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Microbiology and Molecular Biology Reviews, March 2003, p. 16-37, Vol. 67, No. 1
1092-2172/03/$08.00+0 DOI: 10.1128/MMBR.67.1.16-37.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907-1392
SUMMARY INTRODUCTION AGROBACTERIUM ''SPECIES'' AND HOST RANGE MOLECULAR BASIS OF AGROBACTERIUM-MEDIATED TRANSFORMATION What Is T-DNA? How Is T-DNA Transferred from Agrobacterium to Plant Cells? MANIPULATION OF AGROBACTERIUM FOR GENETIC ENGINEERING PURPOSES Introduction of Genes into Plants by Using Agrobacterium How Much DNA Can Be Transferred from Agrobacterium to Plants? What DNA Is Transferred from Agrobacterium to Plants? Transfer of Multiple T-DNAs into the Same Plant Cell, and Generation of ''Marker-Free'' Transgenic Plants Virulence Gene Expression and Plant Transformation T-DNA Integration and Transgene Expression Use of Matrix Attachment Regions To Ameliorate Transgene Silencing Use of Viral Suppressors of Gene Silencing To Increase Transgene Expression When Transgene Expression Is Not Forever MANIPULATION OF PLANT GENES TO IMPROVE TRANSFORMATION Plant Response to Agrobacterium Infection Identification of Plant Genes Encoding Proteins That Interact with Agrobacterium Virulence Proteins Forward Genetic Screening To Identify Plant Genes Involved in Agrobacterium-Mediated Transformation Reverse Genetic Screening for Plant Genes Involved in Agrobacterium-Mediated Transformation Genomics Approaches To Identify Plant Genes That Respond to Agrobacterium Infection PROSPECTS ACKNOWLEDGMENTS REFERENCES
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| INTRODUCTION |
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| AGROBACTERIUM "SPECIES" AND HOST RANGE |
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Regardless of the current confusion in species classification, for the purposes of plant genetic engineering, the most important aspect may be the host range of different Agrobacterium strains. As a genus, Agrobacterium can transfer DNA to a remarkably broad group of organisms including numerous dicot and monocot angiosperm species (12, 68, 262, 341) and gymnosperms (198, 206, 215, 228, 307, 357, 371). In addition, Agrobacterium can transform fungi, including yeasts (32, 33, 260), ascomycetes (1, 71), and basidiomycetes (71). Recently, Agrobacterium was reported to transfer DNA to human cells (187).
The molecular and genetic basis for the host range of a given Agrobacterium strain remains unclear. Early work indicated that the Ti plasmid, rather than chromosomal genes, was the major genetic determinant of host range (207, 315). Several virulence (vir) loci on the Ti plasmid, including virC (367, 368) and virF (220, 267), were shown to determine the range of plant species that could be transformed to yield crown gall tumors. The virH (formerly called pinF) locus appeared to be involved in the ability of Agrobacterium to transform maize, as established by an assay in which symptoms of maize streak virus infection were determined following agroinoculation of maize plants (153). Other vir genes, including virG, contribute to the "hypervirulence" of particular strains (41, 146).
However, it is now clear that host range is a much more complex process, which is under the genetic control of multiple factors within both the bacterium and the plant host. The way one assays for transformation can affect the way one views host range. For example, many monocot plant species, including some cultivars of grasses such as maize (152), rice (39, 40, 85, 139, 265, 321), barley (317), and wheat (42), can now be genetically transformed by many Agrobacterium strains to the phenotype of antibiotic or herbicide resistance. However, these plant species do not support the growth of crown gall tumors. Host range may further result from an interaction of particular Ti plasmids with certain bacterial chromosomal backgrounds. For example, the Ti plasmid pTiBo542, when in its natural host strain A. tumefaciens Bo542, directs limited tumorigenic potential when assayed on many leguminous plant species. However, when placed in the C58 chromosomal background, pTiBo542 directs strong virulence toward soybeans and other legumes (143). Finally, susceptibility to crown gall disease has a genetic basis in cucurbits (292), peas (272), soybeans (15, 214, 246), and grapevines (312) and even among various ecotypes of Arabidopsis thaliana (231). The roles of both bacterial virulence genes and host genes in the transformation process, and the ways in which they may possibly be manipulated for genetic engineering purposes, are discussed below.
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Second, the presence of T-DNA "overdrive" sequences near many T-DNA right borders, but not left borders, may also help establish the functional polarity of right and left borders. Overdrive sequences enhance the transmission of T-strands to plants, although the molecular mechanism of how this occurs remains unknown (131, 156, 256, 291, 336, 337, 345). Early reports suggested that the VirC1 protein binds to the overdrive sequence and may enhance T-DNA border cleavage by the VirD1/VirD2 endonuclease (322). virC1 and virC2 functions are important for virulence; mutation of these genes results in loss of virulence on many plant species (299). However, several laboratories have noted that T-strand production in virC mutant Agrobacterium strains occurs at wild-type levels (301, 344). Thus, any effect of VirC must occur after T-DNA processing.
VirA and VirG proteins function as members of a two-component sensory-signal transduction genetic regulatory system. VirA is a periplasmic antenna that senses the presence of particular plant phenolic compounds that are induced on wounding (3, 87, 162, 195, 303, 324, 359). In coordination with the monosaccharide transporter ChvE and in the presence of the appropriate phenolic and sugar molecules, VirA autophosphorylates and subsequently transphosphorylates the VirG protein (160, 161). VirG in the nonphosphorylated form is inactive; however, on phosphorylation, the protein helps activate or increase the level of transcription of the vir genes, most probably by interaction with vir-box sequences that form a component of vir gene promoters (59, 60, 252). Constitutively active VirA and VirG proteins that do not require phenolic inducers for activity, or VirG proteins that interact more productively with vir-box sequences to activate vir gene expression, may be useful to increase Agrobacterium transformation efficiency or host range. Experiments describing some attempts to manipulate VirA and/or VirG for these purposes are discussed below.
Together with the VirD4 protein, the 11 VirB proteins make up a type IV secretion system necessary for transfer of the T-DNA and several other Vir proteins, including VirE2 and VirF (44, 349). VirD4 may serve as a "linker" to promote the interaction of the processed T-DNA/VirD2 complex with the VirB-encoded secretion apparatus (126). Most VirB proteins either form the membrane channel or serve as ATPases to provide energy for channel assembly or export processes. Several proteins, including VirB2, VirB5, and possibly VirB7, make up the T-pilus (94, 163, 189, 190, 278, 283). VirB2, which is processed and cyclized, is the major pilin protein (94, 163, 189, 190). The function of the pilus in T-DNA transfer remains unclear; it may serve as the conduit for T-DNA and Vir protein transfer, or it may merely function as a "hook" to seize the recipient plant cell and bring the bacterium and plant into close proximity to effect molecular transfer. One aspect of pilus biology that may be important for transformation is its temperature lability. Although vir gene induction is maximal at approximately 25 to 27°C (8, 162, 323), the pilus of some, but not all, Agrobacterium strains is most stable at lower temperatures (approximately 18 to 20°C) (18, 105, 188). Early experiments by Riker indicated a temperature effect on transformation (268). Thus, one may consider cocultivating Agrobacterium with plant cells at lower temperatures during the initial few days of the transformation process.
The VirD2 and VirE2 proteins play essential and perhaps complementary roles in Agrobacterium-mediated transformation. These two proteins have been proposed to constitute, with the T-strand, a "T-complex" that is the transferred form of the T-DNA (149). Whether this complex assembles within the bacterium remains controversial. Citovsky et al. (50) showed that VirE2 could function in a plant cell: transgenic VirE2-expressing tobacco plants could "complement" infection by a virE2 mutant Agrobacterium strain. Several laboratories have shown that VirE2 can transfer to the plant cell in the absence of a T-strand (27, 193, 244, 309, 349), and it is possible that VirE2 complexes with the T-strand either in the bacterial export channel or within the plant cell. A recent report suggests perhaps another role for VirE2 early in the export process: Dumas et al. (90) showed that VirE2 could associate with artificial membranes in vitro and create a channel for the transport of DNA molecules. Thus, it is possible that one function of VirE2 is to form a pore in the plant cytoplasmic membrane to facilitate the passage of the T-strand.
Because of its attachment to the 5' end of the T-strand, VirD2 may serve as a pilot protein to guide the T-strand to and through the type IV export apparatus. Once in the plant cell, VirD2 may function in additional steps of the transformation process. VirD2 contains nuclear localization signal (NLS) sequences that may help direct it and the attached T-DNA to the plant nucleus. The NLS of VirD2 can direct fused reporter proteins and in vitro-assembled T-complexes to the nuclei of plant, animal, and yeast cells (48, 119, 138, 151, 185, 186, 229, 319, 326, 381, 382). Furthermore, VirD2 can associate with a number of Arabidopsis importin-
proteins in an NLS-dependent manner, both in yeast and in vitro (16; S. Bhattacharjee and S. B. Gelvin, unpublished data). Importin- is a component of one of the protein nuclear transport pathways found in eukaryotes. Recent data, however, suggest that VirD2 may not be sufficient to direct T-strands to the nucleus. Ziemienowicz et al. (382) showed that in permeabilized cells, VirD2 could effect the nuclear targeting of small linked oligonucleotides generated in vitro but could not direct the nuclear transport of larger linked molecules. To achieve nuclear targeting of these larger molecules, VirE2 additionally had to be associated with the T-strands. Finally, VirD2 may play a role in integration of the T-DNA into the plant genome. Various mutations in VirD2 can affect either the efficiency (229) or the "precision" (320) of T-DNA integration.
The role of VirE2 in T-DNA nuclear transport also remains controversial. VirE2 is a non-sequence-specific single-stranded DNA binding protein (45, 46, 49, 112, 286). In Agrobacterium cells, VirE2 probably interacts with the VirE1 molecular chaperone and may therefore not be available to bind T-strands (77, 84, 310, 380). However, when bound to single-stranded DNA (perhaps in the plant cell?), VirE2 can alter the DNA from a random-coil conformation to a shape that resembles a coiled telephone cord (47). This elongated shape may help direct the T-strand through the nuclear pore. VirE2 also contains NLS sequences that can direct fused reporter proteins to plant nuclei (48, 50, 326, 383). As with VirD2, VirE2 interacts in yeast with Arabidopsis importin- proteins in an NLS-dependent manner (Bhattacharjee and Gelvin, unpublished). One report indicates that VirE2 bound to single-stranded DNA and microinjected into plant cells can direct the DNA to the nucleus (381). However, other reports demonstrate that VirE2 cannot direct bound single-stranded DNA to the nuclei of either plant or animal cells that are permeabilized in order to effect DNA uptake (380). The cause of these contradictory results remains unclear but may reflect differences in the cell types and DNA delivery systems used by the two groups. When T-DNA is delivered to plant cells from Agrobacterium strains that encode a mutant form of VirD2 containing a precise deletion of the NLS, there is at most only a 40% decrease in transformation efficiency (229, 233, 290). Transgenic plants expressing VirE2 can complement a double-mutant Agrobacterium strain that lacks virE2 and contains a deletion in the NLS-encoding region of virD2 (110). These results suggest that in the absence of NLS sequences in VirD2, some other nuclear targeting mechanism (perhaps involving VirE2) may take place.
When bound to DNA, the NLS motifs of VirE2 may be occluded and inactive. This is because the NLS and DNA binding domains of VirE2 overlap (50). Hohn's group has hypothesized that the primary role of VirE2 in nuclear transport is NLS independent and that VirE2 merely shapes the T-strand so that it can snake through the nuclear pores (274).
Further controversy involves the ability of VirE2 protein to localize to the nuclei of animal cells. Ziemienowicz et al. (381) showed that in permeabilized HeLa cells, octopine-type VirE2 could target to the nucleus, whereas in microinjected Drosophila and Xenopus cells, the NLS sequence of nopaline-type VirE2 had to be changed in order to effect nuclear localization of the altered protein (50). Although the reason for this discrepancy is not known, it is not likely that it results from the use of octopine- versus nopaline-type VirE2 by the two groups (326).
Finally, VirE2 may protect T-strands from nucleolytic degradation that can occur both in the plant cytoplasm and perhaps in the nucleus (274, 374).
The existence of a T-complex composed of a single molecule of VirD2 covalently attached to the 5' end of the T-strand, which in turn is coated by VirE2 molecules, has generally been accepted by the Agrobacterium research community (149). However, the reader should be aware that such a complex has not yet been identified in either Agrobacterium or plant cells. It is possible that other proteins, such as importins (16), VIP1 (329), and even VirF (285), may additionally interact, either directly or indirectly, with the T-strand to form larger T-complexes in the plant cell.
Although the role of Ti plasmid-encoded vir genes has often been considered of primary importance for transformation, many Agrobacterium chromosomal genes are also essential for this process. The role of chromosomal genes was first established by random insertional mutagenesis of the entire Agrobacterium genome (106). Further research defined the roles of many of these genes. Included among these functions are exopolysaccharide production, modification, and secretion (pscA/exoC, chvA, and chvB [36, 37, 88, 89, 313]) and other roles in bacterial attachment to plant cells (att genes [212, 213]), sugar transporters involved in coinduction of vir genes (chvE [86, 172, 289]), regulation of vir gene induction (chvD [204]), and T-DNA transport (acvB [168, 248, 360, 361, 362]). Other genes, such as miaA (116), may also play a more minor role in the transformation process. The recent elucidation of the entire A. tumefaciens C58 sequence (114, 363) will surely provide fertile ground for the discovery of additional genes involved in Agrobacterium-mediated transformation.
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However, Ti plasmids are very large and T-DNA regions do not generally contain unique restriction endonuclease sites not found elsewhere on the Ti plasmid. Therefore, one cannot simply clone a gene of interest into the T-region. Scientists therefore developed a number of strategies to introduce foreign genes into the T-DNA. These strategies involved two different approaches: cloning the gene, by indirect means, into the Ti plasmid such that the new gene was in cis with the virulence genes on the same plasmid, or cloning the gene into a T-region that was on a separate replicon from the vir genes (T-DNA binary vectors).
Two methods were used for cloning foreign DNA into the Ti plasmid. The first method was based on a strategy developed by Ruvkin and Ausubel (277) (Fig. 2). A region of DNA (either the T-region or any region of DNA targeted for disruption) containing unique restriction endonuclease sites is cloned into a broad-host-range plasmid, such as an IncP-based vector. These plasmids can replicate both in Escherichia coli, in which the initial cloning is performed, and in Agrobacterium. The exogenous gene of interest, along with an antibiotic resistance marker, is next cloned into a unique restriction endonuclease site within the target region of DNA. Alternatively, an antibiotic resistance gene can be introduced into the DNA fragment of interest by transposition (107, 297). The resulting plasmid is introduced into Agrobacterium by conjugation or transformation. The presence of this plasmid in Agrobacterium is confirmed by selection for resistance to antibiotics encoded by both the plasmid vector backbone and the resistance marker near the gene of interest. Next, another plasmid of the same incompatibility group as the first plasmid, but harboring yet another antibiotic resistance marker, is introduced into the Agrobacterium strain containing the first plasmid. The resulting bacteria are plated on medium containing antibiotics to select for the second (eviction) plasmid and the resistance marker next to the gene of interest. Because plasmids of the same incompatibility group cannot usually coreside within the same bacterial cell, the bacteria can become resistant to both these antibiotics only if either (i) the first plasmid cointegrates into the Ti plasmid and uses the oriV of the Ti plasmid to replicate or (ii) an exchange of DNA on the first plasmid and the Ti plasmid occurs by double homologous recombination (homogenotization) using homologous sequences on the Ti plasmid flanking both sides of the gene of interest plus the resistance marker. In the first case (cointegration of the entire plasmid with the Ti plasmid), the resistance marker of the plasmid backbone would be expressed; these bacteria are screened for and discarded. In the second instance (homogenotization), the resistance marker encoded by the plasmid backbone is lost. Double homologous recombination can be confirmed by DNA blot analysis of total DNA from the resulting strain (107). A variant of this procedure utilizes a sacB gene on the plasmid backbone of the first plasmid. Only elimination of the plasmid backbone after homogenotization renders the bacterium resistant to growth on sucrose (194).
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Each of these cis-integration methods has advantages and disadvantages. The first strategy can target the foreign gene to any part of the T-region (or other region in the Ti plasmid). However, it is cumbersome to perform and involves somewhat sophisticated microbial genetic procedures that many laboratories shunned. The second method is technically easier but allows cointegration of the foreign gene only into Ti-plasmid locations where pBR322 had previously been placed. However, a modification of this procedure allows cointegration of a pBR322-based plasmid into any region of the Ti plasmid (338, 377). An advantage of both of these systems is that they maintain the foreign gene at the same low copy number as that of the Ti plasmid in Agrobacterium.
Because of the complexity of introducing foreign genes directly into the T-region of a Ti plasmid, several laboratories developed an alternative strategy to use Agrobacterium to deliver foreign genes to plants. This strategy was based on seminal findings of Hoekema et al. (140) and de Frammond et al. (70). These authors determined that the T-region and the vir genes could be separated into two different replicons. When these replicons were within the same Agrobacterium cell, products of the vir genes could act in trans on the T-region to effect T-DNA processing and transfer to a plant cell. Hoekema et al. called this a binary-vector system; the replicon harboring the T-region constituted the binary vector, whereas the replicon containing the vir genes became known as the vir helper. The vir helper plasmid generally contained a complete or partial deletion of the T-region, rendering strains containing this plasmid unable to incite tumors. A number of Agrobacterium strains containing nononcogenic vir helper plasmids have been developed, including LBA4404 (242), GV3101 MP90 (181), AGL0 (192), EHA101 and its derivative strain EHA105 (144, 146), and NT1 (pKPSF2) (247).
T-DNA binary vectors revolutionized the use of Agrobacterium to introduce genes into plants. Scientists without specialized training in microbial genetics could now easily manipulate Agrobacterium to create transgenic plants. These plasmids are small and easy to manipulate in both E. coli and Agrobacterium and generally contain multiple unique restriction endonuclease sites within the T-region into which genes of interest could be cloned. Many vectors were designed for specialized purposes, containing different plant selectable markers, promoters, and poly(A) addition signals between which genes of interest could be inserted, translational enhancers to boost the expression of transgenes, and protein-targeting signals to direct the transgene-encoded protein to particular locations within the plant cell (some representative T-DNA binary vector systems are described in references 10, 20, 25, 26, 62, 113, 120, 216, 364, and 386 and at http://www.cambia.org). Hellens et al. (134) provide a summary of many A. tumefaciens strains and vectors commonly used for plant genetic engineering.
Although the term "binary vector system" is usually used to describe two constituents (a T-DNA component and a vir helper component), each located on a separate plasmid, the original definition placed the two modules only on different replicons. These replicons do not necessarily have to be plasmids. Several groups have shown that T-DNA, when located in the Agrobacterium chromosome, can be mobilized to plant cells by a vir helper plasmid (141, 224).
Miranda et al. (224) showed that by reversing the orientation of a T-DNA right border, they could mobilize an entire Ti plasmid, approximately 200 kbp, into plants. Although the event was rare, this study showed that very large DNA molecules could be introduced into plants using Agrobacterium-mediated transformation. Hamilton et al. (124) first demonstrated the directed transfer of large DNA molecules from Agrobacterium to plants by the development of a binary BAC (BIBAC) system. These authors showed that a 150-kbp cloned insert of human DNA could be introduced into plant cells by using this system. However, the efficient transfer of such a large DNA segment required the overexpression of either virG or both virG and virE. VirE2 encodes a single-stranded DNA binding protein that protects the T-DNA from degradation in the plant cell (275). Because virG is a transcriptional activator of the vir operons (303), expression of additional copies of this regulatory vir gene was thought to enhance the expression of VirE2 and other Vir proteins involved in T-DNA transfer. Overexpression of virE formed part of the BIBAC system that was used to transform large (30- to 150-kbp) DNA fragments into tobacco and the more recalcitrant tomato and Brassica (56, 103, 123, 125). However, the transfer of different-size T-DNAs from various Agrobacterium strains had different requirements for overexpression of virG and virE (103). Liu et al. (203, 204) developed a transformation-competent artificial chromosome vector system based on a P1 origin of replication and used this system to generate libraries of large (40- to 120-kbp) Arabidopsis and wheat DNA molecules. This system did not require overexpression of virG or virE to effect the accurate transfer of large fragments to Arabidopsis.
The use of relatively small T-DNA binary vectors made it easier for scientists to evaluate the transfer of "non-T-DNA" regions to plants. Martineau et al. (211) first reported the transfer of binary vector backbone sequences into transgenic plant DNA and questioned the definition of T-DNA. Wenck et al. (356) found that the entire binary vector, including backbone sequences as well as T-DNA sequences, could frequently be transferred to Nicotiana plumbaginifolia and Arabidopsis thaliana cells. Kononov et al. (182) carefully examined the structure of binary vector backbone sequences that could be found in up to 75% of transgenic tobacco plants and concluded that such transfer could result from either skipping the left T-DNA border when T-DNA was processed from the binary vector or initiation of T-DNA transfer from the left border to bring vector backbone sequences into plant cells. Considering the previous observation by Durrenberger et al. (91) that VirD2 protein could covalently attach to the 5' end of the non-T-DNA strand, Kononov et al. suggested that transfer of vector backbone sequences to plants was a natural consequence of the mechanism of VirD2 function. Thus, the definition of T-DNA and vector backbone constitutes a semantic argument. It would thus appear that the transfer of non-T-DNA sequences to plants may be an unavoidable, but frequently unobserved and untested, result of transformation. Indeed, Frary and Hamilton (103) observed incorporation of BIBAC plasmid sequences into 9 to 38% of tested tomato transformants.
Although the transfer of plasmid backbone sequences may be an unavoidable consequence of the mechanism of Agrobacterium-mediated transformation, it may be possible to select against transgenic plants containing this unwanted DNA. Hanson et al. (132) showed that the incorporation of a toxic "killer" gene into the binary vector backbone sequences could severely reduce the percentage of transgenic plants containing such extra sequences. Remarkably, the transformation frequency of tobacco, tomato, and grape plants infected using this modified binary vector did not substantially differ from that of plants infected using a binary vector lacking the killer gene. Because the presence of uncharacterized DNA in transgenic plants is important for regulatory concerns, such an approach may be useful in the future for the production of plants (especially difficult to transform species) with a more highly defined transgenic composition.
Early research that characterized the integration pattern of T-DNAs in crown gall tumors indicated that each of the two T-DNAs encoded by an octopine-type Ti plasmid could independently integrate into the plant genome, sometimes in multiple copies (43, 63, 314). The molecular analyses suggested that these T-DNAs could be integrated into unlinked sites. These results suggested that cotransformation could be performed to integrate transgenes carried by two different T-DNAs and that perhaps these T-DNAs would segregate in subsequent generations. Three approaches were subsequently used for cotransformation: (i) the introduction of two T-DNAs, each from a different bacterium; (ii) the introduction of two T-DNAs carried by different replicons within the same bacterium; and (iii) the introduction of two T-DNAs located on the same replicon within a bacterium.
Early experiments using these various approaches indicated that cotransformation could be a frequent event. An et al. (9) showed that tobacco cells could be cotransformed to two different phenotypes by a single Agrobacterium strain containing both a Ti plasmid (phytohormone-independent growth) and a T-DNA binary vector (kanamycin-resistant growth). This experiment represents a "one-strain, two-replicon" approach to cotransformation. When the cells were first selected for kanamycin resistance, 10 to 20% of them also displayed phytohormone-independent growth; when the cells were first selected for phytohormone-independent growth, 60% of the resulting calli were also kanamycin resistant. The authors credited these differing frequencies to the higher copy number (5 to 10) of the binary vector in the bacterium relative to the single copy Ti plasmid.
These experiments were extended by de Frammond et al. (69), who showed that fertile transgenic plants could be regenerated from cloned tobacco tissue that was cotransformed by T-DNA from a Ti plasmid and from a micro-Ti (the one-strain, two-replicon approach). The two T-DNAs segregated in progeny plants, indicating that the T-DNAs had integrated into genetically separable loci. Other groups have used the one-strain, two-replicon approach to generate transgenic plants which initially expressed both T-DNA markers but could subsequently segregate the markers from each other (58).
Depicker et al. (80) performed a similar experiment in which the selection markers were phytohormone-independent growth and nopaline synthesis (encoded by a Ti plasmid) and kanamycin-resistant growth (encoded by a T-DNA binary vector). They performed the experiment in two ways: either the two T-DNAs were delivered by two different Agrobacterium strains (the two-strains, two-replicons approach), or both T-DNAs were delivered from a single replicon in one strain (the one-strain, one-replicon approach). The results of these experiments indicated that cotransfer of T-DNAs from the same plasmid in one strain was considerably more efficient than was transfer from two different strains. The use of a single Agrobacterium strain to cotransform plants with two T-DNAs from the same replicon, followed by segregation of the selection gene to generate marker-free transgenic plants, has been described by Komari et al. (178) and Xing et al. (365). In each of these studies, the authors were able to generate marker-free transgenic plants at high frequency.
The use of two Agrobacterium strains to deliver different T-DNAs to the same plant cells was studied by a number of groups (65, 66, 67, 76, 217). Although cotransfer of T-DNAs to genetically unlinked sites was reported, some authors also reported close linkage of the two different T-DNAs in many instances. It thus remains unclear which of the three cotransformation protocols will be reproducibly best for the generation of marker-free transgenic plants.
Extensive genetic studies resulted in the identification of a number of mutations that render the VirG protein active in the absence of phenolic inducing compounds (127, 254). These altered proteins contain mutations that converted either asparagine-54 to aspartic acid (virGN54D) or isoleucine-106 to leucine (virGI106L). Both of these mutant proteins stimulated a high level of vir gene expression, especially when expressed from a high-copy-number plasmid (118). When tested in transient tobacco and maize transformation assays, strains containing the virGN54D mutant effected a higher level of transformation than did strains encoding the wild-type virG gene (130). An even greater effect was seen when the virGN54D allele was harbored on a high-copy-number plasmid; the presence of this mutant gene in Agrobacterium increased the transient transformation of rice and soybean two- to sevenfold (170).
Several laboratories have determined the effect of additional copies of wild-type virG genes on vir gene induction and plant transformation. Rogowsky et al. (273) showed that additional copies of nopaline-type virG resulted in increased vir gene expression. Liu et al. (200) showed that multiple copies of virG altered the pH response profile for vir gene induction. Normally, vir gene induction is very poor at neutral or alkaline pH or in rich medium; additional copies of virG permitted a substantial degree of induction in rich medium even at pH 8.5. Additional copies of virG also increased the transient transformation frequency of rice, celery, and carrot tissues (199).
Given these results in toto, one may conclude that increasing the copy number of virA or virG or decreasing the dependence of the encoded proteins on phenolic inducers would generally increase the transformation efficiency of the resulting strains. However, the situation is likely to be more complex. Belanger et al. (23) showed that individual virA genes may be particularly suited to function in certain genetic backgrounds, and Krishnamohan et al. (183) recently demonstrated that Ti plasmids may have evolved to optimize specific combinations of virA, virG, and vir boxes. As noted above, the Ti-plasmid pTiBo542 in the C58 chromosomal background is hypervirulent on certain legume species, possibly because of the associated virG gene (41, 146, 159), but not in its native Bo542 chromosomal background (143). Recent results from my laboratory indicate that vir gene induction and T-strand production by and transformation efficiency of particular Agrobacterium strains may not correlate well. A. tumefaciens A277, containing the Ti plasmid pTiBo542 within the C58 chromosomal background, is considerably more virulent than are strains A348 and A208, containing the Ti plasmids pTiA6 and pTiT37, respectively, in the same chromosomal background. However, vir gene induction by plant exudates and T-strand production are highest in A. tumefaciens A208 (L.-Y. Lee and S. B. Gelvin, unpublished data). These data further suggest that increased vir gene induction and T-strand production may not necessarily be reliable predictors of transformation efficiency.
An obvious way to circumvent the presumed problems of position effect is to integrate T-DNA into known transcriptionally active regions of the plant genome. However, gene targeting in plants by homologous recombination has been at best extremely inefficient (72, 173, 223, 237, 238, 269, 270). An alternative system for gene targeting is the use of site-specific integration systems such as Cre-lox. However, single-copy transgenes introduced into a lox site in the same position of the plant genome also showed variable levels of expression in independent transformants. Transgene silencing in these instances may have resulted from transgene DNA methylation (61). Such methylation-associated silencing was reported earlier for naturally occurring T-DNA genes (135, 340). Thus, transcriptional silencing may result from integration of transgenes into regions of the plant genome susceptible to DNA methylation and may be a natural consequence of the process of plant transformation.
We now know not only that transgene silencing results from "transcriptional" mechanisms, usually associated with methylation of the transgene promoter (222), but also that transgene silencing is often "posttranscriptional"; i.e., the transgene is transcribed, but the resulting RNA is unstable (219). Such posttranscriptional gene silencing is frequently associated with multiple transgene copies within a cell. Transgenic plants generated by direct DNA transfer methods (e.g., polyethylene glycol- or liposome-mediated transformation, electroporation, or particle bombardment) often integrate a large number of copies of the transgene in tandem or inverted repeat arrays, in either multiple or single loci (176). Although Agrobacterium-mediated transformation usually results in a lower copy number of integrated transgenes, it is common to find tandem copies of a few T-DNAs integrated at a single locus (165). Transgene silencing can occur in plants harboring a single integrated T-DNA (95). However, integration of T-DNA repeats, especially "head-to-head' inverted repeats around the T-DNA right border, frequently results in transgene silencing (51, 164, 304). Thus, a procedure or Agrobacterium strain that could be used to generate transgenic plants with a single integrated T-DNA would be a boon to the agricultural biotechnology industry and to plant molecular biology in general. Grevelding et al. (117) noted that transgenic Arabidopsis plants derived from a root transformation procedure tended to have fewer T-DNA insertions than did plants derived from leaf disks. However, it is not clear if this observation can be generally applicable to other plant species. Anecdotal information from several laboratories suggests that Agrobacterium strains that are less efficient in delivering T-DNA may be more efficient in producing single-copy T-DNA insertions. However, these findings need to be tested rigorously; it is possible that T-DNA copy number may also correlate with the growth state of the bacterium or the plants to be transformed.
When they flank transgenes delivered to plants via Agrobacterium-mediated transformation, MARs appear to have only a small effect on transgene expression (128, 198, 201, 225, 226, 227, 334). Larger increases in transgene expression have been observed using particle bombardment-mediated transformation (5, 6, 236). However, this increase is generally associated with expression of transgenes in plant cells rather than in whole plants (330, 333). It is possible that the higher transgene expression effects of MARs using particle bombardment reflects the higher integrated transgene copy number resulting from this technique as opposed to the relatively lower copy number of integrated T-DNAs delivered by Agrobacterium (7). As such, it is not clear whether MARs will be, on their own, highly useful for decreasing the silencing of transgenes delivered to plants by Agrobacterium-mediated transformation.
As indicated in some of the references cited above, viral suppressors of gene silencing can activate a previously silenced stable transgene. One would then wonder whether such silencing suppressors could prevent the silencing of transgenes stably introduced into plants by Agrobacterium-mediated transformation. Although this hypothesis has not yet been tested and possible negative consequences (such as increased viral susceptibility) may ensue from the stable incorporation of antisilencing genes into a plant genome, experiments in which viral silencing suppressors have been used to increase the levels of transient expression of Agrobacterium-introduced transgenes appear promising. O. Voinnet and colleagues (unpublished data) have recently demonstrated that when cotransformed with various transgenes encoding green fluorescent protein, the potato virus Y Nia protein, or tomato Cf-9 and Cf-4 disease resistance proteins, various viral silencing suppressors dramatically increased the expression of these other proteins. Expression levels up to 50-fold higher than those achieved in control transformations (lacking the viral silencing suppressor genes) were obtained. Several different viral silencing suppressors, including the p25 protein of PVX, the P1-HcPro protein of tobacco etch virus, and the p19 protein of tomato bushy stunt virus, were able to enhance transient transgene expression from both the cauliflower mosaic virus 35S promoter and from native transgene promoters. Of these, the p19 protein was most effective in both increasing transient transgene expression and decreasing the levels of small (21- to 25-bp) RNA molecules associated with posttranscriptional gene silencing. The authors concluded that viral suppressors of gene silencing could be useful for the production of large amounts of proteins in plants.
There may be instances, however, when one would not want a transgene or its product to be present after the initial few hours or days following transformation. Such traits include those that would aid in the transformation process itself or would effect plant DNA rearrangements desired only during the initial transformation event (e.g., gene targeting using site-specific recombinase systems). Two strategies are currently being developed to permit only transient expression of gene products in plants. These are the use of "nonintegrating" T-DNA systems and the transfer of proteins, rather than DNA molecules, from Agrobacterium to plant cells.
Nonintegrating T-DNA systems include the use of mutant Agrobacterium strains and/or plant cells that are proficient in T-DNA nuclear transfer but deficient in T-DNA integration. During a search for domains of VirD2 necessary for nuclear targeting of the T-DNA, Shurvinton et al. (290) defined a C-terminal domain, termed 
, that showed high amino acid sequence homology among virD2 genes. Although this domain was not required for either VirD2 endonuclease activity or nuclear targeting of the T-DNA, replacement of four conserved amino acids by two serine residues resulted in a mutant protein that rendered the encoding Agrobacterium strain highly attenuated in virulence. Narasimhulu et al. (233) and Mysore et al. (229) further showed that Agrobacterium strains harboring this VirD2 substitution were highly deficient in stable transformation (with 2% of the efficiency of wild-type strains) but were still able to transform plant cells transiently at 20% of the efficiency of wild-type strains. Thus, this mutation rendered Agrobacterium strains highly deficient in T-DNA integration but still relatively proficient in delivering T-DNA to the plant nucleus. This mutant VirD2 protein can therefore be used to target T-strands to the nucleus, where they can transiently express but not efficiently integrate.
Nam et al. (231) used a root assay to screen almost 40 A. thaliana ecotypes for their ability to be transformed by Agrobacterium. Among these ecotypes, UE-1 was easily transiently transformed but poorly stably transformed. Genetic and molecular characterization of this ecotype indicated that the block in transformation occurred at the T-DNA integration step. Nam et al. (232) further identified a large number of Arabidopsis T-DNA insertion mutants that were resistant to Agrobacterium transformation (rat mutants). Of the initial 21 mutants, 5 were efficiently transiently transformed but were highly recalcitrant to stable transformation, a phenotype associated with a deficiency in T-DNA integration. Mysore et al. (230) characterized one of these mutants, the rat5 mutant, in greater detail. This mutant was generated by the insertion of T-DNA into the 3' untranslated region of a histone H2A gene (HTA1). Biochemical and molecular data indicated that this mutant could be transiently transformed efficiently but that T-DNA integration was disrupted. Although the precise role of the HTA1 gene in T-DNA integration has yet to be elucidated, root transformation of this mutant (and perhaps other T-DNA integration-deficient mutants) can be used for the transient delivery of T-DNA without efficient subsequent T-DNA integration. The use of the HTA1 gene to improve the transformation efficiency of wild-type plants is discussed below.
Vergunst et al. (349) recently described a novel procedure to transfer proteins directly from Agrobacterium to plant cells. This system relies on the ability of the type IV protein secretion system encoded by the Agrobacterium virB and virD4 genes to transfer certain Vir proteins to plant cells. VirD2, VirE2, and VirF are the three proteins identified to date that can be transferred by this system. These authors showed that translational fusions of the Cre recombinase protein to the N terminus of either VirE2 or VirF could be transferred to plant cells and effect recombination at lox sites. They further showed that the C-terminal 37 amino acids of VirF were sufficient to transfer the fusion protein. These experiments lead to the possibility of using Vir proteins as carriers to introduce other proteins transiently into plant cells.
| MANIPULATION OF PLANT GENES TO IMPROVE TRANSFORMATION |
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Agrobacterium infection of plant tissues may in some instances result in plant tissue necrosis. Several groups have described a slow, spreading necrosis in grape infected by particular Agrobacterium strains (79, 263). More recently, Hansen (129) described an apoptotic response of maize to Agrobacterium infection. The response included both rapid tissue necrosis and cleavage of nuclear DNA into oligonucleosome-sized fragments by endogenous nucleases and is characteristic of a caspase-protease cascade. The expression of two baculovirus cell death suppressor genes, p35 and iap, greatly inhibited both tissue necrosis and endogenous DNA cleavage. Manipulation of these genes during the Agrobacterium-mediated transformation process may thus be useful to increase both plant cell viability and transformation efficiency in plant species with an apoptotic response to Agrobacterium.
Several groups have recently begun to identify plant genes and protein products involved in the transformation process. One of the rationales for these experiments is the hope that identification of these genes may eventually result in their manipulation either to improve transformation or to make plants resistant to crown gall disease. A number of approaches have been employed to identify these plant genes, including (i) use of yeast two-hybrid systems to identify plant proteins that may interact with virulence proteins, (ii) direct "forward genetic" screening to identify plant mutants that cannot be transformed, (iii) "reverse genetic" screening to test whether particular genes of interest may be involved in transformation, and (iv) various genomics approaches to identify plant genes that may be induced or repressed soon after infection by Agrobacterium.
Early work (16) utilized VirD2 as the bait protein in a yeast two-hybrid system to identify an A. thaliana importin- (AtKAP, now known as importin-1) as an interacting partner. Importin- proteins are involved in the nuclear translocation of many proteins harboring NLS sequences, and Arabidopsis encodes at least nine of these proteins (Bhattacharjee and Gelvin, unpublished). Ballas and Citovsky (16) showed that interaction of VirD2 with importin- AtKAP was NLS dependent both in yeast and in vitro. The importance of importin- proteins in the Agrobacterium transformation process has recently been suggested by demonstrating that a T-DNA insertion into the importin-7 gene, or antisense inhibition of expression of the importin-1 (AtKAP) gene, results in a highly attenuated transformation phenotype (Bhattacharjee and Gelvin, unpublished).
VirD2 also interacts with at least two other plant proteins by using a yeast two-hybrid system. Deng et al. (78) identified three VirD2- and two VirE2-interacting proteins. They characterized more fully one of the VirD2 interactors, an Arabidopsis cyclophilin. This protein, as a glutathione S-transferase fusion, interacted strongly with VirD2 in vitro. The authors further showed that the interaction domain of VirD2 was in the central portion of the protein, a region to which no previous function had been ascribed. Cyclosporin A, an inhibitor of cyclophilins, inhibited Agrobacterium-mediated transformation of Arabidopsis roots and tobacco suspension cell cultures. The authors suggested that this plant protein may serve as a chaperone to help in T-complex trafficking within the plant cell. Experiments in my laboratory identified a tomato type 2C protein phosphatase as an interacting partner with VirD2. This phosphatase may be involved in the phosphorylation and dephosphorylation of a serine residue near the C-terminal NLS motif in VirD2. Overexpression of this phosphatase in transfected tobacco BY-2 cells resulted in decreased nuclear targeting of a GUS-VirD2-NLS fusion protein, suggesting that phosphorylation of the VirD2 NLS region may be involved in nuclear targeting of the VirD2/T-strand complex (Y. Tao, P. Rao, and S. B. Gelvin, submitted for publication).
Using VirE2 as the bait protein in a yeast two-hybrid system, Tzfira et al. (329) identified two interacting proteins from Arabidopsis, VIP1 and VIP2. VIP1 may be involved in nuclear targeting of the T-complex because antisense inhibition of VIP1 expression resulted in a deficiency in nuclear targeting of VirE2. Tobacco VIP1 antisense plants were also highly recalcitrant to Agrobacterium infection. Recent results further suggest a use for VIP1 in improving plant transformation: transgenic plants that overexpress VIP1 are hypersusceptible to Agrobacterium transformation (327, 330). VirE2 also interacts in yeast with several of the Arabidopsis importin- proteins, suggesting that VirD2 and VirE2 may have a common mechanism of nuclear import (Bhattacharjee and Gelvin, unpublished).
Schrammeijer et al. (285) recently identified an Arabidopsis Skp1 protein as an interactor with the F-box domain of VirF. Skp1 proteins may be involved in targeting proteins such as cyclins to the 26S proteosome, suggesting that VirF may function in setting the plant cell cycle to effect better transformation. The interaction of VirF with VIP1, but not VirE2, may also suggest a mechanism for Vir protein turnover: if VirF is targeted to the proteosome, it may help target other Vir proteins for proteolysis.
The T-pilus is essential for Agrobacterium-mediated transformation. Mutations in various VirB proteins disrupt transformation but not T-DNA processing (301, 344). As mentioned above, the major T-pilus component is the processed and cyclized VirB2 protein (189), although other virulence proteins, including VirB5 and possibly VirB7, also are minor T-pilus constituents (278, 283). Although the precise role of the T-pilus remains controversial, it is expected that the T-pilus would interact with the plant cell wall or membrane. Experiments in my laboratory have begun to address possible plant-interacting partners with T-pilus components. Using a yeast two-hybrid system and the processed, but not cyclized, form of VirB2 as a bait protein, we have identified two classes of Arabidopsis proteins that strongly and specifically interact with this major T-pilus constituent (H.-H. Hwang and S. B. Gelvin, unpublished data). One of these classes of plant proteins is encoded by a three-member gene family. Although the identity of these three related proteins is not currently known, their hydropathy profiles suggest that they contain membrane-spanning domains. The other interacting protein is a Rab-type GTPase. Each of t
