Phil McClean, my old friend and colleague, has a great set of web pages up on his site. We'll refer to them here.
Agrobacterium tumefaciens is a gram negative aerobic bacterium which occurs as a free-living soil bacterium. It can infect plant tissues through open wounds. Upon infection, dicots respond to infection by initiating massive cell division at the point of infection. Following the initiation of cell division, plant cells near the site of infection begin production of unusual amino acids (opines). Plant cells are unable to catabolize these unusual metabolites, but Agrobacterium cells can catabolize them, using them as both carbon and nitrogen sources.There are at least three species of Agrobacterium: A. tumefaciens, A. rhizogenes, and A. radiobacter. A. tumefaciens initiates gall formation, generally at around the soil surface near the crown. Hence the name 'crown gall' for the tumor-like structure that forms following infection. When I was 12, I was responsible for disking and tilling our olive orchard. Olives are very shallow-rooted trees. I drove too close to many trees. Big lumps grew from the surface roots and trunk, where I'd damaged them with the disc. These were crown galls generated by A. tumefaciens.
A. rhizogenes initiates root proliferation. This pathogen was utilized in the sugarbeet transformation manuscript you previously received.
A. radiobacter is a non-pathogenic variant of the Agrobacterium genus which, if present in sufficiently high numbers, can inhibit plant infection by either rhizogenes or tumefaciens. Radiobacter is utilized by the horticultural industry in areas in which tumefaciens is a problem. Bare rootstock is treated with an abundance of radiobacter cells prior to planting, saturating the Agrobacterium binding sites in the inevitable small wounds caused by transplantation, storage and planting.
Agrobacterium (tumefaciens and rhizogenes) represent the only known example of
biologically-driven transmission of genetic material between prokaryotes and eukaryotes. In
this lecture we will discuss the process underlying DNA transmission, its limitations and uses.
From the evolutionary perspective, the genes which Agrobacterium introduces have never been observed to be sexually transmitted in nature. They are, from the plant's perspective, an evolutionary dead end. In the short term, however, they serve the needs of the bacterium. As Phil points out, the callus tissue that Agrobacterium infestation initiates has never been observed to become reproductive. The constitutively expressed VirA gene appears to be the sensor for acetosyringone or its derivatives. These are produced by dicot root and stem cells following wounding.
The infection process is composed of:
The 'transfer' stage of this process was pretty well diagrammed in Barbara Baker's Science paper on pathogenesis.
The array of Agrobacterium genes involved in this process sounds pretty daunting initially, but becomes more straightforward over time. The primary difference between radiobacter and its infectious relatives is the presence of a large plasmid (the Ti plasmid). The Ti plasmid carries two significant gene regions, the vir region and the tDNA. The tDNA is the DNA that is transferred. Most of the genes required for infection are carried within the vir region. The chromosome carries the Chv genes.
In the vir region of the plasmid are the vir A, vir B, vir C, vir D, vir E, vir g and VirH operons. As an example of the internal complexity of these, vir B contains 11 characterized open reading frames. The VirB protein products are the major components of the trans-membrane channel used by the tDNA. Vir D binds to the 5'end of the tDNA, while VirE2 proteins coat the tDNA and protect it from degradation. tDNA is targeted to the nucleus by the VirD and VirE proteins
Vir A produces the protein most directly associated with chemotaxis and sensing of plant phenolic compounds (notably acetosyringone) associated with wounding. Vir H may act as a detoxifier of the plant bacteriocidal compounds produced through the wound response.
Practical Transformation
There are two basic routes to the use of A. tumefaciens - cointegrate transformation and binary vector transformation. Cointegrate transformation requires homologous recombination between your tDNA/genes of interest construct and the large Agrobacterium plasmid prior to plant infection. Binary vectors, in which the tDNA resides on one plasmid and the Vir genes on another, came into common use in the 1980s. Many groups have developed their own tDNA binary vectors. Art Hunt, of the University of Kentucky, provides nice graphics and complete sequence information for his.
GMOs and the real world.
Barriers between species are now practically non-existent. This is a great thing- evolution does not come up with every useful response in every lineage. Non-host resistance may conceivably convert crops like wheat into non-hosts of rusts and mildews. Golden rice, rice that produces provitamin A, is a great idea. This is the first example of complex metabolic engineering of a major crop plant, and represents a remarkable tour de force of transgenic technologies. Unfortunately, not all ideas in the business have been as remarkable. If time permits, I'll go over a bit of the controversy surrounding the use of transgenics. Not all of it is emotional claptrap.