Characteristics of Agrobacteria
Characteristics of the Ti-Plasmid
Transfer of T-DNA to Plant CellsThe 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
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
Vir RegionInitially 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.
Types of Ti PlasmidsTwo 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
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 TGGCAGGATATAT N TGTAA NC C T TCIn 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 GenomeA 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.
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
Copyright © 1998. Phillip McClean