VirD2 Protein Research Articles

An Agrobacterium VirE2 channel for transferred-DNA transport into plant cells

Transferred DNA (T-DNA) transfer from Agrobacterium tumefaciens into eukaryotic cells is the only known example of interkingdom DNA transfer. T-DNA is a single-stranded segment of Agrobacterium's tumor-inducing plasmid that enters the plant cell as a complex with the bacterial virulence proteins VirD2 and VirE2. The VirE2 protein is highly induced on contact of A. tumefaciens with a plant host and has been reported to act in late steps of transfer. One of its previously demonstrated functions is binding to the single-stranded (ss) T-DNA and protecting it from degradation. Recent experiments suggest other functions of the protein. A combination of planar lipid bilayer experiments, vesicle swelling assays, and DNA transport experiments demonstrated that VirE2 can insert itself into artificial membranes and form channels. These channels are voltage gated, anion selective, and single-stranded DNA-specific and can facilitate the efficient transport of single-stranded DNA through membranes. These experiments demonstrate a VirE2 function as a transmembrane DNA transporter, which could have applications in gene delivery systems.

A conduit for T-DNA through the plant cell membrane: dig it yourself!

Agrobacterium tumefaciens is a plant-pathogenic bacterium notorious for its ability to redirect the plant metabolism to meet the pathogen's gourmandise. This is achieved by the stable genetic modification of the host plant after integration of the bacterial T-DNA into the plant genome, a unique example of inter-kingdom DNA transfer. The T-DNA stems from the bacterial tumor-inducing (Ti) plasmid and travels as a single-stranded molecule from the bacterial to the plant cell, tipped with a covalently linked single molecule of VirD2 protein. Once inside the plant cell, many, cooperatively bound VirE2 protein molecules coat the T-DNA. Laboratory strains of Agrobacterium have had to endure significant disarmament of their pathogenicity and subsequent widespread exploitation as a tool in plant transformation. The genes on the Ti plasmid can be reduced to those that are essential for a successful T-DNA transfer, and sequences intended for plant genome integration are routinely inserted into the T-DNA as ‘stowaways’.

Plant enzymes but not Agrobacterium VirD2 mediate T-DNA ligation in vitro.

Agrobacterium tumefaciens, a gram-negative soil bacterium, transfers DNA to many plant species. In the plant cell, the transferred DNA (T-DNA) is integrated into the genome. An in vitro ligation-integration assay has been designed to investigate the mechanism of T-DNA ligation and the factors involved in this process. The VirD2 protein, which is produced in Agrobacterium and is covalently attached to T-DNA, did not, under our assay conditions, ligate T-DNA to a model target sequence in vitro. We tested whether plant extracts could ligate T-DNA to target oligonucleotides in our test system. The in vitro ligation-integration reaction did indeed take place in the presence of plant extracts. This reaction was inhibited by dTTP, indicating involvement of a plant DNA ligase. We found that prokaryotic DNA ligases could substitute for plant extracts in this reaction. Ligation of the VirD2-bound oligonucleotide to the target sequence mediated by T4 DNA ligase was less efficient than ligation of a free oligonucleotide to the target. T-DNA ligation mediated by a plant enzyme(s) or T4 DNA ligase requires ATP.

Transferred DNA (T-DNA)-associated proteins of Agrobacterium tumefaciens are exported independently of virB.

The transfer of T-DNA from Agrobacterium to plant cells is mediated by a system which involves the virB operon of the Ti plasmid. We report that VirE2 and VirD2, two T-DNA-associated proteins, as well as VirF, a protein known to be secreted into plant cells, are present in the periplasm and supernatant fractions of growing cells of Agrobacterium as are VirJ and ChvE, two known periplasmic proteins. Two cytoplasmic proteins, Ros and chloramphenicol acetyl transferase, and a VirE2green fluorescent protein construct were not detected in the above fraction. Export of VirE2 into the culture supernatant did not require any Ti plasmid genes, except for VirE1, a specific chaperone for VirE2. The levels of the VirE2 and VirD2 proteins in the supernatant increased significantly when cells were grown at 19 degrees C as compared with 28 degrees C. When Agrobacterium expressed the oncogenic suppressive activity protein (Osa), VirE2 and VirF proteins could not be detected in the supernatant or the periplasm and the level of VirD2 was greatly reduced. However, oncogenic suppressive activity protein did not block the accumulation of VirJ and ChvE in the periplasm. Our data suggest that VirD2, VirE2, and VirF are transported across the cytoplasmic membrane by a specific pathway, independent of virB. Thus, transfer of the T-complex of Agrobacterium may take place in two steps, the first mediated by an unidentified pathway and the second by the virB system.

Import of DNA into mammalian nuclei by proteins originating from a plant pathogenic bacterium.

Import of DNA into mammalian nuclei is generally inefficient. Therefore, one of the current challenges in human gene therapy is the development of efficient DNA delivery systems. Here we tested whether bacterial proteins could be used to target DNA to mammalian cells. Agrobacterium tumefaciens, a plant pathogen, efficiently transfers DNA as a nucleoprotein complex to plant cells. Agrobacterium-mediated T-DNA transfer to plant cells is the only known example for interkingdom DNA transfer and is widely used for plant transformation. Agrobacterium virulence proteins VirD2 and VirE2 perform important functions in this process. We reconstituted complexes consisting of the bacterial virulence proteins VirD2, VirE2, and single-stranded DNA (ssDNA) in vitro. These complexes were tested for import into HeLa cell nuclei. Import of ssDNA required both VirD2 and VirE2 proteins. A VirD2 mutant lacking its C-terminal nuclear localization signal was deficient in import of the ssDNA-protein complexes into nuclei. Import of VirD2-ssDNA-VirE2 complexes was fast and efficient, and was shown to depended on importin alpha, Ran, and an energy source. We report here that the bacterium-derived and plant-adapted protein-DNA complex, made in vitro, can be efficiently imported into mammalian nuclei following the classical importin-dependent nuclear import pathway. This demonstrates the potential of our approach to enhance gene transfer to animal cells.

Viruses, microorganisms and scientists meet the nuclear pore. Leysin, VD, Switzerland, February 26-March 1, 1998.

The modular architecture of the nuclear pore complex (NPC) consists of a membrane‐embedded basic framework (the ‘spoke complex’) made up of eight multidomain spokes with two rings on each face (Figure 1A). The ring facing the cytoplasm is decorated with eight ∼50 nm fibrils extending into the cytosol, and the nuclear ring is capped with a basket‐like assembly of eight thin, 50–100 nm filaments joined distally by a 30–40 nm diameter terminal ring. The center of the basic framework harbors the ‘gated transport channel’, which is involved in signal‐mediated bidirectional transport of macromolecules and is plugged to the central ‘transporter’, whose ultimate structure and functional role in mediated nuclear transport remains to be established. N.Pante and U.Aebi (Basel) reported their approaches to the functional roles of the different NPC components and to dissection of the steps of nuclear import and export at the NPC by direct visualization of gold‐labeled proteins and RNAs moving in or out of the cell nucleus through the NPC (Figure 1B). Figure 1. ( A ) Current consensus model of the membrane‐bound NPC. Its major structural components include the basic framework, the central plug or gated channel, the cytoplasmic and nuclear rings, and the cytoplasmic fibrils and nuclear basket. ( B ) Visualization of the different steps of nuclear import of an NLS‐bearing protein through the NPC. At the bottom are selected examples of cross‐sectioned NPCs with associated nucleoplasmin–gold particles (8 nm diameter), depicted at different stages of transport; at the top is a schematic diagram of the distinct steps visualized by electron microscopy. In the first step of transport, the NLS protein to be imported associates with the NLS receptor (importins α and β) (1). This step takes place in the cytoplasm and does not require physical interaction with any NPC component. In a second step, this ‘targeting complex’ docks …

Agrobacterium VirE2 proteins can form a complex with T strands in the plant cytoplasm.

Wild-type VirE2 and VirD2 proteins from Agrobacterium tumefaciens contain nuclear targeting sequences (NLS) that are likely involved in directing transferred T strands to the plant nucleus. An A. tumefaciens virE2 virD2DeltaNLS double mutant was able to form tumors on VirE2-producing transgenic tobacco but not on wild-type tobacco. Because this mutant bacterial strain contains no known T-strand nuclear targeting signal, the data indicate that wild-type VirE2 proteins produced by the plant can interact with the T strands in the plant cytoplasm and direct them to the nucleus.

Interaction of the DNA modifying proteins VirD1 and VirD2 of Agrobacterium tumefaciens: analysis by subcellular localization in mammalian cells.

Interaction between Agrobacterium tumefaciens and plants provides a unique example of interkingdom gene transfer. Agrobacterium, a plant pathogen, is capable to stably transform the plant cell with a segment of its own DNA called T-DNA (transferred DNA). This process depends, among others, on the specialized bacterial virulence proteins VirD1 and VirD2 that excise the T-DNA from its adjacent sequences. Subsequent to transfer to the plant cell, the virulence protein VirD2, through its nuclear localization signal (NLS), is believed to guide the T-DNA to the nucleus. The T-DNA then is integrated into the plant genome. Although both of these proteins are essential for bacterial virulence, physical interaction of them has not been analyzed so far. We studied associations between these proteins by expressing them in mammalian cells and by testing for intracellular localization and colocalization. When expressed in human cells [HeLa, human embryo kidney (HEK) 293], the VirD2 protein homogeneously distributed over the nucleoplasm. The presence of any of two NLSs, on the N and C termini of VirD2, was sufficient for its efficient nuclear localization whereas deletion of both NLSs rendered the protein cytoplasmic. However, this double NLS mutant was translocated to the nucleus in the presence of wild-type VirD2 protein, implying VirD2-VirD2 interaction. The VirD1 protein, by itself localized in the cytoplasm, moved to the nucleus when coexpressed with the VirD2 protein, suggesting VirD1-VirD2 interaction. This interaction was confirmed by coimmunoprecipitation tests. Of interest, both proteins coimported to the nucleus showed a similar, peculiar sublocalization. The data are discussed in terms of functions of the VirD proteins. In addition, coimport of proteins into nuclei is suggested as a useful system in studying individual protein-protein interactions.

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.

Agrobacterium VirD2 protein interacts with plant host cyclophilins.

Agrobacterium tumefaciens induces crown gall tumors on plants by transferring a nucleoprotein complex, the T-complex, from the bacterium to the plant cell. The T-complex consists of T-DNA, a single-stranded DNA segment of the tumor-inducing plasmid, VirD2, an endonuclease covalently bound to the 5' end of the T-DNA, and perhaps VirE2, a single-stranded DNA binding protein. The yeast two-hybrid system was used to screen for proteins interacting with VirD2 and VirE2 to identify components in Arabidopsis thaliana that interact with the T-complex. Three VirD2- and two VirE2-interacting proteins were identified. Here we characterize the interactions of VirD2 with two isoforms of Arabidopsis cyclophilins identified by using this analysis. The VirD2 domain interacting with the cyclophilins is distinct from the endonuclease, omega, and the nuclear localization signal domains. The VirD2-cyclophilin interaction is disrupted in vitro by cyclosporin A, which also inhibits Agrobacterium-mediated transformation of Arabidopsis and tobacco. These data strongly suggest that host cyclophilins play a role in T-DNA transfer.

The omega sequence of VirD2 is important but not essential for efficient transfer of T-DNA by Agrobacterium tumefaciens.

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

Nuclear localization signal binding protein from Arabidopsis mediates nuclear import of Agrobacterium VirD2 protein.

T-DNA nuclear import is a central event in genetic transformation of plant cells by Agrobacterium. Presumably, the T-DNA transport intermediate is a single-stranded DNA molecule associated with two bacterial proteins, VirD2 and VirE2, which most likely mediate the transport process. While VirE2 cooperatively coats the transported single-stranded DNA, VirD2 is covalently attached to its 5' end. To better understand the mechanism of VirD2 action, a cellular receptor for VirD2 was identified and its encoding gene cloned from Arabidopsis. The identified protein, designated AtKAPalpha, specifically bound VirD2 in vivo and in vitro. VirD2-AtKAPalpha interaction was absolutely dependent on the carboxyl-terminal bipartite nuclear localization signal sequence of VirD2. The deduced amino acid sequence of AtKAPalpha was homologous to yeast and animal nuclear localization signal-binding proteins belonging to the karyopherin alpha family. Indeed, AtKAPalpha efficiently rescued a yeast mutant defective for nuclear import. Furthermore, AtKAPalpha specifically mediated transport of VirD2 into the nuclei of permeabilized yeast cells.

Suppression of mutant phenotypes of the Agrobacterium tumefaciens VirB11 ATPase by overproduction of VirB proteins.

The Agrobacterium tumefaciens VirB11 ATPase is postulated to assemble with VirB proteins and the VirD4 protein into a transport system which is dedicated to the export of oncogenic nucleoprotein particles to plant cells. To gain genetic evidence for interactions between VirB11 and other subunits of this transport system, we screened a PCR-mutagenized virB11 library for alleles that diminish the virulence of the wild-type strain A348. Two classes of alleles displaying negative dominance were identified. One class failed to complement a delta virB11 mutation, indicating that the corresponding mutant proteins are nonfunctional. The second class complemented the delta virB11 mutation, indicating that the mutant proteins are fully functional in strains devoid of native VirB11. Mutations of both classes of alleles were in codons for residues clustered in two regions of VirB11, both located outside the Walker A nucleotide binding motif. All dominant alleles were suppressed at least to some extent by multicopy expression of the virB9, virB10, and/or virB11 genes. Taken together, results of these investigations indicate that (i) a functional T-complex transporter is composed of more than one VirB11 subunit and (ii) VirB11 undergoes complex formation with VirB9 and VirB10 during transporter biogenesis.

Early transcription of Agrobacterium T-DNA genes in tobacco and maize.

We developed a sensitive procedure to investigate the kinetics of transcription of an Agrobacterium tumefaciens transferred (T)-DNA-encoded beta-glucuronidase gusA (uidA) gene soon after infection of plant suspension culture cells. The procedure uses a reverse transcriptase-polymerase chain reaction and enables detection of gusA transcripts within 18 to 24 hr after cocultivation of the bacteria with either tobacco or maize cells. Detection of gusA transcripts depended absolutely on the intact virulence (vir) genes virB, virD1/virD2, and virD4 within the bacterium. Mutations in virC and virE resulted in delayed and highly attenuated expression of the gusA gene. A nonpolar transposon insertion into the C-terminal coding region of virD2 resulted in only slightly decreased production of gusA mRNA, although this insertion resulted in the loss of the nuclear localization sequence and the important omega region from VirD2 protein and rendered the bacterium avirulent. However, expression of gusA transcripts in tobacco infected by this virD2 mutant was more transient than in cells infected by a wild-type strain. Infection of tobacco cells with an Agrobacterium strain harboring a mutant virD2 allele from which the omega region had been deleted resulted in similar transient expression of gusA mRNA. These data indicate that the C-terminal nuclear localization signal of the VirD2 protein is not essential for nuclear uptake of T-DNA and further suggest that the omega domain of VirD2 may be required for efficient integration of T-DNA into the plant genome. The finding that the initial kinetics of gusA gene expression in maize cells are similar to those shown in infected tobacco cells but that the presence of gusA mRNA in maize is highly transient suggests that the block to maize transformation involves T-DNA integration and not T-DNA entry into the cell or nuclear targeting.

Agrobacterium VirE2 protein mediates nuclear uptake of single-stranded DNA in plant cells.

Agrobacterium genetically transforms plant cells by transferring a single-stranded DNA (ssDNA) copy of the transferred DNA (T-DNA) element, the T-strand, in a complex with Agrobacterium proteins VirD2, bound to the 5' end, and VirE2. VirE2 binds single-stranded nucleic acid cooperatively, fully coating the T-strand, and the protein localizes to the plant cell nucleus when transiently expressed. The coupling of ssDNA binding and nuclear localizing activities suggests that VirE2 alone could mediate nuclear localization of ssDNA. In this study, fluorescently labeled ssDNA accumulated in the plant cell nucleus specifically when microinjected as a complex with VirE2. Microinjected ssDNA alone remained cytoplasmic. Import of VirE2-ssDNA complex into the nucleus via a protein import pathway was supported by (i) the inhibition of VirE2-ssDNA complex import in the presence of wheat germ agglutinin or a nonhydrolyzable GTP analog, both known inhibitors of protein nuclear import, and (ii) the retardation of import when complexes were prepared from a VirE2 mutant impaired in ssDNA binding and nuclear import.

Osa protein encoded by plasmid pSa is located at the inner membrane but does not inhibit membrane association of VirB and VirD virulence proteins in Agrobacterium tumefaciens

The osa gene of IncW plasmid pSa encodes a 21-kDa protein that completely abolishes the oncogenic activity encoded by virulence genes in Agrobacterium tumefaciens. osa is the last gene of a four-gene operon in pSa, the expression of which appears to be highly regulated since the Osa protein is absent when either pSa or the osa operon is present in the Agrobacterium cell. When the osa gene alone or together with upstream genes within the operon are expressed under the control of a constitutive promoter, Osa protein is produced, enabling us to determine its subcellular location. Immunoblot analyses located Osa protein at the inner membrane of both A. tumefaciens and Escherichia coli. Because Osa inhibits oncogenicity of A. tumefaciens, and because alterations of the products of the virB and virD genes affect oncogenicity, studies were conducted to determine if there are changes in their specific association with the membranes in the presence Osa. Immunoblot analyses of VirB2, VirB3, VirB4, VirB9, and VirD4 in the presence and absence of Osa revealed no differences between the two treatments in these Vir protein associations with the membranes. These results indicate that both virB and virD gene products are produced in the presence of Osa; that they appear unaffected in their association with the membranes; and that Osa is associated with the inner membrane, where VirB2, VirB4, and VirD4 proteins are also located.

Osa protein encoded by plasmid pSa is located at the inner membrane but does not inhibit membrane association of VirB and VirD virulence proteins in Agrobacterium tumefaciens.

The osa gene of IncW plasmid pSa encodes a 21-kDa protein that completely abolishes the oncogenic activity encoded by virulence genes in Agrobacterium tumefaciens. osa is the last gene of a four-gene operon in pSa, the expression of which appears to be highly regulated since the Osa protein is absent when either pSa or the osa operon is present in the Agrobacterium cell. When the osa gene alone or together with upstream genes within the operon are expressed under the control of a constitutive promoter. Osa protein is produced, enabling us to determine its subcellular location. Immunoblot analyses located Osa protein at the inner membrane of both A. tumefaciens and Escherichia coli. Because Osa inhibits oncogenicity of A. tumefaciens, and because alterations of the products of the virB and virD genes affect oncogenicity, studies were conducted to determine if there are changes in their specific association with the membranes in the presence Osa. Immunoblot analyses of VirB2, VirB3, VirB4, VirB9, and VirD4 in the presence and absence of Osa revealed no differences between the two treatments in these Vir protein associations with the membranes. These results indicate that both virB and virD gene products are produced in the presence of Osa; that they appear unaffected in their association with the membranes; and that Osa is associated with the inner membrane, where VirB2, VirB4, and VirD4 proteins are also located.

A Chromosomal Virulence Gene (acvB) Product ofAgrobacterium tumefaciensThat Binds to a T-Strand to Mediate Its Transfer to Host Plant Cells

It has been proposed that during tumorigenesis of plants by Agrobacterium tumefaciens, the T-strand is transferred into the host plant cell in the form of a complex consisting of VirD2 and VirE2 proteins. In this paper, we present data supporting the hypothesis that another protein, AcvB (a gene product of a chromosomal virulence gene, acvB), also binds to the T-strand in the periplasm to mediate its transfer into the plant cell. An avirulent mutant strain (B119 strain, acvB) of A. tumefaciens was shown to be impaired in the transfer of T-strands into the cytoplasm of host plant cell by an experiment using a binary Ti plasmid containing a i-glucuronidase (GUS) reporter gene with an intron. The immunoprecipitation experiment demonstrated the in vitro formation of a complex of single stranded (ss)T-DNA and AcvB. In the periplasm of A. tumefaciens (A208 strain, virulent, acvB+)induced with acetosyringone, the T-strand complex containing AcvB and VirE2 proteins was detected immunochemically, while such a comp...

Inhibition of VirB-mediated transfer of diverse substrates from Agrobacterium tumefaciens by the IncQ plasmid RSF1010

The transfer of DNA from Agrobacterium tumefaciens into a plant cell requires the activities of several virulence (vir) genes that reside on the tumor-inducing (Ti) plasmid. The putative transferred intermediate is a single-stranded DNA (T strand), covalently attached to the VirD2 protein and coated with the single-stranded DNA-binding protein, VirE2. The movement of this intermediate out of Agrobacterium cells and into plant cells requires the expression of the virB operon, which encodes 11 proteins that localize to the membrane system. Our earlier studies showed that the IncQ broad-host-range plasmid RSF1010, which can be transferred from Agrobacterium cells to plant cells, inhibits the transfer of T-DNA from pTiA6 in a fashion that is reversed by overexpression of virB9, virB10, and virB11. Here, we examined the specificity of this inhibition by following the transfer of other T-DNA molecules. By using extracellular complementation assays, the effects of RSF1010 on movement of either VirE2 or an uncoated T strand from A. tumefaciens were also monitored. The RSF1010 derivative plasmid pJW323 drastically inhibited the capacity of strains to serve as VirE2 donors but only partially inhibited T-strand transfer from virE2 mutants. Further, we show that all the virB genes tested are required for the movement of VirE2 and the uncoated T strand as assayed by extracellular complementation. Our results are consistent with a model in which the RSF1010 plasmid, or intermediates from it, compete with the T strand and VirE2 for a common transport site.

Universal PCR primers for detection of phytopathogenic Agrobacterium strains.

Two PCR primer pairs, based on the virD2 and ipt genes, detected a wide variety of pathogenic Agrobacterium strains. The endonuclease domain of VirD2 protein, which cleaves transferred DNA (T-DNA) border sequences, is highly conserved; primer oligonucleotides specific for the endonuclease portion of virD2 detected all pathogenic strains of Agrobacterium tested. PCR primers corresponding to conserved sequences in ipt, the T-DNA-borne cytokinin synthesis gene, detected only Agrobacterium tumefaciens and distinguished it from Agrobacterium rhizogenes. The virD2 and ipt primer pairs did not interfere with each other when included in the same PCR amplification, and this permitted simultaneous detection of both genes in a single reaction. One nonpathogenic Agrobacterium radiobacter strain contained virD2 but not ipt; we speculate that this strain arose from a pathogenic progenitor through a deletion in the T-DNA. The virD2 primer pair appears to be universal for all pathogenic Agrobacterium species; used together, the primer sets reported here should allow unambiguous identification of Ti plasmid DNA in bacteria isolated from soil and plants.