Rhizobium-legume Symbiosis: Molecular Determinants and Geospecificity

Symbiosis in legume plants is an ever evolving research as the factors that initiate, regulate and accomplish the complex relationship between the plant and microorganisms are very dynamic. Rhizobium is a common symbiont in legumes which throughout the process of evolution, has undergone multiple phenotypic and genotypic manifestations. Crop specificity based on the compatible combination of plant and rhizobium is, of course, the modern concept in advanced biofertilizer research. Yet, the biofertilizers are not being considered as replacement of synthetic fertilizers, but just an alternative to the chemical fertilizers. It is clearly evident from both the usage and yielding perspectives that biofertilizers are not the priority of the farming communities. A serious fundamental amendment in the research of biofertilizers is proposed in this review article in the name of Geo specificity. Exploring the natural combination of rhizobium and legume plants with respect to geography and reinoculating in the same geographical region is the central dogma of the concept. Rhizobium of one geography could be sensitive to other geographies even though the crop remains the same. Therefore a new consideration in the biofertilizer research is proposed, presented and illustrated in this review to give more insights about Bio-Geo Specificity.


iNtRODuCtiON
Plant interacting microbes vary in the nature of the relationship they establish with their hosts. For example, pathogenic microorganisms establish interactions that can severely impair the development/survival of the susceptible hosts. In distinction to pathogenic bacteria, symbiotic bacteria example; rhizobia are beneficial to their hosts. Rhizobia, the soil bacteria interact symbiotically with the leguminous plants forming nitrogen-fixing nodules 1 . The process initiates on the root, beginning with the exchange of signal molecules between the host and microbe 2 . It then invades the plant by forming infection threads. Initiation begins from the curled root hair ends and grows towards the plant cortex 3 . Located just in front of these growing threads are the inner cortical cells. Dedifferentiation of these cells leads to the induction of primordium of the nodule. Further, the activity of the nodule meristem is initiated on the outer side of the primordium leading to the further growth of the nodule 4 .
The process of nitrogen fixation occurs naturally by which the atmospheric nitrogen is converted into ammonia (readily available form for plants) with the help of the nitrogenase enzyme 5 . The bacteria present inside the nodules help to fix this nitrogen and the process results in a complex interaction between the host plant and the rhizobia. This mutualism has found to be advantageous for both the host; in which plants provide rhizobacteria with carbon as a source and energy that is necessary for its growth and functions and the microbes, in turn, provide reduced nitrogen to the host in the form of ammonium 6 . This process of nodulation and nitrogen fixing requires several rhizobia bacteria as well as symbiotic genes for effective symbiosis between the host and the microbes. Rhizobial genes present in rhizobacteria will play an important role in nodule development and bacterioid metabolism. Some of them, "nod, nol, noe" genes involves in nodulation, "nif, fix" for nitrogen fixation, and the other plant genes that are expressed in the root tissue are nodulin genes 7 .
One conspicuous feature of legumerhizobialsymbios is a highly specific feature that may occur both at initial and final stages like bacterial infection and nitrogen fixation respectively. Domesticated crop species were found to have higher specificity i.e. fewer compatible symbionts than their wild counterparts 8 . Such constraints could decrease the yield in conditions where no favorable strains are available. This knowledge concerning genetic control of symbiotic specificity would help manipulate the key genetic factors controlling the interaction and improve the agronomic potential of the root nodule symbiosis. Hence the present review will give a brief overview of the various factors accounting for this to the symbiotic specificity and prove that nodules occupy a competitive niche for microbial accommodation.

Review Flavonoid-NodD interaction
In the early course of legume-rhizobial symbiosis, the legumes produce flavonoids, which diffuse across the bacterial membrane resulting in the activation of nodulation genes (nod). These genes encode enzymes that produce bacterial Nod factors, thereby initiating a symbiotic development for the most part of leguminous plants 9 . Once symbiosis is initiated, the process of transcription occurs furthering this mutualism. Flavonoid activates bacterial proteins (NodD) belongs to the LysR family of transcription regulators mediate this transcription of nod genes. These NodD genes bind to the nod boxes (DNA motifs), which are found in the nod genes promoter region 10 .
Broughton and Peck, et al. demonstrated that these NodDs from rhizobial groups respond differently to different flavonoids 11,12 . In view of the fact that legumes produce various types of flavonoids, nod genes that are regulated by NodD will respond to the specific flavonoids at an early stage of symbiosis. For example, mutations in the nodD from R. leguminosarumbv. trifoli results host range extension owing to the expression of nod genes with flavonoid inducers that are generally dormant in nature 13 .The role of NodD regulation by nodD gene expression of Sinorhizobiummeliloti and the mutated expression of nodD of S. meliloti was explained in Peck et al. 12 research. It is henceforth established that flavonoid induced nod gene expression depends on the source of NodD.

Nod factors
The process of nodulation in most part of the legume plants occurs by producing Nod factors. In contrast, photosynthetic bacteria nodulate without producing Nod factors, the reason being the absence of Nod genes 14 . The Nod factors possess a backbone structure called N-acetyl glucosamine oligosaccharide with a fatty chain at the non reducing end which is similar in different rhizobia but vary in size and saturation, including supplementary additions at each ends, such as glycosylation and sulfation 15 . These modifications help us to determine if a particular Nod factor be able to perceive by a particular host 16 .
The nodABC genes usually present in all rhizobium bacteria required for the synthesis of Nod factors (NF's) which are made up of lipo-chitooligosaccharides. A mutation occurs in nodABC genes that interrupt and abolish the entire process of symbiosis. Apart from this, any modifications in the additional genes that determine the Nod factor corealters this specificity. For instance, obliterating the nodE gene present in R. leguminosarum bv. trifolii changes the uniqueness of the fatty acyl chain attached to the Nod factor and this modification affects symbiosis with Trifolium species 17 .
Perception of signals from the Nod factor is mediated by NFRs i.e. Nod factor signals. They are basically serine/threonine receptor kinases containing LysM motifs in the extracellular component 18 . These motifs were demonstrated by molecular and genetic analyses in L. japonicas as host determinants of symbiosis specificity. It was observed that by transferring Lj-NFR1 and Lj-NFR5 receptors into a legume M. truncatula have enabled nodulation by a Mesorhizobium lotisymbiont in the L. japonicus legume plant 19 .

Rhizobial surface polysaccharides & plant lectins
Added classes of bacterial components that are the key factors to interact with the host include the bacterial surface polysaccharides, reported in numerous studies as having a symbiotic role. A various group of bacterial polysaccharides such as lipopolysaccharides (LPS), cyclic glucans, Exopolysaccharides (EPS), and capsular polysaccharides is the bacterial groups of polysaccharides that interact with the host and any defect in any of these components results in the immediate failure of the early or later stages of symbiosis. It was noticed that defects in the EPS production resulted in the arrest at the infection thread stage in the S. meliloti group of microorganisms 20,21 . Furthermore, the severity of these defects had a correlation with the degree of change in the structure of EPS 22 . Likewise, an observed failure in the production or export of cyclic b-glucansled to a drastic problem of infection thread malformation or no formation during symbiosis in the host plant 23 .
Numerous strains of S. meliloti distinguished ecotypes of M. truncatula by producing a normal nodule on one and defective ones on the other 24 . It was demonstrated from the above-mentioned study that by an exchange of EPS locus the phenotype can be switched, by supporting this concept that the surface polysaccharides are the determinants of specificity in the symbiosis process of legume vs.rhizobium. At the nitrogen fixing level, bacterial polysaccharides specificity was expressed in various other legume-rhizobial interactions. For example, Exopolysaccharides mutants of M. Loti produced functional nodules on L. pedunculatus and non-functional ones on L. leucocephala 25 . In R. leguminosarum strain 3841 where some lipopolysaccharide mutants behaved normally on a pea, whereas other mutants behaved a defective mechanism in nitrogen fixation 26 .Lectins belonging to the family of carbohydrate binding proteins are normally found in the legume seeds. Their role as receptors for rhizobial polysaccharides was speculated long ago and thus acts as a determinant of host range 27 . They bind to the surface polysaccharides and promote the addition of rhizobia to root hairs, consequently, initiating nodule formation by enhancing the delivery of Nod factors towards root hairs 28,29 . An observation says that host specificity depends on the -surface polysaccharides interactions in which lectin genes would support a host range expansion 30 .

Host immunity
The plant immune system helps fight against any microbial attack by forming structural barriers preventing invasion and subsequent infection. In the initial phases, they respond to the attack by triggering pathogen-associated molecular pattern immunity (PAMP). In order to dampen this immunity and gain to the host access, gram-negative microbes use a type III secretion system (T3SS). Further, as the second line of defense, plant genes perceive the effector protein of the invading microbe to initiate effectortriggered immunity 31 . Therefore, microbe-encoded effector and plant defense act as determinants of host range for pathogens.
In the preliminary period of compatible legume-rhizobial interaction, defense responses do occur but become less pronounced as symbiosis is established 32 . Microbe-associated molecular patterns (MAMPs) i.e. nod factors plus surface polysaccharides could have played a possible role in the inhibition of defense responses during compatible legume hosts [33][34][35][36] . Contrastingly, these MAMPs can bring for the defense response in nonleguminous plants 37 . This suggests that rhizobia could have developed their specific MAMPs recognized definitive compatible legume hosts for the development of symbiosis.
Infection thread formation initiates when rhizobia get entrapped between the two root hair cells. The number of threads that are initiated with a rhizobial strain generally outnumbers the number of developing nodules 38 . Nutman documentation of infection and nodular dynamics observed that infection proceeds in two phases, First the early phase in which the higher infection rate and the second phase related to the time of nodule formation had a much lower infection rate. He furthermore noticed that the early stage of infections was not uniformly disseminated along the seedling root. There were zones that predominantly susceptible to early infection (root hairs), in which the infection occurred preceding to their occurrence in the farther zone 39 . Apart from these experiments involving split root systems and nodule excision found that nodules accountable for this decline in infection rates (40)(41)(42)(43) . These findings indicate that the plant itself controls its infection rates and, also its numbers of infections to permit the progress of nodule developmental pathways.

Strain-specific nitrogen fixation
L e g u m e p l a n t n a t u ra l l y b u i l d s a fundamental capacity in its nodules to accommodate both the symbiotic as well as endophytic bacteria for their respective roles to perform. However co-inoculation studies revealed that symbiotic bacteria occupy a majority portion of the nodule and the endophytes remained in a small distinctive segment of the nodule 44 . This demonstrates the ability of the symbiont to adopt the nodule environment and compete with the endophytes. It also reveals the fact that symbiotic bacteria effectively perform the communication and improves its mechanical capacity with the host while infecting the rizho zone of the plant. As such no definitive rate of nitrogen fixing efficiency of plant-rhizobial combinations, it varies with a variety of combinations. In some extreme conditions though the rhizobial strains nodulate a host plant but were unable to fix nitrogen. On the other hand, the same strain could have the capacity to fix nitrogen with alternative host genotypes 45,46 .
Recent advances in the literature have led to an increased understanding of the process of nodule development; however, mechanisms involving nitrogen fixation have largely been unknown. But the nodulation specificity and interaction was well understood and published in between the M. truncatula host and S. melilotisps. to achieve the identification of precise genes involved [47][48][49] . Genetic analysis recognized a distinct gene called "Mt-Sym6" which is a conditioned host specific gene in S. meliloti strain A145 for nitrogen fixation immediately upon inoculation into the host plant 45 . Studies performed on strains of R. leguminosarum bv. trifolii found strange results of varying compatibility with white clover plant and caucasian clover plant. One of the T. ambiguum rhizobial strains, ICC105 produced non-fixing nodules when inoculated in white clover whereas produced nodulating genes efficiently while inoculated on to the caucasian clover 50 . The above genetic experiments illustrated that non nodulation is because a unique sequence of ICC105 promoter regions prevented nifA protein which is necessary for activating nitrogenase enzyme assembly.

Bio-geo specific rhizobacteria
The Leguminosae comprising of 19,500 species and 751 genera that were populated major surface portion of land on the earth. Their respective species were adapted all most all terrestrial ecosystems including deserts, tropical rain forests and habituated to tropical as well as arctic habitats [51][52][53] . A papilionoid tribe having 65 native Fynbos legumes was analyzed with rhizobial diversity and their specific host preferences. For further deep analysis, sequencing of 16S rRNA, recA, atp Dchromosomal genes and nodA, nif Hsymbiosis related genes recognized and named those Mesorhizobium as alpha symbionts and Burkholderia as a betasymbiont. Though the host genotype was one of the factors influencing rhizobial diversity, environmental factors like the acidic nature of the soil and site elevation showed a positive association with genetic variations in the Mesorhizobium and Burkholderia. These genetic variations demonstrated host and environmental interactions for the distribution of Fynbosrhizobia in various locations 54 .
A study on Australian Acacia with rhizobia interactions was conducted for understanding the geographic patterns of symbiont abundance and adaptation. Among those 58 different sites were characterized like the size of symbiont population, environmental parameters, and soil chemistry. Further better understanding greenhouse soil experiments were conducted with native soils filled in pots, minimal apparent differences were found between host and seeding responses. When another species A. salicinagrew in A. stenophylla and A. salicinasoils in a similar fashion to check for the same behavior, no similar results were found as A. stenophylla yielded. This signifies that the plants of one geography have a broad adaptation to its own rhizosphere bacteria and perform the best symbiotic compatibility. The soil chemistry also plays a prominent role in nodulation and host growth 55 .
Studies conducted to identify the involvement in the segregation and detection of crop specific rhizobium strains for Glycine max, Cicer aritenum, and Arachis hypogaea from Bhadrachalam forest region. One best rhizobia sps. among the 45 soil samples were analyzed with biochemical, physical and in silico parameters and employed to various unfertile soil samples of the same geography showed an improvement in the plant growth and yield when compared to its counterpart control samples. Further phylogenetic analysis of Glycine max, Cicer aritenum, and Arachis hypogaea was revealed that B. japonicum, M. ciceri, and R. leguminosarum species respectively supported the plant growth by means of increasing IAA, ACC, and nitrogen compounds as well as increasing the growth and nodulation 56-58 . In silico analysis revealed that nifA protein is the root cause for the activation of nitrogenase enzyme which has a derivative capacity of nitrogen fixing 59 . If found any defect in the nifA protein structure usually could not fix atmospheric nitrogen. Though a deficiency found in the M.ciceri of nifA protein, it could, fortunately,manage to promote the growth and produce a good amount of nitrogen through its missing amino terminal domain. The literature says that the activity of the amino terminal domain of nifA protein is hidden in a few special conditions. Further, these studies said that geographical acclimatization and adaptability could a reason for the greater stability of the nifA protein. It is very important to identify cultivable growth promoting Rhizobium strains for selected legume plants inconfined geography.

CONCluSiON
The effective symbiosis between rhizobia and legumes depends on the Nod factors, flavonoids, bacterial surface polysaccharides and plant lectins. They are known to play a prominent role and any alterations in their structure alter the host specificity. Nod factors secreted by the rhizobia are synthesized by common nodulation genes specific to the host. Symbiotic nodulation genes could have been identified and used as genetic markers for the determination of host specificity as well as symbiotic diversity. The abundance rhizobacteria in the native soil samples could play a vital role in the nodulation and also act as an indicator of the effect of the rhizobial population towards its symbiotic plant. Understanding the differences in effectiveness and specificity of plant symbiosis with relevant species at multiple special scales greatly influences the evolution and ecology of plant, soil communities. This also potentially increases the cost effectiveness in a better natural environment. Further, the native or indigenous strains isolated from a geographical area are found to be more specific towards the crop in the same geography. A serious need to explore the biofertilizers with more variable specifications like geographical acclimatization to optimize the crop yield by sustaining the efficacy and shelf life in order to reduce the use of chemical fertilizers.