During the symbiosis of legumes with nitrogen-fixing bacteria, collectively called rhizobia, suppression of excessive rhizobial infection by host plants is important to maximize the benefits of symbiotic nitrogen fixation. However, the molecular mechanism involved in the suppression remains relatively poorly understood. We performed LC-MS and RNA-Seq analysis using rhizobia-infected Lotus japonicus roots and investigated the role of phosphatidylinositol (PI) and phosphatidylinositol phosphates (PIPs) in the symbiosis. Phosphatidylinositol transfer protein (PITP)-like proteins 4 (PLP4), phosphatidylinositol 3-phosphate 5-kinase 4 (PIP5K4), and PIP5K6 mutants, which are involved in the vesicular transport of lipids and phosphorylation of PIPs, were used to show the involvement of the signaling of PI and PIPs. Accumulation of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] during rhizobial infection was examined by a fluorescent marker 1×TUBBY-C (TUBBY). We found that PI signaling-related genes were upregulated, and the amount of PIP2 increased in L. japonicus roots during rhizobial infection. In the PLP4, PIP5K4, and PIP5K6 mutants, rhizobial infection increased, while PIP2 accumulation failed. Furthermore, the observation of PI(4,5)P2 in rhizobia-infected roots revealed that ectopic accumulation was closely related to the suppression of rhizobial infection. Our findings indicate that the accumulation of PI(4,5)P2, mediated by PLP and PIP5Ks, suppresses excessive rhizobial infection in the root epidermis and cortex, leading to the optimal number of nodules.

Chitin, an N-acetyl-D-glucosamine polymer, has multiple functions in living organisms, including the induction of disease resistance and growth promotion in plants. In addition, chitin oligosaccharides (COs) are used as the backbone of the signaling molecule Nod factor secreted by soil bacteria rhizobia to establish a mutual symbiosis with leguminous plants. Nod factor perception triggers host plant responses for rhizobial symbiosis. In this study, the effects of chitins on rhizobial symbiosis were examined in the leguminous plants Lotus japonicus and soybean. Chitin nanofiber (CNF), retained with polymeric structures, and COs elicited calcium spiking in L. japonicus roots expressing a nuclear-localized cameleon reporter. Shoot growth and symbiotic nitrogen fixation were significantly increased by CNF but not COs in L. japonicus and soybean. However, treatments with chitin and cellulose nanofiber, structurally similar polymers to CNF, did not affect shoot growth and nitrogen fixation in L. japonicus. Transcriptome analysis also supported the specific effects of CNF on rhizobial symbiosis in L. japonicus. Although chitins comprise the same monosaccharides and nanofibers share similar physical properties, only CNF can promote rhizobial nitrogen fixation in leguminous plants. Taking the advantages on physical properties, CNF could be a promising material for improving legume yield by enhancing rhizobial symbiosis.

Host plants benefit from legume root nodule symbiosis with nitrogen-fixing bacteria under nitrogen-limiting conditions. In this interaction, the hosts must regulate nodule numbers and distribution patterns to control the degree of symbiosis and maintain root growth functions. The host response to symbiotic bacteria occurs discontinuously but repeatedly at the region behind the tip of the growing roots. Here, live-imaging and transcriptome analyses revealed oscillating host gene expression with approximately 6-hour intervals upon bacterial inoculation. Cytokinin response also exhibited a similar oscillation pattern. Cytokinin signaling is crucial to maintaining the periodicity, as observed in cytokinin receptor mutants displaying altered infection foci distribution. This periodic regulation influences the size of the root region responsive to bacteria, as well as the nodulation process progression.

Arbuscular mycorrhiza (AM) is a mutualistic plant–fungal interaction that greatly benefits the growth of both organisms (Parniske, 2008; Smith & Read, 2010). AM fungi enter the host root, and their hyphae elongate through the cortical cell layer, forming a tree-like symbiotic structure called 'arbuscule', which facilitates nutrient supply to the plant (Harrison, 2012). AM fungi provide nutrients, including phosphate, minerals, and water to the host plant. In return, the fungi receive photosynthetic products from the host plant via the arbuscules (Bago et al., 2003; Zhu & Miller, 2003). AM fungi are known to secrete multiple symbiotic signaling molecules recognized by the host plant, such as chito-oligosaccharides (COs) and lipo-chito-oligosaccharides (Maillet et al., 2011; Genre et al., 2013). Signal perception by the host plant activates symbiosis-related signaling and responses, such as symbiosis-related gene expression and the oscillation of intracellular calcium (Ca) concentration, called Ca spiking (Genre et al., 2013). Lysin motif (LysM)-type receptor proteins are known to perceive COs and regulate AM in rice (Oryza sativa), tomato (Solanum lycopersicum), legumes, and many other plants (Miyata et al., 2014; Buendia et al., 2016; Liao et al., 2018; Feng et al., 2019; Girardin et al., 2019). In rice, chitin elicitor-receptor kinase 1 (OsCERK1) and chitin elicitor-binding protein (CEBiP) form heterodimers that mediate the long-chain CO perception and signaling (Kaku et al., 2006; Shimizu et al., 2010). The perception of COs by the LysM receptor plays an important role in triggering plant immunity (Desaki et al., 2018). In the leguminous plant Lotus japonicus, LYS6 (CERK6) is a LysM receptor involved in CO perception and pathogenic responses; however, its relationship with AM has not yet been demonstrated (Bozsoki et al., 2017). Furthermore, AM fungi infection has been shown to induce LYS11 expression in L. japonicus; however, the phenotype of the lys11 mutant was comparable to that of the wild-type (WT) (Rasmussen et al., 2016). In this study, we searched for receptors involved in AM among CERK1 homologs in L. japonicus, revealing the involvement of two LysM receptors in regulating AM. Seeds of WT L. japonicus MG-20 and the LysM receptor mutant lines were scarified with sandpaper and sterilized with sodium hypochlorite (effective chloride 1%). Once sterilized, the seeds were soaked overnight in sterilized water, germinated on 0.8% agar plates, and grown in a growth chamber (16 h : 8 h, 24°C, light : dark). Spores of Rhizophagus irregularis (DAOM197198; PremierTech, Rivière-du-Loup, QC, Canada) were inoculated on the host plant (> 100 spores per plant) planted in pots containing 300 ml of soil supplied with ½-strength Hoagland solution containing 0.1 mM KNO3 (100 ml per pot). A chive (Allium schoenoprasum) nurse pot system was used to count hyphal attachments and entry points in the host root (Demchenko et al., 2004). The LysM receptors homologous to OsCERK1 in L. japonicus (phylogenetic group LYS-1 in Lohmann et al., 2010) and other homologs known to be involved in AM and root nodule symbiosis were selected for phylogenetic analysis. Amino acid sequences of the LysM receptors were obtained from the National Center for Biotechnology Information and genome database of L. japonicus (http://viewer.shigen.info/lotus/index-j.php). Full-length amino acid sequences were aligned using ClustalW, and a phylogenetic tree was constructed using the neighbor-joining method implemented in the Mega11 software (v.11.0.13, https://www.megasoftware.net/) with a bootstrap value of 1000 replicates (Tamura et al., 2021). The CRISPR/Cas9 system was used to mutate the LYS6 and LYS7 genes of L. japonicus MG-20 plants. Two target sites were selected for each target gene using the CRISPR-P program (http://cbi.hzau.edu.cn/crispr/) (Lei et al., 2014). Each target site sequence harbored > 3 bp mismatches with potential off-target sites in the L. japonicus genome. Target oligonucleotides (primer sets 1–8 in Supporting Information Table S1) were annealed and cloned into the BbsI site of the single-guide RNA (sgRNA) vector pUC19_AtU6oligo (sgRNA targets 1–4; Fig. S1) (Ito et al., 2015). The sgRNA cassette was amplified, and the amplicon was ligated to the PCR product of the other sgRNA (primer sets 9–12 in Table S1). It yielded a vector with the four sgRNA cassettes arranged in tandem. The sgRNA cassettes were then subcloned into the I-SceI site of pZH_gYSA_FFCas9, which already contained the Cas9 and HPT expression cassettes (Ito et al., 2015). CRISPR/Cas9 constructs were introduced into L. japonicus MG-20 using Agrobacterium tumefaciens AGL1, as previously described (Stiller et al., 1997). Mutations around the target sites in the transgenic plants were first evaluated by PCR and then confirmed by sequencing. Homozygous mutants were selected from T1 transgenic plants, and lys6lys7 double mutants (#24, #28, #29) and a lys7 single mutant (#15) were obtained. Progeny (T2) was used in this study. Subsequently, the double mutants were crossed with the WT, and lys6 (lys6#24, #28, #29) and lys7 (lys7#24, #28) single mutants were obtained from the F2 plants. The F3 generation of the single mutants was also used for subsequent analyses. WT seeds from the same parent plants used for the transformation or crossing were used as WT controls in all experiments. Ca spiking was observed using nuclear-localized yellow-cameleon 2.60 (NLS-YC) (Nagai et al., 2004). NLS-YC, under the control of the Ubiquitin promoter, was introduced into L. japonicus via hairy root transformation using A. rhizogenes AR1193 (Takeda et al., 2009). Transgenic roots were treated with a CO mixture (1–8 mers, 1 mg ml−1) (Seikagaku Corporation, Tokyo, Japan), chitin tetramer (CO4, 10−6 M) (IsoSep AB, Tullinge, Sweden), or chitin octamer (CO8, 10−6 M) (IsoSep AB) in BNM medium (Ehrhardt et al., 1996). Ca imaging was performed using a Nikon microscope TE2000-U equipped with 20× dry objectives and imaging systems (MAC5000; Ludl electronic products, Hawthorne, NY, USA, and CoolSNAP Dyno; Teledyne Photometrics, Tuscon, AZ, USA). Fluorescent images of the NLS-YC root were acquired every 5 s with CFP (CFP ex. 440 nm/CFP em. 480 nm) and fluorescence resonance energy transfer (FRET; CFP ex. 440 nm/YFP em. 535 nm) filter sets. Fluorescence intensities of FRET and CFP around the nucleus were measured, and the ratio (FRET int. : CFP int.) was calculated using NIS-Elements AR (Nikon, Tokyo, Japan). Cells with more than two Ca increases were counted as spiking+ cells, and the ratio (spike+ cells : total cells) was calculated. More than three roots (5–15 cells per root) were analyzed in WT and each mutant line. Total RNA was extracted from whole root samples obtained from 5 to 7 plants using the PureLink™ Plant RNA Reagent (Thermo Fisher Scientific, Waltham, MA, USA). Total RNA concentration was quantified using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific). Reverse transcription and real-time PCR were performed using the ReverTra Ace qPCR RT kit (Toyobo, Osaka, Japan) and the Thunderbird qPCR Mix (Toyobo) on the AriaMx Real-Time PCR System (Agilent, Santa Clara, CA, USA), according to the manufacturers' instructions. cDNA was synthesized from 250 ng of total RNA, and each reaction was performed in duplicate or triplicate. CO-induced genes were identified through transcriptome analysis of the root infected with AM fungi (Takeda et al., 2015), and of the roots treated with chitin pentamer (data not shown). The primer sets used for this analysis are listed in Table S1 (primer sets 13–26). PCR conditions were set as described previously by Takeda et al. (2013). Relative gene expressions were compared against Ubiquitin and Elongation factor1 transcript levels using the 2−ΔΔCt method. The relative expression levels did not significantly change between both internal controls. The relative values corrected by Ubiquitin are shown in the graphs of qRT polymerase chain reaction analysis. The fungal structures in the host roots were stained using the ink-staining method (Demchenko et al., 2004) or with a wheat germ agglutinin (WGA)-Alexa Fluor 594 conjugate (Invitrogen) (Harrison et al., 2002). Briefly, the root samples were heated in 5% KOH at 95°C for 1 h (for ink staining) or 15 min (for WGA-Alexa Fluor staining), and washed three times with water or phosphate-buffered saline (PBS). For ink staining, the roots were incubated at 95°C in a solution containing 3% black ink and 5% acetic acid for 20 min. For fluorescent staining, the roots were immersed in PBS containing WGA-Alexa Fluor 594 (1 μg ml−1 final concentration) and kept at room temperature for 1 h. Bright-field and fluorescence microscopy were performed with a stereomicroscope (SZX16; Olympus, Tokyo, Japan) and a confocal microscope (A1; Nikon). The root infected with AM fungi was stained with either ink or WGA-Alexa Fluor 594, and the frequency of colonization (the intercellular fungal structures per unit of root length) was calculated using the magnified intersection method (McGonigle et al., 1990). Finally, the number of hyphal entries in the infected roots was counted and divided by the root length. NFR1 and LYS1 (NFRe), which are involved in root nodule symbiosis (Radutoiu et al., 2003; Murakami et al., 2018), LYS2, LYS6 (CERK6), and LYS7 are close homologs of OsCERK1 in L. japonicus (Fig. 1a). Of these, LYS6 and LYS7 showed a high amino acid identity with OsCERK1 (59.2% and 57.4%, respectively). Furthermore, qRT polymerase chain reaction of these LysM receptors in the roots infected with AM fungi (Rhizophagus irregularis) revealed LYS7 upregulation in the host root, indicating potential involvement of LYS7 in AM (Fig. 1b). Previous studies involving transcriptome analyses of the roots infected with AM fungi also showed either LYS7 induction and no upregulation of the other LysM receptors (Handa et al., 2015; Takeda et al., 2015). Next, we created lys6 and lys7 single mutants and lys6lys7 double mutants using the CRISPR/Cas9 system in L. japonicus MG-20 Miyakojima to assess the function of LysM receptors in AM (Figs S1–S3). The AM fungi-infected roots of the single mutant lines exhibited significantly reduced hyphal colonization and arbuscule formation than WT roots (Figs 1c, S4). This result was consistent with the finding of a previous study reporting the involvement of OsCERK1 and MtLYK9, a close homolog of LYS6, in AM responses in O. sativa and Medicago truncatula, respectively (Miyata et al., 2014; Gibelin-Viala et al., 2019). Another study reported that OsRLK10 (OsCERK2) knockout in rice or RNAi knockdown of SlLYK12 in tomato notably reduced AM fungi colonization in the infected roots (Liao et al., 2018; Miyata et al., 2022). Both proteins had a high amino acid sequence identity with LYS7 in L. japonicus (52.0% and 65.5%, respectively). These findings suggested that LYS6 and LYS7 play a key role in AM in L. japonicus. However, despite reduced fungi colonization in the roots of single mutants compared with WT roots, the single mutants exhibited normal inner hyphae morphology and arbuscule formation (Figs 1d, S5). By contrast, the roots of double mutant plants did not exhibit fungal infection in the roots at 4 wk (Figs 1c, S6a,b) and 6 wk (Fig. S6c,d) after infection with AM fungi. In the double mutant plants, AM fungal hyphae were attached to the root surface, and the morphology of the hyphae was comparable to that in the WT roots (Fig. S7). However, the double mutant roots did not exhibit any hyphal colonization, similar to common symbiosis mutants such as pollux and ccamk (Figs 1c, S4) (Parniske, 2008). The similar phenotype was also observed in the double mutants of the M. truncatula orthologs MtLYK8 and MtCERK1 (Zhang et al., 2024). OsCEKR1 and OsRLK10 (OsCERK2) are considered functional homologs of LjLYS6/LjLYS7; however, the Oscerk1/Oscerk2 double mutant does not exhibit an additive phenotype (Miyata et al., 2022). The LysM receptor genes are considered rapidly evolving genes, and differences in the number of LysM receptor genes have been reported among M. truncatula ecotypes (Luu et al., 2022). These findings suggest that the mechanisms underlying LysM receptor-mediated AM signaling pathway might differ between the monocotyledonous rice and the dicotyledonous L. japonicus. In addition, the additive phenotype of the double mutants suggests that LYS6 and LYS7 regulate AM fungi infection via genetically distinct pathways. However, the impaired fungal infection in the double mutants might be attributed to the redundancy in the functions of these two receptors. Therefore, we investigated the differences in the symbiotic phenotypes of lys6 and lys7 mutants. A previous study reported that the hyphal colonization in the lys6 mutant root was not significantly different from the WT in the longer term (6 wk) infection with AM fungi (Bozsoki et al., 2017). Therefore, we speculated that AM fungal infection in the mutants was aborted in the early stages. To examine the effects of loss-of-function mutations in LYS6 and LYS7 on the initial stage of AM fungi infection, we assessed the number of hyphopodia and the number of hyphal entries into the host roots (Fig. 1e). The number of hyphopodia in the single mutant roots was comparable to that of the WT. Moreover, no morphological abnormalities of hyphopodia, inner hyphae, or arbuscules were observed in the roots of any single mutants (Fig. S5a–c). However, the lys6 mutants exhibited significantly fewer hyphal entries, with a lower entry/hyphopodia ratio than in WT (Fig. 1f). This result indicated that LYS6 knockout restricted hyphae penetration into the host root, suggesting that LYS6 mediates hyphal entry into the host root via the hyphopodia. By contrast, the entry/hyphopodia ratio for lys7 mutants was comparable to that for WT (Fig. 1e,f). The phenotypic difference between lys6 and lys7 mutants during the early stage of infection demonstrates the distinct function of these receptors in AM-related signaling pathways. The expression of AM-induced genes, including RAM1, RAM2, STR1, SbtM1, PT4, VAPYRIN1(VPY1), and EXO70I, was analyzed in mutant roots inoculated with AM fungi (Figs 1g, S8). None of these genes were induced in the lys6lys7 double mutant, reflecting the absence of an infection phenotype. On the contrary, RAM1, RAM2, STR1, SbtM1, and PT4 were induced in the single mutants after AM fungi infection. In the lys7 mutants, all the AM marker genes were induced by AM fungi infection (Fig. 1g); however, it exhibited significantly lower RAM1 and EXO70I expression levels than WT (Fig. S8). The decrease in expression could be reflected by the reduced hyphal colonization and arbuscule formation in the lys7 root. Notably, the lys6 mutants did not exhibit VPY1 and EXO70I induction, suggesting that VPY1 and EXO70I might be located downstream of the LYS6-mediated perception and signaling pathways. The normal arbuscule formation with PT4 induction in the lys6 mutants observed in this study was not consistent with the abnormal arbuscule development previously reported in the M. truncatula exo70I mutant (Fig. 1d,g) (Zhang et al., 2015). The basal expression level of EXO70I in the lys6 mutants may be sufficient to induce arbuscule formation. Meanwhile, VAPYRIN gene knockdown has been shown to reduce hyphal entry in M. truncatula (Pumplin et al., 2010). Although the lys6 mutants did not show the aberrant hyphopodia structures such as the swollen hyphopodia previously reported in the MtVAPYRIN knockdown plants (Fig. S5b), the reduced AM fungi infection in the lys6 mutants might be attributed to the lack of induction of VPY1 and other AM-induced genes regulated by the LYS6-mediated signaling pathway. Chito-oligosaccharides are AM signaling molecules, and LYS6 mediates CO perception and subsequent signaling pathways (Bozsoki et al., 2017). A previous report showed a lack of periodic oscillation in Ca concentration 'Ca spiking' in the rice Oscerk1 mutant (Carotenuto et al., 2017). Therefore, in this study, we analyzed the Ca spiking after CO perception in the mutants. Treatment with high concentrations of CO mixture (1–8 mers) induced Ca spiking in the lys7 mutants, with Ca spiking patterns and spike+ cell/total cell ratios comparable to those in WT (Fig. 2a,b). By contrast, the CO-treated lys6 roots did not exhibit a typical Ca spiking (Fig. 2a,b; lower pattern of lys6). Similarly, the lys6lys7 double mutant did not show a typical Ca spiking pattern by CO treatment (Fig. S9). The residual Ca concentration changes observed in the lys6 and lys6lys7mutants, which were counted in spiking + cells (Figs 2a,b, S9 top and middle pattern of lys6), differed from normal Ca spiking in the shape and frequency of Ca concentration increases, suggesting that they represent other physiological Ca responses in the root cells. A previous study using aequorin-based Ca tracing revealed an increase in nuclear Ca concentration in the lys6 mutants of L. japonicus by the CO treatment, which was suggested to be a CO-induced Ca spiking (Binci et al., 2024). However, this study also reported a marked reduction in the number of responding cells. Thus, this increase in nuclear Ca concentration would correspond to the residual Ca response in the lys6 mutant cells observed in our study (Fig. 2b). These results indicate that the lys6 mutant cells lost the ability to induce Ca spiking in response to CO treatment. Previously, rice has been shown to exhibit different Ca spiking responses between relatively short-chain chitin chains (tetramers or pentamers) and long-chain chitin (heptamers or octamers) (Genre et al., 2013). In this study, WT and lys7 mutants exhibited Ca spiking after treatment with chitin tetramer (CO4) and octamer (CO8), with no significant difference observed between Ca spiking patterns after CO4 and CO8 treatments (Fig. 2b). This result indicated the degree of chitin polymerization did not impact the Ca spiking response in L. japonicus and that LYS7 is not involved in CO-induced Ca spiking. By contrast, the lys6 mutants did not exhibit any Ca spiking after either CO4 or CO8 treatment, indicating that LYS6 is involved in the recognition or signaling of both short- and long-chain chitin and activates the signaling pathways related to Ca spiking. We examined the expression of chitin-induced genes that were also induced after AM fungi infection (Fig. S10). Treatment with the CO mixture (1–8 mers) induced these genes in WT and lys7 mutants but not in lys6 mutants (Fig. 2c), indicating the key role of LYS6 in chitin- and AM-induced gene expression. We found that LYS6 (CERK6), involved in the pathogenic response to chitin (Bozsoki et al., 2017), is also involved in the expression of AM-induced genes and the induction of Ca spiking. LYS6-mediated symbiotic responses play an important role in the entry of AM fungi via the root surface. The hyphal entry process was comparable between lys7 mutants and WT, indicating that LYS7 might be involved in the infection processes after the hyphal entry (Fig. 1c). However, the residual entry of AM fungi in the lys6 mutants and the complete absence of the entry in the lys6lys7 mutants suggest partial regulation of hyphal entry process by LYS7-mediated symbiotic responses. LysM receptors other than LYS6 and LYS7 are also involved in AM and chitin responses in L. japonicus (Rasmussen et al., 2016; Kelly et al., 2023). Similar to the chitin receptor complexes formed by OsCERK1 and OsCEBiP, LYS6 and LYS7 might form separate receptor complexes, mediating the AM and chitin responses. In addition, it cannot be excluded that the LYS6–LYS7 receptor complex forms and regulates the AM signaling pathway. Further analysis is required to elucidate the precise functions of LYS6 and LYS7 and their relationships with other AM-related signaling factors. In conclusion, we demonstrated that the LysM-type receptors LYS6 and LYS7 and their downstream signaling pathways are essential for the initial response to AM in L. japonicus. CRISPR/Cas9 vector was kindly provided by Dr Masaki Endo (NARO, Japan). M Hayata and K Isoshima (Kwansei-Gakuin Univ., Japan) supported plant growth and preparation of samples. NT is supported by the Japan Society for the Promotion of Science KAKENHI (grant no. 22K06288). None declared. NT designed this work. HF prepared the mutant plants and HF, RM, AA and NT analyzed the mutants. The data that support the findings of this study are available in the Supporting Information of this article. Fig. S1 CRISPR/Cas9 construct targeting lysin motif receptor genes. Fig. S2 Schematic diagram of mutations in each mutant line obtained by genome editing. Fig. S3 Genome sequences around the mutation site of each mutant line obtained by genome editing. Fig. S4 Arbuscular mycorrhiza fungi colonization in the roots of lys6lys7 double mutants. Fig. S5 Arbuscular mycorrhiza fungi structures in the roots of lys6 and lys7 mutants. Fig. S6 Arbuscular mycorrhiza fungi colonization in the roots of lys6lys7 double mutants of Lotus japonicus. Fig. S7 Arbuscular mycorrhiza fungi structures on the root surface of lys6lys7 double mutant of Lotus japonicus. Fig. S8 Expression analysis of arbuscular mycorrhiza-induced genes in lys6, lys7, and lys6lys7 mutants. Fig. S9 Calcium spiking responses in lys6lys7 double mutants of Lotus japonicus. Fig. S10 Expression of chitin- and arbuscular mycorrhiza (AM)-induced genes in the roots of Lotus japonicus wild-type at 4 wk after AM fungi infection. Table S1 Primer sets used in this study. Please note: Wiley is not responsible for the content or functionality of any Supporting Information supplied by the authors. Any queries (other than missing material) should be directed to the New Phytologist Central Office. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Arbuscular mycorrhizal (AM) fungi establish mutualistic symbiosis with a wide range of terrestrial plants, including rice. However, the mechanisms underlying the initiation of AM symbiosis are yet to be elucidated, particularly in nonleguminous plants. We previously demonstrated that chitin elicitor receptor kinase 1 (OsCERK1), a lysin motif receptor-like kinase essential for chitin-triggered immunity, also plays a key role in AM symbiosis in rice. However, the mechanisms underlying the regulation of switching between immunity and symbiosis by OsCERK1 are yet to be fully elucidated. SYMBIOSIS RECEPTOR-LIKE KINASE (SYMRK)/DOES NOT MAKE INFECTIONS 2 (DMI2) is a leucine-rich repeat receptor-like kinase associated with both root nodule symbiosis and AM symbiosis in legumes. The homolog of SYMRK in rice, OsSYMRK, has a shorter form than that in legumes because OsSYMRK lacks a malectin-like domain (MLD). The MLD reportedly contributes to symbiosis in Lotus japonicus; however, the contribution of OsSYMRK to AM symbiosis in rice remains unclear. Phylogenetic analyses indicated that the MLD of SYMRK/DMI2 is widely conserved even in mosses and ferns but absent in commelinids, including rice. To understand the function of OsSYMRK, we produced an Ossymrk knockout mutant using genome editing technology. AM colonization was mostly abolished in Ossymrk with a more severe phenotype than Oscerk1. Ca2+ spiking against chitin tetramer was also diminished in Ossymrk. In contrast, comparable defense responses against chitin heptamer to the wild type were observed in Ossymrk. Bimolecular fluorescence complementation studies demonstrating an interaction between OsSYMRK and OsCERK1 indicate that OsSYMRK may play an important role in switching from immunity to symbiosis through the interaction with OsCERK1 in rice.

The establishment of the legume-rhizobia symbiosis, termed the root-nodule symbiosis (RNS), requires elaborate interactions at the molecular level. The host plant-derived transcription factor NODULE INCEPTION (NIN) is known to be crucial for RNS, regulating associated processes such as alteration of root hair morphology, infection thread formation, and cell division during nodulation. This emphasizes the importance of the precise spatiotemporal regulation of NIN expression for the establishment of RNS; however, the detailed role of NIN promoter sequences in this process remains unclear. The daphne mutant, a nin mutant allele containing a chromosomal translocation approximately 7 kb upstream of the start codon, does not form nodules but does form infection threads, indicating that the region within 7 kb of the NIN start codon contributes to NIN expression during infection thread formation. CYCLOPS binds to a CYCLOPS response element (CYC-RE) in the NIN promoter, and cyclops mutants are defective in infection thread formation. Here, we performed complementation analysis in nin mutants, using various truncated forms of the NIN promoter, and found that the CYC-RE is important for infection thread formation. Additionally, the CYC-RE deletion mutant, generated through CRISPR/Cas9 technology, displayed a significant reduction in infection thread formation, indicating that the CYC-RE is important for the fine-tuning of NIN expression during this process. However, the fact that infection thread formation is not completely abolished in the CYC-RE deletion mutant suggests that cis and trans factors other than CYCLOPS and the CYC-RE may cooperatively regulate NIN expression for the induction of infection thread formation. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Legumes and nitrogen-fixing rhizobial bacteria establish root nodule symbiosis, which is orchestrated by several plant hormones. Exogenous addition of biologically active gibberellic acid (GA) is known to inhibit root nodule symbiosis. However, the precise role of GA has not been elucidated because of the trace amounts of these hormones in plants and the multiple functions of GAs. Here, we found that GA signaling acts as a key regulator in a long-distance negative-feedback system of root nodule symbiosis called autoregulation of nodulation (AON). GA biosynthesis is activated during nodule formation in and around the nodule vascular bundles, and bioactive GAs accumulate in the nodule. In addition, GA signaling induces expression of the symbiotic transcription factor NODULE INCEPTION (NIN) via a cis-acting region on the NIN promoter. Mutants with deletions of this cis-acting region have increased susceptibility to rhizobial infection and reduced GA-induced CLE-RS1 and CLE-RS2 expression, suggesting that the inhibitory effect of GAs occurs through AON. This is supported by the GA-insensitive phenotypes of an AON-defective mutant of HYPERNODULATION ABERRANT ROOT FORMATION1 (HAR1) and a reciprocal grafting experiment. Thus, endogenous GAs induce NIN expression via its GA-responsive cis-acting region, and subsequently the GA-induced NIN activates the AON system to regulate nodule formation.

Nitrogen-fixing rhizobia and arbuscular mycorrhizal fungi (AMF) form symbioses with plant roots and these are established by precise regulation of symbiont accommodation within host plant cells. In model legumes such as Lotus japonicus and Medicago truncatula, rhizobia enter into roots through an intracellular invasion system that depends on the formation of a root-hair infection thread (IT). While IT-mediated intracellular rhizobia invasion is thought to be the most evolutionarily derived invasion system, some studies have indicated that a basal intercellular invasion system can replace it when some nodulation-related factors are genetically modified. In addition, intracellular rhizobia accommodation is suggested to have a similar mechanism as AMF accommodation. Nevertheless, our understanding of the underlying genetic mechanisms is incomplete. Here we identify a L. japonicus nodulation-deficient mutant, with a mutation in the LACK OF SYMBIONT ACCOMMODATION (LAN) gene, in which root-hair IT formation is strongly reduced, but intercellular rhizobial invasion eventually results in functional nodule formation. LjLAN encodes a protein that is homologous to Arabidopsis MEDIATOR 2/29/32 possibly acting as a subunit of a Mediator complex, a multiprotein complex required for gene transcription. We also show that LjLAN acts in parallel with a signaling pathway including LjCYCLOPS. In addition, the lan mutation drastically reduces the colonization levels of AMF. Taken together, our data provide a new factor that has a common role in symbiont accommodation process during root nodule and AM symbiosis.

[This corrects the article DOI: 10.1371/journal.pgen.1007865.].

Arbuscular mycorrhizas (AMs) are divided into two types according to morphology: Arum- and Paris-type AMs. Gibberellins (GAs) mainly inhibit the establishment of Arum-type AM symbiosis in most model plants, whereas the effects of GAs on Paris-type AM symbiosis are unclear. To provide insight into the mechanism underlying this type of symbiosis, the roles of GAs were investigated in Eustoma grandiflorum when used as the host plant for Paris-type AM establishment. Eustoma grandiflorum seedlings were inoculated with the model AM fungus, Rhizophagus irregularis, and the effects of GA and the GA biosynthesis inhibitor uniconazole-P on the symbiosis were quantitatively evaluated. Exogenous GA significantly increased hyphopodium formation at the epidermis, thus leading to the promotion of fungal colonization and arbuscule formation in the root cortex. By contrast, the suppression of GA biosynthesis and signaling attenuated fungal entry to E. grandiflorum roots. Moreover, the exudates from GA-treated roots strongly induced the hyphal branching of R. irregularis. Our results show that GA has an contrasting effect on Paris-type AM symbiosis in E. grandiflorum compared with Arum-type AM symbiosis. This finding could be explained by the differential regulation of the early colonization stage, where fungal hyphae make contact with and penetrate the epidermis.

Arbuscular mycorrhizal fungus (AMF) species are some of the most widespread symbionts of land plants. Our much improved reference genome assembly of a model AMF, Rhizophagus irregularis DAOM-181602 (total contigs = 210), facilitated a discovery of repetitive elements with unusual characteristics. R. irregularis has only ten or 11 copies of complete 45S rDNAs, whereas the general eukaryotic genome has tens to thousands of rDNA copies. R. irregularis rDNAs are highly heterogeneous and lack a tandem repeat structure. These findings provide evidence for the hypothesis that rDNA heterogeneity depends on the lack of tandem repeat structures. RNA-Seq analysis confirmed that all rDNA variants are actively transcribed. Observed rDNA/rRNA polymorphisms may modulate translation by using different ribosomes depending on biotic and abiotic interactions. The non-tandem repeat structure and intragenomic heterogeneity of AMF rDNA/rRNA may facilitate successful adaptation to various environmental conditions, increasing host compatibility of these symbiotic fungi.

The rice lysin-motif (LysM) receptor-like kinase OsCERK1 is now known to have a dual role in both pathogenic and symbiotic interactions. Following the recent discovery that the Oscerk1 mutant is unable to host arbuscular mycorrhizal (AM) fungi, we have examined whether OsCERK1 is directly involved in the perception of the short-chain chitin oligomers (Myc-COs) identified in AM fungal exudates and shown to activate nuclear calcium (Ca2+ ) spiking in the rice root epidermis. An Oscerk1 knockout mutant expressing the cameleon NLS-YC2.60 was used to monitor nuclear Ca2+ signaling following root treatment with either crude fungal exudates or purified Myc-COs. Compared with wild-type rice, Ca2+ spiking responses to AM fungal elicitation were absent in root atrichoblasts of the Oscerk1 mutant. By contrast, rice lines mutated in OsCEBiP, encoding the LysM receptor-like protein which associates with OsCERK1 to perceive chitin elicitors of the host immune defense pathway, responded positively to Myc-COs. These findings provide direct evidence that the bi-functional OsCERK1 plays a central role in perceiving short-chain Myc-CO signals and activating the downstream conserved symbiotic signal transduction pathway.

Lotus japonicus THIC is expressed in all organs, and the encoded protein catalyzes thiamine biosynthesis. Loss of function produces chlorosis, a typical thiamine-deficiency phenotype, and mortality. To investigate thiamine's role in symbiosis, we focused on THI1, a thiamine-biosynthesis gene expressed in roots, nodules, and seeds. The thi1 mutant had green leaves, but formed small nodules and immature seeds. These phenotypes were rescued by THI1 complementation and by exogenous thiamine. Thus, THI1 is required for nodule enlargement and seed maturation. On the other hand, colonization by arbuscular mycorrhiza (AM) fungus Rhizophagus irregularis was not affected in the thi1 mutant or by exogenous thiamine. However, spores of R. irregularis stored more thiamine than the source (host plants), despite lacking thiamine biosynthesis genes. Therefore, disturbance of the thiamine supply would affect progeny phenotypes such as spore formation and hyphal growth. Further investigation will be required to elucidate thiamine's effect on AM.

Thiamine (vitamin B1) is essential for living organisms. Unlike animals, plants can synthesize thiamine. In Lotus japonicus, the expression of two thiamine biosynthesis genes, THI1 and THIC, was enhanced by inoculation with rhizobia but not by inoculation with arbuscular mycorrhizal fungi. THIC and THI2 (a THI1 paralog) were expressed in uninoculated leaves. THI2-knockdown plants and the transposon insertion mutant thiC had chlorotic leaves. This typical phenotype of thiamine deficiency was rescued by an exogenous supply of thiamine. In wild-type plants, THI1 was expressed mainly in roots and nodules, and the thi1 mutant had green leaves even in the absence of exogenous thiamine. THI1 was highly expressed in actively dividing cells of nodule primordia. The thi1 mutant had small nodules, and this phenotype was rescued by exogenous thiamine and by THI1 complementation. Exogenous thiamine increased nodule diameter, but the level of arbuscular mycorrhizal colonization was unaffected in the thi1 mutant or by exogenous thiamine. Expression of symbiotic marker genes was induced normally, implying that mainly nodule growth was delayed in the thi1 mutant. Furthermore, this mutant formed many immature seeds with reduced seed weight. These results indicate that thiamine biosynthesis mediated by THI1 enhances nodule enlargement and is required for seed development in L. japonicus.

Arbuscular mycorrhizal (AM) symbiosis is the most widespread association between plants and fungi. To provide novel insights into the molecular mechanisms of AM symbiosis, we screened and investigated genes of the AM fungus Rhizophagus irregularis that contribute to the infection of host plants. R. irregularis genes involved in the infection were explored by RNA-sequencing (RNA-seq) analysis. One of the identified genes was then characterized by a reverse genetic approach using host-induced gene silencing (HIGS), which causes RNA interference in the fungus via the host plant. The RNA-seq analysis revealed that 19 genes are up-regulated by both treatment with strigolactone (SL) (a plant symbiotic signal) and symbiosis. Eleven of the 19 genes were predicted to encode secreted proteins and, of these, SL-induced putative secreted protein 1 (SIS1) showed the largest induction under both conditions. In hairy roots of Medicago truncatula, SIS1 expression is knocked down by HIGS, resulting in significant suppression of colonization and formation of stunted arbuscules. These results suggest that SIS1 is a putative secreted protein that is induced in a wide spatiotemporal range including both the presymbiotic and symbiotic stages and that SIS1 positively regulates colonization of host plants by R. irregularis.

Arbuscular mycorrhiza (AM) is established by the entry of AM fungi into the host plant roots and the formation of symbiotic structures called arbuscules. The host plant supplies photosynthetic products to the AM fungi, which in return provide phosphate and other minerals to the host through the arbuscules. Both partners gain great advantages from this symbiotic interaction, and both regulate AM development. Our recent work revealed that gibberellic acids (GAs) are required for AM development in the legume Lotus japonicus. GA signaling interact with symbiosis signaling pathways, directing AM fungal colonization in host roots. Expression analysis showed that genes for GA biosynthesis and metabolism were induced in host roots around AM fungal hyphae, suggesting that the GA signaling changes with both location and time during AM development. The fluctuating GA concentrations sometimes positively and sometimes negatively affect the expression of AM-induced genes that regulate AM fungal infection and colonization.

Gene expression during arbuscular mycorrhizal development is highly orchestrated in both plants and arbuscular mycorrhizal fungi. To elucidate the gene expression profiles of the symbiotic association, we performed a digital gene expression analysis of Lotus japonicus and Rhizophagus irregularis using a HiSeq 2000 next-generation sequencer with a Cufflinks assembly and de novo transcriptome assembly. There were 3,641 genes differentially expressed during arbuscular mycorrhizal development in L. japonicus, approximately 80% of which were up-regulated. The up-regulated genes included secreted proteins, transporters, proteins involved in lipid and amino acid metabolism, ribosomes and histones. We also detected many genes that were differentially expressed in small-secreted peptides and transcription factors, which may be involved in signal transduction or transcription regulation during symbiosis. Co-regulated genes between arbuscular mycorrhizal and root nodule symbiosis were not particularly abundant, but transcripts encoding for membrane traffic-related proteins, transporters and iron transport-related proteins were found to be highly co-up-regulated. In transcripts of arbuscular mycorrhizal fungi, expansion of cytochrome P450 was observed, which may contribute to various metabolic pathways required to accommodate roots and soil. The comprehensive gene expression data of both plants and arbuscular mycorrhizal fungi provide a powerful platform for investigating the functional and molecular mechanisms underlying arbuscular mycorrhizal symbiosis.

Arbuscular mycorrhiza is a mutualistic plant-fungus interaction that confers great advantages for plant growth. Arbuscular mycorrhizal (AM) fungi enter the host root and form symbiotic structures that facilitate nutrient supplies between the symbionts. The gibberellins (GAs) are phytohormones known to inhibit AM fungal infection. However, our transcriptome analysis and phytohormone quantification revealed GA accumulation in the roots of Lotus japonicus infected with AM fungi, suggesting that de novo GA synthesis plays a role in arbuscular mycorrhiza development. We found pleiotropic effects of GAs on the AM fungal infection. In particular, the morphology of AM fungal colonization was drastically altered by the status of GA signaling in the host root. Exogenous GA treatment inhibited AM hyphal entry into the host root and suppressed the expression of Reduced Arbuscular Mycorrhization1 (RAM1) and RAM2 homologs that function in hyphal entry and arbuscule formation. On the other hand, inhibition of GA biosynthesis or suppression of GA signaling also affected arbuscular mycorrhiza development in the host root. Low-GA conditions suppressed arbuscular mycorrhiza-induced subtilisin-like serine protease1 (SbtM1) expression that is required for AM fungal colonization and reduced hyphal branching in the host root. The reduced hyphal branching and SbtM1 expression caused by the inhibition of GA biosynthesis were recovered by GA treatment, supporting the theory that insufficient GA signaling causes the inhibitory effects on arbuscular mycorrhiza development. Most studies have focused on the negative role of GA signaling, whereas our study demonstrates that GA signaling also positively interacts with symbiotic responses and promotes AM colonization of the host root.

Many leguminous plants have a unique ability to reset and alter the fate of differentiated root cortical cells to form new organs of nitrogen-fixing root nodules during legume-Rhizobium symbiosis. Recent genetic studies on the role of cytokinin signaling reveal that activation of cytokinin signaling is crucial to the nodule organogenesis process. However, the genetic mechanism underlying the initiation of nodule organogenesis is poorly understood due to the low number of genes that have been identified. Here, we have identified a novel nodulation-deficient mutant named vagrant infection thread 1 (vag1) after suppressor mutant screening of spontaneous nodule formation 2, a cytokinin receptor gain-of-function mutant in Lotus japonicus. The VAG1 gene encodes a protein that is putatively orthologous to Arabidopsis ROOT HAIRLESS 1/HYPOCOTYL 7, a component of the plant DNA topoisomerase VI that is involved in the control of endoreduplication. Nodule phenotype of the vag1 mutant shows that VAG1 is required for the ploidy-dependent cell growth of rhizobial-infected cells. Furthermore, VAG1 mediates the onset of endoreduplication in cortical cells during early nodule development, which may be essential for the initiation of cortical cell proliferation that leads to nodule primordium formation. In addition, cortical infection is severely impaired in the vag1 mutants, whereas the epidermal infection threads formation is normal. This suggests that the VAG1-mediated endoreduplication of cortical cells may be required for the guidance of symbiotic bacteria to host meristematic cells.