The resistant soybean cultivar ‘Suinong 10’ against the predominant race of P. sojae (race 1) in Heilongjiang, China (Zhang et al., 2010), was used for gene isolation and the seedlings at the first-node stage (V1; Fehr et al., 1971) were used for various treatments. P. sojae race 1, PSR01, isolated from infected soybean plants in Heilongjiang (Zhang et al., 2010), was used in the experiment. ‘Suinong 10’ seeds were grown with a photoperiod of 16/8 h light/dark and maintained at 22°C and 70% relative humidity in a greenhouse. The susceptible soybean cultivar ‘Dongnong 50’ was obtained from Key Laboratory of Soybean Biology of Chinese Education Ministry, Harbin, and used for gene transformation experiments.

Rapid amplification of cDNA ends was performed to isolate the full-length cDNA, corresponding to an EST up-regulated in soybean ‘Suinong 10’ in response to P. sojae infection, using the SMART RACE cDNA amplification Kit (Clontech, CA, United States). The gene-specific primers GSP1 for 5′- RACE and GSP2 for 3′-RACE (and) were designed to produce the antisense and sense strands, respectively. The full-length cDNA sequence of GmDIR22 was obtained by PCR amplification from ‘Suinong 10’ cDNA using the primer pair GmDIR22F and GmDIR22R (Supplementary Table S1). PCR products were ligated into the pMDTM18-T vector (Takara Biotech Inc, Dalian, China), then transformed into E. coli DH5α cells (Shanghai Biotech Inc, Shanghai, China) and sequenced (GENEWIZ, Beijing, China).

NCBI bioinformatics tools were used to analyze nucleotide and protein sequence data¹. The predicted protein structure was analyzed using SMART². Sequence alignments were performed using DNAMAN software³. A phylogenetic tree, based on the nucleotide sequences of GmDIR22 and other DIR members, was generated using MEGA 5.1 software⁴. The three-dimensional structure of GmDIR22 was predicted using the online program, Phyre2⁵.

To investigate GmDIR22 gene expression patterns in response to various stress factors, ‘Suinong 10’ soybean seedlings were treated with 50 mg.L^-1 gibberellic acid (GA₃), 100 μM methyl jasmonic acid (MeJA), 0.5 mM salicylic acid (SA), and 50 mM ABA. The unifoliolate leaves were harvested at 0, 3, 6, 9, 12, and 24 h after the treatment, wrapped in aluminum foil, immediately frozen in liquid nitrogen, and stored at –80°C until RNA extraction and cDNA synthesis. The untreated soybean leaves were used as controls. For P. sojae treatment, soybean plants were infected with P. sojae zoospores following the method described by Ward et al. (1979). The unifoliolate leaves were harvested for RNA isolation at 0, 6, 12, 24, 48, and 72 h after the treatments, wrapped in aluminum foil, immediately frozen in liquid nitrogen and stored at –80°C.

To determine the abundance of the GmDIR22 transcript, qRT-PCR analysis was performed. Total RNA was extracted from ‘Suinong 10’ soybean leaves at different time points after diverse stress treatments using TRIzol reagent (Invitrogen, Shanghai, China). First-strand cDNA was synthesized from 1 μg RNA using a Moloney murine leukemia virus reverse transcriptase kit (Takara, Dalian, China), according to the manufacturer’s protocol. qRT-PCR analysis was performed using a real-time RT-PCR kit (Takara, Japan) on a CFX96 Touch^TM Real-Time PCR Detection System (Bio-Rad, United States). DNA levels were determined using SYBR Green dye. The soybean housekeeping gene, GmActin4 (GenBank accession no. AF049106), was used as an internal control (see Supplementary Table S1 for primer sequences). For tissue distribution analysis, the transcript levels of the GmEF1β gene (GenBank accession no. NM_001248778) was used as a quantitative control (see Supplementary Table S1 for primer sequences). Relative expression levels were determined using the comparative threshold method (2^-ΔΔCT). Three biological replicates with three technical replicates of each qRT-PCR were performed.

To determine the subcellular localization of GmDIR22, the full-length GmDIR22 was cloned in frame at the 5′-terminus of the green fluorescent protein (GFP) coding sequence in the 35S:GFP vector using GmDIR22-GF and GmDIR22-GR (Supplementary Table S1) as the primer pair, to generate the fusion construct 35S:GmDIR22-GFP. GFP-fused GmDIR22 was transiently expressed in Arabidopsis protoplast cells, following the instructions described by Yoo et al. (2007) with minor modifications.

The full-length GmDIR22 cDNA was framed into the NcoI/XhoI site of the pET29b(+) vector (Novagen, Germany), to generate pET29b(+)-GmDIR22. The recombinant fusion plasmid was transformed into E. coli BL21 (DE3) cells (TransGen Biotech, China). His-tagged GmDIR22 protein expression was induced using 0.5 mM isopropyl-β-D-thiogalactoside (IPTG) at 37°C for 6 h in LB medium containing 50 mg. mL^-1 kanamycin. The analysis and purification of the recombinant protein were performed by SDS–PAGE.

Coupling assays were performed as described by Davin et al. (1997) with minor modifications. The reaction mixtures consisted of ammonium peroxydisulfate (2 μmol.mL^-1) as an oxidant, recombinant protein (1.5 nmol.mL^-1), and E-coniferyl alcohol (10 μmol.mL^-1) in MES-NaOH buffer (0.1 M, pH 5.0) to a total volume of 250 μL. After incubation for 3 h at 30°C, the mixture was extracted three times with ethyl acetate. The extract was evaporated to dryness under vacuum, and the residue re-dissolved in 50% methanol. High Performance Liquid Chromatography (HPLC) was used to separate the substrate and product.

The full-length coding region of GmDIR22 was PCR amplified and inserted into the NcoI/ApalI site of pCAMBIA3301 vector containing the bar gene as the selective marker with the primer pairs GmDIR22-oF and GmDIR22-oR (Supplementary Table S1). The recombinant construct 35S:GmDIR22 was introduced into Agrobacterium tumefaciens strain LBA4404 using the freeze-thaw method, as described by Holsters et al. (1978). The susceptible soybean ‘Dongnong 50’ was used for gene transformation using the Agrobacterium-mediated method described by Paz et al. (2004). Transgenic soybean plants (T₄) were identified by PCR amplification, and developed to T₅ transgenic soybean plants for further analysis.

The response assays of GmDIR22-transformed plants to P. sojae, the living cotyledons and detached leaves of T₅ transgenic soybean plants were treated with pathogen inoculum following the methods described by Dou et al. (2003) with minor modifications. Non-transformed plants treated in the same way served as controls. Disease symptoms on each cotyledon were photographed after inoculation using a Nikon D7000 camera. The relative biomass of P. sojae in infected cotyledons was assessed after 72 h based on the transcript levels of the P. sojae TEF1 gene (GenBank accession no. EU079791) using soybean GmEF1β as a reference gene, determined according to the method described by Chacón et al. (2010) (see Supplementary Table S1 for TEF1 and GmEF1β primer sequences). The pathogen response assays were performed on three biological replicates with their respective three technical replicates.

Samples from air-dried stems were subjected to quantitative analysis for ligninreferred to the Klason method by Kirk and Obst (1988). The cell wall residue (% CWR) was expressed relative to lignin content. Total lignan was extracted from leaf samples referred to the method by Chen et al. (2010). The extract was dried under vacuum, and the residue was re-dissolved in ethanol buffer. The chromotropic acid spectrophotometric method as described by Lu et al. (2011) was used to determine the total lignan.

The antimicrobial activities of lignan extracts were assayed using the hyphal growth inhibition method, as described by Schlumbaum et al. (1986). P. sojae race 1 was cultured on V8 juice agar plates at 25°C for 72 h and the sterile filter-paper disks containing lignan extracts from different transgenic plants were placed around the colony. After incubation for 24 h at 25°C, the pathogen growth zones were observed. In further experiments, the inhibition of the hyphal growth of P. sojae with various concentrations of lignan were Researched, as described by Giannakopoulou et al. (2014) with minor modifications.

The full-length GmDIR22 cDNA (GenBank accession no. HQ_993047) was isolated from ‘Suinong10’ soybean by RACE. The sequence analysis showed that GmDIR22 has an open reading frame of 567 bp and encodes a polypeptide of 188 amino acids (Supplementary Figure S1) with a predicted molecular mass of 20.88 kDa and a pI of 9.02. The deduced protein has a central 145 amino acid dirigent domain at amino acid residues 43–187 (Supplementary Figure S1). The predicted three-dimensional model of the GmDIR22 protein consists of eight β-strands, forming an eight-stranded antiparallel β-barrel (Supplementary Figure S2), similar to the previously reported (+)-pinoresinol-forming DIR from pea, DDR206 (Kim et al., 2015). A phylogenetic tree was constructed using the neighbor-joining algorithm based on the nucleotide sequences of GmDIR22 that contained other members of the DIR family, indicating that these neighboring genes might display similar functions (Figure 1). A total of 101 dir or dir-like nucleotide sequences from Glycine max (1), Arabidopsis thaliana (19), Oryza sativa (28), Picea glauca (26), Thuja plicata (9), and an additional 18 dir genes identified from a variety of species, including pea, cotton, corn, and sesame, etc. (see Supplementary Table S2) were used to construct the phylogenetic tree. In the phylogenetic tree, GmDIR22 is close to five AtDIR members (belonging to dir-b/d subfamily) and shares 55.15, 54.70, 53.66, 52.95, and 47.97 % nucleotide identity with AtDIR20, AtDIR7, AtDIR19, AtDIR3, and AtDIR8, respectively (Figure 1). We then performed sequence comparison between GmDIR22 and the five AtDIR nucleotides to analyze the homology among them. The alignments of the nucleotide sequences show five well-conserved motifs (Supplementary Figure S3).

To determine the expression pattern of GmDIR22 in different soybean cultivars, qRT-PCR was performed to examine the transcript levels of GmDIR22 (Supplementary Table S3). The expression of GmDIR22 was higher in resistant cultivars (‘Suinong 10,’ ‘Williams 82,’ ‘Hefeng 34,’ ‘Nenfeng 15,’ and ‘Kangxian 1’) than that in susceptible cultivars (‘Hefeng 25,’ ‘Heinong 37,’ ‘Dongnong 50,’ ‘Kendou 18,’ and ‘Hefeng 35’) (Figure 2). The responsiveness of GmDIR22 mRNA transcript levels to biotic and abiotic stresses in ‘Suinong10’ soybean plants was evaluated by qRT-PCR (Supplementary Tables S5, S6, S7, S8, S9). The investigation of tissue-specific transcript abundance in ‘Suinong 10’ soybean (Supplementary Table S4) demonstrated that GmDIR22 is constitutively and highly expressed in the stems, followed by the roots and leaves (Figure 3A). After P. sojae treatment, the levels of GmDIR22 mRNA rapidly increases, reaching a maximum level at 48 h after treatment, followed by a decline (Figure 3B). GmDIR22 expression is also induced with the treatments of GA₃, SA, MeJA, and ABA. The levels of GmDIR22 mRNA increases rapidly in response to GA₃ treatment, reaching maximum levels at 9 h, followed by a rapid decline. With SA, MeJA, and ABA treatment, GmDIR22 transcripts accumulates and reaches a maximum level at 6, 6, and 9 h, respectively, but the expression of GmDIR22 is relatively lower than that under P. sojae and GA₃ stresses (Figure 3C).

The subcellular localization of the GmDIR22 protein was analyzed by expressing a gene encoding a GmDIR22-GFP fusion protein under the control of the 35S promoter in Arabidopsis protoplasts. As shown in Figure 4, the observation by confocal microscopy demonstrated that GFP fluorescence is dispersed throughout the entire cells expressing the control 35S-GFP plasmid. In contrast, the GmDIR22-GFP fusion protein is localized exclusively to the membrane of the Arabidopsis cells, indicating that GmDIR22 is a membrane-localized protein.

The expression of recombinant GmDIR22 protein is remarkably enhanced by adding 0.5 mM IPTG at 37°C and increases from 1 to 6 h (Figure 5A). The maximum expression of the protein is achieved at 4 h. The recombinant protein was purified using His-Bind Kits (EMD Millipore, Billerica, MA, USA), and the molecular weight of the purified protein is determined as approximately 23 kDa by SDS–PAGE (Figure 5A). Furthermore, the dirigent ability of recombinant GmDIR22 to couple monolignols was investigated. As shown in Figures 5B,C, GmDIR22 could direct conversion of E-coniferyl alcohol into lignan (+)-pinoresinol, in the presence of ammonium peroxydisulfate as oxidant. The result is similar to that reported for FiDIR (Davin et al., 1997).

To investigate whether overexpression of GmDIR22 could enhance resistance to P. sojae in transgenic plants, the T₅ transgenic soybean plants were selected by qRT-PCR (Supplementary Table S11) to assay the pathogen response (Figure 6A). The living cotyledons and detached leaves were treated with a P. sojae inoculum. As shown in Figure 6B, the cotyledons of the non-transgenic soybean plants exhibits clearer and larger water-soaked lesions compared with those of the transgenic plants after 72 h of incubation with P. sojae, and the lesion areas are significantly smaller in the transgenic lines than those of non-transgenic soybean plants 72 h after inoculation (P < 0.01) (Figure 6C). After 96 h incubation with P. sojae, the similar results are observed in the detached leaves (Figure 6E), and the lesion areas in transgenic soybean lines were significantly smaller than those in non-transgenic plants (P < 0.01) (Figure 6D). The biomass of P. sojae, based on the transcript levels of the P. sojae TEF1 gene (Supplementary Table S10), was significantly lower in transgenic GmDIR22-overexpressing plants than that in non-transgenics (P < 0.01) (Figure 6F). These data indicated that the over-expression of GmDIR22 in soybean plants in a certain extent improves their resistance to P. sojae.

The dirigent phenomenon was discovered by Davin et al. (1997), and then Davin and Lewis (2000) studied the coupling of two E-coniferyl alcohol molecules to produce a (+)-pinoresinol as the first step in the biosynthesis of lignans. Since coniferyl and sinapyl alcohols, together with other monolignol p-coumaryl alcohols, are also polymerized into the macromolecule, lignin, there is some overlap in the biosynthetic pathways involved in lignans and lignin production (Davin and Lewis, 2003). Thus, the changes in the DIR expression levels may cause alterations in the lignan and lignin content of soybean plants. As shown in Figure 7A, the relative lignan content of transgenic plants overexpressing GmDIR22 is significantly higher than that of non-transgenic control plants, indicating an important role for GmDIR22 in the synthesis of lignan in soybean. In contrast, the levels of acid-insoluble lignin remain almost unchanged in transgenic plants compared with those of controls (Figure 7B).

To examine the antimicrobial effects of lignan extracts on P. sojae race 1, the filter-paper disks containing lignan extracts from different transgenic plants were placed around a P. sojae colony. After incubation for 72 h, the results showed that the lignan extracts from transgenic plant lines T5-2 and T5-19 inhibit P. sojae hyphal growth, leading to 1 and 2 mm inhibition zones, respectively, indicating that they exhibit an enhanced antimicrobial effect compared to those from non-transgenic plants (Figure 8A). To further examine the effect of lignan extracts on P. sojae, we conducted inhibition assays using an equal amount hyphal plugs of 1-week-old of P. sojae race 1, which were placed in sterilized plates with rich medium, supplemented with varying concentrations of lignans. Hyphal growth was assessed by visual inspection after 7 days, the results confirmed that the antibacterial effect gradually increased with increasing of the concentration of lignans (Figure 8B). These results demonstrated that lignans from soybean possess antimicrobial activity against P. sojae.