Abstract
Agrobacterium tumefaciens infects plant cells by a unique mechanism involving an interkingdom genetic transfer. A single-stranded DNA substrate is transported across the two cell walls along with the bacterial virulence proteins VirD2 and VirE2. A single VirD2 molecule covalently binds to the 5'-end of the single-stranded DNA, while the VirE2 protein binds stoichiometrically along the length of the DNA, without sequence specificity. An earlier transmission/scanning transmission electron microscopy study indicated a solenoidal ("telephone coil") organization of the VirE2-DNA complex. Here we report a three-dimensional reconstruction of this complex using electron microscopy and single-particle image-processing methods. We find a hollow helical structure of 15.7-nm outer diameter, with a helical rise of 51.5 nm and 4.25 VirE2 proteins/turn. The inner face of the protein units contains a continuous wall and an inward protruding shelf. These structures appear to accommodate the DNA binding. Such a quaternary arrangement naturally sequesters the DNA from cytoplasmic nucleases and suggests a mechanism for its nuclear import by decoration with host cell factors. Coexisting with the helices, we also found VirE2 tetrameric ring structures. A two-dimensional average of the latter confirms the major features of the three-dimensional reconstruction.
Highlights
Agrobacterium tumefaciens is the causative agent of the crown gall disease in many plant species [1, 2]
The T-strand-VirD2 complex and the VirE2 molecules may be exported from the Agrobacterium independently and meet only later in the host cytoplasm, where they assemble into a mature T-complex
The T-complex is imported into the host cell nucleus, where it may be expressed transiently or integrated into the host genome, finalizing the transformation process
Summary
Agrobacterium tumefaciens is the causative agent of the crown gall disease in many plant species [1, 2]. A single-stranded DNA substrate is transported across the two cell walls along with the bacterial virulence proteins VirD2 and VirE2. We report a three-dimensional reconstruction of this complex using electron microscopy and single-particle image-processing methods.
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