NON-AGROBACTERIAL SPECIES FOR GENE TRANSFER TO PLANTS

P. Janaki Krishna
August, 2005

Agrobacterium tumefaciens is a common soil bacterium that causes crown gall disease by transferring some of its DNA to the plant host. This unique mode of action has enabled this bacterium to be used as a tool in plant breeding1. Many desired genes of agronomic importance are engineered into this bacterial DNA and thereby inserted into plant genomes. Though close relatives of Agrobacterium, such as Rhizobium trifolii and Phyllobacterium myrsinacearum, display the gall producing ability by harboring a Ti (Tumor-inducing) plasmid, no direct molecular evidence of gene transfer to plants by these bacteria has been reported. In fact, until now, the body of research has focused on using Agrobacterium as a vehicle for gene transfer. However, researchers are now attempting to use other Agrobacteria-related species, such as Sinorhizobium and Mesorhizobium, to augment gene transfer techniques.

A research team from CAMBIA (http://www.cambia.org/daisy/cambia/563) has investigated whether a non-Agrobacterial species of bacteria can competently transfer genes in plants2. To do this, a disarmed Ti plasmid (pEHA105) from a hypervirulent Agrobacterium strain was introduced into several species of bacteria. To facilitate transfer of this large plasmid, the origin of transfer (oriT) of a broad host range IncP plasmid was integrated into the Ti plasmid of EHA105 at two different locations (pTiWB1, pTiWB3). The modified plasmids were then mobilized into 1) a Rhizobium species (NGR234) that has an exceptionally broad host range, capable of nodulating over 100 different plants3; 2) the alfalfa-symbiont Sinorhizobium meliloti; and 3) Mesorhizobium loti, a representative of a different family (Phyllobacteriaceae). In order to check the genotype of engineered strains by PCR and confirm that the strains were free of contaminating Agrobacterium by selective plating, additional primers were developed. In addition, the transfer rate and replication potential were enhanced by incorporating two broad-spectrum replication origins (sites) to the disarmed Ti plasmid. To assay for gene transfer, three binary vectors were prepared: pCAMBIA1105.1R was introduced into the Rhizobia bacteria, and either pCAMBIA1105.1 or pCAMBIA1405.1 into Agrobacterium.

The plant transformation events were analyzed through GUS activity, Southern blotting, and PCR assays. First, the GUS assay tested the transformation rate in tobacco. The transformation rate using Sinorhizobium meliloti was about 25% that of Agrobacterium, and M. loti had a rate approximately one third of Sinorhizobium. However, that value is still significant enough to get the attention of researchers interested in plant transformation.

The researchers also tested the non-Agrobacterium bacteria for their ability to transform other plant species, namely Arabidopsis and rice. Arabidopsis was transformed with S. meliloti using the floral dip method, producing six transgenic plants from 70,000 T0 seeds, which is 5–10% of the normal efficiency of A. tumefaciens. Interestingly, in all cases, T-DNA was integrated in a manner identical to that of Agrobacterium. Also, an effort was made to increase the transformation efficiency of the floral dip technique by modifying the infiltration medium, whereby a four-fold improvement was obtained. In rice, the transformation efficiency was considerably lower (0.6%) when compared with Agrobacterium tumefaciens (50–80%). One transformed rice plant from a total of 695 calli was regenerated and rooted. T-DNA integration analysis in this rice plant revealed that the T-DNA had integrated into rice chromosome 11.

Thus, though the transformation efficiency was considerably lower when compared to Agrobacterium mediated transformation, the results confirm that all three non-Agrobacterium species, Rhizobium, Sinorhizobium, and Mesorhizobium strains, belonging to two families of bacteria, can transform plants. Of these, S. meliloti is the most competent to transfer genes into both monocots and dicots and into a range of tissues, including leaf tissue, undifferentiated calli, and immature embryo ovules.

Albeit, at a lower frequency, T-DNA transformation appeared to proceed normally, but most notable is the fact that transformation occurred at all using non-virulent, non-Agrobacterium microbes. Though a number of factors that reside on Ti plasmids (as acting genes and DNA elements) play an important role in DNA transfer, it has been noted that other transacting elements are located on Agrobacterial chromosomes4. In addition, the researchers suggest that if there are gene functions necessary for gene transfer that are not encoded by the Ti plasmid, they must have equivalents or homologues in multiple Rhizobial species. It is likewise possible that the small number of vir-related genes on the Ti plasmid is sufficient to confer gene transfer competence to any bacterium. It is also noted that homologues of these transacting Agrobacterium genes exist in other bacteria and it is suggested they could have evolved from DNA transfer to plants in the past.

This study is a breakthrough in research concerning the exploitation of non-Agrobacterial bacteria for gene transfer to plants. It appears that when it comes to acquiring or transferring genetic information, interestingly, bacteria always show promise.

In addition, it is heartening to note that this alternative technology is available to the public in a "protected technology commons," optimized and improved as a ‘Bioforge’ project (http://www.bioforge.net).

References

1. Gelvin SB (2003) Agrobacterium- mediated plant transformation: the biology behind "gene-jockeying" tool. Microbial. Mol. Biol. Rev. 67, 16–37

2. Broothaerts W et al. (2005) Gene transfer to plants by diverse species of bacteria. Nature 433, 629–633

3. Pueppke SG & Broughton WJ (1999) Rhizobium sp. strain NGR234 and R. fredii USDA257 share exceptionally broad, nested host ranges. Mol. Plant Microbe Interact. 12, 293–318

4. Van Montagu M & Schell J. (2003) (1935–2003): Steering Agrobacterium-mediated plant gene engineering. Trends Plant Sci. 8, 353–354

P S Janaki Krishna
Institute of Public Enterprise
Osmania University Campus
Hyderabad, India
jankrisp@yahoo.com