Identification and Characterization of Agrobacterium and Plant Proteins Interacting with Proteins of the Agrobacterium T-complex

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Nester 9723735 The interaction between Agrobacterium tumefaciens and plants is the only documented case of genetic engineering in nature. Several aspects of the molecular mechanism by which Agrobacterium tumefaciens interacts with and genetically transforms a wide variety of higher plants are well understood. The virulence (vir) regulon of a large tumor-inducing (Ti) plasmid of Agrobacterium is activated by a number of signal molecules synthesized by wounded plants, resulting in the production and transfer of a nucleoprotein complex, the T-complex, from the bacterium to the plant cell. The T-complex consists of the T-DNA (a single-stranded DNA segment of the Ti plasmid), VirD2 (the pilot protein covalently bonded to the 5' end of the T-DNA), and VirE2 (the single-stranded DNA binding protein which envelops the T-DNA). Once transferred into the plant cell, the T-complex finds its way into the plant nucleus where the T-DNA is integrated and expressed, leading to the overproduction of plant growth regulators and ultimately tumor formation. However, how the T-complex is transferred across the bacterial envelope, how the T-complex is trafficked once within the plant cell, and how the T-DNA is integrated into plant chromosomes through non-homologous recombination remain poorly understood. This project initiates a research program that has two objectives: to understand the interaction between the T-complex and its transport apparatus within Agrobacterium, and to identify plant factors involved in the transfer and integration of the T-DNA inside the plant cell. Since the specificity of the T-DNA transfer process is determined by the protein components of the T-complex, efforts will focus on identifying the Agrobacterium as well as the plant proteins interacting with the two protein components of the T-complex, VirD2 and VirE2. Several molecular tools for studying protein-protein interactions will be used to identify and study Agrobacterium proteins which recognize and interact with either VirD2 or VirE2 or both and thereby facilitate the transfer of the T-complex from the bacterium to the plant cell. These include the yeast two-hybrid system, cross linking, and co-immunoprecipitation. This study will also identify the signal domains of VirD2 and VirE2 which interact with the bacterial transport apparatus. These studies are expected to provide insights into unexplored areas of the molecular interaction between Agrobacterium and its host, such as the sequential events of the T-complex transfer in both the bacterium and the host plant cell. This will make the Agrobacterium-plant crown gall system a model system not only for studying microorganism-eukaryote interactions but also for studying and probing nucleoprotein trafficking and translocation in bacteria as well as higher plants. The understanding of the host factors involved in the T-DNA transfer and integration process will also likely lead to improved methods for genetically engineering recalcitrant plant species.