A rhizobium-induced FT-FD module locally activates nodule stem cell gene

A rhizobium-induced FT-FD module locally activates nodule stem cell gene | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Biological Sciences - Article A rhizobium-induced FT-FD module locally activates nodule stem cell gene Yong-Fu Fu This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5006581/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Functional divergence of gene homologs enables species to evolve unique physiological processes 1,2 , such as nodulation for the leguminous plant to fix inorganic nitrogen into organic nitrogen 3 . The first step of nodule anlagen formation is to re-activate root cortex cells by rhizobia into stem cells, however, the mechanism of that is unknown. In soybean genome there are six florigen homologs 4-6 , two of which, GmFT2a and GmFT5a are produced in leaves and imported into roots to enhance nodulation 7 . Here, we demonstrated that another florigen homolog GmFT5b and its partner GmFDL23 are induced in root cortex by rhizobia and locally inhibit GmCLV3 expression. Subsequently, GmWUS is released from GmCLV3-1 repression to re-activate cortex cells into stem cells and finally to trigger nodulation. Our results identify a signaling pathway of spatiotemporal re-formation of stem cells in roots for nodule organogenesis mediated by a local florigen module in soybean. Therefore, florigen participates in nodulation at least in two processes as two different signals: stem cell formation (local signal, this study) and primordium initiation or development (systemic signal from leaves, a previous report 7 ). One sentence summary： A florigen homolog GmFT5b and its partner GmFDL23 are induced by rhizobia in roots and locally activates stem cell identity gene GmWUS to trigger nodulation in soybean. Biological sciences/Plant sciences/Plant symbiosis/Rhizobial symbiosis Biological sciences/Stem cells/Reprogramming Biological sciences/Developmental biology/Organogenesis Biological sciences/Evolution/Molecular evolution Figures Figure 1 Figure 2 Figure 3 Figure 4 Main text Florigen is well known as a movable signal being produced in leaves and then transported not only to apexes to initiate flower formation 8,9 or bud dormancy and burst 10 , but also to underground organs to enhance tuberization 11 or nodulation 7 . Gene duplications in plant genomes supply a wonderful opportunity for new functional assignments of homologous genes to adapt to unique environmental stresses and developmental machine of a given plant 12-14 . In soybean genome, at least six florigen homologous genes survive from the evolutionary process and encode proteins sharing similar molecular functions of flowering activity 4-6 . However, their biological functions diverge in soybean evolutional process, partially due to their different expression patterns 4 . Recently, we found that one soybean florigen homolog, GmFT5b (Glyma.19G108200), expressed in roots and nodules (Extended Data Fig. 1), supposing that it may locally function in these organs, different from its siblings, GmFT2a (Glyma.16G150700) and GmFT5a (Glyma.16G044100), both of which are produced in leaves and then move upward to apexes to initiate floral primordia 6 or downward to roots to regulate nodulation 7 . To test our hypothesis, we first investigated the expression pattern of GmFT5b promoter ( GmFT5b pro ), around 10 kb of ATG upstream sequence which may contain potential cis -elements since there are regulating elements in over 8 kb of ATG upstream sequence of Arabidopsis florigen FT promoter 15,16 , using GmFT5b pro :GUS transgenic plants and hairy roots. Results showed that GUS signals were clearly found in most organs with high signals in vascular tissues of roots, nodules, and leaves (Fig. 1a, and Extended Data Fig. 1a). RT-qPCR confirmed that leaves, shoots, flowers, pods, roots, and nodules accumulated GmFT5b transcripts, different from website data (Extended Data Fig. 1b). The expression activity of GmFT5b pro in roots suggested that GmFT5b could be locally produced in roots other than transported from other organs. Interestingly, after rhizobia infection the GUS signal gradually, even though 12 HAI (hour after infection), extended to endodermis and cortex (inner side, next to stele) (Fig. 1a), potential sites for nodule primordia initiation 3,17 . At 5 DAI (day after infection), cortex had strong GUS signals. RT-qPCR results supported that GmFT5b expression was induced by rhizobium infection (Fig. 1b). At 10 DAI, endodermis and bundle sheath kept strong signals, whereas the nodule primordium did not detect strong GUS activity (Extended Data Fig. 1c). In latter nodule developmental stages, vascular system was also the tissue with high GUS signals, but nitrogen fixation zone showed weak signal (Extended Data Fig. 1c). The results suggested that GmFT5b took its roles mainly at two stages of nodulation: initiation and later development stage (vascular tissues). Next, we generated CRISPR/Cas9 edited mutants of GmFT5b gene, Gmft5b-1 and Gmft5b-9 , which had pre-mature mutated sites in exon 1 and 4, respectively (Extended Data Fig. 2a). Both Gmft5b-1 and Gmft5b-9 mutants displayed less nodules, especially small nodules, compared to wild type plants (Fig. 1c and Extended Data Fig. 2b), while overexpressing GmFT5b increased nodule number (Fig. 1d and Extended Data Fig. 2c and 3a). Because GmFT2a and GmFT5a proteins are transported from shoots to roots to enhance nodulation in soybean 7 , we supposed that all members of GmFT family were involved in nodulation. Then, we analyzed the function of FT -like genes on nodulation using GmFT family RNAi transgenic lines, RNAi -GmFTs-1 and -3 5 , and found that silencing GmFT family slightly increased nodule number, especially small nodules (Extended Data Fig. 2d), indicating that there may exist antagonistic effects among different members of GmFT family on nodulation. To make it clear whether GmFT5b functioned locally in roots, we carried out grating experiment. Amazingly, the results demonstrated that it was not Gmft5b-1 scion but Gmft5b-1 stock to determine the number of nodules of the graft chimeras (Fig. 1e and Extended Data Fig. 2e). Taken together, GmFT5b locally took a role in roots in enhancing nodulation in soybean. FT regulates flower anlagen initiation through its interaction with FD to activate flower identity gene, such as AP1 18,19 . We wondered that GmFT5b functioned in soybean nodulation in a similar molecular mechanism. Firstly, we checked if GmFT5b interacted with GmFDL23 or GmFDL20 proteins, both of which showed high expression in roots (Extended Data Fig. 4a and 4b). GmFT5b proteins distributed in whole cells, while GmFDL23 or GmFDL20 proteins localized in nuclei (Fig. 2a and Extended Data Fig. 4c and 4d). As expected, their interactions in nuclei were verified by BiFC (bimolecular fluorescence complementation), Co-IP (co-immunoprecipitation), and Y2H (yeast-two-hybrid) experiments (Fig. 2b, 2c and Extended Data Fig. 4e to 4i). GmFDL23 also showed rhizobium-induced expression pattern in roots and nodules (Fig. 2d, and Extended Data Fig. 4j), but GmFDL20 expression decreased after rhizobium infection (Extended Data Fig. 4k). Without rhizobium infection, GmFDL23 expressed weakly in root stele; once infection, root stele displayed high level of GmFDL23 expression,even some pericycle or endodermis cells expressed GmFDL23 . Loss of function of GmFDL23 led to low number of nodules, while overexpression of GmFDL23 increased nodule number (Fig. 2e, 2f, and Extended Data Fig. 3b, 3e and 6a). Additionally, the expression of early marker genes of nodulation in both Gmft5b and Gmfdl23 mutants was similarly decreased (Fig. 2g). These results suggested thatGmFDL23 interacted with GmFT5b proteins to participate in nodulation. Furthermore, we found that GmFT and GmFDL did not significantly activate GmAP1 promoter (Extended Data Fig. 5a), indicating the GmFT - GmFDL module may regulate nodulation in a novel mechanism, independent of GmAP1 gene. Stem cell activation is a key step for new organ formation. During this process, the stem cell identity gene WUS plays a central role 20-23 , and high WUS activity increases globally organogenesis 24,25 . WUS not only is a direct target of CLV3 , but directly inhibits CLV3 expression 26-28 ; such a regulatory dynamics of this loop drives de novo stem cell formation to initiate a meristem or anlagen 29,30 . Bioinformatic analysis identified binding motifs of bZIP transcription factor in promoters of GmCLV3-1 and GmWUS1 (AGCT 31 , Fig. 3a). We wondered if GmFT5b and GmFDL would act directly in a WUS-CLV3 feedback loop. ChIP-qPCR confirmed that GmFDL23, a bZIP transcription factor, would enrich GmCLV3-1 , but not GmWUS1 , specific fragments of promoters (Fig. 3a and Extended Data Fig. 5b), and EMSA test supported that GmFDL23 proteins physically interacted with the corresponding fragment (Fig. 3b). GmFDL23 and GmFT5b synergistically inhibited the promoter activity of GmCLV3-1 (Fig. 3c) and in the Gmft5b mutant GmCLV3-1 pro promoter activities were significantly enhanced compared to that in wild type plants (Fig. 3d). Furthermore, the mutation of GmFDL23 or GmFT5b genes resulted in increasing GmCLV3-1 expression (Fig. 3e and Extended Data Fig. 2a and 6a), while overexpression of GmFT5b or GmFDL23 repressed GmCLV3-1 expression (Fig. 3f and Extended Data Fig. 3e). Together, the GmFT5b-GmFDL23 module may directly inhibit GmCLV3-1 expression in roots. Interestingly, GmCLV3-1 expression in roots was strongly inhibited by rhizobium infection (Fig. 1b), and after 3 DAI (day after infection) no GUS signals were detected in root cortex (Fig. 4a). Loss of GmCLV3-1 function not only increased the nodule number per plant, but rescued Gmft5b mutant phenotypes on nodule number (Fig. 4b and Extended Data Fig. 3c and 7). Unsurprisingly, overexpression of GmCLV3-1 decreased nodule number (Fig. 4b and Extended Data Fig. 3c and 3e). As what happens in apexes, GmCLV3-1 inhibited GmWUS activity, because overexpression of GmCLV3-1 inhibited GmWUS expression (Fig. 4c) and in Gmclv3-1 mutant GmWUS expression was enhanced (Fig. 2g). Similarly, GmWUS expression was induced by rhizobia (Fig. 1b), and GmWUS displayed as a positive regulator of nodulation (Fig. 4d and Extended Data Fig. 3d, 3e and 6b). Previous reports showed WUSCHEL-RELATEDHOMEOBOX 5 ( WOX5 ), a homolog of GmWUS , might control cell proliferation in nodule meristems 32,33 . In our experiment, GmFT5b plus GmFDL23 inhibited GmWOX5 promoter activity (Fig. 4e), suggesting that GmWUS and GmWOX5 played their roles in different pathways or different stages of nodulation. The type III secretion system (T3SS) is a conserved apparatus employed by rhizobia 34,35 to deliver the type III effectors (T3Es), also named Nop (nodulation outer protein), into the host cell to activate the symbiosis. ErnA, a T3E from Bradyrhizobium ORS3257 and targeted to the plant nucleus, confers the ability to nodulate Aeschynomene indica and ectopic expression of ErnA activates organogenesis of nodule-like structures in A. indica roots 36 . It is postulated that ErnA most probably trigger nodulation via targeting to key regulators in the nodulation signaling pathway, such as NIN and NF-Y 37 . We wondered if T3E had direct effect on GmFT5b and GmFDL23 . Interestingly, soybean codon optimized ErnA gene indeed activated not only GmFT5b and GmFDL23 genes, but also nodulation marker genes (Fig. 4f and 4g). However, we did not observe nodule-like structure in 35S:ErnA hairy roots as in A. indica roots 36 , maybe due to that only ErnA is not enough to induce nodulation in soybean 38 , because ErnA is from Bradyrhizobium strain ORS3257, which does not elicit nodules on soybean 38 . In summary, we proposed a working model (Fig. 4h). In soybean roots, without rhizobium infection, GmFT5b and GmFDL expression was limited in stele cells, while GmCLV3 expressed in cortex cells and thereby inhibited GmWUS expression. Once infection, rhizobium signals (T3E and others) activated the expression of GmFT5b and GmFDL in root cortex cells, which locally inhibited GmCLV3 expression and then release the repression of GmWUS by GmCLV3 . High GmWUS activity triggers de-novo stem cell formation and thereby nodule primordia initiation 3,17 . Limitation In our study, we revealed a pathway for de-novo activation of stem cell genes in root cortex in soybean mediated by a FT-FD module. This may be the very early and indispensable step for nodulation. However, there exist some important issues uncovered. de novo stem cell formation is a progressive process starting with a transient regulatory network in a small group of cells 30 , but we did not both identify such a group of cells and capture spatio-temporal dynamics of GmWUS activity in root cortex. We also did not know the role of GmWUS in nodule cell proliferation, which is different from stem cell proliferation 17 . It is interesting to explore the relationship between GmWUS andits homolog WOX5 in nodulation. And CLV3 is not the only gene controlling WUS expression 39 , other signals also confer regulation of WUS activity. For example, nitrate, an important signal for nodulation, can modulate WUS expression in shoot meristems through cytokinins 24 . What is more, an WUS-independent pathway is identified for regulation of stem cell proliferation 40-42 and the effect of interaction between GmFT5b and other two siblings ( GmFT5a and GmFT2a ) on nodulation is unknown, indicating more complex mechanism of nodulation is waiting to be explained. Declarations Acknowledgements We thank Drs. Dawei Xin, Chao Ma, and Eric Giraud for helpful suggestions on rhizobia and T3E. This work was supported by the National Key Research and Development Program of China (2021YFF1001202), the National Natural Science Foundation of China (32372028, 31771714, and 32360797), and the Innovation Program of Chinese Academy of Agricultural Sciences. Author contributions Y.F. and X.Z conceived, supervised the project, and drafted the manuscript; H.S. conducted most of experiments, including root nodule phenotype analysis, grafting experiments, vector construction, mutant identification, RT-qPCR, GUS histochemical staining, co-immunoprecipitation, subcellular localization, BiFC, transcriptional activation assays, EMSA, and ChIP-qPCR; X.Z. conducted soybean transformation and hairy root transformation, phenotype investigation, and project and lab management; P.H. conducted some root transformation; Q.S. performed the yeast two-hybrid experiments; G.Y., L.L., M.L., L.H., and K.Q. cloned some gene and promoter and constructed related vectors; Y.F. and H.S. analyzed data and plotted figures and tables; X.F, Y.M., Q.C., and J.Y. analyzed some data and reviewed the manuscript; all authors contributed to data analysis and manuscript preparation. Competing interest declaration The authors declare no competing interests. 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ERECTA-family genes coordinate stem cell functions between the epidermal and internal layers of the shoot apical meristem. Development 145 (2018). https://doi.org/10.1242/dev.156380 Lee, C. & Clark, S. E. A WUSCHEL-Independent Stem Cell Specification Pathway Is Repressed by PHB, PHV and CNA in Arabidopsis. PloS one 10 , e0126006 (2015). https://doi.org/10.1371/journal.pone.0126006 Methods Plant materials and growth conditions The genetic background of Gmft5b mutants and GmFT5bPro:GUS transgenic plants is soybean ( Glycine max (Merr.) L.) Williams 82 (WS82), and the genetic background of GmFTs RNAi lines is soybean Tianlong 1 (TL1). Hairy root transformation experiment was performed with WS82 and relative transgenic plants. All materials were planted in vermiculite and grown in a growth chamber at 25°C in a 16 h light / 8 h dark cycle with the light intensity of 400 μM mol m -2 s -1 . During the growth period, nutrient solution was applied as 1 mM KNO 3 , 1 mM CaCl 2 , 10 μM Fe-citrate, 0.25 μM MgSO 4 , 0.25 μM K 2 SO 4 , 1 μM MnSO 4 , 2 μM H 3 BO 3 , 0.5 μM ZnSO 4 , 0.2 μM CuSO 4 , 0.1 μM CoSO 4 , 0.1 μM Na 2 MoO 4 , 5 μM KH 2 PO 4 . The rhizobia ( Sinorhizobium fredii HH103) were applied to soils after three days of transplanting. HH103 was previously cultured in 28°C TY liquid medium (3 g/L yeast extract, 5 g/L tryptone, 0.4 g/L CaCl 2 ) for 2-3 days. Vector construction For promoter activity analysis and transcriptional activity analysis, promoters ( GmFT5b , 10 kb; GmFDL23 , 3 kb; GmAP1-2 , 2.6 kb; GmAP1-3 , 2.5 kb; GmCLV3-1 , 3 kb; GmWUS1 , 3 kb; GmWOX5 , 3 kb) were cloned from WS82 and into the Fu76 entry vector 43 by enzyme digestion and ligation. Subsequently, along with the Fu79-GUS or Fu79-LUC entry vectors, they were transferred into the pSoy10 binary vector by LR reactions. For the construction of vectors for overexpressing hairy roots and transcriptional activity analysis in Nicotiana benthamiana leaves, the coding sequences (CDS) of GmFT5b , GmFDL9 , GmFDL16 , GmFDL20 , GmFDL23 , GmCLV3-1 , and GmWUS1 were cloned from WS82, while the ErnA CDS sequence was synthesized by Genecreate company. All CDS sequences were ligated into the FU28 vector through enzymatic digestion 43 . Subsequently, along with the Fu76- 35S pro vector they were transferred to the pSoy10 binary vector by LR reactions. For gene editing, two sgRNA sequences were designed using CRISPR-P 2.0 software ( http://crispr.hzau.edu.cn/CRISPR2/ ) and ligated into the Fu79 entry vector containing the U6 promoter at the BspQ Ⅰ and Bsa Ⅰ sites. Along with the Fu76-Cas9 vector by LR reactions, theFu79-sgRNAs were then transferred to the pSoy10 or pSoy13 vector. Hairy root transformation WS82 and different mutants were used for hairy root transformation. The constructs were transformed into the Agrobacterium rhizogenesis strain K599 and cultured in 50 mL of liquid LB with selection marker kanamycin until OD 600 = 0.8. Root transformation was performed following a previously reported method 44 . Briefly, sterilized soybean seeds were soaked and germinated, and the elongated radicles were excised. The explants were infected in the resuspension solution for 30 minutes and then transferred to co-cultivation medium (1/10× Gamborg B5 salts, 30 g/L sucrose, 3.9 g/L MES, 4.25 g/L agar, pH 5.4, supplemented with 400 mg/L cysteine, 154.2 mg/L dithiothrietol, and 40 mg/L acetosyringone after sterilization). The explants were maintained in the dark for 3 days and then replaced into hairy root induction medium under 12 h light / 12 h dark cycle conditions. After three days, explants were rolled up in moistened germination paper and cultured for an additional 10 days until roots emerged. Transgenic hairy roots were selected by detecting the DsRed signal under a fluorescent stereomicroscope and non-transgenic hairy roots were removed. The composite plants with positive hairy roots were then transplanted into pots (7.5×7.5 cm) containing vermiculite with nutrient solution and grown in a growth chamber at 25°C under a 16 h light / 8 h dark cycle. Five days later, they were inoculated with rhizobia strain HH103 suspension (OD 600 = 0.1) for nodule phenotype and GUS histochemical analysis. GUS histochemical staining and microscopic observation Transgenic hairy roots and nodules harvested at different stages after rhizobium inoculation, as well as leaves of stable transgenic lines were collected into the GUS staining solution (50 mM sodium phosphate buffer, 0.2% Triton-X-100, 5 mM K 4 Fe(CN) 6 , 5 mM K 3 Fe(CN) 6 , and 1~2 mM X-gluc). The samples were subjected to vacuum for 15 minutes and incubated in dark at 37°C for 12 hours. Subsequently, the samples were cleaned three times with 70% ethanol and observed under an Olympus stereo microscope (SZX7). For samples sectioning, they were embedded in 4% agarose and cut into 80-100 μm slices using a Leica vibrating microtome (VT1000 S). Finally, photos were taken using an Olympus optical microscope (IX71). DNA extraction Plant or hairy root samples were harvested and used for DNA extraction using the SDS method 45 . Briefly, plant tissues were ground in a lysis buffer containing 2% sodium dodecyl sulfate (SDS), 50 mM Tris-HCl (pH 8.0), 10 mM ethylenediaminetetraacetic acid (EDTA), and 200 mM NaCl. The homogenate was incubated at 65°C for 10 minutes and then on ice with one-half volume of 3 M potassium acetate. After incubation, DNA was precipitated with an equal volume of isopropanol and washed with 70% ethanol. The obtained DNA was further subjected to PCR for identification of plant materials or hairy roots. RNA extraction and RT-qPCR The plant tissues were ground to powder in liquid nitrogen, and total RNA was extracted using HiPure Total RNA Mini Kit (Magen). The interference of genomic DNA was removed and the first strand of cDNA was reversely transcribed using the PrimeScript RT Reagent Kit with gDNA Eraser (TaKaRa). RT-qPCR was performed using the ChamQTM Universal SYBR qPCR Master Mix (Vazyme). Each RT-qPCR experiment was conducted with at least three technical replicates. GmUKN1 was used as the internal reference gene 46 . The RT-qPCR primers used are listed in Table S1. Grafting experiment Seeds of WS82 and Gmft5b-1 were sown in vermiculite with nutrient solution for germination (16 h light/8 h dark, 25℃). The hypocotyls of the seven-day-old seedlings were split into two halves, the scion was then gently thinned and grafted to the reciprocal rootstock with grafting clips and sealing film used to assist in fixation. The grafted plants were cultured in dark, warm, and humid environment for 5 days to minimize transpiration. Successfully grafted plants were inoculated with rhizobium strain HH103, and nodule phenotypes were assessed at 21 days after inoculation. Subcellular Localization and bimolecular fluorescence complementation (BiFC) The coding sequences of GmFT5b , GmFDL20 , and GmFDL23 were cloned into the pGWC entry vector 47 , and then recombined into the pGWB5-GFP expression vector for subcellular localization experiments using LR reactions 47 . Additionally, pGWC- GmFDL20 , and pGWC-GmFDL23 were recombined into the pEARLYGAYE201-YN vector by LR reactions, while pGWC- GmFT5b was recombined into the pEARLYGAYE202-YC vector for BiFC experiments. These vectors were transformed into Agrobacterium tumefaciens strain EHA105. When the bacterial culture reached an OD 600 = 1.0, it was resuspended in infiltration buffer (10 mM MES, 150 μM Acetosyringone, 10 mM MgCl 2 ) and used to infiltrate Nicotiana benthamiana leaves. Fluorescence signals were observed and recorded using a Zeiss LSM980 confocal laser scanning microscope. Co-Immunoprecipitation Through LR reactions, the pGWC- GmFDL20 and pGWC- GmFDL23 were recombined into the pGWB20-Myc vector. The resulting binary vectors were transformed into Agrobacterium tumefaciens strain EHA105. Subsequently, they were co-infiltrated with EHA105 containing pGWB5- GmFT5b:GFP construct into Nicotiana benthamiana leaves. Two days post-infiltration, proteins were extracted from the Nicotiana benthamiana leaves. Immunoprecipitation was performed at 4°C for 1 hour with anti-Myc magnetic beads (M047, Anti-Myc-tag mAb-Magnetic Agarose, MBL) and western blot analysis was then performed using anti-Myc (M192-3, MBL) and anti-GFP (M048-3, MBL) antibodies. Yeast two-hybrid The coding sequences of GmFDL20 and GmFDL23 were cloned into the pGADT7 vector and the coding sequence of GmFT5b was cloned into the pGBKT7 vector through restriction enzyme digestion and ligation. These constructs were co-transformed into the yeast strain AH109. Initially, yeasts were grown on SD-Leu/-Trp medium. After colonies formed, they were transferred to SD-Leu/-Trp/-His/-Ade medium and cultured for 3-4 days. The interaction between the proteins was evaluated based on the growth of yeasts under the selective conditions. Transcriptional activity assay pSoy10- AP1-2 pro :LUC , pSoy10- AP1-3 pro :LUC , pSoy10- GmCLV3-1 pro :LUC , pSoy10- GmWUS1 pro :LUC , and pSoy10- GmWOX5 pro :LUC were used as reporters, while pSoy10- 35S :GmFT5b , pSoy10- 35S:GmFDL9 , pSoy10- 35S:GmFDL16 , pSoy10- 35S:GmFDL20 , pSoy10 -35S:GmFDL23 , and pSoy10- 35S:GmCLV3-1 were used as effectors. All constructs were transformed into Agrobacterium tumefaciens strain EHA105 and co-infiltrated into Nicotiana benthamiana leaves, respectively. After 2 days, leaves were treated with 3 mM D-luciferin substrate (Provenand Published, LUCK-1G). Luciferase signals were detected using a Tanon 5200 imaging system, and the intensity was quantified using Image J. ChIP-qPCR assay Chromatin Immunoprecipitation (ChIP) was performed following the previous method 48 . Four grams of 35s:GmFDL23:GFP transgenic soybean roots were harvested three days post rhizobium inoculation and minced. The minced tissue was crosslinked with 1% formaldehyde, followed by quenching with 0.4 M glycine to terminate the crosslinking reaction. The material was then ground into fine powder under liquid nitrogen for chromatin extraction. Chromatin was sonicated using a Bioruptor UCD-200 to shear DNA into fragments ranging from 200 bp to 1000 bp. The sonicated chromatin was incubated with anti-GFP antibodies and IgG (as a negative control) and immunoprecipitated using a 1:1 volume mixture of protein A and protein G beads. Crosslinks were reversed with 215 mM NaCl, and the DNA was purified using the QIAquick PCR Purification Kit (QIAGEN). Quantitative PCR (qPCR) was then employed to analyze the precipitated DNA, using primers specified in the Table S1. EMSA The full-length coding sequence of GmFDL23 was cloned into the pET28a-His expression vector using Bam H I and Sal I restriction sites. The construct was then transformed into Escherichia coli BL21 competent cells for protein expression and purification. Biotin-labeled and unlabeled probes were synthesized by BGI as listed in Table S1. Electrophoretic Mobility Shift Assays (EMSAs) were performed following the protocol of the Light-Shift Chemiluminescent EMSA Kit (Thermo Scientific). For DNA binding, purified recombinant protein was mixed with 50 fM of the labeled probe in a reaction buffer (2.5% (v/v) glycerol, 5 mM MgCl 2 , 50 ng/mL poly (dI, dC), 0.05% (v/v) Nonidet P-40). To perform the competition assay, at least 50-folds of unlabeled probe were added to the mixture. The samples were incubated at room temperature for 20 minutes before being analyzed by non-denaturing 8% polyacrylamide gel electrophoresis. Identification of CRISPR/Cas9 hairy roots Identification of positive hairy roots is followed previous methods 49,50 . PCR amplification was performed using positive transgenic hairy root DNA as a template, with primers (Table S1) designed within 400 bp of both sides of the two sgRNA target sites. The amplified DNA fragments were subsequently sequenced. Heterozygous biallelic mutations, which appear as double peaks, were analyzed using CRISPR-GE (http://skl.scau.edu.cn/) for decoding. All sequencing data of CRISPR/Cas9 hairy roots were listed in Table S3. Accession number All gene identities were listed in Table S2. Statistical analysis All statistical graphs were generated and analyzed using GraphPad Prism 9.0.0 (GraphPad Software). Individual data points were represented by dots. Significance of differences were examined by One-way or Two-way ANOVA with Bonferroni post hoc test or two-tailed Student’s t -test, with p -values over than 0.05 considered as not statistically significant. Types of significant differences were indicated in the figure legends. Reference 43 Wang, X., Fan, C., Zhang, X., Zhu, J. & Fu, Y. F. BioVector, a flexible system for gene specific-expression in plants. BMC plant biology 13 , 198 (2013). https://doi.org/10.1186/1471-2229-13-198 44 Huang, P. et al. An Efficient Agrobacterium rhizogenes-Mediated Hairy Root Transformation Method in a Soybean Root Biology Study. International journal of molecular sciences 23 , 12261 (2022). https://doi.org/10.3390/ijms232012261 45 Ahmed, I. et al. High-quality plant DNA extraction for PCR: an easy approach. Journal of Applied Genetics 50 , 105-107 (2009). https://doi.org/10.1007/BF03195661 46 Huang, G., Ma, J., Han, Y., Chen, X. & Fu, Y. F. J. P. M. B. R. Cloning and Expression Analysis of the Soybean CO-Like Gene GmCOL9. 29 , 352-359 (2011). 47 Chen, Q. J., Zhou, H. M., Chen, J. & Wang, X. C. Using a modified TA cloning method to create entry clones. Anal Biochem 358 , 120-125 (2006). 48 Malapeira, J. & Mas, P. ChIP-seq analysis of histone modifications at the core of the Arabidopsis circadian clock. Methods Mol Biol 1158 , 57-69 (2014). https://doi.org/10.1007/978-1-4939-0700-7_4 49 Liu, W. et al. DSDecode: A Web-Based Tool for Decoding of Sequencing Chromatograms for Genotyping of Targeted Mutations. Molecular plant 8 , 1431-1433 (2015). https://doi.org/10.1016/j.molp.2015.05.009 50 Xie, X. et al. CRISPR-GE: A Convenient Software Toolkit for CRISPR-Based Genome Editing. Molecular plant 10 , 1246-1249 (2017). https://doi.org/10.1016/j.molp.2017.06.004 Additional Declarations There is NO Competing Interest. Supplementary Files TableS1Alistofprimers.xlsx Supplementary Information Table S1 A list of primers TableS2GenemanesandIDs.xlsx Table S2 Gene manes and IDs TableS3SequencingdataforCas9mutants.xlsx Table S3 Sequencing data for Cas9-mutants ExtendedDataFig.17.docx Extended data Figures Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5006581","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Biological Sciences - Article","associatedPublications":[],"authors":[{"id":355174143,"identity":"b8ded912-8402-4bec-a5c8-7dfa7989243a","order_by":0,"name":"Yong-Fu Fu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAv0lEQVRIiWNgGAWjYBAC9gYwJcfAT7QWngNgyphBsoFkLQYHiNYikWP4uOCXQeLm44cffvzBYJdHjBZj45l9BonbzqQZS/MwJBcT1GIvkWMmzdvzJ3HbDQYDaQaGA4kNRNgC0gJ02Az2zz9/EK2F54dB4gYJHjMJHqK08DwrNuZtMDCecSanzJrHIJkILezJGx/z/DGQ7W8/vvnmjwo7wloYGDgMGBjbYBwDwuqBgP0BA8MfolSOglEwCkbBSAUAar45vS+0xtsAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0001-7339-263X","institution":"Insitute of Crop Sciences, CAAS, China","correspondingAuthor":true,"prefix":"","firstName":"Yong-Fu","middleName":"","lastName":"Fu","suffix":""}],"badges":[],"createdAt":"2024-08-31 02:45:08","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5006581/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5006581/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":78742898,"identity":"c0919659-c577-46f6-8c9f-dc27c639c674","added_by":"auto","created_at":"2025-03-18 09:47:07","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":650147,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eGmFT5b\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e enhances nodulation in soybean. a\u003c/strong\u003e, \u003cem\u003eGmFT5b\u003c/em\u003e\u003csub\u003e\u003cem\u003epro\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e:GUS\u003c/em\u003e activity in roots after rhizobia HH103 inoculation. Violet arrows indicate pericycle cells, blue arrows indicate endodermal cells, and violet asterisk indicates cortex cells. \u003cstrong\u003eb\u003c/strong\u003e, Time-course expression of \u003cem\u003eGmFT5b\u003c/em\u003e, \u003cem\u003eGmFDL23\u003c/em\u003e, \u003cem\u003eGmWUS1\u003c/em\u003e, and\u003cem\u003e GmCLV3-1\u003c/em\u003e genes in roots after rhizobia HH103 inoculation.\u003cem\u003e GmUKN1\u003c/em\u003e is used as the reference gene. The left Y-axis is for \u003cem\u003eGmFT5b\u003c/em\u003e, \u003cem\u003eGmFDL23\u003c/em\u003e, and \u003cem\u003eGmWUS1\u003c/em\u003e genes, while the right Y-axis is for \u003cem\u003eGmCLV3-1\u003c/em\u003e gene. Data are normalized to that in the uninfected roots. \u003cstrong\u003ec\u003c/strong\u003e, Images and the number of big (≧ 2 mm) or small (\u0026lt; 2 mm) nodules per plant in WT and\u003cem\u003e Gmft5b\u003c/em\u003e mutants at 21 DAI. \u003cstrong\u003ed\u003c/strong\u003e, Nodule number of hairy roots\u003cstrong\u003e \u003c/strong\u003eexpressing EV and\u003cem\u003e 35S:GmFT5b\u003c/em\u003e line at 21 DAI. \u003cstrong\u003ee\u003c/strong\u003e, Images and the number of big (≧ 2 mm) or small (\u0026lt; 2 mm) nodules per plant in reciprocal grafting between WT and \u003cem\u003eGmft5b-1\u003c/em\u003e mutant at 21 DAI. Scale bar, 100 μm (\u003cstrong\u003ea\u003c/strong\u003e) or 5 mm (\u003cstrong\u003ec\u003c/strong\u003e, \u003cstrong\u003ee\u003c/strong\u003e). HAI, hours after inoculation; DAI, days after inoculation; WT, wild-type; EV, empty vector. Data are mean ± s.d. with \u003cem\u003en\u003c/em\u003e = 3 (\u003cstrong\u003eb\u003c/strong\u003e), \u003cem\u003en\u003c/em\u003e = 11 (\u003cstrong\u003ec\u003c/strong\u003e), \u003cem\u003en\u003c/em\u003e =13 (\u003cstrong\u003ed\u003c/strong\u003e) or \u003cem\u003en\u003c/em\u003e = 14 (\u003cstrong\u003ee\u003c/strong\u003e) biological replicates; Two-way ANOVA (\u003cstrong\u003ec\u003c/strong\u003e, \u003cstrong\u003ee\u003c/strong\u003e), different letters indicate \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05; or two-tailed Student’s \u003cem\u003et\u003c/em\u003e-test (\u003cstrong\u003ed\u003c/strong\u003e), ** \u003cem\u003ep\u003c/em\u003e \u0026lt;0.01.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5006581/v1/5c34e929d4d84fcce801f114.png"},{"id":78742502,"identity":"cba75519-2416-4998-98c7-8d797bf6727e","added_by":"auto","created_at":"2025-03-18 09:39:07","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":419684,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGmFT5b interacts with GmFDL23 to enhance nodulation. a\u003c/strong\u003e, Subcellular localization of GmFT5b-GFP and GmFDL23-GFP proteins in \u003cem\u003eN. benthamiana\u003c/em\u003e leaves. \u003cstrong\u003eb\u003c/strong\u003e, BiFC assay for GmFDL23-nYFP and GmFT5b-cYFP in \u003cem\u003eN. benthamiana\u003c/em\u003e leaves. \u003cstrong\u003ec\u003c/strong\u003e, Co-IP assay for GmFDL23-Myc and GmFT5b-GFP. Proteins were immunoprecipitated (IP) with anti-Myc beads and probed by anti-Myc or anti-GFP antibodies. \u003cstrong\u003ed\u003c/strong\u003e, \u003cem\u003eGmFDL23\u003c/em\u003e\u003csub\u003e\u003cem\u003epro\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e:GUS\u003c/em\u003e activity in roots after rhizobia HH103 inoculation. Violet arrows indicate pericycle or endodermis cells, and red arrow indicates cortex cells. \u003cstrong\u003ee\u003c/strong\u003e and \u003cstrong\u003ef\u003c/strong\u003e, Nodule number per plant in EV and \u003cem\u003eCas9\u003c/em\u003e-\u003cem\u003eGmFDL23\u003c/em\u003e hairy roots (\u003cstrong\u003ee\u003c/strong\u003e) or in EV and \u003cem\u003e35S:GmFDL23\u003c/em\u003e hairy roots (\u003cstrong\u003ef\u003c/strong\u003e) at 21 DAI. \u003cstrong\u003eg\u003c/strong\u003e, Relative expression of marker genes of nodulation in different mutant roots at 5 DAI. \u003cem\u003eGmUKN1\u003c/em\u003e is used as the reference gene. Data are normalized to the expression level in WT or EV. DAI, days after inoculation; WT, wild-type; EV, empty vector. Scale bar, 50 μm (\u003cstrong\u003ea\u003c/strong\u003e,\u003cstrong\u003e b\u003c/strong\u003e) or 100 μm (\u003cstrong\u003ed\u003c/strong\u003e). Data are mean ± s.d. with \u003cem\u003en\u003c/em\u003e = 10 (\u003cstrong\u003ee\u003c/strong\u003e), \u003cem\u003en\u003c/em\u003e = 13 (\u003cstrong\u003ef\u003c/strong\u003e) or \u003cem\u003en\u003c/em\u003e = 3 (\u003cstrong\u003eg\u003c/strong\u003e) biological replicates; Two-tailed Student’s \u003cem\u003et\u003c/em\u003e-test, *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt;0.01.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5006581/v1/10d7f14dc4604789b6845b8a.png"},{"id":78742506,"identity":"9c17ea0f-4396-4511-ad58-08494237549c","added_by":"auto","created_at":"2025-03-18 09:39:07","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":317307,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eGmFT5b\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eGmFDL23\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e synergistically inhibit \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eGmCLV3-1 \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eexpression. a\u003c/strong\u003e, ChIP–qPCR assay to verify binding sites of GmFDL23 on \u003cem\u003eGmCLV3-1\u003c/em\u003e\u003csub\u003e\u003cem\u003epro\u003c/em\u003e\u003c/sub\u003e. Red vertical bars indicate the potential binding site of \u003cem\u003eGmCLV3-1\u003c/em\u003e promoter. Blue letters indicate the site of probes in EMSA assays. \u003cstrong\u003eb\u003c/strong\u003e, EMSA assays. Probe falls at P4 (\u003cstrong\u003ea\u003c/strong\u003e). \u003cstrong\u003ec\u003c/strong\u003e, \u003cem\u003eGmCLV3-1\u003c/em\u003e\u003csub\u003e\u003cem\u003epro\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e:LUC\u003c/em\u003e activity in \u003cem\u003eN. benthamiana\u003c/em\u003e leaves. \u003cstrong\u003ed\u003c/strong\u003e, GUS staining and RT-qPCR analysis in \u003cem\u003eGmCLV3-1\u003c/em\u003e\u003csub\u003e\u003cem\u003epro\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e:GUS\u003c/em\u003e hairy roots in WT or the \u003cem\u003eGmft5b-1\u003c/em\u003e mutant. Scale bar, 100 μm\u003cstrong\u003e. e\u003c/strong\u003e, Relative expression level of \u003cem\u003eGmCLV3-1\u003c/em\u003e in the \u003cem\u003eGmFT5b\u003c/em\u003e mutant and \u003cem\u003eCas9-GmFDL23 \u003c/em\u003ehairy roots. \u003cstrong\u003ef\u003c/strong\u003e, Relative expression level of \u003cem\u003eGmCLV3-1 \u003c/em\u003ein \u003cem\u003e35S:GmFT5b\u003c/em\u003e and\u003cem\u003e 35:GmFDL23 \u003c/em\u003ehairy roots. CK, \u003cem\u003e35S:GFP\u003c/em\u003e; WT, wild-type; EV, empty vector. \u003cem\u003eGmUKN1\u003c/em\u003e is used as the reference gene (\u003cstrong\u003ed, e, f\u003c/strong\u003e). Data are normalized to CK in luciferase activity analysis (\u003cstrong\u003ec\u003c/strong\u003e) or expression levels in WT or EV in RT-qPCR analysis (\u003cstrong\u003ed, e, f\u003c/strong\u003e). Data are mean ± s.d. with \u003cem\u003en\u003c/em\u003e = 3 (\u003cstrong\u003ea, d, e, f\u003c/strong\u003e) or \u003cem\u003en\u003c/em\u003e = 10 (\u003cstrong\u003ec\u003c/strong\u003e); One-way ANOVA (\u003cstrong\u003ec\u003c/strong\u003e), different letters indicate \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05; two-tailed Student’s \u003cem\u003et\u003c/em\u003e-test (\u003cstrong\u003ea\u003c/strong\u003e, \u003cstrong\u003ed\u003c/strong\u003e, \u003cstrong\u003ee\u003c/strong\u003e,\u003cstrong\u003e f\u003c/strong\u003e), **\u003cem\u003ep\u003c/em\u003e \u0026lt;0.01;\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5006581/v1/4caa6a3d9c547cad228fa4b1.png"},{"id":78742507,"identity":"b8132051-7700-46f2-a878-0e25b82d2456","added_by":"auto","created_at":"2025-03-18 09:39:07","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":607951,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRhizobia repress\u003c/strong\u003e \u003cem\u003e\u003cstrong\u003eGmCLV3-1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e activity and then activate \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eGmWUS1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e to trigger nodulation. a\u003c/strong\u003e,\u003cem\u003e GmCLV3-1\u003c/em\u003e\u003csub\u003e\u003cem\u003epro\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e:GUS\u003c/em\u003e activity in roots. \u003cstrong\u003eb\u003c/strong\u003e, Nodule number in WT and \u003cem\u003eGmft5b-1\u003c/em\u003e hairy roots expressing EV, \u003cem\u003eCas9-GmCLV3-1\u003c/em\u003e or\u003cem\u003e 35S:GmCLV3-1\u003c/em\u003e at 21 DAI. \u003cstrong\u003ec\u003c/strong\u003e, \u003cem\u003eGmWUS1\u003c/em\u003e\u003csub\u003e\u003cem\u003epro\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e:LUC\u003c/em\u003e activity in \u003cem\u003eN. benthamiana\u003c/em\u003e leaves. \u003cstrong\u003ed\u003c/strong\u003e, Nodule number in EV, \u003cem\u003eCas9\u003c/em\u003e-\u003cem\u003eGmWUS1\u003c/em\u003e, and \u003cem\u003e35S:GmWUS1\u003c/em\u003e hairy roots at 21 DAI. \u003cstrong\u003ee\u003c/strong\u003e, \u003cem\u003eGmWOX5\u003c/em\u003e\u003csub\u003e\u003cem\u003epro\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e:LUC\u003c/em\u003e activity in \u003cem\u003eN. benthamiana\u003c/em\u003e leaves. \u003cstrong\u003ef \u003c/strong\u003eand\u003cstrong\u003e g\u003c/strong\u003e, The expression of \u003cem\u003eErnA\u003c/em\u003e (\u003cstrong\u003ef\u003c/strong\u003e), \u003cem\u003eGmFT5b\u003c/em\u003e, \u003cem\u003eGmFDL23\u003c/em\u003e, and nodulation marker genes (\u003cstrong\u003eg\u003c/strong\u003e) in \u003cem\u003e35S:ErnA\u003c/em\u003e hairy roots at 7 DAI. \u003cstrong\u003eh\u003c/strong\u003e, A proposed model of \u003cem\u003eGmFT5b\u003c/em\u003e-\u003cem\u003eGmFDL23\u003c/em\u003e promoting nodule formation. In uninfection conditions, \u003cem\u003eGmCLV3-1\u003c/em\u003e dominants in root cortex to inhibit \u003cem\u003eGmWUS1\u003c/em\u003e expression and no nodules form. Once rhizobium infection, \u003cem\u003eGmFDL23\u003c/em\u003e and \u003cem\u003eGmFT5b\u003c/em\u003e are activated by Type III effectors in cortex, that leads to repression of \u003cem\u003eGmCLV3-1\u003c/em\u003e and in turn triggers \u003cem\u003eGmWUS1\u003c/em\u003e activation, which elicits formation of nodules. Gray line/word, inactivation; black line/word, activation; T3E, type III effector. DAI, days after inoculation; WT, wild-type; EV, empty vector; CK, \u003cem\u003e35S:GFP\u003c/em\u003e. \u003cem\u003eGmUKN1\u003c/em\u003e is used as the reference gene (f, g). Data are normalized to CK of luciferase activity analysis (\u003cstrong\u003ec, e\u003c/strong\u003e) and expression levels in WT-EV of RT-qPCR analysis (\u003cstrong\u003ef, g\u003c/strong\u003e); Data are mean ± s.d. with \u003cem\u003en\u003c/em\u003e = 12 (\u003cstrong\u003eb, c\u003c/strong\u003e), \u003cem\u003en\u003c/em\u003e = 10 (\u003cstrong\u003ed\u003c/strong\u003e) \u003cem\u003en\u003c/em\u003e = 8 (\u003cstrong\u003ee\u003c/strong\u003e),\u003cem\u003e \u003c/em\u003eor\u003cem\u003e n\u003c/em\u003e = 3 (\u003cstrong\u003ef, g\u003c/strong\u003e); Two-way ANOVA (\u003cstrong\u003eb, g\u003c/strong\u003e) or One-way ANOVA (\u003cstrong\u003ee\u003c/strong\u003e), different letters indicate \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05; two-tailed Student’s \u003cem\u003et\u003c/em\u003e-test (\u003cstrong\u003ec\u003c/strong\u003e, \u003cstrong\u003ed\u003c/strong\u003e,\u003cstrong\u003e f\u003c/strong\u003e) , *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt;0.01.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-5006581/v1/2bfb083ae3f33fc313efe1c5.png"},{"id":78744098,"identity":"29894d09-88cd-47c3-8548-5d87f12af2bb","added_by":"auto","created_at":"2025-03-18 10:03:13","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3001716,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5006581/v1/b5bb5ea2-4f9a-4664-9463-7f29af196d7d.pdf"},{"id":78742504,"identity":"0abfc2d1-e7fb-41e1-9c93-b56967313a76","added_by":"auto","created_at":"2025-03-18 09:39:07","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":14705,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTable S1 A list of primers\u003c/p\u003e","description":"","filename":"TableS1Alistofprimers.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5006581/v1/fbb97d88c3f0f583454051b0.xlsx"},{"id":78743709,"identity":"8f9a595b-725e-46ee-8e65-35a0393943d8","added_by":"auto","created_at":"2025-03-18 09:55:07","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":11235,"visible":true,"origin":"","legend":"\u003cp\u003eTable S2 Gene manes and IDs\u003c/p\u003e","description":"","filename":"TableS2GenemanesandIDs.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5006581/v1/156caa46ce5dbbe637345647.xlsx"},{"id":78742508,"identity":"e11b8ebd-f7b2-41a4-833a-e3e27967aadf","added_by":"auto","created_at":"2025-03-18 09:39:07","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":27329,"visible":true,"origin":"","legend":"\u003cp\u003eTable S3 Sequencing data for Cas9-mutants\u003c/p\u003e","description":"","filename":"TableS3SequencingdataforCas9mutants.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5006581/v1/4fb4b19eea6d6895d14d8b28.xlsx"},{"id":78742509,"identity":"dae42e98-befc-4e79-9465-46ec433151c6","added_by":"auto","created_at":"2025-03-18 09:39:07","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":3822464,"visible":true,"origin":"","legend":"Extended data Figures","description":"","filename":"ExtendedDataFig.17.docx","url":"https://assets-eu.researchsquare.com/files/rs-5006581/v1/59a0da392f6ee26e03bf516d.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"A rhizobium-induced FT-FD module locally activates nodule stem cell gene","fulltext":[{"header":"Main text","content":"\u003cp\u003eFlorigen is well known as a movable signal being produced in leaves and then transported not only to apexes to initiate flower formation\u003csup\u003e8,9\u003c/sup\u003e or bud dormancy and burst\u003csup\u003e10\u003c/sup\u003e, but also to underground organs to enhance tuberization\u003csup\u003e11\u003c/sup\u003e or nodulation\u003csup\u003e7\u003c/sup\u003e. Gene duplications in plant genomes supply a wonderful opportunity for new functional assignments of homologous genes to adapt to unique environmental stresses and developmental machine of a given plant\u003csup\u003e12-14\u003c/sup\u003e. In soybean genome, at least six florigen homologous genes survive from the evolutionary process and encode proteins sharing similar molecular functions of flowering activity\u003csup\u003e4-6\u003c/sup\u003e. However, their biological functions diverge in soybean evolutional process, partially due to their different expression patterns\u003csup\u003e4\u003c/sup\u003e. Recently, we found that one soybean florigen homolog, \u003cem\u003eGmFT5b\u003c/em\u003e (Glyma.19G108200), expressed in roots and nodules (Extended Data Fig. 1), supposing that it may locally function in these organs, different from its siblings, \u003cem\u003eGmFT2a\u003c/em\u003e (Glyma.16G150700) and\u003cem\u003e\u0026nbsp;GmFT5a\u003c/em\u003e (Glyma.16G044100), both of which are produced in leaves and then move upward to apexes to initiate floral primordia\u003csup\u003e6\u003c/sup\u003e or downward to roots to regulate nodulation\u003csup\u003e7\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eTo test our hypothesis, we first investigated the expression pattern of \u003cem\u003eGmFT5b\u003c/em\u003e promoter (\u003cem\u003eGmFT5b\u003csub\u003epro\u003c/sub\u003e\u003c/em\u003e), around 10 kb of ATG upstream sequence which may contain potential \u003cem\u003ecis\u003c/em\u003e-elements since there are regulating elements in over 8 kb of ATG upstream sequence of \u003cem\u003eArabidopsis\u003c/em\u003e florigen \u003cem\u003eFT\u003c/em\u003e promoter\u003csup\u003e15,16\u003c/sup\u003e, using \u003cem\u003eGmFT5b\u003csub\u003epro\u003c/sub\u003e:GUS\u003c/em\u003e transgenic plants and hairy roots. Results showed that GUS signals were clearly found in most organs with high signals in vascular tissues of roots, nodules, and leaves (Fig. 1a, and Extended Data Fig. 1a). RT-qPCR confirmed that leaves, shoots, flowers, pods, roots, and nodules accumulated\u003cem\u003e\u0026nbsp;GmFT5b\u003c/em\u003e transcripts, different from website data (Extended Data Fig. 1b). The expression activity of \u003cem\u003eGmFT5b\u003csub\u003epro\u003c/sub\u003e\u003c/em\u003e in roots suggested that \u003cem\u003eGmFT5b\u003c/em\u003e could be locally produced in roots other than transported from other organs. Interestingly, after rhizobia infection the GUS signal gradually, even though 12 HAI (hour after infection), extended to endodermis and cortex (inner side, next to stele) (Fig. 1a), potential sites for nodule primordia initiation\u003csup\u003e3,17\u003c/sup\u003e. At 5 DAI (day after infection), cortex had strong GUS signals. RT-qPCR results supported that \u003cem\u003eGmFT5b\u003c/em\u003e expression was induced by rhizobium infection (Fig. 1b). At 10 DAI, endodermis and bundle sheath kept strong signals, whereas the nodule primordium did not detect strong GUS activity (Extended Data Fig. 1c). In latter nodule developmental stages, vascular system was also the tissue with high GUS signals, but nitrogen fixation zone showed weak signal (Extended Data Fig. 1c). The results suggested that \u003cem\u003eGmFT5b\u003c/em\u003e took its roles mainly at two stages of nodulation: initiation and later development stage (vascular tissues).\u003c/p\u003e\n\u003cp\u003eNext, we generated CRISPR/Cas9 edited mutants of \u003cem\u003eGmFT5b\u003c/em\u003e gene, \u003cem\u003eGmft5b-1\u003c/em\u003e and \u003cem\u003eGmft5b-9\u003c/em\u003e, which had pre-mature mutated sites in exon 1 and 4, respectively (Extended Data Fig. 2a). Both \u003cem\u003eGmft5b-1\u003c/em\u003e and \u003cem\u003eGmft5b-9\u0026nbsp;\u003c/em\u003emutants displayed less nodules, especially small nodules, compared to wild type plants (Fig. 1c and Extended Data Fig. 2b), while overexpressing \u003cem\u003eGmFT5b\u003c/em\u003e increased nodule number (Fig. 1d and Extended Data Fig. 2c and 3a). Because GmFT2a and GmFT5a proteins are transported from shoots to roots to enhance nodulation in soybean\u003csup\u003e7\u003c/sup\u003e, we supposed that all members of \u003cem\u003eGmFT\u003c/em\u003e family were involved in nodulation. Then, we analyzed the function of \u003cem\u003eFT\u003c/em\u003e-like genes on nodulation using \u003cem\u003eGmFT\u003c/em\u003e family RNAi transgenic lines, RNAi\u003cem\u003e-GmFTs-1\u003c/em\u003e and \u003cem\u003e-3\u003c/em\u003e\u003csup\u003e5\u003c/sup\u003e, and found that silencing \u003cem\u003eGmFT\u003c/em\u003e family slightly increased nodule number, especially small nodules (Extended Data Fig. 2d), indicating that there may exist antagonistic effects among different members of \u003cem\u003eGmFT\u003c/em\u003e family on nodulation. To make it clear whether \u003cem\u003eGmFT5b\u003c/em\u003e functioned locally in roots, we carried out grating experiment. Amazingly, the results demonstrated that it was not \u003cem\u003eGmft5b-1\u003c/em\u003e scion but\u003cem\u003e\u0026nbsp;Gmft5b-1\u003c/em\u003e stock to determine the number of nodules of the graft chimeras (Fig. 1e and Extended Data Fig. 2e). Taken together, \u003cem\u003eGmFT5b\u003c/em\u003e locally took a role in roots in enhancing nodulation in soybean.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eFT\u003c/em\u003e regulates flower anlagen initiation through its interaction with FD to activate flower identity gene, such as \u003cem\u003eAP1\u003c/em\u003e\u003csup\u003e18,19\u003c/sup\u003e. We wondered that \u003cem\u003eGmFT5b\u003c/em\u003e functioned in soybean nodulation in a similar molecular mechanism. Firstly, we checked if GmFT5b interacted with GmFDL23 or GmFDL20 proteins, both of which showed high expression in roots (Extended Data Fig. 4a and 4b). GmFT5b proteins distributed in whole cells, while GmFDL23 or GmFDL20 proteins localized in nuclei (Fig. 2a and Extended Data Fig. 4c and 4d). As expected, their interactions in nuclei were verified by BiFC (bimolecular fluorescence complementation), Co-IP (co-immunoprecipitation), and Y2H (yeast-two-hybrid) experiments (Fig. 2b, 2c and Extended Data Fig. 4e to 4i). \u003cem\u003eGmFDL23\u003c/em\u003e also showed rhizobium-induced expression pattern in roots and nodules (Fig. 2d, and Extended Data Fig. 4j), but \u003cem\u003eGmFDL20\u0026nbsp;\u003c/em\u003eexpression decreased after rhizobium infection (Extended Data Fig. 4k). Without rhizobium infection, \u003cem\u003eGmFDL23\u0026nbsp;\u003c/em\u003eexpressed weakly in root stele; once infection, root stele displayed high level of \u003cem\u003eGmFDL23\u003c/em\u003e expression,even some pericycle or endodermis cells expressed \u003cem\u003eGmFDL23\u003c/em\u003e. Loss of function of \u003cem\u003eGmFDL23\u003c/em\u003e led to low number of nodules, while overexpression of \u003cem\u003eGmFDL23\u003c/em\u003e increased nodule number (Fig. 2e, 2f, and Extended Data Fig. 3b, 3e and 6a). Additionally, the expression of early marker genes of nodulation in both \u003cem\u003eGmft5b\u003c/em\u003e and \u003cem\u003eGmfdl23\u003c/em\u003e mutants was similarly decreased (Fig. 2g). These results suggested thatGmFDL23 interacted with GmFT5b proteins to participate in nodulation. Furthermore, we found that \u003cem\u003eGmFT\u003c/em\u003e and \u003cem\u003eGmFDL\u003c/em\u003e did not significantly activate \u003cem\u003eGmAP1\u003c/em\u003e promoter (Extended Data Fig. 5a), indicating the\u003cem\u003e\u0026nbsp;GmFT\u003c/em\u003e-\u003cem\u003eGmFDL\u003c/em\u003e module may regulate nodulation in a novel mechanism, independent of \u003cem\u003eGmAP1\u003c/em\u003e gene.\u003c/p\u003e\n\u003cp\u003eStem cell activation is a key step for new organ formation. During this process, the stem cell identity gene \u003cem\u003eWUS\u003c/em\u003e plays a central role\u003csup\u003e20-23\u003c/sup\u003e, and high \u003cem\u003eWUS\u0026nbsp;\u003c/em\u003eactivity increases globally organogenesis\u003csup\u003e24,25\u003c/sup\u003e. \u003cem\u003eWUS\u003c/em\u003e not only is a direct target of \u003cem\u003eCLV3\u003c/em\u003e, but directly inhibits \u003cem\u003eCLV3\u003c/em\u003e expression\u003csup\u003e26-28\u003c/sup\u003e; such a regulatory dynamics of this loop drives \u003cem\u003ede novo\u003c/em\u003e stem cell formation to initiate a meristem or anlagen\u003csup\u003e29,30\u003c/sup\u003e. Bioinformatic analysis identified binding motifs of bZIP transcription factor in promoters of \u003cem\u003eGmCLV3-1\u003c/em\u003e and \u003cem\u003eGmWUS1\u003c/em\u003e (AGCT\u003csup\u003e31\u003c/sup\u003e, Fig. 3a). We wondered if \u003cem\u003eGmFT5b\u003c/em\u003e and \u003cem\u003eGmFDL\u003c/em\u003e would act directly in a WUS-CLV3 feedback loop. ChIP-qPCR confirmed that GmFDL23, a bZIP transcription factor, would enrich \u003cem\u003eGmCLV3-1\u003c/em\u003e, but not \u003cem\u003eGmWUS1\u003c/em\u003e, specific fragments of promoters (Fig. 3a and Extended Data Fig. 5b), and EMSA test supported that GmFDL23 proteins physically interacted with the corresponding fragment (Fig. 3b). \u003cem\u003eGmFDL23\u003c/em\u003e and \u003cem\u003eGmFT5b\u003c/em\u003e synergistically inhibited the promoter activity of\u003cem\u003e\u0026nbsp;GmCLV3-1\u003c/em\u003e (Fig. 3c) and in the \u003cem\u003eGmft5b\u003c/em\u003e mutant \u003cem\u003eGmCLV3-1\u003csub\u003epro\u003c/sub\u003e\u003c/em\u003e promoter activities were significantly enhanced compared to that in wild type plants (Fig. 3d). Furthermore, the mutation of \u003cem\u003eGmFDL23\u003c/em\u003e or \u003cem\u003eGmFT5b\u003c/em\u003e genes resulted in increasing \u003cem\u003eGmCLV3-1\u003c/em\u003e expression (Fig. 3e and Extended Data Fig. 2a and 6a), while overexpression of \u003cem\u003eGmFT5b\u003c/em\u003e or \u003cem\u003eGmFDL23\u003c/em\u003e repressed \u003cem\u003eGmCLV3-1\u003c/em\u003e expression (Fig. 3f and Extended Data Fig. 3e). Together, the \u003cem\u003eGmFT5b-GmFDL23\u003c/em\u003e module may directly inhibit\u003cem\u003e\u0026nbsp;GmCLV3-1\u003c/em\u003e expression in roots.\u003c/p\u003e\n\u003cp\u003eInterestingly, \u003cem\u003eGmCLV3-1\u003c/em\u003e expression in roots was strongly inhibited by rhizobium infection (Fig. 1b), and after 3 DAI (day after infection) no GUS signals were detected in root cortex (Fig. 4a). Loss of\u003cem\u003e\u0026nbsp;GmCLV3-1\u003c/em\u003e function not only increased the nodule number per plant, but rescued \u003cem\u003eGmft5b\u003c/em\u003e mutant phenotypes on nodule number (Fig. 4b and Extended Data Fig. 3c and 7). Unsurprisingly, overexpression of \u003cem\u003eGmCLV3-1\u003c/em\u003e decreased nodule number (Fig. 4b and Extended Data Fig. 3c and 3e). As what happens in apexes, \u003cem\u003eGmCLV3-1\u003c/em\u003e inhibited\u003cem\u003e\u0026nbsp;GmWUS\u003c/em\u003e activity, because overexpression of \u003cem\u003eGmCLV3-1\u003c/em\u003e inhibited \u003cem\u003eGmWUS\u003c/em\u003e expression (Fig. 4c) and in \u003cem\u003eGmclv3-1\u003c/em\u003e mutant \u003cem\u003eGmWUS\u003c/em\u003e expression was enhanced (Fig. 2g). Similarly, \u003cem\u003eGmWUS\u003c/em\u003e expression was induced by rhizobia (Fig. 1b), and \u003cem\u003eGmWUS\u003c/em\u003e displayed as a positive regulator of nodulation (Fig. 4d and Extended Data Fig. 3d, 3e and 6b). Previous reports showed \u003cem\u003eWUSCHEL-RELATEDHOMEOBOX 5\u003c/em\u003e (\u003cem\u003eWOX5\u003c/em\u003e), a homolog of \u003cem\u003eGmWUS\u003c/em\u003e, might control cell proliferation in nodule meristems\u003csup\u003e32,33\u003c/sup\u003e. In our experiment, \u003cem\u003eGmFT5b\u003c/em\u003e plus \u003cem\u003eGmFDL23\u003c/em\u003e inhibited \u003cem\u003eGmWOX5\u003c/em\u003e promoter activity (Fig. 4e), suggesting that \u003cem\u003eGmWUS\u003c/em\u003e and \u003cem\u003eGmWOX5\u003c/em\u003e played their roles in different pathways or different stages of nodulation.\u003c/p\u003e\n\u003cp\u003eThe type III secretion system (T3SS) is a conserved apparatus employed by rhizobia\u003csup\u003e34,35\u003c/sup\u003e to deliver the type III effectors (T3Es), also named Nop (nodulation outer protein), into the host cell to activate the symbiosis. ErnA, a T3E from \u003cem\u003eBradyrhizobium\u003c/em\u003e ORS3257 and targeted to the plant nucleus, confers the ability to nodulate \u003cem\u003eAeschynomene indica\u003c/em\u003e and ectopic expression of \u003cem\u003eErnA\u003c/em\u003e activates organogenesis of nodule-like structures in \u003cem\u003eA. indica\u003c/em\u003e roots\u003csup\u003e36\u003c/sup\u003e. It is postulated that ErnA most probably trigger nodulation via targeting to key regulators in the nodulation signaling pathway, such as NIN and NF-Y\u003csup\u003e37\u003c/sup\u003e. We wondered if T3E had direct effect on \u003cem\u003eGmFT5b\u003c/em\u003e and \u003cem\u003eGmFDL23\u003c/em\u003e. Interestingly, soybean codon optimized \u003cem\u003eErnA\u003c/em\u003e gene indeed activated not only\u003cem\u003e\u0026nbsp;GmFT5b\u003c/em\u003e and\u003cem\u003e\u0026nbsp;GmFDL23\u003c/em\u003e genes, but also nodulation marker genes (Fig. 4f and 4g). However, we did not observe nodule-like structure in \u003cem\u003e35S:ErnA\u003c/em\u003e hairy roots as in \u003cem\u003eA. indica\u003c/em\u003e roots\u003csup\u003e36\u003c/sup\u003e, maybe due to that only \u003cem\u003eErnA\u003c/em\u003e is not enough to induce nodulation in soybean\u003csup\u003e38\u003c/sup\u003e, because \u003cem\u003eErnA\u003c/em\u003e is from \u003cem\u003eBradyrhizobium\u003c/em\u003e strain ORS3257, which does not elicit nodules on soybean\u003csup\u003e38\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eIn summary, we proposed a working model (Fig. 4h). In soybean roots, without rhizobium infection, \u003cem\u003eGmFT5b\u003c/em\u003e and \u003cem\u003eGmFDL\u003c/em\u003e expression was limited in stele cells, while \u003cem\u003eGmCLV3\u003c/em\u003e expressed in cortex cells and thereby inhibited \u003cem\u003eGmWUS\u003c/em\u003e expression. Once infection, rhizobium signals (T3E and others) activated the expression of\u003cem\u003e\u0026nbsp;GmFT5b\u003c/em\u003e and \u003cem\u003eGmFDL\u003c/em\u003e in root cortex cells, which locally inhibited \u003cem\u003eGmCLV3\u003c/em\u003e expression and then release the repression of \u003cem\u003eGmWUS\u003c/em\u003e by \u003cem\u003eGmCLV3\u003c/em\u003e. High GmWUS activity triggers \u003cem\u003ede-novo\u003c/em\u003e stem cell formation and thereby nodule primordia initiation\u003csup\u003e3,17\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\n"},{"header":"Limitation","content":"\u003cp\u003eIn our study, we revealed a pathway for \u003cem\u003ede-novo\u003c/em\u003e activation of stem cell genes in root cortex in soybean mediated by a \u003cem\u003eFT-FD\u003c/em\u003e module. This may be the very early and indispensable step for nodulation. However, there exist some important issues uncovered. \u003cem\u003ede novo\u003c/em\u003e stem cell formation is a progressive process starting with a transient regulatory network in a small group of cells\u003csup\u003e30\u003c/sup\u003e, but we did not both identify such a group of cells and capture spatio-temporal dynamics of \u003cem\u003eGmWUS\u003c/em\u003e activity in root cortex. We also did not know the role of \u003cem\u003eGmWUS\u003c/em\u003e in nodule cell proliferation, which is different from stem cell proliferation\u003csup\u003e17\u003c/sup\u003e. It is interesting to explore the relationship between \u003cem\u003eGmWUS\u003c/em\u003e andits homolog \u003cem\u003eWOX5\u003c/em\u003e in nodulation. And \u003cem\u003eCLV3\u003c/em\u003e is not the only gene controlling \u003cem\u003eWUS\u003c/em\u003e expression\u003csup\u003e39\u003c/sup\u003e, other signals also confer regulation of \u003cem\u003eWUS\u003c/em\u003e activity. For example, nitrate, an important signal for nodulation, can modulate \u003cem\u003eWUS\u003c/em\u003e expression in shoot meristems through cytokinins\u003csup\u003e24\u003c/sup\u003e. What is more, an WUS-independent pathway is identified for regulation of stem cell proliferation\u003csup\u003e40-42\u003c/sup\u003e and the effect of interaction between \u003cem\u003eGmFT5b\u003c/em\u003e and other two siblings (\u003cem\u003eGmFT5a\u003c/em\u003e and \u003cem\u003eGmFT2a\u003c/em\u003e) on nodulation is unknown, indicating more complex mechanism of nodulation is waiting to be explained.\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Drs. Dawei Xin, Chao Ma, and Eric Giraud for helpful suggestions on rhizobia and T3E. This work was supported by the National Key Research and Development Program of China (2021YFF1001202), the National Natural Science Foundation of China (32372028, 31771714, and 32360797), and the Innovation Program of Chinese Academy of Agricultural Sciences.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eY.F. and X.Z conceived, supervised the project, and drafted the manuscript; H.S. conducted most of experiments, including root nodule phenotype analysis, grafting experiments, vector construction, mutant identification, RT-qPCR, GUS histochemical staining, co-immunoprecipitation, subcellular localization, BiFC, transcriptional activation assays, EMSA, and ChIP-qPCR; X.Z. conducted soybean transformation and hairy root transformation, phenotype investigation, and project and lab management; P.H. conducted some root transformation; Q.S. performed the yeast two-hybrid experiments; G.Y., L.L., M.L., L.H., and K.Q. cloned some gene and promoter and constructed related vectors; Y.F. and H.S. analyzed data and plotted figures and tables; X.F, Y.M., Q.C., and J.Y. analyzed some data and reviewed the manuscript; all authors contributed to data analysis and manuscript preparation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interest declaration\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMaterials \u0026amp; Correspondence\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYong-Fu Fu: to whom correspondence and material requests should be addressed at [email protected].\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eStorz, J. 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The Versatile Roles of Type III Secretion Systems in Rhizobium-Legume Symbioses. \u003cem\u003eAnnu Rev Microbiol\u003c/em\u003e \u003cstrong\u003e76\u003c/strong\u003e, 45-65 (2022). https://doi.org/10.1146/annurev-micro-041020-032624\u003c/li\u003e\n\u003cli\u003eRatu, S. T. N.\u003cem\u003e et al.\u003c/em\u003e Rhizobia use a pathogenic-like effector to hijack leguminous nodulation signalling. \u003cem\u003eScientific reports\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, 2034 (2021). https://doi.org/10.1038/s41598-021-81598-6\u003c/li\u003e\n\u003cli\u003eBaurle, I. \u0026amp; Laux, T. Regulation of WUSCHEL transcription in the stem cell niche of the Arabidopsis shoot meristem. \u003cem\u003ePlant Cell\u003c/em\u003e \u003cstrong\u003e17\u003c/strong\u003e, 2271-2280 (2005). https://doi.org/10.1105/tpc.105.032623\u003c/li\u003e\n\u003cli\u003eHuang, W.\u003cem\u003e et al.\u003c/em\u003e ALTERED MERISTEM PROGRAM1 suppresses ectopic stem cell niche formation in the shoot apical meristem in a largely cytokinin-independent manner. \u003cem\u003ePlant Physiol\u003c/em\u003e \u003cstrong\u003e167\u003c/strong\u003e, 1471-1486 (2015). https://doi.org/10.1104/pp.114.254623\u003c/li\u003e\n\u003cli\u003eKimura, Y., Tasaka, M., Torii, K. U. \u0026amp; Uchida, N. ERECTA-family genes coordinate stem cell functions between the epidermal and internal layers of the shoot apical meristem. \u003cem\u003eDevelopment\u003c/em\u003e \u003cstrong\u003e145\u003c/strong\u003e (2018). https://doi.org/10.1242/dev.156380\u003c/li\u003e\n\u003cli\u003eLee, C. \u0026amp; Clark, S. E. A WUSCHEL-Independent Stem Cell Specification Pathway Is Repressed by PHB, PHV and CNA in Arabidopsis. \u003cem\u003ePloS one\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e, e0126006 (2015). https://doi.org/10.1371/journal.pone.0126006\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003ePlant materials and growth conditions\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe genetic background of \u003cem\u003eGmft5b\u003c/em\u003e mutants and \u003cem\u003eGmFT5bPro:GUS\u003c/em\u003e transgenic plants is soybean (\u003cem\u003eGlycine max\u0026nbsp;\u003c/em\u003e(Merr.) L.) Williams 82 (WS82), and the genetic background of \u003cem\u003eGmFTs\u003c/em\u003e RNAi lines is soybean Tianlong 1 (TL1). Hairy root transformation experiment was performed with WS82 and relative transgenic plants. All materials were planted in vermiculite and grown in a growth chamber at 25°C in a 16 h light / 8 h dark cycle with the light intensity of 400 μM mol m\u003csup\u003e-2\u003c/sup\u003e s\u003csup\u003e-1\u003c/sup\u003e. During the growth period, nutrient solution was applied as 1 mM KNO\u003csub\u003e3\u003c/sub\u003e, 1 mM CaCl\u003csub\u003e2\u003c/sub\u003e, 10 μM Fe-citrate, 0.25 μM MgSO\u003csub\u003e4\u003c/sub\u003e, 0.25 μM K\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e, 1 μM MnSO\u003csub\u003e4\u003c/sub\u003e, 2 μM H\u003csub\u003e3\u003c/sub\u003eBO\u003csub\u003e3\u003c/sub\u003e, 0.5 μM ZnSO\u003csub\u003e4\u003c/sub\u003e, 0.2 μM CuSO\u003csub\u003e4\u003c/sub\u003e, 0.1 μM CoSO\u003csub\u003e4\u003c/sub\u003e, 0.1 μM Na\u003csub\u003e2\u003c/sub\u003eMoO\u003csub\u003e4\u003c/sub\u003e, 5 μM KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e. The rhizobia (\u003cem\u003eSinorhizobium fredii\u0026nbsp;\u003c/em\u003eHH103) were applied to soils after three days of transplanting. HH103 was previously cultured in 28°C TY liquid medium (3 g/L yeast extract, 5 g/L tryptone, 0.4 g/L CaCl\u003csub\u003e2\u003c/sub\u003e) for 2-3 days.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eVector construction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor promoter activity analysis and transcriptional activity analysis, promoters (\u003cem\u003eGmFT5b\u003c/em\u003e, 10 kb; \u003cem\u003eGmFDL23\u003c/em\u003e, 3 kb; \u003cem\u003eGmAP1-2\u003c/em\u003e, 2.6 kb; \u003cem\u003eGmAP1-3\u003c/em\u003e, 2.5 kb; \u003cem\u003eGmCLV3-1\u003c/em\u003e, 3 kb; \u003cem\u003eGmWUS1\u003c/em\u003e, 3 kb; \u003cem\u003eGmWOX5\u003c/em\u003e, 3 kb) were cloned from WS82 and into the Fu76 entry vector\u003csup\u003e43\u003c/sup\u003e by enzyme digestion and ligation. Subsequently, along with the Fu79-GUS or Fu79-LUC entry vectors, they were transferred into the pSoy10 binary vector by LR reactions.\u003c/p\u003e\n\u003cp\u003eFor the construction of vectors for overexpressing hairy roots and transcriptional activity analysis in \u003cem\u003eNicotiana benthamiana\u003c/em\u003e leaves, the coding sequences (CDS) of\u003cem\u003e\u0026nbsp;GmFT5b\u003c/em\u003e, \u003cem\u003eGmFDL9\u003c/em\u003e, \u003cem\u003eGmFDL16\u003c/em\u003e, \u003cem\u003eGmFDL20\u003c/em\u003e, \u003cem\u003eGmFDL23\u003c/em\u003e, \u003cem\u003eGmCLV3-1\u003c/em\u003e, and \u003cem\u003eGmWUS1\u003c/em\u003e were cloned from WS82, while the ErnA CDS sequence was synthesized by Genecreate company. All CDS sequences were ligated into the FU28 vector through enzymatic digestion\u003csup\u003e43\u003c/sup\u003e. Subsequently, along with the Fu76-\u003cem\u003e35S\u003csub\u003epro\u003c/sub\u003e\u003c/em\u003e vector they were transferred to the pSoy10 binary vector by LR reactions.\u003c/p\u003e\n\u003cp\u003eFor gene editing, two sgRNA sequences were designed using CRISPR-P 2.0 software (\u003cu\u003ehttp://crispr.hzau.edu.cn/CRISPR2/\u003c/u\u003e) and ligated into the Fu79 entry vector containing the U6 promoter at the \u003cem\u003eBspQ\u003c/em\u003eⅠ\u0026nbsp;and \u003cem\u003eBsa\u003c/em\u003eⅠ\u0026nbsp;sites. Along with the Fu76-Cas9 vector by LR reactions, theFu79-sgRNAs were then transferred to the pSoy10 or pSoy13 vector.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHairy root transformation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWS82 and different mutants were used for hairy root transformation. The constructs were transformed into the \u003cem\u003eAgrobacterium rhizogenesis\u003c/em\u003e strain K599 and cultured in 50 mL of liquid LB with selection marker kanamycin until OD\u003csub\u003e600\u0026nbsp;\u003c/sub\u003e= 0.8. Root transformation was performed following a previously reported method\u003csup\u003e44\u003c/sup\u003e. Briefly, sterilized soybean seeds were soaked and germinated, and the elongated radicles were excised. The explants were infected in the resuspension solution for 30 minutes and then transferred to co-cultivation medium (1/10× Gamborg B5 salts, 30 g/L sucrose, 3.9 g/L MES, 4.25 g/L agar, pH 5.4, supplemented with 400 mg/L cysteine, 154.2 mg/L dithiothrietol, and 40 mg/L acetosyringone after sterilization). The explants were maintained in the dark for 3 days and then replaced into hairy root induction medium under 12 h light / 12 h dark cycle conditions. After three days, explants were rolled up in moistened germination paper and cultured for an additional 10 days until roots emerged. Transgenic hairy roots were selected by detecting the DsRed signal under a fluorescent stereomicroscope and non-transgenic hairy roots were removed. The composite plants with positive hairy roots were then transplanted into pots (7.5×7.5 cm) containing vermiculite with nutrient solution and grown in a growth chamber at 25°C under a 16 h light / 8 h dark cycle. Five days later, they were inoculated with rhizobia strain HH103 suspension (OD\u003csub\u003e600\u0026nbsp;\u003c/sub\u003e= 0.1) for nodule phenotype and GUS histochemical analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGUS histochemical staining and microscopic observation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTransgenic hairy roots and nodules harvested at different stages after rhizobium inoculation, as well as leaves of stable transgenic lines were collected into the GUS staining solution (50 mM sodium phosphate buffer, 0.2% Triton-X-100, 5 mM K\u003csub\u003e4\u003c/sub\u003eFe(CN)\u003csub\u003e6\u003c/sub\u003e, 5 mM K\u003csub\u003e3\u003c/sub\u003eFe(CN)\u003csub\u003e6\u003c/sub\u003e, and 1~2 mM X-gluc). The samples were subjected to vacuum for 15 minutes and incubated in dark at 37°C for 12 hours. Subsequently, the samples were cleaned three times with 70% ethanol and observed under an Olympus stereo microscope (SZX7). For samples sectioning, they were embedded in 4% agarose and cut into 80-100 μm slices using a Leica vibrating microtome (VT1000 S). Finally, photos were taken using an Olympus optical microscope (IX71).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDNA extraction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePlant or hairy root samples were harvested and used for DNA extraction using the SDS method\u003csup\u003e45\u003c/sup\u003e. Briefly, plant tissues were ground in a lysis buffer containing 2% sodium dodecyl sulfate (SDS), 50 mM Tris-HCl (pH 8.0), 10 mM ethylenediaminetetraacetic acid (EDTA), and 200 mM NaCl. The homogenate was incubated at 65°C for 10 minutes and then on ice with one-half volume of 3 M potassium acetate. After incubation, DNA was precipitated with an equal volume of isopropanol and washed with 70% ethanol. The obtained DNA was further subjected to PCR for identification of plant materials or hairy roots.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRNA extraction and RT-qPCR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe plant tissues were ground to powder in liquid nitrogen, and total RNA was extracted using HiPure Total RNA Mini Kit (Magen). The interference of genomic DNA was removed and the first strand of cDNA was reversely transcribed using the PrimeScript RT Reagent Kit with gDNA Eraser (TaKaRa). RT-qPCR was performed using the ChamQTM Universal SYBR qPCR Master Mix (Vazyme). Each RT-qPCR experiment was conducted with at least three technical replicates. \u003cem\u003eGmUKN1\u003c/em\u003e was used as the internal reference gene\u003csup\u003e46\u003c/sup\u003e. The RT-qPCR primers used are listed in Table S1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGrafting experiment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSeeds of WS82 and \u003cem\u003eGmft5b-1\u003c/em\u003e were sown in vermiculite with nutrient solution for germination (16 h light/8 h dark, 25℃). The hypocotyls of the seven-day-old seedlings were split into two halves, the scion was then gently thinned and grafted to the reciprocal rootstock with grafting clips and sealing film used to assist in fixation. The grafted plants were cultured in dark, warm, and humid environment for 5 days to minimize transpiration. Successfully grafted plants were inoculated with rhizobium strain HH103, and nodule phenotypes were assessed at 21 days after inoculation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSubcellular Localization and bimolecular fluorescence complementation (BiFC)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe coding sequences of \u003cem\u003eGmFT5b\u003c/em\u003e,\u003cem\u003e\u0026nbsp;GmFDL20\u003c/em\u003e, and\u003cem\u003e\u0026nbsp;GmFDL23\u003c/em\u003ewere cloned into the pGWC entry vector\u003csup\u003e47\u003c/sup\u003e, and then recombined into the pGWB5-GFP expression vector for subcellular localization experiments using LR reactions\u003csup\u003e47\u003c/sup\u003e. Additionally, pGWC-\u003cem\u003eGmFDL20\u003c/em\u003e, and pGWC-GmFDL23 were recombined into the pEARLYGAYE201-YN vector by LR reactions, while pGWC-\u003cem\u003eGmFT5b\u0026nbsp;\u003c/em\u003ewas recombined into the pEARLYGAYE202-YC vector for BiFC experiments. These vectors were transformed into \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e strain EHA105. When the bacterial culture reached an OD\u003csub\u003e600\u003c/sub\u003e = 1.0, it was resuspended in infiltration buffer (10 mM MES, 150 μM Acetosyringone, 10 mM MgCl\u003csub\u003e2\u003c/sub\u003e) and used to infiltrate \u003cem\u003eNicotiana benthamiana\u003c/em\u003e leaves. Fluorescence signals were observed and recorded using a Zeiss LSM980 confocal laser scanning microscope.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCo-Immunoprecipitation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThrough LR reactions, the pGWC-\u003cem\u003eGmFDL20\u003c/em\u003e and pGWC-\u003cem\u003eGmFDL23\u003c/em\u003ewere recombined into the pGWB20-Myc vector. The resulting binary vectors were transformed into \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e strain EHA105. Subsequently, they were co-infiltrated with EHA105 containing pGWB5-\u003cem\u003eGmFT5b:GFP\u003c/em\u003e construct into \u003cem\u003eNicotiana benthamiana\u003c/em\u003e leaves. Two days post-infiltration, proteins were extracted from the \u003cem\u003eNicotiana benthamiana\u003c/em\u003e leaves. Immunoprecipitation was performed at 4°C for 1 hour with anti-Myc magnetic beads (M047, Anti-Myc-tag mAb-Magnetic Agarose, MBL) and western blot analysis was then performed using anti-Myc (M192-3, MBL) and anti-GFP (M048-3, MBL) antibodies.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eYeast two-hybrid\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe coding sequences of \u003cem\u003eGmFDL20\u003c/em\u003e and \u003cem\u003eGmFDL23\u003c/em\u003e were cloned into the pGADT7 vector and the coding sequence of \u003cem\u003eGmFT5b\u003c/em\u003e was cloned into the pGBKT7 vector through restriction enzyme digestion and ligation. These constructs were co-transformed into the yeast strain AH109. Initially, yeasts were grown on SD-Leu/-Trp medium. After colonies formed, they were transferred to SD-Leu/-Trp/-His/-Ade medium and cultured for 3-4 days. The interaction between the proteins was evaluated based on the growth of yeasts under the selective conditions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTranscriptional activity assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003epSoy10-\u003cem\u003eAP1-2\u003csub\u003epro\u003c/sub\u003e:LUC\u003c/em\u003e, pSoy10-\u003cem\u003eAP1-3\u003csub\u003epro\u003c/sub\u003e:LUC\u003c/em\u003e, pSoy10-\u003cem\u003eGmCLV3-1\u003csub\u003epro\u003c/sub\u003e:LUC\u003c/em\u003e, pSoy10-\u003cem\u003eGmWUS1\u003csub\u003epro\u003c/sub\u003e:LUC\u003c/em\u003e, \u0026nbsp;and pSoy10-\u003cem\u003eGmWOX5\u003csub\u003epro\u003c/sub\u003e:LUC\u003c/em\u003e were used as reporters, while pSoy10-\u003cem\u003e35S\u003c/em\u003e\u003cem\u003e:GmFT5b\u003c/em\u003e, pSoy10-\u003cem\u003e35S:GmFDL9\u003c/em\u003e, pSoy10-\u003cem\u003e35S:GmFDL16\u003c/em\u003e, pSoy10-\u003cem\u003e35S:GmFDL20\u003c/em\u003e, pSoy10\u003cem\u003e-35S:GmFDL23\u003c/em\u003e, and pSoy10-\u003cem\u003e35S:GmCLV3-1\u003c/em\u003e were used as effectors. All constructs were transformed into \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e strain EHA105 and co-infiltrated into \u003cem\u003eNicotiana benthamiana\u003c/em\u003e leaves, respectively. After 2 days, leaves were treated with 3 mM D-luciferin substrate (Provenand Published, LUCK-1G). Luciferase signals were detected using a Tanon 5200 imaging system, and the intensity was quantified using Image J.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eChIP-qPCR assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eChromatin Immunoprecipitation (ChIP) was performed following the previous method\u003csup\u003e48\u003c/sup\u003e. Four grams of \u003cem\u003e35s:GmFDL23:GFP\u0026nbsp;\u003c/em\u003etransgenic soybean roots were harvested three days post rhizobium inoculation and minced. The minced tissue was crosslinked with 1% formaldehyde, followed by quenching with 0.4 M glycine to terminate the crosslinking reaction. The material was then ground into fine powder under liquid nitrogen for chromatin extraction. Chromatin was sonicated using a Bioruptor UCD-200 to shear DNA into fragments ranging from 200 bp to 1000 bp. The sonicated chromatin was incubated with anti-GFP antibodies and IgG (as a negative control) and immunoprecipitated using a 1:1 volume mixture of protein A and protein G beads. Crosslinks were reversed with 215 mM NaCl, and the DNA was purified using the QIAquick PCR Purification Kit (QIAGEN). Quantitative PCR (qPCR) was then employed to analyze the precipitated DNA, using primers specified in the Table S1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEMSA\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe full-length coding sequence of \u003cem\u003eGmFDL23\u003c/em\u003e was cloned into the pET28a-His expression vector using \u003cem\u003eBam\u003c/em\u003eH I and \u003cem\u003eSal\u0026nbsp;\u003c/em\u003eI restriction sites. The construct was then transformed into \u003cem\u003eEscherichia coli\u003c/em\u003e BL21 competent cells for protein expression and purification. Biotin-labeled and unlabeled probes were synthesized by BGI as listed in Table S1. Electrophoretic Mobility Shift Assays (EMSAs) were performed following the protocol of the Light-Shift Chemiluminescent EMSA Kit (Thermo Scientific). For DNA binding, purified recombinant protein was mixed with 50 fM of the labeled probe in a reaction buffer (2.5% (v/v) glycerol, 5 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 50 ng/mL poly (dI, dC), 0.05% (v/v) Nonidet P-40). To perform the competition assay, at least 50-folds of unlabeled probe were added to the mixture. The samples were incubated at room temperature for 20 minutes before being analyzed by non-denaturing 8% polyacrylamide gel electrophoresis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIdentification of CRISPR/Cas9 hairy roots\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIdentification of positive hairy roots is followed previous methods\u003csup\u003e49,50\u003c/sup\u003e. PCR amplification was performed using positive transgenic hairy root DNA as a template, with primers (Table S1) designed within 400 bp of both sides of the two sgRNA target sites. The amplified DNA fragments were subsequently sequenced. Heterozygous biallelic mutations, which appear as double peaks, were analyzed using CRISPR-GE (http://skl.scau.edu.cn/) for decoding. All sequencing data of CRISPR/Cas9 hairy roots were listed in Table S3.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAccession number\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll gene identities were listed in Table S2.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll statistical graphs were generated and analyzed using GraphPad Prism 9.0.0 (GraphPad Software). Individual data points were represented by dots. Significance of differences were examined by One-way or Two-way ANOVA with Bonferroni post hoc test or two-tailed Student’s \u003cem\u003et\u003c/em\u003e-test, with \u003cem\u003ep\u003c/em\u003e-values over than 0.05 considered as not statistically significant. Types of significant differences were indicated in the figure legends.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eReference\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e43\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Wang, X., Fan, C., Zhang, X., Zhu, J. \u0026amp; Fu, Y. F. 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The first step of nodule anlagen formation is to re-activate root cortex cells by rhizobia into stem cells, however, the mechanism of that is unknown. In soybean genome there are six florigen homologs\u003csup\u003e4-6\u003c/sup\u003e, two of which, \u003cem\u003eGmFT2a\u003c/em\u003e and \u003cem\u003eGmFT5a\u003c/em\u003e are produced in leaves and imported into roots to enhance nodulation\u003csup\u003e7\u003c/sup\u003e. Here, we demonstrated that another florigen homolog \u003cem\u003eGmFT5b\u003c/em\u003e and its partner \u003cem\u003eGmFDL23\u003c/em\u003e are induced in root cortex by rhizobia and locally inhibit \u003cem\u003eGmCLV3\u003c/em\u003e expression. Subsequently, \u003cem\u003eGmWUS \u003c/em\u003eis released from \u003cem\u003eGmCLV3-1\u003c/em\u003e repression to re-activate cortex cells into stem cells and finally to trigger nodulation. Our results identify a signaling pathway of spatiotemporal re-formation of stem cells in roots for nodule organogenesis mediated by a local florigen module in soybean. Therefore, florigen participates in nodulation at least in two processes as two different signals: stem cell formation (local signal, this study) and primordium initiation or development (systemic signal from leaves, a previous report\u003csup\u003e7\u003c/sup\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOne sentence summary：\u003c/strong\u003eA florigen homolog \u003cem\u003eGmFT5b\u003c/em\u003eand its partner \u003cem\u003eGmFDL23 \u003c/em\u003eare induced by rhizobia in roots and locally activates stem cell identity gene \u003cem\u003eGmWUS\u003c/em\u003e to trigger nodulation in soybean.\u003c/p\u003e","manuscriptTitle":"A rhizobium-induced FT-FD module locally activates nodule stem cell gene","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-18 09:39:02","doi":"10.21203/rs.3.rs-5006581/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"ede5ec46-b2a7-4500-ad0b-713a94d82b2f","owner":[],"postedDate":"March 18th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":37753760,"name":"Biological sciences/Plant sciences/Plant symbiosis/Rhizobial symbiosis"},{"id":37753761,"name":"Biological sciences/Stem cells/Reprogramming"},{"id":37753762,"name":"Biological sciences/Developmental biology/Organogenesis"},{"id":37753763,"name":"Biological sciences/Evolution/Molecular evolution"}],"tags":[],"updatedAt":"2025-03-18T09:39:02+00:00","versionOfRecord":[],"versionCreatedAt":"2025-03-18 09:39:02","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5006581","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5006581","identity":"rs-5006581","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}