Agrobacterium T-strand production in vitro: sequence-specific cleavage and 5'' protection of single-stranded DNA templates by purified VirD2 protein.

Virulence proteins VirD1 and VirD2 are subunits of a relaxosome-like protein complex that mediates conjugational transfer of a Ti plasmid segment, the T-DNA, from Agrobacterium into higher plants. The VirD1-VirD2 complex binds to 25-bp repeats at the borders of the T-DNA and catalyzes sequence-specific nicking of the conjugative DNA strand (the T-strand) at the third base of these repeats. Nuclear localization signals present in VirD2 target the T-strand to plant cell nuclei. In addition, VirD2 probably plays a role in the high-frequency integration of the T-DNA into the plant genome by illegitimate recombination. Whereas Agrobacterium transformation of dicots is very efficient, T-DNA integration in most monocots can barely be detected. To develop an artificial T-DNA delivery system for monocots, a technique for efficient in vitro production of T-strand DNAs was established by using VirD1 and VirD2 proteins purified from overexpressing Escherichia coli strains. The topoisomerase-like VirD2 enzyme was shown to mediate precise, sequence-specific cleavage of T-DNA border sequences carried by single-stranded DNA templates, even in the absence of VirD1 protein. During this reaction, VirD2 remains covalently bound to the 5' end of artificial T-strand DNAs. In contrast, VirD2, alone or in complex with VirD1, fails to nick linear double-stranded DNA templates in vitro.     

Site-specific cleavage of single-stranded DNA by a Hemophilus restriction endonuclease.

Single-stranded viral DNA of bacteriophage f1 is cleaved into specific fragments by endo R-HaeIII, a restriction endonuclease isolated from Hemophilus aegyptius. The sites of the single strand cleavage correspond to those of the double strand cleavage. A single-stranded DNA fragment containing only one HaeIII site is also cleaved by this enzyme. This observation suggests that the reaction of single-stranded DNA cleavage does not require the formation of a symmetrical double-stranded structure that would result from the intramolecular base-pairing between two different HaeIII sites. Other restriction endonucleases may also cleave single-stranded DNA.     

Covalently bound VirD2 protein of Agrobacterium tumefaciens protects the T-DNA from exonucleolytic degradation.

We show that upon induction of Agrobacterium tumefaciens, free linear double-stranded T-DNA molecules as well as the previously described T-strands are generated from the Ti plasmid. A majority of these molecules are bound to a protein. We show that this protein is the product of the virulence gene virD2. This protein was found to be attached to the 5' terminus of processed T-DNA at the right border and to the rest of the Ti plasmid at the left border. The protein remnant after Pronase digestion rendered the right end of the double-stranded T-DNA resistant to 5'----3' exonucleolytic attack in vitro. The protein-DNA association was resistant to SDS, mercaptoethanol, mild alkali, piperidine, and hydroxylamine, indicating that it involves a covalent linkage. The possible involvement of this T-DNA-protein complex in replication, transduction to the plant, nuclear targeting, and integration into the plant nuclear DNA is discussed.     

Association of single-stranded transferred DNA from Agrobacterium tumefaciens with tobacco cells.

During the inception of crown gall tumorigenesis, the transferred DNA (T-DNA) is processed from the Ti (tumor inducing) plasmid of Agrobacterium tumefaciens and is transferred to plant cells. T-DNA processing and transfer require the induction of vir (virulence) genes by phenolic compounds secreted by wounded plant cells. After vir gene induction, both single-stranded (T-strands) and double-stranded forms of processed T-DNA accumulate in the bacteria. Although current models favor the transfer of T-strands to plants, there has yet been no experimental evidence to show this. In this paper, we show that T-strands disappear from acetosyringone-induced A. tumefaciens within 30 min of bacterial cocultivation with tobacco protoplasts. PCR analysis of T-DNA associated with protoplasts indicates that single-stranded, but not double-stranded, T-DNA can be detected in the plant cells within 30 min of bacterial cocultivation. Control experiments show that this T-DNA does not originate from lysed contaminating bacterial cells. T-DNA transfer depends on a functional bacterial virB operon. Protoplast infections using an A. tumefaciens virE mutant result in a low level of accumulation of T-strands in the plant cells.     

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.     

Recombination between higher plant DNA and the Ti plasmid of Agrobacterium tumefaciens

The Ti plasmid sequences (T-DNA) from the octopine-producing crown gall tumor A6S/2 were isolated by molecular cloning, using the bacteriophage λ vector Charon 4A. Analysis of the cloned DNA segments indicates that the Ti plasmid sequences are covalently joined to plant nuclear DNA. These data demonstrate that genetic recombination between a eukaryote and a prokaryote can occur as a natural phenomenon.     

Cooperative interaction of Agrobacterium VirE2 protein with single-stranded DNA: implications for the T-DNA transfer process.

Induction of Agrobacterium tumefaciens vir gene expression by wounded plant cells results in production of a free transferable single-stranded (ss) copy of T-DNA, the T-strand. One of the Vir proteins, the VirE2 polypeptide, is a ssDNA-binding protein. In the present work, interaction of nopaline-specific VirE2 protein (Mr 69,000) with ssDNA was studied by using nitrocellulose filter binding, gel retardation, and electron microscopy techniques. The VirE2 protein was found to bind to ssDNA molecules with strong cooperativity, forming VirE2-ssDNA complexes with a binding site of 28-30 nucleotides. The VirE2-ssDNA complexes are stable at high salt concentrations and resistant to exonucleolytic activity. When examined under the electron microscope, the VirE2 protein converted collapsed free ssDNA molecules into unfolded and extended structures. The structure and properties of VirE2-ssDNA complexes predict possible functions in Agrobacterium virulence to (i) protect the T-strands from cellular nucleases and (ii) facilitate transfer of the T-strands through bacterial membranes possibly by specific interaction with putative membrane pores formed in plant-induced Agrobacterium cells.     

Mobilization of T-DNA from Agrobacterium to plant cells involves a protein that binds single-stranded DNA.

Crude protein extracts of induced and uninduced octopine wild-type strain of Agrobacterium tumefaciens, as well as several mutants of the virulence loci virA, -B, -G, -C, -D, and -E, were probed with single- and double-stranded synthetic oligodeoxynucleotides of different sequence and length in an electrophoretic retardation assay. Four complexes involving sequence-nonspecific, single-stranded-DNA-binding proteins were recognized. One inducible complex is determined by the virE locus, two Ti-plasmid-dependent complexes are constitutively expressed, and a fourth one is controlled by chromosomal genes. The protein-DNA complexes were characterized by sucrose density gradient centrifugation and by determination of the length of single-stranded DNA required for their formation. It is hypothesized that the single-stranded-DNA-binding proteins are involved in the production of T-DNA intermediates or have a carrier or protective function during T-DNA transfer.     

A bacterial peptide acting as a plant nuclear targeting signal: the amino-terminal portion of Agrobacterium VirD2 protein directs a beta-galactosidase fusion protein into tobacco nuclei.

Agrobacterium tumefaciens is a soil bacterium capable of transferring DNA to the genome of higher plants. Of the virulence region-encoded proteins of the tumor-inducing (Ti) plasmid of A. tumefaciens, the VirD1 and VirD2 proteins are essential for T-DNA transfer to plant cells. These two proteins have been shown to be directly responsible for the formation of T-strands. VirD2 was also shown to be firmly attached to the 5' termini of T-strands; these facts have led to its postulation as a pilot protein in the T-DNA transfer process and as a nucleus-targeting signal in plants. We have constructed a chimeric gene by fusing the virD2 gene and the Escherichia coli lacZ gene. Cell fractionation and electron microscopy studies with transgenic tobacco plants containing the VirD2-LacZ fusion protein indicate that the first 292 amino acids of VirD2 are able to direct the cytoplasmic protein beta-galactosidase to the plant nucleus. This provides an example of cross-kingdom nuclear localization between two free-living organisms: a bacterial peptide is capable of acting as a eukaryotic (plant) nuclear targeting signal.     

A nuclear localization signal and the C-terminal omega sequence in the Agrobacterium tumefaciens VirD2 endonuclease are important for tumor formation.

The T-DNA portion of the Agrobacterium tumefaciens tumor-inducing (Ti) plasmid integrates into plant nuclear DNA. Direct repeats define the T-DNA ends; transfer begins when the VirD2 endonuclease produces a site-specific nick in the right-hand border repeat and attaches to the 5' end of the nicked strand. Subsequent events generate linear single-stranded VirD2-bound DNA molecules that include the entire T-DNA (T-strands). VirD2 protein contains a nuclear localization signal (NLS) near the C terminus and may direct bound T-strands to plant nuclei. We constructed mutations in virD2 and showed that the NLS was important for tumorigenesis, although T-strand production occurred normally in its absence. A tobacco etch virus NLS, substituted for the VirD2 NLS, restored tumor-inducing activity. Amino acids (the omega sequence) at the C terminus of VirD2, outside the NLS and the endonuclease domain, contributed significantly to tumorigenesis, suggesting that VirD2 may serve a third important function in T-DNA transfer.     

Nuclear import of Agrobacterium VirD2 and VirE2 proteins in maize and tobacco.

Previously, we have shown that Agrobacterium-plant cell transferred DNA (T-DNA) transport into the host cell nucleus is likely mediated by two specific bacterial proteins, VirD2 and VirE2. Here, we used these proteins to study molecular pathways of nuclear import. First, the role of VirE2 nuclear localization signals (NLSs) in the T-DNA transport pathway was examined by using tobacco plants transgenic for deletion mutants of VirE2. In these plants, the virulence of wild-type Agrobacterium was reduced possibly by competition for the cellular nuclear import machinery. Second, we analyzed the nuclear localization of VirE2 and VirD2 in the nonhost monocot maize. Part of the known recalcitrance of monocots to transformation by Agrobacterium could be due to a potential selectivity in nuclear import pathways in monocotyledonous and dicotyledonous plants. Nuclear transport of VirD2 and VirE2 in maize leaves and roots was compared to that in tobacco protoplasts and roots. Both proteins accumulated in maize leaf and tobacco protoplast nuclei as well as in nuclei of immature root cells. In contrast, VirD2 and VirE2 expressed in mature roots of maize and tobacco remained cytoplasmic. Point mutations of VirE2 nuclear localization signals, NSE 1 and NSE 2, also revealed that, in maize, the NSE 1 signal was mainly responsible for nuclear import; in contrast, both signals functioned independently in tobacco protoplasts.     

Integration of complete transferred DNA units is dependent on the activity of virulence E2 protein of Agrobacterium tumefaciens.

Agrobacterium tumefaciens transfers transferred DNA (T-DNA), a single-stranded segment of its tumor-inducing (Ti) plasmid, to the plant cell nucleus. The Ti-plasmid-encoded virulence E2 (VirE2) protein expressed in the bacterium has single-stranded DNA (ssDNA)-binding properties and has been reported to act in the plant cell. This protein is thought to exert its influence on transfer efficiency by coating and accompanying the single-stranded T-DNA (ss-T-DNA) to the plant cell genome. Here, we analyze different putative roles of the VirE2 protein in the plant cell. In the absence of VirE2 protein, mainly truncated versions of the T-DNA are integrated. We infer that VirE2 protects the ss-T-DNA against nucleolytic attack during the transfer process and that it is interacting with the ss-T-DNA on its way to the plant cell nucleus. Furthermore, the VirE2 protein was found not to be involved in directing the ss-T-DNA to the plant cell nucleus in a manner dependent on a nuclear localization signal, a function which is carried by the NLS of VirD2. In addition, the efficiency of T-DNA integration into the plant genome was found to be VirE2 independent. We conclude that the VirE2 protein of A. tumefaciens is required to preserve the integrity of the T-DNA but does not contribute to the efficiency of the integration step per se.     

Site-specific cleavage of duplex DNA by a semisynthetic nuclease via triple-helix formation.

A Lys-84----Cys mutant staphylococcal nuclease was selectively linked to the 5' and/or 3' terminus of a thiol-containing polypyrimidine oligonucleotide via a disulfide bond. The oligonucleotide-staphylococcal nuclease adduct is capable of binding to a homopurine-homopyrimidine region of Watson-Crick duplex DNA by the formation of a triple-helical structure. Upon the addition of Ca2+, the nuclease cleaves DNA at sites adjacent to the homopurine tract. Specific double-strand cleavage occurred predominantly at A + T-rich sites to the 5' side of the homopurine tract for both the 5'-derivatized and the 5',3'-diderivatized nucleases; the 3'-derivatized nuclease gave no cleavage. The cleavage pattern is asymmetric and consists of multiple cleavage sites shifted to the 5' side on each strand, centered at the terminal base pair of the binding site. Microgram amounts of plasmid pDP20 DNA (4433 base pairs) containing a homopurine-homopyrimidine tract were selectively cleaved by a semisynthetic nuclease with greater than 75% efficiency at room temperature within 1 hr. Cleavage reaction conditions were optimized with respect to pH, temperature, reaction times, and reaction components. Semisynthetic nucleases of this type should provide a powerful tool in chromosomal DNA manipulations.     

Site-specific cleavage of DNA at 8- and 10-base-pair sequences.

A method is described for cutting DNA at specific sites that are 8 and 10 base pairs long. The DNA is first treated with a specific methylase, either the restriction-modification enzyme M. Taq I, which converts the 4-base sequence T-C-G-A to T-C-G-mA, or the similar enzyme M. Cla I, which converts the 6-base sequence A-T-C-G-A-T to A-T-C-G-mA-T. The DNA is then cleaved with Dpn I, a restriction endonuclease that recognizes the sequence G-mA-T-C. Dpn I is unique in that it cuts only DNA that is methylated at adenine in both strands of its recognition sequence. In DNAs that are not otherwise methylated at adenine in both strands of the sequence G-A-T-C, cleavage by Dpn I occurs only at the following sequences: in the case of M. Taq I methylation, 5' T-C-G-mA - T-C-G-mA 3' 3' mA-G-C - T-mA-G-C - T 5'; in the case of M. Cla I methylation, 5' A - T-C-G-mA - T-C-G-mA-T 3' 3' T-mA-G-C - T-mA-G-C - T-A 5'. Specific cutting and cloning at these methylase/Dpn I-generated sites is shown experimentally. Further, we describe how the above technique can be extended to generate Dpn I cleavage sites of up to 12 base pairs. In DNA that contains equal amounts of each base distributed at random, 8- and 10-base-pair recognition sequences occur, on the average, approximately once every 65,000 and 1,000,000 base pairs, respectively. Potential applications, including the development of cloning vectors and a rapid method for chromosome walking, are discussed.     

Recognition of duplex DNA containing single-stranded regions by recA protein.

Genetic recombination in Escherichia coli requires recA protein, the product of the recA+ gene. In this paper we show that purified recA protein, which binds strongly to denatured DNA, cooperatively recognizes DNA containing short single-stranded regions. The interaction of varying amounts of recA protein with DNA molecules was investigated by measuring its DNA-dependent ATPase activity. In 3mM Mg2+, the ATPase activity was stimulated by excess single-stranded DNA and was minimal with either intact circular or blunt-ended linear duplexes. Single-strand gaps of about 30 nucleotides were sufficient to increase the ATPase activity to a level almost as great as that observed with single-stranded DNA. Sedimentation studies at neutral pH showed cooperative binding of recA protein to single-stranded DNA or to duplex DNA containing single-stranded regions. In the presence of ATP, an intermediate rate of sedimentation was observed; in contrast, adenosine 5'-gamma-thiotriphosphate (ATP[S]) caused the formation of fast-sedimenting DNA-protein complexes. Gapped plasmid DNA plus recA protein and ATP[S] formed large aggregates containing thousands of molecules. Complex formation and stimulation of the ATPase activity of recA protein with duplex DNA containing single-stranded regions indicates that recA protein may change the conformation of the normally duplex molecules to a conformation prepared for homologous pairing.     

Genetic evidence for direct sensing of phenolic compounds by the VirA protein of Agrobacterium tumefaciens.

The virulence (vir) genes of Agrobacterium tumefaciens are induced by low-molecular-weight phenolic compounds and monosaccharides through a two-component regulatory system consisting of the VirA and VirG proteins. However, it is not clear how the phenolic compounds are sensed by the VirA/VirG system. We tested the vir-inducing abilities of 15 different phenolic compounds using four wild-type strains of A. tumefaciens--KU12, C58, A6, and Bo542. We analyzed the relationship between structures of the phenolic compounds and levels of vir gene expression in these strains. In strain KU12, vir genes were not induced by phenolic compounds containing 4'-hydroxy, 3'-methoxy, and 5'-methoxy groups, such as acetosyringone, which strongly induced vir genes of the other three strains. On the other hand, vir genes of strain KU12 were induced by phenolic compounds containing only a 4'-hydroxy group, such as 4-hydroxyacetophenone, which did not induce vir genes of the other three strains. The vir genes of strains KU12, A6, and Bo542 were all induced by phenolic compounds containing 4'-hydroxy and 3'-methoxy groups, such as acetovanillone. By transferring different Ti plasmids into isogenic chromosomal backgrounds, we showed that the phenolic-sensing determinant is associated with Ti plasmid. Subcloning of Ti plasmid indicates that the virA locus determines which phenolic compounds can function as vir gene inducers. These results suggest that the VirA protein directly senses the phenolic compounds for vir gene activation.     

DNA modifying enzymes of Agrobacterium tumefaciens: effect of DNA topoisomerase, restriction endonuclease, and unique DNA endonuclease on plasmid and plant DNA.

Extracts from Agrobacterium tumefaciens strain ID135 contain three enzymes that have been characterized and partially purified. The first enzyme, a DNA topoisomerase, appeared to relax only negatively twisted DNA. The second enzyme, Atu I, a type II restriction endonuclease, generated the identical DNA digestion pattern as EcoRII when several DNAs were used. The third enzyme, endonuclease A, showed a preference for superhelical DNAs as substrates. When plasmid pCK135DNA, obtained from the virulent strain IDI135 of A. tumefaciens, or plant DNA was exposed to the three enzymes, changes in DNA patterns were observed due to either conformational changes or digestion of the DNAs. These enzymes may function in vivo in the processing and incorporation of bacterial DNA in plant cells.     

Site-specific DNA condensation and pairing mediated by the int protein of bacteriophage lambda.

The int protein of bacteriophage lambda catalyzes the site-specific integrative recombination that inserts lambda DNA into the host chromosome. The attachment site region of lambda DNA required for this reaction spans 230 base pairs and includes four separable binding sites for int protein. We have used the electron microscope to determine the functional consequences of the interaction of int with its multiple binding sites. We find that int condenses a 230-base pair segment of DNA into a compact structure about 14 nm in diameter; the condensed region includes all of the four binding sites for int. Condensed segments will form paired structures between attachments sites. We suggest that a sequential cooperative interaction between bound int molecules provides for a distinct reactive DNA conformation and for pairing between substrate sites.     

Insertion sequence elements of Pseudomonas savastanoi: Nucleotide sequence and homology with Agrobacterium tumefaciens transfer DNA

Two types of transposable elements, IS51 and IS52 (IS, insertion sequence), were found in Pseudomonas syringae subsp. savastanoi (P. savastanoi) that spontaneously insert into and inactivate iaaM; the insertion results in the loss of indoleacetic acid production and attenuation of virulence. The nucleotide sequences of both IS elements have sizes and structural features common to other prokaryotic IS elements; IS51 is 1311 base pairs (bp) long and has terminal inverted repeats of 26 bp; IS52 is 1209 bp long and has terminal inverted repeats of 10 bp with a 1 bp mismatch. In the insertion involving IS51, the trinucleotide sequence CAG is duplicated within iaaM sequences at the recombination junction; in those involving IS52 the tetranucleotide sequences TTAG or CTAG are duplicated within iaaM sequences at the recombination junction. A copy of IS51 occurs 2.5 kilobases downstream from IaaH. In contrast to the high copy number of IS51 in the genome of the bacterium, only a few copies of IS52 are present. No nucleotide sequence homology was found between IS51 and IS52. However, a striking nucleotide sequence homology was found between a 531-bp region of IS51 and a portion of the central region of transfer DNA (T-DNA) in the octopine plasmid pTi15955 from Agrobacterium tumefaciens. These observations, together with our earlier finding on the homology between iaaM and iaaH and between gene 1 and gene 2 of transfer DNA, further suggest that genes for indoleacetic acid production in the two systems have a common origin.     

Sequence identity in the nick regions of IncP plasmid transfer origins and T-DNA borders of Agrobacterium Ti plasmids.

The IncP antibiotic-resistance plasmids transfer to a broad range of bacterial species. The RK2 origin of DNA transfer (oriT) consists of a 250-base-pair segment including the single-stranded cleavage site (nic) needed to generate the DNA strand believed to be transferred. Deletion derivatives and a bank of hydroxylamine-generated oriT mutants were screened for loss of transferability. DNA regions flanking both sides of nic are required for optimal transfer of the oriT clone. Of the chemically induced mutants, critical base-pair changes that dramatically reduced transfer frequency were found in a 10-base-pair region adjacent to nic. Relaxation (nicking) assays performed with these point mutants using protein-DNA complexes reconstituted in vitro revealed a correlation between DNA nicking and transfer frequency. Base-pair changes within the proximal arm of an inverted repeat upstream from the nick site resulted in reduced binding of the essential transfer protein TraJ and correspondingly reduced transfer frequencies. The results support a model of relaxosome formation involving at least two essential proteins: TraI and TraJ. The nick region defined by the point mutants was located in a segment known to be nearly identical in the related plasmid R751. This sequence was also found to be highly conserved in both border junctions of the transfer DNA (T-DNA) of plant tumor-inducing plasmids of Agrobacterium tumefaciens, indicating a relationship between IncP-mediated broad-host-range bacterial conjugation and T-DNA transfer to plants.