Identification of plant proteins that interact with the Agrobacterium pilin protein, VirB2

Hau-Hsuan Hwang, Purdue University

Abstract

Agrobacterium tumefaciens is an organism capable of trans-kingdom DNA transfer, transforming mainly plants but also other eukaryotic species, from fungi to human cells. Genetic transformation by A. tumefaciens, which in plants causes neoplastic growths called "crown gall", results from the transfer and integration of a specific DNA fragment (transferred DNA or T-DNA) from the bacterium into the plant genome. The T-DNA export apparatus is composed of two major structural components: the T-pilus and a membrane associated complex that is responsible for translocating substrates across both bacterial cell membranes. Although the T-pilus is essential for T-DNA transfer and virulence, understanding its specific mechanistic functions remains a challenge. My experiments identified plant proteins that interact with VlrB2 protein, the major component of the A. tumefaciens T-pilus, and may therefore be a "receptor" for the T-pilus. Using the C-terminal, processed portion of V1rB2 protein as a bait in a yeast two hybrid screen of an Arabidopsis cDNA library, I identified three related plant proteins, VirB2 Interacting Protein (BTI) 1, (BTI1), BTI2, and BTI3, and a membrane-associated GTPase, AtRAB8. Both yeast two-hybrid assays and in vitro interaction studies demonstrated that the three BTI proteins interact with themselves, each other, and AtRAB8, suggesting that these proteins may form a multimeric complex. The three BTI proteins also interact with GST-VirB2 fusion proteins in GST pull-down assays. Both anti-sense and RNA interference (RNAi) transgenic plants of BTI and AtR4B8 were less susceptible to stable and transient A. tumefaciens transformation than were wild-type plants. Arabidopsis mutant plants containing a T-DNA insertion either in the upstream region or the downstream region of the BTI1 gene were recalcitrant to A. tumefaciens transformation. These data indicate that both BTI and AtRAB8 proteins are involved in the Agrobacterium-mediated root transformation process. Confocal microscopic data indicate that BTI proteins preferentially localize to the cell periphery in the root cells of Arabidopsis plants, suggesting that BTI proteins may contact the A. tumefaciens T-pilus. Finally, the level of BTI1 protein was transiently induced at an early time point following A. tumefaciens infection of Arabidopsis suspension cells. In summary, the BTI and AtRAB8 proteins interact with VirB2 and are involved in the Agrobacterium -mediated transformation process.

Degree

Ph.D.

Advisors

Gelvin, Purdue University.

Subject Area

Molecular biology

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