Symbiotic interactions between some soil microbes and plants are formed to ensure a consistent supply of the inorganic nutrients needed for optimum growth and development of the host plants. Rhizobia bacteria, a nitrogen-fixing bacterium specific to legumes, and arbuscular mycorrhizal, which aids phosphorus absorption, comprise a well-known symbiotic interaction (Cao et al. 2017; Ryu et al. 2012). The relationship between legumes and bacteria that fix nitrogen is crucial to the global nitrogen cycle. 350 to 440 million tons of nitrogen are fixed annually by symbiosis with legumes, and research has shown that this amount contributes for 25 to 30 percent of the total nitrogen fixed by the global nitrogen cycle (Zahran 1999). There is almost a tenfold difference in the efficiency with which these legumes are able to fix nitrogen depending on the host species and the bacteria that fix nitrogen (Zahran 1999).

The establishment of the nitrogen fixing symbiosis is contingent upon the effective formation of a root nodule, which is a specialized organ of the host legume plants that are responsible for supplying an ideal environment for symbiotic rhizobia (Cao et al. 2017). Recent advances in our understanding of the molecular mechanisms underlying the symbiotic interactions between legumes and rhizobia have led to the discovery of critical plant signaling components that are involved in the sensing of nod factors (NF) and its downstream signal transduction pathways (Ghantasala and Roy Choudhury 2022; Minguillon et al. 2022). The characterization of nodulation mutants in model legume plants and the molecular cloning of the key genes encoding nodulation signaling components suggests that the signaling pathways for the nitrogen fixing symbiosis are evolutionary conserved in the legume family (Cao et al. 2017; Ghantasala and Roy Choudhury 2022).

Nodulation is the process by which legume hosts recognize the nod factors releasing by nitrogen-fixing rhizobia and set in motion the symbiotic nitrogen-fixing process. The nod factor accomplishes direct activation of its receptor complex, MtLYK3/NFP (LysM-receptor like kinase 3/Nod Factor Perception), and subsequent activation of downstream pathways (Geurts and Bisseling 2002; Limpens et al. 2003; Moling et al. 2014). These events rapidly induce nuclear Ca²⁺ spiking and direct activation of MtDMI3 (Does-not-Make-Infections 3, a Ca²⁺-calmodulin-dependent kinase) to properly modulate the transcriptional network mediated by nodulation-related transcription factors including NSP1/2 (Nodulation Signaling Pathway 1/2), NIN and ERN1/2 (ERF Required for Nodulation 1/2, (Ane et al. 2004; Ghantasala and Roy Choudhury 2022; Kalo et al. 2005; Levy et al. 2004; Minguillon et al. 2022).

A negative regulation of nodulation, also known as AON (Autoregulaton of nodulation), is also essential for deciding the optimal number of nodule development in parallel with positive nodule formation signals (Nishimura et al. 2002; Oldroyd and Downie 2008; Soyano et al. 2014; Tsikou et al. 2018). These strategies are important contributors to the overall effort to prevent excessive energy drains caused by marginal nitrogen fixing activities (Nishimura et al. 2002; Tsikou et al. 2018). Indeed, hyper-nodule formation mutants, which are created when negative regulatory mechanisms are disrupted, typically suffer from growth defect abnormalities (Nishimura et al. 2002). Most abiotic and biotic stresses, in addition to the hormones that are associated with them, have a significant role in the inhibition of nodule formation (Ryu et al. 2012; Ryu et al. 2017). It is well known that stress hormones such as salicylate, jasmonate, ABA, and ethylene play negative effects in the nodulation process (Cao et al. 2017; Minguillon et al. 2022). In plants, MAPK signaling cascades serve a central signal signaling cues for a variety of stress responses (Meng and Zhang 2013). Symbiotic rhizobia bacterial infections also quickly activated MAPK signaling cascades in legume plants, which led to the activation of a large number of defense-related gene networks and stress-related hormones (Ouaked et al. 2003; Ryu et al. 2017). In addition, I found previously unidentified negative effects that were driven on by MtMKK5-MtMPK3/6-MtERN1 signaling cascades in the formation of nitrogen-fixing nodules in M. truncatula (Ryu et al. 2017). However, the physiological functions of the MtMKK5-trggered defense signaling pathways in the development of nitrogen-fixing nodules are still unknown. In this study, I present molecular evidence of defensive signaling pathways activated by the MtMKK5-MtMPK3/6 signaling module having detrimental effects on the development of nodules.

Plant Materials and nodulation assay

Medicago truncatula cv. Jemalong A17 and Arabidopsis thaliana Col-0 were served as the genetic background and wild-type control in this study. For Medicago plants, after being gently stirred for 5 minutes in strong sulfuric acid, medicago seeds were rinsed with sterile water. Sodium hypochlorite was used to further sterilize the seeds for 2 minutes. The surface-sterilized seeds were placed on upside-down agar plates and kept in the dark at 4°C for two days, and then they further grew in two days at 23°C. Germination seedlings were transferred to Farhaeus agar plates containing 1mM NH₄NO₃ for 2 weeks, and then they were switched to Farhaeus agar plates devoid of a nitrogen source as instructed in the Medicago handbook (http://www.noble.org/MedicagoHandbook/) for 1 week. Nitrogen-starved Medicago seedlings were infected with 200 mL of Sinorhizobium meliloti ABS7M (expressing an aminolevulinic acid synthetase-lacZ fusion) suspension at an OD600 = 0.02 with/without 5 mM of U0126 for nodulation tests, and they were then grown at 23°C (light-dark photoperiod: 16 h/8 h) for an additional two weeks.

Plasmid construction and transgenic plants

The 35S C4PPDK promoter was used to clone the full-length cDNAs (open reading frames (ORFs)) of MtMKK5, MtMKK5a (T229E and S235E; Ryu et al. 2017), MtNIN, MtMPK3, and MtMPK6 into plant specific expression vectors that contained hemagglutinin (HA) or MYC (Ryu et al. 2007). All phenotypic studies were performed on homozygous T3 plants, and the floral dip method was used to generate Arabidopsis overexpressing plants.

qRT-PCR analysis

Total RNA was extracted using the Trizol reagent (Invitrogen) to determine the transcripts’ expression levels. With the use of ImProm-II reverse transcriptase (Promega) and oligo dT primers, double strand cDNA was created from 1mg of RNA. I utilized the gene-specific primers 5’-CAGTGTCTGGATCGGAGGAT-3’ and 5’-TGAACAA TCGATGGACCTGA-3’ for AtACT2 in Quantitative Real-Time PCR. The gene-specific primer sets for AtFRK1 and WRKY29 (Chung and Sheen 2017; Shan et al. 2007) and MtMPK3/ MtMPK6 (Ryu et al. 2017) were previously reported.

Protein-protein interaction and immunoblotting assays

Yeast-two-hybrid assay was performed as described by our previous study (Hong et al. 2021a; Kim et al. 2021). Myc-tagged MtMPK3/6 was transfected into Arabidopsis protoplasts with or without HA-tagged MtMKK5a plasmid DNA to conduct immuno precipitation (IP) experiments. Following that, protoplasts were incubated for 6 hours to allow the transgenes to express properly. Using IP buffer [50 mM Tris-HCl (pH 7.5), 75 mM NaCl, 5 mM EDTA, 1 mM DTT, 1 X protease inhibitor cocktail (Roche), and 1% Triton X-100], total proteins were extracted from the transfected protoplasts. Anti-myc monoclonal antibody (Cell signaling) was added to the extracted proteins for 1 hour, after which the protein complexes were precipitated using ProteinA/G agarose beads (GE healthcare). The precipitated proteins were identified using high-affinity anti-HA (Roche), anti-pTEpY (Cell signaling), or anti-myc antibodies (Cell signaling). Total proteins from protoplasts or seedlings were separated with a 7.5% and 10% SDS-PAGE gel for the immunoblotting studies, and the horseradish peroxidase-conjugated anti-HA (Roche) antibodies were used to detect the protein expression level.

Histological Analysis

The nitrogen-fixing nodule’s histological section was performed in accordance with our earlier report (Hejatko et al. 2009; Hong et al. 2021b). Samples of nodule tissue were fixed in an FAA (Formalin-Aceto-Alcohol) solution containing 5% acetic acid, 45% ethanol, and 5% formaldehyde for 24 hours. The fixed samples were then dehydrated using a graduated ethanol series after being washed with 0.1M phosphate buffer, pH 7.2. The samples were embedded into Spurr’s resin (Ted Pella) for 48 hours. Leica’s RM2065 ultramicrotome was used to cut sections (0.5 or 4 mm), which were then stained with 0.1% toluidine blue and captured on camera using an Olympus BX 53 microscope.

Our previous study revealed that the MtMKK5-MtMPK3/6 signaling module is essential in preventing the formation of symbiotic nodules under stress conditions, although rhizobia infection tread formation remained unaffected (Ryu et al. 2017). These impose that particular developmental signaling pathways involved in symbiotic nodule organogenesis would interact with stress-activated MAPK signaling pathways. To more specifically examine the putative connection between MAPK signaling cascades and the development of symbiotic nodules, I first examined the effects of the MAPKK-specific inhibitor U0126 on the development of nitrogen-fixing nodules in M. truncatula. The lac-Z expressing S. meliloti ABS7M strain successfully colonized and established symbiotic nodules in Medicago roots regardless of U0126 treatment (Fig. 1A). Because the U0126 treatment resulted in a slightly larger nodule (Fig. 1A), I tested the histological analysis using plastic resin to examine the developmental stage of the symbiotic nodule. In M. trucatula, the development of an indeterminate nodule consists of an infection zone (zone I) where rhizobia invade, a meristematic zone II where cell division is active, and a zone III and IV where bacteroid differentiation and nitrogen fixation occur (Pan and Wang 2017; Wang et al. 2018). Specific inhibition of MAPK signaling activities during nitrogen fixing nodule development increased cell division in zones I-II (Fig. 1B). However, U0126 treatment had no effect on bacteroid differentiation in zone III and IV (Fig. 1B). These findings suggest that symbiotic rhizobia infections inhibit the cell proliferation activity of meristem cells in nitrogen-fixing indeterminate nodules by activating MAPK signaling.

Figure 1. MAPK signaling inhibits the proliferation of nodule meristem cells during nitrogen-fixing nodule development. (A) U0126 treatment results in the formation of slightly large nodules in M. truncatula. The phenotypes of nodules on M. truncatula roots infected with Sinorhizobium meliloti for 3 weeks with/without 5 μM U0126. All inoculated S. meliloti strains containing a hemA-lacZ reporter gene for visualizing bacterial cells appear as blue in the β-galactosidase assay. (B) Microscopy analysis for the characterization of symbiotic nodules induced in the absence (left) or presence (right) of 5 μM U0126. Two-micrometer sections of 7-day-old nodules are shown. Sections of nodule zone I–II and zone III–IV are presented. Scale bars = 500 μm (A) and 200 μm (B)

I then investigated whether rhizobia infection or its induced defense signaling cues directly influence the transcriptional regulation of MtMPKs. Under stress conditions, MKK4/5 and its homologous proteins enhance the transcription level and spontaneously induce the phosphorylation of MPK3/6 to stimulate downstream signaling pathways (Meng and Zhang 2013). Because U0126 treatment modulated the upregulation of early symbiotic related transcription factors (Ryu et al. 2017), I examined the early expression patterns of MtMPK3 and MtMPK6 after inoculating S. meliloti to Medicago roots in the presence or absence of U0126 for 1, 6, and 12 hours (Fig. 2A and 2B). Interestingly, symbiotic bacterial infection only increased slightly MtMPK3 but not MtMPK6 until 6 hpi (hour post inoculation), and this expression pattern vanished in the presence of U0126 (Fig. 2A and 2B). Next, I transfected MtMPK3/6 with or without MtMKK5a into protoplasts to investigate the possibility that MtMKK5 could directly phosphorylate the MtMPK3/6. Using an anti-pTEpY antibody in immunoblotting, I confirmed that the phosphorylation of the TEY amino acid motif in the MtMPK3/6 activation loop enhanced the protein-protein interaction with MtMKK5a (Ryu et al. 2017; Fig. 2C). Additionally, I determined that MtMPK3 and MtMPK6, but not MtMPK13, physically interact with the MtNIN protein (Fig. 2D). These findings suggest that MtMKK5-MtMPK3/6-based MAPK signaling cascades negatively influence the early stage of symbiotic interactions between legumes and nitrogen-fixing rhizobia bacteria.

Figure 2. The MtMKK5-MtMPK3/6 module directly interacts with MtNIN. (A, B) MtMPK3 is a Rhizobium-response gene, and its induction is required for MAPK signaling activation. After incubation for 1 hr with/without 5 μM U0126, M. truncatula roots were infected with S. meliloti for the indicated time durations. The levels of the transcripts of MtMPK3 (A) and MtMPK6 (B) were assessed by qRT-PCR. The error bars indicate the S.E. (n = 3); a Student’s t-test was performed (**, P < 0.01; n.s.: no significant). (C) MtMPK3 and 6 are phosphorylated by MaMKK5a. Myc-tagged MtMPK3 and 6 were transfected with/without MtMKK5a-HA into Arabidopsis protoplasts. The MtMPK3 and 6 proteins were immunoprecipitated using anti-myc antibodies and subjected to SDS-PAGE. The dual TEY motif phosphorylation of MtMPK3 and 6 were detected using anti-pTEpY monoclonal antibodies. The expression levels of MtMPKs and MtMKK5a were determined via immunoblotting. (D) MtNIN and MtMPK3/6 interact physically, as demonstrated via a yeast two hybrid assay. The yeasts were selected on synthetic medium lacking Leu, Trp, and His (-LTH) containing 5 mM 3-AT or medium lacking Leu and Trp (-LT)

The MKK5-MPK3/6 signaling cascade is required for a wide range of stress and its related hormone signaling outputs (Meng and Zhang 2013). This module may also contain critical components for ethylene and defense signaling pathways, which are major negative signals for nitrogen-fixing symbiosis (Ryu et al. 2012; Wood 2001). To determine whether MtMKK5-mediated MAPK signaling cascades might trigger both ethylene and defensive responses, MtMKK5 and MtMKK5a-overexpressing Arabidopsis plants were generated. I first verified the levels of MtMKK5(a) protein expression and selected each of the four separate T3 homozygote lines (Fig. 3A upper). Although MKK9 and SIMKK overexpression displayed to induce strong ethylene-related triple responses in Arabidopsis seedlings (Ouaked et al. 2003; Yoo et al. 2008), the 35S-MtMKK5 and 35S-MtMKK5a lines did not exhibit any differences from Col-0 seedlings (Fig. 3A). This implies that the MtMKK5-mediated MAPK signaling cascades would be not involved in the transduction of the ethylene signals in plants. The 35S-MtMKK5a lines, on the other hand, demonstrated stronger defense phenotypes against the Pst DC3000 pathogen in a protein expression dependent manner than Col-0 (Fig. 3B). Defense-related MAPK responsive FRK1 and WRKY29 expression levels were consistently highly correlated with pathogen defense responses (Fig. 3C). Overall, these findings support a central function for the MtMKK5-MtMPK3/6 module in plant defensive signaling pathways.

Figure 3. MtMKK5 enhances plant defense signaling pathways, but not ethylene signaling. (A) Overexpression of MtMKK5-HA and MtMKK5a-HA in Arabidopsis. Protein expression levels of the MtMKK5/MtMKK5a were determined using HRP-conjugated anti-HA antibodies. Actin was used as the internal control (upper panel). Seven-day-old seedlings with the Col-0, 35S-MtMKK5-HA, and 35S-MtMKK5a-HA phenotypes were grown under dark conditions (lower panel). Scale bars = 1 cm. (B) Disease symptoms in the Col-0, 35S-MtMKK5-HA, and 35S-MtMKK5a-HA seedlings. The direct spray method was used to infiltrate 3-week-old Arabidopsis with Pst DC3000 (OD600 = 0.002). The phenotypes were observed three days after inoculation. (C) Overexpression of MtMKK5a enhances the expression levels of the defense-related genes FRK1 and WRKY29. The transcript levels in the 35S-MtMKK5a-HA lines were determined using qRT-PCR. The error bars indicate the S.E. (n = 3); a Student’s t-test was performed (**, P < 0.01). Scale bars=1cm

The study of nitrogen-fixing root nodules is one of the essential areas of research for comprehending not only the agricultural significance of nitrogen fixation, but also the cellular response that determines the new fate of cells for novel differentiation (Ghantasala and Roy Choudhury 2022; Oldroyd and Downie 2008; Wang et al. 2018). Numerous studies have been conducted to identify the receptor protein complex that recognizes the nod factor released by nitrogen-fixing rhizobia bacteria. Furthermore, key signaling components and genes involved in the connection with symbiotic signaling pathways have been identified exclusively (Cao et al. 2017; Ghantasala and Roy Choudhury 2022). Plant hormones such as cytokinin and ethylene, as well as Ca²⁺-mediated transcriptional networks, have been identified as key initial cues for root nodules in these studies (Hamel et al. 2006; Oldroyd and Downie 2008; Ryu et al. 2012; Tsikou et al. 2018). This study reveals that the MtMKK5-MtMPK3/6 module, which is rapidly activated in the early symbiotic process of legumes, suppresses the formation of nitrogen-fixing nodules by interacting with NIN, a crucial transcription factor of symbiotic signaling, and defense signaling against pathogens. One particularly intriguing discovery of this study is that symbiotic signaling and defense against pathogens can both be regulated by the MtMKK5-mediated MAPK signaling module. These findings highlight the requirements for additional research into which transcription factors regulated by MAPK signaling govern the expression of symbiotic genes. In addition, this study supplied a crucial insight regarding the outcomes of studies that can enhance the efficiency of nitrogen fixation by modulating the activity of MAPK signaling.

This work was supported by the National Research Foundation (NRF-2021R1I1A3050947).

Author has read the manuscript and declared that he has no conflict of interest.