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

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中促进定殖和生长的细胞过程。水稻重金属相关植物蛋白（HPPs），包括重金属相关异戊二烯化植物蛋白（HIPPs），是稻瘟病菌（Magnaporthe）效应蛋白的作用靶点，可能具有促进侵染的作用。HPPs/HIPPs在作物和其他植物中形成了一个大的多样性蛋白质家族，但对其生理功能和在疾病中的作用知之甚少。HPP/HIPPs具有重金属相关结构域（HMA），其在结合金属的蛋白质中通常具有N-末端CXXC（C=半胱氨酸）基序。我们将检验的假设是，HPPs/HIPPs的金属结合对于它们的细胞功能和病原体效应物的干扰是重要的。HPP/HIPPs的金属结合数据有限。在纽卡斯尔进行的初步体外研究表明，HIPP 19对铜的偏好超过锌。CXXC基序中的一个半胱氨酸被HIPP 19中的丝氨酸取代，但存在其他半胱氨酸，并且定点诱变被用于鉴定金属结合的位置。为了进一步研究该蛋白家族中的金属结合，将在细菌中过表达精心选择的具有和不具有完整CXXC基序的水稻HPPs/HIPPs HMA结构域。随着研究的进展，这种选择将得到蛋白质生物信息学（Dan Rigden教授，利物浦）的帮助，以预测结合不同金属的HMA，以及那些没有金属结合能力的HMA作为对照。将使用一系列方法实现金属结合的体外表征。还将制作和分析全长HPP/HIPPs。这已被证明具有挑战性的一个例子测试，并将协助协方差分析（利物浦），以更好地定义完整的折叠单位和区域的完整范围克隆。一旦金属结合已在体外证明，其对HPPs/HIPPs的结构和功能的影响将与马克班菲尔德教授（约翰英纳斯中心）进行调查。这将包括蛋白质晶体学，测试金属结合如何影响与M的相互作用。研究了HPPs/HIPPs对ROS产生的干扰效应蛋白和效应蛋白的能力。将金属结合引入水稻免疫受体蛋白（例如Pik），这些蛋白具有HMA作为诱饵结构域，直接检测效应物的存在，也将进行测试。该项目符合BBSRC“农业和粮食安全”战略框架的职权范围。随着全球对粮食的需求不断增加，病原体造成的威胁可能大大减少作物收成，这是一个主要问题。稻瘟病病原体Magnaporthe magnaporthe是水稻最具破坏性的疾病，估计每年摧毁的这种作物足以养活2.12 - 7.42亿人。解决这类植物病害的一种方法是研究病原体和宿主之间通讯的分子基础，如本项目所述。特别是，了解病原体如何为自身利益而靶向宿主细胞过程是关键。迄今为止的证据表明，水稻HPPs和HIPPs是M的靶标。双效应器。HPPs和HIPPs也存在于许多其他主要作物中，包括小麦，因此了解它们的功能以及病原体效应物如何影响它们，对粮食安全具有广泛的影响。由于HMA也存在于一些植物免疫受体中，这项工作可能会导致受体的发展，可以检测更广泛的效应物。这将有助于保护世界上最重要的作物免受植物病害的影响。

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.