ENGINEERING SYNTHETIC SYMBIOSES BETWEEN PLANTS AND BACTERIA TO DELIVER NITROGEN TO CROPS

Nitrogen is an essential element of biological molecules and life on earth. Lack of usable
nitrogen limits growth of microbes, plants, and animals. Lack of nitrogen in agricultural
soils limits plant production of food, feed, fiber and fuel. Nature solved the nitrogen limitation
problem via evolution of biological nitrogen fixation in diazotrophic bacteria that reduce
atmospheric N2 to NH3, which is readily assimilated into biological molecules. Biological
nitrogen fixation is catalyzed by a complex metalloenzyme called nitrogenase whose oxygen-sensitivity
may explain its restricted distribution amongst prokaryotes. Some plants, including most
legumes and a few non-legumes form intimate, nitrogen-fixing symbioses with diazotrophs that
provide the plants with ammonia. As a consequence, legumes have been an integral part of
sustainable agricultural systems for thousands of years. Unfortunately, many important food
species, including the grasses maize/corn, rice, and wheat cannot establish effective nitrogen-fixing
symbioses with diazotrophs, which means that they are dependent on nitrogenous fertilizers
for high yield. Large-scale use of industrially-produced N-fertilizer has doubled the influx
of N into the terrestrial biogeochemical N-cycle, with serious negative consequences for human
health and the natural environment. Therefore, the long-term sustainability of massive N-fertilizer
inputs in agriculture has come into question.
A team of six investigators has come together to solve the dual nitrogen problems of N-fertilizer
over-use in developed countries and soil N-paucity in developing countries by developing effective
endophytic and associative nitrogen-fixing symbioses in a model and a crop plant species.
The team brings together expertise in bacterial and plant genetics, genomics, biochemistry,
molecular and cell biology, physiology and synthetic biology with a deep knowledge of biological
nitrogen fixation. The overarching goal of the project proposed here is to develop effective
N2-fixing symbioses between the model C4-grass, Setaria viridis, or the related crop species,
Zea mays, and the endophytic bacterium, Rhizobium sp. IRBG74, or the associative bacterium,
Pseudomonas fluorescenes Pf5. Successful deployment of biological nitrogen fixation in model
or crop grass species will pave the way for a second Green Revolution that will increase crop
yields for resource-poor farmers and decrease the use and environmental-impact of industrial
N-fertilizers by wealthier farmers.

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.