CopG1, a Novel Transcriptional Regulator Affecting Symbiosis in Bradyrhizobium sp. SUTN9-2

Simple Summary In the process of symbiosis, ΔcopG1 in the type IV secretion system (T4SS) demonstrated the ability to invade root cells but was unable to survive and multiply within root cells. Conversely, traG1 and virD21 were found to be essential in the early stages of nodule formation. Intriguingly, copG1 is required for nod gene expression and acts as a repressor of T4SS genes. Moreover, the absence of copG1 results in certain proteins not being produced, especially T3SS (nopX and nopP) and C4-dicarboxylic acid (dct), which affects the symbiosis between Bradyrhizobium sp. SUTN9-2 and legumes. These findings support the hypothesis that the copG1 gene may serve as a new regulator of the symbiotic process. Abstract The symbiotic interaction between leguminous and Bradyrhizobium sp. SUTN9-2 mainly relies on the nodulation process through Nod factors (NFs), while the type IV secretion system (T4SS) acts as an alternative pathway in this symbiosis. Two copies of T4SS (T4SS1 and T4SS2) are located on the chromosome of SUTN9-2. ΔT4SS1 reduces both nodule number and nitrogenase activity in all SUTN9-2 nodulating legumes. The functions of three selected genes (copG1, traG1, and virD21) within the region of T4SS1 were examined. We generated deleted mutants and tested them in Vigna radiata cv. SUT4. ΔtraG1 and ΔvirD21 exhibited lower invasion efficiency at the early stages of root infection but could be recently restored. In contrast, ΔcopG1 completely hindered nodule organogenesis and nitrogenase activity in all tested legumes. ΔcopG1 showed low expression of the nodulation gene and ttsI but exhibited high expression levels of the T4SS genes, traG1 and trbE1. The secreted proteins from ΔT4SS1 were down-regulated compared to the wild-type. Although ΔcopG1 secreted several proteins after flavonoid induction, T3SS (nopP and nopX) and the C4-dicarboxylate transporter (dct) were not detected. These results confirm the crucial role of the copG1 gene as a novel key regulator in the symbiotic relationship between SUTN9-2 and legumes.


Introduction
Rhizobia-legume symbiosis is a key process of mutually beneficial relationships where nitrogen-fixing Rhizobia bacteria form root nodules and convert atmospheric nitrogen into ammonia that can be used by the plant, while the plant provides the bacteria with carbohydrates [1].This symbiosis is ecologically significant for providing a major input of nitrogen into ecosystems.It also benefits agriculture by reducing reliance on synthetic nitrogen fertilizers, which can have negative environmental impacts [2].However, specific Biology 2024, 13, 415 3 of 19 elucidate the role of T4SS 1 in symbiosis, this study investigated the functions of individual copG 1 , traG 1 , and virD2 1 genes during their interaction with V. radiata cv.SUT4.Notably, this study reveals a crucial function of copG 1 in regulating symbiosis not only in V. radiata but potentially across the Genistoids, Dalbergioids, and Millettioids lineages.This knowledge could be applied to develop future rhizobial inoculants that enhance nitrogen fixation capabilities in a wide range of legumes.

Plasmid Construction and Gene Deletion
The deletion mutants of copG 1 , copG 2 , traG 1 , and virD2 1 genes in Bradyrhizobium sp.SUTN9-2 (GeneBank accession number LAXE00000001) were obtained as follows: The upstream and downstream regions of copG 1 (up: 575 bp, dw: 841 bp), copG 2 (up: 874 bp, dw:1043 bp), traG 1 (up: 1060 bp, dw:921 bp), and virD2 1 (up: 944 bp, dw: 738 bp) genes were obtained by PCR using the primers listed in Table 1.The target deletion genes in SUTN9-2 were obtained by double crossover.PCR fragments corresponding to the upstream and downstream flanking regions of the gene of interest were merged by overlap extension and introduced into a pNTPS129 plasmid harboring the sacB gene [28].Then, an Ω cassette fragment (spectinomycin/streptomycin resistance genes) from pHP45 (omega) [29] was introduced between the upstream and downstream flanking regions, which were already cloned into pNTPS129.The restriction sites for antibiotic insertion were HindIII for copG 1 and BamHI for copG 2 , traG 1 , and virD2 1 .The recombinant plasmids were transferred into SUTN9-2 by triparental mating using pRK2013 as a helper plasmid [30], as described previously [24].A single recombinant clone was obtained from antibiotic selection and PCR verification.Double recombinant clones were selected by culture on AG medium supplemented with 10% sucrose and 200 µg/mL sm.Candidate clones were verified for the loss of the sacB gene from pNTPS129, and the replacement of the Ω cassette was verified by PCR.All mutant strains were further investigated for nodulation efficiency in V. radiata cv.SUT4.
Table 1.Primers used in this study.

XbaI.F
CCT TGA GAT CTA GAT GTA GTC TGC CCC GAA GTA GC These primer sets were used to obtain the deletion of the copG 1 gene of Bradyrhizobium sp.SUTN9-2 by double crossing over.

XbaI.F
GCC GTT TCT AGA ATT GCG ACA ACG GAC CAG GGC AA These primer sets were used to obtain the deletion of the copG 2 gene of Bradyrhizobium sp.SUTN9-2 by double crossing over.
EcoRI.R CTG TCC GAA TTC ATG TCG TTC CTC GGG TTG TAC C V. radiata cv.SUT4 seeds were surface sterilized and germinated as previously described [32] and placed on 0.85% water agar at 28 • C overnight.One-day-old germinated seedlings were transferred into Leonard's jars containing sterilized vermiculite and liquid buffered nodulation media (BNM) [33].Seven days after gemination, seedlings were inoculated with a bacterial suspension of Bradyrhizobium sp.SUTN9-2 or derivative mutants (1 mL per seedling; adjusted to OD 600 = 0.8).Five plants per treatment were selected for nodule counting.Symbiotic phenotypes and nitrogen activity were measured at 7, 14, and 21 dpi.
Acetylene reduction assays (ARAs) were used to evaluate nitrogenase activity.The root samples were transferred into test tubes, which were closed with a plastic stopper.The samples were then incubated with 10% (v/v) pure acetylene instead of air, which was withdrawn for 1 h at room temperature.A 1 mL sample was examined using gas chromatography (GC) with a PE-alumina-packed column to measure the conversion of  C for flame ionization detection (FID) [34].The experiment was conducted with five biological replicates per treatment.Nitrogenase activity is presented in nmol ethylene/h/plant dry weight [35].

Bacterial Induction, RNA Isolation, and qRT-PCR Analysis of Gene Expression
For bacterial induction, the mid-log phase of bacterial cultures, including Bradyrhizobium sp.SUTN9-2 and copG 1 mutant strains (OD 600 = 0.4), was induced by 20 µM genistein at 28 • C for 24 h.Then, bacterial pellets were collected by centrifugation (4000× g, at 4 • C) for total RNA isolation.Total RNA was isolated from bacterial pellets using an RNeasy ® Protect Cell Mini Kit (Qiagen, Chatsworth, CA, USA) according to the manufacturer's instructions.Total RNA was treated at 37 • C for 30 min with RNase-free DNase I (New England Biolabs, Ipswich, MA, USA).cDNA was synthesized using iScript™ Reverse Transcription Supermix for RT-qPCR (Bio-Rad Laboratories, Inc., Hercules, CA, USA) according to the manufacturer's protocol.A cDNA concentration of 50 ng/µL was subjected to real-time PCR using specific primers (Table 1) for nodulation genes (nodA, nodB, nodC, nodD1, and nodD2), transcriptional regulator of T3SS (ttsI), T4SS structural genes (traG 1 and trbE 1 ), and other genes.qRT-PCR reactions were performed with Luna ® Universal qPCR Master Mix (NEB, Ipswich, MA, USA) according to the manufacturer's protocol, and thermal cycling was conducted in a CFX Opus 96 Real-Time PCR System (Bio-Rad Laboratories, Inc.).The reactions were performed in triplicate for each of the three biological replicates.Relative gene expression was analyzed by the comparative Ct method 2(−∆∆CT), and 16s rRNA (accession number: JN578804) was used as an internal control [18].Three biological replicates were analyzed.

Protein Preparation, Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) Analysis, and Protein Identification
Wild-type (WT) and ∆copG 1 Bradyrhizobium sp.SUTN9-2 were grown in AG medium with shaking at 200 rpm and 30 • C until they reached an OD 600 of 1.One percent (v/v) of each starter was inoculated into 100 mL of AG medium with and without 20 µM genistein induction.The cultures were then incubated at 30 • C until they reached an OD 600 of approximately 1.0.The bacterial supernatants were harvested by centrifugation at 4000× g and 4 • C for 1 h, followed by 8000× g for 30 min.One milliliter of 1 M dithiothreitol (DTT) and 7.5 mL of phenol solution (equilibrated with 10 mM Tris HCl at pH 8.0 with ESTA) were added into 25 mL of fresh supernatant.The solution was vigorously mixed with a vortex before centrifugation at 8000× g and 4 • C for 30 min.The water phase was discarded, and the phenol phase was added into another 25 mL of supernatant, followed by vigorous mixing and centrifugation at 8000× g and 4 • C for 30 min.Next, 20 mL of methanol containing 300 µL of 8 M ammonium acetate and 400 µL of 1 M dithiothreitol were added to remove the phenol phase.The secreted protein was precipitated overnight at 20 • C. The solution was then centrifuged at 8000× g and 4 • C for 1 h, and the supernatant was discarded.After precipitation, the protein was washed with chilled 70% (v/v) ethanol and air-dried in a laminar flow before being dissolved in phosphate-buffered saline (PBS).Protein concentrations were determined using a plate reader and the manufacturer's protocol (PanReac, Barcelona, Spain) according to the Bradford method [37].A standard calibration curve was constructed using 0 to 2 µg of bovine serum albumin (BSA).Denaturing SDS-PAGE was performed according to the method of Laemmli [38], in which 10 µg of each lane of protein was analyzed on a 12% SDS-PAGE gel.The protein samples were mixed with loading buffer containing β-mercaptoethanol and heated for 10 min before loading.Protein bands were stained with colloidal Coomassie brilliant blue R-250 to visualize the expression of secreted protein.The protein bands that were observed on the WT lane but not observed on the ∆T4SS 1 lane were cut for protein identification by mass spectrometry.Breiftly, the protein bands were performed ingel digestion by 12.5 ng/µL trypsin (mass spectrometry grade; Promega, Madison, WI, USA).The extracted peptides were collected and dried in the Nitrogen Evaporator (Organomation, Berlin, MA, USA).The peptides were then reconstituted in 15 µL of 0.1% formic acid (FA) for LC/MS analysis.The LC-MS/MS system consists of a liquid chromatography part (Dionex Ultimate 3000, RSLCnano System, Thermo Fisher Scientific, Waltham, MA, USA) in combination with a captivespray ionization/mass spectrometer (Model Q-ToF Compact, Bruker, Germany) at the Proteomics Services, Faculty of Medical Technology, Mahidol University (Salaya Campus, Mahidol University, Nakhon Pathom, Thailand).Mass spectral data from 300 to 1500 m/z were collected in the positive ionization mode.The most abundant peptide ions were analyzed using MS/MS to determine the peptide sequence.The peptide sequence was searched on the UniProt database using the Mascot Daemon version 2.6.0 (Matrix Science, London, UK) search engine.The search parameters in the Mascot daemon MS/MS Ions search included carbamidomethyl at cysteine residues as a fixed modification and oxidation on methionine as a variable modification.The peptide tolerance was set at ±1.6 Da, and the MS/MS fragment tolerance was set at ±0.8 Da.Protein hits were selected with a p-value of ≤0.05.The obtained results were examined against the protein-NCBI database to identify and annotate proteins.

Microscopy
Nodule phenotypes and cross sections of representative nodules generated by the wild-type (WT) or mutants were examined under a stereomicroscope LEIGA EZ4 (Leica Microsystems, Wetzlar, Germany).For in-situ live or dead cell staining, the nodules were harvested and embedded in 5% agarose [39].Three plants per treatment were selected for nodule sections with a thickness of 40-50 µm.They were prepared with a VT1000S vibratome (Leica, Nanterre, France) and incubated with live/dead staining solution (5 µM SYTO9 and 30 µM propidium iodide (PI) in PBS pH 7.0 buffer) for 30 min, followed by staining with 1 calcofluor-white stain for 20 min.Sections were washed to remove the staining solution and mounted in 10% glycerol in PBS buffer.After staining, nodules were observed by confocal microscopy using a Nikon Inverted Eclipse Ti-E Confocal Laser Scanning Microscope.Calcofluor-white was detected with emission at 460-500 nm, while SYTO9 and PI were detected at 510-570 nm and 600-650 nm, respectively [24].Three nodules were randomly selected for imaging and bacteroid observation.

Statistical Analysis
All data were obtained from experiments performed in triplicate.For statistical analyses, one-way analysis of variance (ANOVA) followed by Tukey's honestly significant difference (HSD) test (Tukey's tests at p ≤ 0.05) and Student's t-tests (p ≤ 0.05) were performed using SPSS software (SPSS version 22.0 windows: SPSS Inc., Chicago, IL, USA) and GraphPad Prism statistical software (Version 10.0.3).

Results
3.1.Symbiotic Properties of ∆copG 1 , ∆traG 1 , and ∆virD2 1 in Vigna radiata cv.SUT4 Differences between the wild-type and mutants of Bradyrhizobium sp.SUTN9-2 were observed in terms of nodulation and nitrogenase activity in V. radiata cv.SUT4 (Figure 1).∆traG 1 and ∆virD2 1 induced a higher number of nodules on the plant tested than the wildtype at 7 days post-inoculation (dpi) (Figure 1Q), although the nodules produced displayed a white color (Figure 1I,M) instead of pink, indicating problems in nodule development.Moreover, there were higher numbers of dead cells in nodules inoculated with ∆traG 1 and ∆virD2 1 in the symbiosome area (Figure 1J,N).At 21 dpi, there was no difference in the number of nodules obtained using ∆traG 1 , ∆virD2 1 , or the wild-type (Figure 1R).Interestingly, copG1 has the potential to regulate symbiosis not only in V. radiata but also across diverse lineages such as Genistoids, Dalbergioids, and Millettioids (Table S2).These findings suggest that copG1 may have a conserved role in governing symbiosis interactions across various plant species by controlling the primary symbiotic interaction system.

The copG Genes Are Involved in Nodulation Efficiency of Bradyrhizobium sp. SUTN9-2
The copG gene typically encodes the CopG protein, which is a transcription factor consisting of ribbon helix-turn-helix (RHH) motifs.Bradyrhizobium sp.SUTN9-2 has two copies of the copG gene located downstream of the traG and virD2 genes.These gene clus- In addition, there were different results of nitrogenase activities in each mutant at 21 dpi; ∆traG 1 was identical to that of the wild-type, whereas low nitrogenase activity was obtained with ∆virD2 1 (Figure 1T).Interestingly, ∆copG 1 showed a significant effect on nodulation in that nodule formation was abolished (Figure 1E,G,Q,R).Although nodule organogenesis was not observed in the plant inoculated with ∆copG 1 , both live and dead cells were detected in the cortex and vascular tissue instead (Figure 1H).According to the results, ∆copG 1 could infect plant cells, but it was no longer capable of surviving in host cells.
Interestingly, copG 1 has the potential to regulate symbiosis not only in V. radiata but also across diverse lineages such as Genistoids, Dalbergioids, and Millettioids (Table S2).These findings suggest that copG 1 may have a conserved role in governing symbiosis interactions across various plant species by controlling the primary symbiotic interaction system.The copG gene typically encodes the CopG protein, which is a transcription factor consisting of ribbon helix-turn-helix (RHH) motifs.Bradyrhizobium sp.SUTN9-2 has two copies of the copG gene located downstream of the traG and virD2 genes.These gene clusters are located in distinct locations on the SUTN9-2 chromosome.Although two copies of the copG genes were present on the chromosome, they did not share the same gene sequences.The copG gene copies 1 and 2 (copG 1 and copG 2 ) revealed a low degree of similarity, with 52.35%DNA sequence identity (Figure S2) and 51.77% amino acid sequence identity (Figure S3), which differ in both the N-and C-terminals.Domain architecture analysis identified CopG 1 as an unidentified domain, which shows similarities with B. yuanmingense BRP09, CCBAU05623, and, more distantly, in P10 130. While, CopG 2 was identified as a Pfam:RHH domain that is also found in B. diazoefficiens USDA110, B. diazoefficiens SEMIA5080, and B. japonicum J5 (Figure S4).
To gain a more complete understanding of the functions of copG 1 and copG 2 , we constructed ∆copG 2 , inoculated it into V. radiata cv.SUT4, and compared it with ∆copG 1 and wild-type strains (Figure 2).Contrary to ∆copG 1 (Figure 2B), ∆copG 2 was able to produce pink nodules (Figure 2C) that were smaller than those generated by the wild-type (Figure 2A).At 14 and 21 dpi, ∆copG 2 produced the highest number of nodules compared with the other strains (Figure 2G,H).Despite the high number of nodules generated by ∆copG 2 , the nitrogenase activity was significantly lower than that of the wild-type (Figure 2I,J).Confocal microscopic examination showed dead cells in nodules generated by ∆copG 2 (Figure 2F) in the symbiosome, as seen in red after staining with PI, in contrast with the wild-type (Figure 2D), which contains more live cells as seen in green by SYTO9 staining.These findings indicate that both copG 1 and copG 2 genes are essential for the symbiotic relationship between SUTN9-2 and legume plants.copG 1 is necessary for nodulation, whereas copG 2 is crucial for nitrogenase efficiency.Lack of copG 2 leads to decreased nitrogenase activity, despite the presence of high nodule numbers.

The copG 1 Gene Plays a Crucial Role in the Expression of Nodulation (nod) Genes and Transcriptional Regulator TtsI (ttsI)
To examine whether copG 1 affects the structuring of the NF backbone, transcript levels of nodABC and transcriptional activator nodD (nodD1 and nodD2) were examined with and without 20 µM genistein induction (Figure 3).The nodA, nodC, nodD1, and nodD2 genes were almost not expressed in ∆copG 1 (Figure 3A,C-E), whereas the expression of nodB was not affected by a mutation in copG 1 (Figure 3B).The results indicated that copG 1 modulates, either directly or indirectly, the expression of nod genes, especially the nodD gene (Figure 3D,E), which is a transcriptional activator of NFs [43].Beside NF biosynthesis, NodD1 also activates the transcriptional regulator TtsI (ttsI), a gene encoding for T3SS secretion and synthesis [44].Similar to the nod genes, the expression level of ttsI was not determined in ∆copG 1 in all conditions (Figure 3F).The loss of nodule formation in ∆copG 1 may be caused by the suppression of NF synthesis and T3SS due to the absence of nodD expression.
(Figure 2D), which contains more live cells as seen in green by SYTO9 staining findings indicate that both copG1 and copG2 genes are essential for the symbiotic r ship between SUTN9-2 and legume plants.copG1 is necessary for nodulation, w copG2 is crucial for nitrogenase efficiency.Lack of copG2 leads to decreased nitr activity, despite the presence of high nodule numbers.gene (Figure 3D,E), which is a transcriptional activator of NFs [43].Beside NF biosynthesis, NodD1 also activates the transcriptional regulator TtsI (ttsI), a gene encoding for T3SS secretion and synthesis [44].Similar to the nod genes, the expression level of ttsI was not determined in ΔcopG1 in all conditions (Figure 3F).The loss of nodule formation in ΔcopG1 may be caused by the suppression of NF synthesis and T3SS due to the absence of nodD expression.

Bradyrhizobium sp. SUTN9-2 copG1 Is Involved in the Repression of the T4SS Structural Genes traG1 and trbE1
To understand the relationship between the T4SS and copG1 genes more clearly, the gene expression fold changes were examined.T4SS with the trbE1 and traG1 genes showed

Bradyrhizobium sp. SUTN9-2 copG 1 Is Involved in the Repression of the T4SS Structural Genes traG 1 and trbE 1
To understand the relationship between the T4SS and copG 1 genes more clearly, the gene expression fold changes were examined.T4SS with the trbE 1 and traG 1 genes showed high expression levels under non-symbiotic conditions when copG 1 was deleted (Figure 4A,B).These results indicate that copG 1 of SUTN9-2 may act as a suppressor of trbE 1 and traG 1 under non-symbiotic conditions.However, under mimicked symbiotic conditions with 20 µM genistein induction, the expression of the trbE 1 gene significantly decreased in ∆copG 1 compared to the wild-type.The expression of the traG 1 gene did not differ between the wild-type and the ∆copG 1 (Figure 4C,D).However, it is noteworthy that under mimicked symbiosis conditions, CopG 1 can induce the expression of trbE 1 and traG 1 , resulting in significantly higher levels than those observed under non-induction conditions.These results suggest that copG 1 might act as a synergistic regulator of the T4SS gene in SUTN9-2 under flavonoid induction (Figure 4C,D).
in ΔcopG1 compared to the wild-type.The expression of the traG1 gene did not differ between the wild-type and the ΔcopG1 (Figure 4C,D).However, it is noteworthy that under mimicked symbiosis conditions, CopG1 can induce the expression of trbE1 and traG1, resulting in significantly higher levels than those observed under non-induction conditions.These results suggest that copG1 might act as a synergistic regulator of the T4SS gene in SUTN9-2 under flavonoid induction (Figure 4C,D).

Effect of T4SS and copG1 on the Secreted Protein Pattern after 48 h of Genistein Induction
The secreted protein patterns of Bradyrhizobium sp.SUTN9-2, ∆T4SS1, and ∆copG1 with 20 µM genistein after 48 h of induction were analyzed by SDS-PAGE.The results

Effect of T4SS and copG 1 on the Secreted Protein Pattern after 48 h of Genistein Induction
The secreted protein patterns of Bradyrhizobium sp.SUTN9-2, ∆T4SS 1 , and ∆copG 1 with 20 µM genistein after 48 h of induction were analyzed by SDS-PAGE.The results revealed distinct protein band patterns under the different conditions (Figure 5).The amino acid sequences of each selected band were examined using mass spectrometry (MS) with the MASCOT program (Table S3 and Figures S5-S8).In the wild-type, genistein induction (band 1) contained a protein matched with the T3SS translocon protein NopX (27%), whereas this band was absent at the same position as ∆T4SS 1 .Similarly, a protein band was observed in the wild-type with genistein induction (band 6), and a match was found with the T3SS host specificity protein NopP (18%).This protein was not observed in ∆T4SS 1 (band 7) at the same position.While protein bands from the wild-type (band 3) and ∆T4SS 1 (band 4) were found to be proteins matched with Dct; C 4 -dicarboxylate ABC transporter (31%), it was not observed in ∆copG 1 (band 5).Nevertheless, ∆copG 1 (band 5) was associated with the amino acid ABC transporter substrate-binding protein glutamate/aspartate transporter subunit (38%).
(27%), whereas this band was absent at the same position as ∆T4SS1.Similarly, a protein band was observed in the wild-type with genistein induction (band 6), and a match was found with the T3SS host specificity protein NopP (18%).This protein was not observed in ∆T4SS1 (band 7) at the same position.While protein bands from the wild-type (band 3) and ∆T4SS1 (band 4) were found to be proteins matched with Dct; C4-dicarboxylate ABC transporter (31%), it was not observed in ∆copG1 (band 5).Nevertheless, ∆copG1 (band 5) was associated with the amino acid ABC transporter substrate-binding protein glutamate/aspartate transporter subunit (38%).qRT-PCR was performed to identify the gene expression of T3SS (nopP and nopX) and C4-dicarboxylate transporter (dct), which was not in the secreted protein from ∆copG1.The results showed that the expression of T3Es (nopP and nopX) and the C4-dicarboxylate transporter (dct) under genistein induction was down-regulated in ∆copG1 (Figure 6).This qRT-PCR was performed to identify the gene expression of T3SS (nopP and nopX) and C 4dicarboxylate transporter (dct), which was not in the secreted protein from ∆copG 1 .The results showed that the expression of T3Es (nopP and nopX) and the C 4 -dicarboxylate transporter (dct) under genistein induction was down-regulated in ∆copG 1 (Figure 6).This indicated that these genes required copG 1 to mediate the regulation under genistein induction.indicated that these genes required copG1 to mediate the regulation under genistein induction.

Discussion
At an early nodulation stage of V. radiata cv.SUT4, ΔtraG1 and ΔvirD21 generated a high number of nodules with smaller sizes compared with the wild-type (Figure 1I,M,Q,R).The symbiosome space of ΔtraG1 and ΔvirD21 infecting nodules revealed some dead cells that were not found in the wild-type under confocal microscopy (Figure 1J,N).According to these findings, T4SS is beneficial in the early stages of symbiotic interactions between SUTN9-2 and legumes.Bradyrhizobia have a TraG/Trb operon on the chromosome in the symbiosis island that is similar to that of mesorhizobia based on the traG gene's phylogenetic and gene organization [24].Beside the structural protein, various bacteria containing T4SS also identified ATPase/Coupling protein, VirD4/TraG, and relaxase VirD2 [45].The traG is commonly found in conjugative plasmids that are responsible for horizontal gene transfer between bacteria.The traG gene required to encode the T4SS component served as an ATPase to generate energy during secretion [46].In addition, TraG also acts as a substrate receptor of T4SS called coupling protein, a substrate receptor that mediates the substrate such as effector proteins, DNA, or a DNA-protein complex through the T4SS channel [16,[47][48][49].In the Pfam prediction, the TraG protein was matched with the Pfam family T4SS-DNA_transfer (PF02534), TrwB_AAD_bound (PF10412), and TraG-D_C (PF12696) (Figure S8A).The C-terminal of this protein can interact with the relaxosome, which is essential for DNA transfer and conjugation in bacteria [46][47][48].In mesorhizobia, traG plays an important role in the early stage of infection, and its expression was observed during induction with root exudate and early nodules generated by M. mediterraneum Ca36 T .Corresponding to mesorhizobia, traG1 of SUTN9-2 may play a crucial role in the initiation of symbiotic interaction with legumes [49].In Agrobacterium, the VirD2 protein is a part of the relaxase family that plays a crucial role in conjugating and mobilizing plasmids that are required for translocation and integration of T-strands into recipient plant cells [50,51].The conjugative transfer of ICEMlSymR7A in M. loti R7A requires VirD2 relaxase to initiate rolling-circle replication [52].VirD21 of SUTN9-2 possesses a domain of unknown function (DUF), DUF3363, which is an uncharacterized protein (Figure S8B).Although ΔvirD21 had no effect on the number of nodules in V. radiata cv.SUT4, nitrogen fixation activity was reduced (Figure 1S,T).

Discussion
At an early nodulation stage of V. radiata cv.SUT4, ∆traG 1 and ∆virD2 1 generated a high number of nodules with smaller sizes compared with the wild-type (Figure 1I,M,Q,R).The symbiosome space of ∆traG 1 and ∆virD2 1 infecting nodules revealed some dead cells that were not found in the wild-type under confocal microscopy (Figure 1J,N).According to these findings, T4SS is beneficial in the early stages of symbiotic interactions between SUTN9-2 and legumes.Bradyrhizobia have a TraG/Trb operon on the chromosome in the symbiosis island that is similar to that of mesorhizobia based on the traG gene's phylogenetic and gene organization [24].Beside the structural protein, various bacteria containing T4SS also identified ATPase/Coupling protein, VirD4/TraG, and relaxase VirD2 [45].The traG is commonly found in conjugative plasmids that are responsible for horizontal gene transfer between bacteria.The traG gene required to encode the T4SS component served as an ATPase to generate energy during secretion [46].In addition, TraG also acts as a substrate receptor of T4SS called coupling protein, a substrate receptor that mediates the substrate such as effector proteins, DNA, or a DNA-protein complex through the T4SS channel [16,[47][48][49].In the Pfam prediction, the TraG protein was matched with the Pfam family T4SS-DNA_transfer (PF02534), TrwB_AAD_bound (PF10412), and TraG-D_C (PF12696) (Figure S8A).The C-terminal of this protein can interact with the relaxosome, which is essential for DNA transfer and conjugation in bacteria [46][47][48].In mesorhizobia, traG plays an important role in the early stage of infection, and its expression was observed during induction with root exudate and early nodules generated by M. mediterraneum Ca36 T .Corresponding to mesorhizobia, traG 1 of SUTN9-2 may play a crucial role in the initiation of symbiotic interaction with legumes [49].In Agrobacterium, the VirD2 protein is a part of the relaxase family that plays a crucial role in conjugating and mobilizing plasmids that are required for translocation and integration of T-strands into recipient plant cells [50,51].The conjugative transfer of ICEMlSymR7A in M. loti R7A requires VirD2 relaxase to initiate rolling-circle replication [52].VirD2 1 of SUTN9-2 possesses a domain of unknown function (DUF), DUF3363, which is an uncharacterized protein (Figure S8B).Although ∆virD2 1 had no effect on the number of nodules in V. radiata cv.SUT4, nitrogen fixation activity was reduced (Figure 1S,T).
Nodules generated by ∆virD2 1 showed many uninfected cells (Figure 1N,P).This finding showed that the communication between SUTN9-2 and the legume at the beginning of nodule organogenesis plays an important role in enhancing infection efficiency and nitrogenase activity after infection.These results strongly indicate that the traG 1 and virD2 1 genes may be necessary for symbiotic interaction during the early infection stage.Unlike ∆traG 1 and ∆virD2 1 , ∆copG 1 has an impact on the symbiotic interaction between SUTN9-2 and legumes because this mutant was unable to generate nodules with the tested plant (Figure 1E,G).For that reason, copG 1 was located downstream of traG 1 and virD2 1 within the same cluster, it is assumed that copG 1 shares a common promoter with traG 1 and virD2 1 .This observation was supported by a previous study in which T4SS complementation successfully restored nodule formation [24].Several bacteria, such as Pseudomonas aeruginosa [53], Streptococcus agalactiae [54], Vibrio cholerae [55], Bradyrhizobium sp., and Mesorhizobium sp., contain the copG gene in their genomes [24].This gene encodes CopG protein, a small transcriptional repressor containing a helix-turn-helix motif domain, which is similar to that of regulatory repressors such as Mnt, Arc, and MetJ in Salmonella typhimurium bacteriophage P22 and Escherichia coli [45,56,57].The CopG protein was first discovered in the streptococcal plasmid pMV158 as a transcriptional repressor that interacts with RepB to control the copy number of the plasmid [44,46,58].In addition, copper resistance was also demonstrated to be influenced by CopG in P. aeruginosa and V. cholerae [53,55].
In SUTN9-2, the CopG 1 protein was classified as an uncharacterized conserved protein, whereas CopG 2 was annotated as the Pfam;RHH_1 domain which may serve as a transcriptional regulator within the CopG family (Figure S4) [59].The removal of both copG genes from SUTN9-2 resulted in distinct nodulation efficiency.Even without a nodule generated by ∆copG 1 , it can still infect plants because we can monitor both live and dead cells within plant tissues.Surprisingly, live cells were found mostly in the vascular bundle tissue, which is similar to the way of endophytic bacteria behave.These findings imply that copG 1 may be crucial for SUTN9-2 in protecting the survival of bacterial cells in the host plant.Bacteria can evolve and adapt to their environment through horizontal gene transfer, usually facilitated by conjugation.Conjugation is a significant biological process because it is the primary way to spread antibiotic resistance genes [60].Integrative and conjugative elements (ICEs) are another essential mechanism that contributes to conjugation.ICEs are recognized as elements encoded for excision and transferred by conjugation and integration, regardless of the specific mechanisms involved [61].The T4SS found in SUTN9-2 is classified as a tra/trb operon and is recognized for its crucial role in facilitating conjugal transfer.Although SUTN9-2 lacks a conjugation plasmid, ICE is still present on the chromosome.The genes encoding the T4SS 1 cluster are presented in this ICE, which is an alternative mechanism of genetic exchange in this bacterial strain [24].To study the impaired nodulation phenotype of ∆copG 1 in V. radiata cv.SUT4, we analyzed the expression of nod genes with and without genistein induction.Common nod genes in SUTN9-2, including nodA and nodC genes, were not expressed even with a lack of copG 1 under the flavonoid induction condition, but this did not affect nodB expression.In addition, copG 1 acts as a stimulator for nodD1 and nodD2, which are the primary transcription factors responsible for NF production.In addition to nod genes, the expression level of ttsI was not determined in ∆copG 1 .The TtsI protein is a transcriptional regulator (previously called y4xI) that is activated by flavonoids and NodD1 that bind to conserved sequences called tts-boxes [44,56,62].These proteins are predominantly expressed during the initial infection stages and within mature nodules, and they play a crucial role in enhancing nodulation [57].During symbiotic interaction, SUTN9-2 required copG 1 to mediate the expression of nod genes and ttsI under flavonoid induction.These results indicate that CopG 1 may positively mediate the expression of nod genes via NodD activation before stimulating NF production, nodule organogenesis, and T3SS.Furthermore, copG 1 plays a role as a repressor in T4SS gene expression, suppressing trbE and traG expression under flavonoid stimulation (Figure 4C,D).In contrast, these genes were not affected by flavonoids in the absence of the copG 1 gene.
The protein expression profiles of SUTN9-2, ∆T4SS 1, and ∆copG 1 with genistein treatment were analyzed by SDS-PAGE.The results revealed distinct protein band patterns under different conditions (Figure 5).∆copG 1 exhibited a deficiency in producing nodules in various plant species and a striking increase in protein expression compared with the wild-type.According to the analysis of the copG 1 domain protein (Figure S4), CopG 1 was predicted to be a transcriptional regulator that might play a role in the regulation of gene expression.The ∆copG 1 lane appears to have much more protein intensity overall because the proportion of protein in this lane might be less than in other lanes.Therefore, 10 µg might show a higher band intensity.A comparative proteomic analysis of the whole secretome should be conducted further to identify additional target proteins involved in this interaction.It was found that several proteins were secreted, but the C 4 -dicarboxylate transport system (dct) protein was not identified in ∆copG 1 , and this result corresponded to the downregulation of the dct gene quantified by qRT-PCR (Figure 6).The dct gene plays a crucial role in symbiosis numerous rhizobia [58].For example, the dct mutant of S. meliloti and R. trifolii can generate ineffective nodules with the host legume [58,63].In addition to the dct gene, other genes are expressed in the same pattern, including nopX and nopP, which are also essential for symbiosis (Figure 6).NopX is a component of T3SS as a translocation pore (translocon) apparatus that is important for host-specific interaction between the rhizobium and host plant.The NGR∆nopX has a significant effect on nodule number because this mutant forms fewer nodules in all plant species tested, including Flemingia congesta, Tephrosia vogelii, Pachyrhizus tuberosus, and Lablab purpureus [64].NopP is a T3SS-effector protein that is phosphorylated by plant kinases [65].Lack of NopP in Rhizobium sp.NGR234 reduces the capacity of nodule organogenesis in tropical legumes.This indicates a positive effect of NopP on symbiosis [64].NopP of B. diazoefficiens USDA122 is necessary and causes Rj2-dependent incompatibility [66].The T3SS of SUTN9-2 has no impact on its symbiotic relationship with V. radiata [18].However, based on the protein secretion results of T3Es (NopP and NopX) and nodD gene expression, it is evident that copG 1 regulates the function of nodD and T3SS.Previous reports indicated that nodD controls the function of the nod cluster by binding to the nod box region.Similarly, nodD can regulate T3SS function by binding to ttsI [62].Therefore, the results of this experiment confirm that CopG 1 controls the function of nodD, influencing the expression of nod cluster genes and T3SS.Perhaps CopG 1 is a crucial factor in the early stages of legume and SUTN9-2 communication.It is plausible that the regulatory system governing the expression of nod genes does not solely depend on the interaction between flavonoids and NodD.Another factor, CopG 1 , also collaborates with flavonoids and NodD to regulate the expression of nod genes and T3SS.Carbon and nitrogen metabolism are the primary mechanisms necessary for the exchange of nutrients between plant and bacterial partners.The proteins secreted from ∆copG 1 matched the periplasmic binding proteins of the glutamate/aspartate ABC transporter.Glutamate is a significant contributor to the total metabolite content, which plays an essential role in nitrogen metabolism, amino acid metabolism, transamination, and carbon sources [67,68].During symbiosis, the main carbon source utilized by rhizobia is C 4 -dicarboxylic acid [69,70].In the mimicked symbiotic conditions, ∆copG 1 lost the ability to establish a symbiotic interaction.Thereafter, increasing the glutamate/aspartate ABC transporter may promote carbon and nitrogen uptake to support bacterial cell survival, but it is not necessary for symbiotic interaction.This again suggests that copG 1 may act as a regulator of nodD and T4SS gene expression under symbiotic conditions.The deeper insights into the genetic mechanisms of T4SS genes involved in rhizobia and legume symbiosis should be further investigated through the transcriptomics analysis to obtain the comprehensive gene expression profiles as well as may explore gene expression on the host side for more understanding.

Conclusions
This is the first report about genes related T4SS in Bradyrhizobium sp.SUTN9-2 that involved in the symbiosis interaction with legumes.This finding reveals that T4SS 1 containing copG 1 , traG 1 , and virD2 1 has beneficial effects on symbiotic interactions with diverse legumes.The early stage of infection and nodulation is influenced by traG 1 and virD2 1 .While, copG 1 is necessary for nodulation, the essential role of copG 2 is nitrogen fixation efficiency.Additionally, copG 1 served as a suppressor of T4SS genes under noninduction conditions and was required to stimulate the expression of T4SS genes through flavonoid induction.copG 1 also acted as a suppressor of secreted protein under flavonoid induction conditions.In addition, a lack of copG 1 led to suppressed expression of nopX, nopP, and dct, which are important for infection and nodulation during symbiosis.Thus, copG 1 is most likely responsible for regulation via functions in T3SS, nodD regulation, and the carbon and nitrogen exchange systems, which are significant for SUTN9-2 during symbiosis.In this study, copG 1 was discovered as a new transcriptional factor in the T4SS cluster and is important for host specificity and competition during symbiosis with their host.Knowledge from this research serves as a model for studying the interaction between the host plant and the secretion systems of Bradyrhizobium that further facilitates scientists to identify effectors required for better colonization, enhancing nodulation and nitrogen fixation, which lead to an increase in legume crop yields under sustainable agriculture.

Figure 4 .
Figure 4. Relative expression of representative T4SS structural genes, including trbE1 (A,C) and traG1 genes (B,D) in Bradyrhizobium sp.SUTN9-2 (WT) and ΔcopG1 with and without 20 µM genistein (G) induction.The 16S rRNA gene was used as an internal control.Values represent the mean ± SD (n = 3).p values based on the student's t-test (ns p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001).The green and red arrows represent a statistical increase and decrease, respectively, in gene expression when comparing experiments with and without genistein.
H 2 ) to ethylene (C 2 H 4 ).Detection was performed at an injection temperature of 150 • C and oven temperatures of 200 • C and 50