Effects Of Culture Conditions On Agrobacterium VBP Protein Paralogous Gene Expression

Agrobacterium tumefaciens is a phytopathogenic bacterium that causes the crown gall tumor disease in some dicotyledonous plants by transferring a section of DNA (called T-DNA) from its Ti-plasmid to plant cells and genetically transforming the host cells. When the T-DNA of Ti plasmid has been replaced by exogenous DNA, Agrobacterium tumefaciens is still able to transfer the exogenous T-DNA to the host plant and cause genetic transformation. The T-DNA is transferred to the host cell in the form of protein-single-stranded-T-DNA (called T-complex) through the type IV secretion system (T4SS). The recognition between T-complex and T4SS is fulfilled by a protein called T-complex recruiting protein. Agrobacterium tumefaciens T-DNA transfering requires a lot of proteins to participate in; one of the most important proteins is VirD2. By useing the VirD2as an affinity ligand, an VirD2-binding protein (VBP) was identified. Genome-wide sequence analysis shows that Agrobacterium tumefaciens has two additional genes which can encode proteins highly similar to VBP1. These two paralogous proteins were designated VBP2and VBP3. Like VBP1, both VBP2and VBP3can also bind to VirD2.vbpl is harbored on the cryptical plasmid.vbp2and vbp3reside on the linear chromosome close to each other. VBPs are proved to play a role in recruiting T-complex to the T4SS. Previous study demonstrated that three vbp genes can complement each other in T-complex recruiting. One vbp gene is sufficient for recruiting T-complex. Only Agrobacterium tumefaciens mutant strains in which all three vbp genes are knocked out lose the function of recruiting T-complex, implying that all three vbp genes existing in wild-type cells can translate functional proteins. It is a waste for the bacterial genome if all three paralogous vbp genes existing in the wild type cells are expressed simultaneously. It is possible that the expressions of three paralogous vbp genes might be affected by the different growth environment or physiological conditions. It is also unknown whether the expression of one vbp gene would regulate the expression of other vbp genes. To answer these questions, Agrobacterium wild type strain C58and mutant strains with vbp deletion were cultured in IB medium at different pH, temperature and AS concentration; and the expressions of three paralogous vbp genes in different strains cultured different IB medium were determined by using Western blotting. Our results showed that the expressions of both vbpl and vbp3genes were not affected by the tested tumor induction factors, whereas, the expression of vbp2gene was dependent on the induction factors. It was expressed in high temperature, high pH and low concentration of AS. There is a regulatory relationship between vbp2and vbpl genes. In GMI9017without vbpl gene, VBP2protein is expressed only in high pH conditions.