Conserved cis-elements enable NODULES WITH ACTIVATED DEFENSE1 regulation by NODULE INCEPTION during nodulation.

Root nodule symbiosis within nitrogen-fixing clade (NFC) plants is thought to have arisen from a single gain followed by massive losses in the genomes of ancestral non-nodulating plants. However, molecular evidence supporting this model is limited. Here, we confirm through bioinformatic analysis that NODULES WITH ACTIVATED DEFENSE1 (NAD1) is present only in NFC plants and is thus an NFC-specific gene. Moreover, NAD1 was specifically expressed in nodules. We identified three conserved nodulation-associated cis-regulatory elements (NACE1-3) in the promoter of LjNAD1 from Lotus japonicus that are required for its nodule specific expression. A survey of NFC plants revealed that NACE1 and NACE2 are specific to the Fabales and Papilionoideae, respectively, while NACE3 is present in all NFC plants. Moreover, we found that Nodule inception (NIN) directly binds to all three NACEs to activate NAD1 expression. Mutation of L. japonicus LjNAD1 resulted in the formation of abnormal symbiosomes with enlarged symbiosome space and frequent breakdown of bacteroids in nodules, resembling phenotypes reported for Medicago truncatula Mtnad1 and Mtnin mutants. These data point to NIN-NAD1 as an important module regulating rhizobial accommodation in nodules. The regulation of NAD1 by NIN in the NFC ancestor represent an important evolutionary adaptation for nodulation.

Soybean ethylene response factors GmENS1 and GmENS2 promote nodule senescence.

The final phase in root nodule development is nodule senescence. The mechanism underlying the initiation of nodule senescence requires further elucidation. Here, we investigated the intrinsic signals governing soybean (Glycine max L. Merr.) nodule senescence, uncovering ethylene as a key signal in this intricate mechanism. Two AP2/ERF transcription factor genes, GmENS1 and GmENS2 (Ethylene-responsive transcription factors required for Nodule Senescence), exhibit heightened expression levels in both aged nodules and nodules treated with ethylene. Overexpression of either GmENS1 or GmENS2 accelerated senescence in soybean nodules, whereas the knockout or knockdown of both genes delayed senescence and enhanced nitrogenase activity. Furthermore, our findings indicated that GmENS1 and GmENS2 directly bind to the promoters of GmNAC039, GmNAC018, and GmNAC030, encoding three NAC transcription factors essential for activating soybean nodule senescence. Notably, the nodule senescence process mediated by GmENS1 or GmENS2 overexpression was suppressed in the soybean nac039/018/030 triple mutant compared with the wild-type control. These data indicate GmENS1 and GmENS2 as pivotal transcription factors mediating ethylene-induced nodule senescence through the direct activation of GmNAC039/GmNAC018/GmNAC030 expression in soybean.

Dual RNA-Seq Analysis Pinpoints a Balanced Regulation between Symbiosis and Immunity in Medicago truncatula-Sinorhizobium meliloti Symbiotic Nodules.

Legume-rhizobial symbiosis initiates the formation of root nodules, within which rhizobia reside and differentiate into bacteroids to convert nitrogen into ammonium, facilitating plant growth. This process raises a fundamental question: how is plant immunity modulated within nodules when exposed to a substantial number of foreign bacteria? In Medicago truncatula, a mutation in the NAD1 (Nodules with Activated Defense 1) gene exclusively results in the formation of necrotic nodules combined with activated immunity, underscoring the critical role of NAD1 in suppressing immunity within nodules. In this study, we employed a dual RNA-seq transcriptomic technology to comprehensively analyze gene expression from both hosts and symbionts in the nad1-1 mutant nodules at different developmental stages (6 dpi and 10 dpi). We identified 89 differentially expressed genes (DEGs) related to symbiotic nitrogen fixation and 89 DEGs from M. truncatula associated with immunity in the nad1-1 nodules. Concurrently, we identified 27 rhizobial DEGs in the fix and nif genes of Sinorhizobium meliloti. Furthermore, we identified 56 DEGs from S. meliloti that are related to stress responses to ROS and NO. Our analyses of nitrogen fixation-defective plant nad1-1 mutants with overactivated defenses suggest that the host employs plant immunity to regulate the substantial bacterial colonization in nodules. These findings shed light on the role of NAD1 in inhibiting the plant's immune response to maintain numerous rhizobial endosymbiosis in nodules.

GmNAC039 and GmNAC018 activate the expression of cysteine protease genes to promote soybean nodule senescence.

Root nodules are major sources of nitrogen for soybean (Glycine max (L.) Merr.) growth, development, production, and seed quality. Symbiotic nitrogen fixation is time-limited, as the root nodule senesces during the reproductive stage of plant development, specifically during seed development. Nodule senescence is characterized by the induction of senescence-related genes, such as papain-like cysteine proteases (CYPs), which ultimately leads to the degradation of both bacteroids and plant cells. However, how nodule senescence-related genes are activated in soybean is unknown. Here, we identified 2 paralogous NAC transcription factors, GmNAC039 and GmNAC018, as master regulators of nodule senescence. Overexpression of either gene induced soybean nodule senescence with increased cell death as detected using a TUNEL assay, whereas their knockout delayed senescence and increased nitrogenase activity. Transcriptome analysis and nCUT&Tag-qPCR assays revealed that GmNAC039 directly binds to the core motif CAC(A)A and activates the expression of 4 GmCYP genes (GmCYP35, GmCYP37, GmCYP39, and GmCYP45). Similar to GmNAC039 and GmNAC018, overexpression or knockout of GmCYP genes in nodules resulted in precocious or delayed senescence, respectively. These data provide essential insights into the regulatory mechanisms of nodule senescence, in which GmNAC039 and GmNAC018 directly activate the expression of GmCYP genes to promote nodule senescence.

Upregulation of long noncoding RNA XIST has anticancer effects on ovarian cancer through sponging miR-106a.

Ovarian cancer (OC) is a highly malignant tumor. X inactive specific transcript (XIST) was identified as a cancer-related gene, while its therapeutic effect in OC was poorly defined. The present study was designed to investigate the effectual corollary of the lncRNA XIST in OC. RT-qPCR was used to detect the XIST and miR-106a expression levels of OC tissues and cell lines. OC cell apoptosis and proliferation were detected by flow cytometry, colony formation, and CCK-8 assays. Moreover, bioinformatics analysis was used to predict the targeted miRNA of XIST. The dual-luciferase reporter and RNA pull-down assays were then used to verify the interaction between miR-106a and XIST. OC xenograft nude mice were raised to measure tumor growth. Notably, OC tissues and cells exhibited low XIST levels and high miR-106a levels. The XIST upregulation decreased the OVCAR3 and CAOV3 cell proliferation and inversely promoted cell apoptosis. miR-106a targeted the XIST. Also, the miR-106a overexpression reversed the inhibitory effects of XIST on OC cell proliferation and apoptosis. Our in vivo results suggested that XIST was involved in tumor growth deceleration, while the miR-106a reversed the effect. To conclusion, the present study demonstrated that XIST suppressed OC development via sponging miR-106a both in vitro and in vivo.

Transcriptional regulation of NIN expression by IPN2 is required for root nodule symbiosis in Lotus japonicus.

Expression of Nodule Inception (NIN) is essential for initiation of legume-rhizobial symbiosis. An existing model regarding the regulation of NIN expression involves two GRAS transcription factors - NSP1 (Nodulation Signaling Pathway 1) and NSP2. NSP2 forms a complex with NSP1 to directly bind to NIN promoter. However, rhizobial treatment-induced NIN expression could still be detected in the nsp1 mutant plants, suggesting that other proteins must be involved in the regulation of NIN expression. A combination of molecular, biochemical and genetic analyses was used to investigate the molecular basis of IPN2 in regulating root development and NIN expression in Lotus japonicus. In this study, we identified that IPN2 is a close homolog of Arabidopsis APL (ALTERED PHLOEM DEVELOPMENT) with essential function in root development. However, Lotus IPN2 has a different expression pattern compared with the Arabidopsis APL gene. IPN2 binds to the IPN2-responsive cis element (IPN2-RE) of NIN promoter and activates NIN expression. IPN2, NSP1 and NSP2 form a protein complex to directly target NIN promoter and activate NIN expression in the legume-rhizobial symbiosis. Our data refine the regulatory model of NIN expression that NSP2 works together with NSP1 and IPN2 to activate the NIN gene allowing nodulation in L. japonicus.

Suppression of innate immunity mediated by the CDPK-Rboh complex is required for rhizobial colonization in Medicago truncatula nodules.

Suppression of innate immunity is essential for rhizobial infection and colonization in compatible interactions with leguminous plants. In Medicago nad1 mutant plants, innate immunity is excessively activated, resulting in necrotic cell death after rhizobia are released from infection threads into symbiotic cells, suggesting that innate immunity plays a critical role in regulating bacteroid persistence. In this study, we identified three respiratory burst oxidase homologs (Rboh) and one calcium-dependent protein kinase (CDPK) as key factors for the activation of immunity in Medicago nodules using genetic and biochemical methods. Knock-out of either MtRbohB or MtRbohD in nad1-1 mutant plants produced effective nodules with intact symbiotic cells, while knock-out of MtRbohC decreased brown pigment deposition, leading to less necrosis in nad1-1 mutant nodules. MtCDPK5 directly phosphorylated MtRbohB, MtRbohC and MtRbohD, which triggered immune responses in plants. Knock-out of MtCDPK5 in nad1-1 mutant plants partially restored nitrogen-fixing nodules. Overexpression of the constitutively activated variant MtCDPK5VK under the control of the NAD1 promoter elicited strong immune responses, resulting in ineffective nodules in wild-type plants. Our data provide direct evidence that host plants utilize innate immunity to regulate rhizobial colonization in symbiotic cells in Medicago truncatula.

NODULES WITH ACTIVATED DEFENSE 1 is required for maintenance of rhizobial endosymbiosis in Medicago truncatula.

The symbiotic interaction between legume plants and rhizobia results in the formation of root nodules, in which symbiotic plant cells host and harbor thousands of nitrogen-fixing rhizobia. Here, a Medicago truncatula nodules with activated defense 1 (nad1) mutant was identified using reverse genetics methods. The mutant phenotype was characterized using cell and molecular biology approaches. An RNA-sequencing technique was used to analyze the transcriptomic reprogramming of nad1 mutant nodules. In the nad1 mutant plants, rhizobial infection and propagation in infection threads are normal, whereas rhizobia and their symbiotic plant cells become necrotic immediately after rhizobia are released from infection threads into symbiotic cells of nodules. Defense-associated responses were detected in nad1 nodules. NAD1 is specifically present in root nodule symbiosis plants with the exception of Morus notabilis, and the transcript is highly induced in nodules. NAD1 encodes a small uncharacterized protein with two predicted transmembrane helices and is localized at the endoplasmic reticulum. Our data demonstrate a positive role for NAD1 in the maintenance of rhizobial endosymbiosis during nodulation.

A Lotus japonicus Cochaperone Protein Interacts With the Ubiquitin-Like Domain Protein CIP73 and Plays a Negative Regulatory Role in Nodulation.

The calcium/calmodulin-dependent protein kinase CCaMK forms a complex with its phosphorylation target CIP73 (CCaMK-interacting protein of 73 kDa). In this work, a homolog of the animal HSC/HSP70 interacting protein (HIP) was identified as an interacting partner of CIP73 in Lotus japonicus. L. japonicus HIP contains all functional domains characteristic of animal HIP proteins. The C-terminal STI1-like domain of L. japonicus HIP was found to be necessary and sufficient for interaction with CIP73. The interaction between CIP73 and HIP occurred in both the nuclei and cytoplasm in Nicotiana benthamiana leaf cells. The interactions between CIP73 and HIP and between CIP73 and CCaMK could take place simultaneously in the same nuclei. HIP transcripts were detected in all plant tissues tested. As nodule primordia developed into young nodules, the expression of HIP was down-regulated and the HIP transcript level became very low in mature nodules. More nodules were formed in transgenic hairy roots of L. japonicus expressing HIP RNA interference at 16 days postinoculation as compared with the control hairy roots expressing the empty vector. It appears that HIP may play a role as a negative regulator for nodulation.

Deletion of meso-2,3-butanediol dehydrogenase gene budC for enhanced D-2,3-butanediol production in Bacillus licheniformis.

BACKGROUND: D-2,3-butanediol has many industrial applications such as chiral reagents, solvents, anti-freeze agents, and low freezing point fuels. Traditional D-2,3-butanediol producing microorganisms, such as Klebsiella pneumonia and K. xoytoca, are pathogenic and not capable of producing D-2,3-butanediol at high optical purity. Bacillus licheniformis is a potential 2,3-butanediol producer but the wild type strain (WX-02) produces a mix of D- and meso-type isomers. BudC in B. licheniformis is annotated as 2,3-butanediol dehydrogenase or acetoin reductase, but no pervious experiment was performed to verify this hypothesis. RESULTS: We developed a genetically modified strain of B. licheniformis (WX-02 ΔbudC) as a D-2,3-butanediol producer with high optimal purity. A marker-less gene deletion protocol based on a temperature sensitive knock-out plasmid T2-Ori was used to knock out the budC gene in B. licheniformis WX-02. The budC knock-out strain successfully abolished meso-2,3-butanediol production with enhanced D-2,3-butanediol production. No meso-BDH activity was detectable in cells of this strain. On the other hand, the complementary strain restored the characteristics of wild strain, and produced meso-2,3-butanediol and possessed meso-BDH activity. All of these data suggested that budC encoded the major meso-BDH catalyzing the reversible reaction from acetoin to meso-2,3-butanediol in B. licheniformis. The budC knock-out strain produced D-2,3-butanediol isomer only with a high yield of 30.76 g/L and a productivity of 1.28 g/L-h. CONCLUSIONS: We confirmed the hypothesis that budC gene is responsible to reversibly transfer acetoin to meso-2,3-butanediol in B. licheniformis. A mutant strain of B. licheniformis with depleted budC gene was successfully developed and produced high level of the D-2,3-butanediol with high optimal purity.

A MYB coiled-coil transcription factor interacts with NSP2 and is involved in nodulation in Lotus japonicus.

Transcription factor complex formation is a central step in regulating gene expression. In this report, a novel MYB coiled-coil transcription factor referred to as IPN2, for Interacting Protein of NSP2, is described. The interaction between IPN2 and NSP2 was examined by protein pull-down assays and bimolecular fluorescence complementation (BiFC). Subcellular localization of proteins, gene expression and gene function were assessed in transgenic hairy roots expressing tagged recombinant proteins, promoter-reporter and RNA interference (RNAi) constructs, respectively. The GRAS domain of NSP2 and the coiled-coil domain of IPN2 were found to be responsible for the interaction between the two proteins. IPN2 had strong transcription activation activity, bound directly to the NIN gene promoter, and was localized to the nuclei of Lotus japonicus root cells. The expression of IPN2 was elevated during nodule development, coinciding with increased NSP2 gene expression during nodule organogenesis. RNAi-mediated knockdown expression of IPN2 did not affect arbuscular mycorrhizal development, but had deleterious effects on rhizobial infection and nodule formation in L. japonicus. These results demonstrate an important role of IPN2 in nodule organogenesis and place a new MYB transcription factor in the Nod signaling pathway.