Structure-function analyses of aphid effector-target interaction

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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.