Factors controlling N2-fixing ability and competitiveness of rhizobia to nodulate legumes

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. Rhizobia have been studied for more than 100 years because of this ability to increase yields of legumes crops and rhizobia are routinely applied as inoculants as an alternative to economically expensive chemical fertilizers. The bioavailable nitrogen that is generated in nodules of legumes benefits nonlegume crops grown in rotation or at the same time. However, rhizobial inoculants that have high rates of N2-fixation (i.e. effective strains) when inoculated onto legumes under laboratory conditions often fail to compete in soil for colonisation of legumes against native rhizobia with inferior N2 fixing abilities. This is known as the "rhizobial competition problem". The holy grail of inoculant selection has therefore been to identify elite strains that are both highly effective and competitive. This competition-effectivity problem is of enormous practical importance to use of legumes, but it also highlights the fundamental biological question of what determines the competitiveness of bacteria for colonisation of plant roots. Understanding rhizobial competitiveness is a therefore a prime example of a question that is of both fundamental and applied importance. For the first time in rhizobial research, we are able to assess the bacterial genetic potential and factors rhizobia need for their competitiveness and N2-fixation efficiency in real soil. Research has been conducted under sterile conditions and/or focusing solely on the nodules. Here we propose to step-by-step fully assess the rhizobial life cycle. Our findings will explain why efficient N2 fixers are not necessarily good colonisers. Identifying the essential genes and regulatory pathways will give us detailed knowledge about strain behaviour in different soils and symbiotic success with different plant varieties, allowing us to better select the best inoculants.

Rhizobia fix N2 once they form bacteroids inside mature legume nodules. N2 fixation is therefore assessed on one rhizobial strain and plant cultivar at a time. To overcome these limitations, we developed a barcoded plasmid library to allow simultaneous assessment of competitiveness and effectiveness of rhizobial strains (BarComp). A nifH promoter-Gfp fusion reports on the N2-fixing ability of up to 96 rhizobial strains in a single experiment. We therefore established an indexed library of 84 rhizobia for competitiveness and effectiveness (PNAS 2020, 117:9822). Alongside BarComp we adapted Insertion Sequencing (InSeq) to allow identification of competitiveness and effectiveness genes for pea nodulation (PNAS 2020, 117:23823). In this proposal we bring all these developments together in SorBarSeq, enabling us to generate mutant libraries where rhizobia recovered from pea nodules have a Gfp read out of their N2 fixation rate. Rhizobia will be FAC sorted into high and low Nif expression libraries, allowing identification of the genetic loci controlling effectiveness. Furthermore, by comparing the loci that alter both competitiveness and effectiveness of several elite strains inoculated on peas in soil we will be able to map the shared determinants of symbiotic traits for the first time. After identifying the genetic determinants for competitiveness and effectiveness their transcriptional networks will be mapped by RNASeq. Next their spatiotemporal timing of action will be determined by Lux mapping of gene expression at multiple stages in the rhizobium infection and nodulation life cycle. Finally, the effects of soil and pea cultivar on rhizobial competitiveness will be determined. The ability to bring together multiple elite strains, growth in soil coupled with SorBarSeq and spatiotemporal mapping is particularly timely and enables the rhizobial competition and effectiveness problems to be analysed as a problem of both applied and fundamental importance.