Structure-function analyses of aphid effector-target interaction

蚜虫是一种以韧皮部为食的昆虫，可引起广泛的取食损害，种群密度高，并传播经济上重要的植物病毒。随着杀虫剂使用限制的增加，以及杀虫剂耐药性问题的出现，迫切需要开发新的蚜虫控制策略。最近出现了一个相对较新的研究领域，可以促进这一点，重点是了解蚜虫与其宿主之间发生的分子对话。像植物病原微生物和线虫一样，蚜虫与寄主植物有着密切的联系。这些昆虫将高度特化的口器（称为“柱头”）插入宿主组织中，用于进食和探探，在此期间分泌唾液。这种唾液被输送到不同类型的植物细胞中，含有一系列被称为效应器的蛋白质，这些蛋白质参与操纵宿主细胞的过程，以使蚜虫受益。表征这些效应物以剖析它们在毒力中的作用是了解寄主对蚜虫易感性的重要一步。我们之前在BBSRC EASTBIO博士培训伙伴关系2021年研究项目提案2中描述了几种桃蚜（绿桃蚜）效应物的毒力活性，并确定了几种相互作用的宿主蛋白作为潜在的毒力靶点。在这个项目中，学生将特别专注于了解桃干分枝杆菌效应物如何靶向宿主蛋白来促进植物的易感性。本项目的主要目的是研究蚜虫效应物-宿主靶点相互作用的结构-功能关系，以深入了解相互作用的特异性和分子协同进化。这将通过两种互补的方法来实现：1)结构生物学揭示蚜虫效应物及其宿主蛋白靶点在结合和非结合状态下的蛋白质三维结构，并测试突变如何影响相互作用。重组蛋白将产生结晶，然后进行x射线衍射和体外结合试验。将产生相关的效应体和靶突变体，以确定蛋白质相互作用所需的氨基酸位点和区域。2)效应和靶（缺失）诱变与蛋白相互作用分析相结合。该学生将结合共免疫沉淀法对效应物及其宿主植物靶点进行广泛（删除）诱变，以确定对蛋白质相互作用重要的结构域和氨基酸位点，并在可能的情况下包括自然变异。在这两种方法中产生的效应体和宿主靶突变体将进行功能分析，以确定对宿主对蚜虫敏感性的影响。基于这些结构-功能分析，我们将最终能够研究是否通过干扰蚜虫介导的寄主靶点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以及结构生物学方法的培训，以评估蛋白质-蛋白质相互作用和3D结构的动态。此外，学生将发展分子植物生物学和昆虫学的技能。

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.