Expression of Agrobacterium Homolog Genes Encoding T-complex Recruiting Protein under Virulence Induction Conditions

Abstract

The proteins encoded by three Agrobacterial genes, atu5117, atu4860, and atu4856, are highly homologous to each other in amino acid sequence. All three proteins can bind to VirD2 and are named VBP1, VBP2, and VBP3 (VirD2-binding protein), respectively. VBP is involved in T-DNA transfer by recruiting the T-complex from the cytosol to the polar transport apparatus T4SS (type IV secretion system) and is defined as the "T-complex recruiting protein." However, it remains unknown how these three homologous genes co-exist in a relatively small prokaryotic genome. To understand whether these three homologous genes are expressed differentially under virulence induction conditions, we examined the effects of virulence induction conditions, including various pH values, temperatures and acetosyringone (AS, an effective virulence inducer to Agrobacterium tumefaciens) concentrations, on the expression of the three VBP-encoding genes. Our data showed that vbp1 (atu5117) and vbp3 (atu4856) maintained constant expression under the tested induction conditions, whereas the expression of vbp2 (atu4860) was affected by the conditions. Culture conditions favorable to the expression of vbp2 differed from the reported induction conditions for other virulence proteins. In particular, the pH value was a crucial factor for the expression of vbp2. In addition, the deletion of vbp1 affected the expression of vbp2. Taken together, these results suggest that the mechanisms regulating the expression of these three homologous genes are different from the virulence induction mechanism and that VBP homologs are presumably involved in other biological processes in addition to T-complex recruitment.

Keywords: Agrobacterium; T-complex recruiting protein; gene expression; virD2-binding protein (VBP); virulence induction.

FIGURE 1

Discrimination of three virD2-binding proteins (VBPs) by Western blot assay. Crude extracts from Escherichia coli cells expressing different vbp genes were separated using SDS-PAGE in the indicated lanes and then analyzed by Western blotting using antibodies against the variable sequence regions of the three VBPs. M: Molecular weight marker. The antibodies used for each assay are listed on the right. Lane 1: crude extracts from E. coli cells expressing vbp1. Lane 2: crude extracts from E. coli cells expressing vbp2. Lane 3: crude extracts from E. coli cells expressing vbp3.

FIGURE 2

Expression of three vbp genes in Agrobacterium tumefaciens strain C58 at different pHs (A), temperatures (B) and AS concentrations (C). A. tumefaciens strain C58 cells were induced under the indicated pH, temperature or AS concentration. Crude extracts from the differentially induced C58 cells were separated using SDS-PAGE and then analyzed by Western blotting. Crude extracts from E. coli cells expressing different vbp genes with His-tag were used as positive controls. The antibodies used in this study are listed on the left. NI: crude extracts from C58 cells not induced in IB medium. (A) Effect of pH on the expression of vbps in A. tumefaciens C58. The temperature is 25°C and AS concentration is 100 μg/ml. (B) Effect of temperature on the expression of vbps in A. tumefaciens C58. The pH is 5.5 and AS concentration is 100 μg/ml. (C) Effect of AS concentration on the expression of vbps in A. tumefaciens C58. The pH is 5.5 and temperature is 25°C. Some differences in protein size of E. coli recombinant VBP2 protein (the positive control band in panel C immigrated faster than that in other panels) are likely attributable to prolonged storage.

FIGURE 3

Expression of vbp2 and vbp3 in A. tumefaciens strain GMI9017 at different pHs (A), temperatures (B), and AS concentrations (C). A. tumefaciens strain GMI9017 cells were induced under the indicated pH, temperature or AS concentration. Crude extracts from the differentially induced GMI9017 cells were separated using SDS-PAGE and then analyzed by Western blotting. PC, positive control, crude extracts from E. coli cells expressing vbp2 (up) or vbp3 (down) with His-tag. The antibodies used in this study are listed on the left. NI: crude extracts from GMI9017 cells not induced in IB medium. (A) Effect of pH on the expression of vbp2 and vbp3 in A. tumefaciens GMI9017. The temperature is 25°C and AS concentration is 100 μg/ml. (B) Effect of temperature on the expression of vbp2 and vbp3 in A. tumefaciens GMI9017. The pH is 5.5 and AS concentration is 100 μg/ml. (C) Effect of AS concentration on the expression of vbp2 and vbp3 in A. tumefaciens GMI9017. The pH is 5.5 and temperature is 25°C.

FIGURE 4

Comparison of the expression of vbp2 in A. tumefaciens C58 and A. tumefaciens GMI9017 at different pHs, temperatures and AS concentrations. Both the A. tumefaciens strain C58 cells and GMI9017 cells were induced in parallel under the indicated pH, temperature or AS concentration. Crude extracts from the differentially induced Agrobacterium cells were separated using SDS-PAGE and then analyzed by Western blotting using antibody against VBP2. PC, positive control, crude extract from E. coli cells expressing vbp2 with His-tag.

FIGURE 5

Expression of vbp3 in the double-mutant (vbp1 and vbp2) strain GMV12 at different pHs, temperatures and AS concentrations. A. tumefaciens strain GMV12 cells were induced under the indicated pH, temperature or AS concentration. Crude extracts from the differentially induced GMV12 cells were separated using SDS-PAGE and then analyzed by Western blotting with antibody against VBP3. PC, positive control, crude extract from E. coli cells expressing vbp3 with His-tag. NI: crude extracts from GMV12 cells not induced in IB medium.