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Review on Development of Nitrogen Fixing Rhizobia Through Adaptive Evolution

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Published: 8th Feb 2020 in Biology

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Nitrogen is a building block of life. Molecular nitrogen is a relatively inert form of atmospheric nitrogen and it must be fixed into biologically accessible form in order to be used for organic processes. The root nodule symbiosis established between legumes and rhizobia is an important biological interaction responsible for fixing a significant amount of atmospheric nitrogen. The transfer of symbiotic proficiency between phylogenetically distant bacteria requires genome remodeling and this is supported by experimental evolution strategies, converting soil bacteria into legume symbionts. This approach could enhance global food productivity and environmental security. Since this is an area that has a great deal of interest, in this review recent progress in the understanding the process of Legume-Rhizobium symbiosis as well as adaptive evolution strategies will be discussed.

  1. Introduction

Nitrogen is an essential plant nutrient that is required for plant growth. Although nitrogen is abundant in the earth’s atmosphere but is metabolically unavailable to the higher plants. Nitrogen use efficiencies of crop plants indicated that about 66% of this nitrogen is lost from the soil-plant system (Raun and Johnson 1999) either in the form of nitrous oxides, which are potent greenhouse gases, or as soluble nitrates that find their way into aquatic systems (Glendining et al. 2009). This is not only causing pollution to the environment but also causing an annual economic loss of US $3 billion (Saikia and Vanita 2007). In addition, large scale production of nitrogen fertilizers is energy-intensive process and requires the use of fossil fuels in agriculture (Glendining et al. 2009). For all these reasons, it is desirable to reduce agricultural reliance on nitrogen fertilizers.

However, to reduce reliance on chemical fertilizers, it is necessary to cope with the predicted requirement of increasing agricultural productivity to meet the demands of a growing global population (Beddington 2010). Even today, nearly a billion people remain chronically malnourished across the globe after the agricultural intensification (Foley et al. 2011). The primary reason behind this is the limitations in the supply of nitrogenous fertilizers (Mueller et al. 2012). Increasing crop yields through the increased availability of nitrogen to crops will help to meet the global demand for increased food production (Tilman et al. 2011).

Though plants cannot use atmospheric nitrogen directly, but some bacteria and archaea have evolved the capability to convert atmospheric nitrogen into ammonia, a form readily usable in biological processes.

  1. Biological Nitrogen Fixation

Biological nitrogen fixation (BNF) is the process by which nitrogenous compounds are synthesized from atmospheric N2 by the activities of certain symbiotic bacteria. These bacteria are associated with leguminous plant forming nodules on their roots. In total, approximately 239 teragrams of nitrogen is fixed annually through the process of BNF (Galloway et al. 2008). Biological nitrogen fixation is an ecologically safe alternative but is restricted to the symbiotic interaction of a small group of plants (mainly legumes) with nitrogen-fixing microorganisms (Clua et al. 2018). It is a low-cost strategy for improving crop productivity (Ramaekers et al. 2013).

Legume crops play a significant role in providing nutritional security and sustainable agriculture worldwide. Among all the food legumes, dry beans (all species of Phaseolus) cover 46% of total area followed by chickpea (18%) and cowpea (18%). In terms of total production also, dry beans dominate at 46%, followed by chickpea with 22% of the world total production (Das et al. 2017).


  1. Rhizobia

Rhizobia are Gram-negative α- and β-proteobacteria that have ability to establish nitrogen fixing symbioses with legumes (Remigi et al. 2016). All the rhizobia have Mo-Fe containing variant of the nitrogenase enzyme system, which is the only biological system able to fix nitrogen (Raymond et al. 2004). In legume-rhizobium symbiosis, the interaction is based on the capacity of rhizobia to convert atmospheric N2 into usable form that can be utilized by the plants. Currently, rhizobia belong to 12 genera and have more than 70 species. They have large complex genomes (5.4-9.2 Mb) with one to seven replicons called megaplasmids. Among the most studied symbiotic bacteria are Rhizobium leguminosarum (pea), Bradyrhizobium japonicum (soybean), Sinorhizobium meliloti (alfalfa)and Mesorhizobium loti (lotus)(Hayat et al. 2008). Owing to their relevance in agriculture, most of the research is focused on rhizobia (Masson-Biovin et al. 2009).

  1. Host Range Specificity

Nodulation in legumes evolved as an interaction between the legumes and soil bacteria is highly specific. Nod factor recognition is a key determinant of host range specificity. Rhizobial genes involved in curling the root-hairs of legumes (nodDABC) are physically and functionally served in different Rhizobium species (Lewin et al. 1987). Loss of the ability to produce either Nod factors or flavonoids prevents nodulation. The effective rhizobial stain has to compete with the other indigenous bacteria present in the rhizosphere. Legumes will encounter a more favorable rhizobial strains (Zhao et al. 2017). Effective strains of rhizobia help to form effective nodules which are pink in colour indicating the presence of leghaemoglobin. The ineffective rhizobial strains form ineffective nodules which are small and contain poorly developed bacteroid tissue showing accumulation of starch in host cells (Kukkamalla 2016). The mechanisms by which rhizobia and legume choose their partners are not fully understood. Some authors suggest that the evolution of legume-rhizobium association may be host driven (Sachs et al. 2011). However, legumes can be nodulated by rhizobia even if the association results in low symbiotic nitrogen fixation (Den Herder and Parniske 2009).

  1. Legume- Rhizobium Symbiosis

The legume-rhizobium symbiosis has been widely studied as a model of mutualistic associations and beneficial for sustainable agriculture. The increasing global concern regarding the production of enough food to uphold the growing human population has been reinforcing the importance of sustainable intensification of plant production. Some bacteria are able to promote plant growth through different mechanisms and they can do so endophytically, in symbiosis or as free-living cells (Glick, 2012). Several plants species, mostly legumes, facilitate colonization by nitrogen-fixing rhizobia bacteria and form specialized organs called nodules (Rogers and Oldroyd, 2014). These specialized organs accommodate the bacteria, exhibit an extremely low free oxygen atmosphere and synthesize a form of plant hemoglobin called leghemoglobin, that facilitates O2 diffusion to bacteroids (Masson-Biovin  and Sachs 2018). In most of the rhizobia, mainly set of two genes encode for these functions known as the nod (nodulation) genes and the nif (nitrogen fixation) genes. The formation of symbiotic N2-fixing nodules requires two developmental processes: bacterial infection and nodule organogenesis, which occur in the epidermal and cortical cell of the root respectively. Rhizobium symbiosis with legume species is of special importance, producing 50% of 175 million tons of total biological nitrogen fixation annually worldwide (Yadav et al. 2010).

  1. Signal exchange in the rhizosphere

The symbiotic interaction is initiated after an initial exchange of signals between the legume host and the rhizobia. When nitrogen present in the soil is scarce, legumes secrete a series of phenolic compounds into the rhizosphere, mainly flavonoids (Peters et al. 1990). These molecules are recognized by rhizobia, activating the transcriptional regulator nodD protein, which in turn activates the transcription of genes required for the synthesis of the Nod factor (D’Haeze et al. 2002). Nod factors are lipochito-oligosaccharides consisting of 4-5 residue chitin backbone of β-1,4-linked N-acetyl-D-glucosamine subunits with an N-linked acyl tail attached to the non-reducing end secreted by rhizobia. Nod factors are perceived by receptors present on the plasma membrane of root cells. Perception of Nod factors is necessary and usually sufficient to initiate the molecular and physiological responses in the plant, such as calcium spiking, accumulation of early nodulin transcripts (i.e., genes induced in the plant during nitrogen-fixing symbiosis), root hair curling and the formation of the infection thread, where bacteria can divide and grow towards the cortical cells (Gage et al. 2004). This perception leads to development of two distinct cell types- the development of infection thread (IT) in root epidermis and the initiation of cell divisions in the root cortex (Madsen et al. 2003). The IT carrying growing bacteria develops from the root tip extending towards the root cortex and releases the bacteria in the newly developing nodule (Arrighi et al. 2006). In the nodule, bacteria differentiate into bacteroids (Smit et al. 2007). Bacteroids are the endosymbiotic form of rhizobia, which can reduce atmospheric nitrogen into ammonia to support plant growth and obtain carbon source from plant hosts to maintain cellular activity (Zhao et al. 2017). The symbiosis of legume-rhizobium also depends upon the production of Exopolysaccharides (EPS). Rhizobia are able to produce large amount of EPS. EPS are the active signaling molecules for the release of bacteria from infection threads (Mukherjee et al 2011). The production of a variety of symbiotically active polysaccharides may allow rhizobial strains to adapt to changing environmental conditions and interact efficiently with legumes.

  1. Transfer of Symbiotic Traits

Horizontal gene transfer (HGT) plays a key role in the biodiversity and ecology of bacteria by contributing to their adaptability, fitness, and competitiveness. Evolutionary trends show that there are three genetically different nodulation strategies by which the nodulation traits have evolved in the rhizobia. The nodulation strategies identified in rhizobia to date are the Nod, T3SS and non-Nod/non-T3SS strategies. How these strategies emerged and in which order is still a challenging question. Among the several methods available for inducing nodule formation, the Nod strategy has been most successful as it is used by most of the rhizobia. Other reason for the success of this strategy can be the nod and nif genes which are tightly linked on mobile genetic elements (Lee et al. 2008). Gene acquisition comprises mobile genetic elements (MGEs), such as plasmids and genomic islands. Large MGEs can provide novel traits, such as virulence and mutualistic traits that enable bacteria to interact with eukaryotic hosts. During the free-living rhizospheric stage of bacteria, transfer of MGEs is predicted to be most frequent (Edwards et al. 2015). The first step on the way to symbiosis is transfer of a limited set of key genes probably coupled with the genetic renovating of the recipient genome. This ecological switch typically occurs through the acquisition of horizontally acquired genes.

Rhizobia, are an excellent living system to investigate the molecular mechanisms underlying post- Horizontal Gene Transfer adaptation. These bacteria are facultative endosymbionts that can alternate between a saprophytic life in the soil and a symbiotic life when they come across a compatible legume host (Masson Biovin et al. 2009).The horizontal transfer of essential symbiotic genes, the nod and the nif genes that are carried on plasmids or genomic islands, has played a major role in spreading symbiotic proficiency among diverse group of soil bacteria (Nandasena et al. 2007).

  1. Adaptive Evolution

Experimental evolution studies suggest that a step by step evolution, including MGE transfer followed by genomic remodeling under plant selection pressure (Fig 1), is supported by the existence of clear phenotypic shifts driven by adaptive mutations (Capela et al. 2017). Various phylogenetic studies have suggested that successful nod and nif transfers had been more frequent within genera but rare between genera (Remigi et al. 2016).

Figure 1: Model for the evolution of N2 fixing rhizobia.  nod/nif-containing MGEs are horizontally transferred to diverse soil bacteria, conferring symbiotic potential that depends on the recipient genome (Taken from Masson Biovin et al. 2009).

An experiment conducted by Marchetti et al. (2014) tentatively replays the colonization of a new bacterial genus by a nod–nif symbiosis module. Two members of the β-proteobacteria, the Mimosa symbiont Cupriavidus taiwanensis and the root-infecting pathogen Ralstonia solanacearum, were selected to act as symbiosis gene provider and recipient, respectively. The experiment uses a two-step strategy imitating natural events: horizontal transfer of key genes followed by symbiotic adaptation under legume selection pressure. This experiment has revealed the strong selection imposed by the legume for nodulation and intracellular infection, and a mechanism facilitating the adaptive evolution of the recipient genome (Remigi et al. 2014).

  1. Problem Statement

Currently, there is no effective rhizobial strain available in North America that can form symbiotic association with Phaseolus vulgarisPhaseolus vulgaris, also known as dry-bean, is an important legume that is grown in North America as major source of dietary protein.  There are different bacterial species that can form effective nitrogen fixing nodules on beans, with Rhizobium etli generally being considered as the predominant species (Peèrez-Ramèrez et al. 1998). Rhizobium etli is Gram negative bacterium that is capable of entering into a symbiotic relationship with Phaseolus vulgaris providing the plant with a source of biologically reduced nitrogen.  R. etli CFN42 is a sequenced strain that is being used as a model organism to study plant-microbe interactions and nitrogen fixation. Although it interacts with current bean cultivars, but it does not form an effective symbiotic association. Therefore, beans that are grown as a crop in Canada are typically fertilized using industrially derived nitrogen fertilizers. 

To solve this problem, as a proof of principle, the adaptive evolution strategies can be used to develop inoculum strains that can be used with varieties of beans that are currently grown in Canada.


In past decades, a lot of research is done on understanding the Legume-Rhizobium symbiosis. Nitrogen fixation is the ultimate adaptive trait that turns an ineffective bacterium into an effective one. Host selective pressures and horizontal gene transfers are key mechanisms that shape the genetic structure of symbiotic microorganisms. Evolution of symbiotic plasmids or symbiotic genes may occur independent of the chromosome and account for plant specificity. Understanding the selective forces that govern emergence of symbiotic nitrogen fixation is crucial for the challenging goal of transferring nitrogen-fixation capacities to different legume plants as well as cereal crops.


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