-mediated Transformation

Introduction of
Foreign Genes via
mediated Transformation

Analyzing Gene Expression
With Transgenic Plants

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Agrobacterium-mediated Transformation

Agrobacteria are soil-borne, bacterial plant pathogens which cause tumorous growths or roots to develop on infected plants. The opine concept explains these growths by the presence of host (plant)-synthesized opines incited by the parasitic agent (bacteria). These opines create a chemical environment which favors the continued proliferation of the agent (bacteria). How do the opines allow the bacteria to proliferate? They supply the carbon and nitrogen needed by the bacteria for growth and incite conjugal transfer of it plasmid to neighboring bacteria (genetic colonization).

Characteristics of Agrobacteria

  • related to Rhizobium
  • contain large plasmids: Ti (A. tumefaciens); Ri (A. rhizogenes)
  • integration of a part of the plasmid (T-DNA) induces the tumorous or rooty growth T-DNA;
  • this DNA directs the synthesis of opines, carbon and nitrogen containing molecules used for the continued bacterial growth
  • each bacterial strain can utilize one of the three opine classes: octopine, nopaline and agropine
  • host range of A. tumefaciens
    • 331 genera; 643 species
  • variablilty is seen for infection between
    • varieties of a species
    • the organ of the plant
  • monocot species except for some members of the Liliales and Arales genera are not susceptible

Characteristics of the Ti-Plasmid

  • large circular plasmid contains genes for:
    • virulence
    • catabolism of specific opines
    • host-directed opine synthesis
    • host-directed, bacterial-type plant hormones
  • virulence genes are grouped genes for synthesis of opines and plant hormones are contained on the T-DNA; T-DNA is bordered by 25 bp direct repeats; only the TR border is required for transfer although the Ti border increased the efficiency when it is present; no sequences other that the borders are required for transfer

Transfer of T-DNA to Plant Cells

The T-DNA that is transferred to the plant cells contains genes which encode for proteins involved in opine and plant-type phytohormone biosynthesis. Although these genes reside on a bacterial plasmid they are only active in a plant cell. To be transcribed in the plant cell, though, they must contain typical eukaryotic controlling sequences. All of these genes have been shown to contain TATA and CAAT boxes and typical plant polyadenylation signals. Further, portions of the promoters and the polyadenylation regions have been added to marker genes as a method of following the transformation events.

Two primary steps in transformation

  • 1.binding of Agrobacterium to a plant cell (up to 200/cell can attach)
  • transfer of DNA to the plant cell (multiple T-DNAs can be transferred)
Each step involves a different set of genes. Binding to plant cell requires three chromosomal Agrobacterium genes:

chvA and chvB - mutants at these two loci result in a marked reduction in Agrobacterium binding to plant cells; chvA may encode a transport factor and chvB encodes a protein involved in 2-linked beta-glucan synthesis
pscA - required for the synthesis of the major neutral and acid extracellular polysaccharides

Vir Region

Initially it was thought that wounding, an absolute prerequisite for Agrobacterium transformation, was required for the plant cell and bacteria to come in contact with each other. Actually, wounded cells secrete low-molecular weight molecules that stimulate the vir genes. These molecules are acetosyringone and hydroxy-acetosyringone. These two molecules stimulate the synthesis of several vir genes. Further, acetosyringone can act as a chemical attractant in vitro and thus may act as chemotactic agent in nature.

The vir region contains six genes. virA and virG are the only two monocistronic loci, the other four are polycistronic and encode several proteins. The regulation of these important genes is integrated and involves a cascade of transcriptional events. The cascade begins with virA which is constitutively expressed. The protein encodes a transmembrane protein that senses the chemical environment. It is assumed that this protein senses the presence of acetosyringone. VirG produces two transcripts that differ at the 5' end of the mRNA, one which is constitutively expressed and a longer mRNA that only is expressed in the presence of plant inducible compounds. VirB and virE polypedtides are the two vir products that are highly induced by the wound induced plant phenolic compounds.

Vir Gene Function
VirA encodes a single protein which resembles a transmembrane chemoreceptor found in other bacteria; constitutively expressed; monocistronic
VirG a positive regulatory protein which relays environmental information (when plant inducible factors are present) to other vir loci; requires virA; strongly affects virB,C,D, E: monocistronic
VirE a single-stranded DNA binding protein that appears to coat the T-strand during transfer to the plant cell; polycistronic
VirC and virD site-specific endonuclease that cleaves at the 25 bp direct repeats borders of the T-DNA; produces a T-strand that is the intermediate molecule that is transported to the plant cell; polycistronic
VirB may play a role in directingT-DNA transfer events at the bacterial cell surface

Types of Ti Plasmids

Two classes of Ti-plasmids exists. The nopaline and octopine Ti-plasmids have T- DNAregions but the structure of these regions differs.

Nopaline T-DNA - single continuous segment of about 22kb
Octopine T-DNA - three segments; left DNA (TL) = 13 kb; center DNA (TC) DNA = 1.5 kb; right DNA (TR) = 7.8 kb; TL DNA contains the oncogenic function and TR contains the opine synthetic genes

The only structural requirement for T-DNA transfer is the TR direct repeat border, but the T-DNA region also contains a TL border repeat. The consensus sequence of the TR repeat is:

             TG Pu G     AT
             NC C  T     TC
In this repeat, two domains of 13 and 5-7 bp are conserved. But deletion of the first 6 bp or the last 10 bp blocks T-DNA transfer.

If the TL border is deleted transformation can be detected, but if the TR border is deleted transformation is eliminated. This suggested that transfer was in the right to left direction. Further the orientation of the repeat is important because if the border is reversed, transformation is greatly attenuated. Thus, when the right border is used for transfer, the genes required for the oncogenic functions are transferred, but when the left border is used in lieu of the right border, transfer does not include these gene and the transformation phenotype is not detected.

T-strand - a single stranded product that is derived from the T-DNA; on average, one strand per bacterium is produced; T-strands are derived from the bottom strand of the nopaline plasmid; the 5' end corresponds to the TR border and the 3' end corresponds to the TL border; the nick occurs between the 3rd and 4th base; the 5' end is always within a few bases of the TR border but the 3' border ranges from 100 bases before TL to right at the TL border

Integration into the Plant Genome

A single integrationis the most frequent event, but on average 3 copies are integrated. An analysis of 161 transformants of Arabidopsis was performed to determine the type of integration events.

  • 55% single event
  • 20% two unlinked events
  • 6% three unlinked events
  • 1% four unlinked events
  • 12% non-Mendelian ratios
In tomato it was shown that in ten different transformants, integration occurred at nine different chromosomes. The target DNA sites does not appear specific except for the fact they are AT rich.

Only one detailed analysis of a T-DNA insertion site has been performed. It was determined that a 158 bp repeat was developed at both the TR and TL borders. Other events that occur are short deletions and insertions at the end of the 158 bp segment.

Generalized Model of T-DNA Transfer and Integration

  1. T-strand is produced and transferred to the plant cell as a DNA/protein complex. Reguires vir (Ti-plasmid) and chromosomal genes
  2. A protein that is at the 5' end of the T-strand interacts with a nick in the plant DNA.
  3. A single-stranded DNA attaches to one strand of the plant DNA and a torsional change occurs in the plant DNA which generates a second nick.
  4. Each strand of the T-strand is ligated to the plant DNA and the homologous strand is produced.
  5. Repair and replication of the staggered nick in the plant DNA results in duplication and rearrangement of the target DNA.

Copyright © 1998. Phillip McClean