The importance of metal binding for the function of rice proteins that interact with pathogen effectors

金属结合对于与病原体效应子相互作用的水稻蛋白功能的重要性

• 批准号: 2306772
• 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: 2019
• 资助国家: 英国
• 起止时间: 2019 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2306772
• 关键词: importance; metal; binding; function; rice

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. In recent studies, rice heavy metal-associated plant proteins (HPPs), including the heavy metal-associated isoprenylated plant proteins (HIPPs), were shown to be targets of effector proteins from the rice blast pathogen Magnaporthe oryzae, presumably to promote infection. HPPs/HIPPs form a large diverse family of proteins in crops and other plants, but little is known about their physiological function and role in disease. HPPs/HIPPs possess heavy metal-associated domains (HMAs), 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 data on metal binding by HPPs/HIPPs. Preliminary in vitro studies in Newcastle have shown that HIPP19 has a preference for copper over zinc. One of the cysteines in the CXXC-motif is replaced with a serine in HIPP19, but other cysteines are present, and site-directed mutagenesis is being used to identify the location of metal binding. To study metal binding further in this family of proteins a carefully chosen selection of rice HPPs/HIPPs HMA domains, both with and without the full CXXC motif, will be over-expressed in bacteria. As studies progress, this choice will be assisted by protein bioinformatics (Prof. Dan Rigden, Liverpool) to predict HMAs that bind different metals, and also those without metal-binding capability, as controls. In vitro characterization of metal binding will be achieved using an array of approaches. Full length HPPs/HIPPs will also be produced and analysed. This has proved challenging for the one example tested and will be assisted by covariance analysis (Liverpool) to better define complete folding units and the full extent of regions to clone.Once metal binding has been demonstrated in vitro, its influence on the structure and function of HPPs/HIPPs will be investigated with Prof. Mark Banfield (John Innes Centre). This will include protein crystallography, testing how metal binding influences interactions with M. oryzae 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 HMAs that act as bait domains to directly detect the presence of effectors, will also be tested.This project fits within the remit of the 'Agriculture and Food Security' BBSRC strategic framework. As global demand for food is rising the threat posed by pathogens that can dramatically reduce crop harvests is a major concern. The rice blast pathogen Magnaporthe oryzae is the most devastating disease of rice, estimated to destroy enough of this crop to feed 212-742 million people annually. One approach to address such plant diseases is to investigate the molecular basis of communication between pathogen and host as outlined in this project. In particular, an understanding of how pathogens target host cell processes for their own benefit is key. Evidence to date suggests that rice HPPs and HIPPs are the targets of M. oryzae effectors. HPPs and HIPPs are also present in a number of other major crops, including wheat, therefore understanding their function, and how this can be influenced by pathogen effectors, has wide ranging impact on food security. As HMAs are also found in some plant immune receptors, this work may lead to the development of receptors that can detect a wider range of effectors. This will contribute to efforts to protect the world's most important crops from plant diseases.

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。在最近的研究中，水稻重金属相关植物蛋白（HPPs），包括重金属相关异丙烯化植物蛋白（HIPPs），被证明是水稻稻瘟病菌Magnaporthe oryzae效应蛋白的靶标，可能促进感染。在作物和其他植物中，HPPs/HIPPs形成了一个大而多样的蛋白质家族，但对其生理功能和在疾病中的作用知之甚少。HPPs/HIPPs具有重金属相关结构域（HMAs），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HPPs/HIPPs的金属结合的数据有限。纽卡斯尔的初步体外研究表明，hip19对铜的偏好超过对锌的偏好。在hip19中，cxxc基序中的一个半胱氨酸被丝氨酸取代，但其他半胱氨酸存在，并且正在使用位点定向诱变来确定金属结合的位置。为了进一步研究该蛋白家族的金属结合，我们精心挑选了水稻HPPs/HIPPs HMA结构域，无论是否含有完整的CXXC基序，都将在细菌中过表达。随着研究的进展，这种选择将得到蛋白质生物信息学（教授Dan Rigden，利物浦）的帮助，以预测结合不同金属的HMAs，以及那些没有金属结合能力的HMAs，作为对照。金属结合的体外表征将使用一系列方法来实现。还将制作和分析完整长度的hpp / hipp。事实证明，对于测试的一个例子来说，这是具有挑战性的，协方差分析（Liverpool）将有助于更好地定义完整的折叠单元和要克隆的区域的全部范围。一旦在体外证明了金属结合，将与Mark Banfield教授（John Innes中心）一起研究其对HPPs/HIPPs结构和功能的影响。这将包括蛋白质晶体学，测试金属结合如何影响m.o ryzae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），其具有HMAs作为诱饵结构域直接检测效应物的存在。该项目符合BBSRC“农业与粮食安全”战略框架的职责范围。随着全球对粮食的需求不断上升，病原体造成的威胁可能会大大减少作物收成，这是一个主要问题。稻瘟病病菌稻瘟病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。解决这类植物病害的途径之一是研究病原菌与寄主之间交流的分子基础。特别是，了解病原体如何针对宿主细胞过程以获取自身利益是关键。迄今为止的证据表明，水稻HPPs和HIPPs是M. oryzae效应剂的目标。高致病性病原菌和高致病性病原菌也存在于包括小麦在内的许多其他主要作物中，因此，了解它们的功能以及它们如何受到病原体效应物的影响，对粮食安全具有广泛的影响。由于HMAs也存在于一些植物免疫受体中，因此这项工作可能会导致能够检测更广泛效应物的受体的开发。这将有助于保护世界上最重要的作物免受植物病害的侵害。