Analysis of Vir protein translocation from Agrobacterium tumefaciens using Saccharomyces cerevisiae as a model: evidence for transport of a novel effector protein VirE3

Agrobacterium tumefaciens causes crown gall disease on a variety of plants. During the infection process Agrobacterium transfers a nucleoprotein complex, the VirD2 T-complex, and at least two Vir proteins, VirE2 and VirF, into the plant cell via the VirB/VirD4 type IV secretion system. Recently, we found that T-DNA could also be transferred from Agrobacterium to Saccharomyces cerevisiae. Here, we describe a novel method to also detect trans-kingdom Vir protein transfer from Agrobacterium to yeast, using the Cre/lox system. Protein fusions between Cre and VirE2 or VirF were expressed in Agrobacterium. Transfer of the Cre-Vir fusion proteins from Agrobacterium to yeast was monitored by a selectable excision event resulting from site-specific recombination mediated by Cre on a lox-flanked transgene in yeast. The VirE2 and VirF proteins were transported to yeast via the virB-encoded transfer system in the presence of coupling factor VirD4, analogous to translocation into plant cells. The yeast system therefore provides a suitable and fast model system to study basic aspects of trans-kingdom protein transport from Agrobacterium into host cells. Using this method we showed that VirE2 and VirF protein transfer was inhibited by the presence of the Osa protein. Besides, we found evidence for a novel third effector protein, VirE3, which has a similar C-terminal signature to VirE2 and VirF.     

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.     

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.     

Elevated temperature differentially affects virulence, VirB protein accumulation, and T-pilus formation in different Agrobacterium tumefaciens and Agrobacterium vitis strains.

That gene transfer to plant cells is a temperature-sensitive process has been known for more than 50 years. Previous work indicated that this sensitivity results from the inability to assemble a functional T pilus required for T-DNA and protein transfer to recipient cells. The studies reported here extend these observations and more clearly define the molecular basis of this assembly and transfer defect. T-pilus assembly and virulence protein accumulation were monitored in Agrobacterium tumefaciens strain C58 at different temperatures ranging from 20 degrees C to growth-inhibitory 37 degrees C. Incubation at 28 degrees C but not at 26 degrees C strongly inhibited extracellular assembly of the major T-pilus component VirB2 as well as of pilus-associated protein VirB5, and the highest amounts of T pili were detected at 20 degrees C. Analysis of temperature effects on the cell-bound virulence machinery revealed three classes of virulence proteins. Whereas class I proteins (VirB2, VirB7, VirB9, and VirB10) were readily detected at 28 degrees C, class II proteins (VirB1, VirB4, VirB5, VirB6, VirB8, VirB11, VirD2, and VirE2) were only detected after cell growth below 26 degrees C. Significant levels of class III proteins (VirB3 and VirD4) were only detected at 20 degrees C and not at higher temperatures. Shift of virulence-induced agrobacteria from 20 to 28 or 37 degrees C had no immediate effect on cell-bound T pili or on stability of most virulence proteins. However, the temperature shift caused a rapid decrease in the amount of cell-bound VirB3 and VirD4, and VirB4 and VirB11 levels decreased next. To assess whether destabilization of virulence proteins constitutes a general phenomenon, levels of virulence proteins and of extracellular T pili were monitored in different A. tumefaciens and Agrobacterium vitis strains grown at 20 and 28 degrees C. Levels of many virulence proteins were strongly reduced at 28 degrees C compared to 20 degrees C, and T-pilus assembly did not occur in all strains except "temperature-resistant" Ach5 and Chry5. Virulence protein levels correlated well with bacterial virulence at elevated temperature, suggesting that degradation of a limited set of virulence proteins accounts for the temperature sensitivity of gene transfer to plants.     

Interaction of the virulence protein VirF of Agrobacterium tumefaciens with plant homologs of the yeast Skp1 protein

The infection of plants by Agrobacterium tumefaciens leads to the formation of crown gall tumors due to the transfer of a nucleoprotein complex into plant cells that is mediated by the virulence (vir) region-encoded transport system (reviewed in [1-5]). In addition, A. tumefaciens secretes the Vir proteins, VirE2 and VirF, directly into plant cells via the same VirB/VirD4 transport system [6], and both assist there in the transformation of normal cells into tumor cells. The function of the 22 kDa VirF protein is not clear. Deletion of the virF gene in A. tumefaciens leads to diminished virulence [7, 8] and can be complemented by the expression of the virF gene in the host plant. This finding indicates that VirF functions within the plant cell [8]. Here, we report that the VirF protein is the first prokaryotic protein with an F box by which it can interact with plant homologs of the yeast Skp1 protein. The presence of the F box turned out to be essential for the biological function of VirF. F box proteins and Skp1p are both subunits of a class of E3 ubiquitin ligases referred to as SCF complexes. Thus, VirF may be involved in the targeted proteolysis of specific host proteins in early stages of the transformation process.     

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.     

Role of bacterial virulence proteins in Agrobacterium-mediated transformation of Aspergillus awamori

The Agrobacterium-mediated transformation of Aspergillus awamori was optimized using defined co-cultivation conditions, which resulted in a reproducible and efficient transformation system. Optimal co-cultivation conditions were used to study the role of Agrobacterium tumefaciens virulence proteins in T-DNA transfer. This study revealed that inactivation of either of the regulatory proteins (VirA, VirG), any of the transport pore proteins (VirB), proteins involved in generation of the T-strand (VirD, VirC) or T-strand protection and targeting (VirE2) abolishes or severely reduces the formation of transformants. The results indicate that the Agrobacterium-mediated transformation of A. awamori requires an intact T-DNA machinery for efficient transformation; however, the plant host range factors, like VirE3, VirH, and VirF, are not important.     

Transport of DNA into the nuclei of xenopus oocytes by a modified VirE2 protein of Agrobacterium.

We used Agrobacterium T-DNA nuclear transport to examine the specificity of nuclear targeting between plants and animals and the nuclear import of DNA by a specialized transport protein. Two karyophilic Agrobacterium virulence (Vir) proteins, VirD2 and VirE2, which presumably associate with the transported T-DNA and function in many plant species, were microinjected into Drosophila embryos and Xenopus oocytes. In both animal systems, VirD2 localized to the cell nuclei and VirE2 remained exclusively cytoplasmic, suggesting that VirE2 nuclear localization signals may be plant specific. Repositioning one amino acid residue within VirE2 nuclear localization signals enabled them to function in animal cells. The modified VirE2 protein bound DNA and actively transported it into the nuclei of Xenopus oocytes. These observations suggest a functional difference in nuclear import between animals and plants and show that DNA can be transported into the cell nucleus via a protein-specific pathway.     

DNA Substrate-Induced Activation of the Agrobacterium VirB/VirD4 Type IV Secretion System

The bitopic membrane protein VirB10 of the Agrobacterium VirB/VirD4 type IV secretion system (T4SS) undergoes a structural transition in response to sensing of ATP binding or hydrolysis by the channel ATPases VirD4 and VirB11. This transition, detectable as a change in protease susceptibility, is required for DNA substrate passage through the translocation channel. Here, we present evidence that DNA substrate engagement with VirD4 and VirB11 also is required for activation of VirB10. Several DNA substrates (oncogenic T-DNA and plasmids RSF1010 and pCloDF13) induced the VirB10 conformational change, each by mechanisms requiring relaxase processing at cognate oriT sequences. VirD2 relaxase deleted of its translocation signal or any of the characterized relaxases produced in the absence of cognate DNA substrates did not induce the structural transition. Translocated effector proteins, e.g., VirE2, VirE3, and VirF, also did not induce the transition. By mutational analyses, we supplied evidence that the N-terminal periplasmic loop of VirD4, in addition to its catalytic site, is essential for early-stage DNA substrate transfer and the VirB10 conformational change. Further studies of VirB11 mutants established that three T4SS-mediated processes, DNA transfer, protein transfer, and pilus production, can be uncoupled and that the latter two processes proceed independently of the VirB10 conformational change. Our findings support a general model whereby DNA ligand binding with VirD4 and VirB11 stimulates ATP binding/hydrolysis, which in turn activates VirB10 through a structural transition. This transition confers an open-channel configuration enabling passage of the DNA substrate to the cell surface.     

Agrobacterium proteins VirD2 and VirE2 mediate precise integration of synthetic T-DNA complexes in mammalian cells

Agrobacterium tumefaciens-mediated plant transformation, a unique example of interkingdom gene transfer, has been widely adopted for the generation of transgenic plants. In vitro synthesized transferred DNA (T-DNA) complexes comprising single-stranded DNA and Agrobacterium virulence proteins VirD2 and VirE2, essential for plant transformation, were used to stably transfect HeLa cells. Both proteins positively influenced efficiency and precision of transgene integration by increasing overall transformation rates and by promoting full-length single-copy integration events. These findings demonstrate that the virulence proteins are sufficient for the integration of a T-DNA into a eukaryotic genome in the absence of other bacterial or plant factors. Synthetic T-DNA complexes are therefore unique protein:DNA delivery vectors with potential applications in the field of mammalian transgenesis.     

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.     

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.     

Several components of SKP1/Cullin/F-box E3 ubiquitin ligase complex and associated factors play a role in Agrobacterium-mediated plant transformation

? Successful genetic transformation of plants by Agrobacterium tumefaciens requires the import of bacterial T-DNA and virulence proteins into the plant cell that eventually form a complex (T-complex). The essential components of the T-complex include the single stranded T-DNA, bacterial virulence proteins (VirD2, VirE2, VirE3 and VirF) and associated host proteins that facilitate the transfer and integration of T-DNA. The removal of the proteins from the T-complex is likely achieved by targeted proteolysis mediated by VirF and the plant ubiquitin proteasome complex. ? We evaluated the involvement of the host SKP1/culin/F-box (SCF)-E3 ligase complex and its role in plant transformation. Gene silencing, mutant screening and gene expression studies suggested that the Arabidopsis homologs of yeast SKP1 (suppressor of kinetochore protein 1) protein, ASK1 and ASK2, are required for Agrobacterium-mediated plant transformation. ? We identified the role for SGT1b (suppressor of the G2 allele of SKP1), an accessory protein that associates with SCF-complex, in plant transformation. We also report the differential expression of many genes that encode F-box motif containing SKP1-interacting proteins (SKIP) upon Agrobacterium infection. ? We speculate that these SKIP genes could encode the plant specific F-box proteins that target the T-complex associated proteins for polyubiquitination and subsequent degradation by the 26S proteasome.     

T-DNA转移及整合的分子机制研究进展

农杆菌介导的外源基因转化是目前植物转基因应用比较广泛的方法。近年来关于农杆菌介导的整合机制的研究已经取得了很大的进步。研究表明,在VirD2和VirE2协助下,农杆菌转移T-DNA进入植物细胞,这两种蛋白共同协助T-DNA完成转移、核定位及在植物基因组中的整合。近年来关于拟南芥突变体的研究表明,被转化植物的宿主基因在T-DNA转移及整合过程中发挥主要的作用。通过对现有研究成果详细讨论了Vir’蛋白(VirD2和VirE2)及植物基因在农杆菌介导植物转化中的作用,详细讨论了依靠VirD2蛋白和SDSA(synthesis-dependent strand-annealing)整合的两类不同的整合模式,根据最新的研究成果阐述了以基因组的双链断裂修复为基础的整合模型,并提出新的观点。     

The Agrobacterium DNA Transfer Complex

Agrobacterium tumefaciens elicits tumorous growths on plants by transforming plant cells with a segment of its own DNA. This trait led to development of Agrobacterium as a vector for genetic transformation of flowering plants. The transformation process is a unique mixture of several distinct steps, some of which are evolutionarily and functionally related to bacterial conjugation, and some of which converge with eukaryotic cellular processes. Recent work has advanced our understanding of each of these steps. The early reactions in the production of an ssDNA transfer intermediate (T-strand), mediated by the VirD1/D2 proteins, are chemically similar to formation of a relaxosome in bacterial conjugation. The T-strand is coated by the ssDNA binding protein VirE2; however, whether this binding occurs in the bacterium or in planta is disputed. VirB proteins, related to proteins for the conjugal transfer of DNA between bacteria, most likely form the transfer apparatus. VirD2, which remains covalently bound to the 5′ end of the T-strand, and VirE2 both localize to the nucleus of plant cells. VirE2 also mediates the nuclear accumulation of ssDNA but only when the protein is bound to the ssDNA. Genetically, VirD2 is required for faithful integration of the 5′ end and VirE2 for the 3′ end of the T-strand. The steps of the process currently under active investigation are the assembly of the export apparatus and the enzymology of integration.     

The carboxy-terminus of VirE2 from Agrobacterium tumefaciens is required for its transport to host cells by the virB-encoded type IV transport system

Agrobacterium tumefaciens transfers DNA from the resident 'tumour-inducing' (Ti) plasmid into plant cells, where it can be stably integrated into the plant genome, ultimately resulting in crown gall tumour formation. The mobilized DNA molecule is a single-stranded intermediate with VirD2 covalently bound to its 5' end. Successful transport of the transferred DNA (T-DNA) and integration of the DNA into the genome requires that additional proteins be transported to the plant as well, including the single-stranded (ss)DNA-binding protein, VirE2. The transport of these two different substrates occurs as a result of the activities of a type IV secretion system encoded by the virB operon. Although the substrates have been identified, the mechanism of their transport remains unknown. In the experiments described here, a region in one of these substrates, VirE2, necessary for transport is identified. The addition of a C-terminal FLAG epitope tag to VirE2, or the deletion of its C-terminal 18 amino acids, renders it non-functional in A. tumefaciens. However, transgenic plants expressing either of these virE2 genes respond to virE2 mutants of A. tumefaciens by forming wild-type tumours. These results indicate that this region of VirE2 is necessary for the protein to be transported into the plant cells, but is not necessary for its function within the plant. Additionally, these studies demonstrate that mutant forms of VirE2 lacking this region do not disrupt the activities of the VirB transporter and support the hypothesis that VirE2 and the VirD2 T-strand are transported independently, even when they co-exist in the same cell.     

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.     

The role of the T-pilus in horizontal gene transfer and tumorigenesis

The T-pilus is a flexuous filamentous appendage that is essential for Agrobacterium tumefaciens virulence. T-pilus subunits are derived from a VirB2-processing reaction that generates cyclized polypeptide subunits. The T-pilus filament has a diameter of 10 nm and contains a lumen approximately 2 nm in diameter. Biogenesis of the T-pilus requires all 11 VirB proteins, but not the VirD4 protein, which is used in conjugal plasmid transfer. VirB4 and VirB11 are two ATPases that may form homohexameric rings within the transport apparatus, which is composed of VirB6-10 proteins.     

Protein interactions involved in nuclear import of the Agrobacterium VirE2 protein in vivo and in vitro

Agrobacterium, the only known organism capable of trans-kingdom DNA transfer, genetically transforms plants by transferring a segment of its DNA, T-DNA, into the nucleus of the host cell where it integrates into the plant genome. One of the central events in this genetic transformation process is nuclear import of the T-DNA molecule, which to a large degree is mediated by the bacterial virulence protein VirE2. VirE2 is distinguished by its nuclear targeting, which occurs only in plant but not in animal cells and is facilitated by the cellular VIP1 protein. The molecular mechanism of the VIP1 function is still unclear. Here, we used in vitro assays for nuclear import and quantification of protein-protein interactions to directly demonstrate formation of ternary complexes between VirE2, VIP1, and a component of the cellular nuclear import machinery, karyopherin alpha. Our results indicate that VIP1 functions as a molecular bridge between VirE2 and karyopherin alpha, allowing VirE2 to utilize the host cell nuclear import machinery even without being directly recognized by its components.     

Dimerization of VirD2 Binding Protein Is Essential for Agrobacterium Induced Tumor Formation in Plants

The Type IV Secretion System (T4SS) is the only bacterial secretion system known to translocate both DNA and protein substrates. The VirB/D4 system from Agrobacterium tumefaciens is a typical T4SS. It facilitates the bacteria to translocate the VirD2-T-DNA complex to the host cell cytoplasm. In addition to protein-DNA complexes, the VirB/D4 system is also involved in the translocation of several effector proteins, including VirE2, VirE3 and VirF into the host cell cytoplasm. These effector proteins aid in the proper integration of the translocated DNA into the host genome. The VirD2-binding protein (VBP) is a key cytoplasmic protein that recruits the VirD2–T-DNA complex to the VirD4-coupling protein (VirD4 CP) of the VirB/D4 T4SS apparatus. Here, we report the crystal structure and associated functional studies of the C-terminal domain of VBP. This domain mainly consists of α-helices, and the two monomers of the asymmetric unit form a tight dimer. The structural analysis of this domain confirms the presence of a HEPN (higher eukaryotes and prokaryotes nucleotide-binding) fold. Biophysical studies show that VBP is a dimer in solution and that the HEPN domain is the dimerization domain. Based on structural and mutagenesis analyses, we show that substitution of key residues at the interface disrupts the dimerization of both the HEPN domain and full-length VBP. In addition, pull-down analyses show that only dimeric VBP can interact with VirD2 and VirD4 CP. Finally, we show that only Agrobacterium harboring dimeric full-length VBP can induce tumors in plants. This study sheds light on the structural basis of the substrate recruiting function of VBP in the T4SS pathway of A. tumefaciens and in other pathogenic bacteria employing similar systems.