Genome-wide identification of WRKY genes in soybean and functional analysis of GmWRKY162-GmGSL7c in resistance to soybean mosaic virus

doi:10.21203/rs.3.rs-3749133/v1

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Research Article

Genome-wide identification of WRKY genes in soybean and functional analysis of GmWRKY162-GmGSL7c in resistance to soybean mosaic virus

https://doi.org/10.21203/rs.3.rs-3749133/v1

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Background

Soybean mosaic disease caused by soybean mosaic virus (SMV) is one of the most devastating and widespread diseases in soybean producing areas worldwide. The WRKY transcription factors (TFs) are widely involved in plant development and stress responses. However, the roles of GmWRKY TFs in resistance to SMV infection are largely unclear.

Results

Here, a total of 185 GmWRKYs were identified from soybean (Glycine max), and were clustered into 3 groups and 7 subgroups. The characteristics, such as gene structure, conserved motifs, cis-regulatory elements, and selective pressure, were systematically analyzed. Furthermore, 60 GmWRKY genes were differentially expressed during SMV infection according to the transcriptome data. Among these genes, the expression of GmWRKY162 decreased after imidazole treatment in the incompatibility combination in soybean varieties Jidou 7 with SMV strain N3, which was confirmed by RT‒qPCR. Remarkably, the silencing of GmWRKY162 reduced callose deposition and enhanced virus spread during SMV infection. Next, EMSA and ChIP‒qPCR revealed that GmWRKY162 could directly bind to the promoter of GmGSL7c, which was reported to be involved in callose synthesis in our previous study.

Conclusion

Our study revealed that GmWRKY162 play a positive role in increasing callose deposition and suppressing virus spread during SMV infection by targeting GmGSL7c, which providing valuable insights into the roles of WRKY family genes in response to SMV and guidance for future studies in understanding virus-resistance mechanisms in soybean.

soybean

SMV

WRKY gene family

callose deposition

Soybean mosaic disease caused by SMV is one of the most common viral diseases worldwide. SMV can cause severe soybean yield decline and seed quality deterioration [1]. Although several SMV strains and quantitative trait loci (QTLs) related to SMV resistance in soybean have been identified, mechanistic studies on the function and regulation of antiviral genes are rare [2, 3]. Plasmodesmata (PDs) are plasma membrane-lined channels that connect adjacent cells to mediate symplastic communication in plants [4]. Callose is a beta-1,3 glucan that can be reversibly deposited around the neck of PD, which modifies cell wall properties and restricts PD aperture that affects the transport of signaling elements and pathogenic elicitors in a cell-to-cell manner [5, 6]. The levels of callose at PD are controlled by two groups of enzymes, callose synthases and beta-1,3 glucanases, which synthesize and degrade callose respectively [7]. A number of studies have shown that resistance-inducing chemicals, such as abscisic acid (ABA) [8, 9], salicylic acid (SA) [10], auxin [11], and reactive oxygen species (ROS) [12], contribute to the regulation of callose balance at PDs. Previous studies in our laboratiory showed that callose deposition in the neck of PD and phloem plays key roles in restricting virus transport between cells and long-distance transport of SMV in soybean, respectively [13, 14]. Additionally, as an intracellular signaling molecule, nitric oxide (NO), which acts upstream of hydrogen peroxide (H₂O₂), was reported to play synergistic roles during SMV-induced callose deposition in soybean, [15, 16]. Therefore, callose deposition is believed to play a critical role in preventing virus transport between cells as a physical barrier.

The WRKY family constitutes one of the ten largest TF families and exists exclusively in higher plants, which is involved in various response pathways of biotic and abiotic stress [17, 18]. Members of the WRKY family have been identified in many species, such as Arabidopsis thaliana [19], Nicotiana tabacum [20], Zea mays [21], Triticum aestivum [22], Cymbidium sinense [23], and Akebia trifoliata [24]. WRKY TFs usually contain a highly conserved WRKY domain at the N-terminus and a zinc finger-like motif at the C-terminus [17]. As the representative domain of WRKY TFs, the WRKY domain shows sequence variation across plant species, such as WRKYGQK, WRKYGEK, and WRKYGKK [25]. WRKY domains are necessary for WRKY TFs to recognize and bind to the W-box cis-elements (TTGACC/T) of their target genes [26]. Two main types of zinc finger-like motifs have been identified in WRKY TFs, namely, C2H2 (C-X4-5-C-X22-23-HXH) and C2HC (C-X7-C-X23-HXC) [27]. WRKY TFs can be categorized into three groups (I, II, and III) according to the number of WRKY domains and the types of zinc finger-like motifs [28]. WRKY TFs in Groups I and II harbor two and one WRKY domain, respectively, but all have a C2H2 type zinc finger-like motif; while those in Group III contain one WRKY domain and a C2HC type zinc finger-like motif [25]. Furthermore, Group II WRKY TFs can be divided into five subgroups according to phylogenetic analysis, including IIa, IIb, IIc, IId, and IIe [29].

Several studies have shown that a large number of WRKY genes can be induced by defense-related phytohormones, low temperature, and pathogens, which participate in pattern-triggered immunity (PTI) and effector-triggered immunity (ETI) through regulating ROS production [30]. These results indicate that WRKY genes play important roles in the immune response to biotic and abiotic stress [31]. For example, CmWRKY15-1 interacted with CmNPR1 to promote the expression levels of pathogenesis-related genes which enhance resistance to chrysanthemum white rust through SA pathway in chrysanthemum [32]. In Malus domestica, MdWRKY75e and MdWRKY100 overexpression enhanced resistance to Colletotrichum gloeosporioides and Alternaria alternata [33, 34]. In cotton, overexpression of GhWRKY41 enhanced resistance to Verticillium dahliae by regulating phenylpropanoid metabolism [35]. In tobacco, overexpression of cotton GhWRKY15 enhanced its resistance to tobacco mosaic virus (TMV) and cucumber mosaic virus (CMV) by reducing ROS accumulation [36]. In Arabidopsis, AtWRKY8 improves plant defense against TMV through mediating ABA and ethylene (ET) signaling pathways by up-regulating ABI4, which further inhibits the expression levels of ACS6 and ERF104 [37]. In soybean, the identification of WRKY TFs have been reported under both biotic and abiotic stress. For example, 176 and 185 GmWRKY genes were identified using the Phytozome10.0 and 10.2 databases (Wm82.a2.v1) under drought and salt stress, respectively [37, 38]. Dong et al analyzed soybean WRKY genes in response to Peronospora manshurica infection through transcriptome analysis and found that 22 WRKY genes were differentially expressed between resistant and susceptible genotypes [39]. However, the molecular mechanism underlying WRKY TF-mediated SMV resistance in soybean has rarely been reported.

In this study, 185 soybean WRKY genes were identified genome-wide, and their bioinformatics were analyzed. Among them, 60 GmWRKY genes were differentially expressed during SMV infection according to the transcriptome data. Notably, GmWRKY162 exhibited high expression levels in the incompatibility combination in soybean with SMV strain N3, and the silencing of GmWRKY162 led to reduced callose deposition and enhanced virus spread during SMV infection by targeting GmGSL7c. Taken together, these results provide a fundamental understanding of the molecular mechanism underlying SMV infection and new clues for further research into the functions of soybean WRKY genes.

Soybean planting and inoculation

The soybean varieties, Nannong 1138-2 and Jidou 7, were used in this study, which directly acquired from Dr. Haijian Zhi (Nanjing Agricultural University, China) and Prof. Mengchen Zhang (Institute of Cereal and Oil Crops, Hebei Academy of Agriculture and Forestry Sciences, China), respectively. Soybean seedlings were planted in pots and placed in greenhouses. The temperature was 25℃, and a 14/10 (day/night) photoperiod was used with a high-pressure sodium lamp as the light source. The SMV strains (N3 and SC8) were used in this study, which acquired from Dr. Haijian Zhi (Nanjing Agricultural University, China). Jidou 7 is resistant to the SMV strain N3 (to form incompatible combination) and susceptible to the SMV strain SC8 (to form compatible combination).

The leaf sap from SMV-infected Nanong 1138-2 plants was mixed with emery and gently applied on the soybean leaves of Jidou 7 plants using a brush, according to previous report [16]. Two-week-old seedlings were harvested at 0, 4, 12, 24, and 48 hours after inoculation with SMV strains N3 and SC8, respectively. The leaves without veins were immediately frozen in liquid nitrogen and stored at -80℃ for further experiments.

Identification and chromosomal mapping of WRKY genes in soybean

The latest version of the soybean genome file was downloaded from the Ensembl database (https://plants.ensembl.org/index.html). The pfam seed model WRKY (PF03106) was used for building the Hidden Markov Model (HMM) file using HMMER3 with default parameters. To verify the reliability and exclude false positives, the presence of WRKY domains were confirmed using the SMART database (http://smart.embl-heidelberg.de) and the CDD database (https://www.ncbi.nlm.nih.gov/cdd). In addition, GmWRKY transcription factor sequence were downloaded from PlantTFDB (http://planttfdb.gao-lab.org/) and compared with our data. The MG2C online website (http://mg2c.iask.in/mg2c_v2.0) was used to determine the chromosomal location of each GmWRKY gene.

Phylogenetic analysis, gene structure, conserved motif, and cis-element analyses

The 72 Arabidopsis WRKY proteins described previously were obtained from TAIR (http://www.arabidopsis.org/). The sequences of the WRKY proteins in Arabidopsis and soybean were aligned using ClustalW and used for phylogenetic analysis via the Neighbor-Joining model with 1000 replicated bootstrap values (MEGA7.0). The multiple sequence alignment results were plotted and optimized using the GeneDoc software. The exon/intron position information was obtained from genomic annotation files (GFF/GTF files) in the soybean species. The conserved motif of WRKY protein was predicted using MEME (http://meme-suite.org/tools/meme). The cis-acting elements were analyzed by the PlantCARE software using the 2000 bp sequences upstream of the GmWRKY genes. The phylogenetic tree, conserved motifs, and gene structures were visualized with TBtools [40].

Gene collinearity, duplication events, and protein interaction analysis

The genome data of soybean and Arabidopsis were downloaded from the Ensembl database and the syntenic analysis maps were generated using MCScanX and Circos. All GmWRKY genes were classified into five different categories, namely, singleton, dispersed, proximal, tandem, and WGD/segmental by the duplicate_gene_classifier tool in the Multiple Collinearity Scan toolkit (MCScanX) program [41]. The protein sequences of the GmWRKYs were uploaded to the STRING database (https://cn.string-db.org/), and the interaction networks were constructed based on the orthologous genes between soybean and Arabidopsis.

Total RNA isolation, transcriptome data, and RT‒qPCR

Total RNA was extracted using RNA extraction kit (UNlQ-10 Column TRIzol Total RNA Isolation Kit, Sangon Biotech, Shanghai, China), and the synthesis of the first-strand cDNA was carried out with PrimeScript RT Reagent Kit with gDNA Eraser (TaKaRa, Dalian, China). In our previous studies, H₂O₂-associated transcriptome data were obtained with imidazole (an NADPH oxidase-specific inhibitor) treatment during SMV infection [16]. Based on the transcriptome data, the differentially expressed GmWRKYs were identified during SMV infection. GO functional enrichment analysis ofall differentially expressed genes were performed by the GO EnrichMent tool in TBtools [40]. RT‒qPCR was performed to confirm the response of differentially expressed GmWRKY genes to SMV. GmEF1b was used as the internal control. The primers used are listed in Table S1.

Generation of GmWRKY162-silenced plants

Tobacco rattle virus (TRV) mediated virus-induced gene silencing (VIGS) was used to generate GmWRKY162-silenced plants. The specific fragment of GmWRKY162 was amplified from the cDNA of Jidou 7 using the primers listed in Table S1 and inserted into pTRV2 plasmid between BamH I and Xho I. The empty plasmid of pTRV2 was used as the control (TRV:00). Agrobacterium tumefaciens carrying pTRV1 and the recombinant pTRV2 (TRV:GmWRKY162) were resuspended in the infection buffer (50 mM MES, 2 mM Na₃PO₄, 28 mM D-glucose, 0.1 mM acetosyringone, 4.1 mM L-Cys, and 0.02% (w/v) Silwet L-77) until the cell density (OD600) reached 0.5, respectively. Then, the mixture of the resuspended pTRV1 and the recombinant pTRV2 (1:1) was poured into the roots of the soybean seedlings for each plant. The compound leaves from the first round were harvested after SMV infection. The silencing efficiency of the target genes was examined by RT‒qPCR. The VIGS experiment and the calculation of silencing efficiency were conducted according to the previously reported protocols [16, 42].

Observation of SMV-induced callose and detection of SMV CP transcript products

The soybean leaves were collected at 48, 72, and 120 hours after SMV infection for aniline blue staining and fluorescence observation. The soybean leaves were placed in a fixative solution (50% ethanol, 16.67% glycerol, 16.67% phenol, and 8.33% lactic acid, v/v) and boiled for 2 min, washed for 5 min with ddH₂O for three times, and then treated with the dye solution (0.01% aniline blue dissolved in 0.1 M PBS, pH 7.0) for 15 min. The leaves were washed for 5 min with ddH₂O for 3 times again and observed with a fluorescence microscope (BX53, Olympus, Tokyo, Japan) with Ex/Em = 385 nm/495 nm. ImageJ [43] was used to analyze the microscopic images of the fluorescence of callose and the callose area of 30 inoculation sites were quantified. The upper leaves were observed 15 days after SMV inoculation. RT‒PCR was used to detect the expression of SMV CP according to the previously reported protocol [42].

Chromatin immunoprecipitation (ChIP) and ChIP‒qPCR

ChIP assays were carried out as previously described [44]. The leaves of Jidou 7 were infiltrated with Agrobacterium tumefacien (GV3101) containing 35S:GmWRKY162-GFP or 35S:GFP, respectively. 2 g leaves of three-week-old were sampled at 40 hpi with N3 and crosslinked with 1% (w/v) formaldehyde for 30 min under vacuum and quenched with 2.0 M glycine for 5 min. The chromatin was sheared to 100–500 bp fragments via ultrasonic disruption (Diagenode, Belgium). Immunoprecipitation was performed with Dynabeads Protein G (Sigma-Aldrich, Germany) and anti-GFP antibody (Novus, USA). For the ChIP‒qPCR assay, the amount of immunoprecipitated GmGSL7c chromatin in the P1 and P2 regions on its promoter was detected by qPCR. The primers used are listed in Table S1.

Electrophoretic mobility shift assay

Electrophoretic mobility shift assay (EMSA) was performed using the Light Shift Chemiluminescent EMSA Kit (Thermo Fisher Scientific, USA). The double-stranded DNA probe containing W-box was labeled with biotin at 5’ end (Table S1). Nonlabeled probes (200-fold) were used as competitors. The biotinylated probes (Table S1) were synthesized by Sangon Biotech (Shanghai, China). The biotinylated and unlabeled probers were incubated with 6 µg of His-tagged GmWRKY162 protein in binding reactions (provided with the EMSA kit) for 20 min. The reactions were stopped by the addition of loading buffer and the proteins were resolved on a 6% (w/v) native polyacrylamide gel. Then, the complex was electrophoretically transferred to a Hybond-N⁺ nylon membrane (Solarbio, China), which was subsequently crosslinked under ultraviolet light. The signals were detected using a charge-coupled device camera.

Identification and chromosomal distribution of WRKYs in soybean

To identify WRKY family genes in soybean, we used the WRKY domain (PF03106) as a query for the BLAST search in soybean, and the results were further confirmed in the SMART and NCBI CDD databases. A total of 185 GmWRKY members were identified in soybean, which is consistent with the number recorded in the PlantTFDB. The 185 GmWRKYs were unevenly positioned among the 20 soybean chromosomes (Chrs) and were named from GmWRKY1 to GmWRKY185 according to their physical location on the Chrs (Table S2 and Figure S1). Of the 185 WRKYs, 15 (8.1%) were located on Chr06, which harbored the greatest number of WRKY genes, followed by 14 genes (7.6%) on Chr09, while only 3 genes were mapped on Chr11, Chr12, and Chr20 (Figure S1).

Phylogenetic classification of the GmWRKYs

To reveal the phylogenetic relationship and evolutionary history of soybean GmWRKY proteins, a phylogenetic tree was constructed based on the full-length sequence alignment of 185 GmWRKYs and 72 Arabidopsis AtWRKYs (Fig. 1). Phylogenetic tree analysis reviewed that all the GmWRKYs were clustered into three groups (Group I, Group II and Group III) according to previously reported classification criteria [45], and Group II was further divided into five subgroups in this neighbor-joining (NJ) tree, designated as Group IIa to IIe (Table S2 and Fig. 1). Group I included 30 GmWRKYs (16.2%), featuring two WRKY domains and one C2H2 motif. Among them, GmWRKY4 and GmWRKY21 contained the WRKYGEK heptapeptide, while the others contained WRKYGQK (Table S2 and Figure S2). However, most of Group I members contained C2H2 (C-X4-C-X23-HXH) zinc finger structure apart from GmWRKY2, GmWRKY126 and GmWRKY127, which contained C2H2 (C-X5-C-X23-HXH, Figure S2). Group II included 130 GmWRKYs (70.3%), harboring one WRKY domain and one C2H2 motif, including Group IIa (7.6%), Group IIb (17.8%), Group IIc (23.8%), Group IId (10.3%), and Group IIe (10.8%). Among them, 115 GmWRKYs (88.5%) contained WRKYGQK, whereas GmWRKY105 in IIa and GmWRKY130/162/29/36/47/52/58/60/82 in IIc contained WRKYGKK, GmWRKY75, and GmWRKY108 in IIc contained WRKYGEK and WRKYGLK, respectively (Table S2). Additionally, 72 and 51 GmWRKYs (55.4% and 39.2%, respectively) contained C2H2 (C-X4-C-X23-HXH) and C2H2 (C-X5-C-X23-HXH), respectively (Table S2). Group III included 25 GmWRKYs (13.5%), harboring one WRKY domain and one C2HC motif. Except for GmWRKY46, the others all contained the WRKYGQK heptapeptide. Additionally, AtWRKY49 and GmWRKY70/168, AtWRKY40 and GmWRKY71, which are supposed to belong to Group IIc and Group IIa respectively, were all classified into Group I, indicating that they might be more homologous to members in Group I. Strikingly, a WRKYGQK-like stretch was lost in GmWRKY20, -104, -129; whereas C2H2 or C2HC was missing in GmWRKY20, -42, -71, -78, -93, -105, -111, -163 (Table S2 and Figure S2), however, the effects on functions need further investigation.

To further investigate the number and proportion of WRKY members in the plant genome, eight representative plant species, namely, Glycine max, Arabidopsis thaliana, Triticum aestivum, Cucumis sativus, Nicotiana tabacum, Zea mays, Akebia trifoliata, and Cymbidium sinense, were counted (Table S3 and Figure S3). The number of WRKY genes in Glycine max far exceeded that in other species, except for Triticum aestivum and Nicotiana tabacum, which are both allopolytraploid plant species. The proportions of GmWRKY proteins in Group I was generally the same in different species except for Akebia trifoliata and Cymbidium sinense. The number of WRKY proteins in Group II was generally greater than that in Group I and Group III for the eight plant species, ranging from 54.8–70.5%. Additionally, Group IIc had the most WRKY members in the eight representative plant species. These results suggested that the majority of members of Group II might experience a significant expansion in soybean and other species, which could be caused by whole-genome duplications, tandem duplications, or segmental duplications of genomes.

Conserved motif analysis and gene structure of GmWRKY genes

To further analyze the structural diversity of GmWRKYs in soybean, the phylogenetic tree has been constructed with only GmWRKY members (Fig. 2A). Each GmWRKY was clustered into the corresponding group, as shown in Fig. 1, suggesting that the phylogenetic tree was reasonable. The conserved motifs were subsequently analyzed using the MEME-suite (Fig. 2B). A total of ten distinct motifs (designated Motif 1to Motif 10) were identified in the GmWRKY proteins (Figure S4). Among them, motif 1 contains a WRKY domain (Figure S4), which is present in all the GmWRKY proteins except for GmWRKY62/30/104/49/129/119/20. The types and orders of motifs in the different groups appeared to be similar (Fig. 2B). For example, most of the GmWRKYs in Group I contained more than five motifs, namely, Motif 1, -2, -3, -4, and − 10, and the GmWRKYs in Group IIIcontained Motif 1, -2, -9 or -8 (Table S2). In addition, the GmWRKYs in Group II had various motif types and orders, for example, Group IIa contained Motif 1, -2, -5, and − 6, Group IIb contained Motif 1, -2, -3, -4, -5, -6, and − 7, Group IIc contained Motif 1, -2, -3, and − 4, Group IId contained Motif 1, -2, -4, -8, and − 9, Group IIe contained Motif 1, -2 and − 4 (Table S2), indicating that the GmWRKYs in Group II might have diverse functions in soybean.

To gain insight into the structural diversity and evolution of GmWRKYs in soybean, the number and arrangement of exons and introns were analyzed. The results showed that the GmWRKYs clustered into the same group had similar exon-intron structures (Fig. 2C). The number of introns varied from zero to seven, for example, GmWRKYs in Group I had three to five introns, GmWRKYs in Group II had zero to seven introns, and GmWRKYs in Group III had one, two or five introns (Table S2 and Figure S5A). Notably, GmWRKYs in IIb generally had more introns and more complex exon-intron structures than did those in the other groups (Table S2 and Figure S5B). However, the effects of diverse exon-intron organizations on the function of GmWRKYs need further study.

Duplication events and syntenic analysis of GmWRKY members

Two diploid soybean (Glycine max) species experienced two sequential WGD events, including a polyploidy event at ~ 59 MYA and a Glycine-specific WGD at ~ 8–13 MYA [46, 47]. Duplicated genes in soybean have been classified into five categories, namely, singletons, dispersed, proximal, tandem, and WGD (including segmental duplications), which strongly correlate with gene function and mode of duplication [46]. Therefore, we analyzed the duplication types of GmWRKY genes via MCScanX. The results showed that WGD events accounted for 96.7% (29/30), 90.9% (118/130), and 72.0% (18/25) in Group I, II, and III, respectively (Fig. 3A). In addition, dispersed duplication events accounted for 3.3% (1/30) and 5.4% (7/130) in Group I and II, respectively. Tandem and proximal duplication events occurred mainly in Group III, accounting for 24.0% (6/25) and 4.0% (1/25), respectively. According to previous report, tandem duplication events occur when genes are in a chromosomal region within 200 kb [48]. In this study, three sets of GmWRKYs (GmWRKY107/108/109, GmWRKY119/120/121/122, and GmWRKY135/136) in Groups IIc and III, were identified as tandem duplicate pairs on Chr10, 13 and 14 (Figure S1 and Table S2). WGD events were subsequently visualized within the soybean genome, and 220 segmental duplication pairs were found between 165 GmWRKY genes that were unevenly distributed on 20 chromosomes (Fig. 3B and Table S4). The Ka/Ks (nonsynonymous/synonymous substitutions) of 220 WGD gene pairs were calculated to be less than 1 (Table S4). Therefore, these findings indicated that the expansion of GmWRKYs was caused mainly by the WGD events under negative selection pressure during their evolution.

To further investigate the evolutionary mechanisms of GmWRKYs, the synthetic analysis of WRKYs in Arabidopsis was performed with GmWRKYs (Fig. 3C). The results showed that 76 GmWRKYs (41.1%) displayed syntenic relationships with 35 AtWRKYs (48.6%) in Arabidopsis, and 96 orthologous pairs were identified according to the whole-genome-wide comparative analysis (Table S5), indicating that these WRKYs may be derived from the same ancestor with similar functions.

Promoter analysis of GmWRKY genes

To explore the regulatory mechanisms of GmWRKYs, 32 types of cis-elements were identified in the promoters of GmWRKYs [49, 50], which were involved in hormone responses, stress responses, plant growth, and developmental response, and light responses (Fig. 4 and Table S6). Among them, twelve hormone-responsive elements were found, including Abscisic Acid (ABRE), Auxin (AuxRR-core, TGA-element and TGA-box), Gibberellin (GARE-motif, P-box and TATC-box), MeJA (CGTCA-motif and TGACG-motif), and Salicylic Acid (TCA-element and SARE), and Ethylene (ERE). Among the hormone-responsive elements, the ERE and ABRE were widely distributed among the promoters of GmWRKYs (Fig. 4A) and occupied the largest proportions (28.53% and 26.25% respectively) (Fig. 4B). Eight stress-responsive related elements were identified, including ARE (anaerobic induction), GC-motif, LTR (low-temperature), MBS (drought), TC-rich repeats (stress-responsive), W-box, WUN-motif (wound), and STRE (heat shock response). Remarkably, the W-box existed in 94 GmWRKYs, and can be specifically recognized and bound by WRKY TFs, indicating that these GmWRKYs may be regulated by themselves or other WRKY TFs at the transcriptional level. Eight elements were involved in plant growth and developmental response, including MBSI (MYB binding site), CAT-box (meristem specific element), CCGTCC-motif (meristem specific activation), GCN4-motif (endosperm-specific element), circadian (circadian control responsiveness), HD-Zip 1 (palisade mesophyll cells differentiation), O2-site (zein metabolism regulation), and RY-element (seed-specific regulation). Four elements involved in light response were detected, including Box 4, G-box, AE-box and MRE. Among them, the Box4 elements have a wide distribution in GmWRKY genes, which were identified in 177 (95.7%) members of GmWRKYs and occupied the largest proportions among light response-related motifs (68.33%), providing a reference for further study the functions of GmWRKYs in light response. Additionally, no cis-elements were found in the promoters of GmWRKY30 and GmWRKY78. Taken together, these findings suggested that GmWRKYs not only play vital roles in stress responses, but also in plant growth and development.

Expression patterns of GmWRKYs during SMV infection

In our previous studies, we found that NO and H₂O₂ played synergistic roles in callose deposition during SMV infection, which is crucial for restricting virus transport [15, 16]. To investigate the roles of GmWRKYs in response to SMV infection, we identified 60 GmWRKY genes (Fig. 5) that were differentially expressed during SMV infection in H₂O₂-associated transcriptome data [16]. Among them, seven and eight GmWRKYs belonged to Group I and Group III, respectively. In addition, 45 members belonged to Group II, including eight in Group IIa, nine in Group IIb, eighteen in Group IIc, three in Group IId, and eitht in Group IIe (Fig. 5A). Among them, GmWRKY162 had high expression levels in the incompatibility combination and was downregulated after imidazole treatment (Fig. 5A and 5B), indicating that GmWRKY162 play a positive role in response to SMV. Therefore, GmWRKY162 was selected for further functional analysis.

GmWRKY162 directly targets GmGSL7c to suppress virus spread through increasing callose deposition

To further investigate the function of GmWRKY162 in response to SMV infection, GmWRKY162-silenced Jidou 7 plants were generated via TRV-mediated VIGS, the results of which revealed an efficiency of more than 70% (Fig. 6A). Compared to that of the control (TRV:00), the area of cells with callose fluorescence under SMV infection was significantly decreased at 48, 72, and 120 dpi in GmWRKY162-silenced plants (Fig. 6B and 6C). Furthermore, compared to those in the control, disease symptoms were observed in the non-inoculated upper leaves of GmWRKY162-silenced plants at 15 dpi, and the expression of the coat protein (CP) gene of SMV was significantly induced (Fig. 6D and 6E), indicating that the silencing of GmWRKY162 reduces callose deposition and enhances virus spreading during SMV infection.

To further understand how GmWRKY162 works during SMV infection, 693 differentially expressed genes (DEGs) were identified from the transcriptome database [16], which have similar expression trends with GmWRKY162 (Fig. 7A and Table S7). Functional enrichment analysis revealed that genes associated with catalytic activity, oxidoreductase activity, membrane, and obsolete oxidation-reduction process were enriched (Fig. 7B). Among them, the catalytic activity term enriched the most genes (Table S8), which contained a glucan synthase-like gene (named GmGSL7c) involved in callose synthesis; the functional study of GmGSL7c was carried out in our previous report [51]. Furthermore, a W-box (GTCAA, -1901 bp to -1905 bp) was found through the prediction of the cis-element in the GmGSL7c promoter region (Fig. 7C and Table S9). To test this hypothesis, electrophoretic mobility shift assays (EMSAs) in vitro were performed, which revealed that GmWRKY162 could directly bind to the W-box motif of the GmGSL7c promoter (Fig. 7D). In this, the biotin-labeled probe containing a combination of the W-box from the GmGSL7c promoter was constructed and incubated with the recombinant protein His-tagged GmWRKY162, which caused a mobility shift, indicating that GmWRKY162 bound to W-box motif of the GmGSL7c promoter in vitro (Fig. 7D). Next, ChIP-qPCR was used to determine the enrichment of GmWRKY162-GFP in the P1 region (-1947 bp to -1879 bp, containing a W-box motif) and the P2 region (-781 bp to -735 bp, without a W-box motif) of the GmGSL7c promoter (Fig. 7E). These results showed that the enrichment of GmWRKY162-GFP was markedly greater in the P1 region than in the P2 region in the 35S:GmWRKY162-GFP plants, but not in the 35S:GFP plants (Fig. 7E). In summary, these results demonstrated that GmWRKY162 directly binds to the promoter of GmGSL7c in vitro and in vivo to promote its transcription, which increases callose deposition and suppresses virus spread (Fig. 7F).

Plant WRKY transcription factors play important roles in the process of plant resistance to biotic and abiotic stresses [31, 52–55]. Many WRKY genes have been identified in various species including Arabidopsis thaliana [19], Nicotiana tabacum [20], Zea mays [21], Triticum aestivum [22], Cymbidium sinense [23], Akebia trifoliata [24], and many other plants. However, the findings concerning the WRKY gene family in soybean were not consistent with previous studies and mostly focused on abiotic stresses [37–39]. In addition, the molecular mechanism underlying WRKY TF-mediated SMV resistance in soybean has rarely been characterized.

In this study, a total of 185 GmWRKY genes were identified, which is more than that previously reported in Dong et al (2019) and Yang et al (2017) [56]. Among them, 130 Group II GmWRKY members were identified, and according to chromosomal distribution and sequence duplication event analysis, WGD (118/130), dispersed duplication events (7/130) and tandem duplication events (5/130) contributed to the expansion of GmWRKY genes in Group II, which have been reported to play variety roles in response to biotic and abiotic stresses. The plant WRKY family has been conserved during evolution, and their WRKY domains are similar in each group, while the types and positions of conserved domains differ slightly between groups [24, 57–59]. In our study, the positions of zinc finger motifs in the GmWRKYs in Group II was different from that in Group I, which is consistent with the finding of previous studies in Cucumis sativus [59]. Previously, studies have shown that introns are ubiquitous features of all eukaryotic cells, and need to be removed to produce functional proteins, which results in survival pressure on eukaryotic cells [60]. In addition to encoding elements, such as snoRNAs and miRNAs, introns also play roles in the response to environmental nutrients, which may be the reason for the retention of introns during evolution [60]. In our study, the arrangement and number of introns in GmWRKYs in each group were similar, indicating that they may have synergistic or similar functions.

Previously studies have shown that phytohormones, low temperature, pathogens and other factors can induce the expression of WRKY genes to participate in various physiological processes in plants [61, 62]. However, the regulatory mechanism involved is still unclear. In this study, numerous cis-acting elements were identified in the promoters of GmWRKY genes, especially elements related to phytohormones, including ABA (ABRE), auxin (AuxRR-core, TGA element), gibberellin (GARE motif, P-box, and TATC-box), ethylene (ERE), MeJA (CGTCA motif and TGACG motif) and salicylic acid (TCA element and SARE). In rice, OsARF12 can bind to the AuxRE in the OsWRKY13 promoter to activate its expression, which play a role in rice antiviral immune response to rice dwarf virus [63]. In cotton, GhTINY2 could promote the expression of WRKY51 through binding to the ERE in its promoter, thereby improving resistance to Verticillium dahlia [64]. In tomato, SIWRKY33 can bind to the TCA element at its own promoter to activate its transcription, and enhance the response to cold stress [65]. Additionally, many other cis-elements, such as W-box, LTR, and MBS, also occur in the promoters of GmWRKYs. Therefore, these findings suggest that a variety of TFs perform functions through regulating the expression levels of GmWRKY genes in plant development and stress responses, but their regulatory mechanism needs to be further explored.

Soybean mosaic disease caused by SMV is an important factor leading to severe soybean yield decline and seed quality deterioration [1]. However, the molecular mechanism of soybean resistance to SMV infection is unclear. Numerous studies have shown that the deposition of callose on the plasmodesmata is the main way to against virus invasion [13, 66]. Additionally, H₂O₂ also play important roles in the plant immune response [29, 62, 67]. In our previous study, it was shown that the decreased callose deposition on the plasmodesmata, due to the inhibition of H₂O₂ production, elevated the diffusion of SMV [16]. In this study, 60 GmWRKY genes were differentially expressed during SMV infection according to the H₂O₂-associated transcriptome data; among them, 45 belonged to Group II, indicating that WRKY genes in Group II play key roles in resistance to biotic stresses. For example, the overexpression of OsWRKY genes in Group IIa in rice enhances resistance to Xanthomonas oryzae [68]. In Arabidopsis, WRKY IIa members, including WRKY18, WRKY40, and WRKY60, play positive roles in resistance to Pseudomonas syringae [69]. In our study, we found that GmWRKY162 play a positive role in suppressing virus spread and increasing callose deposition during SMV infection through targeting GmGSL7c. In summary, this study provides valuable insights into the roles of the WRKY gene family in response to SMV, as well as guidance for future studies in understanding virus-resistance mechanisms in soybean.

In conclusion, we identified and comprehensively analyzed 185 GmWRKY genes in soybean, which were divided into 7 GmWRKY subfamilies and unevenly positioned on 20 chromosomes. WRKY members in the same subfamily or evolutionary branch exhibit extensive similarity. Cis-acting element and gene expression analyses revealed the possible involvement of the identified GmWRKY genes in response to SMV infection. Sixty WRKY genes were differentially expressed during SMV infection according to RNA-seq data. Among them, the expression of GmWRKY162 decreased after imidazole treatment in incompatibility combination in soybean with SMV strain N3, which was confirmed by RT-qPCR. Remarkably, the silencing of GmWRKY162 reduced callose deposition and enhanced virus spreading during SMV infection. Next, EMSA and ChIP-qPCR revealed that GmWRKY162 can directly bind to the promoter of GmGSL7c, which is involved in callose synthesis, as proven in our previous published study. Our findings highlighted the important role of GmWRKY162 in resistance to SMV, demonstrating its involvement in callose deposition and virus spreading.

Acknowledgments

We thank Dr. Haijian Zhi (Nanjing Agricultural University, China) for giving the virus strains and the soybean Nannong 1138-2, and Prof. Mengchen Zhang (Institute of Cereal and Oil Crops, Hebei Academy of Agriculture and Forestry Sciences, China) for giving the soybean Jidou 7. We also give special thanks to Prof. Chunji Liu (CSIRO Plant Industry, Australia) for the suggestions in the writing and revision of the manuscript.

Authors’ contributions

DMW and JZ designed the research and supervised the work. ZHZ, RNW, WHS, TJS, FJW, performed the experiments. ZHZ, RNW, WHS, ZLY, FMX, LY and CYY analyzed the data. ZHZ, RNW, WHS, TJS, and ZLY wrote and revised the manuscript. All authors read and approved the final version for publication.

Funding

This work was supported by the Natural Science Foundation of Hebei (grant no. C2023204093; C2020204132), the Industry and Technology System for Modern Agriculture in Hebei province (grant no. HBCT2023040101, HBCT2023040202, and HBCT2023040203), the Special Subproject of Scientific and Technological Innovation of Modern Seed Industry in Hebei Province (grant no. 21326313D-1).

Availability of data and materials

The latest version of the soybean genome file was downloaded from the Ensembl database (https://plants.ensembl.org/index.html). The seed used in this experiment were Nannong 1138-2 and Jidou 7. The SMV strains were N3 and SC8. The datasets supporting the conclusions of this study are included in the article and in additional files.

Ethics approval and consent to participate

Not applicable

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

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Genome-wide identification of WRKY genes in soybean and functional analysis of GmWRKY162-GmGSL7c in resistance to soybean mosaic virus

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Abstract

Figures

Background

Materials and Methods

Soybean planting and inoculation

Identification and chromosomal mapping of WRKY genes in soybean

Phylogenetic analysis, gene structure, conserved motif, and cis-element analyses

Gene collinearity, duplication events, and protein interaction analysis

Total RNA isolation, transcriptome data, and RT‒qPCR

Observation of SMV-induced callose and detection of SMV CP transcript products

Electrophoretic mobility shift assay

Results

Phylogenetic classification of the GmWRKYs

Duplication events and syntenic analysis of GmWRKY members

Discussion

Conclusion

Declarations

References

Additional Declarations

Supplementary Files

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Version 1