EAGER: Biochemical Mechanism of Oomycete RXLR Effector Binding to PI3P

有益微生物和致病微生物对植物、动物和人类以及自然和管理的生态系统都有重大影响。与动植物建立密切互动的微生物使用各种各样的分子来抑制或与宿主的免疫系统谈判，包括可以进入宿主细胞直接重新编程新陈代谢的效应蛋白。一些微生物会导致植物的破坏性疾病，而其他物种则提供了主要的好处。在所有情况下，人们对微生物的效应蛋白如何穿过宿主细胞周围的膜进入细胞内部知之甚少。虽然在这一问题上取得了实质性进展，但研究的技术挑战减缓了进展，并导致了相互矛盾的结论和争议。该项目将致力于解决这一领域最严峻的技术挑战，即效应蛋白如何与宿主膜脂质相互作用的问题，以减少争议并推动该领域的发展。更具体地说，研究人员将进行详细的生化实验，旨在解决关于卵菌效应物与磷脂酰肌醇-3-磷酸结合的相互矛盾的发现。这项工作将集中在目前获得最多结构和生化信息的卵菌的两个效应因子，即大豆疫霉Avh5和辣椒疫霉Avr3a4。该项目有三个目标。1.改进效应器蛋白的表达和纯化方法，以确定获得具有宿主细胞进入和脂质结合活性的蛋白质制剂所需的精确细节。在非常详细的水平上确定重复观察效应器与磷脂酰肌醇-3-磷酸结合所需的生化条件。为了确定与结合有关的残基，使用精确和可量化的方法来测试纯化的效应器及其突变体的脂质结合，包括表面等离子体共振和等温量热法，以及圆二色谱。除了促进对微生物与植物、动物和人类相互作用的理解外，该项目的更广泛影响将包括培训一名博士后学者和几名本科生研究人员，以及指导一名初级研究人员。

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.