ISSN 00062979, Biochemistry (Moscow), 2013, Vol. 78, No. 12, pp. 13211332. © Pleiades Publishing, Ltd., 2013. Original Russian Text © M. I. Chumakov, 2013, published in Biokhimiya, 2013, Vol. 78, No. 12, pp. 16701683.
REVIEW
Protein Apparatus for Horizontal Transfer of Agrobacterial TDNA to Eukaryotic Cells M. I. Chumakov Institute of Biochemistry and Physiology of Plants and Microorganisms, Russian Academy of Sciences, pr. Entuziastov 13, 410049 Saratov, Russia; fax: (8452) 970383; Email:
[email protected] Received April 9, 2013 Revision received September 5, 2013 Abstract—This review analyzes agrobacterial virulence proteins and recipient cell proteins involved in horizontal transfer of a TDNA–protein complex. Specifically, it considers the early stages of the interactions of partners (signal exchange, attachment, close contact); TDNA release from bacterial cells; channel formation for the transfer of ssDNA between the partners; transfer of agrobacterial TDNA through the membrane, cytoplasm, and nuclear membrane of the recipient cell and its incorporation into the recipient cell genome. It further discusses possible pathways of agrobacterial ssDNA transfer to the recipient cells. In particular, the possible role of Tpili and VirE2 protein during conjugative transfer of agrobacterial ssDNA between donor and recipient cells is discussed. DOI: 10.1134/S000629791312002X Key words: horizontal transfer, Agrobacterium, TDNA, virulence proteins, conjugation, Tpili, VirE2
TDNA transfer from agrobacteria to eukaryotic organisms is an example of horizontal DNA transfer between pro and eukaryotes in natural conditions. Soil inhabiting bacteria of the Agrobacterium genus are capa ble of transferring a Ti (tumorinducing) or Ri (root hair inducing) fragment of TDNA plasmids into the genome of a wide range of plants under in planta and in vitro con ditions; prokaryotic virulence proteins and proteins of eukaryotic transport systems participate in this process [1, 2]. Animal cells under in vitro conditions also undergo agrobacterial transformation if virulence genes are induced in the agrobacteria [3] or if a TDNA prepara tion together with virulence proteins VirD2 and VirE2 is artificially introduced into animal cells (HeLa) [4]. Agrobacteria are also capable of transformation of sea urchin [5], fungal, and yeast genomes [68]. TDNA ends are bounded by two direct repeats of 23 nucleotides, and it is transferred with the participation of virulence locus gene products (vir) [9]. Any DNA placed between these boundary sequences can be transferred and built into the plant cell nucleus. Unlike transposons, once incorporat ed, TDNA cannot be transferred again since it has no genes responsible for transfer. TDNA is transferred polarly; deletion of the right border disrupts the transfor Abbreviations: ssDNA, singlestranded DNA; TDNA, trans ferred singlestranded DNA (transfer DNA).
mation process, whereas deletion of the left border has a smaller effect [10]. Genes responsible for the activation of division of recipient cells [11] and biosynthesis of opines [12] are part of TDNA. The virregion (vir, virulence inducing region, 35 kb), located on the Ti plasmid, is not part of TDNA and consists of seven complimentary groups virA, virB, virC, virD, virE, virF, and virG whose expression is induced by signal molecules. In addition to virulence genes located on a Tiplasmid, a number of constitutively expressed chromosomal genes are also involved in agrobacterial transformation: (chvA, chvB) [13], pscA [14], chvE [15], chvD [16], chvG [17], chvI [18], miaA [19], ros [20], and ivr [21]. Generally, the process of TDNA transfer is as fol lows (Fig. 1): signaling molecules from plant wound exu dates interact with agrobacterial receptor membrane pro tein VirA (Fig. 1, stage 1). The signal is further carried into the bacterial cell by means of a twocomponent sys tem of VirA–VirG proteins, launching protein synthesis in the virregion. Then a fragment of TDNA (Tstrand) is cut from the Tiplasmid (virulence proteins VirD1 and VirD2 participate in this process) (Fig. 1, stage 2); the T strand is released from the bacterial cell together with the proteins providing its transfer and integration (VirE1, VirE2, VirE3, VirD2, VirD5, VirF) into the genome of the host cell. The Tstrand and virulence proteins are transferred across the bacterial and plant cell membranes
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via a virBdependent channel (Fig. 1, stage 3). The T complex is then formed in the recipient cell cytoplasm: one Tstrand is covered with 600 molecules of VirE2 pro tein (Fig. 1, stage 4). The transport system of the host cell is involved in Tcomplex transfer towards the nucleus in the cytoplasm of the recipient cell (Fig. 1, stage 5); T DNA incorporation into the recipient cell genome is mediated by the host repair system and agrobacterial vir ulence proteins (VirD2, VirF) (Fig. 1, stage 6). Once integration is complete, the TDNA genes are expressed (Tiplasmid: tms1, tms2 (iaaH), tmr, tm1, ipt, osc; Ri plasmid: aux, rolA, rolB, rolC, rolD); these genes are responsible for the regulation of biosynthesis of phyto hormones (auxin, cytokinin), which determine the shift in plant cell hormonal balance and tumor formation. In addition, the accAaccG genes of the TDNA Tiplasmid are expressed in the plant host. These genes are responsi ble for catabolism of special agrocinopine molecules, which are the source of carbon and nitrogen for agrobac
teria and can be metabolized only by them. This creates selective advantages for the parasite. Let us consider the mechanism of agrobacterial trans formation in more detail. Wounding, division, and differ entiation of plant tissues in dicotyledonous [9, 23] and monocotyledonous [24] plants launch the mechanism of cell wall synthesis and repair; lignin synthesis also begins. A number of phenolic compounds are lignin precursors, including acetosyringone and hydroxyacetosyringone, which act as signaling molecules, chemoattractants for Agrobacterium tumefaciens, capable of activating agrobac terial virgenes expression when added in low concentra tion (10–7 M) [25]. Agrobacteria move in the direction of signaling molecule increasing gradient (up to 50 μm/sec) due to a chemotactic mechanism towards the site of wounding or cell wall expansion. Signaling molecules affect the product of constitutively functioning virA gene, membrane receptor protein VirA encoded by the plasmid [26], and receptor protein ChvE encoded by the chromo
Plant cell
Tcomplex Nuclear pore Ti plasmid proteins proteins RNA
Nucleus
Fig. 1. General scheme of TDNA transfer from agrobacteria to plants (modified from [22]): 1) activation of VirA–VirG twocomponent sig naling system by low molecular weight components of the plant cell wall; 2) TDNA excision, Tstrand formation; 3) independent Tstrand and VirE2 protein transfer into the plant cell, piloted by VirD2 protein, through the virB channel of an agrobacterial cell and an unknown channel of a plant cell; 4) Tstrand covering with VirE2 protein and formation of Tcomplex; 5) Tcomplex transfer through the plant cell cytoplasm, entering the plant cell nucleus; 6) TDNA incorporation into the plant chromosome.
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PROTEINS IN HORIZONTAL TRANSFER OF AGROBACTERIAL TDNA some [15]. Furthermore, wound fluid has lower pH and contains sugars and amino acids, which may also (though to a lesser extent) induce the virA gene and act as chemoat tractants [27, 28]. Under acidic conditions (wounding causes lysosomes to leave the plant cell) the level of ChvG protein significantly increases in A. tumefaciens as a result of degradation of repressor protein ExoR [29]. ChvG together with ChvI protein trigger the system of secretion of agrobacterial virulence proteins [29]. VirA protein is integrated into the inner membrane; it has periplasmic and cytoplasmic domains involved in recognition of signaling molecules of phenolic nature. Interaction between signal ing molecule and receptor changes the receptor conforma tion, inducing the virG gene [3032], whose product trig gers the expression of all the other vir genes (Fig. 1). In the presence of certain monosaccharides, the ChvE protein domain facing the periplasm interacts with the periplasmic domain of VirA protein, causing in agrobacteria a reaction similar to the one caused by phenolic inducers [15, 33]. ChvE protein is also involved in agrobacterial chemotaxis to various monosaccharides. Besides plant cells in wounding sites, there are other cells of plant tissues, i.e. cells of germinating seeds [34], leaves of young seedlings [35], female gametophyte of wheat [36] and corn [37], and other plant organs that suc cessfully undergo agrobacterial transformation on treat ment of intact plants with bacterial suspension by the in planta method [38]. Data on agrobacterial transformation of intact plants presented in a review by Chumakov and Moiseeva [39] indicate that TDNA transfer from agrobacteria with expressed virulence proteins into intact plant tissues and organs under in planta conditions pro ceeds with rather high frequency. Having reached the plant cell surface due to chemotaxis, agrobacteria start colonizing the surface and form a tight contact with the recipient cell, which is described in detail in reviews by Romanchuk and Chumakov [40, 41]. Agrobacterial extracellular structures involved in col onization and contact with the recipient cell surface. Various surfacelocated agrobacterial molecules are involved in the process of attachment and contact with the plant cell surface. Not all of them play a significant role in further infection. Absence of surface molecules causes a variety of reactions in agrobacteria, starting from slight reduction of tumor formation (as in the case of cel lulose fibrils and cyclic 1,2βglucan) to complete block ing of TDNA transfer (as in the case of VirB2 virulence protein) [41, 42]. Polysaccharide structures. In 1982, E. Nester’s labo ratory first presented genetic evidence of the importance of agrobacterial attachment to the plant cell surface in the process of TDNA transfer: A. tumefaciens mutants with impaired attachment ability were shown to lose their vir ulence [43]. Three years later, two sites (chvA, chvB) of 1.5 and 5.0 kb were identified in the A. tumefaciens chro mosome; transposon insertions in these sites affected vir BIOCHEMISTRY (Moscow) Vol. 78 No. 12 2013
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ulence and agrobacterial attachment to the plant cell sur face (the work was carried out in the same laboratory) [44]. Four years later, Nester et al. found that the chvA gene encodes a protein with molecular weight of 65 kDa [45], this protein being closely homological with the E. coli export protein HlyB, and NdvA of rhizobia [46]. The chvB gene controls the formation of a membrane protein with molecular weight of 235 kDa, which covalently binds (1,2)βglucan, thus forming cycloglucan [48]. Mutations in the chvB gene are pleiotropic: mutants lose flagella, their resistance to certain phages changes, and they cannot produce cycloglucan [48, 49]. It was also shown that chvB of A. tumefaciens synthesizes an inactive form of rhicadhesin, a protein involved in the initial stages of agrobacterial attachment to a plant tissue sur face; this protein also stimulates transfer of IncQ plas mids between bacteria [50, 51]. In 1987, Tomashov et al. isolated an A. tumefaciens mutant with mutation in the exoC (pscA) gene [52] encoding phosphoglucomutase [53]. This mutant pro duced little cycloglucan, but it differed from the chvB gene mutant [52]. It has problems with the synthesis of a number of polysaccharides (cyclic glucan, capsular poly saccharide, lipopolysaccharide, succinoglucan) [53]; its ability to attach to the plant surface [50] and virulence [40] are reduced, but is can normally transfer TDNA into the plant cell nucleus when agrobacteria are injected into the plant cell [54]. Based on these observations, Hohn et al. suggested in 1995 that agrobacterial transfor mation does not necessarily involve attachment of agrobacteria to the plant cell surface [54]. However, data presented in the same article indicate that cells that are mutant in their attachment ability cannot transfer T DNA without its artificial injection into the cell. Protein structures. Flagella. Agrobacteria have a polar flagellum (a bunch of flagella) on one of the cell poles. According to Bradley et al., its absence in A. tume faciens mutants does not affect virulence and ability to attach to mesophilic Zinnia cells [55]. According to other authors, agrobacterial mutants without flagella have reduced virulence [56], reduced ability to attach to root epidermis [57], and cannot infect plants in the soil [58]. Pili. Many soil bacteria form fimbriae (pili), which consist of repeating, noncovalently bound protein sub units forming spirally twisted structures in the form of long (15 nm) hollow tubes or nonhollow filaments dis posed perpendicularly to the bacterial cell surface [59]. Escherichia coli has from six [60] to nine different pili types [59]. Escherichia coli conjugative (F) pili are thought to have two hypothetical functions in conjugative plasmid transfer: 1) anchoring on the surface of the recip ient cell so that membranes of the partners would con verge [61]; 2) ssDNA transfer through the pili channel [62]. A hypothesis proposed by Marvin and Hohn in 1969 [61] is the most common, although direct evidence sup porting it has never been found.
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By now, there are three pili types identified in agrobacteria: a) vir, traindependent type (adhesive pili) [63]; b) virindependent, tradependent pili involved in mobility and plasmid DNA transfer between agrobacteria [64]; c) virdependent pili type (Tpili) involved in T DNA transfer [65, 66]. Can agrobacterial pili participate in the attachment to the surface of a recipient cell? In 1987, Stemmer and Sequeira described the formation of vir, traindependent pili in A. tumefaciens [63], which, according to the authors, are adhesive. However, this property was not proven in the quoted work. Electron microscopic analysis of crossbreeding agrobacterial cultures with induced tragenes and previ ously blocked Tpili synthesis showed the formation of thin straight fibrils (tradependent pili), which were absent from the traR mutant [64]. The traR mutant could not form extracellular proteins with molecular weight of 63 and 67 kDa that have agglutinative activity and unknown function [64]. The tradependent pili connect crossbreeding agrobacterial cells, which were destroyed after SDStreatment; they probably also participate in tradependent Tplasmid transfer between agrobacteria. virB1dependent structures. Mutation in virB1 gene reduces virulence, but it does completely block TDNA transfer [67]. In 1996, Nester et al. presented data on the Cterminal part of VirB1 protein having a transglycosy lase function. This Cterminal part has structural similar ity to lysozyme [68] and can be secreted outside, being possibly involved in cell wall degradation at the contact site; it also forms aggregates [69] or short structures on the cell surface [70]. In 2007, in the laboratory of Zambryski, it was shown that the Cterminal, secreted part of VirB1 protein is required for the formation of T pili in the course of cyclization of the major (VirB2 pro tein) and minor (VirB5 protein) subunits of agrobacterial Tpili [71]. virBdependent pili (Tpili). In 1987, Engstrom et al. were the first to suggest that agrobacterial VirB proteins are involved in conjugative contact and pili formation [72]. Ten years later agrobacterial pili were visualized, and their involvement conjugative transfer of plasmids pML122 [73] and pTd33 [74] was demonstrated. However, there is not yet any direct evidence for the par ticipation of Tpili in conjugative contacts between agrobacterial and plant cells. In 1993, Kado et al. first suggested [75] and later [76, 77] submitted evidence of virB2 gene encoding the syn thesis of propilin, a structural Tpili protein. VirB2 pro tein has a large number (100 of 121) of amino acids con taining hydrophobic regions. The Nterminal end of VirB2 protein faces the cytosol; a signal sequence is absent. By the end of the 1990s, it was found that VirB2 protein is secreted from agrobacteria and forms long flex ible hollow structures (Tpili) [72, 7880], which consist mainly from VirB2 protein subunits with molecular
weight of 6.57.2 kDa [72, 78, 80]. In addition to the major VirB2 protein, Tpili include alsoVirB5 and VirB7 proteins located at the end and at the base of pili, respec tively [81, 82]. Tpili are localized on one of the poles of A. tumefaciens cells and play a key role in agrobacterial infection and ssDNA transfer into recipient cells [80, 83]. Such structures are not observed in agrobacterial cells without Tiplasmid or in agrobacteria with inactivated virulence genes. Electron microscopy revealed a signifi cant morphological similarity between agrobacterial T pili and conjugative (F) pili of E. coli [79]. It is believed that in E. coli the end of donor cell pili finds a specific site (possibly lipopolysaccharide) on the surface of the recip ient cell and becomes attached to it with a special protein (the product of the fimH gene). Pili retraction leads to the development of cell–cell contact between the outer membranes, which is stabilized by specialized proteins. Sites of pili contact with the membrane are very similar to the adhesion zones between inner and outer membranes, where pili are formed [84, 85]. Such adhesion zones (“Bayer bridges”) develop as a result of local contact of outer and inner membranes. However, it should be noted that there are fundamental objections to the existence of “Bayer bridges”. Their appearance is treated as an artifact of the preparation of the material for microscopy. When chemical fixation is replaced by cryofixation, adhesion zones are not formed. As the mechanisms of ssDNA transfer by conjuga tion and agrobacterial transformation are analogous, as shown by Lessl and Lanka [86], Kado’s assumption [78] of agrobacterial Tpili function being similar to the func tion of similar Fpili of E. coli seems to be quite reason able. However, the mechanism of contact involving agrobacterial Tpili remains unclear. In particular, it is unclear what might be the role of Tpili in TDNA trans fer into a plant and whether observed structures are involved in bringing together the membranes of cross breeding agrobacteria, or that TDNA is transferred via a Tpili channel. Are virdependent agrobacterial surface structures involved in contact with the recipient cell surface? Kurbanova et al. have shown that acetosyringonemediat ed induction of virulence genes changed the ability of agrobacterial to attach to the plant cell surface [87]. No significant effect of acetosyringone and temperatures unfavorable for Tpili synthesis on agrobacterial adhesion indicates that Tpili do not play a significant role in the attachment to the plant cell surface [87]. Possible role of Tpili in Tstrand transfer. Brinton suggested in 1971 that plasmid ssDNA is transferred via an internal pili channel in E. coli [62]. After nearly two decades, a similar assumption about agrobacterial ssT DNA transfer via an internal Tpili channel was made by Kado [78]. According to electron microscopy estimation, the outer diameter (8.5 nm) of conjugative (F) pili in E. coli BIOCHEMISTRY (Moscow) Vol. 78 No. 12 2013
PROTEINS IN HORIZONTAL TRANSFER OF AGROBACTERIAL TDNA [88] is similar to the outer diameter (810 nm) of agrobacterial Tpili [79, 81]. According to theoretical calculations of Silverman, the inner diameter of the E. coli conjugative pili is 2 nm [89], which is close to Kado’s estimation of the inner diameter of Tpili channel [78], and this value theoretically allows for ssDNA (1.2 nm) passage through the pili channel. However, according to our estimation, the hydrodynamic diameter of Tstrand (TDNA with a piloting VirD2 protein) is 4 nm, which seems to be not enough for Tstand passage in the con jugative pili in a normal (nonretracted) state. But per haps pili retraction caused by partners approaching each other can increase the channel lumen to the size required for the passage of ssDNA with a pilot protein. As pili assembly proceeds rather rapidly and their length exceeds the thickness of the cell wall of plant cells, it can be assumed that attachment to the cell wall is followed by the agrobacterium forming the pili structure starting from the base; later it penetrates the cell wall and reaches the sur face of the plant cell endoplasmic reticulum. Agrobacteria can theoretically use the pili channel to ensure delivery of virulence proteins (VirE1, VirE2, VirE3, VirD2, VirD5, VirF) into the plant cell cytoplasm, as occurs in pseudomonads in the course of pilimediated plant tissue infection [90]. It should be noted that we dis covered cells connected by straight short structures (“bridges”) of unknown nature in a suspension of cross breeding agrobacteria (Fig. 2) [80]. Morphologically similar contact structures (30 40 nm in diameter) were described in 2002 by Kelly and Kado in contact between agrobacterial cells and Streptomyces lividans hyphae [8]. In addition, a number of studies have shown that plasmid DNA transfer between bacteria of one species as well as horizontal transfer between different bacterial species (genera) can proceed without direct contact (crossbreeding cells divided by a membrane) [91] or without direct (visible) contact of cells crossbreeding on solid medium [92]. In 1998, we showed using electronic immunomicroscopy that VirB1 protein was part of short structures on the cell pole, but it could not be found in long, thick contact structures (“bridges”) in conjugating agrobacteria [70]. Thus, at this point the mechanism of conjugative transfer of TDNA and virulence proteins after their leav ing the agrobacterial membrane channel and entering the recipient cell remains unclear; in particular, we do not know whether Tpili are used for TDNA and virulence protein delivery across the recipient cell membrane, or that some other structures are used for this purpose. Plant cell receptors involved in contact with agrobac teria. The range of plant hosts for agrobacteria is extremely wide [2, 93, 94], which seems to exclude any specificity at the stage of attachment. However, certain specificity of interaction at the stage of agrobacterial attachment to the plant surface may exist, as a glycopep tide with molecular weight of 2932 kDa with RGD sites BIOCHEMISTRY (Moscow) Vol. 78 No. 12 2013
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Fig. 2. Formation of “bridges” between conjugating agrobacteria of A. tumefaciens C58 strain and A. tumefaciens UBAPF2 strain (without Tiplasmid). Transmission electron microscopy. Magnification ×950,000; contrasting with 1% uranyl acetate. Co incubation temperature (30°C) and acetosyringone absence in the crossbreeding medium eliminates Tpili formation. The arrow indicates polysaccharide capsule on the bacterial surface.
for ricadhesin protein attachment has been discovered in the cell wall of pea root cells [95]. Plant vitronectinlike protein participates in agrobacterial attachment to carrot protoplasts. This protein is located in the plant cell wall; it is involved in different biological processes, such as plasma membrane adhesion to the cell wall, stretching of the pollen tube, and bacterial interaction with the plant [96]. In 1994, Zhu et al. found that the amino acid sequences of vitronectinlike protein and animal cell elongation factor (EF1α) were very similar; these pro teins were also shown to be immunologically identical [97]. Tstrand formation. Conformational changes in agrobacterial VirA protein activate the virG gene, whose product launches all the other vir genes [14, 98]. A histi dine residue (His474) of VirA protein is phosphorylated in response to plant phenolic compounds; then the phos phate is transferred to aspartic acid in position 52 of the Nterminal domain of the VirG protein. VirG specifical ly binds to the virbox, and virulence proteins are synthe sized as a result of virgene induction. Virulence proteins form the Tcomplex composed of ssTDNA and VirE2 and VirD2 proteins, and ensure its transfer. Mutations in the virD1 and virD2 genes lead to disruptions in TDNA formation, and a high level of virD1 and virD2 gene expression increases the number of formed Tcomplexes and frequency of transformation [99]. In 1992, two independent laboratories showed that VirD1 protein unwinds the DNA strand in the area of a 25bp repeat, and VirD2, being an endonuclease, becomes attached to DNA doublestrand and breaks in one of the chains [10, 100]. Furthermore, VirD2 can also be attached to the 5′end of TDNA [101, 102], and it has two signal sequences at the Cend, which allow it to iden tify the nuclear pore [102, 103]. VirD2 also provides T DNA incorporation into the recipient cell chromosome. Tstrand and virulence protein transfer into a recipi ent cell. Release of Tstrand from the agrobacterial donor
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cell. In 19961997, data appeared on Tstrand and VirE2 protein being transported independently from agrobacte rial cells [104, 105]. In addition to VirE2, virulence pro teins VirE1, VirE3, VirD2, VirD5, and VirF are trans ferred into recipient cells [106, 107]. Secretion of viru lence proteins belongs to VI (T6SS) type; in A. tumefa ciens it is regulated by the ExoRChvGChvI protein cas cade. It is interesting to note that acidic environment promotes degradation of ExoR protein, which physically blocks ChvG protein, whose depression activates the T6SS system [28]. Once attached to the recipient cell surface, agrobac teria transfer the complex of Tstrand and virulence pro teins from the donor to recipient cell during close contact of the partner cells [108], which suggests attributing it to a system of the 4thtype transfer (T4SS). Here are sever al examples of the T4S systems: transfer of plasmid DNA of different incompatibility groups (IncW, IncP, IncN); transfer of ptl operon from Bordetella pertussis [109]; the secretion system of interleukin8 inducing factor in Helicobacter pylori, which is homological to VirB4 protein from A. tumefaciens [110]. Previously, there was a view that Tstrand becomes covered with VirE2 protein in the bacterial cell [10, 111], but in 1998 Fullner showed the Tcomplex is formed in the recipient cell cytoplasm [73]; it was also found that the Tstrand and VirE2 protein were transported from agrobacteria independently [104, 105, 112]. Agrobacterial virB operon has 11 open reading frames; it encodes 11 proteins required for the formation of the membraneassociated export apparatus [10, 113]. Products of the virB2virB11 genes are absolutely required for virulence, while virulence of virB1 gene mutants was only attenuated [72, 114, 115]. The virB operon is probably evolutionarily related to other groups of bacterial genes whose products form membrane com plexes involved in protein secretion, DNA transfer, and pili assembly [86, 116]. Comparison of genetic elements of the Tra2 region of the RP4 plasmid and virB operon of Ti plasmid indicates certain similarities between the processes of DNA transfer in these two systems. The Tra2 region encodes 11 proteins involved in conjugative DNA transfer and pili formation. Six Tra2 proteins are very similar to VirB proteins; their membrane localization (i.e. hydrophobic regions) is particularly characteristic and similar [117]. The virB operon also shows a close rela tionship with the ptl operon of B. pertussis, which encodes products responsible for toxin protein export [109]. Perhaps the transport system for conjugation and TDNA originates from the proteinexporting system. Study of the location of VirB proteins showed mem brane localization of seven out of them [118, 119]. It was suggested that VirB proteins are associated in complexes and together with VirD4 protein form a channel for T DNA transfer across the bacterial membrane. In particu lar, VirB4 and VirB11 exhibit ATPase activity to provide
energy for Tstrand transfer. Both proteins are located on the inner membrane according to their presumed func tion [115, 116]. VirB11 protein is related in its amino acid sequence to TrbBprotein (from the Tra2 operon), which has similar enzymatic activity. Bacterial proteins similar to TrbB are usually membraneassociated and are gath ered into multiprotein complexes that serve for import or export of proteins or DNA. VirB4 protein is located in the inner agrobacterial membrane; it stabilizes VirB8 protein in the periplasm [121]. VirB5 protein is located in the membrane and periplasm, at the end and within Tpili, and is probably a binding protein between Tpili and recipient cell surface [81, 122]. However, if VirB5 protein is added to the medi um during transformation, the frequency of plant trans formation increases, while agrobacterial attachment to the plant surface remains unchanged [122]. VirB6 protein is the most hydrophobic of all VirB proteins. It contains six membranebound regions. It is assumed that VirB6 protein is required for the regulation of Tpili synthesis by interacting with VirB3, VirB5, and VirB7 [123]. VirB8 has no signal sequences, it includes a membranebound region, most charged amino acid residues are located at the Cterminal end, it is localized in the inner membrane [124, 125], and it can interact with VirB5 in Tpili biogenesis [121]. In 2009, Chandran et al. determined the structure of the virBdependent agrobacterial complex [126]. They found that 14 copies of each of the three VirB7, VirB9, and VirB10 proteins form a multiprotein complex in the agrobacterial outer membrane, a ring structure of 185 Å height, and width consisting of two layers surrounding a central chamber of 80 Å at the widest point and inner channel with diameter of 32 Å [126]. The crystal structure of this complex consisting of VirB10 (similar to TraF) is located in the outer membrane; it is identified as a pore formed by αhelices with diameter of 30 Å, which form a pore in the inner membrane [126, 127]. It was postulated that VirB9 in the pore structure can recognize and bind to the 5′end of DNA, being responsible for selective recog nition of DNA (TDNA or plasmids of IncQ compatibil ity group) during its passage through the pore, while VirB10 regulates secretion of TDNA and virulence pro teins through the pore [128]. In particular, the G272R mutation in VirB10, which is part of the channel provid ing ssDNA secretion (TDNA, plasmids of IncQ group), has no effect of the secretion of VirE2 protein from agrobacteria [127]. In 2011, Christie et al. demonstrated that the secre tion system (T4SS) can exist in two states: DNA and vir ulence protein secretion, and pili assembly [127]. When in the state of DNA and virulence protein secretion, the pili are formed short. These pili provide mechanical clo sure of the VirB7VirB9VirB10 channel in the outer membrane. When in the biogenesis state, long Tpili are formed starting from the basis located in the agrobacteri BIOCHEMISTRY (Moscow) Vol. 78 No. 12 2013
PROTEINS IN HORIZONTAL TRANSFER OF AGROBACTERIAL TDNA al outer membrane [127]. The architecture of the channel for TDNA transport of type 4 is not entirely clear, but there is some evidence that the G272R mutation affecting extracellular accumulation of Tpili pilin does not affect Tpili assembly. This indicates that Tpili are not required for the formation of VirB/VirD4 channel between donor and recipient cells [127]. VirD4 is not required for the formation of Tstrand or Tcomplex; it is the ATPase required for Tstrand transport [129]. VirE2 protein and its involvement in Tstrand transfer across the recipient cell membrane. VirE2 is the major vir ulence protein in agrobacteria; about 600 molecules of this protein are synthesized per cell [111]. In agrobacter ial cells, TDNAbinding sites of VirE2, located in the C domain [130], are specifically blocked by VirE1 chaper one protein, which prevents formation of aggregates of VirE2 and helps to keep it in the form required for trans port [131, 132]. As a result, Tcomplex cannot be formed in a bacterial cell and complexing takes place in the plant cell cytoplasm [1, 66]. VirE2 can interact not only with T DNA, but also with other ssDNAs in vitro (Fig. 3). The place and conditions of VirE2–VirE1 complex dissociation remain unknown, but it probably takes place in the plant cell. VirE2 protein, in addition to protecting singlestranded TDNA from the host cell endonucleas es, probably also performs some other functions, since RecA protein, which can bind singlestranded DNA, cannot completely replace VirE2 protein. This function is necessary for TDNA transfer across the membranes or for Tcomplex transport in the cytoplasm, because RecA can replace VirE2 at the stage of transport through the plant cell nuclear pore. In 2001, Hohn et al. found that VirE2 can interact with artificial lipid membrane, increasing its electrical conductivity in the presence of an electric field [133]. This observation was later confirmed in an independent study by Chumakov et al. [135]. As a result, a hypothesis was formulated about VirE2 being able to form complex es [134], which probably participate in the formation of pores for ssDNA transfer [135]. Pore durability in the presence of VirE2 affecting the membrane is 1.57 sec [134, 135], which theoretically allows for the passage of ssDNA of several thousand nucleotides long. For exam ple, the passage of TDNA composed of 1000 nucleotide residues requires about 2 sec if we assume the rate of T DNA passage to be comparable to the rate of plasmid DNA passage through E. coli protein pore during conju gation [136]. The reasons for the pore (channel) opening and its maintenance in an open state remain unknown, and it is certainly interesting to learn of them in the future. Purified recombinant VirE2 stimulates accumula tion of short synthetic oligonucleotides in HeLa line cells treated with compounds that support transmembrane transport [133], as well as in native HeLa cells [134], but not in pig embryo kidney cells (PEK) [137]. However, BIOCHEMISTRY (Moscow) Vol. 78 No. 12 2013
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20 nm
Fig. 3. ssDNA–VirE2 complex formation under in vitro condi tions. Average length of the complex was 208 ± 10 nm (50 meas urements); ssDNA (PCR product of gfp gene, 700 bp) to VirE2 ratio was 1 : 10. Transmission electron microscopy, contrasting with 2% uranyl acetate (from [134]).
ssDNA consisting of 200 nucleotide residues is accumu lated in native PEK cells in the presence of VirE2 in a clathrin, caveolinindependent mode more intensely when compared to control (Volokhina, personal commu nication). If in vivo Tstrand can use a pore consisting of VirE2 protein, then the inner diameter of this pore should be sufficient for its passage. Model calculations show that a complex of four VirE2 molecules may be located in the membrane and have an inner channel of up to 4.6nm diameter [134]. A channel of such size allows the passage of ssDNA in linear form with attached piloting VirD2 protein (its hydrodynamic diameter is about 4 nm) [134]. If TDNA is transferred through a pore formed by VirE2 protein in the recipient cell membrane, then the transfer should proceeds without the involvement of VirD2, because changes its signal sequence responsible for nuclear pore recognition does not affect Tcomplex accumulation in plant cell cytoplasm [103, 108]. VirE2 contains two signal sequences and interacts with plant cell cyclophilin [103, 138]. Endocytosismediated TDNA transfer across recipi ent cell membranes. Plant cells are known to be capable of endocytosis (absorption of compounds through mem brane invagination followed by transport and absorption in the cytoplasm) [139, 140]. At this point, we cannot exclude the possibility of TDNA penetration into the plant cell after secretion from the recipient cell by endo
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cytosis, since artificial stimulation of endocytosis by PEG6000 increases plant transformability by DNA [141]. Plant and animal cell cytoskeleton performs multiple functions, including involvement in the transport of mol ecules into and out of cells. In the case of damage or divi sion, a plant cell synthesizes materials for the construc tion of cell wall. Cytoskeleton is formed for the transport of compounds to the damage site or the place of cell wall construction. Chromosome regions controlling these processes are despiralized. Probably these facts can be the basis for searching for Tcomplex interaction with cytoskeleton elements. Actin filaments (microfilaments) and microtubules are the main components of the cyto plasmic network of eukaryotic cells. Electron microscopy data indicate that actin filaments form a double helix (4 nm in diameter) capable of interacting (associating) with many cell proteins. Interaction of pathogenic bacte ria (Chlamydiae [142], E. coli [143], Salmonella [144]) and symbiotic bacteria (Rhizobium etli) [145] with the host cell surface causes reorganization of its cytoskeleton, which can lead to transfer of the nucleus to the place of bacterial invasion. It should be noted that the nucleus of a damaged cell is located closer to the cell wall, which can significantly accelerate TDNA transfer to the nucleus. Light is one of the most important factors affecting the state of the endoplasmic network, the cytoskeleton. Even minor photosynthetic pauses for 23 days affect the state of endoplasmic reticulum and cause its reduction and reorganization [146]. Agrobacterial transformation of intact tissues of tobacco seedlings does not proceed in the absence of light for 23 days [35]. Perhaps the absence of light causes degradation of endoplasmic reticulum and/or cytoskeleton, as indicated by Gamaley [147]. An actin2 mutant of arabidopsis is resistant to agrobacterial trans formation [148]. Plant control of TDNA transfer. How does Tcomplex enter the plant nucleus? To integrate into the plant chro mosome, the Tstrand has to get to the nucleus, and this means it needs to cross the nuclear membrane via a nuclear pore. Proteins of over 40 kDa need signal sequences for nuclear pore recognition so that transfer through the nuclear pore can proceed. The nuclear mem brane has special receptors that recognize such signals. These receptors are responsible for nuclear localization of such proteins, and the nucleuslocated receptor importin α is responsible for the transfer of plant proteins with sig nal sequences through the nuclear pore. After getting through the plant cell membrane, TDNA is covered with VirE2 protein and Tcomplex is formed. The Tcomplex, with the aid of VirD2 piloting protein, which has sequences for nuclear pore recognition, is transferred from the outer membrane to the nucleus of the plant cell; in the cytoplasm it interacts with cyclophilin proteins [149]. Changes in one of the signal sequences of VirD2 cause problems in Tcomplex transport into the nucleus [4].
VirD2 protein interacts with several cyclophilins in a plant cell [148] and with all tested importin isoforms, CAK2Ms kinase, TATAbinding proteins, and PP2C phosphatase [150]. Importin and PP2C phosphatase interact with VirD2 at the stage of Tcomplex transfer to the nucleus, while interaction between VirD2 and CAK2Ms kinase and TATAbinding proteins occurs at the stage of Tstrand interaction with the recipient cell chromatin [150]. It appears that Tcomplex, guided by piloting VirD2 protein, can be transferred to the nucleus by α/β importin [138], which can interact with cytoskele ton microtubules and microfilaments in vitro. Treatment with cytochalasin B leads to depolymerization of actin microfilaments, violating the connection of importin α to cytoskeleton [151]. The plant protein VIP1, which can bind to VirE2 protein, is required for its import into nucleus and for agrobacterial virulence [137]. VirE2 can not interact directly with importin α; the interaction pro ceeds via adaptor protein VIP1 [152]. VirF protein, attaching to VIP1, destabilizes the VIP1–VirE2 complex by proteolysis [153]. In turn, VIP1 can interact with nucleosome histones [154] and karyopherin α [152]. TDNA incorporation into plant cell chromosome. VirD2 probably initiates transfer and ensures entry of the TDNA 5′end into the nucleus. The leading role of the 5′end may be related to translocation of singlestranded nucleic acids through the nuclear pore. Nuclear import of TDNA is connected to cellular processes that involve transport proteins [152]. Before being incorporated into a recipient cell chromosome, agrobacterial protein VirF releases Tcomplex, which is part of a complex with nucleosome, through VIP1 protein from the covering VirE2 protein [154]. Magori and Citovsky [155] discuss three models of TDNA integration: repair of single strand breaks; repair of doublestrand breaks; single strand repair dependent on microhomology sites. T DNA mainly integrates into plant DNA, which is in de spiralized state. There piloting VirD2 protein, having properties of DNase, breaks one of plant DNA strands in transcribing sites. Next, the TDNA 3′end finds homol ogy with a complementary DNA region of the plant chro mosome. In the next stage, the second DNA strand is composed with the help of plant repair enzymes [154]. A model has also been suggested according to which ssT DNA becomes doublestranded prior to incorporation [155]. In the case of TDNA integration in yeast, two protein types are involved in the process of repair. These proteins participate in the repair of both homologous and nonhomologous recombination of doublestranded DNA [155]. The mechanism of TDNA incorporation probably differs significantly from the mechanism of incorporation of retroviruses; plant proteins of damaged DNA repair are involved. This hypothesis was confirmed by experiments on plants sensitive to UV and γradiation that had reduced ability for TDNA integration [156]. It was BIOCHEMISTRY (Moscow) Vol. 78 No. 12 2013
PROTEINS IN HORIZONTAL TRANSFER OF AGROBACTERIAL TDNA found that after incorporation, the TDNA was shortened by 1070 nucleotide pairs. In this respect, the mechanism of TDNA incorporation is similar to the mechanism of DNA repair in yeasts [157] and humans. There is some evidence confirming this similarity [158]. Analysis of a plant DNA sequence at the site of TDNA incorporation has shown that VirD2 finds similar sequences and becomes covalently attached to them. The left end usual ly loses more nucleotides (up to 50) than the right end in the course of incorporation. It is due to this observation that Hohn et al. suggested that VirD2 protects the right 5′end of TDNA and incorporate it into the plant chro mosome. VirD2 was shown to participate in TDNA 5′ end ligation into the plant chromosome [158]. Inactivation of VirD2 does not prevent TDNA incorpo ration, which indicates that the 5′end ligation is inde pendent from VirD2. In connection to this, it was sug gested that the first bonds are established by the TDNA 3′end by finding corresponding homology in the genome [158]. The efficiency of TDNA incorporation was simi lar in the absence of VirE2 protein, indicating independ ence of this process from the involvement of VirE2 and VirE2 in other processes – protection of ssDNA, entering the nucleus, and transfer across the membrane of the plant cell endoplasmic reticulum. Furthermore, VirE2 interacts with the Nterminal end of VIP1, whose Cter minal end interacts with chromatin [159]. Prior to T DNA integration, agrobacterial VirF protein participates in chromatin modification [159]. This protein degrades VirE2 from the Tcomplex before TDNA integration into the host chromosome [153]. Overexpression of Saccharomyces cerevisiae Rad54 protein, which influ ences chromatin, in Arabidopsis thaliana results in increased frequency of agrobacterial transformation of plants [160]. Absence of histoneacetylating yeast pro teins also increases the frequency of TDNA integration [160]. However, many issues related to the mechanism of TDNA integration remain not fully understood, in par ticular, the precise role of chromatin and host cell repair proteins in TDNA integration. During the 25 years that have passed since its discov ery [161], there has been considerable progress achieved in understanding the moleculargenetic mechanism ensuring agrobacterial TDNA transfer and incorpora tion into the genome of eukaryotic cells; this phenome non has been also widely used in agriculture. However, many aspects of this process remain unknown or exist at the level of hypotheses. Nevertheless, it should be stated that the main agrobacterial and recipient cell proteins providing horizontal transfer of the TDNA–protein complex have been described, and their role in the various stages of interaction of the partners (exchanging of sig nals, attaching, establishing contact, TDNA leaving the bacterial cell, incorporation into recipient cell genome) have been determined. Pathways for transfer of agrobac BIOCHEMISTRY (Moscow) Vol. 78 No. 12 2013
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terial ssDNA into a recipient cell remain incompletely established. In particular, the role of Tpili and agrobac terial VirE2 protein in conjugative transfer of agrobacter ial DNA between donor and recipient cells is being clari fied. Further research is needed to understand the mech anism of movement of Tcomplex in the recipient cell cytoplasm and Tstrand integration into the eukaryotic cell genome. This work was supported in part by the Russian Foundation for Basic Research (grant 110401331a) and the Ministry of Education and Science of the Russian Federation (agreements 8592, 8728).
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