RESUMO
Legumes acquire fixed nitrogen (N) from the soil and through endosymbiotic association with diazotrophic bacteria. However, establishing and maintaining N2-fixing nodules are expensive for the host plant, relative to taking up N from the soil. Therefore, plants suppress symbiosis when N is plentiful and enhance symbiosis when N is sparse. Here, we show that the nitrate transporter MtNRT2.1 is required for optimal nodule establishment in Medicago truncatula under low-nitrate conditions and the repression of nodulation under high-nitrate conditions. The NIN-like protein (NLP) MtNLP1 is required for MtNRT2.1 expression and regulation of nitrate uptake/transport under low- and high-nitrate conditions. Under low nitrate, the gene encoding the C-terminally encoded peptide (CEP) MtCEP1 was more highly expressed, and the exogenous application of MtCEP1 systemically promoted MtNRT2.1 expression in a compact root architecture 2 (MtCRA2)-dependent manner. The enhancement of nodulation by MtCEP1 and nitrate uptake were both impaired in the Mtnrt2.1 mutant under low nitrate. Our study demonstrates that nitrate uptake by MtNRT2.1 differentially affects nodulation at low- and high-nitrate conditions through the actions of MtCEP1 and MtNLP1.
Assuntos
Medicago truncatula , Nitratos , Regulação da Expressão Gênica de Plantas , Medicago truncatula/metabolismo , Nitratos/farmacologia , Nitratos/metabolismo , Peptídeos/genética , Peptídeos/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Nodulação/genética , Nódulos Radiculares de Plantas/genética , Nódulos Radiculares de Plantas/metabolismo , Simbiose/fisiologiaRESUMO
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 ß-1,3/ß-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.
Assuntos
Lotus , Micorrizas , Rhizobium , Micorrizas/genética , Lotus/genética , Lotus/metabolismo , Lotus/microbiologia , Nódulos Radiculares de Plantas/genética , Nódulos Radiculares de Plantas/metabolismo , Nódulos Radiculares de Plantas/microbiologia , Rhizobium/metabolismo , Raízes de Plantas/metabolismo , Mutação , Simbiose/genética , Fosfotransferases/metabolismo , Polissacarídeos/metabolismo , Glucanos/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Regulação da Expressão Gênica de PlantasRESUMO
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.
Assuntos
Lotus , Rhizobium , Rhizobium/genética , Lotus/genética , Lotus/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Simbiose/genética , Regulação da Expressão Gênica de Plantas , Nódulos Radiculares de Plantas/genética , Raízes de PlantasRESUMO
Legumes have evolved a nitrogen-fixing symbiotic interaction with rhizobia, and this association helps them to cope with the limited nitrogen conditions in soil. The compatible interaction between the host plant and rhizobia leads to the formation of root nodules, wherein internalization and transition of rhizobia into their symbiotic form, termed bacteroids, occur. Rhizobia in the nodules of the Inverted Repeat-Lacking Clade legumes, including Medicago truncatula, undergo terminal differentiation, resulting in elongated and endoreduplicated bacteroids. This transition of endocytosed rhizobia is mediated by a large gene family of host-produced nodule-specific cysteine-rich (NCR) peptides in M. truncatula. Few NCRs have been recently found to be essential for complete differentiation and persistence of bacteroids. Here, we show that a M. truncatula symbiotic mutant FN9285, defective in the complete transition of rhizobia, is deficient in a cluster of NCR genes. More specifically, we show that the loss of the duplicated genes NCR086 and NCR314 in the A17 genotype, found in a single copy in Medicago littoralis R108, is responsible for the ineffective symbiotic phenotype of FN9285. The NCR086 and NCR314 gene pair encodes the same mature peptide but their transcriptional activity varies considerably. Nevertheless, both genes can restore the effective symbiosis in FN9285 indicating that their complementation ability does not depend on the strength of their expression activity. The identification of the NCR086/NCR314 peptide, essential for complete bacteroid differentiation, has extended the list of peptides, from a gene family of several hundred members, that are essential for effective nitrogen-fixing symbiosis in M. truncatula.
Assuntos
Medicago truncatula , Família Multigênica , Proteínas de Plantas , Nódulos Radiculares de Plantas , Simbiose , Medicago truncatula/microbiologia , Medicago truncatula/genética , Medicago truncatula/fisiologia , Nódulos Radiculares de Plantas/microbiologia , Nódulos Radiculares de Plantas/genética , Simbiose/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Regulação da Expressão Gênica de Plantas , Rhizobium/fisiologia , Rhizobium/genética , Fixação de Nitrogênio/genética , Peptídeos/metabolismo , Peptídeos/genética , Sinorhizobium meliloti/fisiologia , Sinorhizobium meliloti/genética , Cisteína/metabolismoRESUMO
Rhizobial phosphatidylcholine (PC) is thought to be a critical phospholipid for the symbiotic relationship between rhizobia and legume host plants. A PC-deficient mutant of Sinorhizobium meliloti overproduces succinoglycan, is unable to swim, and lacks the ability to form nodules on alfalfa (Medicago sativa) host roots. Suppressor mutants had been obtained which did not overproduce succinoglycan and regained the ability to swim. Previously, we showed that point mutations leading to altered ExoS proteins can reverse the succinoglycan and swimming phenotypes of a PC-deficient mutant. Here, we report that other point mutations leading to altered ExoS, ChvI, FabA, or RpoH1 proteins also revert the succinoglycan and swimming phenotypes of PC-deficient mutants. Notably, the suppressor mutants also restore the ability to form nodule organs on alfalfa roots. However, nodules generated by these suppressor mutants express only low levels of an early nodulin, do not induce leghemoglobin transcript accumulation, thus remain white, and are unable to fix nitrogen. Among these suppressor mutants, we detected a reduced function mutant of the 3-hydoxydecanoyl-acyl carrier protein dehydratase FabA that produces reduced amounts of unsaturated and increased amounts of shorter chain fatty acids. This alteration of fatty acid composition probably affects lipid packing thereby partially compensating for the previous loss of PC and contributing to the restoration of membrane homeostasis.
Assuntos
Ácidos Graxos , Medicago sativa , Fosfatidilcolinas , Nodulação , Sinorhizobium meliloti , Simbiose , Sinorhizobium meliloti/fisiologia , Sinorhizobium meliloti/genética , Medicago sativa/microbiologia , Medicago sativa/genética , Nodulação/genética , Ácidos Graxos/metabolismo , Ácidos Graxos/biossíntese , Fosfatidilcolinas/metabolismo , Fosfatidilcolinas/biossíntese , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Nódulos Radiculares de Plantas/microbiologia , Nódulos Radiculares de Plantas/genética , Nódulos Radiculares de Plantas/metabolismo , Mutação , Polissacarídeos Bacterianos/metabolismo , Polissacarídeos Bacterianos/biossíntese , Fixação de NitrogênioRESUMO
The conservation of GOLVEN (GLV)/ROOT MERISTEM GROWTH FACTOR (RGF) peptide encoding genes across plant genomes capable of forming roots or root-like structures underscores their potential significance in the terrestrial adaptation of plants. This study investigates the function and role of GOLVEN peptide-coding genes in Medicago truncatula. Five out of fifteen GLV/RGF genes were notably upregulated during nodule organogenesis and were differentially responsive to nitrogen deficiency and auxin treatment. Specifically, the expression of MtGLV9 and MtGLV10 at nodule initiation sites was contingent upon the NODULE INCEPTION transcription factor. Overexpression of these five nodule-induced GLV genes in hairy roots of M. truncatula and application of their synthetic peptide analogues led to a decrease in nodule count by 25-50%. Uniquely, the GOLVEN10 peptide altered the positioning of the first formed lateral root and nodule on the primary root axis, an observation we term 'noduletaxis'; this decreased the length of the lateral organ formation zone on roots. Histological section of roots treated with synthetic GOLVEN10 peptide revealed an increased cell number within the root cortical cell layers without a corresponding increase in cell length, leading to an elongation of the root likely introducing a spatiotemporal delay in organ formation. At the transcription level, the GOLVEN10 peptide suppressed expression of microtubule-related genes and exerted its effects by changing expression of a large subset of Auxin responsive genes. These findings advance our understanding of the molecular mechanisms by which GOLVEN peptides modulate root morphology, nodule ontogeny, and interactions with key transcriptional pathways.
Assuntos
Regulação da Expressão Gênica de Plantas , Medicago truncatula , Proteínas de Plantas , Raízes de Plantas , Nódulos Radiculares de Plantas , Medicago truncatula/genética , Medicago truncatula/crescimento & desenvolvimento , Medicago truncatula/metabolismo , Medicago truncatula/efeitos dos fármacos , Medicago truncatula/fisiologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/genética , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/metabolismo , Nódulos Radiculares de Plantas/genética , Nódulos Radiculares de Plantas/crescimento & desenvolvimento , Nódulos Radiculares de Plantas/metabolismo , Nódulos Radiculares de Plantas/efeitos dos fármacos , Ácidos Indolacéticos/metabolismo , Ácidos Indolacéticos/farmacologia , Nodulação/genética , Meristema/genética , Meristema/crescimento & desenvolvimento , Meristema/efeitos dos fármacos , Peptídeos/metabolismo , Peptídeos/genéticaRESUMO
Leguminous plants provide carbon to symbiotic rhizobia in root nodules to fuel the energy-consuming process of nitrogen fixation. The carbon investment pattern from the acquired sources is crucial for shaping the growth regime of the host plants. The autoregulation of nodulation (AON) signaling pathway tightly regulates the number of nodules that form. AON disruption leads to excessive nodule formation and stunted shoot growth. However, the physiological role of AON in adjusting the carbon investment pattern is unknown. Here, we show that AON plays an important role in sustaining shoot water availability, which is essential for promoting carbon investment in shoot growth in Lotus japonicus. We found that AON-defective mutants exhibit substantial accumulation of nonstructural carbohydrates, such as sucrose. Consistent with this metabolic signature, resilience against water-deficit stress was enhanced in the shoots of the AON-defective mutants. Furthermore, the water uptake ability was attenuated in the AON-defective mutants, likely due to the increased ratio of nodulation zone, which is covered with hydrophobic surfaces, on the roots. These results increase our physiological understanding of legume-rhizobia symbiosis by revealing a trade-off between root nodule formation and shoot water availability.
Assuntos
Lotus , Brotos de Planta , Nódulos Radiculares de Plantas , Água , Lotus/genética , Lotus/metabolismo , Lotus/crescimento & desenvolvimento , Lotus/microbiologia , Água/metabolismo , Nódulos Radiculares de Plantas/metabolismo , Nódulos Radiculares de Plantas/crescimento & desenvolvimento , Nódulos Radiculares de Plantas/genética , Brotos de Planta/metabolismo , Brotos de Planta/crescimento & desenvolvimento , Brotos de Planta/genética , Nodulação , Mutação/genética , Simbiose/fisiologia , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Carbono/metabolismo , Raízes de Plantas/metabolismo , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/microbiologia , Fixação de NitrogênioRESUMO
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.
Assuntos
Lotus , Regulação da Expressão Gênica de Plantas , Lotus/genética , Lotus/metabolismo , Nitratos/metabolismo , Nitrogênio/metabolismo , Proteínas de Plantas/metabolismo , Nodulação/genética , Nódulos Radiculares de Plantas/genética , Nódulos Radiculares de Plantas/metabolismo , Solo , Simbiose/fisiologiaRESUMO
BACKGROUND: The symbiosis among plants, rhizobia, and arbuscular mycorrhizal fungi (AMF) is one of the most well-known symbiotic relationships in nature. However, it is still unclear how bilateral/tripartite symbiosis works under resource-limited conditions and the diverse genetic backgrounds of the host. RESULTS: Using a full factorial design, we manipulated mungbean accessions/subspecies, rhizobia, and AMF to test their effects on each other. Rhizobia functions as a typical facilitator by increasing plant nitrogen content, plant weight, chlorophyll content, and AMF colonization. In contrast, AMF resulted in a tradeoff in plants (reducing biomass for phosphorus acquisition) and behaved as a competitor in reducing rhizobia fitness (nodule weight). Plant genotype did not have a significant effect on AMF fitness, but different mungbean accessions had distinct rhizobia affinities. In contrast to previous studies, the positive relationship between plant and rhizobia fitness was attenuated in the presence of AMF, with wild mungbean being more responsive to the beneficial effect of rhizobia and attenuation by AMF. CONCLUSIONS: We showed that this complex tripartite relationship does not unconditionally benefit all parties. Moreover, rhizobia species and host genetic background affect the symbiotic relationship significantly. This study provides a new opportunity to re-evaluate the relationships between legume plants and their symbiotic partners.
Assuntos
Micorrizas , Rhizobium , Simbiose , Vigna , Micorrizas/fisiologia , Vigna/microbiologia , Vigna/genética , Vigna/fisiologia , Rhizobium/fisiologia , Nódulos Radiculares de Plantas/microbiologia , Nódulos Radiculares de Plantas/genética , Nódulos Radiculares de Plantas/fisiologiaRESUMO
BACKGROUND: Legumes utilize a long-distance signaling feedback pathway, termed Autoregulation of Nodulation (AON), to regulate the establishment and maintenance of their symbiosis with rhizobia. Several proteins key to this pathway have been discovered, but the AON pathway is not completely understood. RESULTS: We report a new hypernodulating mutant, defective in autoregulation, with disruption of a gene, DAR (Medtr2g450550/MtrunA17_Chr2g0304631), previously unknown to play a role in AON. The dar-1 mutant produces ten-fold more nodules than wild type, similar to AON mutants with disrupted SUNN gene function. As in sunn mutants, suppression of nodulation by CLE peptides MtCLE12 and MtCLE13 is abolished in dar. Furthermore, dar-1 also shows increased root length colonization by an arbuscular mycorrhizal fungus, suggesting a role for DAR in autoregulation of mycorrhizal symbiosis (AOM). However, unlike SUNN which functions in the shoot to control nodulation, DAR functions in the root. CONCLUSIONS: DAR encodes a membrane protein that is a member of a small protein family in M. truncatula. Our results suggest that DAR could be involved in the subcellular transport of signals involved in symbiosis regulation, but it is not upregulated during symbiosis. DAR gene family members are also present in Arabidopsis, lycophytes, mosses, and microalgae, suggesting the AON and AOM may use pathway components common to other plants, even those that do not undergo either symbiosis.
Assuntos
Medicago truncatula , Micorrizas , Proteínas de Plantas , Nodulação , Simbiose , Medicago truncatula/genética , Medicago truncatula/microbiologia , Medicago truncatula/fisiologia , Micorrizas/fisiologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Nodulação/genética , Simbiose/genética , Regulação da Expressão Gênica de Plantas , Mutação , Genes de Plantas , Raízes de Plantas/microbiologia , Raízes de Plantas/genética , Homeostase , Nódulos Radiculares de Plantas/microbiologia , Nódulos Radiculares de Plantas/genética , Nódulos Radiculares de Plantas/metabolismoRESUMO
Legume plants develop two types of root postembryonic organs, lateral roots and symbiotic nodules, using shared regulatory components. The module composed by the microRNA390, the Trans-Acting SIRNA3 (TAS3) RNA and the Auxin Response Factors (ARF)2, ARF3, and ARF4 (miR390/TAS3/ARFs) mediates the control of both lateral roots and symbiotic nodules in legumes. Here, a transcriptomic approach identified a member of the Lateral Organ Boundaries Domain (LBD) family of transcription factors in Medicago truncatula, designated MtLBD17/29a, which is regulated by the miR390/TAS3/ARFs module. ChIP-PCR experiments evidenced that MtARF2 binds to an Auxin Response Element present in the MtLBD17/29a promoter. MtLBD17/29a is expressed in root meristems, lateral root primordia, and noninfected cells of symbiotic nodules. Knockdown of MtLBD17/29a reduced the length of primary and lateral roots and enhanced lateral root formation, whereas overexpression of MtLBD17/29a produced the opposite phenotype. Interestingly, both knockdown and overexpression of MtLBD17/29a reduced nodule number and infection events and impaired the induction of the symbiotic genes Nodulation Signaling Pathway (NSP) 1 and 2. Our results demonstrate that MtLBD17/29a is regulated by the miR390/TAS3/ARFs module and a direct target of MtARF2, revealing a new lateral root regulatory hub recruited by legumes to act in the root nodule symbiotic program.
Assuntos
Medicago truncatula , Proteínas de Plantas , Nodulação , Raízes de Plantas , Fatores de Transcrição , Regulação da Expressão Gênica de Plantas , Técnicas de Silenciamento de Genes , Ácidos Indolacéticos/metabolismo , Medicago truncatula/genética , Medicago truncatula/crescimento & desenvolvimento , Medicago truncatula/microbiologia , MicroRNAs/genética , MicroRNAs/metabolismo , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Nodulação/genética , Raízes de Plantas/genética , Raízes de Plantas/crescimento & desenvolvimento , Regiões Promotoras Genéticas/genética , Nódulos Radiculares de Plantas/genética , Nódulos Radiculares de Plantas/crescimento & desenvolvimento , Simbiose/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genéticaRESUMO
The lipid transport protein (LTP) product of the AsE246 gene of Chinese milk vetch (Astragalus sinicus) contributes to the transport of plant-synthesized lipids to the symbiosome membranes (SMs) that are required for nodule organogenesis in this legume. However, the mechanisms used by nodule-specific LTPs remain unknown. In this study, a functional protein in the DnaJ-like family, designated AsDJL1, was identified and shown to interact with AsE246. Immunofluorescence showed that AsDJL1 was expressed in infection threads (ITs) and in nodule cells and that it co-localized with rhizobium, and an immunoelectron microscopy assay localized the protein to SMs. Via co-transformation into Nicotiana benthamiana cells, AsDJL1 and AsE246 displayed subcellular co-localization in the cells of this heterologous host. Co-immunoprecipitation assays confirmed that AsDJL1 interacted with AsE246 in nodules. The essential interacting region of AsDJL1 was determined to be the zinc finger domain at its C-terminus. Chinese milk vetch plants transfected with AsDJL1-RNAi had significantly decreased numbers of ITs, nodule primordia and nodules as well as reduced (by 83%) nodule nitrogenase activity compared with the controls. By contrast, AsDJL1 overexpression led to increased nodule fresh weight and nitrogenase activity. RNAi-AsDJL1 also significantly affected the abundance of lipids, especially digalactosyldiacylglycerol, in early-infected roots and transgenic nodules. Taken together, the results of this study provide insights into the symbiotic functions of AsDJL1, which may participate in lipid transport to SMs and play an essential role in rhizobial infection and nodule organogenesis.
Assuntos
Astrágalo , Fabaceae , Rhizobium , Fixação de Nitrogênio/genética , Nódulos Radiculares de Plantas/genética , Nódulos Radiculares de Plantas/metabolismo , Proteínas de Transporte/metabolismo , Astrágalo/metabolismo , Nitrogenase/metabolismo , Lipídeos , Simbiose/genética , Nodulação , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismoRESUMO
The legume-rhizobium symbiosis represents a unique model within the realm of plant-microbe interactions. Unlike typical cases of pathogenic invasion, the infection of rhizobia and their residence within symbiotic cells do not elicit a noticeable immune response in plants. Nevertheless, there is still much to uncover regarding the mechanisms through which plant immunity influences rhizobial symbiosis. In this study, we identify an important player in this intricate interplay: Lotus japonicus PRP1, which serves as a positive regulator of plant immunity but also exhibits the capacity to decrease rhizobial colonization and nitrogen fixation within nodules. The PRP1 gene encodes an uncharacterized protein and is named Pathogenesis-Related Protein1, owing to its orthologue in Arabidopsis thaliana, a pathogenesis-related family protein (At1g78780). The PRP1 gene displays high expression levels in nodules compared to other tissues. We observed an increase in rhizobium infection in the L. japonicus prp1 mutants, whereas PRP1-overexpressing plants exhibited a reduction in rhizobium infection compared to control plants. Intriguingly, L. japonicus prp1 mutants produced nodules with a pinker colour compared to wild-type controls, accompanied by elevated levels of leghaemoglobin and an increased proportion of infected cells within the prp1 nodules. The transcription factor Nodule Inception (NIN) can directly bind to the PRP1 promoter, activating PRP1 gene expression. Furthermore, we found that PRP1 is a positive mediator of innate immunity in plants. In summary, our study provides clear evidence of the intricate relationship between plant immunity and symbiosis. PRP1, acting as a positive regulator of plant immunity, simultaneously exerts suppressive effects on rhizobial infection and colonization within nodules.
Assuntos
Lotus , Proteínas de Plantas , Nódulos Radiculares de Plantas , Simbiose , Lotus/genética , Lotus/microbiologia , Lotus/fisiologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Nódulos Radiculares de Plantas/microbiologia , Nódulos Radiculares de Plantas/genética , Nódulos Radiculares de Plantas/metabolismo , Rhizobium/fisiologia , Regulação da Expressão Gênica de PlantasRESUMO
Leguminous plants produce nodules for nitrogen fixation; however, nodule production incurs an energy cost. Therefore, as an adaptive strategy, leguminous plants halt root nodule development when sufficient amounts of nitrogen nutrients, such as nitrate, are present in the environment. Although legume NODULE INCEPTION (NIN)-LIKE PROTEIN (NLP) transcription factors have recently been identified, understanding how nodulation is controlled by nitrate, a fundamental question for nitrate-mediated transcriptional regulation of symbiotic genes, remains elusive. Here, we show that two Lotus japonicus NLPs, NITRATE UNRESPONSIVE SYMBIOSIS 1 (NRSYM1)/LjNLP4 and NRSYM2/LjNLP1, have overlapping functions in the nitrate-induced control of nodulation and act as master regulators for nitrate-dependent gene expression. We further identify candidate target genes of LjNLP4 by combining transcriptome analysis with a DNA affinity purification-seq approach. We then demonstrate that LjNLP4 and LjNIN, a key nodulation-specific regulator and paralog of LjNLP4, have different DNA-binding specificities. Moreover, LjNLP4-LjNIN dimerization underlies LjNLP4-mediated bifunctional transcriptional regulation. These data provide a basic principle for how nitrate controls nodulation through positive and negative regulation of symbiotic genes.
Assuntos
Fatores de Transcrição/metabolismo , Regulação da Expressão Gênica de Plantas , Lotus/genética , Lotus/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Nodulação/genética , Nodulação/fisiologia , Nódulos Radiculares de Plantas/genética , Nódulos Radiculares de Plantas/metabolismo , Simbiose/genética , Simbiose/fisiologia , Fatores de Transcrição/genéticaRESUMO
Symbiotic nitrogen fixation (SNF) is crucial for legumes, providing them with the nitrogen necessary for plant growth and development. Nodulation is the first step in the establishment of SNF. However, the determinant genes in soybean nodulation and the understanding of the underlying molecular mechanisms governing nodulation are still limited. Herein, we identified a phosphatase, GmPP2C61A, which was specifically induced by rhizobia inoculation. Using transgenic hairy roots harboring GmPP2C61A::GUS, we showed that GmPP2C61A was mainly induced in epidermal cells following rhizobia inoculation. Functional analysis revealed that knockdown or knock-out of GmPP2C61A significantly reduced the number of nodules, while overexpression of GmPP2C61A promoted nodule formation. Additionally, GmPP2C61A protein was mainly localized in the cytoplasm and exhibited conserved phosphatase activity in vitro. Our findings suggest that phosphatase GmPP2C61A serves as a critical regulator in soybean nodulation, highlighting its potential significance in enhancing symbiotic nitrogen fixation.
Assuntos
Regulação da Expressão Gênica de Plantas , Glycine max , Proteínas de Plantas , Nodulação , Glycine max/genética , Glycine max/microbiologia , Glycine max/fisiologia , Fixação de Nitrogênio , Monoéster Fosfórico Hidrolases/metabolismo , Monoéster Fosfórico Hidrolases/genética , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Nodulação/genética , Raízes de Plantas/genética , Raízes de Plantas/microbiologia , Raízes de Plantas/metabolismo , Plantas Geneticamente Modificadas , Rhizobium/fisiologia , Nódulos Radiculares de Plantas/genética , Nódulos Radiculares de Plantas/microbiologia , Nódulos Radiculares de Plantas/metabolismo , Simbiose/genéticaRESUMO
Symbiotic nitrogen fixation (SNF) can play a key role in agroecosystems to reduce the negative impact of nitrogen fertilizers. Its efficiency is strongly affected by the combination of bacterial and plant genotypes, but the mechanisms responsible for the differences in the efficiency of rhizobium strains are not well documented. In Medicago truncatula, SNF has been mostly studied using model systems, such as M. truncatula A17 in interaction with Sinorhizobium meliloti Sm2011. Here we analyzed both the wild-type (wt) A17 and the Mtefd-1 mutant in interaction with five S. meliloti and two Sinorhizobium medicae strains. ETHYLENE RESPONSE FACTOR REQUIRED FOR NODULE DIFFERENTIATION (MtEFD) encodes a transcription factor, which contributes to the control of nodule number and differentiation in M. truncatula. We found that, in contrast to Sm2011, four strains induce functional (Fix+) nodules in Mtefd-1, although less efficient for SNF than in wt A17. In contrast, the Mtefd-1 hypernodulation phenotype is not strain-dependent. We compared the plant nodule transcriptomes in response to SmBL225C, a highly efficient strain with A17, versus Sm2011, in wt and Mtefd-1 backgrounds. This revealed faster nodule development with SmBL225C and early nodule senescence with Sm2011. These RNA sequencing analyses allowed us to identify candidate plant factors that could drive the differential nodule phenotype. In conclusion, this work shows the value of having a set of rhizobium strains to fully evaluate the biological importance of a plant symbiotic gene.
Assuntos
Medicago truncatula , Sinorhizobium meliloti , Sinorhizobium , Sinorhizobium/genética , Sinorhizobium meliloti/genética , Fixação de Nitrogênio/genética , Fatores de Transcrição/genética , Simbiose/genética , Nódulos Radiculares de Plantas/genética , Nódulos Radiculares de Plantas/microbiologiaRESUMO
MAIN CONCLUSION: A comprehensive understanding of nitrogen signaling cascades involving heterotrimeric G-proteins and their putative receptors can assist in the production of nitrogen-efficient plants. Plants are immobile in nature, so they must endure abiotic stresses including nutrient stress. Plant development and agricultural productivity are frequently constrained by the restricted availability of nitrogen in the soil. Non-legume plants acquire nitrogen from the soil through root membrane-bound transporters. In depleted soil nitrogen conditions, legumes are naturally conditioned to fix atmospheric nitrogen with the aid of nodulation elicited by nitrogen-fixing bacteria. Moreover, apart from the symbiotic nitrogen fixation process, nitrogen uptake from the soil can also be a significant secondary source to satisfy the nitrogen requirements of legumes. Heterotrimeric G-proteins function as molecular switches to help plant cells relay diverse stimuli emanating from external stress conditions. They are comprised of Gα, Gß and Gγ subunits, which cooperate with several downstream effectors to regulate multiple plant signaling events. In the present review, we concentrate on signaling mechanisms that regulate plant nitrogen nutrition. Our review highlights the potential of heterotrimeric G-proteins, together with their putative receptors, to assist the legume root nodule symbiosis (RNS) cascade, particularly during calcium spiking and nodulation. Additionally, the functions of heterotrimeric G-proteins in nitrogen acquisition by plant roots as well as in improving nitrogen use efficiency (NUE) have also been discussed. Future research oriented towards heterotrimeric G-proteins through genome editing tools can be a game changer in the enhancement of the nitrogen fixation process. This will foster the precise manipulation and production of plants to ensure global food security in an era of climate change by enhancing crop productivity and minimizing reliance on external inputs.
Assuntos
Fabaceae , Proteínas Heterotriméricas de Ligação ao GTP , Rhizobium , Nódulos Radiculares de Plantas/genética , Nodulação/fisiologia , Nitrogênio/metabolismo , Fixação de Nitrogênio , Fabaceae/genética , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Simbiose/fisiologia , Plantas/metabolismo , Verduras/metabolismo , Solo , Rhizobium/fisiologiaRESUMO
Nodule Inception (NIN) is one of the most important root nodule symbiotic genes as it is required for both infection and nodule organogenesis in legumes. Unlike most legumes with a sole NIN gene, there are four putative orthologous NIN genes in soybean (Glycine max). Whether and how these NIN genes contribute to soybean-rhizobia symbiotic interaction remain unknown. In this study, we found that all four GmNIN genes are induced by rhizobia and that conserved CE and CYC binding motifs in their promoter regions are required for their expression in the nodule formation process. By generation of multiplex Gmnin mutants, we found that the Gmnin1a nin2a nin2b triple mutant and Gmnin1a nin1b nin2a nin2b quadruple mutant displayed similar defects in rhizobia infection and root nodule formation, Gmnin2a nin2b produced fewer nodules but displayed a hyper infection phenotype compared to wild type (WT), while the Gmnin1a nin1b double mutant nodulated similar to WT. Overexpression of GmNIN1a, GmNIN1b, GmNIN2a, and GmNIN2b reduced nodule numbers after rhizobia inoculation, with GmNIN1b overexpression having the weakest effect. In addition, overexpression of GmNIN1a, GmNIN2a, or GmNIN2b, but not GmNIN1b, produced malformed pseudo-nodule-like structures without rhizobia inoculation. In conclusion, GmNIN1a, GmNIN2a, and GmNIN2b play functionally redundant yet complicated roles in soybean nodulation. GmNIN1b, although expressed at a comparable level with the other homologs, plays a minor role in root nodule symbiosis. Our work provides insight into the understanding of the asymmetrically redundant function of GmNIN genes in soybean.
Assuntos
Glycine max/crescimento & desenvolvimento , Glycine max/genética , Glycine max/metabolismo , Nódulos Radiculares de Plantas/crescimento & desenvolvimento , Nódulos Radiculares de Plantas/genética , Nódulos Radiculares de Plantas/metabolismo , Simbiose/genética , Produtos Agrícolas/genética , Produtos Agrícolas/crescimento & desenvolvimento , Produtos Agrícolas/metabolismo , Produtos Agrícolas/microbiologia , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Variação Genética , Genótipo , Rhizobium , Nódulos Radiculares de Plantas/microbiologia , Glycine max/microbiologiaRESUMO
Most legumes can establish a symbiotic association with soil rhizobia that trigger the development of root nodules. These nodules host the rhizobia and allow them to fix nitrogen efficiently. The perception of bacterial lipo-chitooligosaccharides (LCOs) in the epidermis initiates a signaling cascade that allows rhizobial intracellular infection in the root and de-differentiation and activation of cell division that gives rise to the nodule. Thus, nodule organogenesis and rhizobial infection need to be coupled in space and time for successful nodulation. The plant hormone cytokinin (CK) contributes to the coordination of this process, acting as an essential positive regulator of nodule organogenesis. However, the temporal regulation of tissue-specific CK signaling and biosynthesis in response to LCOs or Sinorhizobium meliloti inoculation in Medicago truncatula remains poorly understood. In this study, using a fluorescence-based CK sensor (pTCSn::nls:tGFP), we performed a high-resolution tissue-specific temporal characterization of the sequential activation of CK response during root infection and nodule development in M. truncatula after inoculation with S. meliloti. Loss-of-function mutants of the CK-biosynthetic gene ISOPENTENYLTRANSFERASE 3 (IPT3) showed impairment of nodulation, suggesting that IPT3 is required for nodule development in M. truncatula. Simultaneous live imaging of pIPT3::nls:tdTOMATO and the CK sensor showed that IPT3 induction in the pericycle at the base of nodule primordium contributes to CK biosynthesis, which in turn promotes expression of positive regulators of nodule organogenesis in M. truncatula.
Assuntos
Alquil e Aril Transferases/metabolismo , Citocininas/genética , Citocininas/metabolismo , Medicago truncatula/genética , Medicago truncatula/fisiologia , Nodulação/genética , Nódulos Radiculares de Plantas/metabolismo , Simbiose/genética , Alquil e Aril Transferases/genética , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Fixação de Nitrogênio/genética , Fixação de Nitrogênio/fisiologia , Organogênese/genética , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Nódulos Radiculares de Plantas/genética , Nódulos Radiculares de Plantas/crescimento & desenvolvimento , Sinorhizobium meliloti/fisiologia , Simbiose/fisiologiaRESUMO
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.