The Agrobacterium T-DNA transport pore proteins VirB8, VirB9, and VirB10 interact with one another

The VirB proteins of Agrobacterium tumefaciens form a transport pore to transfer DNA from bacteria to plants. The assembly of the transport pore will require interaction among the constituent proteins. The identification of proteins that interact with one another can provide clues to the assembly of the transport pore. We studied interaction among four putative transport pore proteins, VirB7, VirB8, VirB9 and VirB10. Using the yeast two-hybrid assay, we observed that VirB8, VirB9, and VirB10 interact with one another. In vitro studies using protein fusions demonstrated that VirB10 interacts with VirB9 and itself. These results suggest that the outer membrane VirB7-VirB9 complex interacts with the inner membrane proteins VirB8 and VirB10 for the assembly of the transport pore. Fusions that contain small, defined segments of the proteins were used to define the interaction domains of VirB8 and VirB9. All interaction domains of both proteins mapped to the N-terminal half of the proteins. Two separate domains at the N- and C-terminal ends of VirB9 are involved in its homotypic interaction, suggesting that VirB9 forms a higher oligomer. We observed that the alteration of serine at position 87 of VirB8 to leucine abolished its DNA transfer function. Studies on the interaction of the mutant protein with the other VirB proteins showed that the VirB8S87L mutant is defective in interaction with VirB9. The mutant, however, interacted efficiently with VirB8 and VirB10, suggesting that the VirB8-VirB9 interaction is essential for DNA transfer.     

Identification of the VirB4-VirB8-VirB5-VirB2 pilus assembly sequence of type IV secretion systems

Type IV secretion systems mediate the translocation of virulence factors (proteins and/or DNA) from Gram-negative bacteria into eukaryotic cells. A complex of 11 conserved proteins (VirB1-VirB11) spans the inner and the outer membrane and assembles extracellular T-pili in Agrobacterium tumefaciens. Here we report a sequence of protein interactions required for the formation of complexes between VirB2 and VirB5, which precedes their incorporation into pili. The NTPase Walker A active site of the inner membrane protein VirB4 is required for virulence, but an active site VirB4 variant stabilized VirB3 and VirB8 and enabled T-pilus formation. Analysis of VirB protein complexes extracted from the membranes with mild detergent revealed that VirB2-VirB5 complex formation depended on VirB4, which identified a novel T-pilus assembly step. Bicistron expression demonstrated direct interaction of VirB4 with VirB8, and analyses with purified proteins showed that VirB5 bound to VirB8 and VirB10. VirB4 therefore localizes at the basis of a trans-envelope interaction sequence, and by stabilization of VirB8 it mediates the incorporation of VirB5 and VirB2 into extracellular pili.     

Agrobacterium tumefaciens VirB6 protein participates in formation of VirB7 and VirB9 complexes required for type IV secretion

This study characterized the contribution of Agrobacterium tumefaciens VirB6, a polytopic inner membrane protein, to the formation of outer membrane VirB7 lipoprotein and VirB9 protein multimers required for type IV secretion. VirB7 assembles as a disulfide cross-linked homodimer that associates with the T pilus and a VirB7-VirB9 heterodimer that stabilizes other VirB proteins during biogenesis of the secretion machine. Two presumptive VirB protein complexes, composed of VirB6, VirB7, and VirB9 and of VirB7, VirB9, and VirB10, were isolated by immunoprecipitation or glutathione S-transferase pulldown assays from detergent-solubilized membrane extracts of wild-type A348 and a strain producing only VirB6 through VirB10 among the VirB proteins. To examine the biological importance of VirB6 complex formation for type IV secretion, we monitored the effects of nonstoichiometric VirB6 production and the synthesis of VirB6 derivatives with 4-residue insertions (VirB6.i4) on VirB7 and VirB9 multimerization, T-pilus assembly, and substrate transfer. A virB6 gene deletion mutant accumulated VirB7 dimers at diminished steady-state levels, whereas complementation with a plasmid bearing wild-type virB6 partially restored accumulation of the dimers. VirB6 overproduction was correlated with formation of higher-order VirB9 complexes or aggregates and also blocked substrate transfer without a detectable disruption of T-pilus production; these phenotypes were displayed by cells grown at 28 degrees C, a temperature that favors VirB protein turnover, but not by cells grown at 20 degrees C. Strains producing several VirB6.i4 mutant proteins assembled novel VirB7 and VirB9 complexes detectable by nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and two strains producing the D60.i4 and L191.i4 mutant proteins translocated IncQ plasmid and VirE2 effector protein substrates in the absence of a detectable T pilus. Our findings support a model that VirB6 mediates formation of VirB7 and VirB9 complexes required for biogenesis of the T pilus and the secretion channel.     

The Agrobacterium tumefaciens VirB7 lipoprotein is required for stabilization of VirB proteins during assembly of the T-complex transport apparatus.

The Agrobacterium tumefaciens virB7 gene product is a lipoprotein whose function is required for the transmission of oncogenic T-DNA to susceptible plant cells. Three lines of study provided evidence that VirB7 interacts with and stabilizes other VirB proteins during the assembly of the putative T-complex transport apparatus. First, a precise deletion of virB7 from the pTiA6NC plasmid of wild-type strain A348 was correlated with significant reductions in the steady-state levels of several VirB proteins, including VirB4, VirB9, VirB10, and VirB11; trans expression of virB7 in the delta virB7 mutant partially restored the levels of these proteins, and trans coexpression of virB7 and virB8 fully restored the levels of these proteins to wild-type levels. Second, modulation of VirB7 levels resulted in corresponding changes in the levels of other VirB proteins in the following cell types: (i) a delta virB7 mutant expressing virB7 and virB8 from isopropyl-beta-D-thiogalactopyranoside (IPTG)-inducible Plac and other virB genes from acetosyringone (AS)-inducible PvirB; (ii) a delta virB operon mutant expressing virB7 and virB8 from Plac and virB9, virB10, and virB11 from PvirB; and (iii) a delta virB operon mutant expressing virB7 from IPTG-inducible Pklac and virB9 from an AS-inducible PvirB. Third, the synthesis of a VirB7::PhoA fusion protein in strain A348 was correlated with a significant reduction in the steady-state levels of VirB4, VirB5, and VirB7 through VirB11; these cells also exhibited a severely attenuated virulence phenotype, indicating that synthesis of the fusion protein perturbs the assembly of VirB proteins into a stabilized protein complex required for T-complex transport. Extracts of AS-induced cells electrophoresed under nonreducing conditions possessed undetectable levels of the 32-kDa VirB9 and 4.5-kDa VirB7 monomers and instead possessed a 36-kDa complex that cross-reacted with both VirB7 and VirB9 antisera and accumulated as a function of virB7 expression. Our results are consistent with a model in which VirB7 stabilizes VirB9 by formation of a covalent intermolecular cross-link; in turn, the VirB7-VirB9 heterodimer promotes the assembly of a functional T-complex transport machinery.     

VirB6 is required for stabilization of VirB5 and VirB3 and formation of VirB7 homodimers in Agrobacterium tumefaciens

VirB6 from Agrobacterium tumefaciens is an essential component of the type IV secretion machinery for T pilus formation and genetic transformation of plants. Due to its predicted topology as a polytopic inner membrane protein, it was proposed to form the transport pore for cell-to-cell transfer of genetic material and proteinaceous virulence factors. Here, we show that the absence of VirB6 leads to reduced cellular levels of VirB5 and VirB3, which were proposed to assist T pilus formation as minor component(s) or assembly factor(s), respectively. Overexpression of virB6 in trans restored levels of cell-bound and T pilus-associated VirB5 to wild type but did not restore VirB3 levels. Thus, VirB6 has a stabilizing effect on VirB5 accumulation, thereby regulating T pilus assembly. In the absence of VirB6, cell-bound VirB7 monomers and VirB7-VirB9 heterodimers were reduced and VirB7 homodimer formation was abolished. This effect could not be restored by expression of VirB6 in trans. Expression of TraD, a component of the transfer machinery of the IncN plasmid pKM101, with significant sequence similarity to VirB6, restored neither protein levels nor bacterial virulence but partly permitted T pilus formation in a virB6 deletion strain. VirB6 may therefore regulate T pilus formation by direct interaction with VirB5, and wild-type levels of VirB3 and VirB7 homodimers are not required.     

VirB1, a component of the T-complex transfer machinery of Agrobacterium tumefaciens, is processed to a C-terminal secreted product, VirB1.

During genetic transformation of plant cells by Agrobacterium tumefaciens, 11 VirB proteins and VirD4 are proposed to form a transmembrane bridge to transfer a DNA-protein complex (T-complex) into the plant cytoplasm. In this study, the localization of the first product of the virB operon, VirB1, was studied in detail. While full-length VirB1 localized mostly to the inner membrane, an immunoreactive VirB1 product was found as soluble processed form, designated VirB1*. Equal amounts of VirB1* could be detected in concentrated culture supernatants versus associated with the cell. VirB1* was purified from the supernatant of vir-induced cells by ammonium sulfate precipitation and Q-Sepharose chromatography. Sequence analysis of the N terminus of VirB1* localized the processing site after amino acid 172 of VirB1. Cell-associated VirB1* was partly removed by vortexing, suggesting a loose association with the cell or active secretion. However, cross-linking and coimmunoprecipitation showed a close association of cell-bound VirB1* with the VirB9-VirB7 heterodimer, a membrane-associated component of the T-complex transfer machinery. Homologies of the N-terminal part of VirB1 to bacterial transglycosylases suggest that it may assist T-complex transfer by local lysis of the bacterial cell wall, whereas the exposed localization of the C-terminal processing product VirB1* predicts direct interaction with the plant. Thus, VirB1 may be a bifunctional protein where both parts have different functions in T-complex transfer from Agrobacterium to plant cells.     

Delineation of the interaction domains of Agrobacterium tumefaciens VirB7 and VirB9 by use of the yeast two-hybrid assay.

The Agrobacterium tumefaciens VirB proteins are postulated to form a transport pore for the transfer of T-DNA. Formation of the transport pore will involve interactions among the VirB proteins. A powerful genetic method to study protein-protein interaction is the yeast two-hybrid assay. To test whether this method can be used to study interactions among the VirB membrane proteins, we studied the interaction of VirB7 and VirB9 in yeast. We recently demonstrated that VirB7 and VirB9 form a protein complex linked by a disulfide bond between cysteine 24 of VirB7 and cysteine 262 of VirB9 (L. Anderson, A. Hertzel, and A. Das, Proc. Natl. Acad. Sci. USA 93:8889-8894, 1996). We now demonstrate that VirB7 and VirB9 interact in yeast, and this interaction does not require the cysteine residues essential for the disulfide linkage. By using defined segments in fusion constructions, we mapped the VirB7 interaction domain of VirB9 to residues 173 to 275. In tumor formation assays, both virB7C24S and virB9C262S expressed from a multicopy plasmid complemented the respective deletion mutation, indicating that the cysteine residues may not be essential for DNA transfer.     

Interactions between VirB9 and VirB10 membrane proteins involved in movement of DNA from Agrobacterium tumefaciens into plant cells.

The 11 VirB proteins from Agrobacterium tumefaciens are predicted to form a membrane-bound complex that mediates the movement of DNA from the bacterium into plant cells. The studies reported here on the possible VirB protein interactions in such a complex demonstrate that VirB9 and VirB10 can each form high-molecular-weight complexes after treatment with a chemical cross-linker. Analysis of nonpolar virB mutants showed that the formation of the VirB10 complexes does not occur in a virB9 mutant and that VirB9 and VirB10 are not components of the same cross-linked complex. VirB9, when stabilized by the concurrent expression of VirB7, was shown to be sufficient to permit VirB10 to cross-link into its usual high-molecular-weight forms in the absence of other Vir proteins. Randomly introduced single point mutations in virB9 resulted in Agrobacterium strains with severely attenuated virulence. Although some of the mutants contained wild-type levels of VirB9 and displayed an unaltered VirB9 cross-linking pattern, VirB10 cross-linking was drastically reduced. We conclude that specific amino acid residues in VirB9 are necessary for interaction with VirB10 resulting in the capacity of VirB10 to participate in high-molecular-weight complexes that can be visualized by chemical cross-linking.     

The type IV secretion apparatus protein VirB6 of Agrobacterium tumefaciens localizes to a cell pole

Agrobacterium tumefaciens VirB proteins assemble a type IV secretion apparatus for the transfer of DNA and proteins to plant cells. To study the role of the VirB6 protein in the assembly and function of the type IV apparatus, we determined its subcellular location by immunofluorescence microscopy. In wild-type bacteria VirB6 localized to the cell poles but in the absence of the tumour-inducing plasmid it localized to random sites on the cell membranes. Five of the 11 VirB proteins, VirB7-VirB11, are required for the polar localization of VirB6. We identified two regions of VirB6, a conserved tryptophan residue at position 197 and the extreme C-terminus, that are essential for its polar localization. Topology determination by PhoA fusion analysis placed both regions in the cell cytoplasm. Alteration of tryptophan 197 or the deletion of the extreme C-terminus led to the mislocalization of the mutant protein. The mutations abolished the DNA transfer function of the protein as well. The C-terminus of VirB6, in silico, can form an amphipathic helix that may encode a protein-protein interaction domain essential for targeting the protein to a cell pole. We previously reported that another DNA transfer protein, VirD4, localizes to a cell pole. To determine whether VirB6 and VirD4 localize to the same pole, we performed colocalization experiments. Both proteins localized to the same pole indicating that VirB6 and VirD4 are in close proximity and VirB6 is probably a component of the transport apparatus.     

VirB7 lipoprotein is exocellular and associates with the Agrobacterium tumefaciens T pilus

Agrobacterium tumefaciens transfers oncogenic T-DNA and effector proteins to plant cells via a type IV secretion pathway. This transfer system, assembled from the products of the virB operon, is thought to consist of a transenvelope mating channel and the T pilus. When screened for the presence of VirB and VirE proteins, material sheared from the cell surface of octopine strain A348 was seen to possess detectable levels of VirB2 pilin, VirB5, and the VirB7 outer membrane lipoprotein. Material sheared from the cell surface of most virB gene deletion mutants also possessed VirB7, but not VirB2 or VirB5. During purification of the T pilus from wild-type cells, VirB2, VirB5, and VirB7 cofractionated through successive steps of gel filtration chromatography and sucrose density gradient centrifugation. A complex containing VirB2 and VirB7 was precipitated from a gel filtration fraction enriched for T pilus with both anti-VirB2 and anti-VirB7 antiserum. Both the exocellular and cellular forms of VirB7 migrated as disulfide-cross-linked dimers and monomers when samples were electrophoresed under nonreducing conditions. A mutant synthesizing VirB7 with a Ser substitution of the lipid-modified Cys15 residue failed to elaborate the T pilus, whereas a mutant synthesizing VirB7 with a Ser substitution for the disulfide-reactive Cys24 residue produced very low levels of T pilus. Together, these findings establish that the VirB7 lipoprotein localizes exocellularly, it associates with the T pilus, and both VirB7 lipid modification and disulfide cross-linking are important for T-pilus assembly. T-pilus-associated VirB2 migrated in nonreducing gels as a monomer and a disulfide-cross-linked homodimer, whereas cellular VirB2 migrated as a monomer. A strain synthesizing a VirB2 mutant with a Ser substitution for the reactive Cys64 residue elaborated T pilus but exhibited an attenuated virulence phenotype. Dithiothreitol-treated T pilus composed of native VirB2 pilin and untreated T pilus composed of the VirB2C64S mutant pilin distributed in sucrose gradients more predominantly in regions of lower sucrose density than untreated, native T pili. These findings indicate that intermolecular cross-linking of pilin monomers is not required for T-pilus production, but cross-linking does contribute to T-pilus stabilization.     

Assembly of the VirB transport complex for DNA transfer from Agrobacterium tumefaciens to plant cells

The VirB transporter is a type IV secretion system that mediates the genetic transformation of plant cells by Agrobacterium tumefaciens. Assembly of this transporter depends on, first, formation of a VirB7/B9 complex that stabilizes many of the VirB proteins, second, formation of a virulence-specific pilus composed primarily of VirB2 and VirB5, and, third, post-translational processing of VirB1 and VirB2.     

Agrobacterium tumefaciens VirB9, an outer-membrane-associated component of a type IV secretion system, regulates substrate selection and T-pilus biogenesis

Agrobacterium tumefaciens translocates DNA and protein substrates between cells via a type IV secretion system (T4SS) whose channel subunits include the VirD4 coupling protein, VirB11 ATPase, VirB6, VirB8, VirB2, and VirB9. In this study, we used linker insertion mutagenesis to characterize the contribution of the outer-membrane-associated VirB9 to assembly and function of the VirB/D4 T4SS. Twenty-five dipeptide insertion mutations were classified as permissive for intercellular substrate transfer (Tra+), completely transfer defective (Tra-), or substrate discriminating, e.g., selectively permissive for transfer only of the oncogenic transfer DNA and the VirE2 protein substrates or of a mobilizable IncQ plasmid substrate. Mutations inhibiting transfer of DNA substrates did not affect formation of close contacts of the substrate with inner membrane channel subunits but blocked formation of contacts with the VirB2 and VirB9 channel subunits, which is indicative of a defect in assembly or function of the distal portion of the secretion channel. Several mutations in the N- and C-terminal regions disrupted VirB9 complex formation with the outer-membrane-associated lipoprotein VirB7 or the inner membrane energy sensor VirB10. Several VirB9.i2-producing Tra+ strains failed to elaborate T pilus at detectable levels (Pil-), and three such Tra+ Pil- mutant strains were rendered Tra- upon deletion of virB2, indicating that the cellular form of pilin protein is essential for substrate translocation. Our findings, together with computer-based analyses, support a model in which distinct domains of VirB9 contribute to substrate selection and translocation, establishment of channel subunit contacts, and T-pilus biogenesis.     

Membrane location of the Ti plasmid VirB proteins involved in the biosynthesis of a pilin-like conjugative structure on Agrobacterium tumefaciens

Abstract The virB operon of the Agrobacterium tumefaciens Ti plasmid encodes 11 proteins. Specific antisera to VirB2, VirB3 and VirB9 were used to locate these virulence proteins in the A. tumefaciens cell. Immunoblot analysis located VirB2 protein to the inner and outer membranes; VirB3 and VirB9 were likewise associated with both membranes, but mainly in the outer membrane. VirB2 is processed from a 12.3-kDa protein into a 7.2-kDa polypeptide. Such sized protein results from cleavage at residue Ala⁴⁷, upstream of which two additional alanine residues Ala⁴⁵-Ala⁴⁶ are contained and bearing resemblance to a signal peptide peptidase-I cleavage sequence. VirB2 and VirB3 sequences are strikingly similar to the pilin biosynthetic proteins TraA and TraL encoded by the tra operon of F and R1-19 plasmids. Since traA encodes a propilin that is cleaved into a 7.2-kDa conjugative pilin product and since this cleavage site is present in both TraA and VirB2, we propose that virB2 encodes a pilin-like protein which together with VirB3 and VirB9 as well as other VirB proteins may be used for interkingdom T-DNA transfer between bacteria and plants.     

Self-assembly of the Agrobacterium tumefaciens VirB11 traffic ATPase

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 lambda cI repressor protein formed dimers, as shown by immunity of Escherichia coli to lambda 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. tumefaciens cells. 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 (Delta GKT174-176). 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.     

Four VirB6 paralogs and VirB9 are expressed and interact in Ehrlichia chaffeensis-containing vacuoles

The type IV secretion system is an important virulence factor in several host cell-associated pathogens, as it delivers various bacterial macromolecules to target eukaryotic cells. Genes homologous to several virB genes and virD4 of Agrobacterium tumefaciens are found in an intravacuolar pathogen Ehrlichia chaffeensis, the tick-borne causative agent of human monocytic ehrlichiosis. In particular, despite its small genome size, E. chaffeensis has four tandem virB6 paralogs (virB6-1, -2, -3, and -4) that are 3- to 10-fold larger than A. tumefaciens virB6. The present study for the first time illustrates the relevance of the larger quadruple VirB6 paralogs by demonstrating the protein expression and interaction in E. chaffeensis. All four virB6 paralogs were cotranscribed in THP-1 human leukemia and ISE6 tick cell cultures. The four VirB6 proteins and VirB9 were expressed by E. chaffeensis in THP-1 cells, and amounts of these five proteins were similar in isolated E. chaffeensis-containing vacuoles and vacuole-free E. chaffeensis. In addition, an 80-kDa fragment of VirB6-2 was detected, which was strikingly more prevalent in E. chaffeensis-containing vacuoles than in vacuole-free E. chaffeensis. Coimmunoprecipitation analysis revealed VirB9 interaction with VirB6-1 and VirB6-2; VirB6-4 interaction with VirB6-1, VirB6-2, and VirB6-3; and VirB6-2 80-kDa fragment interaction with VirB6-3 and VirB6-4. The interaction of VirB9 and VirB6-2 was confirmed by far-Western blotting. The results suggest that E. chaffeensis VirB9, the quadruple VirB6 proteins, and the VirB6-2 80-kDa fragment form a unique molecular subassembly to cooperate in type IV secretion.     

Functional analysis of the Agrobacterium tumefaciens T-DNA transport pore protein VirB8

The VirB8 protein of Agrobacterium tumefaciens is essential for DNA transfer to plants. VirB8, a 237-residue polypeptide, is an integral membrane protein with a short N-terminal cytoplasmic domain. It interacts with two transport pore proteins, VirB9 and VirB10, in addition to itself. To study the role of these interactions in DNA transfer and to identify essential amino acids of VirB8, we introduced random mutations in virB8 by the mutagenic PCR method. The putative mutants were tested for VirB8 function by the ability to complement a virB8 deletion mutant in tumor formation assays. After multiple rounds of screening 13 mutants that failed to complement the virB8 deletion mutation were identified. Analysis of the mutant strains by DNA sequence analysis, Western blot assays, and reconstruction of new point mutations led to the identification of five amino acid residues that are essential for VirB8 function. The substitution of glycine-78 to serine, serine-87 to leucine, alanine-100 to valine, arginine-107 to proline or alanine, and threonine-192 to methionine led to the loss of VirB8 activity. When introduced into the wild-type strain, virB8(S87L) partially suppressed the tumor forming ability of the wild-type protein. Analysis of protein-protein interaction by the yeast two-hybrid assay indicated that VirB8(R107P) is defective in interactions with both VirB9 and VirB10. A second mutant VirB8(S87L) is defective in interaction with VirB9.     

The type IV secretion system component VirB5 binds to the trans-zeatin biosynthetic enzyme Tzs and enables its translocation to the cell surface of Agrobacterium tumefaciens

VirB5 is a minor component of the extracellular T pilus determined by the Agrobacterium tumefaciens type IV secretion system. To identify proteins that interact with VirB5 during the pilus assembly process, we purified VirB5 as a recombinant fusion protein and, by using a gel overlay assay, we detected a 26-kDa interacting protein in Agrobacterium cell lysates. The VirB5-binding protein was purified from A. tumefaciens and identified as the cytokinin biosynthetic enzyme Tzs. The VirB5-Tzs interaction was confirmed using pulldown assays with purified proteins and the yeast two-hybrid system. An analysis of the subcellular localization in A. tumefaciens showed that Tzs was present in the soluble as well as the membrane fraction. Tzs was extracted from the membranes with the mild detergent dodecyl-beta-D-maltoside in complexes of different molecular masses, and this association was strongly reduced in the absence of VirB5. Using immunoelectron microscopy, we also detected Tzs on the Agrobacterium cell surface. A functional type IV secretion system was required for efficient translocation to the surface, but Tzs was not secreted into the cell supernatant. The fact that Tzs localizes on the cell surface suggests that it may contribute to the interaction of Agrobacterium with plants.     

A component of the Xanthomonadaceae type IV secretion system combines a VirB7 motif with a N0 domain found in outer membrane transport proteins

Type IV secretion systems (T4SS) are used by Gram-negative bacteria to translocate protein and DNA substrates across the cell envelope and into target cells. Translocation across the outer membrane is achieved via a ringed tetradecameric outer membrane complex made up of a small VirB7 lipoprotein (normally 30 to 45 residues in the mature form) and the C-terminal domains of the VirB9 and VirB10 subunits. Several species from the genera of Xanthomonas phytopathogens possess an uncharacterized type IV secretion system with some distinguishing features, one of which is an unusually large VirB7 subunit (118 residues in the mature form). Here, we report the NMR and 1.0 Å X-ray structures of the VirB7 subunit from Xanthomonas citri subsp. citri (VirB7_XAC2622) and its interaction with VirB9. NMR solution studies show that residues 27–41 of the disordered flexible N-terminal region of VirB7_XAC2622 interact specifically with the VirB9 C-terminal domain, resulting in a significant reduction in the conformational freedom of both regions. VirB7_XAC2622 has a unique C-terminal domain whose topology is strikingly similar to that of N0 domains found in proteins from different systems involved in transport across the bacterial outer membrane. We show that VirB7_XAC2622 oligomerizes through interactions involving conserved residues in the N0 domain and residues 42–49 within the flexible N-terminal region and that these homotropic interactions can persist in the presence of heterotropic interactions with VirB9. Finally, we propose that VirB7_XAC2622 oligomerization is compatible with the core complex structure in a manner such that the N0 domains form an extra layer on the perimeter of the tetradecameric ring.     

Symbiotic phenotypes and translocated effector proteins of the Mesorhizobium loti strain R7A VirB/D4 type IV secretion system

The symbiosis island of Mesorhizobium loti strain R7A contains genes with strong similarity to the structural vir genes (virB1-11; virD4) of Agrobacterium tumefaciens that encode the type IV secretion system (T4SS) required for T-DNA transfer to plants. In contrast, M. loti strain MAFF303099 lacks these genes but contains genes not present in strain R7A that encode a type III secretion system (T3SS). Here we show by hybridization analysis that most M. loti strains contain the VirB/D4 T4SS and not the T3SS. Strikingly, strain R7A vir gene mutants formed large nodules containing bacteroids on Leucaena leucocephala in contrast to the wild-type strain that formed only uninfected tumour-like structures. A rhcJ T3SS mutant of strain MAFF303099 also nodulated L. leucocephala, unlike the wild type. On Lotus corniculatus, the vir mutants were delayed in nodulation and were less competitive compared with the wild type. Two strain R7A genes, msi059 and msi061, were identified through their mutant phenotypes as possibly encoding translocated effector proteins. Both Msi059 and Msi061 were translocated through the A. tumefaciens VirB/D4 system into Saccharomyces cerevisiae and Arabidopsis thaliana, as shown using the Cre recombinase Reporter Assay for Translocation (CRAfT). Taken together, these results suggest that the VirB/D4 T4SS of M. loti R7A plays an analogous symbiotic role to that of T3SS found in other rhizobia. The heterologous translocation of rhizobial proteins by the Agrobacterium VirB/D4 T4SS is the first demonstration that rhizobial effector proteins are translocated into plant cells and confirms functional conservation between the M. loti and A. tumefaciens T4SS.