Cooperative binding of Agrobacterium tumefaciens VirE2 protein to single-stranded DNA.

The VirE2 protein of Agrobacterium tumefaciens Ti plasmid pTiA6 is a single-stranded-DNA-binding protein. Density gradient centrifugation studies showed that it exists as a tetramer in solution. Monomeric VirE2 active in DNA binding could also be obtained by using a different protein isolation procedure. VirE2 was found to be thermolabile; brief incubation at 37 degrees C abolished its DNA-binding activity. It was insensitive to the sulfhydryl-specific reagent N-ethylmaleimide. Removal of the carboxy-terminal 37 residues of the 533-residue VirE2 polypeptide led to complete loss of DNA-binding activity; however, chimeric fusion proteins containing up to 125 residues of the VirE2 C terminus were inactive in DNA binding. In nuclease protection studies, VirE2 protected single-stranded DNA against degradation by DNase I. Analysis of the DNA-VirE2 complex by electron microscopy demonstrated that VirE2 coats a single-stranded DNA molecule and that the binding of VirE2 to its substrate is cooperative.     

Plant transformation by Agrobacterium tumefaciens: modulation of single-stranded DNA-VirE2 complex assembly by VirE1

Agrobacterium tumefaciens infects plant cells by the transfer of DNA. A key factor in this process is the bacterial virulence protein VirE2, which associates stoichiometrically with the transported single-stranded (ss) DNA molecule (T-strand). As observed in vitro by transmission electron microscopy, VirE2-ssDNA readily forms an extended helical complex with a structure well suited to the tasks of DNA protection and nuclear import. Here we have elucidated the role of the specific molecular chaperone VirE1 in regulating VireE2-VirE2 and VirE2-ssDNA interactions. VirE2 alone formed functional filamentous aggregates capable of ssDNA binding. In contrast, co-expression with VirE1 yielded monodisperse VirE1-VirE2 complexes. Cooperative binding of VirE2 to ssDNA released VirE1, resulting in a controlled formation mechanism for the helical complex that is further promoted by macromolecular crowding. Based on this in vitro evidence, we suggest that the constrained volume of the VirB channel provides a natural site for the exchange of VirE2 binding from VirE1 to the T-strand.     

Functional domains of Agrobacterium tumefaciens single-stranded DNA-binding protein VirE2.

The transferred DNA (T-DNA) portion of the Agrobacterium tumefaciens tumor-inducing (Ti) plasmid enters infected plant cells and integrates into plant nuclear DNA. Direct repeats define the T-DNA ends; transfer begins when the VirD2 endonuclease produces a site-specific nick in the right-hand border repeat and attaches to the 5' end of the nicked strand. Subsequent events liberate the lower strand of the T-DNA from the Ti plasmid, producing single-stranded DNA molecules (T strands) that are covalently linked to VirD2 at their 5' ends. A. tumefaciens appears to transfer T-DNA into plant cells as a T-strand-VirD2 complex. The bacterium also transports VirE2, a cooperative single-stranded DNA-binding protein, into plant cells during infection. Both VirD2 and VirE2 contain nuclear localization signals that may direct these proteins, and bound T strands, into plant nuclei. Here we report the locations of functional regions of VirE2 identified by eight insertions of XhoI linker oligonucleotides, and one deletion mutation, throughout virE2. We examined the effects of these mutations on virulence, single-stranded DNA (ssDNA) binding, and accumulation of VirE2 in A. tumefaciens. Two of the mutations in the C-terminal half of VirE2 eliminated ssDNA binding, whereas two insertions in the N-terminal half altered cooperativity. Four of the mutations, distributed throughout virE2, decreased the stability of VirE2 in A. tumefaciens. In addition, we isolated a mutation in the central region of VirE2 that decreased tumorigenicity but did not affect ssDNA binding or VirE2 accumulation. This mutation may affect export of VirE2 into plant cells or nuclear localization of VirE2, or it may affect an uncharacterized activity of VirE2.     

Agrobacterium rhizogenes GALLS protein substitutes for Agrobacterium tumefaciens single-stranded DNA-binding protein VirE2

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.     

Three-dimensional reconstruction of Agrobacterium VirE2 protein with single-stranded DNA

Agrobacterium tumefaciens infects plant cells by a unique mechanism involving an interkingdom genetic transfer. A single-stranded DNA substrate is transported across the two cell walls along with the bacterial virulence proteins VirD2 and VirE2. A single VirD2 molecule covalently binds to the 5'-end of the single-stranded DNA, while the VirE2 protein binds stoichiometrically along the length of the DNA, without sequence specificity. An earlier transmission/scanning transmission electron microscopy study indicated a solenoidal ("telephone coil") organization of the VirE2-DNA complex. Here we report a three-dimensional reconstruction of this complex using electron microscopy and single-particle image-processing methods. We find a hollow helical structure of 15.7-nm outer diameter, with a helical rise of 51.5 nm and 4.25 VirE2 proteins/turn. The inner face of the protein units contains a continuous wall and an inward protruding shelf. These structures appear to accommodate the DNA binding. Such a quaternary arrangement naturally sequesters the DNA from cytoplasmic nucleases and suggests a mechanism for its nuclear import by decoration with host cell factors. Coexisting with the helices, we also found VirE2 tetrameric ring structures. A two-dimensional average of the latter confirms the major features of the three-dimensional reconstruction.     

VirE1 protein mediates export of the single-stranded DNA-binding protein VirE2 from Agrobacterium tumefaciens into plant cells.

Agrobacterium tumefaciens transfers single-stranded DNAs (T strands) into plant cells. VirE1 and VirE2, which is a single-stranded DNA binding protein, are important for tumorigenesis. We show that T strands and VirE2 can enter plant cells independently and that export of VirE2, but not of T strands, depends on VirE1.     

The Agrobacterium tumefaciens Chaperone-Like Protein, VirE1, Interacts with VirE2 at Domains Required for Single-Stranded DNA Binding and Cooperative Interaction

Agrobacterium tumefaciens transfers single-stranded DNA (ssDNA) into plants. Efficient tumorigenesis requires VirE1-dependent export of ssDNA-binding (SSB) protein VirE2. VirE1 binds VirE2 domains involved in SSB and self-association, and VirE1 may facilitate VirE2 export by preventing VirE2 aggregation and the premature binding of VirE2 to ssDNA.     

VIP1, an Arabidopsis protein that interacts with Agrobacterium VirE2, is involved in VirE2 nuclear import and Agrobacterium infectivity.

T-DNA nuclear import is a central event in genetic transformation of plant cells by Agrobacterium. This event is thought to be mediated by two bacterial proteins, VirD2 and VirE2, which are associated with the transported T-DNA molecule. While VirD2 is imported into the nuclei of plant, animal and yeast cells, nuclear uptake of VirE2 occurs most efficiently in plant cells. To understand better the mechanism of VirE2 action, a cellular interactor of VirE2 was identified and its encoding gene cloned from Arabidopsis. The identified plant protein, designated VIP1, specifically bound VirE2 and allowed its nuclear import in non-plant systems. In plants, VIP1 was required for VirE2 nuclear import and Agrobacterium tumorigenicity, participating in early stages of T-DNA expression.     

VirE2, a type IV secretion substrate,interacts with the VirD4 transfer protein at cell poles of Agrobacterium tumefaciens

Agrobacterium tumefaciens transfers oncogenic DNA and effector proteins to plant cells during the course of infection. Substrate translocation across the bacterial cell envelope is mediated by a type IV secretion (TFS) system composed of the VirB proteins, as well as VirD4, a member of a large family of inner membrane proteins implicated in the coupling of DNA transfer intermediates to the secretion machine. In this study, we demonstrate with novel cytological screens - a two-hybrid (C2H) assay and bimolecular fluorescence complementation (BiFC) - and by immunoprecipitation of chemically cross-linked protein complexes that the VirE2 effector protein interacts directly with the VirD4 coupling protein at cell poles of A. tumefaciens. Analyses of truncation derivatives showed that VirE2 interacts via its C terminus with VirD4, and, further, an NH2-terminal membrane-spanning domain of VirD4 is dispensable for complex formation. VirE2 interacts with VirD4 independently of the virB-encoded transfer machine and T pilus, the putative periplasmic chaperones AcvB and VirJ, and the T-DNA transfer intermediate. Finally, VirE2 is recruited to polar-localized VirD4 as a complex with its stabilizing secretion chaperone VirE1, yet the effector-coupling protein interaction is not dependent on chaperone binding. Together, our findings establish for the first time that a protein substrate of a type IV secretion system is recruited to a member of the coupling protein superfamily.     

The Conjugal Intermediate of Plasmid RSF1010 Inhibits Agrobacterium tumefaciens Virulence and VirB-Dependent Export of VirE2

Agrobacterium tumefaciens causes crown gall disease by transferring oncogenic, single-stranded DNA (T strand), covalently attached to the VirD2 protein, across the bacterial envelope into plant cells where its expression results in tumor formation. The single-stranded DNA binding protein VirE2 is also transferred into the plant cell, though the location at which VirE2 interacts with the T strand is still under investigation. The movement of the transferred DNA and VirE2 from A. tumefaciens to the plant cell depends on the membrane-localized VirB and VirD4 proteins. Further, the movement of the IncQ broad-host-range plasmid RSF1010 between Agrobacterium strains or from Agrobacterium to plants also requires the virB-encoded transfer system. Our earlier studies showed that the presence of the RSF1010 plasmid in wild-type strains of Agrobacterium inhibits both their virulence and their capacity to transport VirE2, as assayed by coinfection with virE mutants. Here we demonstrate that the capacity to form a conjugal intermediate of RSF1010 is necessary for this inhibition, suggesting that the transferred form of the plasmid competes with the VirD2-T strand and/or VirE2 for a common export site.     

Isolation, purification, and identification of virulence protein VirE2 from Agrobacterium tumefaciens

Bacteria of the genus Agrobacterium are capable of transferring a fragment of their Ti-plasmid, T-DNA, in a complex with the proteins VirE2 and VirD2, into the nuclei of plant cells and incorporating it into the chromosome of the host. The mechanisms of T-DNA transportation through membrane and cytoplasm of the plant cell are unknown. The aim of this work was isolation of virulence protein VirE2 for studying its role in T-DNA transportation through the membrane and cytoplasm of eukaryotic cells. For VirE2 accumulation, virE2 gene was cloned into plasmid pQE31. VirE2 was isolated from the cells of E. coli strain XL1-blue, containing the recombinant plasmid pQE31-virE2. The cells were disrupted ultrasonically, and the protein with six histidine residues at the N-end was isolated by means of affinity chromatography on a Ni-NTA-superose column. The purified protein was tested by the immunodot method using polyclonal rabbit antibodies and anti-VirE2 miniantibodies. The ability of the recombinant protein VirE2 to bind to single-stranded DNA was judged from the formation of complexes detected by electrophoresis in agarose gel. Thus, we isolated, purified, and partially characterized the Agrobacterium tumefaciens virulence protein VirE2 which is capable of binding to single-stranded T-DNA upon transfer to the plant cell.     

Recognition of the Agrobacterium tumefaciens VirE2 translocation signal by the VirB/D4 transport system does not require VirE1

Agrobacterium tumefaciens uses a type IV secretion system to deliver a nucleoprotein complex and effector proteins directly into plant cells. The single-stranded DNA-binding protein VirE2, the F-box protein VirF and VirE3 are delivered into host cells via this VirB/D4 encoded translocation system. VirE1 functions as a chaperone of VirE2 by regulating its efficient translation and preventing VirE2-VirE2 aggregation in the bacterial cell. We analyzed whether the VirE1 chaperone is also essential for transport recognition of VirE2 by the VirB/D4 encoded type IV secretion system. In addition, we assayed whether translocation of VirF and VirE3, which also forms part of the virE operon, is affected by the absence of VirE1. We employed the earlier developed CRAFT (Cre recombinase Reporter Assay For Translocation) assay to detect transfer of Cre::Vir fusion proteins from A. tumefaciens into plants, monitored by stable reconstitution of a kanamycin resistance marker, and into yeast, screened by loss of the URA3 gene. We show that the C-terminal 50 amino acids of VirE2 and VirE3 are sufficient to mediate Cre translocation into host cells, confirming earlier indications of a C-terminal transport signal. This transfer was independent of the presence or absence of VirE1. Besides, the translocation efficiency of VirF is not altered in a virE1 mutant. The results unambiguously show that the VirE1 chaperone is not essential for the recognition of the VirE2 transport signal by the transport system and the subsequent translocation across the bacterial envelope into host cells.     

Isolation, purification, and identification of the virulence protein VirE2 from Agrobacterium tumefaciens

Bacteria of the genus Agrobacterium can transfer a portion of their Ti plasmid (T-DNA) in complex with the VirE2 and VirD2 proteins into the plant-cell nucleus and cause it to be integrated in the host-cell chromosomes. The mechanism of T-DNA transfer across the plant-cell membrane and cytoplasm is unknown. The aim of this study was to isolate the virulence protein VirE2 in order to explore its role in T-DNA transfer across the eukaryotic-cell membrane and cytoplasm. To obtain VirE2, we cloned the virE2 gene into plasmid pQE31 in Escherichia coli cells. VirE2 protein was isolated from E. coli XL-1 blue cells containing a recombinant plasmid, pQE31-virE2. The cells were ultrasonically disrupted, and the protein containing six histidine residues at the N-terminal end was isolated by affinity chromatography on Ni-NTA agarose. The purified preparation was tested by immunodot, by using polyclonal rabbit antibodies and miniantibodies produced toward VirE2. The capacity of the recombinant protein VirE2 for interacting with single-stranded DNA was tested by the formation of complexes, recorded by agarose-gel electrophoresis. In summary, A. tumefaciens virulence protein VirE2, capable of forming a complex with single-stranded T-DNA during transfer into the plant cell, was isolated, purified, and partially characterized. Anti-VirE2 miniantibodies were obtained, and direct labeling of VirE2 with colloidal gold was done for the first time.     

Production of miniantibodies to Agrobacterium tumefaciens VirE2 virulence protein by the method of phage display

The scFv miniantibodies to the recombinant protein VirE2 from Agrobacterium tumefaciens were obtained by the method of phage display. The miniantibodies were purified and tested using timmunodot method for binding to a recombinant protein from Escherichia coli and to the native protein VirE2 from A. tumefaciens. The functional activity of the miniantibodies was comparable to the activity of mouse polyclonal antibodies against the VirE2 protein.     

VirE1 is a specific molecular chaperone for the exported single-stranded-DNA-binding protein VirE2 in Agrobacterium

Agrobacterium tumefaciens induces tumours on plants by transferring a nucleoprotein complex, the T-complex, from the bacterium to the plant cell. The T-complex consists of a single-stranded DNA (ssDNA) segment, the T-DNA, and VirD2, an endonuclease covalently attached to the 5' end of the T-DNA. A type IV secretion system encoded by the virB operon and virD4 is required for the entry of the T-complex and VirE2, a ssDNA-binding protein, into plant cells. The VirE1 protein is specifically required for the export of the VirE2 protein, as demonstrated by extracellular complementation and tumour formation. In this report, using a yeast two-hybrid system, we demonstrated that the VirE1 and VirE2 proteins interact and confirmed this interaction by in vitro binding assays. Although VirE2 is a ssDNA-binding protein, addition of ssDNA into the binding buffer did not interfere with the interaction of VirE1 and VirE2. VirE2 also interacts with itself, but the interaction between VirE1 and VirE2 is stronger than the VirE2 self-interaction, as measured in a lacZ reporter gene assay. In addition, the interaction of VirE2 with itself is inhibited by VirE1, indicating that VirE2 binds VirE1 preferentially. Analysis of various virE2 deletions indicated that the VirE1 interaction domain of VirE2 overlaps the VirE2 self-interaction domain. Incubation of extracts from Escherichia coli overexpressing His-VirE1 with the extracts of E. coli overexpressing His-VirE2 increased the yield of His-VirE2 in the soluble fraction. In a similar purified protein solubility assay, His-VirE1 increased the amount of His-VirE2 partitioning into the soluble fraction. In Agrobacterium, VirE2 was undetectable in the soluble protein fraction unless VirE1 was co-expressed. When urea was added to solubilize any large protein aggregates, a low level of VirE2 was detected. These results indicate that VirE1 prevents VirE2 from aggregating, enhances the stability of VirE2 and, perhaps, maintains VirE2 in an export-competent state. Analysis of the deduced amino acid sequence of the VirE1 protein revealed that the VirE1 protein shares a number of properties with molecular chaperones that are involved in the transport of specific proteins into animal and plant cells using type III secretion systems. We suggest that VirE1 functions as a specific molecular chaperone for VirE2, the first such chaperone linked to the presumed type IV secretion system.     

Agrobacterium tumefaciens oncogenic suppressors inhibit T-DNA and VirE2 protein substrate binding to the VirD4 coupling protein

Agrobacterium tumefaciens uses a type IV secretion (T4S) system composed of VirB proteins and VirD4 to deliver oncogenic DNA (T-DNA) and protein substrates to susceptible plant cells during the course of infection. Here, by use of the Transfer DNA ImmunoPrecipitation (TrIP) assay, we present evidence that the mobilizable plasmid RSF1010 (IncQ) follows the same translocation pathway through the VirB/D4 secretion channel as described previously for the T-DNA. The RSF1010 transfer intermediate and the Osa protein of plasmid pSa (IncW), related in sequence to the FiwA fertility inhibition factor of plasmid RP1 (IncPalpha), render A. tumefaciens host cells nearly avirulent. By use of a semi-quantitative TrIP assay, we show that both of these 'oncogenic suppressor factors' inhibit binding of T-DNA to the VirD4 substrate receptor. Both factors also inhibit binding of the VirE2 protein substrate to VirD4, as shown by coimmunoprecipitation and bimolecular fluorescence complementation assays. Osa fused to the green fluorescent protein (GFP) also blocks T-DNA and VirE2 binding to VirD4, and Osa-GFP colocalizes with VirD4 at A. tumefaciens cell poles. RSF1010 and Osa interfere specifically with VirD4 receptor function and not with VirB channel activity, as shown by (i) TrIP and (ii) a genetic screen for effects of the oncogenic suppressors on pCloDF13 translocation through a chimeric secretion channel composed of the pCloDF13-encoded MobB receptor and VirB channel subunits. Our findings establish that a competing plasmid substrate and a plasmid fertility inhibition factor act on a common target, the T4S receptor, to inhibit docking of DNA and protein substrates to the translocation apparatus.     

Right-border sequences enable the left border of an Agrobacterium tumefaciens nopaline Ti-plasmid to produce single-stranded DNA

The T-region of nopaline-type Ti-plasmids (the portion of the plasmid that is transferred to plant cells) of Agrobacterium tumefaciens is delimited by 23–25 bp direct repeats. They are nicked by the products of the virD locus and the presence of these nicked sites is correlated with the synthesis of single-stranded T-region copies. Despite previous indications to the contrary, we show that the pTiT37 T-region left border is capable of producing single-stranded DNA with high efficiency and that its ability to do so is totally dependent on right border-proximal cis-acting sequences, most probably overdrive, located several kilobases from the border. The absence of overdrive does not affect the single-strand nicking activity of the virD product but only the production of single-stranded copies from the nicked substrate.     

The lipoprotein VirB7 interacts with VirB9 in the membranes of Agrobacterium tumefaciens.

VirB9 and VirB7 are essential components of the putative VirB membrane channel required for transfer of the T-complex from Agrobacterium tumefaciens into plants. In this report, we present a biochemical analysis of their interaction and cellular localization. A comparison of relative electrophoretic mobilities under nonreducing and reducing conditions suggested that they form thiol-sensitive complexes with other proteins. Two-dimensional gel electrophoresis identified one complex as a heterodimer of VirB9 and VirB7 covalently linked by a disulfide bond, as well as VirB7 homodimers and monomers. Immunoprecipitation with VirB9-specific antiserum isolated the heterodimeric VirB9-VirB7 complex. Incubation with reducing agent split the complex into its constituent VirB9 and VirB7, which further confirmed linkage via cysteine residues. The interaction between VirB9 and VirB7 also was observed in the yeast two-hybrid system. Membrane attachment of VirB9-VirB7 may be conferred by lipoprotein modification, since labeling with [3H]palmitic acid in A. tumefaciens verified that VirB7 is a lipoprotein associated with VirB9. VirB9 and VirB7 showed equal distribution between inner and outer membranes, in accord with their proposed association with the transmembrane VirB complex.