UC Berkeley

The interaction between Agrobacterium tumefaciens and its plant host is a classic model system for investigating host pathogen interactions. Significant progress has been made in various aspects of A. tumefaciens mediated genetic transformation of plants, which leads to the formation of crown gall tumors. It is known that wounded plant cells release phenolic compounds induces the expression of the transformation products, the T-strand and virulence (Vir) proteins, which are encoded on the tumor-inducing plasmid (pTi). The Vir proteins assemble a translocation channel called the virulence-induced type IV secretion system (vir-T4SS). The T-strand is a single stranded segment of DNA that gets integrated into the host plant cell and the expression of the genes on the T-strand causes crown gall tumors.

The vir-T4SS is composed of 11 VirB proteins and VirD4, and all 12 components are essential for maximal DNA and protein transport. These components can be divided into 3 major groups. The first group consists of the T-pilus and its associated components (VirB1, VirB2, VirB3, and VirB5). VirB1 has homology to lytic transglycolases and is likely to cleave the peptidoglycan to facilitate assembly of the vir-T4SS. VirB2 is the major component of T-pilus, and VirB5 is localized at the T-pilus tip. VirB1 and VirB3 are required for T-pilus assembly. The second group consists of the core transport complex that spans the inner and outer membranes and periplasm (VirB6-VirB10). The third group consists of the energetic components (VirB4, VirB11, and VirD4). These components have ATPase homology and ATP-binding motifs, and may energize the assembly and transfer of DNA and proteins through the vir-T4SS. VirD4 is also the coupling protein that brings DNA and vir-T4SS substrate proteins to the vir-T4SS. The vir-T4SS transports the T-strand, and at least 4 Vir protein substrates (VirD2, VirE2, VirE3, and VirF) into the host cell. It is estimated that 50 T-strands and thousands of VirE2, VirE3, and VirF proteins are all transported through the vir-T4SS. However, it still remains unclear how many vir -T4SS are localized on the bacterial cell and how the vir -T4SS interacts with the host cell to transport the T-strand and the vir-T4SS protein substrates.

It was proposed that most Vir proteins were localized at the pole of A. tumefaciens and that A. tumefaciens attached to host cells via its poles. However, early studies from the Zambryski lab suggested that at least one component of the T4SS, VirB8, localized around the bacterial perimeter in transverse-section and in multiple sites in longitudinal section of vir-induced cells. Other studies suggested that not all T4SS components localize to the same position perhaps suggesting subassemblies. Therefore, to clear these inconsistencies and to identify how many vir-T4SS are localized on the bacterial cell and to better understand how the vir-T4SS interact with the host cell to transport the T-strand and the vir-T4SS protein substrates, I analyzed the localization of the vir-T4SS components and substrates in the presence and absence of its host.

I cloned the structural components of the vir-T4SS and the vir-T4SS substrates as GFP fusion vectors for induced expression in the two most common lab strains of A. tumefaciens. After induction, GFP fusions were imaged by high-resolution deconvolution microscopy. Tumorigenesis assays were used to test the functionality of fusion proteins. I found that those that carried a cytoplasmically localized GFP retained their functions, while those with predicted periplasmic GFP and GFP fusions to VirB10 had a dominant-negative effect on tumor formation. Thus, suggesting the addition of GFP to these proteins may interfere with the correct assembly and proper function of the vir-T4SS. Three-dimensional reconstruction of the deconvolved images revealed that most of the proteins were found in multiple foci around the cell periphery. I confirmed these findings by examining endogenous protein patterns by immunofluorescence microscopy. Extensive quantitative analyses also verified that the Vir proteins of the vir-T4SS are localized to multiple foci around the periphery of the Agrobacterial cell. In support, I found multiple T-pili on bacterial cells. Interestingly, the localization of the vir-T4SS components resembled a helical pattern. Other proteins that form helical patterns are bacterial cytoskeletal components, such as MinD. In fact, VirB8 and MinD colocalize suggesting that cytoskeletal components may provide an existing scaffold for vir-T4SS assembly. Furthermore, I found that Agrobacteria attaches predominantly laterally to host cells and few cells attach at their poles. Also, the localization of the vir-T4SS does not change upon contact to host cells suggesting that perhaps multiple vir-T4SS are utilized.

In summary, my results revealed that the vir-T4SS is localized around the perimeter of the cell resembling a helical pattern, and that A. tumefaciens attached to host cells predominantly along their sides. My work challenges the existing paradigm of polar localization and orientation of attachment and has led to a novel model with multiple vir-T4SS around the bacteria. This would maximize the possibility for effective attachment along the bacterial length, which may allow efficient transfer of numerous amounts of T-strands and thousands of protein substrates into the host cell.