Nitrogen fixation and agarobacterium

1. Plant Biotechnology
Transformation techniques
Actual cloning in plants:
• Agrobacterium
• Particle bombardment
• PEG
• Electroporation
• Silicon carbide fibers (Whiskers)
• Chloroplast transformation
How to insert the DNA of your choice into the host plant?
Cloning into vector:
• Recombinant DNA techniques
• PCR, restriction enzymes
• Ligation in vector
• Transformation of bacterial host
• Often several rounds!

2. Plant Transformation: the Agrobacterium method
Plant transformation:
Agrobacterium-mediated gene transfer
T-DNA
Binary vectors
Selectable markers
What to achieve with/expect after transformation?

3. Agrobacterium-mediated plant transformation
Agrobacterium
• Gram negative soil bacteria
• Flagellum; motile
• Infect plant at wounds in root
• Chemotaxis
• Transfer of part of DNA from Agrobacterium to plant
• Inter-Kingdom horizontal gene transfer
• Result: crown gall (in case of A. tumefaciens)
• Bacterium uses plant to produce shelter and food
• Parasitic
Most Agrobacterium species are free living and
saprophytic.
Four exceptions:
• A. tumefaciens crown gall
• A. rhizogenes hairy roots
• A. rubi cane gall
• A. vitis tumours on grape vine?

4. Crown galls on
peach seedlings
Gene transfer causes tumours
1. How does gene transfer work?
(“oncogenes” introduced)
2. Can we manipulate
Agrobacterium and introduce
genes of interest?

5. Mechanism of Agrobacterium transformation
Agrobacterium in rhizosphere
•Wound in plant tissue
•Chemicals released
•Agrobacterium moves towards wound (“chemotaxis”)
•Infects plant cells
•Part of DNA is transferred to host plant cell:
•T-DNA
•Genes on T-DNA induce tumour
•Beneficious for Agrobacterium
(not for plant!)

6. Ti-plasmid of Agrobacterium
Ti-plasmid
•Large circular plasmid (200 kb)
•Vir-region
•Opine production
•Origin of replication
•Transfer region
•T-DNA region
•LB/RB

7. The Vir genes are instrumental in the transformation process
• VirA senses phenolics / sugars (wound)
• Vir A is a kinase:
• It first autophosphorylates
• phosphorylates/activates VirG
• Activates VirG
• VirG induces expression of all Vir genes
• Vir G is a transcription factor
• VirD1/VirD2 recognise and excise T-DNA
• VirD2 nicks DNA (single strand) at LB and RB
• VirB forms channel through plant cell membrane
• VirD2/virE2 transport T-DNA to nucleus (NLS) through
nuclear pore
Transcriptional regulation
Post-translational
regulation

8. Excision of T-DNA and transport by virD/virE2
• Single-strand nicks are made at LB
and RB
• A single stranded T-DNA is excised
• The remaining ss part of the T-DNA
on the Ti-plasmid is repaired to
double stranded DNA

9. Transfer of T-DNA / vir complex to plant cell

10. 3’
LB
5’
RB
T-DNA excision and transport
• RB nicked first
• If excision incomplete: 3’ end missing
• Early T-DNA design: SM at 3’ end
However…
• 5’ end protected by VirD1
• 3’ end unprotected
• 100s of nucleotides may be deleted by exonucleases
• Modern design: SM at 3’ end of T-DNA
Dynamics of T-DNA excision – consequences for construct design

11. Transfer of T-DNA into the nucleus of plant cell
Note that
integration is
random!
Directed
integration
possible in other
organisms
How to obtain
directed
intergration in
plants…?

12. Once integrated in the plant genome…
T-DNA:
•Auxin
•Cytokinin
•Opine/agropine
•LB/RB: 24 bp repeats:
Plant hormones – growth – “oncogenes”
Carbon source – energy source
Necessary for integration in plant genome

13. Agrobacterium lives in crown gall

14. How to use this system?
Not interested in:
-Tumour
-Opine synthesis
Replace tumour + opine genes with
genes of interest; include selectable
marker
Leave LB + RB in place!
Vir genes still necessary

15. Development of transformation vector
Binary vector
•Shuttle vector: E. coli – Agrobacterium – plant
•Smaller than Ti-plasmid (10 kb vs 200 kb)
•No tumour induction
•Stable integration of T_DNA in plant genome
Widely used approach:
Agrobacterium
-”disabled Ti-vector” (+ vir genes; - tumour genes)
-binary vector (LB/RB; gene of interest; selectable marker

16. Agrobacterium mediated transformation
A combination of vectors is used:
• Disarmed Ti-plasmid: vir region present, but T-DNA deleted
• Binary vector with T-DNA

17. What’s on a Binary vector?

18. A bit more detailed – what do we need?
Promoter (homologous/heterologous)
Gene (cDNA or genomic; intron 1 included?)
Terminator of transcription (for correct poly-A addition)

19. pBin19: the Mother of all binary vectors
What’s on T-DNA and
why…?
•LB / RB
•Pnos
•npt2
•nosA
•lacZ
•mcs
What to insert?
•Promoter
•Gene of interest

20. How to transform plants with Agrobacterium?
Root transformation
Protoplast
transformation
“Floral dipping”;
whole plant
submerged in
Agrobacterium-
solution
Selection (callus
growth or
seedling/plant
growth on selective
medium)

21. Selection: why and how?
Transformation efficiency usually low
•Most plants / cells have not taken up T-DNA
•Selection for positive transformants needed
Plant selectable markers:
•Antibiotics npt2 (kanamycin)
•Herbicide resistance bar, EPSP
•Other markers
Problems: do we want to introduce these genes in our
crops? Resistance? Escape in wild species? “superweeds?”

22. Transgene expression
Possibilities for transgenes in plants:
•Over-express a gene
•Introduce extra copies
•Express from strong promoter
•Ectopic expression
•Express a novel gene (Cry-genes in Bt, herbicide)
•Silence a gene
•Introduce anti-sense gene (Flavr Savr tomato)
•RNA-i technique (RNA-interference) [GA20-oxidase]
•Knock out endogenous gene (Arabidopsis) [Ceres-example]
Your transgene may knock out an
important gene!
=> Always screen several
transformants

23. Agrobacterium mediated transformation
• Efficient in dicots
• However, increasingly used in monocots as well
• Adapted for use in transforming fungal, yeast and human cells as well!
• Hypervirulent strains: extra Vir genes, higher Vir gene activity
• Mainly A. tumefaciens; other species useful?
• Transformation of protoplasts; regeneration of plants
• Leaf disc transformation
• Floral dip (aim for transformation of gametophytes)
• In all cases: hemizygotes
• Often back-crossed in desired crop varieties
• End with selfing to obtain homozygous plants

24. Summary
Agrobacterium-mediated transformation
•Ti-plasmid
•Vir-genes
•T-DNA
•LB/RB
•Oncogenes
•Opine synthesis
Binary vectors:
•T-DNA (LB/RB)
•Selectable marker
•Mcs
Combine with disarmed Ti-plasmid
•Vir-genes
=> Vector for transformation &
expression of foreign DNA into plant cell