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

植物病害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的细胞过程，促进定植和生长。水稻重金属相关植物蛋白（HPPs），包括重金属相关异丙烯酰化植物蛋白（HIPPs），是水稻稻瘟病菌稻瘟病菌效应蛋白的靶标，可能促进侵染。HPPs/HIPPs在作物和其他植物中形成了一个多样化的蛋白质家族，但对它们在疾病中的功能和作用知之甚少。HPPs/HIPPs具有重金属相关（HMA）结构域，其在结合金属的蛋白质中通常具有n端CXXC （C=半胱氨酸）基序。我们将验证的假设是，HPPs/HIPPs的金属结合对它们的细胞功能和病原体效应物的扰动很重要。关于金属在HPPs/HIPPs的结构和功能中的作用的信息有限。为了研究该蛋白家族的金属结合，我们精心挑选了具有和不具有完整CXXC基序的水稻HPPs/HIPPs HMA结构域。金属结合的体外表征将通过一系列光谱和生物物理方法来实现。随着研究的进展，蛋白质生物信息学将辅助这种选择。HMA结构域的结构，通过蛋白质晶体学或建模来确定，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。还将制作和分析完整长度的hpp / hipp。一些HPPs/HIPPs相对较小，大约有120个残基，在大肠杆菌中过表达。然而，事实证明，提纯具有挑战性。AlphaFold2模型的可用性可以帮助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。一旦在体外证明了金属结合，将研究其对HPPs/HIPPs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效应的hip19 - hma的结构），测试金属结合如何影响与效应蛋白的相互作用，以及效应蛋白干扰HPPs/HIPPs产生ROS的能力。还将测试将金属结合引入水稻免疫受体蛋白（例如Pik），它具有HMA结构域，作为诱饵结构域直接检测效应器的存在。参与这个项目的跨学科团队将教授一系列技能。这将包括现代分子生物学技术以及如何纯化蛋白质，特别是HPPs/HIPPs。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器相互作用的影响，包括使用x射线晶体学，这也将提供关于金属结合如何改变HPPs/HIPPs结构的详细信息。体内技术将用于研究金属对HPPs/HIPPs功能的作用及其在发病机制中的参与。一系列蛋白质生物信息学将补充这些研究。该项目符合生物科学促进可持续农业和粮食（原农业和粮食安全）优先项目下的“应对战略挑战”目标。稻瘟病病原体是水稻最具破坏性的疾病，据估计，每年摧毁的水稻足以养活2.12亿至7.42亿人。这种疾病可以通过研究病原体-宿主通讯的分子基础来解决，如本项目所述。高效磷/高效磷存在于包括小麦在内的其他作物中，因此这项工作对粮食安全具有广泛影响。这些研究有助于保护世界上最重要的作物免受植物病害的侵害。

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.