Genetic analysis of the virD operon of Agrobacterium tumefaciens: a search for functions involved in transport of T-DNA into the plant cell nucleus and in T-DNA integration.

The transferred DNA (T-DNA) is transported from Agrobacterium tumefaciens to the nucleus and is stably integrated into the genome of many plant species. It has been proposed that the VirD2 protein, tightly attached to the T-DNA, pilots the T-DNA into the plant cell nucleus and that it is involved in integration. Using agroinfection and beta-glucuronidase expression as two different very sensitive transient assays for T-DNA transfer, together with assays for stable integration, we have shown that the C-terminal half of the VirD2 protein and the VirD3 protein are not involved in T-DNA integration. However, the bipartite nuclear localization signal, which is located within the C terminus of the VirD2 protein and which has previously been shown to be able to target a foreign protein into the plant cell nucleus, was shown to be required for efficient T-DNA transfer. virD4 mutants were shown by agroinfection to be completely inactive in T-DNA transfer.     

Caspase-resistant VirD2 protein provides enhanced gene delivery and expression in plants

Agrobacterium tumefaciens VirD2 protein is one of the key elements of Agrobacterium-mediated plant transformation, a process of transfer of T-DNA sequence from the Agrobacterium tumour inducing plasmid into the nucleus of infected plant cells and its integration into the host genome. The VirD2 protein has been shown to be a substrate for a plant caspase-like protease activity (PCLP) in tobacco. We demonstrate here that mutagenesis of the VirD2 protein to prevent cleavage by PCLP increases the efficiency of reporter gene transfer and expression. These results indicate that PCLP cleavage of the Agrobacterium VirD2 protein acts to limit the effectiveness of T-DNA transfer and is a novel resistance mechanism that plants utilise to combat Agrobacterium infection. Brian Reavy and Svetlana Bagirova contributed equally to this work.     

Bacterial Conjugation Protein MobA Mediates Integration of Complex DNA Structures into Plant Cells

Agrobacterium tumefaciens transfers T-DNA to plant cells, where it integrates into the genome, a property that is ensured by bacterial proteins VirD2 and VirE2. Under natural conditions, the protein MobA mobilizes its encoding plasmid, RSF1010, between different bacteria. A detailed analysis of MobA-mediated DNA mobilization by Agrobacterium to plants was performed. We compared the ability of MobA to transfer DNA and integrate it into the plant genome to that of pilot protein VirD2. MobA was found to be about 100-fold less efficient than VirD2 in conducting the DNA from the pTi plasmid to the plant cell nucleus. However, interestingly, DNAs transferred by the two proteins were integrated into the plant cell genome with similar efficiencies. In contrast, most of the integrated DNA copies transferred from a MobA-containing strain were truncated at the 5' end. Isolation and analysis of the most conserved 5' ends revealed patterns which resulted from the illegitimate integration of one transferred DNA within another. These complex integration patterns indicate a specific deficiency in MobA. The data conform to a model according to which efficiency of T-DNA integration is determined by plant enzymes and integrity is determined by bacterial proteins.     

The VirD2 protein of Agrobacterlum tumefaciens carries nuclear localization signals important for transfer of T-DNA to plants

Agrobacterium tumefaciens is able to transfer a piece of DNA, the T-DNA, to the nucleus of the plant cell. The VirD2 protein is required for the production of the T-DNA, it is tightly linked to the T-DNA and it is thought to direct it to the plant genome. Two nuclear localization signals (NLS), one in the N-terminal part and one in the C-terminal part of the VirD2 protein, have been shown to be able to target marker proteins to the plant nucleus. Here we analyze nuclear entry of the T-DNA complex using a new and very sensitive assay for T-DNA transfer. We show that optimal T-DNA transfer requires the VirD2 NLS located in the C-terminal part of the protein, whereas mutations in the N-terminal NLS coding sequence seem to have no effect on T-DNA transfer.     

The VirD2 protein of Agrobacterlum tumefaciens carries nuclear localization signals important for transfer of T-DNA to plants

Agrobacterium tumefaciens is able to transfer a piece of DNA, the T-DNA, to the nucleus of the plant cell. The VirD2 protein is required for the production of the T-DNA, it is tightly linked to the T-DNA and it is thought to direct it to the plant genome. Two nuclear localization signals (NLS), one in the N-terminal part and one in the C-terminal part of the VirD2 protein, have been shown to be able to target marker proteins to the plant nucleus. Here we analyze nuclear entry of the T-DNA complex using a new and very sensitive assay for T-DNA transfer. We show that optimal T-DNA transfer requires the VirD2 NLS located in the C-terminal part of the protein, whereas mutations in the N-terminal NLS coding sequence seem to have no effect on T-DNA transfer.     

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.     

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.     

<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation of plants by the pTF-FC2 plasmid is efficient and strictly dependent on the MobA protein

In the transformation of plants by Agrobacterium tumefaciens the VirD2 protein has been shown to pilot T-DNA during its transfer to the plant cell nucleus. Other studies have shown that the MobA protein of plasmid RSF1010 is capable of mediating its transfer from Agrobacterium cells to plant cells by a similar process. We have demonstrated previously that plasmid pTF-FC2, which has some similarity to RSF1010, is also able to transfer DNA efficiently. In this study, we performed a mutational analysis of the roles played by A. tumefaciens VirD2 and pTF-FC2 MobA in DNA transfer-mediated by A. tumefaciens carrying pTF-FC2. We show that MobA+/VirD2+ and MobA+/VirD2– strains were equally proficient in their ability to transfer a pTF-FC2-derived plasmid DNA to plants and to transform them. However, the MobA–/VirD2+ strain showed a DNA transfer efficiency of 0.03% compared with that of the other two strains. This sharply contrasts with our results that VirD2 can rather efficiently cleave the oriT sequence of pFT-FC2 in vitro. We therefore conclude that MobA plays a major VirD2-independent role in plant transformation by pTF-FC2.     

Import of Agrobacterium T-DNA into plant nuclei: two distinct functions of VirD2 and VirE2 proteins

To study the mechanism of nuclear import of T-DNA, complexes consisting of the virulence proteins VirD2 and VirE2 as well as single-stranded DNA (ssDNA) were tested for import into plant nuclei in vitro. Import of these complexes was fast and efficient and could be inhibited by a competitor, a nuclear localization signal (NLS) coupled to BSA. For import of short ssDNA, VirD2 was sufficient, whereas import of long ssDNA additionally required VirE2. A VirD2 mutant lacking its C-terminal NLS was unable to mediate import of the T-DNA complexes into nuclei. Although free VirE2 molecules were imported into nuclei, once bound to ssDNA they were not imported, implying that when complexed to DNA, the NLSs of VirE2 are not exposed and thus do not function. RecA, another ssDNA binding protein, could substitute for VirE2 in the nuclear import of T-DNA but not in earlier events of T-DNA transfer to plant cells. We propose that VirD2 directs the T-DNA complex to the nuclear pore, whereas both proteins mediate its passage through the pore. Therefore, by binding to ssDNA, VirE2 may shape the T-DNA complex such that it is accepted for translocation into the nucleus.     

Agrobacterium VirD2-binding protein is involved in tumorigenesis and redundantly encoded in conjugative transfer gene clusters

Agrobacterium tumefaciens can transfer oncogenic T-DNA into plant cells; T-DNA transfer is mechanistically similar to a conjugation process. VirD2 is the pilot protein that guides the transfer, because it is covalently associated with single-stranded T-DNA to form the transfer substrate T-complex. We used the VirD2 protein as an affinity ligand to isolate VirD2-binding proteins (VBPs). By pull-down assays and peptide-mass-fingerprint matching, we identified an A. tumefaciens protein designated VBP1 that could bind VirD2 directly. Genome-wide sequence analysis showed that A. tumefaciens has two additional genes encoding proteins highly similar to VBP1, designated vbp2 and vbp3. Like VBP1, both VBP2 and VBP3 also could bind VirD2; all three VBPs contain a putative nucleotidyltransferase motif. Mutational analysis of vbp demonstrated that the three vbp genes could functionally complement each other. Consequently, only inactivation of all three vbp genes highly attenuated the bacterial ability to cause tumors on plants. Although vbp1 is harbored on the megaplasmid pAtC58, vbp2 and vbp3 reside on the linear chromosome. The vbp genes are clustered with conjugative transfer genes, suggesting linkage between the conjugation and virulence factor. The three VBPs appear to contain C-terminal positively charged residues, often present in the transfer substrate proteins of type IV secretion systems. Inactivation of the three vbp genes did not affect the T-strand production. Our data indicate that VBP is a newly identified virulence factor that may affect the transfer process subsequent to T-DNA production.     

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.     

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.     

VirD proteins of Agrobacterium tumefaciens are required for the formation of a covalent DNA--protein complex at the 5'' terminus of T-strand molecules.

The T-DNA transfer process of Agrobacterium tumefaciens is activated by the induction of the Ti plasmid virulence (vir) loci by plant signal molecules such as acetosyringone. Upon initiation of the T-DNA transfer process, site-specific nicks occur at the 25-bp border sequences. This cleavage leads to the generation of a free, linear ssT-DNA molecule which is bound by sequence non-specific VirE proteins. Here we present evidence for the involvement of other acetosyringone-induced proteins in the formation of a covalent complex between the T-strand and protein, designated the T-complex. Alkaline gel-electrophoretic analysis showed that proteins specifically bind to the 5' termini of nicked T-DNA molecules. The T-complex can be formed in Escherichia coli when the VirD1 and VirD2 proteins are expressed.     

Transgene integration in aspen: structures of integration sites and mechanism of T-DNA integration

To obtain insight into the mechanism of transferred DNA (T-DNA) integration in a long-lived tree system, we analysed 30 transgenic aspen lines. In total, 27 right T-DNA/plant junctions, 20 left T-DNA/plant junctions, and 10 target insertions from control plants were obtained. At the right end, the T-DNA was conserved up to the cleavage site in 18 transgenic lines (67%), and the right border repeat was deleted in nine junctions. Nucleotides from the left border repeat were present in 19 transgenic lines out of 20 cases analysed. However, only four (20%) of the left border ends were conserved to the processing end, indicating that the T-DNA left and right ends are treated mechanistically differently during the T-DNA integration process. Comparison of the genomic target sites prior to integration to the T-DNA revealed that the T-DNA inserted into the plant genome without any notable deletion of genomic sequence in three out of 10 transgenic lines analysed. However, deletions of DNA ranging in length from a few nucleotides to more than 500 bp were observed in other transgenic lines. Filler DNAs of up to 235 bp were observed on left and/or right junctions of six transgenic lines, which in most cases originated from the nearby host genomic sequence or from the T-DNA. Short sequence similarities between recombining strands near break points, in particular for the left T-DNA end, were observed in most of the lines analysed. These results confirm the well-accepted T-DNA integration model based on single-stranded annealing followed by ligation of the right border which is preserved by the VirD2 protein. However, a second category of T-DNA integration was also identified in nine transgenic lines, in which the right border of the T-DNA was partly truncated. Such integration events are described via a model for the repair of genomic double-strand breaks in somatic plant cells based on synthesis-dependent strand-annealing. This report in a long-lived tree system provides major insight into the mechanism of transgene integration.     

VirD4-independent transformation by CloDF13 evidences an unknown factor required for the genetic colonization of plants via Agrobacterium

Agrobacterium uses a mechanism similar to conjugation for trans-kingdom transfer of its oncogenic T-DNA. A defined VirB/VirD4 Type IV secretion system is responsible for such a genetic transfer. In addition, certain virulence proteins as VirE2 can be mobilized into host cells by the same apparatus. VirE2 is essential to achieve plant but not yeast transformation. We found that the limited host range plasmid CloDF13 can be recruited by the virulence apparatus of Agrobacterium for transfer to eukaryotic hosts. As expected the VirB transport complex was required for such trans-kingdom DNA transfer. However, unexpectedly, the coupling factor VirD4 turned out to be necessary for transfer to plants but not for transport into yeast. The CloDF13 encoded coupling factor (Mob) was essential for transfer to both plants and yeast though. This is interpreted by the different specificities of Mob and VirD4. Hence, Mob being required for the transport of the CloDF13 transferred DNA (to both plants and yeast) and VirD4 being required for transport of virulence proteins such as VirE2. Nevertheless, the presence of the VirE2 protein in the host plant was not sufficient to restore the deficiency for VirD4 in the transforming bacteria. We propose that Mob functions encoded by the plasmid CloDF13 are sufficient for DNA mobilization to eukaryotic cells but that the VirD4-mediated pathway is essential to achieve DNA nuclear establishment specifically in plants. This suggests that other Agrobacterium virulence proteins besides VirE2 are translocated and essential for plant transformation.     

Analysis of junctions between T-DNA and plant genome in transgenic<Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

To understand the mechanism forAgrobacterium- mediated transformation of plants, we analyzed the junctions between T-DNA and plant genome, using 12 individual transgenic lines transformed with 7 different plant expression constructs. After performing TAIL-PCR, we sequenced 42 PCR products for analysis. All of the RBs were nicked by VirD1/VirD2 proteins whereas only 62% of the LBs were. Additional deletions of the adjacent T-DNA region were found in 50% of the RBs. For the LBs, only two showed such additional deletions. Filler DNAs were observed in 60% of the RBs (ranging from 1 to 132 nucleotides) versus 54% for the LBs. We also found that only 25% of the RBs were integrated into the plant genome while the rest showed integration into the expression constructs. In comparison, all of the LBs were integrated, except for one that was con-sidered intact. Our results suggest that the origin for a binary vector backbone (BVB) in the plant genome is due not only to a mistake in the VirD1/VirD2 proteins within the T-DNA borders but also because of the linkage of RBs to either the T-DNA or BVB.     

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.     

Expression of Agrobacterium nopaline-specific VirD1, VirD2, and VirC1 proteins and their requirement for T-strand production in E. coli

Induction of Ti plasmid virulence (vir) genes during early stages of the genetic transformation of plant cells by Agrobacterium tumefaciens results in several molecular events that are involved in generating a transferable T-DNA copy. These events include site-specific nicking at the T-DNA borders and synthesis of free, unipolar, linear, single-stranded copies of the T-DNA (T-strands). Here E. coli was used as a heterologous cell to assay the requirements for T-strand synthesis. Cells of E. coli harbored two compatible plasmids, one containing coding sequences overlapping the virC and virD regions of the nopaline Ti plasmid, and a second plasmid containing a T-DNA region. The amount of vir proteins produced was varied by placing their expression under the control of either native Agrobacterium, tac, or T7 promoters. The data show that VirD1 and VirD2 proteins are absolutely essential for T-strand production in E. coli, and the relative amounts of these polypeptides produced correlate with the amounts of T-strand observed. When VirD1 and VirD2 products are limiting, the VirC1 protein increases T-strand production. The yield of T-strands also varies as a function of the plasmid vector used to clone the T-DNA region substrate; the same T-DNA cloned into pLAFR1 produces more T-strands than that cloned into the higher copy number plasmid pACYC184. In summary, VirD1 and VirD2 proteins are the minimal requirements for T-strand production; however, other factors such as VirC1, the relative concentration of VirD1, VirD2, and the T-DNA substrate, and possibly additional functions (e.g., those specified by pLAFR1) influence the efficiency of T-strand production. Additional results regarding the requirements for expression of VirD1 and VirD2 polypeptides are presented.     

Association of the virD2 protein with the 5'' end of T strands in Agrobacterium tumefaciens.

The soil bacterium Agrobacterium tumefaciens can incite tumors in many dicotyledonous plants by transferring a portion (T-DNA) of its Ti plasmid into susceptible plant cells. The T-DNA is flanked by border sequences that serve as recognition sites for specific cleavage by an endonuclease that comprises two virD-encoded proteins (VirD1 and VirD2). After cleavage, both double-stranded, nicked T-DNA molecules and single-stranded T-DNA molecules (T strands) were present. We have determined that a protein is tightly associated with, and probably covalently attached to, the 5' end of the T strands. Analysis of deletion derivatives in Escherichia coli, immunoprecipitation, and a procedure combining immunoblot and nucleic acid hybridization data identified this protein as the gene product of virD2.     

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.