**5.** *Rj-***gene specific nodulation genes (***Rj-gsn* **genes) of** *Bradyrhizobium* **strains**

Genotype-specific nodulation (*gsn*) genes are reported in *B. japonicum.* The *nolA* gene al‐ lowed serogroup 123 strains to nodulate soybean plants having USDA123 restricting Plant Introduction (PI) genotypes [46]. The *noeD* gene restricted nodulation of soybean genotype PI 417566 with USDA110 [47]. There have been, however, no reports of the identification of the *Rj-genotype* specific nodulation (*Rj-gsn*) gene that is related to the incompatible combina‐ tions with *Rj* soybeans. This study reports the isolation and characterization of Tn*5* mutants of *B. japonicum* strain Is-1 with the ability to nodulate cv. CNS (*Rj*2*Rj*3) and shows the candi‐ dates of the *Rj-gsn* genes with the identifications of Tn5-flanking sequences in these mu‐ tants. As this is the first report to overcome the nodulation restriction conditioned by *Rj*2 cultivars, these mutants will be helpful in identifying the *Rj-gsn* gene in further studies.

### **5.1. Materials and methods**

**Figure 1.** Difference in beta diversity compared to gamma diversity (*H*'β/*H*'γ) among pairs of cultivation temperatures.

A Comprehensive Survey of International Soybean Research - Genetics, Physiology, Agronomy and Nitrogen

We also investigated the differences in bradyrhizobial communities for the pairs of cultiva‐ tion temperature. The nodulation tendencies of soybean cultivars were similar for each culti‐ vation temperature, and differences in the community structures between low and high cultivation temperatures were relatively larger than the other comparisons, although the statistical significant difference was not detected. This possible reason is that responses of soybean cultivars for cultivation temperatures on soybean nodulating bradyrhizobial com‐ munities are different among each soybean cultivar even in same *Rj*-genotypes. Therefore, analyses of soybean-nodulating rhizobial communities on not only *Rj*-genotypes but also ev‐ ery soybean cultivars must be conducted for environmental factors affecting soybean-nodu‐ lating rhizobial community structures such as cultivation temperature in further studies. The responses of host soybean and soybean-nodulating bradyrhizobia under cultivation conditions such as a suboptimal root zone temperature were reported previously. The low‐ ering of temperature delayed bradyrhizobia infection of soybean roots and lowered the gen‐ istein secretion from soybean roots [39, 40]. It also appeared to inhibit the expression of the nodulation (*nod*) gene of soybean-nodulating bradyrhizobia [41]. However, Pan and Smith [42] reported that the concentration of daidzein secreted from soybean roots increased with decreasing root zone temperature. The physiological factors of bradyrhizobia involved in the nodulation are the expression of the nod gene and growth capability in soil and rhizo‐ sphere. Yokoyama [43] demonstrated that the expression level of the nod gene of three *Bra‐ dyrhizobium* strains, *B. japonicum* USDA110 strain, *B. elkanii* USDA76 strain, and *Bradyrhizobium* sp. TARC 64 strain (isolates from Thailand soil [44]), which were mutants of *nodY-lacZ* fusion, were different depending on the incubation temperature (20, 23, 26, 30, 33, 35, 37, and 40˚C) and suggested that the transcriptional responses of the *nod* gene of US‐ DA110 strain and USDA76 strain were distinctly different at 23 to 35°C. Saeki et al. [28] demonstrated that the population occupancy of four *Bradyrhizobium* USDA strains, *B. japoni‐ cum* USDA6T, 38, and 123 and *B. elkanii* USDA76T, in soil microcosms changed with different

Each value is expressed as the mean ± the standard error (n = 3 or 4].

Relationships

100

**Plants:** Soybean (*Glycine max* L. Merr.) cvs. CNS (*Rj*2*Rj*3), D- 51 (*Rj*3), Hardee (*Rj*2*Rj*3), Hill (*Rj*4) and IAC-2 (*Rj*2*Rj*3) were used. No soybean cultivar harboring only the *Rj*2-gene has been reported to date. The *Rj-*genotypes are indicated in parentheses [11, 14].

**Bacterial strains and plasmids:** The bacterial strains and plasmids used in this study are de‐ scribed in Table 9. The *B. japonicum* strain Is-1 [13] and Tn5 mutants were grown on HM salt medium [48] supplemented with 0.1% arabinose at 30˚C. *Escherichia coli* strains were grown on Luria–Bertani medium [49] at 37˚C. Antibiotics were added to the media at the following final concentrations: kanamycin at 50 µg mL-1 for Tn5 mutant and ampicillin at 100 µg mL-1 and kanamycin at 50 µg mL-1 for *E. coli* S17-1 harboring pUTKm (Table 9).

**Tn5 mutagenesis:** Transposon mini-Tn*5* was introduced into *B. japonicum* strain Is-1 by mat‐ ing with *E. coli* strain S17-1 [50] carrying the suicide vector pUTKm [51]. The *E. coli* donor strain S17-1 (pUTKm) was grown with kanamycin and ampicillin for 12 h at 100 rpm in a rotatory shaker, and then 1 mL of culture was spun down for 2 min at 4,000 × g, and then rinsed three times with HM salts medium [52] to remove the antibiotics, and then suspend‐ ed into 50 µL of HM salts medium. The *B. japonicum* strain Is-1 was grown for 6 days at 100 rpm in a rotatory shaker, and then 2 mL of the culture was spun down for 3 min at 9,000 × g

and then suspended into 50 µL of HM salt medium. Equal volumes (50 µL) of *E. coli* donor and *B. japonicum* recipient cells prepared as described above were mixed in a 1.5-mL eppen‐ dorf tube. The mating mixture was spread onto a cellulose acetate membrane filter (Advan‐ tec, Tokyo, Japan; pore size 0.45 µm). The mating was carried out on HM plates at 30˚C. The incubation time of the mating was 2 days. After mating, cells were suspended in 1 mL of HM salts medium and 100 µL aliquots of this suspension were spread onto 10 HM plates. Kanamycin was added to select for kanamycine-resistant (Kmr ) transconjugants.


Km, kanamycin; Amp, ampicillin.

**Table 9.** Bacterial strains and plasmids used in this study.

**Isolation of Tn5 mutants:** All Kmr transconjugant colonies in each HM plate were suspend‐ ed in 20 mL of HM medium and cultivated for 7 days at 30˚C. These 10 cultures containing Kmr transconjugants were separately spun down for 10 min at 6,000 × g and then rinsed twice with half-strength modified Hoagland nutrient (MHN) solution [53] to remove the an‐ tibiotic. Each pellet of Kmr transconjugants was suspended in 60 mL of MHN solution. These 10 suspensions were separately inoculated onto CNS (*Rj*2*Rj*3) at a rate of 5 mL per seed in each pot. The pots and seeds were prepared as follows. Both vermiculite (2.8 L) and MHN solution (1.4 L) were added to a 3.0-L porcelain pot and autoclaved at 121˚C for 20 min. The seeds were sterilized with 5% sodium hypochlorite for 5 min and then rinsed with ethanol five times, and then rinsed with MHN solution five times. The surface-sterilized seeds of CNS were planted in a vermiculite medium at a rate of 12 seeds per pot. The inocu‐ lated plants were grown in a phytotron controlled at 25 ± 1˚C under natural light conditions and sterilized water was supplied to maintain the moisture content. After 4 weeks of culti‐ vation, Tn5 mutants were isolated from nodules produced on the root of CNS. The nodules were washed to remove vermiculite and immersed in 95% ethanol for 1 min, and then in 5% hydrogen peroxide solution for 5 min. The sterilized nodule was crushed and suspended in 5 mL of autoclaved 0.9% saline water. A drop of the turbid suspension was transferred to a yeast extract mannitol agar (YMA) plate [16] containing Congo Red (25 µg mL–1) and streaked. These plates were incubated for 7 days at 30˚C.

**Amplification of the Tn5-flanking sequence:** Kwon and Ricke [54] developed an efficient method to specifically amplify the transposon-flanking sequences with unique Y-shape link‐ ers. The amplification of Tn5- flanking sequence was carried out according to their method. Because pUTKm was used to carry out Tn5 mutagenesis in both our study and the study by Kwon and Ricke [54], the same oligonucleotides are used in these two experiments: Linker 1, 5′- TTTCTGCTCGAATTCAAGCTTCTAACGATGTACGGGGACACATG-3′; Linker 2, 5′- TGTCCCCGTACATCGTTAGAACTACTCGTACCATCCACAT-3′; Y linker primer, 5′- CTGCTCGAATTCAAGCTTCT-3′; Tn5 primer, 5′- GGCCAGATCTGATCAAGAGA-3′. The genomic DNA was prepared using ISOPLANT (Nippon gene, Toyama, Japan). The Y linker was designed to have a 3′ overhang complementary to the sticky end generated by the *Nla* III or *Sph* I. In the present study, *Sph* I (Toyobo, Osaka, Japan) was used for digestion of the genomic DNA. Polymerase chain reactions (PCR) were carried out using the Program Temp Control System (ASTEC, Fukuoka, Japan) and TaKaRa Ex Taq Hot Start Version (Takara Bio, Shiga, Japan). Thermal cycling conditions were as follows: initial denaturing was car‐ ried out for 2 min at 95˚C followed by 30 cycles of denaturing (30 s at 95˚C), annealing (30 s at 60˚C) and extension (90 s at 70˚C). After the final cycle an extension step of 5 min at 70˚C was added. The PCR products were analyzed on a 3% agarose gel (Nacalai Tesque, Kyoto, Japan) and stained with ethidium bromide (Nacalai Tesque, Kyoto, Japan).

**Cloning, DNA sequencing and sequence analysis:** The PCR products were cloned with pGEM-T Easy Vector System (Promega, Madison, WI, USA) (Table 9). Cloned DNA frag‐ ments were sequenced commercially by Macrogen (Seoul, Korea). Homology searches were carried out using the BLASTN program in the RhizoBase, which is a database for the ge‐ nome of *B. japonicum* USDA110 (http://www.kazusa.or.jp/rhizobase/Bradyrhizobium/).

#### **5.2. Results and discussion**

and then suspended into 50 µL of HM salt medium. Equal volumes (50 µL) of *E. coli* donor and *B. japonicum* recipient cells prepared as described above were mixed in a 1.5-mL eppen‐ dorf tube. The mating mixture was spread onto a cellulose acetate membrane filter (Advan‐ tec, Tokyo, Japan; pore size 0.45 µm). The mating was carried out on HM plates at 30˚C. The incubation time of the mating was 2 days. After mating, cells were suspended in 1 mL of HM salts medium and 100 µL aliquots of this suspension were spread onto 10 HM plates.

A Comprehensive Survey of International Soybean Research - Genetics, Physiology, Agronomy and Nitrogen

Strain and plasmid Relevant chracteristics Reference or source

Table 9. Bacterial strains and plasmids used in this study

 Is-1 Incompatible with *Rj*2*Rj*3 soybean and compatible with *Rj*4 Ishizuka et al. (1991a) 1C1 Is-1::Tn*5*, Km<sup>r</sup> This study 1C2 Is-1::Tn*5*, Kmr This study 5C1 Is-1::Tn*5*, Kmr This study

 S17-1 *pro thi recA hsdR*; chromosomal RP4-2(Tn*l*::ISRI *tet*::Mu Km::Tn7) Simon et al. ( ) 1983 JM109 *rec*A1; cloning strain Takara Bio, Shiga, Japan

ed in 20 mL of HM medium and cultivated for 7 days at 30˚C. These 10 cultures containing

These 10 suspensions were separately inoculated onto CNS (*Rj*2*Rj*3) at a rate of 5 mL per seed in each pot. The pots and seeds were prepared as follows. Both vermiculite (2.8 L) and MHN solution (1.4 L) were added to a 3.0-L porcelain pot and autoclaved at 121˚C for 20 min. The seeds were sterilized with 5% sodium hypochlorite for 5 min and then rinsed with ethanol five times, and then rinsed with MHN solution five times. The surface-sterilized seeds of CNS were planted in a vermiculite medium at a rate of 12 seeds per pot. The inocu‐ lated plants were grown in a phytotron controlled at 25 ± 1˚C under natural light conditions and sterilized water was supplied to maintain the moisture content. After 4 weeks of culti‐ vation, Tn5 mutants were isolated from nodules produced on the root of CNS. The nodules were washed to remove vermiculite and immersed in 95% ethanol for 1 min, and then in 5% hydrogen peroxide solution for 5 min. The sterilized nodule was crushed and suspended in 5 mL of autoclaved 0.9% saline water. A drop of the turbid suspension was transferred to a yeast extract mannitol agar (YMA) plate [16] containing Congo Red (25 µg mL–1) and

 transconjugants were separately spun down for 10 min at 6,000 × g and then rinsed twice with half-strength modified Hoagland nutrient (MHN) solution [53] to remove the an‐

y 6C1 Is-1::Tn*<sup>5</sup>*, Kmr This study 7C1 Is-1::Tn*5*, Kmr This study 7C2 Is-1::Tn*5*, Kmr This study 10C1 Tn*5*-induced spontaneous mutant, Kmr This study 10C2 Is-1::Tn*5*, Kmr This study

) transconjugants.

, Apr Herrero et al. 1990

transconjugant colonies in each HM plate were suspend‐

transconjugants was suspended in 60 mL of MHN solution.

Kanamycin was added to select for kanamycine-resistant (Kmr

pUTKm Tn5-based delivery plasmid with Km<sup>r</sup>

streaked. These plates were incubated for 7 days at 30˚C.

**Table 9.** Bacterial strains and plasmids used in this study.

**Isolation of Tn5 mutants:** All Kmr

tibiotic. Each pellet of Kmr

*Bradyrhizobium japonicum*

Relationships

102

*Escherichia coli*

Km, kanamycin; Amp, ampicillin.

Plasmid

Kmr

Eight nodules and two popcorn-like nodules were produced on cv. CNS (*Rj*2*Rj*3) inoculated with the Kmr transconjugants. Tn*5* mutants (1C1, 1C2, 5C1, 6C1, 7C1, 7C2, 10C1 and 10C2) were isolated from the eight nodules and no Tn*5* mutants were isolated from the popcornlike nodules. The nodulation type [13] of Tn*5* mutants was determined with CNS (*Rj*2*Rj*3) and Hill (*Rj*4). All of them nodulated CNS effectively and retained the ability to nodulate Hill effectively [55]. Nodule effectiveness was judged by whether or not the nodule section indicated a section of red color. All Tn5 mutants were classified into nodulation type A. Al‐ though all nodules formed by Tn*5* mutants indicated a red color section, the nitrogen fixa‐ tion activities were different for each one. In particular, the nitrogen fixation activities of 6C1 and 7C1 were drastically low. The nodulation profile test was carried out with D-51 (*Rj*3), Hardee (*Rj*2*Rj*3) and IAC-2 (*Rj*2*Rj*3). All Tn*5* mutants nodulated both the *Rj*2*Rj*3- and the *Rj*3 cultivars effectively.

The results of the electrophoresis of the PCR products showed that all mutants except for 10C1 contained a single copy of Tn*5* and that the fragment size of each PCR product was different. The PCR product amplified from 10C1 was not detected [55]. These results are equal to those obtained by genomic southern blot analysis (data not shown).

A Comprehensive Survey of International Soybean Research - Genetics, Physiology, Agronomy and Nitrogen Relationships 104


† The location in the genome of USDA110 homologous to the Tn*5* flanking sequence is shown. ‡ The gene of USDA110 that contains the homologous sequence to the Tn*5*-flanking sequence inside or upstream of itself is shown. § The location in the genome of USDA110 homologous to the Tn*5* flanking sequence of 1C2 is upstream of *bsr7468*.

**Table 10.** Results of the blast searches of Tn*5*-flanking sequences based on RhizoBase.

The results of blast searches of cloned PCR products against the RhizoBase revealed that all homologous sequences to Tn*5*-flanking sequences were found in the complete genome se‐ quence of *B. japonicum* USDA110 [56] (Table 10). All of them were located in different re‐ gions in the genome of *B. japonicum* USDA110 and all of them except for one in mutant 1C2 are within the open reading frames predicted by the USDA110 genome sequence project (Table 10). Using the "plasmid rescue" method [57], we have already determined Tn*5*-flank‐ ing sequences longer than the ones found in this report [58]. These sequences have been de‐ posited into the DNA Data Bank of Japan (DDBJ) database (sequential accession numbers AB243409 through to AB243415].

*B. japonicum* strain USDA110 has an ability to form root nodules on soybeans (*Glycine max* L. Merr.) and is superior at symbiotic nitrogen fixation with soybeans compared with other strains [2]. The complete genome sequence of this strain has been determined [56]. This se‐ quence information is a useful tool to determine the mechanism of nodulation and nitrogen fixation in *B. japonicum*. The effect of rhizobial inoculation depends largely on the nitrogen fixation ability of the strain that forms most of the nodules. However, in soils containing in‐ digenous bradyrhizobia, the inoculation of highly effective rhizobia does not always result in the formation of effective nodules and in high nitrogen fixation [59]. To improve the effec‐ tiveness of the inoculation, the molecular genetic information of various strains, as well as USDA110, is believed to be required. The *B. japonicum* strain Is-1 nodulated Hill (*Rj*4) but did not nodulate CNS (*Rj*2*Rj*3) and was classified into the nodulation type B. Although the *gsn* genes have been reported [46, 60], there have been no reports of the identification of an *Rjgsn* gene. In this study, Tn*5* mutants were isolated from nodules produced on CNS inoculat‐ ed with the Kmr transconjugants. It is confirmed that these mutants are derived from Is-1 using amplified fragment length polymorphism (AFLP) analysis [61].

Tn*5*-flanking sequences were specifically amplified from Tn*5* mutants except for 10C1 [58]. These results show that all mutants except for 10C1 contained a single copy of Tn*5* and that each Tn*5* insertion site was different and that 10C1 is probably a spontaneous kanamycinresistant mutant. Wei and Bauer [62] also reported that Tn*5*-induced mutants with different phenotypes from the wild strain did not contain Tn*5* insertion. The analyses of these sequen‐ ces showed that all Tn*5* mutants contained a single copy of Tn*5* and that each Tn*5* insertion site was different. There was no clear relationship among the Tn*5*-inserted gene products. However, most of the Tn*5*-inserted gene products related to the cell membrane structure (e.g. the probable rare lipoprotein A, cold shock protein, the transcriptional regulatory pro‐ tein ArsR family, integral inner-membrane metabolite transport protein and putative inte‐ gral membrane protein) (Table 10). The probable rare lipoprotein A, integral innermembrane metabolite transport protein and putative integral membrane protein are a part of the membrane structure. Meanwhile, it is thought that cold shock protein and the tran‐ scriptional regulatory protein ArsR family may be indirectly related to the membrane struc‐ ture. The membrane-bound pump is regulated by the ArsR family [63] and the relationship between cold shock protein and membrane composition was reviewed by Ulusu et al. [64]. Therefore, changes or damage to the cell membrane structure in mutants may overcome the nodulation restriction conditioned by *Rj*2-soybean. Judd et al. [65], however, reported that the transposon was not responsible for host range extension in the Tn*5* mutant of USDA 438. In the future, we must confirm that the Tn*5* insertion is responsible for the acquisition of the ability to nodulate *Rj*2-soybean.

Tn5 mutant Length Identity Location† Gene ‡ Deduced gene product 1C1 773 769/773(99%) 5009532–5010304 b *bll4521* Probable rare lipoprotein A 1C2§ 1484 1478/1484(99%) 8203627–8205110 b *bsr7468* Cold shock protein

7C1 1896 1892/1896(99%) 6149473–6151368 b *bll5593* Unknown protein

**Table 10.** Results of the blast searches of Tn*5*-flanking sequences based on RhizoBase.

using amplified fragment length polymorphism (AFLP) analysis [61].

The location in the genome of USDA110 homologous to the Tn*5* flanking sequence is shown. ‡

sequence to the Tn*5*-flanking sequence inside or upstream of itself is shown. §

flanking sequence of 1C2 is upstream of *bsr7468*.

AB243409 through to AB243415].

ed with the Kmr

†

Relationships

104

 5C1 756 752/756(99%) 1540572–1541327 b *blr1414* Transcriptional regulatory protein ArsR family 6C1 227 227/227(100%) 1308155 1307929 b *bll1193* I l i b b li i

A Comprehensive Survey of International Soybean Research - Genetics, Physiology, Agronomy and Nitrogen

 7C2 2092 2070/2092(98%) 6353378–6355469 b *blr5786* Putative integral membrane protein 10C2 2092 2070/2092(98%) 6353378–6355469 b *blr5786* Putative integral membrane protein

Table 10. Results of the blast searches of Tn5-flanking sequences based on RhizoBase

1308155–1307929 Integralinner membrane metabolite transport protein

The results of blast searches of cloned PCR products against the RhizoBase revealed that all homologous sequences to Tn*5*-flanking sequences were found in the complete genome se‐ quence of *B. japonicum* USDA110 [56] (Table 10). All of them were located in different re‐ gions in the genome of *B. japonicum* USDA110 and all of them except for one in mutant 1C2 are within the open reading frames predicted by the USDA110 genome sequence project (Table 10). Using the "plasmid rescue" method [57], we have already determined Tn*5*-flank‐ ing sequences longer than the ones found in this report [58]. These sequences have been de‐ posited into the DNA Data Bank of Japan (DDBJ) database (sequential accession numbers

*B. japonicum* strain USDA110 has an ability to form root nodules on soybeans (*Glycine max* L. Merr.) and is superior at symbiotic nitrogen fixation with soybeans compared with other strains [2]. The complete genome sequence of this strain has been determined [56]. This se‐ quence information is a useful tool to determine the mechanism of nodulation and nitrogen fixation in *B. japonicum*. The effect of rhizobial inoculation depends largely on the nitrogen fixation ability of the strain that forms most of the nodules. However, in soils containing in‐ digenous bradyrhizobia, the inoculation of highly effective rhizobia does not always result in the formation of effective nodules and in high nitrogen fixation [59]. To improve the effec‐ tiveness of the inoculation, the molecular genetic information of various strains, as well as USDA110, is believed to be required. The *B. japonicum* strain Is-1 nodulated Hill (*Rj*4) but did not nodulate CNS (*Rj*2*Rj*3) and was classified into the nodulation type B. Although the *gsn* genes have been reported [46, 60], there have been no reports of the identification of an *Rjgsn* gene. In this study, Tn*5* mutants were isolated from nodules produced on CNS inoculat‐

Tn*5*-flanking sequences were specifically amplified from Tn*5* mutants except for 10C1 [58]. These results show that all mutants except for 10C1 contained a single copy of Tn*5* and that each Tn*5* insertion site was different and that 10C1 is probably a spontaneous kanamycinresistant mutant. Wei and Bauer [62] also reported that Tn*5*-induced mutants with different phenotypes from the wild strain did not contain Tn*5* insertion. The analyses of these sequen‐ ces showed that all Tn*5* mutants contained a single copy of Tn*5* and that each Tn*5* insertion site was different. There was no clear relationship among the Tn*5*-inserted gene products. However, most of the Tn*5*-inserted gene products related to the cell membrane structure

transconjugants. It is confirmed that these mutants are derived from Is-1

The gene of USDA110 that contains the homologous

The location in the genome of USDA110 homologous to the Tn*5*
