Expression and functional characterization of the Agrobacterium VirB2 amino acid substitution variants in T-pilus biogenesis, virulence, and transient transformation efficiency.

Hung-Yi Wu,Chao-Ying Chen,Erh-Min Lai,Ching-Hong Yang

doi:10.1371/journal.pone.0101142

Abstract

Agrobacterium tumefaciens is a phytopathogenic bacterium that causes crown gall disease by transferring transferred DNA (T-DNA) into the plant genome. The translocation process is mediated by the type IV secretion system (T4SS) consisting of the VirD4 coupling protein and 11 VirB proteins (VirB1 to VirB11). All VirB proteins are required for the production of T-pilus, which consists of processed VirB2 (T-pilin) and VirB5 as major and minor subunits, respectively. VirB2 is an essential component of T4SS, but the roles of VirB2 and the assembled T-pilus in Agrobacterium virulence and the T-DNA transfer process remain unknown. Here, we generated 34 VirB2 amino acid substitution variants to study the functions of VirB2 involved in VirB2 stability, extracellular VirB2/T-pilus production and virulence of A. tumefaciens. From the capacity for extracellular VirB2 production (ExB2+ or ExB2−) and tumorigenesis on tomato stems (Vir+ or Vir−), the mutants could be classified into three groups: ExB2−/Vir−, ExB2−/Vir+, and ExB2+/Vir+. We also confirmed by electron microscopy that five ExB2−/Vir+ mutants exhibited a wild-type level of virulence with their deficiency in T-pilus formation. Interestingly, although the five T-pilus−/Vir+ uncoupling mutants retained a wild-type level of tumorigenesis efficiency on tomato stems and/or potato tuber discs, their transient transformation efficiency in Arabidopsis seedlings was highly attenuated. In conclusion, we have provided evidence for a role of T-pilus in Agrobacterium transformation process and have identified the domains and amino acid residues critical for VirB2 stability, T-pilus biogenesis, tumorigenesis, and transient transformation efficiency.

Highlights

Agrobacterium tumefaciens is a Gram-negative plant pathogenic bacterium that causes crown gall disease in a wide range of plants [1]
The Transfer DNA (T-DNA) immunoprecipitation (TrIP) technique revealed that T-DNA was first recruited by the VirD4 coupling protein, the substrate passed to the innermembrane–associated ATPase VirB11
Cell pellets were resuspended again and subjected to shearing to obtain the S2 fraction enriched for T-pilus. Both intracellular and extracellular VirB2 levels were restored to wild-type levels in DvirB2 complemented with wild-type VirB2, which was consistent with its full complementation by tumorigenesis analysis on tomato stems (Figure 2)

Summary

Introduction

Agrobacterium tumefaciens is a Gram-negative plant pathogenic bacterium that causes crown gall disease in a wide range of plants [1]. A. tumefaciens can sense plant-released phenolic compounds (e.g., acetosyringone; AS) to activate the expression of virulence factors for infection. The VirA/VirG two-component system is responsible for the phenolics-induced virulence (vir) gene expression [2]. The T-DNA and effector protein substrates are transferred through the VirB/VirD4 assembled type IV secretion system (T4SS) into host plant cells [2,3,4]. The A. tumefaciens VirB/VirD4 T4SS consists of the envelopespanning translocation channel and the extracellular T-pilus structure [5,6,7,8]. Accumulating biochemical and genetic data suggest a possible VirB/D4 T4SS assembly and T-DNA translocation pathway [9,10]. The T-DNA immunoprecipitation (TrIP) technique revealed that T-DNA was first recruited by the VirD4 coupling protein, the substrate passed to the innermembrane–associated ATPase VirB11.

Methods

Results

Conclusion