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Loss of Lateral suppressor gene is associated with evolution of root nodule symbiosis in Leguminosae

Genome Biology volume 25, Article number: 250 (2024) Cite this article

Abstract

Background

Root nodule symbiosis (RNS) is a fascinating evolutionary event. Given that limited genes conferring the evolution of RNS in Leguminosae have been functionally validated, the genetic basis of the evolution of RNS remains largely unknown. Identifying the genes involved in the evolution of RNS will help to reveal the mystery.

Results

Here, we investigate the gene loss event during the evolution of RNS in Leguminosae through phylogenomic and synteny analyses in 48 species including 16 Leguminosae species. We reveal that loss of the Lateral suppressor gene, a member of the GRAS-domain protein family, is associated with the evolution of RNS in Leguminosae. Ectopic expression of the Lateral suppressor (Ls) gene from tomato and its homolog MONOCULM 1 (MOC1) and Os7 from rice in soybean and Medicago truncatula result in almost completely lost nodulation capability. Further investigation shows that Lateral suppressor protein, Ls, MOC1, and Os7 might function through an interaction with NODULATION SIGNALING PATHWAY 2 (NSP2) and CYCLOPS to repress the transcription of NODULE INCEPTION (NIN) to inhibit the nodulation in Leguminosae. Additionally, we find that the cathepsin H (CTSH), a conserved protein, could interact with Lateral suppressor protein, Ls, MOC1, and Os7 and affect the nodulation.

Conclusions

This study sheds light on uncovering the genetic basis of the evolution of RNS in Leguminosae and suggests that gene loss plays an essential role.

Background

Nitrogen (N) is an essential nutrient for plant growth, but plants cannot obtain it directly from the atmosphere. Large quantities of nitrogen fertilizers are applied in agricultural practices each year, which significantly improves crop production but also results in great economic and environmental costs. Nitrogen-fixing root nodule symbiosis (RNS) is a remarkable specialized trait that allows plants to acquire nitrogen from bacteria capable of fixing atmospheric nitrogen. Plants signal to bacteria, recognize compatible partners, allow their entry, accommodate them within novel organs (nodules, typically produced on roots), and provide them with a carbon source. Genetic engineering of RNS in non-nodulating crops has been a long-standing goal in sustainable agriculture [1, 2]. A better understanding of the genomic underpinnings governing RNS assembly and evolution could facilitate such efforts.

RNS is limited to plants belonging to the four orders that comprise the so-called Nitrogen Fixing Nodulation Clade (NFNC) that diverged approximately 115 million years ago (MYA) within the rosid clade, namely Fabales (including Leguminosae), Fagales, Cucurbitales, and Rosales [3]. Nodulation is rare in three of the four orders of NFNC with an exception of Fabales, where nodulation is found in the majority of species comprising the third largest family of flowering plants, Leguminosae [4]. It is widely (though not universally) accepted that nodulation evolved in the ancestor of the NFNC [5], which means that nodulating lineages have had approximately 115 million years to modify the original nodulation program inherited from that ancestor. Because of the rapid radiation/proliferation of the NFNC [6, 7], considerable divergence is expected in the nodulation symbiosis.

Gene loss is an important source of genetic variation that takes part in evolution [8,9,10,11,12,13,14,15]. In bacteria, to adapt various environmental conditions, gene loss events are frequently occurred [16]. In contrast, the importance of gene loss involving in phenotypic adaptations or evolution in plants and mammals is largely undisclosed although several cases were reported. For instance, it was found the loss of S locus cysteine-rich protein (SCR) [17] and/or S locus receptor kinase (SRK) [18] genes is essential for the evolution of outcrossing based on self-incompatibility (SI) to a selfing system in Arabidopsis [19]; loss of ANTHOCYANIN2 (AN2) and flavonoid 3'‑hydroxylase (F3'h) gene leads to flower color changes in Petunia axillaris and Ipomoea quamoclit, respectively, which is helpful for the pollination adaptation [20, 21]. For the evolution of RNS, it was reported that the parallel loss or loss-of-function of NODULE INCEPTION (NIN), RHIZOBIUM-DIRECTED POLAR GROWTH (RPG) and NOD FACTOR PERCEPTION (NFP) genes was associated with the evolution of RNS [22, 23]. However, it was also found that NIN was not completely lost and its homologs also presented in the non-NFNC species [22]. Nevertheless, the absent/present of the Predisposition-Associated Cis-regulatory Element (PACE) in the NIN promoter may play an important role in its spatial expression [24]. Though much progress on the evolution of RNS has been made, large remains to be learned, especially any other gene loss event that is also involved in its evolution.

Here, through comprehensive phylogenomic analyses and functional validation, we found that loss of the Lateral suppressor gene was essential for the evolution of RNS in Leguminosae and revealed a possible working model of the Lateral suppressor gene in controlling nodulation.

Results

Lateral suppressor and SULFUR DEFICIENCY INDUCED 2 were lost in Leguminosae

To investigate the candidate gene loss events potentially involved in the evolution of RNS in Leguminosae, we conducted phylogenomic analyses using 48 representative species among angiosperms with high-quality genome sequences (Fig. 1a, Additional file 2: Table S1). In this study, 16 species from three of the six subfamilies of Leguminosae [25] were sampled, including members of both subfamilies with nodulating species (Caesalpinioideae and Papilionoideae) and Cercis canadensis, representing the non-nodulating Cercidoideae, which is likely sister to the remaining subfamilies. Caesalpinioideae was represented by a single species each from the nodulating genus, Chamaecrista, and the non-nodulating Senna, sister to the Chamaecrista clade (Fig. 1a). The largest and best-known legume subfamily, Papilionoideae [4], was represented by 13 species, all of them from nodulating genera except for Nissolia, which has lost the ability to nodulate (Fig. 1a).

Fig. 1
figure 1

Lateral suppressor was lost in Leguminosae. a The presence and absence of variation of Leguminosae-loss genes identified by phylogenomic analyses of 48 species. The species tree was constructed using 267 conserved and low-copy orthologous genes. Bootstrap values (1000 replicates) lower than 100% are indicated on the nodes. NFNC, Nitrogen Fixing Nodulation Clade; SDI2, SULFUR DEFICIENCY INDUCED 2; Ls, Lateral suppressor. b Phylogenetic analyses of the GRAS families from 33 species (16 Leguminosae and 17 non-Leguminosae species). A human (Homo sapiens) signal transducers and activators of transcription (STAT) protein (AAH02704) were used as an outgroup. Subfamilies (Ls, DELLA, etc.) are color-coded. The genes of Leguminosae and non-Leguminosae species are indicated with blue and red branches, respectively. SCL, SCARECROW like; DLT, DWARF AND LOW TILLERING; HAM, Hairy Meristem; SCR, SCARECROW; RAM1, Reduced Arbuscular Mycorrhization 1; NSP2, NODULATION SIGNALING PATHWAY 2; SHR, SHORTROOT; RAD1, REQUIRED FOR ARBUSCULE DEVELOPMENT 1; PAT1, phytochrome A signal transduction 1; LISCL, Lilium longiflorum SCARECROW like. c Synteny analysis of Lateral suppressor gene in representative non-Leguminosae and Leguminosae species. Leguminosae species names are highlighted in green. Homologous genes in each specific block are connected by lines of specific colors. The homologous genes of the Lateral suppressor gene in non-Leguminosae species are connected by magenta lines. Asterisks indicate the end of the chromosome or scaffold. d The heatmap of the genewise score of Ls orthologous groups. Full-length protein sequences of Ls orthogroups were used as baits to perform tblastn against the genome sequences of the 48 species. The best hit sequences were used to perform gene prediction using genewise, and the scores were used to plot the heatmap. The x-axis represents Ls orthogroups and y-axis represents the best hit of 48 species

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Phylogenetic analysis discovered 2 orthologous groups, SULFUR DEFICIENCY INDUCED 2 (SDI2) and Lateral suppressor (Ls), which were found in all non-Leguminosae but absent, and therefore presumably lost, in Leguminosae (Fig. 1a, Additional file 2: Table S2). SDI2 belongs to the tetratricopeptide repeat (TPR)-like superfamily and the gene in Arabidopsis thaliana is designated as SDI2 which was reported to play an important role in sulfur metabolism by reducing glucosinolates biosynthesis under sulfur-deprived conditions [26]. The Lateral suppressor gene is a member of the GRAS-domain protein family of plant-specific transcription regulators [27,28,29], which can be further classified into several subfamilies [30]. The Lateral suppressor (Ls) gene in tomato [27], LATERAL SUPPRESSOR (LAS) gene in Arabidopsis thaliana [28], MONOCULM 1 (MOC1) gene in rice [29], ERAMOSA in Antirrhinum [31], AaLAS in Arabis alpine [32], LOSS OF AXILLARY MERISTEMS (LAM) in strawberry [33], and ClLs in watermelon [34] are members of the Ls subfamily controlling axillary branching. We found that except for the Ls or SDI2 subfamily, other major GRAS or TPR subfamilies were universally present among Leguminosae and non-Leguminosae plant species (Fig. 1b, Additional file 1: Fig. S1a, Additional file 2: Table S3).

To confirm the loss of Lateral suppressor gene and SDI2 in Leguminosae, we further performed synteny analysis and blasted the nucleotide sequences at a whole-genome level in representative Leguminosae and non-Leguminosae species. Synteny analysis revealed that although the neighboring genes of the Lateral suppressor gene or SDI2 showed preservation in non-Leguminosae and Leguminosae, the Lateral suppressor gene and SDI2 were absence at the expected location in Leguminosae (Fig. 1c, Additional file 1: Fig. S1b). Nucleotide sequences blast showed that no gene predicted in the best hit sequences in the Leguminosae species (Fig. 1d, Additional file 1: Fig. S1c). These results suggested that the Lateral suppressor gene and SDI2 were lost in the Leguminosae species we investigated.

Ectopic expression of the non-Leguminosae Lateral suppressor gene inhibits nodulation in Leguminosae

To determine whether the loss of the Lateral suppressor gene and SDI2 was associated with the evolution of RNS in Leguminosae, we conducted hairy root transformations. Firstly, we introduced one SDI2 gene from rice, a non-Leguminosae species, into soybean. Although the gene was expressed, we did not observe significant nodulation differences (Additional file 1: Fig. S2a–c), suggesting that loss of SDI2 is unlikely to be involved in the evolution of RNS in Leguminosae and it may be associated with other traits in Leguminosae, such as seed component accumulation as it has been reported that SDI2 may affect seed protein accumulation under sulfur-deprived conditions [26, 35].

Then, we introduced MOC1 and its homolog Os7 in rice, and Ls gene from tomato, into soybean using hairy root transformation driven by the cauliflower mosaic virus (CaMV) 35S promoter. We found that the transgenic soybean roots carrying each of the three orthologous genes almost completely lost nodulation capability (Fig. 2a–d, Additional file 1: Fig. S3a). The same results were obtained when the three genes were individually introduced into Medicago truncatula (M. truncatula) using hairy root transformation (Fig. 2e–h, Additional file 1: Fig. S3b). The infection events were impaired when Os7 or MOC1 was introduced into M. truncatula (Additional file 1: Fig. S4a, b). Cultured with adequate N (treated with 5 mM KNO3), no significant differences in growth habits were observed between the transgenic plants carrying empty vector and 35S:MOC1 (Additional file 1: Fig. S4c). In contrast, in the absence of N (treated with 5 mM KCl), the 35S:MOC1 transgenic plants exhibited significantly dwarfed and yellowed leaves compared to those carrying the empty vector after 4 weeks of inoculation with Bradyrhizobium diazoefficiens strain USDA110 (Additional file 1: Fig. S4c). These results suggested that the expression of Lateral suppressor gene repressed nodulation but did not affect plant growth in the presence of N nutrients. However, overexpressing members of the other GRAS subfamilies in soybean did not yield the same nodulation inhibitory effect as the Lateral suppressor gene (Fig. 2i–l, Additional file 1: Fig. S3c). Above results indicated that ectopic expression of Lateral suppressor gene rather than other GRAS members was responsible for inhibiting nodulation in Leguminosae.

Fig. 2
figure 2

Effects of Leguminosae-loss Lateral suppressor gene on RNS. a, c Nodulation phenotypes produced by transformation of Os7 and MONOCULM 1 (MOC1) (a) and Lateral suppressor (Ls) (c) in soybean hairy root. Bar, 1 cm. b, d Number of nodules per hairy root and number of nodules per root length (cm) after the transformation of Os7 and MOC1 (b) and Ls (d) in soybean. e, g Nodulation phenotypes produced by transformation of Os7 and MOC1 (e) and Ls (g) in M. truncatula hairy root. Bar, 2 mm. f, h Number of nodules per plant and number of nodules per root length (cm) after transformation of Os7 and MOC1 (f) and Ls (h) in M. truncatula. i–l Number of nodules per hairy root and number of nodules per root fresh weight (FW, g) of overexpression GmHAM (i), GmSCL3a and GmLISCLa (j), GmNSP1a and GmNSP1b (k), and GmNSP2a and GmNSP2b (l) in soybean. Hairy roots were collected at 21 days after inoculation (DAI) for nodulation phenotype. EV indicates transformation with an empty vector. Asterisks indicate significant differences from the empty vector controls and the letter “ns” indicates that there is no significant difference compared to EV. Statistical significance was determined by a two-sided t-test in d, h, and i–l. The lowercase letters in b and f indicate significant differences between samples [analysis of variance (ANOVA), least significant difference (LSD) method]

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Lateral suppressor protein inhibits NIN transcription by interacting with NSP2 and CYCLOPS

NIN is the master transcription factor that regulates Leguminosae nodulation [36, 37]. We found that the transcription of reported MtNIN was significantly suppressed in the Os7, MOC1, or Ls gene overexpression hairy roots in M. truncatula (Additional file 1: Fig. S3d, e). The soybean genome contains four NIN homologs that are all expressed on induction by rhizobia, although at very different levels [38]. Knockdown of the four NIN genes almost completely eliminated nodulation ability in soybean (Additional file 1: Fig. S2d–f), confirming their roles in soybean nodulation. Our findings revealed that transformation with Os7, MOC1, or Ls gene also significantly reduced the expression levels of all four NIN genes in soybean (Additional file 1: Fig. S3f, g), suggesting that the Lateral suppressor gene inhibits RNS by suppressing NIN transcription.

NIN transcription in M. truncatula can be induced by NSP1 through interaction with NSP2 [39]. Yeast two-hybrid, split firefly luciferase complementation (SFLC) assays and coimmunoprecipitation (Co-IP) assays determined that Os7, MOC1, or Ls protein could interact with MtNSP2 (Fig. 3a–c). Further investigation showed that Os7, MOC1, or Ls protein could repress the transcriptional activity of the MtNSP1/MtNSP2 complex on MtNIN (Fig. 3d). In soybean, there are two NSP2 members, GmNSP2a and GmNSP2b, and two NSP1 members, GmNSP1a and GmNSP1b [40]. We found Os7, MOC1 or Ls protein could interact with both GmNSP2a and GmNSP2b (Additional file 1: Fig. S5a–f). In addition, we determined that MOC1 and Os7 could also interact with OsNSP2 in rice (Fig. 3e–g). However, no interaction was detected between Os7, MOC1, or Ls protein and GmNSP1 by the yeast two-hybrid system (Additional file 1: Fig. S5g).

Fig. 3
figure 3

Lateral suppressor protein interacts with MtNSP2 to inhibit the expression of NIN. a Yeast two-hybrid assays between MtNSP2 and Os7, MOC1 or Ls protein. AD, activation domain; BD, binding domain. b Split firefly luciferase complementation (SFLC) assays between MtNSP2 and Os7, MOC1, or Ls protein. c Coimmunoprecipitation (Co-IP) assays between MtNSP2 and Os7, MOC1 or Ls protein. d The effect of Os7, MOC1, or Ls protein on inhibiting MtNSP2-MtNSP1 activation to induce MtNIN transcription. The lowercase letters indicate significant differences between samples [ANOVA, LSD method]. REN, Renilla luciferase; LUC, firefly luciferase. e Yeast two-hybrid assays between OsNSP2 and Os7 or MOC1. f SFLC assays between OsNSP2 and Os7 or MOC1. g Co-IP assays between OsNSP2 and Os7 or MOC1

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Additionally, phosphorylated CYCLOPS, a DNA binding transcriptional activator, is known to regulate NIN transcription by binding to its promoter in Lotus japonicus [24, 41], and the replacement of phosphorylation serine sites (S50 and S154) with aspartate (D) (S50D and S154D, CYCLOPS-DD) could result in a gain-of-function activity of CYCLOPS [41]. We discovered that Os7, MOC1, or Ls protein not only interact with CYCLOPS (Fig. 4a, b), but also exert additional repression on the transcriptional activation of LjNIN by CYCLOPS-DD (Fig. 4c). Hence, our results suggest that Os7, MOC1, or Ls protein interacts with both NSP2 and CYCLOPS, inhibiting NIN transcription and repressing nodulation in Leguminosae.

Fig. 4
figure 4

Lateral suppressor protein interacts with CYCLOPS to inhibit the expression of NIN. a SFLC assays between CYCLOPS and Os7, MOC1, or Ls protein. b Co-IP assays between CYCLOPS and Os7, MOC1 or Ls protein. c The effect of Os7, MOC1, or Ls protein on the inhibition of LjNIN transcription activation by CYCLOPS-DD. The lowercase letters indicate significant differences between samples [ANOVA, LSD method]

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Lateral suppressor protein interacts with cathepsin H

The way in which cells adapt to gene loss can be linked to the functional effect of the lost gene [42,43,44,45]. To identify other factors with which Os7, MOC1 or Ls protein interact that affect nodulation in Leguminosae, we screened a yeast two-hybrid library in soybean using either MOC1, Os7, or Ls protein as bait, respectively. We found that FW2.2-like (FWL), nodulin-22, nodulin-20a, nodulin-C51, and cathepsin H (GmCTSH) proteins could interact with each bait (Additional file 1: Fig. S6a). Nodulin genes by definition are highly expressed in nodules [46] and the FWL family is essential for soybean nodule organogenesis [47, 48]. GmCTSH encodes a cysteine proteinase with cathepsin-H-like activity belonging to the plant papain-like cysteine protease (PLCPs) family [49]. Some cysteine proteinase families, such as legumain from clan CD [50], papain from clan CA [51], and cysteine proteinase with cathepsin-L-like activity [52] have been reported to be involved in nodule development, which provides insights into GmCTSH’s putative symbiotic functions.

We confirmed the interaction between Os7, MOC1, or Ls protein and GmCTSH using yeast two-hybrid assays, SFLC assays, and Co-IP assays (Fig. 5a–c). We also detected an interaction between Os7, MOC1, or Ls protein and CTSH in M. truncatula (the best hit of GmCTSH in M. truncatula, termed MtCTSH) (Fig. 5d, e). To investigate whether CTSH is involved in nodulation, we specifically knocked down GmCTSH in soybean and MtCTSH in M. truncatula, and observed a significant reduction in nodule numbers in both GmCTSH-RNAi and MtCTSH-RNAi transgenic lines (Fig. 6a–f).

Fig. 5
figure 5

Lateral suppressor protein interacts with soybean and M. truncatula cathepsin H (CTSH) protein. a Yeast two-hybrid assays between GmCTSH and Os7, MOC1 or Ls protein. b SFLC assays between GmCTSH and Os7, MOC1 or Ls protein. c Co-IP assays between GmCTSH and Os7, MOC1 or Ls protein. d SFLC assays between MtCTSH and Os7, MOC1 or Ls protein. e Co-IP assays between MtCTSH and Os7, MOC1, or Ls protein

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Fig. 6
figure 6

CTSH affects RNS in Leguminosae. a Nodulation phenotypes produced by knocking down GmCTSH in soybean. Bar, 1 cm. b Number of nodules per hairy root and number of nodules per root length (cm) after GmCTSH-RNAi transformation in soybean. c qRT-PCR analysis of GmCTSH expression in transgenic soybean hairy roots carrying the EV and GmCTSH-RNAi. d Nodulation phenotypes produced by knocking down MtCTSH in M. truncatula. Bar, 2 mm. e Number of nodules per plant and number of nodules per root length (cm) after MtCTSH-RNAi transformation in M. truncatula. f qRT-PCR analysis of MtCTSH expression in transgenic M. truncatula hairy roots carrying the EV and MtCTSH-RNAi. EV indicates transformation with an empty vector. Asterisks indicate significant differences from the empty vector controls. Statistical significance was determined by a two-sided t-test in b, c, e, and f. The expression of soybean Actin gene or M. truncatula Ubiquitin gene was used as a normalization control. Data represent the mean ± s.e. in c and f. Hairy roots were collected at 21 DAI for expression experiments

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Phylogenetic analysis showed that CTSH was a species-conserved orthologous group (Additional file 1: Fig. S6b, c, Additional file 2: Table S4). Further investigation showed that of the 5567 conserved orthologous groups in the plant species we sampled, more than half (2799 of the 5567) were differentially expressed following infection with Bradyrhizobium diazoefficiens strain USDA110 in soybean [53] (Additional file 2: Table S4). This suggests that conserved orthologous groups could be candidates for subsequent investigation by reverse genetics in multiple species.

Therefore, it is likely that in Leguminosae, without Lateral suppressor gene, NSP1-NSP2 complex and calcium- and calmodulin-dependent protein kinase (CCaMK)-CYCLOPS complex activate NIN expression by binding to its promoter (Fig. 7). When Lateral suppressor gene is in the Leguminosae, it negatively affects nodulation through several different pathways, including interacting with NSP2, CYCLOPS and CTSH (Fig. 7).

Fig. 7
figure 7

Working model of Lateral suppressor gene inhibiting nodulation when introduced in Leguminosae. In Leguminosae, the NSP1-NSP2 complex and CCaMK-CYCLOPS complex activate NIN expression by binding to its promoter. When the Lateral suppressor gene, the Leguminosae-loss gene introduced in Leguminosae, it inhibits NIN transcription by interacting with NSP2 and CYCLOPS resulting in almost non-nodulation. Besides, Lateral suppressor protein can interact with proteins of other functional RNS genes, such as CTSH, to affect RNS in Leguminosae. PACE, Predisposition-associated Cis-regulatory Element; NRE, NODULATION RESPONSIVE ELEMENT

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Discussion

Previous studies have determined that members of several different GRAS subfamilies play important roles in RNS, including NODULATION SIGNALING PATHWAY 1 (NSP1) [54], NSP2 [55], DELLA [56, 57], Hairy Meristem 4 (HAM4) [58], SHORTROOT-SCARECROW (SHR-SCR) module [59], and Scarecrow like 13 (SCL13) Involved in Nodulation (SIN1) [60]. Interestingly, here we found that the ectopic expression of the Lateral suppressor gene, a Leguminosae-loss gene and that also belongs to GRAS subfamilies, almost completely inhibited nodulation in Leguminosae. Our results suggested that the ancestor of Os7, MOC1, or Ls protein may interact with NSP2, CYCLOPS, and CTSH to repress the transcription of NIN to inhibit the nodulation; whereas, loss of the Lateral suppressor gene in Leguminosae released the inhibition and provided a fundamental base for RNS origination (Fig. 7).

The evolutionary model of the Lateral suppressor gene in RNS may also be supported by the functions of the Lateral suppressor gene and its interaction proteins. MOC1 can be targeted for degradation by Tillering and Dwarf 1 (TAD1) [61,62,63], which encodes a Cdh1-type activator of APC/C that is orthologous to CELL CYCLE SWITCH52 (CCS52A) in M. truncatula, a gene that is indispensable for symbiotic cell differentiation [64]. In addition, MOC1 is protected from degradation by binding to the DELLA protein SLENDER RICE 1 (SLR1) [65]; the M. truncatula DELLA protein encoded by MtDELLA1, MtDELLA2, and MtDELLA3 forms a complex with Interacting Protein of DMI3 (IPD3, orthologue of Lotus japonicus CYCLOPS) and NSP2 to positively regulate rhizobial symbiosis [56, 57]. Furthermore, MOC1 and MONOCULM 3 (MOC3) physically interact to regulate tiller bud outgrowth through upregulating the expression of FLORAL ORGAN NUMBER1 (FON1), the homolog of CLAVATA1 in rice [66]; and CLAVATA1 homologs nodule autoregulation receptor kinase (NARK) in soybean, HYPERNODULATION ABERRANT ROOT FORMATION1 (HAR1) in Lotus, and SUPER NUMERIC NODULES (SUNN) in M. truncatula, are needed for control of the autoregulation of nodulation (AON) pathway [67,68,69]. The similar gene regulatory networks of the Lateral suppressor gene in Leguminosae and non-Leguminosae and the fact that the Lateral suppressor protein could interact with functional RNS genes may indeed suggest that the ancestor of the Lateral suppressor gene may also be involved in nodulation.

Additionally, the function of the Lateral suppressor gene as a transcriptional factor in RNS remains elusive. In Arabidopsis, SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE (SPL9) directly binds and represses the transcription of LAS [70, 71] while CUP-SHAPED COTYLEDON 2 (CUC2) and B-type ARABIDOPSIS RESPONSE REGULATOR1 (ARR1) function as positive regulators of LAS expression [70, 72, 73]. Besides, LAS binds directly to the promoter of GA 2-oxidase 4 (GA2ox4) to induce its expression, which generates a low-GA content region [71]. In rice, Oryza sativa homeobox 1 (OSH1) and OsTB1 might be possible downstream genes regulated by MOC1 as their reduced expression in the moc1 mutant [29]. Taking these results together, the Lateral suppressor gene integrates multiple genes to regulate axillary meristem initiation. Thus, although it has been shown that Lateral suppressor protein can interact with NSP2, CYCLOPS, and CTSH in Leguminosae to repress nodulation, how the Lateral suppressor gene regulates RNS functional genes as a transcriptional factor needs to be further investigated.

Divergence of any phenotype involves changes to the gene repertoire underlying it, including the removal of some genes from participation in the phenotype. If the only role of the gene was its contribution to the phenotype in which it no longer was needed, the gene would no longer contribute to organismal fitness. This would release it from purifying natural selection and allow it to accumulate mutations, likely resulting in it being pseudogenized and/or deleted. For example, the loss or pseudogenization of the master nodulation transcription factor, NIN, from diverse non-nodulating lineages in the NFNC following environmentally-induced cessation of nodulation constitutes the empirical evidence for the single gain, massive parallel loss theory of nodulation evolution [22, 23]. Gene loss can also be driven by positive selection. When a phenotype is conserved across taxa but genes contributing to it are absent from a lineage, that lineage presumably has found an alternative way, involving different genes, to maintain the phenotype. The absence of genes representing the Ls class of GRAS transcription regulators in Leguminosae means that the critical roles of these genes in other flowering plants, such as control of lateral shoot formation [30], have been taken over by other genes in Leguminosae, making Lateral suppressor gene evolutionarily dispensable.

Our study demonstrated that loss of the Lateral suppressor gene was associated with the evolution of RNS in Leguminosae, however, there remain many other unanswered questions. For example, genes belonging to the Ls subfamily are found in members of the other three orders of the NFNC in both nodulating and non-nodulating species. Interestingly, when the expression of the Lateral suppressor gene was investigated using reported transcriptome data [23, 74] (Additional file 1: Table S5) in the actinorhizal plant Datisca glomerata and Parasponia andersonii, it was found that although they showed low expression in most tissues, but exhibited significantly reduced expression value in inoculated nodules comparing with the root (Additional file 1: Tables S6, 7), indicating the Lateral suppressor gene may suppress the release of rhizobia from infection threads into symbiotic cells. However, further detailed functional investigation works are needed to better understand the role of the Lateral suppressor gene in nodulation in actinorhizal plants. Besides, although ectopic expression of SDI2 and other GRAS members in soybean did not cause the reduction number of nodules (Fig. 2i–l, Additional file 1: Fig. S2a–c), it is possible that ectopic expression of the Lateral suppressor gene would lead to dysfunction. These questions provide opportunities for further research on the mechanism of Lateral suppressor gene function in Leguminosae and non-Leguminosae. Moreover, gene gains and losses are not the only drivers, as gene expression variations caused by mutations of cis or trans elements in the promoters and domain changes caused by amino acid mutations also result in significant phenotypic changes [59, 75, 76]. For example, NIN-binding nucleotide sequences were only found in leguminous ASYMMETRIC LEAVES 2-LIKE 18 (ASL18) introns, not in homologues of non-Leguminosae [75, 76]. Besides, an AT1-box and an enhancer, driving the cortical expression of SCR, were typically found within 100 bp of each other in the SCR promoters from Leguminosae, whereas they were further from each other or one was absent in the SCR promoters from species outside the NFNC [59]. Thus, the genes triggering the evolution of RNS may be more complicated than we have found to date.

Conclusions

In our study, we disclosed the role of gene loss in the evolution of RNS in Leguminosae. First, we determined that two orthogroups were lost in the evolution of Leguminosae. Subsequently, we validated the roles of gene loss in RNS by transgenic experiments. Notably and surprisingly, when the Lateral suppressor gene, the lost gene in Leguminosae during evolution, was introduced from non-RNS species (rice and tomato) into soybean and Medicago (two typical Leguminsoae plants), the transgenic plants completely lost the RNS ability. Furthermore, through a systematic functional analysis, we revealed the clear working model of the Lateral suppressor gene in inhibiting RNS: interacts with NSP2 and CYCLOPS and suppresses the transcript activity of NSP2/NSP1 and CCaMK/CYCLOPS complex on NIN. We also revealed a novel RNS functional gene, CTSH, by screening Lateral suppressor protein-interacting proteins. In short, our study dissected gene loss events in the evolution of RNS in Leguminosae and suggests that loss of the Lateral suppressor gene plays a vital role in Leguminosae harboring the RNS ability.

Methods

Plant and materials

Glycine max [L.] Merr., cv. Williams 82 was used for RNA isolation and hairy root transformation. Bradyrhizobium diazoefficiens strain USDA110 was used for soybean root inoculation. M. truncatula Jemalong A17 and Sinorhizobium meliloti (S. meliloti) strain 1021 carrying pXLGD4 were used in the M. truncatula inoculation assays. Agrobacterium rhizogenes (A. rhizogenes) strains K599 and AR1193 were used for hairy root transformation in soybean and M. truncatula respectively. Saccharomyces cerevisiae strain AH109 was used for the yeast two-hybrid assay and the yeast two-hybrid mating system. Agrobacterium tumefaciens (A. tumefaciens) GV3101 was used for expression in Nicotiana benthamiana (N. benthamiana).

Growth condition

All the soybean materials are grown in a chamber with a 16-/8-h day/night cycle at 28 °C and 60% relative humidity. The inoculated plants were supplied with B&D nutrient solution (CaCl2, 1 mM; KH2PO4, 0.5 mM; ferric citrate, 10 µM; MgSO4, 0.25 mM; K2SO4, 0.25 mM; MnSO4, 1 µM; H3BO3, 2 µM; ZnSO4, 0.5 µM; CuSO4, 0.2 µM; CoSO4, 0.1 µM; Na2MoO4, 0.1 µM) [77] or tap water every other time.

M. truncatula materials were grown in pots filled with a 1:1 mixture soil of vermiculite and perlite. The plants were incubated in controlled-environment rooms at 22 ℃/18 ℃ (day/night), with a light period of 16 h/8 h (day/night), a light intensity of 250 μmol·m−2·s−1, and a relative humidity of 70%. The inoculated plants were supplied with nitrogen-free water.

Identification of orthologous groups

To cluster gene families and investigate the phylogenetic relationships of these studied species, we selected 48 species with high-quality genomes requiring that their scaffold N50 was longer than 95 kb and that the assembled genomic sequences cover more than 65% of the estimated genome size (Additional file 2: Table S1). For the phylogenomic analyses, we first filtered the protein sequences with the following criteria: if a gene had multiple encoded protein isoforms, only the longest protein isoform was retained; if the protein had less than 10 amino acids, it was removed. An all-against-all BLASTP (v2.7.1 +) [78, 79] was performed using the protein sequences from the 48 species, and pairwise similarities were obtained. The results were further filtered: query-subject-pairs with sequence similarities below 20% identity were removed; query-subject-pairs with BLAST e-values higher than 1e-5 were removed; and alignment coverage less than 30% of either the total query or subject sequence length in the high-scoring pairs (HSPs) were removed [80,81,82,83]. Subsequently, by using OrthoFinder (v2.2.6) [84, 85] under default parameters, we identified 40059 orthologous groups (each group contained at least 2 genes). The previously identified orthologous groups were further clustered using OrthoMCL (v2.0.9; MySQL v5.7.12) [86] with the default parameters (percent match cutoff: 50%; e value cutoff: 1e-5; inflation: 2; scheme = 4). Among the finally identified orthologous groups, there are 2 orthologous groups with genes present in all non-Leguminosae species and absent in all 16 species of Leguminosae and 5567 conserved orthologous groups with genes existing in at least 47 species.

Species tree construction

To construct the species tree, only orthologous groups with conserved and single/low-copy genes were used. The selection criteria were that all 48 species had genes in the orthologous group, and the genes were single copies in at least 43 species. After selection, a total of 309 orthologous groups were chosen for subsequent species tree construction. If there were two or more copies in one orthologous group in a species, only the copy having the highest similarity with the gene from Amborella trichopoda was retained for further analyses. Then we performed phylogenetic analysis for each of these orthologous groups. Forty-two orthologous groups failed to construct trees using coding DNA sequence (CDS) sequences. Therefore, 267 orthologous groups were retained to construct a species tree. Then, alignments of the amino acid sequences from each orthologous group were performed using MAFFT (v7.490) with the default parameters. The generated alignments were adjusted using trimAl (v1.4.rev15) [87] by trimming the low-quality sequences and the poorly aligned regions and then were transformed into nucleotide matrices by PAL2NAL [88]. After trimming and filtering, the nucleotide matrices from each orthologous group were concatenated and used for species tree construction, which was performed with RAxML (v8.2.12) [89] by the maximum likelihood method based on the model GTRGAMMAIX model, which was chosen by ModelTest-NG [90]. MEGA (v11.0.13) [91] and FigTree (v1.4.3) (http://tree.bio.ed.ac.uk/software/figtree/) was used for editing and displaying the species tree.

Gene family phylogenetic analyses

The GRAS members of 7 Leguminosae species were identified by HMMER (v3.1b2) (www.hmmer.org) search by GRAS domain (PF03514) in the Pfam database (v35.0). The other GRAS members of 26 species were extracted from the PlantTFDB database [92]. The subfamily classification is listed in Additional file 2: Table S3. To build the phylogenetic trees, the protein sequences from each gene family were aligned using MAFFT (v7.490) and then subjected to automated trimming by trimAl (v1.4.rev15). The trimmed alignments were used for phylogenetic tree construction. The phylogenetic trees of GRAS and SDI2 families were constructed by the IQ-TREE (v2.2.0.3) [93]; the model used was chosen by ModelFinder [94]. The phylogenetic tree of the CTSH orthologous group was constructed by RAxML (v8.2.12) [89] or PhyloBayes MPI version 2 [95]. The model was chosen by ModelTest-NG [90]. The phylogenetic tree was edited and visualized by using the online application iTOL v6.6 (Interactive Tree of Life, http://itol.embl.de/) [96].

Validation of the absence of Lateral suppressor gene and SDI2 on the genome of Leguminosae species

To check the absence of Leguminosae-loss genes in Leguminosae, we used the protein sequences of the genes in Ls or SDI2 orthogroups to blast the genome of the 48 species using tblastn and predicated the open reading frame of the best-hit sequences using genewise in wise (v2.4.1) (https://www.ebi.ac.uk/~birney/wise2/). The score value calculated by genewise was used to build a heatmap.

Synteny analysis

We used JCVI utility libraries (v1.2.4) [97] to identify syntenic genes of the Lateral suppressor gene or SDI2 in representative Leguminosae and non-Leguminosae species based on CDS. The syntenic genes of each pair of compared genomes were concatenated together, and the synteny blocks with Lateral suppressor gene or SDI2 genes were selected for plotting.

RNA-seq analysis

We exploited previously reported transcriptome datasets [23, 74] (Additional file 2: Table S5) of Datisca glomerata and Parasponia andersonii. To analyze transcriptional profiles, we trimmed the sequence reads using trimmomatic (V0.39) [98] and aligned the trimmed reads to the reference genome of Datisca glomerata (C. Presl) Baill. (GCA_003255025.1) [22] or Parasponia andersonii WU1 (GCA_002914805.1) [23] using HISAT2 (v2.0.0-beta) [99]. Gene expression level inferred by fragments per kilobase per million base pairs sequenced (FPKM) was estimated using StringTie (v1.2.4) [100].

Soybean hairy root transformation and transgenic root analysis

For 35S:SDI2, 35S:Os7, 35S:MOC1, 35S:Ls, 35S:GmHAM, 35S:GmSCL3a, 35S:GmLISCLa, 35S:GmNSP1a, 35S:GmNSP1b, 35S:GmNSP2a, 35S:GmNSP2b, GmNINs-RNAi, and GmCTSH-RNAi constructs, the sequences of candidate genes were amplified from cDNA or DNA templates using KOD Fx Neo polymerase (TOYOBO KFX-201). The appropriate sequences were first cloned and inserted into the Gateway donor vector pDONOR221 using BP ClonaseTM II enzyme mix (Invitrogen 11789–020) and then were recombined into Gateway destination vectors using LR CLONASE II ENZYME MIX (Invitrogen 11791–020). pB7WG2D.1 and pK7GWIWGIIRR destination vectors were used for overexpression and RNA silencing experiments, respectively. The primers used in this study are listed in Additional file 2: Table S8.

For hairy root transformation in soybean, the seedling plants were transformed with A. rhizogenes strain K599 carrying the appropriate binary vector based on a previously described protocol [101, 102]. To inoculate the hairy roots, B. diazoefficiens was grown in TY liquid medium containing tryptone 5 g/L, yeast extract 3 g/L, and CaCl2·2H2O 0.7 g/L at 28 °C for 4–5 days in the presence of the spectinomycin antibiotic to OD600 at 0.8 to 1. Bacteria were pelleted and diluted using ddH2O to an OD600 of 0.08 to 0.1. Then, each plant was inoculated with a 30 mL suspension of B. diazoefficiens. Nodule numbers, root length, and root fresh weight were scored 21 days after inoculation. To check the gene expression levels, total RNA was extracted using Quick RNA Isolation Kit (Huayueyang 0416–50) according to the manufacturer’s instructions.

M. truncatula hairy root transformation and nodulation phenotype observation

For the MtCTSH-RNAi construct in M. truncatula Jemalong A17, a 200 bp CDS of Medtr4g125300.1 (MtCTSH) was amplified from the cDNA library using Phanta® Max Super-Fidelity DNA Polymerase (Vazyme Biotech) according to the manufacturer’s instructions. The PCR products were inserted into pDONR207 using BP ClonaseTM II enzyme mix and then combined with pUB-GWS-GFP to generate the construct using LR CLONASE II ENZYME MIX. The constructs in the destination vector were introduced into A. rhizogenes AR1193 by electroporation and then transformed into M. truncatula A17 by hairy root transformation. The transgenic plants were transferred to a vermiculite and perlite mixture and inoculated with S. meliloti 1021 carrying the pXLGD4 (lacZ) plasmid after 1 week. The infection events, nodule numbers, and root length were analyzed at 21 DAI. The infection events were scored using light microscopy (ECLIPSE Ni, Nikon) after histochemical staining of the roots with X-Gal. Digital images were captured using a digital camera mounted on the light microscope. The primers used in this study are listed in Additional file 2: Table S8.

Gene expression assay

A total of 2 μg of RNA was used for first-strand cDNA synthesis using TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix (TRANS, AT311-02) following the manufacturer’s instructions. Real-time PCR was performed on a Roche LightCycler 480 system with LightCycler 480 SYBR Green I Master Mix (Roche, 04887352001) or TransStart Top Green qPCR SuperMix Top Green (TRANS, AQ131-04). Gene expression was normalized to the expression of Actin or Ubiquitin and the relative expression level was calculated using the 2−ΔΔCT method. The primers used in this study are listed in Additional file 2: Table S8.

Yeast two-hybrid assays

The yeast two-hybrid assay was performed using the Matchmaker GAL4 Two Hybrid System 3 (Clontech) according to the yeast protocols handbook (Clontech). The CDSs of MOC1, Os7, Ls, OsNSP2, MtNSP2, GmNSP2a, GmNSP2b, GmNSP1a, GmNSP1b, and GmCTSH were fused with the GAL4 activation domain (AD) in pGADT7 (Takara Bio Inc.) or the GAL4 binding domain (BD) in pGBKT7 (Takara Bio Inc.). The relevant primer sequences are given in Additional file 2: Table S8. Different combinations of plasmids were then co-transformed into the yeast (Saccharomyces cerevisiae) strain AH109. The cotransformed yeast was screened on the SD-Trp/-Leu and interactions were screened on SD-Trp/-Leu/-His/-Ade at 30 °C.

The yeast two-hybrid mating system was performed according to the Matchmaker® Gold Yeast Two-Hybrid System (Clontech). The full-length of Os7, MOC1, and Ls CDSs were amplified and subcloned and inserted into pGBKT7, and then transformed into yeast strain AH109. Each Os7, MOC1, and Ls protein was used as bait to screen a cDNA library prepared from poly(A)-containing RNA sampled from root and root nodules. Experimental procedures for screening and plasmid identification were performed according to the manufacturer’s user guide.

Split firefly luciferase complementation (SFLC) assays

The CDSs of MOC1, Os7, Ls, OsNSP2, MtNSP2, GmNSP2a, GmNSP2b, CYCLOPS, GmCTSH, and MtCTSH were cloned and inserted into pCAMBIA-split_cLUC or pCAMBIA-split_nLUC to generate the needed fusion transgenes. The primers used for vector construction are listed in Additional file 2: Table S8. Different combinations of A. tumefaciens strain GV3101 cells containing the above plasmids were transformed into N. benthamiana leaves, according to a previous study [103]. After 2 days, the N. benthamiana leaves were smeared with 1 mM luciferin to measure luciferase intensity in a NightOWL II LB 983 imaging apparatus [104].

Coimmunoprecipitation (Co-IP) assays

The CDSs of MOC1, Os7, Ls, OsNSP2, MtNSP2, CYCLOPS, GmNSP2a, GmNSP2b, GmCTSH, and MtCTSH were amplified and then inserted into either the PUC-35S-HA or the PUC-35S-FLAG vector. The primers used for vector construction are listed in Additional file 2: Table S8. Each plasmid combination was introduced into Arabidopsis protoplasts and incubated at 22 °C in the dark for 16 h. Total protein was extracted from harvested protoplasts by treatment with extraction buffer (50 mM HEPES, 1 mM EDTA, 150 mM KCl, 0.3% Trion X-100, 1 mM DTT, 1 mM PMSF, and 1 × complete protease inhibitor cocktail (Roche)). Protoplasts were broken down by vortexing and were then incubated for 10 min on ice. The samples were centrifuged at 15000 rcf for 20 min at 4 °C. Partial supernatant (40 μL) was retained for input samples to ensure that proteins were normally expressed in all samples. The remaining supernatant was incubated with FLAG magnetic beads (MBL) for 2 h at 4 °C. The beads were washed six times with a wash buffer. The immunoprecipitates were separated by electrophoresis in 12% SDS–polyacrylamide gels and transferred to a nitrocellulose membrane (GE Healthcare). Proteins were detected by treating the membranes with anti-HA-tagged mAb-HRP-DirectT (1:5000, MBL, M180-7) or anti-DDDDK-tagged mAb-HRP-DirectT antibodies (1:2000, MBL, M185-7).

Transient dual luciferase reporter assay

The remote upstream cis-regulatory conserved region with putative cytokinin response elements (CE region) in combination with the −5 kb region is sufficient to induce wild-type-like nodule organogenesis in M. truncatula [105]. Therefore, the sequence of the CE region fused to the −5 kb region (ProNINCE-5 kb [105]) of MtNIN was amplified from M. truncatula A17 genomic DNA. 4831 bp promoter sequence of LjNIN was amplified from pNIN:GUS [106]. These sequences of the promoters of MtNIN and LjNIN were inserted into the pGreen II 0800-LUC vector to be used as a transcriptional activity reporter plasmid (10 μg of plasmid per transfection). CYCLOPS-DD (S50D and S154D) sequence was generated using Fast MultiSite Mutagenesis System kit (TRANS, FM201-01). The full-length CDSs of MtNSP1, MtNSP2, CYCLOPS, CYCLOPS-DD, Os7, MOC1, and Ls were cloned and inserted into the PUC-35S-Flag or PUC-35S-HA vector, and these plasmids were used as the effector (12 μg per transfection). The primers used in this study are listed in Additional file 2: Table S8. The reporter plasmid and corresponding effector plasmids were co-transformed into Arabidopsis protoplasts as described previously [107]. The activities of firefly luciferase (LUC) and Renilla luciferase (REN) were examined using a Dual-Luciferase Reporter Assay System kit (Promega, E1960).

Statistical analysis

All details of the statistics applied are provided alongside the figure and corresponding legends. Statistical analyses were performed in R.

Accession numbers

Sequence data from this article can be found in the Phytozome under the following accession numbers: SDI2, LOC_Os05g43040; MOC1, LOC_Os06g40780; Os7, LOC_Os02g10360; Ls, Solyc07g066250.1; GmHAM, Glyma.U013800; GmSCL3a, Glyma.11G150200; GmLISCLa, Glyma.11G138300; GmNSP1a, Glyma.07G039400; GmNSP1b, Glyma.16G008200; GmNSP2a, Glyma.06G110800; GmNSP2b, Glyma.04G251900; GmCTSH, Glyma.15G177800; MtCTSH, Medtr4g125300; MtUbiquitin, Medtr4g088485; GmActin, Glyma.18G290800; GmNIN1a, Glyma.04G000600; GmNIN1b, Glyma.06G000400; GmNIN2a, Glyma.02G311000; GmNIN2b, Glyma.14G001600; OsNSP2, LOC_Os03g15680; MtNSP2, Medtr3g072710; CYCLOPS, Lj2g0008424; MtNSP1, Medtr8g020840; MtNIN, Medtr5g099060; LjNIN, LjContig00081g0005881.

Availability of data and materials

Previously published soybean RNA-seq datasets analyzed here were from the Genome Sequence Archive [108] in the National Genomics Data Center [109], China National Center for Bioinformation / Beijing Institute of Genomics, Chinese Academy of Sciences under the accession number CRA003430 [53]. The published RNA-seq data for the Datisca glomerata was downloaded from the CNGB Sequence Archive (CNSA) of the China National Gene Bank DataBase (CNGBdb) under CNP0004055 (Additional file 1: Table S5) [74]. The published RNA-seq data for the Parasponia andersonii was downloaded from the NCBI SRA database under PRJNA272473 (Additional file 1: Table S5) [23]. The datasets of 48 species analyzed here are noted in Additional file 2: Table S1 [22, 23, 80, 81, 110,111,112,113,114,115,116,117,118,119,120,121,122,123,124,125,126,127,128,129,130,131,132,133,134,135,136,137,138,139,140,141,142,143,144,145]. The protein alignments used to make the species tree (OMIX003873) [146] and GRAS gene family phylogenies (OMIX003876) [147] have been deposited in the OMIX, China National Center for Bioinformation / Beijing Institute of Genomics, Chinese Academy of Sciences (https://ngdc.cncb.ac.cn/omix) [109]. No customized code was generated in this study. All bioinformatic tools and software used in this study are cited in the text.

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Acknowledgements

We thank Dr. Jeremy Murray (CAS Center for Excellence in Molecular Plant Sciences, Shanghai Institute of Plant Physiology and Ecology, Chinese Academy of Sciences) and Ms. Anita K. Snyder for revising the manuscript; Dr. Xia Li (Huazhong Agricultural University) for sharing hairy root transformation method in soybean and providing the strains; Dr. Weicai Yang (Institute of Genetics and Developmental Biology, Chinese Academy of Sciences) for providing pK7GWIWGIIRR destination vector for RNAi silencing experiments; Dr. Cao Xu (Institute of Genetics and Developmental Biology, Chinese Academy of Sciences) for providing the sample of Solanum lycopersicum Heinz 1706; Ms. Min Shi for her assistance on the functional study.

Peer review information

Shuangxia Jin and Wenjing She were the primary editors of this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

Review history

The review history is available as Additional file 4.

Funding

This work was supported by: The National Natural Science Foundation of China (grant nos. 32388201 and 32300512), “Strategic Priority Research Program” of the Chinese Academy of Sciences (grant no. XDA24030501), CAS Project for Young Scientists in Basic Research (YSBR-078), and the Xplorer Prize.

Author information

Author notes

  1. Tengfei Liu, Zhi Liu, Jingwei Fan, Yaqin Yuan, and Haiyue Liu contributed equally to this work.

Authors and Affiliations

  1. Key Laboratory of Seed Innovation, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China

    Tengfei Liu, Zhi Liu, Jingwei Fan, Yaqin Yuan, Shuaiying Xiang, Xia Yang, Yucheng Liu, Shulin Liu, Min Zhang & Zhixi Tian

  2. Key Laboratory of Plant Carbon Capture, CAS Center for Excellence in Molecular Plant Sciences, Shanghai Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, Shanghai, China

    Haiyue Liu & Fang Xie

  3. Shenzhen Branch, Guangdong Laboratory for Lingnan Modern Agriculture, Genome Analysis Laboratory of the Ministry of Agriculture, Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences, Shenzhen, China

    Wenfei Xian & Shifeng Cheng

  4. State Key Laboratory of Plant Diversity and Specialty Crops, Institute of Botany, the Chinese Academy of Sciences, Beijing, China

    Yuannian Jiao

  5. School of Integrative Plant Science, Sections of Plant Biology and Plant Breeding & Genetics, Cornell University, Ithaca, NY, USA

    Jeff J. Doyle

  6. State Key Laboratory of Plant Genomics and National Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China

    Jiayang Li

  7. University of Chinese Academy of Sciences, Beijing, China

    Tengfei Liu, Zhi Liu, Yaqin Yuan, Haiyue Liu, Shuaiying Xiang, Xia Yang, Yuannian Jiao, Fang Xie, Jiayang Li & Zhixi Tian

  8. Department of Molecular Biology, Max Planck Institute for Biology Tübingen, Tübingen, Germany

    Wenfei Xian

  9. Hebei Key Laboratory of Crop Genetics and Breeding, Institute of Cereal and Oil Crops, Hebei Academy of Agricultural and Forestry Sciences, Shi-Jiazhuang, China

    Zhi Liu

  10. Yazhouwan National Laboratory, Sanya, Hainan, China

    Jiayang Li & Zhixi Tian

Authors
  1. Tengfei Liu
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  2. Zhi Liu
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Contributions

Z.T. designed the experiments and managed the project. T.L., W.X., S.C., J.J.D., Y.J., and Z.T. performed the phylogenomic analyses and evolutionary analyses. T.L., Z.L., J.F., Y.Y., H.L., S.X., X.Y., Y.L., S.L., M.Z., F.X., J.L., and Z.T. performed functional validation and data analysis. Z.T., J.J.D., S.C., F.X., T.L., J.F., Y.Y., H.L., and W.X. wrote the manuscript. All authors discussed the results and commented on the manuscript.

Corresponding authors

Correspondence to Jeff J. Doyle, Fang Xie, Jiayang Li or Zhixi Tian.

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Supplementary Information

13059_2024_3393_MOESM1_ESM.pdf

Additional file 1: Supplementary material: figures S1-S6. Fig. S1 SULFUR DEFICIENCY INDUCED 2 was lost in Leguminosae. Fig. S2 The nodulation phenotype of SDI2 overexpression and NINs knockdown transformation. Fig. S3 Expression of Lateral suppressor gene transformation. Fig. S4 The infection phenotype and growth phenotype of Lateral suppressor gene transformation. Fig. S5 Lateral suppressor protein interacts with GmNSP2. Fig. S6 Phylogenetic analyses of CTSH orthologous group.

13059_2024_3393_MOESM2_ESM.xlsx

Additional file 2: Supplementary tables: Supplementary Tables S1-S8. Table S1. The 48 species used in this study. Table S2. Information of Leguminosae-loss orthologous groups. Table S3. Subfamily classification of GRAS members. Table S4. Information of conserved orthologous groups. Table S5. The list of RNA-seq samples analyzed in this study. Table S6. FPKM value of dgLs among different tissues. Table S7. FPKM value of paLs among different tissues. Table S8. Primers used in this study.

13059_2024_3393_MOESM3_ESM.docx

Additional file 3. Uncropped blot images related to Figs. 3, 4 and 5 and Additional file 1: Figs. S2, S3 and S5.

Additional file 4. Review history.

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Liu, T., Liu, Z., Fan, J. et al. Loss of Lateral suppressor gene is associated with evolution of root nodule symbiosis in Leguminosae. Genome Biol 25, 250 (2024). https://doi.org/10.1186/s13059-024-03393-6

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Keywords

Genome Biology

ISSN: 1474-760X

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