Genetic Determinants of Microbiome Assembly on Plant Roots

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. The area directly around roots that is occupied by these organisms is known as the rhizosphere and the collective name for the organisms is the rhizosphere microbiota. Microorganisms also reside inside plant roots, usually between plant cells and are knows as endophytes. Together the rhizosphere and endosphere microbiotas makes up the root microbiota of a plant. It has been shown over the last few years that the root microbiota is critical for the health and growth of plants, with many microorganisms shown to be plant growth promoting. 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. Other bacteria are crucial to make phosphate available to plants which with nitrogen are the two main nutrients limiting plant growth. However, we now realise that bacteria do not work alone but rather they work as complex communities, also known as a microbiome, with hundreds or even thousands of members interacting with each other and host plants. Just as for the human gut and body the health and growth of plants is profoundly altered by the bacterial microbiome. A recent breakthrough is the demonstration that we can study how microbiomes develop using just 6 or 7 key members which simplifies the analysis. In this proposal we have developed ways to track how 7 bacteria interact and to study the underlying genetic causes. This enables us to move beyond simple characterisation of the components of the microbiota to examine the genetic mechanisms of control and how a microbiome stably colonises plant roots. This research will lead to a step change in characterisation of plant microbiota of agriculturally critical crops, including cereals such as barley and wheat.

Bacterial communities are crucial to plant growth as shown by numerous phylogenetic analyses of plant roots revealing bacterial communities are strongly shaped by the plant and in turn bacteria act at the community level to promote growth, and for example, can establish suppressive communities that inhibit pathogens. What is missing are molecular studies on how bacterial microbiomes assemble on roots and interact with each other and the plant, leading to mechanistic understanding of microbiome assembly. The problem is the scale of complexity and diversity of bacteria that colonise roots. However, this has been transformed by recent advances showing that the underlying principles of microbiome assembly can be determined using bacterial Synthetic Communities (SynComs) of 6-7 members. However, for molecular analysis of even 7 membered SynComs there needed to be a new raft of imaging and analysis techniques to facilitate their study. We therefore developed differential fluorescence measurement (DFM), of up to 7 different bacteria, adapted the global bacterial mutagenesis strategy, Barseq, and established methods for meta-transcriptional analysis of bacterial communities enabling elucidation of the genetic determinants of microbiome assembly during root colonisation This transforms our ability to study communities of bacteria with profound implications for understanding how bacteria promote or inhibit plant growth and colonise roots. It can lead to understanding of how to rationally develop inocula for plants that will be stable in the environment. Given the recent enormous international investment in bacterial inoculants for plant growth makes understanding the basic biology of microbiome assembly particularly timely. However, the techniques will be applicable to all areas of animal, plant and human microbiome analysis so that its importance to both fundamental and applied biology is difficult to overstate

Within this proposal, we will extend established work within our groups to develop techniques to identify the genetic determinants of root colonisation and stability of the microbiome. This is particularly important to plant growth, nitrogen and phosphate utilisation, but also has relevance to disease resistance and herbicide and pesticide use. In the bigger context the importance of the microbiome in plant growth and for agrochemical exploitation has recently been recognised. Large scale international funding has gone into this area with companies such as Bayer and Ginko forming Joyn Bio and start-ups including AgBiome and PIVOT focussed on microbial inocula and consortia for plants in agriculture. This is particularly important to nitrogen and phosphate utilisation and also to disease resistance and herbicide and pesticide use in plants. Nitrogen is at double its preindustrial level and now beyond the safe operating boundary of the earth, with widespread pollution of groundwater and ocean coastal zones by nitrates and phosphates leading to eutrophication and costal dead zones. These nutrients are leading players in the perfect storm, demanding increased agricultural production but requiring changes in agricultural practice to avoid environmental carnage. Understanding how synthetic community of bacteria (SynComs) assemble and are stabilised is a powerful resource for the use of microbes to help tackle these issues and therefore has over-arching relevance to society and government policy. Furthermore, in a regulatory environment where less fertilizer and pesticide use are becoming mandatory, this work will offer tangible results to help meet these targets and assist the competitiveness of UK industry. Currently we have strong links with UK Legume technology, including two iCase Ph.D students, working on legume and other inoculants. 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), who will directly benefit from our findings. 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 stable microbial inoculants.