Rhizobial Infection Research Articles

The Bax inhibitor GmBI-1α interacts with a Nod factor receptor and plays a dual role in the legume-rhizobia symbiosis.

The gene networks surrounding Nod factor receptors that govern the symbiotic process between legumes and rhizobia remain largely unexplored. Here, we identify 13 novel GmNFR1α-associated proteins by yeast two-hybrid screening, and describe a potential interacting protein, GmBI-1α. GmBI-1α had the highest positive correlation with GmNFR1α in a co-expression network analysis, and its expression at the mRNA level in roots was enhanced by rhizobial infection. Moreover, GmBI-1α-GmNFR1α interaction was shown to occur in vitro and in vivo. The GmBI-1α protein was localized to multiple subcellular locations, including the endoplasmic reticulum and plasma membrane. Overexpression of GmBI-1α increased the nodule number in transgenic hairy roots or transgenic soybean, whereas down-regulation of GmBI-1α transcripts by RNA interference reduced the nodule number. In addition, the nodules in GmBI-1α-overexpressing plants became smaller in size and infected area with reduced nitrogenase activity. In GmBI-1α-overexpressing transgenic soybean, the elevated GmBI-1α also promoted plant growth and suppressed the expression of defense signaling-related genes. Infection thread analysis of GmBI-1α-overexpressing plants showed that GmBI-1α promoted rhizobial infection. Collectively, our findings support a GmNFR1α-associated protein in the Nod factor signaling pathway and shed new light on the regulatory mechanism of GmNFR1α in rhizobial symbiosis.

RNA-Seq analysis of mung bean (Vigna radiata L.) roots shows differential gene expression and predicts regulatory pathways responding to taxonomically different rhizobia

Symbiotic interaction among legume and rhizobia is a complex phenomenon which results in the formation of nitrogen-fixing nodules. Mung bean is promiscuous host however expression profile of this important legume plant in response to rhizobial infection was particularly lacking and urgently needed. We have demonstrated the pattern of gene expression of mung bean roots inoculated with two symbionts Bradyrhizobium yuanmingense Vr50 and Sinorhizobium (Ensifer) aridi Vr33 and non-inoculated control (CK). The RNA-Seq data analyzed at two growth stages i.e., 1–3 h and 10–16 days post inoculation revealed significantly higher number of differentially expressed genes (DEGs) at nodulation stage. The DEGs encoding receptor kinases identified at early stage might be involved in perception of Nod factors produced by different rhizobia. At nodulation stage important genes involved in plant hormone signal transduction, nitrogen and sulfur metabolism were identified. KEGG pathway enrichment analysis showed that metabolic pathways were most prominent in both groups (Group 1: Vr33 vs CK; Group 2: Vr50 vs CK), followed by biosynthesis of secondary metabolites, plant hormone signal transduction and biosynthesis of amino acids. Furthermore, DEGs involved in cell communication and plant hormone signal transduction were found to be different among two symbiotic systems while DEGs involved in carbon, nitrogen and sulfur metabolism were similar but their expression varied in response to two rhizobial strains. This study provides the first insight into the mechanisms underlying interactions of mung bean host with two taxonomically different symbionts (Bradyrhizobium and Sinorhizobium) and the candidate genes for better understanding the mechanisms of symbiotic host-specificity.

G-type receptor-like kinase AsNIP43 interacts with rhizobia effector nodulation outer protein P and is required for symbiosis.

In the Rhizobium-Legume symbiosis, the nodulation outer protein P (NopP) effector is one of the key regulators for rhizobial infection and nodule organogenesis. However, the molecular mechanism through which host legume plants sense NopP remains largely unknown. Here, we constructed an nopP deletion mutant of Mesorhizobium huakuii and found that nopP negatively regulates nodulation on Chinese milk vetch (Astragalus sinicus). Screening for NopP interacting proteins in host plants using the yeast 2-hybrid system identified NopP interacting protein 43 (AsNIP43), which encodes a G-type receptor-like kinase (LecRLK). The B-lectin domain at the N terminus of AsNIP43 was essential in mediating its interaction with NopP, which was confirmed in vitro and in vivo. Subcellular localization, co-localization, and gene expression analyses showed that AsNIP43 and NopP function tightly associated with earlier infection events. RNA interference (RNAi) knockdown of AsNIP43 expression by hairy root transformation led to decreased nodule formation. AsNIP43 plays a positive role in symbiosis, which was further verified in the model legume Medicago truncatula. Transcriptome analysis indicated that MtRLK (a homolog of AsNIP43 in M. truncatula) may function to affect defense gene expression and thus to regulate early nodulation. Taken together, we show that LecRLK AsNIP43 is a legume host target that interacts with rhizobia effector NopP is essential for rhizobial infection and nodulation.

Aromatic amino acid biosynthesis impacts root hair development and symbiotic associations in Lotus japonicus.

Legume roots can be symbiotically colonized by arbuscular mycorrhizal (AM) fungi and nitrogen-fixing bacteria. In Lotus japonicus, the latter occurs intracellularly by the cognate rhizobial partner Mesorhizobium loti or intercellularly with the Agrobacterium pusense strain IRBG74. Although these symbiotic programs show distinctive cellular and transcriptome signatures, some molecular components are shared. In this study, we demonstrate that 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase 1 (DAHPS1), the first enzyme in the biosynthetic pathway of aromatic amino acids (AAAs), plays a critical role in root hair development and for AM and rhizobial symbioses in Lotus. Two homozygous DAHPS1 mutants (dahps1-1 and dahps1-2) showed drastic alterations in root hair morphology, associated with alterations in cell wall dynamics and a progressive disruption of the actin cytoskeleton. The altered root hair structure was prevented by pharmacological and genetic complementation. dahps1-1 and dahps1-2 showed significant reductions in rhizobial infection (intracellular and intercellular) and nodule organogenesis and a delay in AM colonization. RNAseq analysis of dahps1-2 roots suggested that these phenotypes are associated with downregulation of several cell wall-related genes, and with an attenuated signaling response. Interestingly, the dahps1 mutants showed no detectable pleiotropic effects, suggesting a more selective recruitment of this gene in certain biological processes. This work provides robust evidence linking AAA metabolism to root hair development and successful symbiotic associations.

Roles of a CCR4-NOT complex component GmNOT4-1 in regulating soybean nodulation.

Legume-rhizobial symbiotic nitrogen fixation is the most efficient nitrogen assimilation system in the ecosystem. In the special interaction between organ-root nodules, legumes supply rhizobial carbohydrates for their proliferation, while rhizobials provide host plants with absorbable nitrogen. Nodule initiation and formation require a complex molecular dialogue between legumes and rhizobia, which involves the accurate regulation of a series of legume genes. The CCR4-NOT complex is a conserved multi-subunit complex with functions regulating gene expression in many cellular processes. However, the functions of the CCR4-NOT complex in rhizobia-host interactions remain unclear. In this study, we identified seven members of the NOT4 family in soybean and further classified them into three subgroups. Bioinformatic analysis showed that NOT4s shared relatively conserved motifs and gene structures in each subgroup, while there were significant differences between NOT4s in the different subgroups. Expression profile analysis indicated that NOT4s may be involved in nodulation in soybean, as most of them were induced by Rhizobium infection and highly expressed in nodules. We further selected GmNOT4-1 to clarify the biological function of these genes in soybean nodulation. Interestingly, we found that either GmNOT4-1 overexpression or down-regulation of GmNOT4-1 by RNAi or CRISPR/Cas9 gene editing would suppress the number of nodules in soybean. Intriguingly, alterations in the expression of GmNOT4-1 repressed the expression of genes in the Nod factor signaling pathway. This research provides new insight into the function of the CCR4-NOT family in legumes and reveals GmNOT4-1 to be a potent gene for regulating symbiotic nodulation.

GFP labeling of a Bradyrhizobium strain and an attempt to track the crack entry process during symbiosis with peanuts.

Compared to the well-studied model legumes, where symbiosis is established via root hair entry, the peanut is infected by Bradyrhizobium through the crack entry, which is less common and not fully understood. Crack entry is, however, considered a primitive symbiotic infection pathway, which could be potentially utilized for engineering non-legume species with nitrogen fixation ability. We utilized a fluorescence-labeled Bradyrhizobium strain to help in understanding the crack entry process at the cellular level. A modified plasmid pRJPaph-bjGFP, harboring the codon-optimized GFP gene and tetracycline resistance gene, was created and conjugated into Bradyrhizobium strain Lb8, an isolate from peanut nodules, through tri-parental mating. Microscopic observation and peanut inoculation assays confirmed the successful GFP tagging of Lb8, which is capable of generating root nodules. A marking system for peanut root potential infection sites and an optimized sample preparation protocol for cryostat sectioning was developed. The feasibility of using the GFP-tagged Lb8 for observing crack entry was examined. GFP signal was detected at the nodule primordial stage and the following nodule developmental stages with robust GFP signals observed in infected cells in the mature nodules. Spherical bacteroids in the root tissue were visualized at the nodules' inner cortex under higher magnification, reflecting the trace along the rhizobial infection path. The GFP labeled Lb8 can serve as an essential tool for plant-microbe studies between the cultivated peanut and Bradyrhizobium, which could facilitate further study of the crack entry process during the legume-rhizobia symbiosis.

A glycan receptor kinase facilitates intracellular accommodation of arbuscular mycorrhiza and symbiotic rhizobia in the legume Lotus japonicus.

Receptors that distinguish the multitude of microbes surrounding plants in the environment enable dynamic responses to the biotic and abiotic conditions encountered. In this study, we identify and characterise a glycan receptor kinase, EPR3a, closely related to the exopolysaccharide receptor EPR3. Epr3a is up-regulated in roots colonised by arbuscular mycorrhizal (AM) fungi and is able to bind glucans with a branching pattern characteristic of surface-exposed fungal glucans. Expression studies with cellular resolution show localised activation of the Epr3a promoter in cortical root cells containing arbuscules. Fungal infection and intracellular arbuscule formation are reduced in epr3a mutants. In vitro, the EPR3a ectodomain binds cell wall glucans in affinity gel electrophoresis assays. In microscale thermophoresis (MST) assays, rhizobial exopolysaccharide binding is detected with affinities comparable to those observed for EPR3, and both EPR3a and EPR3 bind a well-defined β-1,3/β-1,6 decasaccharide derived from exopolysaccharides of endophytic and pathogenic fungi. Both EPR3a and EPR3 function in the intracellular accommodation of microbes. However, contrasting expression patterns and divergent ligand affinities result in distinct functions in AM colonisation and rhizobial infection in Lotus japonicus. The presence of Epr3a and Epr3 genes in both eudicot and monocot plant genomes suggest a conserved function of these receptor kinases in glycan perception.

NODULE INCEPTION: a direct regulator of gibberellin biosynthesis during symbiotic nodulation.

This article is a Commentary on Gao et al. (2023), 239: 459–465.

GmNLP7a inhibits soybean nodulation by interacting with GmNIN1a

Nitrogen (N) is an essential macronutrient for plant growth and productivity. Leguminous plants establish symbiotic relationships with nitrogen-fixing rhizobial bacteria to use atmospheric dinitrogen gas to meet high N demand under low-N conditions. Nodule formation and N fixation are energy-consuming processes and are inhibited by nitrate present in the environment. Previous studies in model leguminous plants characterized NIN-LIKE PROTEIN (NLP) proteins that mediate nitrate control of root nodule symbiosis, but the mechanism by which nitrate regulates soybean root nodules via NLP remains unclear. In the soybean genome we found four homologs of AtNLP7, named GmNLP7a–GmNLP7d. We showed that the expression of GmNLP7s is responsive to nitrate but not to rhizobial infection and localized GmNLP7a to the nucleus. Downregulation of GmNLP7s increased nodule number, and overexpression of GmNLP7a (GmNLP7aOE) reduced nodule number regardless of nitrate availability, suggesting a negative role for GmNLP7s in nodulation. Nitrogenase activity in the GmNLP7aOE line was comparable to that of the wild type, indicating that GmNLP7a does not affect mature nodule activity. Overexpression of GmNLP7a downregulated the expression of GmNIN1a and GmENOD40-1. GmNLP7a interacted with GmNIN1a via the PB1 domain. Our results reveal a new regulator of GmNLP7 in nodulation and a molecular mechanism by which nitrate affects nodule number in soybean.

Genome-Wide Identification and Characterization of the Soybean Snf2 Gene Family and Expression Response to Rhizobia.

Sucrose nonfermenting 2 (Snf2) family proteins are the core component of chromatin remodeling complexes that can alter chromatin structure and nucleosome position by utilizing the energy of ATP, playing a vital role in transcription regulation, DNA replication, and DNA damage repair. Snf2 family proteins have been characterized in various species including plants, and they have been found to regulate development and stress responses in Arabidopsis. Soybean (Glycine max) is an important food and economic crop worldwide, unlike other non-leguminous crops, soybeans can form a symbiotic relationship with rhizobia for biological nitrogen fixation. However, little is known about Snf2 family proteins in soybean. In this study, we identified 66 Snf2 family genes in soybean that could be classified into six groups like Arabidopsis, unevenly distributed on 20 soybean chromosomes. Phylogenetic analysis with Arabidopsis revealed that these 66 Snf2 family genes could be divided into 18 subfamilies. Collinear analysis showed that segmental duplication was the main mechanism for expansion of Snf2 genes rather than tandem repeats. Further evolutionary analysis indicated that the duplicated gene pairs had undergone purifying selection. All Snf2 proteins contained seven domains, and each Snf2 protein had at least one SNF2_N domain and one Helicase_C domain. Promoter analysis revealed that most Snf2 genes had cis-elements associated with jasmonic acid, abscisic acid, and nodule specificity in their promoter regions. Microarray data and real-time quantitative PCR (qPCR) analysis revealed that the expression profiles of most Snf2 family genes were detected in both root and nodule tissues, and some of them were found to be significantly downregulated after rhizobial infection. In this study, we conducted a comprehensive analysis of the soybean Snf2 family genes and demonstrated their responsiveness to Rhizobia infection. This provides insight into the potential roles of Snf2 family genes in soybean symbiotic nodulation.

Identification and Characterization of Common Bean (Phaseolus vulgaris) Non-Nodulating Mutants Altered in Rhizobial Infection.

The symbiotic N2-fixation process in the legume-rhizobia interaction is relevant for sustainable agriculture. The characterization of symbiotic mutants, mainly in model legumes, has been instrumental for the discovery of symbiotic genes, but similar studies in crop legumes are scant. To isolate and characterize common bean (Phaseolus vulgaris) symbiotic mutants, an ethyl methanesulphonate-induced mutant population from the BAT 93 genotype was analyzed. Our initial screening of Rhizobium etli CE3-inoculated mutant plants revealed different alterations in nodulation. We proceeded with the characterization of three non-nodulating (nnod), apparently monogenic/recessive mutants: nnod(1895), nnod(2353) and nnod(2114). Their reduced growth in a symbiotic condition was restored when the nitrate was added. A similar nnod phenotype was observed upon inoculation with other efficient rhizobia species. A microscopic analysis revealed a different impairment for each mutant in an early symbiotic step. nnod(1895) formed decreased root hair curling but had increased non-effective root hair deformation and no rhizobia infection. nnod(2353) produced normal root hair curling and rhizobia entrapment to form infection chambers, but the development of the latter was blocked. nnod(2114) formed infection threads that did not elongate and thus did not reach the root cortex level; it occasionally formed non-infected pseudo-nodules. The current research is aimed at mapping the responsible mutated gene for a better understanding of SNF in this critical food crop.

RPG acts as a central determinant for infectosome formation and cellular polarization during intracellular rhizobial infections.

Host-controlled intracellular accommodation of nitrogen-fixing bacteria is essential for the establishment of a functional Root Nodule Symbiosis (RNS). In many host plants, this occurs via transcellular tubular structures (infection threads - ITs) that extend across cell layers via polar tip-growth. Comparative phylogenomic studies have identified RPG (RHIZOBIUM-DIRECTED POLAR GROWTH) among the critical genetic determinants for bacterial infection. In Medicago truncatula, RPG is required for effective IT progression within root hairs but the cellular and molecular function of the encoded protein remains elusive. Here, we show that RPG resides in the protein complex formed by the core endosymbiotic components VAPYRIN (VPY) and LUMPY INFECTION (LIN) required for IT polar growth, co-localizes with both VPY and LIN in IT tip- and perinuclear-associated puncta of M. truncatula root hairs undergoing infection and is necessary for VPY recruitment into these structures. Fluorescence Lifetime Imaging Microscopy (FLIM) of phosphoinositide species during bacterial infection revealed that functional RPG is required to sustain strong membrane polarization at the advancing tip of the IT. In addition, loss of RPG functionality alters the cytoskeleton-mediated connectivity between the IT tip and the nucleus and affects the polar secretion of the cell wall modifying enzyme NODULE PECTATE LYASE (NPL). Our results integrate RPG into a core host machinery required to support symbiont accommodation, suggesting that its occurrence in plant host genomes is essential to co-opt a multimeric protein module committed to endosymbiosis to sustain IT-mediated bacterial infection.

RPG interacts with E3-ligase CERBERUS to mediate rhizobial infection in Lotus japonicus.

Symbiotic interactions between rhizobia and legumes result in the formation of root nodules, which fix nitrogen that can be used for plant growth. Rhizobia usually invade legume roots through a plant-made tunnel-like structure called an infection thread (IT). RPG (Rhizobium-directed polar growth) encodes a coiled-coil protein that has been identified in Medicago truncatula as required for root nodule infection, but the function of RPG remains poorly understood. In this study, we identified and characterized RPG in Lotus japonicus and determined that it is required for IT formation. RPG was induced by Mesorhizobium loti or purified Nodulation factor and displayed an infection-specific expression pattern. Nodule inception (NIN) bound to the RPG promoter and induced its expression. We showed that RPG displayed punctate subcellular localization in L. japonicus root protoplasts and in root hairs infected by M. loti. The N-terminal predicted C2 lipid-binding domain of RPG was not required for this subcellular localization or for function. CERBERUS, a U-box E3 ligase which is also required for rhizobial infection, was found to be localized similarly in puncta. RPG co-localized and directly interacted with CERBERUS in the early endosome (TGN/EE) compartment and near the nuclei in root hairs after rhizobial inoculation. Our study sheds light on an RPG-CERBERUS protein complex that is involved in an exocytotic pathway mediating IT elongation.

Exogenously Applied Cytokinin Altered the Bacterial Release and Subsequent Stages of Nodule Development in Pea Ipd3/Cyclops Mutant.

Regulation of plant hormonal status is one of the major targets of symbiotic signaling during nodule formation in legume plants. However, the genetic and hormonal networks that regulate transition to differentiation of nodules are not well-characterized in legume plants. Analysis of plant mutants forming nodules impaired in rhizobial infection allowed us to identify some regulators involved in the control of the later stages of nodule development. In the current work, we extend our earlier studies on the influence of exogenously applied cytokinin on the later stages of nodule morphogenesis using pea sym33 (ipd3/cyclops) mutants impaired in the gene encoding IPD3/CYCLOPS transcription factor. One of the noticeable effects of the influence of exogenously applied cytokinin on nodules in the sym33-3 mutant was an increasing size of these structures. Cytokinin treatment was shown to stimulate bacterial release and increase the percentage of infected cells in nodules. To explore the role of possible regulators of nodule differentiation, we performed searching in pea transcriptome. The transcriptome study in pea P. sativum revealed the importance of the CCS52 regulator, EFD transcription factor, SYMREM regulator, RSD, the MADS-domain/AGL, and SHORT INTERNODE/STYLISH gene families encoding transcription factors in the control of nodule differentiation. Analysis of the expression patterns was verified by real-time PCR in response to exogenously applied cytokinin treatment.

Identification and Molecular Characterization of RWP-RK Transcription Factors in Soybean.

The RWP-RK is a small family of plant-specific transcription factors that are mainly involved in nitrate starvation responses, gametogenesis, and root nodulation. To date, the molecular mechanisms underpinning nitrate-regulated gene expression in many plant species have been extensively studied. However, the regulation of nodulation-specific NIN proteins during nodulation and rhizobial infection under nitrogen starvation in soybean still remain unclear. Here, we investigated the genome-wide identification of RWP-RK transcription factors and their essential role in nitrate-inducible and stress-responsive gene expression in soybean. In total, 28 RWP-RK genes were identified from the soybean genome, which were unevenly distributed on 20 chromosomes from 5 distinct groups during phylogeny classification. The conserved topology of RWP-RK protein motifs, cis-acting elements, and functional annotation has led to their potential as key regulators during plant growth, development, and diverse stress responses. The RNA-seq data revealed that the up-regulation of GmRWP-RK genes in the nodules indicated that these genes might play crucial roles during root nodulation in soybean. Furthermore, qRT-PCR analysis revealed that most GmRWP-RK genes under Phytophthora sojae infection and diverse environmental conditions (such as heat, nitrogen, and salt) were significantly induced, thus opening a new window of possibilities into their regulatory roles in adaptation mechanisms that allow soybean to tolerate biotic and abiotic stress. In addition, the dual luciferase assay indicated that GmRWP-RK1 and GmRWP-RK2 efficiently bind to the promoters of GmYUC2, GmSPL9, and GmNIN, highlighting their possible involvement in nodule formation. Together, our findings provide novel insights into the functional role of the RWP-RK family during defense responses and root nodulation in soybean.

The effects of ERN1 on gene expression during early rhizobial infection in Lotus japonicus.

Legumes develop root nodules in association with compatible rhizobia to overcome nitrogen deficiency. Rhizobia enter the host legume, mainly through infection threads, and induce nodule primordium formation in the root cortex. Multiple transcription factors have been identified to be involved in the regulation of the establishment of root nodule symbiosis, including ERF Required for Nodulation1 (ERN1). ERN1 is involved in a transcription network with CYCLOPS and NODULE INCEPTION (NIN). Mutation of ERN1 often results in misshapen root hair tips, deficient infection thread formation, and immature root nodules. ERN1 directly activates the expression of ENOD11 in Medicago truncatula to assist cell wall remodeling and Epr3 in Lotus japonicus to distinguish rhizobial exopolysaccharide signals. However, aside from these two genes, it remains unclear which genes are regulated by LjERN1 or what role LjERN1 plays during root nodule symbiosis. Thus, we conducted RNA sequencing to compare the gene expression profiles of wild-type L. japonicus and Ljern1-6 mutants. In total, 234 differentially expressed genes were identified as candidate LjERN1 target genes. These genes were found to be associated with cell wall remodeling, signal transduction, phytohormone metabolism, and transcription regulation, suggesting that LjERN1 is involved in multiple processes during the early stages of the establishment of root nodule symbiosis. Many of these candidate genes including RINRK1 showed decreased expression levels in Ljnin-2 mutants based on a search of a public database, suggesting that LjERN1 and LjNIN coordinately regulate gene expression. Our data extend the current understanding of the pleiotropic role of LjERN1 in root nodule symbiosis.

A novel secreted protein, NISP1, is phosphorylated by soybean Nodulation Receptor Kinase to promote nodule symbiosis.

Nodulation Receptor Kinase (NORK) functions as a co-receptor of Nod factor receptors to mediate rhizobial symbiosis in legumes, but its direct phosphorylation substrates that positively mediate root nodulation remain to be fully identified. Here, we identified a GmNORK-Interacting Small Protein (GmNISP1) that functions as a phosphorylation target of GmNORK to promote soybean nodulation. GmNORKα directly interacted with and phosphorylated GmNISP1. Transcription of GmNISP1 was strongly induced after rhizobial infection in soybean roots and nodules. GmNISP1 encodes a peptide containing 90 amino acids with a "DY" consensus motif at its N-terminus. GmNISP1 protein was detected to be present in the apoplastic space. Phosphorylation of GmNISP1 by GmNORKα could enhance its secretion into the apoplast. Pretreatment with either purified GmNISP1 or phosphorylation-mimic GmNISP112D on the roots could significantly increase nodule numbers compared with the treatment with phosphorylation-inactive GmNISP112A . The data suggested a model that soybean GmNORK phosphorylates GmNISP1 to promote its secretion into the apoplast, which might function as a potential peptide hormone to promote root nodulation.

The Role of GmSnRK1-GmNodH Module in Regulating Soybean Nodulation Capacity.

SnRK1 protein kinase plays hub roles in plant carbon and nitrogen metabolism. However, the function of SnRK1 in legume nodulation and symbiotic nitrogen fixation is still elusive. In this study, we identified GmNodH, a putative sulfotransferase, as an interacting protein of GmSnRK1 by yeast two-hybrid screen. The qRT-PCR assays indicate that GmNodH gene is highly expressed in soybean roots and could be induced by rhizobial infection and nitrate stress. Fluorescence microscopic analyses showed that GmNodH was colocalized with GsSnRK1 on plasma membrane. The physical interaction between GmNodH and GmSnRK1 was further verified by using split-luciferase complementary assay and pull-down approaches. In vitro phosphorylation assay showed that GmSnRK1 could phosphorylate GmNodH at Ser193. To dissect the function and genetic relationship of GmSnRK1 and GmNodH in soybean, we co-expressed the wild-type and mutated GmSnRK1 and GmNodH genes in soybean hairy roots and found that co-expression of GmSnRK1/GmNodH genes significantly promoted soybean nodulation rates and the expression levels of nodulation-related GmNF5α and GmNSP1 genes. Taken together, this study provides the first biological evidence that GmSnRK1 may interact with and phosphorylate GmNodH to synergistically regulate soybean nodulation.

Discovering the genetic modules controlling root nodule symbiosis under abiotic stresses: salinity as a case study.

Legumes form a symbiotic association with rhizobia and fix atmospheric nitrogen in specialized root organs known as nodules. It is well known that salt stress inhibits root nodule symbiosis by decreasing rhizobial growth, rhizobial infection, nodule number, and nitrogenase activity in diverse legumes. Despite this knowledge, the genetic and molecular mechanisms governing salt stress's inhibition of nodulation and nitrogen fixation are still elusive. In this Viewpoint, we summarize the most recent knowledge of the genetic mechanisms that shape this symbiosis according to the salt levels in the soil. We emphasize the relevance of modulating the activity of the transcription factor Nodule Inception to properly shape the symbiosis with rhizobia accordingly. We also highlight the knowledge gaps that are critical for gaining a deeper understanding of the molecular mechanisms underlying the adaptation of the root nodule symbiosis to salt-stress conditions. We consider that filling these gaps can help to improve legume nodulation and harness its ecological benefits even under salt-stress conditions.

Overexpression of GmPHR1 Promotes Soybean Yield through Global Regulation of Nutrient Acquisition and Root Development

MYB-CC transcription factors (TFs) are essential for plant growth and development. Members of the MYB-CC subfamily with long N terminal domains, such as phosphate starvation response 1 (PHR1) or PHR1-like TFs, have well documented functions, while those with short N terminal domains remain less understood. In this study, we identified a nodule specific MYB-CC transcription factor 1 (GmPHR1) in soybean that is different from other canonical PHR family genes in that GmPHR1 harbors a short N terminal ahead of its MYB-CC domain and was highly induced by rhizobium infection. The overexpression of GmPHR1 dramatically increased the ratio of deformed root hairs, enhanced subsequent soybean nodulation, and promoted soybean growth in pot experiments. The growth promotion effects of GmPHR1 overexpression were further demonstrated in field trails in which two GmPHR1-OE lines yielded 10.78% and 8.19% more than the wild type line. Transcriptome analysis suggested that GmPHR1 overexpression led to global reprogramming, with 749 genes upregulated and 279 genes downregulated, especially for genes involved in MYB transcription factor activities, root growth, and nutrient acquisition. Taken together, we conclude that GmPHR1 is a key gene involved in the global regulation of nodulation, root growth, and nutrient acquisition in soybeans, and is thus a promising candidate gene to target for soybean yield enhancement.