Prepared by: Darshan T. Dharajiya Ph.D. (Plant Molecular Biology and Biotechnology)

Dept. of Plant Molecular Biology and Biotechnology,CPCA, S. D. Agricultural University.

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Content

o Agrobacterium tumefacienso Molecular Biology of Agrobacterium Infection• The Ti-Plasmid• Organization of T-DNA• Steps of Agrobacterium-plant cell interactiono References

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Biology of the Agrobacterium - plant interaction

The only known natural example of inter-kingdom DNA transfer

The only known natural example of inter-kingdom DNA transfer

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Crown gall disease 5

Agrobacterium tumefaciens

• Gram negative, short rod, soil bacterium

• established by H. J. Conn

• broad-host range pathogen,

• initiating tumours on plants of most dicotyledonous genera and some monocots

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The genus Agrobacterium hasa wide host range

• Overall, Agrobacterium can transfer T-DNA to a broad group of plants.

• Yet, individual Agrobacterium strains have a limited host range.

• The molecular basis for the strain-specific host range is unknown.

• Many monocot plants can be transformed (now), although they do not form crown gall tumors.

• Under lab conditions, T-DNA can be transferred to yeast, other fungi, and even animal and human cells.

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Why Agrobacterium is used forproducing transgenic plants?

• The T-DNA element is defined by its borders but not the sequences within.

• So researchers can substitute the T-DNA coding region with any DNA sequence without any effect on its transfer from Agrobacterium into the plant.

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Molecular Biology of Agrobacterium Infection

• The process of infection by Agrobacterium tumefaciens culminates in the transfer of a small part of pTi into the plant cell genome; this DNA sequence is called T-DNA.

• The infection process is governed by both chromosomal and plasmid-borne genes of Agrobacterium tumefaciens.

• Attachment of bacteria to plant cells begins the infection, governed by chromosomal virulence genes (chv); which are expressed constitutively.

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The Ti Plasmid• The Ti plasmid is a large conjugative plasmid or megaplasmid

of about 200 kb (range 150-250 kb).• The pTi is unique bacterial plasmids in the following two

respects:– They contain some genes, located within their T-DNA,

which have regulatory sequences recognized by plant cells, while their remaining genes have prokaryotic regulatory sequences.

– These plasmids naturally transfer a part of their DNA, the T-DNA, into the host plant genome, which makes Agrobacterium a natural genetic engineer.

• The Ti plasmids are classified into different types based on the type of opine, namely, octopine, nopaline, succinamopine and leucinopine, produced by their genes.

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The Ti Plasmid

• The different Ti plasmids can be grouped into two general categories: octopine type and nopaline type.

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The Ti Plasmid

• Ti Plasmids contain the following important functional regions:

1. T-DNA contains oncogenes and opine synthesis genes, and is transferred into the host plant genome.

2. vir region regulates the transfer of T-DNA into plant cells.

3. Opine catabolism regions produce enzymes necessary for the utilization of opines by Agrobacterium.

4. Conjugative transfer (oriT or tra) region functions in conjugative transfer of the plasmid.

5. Origin of replication (ori) for propagation in Agrobacterium.

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Agrobacterium-induced plant tumors contain high concentrations of

• Plant hormones (auxin, cytokinin)• Opines (octopine, nopaline)

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Organization of T-DNA

• T-DNA (transferred DNA) is the 23 kb segment of Ti plasmids, which is transferred into the plant genome during Agrobacterium infection.

• T-DNA is defined on both its sides by a 24 bp direct repeat border sequence, and contains the genes for tumour/hairy root induction and those for opine biosynthesis.

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Organization of T-DNA

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T-DNA

• T-DNA carries genes involved in the synthesis of plant growth hormones (auxin, auxin synthesis; cyt, cytokinin synthesis) and the production of low molecular weight amino acid and sugar phosphate derivatives called opines (ocs, octopine; mas, mannopine; and ags, agropine).

• Agrobacterium are usually classified based on the type of opines specified by the bacterial T-DNA.

• All the genes present in T-DNA contain eukaryotic regulatory sequences. As a result, these genes are expressed only in plant cells, and they are not expressed in the Agrobacterium.

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Organization of vir Region

• The vir region contains 8 operons (designated as virA, virB, virC, virD, virE, virF, virG and virH), which together span about 40 kb of DNA and have 25 genes.

• This region mediates the transfer of T-DNA into plant genomes, and hence is essential for virulence or production of crowngall/hairy root disease; therefore, it is called the virulence region or vir region.

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• On the other hand, the genes present in T-DNA are not required for its transfer; only the 24 bp direct repeat left and right borders of T-DNA are essential for the transfer.

• Of the 8 vir operons, 4 operons, viz., virA, virB, virD and virG, are essential for virulence, while the remaining 4 operons play an accessory role.

• The operons vir A and virG are constitutive, encode one protein each, and are concerned with the regulation of all the vir operons.

Organization of vir Region

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Gene/Operon Function

T-DNA

iaaM (auxL tins I)*Auxin biosynthesis; encodes enzyme tryptophan-2-mono-oxygenase, which converts tryptophan into indole-3-acctamide (IAM).

iaaH (aux2, tms2)Auxin biosynthesis; encodes enzyme indole-3-acetamide hydrolase, which converts IAM into IAA (indole-3-acetic acid).

ipt (tmr, Cyt )Cytokinin biosynthesis; encodes enzyme isopentenyl transferase, which catalyzes the formation of isopentenyl adenine.

NosNopaline biosynthesis; encodes the enzyme nopaline synthase, which produces nopaline from arginine and pyruvic acid.

24 bp left and right border sequences

Site of endonuclease action during T-DNA transfer; the only sequences of T-DNA essential for its transfer.

vir Region

vir A(1)Encodes a receptor for acetosyringone that functions as an autokinase; also phosphorylates VirG protein; constitutive expression.

virB (11)Membrane proteins; possibly form a channel for T-DNA transport (conjugal tube formation); VirB 11 has ATPase activity.

virC (2)Helicase; binds to the overdrive region just outside the right border; involved in unwinding of T-DNA.

virD (4)VirDl has topoisomerase activity; it binds to the right border of T-DNA; VirD2 is an endonuclease; it nicks the right border.

virE (2) Single-strand binding proteins (SSBP); bind to T-DNA during its transfer.

virF(l) Not well understood.

virG (l)DNA binding protein; probably forms dimer after phosphorylation by VirA, and induces the expression of all vir operons; constitutive expression.

virH (2) Not well known.20

Steps of Agrobacterium-plant cell interaction

• Cell-cell recognition and binding• Signal transduction and transcriptional

activation of vir genes• Conjugal DNA metabolism• Intercellular transport• Nuclear import• T-DNA integration

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Agrobacterium-host cell recognition is a two-step process

1. Loosely bound step: acetylated polysaccharides are synthesized.

2. Strong binding step: bound bacteria synthesize cellulose filaments to stabilize the initial binding, resulting in a tight association between Agrobacterium and the host cell.

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Receptors are involved in initial binding

• Plant vitronectin-like protein (PVN, 55kDa) was found on the surface of plant cell. This protein is probably involved in initial bacteria/plant cell binding.

• PVN is only immunologically related to animal vitronectin.

• Animal vitronectin is an important component of the extracellular matrix and is also an receptor for several bacterial strains.

• Aside from PVN, rhicadhesin-binding protein was found in pea roots.

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30Electron microscopy of Attachment of Agrobacterium cells to plant cell

Plant signals

• Wounded plants secrete sap with acidic pH (5.0 to 5.8) and a high content of various phenolic compounds (lignin, flavonoid precursors) serving as chemical attractants to Agrobacterium and stimulants for vir gene expression.

• Among these phenolic compounds, acetosyringone (AS) is the most effective.

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Representative phenols and sugars (pyranose forms) capable of serving as signals to induce vir gene expression. Active versus inactive enantiomers of DDF and MMCP are shown. AS, acetosyringone; DDF dehydrodiferulate dimethylester; MMCP, 1-hydroxymethyl-2-(4-hydroxy-3-methoxyphenyl)-cyclopropane. 32

• Sugars like glucose and galactose also stimulate vir gene expression when AS is limited or absent. These sugars are probably acting through the chvE gene to activate vir genes.

• Low opine levels further enhance vir gene expression in the presence of AS.

Plant signals

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• These compounds stimulate the autophosphorylation of a transmembrane receptor kinase VirA at its His-474.

• It in turn transfers its phosphate group to the Asp-52 of the cytoplasmic VirG protein.

Plant signals

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Production of T-strand

• Every induced Agrobacterium cell produces one T-strand.

• VirD1 and VirD2 are involved in the initial T-strand processing, acting as site-and strand-specific endonucleases.

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• After cleavage, VirD2 covalently attaches to the 5’ end of the T-strand at the right border nick and to the 5’-end of the remaining bottom strand of the Ti plasmid at the left border nick by its tyrosine 29.

Production of T-strand

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Formation of the T-complex

• The T-complex is composed of at least three components: one T-strand DNA molecule, one VirD2 protein, and around 600 VirE2 proteins.

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• Whether VirE2 associates with T-strand before or after the intercellular transport is not clear.

Formation of the T-complex

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• If VirE2 associates with the T-strand after intercellular transport, VirE1 is probably involved in preventing VirE2-T-strand binding.

• Judging from the size of the mature T-complex (13nm in diameter) and the inner dimension of T-pilus (10nm width), the T-strand is probably associated with VirE2 after intercellular transport.

Formation of the T-complex

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Intercellular transport

• Transport of the T-complex into the host cell most likely occurs through a type IV secretion system.

• In Agrobacterium, the type IV transporter (called T-pilus) comprises proteins encoded by virD4 and by the 11 open reading frames of the virB operon.

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• Intercellular transport of T-DNA is probably energy dependent, requiring ATPase activities from VirB4 and VirB11.

• Physical contact between Agrobacterium and the plant cell is required to initiate T-complex export. Without recipient plant cells, T-strands accumulate when vir genes are induced.

Intercellular transport

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Nuclear Import

• Because the large size of T-complex (50,000 kD, ~13nm in diameter), the nuclear import of T-complex requires active nuclear import.

• The T-complex nuclear import is presumably mediated by the T-complex proteins, VirD2 and VirE2. Both of them have nuclear-localizing activities.

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• VirD2 is imported into the cell nucleus by a mechanism conserved between animal, yeast and plant cells.

• VirE2 has a plant-specific nuclear localization mechanism. It does not localize to the nucleus of yeast or animal cells.

Nuclear Import

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• In host plant cells VirD2 and VirE2 likely cooperate with cellular factors to mediate T-complex nuclear import and integration into the host genome.

Nuclear Import

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Chromatin targeting of T-Complex

• CAK2M and TATA-Box binding protein (TBP) both of which bind to VirD2 and VIP1 binds to VirE2.

• Which leads in chromatin targeting of T-Complex.

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Targeted proteolysis and T-DNA uncoating

• It is necessary to expose T-strand for integration.

• Vir F2- a bacterial host range factor exported into host cell.

• Vir F2 possess F-box domain which binds to Ask protein family.

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• F-box and Skp1 represent conserved components of E3 ubiquitin ligases called SCF (Skp1-Cullin-F-box Protein) complex that mediate protein destabilization by targeted proteosomal degradation.

Targeted proteolysis and T-DNA uncoating

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• Annealing of LB and RB of T-DNA to the host genome.

• Insertion & ligation of T-strand into nick.

• Conversion of T-strand into double stranded by gap repair mechanism.

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DNA repair and Integration

DNA repair and Integration

• DSB induction at specific site in host DNA by rare cutting restriction enzymes.

• Insertion of T-DNA in this site.

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DNA repair and Integration

o In Arabidopsis,• KU80 – factor of NHEJ,

Form complex with KU70 and DNA protein Kinase during integration.

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T-DNA integration is not highly sequence-specific

• There is no obvious site preference for integration of T-DNA throughout the genome.

• About 40% of the integrations are in genes and more of them are in introns.

• The mechanism of NHEJ makes deletions after T-DNA integration a common phenomenon.

• DSB mechanism

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Events of NHEJ in Agrobacterium T-DNA integration

• Integration is initiated by the 3’(LB) of the T-DNA invading a poly T-rich site of the host DNA.

• A duplex is formed between the upstream region of the 3’-end of T-DNA and the top strand of the host DNA.

• The 3’-end of T-DNA is ligated to the host DNA after a region downstream of the duplex is degraded.

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Events of NHEJ in Agrobacterium T-DNA integration

• A nick in the upper host DNA strand is created downstream of the duplex and used to initiate the synthesis of the complementary strand of the invading T-DNA.

• The right end of the T-DNA is ligated to the bottom strand of the host DNA. This pairing frequently involves a G and another nucleotide upstream of it.

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Importance to Biotechnology & Genetically Modified Organisms

• Oncogenes and opine-creating genes can be removed from the Ti-Plasmid that is transferred to the plant cell by T-DNA.

• Scientists can insert any gene they want into the plasmid in place of the tumor causing genes and subsequently into the plant cell genome.

• Original problems existed in that Agrobacterium tumefaciens only affects dicotyledonous plants.

• Monocotyledon plants like corn are not very susceptible to the bacterial infection. • By varying experimental materials, culture conditions, bacterial strains, etc.

scientists have successfully used A. tumefaciens Gene Transfer to produce BT Corn .

• This method of gene transfer enables large DNA strands to be transferred into the plant cell without risk of rearrangement whereas other methods like the Gene Gun have trouble doing this.

• The vast majority of approved genetically engineered agriculture has been transformed by means of Agrobacterium tumefaciens Mediated Gene Transfer.

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References

• Tzvi T. and Vitaly C., (2006). Agrobacterium – mediated genetic transformation of plants biology and biotechnology. Current Opinion in Biotechnology. 17: 147-154.

• Colleen A.M. and Andrew N.B., (2006). Agrobacterium tumefaciens and plant cell interactions and activities required for interkingdom macromolecular transfer. Annu. Rev. Cell Dev. Biol. 22: 101-127.

• Vitaly C., Stanislav V.K., Benoit L., Adi Z., Mery D.Y., Sachi V., Andriy T. and Tzvi T., (2006). Biological systems of the host cell involved in Agrobacterium infection. Cellular Microbiology. 9: 9-20.

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• De la Riva,G.A., Gonzalez-Cabrera,J., Vazquez-Padron,R., Ayra-Pardo,C. (1998). Agrobacterium tumefaciens: a natural tool for plant transformation. Electronic J of Biotech. 1(3): 1-18.

• http://en.wikipedia.org/wiki/Agrobacterium• http://www.bio.davidson.edu/people/kabernd/semi

nar/2002/method/dsmeth/ds.htm• B.D. Singh. (2008). Biotechnology. Third edition.

Kalyani Publisher, New Delhi (India). pp: 355-363.

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References

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