Genome-wide survey of soybean papain-like cysteine proteases and their expression analysis in root nodule symbiosis

Plant papain-like cysteine proteases (PLCPs) are a large class of proteolytic enzymes and play important roles in root nodule symbiosis (RNS), while the whole-genome studies of PLCP family genes in legume are quite limited, and the roles of Glycine max PLCPs (GmPLCPs) in nodulation, nodule development and senescence are not fully understood. In the present study, we identified 97 GmPLCPs and performed a genome-wide survey to explore the expansion of soybean PLCP family genes and their relationships to RNS. Nineteen paralogous pairs of genomic segments, consisting of 77 GmPLCPs, formed by whole-genome duplication (WGD) events were identified, showing a high degree of complexity in duplication. Phylogenetic analysis among different species showed that the lineage differentiation of GmPLCPs occurred after family expansion, and large tandem repeat segment were specifically in soybean. The expression patterns of GmPLCPs in symbiosis-related tissues and nodules identified RNS-related GmPLCPs and provided insights into their putative symbiotic functions in soybean. The symbiotic function analyses showed that a RNS-related GmPLCP gene (Glyma.04G190700) really participate in nodulation and nodule development. Our findings improved our understanding of the functional diversity of legume PLCP family genes, and provided insights into the putative roles of the legume PLCPs in nodulation, nodule development and senescence.

senescence in soybean, Astragalus sinicus, Pisum sativum and Medicago truncatula [17]. For example, PsCYP1 is expressed at the onset of senescence in the indeterminate nodules, and PsCyp15A and MsCYP15A are expressed in nodules [18,19]. MtCP6 is induced to express during both developmental and stress-induced nodule senescence, and its early expression promoted nodule senescence [20]. MtCP77 positively regulates nodule senescence by accelerating plant PCD and ROS accumulation [21]. Asnodf32, a nodule-specific cysteine protease [22], negatively regulate nodule development, bacteroid senescence and nodule lifespan in A. sinicus [23]. Glycine max CYSP1(GmCYSP1) may participate in nodule development and senescence [24]. However, these studies are mainly based on individual members of PLCPs. In soybean, dozens of PLCPs are associated with root nodule symbiosis (RNS) [25][26][27], while the role of GmPLCP in nodulation, nodule development and senescence is not fully understood.
In the present study, a whole-genome survey was performed to explore the special characteristics and the expansion of soybean PLCP family genes. The expression profiles of GmPLCPs in soybean root nodule symbiosis were analyzed to identify RNS-associated PLCPs. The symbiotic function analysis showed that a RNS-related GmPLCP gene (Glyma.04G190700) was likely to play roles in nodulation and nodule development. Our findings improve our understanding of the functional diversity of legume PLCP family genes, and provide insights into the putative roles of the legume PLCPs in nodulation, nodule development and senescence.

Identification of PLCP gene family in soybean
Surveys of the soybean genomes preliminary identified 106 gene loci encoding putative PLCPs in the Glycine max var. Williams 82 genome (Table S1). The identified soybean PLCPs had various molecular masses ranging from 6128.82 to 57,479.13 Da, and they encoded peptides with 55~517 amino acid residues and isoelectric point (pI) of 4.3~9.32. Conserved domains in these 106 PLCPs were analyzed by NIH/NLM/NCBI CD-search tool, and Table S2 lists the detailed information. Inhibi-tor_I29 and peptidase_C1 motifs are commonly conserved in soybean PLCPs, and five PLCPs (Glyma.04G028300, Glyma.10G120700, Glyma.13G229100, Glyma.14G085800 and Glyma.17G239000) also had a GRAN motif. Besides, among these 106 soybean PLCPs, nine of them were considered as putative pseudogenes based on their sequence length (Table S1) and the absence of peptidase_C1 domain or the presence of large fragment deletion in the peptidase_C1 domain (Table S2), so we identified a total of 97 GmPLCPs in soybean.

Chromosome location and duplication of soybean PLCPs
To survey the potential duplications of soybean PLCPs, firstly, candidate GmPLCP duplicate pairs located in a pair of paralogous blocks formed by Glycine WGD. As shown in Fig. 1, 19 candidate paralogous segment pairs containing 77 soybean PLCPs were observed on 17 soybean chromosomes. Besides, two gene clusters (Glyma.06G 275100, Glyma.06G275200 and Glyma.06G275300; Glyma.06G283000 and Glyma.06G283100) were observed on chromosome 6, and Glyma.12G130300 was localized in the big gene cluster on chromosome 12. Secondly, collinearity analysis was further carried out among candidate duplicate segment pairs, and Fig. 2 shows the flanking regions of the candidate duplicate segment pairs. Finally, 19 duplicate segment pairs formed by Glycine WGD events were identified (Table 1). Among them, it was worth noting that pairs 10 and 11 included large tandem repeat segments located on chromosome 6 or chromosome 12, indicating that a high degree of complexity existed in these soybean PLCP duplications.
The Soybean Genome Database was used to search the synonymous mutation rate (Ks) values of these PLCPs. All Ks values ranged from 0.1028 to 0.3374, and the divergence times of these 19 duplicate paralogous pairs ranged from 8.43 and 27.66 Mya, with an average of 14.2 Mya (Table 1), which was consistent with WGD events (10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20). The ratio of non-synonymous mutation rate (Ka) to Ks (ω = Ka/Ks) is usually used to measure the history of selection acting on coding sequences [28]. When ω < 1, at least one gene is under purifying selection, whereas ω > 1 suggests directional selection [29,30]. Table 1 shows that ω ranged from 0.069 to 0.643 for 17 duplicate segment pairs (except for No. 10 and No. 11), suggesting that these genes were constrained by purifying selection.
Phylogenetic and exon-intron structure analysis of PLCPs in Arabidopsis thaliana, M. trunctula, Lotus japonicus and soybean To investigate the phylogenetic relationship of soybean PLCPs with those of other legume plants and nonlegume plants, we conducted a full-length peptide sequence alignment among 97 GmPLCPs (Soybean), 26 AtPLCPs (A. thaliana), 33 MtPLCPs (M. trunctula), and 25 LjPLCPs (L. japonicus) using the MEGA (version 6.0) (Fig. 3). The PLCPs from these four different species were distributed in nine groups (Group A to Group I). Among them, soybean PLCPs and 19 duplicate segment pairs formed by Glycine WGD events were distributed in all the nine groups, indicating that the lineage differentiation of soybean PLCPs occurred after family expansion. The soybean PLCP members were not evenly distributed in these nine groups, and among them, Group A, B and Group D were formed with only legume PLCPs (Fig. 3), indicating that these three groups occurred before the differentiation between A. thaliana and legumes or were lost in A. thaliana or specific in legumes. Besides, AT5G45890 was independent of each group, indicating that this gene had species-specific characteristics and new functions.
Among the PLCPs from the three legume plants, although tandem repeat events were found in M. trunctula and L. japonicus, large tandem repeat segment pairs only existed in soybean PLCPs (pairs No. 10 and 11 in group A and B), indicating that this complex replication of PLCPs was specific for soybean, not for legume. The number of PLCPs in soybean was significantly more than that in M. trunctula and L. japonicus, in addition to the above-mentioned large tandem repeat segment events, the reason could be mainly attributed to fragment repeat events in soybean, especially in groups G and I (Fig. 3). In these two groups, there were only three MtPLCPs and three LjPLCPs, while there were 20 paralogous soybean PLCPs. However, it remained largely unexplored why these paralogous genes were retained in soybean, which might be associated with some biological functions.
The cDNA sequence of each PLCP in above-mentioned nine groups (Fig. 3) was compared with their genomic sequences to analyze their UTR/exon/intron structures, and similar gene structures were found within each group among these PLCPs (Fig. 4). Most of the PLCPs in Group A to Group D exhibited relatively simple gene structures, among them, 70% PLCPs contained only one intron. While in Group E to Group I, all PLCPs in these five groups harbored two or more introns (Fig. 4). These group-specific gene structures were consistence with the relationships between PLCPs across species and further supported functional divergence among these PLCPs.

Go analysis of soybean PLCPs
Go of the soybean PLCPs was investigated based on the putative assignment of 42 Go terms using the data in Soybean Genome Database. These Go function terms were divided into three categories: biological process, cellular components, and molecular function (Fig. 5), and the detailed gene ID information of them was shown in Table S3. Among them, five genes (Glyma.06G042600, Glyma.06G282300, Glyma.12G124300, Glyma.12G126000 and Glyma.12G131000) had no predicted Go function. All of the rest had cysteine-type peptidase activity and participated in the proteolysis process, and most of the soybean PLCPs were localized in extracellular region (87 genes). For the duplicate segment pairs, two genes in pair 1, three genes in pair 15 and 23 genes in the two most complex pairs (pairs NO. 10 and 11) participated in the aging, response to ethylene, defense response to fungus, incompatible interaction and leaf senescence processes, and had senescence-associated vacuole location. For the four pairs (No. 1, 9, 17 and 19), one of the tandem repeat genes of them has different function from the others. Besides, for the rest 12 gene pairs, except for pair 6 (Glyma.06G042600 had no predicted Go function), both of the two genes participated in the same biological processes.

Expression patterns of soybean PLCPs in RNS
To determine the phylogenetic relationships among the different members of soybean PLCPs, we performed a phylogenetic analysis based on the 97 full-length PLCP peptide sequence alignments. Combining with the classification in Fig. 2, these 97 soybean PLCPs were divided into 9 classes, for example, the soybean PLCPs in Group A were classified in class 1, the soybean PLCPs in Group B were classified in class 2, and so on (Fig. 6a).
To determine which soybean PLCPs were involved in RNS, we firstly investigated the expression profiles of the 97 soybean PLCPs in symbiosis-related tissues based on plant phytozome database, and these tissues included roots, root hairs, nodules, nodules. Symbiotic condition and root. Symiotic condition. The results showed that soybean PLCPs have distinct expression patterns in these tissues, and most of the highly expressed genes mainly focused on class 6 to class 10 ( Fig. 6b). Then, we used our previous RNA-Seq data [18] to analyse the expression profiles of these 97 soybean PLCPs in five different nodules according to our previous RNA-Seq data (Fig. 6c). We compared the expression levels of these 97 soybean PLCPs in above-mentioned symbiosis-related tissues and nodule samples, and founded that among the highly expressed genes in symbiosis-related tissues, some PLCPs also had high expression in nodule samples ( Fig. 6b and c), indicating that these PLCPs may participate in RNS or nodule development.
To exam whether these PLCPs with high expression both in symbiosis-related tissues and nodule samples have roles in nodule development and/or senescence, we analyzed the expression difference of 28 selected PLCPs between different nodule development time points by qPCR. Firstly, the expression stability of four reference genes (ELF1b, QACT, G6PD and Ubiquitin) was evaluated, of which, ELF1b and QACT were most stable in all samples, while GmG6PD and Ubiquitin were consistently unstable (Fig. S1). Then, ELF1b and QACT were  selected as reference genes for qPCR experiment and the results showed that nearly all of these 28 PLCPs were differentially expressed between the five nodules (Fig. 7). Among them, 12 PLCPs were up-regulated during nodule development and/or senescence, and reached their peaks at nodules collected at 84 days of post inoculation (84dN) (Fig. 7a, d, g, l, n, r, s, u, v, x, y and ab). Four PLCPs were down-regulated during nodule development, and had low expression at 64dN or 84dN (Fig. 7b, c, f and h). Seven PLCPs reached their peaks at 30dN or 42dN (Fig.  7e, I, k, m, o, w and aa). Three PLCPs were down-regulated then up-regulated during nodule development and/or senescence (Fig. 7p, q and z). Glyma.06G014800 was upregulated then down-regulated during nodule development and/or senescence (Fig.7j), and Glyma.14G216300 had high expression at 30dN and 84dN (Fig. 7t).
Functional analysis of Glyma.04G190700 in soybean by hairy root transformation As described above, Glyma.04G190700 was highly expressed in symbiosis-related tissues (Fig. 6b) and upregulated during nodule development ( Fig. 6c and Fig. 7g), suggesting that Glyma.04G190700 may play a role in nodulation and nodule development. To confirm this result, RNA interference (RNAi) of Glyma.04G190700 was performed using the soybean hairy root transformation method (Fig. 8). The symbiotic phenotypes were scored at 50 days after inoculation with BXYD3. RNAi of Glyma.04G190700 resulted in increase in nodule number (Fig. 6a). Nodules in RNAi of Glyma.04G190700 showed significantly higher nitrogenase activities than in the control (Fig. 6b). The expression levels of Glyma.04G190700 and four nodulin genes (ENOD40, Nodulin35, Calmodulin and Lb1) [31][32][33] were examined by qPCR in hairy roots and nodules ( Fig. 8c and d). The Glyma.04G190700 transcript was reduced to less than 50% in the RNAi hairy roots and nodules as compared with that in the control ( Fig. 8c and d). The expression levels of the four nodulin genes (ENOD40, Nodulin35, Calmodulin and Lb1) were increased drastically in the Glyma.04G190700 RNAi hairy roots and nodules as compared with those in the control hairy roots and nodules ( Fig. 8c and d). Together, these results strongly indicate that Glyma.04G190700 participate in nodulation and nodule development.

Discussion
PLCPs are a large class of proteolytic enzymes and play important roles in RNS [7,8], while the whole-genome studies of PLCP family genes in legume are quite limited.
In the present study, we firstly performed the wholegenome survey of soybean PLCP genes and explored the expansion of soybean PLCP family genes. The resultant expression patterns of GmPLCPs in soybean RNS provided insights into the putative roles of the legume PLCPs in nodulation, nodule development and senescence.
Genome-wide scan and the expansion of soybean PLCP family genes Genome-wide identify of PLCPs has been systematically performed in Arabidopsis, rubber, cassava, castor, poplar, grapevine, G. hirsutum, C. papaya and rice [1,[11][12][13][14]. In the present study, 97 GmPLCPs were identified in soybean, and the number is more than that of the other plants in the previous studies. According to the phylogenetic clade and structure features, these soybean PLCPs were classed into nine subfamilies, which were similar to previous studies [11,[34][35][36]. However, among these nine Groups, three Groups were formed with only legume PLCPs and only 26 At PLCPs in the Groups, which is greatly different from other studies [1,11,12]. Two whole genome duplication (WGD) events, occurring approximately 59 and 13 Ma, have been undergone in the soybean genome [37][38][39], and have significant contribution to the expansion of many multigene families in soybean [28,40,41]. Different gene families may have distinct polyploidy-derived duplicate events and the evolutionary mechanisms of the retention of duplicate genes [28], which play important roles in adaptive evolution and biological functions of families [42][43][44]. For soybean PLCP gene family, the number was significantly more than that in A. thaliana, M. trunctula and L. japonicus, which may result from WGD events, tandem duplication and large-scale segmental duplication [11,12]. In the duplicate events of the soybean PLCP gene family two large-scale segmental duplication pairs included 37 GmPLCPs and greatly contributed to the family expansion, small tandem duplication pairs and single-gene segmental duplication pairs also have contribution in the expansion of soybean PLCP family genes. Besides, the retention of these repeat clusters genes might be mainly attributed to their different expression patterns, special structures and functions among these pairs, which was similar to the previous studies [45][46][47].

Potential symbiotic function of GmPLCPs in soybean
Previous studies have shown that PLCPs play important roles in endogenous protein turnover [48], seed traits, germination and maturation [49,50], abiotic environmental stresses [11,49] and protection of plants against mites [51], fungi [52,53], bacteria [54] and viruses [55]. The type III secretion system (T3SS) of rhizobia, an introducer of virulence factors from plant pathogens, can be induced by legume-derived flavonoid and has been reported to modulate nodulation process through recognition by the host defence system [56]. Besides, our previous research has shown that nodule development and senescence are directly associated with the  plant immunity defence [26]. PLCPs have been reported to involve in biotic and abiotic stresses [11] and responsible for defense against pathogen bacteria and regulate plant immunity [54], and more and more molecular mechanisms were discovered in recent years [11]. In the present study, it was worth noting that no large tandem duplication pairs and only two small tandem duplication pairs of GmPLCPs were defferent expression in nodule development, suggesting that the RNSrelated GmPLCP duplicate genes were mainly derived from single-gene segmental duplication rather than tandem duplication. Both homologous genes in the RNSrelated duplicate pairs showed similar expression pattern in the RNS, which is different from the previous research [11]. Previous studies have been suggested that PLCPs play important roles in the development and senescence of several legume root nodules [17,[20][21][22][23][24]57]. In soybean, PLCPs may play important roles in nodulation [15,27] as well as nodule development and/or senescence [25,26]. However, the roles of GmPLCPs in nodulation, nodule development and senescence are not fully understood. In the present study, the expression profiles of GmPLCPs in five symbiosis-related tissues and five different nodule samples [18] were comprehensively analyzed, and the results identified dozens RNS-related PLCPs, suggesting that multiple GmPLCPs may participate in nodulation signal recognition and immunity and/ or nodule development and senescence. In the previous transcriptional profile data, the expressions of 14 GmPLCPs were particularly increased during the onset of senescence [25]. Among these GmPLCPs, 12 genes were up-regulated during nodule senescence, while the rest two genes had low expression at 64dN or 84dN. In this study, the other four GmPLCPs may also play roles in nodule development and senescence. Besides, five (See figure on previous page.) Fig. 6 Phylogenetic and expression analysis of the identified 97 GmPLCPs. a Phylogenetic analysis of the identified 97 GmPLCPs. Phylogenetic tree construction of GmPLCPs is based on the full-length deduced amino acid sequences using the programs RAxML, MEGA version 6.0 and MrBayes 3.2. The tree shows nine major phylogenetic classes (class 1 to class 9) indicated with different shadows. b Expression analysis of the identified 97 GmPLCPs in five RNS-related tissues in the Phytozome database. The expression values of GmPLCPs in the Phytozome database and the pheatmap packages in R were used to product the Heatmap. These tissues include Nodules (1), Root Hairs (2), Roots (3), Nodules. Symbiotic. Condition (4) and Roots. Symbiotic. Condition (5). c RNA-seq analysis of the identified 97 GmPLCPs in five different nodules of soybean. Our previous RNA-Seq data [18] and the pheatmap packages in R were used to product this Heatmap. These five nodule samples include the nodules in branching stage (6), flowering stage (7), fruiting stage (8), pod stage (9) and harvest stage (10), and the description of these five important developmental stages were shown in our previous research [18] Fig. 7 qPCR analysis of 28 selected GmPLCPs in nodule development. Soybean ELF1b and QACT were selected as reference genes to normalize the relative expression levels of each GmPLCP gene, and the expression level in 12dN was used as a starting point control to calculate the relative expression levels of other nodule samples. All qPCR reactions were repeated three times and the data are presented as the mean ± SD PLCPs may participate in the nitrogen-fixation process, and eight PLCPs may participate in the early and/or middle stage of nodule development. These data indicated that GmPLCPs may not only have roles in nodule senescence, but also participate in nodulation and nodule development. In this study, the symbiosis function analyses of Glyma.04G190700 showed that it really participates in nodulation and nodule development. The specific regulatory role and distinct functions of RNSrelated GmPLCPs need to be further studied.

Characteristics of the RNS-related GmPLCPs
Previous studies have shown that polyploidy-derived duplicate genes lead to enhanced RNS in legumes [58,59].
In the present study, RNS-related GmPLCPs were mainly focused on class 5 to class 9, and most of these genes participated in single-gene segmental duplication, indicating that polyploidy-derived duplicate event of GmPLCPs also played important roles in RNS. Besides, RNS-related GmPLCPs possessed relatively complex gene structures containing UTR sequences and three or more introns. Three divergent motif patterns were observed in these RNS-related GmPLCPs. The first pattern contained a pro-peptide_C1 domain [60] and a peptidase _C1A_cathepsin B domain [61] (only for Glyma.03G226300 and Glyma.19G223300). In the second pattern, which was found in three proteins (Glyma.04G028300, Glyma.14G085800 and Glyma.17G239000), had not only two typical motifs (an inhibitor _I29 domain and a peptidase _C1 domain) [62], but also a GRAN domain in their C-terminal regions, which was similar to some known PLCPs [10]. The rest or the most of these RNS-related GmPLCPs were generally categorized into the third pattern, which contained two typical motifs. These results indicated that there was no special protein structure in RNS-related GmPLCPs, which was similar to the nodulation and nodule developmentrelated soybean cystatins [63].

Conclusions
In summary, we conducted a genome-wide survey and identified 97 GmPLCPs. A total of 19 segmental duplication pairs created by WGD event were identified and analyzed, suggesting a high degree of complexity in the duplications of soybean PLCPs. Expression profiles of GmPLCPs in soybean root nodule symbiosis were used to identify the RNSrelated PLCPs. The symbiosis function analyses showed that a RNS-related GmPLCP gene (Glyma.04G190700) really participate in nodulation and nodule development.
Our findings improve our understanding of the functional diversity of legume PLCP family genes, and provide insights into the putative roles of the legume PLCPs in nodulation, nodule development and senescence.

Methods
Identification of PLCPs in soybean, M. truncatula, L. japonicus and A. thaliana and gene structure analysis The PLCP family genes in soybean were identified from the Soybean Genome Database [http://soybase.org/] and the Glycine max Wm82.a2.v1 Phytozome Database [http://www.phytozome.net/soybean]. All of the identified GmPLCPs were then analyzed by NIH/NLM/NCBI CD-search tool, sequences without peptidase_C1 domain or the presence of large fragment deletion in the peptid-ase_C1 domain and/or the sequence length of < 150 amino acids were considered as putative pseudogenes and removed manually (Table S1 and Table S2). Basic Local Alignment Search Tool algorithms (BLASTP) with a threshold of e-value <1e-10 was used to identify the homologues of GmPLCPs in M. truncatula, L. japonicus and A. thaliana. The exon/intron/UTR structures of PLCPs were analyzed by using the gene structure display server program GSDS2.0 (http://gsds.cbi.pku.edu.cn/).

Soybean PLCPs sequence
Phytozome v12.0 Database was searched to download the sequences of GmPLCPs. ExPasy site (http://web. expasy.org/protparam/) was used to calculate the isoelectric point (pI) and molecular weight of GmPLCPs. Map Chart software and the soybean genome annotation file (Gmax_275_Wm82.a2.v1.gene.gff3) were used to analysis the locations of GmPLCPs on chromosome. The SoyBase and the Soybean Breeder's Toolbox (https://soybase.org/gb2/gbrowse/gmax2.0/) was used to obtain the blocks regarded as recent duplications.

Phylogenetic analysis
The different PLCPs were applied for multi-species phylogenetic analysis including 97 GmPLCPs, 26 PLCPs from A. thaliana, 33 PLCPs from M. trunctula, and 25 from L. japonicus. Clustal W program was used to conduct the full-length peptide sequence alignments. MEGA6 software [64], Neighbor-Joining (NJ) method and 1000 bootstrap replicates analysis with the pdistance model were used to perform the multi-species phylogenetic tree. The programs RAxML, MEGA version 6.0 and MrBayes 3.2 (http://www.megasoftware.net) [64][65][66] were used to perform the phylogenetic of 97  (a and b). c qPCR analysis of the transcript levels of Glyma.04G190700, ENOD40, Nodulin35, Calmodulin and Lb1 in the control and transgenic hairy roots. d qPCR analysis of the transcript levels of Glyma.04G190700, ENOD40, Nodulin35, Calmodulin and Lb1 in the control and transgenic nodules. Total mixed RNA isolated from root system (including small nodules) or nodules of multiple independent transgenic plants was used for qPCR analysis. The references in the control hairy roots and nodules were used to calculate relative expression levels of these five genes in transgenic hairy roots and nodules. The data are presented as the mean ± SD and " " indicates statistical difference between different inoculated hair roots (t test, p < 0.01) GmPLCPs. RAxML 8.0.0 [67] was used to perform the Maximum likelihood (ML) analysis, the 1000 bootstrap replicates convergence test using the extended majorityrule consensus tree criterion (auto MRE) in RAxML was used to perform rapid 1000 bootstrap replicates analysis, and mixed model was used to construct the MrBayes analysis.

Identification and analysis of duplicate segments pairs formed by soybean WGD events
To identify the duplicate segments pairs formed by soybean WGD events, firstly, the synonymous (Ks) of each GmPLCP or the duplicate gene pairs of GmPLCPs was identified from the SoyBase and the Soybean Breeder's Toolbox. Secondly, according to the distribution of the GmPLCPs on the soybean chromosomes and their values of synonymous, eleven tandem repeat gene clusters were identified (Fig. 1). Thirdly, two big paralogous clusters (pairs No. 10 and 11) with two big tandem repeat gene clusters in each pair, five paralogous clusters (pairs No. 1, 9, 15, 17 and 19) with one tandem repeat gene cluster in each pair and 12 paralogous gene pairs were preliminary identified. Then to examine these preliminary identified 19 candidate paralogous segments pairs, the following two criterions were used in this study: 1) Duplicated segments pairs were grouped together in the GmPLCPs phylogenetic tree ( Fig. 3 and Fig. 6a), and 2) the flanking regions of candidate duplicated segments pairs were showed in the collinearity analysis (Fig. 2). The divergence times (T) were calculated using T = Ks/ (2 × 6.1 × 10 − 9 ) × 10 − 6 Mya [68] to estimate the date of the duplication pairs. Besides, the non-synonymous (Ka) of the paralog pairs was calculated using theYN00 method of the PAML program [69] to investigate the positive Darwinian selection in divergence following duplication. The ratio of Ka to Ks (ω = Ka/Ks) was calculated to measure the history of selection acting on coding sequences [28].

Go annotation and gene expression analysis
The Go annotation of GmPLCPs was conducted by using the "Go Term Enrichment Tool" in Soybean Genome Database [http://soybase.org/]. The soybean database (https://soybase.org/ goslimgraphic_v2 /dashboard. php) was searched to download the detail gene information of these Go terms. The plant Phytozome database (Phytozome 12, http://www.phytozome.net/ soybean) was used to download the expression patterns data of GmPLCPs in five symbiosis-related tissues. The expression patterns of GmPLCPs in five nodule samples were analyzed by searching our previous RNA-seq data [26]. The pheatmap packages in R [41] were used to produce the heatmaps of these GmPLCPs.

Plant materials and growth conditions
The surface-sterilized soybean Tianlong No.1 seedlings were germinated on moistened filter paper in a greenhouse, in which the day/night cycle was maintained at 16/8 h and the relative humidity (RH) at 70%, at 28°C for 2-3 d. The seedlings were then grown in pots filled with sterilized perlite and vermiculite in proportion of 1: 1, and watered with half-strength B&D medium [63]. After 4-5 d, soybean rhizobium 113-2 strain (stored in our lab) was used to inoculate the seedlings. Samples for RNA isolation were collected from soybean nodules at five time points: 12dN (nodules at 12 days after inoculation), 30dN (nodules at 30 days after inoculation), 42dN (nodules at 42 days after inoculation), 64dN (nodules at 64 days after inoculation) and 84dN (nodules at 84 days after inoculation). Nodules from different time points were separately collected with three biological replicates and were frozen at − 80°C for RNA isolation.

RNA extraction and qPCR
We used TRIzol reagent (Invitrogen, USA) to extract the total RNA of nodules, DNase I (Takara) to digest the total RNA, and a Prime Script RT reagent Kit (Perfect Real Time) with gDNA Eraser (Takara Bio, Inc) to perform the reverse-transcribed analysis. RNA quantity and quality were measured using an Epoch Multi-Volume Spectrophotometer system, NanoDrop and Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA), and qPCR reactions on the five RNA samples were used to confirm the absence of gDNA in these RNA samples. cDNA from the reverse transcription of approximately 1 μg of RNA was used as the template for qPCR using primer sets listed in Table S4. RT-PCR amplification mixtures (20 μl) contained 2 μl template cDNA, iTaq Universial SYBR Green Supermix (10 μl) (Applied Biosystems) and 0.5 μl forward and reverse primer. Reactions were run on a CFX Connect Real-Time System (Applied Biosystems), and each assay included a notemplate control (negative control). The cycling conditions of 30 s at 95°C followed by 40 cycles of 5 s at 95°C, 15 s at 60°C and 12 s at 72°C and final 5 s at 72°C. After PCR amplification, a melting curve was generated for every PCR product to check the specificity of the PCR reaction (Fig. S2). The expression stability of four reference genes (ELF1b, QACT, G6PD and Ubiquitin) was evaluated, and ELF1b and QACT were selected as reference genes for the qPCR analyses of 28 selected GmPLCPs. Sample cycle threshold (CT) values were standardized for each template using the two reference gene as control, and the geNorm method [70] ; E, PCR efficiency) was used to analyze the relative changes in gene expression from the qPCR experiments. Three biological replica samples and three or more technical replicate reactions per sample were used to ensure statistical credibility.
A. rhizogenes cells K599 harboring pGlyma.04G190700-RNAi-1, pGlyma.04G190700-RNAi-2 and empty vector were used to induce formation of transgenic hairy roots in soybean. Transgenic hairy roots expressing the empty vector were used as a control. After inoculation with BXYD3, plants with positive transgenic hairy roots were grown for 50 days and nodulation phenotypes were scored. The expression level of Glyma.04G190700, ENOD40, Nodulin35, Calmodulin and Lb1 in pGlyma.04G190700-RNAi or control was determined by qPCR using the primers listed in Table S5 and the procedure as described previously [71,72]. Nitrogenase activity was determined by the acetylene reduction assay (ARA) as described by gas chromatography (GC-14, Japan) [73].

Soybean hairy root transformation
Glyma.04G190700-specific RNAi constructs were transferred into A. rhizogenes K599 by electroporation, and then an A. rhizogenes-mediated procedure [74] was used to induce soybean hairy root formation. After infection, the soybean seedlings were transplanted to hydroponics containing soybean total nitrogen nutrition solution and covered with a transparent plastic to ensure high humidity. Removed the cotyledons after callus formation (about 5 days) and transplanted the seedlings to large hydroponic tanks. Within two weeks, hairy roots started to sprout from the site of infection and were screened with a fluorescence microscopy (Fig. S3). We then extracted the genomic DNA from the transgenic hairy roots to confirm CK and RNAi through gene specific primers. Each seedling was allowed to have only one transgenic hairy root and wrapped with ropes. For nodulation assays, transgenic composite plants were inoculated with Rhizobium BXYD3 and transferred to pots filled with vermiculite with 1/10 N (530 mmol/LN) and grown in a chamber in a 16−/8-h day/night cycle at 26°C. We scored the nodulation phenotypes of these transgenic composite plants at 50 days after BXYD3 inoculation and used empty vector transgenic hairy roots as the control.