Factors affecting the rate of T-DNA transfer from Agrobacterium tumefaciens to Nicotiana glauca plant cells

1992 ◽  
Vol 19 (6) ◽  
pp. 1019-1030 ◽  
Author(s):  
Teresa Mozo ◽  
J. J. Hooykaas
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Plant Cells ◽  
Dna Transfer ◽  
Nicotiana Glauca ◽  
Factors Affecting

Identification of the signal molecules produced by wounded plant cells that activate T-DNA transfer in Agrobacterium tumefaciens

Nature ◽  
1985 ◽  
Vol 318 (6047) ◽  
pp. 624-629 ◽  
Author(s):  
Scott E. Stachel ◽  
Eric Messens ◽  
Marc Van Montagu ◽  
Patricia Zambryski
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Plant Cells ◽  
Dna Transfer ◽  
Signal Molecules

Gene-Expression Following T-DNA Transfer Into Plant Cells Is Aphidicolin-Sensitive

1994 ◽  
Vol 21 (2) ◽  
pp. 125 ◽  
Author(s):  
AM Chaudhury ◽  
ES Dennis ◽  
RIS Brettell
Keyword(s):  
Gene Expression ◽  
Dna Replication ◽  
Agrobacterium Tumefaciens ◽  
Plant Cells ◽  
Nicotiana Plumbaginifolia ◽  
Dna Transfer ◽  
Host Cells ◽  
35S Promoter ◽  
Glucuronidase Activity ◽  
Pass Through

A transient assay for gene-expression was used to study the early events of T-DNA transfer. Particularly, it was asked if gene expression following T-DNA transfer required DNA replication in the host cell. A β-glucuronidase gene, linked to a CaMV 35S promoter (35S-GUS, engineered so that it was inactive in Agrobacterium tumefaciens) was introduced into Nicotiana plumbaginifolia protoplasts via a disarmed supervirulent strain of Agrobacterium tumefaciens. High β-glucuronidase activity appeared after 3 days of co-cultivation. The activity required the presence of the vir functions of agrobacteria. The activity was drastically reduced if the plant cells were treated with aphidicolin, an inhibitor of DNA replication in eukaryotic cells. While double-stranded (ds) 35S-GUS DNA, introduced by electroporation, showed undiminished expression in the presence of aphidicolin, gene expression from single-stranded (ss) 35S-GUS DNA was inhibited by aphidicolin. These results suggest that DNA replication in host cells is not required for gene expression if ds-DNA is introduced by electroporation, but is required if ss-DNA is introduced by electroporation, or if DNA is transferred via A. tumefaciens. The findings are consistent with a model of T-DNA transfer in which ss-DNA molecules, once introduced into plant cells, must pass through an aphidicolin sensitive step before they can be transcribed. The simplest interpretation is that the ss-DNA is replicated by the host cell's aphidicolin-sensitive DNA polymerase before being integrated into the host genome.

scholarly journals The Omega Sequence of VirD2 Is Important but Not Essential for Efficient Transfer of T-DNA by Agrobacterium tumefaciens

1998 ◽  
Vol 11 (1) ◽  
pp. 57-63 ◽  
Author(s):  
Ana María Bravo-Angel ◽  
Barbara Hohn ◽  
Bruno Tinland
Keyword(s):  
Amino Acids ◽  
Agrobacterium Tumefaciens ◽  
Mutational Analysis ◽  
Plant Cells ◽  
Dna Transfer ◽  
Wild Type ◽  
C Terminus ◽  
Efficient Transfer

The VirD2 protein of Agrobacterium tumefaciens contains defined sequences necessary for processing and transferring the T-DNA during transformation of plant cells. We performed a mutational analysis of the conserved omega sequence of VirD2, whose role has proven to be difficult to elucidate so far. In this report, we show that a deletion of these 5 amino acids or their replacement by 5 glycines reduced T-DNA transfer considerably, compared with wild type, demonstrating that the omega sequence is important for the efficient transfer of T-DNAs. However, the efficiency and pattern of integration of the T-DNAs were not affected by any modifications of the omega sequence. The importance of the C terminus of VirD2 for T-DNA transfer is discussed.

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

Nature ◽  
1986 ◽  
Vol 322 (6081) ◽  
pp. 706-712 ◽  
Author(s):  
Scott E. Stachel ◽  
Benedikt Timmerman ◽  
Patricia Zambryski
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Plant Cells ◽  
Dna Transfer ◽  
Dna Molecules

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

2004 ◽  
Vol 186 (10) ◽  
pp. 3065-3077 ◽  
Author(s):  
Larry D. Hodges ◽  
Josh Cuperus ◽  
Walt Ream
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Agrobacterium Rhizogenes ◽  
Binding Protein ◽  
Plant Cells ◽  
Dna Transfer ◽  
Nucleoside Triphosphate ◽  
Binding Motif ◽  
Single Stranded Dna ◽  
Type Iv ◽  
Vir Proteins

ABSTRACT Agrobacterium tumefaciens and Agrobacterium rhizogenes transfer plasmid-encoded genes and virulence (Vir) proteins into plant cells. The transferred DNA (T-DNA) is stably inherited and expressed in plant cells, causing crown gall or hairy root disease. DNA transfer from A. tumefaciens into plant cells resembles plasmid conjugation; single-stranded DNA (ssDNA) is exported from the bacteria via a type IV secretion system comprised of VirB1 through VirB11 and VirD4. Bacteria also secrete certain Vir proteins into plant cells via this pore. One of these, VirE2, is an ssDNA-binding protein crucial for efficient T-DNA transfer and integration. VirE2 binds incoming ssT-DNA and helps target it into the nucleus. Some strains of A. rhizogenes lack VirE2, but they still transfer T-DNA efficiently. We isolated a novel gene from A. rhizogenes that restored pathogenicity to virE2 mutant A. tumefaciens. The GALLS gene was essential for pathogenicity of A. rhizogenes. Unlike VirE2, GALLS contains a nucleoside triphosphate binding motif similar to one in TraA, a strand transferase conjugation protein. Despite their lack of similarity, GALLS substituted for VirE2.

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

1998 ◽  
Vol 11 (5) ◽  
pp. 429-433 ◽  
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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