Engineering synthetic signalling between plants and microbes

Plant roots are critical for the uptake of mineral nutrients by plants. In addition, they interact with the soil environment and a complex assemblage of bacteria, fungi, single celled animal cells, nematodes and other organisms. Bacteria are simple single celled microorganisms that lack the membrane bound structures found in higher cells of plants and animals. However, while bacteria may have a less complex cellular organisation, they carry out a huge range of chemical reactions not found in plants and animals. Bacteria are responsible for the cycling of many nutrients such as N2 (N2 is also known as nitrogen gas and consists of two nitrogen atoms bound by a strong triple bond), which is a very inert atmospheric gas. N2 makes up 78% of the atmosphere but is very unreactive and cannot be used directly as a source of nitrogen, which is needed for amino acid, protein and DNA synthesis. However, a small number of bacteria can reduce (add hydrogen) to N2 and convert it into ammonia (NH3), which is readily incorporated into amino acids and then all the other building blocks of life, by a wide range of organisms including bacteria and plants. In many parts of the world the limitation to growth of plants, which in turn support animal life, is the supply of nitrogen as ammonia or nitrate. In the past, much of the nitrogen was provided by biological nitrogen fixation, particularly by a group of plants known as legumes. The legumes form nodules on their roots which house bacteria, called rhizobia, which reduce N2 to ammonia and supply it to plants in return for a carbon and energy source. This legume-rhizobia symbiosis is responsible for providing up to 50-60% of the biosphere's biologically available nitrogen (i.e. ammonia) and is therefore essential to life on earth. However, in spite of the importance of legumes more recently their use has declined and nitrogen is mainly provided to crops by chemically synthesised fertiliser. This has major negative impacts on the environment as much of this nitrogen is lost to the environment as pollution causing algal blooms and contributing to greenhouse gases. Part of the decline in legume use is also because cereals such as wheat, maize, rice and barley have much higher grain yields than legumes and modern agricultural practise has been optimised for their growth. We are therefore developing ways to use bacteria that reduce N2 to ammonia (also called nitrogen-fixing bacteria) to inoculate cereal roots to enable the plants to obtain ammonia without external fertiliser application. To control this process, we have developed plants that produce a signal, called rhizopine, that the bacteria on their roots can detect. We are now developing the control systems to fine tune this process so that rhizopine is able to control the synthesis and secretion of the ammonia by the bacteria to feed the roots of plants.

Using legumes in crop rotation can be of great benefit, but agricultural productivity depends on high-yielding cereals. Attempts are therefore underway to utilise N2-fixing bacteria on roots or inside engineered nodules of cereals to provide ammonia to plants, in order to reduce external fertiliser application. Since, currently there is no means of control of bacterial infection of cereals, N2-fixation and N release from bacteria to plants, it is not possible to prevent promiscuous transfer of N2-fixing bacteria to provide N for weeds. This led us in previous work to engineer Medicago truncatula and barley to produce a unique signalling molecule, rhizopine (an aminated inositol derivative), which can regulate gene expression in microbes containing the MocR regulator. This opens up entirely novel approaches to controlling interactions between plants and their microbiome. To maximise control of rhizopine signalling an extensive toolkit for rhizopine-dependent expression of target genes will be developed. This includes a library of fine-tuned -10 promoter variants of PmocB and PmocD to widen the induction range, amplifier circuits using TraR1 and T7 polymerase for increased induction and repression circuits using TetR, LacI or dCas9 to reduce gene expression. These tools will allow optimised rhizopine-dependent expression of nifA and partial repression of glnA in Azorhizobium caulinodans ORS57, promoting N2 fixation and ammonia secretion. The ability of this strain to promote growth of rhizopine-secreting barley will then be determined in sterile medium and soil. A flavonoid-independent nodDFI under rhizopine control will be used to induce lipochitooligosaccharides (LCO) production, creating a two-way signalling cascade from plant to bacteria and back again. Rhizopine control of bacterial production of LCO and chitooligosaccharides (CO) signalling molecules lays the foundation for two-way coordinated control of plant responses to engineered symbiotic bacteria.

We have established a novel rhizopine signalling pathway by which plants can control bacteria within their root microbiome. Our objective is to develop fine-tuned pathways for induction and repression of genetic circuits in bacteria. This will enable plants to use rhizopine to control expression of plant growth promoting properties in the bacteria. To make full use of synthetic signalling between plants and bacteria we will also develop a reciprocal rhizopine induced signal from the bacteria to the plant based on lipochitooligosaccharide and chitooligosaccharides synthesis using rhizobial nodulation genes. Our initial aim is to develop Azorhizobium caulinodans ORS571 as a model to establish a nitrogen-fixing symbiont for use in provision of nitrogen to cereals. This can be as an epiphyte on the surface of roots, as an endophyte inside roots or, for a long-term aim, as a symbiont inside engineered cereal nodules. While this is undoubtedly ambitious it represents a grand-challenge for scientists to reduce nitrogen fertilizer application. This is because the use of massive fertilizer inputs into agriculture is the main reason reactive nitrogen in the environment is at double its preindustrial level and now beyond the safe operating boundary of the earth. It has led to widespread pollution of groundwater and ocean coastal zones by nitrates leading to eutrophication and costal dead zones. Nitrogen runoff from chemical fertilizer is a leading player in the perfect storm, demanding increased agricultural production but requiring changes in agricultural practice to avoid environmental carnage. Developing control mechanisms to enable cereals to control bacteria and their fixation of N2 is a critical part of enabling more efficient provision of nitrogen to crops in a sustainable way. This in turn can help to reduce the over use of chemical fertilizers and mitigate the negative environmental impact. Furthermore, in a regulatory environment where less fertilizer and pesticide use are becoming mandatory, this work will offer in the long-term tangible results to help meet these targets and assist the competitiveness of UK industry. We will maximize the potential impact of our research by directly engaging with a range of stakeholders, including crop breeders, policymakers and farmers via our existing knowledge transfer networks, including the UK Wheat Genetic Improvement Network (WGIN), landowner/farmers groups (e.g. NFU, SNFU, NFUW, HCC, Growers association, Soil Association), academic societies (BES, BSSS), conservation bodies and local/national government departments (Defra; EA), agencies (SEPA; Natural England; Natural Resources Wales, SNH). We will also expand our current collaborations with our industrial partners, Legume Technology and Pivot Bioscience as part of considering how engineered microbes might be used as inoculants. Reducing inputs into agriculture while maintaining yields has direct benefits to British farming but also to maintenance of the countryside and its use and recreation by the British public. It will help the UK meet local and European environmental targets and help the long-term sustainability and stability of our environment. We will also be training the next generation of scientists to develop practical solutions to environmental problems and develop microbes for the provision of nitrogen to crops and promote plant growth.