Symbiotic characteristics of Bradyrhizobium diazoefficiens USDA 110 mutants associated with shrubby sophora (Sophora flavescens) and soybean (Glycine max)

Microbiological Research 214 (2018) 19–27

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Symbiotic characteristics of Bradyrhizobium diazoefficiens USDA 110 mutants associated with shrubby sophora (Sophora flavescens) and soybean (Glycine max)

T

Yuan Hui Liua,b, En Tao Wangc, Yin Shan Jiaoa,b, Chang Fu Tiana,b, Lei Wanga,b, Zi Jian Wanga,b, ⁎ Jia Jing Guana,b, Raghvendra Pratap Singhd, Wen Xin Chena,b, Wen Feng Chena,b, a

State Key Laboratory of Agrobiotechnology, Beijing 100193, China College of Biological Sciences and Rhizobium Research Center, China Agricultural University, Beijing 100193, China Departamento de Microbiología, Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, México D. F. 11340, México d Microbial Genomics Laboratory, National Bureau of Agriculturally Important Microorganisms, Maunath Bhanjan, Uttar Pradesh 275101, India b c

A R T I C LE I N FO

A B S T R A C T

Keywords: Bradyrhizobium diazoefficiens USDA 110 Sophora flavescens Nodulation Tn5 mutation Soybean (Glycine max) Symbiotic characterization

Site-specific insertion plasmid pVO155 was used to knockout the genes involved in the alternation of host range of strain Bradyrhizobium diazoefficiens USDA 110 from its original determinate-nodule-forming host soybean (Glycine max), to promiscuous and indeterminate-nodule-forming shrubby legume sophora (Sophora flavescens). Symbiotic phenotypes of these mutants inoculated to these two legumes, were compared to those infected by wild-type strain USDA 110. Six genes of the total fourteen Tn5 transposon mutated genes were broken using the pVO155 plasmid. Both Tn5 and pVO155-inserted mutants could nodulate S. flavescens with different morphologies of low-efficient indeterminate nodules. One to several rod or irregular bacteroids, containing different contents of poly-β-hydroxybutyrate or polyphosphate were found within the symbiosomes in nodulated cells of S. flavescens infected by the pVO155-inserted mutants. Moreover, none of bacteroids were observed in the pseudonodules of S. flavescens, infected by wild-type strain USDA 110. These mutants had the nodulation ability with soybean but the symbiotic efficiency reduced to diverse extents. These findings enlighten the complicated interactions between rhizobia and legumes, i. e., mutation of genes involved in metabolic pathways, transporters, chemotaxis and mobility could alter the rhizobial entry and development of the bacteroid inside the nodules of a new host legume.

1. Introduction Sophora flavescens, the shrubby sophora (“Ku Shen” in Chinese), is a medicinal leguminous herb, whose roots secrete nitrogen-containing sophocarpidine or matrine, which could be used as insecticide or antihepatoma agents. This legume can establish effective symbioses with various rhizobia belonging to different genera and cross-nodulation groups (Jiao et al., 2015) including with Sinorhizobium fredii but not with Bradyrhizobium diazoefficiens, two well-known soybean-nodulating rhizobia (Zhang et al., 2011; Tian et al., 2012; Delamuta et al., 2013). Although the identical structures of lipochitooligosaccharides (LCOs) secreted by these two rhizobial species may endow nodulation on the common host, soybean (Glycine max), they still have their distinct symbiotic partners. Comparatively, S. fredii presents broader host ranges forming either indeterminate nodules on Chinese liquorice

(Glycyrrhiza uralensis) (Crespo-Rivas et al., 2016), pigeonpea (Cajanus cajan) (Li et al., 2015) and shrubby sophora (S. flavescens) (Jiao et al., 2015); or determinate nodules on wild soybean (Glycine soja) (Scholla and Elkan, 1984; Wu et al., 2011), cowpea (Vigna unguiculata) (Scholla and Elkan, 1984), bird's-foot trefoils (Lotus burttii) (Acosta-Jurado et al., 2016) as well as ineffective symbiosis with purple bush-bean (Macroptilium atropurpureum), phasey bean (Macroptilium lathyroides), prickly sesban (Sesbania cannahina), mung bean (Vigna radiata), and different cultivars of soybean (Keyser et al., 1982). While B. diazoefficiens is specific for purple bush-bean, cowpea and mung bean (Göttfert et al., 1990; Lardi et al., 2016) besides soybean as its limited partners usually forming determinate nodules. Affiliated to Bradyrhizobium japonicum, wild-type strain USDA 110 was originally isolated from root nodules of soybean grown in Florida, USA (Mathis et al., 1997) and was renamed as B. diazoefficiens by

⁎ Corresponding author at: State Key Laboratory of Agrobiotechnology, Beijing, 100193, China; College of Biological Sciences and Rhizobium Research Center, China Agricultural University, Beijing 100193, China. E-mail address: [email protected] (W.F. Chen).

https://doi.org/10.1016/j.micres.2018.05.012 Received 9 December 2017; Received in revised form 9 April 2018; Accepted 14 May 2018 0944-5013/ © 2018 Elsevier GmbH. All rights reserved.

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2.3. Nodulation characterization of wild-type strain and mutants

Delamuta et al. (2013). Strain USDA 110 is one of the most agriculturally important N2-fixing bacterium associated with soybean (Narozna et al., 2015) and it has been widely employed as a type strain in various studies of genetics, genome (Kaneko et al., 2002), and molecular interaction and host specificity with soybean (van Berkum and Fuhrmann, 2009; Quelas et al., 2010; Ledermann et al., 2015). Previous reports have suggested that soybean controls the specific nodulation with Bradyrhizobium or Sinorhizobium by specific genes (Yang et al., 2010; Fan et al., 2017), while several genes in rhizobia also compete the soybean cultivar compatibility (Bellato et al., 1997; Annapurna and Krishnan, 2003). In our most recent study (Liu et al., 2018), symbiotic promiscuity between S. flavescens and six different rhizobia was explored molecularly, and the functional nodules, formed on S. flavescens by Tn5 transposon mutagenesis of strain USDA 110, were reported. However, the genetic characterization of the mutated genes as well as the symbiotic properties of these Tn5 mutants in interacting with S. flavescens and soybean have not been studied in details. In addition, whether the pseudonodules induced by the wild-type USDA 110 and the indeterminate nodules induced by the mutants contained bacteroids or not are still not known. The aim of present study was to identify the mutated genes and analyze the symbiotic characteristics of the mutants of B. diazoefficiens on S. flavescens, and soybean, as well as to explore the potential factor (s) causing negative response from the host plant.

Surface sterilization and germination of S. flavescens (cultivar Zhenku No. 2) and soybean (cultivar Jidou No. 17) seeds were performed as reported by Jiao et al. (2015). Seedlings were transferred to a Leonard jar assembly containing sterile vermiculite moistened with low-nitrogen plant nutrient solution (Vincent, 1970). Each treatment consisted of 20 plants, with one plant per jar. Each plant was inoculated with 1 mL of rhizobial or their Tn5 or pVO155 mutant culture (concentration of OD600 = 0.2, diluted with 0.8% (w/v) NaCl). Nodulation test was repeated three times. Plants were grown in a greenhouse under a 16-h light (25 °C)/8-h dark (16 °C) photoperiod, and were harvested for checking the phenotype from 26 to 45 days post-inoculation (dpi). Biological characteristics, including nodule number, fresh nodule weight, shoot dry weight and chlorophyll content, were determined and compared among plants inoculated with wild-type strain and the mutants. Leaf chlorophyll concentration was determined with a SPAD-502 plus Meter (Konica Minolta, Osaka, Japan) (Ling et al., 2011). Plant shoots were dried at 65 °C for 5 days and then weighed. The number of root nodules per plant was counted. To visualize the morphology of bacteroids inside nodules, paraffin section or ultrathin sections of nodules were prepared and observed under a light microscope or transmission electron microscope (TEM) (Model: JEM-1230, Tokyo, Japan) as described previously (Li et al., 2013).

2. Materials and methods

2.4. Motility assay

2.1. Bacterial strains and growth conditions

Wild-type strain USDA 110 and BDT-7pvo mutant (the flgE gene encoding for flagellar hook-basal body protein was knocked out) were grown in the TY broth (Beringer, 1974) to mid-log phase. The cell optical densities at 600 nm (OD600) of each culture was standardized, and equal amounts of inoculum were inoculated with a inoculating needle into a test tube containing TY soft agar (0.3%) medium. The motility and spread of the strains grown in the soft agar tube was observed 4 days after inoculation at 28 °C.

The bacterial strains, Tn5 mutants, and plasmids used in this study are listed in Supplementary Table S1. B. diazoefficiens USDA 110 was cultured at 28 °C on TY medium (Beringer, 1974) or yeast extract mannitol (YEM) medium (Vincent, 1970) supplemented with 10 μg mL−1 trimethoprim (TMP). Escherichia coli DH5α was grown at 37 °C in Luria–Bertani (LB) medium (Miller, 1972) supplemented with 50 μg mL−1 kanamycin (Km).

2.5. Growth determination 2.2. Identification of Tn5 inserted genes using pVO155 plasmid Wild-type strain USDA 110 and the BDT-28pvo mutant (the corA gene encoding magnesium transporter was mutated) were cultured on TY medium at 28 °C to mid-log phase, and then were inoculated into liquid defined minimal medium (Krol and Becker, 2004) supplemented with either 0.01 mM (Mg2+-limiting) or 1 mM (Mg2+-sufficient) MgSO4. The starting density (OD600) of the culture was adjusted to 0.05. The growth was measured using spectrophotometer. The growth phenotype of BDT-2pvo mutant (ppsA gene encoding for phosphoenolpyruvate synthase was knocked out) was tested in same liquid defined minimal medium (Krol and Becker, 2004), but the carbon source (mannitol) was replaced by pyruvic acid. Growth was detected same to the above method using spectrophotometer.

The construction of Tn5 mutants was described previously (Liu et al., 2018). To verify the symbiotic phenotypes caused by Tn5 transposon insertion, six genes encoding for phosphoenolpyruvate (PEP) synthase, hypothetical membrane protein, flagellar hook-basal body protein, NAD(P)H-quinone oxidoreductase, SH3 domain-containing protein and magnesium transporter, which were defined as BDT-2, BDT-4, BDT-7, BDT-10, BDT-18 and BDT-28, respectively, were mutated via single-crossover recombination using site-specific insertion plasmid pVO155 (Table 1 and Supplementary Table S1). To knockout these six genes, internal regions with different lengths of them were amplified respectively using corresponding primer pairs and PCR annealing conditions listed in Supplementary Table S2. Then, the obtained PCR product was ligated into digested pVO155 plasmid with XbaI and BamHI restriction endonucleases according to the manufacturer’s instructions of seamless clone kit (No. C5891, Clone Smarter Technologies Co.). The recombined plasmid was transformed into heat-shocked E. coli DH5α strains and triparentally conjugated with the wild-type strain USDA 110 and the helper strain DH5α containing pRK2013 (Kmr) plasmid. The recombined conjugants were selected on TY agar plates containing two antibiotics of Km (50 μg mL−1) and TMP (10 μg mL−1) simultaneously. Genetic modification in the single crossover was verified by PCR amplification, using the corresponding detection primer and universal primer M13R, listed in Supplementary Table S2, and DNA sequencing by Tsingke Biological Technology Co. Ltd. Sequence analyses and comparison were carried out referring to the whole genome of strain USDA 110 (GenBank No. BA000040.2) and related bacterial sequence data in GenBank database.

3. Results 3.1. Characterization of the mutated genes The inserted positions by Tn5 transposons in the 14 genes in USDA 110 genome (GenBank No. NZ_CP011360) was shown in Table 1 and Fig. 1. The locus, length, deduced gene product and percentage of amino acid identity using BLASTP were shown in Table 1. The amino acid sequences of the annotated genes had identities ranging from 55 to 100% with those deduced from other bacteria (Table 1). Based on the highest identity of each sequence, we proposed and revised the functions of these 14 genes (Table 1). Among them, functions of three mutated genes in mutants BDT-4, BDT-5 and BDT-16 were unknown and were annotated as hypothetical membrane protein and hypothetical proteins. Another three mutants (BDT-18, −19 and −22) were 20

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Table 1 Characterization of the Tn5 inserted genes. Strain

Locus_taga (gene)

Length (bp)

Annotation

BDT-2, BDT-2pvo

blr4655 (ppsA)

2430

Phosphoenolpyruvate synthase

BDT-4, BDT-4pvo

blr3848

420

Hypothetical membrane protein

BDT-5

bll6035

3948

Hypothetical protein

BDT-7, BDT-7pvo

bll5854 (flgE)

2265

Flagellar hook-basal body protein

BDT-9

bll0096

2076

Chemotaxis protein

BDT-10, BDT10pvo

bll1503 (qor)

999

NAD(P)H-quinone oxidoreductase

BDT-11

blr3961

1179

NAD(FAD)-utilizing dehydrogenase

BDT-16

bll2373

4362

Hypothetical protein

BDT-18, −19, −22, BDT18pvo

blr0767

531

SH3-like domain-containing protein

BDT-21

bll7899

BDT-25

BDT-28, BDT28pvo a b c

bll4072 (dnaC)

blr2622 (corA)

1785

1251

978

Isovaleryl-CoA dehydrogenase (IVD)

Replicative DNA helicase

Magnesium transporter

BLASTP analysis with closely related strains based on amino acid identity Strainb

Locus_tagc

Annotation

Identity

B. sp. 3(2017) B. sp. BTAi1 B. sp. ORS 285 B. diazoefficiens SEMIA 5080 B. sp. CCBAU 43298

CIT39_RS12655 BBTA_RS07450 BRAO285_RS32510 BJA5080_04836

Phosphoenolpyruvate synthase Phosphoenolpyruvate synthase Phosphoenolpyruvate synthase Hypothetical protein

93% 70% 69% 100%

IC50_RS35160

94%

B. sp. WSM1417 B. sp. 2(2017) B. sp. OK095 B. sp. BR10280 B. sp. Rc2d N. hamburgensis X14 B. sp. cf659

BRA1417_RS0120035 CIT40_RS06150 SAMN05443254_101805 A5906_RS39395 BLV07_RS44680 NHAM_RS06800 BM118_RS19400

B. sp. ORS 278 B. sp. WSM471

BRADO_RS07130 BRA471DRAFT_RS04505

B. sp. WSM2254 B. japonicum SEMIA 5079 A. sp. GAS231 B. sp. WSM2254

A3M7_RS0112135 BJS_08099

Nuclear transport factor 2 family protein Membrane protein Hypothetical protein VCBS repeat-containing protein Flagellar hook-basal body protein Flagellar hook-basal body protein Flagellar hook protein FlgE Methyl-accepting chemotaxis protein HAMP domain-containing protein Methyl-accepting chemotaxis protein NAD(P)H-quinone oxidoreductase Methionyl-tRNA synthetase

88% 93%

B. B. B. B. B.

BM118_RS05795 UB31_01205 BM118_RS23305 N554_RS0131470 TSA1_RS24715

NAD(P)H-quinone oxidoreductase Aminoacetone oxidase family FADbinding enzyme TIGR03862 family flavoprotein NAD(FAD)-utilizing dehydrogenase Hypothetical protein RHS repeat protein Aspartyl-tRNA synthetase (containing SH3-like domain) SH3-like domain-containing protein

80% 97%

sp. sp. sp. sp. sp.

cf659 LTSP849 cf659 URHD0069 TSA1

BLS26_RS13080 A3M7_RS0105990

90% 90% 80% 97% 94% 64% 98% 60% 92% 99% 97%

93% 87% 94% 55% 98%

B. canariense GAS369 B. sp. R5 B. sp. WSM2254

SAMN05444158_5318

B. canariense UBMA052 B. sp. G22 B. diazoefficiens SEMIA 5080 B. sp.

BSZ20_RS14840

SH3-like domain-containing protein Isovaleryl-CoA dehydrogenase (IVD) DNA alkylation response protein

BN2626_G22_1516 KGJ69623.1

Acyl-CoA dehydrogenase Putative replicative DNA helicase

95% 100%

WP_011086848.1

100%

B. sp. WSM2254 B. sp. TSA1 B. sp. LTSP857

A3M7_RS0124140 TSA1_RS34945 UP06_RS13055

Class I SAM-dependent methyltransferase Magnesium transporter Magnesium transporter Magnesium transporter

SAMN05216337_100595 A3M7_RS0107655

81%

96%

99% 96% 93%

The locus_tag is based on the genomic sequence of B. diazoefficiens USDA 110 (GenBank No. BA000040.2). B., Bradyrhizobium. N., Nitrobacter. A., Afipia. The locus_tag could be found in GenBank. Fig. 1. Physical maps of the inserted genes by Tn5 (inverted triangle) and pVO155 (diamond) in B. diazoefficiens USDA 110 genome. The inserted genes are surrounded by two adjacent genes. Direction of arrows indicated transcriptional direction of genes. Functions of the genes were shown under arrows. bar, 500 bp length.

21

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on soybean (Fig. 5). The leaf chlorophyll content (Fig. 5A) and shoot dry weight (Fig. 5B) of the soybean plants inoculated by these pVO155 mutants were not significantly different, except BDT-18pvo-induced plant that shoot dry weight decreased greatly. There was a significantly decrease in the number of nodules on soybean inoculated with mutants compared to the wild-type strain USDA 110, except of BDT-7pvo and BDT-28pvo (Fig. 5C). Nodule fresh weight induced by BDT-2pvo, BDT10pvo and BDT-18pvo also significantly reduced compared with those induced by BDT-4pvo, BDT-7pvo, BDT-28pvo and the wild-type strains (Fig. 5D).

found to be mutated in a single gene (locus at blr0767) and the gene was annotated automatically using computer prediction as “aspartyltRNA synthetase”. By BLAST searching of the protein sequence of this gene, we found it contained two SH3 domains (47–103 and 114–168 in amino acid sequence). These three Tn5 mutants (BDT-18, −19 and −22) had same insertion site and located at down-streaming of the gene (Fig. 1). Only 6 insertion mutants designated as BDT-2pvo, BDT-4pvo, BDT7pvo, BDT-10pvo, BDT-18pvo and BDT-28pvo, respectively, by pVO155 plasmid were obtained and their insertion positions were shown in Fig. 1. The positions of pVO155 inserted genes were confirmed to be in the same Tn5 mutated genes based on sequence comparisons but they had different insertion sites (Fig. 1). The mutated genes had their own individual promoters and most of their transcriptional directions were different from up/down-streaming genes (Fig. 1).

3.5. Phenotypes of the free-living of mutants Under free-living conditions, the mutant BDT-7pvo (mutation in flagellar hook-basal body protein) showed decreased motility in 0.3% soft agar TY medium with only a thin growth line along the inoculation area, whereas the wild-type strain USDA 110 exhibited diffused funnelshaped growth in the medium (Supplementary Fig. S4). The mutant BDT-28pvo (mutation in magnesium transporter) could not grow in both Mg2+ concentrations (0.01 mM and 1 mM Mg2+), while the wildtype strain USDA 110 grew normally in 1 mM Mg2+ concentration. Though the wild-type strain grew poorly in 0.1 mM Mg2+, it grew much better than the mutant strain (Supplementary Fig. S5). Both the BDT2pvo mutant (mutation in phosphoenolpyruvate synthase) and wildtype strain USDA 110 could grow in the pyruvic acid-containing medium, but there was no difference between them.

3.2. Symbiotic phenotype of Tn5 mutants on S. flavescens Different nodulation phenotypes of S. flavescens inoculated by these 14 Tn5 mutants were observed and compared with Student’s t-test. Wild-type strain USDA 110 only induced pseudonodules on the roots of S. flavescens and none of bacteroids were found inside the pseudonodules (Supplementary Fig. S1). With the exception of BDT-2, BDT-9 and BDT-28 that formed a few black senescent nodules, and BDT-21 that formed pink nodules, the other mutants formed white or light pink nodules on S. flavescens (Supplementary Fig. S2). The shapes of the root nodules varied from small round (2 mm in diameter) to long rob (4 mm in length). The shoot dry weight of the plant inoculated with the Tn5 mutants had no significant difference (except BDT-28) compared with the plants inoculated with the wild-type strain and the uninoculated control (Supplementary Fig. S3). The color of leaves of the plants inoculated with Tn5 mutants (except BDT-9, BDT-19, BDT-25 and BDT-28 mutants) were significantly greener than those inoculated with wildtype (WT) strain USDA 110 and the uninoculated control (Supplementary Fig. S2). Similarly, the chlorophyll contents of the plants inoculated with Tn5 mutants (except BDT-9, BDT-19, BDT-25 and BDT-28) were higher than the plants inoculated with WT strain and the control (Fig. 2A). The symbiotic phenotypes of the plants inoculated with three mutants of BDT-18, −19 and −22 displayed some differences though they were mutated in a common gene.

4. Discussion This study further investigated symbiotic properties of restrictive nodulation of B. diazoefficiens USDA 110 on S. flavescens by using the Tn5 and pVO155 inserted mutants. The wild-type strain USDA 110 secretes LCOs or the Nod factors structurally identical to those of S. fredii (Sanjuan et al., 1992; Bec-Ferte et al., 1994; Duzan et al., 2006), but only S. fredii is able to nodulate S. flavescens (Liu et al., 2018). This suggests that there may be some other regulative mechanisms in addition to structure of LCOs in strain USDA 110 to prevent its symbiotic establishment with S. flavescens. This wild-type strain formed small pseudonodules on S. flavescens without bacteroid inside (Supplementary Fig. S1) meaning the LCOs signals produced by strain USDA 110 could be recognized by the plant S. flavescens and therefore stimulating the formation of nodule primordium (Rightmyer and Long, 2011). But its further entry into the nodule cell may be inhibited by some negative regulators. The formation of bigger nodules on S. flavescens by the Tn5 and pVO155 mutants suggested that genes involved in negative regulation had been disrupted. The identification of these negative regulatory genes in strain USDA 110 provides novel insights into the genes related to the function of host-specific nodulation (hsn) (Kondorosi et al., 1984). Our findings can also explain the previous results stating that gene mutations could lead to an expanded host range (Brewin et al., 1980). Three Tn5 insertion mutants (BDT-18, BDT-19, and BDT-22) were found in common gene encoding for a protein with two Src homology 3 domains (or SH3 domains), each containing about 60 amino acid residues. Although, SH3 domain was described firstly for the conserved sequence in a viral adaptor protein, it is also found in phospholipase and some cytoplasmic tyrosine kinases. SH3 domain-containing proteins are believed to recognize and interact with proline-rich sequence motifs (Weng et al., 1995). SH3 domain was widely involved in the intracellular signaling transduction and was predicted to contribute to the pathogen-host interaction (Ponting et al., 1999). Few reports described the exact function of the SH3 domain in rhizobia (Xi et al., 2000) while this domain has been studied extensively in other bacteria. Report ever showed that the SH3 domain engaged an adaptor which facilitated the infection of the pathogen Listeria monocytogenes on the mammalian host via changing the host signaling transduction

3.3. Symbiotic phenotype of pVO155 inserted mutants on S. flavescens Of the 14 Tn5 mutants, 6 of them were confirmed using site-specific plasmid pVO155. The symbiotic phenotypes of S. flavescens inoculated by these 6 pVO155 inserted mutants were shown in Figs. 2B–D and 3 . The nodules formed by these pVO155-inserted mutants on S. flavescens showed different shape, size, number and weight (Fig. 3). The mutants of BDT-2pvo, BDT-7pvo and BDT-10pvo formed light pink nodules while the BDT-4pvo, BDT-18pvo and BDT-28pvo produced white nodules on the S. flavescens (Fig. 3). The shoot dry weight, nodule number per plant and leaf chlorophyll content of plants inoculated with pVO155 inserted mutants showed a tendency of higher (not significant) than those inoculated with the wild-type strain (Figs. 2B–D and 3). The ultrathin sections of nodules formed by pVO155 insertion mutants on S. flavescens under TEM showed that many bacteroids were observed (Fig. 4). One to several bacteroids was/were found inside each symbiosome (Fig. 4). Different size of clavate or irregular bacteroids containing inclusions of poly-β-hydroxybutyrate (PHB) (white particles) or polyphosphate (PP) (black particles or aligned into line) were found inside the nodules infected by the pVO155 insertion mutants (Fig. 4). 3.4. Nodulation characterization on soybean These pVO155 insertion mutants did not affect the nodule formation 22

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Fig. 2. Symbiotic properties of S. flavescens inoculated with B. diazoefficiens USDA 110 wild type (WT), Tn5 and pVO155 inserted mutants. A, Tn5 mutants. B, C, D, pVO155 inserted mutants. Student’s t-test, *, p < 0.05; **, p < 0.01.

more preponderant mutualism with the host plant (Mellor et al., 1987; Caetano-Anollés et al., 1988). Moreover, flagellin served as general elicitor involved in stimulating innate immune responses in thale cress (Arabidopsis) (Abramovitch et al., 2006). Previous study indicated that amino-acid variations in flagellin allow Xanthomonas campestris pv. campestris to avoid pattern recognition receptors (PRRs)-mediated detection by Arabidopsis (Sun et al., 2006). Flg22, a 22-amino acid peptide conserved in the N-terminus of bacterial flagellin, was found to be recognized by the Arabidopsis FLS2 leucine-rich repeat receptor (LRR) (Gomez-Gomez et al., 1999; Gomez-Gomez and Boller, 2000), and Arabidopsis that lacks LRR is more susceptible to Pseudomonas infection when the pathogen is sprayed on plant leaves (Zipfel et al., 2004). Flagellum hook protein-encoding gene flgE of Mesorhizobium tianshanense was reported to be critical for the recognition and infection in the initial stage of the symbiosis (Zheng et al., 2015). When Sinorhizobium meliloti strain Sm2011 and Pseudomonas syringae pv. tomato were co-inoculated to Medicago truncatula, strain Sm2011 specifically

(Dokainish et al., 2007). In our current study, three Tn5 inserted mutants (BDT-18, −19, −22) were obtained, confirming the importance of this gene in determining the rhizobial entry to nodule cells of S. flavescens, though the symbiotic phenotypes of them displayed some differences because of unknown reasons. Disruption of this gene by pVO155 insertion (BDT-18pvo) in another site of the gene produced same function endowing the mutant the capability of entry to nodule cells of S. flavescens. The potential role of SH3 domain-containing protein in inhibiting the infection and entry of rhizobia into nodule cells of a new legume host is needed to be explored further in the future. Two of the S. flavescens-nodulating mutants were identified to be related to the flagellum hook protein (BDT-7) and chemotaxis (BDT-9) genes. On the one hand, the flagellum hook protein and chemotaxis protein are involved in bacterial motility (Supplementary Fig. S4), which is crucial for bacteria to colonize and invade the hosts. Previous studies have shown that motile rhizobium have obvious advantages in symbiosis compared to nonmotile one (Soby and Bergman, 1983) or is

Fig. 3. Plant shoot, roots and nodules of S. flavescens inoculated with B. diazoefficiens USDA 110 and pVO155 insertion mutants. Plants were photographed 40 days after inoculation (dai). Bars for shoot and root, 1 cm; bars for nodule section, 1 mm. 23

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Fig. 4. Transmission electron microscope (TEM) image of root nodule section of S. flavescens inoculated with pVO155 insertion mutants. Poly-β-hydroxybutyrate (PHB) particle was marked with black solid arrow. Polyphosphate (PP) was indicated with white arrow. Bars, 2 μm. Fig. 5. Symbiotic properties of soybean inoculated with B. diazoefficiens USDA 110 and pVO155 insertion mutants. (A) chlorophyll content, (B) shoot dry weight, (C) number of nodules and (D) weight of fresh nodules measured 35 days after inoculation. WT, wild-type strain USDA 110. Control, no inoculation. Student’s t-test, *, p < 0.05; **, p < 0.01.

suppressed the transcription of flagellin-related defense responses induced by P. syringae pv. tomato (Chen et al., 2017). Based upon the results in our present study, flagellin of strain USDA 110 may elicit innate immune responses from S. flavescens. So mutation of flagellum hook protein in strain USDA 110 can avoid immune responses from S. flavescens, resulting in the entry into nodule cells of S. flavescens. While flagellin of strain USDA 110 may coevolve with recognition receptors in soybean, which leads to symbiotic compatibility between them. The identification of flagellin recognition receptors and detailed mechanism involved in preventing wild-type strain USDA 110 from entry into nodule cells of S. flavescens is interesting to be uncovered. Mutant BDT-28 was identified as a mutation in corA gene encoding for Mg2+ transporter. Four types of Mg2+ transports, CorA, MgtA/B and MgtE, in prokaryotes have been found (Kobayashi and Tanoi,

2015). Of these transports, CorA and MgtE are the most widespread and considered as primary Mg2+ transporters in bacteria (Armitano et al., 2016). Since Mg2+ is crucial to the functions of many enzymes including nitrogenase and energy ATP, it is easy to speculate how this mutation leads to the formation of lower efficient N2-fixing nodules (Fig. 3 and Supplementary Fig. S2). As reported, a miniTn5 mutant of strain Rhizobium RU4107 in a gene of mgtE-like failed to fix nitrogen (Fix−) on pea (Pisum sativum) (Karunakaran et al., 2009). Legume hairy vetch (Vicia hirsute) inoculated with MgtE mutant of Rhizobium leguminosarum presented significantly reduced N2-fixation efficiency compared to that inoculated with wild-type strain (Hood et al., 2015). In addition, MgtE in pathogenic bacterium Borrelia burgdorferi, the causative agent of Lyme disease, was relevant to its virulence (Aron et al., 1996). And lacking of corA gene in Salmonella enterica serovar 24

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(Abramovitch et al., 2006). Chemotaxis protein and flagellum hook protein is possibly involved in stimulating innate immune responses in legumes. Flagellin was reported to affect the development and extension of symbiosis in Mesorhizobium tianshanense (Zheng et al., 2015). LCOs secreted by rhizobia can suppress the immune system of legume to permit the rhizobia to enter the nodule cells (Gourion et al., 2015). On the contrary, the original products of the mutated genes in current study may activate the immune system of S. flavescens preventing the invasion of the wide-type strain USDA 110. Abortion of the products of the gene mutants gives their chance to invade the nodule cells successfully.

Typhimurium was attenuated in mice and also defective for invasion and replication within Caco-2 epithelial cells (Papp-Wallace et al., 2008). In our current study, we found the Mg2+ transporter mutants (BDT-28pvo) could form nodules (Fig. 3) on S. flavescens with bacteroids inside the nodule cells (Fig. 4), while the N2-fixation efficiency was lower characterized by white color of nodule section and lower chlorophyll content. Therefore, the function of Mg2+ transporter in strain USDA 110 may has many aspects, regulating the Mg2+ absorption, influencing the cell growth under free-living condition (Supplementary Fig. S5), changing the invasion in a new host, and reducing the nitrogen fixation in symbiotic nodules. Four mutants obtained in this study had the Tn5 insertion in genes encoding for NAD(P)H-quinone oxidoreductase (BDT-10), NAD(FAD)utilizing dehydrogenase (BDT-11), isovalery-CoA dehydrogenase (BDT21) and phosphoenolpyruvate synthase (BDT-2). The common features of them were that all were involved in the energy metabolism or electron transport. Sufficient ATP is needed to ensure the reduction of N2 into ammonia (Yang et al., 2011). Rhizobia begin to fix nitrogen by expressing the ATP-requiring nitrogenase in the differentiated bacteroid (Fischer, 1994). Pyruvic acid has a dual role of ATP-generator and a reductant in the nitrogenase system (Hardy and D'Eustachio, 1964). The inactivation of phosphoenolpyruvate synthase (BDT-2 and BDT-2pvo) may be complemented by other anaplerotic pathways to produce PEP from pyruvic acid, therefore both BDT-2pvo mutant and wild-type strain could grow in pyruvic acid-containing defined medium under free-living conditions. Other reports showed that phosphoenolpyruvate synthase and NAD(P)H-quinone oxidoreductase was linked to bacterial virulence and host immunity (Ryan et al., 2014; McCormick and Jakeman, 2015). Thus, we speculate that these metabolism-related genes may regulate the infection and N2-fixing ability of strain USDA 110, and resulted in the entry and formation of inefficient N2-fixing nodules on the S. flavescens. Mutant BDT-25 was found in insertion of dnaC gene encoding for putative replicative DNA helicase (bll4072 in USDA 110; KGJ69623.1 in B. diazoefficiens SEMIA 5080). It was ever reported that the expression of the dnaC gene was down-regulated in the non-growing state of nitrogen-fixing bacteroids of strain Sinorhizobium sp. NGR234 (Li et al., 2013). The function of this replicative DNA helicase in rhizobial interaction with legume has not yet been explored. In addition, the roles of three hypothetical proteins (BDT-4, BDT-5 and BDT-16) were not clear. More interestingly, we found different effects of these mutated genes on nodulation of S. flavescens and of the original host soybean. The mutated genes involved in metabolism may act as negative regulator in symbiosis with S. flavescens, while they served as positive regulator when inoculated to soybean. One possible explanation is that different pathway or different symbiotic mechanism of one gene functions in the two legumes. For the same reason, different symbiotic phenotypes exist in a single strain associated with different host legumes. For example, Mg2+ transporter is required for N2-fixation in nodules of pea (Pisum sativum) and hairy vetch (Vicia hirsute), but not in nodules of broad bean (V. faba) (Hood et al., 2015). It is still remained to be resolved how the rhizobial mutants escape the plant defense systems and develop symbiotic interaction with S. flavescens. It is known that host plant can activate innate immune response when subjected to microbial invasion (Ausubel, 2005). Microbial factors belonging to microbe-associated molecular patterns (MAMPs) were identified as general or host-specific elicitors in plants (Abramovitch et al., 2006; Cao et al., 2017). General elicitors are conserved in bacteria and include flagellin, elongation factor Tu (EFTu), cold shock protein (CSP), chitin and lipopolysaccharide (LPS) (Shibuya and Minami, 2001; Zeidler et al., 2004; Boller, 2005). Plants respond to general elicitors with basal defense system which regulate metabolism pathways or change the signaling cascades (Abramovitch et al., 2006). Specially, most host-specific elicitors lead to hypersensitive response or cell death by injecting effector proteins into plant cells

5. Conclusions In conclusion, the potential mechanism underlying the symbiotic incompatibility between the wild-type strain USDA 110 and S. flavescens was studied by Tn5 insertion mutant screening and the identification of the insertion sites using pVO155 plasmid. Wild-type strain USDA 110 only induce pseudonodules on S. flavescens with no bacteroid inside, while the gene mutants related to metabolism, transportation, regulation, chemotaxis and mobility extend the host range from soybean to promiscuous legume S. flavescens, forming various indeterminate nodules with bacteroids inside though the N2-fixation efficiency was lower. Genes involved in these pathways may act as the negative factors rejecting the entry of wild-type strain USDA 110 into nodule cell of S. flavescens. The original products encoded by these mutated genes may be immunoactivators to stimulate the plant to inhibit rhizobial infection into the nodule cells. The disruption of these genes may endow the mutants to escape the MAMPs-triggered immunity from the host plant resulting in the entry of less-virulent rhizobial strains into the nodule cells (Denison and Kiers, 2004). How do the mutations lead to the entry and infection, bacteroid development inside nodule cells, and balanced regulation of defense immunity from the host plant need to be further explored in the future. Acknowledgements This work was financially supported by the National Natural Science Foundation of China (31770039), the National Basic Research Program of China (973 program 2015CB158300), URP and Innovation Program for Undergraduates from CBS and China Agricultural University. Thanks for Shangying Wu for kind providing seeds of Ku Shen (Sophora flavescens) (cultivar Zhenku No. 2). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.micres.2018.05.012. References Abramovitch, R.B., Anderson, J.C., Martin, G.B., 2006. Bacterial elicitation and evasion of plant innate immunity. Nat. Rev. Mol. Cell Biol. 7 (8), 601–611. Acosta-Jurado, S., Rodríguez-Navarro, D.-N., Kawaharada, Y., Perea, J.F., Gil-Serrano, A., Jin, H., An, Q., Rodríguez-Carvajal, M.A., Andersen, S.U., Sandal, N., Stougaard, J., Vinardell, J.-M., Ruiz-Sainz, J.E., 2016. Sinorhizobium fredii HH103 invades lotus burttii by crack entry in a Nod factor–and surface polysaccharide–dependent manner. Mol. Plant-Microbe Int. 29 (12), 925–937. Annapurna, K., Krishnan, H.B., 2003. Molecular aspects of soybean cultivar-specific nodulation by Sinorhizobium fredii USDA257. Ind. J. Exp. Biol. 41, 1114–1123. Armitano, J., Redder, P., Guimarães, V.A., Linder, P., 2016. An essential factor for high Mg2+ tolerance of Staphylococcus aureus. Front. Microbiol. 7 (1888). Aron, L., Toth, C., Godfrey, H.P., Cabello, F.C., 1996. Identification and mapping of a chromosomal gene cluster of Borrelia burgdorferi containing genes expressed in vivo. FEMS Microbiol. Lett. 145, 309–314. Ausubel, F.M., 2005. Are innate immune signaling pathways in plants and animals conserved? Nat. Immunol. 6 (10), 973–979. Bec-Ferte, Krishnan, M.P., Prome, D., Savagnac, A., Pueppke, S.G., Prome, J.C., 1994. Structures of nodulation factors from the nitrogen-fixing soybean symbiont Rhizobium fredii USDA257. Biochemistry 33, 11782–11788.

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