1. Introduction
A complex exchange of signals between legumes and rhizobia bacteria is the key factor in the successful symbiosis between two partners. The establishment of nitrogen-fixing nodules is ensured by this intricate communication system, which is crucial for legumes to thrive in nitrogen-limited environments [
1,
2]. Two crucial mechanisms are necessary for the nodulation process of many rhizobia: (i) the classical mechanism of perception of the Nod factor (NF) and (ii) the type III secretion system (T3SS) which deliver effector proteins into plant cells [
3,
4]. NFs are well-studied while, we are just beginning to understand the roles of effectors/secretion systems. The secreted effector proteins from T3SS (T3Es) can have either a positive or negative impact on the symbiosis efficiency, depending on the plant species [
5]. For example,
Bradyrhizobium vignae ORS3257 contains multiple effector proteins crucial for modulating symbiotic properties in different
Vigna species. NopT and NopAB play essential roles in nodulation in
V. unguiculata and
V. mungo. Whereas, NopP2 displayed incompatibility with
V. radiata [
6]. Beside the T3SS, others secretion systems have been identified in several rhizobia genera, including type IV and VI secretion systems (T4SS and T6SS) and these systems have also reported as the key determinant to the symbiotic interactions during infection processes. For instance, the T6SS of
Rhizobium etli Mim1 and
Bradyrhizobium sp. LmicA16 (A16) exhibit a positive effect on nodulation with their host [
8,
9]. In the case of T4SS, this secretion machinery is found in various bacteria, including
Rhizobium. It functions as a molecular channel, allowing bacteria to transport diverse molecules across their cell envelope [
10]. Various rhizobia, including
Bradyrhizobium,
Rhizobium,
Sinorhizobium, and
Mesorhizobium, possess T4SS which belongs to the
tra/
trb operon and could be found either in chromosome or plasmid depending on the bacterial strain [
11,
12,
13,
14]. Interestingly, most of bradyrhizobia harbor the
tra/
trb operon on the chromosome. Few studies about the role of T4SS of
Bradyrhizobium in the symbiotic process have been reported. Previously, we found two clusters of T4SS (T4SS
1 and T4SS
2) located on the chromosome of
Bradyrhizobium sp. SUTN9-2 with different gene arrangements (
Figure S1). Additionally, a specialized gene arrangement consisting of
copG,
traG, and
virD2 genes was observed in the T4SS gene cluster of
Bradyrhizobium species. The T4SS evolutionary analysis of the
Rhizobiales order, encompassing
Bradyrhizobium,
Rhizobium,
Sinorhizobium, and
Mesorhizobium through the
traG gene phylogenetic tree, demonstrates a co-evolutionary trend between
Bradyrhizobium and
Mesorhizobium. Upon phylogenetic examination of the
copG,
traG, and
virD2 combination genes in
Bradyrhizobium, two copies of these clusters were divided into two clades within the bradyrhizobia group [
15]. Moreover, copy 1 of
copG,
traG, and
virD2 genes exhibits a close evolutionary association with
B. yuanmingense BRP09, which are main rhizobia associated with cowpea and mung bean in the subtropical region of China [
16], as well as with
B. diazoefficiens USDA110, soybean inoculant [
17]. Interestingly, T4SS
1 mutant (deleted
copG1,
traG1, and
virD21 fragment) retard nodulation in
V. radiata cv. SUT4 and
Crotalaria juncea and both the number of nodules and nitrogenase activity were decreased compared with the wild type. The results indicated that T4SS
1 has a positive effect on the symbiotic interaction with tested plants [
15]. To understand the role of T4SS
1 in the symbiotic process, individual deletion mutants were generated in
copG1,
traG1 and
virD21 genes (encoding a putative transcriptional factor gene, T4SS structural gene, and relaxase gene, respectively), and examined for their roles in the symbiotic interaction with
V. radiata cv. SUT4 [
18,
19,
20]. In addition to
V. radiata cv. SUT4, the loss of nodulation ability was observed in different plant species of Genistoids, Dalbergioids, and Millettioids upon infection with Δ
copG1 (
Table S1).
4. Discussion
At an early nodulation stage of
V. radiata cv. SUT4, Δ
traG1 and Δ
virD21 generated a high number of nodules with smaller size compared to the wild type (
Figure 1I,M,Q-R). The symbiosome space of Δ
traG1 and Δ
virD21 infecting nodules revealed some dead cells that were not found in the wild type under confocal microscopy (
Figure 1J,N). According to the findings, T4SS is beneficial in the early stages of symbiotic interaction 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 [
15]. Beside the structural protein, various bacteria containing T4SS also identified ATPase/Coupling protein, VirD4/TraG and relaxase, VirD2 [
20]. The
traG is commonly found in conjugative plasmids that are responsible for horizontal gene transfer between bacteria. The
traG gene required for encode T4SS component served as ATPase to generate energy during secretion [
19]. In addition, TraG also act as a substrate receptor of T4SS called coupling protein, a substrate receptor that mediate the substrate such as effector proteins, DNA or DNA-protein complex through T4SS channel [
7,
10,
41,
42]. In the Pfam prediction, TraG protein was matched with Pfam family T4SS-DNA_transfer (PF02534), TrwB_AAD_bound (PF10412), and TraG-D_C (PF12696) (
Figure S8A). The C-terminal of this protein is able to interact with the relaxosome, which is essential for DNA transfer and conjugation in bacteria [
19,
43,
44]. In mesorhizobia,
traG plays an important role in an 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 beginning of symbiotic interaction with legumes [
7]. In
Agrobacterium, VirD2 protein is a part of the relaxase family that plays a crucial in conjugating and mobilizing plasmids that are required for translocation and integration of T-strands into recipient plant cells [
45,
46]. The conjugative transfer of ICEMlSymR7A in
M. loti R7A requires VirD2 relaxase to initiate the rolling-circle replication [
47]. VirD2
1 of SUTN9-2 possesses a domain of unknown function (DUF), DUF3363, which is an uncharacterized protein (
Figure S8B). Although Δ
virD21 had no effect on the number of nodules of
V. radiata cv. SUT4, but the activity of nitrogen fixation was reduced (
Figure 1S,T).
Nodules generated by Δ
virD21 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 indicated that the
traG1 and
virD21 genes may be necessary for symbiotic interaction during an early infection stage. Unlike Δ
traG1 and Δ
virD21, Δ
copG1 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). Since,
copG1 was located downstream of
traG1 and
virD21 within the same cluster, it is assumed that
copG1 may share a common promoter with
traG1 and
virD21. This observation was supported by the previous study, where T4SS complementation successfully restored nodule formation [
15]. Several bacteria, such as
Pseudomonas aeruginosa [
48],
Streptococcus agalactiae [
38],
Vibrio cholerae [
49],
Bradyrhizobium sp., and
Mesorhizobium sp. contain the
copG gene in their genomes [
15]. 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 [
18,
50,
51]. The CopG protein was first discovered in streptococcal plasmid pMV158 as a transcriptional repressor that interacts with RepB to control the copy number of the plasmid [
34,
38,
52]. In addition, copper resistance was also demonstrated to be influenced by CopG in
P. aeruginosa and
V. cholerae [
48,
49].
In SUTN9-2, the CopG
1 protein was classified as an uncharacterized conserved protein, while CopG
2 was annotated as the Pfam;RHH_1 domain (PF01402), which may serve as a transcriptional regulator within the CopG family (
Figure S4) [
34]. The removal of both
copG genes in SUTN9-2 resulted in distinct nodulation efficiency. Even without a nodule generated by Δ
copG1, it can still infect plants because we could monitor both live and dead cells within plant tissues. Surprisingly, live cells were found mostly in the vascular bundle tissue, which is similar to how endophytic bacteria behave. These findings imply that
copG1 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, which is usually facilitated by conjugation. Conjugation is a significant biological process as it is the primary way to spread antibiotic resistance genes [
53]. 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 [
54]. 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 [
15]. In order to study the impaired nodulation phenotype of Δ
copG1 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
copG1 under flavonoid induction condition, but it did not affect
nodB expression. Additionally,
copG1 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, which was not determined in Δ
copG1. TtsI protein is a transcriptional regulator (previously called
y4xI) is activated by flavonoids and NodD1 that bind to conserved sequences called
tts-boxes [
40,
55,
56]. This protein controlled the genes responsible for T3SS synthesis and effector protein secretion which have an effect for symbiosis interaction between rhizobium-legumes interaction depends on plant host [
6,
57]. During symbiotic interaction, SUTN9-2 required
copG1 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,
copG1 plays a role as a repressor in T4SS gene expression, suppressing
trbE and
traG gene expression under flavonoid stimulation (
Figure 4C,D). In contrast, these genes were not affected by flavonoids in the absence of the
copG1 gene.
The protein expression profiles of SUTN9-2, ∆T4SS
1 and ∆
copG1 with genistein treatment were analyzed by SDS-PAGE. The results revealed distinct protein band patterns in the different conditions (
Figure 5). ∆
copG1 exhibited a deficiency in producing nodules in various plant species and a striking increase in protein expression compared to the wild type. According to the analysis of
copG1 domain protein (
Figure S4),
CopG1 was predicted to be a transcriptional regulator which might play a role in the regulation of gene expression. The Δ
copG1 lane appears to have much more protein intensity overall because the proportion of protein in this lane might be less than other lanes. So, 10 µg might be shown 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 ∆
copG1 and this result was corresponded to the down-regulated of
dct gene quantified by qRT-PCR (
Figure 6). The
dct gene play a crucial role for symbiosis in numerous rhizobia [
58]. For example, the
dct mutant of
S. meliloti and
R. trifolii can generate ineffective nodules with the host legume [
59,
60]. Besides the
dct gene, there are other genes that 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 NGRΔ
nopX has a significant effect on the nodule number because this mutant forms fewer nodules in all plant species tested, including
Flemingia congesta,
Tephrosia vogelii,
Pachyrhizus tuberosus, and
Lablab purpureus [
61]. NopP is a T3SS effector protein that is phosphorylated by plant kinases [
62]. A 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 [
61]. NopP of
B. diazoefficiens USDA122 is necessary and causes Rj2-dependent incompatibility [
63]. The T3SS of SUTN9-2 has no impact on the symbiotic relationship with
V. radiata [
11]. However, based on the protein secretion results of T3Es (NopP and NopX) and
nodD gene expression, it is evident that
copG1 regulates the function of
nodD and T3SS. Previous reports indicated that
nodD controls the function of
nod cluster by binding to the nod box region. Similarly,
nodD can regulate the T3SS function by binding to
ttsI [
55]. Therefore, the results of this experiment confirm that CopG
1 controls the function of
nodD, influencing the expression of the
nod cluster genes and T3SS. Perhaps, CopG
1 is a crucial factor in the early stage 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 in regulating the expression of
nod genes and T3SS. Carbon and nitrogen metabolism are the primary mechanisms that are necessary for the exchange of nutrients between plant and bacteria partners. The proteins secreted from ∆
copG1 matched with 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 [
64,
65]. During symbiosis, the main carbon source utilized by rhizobia is C
4-dicarboxylic acid [
66,
67]. In the mimicked symbiotic conditions, ∆
copG1 lost the ability to establish symbiotic interaction. Thereafter, the increasing of 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
copG1 may act as a regulator of
nodD and T4SS gene expression under symbiosis conditions.
Figure 1.
Symbiotic phenotype of Bradyrhizobium sp. SUTN9-2 mutants during symbiosis with Vigna radiata cv. SUT4. Nodule phenotype at 7 and 21 dpi generated by wild type (A, C), ΔcopG1 (E, G), ΔtraG1 (I, K) and ΔvirD21 (M, O). Cytological analysis of the nodules (at 7 dpi and 21 dpi) induced by SUTN9-2 with wild type (B, D), ΔcopG1 (F, H), ΔtraG1 (J, L) and ΔvirD21 (N, P) observed by confocal microscopy after staining with propidium iodide, PI (red; infected plant nuclei and dead bacteria), SYTO9 (green: live bacteria), and calcofluor (blue: plant cell wall). Number of nodules at 7 dpi (Q) and 21 dpi (R). Nitrogen fixation activity determined by the acetylene reduction assay (ARA) of plants infected with the indicated bacterial mutants at 7 dpi (S) and 21 dpi (T). Values represent mean ± SD (n = 5). Scale bars; white bars indicate 1 mm, yellow bars indicate 100 µm (20X) and 50 µm (40X). P values based on Tukey’s test (* P <0.05,** P<0.01,*** P < 0.001).
Figure 1.
Symbiotic phenotype of Bradyrhizobium sp. SUTN9-2 mutants during symbiosis with Vigna radiata cv. SUT4. Nodule phenotype at 7 and 21 dpi generated by wild type (A, C), ΔcopG1 (E, G), ΔtraG1 (I, K) and ΔvirD21 (M, O). Cytological analysis of the nodules (at 7 dpi and 21 dpi) induced by SUTN9-2 with wild type (B, D), ΔcopG1 (F, H), ΔtraG1 (J, L) and ΔvirD21 (N, P) observed by confocal microscopy after staining with propidium iodide, PI (red; infected plant nuclei and dead bacteria), SYTO9 (green: live bacteria), and calcofluor (blue: plant cell wall). Number of nodules at 7 dpi (Q) and 21 dpi (R). Nitrogen fixation activity determined by the acetylene reduction assay (ARA) of plants infected with the indicated bacterial mutants at 7 dpi (S) and 21 dpi (T). Values represent mean ± SD (n = 5). Scale bars; white bars indicate 1 mm, yellow bars indicate 100 µm (20X) and 50 µm (40X). P values based on Tukey’s test (* P <0.05,** P<0.01,*** P < 0.001).
Figure 2.
Derivative copG mutants of Bradyrhizobium sp. SUTN9-2 presenting different symbiotic interaction with Vigna radiata cv. SUT4. Nodule phenotypes induced by wild type (A), ΔcopG1 (B), and ΔcopG2 (C). Cytological analysis of live/dead cells of section nodule infected with wild type (D), ΔcopG1 (E), and ΔcopG2 (F) at 14 dpi were observed with confocal microscopy, and bacteroids were stained with PI, SYTO9, and calcofluor-white. Number of nodules at 14 dpi (G) and 21 dpi (H). Nitrogen fixation activity determined by ARA of plants infected with the indicated bacterial mutants at 14 (I) and 21 dpi (J). Values represent mean ± SD (n = 5). Scale bars; white bars indicate 1 mm and yellow bars indicate 50 µm (40X). P values based on Tukey’s test (** P <0.01, *** P <0.001).
Figure 2.
Derivative copG mutants of Bradyrhizobium sp. SUTN9-2 presenting different symbiotic interaction with Vigna radiata cv. SUT4. Nodule phenotypes induced by wild type (A), ΔcopG1 (B), and ΔcopG2 (C). Cytological analysis of live/dead cells of section nodule infected with wild type (D), ΔcopG1 (E), and ΔcopG2 (F) at 14 dpi were observed with confocal microscopy, and bacteroids were stained with PI, SYTO9, and calcofluor-white. Number of nodules at 14 dpi (G) and 21 dpi (H). Nitrogen fixation activity determined by ARA of plants infected with the indicated bacterial mutants at 14 (I) and 21 dpi (J). Values represent mean ± SD (n = 5). Scale bars; white bars indicate 1 mm and yellow bars indicate 50 µm (40X). P values based on Tukey’s test (** P <0.01, *** P <0.001).
Figure 3.
qRT-PCR analysis of nod genes from wild type Bradyrhizobium sp. SUTN9-2 (WT) and ΔcopG1 grown in absence and presence of 20 μM genistein (G). Expression of structural genes nodA (A), nodB (B), and nodC (C) and regulatory genes nodD1 (D), nodD2 (E) and transcriptional regulator of T3SS, ttsI (F). Data were normalized in relation to the endogenous control (16S rRNA). Values represent mean ± SD (n = 3). P values based on Tukey’s test (*** P < 0.001).
Figure 3.
qRT-PCR analysis of nod genes from wild type Bradyrhizobium sp. SUTN9-2 (WT) and ΔcopG1 grown in absence and presence of 20 μM genistein (G). Expression of structural genes nodA (A), nodB (B), and nodC (C) and regulatory genes nodD1 (D), nodD2 (E) and transcriptional regulator of T3SS, ttsI (F). Data were normalized in relation to the endogenous control (16S rRNA). Values represent mean ± SD (n = 3). P values based on Tukey’s test (*** P < 0.001).
Figure 4.
Relative expression of representatives 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 mean ± SD (n = 3). P values based on 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 expressions when comparing experiments with the presence and absence of genistein.
Figure 4.
Relative expression of representatives 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 mean ± SD (n = 3). P values based on 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 expressions when comparing experiments with the presence and absence of genistein.
Figure 5.
SDA-PAGE analysis of proteins secretion into the external medium of Bradyrhizobium sp. SUTN9-2 (WT), ΔT4SS1, and ΔcopG1 with 20 μM genistein (+Gen) and without 20 μM genistein (-Gen) induction. Numbers on the left indicate molecular size markers in kilodaltons. The arrowhead indicates bands identified by mass spectrometry (MS) analysis of WT (1, 3, 6), ΔT4SS1 (2, 4, 7), and ΔcopG1 (5).
Figure 5.
SDA-PAGE analysis of proteins secretion into the external medium of Bradyrhizobium sp. SUTN9-2 (WT), ΔT4SS1, and ΔcopG1 with 20 μM genistein (+Gen) and without 20 μM genistein (-Gen) induction. Numbers on the left indicate molecular size markers in kilodaltons. The arrowhead indicates bands identified by mass spectrometry (MS) analysis of WT (1, 3, 6), ΔT4SS1 (2, 4, 7), and ΔcopG1 (5).
Figure 6.
Relative expression of Nodulation outer protein X (nopX) (A), C4-dicarboxylate transporter (dct) (B), and nopP genes (C) in Bradyhizobium sp. SUTN9-2 (WT) and ΔcopG1 under 20 μM genistein induction (+G). Data were normalized in relation to endogenous control (16S rRNA). Values represent mean ± SD (n = 3). P values based on Student’s t-test (* P <0.05, ** P < 0.01,*** P < 0.001).
Figure 6.
Relative expression of Nodulation outer protein X (nopX) (A), C4-dicarboxylate transporter (dct) (B), and nopP genes (C) in Bradyhizobium sp. SUTN9-2 (WT) and ΔcopG1 under 20 μM genistein induction (+G). Data were normalized in relation to endogenous control (16S rRNA). Values represent mean ± SD (n = 3). P values based on Student’s t-test (* P <0.05, ** P < 0.01,*** P < 0.001).
Table 1.
Bacterial strains and plasmids used in this study.
Table 1.
Bacterial strains and plasmids used in this study.
Strain or Plasmid |
Relevant Characteristics |
Reference or |
Source |
Strain |
|
|
Bradyrhizobium sp. |
|
|
SUTN9-2 |
A. americana nodule isolate (paddy crop) |
|
∆copG1
|
SUTN9-2 derivative containing an Ω cassette insertion at HindIII site, copG copy 1::sm/sp; Smr, Spr
|
This study |
∆copG2
|
SUTN9-2 derivative containing an Ω cassette insertion at BamHI site, copG copy 2::sm/sp; Smr, Spr
|
This study |
∆traG1
|
SUTN9-2 derivative containing an Ω cassette insertion at BamHI site, traG copy 1::sm/sp; Smr, Spr
|
This study |
∆virD21
|
SUTN9-2 derivative containing an Ω cassette insertion at BamHI site, virD2 copy 1::sm/sp; Smr, Spr
|
This study |
Escherichia coli |
|
|
DH5α |
supE44 ΔlacU169 hsdR17 recA1 endA1 gyrA96 thi-1 relA1 |
Toyobo Inc. |
Plasmid |
|
|
pRK2013 |
ColE1 replicon carrying RK2 transfer genes; Kmr; Helper plasmid |
[22] |
pNTPS129 |
Cloning vector harboring sacB gene under the control of the constitutive npt2 promoter; Kmr
|
[23] |
pNTPS129-∆copG1
|
pNTPS129-npt2-sacB containing the flanking region of copG copy 1 |
This study |
pNTPS129-∆copG2
|
pNTPS129-npt2-sacB containing the flanking region of copG copy 2 |
This study |
pNTPS129-∆traG1
|
pNTPS129-npt2-sacB containing the flanking region of traG copy 1 |
This study |
pNTPS129-∆virD21
|
pNTPS129-npt2-sacB containing the flanking region of virD2 copy 1 |
This study |
Table 2.
Primers used in this study.
Table 2.
Primers used in this study.
Name |
Sequences (5’-3’) |
Descriptions |
Primers for gene deletion |
|
|
Up.copG1. XbaI.F |
CCT TGA GAT CTA GAT GTA GTC TGC CCC GAA GTA GC |
This primer sets used to obtain the deletion of copG1 gene of Bradyrhizobium sp. SUTN9-2 by double crossing over. |
Up. copG1. overl. HindIII. R |
GAG GCG GAC ATG AAA GCT TAA TGA AGG CGG ACG GCC ACT AG |
Dw. copG1. overl. HindIII. F |
GTC CGC CTT CAT TAA GCT TTC ATG TCC GCC TCA CAG TCC GA |
Dw. copG1. EcoRI.R |
AGA TCG GGA ATT CGT TGA CCG AGG ATC TTC AGG CCA |
Up. copG2. XbaI.F |
GCC GTT TCT AGA ATT GCG ACA ACG GAC CAG GGC AA |
This primer sets used to obtain the deletion of copG2 gene of Bradyrhizobium sp. SUTN9-2 by double crossing over. |
Up. copG2. overl. HindIII. R |
GCG CGA CCG AAT GAA GCT TAA GCT GGT CAC GCT ATC GGC T |
Dw. copG2. overl. HindIII. F |
GCG TGA CCA GCT TAA GCT TCA TTC GGT CGC GCA TAT TGC C |
Dw. copG2. EcoRI. R |
CTG TCC GAA TTC ATG TCG TTC CTC GGG TTG TAC C |
Up. traG1. XbaI. F |
TTC GGG TCT AGA TGT AGT CTG CCC CGA AGT AGC |
This primer sets used to obtain the deletion of traG1 gene of Bradyrhizobium sp. SUTN9-2 by double crossing over. |
Up. traG1. overl. BamHI
|
TCC CTC CAA TCA CGG ATC CAT CCT GGT GAC GAT CTC GGA C |
Dw. traG1. overl. BamHI
|
TCG TCA CCA GGA TGG ATC CGT GAT TGG AGG GAT CGT TCA CAG |
Dw. traG1.EcoRI.R |
CCG GCT GAA TTC CTT GGA AAG CCT TGG TCT CG |
Up. virD21. XbaI. F |
ACC GGC TTC TAG AAG ATG CGC AGT CCG CAT CAT C |
This primer sets used to obtain the deletion of virD21 gene of Bradyrhizobium sp. SUTN9-2 by double crossing over. |
Up. virD21. overl. BamHI
|
GAG GAG AAG GAA TGG ATC CTG AAC GAT CCC TCC AAT CAC CG |
Dw. virD21. overl. BamHI
|
GAG GGA TCG TTC AGG ATC CAT TCC TTC TCC TCA GCC ATG GC |
Dw. virD21. EcoRI. R |
CCA TCG GAA TTC TTG TCG ATG CGG AGG AGG CAT C |
Primers for qRT-PCR analysis
|
|
|
SUTN9-2. nodA. F |
GTT CAA TGC GCA GCC CTT TGA G |
Specific primers for nodA gene expression in SUTN9-2 on chromosome
|
SUTN9-2. nodA. R |
ATT CCG AGT CCT TCG AGA TCC G |
SUTN9-2. nodC. F |
ATT GGC TCG CGT GCA ACG AAG A |
Specific primers for nodC gene expression in SUTN9-2 on chromosome
|
SUTN9-2. nodC. R |
AAT CAC TCG GCT TCC CAC GGA A |
SUTN9-2. nodD1. F |
ATT CGT CTC CTC AGA CCG TGC T |
Specific primers for nodD1 gene expression in SUTN9-2 on chromosome
|
SUTN9-2. nodD1. R |
TTC ATG TCG AGT GCG CAC CCT A |
SUTN9-2. nodD2. F |
TGC TTA ACT GCA ACG TGA CCC |
Specific primers for nodD2 gene expression in SUTN9-2 on chromosome
|
SUTN9-2. nodD2. R |
ATG AGC ACG AGG AGC TTC TC |
SUTN9-2. trbE1. F |
GAT TGC AGG AGA ACC GTG AGG C |
Specific primers for trbE1 gene expression in SUTN9-2 on chromosome
|
SUTN9-2. trbE1. R |
AAC AGC GCC GAG GAT TCA GTC T |
SUTN9-2. traG1. F |
TTC TCG ATC TGG TTC AGC GAC TG |
Specific primers for traG1 gene expression in SUTN9-2 on chromosome
|
SUTN9-2. traG1. R |
TTG ACC GAG GAT CTT CAG GCC A |
SUTN9-2. ttsI. F |
ATG AGT TCG TCG GTG GAC AC |
Specific primers for transcriptional regulator TtsI (ttsI) gene expression in SUTN9-2 on chromosome |
SUTN9-2. ttsI. R |
CCA CAT GGT CCT GCT CGA AT |
|
16s. F |
ATT ACC GCG GCT GCT GG |
Universal primers for 16S rRNA used as internal control for bacterial gene expression [25] |
16s. R |
ACT CCT ACG CGA GGC AGC AG |
dct. F |
CGA CTA TCA GGG CGT GAA AT |
Specific primers for C4-dicarboxylate transport (dct) gene expression in SUTN9-2 on chromosome
|
dct. R |
TCC AGC AAT CAG ACC TGT G |
nopX. F |
GGGTGGTCGAGGAAGTATTG |
Specific primers for Type III secretion system (T3SS) gene expression in SUTN9-2 on chromosome |
nopX. R |
GGTTATGACCCAGACCGATG |
nopP. F |
GGTCACACCGACGAAGATAC |
nopP. R |
CCGAAGATCCACTTGGGATG |