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Agrobacterium-mediated transformation, pathogenesis by trans-kingdom conjugation

Van Leeuwenhoek Lecture on BioScience.

Paul Hooykaas is Professor of Genetics at the Institute of Biology Leiden (IBL). He studied chemistry and subsequently obtained his PhD in Leiden, for which he was awarded with the C.J. Cock prize. After initial studies demonstrating that large plasmids in bacteria of the Rhizobiaceae family largely determine whether the bacterial host behaves as a saprophyte, symbiont or pathogen, he became involved in unraveling the intricacies of how the tumor-inducing Agrobacterium tumefaciens infects and benefits from plants and how this can be exploited for genome engineering. Hooykaas is one of the inventors of the binary vector system that still is the preferred vector system for plant transformation nowadays. For his work Hooykaas received the AKZO prize and he had the honor to be appointed as Academy professor in 2009. He is a member of the Royal Netherlands Academy of Sciences (KNAW) and of Academia Europaea.

Agrobacterium induces tumor formation in plants by transferring a segment of oncogenic T-DNA. Two-way chemical signaling initiates the cascade of events ultimately leading to T-DNA transfer. This restricts transfer specifically to plants, but adoption of the proper "plant-like" conditions allow the system to transfer DNA into other hosts such as yeast and fungi. The virulence genes responsible for T-DNA transfer are related to genes responsible for conjugative DNA transfer between bacteria.  An unexpected discovery was that Agrobacterium not only locates DNA molecules into host cells, but also a set of virulence (effector) proteins. Translocation is mediated by a nanomachine, which is now called a Type4Secretion System and which is not only present in Agrobacterium, but also in human pathogens such as Legionella, Helicobacter and Brucella. Translocated proteins are recognized by a specific signature in their C-terminal domain. The T4SS can transfer DNA molecules, but only when these are covalently linked to a relaxase protein with a C-terminal translocation domain, which indicated that conjugative DNA transfer is evolutionary derived from protein secretion. The translocated virulence proteins promote infection in a multitude of ways and are responsible for the extremely high transformation efficiency of Agrobacterium. They promote nuclear targeting of the transferred DNA molecules, but also inhibit defense responses of the plant and interfere with the chromosome segregation apparatus to promote transformation and tumorigenesis. The integration of DNA molecules is largely mediated by recombination enzymes of the host and these may be modulated for targeted integration and genome engineering. In the lecture it will be shown how genetic and genomic tools and knowledge of model organisms such as yeast and Arabidopsis thaliana have played a crucial role in this research.