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

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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.