Scientific References and Molecular Mechanisms of Symbiosis (2025 Update)

Overview of Bradyrhizobium elkanii Symbiotic Signaling

The establishment of B. elkanii-legume symbiosis is a sophisticated molecular dialogue involving plant-derived signals (flavonoids), bacterial Nod factors (NFs), Type III secretion system (T3SS) effectors, and host-encoded resistance proteins. This intricate regulatory network determines host specificity, nodule organogenesis, and nitrogen fixation efficiency.

1. Molecular Signaling Initiation

Flavonoid-Mediated Activation

Host-to-Bacterium Signal:Legume roots experiencing nitrogen starvation exude flavonoid compounds (e.g., genistein, daidzein, luteolin) into the rhizosphere. These flavonoids penetrate the B. elkanii cell membrane and bind to the NodD regulatory protein, a member of the LysR family of transcriptional regulators.

Key Research Findings:

• Flavonoid concentrations as low as 10⁻⁸ M activate nod gene expression in B. elkanii

• Different legume species exude distinct flavonoid profiles, contributing to host specificity

• Transcription of the nodYABCSUIJnolMNOnodZ operon is directly dependent upon NodD-flavonoid complexes

• TtsI (transcriptional activator of T3SS) is also responsive to flavonoids and coordinates both Nod factor and T3SS expression

Regulatory Architecture

The B. elkanii regulatory circuit involves:

• NodD: LysR-type regulator controlling nod gene expression

• NodW: Regulatory protein modulating flavonoid recognition

• TtsI: Transcriptional regulator of T3SS genes, activated by plant flavonoids

• Coordination of these regulators ensures spatiotemporal expression of symbiotic genes

2. Nod Factor Biosynthesis and Host Recognition

Structure and Function

Nod Factors (NFs):Nod factors are lipochitooligosaccharides (LCOs) comprising a backbone of 3–5 N-acetyl-D-glucosamine (GlcNAc) units with a long-chain fatty acyl group (C16–C18) attached to the non-reducing terminus.

Nod Gene Clusters in B. elkanii:

• nodA: Encodes N-acetyl transferase; transfers the acyl chain to the GlcNAc backbone

• nodB: N-acetyl lyase; removes N-acetyl group from the non-reducing terminus

• nodC: Chitin synthase; synthesizes the GlcNAc backbone

• nodS, nodU, nodI, nodJ: Involved in modification and transport of Nod factors

• nodZ: Encodes a glucosidase involved in Nod factor modification for B. elkanii-specific legume recognition

Nod Factor Modification

B. elkanii produces modified Nod factors unique to this species:

• Acetyl substitution patterns differ between strains

• Host-specific decorations on the oligosaccharide backbone determine compatibility with legume receptors (NFRs: Nod Factor Receptors)

• Molecular recognition is highly specific; B. elkanii NF structure triggers nodulation in soybean (Glycine max), but not in hosts compatible with other rhizobia

Structural Variations and Host Specificity

B. elkanii genomes harbor extensive nodulation gene repertoires:

• Multiple nod gene variants on symbiotic islands allow synthesis of a spectrum of Nod factor structures

• Comparative genomic analysis reveals gene duplications and deletions affecting Nod factor decoration

• These variations contribute to the competitive nodulation phenotype of B. elkanii and its ability to nodulate multiple legume hosts at variable efficiency

3. Type III Secretion System (T3SS) and Effector Proteins

T3SS Architecture

The T3SS is a molecular syringe-like apparatus embedded in the bacterial cell envelope that delivers effector proteins (Nops: nodulation outer proteins) directly into host plant cells.

T3SS Components in B. elkanii:

• RhcJ: Outer membrane channel protein

• RhcV: Inner membrane channel protein

• RhcQ: ATPase providing energy for protein secretion

• RhcC, RhcD, RhcE, RhcF: Basal body proteins

• FlhA, FliK, FliP: Apparatus assembly proteins

Transcriptional Control:

• T3SS gene expression is controlled by TtsI (transcriptional activator)

• TtsI is activated by plant flavonoids, creating a coordinated response with Nod factor synthesis

• The T3SS is activated only in the presence of compatible plant roots, preventing wasteful energy expenditure in the soil

T3SS Effector Proteins and Functions

NopL: Key Determinant for Nodule Organogenesis

Function: NopL is among the most critical T3SS effectors, particularly for B. elkanii USDA61 symbiosis with certain legume species (e.g., Vigna mungo).

• NopL-deleted mutants form infection threads on Vigna mungo roots but fail to establish nodules, indicating its essential role in nodule primordia formation

• NopL is exclusively conserved among Bradyrhizobium and Sinorhizobium genera, suggesting ancient evolutionary origin

• Phylogenetic analysis indicates NopL diverged from the canonical T3SS lineage, suggesting specialized symbiotic function

Mechanism:

• NopL enters host cell nuclei and likely interacts with plant transcription factors

• Suppresses host immune responses that would otherwise block infection

• Triggers expression of early nodulation genes required for meristem initiation

Bel2-5: NF-Independent Nodulation Effector

Dual Functions:

• In some legumes (e.g., soybean nfr1 mutants), Bel2-5 can trigger nodulation independently of Nod factors

• In soybean carrying the Rj4 allele (dominant resistance gene), Bel2-5 acts as a virulence factor, triggering immune responses that prevent infection

Structural Features:

• Contains ubiquitin-like protease (ULP) domain

• Two EAR (ethylene-responsive element-binding factor-associated amphiphilic repression) motifs for transcriptional regulation

• Nuclear localization signal (NLS) enabling entry into plant cell nuclei

• Internal repeat sequences with unknown function

• Shares structural similarity with XopD from the plant pathogen Xanthomonas campestris pv. vesicatoria

Domain-Function Correlation:

• The C-terminal ULP domain and upstream regions are critical for Bel2-5-dependent nodulation phenotypes

• Mutations in EAR motifs abolish nodulation ability

• Deletion of NLS impairs nuclear targeting and symbiotic function

InnB: Strain-Specific Symbiotic Modulator

Host-Specific Effects:

• InnB promotes nodulation on Vigna mungo cultivars

• InnB restricts nodulation on Vigna radiata cv. KPS1

• This differential phenotype reflects distinct recognition mechanisms in different legume species

Expression and Localization:

• innB expression is flavonoid-dependent and TtsI-regulated

• InnB protein is secreted via T3SS and translocated into host cells

• Adenylate cyclase assays confirm T3SS-dependent translocation into nodule cells

NopM: Ubiquitin Ligase Triggering Senescence

Function: NopM triggers early senescence-like responses in incompatible hosts (e.g., Lotus species).

• Possesses E3 ubiquitin ligase domain and leucine-rich-repeat domain

• Acts similarly to PAMP-triggered immunity (PTI) and effector-triggered immunity (ETI) in pathogenic bacteria

• Mediates ubiquitination of host target proteins, leading to degradation and immune responses

• Results in browning of nodules and disrupted symbiosis

Phylogenetic Conservation:

• NopM homologs are found in both pathogenic and symbiotic bacteria, highlighting the evolutionary relatedness of virulence and symbiotic mechanisms

NopF: Infection Thread Inhibitor

Role in Host Specificity:

• NopF triggers inhibition of infection thread formation in Lotus japonicus Gifu

• Represents a post-recognition checkpoint for host-pathogen compatibility

• Allows alternative legume accessions (L. burttii, L. japonicum MG-20) to proceed with symbiosis, despite presence of NopF

NopP2: Fine-Tuning Symbiotic Efficiency

Function: NopP2 fine-tunes symbiotic effectiveness with Vigna radiata.

• Located within the symbiotic island near the nif cluster

• Differential effects depending on host genotype and strain background

• Contributes to variable nodulation phenotypes among B. elkanii strains

4. Host Specificity and Rj Gene-Mediated Resistance

The Rj Gene System in Soybean

Soybean (Glycine max) possesses a dominant host resistance system controlled by Rj (Rejection) genes that restrict nodulation by specific Bradyrhizobium strains.

Rj4 Gene:

• Encodes a thaumatin-like protein (TLP), a member of the pathogenesis-related (PR-5) protein family

• Structurally similar to plant anti-fungal proteins

• Restricts nodulation by many B. elkanii strains, particularly Type B strains (e.g., USDA61)

• Soybean cultivars carrying Rj4 are incompatible with B. elkanii but compatible with Bradyrhizobium diazoefficiens USDA110

Rj2 Gene:

• Encodes a TIR-NBS-LRR protein (Toll-interleukin receptor/nucleotide-binding site/leucine-rich repeat)

• Represents a receptor-like immune protein structurally similar to plant R proteins for pathogen resistance

• Critical amino acid I490 (isoleucine) in Rj2 determines incompatibility with Bradyrhizobium diazoefficiens USDA122

• Restricts specific rhizobial strains but allows infection by compatible strains

Rj3 Gene:

• Restricts B. elkanii Type B strains (e.g., BLY3-8, BLY6-1, USDA33) despite allowing nodulation by B. japonicum USDA110

• T3SS and its effectors are critical for Rj3-mediated incompatibility

• Mutations in T3SS components (TtsI, RhcJ) overcome Rj3 restriction, confirming T3SS involvement

Gene-for-Gene Model of Symbiotic Specificity

The B. elkanii-soybean system exemplifies a gene-for-gene interaction:

• Bacterial avirulence gene (avr): T3SS effector genes (e.g., nopL, bel2-5, nopM) function as avirulence determinants

• Plant resistance gene (R): Soybean Rj genes encode receptors recognizing effector-triggered immune responses

• Incompatibility occurs when bacterial effector matches soybean R gene recognition specificity

• Compatibility requires bacterial effectors that evade or suppress Rj-mediated immunity

5. Infection and Nodule Development

Infection Thread Formation

Stages:

1. Pre-infection: Nod factors bind to NFR1/NFR5 receptors on legume root epidermis, activating early symbiotic signaling

2. Infection initiation: B. elkanii invades through root hair curling (Nod factor-dependent) or via crack entry (T3SS-dependent in certain genotypes)

3. Intercellular infection: Bacteria travel through infection threads (wall-bound tubular structures) into the cortex

4. Release and bacteroid formation: Bacteria are released into cortical cells and enclosed within plant-derived peribacteroid membranes

Role of T3SS in Infection

• Nod factor-independent nodulation: B. elkanii T3SS effectors (particularly Bel2-5) can trigger nodulation of soybean nfr1 mutants lacking functional Nod factor receptors

• Infection thread progression: T3SS suppresses plant defense responses (ROS production, ethylene synthesis) that normally block infection thread elongation

• Bacterial release: T3SS effectors facilitate bacterial transition from infection threads into cortical cells for bacteroid development

Nodule Organogenesis and Development

Transcriptional Reprogramming:

• B. elkanii T3SS effectors and Nod factors activate soybean early nodulation genes: ENOD40, ENOD93, NIN (Nodule Inception), NSP1, NSP2

• These plant genes activate meristem-like programs in cortical cells, initiating nodule primordia

• Coordinated T3E activity (NopL, Bel2-5, NopP2) is essential for primordia formation

Nodule Maturation:

• Infected cells undergo endoreduplication (multiple rounds of DNA replication without cell division)

• Cortical cells expand to accommodate dividing bacterial cells

• Peribacteroid membranes establish nutrient exchange compartments

Gibberellin Role:

• B. elkanii synthesizes gibberellin precursor (GA₉) via cytochrome P450 monooxygenase

• Host soybean expresses GA 3-oxidases (GA3ox) within nodules, converting GA₉ to bioactive GA₄

• GA₄ regulates nodule size, influences meristem bifurcation, and modulates senescence

• Higher GA levels correlate with increased nodule size and bacterial progeny, providing selective advantage to GA-producing strains

6. Nitrogen Fixation Biochemistry

Nitrogenase Enzyme Complex

Components:

• Component I (MoFe protein): Contains molybdenum and iron clusters

• Component II (Fe protein): Contains iron-sulfur cluster; transfers electrons to Component I

• Electron donors: Bacteroid respiration provides reducing power; organic acids (malate, α-ketoglutarate) drive electron transport

Catalytic Reaction:[ \text{N}_2 + 8 e^- + 16 \text{ATP} \to 2 \text{NH}_3 + \text{H}_2 + 16 \text{ADP} + 16 P_i ]

Key Features:

• Requires strictly anaerobic conditions (oxygen sensitivity)

• Demands substantial ATP input (~16 molecules ATP per N₂ molecule fixed)

• B. elkanii bacteroids express oxygen-scavenging mechanisms including leghemoglobin synthesis

Oxygen Management in Nodules

Oxygen Gradient:

• Outer nodule layers maintain aerobic respiration for ATP generation

• Interior nodule zones remain anaerobic for nitrogenase activity

• B. elkanii respiration consumes oxygen in bacterial layers, maintaining hypoxia in nitrogenase-active compartments

Oxygen-Protective Mechanisms:

• Leghemoglobin (plant-encoded, bacteroid-synthesized iron-containing protein) buffers oxygen at nanomolar levels, preventing nitrogenase inactivation

• Bacteroid differentiation produces enlarged, polyploid cells with reduced permeability to oxygen

• Expressed late nodulation proteins (Nols) contribute to oxygen protection

Metabolic Integration

Carbon-Nitrogen Balance:

• Host plants provide carbohydrates (photosynthetically-derived organic acids) to bacteroids

• B. elkanii oxidizes organic acids via citric acid cycle and electron transport chains, generating ATP and reducing equivalents for nitrogenase

• Efficient strains (e.g., B. elkanii USDA76) show higher enzyme levels for Nod factor synthesis and metabolic integration

Ammonia Utilization:

• Ammonia fixed by nitrogenase is rapidly assimilated via glutamine synthetase (GS) in bacteroids

• However, much ammonia is excreted to host cells, where plants incorporate it into amino acids (glutamine, aspartate)

• Plant cells return nitrogen to bacteroids as amino acids and organic compounds, establishing exchange equilibrium

7. Regulatory Networks and Gene Expression

NifA-RpoN Regulatory Circuit

NifA: Sigma-54-dependent transcriptional activator controlling expression of nitrogen fixation (nif) and related genes

• Activates nifHDK genes encoding nitrogenase structural proteins

• Responsive to oxygen levels; activated under microoxic conditions characteristic of nodule interiors

• Coordinates temporal expression of nif genes with nodule development progression

RpoN: Sigma-54 RNA polymerase recognizing NifA-bound promoters

• Directs transcription from nif promoters bearing NifA-binding sites

• Links nitrogen fixation gene expression to nodule maturation stage

GlnR Regulatory Protein

Function: Controls nitrogen assimilation genes and cross-talks with symbiotic signaling

• Represses genes for nitrogen scavenging (e.g., ABC transporters) when ammonia is abundant

• Releases repression when ammonia becomes limiting, activating alternative nitrogen acquisition pathways

• Prevents metabolic conflict during high nitrogen fixation rates

AdeR (Adenine Deaminase Regulator)

Role: Modulates purine metabolism and symbiotic efficiency

• Controls genes involved in nucleotide synthesis

• Adjusted expression enables rapid bacterial replication in nodules while supporting biosynthesis of symbiotic proteins

8. Comparative Genomics: Symbiotic Island Architecture

Symbiotic Island Composition

B. elkanii genomes contain low GC-content regions (symbiotic islands) harboring symbiosis-essential genes:

Island A (Main symbiotic island): ~690 kb

• Contains nod cluster: nodABC, nodD, nodZ, regulatory sequences

• Contains nif cluster: nifHDK, nifENX, fixABCX

• Contains fix genes (flavoproteins, cytochromes) for electron transport

Island B (Small region): ~4–44 kb

• Variable across strains; minimal genes

Island C: ~200–518 kb

• Contains additional metabolic and regulatory genes

• Variable gene content among B. elkanii strains

Lateral Gene Transfer and Evolutionary Plasticity

Pangenome Analysis:

• Bradyrhizobium pangenome: 84,078 gene families across species

• Core genome: 824 genes (essential cell processes)

• Accessory genome: 42,409 genes (including symbiotic, metabolic, stress response functions)

• B. elkanii genomes are moderately stable compared to highly plastic genomes of some Sinorhizobium species

Genetic Variations:

• SNPs and indels in symbiotic islands correlate with symbiotic phenotype differences

• Polymorphisms in nif, fix, and nodulation regulatory genes drive intraspecific variation

• Integrative conjugative elements (ICEs) facilitate horizontal transfer of symbiotic genes between Bradyrhizobium strains

9. Stress Response and Environmental Adaptation

Osmotic Stress Tolerance

Mechanisms:

• Production of exopolysaccharides (EPS) and trehalose

• Upregulation of osmolyte synthesis under salt stress

• Maintenance of cell membrane integrity under water deficit

Acid-Soil Adaptation

pH Tolerance:

• Many B. elkanii strains tolerate pH 4.5–6.5, though optimal nodulation occurs at pH 6.0–7.5

• Expression of acid-tolerance proteins enables survival in acidic soils

• Selection pressure in Brazilian Cerrado soils (naturally acidic) has generated acid-adapted B. elkanii strains