The Ti Plasmid from the Wide Host Range Agrobacterium vitis Strain Tm4: Map and Homology with Other Ti Plasmids

Agrobacterium tumefaciens strain AB2/73 isolated from Lippia canescens has been described as a limited-host-range strain. Its tumor-inducing (Ti) plasmid has been found to lack DNA homology to known T-DNAs (L. Unger, S. F. Ziegler, G. A. Huffman, V. C. Knauf, R. Peet, L. W. Moore, M. P. Gordon, and E. W. Nester. J. Bacteriol. 164:723–730, 1985). We have isolated a T-DNA from AB2/73 by using a heterologous border sequence as a probe. The AB2/73 T-DNA sequence (3,504 bp) is flanked by canonical border sequences, has no detectable DNA homology with other T-DNAs, and contains only two genes: lsn ( Lippia strain nopaline synthaselike gene) and lso ( Lippia strain oncogene). The lso gene induces nondif-ferentiating tumors on a limited number of hosts when transferred by a Ti plasmid from a wide-host-range strain. Part of the predicted Lso protein is weakly homologous to other Agrobacterium oncoproteins encoded by rolB, rolBTR, orf13, gene e, gene 5, and gene 3′. A 28-kb fragment corresponding to the virA to virE region was cloned by using a heterologous vir fragment as probe. The AB2/73 vir region is homologous to most of the C58 virulence region; however, the virA gene is most related to the virA gene of the Agrobacterium vitis limited-host-range strain Ag162.

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Limited Host Range Ti Plasmids: Recent Origin from Wide Host Range Ti Plasmids and Involvement of a Novel IS Element, IS868

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-4-190 ◽

1991 ◽

Vol 4 (2) ◽

pp. 190 ◽

Cited By ~ 15

Author(s):

François Paulus

Keyword(s):

Host Range ◽

Is Element ◽

Wide Host Range ◽

Ti Plasmids ◽

Limited Host Range ◽

Recent Origin

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Construction of Disarmed Ti Plasmids Transferable between Escherichia coli and Agrobacterium Species

Applied and Environmental Microbiology ◽

10.1128/aem.01856-08 ◽

2009 ◽

Vol 75 (7) ◽

pp. 1845-1851 ◽

Cited By ~ 7

Author(s):

Kazuya Kiyokawa ◽

Shinji Yamamoto ◽

Kei Sakuma ◽

Katsuyuki Tanaka ◽

Kazuki Moriguchi ◽

...

Keyword(s):

Escherichia Coli ◽

Host Range ◽

Plant Transformation ◽

Ti Plasmid ◽

Broad Host Range ◽

Bacterial Strains ◽

E Coli ◽

Ti Plasmids ◽

The Right ◽

Border Sequence

ABSTRACT Agrobacterium-mediated plant transformation has been used widely, but there are plants that are recalcitrant to this type of transformation. This transformation method uses bacterial strains harboring a modified tumor-inducing (Ti) plasmid that lacks the transfer DNA (T-DNA) region (disarmed Ti plasmid). It is desirable to develop strains that can broaden the host range. A large number of Agrobacterium strains have not been tested yet to determine whether they can be used in transformation. In order to improve the disarming method and to obtain strains disarmed and ready for the plant transformation test, we developed a simple scheme to make certain Ti plasmids disarmed and simultaneously maintainable in Escherichia coli and mobilizable between E. coli and Agrobacterium. To establish the scheme in nopaline-type Ti plasmids, a neighboring segment to the left of the left border sequence, a neighboring segment to the right of the right border sequence of pTi-SAKURA, a cassette harboring the pSC101 replication gene between these two segments, the broad-host-range IncP-type oriT, and the gentamicin resistance gene were inserted into a suicide-type sacB-containing vector. Replacement of T-DNA with the cassette in pTiC58 and pTi-SAKURA occurred at a high frequency and with high accuracy when the tool plasmid was used. We confirmed that there was stable maintenance of the modified Ti plasmids in E. coli strain S17-1λpir and conjugal transfer from E. coli to Ti-less Agrobacterium strains and that the reconstituted Agrobacterium strains were competent to transfer DNA into plant cells. As the modified plasmid delivery system was simple and efficient, conversion of strains to the disarmed type was easy and should be applicable in studies to screen for useful strains.

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The Presence and Characterization of a virF Gene on Agrobacterium vitis Ti Plasmids

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.1998.11.5.429 ◽

1998 ◽

Vol 11 (5) ◽

pp. 429-433 ◽

Cited By ~ 14

Author(s):

B. Schrammeijer ◽

J. Hemelaar ◽

P. J. J. Hooykaas

Keyword(s):

Amino Acids ◽

Agrobacterium Tumefaciens ◽

Molecular Mass ◽

Promoter Region ◽

Consensus Sequence ◽

Tumor Formation ◽

Nicotiana Glauca ◽

Agrobacterium Vitis ◽

Ti Plasmids

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 plas-mids 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 pTiAg57 virF is inducible by the plant phenolic compound acetosyringone through the presence of a vir-box consensus sequence in its promoter region. The VirF protein from pTiAg57 can complement octopine A. tumefaciens strains deleted for virF as shown by tumor formation on N. glauca.

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