Structure-function analyses of aphid effector-target interaction

蚜虫效应子-靶标相互作用的结构-功能分析

• 批准号: 2593179
• 金额: --
• 依托单位: University of Dundee
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2021
• 资助国家: 英国
• 起止时间: 2021 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2593179
• 关键词: Structure; function; analyses; aphid; effector

Aphids are phloem-feeding insects that induce extensive feeding damage, achieve high population densities, and transmit economically important plant viruses. With increasing restrictions on the use of insecticides, as well as problems with insecticide resistance, there is an urgent need to develop novel aphid control strategies. A relatively new area of research that can facilitate this has recently emerged and focuses on understanding the molecular dialogue that takesplace between aphids and their hosts. Like plant pathogenic microbes and nematodes, aphids form close associations with host plants. These insects insert highly specialized mouthparts, called stylets, inside host tissue for feeding and probing, during which saliva is secreted. This saliva is delivered intodifferent plant cell types and contains a wide array of proteins, called effectors, which are involved in manipulation of host cell processes to the benefit of the aphid. Characterization of these effectorsto dissect their role in virulence is an important step forward in understanding host susceptibility to aphids.We previously characterized several Myzus persicae(green peach aphid) effectors with BBSRC EASTBIO Doctoral Training Partnership 2021 Cohort: PhD Project Proposal2regards to their virulence activity and identified several interacting host proteins as potential virulence targets. In this project, the student will specifically focus on understanding how M. persicaeeffectors target host proteins to promote plant susceptibility. The key objective of this project is to investigate the structure-function relationship of aphid effector-host target interactions to gain insight into interaction specificity and molecular co-evolution. This will be achieved by applying 2 complementary approaches:1)Structural biology to reveal protein 3D structures of aphid effectors and their host protein targets in bound and unbound state and test how mutations affect interaction. Recombinant proteins will be produced for crystallization followed by X-ray diffraction and in vitro binding assays. Where relevant effector and target mutants will be generated to define amino acid sites and regions required for protein-protein interactions. 2)Effector and target (deletion) mutagenesis in combination with protein-protein interactions assays. The student will perform extensive (deletion) mutagenesis of effectors and their host plant targets in combination with co-immunoprecipitation assays to identify domains and amino acid sites important for protein-protein interactions, and also include natural variants where available.Effector and host target mutants generated in these 2 approaches will be subjected to functional assays to determine the impact on host susceptibility to aphids. Based on these structure-function analyses, we will ultimately be able to investigate whether by interfering with aphid-mediated targeting of the host targets we can reduce plant susceptibility to aphids, which would have important implications for development of novel crop protection strategies.The student will receivetraining in protein expression and purification as well as structural biology approaches to assess dynamics of protein-protein interactions and 3D structures. In addition, the student will develop skills in molecular plant biologyand entomology.

蚜虫是一种以韧皮部为食的昆虫，会造成广泛的取食危害，达到高种群密度，并传播经济上重要的植物病毒。随着对杀虫剂使用的限制越来越多，以及抗药性问题的出现，迫切需要开发新的防治策略。最近出现了一个相对较新的研究领域，可以促进这一点，该领域的重点是了解蚜虫和它们的宿主之间发生的分子对话。与植物病原微生物和线虫一样，蚜虫与寄主植物形成了密切的联系。这些昆虫在寄主组织内插入高度专门化的口器，称为口针，用于取食和探测，在此期间唾液会分泌。这种唾液被输送到不同类型的植物细胞中，并含有一系列被称为效应器的蛋白质，这些蛋白质参与了对寄主细胞过程的操纵，以造福于蚜虫。研究这些效应器在毒力中的作用是了解寄主对蚜虫敏感性的重要一步。我们以前与BBSRC EASTBIO博士培训伙伴2021 Cohort：PHD Project Proposal2研究了几个桃蚜(桃蚜)效应器的毒力活性，并确定了几个相互作用的宿主蛋白作为潜在的毒力靶标。在这个项目中，学生将特别专注于了解桃红假单胞菌是如何以宿主蛋白为目标来促进植物敏感性的。该项目的主要目标是研究蚜虫效应器-寄主靶标相互作用的结构-功能关系，以深入了解相互作用的专一性和分子共同进化。这将通过应用两种互补的方法来实现：1)结构生物学，揭示结合和非结合状态下的蚜虫效应器及其寄主蛋白靶标的蛋白质3D结构，并测试突变如何影响相互作用。重组蛋白将被生产出来用于结晶，随后将进行X射线衍射和体外结合试验。相关的效应子和靶突变体将被产生来定义蛋白质-蛋白质相互作用所需的氨基酸位置和区域。2)效应子和靶(缺失)突变结合蛋白质-蛋白质相互作用分析。学生将对效应器和它们的寄主植物目标进行广泛的(缺失)突变，结合免疫共沉淀分析来确定对蛋白质-蛋白质相互作用重要的结构域和氨基酸位置，并在可用的情况下包括自然变异。在这两种方法中产生的效应器和寄主目标突变体将接受功能分析，以确定对寄主对蚜虫的易感性的影响。基于这些结构-功能分析，我们最终将能够调查是否可以通过干扰蚜虫介导的靶标来降低植物对蚜虫的敏感性，这将对开发新的作物保护策略具有重要意义。学生将接受蛋白质表达和纯化方面的培训，以及评估蛋白质-蛋白质相互作用和3D结构动态的结构生物学方法。此外，学生还将发展分子植物生物学和昆虫学方面的技能。