Agrobacterium tumifaciens Horizontal gene transfer Interkingdom gene transfer Virulence or Vir a b c d e f g genes Crown gall disease Regulation of vir genes Relaxosome

1. Dr. IshanY. Pandya (Academic Faculty, Head and Fellow)
PhD BIOTECHNOLOGY, M.Sc., MBA Biotechnology
E-mail: genomes.world37@gmail.com
GEER Foundation, DST-IUCN (Member)
Agrobacterium Tumifaciens: Fundamental
concepts, and Application in Plant biotechnology

2. AGROBACTREIUM-Nature’s genetic engineer
Agrobacterium tumefaciens is a gram negative, motile, rod shaped
bacterium which is non sporing, and is closely related to the N-
fixing rhizobium bacteria which form root nodules on leguminous
plants. The bacterium is surrounded by a small number of
peritricious flagella.
Virulent bacteria contain one or more large plasmids, one of
which carries the genes for tumor induction and is known as the
Ti (tumor inducing) plasmid. The Ti plasmid also contains the
genes that determine the host range and the symptoms, which the
infection will produce. Without this Ti plasmid, the bacterium is
described as being non virulent and will not be able to cause
disease on the plant.
WHY CALLED NATURE’S GENETIC ENGINEER?

3. Crown galls-what are they? Crown Galls first appear as small, white,
soft protrusions, initially found at the base of the plant stem. As the
tumors enlarge, the surface takes on a mottled dark brown appearance
due to the death and decay of the peripheral cells.
When infected with the bacterium, plants may also become stunted,
produce small chlorotic leaves, and are more susceptible to extreme
environmental conditions.
A. tumefaciens is most well known for its ability to integrate a small
part of the Ti plasmid into the host plant genome, which causes the
plant cells to become cancer cells and produce specific compounds
called Opines, which the bacterium utilize as a carbon source. The
bacterium redirects the metabolic activities of the plant to produce
compounds specific to the bacterium. It is this process which gives
A.Tumefaciens its potential to be used as a tool for plant transformation

4. CROWN GALL TUMOR

5. ATTACHMENT AND PENETRATION
 The initial pre-penetration event in the soil rhizosphere is the conjugal transfer of the Ti plasmid,
therefore increasing the number of pathogenic isolates in the soil. Quorum sensing proteins TraI and TraR
induce the expression of genes required for bacterial cell mating and mobilization of the plasmid. This
response is also affected by opines produced by infected plants, which either suppress or activate a
repressor of the TraR gene
 A. tumefaciens possess swimming motility which is mediated by flagella. It is thought that migration occurs
towards sugars and amino acids which accumulate around plant roots in the rhizosphere. Some strains
may also be attracted to specific plant compounds released from wounded plants such as acetosyringone,
and also to opines.
 Attachment to the plant is a two stage process, firstly involving a weak initial adhesion, then the bacteria
synthesize cellulose fibrils which anchor them to the wounded plant cell surface. Some of the bacterial
genes required for this process have been identified, namely chvA, chvB, pscA and att, as a mutation in any
of these genes leaves the bacterium unable to attach to the plant. There are also molecules within the
plant which are thought to be involved in the attachment process. One such molecule is vitronectin; an
adhesive glycoprotein which is a component of the plant extracellular matrix (ECM). Vitronectin is more
commonly associated with the cohesion of plant cells, thus having a role in plant structure and rigidity

6. INTERKINGDOM GENE TRANSFER
 During crown gall tumerogenesis, only the T-DNA and
some proteins are transferred into the host cell.
 The T-DNA itself is not sequence-specific and is defined
exclusively by its left and right borders which are two 25-
bp direct repeats
 SOME OBSERVATIONS –
1. Crown gall tumor cells continue to grow and produce
opines even after the bacteria are killed by antibiotics.
2. When hybridization is done between T-DNA and dna
isolated from bacteria free tumor cells, dna from non-
transformed cells didn’t hybridize

7. Vir Gene Function
Vir A, Vir G Sense phenolic compounds from wounded plant cells and induce expression
of other virulence genes
VirD2 Endonuclease; cuts T-DNA at right border to initiate T-strand synthesis
Vir D1 Topiosomerase; Helps Vir D2 to recognize and cleave within the 25bp
border sequence
Vir D2 Covalently attaches to the 5I end of the T-strand, thus forming the
T-DNA Complex. Also guides the T-DNA complex through the nuclear pores
Vir C Binds to the 'overdrive' region to promote high efficiency T-strand
Synthesis
Vir E2 Binds to T-strand protecting it from nuclease attack, and intercalates
with lipids to form channels in the plant membranes through which the
T-complex passes
Vir E1 Acts as a chaperone which stabilizes Vir E2 in the Agrobacterium
Vir B & Vir D4 Assemble into a secretion system which spans the inner and outer bacterial
membranes. Required for Export of the T-complex and Vir E2 into the
plant cell

8. REGULATION OF VIR GENES

9.  The transformation process begins with the induction of the Agrobacterium VirA-VirG sensory machinery
and subsequently the virulence (Vir) proteins by host-specific small phenolic signal molecules.
 VirA acts as a membrane sensor protein, whereas VirG regulates the cytoplasmic response to wounded
plant cell phenolic compounds and promotes activation of all the Vir genes. VirG specifically interacts
with the vir box; a conserved 12 base pair sequence located in the promoter sequence of all the vir
genes.
 Plant phenolics are known to be bacteriostatic at higher concentrations, a well known plant defense
mechanism. To overcome this, a VirH protein is expressed as a result of VirG, which is believed to
detoxify these harmful compounds
 The VirD1-VirD2 protein complex acts as an endonuclease which nicks both borders on the noncoding
strand of the T-DNA region.
 By strand replacement-ss T-dna with virD2 at 5’ end is released – gets coated with virE2 – forms
MATURE T-DNA COMPLEX.
 The T-complex requires a specific export system to deliver it across both the bacterial envelope and the
plant cell membrane and into the plant cell cytoplasm. T-complex export occurs via a type IV secretion
mechanism, comprising of a filamentous pilus and a transporter complex that translocates substances
through the cell membranes. In A. tumefaciens, the type IV secretion system is assembled from proteins
encoded by the virD4 gene and the virB operon. Eleven VirB proteins are encoded by the VirB operon,
all of which have a role to play in the transport of the T-complex across the membrane. VirB1 initiates
the assembly, and VirB2 is the main structural protein in the pilus. The pilus was proposed by Zupan et
al.
 This type IV secretion system and the genes are homologous in many spp. And its involvement in
CONJUGATION and PROTEIN EXPORT suggests that conjugation maybe a specialized form of
protein export

11. VirB pilus
 Agrobacterium produces a virB/D4 dependent pilus on induction by vir regulon. The several proteins and
functions are listed in the figure.
 virB2-major component of the pilus. has sequence similarity to traA of F PLASMID.
 virB4-innermembrane protein.sequence similarity to TraC. Has ATPase actiity and required for transfer
of VirB3 to its location on outer membrane.
 virB5-innermembrane protein. Sequence similarity with TraE and is involved in Pilus assembly
 virB10+B9+B11-elevated levels of all 3 are required to overcome RSF mediated inhibition. virB11 also
has ATPase and kinase activity. ATPase activity-drives the assembly of B9+B10 and also provides force to
the macromolecules to move through the pore after assembly.
 virB10-forms high molecular weight aggregates which form the transmembrane transporter.
 virB7-outer membrane lipoprotein which stabilizes B9 by forming disulfide bonds with it.
 virB8-affects in accumulation of B9.
 virB1-requiredfor the export of E2 but not the t-strands. It has a sequence similarity with LYSOZYME
and has GLYCOSIDASE activity.it also occurs on both, inner and outer, membranes. All these properties
allow it to create openings in bacterial cell wall.

13. CONJUGATION MODEL OF T-DNA TRANSFER
 In many ways,T-DNA transfer resembles broad host range plasmid conjugation
process-@binding of a multi-subunit endonuclease.. @nicking DNA at 5’end..
@transfer of a ss-DNA from a donor to recepient@ Border sequences share
homology with ORiT of plasmids
 Support for conjugation model-RSF1010 transfer into plant cells by the bacterium
 An essential protein called COUPLING PROTEIN links the relaxosome to the
transmembrane secretion apparatus.this is also called MATING PAIR FORMATION
SYSTEM.
 BORDER SEQUENCES-right and left,23b.p.
 OVERDRIVE sequence-flanks right border, increases transfer process 100 fold

14. RELAXOSOME
 Several vir operons take part in T-DNA transfer.virD1 and virD2 encode a site specific
endonuclease- nicks the bottom strand of border b/w 3rd
and 4th
base
 Vird2-attaches to 5’end of nicked DNA via phosphodiester bond with specific tyrosine
residue.
 OVERDRIVE sequence is near the right border and together with virC1 and virC2,
stimulates tumorigenesis 100 fold. The T-DNA transfer is unidirectional and begins at the
right border and this distinguishment is made possible with overdrive sequence.
 Induction of vir genes and border nicking leads to formation of T-STRANDS. These are full
length, linear, ss-DNA comprised of bottom strand of T-DNA.
 QUE-HOW DO WE KNOW THAT IT’S THE BOTTOM STRAND THAT IS GETTING
TRANSFERRED AND THAT ONLY SS-DNA GETS TRANSFERRED??
 T-STRAND displacement occurs 5’-3’, requires helicase activity and is accompanied by the
synthesis of a new copy of the bottom strand.

15. VirE2
 VirE2, a ssDNA-binding protein of 533a.a, is presumed to coat the T-strand along its length.
As do most ssDNA-binding proteins, VirE2 binds ssDNA cooperatively and without sequence
specificity
 Mutational analysis of VirE2 revealed that the amino-terminal part of the protein is important
for its binding cooperatively, while its carboxy -terminal portion is essential for ssDNA
binding. Importantly, the carboxy-terminal part of VirE2 also contains an RPR motif, which
likely functions as a signal for protein export from Agrobacterium into plant cells through the
VirB/VirD4 channel
 What’s the length of T-dna which can be transferred??
 The T-complex matures in the bacterial or the host cell??
 There is the presence of nuclear localizing signals {NLS} in virE2 suggesting that it enters
plant nuclei during infection. proved by an experiment.
 T-strands are most probably associated with VirE2—which provides them with the necessary
protection within the host cell cytoplasm—and imported into the nucleus as a mature T-
complex.

16.  COMPLEMENTATION by mixed infection.
 Interaction cloning experiments identified protein contacts b/w virE2 and virE1. virE1 binds to
domains of virE2 involved in binding ss-DNA and it prevents virE2 aggregation and premature
binding of virE2 to ss-DNA.
The export model seeks to explain several observations like:
1. virE2 made in 1 strain can interact with T-STRANDS generated in other
2. Export of E2 requires E1 but t-strand transfer doesn’t
3. Presence of plasmid RSF1010 blocks virE2 export while only reduces t-strands transfer
 Thus, from the above studies we can conclude that agrobacterium can export virE2 and t-strands
seperately and that virE2 and T-strands are exported seperately, although the latter part is still vague
and subjected to ambiguity.

17. VirD2-the pilot
 virD2 has a conserved tyrosine residue in its domain through which it attaches to the t-strand.
 The N-terminal half has the endonuclease activity while the C-terminal is required for tumorigenesis.
This domain also has a NLS for targetion in nucleus.
 virD2 also participates in INTEGRATION. sequences indicate that right hand ends of the integrated
DNA correspond excatly to the base at which the t-strands attach to virD2.
 Also, MUTATIONS in virD2 reduce integration or result in t-dna with aberrant right hands.
 virD2 also shows LIGASE activity. It can ligate the cut ss-Dnas containing the bottom strand toreform
the orignal substrate or join virD2 bound portion to an OLIGONUCLEOTIDE. This ligase activity is
responsible for joining the 5’ ends of t-strand to the plant DNA.
 Thus, virD2 shows ENDONUCLEASE,TARGETION,INTEGRATION & LIGATION activity….

18. VirD4-gateway to the pore
 virD4 is similar to the pTi encoded TraG and can substitute the latter allowing
conjugal transfer of RSF1010 through the pilus.
 Thus, VirD4 is an interface between the relaxosome and the transmembrane pore.
 Besides, export of virE2 also requires virD4 astablishing its role in protein transport.
 Formation of pilus also requires virD4 indicating that it participates in translocation
of pilus proteins as well.

19. Molecular Structure of the Mature T-complex
 Complexes, formed in vitro by interaction between purified VirE2 and the bacteriophage M13
ssDNA, were examined by scanning transmission electron microscopy, followed by mass
analysis. These analyses revealed that the VirE2-ssDNA association produces rigid and coiled
filaments that are 12.6 nm-wide, with a density of 58 kDa/nm, and that each turn of the
filament coil contains an average of 3.4 molecules of VirE2 and 63.6 bases of ssDNA.
 Based on these parameters, a 22-kb T-strand of the wild-type nopaline-specific Agrobacterium is
calculated to associate with 1,176 molecules of VirE2
 The length of the mobilized T-strand, when coated with VirE2 molecules, is estimated to
range between 40 nm and 80 microns for T-DNA regions between 20 Kb and 150 Kb
 the T-DNA outer diameter(12.6 nm) exceeds the orifice of the nuclear pore diffusion channels
(9 nm), but it is easily compatible with the size-exclusion limit of the nuclear pore, which
reaches 23-39 nm during the process of active nuclear uptake.
 Thus, packaging (by VirE2) is essential for nuclear import.
 WHAT IF VirE2 WERE ABSENT??

20. Molecular structure of the T-DNA region and T-complex

21. T-complex nuclear import
 T-complexes are polar molecules and their nuclear import is thought to occur in a polar fashion.
virD2 contains a NLS at the C terminal while the virE2 has a NLS located in the middle of the
molecule. (Mutations within the central region of the VirE2 sequence decreased Agrobacterium
tumorigenicity but did not affect the ssDNA-binding activity or stability of the protein)
 Using microinjection of in vitro-formed T-complexes, the ability of VirE2 to direct fluorescently
labeled ssDNA into the plant cell nucleus was studied. EXPERIMENT TO DEMONSTATE ROLE
OF E2 IN NUCLEAR IMPORT:
1. Microinject fluorescently labelled ds and ss DNA into nucleus-only cytoplasmic fluorescence seen.
2. Microinjection of in-vitro-formed VirE2-ssDNA complexes and VirE2-dsDNA-only ss-DNA
showed accumulation in the nucleus
3. Add nuclear import-specific inhibitors such as wheat germ agglutinin and nonhydrolyzable analogs of
GTP-nuclear import of VirE2-ssDNA was blocked

22. VirD2 & VirE2-COMPLEMENTARY ACTION
 Several approaches have been utilized to investigate whether VirD2 and VirE2 may
perform different but complementary functions during nuclear import of the T-
complexes
1. using a heterologous living nonplant cell system which lacks nuclear transport machinery
of the Agrobacterium natural host cells, differences between VirD2 and VirE2 nuclear
import could be discerned - VirD2 localized in the nucleus of Xenopus oocytes, Drosophila
embryos, 83 human kidney and HeLa cells. VirE2, on the other hand, did not.
2. in vitro-formed VirE2-VirD2-ssDNA complexes were tested for their import into plant
nuclei in vitro
3. RecA, a protein that can bind ssDNA is capable of replacing VirE2 during nuclear import
of long T-DNAs, but not during earlier events of T-DNA transfer to plant cells.
 CONCLUSIONS - It was thus suggested that VirD2 and VirE2 perform complementary
functions in T-complex nuclear import. 70 While VirD2 initially directs the T-complex
into the nuclear pore, VirE2 may shape it in a transferable form and assist translocation of
the entire T-complex into the host cell nucleus. Collectively, functional differences
between VirD2 and VirE2 suggest that (i) in plant cells, VirE2 and VirD2 employ
different cellular factors for their nuclear entry, and (ii) animal cells lack the subset of
factors that recognize VirE2 and help its nuclear uptake in plant cells

23. HOST PROTEINS THAT INTERACT WITH VirD2 and VirE2
 One set of VirD2-interacting host proteins are members of a large cyclophilin family of
peptidyl-prolyl cis-trans isomerases (PPIases)- cyclophilin DIP1, Roc1, Roc4, 98 and
CypA They have been proposed to maintain the proper conformation of VirD2 within the
host cell cytoplasm and/or nucleus during T-DNA nuclear import and/or integration. may
act as molecular chaperones
 Another host cellular factor that binds VirD2 is the tomato DIG3 protein. This protein, a
type 2C serine/threonine protein phosphatase (PP2C), was found to interact specifically with
the VirD2 NLS region - An Arabidopsis abi1 mutant, knocked out in a PP2C homolog,
exhibited higher sensitivity to Agrobacterium-mediated genetic transformation, than did wild-
type plants. In addition, over-expression of DIG3 in tobacco protoplasts specifically inhibited
nuclear import of a GUS-VirD2 NLS fusion protein
 A third type of VirD2-interacting host protein is a member of the growing karyopherin α
family, known to mediate nuclear import of many NLS-containing proteins-AtKAPα was
found to possess the classical features typical of karyopherin proteins: it contains eightα
contiguous repeats of the “arm” motif 107 and four amino-terminal clusters of basic amino
acids.-- arm” motifs are thought to recognize the imported cargo through its NLSs while the
amino terminal basic domain is thought to interact with the karyopherin ß proteins

24.  unlike VirD2, VirE2 did not interact with AtKAP in the yeast two-hybrid assay but wasα
found to interact with another Arabidopsis protein, VIP1.VIP1 contains a conserved stretch of
basic amino acids (basic domain) abutting a heptad leucine repeat (leucine zipper)-structural
features characteristic of the basic-zipper (b-ZIP) proteins. Indeed, VIP1 expression was
shown to promote nuclear import of VirE2 in yeast cells.Down-regulation of VIP1 in plant
cells, using antisense transgenic plants, blocked the nuclear uptake of GUS-tagged VirE2, but
not of GUS-VirD2, thus demonstrating the specific role of VIP1 in the nuclear import of
VirE2 molecules in plant cells.
 VIP1 nuclear import depended on the presence of the cellular Srp1 protein, indicating that
VIP1 is imported into the cell nucleus via the karyopherin -dependent pathwayα
 Moreover, the low cellular levels of VIP1 found in various plant tissues suggest that, in
nature, Agrobacterium-mediated transformation may not occur at its maximal possible
efficiency. In fact, inoculation of various plant tissues of most Agrobacterium-susceptible plant
species results in the transformation of an extremely low number of cells, even with very
dense Agrobacterium inoculum. Over-expression of VIP1 in transgenic plants resulted not only
in higher susceptibility to Agrobacterium infection, but also in faster nuclear import of the T-
DNA
 VIP1 may perform a dual function: facilitating nuclear targeting of VirE2 and playing a role in
the intranuclear transport of VirE2 and its cognate T-strand to the site of integration

25. A Model for T-DNA Nuclear Import and Intranuclear Transport
 Once inside the cytoplasm, the T-strand, shaped and protected by its chaperones VirD2 and
VirE2, begins the journey to the host cell nucleus and its resident genome
 VirE2, cooperatively coats the T-strand, shapes it into a coiled filament and protects it from
cellular nucleases
 VirD2, on the other hand, acts as the T-complex pilot and guides it to the nuclear pore.
 Because of their very large size and rigid coiled shape, T-complexes cannot move through the
cytoplasm in a simple Brownian motion, let alone passively diffuse through the nuclear pore.
this coiled “telephone cord”-like complex may stretch, thus reducing its outer diameter and
facilitating the import process, once it arrives to the nuclear pore.
 T-complex bacterial chaperones, VirD2 and VirE2, presumably interact specifically with their
respective cellular factors as well as with the Agrobacterium VirE3 protein

26.  Once inside the nucleus, perhaps even before the entire t-complex molecule has
completely traversed the nuclear pore, the VirD2 NLS region may become
dephosphorylated by PP2C ; this VirD2 dephosphorylation has been proposed to
regulate its nuclear import. Within the cell nucleus, VirD2 may also interact with
CAK2M and TBP. Because both CAK2M and TBP are members of the plant RNA
transcription machinery, their interactions with VirD2 may further guide the entire
T-complex into the site of integration in the host chromosome.
 Similar to VirD2, VirE2 also associates with a putative member of plant
transcriptional complexes, VIP1. In addition to facilitating VirE2 nuclear import,
VIP1 may also function in the intranuclear transport of the T-complex, leading it to
chromosomal regions where the host DNA is more exposed and, thus, more suitable
for T-DNA integration. Here again, the combined, and noncompetitive action of
VirD2 and VirE2, through their interaction with different host factors, may
represent the molecular basis for the polar nature of T-DNA integration

28. POTENTIAL USES OF TRANSFORMANTS

29. CONCLUSIONS
 During the last 15 years, improvements in biotechnology have come a long way since the
realisation that plants can be genetically modified to give desirable phoentypic variations. Now that
we are able to make transgenic plants, the main questions facing plant scientists are how to regulate
gene expression, how can transformation be made more efficient and consistent, and perhaps most
importantly, what are the environmental implications of this technology.
 One of the main drawbacks of A. tumefaciens is its inability to effectively transform many
monocotyledons, although current research by Ke et al. (2001) suggest that genetically engineered
"supervirulent" strains may be effective in transforming many different plant species.
 In a study carried out in 1994 by Hiei et al., it was found that almost all of the transgenic Japonica
rice plants had normal morphology, and 70% were fully fertile. Similar results were found when
Indica varieties were also investigated. Delivery of foreign DNA into rice by A. tumefaciens is
becoming standard practice in a growing number of laboratories, thus allowing the genetic
improvement of many ovarieties of this fundamentally important crop plant.
 Important problems facing plant transformation which still remain to be solved include regulation
of the DNA integration, and achieving the holy grail of plant transformation technology, that is
targeted gene disruption and gene replacement By homologous recombination. Recent reports of
efficient targeting in Arabidopsis thaliana suggest that this breakthrough is closer than we might think
(Gelvin, 1998).
 It seems probable that Agrobacterium mediated transfer techniques will soon be extended to other
recalcitrant species of commercially important plants as soon as the methodologies are optimized.

30. REFERENCES
Madame Curie Bioscience Database – NCBS e-books
MOLECULAR GENETICS-STRIPES
www.Agrobacteriumtumefaciens.com