Andrew Binns

Genetic and biochemical analysis of Agrobacterium-plant cell interaction

Agrobacterium tumefaciens is a gram-negative soil bacterium that has the unique capacity to transfer DNA from its resident Ti (tumor-inducing) plasmid, and proteins encoded by this plasmid, into plant cells. The transferred DNA (the T-DNA) is ultimately moved into the nucleus, integrated into the chromosomal DNA and expressed. T-DNA expression results in the production of i) growth factors that cause uncontrolled cell proliferation and ii) novel amino acid-sugar conjugates that cannot be metabolized by the plant cell but will serve as a carbon and nitrogen source for the inciting bacteria. Thus, Agrobacterium engineers the plant cell so that it proliferates indefinitely while producing nutrients that are dedicated to bacterial growth. This system can also be manipulated so that rather than transferring the wild type T-DNA the Agrobacterium will transfer any DNA cloned into an appropriate vector, thus allowing for routine genetic engineering of plants.

The research in our lab is focused currently on two major questions in relation to the Agrobacterium-mediated transformation process:

How are plant derived signals recognized and how does this recognition activate the expression of the virulence genes?

Initiation of plant transformation by Agrobacterium occurs when the virulence (vir) genes of the Ti plasmid are activated. This occurs as a result of the activities of a classic two component regulatory system. The sensor kinase (VirA), a membrane bound dimeric histidine kinase responds to plant derived compounds (certain sugars, phenolics and low pH) by phosphorylating the response regulator (VirG) which, in turn, activates transcription of the vir genes. We are particularly interested in how the different signals are recognized by and activate the VirA sensor kinase. Our recent studies have shown that a diverse set of sugars bind to a periplasmic protein (ChvE) that subsequently interacts with the periplasmic domain of VirA, resulting in an increase in the sensitivity of the system to phenolic derivatives produced by the plant. We have identified regions of ChvE that are necessary for sugar binding and regions that are necessary for activation of VirA.  We have also demonstrated that ChvE is part of an operon that encodes and ABC transporter that is involved in the utilization of sugars as carbon sources by Agrobacterium.  Currently, the structure of ChvE, bound to different sugars, is being solved and regions of the periplasmic domain of VirA that interacts with ChvE is being identified.  The ultimate goal of these studies is to understand how the binding of sugar-bound ChvE to the periplasmic domain of VirA results in the increase in sensitivity of this kinase to the phenolic molecules, which appear to be recognized by a cytoplasmic domain of VirA.

What is the spatial and temporal distribution of the plant derived signals and does this distribution provide information to the pathogen about the susceptibility of the nearby plant cells to the transformation process?

Agrobacterium recognizes an extremely diverse set of plant-derived molecules in the control of its virulence genes that are required for DNA transfer. We are currently utilizing a variety of  ChvE and VirA variants that are have either more or less restrictive signal binding properties, along with fluorescent protein reporters of vir gene expression, to address the following questions: 1) is the ‘promiscuity’ (in relation to signal perception) of the signal receptors related to the capacity of Agrobacterium to infect such a broad host range of plants and plant tissues? and 2) is the complexity of the signaling and the distribution of multiple signals within the plant – that is the signal landscape – related to the identification by the bacteria of plant cells ‘competent’ for tumor formation in contrast to competent for DNA transfer?  Together, these studies will correlate signal complexity in the host plant with two different aspects of the infectious process (vir gene expression and/or cell transformation) and answer the question of whether vir gene induction is the limiting factor for plant transformation.