Comparative genome analysis of Sesbania cannabina-nodulating Rhizobium spp. revealing the symbiotic and transferrable characteristics of symbiosis plasmids

Symbiotic nitrogen fixation between legumes and rhizobia makes a great contribution to the terrestrial ecosystem. The successful symbiosis between the partners mainly depends on the nod and nif genes in rhizobia, while the specific symbiosis is mainly determined by the structure of Nod factors and the corresponding secretion systems (type III secretion system; T3SS), etc. These symbiosis genes are usually located on symbiotic plasmids or a chromosomal symbiotic island, both could be transferred interspecies. In our previous studies, Sesbania cannabina-nodulating rhizobia across the world were classified into 16 species of four genera and all the strains, especially those of Rhizobium spp., harboured extraordinarily highly conserved symbiosis genes, suggesting that horizontal transfer of symbiosis genes might have happened among them. In order to learn the genomic basis of diversification of rhizobia under the selection of host specificity, we performed this study to compare the complete genome sequences of four Rhizobium strains associated with S. cannabina, YTUBH007, YTUZZ027, YTUHZ044 and YTUHZ045. Their complete genomes were sequenced and assembled at the replicon level. Each strain represents a different species according to the average nucleotide identity (ANI) values calculated using the whole-genome sequences; furthermore, except for YTUBH007, which was classified as Rhizobium binae , the remaining three strains were identified as new candidate species. A single symbiotic plasmid sized 345–402 kb containing complete nod, nif, fix, T3SS and conjugal transfer genes was detected in each strain. The high ANI and amino acid identity (AAI) values, as well as the close phylogenetic relationships among the entire symbiotic plasmid sequences, indicate that they have the same origin and the entire plasmid has been transferred among different Rhizobium species. These results indicate that S. cannabina stringently selects a certain symbiosis gene background of the rhizobia for nodulation, which might have forced the symbiosis genes to transfer from some introduced rhizobia to the related native or local-condition-adapted bacteria. The existence of almost complete conjugal transfer related elements, but not the gene virD, indicated that the self-transfer of the symbiotic plasmid in these rhizobial strains may be realized via a virD-independent pathway or through another unidentified gene. This study provides insight for the better understanding of high-frequency symbiotic plasmid transfer, host-specific nodulation and the host shift for rhizobia.


DATA SummARy
The genome sequences of the four Rhizobium strains YTUBH007, YTUZZ027, YTUHZ044 and YTUHZ045 sequenced in this study were deposited in the National Center for Biotechnology Information database under the accession numbers PRJNA856439, PRJNA843512, PRJNA856444 and PRJNA856733, respectively.

InTRoDuCTIon
Rhizobia are Gram-negative bacteria known for their ability to elicit root or stem nodules on legume hosts to establish nitrogenfixing symbiosis. This kind of symbiosis is important to the terrestrial ecosystem for contributing half of global terrestrial nitrogen compounds [1]. The nodulation process is elicited through the rhizobial synthesis of Nod factors after detection of the flavonoid compounds secreted by the legume host, then the Nod factors induce root hair curling, formation of the infection thread and division of the cortical cells. Finally, the rhizobial cells enter the cortical cells and differentiate to nitrogen-fixing bacteroids [2]. In this mutualism association, the legume host benefits from absorbing ammonium compounds synthesized by rhizobia, while the bacteria receive other nutrients, mainly carbon sources, produced by the host [3].
The nodulation process involves a highly complex molecule exchange interaction, in which the products of nod, nif and secretionsystem-related genes play key roles [3]. These genes are usually located on a large symbiotic plasmid, the so called symbiotic plasmid (pSym), in fast-growing Rhizobium and Sinorhizobium species, and on the symbiotic island in slow growing Bradyrhizobium species and some species of Mesorhizobium [4][5][6]. Both pSym or symbiotic island could be transferred interspecies or even inter-genera through conjugal transfer [4,6,7], so called horizontal gene transfer (HGT), resulting in a symbiotic phenotype shift. The successful self-transfer of a plasmid needs some essential elements: an oriT site and a conjugative transfer system. The oriT region is usually tens to hundreds of base pairs in length, contains a conserved nick region (flanking the nic site) and variable numbers of inverted repeats [8]. The nic site can be recognized and cleaved by a relaxase; however, the inverted repeats are involved in the localization to a precise nic site as well as the termination of ssDNA transfer [8,9]. The conjugative transfer system encompasses two parts: the transfer (tra) genes involved in DNA transfer and replication (Dtr), and the mating pair formation (Mpf) component [10]. The Dtr set includes proteins participating in DNA relaxation (traA), pilus formation and ssDNA transfer (traCDG) [11,12], while the Mpf is composed of trb or virD4 (type IV secretion system; T4SS) related genes [10,13,14]. However, the expression of both Dtr and Mpf genes is regulated by TraI, CinI, TraR and CinR [15]. The quorum-sensing regulation is generated by the product of cinI or traI, which produces l-homoserine lactone to form complexes with TraI or CinI regulators, then the transcription of Dtr and Mpf genes involved in conjugation is induced [15,16].
As a legume species belonging to the tribe Sesbanieae of subfamily Papilionoideae, Sesbania cannabina is an annual semi-shrub native to the South Pacific Islands and has spread in Asia, Africa and Europe (https://www.iucnredlist.org/species/168726/ 20141760). It forms nitrogen-fixing root nodules with rhizobia belonging to 16 species in four genera [17]. The rhizobia associating with S. cannabina showed biogeographical patterns: the Rhizobium species were mainly distributed in acid environments; whereas the Sinorhizobium species were dominant in alkaline saline conditions [17,18]. Interestingly, all the Sesbania-nodulating rhizobia harboured highly conserved symbiosis genes and formed a mono clade in the phylogenetic tree of these genes, indicating HGT events [17]. Among the S. cannabina-nodulating rhizobia, the strains of Rhizobium spp. contained extraordinarily conserved symbiosis genes, indicating the possibility that they may have acquire these genes recently.
Previously, events of horizontal transfer of symbiosis genes have been estimated among the rhizobia nodulating with chickpea [19], common bean [20,21], soybean [22] and some other plants grown in China. Comparative genomic studies on genes inside and outside the symbiosis islands have revealed the possibility that successful HGT needs compatibility between the transferred genes and the genomic background [22,23]. In this study, genomes of four S. cannabina-nodulating Rhizobium strains were sequenced by combining the PacBio and Illumina HiSeq platforms, and were assembled at the replicon level; the genome characteristics of the pSyms were analysed. The identities, phylogenetic relationships, symbiotic properties and the transferrable abilities among

Impact Statement
Sesbania cannabina-nodulating rhizobia were classified into 16 species of four genera but with highly conserved symbiotic genes, especially for Rhizobium spp., indicating they acquired the genes through horizontal transfer. To uncover the transfer mechanism of these genes, the complete genome sequences of four Rhizobium strains were obtained and analysed. We revealed the rhizobia acquired the symbiotic plasmid through a conjugal transfer process but using a virD-independent pathway. In addition, we found the symbiotic plasmid of Agrobacterium pusense IRBG74 could not be transferred due to the absence of necessary conjugal transfer elements. This study provides insight for the better understanding of rhizobial symbiotic plasmid transfer and evolution. these pSyms were characterized and compared. We aimed to determine the genomic features and evolution for the transferrable symbiotic plasmids.

Rhizobial strains
In this study, four representative strains for S. cannabina-nodulating rhizobia isolated from weakly acidic-neutral soils (pH 5.92-7.34), including Rhizobium binae YTUBH007, Rhizobium sp. YTUHZ045, Rhizobium sp. YTUHZ044 and Rhizobium sp. YTUZZ027 [17], were used for a comparative genomic study with two other genome sequences of S. cannabina-nodulating strains Sinorhizobium alkalisoli YIC4027 T [24] and Agrobacterium pusense IRBG74 [25], which were download from GenBank. Almost identical nodA gene sequences (98.8-100 % similarities) were detected among the Rhizobium and Sinorhizobium strains, and the nodA gene of the A. pusense strain shared similarities of 92.2-92.4 % with those of the other five strains [17]. These strains formed a sample set with similar symbiosis genes, but different species-genus backgrounds.

Genome sequencing, assembly and annotation
Genomic DNA was extracted separately from strains R. binae YTUBH007, Rhizobium sp. YTUZZ027, Rhizobium sp. YTUHZ044 and Rhizobium sp. YTUHZ045 by using a genome extraction kit for bacteria (Sangon Biotech), according to the manufacturer's instructions. The DNA samples were examined by a NanoDrop spectrophotometer (Thermo) and then fragmented for genomic DNA library preparation. Then, the Illumina PE library (400 bp) and PacBio library (20 kb) were constructed and sequenced with the Illumina HiSeq 2500 platform with 150 bp paired-end technology and the PacBio RSII platform, respectively, at Biozeron Biotechnology.
The raw reads were checked using FastQC and then pruned/filtered by Trimmomatic (v0.38) [26] to obtain clean reads. The clean Illumina reads were assembled by using SOAPdenovo (v2.04) [27], and blasR [28] was used to compare the PacBio reads, then the Celera Assembler 8.0 [29] was selected to connect scaffolds to obtain the complete genome sequences. The complete genome sequences were annotated by using Prokka (v1.14.6) [30] and eggNOG (v2.1.4) [31]. The genome sequences of strains Sinorhizobium alkalisoli YIC4027 T [18] and A. pusense IRBG74 were extracted from the GenBank database for comparative study. The genome characteristics were counted for all the six tested strains by using Python (v3.9.12). The plasmids with nodulation genes nodABC and iron-nitrogenase-encoding gene nifH were classified as pSyms in this study.

Comparison of the complete genome and plasmid sequences
For this comparison, the genome sequences of several reference strains representing other rhizobia were extracted from the GenBank database, and were analysed together with those acquired in this study. The core genes among either the complete genome or pSyms were analysed by Orthofinder (v2.5.4) [32]. A maximum-likelihood phylogenetic tree was reconstructed based on the concatenated single copy core sequences by using iq-tree 2.2 [33] and then visualized by iTOL (https://itol.embl.de/). Average nucleotide identity (ANI) values between the pair of complete genomes or pSym sequences were calculated by using the OrthoANI program [34], and the amino acid identity (AAI) values were calculated by CompareM (v0.1.2) (https://github.com/ dparks1134/CompareM). The heatmaps were generated according to the ANI and AAI values by using R (v4.1.0).

Gene annotation for pSyms
eggNOG (v2.1.4) [31] was used to annotate the pSym sequences. The results were assessed based on Clusters of Orthologous Groups (COGs) using 'stringr' and 'dplyr' packages from the R software. The Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation process for pSyms was performed by using the online program (https://www.genome.jp/kegg/mapper/reconstruct. html).

Analyses of symbiosis, nitrogen fixation and conjugal transfer related genes
The protein sequence database [including nod, nif, fix, type III secretion system (T3SS) and conjugative transfer related genes] was constructed according to previous studies [35]. The alignment for each pSym was performed by using each reference sequence in the database using blast software version 2.13.0. The genetic organization plots for these genes were drawn using the online program 'gene cluster' (https://xiaochi.chifei3d.com/static/xiaochiPlot/src/gene_cluster.html). The oriT for each pSym was predicted using the online oriTfinder prediction program [36].

Genomic characteristics of S. cannabina-nodulating Rhizobium spp.
The complete genome sequences of the four tested Rhizobium strains obtained in this study were at the replicon level, with genome sizes of 6.55 to 6.75 Mb and 3-7 plasmids (Figs 1 and S1, Table S1, available with the online version of this article). The symbiotic plasmid sizes range from 344 to 402 kb and the G+C content is 58.43-59.09 mol%, which is obviously lower than for the complete genome sequences (Figs 1 and S1, Table S1). The gene functions of pSym coding sequences (CDSs) detected in all the six tested strains were classified into COGs families composed of 19 categories (Table S2) and three main function classes (>10 % of the total CDSs): replication, recombination and repair (L); energy production and conversion (C); amino acid transport and metabolism (E); which accounted for 20.57, 10 and 10 % of the total classified CDSs, respectively. Meanwhile, 17 % of the CDSs were classified as function unknown (S). In addition, the CDSs of the pSyms were classified into 27 KEGG pathways (Table S3), with the major pathways of membrane transport, amino acid metabolism, carbohydrate metabolism and xenobiotics biodegradation and metabolism, representing 17, 16, 15 and 15 % of total CDSs, respectively. These prediction results indicate that these rhizobia harbour many genes corresponding to the transportation of amino acids and carbohydrate and energy metabolism, which are related to the function of the high energy consuming nitrogen fixation process and the nutrient exchange between rhizobia and the host plant in nodule symbiosis.

Phylogenetic and homologue analyses of the whole genomes and pSyms
According to the phylogenetic tree reconstructed using the whole-genome sequences, the four strains sequenced in this study were clustered within Rhizobium (Fig. 2a). The ANI values were 99.0 % between R. binae YTUBH007 and the type strain R. binae BLR195 T , and less than 95 % among the three strains representing the unnamed genospecies and the reference strains of defined species (Fig. 3). However, the phylogenetic tree reconstructed using the complete sequences of pSyms ( Fig. 2b) showed different topology relationships with the complete genome sequences. All the pSyms of S. cannabina-nodulating rhizobia, including Sinorhizobium alkalisoli YIC4027, A. pusense IRBG74 and the four Rhizobium strains sequenced in this study, formed a cluster sharing ANI values between 86.15 and 99.97 % (Fig. 2b). The highest ANI values occurred among Rhizobium sp. YTUHZ045, Rhizobium sp. YTUZZ027 and R. binae YTUBH007 (>99.42 %), which were consistent with their genome phylogenetic relationships (Fig. 2b). The AAI values showed the same tendencies as for ANI values (Fig. 3).
A total of 130 core genes were determined among the six S. cannabina-nodulating rhizobial strains (Fig. 4), including 16 nod, 15 nif and 10 transposition genes (Table S4). In addition, another 82 common genes were shared by the four S. cannabina-nodulating Rhizobium strains (Fig. 4, Table S4), including 17 genes related to the T3SS and 17 genes related to conjugal transfer (Table S4), which were absent in A. pusense IRBG74. For strain-specific genes, A. pusense IRBG74 presented the highest number (371), followed by Sinorhizobium alkalisoli YIC4027 (158); while only one (R. binae YTUBH007 and Rhizobium sp. YTUZZ027) or two (Rhizobium sp. YTUHZ045) were detected for the Rhizobium strains. In addition, the common shared genes between each pair of the Rhizobium pSyms indicating that YTUBH007, YTUZZ027 and YTUHZ045 have closer relationships than that with YTUHZ044 ( Fig. 4), consistent with the phylogenetic relationships and ANI values (Figs 2b and 3).
In our previous study, the S. cannabina-nodulating rhizobia across the world were classified as 16 species in four genera, and the symbiosis genes of all the strains formed a unique cluster [17]. Among them, the strains within different Rhizobium species presented extraordinarily highly conserved symbiosis genes [17], indicating that they might have acquired the symbiosis genes        through HGT in a short evolutionary history. The symbiosis genes are usually located on pSyms [4,[37][38][39] or chromosomal symbiotic islands [40][41][42][43][44]. As for other fast-growing rhizobia, the symbiosis genes of S. cannabina-nodulating rhizobia A. pusense IRBG74 and Sinorhizobium alkalisoli YIC4027 were also located in a pSym [24,45]. In the present study, we firstly evidenced the existence of pSym in the S. cannabina-nodulating Rhizobium species. According to the phylogenetic relationships (Fig. 2a) and the ANI values (Fig. 3), the four Rhizobium strains were classified as different species with one validly published (R. binae) and three new candidate species, which supported the previously reported species affiliation based upon MLSA (multilocus sequence analysis) [17,18].
A single symbiosis plasmid was identified in each strain and both the genes nodABC and nifHDK essential for Nod factor synthesis and nitrogenase are located in the pSym, similar to the previously described pSyms [46]. The assembled pSyms in this study presented sizes of 345 to 402 kb and G+C content of 58.43-59.09 mol%, which was obviously lower than that of the chromosome 59.65-61.24 mol%, and was consistent with other Rhizobium strains [47]. The sizes of the detected pSyms were much smaller than those of the previously published S. cannabina-nodulating rhizobia A. pusense IRBG74 (585 kb) and Sinorhizobium alkalisoli YIC4027 (456 kb).

Relationships among the pSyms of S. cannabina-nodulating rhizobia
The ANI and AAI values of the pSyms were consistent with the corresponding phylogenetic relationships based on the pSym sequences (Figs 2b and 3) and on the symbiosis genes [17,18], indicating that the pSyms of S. cannabina-nodulating rhizobia have the same origin. Due to the greatly high identities, fewer strain-specific genes and more core genes among the pSyms in the four tested Rhizobium strains (Figs 3 and 4), it could be estimated that these strains acquired the plasmid in a short evolutionary history. Among the four Rhizobium strains, YTUHZ044 has the smallest pSym with the most strain-specific genes, supporting it having a more distant relationship with the other three Rhizobium strains (Fig. 4). Among the 130 core genes present in all the six pSyms, ten genes were related to transposition, which is consistent with the high abundance of insertion sequences in pSyms [48]. Other types of core genes in the pSyms of tested Rhizobium strains were those related to T3SS and conjugal transfer genes (Dtr-and Mpf-encoding genes), which were absent in A. pusense (Fig. 5c, d, Table S4). No identical pSyms were found among the tested strains, indicating that the pSyms might have been modified by the acceptor rhizobia after the HGT.

Symbiotic characteristics of the pSyms
The nodulation process between legume and rhizobia is primarily incited by the Nod factor. In this study, the common nod genes were very similar among the six S. cannabina-nodulating strains, and the Nod factors they produce may have a complicated chemical structure modified by fucosylation, N-methylation, carbamoylation and arabinosylation, catabolized by NodZ, NodS, NodU and NeoP [49]. Thus, all the Sesbania rhizobia may secrete very similar decorated Nod factors, and the chemical structures may be strongly selected by the host, since S. cannabina only nodulated with rhizobia harbouring similar symbiosis genes [17,50].  All the six Sesbania-nodulating rhizobia contained 15 complete nif and fix genes (Fig. 5b), including the nif core genes nifHDK encoding the full nitrogenase complex [51] and other nitrogen-fixation-related genes such as: nifB, which is needed for synthesis of the iron-molybdenum cofactor [52]; nifW and nifZ, whose products may be required for protection of the nitrogenase from oxygen [53]; the genes for the regulatory protein NifA [52]; the ferredoxin gene fdxN, which is essential for nitrogen fixation in Sinorhizobium meliloti [52]; nifU and nifS involved in the formation of the mature Fe-S cluster required for nitrogenase complex function [54]. However, nifV involved in synthesis of the iron-molybdenum cofactor [55] was absent in all the six strains; so, this cofactor might be provided by the host during symbiotic nitrogen fixation [56] in S. cannabina.
The T3SS widely distributed in rhizobial genomes determines the symbiotic efficiency, it can be completely necessary for nodule formation or can block nodulation during the rhizobium-legume symbiosis process [57]. It is a protein complex and secretes effector proteins from the cytosol of the bacteria into the host cell through a tube spanning the bacterial and host membranes, and it consists of needle structure proteins and effector proteins secreted by the needle [58]. In this study, except for A. pusense IRBG74, which is without any related genes, the other five strains contain genes encoding 14 structural genes: sctQ, sctL, sctO and sctN encoding proteins that form the cytoplasmic complex in the bacteria; sctR, sctS, sctT, sctU and sctV encoding proteins that form the export apparatus; sctC1, sctC2, sctD and sctJ encoding proteins of the basal body part; sctI encoding proteins of the needle part [58]. In addition, nopA encodes a key component of the extracellular part of the secretion machinery, whereas nopX encodes a part of the translocon that directs effector proteins across the plant plasma membrane [59,60]. The only common effector protein shared by the five strains was NopC, which positively affects the symbiosis of Sinorhizobium fredii HH103 with soybean but negatively regulates its symbiosis with Lotus japonicus [61,62]. Interestingly, A. pusense IRBG74 was successful for nodulation [63,64] without any T3SS-related gene (Fig. 5), indicating that T3SS is unnecessary for S. cannabina nodulation.

Transfer ability prediction for pSyms
For rhizobia, the symbiosis varieties (symbiovars) and effectiveness (nitrogen fixation) were mainly determined by the symbiosis genes located on pSyms or symbiotic islands [65]. However, the symbiosis genes could be transferred through transfer of the entire plasmid [4] or symbiotic island by integrative and conjugative elements (ICEs) to the receptor strain [4,6,7]. Comparison between the phylogenetic relationships, ANI values and AAI values of the whole genome and pSym (Table 1), and the Venn diagram (Fig. 4), clearly evidenced that the pSyms in the tested S. cannabina-nodulating rhizobia were acquired through plasmid transfer.
The transfer of pSym is mainly through conjugative transfer [4], and genes encoding Dtr and Mpf and the oriT sequence region are necessary for successful conjugative transfer [10,36]. The oriT sequence is recognized by Dtr and then the transfer is elicited [14,36]. The Dtr usually comprises necessary tra genes (traACDG) [12] and the regulatory genes cinIR or traIR [15]. traA encodes relaxase that interacts with oriT [12]; traC encodes ATPase, which is involved in pilus assembly and contributes to conjugation at the cell-cell contact stage [66,67]; traD encodes the coupling protein TraD, a hexameric ring ATPase that forms the cytoplasmic face of the conjugative pore [11]; and traG encodes a large transfer protein, which consists of an inner membrane domain and a large periplasmic domain involved in pilus tip assembly and mating pair stabilization [11,66]. Except for the A. pusense IRBG74 without any Dtr genes, the other five tested strains harboured traA, traCDG and the regulatory genes cinI and cinR (Fig. 5d), indicating they encompass the necessary tra genes.
The Mpf genes in rhizobial pSyms have two types, the complete trb-like systems such as that in Sinorhizobium sp. NGR234 [68], and virB T4SSs such as that in Rhizobium etli CFN42 and Sinorhizobium meliloti 1021 [12,52]. The virB T4SS is usually composed of 12 genes (virB1-11/D4): virB4 and virB11 encode ATPases that coordinate the recruitment and processing of substrates, catalyse structural changes in the T4SS channel necessary for substrate passage and also regulate pilus biogenesis [13,14,69]; virB3, virB6 and virB8 encode integral inner membrane (IM) subunits that presumptively form an IM channel [14]; virB1 encodes a transglycosylase that contributes to assembly of the channel across the murein layer [14]; and virB7, virB9 and virB10 encode outer membrane (OM)-associated subunits that form a structural scaffold for the portion of the channel spanning the periplasm and OM [70]. Except for A. pusense IRBG74 without virB1, virB3 and virB5, the other five Sesbania-nodulating strains harboured a complete set of virB genes (Fig. 5d). However, virD4 was absent in all the pSyms of Sesbania-nodulating rhizobia; furthermore, this gene was also absent in the self-transferrable pSyms in Sinorhizobium meliloti 1021 [71] and R. etli CFN42 [72] (data not shown). Thus, the transfer of pSyms in the Sesbania-nodulating rhizobia may be via a virD-independent way or by an unidentified gene with the same function.
Without oriT, tra and incomplete vir genes, the pSym of strain A. pusense IRBG74 seemed not self-transferable. However, the other five tested strains had the necessary genes of conjugal transfer including oriT, tra and the complete virB, indicating they could be transferred as the donor strain. The existence of diverse S. cannabina-nodulating rhizobia indicates their transferrable pSym could be transferred frequently, and the receptor strains adapted to the local conditions could establish symbiosis with S. cannabina. In this case, the receptor strains might be native bacteria without symbiosis genes [42] or could have an original pSym corresponding to other hosts. In the latter case, the original pSym may have been lost due to the incompatibility of two similar pSyms [73], and finally resulted in the receptor strain becoming the S. cannabina-specific rhizobium, such as R. binae YTUBH007. It is interesting to note A. pusense IRBG74 is the only Agrobacterium strain with symbiotic nitrogen fixation ability according to the genome sequences (578 Agrobacterium genomes from GenBank, data not shown), which was presumed to be loss of Ti plasmid and acquisition of the symbiotic plasmid from rhizobium [25,64]. However, according to our study, the symbiotic plasmid of A. pusense IRBG74 is not self-transferrable, which is not consistent with any other species sharing the same symbiotic gene type with it [17]. Thus, we speculate the transfer-related element of the plasmid may have been lost in the evolutionary history of this species.

Conclusion
Four S. cannabina-nodulating Rhizobium strains were sequenced and assembled at the replicon level and compared with another two related strains in this study. Each of the six strains harboured a pSym with size (345 554-402 479 bp) and genomic organization different from each other. The phylogenetic relationships, ANI and AAI values, and the shared genes among the pSyms indicated that they acquired the entire plasmid with a short evolutionary history. The nod, nif, T3SS and conjugal transfer related genes were located on the pSyms. All the S. cannabina-nodulating rhizobia presented very similar nod gene clusters, indicating that the legume rigorously selected the chemical structure of the Nod factor of the rhizobia; however, the T3SS might be unnecessary for the symbiosis with S. cannabina. The transferrable pSym of each strain contained oriT, cinIR, traACDG and complete virB1-11 genes but without virD, indicating that they may be transferred through a virD-independent mode or by another unidentified gene.

Funding information
This work was supported by the Natural Science Foundation of Shandong Province (ZR202102280248 and ZR2020MC043).
Author contribution K.M.H., Y.L. and Y.L. contributed to conception and design of the study. K.M.H. and Z.P.Z. implemented DNA extraction, and sequence assembly. K.M.H. and Y.L (the correspondence). performed data analysis and wrote the manuscript with help from L.S. and E.T.W. All authors contributed to manuscript revision, and read and approved the submitted version.

Conflicts of interest
The authors declare that there are no conflicts of interest