Novel constructions to enable the integration of genes into the Agrobacterium tumefaciens C58 chromosome.

We constructed several versatile sets of vectors that can be used to introduce any gene into the pgl/picA locus of the Agrobacterium tumefaciens C58 chromosome without affecting T-DNA transfer. One set contains a fragment containing the lacIq and lacZ genes and a multiple cloning site from pBluescriptII SK(+) inserted into a PstI site between the pgl and picA genes on an incPalpha plasmid. The resulting plasmid contains eight unique restriction endonuclease sites and the ability to use blue-white screening for the presence of an insert. A second plasmid also contains a beta-lactamase gene within this locus and provides a convenient ampicillin-carbenicillin resistance marker for the selection of genes integrated into the chromosome following double homologous recombination (homogenotization). A third plasmid contains, in addition to the lacZ, lacIq, and beta-lactamase genes within the pgl/picA locus, a sacRB gene cassette within the vector to counterselect against the presence of the vector within A. tumefaciens. To test this system, we introduced a wild-type virD2 gene into the A. tumefaciens chromosome at the pgl/picA locus. When a Ti plasmid harboring a deletion of virD2 was in this strain, the integrated virD2 gene complemented the virD2 deletion and the resulting transformation phenotype was identical to that resulting from A. tumefaciens strains harboring a wild-type virD2 gene located on a replicating plasmid.

Arabidopsis ecotypes and mutants that are recalcitrant to Agrobacterium root transformation are susceptible to germ-line transformation.

Germ-line transformation (vacuum infiltration) is frequently used to transform Arabidopsis thaliana using Agrobacterium tumefaciens. We have recently identified several Arabidopsis ecotypes and T-DNA-tagged mutants that are recalcitrant to Agrobacterium-mediated transformation of cut root segments. Some of these ecotypes and mutants are deficient in their ability to bind bacteria. Some are deficient in T-DNA integration. We report here that using a germ-line transformation protocol we transformed these ecotypes and mutants, including attachment- and integration-defective Arabidopsis plants, with a frequency similar to that of highly susceptible wild-type plants. However, we could not transform otherwise highly susceptible Arabidopsis plants by germ-line or root transformation using several vir and attachment-deficient Agrobacterium mutants. These results indicate that certain plant factors important for transformation may exist in germ-line tissue but may be lacking in some somatic cells.

An Arabidopsis histone H2A mutant is deficient in Agrobacterium T-DNA integration.

Agrobacterium tumefaciens genetically transforms plant cells by transferring a portion of the bacterial Ti-plasmid, the T-DNA, to the plant and integrating the T-DNA into the plant genome. Little is known about the T-DNA integration process, and no plant genes involved in integration have yet been identified. We characterized an Arabidopsis mutant generated by T-DNA insertional mutagenesis, rat5, that is resistant to Agrobacterium root transformation. rat5 contains two copies of T-DNA integrated as a tandem direct repeat into the 3' untranslated region of a histone H2A gene, upstream of the polyadenylation signal sequence. Transient and stable beta-glucuronidase expression data and assessment of the amount of T-DNA integrated into the genomes of wild-type and rat5 Arabidopsis plants indicated that the rat5 mutant is deficient in T-DNA integration. We complemented the rat5 mutation by expressing the RAT5 histone H2A gene in the mutant plant. Overexpression of RAT5 in wild-type plants increased Agrobacterium transformation efficiency. Furthermore, transient expression of a RAT5 gene from the incoming T-DNA was sufficient to complement the rat5 mutant and to increase the transformation efficiency of wild-type Arabidopsis plants.

Identification of T-DNA tagged Arabidopsis mutants that are resistant to transformation by Agrobacterium.

We have identified T-DNA tagged Arabidopsis mutants that are resistant to transformation by Agrobacterium tumefaciens (rat mutants). These mutants are highly recalcitrant to the induction of both crown gall tumors and phosphinothricin-resistant calli. The results of transient GUS (beta-glucuronidase) assays suggest that some of these mutants are blocked at an early step in the Agrobacterium-mediated transformation process, whereas others are blocked at a step subsequent to translocation of T-DNA into the nucleus. Attachment of Agrobacterium to roots of the mutants rat1 and rat3 was decreased under various incubation conditions. In most mutants, the transformation-deficient phenotype co-segregated with the kanamycin resistance encoded by the mutagenizing T-DNA. In crosses with susceptible wild-type plants, the resistance phenotype of many of these mutants segregated either as a semi-dominant or dominant trait.

pSa causes oncogenic suppression of Agrobacterium by inhibiting VirE2 protein export.

When coresident with the Ti (tumor-inducing) plasmid, the 21-kDa product of the osa gene of the plasmid pSa can suppress crown gall tumorigenesis incited by Agrobacterium tumefaciens. Neither T-DNA processing nor vir (virulence) gene induction is affected by the presence of osa in the bacterium. We used Arabidopsis thaliana root segments and tobacco leaf discs to demonstrate that Osa inhibits A. tumefaciens from transforming these plants to the stable phenotypes of tumorigenesis, kanamycin resistance, and stable beta-glucuronidase (GUS) expression. When A. tumefaciens contained osa, the lack of expression of transient GUS activity in infected plant tissues, as well as the lack of systemic viral symptoms following agroinfection of Nicotiana benthamiana by tomato mottle virus, suggested that oncogenic suppression by Osa occurs before T-DNA enters the plant nucleus. The extracellular complementation of an A. tumefaciens virE2 mutant (the T-DNA donor strain) by an A. tumefaciens strain lacking T-DNA but containing a wild-type virE2 gene (the VirE2 donor strain) was blocked when osa was present in the VirE2 donor strain, but not when osa was present in the T-DNA donor strain. These data indicate that osa inhibits VirE2 protein, but not T-DNA export from A. tumefaciens. These data further suggest that VirE2 protein and T-DNA are separately exported from the bacterium. The successful infection of Datura stramonium plants and leaf discs of transgenic tobacco plants expressing VirE2 protein by an A. tumefaciens virE2 mutant carrying osa confirmed that oncogenic suppression by osa does not occur by blocking T-DNA transfer. Overexpression of virB9, virB10, and virB11 in A. tumefaciens did not overcome oncogenic suppression by osa. The finding that the expression of the osa gene by itself, rather than the formation of a conjugal intermediate with pSa, blocks transformation suggests that the mechanism of oncogenic suppression by osa may differ from that of the IncQ plasmid RSF1010.

Agrobacterium tumefaciens transformation of the radiation hypersensitive Arabidopsis thaliana mutants uvh1 and rad5.

The Arabidopsis thaliana mutants uvh1 and rad5, originally identified as radiation hypersensitive, were reported to be deficient in T-DNA integration based on the relative efficiencies of stable transformation and T-DNA transfer. We reassessed these mutants for susceptibility to transformation by Agrobacterium tumefaciens. The mutant rad5 showed a significant reduction in the efficiency of transient as well as stable transformation, compared with its wild-type progenitor. These data indicate that rad5 is blocked at a step in the transformation process prior to T-DNA integration. We additionally found, using both an in vitro root inoculation and an in vivo flower bolt inoculation assay, that the mutant uvh1 is as susceptible to A. tumefaciens-mediated transformation as is its wild-type progenitor, C10.

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.

Role of the Agrobacterium tumefaciens VirD2 protein in T-DNA transfer and integration.

VirD2 is one of the key Agrobacterium tumefaciens proteins involved in T-DNA processing and transfer. In addition to its endonuclease domain, VirD2 contains a bipartite C-terminal nuclear localization sequence (NLS) and a conserved region called omega that is important for virulence. Previous results from our laboratory indicated that the C-terminal, bipartite NLS and the omega region are not essential for nuclear uptake of T-DNA, and further suggested that the omega domain may be required for efficient integration of T-DNA into the plant genome. In this study, we took two approaches to investigate the importance of the omega domain in T-DNA integration. Using the first approach, we constructed a T-DNA binary vector containing a promoterless gusA-intron gene just inside the right T-DNA border. The expression of beta-glucuronidase (GUS) activity in plant cells transformed by this T-DNA would indicate that the T-DNA integrated downstream of a plant promoter. Approximately 0.4% of the tobacco cell clusters infected by a wild-type A. tumefaciens strain harboring this vector stained blue with 5-bromo-4-chloro-3-indolyl beta-D-glucuronic acid (X-gluc). However, using an omega-mutant A. tumefaciens strain harboring the same binary vector, we did not detect any blue staining. Using the second approach, we directly demonstrated that more T-DNA is integrated into high-molecular-weight plant DNA after infection of Arabidopsis thaliana cells with a wild-type A. tumefaciens strain than with a strain containing a VirD2 omega deletion/substitution. Taken together, these data indicate that the VirD2 omega domain is important for efficient T-DNA integration. To determine whether the use of the T-DNA right border is altered in those few tumors generated by A. tumefaciens strains harboring the omega mutation, we analyzed DNA extracted from these tumors. Our data indicate that the right border was used to integrate the T-DNA in a similar manner regardless of whether the VirD2 protein encoded by the inciting A. tumefaciens was wild-type or contained an omega mutation. In addition, a mutant VirD2 protein lacking the omega domain was as least as active in cleaving a T-DNA border in vitro as was the wild-type protein. Finally, we investigated the role of various amino acids of the omega and bipartite NLS domains in the targeting of a GUS-VirD2 fusion protein to the nucleus of electroporated tobacco protoplasts. Deletion of the omega domain, or mutation of the 10-amino-acid region between the two components of the bipartite NLS, had little effect upon the nuclear targeting of the GUS-VirD2 fusion protein. Mutation of both components of the NLS reduced, but did not eliminate, targeting of the fusion protein to the nucleus.

Integration of T-DNA binary vector 'backbone' sequences into the tobacco genome: evidence for multiple complex patterns of integration.

During the process of crown gall tumorigenesis, Agrobacterium tumefaciens transfers part of the tumor-inducing (Ti) plasmid, the T-DNA, to a plant cell where it eventually becomes stably integrated into the plant genome. Directly repeated DNA sequences, called T-DNA borders, define the left and the right ends of the T-DNA. The T-DNA can be physically separated from the remainder of the Ti-plasmid, creating a 'binary vector' system; this system is frequently used to generate transgenic plants. Scientists initially thought that only those sequences located between T-DNA left and right borders transferred to the plant. More recently, however, several reports have appeared describing the integration of the non-T-DNA binary vector 'backbone' sequences into the genome of transgenic plants. In order to investigate this phenomenon, we constructed T-DNA binary vectors containing a nos-nptll gene within the T-DNA and a mas2'-gusA (beta-glucuronidase) gene outside the T-DNA borders. We regenerated kanamycin-resistant transgenic tobacco plants and analyzed these plants for the expression of the vector-localized gusA gene and for the presence of binary vector backbone sequences. Approximately one-fifth of the plants expressed detectable GUS activity. PCR analysis indicated that approximately 75% of the plants contained the gusA gene. Southern blot analysis indicated that the vector backbone sequences could integrate into the tobacco genome linked either to the left or to the right T-DNA border. The vector backbone sequences could also integrate into the plant genome independently of (unlinked to) the T-DNA. Although we could readily detect T-strands containing the T-DNA within the bacterium, we could not detect T-strands containing only the vector backbone sequences or these vector sequences linked to the T-DNA.

Differences in susceptibility of Arabidopsis ecotypes to crown gall disease may result from a deficiency in T-DNA integration.

We show that among ecotypes of Arabidopsis, there is considerable variation in their susceptibility to crown gall disease. Differences in susceptibility are heritable and, in one ecotype, segregate as a single major contributing locus. In several ecotypes, recalcitrance to tumorigenesis results from decreased binding of Agrobacterium to inoculated root explants. The recalcitrance of another ecotype occurs at a late step in T-DNA transfer. Transient expression of a T-DNA-encoded beta-glucuronidase gusA gene is efficient, but the ecotype is deficient in crown gall tumorigenesis, transformation to kanamycin resistance, and stable GUS expression. This ecotype is also more sensitive to gamma radiation than is a susceptible ecotype. DNA gel blot analysis showed that after infection by Agrobacterium, less T-DNA was integrated into the genome of the recalcitrant ecotype than was integrated into the genome of a highly susceptible ecotype.

A cytoplasmic male sterility-associated mitochondrial protein causes pollen disruption in transgenic tobacco.

In higher plants, dominant mitochondrial mutations are associated with pollen sterility. This phenomenon is known as cytoplasmic male sterility (CMS). It is thought that the disruption in pollen development is a consequence of mitochondrial dysfunction. To provide definitive evidence that expression of an abnormal mitochondrial gene can interrupt pollen development, a CMS-associated mitochondrial DNA sequence from common bean, orf239, was introduced into the tobacco nuclear genome. Several transformants containing the orf239 gene constructs, with or without a mitochondrial targeting sequence, exhibited a semi sterile or male-sterile phenotype. Expression of the gene fusions in transformed anthers was confirmed using RNA gel blotting, ELISA, and light and electron microscopic immunocytochemistry. Immunocytological analysis showed that the ORF239 protein could associate with the cell wall of aberrant developing microspores. This pattern of extracellular localization was earlier observed in the CMS common bean line containing orf239 in the mitochondrial genome. Results presented here demonstrate that ORF239 causes pollen disruption in transgenic tobacco plants and may do so without targeting of the protein to the mitochondrion.

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.

Transfer RNA Is the Source of Extracellular Isopentenyladenine in a Ti-Plasmidless Strain of Agrobacterium tumefaciens.

Even in the absence of the classical Ti plasmid-encoded cytokinin biosynthetic genes ipt and tzs, Agrobacterium tumefaciens strains still release significant amounts of the cytokinin isopentenyladenine (iP) into the culture medium (R.W. Kaiss-Chapman and R.O. Morris [1977] Biochem Biophys Res Commun 76: 453-459). A potential source of the iP is isopentenylated transfer RNA (tRNA), which, in turn, is synthesized by the activity of tRNA:isopentenyltransferase encoded by the bacterial miaA gene. To determine whether secreted iP had its origin in isopentenylated tRNA, a miaA- deletion/insertion mutant was prepared and reconstructed in Agrobacterium tumefaciens in vivo. The mutant no longer possessed tRNA:isopentenylation activity and no longer released iP into the extracellular medium. Transfer RNA therefore makes a small but significant contribution to the total amount of cytokinin normally secreted by Agrobacterium strains. tRNA-mediated synthesis may also account for cytokinin production by other plant-associated bacteria, such as Rhizobia, that have been reported to secrete similarly low levels of nonhydroxylated cytokinins.

Sequence-specific interactions of wound-inducible nuclear factors with mannopine synthase 2' promoter wound-responsive elements.

A 318 bp mannopine synthase 2' (mas2') promoter element from the T-DNA of Agrobacterium tumefacians can direct wound-inducible and root-preferential expression of a linked uidA gene in transgenic tobacco plants. Wound inducibility is further enhanced by sucrose in the medium. Promoter deletion analysis indicated that the sucrose enhancement is conferred by a region extending from -318 to -213. DNase I footprinting indicated that an A/T-rich DNA sequence in this region is protected by tobacco nuclear factors. Regions extending from -103 to +66 and from -213 to -138 directed wound-inducibile expression of a linked uidA gene when placed downstream of a CaMV 35S enhancer or upstream of a truncated (-209) CaMV 35S promoter, respectively. DNase I footprinting analyses indicated that proteins from wounded tobacco leaves specifically bound to three contiguous motifs downstream of the mas2' TATA box. In addition to a common retarded band formed by the upstream wound-responsive element complexed with proteins from either wounded or unwounded tobacco leaves, two unique retarded bands were observed when this element was incubated with protein from wounded leaves. Methylation interference analysis additionally identified an unique motif composed of promoter elements and nuclear factors derived specifically from wounded tobacco leaves. We propose a model to describe the involvement of nuclear factors with mas2' promoter elements in wound-inducible gene expression.

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.

Formation of a putative relaxation intermediate during T-DNA processing directed by the Agrobacterium tumefaciens VirD1,D2 endonuclease.

During the initial stages of crown gall tumorigenesis, the T-DNA region of the Agrobacterium tumefaciens Ti-plasmid is processed, resulting in the production of T-DNA molecules that are subsequently transferred to the plant cell. Processing of the T-DNA in the bacterium involves the nicking of T-DNA border sequences by an endonuclease encoded by the virD locus, and the subsequent tight (possibly covalent) association of the VirD2 protein with the 5' end of the processed single-stranded or double-stranded T-DNA molecule. To investigate the interaction of the VirD1,D2 endonuclease with a right T-DNA border, a set of plasmids containing both the border and virD sequences on the same high-copy-number replicon has been constructed and introduced into Escherichia coli. In this model system a tight nucleoprotein complex is formed between the relaxed double-stranded substrate plasmid and the VirD2 protein. This putative T-DNA processing complex may be analogous to the covalent relaxation complex formed between the pilot protein and plasmid DNA during bacterial conjugation. VirD2 attachment to the relaxed substrate plasmid was resistant to denaturing agents but sensitive to S1 nuclease digestion, indicating a single-stranded region near the site of protein attachment. We speculate that this structure may be an intermediate formed prior to T-strand unwinding from the substrate plasmid in a host bacterium.

Factors influencing Agrobacterium-mediated transient expression of gusA in rice.

Transient expression of GUS in rice (Oryza sativa L.) mediated by Agrobacterium tumefaciens was characterized using binary vectors containing gusA genes that express minimal (pKIWI105 and pCNL1) or no (p35S-GUS-INT and pCNL56) GUS activity in bacteria. Four-day old seedlings obtained from seeds or immature embryos of rice were cut into shoot, root, and seed remnants and inoculated with various strains of A. tumefaciens. Transient GUS expression events were quantitated histochemically by determining the frequency of explants exhibiting blue spots indicative of GUS at four to six days after cocultivation with A. tumefaciens. A. tumefaciens strains that did not contain the gusA gene (At643) or a Ti-plasmid (At563 and At657) did not elicit any blue staining characteristic of GUS activity. Several parameters were important in obtaining efficient transient expression of GUS in rice mediated by A. tumefaciens. The growth regulator 2,4-D inhibited GUS expression if present during the seed germination period, but the presence of 6 mg/l 2,4-D during cocultivation of the explants with A. tumefaciens slightly enhanced GUS expression efficiency. All 21 rice cultivars tested expressed GUS after co-cultivation with A. tumefaciens. The GUS expression frequency was highest amongst the indica cultivars. The frequencies of GUS expression in japonica cultivars and in Oryza glaberrima cultivars (grown primarily in Africa) were generally one-half to one-third the level found for indica varieties. Leaf explants were more susceptible to A. tumefaciens-facilitated GUS expression than were roots or seed remnants. The vir genes of an agropine-type Ti-plasmid of A. tumefaciens were most effective in directing transient GUS expression in rice, whereas those of a nopaline-type and an octopine-type plasmid were less effective. We have also found that the frequency of transient expression of GUS was higher with pBIN19 as the precursor cloning vector than with pEND4K as the precursor cloning vector. Reasons for differences in effectiveness of these binary vectors are discussed. Using the conditions described here, A. tumefaciens-mediated frequencies of transient GUS expression in four-day old shoots of several rice cultivars were routinely in excess of 50%.

Multiple copies of virG enhance the transient transformation of celery, carrot and rice tissues by Agrobacterium tumefaciens.

In an effort to improve the T-DNA-mediated transformation frequency of economically important crops, we investigated the possible enhancement effect of multiple copies of virG genes contained in Agrobacterium tumefaciens strains upon the transient transformation of celery, carrot and rice tissues. Four days after A. tumefaciens infection, we performed histochemical beta-glucuronidase (GUS) assays to determine the frequency of transient transformation of calli from celery and carrot, and explants from rice and celery. Additional copies of octopine- and agropine-type virG genes in A. tumefaciens strains containing an agropine-type Ti-plasmid enhanced the frequency of transient transformation of celery and rice. This enhancement ranged from 25% to five-fold, depending upon the source of the virG gene and the plant tissues inoculated. For both rice and celery, we observed a greater enhancement of transformation using A. tumefaciens strains containing additional copies of an octopine-type virG gene than with strains harboring additional copies of an agropine-type virG gene. Multiple copies of virG genes contained in A. tumefaciens strains harboring a nopaline-type Ti-plasmid had a smaller enhancing effect upon the transformation of celery tissues, and no enhancing effect upon the transformation of rice. In contrast, we obtained a three-fold increase in the transient transformation frequency of carrot calli using an A. tumefaciens strain harboring a nopaline-type Ti-plasmid and additional copies of an octopine-type virG gene. Our results show that multiple copies of virG in A. tumefaciens can greatly enhance the transient transformation frequency of celery, carrot and rice tissues, and that this enhancement is influenced by both the type of Ti-plasmid harbored by A. tumefaciens and by the infected plant species.