Non-pathogenic Type III Secretion system-based tools for delivery of bioactive proteins into plant cells

基于非致病性 III 型分泌系统的工具，用于将生物活性蛋白递送到植物细胞中

• 批准号: 1960533
• 金额: --
• 依托单位: Imperial College London
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2017
• 资助国家: 英国
• 起止时间: 2017 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-1960533
• 关键词: Non; pathogenic; Type; III; Secretion

The global population is predicted to rise dramatically over the next three decades, requiring a concurrent increase in food production (FAO, 2011). Consequently, there is growing interest in the use of plant biotechnology to increase crop yield, often through genetic manipulation to include desirable traits such as resistance to pests, drought and herbicides (Halford, 2012). However, modifying the plant genome necessitates the stable expression of transgenes in the plant, whereas it may be desirable for proteins to only be present transiently. Working with Bayer CropScience, the aim of this PhD project is to develop a system utilising type III secretion systems in order to deliver proteins directly to the plant, avoiding the laborious process of creating transgenic plant strains. The virulence of the plant pathogen Pseudomonas syringae relies on type III secretion system (T3SS)-mediated delivery of effector proteins directly into plant cells. To avoid the disease-causing phenotype conferred by P. syringae, this project will utilise a strain of the non-pathogenic bacterium P. fluorescens that has been previously engineered to express a functional T3SS of P. syringae origin (Thomas et al., 2009). Studies have shown the utility of this system for non-native effector protein delivery, including those of fungal origin (Upadhyaya et al., 2014). The ability of this system to traverse the thick plant cell wall and the relative ease of genetic manipulation in bacterial cells make T3SSs a potentially powerful mode of heterologous protein delivery, and this project will focus on the delivery of non-effector proteins which may be of agricultural interest, such as those with effects on plant development, immunity and stress tolerance.Initially, the project will focus on the delivery of a single plant-native protein to the model plant species Arabidopsis thaliana. The protein will be expressed as a fusion protein with a P. syringae-native effector carrying the necessary signal for T3SS-mediated translocation into the plant cell. Bacterial expression and secretion, in addition to delivery into A. thaliana and subsequent biological activity, will be then assayed to ensure the system is functional. Should this technique be successful, fluorescent technology will be employed to track in planta localisation and movement of translocated protein, to support understanding of in planta protein movement. Finally, the efficiency of delivering other non-effector proteins into plants will be examined, as well as the possibility of delivery to other plant species, with the view of developing a system in which a gene sequence can be quickly cloned into a plasmid and expressed in P. fluorescens in order to deliver proteins to plants in a cost-effective, rapid manner. References:FAO. (2011) The state of the world's land and water resources for food and agriculture (SOLAW) - Managing systems at risk. Food and Agriculture Organization of the United Nations, Rome and Earthscan, London.Halford, N.G. (2012) 'Towards two decades of plant biotechnology: successes, failures, and prospects.', Food and Energy Security, 1(1): pp 9-28.Thomas, W.J., Thireault, C.A., Kimbrel, J.A., Chang, J.H. (2009) 'Recombineering and stable integration of the Pseudomonas syringae pv. syringae 61 hrp/hrc cluster into the genome of the soil bacterium Pseudomonas fluorescens Pf0-1.', The Plant Journal, 60(5): pp 919-28.

预计全球人口在未来三十年将急剧增加，需要粮食产量同时增加（粮农组织，2011）。因此，人们对利用植物生物技术来提高作物产量越来越感兴趣，通常是通过基因操作来获得所需的性状，如抗害虫、干旱和除草剂（Halford，2012）。然而，修饰植物基因组需要转基因在植物中稳定表达，而蛋白质可能需要仅短暂存在。该博士项目与拜耳作物科学合作，旨在开发一种利用 III 型分泌系统的系统，以便将蛋白质直接输送到植物中，从而避免创建转基因植物菌株的繁琐过程。植物病原体丁香假单胞菌的毒力依赖于 III 型分泌系统 (T3SS) 介导的效应蛋白直接递送到植物细胞中。为了避免丁香假单胞菌带来的致病表型，该项目将利用一种非致病性细菌荧光假单胞菌菌株，该菌株先前已被改造为表达丁香假单胞菌来源的功能性 T3SS（Thomas 等，2009）。研究表明该系统可用于非天然效应蛋白递送，包括真菌来源的效应蛋白（Upadhyaya 等，2014）。该系统能够穿越厚厚的植物细胞壁，并且在细菌细胞中相对容易进行遗传操作，使 T3SS 成为一种潜在的强大的异源蛋白质递送模式，该项目将重点关注可能具有农业意义的非效应蛋白的递送，例如那些对植物发育、免疫和胁迫耐受性有影响的蛋白。最初，该项目将重点关注向模型植物物种递送单一植物天然蛋白。 拟南芥。该蛋白将表达为与丁香假单胞菌天然效应子的融合蛋白，该效应子携带 T3SS 介导的易位进入植物细胞所需的信号。然后，除了输送到拟南芥和随后的生物活性之外，还将分析细菌的表达和分泌，以确保系统正常运行。如果这项技术成功，将采用荧光技术来追踪易位蛋白在植物体内的定位和运动，以支持对植物体内蛋白质运动的理解。最后，将检查将其他非效应蛋白递送到植物中的效率，以及递送到其他植物物种的可能性，以开发一种系统，在该系统中，基因序列可以快速克隆到质粒中并在荧光假单胞菌中表达，以便以经济高效的方式将蛋白质递送到植物中，快速的方式。参考文献：粮农组织。 (2011) 世界粮食和农业土地和水资源状况 (SOLAW) - 管理面临风险的系统。联合国粮食及农业组织，罗马和 Earthscan，伦敦。哈尔福德，N.G. (2012)“植物生物技术的二十年：成功、失败和前景。”，食品和能源安全，1(1)：第 9-28 页。Thomas, W.J.、Thireault, C.A.、Kimbrel, J.A.、Chang, J.H. (2009) '丁香假单胞菌 pv. 的重组工程和稳定整合。丁香菌 61 hrp/hrc 簇进入土壤细菌荧光假单胞菌 Pf0-1 的基因组中。'，《植物杂志》，60(5)：第 919-28 页。