Abstract
VirB5 is a type 4 secretion system protein of Agrobacterium located on the surface of the bacterial cell. This localization pattern suggests a function for VirB5 which is beyond its known role in biogenesis and/or stabilization of the T-pilus and which may involve early interactions between Agrobacterium and the host cell. Here, we identify VirB5 as the first Agrobacterium virulence protein that can enhance infectivity extracellularly. Specifically, we show that elevating the amounts of the extracellular VirB5—by exogenous addition of the purified protein, its overexpression in the bacterium, or transgenic expression in and secretion out of the host cell—enhances the efficiency the Agrobacterium-mediated T-DNA transfer, as measured by transient expression of genes contained on the transferred T-DNA molecule. Importantly, the exogenous VirB5 enhanced transient T-DNA expression in sugar beet, a major crop recalcitrant to genetic manipulation. Increasing the pool of the extracellular VirB5 did not complement an Agrobacterium virB5 mutant, suggesting a dual function for VirB5: in the bacterium and at the bacterium-host cell interface. Consistent with this idea, VirB5 expressed in the host cell, but not secreted, had no effect on the transformation efficiency. That the increase in T-DNA expression promoted by the exogenous VirB5 was not due to its effects on bacterial growth, virulence gene induction, bacterial attachment to plant tissue, or host cell defense response suggests that VirB5 participates in the early steps of the T-DNA transfer to the plant cell.
Highlights
Agrobacterium tumefaciens is a phytopathogenic bacterium that ‘‘genetically engineers’’ its hosts by transferring T-DNA from the Ti plasmid to the host cell [1,2,3]
If extracellular VirB5 plays a role in Agrobacterium infection which is beyond its known intracellular functions, it might do so via five major mechanisms that affect early stages of the transformation process: bacterial cell growth, virulence gene induction, binding to the host cells, suppression of host defense, and enhancement of the T-DNA transfer
We examined whether exogenous VirB5 can alter attachment of Agrobacterium cells to the surface of the host plant cell
Summary
Agrobacterium tumefaciens is a phytopathogenic bacterium that ‘‘genetically engineers’’ its hosts by transferring T-DNA from the Ti plasmid to the host cell [1,2,3]. In nature, Agrobacterium infects plants, causing neoplastic growths at the sites of infection, under laboratory conditions, this pathogen can genetically transform eukaryotic cells from diverse origins, from plant to yeast [4,5] to human [6], indicating the conserved nature of the transformation process [7] This transformation represents the only known natural example of trans-kingdom DNA transfer, making Agrobacterium a tool-of-choice to produce transgenic plants for research and biotechnology, and a unique experimental system to study general aspects of interactions between pathogenic bacteria and their eukaryotic hosts. Whereas a substantial body of evidence suggests potential functions for many of the T4SS proteins [9], their roles in the interaction with and T-DNA transport to the host cell remain largely obscure. To begin exploring these roles, we focused on one of the exported T4SS proteins, VirB5, the function of which outside of the bacterial cell is unknown
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