Differential Expression of Crown Gall Tumor Markers in Transformants Obtained after in vitro Agrobacterium tumefaciens-Induced Transformation of Cell Wall Regenerating Protoplasts Derived from Nicotiana tabacum  

Microsoft Academic Search

To obtain transformation of plant cells, we incubated 3-day-old cell wall-regenerating protoplasts from tobacco with Agrobacterium tumefaciens harboring tumor-inducing plasmids. Putative transformed tobacco cells were selected by phytohormone autotrophic growth and were shown to be transformed by the detection of the tumor cell specific enzymes lysopine dehydrogenase or nopaline dehydrogenase. This was substantiated by the detection, in transformed tumor tissues,

George J. Wullems; Lucy Molendijk; Gert Ooms; Robbert A. Schilperoort

1981-01-01

315

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

316

Agrobacterium tumefaciens-mediated high frequency genetic transformation of an Indian cowpea ( Vigna unguiculata L. Walp.) cultivar and transmission of transgenes into progeny  

Microsoft Academic Search

A reproducible Agrobacterium tumefaciens-mediated genetic transformation system for the production of fertile transgenic plants of cowpea (Vigna unguiculata) that transmits transgenes into progeny in Mendelian fashion has been developed. The cotyledonary node explants excised from 4-d-old seedlings, raised in vitro on medium containing salts of Murashige and Skoog's and vitamins of Gamborg's media (MBM) and BAP (10?M), were co-cultured with

Darshna Chaudhury; Seema Madanpotra; Ranjana Jaiwal; Raman Saini; P. Ananda Kumar; Pawan K. Jaiwal

2007-01-01

317

Transformation of a recalcitrant grain legume, Vigna mungo L. Hepper, using Agrobacterium tumefaciens -mediated gene transfer to shoot apical meristem cultures  

Microsoft Academic Search

The efficiency of Vigna mungo L. Hepper transformation was significantly increased from an average of 1% to 6.5% by using shoot apices excised from embryonic axes precultured on 10 µM benzyl-6-aminopurine (BAP) for 3 days and wounded prior to inoculation in Agrobacterium tumefaciens strain EHA105 carrying the binary vector pCAMBIA2301, which contains a neomycin phosphotransferase gene (nptII) and a ß-glucuronidase (GUS) gene

Raman Saini; Pawan K. Jaiwal

2005-01-01

318

Agrobacterium tumefaciens mediated transfer of Phaseolus vulgaris ?-amylase inhibitor-1 gene into mungbean Vigna radiata (L.) Wilczek using bar as selectable marker  

Microsoft Academic Search

Morphologically normal and fertile transgenic plants of mungbean with two transgenes, bar and ?-amylase inhibitor, have been developed for the first time. Cotyledonary node explants were transformed by cocultivation\\u000a with Agrobacterium tumefaciens strain EHA105 harboring a binary vector pKSB that carried bialaphos resistance (bar) gene and Phaseolus vulgaris ?-amylase inhibitor-1 (?AI-1) gene. Green transformed shoots were regenerated and rooted on

Sonia; Raman Saini; Rana P. Singh; Pawan K. Jaiwal

2007-01-01

319

Efficient Agrobacterium tumefaciens -mediated transformation of sweet potato ( Ipomoea batatas (L.) Lam.) from stem explants using a two-step kanamycin-hygromycin selection method  

Microsoft Academic Search

Summary  To achieve reliable stable transformation of sweet potato, we first developed efficient shoot regeneration for stem explants,\\u000a leaf disks, and petioles of sweet potato (Ipomoea batatas (L.) Lam.) cultivar Beniazuma. The shoot regeneration protocol enabled reproducible stable transformation mediated by Agrobacterium tumefaciens strain EHA105. The binary vector pIG121Hm contains the npt II (pnos) gene for kanamycin (Km) resistance, the hpt

Guo-Qing Song; Hideo Honda; Ken-Ichi Yamaguchi

2004-01-01

320

Production of stable transgenic sunflowers (Helianthus annuus L.) from wounded immature embryos by particle bombardment and co-cultivation with Agrobacterium tumefaciens  

Microsoft Academic Search

Split embryonic axes of 21-day old immature sunflower (Helianthus annuus L.) embryos were bombarded by microparticles and then co-cultured with disarmed Agrobacterium tumefaciens strain EHA105 bearing a binary vector carrying nptII and uidA genes. Apical shoot bud development and organogenesis induced on the explants led T0 transgenic plants. Southern blot analysis revealed complex integration patterns in T0 plants. The uidA

Olivier Lucas; Jean Kallerhoff; Gilbert Alibert

2000-01-01

321

A biosystem for alginate metabolism in Agrobacterium tumefaciens strain C58: Molecular identification of Atu3025 as an exotype family PL15 alginate lyase  

Microsoft Academic Search

The Gram-negative bacterium Sphingomonas sp. strain A1 (strain A1) has a peculiar biosystem to directly import and depolymerize a macromolecule, alginate, which is encoded by a cluster of genes on the genome. We identified five clustered ORFs homologous to some genes of the strain A1 cluster in the genome of Agrobacterium tumefaciens strain C58 (strain C58). These ORFs are Atu3021,

Akihito Ochiai; Wataru Hashimoto; Kousaku Murata

2006-01-01

322

Antitumor activity of some complex preparations in the culture of potato cells transformed by Agrobacterium tumefaciens  

Microsoft Academic Search

The complex preparations (CP) containing yeast mannan, diacetyl-2,4-dioxihexahydro-1,3,5-triasyne, cyanoguanidine, alcan-sulfo-acids,\\u000a or secondary metabolites of Pseudomonas sp. manifest in the inhibitory activity against tumors induced by A. tumefaciens in the parenchymatous tissue of potato tubers. As a rule, antitumor activity of CP is higher in the prophylactic application\\u000a compared to the therapeutic application of the preparations.

O. G. Kovalenko; O. M. Polishchuk; V. O. Isakova

2009-01-01

323

Celery transformation by Agrobacterium tumefaciens: cytological and genetic analysis of transgenic plants  

Microsoft Academic Search

Transgenic celery plants were obtained following co-cultivation of petiole explants with Agrobacterlum tumefaciens containing pMON200, a cointegrate vector carrying genes for kanamycin resistance and nopaline synthase. Transformants were selected by ability of callus to grow in the presence of 50mg\\/l kanamycin. Transformation was confirmed either by the presence of nopaline or by Southern blots. Cytological analysis of 14 transformed plants

D. Catlin; O. Ochoa; S. McCormick; C. F. Quiros

1988-01-01

324

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

325

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

326

Presence of a plant cell wall is not required for transformation of Nicotiana by Agrobacterium tumefaciens  

Microsoft Academic Search

Agrobacterium has been used to transform zero to six-day-old cell wall nonregenerating (CWNR) and cell wall regenerating (CWR) leaf protoplasts of tobacco. Transformed cells were selected by phoytohormone autotrophic growth and were verified by detection of the presence of lysopine dehydrogenase. Transformation frequencies in CWNR protoplasts were at least as high as those in CWR protoplasts, indicating that a plant

Ebrahim Firoozabady; David W. Galbraith

1984-01-01

327

Study on the Genetic Transformation of Gentian by Gene Recombinant  

Microsoft Academic Search

Transformation recombinant vector pMHL7133-Gus linked with rol gene which be cloned from Agrobacterium Rhizogenes R1000 through Agrobacterium tumefaciens LBA4404 into explant of gentian lamina, inducing rol gene express and producing hair root. Meanwhile, using the Agrobacterium Rhizogenes R1000 infect gentian directly as a comparison, we built two sets of transform system of Agrobacterium for hairy root through researching on all

Yan Yu Qing; Wang Yang; Xu Xiang Ling

328

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

PubMed Central

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

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

2010-01-01

329

Effects of tomato cultivar, leaf age, and bacterial strain on transformation by Agrobacterium tumefaciens  

Microsoft Academic Search

Differences in transformation of the tomato cultivar (Ohio 7870, Roma, UCD82b) by wild-type Agrobacterium strains (A6, A66, A281) were identified in a leaf disk assay system. Transformation was expressed as the percentage of explants producing callus on hormone-free medium and was confirmed by opine production. Ohio 7870 and Roma were more readily transformed than UCD82b by all three strains of

Melanie E. Davis; R. Daniel Lineberger; A. Raymond Miller

1991-01-01

330

Efficient regeneration of Brassica napus L. hypocotyls and genetic transformation by Agrobacterium tumefaciens  

Microsoft Academic Search

An efficient system for shoot regeneration and Agrobacterium-mediated gene transfer into Brassica napus was developed through the modification of the culture conditions. Different concentrations of benzyladenine (1.5, 3.0 and 4.5 mg dm–3) and thidiazuron (0.0, 0.15 and 0.30 mg dm–3) were evaluated for shoot regeneration of 7, 14 and 21-d-old hypocotyl explants. Maximum shoot regeneration frequency was obtained in 21-d-old

P. Jonoubi; A. Mousavi; A. Majd; A. H. Salmanian; M. Jalali Javaran; J. Daneshian

2005-01-01

331

Transgenic peppermint (Mentha×piperita L.) plants obtained by cocultivation with Agrobacterium tumefaciens  

Microsoft Academic Search

The first transgenic peppermint (Mentha×piperita L. cultivar Black Mitcham) plants have been obtained by Agrobacterium-mediated transformation by cocultivation with morphogenically responsive leaf explants. Basal leaf explants with petioles,\\u000a from leaves closest to the apex of in-vitro-culture-maintained shoots (5 cm), exhibited optimal shoot organogenetic responsiveness\\u000a on medium supplemented with thidiazuron (8.4 µm). Shoot formation occurred at sites of excision on the

X. Niu; K. Lin; P. M. Hasegawa; R. A. Bressan; S. C. Weller

1998-01-01

332

Presence of Unintended Agrobacterium tumefaciens Cloning Vector Sequences in Genetically Modified Plants  

Microsoft Academic Search

Agrobacterium transformation was used in the production of genetically modified plants from oilseed rape (Brassica napus) and tobacco (Nicotiana tabacum). After inoculation stop with the antibiotic timentin, a subsequent one-week treatment eliminated the vector bacterium from\\u000a the oilseed rape plate explant cultures. From the tobacco, however, we recorded vector-derived signals one week after potting\\u000a the regenerants in the greenhouse and

Katarina Björklöf; Michael Färdig; Kirsten S. Jřrgensen

2006-01-01

333

The assimilation of gamma-butyrolactone in Agrobacterium tumefaciens C58 interferes with the accumulation of the N-acyl-homoserine lactone signal.  

PubMed

Agrobacterium tumefaciens C58 communicates using N-acyl-homoserine lactones (acyl-HSL) and contains two lactonase-encoding genes, attM and aiiB, the products of which are capable of inactivating the acyl-HSL signal. In A. tumefaciens A6, the expression of the attKLM operon is controlled by the transcriptional repressor encoded by an adjacent gene, attJ. An attJ::Tn5 mutant does not accumulate acyl-HSL because of the constitutive expression of the lactonase AttM, the activity of which inactivates acyl-HSL. In this work, the attKLM operon of A. tumefaciens C58 was shown to be involved in an assimilative pathway of gamma-butyrolactone (GBL), gamma-hydroxybutyrate (GHB), and succinate semialdehyde (SSA), in which AttM and AttL are key enzymes for GBL and GHB assimilation. The expression of the attKLM promoter was activated in the presence of GBL, GHB, and SSA. Under these conditions, A. tumefaciens C58 did not accumulate the acyl-HSL that it naturally synthesizes, and also became able to inactivate exogenous acyl-HSL signals. Therefore, in A. tumefaciens C58, the assimilative pathway of gamma-butyrolactone interferes with the acyl-HSL signaling. PMID:15384485

Carlier, Aurélien; Chevrot, Romain; Dessaux, Yves; Faure, Denis

2004-09-01

334

Highly efficient transformation and plant regeneration of tall fescue mediated by Agrobacterium tumefaciens.  

PubMed

An efficient and reproducible system has been developed for the production of transgenic plants in tall fescue (Festuca arundinacea Schreb.) using A. tumefaciens-mediated transformation. Two-months-old suspension cultures served as excellent explants for transformation. The explants were inoculated with an A. tumefaciens strain EHA105 carrying a plasmid pDBA121 containing genes for hygromycin phosphotransferase (hpt) and phosphinotricin acetyltransferase (bar). The commercial herbicide Basta was used as a selective agent. Inclusion of acetosyringone (ACS) 20 mg/L in the co-culture medium led to an increase in transformation efficiency. The efficiency of transformation was highly dependent on the genotype, the explant, the culture medium and selective agent used. Tall fescue transformation efficiency is 2.85-10.9 plants per gram fresh weight (FW) of suspension cultures. This is much higher than the corresponding values reported before (2-5 plants). So far more than 300 transgenic plants have been obtained, the fertility of some transgenic plants had decreased. Stable integration and high expression of the transgenes were confirmed by PCR analysis and Southern blot hybridization or herbicide Basta spraying test. PMID:15840933

Hu, Zhang-Hua; Chen, Jin-Qing; Wu, Guan-Ting; Jin, Wei; Lang, Chun-Xiu; Huang, Rui-Zhi; Wang, Fu-Lin; Liu, Zhi-Hong; Chen, Xiao-Yun

2005-04-01

335

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

PubMed

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

1994-07-01

336

[Cotransformation of aspen and birch with three T-DNA regions from two different replicons in one Agrobacterium tumefaciens strain].  

PubMed

The cointegration rate into the aspen and birch genomes of foreign genes from a binary vector and a disarmed Ti plasmid pCBE21 carried by the same Agrobacterium tumefaciens strain was studied. The cotransformation rate for the genes within the Ti plasmid varied from 30 to 100%; while the transformation rate for the gene from T(L) region was twofold higher as compared with the T(R) region. On the average, the gene transfer from all three T-DNAs was recorded in 10.9% of the transgenic lines. For the vector pBI121, the cotransformation rates for the genes from both regions of pCBE21 T-DNA were higher as compared with the vector pGS. In addition, a concurrent transfer of the genes from the Ti plasmid T(L) and T(R) regions was recorded only after the transformation with the vector pBI121. These results can be used for constructing woody plants containing several genes. PMID:21261057

Lebedev, V G; Shestibratov, K A; Shadrina, T E; Bulatova, I V; Abramochkin, D G; Miroshnikov, A I

2010-11-01

337

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

PubMed Central

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

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

1984-01-01

338

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

PubMed Central

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

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

2011-01-01

339

Characterization of an Agrobacterium tumefaciens d-Psicose 3-Epimerase That Converts d-Fructose to d-Psicose  

PubMed Central

The noncharacterized gene previously proposed as the d-tagatose 3-epimerase gene from Agrobacterium tumefaciens was cloned and expressed in Escherichia coli. The expressed enzyme was purified by three-step chromatography with a final specific activity of 8.89 U/mg. The molecular mass of the purified protein was estimated to be 132 kDa of four identical subunits. Mn2+ significantly increased the epimerization rate from d-fructose to d-psicose. The enzyme exhibited maximal activity at 50°C and pH 8.0 with Mn2+. The turnover number (kcat) and catalytic efficiency (kcat/Km) of the enzyme for d-psicose were markedly higher than those for d-tagatose, suggesting that the enzyme is not d-tagatose 3-epimerase but d-psicose 3-epimerase. The equilibrium ratio between d-psicose and d-fructose was 32:68 at 30°C. d-Psicose was produced at 230 g/liter from 700-g/liter d-fructose at 50°C after 100 min, corresponding to a conversion yield of 32.9%.

Kim, Hye-Jung; Hyun, Eun-Kyung; Kim, Yeong-Su; Lee, Yong-Joo; Oh, Deok-Kun

2006-01-01

340

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.

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

1995-01-01

341

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.

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

1994-01-01

342

Efficient Agrobacterium tumefaciens-mediated transformation of embryogenic calli and regeneration of Hevea brasiliensis Müll Arg. plants.  

PubMed

An efficient procedure for producing transgenic Hevea brasiliensis callus and plant lines from clone PB 260 was established with Agrobacterium tumefaciens using strain EHA105 harbouring the vector pCAMBIA2301. Transformation capacity and competence of the embryogenic calli were improved after two cycles of cryopreservation. When the cocultivation temperature was reduced from 27 to 20 degrees C, the duration of this phase could be increased up to 7 days, promoting an increase in GUS activity. These transformation conditions led to the isolation of 24 callus lines resistant to paromomycin, which is used as a selection agent. Nineteen of these lines revealed the existence of one to four copies of T-DNA by Southern-blot analysis. Nine of them were transferred for regeneration by somatic embryogenesis. Three hundred seventy-four transgenic plants have thus been generated from six independent lines bearing 1, 2 or 3 copies of T-DNA. The efficiency and reproducibility of this method means that functional characterization of genes involved in natural rubber production can be envisaged. PMID:16136315

Blanc, Géraldine; Baptiste, Christelle; Oliver, Gérald; Martin, Florence; Montoro, Pascal

2005-08-31

343

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

344

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

2013-01-10

345

The Brucella suis Homologue of the Agrobacterium tumefaciens Chromosomal Virulence Operon chvE Is Essential for Sugar Utilization but Not for Survival in Macrophages  

PubMed Central

Brucella strains possess an operon encoding type IV secretion machinery very similar to that coded by the Agrobacterium tumefaciens virB operon. Here we describe cloning of the Brucella suis homologue of the chvE-gguA-gguB operon of A. tumefaciens and characterize the sugar binding protein ChvE (78% identity), which in A. tumefaciens is involved in virulence gene expression. B. suis chvE is upstream of the putative sugar transporter-encoding genes gguA and gguB, also present in A. tumefaciens, but not adjacent to that of a LysR-type transcription regulator. Although results of Southern hybridization experiments suggested that the gene is present in all Brucella strains, the ChvE protein was detected only in B. suis and Brucella canis with A. tumefaciens ChvE-specific antisera, suggesting that chvE genes are differently expressed in different Brucella species. Analysis of cell growth of B. suis and of its chvE or gguA mutants in different media revealed that ChvE exhibited a sugar specificity similar to that of its A. tumefaciens homologue and that both ChvE and GguA were necessary for utilization of these sugars. Murine or human macrophage infections with B. suis chvE and gguA mutants resulted in multiplication similar to that of the wild-type strain, suggesting that virB expression was unaffected. These data indicate that the ChvE and GguA homologous proteins of B. suis are essential for the utilization of certain sugars but are not necessary for survival and replication inside macrophages.

Alvarez-Martinez, Maria-Teresa; Machold, Jan; Weise, Christoph; Schmidt-Eisenlohr, Heike; Baron, Christian; Rouot, Bruno

2001-01-01

346

Agrobacterium tumefaciens can Obtain Sulfur from an Opine that is Synthesized by Octopine Synthase Using S-methylmethionine as a Substrate  

PubMed Central

Summary Agrobacterium tumefaciens incites plant tumors that produce nutrients called opines, which are utilized by the bacteria during host colonization. Various opines provide sources of carbon, nitrogen, and phosphorous, but virtually nothing was previously known about how A. tumefaciens acquires sulfur during colonization. Some strains encode an operon required for the catabolism of the opine octopine. This operon contains a gene, msh, that is predicted to direct the conversion of S-methylmethionine (SMM) and homocysteine (HCys) to two equivalents of methionine. Purified Msh carried out this reaction, suggesting that SMM could be an intermediate in opine catabolism. Purified octopine synthase (Ocs, normally expressed in plant tumors) utilized SMM and pyruvate to produce a novel opine, designated sulfonopine, whose catabolism by the bacteria would regenerate SMM. Sulfonopine was produced by tobacco and Arabidopsis when colonized by A. tumefaciens and was utilized as sole source of sulfur by A. tumefaciens. Purified Ocs also used thirteen other proteogenic and nonproteogenic amino acids as substrates, including three that contain sulfur. Sulfonopine and eleven other opines were tested for induction of octopine catabolic operon and all were able to do so. This is the first study of the acquisition of sulfur, an essential element, by this pathogen.

Flores-Mireles, Ana Lidia; Eberhard, Anatol; Winans, Stephen C.

2012-01-01

347

Complementation of a threonine dehydratase-deficient Nicotiana plumbaginifolia mutant after Agrobacterium tumefaciens-mediated transfer of the Saccharomyces cerevisiae ILV1 gene.  

PubMed

The Saccharomyces cerevisiae ILV1 gene, encoding threonine dehydratase (EC 4.2.1.16) was fused to the transferred DNA nopaline synthase promoter and the 3' noncoding region of the octopine synthase gene. It was introduced, by Agrobacterium tumefaciens-mediated gene transfer, into an isoleucine-requiring Nicotiana plumbaginifolia auxotroph deficient in threonine dehydratase. Functional complementation by the ILV1 gene product was demonstrated by the selection of several transformed lines on a medium without isoleucine and by the identification in these lines of the yeast threonine dehydratase activity. This is the first example illustrating the complementation of a plant auxotroph by transfection with a cloned gene. PMID:3302681

Colau, D; Negrutiu, I; Van Montagu, M; Hernalsteens, J P

1987-07-01

348

Crystallization and preliminary X-ray analysis of an exotype alginate lyase Atu3025 from Agrobacterium tumefaciens strain C58, a member of polysaccharide lyase family 15  

PubMed Central

Almost all alginate lyases depolymerize alginate in an endolytical fashion via a ?-elimination reaction. The alginate lyase Atu3025 from Agrobacterium tumefaciens strain C58, consisting of 776 amino-acid residues, is a novel exotype alginate lyase classified into polysaccharide lyase family 15. The enzyme was crystallized at 293?K by sitting-drop vapour diffusion with polyethylene glycol 4000 as a precipitant. Preliminary X-ray analysis showed that the Atu3025 crystal belonged to space group P21 and diffracted to 2.8?Ĺ resolution, with unit-cell parameters a = 107.7, b = 108.3, c = 149.5?Ĺ, ? = 91.5°.

Ochiai, Akihito; Yamasaki, Masayuki; Mikami, Bunzo; Hashimoto, Wataru; Murata, Kousaku

2006-01-01

349

The Ti plasmid increases the efficiency of Agrobacterium tumefaciens as a recipient in virB-mediated conjugal transfer of an IncQ plasmid  

PubMed Central

The T-DNA transfer apparatus of Agrobacterium tumefaciens mediates the delivery of the T-DNA into plant cells, the transfer of the IncQ plasmid RSF1010 into plant cells, and the conjugal transfer of RSF1010 between Agrobacteria. We show in this report that the Agrobacterium-to-Agrobacterium conjugal transfer efficiencies of RSF1010 increase dramatically if the recipient strain, as well as the donor strain, carries a wild-type Ti plasmid and is capable of vir gene expression. Investigation of possible mechanisms that could account for this increased efficiency revealed that the VirB proteins encoded by the Ti plasmid were required. Although, with the exception of VirB1, all of the proteins that form the putative T-DNA transfer apparatus (VirB1–11, VirD4) are required for an Agrobacterium strain to serve as an RSF1010 donor, expression of only a subset of these proteins is required for the increase in conjugal transfer mediated by the recipient. Specifically, VirB5, 6, 11, and VirD4 are essential donor components but are dispensable for the increased recipient capacity. Defined point mutations in virB9 affected donor and recipient capacities to the same relative extent, suggesting that similar functions of VirB9 are important in both of these contexts.

Bohne, Jutta; Yim, Andy; Binns, Andrew N.

1998-01-01

350

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

PubMed

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

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

2012-05-25

351

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

2012-02-02

352

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

PubMed

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

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

2010-07-14

353

Positive regulation of phenolic catabolism in Agrobacterium tumefaciens by the pcaQ gene in response to beta-carboxy-cis,cis-muconate.  

PubMed

An Escherichia coli system for generating a commercially unavailable catabolite in vivo was developed and was used to facilitate molecular genetic studies of phenolic catabolism. Introduction of the plasmid-borne Acinetobacter pcaHG genes, encoding the 3,4-dioxygenase which acts on protocatechuate, into E. coli resulted in bioconversion of exogenously supplied protocatechuate into beta-carboxy-cis,cis-muconate. This compound has been shown to be an inducer of the protocatechuate (pca) genes required for catabolism of protocatechuate to tricarboxylic acid cycle intermediates in Rhizobium leguminosarum biovar trifolii. The E. coli bioconversion system was used to explore regulation of the pca genes in a related bacterium, Agrobacterium tumefaciens. The pcaD gene, which encodes beta-ketoadipate enol-lactone hydrolase, from A. tumefaciens A348 was cloned and was shown to be adjacent to a regulatory region which responds strongly to beta-carboxy-cis,cis-muconate in E. coli. Site-specific insertional mutagenesis of the regulatory region eliminated expression of the pcaD gene in E. coli. When the mutation was incorporated into the A. tumefaciens chromosome, it eliminated expression of the pcaD gene and at least three other pca genes as well. The regulatory region was shown to activate gene expression in trans. The novel regulatory gene was termed pcaQ to differentiate it from pca regulatory genes identified in other microbes, which bind different metabolites. PMID:8501056

Parke, D

1993-06-01

354

Attachment of Agrobacterium tumefaciens to carrot cells and Arabidopsis wound sites is correlated with the presence of a cell-associated, acidic polysaccharide.  

PubMed Central

An early step in crown gall tumor formation involves the attachment of Agrobacterium tumefaciens to host plant cells. A. tumefaciens C58::A205 (C58 attR) is a Tn3HoHo1 insertion mutant that was found to be avirulent on Bryophyllum daigremontiana and unable to attach to carrot suspension cells. The mutation mapped to an open reading frame encoding a putative protein of 247 amino acids which has significant homology to transacetylases from many bacteria. Biochemical analysis of polysaccharide extracts from wild-type strain C58 and the C58::A205 mutant showed that the latter was deficient in the production of a cell-associated polysaccharide. Anion-exchange chromatography followed by 1H nuclear magnetic resonance and gas chromatography-mass spectrometry analyses showed that the polysaccharide produced by strain C58 was an acetylated, acidic polysaccharide and that the polysaccharide preparation contained three sugars: glucose, glucosamine, and an unidentified deoxy-sugar. Application of the polysaccharide preparation from strain C58 to carrot suspension cells prior to inoculation with the bacteria effectively inhibited attachment of the bacteria to the carrot cells, whereas an identical preparation from strain C58::A205 had no inhibitory effect and did not contain the acidic polysaccharide. Similarly, preincubation of Arabidopsis thaliana root segments with the polysaccharide prevented attachment of strain C58 to that plant. This indicates that the acidic polysaccharide may play a role in the attachment of A. tumefaciens to host soma plant cells.

Reuhs, B L; Kim, J S; Matthysse, A G

1997-01-01

355

Interaction of Root-knot Nematodes (RKN) and the Bacterium Agrobacterium Tumefaciens in Roots of Prunus Cerasifera: Evidence of the Protective Effect of the Ma RKN Resistance Genes Against Expression of Crown Gall Symptoms  

Microsoft Academic Search

Agrobacterium tumefaciens (AT) is the causal agent of crown gall, a major problem in the family Rosaceae and particularly for Prunus spp. Crown gall symptoms result from the bacterial infection of the cells damaged mechanically at the collar or by root parasitic nematodes. Myrobalan plum (P. cerasifera) is susceptible to AT and is not a host for the root-knot nematode

Maria-Jose Rubio-Cabetas; Jean-Claude Minot; Roger Voisin; Daniel Esmenjaud

2001-01-01

356

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

2008-02-27

357

Genetic transformation of Begonia tuberhybrida by Ri rol genes  

Microsoft Academic Search

We have developed an Agrobacterium -mediated transformation system for commercial Begonia species. The leaf explants of Begonia semperflorens, Begonia x hiemalis and B. tuberhybrida were inoculated with Agrobacterium tumefaciens LBA4404 harboring a binary vector pBI121 which contains rolA, B and C genes of an agropine type Ri plasmid (pRiA4b). Kanamycin resistant shoots of B. tuberhybrida were obtained on MS agar

Shigeto Kiyokawa; Yasuhiro Kikuchi; Hiroshi Kamada; Hiroshi Harada

1996-01-01

358

Virulence genes, borders, and overdrive generate single-stranded T-DNA molecules from the A6 Ti plasmid of Agrobacterium tumefaciens.  

PubMed Central

Agrobacterium tumefaciens transfers the T-DNA portion of its Ti plasmid to the nuclear genome of plant cells. Upon cocultivation of A. tumefaciens A348 with regenerating tobacco leaf protoplasts, six distinct single-stranded T-DNA molecules (T strands) were generated in addition to double-stranded T-DNA border cleavages which we have previously reported (K. Veluthambi, R.K. Jayaswal, and S.B. Gelvin, Proc. Natl. Acad. Sci. USA 84:1881-1885, 1987). The T region of an octopine-type Ti plasmid has four border repeats delimiting three T-DNA regions defined as T left (TL), T center (TC), and T right (TR). The six T strands generated upon induction corresponded to the TL, TC, TR, TL + TC, TC + TR, and TL + TC + TR regions, suggesting that the initiation and termination of T-strand synthesis can occur at each of the four borders. Most TL + TC + TR T-strand molecules corresponded to the top T-DNA strand, whereas the other five T strands corresponded to the bottom T-DNA strand. Generation of T strands required the virA, virG, and virD operons. Extra copies of vir genes, harbored on cosmids within derivatives of A. tumefaciens A348, enhanced production of T strands. The presence of right and left border repeats in their native orientation is important for the generation of full-length T strands. When a right border repeat was placed in the opposite orientation, single-stranded T-DNA molecules that corresponded to the top strand were generated. Deletion of overdrive, a sequence that flanks right border repeats and functions as a T-DNA transmission enhancer, reduced the level of T-strand generation. Induction of A. tumefaciens cells by regenerating tobacco protoplasts increased the copy number of the Ti plasmid relative to the bacterial chromosome. Images

Veluthambi, K; Ream, W; Gelvin, S B

1988-01-01

359

Enhanced production of single copy backbone-free transgenic plants in multiple crop species using binary vectors with a pRi replication origin in Agrobacterium tumefaciens.  

PubMed

Single transgene copy, vector backbone-free transgenic crop plants are highly desired for functional genomics and many biotechnological applications. We demonstrate that binary vectors that use a replication origin derived from the Ri plasmid of Agrobacterium rhizogenes (oriRi) increase the frequency of single copy, backbone-free transgenic plants in Agrobacterium tumefaciens mediated transformation of soybean, canola, and corn, compared to RK2-derived binary vectors (RK2 oriV). In large scale soybean transformation experiments, the frequency of single copy, backbone-free transgenic plants was nearly doubled in two versions of the oriRi vectors compared to the RK2 oriV control vector. In canola transformation experiments, the oriRi vector produced more single copy, backbone-free transgenic plants than did the RK2 oriV vector. In corn transformation experiments, the frequency of single copy backbone-free transgenic plants was also significantly increased when using the oriRi vector, although the transformation frequency dropped. These results, derived from transformation experiments using three crops, indicate the advantage of oriRi vectors over RK2 oriV binary vectors for the production of single copy, backbone-free transgenic plants using Agrobacterium-mediated transformation. PMID:21042934

Ye, Xudong; Williams, Edward J; Shen, Junjiang; Johnson, Susan; Lowe, Brenda; Radke, Sharon; Strickland, Steve; Esser, James A; Petersen, Michael W; Gilbertson, Larry A

2010-11-02

360

Efficiency of transformation of Polish cultivars of pea (Pisum sativum L.) with various regeneration capacity by using hypervirulent Agrobacterium tumefaciens strains.  

PubMed

An Agrobacterium-mediated transformation method of pea has been developed for several edible and fodder cultivars of pea (Pisum sativum L.), characterized previously in their potential for regeneration via organogenesis. The most appropriate explant, which was susceptible to Agrobacterium infection and capable of regenerating transgenic plants, turned out to be a slice of an immature embryo, including the embryo axis and the basal part of a cotyledon. Three hypervirulent strains of A. tumefaciens were tested: AgL0, AgL1 and EHA105. Each carried the binary vector pP35SGIB containing the uid gene, with an intron under control of the 35S promoter, and the bar gene conferring resistance to phosphinotricin. Strain AgL0 was found to be efficient for the majority of cultivars, followed by AgL1 and EHA105. Transformation efficiency varied from 0.7 to 4.1%, depending on cultivar and Agrobacterium strain. The transformation efficiency of particular pea cultivars did not clearly correspond to their regeneration capacity, which--although indispensable--was not a critical parameter of successful transformation. The presence of integrated genes in pea genomic DNA was detected by the PCR. T-DNA was stably transmitted to the progeny, as it was confirmed by Southern hybridization. The activity of introduced genes was analysed by the histochemical GUS assay and by painting leaves or by spraying transgenic plants with the herbicide Basta. PMID:15876681

Pniewski, Tomasz; Kapusta, Józef

2005-01-01

361

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

PubMed

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

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

2013-08-08

362

Overexpression of virD1 and virD2 genes in Agrobacterium tumefaciens enhances T-complex formation and plant transformation.  

PubMed Central

The VirD1 and VirD2 proteins encoded by an inducible locus of the virulence (vir) region of the Agrobacterium tumefaciens Ti plasmid are required for site-specific nicking at T-DNA border sites. We have determined the nucleotide sequence of a 3.6-kilobase-pair fragment carrying the virD locus from nopaline Ti plasmid pTiC58. In contrast to the previous report (Hagiya et al., Proc. Natl. Acad. Sci. USA 82:2669-2673, 1985), we found that the first three open reading frames were capable of encoding polypeptides of 16.1, 49.7, and 21.4 kilodaltons. Deletion analysis demonstrated that the N-terminal conserved domain of VirD2 was absolutely essential for its endonuclease activity. When extra copies of the virD1 and virD2 genes were present in an A. tumefaciens strain carrying a Ti plasmid, increased amounts of T-strand and nicked molecules could be detected at early stages of vir induction. Such strains possessed the ability to transform plants with higher efficiency. Images

Wang, K; Herrera-Estrella, A; Van Montagu, M

1990-01-01

363

The Never ripe Mutant Provides Evidence That Tumor-Induced Ethylene Controls the Morphogenesis of Agrobacterium tumefaciens-Induced Crown Galls on Tomato Stems12  

PubMed Central

We confirm the hypothesis that Agrobacterium tumefaciens-induced galls produce ethylene that controls vessel differentiation in the host stem of tomato (Lycopersicon esculentum Mill.). Using an ethylene-insensitive mutant, Never ripe (Nr), and its isogenic wild-type parent we show that infection by A. tumefaciens results in high rates of ethylene evolution from the developing crown galls. Ethylene evolution from isolated internodes carrying galls was up to 50-fold greater than from isolated internodes of control plants when measured 21 and 28 d after infection. Tumor-induced ethylene substantially decreased vessel diameter in the host tissues beside the tumor in wild-type stems but had a very limited effect in the Nr stems. Ethylene promoted the typical unorganized callus shape of the gall, which maximized the tumor surface in wild-type stems, whereas the galls on the Nr stems had a smooth surface. The combination of decreased vessel diameter in the host and increased tumor surface ensured water-supply priority to the growing gall over the host shoot. These results indicate that in addition to the well-defined roles of auxin and cytokinin, there is a critical role for ethylene in determining crown-gall morphogenesis.

Aloni, Roni; Wolf, Asnat; Feigenbaum, Pua; Avni, Adi; Klee, Harry J.

1998-01-01

364

Agrobacterium tumefaciens mediated transfer of Phaseolus vulgaris alpha-amylase inhibitor-1 gene into mungbean Vigna radiata (L.) Wilczek using bar as selectable marker.  

PubMed

Morphologically normal and fertile transgenic plants of mungbean with two transgenes, bar and alpha-amylase inhibitor, have been developed for the first time. Cotyledonary node explants were transformed by cocultivation with Agrobacterium tumefaciens strain EHA105 harboring a binary vector pKSB that carried bialaphos resistance (bar) gene and Phaseolus vulgaris alpha-amylase inhibitor-1 (alphaAI-1) gene. Green transformed shoots were regenerated and rooted on medium containing phosphinothricin (PPT). Preculture and wounding of the explants, presence of acetosyringone and PPT-based selection of transformants played significant role in enhancing transformation frequency. Presence and expression of the bar gene in primary transformants was evidenced by PCR-Southern analysis and PPT leaf paint assay, respectively. Integration of the Phaseolus vulgaris alpha-amylase inhibitor gene was confirmed by Southern blot analysis. PCR analysis revealed inheritance of both the transgenes in most of the T(1) lines. Tolerance to herbicide was evidenced from seed germination test and chlorophenol red assay in T(1) plants. Transgenic plants could be recovered after 8-10 weeks of cocultivation with Agrobacterium. An overall transformation frequency of 1.51% was achieved. PMID:16983450

Sonia; Saini, Raman; Singh, Rana P; Jaiwal, Pawan K

2006-09-16

365

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

2011-05-17

366

The Agrobacterium tumefaciens Chaperone-Like Protein, VirE1, Interacts with VirE2 at Domains Required for Single-Stranded DNA Binding and Cooperative Interaction†  

PubMed Central

Agrobacterium tumefaciens transfers single-stranded DNA (ssDNA) into plants. Efficient tumorigenesis requires VirE1-dependent export of ssDNA-binding (SSB) protein VirE2. VirE1 binds VirE2 domains involved in SSB and self-association, and VirE1 may facilitate VirE2 export by preventing VirE2 aggregation and the premature binding of VirE2 to ssDNA.

Sundberg, Christopher D.; Ream, Walt

1999-01-01

367

In planta analysis of the Agrobacterium tumefaciens T-cyt gene promoter: identification of an upstream region essential for promoter activity in leaf, stem and root cells of transgenic tobacco  

Microsoft Academic Search

The promoter region of the Agrobacterium tumefaciens T-cyt gene was fused to a ß-glucuronidase (gusA) reporter gene and introduced into tobacco plants. Detection of gusA expression in transgenic F1 progeny revealed that the T-cyt promoter is active in many, if not all, cell types in leaves, stems and roots of fully developed plants. Developmental stage-dependent promoter activity was observed in

Saskia T. C. Neuteboom; Esther Hulleman; Rob A. Schilperoort; J. Harry C. Hoge

1993-01-01

368

Transgenic crop plants expressing synthetic cry9Aa gene are protected against insect damage  

Microsoft Academic Search

A synthetic gene sequence of cry9Aa was made to achieve high expression levels in a plant cell. Tobacco, potato, cauliflower and turnip rape plants were transformed with this synthetic gene driven by the double 35S promoter using Agrobacterium tumefaciens LBA4404. The presence and expression of the synthetic cry9Aa gene was evaluated in Southern, Northern and Western analysis and with insect

Viktor Kuvshinov; Kimmo Koivu; Anne Kanerva; Eija Pehu

2001-01-01

369

Enhancement of Trichoderma endochitinase secretion in tobacco cell cultures using an ?- amylase signal peptide  

Microsoft Academic Search

An ?-amylase signal peptide from rice was synthesized and fused with endochitinase (ech42) gene cloned from Trichoderma virens. The chimeric gene was designated as PSP?-amyech42, and this was transferred to a plant transformation vector, referred to as pMASGK. Leaf explants of tobacco cv White Burley\\u000a were co-cultivated with Agrobacterium tumefaciens strain LBA4404 carrying ech42 with its own signal peptide(ech42SP) and

Archana Kumari; Gaurav Sharma; Sumangala Bhat; Ramesh S. Bhat; P. U. Krishnaraj; M. S. Kuruvinashetti

370

Establishment of an Agrobacteriuim -mediated cotyledon disc transformation method for Jatropha curcas  

Microsoft Academic Search

Jatropha curcas contains high amounts of oil in its seed and has been considered for bio-diesel production. A transformation procedure for\\u000a J. curcas has been established for the first time via\\u000a Agrobacterium tumefaciens infection of cotyledon disc explants. The results indicated that the efficiency of transformation using the strain LBA4404\\u000a and phosphinothricin for selection was an improvement over that with

Meiru Li; Hongqing Li; Huawu Jiang; Xiaoping Pan; Guojiang Wu

2008-01-01

371

Modification of plant architecture in Limonium spp. induced by rol genes  

Microsoft Academic Search

An Agrobacterium-mediated transformation system for Limonium has been developed. The leaf explants of the sterile hybrid L116 (Limonium otolepis, Kuntze × Limonium latifolium, Kuntze) were inoculated with A. tumefaciens LBA4404 harboring the binary vector pBin19 containing a T-DNA fragment encompassing rol A,B and C genes of A. rhizogenes Ri plasmid (pRi1855). Transgenic shoots, regenerated on selection medium, were micropropagated, rooted,

Antonio Mercuri; Simona Bruna; Laura De Benedetti; Gianluca Burchi; Tito Schiva

2001-01-01

372

Conditions of transformation and regeneration of `Induka' and `Elista' strawberry plants  

Microsoft Academic Search

Efficiency of plants' transformation depends on many factors. The genotype, applied techniques and conditions of plant's modification and modified plant regeneration are the most important among them. In our studies regeneration and transformation conditions for two strawberry cultivars were determined and compared. Plants were transformed by Agrobacterium tumefaciens LBA4404 strain containing plasmid pBIN19 with nptII and gus-reporter genes. Experiment was

Agnieszka Grucha?a; Ma?gorzata Korbin; Edward ?urawicz

2004-01-01

373

Genetically modified coffee plants expressing the Bacillus thuringiensis cry 1Ac gene for resistance to leaf miner  

Microsoft Academic Search

A synthetic version of the cry1Ac gene of Bacillus thuringiensis has been used for the transformation of coffee species (Coffea canephora and C. arabica) to confer resistance to an important pest, the coffee leaf miner (Perileucoptera coffeella and other Leucoptera spp). Somatic embryos were co-cultivated with the LBA4404 strain of Agrobacterium tumefaciens containing the cry1Ac gene. More than 100 transformed

T. Leroy; A.-M. Henry; M. Royer; I. Altosaar; R. Frutos; D. Duris; R. Philippe

2000-01-01

374

Overexpression of a 10-deacetylbaccatin III-10 ?- O -acetyltransferase gene leads to increased taxol yield in cells of Taxus chinensis  

Microsoft Academic Search

Taxus chinensis suspension cells were transformed by the Agrobacterium-mediated transformation method to overexpress the dbat gene coding for 10-deacetylbaccatin III-10 ?-O-acetyltransferase, a key enzyme for taxol biosynthesis. A. tumefaciens strain LBA4404 harboring either pCAMBIA1303 or the recombinant plasmid p1303-SdbatN was used. Both plasmids harbored the hygromycin phosphotransferase gene (hptII) and gusA-mgfp5 gene as selectable markers, but the latter plasmid also

Peng Zhang; Shu-Tao Li; Ting-Ting Liu; Chun-Hua Fu; Peng-Peng Zhou; Chun-Fang Zhao; Long-Jiang Yu

2011-01-01

375

Cell-Autonomous Cytokinin-lndependent Crowth of Tobacco Cells Transformed by Agrobacterium tumefaciens Strains Lacking the Cytokinin Biosynthesis Gene  

Microsoft Academic Search

Mutations at the cytokinin biosynthesis locus (tmr) of Agrobac- terium 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 tmf mutant strain A356 by producing rapidly growing, unorganized tumors, indicating that these lines can grow in a cytokinin-independent fashion

Robert C. Black; Andrew N. Binns; Chi-Feng Chang; David C. Lynn

1994-01-01

376

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

2009-06-16

377

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

378

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

2005-12-21

379

Units of genetic expression in the virulence region of a plant tumor-inducing plasmid of Agrobacterium tumefaciens  

Microsoft Academic Search

The effect of a large number of Tn3 insertions in the vir region of the Ti plasmid pTiA6NC on the virulence of Agrobacterium was determined. The Vir- insertions were mapped in three of the five loci that have been defined previously. Merodiploid Rec- strains carrying one insertion mutation on the Ti plasmid and another insertion mutation (or the homologous wild-type

V. N. Iyer; H. J. Klee; E. W. Nester

1982-01-01

380

Development of a rapid and simple Agrobacterium tumefaciens- mediated transformation system for the fungal pathogen Heterobasidion annosum  

Microsoft Academic Search

Heterobasidion annosum causes root and butt-rot in trees and is the most serious forest pathogen in the northern hemisphere. We developed a rapid and simple Agrobacterium-mediated method of gene delivery into H. annosum to be used in functional studies of candidate genes and for visualization of mycelial interactions. Heterobasidion annosum TC 32-1 was cocultivated at pH 5.6 and 20 1C

Nicklas Samils; Malin Elfstrand; Daniel L. Lindner Czederpiltz; Jan Fahleson; Christina Dixelius; Jan Stenlid

2006-01-01

381

Effects of antioxidants on the plant regeneration and GUS expressive frequency of peanut ( Arachis hypogaea ) explants by Agrobacterium tumefaciens  

Microsoft Academic Search

Our objective is to develop an Agrobacterium-based transformation system for peanut. Ascorbic acid (AA), sodium selenite (Se), DL-a-tocopherol (TOC) and glutathione (GSH) were used as antioxidants during the plant regeneration and co-cultivation with Agrobacteriumtumefaciens. Percentage of explants with buds or shoots increased from 50 in control group to 88, 90, 87 and 76 in GSH, TOC, Se or AA treated

Zheng Qiusheng; Ju Bao; Liang Likun; Xiao Xianhua

2005-01-01

382

The Quorum-sensing Protein TraR of Agrobacterium tumefaciens is Susceptible to Intrinsic and TraM-mediated Proteolytic Instability  

PubMed Central

Summary TraR of Agrobacterium tumefaciens is a LuxR-type transcription factor that regulates genes required for replication and conjugation of the tumor-inducing (Ti) plasmid. TraR binds the pheromone 3-oxo-octanoylhomoserine lactone (OOHL) and requires this molecule for folding into a protease-resistant, soluble conformation. Even after binding to OOHL, TraR is degraded at readily detectable rates. Here we show that the N-terminal domain of TraR, which binds OOHL, is more resistant to degradation than the full length protein, suggesting that sites on the C-terminal DNA binding domain (TraR(171-234)) enhance protein turnover. A fusion between GFP and TraR-(171-234) was poorly fluorescent, and truncations of this fusion protein allowed us to identify residues in this domain that contribute to protein degradation. TraR activity was previously shown to be inhibited by the antiactivator TraM. These proteins form 2:2 complexes that fail to bind DNA sequences. Here we show that TraM sharply decreased the accumulation of TraR in whole cells, indicating that TraM facilitates proteolysis of TraR. The TraM component of these complexes is spared from proteolysis, and could therefore act catalytically.

Costa, Esther D.; Chai, Yunrong; Winans, Stephen C.

2012-01-01

383

Transformation of a recalcitrant grain legume, Vigna mungo L. Hepper, using Agrobacterium tumefaciens-mediated gene transfer to shoot apical meristem cultures.  

PubMed

The efficiency of Vigna mungo L. Hepper transformation was significantly increased from an average of 1% to 6.5% by using shoot apices excised from embryonic axes precultured on 10 microM benzyl-6-aminopurine (BAP) for 3 days and wounded prior to inoculation in Agrobacterium tumefaciens strain EHA105 carrying the binary vector pCAMBIA2301, which contains a neomycin phosphotransferase gene (nptII) and a beta-glucuronidase (GUS) gene (gusA) interrupted by an intron. The transformed green shoots that were selected and rooted on medium containing kanamycin, and which tested positive for nptII gene by polymerase chain reaction, were established in soil to collect seeds. GUS activity was detected in whole T(0) shoots and T(1) seedlings. All T(0) plants were morphologically normal, fertile and the majority of them transmitted transgenes in a 3:1 ratio to their progenies. Southern analysis of T(1) plants showed integration of nptII into the plant genome. PMID:15815929

Saini, Raman; Jaiwal, Pawan K

2005-04-07

384

Stable genetic transformation of castor (Ricinus communis L.) via Agrobacterium tumefaciens-mediated gene transfer using embryo axes from mature seeds.  

PubMed

A protocol for the transformation of castor embryo axes using the pCAMBIA vector 1304 in disarmed Agrobacterium tumefaciens strain EHA105 is presented. Co-cultivated explants were initially subjected to expansion and proliferation on MS medium with 0.5 mg l(-1) TDZ followed by three cycles of selection on medium with 0.5 mg l(-1) BA and increasing concentrations of hygromycin (20-40-60 mg l(-1)). Selected shoot clusters were transferred to medium with 0.5 mg l(-1) BA for proliferation and 0.2 mg l(-1) BA for shoot elongation. Elongated shoots were rooted on half-strength MS medium with 2.0 mg l(-1) NAA. The presence and stable integration of the hpt gene was confirmed through PCR, RT-PCR, PCR-Southern blot, sequence analysis, Southern blot analysis and PCR analysis of progeny. Southern blot analysis of the primary transformants showed single copy integration and progeny analysis revealed monogenic inheritance of the introduced gene. This paper reports the first successful attempt at producing transgenic castor. PMID:15580353

Sujatha, M; Sailaja, M

2004-12-03

385

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

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

1993-01-01

386

A nuclear localization signal and the C-terminal omega sequence in the Agrobacterium tumefaciens VirD2 endonuclease are important for tumor formation.  

PubMed Central

The T-DNA portion of the Agrobacterium tumefaciens tumor-inducing (Ti) plasmid integrates into plant nuclear DNA. Direct repeats define the T-DNA ends; transfer begins when the VirD2 endonuclease produces a site-specific nick in the right-hand border repeat and attaches to the 5' end of the nicked strand. Subsequent events generate linear single-stranded VirD2-bound DNA molecules that include the entire T-DNA (T-strands). VirD2 protein contains a nuclear localization signal (NLS) near the C terminus and may direct bound T-strands to plant nuclei. We constructed mutations in virD2 and showed that the NLS was important for tumorigenesis, although T-strand production occurred normally in its absence. A tobacco etch virus NLS, substituted for the VirD2 NLS, restored tumor-inducing activity. Amino acids (the omega sequence) at the C terminus of VirD2, outside the NLS and the endonuclease domain, contributed significantly to tumorigenesis, suggesting that VirD2 may serve a third important function in T-DNA transfer. Images

Shurvinton, C E; Hodges, L; Ream, W

1992-01-01

387

Integration of a bacterial ?-carotene ketolase gene into the Mucor circinelloides genome by the Agrobacterium tumefaciens-mediated transformation method.  

PubMed

Plasmids introduced in Mucor circinelloides (and most transformable Mucorales) tend to replicate autonomously, and hardly ever integrate in the genome. This is critical if we want to express exogenous genes, because plasmids are easily lost during vegetative growth, and the ratio of plasmid molecules/nuclei is invariably low. Linearized molecules of DNA have been used to get their genomic integration but the transformation efficiency drops extremely. We have developed and highly optimized an efficient Agrobacterium-mediated transformation system for M. circinelloides to facilitate the integration of transforming DNA in the genome of the recipient strain that could also be used for other Mucorales. PMID:22711121

Papp, Tamás; Csernetics, Arpád; Nyilasi, Ildikó; Vágvölgyi, Csaba; Iturriaga, Enrique A

2012-01-01

388

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

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; Jensen, John Beck; Bhuvaneswari, T V

2013-06-14

389

One out of Four: HspL but No Other Small Heat Shock Protein of Agrobacterium tumefaciens Acts as Efficient Virulence-Promoting VirB8 Chaperone  

PubMed Central

Alpha-crystallin-type small heat shock proteins (sHsps) are ubiquitously distributed in most eukaryotes and prokaryotes. Four sHsp genes named hspL, hspC, hspAT1, and hspAT2 were identified in Agrobacterium tumefaciens, a plant pathogenic bacterium capable of unique interkingdom DNA transfer via type IV secretion system (T4SS). HspL is highly expressed in virulence-induced growth condition and functions as a VirB8 chaperone to promote T4SS-mediated DNA transfer. Here, we used genetic and biochemical approaches to investigate the involvement of the other three sHsps in T4SS and discovered the molecular basis underlying the dominant function of HspL in promoting T4SS function. While single deletion of hspL but no other sHsp gene reduced T4SS-mediated DNA transfer and tumorigenesis efficiency, additional deletion of other sHsp genes in the hspL deletion background caused synergistic effects in the virulence phenotypes. This is correlated with the high induction of hspL and only modest increase of hspC, hspAT1, and hspAT2 at their mRNA and protein abundance in virulence-induced growth condition. Interestingly, overexpression of any single sHsp gene alone in the quadruple mutant caused increased T4SS-mediated DNA transfer and tumorigenesis. Thermal aggregation protecting assays in vitro indicated that all four sHsps exhibit chaperone activity for the model substrate citrate synthase but only HspL functions as efficient chaperone for VirB8. The higher VirB8 chaperone activity of HspL was also demonstrated in vivo, in which lower amounts of HspL than other sHsps were sufficient in maintaining VirB8 homeostasis in A. tumefaciens. Domain swapping between HspL and HspAT2 indicated that N-terminal, central alpha-crystallin, and C-terminal domains of HspL all contribute to HspL function as an efficient VirB8 chaperone. Taken together, we suggest that the dominant role of HspL in promoting T4SS function is based on its higher expression in virulence-induced condition and its more efficient VirB8 chaperone activity as compared to other sHsps.

Wu, Chih-Feng; Narberhaus, Franz; Lai, Erh-Min

2012-01-01

390

Ectopic localization of auxin and cytokinin in tobacco seedlings by the plant-oncogenic AK-6b gene of Agrobacterium tumefaciens AKE10.  

PubMed

The oncogenic 6b gene of Agrobacterium tumefaciens induces a number of morphological and metabolic alterations in plants. Although molecular functions associated with the 6b genes have been proposed, including auxin transport, sugar transport, transcriptional regulation, and miRNA metabolism, so far an unequivocal conclusion has not been obtained. We investigated the association between auxin accumulation and tumor development of the tobacco seedlings expressing the AK-6b gene under the control of the dexamethasone-inducible promoter. Indole-3-acetic acid (IAA) localization was examined by immunochemical staining with monoclonal antibody against IAA and by histochemical analysis using the IAA-specific induced construct, DR5::GUS (?-glucuronidase). Both procedures indicated that IAA preferentially accumulated in the tumorous protrusions as well as in newly developing vascular bundles in the tumors. Furthermore, true leaves also showed abaxial IAA localization, leading to altered leaves in which the adaxial and abaxial identities were no longer evident. Co-localization of cytokinin and auxin in the abaxial tumors was verified by immunochemical staining with an antibody against cytokinin. Treatment of AK-6b-seedlings with N-1-naphthylphthalamic acid, an inhibitor of polar auxin transport, promoted the morphological severity of phenotypes, whereas 1-naphthoxyacetic acid, a specific auxin influx carrier inhibitor, induced tumor regression on cotyledons and new tumorous proliferations on hypocotyls. Prominent accumulation of both auxin and cytokinin was observed in both regressed and newly developing tumors. We suggest from these results that modulation of auxin/cytokinin localization as a result of AK-6b gene expression is responsible for the tumorous proliferation. PMID:23873395

Takahashi, Sachiko; Sato, Rui; Takahashi, Miho; Hashiba, Noriko; Ogawa, Atsushi; Toyofuku, Kyoko; Sawata, Taiki; Ohsawa, Yuki; Ueda, Kenji; Wabiko, Hiroetsu

2013-07-20

391

An efficient method for the production of transgenic plants of peanut (Arachis hypogaea L.) through Agrobacterium tumefaciens-mediated genetic transformation.  

PubMed

Cotyledon explants from mature peanut seeds (Arachis hypogaea L.) were optimized to obtain adventitious shoot buds with high frequencies (>90%). Efficient transformation of these cotyledons by using Agrobacterium tumefaciens strain C58 carrying neomycin phosphotransferase II (nptII) and ß-glucuronidase (GUS; uidA), or coat protein gene of the Indian peanut clump virus (IPCVcp) and nptII on binary vectors (pBI121; pROKII:IPCVcp) led to the production of a large percentage (55%) of transgenic plants. Transformed individuals were obtained through selection on medium containing 125 mg l(-1) kanamycin. A large number of independently transformed plants (over 75) were successfully transplanted to the glasshouse. Integration of the transgenes and stable genetic transformants in the progeny were assessed by PCR amplification of 700-bp fragment of nptII and 585-bp of IPCVcp genes, and Southern blot hybridizations in the T1 generation of transgenic plants. Analysis of 35 transgenic plants of T1 generation from the progeny of a single transformation event suggested the segregation of a single copy insert in a 3:1 Mendelian ratio. On an average, 120-150 days were required between the initiation of explant transformation and transfer of rooted plants to the greenhouse. The cotyledon regeneration system proved to be an excellent vehicle for the production of a large number of independently transformed peanut plants. Shoot formation was rapid and prolific, and a large proportion of these shoots developed into fertile plants. The method reported here provides new opportunities for the crop improvement of peanut via genetic transformation. PMID:11011088

Sharma; Anjaiah

2000-10-16

392

The Phenolic Recognition Profiles of the Agrobacterium tumefaciens VirA Protein Are Broadened by a High Level of the Sugar Binding Protein ChvE  

PubMed Central

The formation of crown gall tumors by Agrobacterium tumefaciens requires that the virulence (vir) genes be induced by chemical signals which consist of specific phenolic compounds and monosaccharides, synthesized at plant wound sites. Signal transduction in the activation of these genes is mediated by the VirA-VirG two-component regulatory system, together with ChvE, a glucose-galactose binding protein which interacts with VirA. We have previously presented genetic evidence that virA senses phenolic compounds directly (Y.-W. Lee, S. Jin, W.-S. Sim, and E. W. Nester, Proc. Natl. Acad. Sci. USA 92:12245–12249, 1995). The vir genes of strain KU12 can be induced by 4-hydroxyacetophenone, p-coumaric acid, and phenol, whereas these same phenolic compounds are weak inducers of the vir genes of strain A6. In this report, we show that a specific inducing sugar can broaden the specificity of the phenolic compound which VirA senses. 4-Hydroxyacetophenone and other related phenolic compounds function as inducing phenolic compounds with the virA gene of A6 if arabinose replaces glucose as the inducing sugar. We further demonstrate that this broadened specificity for phenolic inducers results from the increased level of ChvE through induction by arabinose via the regulatory protein GbpR. If high levels of ChvE are present, then poorly inducing phenolic compounds can induce the vir genes to high levels in combination with glucose. Comparing the induction response of the wild type and that of a VirA mutant with a mutation in its receiver domain revealed that the activity of the receiver domain is controlled by the periplasmic domain. We discuss these observations in terms of how VirA senses and transduces signals elicited by the two classes of plant signal molecules.

Peng, Wen-Tao; Lee, Yong-Woog; Nester, Eugene W.

1998-01-01

393

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

2013-05-14

394

Generation of selectable marker-free sheath blight resistant transgenic rice plants by efficient co-transformation of a cointegrate vector T-DNA and a binary vector T-DNA in one Agrobacterium tumefaciens strain  

Microsoft Academic Search

Co-transformation of Oryza sativa L. var. Pusa Basmati1 was done using an Agrobacterium\\u000a tumefaciens strain harbouring a single-copy cointegrate vector and a multi-copy binary vector in the same cell. The T-DNA of the cointegrate\\u000a vector pGV2260::pSSJ1 carried the hygromycin phosphotransferase (hph) and ?-glucuronidase (gus) genes. The binary vector pCam-chi11, without a plant selectable marker gene, harboured the rice chitinase (chi11)

Rajasekaran Sripriya; Vengoji Raghupathy; Karuppannan Veluthambi

2008-01-01

395

The Protein Encoded by Oncogene 6b from Agrobacterium tumefaciens Interacts with a Nuclear Protein of Tobacco  

PubMed Central

The 6b gene in the T-DNA from Agrobacterium has oncogenic activity in plant cells, inducing tumor formation, the phytohormone-independent division of cells, and alterations in leaf morphology. The product of the 6b gene appears to promote some aspects of the proliferation of plant cells, but the molecular mechanism of its action remains unknown. We report here that the 6b protein associates with a nuclear protein in tobacco that we have designated NtSIP1 (for Nicotiana tabacum 6b–interacting protein 1). NtSIP1 appears to be a transcription factor because its predicted amino acid sequence includes two regions that resemble a nuclear localization signal and a putative DNA binding motif, which is similar in terms of amino acid sequence to the triple helix motif of rice transcription factor GT-2. Expression in tobacco cells of a fusion protein composed of the DNA binding domain of the yeast GAL4 protein and the 6b protein activated the transcription of a reporter gene that was under the control of a chimeric promoter that included the GAL4 upstream activating sequence and the 35S minimal promoter of Cauliflower mosaic virus. Furthermore, nuclear localization of green fluorescent protein–fused 6b protein was enhanced by NtSIP1. A cluster of acidic residues in the 6b protein appeared to be essential for nuclear localization and for transactivation as well as for the hormone-independent growth of tobacco cells. Thus, it seems possible that the 6b protein might function in the proliferation of plant cells, at least in part, through an association with NtSIP1.

Kitakura, Saeko; Fujita, Tomomichi; Ueno, Yoshihisa; Terakura, Shinji; Wabiko, Hiroetsu; Machida, Yasunori

2002-01-01

396

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

2004-05-05

397

Comparative transcriptome analysis of Agrobacterium tumefaciens in response to plant signal salicylic acid, indole-3-acetic acid and gamma-amino butyric acid reveals signalling cross-talk and Agrobacterium--plant co-evolution.  

PubMed

Agrobacterium has evolved sophisticated strategies to perceive and transduce plant-derived cues. Recent studies have found that numerous plant signals, including salicylic acid (SA), indole-3-acetic acid (IAA) and gamma-amino butyric acid (GABA), profoundly affect Agrobacterium-plant interactions. Here we determine and compare the transcriptome profiles of Agrobacterium in response to these three plant signals. Collectively, the transcription of 103, 115 and 95 genes was significantly altered by SA, IAA and GABA respectively. Both distinct cellular responses and overlapping signalling pathways were elicited by these three plant signals. Interestingly, these three plant compounds function additively to shut off the Agrobacterium virulence programme and activate the quorum-quenching machinery. Moreover, the repression of the virulence programme by SA and IAA and the inactivation of quorum-sensing signals by SA and GABA are regulated through independent pathways. Our data indicate that these plant signals, while cross-talk in plant signalling networks, also act as cross-kingdom signals and play redundant roles in tailoring Agrobacterium regulatory pathways, resulting in intensive signalling cross-talk in Agrobacterium. Our results support the notion that Agrobacterium has evolved the ability to hijack plant signals for its own benefit. The complex signalling interplay between Agrobacterium and its plant hosts reflects an exquisite co-evolutionary balance. PMID:18671824

Yuan, Ze-Chun; Haudecoeur, Elise; Faure, Denis; Kerr, Kathleen F; Nester, Eugene W

2008-08-15

398

Centrifugation Assisted Agrobacterium tumefaciens- mediated Transformation (CAAT) of embryogenic cell suspensions of banana ( Musa spp. Cavendish AAA and Lady finger AAB)  

Microsoft Academic Search

Centrifugation-assisted Agrobacterium-mediated transformation (CAAT) protocol, developed using banana cultivars from two economically important genomic groups (AAA and AAB) of cultivated Musa, is described. This protocol resulted in 25-65 plants\\/50mg of settled cell volume of embryogenic suspension cells, depending upon the Agrobacterium strain used, and gave rise to hundreds of morphologically normal, transgenic plants in two banana cultivars from the two

Harjeet Khanna; Doug Becker; Jennifer Kleidon; James Dale

2004-01-01

399

VirB1, a component of the T-complex transfer machinery of Agrobacterium tumefaciens, is processed to a C-terminal secreted product, VirB1.  

PubMed Central

During genetic transformation of plant cells by Agrobacterium tumefaciens, 11 VirB proteins and VirD4 are proposed to form a transmembrane bridge to transfer a DNA-protein complex (T-complex) into the plant cytoplasm. In this study, the localization of the first product of the virB operon, VirB1, was studied in detail. While full-length VirB1 localized mostly to the inner membrane, an immunoreactive VirB1 product was found as soluble processed form, designated VirB1*. Equal amounts of VirB1* could be detected in concentrated culture supernatants versus associated with the cell. VirB1* was purified from the supernatant of vir-induced cells by ammonium sulfate precipitation and Q-Sepharose chromatography. Sequence analysis of the N terminus of VirB1* localized the processing site after amino acid 172 of VirB1. Cell-associated VirB1* was partly removed by vortexing, suggesting a loose association with the cell or active secretion. However, cross-linking and coimmunoprecipitation showed a close association of cell-bound VirB1* with the VirB9-VirB7 heterodimer, a membrane-associated component of the T-complex transfer machinery. Homologies of the N-terminal part of VirB1 to bacterial transglycosylases suggest that it may assist T-complex transfer by local lysis of the bacterial cell wall, whereas the exposed localization of the C-terminal processing product VirB1* predicts direct interaction with the plant. Thus, VirB1 may be a bifunctional protein where both parts have different functions in T-complex transfer from Agrobacterium to plant cells.

Baron, C; Llosa, M; Zhou, S; Zambryski, P C

1997-01-01

400

Two T-DNA's co-transformed into Brassica napus by a double Agrobacterium tumefaciens infection are mainly integrated at the same locus  

Microsoft Academic Search

Summary Hypocotyl explants of threeBrassica napus varieties were infected with two nopaline typeAgrobacterium strains each carrying a distinct disarmed T-DNA containing different selectable markers. Selection was done for only one of the markers, after which the regenerated plants were screened for the presence of the second marker. High co-transformation frequencies of both T-DNA's were obtained (39%–85% of the transformants). Where

Marc De Block; Dirk Debrouwer

1991-01-01

401

Cytokinin production by Agrobacterium and Pseudomonas spp.  

PubMed Central

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

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

1987-01-01

402

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

PubMed Central

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

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

1989-01-01

403

Agrobacterium-mediated transformation of oat (Avena sativa L.) cultivars via immature embryo and leaf explants.  

PubMed

This paper reports on the successful Agrobacterium-mediated transformation of oat, and on some factors influencing this process. In the first step of the experiments, three cultivars, two types of explant, and three combinations of strain/vectors, which were successfully used for transformation of other cereals were tested. Transgenic plants were obtained from the immature embryos of cvs. Bajka, Slawko and Akt and from leaf base explants of cv. Bajka after transformation with A. thumefaciens strain LBA4404(pTOK233). The highest transformation rate (12.3%) was obtained for immature embryos of cv. Bajka. About 79% of the selected plants proved to be transgenic; however, only 14.3% of the T(0) plants and 27.5% of the T(1) showed GUS expression. Cell competence of both types of explant differed in terms of their transformation ability and transgene expression. The next step of the study was to test the suitability for oat transformation of the pGreen binary vector combined with different selection cassettes: nptII or bar under the nos or 35S promoter. Transgenic plants were selected in combinations transformed with nos::nptII, 35S::nptII and nos::bar. The highest transformation efficiency (5.3%) was obtained for cv. Akt transformed with nos::nptII. A detailed analysis of the T(0) plants selected from a given callus line and their progeny revealed that they were the mixture of transgenic, chimeric-transgenic and non-transgenic individuals. Southern blot analysis of T(0) and T(1) showed simple integration pattern with the low copy number of the introduced transgenes. PMID:18690445

Gasparis, Sebastian; Bregier, Cezary; Orczyk, Waclaw; Nadolska-Orczyk, Anna

2008-08-09

404

Intermolecular disulfide bonds stabilize VirB7 homodimers and VirB7/VirB9 heterodimers during biogenesis of the Agrobacterium tumefaciens T-complex transport apparatus.  

PubMed Central

The Agrobacterium tumefaciens VirB7 lipoprotein contributes to the stabilization of VirB proteins during biogenesis of the putative T-complex transport apparatus. Here, we report that stabilization of VirB7 itself is correlated with its ability to form disulfide cross-linked homodimers via a reactive Cys-24 residue. Three types of beta-mercaptoethanol-dissociable complexes were visualized with VirB7 and/or a VirB7::PhoA41 fusion protein: (i) a 9-kDa complex corresponding in size to a VirB7 homodimer, (ii) a 54-kDa complex corresponding in size to a VirB7/VirB7::PhoA41 mixed dimer, and (iii) a 102-kDa complex corresponding to a VirB7::PhoA41 homodimer. A VirB7C24S mutant protein was immunologically undetectable, whereas the corresponding VirB7C24S::PhoA41 derivative accumulated to detectable levels but failed to form dissociable homodimers or mixed dimers with wild-type VirB7. We further report that VirB7-dependent stabilization of VirB9 is correlated with the ability of these two proteins to dimerize via formation of a disulfide bridge between reactive Cys-24 and Cys-262 residues, respectively. Two types of dissociable complexes were visualized: (i) a 36-kDa complex corresponding in size to a VirB7/VirB9 heterodimer and (ii) an 84-kDa complex corresponding in size to a VirB7/VirB9::PhoA293 heterodimer. A VirB9C262S mutant protein was immunologically undetectable, whereas the corresponding VirB9C262S::PhoA293 derivative accumulated to detectable levels but failed to form dissociable heterodimers with wild-type VirB7. Taken together, these results support a model in which the formation of disulfide cross-linked VirB7 dimers represent critical early steps in the biogenesis of the T-complex transport apparatus. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4

Spudich, G M; Fernandez, D; Zhou, X R; Christie, P J

1996-01-01

405

Agrobacterium-Host Attachment and Biofilm Formation  

Microsoft Academic Search

Physical association with host plant tissue is a prerequisite to Agrobacterium tumefaciens infection and subsequent disease. Mechanisms of tissue adherence have been extensively studied in mammalian pathogens, but\\u000a less so in plant-associated bacteria. Cells of A. tumefaciens often attach to plant tissue by a single pole. In the appropriate environment, these attached bacteria eventually develop\\u000a into multicellular assemblies called biofilms,

Clay Fuqua

406

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

2004-06-08

407

Agrobacterium -Mediated Transformation of Common Bermudagrass ( Cynodon dactylon )  

Microsoft Academic Search

Common bermudagrass, Cynodon dactylon, is a widely used warm-season turf and forage species in the temperate and tropical regions of the world. We have been able to transform the species using Agrobacterium-mediated approach. In seven experiments reported here, a total of 67 plates of calluses and suspensions were infected with Agrobacterium tumefaciens strains, and nine hygromycin B resistant calluses were

L. Li; R. Li; S. Fei; R. Qu

2005-01-01

408

Agrobacterium -mediated transformation of Perilla ( Perilla frutescens )  

Microsoft Academic Search

Agrobacterium tumefaciens-mediated transformation system for perilla (Perilla frutescens Britt) was developed. Agrobacterium strain EHA105 harboring binary vector pBK I containing bar and ?-tmt cassettes or pIG121Hm containing nptII, hpt, and gusA cassettes were used for transformation. Three different types of explant, hypocotyl, cotyledon and leaf, were evaluated for transformation and hypocotyl explants resulted in the highest transformation efficiency with an

Byoung-Kyu Lee; Seung-Hee Yu; Yul-Ho Kim; Byung-Ohg Ahn; Han-Sun Hur; Sang-Chul Lee; Zhanyuan Zhang; Jang-Yong Lee

2005-01-01

409

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.

410

An Agrobacterium VirE2 channel for transferred-DNA transport into plant cells  

Microsoft Academic Search

Transferred DNA (T-DNA) transfer from Agrobacterium tumefaciens into eukaryotic cells is the only known example of interkingdom DNA transfer. T-DNA is a single-stranded segment of Agrobacterium's tumor-inducing plasmid that enters the plant cell as a complex with the bacterial virulence proteins VirD2 and VirE2. The VirE2 protein is highly induced on contact of A. tumefaciens with a plant host and

Fabrice Dumas; Myriam Duckely; Pawel Pelczar; Patrick van Gelder; Barbara Hohn

2001-01-01

411

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

412

Susceptibility of dry bean (Phaseolus vulgaris L.) to Agrobacterium infection: Transformation of cotyledonary and hypocotyl tissues  

Microsoft Academic Search

A germinating-seed assay was developed to determine the susceptibility of dry bean (Phaseolus vulgaris L.) to infection by Agrobacterium tumefaciens. Seedlings infected one to three days after germination were more susceptible to A. tumefaciens infection than seedlings germinated for five to seven days and the galls that formed on the one to three day seedlings were significantly larger. Nineteen genotypes

Phillip McClean; Paula Chee; Bruce Held I; Jorge Simental; Roger F. Drong; Jerry Slightom

1991-01-01

413

A novel system for Agrobacterium-mediated transformation of wheat (Triticum aestivum L.) cells  

Microsoft Academic Search

A new approach for transforming the cultured cells of wheat (Triticum aestivum L. cv. Ganmai 8) was developed using Agrobacterium tumefaciens. The features of the optimum procedure were: (a) both combined synthetic signal molecules and multiple natural extracts from susceptible plants were used to pretreat the primary vigorous Agrobacterium (PVA) cells for approximately 16 h; (b) the gyratory magnetic field

Yao Xu; Baojian Li; Jingfen Jia

1993-01-01

414

Genetic manipulation in cultivars of oilseed rape ( Brassica napus ) using Agrobacterium  

Microsoft Academic Search

The response of oilseed rape cultivars to infection with Agrobacterium tumefaciens and A. rhizogenes and the possibility of regenerating genetically transformed oilseed rape plants were examined. The frequency at which Agrobacterium induced galls or hairy-roots on in vitro cultured plants ranged from 10% to 70%, depending on the cultivar. From galls induced by the tumorigenic strain T37, known to be

G. Ooms; A. Bains; M. Burrell; A. Karp; D. Twell; E. Wilcox

1985-01-01

415

VIP1: linking Agrobacterium-mediated transformation to plant immunity?  

PubMed

Agrobacterium tumefaciens is the most efficient vehicle used today for the production of transgenic plants and plays an essential role in basic scientific research and in agricultural biotechnology. Previously, plant VirE2-interacting protein 1 (VIP1) was shown to play a role in Agrobacterium-mediated transformation. Recent reports demonstrate that VIP1, as one of the bZIP transcription factors, is also involved in plant immunity responses. Agrobacterium is able to activate and abuse VIP1 for transformation. These findings highlight Agrobacterium-host interaction and unveil how Agrobacterium hijacks host cellular mechanism for its own benefit. This review focuses on the roles played by VIP1 in Agrobacterium-mediated transformation and plant immunity. PMID:20473505

Liu, Yukun; Kong, Xiangpei; Pan, Jiaowen; Li, Dequan

2010-05-15

416

VIP1: linking Agrobacterium -mediated transformation to plant immunity?  

Microsoft Academic Search

Agrobacterium tumefaciens is the most efficient vehicle used today for the production of transgenic plants and plays an essential role in basic scientific\\u000a research and in agricultural biotechnology. Previously, plant VirE2-interacting protein 1 (VIP1) was shown to play a role\\u000a in Agrobacterium-mediated transformation. Recent reports demonstrate that VIP1, as one of the bZIP transcription factors, is also involved\\u000a in plant

Yukun Liu; Xiangpei Kong; Jiaowen Pan; Dequan Li

2010-01-01

417

Agrobacterium -Mediated Transformation of Switchgrass and Inheritance of the Transgenes  

Microsoft Academic Search

Switchgrass (Panicum virgatum L.) has been developed into an important biofuel crop. Embryogenic calli induced from caryopses or inflorescences of the\\u000a lowland switchgrass cultivar Alamo were used for Agrobacterium-mediated transformation. A chimeric hygromycin phosphotransferase gene (hph) was used as the selectable marker and hygromycin as the selection agent. Embryogenic calli were infected with Agrobacterium tumefaciens strain EHA105. Calli resistant to

Yajun Xi; Chunxiang Fu; Yaxin Ge; Rangaraj Nandakumar; Hiroshi Hisano; Joseph Bouton; Zeng-Yu Wang

2009-01-01

418

Assessment of conditions affecting Agrobacterium   rhizogenes -mediated transformation of soybean  

Microsoft Academic Search

Agrobacterium rhizogenes-mediated transformation has become a powerful tool for studying gene function and root biology due to its quick and simple\\u000a methodology. This transformation method is particularly suitable for those plants, including legumes, whose transformation\\u000a using Agrobacterium tumefaciens has been challenging. Although there are some reports on A. rhizogenes-mediated transformation of legumes to produce ‘composite’ plants, conditions influencing A. rhizogenes-mediated transformation of soybean [Glycine

Dong Cao; Wensheng Hou; Shikui Song; Hongbo Sun; Cunxiang Wu; Yongsheng Gao; Tianfu Han

2009-01-01

419

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

420

Agrobacterium-mediated transformation of African violet  

Microsoft Academic Search

A method for genetic transformation of Saintpaulia ionantha by co-cultivation of in vitro-grown leaves and petioles with Agrobacterium tumefaciens is described. Two bacterial strains, EHA105 and A281 both harbouring the binary plasmid pKIWI105 carrying the genes uidA and nptII, were used in the experiments. Regenerants were not obtained using the disarmed strain EHA105. The oncogenic strain A281 resulted in efficient

Antonio Mercuri; Laura De Benedetti; Gianluca Burchi; Tito Schiva

2000-01-01

421

Colonization of Phaseolus vulgaris nodules by Agrobacterium-like strains.  

PubMed

Non-nodulating Agrobacterium-like strains identified among root nodule isolates of common bean were labeled with gusA, a reporter gene encoding beta-glucuronidase (GUS). Bean plants were then co-inoculated with an infective Rhizobium strain and labeled transconjugants of Agrobacterium-like strains. Blue staining of nodules showed that Agrobacterium-like strains were able to colonize these symbiotic organs. Isolation and characterization by restriction fragment length polymorphism analysis of PCR-amplified 16S rRNA genes revealed a mixed population of Rhizobium and Agrobacterium-like strains in all nodules showing GUS activity. PCR amplification of the nifH gene and nodulation tests did not show any evidence of acquisition of symbiotic gene by lateral transfer from Rhizobium to Agrobacterium-like strains. Moreover, these strains were able to invade mature nodules. Based on sequencing of the 16S rRNA gene, one of these Agrobacterium-like strains showed 99.4% sequence similarity with Agrobacterium bv. 1 reference strains and 99% similarity with an Agrobacterium bv. 1 strain isolated from Acacia mollisima in Senegal. Agrobacterium tumefaciens C58 and the disarmed variant AT123 did not show any ability to colonize nodules. Co-inoculation of bean seeds with Agrobacterium and Rhizobium strains did not enhance nodulation and plant yield under controlled conditions. PMID:16091768

Mhamdi, Ridha; Mrabet, Moncef; Laguerre, Gisčle; Tiwari, Ravi; Aouani, Mohamed Elarbi

2005-02-01

422

AGROBACTERIUM-MEDIATED TRANSFORMATION OF BOTTEL GOURD (LAGENARIA SICERARIA STANDL.)  

Technology Transfer Automated Retrieval System (TEKTRAN)

We describe a procedure for producing transgenic bottle gourd plants by inoculating cotyledon explants with Agrobacterium tumefaciens strain AGL1 that carries the binary vector pCAMBIA3301 containing a glufosinate ammonium-resistance ( bar) gene and the beta- d-glucuronidase (GUS) reporter gene. The...

423

Agrobacterium -mediated transient transformation of marigold ( Tagetes erecta )  

Microsoft Academic Search

Hypocotyls, roots, leaf sections and shoot tips from Tagetes erecta plantlets were inoculated with Agrobacterium tumefaciens, harboring the binary vector pCAMBIA2301, containing the ?-glucuronidase gene. Histochemical GUS assays of infected tissues\\u000a showed transient gus gene expression after 3 days.

Gregorio Godoy-Hernández; Elide Avilés Berzunza; Lizbeth Castro Concha; María de Lourdes Miranda-Ham

2006-01-01

424

EFFICIENT AND GENOTYPE-INDEPENDENT AGROBACTERIUM - MEDIATED TOMATO TRANSFORMATION  

Technology Transfer Automated Retrieval System (TEKTRAN)

An efficient method to transform five cultivars of tomato (Lycopersicon esculentum). Micro-Tom, Red Cherry, Rubion, Piedmont, and E6203 is reported. A comparison was made of leaf, cotyledon, and hypocotyl explants on 7 different regeneration media without Agrobacterium tumefaciens cocultivation and ...

425

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...

426

UNIT 3D.3: PHENOTYPIC ANALYSES OF AGROBACTERIUM  

PubMed Central

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

Morton, Elise R.; Fuqua, Clay

2012-01-01

427

UNIT 3D.1 Laboratory Maintenance of Agrobacterium  

PubMed Central

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

Morton, Elise R.; Fuqua, Clay

2012-01-01

428

Agrobacterium -mediated transformation (AMT) of Trichoderma reesei as an efficient tool for random insertional mutagenesis  

Microsoft Academic Search

Filamentous fungus Trichoderma reesei QM9414 was successfully transformed with Agrobacterium tumefaciens AGL-1 for random integration of transforming DNA (T-DNA). Co-cultivation of T. reesei conidia or protoplasts with A. tumefaciens in the presence of acetosyringone resulted in the formation of hygromycin B-resistant fungal colonies with high transformation\\u000a frequency. Nine randomly selected resistant clones were proved to be stable through mitotic cell

Yao Hua Zhong; Xiao Li Wang; Tian Hong Wang; Qiao Jiang

2007-01-01

429

Construction of an intron-containing marker gene: Splicing of the intron in transgenic plants and its use in monitoring early events in Agrobacterium -mediated plant transformation  

Microsoft Academic Search

Agrobacterium tumefaciens is a commonly used tool for transforming dicotyledonous plants. The underlying mechanism of transformation however is not very well understood. One problem complicating the analysis of this mechanism is the fact that most indicator genes are already active in Agrobacterium, thereby preventing the precise determination of timing and localisation of T-DNA transfer to plant cells. In order to

G. Vancanneyt; R. Schmidt; A. O'Connor-Sanchez; L. Willmitzer; M. Rocha-Sosa

1990-01-01

430

Agrobacterium -mediated transformation as a tool for functional genomics in fungi  

Microsoft Academic Search

In the era of functional genomics, the need for tools to perform large-scale targeted and random mutagenesis is increasing. A potential tool is Agrobacterium-mediated fungal transformation. A. tumefaciens is able to transfer a part of its DNA (transferred DNA; T-DNA) to a wide variety of fungi and the number of fungi that can be transformed by Agrobacterium-mediated transformation (AMT) is

Caroline B. Michielse; Paul J. J. Hooykaas; Cees A. M. J. J. van den Hondel; Arthur F. J. Ram

2005-01-01

431

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

432

Regeneration of plants from “habituated” and “Agrobacterium-transformed” single-cell clones of tobacco  

Microsoft Academic Search

One “habituated” and three “Agrobacterium-transformed crown gall” callus strains of tobacco, all hormone-autotrophic, were cloned and tested for regeneration to plants. The crown gall strains originated from “unorganized” tumors induced by highly virulent strains of Agrobacterium tumefaciens. The regeneration of complete plants from a great number of habituated clones as well as from three fully transformed single-cell clones isolated from

M. D. Sacristán; G. Melchers

1977-01-01

433

Agrobacterium rhizogenes inserts T-DNA into the genomes of the host plant root cells  

Microsoft Academic Search

Agrobacterium rhizogenes, which induces hairy root disease of dicotyledonous plants1, is closely related to Agrobacterium tumefaciens, the causative agent of crown gall disease1-3. Virulence in both species is conferred by large plasmids4-7. Infected plant tissue synthesizes novel metabolites, opines8-11, that are not found in normal plant tissues. The pattern of opines synthesized is determined by the type of virulence plasmid

Mary-Dell Chilton; David A. Tepfer; Annik Petit; Chantal David; Francine Casse-Delbart; Jacques Tempé

1982-01-01

434

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

Microsoft Academic Search

Agrobacterium tumefaciens-mediated genetic transformation is an efficient tool for genetic engineering of plants. VirE2 is a single-stranded DNA binding Agrobacterium protein that is transported into the plant cell and presumably protects the T-DNA from degradation. Using a yeast two-hybrid system, we identified Arabidopsis thaliana VIRE2-INTERACTING PROTEIN2 (VIP2) with a NOT domain that is conserved in both plants and animals. Furthermore,

Ajith Anand; Alexander Krichevsky; Sebastian Schornack; Thomas Lahaye; Tzvi Tzfira; Yuhong Tang; Vitaly Citovsky; K. S. Mysore

2007-01-01

435

GUS expression in blueberry (Vaccinium spp.): factors influencing Agrobacterium-mediated gene transfer efficiency  

Microsoft Academic Search

Several factors were investigated for their influence on the transfer of an intron-containing ?-glucuronidase (GUS) gene into blueberry (Vaccinium spp.) leaf explants during the early stages of Agrobacterium-mediated gene transfer, including days of cocultivation, strain of Agrobacterium tumefaciens, explant age and genotype. The number of GUS-expressing leaf zones and calli were counted immediately and 2 weeks after cocultivation,\\u000a respectively, to

X. Cao; Q. Liu; L. J. Rowland; F. A. Hammerschlag

1998-01-01

436

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

437

Agrobacterium -mediated transformation using embryogenic calli in Satsuma mandarin ( Citrus unshiu Marc.) cv. Miyagawa wase  

Microsoft Academic Search

Agrobacterium-mediated transformation in Satsuma mandarin (Citrus unshiu Marc.) cv. Miyagawa wase was achieved with reasonable transformation efficiency of about 22%, which was the percentage of\\u000a transgenic plantlets confirmed by genomic PCR (37 plantlets\\/168 hygromycin-resistant calli). Embryogenic calli of Miyagawa\\u000a wase were infected with Agrobacterium tumefaciens strain EHA105 harboring binary vector pCAMBIA1300 that contained miraculin gene (a taste-modifying protein) and hygromycin

Seong Beom Jin; Jeong Won Park; Hyeon Jin Sun; Su Hyun Yun; Hyo Yeon Lee; Dong Sun Lee; Quan Chun Hong; Yong Woo Kim; Key Zung Riu; Jae Hoon Kim

2011-01-01

438

Agrobacterium-mediated Japonica rice transformation: a procedure assisted by an antinecrotic treatment  

Microsoft Academic Search

An Agrobacterium-mediated transformation protocol for Japonica rice (cv. R321), using conventional genetic vectors and explants pretreated\\u000a with antinecrotic compounds is presented. We evaluated the effect of two compounds with known antioxidant activity (ascorbic\\u000a acid and cysteine) and silver nitrate on the viability of stem sections taken from in vitro rice plantlets, and on their interaction with Agrobacterium tumefaciens (At 2260)

Gil A. Enríquez-Obregón; Dmitri L. Prieto-Samsónov; Gustavo A. de la Riva; Marlene Pérez; Guillermo Selman-Housein; Roberto I. Vázquez-Padrón

1999-01-01

439

Agrobacterium-mediated Transformation of Mungbean (Vigna radiata (L.) Wilczek)  

Microsoft Academic Search

Plasmid pCAMBIA 1301-choA was transformed into Agrobacterium rhizogenes strain K599 and A. tumefaciens strain EHA 105 for mungbean transformation. Cotyledons from different ages of mungbean seedlings were inoculated with both bacteria. The two-day-old cotyledons that were co-cultured with hairy root bacteria showed higher ability to produce branched roots than the others. An average of 10 branched roots was formed on

Potjamarn SURANINPONG; Sontichai CHANPRAME; Hyeon-JE CHO; Jack M. WIDHOLM; Aree WARANYUWAT

440

Agrobacterium -mediated transformation of European chestnut embryogenic cultures  

Microsoft Academic Search

An innovative and efficient genetic transformation protocol for European chestnut is described in which embryogenic cultures are used as the target material. When somatic embryos at the globular or early-torpedo stages were cocultured for 4 days with Agrobacterium tumefaciens strain EHA105 harbouring the pUbiGUSINT plasmid containing marker genes, a transformation efficiency of 25% was recorded. Murashige and Skoog culture medium

E. Corredoira; D. Montenegro; A. M. Vieitez; A. Ballester

2004-01-01

441

Agrobacterium -mediated transformation of Fraxinus pennsylvanica hypocotyls and plant regeneration  

Microsoft Academic Search

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

Ningxia Du; Paula M. Pijut

2009-01-01

442

Agrobacterium -mediated transformation of protocorm-like bodies in Cattleya  

Microsoft Academic Search

Protocorm-like bodies (PLBs) of Cattleya orchid CM2450 were cocultivated with Agrobacterium tumefaciens strain EHA101 carrying either plasmid pIG121-Hm harboring genes coding for neomycin phosphotransferase II, hygromycin phosphotransferase,\\u000a and ?-glucuronidase (GUS) or plasmid pBBRacdS harboring these same genes along with a gene coding for 1-aminocyclopropane-1-carboxylate (ACC) deaminase. PLBs were maintained\\u000a in a liquid New Dogashima (ND) medium and then added to

Lin ZhangDong; Dong Poh Chin; Masahiro Mii

2010-01-01

443

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. ...

444

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

445

Agrobacterium -mediated transformation of strawberry calli and recovery of transgenic plants  

Microsoft Academic Search

Transformed calli and shoots of strawberry (Fragaria × ananassa Duch.) cv. Redcoat were obtained using Agrobacterium tumefaciens carrying plasmid pB1121. Inoculated leaf explants produced transgenic calli at a frequency of 3% on selection medium containing 50 µg\\/ml kanamycin. Twenty per cent of selected caili regenerated, giving rise to transgenic shoots. All transgenic calli and shoots expressed substantial amounts of GUS

Narender S. Nehra; Ravindra N. Chibbar; Kutty K. Kartha; Raju S. S. Datla; William L. Crosby; Cecil Stushnoff

1990-01-01

446

The effects of Agrobacterium rhizogenes rolAB genes in lettuce  

Microsoft Academic Search

Agrobacterium rhizogenes rolAB genes were transferred into lettuce (Lactuca sativa L.) cultivar ‘Lake Nyah’ using a supervirulent strain of A. tumefaciens. Southern hybridisation confirmed the presence of the rol genes in kanamycinresistant plants. In culture, transgenic plants exhibited extensive root development and an increased response to auxin. Phenotypic characterisation of transgenic populations indicated significant alterations in plant development, especially in

Ian S. Curtis; Caiping He; J. Brian Power; Domenico Mariotti; Ad de Laat; Michael R. Davey

1996-01-01

447

Cell Walls of Crown-Gall Tumors and Embryonic Plant Tissues Lack Agrobacterium Adherence Sites  

Microsoft Academic Search

Crown-gall tumor initiation by Agrobacterium tumefaciens is inhibited by cell walls from normal dicotyledonous plants but not by cell walls from crown-gall tumors apparently because of bacterial adherence or nonadherence, respectively, to the different cell walls. Cell walls from normal and tumor tissues in culture also show this difference, indicating that the two types of tissue stably maintain this difference

James A. Lippincott; Barbara B. Lippincott

1978-01-01

448

Agrobacterium T-DNA-mediated integration and gene replacement in the brown rot pathogen Monilinia fructicola  

Microsoft Academic Search

A transformation system utilizing Agrobacterium tumefaciens was developed for targeted gene disruption in Monilinia fructicola, a fungal pathogen that causes brown rot disease of stone fruits. Transformation with a vector containing the neomycin phosphotransferase II (nptII) cassette flanked with 4 kb cutinase gene (Mfcut1) flanking sequences resulted in an average of 13 transformants per 105 spores. When assayed by PCR and

Miin-Huey Lee; Richard M. Bostock

2006-01-01

449

Agrobacterium tumefasciens-mediated transformation of the aquatic fungus Blastocladiella emersonii  

Microsoft Academic Search

Agrobacterium tumefaciens is widely used for plant DNA transformation and more recently, has also been used to transform yeast, filamentous fungi and even human cells. Using this technique, we developed the first transformation protocol for the saprobic aquatic fungus Blastocladiella emersonii, a Blastocladiomycete localized at the base of fungal phylogenetic tree, which has been shown as a promising and interesting

André L. G. Vieira; César M. Camilo

2011-01-01

450

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...

451

Study on Flax Genetic Transformation Mediated by Agrobacterium tumefaciens  

Microsoft Academic Search

Flax is an important natural material for the linen spinning industry and as an oil crop in China. Flax products worth US $100 million are exported every year. Recently, with the development of a market economy and China's entry into the WTO, farmers and flax factories have shown a need for new varieties that have a short vegetation period, are

Wang Yu Fu; Kang Qing Hua; Liu Yan; Li Xi Chen; Liu Shao Jun; Xu Ying

2004-01-01

452

Agrobacterium tumefaciens -mediated transformation of a medicinal plant Taraxacum platycarpum  

Microsoft Academic Search

Dandelion plants, the genus Taraxacum, are used in herbal medicine owing to their choleretic, diuretic and anti-carcinogenic activities and several medicinal compounds have been isolated from the roots of these plants. Metabolic manipulation of secondary metabolite biosynthesis is a potential strategy to improve the production of high-value secondary metabolites. The enzyme 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) is known to control a key

Tae Woong Bae; Hae Ryoung Park; Youn Sig Kwak; Hyo Yeon Lee; Stephen B. Ryu

2005-01-01

453

Genetic transformation and plant regeneration of watermelon using Agrobacterium tumefaciens  

Microsoft Academic Search

Adventitious shoots formed on the proximal cut edges of different cotyledonary explants of watermelon [Citrullus lanatus (Thunb.) Matsum. & Nakai; cvs. Sweet Gem and Gold Medal] cultured on Murashige and Skoog's (MS) medium with 1 mgl-1 6-benzyladenine (BA). Light (16-h photoperiod, about 7 Wm-2 cool-white fluorescent lamps) was essential for shoot formation. To obtain transformed plants, cotyledonary explants of Sweet

Pil S. Choi; Wong Y. Soh; Youn S. Kim; Ook J. Yoo; Jang R. Liu

1994-01-01

454

Transfer of citrus tristeza virus (CTV)-derived resistance candidate sequences to four grapefruit cultivars through Agrobacterium -mediated genetic transformation  

Microsoft Academic Search

Transgenic plants of grapefruit (Citrus paradisi Macf.) cvs. ‘Duncan’, ‘Flame’, ‘Marsh’, and ‘Ruby Red’ were obtained using Agrobacterium tumefaciens-mediated transformation of seedling epicotyl tissue. Two citrus tristeza virus (CTV)-derived candidate resistance genes:\\u000a ‘392’ (3? region of the p23 ORF plus 3? untranslated region—UTR) and ‘p23 hairpin’ (sense-p23 ORF plus UTR plus antisense-p23\\u000a ORF) were introduced into grapefruit using Agrobacterium strains

G. Ananthakrishnan; V. Orbovi?; G. Pasquali; M. ?alovi?; J. W. Grosser

2007-01-01

455

Mungbean plants expressing BjNPR1 exhibit enhanced resistance against the seedling rot pathogen, Rhizoctonia solani.  

PubMed

Mungbean, Vigna radiata (L.) Wilczek is an important pulse crop that is widely cultivated in semi- arid tropics. The crop is attacked by various soil-borne pathogens like Rhizoctonia solani, which causes dry rot disease and seriously affects its productivity. Earlier we characterized the non-expressor of pathogenesis related gene-1(BjNPR1) of mustard, Brassica juncea, the counterpart of AtNPR1 of Arabidopsis thaliana. Here, we transformed mungbean with BjNPR1 via Agrobacterium tumefaciens. Because of the recalcitrant nature of mungbean, the effect of some factors like Agrobacterium tumefaciens strains (GV2260 and LBA4404), pH, L: -cysteine and tobacco leaf extract was tested in transformation. The transgenic status of 15 plants was confirmed by PCR using primers for nptII. The independent integration of T-DNA in transgenic plants was analyzed by Southern hybridization with an nptII probe and the expression of BjNPR1 was confirmed by RT-PCR. Some of the T(0) plants were selected for detached leaf anti-fungal bioassay using the fungus Rhizoctonia solani, which showed moderate to high level of resistance depending on the level of expression of BjNPR1. The seedling bioassay of transgenic T(2) plants indicated resistance against dry rot disease caused by R. solani. PMID:21584838

Vijayan, S; Kirti, P B

2011-05-17

456

AGROBACTERIUM CONCENTRATIONS AND SEVERITY OF BRONZE WILT SYMPTOMS IN COTTON CULTIVARS TREATED WITH FUNGAL BIOCONTROL AGENTS AT PLANTING  

Technology Transfer Automated Retrieval System (TEKTRAN)

The fungi, Trichoderma virens (Isolates GV4 and GV6), Trichoderma koningii x T. virens fusant #12, Gliocladium catenulatum, Gliocladium roseum, Fusarium oxysporum, and Fusarium solani were trested for their ability to colonize roots, to affect Agrobacterium tumefaciens colonization and bronze wilt s...

457

Towards crop improvement in bell pepper ( Capsicum annuum L.): Transgenics ( uid A:: hpt II) by a tissue-culture-independent Agrobacterium-mediated in planta approach  

Microsoft Academic Search

Agrobacterium tumefaciens-mediated transformation has been one of the methods used to generate transgenic plants in bell pepper. An alternate transformation method that avoids\\/minimizes tissue culture would be beneficial for the improvement of bell pepper due to its recalcitrant nature. In this report, transgenic bell pepper plants have been developed by a tissue-culture-independent A. tumefaciens-mediated in planta transformation procedure. In the

A. Manoj Kumar; Kalpana N. Reddy; Rohini Sreevathsa; Girija Ganeshan; M. Udayakumar

2009-01-01

458

Amenability of castor to an Agrobacterium -mediated in planta transformation strategy using a cry1AcF gene for insect tolerance  

Microsoft Academic Search

Agrobacterium tumefaciens mediated in planta transformation protocol was developed for castor, Ricinus communis. Two-day-old seedlings were infected with Agrobacterium strain EHA105\\/pBinBt8 harboring cry1AcF and established in the greenhouse. Screening the T1 generation seedlings on 300 mg L?1 kanamycin identified the putative transformants. Molecular and expression analysis confirmed the transgenic nature and identified\\u000a high-expressing plants. Western blot analysis confirmed the co-integration

Arthikala Manoj Kumar; Rohini Sreevathsa; Kalpana Nanja Reddy; Prasa Trichy Ganesh; Makarla Udayakumar

2011-01-01