Owen Patterson’s (UK Secretary of State for Environment, Food and Rural Affairs) recent speech on GM crops has sparked much debate. Seems like good time to write about a bacterial pathogen (disease-causing) that has been genetically modifying plants to produce its favourite food source long before us humans even thought of it.
Agrobacterium tumefaciens is able to infect and cause crown gall disease in about 60% of Eudicot species species including trees such as cherry, apple and willow and vegetables such as sugar beet and courgette (1, 2). Infected plants produce develop tumours (galls) on stems and roots (see below) as a result of uncontrolled cell division. Generally most plants with crown gall disease survive and continue to grow but it can lead to yield losses in some crop plants.
When A. tumefaciens infects a plant it transfers some of its DNA into the genome of the plant cells, reprogramming them to divide and produce chemicals called opines. The bacteria use these opines as their primary food source but for the plant they are completely useless. Once the DNA transfer is complete the tumour cells can grow and divide independently of the bacteria.
The virulence (vir) genes required for A. tumefaciens to infect and transfer DNA into the plant cells are contained on the Ti plasmid. Also within the Ti plasmid is the “transfer DNA” or T-DNA, the sequence that is transferred into the plant genome. This is flanked by two 25 bp repeat sequences, which are essential for T-DNA transfer and integration into the plant host genome. Within the T-DNA region there are genes for the synthesis of the plant hormones auxin and cytokinin, which promote plant cell division, and for opine synthesis. The T-DNA genes themselves aren’t required for T-DNA transfer and are only expressed within the plant cells. By promoting cell plant cell division A. tumefaciens increases the number of cells that are producing their preferred food source. All in all a pretty neat system!
A. tumefaciens-mediated DNA transfer has been harnessed as a tool for molecular biology. Removing the hormone and opine synthesis genes from the T-DNA region of the Ti plasmid results in A. tumefaciens that can still transfer the T-DNA into plants but without the disease-causing effects. Scientists can insert genes of interest into this “disarmed” Ti plasmid and by including a marker gene (for example GFP or kanamycin resistance) they can select for plants that have the T-DNA inserted into the genome. One drawback of the Ti plasmid is that it is rather large making it difficult to work with in the lab. A binary vector system has been developed where the vir genes and the T-DNA are contained on separate, smaller plasmids, called the “helper plasmid” and “binary plasmid” respectively.
In nature the T-DNA inserted into plants infected by A. tumefaciens is not passed on to the next generation as only individual cells in the stem and/or root are transformed, not the whole plant.
To generate a truely genetically modified plant in the lab, plants are regenerated from single transformed cells. The seed produced by these regenerated plants has the T-DNA integrated into the genome, thus the introduced genes can be passed on down the generations. Another method of plant transformation involves dipping the flowers in a solution of A. tumefaciens, which leads to the T-DNA being incorporated into developing embryos. This is much easier than regenerating plants but only works on a few species including the model plant Arabidopsis thaliana.
Genetically modified plants generated using A. tumefaciens mediated-transformation are widely used for research purposes (including my PhD!) and grown commercially in a number of countries including USA and China. Under lab conditions it is also possible for A. tumefaciens to transfer T-DNA into non-plants including fungi (Aspergillus niger and Saccharomyces cerevisiae) and human HeLa cells. This opens the possibility of using A. tumefaciens as a tool for transforming non-plants too (2).
1) Royal Horticultural Society website: Crown Gall (accessed 01/07/13) http://apps.rhs.org.uk/advicesearch/profile.aspx?pid=141
2) Plant Cell Plant Science Teaching Tools. A really useful pathogen, Agrobacterium tumefaciens http://www.plantcell.org/site/teachingtools/TTPB23.xhtml