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. 2010 Feb 23;107(8):3758-63.
doi: 10.1073/pnas.0914940107. Epub 2010 Feb 2.

Agrobacterium type IV secretion system and its substrates form helical arrays around the circumference of virulence-induced cells

Julieta Aguilar  1 , John Zupan, Todd A Cameron, Patricia C Zambryski
Affiliations

Agrobacterium type IV secretion system and its substrates form helical arrays around the circumference of virulence-induced cells

Julieta Aguilar et al. Proc Natl Acad Sci U S A. .

Abstract

The genetic transformation of plant cells by Agrobacterium tumefaciens results from the transfer of DNA and proteins via a specific virulence (vir) -induced type IV secretion system (T4SS). To better understand T4SS function, we analyzed the localization of its structural components and substrates by deconvolution fluorescence microscopy. GFP fusions to T4SS proteins with cytoplasmic tails, VirB8 and VirD4, or cytoplasmic T4SS substrate proteins, VirD2, VirE2, and VirF, localize in a helical pattern of fluorescent foci around the perimeter of the bacterial cell. All fusion proteins were expressed at native levels of vir induction. Importantly, most fusion proteins are functional and do not exhibit dominant-negative effects on DNA transfer to plant cells. Further, GFP-VirB8 complements a virB8 deletion strain. We also detect native VirB8 localization as a helical array of foci by immunofluorescence microscopy. T4SS foci likely use an existing helical scaffold during their assembly. Indeed, the bacterial cytoskeletal component MinD colocalizes with GFP-VirB8. Helical arrays of foci are found at all times investigated between 12 and 48 h post vir induction at 19 degrees C. These data lead to a model with multiple T4SSs around the bacterial cell that likely facilitate host cell attachment and DNA transfer. In support, we find multiple T pili around vir-induced bacterial cells.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
GFP-VirB8 localizes to a helical array of foci in vir-induced Agrobacterium. (A–C) GFP in wild-type Agrobacterium. (D–F) GFP-VirB8 in wild-type Agrobacterium. (G–I) GFP-VirB8 in ΔvirB8. (J–L) Native VirB8 detected with primary rabbit antibodies to VirB8 followed by detection with fluorescent secondary anti-rabbit antibodies. A, D, G, and J are widefield fluorescence images of representative populations of vir-induced cells. B, E, H, and K are images of a deconvolved 3D stack. C, F, I, and K show five sections from top to bottom through the 3D deconvolved images. (M and N) Tumor assays on carrot discs for control (Ti plasmid cured and wild-type C58, respectively). (O and P) tumor assays for wild type and ΔvirB8 strains carrying GFP-VirB8, respectively. (Q) Protein levels of GFP-VirB8 in wild type and ΔvirB8 strains by western blot using anti-VirB8 antibodies. Lane 1, wild type (C58); lane 2, GFP-VirB8 in C58; lane 3, GFP-VirB8 in ΔvirB8. * VirB8, 26 kDa; GFP-VirB8, 54 kDa.
Fig. 2.
Fig. 2.
GFP-VirB8 colocalizes with RFP-MinD. (A) A deconvolved stack of RFP-MinD, and three different orientations of a deconvolved 3D image. (B–D) GFP-VirB8 and RFP-MinD expressed in the same cells. (B) GFP fluorescence. (C) RFP fluorescence. (D) GFP and RFP colocalization. (E) Scatterplot of green (x axis) and red (y axis) pixel intensities used to determine the threshold for colocalization.
Fig. 3.
Fig. 3.
T4SS substrates VirD2, VirF, and VirE2 localize to a helical array of foci in vir-induced Agrobacterium. (A and B) GFP-VirD2. (D and E) GFP-VirF. (G and H) GFP-VirE2. (J and K) VirD4-GFP. A, D, G, and J are widefield images of representative populations of vir-induced cells. B, E, H, and K are 3D deconvolved images. C, F, I, and L show tumor assays on carrot discs for each strain.
Fig. 4.
Fig. 4.
Time course of T4SS and T4SS substrate localization. GFP-VirB8 was localized in wild-type C58 Agrobacterium and ΔvirB8. GFP-VirB10 and GFP-VirF were assayed in wild-type C58 Agrobacterium. The cells shown are representative of the localization patterns of fields of cells at 12, 18, 24, and 48 h post vir induction. All images are widefield fluorescence. (A) GFP-VirB8 in C58. (B) GFP-VirB8 in CB1008. (C) GFP-VirB10 in C58. (D) GFP-VirF in C58.
Fig. 5.
Fig. 5.
Detection of T pili in vir-induced Agrobacterium and during attachment to the plant cell surface. (A) Arrows indicate multiple T pili around the circumference of vir-induced Agrobacterium. Top right shows flagellum for comparison (arrowhead). (B) Multiple T pili of the vir-induced bald strain of Agrobacterium contact the surface of a tobacco leaf cell.
Fig. 6.
Fig. 6.
Model for T4SS localization and function during binding to plant cells. Agrobacterium with polar and helically localized T4SSs and T pili make initial contact with a plant cell membrane (thick curved line) (A), and following T pili retraction multiple T4SSs attach to the plant cell surface (B). The T4SS associates with an endogenous helix in the cell.
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