How does metal binding affect the function of proteins targeted by a devastating pathogen of cereal crops?

金属结合如何影响谷类作物毁灭性病原体靶向的蛋白质的功能？

• 批准号: 2901648
• 金额: --
• 依托单位: Newcastle University
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2024
• 资助国家: 英国
• 起止时间: 2024 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2901648
• 关键词: How; does; metal; binding; affect

Plant diseases are a continuous threat to global food production and security. Many plant pathogens use effector proteins to interfere with cellular processes in the host promoting colonisation and growth. Rice heavy metal-associated plant proteins (HPPs), including the heavy metal-associated isoprenylated plant proteins (HIPPs), are targets of effector proteins from the rice blast pathogen Magnaporthe oryzae, presumably to promote infection. HPPs/HIPPs form a diverse family of proteins in crops and other plants, but little is known about their function and role in disease. HPPs/HIPPs possess heavy metal-associated (HMA) domains, which in proteins that bind metals typically have an N-terminal CXXC (C=cysteine) motif. The hypothesis we will test is that metal binding by HPPs/HIPPs is important for their cellular functions and perturbation by pathogen effectors.There is limited information about the role of metals for the structure and function of HPPs/HIPPs. To study metal binding in this protein family, a carefully chosen selection of rice HPPs/HIPPs HMA domains, both with and without the full CXXC motif, will be produced. In vitro characterisation of metal binding will be achieved with an array of spectroscopic and biophysical approaches. As studies progress, this choice will be assisted by protein bioinformatics. The structures of HMA domains, either determined by protein crystallography or modelling, will be used in conjunction with new deep learning-based methods to predict metal-binding capability and specificity. Full length HPPs/HIPPs will also be produced and analysed. Some HPPs/HIPPs are relatively small possessing approximately 120 residues, and over-express in E. coli. However, purification has proved challenging. The availability of AlphaFold2 models could assist by better defining the boundaries of folded units and allow the elimination of unnecessary terminal regions. Once metal binding has been demonstrated in vitro, its influence on the structure and function of HPPs/HIPPs will be investigated. This will include protein crystallography (the structure of HIPP19-HMA with an M. oryzae effector has been determined), testing how metal binding influences interactions with effector proteins and the ability of effectors to perturb ROS production by HPPs/HIPPs. Introducing metal binding into rice immune receptor proteins (e.g. Pik), which have HMA domains that act as bait domains to directly detect the presence of effectors, will also be tested.The interdisciplinary team involved in this project will teach a range of skills. This will include modern molecular biology techniques as well as how to purify proteins, particularly HPPs/HIPPs. Many approaches will be used to investigate metal binding, mostly under strict anaerobic conditions. The influence of metals on interactions with effectors will be studied including using X-ray crystallography, which will also provide detailed information about how metal binding alters the structures of HPPs/HIPPs. In vivo techniques will be used to investigate the role of metals on the function of HPPs/HIPPs and their involvement in pathogenesis. A range of protein bioinformatics will complement these studies.This project fits within BBSRCs Tacking Strategic Challenges objective under the Bioscience for Sustainable Agriculture and Food (previously Agriculture and Food Security) priority. The rice blast pathogen is the most devastating disease of rice, estimated to destroy enough of this crop to feed 212-742 million people annually. This disease can be addressed by investigating the molecular basis of pathogen-host communication as outlined in this project. HPPs/HIPPs are present in other crops including wheat and this work therefore has wide-ranging impact on food security. Such studies can contribute to efforts to protect the world's most important crops from plant diseases.

植物疾病是对全球粮食生产和安全的持续威胁。许多植物病原体使用效应蛋白来干扰宿主促进定殖和生长的细胞过程。水稻重金属相关的植物蛋白（HPP），包括重金属相关的异on-植物蛋白（嬉皮），是稻米病原体蛋白质的效应蛋白的靶标，大概是促进感染的。 HPP/Hipps在农作物和其他植物中形成了多种蛋白质家族，但对它们在疾病中的功能和作用知之甚少。 HPP/HIPP具有重金属相关（HMA）结构域，在结合金属通常具有N末端CXXC（C =半胱氨酸）基序的蛋白质中。我们将检验的假设是，HPP/HIPP的金属结合对于它们的细胞功能和病原体效应子的扰动很重要。关于金属在HPP/HIPP的结构和功能中的作用的信息有限。为了研究该蛋白质家族中的金属结合，将产生精心选择的水稻HPPS/HIPPS HMA结构域，无论有没有完整的CXXC基序。通过一系列光谱和生物物理方法，将实现金属结合的体外表征。随着研究的进展，这种选择将由蛋白质生物信息学协助。由蛋白质晶体学或建模确定的HMA结构域的结构将与新的基于深度学习的方法结合使用，以预测金属结合能力和特异性。还将生产和分析全长的HPP/嬉皮士。一些HPP/HIPP相对较小，拥有约120个残基，并且在大肠杆菌中过表达。但是，纯化已被证明具有挑战性。 AlphaFold2模型的可用性可以通过更好地定义折叠单元的边界并允许消除不必要的终端区域来有助于。一旦在体外证明了金属结合，将研究其对HPP/HIPP的结构和功能的影响。这将包括蛋白质晶体学（已经确定了带有M. oryzae效应子的HIPP19-HMA的结构），测试金属结合如何影响与效应子蛋白的相互作用以及效应子通过HPPS/HIPP扰动ROS的能力。将金属结合到水稻免疫受体蛋白（例如PIK）中，它们的HMA结构域充当诱饵域直接检测效应子的存在。这将包括现代分子生物学技术以及如何纯化蛋白质，尤其是HPP/HIPP。许多方法将用于研究金属结合，主要是在严格的厌氧条件下。将研究金属对与效应子相互作用的影响，包括使用X射线晶体学，这还将提供有关金属结合如何改变HPP/HIPP结构的详细信息。体内技术将用于研究金属对HPP/HIPP功能及其在发病机理中的作用的作用。一系列蛋白质生物信息学将补充这些研究。该项目适合在可持续农业和粮食（以前的农业和粮食安全）优先事项的生物科学下解决战略挑战的目标。水稻爆炸病原体是大米最具破坏性的疾病，估计会破坏足够多的农作物，每年为212-7.42亿人提供食物。该疾病可以通过研究该项目中概述的病原体宿主通信的分子基础来解决。 HPP/HIPP在包括小麦在内的其他农作物中都存在，因此这项工作对粮食安全产生了广泛的影响。这样的研究可以促进保护世界上最重要的农作物免受植物疾病的影响。