Efficient vir gene induction in Agrobacterium tumefaciens requires virA, virG, and vir box from the same Ti plasmid

The vir genes of octopine, nopaline, and L,L-succinamopine Ti plasmids exhibit structural and functional similarities. However, we observed differences in the interactions between octopine and nopaline vir components. The induction of an octopine virE(A6)::lacZ fusion (pSM358cd) was 2.3-fold higher in an octopine strain (A348) than in a nopaline strain (C58). Supplementation of the octopine virG(A6) in a nopaline strain with pSM358 did not completely restore virE(A6) induction. However, addition of the octopine virA(A6) to the above strain increased virE(A6) induction to a level almost comparable to that in octopine strains. In a reciprocal analysis, the induction of a nopaline virE(C58)::cat fusion (pUCD1553) was two- to threefold higher in nopaline (C58 and T37) strains than in octopine (A348 and Ach5) and L,L-succinamopine (A281) strains. Supplementation of nopaline virA(C58) and virG(C58) in an octopine strain (A348) harboring pUCD1553 increased induction levels of virE(C58)::cat fusion to a level comparable to that in a nopaline strain (C58). Our results suggest that octopine and L,L-succinamopine VirG proteins induce the octopine virE(A6) more efficiently than they do the nopaline virE(C58). Conversely, the nopaline VirG protein induces the nopaline virE(C58) more efficiently than it does the octopine virE(A6). The ability of Bo542 virG to bring about supervirulence in tobacco is observed for an octopine vir helper (LBA4404) but not for a nopaline vir helper (PMP90). Our analyses reveal that quantitative differences exist in the interactions between VirG and vir boxes of different Ti plasmids. Efficient vir gene induction in octopine and nopaline strains requires virA, virG, and vir boxes from the respective Ti plasmids.     

Limited-host-range plasmid of Agrobacterium tumefaciens: molecular and genetic analyses of transferred DNA.

A tumor-inducing (Ti) plasmid from a strain of Agrobacterium tumefaciens that induces tumors on only a limited range of plants was characterized and compared with the Ti plasmids from strains that induce tumors on a wide range of plants. Whereas all wide-host-range Ti plasmids characterized to date contain closely linked oncogenic loci within a single transferred DNA (T-DNA) region, homology to these loci is divided into two widely separated T-DNA regions on the limited-host-range plasmid. These two plasmid regions, TA-DNA and TB-DNA, are separated by approximately 25 kilobases of DNA which is not maintained in the tumor. The TA-DNA region resembles a deleted form of the wide-host-range TL-DNA and contains a region homologous to the cytokinin biosynthetic gene. However, a region homologous to the two auxin biosynthetic loci of the wide-host-range plasmid mapped within the TB-DNA region. These latter genes play an important role in tumor formation because mutations in these loci result in a loss of virulence on Nicotiana plants. Furthermore, the TB-DNA region alone conferred tumorigenicity onto strains with an intact set of vir genes. Our results suggest that factors within both the T-DNA and the vir regions contribute to the expression of host range in Agrobacterium species. There was a tremendous variation among plants in susceptibility to tumor formation by various A. tumefaciens strains. This variation occurred not only among different plant species, but also among different varieties of plants within the same genus.     

Virulence of Agrobacterium tumefaciens Strain A281 on Legumes

This study addresses the basis of host range on legumes of Agrobacterium tumefaciens strain A281, an l,l-succinamopine strain. We tested virulence of T-DNA and vir region constructs from this tumor-inducing (Ti) plasmid with complementary Ti plasmid regions from heterologous nopaline and octopine strains.     

Analysis of unique variable region of a plant root inducing plasmid, pRi1724, by the construction of its physical map and library.

Ri plasmids in Agrobacterium rhizogenes specifically induce the hairy root syndrome on various dicotyledonous plants. Its T-DNA transfer system as well as those of Ti plasmids have successfully provided the fundamental technique to introduce exogenous genes into plants. To study the Ri genome structure, we constructed a complete BamHI physical map and a lambda library of pRi1724 of A. rhizogenes strain 1724. By using these, we carried out the complete sequence of the 74-kb region between the right border of T-DNA and tra operon, which is the highly variable region (VAR) among Ri and Ti plasmids. As a result, we found three kinds of putative ABC-type transport operons, histidine utilization operon, glycerol utilization operon and two chemoreceptor genes. In addition, a virulence-related gene, tzs was located independently of the vir region.     

The octopine-type Ti plasmid pTiA6 of Agrobacterium tumefaciens contains a gene homologous to the chromosomal virulence gene acvB.

Although the majority of genes required for the transfer of T-DNA from Agrobacterium tumefaciens to plant nuclei are located on the Ti plasmid, some chromosomal genes, including the recently described acvB gene, are also required. We show that AcvB shows 50% identity with the product of an open reading frame, designated virJ, that is found between the virA and virB genes in the octopine-type Ti plasmid pTiA6. This reading frame is not found in the nopaline-type Ti plasmid pTiC58. acvB is required for tumorigenesis by a strain carrying a nopaline-type Ti plasmid, and virJ complements this nontumorigenic phenotype, indicating that the products of these genes have similar functions. A virJ-phoA fusion expressed enzymatically active alkaline phosphatase, indicating that VirJ is at least partially exported. virJ is induced in a VirA/VirG-dependent fashion by the vir gene inducer acetosyringone. Primer extension analysis and subcloning of the virJ-phoA fusion indicate that the acetosyringone-inducible promoter lies directly upstream of the virJ structural gene. Although the roles of the two homologous genes in tumorigenesis remain to be elucidated, strains lacking acvB and virJ (i) are proficient for induction of the vir regulon, (ii) are able to transfer their Ti plasmids by conjugation, and (iii) are resistant to plant wound extracts. Finally, mutations in these genes cannot be complemented extracellularly.     

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.     

NewAgrobacterium helper plasmids for gene transfer to plants

We describe the construction of new helper Ti plasmids forAgrobacterium-mediated plant transformation. These plasmids are derived from three differentAgrobacterium tumefaciens Ti plasmids, the octopine plasmid pTiB6, the nopaline plasmid pTiC58, and the L,L-succinamopine plasmid pTiBo542. The T-DNA regions of these plasmids were deleted using site-directed mutagenesis to yield replicons carrying thevir genes that will complement binary vectorsin trans. Data are included that demonstrate strain utility. The advantages ofAgrobacterium strains harbouring these disamed Ti plasmids for plant transformation viaAgrobacterium are discussed.     

Structure and function of root-inducing (Ri) plasmids and their relation to tumor-inducing (Ti) plasmids

The abilities of Agrobacterium tumefaciens and A. rhizogenes to transform dicotyle-dons and cause crown gall and hairy root disease are caused by the presence of tumor inducing (Ti) and root inducing (Ri) plasmids. During transformation plasmid T-DNA (transferred DNA) is inserted into the plant genome. The T-region is flanked by 25 bp direct repeats, which are essential for transfer. The T-regions contain oncogenes that are expressed in the plants. Some of these code for enzymes that synthesize auxin or cytokinin. Another type, present in Ri plasmids only, appears to impose a high hormone sensitivity on the infected tissue. The T-DNA also contains genes for enzymes synthesizing opines, which the bacteria catabolize. The T-DNA transfer is initiated by the induction of genes in the virulence (vir) region of the plasmid by phenolic compounds secreted by wounded tissue. The products of the vir -genes and of chromosomal genes mediate transfer of T-DNA to the plant cells. Crown gall disease is caused by production of auxin and cytokinin by the transferred T-DNA. The T-DNA of Ri plasmids codes for at least three genes that each can induce root formation, and that together cause hairy root formation from plant tissue. Current results indicate that the products of these genes induce a potential for increased auxin sensitivity that is expressed when the transformed cells are subjected to a certain level of auxin. After this stage the transformed roots can be grown in culture without exogenous supply of hormones.     

Characteristics of Ti plasmids from broad-host-range and ecologically specific biotype 2 and 3 strains of Agrobacterium tumefaciens.

Agrobacterium tumefaciens strains isolated from crown gall tumors on grapevines in California were consistently of the biotype 3 group. All 11 of these strains were limited in their host range and harbored Ti plasmids with molecular masses between 119 and 142 megadaltons (Mdal) as well as a larger cryptic plasmid of greater than 200 Mdal; occasionally a smaller cryptic plasmid of 65 Mdal was also present. Ti plasmids o these strains have DNA sequences in common with Ti plasmids of octopine and nopaline strains belonging to the biotype 1 group and exhibited sequence homologies with the conserved region of the T-DNA. Ten of the 11 strains utilized octopine as a sole source of carbon and nitrogen and 3 strains catabolized both octopine and nopaline, whereas 1 strain catabolized only nopaline. All of these strains were resistant to the bacteriocin agrocin-84, except one grapevine strain that belonged to the biotype 1 group and was agrocin sensitive; it is also differed in its plasmid and virulence characteristics. Isolations from Rubus ursinus ollalieberry galls yielded exclusively biotype 2 strains. These strans were insensitive to agrocin-84, utilized nopaline as a sole carbon and nitrogen source, and were highly virulent on all host plants tested. They contained Ti plasmids ranging between 100 and 130 Mdal and occasionally a cryptic plasmid of 69 Mdal. Their Ti plasmids have DNA sequences in common with Ti plasmids of biotype 1 strains and with the conserved region of the T-DNA.     

Efficient octopine Ti plasmid-derived vectors for Agrobacterium-mediated gene transfer to plants.

A two-component cloning system to transfer foreign DNA into plants was derived from the octopine Ti plasmid pTiB6S3. pGV2260 is a non-oncogenic Ti plasmid from which the T-region is deleted and substituted by pBR322. pGV831 is a streptomycin-resistant pBR325 derivative that contains a kanamycin resistance marker gene for plant cells and a site for cloning foreign genes between the 25-bp border sequences of the octopine T-region. Conjugative transfer of pGV831 derivatives to Agrobacterium and cointegration by homologous recombination between the pBR322 sequences present on pGV831 and pGV2260, can be obtained in a single step. Strains carrying the resulting cointegrated plasmids transfer and integrate T-DNA into the genome of tobacco protoplasts, and transformed tobacco calli are readily selected as resistant to kanamycin. Intact plants containing the entire DNA region between the T-DNA borders have been regenerated from such clones. In view of these properties we present pGV831 and its derivatives as vectors for efficient integration of foreign genes into plants.     

Dynamic structure of Agrobacterium tumefaciens Ti plasmids.

Agrobacterium tumefaciens C58F is a variant of strain C58 which generates a high proportion of avirulent mutants in the presence of the virulence (vir) gene inducer acetosyringone. These mutants are altered in the Ti plasmid and do not respond to the acetosyringone signal (C. Fortin, E. W. Nester, and P. Dion, J. Bacteriol. 174:5676-5685, 1992). The physical organization of the Ti plasmid was compared in strain C58 and its variant. One feature distinguishing pTiC58F from its parent plasmid was the presence of the insertion element IS426. Three copies of this element were detected in the strain C58 chromosome, whereas two additional copies were found in strain C58F, including one copy in the Ti plasmid. This particular copy of IS426 was associated with the region of arginine and nopaline catabolism of pTiC58F. Most of the avirulent mutants recovered following growth of strain C58F in the presence of acetosyringone were complemented by clones carrying either virA or virG. Element IS426 was no longer found in the arginine and nopaline catabolism region of the Ti plasmids from the virA and virG mutants, but it resided in the particular KpnI fragment containing the modified vir locus. Behavior of a strain C58F derivative, which was inactivated in a chromosomal component required for the response to acetosyringone, was consistent with the possibility that vir gene induction is essential to the massive production of avirulent mutants.     

Overexpression of virD1 and virD2 genes in Agrobacterium tumefaciens enhances T-complex formation and plant transformation.

The VirD1 and VirD2 proteins encoded by an inducible locus of the virulence (vir) region of the Agrobacterium tumefaciens Ti plasmid are required for site-specific nicking at T-DNA border sites. We have determined the nucleotide sequence of a 3.6-kilobase-pair fragment carrying the virD locus from nopaline Ti plasmid pTiC58. In contrast to the previous report (Hagiya et al., Proc. Natl. Acad. Sci. USA 82:2669-2673, 1985), we found that the first three open reading frames were capable of encoding polypeptides of 16.1, 49.7, and 21.4 kilodaltons. Deletion analysis demonstrated that the N-terminal conserved domain of VirD2 was absolutely essential for its endonuclease activity. When extra copies of the virD1 and virD2 genes were present in an A. tumefaciens strain carrying a Ti plasmid, increased amounts of T-strand and nicked molecules could be detected at early stages of vir induction. Such strains possessed the ability to transform plants with higher efficiency.     

Virulence genes, borders, and overdrive generate single-stranded T-DNA molecules from the A6 Ti plasmid of Agrobacterium tumefaciens.

Agrobacterium tumefaciens transfers the T-DNA portion of its Ti plasmid to the nuclear genome of plant cells. Upon cocultivation of A. tumefaciens A348 with regenerating tobacco leaf protoplasts, six distinct single-stranded T-DNA molecules (T strands) were generated in addition to double-stranded T-DNA border cleavages which we have previously reported (K. Veluthambi, R.K. Jayaswal, and S.B. Gelvin, Proc. Natl. Acad. Sci. USA 84:1881-1885, 1987). The T region of an octopine-type Ti plasmid has four border repeats delimiting three T-DNA regions defined as T left (TL), T center (TC), and T right (TR). The six T strands generated upon induction corresponded to the TL, TC, TR, TL + TC, TC + TR, and TL + TC + TR regions, suggesting that the initiation and termination of T-strand synthesis can occur at each of the four borders. Most TL + TC + TR T-strand molecules corresponded to the top T-DNA strand, whereas the other five T strands corresponded to the bottom T-DNA strand. Generation of T strands required the virA, virG, and virD operons. Extra copies of vir genes, harbored on cosmids within derivatives of A. tumefaciens A348, enhanced production of T strands. The presence of right and left border repeats in their native orientation is important for the generation of full-length T strands. When a right border repeat was placed in the opposite orientation, single-stranded T-DNA molecules that corresponded to the top strand were generated. Deletion of overdrive, a sequence that flanks right border repeats and functions as a T-DNA transmission enhancer, reduced the level of T-strand generation. Induction of A. tumefaciens cells by regenerating tobacco protoplasts increased the copy number of the Ti plasmid relative to the bacterial chromosome.     

Sequence characterization of the vir region of a nopaline type Ti plasmid, pTi-SAKURA.

We isolated a crown gall tumor-inducing nopaline type Ti plasmid from Agrobacterium tumefaciens on a Sakura Japanese cherry tree, and designated it as pTi-SAKURA. By primer walking sequencing with long PCR and a newly developed PCR subcloning technique for long insert DNA, we completed DNA sequencing of the most important functional unit, the virulence (vir) region of pTi-SAKURA, which is indispensable for T-DNA transfer into the plant's chromosomes. By homology searches with the vir genes of other bacterial plasmids, we identified 11 open reading frames (orfs) and 31 genes and 11 vir box, which are 6 bp regulatory sequences. In total, 26 vir genes, including the putative virF and virK and the main vir region, were present as the vir gene cluster. The presence of vir box, GC content, codon usage and expression analysis in these genes led us to propose a new vir region.     

Independent induction of transformed roots by the TL and TR regions of the Ri plasmid of agropine type Agrobacterium rhizogenes

Summary To analyse the respective role of TL- and TR-DNA in root induction by agropine-type Agrobacterium rhizogenes Ri plasmids, deletions covering the TL- or the TR-regions were constructed in vitro and introduced into pRiA4 by marker exchange. Each T-region of pRiHRI was also cloned separately on an independent replicon and used in a binary system with the virulence functions of either an Ri or a Ti plasmid provided in trans. Transformed roots were induced on tobacco and tomato explants by TL-DNA as well as by TR-DNA, suggesting that agropine type Ri plasmids from strains A4 and HRI can induce root proliferation by two independent transformation mechanisms. The root induction by the TR-DNA is probably due to auxin biosynthesis by gene products of aux loci homologous to the tms genes of Ti plasmid T-DNA. The molecular mechanism of root proliferation induced by the TL-DNA is probably equivalent to that of mannopine type Ri plasmid T-DNA.     

Complementation analysis of Agrobacterium tumefaciens Ti plasmid mutations affecting oncogenicity

A wide host range cosmid vector has been constructed by insertion of the lambda cos site into the plasmid pRK2501. This cosmid, which is maintained in Agrobacterium tumefaciens and is compatible with the Ti plasmid, has been used to make a clone bank of the A. tumefaciens pTiA6 plasmid. Several pTiA6 cosmids have been used to complement Tn5-induced Ti plasmid mutations. Five avirulent mutations which map outside of the region of the plasmid maintained in plant tumours (T-DNA) could be complemented in a trans orientation. Two mutations which are located on a single HpaI restriction fragment outside of the T-DNA, as well as three mutations which map within the T-DNA region, could not be complemented in a trans orientation in a REC- host.     

Transgenic tomato (Lycopersicon esculentum L.) transformed with a binary vector in Agrobacterium rhizogenes: Non-chimeric origin of callus clone and low copy numbers of integrated vector T-DNA

Summary We transformed tomato (Lycopersicon esculentum L.) by using Agrobacterium rhizogenes containing two independent plasmids: the wild-type Ri-plasmid, and the vector plasmid, pARC8. The T-DNA of the vector plasmid contained a marker gene (Nos/Kan) encoding neomycin phosphotransferase which conferred resistance to kanamycin in transformed plant cells. Transgenic plants (R ₀) with normal phenotype were regenerated from transformed organogenic calli by the punctured cotyledon transformation method. Southern blot analysis of the DNA from these transgenic plants showed that one or two copies of the vector plasmid T-DNA, but none of the Ri-plamid T-DNA, were integrated into the plant genome. Different transgenic plants derived from the same callus clone showed an identical DNA banding pattern, indicating the non-chimeric origin of these plants. We also transformed tomato by using A. tumefaciens strain LBA4404 containing a disarmed Ti-plasmid (pAL4404), and a vector plasmid (pARC8). Transgenic plants derived via A. tumefaciens transformation, like those via A. rhizogenes, contained one to two copies of the integrated vector T-DNA. The kanamycin resistance trait in the progeny (R ₁) of most transgenic plants segregated at a ratio of 3:1, suggesting that the vector T-DNAs were integrated at a single site on a tomato chromosome. In some cases, the expression of the marker gene (Nos/Kan) seemed to be suppressed or lost in the progeny.     

Molecular characterization of the vir regulon of Agrobacterium tumefaciens: complete nucleotide sequence and gene organization of the 28.63-kbp regulon cloned as a single unit

The entire vir regulon of Agrobacterium tumefaciens was subcloned and the complete 28.6-kbp nucleotide sequence was determined. The regulon was cloned as a single unit into two replicons, one of which replicates at a high copy number in this bacterium, and a second which has broad-host-range features to replicate in other Gram-negative bacteria. These vir region plasmids are able to confer in trans the processing and transfer activities on a second plasmid containing the T-DNA. In the high copy number vir region plasmid pUCD2614, a moderate increase in basal vir gene expression was observed as judged by virE::cat fusion expression assays relative to the wild-type control plasmid. Furthermore, higher efficiencies of tobacco leaf disk transformation were observed than with the widely used vir helper plasmid pAL4404. The nucleotide sequence studies showed that the vir region consists of 28,631 bp comprising 24 open reading frames which encode proteins involved in tumorigenicity. Two open reading frames not previously characterized, virH and ORF5, were uncovered within the virD/virE intervening spacer region. Together these studies more completely characterize the structure and function of the vir regulon.