Beneficial and pathogenic microbes have major impacts on plants, animals, and humans, and on natural and managed ecosystems. Microbes that establish close interactions with plants and animals use a wide diversity of molecules to suppress or negotiate with their hosts' immune systems, including effector proteins that can enter host cells to directly re-program metabolism. Some microbes cause destructive diseases of plants, while other species provide major benefits. In all cases, it is poorly understood how effector proteins from microbes cross the membrane surrounding host cells to enter the cell interior. Although substantial progress has been made on this question, the technical challenges of the studies have slowed progress and have resulted in conflicting findings and controversy. This project will focus on solving the most severe technical challenges in this area, namely the question of how effector proteins interact with host membrane lipids, in order to reduce controversy and advance the field.
More specifically, the investigators will carry out detailed biochemical experiments aimed at resolving conflicting findings regarding the binding of oomycete effectors to the lipid phosphatidylinositol-3-phosphate. The work will focus on the two effectors from oomycetes for which the most structural and biochemical information is currently available, namely Phytophthora sojae Avh5 and Phytophthora capsici Avr3a4.
The project has three objectives.
1. Refine the effector protein expression and purification methods in order to identify the precise details required for obtaining preparations of the proteins that are active in host cell entry and lipid binding.
2. Identify at a very detailed level the biochemical conditions required to reproducibly observe binding of the effectors to phosphatidylinositol-3-phosphate.
3. In order to identify residues involved in binding, use precise and quantifiable methods to assay lipid binding by the purified effectors and their mutants, including Surface Plasmon Resonance and Isothermal Calorimetry, and circular dichroism spectroscopy.
In addition to advancing understanding of how microbes interact with plants, animals and humans, the broader impacts of this project will include the training of a postdoctoral scholar and several undergraduate researchers, and the mentoring of a junior investigator.
