Supramolecular complexes of the Agrobacterium tumefaciens virulence protein VirE2

Abstract

VirE2 is a ssDNA binding protein essential for virulence in Agrobacterium tumefaciens. A tetracysteine mutant (VirE2-TC) was prepared for in vitro and in vivo fluorescence imaging based on the ReAsH reagent. VirE2-TC was found to be biochemically active as it binds both ssDNA and the acidic secretion chaperone VirE1. It was also biologically functional in complementing virE2 null strains transforming Arabidopsis thaliana roots and Nicotiana tabacum leaves. In vitro experiments demonstrated a two-color fluorescent complex using VirE2-TC/ReAsH and Alexa Fluor 488 labeled ssDNA. In vivo, fluorescent VirE2-TC/ReAsH was detected in bacteria and in plant cells at time frames relevant to transformation. Importance Cell to cell transfer of proteins and nucleic acids lies at the heart of many biological processes. Detecting such transfer is often problematic or indirect. We show here that the biarsenical fluorescent reagent ReAsH can be used to reveal the presence of the secreted effector VirE2 from Agrobacterium tumefaciens in the cytoplasm of host plant cells. Our studies establish a new method for monitoring translocation of the VirE2 effector from A. tumefaciens to plant target cells during the infection process.

VirE2-DEPENDENT PORES FOR ssDNA TRANSFER ACROSS ARTIFICIAL AND CELL MEMBRANES

“…VirE2 was isolated from cells of Escherichia coli strain XL1-blue, containing recombinant plasmid pQE31ÀvirE2, and was purified as described in Volokhina et al 19 The average diameter and the size distribution for the VirE2 complex in aqueous solutions were determined by the method of dynamic light scattering (DLS) by using a Zetasizer Nano instrument (model ZEN 3500; Malvern Instruments Ltd, Malvern, UK) equipped with a 25 mW helium/neon laser and with the polydispersity analysis software package Dispersion Technology Software 4.2 (Malvern). .…”