The genetic and transcriptional organization of the vir region of the A6 Ti plasmid of Agrobacterium tumefaciens.

Stachel, Scott E.; Nester, Eugene W.

doi:10.1002/j.1460-2075.1986.tb04381.x

Cited by 416 publications

(412 citation statements)

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“…Since VirB11 is required for virulence of A. tumefaciens (12,54), virB11 deletion strain A10011 expressing MBP or MBPVirB11 was tested for virulence. Strain A10011 expressing MBP-VirB11 was fully virulent on K. diagremontiana leaves when assayed at several dilutions (Fig.…”

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

“…The genes required for the transfer of T-DNA into the recipient plant cells are divided into eight vir operons on the octopine-type Ti plasmid (54). These genes are expressed only when the bacteria are exposed to wounded plant cells as a result of the action of several plant signal molecules acting in concert with the virA and virG gene products.…”

mentioning

confidence: 99%

“…The 11 genes of the virB operon and virD4 are required for this transport step in the tumor formation process (1,14). virD4 and the virB operon are not required for any of the early steps such as attachment, induction, or T-DNA processing but are absolutely essential for tumor formation (7,20,51,54,66). VirD4, VirB1, VirB2, VirB3, VirB4, VirB5, VirB8, VirB9, VirB10, and VirB11 have all been localized to the membrane fraction by immunological techniques (12,41,50,51,57,58,67).…”

mentioning

confidence: 99%

“…The two potential nucleotide-binding proteins in the virB operon, VirB4 and VirB11, are both absolutely required for virulence (7,20,51,54). Both proteins are also localized to the inner membrane of A. tumefaciens even though VirB11 does not contain an N-terminal export sequence or long hydrophobic stretches of amino acids (12,57,65).…”

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Agrobacterium tumefaciens VirB11 protein requires a consensus nucleotide-binding site for function in virulence

1995

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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 importance of the Walker nucleotide-binding site present in VirB11, mutations were generated to replace the conserved lysine residue with either alanine or arginine. Expression of the virB11K175A mutant gene resulted in an avirulent phenotype, and expression of the virB11K175R mutant gene gave rise to an attenuated virulence phenotype. Both mutant proteins were present at levels three to four times higher than that of VirB11 in the wild-type strain. The mutant genes did not exhibit a transdominant phenotype on tumor formation in bacteria that were expressing wild-type virB11. The mutant proteins also localized properly to the inner membrane of A. tumefaciens, but the VirB11K175R protein appeared to be unstable after lysis of the cells.The soil bacterium Agrobacterium tumefaciens causes the plant disease crown gall. The bacteria can infect dicotyledonous plants at a wound site and induce the formation of tumors at the site of infection. Virulent strains of A. tumefaciens contain a large tumor-inducing (Ti) plasmid that carries the factors responsible for formation of the crown gall tumors. The bacteria have the unusual property of being able to transfer a segment of DNA (T-DNA) from the Ti plasmid into the host plant cells. The T-DNA is integrated into the plant genome, and expression of the oncogenes present in the T-DNA results in formation of the tumor (for recent reviews, see references 26, 70, and 73).The genes required for the transfer of T-DNA into the recipient plant cells are divided into eight vir operons on the octopine-type Ti plasmid (54). These genes are expressed only when the bacteria are exposed to wounded plant cells as a result of the action of several plant signal molecules acting in concert with the virA and virG gene products. Once the vir genes have been expressed, the gene products act in a coordinated fashion to synthesize T-DNA strands and direct the transfer of the T-DNA into a recipient plant cell.The T-DNA and associated proteins are transported through the bacterial inner and outer membranes and thr...

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Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Agrobacterium tumefaciens VirB11 protein requires a consensus nucleotide-binding site for function in virulence

1995

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Synthesis of high specific radioactivity 3,5-[3H6]dimethoxy-4-hydroxyacetophenone, an inducing compound of the vir gene in Agrobacterium tumefaciens

Lee

Morimoto

Williams

1997

J Label Compd Radiopharm

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Acetosyringone (3,5‐dimethoxy‐4‐hydroxyacetophenone, 1) is one of the plant signals that induce the vir genes of Agrobacterium tumefaciens which causes crown gall tumours on dicotyledonous plants. Vir gene induction is enhanced by other environmental factors like specific monosaccarides and acidic pH. However, it is still unclear how the inducer interacts with Agrobacterium. To identify the receptor(s) in Agrobacterium tumefaciens to which the plant signal binds, highly radioactive acetosyringone (3,5‐[3H6]dimethoxy‐4‐hydroxyacetophenone 6) was synthesized using C3H3I which was generated by two different methods. In Method 1, C3H3I was generated from 4‐[methyl‐3H]methoxy biphenyl to give methyl species with tritium NMR peak intensity ratios (C3H3: C3H2: C3HH2 = 44 : 42 : 14), and a calculated specific radioactivity of 56.1 Ci/mmole. Method 2 gave higher specific radioactivity reagent (72.6 Ci/mmole), with measured NMR peak intensity ratios of (3H3: C3H2: C3HH2 = 87 : 7 : 6). © John Wiley & Sons, Ltd.

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Plant Transformation Technology: Agrobacterium ‐Mediated Transformation

Komari

Ishida

Hiei

2004

Handbook of Plant Biotechnology

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The soil phytopathogen Agrobacterium tumefaciens induces tumours, known as crown galls, mainly on dicotyledonous plants. Such tumours are generated by a complex, multi‐step transformation process. Another species, A. rhizogenes , causes hairy root disease in higher plants via an identical process. Agrobacterium has been utilised for the transfer of genes to dicotyledonous plants. Now, monocotyledonous plants are routinely transformed by Agrobacterium despite the fact that these plants, including important cereals, were thought until recently to be outside the range of this technology. Most of the common transformation methods heavily depend on tissue culture technology and we refer to such methods as in vitro transformation. The only case of a routine tissue culture free method is the ‘ in planta ’ transformation of Arabidopsis thaliana . Numerous critical factors are involved in both approaches. In transformation in vitro , key factors include choice of vectors and bacterial strains, types of plant tissues to be infected, procedures of preparation the tissues, protocols of infection and co‐cultivation, methods for subsequent culture and selection of transformed cells, antibiotics to remove infecting bacteria, techniques for regeneration of transgenic plants and genotypes of plants. It is our opinion that the type and quality of the starting material is the most important one among them. The capacity to serve as a host plant for crown gall disease is no longer a prerequisite for a host of vector systems based on Agrobacterium . The real prerequisite for in vitro transformation is the availability of technology for dedifferentiation of tissues and regeneration of plants in a given species. On the other hand, the biological processes involved in in planta transformation are yet to be elucidated. Recent evidence suggested that ovules are the primary target, and further basic understanding is likely to help extend the number of species transformed by the method. The plant species that are routinely transformed by Agrobacterium are expanding rapidly. Some gymnosperms, several forest trees and fruit trees, various legumes, and cereal and non‐cereal monocotyledons, which were once considered very recalcitrant, are now in a long list of transformable plants. Arabidopsis , tobacco and rice are the top three species that were transformed during the last two years. Although it is only half a dozen years since the current procedure of rice transformation mediated by Agrobacterium was published, the economic importance and the accumulation of genomic information has made rice the species of focus in plant biotechnology. The advantages of Agrobacterium ‐mediated transformation include the transfer of pieces of DNA with defined ends and minimal rearrangement, the transfer of relatively large segments of DNA, the integration of small numbers of copies of genes into plant chromosomes and the high quality and fertility of transgenic plants. However, transformation does not always produce such ‘clean’ events. Formation of repeats of transgenes, certain rearrangements, integration of non‐target DNA segments and unstable expression of transgenes are among the complications. Although the majority of transgenic plants usually appear ‘good’ in a particular test, accumulation of ‘dropped’ plants is significant after multiple rounds of characterisation and screening. Therefore, further improvement in each of steps is highly desired. Application of the gene transfer mediated by Agrobacterium is further expanding. Transient expression of genes delivered by Agrobacterium is now a useful tool in the study of promoters and gene function. Vectors specifically designed to carry very large segments of DNA have been developed and extensively tested. Insertional mutagenesis by DNA transferred by Agrobacterium is a routine technique in genomics study in Arabidopsis and rice. Methods for targeted integration of transgenes to genomes of higher plants have been drawing considerable attention. Various technologies for the production of ‘selection marker free’ transgenic plants are now in place for better public acceptance of biotechnology products.

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The genetic and transcriptional organization of the vir region of the A6 Ti plasmid of Agrobacterium tumefaciens.

Cited by 416 publications

References 32 publications

Agrobacterium tumefaciens VirB11 protein requires a consensus nucleotide-binding site for function in virulence

Agrobacterium tumefaciens VirB11 protein requires a consensus nucleotide-binding site for function in virulence

Synthesis of high specific radioactivity 3,5-[3H6]dimethoxy-4-hydroxyacetophenone, an inducing compound of the vir gene in Agrobacterium tumefaciens

Plant Transformation Technology: Agrobacterium ‐Mediated Transformation

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