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 Agrobacterium rhizogenes GALLS Protein Contains Domains for ATP Binding, Nuclear Localization, and Type IV Secretion

2006 ◽  
Vol 188 (23) ◽  
pp. 8222-8230 ◽  
Author(s):  
Larry D. Hodges ◽  
Annette C. Vergunst ◽  
Jason Neal-McKinney ◽  
Amke den Dulk-Ras ◽  
Deborah M. Moyer ◽  
...  
Keyword(s):  
Agrobacterium Rhizogenes ◽  
Nuclear Localization ◽  
Plant Pathogens ◽  
Plant Cells ◽  
Tumor Formation ◽  
Type Iv Secretion ◽  
Plant Genome ◽  
Atp Binding ◽  
Type Iv ◽  
Vir Proteins

ABSTRACT Agrobacterium tumefaciens and Agrobacterium rhizogenes are closely related plant pathogens that cause different diseases, crown gall and hairy root. Both diseases result from transfer, integration, and expression of plasmid-encoded bacterial genes located on the transferred DNA (T-DNA) in the plant genome. Bacterial virulence (Vir) proteins necessary for infection are also translocated into plant cells. Transfer of single-stranded DNA (ssDNA) and Vir proteins requires a type IV secretion system, a protein complex spanning the bacterial envelope. A. tumefaciens translocates the ssDNA-binding protein VirE2 into plant cells, where it binds single-stranded T-DNA and helps target it to the nucleus. Although some strains of A. rhizogenes lack VirE2, they are pathogenic and transfer T-DNA efficiently. Instead, these bacteria express the GALLS protein, which is essential for their virulence. The GALLS protein can complement an A. tumefaciens virE2 mutant for tumor formation, indicating that GALLS can substitute for VirE2. Unlike VirE2, GALLS contains ATP-binding and helicase motifs similar to those in TraA, a strand transferase involved in conjugation. Both GALLS and VirE2 contain nuclear localization sequences and a C-terminal type IV secretion signal. Here we show that mutations in any of these domains abolished the ability of GALLS to substitute for VirE2.

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 Membrane and Core Periplasmic Agrobacterium tumefaciens Virulence Type IV Secretion System Components Localize to Multiple Sites around the Bacterial Perimeter during Lateral Attachment to Plant Cells

mBio ◽  
2011 ◽  
Vol 2 (6) ◽  
Author(s):  
Julieta Aguilar ◽  
Todd A. Cameron ◽  
John Zupan ◽  
Patricia Zambryski
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Plant Cells ◽  
Secretion System ◽  
Type Iv Secretion ◽  
Host Cells ◽  
Type Iv Secretion System ◽  
Secretion Systems ◽  
Content Type ◽  
Protein Substrates ◽  
Type Iv

ABSTRACTType IV secretion systems (T4SS) transfer DNA and/or proteins into recipient cells. Here we performed immunofluorescence deconvolution microscopy to localize the assembled T4SS by detection of its native components VirB1, VirB2, VirB4, VirB5, VirB7, VirB8, VirB9, VirB10, and VirB11 in the C58 nopaline strain ofAgrobacterium tumefaciens, following induction of virulence (vir) gene expression. These different proteins represent T4SS components spanning the inner membrane, periplasm, or outer membrane. Native VirB2, VirB5, VirB7, and VirB8 were also localized in theA. tumefaciensoctopine strain A348. Quantitative analyses of the localization of all the above Vir proteins in nopaline and octopine strains revealed multiple foci in single optical sections in over 80% and 70% of the bacterial cells, respectively. Green fluorescent protein (GFP)-VirB8 expression followingvirinduction was used to monitor bacterial binding to live host plant cells; bacteria bind predominantly along their lengths, with few bacteria binding via their poles or subpoles.vir-induced attachment-defective bacteria or bacteria without the Ti plasmid do not bind to plant cells. These data support a model where multiplevir-T4SS around the perimeter of the bacterium maximize effective contact with the host to facilitate efficient transfer of DNA and protein substrates.IMPORTANCETransfer of DNA and/or proteins to host cells through multiprotein type IV secretion system (T4SS) complexes that span the bacterial cell envelope is critical to bacterial pathogenesis. Early reports suggested that T4SS components localized at the cell poles. Now, higher-resolution deconvolution fluorescence microscopy reveals that all structural components of theAgrobacterium tumefaciens vir-T4SS, as well as its transported protein substrates, localize to multiple foci around the cell perimeter. These results lead to a new model ofA. tumefaciensattachment to a plant cell, whereA. tumefacienstakes advantage of the multiplevir-T4SS along its length to make intimate lateral contact with plant cells and thereby effectively transfer DNA and/or proteins through thevir-T4SS. The T4SS ofA. tumefaciensis among the best-studied T4SS, and the majority of its components are highly conserved in different pathogenic bacterial species. Thus, the results presented can be applied to a broad range of pathogens that utilize T4SS.

scholarly journals Self-Assembly of the Agrobacterium tumefaciens VirB11 Traffic ATPase

2000 ◽  
Vol 182 (15) ◽  
pp. 4137-4145 ◽  
Author(s):  
Svetlana Rashkova ◽  
Xue-Rong Zhou ◽  
Jun Chen ◽  
Peter J. Christie
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Self Assembly ◽  
Fluorescent Protein ◽  
Chimeric Protein ◽  
Dominant Negative ◽  
Binding Motif ◽  
Hybrid Protein ◽  
Type Iv ◽  
Cell Extracts

ABSTRACT The Agrobacterium tumefaciens VirB11 ATPase is a component of a type IV transporter dedicated to T-DNA delivery to plant cells. In this study, we tested a prediction from genetic findings that VirB11 self-associates in vivo. A chimeric protein composed of VirB11 fused to the DNA binding domain of λ cI repressor protein formed dimers, as shown by immunity of Escherichia coli to λ superinfection. An allele encoding VirB11 fused at its C terminus to the green fluorescent protein (GFP) exerted strong negative dominance when synthesized in wild-type A. tumefacienscells. Dominance was suppressed by overproduction of native VirB11, suggestive of titrating or competitive interactions between VirB11 and VirB11::GFP. In support of the titration model, a complex of native VirB11 and VirB11::GFP was recovered by precipitation with anti-GFP antibodies from detergent-solubilized A. tumefaciens cell extracts. VirB11 was shown by cI repressor fusion and immunoprecipitation assays to interact with VirB11 derivatives encoded by (i) 11 dominant negative alleles, (ii) recessive alleles bearing codon substitutions or deletions in the Walker A nucleotide binding motif, and (iii) alleles corresponding to the 5′ and 3′ halves of virB11. Further immunoprecipitation studies showed a hybrid protein composed of the N-terminal half of VirB11 fused to GFP interacted with mutant proteins exerting dominant effects and with a recessive Walker A deletion mutant (ΔGKT174-176). By contrast, a hybrid protein composed of the C-terminal half fused to GFP interacted with mutants exerting dominant effects but not the Walker A mutant protein. Together, these studies establish that VirB11 assembles as homomultimers in vivo via domains residing in each half of the protein. Furthermore, ATP binding appears to be critical for C-terminal interactions required for assembly of productive homomultimers.

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