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Nod factor

Nod factors are lipo-chitooligosaccharide signaling molecules secreted by rhizobial bacteria that initiate and regulate the symbiotic interaction with legume host plants, triggering the formation of root nodules essential for biological nitrogen fixation.^[1] These molecules enable specific recognition between compatible partners, allowing rhizobia to enter root cells and convert atmospheric nitrogen into ammonia usable by the plant, in exchange for carbon compounds from photosynthesis.^[2] Discovered in the early 1990s, Nod factors represent a cornerstone of plant-microbe communication, with their activity effective at concentrations as low as 10^{-12} M.^[3] Structurally, Nod factors consist of a chitooligosaccharide core of three to five N-acetyl-D-glucosamine residues linked via β-1,4 glycosidic bonds, with an N-linked acyl chain (typically C16 to C18 fatty acid) at the non-reducing end and various decorations such as sulfation, methylation, or carbamoylation at the reducing end.^[1] These modifications, encoded by host-specific rhizobial nod genes (e.g., nodH for sulfation in Sinorhizobium meliloti), determine the molecule's ability to elicit responses in particular legume species, ensuring symbiotic specificity.^[2] Production occurs in rhizobia upon perception of plant-root exudates, primarily flavonoids, which activate the transcriptional regulator NodD to induce the nod gene cluster.^[4] Upon release into the rhizosphere, Nod factors diffuse to the root epidermis where they bind to LysM domain-containing receptor-like kinases, such as NFR1/NFR5 in Lotus japonicus or LYK3/NFP in Medicago truncatula, with high affinity (dissociation constant in the nanomolar range).^[1] This binding initiates a rapid signaling cascade involving calcium oscillations, reactive oxygen species production, and activation of transcription factors like NIN (nodule inception), culminating in root hair curling to entrap bacteria, infection thread formation, and dedifferentiation of cortical cells into nodule primordia.^[2] The process is tightly regulated by plant hormones, including auxins, cytokinins, and ethylene, which Nod factors modulate to balance symbiosis establishment and prevent over-nodulation.^[4] The legume-rhizobia symbiosis mediated by Nod factors is ecologically and agriculturally vital, supporting nitrogen input in natural ecosystems and reducing the need for synthetic fertilizers in legume crops like soybeans, alfalfa, and peas, which account for a significant portion of global protein production.^[5] Beyond legumes, Nod factor-like LCOs occur in other microbes, suggesting an ancient evolutionary origin shared with mycorrhizal associations, potentially over 400 million years old.^[1] Research continues to explore Nod factor analogs for engineering nitrogen-fixing symbioses in non-legumes, aiming to enhance sustainable agriculture.^[2]

Discovery and Background

Historical Context

The study of legume-rhizobia symbiosis originated in the late 19th century, when researchers began investigating the mechanisms behind the enhanced growth of leguminous plants in nitrogen-poor soils. In 1888, Hermann Hellriegel and Hermann Wilfarth conducted pivotal experiments demonstrating that legumes, such as peas and beans, could assimilate atmospheric nitrogen through associations with soil microorganisms, leading to the formation of root nodules that facilitate biological nitrogen fixation. This discovery shifted agricultural practices and laid the groundwork for understanding symbiotic nitrogen fixation, with Hellriegel hypothesizing microbial involvement in nodule development.^[6] Concurrently, Martinus Beijerinck isolated the first Rhizobium bacterium from lupin nodules in 1888, confirming the microbial basis of the symbiosis and naming it Bacillus radicicola. In the early to mid-20th century, research focused on characterizing the nodule-forming process and bacterial-host specificity. Edwin Broun Fred, Ira Lawrence Baldwin, and Elizabeth McCoy's 1932 monograph, Root Nodule Bacteria and Leguminous Plants, synthesized decades of observations, detailing the infection threads, nodule morphology, and environmental factors influencing symbiosis across various legumes.^[7] Their work emphasized the role of root exudates in attracting bacteria but did not yet identify specific chemical signals. Progress accelerated in the 1970s and 1980s with genetic analyses; Peter Gresshoff and colleagues identified regulatory mutants affecting nodulation in soybeans during the early 1980s, revealing genetic controls on symbiosis efficiency. Similarly, Roland Rolfe contributed to early studies on host-bacteria interactions, including phenolic compounds from clover roots that modulated rhizobial activity in the late 1980s. The 1980s marked a molecular breakthrough with the identification of nodulation (nod) genes in rhizobia, essential for initiating symbiosis. These genes were first cloned from Rhizobium meliloti in 1984, encoding proteins involved in signal production. In 1986, flavonoids such as luteolin were shown to induce nod gene expression in rhizobia, acting as plant-derived signals that trigger bacterial responses specific to host legumes. The culmination came in the early 1990s, when Jean Dénarié and collaborators purified and structurally characterized Nod factors from R. meliloti as acylated lipo-chitooligosaccharides, demonstrating their role in eliciting root hair curling and cortical cell division in alfalfa. This 1990 identification, built on prior genetic work, confirmed Nod factors as the key symbiotic signals, revolutionizing understanding of host specificity and signaling in the symbiosis.

Initial Identification

The initial identification of Nod factors relied on bioassays monitoring root hair deformation in compatible legume hosts, such as alfalfa (Medicago sativa) and white clover (Trifolium repens), as a rapid indicator of symbiotic signaling activity. In these assays, root hairs from young seedlings were exposed to bacterial culture supernatants or fractions, with deformation—characterized by swelling, branching, or curling—observed within hours at sub-nanomolar concentrations, distinguishing active symbiotic signals from non-specific factors.^[8]^[9] This approach, developed in the late 1980s and refined in the early 1990s, allowed fractionation of bacterial exudates to isolate bioactive components from rhizobial strains like Sinorhizobium meliloti and Rhizobium trifolii. Purification of these signals involved sequential chromatographic techniques, including gel permeation, ion-exchange, and reversed-phase high-performance liquid chromatography (HPLC), followed by structural characterization using mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy. In 1990, Lerouge et al. isolated the major alfalfa-specific signal, NodRm-1, from an overproducing S. meliloti strain, elucidating its structure as a sulfated β-1,4-linked tetrasaccharide of N-acetyl-D-glucosamine, with an N-linked C16:2 acyl chain on the non-reducing end and a sulfate group on the 6-position of the terminal N-acetyl-D-glucosamine residue at the reducing end.^[8] Subsequent work in 1992 expanded this to a family of related sulfated lipo-chitooligosaccharides varying in chain length and substitutions, confirming their role as core signaling molecules. Activity confirmation demonstrated that purified Nod factors elicit nodulation responses at picomolar concentrations (e.g., 10^{-10} M for root hair deformation), far exceeding bacterial densities needed for symbiosis. Host-specificity was evident in comparative tests: NodRm factors induced deformation and cortical cell divisions in alfalfa but not in non-hosts like pea (Pisum sativum) or soybean (Glycine max), underscoring structural determinants like sulfation for recognition.^[8]^[10] Plant root exudates, particularly flavonoids such as luteolin, initiate Nod factor production by inducing bacterial nod gene expression in a concentration-dependent manner (e.g., 10^{-6} M luteolin activates nodABC in S. meliloti). This triggers the regulated release of Nod factors into the rhizosphere, establishing the initial dialog for host-specific symbiosis.^[11]

Chemical Composition

Core Molecular Structure

Nod factors are lipo-chitooligosaccharides characterized by a core backbone consisting of a β-1,4-linked oligomer of N-acetyl-D-glucosamine residues, typically comprising 3 to 5 units. This chitooligosaccharide structure is acylated at the nitrogen atom of the non-reducing terminal glucosamine residue with a fatty acid chain, most commonly a C16 or C18 acyl group, which confers the lipophilic nature essential for membrane interactions and biological activity. The seminal identification of this core in Rhizobium meliloti revealed a tetrameric form with a C16:2 acyl chain, establishing the foundational architecture shared across rhizobial species.^[12]^[5] Key functional groups on the core structure include the reducing end of the terminal glucosamine, which remains available for potential interactions, and modifications such as sulfate or carbamoyl groups on the reducing end glucosamine that can influence specificity without altering the fundamental scaffold. A representative general formula for the core trimeric structure is depicted as:

\text{R-C(O)-NH-GlcNAc-}(\beta 1,4)\text{-GlcNAc-}(\beta 1,4)\text{-GlcNAc}

where R denotes the acyl chain (e.g., a 16-carbon fatty acid) and GlcNAc represents N-acetyl-D-glucosamine. Longer oligomers follow the same linkage pattern with the acyl attachment at the non-reducing terminus.^[12]^[5] Physicochemically, the acyl chain renders Nod factors amphipathic, with lipophilicity facilitating diffusion across bacterial membranes and interaction with plant receptors, while the hydrophilic oligosaccharide portion enables specific binding. Their molecular weight generally ranges from 1,200 to 1,500 Da, depending on oligomer length and substitutions, allowing high potency at nanomolar concentrations in symbiotic signaling.^[5]^[13]

Structural Diversity Across Rhizobia

Nod factors exhibit significant structural diversity across different rhizobial species, primarily in the acyl chain and oligosaccharide decorations, which underpin host specificity in legume symbiosis.^[14] The core chitooligosaccharide backbone consists of 3-5 N-acetylglucosamine (GlcNAc) units, but variations in attached substituents modulate recognition by host plants.^[14] The acyl chain, attached to the terminal GlcNAc, varies in length from C14 to C26 carbons and degree of unsaturation, influencing the molecule's hydrophobicity, solubility, and interaction with plant receptors.^[14] For instance, many rhizobia, including Rhizobium species, incorporate cis-vaccenic acid (C18:1), a monounsaturated fatty acid that enhances membrane association and signaling efficiency.^[14] Longer chains, such as C20 or C26 variants with multiple unsaturations (e.g., C20:3), are common in slow-growing rhizobia like Bradyrhizobium, contributing to specificity for tropical legumes.^[14] Oligosaccharide decorations further diversify Nod factor structure, with additions like O-methyl groups, fucose, arabinose, or sulfate on the reducing or non-reducing ends.^[14] These modifications, often species-specific, fine-tune host recognition; for example, in Rhizobium leguminosarum, the NodFe factor features fucosyl residues on the terminal GlcNAc, essential for nodulation of peas (Pisum sativum).^[15] Similarly, Sinorhizobium meliloti produces NodSm factors with a C16:2 polyunsaturated acyl chain and a sulfate group at the reducing end, conferring specificity for alfalfa (Medicago sativa).^[14] In Bradyrhizobium spp., such as those nodulating soybeans, fucose decorations combined with longer acyl chains (e.g., C18:1) enable infection of tropical legumes.^[14] Structure-activity relationship studies from the 1990s, including NMR analyses, have elucidated how these variations impact biological activity.^[16] Acyl chain length and unsaturation affect solubility and diffusion to root hairs, while decorations like sulfate or fucose lower recognition thresholds by enhancing receptor affinity, sometimes by orders of magnitude in nodulation assays.^[14] For instance, desulfated NodSm analogs show reduced activity on alfalfa roots, highlighting the sulfate's role in precise host discrimination.^[16] These findings underscore the evolutionary adaptation of Nod factor structures to match specific legume receptors, ensuring symbiotic fidelity.^[14]

Biosynthesis and Regulation

Genetic Basis in Bacteria

The nod gene clusters in rhizobia are typically organized as conserved loci responsible for the synthesis of Nod factors, key signaling molecules in legume symbiosis. These clusters are commonly located on large symbiotic plasmids, such as the pSymA megaplasmid in Sinorhizobium meliloti, though some species carry them on the chromosome. The core genes, nodA, nodB, and nodC, form the foundational operon essential for Nod factor backbone assembly: nodC encodes a chitin synthase that polymerizes β-1,4-linked N-acetyl-D-glucosamine oligomers, nodB encodes a chitooligosaccharide deacetylase that removes acetyl groups from the non-reducing end, and nodA encodes an acyltransferase that attaches a fatty acyl chain to the terminal sugar, conferring lipochitooligosaccharide structure.^[17]^[18] Accessory genes within the cluster add host-specific modifications to the core Nod factor structure, enhancing recognition by particular legumes. For instance, nodH and nodI in S. meliloti encode sulfotransferases for O-sulfation at the reducing end and an ATP sulfurylase, respectively, while nodL encodes an O-acetyltransferase for acetylation on the terminal residue. Host-specificity determinants include nodF and nodE in Rhizobium leguminosarum, which direct fucosylation and the incorporation of specific acyl chain lengths, respectively. These modifications, produced by the accessory genes, allow Nod factors to elicit tailored symbiotic responses in host plants.^[17]^[19] Regulation of nod gene expression is primarily controlled by the NodD protein, a LysR-type transcriptional activator present in multiple copies (e.g., nodD1, nodD2, nodD3 in S. meliloti). In the presence of plant-derived flavonoids, NodD binds to conserved nod box promoter sequences upstream of the nod operons, inducing transcription of the biosynthetic genes. This flavonoid-NodD interaction ensures nod gene activation only in proximity to compatible host roots.^[17] In S. meliloti, the full nod cluster spans over 20 genes organized in multiple operons on pSymA, encompassing core and accessory loci as well as regulatory elements. Mutational studies confirm their essentiality: disruptions in nodABC abolish Nod factor production and prevent nodule formation on alfalfa, while accessory gene mutants (e.g., nodH) produce unmodified factors that fail to induce effective symbiosis, underscoring the cluster's role in host specificity.^[17]^[19]

Environmental Influences on Production

Acidic soil conditions significantly influence Nod factor production in rhizobia, often enhancing yield and modifying molecular profiles to adapt to stress. In Rhizobium tropici CIAT899, exposure to low pH (specifically pH 4.5) increases overall Nod factor production compared to neutral conditions (pH 7.0), with the bacterium synthesizing 52 distinct Nod factor variants under acidic stress versus 29 at neutral pH. ^[20] This adaptation is particularly relevant for symbioses in acidic soils common to bean cultivation, where the altered profiles include structural variations that may improve host recognition and infection efficiency. Studies from 2005 indicate that such changes under pH 5-6 promote higher Nod factor yields and shifts toward more saturated acyl chains, enabling better resilience in low-pH environments. ^[21] Nutrient availability, particularly limitations in key elements like phosphate, iron, and molybdenum, modulates Nod factor synthesis by impacting nod gene expression in rhizobia. Iron limitation disrupts nod gene regulation due to its role in essential enzymatic processes and regulatory proteins, reducing expression and altering production profiles in strains like Bradyrhizobium japonicum. ^[22] Temperature and oxygen levels further regulate Nod factor production, with optimal conditions aligning to soil microenvironments during root colonization. Nod factor yields peak at around 28°C in species such as Bradyrhizobium japonicum and Rhizobium leguminosarum, where lower temperatures (e.g., 15-17°C) suppress synthesis while higher ones inhibit overall rhizobial viability. ^[23]

Perception and Signaling

Receptor Recognition in Plants

Nod factors are perceived by plants through lysin motif (LysM) receptor-like kinases (RLKs), which feature extracellular LysM domains that bind the chitooligosaccharide backbone and the acyl chain of the Nod factor. In the model legume Lotus japonicus, the receptors NFR1 and NFR5 were identified in 2003 as essential LysM-RLKs required for Nod factor perception and symbiotic signaling upstream of the common symbiosis pathway. Similarly, in Medicago truncatula, the LysM-RLKs LYK3 and NFP perform analogous roles, with LYK3 acting as an entry receptor for rhizobial infection and NFP facilitating Nod factor binding. The extracellular LysM domains of these receptors specifically recognize the Nod factor's core structure, including the β-1,4-linked N-acetylglucosamine oligomers and the N-linked acyl moiety, enabling host-specific symbiotic interactions.^[24] The binding mechanism involves high-affinity interactions in the nanomolar range, as demonstrated by surface plasmon resonance and microscale thermophoresis assays with purified NFR1 and NFR5 ectodomains from L. japonicus, where dissociation constants (K_d) were measured at 0.61–10.1 nM for compatible Nod factors.^[24] Decorations such as sulfate groups on the reducing-end glucosamine (critical for S. meliloti Nod factors in M. truncatula) or fucose residues on the non-reducing end (key for M. loti Nod factors in L. japonicus) enhance specificity by fitting into dedicated pockets within the LysM domains, as revealed by structural analyses including NMR spectroscopy and motif mapping in the 2010s.^[25]^[26] These modifications discriminate compatible symbionts, with desulfated or defucosylated Nod factors showing reduced affinity and eliciting weaker or no symbiotic responses.^[24] LysM receptors localize to the plasma membrane of root epidermal cells. An immediate downstream response is calcium influx at the root hair tip, which precedes sustained calcium spiking patterns essential for symbiosis initiation.^[24] Recent studies have elucidated the receptor complex assembly, confirming that NFR1 and NFR5 form a core heterocomplex essential for initiating symbiotic signaling, with a conserved juxtamembrane motif in NFR5 promoting these interactions (as of 2024).^[27]^[28] Genetic evidence confirms the essentiality of these receptors: knockout mutants of nfr1 or nfr5 in L. japonicus fail to respond to Nod factors, showing no root hair deformation, calcium responses, or nodulation, even with compatible rhizobia. Analogous lyk3 and nfp mutants in M. truncatula abolish infection thread formation and nodule development, underscoring their non-redundant roles in establishing host-symbiont compatibility.

Downstream Signal Transduction

Upon perception of Nod factors by plant receptors, one of the earliest intracellular responses in root hair cells is a rapid influx of calcium ions (Ca²⁺) through plasma membrane channels, including the cyclic nucleotide-gated channel CNGC15, which contributes to the initial depolarization and subsequent nuclear-localized Ca²⁺ oscillations.^[29] These oscillations, characterized by periodic spikes in nuclear Ca²⁺ concentration, are a hallmark of symbiotic signaling and are essential for decoding the Nod factor signal.^[30] In legumes such as Medicago truncatula and Lotus japonicus, these Ca²⁺ signatures differ in frequency and amplitude depending on the symbiotic partner, enabling specificity in the response.^[30] The Ca²⁺ oscillations are transduced into downstream signaling by calcium- and calmodulin-dependent protein kinase (CCaMK), a key decoder that autophosphorylates upon binding calmodulin in response to the spiking pattern. Identified in 2007 through genetic studies in Lotus japonicus, CCaMK is required for Nod factor-induced gene expression and nodule organogenesis, as mutants lacking functional CCaMK fail to progress beyond early calcium responses. This kinase integrates the symbiotic Ca²⁺ code to activate transcriptional regulators, linking perception to cellular reprogramming.^[31] The core symbiotic signaling pathway, often referred to as the common SYM pathway, involves conserved genes shared between rhizobial nodulation and arbuscular mycorrhizal symbiosis, such as DMI1 and DMI3 in Medicago truncatula.^[32] DMI1 encodes a nuclear envelope-localized ion channel that facilitates Ca²⁺ release from intracellular stores to initiate oscillations, while DMI3 is the Medicago ortholog of CCaMK, functioning downstream to propagate the signal.^[32] Specific components within this pathway, including formins like those mediating cortical microtubule dynamics, support localized cellular responses in the root cortex, such as rearrangements necessary for infection structure accommodation.^[33] Downstream of Ca²⁺ decoding, transcriptional regulation is orchestrated by the NIN (Nodule Inception) transcription factor, a RWP-RK domain protein directly induced by Nod factor signaling.^[34] NIN integrates inputs from the symbiotic pathway with cytokinin signaling, binding promoter regions to activate early nodulin (ENOD) genes, including ENOD11 and ENOD40, which drive cortical cell divisions and primordium formation.^[34] This integration ensures coordinated epidermal and cortical responses, with NIN restricting certain genes to specific tissues for precise nodule patterning.^[35] To prevent excessive nodulation and maintain resource balance, feedback autoregulation occurs via CLE (CLAVATA3/EMBRYO SURROUNDING REGION-related) peptides, small signaling molecules produced in roots upon Nod factor stimulation.^[36] These root-derived CLE peptides, such as MtCLE12 and MtCLE13 in Medicago, are transported to the shoot, where they bind leucine-rich repeat receptor kinases like SUNN (SHOOT-RUNNING NODULATION), triggering a shoot-to-root inhibitory signal that limits further nodule initiation.^[36] This long-distance shoot-root communication fine-tunes nodule number in response to environmental nitrogen levels, ensuring symbiotic efficiency.^[37]

Role in Nodulation Process

Initiation of Symbiosis

Upon exposure to Nod factors produced by compatible rhizobia, root hairs of legume plants exhibit rapid deformation within hours, beginning with tip swelling and progressing to curling that forms a characteristic shepherd's crook structure, which entraps bacteria to initiate infection. This morphological change is driven by reorganization of the actin cytoskeleton, where Nod factors trigger fragmentation of actin bundles into diffuse networks and short filaments at the growing tip, redirecting polar growth toward the signal source.^[38]^[39] A key early intracellular response is the generation of calcium spiking patterns, with nuclear- and perinuclear-localized oscillations emerging approximately 10-15 minutes after Nod factor application. These rhythmic calcium fluxes are distinctive to Nod factor perception, differing from those elicited by other microbial signals in frequency and localization, and are subsequently decoded by the calcium- and calmodulin-dependent protein kinase CCaMK to activate downstream symbiotic pathways.^[39]^[40] Within 3-8 hours of Nod factor treatment, early nodulin genes such as ENOD40 and the root hair infection peroxidase gene RIP1 are transcriptionally activated, particularly in dividing cortical cells adjacent to deforming root hairs. ENOD40, encoding a small regulatory RNA, promotes cortical cell divisions essential for primordium initiation, while RIP1 contributes to reactive oxygen species management during early infection site preparation.^[39]^[41] Host specificity is evident in the partial nature of these responses, as Nod factors from incompatible rhizobia often induce root hair tip swelling in non-host legumes but fail to elicit full curling or shepherd's crook formation, preventing progression to infection.^[42]

Infection and Nodule Development

Upon recognition of Nod factors by root hairs, which have undergone deformation and curling as an early symbiotic response, the infection thread (IT) initiates through polar growth from the curled root hair tip. This structure elongates inward via inverted tip growth, guided by localized Nod factor signaling that sustains cytoskeletal reorganization involving actin and microtubules, as well as ion fluxes and reactive oxygen species production.^[43] The IT is lined by a plant-derived membrane, facilitating bacterial invasion without breaching the host cell wall, and its progression depends on continuous low-concentration Nod factor activity to maintain elongation and prevent premature termination.^[44] Concurrent with IT formation, Nod factors induce cell division in the root cortex and pericycle, establishing the nodule primordium through a meristematic zone. In indeterminate nodules, typical of species like Medicago truncatula and Pisum sativum, a persistent apical meristem drives longitudinal growth with ongoing IT branching and cortical divisions, resulting in a zoned structure with a developmental gradient.^[45] In contrast, determinate nodules, as in Lotus japonicus and soybean (Glycine max), feature a transient meristem without persistent activity, leading to spherical nodules where cell divisions occur more uniformly and IT growth ceases earlier, influenced by Nod factor-mediated cytokinin signaling.^[46] These differences arise from variations in Nod factor perception and downstream transcriptional responses, such as those involving NSP1 and NSP2 transcription factors, which promote meristem initiation.^[47] As the IT reaches the cortical cells of the primordium, rhizobia are released from the thread tip or infection droplets into the plant cytoplasm via endocytosis-like processes, forming organelle-like symbiosomes enclosed by a host-derived peribacteroid membrane. This release involves the shedding of bacterial exopolysaccharide capsules and fusion with plant vesicular trafficking components, such as VAMP721, ensuring enclosed differentiation without free bacterial proliferation.^[48] Within symbiosomes, rhizobia terminally differentiate into nitrogen-fixing bacteroids, a process regulated by symbiotic signals including Nod factor-induced gene expression.^[49] Nitrogen fixation commences in mature infected cells approximately 7–14 days post-infection, coinciding with bacteroid activation and the production of leghemoglobin, a plant hemoglobin that buffers oxygen levels to protect the oxygen-sensitive nitrogenase enzyme while facilitating its diffusion. To prevent excessive nodule formation and resource depletion, autoregulation of nodulation integrates Nod factor responses via miR172, which targets the AP2 transcription factor NNC1 in soybean, repressing excessive primordia development and maintaining symbiotic balance.^[50]

Evolutionary and Applied Aspects

Evolutionary Origins

The Nod factor-mediated symbiosis between rhizobial bacteria and legumes represents a relatively recent evolutionary innovation, building upon an ancient foundation of chitin-based signaling that dates back approximately 400 million years to the origin of land plants and their association with arbuscular mycorrhizal (AM) fungi.^[51] Nod factors, which are lipochitooligosaccharides structurally derived from chitin oligomers produced by AM fungi (known as Myc factors), exploit a conserved common symbiosis (SYM) pathway in plants to initiate mutualistic interactions.^[52] This pathway, involving key components like the calcium-calmodulin-dependent kinase CCaMK and the nuclear ion channel CASTOR/POLLUX, was originally co-opted for nutrient exchange with fungi and later adapted for bacterial nodulation, highlighting a shared evolutionary module that predates legume-specific traits by hundreds of millions of years.^[53] Rhizobial diversification and the spread of Nod factor production were driven by horizontal gene transfer (HGT) of nod gene clusters, which are often located on mobile plasmids or symbiotic islands, allowing rapid dissemination across bacterial lineages.^[54] Phylogenetic analyses indicate that nod genes originated in alpha-proteobacteria (Rhizobiaceae family, around 51 million years ago), with subsequent HGT facilitating spread to other alpha- and beta-proteobacteria lineages and broader host colonization.^[55] This HGT mechanism underscores the dynamic, opportunistic nature of symbiotic evolution, where nod genes were acquired and refined multiple times to match diverse legume hosts. Extensions of Nod factor responsiveness beyond legumes are evident in non-legume plants like Parasponia, the sole non-leguminous genus capable of rhizobial nodulation, which exhibits partial responses to Nod factors through conserved LysM receptor kinases that trigger root hair deformation and cortical responses.^[56] Genomic studies from the 2020s reveal that these symbiotic pathways arose via co-option of pre-existing plant defense mechanisms, particularly those involving chitin perception for pathogen resistance, allowing Nod factors to suppress immunity and promote accommodation of beneficial microbes.^[57] A pivotal event in legume evolution was the duplication of LysM receptor genes around 60 million years ago, coinciding with the diversification of the Fabaceae family, which enabled the evolution of receptor specificity for distinct Nod factor structures and restricted nodulation to compatible rhizobial partners.^[58]

Implications for Agriculture

Research on Nod factors has led to the development of commercial rhizobial inoculants engineered to produce higher levels of these signaling molecules, enhancing nodulation efficiency in nutrient-poor soils. For instance, modified Rhizobium strains with upregulated nod gene expression have demonstrated improved nodule occupancy and nitrogen fixation in legumes like soybean, allowing better establishment in acidic or low-fertility environments.^[59] These inoculants are particularly valuable for sustainable farming in regions with marginal soils, where native rhizobia may underperform.^[60] Synthetic Nod factors, often applied as foliar sprays or seed treatments, have shown promise in boosting legume productivity by mimicking natural signaling to accelerate nodulation and growth. In greenhouse trials with peas (Pisum sativum), foliar application of lipo-chitooligosaccharides (LCOs, a class of Nod factors) at concentrations around 10^{-11} M increased seed yield by 22-32% compared to controls, attributed to enhanced nodule formation and nitrogen uptake.^[61] Field studies from the 2010s, such as those on soybeans, reported similar applications leading to 20-30% improvements in biomass and nitrogen fixation under stress conditions like drought, supporting reduced reliance on synthetic fertilizers.^[62] These approaches promote early symbiotic responses, as Nod factors initiate root hair deformation and cortical cell division essential for nodule development.^[63] Advances in genetic engineering have extended Nod factor perception to non-legume crops, aiming to enable nitrogen-fixing symbioses in cereals. Using CRISPR/Cas9, researchers have introduced legume-derived Nod factor receptors (e.g., NFR1 and NFR5 from Lotus japonicus) into rice and maize, allowing these plants to respond to rhizobial signals with root hair curling and infection thread formation.^[64] In 2020s studies, such edited rice lines exhibited partial symbiotic responses, with potential to reduce nitrogen fertilizer needs by up to 40-90% based on modeling, though full field trials demonstrating these reductions while maintaining yields are ongoing as of 2025. Similar efforts in maize have focused on integrating Myc factor receptors alongside Nod pathways to support dual symbioses with arbuscular mycorrhizae and rhizobia.^[65] Recent 2024-2025 research highlights persistent challenges, including oxygen sensitivity of nitrogenase and high energy demands, indicating that while signaling is partially established, complete functional nodules in cereals remain elusive.^[66] Despite these innovations, practical adoption faces challenges including Nod factor instability in diverse soil conditions, where microbial degradation and environmental factors like pH can reduce efficacy.^[60] Regulatory hurdles for genetically modified rhizobia and receptor-engineered crops, particularly concerning biosafety and gene flow, also limit commercialization, though non-GM synthetic Nod factor products offer a pathway for faster integration into sustainable agriculture.^[67] Overall, these applications could significantly cut global fertilizer use, enhancing soil health and crop resilience.^[64]

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The symbiotic nitrogen-fixing soil bacterium Sinorhizobium meliloti contains three replicons: pSymA, pSymB, and the chromosome. We report here the complete ...
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Low pH changes the profile of nodulation factors produced by ...
We report that acidity increases rhizobial Nod factors production, and we exhaustively study the nodulation factor structures produced under abiotic stress.
[21]
Low pH Changes the Profile of Nodulation Factors Produced by ...
We report that acidity increases rhizobial Nod factors production, and we exhaustively study the nodulation factor structures produced under abiotic stress.
[22]
Phosphate Deficiency Negatively Affects Early Steps of the ...
Phosphate (Pi) deficiency reduces nodule formation and development in different legume species including common bean. Despite significant progress in the ...
[23]
Iron: an essential micronutrient for the legume-rhizobium symbiosis
This review will focus on the role of iron in the legume–rhizobium symbiosis, specifically iron movement within the symbiotic organ, the nodule.
[24]
Soybean-Nodulating Rhizobia: Ecology, Characterization, Diversity ...
Soil phosphorus (P), acidity, alkalinity, and salinity are among the abiotic factors which determine biogeographical patterns of soybean nodulating rhizobia ( ...
[25]
Nod Bj-V (C18:1, MeFuc) production by Bradyrhizobium japonicum ...
The greatest amounts of the major Nod factor, Nod Bj-V (C18:1, MeFuc), were produced at 28 °C for both strains. At 17 and 15 °C, the Nod factor production ...
[26]
Transcriptomic Studies of the Effect of nod Gene-Inducing Molecules ...
This review is specially focused on transcriptomic changes that takes place when five rhizobial species, Bradyrhizobium japonicum (=diazoefficiens) USDA 110,
[27]
Rhizobial symbiosis promotes drought tolerance in Vicia sativa and ...
Drought is a factor that most significantly influences nodulation and nitrogen fixation by legume-rhizobium symbiosis. Effects of drought stress on legume ...Rhizobial Symbiosis Promotes... · 3. Results · 3.4. Analysis Of...
[28]
Characterization of drought stress-mitigating Rhizobium from faba ...
Aug 24, 2023 · Here, we phylogenetically and biochemically characterized a novel strain of Rhizobium ('QHCD11') sampled from the root nodules of faba beans ...Missing: 2020s | Show results with:2020s
[29]
Legume receptors perceive the rhizobial lipochitin oligosaccharide signal molecules by direct binding | PNAS
### Summary of Binding Affinity, Mechanism, and Role of Sulfate/Fucose Decorations
[30]
LysM domains of Medicago truncatula NFP protein involved in Nod ...
The extracellular region of this protein contains three tandem lysin motifs (LysMs), a short peptide domain that is implicated in peptidoglycan or chitin ...
[31]
Ligand-recognizing motifs in plant LysM receptors are ... - Science
Aug 7, 2020 · Two motifs in the LysM1 domains of these receptors determine specific recognition of ligands and discriminate between their in planta functions.
[32]
Autoactive CNGC15 enhances root endosymbiosis in legume and ...
Jan 15, 2025 · Notably, Nod factors induce higher-frequency Ca2+ oscillations in cngc15 ... calcium influx and calcium spiking in root hairs. New Phytol ...
[33]
Differential and chaotic calcium signatures in the symbiosis ... - PNAS
Jul 15, 2008 · We have shown that Nod factor and mycorrhizal fungi can induce differential calcium responses by using the same signal transduction pathway.Results · Mycorrhizal-Induced Calcium... · Rhizobial Bacteria And...
[34]
and calmodulin-dependent protein kinase (CCaMK), the central ...
Epistasis tests demonstrated that a main function of signaling components upstream of calcium spiking is the activation of CCaMK. Despite CCaMK being a central ...
[35]
Genetic and Cytogenetic Mapping of DMI1, DMI2, and DMI3 Genes ...
The DMI1, DMI2, and DMI3 genes of Medicago truncatula, which are required for both nodulation and mycorrhization, control early steps of Nod factor signal ...Missing: SYM | Show results with:SYM
[36]
Calcium/calmodulin-mediated microbial symbiotic interactions in ...
Oct 17, 2022 · Following Nod factor induced Ca2+ influxes, some Ca2+ binding proteins, such as CCaMK, decode the symbiotic Ca2+ signal into down-stream ...
[37]
The NIN Transcription Factor Coordinates Diverse Nodulation ...
Dec 15, 2015 · In contrast, NIN is sufficient to promote the expression of the cytokinin receptor Cytokinin Response 1 (CRE1), which is restricted to the root ...
[38]
NIN—at the heart of NItrogen-fixing Nodule symbiosis - Frontiers
Jan 11, 2024 · The transcription factor NIN belongs to a plant-specific RWP-RK protein family. NIN controls all aspects of symbiotic nodulation in legumes: ...Abstract · Introduction · The transcription factor NIN... · NIN is essential for nodule...
[39]
CLE peptides control Medicago truncatula nodulation locally and ...
The CLAVATA3/embryo-surrounding region (CLE) peptides control the fine balance between proliferation and differentiation in plant development.
[40]
Characterisation of Medicago truncatula CLE34 and CLE35 in ...
Oct 17, 2020 · Autoregulation of nodulation is a systemic feedback mechanism requiring extensive signalling between the roots and shoot. Moreover, when ...Introduction · Materials and Methods · Results · Discussion
[41]
Rearrangement of Actin Microfilaments in Plant Root Hairs ...
These results indicate that Nod factors alter the organization of actin microfilaments in root hair cells, and this could be a prelude for the formation of ...
[42]
Genetic analysis of calcium spiking responses in nodulation mutants ...
Mutants in three complementation groups, dmi1, dmi2, and dmi3, are characterized by root hair swelling but accomplish no further response: they do not display ...Missing: SYM | Show results with:SYM
[43]
A Ca2+/calmodulin-dependent protein kinase required for symbiotic ...
We report that DMI3 encodes a calcium/CaM-dependent protein kinase (CCaMK) that functions downstream of calcium spiking and is required for Nod factor signal ...
[44]
Studying Early Nodulin Gene ENOD40 Expression and Induction by ...
ENOD40, an early nodulin gene, is expressed following inoculation with Rhizobium meliloti or by adding R. meliloti-produced nodulation (Nod) factors or the ...
[45]
Early nodulin gene expression during Nod factorâ
The expression of both genes was first detectable at 8 h after addition of Nod factor which is ample time after root hair deformation. ... Plant growth conditions ...
[46]
Infection and Invasion of Roots by Symbiotic, Nitrogen-Fixing ...
Jun 1, 2004 · Root Hair Deformation and Curling. Root hairs of host plants deform under the influence of purified Nod factors from compatible rhizobial ...<|control11|><|separator|>
[47]
https://doi.org/10.1093/jxb/ery046
[48]
https://doi.org/10.1111/nph.13837
[49]
https://doi.org/10.1038/s41477-020-0613-7
[50]
https://pmc.ncbi.nlm.nih.gov/articles/PMC4311200/
[51]
https://pmc.ncbi.nlm.nih.gov/articles/PMC5940448/
[52]
https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2017.02229/full
[53]
https://academic.oup.com/plphys/article/149/1/306/6107804
[54]
Plant Signaling and Metabolic Pathways Enabling Arbuscular ...
Plants have lived in close association with arbuscular mycorrhizal (AM) fungi for over 400 million years. Today, this endosymbiosis occurs broadly in the plant ...
[55]
The Symbiosome: Legume and Rhizobia Co-evolution toward a ...
In legume nodules, symbiosomes containing endosymbiotic rhizobial bacteria act as temporary plant organelles that are responsible for nitrogen fixation.Introduction · The Origin of Mitochondria and... · Evolutionary Considerations...
[56]
Antiquity and Function of CASTOR and POLLUX, the Twin Ion ...
Analysis of these genes has begun to reveal the Nod factor and mycorrhizal signaling pathways. The Nod factors are likely perceived directly by the receptor ...
[57]
Phylogenetic distribution and evolutionary dynamics of nod and ...
The nod genes, which remain the main determinants conferring the ability to nodulate plants to rhizobia, can be transferred from strain to strain by horizontal ...
[58]
From β- to α-Proteobacteria: The Origin and Evolution of Rhizobial ...
Sep 11, 2013 · These common nod genes were first described as the nodA, nodB, and nodC genes and are involved in the biosynthesis of the core of the Nod ...Missing: timeline | Show results with:timeline
[59]
The Non-Legume Parasponia andersonii Mediates the Fitness of ...
Here, we focused on the symbiosis between Parasponia hosts and N2-fixing rhizobium bacteria. Parasponia is the only non-legume lineage to have evolved a ...
[60]
Convergent evolution of NFP-facilitated root nodule symbiosis - PNAS
Sep 9, 2025 · The evolutionary origins of this symbiosis remain uncertain due to conflicting phylogenetic and genomic evidence. Detailed evolutionary ...Abstract · Sign Up For Pnas Alerts · Results<|control11|><|separator|>
[61]
Evolution of a symbiotic receptor through gene duplications in the ...
Oct 28, 2013 · The symbiosis between plants of the legume family (Fabaceae) and the nitrogen-fixing bacteria referred to as rhizobia appeared c. 60 million yr ...
[62]
[PDF] Engineering Rhizobium Strains for Enhanced Nitrogen Fixation in ...
Engineered strains maintained high nodulation rates across different soybean varieties and soil types. Nodule structure analysis showed larger and darker ...
[63]
Competition, Nodule Occupancy, and Persistence of Inoculant Strains
Inoculants often fail to compete for nodule occupancy against native rhizobia with inferior nitrogen-fixing abilities, resulting in low yields.
[64]
The Influence of Foliar Application of Nod Factors (LCOs) and ...
As a result, an over 30% seed yield increase was obtained, compared to the control group. ... Nod Factor Structures, Responses, and Perception during Initiation ...<|control11|><|separator|>
[65]
Effect of Nod factor sprays on soybean growth and productivity ...
Nod factors may constitute a new agricultural technology that increases soybean yield, contributing to increased biomass production, for increased world food ...Missing: implications | Show results with:implications
[66]
Nod factors stimulate seed germination and promote growth and ...
Mar 20, 2012 · Nod factors and artificial LCOs (but not the chitin pentamer) act as phytohormones enhancing seed germination, promoting plant growth and ...Missing: NodRm | Show results with:NodRm
[67]
Biological nitrogen fixation in cereal crops: Progress, strategies, and ...
Mar 13, 2023 · These bacteria fix N under hypoxic or microaerobic conditions. Examples of nodule-inducing rhizobia are Bradyrhizobium (which forms nodules on ...
[68]
A Roadmap toward Engineered Nitrogen-Fixing Nodule Symbiosis
Jan 13, 2020 · In the late 19th century, it was discovered that legumes can establish a root nodule endosymbiosis with nitrogen-fixing rhizobia.<|control11|><|separator|>
[69]
Biological nitrogen fixation in cereal crops: Progress, strategies, and ...
Nov 28, 2022 · Non-legume crops (such as maize, right) are engineered for symbiotic nitrogen fixation by replacing the extracellular domains of the Myc factor ...