Engineering synthetic symbioses between plants and bacteria to deliver nitrogen to crops

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We propose to develop a novel model system using the C4-grass Setaria viridis (Setaria), and its interaction with both a model endophyte (Rhizobium sp. IRBG74) and an associative bacterium, (Pseudomonas fluorescens Pf-5). Synthetic biology will be used to genetically alter Setaria and the two bacteria to ensure a lock and key interaction between plant and microbe, while maximizing nitrogen fixation by the bacteria and delivery of ammonium to the plant. A genetic system will be introduced into the bacteria that responds to environmental signals, a plant metabolite, and nutritional needs. These sensors will be integrated by synthetic circuits and used to control the expression of nod genes and the production of ammonia via a heterologous refactored nitrogen fixation pathway. Using a refactored system will enable the induction of this pathway in response to desired signals and will eliminate unwanted regulation, for example by oxygen and ammonia. This will be accomplished by designing assemblies of nif genes based on the nif gene clusters of model free-living nitrogen fixers exploiting the most robust nitrogen-fixing metabolism that allows oxygen sensitive nitrogenase to function during aerobic respiration.
A dual approach using an endophyte and an associative bacterium is proposed because the challenges to optimise nitrogen fixation faced by endophytes and associative bacteria are similar. Both approaches are essential because whether a nitrogen-fixing endophyte or an associative bacterium is ultimately deployed in the field will depend on different circumstances. Finally, while we will develop a model system based around Setaria in parallel we will genetically manipulate the ancient and important crop species, Zea mays (maize). This will allow us to advance from proof-of-concept to practical application in a crop species that is central to agriculture in both the developing and developed world.

Nitrogen is one of the main constraints on agricultural productivity so its use is essential for high crop yields. In a world where food security is now considered a national priority crop yield is of critical importance. However, the drive for yield alone has led to very high application of nitrogen with consequent nitrate contamination of groundwater and problems of eutrophication. The problem is so serious that reactive nitrogen in the biosphere has doubled from preindustrial levels primarily through massive inputs into agriculture. It also results in the production of N2O which is around 300 times more potent than CO2 as a greenhouse gas. These are problems of regional, national and international scope that require urgent amelioration and are at the forefront of the grand challenges for UK science. By improving our understanding of how rhizobia develop into N2 fixing bacteroids in legume nodule we acquire the understanding to improve the competitive success of desirable strains of Rhizobium. It also lays down a foundation of understanding for the transfer of bacteria to nodules in other plants such as cereals. These aims are long term but ultimately this work has relevance to farming practice as well as government policy in decisions about nitrogen utilization in agriculture. It is also relevant to UK attempts to reduce greenhouse emissions and produce a low carbon economy. Understanding the nitrogen fixation and its role in the nitrogen cycle in agricultural has wider benefits applicable to the UK public because of its importance in food security and meeting international obligations for mitigating the effects of climate change. We propose to reach a wide audience of farmers, the public, national and international policy makers and charitable institutions through active outreach (Friends of John Innes). We also have strong links with "The Nitrous Oxide Focus Group" and the newly formed "Consortium for Legumes in Agriculture, Society and Environment", which is an international consortium to promote understanding on the use of legumes. In addition we have broad links to the environmenral impact of this work through the Earth and Life Systems Alliance between JIC and UEA (ELSA) and to the UK government via the "Living With Environmental Change program (LWEC)" which has its secretariat at UEA.