Agrobacterium

Agrobacteria is a group of Gram-negative, non-sporing, aerobic, soil bacteria. The genus Agrobacterium belongs to a family of Rhizobiaceae which is present in a widespread endosymbiotic interactions with dicotyledonous plants in particular such as rose (e.g. apple, pear, peach), ficus, rubus families, and grapes. A. tumefaciens was initially detected in large numbers within growing tissues in fruit trees. These growing tissues found in branches and stems were so-called the “crown gall”. In 1940s, Armin Braun from Rockefeller University observed that crown galls, once formed, can propagate in vitro culturing conditions. This observation was later confirmed and the bacterium was found responsible of inducing tumor formation whereas it was not particularly involved in tissue proliferation. Indeed, years later, Agrobacterium was found beholding, aside from its chromosomal DNA, a mega-plasmid (Ti plasmid; tumor-inducing). Ti plasmid enables the transfer a piece of its DNA into the host plant cell, once infection occurs (Megaplasmids are large circular plasmids up to 200-kb in length).

Agrobacterium is a bacterium that enables horizontal gene transfer in nature through a segment of its Ti plasmid that is transferred to plant cells,  known as “T-DNA” (Transferred DNA). Genes carried by T-DNA (e.g. iaaM, ipt, tml) are integrated into plant chromosome and their expression is resulted in disruption of Cytokinin/Auxin balance in cells due to synthesized metabolites. Disruption of this balance leads to unregulated cell divisions and ultimately formation of crown galls. George Morel discovered the presence of uncommon amino acids (called “opines”) in these tissues. Opine synthesis is specific to tumorous cells, as plants do not usually produce these compounds, and are useful to the bacterium serving as carbon and nitrogen source.  DNA transfer from bacteria to plant cells has been investigated for nearly 50 years but is mostly elucidated today.

Ti plasmids can cause the production of different types of opines chemically, therefore Agrobacterium strains are classified for the Ti plasmids they carry, including octopine type or nopaline type. T-DNAs carried on Ti plasmids vary in their size from 8 to 23-kb (e.g. Nopaline type Ti plasmid has a single 23-kb T-DNA). These plasmids also contain replication origin, and regions for virulence, opine catabolism and conjugation.

Structure:

The genome sequence of Agrobacterium strain (C58) was published in 2001 (by two groups). Accordingly, bacterium has two chromosomes (circular and linear) and two different plasmids (Ti plasmid, and the cryptic plasmid pATC58). The total genome is of 5.67-Mb. Genes responsible for plant infection in Agrobacterium can be found in all genomic parts.

 

The ability of transferring its DNA into plant genome made Agrobacterium one of the most important tools in plant genetic engineering during 90s. Its basic mechanism relies on the DNA-transfer to plant species. T-DNA carried by Ti plasmid contains 25-bp border sequences which are called right (RB) and left border (LB) mediating T-DNA excision.

Figure 0

T-DNA excision from Ti plasmid.

 

T-DNA is covered by bacterial proteins later on during the infection initiation stage, and a DNA-protein complex is formed. Ti plasmid carries a 35-kb region called Vir (virulence) region encoding proteins of highly functional importance during each steps of infection. This region has 8 operons (VirA-VirH) including nearly 25 genes. All genes except for VirA, Vir F and VirG, are in operon and polycictronics.  Virulence genes are not expressed under normal conditions except for VirA and VirG. However, virulence operon becomes activated after the induction of VirA and VirG proteins sensing plant signals (e.g. phenolic compounds excreted from wounding sites).

Infection:

Signal transduction mediated by VirA and VirG is the initial step of infection. Second step involves the excision of a single strand T-DNA, which will be bound by virulence proteins of VirE2 and VirD2; thus forming together what is called the T-complex. Encoded VirD1 and VirD2 by Vir regions are specific endonucleases and cut T-DNA from left and right borders. VirD2 later binds the 5’ end of nicked T-DNA, covalently while the rest of the strand is coated by a single-strand DNA binding protein, VirE2. T-complex is transferred to plant cells by an extracellular pilus formed by bacterial proteins. A number of proteins including VirB1-11 and VirD4, encoded by VirB and VirD operons play role in the formation of this pilus. Essentially, the pilus is a Type IV secretion system which is widely employed in bacteria, and is known as VirB/D4 in Agrobacterium. T-complex passes through VirB/D4 channel and reaches plant cell nucleus.

Gene transfer tool:

Agrobacterium mediated gene transfer in plants is based on replacing the original genes within the T-DNA segment by other gene(s) of interest between its left and right border sequences. For this purpose, oncogenes between border sequences were eliminated to produce disarmed Ti plasmids. A lot of strains carrying such plasmids are routinely used for plant transformation (e.g. AGL-1, LBA4404, C58-Z707).

Agrobacteria can be classified according to their growth characteristics; Biotype 1 strains (e.g. Ach5, A6, C58) can produce Ketolactose from lactose. These strains proliferate at temperatures up to 37oC (under lab conditions at 28-29oC). On the other hand, Biotype 2 strains (e.g. ATCC 23834, A4) have the capability of grow on Erythritol as carbon source. These strains poorly grow at temperatures higher than 30oC. They require special media other than of standard E. coli media.  Two vector systems developed for exploiting Agrobacterium-mediated gene transfer are “binary vectors” and “integrative vectors”. Reasons in developing such systems were (1) Natural Ti plasmids are quite large to use in gene transfer and have low copy numbers; (2) Ti plasmids cannot be propagated in E. coli and have not restriction sites necessary for cloning. Up to date, a number of modifications have been made in Agrobacterium strains and vectors in order to increase transformation efficiency.

Fig 1

A simplified procedure for Agrobacterium-mediated transformation of plants.

 

 

Further Reading:

Sheng J, Citovsky V (1996). Agrobacterium-plant cell DNA transport: have virulence proteins, will travel Plant Cell 8(10):1699-710.

Gelvin SB (2006) Agrobacterium virulence gene induction. Methods in Molecular Biology vol. 343 pp 77-85. doi 10.1385/1-59745-130-4:77

Ream W. (2008) Production of a mobile T-DNA by Agrobacterium tumefaciens. In: Tzfira T., Citovsky V. (eds) Agrobacterium: From Biology to Biotechnology. Springer, New York, NY.

Bourras S (2015) Agrobacterium tumefaciens gene transfer: how a plant pathogen hacks the nuclei of plant and non plant organisms. Phytopathology 105(10):1288-1301.

Agro resim for blog jpg

SEM image of Agrobacterium on the root surface of barley.

 

Fig 2

Agrobacterium-mediated transformation of barley callus.

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