BASIC PROCESSES UNDERLYING Agrobacterium Mediated DNA Transfer To Plant Cells

ANNUAL
REVIEWS Further
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Annu. Rev. Genet. 1988. 22:1-30
Copyright © 1988 by Annual Reviews 1nc. All rights reserved
BASIC PROCESSES UNDERLYING

AGROBACTERIUM-MEDIATED DNA
TRANSFER TO PLANT CELLS
Annu. Rev. Genet. 1988.22:1-30. Downloaded from www.annualreviews.org
Patricia Zambryski
by Laurentian University on 03/11/13. For personal use only.
Division of Molecular Plant Biology, Hilgard Hall, University of California,

Berkeley, California 94720
CONTENTS
INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
GENERAL CONCEPTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
ACTIVATION AND EXPRESSION OF GENES UNDER VIR CONTROL. . . . . . . . . . . . . . 3
GENERATION OF A TRANSFERABLE T-DNA COpy. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... 6
Structural requirements for T-DNA transfer................................................. 6
The T-DNA transfer intermediate: circular. linear. single- or double-stranded? .... 9
The T-strand as the transfer intermediate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Protein produ cts which function to generate transferable T-DNA copies . . . . . . . . . . . . . 12
Model for the mechanism of T-DNA transfer..... . . . ...... . . . . . . .. . . . . . ..... . . . ... . . . . . . . . . 15
Bacterial conjugation versus T-DNA transfer.. .. . . . . .. . . . . . .. . . . . . . . .. .. .
. . . . . . . . . . . . . . . . . 16
INTEGRATION O F THE T-DNA INTO THE PLANT GENOME. . . . . . . . . . . . . . . . . . . . . . . . . 19
SUMMARY AND PROSPECTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
INTRODUCTION
Agrob acterium tumefaciens is a soil phytopathogen that genetically trans

forms plant cells. This fact forms the basis of both the renown and utility of
the Agrobacterium system. In nature, this transformation results in crown gall
tumors, an agronomically important disease which affects most di
cotyledonous plants. The disease is the direct result of the transfer of a
particular DNA segment, the T-DNA (transferred DNA) , from the tumor
inducing (Ti) plasmid within the bacterium to the plant cell genome where its
integration and expression result in the crown gall phenotype (reviewed in 50,
57, 114). An understanding of the basic principles involved in this
transformation process has led to the design of modified, nononcogenic
0066-4197/88/1215-0001$02.00
2 ZAMBRYSKI
Agrobacterium strains which can now be used to transfer any DNA of interest
to plant cells without interfering with their normal growth and regeneration
properties.
While Agrobacterium can be used routinely as a vector to transfer DNA to
plant cells without need to understand the underlying mechanisms involved,
the "biology" of the system presents a number of interesting topics, from how
bacterial plant cell recognition occurs in the complex soil microenvironment
to how the T-DNA determines the neoplastic crown gall phenotype. The steps
inbetween plant cell recognition and transformation represent the T-DNA
transfer process; this aspect is presented here. For the reader interested in the
more physiological aspects, other reviews present the evidence that the
T-DNA encodes novel enzymatic pathways for plant growth hormones whose
overproduction results in the perturbation of normal plant cell growth and
differentiation (40, 62). For those with more applied incentives and goals,
numerous reports summarize and offer Ti-based vectors and methodologies
for directed transfer of DNA of interest to plant cells (57, 72, 113).
GENERAL CONCEPTS
Agrobacterium carries three genetic components required for plant cell

transformation. Two components, the T-DNA and the virulence (vir) region,
are located on the large (roughly 200 kbp) Ti plasmid (for example, 20, 38).
The T-DNA is the DNA segment that is transferred from Agrobacterium to
the plant cell. In contrast to transposable elements that can move repeatedly,
the T-DNA is stable following its integration into the plant genome; the
T-DNA, unlike transposons, does not encode the products that mediate its
transfer (54, 112). The vir region is a 35 kbp non T-DNA linked region which
provides most of the trans-acting functions for T-DNA transit. The vir region
is organized into six complementation groups that are either absolutely es
sential for (virA, virB, virD, and virG) or that enhance the efficiency of (virC
and vir£) plant cell transformation (82). The vir region is the master switch
for the transformation process since the expression of the vir loci is regulated
to occur only when Agrobacterium is in the presence of susceptible plant cells
(80-82).
The third bacterial component of the T-DNA transfer process resides in the
Agrobacterium chromosome. Three chromosomal virulence loci, chvA and
chvB (23, 24) and pscA (89), encode products involved in the binding of
Agrobacterium to plant cells during the infection process. The chvA and chvB
are located on a 15.5 kbp segment of the Agrobacterium chromosome. The
8.5 kbp chvB portion encodes a membrane protein of approximately 235
kilodaltons (kd) that acts an an intermediate in the synthesis of cyclic /3-1,2-
glucan (77, 115) , and chvA may encode a transport function (E. Nester,
AGROBACTERIUM DNA TRANSFER 3
personal communication) . The pscA locus is approximately 3.0 kbp and is

required for the synthesis of the major neutral and acidic extracellular poly
saccharides (89) . All three loci have dramatic effects on the surface composi
tion of the bacterial cells, but it is not known exactly how their products
enhance attachment to plant cells. Chromosomal virulence loci are con
stitutively expressed, perhaps reflecting an additional role of surface polysac
charides in mediating general bacterial-plant cell interactions. Other soil
bacteria have genes that are functionally and structurally related to the Agro
bacterium chromosomal virulence loci; the ndvA, ndvB, and exoC loci of
Rhizobium meliloti, required to initiate nitrogen fixing nodule development,
are related to the chvA , chvB, and pscA loci of Agrobacterium (26, 60) .
ACTIVATION AND EXPRESSION OF GENES UNDER

VIR CONTROL
The discovery of how Agrobacterium has evolved to control and activate the
T-DNA transfer process reveals both the logic and conservatism of biological
processes. Initially , it was assumed that Agrobacterium only infected wound
ed plants because their cells presented less of a physical barrier to penetration
than did unwounded cells which have thick cell walls. However, wounded but
otherwise metabolically active cells excrete low molecular weight signal
molecules recognized specifically by Agrobacterium to induce vir gene ex
pression, thereby to activate T-DNA transfer (80, 8 1 ) . The vir inducing
molecules were purified from the culture media of tobacco cells and identified
as acetosyringone (AS), and hydroxy-acetosyringone (OH-AS) (80). AS and
OH-AS most resemble products of phenylpropanoid metabolism, the major
pathway to produce plant secondary metabolites, lignins, and flavonoids;
such compounds are important to plants under stress or injury (15). Thus, the
activation of vir gene expression makes sense. In nature Agrobacterium only
infects wounded plants, and the production of AS and OH-AS is stimulated by
wounding . Also AS can act as a chemical attractant for Agrobacterium in
vitro, suggesting that its presence at plant wound sites in nature may serve a
chemotactic role (4). Other soil bacteria also use plant signals to initiate
specialized interactions with plant cells. Rhizobium specific genes involved in
the formation of nitrogen-fixing nodules are induced by flavonoid molecules
of their plant hosts (29, 67, 7 1 ) .
The induction of vir gene expression was shown to be at the level of
transcription ( 1 6, 42, 83). How does Agrobacterium link the recognition of
the plant phenolics to the expression of vir specific transcripts? At least two
components are required, extracellular recognition and intracellular response .
These two steps are proposed to be mediated by the products of virA and virG,
4 ZAMBRYSKI
and mutants in these genes are severely attenuated (virA ) or totally deficient
(virG) for induction of the other vir(B,C,D, E,) loci (83) .Current speculation
on how virA and virG function to activate the expression of the other vir loci is
based on the fact that each polypeptide shares significant amino acid homolo
gy to other pairs of bacterial proteins which act as sensor-regulators of gene
expression in response to environmental stimuli (73 , see also Albright &
Ronson, Annu . Rev. Gen. 23, submitted). For example, the envZI ompR,
ntrBI ntrC, phoRI phoB pairs of genes allow E. coli to respond to changes in
osmolarity, nitrogen and phosphate concentrations, respectively. The first
gene in each pair is a membrane protein that directly senses the external
environment. The second :nember acts as a positive activator of the transcrip
tion of gene(s) representing the cell's response to the stimulus. How the
presence of the signal is transduced from the sensor to the regulator protein is
best known in the ntrBlntrC system; ntrB converts ntrC between active and
inactive forms by phosphorylation and dephosphorylation (52, 63) .
Thus, A grobacterium has adapted a preexisting bacterial process to serve
its unique interaction with plant cells. By analogy to the E. coli systems, virA
most likely functions as a chemoreceptor which senses the presence of plant
wound factors such as AS and transmits this information to the inside of the
bacterium (potentially by modification of the virG protein ). Like its
homologous E.coli counterparts, virA contains a transmembrane domain, and
cell fractionation experiments have localized virA to the inner membrane (56) .
How virG transcriptionally activates vir genes is unknown . The inducible vir
loci lack a minus 35 consensus sequence in their promoter regions. Yet they
possess other hexanucleotide sequences in common which might function as
cis-regulatory sequences for vir specific transcription ( 1 6) mediated by the
virG product alone or in concert with RNA polymerase holoenzyme.
The regulation of virA is simple; it is constitutively expressed. Thus, it is
available to respond to plant signals. The levels of induction of virA gene
fusions as well as the virA transcript itself are identical, whether A grobacte
rium is grown vegetatively or in the presence of plant cells (83) . In contrast,
the regulation of virG is complex (83). There are two distinct virG messages
that differ at their 5' termini. A constitutive message is produced under both
vegetative and plant induced conditions; and an induced message is produced
only during plant induction. The latter message is 50 bases longer at its
5' terminus, and it is present at ten-fold higher levels than is the constitutive
transcript. virG expression is further complicated by the fact that virG induc
tion by plant cells is regulated at two distinct levels. One level is (apparently)
independent of the presence of a wild-type copy of virG, since virG mutants
show a significant (roughly 25%) level of plant-induced virG transcription.
The second level of virG induction is, as with other inducible vir loci,
dependent on intact virG; thus , virG positively autoregulates its own expres
sion (83). Further, virG is produced at high levels, an unusual property for an
activator protein (83). Large quantities may be needed if virG is not very
efficient in its role . Indeed, a so-called supervirulent strain of Agrobacterium
produces even more elevated levels of virG product (45).
Figure 1 summarizes the vir gene products of the octopine Ti plasmid . Less
detailed information exists for the vir region of the nopaline Ti plasmid;
however, the high homology betwccn octopine and nopaline vir regions
shown by heteroduplex analysis (27) predicts that their polypeptide products
will prove to be highly similar. The complete nucleotide sequence is available
for each of the loci, and the predicted sizes of their specified proteins are
given, along with their most probable functions where these are known. virA
and virG are the only monocistronic vir loci, specifying polypeptides of 70 kd
(56) and 30 kd (6 1 , 69 , 1 04) . virC is 2.0 kbp and its nucleotide sequence
predicts two polypetides of 26 kd and 23kd ( 109); this is the only vir locus that
at present has no assigned or probable function. The sequence of the 2.0 kbp
virE region predicts two polypetides of 7 . 0 kd and 60.5 kd ( 1 05; see also 35) .
The sequence of the 4 . 5 kbp virD predicts four polypeptides of 16 kd, 47 kd,
2 1 kd, and 75 kd (43 , 68, 1 1 0) . virB is the largest vir locus, roughly 9 . 5 kbp
in size. The nucleotide sequence of the virB region predicts eleven open
reading frames specifying from 5' to 3 ' polypeptides of 25 . 9 kd, 1 2 kd, 1 1 . 7
kd, 2 l . 6 kd, 65 . 7 kd, 23 . 5 kd , 3 l . 7 kd, 5 .9 kd , 26. 1 kd, 7 2 . 7 kd, and 38 kd
( 1 02). However, these data do not agree with genetic studies using AS
induced Agrobacterium which identified polypeptides of 33 kd, 80 kd , and 25
kd encoded by the N-terminal half of the locus (28) . This apparent conflict
remains to be resolved. The sizes of the virB polypeptides shown in Figure 1
are taken from the in vivo data. Only the three virB polypeptides and the
larger of the two virE polypeptides have been directly observed in vir induced -
Agrobacterium (28); all the other protein assignments are from sequencing
data as well as expression of cloned vir specific sequences in E.coli.
The relative amounts and intracellular locations of some of the vir proteins
have also been determined; these assignments either confirm, or stimulate,
predictions for vir protein function. For example , the virA protein is localized
to the membrane (56) as would be predicted for a sensor of plant signal
molecules. The most abundant vir proteins produced in AS-induced Agrobac
terium are virE and virB specific polypeptides (28). virE encodes a single
stranded (ss) DNA binding protein ( 1 1 , 1 3 , 1 7) which could stoichiometrical
ly cover single stranded T-DNA molecules; this fits with its relative abun
dance in AS induced cells. Fractionation of this polypeptide to either the
membrane or soluble fraction (28) also suggests a role in T-DNA transfer at
these two locations. It is interesting that while the function(s) of the virB
products are unknown, their fractionation to the cell envelope (28) predicts
that they play a role in directing T-DNA transfer events which occur at the
bacterial cell surface , and their high levels of production further suggest a
structural rather than an enzymatic function.
6 ZAMBRYSKI
vir A vir B virG vlre vir D virE LocuS

'2.(j"l 9.5
1'11)1 �I 4.5 � Size (kbp)
=:t> C>ct> <3:=' [> ==t>

� �� 760.5
1--;
Proteins (kDa)
T � Y. T
border endo-
c...r Probable
plant transfer transcnpllonal SS DNA
sensor structure activator nuclease binding function
M M e? ? e? elM Location
Figure 1. The physical and functional organization of the virulence region of the octopine Ti
plasmid. The large open arrows indicate the transcriptional orientation and the genetically defined
limits of the octopine vir loci (from Stachel & Nester, 82). The references for the assignments of
polypeptide sizes and probable functions of vir specific products can be found in the text. C, and
M, cytoplasm and membrane, respectively.
At least five additional proteins, designated virulence-related-proteins

(VRPs), are induced in Agrobacterium grown in the presence of AS (28). The
production of the VRPs depends on intact virA and virG, i.e. they are under
the control of the vir regulatory system. One VRP of apparent molecular
weight 45 kd is encoded by pinF [plant inducible locus F (82)] which maps
immediately adjacent to the virA locus; another VRP of apparent molecular
weight 27 kd is encoded by an unknown region of the Ti plasmid, while the
other VRPs are most likely chromosomally encoded. The existence of the
VRPs indicates another level of genetic and functional complexity to the
T-DNA transfer process. However, their roles remain to be determined.
While pinF is nonessential (82), other VRPs may directly function in T-DNA
transfer by providing a component either of the regulatory system (such as a
sigma factor for RNA polymerase) or of the transfer apparatus (such as a
DNA or membrane associated protein). VRPs may also function indirectly in
T-DNA transfer per se, by affecting the general metabolism of the bacterial
cell during the plant cell transformation process. Basic biosynthetic or energy
transfer pathways may be redirected from vegetative growth to providing
substrates more specific for the efficient transfer of the T-DNA element.
GENERATION OF A TRANSFERABLE T-DNA COpy
Structural Requirements for T-DNA Transfer

The T-DNA region is defined as that segment of the Ti plasmid that is
homologous to sequences present in transformed plant cells. The actual sizes
of the T-DNA elements vary in different, naturally occurring Ti plasmids. Ti
plasmids are classified according to the type of opine (sugar-amino acid
derivative) they induce in crown gall tumor cells (reviewed in 87). Two
commonly studied Ti plasmids , nopaline and octopine, result in nopaline- or
octopine-producing tumor tissues, respectively. The nopaline T-DNA is one

large continuous segment roughly 22 kbp in size, and the octopine Ti plasmid
contains three adjacent T-DNAs, a left T-DNA (TL) element of 13 kbp, a
central T-DNA (TC) element of 1.5 kbp, and a right T-DNA (TR) element of
7.8 kbp (Figure 2 below). The TL element contains oncogenic functions for
tumor initiation and maintenance, TR contains several opine synthetic genes,
and TC does not specify a phenotype in transformed plant cells.
The structural limits of different T-DNAs were defined by comparing the
nucleotide sequence at the ends of the T-DNA element following integration
into the plant genome with the nucleotide sequence of the corresponding
region of the Ti plasmid. These analyses revealed that in all cases the
homology between sequences present in the Ti plasmid and those in trans

formed plant cell DNA ends within or proximal to a 25 bp sequence that
flanks the T-DNA region of the Ti plasmids as direct (albeit, imperfect)

repeats (39, 53, 77, 78, 106, 111). These repeats delimit all T-DNAs an
alyzed to date. A consensus of six T-DNA terminal repeats from the most
studied Ti plasmids, two from nopaline and four from octopine (6,31), shows
two conserved domains of 13 and 5 to 7 bp as follows:
T Pu G AT
TGGCAGGATATAT � TGTAA
NC C T TC
Only these 25 bp direct repeats at the ends of the T-DNA are required in cis
for its mobilization to the plant cell because its transfer is unaffected by (a)
deletions of the internal portion of native T-DNAs (54, 1 1 2) or (b) placement
of cloned fragments carrying only T-DNA border repeats, either on a separate
plasmid (19, 36) or on the Agrobacterium chromosome (37; S. Stachel , D.
Corbin, unpublished results) in trans to the Ti plasmid vir region. These
findings form the basis of the design of modified and simplified T-DNA
molecules that are useful as vectors to transform plant cells with cloned DNA
fragments of interest (57, 72, 113). Furthermore, deletion of the first 6 bp or
the last 10 bp of the 25 bp sequences blocks T-DNA transfer (101).
Early genetic studies showed that while deletion of the region overlapping
the left border 25 bp repeat had little effect on Agrobacterium pathogenicity,
deletion of the right border region abolished crown gall tumor formation (47,
76). When restriction fragments overlapping the native right border, or a
clone carrying a synthetic 25 bp repeat sequence, were reintroduced at the
right border deletion site, tumor forming activity was restored (65, 99).
Further, if the orientation of the right border fragment (or 25 bp sequence) is
reversed with respect to its natural orientation, the efficiency of T-DNA
transfer is greatly attenuated (65,99). These results suggested that the T-DNA
element might be transferred in a rightward to leftward direction, determined
by the orientation of the border repeats. Thus, when the right border is present
8 ZAMBRYSKI
in its natural orientation, transfer will include the T-DNA tumor specifying
genes internal to the T-DNA element; sequences with homology to the 25 bp
repeat (95) could serve as "left" borders. When only the left border is present,
transfer would only occur away from the T-DNA element, and no tumor
forming genes would be transferred to the plant cell. Recent molecular studies
provide a confirmation and an explanation for the polar function of the 25 bp
T-DNA border repeats (see below).
As noted above, all the 25 bp sequences identified to date are highly related
to each other; this suggests they might all be capable of directing T-DNA
transfer. However, nonselective use of all T-DNA borders would lead to
nonproductive T-DNA transfer events. For example, in the simple case of the
two border nopaline Ti plasmid, activity of the left T-DNA border in a polar
manner would lead to transfer away from the T-DNA element. Recent data
suggest that there are sequences adjacent to the 25 bp border repeats that
influence their efficiency to promote T-DNA transfer. Cloned DNA frag
ments containing chemically synthesized native right or left border sequences
alone are equally active (at levels corresponding to, on average, 30% of wild
type) in T-DNA transfer; however, fragments that contain several kbp of
DNA sequences bracketing the native right or left nopaline Ti plasmid borders
were very (95%) active and less ( 1 0-40%) active, respectively ( 1 0 1 ; see also
44).
The situation regarding the four border T-DNA region of the octopine Ti
plasmid is more complex. A 24 bp DNA sequence (5 'TAAPuTPyNCTGT
PuTNTGTTTGTITG 3'), designated overdrive (66) , situated to the right and
adjacent (within 60 bp) to the right copies of the native 25 bp border repeats of
the TL and TR T-DNA elements of the octopine Ti plasmid is essential for
efficient transfer of constructs carrying only synthetic 25 bp repeats. In
contrast to the nopaline case, little T-DNA transfer occurred in the absence of
the overdrive sequence, and this sequence alone was fully active in the
absence of its neighboring natural sequences. These data initially were used to
explain the transfer of TL and TR elements; however, there are other possible
(TC, or TR-TC or TC-TL or TR-TC-TL hybrids) octopine specific T-DNAs
(86). Overdrive acts like a true enhancer; it can stimulate T-DNA transfer
when placed in either orientation, on either side, and at a variable distance (up
to 6 kbp) from synthetic border repeat sequences (66, 94; W. Ream, un
published results). That overdrive can act at a distance would allow it to
function in the transfer of the above mentioned additional octopine T-DNAs.
Further, the rightward to leftward polarity of T-DNA transfer could be
maintained as long as natural overdrive sequences were located primarily on
the right side of the T-DNA region.
While some data indicate a specific role to the overdrive sequence, any
models to explain the activity of the T-DNA borders must take into account
that the sequences immediately surrounding octopine or nopaline borders are
highly dissimilar (see for example, 6). In addition, there are no sequences
with good homology to the octopine 24 bp overdrive sequence adjacent to the
right nopaline border (101). Thus, any potential overdrive sequences in the
nopaline Ti plasmid must be either different from those in the octopine Ti
plasmid or farther away from the borders . Another possibility is that each Ti
plasmid has evolved slightly modified mechanisms to accommodate different
T-DNA border regions . For example, the (vir specific) proteins that recognize
and use the T-DNA borders may have altered their specificities to accommo
date different contextual environments of nucleotide sequences. Or the nopa
line vir proteins that react with the border sequences may be inherently more
active than their octopine counterparts . Support for this hypothesis is that the
presence of overdrive is not required for efficient T-DNA transfer from the
octopine Ti plasmid if the concentration of vir specific products is elevated by
increasing the copy number of DNA sequences overlapping the vir region (W.
Ream, unpublished results) .
The T-DNA Transfer Intermediate: Circular, Linear, Single

or Double- Stranded?
While the above studies clearly demonstrate that the T-DNA borders are
essential for T-DNA transfer (and, potentially, integration), they do not
address how the borders are used to generate a T-DNA copy that will
ultimately be transferred to the plant cell. Somehow the T-DNA must be
removed or copied from the Ti plasmid, and the T-DNA borders might be
expected to play a role in such a process. The first studies suggested that the
T-DNA intermediate might be a double-stranded circular molecule; however,
this work was designed to select specifically for circular T-DNA molecules
(49). T-circles (engineered to contain an E.coli replicon with or without a
lambda cos site in their T-DNA segment) were recovered following transfec
tion or transformation of E. coli with total DNA prepared from vir induced
Agrobacterium; no other T-DNA homologous structure would exist and
stably replicate in the heterologous E. coli host. These T-circles were found to
contain a single hybrid T-DNA border sequence to suggest that they are the
result of a site-specific recombination between the left and right copies of the
border sequences (49). T-circles were initially attractive as candidates for the
T-DNA intermediate molecule; their existence seemed to support the ex
periments of the times, that is, the construction of small T-DNA transfer
vectors capable of DNA transfer when placed in trans to the vir region. Such
vectors might be expected to be ready for transfer without need to be excised
from the Ti plasmid. However, the production of T-circles in Ti plasmid
containing Agrobacterium is a rare event occurring at a frequency between 3
X 10-3 (90) and 10-5 (2, 59), an unlikely property for an intermediate in an
efficient transfer system.
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Recent studies further characterize the (albeit rare) formation of T-circles in

vir -induced Agrobacterium (90). Two types of recombinant T-DNA contain
ing circles were observed, a major class representing precise site-specific
recombination between both T-DNA borders, and a second minor class
representing recombination events either using only one T-DNA border and
other Ti plasmid sequences or involving Ti plasmid sequences other than the
T-DNA borders . The observed T-DNA homologous circles are proposed to
result from single strand nicks at the T-DNA border sequences (see below)
which unwind sufficiently to promote recombination over short (less than 20
bp) stretches of DNA with local homology to border contiguous sequences.
The second candidate T-DNA intermediate is a double stranded linear
molecule . This structure is hinted at by data that detected double stranded

breaks at the T-DNA borders of the Ti plasmid following the activation of vir
gene expression (96, and see below). Also, the recovery of T-circles by the
lambda transfection system was eliminated if the DNA sample was treated
with moderate heat; these results were interpreted to indicate that the T-DNA
ends were linear prior to packaging (50). However, there are two strong
arguments against either a linear or circular double stranded T-DNA in
termediate . First, T-circles produced by recombination , or linear double
stranded T-DNAs produced by cleavage at the borders, would result in the
loss of the T-DNA region from the Ti plasmid. It seems unlikely that the
T-DNA transfer process would have evolved to be suicidal. Secondly, and
most importantly, the major free T-DNA homologous molecule, observed
directly in hybridization analyses using Agrobacterium DNA prepared after
activation of vir gene expression, is a linear single-stranded copy of the
T-DNA region , designated the T-strand (84, 86 , 97) .
The T-Strand as the Transfer Intermediate

Hybridization techniques were used to directly characterize the novel struc
tures associated with the T-DNA and its borders in Agrobacterium soon after
the induction of vir gene expression by AS (84) . Total AS-induced DNA was
electrophoresed directly , i.e. in the absence of restriction digestion , to assay
for the presence of potential T-DNA homologous molecules which had a
higher mobility than did total uncut DNA. A variation of the Southern blotting
technique was used to distinguish between single stranded (ss) and double
stranded (ds) DNA molecules present. By use of the ,two border nopaline Ti
plasmid, a linear ssDNA copy of the T-DNA region was detected (84). This
novel molecule, designated the T-strand, is produced at about one copy per
bacterium, and it corresponds to the bottom strand of the nopaline T-DNA
region such that its 5 ' and 3 ' ends map to the right and left T-DNA border
repeats. The discovery of the T-strand and its confirmation by others ( 1 , 97)
uncovered a unique and interesting molecule, whose properties both ex-
plained earlier genetic studies for polar T-DNA transfer and suggested a
possible mechanism for the T-DNA transfer process (see below).
Whereas the T -strand can be observed as a free, non Ti plasmid linked
molecule, AS-induced changes were expected to occur to the T-DNA region
while it is still linked to the Ti plasmid. Two types of structures were observed
(84):(a) single stranded endonucleolytic cleavages within the bottom strand of
the T-DNA border repeats, designated border nicks , and (b) Ti plasmid
molecules containing T-DNA regions where the internal portions exhibit
partial single stranded and/or triple stranded character (designated T-region
structures) . Detailed studies localized the border nicks to between the third
and fourth base of the bottom strand of the 25 bp border repeat sequence
(1,100), confirming that these sites must be directly involved in the T-DNA
transfer process. Border nicks were proposed to represent initiation and
termination sites of T -strand synthesis, respectively , while T -region structures

possibly correspond to intermediate steps in this synthesis (84).
The detection of defined T -strands and border nicks provides a means to
dissect and understand the early steps of the T-DNA transfer process. One of
the most obvious questions is whether the levels of these molecular reactions
correlate with the efficiency of T-DNA transfer to plant cells as measured by
transformation frequency . Wang et al compared nopaline Ti plasmids contain
ing either a synthetic 25 bp T-DNA border or the native right T-DNA border
region for their ability to act as substrates for vir -induced border nicking
(100) or to promote tumor formation (101). It is interesting that the synthetic
25 bp border repeat was as good as , and even slightly better than, the native
right border fragment in the nicking assay, but the native border sequence was
more active in promoting plant cell transformation . These results suggest that
the sequence context surrounding the right border is important in mediating a
step subsequent to border nicking to promote T-DNA transfer. For example,
to generate the T-strand likely requires proteins in addition to the border
endonuclease (see below); these latter proteins may have a requirement for a
sequence context, or specific overdrive-like sequence, to unwind the T-strand
from the Ti plasmid. Recent experiments with Agrobacterium carrying octo
pine Ti plasmid derivatives confirm that constructs lacking the overdrive
sequence are only partially reduced in vir induced border cleavage (97);
however, the absence of the overdrive more dramatically affects T-strand
production (94, 97). Furthermore, the lack of T-strands correlates directly
with the loss of tumorigenicity of the overdrive-minus strains (94). Thus,
border nicks are a necessary but insufficient prerequisite for T-strand produc
tion .
The production of single T-strand molecules from nopaline type Ti plas
mids carrying two border sequences is easy to envisage . The situation in four
border Ti plasmids such as octopine is much more complex. Figure 2 dia-
12 ZAMBRYSKI
grams the T-strands produced from the octopine T-DNA regions; the four
borders A,B ,C,D, bracketing the three T-DNAs TL, TC, and TR, produce six
distinct T-strands. These linear single stranded molecules correspond exactly
to the molecules expected if all border-to-border combinations are used for
T-strand production (86, 97). Thus, left and right borders can be used to both
initiate and terminate T-strand production. Also, borders can be "skipped" to
produce composite molecules (i.e. , TRl TCl TL, TRl TC, and TCiTL T
strands). Since each of the four borders are observed to be nicked in
dependently of each other, most likely the T-strand made in a particular
induced cell is a function of which borders are cleaved on its particular Ti
plasmid. Thus, T-strand synthesis would initiate at a border nick and then
proceed 5 ' to 3' until it encountered a second nick. The "skipped" borders of
composite T-strands would be expected to be uncleaved on the parent Ti
plasmid. The copy number of T-strands is approximately one per AS-induced

bacterium [note that each of the six octopine T-strands is present at one sixth
the concentration (86) of the single nopaline T-strand (84)], and this supports
the model that the distribution of border nicks in the Ti plasmid population
determines which T -strands are produced. That T-strands (TC and TCl TL) are
produced from a border that lacks an adjacent overdrive sequence can be
resolved (as discussed above) if the overdrive sequence does not need to be
adjacent to a border site to act.
Protein Products Which Function To Generate Transferable

T-DNA Copies
To date several vir specific products have been identified as requirements for
border nicking and T-strand production. The products of virA and virG are
required for overall vir expression, i.e. in their absence no T-DNA associated
molecular reactions occur (86) . More specifically, of the other vir loci, only
TL TC TR
1<1 J'<II d
A B C D
t.--------------------------------t D �A
t--------------1' D �B
t----------t D�C
t.---------------------t C�A
t.---t C�B
t-------------------t B �A
Figure 2. Generation of multiple T-strands from the four border T -DNA region of the octopine
Ti plasmid. The three adjacent T-DNAs of the octopine Ti plasmid, TL (T-DNA left), TC
(T-DNA center), and TR (T-DNA right), are delimited by the four T-DNA borders (open leftward
pointing arrows) A,B,C, and D. Below are the six possible combinations of border nicks (short
vertical arrows) resulting in six distinct T-strands (long horizontal arrows).
mutants located within the first half of the 4.S kbp virD locus , encoding the
VirDl and VirD2 polypeptides, block the production of both border nicks (2,
86, 96, 1 07) and T-strands (86, 97). The expression of virDl and virD2 in
E.coli directs border nicking as well as T-strand production from plasmid
constructs carrying T-DNA regions (43, and G. De Vos and P. Zambryski ,
unpublished results). Thus, it is assumed that VirDl and VirD2 act as a site
specific endonuclease which recognizes and cleaves the lower strand of the 25
bp T-DNA border repeat sequences; the nicked borders are then used for
T-strand production. Constructs containing VirD l and as little as 50% of the
N-terminal half of VirD2 (G. De Vos, and P. Zambryski, op cit) are also
active in nicking and T-strand production; thus, the C-terminal half of VirD2
may be nonessential or have another function.

Several activities other than the border endonuclease, including helicase(s),
polymerase(s), and repair enzymes, are expected to be necessary components

for the production of T-strand molecules. Since no other vir or chromosomal
virulence mutants are deficient for T-strand production alone, and since it
seems unlikely that the VirDl and VirD2 polypeptides could specify all these
activities (in addition to the border endonuclease), it is speculated that some
functions necessary to complete the generation of the T-strand molecule might
be essential bacterial functions encoded by the Ag robacterium chromosome .
Alternatively, the VirD 1/virD2 endonuclease may possess an additional
activity , such as helicase .
Since the T-strand is a linear ssDNA, it was also expected that its synthesis
and/or processing could be facilitated by ssDNA binding proteins. Recently,
the largest open reading frame of the virE locus of both the octopine and
nopaline Ti plasmids, virE2, has been determined to specify such a function
(11, 1 3 , 17) . Radioactively labeled ssDNA (but not dsDNA or RNA) probes
were retarded on polyacrylamide gels specifically following incubation with
extracts prepared from vir induced cells. A variety of different ssDNAs were
all effective substrates for the gel retardation assay; this suggests that the
binding occurred independent of sequence composition. The nucleotide se
quences of the octopine and nopaline virE2 regions predict polypeptides of
60.S kd (lOS) and 64 kd (3S), and the relative mobilities of these polypetides
on denaturing protein gels are 65 kd and 69 kd respectively.
That Agrobacterium has evolved an ssDNA binding protein specifically
under vir control strengthens the argument that an ssDNA (i.e. T-strand) is
indeed the T-DNA intermediate molecule transferred by Agrobacterium to
plant cells. Furthermore, the 65 (or 69) kd VirE2 polypeptide is the most
abundant protein produced in AS-induced cells ( 1 3, 28); the relative amount
of VirE2 produced suggests it has a structural rather than an enzymatic role in
the T-DNA transfer process. To completely cover a single stranded 20 kb
T-DNA molecule would require 700 to 350 protein molecules, assuming a
14 ZAMBRYSKI
30-60 nucleotide substrate. This number of molecules would represent ap

proximately 0. 1 % of the total cell protein, which is consistent with the
amount of VirE2 protein observed. The size of the substrate binding site is
estimated by analogy to the size of the binding sites for the smooth contoured
or beaded structure of the E. coli SSB (ssDNA binding protein) tetramer (9);
this estimate is supported by additional data indicating that synthetic oligonu
cleotides at least 36 nucleotides long were required to be effectively retarded
on gels following incubation specifically with extracts from vir induced cells
(33).
Single stranded DNA binding proteins can be classified into two major
groups, enzymes and cofactors (RecA protein, DNA polymerase, lactate
dehydrogenase, RNA polymerase, C1C2 polymerase, and others), and true

SSB (9). The latter class designation implies modes of action similar to those
of E.coli S SB and bacteriophage T4 gene 32 protein: (i) ssDNA binding, (ii)

requirement in stoichiometric rather that catalytic amounts, (iii) cooperative
DNA binding, (iv) stimulation of DNA polymerase , and (v) absence of
intrinsic enzymatic activity . The VirE2 protein clearly fulfills the first crite
rion, and its relative abundance fits with a potential requirement in
stoichiometric amounts to cover vir induced ssDNAs. In addition , VirE2
protein binds tightly to DNA , requiring salts of 1.0 molar or greater to
completely dissociate itself from its complex with ssDNA probes (V. Citov
sky, and P. Zambryski, manuscript in preparation). Biochemical analyses of
the binding of purified VirE2 protein to DNA substrates in vitro suggest that
this binding is highly cooperative. Also, cooperativity can be visualized under
the electron microscope (V. Citovsky, M. L. Wong , and P. Zambryski ,
manuscript in preparation), where VirE2 coats linear ssDNAs, resulting in the
rod-like DNA-protein complexes. Further, under low VirE2: ssDNA ratios,
few ssDNAs are coated to completion with VirE2, while the majority of the
DNA is condensed and unassociated with protein . Thus, the VirE2 protein
fulfills three characteristics of a true SSB . virE2 does not complement E.coli
S SB mutants ( 1 1 ), and this suggests that it does not have a role in DNA
replication per se . Whether VirE2 has any enzymatic activities such as
ATPase or helicase remains to be tested.
The assignment of ssDNA binding function to the virE locus may help to
explain the unusual phenotype of virE mutants. In the first place, virE mutants
are avirulent only on some plant hosts (30, 34, 82, 1 08) . Since ssDNA
binding proteins have a high affinity for ssDNA without regard to sequence
composition (9), Agrobacterium chromosomally specified ssDNA binding
protein may substitute for the vir ssDNA binding protein in virE mutants.
These chromosomally encoded ssDNA binding proteins may be less abundant
than the VirE2 protein; thus they may be able to protect the T-strand from
degradation in some, but not all , plant hosts. Furthermore, virE mutants can
be complemented to wild-type virulence if they are coinoculated on plants

with an Agrobacterium strain carrying a wild-type vir region (64; D. Corbin,
S. Stachel, unpublished results), and they suggest that the product of the virE
locus might function extracellulary, potentially as part of a T-strand/protein
complex. That virE mutants are not blocked in T-strand synthesis (86, 97)
also suggests that virE function comes into play at a later stage of the transfer
process. Several possible roles can be envisioned for the VirE2 protein,
including packaging of the T-strand, protecting the T-strand from nucleases
during its transit, and potentially even participating in active transport and
integration of the T-strand into plant genomic DNA. The virE locus has the
additional capacity to encode a short polypeptide of 7. 0 kd at its 5' end (105);
the role of such a polypeptide and whether it might interact with VirE2 are
unknown.
Model for the Mechanism of T-DNA Transfer

While the T-strand may end up packaged into a viral-like particle potentially
coated with VirE2 protein, the fact that T-strands are not produced in abun
dant quantities and that T-DNA transfer requires close physical contact
between Agrobacterium and the plant cell suggests that the process is not
analogous to viral infection per se. However, if we postulate that Agrobacte
rium uses evolutionarily conserved mechanisms to suit its ends, then the
known characteristics of T-DNA transfer are most similar to DNA transfer
between bacterial cells via conjugation. Indeed, border nicks are analogous to
nicks at the origin of conjugal DNA transfer. The T-strand is analogous to the
linear donor ssDNA molecule during bacterial conjugation, and AS-induced
internal T -region structures may be analogous to replacement strand synthesis
intermediates of conjugal donor DNA. Furthermore, bacterial conjugation
requires direct contact between donor and recipient cells, and the same
requirement holds for Agrobacterium mediated T-DNA transfer.
Strong support for the model that T-DNA transfer is bacterial conjugation
applied to plant cells (8) derives from experiments which show that the origin
of transfer (oriT) from a conjugative E. coli plasmid, pRSFIO lO, can sub
stitute for the T-DNA borders in directing DNA transfer to plant cells from
Agrobacterium. This hybrid transfer system also requires an intact Ti plasmid
vir region, and a region of pRSFlOlO encoding polypeptides involved in
plasmid mobilization. The oriT of pRSFIOlO and its cognate mobilization
proteins presumably generate a conjugative DNA transfer intermediate which
is then transferred to plant cells using the Agrobacterium vir specific transfer
machinery. It will be interesting to determine whether all or only some of the
vir polypeptides are required for this transfer system. Those that are not
essential may be those that have particular substrate requirements, such as the
virD specific border endonuclease. Thus, the heterologous pRSFIOIO-vir
16 ZAMBRYSKI
transfer system can provide insight into the function of Agrobacterium specif
ic products .
Bacterial Conjugation Versus T-DNA Transfer

The best studied example of bacterial conjugation is that of the F (fertility)
conjugative plasmid, and several excellent recent reviews summarize greater
than 40 years of research on this vanguard system (4 1 , 103). The F plasmid is
100 kbp in size, and 33.3 kbp of contiguous sequences, encoding 23 identi
fied functions, are required to mediate plasmid fertility. Seven salient features
of conjugation, three related to the processing of conjugal DNA and four
related to the interaction between the donor and recipient cells, are summa
rized and compared to T-DNA transfer (see also 1 14) .

1 . INITIATION OF CONJUGAL DNA TRANSFER. Conjugative DNA processing is
initiated at a specific site on the F-plasmid, the origin of transfer (oriD. This
site is immediately adjacent to the F-region, and it is oriented to direct transfer
away from this region, i . e . , the F-coding sequences are transferred last. The
oriT site is recognized by the Tral/TraY endonuclease (9 1 ) which introduces a
nick on the strand destined for transfer. N icking at the T-DNA border
sequences by the VirD IIVirD2 endonuclease is analogous to oriT nicking by
the Trall TraY cndonuclease.
2. DIRECTIONAL TRANSFER OF DONOR DNA. The 5' end at the oriT nick
provides the leading terminus for the linear transfer of F plasmid DNA out of
the donor cell. The Tral protein also has helicase and ATPase activities
required for unwinding the donor DNA strand. It has been proposed that the
oriT endonuclease may have an additional function as part of a protein-DNA
complex that acts to "pilot" the F strand into the donor cell . For example, the
TraY protein is localized to the bacterial membrane , and its association with
Tral bound to oriT may direct the transferred strand through the donor cell
envelope . The T-DNA transfer process has been shown to be polar, in the
direction right border to left border. The 5' end of the transferable T-DNA
copy, the T-strand, is at the right T-DNA border. Thus, T-DNA transfer is
probably 5' to 3' as in bacterial conjugation. By analogy to F, the VirD l!
VirD2 endonuclease may remain attached to the 5' end of the T-strand to
facilitate T-DNA transfer. Experiments in progress (E. Howard, V. Citovsky,
B . Winsor, and P . Zambryski) suggest that at least the VirD2 polypeptide can
be found tightly associated with the T-strand in vir induced Agro bacterium.
3. CONJUGATIVE DNA SYNTHESIS. Transfer of donor F-specific single
stranded DNA is associated with synthesis of a replacement strand in the
donor cell, and of a complementary strand in the recipient cell. Replacement
strand synthesis in the donor cell is mediated by the E.coli DNA polymerase
holoenzyme, and the 3' OH at the oriT nick site is the priming site. It is not
known how replacement strand synthesis occurs in the recipient cell;
potentially an RNA primer is synthesized de novo. Some conjugative plas

mids (but not F) transfer proteins with primase function into the recipient; this
transmission is selective since donor cell proteins are not generally transferred
(10). In Agrobacterium, replacement strand synthesis of the lower strand of
the T-DNA region presumably uses the 3'OH of the nicked right T-DNA
border as a priming site; whether or not vir proteins are involved in this
reaction is unknown, but it is not unlikely that chromosomally encoded
enzymes could perform this step. While the T-DNA -protein transfer complex
may carry proteins that stimulate transport and integration of the T-strand into
the plant cell genome, replacement synthesis of the other T-DNA strand may
occur by plant cell enzymes. The Agrobacterium system differs from F

transfer in that the T-strand can be produced in the absence of plant cells,
following addition of the vir inducer AS . However, this fact may reflect
laboratory conditions which provide Agrobacterium with an excess of AS to

optimize vir induction; in nature Agrobacterium may associate closely with
plant cells to obtain sufficient quantities of vir inducer(s).
There are further characteristics of conjugal DNA metabolism to compare
with T-DNA transfer. Whereas the single oriT of F results in the un
idirectional transfer of the entire plasmid replicon, T-DNA transfer only
mobilizes the T-DNA region. The T-DNA region is essentially bracketed by
two oriTs. To minimize transfer leftward away from the T-DNA element
poses one additional requirement in the T-DNA system, that the rightmost
"oriT' , (T-DNA border) is more active than the left. Potentially F-derived
plasmid constructs carrying multiple, directly repeated copies of oriT would
transfer DNA segments in between the oriT sites; in this case, F transfer might
resemble T-DNA transfer. Moreover, defective T-DNA transfer from Ti
plasmids containing reverse oriented right borders may be comparable to
interrupted transfer of the E.coli chromosome mediated by integrated copies
of F plasmid, i.e., T-strand synthesis occurs (rightward) away from the
T-DNA, so that the tumor forming genes are only reached if the entire 200
kbp Ti plasmid is traversed. When the T-DNA is placed on smaller plasmid
replicons up to 50 kbp in size, transfer is unaffected by the orientation of the
borders (44, 74) .
There are few F-specific or vir -specific products involved in F-strand or
T-strand synthesis and processing. Besides the Tral/TraY endonuclease, only
two other F tra genes, D and M, are needed for conjugation. TraD protein
binds nonspecifically to single and double stranded DNA and is located in the
inner bacterial membrane; it is hypothesized to form part of a pore for DNA
export. TraM is also an inner membrane protein which binds specifically to
the oriT region of F-strand DNA. TraM may activate donor strand unwinding
and concomitant replacement strand synthesis in response to the signal that a
stable mating pair has formed (see below). So far only two vir proteins, VirDI
18 ZAMBRYSKI
and VirD2, appear to be required for T-strand synthesis. Whether any of the
vir products, such as the 3 ' virD products VirD3 and VirD4 , are involved in
targeting the T-strand to the bacterial (or plant cell) membranes remains to be
determined.
The F plasmid also encodes its own SSB protein; however, the locus is
located outside the tra region , in the segment of DNA first transferred to the
recipient cell. This location may serve to provide a template for the synthesis
of SSB in the recipient to protect the newly arrived single-stranded DNA.
F-specific SSB is not essential for conjugal DNA transfer; however, chromo
somally encoded SSB may replace F-SSB . SSB might be expected to (i)
maintain the DNA at the nicked site in a non duplex state to enhance helicase
activity, (ii) promote correct initiation and elongation during replacement
strand synthesis, and (iii) protect and stabilize donor DNA. As mentioned,
Agro bacterium also synthesizes a T-DNA transfer specific ssDNA binding

protein from the virE locus .
4. MATING SIGNAL. The recognition of a recipient cell and the formation of
a stable mating pair stimulates F DNA transfer and concomitant replacement
strand synthesis. Since the tra genes are constitutively expressed in the F
system, nicking and religation at oriT probably occur continuously in donor
cells; however, following stable contact with a recipient cell, some (as yet
unknown) signal is transmitted from the recipient to stimulate donor DNA
transfer. Plant phenolics such as AS are essentially a mating signal to Agro
bacterium to begin the T-DNA transfer process . In contrast to the F system,
vir gene expression encoding nicking and transfer functions is under tight
control dependent on the presence of AS .
5 . F-PILUS FORMATION. F-pili establish the physical contact between donor
and recipient cells . Pili are composed of a single 7 .0 kd protein, pilin ,
arranged as repeating units elongated along four intertwined helixes. While
F-pili themselves are simple in composition, at least 40% of the coding
capacity of the F tra region is needed for the synthesis (traA ) , processing
(traQ), and erection of the pilus structure (traL, E, K, B, V, C, W, U, F, H,
and G) .
Elongated pili are not a formal requirement for conjugal DNA transfer, as
many Gram positive bacteria transfer DNA without pili (14). F pili primarily
function to locate an appropriate recipient cell; once a stable pair has formed,
F pili retract. Actual DNA transfer may then proceed through a transmem
brane pore near the base of the pilus. The Agrobacterium chromosomal
virulence loci, chvA , chvB, and pscA , may provide the pilus-analogous
function which allows Agrobacterium to form an effective contact with
recipient plant cells; in this case bacterial polysaccharide projections may
attach to surface components of the plant cell. In both F and T-DNA transfer,
some specialized pore structure must mediate actual DNA transfer. The
abundant virB specific polypeptides which fractionate to the cell envelope in

vir induced cells may provide some of the structural components of a transfer
channel.
6. STABILIZATION OF THE MATING PAIR. Mutations in traN or the C-terminal
region of traG are defective in the formation of stable aggregates with
recipient cells and conjugative DNA transfer. Neither mutation affects pro
cessing of donor DNA, mating signal reception, or F-pilus formation. The
TraG and N products may anchor donor to the major outer membrane protein,
OmpA, of recipient cells , since ompA mutants are also defective in the
formation of stable mating pairs. No Agrobacterium function has been de
fined to stabilize its association with plant cells during T-DNA transfer;
however, it is not unlikely that such a function exists .
7. PREVENTION OF NONPRODUCTIVE MATING. The F specific genes traS and
traT encode products that prevent sibling mating. The TraS protein is local
ized to the inner bacterial membrane and may block DNA transfer directly.
The TraT protein is a lipoprotein located in the outer bacterial membrane that
binds the pilus tip in a competitive fashion. It is presumed, but not proven,
that the T-DNA transfer process is restricted to plant cells; to accomplish this
exclusive interaction requires that T-DNA transfer specific proteins (either at
the bacterial surface or as part of the T-DNA-protein complex) recognize
plant rather than bacterial cells. A plant-specific mating function is also
implied by the existence of a separate conjugative operon , located outside of
the T-DNA and vir regions on the Ti plasmid, for transfer of Ti plasmids
between bacterial cells (20, 38) .
Thus, except for the elaboration of an extended pilus structure, most
characteristics of F-conjugation are shared by the T-DNA transfer process.
Only a few of the loci essential for either process are required to modify donor
DNA into transferable DNA; most proteins for conjugal (or T-DNA) metabo
lism are chromosomally specified. Just as most F specific tra functions are for
assembly of a transfer structure, most vir products may have a similar role.
The future will tell how well the parallel between bacterial conjugation and
T-DNA transfer holds. The T-DNA transfer system may repay some of its
debt to insights gained from earlier analyses of F-factor, since certain aspects
of conjugation may be easier to study in the T-DNA transfer process. For
example, it is difficult to distinguish donor and recipient bacterial cells ,
whereas donor Agrobacterium clearly can be separated from plant cells.
INTEGRATION OF THE T-DNA INTO THE PLANT

GENOME
Single integrated T-DNA copies are frequent, but on average three copies of
the T-DNA are found incorporated into the genomes of various di-
20 ZAMBRYSKI
cotyledonous plant species including tobacco ( 18 , 55 , 79, 88, 95) , petunia

(46, 48 , 98), tomato ( 12) , sunflower (92) , morning glory (78) , and Crepis
cappilaris (3); occasionally, 20--50 copies have been observed (7). An exten
sive study of the segregation patterns of 16 1 individual transformants of
Arabidopsis thaliana (K. Feldman , M. Christianson , manuscript in prepara
tion) revealed that 55% of the transformants segregated for one, 20% for two
unlinked, 6% for three unlinked, and 1 % for four unlinked T-DNA elements,
respectively. The remaining 12% did not segregate in a simple Mendelian
fashion. In a separate study , 10 independent transformed lines of tomato ( 12)
were analyzed for linkage between T-DNA markers and either isozyme
patterns or restriction fragment length polymorphisms. Nine out of the ten

inserts mapped to different chromosomal locations. Furthermore, the inser
tion sites of the T-DNA in four transformed lines of Crepis capillaris were
directly visualized by in situ hybridization and mapped to four out of the six
possible Crepis chromosomes (3). The fact that Agrobacterium can transform
a wide range of dicotyledonous plant species also supports that T-DNA
insertion is not dependent on a specific target DNA sequence.
It is interesting that tandem arrays of integrated T-DNA copies can contain
either direct or indirect repeats (55 , 48, 79) , and both types of repeats can be
found in a single array. As no multimeric T-DNA forms have been observed
in vir induced Agrobacterium, it is more likely that they arise in the plant cell.
Potentially T-DNA repeats are the result of replication and repair of the
T-DNA during insertion into plant DNA; or the T-DNA may be replicated and
ligated in a random (i . e . , head to tail , or head to head) fashion prior to
integration. Either single copy or repeated T-DNA arrangements are equally
capable of expressing internal T-DNA sequences.
Nucleotide sequencing of cloned T-DNA -plant junctions reveals that the
plant sequences immediately adjacent to the T-DNA borders are unrelated
( 3 9 , 53 , 77 , 106, 1 1 1 ); the only shared feature between these plant target
sequences is that they are enriched for A's and T's. On the T-DNA side, these
studies reveal that junction points on the right end are within or a few bases
from the right 25 bp border repeat sequence, and the junction points at the left
end are spread over 100 bp internal to and including the left 25 bp border
sequence . Thus, T-DNA insertion is more precise on its right side than on its
left, and this suggests that T-DNA integration, like the generation of the
transferable T-strand copy, is directed by the right T-DNA border .
Some clues t o the T-DNA integration process can b e gained from analyses
of plant target sites before and after T-DNA insertion. While there has only
been one study to date, the results show that plant DNA can undergo a variety
of rearrangements as a consequence of the T-DNA insertion event (32). The
most striking alteration of target sequences is the generation of a direct repeat
of 1 5 8 bp at the right and left T-DNA junctions. Besides this duplication
event, there are short deletion and insertion events at the ends of the 1 58 bp
segment. Additional analyses of this type will reveal whether or not long
duplications of target sequences are a general property of the T-DNA integra
tion process. Potentially the duplication of the T-DNA element during the
generation of tandem arrays and the duplication of target plant DNA se
quences reflect a common process.
The integration of the T-DNA into plant DNA shares features with other
examples of DNA insertion in eukaryotic cells. In plant cells, the maize
transposon Ac is the best studied (75 ). Ac insertion also involved duplication
of target sequences at the insertion site; however Ac insertion events are less
disruptive; usually only a short segment (up to 10 bp) of target DNA sequence
is duplicated at the ends of the element. The relative precision of Ac insertion
may reflect that Ac itself encodes a transposase function. T-DNA integration
may be better compared to more disruptive integration events exemplified by

some viral insertions. For example , deletion of target DNA is typical, and the
extent may vary from a few (2 1 ) to several thousand (22) bp. Also, in a
particular polyoma virus/host DNA recombinant joint, a 37 bp filler sequence
was found to be homologous to an inverted single copy host sequence found
650 bp away from the junction site (22). Similarly, in the plant target
analyzed (32) , a 33 bp filler sequence at the left of the T-DNA-plant junction
was observed to be homologous to an inverted host sequence 200 bp from the
junction site.
The variety of T-DNA as well as target DNA rearrangements which occur
suggest that T-DNA insertion is a multistep process involving several differ
ent types of recombination, replication , and repair activities most likely
mediated by host plant-encoded enzymes. However, the efficiency of the
Agrobacterium transformation process suggests that additional factors play a
role. Possibly the T-DNA complex may be directed preferentially into the
nucleus. Polar T-DNA transfer as well as targeting of the T-DNA/protein
complex to the plant cell nucleus may be mediated by proteins linked to the
right end of the T-DNA. The efficiency of T-DNA integration may also be in
part due to the nature of the proposed T-DNA transfer intermediate molecule,
the T-strand. While it is unknown whether the T-strand is altered (e .g. into a
duplex form) prior to transfer and/or integration, its single strandedness does
not conflict with a role during its integration. In fact, most models for general
recombination involve invasion of target sequences by single stranded donor
DNA (25). Linear duplex DNAs are thought to be inactive as recombination
intermediates until rendered single stranded at their ends by cellular exonucle
ases (5, 5 1 , 58).
It is still too early to formulate a precise model for T-DNA insertion into
plant DNA; however, the following steps are consistent with the available
information. First, the T-strand is transferred to the plant cell as a DNA/
22 ZAMBRYSKI
protein complex. Second, following localization of the complex to the cell

nucleus, a protein at the 5 ' (right) end of the T-strand interacts with a nicked
sequence in plant DNA. Third, the attachment of a ssDNA to one strand of
plant DNA results in a local torsional strain which is relieved by producing a
second nick on the opposite strand of the target site; the position of the second
nick may vary with the overall three dimensional structure of the plant
chromatin at the insertion site. Fourth, each end of the T-strand is ligated to
plant DNA, and its homologous strand is copied by cellular enzymes . Fifth,
repair and replication of the staggered nick in the plant target result in the
production of a repeated sequence (of variable length), plus additional se
quence rearrangements ("filler" DNA, deletion, etc) at the ends of the inserted
T-DNA element. Insertion of the large T-DNA segment likely is disruptive to
the integrity of the target DNA and may result in errors in replication and
repair at the insertion site, leading occasionally to the formation of direct and
indirect T-DNA repeats .
SUMMARY AND PROSPECTS
There are at least seven steps in the transfer of the T-DNA molecule from A .
tumefaciens to the plant cell; (a) recognition of a susceptible plant cell, (b)
induction of vir gene expression, (c) production of a transferable T-DNA
copy, (d) transfer of the T-DNA complex to the bacterial membrane, (e)
transfer of the T-DNA complex through the bacterial membrane and the plant
cell membrane, (f) transfer of the T-DNA complex through the plant cyto
plasm and through the nuclear membrane, and finally (g) integration of the
T-DNA element as a linear nonpermuted fragment into the plant nuclear
genome.
We are part way through understanding step (a), and we hope the compari
son of the virAl virG system to other bacterial regulatory circuits will assist in
defining exactly how they work to induce vir transcription inside the bacterial
cell. It is also important to determine the events on the outside of the cell
during the interaction of the VirA protein with plant phenolics. For example,
differences in the primary structure of VirA proteins are directly related to
differences in vir expression and infectivity between limited and wide host
range strains of Agrobacterium (56, 83); however, the domains of the protein
responsible for differential activity are unknown . Infectivity may be a func
tion of the vir inducing activity of plant specified phenolic compounds.
However, since vir induction can be mediated by a variety of phenolics
present in all plant species (80), other factors must play a role in the initiation
of the T-DNA transfer process . While it has been proposed that a deficiency
of vir inducers may explain why monocotyledonous plant cells are not
susceptible to Agrobacterium infection (93), some monocot cultures, notably
wheat, produce significant levels of vir inducing compounds whose chemical

structure is related to AS (E. Messens, H. Lorz, S. Stachel , P. Zambryski ,
unpublished results) .
Future work on step (b) will be mainly to assign functions to the rest of the
vir proteins; it is a direct part of the analysis of the actual transfer of the
T-DNA. Regarding step (c) , a major puzzle is the observation of double
stranded breaks at the T-DNA borders in restriction digests of vir induced
DNA (96) . Further, internal T-DNA homologous double stranded cleavage
products are more abundant than external fragments (84 , 86). These data can
be interpreted as support that the T-DNA excises from the T-region as a linear
double stranded molecule. However, electrophoresis of undigested AS
induced DNA does not reveal DNA migrating at the size expected for a free
linear double stranded T-DNA copy . Linear T-DNAs are found in unrestricted
DNA only if it is treated with S 1 nuclease prior to electrophoresis (84); this is

more in agreement with single strand nicks at the T-DNA borders .
Potentially the double stranded breaks seen in enzymatically restricted
DNA are produced artifactually following enzymatic treatment. The Ti plas
mid linked T-DNA region undergoing reactions to form a free T-strand copy
must be a multistranded and topologically complex structure; that double
stranded cleavage products are predominantly internal to the T-DNA region
supports this concept. Also some of these T-DNA homologous (i.e. , internal)
restriction fragments, designated T-region structures, bind to nitrocellulose
filters in the absence of DNA denaturation (84). This binding suggests that
T-region structures either represent fragments with partial single stranded
character, or fragments attached to a polypeptide moiety . Since the DNA is
prepared following treatment with protease, this latter suggestion would
imply attachment of a residual protein fragment to some part of the T-DNA
region . Thus, the topology of the T-DNA region during the production of a
transferable T-DNA copy is an area sorely lacking in information . There are
several additional interrelated questions. Is the T-strand displaced by DNA
synthesis initiating at the 5' end of the nicked right T-DNA border? Or, is the
T-strand first unwound from the T-DNA region prior to replacement strand
synthesis of the bottom strand? So far the data suggest that the T-strand is
synthesized at approximately one copy per AS-induced cel l . However, is this
the maximum level of T-strand production, or can additional T-strands be
made?
How the sequence context of the T-DNA borders influences their use in
T-DNA transfer is far from resolved. Most experiments have tried to address
how the borders might be more efficiently recognized, using sequences that
enhance their affinity for enzymes which act at these sites to generate a
transferable T-DNA copy. Thus, most studies focus on right border activity.
But what about the left border; how is it used? Is the T-DNA transfer process
24 ZAMBRYSKI
sloppy , generating T-strands leftward away from the T -DNA element? Left
border fragments are estimated to be 10-- 40% as active as right border
fragments ( 1 0 1 ) . But why generate any leftward T-DNA copies at all; such
events would lead to the transfer of the vir region and might interfere with the
capacity to encode the machinery for T-DNA transfer. In fact there has been
no systematic and quantitative analysis of potential leftward T-strands , and of
whether there are DNA sequences or protein factors that reduce left border
activity.
In the simple two border nopaline Ti plasmid, there are two seemingly
contradictory results: (i) right borders are more active than left borders in
T-DNA transfer (4 1 , 1 0 1 ) ; and (ii) constructs carrying only 25 bp repeats or
restriction fragments overlapping the left border are more frequently nicked
than those carrying restriction fragments overlapping the native right T-DNA
border ( l 00) . These differences might be explained if there are two possible
reactions that occur at the T -DNA borders, either nicking (at left borders) or
nicking plus sticking of protein factors (at right borders). For example, the
virD endonuclease might nick and stick to those borders that have an adjacent
overdrive element which binds a factor to promote the sticking reaction.
Binding of protein to the 5 ' end would stimulate T-strand displacement,
leaving the 3' end of the nicked site available for replacement strand syn
thesis; hence the right border would appear less nicked. This "nick" versus
"nick and stick" hypothesis would also apply if there were vir specific or host
protein factors other than the VirD l /virD2 endonuclease which were induced
to attach to the 5 ' end of right border nicked sites.
To sort out steps (d) and (e) , and potentially step (J) , will require a
significant commitment to studying membrane structure and function both in
bacterial and plant cells. Step (g) , the end result of the coordinated effort of 20
or more bacterial gene products, is less tractable for the present since it
depends largely on the identification of the Agrobacterium products that are
part of the TooDNA transfer complex. Bacterial protein products may exist that
either localize the T-DNA complex to the nucleus or act to insert the T-DNA
molecule into plant DNA. Also, several plant products may be involved in the
last steps of T-DNA transfer. One of the effects of wounding a plant cell is to
stimulate DNA replication and cell proliferation, and such processes typically
involve recombination and/or repair enzyme activities which may enhance
T-DNA integration. For these last steps the A grobacterium plant cell interac
-
tion provides an avenue to explore several fundamental plant processes, from

those regulating uptake of molecules at the cell surface, to mechanisms
determining transport through the internal network of plant cell membranes,
to the replication and rearrangment of DNA sequences and chromatin struc
ture in the nucleus.
Although the T-DNA transfer process is the only known example of
interkingdom DNA transfer, the underlying processes may be neither exotic

or unique. Agrobacterium might best be considered an evolutionary pragma
tist, borrowing existing processes to suit its ends . So far, data suggest that the
early steps of the T-DNA transfer process use well conserved bacterial
processes, from signal recognition to conjugative DNA transfer (85). It will
be interesting to determine what other examples of evolutionary economy
may be called into play for the later steps of the T-DNA transfer process.
ACKNOWLEDGMENTS
I thank my many coworkers for obtaining the results that make this review
possible, and their names are cited in the articles I coauthor. I especially thank
Scott Stachel for his determination to perform a variety of analyses to help
solve many questions related to the biology of the Agrobacterium-plant cell

interaction. I am grateful to my colleagues for sending preprints and sharing
their unpublished observations. I thank Marc Van Montagu and Jeff Schell of
the University of Gent, Belgium, for support of my research during the many
years of my stay in their lab. The work in my laboratory is currently supported
by grant DMB-86 1 7772 from the National Science Foundation.
Literature Cited
I . Albright, L. M . , Yanofsky, M. F . , A . tumefaciens octopine Ti plasmid

Leroux, B . , M a , D . , Nester, E . W . pTi 15955 . Plant Mol. B ioi. 2: 335-50
1987 . Processing o f the T-DNA o f A . 7. Barton, K. A . , Binns, A. N . , Matzke,
tumefaciens generates border nicks and A.J. M . , Chilton, M. D. 1983 . Regener
linear, single stranded T-DNA 1. Bac ation of intact tobacco plants containing
terial. 1 69 : 1 046-55 full length copies of genetically en
2. Alt-Moerbe, J . , Rak, B . , Schroeder, J . gineered T-DNA and transmission of T
1 986. A 3 . 6 kbp segment from the vi r DNA to RI progeny. Cell 32: 1 033-
region of Ti plasmids contains genes 43
responsible for border sequence-directed 8. Buchanan-WollasWn , V . , Passiatore, J.
production of T-region circles in E. coli. E . , Cannon, F . 1987 . The mob and oriT
EMBO 1. 5 : 1 1 29-35 mobilization functions of a bacterial
3. Ambros, P. F . , Matzke, A. J. M . , Matz plasmid promote its transfer to plants.
ke, M. A. 1986. Localization of Agro Nature 328: 172-75
bacterium rhizogenes T-DNA in plant 9. Chase, J. W . , Williams, K. R. 1986.
chromosomes by in s itu hybridization. Single stranded DNA binding proteins
EMBO 1. 5 :2073-77 required for DNA replication. Annu.
4. Ashby, A. M . , Watson, M. D . , Shaw, R e v. Biochem . 5 5 : 1 03-36
C. H. 1987 . A Ti plasmid determined 10. Chatfield, L. K . , Wilkins, B. M. 1984.
function is responsible for chemotaxis of Conjugative transfer of IncH plasmid
A . tumefaciens towards the plant wound DNA primase. Mol. Gen . Genet.
compound acetosyringone. FEMS Mi 197:461-66
crobiol. L ett. 4 1 : 1 89-92 11. Christie, P. J . , Ward, J. E. , Winans, S . ,
5. Ayares, D . , Chekuri, L. , Song, K. Y . , Nester, E . W . 1988. The Agrobacterium
Kucheriapati, R. 1986. Sequence tumefaciens virE2 gene product is a
homology requirements for intermolecu single stranded DNA binding protein
lar recombination in mammalian cells. that associates with T-strands. J. B a c
Proc. Natl. Acad. Sci. USA 83:5 1 99- teriol. 1 70:2659-67
5203 12. Chyi , Y. S . , Jorgensen, R. A . , Gold
6. Barker, R. F . , Idler, K. B . , Thompson, stein, D . Tanksley, S. D . , Loaiza
D. V . , Kemp, J. D. 1983. Nucleotide Figueroa, F. 1986. Locations and stabil
sequence of the T-DNA region from the ity of Agrobacterium -mediated T-DNA
26 ZAMBRYSKI
insertions in the Lycopersicon genome. Kashyap, L . , Douglas, C. , e t al . 1 986.

Mol. Gen. Genet. 204:64-69 Rhizobium meliloti genes required for
13. Citovsky, V . , DeVos, G . , Zambryski, nodule development are related to chro
P. 1 988. Single stranded DNA binding mosomal virulence genes in A . tumefa
protein encoded by the virE locus of ciens. Proc. Natl. Acad. Sci. USA 83:
Agrobacterium tumefaciens. Science. 4403-07
240:50 1 -4 27. Engler, G . , Depicker, A. , Maenhaut,
14. Clewell D. G . , Gawron-Burke, C. 1 986. R. , Villarroel, R . , Van Montagu, M. et
Conjugative transposons and the dis al. 1 98 1 . Physical mapping of DNA
semination of antibiotic resistance in base sequence homologies between a
Streptococci. Annu. Rev. Microbiol. octopine and a nopaline Ti plasmid. J.
40;635-59 Mol. Bioi. 1 52: 1 83-208
15. Darvill, A. , Albersheim, P. 1 984. Phy 28. Engstrom, P. , Zambryski, P . , Van Mon
toalexins and their elicitors-a defense tagu, M . , Stachel, S. E. 1987.
against microbial infection in plants. Characterization of Agrobacterium
Annu. Rev. Plant Physiol. 35:243-75 tumefaciens virulence proteins induced

16. Das, A . , Stachel, S. E . , Ebert, P . , by the plant factor acetosyringone. J.
Allenza, P. , Montoya, A. , Nester, E . Mol. Bioi. 1 97:635-45
1 986. Promoters o f A . tumefaciens Ti 29. Firmin, 1. L . , Wilson, K. E . , Rossen ,

plasmid virulence genes. Nucl. Acids. L . , 10hnston, A . W . B . 1986. Flavonoid
Res. 1 4 : 1355-64 activation of nodulation genes in Rhizo
17. Das, A. 1988. The A . tumefaciens virE bium reversed by other compuunds
operon encodes a single stranded DNA present in plants. Nature 423:90--9 2
binding protein . Proc. Natl. Acad. Sci. 30. Gardener, R. C, Knauf, V. 1986.
USA 85:2509- 1 3 Transfer of Agrobacterium DNA to
18. DeBeuckeleer, M . , Lemmers , M . , De plants requires a T-DNA border but not
Vos, G . , Willmitzer, L. , Van Montagu, the virE locus. Science 23 1 :725-27
M . , et al. 1 9l H . Further insight on the 31. Geilan, J., De Beuckeleer, M.,
transferred DNA of octopine crown gall. Seurinck, J . , DeBoeck, F . , D e Greve,
Mol. Gen. Genet. 1 83 : 283-88 H. , et al. 1 984. The complete nucleotide
19. De Framond, A . , Barton, K. A . , Chil sequence of the TL-DNA of the A .
ton, M-D. 1 983. Mini-Ti: a new vector tumefaciens plasmid pTiAch5 . EMBO J.
strategy for plant genetic engineering. 3, 835-846.
Biotechnology 1 :262-69 32. Gheysen , G . , Van Montagu , M . , Zam
20. DeGreve, H . , Decraemer, H . , Seurinck, bryski , P. 1 987. Integration of A .
1 . , Van Montagu, M . , Schell, 1. 1 98 1 . tumefaciens T-DNA involves rearrange
The functional organization of the octo ments of target plant DNA sequences.
pine A. tumefaciens plasmid pTiB6S3. Proc. Natl. Acad. Sci. USA 84: 6 1 69-73
Plasmid 6:235-48 33. Gietl, c . , Koukolikova-Nicola, Z. ,
21. Dejean, A . , Bougueleret, L . , Grzeschik, Hohn, B . 1 987. Mobilization of T-DNA
K. H . , Tiollais, P. 1 9l!6. Hepatitis B from Agrobacterium to plant cells in
virus DNA integration in a sequence volves a protein that binds single
homologous to v-erb-A and steroid re stranded DNA. Proc. Natl. Acad. Sci.
ceptor genes in a hepatocellular carcino USA 84:9006- 1 0
ma. Nature 322:70--7 2 34. Hirooka, T . , Kado, C. I . 1 986. Location
22. Della Valle, G . , Fenton, R. G . , Basili of the right boundary of the virulence
co, C 1 98 1 . Polyoma large T antigen region on A. tumefaciens plasmid
regulates the integratiun uf viral DNA pTiC58 and a hust specifying gene next
sequences into the genome of trans to the boundary. J; Bacterial. 168:237-
formed cells. Cell 23: 347-55 43
23. Douglas, C. 1 . , Halperin, W . , Nester, 35. Hirooka, T . , Rogowsky, P . M . , Kado,
W. W. 1982. Agrobacterium tumefa C . I. 1 987. Characterization of the virE
ciens mutants affected in attachment to locus of A. tumefaciens plasmid pTiC58
plant cells. J. Bacteriol. 1 52: 1 265-75 J. Bacteriol. 1 69: 1 529-36
24. Douglas, C. l . , Staneloni , R . I . , Rubin, 36. Hoekema, A . , Hirsch, P. R. , Hooykaas,
R. A . , Nester, W. W. 1 985. Identifica P.J.J . , Schilperoort, R. A. 1983. A bi
tion and genetic analysis of an Agrobac nary plant vector strategy based on sepa
terium tumefaciens chromosomal viru ration of vir - and T-region of the A .
lence region. J. Bacteriol. 1 6 1 : 850--60 tumefaciens T i plasmid. Nature 3 1 0:
25. Dressler, D . , Potter, H . 1982. Molecu 1 1 5-20
lar mechanisms in genetic recombina 37. Hoekema, A . , Roelvink, P. W.,
tion. Annu. Rev. Biochem. 5 1 :727-6 1 Hooykaas, P.I.J . , Schilperoort, R. A .
26. Dylan, T. , Ielpi, L . , Stanfield, S . , 1984. Delivery o f T-DNA from the A .
tumefaciens chromosome into plant dens C58 derivatives. Mol. Gen . Genet.
cells. EMBO J. 3:2485-90 207:47 1-77
3 8 . Holsters, M . , Silva, B . , Van Vliet, F. , 49. Koukolikova-Nicola, Z . , Shillito, R.
Genetello, c . , De Block, M . , et al. D . , Hohn, B . , Wang, K . , Van Montagu,
1 980. The functional organization of M . , et al. 1 985. Involvement of circular
the nopaline A. tumefaciens plasmid intermediates in the transfer of T-DNA
pTiC58. Plasmid 3:2 1 2-30 from Agrobacterium tumefaciens to
39. Holsters, M . , Villarroel, R . , Gielen, J . , plant cells. Nature 3 1 3 : 1 91-96
Seurinck, J. , D e Greve, H . , e t al. 1 983. 50. Koukolikova-Nicola, Z. , Albright, L. ,
An analysis of the boundaries of the Hohn, B. 1 987. The mechanism of T
octopine TL-DNA in tumors induced by DNA transfer from Agrobacterium
Agrobacterillm tllmefaciens. Mol. Gen. tumefaciens to the plant cell. In Plant
Genet. 1 90: 35-4 1 DNA Infectious Agents, ed. Th. Hohn,
40. Inze, D . , Follin, A . , Van Onckelen, H . , J. Schell, pp. 1 09-48 . New York:
Rudelsheim, P . , Schell, J . , et al 1 987 . Springer-Verlag
5 1 . Kucherlapati, R. S . , Eves, E . M . , Song,
Functional Analysis of the T-DNA onc

genes. In Molecular Biology of Plant K . Y . , Murse, B. S . , Smithies, 0. 1 984.
Growth Control, ed. J. E. Fox, M Homologous recombination between
Jacobs, pp. 1 8 1-96. New York: Liss plasmids in mammalian cells can be en
4 1 . Ippen-Ihler, K. A . , Minkley, E . G. hanced by pretreatment of input DNA.

1986. The conjugation system of F, the Proc. Natl. Acad. Sci. USA 8 1 :3 1 53-57
fertility factor of Escherichia coli. Annu. 52. Kustu, S . , Sei, K . , Keener, J. 1 986.
Rev. Genet. 20:593-624 Nitrogen regulation in enteric bacteria.
42. Janssens, A . , Genetello, C . , Van Mon Symp . Soc. Gen. Microbiol. 39: 1 39-54
tagu, M . , Zambryski, P. 1 986. Plant 5 3 . Kwok, W. W . , Nester, E. W . , Gordon,
cells induce transcription of the Agro M. P. 1 985. Unusual plasmid DNA
bacterium tumefadens nopaline pTiC58 organization in an octopine crown gall
virulence region, Plant Science 47: 1 85- tumor. Nue!. Acids Res. 1 3 :459-7 1
93 54. Leemans, J . , Deblaere, R . , Willmitzer,
43. Jayaswal, R. K . , Veluthambi, K . , Gel L . , De Greve, H . , Hemalsteens, 1. P . , et
vin, S . B . , Slightom, J. L. 1 987. Double al. 1 982. Genetic identification of func
stranded T-DNA cleavage and the tions of TL-transcripts in octopine crown
generation of single stranded T -DNA galls. EMBO J. 1 : 1 47-52
molecules in E. coli by a virD encoded 5 5 . Lemmers, M . , DeBeuckeleer, M . ,
border specific endonuclease from A . Holsters, M . , Zambryski, P. , Depicker,
tumefaciens. J . Bacterial. 1 69:5035- A . , et al. 1 980. Internal organization,
45 boundaries and integration of the Ti
44. Jen, G . , Chilton, M. D. 1 986. The right plasmid DNA in nopaline crown gall
border region of pTiT37 T-DNA is in tumors. J. Mol. Bioi. 144:353-76
trinsically more active than the left bor 56. Leroux, B . . , Yanofsky , M. J . , Winans,
der region in promoting T-DNA S. C . , Ward, J . E . , Ziegler, S . F . , et al.
transformation. Proc. Natl. Acad. Sci. 1987. Characterization uf the virA lucus
USA 83:3895-99 of Agrobacterium tumefaciens: a trans
45 . Jin, S . , Komari , T . , Gordon, M. P . , criptional regulator and host range deter
Nester, E. W . 1 987 . Genes responsible minant. EMBO J. 6:849-56
for the supervirulence phenotype of A . 57. Lichtenstein, C. P. , Fuller, S. L. 1 987 .
tumefaciens A28 1 . J. Bacterial. 1 69 : Vectors for the genetic engineering of
44 1 7-25 plants. In Genetic Engineering, Vol. 6.
46. Jones, J . D . G . , Gilbert, D. E. , Grady, pp. 103-83. New York: Academic Press
K. L . , Jorgensen, R. A. 1 987. T-DNA 58. Lin, F. W . , Sperle, K . , Sternberg, N .
structure and gene expression in petunia 1 984. Model for homologous recombi
plants transformed by A. tumefaciens nation during transfer of DNA into
C58 derivatives. Mol. Gen. Genet. mouse L cells: role for DNA ends in the
207:478-85 recombination process . Molec. Cell.
47. Joos, H . , Inze, D . , Caplan, A . , Sor BioI. 4: 1 020-34
mann, M . , Van Montagu, M . , Schell, J . 59. Machida, Y . , Usami, S . , Yamamoto,
1 983. Genetic analysis o f T-DNA tran A . , Niwa, Y . , Takebe, l. 1 986. Plant
scripts in nopaline crown galls. Cell inducible recombination between the 25
32: 1 057-67 bp border sequences of T-DNA in A .
48. Jorgensen, R. A . , Snyder, c . , Jones, tumefaciens. Mol. Gen. Genet. 204:
1 . D.G. 1 987. T-DNA is organized pre 374-82
dominantly in inverted repeat structures 60. Marks, J. R . , Lynch, T. J . , Karlinsey, J .
in plants transformed with A . tumefa- E . , Thomashow, M . F. 1987. A . tumefa-
28 ZAMBRYSKI
ciens virulence locus pscA is related to Agrobacterium. In Plant DNA Infectious

the Rhizobium meliloti exoC locus . 1. Agents, ed. Th. Hohn, 1. Schell , pp.
Bacteriol. 1 69:5835-37 179-203. New York: Springer-Verlag
61. Melchers, L. S . , Thompson, D. V . , 73 . Ronson, C. W . , Nixon, B. T . , Ausubel,
Idler, K . B . , Schilperoort, R. A . , F. M. 1987. Conserved domains in
Hooykaas, P . J . J . 1 986. Nucleotide se bacterial regulatory proteins that respond
quence of the virulence gene virG of to environmental stimuli. Cell 5:579-
Agrobacterium tumefaciens octopine Ti 81
plasmid: significant homology between 74. Rubin, R . A . 1 986. Genetic studies on
virG and the regulatory genes ompR, the role of octopine T-DNA border re
phoB, and dye of E.coli. Nucl. Acids gions in crown gall tumor formation.
Res. 24:9933-42 Mol. Gen. Genet. 202:3 1 2-20
62. Morris, R. O. 1 986. Genes specifying 75. Saedler, H., Nevers, P. 1985.
auxin and cytokinin biosynthesis in phy Transposition in plants : a molecular
topathogens. Annu. Rev. Plant Physiol. model. EMBO 1. 4:585-90
37:509-38 76. Shaw, C. H . , Watson, M. D . , Carter,

63. Ninfa, A . , Magasanik, B. 1 986. Cova G. H . , Shaw, C. H. 1984. The right
lent modification of the glnG product, hand copy of the nopaline Ti plasmid 25
NR 1 , by the glnL product, NRll, reg bp repeat is required for tumor forma
ulates transcription of the glnALG op tion. Nuc/. Acids Res. 1 2:603 1 -4 1
eron in E.coli. Proc. Natl. Acad. Sci. 77. S impson, R . B . , O'Hara, P . 1 . , Kwok,
USA 83:5909- 1 3 W . , Montoya, A. L . , Lichtenstein, C . ,
64. Otten, L . , DeGreve, H . , Leemans, J . , et a l . 1 982. DNA from the A6S/2 crown
Hain, R . , Hooykass, P . , e t al. 1 984. gall tumor contains scrambled Ti plas
Restoration of virulence of vir region mid sequences near its junctions with the
mutants of A . tumefaciens strain B6S3 plant DNA. Cell 29: 1 005- 1 4
by coinfection with normal and mutant 78. Slightom, J . L . , Jouanin, L . , Leach, F . ,
Agrobacterium strains. Mol. Gen. Drong, R . F. , Tepfer, D. 1 98 5 . Isolation
Genet. 1 95: 1 59-63 and identification of TL-T-DNA Iplant
65 . Peralta, E. G . , Ream , L. W . 1985 . T ju nction s in Convolvulus arvenis trans
DNA border sequences required for formed by Agrobacterium rhizogenes
crown gall tumorigenesis. Proc. Natl. strain A4. EMBO 1. 4:3069-77
Acad. Sci. USA 82:5 1 1 2- 1 6 79. Spielman, A . , Simpson, R. B . 1 986.
66. Peralta, E . G . , Hellmiss, R . , Ream, R. T-DNA structure in transgenic tobacco
1 986. Overdrive, a T-DNA transmission plants with mUltiple independent inte
enhancer on the A . fumefaciens tumor gration sites. Mol. Gen. Genet. 205:34-
inducing plasmid. EMBO 1. 5 : 1 1 37-42 41
67 . Peters, K . N . , Frost, J . W . , Long, S. R . 80. Staehel , S . E . , Messens, E . , Van Mon
1 986. A plant flavone, luteolin, in duces tagu , M., Zambryski, P. 1985.
expression of Rhizobium meli/oti Identification of the signal molecules
nodulation genes. Science 233:977-80 produced by wounded plant cells which
68. Porter, S. G . , Yanofsky, M. f. , Nester, activate the T-DNA transfer process in
E. W. 1 987. Molecular characterization Agrobacterium tumefaciens. Nature
of the virD operon from A. tumejaciens. 3 1 8:624-29
Nucl. Acids Res. 1 5:7503- 1 7 81. Stachel, S . E . , Nester, E. W . , Zambry
69. Powell, B . S . , Powell, G . K . , Morris, ski, P. 1986. A plant cell factor induces
R. 0 . , Rogowsky, P. M . , Kado, C. I . Agrobacterium tume/aciens vir gene ex
1987. Nucleotide sequence o f the virG pression. Proc. Nat!. Acad. Sci. USA
locus of the Agrobacferium fumejaciens 83:379-83
plasmid pTiC58 . Mol. Microbiol. I : 82. Stachel, S . E. , Nester, E. W. 1 986. The
309- 1 6 genetic and transcriptional organization
70. Puvanesarajah , V . , Schell, F . M . , of the vir region of the A6 Ti plasmid of
Stacey, G . , Douglas, C . J . , Nester, E . Agrobacterium. EMBO 1. 5 : 1 445-
W . 1 985. Role for 2-linked beta-D 54
glucan in the virulence of A . tumeja 8 3 . Staehel, S. E . , Zambryski, P. 1 986.
ciens. 1. Bacteriol. 164: 1 02-6 virA and virG control the plant-induced
71. Redmond, J. W., Bartley, M., activation of the T -DNA transfer process
Djordjevic, M . A . , I nne s R . W. ,
, of Agrobacterium tumejaciens. Cell
Kuempel, P. L . , et al. 1 986. Flavones 46:325-33
induce expression of nodulation genes in 84. Stachel, S. E. Timmerman, B . , Zam
Rhizobium. Nature 323 :632-35 bryski , P. 1 986. Generation of single
72. Rogers, S. G . , Klee, H. 1 987. Pathways stranded T-DNA molecules during the
to plant genetic manipulation employing initial stages of T-DNA transfer from
Agrobacterium tumefaciens to plant bacterium tumefaciens. 1. Mol. Bioi.

cells. Nature 322:706- 1 2 1 88: 1 29--45
85. Stachel, S . E . , Zambryski, P . C . 1986. 96. Veluthambi, K . , Jayaswal, R. K . , Gel
Agrobacterium tumefaciens and the sus vin, S. B. 1987. Virulence genes A, G ,
ceptible plant cell: a novel adaptation of and D mediate the double stranded bor
extracellular recognition and DNA con der cleavage of T-DNA from the Agro
jugation . Cell 47: 1 5 5-57 bacterium Ti plasmid. Proc. Nat!. Acad.
86. Stachel, S. E . , Timmerman, B . , Zam Sci. USA 84: 1 88 1-85
bryski, P. 1987. Activation of Agrobac 97. Veluthambi, K . , Ream, W . , Gelvin, S .
terium tumefaciens vir gene expression 1 988. Virulence genes, borders, and
generates multiple single-stranded T overdrive generate single stranded T
strand molecules from the pTiA6 T DNA molecules from the A6 Ti plas
region: requirement for 5 ' virD prod mid of Agrobacterium. J. Bacteriol.
ucts. EMBO J. 6:857-63 170: 1 523-32
87. Tempe, J . , Petit, A. 1 982. Opine utiliza 98. Wallroth, M . , Gerats, A.G. M . , Rogers,
tion by Agrobacterium. In Molecular Bi S. G . , Fraley , R. T . . Horsch. R. B .
ology of Plant Tumors, G. Kahl, J . 1986. Chromosomal localization o f for

Schell, p p . 451-59. New York: Aca eign genes in Petunia hybrida . Mol.
demic Press Gen. Genet. 202:6- 1 5
88. Thomashow, M. F . , Nutter, R . , Mon 9 9 . Wang, K . , Herrera-Estrella, L . , Van

toya, A . , Gordon, M. P . , Nester, E. W . Montagu, M . . Zambryski, P. 1984.
1980. Integration and organization o f Ti Right 25 bp terminus sequences of the
plasmid sequences in crown gall tumors. nopaline T-DNA is essential for and de
Cell 19:729-39 termines direction of DNA transfer from
89. Thomashow, M. F . , Karlinsey, J. E . , Agrobacterium to the plant genome. Cell
Marks, J . R . , Hurlbert, R. E. 1 987. 38:35--4 1
Identification of a new virulence locus in 100. Wang, K . , Stachel, S. E . , Timmerman,
A. tumefaciens that affects polysaccha B . , Van Montagu, M . , Zambryski, P.
ride composition and plant-cell attach 1987. Site-specific nick occurs within
ment. J. Bacteriol. 1 69:3209- 1 6 the 25 bp transfer promoting border se
9 0 . Timmerman, B . , Van Montagu, M . , quence following induction of vir gene
Zambryski, P . 1988. Vir-induced re expression in Agrobacterium tumefa
combination in Agrobacterium: physical ciens. Science 235:587-91
characterization of precise and imprecise 1 0 1 . Wang, K . , Genetello, c . , Van Monta
T-circle formation. 1. Mol. Bioi. In gu, M. , Zambryski, p, 1987. Sequence
press context of the T-DNA border repeat ele
9 1 . Traxler, B. A . , Minkley, E. G. 1987. ment determines its relative activity dur
Revised genetic map of the distal end of ing T-DNA transfer to plant cells. Mol.
the F transfer operon: implications for Gen. Genet. 2 1 0:338-46
DNA helicase I , nicking at onT, and \02. Ward, J. E . , Akiyoski, D. , Regier, D . ,
conjugal DNA transport. J. Bacteriol. Datta, A . , Gordon , M . P . , et al. 1988.
169:325 1-59 Characterization of the virB operon from
92. Ursie, D . , Slightom, J. L . , Kemp, J. D. an Agrobacterium tumefaciens Ti plas
1 983. A. tumefaciens T-DNA integrates mid. J. Bioi. Chem. 263:5804- 1 4
into mulitple sites of the sunflower 1 0 3 . Willetts, N . , Skurray, R. 1987. Struc
crown gall genome. Mol. Gen. Genet. ture and function of the F factor and
1 90:494-503 mechanism of conjugation. In Es
93. Usami, S . , Morikawa, S . , Takebe, 1 . , cherichia coli and Salmonella typhimur
Machida, Y . 1987. Absence in monoco ium. Cellular and Molecular Biology,
tyledonous plants of the diffusible plant Vol. 2, ed. F. C. Neidhardt, J. L. In
factors inducing T-DNA circularization graham, K. B . Low, B. Magasanik, M .
and vir gene expression in Agrobacter Schaechter, and H . E . Umbarger, pp.
ium. Mol. Gen. Genet. 209:221-26 1 1 1 0-33. Am. Soc. Microbiol. Wash
94. Van Haaren, M . J .J . , Sedee, N J . A . , ington, DC
Schilperoort, R . A . , Hooykaas, P.J.J. \04. Winans, S. C . , Ebert. P. R . , Stachel, S .
1987 . Overdrive is a T-region transfer E . , Gordon, M . P. , Nester, E. W . 1986.
enchancer which stimulates T-strand A gene essential for Agrobacterium viru
production in A . tumefaciens. Nucl. lence is homologous to a family of posi
Acids Res. 1 5 :8983-97 tive regulatory loci. Proc. Nat!. Acad.
95. Van Lijsebettens, M . , Inze, D . , Schell , Sci. USA 83:8278-82
J. , Van Montagu, M. 1 986. Trans \05. Winans, S. c . , Allenza, P . , Stachel, S .
formed cell clones as a tool to study E . , McBride, K . E . , Nester, E. W .
T-DNA integration mediated by Agro- 1987. Characterization of the virE
30 ZAMBRYSKl
operon of the Agrobacterium Ti plasmid K . , Goodman, H. 1982. Tumor induc

pTiA6. Nucl. Acids. Res. 15:825-37 tion by Agrobacterium tumefaciens:
106. Yadav, N. S . , VanderJeyden, J . , Ben analysis of the boundaries of T-DNA. J.
nett, D. R. , Barnes, W. M . , Chilton, Mol. Appl. Genet. 1 :36 1-70
M . -D . 1982. Short direct repeats flank 1 1 2. Zambryski, P . , Joos, H . , Genetello, C . ,
the T-DNA on a nopaline Ti plasmid. Leemans, J . , Van Montagu, M . , Schell,
Proc. Natl. Acad. Sci. USA 79:6322-26 J. 1983 . Ti plasmid vector for the in
1 07 . Yamamoto, A . , Iwahashi, M. , Yanov troduction of DNA into plant cells with
sky, M . F. , Nester, E. W . , Takebe, I . , out alteration of their normal regenera
Machida, Y . 1987. The proximal region tion capacity. EMBO J. 2:2 1 43-50
in the virD locus of Agrobacterium 1 13. Zambryski, P . , Herrera-Estrella, L . , De
tumefaciens is necessary for the plant Block, M . , Van Montagu, M . , Schell, J.
inducible circularization of T-DNA. 1984. The use of the Ti plasmid of Agro
Molec. Gen. Genet. 206: 174-77 bacterium to study the transfer and ex
108. Yanofsky, M . , Lowe, B . , Montoya, A . , pression of foreign DNA in plant cells:
Rubin, R . , Krul, e t al. 1985. Molecular new vectors and methods. In Genetic
and genetic analysis of factor controlling Engineering, Principles and Methods,
host range in Agrobacterium tumefa Vol. 6, ed. J. SetJow, A. Hollaender,
ciens. Mol. Gen. Genet. 201 :237-46 pp. 253-78. New York: Plenum
109. Yanofsky, M. F. , Nester, E. W. 1986. 1 1 4. Zambryski, P. 1988 . Agrobacterium

Molecular characterization of a host plant cell DNA transfer. In Mobile DNA,
range determining locus from Agrobac ed. D. Berg, M. Howe Washington,
terium tumefaciens. J. Bacteriol. DC: Am. Soc. Microbiol. In press
168:244-50 115. Zorreguieta, A . , Geremia, R. A . ,
1 1 0. Yanofsky, M. F . , Porter, S. G . , Young, Cavaignac, S . , Cangelosi. G . A . , Nes
C . , Albright, L. M . , Gordon, M. P . , et ter, E. W . , Ugalde. R. A . 1988. A chro
al. 1986. The virD operon of Agrobac mosomal virulence locus of Agrobacte
terium tumefaciens encodes a site rium tumefaciens is the structural gene
specific endonuclease. Cell 47:471-77 for an intermediate in f3- 1 , 2-glucan syn
1 1 1. Zambryski, P . , Depicker, A . , Kruger, thesis. J. Bacteriol. In press

BASIC PROCESSES UNDERLYING Agrobacterium Mediated DNA Transfer To Plant Cells

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BASIC PROCESSES UNDERLYING Agrobacterium Mediated DNA Transfer To Plant Cells

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ANNUAL

BASIC PROCESSES UNDERLYING

Division of Molecular Plant Biology, Hilgard Hall, University of California,

Agrob acterium tumefaciens is a soil phytopathogen that genetically trans­

Agrobacterium carries three genetic components required for plant cell

personal communication) . The pscA locus is approximately 3.0 kbp and is

ACTIVATION AND EXPRESSION OF GENES UNDER

vir A vir B virG vlre vir D virE LocuS

=:t> C>ct> <3:=' [> ==t>

At least five additional proteins, designated virulence-related-proteins

GENERATION OF A TRANSFERABLE T-DNA COpy

Structural Requirements for T-DNA Transfer

octopine-producing tumor tissues, respectively. The nopaline T-DNA is one

homology between sequences present in the Ti plasmid and those in trans­

flanks the T-DNA region of the Ti plasmids as direct (albeit, imperfect)

The T-DNA Transfer Intermediate: Circular, Linear, Single­

Recent studies further characterize the (albeit rare) formation of T-circles in

molecule . This structure is hinted at by data that detected double stranded

The T-Strand as the Transfer Intermediate

termination sites of T -strand synthesis, respectively , while T -region structures

plasmid. The copy number of T-strands is approximately one per AS-induced

Protein Products Which Function To Generate Transferable

may be nonessential or have another function.

polymerase(s), and repair enzymes, are expected to be necessary components

30-60 nucleotide substrate. This number of molecules would represent ap­

dehydrogenase, RNA polymerase, C1C2 polymerase, and others), and true

of E.coli S SB and bacteriophage T4 gene 32 protein: (i) ssDNA binding, (ii)

be complemented to wild-type virulence if they are coinoculated on plants

Model for the Mechanism of T-DNA Transfer

Bacterial Conjugation Versus T-DNA Transfer

rized and compared to T-DNA transfer (see also 1 14) .

potentially an RNA primer is synthesized de novo. Some conjugative plas­

occur by plant cell enzymes. The Agrobacterium system differs from F

laboratory conditions which provide Agrobacterium with an excess of AS to

Agro bacterium also synthesizes a T-DNA transfer specific ssDNA binding

abundant virB specific polypeptides which fractionate to the cell envelope in

INTEGRATION OF THE T-DNA INTO THE PLANT

cotyledonous plant species including tobacco ( 18 , 55 , 79, 88, 95) , petunia

patterns or restriction fragment length polymorphisms. Nine out of the ten

may be better compared to more disruptive integration events exemplified by

protein complex. Second, following localization of the complex to the cell

SUMMARY AND PROSPECTS

wheat, produce significant levels of vir inducing compounds whose chemical

DNA only if it is treated with S 1 nuclease prior to electrophoresis (84); this is

tion provides an avenue to explore several fundamental plant processes, from

interkingdom DNA transfer, the underlying processes may be neither exotic

solve many questions related to the biology of the Agrobacterium-plant cell

I . Albright, L. M . , Yanofsky, M. F . , A . tumefaciens octopine Ti plasmid

insertions in the Lycopersicon genome. Kashyap, L . , Douglas, C. , e t al . 1 986.

Annu. Rev. Plant Physiol. 35:243-75 tumefaciens virulence proteins induced

1 986. Promoters o f A . tumefaciens Ti 29. Firmin, 1. L . , Wilson, K. E . , Rossen ,

Functional Analysis of the T-DNA onc

4 1 . Ippen-Ihler, K. A . , Minkley, E . G. hanced by pretreatment of input DNA.

ciens virulence locus pscA is related to Agrobacterium. In Plant DNA Infectious

37:509-38 76. Shaw, C. H . , Watson, M. D . , Carter,

Agrobacterium tumefaciens to plant bacterium tumefaciens. 1. Mol. Bioi.

ology of Plant Tumors, G. Kahl, J . 1986. Chromosomal localization o f for­

88. Thomashow, M. F . , Nutter, R . , Mon­ 9 9 . Wang, K . , Herrera-Estrella, L . , Van

operon of the Agrobacterium Ti plasmid K . , Goodman, H. 1982. Tumor induc­

109. Yanofsky, M. F. , Nester, E. W. 1986. 1 1 4. Zambryski, P. 1988 . Agrobacterium­

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Agrob acterium tumefaciens is a soil phytopathogen that genetically trans

homology between sequences present in the Ti plasmid and those in trans

The T-DNA Transfer Intermediate: Circular, Linear, Single

30-60 nucleotide substrate. This number of molecules would represent ap

potentially an RNA primer is synthesized de novo. Some conjugative plas

ology of Plant Tumors, G. Kahl, J . 1986. Chromosomal localization o f for

88. Thomashow, M. F . , Nutter, R . , Mon 9 9 . Wang, K . , Herrera-Estrella, L . , Van

operon of the Agrobacterium Ti plasmid K . , Goodman, H. 1982. Tumor induc

109. Yanofsky, M. F. , Nester, E. W. 1986. 1 1 4. Zambryski, P. 1988 . Agrobacterium