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

预计未来30年全球人口将急剧增加，粮食产量也将随之增加（粮农组织，2011年）。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，通常是通过基因操纵来增加理想的性状，如抗虫害、抗旱和抗除草剂（Halford, 2012）。然而，修改植物基因组需要转基因在植物中的稳定表达，而蛋白质可能只是暂时存在的。与拜耳作物科学公司合作，这个博士项目的目标是开发一个利用III型分泌系统的系统，以便将蛋白质直接输送到植物中，避免了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进入植物细胞。为了避免紫丁香假单胞菌带来的致病表型，该项目将利用非致病性荧光假单胞菌菌株，该菌株先前已被改造为表达紫丁香假单胞菌起源的功能性T3SS （Thomas et al., 2009）。研究表明，该系统可用于非天然效应蛋白的递送，包括那些真菌来源的效应蛋白（Upadhyaya等，2014）。该系统穿越植物细胞壁的能力以及在细菌细胞中相对容易的遗传操作使t3ss成为一种潜在的强大的异源蛋白递送模式，该项目将重点关注可能具有农业利益的非效应蛋白的递送，例如那些对植物发育，免疫和耐受性有影响的蛋白。最初，该项目将专注于向模式植物拟南芥提供单一植物原生蛋白。该蛋白将以融合蛋白的形式表达，与丁香属植物的天然效应蛋白结合，携带t3ss介导的植物细胞易位所需的信号。细菌的表达和分泌，以及在拟南芥中的传递和随后的生物活性，将被检测以确保系统的功能。如果这项技术取得成功，荧光技术将用于跟踪植物中易位蛋白的定位和运动，以支持对植物中蛋白质运动的理解。最后，将研究将其他非效应蛋白传递到植物中的效率，以及传递到其他植物物种的可能性，以期开发一种系统，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，从而以经济、快速的方式将蛋白质传递到植物中。引用:粮农组织。（2011）世界粮食和农业土地和水资源状况（SOLAW）——面临风险的系统管理。联合国粮食及农业组织，罗马和地球扫描，伦敦。Halford， N.G.(2012)，《走向二十年的植物生物技术：成功、失败和前景》。《粮食与能源安全》，第1期，第9- 12页。Thomas, w.j., Thireault, c.a., Kimbrel, j.a., Chang， J.H.（2009）“丁香假单胞菌的重组和稳定整合[j]。在土壤细菌荧光假单胞菌Pf0-1基因组中发现了丁香61 hrp/hrc簇。”，植物学报，60(5):pp 919-28。

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.