The agrobacterium tumefaciens gene transfer to plant cell
A. de la Riva*
Running title: Agrobacterium-mediated transformation
Key words: Agrobacterium tumefaciens, T-DNA, plant transformation
The update information of mechanisms of T-DNA transfer to plant cells by Agrobacterium tumefaciens is provided. Characterisation of such mechanisms shows multiple similarities with the bacterial conjugation allowing us to consider the evolutive relation between both genetic transfer processes. The general assessments of this process and its implication for plant genetic engineering are discussed with an emphasis in the application of this methodology to monocotyledonous plants.
Plant transformation mediated by the soil plant pathogen Agrobacterium tumefaciens has become the most used method for plant transformation. A. tumefaciens naturally infects the wound sites in dicotyledonous plant causing the formation of the crown gall tumours. Since the discovery of the bacterial origin of this neoplastic diseases (Smith and Townsend, 1907) a large number of researches have focused on the study of this process, firstly with the hope to understand the mechanisms of oncogenesis in general and applied it to study of cancer disease in animals and humans. Later this hypothesis was discarded and the interest on crown gall disease largely decreased until it was evident that A. tumefaciens is capable to transfer a particular DNA segment (T-DNA) of the tumour-inducing (Ti) plasmid into the nucleus of infected cells where it is subsequently stable integrated into the host genome and transcribed, causing the crown gall disease (Nester et al., 1984; Binns and Thomashaw, 1988). The T-DNA contains two types of genes: the oncogenic genes, encoding for enzymes involved in the synthesis of auxins and cytokinins and responsible for tumour formation; and the genes encoding for the synthesis of opines, a product resulted from condensation between amino acids and sugars, which are produced and excreted by the crown gall cells and consume by A. tumefaciens as carbon and nitrogen sources. Outside the T-DNA, are located the genes for the opine catabolism, the genes involved in the process of T-DNA transfer from the bacterium to the plant cell and for the bacterium-bacterium plasmid conjugative transfer genes (Hooykaas and Schilperoort, 1992; Zupan and Zambrysky, 1995).
Virulent strains of A. tumefaciens and A. rhizogenes contain a large megaplasmid (more than 200 kb) which play a key role in tumour induction and for this reason it was named Ti plasmid, or Ri in the case of A. rhizogenes. The T-DNA fragment is flanked by 25-bp direct repeats, which act as a cis element signal for the transfer apparatus. The transfer is mediated by the co-operative action of proteins encoded by genes determined in the Ti plasmid virulence region (vir genes) and in the bacterial chromosome. The 30 kb virulence (vir) region is a regulon organised in six operons that are essential for the T-DNA transfer (virA, virB, virD, and virG) or for the increasing of transfer efficiency (virC and virE) (Hooykaas and Schilperoort, 1992; Zupan and Zambryski, 1995, Jeon et al., 1998). Different chromosomal-determined genetic elements have shown their functional role in the attachment of A. tumefaciens to the plant cell and bacterial colonisation. The loci chvA and chvB, involved in the synthesis and excretion of the b -1,2 glucan (Cangelosi et al., 1989); the chvE required for the sugar enhancement of vir genes induction and bacterial chemotaxis (Ankenbauer et al., 1990, Cangelosi et al., 1990, 1991); the cel locus, responsible for the synthesis of cellulose fibrils (Matthysse 1983); the pscA (exoC) locus, playing its role in the synthesis of both cyclic glucan and acid succinoglycan (Cangelosi et at., 1987, 1991); and the att locus, which is involved in the cell surface proteins (Matthysse, 1987).
The initial results of the studies on T-DNA transfer process to plant cells demonstrate three important facts for the practical use of this process in plants transformation. Firstly, the tumour formation is a transformation process of plant cells resulted from transfer and integration of T-DNA and the subsequent expression of T-DNA genes. Secondly, the T-DNA genes are transcribed only in plant cells and do not play any role during the transfer process. Thirdly, any foreign DNA placed between the T-DNA borders can be transferred to plant cell, no matter where it comes from. These well-established facts, allowed the construction of the first vector and bacterial strain systems for plant transformation (for review Hooykaas and Schilperoort, 1992; Deblaere et al., 1985; Hamilton, 1998; Torisky et al., 1998).
Agrobacterium tumefaciens T-DNA transfer process
The process of gene transfer from Agrobacterium tumefaciens to plant cells implies several essential steps: (1) bacterial colonisation (2) induction of bacterial virulence system, (3) Generation of T-DNA transfer complex (4) T-DNA transfer and (5) integration of T-DNA into plant genome. Supported by the most recent experimental data and accepted hypothesis on T-DNA transfer we presented a hypothetical model depicting the most important stages of this process (Figure 1).
Bacterial colonisation is an essential and the earliest step in tumour induction and it takes place when A. tumefaciens is attached to the plant cell surfac (Matthysse , 1986). Mutagenesis studies show that non-attaching mutants loss the tumour-inducing capacity (Cangelosi et al., 1987, Douglas et al., 1982, Thomashow et al., 1987, Bradley et al., 1997). The polysaccharides of the A. tumefaciens (lipopolysaccharides (LPS), and capsular polysacharides (K-antigens)) are proposed to play an important role in the colonising process (Whatley and Spiess, 1977). The LPS are integral part of outer membrane and capsular polysaccharides (K-antigens), lacking of lipid anchor, have strong anionic nature. There are evidences that capsular polysaccharides may play specific role during the interaction with the host plant (Bradley et al., 1997).
The chromosomal 20kb att locus contains the genes required for successful bacterium attachment to the plant cell. This locus has been extensively studied (Ames et al., 1990; Higgings et al., 1990,Matthysse et al. 1996). Insertions in the left 10 kb side of this region produce avirulent mutants that could restore its attachment capacity if the culture medium is previously conditioned by the incubation of wild-type virulent bacterium with plant cells. In contrast, mutational insertion in the 10 kb right side of the att locus results in the irreversible loss of attachment capacity, which can not be restored by conditioned medium (Bradley, 1997). These results suggest that genes, placed at att left side, are involved in molecular signalling events, while the right side genes are likely to be responsible for the synthesis of fundamental components.
Induction of bacterial virulence system
The T-DNA transfer is mediated by products encoded by the 30-40 kb vir region of the Ti plasmid. This region is composed by at least six essential operons (vir A, vir B, vir C, vir D, vir E, virG ) and two non-essential (virF, virH). The number of genes per operon differs, virA, virG and virF have only one gene; virE, virC, virH have two genes while virD and virB have four and eleven genes respectively. The only constitutive expressed operons are virA and virG, coding for a two-component (VirA-VirG) system activating the transcription of the other vir genes, and has structural and functional similarities to other already described for other cellular mechanisms (Nixon, 1986, Iuchi, 1993).
VirA is a transmembrane dimeric sensor protein that detects signal molecules, released from wounded plants (Pan et al., 1993). The signals for VirA activation include acidic pH, phenolic compounds, such as acetosyringone (Winans et al., 1992), and certain class of monosaccharides which acts sinergically with phelolic compounds (Ankenbauer et al., 1990; Cangelosi et al., 1990; Shimoda et al., 1990a, 1990b; Doty et al., 1996). VirA protein can be structurally defined into three domains: the periplasmic or input domain and two transmembrane domains (TM1 and TM2). The TM1 and TM2 domains act as a transmitter (signaling) and receiver (sensor) (Parkinson, 1993). The periplasmic domain is important for monosaccharide detection (Chang and Winans, 1992). Within the periplasmic domain, adjacent to the TM2 domain is an amphipatic helix, with strong hydrophilic and hydrophobic region (Heath et al., 1995). This structure is characteristic for other transmembrane sensor proteins and folds the protein to be simultaneously aligned with the inner membrane and anchored in the membrane (Seligman and Manoil, 1994). The TM2 is the kinase domain and plays a crucial role in the activation of VirA, phosphorilating itself on a conserved His-474 residue (Huang et al., 1990; Jin et al., 1990a, 1990b) in response to signalling molecules from wounded plant sites. Monosaccharide detection by VirA is important amplification system to respond to low levels of phenolic compounds. The induction of this system is only possible through the periplasmic sugar (glucose/galactose) binding protein ChvE (Ankenbauer and Nester, 1990; Cangelosi et al., 1990), which interacts with VirA (Shimoda et al., 1990a, 1990b, Turk et al., 1993; Chang and Winans, 1992). Recent studies for determination of VirA regions, important for its sensing activity suggested the position, which may be involved on TM1-TM2 interaction. This interaction causes the exposure of the amphipathic helix to small phenolic compounds and suggests a putative model for the VirA-ChvE interaction (Doty et al., 1996).
Activated VirA has the capacity to transfer its phosphate to a conserved aspartate residue of the cytoplasmic DNA binding protein VirG (Jin et al., 1990; Pan et al., 1993). VirG functions as transcriptional factor regulating the expression of vir genes when it is phosphorilated by VirA (Jin et al., 1990a, 1990b). The C-terminal region is responsible for the DNA binding activity, while the N-terminal is the phosphorilation domain and shows homology with the VirA receiver (sensor) domain.
The activation of vir system also depends on external factors like temperature and pH. At temperatures greater than 32°C the vir genes are not expressed because of a conformational change in the folding of VirA induce the inactivation of its properties (Jin et al., 1993). The effect of temperature on VirA is suppressed by a mutant form of VirG (VirGc), which activates the constitutive expression of the vir genes. However, this mutant can not confer the virulence capacity at that temperature to Agrobacterium, probably because the folding of other proteins actively participate in the T-DNA transfer process are also affected at high temperature (Fullner et al., 1996).
Generation of T-DNA transfer complex
The activation of vir genes carries out the generation of single-stranded (ss) molecules representing the copy of the bottom T-DNA strand. Any DNA placed between T-DNA borders will be transferred to the plant cell, as single strand DNA, and integrated into plant genome. These are the only cis acting elements of the T-DNA transfer system. The proteins VirD1 and VirD2 play the key role in this step are, recognising the T-DNA border sequences and nicking (endonuclease activity) the bottom strand at each border. The nick sites are assumed as the initiation and termination sites for T-strand recovery. After endonucleotidic cleavage VirD2 remains covalently attached to the 5'-end of the ss-T-strand. This association prevents the exonucleolitic attack to the 5'-end of the ss-T-strand (Dürrenberger et al., 1989) and distinguishes the 5'-end as the leading end of the T-DNA transfer complex. VirD1 interacts with the region where the ss-T-strand will be originated. Experiments "in vitro" experiments evidenced that for cleavage of supercoiled stranded substrate by VirD2 is essential the presence of VirD1 (Zupan and Zambryski, 1995; Christie et al., 1997). The simultaneous restoration of the excised ss-T-strand is evolutionarily related to other bacterial conjugative DNA transfer processes, which include the generation of the single strand DNA (Zupan and Zambryski, 1995; Christie, 1997; Lessl et al., 1994).
Extensive mutation or deletion of the right T-DNA border is followed by almost completely loss of T-DNA transfer capacity, while at the left border resulted in lower transfer efficiency (Hille et al., 1983). This fact indicates that T-strand synthesis is in 5' to 3' direction, and it is initiated at the right border and that the termination process take place even when the left border is mutated or completely absent, although at lower efficiency (Filichkin and Gelvin, 1993). The presence of an enhancer or "overdrive" sequence next to the right border (Peralta and Ream, 1985), which specifically recognised by VirC1 protein (Toro et al., 1989) could make the difference between both T-DNA borders (van Haaren at al., 1987; Rogowsky et al., 1990).
Two models for the translocation of T-DNA-complex
The transferring vehicle to the plant nucleus is a ssT-DNA-protein complex. According to the most accepted model, the ssT-DNA-VirD2 complex is coated by the 69 kDa VirE2 protein, a single strand DNA binding protein. This co-operative association prevents the attack of nucleases and, in addition, extends the ssT-DNA strand reducing the complex diameter to approximately 2 nm, making easier the translocation through membrane channels. However, that association does not stabilises T-DNA complex inside Agrobacterium (Zupan et al., 1996). VirE2 contains two plant nuclear location signals (NLS) and VirD2 one (Bravo Angel et al, 1998). This fact indicates that both proteins are presumably to play important role once the complex is in the plant cell mediating the complex uptake to the nucleus (Herrera-Estrella et al., 1990; Shurvinton et al., 1992; Rossi et al., 1993, Tinland et al., 1995, Zupan et al., 1996).
VirE1 is essential for the export of VirE2 to the plant cell (Binns et al., 1995). Bacterial strains mutated in virE1, cannot export VirE2 and resulted in its accumulation inside the bacterium. Such mutants can be complemented if coinfecting with strain that can export VirE2, indicating that this protein can be exported independently and that for transmission event does not necessary the transfer of VirE2 as part of the ss-T-DNA complex (Sundberg et al., 1996) and that is possible the transfer of naked T-DNA to the plant cell (Binns et al., 1995; Sundberg et al., 1996).
From these experimental evidences an alternative model was brought. This model proposes that the transfer complex is a single-strand DNA covalently bound at its 5'-end with VirD2, but uncoated by VirE2. The independent export of VirE2 to plant cell is presented as natural process, and once the naked ssT-DNA-VirD2 complex is inside the plant cell, it is coated by VirE2 (Binns et al., 1995; Lessl et al., 1992).
Previous researches described the role of 9.5 kb virB operon in the generation of a suitable cell surface structure for the ssT-DNA complex transfer from bacterium to plant (Finberg et al., 1995; Stephens et al., 1995; Zhou and Christie, 1997; Dang and Christie, 1997; Rashkova et al., 1997, Fernandez et al., 1996; Beaupré et al., 1997). The VirD4 protein is also required for the ss-T-DNA transport. The function of VirD4 is the ATP-dependent linkage of protein complex necessary for T-DNA translocation (Firth et al., 1996).
VirB are proteins that present the hydrophathy characteristics similar others membrane-associated proteins (Kuldau et al., 1990; Shirasu et al., 1990, 1994; Thompson et al., 1988; Ward et al., 1988). VirD4 protein is a transmembrane protein but predominantly located at the cytoplasmatic side of the cytoplasmic membrane (Okamoto et al., 1991). Comparative studies showed highly degree of homology between the virB operon and transfer regions of broad host range (BHR) plasmid in genetic organisation, nucleotide sequence and protein function (Pohlman et al., 1994; Lessl et al., 1992). Both systems delivery non-self transmissible DNA-protein complex to recipient host cell. In addition, they have the capacity to DNA interkingdom delivery (Heinemann and Sprague, 1989; Bundock et al., 1995; Piers et al., 1996) suggesting that T-DNA transfer apparatus and conjugation systems are related and probably evolved from a common ancestral (Christie et al., 1997; Oger et al., 1998).
The majority of VirB proteins are assembled as a membrane-spanning protein channel involved both membranes (Shirasu and Kado, 1993a, 1993b; Shirasu et al., 1994; Stephens et al., 1995; Das and Xie, 1998). Except for VirB11, they have multiple periplasmic domains (Christie, 1997). VirB1 is the only member of VirB proteins found in the extracellular milieu (Baron et al., 1997) although it is possible that some of the other VirB proteins may be redistributed during the process of biogenesis and functioning of the transcellular conjugal channel (Christie, 1997). That could be the case of the VirB2, which is translated as a 12 kDa proprotein, and later is later proteolically processed to its mature 7 kDa functional form (Jones et al., 1996).
VirB4 and VirB11 are hydrophilic ATPases necessary for active DNA transfer. Vir B11 lacks continuous sequence of hydrophobic residues, motivating of periplasmic domains (Rashkova et al., 1997). These characteristics are atypical for this type of protein and evidence the possible dinamic co-existence of different conformational forms "in vivo". VirB4 tightly associates with the cytoplasmic membrane (Dang and Christie, 1997). It contains two putative extracellular domains conferring transmembrane topology to this protein, which is presumed to allow the ATP-dependent conformational change in the conjugation channel. Probably, the functional forms of VirB4 and VirB11 are homo- and heterodimmers (Dang and Christie, 1997). The VirB7-VirB9 heterodimmer is assumed to stabilises other Vir proteins during assembly of functional transmembrane channel (Fernandez et al., 1996; Spudich et al., 1996).
Recent studies identified some of the initial steps of biogenesis of ssT-DNA complex apparatus. Firstly, VirB7 and VirB9 monomers are exported to the membrane and processed. They interact each other to form covalently cross-linked homo- and heterodimmers. Although it is widely assumed the role of both types of dimmers in the biogenesis of the transfer apparatus, it is likely that only heterodimmers are the essential ones (Fernández et al., 1996; Spudich et al., 1996). Subsequently the VirB7-VirB9 heterodimmer is sorted to the outer membrane. The sorting mechanism has not been elucidated (Christie, 1997) the next step implies the interaction with the other Vir proteins for assembling the transfer channel with the contribution of the transglycosidase (Berger and Christie, 1993, 1994).
Two accessory vir operons, present in the octopine Ti-plasmid are virF and virH. The virF operon encoding for a 23-kDa protein that functions once the T-DNA complex is inside the plant cells via the conjugal channel or independently, as it was assumed for VirE2 export. The role of VirF is seems to be related with the nuclear targeting of the ssT-DNA complex but its contribution is less important than in the case of VirF (Hooykass and Schilperoort, 1992). The virH operon consists in two genes that code for VirH1 and VirH2 proteins. These Vir proteins are no essential but could enhance the transfer efficiency, detoxifying certain plant compounds that can affect the growth of bacterial (Kanemoto et al., 1989). If that is the function of VirH proteins, they play a role in the host range specificity of bacterial strain for different plant species.
Integration of T-DNA into plant genome.
Inside the plant cell, the ssT-DNA complex is targeted to the nucleus crossing the nuclear membrane. Two Vir proteins have been found to be important in this step: VirD2 and VirE2, which are the most important; and probably VirF, which has minor contribution to this process (Hooykaas and Schilperoort, 1992). The two NLS of VirE2 have been considered important for the continuos nuclear import of ss-T-DNA complex, probably by keeping both sides of nuclear pore simultaneously open. The nuclear import is probably also mediated by specific NLS-binding proteins, which are present in plant cytoplasm.
The final step of T-DNA transfer is its integration into plant genome. It is considered that the integration occurs by illegitimate recombination (Gheysen et al., 1991, Lehman et al., 1994; Puchta, 1998). According to this model, paring of a few bases provides just a minimum specificity for the recombination process by positioning VirD2 for the ligation. The 3´-end or adjacent sequences of T-DNA find some low homologies with plant DNA resulting in the first contact (synapses) between the T-strand and plant DNA forms a gap in 3'-5' strand of plant DNA. Displaced plant DNA is subsequently cut at the 3'-end position of the gap by endonucleases, and the first nucleotide of the 5' attached to VirD2 pairs with a nucleotide in the top (5'-3') plant DNA strand. The 3' overhanging part of T-DNA together with displaced plant DNA are digested away, either by endonucleases or by 3'-5' exonucleases. Them, the 5' attached to VirD2 end and other 3'-end of T-strand (paired with plant DNA during since the first step of integration process) joins the nicks in the bottom plant DNA strand. Once the introduction of T-strand in the 3'-5' strand of the plant DNA is completed, a torsion followed by a nick into opposite plant DNA strand is produced. This situation activates the repair mechanism of the plant cell and the complementary strand is synthesised using the early inserted T-DNA strand as a template (Tinland et al., 1995).
VirD2 has an active role in the precise integration on T-strand in the plant chromosome. The release of VirD2 protein may provide the energy containing in its phosphodiester bond, at the Tyr29 residue, with the first nucleotide of T-strand, providing the 5'-end of the T-strand for ligation to the plant DNA. This phosphodiester bound can serve as electrophilic substrate for nucleophilic 3'-OH from nicked plant DNA. (Jayaram, 1994) When the mutant VirD2 protein is transferred attached to the T-strand, the integration process take place with the loss of nucleotides at the 5'-end of the T-strand (Tinland et al., 1995).
Plant transformation mediated by Agrobacterium tumefaciens
The first plant transformed by Agrobacterium tumefaciens was tobacco (Herrera-Estrella, 1983). Since that crucial moment in the development of plant science, a great progress in understanding the Agrobacterium-mediated gene transfer to plant cells has been archived. However, Agrobacterium tumefaciens naturally infects only dicotyledonous plants and many economically important plants, including the cereals, remained accessible for genetic manipulation during long time. For these cases alternative direct transformation methods have been developed (Shillito et al, 1985; Potrykus, 1991, Uchimiya et al., 1986, de la Pena et al., 1987, Fromm et al., 1985, 1986; Lörz et al., 1985; Arencibia 1995, Sanford, 1988). However Agrobacterium-mediated transformation have remarkable advantages over direct transformation methods in reducing the copy number of the transgene, potentially leading to fewer problems with transgene cosuppresion and instability (Koncz et al., 1994, Hansen et al., 1997). In addition, It is a single-cell transformation system and avoids the obtainment of mosaic plants, which are more frequent when direct transformation is used (Enríquez-Obregón et al 1997, 1998).
The monocots have been considered to be outside the Agrobacterium host range and other gene-transfer methods were developed for these plants. To develop this methodologies for a monocot plant it is important to take in consideration the critical aspects in the Agrobacterium tumefaciens-plant interaction, the cellular and tissue culture methodologies developed for that specie. The suitable genetic materials (bacterial strains, binary vectors, reporter and marker genes, promoters) and molecular biology techniques available in the laboratory, are necessary for selection of DNA to be introduce. This DNA must be expressible in plant making possible the identification of transformed plants in selectable medium and using molecular biology techniques test and characterise the transformation events (for review Birch, 1997).
Transformation is currently used for genetic manipulation of more than 120 species of at least 35 families, including the most major economic crops, vegetables, ornamental, medicinal, fruit, tree and pasture plants (Birch, 1997), using Agrobacterium-mediated or direct transformation methods. The idea, that some species can not accept the integration of foreign DNA in its genome and lack the capacity to be transformed is unacceptable under the increasing number species that have been transformed.
The optimisation of Agrobacterium tumefaciens-plant interaction is probably the most important aspect to be considered. It includes the integrity of bacterial strain its correct manipulation as warranty of the virulence machine integrity and the study of reaction in wounded plant tissue, which may develop necrotic process in the wounded tissue or affect the interaction and release compounds inducers or repressors of Agrobacterium virulence system. The type of explant is also important fact and it must be suitable for regeneration allowing the recovering of whole transgenic plants. The establishment of method for efficient regeneration for one particular species is crucial for its transformation.
The mentioned aspects are important to establish transformation procedure for any plant but particularly for those species categorised as recalcitrant. In this category have been included cereals, legumes and woody plants, which are very difficult to transform or remain untransformed. Many species originally considered in this category has been transformed in recent years. One of these species, sugarcane, has been transformed in our laboratory (Figure 2) ( Arencibia et al., 1998).
Agrobacterium tumefaciens is more than the causative agent of crown gall disease affecting dicotyledonous plants. It is also firstly the natural instance for the introduction of foreign gene in plants allowing its genetic manipulation. Similarities have been found between T-DNA and conjugal transfer systems are evolutionally related and apparently evolved from a common ancestral.
Although the gene transfer mechanisms remain largely unknown, great progress has been obtained in practical implementation of transformation protocols for both dicotyledonous and monocotyledonous plants. Particularly important is the extension of this single-cell transformation methodology to monocotyledonous plants. This advance has biological and practical implications. Firstly, because of advances of A. tumefaciens-mediated gene transfer over the direct transformation methods, which where the only way for genetic manipulation of economically important crops as cereals and legumes. Second, it has been demonstrated that T-DNA is transferred to dicot and monocot plants by an identical molecular mechanism. This confirmation implies that any plant can potentially be transformed by this method if suitable transformation protocol is established.
The Agrobacterium-mediated transformation protocols differ from one plant specie to other and, within species, from one cultivar to other. In consequence, the optimisation of Agrobacterium-mediated transformation methodologies requires the considered of several factors that can be determined in the successful transformation of one species. Firstly, the optimisation of Agrobacterium-plant interaction on competent cells from different regenerable tissues. Second, the development of suitable method for regeneration from transformed cells.
Undoubtedly, the development of transformation procedures based on A. tumefaciens-mediated gene transfer for new economically important species are advisable and the results obtained in last years evidence a promising future.
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