CSS451

CSS/HRT 451

Agrobacterium-mediated transformation

Guo-qing Song & David Douches

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

CSS451

OUTLINE
Agrobacterium Tumefaciens & Crown Gall Disease
Mechanism of Gene Transfer using Agrobacterium
f f f f Ti-plasmid Chromosomal and Vir Genes T-DNA Transfer T-DNA Integration

Engineering binary vectors for plant transformation Transformation protocols using Agrobacterium Factors influencing transformation efficiency
3

CSS451

Why Agrobacterium?
f Agrobacterium tumefaciens- or Agrobacterium rhizogenes-mediated transformation is to date
the most commonly used method for obtaining transgenic plants.

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

fAgrobacteria are naturally occurring, ubiquitous soil borne

y A. tumefaciens causes crown gall disease (tumors) y A. rhizogenes causes root hair disease (hairy root) f Other bacterial groups also contain species capable of interkingdom genetic exchange (Gelvin 2005). pathogens.

CSS451

A.Tumefaciens & Crown Gall Disease

CSS451

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

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

Gene Transfer using Agrobacterium Ti-plasmid

GGCAGGATATTCAATTGTAAAT Right T-DNA border GGCAGGATATTCAATTGTAAAT Left T-DNA border

Right and left border (RB, LB) sequences are the only parts of T-DNA needed to enable transfer into plants-removal of other 7 T-DNA genes creates a disarmed plants.

Gene Transfer using Agrobacterium Agro types

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

Carry genes for agropine synthesis and catabolism. Tumors do not differentiate and die out. Carry genes(3 required) to synthesize octopine in the plant and catabolism in the bacteria. Tumors do not differentiate, but remain as callus tissue.

Agropine-type (strain EHA105::pEHA105):

Octopine-type (strain LBA4404::pAL4404):

Carry gene for synthesizing nopaline in the plant and for utilization (catabolism) in the bacteria. Tumors can differentiate into shooty masses (teratomas).
8

Nopaline-type (strain GV3101::pMP90 (pTiC58)):

Gene Transfer using Agrobacterium Agro types

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

EHA105rifampicin (chromosomal) and streptomycin (on

the Ti plasmid)

GV3101: streptomycin 500 mg/l

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

Gene Transfer using Agrobacterium

Chromosomal and Vir Genes

CSS451

Chromosomal and vir genes of bacterial cells are both involved in T-DNA transfer
Virulence genes vir A vir B vir C vir D vir E vir F vir G
Chromosomal genes

Chemoreceptor, activator of vir G Transmembrane complex Host-range specificity Site-specific endonuclease T-DNA processing and protection Host range specificity Positive regulator of vir B, C, D, E, F
Attachment to plant cell, vir gene regulation
10

Disarmed Ti-plasmid

T-DNA LB auxin cytokin opine RB

Oncogenic genes vir genes ori opine catabolism

11

Disarmed Ti-plasmid

LB RB

vir genes

ori

opine catabolism

Disarmed Ti -plasmid

12

A Binary Vector Map

13

A Binary Vector Map

Plant selectable marker

KmR Bacterial selectable marker

14

From Dr. S. Gelvin, Purgue University

Introduction of Binary Vector into Agro

Electroporation Freeze/Thaw Triparental Mating

Agrobacterium Competent Agrobacterial Cells

15

Wise et al., 2006, Agrobacterium Protocols

Introduction of Binary Vector into Agro Electroporation

Freeze/Thaw Liquid nitrogen (196C) 3 min----37C 30 min

16

Binary Vector System

Agro only
Ti Helper Plasmid

Agro/E. Coli
Binary Vector
17

Binary Vector System

Plant selectable marker

KmR Bacterial selectable marker

18

Introduction of Binary Vector into Agro

Agro colonies Agro culture

Agro stock
(-80C with Glycerol)

19

CSS451

Mechanism of Gene Transfer using Agrobacterium

External Signals such as Acetosyringone

Passage of T-DNA from Agrobacterium cells into plant20 genomic DNA Stanton B. Gelvin. Nature 433: 583-584 (2005)

Mechanism of Gene Transfer using Agrobacterium

------ The Plant Cell Step

CSS451

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?

21

CSS451

22

CSS451

Expression of the Transgene

External Signal Cell Receptor Regulatory Elements Promoter Go Transcription Where When How much mRNA
Constitutive promoters: CaMV35S, Actin, Ubi Inducible prompters: rbcS Tissue specific promoters: Cab a stop codon (or termination codon): UAG (in

Gene

Terminator

STOP

Translation

Protein

RNA) / TAG (in DNA) ("amber"), UAA / TAA ("ochre"), and UGA / TGA ("opal" or "umber"

23

CSS451

Summary: T -DNA transfer T-DNA

1.

3.
6.

2. 4.

5.

7.

8.

9.

The plant releases wound signal compounds, such as acetosyringone. Vir C and/or Vir F recognize the host plant cells. The signal binds to vir A on the Agrobacterium membrane. Vir A with signal bound activates vir G. Activated vir G turns on other vir genes, including vir D and E. Vir D cuts at a specific site in the Ti plasmid (tumor-inducing), the left order. 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. Vir B + T-DNA complex is transferred to the plant cell, where it integrates in nuclear DNA.
T-DNA codes for proteins that produce hormones and opines. Hormones encourage growth of the transformed plant tissue. Opines feed bacteria a carbon and nitrogen source.
24

Agrobacteria attach to plant cell surfaces at wound sites.

Zhu et al. Journal of Bacteriology (2000)

Agrobacterium Preparation

Agro-transformation

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

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

25

Agrobacterium Protocols---Transformation
Tobacco Rice 4
Re-growth

CSS451

Tobacco

Rice

Inoculation

Molecular verification of gene presence & expression

PCR

Co-cultivation

Southern Blot

Selection and regeneration

Flowering and setting seeds

26

CSS451

The Floral Dip Method

A good stage for floral dipping Co-cultivation

Infection

Seed setting

Harvest seeds

Selection

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

27
Zhang et al. Nature Protocols 1(2) (2006)

Experiment 2: Tobacco Transformation

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.

28

CSS451

Experiment 2: Tobacco Transformation

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)

NOS-pro

NPT II (KanR)

NOS-ter

35S-pro

GUS

NOS-ter

pBI121
LBA 4404 with the Ach5 chromosomal background exhibits clumping

29

CSS451

Experiment 2
Plant materials: Tobacco cv. Samsun
Seeds Seed germination Seedling Plant

Mature seeds harvested from wild type tobacco plant cv. Samsun 1. Surface sterilization (50% clorox + 0.02% Tween 20, 15 min; 4 washes in sterile water). 2. Seed germination on MS medium.

Sterile seedling was maintained in Magenta box GA7 containing 50 ml MS medium. Subcuture cy cutting the internodes. Culture conditions: 25C, 16 hphotoperiod, 35-50 E m-2s-1.
30

Experiment 2
Explant preparation 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.

Inoculation

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 M 1. Co-cultivation medium: RM 2. Co-cultivation time: 2-4 d 3. Environmental conditions: in the dark 4. Ac: 100 M 1. Selection medium: RM + 100 mg/l Km + 500 mg/l Tn 2. Subculture: every 3 wk
31

Co-cultivation
2-3 d A. tumefaciens 5 d S. meliloti 5 d M. loti 5-11Rhizobium sp. NGR234

Selection and regeneration

CSS451

Explant preparation

1. 2. 3.

Segments from hypocotyl, cotyledons, epicotyl, leaf, internodes and petiole (Dicot) Embryogenic calluses (Monocot) Well developed regeneration system via either organogenesis or somatic embryogenesis

Inoculation

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 M 1. Co-cultivation medium: RM 2. Co-cultivation time: 2-4 d 3. Environmental conditions: in the dark 4. Ac: 100 M 1. Selection medium: RM + 100 mg/l Km + 500 mg/l Tn 2. Subculture: every 3 wk
32

Co-cultivation
2-3 d A. tumefaciens 5 d S. meliloti 5 d M. loti 5-11Rhizobium sp. NGR234

Selection and regeneration