Abstract
Soybean cyst nematode (SCN; Heterodera glycines Ichinohe), one of the most devastating soybean (Glycine max) pathogens, causes significant yield loss in soybean production. Nematode infection triggers plant defense responses; however, the components involved in the upstream signaling cascade remain largely unknown. In this study, we established that a mitogen-activated protein kinase (MAPK) signaling module, activated by nematode infection or wounding, is crucial for soybeans to establish SCN resistance. GmMPK3 and GmMPK6 directly interact with CDG1-LIKE1 (GmCDL1), a member of the receptor-like cytoplasmic kinase (RLCK) subfamily VII. These kinases phosphorylate GmCDL1 at Thr-372 to prevent its proteasome-mediated degradation. Functional analysis demonstrated that GmCDL1 positively regulates immune responses and promotes SCN resistance in soybeans. GmMPK3-mediated and GmMPK6-mediated phosphorylation of GmCDL1 enhances GmMPK3 and GmMPK6 activation and soybean disease resistance, representing a positive feedback mechanism. Additionally, 2 L-type lectin receptor kinases, GmLecRK02g and GmLecRK08g, associate with GmCDL1 to initiate downstream immune signaling. Notably, our study also unveils the potential involvement of GmLecRKs and GmCDL1 in countering other soybean pathogens beyond nematodes. Taken together, our findings reveal the pivotal role of the GmLecRKs–GmCDL1–MAPK regulatory module in triggering soybean basal immune responses.
The soybean receptor-like cytoplasmic kinase GmCDL1 directly links L-type lectin receptor kinases for MAP kinase activation and undergoes MAP kinase–mediated phosphorylation to boost immune signaling.
IN A NUTSHELL.
Background: The soybean cyst nematode (Heterodera glycines Ichinohe) is considered the most economically important soybean (Glycine max) pathogen. It is widely distributed in soybean-producing regions and is responsible for substantial annual yield losses. These nematodes invade the soybean root system, migrate intracellularly, and cause extensive damage to host tissues. The signaling components orchestrating the activation of plant defense responses during the early stages of nematode infection, particularly in roots, remain largely unknown.
Question: What role does phosphorylation-dependent signaling play in soybean resistance against the soybean cyst nematode?
Findings: Here, we identified a signaling module conferring resistance to soybean cyst nematodes. We demonstrated that GmCDL1, a relatively understudied receptor-like cytoplasmic kinase highly expressed in soybean roots, directly links L-type lectin receptor kinases (LecRKs) to mitogen-activated protein kinase (MAPK) activation during cyst nematode resistance. Additionally, we highlighted an important role of MAPK-mediated phosphorylation in regulating GmCDL1 stability, indicating a positive feedback mechanism to boost immune responses. Our data provide mechanistic insights into the early signaling pathways regulating soybean basal immune responses.
Next steps: Phosphorylation of GmCDL1 attenuates its proteasome-mediated degradation; further investigation into the underlying mechanism is warranted. In addition, identifying elicitors for the LecRK receptors presents a challenging yet exciting area for exploration.
Introduction
Soybean (Glycine max [L.] Merr.), one of the most economically important legume crops for oil and protein, is constantly under attack by various biotic and abiotic stresses. Soybean cyst nematode (SCN; Heterodera glycines Ichinohe), an obligate sedentary endoparasite, is widely distributed in soybean-producing regions and is considered as one of the major threats to soybean growth and production (Wrather and Koenning 2009; Jones et al. 2013). The infective 2nd-stage juvenile (J2) of SCN invades soybean roots and migrates toward the vasculature, where a metabolically highly active and multinucleated feeding structure called syncytium is formed, serving as the nutrient sink for the nematode's entire life cycle. Unlike root-knot nematodes (Meloidogyne spp.), cyst nematodes move intracellularly and cause more damage to the host tissues. Nematode effector proteins mainly secreted from the esophageal gland cells are delivered into the plant roots through the protrusible stylet and favor the successful nematode growth and development.
Plants have evolved a multilayered and sophisticated surveillance system to defend against a myriad of invading pathogens, including pathogenic microorganisms, nematodes, insects, and even parasitic plants in natural environments (DeFalco and Zipfel 2021; Eves-van den Akker 2021; Siddique et al. 2022). Cell surface–localized pattern recognition receptors (PRRs), including either membrane-localized receptor-like kinases (RLKs) or receptor-like proteins, are deployed to perceive diverse pathogen-derived or host-derived immunogenic molecules, known as pathogen-associated molecular patterns, damage-associated molecular patterns (DAMPs), and phytocytokines, resulting in the prompt activation of basal immunity referred to as pattern-triggered immunity (PTI).
The highly variable ectodomain of PRRs determines ligand recognition properties, for instance, proteinaceous ligands typically recognized by the leucine-rich repeat (LRR)–containing PRRs, N-acetylglucosamine-containing microbial molecules mainly perceived by the lysin motif (LysM) receptors, and plant cell wall–derived oligogalacturonides (OGs), which can be recognized by the epidermal growth factor-type receptors of the wall-associated kinase family (Zhang and Thomma 2013). The L-type lectin receptor kinases (LecRKs) represent an important class of PRRs responsible for sensing nonproteinaceous elicitors. The G-type LecRKs, LIPOOLIGOSACCHARIDE-SPECIFIC REDUCED ELICITATION (LORE) and RESISTANT TO DFPM-INHIBITION OF ABSCISIC ACID SIGNALING2 (RDA2), were characterized as receptors for pathogen-derived lipids such as 3-OH-FAs and sphingolipids (Kutschera et al. 2019; Kato et al. 2022). The L-type LecRKs, DOES NOT RESPOND TO NUCLEOTIDES1 (DORN1) and LecRK-I.8, can sense host-derived small molecules extracellular ATP and NAD+, respectively (Bouwmeester et al. 2011; Choi et al. 2014). Despite the different origins, immunogenic patterns activate largely conserved cytoplasmic signaling events, including rapid calcium influx, reactive oxygen species (ROS) production, mitogen-activated protein kinases (MAPKs) activation, reinforcement of the cell wall, and the reprogramming of defense-related gene expression.
Currently, the role of basal defenses in plant–nematode interactions is not well understood; especially, the nematode patterns and recognition machinery remain less explored (Sato et al. 2019). The only nematode-associated molecular pattern (NAMP) identified so far is ascaroside #18, which belongs to an evolutionarily conserved family of nematode pheromone (Manosalva et al. 2015; Manohar et al. 2020; Ning et al. 2020). The PRR NEMATODE-INDUCED LRR-RLK1 (NILR1) functions to trigger PTI responses by perceiving currently unknown nematode-derived proteinaceous elicitors in NemaWater (Mendy et al. 2017). Notably, NILR has been recently characterized as an immune receptor for the nematode ascaroside #18 (Huang et al. 2023).
Studies also showed that nematode-triggered PTI responses are mediated by BRI1-ASSOCIATED RECEPTOR KINASE1, a common immune coreceptor for numerous PRRs (Peng and Kaloshian 2014; Teixeira et al. 2016; Mendy et al. 2017). During nematode invasion and migration inside host roots, the physical impact caused by stylet thrust and body movement, in combination with a wide repertoire of cell wall–degrading enzymes secreted from nematode, is also implicated in the release of potential DAMP signals (Bellafiore and Briggs 2010; Vieira et al. 2011; Hewezi and Baum 2013; Shah et al. 2017; Gheysen and Mitchum 2019; Siddique et al. 2022). Plant cell wall receptors polygalacturonase-inhibiting proteins, which inhibit polygalacturonase-mediated pectin degradation, are involved in the generation of OG elicitors and subsequently attenuate cyst nematode infection (Shah et al. 2017). Proline-rich extensin-like receptor kinases (PERKs) mediate OG-triggered immune responses to nematode infection (Siddique et al. 2022). In addition, exogenous treatment with plant elicitor peptides (PEPs) has been reported to improve nematode resistance (Lee et al. 2018; Zhang and Gleason 2020). Although studies indicate that PTI is effective in reducing nematode susceptibility, the signaling components orchestrating the activation of PTI responses during nematode invasion are largely unknown.
Receptor-like cytoplasmic kinases (RLCKs) have emerged as major components of signaling proteins linking PRRs to the key downstream intracellular signaling modules by phosphorelay (Liang and Zhou 2018). Among them, a number of RLCKs belonging to subfamily VII, such as BOTRYTIS-INDUCED KINASE1 (BIK1) and PBS1-LIKE1 (PBL1), have been well studied in Arabidopsis (Arabidopsis thaliana) as key convergent signaling hubs, which integrate signals from multiple PRRs through direct association. Systematic characterization of 9 high-order mutants of 46 RLCK VII members in Arabidopsis reveals the functional divergence and redundancy within and across different RLCK subgroups. It was found that RLCK VII-5, RLCK VII-7, and RLCK VII-8 subgroups, as well as RLCK VII-6 member RPM1-INDUCED PROTEIN KINASE (RIPK)/PBL14, are required for ROS production in response to multiple patterns tested. The RLCK VII-4 subgroup is important for chitin-triggered ROS production and MAPK activation, whereas BIK1 and PBL1 function in PEP-triggered MAPK activation, indicating the diverse contributions of RLCK VII family members to MAPK activation in response to different patterns. Likewise, numerous RLCK VIIs in rice (Oryza sativa) have been reported to be required for OsCERK1-mediated or XA21-mediated resistance, pointing to a conserved mechanism in plants (Yamaguchi et al. 2013; Ao et al. 2014; Zhou et al. 2016; Yamada et al. 2017; Wang et al. 2017b; DeFalco and Zipfel 2021).
Furthermore, the protein activation and turnover of RLCK VIIs are under dynamic regulation during immune signaling. For example, the PLANT U-BOX E3 ligases PUB25 and PUB26 and RING-H2 FINGER A3A (RHA3A) and RHA3B determine the distinct fate of BIK1 through polyubiquitination and monoubiquitination, respectively (Wang et al. 2018; Ma et al. 2020). Despite the remarkable progress in RLCK VII-mediated plant immune signaling, further study is still needed to decipher the activating and regulatory basis of the majority members in this large family during defense signaling triggered by various patterns.
Multiple proteins, which are responsible for triggering key aspects of PTI signaling, have been identified as substrates of RLCK VIIs in past years. The activation of MAPK cascades by RLCKs can transduce extracellular stimuli into intracellular responses, resulting in transcriptional reprogramming and subsequent immunity (Yamada et al. 2016; Bi et al. 2018). Upon activation, the upstream MAPK kinase kinases (MAPKKKs or MEKKs) phosphorylate and activate the middle MAPK kinases (MKKs or MEKs), which, in turn, trigger the activation of the bottom tier MAPKs (MPKs) through phosphorylation (MAPK Group 2002; Rodriguez et al. 2010).
RLCK VII-4 has been found to phosphorylate MAPKKK3/5 and MEKK1, thereby activating MKK4/5–MPK3/6 and MKK1/2–MPK4 cascades, respectively. The activated MPK3/6 and MPK4 subsequently phosphorylate MAPKKK5 and MEKK1 to form a positive feedback loop that amplifies the MAPK cascade. Similarly, PBL27 directly phosphorylates MAPKKK5, and OsRLCK185 phosphorylates OsMAPKKKε and OsMAPKKK18 to regulate CERK1-dependent MAPK activation (Yamada et al. 2017; Wang et al. 2017b). A recent study indicates that the 2 signaling modules, MKK4/5–MPK3/6 and MKK3–MPK1/2/7/14, are activated independently in response to wounding and insect attacks (Sözen et al. 2020). The MPK3/6 pathway is also activated under the attack of beet cyst nematode Heterodera schachtii in Arabidopsis. Increased nematode susceptibility was observed in mpk3 and mpk6 mutant lines, while the Arabidopsis PP2C-type phosphatase AP2C1 attenuates the defense signaling against nematode (Sidonskaya et al. 2016).
In order to understand phosphorylation-dependent signaling during the early stage of soybean–SCN interaction, we investigated quantitative phosphoproteomic changes under nematode infection. Based on our phosphoproteomic data, we found that the pS/pTP motif, a potential MAPK phosphorylation motif, is enriched among the significantly regulated phosphopeptides. Functional analysis revealed that the GmMKK4–GmMPK3/6 module, activated by nematode infection or wounding, confers resistance against SCN. Soybean CDG1-LIKE1 (GmCDL1), a member of RLCK VII-1, can be phosphorylated by GmMPK3 and GmMPK6 at Thr-372 both in vivo and in vitro through direct association, which, in turn, enhances the stability of GmCDL1 to boost key immune signaling and nematode resistance. Meanwhile, we identified 2 LecRKs, GmLecRK02g and GmLecRK08g, associated with GmCDL1 to regulate basal immunity and nematode resistance. Significantly, our study also reveals the broader role of GmLecRKs and GmCDL1 in countering other soybean pathogens beyond nematodes. Collectively, our findings unveil the prominent roles of a signaling module consisting of GmLecRKs–GmCDL1–MAP kinase cascade in conferring basal resistance to soybean pathogens.
Results
GmMPK3 and GmMPK6 phosphorylations are activated by SCN infection and wounding
In order to understand the protein phosphorylation changes during the early stage of soybean–SCN interaction, we have conducted a tandem mass tag (TMT) quantitative phosphoproteomic analysis of soybean roots infected with SCN. Based on our relative quantification data of phosphopeptides, we found that the pS/pTP motif, a potential MAPK phosphorylation motif, was enriched (38.2%, P < 0.0003) in the phosphopeptides significantly regulated by nematode infection (Fig. 1, A and B; Supplementary Data Set 1). The MAPK cascade has been demonstrated to be commonly involved in the interaction between plants and pathogens (Sidonskaya et al. 2016; Yamada et al. 2016; Bi et al. 2018; Sözen et al. 2020). Therefore, we investigated whether SCN infection can trigger MAPK activation by immunoblotting with α-pERK antibody, which recognizes the phosphorylated form of MPK3, MPK4, and MPK6. When soybean roots were inoculated with SCN, GmMPK3 and GmMPK6 activation was observed at 1 dpi (day post inoculation), with the maximum activation detected at 2 dpi, and then decreased to the basal level until 5 dpi (Fig. 1C). Similarly, MAPK activation was observed in resistant soybean varieties, PI88788 and PI548402, when inoculated with different virulent nematode populations, with no visible difference across treatments (Supplementary Fig. S1).
Figure 1.
Infection of SCN activates GmMPK3 and GmMPK6 signaling pathways. A) MAPK-conserved phosphorylation motifs were enriched in the quantitative phosphoproteome of nematode-infected roots. The phosphopeptides significantly regulated by SCN in cultivar Williams 82 were submitted to Motif-x for motif analysis. The significantly enriched motif pS/pTP for MAPKs was shown (P < 0.0003). T/C indicates the SCN treatment group relative to the controls. B) Diagram showing the proportion of phosphopeptides with pS/pTP motif relative to the total phosphopeptides differentially regulated by the nematode. GmMPK3 and GmMPK6 activations in soybean roots upon SCN (SCN) infection C), wound D), 100 μM γATP E), and 10 μM U0126 F) treatments, respectively. Three roots were pooled for each treatment. GmMPK3 and GmMPK6 activations were analyzed by immunoblot with an α-pERK antibody (IB: α-pERK), and equal loading is shown by α-actin immunoblot (IB: α-actin). The representative images from 3 biological replicates were shown. G to I) SCN reproduction in Williams 82 after pretreatment with MAPK pathway inhibitor U0126. Individual soybean roots were treated with 1, 5, and 10 μM U0126 for 24 h at 3 d after germination. After brief washing, the roots were inoculated with 300 J2s. The number of cysts per plant G) and per gram of fresh root H) were obtained at 30 dpi. The total root weight from each transgenic event was measured to evaluate its impact on root growth. After acid fuchsin staining, nematode development I) was analyzed in transgenic roots at 14 dpi (n ≥ 8 independent roots). J2, J3, and J4 indicate 2nd-stage, 3rd-stage, and 4th-stage juveniles. Data are shown as means ± Se from 1 representative experiment (n ≥ 5 pots with 4 plants per pot). Different letters indicate significant differences as determined by one-way ANOVA (P < 0.05). The experiments were repeated twice with similar results.
Since SCN causes severe root damage during penetration and migration, we also tested MAPK activation in response to wounding. We observed rapid and transient activation of GmMPK3 and GmMPK6 in mechanically damaged soybean roots, which peaked within 10 and 15 min after wounding treatment for GmMPK6 and GmMPK3, respectively (Fig. 1D). By immunoblot analysis of clustered regularly interspaced short palindromic repeats (CRISPR)-edited soybean roots with 2 GmMPK3 putative paralogs mutated, we confirmed that the lower band is indeed GmMPK3s (Supplementary Fig. S2A and Data Set 2).
We were unable to obtain GmMPK6-edited roots after several attempts probably owing to its lethality phenotype, but we confirmed that the upper band is GmMPK6s using GmMPK6-silenced roots (Supplementary Fig. S2, B and C). In addition, ATP, which is released upon tissue damage or pathogen attack, similarly activated GmMPK3 and GmMPK6 in soybean roots (Fig. 1E). Taken together, these results showed GmMPK3 and GmMPK6 phosphorylation is induced in response to SCN and wounding treatment.
GmMKK4–GmMPK3/6 cascade positively regulates SCN resistance in soybean
To determine the function of the MAPK signaling pathway in SCN resistance, we evaluate the nematode infection by exogenous application of the MAPK pathway inhibitor U0126 (Yoo et al. 2008; Ryu et al. 2017). As expected, wound-induced MAPK activation was substantially reduced upon U0126 treatment (Fig. 1F). When soybean roots were pretreated with U0126 for 24 h at the final concentrations of 1, 5, and 10 μM, respectively, we observed a 12.2% to 17.0% increase for the number of cysts per root system at 30 dpi (Fig. 1G). Meanwhile, the number of cysts per gram of fresh root also exhibited the same trend (Fig. 1H). In line with this, the nematode development was facilitated in U0126-treated roots compared to the controls (Fig. 1I). Previous studies have revealed that silencing of the soybean MAPK family genes, including GmMPK3 and GmMPK6, resulted in an increased nematode susceptibility (McNeece et al. 2019). These findings indicate that suppression of the MAPK pathway could decrease SCN resistance.
MKK4/5–MPK3/6 constitutes a well-studied MAPK cascade that is implicated in plant defense responses in Arabidopsis (Zhao et al. 2017; Sözen et al. 2020). We verified the interaction between GmMPK6 and its upstream GmMKK4 by luciferase complementation imaging (LCI) assay in Nicotiana benthamiana (Supplementary Fig. S3A). Besides, the expression of GmMPK3, GmMPK6, and GmMKK4 in N. benthamiana leaves and soybean roots displayed an obvious cell death phenotype (Fig. 2, A and B; Supplementary Fig. S3B). 3,3'-diaminobenzidine (DAB) and Evans blue staining showed induced hydrogen peroxide (H2O2) accumulation and the presence of cell death, respectively (Fig. 2B).
Figure 2.
GmMKK4–GmMPK3/6 signaling pathway positively regulates SCN resistance. A) Overexpression of GmMKK4 and GmMPK3/6 induces cell death in soybean roots. White arrows indicate positive transgenic roots. Empty vectors (EVs) were used as controls. OE indicates overexpression of the corresponding genes. Bar = 2 cm (upper panel); 200 μM (lower panel). Results from 1 representative experiment were shown. B) Expression of GmMKK4 and GmMPK3/6 induces cell death in N. benthamiana. DAB and Evans blue staining showed induced hydrogen peroxide (H2O2) accumulation (brown precipitates) and the presence of cell death (blue staining), respectively. C to F) SCN infection phenotype in GmMKK4, GmMPK3, and GmMPK6 transgenic roots. Individual transgenic hairy roots expressing GmMKK4, GmMPK3, and GmMPK6 driven by the SCN-inducible promoter were inoculated with 300 J2s. The number of cysts per plant and per gram of fresh root were obtained at 30 dpi. The total root weight from each transgenic event was measured to evaluate its impact on root growth. Empty vectors were used as controls. Data are shown as means ± Se (n ≥ 8). The average cyst number from individual pots was considered as 1 technical replicate. Different letters and asterisks indicate significant differences as determined by one-way ANOVA (P < 0.05) and Student's t-test (***P < 0.001). The experiments were performed 3 times with similar results. G) Representative images of transgenic roots expressing GmMKK4, GmMPK3, and GmMPK6 at 30 dpi with SCN. Bar = 1 cm (left panel); 1 mm (right panel). H and I) Acid fuchsin staining showing the nematode development in transgenic roots at 14 dpi. Data from 1 representative experiment are shown as means ± Se (n ≥ 8 independent roots). J2, J3, and J4 indicate 2nd-stage, 3rd-stage, and 4th-stage juveniles. Bar = 200 μM.
To confirm the resistance function of the MAPK pathway in soybean nematode resistance, we alternatively employed a previously identified nematode-inducible promoter pGlyma.06g036700 to drive the expression of GmMPK3, GmMPK6, and GmMKK4 in W82 (Zhang et al. 2022). GmMPK3, GmMPK6, and GmMKK4 transgenic roots had a reduced number of cysts relative to the controls, indicating that overexpression of GmMPK3, GmMPK6, and GmMKK4 significantly enhanced soybean resistance to cyst nematode (Fig. 2C to G). In line with this, the nematode development was delayed in the overexpression roots compared to the controls (Fig. 2, H and I). We further evaluated the function of GmMKK4 by silencing analysis, and we observed a consistent susceptibility phenotype in GmMKK4-silenced hairy roots (Supplementary Fig. S4). These results indicate that the GmMKK4–GmMPK3/6 cascade positively regulates soybean resistance against SCN.
GmMPK3 and GmMPK6 interact with GmCDL1
RLCK VII proteins trigger numerous key signaling modules in PTI signaling (Bi et al. 2018; Liang and Zhou 2018). According to our phosphoproteomic data, a member of the RLCKVII-1 subfamily, CDG1-LIKE1 protein (referred to as GmCDL1 hereafter), showed enhanced phosphorylation in response to SCN infection. A phosphopeptide (HAPGpTPPR), corresponding to the C-terminal of GmCDL1, was found to be upregulated 10-fold in SCN-infected roots compared with the controls (Fig. 3, A to C; Supplementary Data Set 1).
Figure 3.
GmMPK3 and GmMPK6 interact with and phosphorylate RLCK GmCDL1. A) Schematic representation of GmCDL1 domain structure. Asterisk represents phosphorylation sites detected from the phosphoproteomic analysis. TP represents threonine 372 and proline 373 residues of GmCDL1, respectively. B) Mass spectrogram showing the phosphorylation of GmCDL1 at Thr-372. The sequence at m/z 456.708 matches HAPGpTPPR of GmCDL1. Red and blue lines indicate matched y-ions and b-ions, respectively. C) Levels of the phosphopeptide containing Thr-372 were upregulated in soybean roots at 3 dpi with SCN. The data were extracted from quantitative phosphoproteomic datasets and were shown as means ± Se (n = 3). Asterisks indicate significant differences as determined by Student's t-test (***P < 0.001). D) Expression levels of GmCDL1 upon SCN infection and wound treatment. Individual samples containing 3 roots were pooled for RT-qPCR assay at 3 d after SCN infection or 15 min after wounding. SKIP16 was used as an internal control. Relative gene expression level was determined using the 2−ΔCt method with normalization to the internal control. Data are means ± Se (n = 3 biological replicates with 2 technical replicates). Different letters indicate significant differences as determined by one-way ANOVA (P < 0.05). E) GmCDL1 interacts with GmMPK6 by split luciferase complementation assay. The indicated constructs were coexpressed in N. benthamiana for 36 h, and the leaves were treated with 1 mM luciferin for fluorescence signal detection. GmMKK4 and GmMPK6 were coexpressed as positive controls. The color scale represents luminescence intensity in counts per second. F) GmCDL1 associates with GmMPK3 and GmMPK6 by Co-IP assay. nGFP-GmCDL1 was coexpressed with GmMPK3- and GmMPK6-FLAG or an empty vector control for 36 h in N. benthamiana leaves. Total protein was extracted for immunoprecipitation (IP) with α-FLAG beads (IP: α-FLAG), and the proteins were detected using immunoblots with α-FLAG or α-GFP antibody (IB: α-FLAG, IB: α-GFP). G) GmCDL1 directly interacts with GmMPK6 in an in vitro GST pull-down assay. GST-GmMPK6 or GST control immobilized on glutathione sepharose beads (IP: α-GST) was incubated with GmCDL1-His. α-His or α-GST antibody was used for immunoblot analysis (IB: α-His, IB: α-GST). GmMPK3 and GmMPK6 phosphorylate GmCDL1 at Thr-372 in an in vitro kinase assay. The purified recombinant proteins were incubated in the kinase buffer with P32-labeled or unlabeled ATP for 30 min and the phosphorylation was detected either by autoradiography H and I) or by α-pT372 immunoblot J). The protein inputs were stained with Coomassie brilliant blue (CBB) or detected by immunoblotting (IB: α-MBP, IB: α-GST). Thr-372 phosphorylation of GmCDL1 was elevated by GmMPK3 or GmMPK6 expression. GmCDL1 or GmCDL1T372A were coexpressed with or without GmMPK6K) and GmMPK3L) for 36 h in N. benthamiana. Leaves were collected for immunoprecipitation with α-FLAG antibody (IP: α-FLAG), and protein phosphorylation was detected by α-pT372 immunoblot (IB: α-pT372). The protein inputs were detected by immunoblotting (IB: α-GFP, IB: α-FLAG). M) Wound-induced phosphorylation of GmCDL1 at Thr-372. GmCDL1 and GmCDL1T372A were expressed for 3 d in N. benthamiana, respectively. Leaves were treated by wounding 10 min before sample collection. The experiment was performed as in K). N) Wound-induced phosphorylation of Thr-372 was reduced by MAPK inhibitor U0126 treatment. Leaves were treated with 50 μM U0126 for 12 h. The experiments were repeated at least twice with similar results. Results from 1 representative experiment were shown.
GmCDL1 consists of 380 amino acids containing a conserved kinase domain (Fig. 3A). Phylogenetic analysis showed that GmCDL1 along with its homologs in soybean named GmCDL1-03g, GmCDL1-13g, and GmCDL1-10g, exhibiting 96.0%, 89.8%, and 89.6% sequence identities to GmCDL1, respectively, were clustered with CDL1 (PBL7) from Arabidopsis. Sequence alignment showed that the Thr-372 phosphorylation site is conserved between GmCDL1 and its homologs in various plant species, except rice and N. benthamiana (Supplementary Fig. S5). Spatial expression data retrieved from the SoyBase database showed GmCDL1 and its putative paralogs are highly expressed in soybean root, indicating their potential function in roots (Supplementary Fig. S6). RT-qPCR was performed to analyze the gene expression changes of GmCDL1 upon wounding and nematode infection, and we observed no changes at the transcriptional level (Fig. 3D).
Interestingly, the phosphosite of GmCDL1 matched the typical phosphorylation sites of MAPK (pS/pTP; http://igps.biocuckoo.org; Pitzschke 2015). In addition, the corresponding phosphosite within CDL1 in Arabidopsis was identified as a potential MPK6 target site through a large-scale identification of direct substrates of protein kinases in plant stress responses (Wang et al. 2020). Therefore, we first examined the ability of GmMPK3 and GmMPK6 to interact with GmCDL1. By using LCI assay in N. benthamiana, we found that GmMPK3 and GmMPK6 interacted with GmCDL1, whereas no interaction was detected for the truncated form of GmCDL1 only containing the kinase domain (GmCDL1KD), indicating that full length of GmCDL1 is required for its interaction with GmMPK3 and GmMPK6 (Fig. 3E; Supplementary Fig. S7, A and B).
The interaction was confirmed by a co-immunoprecipitation (Co-IP) assay in N. benthamiana. Following immunoprecipitation with α-FLAG agarose beads, we successfully detected the bands with the expected mobility of GFP-GmCDL1 with α-GFP antibody in the protein extracts from leaves coexpressing GFP-GmCDL1 with GmMPK3-FLAG or GmMPK6-FLAG but not in the control coexpressing with FLAG-tagged empty vector (Fig. 3F). Finally, we performed a glutathione S-transferase (GST) pull-down assay to test the direct association between recombinant GST-GmMPK3/6 and GmCDL1-His proteins. Glutathione sepharose beads immobilized with GST-GmMPK3 or GST-GmMPK6 could effectively pull down GmCDL1-His, but GST alone could not (Fig. 3G; Supplementary Fig. S7C). These results confirm that GmCDL1 interacts with GmMPK3 and GmMPK6 both in vitro and in vivo.
GmMPK3 and GmMPK6 phosphorylate GmCDL1 at Thr-372
The above results implied that GmCDL1 might be phosphorylated by GmMPK3 and GmMPK6 at Thr-372 via direct interaction. We then tested whether Thr-372 of GmCDL1 is indeed phosphorylated by GmMPK3 and GmMPK6 in vitro. GmCDL1 exhibited autophosphorylation activity, and the GmCDL1K102E, which carries a mutation in the putative ATP-binding site, abolished its autophosphorylation activity (Fig. 3H; Supplementary Fig. S8A). Thus, we use recombinant MBP-tagged GmCDL1K102E for the in vitro MAPK phosphorylation assays.
Recombinant GST-GmMPK3 and GST-GmMPK6 can phosphorylate GmCDL1K102E in the presence of constitutively active GmMKK4DD (T214D/S220D), which is supposed to activate MPK3 and MPK6 (Ren et al. 2002; Liu and Zhang 2004). By contrast, neither GmMPK3/6 nor GmMKK4DD alone is able to phosphorylate GmCDL1K102E, indicating the importance of GmMKK4DD in the activation of GmMPK3 and GmMPK6 (Fig. 3, H and I). When Thr-372 was mutated to Ala in GmCDL1K102E (GmCDL1K102E T372A), the purified protein was no longer phosphorylated by GmMPK3 and GmMPK6 (Fig. 3, H and I). In addition, we developed an antibody that specifically recognizes the phosphopeptide containing Thr-372 (α-pT372) for immunoblot analysis. Strong phosphorylation of Thr-372 was detected for the recombinant GmCDL1K102E when incubated with GmMPK6 in a GmMKK4-dependent manner, but no signal was detected for the GmCDL1K102E T372A variant (Fig. 3J).
To determine whether Thr-372 phosphorylation of GmCDL1 occurs in vivo, we expressed GmCDL1 and GmCDL1T372A in N. benthamiana. Immunoblot analysis with α-pT372 specifically detected phosphorylation on the affinity-purified GmCDL1 but not GmCDL1T372A (Fig. 3K). Additional coexpression with GmMPK3 and GmMPK6 enhanced phosphorylation of GmCDL1 at Thr-372 (Fig. 3, K and L). Notably, wounding resulted in a much stronger signal, demonstrating that wounding triggers phosphorylation of GmCDL1 at Thr-372 (Fig. 3M). To verify whether the wound-induced phosphorylation of Thr-372 requires the MAPK signaling pathway, we treated GmCDL1-expressing leaves with U0126, and the enriched GmCDL1 was analyzed with α-pT372. Both the basal and wound-induced levels of Thr-372 phosphorylation were substantially reduced upon U0126 treatment (Fig. 3N). These results indicate that intact MAPK signaling transduction is required for the wound-induced phosphorylation of GmCDL1 Thr-372.
GmMPK3-mediated and GmMPK6-mediated GmCDL1 phosphorylations positively regulate GmCDL1 stability
To determine whether the function of GmCDL1 is affected by Thr-372 phosphorylation, we investigated the effect of Thr-372 phosphorylation on the autophosphorylation activity of GmCDL1. We found that wild-type GmCDL1, phospho-dead GmCDL1T372A, and phospho-mimetic GmCDL1T372D variants exhibited comparable autophosphorylation activity in the in vitro kinase assay (Supplementary Fig. S8A). We also examined the impact of phosphorylation on the subcellular localization of GmCDL1. Both GmCDL1 and its Thr-372 variants were located in the cytoplasm and nucleus (Supplementary Fig. S8B). In addition, we performed an LCI assay in N. benthamiana in order to evaluate whether the interaction between GmCDL1 and GmMPK3/6 was altered by Thr-372 phosphorylation. When coinfiltrated with GmMPK6, wild-type GmCDL1 and its Thr-372 variants displayed similar luminescence intensity and luciferase activity, suggesting that the Thr-372 phosphorylation does not affect GmCDL1-GmMPK6 association (Supplementary Fig. S8, C and D).
Previous studies have shown that the protein accumulation of RLCK VIIs is under tight control during immune signaling (Wang et al. 2018; Ma et al. 2020). We tested whether the GmCDL1 protein stability is affected by Thr-372 phosphorylation by expressing wild-type GmCDL1 and its Thr-372 variants in N. benthamiana for an in vitro degradation assay. In the time-course experiments, the protein levels of GmCDL1 were determined by α-Myc immunoblot analysis by adding ATP to the crude lysate. In the presence of protein synthesis inhibitor cycloheximide (CHX), the protein levels of GmCDL1T372A were rapidly reduced. Although the protein amounts of wild-type GmCDL1 and GmCDL1T372D were also declined, the degradation was less pronounced compared with the fast degradation of GmCDL1T372A. These results indicate that the Thr-372 phosphorylation promotes GmCDL1 stability in vitro (Fig. 4A). Furthermore, we conducted the degradation assay in vivo. The levels of GmCDL1 and GmCDL1T372A variants, which were transiently expressed in N. benthamiana, were evaluated by immunoblotting after CHX treatment for 6 h. Consistent with the above findings, GmCDL1T372A degraded more rapidly than GmCDL1 upon CHX treatment (Fig. 4B).
Figure 4.
GmMPK3/6-mediated GmCDL1 phosphorylation positively regulates GmCDL1 stability. A) Protein stability analysis of GmCDL1. Wild-type GmCDL1, phospho-mimetic mutant GmCDL1T372D, and phospho-dead mutant GmCDL1T372A were transiently expressed for 3 d in N. benthamiana. Crude protein lysates were treated with 50 μM CHX for the indicated time period. The protein levels of GmCDL1 were detected with α-Myc immunoblot (IB: α-Myc). α-Actin immunoblot indicates equal loading (IB: α-actin). Numbers above the lanes represent relative band intensity quantified by Image J and normalized to the levels of actin. B) Protein stability analysis of GmCDL1 and GmCDL1T372A mutant in vivo. At 3 d post infiltration in N. benthamiana, leaves expressing the indicated proteins were treated with 50 μM CHX for 6 h and were subjected to the immunoblot analysis. The protein levels of GmCDL1 were detected as in A). C)GmMPK3 or GmMPK6 coexpression increased GmCDL1 abundance in N. benthamiana. Leaves were collected at 36 h post infiltration for immunoblot analysis (IB: α-FLAG, IB: α-GFP, or IB: α-actin). The kinase-dead variant of GmMPK6 (GmMPK6KD) was expressed as a control. D) Enhanced GmCDL1 abundance under wound or MG132 treatment. GmCDL1 was transiently expressed in N. benthamiana for 3 d. Following wounding treatment for 15 min or 50 μM MG132 treatment for 2 h, the protein levels of GmCDL1 were detected by α-Myc immunoblot (IB: α-Myc). Equal loading was confirmed by Ponceau S staining (PonC.). Image J software was used to quantify band intensity relative to the levels of Rubisco (RBC). E) Wound-induced GmCDL1 accumulation was compromised by U0126 treatment. The experiment was performed as in D) with 50 μM U0126 treatment for 2 h before wounding. F and G) Effect of Thr-372 phosphorylation on wound-induced GmCDL1 stability. GmCDL1 and GmCDL1T372A mutant were expressed for 3 d in N. benthamiana. The infiltrated leaves were treated with 50 μM CHX for 2 h. After 15 min of wound treatment, samples were subjected to immunoblot analysis (IB: α-Myc, IB: α-actin). Image J software was used to quantify band intensity based on the results of 3 independent experiments G). Error bars represent ± Se (n = 3). Asterisks indicate significant differences as determined by Student's t-test (*P < 0.05). Increased GmCDL1 abundance in soybean roots under SCN H) and wound I) treatment. Individual samples containing 3 independent roots were treated with 300 J2s and wounding for the indicated time period at 3 d post germination. Protein levels were detected by α-GmCDL1 or α-actin immunoblot (IB: α-GmCDL1, IB: α-actin). J) U0126 treatment reduced GmCDL1 accumulation in soybean roots. Individual samples containing 3 independent roots were treated with 10 μM U0126 for 24 h before wounding. Then the samples were subjected to immunoblot analysis (IB: α-GmCDL1, IB: α-actin). K) Enhanced GmCDL1 accumulation in soybean roots under γATP treatment. The experiment was performed as in H) with 100 μM γATP treatment. The experiments were performed 3 times with similar results.
Given that the phosphorylation of Thr-372 by GmMPK3 and GmMPK6 is induced by wounding, we hypothesized that the GmCDL1 stability is affected by nematode and wounding treatment in GmMPK3 and GmMPK6-dependent manners. Indeed, coexpression with GmMPK3 or GmMPK6 in N. benthamiana increased GmCDL1 protein levels, but the kinase-inactive GmMPK6KD (K88E/K89E) showed no impact, indicating that the activation of the MAPK signaling pathway promotes GmCDL1 stability (Fig. 4C). We further expressed GmCDL1 in N. benthamiana followed by wounding treatment. Immunoblot analysis showed that the GmCDL1 protein levels were enhanced under wounding treatment compared with untreated samples (Fig. 4, D to G). Meanwhile, GmCDL1 levels were comparable between 26S proteasome inhibitor MG132 and wounding treatment, suggesting that MG132 could repress the degradation of GmCDL1 (Fig. 4D). However, the basal and wound-induced protein levels were much less by U0126 treatment (Fig. 4E).
Next, we determined the effect of Thr-372 phosphorylation on wound-induced GmCDL1 stability. The GmCDL1T372A protein levels were not significantly affected by wounding treatment compared with the enhanced level of wild-type GmCDL1 (Fig. 4, F and G). Moreover, we verified that the nematode infection and wounding treatment greatly induced GmCDL1 protein levels in soybean roots by α-GmCDL1 immunoblot analysis (Fig. 4, H and I). Accordingly, the wound-induced protein accumulation in soybean roots was blocked by the addition of U0126 (Fig. 4J). We also observed increased levels of GmCDL1 by ATP treatment, suggesting its potential role in soybean responses to DAMPs (Fig. 4K).
Interestingly, we noticed 2 bands when using α-GmCDL1 antibody to analyze the endogenous GmCDL1 levels in soybeans. Among the 4 GmCDL1 putative paralogs in soybean, 2 of them (GmCDL1-10g and GmCDL1-13g) are predicted to have 0.45 kD larger molecular mass than the other 2 (GmCDL1 and GmCDL1-03g). We used CRISPR/CRISPR-associated protein 9 (Cas9)–mediated editing to mutate GmCDL1-10g and GmCDL1-13g simultaneously in soybean roots by using 2 sequence-specific single-guide RNAs (sgRNAs) for these 2 genes (Supplementary Data Set 2). Following α-GmCDL1 immunoblot, we confirmed that the upper band represents GmCDL1-10g and GmCDL1-13g (Supplementary Fig. S9). The consistent induction trend upon treatment indicates that the GmCDL1 putative paralogs might share conserved functions. The aforementioned results further confirm the notion that GmMPK3- and GmMPK6-mediated GmCDL1 phosphorylation positively regulates GmCDL1 stability.
Phosphorylation at Thr-372 of GmCDL1 is required for its function in SCN resistance
To determine the biological role of GmCDL1 in defense responses, we generated GmCDL1-knockout, knockdown, and overexpression of soybean transgenic hairy roots for the following analysis. We confirmed the expression levels of GmCDL1 in the transgenic roots by RT-qPCR (Supplementary Fig. S10, A and B). CRISPR-edited roots for GmCDL1 were validated by sequencing (Supplementary Data Set 2). We first tested GmMPK3 and GmMPK6 activation in the transgenic roots. The wound-triggered GmMPK3 and GmMPK6 activation was substantially reduced in GmCDL1-knockout and knockdown roots relative to the empty vector controls (Fig. 5, A and B). Comparatively, GmCDL1 overexpression showed increased activation of GmMPK3 and GmMPK6 than the controls (Fig. 5C). Notably, GmCDL1T372D overexpression resulted in a more evident GmMPK3 and GmMPK6 activation following wounding treatment, whereas GmCDL1T372A overexpression led to a lower activation compared to the wild-type GmCDL1 (Fig. 5C). These results highlight the importance of GmCDL1 phosphorylation in boosting PTI responses.
Figure 5.
GmCDL1 Thr-372 phosphorylation is essential for its full function in SCN resistance. Reduced GmMPK3 and GmMPK6 activation in GmCDL1-knockout A) and GmCDL1-knockdown B) transgenic hairy roots. Individual samples containing 3 independent transgenic hairy roots were treated with wound for 10 min and then subjected to GmMPK3 and GmMPK6 activation analysis by immunoblot with α-pERK antibody (IB: α-pERK). Equal sample loading was shown by α-actin immunoblot (IB: α-actin). EV represents empty vector control. CR-GmCDL1 or Ri-GmCDL1 indicate CRISPR/Cas9-mediated editing or silencing of GmCDL1. C) Enhanced GmMPK3/6 activation in soybean roots overexpressing (OE) GmCDL1 and its Thr-372 variants. The experiment was performed as in A). D to F) SCN resistance phenotype of GmCDL1. Transgenic hairy roots overexpressing GmCDL1 and Thr-372 mutants were inoculated with 300 J2s. The number of cysts per plant D) and per gram of fresh root E) were obtained at 30 dpi. The total root weight from each transgenic event was measured to evaluate its impact on root growth. Data were shown as means ± Se (n ≥ 9). The average cyst number from individual pots was considered as 1 replicate. Different letters indicate significant differences as determined by one-way ANOVA (P < 0.05). Nematode development F) was analyzed by acid fuchsin staining in transgenic roots at 14 dpi, and data were shown as means ± Se (n ≥ 9 independent roots). J2, J3, and J4 indicate 2nd-, 3rd-, and 4th-stage juveniles. Empty vector (EV) was used as the control. The experiments were repeated twice with similar results. G and H) Increased susceptibility to cyst nematode in GmCDL1-knockout roots. The experiments were performed as in D) and E). Asterisks indicate significant differences as determined by Student's t-test (*P < 0.05; **P < 0.01).
We further investigated the nematode resistance phenotype of GmCDL1 and its Thr-372 variants. Under SCN infection, the transgenic roots overexpressing GmCDL1 and GmCDL1T372D showed enhanced resistance compared with empty vector controls, as measured by cyst number per plant as well as cyst number per gram of fresh root. But for GmCDL1T372A overexpression, the resistance was weakened (Fig. 5, D and E). In line with this, the nematode development was delayed in GmCDL1 and GmCDL1T372D overexpression roots compared to the controls but not in GmCDL1T372A overexpression roots (Fig. 5F). Besides, GmCDL1-knockout roots showed increased susceptibility to cyst nematode (Fig. 5, G and H). Therefore, GmCDL1 played a positive role in nematode disease resistance, and Thr-372 phosphorylation is required for its full function.
Two LecRKs interact with GmCDL1
By association with multiple PRRs, RLCKs transduce signals from the membrane to initiate downstream responses (Lu et al. 2010; Liu et al. 2013; Shi et al. 2013; Shinya et al. 2014; Tang et al. 2015; DeFalco and Zipfel 2021). In Arabidopsis, MAPKKK3/5 are known to be phosphorylated by RLCK VII to mediate pattern-triggered MPK3/6 activation. Similarly, we confirmed the interaction between GmMAPKKK5 and GmCDL1 by LCI and in vitro GST pull-down assays (Supplementary Fig. S11, A and B). GmCDL1 can indeed phosphorylate both the N-terminus and C-terminus of GmMAPKKK5 in in vitro kinase assays (Supplementary Fig. S11C).
To further identify GmCDL1-interacting proteins, we transiently expressed GmCDL1-FLAG in N. benthamiana leaves, and GmCDL1 coimmunoprecipitated proteins were analyzed by liquid chromatography–tandem MS (LC-MS/MS). Compared with the controls, 2 LecRKs (AKV93707 and AKV93685), sharing 60.7% and 52.2% sequence identities with GmLecRK02g and GmLecRK08g in soybean, were considered as candidates (Supplementary Data Set 3). GmLecRK02g, which represents a leguminous LecRK with putative homologs in Solanaceae, consists of 686 amino acids with a signal peptide, a lectin domain, a transmembrane domain, and a kinase domain (Fig. 6A; Zeng et al. 2021). GmLecRK08g, containing 701 amino acids with similar protein structure, is homologous to LecRK-IX.1 in Arabidopsis with 49.2% sequence identity (Fig. 6A). Phylogenetic analysis showed that GmLecRK02g and GmLecRK08g belong to different subclades of L-type lectin RLKs (Zeng et al. 2021). The gene expression levels of GmLecRK02g and GmLecRK08g were induced by SCN infection but not affected by wounding treatment (Fig. 6B).
Figure 6.
GmCDL1 interacts with GmLecRK02g and GmLecRK08g. A) Schematic diagram showing protein structures of GmLecRK02g and GmLecRK08g. Signal peptide (SP), Lectin_legB domain, transmembrane domain (TM), and kinase domain are shown as red, blue, green, and yellow rectangles, respectively. B) RT-qPCR analysis of GmLecRK02g and GmLecRK08g upon wound and SCN treatments. The roots, which were treated with wound for 15 min or SCN for 3 d, were collected for RT-qPCR analysis. SKIP16 was used as the internal control. Relative gene expression level was determined using the 2−ΔCt method with normalization to the internal control. Error bars represent ± Se (n = 3 biological replicates with 2 technical replicates). Different letters indicate significant differences as determined by 1-way ANOVA (P < 0.05). C) Cell death symptoms induced by GmLecRK08g. GmLecRK02g, GmLecRK08g, and GmLecRK08gK398E tagged with FLAG were expressed in N. benthamiana leaves. Representative leaves were photographed at 2 d post infiltration. Evans blue and DAB staining showed the presence of cell death and the induced accumulation of H2O2 at 36 h post infiltration. D and E) GmCDL1 interacts with GmLecRK02g and GmLecRK08g by split luciferase complementation assays. GmCDL1 and GmLecRK02g or GmLecRK08gK398E were coexpressed in N. benthamiana for 36 h, respectively. The leaves were treated with 1 mM luciferin for fluorescence signal detection. GmLecRK09g was selected as a negative control. The color scale indicates luminescence intensity in counts per second. F and G) GmCDL1 interacts with GmLecRK02g and GmLecRK08g by Co-IP assays. nGFP-GmCDL1 was coexpressed with GmLecRK02gK427E-, GmLecRK08gK398E-FLAG, or an empty vector control for 3 d in N. benthamiana leaves with or without wound treatment. Total protein was extracted for immunoprecipitation (IP) with α-FLAG beads (IP: α-FLAG), and the proteins were detected using immunoblots with α-FLAG or α-GFP antibody (IB: α-FLAG, IB: α-GFP). H and I) GmCDL1 directly interacts with GmLecRK02g and GmLecRK08g in GST pull-down assays. Glutathione sepharose beads were used for immunoprecipitation (IP: α-GST). α-His or α-GST antibody was used for immunoblot analysis (IB: α-His, IB: α-GST). The above experiments were performed at least twice with similar results. J) GmLecRK08g phosphorylates GmCDL1 in an in vitro kinase assay. The phosphorylation was detected by autoradiography. The protein inputs were stained with Coomassie brilliant blue (CBB).
We further validated the interactions between GmCDL1 and GmLecRK02g or GmLecRK08g. Because overexpression of GmLecRK08g in N. benthamiana led to an apparent cell death with high H2O2 accumulation, we generated a kinase-inactive mutant GmLecRK08gK398E by introducing a mutation in the putative ATP-binding site in order to get adequate protein amount for the interaction assays (Fig. 6C; Supplementary Fig. S12). LCI assay showed the GmCDL1 can interact with GmLecRK02g or GmLecRK08gK398E, while no interaction was detected between GmCDL1 and a randomly chosen L-type LecRK GmLecRK09g, consolidating the specificity of the interaction between GmCDL1 and certain LecRKs (Fig. 6, D and E). Besides, GmLecRK02gΔSP, with the signal peptide removed, no longer interacted with GmCDL1, indicating that proper subcellular localization is a prerequisite for the interaction (Supplementary Fig. S13).
Then we successfully confirmed the interactions by Co-IP assays using N. benthamiana leaves coexpressing GFP-tagged GmCDL1 and FLAG-tagged kinase-inactive GmLecRK02gK427E or GmLecRK08gK398E, indicating the kinase activity of LecRKs seems not to be required for the association. Interestingly, the association appears to be reduced upon wounding treatment (Fig. 6, F and G), suggesting that GmCDL1 might be released from the receptors upon activation. Furthermore, we detected a direct interaction between GmCDL1-His and the GST-tagged cytosolic domain of GmLecRK02g or GmLecRK08gK398E in vitro (Fig. 6, H and I).
To further test whether GmLecRKs can phosphorylate GmCDL1, we purified MBP-tagged fusion proteins of GmCDL1, GmLecRK02gcd, GmLecRK08gcd, and their kinase-inactive variants for in vitro kinase assays. The results showed that GmLecRK08gcd, but not its kinase-inactive variant GmLecRK08gcdK398E, exhibited autophosphorylation kinase activity (Fig. 6J). Significantly, GmLecRK08gcd directly phosphorylated GmCDL1 and its kinase-inactive variant GmCDL1K102E, while GmLecRK08gcdK398E completely eliminated its phosphorylation activity on GmCDL1 (Fig. 6J). For recombinant GmLecRK02gcd protein, we did not detect any kinase activity, including the autophosphorylation activity, presumably due to its weak or unstable activity (Supplementary Fig. S14).
To determine the potential phosphosites of GmCDL1 by GmLecRK08gcd, the in vitro phosphorylated GmCDL1K102E was subsequently subjected to LC-MS/MS analysis. In total, 3 phosphosites, Thr-231, Ser-234, and Thr-235, were identified within the activation loop of GmCDL1K102E (Supplementary Fig. S15, A and B). Sequence alignment revealed that the Ser-234 and Thr-235 phosphosites in GmCDL1 correspond to Ser-236 and Thr-237 in BIK1, 2 highly conserved residues across related kinases (Supplementary Fig. S16A). Notably, the phosphorylation of these 2 residues in BIK1 has been well studied to be essential for its kinase activity, as well as for the function of BIK1 in a variety of plant immune responses (Lu et al. 2010; Zhang et al. 2010; Laluk et al. 2011; Liu et al. 2013; Xu et al. 2013; Lin et al. 2014; Luo et al. 2020). We subsequently confirmed that the Ser-234 and Thr-235 phosphorylation indeed enhanced GmCDL1-mediated MAPK activation upon wounding treatment in soybean transgenic hairy roots (Supplementary Fig. S16B). These findings reveal that GmLecRKs interact with and phosphorylate GmCDL1 to activate downstream signaling.
GmLecRK02g and GmLecRK08g are required for nematode resistance
To uncover the function of GmLecRK02g and GmLecRK08g in plant immunity, we employed CRISPR/Cas9 to knock out their expression or overexpress them in soybean transgenic hairy roots (Supplementary Fig. S17 and Data Set 2). We carried out the MAPK activation assay in the GmLecRK02g- and GmLecRK08g-knockout roots after wounding challenges. The transgenic roots displayed a substantial reduction in wound-induced MAPK activation (Fig. 7, A and B). Additionally, GmCDL1-mediated MAPK activation was compromised when GmLecRK02g or GmLecRK08 was mutated, indicating these receptors are required to initiate the downstream signaling (Fig. 7, C and D). SCN resistance was compromised, and nematode development was facilitated in the GmLecRK02g-edited and GmLecRK08g-edited roots compared with the control (Fig. 7, E to J; Supplementary Fig. S18, A and B), whereas enhanced resistance and delayed nematode development were observed in the overexpression roots (Fig. 7, K to P; Supplementary Fig. S18C). All the above results indicate that GmLecRK02g and GmLecRK08g contribute to SCN resistance by directly activating GmCDL1-mediated immune signaling.
Figure 7.
GmLecRK02g and GmLecRK08g positively regulate MAPK activation and SCN resistance. A and B) GmLecRK02g and GmLecRK08g are required for GmMPK3 and GmMPK6 activation in soybean roots. Individual samples containing 3 CRISPR-edited (CR) transgenic hairy roots for GmLecRK02g or GmLecRK08g were treated with wound for 10 min and collected for GmMPK3/6 activation analysis by immunoblot with α-pERK antibody (IB: α-pERK). α-Actin immunoblot was conducted to confirm equal sample loading (IB: α-actin). EV represents empty vector control. Similar results were observed in 3 independent experiments. C and D)GmLecRK02g and GmLecRK08g are required for GmCDL1-mediated MAPK activation. Transgenic hairy roots with overexpression of GmCDL1 and knockout of GmLecRK02g or GmLecRK08g were generated. OE or CR indicates overexpression or CRISPR/Cas9-mediated editing of target genes, respectively. The experiments were performed as in A) and B). The data shown are representatives of 3 biological replicates. E to G) Knockout of GmLecRK02g increased nematode susceptibility. H to J) Knockout of GmLecRK08g increased nematode susceptibility. K to M) SCN resistance phenotype in soybean roots expressing GmLecRK02g. N to P) SCN resistance phenotype in soybean roots expressing GmLecRK08g. The experiments were performed as aforementioned. EV represents empty vector control. The number of cysts per plant and per gram of fresh root were obtained at 30 dpi. The total root weight from each transgenic event was measured to evaluate its impact on root growth. Acid fuchsin staining was conducted to analyze the nematode development in transgenic roots at 14 dpi. J2, J3, and J4 indicate 2nd-stage, 3rd-stage, and 4th-stage juveniles. Data were shown as means ± Se (n ≥ 6). The average cyst number from individual pots was considered as 1 replicate. Asterisks indicate significant differences as determined by Student's t-test (*P < 0.05; **P < 0.01; ***P < 0.001). The experiments were independently performed 3 times with similar results.
To determine whether the GmLecRKs–GmCDL1 module may contribute to the responses against other soybean pathogens, we tested the role of GmLecRKs and GmCDL1 in soybean responses against the pathogenic oomycete Phytophthora sojae, a major causal agent of soybean root rot. First, we demonstrated that infection of P. sojae led to MAPK activation and GmCDL1 accumulation (Supplementary Fig. S19, A and B). Subsequently, we inoculated transgenic roots with P. sojae. The expression of GmCDL1 or 2 GmLecRKs in hairy roots enhanced soybean resistance to P. sojae infection, as indicated by the reduced lesion length, while the knockout analysis increased susceptibility (Supplementary Fig. S19, C to H). Together, these results demonstrate that the GmLecRKs–GmCDL1–MAPK may function in countering various pathogens, suggesting a broader role beyond nematode-specific responses.
Discussion
Plant-parasitic nematodes invade the root systems of most economically important crops, causing significant yield losses worldwide. It will be valuable to have a detailed understanding of the signaling networks during the early plant–nematode interactions, thus offering potential means to enhance nematode resistance. In the present study, we established the GmLecRKs–GmCDL1–GmMAPKs–mediated signaling module in soybeans during the early stage of SCN parasitism. We highlighted the crucial role of RLCK VII-1 member GmCDL1, which directly links LecRKs for MAPK activation during soybean basal resistance. Our findings also uncover MAPK-mediated feedback regulation on GmCDL1, pointing to a common mechanism to boost immune signaling (Fig. 8).
Figure 8.
A proposed model for the role of GmLecRKs–GmCDL1–GmMPK3/6 signaling pathways in SCN resistance. Under normal conditions, GmCDL1 remains inactive, and the downstream immune signaling is switched off. Upon nematode stimulation, GmCDL1 is released from the LecRK PRR complex following phosphorylation by PRRs. Then, the GmMAPKKK5–GmMKK4–GmMPK3/6 cascade is activated to confer resistance against SCN. The feedback phosphorylation of GmCDL1 by MAPKs enhances the stability of GmCDL1, further boosting key immune signaling and contributing to nematode resistance. NAMP, nematode-associated molecular pattern. 26S represents the 26S proteasome degradation system. Black and gray arrows indicate activated and inactivated pathways. Solid lines indicate regulation tested in this study, while dashed lines indicate proposed regulation. The red “X” symbol indicates a blocked pathway, and the red “P” dots denote phosphorylation.
Pattern-triggered immunity, activated by host-derived or pathogen-derived molecular patterns, is extensively studied to be essential for plants to fend off diverse pathogens in various model plant–pathogen interaction systems; however, the signaling components involved in nematode-induced PTI responses in roots are far less understood. Our present findings along with previous studies revealed that the basal defense responses are activated in nematode-infected roots, and the PTI signaling components, including several PRRs, RLCKs, RESPIRATORY BURST OXIDASE HOMOLOG (RBOH) genes, and MAPK family genes, contribute to nematode resistance (Siddique et al. 2014; Mendy et al. 2017; Shah et al. 2017; McNeece et al. 2019). Additionally, the penetration and intracellular migration of cyst nematodes cause extensive damage, leading to the generation of danger signals to elicit defense responses. We demonstrated mechanical wounding-induced and SCN-induced activation of MAPKs in soybean roots, corroborating previous observations in Arabidopsis (Sidonskaya et al. 2016; Hõrak 2020).
Notably, the response to nematodes in Arabidopsis occurs more rapidly than in soybeans. We proposed that MAPK activation triggered by cyst nematode seems to be resulted from the mechanical damage caused by nematode invasion, although the reaction to nematode infection is greatly postponed compared to the rapid activation pattern observed with wounding treatment. As nematodes do not behave synchronously during infection, only a portion of the cells may respond to nematode infection, especially in thick soybean roots. The damage caused by the nematodes may reach the maximum at a later time point with the progression of infection. On the other hand, nematode effectors might suppress PTI signaling, contributing to delayed reactions at the early stage of infection. In the future, developing assays that can differentiate the DAMP-triggered response from the NAMP-triggered response would be a valuable endeavor.
MPK3 and MPK6-mediated phosphorylation has been implicated in regulating the activity and turnover of various downstream substrates associated with stress responses. For instance, phosphorylation of WRKY33 by MPK3 and MPK6 enhances the transactivation activity of WRKY33 and positively regulates camalexin biosynthesis (Zhou et al. 2020). MPK3 and MPK6 also phosphorylate various transcription factors, such as ETHYLENE RESPONSE FACTOR6 (ERF6), ERF104, and MYB DOMAIN PROTEIN4 (MYB4), to regulate the activation of defense genes (Bethke et al. 2009; Meng et al. 2013; Lin et al. 2022). ACC SYNTHASE protein is stabilized by MPK6-mediated phosphorylation, leading to induced ethylene biosynthesis and disease resistance (Li et al. 2012). In addition, the MPK3 and MPK6-mediated phosphorylation of INDUCER OF CBF EXPRESSION1 (ICE1), a key regulator in cold-stress responses, negatively regulates ICE1 stability and freezing tolerance in Arabidopsis (Li et al. 2017; Zhao et al. 2017). The identification of upstream component GmCDL1 as a substrate for GmMPK3 and GmMPK6 reveals a positive feedback regulation mechanism, and the phosphorylation of GmCDL1 at Thr-372 is subsequently important for enhancing MAPK activation. Additionally, MAPKKK5 and MEKK1 have been reported to be phosphorylated by MPK6 and MPK4, respectively, indicating the significance of MAPK-mediated feedback regulation as an important mechanism to amplify signal transduction.
RLCK VIIs act as key components of immune signaling, for which the activation and turnover are under complex regulation. E3 ubiquitin ligases PUB25 and PUB26 selectively target underphosphorylated BIK1 for degradation via polyubiquitination, whereas RHA3A and RHA3B monoubiquitinate the phosphorylated form of BIK1 to activate PRR signaling (Wang et al. 2018; Ma et al. 2020). We present evidence indicating that GmCDL1 is subjected to posttranslational regulation. Although GmCDL1 is expressed constantly in roots, the protein levels are hardly detected, especially in soybean roots under normal conditions. Upon stimulation, GmCDL1 is released from the PRR complex, probably following phosphorylation by the PRR complex. The phosphorylation of GmCDL1 by MAPKs or PRRs attenuates proteasome-mediated degradation, resulting in a significant increase in their protein levels. The responsible E3 ligase awaits to be explored in the future.
LecRKs are emerging as important plant immune receptors to perceive nonproteinaceous elicitors, such as eNAD+, eNADP+, eATP, 3-OH-FAs, and sphingolipids (Choi et al. 2014; Wang et al. 2017a; Kutschera et al. 2019; Wang et al. 2019; Kato et al. 2022). However, the cognate ligands remain undetermined for most of them. LecRKs can be classified into 3 types (L, G, and C-type) based on their extracellular lectin domains. Accumulating evidence demonstrates their functions in PTI responses and disease resistance (Desclos-Theveniau et al. 2012; Singh et al. 2012; Wang et al. 2015, 2016; Yekondi et al. 2018; Gouhier-Darimont et al. 2019; Groux et al. 2021; Pi et al. 2022). For example, a gene cluster containing 3 G-type LecRKs confers broad-spectrum resistance to the brown planthopper (Nilaparvata lugens) in rice (Liu et al. 2015). A G-type LecRK, ENHANCED RESISTANCE TO NEMATODES1 (ERN1), was identified as a negative regulator of PTI responses and root-knot nematode resistance (Zhou et al. 2023).
We showed that 2 soybean LecRKs, belonging to different subclades of L-type LecRKs, directly associate with GmCDL1, positively contributing to resistance against cyst nematode and potentially other pathogens. Three residues (Thr-231, Ser-234, and Thr-235), located within the activation loop of GmCDL1, are phosphorylated by LecRKs. The corresponding residues that are highly conserved across RLCK VII subfamily kinases have been shown to be essential for their activities and biological functions in various plant immune responses (Lu et al. 2010; Zhang et al. 2010; Laluk et al. 2011; Liu et al. 2013; Xu et al. 2013; Lin et al. 2014; Luo et al. 2020). Similar regulation mode may not be limited to nematode and wounding stresses. For instance, the phosphorylation of PBL34/35/36 in Arabidopsis and OsRLCK118/176 in rice is required for LORE- and SPL11 cell-death suppressor2 (SDS2)–mediated resistance against bacteria and fungi (Fan et al. 2018; Luo et al. 2020). Our study raises further interest in the relationship between these 2 LecRKs, although it is conceivable that they may recognize different elicitors released during various pathogen infections. Additionally, this highlights the potential role of GmCDL1 in integrating signals from various elicitors. Further efforts are needed to gain a better understanding of how the specificity between PRRs and RLCKs is co-opted by diverse pathogens.
Materials and methods
Plant and nematode materials
The soybean (G. max) cultivar Williams 82 (W82), which is susceptible to SCN, was used in most of our study unless otherwise indicated. The SCN (H. glycines Ichinohe, HG type 0, Race 3) was maintained on W82 with sand:potting soil mixture (3:1) under a 16-h light/8-h dark regime in a 28 °C greenhouse with the white fluorescent light intensity of 200 µmol m−2 s−1 (Pak, T5-28W) according to the standard procedures (Niblack et al. 1993). N. benthamiana plants were grown in a growth room at 22 °C under a 16-h light/8-h dark photoperiod with 100 µmol m−2 s−1 illumination (Pak, T5-28W) and 45% relative humidity. Cysts were extracted from infested soil at 35 d post inoculation using sieves and centrifugation. The cysts were crushed with a rubber stopper, then the collected eggs were purified with 40% (w/v) sucrose aqueous solution. For inoculation experiments, 2nd-stage juveniles (J2s) were hatched by incubating eggs with distilled water at 26 °C for 2 to 3 d. To compare differences in MAPK activation across soybean varieties in response to different nematode populations, 2 HG type 0-resistant varieties PI88788 and PI548402, and 2 virulent nematode populations Race 4 and Race 5 (courtesy of Dr. Shiming Liu at Chinese Academy of Agricultural Sciences and Dr. Congli Wang at Chinese Academy of Sciences), were additionally tested. A major causal agent of soybean root rot, P. sojae, was kindly provided by Dr. Wenwu Ye and Dr. Yuanchao Wang at Nanjing Agricultural University (Qiu et al. 2023).
Vector construction
For overexpression constructs, the sequence information of GmMPK3, GmMPK6, GmMKK4, GmCDL1, GmLecRK02g, and GmLecRK08g was retrieved from Phytozome (https://phytozome-next.jgi.doe.gov), and the coding regions were amplified from W82 using gene-specific primers. The PCR products were subcloned into the binary vectors pSM101-5FLAG with SalI and XbaI digestion (Zhang et al. 2022). To generate a silencing construct, the 611-, 400-, and 301-bp long fragments of GmCDL1, GmMKK4, and GmMPK6 were subcloned into RNAi binary vectors with AscI/SwaI and BamHI/AvrII restriction sites (Zhang et al. 2022). CRISPR/Cas9 technology was used to knock out candidate genes. One or 2 specific sgRNAs were designed for targeted mutagenesis. The assembled polycistronic tRNA-gRNA (PTG) fragments were subsequently cloned into the pCAMBIA1300 binary vector (Yu et al. 2023). The genomic sequences of candidate genes were amplified by PCR using primers flanking the target sites from individual transgenic hairy roots, and mutations were confirmed by sequencing.
For subcellular localization, the coding sequences of GmCDL1 and Thr-372 variants were subcloned into the pGWB5-GFP vector to generate GmCDL1-GFP, GmCDL1T372A-GFP, and GmCDL1T372D-GFP fusion constructs (Wang et al. 2021). GmCDL1 variants were generated by overlap PCR using primers carrying point mutations. For pull-down assays, the full-length coding sequences of GmCDL1, GmMPK3, and GmMPK6, the cytosolic domain-coding sequences of GmLecRK02g and GmLecRK08gK398E, and the C-terminus (567 to 637 aa) or N-terminus (1 to 305 aa) sequences of GmMAPKKK5 were cloned in-frame into pET-28a and pGEX-6P-1 vectors to express recombinant GmCDL1-His, GST-GmMPK3, GST-GmMPK6, GST-GmLecRK02gcd, GST-GmLecRK08gcdK398E, GST-GmMAPKKK5c, and GST-GmMAPKKK5n proteins in Escherichia coli. For in vitro phosphorylation assays, wild-type GmCDL1 and its mutated variants GmCDL1K102E, GmCDL1K102E T372A, GmCDL1T372A, GmCDL1T372D, along with the cytosolic domain-coding sequences of GmLecRK02g, GmLecRK08g, GmLecRK02gK427E, and GmLecRK08gK398E, were cloned in-frame into pMAL-c2X vector to generate MBP-tagged fusion proteins (Huang et al. 2020). For Co-IP assays, the coding sequence of GmCDL1 was inserted into the pCAMBIA2300-nGFP vector to produce an N-terminal GFP-tagged fusion construct (Wang et al. 2023). For LCI assays, the coding sequences of GmCDL1, GmCDL1KD (Kinase domain), GmLecRK02g, GmLecRK08gK398E, GmMPK3, GmMPK6, and GmMAPKKK5c were cloned as indicated into the pCAMBIA-35S-cLUC and pCAMBIA-35S-nLUC vectors to express C-terminal nLUC and N-terminal cLUC fusion constructs, respectively (Chen et al. 2008). For protein degradation assays, GmCDL1 and its phosphorylation variants, including phospho-inactive GmCDL1T372A and phosphor-mimetic GmCDL1T372D, were fused into a pGWB517-4Myc vector (Huang et al. 2020). All the primers used to construct vectors were listed in Supplementary Data Set 4.
Soybean hairy root transformation
Agrobacterium rhizogenes strain K599-mediated transformation was performed to generate soybean transgenic hairy roots using a GFP-selection marker. One GFP-positive root for an individual plant was selected using an Olympus SZX16 fluorescence stereomicroscope (Olympus SZX16, Tokyo, Japan) for subsequent analysis as described previously (Guo et al. 2015; Tóth et al. 2016). Four plants were planted in 1 pot and allowed to recover for an additional 3 d in the sand:soil mixture. For nematode inoculation, 300 preparasitic J2s were applied to each plant. After 30 dpi, cysts from each pot were harvested and counted under a stereomicroscope. The average cyst number per plant or per gram of fresh root in each pot was considered as a technical replicate. The total root weight from each transgenic event was measured to evaluate its impact on root growth. Nematode development was evaluated at 14 dpi by acid fuchsin staining. For wounding treatment, roots (7 cm in length) were wounded 10 times using tweezers, and then samples with or without treatment were collected at the indicated time points. For inoculation with P. sojae, the strain was grown on V8 medium at 25 °C for 5 d, and soybean roots were inoculated with 0.5-cm-diameter medium blocks containing the mycelia for the indicated time points. Lesion length was measured at 48 h post inoculation.
Histochemical staining
The roots were cleared and stained with acid fuchsin. Roots were boiled in 0.8% (v/v) acetic acid and 0.013% (w/v) acid fuchsin for 3 min and then de-stained in acidified glycerol. The various stages of nematodes were photographed under a stereomicroscope. DAB and Evans blue staining were performed to determine H2O2 production and the occurrence of cell death (Wang et al. 2021). Briefly, detached leaves were completely submerged in 1 mg mL−1 DAB and 0.25% Evans blue aqueous solution and then subjected to two 5-min cycles of vacuum followed by a 20-min maintenance under vacuum. The leaves were then incubated and well washed for photography.
Agrobacterium-mediated transient expression in N. benthamiana
Fully expanded leaves of 6-wk-old N. benthamiana plants were used for agroinfiltration assays. The Agrobacterium (Agrobacterium tumefaciens) strain EHA105 harboring the indicated vectors was incubated in a LB liquid medium at 28 °C for 12 h. Then, the cultures were collected and resuspended in an infiltration buffer (10 mM MES pH 5.6, 10 mM MgCl2, and 0.2 mM acetosyringone) to a final concentration of OD600 = 1.0. The suspensions were kept at room temperature for 2 to 5 h without shaking. The A. tumefaciens strain carrying the p19 silencing suppressor was used to enhance gene expression. For coinfiltration, equal volumes of Agrobacterium suspensions carrying the indicated constructs were mixed and syringe-infiltrated into N. benthamiana leaves. After infiltration, plants were cultured at 22 °C for 36 to 48 h at 16-h light/8-h dark photoperiod with light intensity around 100 µmol m−2 s−1. For wounding experiments, the infiltrated leaf discs were wounded 3 times, and then the samples were collected at the indicated time points using a circular cork borer (5 mm in diameter).
Subcellular localization
A. tumefaciens strain EHA105 containing the indicated constructs was infiltrated into N. benthamiana leaves as described above. At 2 d after infiltration, the epidermal cell layer of the leaves was visualized with a Leica TCS SP8 confocal microscope. GFP fluorescence was excited at 488 nm with an argon-ion laser, and emission was recorded via a 500- to 550-nm bandpass filter.
GST pull-down assay
Recombinant GST-GmMPK3, GST-GmMPK6, GST-GmLecRK02gcd, GST-GmLecRK08gcdK398E, and GST-GmMAPKKK5c proteins were expressed and purified from E. coli strain BL21(DE3) with 20 μL glutathione sepharose resin (Genscript, Nanjing, China) in 500 μL lysis buffer [50 mM Tris-HCl, pH 7.4, 400 mM NaCl, 1 mM DTT, and 1 mM phenylmethanesulfonyl fluoride (PMSF)] at 4 °C for 4 h with gentle rotation. Then, the resin was harvested and incubated with cell extracts of GmCDL1-His at 4 °C for 12 h with gentle rotation. After washing 6 times with lysis buffer, the resin was boiled with 2×SDS loading buffer at 95 °C for 8 min. The eluted proteins were separated by SDS-PAGE and subjected to immunoblotting analysis with α-His (ABclonal, Cat. AE003, 1:5000 dilution) or α-GST antibodies (ABclonal, Cat. AE001, 1:5000 dilution). The purified GST protein was used as a control.
Firefly LCI assay
The assay was performed as previously described (Chen et al. 2008). Briefly, A. tumefaciens strains EHA105 containing the desired constructs were infiltrated into N. benthamiana leaves. After 36 or 48 h, the infiltrated leaves were sprayed with 1 mM luciferin in darkness for 5 min. A charge-coupled device imaging system of the chemiluminescence apparatus (Tanon, Shanghai, China) was used to capture the luminescence images. Equal expression of proteins was verified by immunoblotting.
Co-IP assay
The assays were conducted as previously described (Huang et al. 2020). The crude proteins were extracted from N. benthamiana leaves with protein extraction buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 10% glycerol (v/v), 1 mM PMSF, and 1× protease inhibitors from Sigma-Aldrich) at 36 h after infiltration. Samples were incubated on ice for 30 min and centrifuged at 12,000 g at 4 °C for 30 min. The supernatant was incubated with α-FLAG agarose beads (Sigma-Aldrich, Cat. A2220) for 2 h at 4 °C with gentle rotation. The beads were washed at least 6 times with washing buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2 mM EDTA, 10% glycerol (v/v), and 1 mM PMSF), and the proteins were eluted by boiling with 40 μL 1×SDS loading buffer for 8 min. Co-IP proteins were separated by 10% SDS-PAGE and detected by immunoblotting with α-GFP (Sigma-Aldrich, Cat. SAB4701015, 1:5000 dilution) and α-FLAG antibodies (Sigma-Aldrich, Cat. SAB4301135, 1:5000 dilution).
In vitro phosphorylation assay
The in vitro phosphorylation assay was performed as previously described (Li et al. 2017) with slight modifications. Recombinant GST-tagged GmMPK3, GmMPK6, GmMKK4DD, GmMAPKKK5c, and GmMAPKKK5n proteins were affinity-purified by using glutathione sepharose resin. Recombinant MBP-tagged GmCDL1, GmLecRK02gcd/08gcd, and their mutated variants (GmCDL1K102E, GmCDL1T372A, GmCDL1T372D, GmCDL1K102E T372A, GmLecRK02gcdK427E, and GmLecRK08gcdK398E) were purified using amylase resin (Genscript, Nanjing, China). Recombinant GmMPK3 and GmMPK6 were incubated with recombinant GmMKK4DD in the reaction buffer (50 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 10 mM MnCl2, 50 μM ATP, and 1 mM DTT) at 28 °C for 30 min. The activated GmMPK3 and GmMPK6 were then used to phosphorylate recombinant GmCDL1, GmCDL1K102E, and GmCDL1K102E T372A proteins (1:10 enzyme–substrate ratio) in the reaction buffer (50 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 10 mM MnCl2, 25 μM ATP, 1 mM DTT, and 1 µCi of [γ-32P] ATP) at 28 °C for 30 min. Similar reactions were conducted to test the phosphorylation between GmLecRK02g/08 g and GmCDL1 or GmCDL1 and GmMAPKKK5. The reactions were terminated by adding 5×SDS loading buffer followed by boiling for 5 min. Proteins were separated by 8% SDS-PAGE, and phosphorylation was detected by autoradiography using FUJI FLA5100 (Fujifilm, Tokyo, Japan). The SDS-PAGE gels were stained with Coomassie blue as loading controls.
Detection of site-specific phosphorylation in vivo
Phosphosite-specific antibody of GmCDL1, α-pT372, was produced by ABclonal (Wuhan, China). The phosphopeptide (C-HAPG[pT]PPR) and the control peptide (C-HAPGTPPR) were synthesized and conjugated to keyhole limpet hemocyanin carriers for rabbit immunization. The polyclonal antibody was affinity-purified from rabbit antiserum by affinity chromatography using the phosphopeptide or the control peptide. The eluate for the phosphopeptide was passed through the column coupled with control peptides to remove nonspecific antibodies. To detect phosphorylation in vivo, total protein was extracted with protein extraction buffer, and GmCDL1 protein was immunoprecipitated with α-FLAG agarose beads. Site-specific phosphorylation was detected by immunoblotting with the α-pT372 antibody at a 1:1000 dilution. The α-GmCDL1 antibody was used to detect the endogenous protein level of GmCDL1 in soybean by immunoblotting at a 1:1000 dilution.
Protein degradation assay in N. benthamiana
The effect of phosphorylation on the protein stability of GmCDL1 was investigated as previously described with minor modifications (Pan et al. 2020). The N. benthamiana leaves expressing Myc-tagged GmCDL1, GmCDL1T372D, and GmCDL1T372A were harvested at 3 d post infiltration. The total proteins were extracted in a native extraction buffer (50 mM Tris-MES, pH 8.0, 0.5 M sucrose, 1 mM MgCl2, 10 mM EDTA, 5 mM DTT, and protease inhibitor cocktail). The lysate supplemented with 50 μM ATP and 50 μM protein synthesis inhibitor CHX (Sigma-Aldrich) was divided into 4 tubes and incubated at 28 °C for the indicated time period. GmCDL1 protein levels were detected with α-Myc immunoblot (ABclonal, Cat. AE010, 1:5000 dilution). Equal loading was confirmed by an α-actin immunoblot (ABclonal, Cat. AC009, 1:5000 dilution). For the in vivo degradation assay, the N. benthamiana leaves expressing the desired proteins were infiltrated with 50 μM CHX at 6 h before sample collection. After protein extraction, the levels of GmCDL1 protein were determined by immunoblotting. To evaluate the protein levels under wounding treatment, the N. benthamiana leaves expressing GmCDL1 and its phosphorylation variants were infiltrated with 50 μM CHX, in the absence or presence of 50 μM MG132 or MAPK pathway inhibitor U0126 as indicated, at 2 h before wounding treatment (Yoo et al. 2008; Ryu et al. 2017). After 15 min, samples were collected for immunoblotting.
MAPK activation assay
Soybean roots were treated with SCN, wounding, γATP, and P. sojae as described above for the indicated durations. Three 7-cm-long roots were collected, and total proteins were extracted by incubating homogenized samples in the aforementioned protein extraction buffer for 30 min. After centrifugation, equal amounts of proteins were separated by 10% SDS-PAGE, and MAPK activation was detected by immunoblotting with an α-phospho-p44/p42 MAPK monoclonal antibody (α-pERK, Cell Signaling Technology, Cat. 9101S) at a 1:2000 dilution. Ponceau staining of the Rubisco bands or α-actin immunoblot was used as the loading control. Chemical inhibitor U0126 was applied exogenously to soybean roots at the indicated concentrations to suppress the MAPK pathway. For N. benthamiana, U0126 was infiltrated at 50 μM into the leaves at the indicated time points.
RNA extraction and reverse transcription quantitative PCR
Total RNA was extracted from soybean roots using Ultrapure RNA Kit (CWBIO, Beijing, China). First-strand cDNA was synthesized using HiScriptII Q RT Super Mix (Vazyme, Nanjing, China) according to the manufacturer's instructions. To check the expression levels of the target genes, gene-specific primers were designed (Supplementary Data Set 4), and RT-qPCR was conducted using the AceQ qPCR SYBR Green Master Mix (Vazyme, Nanjing, China) with an iCycler iQ5 thermal cycler (Bio-Rad, CA, USA) real-time PCR system. SKP1/ASK-INTERACTING PROTEIN16 (SKIP16) was used as an internal control. The expression of the reference gene has been previously reported to remain unaffected by various stress conditions, including nematode infection (Cook et al. 2012; Guo et al. 2015; Wang et al. 2021). The dissociation curve was generated to verify amplicon specificity. Relative gene expression level was determined using the 2–ΔCt method with normalization to the internal control (Livak and Schmittgen 2001).
TMT labeling phosphoproteomic analysis
Soybean seeds of W82 were cultured on germination paper for 3 d, and the roots were inoculated with 300 J2s. Controls were mock-inoculated with 0.1% agarose alone. One day after inoculation, the roots were rinsed with sterile water to synchronize the infection. At 3 dpi, 3-cm-long root pieces around the infection site were harvested for quantitative phosphoproteomic analysis. Root tissue was homogenized in precooled extraction buffer (10 mM DTT, 1% proteinase inhibitor cocktail, and 1% phosphatase inhibitor) with sonication. An equal volume of Tris-saturated phenols was then added and vortexed for 5 min. The upper phenol phase was collected by centrifugation, and 5 volumes of ammonium sulfate-saturated methanol were added to precipitate the protein. The resulting precipitate was re-dissolved with 8 M urea, and the protein concentration was determined by the BCA Protein Assay Kit (Bio-Rad, Berkeley, CA, USA). The protein solution was subjected to trypsin digestion, TMT labeling, phosphopeptide enrichment, and LC-MS/MS analysis (PTM BIO, Hangzhou, China). The resulting data were processed using MaxQuant software v.1.5.2.8 and searched against the UniProt G. max database (https://www.uniprot.org/). A fold-change of ≥1.5 or ≤0.667 was regarded as significantly upregulated or downregulated. Motif-x software was used for phosphorylation motif enrichment analysis with P < 0.0003.
Phylogenetic analysis
The amino acid sequences of the RLCK VII proteins were obtained from Phytozome (https://phytozome-next.jgi.doe.gov) and Solanaceae Genomics Network (https://solgenomics.net/). The phylogenetic analysis was conducted with MEGA-X as previously described (Kumar et al. 2018; Rao et al. 2018). Full-length amino acid sequences were aligned using the MUSCLE algorithm of MEGA-X. A bootstrap consensus tree inferred from 500 replicates using the neighbor-joining method was constructed. The numbers next to the branches represent the percentage of bootstrap support. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distance used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method and represented in the units of the number of amino acid substitutions per site. The alignment and machine-readable tree files are provided as Supplementary Files S1 to S3.
Statistical analysis
The nematode infection and gene expression data were analyzed by Student's t-test or 1-way ANOVA with Tukey's test using GraphPad Prism v.8.3.0. Details of statistical data are provided in Supplementary Data Set 5.
Accession numbers
Sequence data from this article can be found in the Phytozome database (https://phytozome-next.jgi.doe.gov/) under the following accession numbers: GmCDL1 (Glyma.19G177300), GmCDL1-03g (Glyma.03G176500), GmCDL1-10g (Glyma.10G047900), GmCDL1-13g (Glyma.13G135800), GmLecRK02g (Glyma.02G221900), GmLecRK08g (Glyma.08G065800), GmLecRK09g (Glyma.09G110500), GmMPK3 (Glyma.12G073000), GmMPK6 (Glyma.02G138800), GmMKK4 (Glyma.07G003200), GmMAPKKK5 (Glyma.17G177900), SKIP16 (Glyma.12G025500), LecRK-IX.1 (AT5G10530), and AtBIK1 (AT2G39660).
Supplementary Material
Acknowledgments
We thank Dr. Shiming Liu (CASS, China) and Dr. Congli Wang (CAS, China) for kindly providing nematode populations and Dr. Wenwu Ye and Dr. Yuanchao Wang (Nanjing Agricultural University, China) for providing soybean root rot pathogen. We would like to thank the National Key Laboratory of Agricultural Microbiology Core Facility and the Center for Protein Research (CPR, HZAU) for assistance in microscopy imaging and LC-MS/MS analysis.
Contributor Information
Lei Zhang, National Key Laboratory of Agricultural Microbiology, College of Plant Science and Technology, Huazhong Agricultural University, Wuhan, Hubei 430070, China.
Qun Zhu, National Key Laboratory of Agricultural Microbiology, College of Plant Science and Technology, Huazhong Agricultural University, Wuhan, Hubei 430070, China.
Yuanhua Tan, National Key Laboratory of Agricultural Microbiology, College of Plant Science and Technology, Huazhong Agricultural University, Wuhan, Hubei 430070, China.
Miaomiao Deng, National Key Laboratory of Agricultural Microbiology, College of Plant Science and Technology, Huazhong Agricultural University, Wuhan, Hubei 430070, China.
Lei Zhang, Department of Botany and Plant Pathology, Purdue University, West Lafayette, IN 47907, USA.
Yangrong Cao, National Key Laboratory of Agricultural Microbiology, Hubei Hongshan Laboratory, Huazhong Agricultural University, Wuhan, Hubei 430070, China.
Xiaoli Guo, National Key Laboratory of Agricultural Microbiology, College of Plant Science and Technology, Huazhong Agricultural University, Wuhan, Hubei 430070, China.
Author contributions
X.G. supervised the work; L.Z. and X.G. designed the experiments and wrote the manuscript with input from all coauthors; L.Z. performed the experiments and data analysis; Q.Z., Y.T., and M.D. helped with the phenotyping experiments; Y.C. and L.Z. provided the vectors and helped with the data analysis. All authors approved the submitted version of the manuscript.
Supplementary data
The following materials are available in the online version of this article.
Supplementary Figure S1. GmMPK3 and GmMPK6 activation assay across soybean varieties inoculated with different virulent nematode populations.
Supplementary Figure S2. GmMPK3 and GmMPK6 activation assay in GmMPK3-edited or GmMPK6-silenced transgenic soybean roots.
Supplementary Figure S3. Expressions of GmMKK4, GmMPK3, and GmMPK6 in N. benthamiana.
Supplementary Figure S4. Silencing of GmMKK4 in transgenic hairy roots decreased soybean nematode resistance.
Supplementary Figure S5. GmCDL1 is belonging to the RLCK VII-1 subgroup.
Supplementary Figure S6. Expression pattern of GmCDL1 and its homologous genes in various tissues of soybean.
Supplementary Figure S7. GmMPK3 interacts with GmCDL1.
Supplementary Figure S8. The effect of Thr-372 phosphorylation on the function of GmCDL1.
Supplementary Figure S9. GmCDL1 homologs were detected by α-GmCDL1 immunoblot.
Supplementary Figure S10. Expression analysis of GmCDL1 in transgenic soybean roots.
Supplementary Figure S11. GmCDL1 interacts with GmMAPKKK5 and phosphorylates its N-terminus and C-terminus.
Supplementary Figure S12. Expression of GmLecRK02g and GmLecRK08g in N. benthamiana leaves.
Supplementary Figure S13. Signal peptide deletion in GmLecRK02g abolished the interaction between GmLecRK02g and GmCDL1.
Supplementary Figure S14. Purified GmLecRK02gcd does not exhibit kinase activity.
Supplementary Figure S15. Identification of phosphosites within GmCDL1 by GmLecRK08g.
Supplementary Figure S16. Phosphorylation of Ser-234 and Thr-235 in GmCDL1 enhanced MAPK activation.
Supplementary Figure S17. Expression analysis of transgenic soybean roots.
Supplementary Figure S18. Knockout of GmLecRK02g and GmLecRK08g facilitated cyst nematode development in soybean roots.
Supplementary Figure S19. GmLecRKs–GmCDL1–GmMPK3/6 signaling pathways regulate soybean resistance to P. sojae.
Supplementary Data Set S1. List of phosphopeptides that were significantly regulated by soybean cyst nematode with fold-change ≥1.5 or ≤0.667 as the threshold.
Supplementary Data Set S2. Sequencing results of CRISPR/Cas9 editing sites in target genes.
Supplementary Data Set S3. List of GmCDL1-associated proteins by IP-MS/MS.
Supplementary Data Set S4. Primers used in this study.
Supplementary Data Set S5. Summary of statistical analysis.
Supplementary Data Set S6. Supplementary data of other replicates.
Supplementary File 1. Sequence alignment of RLCK VII-1 subgroup shown in Supplementary Fig. S5.
Supplementary File 2. Phylogenetic tree of RLCK VII-1 subgroup in Newick format for Supplementary Fig. S5.
Supplementary File 3. Sequence alignment of GmCDL1 homologs and AtBIK1 shown in Supplementary Fig. S16.
Funding
We gratefully acknowledge the financial support by the National Key Research and Development Program of China (2023YFD1400400), Huazhong Agricultural University Scientific and Technological Self-Innovation Foundation (2021ZKPY015), China Postdoctoral Science Foundation (2023M731236), and Natural Science Foundation of Hubei Province (2023AFB259).
Data availability
The data supporting the findings of the study are provided in Supplementary Data Set S6.
Dive Curated Terms
The following phenotypic, genotypic, and functional terms are of significance to the work described in this paper:
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Supplementary Materials
Data Availability Statement
The data supporting the findings of the study are provided in Supplementary Data Set S6.