Assaying allosteric modulators of plant immune receptors for innovative crop protection

该项目将解决番茄PSKR1受体变构调节的基本问题，因为我们将能够通过开发和验证基于微尺度热泳术（MST）的强大且易于使用的无细胞分析，准确量化受体-配体相互作用。MST检测能够表征广泛的生物分子的相互作用，即使是具有挑战性的样品，如整个细胞裂解物。该提案的引人注目的目标在于将荧光团标记的PSKR1受体应用于MST无固定技术，而无需蛋白质纯化。我们将在烟草叶片中短暂表达番茄PSKR1，以确保原生微环境和翻译后修饰（如糖基化），并随后测试、验证和优化结合肽和小分子的检测条件。通过这种方法，我们将开发一种新技术，为植物免疫系统应用于作物保护的研究提供基础，并使以靶点为中心的新型保护剂的发现成为可能。我们将建立新的方法来研究植物PSK受体在健康和疾病中的功能，以及植物免疫受体在宿主-病原体相互作用中的作用。我们将了解如何干扰在我们的试点数据中确定的特定变抗结合位点，影响PSKR1和病原体衍生肽之间的相互作用，以及如何利用这些位点增强植物对病原体的免疫反应。本提案中产生的技术和概念验证数据将用于支持我们与项目合作伙伴和合作者计划的更大的资金竞标，并将通过PSKR1的目标验证和新化学实体的发现为商业化提供途径，这些化学实体将共同开发成新型作物保护剂。拟议的研究跨越生物分子相互作用、生物化学、细胞、分子和植物生物学，将对作物保护、植物免疫学、早期职业科学家的培训以及来自不同相关学科的学者、工业合作伙伴和种植者之间的跨部门合作等领域产生影响。总之，这个多学科的提议将产生(i)测定，定量PSKR1和配体之间的相互作用；（ii）通过PSKR1受体的宿主-病原体相互作用的概念验证数据；（iii）变构调节剂的开发有助于开发新的作物保护剂，支持未来的拨款申请，并建立新的知识产权。

This project will address fundamental questions regarding allosteric modulation of tomato PSKR1 receptors, as we will be able to accurately quantify receptor-ligand interactions through development and validation of robust and facile to use cell-free assay, based on microscale thermophoresis (MST). MST detection enables characterisation of interactions for a wide range of biomolecules, even for challenging samples such as whole cell lysates.The compelling goal of this proposal lies in the application of a fluorophore-tagged PSKR1 receptor into MST immobilisation-free technology without a requirement of protein purification. We will transiently express tomato PSKR1 in tobacco leaves to ensure native microenvironment and post-translational modifications (e.g. glycosylations), and subsequently test, validate and optimise the assay conditions for binding peptides and small molecules alike. Through this approach, we will develop a new technology underpinning research in plant immune system applied to crop protection and enabling target-focused discovery of novel protective agents. We will establish new ways to study the function of plant PSK receptors in health and disease, and involvement of plant immune receptors in host-pathogen interactions at unprecedented level of detail. We will understand how interfering with specific allosteric binding site, identified in our pilot data, impacts interactions between PSKR1 and pathogen-derived peptides showed to be involved in the disease, and how these sites can be utilised in boosting plant immune responses against the pathogens. Technology and proof-of-concept data generated in this proposal will be used to support the larger funding bid that we plan with our project partner and collaborators, and will also provide a route for commercialisation through target validation of PSKR1 and discovery of novel chemical entities that will be jointly developed into novel crop protective agents.Proposed research, spanning biomolecular interactions, biochemistry, cell, molecular and plant biology will have an impact on in the areas of crop protection, plant immunology, training of early career scientists, and cross-sectoral engagement between academics from diverse related disciplines, industrial partners, and growers. In conclusion, this multidisciplinary proposal will yield (i) the assay, quantifying interactions between PSKR1 and ligands; (ii) proof-of-concept data on host-pathogen interactions via PSKR1 receptors; (iii) development of allosteric modulator leads to enable development of new agents for crop protection, to underpin future grant applications, and to establish a new intellectual property.