Agrobacterium- Lec 2 - Info - Csb350 Exam

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Agrobacterium tumefaciens

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-its virulence
-its T-DNA structure
-Promoters
-its biosynthesis
-classification
-Opine production
-Transformation in -Monocotyledonous
-For Agrobacterium-mediated transformation
-Agrobacterium for genetic engineering of plants
-Resistant selectable markers
-Transforming procedure (Regeneration) for multicellular organisms in rice and arabidopsis

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Agrobacterium tumefaciens

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is a soil borne gram-negative bacterium, that has a unique ability to introduce part of its DNA into plant cells.
-co-cultured or inoculated with plant tissue
-transfer part of their DNA into plant cells.

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Agrobacterium
Virulence

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nduces crown gall disease. DNA transfer from A.tumefaciens to eukaryotic cells is the
only known example of interkingdom DNA transfer
-contain one or more large plasmids:
one of which carries the genes for tumor induction and is known as the Ti (tumor inducing) plasmid.
A. tumefaciens integrates a small part of the Ti plasmid into the host plant genome, which causes the plant cells to become cancer cells and produce specific compounds called Opines, which the bacterium utilize as a carbon source.

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tumefaciens T-DNA structure is composed 25 bp repeats and

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LB → RB: Shi (L),Shi (R), Roi (R), Nos (L)

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LB: Shi

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Shoot inducing - 2 genes for auxin synth

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Roi

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root inducing gene for cytokin synthesis

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Promoters are Eukaryotic

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This means they are not expressed in the bacteria but in the plant.

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1. bacterium makes a by-pass in auxin biosynth

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Trp → IAA

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2. ‘’ Cytokinin biosynthesis

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DXP → Cytokinin

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Agrobacterium Strains are grouped by

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the type of Opine synthesized.
Agrobacterium-infected plants produce
a particular type of opines, which
becomes nutrient for the bacterium

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Opine(s):
amino acid derivatives.

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Agropine-type, Octopine-type, Nopaline-type

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1. Agropine-type bacteria

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(ex. EHA105::pEHA105) ~EHA

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2. Octopine-type

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(ex. LBA4404::pAL4404) ~LBA- PAL
requires 3 genes for its synthesis

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3. Nopaline-type

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(ex. GV3101::pMP90) ~GV-PMP

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Why is Transformation in Monocotyledonous crops and cereals
(wheat, barley, maize, and rice) a problem?

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-not natural hosts to Agrobacterium.

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Finding to solve problem

enables Agrobacterium-mediated transformation, such as GFP, into monocots

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NEED stronger vir genes and stronger expression:
i)acetosyringone (inducer of vir genes)

ii)hyper-virulent strains (with their high ability to express vir genes)

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For Agrobacterium-mediated transformation

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(1)Ori vector for E. coli replication
(2)Selection marker for E. coli
(3)The gene of interest (or gene that will be introduced into plant genome)
(4)Selection marker for plants (drug resistance
or visible)
(1)~(4) + (stops here for simple particle bombardement)

(additional for agrobacterial transformation)
(5) Ori vector for Agrobacterium
(6) Selection marker for Agrobacterium
(7) Border sequences (LB & RB)
(8) Vir genes

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Agrobacterium strains commonly used in the lab with selction marker

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GV3101, LBA4404, EHA105, AGL-1***
Nopa , octo , agro,

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GV3101 (Produces Nopa )

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Rifampicin resistance (chromosomal)

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LBA4404(Produces octo )

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Rifampicin resistance (chromosomal)

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EHA105 (Produces agro)

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Rifampicin resistance (chromosomal)

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AGL-1 (Produces )

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Rifampicin & Carbenicillin resistance (chromosomal)

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Binary vector system

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Systems in which T-DNA and vir genes are
located on separate replicons (i.e. plasmids)

T-DNA is located on the binary vector (the non-T-DNA region of this vector containing origin(s) of replication that could function both in E. coli and in A. tumefaciens, and antibiotic-resistance genes.)
-these vir genes are also located on Ti plasmid or helper plasmid.

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How can we make the Ti plasmid usefull for transformation?

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How can we take out tumour formations
(1)take out auxin and citokin genes (oncogenes)
(2)Retain vir genes, LB & RBorder, ori
More efficient
(3)Ti plasmid(120Kb) is reduced

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Agrobacterium for genetic engineering of plants

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Ti plasmid (Helper plasmid)

Binary vector

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Ti plasmid (Helper plasmid)

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vir genes and ori

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Binary vector

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-ori (E.coli) and ori (agrobacterium), selectable marker for bacteria.
-Inside the TDNA LB and RB: Cloning site for plant genes( LB ) and selectable marker for plant(RB).

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Selectable markers

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antibiotics and herbicides

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antibiotics

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Kanamycin (NPTII)
(neomycin phosphotransferase II)

Hygromycin (HPTII)
(hygromycin phosphotransferase II)

Gentamicin
(Gentamicin 3-N-acetyltransferase)

Streptomycin
(Aminoglycoside-3-adenyltransferase)

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Herbicides

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-Glyphosate
5-enolpyruvylshikimate-3-phosphate synthase

-Sulfonylurea
Acetplactate synthase (ALS)

-bialaphos/Basta
phosphinothricin N-acetyltransferase

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IN lab 2

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GV3101 producing nopaline Tumefascien

-Ti plasmid: Gentamyacin Resistant gene containing vir genes
-Binary vector: kanmyacin resistant gene on pEGAD binary vector.

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Gentamicin

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is an aminoglycoside antibiotic.
-synthesized by Micromonospora,
(a genus of Gram-positive bacteria widely present in the environment (water and soil)).
-Gen is a bactericidal antibiotic that works by binding the 30S subunit of the bacterial ribosome, interrupting protein synthesis.

-Gentamicin acetyltransferase (AAC) : inactivates antibiotic and the cells expressing the AAC are Gen-resistant.

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Kanamycin sulfate

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aminoglycoside antibiotic
-from Streptomyces kanamyceticus
-interacts with the 30S subunit of prokaryotic ribosomes.
It induces substantial amounts of mistranslation and indirectly inhibits translocation during protein synthesis.
- Neomycin phosphotransferase II (NPT II) inactivates Kanamycin, so the cells expressing NPT II are Kanamycin-resistant

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Transforming multicellular organism

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can only transfrom one cell that will generate an entire organism. (thats why we infected plants at the early stage of seed production)

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Regeneration

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Is the process of generating a whole plant from a single transformed cell

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can be from tissue culture

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placed in Petri dishes.
The response of the plant tissue depends on :
the starting plant materials
and the medium in the Petri dish.

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shoot morphogenesis,

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shoots form,
which must generate roots before
they can be transferred to the soil.

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STEPS for RICE

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-callus induction
-Agrobacterium inoculation (Particle bombardment)
-Antibiotic selection
-Shoot regeneration
-Rooting and Growth

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STEPS for Arabidopsis

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(floral dip method)

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Generation of Transformants using Agrobacterium strain
GV3101 and binary vector pEGAD

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RB- 35S-EGFP(Ala)10 - cDNA polylinker (which also contains restriction sites)- 35S - Basta r, LB