Protein apparatus for horizontal transfer of agrobacterial T-DNA to eukaryotic cells

T�DNA 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 (tumor�inducing) or Ri (root hair�

inducing) fragment of T�DNA 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 T�DNA 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 [6�8]. T�DNA 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, T�DNA cannot be transferred again since it has no

genes responsible for transfer. T�DNA is transferred

polarly; deletion of the right border disrupts the transfor�

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 T�DNA. The vir�region (vir, virulence�

inducing region, 35 kb), located on the Ti plasmid, is not

part of T�DNA 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 Ti�plasmid, 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 T�DNA 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 two�component sys�

tem of VirA–VirG proteins, launching protein synthesis

in the vir�region. Then a fragment of T�DNA (T�strand)

is cut from the Ti�plasmid (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 T�strand and virulence proteins are

transferred across the bacterial and plant cell membranes

ISSN 0006�2979, Biochemistry (Moscow), 2013, Vol. 78, No. 12, pp. 1321�1332. © Pleiades Publishing, Ltd., 2013.

Original Russian Text © M. I. Chumakov, 2013, published in Biokhimiya, 2013, Vol. 78, No. 12, pp. 1670�1683.

REVIEW

1321

Abbreviations: ssDNA, single�stranded DNA; T�DNA, trans�

ferred single�stranded DNA (transfer DNA).

Protein Apparatus for Horizontal Transferof Agrobacterial T�DNA 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) 970�383; E�mail: [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 T�DNA–protein complex. Specifically, it considers the early stages of the interactions of partners (signal exchange,

attachment, close contact); T�DNA release from bacterial cells; channel formation for the transfer of ssDNA between the

partners; transfer of agrobacterial T�DNA 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 T�pili 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, T�DNA, virulence proteins, conjugation, T�pili, VirE2

1322 CHUMAKOV

BIOCHEMISTRY (Moscow) Vol. 78 No. 12 2013

via a virB�dependent channel (Fig. 1, stage 3). The T�

complex is then formed in the recipient cell cytoplasm:

one T�strand is covered with 600 molecules of VirE2 pro�

tein (Fig. 1, stage 4). The transport system of the host cell

is involved in T�complex 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 T�DNA genes are expressed

(Ti�plasmid: 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 accA�accG genes of the T�DNA Ti�plasmid

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 vir�genes 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�

Fig. 1. General scheme of T�DNA transfer from agrobacteria to plants (modified from [22]): 1) activation of VirA–VirG two�component sig�

naling system by low molecular weight components of the plant cell wall; 2) T�DNA excision, T�strand formation; 3) independent T�strand

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) T�strand covering with VirE2 protein and formation of T�complex; 5) T�complex transfer through the plant cell

cytoplasm, entering the plant cell nucleus; 6) T�DNA incorporation into the plant chromosome.

Plant cell

Ti plasmid

proteinsproteins

T�complex

Nuclear pore

RNA

Nucleus

PROTEINS IN HORIZONTAL TRANSFER OF AGROBACTERIAL T�DNA 1323


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 [30�32], 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 T�DNA 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 surface�located 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 1,2�β�glucan) to complete block�

ing of T�DNA 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 T�DNA 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�

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)�β�glucan, 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 T�DNA

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, non�covalently bound protein sub�

units forming spirally twisted structures in the form of

long (1�5 nm) hollow tubes or non�hollow 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.

1324 CHUMAKOV


By now, there are three pili types identified in

agrobacteria: a) vir�, tra�independent type (adhesive pili)

[63]; b) vir�independent, tra�dependent pili involved in

mobility and plasmid DNA transfer between agrobacteria

[64]; c) vir�dependent pili type (T�pili) 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�, tra�independent

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 cross�breeding

agrobacterial cultures with induced tra�genes and previ�

ously blocked T�pili synthesis showed the formation of

thin straight fibrils (tra�dependent 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 tra�dependent pili connect

cross�breeding agrobacterial cells, which were destroyed

after SDS�treatment; they probably also participate in

tra�dependent T�plasmid transfer between agrobacteria.

virB1�dependent structures. Mutation in virB1 gene

reduces virulence, but it does completely block T�DNA

transfer [67]. In 1996, Nester et al. presented data on the

C�terminal part of VirB1 protein having a transglycosy�

lase function. This C�terminal 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 C�terminal, 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

T�pili [71].

virB�dependent pili (T�pili). 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 T�pili 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 T�pili protein. VirB2 pro�

tein has a large number (100 of 121) of amino acids con�

taining hydrophobic regions. The N�terminal 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 (T�pili) [72, 78�80], which consist

mainly from VirB2 protein subunits with molecular

weight of 6.5�7.2 kDa [72, 78, 80]. In addition to the

major VirB2 protein, T�pili include alsoVirB5 and VirB7

proteins located at the end and at the base of pili, respec�

tively [81, 82]. T�pili 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 Ti�plasmid 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 T�pili function being similar to the func�

tion of similar F�pili of E. coli seems to be quite reason�

able. However, the mechanism of contact involving

agrobacterial T�pili remains unclear. In particular, it is

unclear what might be the role of T�pili in T�DNA trans�

fer into a plant and whether observed structures are

involved in bringing together the membranes of cross�

breeding agrobacteria, or that T�DNA is transferred via a

T�pili channel.

Are vir�dependent agrobacterial surface structures

involved in contact with the recipient cell surface?

Kurbanova et al. have shown that acetosyringone�mediat�

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 T�pili synthesis on agrobacterial adhesion

indicates that T�pili do not play a significant role in the

attachment to the plant cell surface [87].

Possible role of T�pili in T�strand 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 T�pili channel was made by

Kado [78].

According to electron microscopy estimation, the

outer diameter (8.5 nm) of conjugative (F) pili in E. coli



[88] is similar to the outer diameter (8�10 nm) of

agrobacterial T�pili [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 T�pili 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 T�strand

(T�DNA with a piloting VirD2 protein) is 4 nm, which

seems to be not enough for T�stand passage in the con�

jugative pili in a normal (non�retracted) 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 pili�mediated

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 (cross�breeding cells divided by a

membrane) [91] or without direct (visible) contact of

cells cross�breeding 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 T�DNA 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 T�pili are used for T�DNA 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 29�32 kDa with RGD sites

for ricadhesin protein attachment has been discovered in

the cell wall of pea root cells [95]. Plant vitronectin�like

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 vitronectin�like protein and animal cell

elongation factor (EF�1α) were very similar; these pro�

teins were also shown to be immunologically identical

[97].

T�strand 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

N�terminal domain of the VirG protein. VirG specifical�

ly binds to the vir�box, and virulence proteins are synthe�

sized as a result of vir�gene induction. Virulence proteins

form the T�complex composed of ssT�DNA and VirE2

and VirD2 proteins, and ensure its transfer. Mutations in

the virD1 and virD2 genes lead to disruptions in T�DNA

formation, and a high level of virD1 and virD2 gene

expression increases the number of formed T�complexes

and frequency of transformation [99].

In 1992, two independent laboratories showed that

VirD1 protein unwinds the DNA strand in the area of a

25�bp repeat, and VirD2, being an endonuclease,

becomes attached to DNA double�strand and breaks in

one of the chains [10, 100]. Furthermore, VirD2 can also

be attached to the 5′�end of T�DNA [101, 102], and it has

two signal sequences at the C�end, which allow it to iden�

tify the nuclear pore [102, 103]. VirD2 also provides T�

DNA incorporation into the recipient cell chromosome.

T�strand and virulence protein transfer into a recipi�ent cell. Release of T�strand from the agrobacterial donor

Fig. 2. Formation of “bridges” between conjugating agrobacteria

of A. tumefaciens C58 strain and A. tumefaciens UBAPF�2 strain

(without Ti�plasmid). Transmission electron microscopy.

Magnification ×950,000; contrasting with 1% uranyl acetate. Co�

incubation temperature (30°C) and acetosyringone absence in

the cross�breeding medium eliminates T�pili formation. The

arrow indicates polysaccharide capsule on the bacterial surface.

1326 CHUMAKOV


cell. In 1996�1997, data appeared on T�strand 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 ExoR�ChvG�ChvI 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 T�strand 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 4th�type 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 interleukin�8 inducing factor in

Helicobacter pylori, which is homological to VirB4 protein

from A. tumefaciens [110].

Previously, there was a view that T�strand becomes

covered with VirE2 protein in the bacterial cell [10, 111],

but in 1998 Fullner showed the T�complex is formed in

the recipient cell cytoplasm [73]; it was also found that

the T�strand 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 membrane�associated export apparatus [10, 113].

Products of the virB2�virB11 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 T�DNA

originates from the protein�exporting 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 T�strand 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 TrbB�protein (from the Tra2 operon), which

has similar enzymatic activity. Bacterial proteins similar

to TrbB are usually membrane�associated and are gath�

ered into multi�protein 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 T�pili, and is probably a

binding protein between T�pili 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 membrane�bound regions. It is

assumed that VirB6 protein is required for the regulation

of T�pili synthesis by interacting with VirB3, VirB5, and

VirB7 [123]. VirB8 has no signal sequences, it includes a

membrane�bound region, most charged amino acid

residues are located at the C�terminal end, it is localized

in the inner membrane [124, 125], and it can interact

with VirB5 in T�pili biogenesis [121].

In 2009, Chandran et al. determined the structure of

the virB�dependent 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 α�helices 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 (T�DNA or plasmids of IncQ compatibil�

ity group) during its passage through the pore, while

VirB10 regulates secretion of T�DNA 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 (T�DNA, 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 VirB7�VirB9�VirB10 channel in the outer

membrane. When in the biogenesis state, long T�pili are

formed starting from the basis located in the agrobacteri�



al outer membrane [127]. The architecture of the channel

for T�DNA transport of type 4 is not entirely clear, but

there is some evidence that the G272R mutation affecting

extracellular accumulation of T�pili pilin does not affect

T�pili assembly. This indicates that T�pili are not required

for the formation of VirB/VirD4 channel between donor

and recipient cells [127]. VirD4 is not required for the

formation of T�strand or T�complex; it is the ATPase

required for T�strand transport [129].

VirE2 protein and its involvement in T�strand transferacross 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, T�DNA�binding 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, T�complex 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

single�stranded T�DNA from the host cell endonucleas�

es, probably also performs some other functions, since

RecA protein, which can bind single�stranded DNA,

cannot completely replace VirE2 protein. This function is

necessary for T�DNA transfer across the membranes or

for T�complex 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 1.5�7 sec

[134, 135], which theoretically allows for the passage of

ssDNA of several thousand nucleotides long. For exam�

ple, the passage of T�DNA 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,

ssDNA consisting of 200 nucleotide residues is accumu�

lated in native PEK cells in the presence of VirE2 in a

clathrin�, caveolin�independent mode more intensely

when compared to control (Volokhina, personal commu�

nication).

If in vivo T�strand 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 4.6�nm

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 T�DNA 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 T�complex

accumulation in plant cell cytoplasm [103, 108]. VirE2

contains two signal sequences and interacts with plant cell

cyclophilin [103, 138].

Endocytosis�mediated T�DNA 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 T�DNA penetration into the

plant cell after secretion from the recipient cell by endo�

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]).

20 nm

1328 CHUMAKOV


cytosis, since artificial stimulation of endocytosis by

PEG�6000 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 T�complex 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 T�DNA 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 2�3 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 2�3 days [35]. Perhaps the absence of

light causes degradation of endoplasmic reticulum and/or

cytoskeleton, as indicated by Gamaley [147]. An actin�2

mutant of arabidopsis is resistant to agrobacterial trans�

formation [148].

Plant control of T�DNA transfer. How does T�complex

enter the plant nucleus? To integrate into the plant chro�

mosome, the T�strand 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 nucleus�located 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, T�DNA is covered with

VirE2 protein and T�complex is formed. The T�complex,

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 T�complex transport into the nucleus [4].

VirD2 protein interacts with several cyclophilins in a

plant cell [148] and with all tested importin isoforms,

CAK2Ms kinase, TATA�binding proteins, and PP2C

phosphatase [150]. Importin and PP2C phosphatase

interact with VirD2 at the stage of T�complex transfer to

the nucleus, while interaction between VirD2 and

CAK2Ms kinase and TATA�binding proteins occurs at

the stage of T�strand interaction with the recipient cell

chromatin [150]. It appears that T�complex, 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].

T�DNA incorporation into plant cell chromosome.VirD2 probably initiates transfer and ensures entry of the

T�DNA 5′�end into the nucleus. The leading role of the

5′�end may be related to translocation of single�stranded

nucleic acids through the nuclear pore. Nuclear import of

T�DNA is connected to cellular processes that involve

transport proteins [152]. Before being incorporated into a

recipient cell chromosome, agrobacterial protein VirF

releases T�complex, 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 T�DNA integration: repair of single�

strand breaks; repair of double�strand 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 T�DNA 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 double�stranded prior to incorporation

[155]. In the case of T�DNA integration in yeast, two

protein types are involved in the process of repair. These

proteins participate in the repair of both homologous and

non�homologous recombination of double�stranded

DNA [155].

The mechanism of T�DNA 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 γ�radiation that had

reduced ability for T�DNA integration [156]. It was



found that after incorporation, the T�DNA was shortened

by 10�70 nucleotide pairs. In this respect, the mechanism

of T�DNA 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 T�DNA 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 T�DNA and incorporate it into the plant chro�

mosome. VirD2 was shown to participate in T�DNA 5′�end ligation into the plant chromosome [158].

Inactivation of VirD2 does not prevent T�DNA 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 T�DNA

3′�end by finding corresponding homology in the genome

[158]. The efficiency of T�DNA 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 N�terminal end of VIP1, whose C�ter�

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 T�complex before T�DNA 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 histone�acetylating yeast pro�

teins also increases the frequency of T�DNA integration

[160]. However, many issues related to the mechanism of

T�DNA integration remain not fully understood, in par�

ticular, the precise role of chromatin and host cell repair

proteins in T�DNA integration.

During the 25 years that have passed since its discov�

ery [161], there has been considerable progress achieved

in understanding the molecular�genetic mechanism

ensuring agrobacterial T�DNA 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 T�DNA–protein

complex have been described, and their role in the various

stages of interaction of the partners (exchanging of sig�

nals, attaching, establishing contact, T�DNA leaving the

bacterial cell, incorporation into recipient cell genome)

have been determined. Pathways for transfer of agrobac�

terial ssDNA into a recipient cell remain incompletely

established. In particular, the role of T�pili 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 T�complex in the recipient cell

cytoplasm and T�strand integration into the eukaryotic

cell genome.

This work was supported in part by the Russian

Foundation for Basic Research (grant 11�04�01331a) and

the Ministry of Education and Science of the Russian

Federation (agreements 8592, 8728).

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