Orientation Programme - PowerPoint Presentation

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Orientation Programme

UGC HRDC AMU ALIGARH

April 20 to May 03, 2020

Assignment #2

Submitted by

Dr. Aamir

Raina

Assistant Professor

Botany Section, Women’s College,

Aligarh Muslim University, Aligarh

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Agrobacterium Mediated Gene Transfer In PlantsAAMIR RAINA M Sc, Ph. D (Genetics and Plant Breeding)

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Gene Transfer

Recombinant DNA technology is based on the insertion of a DNA fragment (Gene of interest) into a suitable cloning vector and then its introduction into a suitable host to propagate the recombinant DNA.

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Gene Carrier Vehicle

If a gene is to be introduced into a host cell,a carrier molecule that can transport the gene into the host cell is required.Such a molecule is called a cloning vehicle or carrier molecule or a vector.

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Gene Carrier Vehicle

Following are few gene carrier vehicles. Plasmids Bacteriophages Cosmids VirusesVirus vector for animal cells. Simian Virus

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Making Transgenic Plants—Why?

Study gene function and regulation Making new organismic tools for other fields of researchCuring genetic diseasesImproving agriculture and related raw materialsNew sources of bioengineered drugs.

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Must get DNA:

into the cellsintegrated into the genome (unless using transient expression assays)expressed (everywhere or controlled) For (1) and (2), two main approaches for plants:Agrobacterium - mediated gene transferDirect gene transfer

For (3), use promoter that will direct expression when and where wanted – may also require other modifications such as removing or replacing introns.

Genetic Engineering of Plants

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Agrobacteria

soil bacteria, gram-negative, related to Rhizobia species: It invades many dicot plants when they are injured at the soil level and causes crown gall disease

Agrobacterium tumefaciens-Nature’s smallest genetic engineer

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The ability to cause crown gall disease is associated with the presence of Ti Plasmid within bacterial cell

A remarkable feature of Ti Plasmid is that after infection part of molecule is integrated into plant chromosomal DNA. This segment called T-DNA is between 15 and 30 kb in sizeIt is maintained in a stable form in plant cellc and is passed onto daughter cells as an integral part of chromosomeAgrobacterium tumefaciens-Nature’s smallest genetic engineer

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Crown galls caused by

A. tumefaciens on nightshade. More about Galls: http://waynesword.palomar.edu/pljuly99.htmhttp://kaweahoaks.com/html/galls_ofthe_voaks.html

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the species of choice for engineering dicot plants; monocots are generally resistantsome dicots more resistant than others (a genetic basis for this)complex bacterium – genome has been sequenced; 4 chromosomes; ~ 5500 genesAgrobacterium tumefaciens

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Agrobacterium tumefaciens

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Infection occurs at wound sites

Involves recognition and chemotaxis of the bacterium toward wounded cellsgalls are “real tumors”, can be removed and will grow indefinitely without hormonesgenetic information must be transferred to plant cellsInfection and tumorigenesis

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Synthesize a unique amino acid, called “opine”

octopine and nopaline - derived from arginineagropine - derived from glutamateOpine depends on the strain of A. tumefaciensOpines are catabolized by the bacteria, which can use only the specific opine that it causes the plant to produce.Has obvious advantages for the bacteria, what about the plant?

Tumor characteristics

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It was recognized early that virulent strains could be cured of virulence, and that cured strains could regain virulence when exposed to virulent strains; suggested an extra- chromosomal element.

Large plasmids were found in A. tumefaciens and their presence correlated with virulence: called tumor-inducing or Ti plasmids.Elucidation of the TIP (tumor-inducing principle)

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An

extrachromosomal double stranded circular DNA molecule Tumour inducing Large (200-kb in size) and conjugative type~10% of plasmid transferred to plant cell after infectionTransferred DNA (called T-DNA) integrates semi-randomly into nuclear DNA

Ti plasmid also encodes:enzymes involved in opine metabolismproteins involved in mobilizing T-DNA (

Vir

genes)

Ti Plasmid

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auxA auxB cyt ocs

LB

RB

LB, RB – left and right borders (direct repeat)

auxA

+

auxB

– enzymes that produce auxin

cyt

– enzyme that produces cytokinin

Ocs – octopine synthase, produces octopine

T-DNA

These genes have typical eukaryotic expression signals!

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auxA auxBTryptophan indoleacetamide  indoleacetic acid (auxin) cyt

AMP + isopentenylpyrophosphate 

isopentyl

-AMP (a cytokinin)

Increased levels of these hormones stimulate cell division.

Explains uncontrolled growth of tumor.

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On the Ti plasmid

Transfer the T-DNA to plant cellAcetosyringone (AS) (a flavonoid) released by wounded plant cells activates vir genes.virA,B,C,D,E,F,G (7 complementation groups, but some have multiple ORFs), span about 30 kb of Ti plasmid.Vir (virulent) genes

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virA

- transports AS into bacterium, activates virG post-translationally (by phosphoryl.)virG - promotes transcription of other vir genes

virD2 - endonuclease/integrase that cuts T- DNA at the borders but only on one strand; attaches to the 5' end of the SS

virE2

- binds SS of T-DNA & can form channels in artificial membranes

virE1

- chaperone for

virE2

virD2

&

virE2

also have NLSs, gets T-DNA to the nucleus of plant cell

virB

- operon of 11 proteins, gets T-DNA through bacterial membranes

Vir

gene functions (cont.)

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From Covey & Grierson

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Gauthier, A. et al. (2003) J. Biol. Chem. 278:25273-25276

Type IV Secretion Sys. many pathogens, also used in conjugation promiscuous forms T-Pilus B7-B10 span OM & IM

B7-B9 in OM interacts w/B8 & B10 of IM to form channel

3 ATPases

D4 promotes specific transport

B2 can form filaments

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VirE2 may get DNA-protein complex across host PM

Dumas et al., (2001), Proc. Natl. Acad. Sci. USA, 98:485

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T-DNA is excised from the TI Plasmid and transferred to the nucleus of the plant cell.

Here the T-DNA gets integrated into the plant DNA .The T-DNA can be passed onto daughter cells as an integral part of plant chromosome Transfer of tumour inducing Principle

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Only small segment of Ti Plasmid is transferred to the host plant cell and gets integrated with the genome

It contains genes for tumour formation(Tum) and nopalina biosynthesis(NOS)Tum genes encode enzymes that catalyse the synthesis of phytoharmones like IAA and the Cytokinin, Isopentenyl adenosine that causes tumourous growth of cells in crown gallsThe T-DNA is bordered by 25 bp repeats, required for the excision and transfer of T-DNAThe T-DNA

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Recognition of susceptible wounded plant cell

Plant exudates act as signals by inducing genes in the Vir genes of the Ti Plasmid Acetosyringone (AS) ,alpha – hydroxy acetosyringone-Binding to wounded cells, controlled by two chromosomal genes of agrobacterium-chv-A and chv-BTumour induction by Agrobacterium

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Excision, transfer and integration

The border repeats of the T-DNA play an important roleAny DNA sequence located between the border repeats is transferred to the post plant The T-DNA region is excised from the plasmid by the enzymes encoded by the Vir regionThese enzymes specifically recognize the T-DNA borders The T-DNA enters the plant cell and integrates into the host genome , mediated by host enzymesTumour induction by Agrobacterium

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The Ti plasmid has an innate ability to transmit bacterial DNA into plant cell.

This potential is exploited by the genetic engineers to use this as a vector The gene of a donnor organism can be introduced into the Ti Plasmid at the TDNA region This plassmid now becomes a recombinant plasmid By Agrobacterium infection , the donor genes can be transferred and integrated into the genotype of host This results in the production of a transgenic plant Ti plasmid as a vector

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A mature plant regenerated from transformed cells will contain the cloned gene in every cell and will pass on the cloned gene onto its

offspringsHowever regeneration of transformed plant can occure only if the Ti plasmid has been disarmed so that transformed cells donot display cancerious properties.Ti plasmid as a vector

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Disarming is possible because cancer genes are not needed for infection

Infectivity is controlled by virulence region of Ti plasmid.Infactonly parts ofTDNA are involved in infection are two 25bp repeat sequance Disarmed Ti plasmid

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Any DNA placed between these two repeat sequences will be treated as T- DNA and transferred to plant

It is therefore possible to remove all the cancer genes from normal T-DNA and replace them with an entirely new set of genes with out disturbing the infection process.Disarmed Ti plasmid

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Removal of genes responsible of auxin cytokinin and nopaline synthesis.

Disarmed Ti plasmidsDeletion of T-DNA REGION.PGV3850 is constructed from pTiC58It has pBR322 with AmpR Has Border repeats and NOS genesDisarmed Ti plasmid

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Agrobacterium having this PGV 3850 can transfer the modified T-DNA into plant cells.

But the recipient cells will not produce tumour, but could produce nopaline.This can be used as an efficient vector for introducing foreign genes into plants.Disarmed Ti plasmid

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The main problem is of course that a unique restriction site is an impossibility with a plasmid 200Kb in size.

Novel strategies have to be developed for inserting new DNA into plasmid.LIMITATIONS

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Strategy:

1. Move T-DNA onto a separate, small plasmid.2. Remove aux and cyt genes.3. Insert selectable marker (kanamycin resistance) gene in T-DNA. 4. Vir genes are retained on a separate plasmid.5. Put foreign gene between T-DNA borders.

6. Co-transform Agrobacterium with both plasmids.7. Infect plant with the transformed bacteria.

Binary vector system

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Binary vector system

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A foreign gene cloned into an appropriate plasmid (pBR322) can be integrated with the disarmed Ti plasmid by a homologous recombination

A compound plasmid called cointegrate is formed. Construction of Cointegrate

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Leaf-disc transformation -

after selection and regeneration with tissue culture, get plants with the introduced gene in every cell Floral Dip – does not require tissue culture. Reproductive tissue is transformed and the resulting seeds are screened for drug-resistant growth. (Clough and Bent (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant Journal 16, 735–743)2 Common Transformation Protocols

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Making a transgenic plant by leaf disc transformation with

Agrobacterium.S.J. Clough, A.F. Bent (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant Journal 16, 735–743.

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Employing method, genes from entirely unrelated plants can be transferred to other dicots transgenic plants with the following attributes were developed.

Resistance –To antibioticsPesticides and insect pests Drought and herbicides Uses

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Thank You