24The rhizobium-legume symbiosis is a major source of fixed nitrogen (ammonia) in the 25 biosphere. The potential for this process to increase agricultural yield while reducing the reliance 26 on nitrogen-based fertilizers has generated interest in understanding and manipulating this process. 27For decades, rhizobium research has benefited from the use of leading techniques from a very 28 broad set of fields, including population genetics, molecular genetics, genomics, and systems 29 biology. In this review, we summarize many of the research strategies that have been employed in 30 the study of rhizobia and the unique knowledge gained from these diverse tools, with a focus on 31 genome and systems-level approaches. We then describe ongoing synthetic biology approaches 32 aimed at improving existing symbioses or engineering completely new symbiotic interactions. The 33 review concludes with our perspective of the future directions and challenges of the field, with an 34 emphasis on how the application of a multi-disciplinary approach and the development of new 35 methods will be necessary to ensure successful biotechnological manipulation of the symbiosis.
SYMBIOTIC NITROGEN FIXATION: CHALLENGES AND PROSPECTS 60Biological nitrogen fixation (BNF) is an agriculturally and ecologically crucial process that 61 is performed by prokaryotes in two ecological groups: i) BNF performed by free-living cells often 62 in close association with plants, or ii) by rhizobia that fix nitrogen during an endosymbiotic 63 relationship with legumes. The fixation of N2-gas into ammonia by root-nodule bacteria (rhizobia) 64 is referred to as symbiotic nitrogen fixation (SNF), and it is a more efficient process in terms of 65 supplying nitrogen to the plant. Phylogenetically, most rhizobia are α-proteobacteria, but some 66 rhizobia are β-proteobacteria ( Figure 1). The various genetic, biochemical, and evolutionary 67 aspects of the symbiotic interaction have been reviewed over the years (Long 1996, Gage 2004, 68 MacLean et al. 2007, Jones et al. 2007, Gibson et al. 2008, Oldroyd and Downie 2008, Masson-69 Boivin et al. 2009, Downie 2010, Oldroyd et al. 2011, Udvardi and Poole 2013, Haag et al. 2013, 70 Remigi et al. 2016, Mus et al. 2016, Poole et al. 2018. In brief, the symbiosis is initiated following 71an exchange of signals between the roots of the plant and free-living rhizobia in root-proximal soil 72 (the rhizosphere). As root nodule tissue develops (Figure 2), the rhizobia enter this specialized 73 tissue through an extracellular infection thread; as these inwardly growing threads reach 74 differentiated nodule cells, the bacteria become surrounded by a plant-derived membrane and 75 taken up into the plant cytosol, where they are known as bacteroids. The nascent bacteroids then 76 undergo major morphological and transcriptional changes, leading to active nitrogen fixation, 77 which is the conversion of atmospheric N2 into NH3 for the plant. 78It has been estimated that BNF contributes several teragrams of nitr...