Molecular engineering of the ubiquitin-proteasome system: A new approach to pathogen resistance in Plants

大麦是世界第四大作物，在英国排名第二。包括由褐锈菌引起的疾病，大麦产量损失高达40%。该项目的重点是改善植物自身的自然防御能力，以减少与疾病相关的损失，作为一种具有成本效益和环境可持续的替代化学处理方法。作为对病原体攻击的反应，植物发起足以抵御大多数病原体的免疫反应。然而，适应的病原体产生的效应蛋白能够抑制宿主的免疫反应，促进成功的感染，造成严重的作物损害。这个项目采用了一种新的工程生物学方法来重新利用植物自身的酶来标记要降解的效应蛋白。通过K48型连接将泛素添加到蛋白质中，使蛋白质被蛋白酶体降解。UKRI BBSRC EASTBIO博士培训伙伴关系2022队列：博士项目提案2这个博士项目的目标是重新设计K48型E3连接酶的底物特异性，从而创建一种合成泛素途径，针对特定的病原体效应因子进行蛋白酶体介导的降解，从而产生增强的抗病能力。我们寻找一个对将基础生物分子工程应用于现实世界问题感兴趣的学生。学生将受益于Regan(蛋白质工程专家)和Orosa(植物科学专家)，以及我们的工业合作伙伴和植物生物技术和作物保护方面的领先公司Syngenta的共同指导。具体地说，该博士项目的目标是i)鉴定大麦中通过K48连接将泛素添加到底物上的E3连接酶；ii)鉴定和分析它们的底物结合结构域--重点是E3连接酶-底物相互作用的特异性；iii)重新设计所选的E3连接酶以结合目标病原体的效应蛋白并使其泛素化。Iv)测试大麦中重新设计的E3连接酶是否提高了对病原菌的抗性。目标i)和ii)将涉及最新的序列和结构分析，以确定底物结合结构域中的特异性决定残基。目标三)和目标四)将涉及湿实验室蛋白质工程和植物生物技术工作。底物结合所需的氨基酸将进行组合突变，并使用我们团队开发的高通量方法筛选突变体，以确定能够与目标病原体的关键效应蛋白(S)结合并泛素化的E3连接酶变异体。新开发的针对病原菌效应蛋白(S)的合成E3连接酶将在大麦中稳定过表达，并在植物中进行测试。人工合成的E3连接酶对叶锈菌诱导的疾病进展的影响将通过感染试验进行测试。PHD项目的一个最终目标是同时针对几个效应器来产生强大和持久的抗病能力。通过这个项目，将开发一种创新的方法和新的工具来操纵植物病原体的相互作用。学生将加入一个充满活力的研究小组，除了学术和公司研究的经验外，还将获得强有力的跨学科培训。如果成功，这项技术将被转移到其他作物和病理系统。减少疾病对作物的影响，将有助于实现联合国目标2：零饥饿方案

Barley (Hordeum vulgare) is the fourth most important crop worldwide and second in the UK. Diseases, including those caused by the brown rust pathogen, Puccinia hordei, cause barley yield losses of up to 40%. This project focuses on improving plant's own natural defences to alleviate disease-related losses as a cost-effective and environmentally sustainable alternative to chemical treatments. In response to pathogen attack, plants initiate immune responses that are sufficient to fend off most pathogens. Adapted pathogens, however, produce effector proteins that are capable of suppressing the host immune response and promote successful infection, causing severe crop damage. This project takes a novel engineering biology approach to re-purpose a plant's own enzymes to mark effector proteins for degradation. Addition of ubiquitin to a protein, via the K48 type linkage, targets that protein for degradation by the proteasome. UKRI BBSRC EASTBIO Doctoral Training Partnership 2022 Cohort: PhD Project Proposal2The goal of this PhD project is to re-engineer the substrate specificity of K48 type E3 ligases, and thus to create a synthetic ubiquitin pathway that targets specific pathogen effectors for proteasome-mediated degradation, thereby generating enhanced disease resistance.We seek a student who is interested in applying fundamental biological molecular engineering to a real-world problem. The student will benefit from the combined mentorship of Regan (expert in protein engineering) and Orosa (expert in plant science), and of Syngenta, our industrial partner and a leading company in plant biotechnology and crop protection.Specifically, the goals of the PhD project are i) identify E3 ligases in barley that add ubiquitin to substrates via K48 linkages; ii) identify and analyse their substrate-binding domains -with emphasis on the specificity of the E3 ligase-substrate interaction; iii) re-engineer chosen E3 ligases to bind and ubiquitinylate effector proteins of the target pathogen. iv) test the re-engineered E3 ligases in barley for increased resistance to the pathogen.Goals i) and ii) will involve state of the art sequence and structural analysis to identify specificity-determining residues in substrate-binding domains. Goals iii) and iv) will involve wet-lab protein engineering and plant biotechnology work. The amino acids essential for substrate binding will be combinatorially mutated and the mutants screened using high throughput methodologies developed by our teamto identify E3 ligase variants of the that are able to bind to and ubiquitinylate key effector(s) protein(s) of the target pathogen. Newly developed synthetic E3 ligases targeting pathogen effector(s) will be tested in planta, by stable overexpression in barley. The impact of the synthetic E3 ligase on the progression of Puccinia induced disease will be tested by infection assays. An ultimate goal of the PhD project will the simultaneous targeting of several effectors to produce robust and durable disease resistance.Through this project an innovative approach and new tools to manipulate plantpathogen interactions will be developed. The student will join a vibrant group if researchers, and will gain strong interdisciplinary training in addition to experience of both academic and company research. If successful, the technology will be transferable to other crops and pathosystems.Reducing the impact of disease on crops, will contribute to addressing the United Nations Goal2: Zero Hunger programme