Indirect transformation: Genetic transformation of plant tissues with the use of Agrobacterium, Ti-plasmid and mechanism of T-DNA transfer (different protein involved and their role, vir region and other genes

involved)

Mitesh Shrestha

Methods of genetic transformation :

Various gene transfer techniques are grouped in two main categories –

Indirect gene transfer or vector-mediated gene transfer.

Direct gene transfer or vector less gene transfer.

The gene transfer methods normally include two categories Direct method

o Electroporation o Microinjection o Transformation by particle gun bombardment o Direct transformation of protoplast by using PEG o Chloroplast transformation o In planta transformation Indirect method

o Virus/Bacteria-mediated transformation. -The gene transfer results can be characterized as transient and stable expression .

Methods of genetic transformation:

The first step of genetic transformation is gene transfer to develop transgenic plants. Various gene transfer techniques are grouped in two main categories:

A. Direct or vector-less gene transfer

B. Indirect or vector-mediated gene transfer

A. Direct or vector-less gene transfer : • Gene transfer occur directly without taking help from any intermediated

system or vector.

• It has been further subdivided into three categories: 1. Physical gene transfer method

Electroporation

Particle bombardment/biolistics

Microinjecton,etc

2. Chemical gene transfer method

PEG-mediated gene transfer

3. DNA imbibition by cell, tissue, and organs.

Indirect gene transfer

• Indirect gene transfer is vector mediated gene transfer that means a

vector is needed for this type of transformation.

The term ‘‘Plant gene vector’’ applies to potential vectors both for

the transfer of genetic information between plants and also from

other organism (bacteria, fungi and animals) to plants.

• Plant gene vectors can be transferred by using plasmids of

Agrobacterium, viruses, and transposable elements.

Agrobacterium-mediated transformation is the mostly used method

for indirect transformation in plants.

Agrobacterium-mediated transformation

• Transfer of DNA from bacteria to plants

• The most promising and widely used technology for indirect transformation

• Agrobacterium-mediated transformation using bacterial plasmid as a vector

• A. tumefaciens contains Ti plasmid which normally infect dicotyledonous

plant cell

• Three types of bacterial species can be used, they are:

A. tumefaciens

A. rhizogenes

A. radiobacter

Fig.1: Agrobacterium-mediated genetic transformation.

Agrobacterium Biology:

• Agrobacterium is a genus of gram-negative, non-sporeforming, rod-shaped bacterium.

• Belongs to the bacterial family Rhizobiaceae

• The bacterium is motile and surrounded by a small number of peritricious flagella.

• These are found near soil level at the junction of stem and root.

• Virulent bacteria contain one or more large plasmids, one of which carries the genes for tumour induction and is known as the Ti (tumour inducing) plasmid.

• Virulent on most dicots, some monocots, and Gymnosperms.

• Requires wound for tumorigenesis.

Fig.: Agrobacterium

Classification of Agrobacteria

• The old system of classification according to their phytopathogenic characteristics is as follows: Agrobacterium tumefaciens: Induces Crown Gall disease

Agrobacterium rhizogenes: Induces Hairy root disease

Agrobacterium radiobacter: An avirulent strain

• Recently, Agrobacteria have been re-classified according to their growth

patterns. This way three different Biotypes are found. Biotype 1 and 2 are used in plant genetic engineering: Biotype 1: Can be distinguished from other bacteria by the production of

ketolactolase from lactose.

Biotype 2 : These are identified by their ability to grow on erythritol as a carbon source.

Biotype 3: Not used in plant genetic experiments. Causes crown gall disease in grapevines.

Comparisons between Biotype I &II:

Biotype-I Biotype-II

1. Strains can be distinguished from other bacteria by the production of ketolactolase from lactose.

1. Strains are identified by their ability to grow on erythritol as a carbon source.

2. Proliferate at temperatures up to 37°c. 2. Proliferate poorly at temperatures higher than 30°c.

3. Classic E. coli media , i.e. LB(Luria Broth), NB(Nutrient broth), TY(Tryptone yeast extract) are used for growth. Also grown in defined minimal media, i.e. YMB(Yeast Maltase broth)

3. Do not grow well in classic E. coli media, instead can be cultured on- TY+Ca, YMB, or in a defined minimal medium(MM).

4. Example: Agrobacterium tumefaciens strains: Ach5, A6, B6, etc.

4. Example: A. rhizogenes strains: NCPPB 1855, ATCC 23834, A4 etc.

Crown Gall Disease

• Agrobacterium tumefaciens is most widely known for causing crown gall disease.

• Crown Gall is a common disease of many woody shrubs and some herbaceous plants.

• Agrobacterium tumefaciens can infect 140 genera in over 90 families of plants.

• It is especially common in the rose family.

• The disease causes the formation of tumor-like swellings called galls that can generally be found on the crown of the plant just above the soil.

Crown Gall Disease of different plants:

Gall

Agrobacterium tumefaciens causes crown gall disease of a wide range of dicotyledonous plants

• Crown galls are tumors, which often arise on the stem where it meets the soil (crown), but they also develop on roots and branches.

• Crown Galls first appear as small, white, soft protrusions.

• As the tumours enlarge, the surface takes on a mottled dark brown appearance due to the death and decay of the peripheral cells.

• The tumour usually appears either as a swelling of the plant tissue, or as a separate mass of tissue close to the plant surface, joined only by a narrow neck of tissue.

• Tumours can either be soft and spongy and may crumble on touch, or can be hard and appear as knobby or knotty.

• Some tumours can reach up to 30cm in diameter.

• Plants may also become stunted, produce small chlorotic leaves, and are more susceptible to extreme environmental conditions such as winter cold and wind.

Crown gall disease symptoms:

Hairy Root Disease Hairy root disease in dicotyledonous plants is caused by Agrobacterium

rhizogenes a gram negative soil bacterium.

A. rhizogenes induces the formation of proliferative multi-branched adventitious roots at the site of infection; so called 'hairy roots‘.

In the rhizosphere, plants may suffer from wounds by soil pathogens or other sources. This leads to the secretion of phenolic compounds like acetosyringone.

These compounds have chemotactic effects that attract the bacteria.

Under such conditions, certain bacterial genes are turned on leading to the transfer of its T-DNA from its root inducing plasmid (Ri plasmid) into the plant through the wound.

After integration and expression, the hairy root phenotype is observed.

The symptoms observed with A.rhizogenes are suggestive of auxin effects.

Hairy root disease symptoms: • Multi-branched adventitious Hair like roots present at the site of

infection.

• Overdevelopment of a root system that is not completely geotropic.

• Altered (wrinkled) leaf morphology, if leaves are present.

Fig: Hairy root disease of different plants

Ti Plasmid

• Ti plasmid or tumor inducing plasmid is a large plasmid found in Agrobacterium tumefaciens.

• It has a T-DNA portion which is transferred into a host cell, and thus induce tumors.

• The infected plant cells in the tumor acquire two new properties-

i. They show phytohormone independent growth

ii. They contain one or more of unusual aminoacid derivatives known as Opines.

• Earlier the bacterial families and the crown gall they establish were divided on the basis of their production of two different aminoacids, called, octopine and nopaline.

1. Large circular DNA molecule, up to 200kb

in length.

2. Exits in bacterial cells as independent

replicating genetic unit.

3. Smaller than the main chromo-

some.

4. Molecular weight is about

1.2× 10^8(3 to 8% of the Agrobacterium

chromosome).

5. Contains a T-DNA region.

Common features of Ti plasmid:

Fig: Agrobacterium cell, containing Ti plasmid

Organization of Ti plasmid

• Ti plasmid have some major regions. Such as-

Virulence region

T-DNA region

Conjugative transfer region

Opine catabolism region

Origin of replication region

Fig.6: Organization of Ti plasmid

Function of these regions:

• Virulence region: This region code for the enzymes responsible for mediating transduction of T-DNA to plant cells.

• T-DNA region: T-DNA or transferred DNA is that region of the plasmid which is transferred into the plant cells. It contains genes that code for enzymes synthesizing opines and phytohormones.

• Conjugative transfer region: Region enabling conjugative transfer.

• Opine catabolism region: Region for opine catabolism.

• Origin of replication region: Region responsible for replication.

Ri Plasmid • Agrobacterium rhizogenes causes hairy root disease of many dicotyledonous

plants .

• The ability of A. rhizogenes to incite hairy root disease is determined by a virulence plasmid known as the Ri-plasmid.

• Ri-plasmid shares extensive functional homology with the Ti–plasmid of A. tumefaciens.

• Ri–plasmid contains a distinct segment (s) of DNA which is transferred to plant genome during infection.

• The transfer of the DNA (T-DNA) to the plant genome is mediated by another segment on the plasmid known as the virulence (vir) region.

• The T-DNA also confers on the transformed tissue the ability to produce modified amino acids (opines).

Organization of Ri plasmid

• Ri plasmid have the same regions as Ti plasmid. Such as-

Virulence region

T-DNA region

Conjugative transfer region

Opine catabolism region

Origin of replication region

Simplified genomic map of Ri plasmid

Origin of replication

Virulence region

Opine catabolism

Conjugative transfer

T-DNA

Left border Right

border Single T-DNA Mannopine -type Two T-DNA Agropine-type TL TR

Ti plasmid

Ri plasmid

1. Tumor inducing plasmid.

1. Rhizoid like root inducing plasmid.

2. Responsible for crown gall disease.

2. Responsible for hairy root disease.

3. T-DNA synthesizes:

Opine

Auxin

Cytokinin

3. T-DNA synthesizes: Auxin Opine

4. Found in A. tumefaciens 4. Found in A. rhizogenes

5. Widely used in plant genetic transformation technique for developing high yielding variety.

5. Widely used in the production of secondary metabolites, found in roots

6. Smaller than Ri plasmid 6. Larger than Ti plasmid.

Agrobacterium

Plasmid

How Does Agrobacterium Gene Transfer Work?

1. Extract DNA from donor

2. Cut DNA into fragments

3. Sort DNA fragments

4. Recombine DNA fragments

5. Transfer plasmids with bonded DNA

6. Grow transformed (recipient) cells

Agrobacterium Transformation – Plant Transformation

Agrobacterium infects plant cells. Protein gene is

transferred into cell chromosome.

Plant tissue is cultured to produce transformed

plantlets with each cell containing transgene. Generated plant will express

resistance if infected with virus.

Kanamycin

Fig. Diagrammatical representation of Plant genetic transformation.

Advantages

• Vectors naturally transfer.

• Simple leaf, nodal segment etc

used as explants.

• Transgenic plant produced in

this manner are quite stable for

several generation.

Disadvantages

• Number of transformed tissue are less.

• Only applicable in case of dicot plants.

• Specific strain is essential for this

technique.

• Without marker genes Agrobacterium-

mediated transformed plant genes not

identified phenotypically.

Ti-Plasmid • Ti plasmid has a central role in crown gall formation

• It is the portion of Ti plasmid that is integrated into the host genome.

• Large plasmid ( ̴200kb) size

• Ti plasmid features- They contain one or more

T-DNA regions. They contain a vir region. They contain an origin of

replication. They contain a region

enabling conjugative transfer.

They contain genes for the catabolism of opines

Types of Ti-Plasmid • Classified based on the types of opines

produced

• Nopaline plasmids: carry gene for synthesizing nopaline in the plant and for utilization (catabolism) in the bacteria. Tumors can differentiate into shooty masses (teratomas). Only one T-DNA ̴20kb size

• Octopine plasmids: carry genes(3 required) to synthesize octopine in the plant and catabolism in the bacteria. Tumors do not differentiate, but remain as callus tissue. Two T-DNA, termed TL and TR of size 14kb and 7kb respectively; only TL is oncogenic and TR carries gene for opine biosynthesis. Octopine strain contain an overdrive or enhancer sequence associated with right border sequence: required for optimal DNA transfer

T-DNA • Responsible for the tumorous phenotype

• The T-DNA region of any Ti-plasmid is defined by the presence of the right and the left border sequences

• Border sequences are 24bp imperfect repeats

• Any DNA between the borders will be transferred into the genome of the plant

Characteristics of T-DNA • Stably integrates (randomly) into the plant genome.

• Expression of genes in wild-type T-DNA results in dramatic physiological changes to the plant cell.

• Synthesis of plant growth hormones (auxins and cytokinins) neoplastic growth (tumor formation)

Functions of some T-DNA genes in A. tumifaciens Ti-Plasmids

Gene Product Function

ocs Octopine synthase Opine synthesis

nos Nopaline synthase Opine synthesis

tms1 (iaaM or auxA) Tryptophan-2-mono-oxygenase

Auxin synthesis

tms2 (iaaH or auxB) Indole acetamide hydrolase

Auxin synthesis

cyt (ipt or tmr) Isopentyl transferase Cytokinin synthesis

tml Unknown Determine tumor size in some spp.

These genes have typical eukaryotic expression signals

Vir genes

• Genes responsible for T-DNA transfer

• Around 40kb region

• Located outside the T-DNA region

• Virulence region

• At least eight vir gene operons

Vir genes • The T-DNA transfer is mediated by products encoded by the 30-40 kb vir

region of the Ti plasmid.

• Composed by at least six essential operons (vir A, vir B, vir C, vir D, vir E, virG ) and two non-essential (virF, virH).

• Number of genes per operon differs, virA, virG and virF have only one gene; virE, virC, virH have two genes while virD and virB have four and eleven genes respectively.

• The only constitutively expressed operons are virA and virG, coding for a two-component (VirA-VirG) system activating the transcription of the other vir genes.

• The VirA-VirG two component system has structural and functional similarities to other already described for other cellular mechanisms

VirA • VirA is a transmembrane dimeric sensor protein that detects signal

molecules, mainly small phenolic compounds, released from wounded plants.

• The signals for VirA activation include acidic pH, phenolic compounds, such as acetosyringone and certain class of monosaccharides which acts synergistically with phenolic compounds.

• VirA protein can be structurally defined into three domains:

• the periplasmic or input domain and two trans membrane domains (TM1 and TM2).

• The TM1 and TM2 domains act as a transmitter (signaling) and receiver (sensor). The periplasmic domain is important for monosaccharide detection. Within the periplasmic domain, adjacent to the TM2 domain is an amphipathic helix, with strong hydrophilic and hydrophobic regions.

VirA • This structure is characteristic for other trans membrane sensor proteins

and folds the protein to be simultaneously aligned with the inner membrane and anchored in the membrane.

• The TM2 is the kinase domain and plays a crucial role in the activation of VirA, phosphorylating itself on a conserved His-474 residue in response to signaling molecules from wounded plant sites.

• Monosaccharide detection by VirA is an important amplification system and responds to low levels of phenolic compounds. The induction of this system is only possible through the periplasmic sugar (glucose/galactose) binding protein ChvE, which interacts with VirA.

• Recent studies for determination of VirA regions, important for its sensing activity suggested the position, which may be involved on TM1-TM2 interaction. This interaction causes the exposure of the amphipathic helix to small phenolic compounds and suggests a putative model for the VirA-ChvE interaction.

VirG • Activated VirA has the capacity to transfer its phosphate to a conserved aspartate

residue of the cytoplasmic DNA binding protein VirG.

• VirG functions as a transcriptional factor regulating the expression of vir genes when it is phosphorylated by VirA.

• The C-terminal region is responsible for the DNA binding activity, while the N-terminal is the phosphorylation domain and shows homology with the VirA receiver (sensor) domain.

External factors

• The activation of vir system also depends on external factors like temperature and pH.

• At temperatures greater than 32°C, the vir genes are not expressed because of a conformational change in the folding of VirA induce the inactivation of its properties. The effect of temperature on VirA is suppressed by a mutant form of VirG (VirGc), which activates the constitutive expression of the vir genes.

• However, this mutant cannot confers the virulence capacity at that temperature to Agrobacterium, probably because the folding of other proteins that actively participate in the T-DNA transfer process are also affected at high temperature

Vir genes/proteins Function

VirA / transmembrane dimeric sensor protein

Encodes phenolic sensor kinase Part of two component system with virG Phosphorylates and activates virG

virG Encodes transcription factor responsible for induction of all vir gene expression

virB1-11 Encode components of transfer apparatus for transfer of T-DNA

virC1 Encodes overdrive binding protein thereby enhancing efficiency of T-DNA transfer

virD1 Modulates activity of virD2 activity

virD2 Nicks T-DNA and direct it through virB/virD4 transfer apparatus Nuclear localization of T-DNA by interacting with importins and cyclophilins Protects 5’ end of T-DNA from nucleases

virD4 Component of transfer apparatus

virE1 Required for virE2 export from the A. tumifaciens

virE2 Single stranded DNA binding protein; therefore prevents T-DNA from degradation Involved in nuclear targeting and passage through NPC

Ti plasmids and the bacterial chromosome act

in concert to transform the plant

1. Agrobacterium tumefaciens chromosomal genes: chvA, chvB, pscA required for initial binding of the bacterium to the plant cell and code for polysaccharide on bacterial cell surface.

2. Virulence region (vir) carried on pTi, but not in the transferred region (T-DNA). Genes code for proteins that prepare the T-DNA and the bacterium for transfer.

3. T-DNA encodes genes for opine synthesis and for tumor production.

4. occ (opine catabolism) genes carried on the pTi allow the bacterium to utilize opines as nutrient.

Process of T-DNA transfer and Integration

1. Signal recognition by Agrobacterium

2. Attachment to Plant cells

3. Induction of vir genes

4. Production of T-strand

5. Transfer of T-DNA out of the bacterial cell

6. Transfer of T-DNA and vir proteins into the plant cell and nuclear localization

1. Signal recognition by Agrobacterium

• The plants at the wound site releases phenolics and sugars like acetosyringone, α-hydroxy acetosyringone : probably as part of the plant defense mechanism

• Called as signal molecules

• Recognised by virA gene

via chemotaxis

2. Attachment to plant cells

• This is a polar two step process:

1. Mediated by cell associated acetylated, acidic polysaccharides encoded by attR locus. attR mutants are avirulent. This step is reversible because sheer forces are sufficient to dislodge bacteria.

2. Involves formation of cellulose fibrils by bacterium, which enmeshes large number of bacteria at the wound site.

3. chvA, chvB and pscA genes are involved in synthesis, processing and export of cyclic beta 1,2 glucans and other sugars, that may be involved indirectly in bacterial attachment

3. Induction of vir genes Vir A

vir A gene is constitutively expressed. Vir A protein is located in the inner

membrane and responds to the chemical environment [acidic pH and

acetosyringone (AS)]. In the presence of the stimulants, it is auto-

phosphorylated.

Linker responds to pH and interacts with ChvE (a sugar-binding protein

encoded by Agrobacterium genome). At sub-optimal AS levels, VirA can

be further stimulated by sugars, opines or amino acids.

NH2

COOH

cytoplasm periplasm

Inner membrane

receiver

sensor kinase

linker

AS (pH)

Auto-phosphorylation

• vir G gene is also constitutively expressed.

• Vir G protein is freely available in the cytoplasm.

• The activated (phosphorylated) Vir A in turn phosphorylates Vir G protein

at aspartic acid residue 52.

• Phosphorylated Vir G becomes the transcriptional activator of the

remaining vir genes as well as that for virA and virG.

AS

P

P

Vir G

P

Vir A Vir G

• Vir gene induction enhanced by glucose, galactose and xylose

4. Production of T- strand • virD1/virD2 complex recognize the left and right

borders

• virD2 produces a single nick in the T-DNA and itself becomes attached to 5’end of the eventually displaced T-DNA strand

overdrive

Right Border

Left Border T-DNA

virD/virC 5’

Right border Left border

D

virD/virC

gap filled in

T-strand

T-DNA

virE

Right border Left border

D

T-strand coated with virE

T-DNA

virD nicks at Left Border sequence

5. Transfer of T-DNA out of the bacterial cell

• T-DNA/virD2 complex is exported from the bacterial cell into the host plant cell through a T-pilus

• T-pilus is a membrane channel secretory system composed of the proteins encoded by virB and virD4

• virE2 also transported along

6. Transfer of T-DNA and vir proteins into the

plant cell and their nuclear localization • virE2 proteins coat the T-DNA thereby protecting it from nucleases

• Also facilitate nuclear localisation by maintaining correct conformation of the T-DNA/virD2 complex

• virD2 possesses a nuclear localization signal (NLS) that interacts with importin protien of the plants

• virE2 is also found to possess NLS

• Plant proteins called cyclophillin bind with virD2 and aid in integration

• Integration is also called “illegitimate recombination”- different from homologous recombination- doesn’t depend on region of sequence similarity: random integration but having some homology with the plant DNA

T-DNA is transferred as a linear , single stranded molecule from Ti-plasmid; passes into the plant cell and eventually becomes integrated into the plant chromosomal DNA

Fig: A simplified process of the T-DNA transfer and integration process

Overview of the Infection Process

• Large size of Ti plasmid(200-800 kb)- difficult to handle

• Presence of oncogenes or tumor causing genes prevents them from being regenerated into mature plants

• Gene encoding opine synthesis not useful to a transgenic plants; instead might lower the plant yield by diverting the resources into opine production

• Ti plasmid doesn’t replicate in E. coli but for manipulation of Ti plasmid it is easier in bacterium so an origin of replication that functions in E. coli should be added

• Lack of unique restriction sites and marker site within T-DNA

Constraints of Wild type Ti-plasmid

Existing problems and future prospects

• Tissue browning and necrosis after Agrobacterium infection are still major

obstacles in the genetic transformation of cereals.

• On pathogen infection, one of the earliest defence mechanisms activated is the

production of reactive oxygen species, referred to as an oxidative burst, which

activates programmed cell death (HR). A correlation between the reduction in cell

death and the improved transformation frequency has been demonstrated. Necrosis

inhibiting agents, such as silver nitrate, increased efficiency of transformation.

• Suppression of the host defence response is a prerequisite to successful plant

transformation.

General scheme for Agrobacterium-mediated transformation of cereal plants

Agrobacterium in humans

• Although generally seen as an infection in plants, Agrobacterium can be responsible for opportunistic infections in humans with weakened immune systems,but has not been shown to be a primary pathogen in otherwise healthy individuals.

• One of the earliest associations of human disease caused by Agrobacterium radiobacter was reported by Dr. J. R. Cain in Scotland (1988).

Assignments

• How is mode of action different for Agrobacterium from Rhizobium? [1]

• Explain the role of various Vir genes for successful infection of plants by Agrobacterium? [7.5]

• Write names of bacteria involved as ‘Non-Agrobacterium’-mediated genetic transformation of plant species. [1]

• Illustrate the structural organization of Ti plasmid. [3]

• How Agrobacterium is removed from the media ? [1]

• What are the problems associated with Agrobacterium mediated transformation? [3]

• Differentiate between Ti and Ri plasmid. [3]

• Write the mode of infection of Agrobacterium showing involvement of genes required for various stages. [7.5]