GmSnRK1.1, a Sucrose Non-fermenting-1(SNF1)-Related Protein Kinase, Promotes Soybean Resistance to Phytophthora sojae

Phytophthora root and stem rot, a destructive disease of soybean [Glycine max (L.) Merr.], is caused by the oomycete Phytophthora sojae. However, how the disease resistance mechanisms of soybean respond to P. sojae infection remains unclear. Previously, we showed that GmWRKY31, which interacts with a sucrose non-fermenting-1(SNF1)-related protein kinase (SnRK), enhances resistance to P. sojae in soybean. Here, we report that the membrane-localized SnRK GmSnRK1.1 is involved in the soybean host response to P. sojae. The overexpression of GmSnRK1.1 (GmSnRK1.1-OE) increased soybean resistance to P. sojae, and the RNA interference (RNAi)-mediated silencing of GmSnRK1.1 (GmSnRK1.1-R) reduced resistance to P. sojae. Moreover, the activities and transcript levels of the antioxidant enzymes superoxide dismutase and peroxidase were markedly higher in the GmSnRK1.1-OE transgenic soybean plants than in the wild type (WT), but were reduced in the GmSnRK1.1-R plants. Several isoflavonoid phytoalexins related genes GmPAL, GmIFR, Gm4CL and GmCHS were significantly higher in “Suinong 10” and GmSnRK1.1-OE lines than these in “Dongnong 50,” and were significantly lower in GmSnRK1.1-R lines. In addition, the accumulation of salicylic acid (SA) and the expression level of the SA biosynthesis-related gene were significantly higher in the GmSnRK1.1-OE plants than in the WT and GmSnRK1.1-R plants, moreover, SA biosynthesis inhibitor treated GmSnRK1.1-R lines plants displayed clearly increased pathogen biomass compared with H2O-treated plants after 24 h post-inoculation. These results showed that GmSnRK1.1 positively regulates soybean resistance to P. sojae, potentially functioning via effects on the expression of SA-related genes and increased accumulation of SA.

SnRK1 regulates carbon metabolism (Halford and Hardie, 1998;Fragoso et al., 2009;Nunes et al., 2013;Zhai et al., 2017) and responds to hormonal signals, particularly abscisic acid (ABA), providing a possible link between the hormone and sugar signaling pathways (Radchuk et al., 2006(Radchuk et al., , 2010Jossier et al., 2009;Coello et al., 2012;Tsai and Gazzarrini, 2012;Rodrigues et al., 2013). ABA negatively regulates resistance to P. sojae and active levels are depleted as part of the response to incompatible soybean genotypes (McDonald and Cahill, 1999;Mohr and Cahill, 2001;Asselbergh et al., 2007). Moreover, in wheat, SnRK1 is negatively regulated by ABA (Patricia et al., 2012). Moreover, SnRK1 regulates plant metabolism in response to stresses such as darkness and flooding, as well as developmental changes such as flowering, seed germination, and seedling growth (Baena-González et al., 2007;Jossier et al., 2009;Lee et al., 2009;Coello et al., 2011;Cho et al., 2012;Wu et al., 2017). In Arabidopsis thaliana, SnRK1 is involved in the responses to sugar and darkness by regulating the expression of stress-responsive genes and ABA signaling (Baena-González et al., 2007;Jossier et al., 2009). SnRK1 activities in rice (Oryza sativa) and Arabidopsis have a decisive influence on the expression of stress-inducible genes and the induction of stress-tolerance processes (Cho et al., 2012); for example, the rice protein kinase CIPK15 regulates carbohydrate catabolism and fermentation via the SnRK1A-MYBS1-mediated sugar signaling pathway, enabling rice to grow under floodwater (Lee et al., 2009). In Arabidopsis, FUS3 interacts with SnRK1.1 to regulate lateral organ development (Tsai and Gazzarrini, 2012), but also promotes dormancy and inhibits germination through crossregulation of the ABA and gibberellin pathways (Gazzarrini and Tsai, 2015). Under low-sugar conditions, Arabidopsis SnRK1 was triggered to phosphorylate and inactivate the INDETERMINATE DOMAIN (IDD)-containing transcription factor IDD8, thereby leading to delayed flowering (Jeong et al., 2015). These discoveries show that SnRK1 coordinates the responses to a wide array of abiotic stresses (Baena-González et al., 2007;Lee et al., 2009;Cho et al., 2012;Jeong et al., 2015). Relatively little is known about the mechanisms by which SnRK1 functions in the responses to biotic stress. The overexpression of SnRK1 in tobacco (Nicotiana sp.) made the transgenic plants more resistant to geminivirus infection (Hao et al., 2003). SnRK1 interacts with the effector AvrBsT, which is involved in suppression of the AvrBs1-specific hypersensitive response in pepper (Capsicum annuum) plants (Szczesny et al., 2010). The rice SnRK1b gene OSK35 was enhanced the plant resistance to fungal and bacterial pathogens (Kim et al., 2015). Despite these insights, no systematic research on the disease-related roles of SnRK1 in another major crop species, soybean (Glycine max), has been reported.
In a previous study, we showed that a novel WRKY transcription factor, GmWRKY31, enhances soybean resistance to P. sojae, and identified 19 putative GmWRKY31-interacting proteins (Fan et al., 2017), of which a Sucrose non-Fermenting-1-Related Protein Kinase (SnRK1) was selected for further study. In the present study, we isolated a GmWRKY31-interacting GmSnRK1.1 (GenBank accession no. XM_006585690), and generated transgenic soybean plants either overexpressing GmSnRK1.1 (GmSnRK1.1-OE) or with an RNA-interference (RNAi)-mediated reduced expression of this gene (GmSnRK1.1-R). Overexpression and RNA interference analysis demonstrates that GmSnRK1.1 positively regulates of soybean resistance to this pathogen, likely via a SA-signaling pathway.

Plant Materials and Growth Conditions
The soybean cultivar used for the various treatments and gene isolation was "Suinong 10, " which is highly resistant to P. sojae race 1 (PSR01) isolated in Heilongjiang, China (Zhang et al., 2010). The susceptible soybean cultivar "Dongnong 50" was used for the gene transformation experiments. These lines were obtained from the Key Laboratory of Soybean Biology in the Chinese Ministry of Education, Harbin. PSR01 was previously isolated from infected soybean plants in Heilongjiang, China (Zhang et al., 2010). This isolate was propagated at 25 • C for 7 days on V8 juice agar in a glass dish. The seeds of "Suinong 10" and "Dongnong 50" were grown at 25 • C and 60% relative ambient humidity in a growth cabinet, with a 16-h light/8-h dark photoperiod. For the P. sojae infection, the hypocotyls of soybean cultivars "Suinong 10" and "Dongnong 50" were inoculated at the first-node stage (V1) (Fehr et al., 1971) using either zoospores of P. sojae or a mock inoculation with sterile water following the procedure described by Kaufmann and Gerdemann (1958), with minor modifications. The P. sojae zoospores were induced as described by Ward et al. (1979), and the concentration of zoospores was estimated to be about 1 × 10 5 spores mL −1 using a hemacytometer. The leaves of the inoculated plants were harvested and immediately frozen in liquid nitrogen at 0, 1, 3, 6, 9, 12, 24, and 48 h after the treatment, and stored at −80 • C until required for RNA extraction.

Isolation of the GmSnRK1.1 Gene
The full-length cDNA of GmSnRK1.1 (GenBank accession no. XM_006585690) was isolated from soybean "Suinong 10" using RT-PCR with the primers GmSnRK1.1-F/R (see Supplementary  Table S1). The extraction of total RNA and reverse transcription were performed using TRIzol reagent (Invitrogen, China) and ReverTra Ace Kit (Toyobo, Japan). The products of the RT-PCR amplification were cloned into a pMD-18T vector (Takara Bio, Japan), transformed into Escherichia coli DH5α cells (TransGen Biotech, China), and sequenced by GENEWIZ (China). DNAMAN software 1 was used for the sequence alignments, and a phylogenetic analysis of GmSnRK1.1 was carried out using MEGA5 software. The GmSnRK1.1 protein structure was analyzed using the online program Phyre2 2 .

qRT-PCR Analysis
A qRT-PCR analysis was performed to confirm the transcript levels of GmSnRK1.1 using a LightCycler96 instrument (Roche, Switzerland) with a real-time PCR kit (TOYOBO, Japan). GmEF1β (GenBank accession no. NM_001248778) was used as the internal control (see Supplementary Table S1 for primers). The relative transcript abundance of the target gene was calculated using the 2 − CT method. Three biological replications were performed for each line in each analysis.

Pull-Down Assays
GmSnRK1.1 was cloned into the pET29b (+) expression vector (Merck Millipore, United States), while GmWRKY31 was cloned into the pGEX-4T-1 expression vector (GE Healthcare, United States). The His-GmSnRK1.1 and glutathione S-transferase-GmWRKY31 proteins were separately produced 2 http://www.sbg.bio.ic.ac.uk/phyre2 in E. coli BL21 (DE3) cells, then harvested and purified using a GST-Sefinose kit (Sangon, China) or a His-bind Purification Kit (Merck Millipore). The pull-down assay was performed as described by Yang et al. (2008), with minor modifications. In a total volume of 1 mL GST binding buffer (Sangon), the GST or GmWRKY31-GST recombinant proteins were incubated for 1 h at 4 • C with 400 µL GST resin (Sangon), after which equal volumes of the GmSnRK1.1-His recombinant protein were added and incubated for 6 h at 4 • C. The binding reaction was washed five times with binding buffer, each for 10 min at 4 • C, then the pulled-down proteins were eluted by boiling, separated on a 12% SDS-PAGE gel, and immunoblotted with anti-His antibody and anti-GST antibody (Abmart, United States).

Subcellular Localization Assays of the GmSnRK1.1 Protein
The full-length GmSnRK1.1 sequence was cloned using RT-PCR with the primers GmSnRK1.1GF and GmSnRK1.1GR (listed in Supplementary Table S1). The coding sequence under the control of the constitutive CaMV 35S promoter was fused to the N-terminus of the green fluorescent protein (GFP). The resulting 35S:GmSnRK1.1-GFP expression plasmid (or the 35S:GFP control) was transformed into Arabidopsis protoplast cells using a PEG-mediated transfection, as described by Yoo et al. (2007). The fluorescence signal was mapped using a TCS SP2 spectral confocal microscopic imaging system (Leica Microsystems). The 514 nm Ar/ArKr laser was used for GFP and Chlorophyll. GFP and Chlorophyll were excitated at 488 nm. The wavelength range of captured light was 500-530 nm for GFP, and 650-750 nm for Chlorophyll.

Vector Construction and Transformation of Soybean
For the generation of the overexpression lines, a 4 × myc sequence was synthesized (GENEWIZ) and inserted into a pCAMBIA3301 vector to generate a pCAMBIA3301-4 × Myc plasmid, the GmSnRK1.1 coding sequence was inserted into the BglII/SpeI site of the plasmid, and the 4 × myc and bar sequences were later used as markers. The GmSnRK1.1 cDNA fragment was amplified using the primers GmSnRK1.1-R-F/R and inserted into the vector PFGC5941 (Kerschen et al., 2004). The constructs were transformed into Agrobacterium tumefaciens (strain LBA4404) using the freeze-thaw method (Holsters et al., 1978). "Dongnong 50" was previously used for Agrobacterium-mediated genetic transformation (Paz et al., 2004). Transgenic soybean plants were preliminarily verified using a PCR amplification and qPCR analysis, after which a Western blot with an anti-myc antibody (Abmart) was used to identify the plants overexpressing GmSnRK1.1.

Assessment of Pathogen Resistance and the Disease Response
For pathogen infection, the living cotyledons of the WT and transgenic soybean plants at the V1 stage of development were infected with P. sojae zoospores (approximately 1 × 10 5 spores mL −1 ) using the methods described by Morrison and Thorne (1978), and the roots inoculation was performed using the procedure described by Zhang et al. (2017). The disease symptoms on the infected cotyledons and roots were observed and photographed with a Nikon D7000 camera. ImageJ 3 was used to measure the lesions of the infected cotyledons. The P. sojae biomass was quantified based on the accumulation of P. sojae TEF1 (GenBank accession no. EU079791) in the soybean plants, relative to the levels of GmEF1β, as previously described by Chacón et al. (2010). The pathogen response assays were performed on three biological replicates, each with three technical replicates.

Determination of Antioxidant Enzyme Activity
For the enzyme assays, the total proteins were extracted from approximately 0.1 g of leaves using 1 mL ice-cold 25 mM HEPES buffer (pH 7.8) containing 0.2 mM EDTA, 2 mM ascorbate, and 2% polyvinylpyrrolidone. The homogenates were centrifuged at 4 • C for 15 min at 12,000 × g, after which the supernatants 3 https://imagej.nih.gov/ij/index.html were carefully removed and used for the enzymatic activity measurements. The superoxide dismutase (SOD) and peroxidase (POD) activities were assayed as described by Wang et al. (2011).

GmSnRK1.1 Interacts With GmWRKY31
Using yeast two-hybrid assays, GmWRKY31 was found to interact with GmSnRK1.1 (Figure 1A), which was further confirmed using a BiFC assay demonstrating that GmSnRK1.1 can interact with GmWRKY31 in the nuclei of Arabidopsis protoplast cells ( Figure 1C). In accordance with the results of the BiFC assay, a glutathione S-transferase pull-down assay showed that the His-tagged GmSnRK1.1 recombinant protein Frontiers in Plant Science | www.frontiersin.org was pulled down by GST-GmWRKY31, but not by GST alone (Figure 1B), further indicating that GmWRKY31 interacts with GmSnRK1.1 in vitro. These results suggest that GmWRKY31 directly interacts with GmSnRK1.1.

Sequence Analysis of GmSnRK1.1
The full-length GmSnRK1.1 cDNA is 1,990 bp long and contains a 1,533 bp open reading frame, which encodes a polypeptide of 510 amino acids (Supplementary Figure S2) Figures S1B,C). The structure of GmSnRK1.1 was analyzed using Phyre, predicting that it functions as a heterotrimer complex, in which the catalytic α subunit combines FIGURE 2 | Expression patterns of GmSnRK1.1 in Phytophthora sojae-resistant and P. sojae-susceptible soybean cultivars. (A) The tissue-specific expression patterns of GmSnRK1.1 in resistant cultivar "Suinong 10" (SN10) and susceptible cultivar "Dongnong 50" (DN50) under normal conditions. (B) Relative expression of GmSnRK1.1 in soybean cultivars 'Suinong 10" and "Dongnong 50" on P. sojae infection. The infected samples were collected at 0, 1, 3, 6, 9, 12, 24, 36 and 48 h after P. sojae infection. The relative expression levels of GmSnRK1.1 were compared with those of mock-treated plants (plants treated with sterile water) at the same time point. Fourteen-day-old soybean plants were used for analysis. The housekeeping gene of soybean GmEF1β (NM_001248778) was used as an internal control to normalize the data. The experiment was performed on three biological replicates, each with three technical replicates, and was statistically analyzed using Student's t-test ( * P < 0.05, * * P < 0.01). Bars indicate the standard error of the mean. with a β regulatory subunit and an activating γ subunit (Supplementary Figure S1C).

GmSnRK1.1 Expression Is Significantly Induced by P. sojae
To evaluate the responsiveness of GmSnRK1.1 to biotic stresses in the "Dongnong 50" and "Suinong 10" soybean cultivars, its temporal and spatial patterns were investigated using qRT-PCR. The examination of the tissue-specific transcript levels in these cultivars revealed that GmSnRK1.1 was highly expressed in the stems, followed by the roots and cotyledons (Figure 2A). In "Suinong 10" plants inoculated with P. sojae, the GmSnRK1.1 mRNA levels increased to a peak level at 9 h after inoculation, followed by a decline (Figure 2B). A similar pattern was observed in "Dongnong 50, " although the relative expression level of GmSnRK1.1 was significantly higher in "Suinong 10" than in "Dongnong 50" (Figure 2B).

Subcellular Localization of the GmSnRK1.1 Protein
The subcellular localization of the GmSnRK1.1 protein was analyzed in Arabidopsis protoplasts producing a GmSnRK1.1-GFP fusion protein under the control of the 35S promoter. As shown in Figure 3, GFP fluorescence was distributed throughout the cells expressing the 35S:GFP control plasmid. In contrast, the GmSnRK1.1-GFP fusion protein was exclusively localized to the Arabidopsis cell membrane, resembling the pattern of the membrane-localized GmDIR22-GFP fusion protein  used as a control. These results indicated that GmSnRK1.1 is a membrane-localized protein.

GmSnRK1.1 Enhances Resistance to P. sojae in Transgenic Soybean Plants
To analyze the function of GmSnRK1.1 in response to infection by P. sojae, we generated GmSnRK1.1-OE and GmSnRK1.1-R FIGURE 3 | Analysis of the subcellular localization of GmSnRK1.1-GFP protein in Arabidopsis protoplasts. Subcellular localization was investigated in Arabidopsis mesophyll protoplasts under a confocal microscope. The fluorescent distribution of humanized hGFP, the fusion protein GmSnRK1.1-hGFP, GmDIR22-hGFP and GmBTB/POZ-hGFP were observed under white light, UV light, and red light, respectively. Bars, 10 µm. transgenic soybean plants, which were developed into transgenic T 3 lines. the expression of GmSnRK1.1 in three positive overexpressing transgenic soybean lines using Western blot ( Figure 1D). The resistance of the T 3 transgenic plants to P. sojae was tested in their cotyledons and roots. A notable difference was observed in the development of disease symptoms after a 96 h incubation with zoospores of P. sojae. In the GmSnRK1.1-R lines, the cotyledons exhibited clear water-soaked lesions and were softer than the WT, however, almost no disease symptoms were observed in the GmSnRK1.1-OE lines (Figure 4A). In addition, the P. sojae biomass (indicated by the relative abundance of TEF1 genomic sequence per area of infected living cotyledon) was significantly (P < 0.01) lower in the GmSnRK1.1-OE lines than in the WT plants, but higher in the GmSnRK1.1-R lines ( Figure 4B). The lesion areas of the GmSnRK1.1-OE lines were significantly (P < 0.01) smaller than that of the WT (Figure 4C), but significantly larger in the GmSnRK1.1-R lines. Similar results were obtained after a 6-d incubation with P. sojae. The living roots of the WT soybean plants and GmSnRK1.1-R soybean lines exhibited watery lesions and even rotting, while those of the GmSnRK1.1-OE lines remained healthy (Figure 5). Similar to the results of infecting living cotyledon, the biomass of P. sojae after 6 days of roots infection was significantly reduced in the GmSnRK1.1-OE lines, but significantly increased in the GmSnRK1.1-R lines, relative to the WT. These results indicated that overexpression of GmSnRK1.1 in soybean plants  .1-R transgenic lines and WT soybean based on P. sojae TEF1 transcript levels. The experiment was performed on three biological replicates, each with three technical replicates, and statistically analyzed using Student's t-test ( * P < 0.05, * * P < 0.01). Bars indicate the standard error of the mean.
Frontiers in Plant Science | www.frontiersin.org enhances their resistance to P. sojae infection. In addition, we have constructed the overexpression vector of kinaseinactive GmSnRK1.1 by synthesizing the mutation sequence of phosphorylation site (Thr157Ala, Thr235Ala, Thr261Ala). Furthermore, the overexpression of kinase-inactive GmSnRK1.1, the overexpression of GmSnRK1.1, and vector control transgenic soybean hairy roots generated by Agrobacterium rhizogenesmediated transformation will be used to investigate the effect of the GmSnRK1.1's kinase activity on resistance to P. sojae in soybean. The results demonstrated that GmSnRK1.1's kinase activity could increase the resistance to P. sojae in soybean (Supplementary Figure S5).

Overexpression of GmSnRK1.1 Affects Antioxidant Enzyme Activity
The antioxidant defense system is well-developed in plants, involving the scavenging of reactive oxygen species (ROS) by SOD and POD (Du et al., 2001). We analyzed the SOD and POD activities in the transgenic and WT soybean plants inoculated with P. sojae, as well as the expression of the associated genes GmSOD1 (NM_001248369) and GmPOD (XM_006575142). Under both the mock treatment and at 24 h after inoculation with P. sojae, the activity levels of SOD and POD, as well as the transcript abundance of the associated genes, were significantly higher in the GmSnRK1.1-OE lines than in the WT, but were significantly reduced in the GmSnRK1.1-R lines (Figure 6). These results suggested that GmSnRK1.1 increases the activities of the antioxidant enzymes in soybean plants in response to P. sojae infection.

GmSnRK1.1 Regulates the Expression of Defense-Associated Genes in Response to P. sojae Infection
Race-specific resistance to P. sojae has been shown to be mediated by isoflavonoid phytoalexins in other soybean varieties (Subramanian et al., 2005;Graham et al., 2007;Cheng et al., 2015;Zhang et al., 2017). We measured the expressions of several isoflavonoid phytoalexins genes, including GmPAL (GenBank accession no. NM_001250027), GmIFR (GenBank accession no. NM_001254100), Gm4CL (GenBank accession no. NM_001256363.1) and GmCHS (GenBank accession no. XM_003518780). These results indicated that isoflavonoid phytoalexins related genes were significantly higher in "Suinong 10" and GmSnRK1.1-OE lines than these in "Dongnong 50, " and were significantly lower in GmSnRK1.1-R lines (Figure 7). Moreover, we next monitored the expression levels of GmSnRK1.1 during P. sojae infection using qRT-PCR. The transcript levels of GmSnRK1.1 were significantly higher in the GmSnRK1.1-OE plants than in the WT under both the mock treatment and a 24 h infection with P. sojae, but were FIGURE 6 | Analysis of antioxidant enzyme activity (A,B) and the relative expression of genes (C,D) under mock treatment and infected by Phytophthora sojae at 24 h post-inoculation (hpi). The activity of the control sample [mock-treated wild-type (WT) plants] was set to unity. The experiment was performed on three biological replicates, each with three technical replicates, and statistically analyzed using Student's t-test ( * P < 0.05, * * P < 0.01). Bars indicate the standard error of the mean. POD, peroxidase; SOD, superoxide dismutase. FIGURE 7 | The relative transcript levels of isoflavonoid phytoalexins related genes in "Suinong 10" (SN10), "Dongnong 50" (DN50) and GmSnRK1.1 transgenic soybean plants. The housekeeping gene of soybean GmEF1β was used as an internal control to normalize the data. The experiment was performed on three biological replicates with their respective three technical replicates and statistically analyzed using Student's t-test ( * P < 0.05, * * P < 0.01). Bars indicate the standard error of the mean.
significantly lower in the GmSnRK1.1-R lines (Supplementary Figure S4). Pathogen-related proteins are key members in the plant response to pathogen infection ( Van Loon and Van Strien, 1999;Loon et al., 2006;Xu et al., 2014). To explore the possible mechanisms of the GmSnRK1.1-regulated resistance to P. sojae, we detected the transcriptional levels of various defense-response genes, including GmWRKY31, GmNPR1 (GenBank accession no. NM_001251745.1), GmPR1 (GenBank accession no. AF136636), and GmPR5 (GenBank accession no. M21297). As shown in Supplementary Figure S4, after 12 h incubation with P. sojae, the expression levels of these resistance-related genes were significantly more highly expressed in the GmSnRK1.1-OE plants than in the WT, but were significantly lower in the GmSnRK1.1-R plants. In contrast, no significant differences in the expression of GmPR10 (GenBank accession no. FJ960440) were detected between these lines. These results indicate that one mechanism by which GmSnRK1.1 enhances soybean defense against P. sojae is by regulating the expression of the defense-related genes.

GmSnRK1.1 Affects SA Accumulation and the Expression of the SA Biosynthesis Genes
To test whether the GmSnRK1.1 could regulate the accumulation of SA, the SA contents of the T 3 GmSnRK1.1 transgenic lines were evaluated. As shown in Figure 8A, the GmSnRK1.1-OE transgenic soybean leaves contained significantly more SA than the WT leaves, while the GmSnRK1.1-R plants accumulated significantly less SA. we also analyzed the transcript levels of GmICS1 (XM_003522145), which plays a key role in SA biosynthesis. GmICS1 was significantly more highly expressed in the GmSnRK1.1-OE transgenic lines than in the WT, while the GmSnRK1.1-R transgenic lines has a significantly lower level of GmICS1 expression ( Figure 8B). Furthermore, whether or not the defense mechanism is dependent on SA, we measured the expression level of GmSnRK1.1 and the relative biomass of P. sojae in "Suinong 10, " "Dongnong 50" and GmSnRK1.1 transgenic soybean lines treat with sterile water and SA biosynthesis inhibitor (100 µM 1-aminobenzotriazole). As expected, SA biosynthesis inhibitor treated plants displayed clearly increased pathogen biomass compared with H 2 O-treated plants after 24 h post-inoculation ( Figure 8D). These results suggested that GmSnRK1.1 plays a positive important role in the response to P. sojae infection, increasing the diseaseresistance of soybean via a possible mechanism involving the SA signaling pathway.

DISCUSSION
Identification and characterization of genes involved in response to P. sojae infection in soybean has contributed to our understanding of the genetic mechanisms of resistance (Xu et al., 2014;Cheng et al., 2015;Kong et al., 2015;Zhang et al., 2017). In our previous study, GmWRKY31 was found to play a key role in increasing the disease resistance of soybean plants to P. sojae infection (Fan et al., 2017). In the present study, we identified and functionally characterized GmSnRK1.1 as an interacting partner of GmWRKY31, demonstrating that it plays a positive role in the response to P. sojae infection. The SnRK1 protein kinases are considered central regulators of the energy metabolism and stress signaling in plants (Baena-González and Sheen, 2008;Radchuk et al., 2010;Tsai and Gazzarrini, 2012), however, the biological functions of the SnRK1s in soybean are poorly understood. Here, we first discovered that the overexpression of GmSnRK1.1 significantly increased the plant responses to P. sojae infection, while RNA-interference mediated reduced expression of this gene significantly increased the susceptibility of the transgenic plants to this pathogen (Figures 4, 5).
Previous studies have confirmed that the SnRK1s play a role in the plant defense against viruses, fungi, and bacteria (Hao et al., 2003;Szczesny et al., 2010;Kim et al., 2015); for example, the overexpression of SnRK1 was shown to significantly increase the resistance of tobacco plants to geminivirus infection (Hao et al., 2003). In rice, OSK35 (a rice SnRK1b gene) positively regulates the disease resistance of plants subjected to the fungal pathogen Magnaporthe oryzae and the bacterial pathogen Xanthomonas oryzae pv. oryzae (Xoo) (Kim et al., 2015). The wheat (Triticum aestivum) alpha subunit of TaSnRK1a interacts with TaFROG (Fusarium Resistance Orphan Gene) to increase the disease resistance response to the mycotoxigenic fungus Fusarium graminearum (Perochon et al., 2015). The hypersensitive response was induced by the Xanthomonas campestris pv. vesicatoria effector AvrBs1 in snrk1 mutant pepper plants (Szczesny et al., 2010). In this work, the temporal and spatial patterns of GmSnRK1.1 expression were analyzed in the P. sojae-resistant soybean cultivar "Suinong 10" and the susceptible cultivar "Dongnong 50, " revealing that GmSnRK1.1 was markedly expressed in the stems of these lines, with lower expression levels in the roots and cotyledons (Figure 2A). We detected that the expression levels of GmSnRK1.1 following P. sojae infection were much higher in "Suinong 10" than in the susceptible cultivar "Dongnong 50, " and that P. sojae infection markedly increased the expression levels of GmSnRK1.1 in both cultivars ( Figure 2B). In addition, GmSnRK1.1 is negatively regulated by ABA (Supplementary Figure S3). We further demonstrated that the GmSnRK1.1-OE transgenic plants had an increased disease resistance to P. sojae infection, while the GmSnRK1.1-R transgenic plants exhibited increased susceptibility (Figures 4, 5).
Further research showed that GmSnRK1.1 positively regulates the activities and transcription levels of antioxidant enzymes SOD and POD (Figure 6). SOD constitutes the first line of defense against ROS within a cell (Alscher et al., 2002), while POD plays a key role in scavenging ROS and preventing cellular damage (Tewari et al., 2006). Therefore, we further analysis the activities and transcription level of SOD and POD, In plants infected with P. sojae, GmSnRK1.1 positively enhanced the activities and transcription levels of SOD and POD, enabling them to scavenge the ROS and provide sufficient protection against oxidative damage.
The phytohormones SA, jasmonic acid, and ethylene play central roles in regulating the plant responses to pathogen attack (Reymond and Farmer, 1998;Kunkel and Brooks, 2002;Spoel et al., 2003;Robert-Seilaniantz et al., 2011;Alazem and Lin, 2015). SA mediates and activates the biotic stress response to pathogenic challenge (Pieterse et al., 2009;Sugano et al., 2013;Alazem and Lin, 2015), with the transcriptional cofactor NPR1 playing a key role in the SAsignaling pathway of several plant species (Vlot et al., 2009). In rice, OsSnRK1a positively regulates plant resistance by linking to the SA pathway (Filipe et al., 2018). In previous studies, the overexpression of GmWRKY31 was found to induce the expression of GmNPR1, increasing the disease resistance of soybean plants in response to P. sojae infection via the activation of the SA-signaling pathway (Fan et al., 2017). The results of this study supported these findings, as the expression levels of GmWRKY31 and GmNPR1 were markedly higher in the GmSnRK1.1-OE transgenic plants in comparison with the WT and lower in the GmSnRK1.1-R transgenic plants (Supplementary Figure S4). A downstream member of the SnRK1 signaling pathway, STOREKEEPER RELATED1/G-Element Binding Protein (STKR1), was previously found to display transcriptional changes which constitutively activated the SA-related defense in transformed Arabidopsis plants (Nietzsche et al., 2018). We also found that the overexpression of GmSnRK1.1 induced the expression of GmPR1 and GmPR5, which are effector genes for the systemic acquired resistance response, a process mediated by SA (Ward et al., 1991;He et al., 2007). The high expression levels of these genes indicated that SA signaling was activated in the GmSnRK1.1-OE plants, which was confirmed by our determination that SA accumulation and the expression of the SA biosynthesis gene GmICS1 were upregulated in these plants relative to the WT (Figure 8). In addition, we examined the expression level of GmSnRK1.1 and relative biomass of P. sojae in "Suinong 10, " "Dongnong 50" and GmSnRK1.1 transgenic soybean lines treated with H 2 O (mock) and SA biosynthesis inhibitor (100 µM ABT) under P. sojae infection, the results showed that SA is involved in GmSnRK1.1-mediating defense to P. sojae (Figure 8). These indicated that GmSnRK1.1 promotes the accumulation of SA and the expression of GmICS1, and suggests that GmSnRK1.1 acts as a positive regulator of the downstream defense pathways and SAdependent defense signaling.
In Glycine soja, a ABA activated calcium independent SnRKtype kinase, GsAPK, was localized in the plasma membrane (Liang et al., 2012). In Arabidopsis, SnRK1 is localized to the plant nucleus and endoplasmic reticulum (Blanco et al., 2019). In this work, GmSnRK1.1 was localized in the plasma membrane (Figure 3), and GmWRKY31 was localized to the plant nucleus (Fan et al., 2017). The BiFC assay showed that GmSnRK1.1 interacted with GmWRKY31 in the nucleus (Figure 1), but the mechanism of nuclear interaction is not clear which require further study and discussion.

DATA AVAILABILITY
All datasets generated for this study are included in the manuscript and/or the Supplementary Files.  Fourteen day-old plants were used for the treatments and analyses. The housekeeping gene of soybean GmEF1β was used as an internal control to normalize the data. The relative transcript levels of GmSnRK1.1 were quantified compared with mock plants at the same time points. The experiment was performed on three biological replicates with their respective three technical replicates and statistically analyzed using Student's t-test ( * P < 0.05, * * P < 0.01). Bars indicate the standard error of the mean. FIGURE S4 | Relative expression levels of defense-associated genes in soybean plants under mock treatment and infected by Phytophthora sojae at 24 h post-inoculation (hpi). The housekeeping gene of soybean GmEF1β was used as an internal control to normalize the data. The expression level of the control sample [mock-treated wild-type (WT) plants] was set to unity. The experiment was performed on three biological replicates, each with three technical replicates, and was statistically analyzed using Student's t-test ( * P < 0.05, * * P < 0.01). Bars indicate the standard error of the mean. FIGURE S5 | The effect of GmSnRK1.1 kinase assay on the resistance to P. sojae. (A) Mutation of GmSnRK1.1 activation sites. (B) Disease symptoms on the hairy roots of the overexpression of GmSnRK1.1 (GmSnRK1.1-OE), the overexpression of kinase-inactive GmSnRK1.1 (GmSnRK1.1 mutation-OE), and empty vector control (3301), GmSnRK1.1 RNA interference (RNAi)-mediated silencing (GmSnRK1.1-R) transgenic soybean at 4 days after inoculation with Phytophthora sojae. (C) Quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis of the relative biomass of P. sojae in GmSnRK1.1-OE, GmSnRK1.1 mutation-OE, GmSnRK1.1-R, and 3301 empty vector transgenic soybean hairy roots based on P. sojae TEF1 transcript levels. The experiment was performed on three biological replicates, each with three technical replicates, and statistically analyzed using Student's t-test ( * P < 0.05, * * P < 0.01). Bars indicate the standard error of the mean. (D) Transgenic soybean hairy roots were tested using Liberty Link strips.

AUTHOR CONTRIBUTIONS
TABLE S1 | Primer sequences used in this study.