CSS/HRT 451 CSS/HRT 451 CSS/HRT 451 CSS451

Agrobacterium-mediated transformation

Guo-qing Song & David Douches

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Learning Objectives

• Understand what key changes had to be made to the Agro Ti plasmid for the experiment transfer of novel genes into plants

• Understand the binary plasmid transformation system

• Understand some mechanisms of gene transfer

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Agrobacterium Tumefaciens & Crown Gall Disease

MechanismMechanism of of Gene Transfer using Gene Transfer using AgrobacteriumAgrobacterium

Engineering binary vectors for plant transformation Engineering binary vectors for plant transformation

Transformation protocols using Transformation protocols using AgrobacteriumAgrobacterium

Factors influencing transformation efficiencyFactors influencing transformation efficiency

OUTLINECSS451

Ti-plasmidChromosomal and Vir GenesT-DNA TransferT-DNA Integration

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Agrobacterium tumefaciens- or Agrobacteriumrhizogenes-mediated transformation is to date the most commonly used method for obtaining transgenic plants.

The tumorigenic host plant species for range A. tumefaciens include: Large number of dicots and some monocots and Gymnosperms.

Agrobacteria are naturally occurring, ubiquitous soil bornepathogens.A. tumefaciens causes crown gall disease (tumors)A. rhizogenes causes root hair disease (hairy root)Other bacterial groups also contain species capable of

interkingdom genetic exchange (Gelvin 2005).

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Why Agrobacterium?

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6Stanton B. Gelvin. Nature 433: 583-584 (2005).

A.Tumefaciens & Crown Gall DiseaseA.Tumefaciens & Crown Gall Disease

http://arabidopsis.info/students/agrobacterium/

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Right and left border (RB, LB) sequences are the only parts of T-DNA needed to enable transfer into plants-removal of other T-DNA genes creates a disarmed plants.

GGCAGGATATTCAATTGTAAAT

GGCAGGATATTCAATTGTAAAT

Left T-DNA border

Right T-DNA border

Gene Transfer using Gene Transfer using AgrobacteriumAgrobacteriumTiTi--plasmidplasmid

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AgropineAgropine--typetype (strain EHA105::pEHA105): (strain EHA105::pEHA105): Carry genes for agropine synthesis and catabolism. Tumors do not differentiate and die out.

OctopineOctopine--typetype (strain LBA4404::pAL4404):(strain LBA4404::pAL4404):Carry genes(3 required) to synthesize octopine in the plant and catabolism in the bacteria. Tumors do not differentiate, but remain as callus tissue.

NopalineNopaline--typetype (strain GV3101::pMP90 (pTiC58)):(strain GV3101::pMP90 (pTiC58)):Carry gene for synthesizing nopaline in the plant and for utilization (catabolism) in the bacteria. Tumors can differentiate into shooty masses (teratomas).

Gene Transfer using Gene Transfer using AgrobacteriumAgrobacterium

Agro typesAgro typesHellens et al (2000; Trends in Plant Science 5:446-451)

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Gene Transfer using Gene Transfer using AgrobacteriumAgrobacterium

Agro typesAgro types

LBA4404: rifampicin (chromosomal) and streptomycin (on the Ti plasmid)

EHA105：rifampicin (chromosomal) and streptomycin (on the Ti plasmid)

GV3101: streptomycin 500 mg/l

Hellens et al (2000; Trends in Plant Science 5:446-451)

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Chemoreceptor, activator of vir G Transmembrane complexHost-range specificitySite-specific endonucleaseT-DNA processing and protectionHost range specificityPositive regulator of vir B, C, D, E, F

Virulence genesvir Avir Bvir Cvir Dvir Evir Fvir G

Attachment to plant cell, vir gene regulationChromosomal genes

Chromosomal and vir genes of bacterial cells are both involved in T-DNA transfer

Gene Transfer using Gene Transfer using AgrobacteriumAgrobacteriumCSS451

Chromosomal and and VirVir GenesGenes

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LB RBauxin cytokin opine

vir genes ori opine catabolism

T-DNA

Oncogenic genes

Disarmed TiDisarmed Ti--plasmidplasmid

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LB RB

vir genes ori opine catabolism

Disarmed Ti -plasmid

Disarmed TiDisarmed Ti--plasmidplasmid

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A Binary Vector MapA Binary Vector Map

14From Dr. S. From Dr. S. GelvinGelvin, , PurguePurgue UniversityUniversity

KmR

Bacterial selectable marker

Plant selectable marker

A Binary Vector MapA Binary Vector Map

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Introduction of Binary Vector into AgroIntroduction of Binary Vector into Agro

Agrobacterium

ElectroporationElectroporationFreeze/ThawFreeze/ThawTriparentalTriparental MatingMating

Competent Agrobacterial CellsWise et al., 2006, Wise et al., 2006, AgrobacteriumAgrobacterium ProtocolsProtocols

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ElectroporationElectroporation

Freeze/ThawFreeze/Thaw

Liquid nitrogen (196C) 3 minLiquid nitrogen (196C) 3 min--------37C 30 min37C 30 min

Introduction of Binary Vector into AgroIntroduction of Binary Vector into Agro

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Binary Vector SystemBinary Vector System

Binary VectorBinary Vector

Binary Vector SystemBinary Vector System

Ti Helper PlasmidTi Helper Plasmid

Agro only Agro/E. Coli

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KmR

Bacterial selectable marker

Plant selectable marker

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Agro culture

Introduction of Binary Vector into AgroIntroduction of Binary Vector into Agro

Agro colonies

Agro stock(-80C with Glycerol)

20Stanton B. Gelvin. Nature 433: 583-584 (2005)

MechanismMechanism of Gene Transfer using of Gene Transfer using AgrobacteriumAgrobacterium

Passage of T-DNA from Agrobacterium cells into plant genomic DNA

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External Signals such as

Acetosyringone

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Chromosomal and vir genes of bacterial cells are both involved in T-DNA transfer

The plant cell step of T-DNA transfer is poorly understood

► Entry into plant cell?

► Nuclear uptake?

► Integration into chromosome?

MechanismMechanism of of Gene Transfer using Gene Transfer using AgrobacteriumAgrobacterium------------ The Plant Cell StepThe Plant Cell Step

►►

►

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Regulatory Elements

Where When How much

GoSTOP

Transcription

Translation

mRNA

Protein

Promoter Gene Terminator

External Signal

Cell Receptor

Expression of the Expression of the TransgeneTransgeneCSS451

Constitutive promoters: CaMV35S, Actin, Ubi

Inducible prompters: rbcS

Tissue specific promoters: Cab

a stop codon (or termination codon): UAG (in RNA) / TAG (in DNA) ("amber"), UAA / TAA ("ochre"), and UGA / TGA ("opal" or "umber"

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1. Agrobacteria attach to plant cell surfaces at wound sites. 2. The plant releases wound signal compounds, such as acetosyringone. 3. Vir C and/or Vir F recognize the host plant cells. 4. The signal binds to vir A on the Agrobacterium membrane. 5. Vir A with signal bound activates vir G. 6. Activated vir G turns on other vir genes, including vir D and E. 7. Vir D cuts at a specific site in the Ti plasmid (tumor-inducing), the left

order. 8. Single stranded T-DNA is bound by vir E product as the DNA unwinds

from the vir D cut site. Binding and unwinding stop at the right border. 9. Vir B + T-DNA complex is transferred to the plant cell, where it

integrates in nuclear DNA.

Zhu et al. Journal of Bacteriology (2000)

Summary: T-DNA transferSummary: TSummary: T--DNA transferDNA transferCSS451

T-DNA codes for proteins that produce hormones and opines. Hormonesencourage growth of the transformed plant tissue. Opines feed bacteria a carbon and nitrogen source.

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Agrobacterium PreparationAgrobacterium Preparation

Temperature: 30C Medium: LB, YEB, or YEPAntibiotic: Km or based on the SMG in the vector. Time: 24-48 hr.Concentration: O.D.600=0.5-1.0

Streak the plateTemperature: 30C Medium: LB Antibiotic: Km vector. Time: 2-3 days.

Agro-transformation

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Inoculation

Selection and regeneration

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Re-growth

Molecular verification of gene presence & expression

Flowering and setting seeds 6

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Agrobacterium Protocols---TransformationAgrobacterium Protocols---Transformation

Co-cultivation

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Tobacco Rice Tobacco Rice

PCR

SouthernBlot

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Zhang et al. Nature Protocols 1(2) (2006)

The Floral Dip Method

Infection

Co-cultivation

A good stage for floral dipping

Agrobacterium-mediated transformation of Arabidopsis thaliana using the floral dip method

Seed setting Harvest seeds Selection

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Objectives:• To get familiar with A. tumefaciens-mediated transformation,

such as explants, T-DNA, infection, co-cultivation, selection, binary vector, right border and left border, selectable marker gene (SMG), markers for screening, regeneration, antibiotics, and etc.

• To understand the differences between the wild-type T-DNA and the disarmed T-DNA.

Experiment 2: Tobacco Transformation

29LBA 4404 with the Ach5 chromosomal background exhibits clumping

NOS-pro NPT II (KanR) NOS-ter 35S-pro GUS NOS-ter

pBI121

Experiment 2: Tobacco Transformation CSS451

Agro strains: LBA4404:pBI121 & ACH5

1. LBA4404 has the Ach5 chromosomal background

2. ACH5 (pTiAch5---a wild-type octopine plasmid)

3. LBA4404 (pAL4404---a disarmed octopine plasmid)

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Experiment 2

Plant materials: Tobacco cv. Samsun

• Sterile seedling was maintained in Magenta box GA7 containing 50 ml MS medium.• Subcuture cy cutting the internodes.• Culture conditions: 25C, 16 h-photoperiod, 35-50 µE m-2s-1.

Seedling

Mature seeds harvested from wild type tobacco plant cv. Samsun1. Surface sterilization (50% clorox + 0.02% Tween 20, 15 min; 4 washes in sterile water).

2. Seed germination on MS medium.

PlantSeed germinationSeeds

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3 1. Selection medium: RM + 100 mg/l Km + 500 mg/l Tn

2. Subculture: every 3 wk

Selection and regeneration

1. Co-cultivation medium: RM2. Co-cultivation time: 2-4 d 3. Environmental conditions: in the dark4. Ac: 100 µM

Co-cultivation2-3 d A. tumefaciens5 d S. meliloti5 d M. loti5-11Rhizobium sp. NGR234

1. Agro concentration: O.D.600 = 0.5-0.8.2. Infection time: 10-15 min.3. Infection medium: Regeneration medium (RM)4. Acetosyringone (Ac): 100 µM

Inoculation

1. Explant size: 0.5-0.8 cm X 0.5-0.8 cm.2. To prepare the explants using sterile techniques. 3. Do not let the explants too dry.

Explant preparation

Experiment 2

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1. Selection medium: RM + 100 mg/l Km + 500 mg/l Tn2. Subculture: every 3 wk

Selection and regeneration

1. Co-cultivation medium: RM2. Co-cultivation time: 2-4 d 3. Environmental conditions: in the dark4. Ac: 100 µM

Co-cultivation2-3 d A. tumefaciens5 d S. meliloti5 d M. loti5-11Rhizobium sp. NGR234

1. Agro concentration: O.D.600 = 0.5-0.8.2. Infection time: 10-15 min.3. Infection medium: Regeneration medium (RM)4. Acetosyringone (Ac): 100 µM

Inoculation

1. Segments from hypocotyl, cotyledons, epicotyl, leaf, internodes and petiole (Dicot)

2. Embryogenic calluses (Monocot)3. Well developed regeneration system via either

organogenesis or somatic embryogenesis

Explant preparation