Symbiotic Gene Expression Research Articles

Effects of a Protease Inhibitor Protein on Buchnera aphidicola and Gene Expression in Pea Aphids (Acyrthosiphon pisum)

ABSTRACTAphids are serious insect pests for agricultural and horticultural crops and may cause the major economic losses. Insecticides used to control aphids have caused environmental pollution and the insecticide residues in agricultural products. A new protease inhibitor gene from bacterium Xenorhabdus bovienii (Xbpi‐1) and the protease inhibitor protein expressed by the gene against the pea aphid (Acyrthosiphon pisum Harris; Hemiptera: Aphididae) have been reported, however, effects of the PIP on the symbiotic bacteria, Buchnera aphidicola, and gene expression in pea aphids are unknown. By assessing the quantity of B. aphidicola, the primary symbiotic bacterium, in the aphid fed on an artificial diet containing 100 and 500 μg/mL Xbpi‐1, we observed a substantial reduction in its population by 27% and 46%, respectively, as analysed by real‐time quantitative reverse transcription PCR (qRT‐PCR). Furthermore, the growth of other aphid‐associated bacteria was also significantly inhibited by Xbpi‐1. To elucidate the mechanisms at the gene level, we conducted transcriptome analysis and identified differentially expressed genes (DEGs). Subsequent Gene Ontology (GO) analysis of the 213 DEGs shed light on the impact of Xbpi‐1 on aphid metabolism processes and gene expression. Notably, the results highlighted several aphid nutrient metabolism pathways affected by Xbpi‐1, which are relevant to vector‐borne diseases. These pathways encompass crucial factors such as heat shock proteins, cuticle proteins and proteases. The results from this study revealed that the PIP had a novel mechanism against pea aphids by having adverse effects on the primary symbiotic bacteria in pea aphids and affecting aphid gene expression, showing that the PIP may be a promise bioinsecticide for aphid control in the future.

The phosphate starvation response regulator PHR2 antagonizes arbuscule maintenance in Medicago.

Phosphate starvation response (PHR) transcription factors play essential roles in regulating phosphate uptake in plants through binding to the P1BS cis-element in the promoter of phosphate starvation response genes. Recently, PHRs were also shown to positively regulate arbuscular mycorrhizal colonization in rice and lotus by controlling the expression of many symbiotic genes. However, their role in arbuscule development has remained unclear. In Medicago, we previously showed that arbuscule degradation is controlled by two SPX proteins that are highly expressed in arbuscule-containing cells. Since SPX proteins bind to PHRs and repress their activity in a phosphate-dependent manner, we investigated whether arbuscule maintenance is also regulated by PHR. Here, we show that PHR2 is a major regulator of the phosphate starvation response in Medicago. Knockout of phr2 showed reduced phosphate starvation response, symbiotic gene expression, and fungal colonization levels. However, the arbuscules that formed showed less degradation, suggesting a negative role for PHR2 in arbuscule maintenance. This was supported by the observation that overexpression of PHR2 led to enhanced degradation of arbuscules. Although many arbuscule-induced genes contain P1BS elements in their promoters, we found that the P1BS cis-elements in the promoter of the symbiotic phosphate transporter PT4 are not required for arbuscule-containing cell expression. Since both PHR2 and SPX1/3 negatively affect arbuscule maintenance, our results indicate that they control arbuscule maintenance partly via different mechanisms. While PHR2 potentiates symbiotic gene expression and colonization, its activity in arbuscule-containing cells needs to be tightly controlled to maintain a successful symbiosis in Medicago.

Identification and characterization of the TetR family transcriptional regulator NffT in Rhizobium johnstonii.

Symbiotic nitrogen fixation (SNF) by rhizobia is not only the main natural bionitrogen-source for organisms but also a green process leveraged to increase the fertility of soil for agricultural production. However, an insufficient understanding of the regulatory mechanism of SNF hinders its practical application. During SNF, nifA-fixA signaling is essential for the biosynthesis of nitrogenases and electron transfer chain proteins. In the present study, the TetR regulator NffT, whose mutation increased fixA expression, was discovered through a fixA-promoter-β-glucuronidase fusion assay performed with Rhizobium johnstonii. Real-time quantitative PCR analysis showed that nffT deletion increased the expression of symbiotic genes including nifA and fixA in nifA-fixA signaling, and fixL, fixK, fnrN, and fixN9 in fixL-fixN signaling. nffT overexpression resulted in disordered nodules and reduced nitrogen-fixing efficiency. Electrophoretic mobility shift assays revealed that NffT directly regulated the transcription of RL0091-93, which encode an ATP-binding ABC transporter predicted to be involved in carbohydrate transport. Purified His-tagged NffT bound to a 68 bp DNA sequence located -32 to -99 bp upstream of RL0091-93 and NffT deletion significantly increased the expression of RL0091-93. nffT-promoter-β-glucuronidase fusion assay indicated that nffT expression was regulated by the cobNTS genes and cobalamin. Mutations in cobNTS significantly decreased the expression of nffT, and cobalamin restored its expression. These results revealed that NffT affects nodule development and nitrogen-fixing reaction by participating in a complex regulatory network of symbiotic and carbohydrate metabolic genes and, thus, plays a pivotal regulatory role during symbiosis of R. johnstonii-Pisum sativum.IMPORTANCESymbiotic nitrogen fixation (SNF) by rhizobia is a green way to maintain soil fertility without causing environmental pollution or consuming chemical energy. A detailed understanding of the regulatory mechanism of this complex process is essential for promoting sustainable agriculture. In this study, we discovered the TetR-type regulator NffT, which suppressed the expression of fixA in Rhizobium johnstonii. Furthermore, NffT was confirmed to play pleiotropic roles in R. johnstonii-Pisum sativum symbiosis; specifically, it inhibited rhizobial growth, nodule differentiation, and nitrogen-fixing reactions. We revealed that NffT indirectly affected R. johnstonii-P. sativum symbiosis by participating in a complex regulatory network of symbiotic and carbohydrate metabolic genes. Furthermore, cobalamin, a chemical molecule, was reported for the first time to be involved in TetR-type protein transcription during symbiosis. Thus, NffT identification connects SNF regulation with genetic, metabolic, and chemical signals and provides new insights into the complex regulation of SNF, laying an experimental basis for the targeted construction of rhizobial strains with highly efficient nitrogen-fixing capacity.

Effects of scale worm parasitism on interactions between the symbiotic gill microbiome and gene regulation in deep sea mussel hosts.

Diverse adaptations to the challenging deep sea environment are expected to be found across all deep sea organisms. Scale worms Branchipolynoe pettiboneae are believed to adapt to the deep sea environment by parasitizing deep sea mussels; this biotic interaction is one of most known in the deep sea chemosynthetic ecosystem. However, the mechanisms underlying the effects of scale worm parasitism on hosts are unclear. Previous studies have revealed that the microbiota plays an important role in host adaptability. Here, we compared gill-microbiota, gene expression and host-microorganism interactions in a group of deep sea mussels (Gigantidas haimaensis) parasitized by scale worm (PA group) and a no parasitic control group (NPA group). The symbiotic microorganism diversity of the PA group significantly decreased than NPA group, while the relative abundance of chemoautotrophic symbiotic bacteria that provide the host with organic carbon compounds significantly increased in PA. Interestingly, RNA-seq revealed that G. haimaensis hosts responded to B. pettiboneaei parasitism through significant upregulation of protein and lipid anabolism related genes, and that this parasitism may enhance host mussel nutrient anabolism but inhibit the host’s ability to absorb nutrients, thus potentially helping the parasite obtain nutrients from the host. In an integrated analysis of the interactions between changes in the microbiota and host gene dysregulation, we found an agreement between the microbiota and transcriptomic responses to B. pettiboneaei parasitism. Together, our findings provide new insights into the effects of parasite scale worms on changes in symbiotic bacteria and gene expression in deep sea mussel hosts. We explored the potential role of host-microorganism interactions between scale worms and deep sea mussels, and revealed the mechanisms through which scale worm parasitism affects hosts in deep sea chemosynthetic ecosystem.

Nitrate transport via NRT2.1 mediates NIN-LIKE PROTEIN-dependent suppression of root nodulation in Lotus japonicus.

Legumes have adaptive mechanisms that regulate nodulation in response to the amount of nitrogen in the soil. In Lotus japonicus, two NODULE INCEPTION (NIN)-LIKE PROTEIN (NLP) transcription factors, LjNLP4 and LjNLP1, play pivotal roles in the negative regulation of nodulation by controlling the expression of symbiotic genes in high nitrate conditions. Despite an improved understanding of the molecular basis for regulating nodulation, how nitrate plays a role in the signaling pathway to negatively regulate this process is largely unknown. Here, we show that nitrate transport via NITRATE TRANSPORTER 2.1 (LjNRT2.1) is a key step in the NLP signaling pathway to control nodulation. A mutation in the LjNRT2.1 gene attenuates the nitrate-induced control of nodulation. LjNLP1 is necessary and sufficient to induce LjNRT2.1 expression, thereby regulating nitrate uptake/transport. Our data suggest that LjNRT2.1-mediated nitrate uptake/transport is required for LjNLP4 nuclear localization and induction/repression of symbiotic genes. We further show that LjNIN, a positive regulator of nodulation, counteracts the LjNLP1-dependent induction of LjNRT2.1 expression, which is linked to a reduction in nitrate uptake. These findings suggest a plant strategy in which nitrogen acquisition switches from obtaining nitrogen from the soil to symbiotic nitrogen fixation.

Salt Stress Enhances Early Symbiotic Gene Expression in Medicago truncatula and Induces a Stress-Specific Set of Rhizobium-Responsive Genes

Salt stress is a major agricultural concern inhibiting not only plant growth but also the symbiotic association between legume roots and the soil bacteria rhizobia. This symbiotic association is initiated by a molecular dialogue between the two partners, leading to the activation of a signaling cascade in the legume host and, ultimately, the formation of nitrogen-fixing root nodules. Here, we show that a moderate salt stress increases the responsiveness of early symbiotic genes in Medicago truncatula to its symbiotic partner, Sinorhizobium meliloti while, conversely, inoculation with S. meliloti counteracts salt-regulated gene expression, restoring one-third to control levels. Our analysis of early nodulin 11 (ENOD11) shows that salt-induced expression is dynamic, Nod-factor dependent, and requires the ionic but not the osmotic component of salt. We demonstrate that salt stimulation of rhizobium-induced gene expression requires NSP2, which functions as a node to integrate the abiotic and biotic signals. In addition, our work reveals that inoculation with S. meliloti succinoglycan mutants also hyperinduces ENOD11 expression in the presence or absence of salt, suggesting a possible link between rhizobial exopolysaccharide and the plant response to salt stress. Finally, we identify an accessory set of genes that are induced by rhizobium only under conditions of salt stress and have not been previously identified as being nodulation-related genes. Our data suggest that interplay of core nodulation genes with different accessory sets, specific for different abiotic conditions, functions to establish the symbiosis. Together, our findings reveal a complex and dynamic interaction between plant, microbe, and environment. [Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY 4.0 International license .

GSK3-mediated stress signaling inhibits legume–rhizobium symbiosis by phosphorylating GmNSP1 in soybean

Legumes establish symbiotic associations with rhizobia for biological nitrogen fixation. This process is highly regulated by various abiotic stresses, but the underlying genetic and molecular mechanisms remain largely unknown. In this study, we discovered that the glycogen synthase kinase 3 (GSK3)-like kinase, GmSK2-8, plays an important role in inhibiting symbiotic signaling and nodule formation in soybean (Glycine max) under salt stress. We found that GmSK2-8 is strongly induced in soybean under high-salt conditions, while GmSK2-8 could interact with two G. max Nodulation Signaling Pathway 1 (GmNSP1) proteins, GmNSP1a and GmNSP1b; these key transcription factors are essential for rhizobial infection, nodule initiation, and symbiotic gene expression in soybean. Furthermore, we demonstrated that GmSK2-8 phosphorylates the LHRI domain of GmNSP1a, inhibits its binding to the promoters of symbiotic genes, and thus suppresses nodule formation under salt stress. Knockdown of GmSK2-8 and its close homologs also resulted in reduced plant sensitivity to salt stress during nodule formation. Taken together, our findings indicate that GSK3-like kinases directly regulate the activities of GmNSP1s to mediate salt-inhibited legume–rhizobium symbiosis, providing novel targets for improving symbiotic nitrogen fixation under environmental stress conditions in soybean and possibly other legumes.

Genome-Wide Identification, Characterization, and Regulation of RWP-RK Gene Family in the Nitrogen-Fixing Clade.

RWP-RK is a plant-specific family of transcription factors, involved in nitrate response, gametogenesis, and nodulation. However, genome-wide characterization, phylogeny, and the regulation of RWP-RK genes in the nodulating and non-nodulating plant species of nitrogen-fixing clade (NFC) are widely unknown. Therefore, we identified a total of 292 RWP-RKs, including 278 RWP-RKs from 25 NFC species and 14 RWP-RKs from the outgroup, Arabidopsis thaliana. We classified the 292 RWP-RKs in two subfamilies: the NIN-like proteins (NLPs) and the RWP-RK domain proteins (RKDs). The transcriptome and phylogenetic analysis of RWP-RKs suggested that, compared to RKD genes, the NLP genes were just upregulated in nitrate response and nodulation. Moreover, nodule-specific NLP genes of some nodulating NFC species may have a common ancestor (OG0002084) with AtNLP genes in A. thaliana. Further, co-expression networks of A.thaliana under N-starvation and N-supplementation conditions revealed that there is a higher correlation between expression of AtNLP genes and symbiotic genes during N-starvation. In P. vulgaris, we confirmed that N-starvation stimulated nodulation by regulating expression of PvNLP2, closely related to AtNLP6 and AtNLP7 with another common origin (OG0004041). Taken together, we concluded that different origins of the NLP genes involved in both N-starvation response and specific expression of nodulation would contribute to the evolution of nodulation in NFC plant species. Our results shed light on the phylogenetic relationships of NLP genes and their differential regulation in nitrate response of A. thaliana and nodulation of NFC.

Sinorhizobium fredii HH103 syrM inactivation affects the expression of a large number of genes, impairs nodulation with soybean and extends the host-range to Lotus japonicus.

Sinorhizobium fredii HH103 RifR is a broad host-range rhizobial strain able to nodulate with soybean and Lotus burttii, but it is ineffective with L. japonicus. Here, we study the role of the HH103 RifR SyrM protein in the regulation of gene expression and its relevance in symbiosis with those three legumes. RNAseq analyses show that HH103 SyrM is an important transcriptional regulator not only in the presence of inducer flavonoids but also in its absence. Lack of SyrM increases Nod factors production and decreases genistein-mediated repression of exopolysaccharide production in HH103. In symbiosis, mutation of syrM partially impaired interaction with soybean but improves effectiveness with L. burttii and extends the host-rage to L. japonicus Gifu. In addition, HH103 syrM mutants enter in both Lotus species by infection threads, whereas HH103 uses the more primitive intercellular infection to enter into L. burttii roots These symbiotic phenotypes were previously observed in two other HH103 mutants affected in symbiotic regulators, nodD2 and nolR, revealing that in S. fredii HH103 numerous transcriptional regulators finely modulate symbiotic gene expression.

A Phosphate-Dependent Requirement for Transcription Factors IPD3 and IPD3L During Arbuscular Mycorrhizal Symbiosis in Medicago truncatula.

During arbuscular mycorrhizal (AM) symbiosis, activation of a symbiosis signaling pathway induces gene expression necessary for accommodation of AM fungi. Here, we focus on pathway components Medicago truncatula INTERACTING PROTEIN OF DOES NOT MAKE INFECTIONS 3 (IPD3) and IPD3 LIKE (IPD3L), which are potential orthologs of Lotus japonicus CYCLOPS, a transcriptional regulator essential for AM symbiosis. In the double mutant ipd3 ipd3l, hyphal entry through the epidermis and overall colonization levels are reduced relative to the wild type but fully developed arbuscules are present in the cortex. In comparison with the wild type, colonization of ipd3 ipd3l is acutely sensitive to higher phosphate levels in the growth medium, with a disproportionate decrease in epidermal penetration, overall colonization, and symbiotic gene expression. When constitutively expressed in ipd3 ipd3l, an autoactive DOES NOT MAKE INFECTIONS 3 induces the expression of transcriptional regulators REDUCED ARBUSCULAR MYCORRHIZA 1 and REQUIRED for ARBUSCULE DEVELOPMENT 1, providing a possible avenue for arbuscule development in the absence of IPD3 and IPD3L. An increased sensitivity of ipd3 ipd3l to GA3 suggests an involvement of DELLA. The data reveal partial redundancy in the symbiosis signaling pathway, which may ensure robust signaling in low-phosphorus environments, while IPD3 and IPD3L maintain signaling in higher-phosphorus environments. The latter may buffer the pathway from short-term variation in phosphorus levels encountered by roots during growth in heterogeneous soil environments.

Whole-genome landscape of Medicago truncatula symbiotic genes.

Advances in deciphering the functional architecture of eukaryotic genomes have been facilitated by recent breakthroughs in sequencing technologies, enabling a more comprehensive representation of genes and repeat elements in genome sequence assemblies, as well as more sensitive and tissue-specific analyses of gene expression. Here we show that PacBio sequencing has led to a substantially improved genome assembly of Medicago truncatula A17, a legume model species notable for endosymbiosis studies1, and has enabled the identification of genome rearrangements between genotypes at a near-base-pair resolution. Annotation of the new M. truncatula genome sequence has allowed for a thorough analysis of transposable elements and their dynamics, as well as the identification of new players involved in symbiotic nodule development, in particular 1,037 upregulated long non-coding RNAs (lncRNAs). We have also discovered that a substantial proportion (~35% and 38%, respectively) of the genes upregulated in nodules or expressed in the nodule differentiation zone colocalize in genomic clusters (270 and 211, respectively), here termed symbiotic islands. These islands contain numerous expressed lncRNA genes and display differentially both DNA methylation and histone marks. Epigenetic regulations and lncRNAs are therefore attractive candidate elements for the orchestration of symbiotic gene expression in the M. truncatula genome.

MtNODULE ROOT1 and MtNODULE ROOT2 Are Essential for Indeterminate Nodule Identity.

Symbiotic interactions between legume plants and rhizobia result in the formation of nitrogen-fixing nodules, but the molecular actors and the mechanisms allowing for the maintenance of nodule identity are poorly understood. Medicago truncatula NODULE ROOT1 (MtNOOT1), Pisum sativum COCHLEATA1 (PsCOCH1), and Lotus japonicus NOOT-BOP-COCH-LIKE1 (LjNBCL1) are orthologs of Arabidopsis (Arabidopsis thaliana) AtBLADE-ON-PETIOLE1/2 and are members of the NBCL gene family, which has conserved roles in plant development and is essential for indeterminate and determinate nodule identity in legumes. The loss of function of MtNOOT1, PsCOCH1, and LjNBCL1 triggers a partial loss of nodule identity characterized by the development of ectopic roots arising from nodule vascular meristems. Here, we report the identification and characterization of a second gene involved in regulating indeterminate nodule identity in M. truncatula, MtNOOT2MtNOOT2 is the paralog of MtNOOT1 and belongs to a second legume-specific NBCL subclade, the NBCL2 clade. MtNOOT2 expression was induced during early nodule formation, and it was expressed primarily in the nodule central meristem. Mtnoot2 mutants did not present any particular symbiotic phenotype; however, the loss of function of both MtNOOT1 and MtNOOT2 resulted in the complete loss of nodule identity and was accompanied by drastic changes in the expression of symbiotic, defense, and root apical meristem marker genes. Mtnoot1 noot2 double mutants developed only nonfixing root-like structures that were no longer able to host symbiotic rhizobia. This study provides original insights into the molecular basis underlying nodule identity in legumes forming indeterminate nodules.

Arbuscular Mycorrhizal Fungal 14-3-3 Proteins Are Involved in Arbuscule Formation and Responses to Abiotic Stresses During AM Symbiosis.

Arbuscular mycorrhizal (AM) fungi are soil-borne fungi belonging to the ancient phylum Glomeromycota and are important symbionts of the arbuscular mycorrhiza, enhancing plant nutrient acquisition and resistance to various abiotic stresses. In contrast to their significant physiological implications, the molecular basis involved is poorly understood, largely due to their obligate biotrophism and complicated genetics. Here, we identify and characterize three genes termed Fm201, Ri14-3-3 and RiBMH2 that encode 14-3-3-like proteins in the AM fungi Funneliformis mosseae and Rhizophagus irregularis, respectively. The transcriptional levels of Fm201, Ri14-3-3 and RiBMH2 are strongly induced in the pre-symbiotic and symbiotic phases, including germinating spores, intraradical hyphae- and arbuscules-enriched roots. To functionally characterize the Fm201, Ri14-3-3 and RiBMH2 genes, we took advantage of a yeast heterologous system owing to the lack of AM fungal transformation systems. Our data suggest that all three genes can restore the lethal Saccharomyces cerevisiae bmh1 bmh2 double mutant on galactose-containing media. Importantly, yeast one-hybrid analysis suggests that the transcription factor RiMsn2 is able to recognize the STRE (CCCCT/AGGGG) element present in the promoter region of Fm201 gene. More importantly, Host-Induced Gene Silencing of both Ri14-3-3 and RiBMH2 in Rhizophagus irregularis impairs the arbuscule formation in AM symbiosis and inhibits the expression of symbiotic PT4 and MST2 genes from plant and fungal partners, respectively. We further subjected the AM fungus-Medicago truncatula association system to drought or salinity stress. Accordingly, the expression profiles in both mycorrhizal roots and extraradical hyphae reveal that these three 14-3-3-like genes are involved in response to drought or salinity stress. Collectively, our results provide new insights into molecular functions of the AM fungal 14-3-3 proteins in abiotic stress responses and arbuscule formation during AM symbiosis.

The Absence of the N-acyl-homoserine-lactone Autoinducer Synthase Genes traI and ngrI Increases the Copy Number of the Symbiotic Plasmid in Sinorhizobium fredii NGR234.

Plant-released flavonoids induce the transcription of symbiotic genes in rhizobia and one of the first bacterial responses is the synthesis of so called Nod factors. They are responsible for the initial root hair curling during onset of root nodule development. This signal exchange is believed to be essential for initiating the plant symbiosis with rhizobia affiliated with the Alphaproteobacteria. Here, we provide evidence that in the broad host range strain Sinorhizobium fredii NGR234 the complete lack of quorum sensing molecules results in an elevated copy number of its symbiotic plasmid (pNGR234a). This in turn triggers the expression of symbiotic genes and the production of Nod factors in the absence of plant signals. Therefore, increasing the copy number of specific plasmids could be a widespread mechanism of specialized bacterial populations to bridge gaps in signaling cascades.

The Thiamine Biosynthesis Gene THI1 Promotes Nodule Growth and Seed Maturation.

Thiamine (vitamin B1) is essential for living organisms. Unlike animals, plants can synthesize thiamine. In Lotus japonicus, the expression of two thiamine biosynthesis genes, THI1 and THIC, was enhanced by inoculation with rhizobia but not by inoculation with arbuscular mycorrhizal fungi. THIC and THI2 (a THI1 paralog) were expressed in uninoculated leaves. THI2-knockdown plants and the transposon insertion mutant thiC had chlorotic leaves. This typical phenotype of thiamine deficiency was rescued by an exogenous supply of thiamine. In wild-type plants, THI1 was expressed mainly in roots and nodules, and the thi1 mutant had green leaves even in the absence of exogenous thiamine. THI1 was highly expressed in actively dividing cells of nodule primordia. The thi1 mutant had small nodules, and this phenotype was rescued by exogenous thiamine and by THI1 complementation. Exogenous thiamine increased nodule diameter, but the level of arbuscular mycorrhizal colonization was unaffected in the thi1 mutant or by exogenous thiamine. Expression of symbiotic marker genes was induced normally, implying that mainly nodule growth was delayed in the thi1 mutant. Furthermore, this mutant formed many immature seeds with reduced seed weight. These results indicate that thiamine biosynthesis mediated by THI1 enhances nodule enlargement and is required for seed development in L. japonicus.

A transcriptomic analysis of the effect of genistein on Sinorhizobium fredii HH103 reveals novel rhizobial genes putatively involved in symbiosis.

Sinorhizobium fredii HH103 is a rhizobial soybean symbiont that exhibits an extremely broad host-range. Flavonoids exuded by legume roots induce the expression of rhizobial symbiotic genes and activate the bacterial protein NodD, which binds to regulatory DNA sequences called nod boxes (NB). NB drive the expression of genes involved in the production of molecular signals (Nod factors) as well as the transcription of ttsI, whose encoded product binds to tts boxes (TB), inducing the secretion of proteins (effectors) through the type 3 secretion system (T3SS). In this work, a S. fredii HH103 global gene expression analysis in the presence of the flavonoid genistein was carried out, revealing a complex regulatory network. Three groups of genes differentially expressed were identified: i) genes controlled by NB, ii) genes regulated by TB, and iii) genes not preceded by a NB or a TB. Interestingly, we have found differentially expressed genes not previously studied in rhizobia, being some of them not related to Nod factors or the T3SS. Future characterization of these putative symbiotic-related genes could shed light on the understanding of the complex molecular dialogue established between rhizobia and legumes.

Opening the "black box" of nodD3, nodD4 and nodD5 genes of Rhizobium tropici strain CIAT 899.

BackgroundTranscription of nodulation genes in rhizobial species is orchestrated by the regulatory nodD gene. Rhizobium tropici strain CIAT 899 is an intriguing species in possessing features such as broad host range, high tolerance of abiotic stresses and, especially, by carrying the highest known number of nodD genes—five—and the greatest diversity of Nod factors (lipochitooligosaccharides, LCOs). Here we shed light on the roles of the multiple nodD genes of CIAT 899 by reporting, for the first time, results obtained with nodD3, nodD4 and nodD5 mutants.MethodsThe three nodD mutants were built by insertion of Ω interposon. Nod factors were purified and identified by LC-MS/MS analyses. In addition, nodD1 and nodC relative gene expressions were measured by quantitative RT-PCR in the wt and derivative mutant strains. Phenotypic traits such as exopolysaccharide (EPS), lipopolysaccharide (LPS), swimming and swarming motilities, biofilm formation and indole acetid acid (IAA) production were also perfomed. All these experiments were carried out in presence of both inducers of CIAT 899, apigenin and salt. Finally, nodulation assays were evaluated in up to six different legumes, including common bean (Phaseolus vulgaris L.).ResultsPhenotypic and symbiotic properties, Nod factors and gene expression of nodD3, nodD4 and nodD5 mutants were compared with those of the wild-type (WT) CIAT 899, both in the presence and in the absence of the nod-gene-inducing molecule apigenin and of saline stress. No differences between the mutants and the WT were observed in exopolysaccharide (EPS) and lipopolysaccharide (LPS) profiles, motility, indole acetic acid (IAA) synthesis or biofilm production, either in the presence, or in the absence of inducers. Nodulation studies demonstrated the most complex regulatory system described so far, requiring from one (Leucaena leucocephala, Lotus burtii) to four (Lotus japonicus) nodD genes. Up to 38 different structures of Nod factors were detected, being higher under salt stress, except for the nodD5 mutant; in addition, a high number of structures was synthesized by the nodD4 mutant in the absence of any inducer. Probable activator (nodD3 and nodD5) or repressor roles (nodD4), possibly via nodD1 and/or nodD2, were attributed to the three nodD genes. Expression of nodC, nodD1 and each nodD studied by RT-qPCR confirmed that nodD3 is an activator of nodD1, both in the presence of apigenin and salt stress. In contrast, nodD4 might be an inducer with apigenin and a repressor under saline stress, whereas nodD5 was an inducer under both conditions.ConclusionsWe report for R. tropici CIAT 899 the most complex model of regulation of nodulation genes described so far. Five nodD genes performed different roles depending on the host plant and the inducing environment. Nodulation required from one to four nodD genes, depending on the host legume. nodD3 and nodD5 were identified as activators of the nodD1 gene, whereas, for the first time, it was shown that a regulatory nodD gene—nodD4—might act as repressor or inducer, depending on the inducing environment, giving support to the hypothesis that nodD roles go beyond nodulation, in terms of responses to abiotic stresses.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-2033-z) contains supplementary material, which is available to authorized users.

A role for the mevalonate pathway in early plant symbiotic signaling

Rhizobia and arbuscular mycorrhizal fungi produce signals that are perceived by host legume receptors at the plasma membrane and trigger sustained oscillations of the nuclear and perinuclear Ca(2+) concentration (Ca(2+) spiking), which in turn leads to gene expression and downstream symbiotic responses. The activation of Ca(2+) spiking requires the plasma membrane-localized receptor-like kinase Does not Make Infections 2 (DMI2) as well as the nuclear cation channel DMI1. A key enzyme regulating the mevalonate (MVA) pathway, 3-Hydroxy-3-Methylglutaryl CoA Reductase 1 (HMGR1), interacts with DMI2 and is required for the legume-rhizobium symbiosis. Here, we show that HMGR1 is required to initiate Ca(2+) spiking and symbiotic gene expression in Medicago truncatula roots in response to rhizobial and arbuscular mycorrhizal fungal signals. Furthermore, MVA, the direct product of HMGR1 activity, is sufficient to induce nuclear-associated Ca(2+) spiking and symbiotic gene expression in both wild-type plants and dmi2 mutants, but interestingly not in dmi1 mutants. Finally, MVA induced Ca(2+) spiking in Human Embryonic Kidney 293 cells expressing DMI1. This demonstrates that the nuclear cation channel DMI1 is sufficient to support MVA-induced Ca(2+) spiking in this heterologous system.

NODULE INCEPTION antagonistically regulates gene expression with nitrate in Lotus japonicus.

Legumes produce root nodules as symbiotic organs where nitrogen-fixing bacteria are accommodated. Lotus japonicus NODULE INCEPTION (NIN) is an essential factor that specifically and positively regulates nodulation processes, and has evolved from a member of the NIN-like proteins, of which Arabidopsis homologs target nitrate-responsive elements (NREs), and activate gene expression in response to nitrate. It is therefore assumed that the NIN-mediated transcriptional network overlaps with those regulated by NLPs, because of their common DNA-binding RWP-RK domains. However, nodulation is inhibited in the presence of nitrate, and involvement of NIN in nitrate responses has remained largely unknown. Here we determined a consensus of NIN-binding nucleotide sequences (NBSs) by in vitro experiments, and revealed that the sequence pattern was very similar to those of NREs. Chromatin immunoprecitiation (ChIP)-PCR analyses showed that NIN targeted NREs in L. japonicus nitrate-inducible gene promoters, including LjNIR1, LjNRT2.1 and LjNRT2.2. Affinities of NIN binding to the NREs were comparable with that to NBS-yB1a, an NBS on the symbiotic LjNF-YB1 promoter, indicating that NREs are potential targets of NIN. However, rhizobial infection did not activate LjNIR1, LjNRT2.1 and LjNRT2.2. NIN ectopic expression interfered with nitrate-dependent activation of these genes. Nitrate treatment followed by NIN activation down-regulated expression of symbiotic NIN target genes. Our results showed that NIN and nitrate antagonistically regulate expression of genes that are activated by nitrate and NIN, respectively. We propose that this antagonistic relationship prevents inappropriate activation of genes in response to nitrate and rhizobial infection.

Two CCAAT-box-binding transcription factors redundantly regulate early steps of the legume-rhizobia endosymbiosis.

During endosymbiotic interactions between legume plants and nitrogen-fixing rhizobia, successful root infection by bacteria and nodule organogenesis requires the perception and transduction of bacterial lipo-chitooligosaccharidic signal called Nod factor (NF). NF perception in legume roots leads to the activation of an early signaling pathway and of a set of symbiotic genes which is controlled by specific early transcription factors (TFs) including CYCLOPS/IPD3, NSP1, NSP2, ERN1 and NIN. In this study, we bring convincing evidence that the Medicago truncatula CCAAT-box-binding NF-YA1 TF, previously associated with later stages of rhizobial infection and nodule meristem formation is, together with its closest homolog NF-YA2, also an essential positive regulator of the NF-signaling pathway. Here we show that NF-YA1 and NF-YA2 are both expressed in epidermal cells responding to NFs and their knock-down by reverse genetic approaches severely affects the NF-induced expression of symbiotic genes and rhizobial infection. Further over-expression, transactivation and ChIP-PCR approaches indicate that NF-YA1 and NF-YA2 function, at least in part, via the direct activation of ERN1. We thus propose a model in which NF-YA1 and NF-YA2 appear as early symbiotic regulators acting downstream of DMI3 and NIN and possibly within the same regulatory complexes as NSP1/2 to directly activate the expression of ERN1.