Agrobacterium-Mediated Plant Transformation PDF

MICROBIOLOGY AND MOLECULAR BIOLOGY REVIEWS, Mar. 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. Agrobacterium-Mediated Plant Transformation: the Biology behind the “Gene-Jockeying” Tool Stanton B. Gelvin* Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907-1392 INTRODUCTION.........................................................................................................................................................16 AGROBACTERIUM “SPECIES” AND HOST RANGE...........................................................................................16 MOLECULAR BASIS OF AGROBACTERIUM-MEDIATED TRANSFORMATION..........................................17 What Is T-DNA?......................................................................................................................................................17 How Is T-DNA Transferred from Agrobacterium to Plant Cells?.....................................................................18 MANIPULATION OF AGROBACTERIUM FOR GENETIC ENGINEERING PURPOSES..............................20 Introduction of Genes into Plants by Using Agrobacterium...............................................................................20 How Much DNA Can Be Transferred from Agrobacterium to Plants?............................................................22 What DNA Is Transferred from Agrobacterium to Plants?................................................................................22 Transfer of Multiple T-DNAs into the Same Plant Cell, and Generation of “Marker-Free” Transgenic Plants................................................................................................................................................23 Virulence Gene Expression and Plant Transformation.....................................................................................23 T-DNA Integration and Transgene Expression...................................................................................................24 Use of Matrix Attachment Regions To Ameliorate Transgene Silencing........................................................25 Use of Viral Suppressors of Gene Silencing To Increase Transgene Expression..........................................25 When Transgene Expression Is Not Forever.......................................................................................................25 MANIPULATION OF PLANT GENES TO IMPROVE TRANSFORMATION..................................................26 Plant Response to Agrobacterium Infection..........................................................................................................26 Identification of Plant Genes Encoding Proteins That Interact with Agrobacterium Virulence Proteins...............................................................................................................................................27 Forward Genetic Screening To Identify Plant Genes Involved in Agrobacterium-Mediated Transformation....................................................................................................................................................27 Reverse Genetic Screening for Plant Genes Involved in Agrobacterium-Mediated Transformation............28 Genomics Approaches To Identify Plant Genes That Respond to Agrobacterium Infection.........................29 PROSPECTS.................................................................................................................................................................29 ACKNOWLEDGMENTS.............................................................................................................................................30 REFERENCES..............................................................................................................................................................30 INTRODUCTION rium biology (44, 73, 109, 325, 327, 328, 384, 385). In this review, I describe how scientists utilized knowledge of basic Twenty-five years ago, the concept of using Agrobacterium Agrobacterium biology to develop Agrobacterium as a “tool” for tumefaciens as a vector to create transgenic plants was viewed plant genetic engineering. I also explore how our increasing as a prospect and a “wish.” Today, many agronomically and understanding of Agrobacterium biology may help extend the horticulturally important species are routinely transformed us- utility of Agrobacterium-mediated transformation. It is my be- ing this bacterium, and the list of species that is susceptible to lief that further improvements in transformation technology Agrobacterium-mediated transformation seems to grow daily. will necessarily involve the manipulation of these fundamental In some developed countries, a high percentage of the acreage biological processes. of such economically important crops as corn, soybeans, cot- ton, canola, potatoes, and tomatoes is transgenic; an increas- ing number of these transgenic varieties are or will soon be AGROBACTERIUM “SPECIES” AND HOST RANGE generated by Agrobacterium-mediated, as opposed to particle bombardment-mediated transformation. There still remain, The genus Agrobacterium has been divided into a number of however, many challenges for genotype-independent transfor- species. However, this division has reflected, for the most part, mation of many economically important crop species, as well as disease symptomology and host range. Thus, A. radiobacter is forest species used for lumber, paper, and pulp production. In an “avirulent” species, A. tumefaciens causes crown gall dis- addition, predictable and stable expression of transgenes re- ease, A. rhizogenes causes hairy root disease, and A. rubi causes mains problematic. Several excellent reviews have appeared cane gall disease. More recently, a new species has been pro- recently that describe in detail various aspects of Agrobacte- posed, A. vitis, which causes galls on grape and a few other plant species (244). Although Bergey’s Manual of Systematic Bacteriology still reflects this nomenclature, classification is * Mailing address: Department of Biological Sciences, Purdue Uni- complex and confusing; we now know that symptoms follow, versity, West Lafayette, IN 47907-1392. Phone: (765) 494-4939. Fax: for the most part, the type of tumorigenic plasmid contained (765) 496-1496. E-mail: [email protected]. within a particular strain. Curing a particular plasmid and 16 VOL. 67, 2003 AGROBACTERIUM-MEDIATED PLANT TRANSFORMATION 17 replacing this plasmid with another type of tumorigenic plas- mid can alter disease symptoms. For example, infection of plants with A. tumefaciens C58, containing the nopaline-type Ti plasmid pTiC58, results in the formation of crown gall terato- mas. When this plasmid is cured, the strain becomes nonpatho- genic. Introduction of Ri plasmids into the cured strain “con- verts” the bacterium into a rhizogenic strain (191, 358). Furthermore, one can introduce a Ti (tumor-inducing) plasmid from A. tumefaciens into A. rhizogenes; the resulting strain incites tumors of altered morphology on Kalanchoe plants (53). Thus, because A. tumefaciens can be “converted” into A. rhi- zogenes simply by substituting one type of oncogenic plasmid for another, the term “species” becomes meaningless. Perhaps a more meaningful classification system divides the genus Agrobacterium into “biovars” based on growth and metabolic characteristics (171). Using this system, most A. tumefaciens and A. rubi (316) strains belong to biovar I, A. rhizogenes strains fit into biovar II, and biovar III is represented by A. vitis strains. More recently, yet another taxonomic classification system for the genus Agrobacterium has been proposed (374). The recent completion of the DNA sequence of the entire A. tumefaciens C58 genome (which is composed of a linear and a circular chromosome, a Ti plasmid, and another large plasmid [114, FIG. 1. Schematic representation of a typical octopine-type Ti plas- 115, 363]) may provide a starting point for reclassification of mid (A) and the T-DNA region of a typical octopine-type Ti plasmid Agrobacterium “strains” into true “species.” (B). (A) The T-DNA is divided into three regions. TL (T-DNA left), Regardless of the current confusion in species classification, TC (T-DNA center), and TR (T-DNA right). The black circles indicate for the purposes of plant genetic engineering, the most impor- T-DNA border repeat sequences. oriV, the vegetative origin of repli- cation of the Ti plasmid, is indicated by a white circle. (B) The various tant aspect may be the host range of different Agrobacterium T-DNA-encoded transcripts, and their direction of transcription, are strains. As a genus, Agrobacterium can transfer DNA to a indicated by arrows. Genes encoding functions involved in auxin syn- remarkably broad group of organisms including numerous di- thesis (auxin), cytokinin synthesis (cyt), and the synthesis of the opines cot and monocot angiosperm species (12, 68, 262, 341) and octopine (ocs), mannopine (mas), and agropine (ags) are indicated. 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, Ti plasmids with certain bacterial chromosomal backgrounds. Agrobacterium was reported to transfer DNA to human cells For example, the Ti plasmid pTiBo542, when in its natural host (187). strain A. tumefaciens Bo542, directs limited tumorigenic po- The molecular and genetic basis for the host range of a given tential when assayed on many leguminous plant species. How- Agrobacterium strain remains unclear. Early work indicated ever, when placed in the C58 chromosomal background, that the Ti plasmid, rather than chromosomal genes, was the pTiBo542 directs strong virulence toward soybeans and other major genetic determinant of host range (207, 315). Several legumes (143). Finally, susceptibility to crown gall disease has virulence (vir) loci on the Ti plasmid, including virC (367, 368) a genetic basis in cucurbits (292), peas (272), soybeans (15, 214, and virF (220, 267), were shown to determine the range of 246), and grapevines (312) and even among various ecotypes of plant species that could be transformed to yield crown gall Arabidopsis thaliana (231). The roles of both bacterial viru- tumors. The virH (formerly called pinF) locus appeared to be lence genes and host genes in the transformation process, and involved in the ability of Agrobacterium to transform maize, as the ways in which they may possibly be manipulated for genetic established by an assay in which symptoms of maize streak engineering purposes, are discussed below. virus infection were determined following agroinoculation of maize plants (153). Other vir genes, including virG, contribute to the “hypervirulence” of particular strains (41, 146). MOLECULAR BASIS OF AGROBACTERIUM-MEDIATED However, it is now clear that host range is a much more TRANSFORMATION complex process, which is under the genetic control of multiple What Is T-DNA? factors within both the bacterium and the plant host. The way one assays for transformation can affect the way one views host The molecular basis of genetic transformation of plant cells range. For example, many monocot plant species, including by Agrobacterium is transfer from the bacterium and integra- some cultivars of grasses such as maize (152), rice (39, 40, 85, tion into the plant nuclear genome of a region of a large 139, 265, 321), barley (317), and wheat (42), can now be ge- tumor-inducing (Ti) or rhizogenic (Ri) plasmid resident in netically transformed by many Agrobacterium strains to the Agrobacterium (Fig. 1A). Ti plasmids are on the order of 200 to phenotype of antibiotic or herbicide resistance. However, these 800 kbp in size (81, 100, 111, 114, 145, 166, 175, 177, 245, 250, plant species do not support the growth of crown gall tumors. 251, 261, 311, 332, 342, 363). The transferred DNA (T-DNA) Host range may further result from an interaction of particular (Fig. 1B) is referred to as the T-region when located on the Ti 18 GELVIN MICROBIOL. MOL. BIOL. REV. or Ri plasmid. T-regions on native Ti and Ri plasmids are loss of virulence on many plant species (299). However, several approximately 10 to 30 kbp in size (17, 34, 197, 311, 378). Thus, laboratories have noted that T-strand production in virC mu- T-regions generally represent less than 10% of the Ti plasmid. tant Agrobacterium strains occurs at wild-type levels (301, 344). Some Ti plasmids contain one T-region, whereas others con- Thus, any effect of VirC must occur after T-DNA processing. tain multiple T-regions (17, 311). The processing of the T- DNA from the Ti plasmid and its subsequent export from the How Is T-DNA Transferred from Agrobacterium bacterium to the plant cell result in large part from the activity to Plant Cells? of virulence (vir) genes carried by the Ti plasmid (106, 147, 148, 174, 208, 303). As indicated above, many proteins encoded by vir genes play T-regions are defined by T-DNA border sequences. These essential roles in the Agrobacterium-mediated transformation borders are 25 bp in length and highly homologous in sequence process. Some of these roles have been discussed in several (167, 366). They flank the T-region in a directly repeated excellent review articles (44, 109, 325, 327, 328, 384), and I orientation (257, 276, 335, 345, 352). In general, the T-DNA shall therefore limit my description to the roles of Vir proteins borders delimit the T-DNA (but see below for exceptions), that may serve as points of manipulation for the improvement because these sequences are the target of the VirD1/VirD2 of the transformation process. border-specific endonuclease that processes the T-DNA from VirA and VirG proteins function as members of a two- the Ti plasmid. There appears to be a polarity established component sensory-signal transduction genetic regulatory sys- among T-DNA borders: right borders initially appeared to be tem. VirA is a periplasmic antenna that senses the presence of more important than left borders (136, 156, 286, 352, 353). We particular plant phenolic compounds that are induced on now know that this polarity may be caused by several factors. wounding (3, 87, 162, 195, 303, 324, 359). In coordination with First, the border sequences not only serve as the target for the the monosaccharide transporter ChvE and in the presence of VirD1/VirD2 endonuclease but also serve as the covalent at- the appropriate phenolic and sugar molecules, VirA autophos- tachment site for VirD2 protein. Within the Ti or Ri plasmid phorylates and subsequently transphosphorylates the VirG (or T-DNA binary vectors [see below]), T-DNA borders are protein (160, 161). VirG in the nonphosphorylated form is made up of double-stranded DNA. Cleavage of these double- inactive; however, on phosphorylation, the protein helps acti- stranded border sequences requires VirD1 and VirD2 pro- vate or increase the level of transcription of the vir genes, most teins, both in vivo (82, 99, 155, 369) and in vitro (281). In vitro, probably by interaction with vir-box sequences that form a however, VirD2 protein alone can cleave a single-stranded component of vir gene promoters (59, 60, 252). Constitutively T-DNA border sequence (154, 249). Cleavage of the 25-bp active VirA and VirG proteins that do not require phenolic T-DNA border results predominantly from the nicking of the inducers for activity, or VirG proteins that interact more pro- T-DNA “lower strand,” as conventionally presented, between ductively with vir-box sequences to activate vir gene expression, nucleotides 3 and 4 of the border sequence (301, 353). How- may be useful to increase Agrobacterium transformation effi- ever, double-strand cleavage of the T-DNA border has also ciency or host range. Experiments describing some attempts to been noted (155, 305, 344). Nicking of the border is associated manipulate VirA and/or VirG for these purposes are discussed with the tight (probably covalent) linkage of the VirD2 protein, below. through tyrosine 29 (351), to the 5⬘ end of the resulting single- Together with the VirD4 protein, the 11 VirB proteins make stranded T-DNA molecule termed the T-strand (91, 99, 137, up a type IV secretion system necessary for transfer of the 150, 355, 373). It is this T-strand, and not a double-stranded T-DNA and several other Vir proteins, including VirE2 and T-DNA molecule, that is transferred to the plant cell (318, VirF (44, 349). VirD4 may serve as a “linker” to promote the 375). Thus, it is the VirD2 protein attached to the right border, interaction of the processed T-DNA/VirD2 complex with the and not the border sequence per se, that establishes polarity VirB-encoded secretion apparatus (126). Most VirB proteins and the importance of right borders relative to left borders. It either form the membrane channel or serve as ATPases to should be noted, however, that because left-border nicking is provide energy for channel assembly or export processes. Sev- also associated with VirD2 attachment to the remaining mol- eral proteins, including VirB2, VirB5, and possibly VirB7, ecule (the “non-T-DNA” portion of the Ti plasmid or “back- make up the T-pilus (94, 163, 189, 190, 278, 283). VirB2, which bone” region of the T-DNA binary vector ), it may be is processed and cyclized, is the major pilin protein (94, 163, possible to process T-strands from these regions of Ti and Ri 189, 190). The function of the pilus in T-DNA transfer remains plasmids and from T-DNA binary vectors (182, 264, 356). The unclear; it may serve as the conduit for T-DNA and Vir protein problem of vector “backbone” sequence transfer to plants is transfer, or it may merely function as a “hook” to seize the discussed below. recipient plant cell and bring the bacterium and plant into Second, the presence of T-DNA “overdrive” sequences near close proximity to effect molecular transfer. One aspect of pilus many T-DNA right borders, but not left borders, may also help biology that may be important for transformation is its tem- establish the functional polarity of right and left borders. Over- perature lability. Although vir gene induction is maximal at drive sequences enhance the transmission of T-strands to approximately 25 to 27°C (8, 162, 323), the pilus of some, but plants, although the molecular mechanism of how this occurs not all, Agrobacterium strains is most stable at lower tempera- remains unknown (131, 156, 256, 291, 336, 337, 345). Early tures (approximately 18 to 20°C) (18, 105, 188). Early experi- reports suggested that the VirC1 protein binds to the overdrive ments by Riker indicated a temperature effect on transforma- sequence and may enhance T-DNA border cleavage by the tion (268). Thus, one may consider cocultivating Agrobacterium VirD1/VirD2 endonuclease (322). virC1 and virC2 functions with plant cells at lower temperatures during the initial few are important for virulence; mutation of these genes results in days of the transformation process. VOL. 67, 2003 AGROBACTERIUM-MEDIATED PLANT TRANSFORMATION 19 The VirD2 and VirE2 proteins play essential and perhaps (381). However, other reports demonstrate that VirE2 cannot complementary roles in Agrobacterium-mediated transforma- direct bound single-stranded DNA to the nuclei of either plant tion. These two proteins have been proposed to constitute, or animal cells that are permeabilized in order to effect DNA with the T-strand, a “T-complex” that is the transferred form uptake (380). The cause of these contradictory results remains of the T-DNA (149). Whether this complex assembles within unclear but may reflect differences in the cell types and DNA the bacterium remains controversial. Citovsky et al. (50) delivery systems used by the two groups. When T-DNA is showed that VirE2 could function in a plant cell: transgenic delivered to plant cells from Agrobacterium strains that encode VirE2-expressing tobacco plants could “complement” infec- a mutant form of VirD2 containing a precise deletion of the tion by a virE2 mutant Agrobacterium strain. Several laborato- NLS, there is at most only a 40% decrease in transformation ries have shown that VirE2 can transfer to the plant cell in the efficiency (229, 233, 290). Transgenic plants expressing VirE2 absence of a T-strand (27, 193, 244, 309, 349), and it is possible can complement a double-mutant Agrobacterium strain that that VirE2 complexes with the T-strand either in the bacterial lacks virE2 and contains a deletion in the NLS-encoding region export channel or within the plant cell. A recent report sug- of virD2 (110). These results suggest that in the absence of NLS gests perhaps another role for VirE2 early in the export pro- sequences in VirD2, some other nuclear targeting mechanism cess: Dumas et al. (90) showed that VirE2 could associate with (perhaps involving VirE2) may take place. artificial membranes in vitro and create a channel for the When bound to DNA, the NLS motifs of VirE2 may be transport of DNA molecules. Thus, it is possible that one occluded and inactive. This is because the NLS and DNA function of VirE2 is to form a pore in the plant cytoplasmic binding domains of VirE2 overlap (50). Hohn’s group has membrane to facilitate the passage of the T-strand. hypothesized that the primary role of VirE2 in nuclear trans- Because of its attachment to the 5⬘ end of the T-strand, port is NLS independent and that VirE2 merely shapes the VirD2 may serve as a pilot protein to guide the T-strand to and T-strand so that it can snake through the nuclear pores (274). through the type IV export apparatus. Once in the plant cell, Further controversy involves the ability of VirE2 protein to VirD2 may function in additional steps of the transformation localize to the nuclei of animal cells. Ziemienowicz et al. (381) process. VirD2 contains nuclear localization signal (NLS) se- showed that in permeabilized HeLa cells, octopine-type VirE2 quences that may help direct it and the attached T-DNA to the could target to the nucleus, whereas in microinjected Drosoph- plant nucleus. The NLS of VirD2 can direct fused reporter ila and Xenopus cells, the NLS sequence of nopaline-type proteins and in vitro-assembled T-complexes to the nuclei of VirE2 had to be changed in order to effect nuclear localization plant, animal, and yeast cells (48, 119, 138, 151, 185, 186, 229, of the altered protein (50). Although the reason for this dis- 319, 326, 381, 382). Furthermore, VirD2 can associate with a crepancy is not known, it is not likely that it results from the number of Arabidopsis importin-␣ proteins in an NLS-depen- use of octopine- versus nopaline-type VirE2 by the two groups dent manner, both in yeast and in vitro (16; S. Bhattacharjee (326). and S. B. Gelvin, unpublished data). Importin-␣ is a compo- Finally, VirE2 may protect T-strands from nucleolytic deg- nent of one of the protein nuclear transport pathways found in radation that can occur both in the plant cytoplasm and per- eukaryotes. Recent data, however, suggest that VirD2 may not haps in the nucleus (274, 374). be sufficient to direct T-strands to the nucleus. Ziemienowicz The existence of a T-complex composed of a single molecule et al. (382) showed that in permeabilized cells, VirD2 could of VirD2 covalently attached to the 5⬘ end of the T-strand, effect the nuclear targeting of small linked oligonucleotides which in turn is coated by VirE2 molecules, has generally been generated in vitro but could not direct the nuclear transport of accepted by the Agrobacterium research community (149). larger linked molecules. To achieve nuclear targeting of these However, the reader should be aware that such a complex has larger molecules, VirE2 additionally had to be associated with not yet been identified in either Agrobacterium or plant cells. It the T-strands. Finally, VirD2 may play a role in integration of is possible that other proteins, such as importins (16), VIP1 the T-DNA into the plant genome. Various mutations in (329), and even VirF (285), may additionally interact, either VirD2 can affect either the efficiency (229) or the “precision” directly or indirectly, with the T-strand to form larger T-com- (320) of T-DNA integration. plexes in the plant cell. The role of VirE2 in T-DNA nuclear transport also remains Although the role of Ti plasmid-encoded vir genes has often controversial. VirE2 is a non-sequence-specific single-stranded been considered of primary importance for transformation, DNA binding protein (45, 46, 49, 112, 286). In Agrobacterium many Agrobacterium chromosomal genes are also essential for cells, VirE2 probably interacts with the VirE1 molecular chap- this process. The role of chromosomal genes was first estab- erone and may therefore not be available to bind T-strands (77, lished by random insertional mutagenesis of the entire 84, 310, 380). However, when bound to single-stranded DNA Agrobacterium genome (106). Further research defined the (perhaps in the plant cell?), VirE2 can alter the DNA from a roles of many of these genes. Included among these functions random-coil conformation to a shape that resembles a coiled are exopolysaccharide production, modification, and secretion telephone cord (47). This elongated shape may help direct the (pscA/exoC, chvA, and chvB [36, 37, 88, 89, 313]) and other T-strand through the nuclear pore. VirE2 also contains NLS roles in bacterial attachment to plant cells (att genes [212, sequences that can direct fused reporter proteins to plant nu- 213]), sugar transporters involved in coinduction of vir genes clei (48, 50, 326, 383). As with VirD2, VirE2 interacts in yeast (chvE [86, 172, 289]), regulation of vir gene induction (chvD with Arabidopsis importin-␣ proteins in an NLS-dependent ), and T-DNA transport (acvB [168, 248, 360, 361, 362]). manner (Bhattacharjee and Gelvin, unpublished). One report Other genes, such as miaA (116), may also play a more minor indicates that VirE2 bound to single-stranded DNA and mi- role in the transformation process. The recent elucidation of croinjected into plant cells can direct the DNA to the nucleus the entire A. tumefaciens C58 sequence (114, 363) will surely 20 GELVIN MICROBIOL. MOL. BIOL. REV. provide fertile ground for the discovery of additional genes bacterial cell, the bacteria can become resistant to both these involved in Agrobacterium-mediated transformation. 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 MANIPULATION OF AGROBACTERIUM FOR GENETIC plasmid occurs by double homologous recombination (homog- ENGINEERING PURPOSES enotization) 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 Introduction of Genes into Plants by with the Ti plasmid), the resistance marker of the plasmid Using Agrobacterium backbone would be expressed; these bacteria are screened for Years before scientists elucidated the molecular mechanism and discarded. In the second instance (homogenotization), the of Agrobacterium-mediated transformation of plants, Armin resistance marker encoded by the plasmid backbone is lost. Braun proposed the concept of a “tumor-inducing principle” Double homologous recombination can be confirmed by DNA that was stably transferred to and propagated in the plant blot analysis of total DNA from the resulting strain (107). A genome (30). Research in the 1970s resulted in the identifica- variant of this procedure utilizes a sacB gene on the plasmid tion of large plasmids in virulent Agrobacterium strains (376), backbone of the first plasmid. Only elimination of the plasmid although we now know that many strains contain plasmids backbone after homogenotization renders the bacterium resis- unrelated to virulence. Genetic experiments indicated that a tant to growth on sucrose (194). particular class of plasmids, the Ti (and later Ri) plasmids, Another method to introduce foreign DNA into the T-re- were responsible for tumorigenesis (339) and that a portion of gion of the Ti plasmid involves first introducing a ColE1 rep- these plasmids, the T-DNA, was transferred to plant cells and licon, such as pBR322, into the T-region of a Ti plasmid. DNA incorporated into the plant genome (43). It was thus obvious to to be integrated into this T-region is cloned into a separate propose that Ti plasmids be used as a vector to introduce pBR322-derived molecule containing a second antibiotic re- foreign genes into plant cells. sistance marker. This plasmid is introduced into the altered However, Ti plasmids are very large and T-DNA regions do Agrobacterium strain, and the resulting strain is selected for not generally contain unique restriction endonuclease sites not resistance to the second antibiotic. Because ColE1 replicons found elsewhere on the Ti plasmid. Therefore, one cannot cannot function in Agrobacterium, the pBR322-based plasmid simply clone a gene of interest into the T-region. Scientists would have to cointegrate into the pBR322 segment of the therefore developed a number of strategies to introduce for- altered T-region for the stable expression of the plasmid-en- eign genes into the T-DNA. These strategies involved two coded resistance gene (379). A modification of this procedure different approaches: cloning the gene, by indirect means, into was used to develop the “split-end vector” system. Using this the Ti plasmid such that the new gene was in cis with the system, a gene of interest is cloned into a pBR322-based vector virulence genes on the same plasmid, or cloning the gene into that contains a T-DNA right border, a nos-nptII chimaeric a T-region that was on a separate replicon from the vir genes gene for selection of transgenic plants, a spectinomycin-strep- (T-DNA binary vectors). tomycin resistance marker to select for the presence of the Two methods were used for cloning foreign DNA into the Ti plasmid in Agrobacterium, and a region of homology with a plasmid. The first method was based on a strategy developed nononcogenic portion of an octopine-type T-region. Cointe- by Ruvkin and Ausubel (277) (Fig. 2). A region of DNA (either gration of this plasmid with a Ti-plasmid lacking a right border the T-region or any region of DNA targeted for disruption) but containing the T-DNA homologous region restores border containing unique restriction endonuclease sites is cloned into activity and localizes the gene of interest and the plant select- a broad-host-range plasmid, such as an IncP␣-based vector. able marker within the reconstituted T-region (101). These plasmids can replicate both in Escherichia coli, in which Each of these cis-integration methods has advantages and the initial cloning is performed, and in Agrobacterium. The disadvantages. The first strategy can target the foreign gene to exogenous gene of interest, along with an antibiotic resistance any part of the T-region (or other region in the Ti plasmid). marker, is next cloned into a unique restriction endonuclease However, it is cumbersome to perform and involves somewhat site within the target region of DNA. Alternatively, an antibi- sophisticated microbial genetic procedures that many labora- otic resistance gene can be introduced into the DNA fragment tories shunned. The second method is technically easier but of interest by transposition (107, 297). The resulting plasmid is allows cointegration of the foreign gene only into Ti-plasmid introduced into Agrobacterium by conjugation or transforma- locations where pBR322 had previously been placed. However, tion. The presence of this plasmid in Agrobacterium is con- a modification of this procedure allows cointegration of a firmed by selection for resistance to antibiotics encoded by pBR322-based plasmid into any region of the Ti plasmid (338, both the plasmid vector backbone and the resistance marker 377). An advantage of both of these systems is that they main- near the gene of interest. Next, another plasmid of the same tain the foreign gene at the same low copy number as that of incompatibility group as the first plasmid, but harboring yet the Ti plasmid in Agrobacterium. another antibiotic resistance marker, is introduced into the Because of the complexity of introducing foreign genes di- Agrobacterium strain containing the first plasmid. The resulting rectly into the T-region of a Ti plasmid, several laboratories bacteria are plated on medium containing antibiotics to select developed an alternative strategy to use Agrobacterium to de- for the second (eviction) plasmid and the resistance marker liver foreign genes to plants. This strategy was based on sem- next to the gene of interest. Because plasmids of the same inal findings of Hoekema et al. (140) and de Frammond et al. incompatibility group cannot usually coreside within the same (70). These authors determined that the T-region and the vir VOL. 67, 2003 AGROBACTERIUM-MEDIATED PLANT TRANSFORMATION 21 FIG. 2. Schematic representation of the steps involved in gene replacement by double homologous recombination (homogenotization [107, 277]). The green lines represent regions targeted for disruption. (A) An antibiotic resistance gene (in this case, encoding a ␤-lactamase that confers resistance to carbenicillin) has been inserted into the targeted gene that has been cloned into an IncP␣ plasmid (containing a kanamycin resistance gene [kan] in its backbone) and introduced into Agrobacterium. Double homologous recombination is allowed to take place. (B) Following double homologous recombination, the disrupted gene is exchanged onto the Ti plasmid (pTi). (C) A plasmid of the same incompatibility group as the first plasmid is introduced into Agrobacterium. An example is the IncP␣ plasmid pPH1JI, containing a gentamicin resistance gene (gent). (D) Because plasmids of the same incompatibility group (in this case IncP␣) cannot replicate independently in the cell at the same time, selection for gentamicin resistance results in eviction of the other IncP␣ plasmid, onto which has been exchanged the wild-type gene. genes could be separated into two different replicons. When rium to introduce genes into plants. Scientists without special- these replicons were within the same Agrobacterium cell, prod- ized training in microbial genetics could now easily manipulate ucts of the vir genes could act in trans on the T-region to effect Agrobacterium to create transgenic plants. These plasmids are T-DNA processing and transfer to a plant cell. Hoekema et al. small and easy to manipulate in both E. coli and Agrobacterium called this a binary-vector system; the replicon harboring the and generally contain multiple unique restriction endonuclease T-region constituted the binary vector, whereas the replicon sites within the T-region into which genes of interest could be containing the vir genes became known as the vir helper. The cloned. Many vectors were designed for specialized purposes, vir helper plasmid generally contained a complete or partial containing different plant selectable markers, promoters, and deletion of the T-region, rendering strains containing this poly(A) addition signals between which genes of interest could plasmid unable to incite tumors. A number of Agrobacterium be inserted, translational enhancers to boost the expression of strains containing nononcogenic vir helper plasmids have been transgenes, and protein-targeting signals to direct the trans- developed, including LBA4404 (242), GV3101 MP90 (181), gene-encoded protein to particular locations within the plant AGL0 (192), EHA101 and its derivative strain EHA105 (144, cell (some representative T-DNA binary vector systems are 146), and NT1 (pKPSF2) (247). described in references 10, 20, 25, 26, 62, 113, 120, 216, 364, T-DNA binary vectors revolutionized the use of Agrobacte- and 386 and at http://www.cambia.org). Hellens et al. (134) 22 GELVIN MICROBIOL. MOL. BIOL. REV. provide a summary of many A. tumefaciens strains and vectors What DNA Is Transferred from Agrobacterium commonly used for plant genetic engineering. to Plants? Although the term “binary vector system” is usually used to T-DNA was initially defined as the portion (the T-region) of describe two constituents (a T-DNA component and a vir the Ti plasmid that was transferred from Agrobacterium to helper component), each located on a separate plasmid, the plant cells to form crown gall tumors. T-DNA border repeat original definition placed the two modules only on different sequences defined the T-region (366), and regions of the Ti replicons. These replicons do not necessarily have to be plas- plasmid outside these borders were not initially found in tumor mids. Several groups have shown that T-DNA, when located in cells (43). However, the transfer of Ti-plasmid sequences out- the Agrobacterium chromosome, can be mobilized to plant cells side the conventional T-region may at first have been missed by a vir helper plasmid (141, 224). because of a lack of known selectable (e.g., tumorigenesis) or screenable (e.g., opine production) markers. Ooms et al. (241) observed the incorporation into plant DNA of regions of the Ti How Much DNA Can Be Transferred from plasmid later shown to be outside the classical T-DNA borders. Agrobacterium to Plants? Ramanathan and Veluthambi (264) also showed that a nos- The T-regions of natural Ti and Ri plasmids can be large nptII cassette, placed outside the T-DNA left border, could be enough to encode tens of genes. For example, the T-region of transferred to and confer kanamycin resistance on infected pTiC58 is approximately 23 kbp in size. In addition, some Ti tobacco cells. and Ri plasmids contain multiple T-regions, each of which can The use of relatively small T-DNA binary vectors made it be transferred to plants individually or in combination (34, easier for scientists to evaluate the transfer of “non-T-DNA” 314). For purposes of plant genetic engineering, scientists may regions to plants. Martineau et al. (211) first reported the wish to introduce into plants large T-DNAs with the capacity transfer of binary vector backbone sequences into transgenic to encode multiple gene products in a biosynthetic pathway. plant DNA and questioned the definition of T-DNA. Wenck et Alternatively, the reintroduction of large regions of a plant al. (356) found that the entire binary vector, including back- genome into a mutant plant may be useful to identify, by bone sequences as well as T-DNA sequences, could frequently genetic complementation, genes responsible for a particular be transferred to Nicotiana plumbaginifolia and Arabidopsis phenotype. How large a T-region can be transferred to plants? thaliana cells. Kononov et al. (182) carefully examined the Miranda et al. (224) showed that by reversing the orientation structure of binary vector backbone sequences that could be of a T-DNA right border, they could mobilize an entire Ti found in up to 75% of transgenic tobacco plants and concluded that such transfer could result from either skipping the left plasmid, approximately 200 kbp, into plants. Although the T-DNA border when T-DNA was processed from the binary event was rare, this study showed that very large DNA mole- vector or initiation of T-DNA transfer from the left border to cules could be introduced into plants using Agrobacterium- bring vector backbone sequences into plant cells. Considering mediated transformation. Hamilton et al. (124) first demon- the previous observation by Durrenberger et al. (91) that strated the directed transfer of large DNA molecules from VirD2 protein could covalently attach to the 5⬘ end of the Agrobacterium to plants by the development of a binary BAC non-T-DNA strand, Kononov et al. suggested that transfer of (BIBAC) system. These authors showed that a 150-kbp cloned vector backbone sequences to plants was a natural conse- insert of human DNA could be introduced into plant cells by quence of the mechanism of VirD2 function. Thus, the defi- using this system. However, the efficient transfer of such a large nition of T-DNA and vector backbone constitutes a semantic DNA segment required the overexpression of either virG or argument. It would thus appear that the transfer of non-T- both virG and virE. VirE2 encodes a single-stranded DNA DNA sequences to plants may be an unavoidable, but fre- binding protein that protects the T-DNA from degradation in quently unobserved and untested, result of transformation. the plant cell (275). Because virG is a transcriptional activator Indeed, Frary and Hamilton (103) observed incorporation of of the vir operons (303), expression of additional copies of this BIBAC plasmid sequences into 9 to 38% of tested tomato regulatory vir gene was thought to enhance the expression of transformants. VirE2 and other Vir proteins involved in T-DNA transfer. Although the transfer of plasmid backbone sequences may Overexpression of virE formed part of the BIBAC system be an unavoidable consequence of the mechanism of Agrobac- that was used to transform large (30- to 150-kbp) DNA terium-mediated transformation, it may be possible to select fragments into tobacco and the more recalcitrant tomato against transgenic plants containing this unwanted DNA. Han- and Brassica (56, 103, 123, 125). However, the transfer of son et al. (132) showed that the incorporation of a toxic “killer” different-size T-DNAs from various Agrobacterium strains gene into the binary vector backbone sequences could severely had different requirements for overexpression of virG and reduce the percentage of transgenic plants containing such virE (103). Liu et al. (203, 204) developed a transformation- extra sequences. Remarkably, the transformation frequency of competent artificial chromosome vector system based on a tobacco, tomato, and grape plants infected using this modified P1 origin of replication and used this system to generate binary vector did not substantially differ from that of plants libraries of large (40- to 120-kbp) Arabidopsis and wheat DNA infected using a binary vector lacking the killer gene. Because molecules. This system did not require overexpression of virG the presence of uncharacterized DNA in transgenic plants is or virE to effect the accurate transfer of large fragments to important for regulatory concerns, such an approach may be Arabidopsis. useful in the future for the production of plants (especially VOL. 67, 2003 AGROBACTERIUM-MEDIATED PLANT TRANSFORMATION 23 difficult to transform species) with a more highly defined trans- These experiments were extended by de Frammond et al. genic composition. (69), who showed that fertile transgenic plants could be regen- erated from cloned tobacco tissue that was cotransformed by T-DNA from a Ti plasmid and from a micro-Ti (the one-strain, Transfer of Multiple T-DNAs into the Same Plant Cell, and two-replicon approach). The two T-DNAs segregated in prog- Generation of “Marker-Free” Transgenic Plants eny plants, indicating that the T-DNAs had integrated into Because of concerns regarding the spread of antibiotic re- genetically separable loci. Other groups have used the one- sistance genes in nature or the escape of herbicide resistance strain, two-replicon approach to generate transgenic plants genes to wild weedy species, scientists have developed several which initially expressed both T-DNA markers but could sub- methods to generate marker-free transgenic plants. These sequently segregate the markers from each other (58). plants would initially be selected for resistance to an antibiotic Depicker et al. (80) performed a similar experiment in which or herbicide, but the selection marker would be removed on the selection markers were phytohormone-independent growth subsequent manipulation and plant growth. Several methods and nopaline synthesis (encoded by a Ti plasmid) and kana- have been proposed to eliminate the selection marker from the mycin-resistant growth (encoded by a T-DNA binary vector). primary transformant. These include use of a site-specific re- They performed the experiment in two ways: either the two combination system, such as Cre-lox or Flp-Frt (2, 19, 57, 209, T-DNAs were delivered by two different Agrobacterium strains 235, 347, 348) to remove the marker, transposon-based move- (the two-strains, two-replicons approach), or both T-DNAs ment of the selection marker from the initial site of insertion were delivered from a single replicon in one strain (the one- from the plant genome entirely or to another unlinked site strain, one-replicon approach). The results of these experi- from which it can be segregated in subsequent generations ments indicated that cotransfer of T-DNAs from the same (93), or the use of multiple T-DNAs which can insert into plasmid in one strain was considerably more efficient than was unlinked sites for future segregation (reviewed in references transfer from two different strains. The use of a single Agrobac- 142 and 372). Each of these systems has advantages and dis- terium strain to cotransform plants with two T-DNAs from the advantages. For example, excision of marker genes using a same replicon, followed by segregation of the selection gene to site-specific recombination system requires introduction of the generate marker-free transgenic plants, has been described by site-specific recombinase into plants, either by transformation Komari et al. (178) and Xing et al. (365). In each of these or by genetic crossing. Segregation of markers can occur only studies, the authors were able to generate marker-free trans- in progeny following the generation of the initial transgenic genic plants at high frequency. plant and is limited to species naturally propagated through The use of two Agrobacterium strains to deliver different seed and not those propagated vegetatively. T-DNAs to the same plant cells was studied by a number of Early research that characterized the integration pattern of groups (65, 66, 67, 76, 217). Although cotransfer of T-DNAs to T-DNAs in crown gall tumors indicated that each of the two genetically unlinked sites was reported, some authors also re- T-DNAs encoded by an octopine-type Ti plasmid could inde- ported close linkage of the two different T-DNAs in many pendently integrate into the plant genome, sometimes in mul- instances. It thus remains unclear which of the three cotrans- tiple copies (43, 63, 314). The molecular analyses suggested formation protocols will be reproducibly best for the genera- that these T-DNAs could be integrated into unlinked sites. tion of marker-free transgenic plants. These results suggested that cotransformation could be performed to integrate transgenes carried by two different Virulence Gene Expression and Plant Transformation T-DNAs and that perhaps these T-DNAs would segregate in subsequent generations. Three approaches were subse- The processing and transfer of T-DNA from Agrobacterium quently used for cotransformation: (i) the introduction of to plant cells is regulated by the activity of the vir genes. two T-DNAs, each from a different bacterium; (ii) the intro- Virulence gene activity is induced by plant wound-induced duction of two T-DNAs carried by different replicons within phenolic compounds such as acetosyringone and related mol- the same bacterium; and (iii) the introduction of two T-DNAs ecules (28, 74, 75, 92, 228, 293, 295, 298, 300). However, there located on the same replicon within a bacterium. may be instances in which scientists would like to induce vir Early experiments using these various approaches indicated genes to levels higher than that accomplished by plant extracts. that cotransformation could be a frequent event. An et al. (9) Several groups have therefore identified virA and virG mutants showed that tobacco cells could be cotransformed to two dif- that function constitutively, in the absence of phenolic induc- ferent phenotypes by a single Agrobacterium strain containing ers. Constitutive virA mutants were characterized by several both a Ti plasmid (phytohormone-independent growth) and a groups (13, 218, 253). However, more emphasis has been T-DNA binary vector (kanamycin-resistant growth). This ex- placed on inducer-independent virG mutants, possibly because periment represents a “one-strain, two-replicon” approach to virG functions downstream of virA. cotransformation. When the cells were first selected for kana- Extensive genetic studies resulted in the identification of a mycin resistance, 10 to 20% of them also displayed phytohor- number of mutations that render the VirG protein active in the mone-independent growth; when the cells were first selected absence of phenolic inducing compounds (127, 254). These for phytohormone-independent growth, 60% of the resulting altered proteins contain mutations that converted either aspar- calli were also kanamycin resistant. The authors credited these agine-54 to aspartic acid (virGN54D) or isoleucine-106 to differing frequencies to the higher copy number (5 to 10) of the leucine (virGI106L). Both of these mutant proteins stimulated binary vector in the bacterium relative to the single copy Ti a high level of vir gene expression, especially when expressed plasmid. from a high-copy-number plasmid (118). When tested in tran- 24 GELVIN MICROBIOL. MOL. BIOL. REV. sient tobacco and maize transformation assays, strains contain- of the plant genome. The high percentage (approximately ing the virGN54D mutant effected a higher level of transfor- 30%) of T-DNA integration events that resulted in activation mation than did strains encoding the wild-type virG gene (130). of a promoterless reporter transgene positioned near a T-DNA An even greater effect was seen when the virGN54D allele was border suggested that T-DNA may preferentially integrate into harbored on a high-copy-number plasmid; the presence of this transcriptionally active regions of the genome. Only integra- mutant gene in Agrobacterium increased the transient transfor- tion events that would link the promoterless transgene with an mation of rice and soybean two- to sevenfold (170). active promoter would result in reporter activity (180). How- Several laboratories have determined the effect of additional ever, a drawback to some of these experiments was that trans- copies of wild-type virG genes on vir gene induction and plant genic events may have been biased by the selection of antibi- transformation. Rogowsky et al. (273) showed that additional otic resistant plants expressing an antibiotic marker gene copies of nopaline-type virG resulted in increased vir gene carried by the T-DNA. It is not clear whether T-DNA inser- expression. Liu et al. (200) showed that multiple copies of virG tions into transcriptionally inert regions of the genome would altered the pH response profile for vir gene induction. Nor- have gone unnoticed because of lack of expression of the mally, vir gene induction is very poor at neutral or alkaline pH antibiotic resistance marker gene. or in rich medium; additional copies of virG permitted a sub- An obvious way to circumvent the presumed problems of stantial degree of induction in rich medium even at pH 8.5. position effect is to integrate T-DNA into known transcription- Additional copies of virG also increased the transient transfor- ally active regions of the plant genome. However, gene target- mation frequency of rice, celery, and carrot tissues (199). ing in plants by homologous recombination has been at best Given these results in toto, one may conclude that increasing extremely inefficient (72, 173, 223, 237, 238, 269, 270). An the copy number of virA or virG or decreasing the dependence alternative system for gene targeting is the use of site-specific of the encoded proteins on phenolic inducers would generally integration systems such as Cre-lox. However, single-copy increase the transformation efficiency of the resulting strains. transgenes introduced into a lox site in the same position of the However, the situation is likely to be more complex. Belanger plant genome also showed variable levels of expression in in- et al. (23) showed that individual virA genes may be particu- dependent transformants. Transgene silencing in these in- larly suited to function in certain genetic backgrounds, and stances may have resulted from transgene DNA methylation Krishnamohan et al. (183) recently demonstrated that Ti plas- (61). Such methylation-associated silencing was reported ear- mids may have evolved to optimize specific combinations of lier for naturally occurring T-DNA genes (135, 340). Thus, virA, virG, and vir boxes. As noted above, the Ti-plasmid transcriptional silencing may result from integration of trans- pTiBo542 in the C58 chromosomal background is hyperviru- genes into regions of the plant genome susceptible to DNA lent on certain legume species, possibly because of the associ- methylation and may be a natural consequence of the process ated virG gene (41, 146, 159), but not in its native Bo542 of plant transformation. chromosomal background (143). Recent results from my lab- We now know not only that transgene silencing results from oratory indicate that vir gene induction and T-strand produc- “transcriptional” mechanisms, usually associated with methyl- tion by and transformation efficiency of particular Agrobacte- ation of the transgene promoter (222), but also that transgene rium strains may not correlate well. A. tumefaciens A277, silencing is often “posttranscriptional”; i.e., the transgene is containing the Ti plasmid pTiBo542 within the C58 chromo- transcribed, but the resulting RNA is unstable (219). Such somal background, is considerably more virulent than are posttranscriptional gene silencing is frequently associated with strains A348 and A208, containing the Ti plasmids pTiA6 and multiple transgene copies within a cell. Transgenic plants gen- pTiT37, respectively, in the same chromosomal background. erated by direct DNA transfer methods (e.g., polyethylene However, vir gene induction by plant exudates and T-strand glycol- or liposome-mediated transformation, electroporation, production are highest in A. tumefaciens A208 (L.-Y. Lee and or particle bombardment) often integrate a large number of S. B. Gelvin, unpublished data). These data further suggest copies of the transgene in tandem or inverted repeat arrays, in that increased vir gene induction and T-strand production may either multiple or single loci (176). Although Agrobacterium- not necessarily be reliable predictors of transformation effi- mediated transformation usually results in a lower copy num- ciency. ber 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 T-DNA Integration and Transgene Expression integrated T-DNA (95). However, integration of T-DNA re- Plant transformation does not always result in efficient trans- peats, especially ⬙head-to-head’ inverted repeats around the gene expression. The literature is replete with examples of T-DNA right border, frequently results in transgene silencing variable expression levels of transgenes, which frequently did (51, 164, 304). Thus, a procedure or Agrobacterium strain that not correlate with transgene copy number (see, for example, could be used to generate transgenic plants with a single inte- reference 255). This lack of correspondence was initially at- grated T-DNA would be a boon to the agricultural biotechnol- tributed to position effects, i.e., the position within the genome ogy industry and to plant molecular biology in general. Grev- into which the T-DNA integrated was credited with the ability elding et al. (117) noted that transgenic Arabidopsis plants of transgenes to express. T-DNA could integrate near to or far derived from a root transformation procedure tended to have from transcriptional activating elements or enhancers, result- fewer T-DNA insertions than did plants derived from leaf ing in the activation (or lack thereof) of T-DNA-carried trans- disks. However, it is not clear if this observation can be gen- genes (22, 35, 296, 308). T-DNA could also integrate into erally applicable to other plant species. Anecdotal information transcriptionally competent or transcriptionally silent regions from several laboratories suggests that Agrobacterium strains VOL. 67, 2003 AGROBACTERIUM-MEDIATED PLANT TRANSFORMATION 25 that are less efficient in delivering T-DNA may be more effi- As indicated in some of the references cited above, viral cient in producing single-copy T-DNA insertions. However, suppressors of gene silencing can activate a previously silenced these findings need to be tested rigorously; it is possible that stable transgene. One would then wonder whether such silenc- T-DNA copy number may also correlate with the growth state ing suppressors could prevent the silencing of transgenes stably of the bacterium or the plants to be transformed. introduced into plants by Agrobacterium-mediated transforma- tion. Although this hypothesis has not yet been tested and possible negative consequences (such as increased viral sus- Use of Matrix Attachment Regions To Ameliorate ceptibility) may ensue from the stable incorporation of antisi- Transgene Silencing lencing genes into a plant genome, experiments in which viral At present, the generation of single-copy transgenic plants is silencing suppressors have been used to increase the levels of still somewhat hit and miss. Scientists usually produce a rela- transient expression of Agrobacterium-introduced transgenes tively large number of independent transformants and screen appear promising. O. Voinnet and colleagues (unpublished them for plants containing a single-copy T-DNA insertion. At data) have recently demonstrated that when cotransformed best, this can be a time-consuming nuisance. However, for with various transgenes encoding green fluorescent protein, agronomically important species, elite cultivars, or lines that the potato virus Y Nia protein, or tomato Cf-9 and Cf-4 disease are recalcitrant to transformation, it can become a rate-limit- resistance proteins, various viral silencing suppressors dramat- ing step. An alternative to this approach may be to generate ically increased the expression of these other proteins. Expres- transgenic plants containing a few copies of T-DNA that are sion levels up to 50-fold higher than those achieved in control insulated from each other. One proposed mechanism to ac- transformations (lacking the viral silencing suppressor genes) complish this is to flank transgenes within the T-DNA with were obtained. Several different viral silencing suppressors, matrix attachment regions (MARs). MARs are DNA se- including the p25 protein of PVX, the P1-HcPro protein of quences that either are associated with chromosome “matri- tobacco etch virus, and the p19 protein of tomato bushy stunt ces” as isolated or can associate with these matrices in vitro virus, were able to enhance transient transgene expression (121, 122, 294, 350). Among other properties, they have been from both the cauliflower mosaic virus 35S promoter and from ascribed the role of insulating genes within a looped chromatin native transgene promoters. Of these, the p19 protein was domain from transcription-activating or -silencing effects of most effective in both increasing transient transgene expression neighboring domains. In animal cells, such insulating effects and decreasing the levels of small (21- to 25-bp) RNA mole- may render transgene expression proportional to transgene cules associated with posttranscriptional gene silencing. The copy number (306). However, some of the MARs initially used authors concluded that viral suppressors of gene silencing in animal experiments may also have contained enhancer ele- could be useful for the production of large amounts of proteins ments, confounding the interpretation of the original experi- in plants. ments (29, 259). When they flank transgenes delivered to plants via Agrobac- When Transgene Expression Is Not Forever terium-mediated transformation, MARs appear to have only a small effect on transgene expression (128, 198, 201, 225, 226, Experiments to express transgenes in plants initially used 227, 334). Larger increases in transgene expression have been elements, such as the cauliflower mosaic virus 35S and 19S observed using particle bombardment-mediated transforma- promoters or opine synthase promoters, that would express the tion (5, 6, 236). However, this increase is generally associated transgene in a relatively constitutive manner (24, 133, 179, 196, with expression of transgenes in plant cells rather than in 234, 280, 343). However, as plant genetic engineering experi- whole plants (330, 333). It is possible that the higher transgene ments became more refined, scientists turned to regulated expression effects of MARs using particle bombardment re- promoters that would express a transgene in a particular de- flects the higher integrated transgene copy number resulting velopmental, environmental, or tissue-specific pattern. Systems from this technique as opposed to the relatively lower copy were also developed that would allow scientists to induce trans- number of integrated T-DNAs delivered by Agrobacterium (7). gene expression at will, allowing for the overexpression of a As such, it is not clear whether MARs will be, on their own, particular product or expression of a product that may be toxic highly useful for decreasing the silencing of transgenes deliv- during certain stages of plant development. Such inducible ered to plants by Agrobacterium-mediated transformation. systems included those regulated by tetracycline (108), alcohol (279), copper (221), heat shock (284), and steroid hormones (14, 282) (see reference 158 for a recent review of chemically Use of Viral Suppressors of Gene Silencing To Increase inducible gene induction systems). Many of these systems were Transgene Expression leaky, permitting the expression of transgenes under nonin- Recent data from a number of laboratories indicates that duced conditions. some plant viruses, both DNA and RNA viruses, contain genes There may be instances, however, when one would not want that suppress gene silencing (4, 11, 21, 31, 38, 55, 169, 210). a transgene or its product to be present after the initial few Several investigators have speculated that viral antisilencing hours or days following transformation. Such traits include has evolved as a mechanism for viruses to evade a plant’s those that would aid in the transformation process itself or defense through viral gene silencing (55, 266). Regardless of would effect plant DNA rearrangements desired only during the reason for and mechanism of antisilencing, viral suppres- the initial transformation event (e.g., gene targeting using site- sors of silencing may be useful to mitigate the silencing of specific recombinase systems). Two strategies are currently transgenes. being developed to permit only transient expression of gene 26 GELVIN MICROBIOL. MOL. BIOL. REV. products in plants. These are the use of “nonintegrating” T- fer the fusion protein. These experiments lead to the possibility DNA systems and the transfer of proteins, rather than DNA of using Vir proteins as carriers to introduce other proteins molecules, from Agrobacterium to plant cells. transiently into 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. MANIPULATION OF PLANT GENES TO IMPROVE During a search for domains of VirD2 necessary for nuclear TRANSFORMATION targeting of the T-DNA, Shurvinton et al. (290) defined a C-terminal domain, termed ␻, that showed high amino acid Plant Response to Agrobacterium Infection sequence homology among virD2 genes. Although this domain was not required for either VirD2 endonuclease activity or Although great advances have been made over the past nuclear targeting of the T-DNA, replacement of four con- decade to increase the number of plant species that can be served amino acids by two serine residues resulted in a mutant transformed and regenerated using Agrobacterium, many im- protein that rendered the encoding Agrobacterium strain highly portant species or inbred lines remain highly recalcitrant to attenuated in virulence. Narasimhulu et al. (233) and Mysore Agrobacterium-mediated transformation. The question has of- et al. (229) further showed that Agrobacterium strains harbor- ten arisen, “Who has the problem with transformation, Agro- ing this VirD2 ␻ substitution were highly deficient in stable bacterium or the researcher?” The very wide host range of transformation (with 2% of the efficiency of wild-type strains) Agrobacterium, including gymnosperms and perhaps lower plant but were still able to transform plant cells transiently at 20% of phyla, a variety of fungi, and even animal cells, suggests that the efficiency of wild-type strains. Thus, this mutation rendered T-DNA transfer to the recipient (i.e., entry exclusion) may not Agrobacterium strains highly deficient in T-DNA integration be the problem. That Agrobacterium can transiently transform but still relatively proficient in delivering T-DNA to the plant a number of these species efficiently, including agronomically nucleus. This mutant VirD2 protein can therefore be used to important species such as maize and soybean (170, 271, 288), target T-strands to the nucleus, where they can transiently suggests that in many instances T-DNA integration may re- express but not efficiently integrate. main the limiting step. Alteration of the tissue culture condi- Nam et al. (231) used a root assay to screen almost 40 A. tions, for example by the use of antioxidants during the trans- thaliana ecotypes for their ability to be transformed by Agro- formation of grape, rice, maize, and soybean, has increased the bacterium. Among these ecotypes, UE-1 was easily transiently probability of stably transforming cell types that can be regen- transformed but poorly stably transformed. Genetic and mo- erated (96, 102, 239, 240, 258). However, such manipulations lecular characterization of this ecotype indicated that the block of the transformation conditions may have limitations. in transformation occurred at the T-DNA integration step. Agrobacterium infection of plant tissues may in some in- Nam et al. (232) further identified a large number of Arabi- stances result in plant tissue necrosis. Several groups have dopsis T-DNA insertion mutants that were resistant to Agro- described a slow, spreading necrosis in grape infected by par- bacterium transformation (rat mutants). Of the initial 21 mu- ticular Agrobacterium strains (79, 263). More recently, Hansen tants, 5 were efficiently transiently transformed but were highly (129) described an apoptotic response of maize to Agrobacte- recalcitrant to stable transformation, a phenotype associated rium infection. The response included both rapid tissue necro- with a deficiency in T-DNA integration. Mysore et al. (230) sis and cleavage of nuclear DNA into oligonucleosome-sized characterized one of these mutants, the rat5 mutant, in greater fragments by endogenous nucleases and is characteristic of a detail. This mutant was generated by the insertion of T-DNA caspase-protease cascade. The expression of two baculovirus into the 3⬘ untranslated region of a histone H2A gene (HTA1). cell death suppressor genes, p35 and iap, greatly inhibited both Biochemical and molecular data indicated that this mutant tissue necrosis and endogenous DNA cleavage. Manipulation could be transiently transformed efficiently but that T-DNA of these genes during the Agrobacterium-mediated transforma- integration was disrupted. Although the precise role of the tion process may thus be useful to increase both plant cell HTA1 gene in T-DNA integration has yet to be elucidated, viability and transformation efficiency in plant species with an root transformation of this mutant (and perhaps other T-DNA apoptotic response to Agrobacterium. integration-deficient mutants) can be used for the transient Several groups have recently begun to identify plant genes delivery of T-DNA without efficient subsequent T-DNA inte- and protein products involved in the transformation process. gration. The use of the HTA1 gene to improve the transfor- One of the rationales for these experiments is the hope that mation efficiency of wild-type plants is discussed below. identification of these genes may eventually result in their Vergunst et al. (349) recently described a novel procedure to manipulation either to improve transformation or to make transfer proteins directly from Agrobacterium to plant cells. plants resistant to crown gall disease. A number of approaches This system relies on the ability of the type IV protein secre- have been employed to identify these plant genes, including (i) tion system encoded by the Agrobacterium virB and virD4 genes use of yeast two-hybrid systems to identify plant proteins that to transfer certain Vir proteins to plant cells. VirD2, VirE2, may interact with virulence proteins, (ii) direct “forward ge- and VirF are the three proteins identified to date that can be netic” screening to identify plant mutants that cannot be trans- transferred by this system. These authors showed that transla- formed, (iii) “reverse genetic” screening to test whether par- tional fusions of the Cre recombinase protein to the N termi- ticular genes of interest may be involved in transformation, and nus of either VirE2 or VirF could be transferred to plant cells (iv) various genomics approaches to identify plant genes that and effect recombination at lox sites. They further showed that may be induced or repressed soon after infection by Agrobac- the C-terminal 37 amino acids of VirF were sufficient to trans- terium. VOL. 67, 2003 AGROBACTERIUM-MEDIATED PLANT TRANSFORMATION 27 Identification of Plant Genes Encoding Proteins That gest a use for VIP1 in improving plant transformation: trans- Interact with Agrobacterium Virulence Proteins genic plants that overexpress VIP1 are hypersusceptible to Agrobacterium transformation (327, 330). VirE2 also interacts Several Agrobacterium virulence proteins would be expected in yeast with several of the Arabidopsis importin-␣ proteins, to interact with plant proteins. These include the processed suggesting that VirD2 and VirE2 may have a common mech- form of VirB2, the major component of the T-pilus that is anism of nuclear import (Bhattacharjee and Gelvin, unpub- required for transformation; VirD2, the protein that caps the lished). 5⬘ end of the transferred T-strand; VirE2, the single-stranded Schrammeijer et al. (285) recently identified an Arabidopsis DNA binding protein that presumably coats the T-strand; and Skp1 protein as an interactor with the F-box domain of VirF. VirF, which is transferred to plant cells but whose function Skp1 proteins may be involved in targeting proteins such as remains unknown. Several other Vir proteins that are on the cyclins to the 26S proteosome, suggesting that VirF may func- bacterial cell surface, such as VirB5 and VirB7 (minor com- tion in setting the plant cell cycle to effect better transforma- ponents of the T-pilus), and VirB1ⴱ (a processed product of tion. The interaction of VirF with VIP1, but not VirE2, may VirB1 that can be found in the extracellular medium), may also also suggest a mechanism for Vir protein turnover: if VirF is interact with proteins on the surface of plant cells. targeted to the proteosome, it may help target other Vir pro- Early work (16) utilized VirD2 as the bait protein in a teins for proteolysis. yeast two-hybrid system to identify an A. thaliana importin-␣ The T-pilus is essential for Agrobacterium-mediated trans- (AtKAP, now known as importin-␣1) as an interacting partner. formation. Mutations in various VirB proteins disrupt trans- Importin-␣ proteins are involved in the nuclear translocation formation but not T-DNA processing (301, 344). As mentioned of many proteins harboring NLS sequences, and Arabidopsis above, the major T-pilus component is the processed and cy- encodes at least nine of these proteins (Bhattacharjee and clized VirB2 protein (189), although other virulence proteins, Gelvin, unpublished). Ballas and Citovsky (16) showed that including VirB5 and possibly VirB7, also are minor T-pilus interaction of VirD2 with importin-␣ AtKAP was NLS depen- constituents (278, 283). Although the precise role of the T- dent both in yeast and in vitro. The importance of importin-␣ pilus remains controversial, it is expected that the T-pilus proteins in the Agrobacterium transformation process has re- would interact with the plant cell wall or membrane. Experi- cently been suggested by demonstrating that a T-DNA inser- ments in my laboratory have begun to address possible plant- tion into the importin-␣7 gene, or antisense inhibition of ex- interacting partners with T-pilus components. Using a yeast pression of the importin-␣1 (AtKAP) gene, results in a highly two-hybrid system and the processed, but not cyclized, form of attenuated transformation phenotype (Bhattacharjee and Gel- VirB2 as a bait protein, we have identified two classes of vin, unpublished). Arabidopsis proteins that strongly and specifically interact with VirD2 also interacts with at least two other plant proteins by this major T-pilus constituent (H.-H. Hwang and S. B. Gelvin, using a yeast two-hybrid system. Deng et al. (78) identified unpublished data). One of these classes of plant proteins is three VirD2- and two VirE2-interacting proteins. They char- encoded by a three-member gene family. Although the identity acterized more fully one of the VirD2 interactors, an Arabi- of these three related proteins is not currently known, their dopsis cyclophilin. This protein, as a glutathione S-transferase hydropathy profiles suggest that they contain membrane-span- fusion, interacted strongly with VirD2 in vitro. The authors ning domains. The other interacting protein is a Rab-type further showed that the interaction domain of VirD2 was in GTPase. Each of these four plant proteins interacts in yeast the central portion of the protein, a region to which no previ- with itself and with each other but not with other tested viru- ous function had been ascribed. Cyclosporin A, an inhibitor of lence proteins, including VirB1, VirB1ⴱ, VirB5, VirD2, VirE2, cyclophilins, inhibited Agrobacterium-mediated transformation and VirF. In vivo data indicate that each of these proteins is of Arabidopsis roots and tobacco suspension cell cultures. The involved in Agrobacterium-mediated transformation. Antisense authors suggested that this plant protein may serve as a chap- or RNAi inhibition of expression of the genes encoding these erone to help in T-complex trafficking within the plant cell. proteins results in a transformation-deficient phenotype. In Experiments in my laboratory identified a tomato type 2C addition, an Arabidopsis mutant line containing a T-DNA in- protein phosphatase as an interacting partner with VirD2. This sertion into the promoter region of one of the “unknown phosphatase may be involved in the phosphorylation and de- protein” genes also is highly recalcitrant to Agrobacterium- phosphorylation of a serine residue near the C-terminal NLS mediated transformation. motif in VirD2. Overexpression of this phosphatase in trans- fected tobacco BY-2 cells resulted in decreased nuclear target- Forward Genetic Screening To Identify Plant Genes ing of a GUS-VirD2-NLS fusion protein, suggesting that phos- Involved in Agrobacterium-Mediated Transformation phorylation of the VirD2 NLS region may be involved in nuclear targeting of the VirD2/T-strand complex (Y. Tao, Scientists have shown a genetic basis for susceptibility to P. Rao, and S. B. Gelvin, submitted for publication). crown gall disease in some plant species (15, 214, 231, 246, 272, Using VirE2 as the bait protein in a yeast two-hybrid system, 292, 312). In an effort to identify plant genes involved in Tzfira et al. (329) identified two interacting proteins from Ara- Agrobacterium-mediated transformation, my laboratory em- bidopsis, VIP1 and VIP2. VIP1 may be involved in nuclear barked on a major project to identify Arabidopsis T-DNA in- targeting of the T-complex because antisense inhibition of sertion mutants that are resistant to Agrobacterium transfor- VIP1 expression resulted in a deficiency in nuclear targeting of mation (rat mutants ). These studies have resulted in the VirE2. Tobacco VIP1 antisense plants were also highly recal- identification of more than 70 such mutants to date. The roles citrant to Agrobacterium infection. Recent results further sug- of many of the mutated genes in the transformation process 28 GELVIN MICROBIOL. MOL. BIOL. REV. have been revealed by various assays. Thus, rat1 (encoding an tants and thus may sensitize plant cells to Agrobacterium- arabinogalactan protein) and rat3 (probably encoding a plant mediated transformation. We suggest that overexpression of cell wall protein) are involved in bacterial attachment to roots the RAT5 histone H2A-1 gene may improve the transformation (23). Other rat genes that may affect cell wall structure include efficiency of recalcitrant plants. a xylan synthase (rat4 ) and a ␤-expansin (A. Kaiser, A. Kopecki, Y. Zhu, and S. B. Gelvin, unpublished data). Because bacterial attachment to the roots of the rat4 mutant appears Reverse Genetic Screening for Plant Genes Involved in nearly normal (A. Matthysse, unpublished data), RAT4 may be Agrobacterium-Mediated Transformation involved in T-DNA transfer to the cytoplasm. Using this forward-genetics approach, we have identified a Plant genes encoding several proteins that interact with vir- number of other rat mutants in later stages of the transforma- ulence proteins have been identified using a yeast two-hybrid tion process. T-DNA insertions into genes encoding ␣- and system. Such interactions are at best suggestive of a role for ␤-importins are probably blocked in the T-DNA nuclear tar- these genes in plant transformation. Their roles must be shown geting process (S. Bhattacharjee, H. Cao, J. Humara, Y. Zhu, directly. One way to accomplish this is to inhibit gene expres- and S. B. Gelvin, unpublished data). Other mutants, including sion in planta using techniques such as antisense RNA, RNAi, the rat5 (a histone H2A mutant [230, 232]), rat17, rat18, rat20, or mutagenesis. I have discussed above the use of antisense and rat22 mutants, are probably involved in T-DNA integra- RNA and RNAi to show that VIP1 (a VirE2 interactor), a Rab tion (232). A T-DNA insertion between two closely spaced GTPase, and several proteins of unidentified function (VirB2 replacement histone H3 genes (histone H3-4 and H3-5) also interactors) are involved in Agrobacterium-mediated transfor- results in the rat phenotype (J. Li, Y. Zhu, and S. B. Gelvin, mation. Suppression of expression of these genes may be one unpublished data). method to generate plants resistant to crown gall disease. The finding that the histone H2A-1 gene affects T-DNA Another method to test the role of a particular gene in integration has led to a more extensive characterization of this transformation would be to mutate that gene and then assay gene. The Arabidopsis histone H2A gene family includes 13 the plant for transformation susceptibility. However, at present members. We have initiated a study of the expression pattern site-directed mutagenesis is not an efficient method for use in of each of these genes and an examination of the role that each plants. An alternative reverse genetic approach is to identify of these genes may play in Agrobacterium-mediated transfor- mutant plants containing transposon or T-DNA insertions in mation (370; H. Yi, T. Fujinuma, and S. B. Gelvin, unpublished genes of interest. Several PCR-based strategies have been de- data). The histone H2A-1 gene, encoded by RAT5, is expressed scribed to identify such knockout mutations in Arabidopsis (98, in numerous cell types, including cells that are not undergoing 104, 184), tomato (52), and rice (157). Using one such strategy, rapid division. This expression pattern is characteristic of a my laboratory has identified Arabidopsis mutant lines contain- “replacement” histone gene. In roots, the gene is expressed in ing disruptions in various importin-␣ and importin-␤ genes lateral root primordia, the meristem region, and the elon- (putatively involved in nuclear transport of the T-complex) and gation zone. Interestingly, the root elongation zone is the various genes involved in plant chromatin structure (putatively region most highly susceptible to Agrobacterium-mediated involved in T-DNA integration into the plant genome). Some transformation (370). Other experiments indicate that histone of these mutants are either moderately or highly resistant to H2A-1 gene expression and susceptibility to Agrobacterium- Agrobacterium-mediated transformation (S. Bhattacharjee, H. mediated transformation are highly correlated. Thus, expres- Cao, H. Humara, A. Kaiser, A. Kopecki, J. Li, X. Zhao, and sion of this gene may be predictive of cell types that are most S. B. Gelvin, unpublished data), and contain T-DNA insertions sensitive to transformation. Knowledge of plant cell transfor- in or near genes encoding importin-␣7 or importin-␤3, various mation competency may be important for the genetic engineer- histones (including histones H2A1, H2A3, H2B5, H2B6, H3-4, ing of recalcitrant plant species and cultivars. H3-5, and H4-1), histone acetyltransferases (including HAC4, Because mutation of the histone H2A-1 gene resulted HAC6, HAC9, HAC10, and HAC11), and a histone deacety- in decreased Arabidopsis root transformation, we examined whether overexpression of this gene would increase the effi- lase (HDA1). We have not yet, however, established the pre- ciency of Agrobacterium-mediated transformation. Transgenic cise roles of these plant genes in the Agrobacterium-mediated Arabidopsis plants containing additional genomic (230) or transformation process. cDNA (H. Yi and S. B. Gelvin, unpublished data) H2A-1 Escobar et al. (97) have recently described a novel reverse copies are two- to sixfold more transformation competent than genetic strategy to produce crown gall-resistant plants. They are plants containing the normal histone H2A-1 gene comple- generated transgenic Arabidopsis and tomato plants expressing ment. In addition, transient expression of the histone H2A-1 double-stranded RNA constructions targeted to T-DNA-en- gene from an incoming T-DNA both complements the rat5 coded auxin and cytokinin biosynthetic oncogenes. These mutant (230) and increases the transformation efficiency of genes are highly homologous among a large variety of different normally susceptible and highly recalcitrant Arabidopsis Agrobacterium strains. Many transgenic plants expressing these ecotypes (L.-Y. Lee and S. B. Gelvin, unpublished data). Fi- RNAi constructions were highly resistant to crown gall disease nally, overexpression of the RAT5 histone H2A-1 gene in var- directed by a broad range of oncogenic strains, although they ious rat mutants (other than the rat5 mutant) also restores were not generally resistant to Agrobacterium-mediated trans- transformation competency (L.-Y. Lee, S. Davis, X. Sui, and formation per se. A similar approach has been used to gen- S. B. Gelvin, unpublished data). Expression of the RAT5 gene erate crown gall disease-resistant tobacco and apple plants is therefore epistatic over the rat phenotype of other rat mu- (W. Ream, unpublished data). VOL. 67, 2003 AGROBACTERIUM-MEDIATED PLANT TRANSFORMATION 29 Genomics Approaches To Identify Plant Genes That ern agricultural biotechnology is heavily dependent on using Respond to Agrobacterium Infection Agrobacterium to create transgenic plants, and it is difficult to think of an area of plant science research that has not bene- As described above (129), plants may respond to infection by fited from this technology. However, there remain many chal- Agrobacterium, and this response may involve differential plant lenges. Many economically important plant species, or elite gene expression. Genes that are induced or repressed during varieties of particular species, remain highly recalcitrant to the early stages of Agrobacterium-mediated transformation Agrobacterium-mediated transformation, and the day has not may provide targets for manipulation of the host to improve yet arrived when flowers will be the only things seen coming the efficiency of transformation of recalcitrant plant species. from the barrels of gene guns. However, I feel that such a day Several laboratories have consequently begun investigations to is not too far in th

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