EASTBIO Targeted genome editing to replace nuclear-encoded Rubisco in rice

Second Supervisor: Dr Elizabete Carmo-Silva, Lancaster University

Overview. Ribulose 1,5-bisphosphate carboxylase/ oxygenase (Rubisco) is the driving force behind CO2 assimilation in all plants. Despite its importance, Rubisco is a relatively slow and error-prone enzyme, and thus a key limiting factor for growth, resilience and productivity. This is particularly evident in key staple C3 crop plants, such as rice, where increasing the operating efficiency of Rubisco and reducing photorespiration are considered promising strategies for improving crop yields. Much work has focused on improving our understanding of the assembly, structure and regulation of this enigmatic enzyme and how we might tailor the performance of Rubisco for crop plants. Two recent developments have helped to open up exciting new avenues to explore Rubisco: i) improvements in our understanding of the natural variability of the catalytic traits of Rubisco and the chemical mechanism of its carboxylation and oxygenation reactions, and ii) advances in our capacity to assemble plant Rubiscos in vitro (i.e. in E. coli (1)) and engineer Rubisco in planta. In particular, the small subunit of Rubisco (SSU) is receiving increasing recognition as an important component regulating the abundance and catalytic characteristics of Rubisco (2). Furthermore, the SSU is an excellent target for nuclear gene editing (e.g. using CRISPR/Cas approaches).

Project Plan. Rice is arguably the most important C3 crop for global food security. Previous exciting work has shown that overexpression of heterologous or native SSUs in rice can modify the catalytic properties and abundance of Rubisco (2). Rice is fully sequenced and diploid, amenable to tissue culturing, and high editing efficiencies have been reported using CRISPR/Cas (3). In this project you will employ the latest molecular tools to modify rice Rubisco and investigate the extent to which its catalytic characteristics can be enhanced or modified for building pyrenoid-based CO2-concentrating mechanisms components (see McCormick lab for further details on the latter). You will generate and characterise mutant rice lines and explore the molecular and physiological implications of modifying Rubisco in this important food crop. This approach will provide an unprecedented capacity to evaluate the role of Rubisco SSU in rice and could open up the prospect of enhancing crop yields in the future through a novel genetic tailoring strategy.

Training: The project is an outstanding training opportunity for developing a multidisciplinary approach to plant science with two leading UK plant science research labs in Edinburgh and Lancaster. You will develop expertise in a wide range of molecular and whole plant analysis skills, including DNA, RNA and protein analyses (e.g. qRT-PCR, Western blot, enzyme activity assays), and photosynthetic physiology using leaf gas exchange and fluorescence techniques. You will gain experience in synthetic biology-based approaches by generating and working with transgenic rice lines using CRISPR/Cas. This work will provide important new information for both fundamental and applied research about the regulation and makeup of Rubisco in rice and the role of the SSU in growth and development. You will interact with crop scientists to develop strategies to take important findings through to application in crops. There will also be abundant opportunity for you to present your research at regular meetings, including national and international conferences.