TY - JOUR. T1 - Genetic analysis of agrobacterium tumefaciens unipolar polysaccharide production reveals complex integrated control of the motile-to-sessile switch. AU - Xu, Jing. AU - Kim, Jinwoo. AU - Koestler, Benjamin J.. AU - Choi, Jeong-Hyeon. AU - Waters, Christopher M.. AU - Fuqua, Clay. PY - 2013/9. Y1 - 2013/9. N2 - 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, ...

virB11, one of the 11 genes of the virB operon, is absolutely required for transport of T-DNA from Agrobacterium tumefaciens into plant cells. Previous studies reported that VirB11 is an ATPase with autophosphorylation activity and localizes to the inner membrane even though the protein does not contain the consensus N-terminal export sequence. In this report, we show that VirB11 localizes to the inner membrane even in the absence of other tumor-inducing (Ti) plasmid-encoded proteins. To facilitate the further characterization of VirB11, we purified this protein from the soluble fraction of an Escherichia coli extract by fusing VirB11 to the maltose-binding protein. The maltose-binding protein-VirB11 fusion was able to complement a virB11 deletion mutant of A. tumefaciens for tumor formation and also localized properly to the inner membrane of A. tumefaciens. The 72-kDa protein, purified from E. coli, exhibited no autophosphorylation, ATPase activity, or ATP-binding activity. To study the ...

Several Agrobacterium tumefaciens strains have been isolated for their ability to produce D-amino acids from D, L-substituted hydantoins. The optically pure D-amino acids are used in the synthesis of pharmaceuticals, as food additives and as insecticides. This hydrolysis of D, L-substituted hydantoins is catalysed by two hydantoin-hydrolyzing enzymes, an hydantoinase and an N-carbamyl amino acid amidohydrolase. While the hydantoin-hydrolyzing enzymes have been studied in detail, the mechanisms that control expression of the hyu genes have not. The research reported in this work elucidates some of the mechanisms involved in the regulation of the hyu genes in A. tumefaciens strains.. The hydantoin-hydrolyzing enzyme activity from the environmental isolate A. tumefaciens RU-AE01 was characterized. A broad host range vector for the simultaneous analysis of divergent promoters was constructed. The promoter regions responsible for the activation of transcription of hyuH and hyuC were identified by ...

TY - CHAP. T1 - Agrobacterium tumefaciens-Mediated Transformation. AU - Frandsen, Rasmus John Normand. PY - 2015. Y1 - 2015. N2 - The use of Agrobacterium tumefaciens-mediated transformation for achieving genetic transformation of fungi has steadily increased over the last decade, and has proven to be almost universally applicable technique once suitable selection markers have been developed. In recent years the major technical advances has been made within the initial steps of the process, more specifically the efficient construction of plasmids for performing targeted genome modifications. This chapter provides a generic protocol for performing genetic transformation of ascomycetes via A. tumefaciens-mediated transformation (AMT) and guidelines for optimizing the AMT process with new fungal species. The chapter also includes a highly efficient vector construction system based on Uracil Specific Excisions Reagent (USER) cloning and specific PCR generated building blocks, which can be combined ...

The type VI secretion system (T6SS) is an effector delivery system used by Gram-negative bacteria to kill other bacteria or eukaryotic hosts to gain fitness. The plant pathogen Agrobacterium tumefaciens utilizes its T6SS to kill other bacteria, such as Escherichia coli. We observed that the A. tumefaciens T6SS-dependent killing outcome differs when using different T6SS-lacking, K-12 E. coli strains as a recipient cell. Thus, we hypothesized that the A. tumefaciens T6SS killing outcome not only relies on the T6SS activity of the attacker cells but also depends on the recipient cells. Here, we developed a high-throughput interbacterial competition platform to test the hypothesis by screening for mutants with reduced killing outcomes caused by A. tumefaciens strain C58. Among the 3,909 strains in the E. coli Keio library screened, 16 mutants with less susceptibility to A. tumefaciens C58 T6SS-dependent killing were identified, and four of them were validated by complementation test. Among the four, the

The acc locus from the classic nopaline-type Ti plasmid pTiC58 confers utilization of agrocinopines A+B and susceptibility to an antibiotic called agrocin 84. DNA sequence analyses revealed that acc is composed of eight genes, acc R, and accABCDEFG. accR codes for the repressor which regulates this locus. The predicted proteins from accABCDE are related to each component of ABC-type transport systems, while the predicted products of accF and accG are related to catabolic enzymes. Genetic analyses of insertion mutations showed that accABCDE are required for uptake of both agrocin 84 and agrocinopines, whereas accF and accG are required for utilization of the opines. The accF mutant was not susceptible to agrocin 84 although it took up the antibiotic. This suggests that agrocin 84 must be activated by functions coded for by accF after it is transported into the bacterium. Analysis of lacZ fusions showed that acc is induced in response to agrocinopines and to phosphate starvation. Analyses of ...

Characterization of a putative periplasmic transport system for octopine accumulation encoded by Agrobacterium tumefaciens Ti plasmid pTiA6.

FIG. 1. Immunoprecipitation studies with anti-VirB6, anti-VirB7, and anti-VirB9 antisera. (A) Isolation of VirB protein complexes from detergent-solubilized membrane extracts of wild-type A348. (B) VirB complexes isolated from PC1000(pSJB610). Lanes: αB6, αB7, and αB9, anti-VirB antisera; PI, preimmune serum; PA, protein A Sepharose (these were all used for precipitation); Sol. Prot., solubilized starting material for the precipitations; MW, molecular weight markers, with sizes in kilodaltons shown at left. Blots were probed with antiserum to the VirB proteins listed at the right. The cross-reactive material in the blot developed with anti-VirB10 antiserum is heavy-chain IgG, but native VirB10 (48 kDa) and VirB10′ (40 kDa) derived from translation from an internal Met were clearly distinguished from this background in the immunoblots. The IgG light chain also was immunoreactive and formed a nonspecific background in blots developed with the anti-VirB6, -VirB8, and -VirB9 antisera. ...

Abstract. The broad host range pathogenic bacterium Agrobacterium tumefaciens has been widely studied as a model system to understand horizontal gene flow, secretion of effector proteins into host cells, and plant-pathogen interactions. Agrobacterium-mediated plant transformation also is the major method for generating transgenic plants for research and biotechnology purposes. Agrobacterium species have the natural ability to conduct interkingdom genetic transfer from bacteria to eukaryotes, including most plant species, yeast, fungi, and even animal cells. In nature, A. tumefaciens causes crown gall disease resulting from expression in plants of auxin and cytokinin biosynthesis genes encoded by the transferred (T-) DNA. Gene transfer from A. tumefaciens to host cells requires virulence (vir) genes that reside on the resident tumor-inducing (Ti) plasmid. In addition to T-DNA, several Virulence (Vir) effector proteins are also translocated to host cells through a bacterial type IV secretion ...

Certain plant-associated prokaryotes such as Corynebacteriurn fascians and Agrobacterium tumefaciens are known to produce substantial levels of cytokinins. In view of the fragmentary and presumptive evidence available on the quantitative and qualitative levels of cytokinins produced by A. tumefaciens, it was decided to conduct a study of the cytokinins produced by A. tumefaciens. In order to accomplish this objective, a sequence of methods was developed which allowed the isolation, purification and rigorous chemical identification of cytokinins at the sub-microgram level. The first step of purification utilized the adsorption of cytokinins onto C18 silica by a process known as trace enrichment. Further purification was effected by high performance liquid chromatography and cytokinin-active fractions were located by bioassay. Material in biologically active fractions was permethylated and analyzed by combined capillary gas-liquid chromatography and mass spectrometry. A. tumefaciens tRNA was found ...

As a special phytopathogen, Agrobacterium tumefaciens infects a wide range of plant hosts and causes plant tumors also known as crown galls. The complexity of Agrobacterium-plant interaction has been studied for several decades. Agrobacterium pathogenicity is largely attributed to its evolved capabilities of precise recognition and response to plant-derived chemical signals. Agrobacterium perceives plant-derived signals to activate its virulence genes, which are responsible for transferring and integrating its Transferred DNA (T-DNA) from its Tumor-inducing (Ti) plasmid into the plant nucleus. The expression of T-DNA in plant hosts leads to the production of a large amount of indole-3-acetic acid (IAA), cytokinin (CK), and opines. IAA and CK stimulate plant growth, resulting in tumor formation. Agrobacterium utilizes opines as nutrient sources as well as signals in order to activate its quorum sensing (QS) to further promote virulence and opine metabolism. Intriguingly, Agrobacterium also ...

TY - JOUR. T1 - The Agrobacterium T-DNA transport pore proteins VirB8, VirB9, and Virb10 interact with one another. AU - Das, Anath. AU - Xie, Yong Hong. PY - 2000/2. Y1 - 2000/2. N2 - The VirB proteins of Agrobacterium tumefaciens form a transport pore to transfer DNA from bacteria to plants. The assembly of the transport pore will require interaction among the constituent proteins. The identification of proteins that interact with one another can provide clues to the assembly of the transport pore. We studied interaction among four putative transport pore proteins, VirB7, VirB8, VirB9 and VirB10. Using the yeast two-hybrid assay, we observed that VirB8, VirB9, and VirB10 interact with one another. In vitro studies using protein fusions demonstrated that VirB10 interacts with VirB9 and itself. These results suggest that the outer membrane VirB7-VirB9 complex interacts with the inner membrane proteins VirB8 and VirB10 for the assembly of the transport pore. Fusions that contain small, defined ...

44,203-229. , Hirsch, P. , Hooykaas, P J J , and Schilperoort, R. A (1983) A binary plant vector strategy based on separation of vir- and T-region of the Agrobacterium tumefaciens Tr-plasmrd. Nature 303, 179,180. 8. Stachel, S. E and Nester, E W. (1986) The genetic and transcriptional organization of the vir region of the A6 TI plasmid of Agrobacterium tumefaciens. EMBO J. $1445-1454 9. Stachel, S E and Zambryski, P (1986) virA and virG control the plant-induced activation of the T-DNA transfer process of Agrobacterium tumefaciens. Biol. Rep. 10, 12-36. &APTER 7 Leaf Disk Transformation Ian S. Curtis, Michael R. Davey, and J. Brian Power 1. Introduction Reliable and efficient methods of transferring cloned genes into plants are essential for engineering crops with desired traits. The Gram-negative soil bacteria, Agrobacterium tumefaciens and A. rhizogenes, are natural genetic engineers, capable of transforming a range of dicotyledonous plants by transferring plasmid-encoded genes into recipient ...

Agrobacterium are Gram-negative, motile, soil-dwelling plant pathogens with the species name given based on the disease phenotype associated with the bacteria. They invade the crown, roots and stems of a great variety of plants via wounds causing tumors. The diseases are crown gall, hairy root, and cane gall. Some strains possess a wide host range, whereas other possess a very limited host range. The tumor is correlated with the presence of a large tumor-inducing plasmid (Ti plasmid) in the bacteria. Thus A.tumefaciens causes crown gall on many dicotyledonous plants; A.rubi causes crown gall on raspberries; A.vitis gall formation on grapes; A.rhizogenes causes hairy roots; A.radiobacter is avirulent. However the ability to cause disease is associated with transmissible plasmids, and this grouping is easily disrupted when plasmids move from one strain to another. More recently Agrobacterium have been classified into 3 biovars based on physiological and biochemical phenotypes without consideration ...

Transformed Arabidopsis thaliana plants have been produced by a modified leaf disk transformation-regeneration method. Leaf pieces from sterilely grown plants were precultured for 2 days and inoculated with an Agrobacterium tumefaciens strain containing an avirulent Ti (tumor-inducing) plasmid with a chimeric gene encoding hygromycin resistance. After cocultivation for 2 days, the leaf pieces were placed on a medium that selects for hygromycin resistance. Shoots regenerated within 3 months and were excised, rooted, and transferred to soil. Transformation was confirmed by opine production, hygromycin resistance, and DNA blot hybridization of both primary transformants and progeny. This process for producing transgenic Arabidopsis plants should enhance the usefulness of the species for experimental biology. |P /|

Plant material and transformation: Plants were grown in peat soil with 18 hr light and 6 hr dark. DNA constructs were transformed into Agrobacterium tumefaciens strain C58 and then transformed into Arabidopsis plants by the floral dip method (Clough and Bent 1998).. Plasmid construction: Tag1 promoter-GUS expression plasmids were constructed as follows. The pTPG2 and pTPG3 plasmids were generated by PCR amplifying 1- to 262-bp and 1- to 548-bp fragments, respectively, from the 5′ end of Tag1 into the XbaI and BamHI restriction sites of the pBI101.3 GUS expression vector (accession no. U12640). pTPG4 was made by ligating a XbaI-ScaI Tag1 fragment (1-1251 bp of Tag1) from the pBT1 plasmid (Liuet al. 2001a), which contains Tag1 in the BlueScript vector (Stratagene, La Jolla, CA), into the XbaI-SmaI sites of pBI101.3 to generate an in-frame fusion between the Tag1 transposase and GUS. pTPG5 was made by ligating a XbaI-NdeI 5′ Tag1 fragment (1-1950 bp of Tag1 in pBT1) into the XbaI and BamHI ...

Nesme, X., Leclerc, M.C. & Bardin, R. 1989 PCR detection of an original endosymbiont: the Ti plasmid of Agrobacterium tumefaciens In. Endocytobiology IV, eds.Nardon, P., Gianinazzi- Peason, V., Greines, A.M., Margulis, L. & Smith, D.C, pp. 47-50. Paris: Institute National de Recherche Agronomique ...

Author: Willmitzer, L.; Genre: Meeting Abstract; Published in Print: 1984; Title: The Ti Plasmid of Agrobacterium Tumefaciens - a Plant-Oriented Gene Vector

The present invention allows large glass substrates to be rapidly moved from one processing station to another. Such movement occurs such that drives in different chambers are synchronized to move the glass substrates on shuttles at appropriate times. In systems according to the invention, at least a first and second chamber are provided. Typically, the first chamber is a load lock and the second chamber is a processing chamber. A substrate transfer shuttle is used to move substrate along a guide path defined by, e.g., guide rollers. Drive mechanisms are employed for most chambers to drive the shuttle along associated portions of the path. A control system is provided which powers the drive mechanism for the first chamber to drive the substrate transfer shuttle from a first position toward a second position and through an intermediate position. At the intermediate position, the substrate transfer shuttle begins to engage and induce movement of the drive mechanism for the second chamber. The control

Abstract: Agrobacterium tumefaciens infects various plants and causes crown gall diseases involving temporal expression of virulence factors. SghA is a newly-identified virulence factor enzymatically releasing salicylic acid from its glucoside conjugate and controlling the plant tumor development. Here we report the structural basis of SghR, a LacI-type transcription factor and highly conserved in Rhizobiaceae family, regulating the expression of SghA and involved in tumorigenesis. We identified and characterized the binding site of SghR on the promoter region of sghA, and then determined the crystal structures of apo-SghR, SghR complexed with its operator DNA and ligand sucrose, respectively. These results provide detailed insights into how SghR recognizes its cognate DNA and shed a mechanistic light on how sucrose attenuates the affinity of SghR with DNA to modulate the expression of SghA. Given the important role of SghR in mediating the signaling crosstalk during Agrobacterium infection, our ...

A series of vectors has been constructed for the purpose of introducing cloned DNAs into plant genomes, using Agrobacterium tumefaciens-mediated transformation methods. One of these vectors, pCIT20, is a plasmid that contains a multiple cloning site (MCS), and a marker (Hph) that confers hygromycin resistance to plant cells. The others are all cosmid vectors which allow insertion of up to 46 kb of plant genomic DNA, and which also contain all of the necessary sequences for A. tumefaciens-mediated plant transformation. The cosmid vectors either contain a Hph marker (pCIT03), or a kanamycin-resistance marker (pCIT101-104). Three of the cosmid vectors (pCIT30, pCIT101, and pCIT103) carry bacteriophage T7 and SP6 promoters flanking the cloning Bg/II site, for synthesis of end-specific RNAs. The end-specific RNAs may be used as probes when labeled with radioactive or biotinylated nucleotides, for example, in a chromosome-walking experiment. The other two cosmid vectors (pCIT102 and pCIT104) carry ...

Agrobacterium are Gram-negative, motile, soil-dwelling plant pathogens with the species name given based on the disease phenotype associated with the bacteria. They invade the crown, roots and stems of a great variety of plants via wounds causing tumors. The diseases are crown gall, hairy root, and cane gall. Some strains possess a wide host range, whereas other possess a very limited host range. The tumor is correlated with the presence of a large tumor-inducing plasmid (Ti plasmid) in the bacteria. Thus A.tumefaciens causes crown gall on many dicotyledonous plants; A.rubi causes crown gall on raspberries; A.vitis gall formation on grapes; A.rhizogenes causes hairy roots; A.radiobacter is avirulent. However the ability to cause disease is associated with transmissible plasmids, and this grouping is easily disrupted when plasmids move from one strain to another. More recently Agrobacterium have been classified into 3 biovars based on physiological and biochemical phenotypes without consideration ...

Agrobacterium are Gram-negative, motile, soil-dwelling plant pathogens with the species name given based on the disease phenotype associated with the bacteria. They invade the crown, roots and stems of a great variety of plants via wounds causing tumors. The diseases are crown gall, hairy root, and cane gall. Some strains possess a wide host range, whereas other possess a very limited host range. The tumor is correlated with the presence of a large tumor-inducing plasmid (Ti plasmid) in the bacteria. Thus A.tumefaciens causes crown gall on many dicotyledonous plants; A.rubi causes crown gall on raspberries; A.vitis gall formation on grapes; A.rhizogenes causes hairy roots; A.radiobacter is avirulent. However the ability to cause disease is associated with transmissible plasmids, and this grouping is easily disrupted when plasmids move from one strain to another. More recently Agrobacterium have been classified into 3 biovars based on physiological and biochemical phenotypes without consideration ...

Monomethylamine can be used by nonmethylotrophs as a sole nitrogen source but not as a carbon source; however, little is known about the genes and enzymes involved. The gamma-glutamylmethylamide/N-methylglutamate pathway for monomethylamine utilization by methylotrophs has recently been resolved. We have identified genes encoding key enzymes of this pathway in nonmethylotrophs (e. g., Agrobacterium tumefaciens) and demonstrated that this pathway is also involved in the utilization of monomethylamine as a nitrogen source by nonmethylotrophs.. ...

Many bacteria, including Agrobacterium tumefaciens, rely on an asymmetric localization, distribution, and orientation of specific appendages when interacting wi...

Previous work on identifying the molecular mechanisms mediating plant-pathogen interactions and reciprocal host responses have little emphasis on developing models that closely resemble host-microbe interaction in planta. This work establishes an amalgamated model of interaction wherein successful pathogens elicit and overcome host defenses activated by microbial signatures and virulence factors. Using a hydroponic co-cultivation model, we assessed the responses of Arabidopsis thaliana Col-0 to Agrobacterium tumefaciens C58 to ameliorate limitations of previous approaches. Comparisons of differential gene expression between directly and indirectly affected host sites by microarray analysis revealed both reactive and pro-active defense responses, respectively. Selected homozygous single-gene knockouts for proactive defenses show variable A. tumefaciens root surface attachment and root secretion profiles. Studying host-microbe responses using hydroponics may improve priming of cash crops against pathogens

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The novel genetic material in the new canola lines was inserted into the canola variety, Westar, using Agrobacterium tumefaciens-mediated transformation with the disarmed Ti-plasmid pMOG625. The T-DNA contained both phytase and nptII genes. The phytase gene is under the control of the cruciferin A seed storage protein transcript promoter which includes a cruciferin signal peptide sequence. Its terminator is also from the cruciferin A seed storage protein transcript. Both controlling sequences were obtained from Brassica napus. The nptII gene is under the control of the NOS promoter and terminator with an Agrobacterium tumefaciens-derived open reading frame inserted between the gene and its terminator. The open reading frame consists of coding for 50 amino acids from the Agrobacterium ornithine-cyclo-deaminase ...

Wood DW, Setubal JC, Kaul R, Monks DE, Kitajima JP, Okura VK, Zhou Y, Chen L, Wood GE, Almeida Jr NF, Woo L, Chen Y, Paulsen IT, Eisen JA, Karp PD, Bovee Sr D, Chapman P, Clendenning J, Deatherage G, Gillet W, Grant C, Kutyavin T, Levy R, Li M-J, McClelland R, Palmieri A, Raymond C, Rouse G, Saenphimmachak C, Wu Z, Romero P, Gordon D, Zhang S, Yoo H, Tao Y, Biddle P, Jung M, Krespan W, Perry M, Gordon-Kamm B, Liao L, Kim S, Hendrick C, Zhao Z-Y, Dolan M, Chumley F, Tingey SC, Tomb J-F, Gordon MP, Olson MV, Nester EW. 2001. The genome of the natural genetic engineer Agrobacterium tumefaciens C58. Science 294: 2317-2323. PMID: 11743193. PDF. ...

Wood DW, Setubal JC, Kaul R, Monks DE, Kitajima JP, Okura VK, Zhou Y, Chen L, Wood GE, Almeida Jr NF, Woo L, Chen Y, Paulsen IT, Eisen JA, Karp PD, Bovee Sr D, Chapman P, Clendenning J, Deatherage G, Gillet W, Grant C, Kutyavin T, Levy R, Li M-J, McClelland R, Palmieri A, Raymond C, Rouse G, Saenphimmachak C, Wu Z, Romero P, Gordon D, Zhang S, Yoo H, Tao Y, Biddle P, Jung M, Krespan W, Perry M, Gordon-Kamm B, Liao L, Kim S, Hendrick C, Zhao Z-Y, Dolan M, Chumley F, Tingey SC, Tomb J-F, Gordon MP, Olson MV, Nester EW. 2001. The genome of the natural genetic engineer Agrobacterium tumefaciens C58. Science 294: 2317-2323. PMID: 11743193. PDF. ...

Wood DW, Setubal JC, Kaul R, Monks DE, Kitajima JP, Okura VK, Zhou Y, Chen L, Wood GE, Almeida Jr NF, Woo L, Chen Y, Paulsen IT, Eisen JA, Karp PD, Bovee Sr D, Chapman P, Clendenning J, Deatherage G, Gillet W, Grant C, Kutyavin T, Levy R, Li M-J, McClelland R, Palmieri A, Raymond C, Rouse G, Saenphimmachak C, Wu Z, Romero P, Gordon D, Zhang S, Yoo H, Tao Y, Biddle P, Jung M, Krespan W, Perry M, Gordon-Kamm B, Liao L, Kim S, Hendrick C, Zhao Z-Y, Dolan M, Chumley F, Tingey SC, Tomb J-F, Gordon MP, Olson MV, Nester EW. 2001. The genome of the natural genetic engineer Agrobacterium tumefaciens C58. Science 294: 2317-2323. PMID: 11743193. PDF. ...

Background: Agrobacterium tumefaciens strain GV3101 (pMP90) is widely used in transient gene expression assays, including assays to study pathogen effectors and plant disease resistance mechanisms. However, inoculation of A. tumefaciens GV3101 into Nicotiana tabacum (tobacco) leaves prior to infiltration with pathogenic and non-host strains of Pseudomonas syringae results in suppression of macroscopic symptoms when compared with leaves pre-treated with a buffer control. Methodology/Findings: To gain further insight into the mechanistic basis of symptom suppression by A. tumefaciens we examined the effect of pre-treatment with A. tumefaciens on the growth of P. syringae, the production of the plant signalling molecules salicylic acid (SA) and abscisic acid (ABA), and the presence of callose deposits. Pre-treatment with A. tumefaciens reduced ABA levels, P. syringae multiplication and P. syringae-elicited SA and ABA production, but promoted increased callose deposition. However, pre-treatment with A.

A forward genetics approach was applied in order to investigate the molecular basis of morphological transition in the wheat pathogenic fungus Zymoseptoria tritici. Z. tritici is a dimorphic plant pathogen displaying environmentally regulated morphogenetic transition between yeast-like and hyphal growth. Considering the infection mode of Z. tritici, the switching to hyphal growth is essential for pathogenicity allowing the fungus the host invasion through natural openings like stomata. We exploited a previously developed Agrobacterium tumefaciens-mediated transformation (ATMT) to generate a mutant library by insertional mutagenesis including more than 10,000 random mutants. To identify genes involved in dimorphic switch, a plate-based screening system was established. With this approach eleven dimorphic switch deficient random mutants were recovered, ten of which exhibited a yeast-like mode of growth and one mutant predominantly growing filamentously, producing high amount of mycelium under different

Although the Agrobacterium tumefaciens-mediated transformation efficiency was only a fraction of 1%, it was possible to exploit the transposition frequency of a single T0 line to initiate the development of a functional resource for activation tagging in tomato. The practice of using micropropagation to produce many clonal plants from a single tissue culture regenerant proved valuable, as it multiplied T1 seed production by up to 25 times. This strategy also capitalized on the behavior of transposase in Ac/Ds-ATag-Bar_gosGFP by isolating chimeric tissue from the original transformant into separate plantlets, allowing germinal transposition from multiple sites of Ds integration. The selection of a self-fertile, true breeding tomato cultivar allowed crossing to nontransgenic cv M82, thus maximizing T1 seed production. Pollen could be collected from transgenic flowers and distributed to multiple nontransgenic plants, all while still obtaining transgenic self-progeny.. Modifications made to the ...

OsARF1 is the first full-length member of auxin response factor (ARF) gene family to be cloned from monocot plant. Using quantitative RT-PCR this study found that, the transcript abundance of OsARF1 was significantly higher in embryonic tissues than in vegetative tissues. To investigate the effect of OsARF1 on the phenotype of rice, a cDNA fragment of OsARF1 was inserted in inverse orientation to the 35S promoter in vector pBin438 to produce an antisense (AS) construction. The AS-OsARF1 construct was transferred into rice (Oryza sativa L. japonica) calli via Agrobacterium tumefaciens-mediated transformation. Molecular analysis of transgenic plants showed that the functional expression of OsARF1 was inhibited at mRNA level efficiently. The AS-OsARF1 plants showed extremely low growth, poor vigor, short curled leaves and tillered but were sterile. Therefore, the OsARF1 was shown to be essential for growth in vegetative organs and seed development.. ...

The major seed storage protein phaseolin of common bean (Phaseolus vulgaris L.) is deficient in methionine, an essential amino acid for human and animal health. To improve the nutritional quality of common bean, we designed methionine enhancement of phaseolin based on the three-dimensional structure of protein, de novo design principles and genetic information. Amino acid substitution and loop insertion were targeted to the interior and exterior, respectively, of the proteins β-barrels. First, we introduced the methionine enhancement mutations into phaseolin cDNA, expressed cDNA in Escherichia coli and purified monomeric non-glycosylated proteins. Biophysical analysis of E. coli-expressed proteins demonstrated a similar structural stability of wild-type and mutant phaseolin monomers. Here, we attempted to test the structural stability of the methionine-enhanced phaseolin by introducing phaseolin cDNA to tobacco via Agrobacterium tumefaciens-mediated transformation of leaf disks, regenerating

Hydrophobins are small, cysteine-rich, secreted proteins, ubiquitously produced by filamentous fungi, and that are speculated to function in fungal growth, cell surface properties, and development, although this has been rigorously tested for only a few species. We identified three hydrophobin genes from the entomopathogenic fungus, Metarhizium brunneum and provided functional characterization of strains lacking these genes. One gene (HYD1/ssgA) encodes a Class I hydrophobin identified previously. Two new genes, HYD3 and HYD2, encode a Class-I and Class-II hydrophobin, respectively. To examine function, we deleted all three, separately, from the M. brunneum strain KTU-60 genome using Agrobacterium tumefaciens-mediated transformation. Deletion strains were screened for alterations in developmental phenotypes including growth, sporulation, pigmentation, colony surface properties, and virulence to insects. All deletion strains were reduced in their ability to sporulate and showed alterations in ...

The transfer DNA (abbreviated T-DNA) is the transferred DNA of the tumor-inducing (Ti) plasmid of some species of bacteria such as Agrobacterium tumefaciens and Agrobacterium rhizogenes. The T-DNA is transferred from bacterium into the host plants nuclear DNA genome. The capability of this specialized tumor-inducing (Ti) plasmid is attributed to two essential regions required for DNA transfer to the host cell. As the T-DNA is bordered by 25-base-pair repeats on each end. Transfer is initiated at the right border and terminated at the left border and requires the vir genes of the Ti plasmid. The bacterial T-DNA is about 24,000 base pairs long and contains genes that code for enzymes synthesizing opines and phytohormones. By transferring the T-DNA into the plant genome, the bacterium essentially reprograms the plant cells to grow into a tumor and produce a unique food source for the bacteria. The synthesis of the plant hormones auxin and cytokinin by enzymes encoded in the T-DNA enables the plant ...

Indeed, taking a gene from a soil bacterium (Bacillus thuringiensis - Bt) that produces a natural pesticide and injecting that gene into the DNA of a soy plant, is hardly Mother Natures way of hybridising plants. Ummmm...FAIL! Sorry, that is EXACTLY how Mother Gaia engineers plants, via Agrobacterium tumefaciens: that bug causes crown gall by inserting DNA into the host plant cells that provides a nice little niche environment for the bacterium to grow.. In fact, if people actually understood just how much other DNA finds its way into plant genomes, they would lose all fear of GMOs. Or, alternatively, not eat anything ever again - which wwould be a fitting Darwinian punishment for stupidity.. Seriously: just the tobacco (Nicotiana tabacum) genome, for example, contains genes or sequences derived from Agrobacterium, from plant-infecting single-stranded DNA geminiviruses, from single-stranded RNA potyviruses, from insects that feed on plants, and from other bacteria and probably from fungi. ...

The plasmid vector pGreenII, which is widely used in the production of stable plant transformants, is shown herein to predispose constructs to the acquisition of mutations (Figure 1) despite its earlier revision (Hellens and Mullineaux 2000). This predisposition arises from pGreenII having an adverse effect on the growth of E. coli. It perturbs normal cell division resulting in the production of long filaments (Figure 1), a phenomenon associated with stressed cells (Justice et al. 2008), and causes a dramatic reduction in cell viability following overnight incubation (Table 1). This is far from ideal as the insertion of DNA into plasmids can itself affect the growth of E. coli through increased metabolic burden and the acquisition of activities that perturb cellular functions (Bentley et al. 2009; Rosano and Ceccarelli 2014). In our case, the insertion of a 4605 bp fragment containing the cDNA of the plant DNA methyltransferase 1 (MET1) into pGreenII generated sufficient selective pressure for ...

Figure 1. Schematic representation of the process of A. tumefaciens-mediated transformation. Phenolic and sugar molecules from wounded plant cells trigger in the bacterium a series of events resulting in the processing of the T-DNA by the VirD1/VirD2 endonuclease and the subsequent transfer of the VirD2/T-strand complex, along with VirE2 and VirF proteins, from the bacterium through the VirB/VirD4 type IV secretion system. Key events in the plant cell include bacterial attachment, T-complex and Vir protein transfer, cytoplasmic trafficking of the T-complex, nuclear targeting, and T-DNA integration. Some of the plant genes necessary for these processes are depicted by representative rat mutants.. ...

A frictionless transport apparatus for transporting an object from a first to a second station is disclosed. The apparatus has a frame extending between the first and second stations and a carriage mounted for movement along said frame, in a levitated condition. A substantial portion of the weight of the carriage and load is supported by biasing magnets on the frame and carriage. The carriage and load are stabilized in a levitated condition by magnetic interactions between a diamagnetic plate on the carriage and a magnetic array extending along the frame. The carriage is moved in its levitated condition by a frictionless drive system, such as an electromagnetic, electrostatic, or pneumatic drive system. Also disclosed is a method of frictionless transport of an object between first and second stations.

As a microbiologist and cell biologist, Dr. Heindls research focuses on the prokaryotic development and bacterial interactions within the environment. He uses the plant pathogen Agrobacterium tumefaciensand related bacteria to understand regulatory principles and molecular mechanisms governing bacterial developmental stages from growth and remodeling, to the morphogenesis, division, and biofilm formation. His work has been published in several renowned scientific journals, such as Journal of Bacteriology, Infection and Immunity, and PLOS ONE-a peer-reviewed open access scientific journal published by the Public Library of Science. In his new role, Dr. Heindls responsibilities include teaching a variety of undergraduate microbiology courses and developing new courses for the program. He is also charged with establishing an active research program with undergraduate and graduate students in the Department of Biological Sciences. Dr. Heindl, a native of Indian Harbour Beach, Florida, received his ...

Agrobacterium vitis and some strains of A. tumefaciens and A. rhizogenes cause a dangerous chronic disease on grape plants-crown gall. Molecular-genetic methods were used in the work to identify and study the diversity of pathogenic agrobacteria that cause crown gall in the vineyards of Krasnodar krai. The research material was 76 samples of young and lignified tumor-like outgrowths of crown gall ...

T4SSs each possess an envelope-spanning channel composed of conserved components termed the core complex. Early biochemical studies showed that VirB7, VirB9 and VirB10 form a transporter subassembly that is both intrinsically stable and stabilizing for other VirB subunits. This core complex from the conjugative pKM101 system provided the first high-resolution images [26]. The core secretion channel is a multimeric VirB7-VirB9-VirB10 complex containing 14 copies of each protein. A cylindrical structure spanning the entire cell envelope is composed of two layers (designated I and O). Each layer forms a double-walled ring-like structure that defines hollow chambers inside the complex (figure 3b). The structure surrounds a central chamber of about 80 Å at its widest point. The N-terminal domains (NTDs) of VirB9 and VirB10 comprise the I layer and this part of the channel is anchored in the IM by an N-terminal transmembrane helix of VirB10. An opening at the base of the I layer spans 55 Å. The O ...

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During infection of plants by Agrobacterium tumefaciens, plants are wounded and then a tumor is induced, which is a source of opines, chemicals that stimulate the production of the quorum-sensing (QS) signal N-(3-oxooctanoyl)homoserine lactone (OC8-HSL). γ-aminobutyric acid (GABA) is produced by plants as part of the response to wounding. Chevrot et al. show that GABA stimulates expression of the attKLM operon in A. tumefaciens, which produces a lactonase that inactivates OC8-HSL. OC8-HSL was undetectable in cultures of A. tumefaciens exposed to GABA. Proteins encoded by the attKLM operon were identified in a proteomic screen for proteins synthesized in response to the addition of GABA to cultures of A. tumefaciens. The induction of the attKLM operon was also monitored using a reporter assay, and in A. tumefaciens deficient for the GABA transporter system, GABA did not induce the reporter. The importance of GABA for the plant response was verified using a transgenic tobacco that expressed a ...

During infection of plants by Agrobacterium tumefaciens, plants are wounded and then a tumor is induced, which becomes a source of opines: chemicals that stimulate the production of the quorum-sensing (QS) signal N-(3-oxooctanoyl) homoserine lactone (OC8-HSL). γ-aminobutyric acid (GABA) is produced by plants as part of the response to wounding. Chevrot et al. show that GABA stimulates expression of the attKLM operon in A. tumefaciens, which produces a lactonase that opens the ring and inactivates OC8-HSL. Consequently, OC8-HSL was undetectable in cultures of A. tumefaciens exposed to GABA. Proteins encoded by the attKLM operon were identified in a screen for proteins synthesized in response to the addition of GABA to cultures of A. tumefaciens. The induction of the attKLM operon was also monitored using a reporter assay, and in A. tumefaciens deficient for the GABA transporter system, GABA did not induce the reporter. The importance of GABA for the plant response was verified using transgenic ...

An efficient variety-independent method for producing transgenic eggplant (Solanum melongena L.) via Agrobacterium tumefaciens-mediated genetic transformation was developed. Root explants were transformed by co-cultivation with Agrobacterium tumefaciens strain LBA4404 harbouring a binary vector pBAL2 carrying the reporter gene \beta-glucuronidase intron (GUS-INT) and the marker gene neomycin phosphotransferase (NPTII). Transgenic calli were induced in media containing 0.1 mg$ l-^{1}$ thidiazuron (TDZ), 3.0 mg $l-^{1} N^{6}$-benzylaminopurine, 100 mg$ l-^{1}$ kanamycin and 500 mg l? cefotaxime. The putative transgenic shoot buds elongated on basal selection medium and rooted efficiently on Soilrite irrigated with water containing 100 mg$ l-^{1} $kanamycin sulphate. Transgenic plants were raised in pots and seeds subsequently collected from mature fruits. Histochemical GUS assay and polymerase vchain reaction analysis of field-established transgenic plants and their offsprings confirmed the ...

Rhizobium, Agrobacterium and Allorhizobium are genera within the bacterial family Rhizobiaceae, together with Sinorhizobium. The species of Agrobacterium, Agrobacterium tumefaciens (syn. Agrobacterium radiobacter), Agrobacterium rhizogenes, Agrobacterium rubi and Agrobacterium vitis, together with Allorhizobium undicola, form a monophyletic group with all Rhizobium species, based on comparative 16S rDNA analyses. Agrobacterium is an artificial genus comprising plant-pathogenic species. The monophyletic nature of Agrobacterium, Allorhizobium and Rhizobium and their common phenotypic generic circumscription support their amalgamation into a single genus, Rhizobium. Agrobacterium tumefaciens was conserved as the type species of Agrobacterium, but the epithet radiobacter would take precedence as Rhizobium radiobacter in the revised genus. The proposed new combinations are Rhizobium radiobacter, Rhizobium rhizogenes, Rhizobium rubi, Rhizobium undicola and Rhizobium vitis.

Other names: A. rhizogenes, ATCC 11325, Agrobacterium biovar 2, Agrobacterium genomic group 10, Agrobacterium genomic species 10, Agrobacterium genomosp. 10, Agrobacterium rhizogenes, Agrobacterium rhizogenes (RI plasmid PRI1724), Agrobacterium rhizogenes (RI plasmid PRI8196), Agrobacterium rhizogenes (RI plasmid PRIA4B), CFBP 5520, CIP 104328, DSM 30148, ICMP 5794, IFO 13257, JCM 20919, LMG 150, NBRC 13257, NCPPB 2991, Rhizobium rhizogenes, Rhizobium sp. LMG 9509 ...

Other names: A. rhizogenes, ATCC 11325, Agrobacterium biovar 2, Agrobacterium genomic group 10, Agrobacterium genomic species 10, Agrobacterium genomosp. 10, Agrobacterium rhizogenes, Agrobacterium rhizogenes (RI plasmid PRI1724), Agrobacterium rhizogenes (RI plasmid PRI8196), Agrobacterium rhizogenes (RI plasmid PRIA4B), CFBP 5520, CIP 104328, DSM 30148, ICMP 5794, IFO 13257, JCM 20919, LMG 150, NBRC 13257, NCPPB 2991, Rhizobium rhizogenes, Rhizobium sp. LMG 9509 ...

The main purpose of this study was to develop a non-tissue culture based Agrobacterium mediated transformation method for chickpea. The influences of several factors were investigated on the transfer of -glucuronidase (GUS) gene into chickpea (Cicer arietinum) seedlings during the early stages of Agrobacterium-mediated gene transfer, including cocultivation period in liquid induction medium (2, 8, 16 and 24 h), strains of Agrobacterium tumefaciens (C58C1, EHA105, KYRT1) containing the plasmid pTJK136, developmental stage (16 h imbibed and 40 h germinated), microwounding, vacuum infiltration (200, 400, 600 mmHg for 20 and 40 min) and genotype (5 different). The number of GUS-expressing foci was counted to evaluate the gene transfer process. The KYRT1/pTJK136 strain of A. tumefaciens was significantly more effective for transformation than the C58C1/pTJK136 and EHA105/pTJK136 strains. The highest transient GUS activity was obtained from 16 h imbibed seedlings of cv.Uzunlu wounded with a needle and ...

Transgenic plum plants expressing the papaya ringspot virus (PRV) coat protein (CP) were produced by Agrobacterium-mediated transformation of hypocotyl slices. Hypocotyl slices were cocultivated with Agrobacterium tumefaciens strain C58/Z707 containing the plasmid pGA482GG/CPPRV-4. This plasmid carries the PRVCP gene construct and chimeric NPTII and GUS genes. Shoots were regenerated on Murashige and Skoog salts, vitamins, 2% sucrose, 2.5 μm indolebutyric acid, 7.5 μm thidiazuron, and appropriate antibiotics for selection. Integration of the foreign genes was verified through kanamycin resistance, GUS assays, polymerase chain reaction (PCR), and Southern blot analyses. Four transgenic clones were identified. Three were vegetatively propagated and graft-inoculated with plum pox virus (PPV)-infected budwood in a quarantine, containment greenhouse. PPV infection was evaluated over a 2- to 4-year period through visual symptoms, enzyme-linked immunosorbent assay, and reverse transcriptase PCR ...

Octopine and nopaline strains of Agrobacterium tumefaciens differ in their ability to induce tumors on Nicotiana glauca. The presence of a virF locus on the octopine Ti plasmid makes N. glauca a host plant for these strains, indicating that the VirF protein is a host-range determinant. Here we show the presence of a virF locus not only on the Agrobacterium vitis octopine/cucumopine plasmids pTiAg57 and pTiTm4, but also on the nopaline Ti plasmids pTiAT1, pTiAT66a, and pTiAT66b. On the octopine Ti plasmids from A. tumefaciens the virF gene is located between the virE locus and the left border of the T-region. In contrast, the virF gene on Ti plasmids of A. vitis is located at the very left end of the vir-region near the virA locus. The virF gene of pTiAg57 has been sequenced and codes for a protein of 202 amino acids with a molecular mass of 22,280 Da. Comparison showed that the virF gene from A. vitis strain Ag57 is almost identical to that from A. tumefaciens octopine strains. The transcription of the

We have achieved efficient transformation system for forage-type tall fescue plants by Agrobacterium tumefaciens. Mature seed-derived embryogenic calli were infected and co-cultivated with each of three A. tumefaciens strains, all of which harbored a standard binary vector pIG121Hm encoding the neomycin phosphotransferase II (NPTII), hygromycin phosphotransferase (HPT) and intron-containing |TEX|$\beta$|/TEX|-glucuronidase (intron-GUS) genes in the T-DNA region. Transformation efficiency was influenced by the A. tumefaciens strain, addition of the phenolic compound acetosyringone and duration of vacuum treatment. Of the three A. tumefaciens strains tested, EHA101/pIG121Hm was found to be most effective followed by GV3101/pIG121Hm and LBA4404/pIG121Hm for transient GUS expression after 3 days co-cultivation. Inclusion of 100 |TEX|$\mu$|/TEX|M acetosyringone in both the inoculation and co-cultivation media lead to an improvement in transient GUS expression observed in targeted calli. Vacuum treatment

Background Agrobacterium tumefaciens has long been known to transform plant tissue in nature as part of its infection process. This natural mechanism has been utilised over the last few decades in laboratories world wide to genetically manipulate many species of plants. More recently this technology has been successfully applied to non-plant organisms in the laboratory, including fungi, where the plant wound hormone acetosyringone, an inducer of transformation, is supplied exogenously. In the natural environment it is possible that Agrobacterium and fungi may encounter each other at plant wound sites, where acetosyringone would be present, raising the possibility of natural gene transfer from bacterium to fungus. Methodology/Principal Findings We investigate this hypothesis through the development of experiments designed to replicate such a situation at a plant wound site. A. tumefaciens harbouring the plasmid pCAMDsRed was co-cultivated with the common plant pathogenic fungus Verticillium albo-atrum

FINAL DIAGNOSIS: AGROBACTERIUM RADIOBACTER. CONTRIBUTORS NOTE:. The genus Agrobacterium are aerobic, gram negative, peritrichous bacilli which are mainly plant pathogens found in soil all over the world. Agrobacterium tumefaciens and Agrobacterium radiobacter are two species that have been isolated from human specimens although only A. radiobacter is associated with clinical symptoms (1).. Despite A. radiobacters association with significant clinical symptoms, it is considered to be of low virulence and there have been no reports of mortality from the organism alone. The only difference between the two species is the presence of a tumor-producing plasmid, the Ti plasmid, in A. tumefaciens which results in a tumorous growth in plants (2).. Agrobacterium radiobacter is infrequently recognized in clinical specimens however it has been associated with immunocompromised patients that have implanted medical devices or transcutaneous catheters (1,3,4). It was first isolated in 1967 but was not ...

The plant architecture has significant effects on grain yield of various crops, including soybean (Glycine max), but the knowledge on optimization of plant architecture in order to increase yield potential is still limited. Recently, CRISPR/Cas9 system has revolutionized genome editing, and has been widely utilized to edit the genomes of a diverse range of crop plants. In the present study, we employed the CRISPR/Cas9 system to mutate four genes encoding SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factors of the SPL9 family in soybean. These four GmSPL9 genes are negatively regulated by GmmiR156b, a target for the improvement of soybean plant architecture and yields. The soybean Williams 82 was transformed with the binary CRISPR/Cas9 plasmid, assembled with four sgRNA expression cassettes driven by the Arabidopsis thaliana U3 or U6 promoter, targeting different sites of these four SPL9 genes via Agrobacterium tumefaciens-mediated transformation. A 1-bp deletion was detected in one target

Vitis aestivalis Norton grapevines are more resistant to disease and stress, but are less productive than Vitis vinifera cultivars. Identification and characterization of V. aestivalis Norton defense genes VaERF3a, VaERF3b, VaERF4, VaERF5, VaMAPKK, VaWRKY4, and VaWRKY30 can unveil mechanisms for resistance and tolerance. Amino acid analyses indicated that VaERF3a, VaERF3b, VaERF4 and VaERF5 contain an AP2 DNA binding domain, VaMAPKK has a serine/threonine kinase domain, and VaWRKY4 and VaWRKY30 include a WRKY domain. Nucleotide and amino acid sequence analysis suggested that VaERF3a and VaERF3b may be paralogous, while VaWRKY4 and VaWRKY30 are two different genes. V vinifera homologues of each gene were also found in the newly sequenced whole genone of PN40024. V. aestivalis Norton defense genes were transformed into Nicotiana tabacum via an Agrobacterium tumefaciens-mediated transformation procedure. Viable shoots were not generated from the VaWRKY4 and VaWRKY30 transformations. RNA blots were

Agrobacterium species that are pathogenic on plants, including Agrobacterium tumefaciens, A. vitis, A. rubi, and A. rhizogenes, all carry megaplasmids. By contrast, nonpathogenic strains either lack these plasmids entirely or carry mutant forms of plasmids. A strict requirement of the Ti plasmid for virulence was established through mutational analyses and by a demonstration that the introduction of Ti plasmids into Rhizobium or Phyllobacterium spp. converts these nonpathogenic species into tumor-inducing pathogens ( 2 , 3 ). Ti plasmids induce a disease called crown gall, which is typified by the formation of undifferentiated plant tumors at the plant crown (the subterranean-to-aerial transition zone). The related root-inducing or Ri megaplasmids carried by A. rhizogenes instead induce hairy root disease, which is typified by the formation of entangled masses of roots at the infection site ( 4 ).

Agrobacterium infection, which is widely used to generate transgenic plants, is often accompanied by T-DNA-linked mutations and transpositions in flowering plants. It is not known if Agrobacterium infection also affects the rates of point mutations, somatic homologous recombinations (SHR) and frame-shift mutations (FSM). We examined the effects of Agrobacterium infection on five types of somatic mutations using a set of mutation detector lines of Arabidopsis thaliana. To verify the effect of secreted factors, we exposed the plants to different Agrobacterium strains, including wild type (Ach5), its derivatives lacking vir genes, oncogenes or T-DNA, and the heat-killed form for 48 h post-infection; also, for a smaller set of strains, we examined the rates of three types of mutations at multiple time-points. The mutation detector lines carried a non-functional β-glucuronidase gene (GUS) and a reversion of mutated GUS to its functional form resulted in blue spots. Based on the

Based on the use of Agrobacterium tumefaciens-mediated transformation commodity crop improvement through genetic engineering is the fastest adopted crop te

PROJECT SUMMARYThe transmission of macromolecules across biological membranes is a fundamental process in all cells. In theearliest studies of genetic exchange in bacteria dating back to the 1940s, the F plasmid (then termed `sexfactor) was shown to self-transfer and, through recombination, mediate the transfer of the entire E. colichromosome to recipient bacteria. In the ensuing ~75 years, studies established the broad medical importanceof F and other mobile genetic elements (MGEs) in the shaping of bacterial genomes and as vectors fordissemination of antibiotic resistance and other fitness traits among bacterial populations. MGEs also encodeconjugative pili or other cell surface adhesins, which promote intercellular contacts necessary for DNA transferand establishment robust, antibiotic-resistant biofilm communities. MGEs are transmitted intercellularly throughnanomachines termed type IV secretion systems (T4SSs). The T4SSs are present in most if not all bacterialspecies, where they have ...

Bifunctional serine/threonine kinase and phosphorylase involved in the regulation of the pyruvate, phosphate dikinase (PPDK) by catalyzing its phosphorylation/dephosphorylation.

Agrobacterium-mediated transformation is the most widely used technique for generating transgenic plants. However, many crops remain recalcitrant. We found that an Arabidopsis myb family transcription factor (MTF1) inhibited plant transformation susceptibility. Mutating MTF1 increased attachment of several Agrobacterium strains to roots and increased both stable and transient transformation in both susceptible and transformation-resistant Arabidopsis ecotypes. Cytokinins from Agrobacterium tumefaciens decreased the expression of MTF1 through activation of the cytokinin response regulator ARR3. Mutating AHK3 and AHK4, genes that encode cytokinin-responsive kinases, increased the expression of MTF1 and impaired plant transformation. Mutant mtf1 plants also had increased expression of AT14A, which encodes a putative transmembrane receptor for cell adhesion molecules. Plants overexpressing AT14A exhibited increased susceptibility to transformation, whereas at14a mutant plants exhibited decreased attachment

As an Agrobacterium tumefaciens T-DNA oncogene, T-6b induces the development of tumors and the enation syndrome in vegetative tissues of transgenic plants. Most of these effects are related to increases in soluble sugar contents. To verify the potential roles of T-6b in the distribution of carbon in developing seeds, not in vegetative tissues, we fused an endosperm-specific promoter to the T-6b gene for expression in transgenic Arabidopsis thaliana plants. The expression of T-6b in reproductive organs did not induce the development of the enation syndrome, and moreover, promoted endosperm expansion, which increased the total seed biomass by more than 10%. Additionally, T-6b also increased oil content in mature seeds by more than 10% accompanied with the decrease of starch and mucilage content at the same time. T-6b enhances seed biomass and helps oil biosynthesis but not polysaccharides in reproductive organs without disturbing vegetative growth and development. Our findings suggest T-6b may be very

Escribed . Briefly, the AtEHD constructs were cloned in pBINplus and introduced by electroporation into Agrobacterium tumefaciens strain GV3101. Agrobacterium

The influence of different antibiotic treatments in soybean genetic transformation was evaluated. First, an assay was performed to verify how different antibiotic treatments affect soybean embryogenic tissues. The effect of carbenicillin at 500 mg L-1 was genotype-dependent. This antibiotic did not affect embryo survival of cv. IAS5, but a three-fold increase of embryo proliferation was observed for cv. Bragg, when compared to the control. On the other hand, cefotaxime at 350 and 500 mg L-1 caused death of embryogenic tissues of both cultivars. Finally, the association of cefotaxime (250 mg L-1) + vancomycin (250 mg L-1) did not affect negatively the somatic embryos of tested cultivars until 63 days of treatment. Thereafter, a second experiment was carried out to determine the efficacy of different antibiotic treatments in suppressing LBA4404 Agrobacterium tumefaciens strain in genetic transformation. On tissue culture conditions, carbenicillin at 500 and 1000 mg L-1 was not active against ...

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 under these conditions. Habituated tissue cultures, which resemble crown-gall tumor cultures, however, form cell walls that are inhibitory like those of the normal cultures from which they are derived. Monocotyledonous plants do not act as hosts for Agrobacterium and bacteria-specific inhibition is not shown by cell walls from several species of grass, a monocot family. Cell wallsfrom embryonic tissues (dicot seedlings less than 2 centimeters long), unlike those from older seedlings, are non-inhibitory. Crown-gall tumors thus resemble embryonic tissues in this respect.. ...

when virulent cells of agrobacterium tumefaciens strain b6 were exposed to low-level microwave radiation at a frequency of 10,000 mhz and an intensity of 0.58 mw/cm2 for 30 to 120 min, a 30 to 60% decrease in their ability to produce tumors on potato and turnip disks was observed. this microwave exposure did not affect the viability of these bacteria or their ability to attach to a tumor-binding site nor did it induce thermal shock. this loss of virulence was reversible within 12 h ...

In this study, we have begun to examine the complex interactions between two common environmental microorganisms, P.a. and A.t., in planktonic and biofilm growth modes. During exponential growth in dispersed, liquid culture, P.a. dominated A.t. because of a higher growth rate. Within biofilms grown on glass surfaces in the same defined medium, P.a. was also found to numerically dominate the population and to cover adherent A.t., a process requiring motility via flagella and type IV pili. Quorum-sensing mutants displayed an impaired competition phenotype in both liquid and flow-cell biofilm cultures. Motility was found to be important for both species in coculture biofilms. Although A.t. was outnumbered after the rapid-growth phase in both growth formats, its population remained viable, leading to a period of coexistence of these two microbes.. Quorum sensing appears to allow P.a. to achieve a slightly higher growth yield in liquid cocultures. Several quorum-sensing-regulated secreted functions ...

Peptidoglycan (PG) is a fundamental feature of nearly all bacteria and plays a primary role in maintaining cell integrity and cell shape. The PG layer is an interconnected mesh in the bacterial cell envelope, and, when isolated intact from a cell, the sacculus it forms retains the overall, albeit deflated, shape of the original cell. PG is composed of glycan strands covalently bonded by short, periodic peptide cross-links. In bacilli, the glycan strands run roughly perpendicularly to the long axis of the cell, with peptide cross-links arranged in parallel to the cell axis (1-4). This architecture allows bacteria to withstand significant osmotic pressure, and thicker cell walls confer greater resistance: typical Gram-negative strains can withstand ~2 to ~5 atm, whereas Gram-positive strains with thicker cell walls can withstand upward of 20 atm (5, 6).. To grow and divide, bacteria must enlarge and remodel this PG mesh. For cell division, most bacteria in which this has been studied share the ...