Rhizobial infection-specific accumulation of phosphatidylinositol 4,5-bisphosphate inhibits the excessive infection of rhizobia inLotus japonicus

Rhizobial infection-specific accumulation of phosphatidylinositol 4,5-bisphosphate inhibits the excessive infection of rhizobia in Lotus japonicus | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Rhizobial infection-specific accumulation of phosphatidylinositol 4,5-bisphosphate inhibits the excessive infection of rhizobia in Lotus japonicus View ORCID Profile Akira Akamatsu , View ORCID Profile Toshiki Ishikawa , Hiroto Tanaka , View ORCID Profile Yoji Kawano , View ORCID Profile Makoto Hayashi , View ORCID Profile Naoya Takeda doi: https://doi.org/10.1101/2025.06.26.661640 Akira Akamatsu 1 Graduate School of Biological and Environmental Sciences, Kwansei Gakuin University, 1 Gakuen-Uegahara , Sanda-City, Hyogo 669-1330, Japan 2 RIKEN Center for Sustainable Resource Science, 1-7-22, Suehiro, Tsurumi , Yokohama, 230-0045 Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Akira Akamatsu For correspondence: akira.akamatsu{at}riken.jp Toshiki Ishikawa 3 Graduate School of Science and Engineering, Saitama University, 225 Shimo-Okubo, Sakura-Ku , Saitama-City, Saitama 338-8570, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Toshiki Ishikawa Hiroto Tanaka 1 Graduate School of Biological and Environmental Sciences, Kwansei Gakuin University, 1 Gakuen-Uegahara , Sanda-City, Hyogo 669-1330, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site Yoji Kawano 4 Institute of Plant Science and Resources, Okayama University, 2-20-1 , Chuo, Kurashiki, Okayama, 710-0046, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Yoji Kawano Makoto Hayashi 2 RIKEN Center for Sustainable Resource Science, 1-7-22, Suehiro, Tsurumi , Yokohama, 230-0045 Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Makoto Hayashi Naoya Takeda 1 Graduate School of Biological and Environmental Sciences, Kwansei Gakuin University, 1 Gakuen-Uegahara , Sanda-City, Hyogo 669-1330, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Naoya Takeda Abstract Full Text Info/History Metrics Supplementary material Preview PDF Summary During the symbiosis of legume 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, it remains relatively poorly understood the molecular mechanism involved in the suppression. 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)P 2 ] during rhizobial infection were examined by a fluorescent marker 1xTUBBY-C (TUBBY). We found that PI signaling-related genes were upregulated, and the amount of PIP 2 increased in L. japonicus roots during rhizobial infection. In the PLP4 , PIP5K4 and PIP5K6 mutants, rhizobial infection increased and PIP 2 accumulation was failed. Furthermore, the observation of PI(4,5)P 2 in rhizobia-infected roots revealed that the ectopic accumulation was closely related to suppression of rhizobial infection. Our findings indicate that the accumulation of PI(4,5)P 2 , which is mediated by PLP and PIP5Ks, suppresses excessive rhizobial infection in the root epidermis and cortex, leading to the optimal number of nodules. Introduction Nitrogen-fixing symbiotic bacteria, collectively called rhizobia, invade legume plant cells, resulting in formation of root nodules in which atmospheric nitrogen is converted to ammonia. The ammonia produced by rhizobia is an essential nutrient for host plant growth when soil nitrogen is poor. For this reason, root nodule symbiosis (RNS) has long been considered to be an agriculturally important trait. During rhizobial infection, nodulation factors (Nod Factors: NFs), lipochito– oligosaccharides produced by rhizobia are important signals that trigger host plant symbiotic responses ( Oldroyd, 2013 ). NF recognition by plasma membrane (PM) receptors in host plants induces rapid changes in Ca 2+ concentration in and around the nucleus, termed Ca 2+ spiking. Ca 2+ spiking is decoded by Ca 2+ /calmodulin-dependent protein kinase (CCaMK), and CYCLOPS phosphorylated by CCaMK induces expression of two transcription factors, Nodule Inception ( NIN ) and ERF REQUIRED FOR NODULATION1 (ERN1) ( Schauser et al ., 1999 ; Singh et al ., 2014 ; Cerri et al ., 2017 ). In the model legumes Lotus japonicus and Medicago truncatula, rhizobia enter the host cell at the tip of root hairs. On receiving NF signals, the root hair tip curls to create an infection chamber, a space to enclose the rhizobia ( Fournier et al ., 2015 ). Rhizobia grow in the chamber, the stage of infection called a microcolony ( Gage, 2004 ), and the cell membrane surrounding the rhizobia swells, creating polarity at the swelling site and initiating cell invasion ( Liu et al ., 2019 ). At the stage of infection thread formation in root hairs, cell division in the cortex is initiated, leading to the development of these cells into nodules ( Suzaki et al., 2012 ). Rhizobia invade deeper into the root through tunnel-like structures called infection threads. Infection threads that progress from the epidermal to the cortical cells often branch their tips and eventually release rhizobia into the nodule cells ( Rae et al ., 2021 ). Although RNS promotes host plant growth, excessive rhizobial infection significantly inhibits plant growth. In supernodulation mutants, numerous nodules are formed, whereas overall plant growth is impaired. This highlights the critical importance of maintaining a symbiotic balance between the host plant and rhizobial bacteria ( Buttery, 1989 ; Wopereis et al ., 2000 ; Ferguson et al ., 2014 ). Rhizobial infection induces suppression of further infection to maintain an optimal level of symbiosis. The suppression system involves long-distance regulation, which regulates rhizobial infection via the shoot. The transcription factor NIN, which promotes RNS, also plays a role in the suppression system. In L. japonicus roots, CLE-RS1/2 peptides induced by NIN are transported from root to shoot via the xylem and are recognized by the HAR1 receptor ( Okamoto et al ., 2009 ; Okamoto et al ., 2013 ; Soyano et al ., 2014 ). In the absence of rhizobial infection, shoot-derived microRNA2111 suppresses the expression of the F-box protein TOO MUCH LOVE (TML) in roots. In contrast, during rhizobial infection, the expression of microRNA2111 is downregulated, resulting in increased TML expression and subsequent suppression of further rhizobial infection ( Magori et al ., 2009 ; Tsikou et al., 2018 ). This cascade is called autoregulation of nodulation (AON). The suppression of rhizobial infection is also regulated by plant hormones. Treatment of either ET precursor ACC or GA inhibit nodule formation ( Oldroyd et al., 2001 ; Maekawa et al., 2009 ). M. truncatula ethylene insensitive 2 ( Mtein2 , also referred to as SICKLE ), in which the ET signaling is compromised, shows increase of the number of nodules ( Penmetsa and Cook, 1997 ). In L. japonicus LjEIN2-1 and LjEIN2-2 cooperatively regulate the nodule number ( Miyata et al ., 2013 ). In addition, M. truncatula ent-copalyl diphosphate synthase 1 (CPS1) and GA3 oxidase 1 (GA3ox1), which are involved in the biosynthesis of bioactive GA, are transcriptionally regulated by NIN ( Gao et al., 2023 ). In a cytokinin perception mutant HYPERINFECTED 1 ( hit1 ) the number of infection threads also increases, suggesting that cytokinin functions to suppress rhizobial infection ( Murray et al., 2007 ). Thus, leguminous plants employ multiple mechanisms to restrict nodulation. Phosphatidylinositol (PI), a membrane phospholipid with anionic polarity, undergoes phosphorylation on the inositol ring to become phosphatidylinositol phosphates (PIP and PIP 2 ), such as PI(3)P, PI(4)P, PI(3,5)P 2 , and PI(4,5)P 2 , each with specific functions ( Noack & Jaillais, 2020 ). The PIP and PIP 2 are produced by the catalysis of specific kinases, including phosphatidylinositol 4-kinase (PI4K) and phosphatidylinositol-4-phosphate 5-kinases (PIP5K). In plants, phosphatidylinositol phosphates are known to be involved in the apical cell expansion of tip-growing cell, such as root hairs and pollen tubes ( Ischebeck et al ., 2008 ; Hirano et al ., 2018 ). Arabidopsis PIP5K3 (AtPIP5K3) and its product PI(4,5)P 2 localize to the apex of the root hair, and they are required for polar tip growth ( Hirano et al ., 2018 ). In Arabidopsis , the PI(4,5)P 2 produced by AtPIP5K4 and AtPIP5K5 regulates the polar tip growth of pollen tubes via regulation of apical pectin deposition ( Ischebeck et al ., 2008 ). Furthermore, phosphatidylinositol phosphates are known to be involved in the host plant infection process by pathogenic microbes. In Arabidopsis , PI(4,5)P 2 accumulates in the extrahaustorial membrane (EHM) of powdery mildew infection sites ( Qin et al ., 2020 ). Loss of function of PIP5Ks results in suppression of infection, indicating that PI(4,5)P 2 accumulation is an essential factor in the infection of powdery mildew ( Qin et al ., 2020 ). It has also been shown that PI(4,5)P 2 accumulation in the extra-invasive hyphal membrane (EIHM) is conducive to Colletotrichum tropicale infection, which causes plant anthracnose ( Shimada et al ., 2019 ). In addition to C. tropicale , during infection of the oomycete Hyaloperonospora arabidopsis , a causal pathogen of downy mildew, PI(4)P is enriched in the EHM ( Shimada et al ., 2019 ). When arbuscular mycorrhizal fungi establish symbiosis with M. truncatula , PI(4,5)P 2 accumulates in periarbuscular membrane at the invading hyphae and at the arbuscule trunks ( Ivanov & Harrison, 2019 ). These findings indicate that phosphatidylinositol phosphates are involved in plant-microbe interactions, especially when tip-growing hyphae invade into host cells. Only little information is available for the behavior of phosphatidylinositol phosphates in rhizobial infection. Phosphatidylinositol 3 kinase (PI3K) has been shown to be involved in the generation of reactive oxygen species when rhizobia invade root hair cells ( Peleg-Grossman et al ., 2007 ; Robert et al ., 2018 ). LjPLP4 (formerly PLP-IV ) encodes a protein with Sec14 and Nodulin domains in which the expression of the mRNA encoding the C-terminus of the protein (LjNOD16) is induced in nodules ( Kapranov et al., 1997 ; Kapranov et al., 2001 ). The Sec14 domain of LjPLP4 has a PI transfer activity ( Kapranov et al ., 2001 ). In addition, AtSFH1, the orthologous gene of LjPLP4 in Arabidopsis , promotes the accumulation of PI(4)P and PI(4,5)P₂ by facilitating the functions of PI4K and PIP5K to the PM. However, it is still unknown whether phosphatidylinositol phosphates, especially PI(4,5)P 2 , are involved in rhizobial infection. Therefore, investigating phosphatidylinositol phosphate dynamics is helpful to understand how they play roles in RNS. In this study, we showed that increased expression levels of PI signaling-related genes led to accumulation of PI and phosphatidylinositol phosphates in a rhizobial inoculation-dependent manner. In the mutants of LjPLP4 , LjPIP5K4 and LjPIP5K6, accumulation of PI and phosphatidylinositol phosphates was not induced by rhizobial inoculation, resulted in promotion of rhizobial infection. PI(4,5)P 2 marker analysis revealed that accumulation of PI(4,5)P 2 correlated with suppression of rhizobial infection, and successful infection eliminated accumulation of PI(4,5)P 2 . The role of PI(4,5)P₂ in RNS appears to differ from its function in other plant–microbe interactions, potentially contributing to the fine-tuning of RNS through the suppression of excessive rhizobial infection. Materials and Methods Plant materials and growth conditions We used the wild type L. japonicus MG-20, and symbiosis mutants in the MG-20 background ( har1-7 , nin-9 , cyclops-6, and tml ) which was described previously ( Magori et al ., 2009 ; Suzaki et al ., 2012 ). The mutants produced by CRISPR/Cas9 in this study were Ljplp4-4-10 , Ljplp4-4-11 , Ljplp4-9 , Ljpip5k4-2 , Ljpip5k4-3 , Ljpip5k4-9 , Ljpip5k6-1 , Ljpip5k6-5 , and Ljpip5k6-6 as described below. The tml har1 , tml Ljplp4-4-10 , har1-7 Ljplp4-4-10 and Ljplp4 YC2.60 mutants were produced by crossing. From the F 2 population, the homozygous double mutants were selected using PCR. To produce the LjPLP4 OE, the full-length of LjPLP4 driven by the Lotus Ubiquitin promoter ( pLjUbiquitin::LjPLP4 ) were expressed in L. japonicus MG-20. The seeds were scarified with sandpaper, sterilized with sodium hypochlorite (effective chloride; approximately 1%) for 10 min, and soaked overnight in sterilized water. The sterilized seeds were germinated on 0.8% agar plates and grown in a growth chamber (24°C, 16-h light/8-h dark). Analysis of rhizobial infection and root hair growth We used Mesorhizobium loti (MAFF303099), or transgenic M. loti strain carrying DsRed ( Maekawa et al ., 2009 ) and GFP ( Akamatsu et al., 2022 ), for the quantitative analysis of infection threads and nodules. These strains were used to inoculate 3-day-old plants (3 days after germination, 2 days dark/1 day light) in plastic pots (SPL Life Sciences, Korea) containing 300 mL vermiculite supplied with B&D medium ( Broughton, 1971 ) containing 0.1 mM KNO 3 (100 mL per pot). A total volume of 1 mL M. loti solution with an absorbance of 0.1 at 600 nm was added to each pot. Root nodule infection phenotypes, including the number of nodules, infection threads, and infection events, were determined by roots inoculated with M. loti carrying DsRed. The number observed for each plant was shown. In the measurement of infection threads, the total number of microcolonies (MCs), infection threads (ITs), and ramifying ITs was shown ( Figs. 2c, e , 4e, g , 5e, g, i ). Download figure Open in new tab Figure 1. Expression of PI signaling-related genes and accumulation of PI, PIP, and PIP 2 a, b . RNA-seq analysis using Lotus japonicus MG-20 roots during symbiotic or pathogenic microbe interaction. Relative expression in log 2 (FC) of ( M. loti / Mock) at 1 week after inoculation (wai) (a) or ( P. palmivora / Mock) at 5 days after inoculation (dpi) (b). (*; log 2 (FC) ≥ 1 or log 2 (FC) ≤ −1 and P ≤ 0.05). Red bars indicate increased expression, and deep blue bars indicate decreased expression. c–e . LC-MS analysis of phosphoinositol (PI) (c), phosphatidylinositol monophosphate (PIP) (d), and phosphatidylinositol bisphosphate (PIP 2 ) (e) in L. japonicus roots under control conditions (white) or inoculated with M. loti for 1 week (Red) (n = 3 biological replicates, the bars show average, and circles show each data point, t -test; *P < 0.01). Download figure Open in new tab Figure 2. LjPLP4 negatively regulates rhizobial infection but is required for P. palmivora infection. a. Rhizobia-infected and non-infected L. japonicus MG-20 wild type (WT) roots transformed with pLjPLP4::GUS . The roots were stained with GUS staining buffer at 2 wai of M. loti carrying DsRed. The image on the right is an enlargement of the framed area of the center image. Scale bars, 100 µm. b–f. The plants were inoculated with M. loti carrying DsRed for 1 week. b . Nodules and infection threads observed in wild type, Ljplp4-4-10, and Ljplp4-9 . Scale bars, 200 µm. c–f. Quantitative analysis of rhizobial infection phenotypes of Ljplp4 mutants (c, d) and LjPLP4 overexpressing (OE) plants (e, f). n ≥ 12, Dunnett’s test, one asterisk (*) indicates P < 0.01. ns: not significant. Similar results were obtained in more than three independent experiments. g–i. P. palmivora infection phenotypes in L. japonicus MG-20 wild type, Ljplp4-4-10 , Ljplp4-4-11 , Ljplp4-9, and har1-7 at 5 dpi. g . Representative image of P. palmivora infected L. japonicus roots in LjPLP4 and har1-7 mutants. Scale bar, 0.5 cm. h, i. Quantitative analysis of P. palmivora infection. Infection ratio (infected lesion/root length) (h). Measurement of PpEF1a expression in P. palmivora infected roots by real-time PCR (i). n ≥ 12, Tukey test, P < 0.01. Similar results were obtained in three independent experiments. Download figure Open in new tab Figure 3. The plasma membrane localization of LjPLP4 in L. japonicus roots. a–d. Hairy roots transformed with the fluorescent constructs. a . Venus-LjPLP4 (green) and Free RFP (magenta), which is a transformation marker, were expressed in L. japonicus wild type roots. The images show the epidermis and the cortex. Abbreviations are Ep, epidermis; Co, cortex. b–d . Venus-LjPLP4 (green) was co-expressed in root hair cells with NFR1-mCherry (magenta) (b), tdTomato-CTT (magenta) (c), and GmMAN49-mCherry (magenta) (d). Scale bars, 50 µm or 10 µm in the enlarged images. The arrowhead indicates a merged signal of green and magenta. e-g . Intensity profiles of Venus and RFP/mCherry/tdTomato signals along the yellow transect shown in (b-d). h . CBB images of purified HIS-LjPLP4 full length (FL) or ΔSEC14. i-k . PIP-strip, membrane binding assays with lipid-spotted membranes. j . PIP Arrays, membranes pre-spotted with a concentration gradient. The membranes were incubated with 1.0 µg/mL of LjPLP4 FL (i, j) or ΔSEC14 protein (k). The lipids shown in red are not present in plants. Abbreviations are LPC, lysophosphatidylcholine; PE, phosphatidylethanolamine; PS, phosphatidylserine; PC, phosphatidylcholine. Download figure Open in new tab Figure 4. LjPIP5K4 and LjPIP5K6 negatively regulates rhizobial infection. a. Rhizobia-infected L. japonicus MG-20 wild type (WT) roots transformed with pLjPIP5K4::GUS (a, b) or pLjPIP5K6 :: GUS (c, d). Infected epidermal cells (a, c), nodule primordium (b, d). The roots were stained with GUS staining buffer at 2 wai of M. loti carrying DsRed. Scale bars; 100 µm. e-h. Quantitative analysis of rhizobial infection phenotypes of Ljpip5k4 and Ljpip5k6 mutants. The plants were inoculated with M. loti carrying DsRed for 1 week (n ≥ 10). Dunnett’s test, *P < 0.05, **P < 0.01, ns; not significant. Similar results were obtained in more than three independent experiments. Download figure Open in new tab Figure 5. NIN regulates the suppression of rhizobial infection by LjPLP4 , LjPIP5K4 and LjPIP5K6 The plants were inoculated with M. loti wild-type (a-c) or carrying DsRed (e-i) for 1 week. a-c . Measurement of LjPLP4 , LjPIP5K4 and LjPIP5K6 expression in nin-9 and cyclops-6 mutants by real-time PCR (n = 4, t-test, *P < 0.05, ns; not significant). d . Measurement of NIN, LjPLP4, LjPIP5K4 and LjPIP5K6 expression in NIN -overexpressing roots by real-time PCR (n = 4, t-test, *P < 0.01). e-f . Quantitative analysis of rhizobial infection phenotypes of NIN -overexpressing roots in wild type, Ljplp4 , Ljpip5k4 and Ljpip5k6 mutants, and wild type roots transformed with empty vector (EV). g-j , plp4-4-10 , tml har1-7 , plp4-4-10 tml and tml (g, h), or plp4-4-10 , tml har1-7 , plp4-4-10 har1-7 and har1-7 (i, j). n ≥ 12, Tukey test, P < 0.05. Similar results were obtained in more than three independent experiments. To measure the root hair length in L. japonicus , root hairs located 5 mm from the root tip of seedlings (2 days after germination) were imaged under a upright microscope (Leica DFC7000 T camera mounted on a Leica DM6 B). The images obtained were used directly for root hair length measurement using ImageJ software ( https://imagej.net/ij/ ). Phytophthora palmivora infection and phenotypic analysis P. palmivora (E.J. Butler) ( Rahman et al ., 2014 ) was grown on V8 juice Original (Campbell’s, USA) agar medium for 6–10 days until mycelium was fully expanded in the 90-mm Petri dishes. The plates were kept in a fume hood for 24 h to dry the medium. A total volume of 10 mL sterilized cool water was poured on each Petri dish, allowed to sit for 1 h to induce the release of zoospores, and the concentration of zoospores was quantified on a hemacytometer. L. japonicus MG-20 wild-type and mutant seedlings were grown on 0.8% agar medium (3 days after germination, 2 days dark/1 day light), then inoculated at 23℃ with or without 1 mL (1 × 10 6 zoospore/mL) of P. palmivora solution. To quantify P. palmivora growth on the roots, roots 5 days post-inoculation were used to measure the lesion size, which was normalized to individual root length. These same root samples were then frozen in liquid nitrogen to extract RNA for real-time PCR analysis using the P. palmivora EF1a gene compared with the L. japonicus Ubiquitin housekeeping gene. Plasmid construction Gene fragments used to create binary vectors for plant transformation and protein expression in Escherichia coli were amplified by PCR using Prime STAR GXL DNA polymerase (Takara, Japan), and the specific primer sets were listed in Table S1. The promoter region (3 kbp) of LjPLP4 , LjPIP5K4 or LjPIP5K6 was amplified using the primer set 1, 2 or 3 (Table S1) from the L. japonicus MG-20 wild-type genome, followed by cloning into the entry vector pENTR D/TOPO (ThermoFisher, USA). For the promoter-GUS fusion construction, the entry clones were transferred into the destination vector pKGWFS7 ( Karimi et al ., 2002 ) by the LR reaction. To construct the pLjUbiquitin::Venus-LjPLP4 , pLjUbiquitin::mCherry-LjPLP4 , pLjUbiquitin::LjPIP5K6-Venus , or pLjUbiquitin::NFR1-mCherry binary vectors, the coding sequence (CDS) of LjPLP4 , LjPIP5K6 or NFR1 was amplified from cDNA synthesized from total RNA of L. japonicus MG-20 wild type using specific primer sets 4, 5, 6, or 7 (Table S1). They were fused with the amplified entry vector containing Venus or mCherry by inverse PCR using the primer set 8, 9, 10, and 11 (Table S1), using the SLiCE method ( Motohashi, 2017 ). For the overexpression construction, the entry clones were transferred into the destination vector pLjUbiquitin ( Maekawa et al ., 2008 ) by the LR reaction. To express HIS-tagged LjPLP4 protein in Escherichia coli , LjPLP4 was amplified using the specific primer set 12 and cloned into amplified pET28b vector using the PCR using the primer set 13 combined with the SLiCE method. To express HIS-tagged LjPLP4 ΔSEC14, pENTR-LjPLP4 ΔSEC14 was generated using the SLiCE method combined with Inverse PCR based on pENTR-LjPLP4. Based on this, LjPLP4 ΔSEC14 was amplified using the primer set 14 and fused with the pET28b vector using the SLiCE method. Real-time PCR analysis Total RNA was extracted from root samples from 10 plants using the RNeasy Plant Mini Kit (QIAGEN, Netherlands). For all analyses, four or more independent biological replicates were obtained through separate samplings. The RNA concentration was quantified using NANODROP ONE (ThermoFisher, USA). Reverse-transcription and real-time PCR were performed using the ReverTra Ace qPCR RT kit (Toyobo, Japan) and the Thunderbird qPCR Mix (Toyobo, Japan) with the AriaMx Real-Time PCR System (Agilent, USA) according to the manufacturer’s instructions. The cDNA was synthesized from 500 ng total RNA, and each reaction was performed in triplicate to correct technical errors. The primer sets used to amplify LjUbi , PpEF1a , CLE-RS1, LjPLP4, PIP5K4 and PIP5K6 were listed in Table S1 (primer sets 15-20). Relative transcript levels were compared to Ubiquitin expression using the Ct value. Three to four biologically independent experiments were performed to calculate averages, and the statistical analysis compared with the control expression levels was performed using either Student’s t-test or Dunnett’s test. RNA-Seq analysis L. japonicus MG-20 wild type was inoculated with M. loti (for 1 week) or P. palmivora (for 5 days) suspended in water on 0.8% agar. Also, for the negative control, roots were treated with water, and this treatment was designated as the mock condition. Total RNA was extracted from root samples from 10 plants using the RNeasy Plant Mini Kit (QIAGEN, Netherlands). To compare expression levels, total RNA was also extracted from plants that were not inoculated with M. loti or P. palmivora . Three biological replicates were conducted for each condition. RNA libraries from L. japonicus MG-20 roots that were infected were prepared for sequencing using the TruSeq Stranded Total RNA (Illumina, San Diego, CA, USA). Whole transcriptome sequencing was applied to RNA samples using the Illumina NovaSeq 6000 platform in the 101-bp single-end mode. Sequenced reads were mapped to the Gifu_1.2 genome ( Kamal et al ., 2020 ) using TopHat ver. 2.0.13 in combination with Bowtie2 ver. 2.2.3 and SAMtools ver. 1.0 ( Li et al ., 2009 ; Langdon, 2015 ). The number of fragments per kilobase of exon per million fragments mapped was calculated using Cufflinks ver. 2.2.1 ( Trapnell et al ., 2010 ). LC-MS analysis Approximately 120 seedlings of L. japonicus MG-20 wild type, Ljpip4 , and Ljpip5k6 , were inoculated with M. loti suspended in water (for 1 week) on 0.8% agar plates, respectively. Also, for the negative control, roots were treated with water. PI, PIP, and PIP 2 were analyzed by a phosphate methylation method coupled with LC-MS according to previous reports ( Clark et al ., 2011 ; Cai et al ., 2016 ). In brief, lyophilized tissues (∼5 mg dry weight) were homogenized in 600 µl methyl-tert-butyl methyl ether (MTBE)/methanol (1:1, by vol). The homogenate was phase-partitioned by adding 700 µl MTBE and 300 µl 0.1 N HCl, and the upper ether layer was collected and dried using a centrifugal concentrator. The residue was dissolved in 200 µl of the upper phase of MTBE/methanol/0.01 N HCl (10:3:3, by vol) and mixed with 20 µl of (trimethylsilyl) diazomethane (2M in hexane, Sigma). Methylation of the phosphate groups was conducted for 20 min at room temperature and stopped by adding 6 µl acetic acid. After dryness, the residue was dissolved in methanol/acetonitrile/2-propanol (1:1:1 by vol) and analyzed by LCMS-8030 (Shimadzu) equipped with a Shim-pack Scepter C18 column (3 µm × 2.0 mm × 75 mm). The column was kept at 40°C and eluted with a binary gradient of solvent A (5 mM ammonium formate and 0.1% formate) and B (methanol/acetonitrile/2-propanol, 1:1:3 by vol, containing 5 mM ammonium formate and 0.1% formate): 0 to 15 min, 30%B to 100%B and maintained for 3 min. Permethylated PIPs were detected by the multiple reaction monitoring with [M+NH4]+ to [diacylglycerol+H−H2O]+. Lipid quantity was shown as normalized peak intensities using 18:0-20:4 PI (Sigma-Aldrich, USA) as an internal standard added to samples at the first extraction. Peak identity was confirmed using authentic standards of 18:1 PIP and 18:1 PIP 2 (Sigma-Aldrich, USA). Hairy root transformation The binary vectors were introduced into L. japonicus MG20 wild type or mutant plants by hairy root transformation using Agrobacterium rhizogenes AR1193 ( Offringa et al ., 1986 ). A. rhizogenes colonies were suspended in sterilized water. Roots of 6-day-old seedlings (3 days dark followed by 3 days in 16-h light/8-h dark, 24℃) were cut off below the hypocotyl in A. rhizogenes suspension. The infected shoots were incubated for 5 days in a growth chamber (24℃, 16-h light/8-h dark) on B5 medium (FUJIFILM, Japan) solidified with 1.0% agar and subsequently transferred to the same medium containing 12.5 μg/mL Meropen (Sumitomo Pharma, Japan) for 10 days to 3 weeks until bacterial growth was suppressed. Transgenic hairy roots were selected based on fluorescence under a stereomicroscope (SZX16; EVIDENT, Japan). Production of Ljplp4, Ljpip5k4 and Ljpip5k6 mutants using the CRISPR/Cas9 system, overexpression plants of LjPLP4 and UBQ10prom :: 2xCHERRY-1xTUBBY-C ( TUBBY ) expressing plants We used the CRISPR/Cas9 system to create mutation within the LjPLP4 (LotjaGi2g1v0386800), LjPIP5K4 (LotjaGi1g1v0664200) or LjPIP5K6 (LotjaGi3g1v0109700) genes in the L . japonicus MG-20 genome. Two target sites were selected on the first exon and second exon of LjPLP4 and the first exon for LjPIP5K4 and LjPIP5K6 using the CRISPR-P program ( http://cbi.hzau.edu.cn/crispr/ ) ( Lei et al ., 2014 ) (Figs. S3a, S8a and S9a). The target oligonucleotides (primers set 21-22 for LjPLP4 , 23-24 for LjPIP5K4 or 25-26 for LjPIP5K6 in Table S1) were annealed and cloned into the Bbs I site of the single guide RNA (sgRNA) vector pUC19_AtU6oligo ( Ito et al ., 2015 ). One sgRNA cassette was amplified using phosphorylated primers set 27 (Table S1), and the amplicon was ligated with the PCR product of the other sgRNA, including the pUC19 vector, amplified using the primer set 28 (Table S1), thereby constructing a vector in which two sgRNA cassettes were arranged in tandem. The sgRNA cassettes were subcloned into the I-SceI site of pZH_gYSA_FFCas9, which contained the Cas9 and HPT expression cassettes ( Stiller et al ., 1997 ; Ito et al ., 2015 ; Nishida et al ., 2018 ). The CRISPR/Cas9 constructs were introduced into L . japonicus MG-20 by Agrobacterium tumefaciens AGL1 as described previously ( Stiller et al ., 1997 ). The deletion or mutation of the target sites in the transgenic plants was examined by PCR using the primer set 29 for LjPLP4 , 30 for LjPIP5K4 or 31 for LjPIP5K6 (Figs. S3b, S8b and S9b; Table S1) and confirmed by sequencing of the region (Figs. S3c, S8c and S9c). Homozygous lines of the deletion mutants were selected from the T1 transgenic plants, and the progeny (T2) were used in this study. For overexpression of LjPLP4 , the pLjUbiquitin::LjPLP4 constructs were introduced into L . japonicus MG-20 by Agrobacterium tumefaciens AGL1. The transgenic plants were examined by PCR using the primer set 32 (Table S1), and the expression level of LjPLP4 was calculated by real-time PCR using the primer set 18 (Table S1). Homozygous overexpression lines were selected from the T2 transgenic plants and used in this study. TUBBY expressing plants were generated by transformation with AGL1 harboring AtUBQ10prom::2xCHERRY-1xTUBBY-C (TUBBY) ( Simon et al ., 2014 ) as described above. GUS staining Transgenic hairy roots carrying the promoter GUS fusions were stained with a GUS staining buffer solution (0.5 mg/mL 5-bromo-4-chloro-3-indolyl-β-D-glucuronide [X-Gluc], 100 mM sodium phosphate buffer [pH 7.0], 100 mM EDTA, 0.5 mM K 4 [Fe(C.N.) 6 ], 0.5 mM K 3 [Fe(C.N.) 6 ], 0.1%v/v) at 30°C overnight. The stained roots were observed using a stereomicroscope (SZX16; EVIDENT, Japan). Subcellular localization and observation of PI(4,5)P₂ Venus-LjPLP4 or LjPIP5K6-Venus expressing roots obtained using hairy root transformation were mounted on glass slides. As organelle markers, pLjUbiquitin::NFR1-mCherry , p35S::tdTomato-CTT , or pMtBCP1::GmMAN49-mCherry were co-expressed, respectively. Observations were performed under a confocal microscope A1 (Nikon, Japan) using a 20× objective lens. Observation of PI(4,5)P 2 was performed using a confocal microscope SP8 (Leica Microsystems, Germany) using a 20× or 40× objective lens at 7 to 10 days after infection with M. loti carrying DsRed. All images in this study were processed with the software ImageJ. Grafting The grafting experiment was performed as described by Magori et al. (2009) . Three-day-old seedlings (MG-20 wild type and Ljplp4 ) were cut at the hypocotyls with a scalpel blade (Feather). Shoot scions were carefully cut at an angle and inserted into vertical slits made in the rootstock. The grafted plants were placed on pre-wet filter paper in a petri dish and covered with an additional filter paper. They were grown for 5 days under a 24°C condition with a 16-hour light/8-hour dark cycle. Then, plants were inoculated with M. loti carrying DsRed as described above, and infection was monitored one week later. Protein extraction and PIP strip HIS-tagged fusion proteins were expressed in E. coli BL21 (DE3) and extracted using CelLytic ™ B Cell Lysis Reagent (Sigma-Aldrich, USA) added 8 M urea. Protein purification with HIS-Select® Spin Columns (Sigma-Aldrich, USA) was performed according to the manufacturer’s protocol. [Equilibration Buffer and Wash Buffer; 0.1 M sodium phosphate and 8 M urea (pH 8.0), Elution Buffer; 0.1 M sodium phosphate and 8 M urea (pH 4.5)]. PIP Strips (Echelon Biosciences, USA) were blocked with Blocking One (Nacalai Tesque, Japan) overnight at 4℃ in a Petri dish. The protein–lipid overlay incubation was performed with 1 mL HIS-tagged protein (1.0 µg/mL protein in PBS) at 4℃ overnight. The strip was washed three times for 10 min with TBS-T (50 mM Tris, 138 mM NaCl, 2.7 mM KCl, 0.1% Tween 20), then incubated with 1:2000 diluted His-tagged Polyclonal antibody (Proteintech, USA) in Solution 1 of Can Get Signal® Immunoreaction Enhancer Solution (TOYOBO, Japan) for 1 hour at room temperature. The strip was washed three times for 10 minutes with TBS-T and incubated for 1 h with 1:5000 Goat Anti-Rabbit IgG H&L (HRP) antibody (Abcam, UK) in Solution 2 of Can Get Signal® Immunoreaction Enhancer Solution. After repeating the wash step three times with TBS-T for 10 min each, protein–lipid binding was detected by ImageQuant 800 (GE Healthcare, USA). Statistical analysis The data were analyzed using BellCurve for Excel (BellCurve, Japan). One-way ANOVA with Tukey test ( Figs. 2h, i , 5e-h and S12a, b), two-way ANOVA with Tukey test (Fig. S13) or Dunnett’s test ( Figs. 2c-f , 4e-h , S3d, e, S4, S5, S8d, e, S9d, e and S10) were applied. Student’s t-test by two-side were carried out ( Figs. 1c-e , 5a-d , S1b and S12c, d). At least three biological repetitions were performed for all experiments. Results Expression of PI signaling-related genes and accumulation of PI and phosphatidylinositol phosphates are induced during rhizobial infection To investigate PI and phosphatidylinositol phosphate dynamics during rhizobial infection ( M. loti ), an RNA-seq analysis was performed using L. japonicus roots 1 week after inoculation ( Fig. 1a ). We focused on the expression of PI signaling-related genes; phosphoinositide 3-kinase ( PI3K ) and phosphoinositide 4-kinase ( PI4K ), which phosphorylate phosphatidylinositol to phosphatidylinositol monophosphate (PIP), phosphatidylinositol 3-phosphate 5-kinase ( PI(3)P5K ) and phosphatidylinositol 4-phosphate 5-kinase ( PI(4)P5K or PIP5K ), which phosphorylate PIP to phosphatidylinositol bisphosphate (PIP 2 ), and phosphatidylinositol transfer protein ( PITP ) -like proteins ( PLP ). The expression levels of LjPI4K (LotjaGi2g1v0442900) and three PIP5Ks (LotjaGi1g1v0664200, LotjaGi3g1v0109700, and LotjaGi3g1v0318000) were significantly upregulated by rhizobial infection ( Fig. 1a ). Due to specific PI(4,5)P 2 or PI(4)P accumulation in Arabidopsis infected by powdery mildew or oomycete downy mildew, respectively, we performed another RNA-seq analysis during infection of Phytophthora palmivora , a pathogenic oomycete for L. japonicus ( Rey et al ., 2015 ; Fuechtbauer et al ., 2018 ) ( Fig. 1b ). The expression of the PI signaling-related genes was generally suppressed upon infection with P. palmivora , especially that of PIP5Ks , which was markedly upregulated during rhizobial infection. Moreover, no PLP genes were significantly upregulated during P. palmivora infection. These results suggest that there is a difference in the role of PI signaling between rhizobial infection and pathogen infection of P. palmivora . To quantify the amounts of PI, PIP, and PIP 2 in L. japonicus roots during rhizobial infection, we performed LC-MS. The results showed that accumulation of PI, PIP, and PIP 2 was induced in a rhizobial infection-dependent manner ( Fig. 1c-e ). We initially focused on LjPLP4 (LotjaGi2g1v0386800), which expression was only significantly induced among homologous genes for a PI transfer protein affecting the accumulation of PI, PIP and PIP 2 ( Fig. 1a ). Quantitative polymerase chain reaction (PCR) confirmed that LjPLP4 is the only PLP gene whose expression is upregulated during rhizobial infection among the eight PLP genes in the L. japonicus genome (Fig. S1). LjPLP4 suppresses rhizobial symbiosis while it has a promoting role in oomycete infection To confirm the tissue-specific expression of LjPLP4 during rhizobial infection, we performed GUS analysis with the promoter region of LjPLP4 and found that the GUS signal was induced by rhizobial infection throughout the root compared with roots that were not infected with rhizobia, but not in nodules ( Figs. 2a and S2). We generated LjPLP4 mutants using the CRISPR/Cas9 system to investigate whether LjPLP4 was required for rhizobial infection (Fig. S3). None of these mutants had any effect on root growth (Fig. S3d-g). Inoculation of LjPLP4 mutants with rhizobia resulted in a marked increase in the number of infection threads when compared with the wild type ( Fig. 2b-c ). Moreover, no significant difference was observed in the number of nodules compared with the wild type ( Fig. 2d ). We investigated at which stage of rhizobial invasion LjPLP4 was possibly involved. The results showed that the number of microcolonies (MCs) was significantly reduced in the LjPLP4 mutants, while the number of infection threads (ITs) that invaded to the epidermal cell increased compared to the wild type (Fig. S4a, b). The number of infection threads that branched from the tip and penetrated the cortical cells (Ramifying infection threads; Ramifying ITs) did not significantly increase overall (Fig. S4c). In addition, LjPLP4 overexpression ( PLP4 OE ) also did not affect root growth (Fig. S5) but suppressed rhizobial infection without altering nodule number ( Fig. 2e, f ). These results strongly suggest that the function of LjPLP4 is to suppress rhizobial infection in the epidermis. When P. palmivora was infected with the L. japonicus wild type and LjPLP4 mutants, infection was suppressed in Ljplp4 compared pathogen infection is opposite to that in rhizobial infection. LjPLP4 regulates rhizobial infection at the plasma membrane LjPLP4 was shown to be localized to the PM in onion epidermal cells based on the observation using fluorescent protein ( Kapranov et al ., 2001 ). To investigate LjPLP4 subcellular localization in L. japonicus , Venus fluorescent protein fused to the N-terminal of LjPLP4 was expressed in roots. The Venus fluorescent pattern differed between the epidermal and cortical cells ( Fig. 3a ). In the cortical cells, dot-like structures were mainly observed at the periphery of the cells. In the epidermal cells, it appeared to be mainly localized at the PM, with a few dot-like structures similar to those in the cortical cells. The co-expression of the NF receptor NFR1 fused with mCherry, which was used as a PM marker, with Venus-LjPLP4 indicated that LjPLP4 was localized at the PM in root hairs ( Fig. 3b, e ) ( Madsen et al ., 2011 ). The dot-like structures were consistent with tdTomato-CTT, an endoplasmic reticulum (ER) marker ( Fig. 3c, f ) ( Nagano et al., 2020 ). In addition, it was partly co-localized with a Golgi apparatus marker ( Fig. 3d, g ) ( Ivanov & Harrison, 2014 ). These findings suggest that LjPLP4 functions differently between the epidermal and cortical cells based on its subcellular localization. To further specify the function of LjPLP4, the binding of LjPLP4 to phospholipids was examined using the PIP-strip assay. We used 6xHIS-LjPLP4 full-length (FL) or ΔSEC14 protein, in the latter the Sec14 domain was deleted, that was purified using the HIS tag ( Fig. 3h ). LjPLP4 FL bound to PI(3)P, PI(4)P, and PI(4,5)P 2 but not to PI(3,5)P 2 among phosphatidylinositol phosphates present in plants ( Fig. 3i ). To confirm the specificity and sensitivity of the LjPLP4 FL protein towards PI and phosphatidylinositol phosphates, array strips were used ( Fig. 3j ). These data indicated that LjPLP4 bound almost equally to PI(3)P, PI(4)P, and PI(4,5)P 2 , and these signals were lost when using ΔSEC14, indicating that binding was mediated by the SEC14 domain ( Fig. 3k ). LjPLP4 is not involved in Ca 2+ spiking induced by NF recognition To test whether LjPLP4, which has been suggested to function at the PM by subcellular localization analysis ( Fig. 3a, b ), affects the perception of NFs, we quantified Ca 2+ spiking induced by NF treatment. We generated wild type and Ljplp4 plants expressing Yellow Cameleon 2.60 (NLS-YC), a calcium indicator ( Nagai et al ., 2004 ; Soyano et al ., 2024 ). NFs were added to the roots, and Ca 2+ spiking was observed (Fig. S6). The results showed that the LjPLP4 did not affect Ca 2+ spiking because Ca 2+ spiking occurred as frequently as in the wild type and no apparent differences in the spiking waveform observed (Fig. S6). This result suggests that LjPLP4 functions downstream of, or independent to, Ca 2+ spiking induced by NFs. LjPIP5K4 and LjPIP5K6 are phosphatidylinositol 4-phosphate 5-kinase responsible for suppressing rhizobial infection Among the three LjPIP5Ks whose expression levels were significantly upregulated during rhizobial infection ( Fig. 1a ), LjPIP5K4 (LotjaGi1g1v0664200) and LjPIP5K6 (LotjaGi3g1v0109700), encoding orthologous genes of Arabidopsis PIP5K4 and PIP5K6, respectively, were selected for further investigation (Fig. S7). To confirm the tissue-specific expression of LjPIP5K4 and LjPIP5K6 during rhizobial infection, we performed GUS analysis with the promoter region of either LjPIP5K4 or LjPIP5K6 , and found that the GUS signal was induced by rhizobial infection in the epidermal cells where infection threads formed, and in the nodule primordia ( Fig. 4a-d ). We generated mutants of LjPIP5K4 and LjPIP5K6, respectively, using CRISPR/Cas9, and confirmed that they did not affect root growth (Figs. S8 and S9). The loss of function in both of the genes resulted in an increase of infection threads compared with the wild type ( Fig. 4e, g ). The number of MCs was significantly reduced in the LjPIP5K4 and LjPIP5K6 mutants, while the number of Ramifying IT significantly increased compared to the wild type (Fig. S10). Subcellular localization analysis using the mCherry fluorescent protein suggested that LjPIP5K6 was co-localized with LjPLP4 at the PM in root hairs (Fig. S11). These results are consistent with the phenotype observed in the Ljplp4 mutants, suggesting that LjPLP4 and LjPIP5K6 function concomitantly in the suppression of rhizobial infection. Furthermore, in both Ljpip5k4 and Ljpip5k6 mutants, the number of nodules was significantly higher than that in the wild type ( Fig. 4f, h ). The above results suggest that PI(4,5)P 2 , which is specifically produced by PIP5K, contributes to rhizobial infection in addition to nodule formation. NIN regulates the suppression of rhizobial infection partly through LjPLP4 , LjPIP5K4 and LjPIP4K6 Because overexpression of NIN in L. japonicus strongly suppresses the rhizobial infection ( Soyano et al ., 2014 ; Yoro et al., 2014 ), we investigated whether the suppression of rhizobial infection by LjPLP4 , LjPIP5K4 and LjPIP4K6 is regulated by NIN. In the nin-9 and cyclops-6 mutant, the expression of LjPLP4, LjPIP5K4 and LjPIP5K 6 was not induced by inoculation of rhizobia ( Fig. 5a-c ), suggesting that the suppression of rhizobial infection by LjPLP4, LjPIP5K4 and LjPIP5K6 may be controlled by NIN and CYCLOPS. Therefore, to further investigate the relationship between NIN and LjPLP4 , LjPIP5K4 or LjPIP5K6 , we generated NIN -overexpressing roots using the L. japonicus MG-20 wild type. In the NIN-overexpressing roots, the expression levels of LjPLP4 , LjPIP5K4 , and LjPIP5K6 were found to be significantly higher compared to the roots expressing empty vector (EV) ( Fig. 5d ). Accordingly, the number of infection threads significantly increased in the LjPLP4 , LjPIP5K4 , and LjPIP5K6 mutants compared to wild type plants overexpressing NIN, but not comparable to wild type plants expressing EV ( Fig 5e ). In Ljpip5k4 and Ljpip5k6 mutants, the suppression of nodule number by overexpression of NIN was weaker compared to the wild type, whereas in Ljplp4 , significant difference was not observed compared to the wild type ( Fig. 5f ). The above results suggest that NIN regulates the suppression of rhizobial infection partly through LjPLP4 , LjPIP5K4 and LjPIP4K6 . The suppression of rhizobial infection observed in Ljplp4 led us to investigate whether LjPLP4 is involved in the AON. It has been reported that the number of infection threads and nodules significantly increases in the AON mutants, har1 and tml ( Magori et al., 2009 ; Miri et al., 2019 ). A comparison of rhizobial infection phenotypes in Ljplp4 , har1-7 , and tml revealed that while there were significant differences in the number of nodules between Ljplp4 and har1-7 or tml , there were no significant differences in the number of infection threads (Fig. S12a, b). We quantified the CLE-RS1 expression in Ljplp4 roots 1 week after rhizobial inoculation and found that the expression level was induced similarly to the wild type but not in nin-9 , indicating that the AON is triggered in Ljplp4 roots (Fig. S12c). Furthermore, a grafting experiment between Ljplp4 and the wild type was conducted to assess whether PLP4 was required in the shoot. It turned out that PLP4 functioned in the root to suppress infection thread formation (Fig. S12e,f). To know whether TML and/or HAR1 regulates LjPLP4 expression, we examined LjPLP4 expression in tml and har1-7 roots 1 week after inoculation. The induction of LjPLP4 expression by rhizobial infection that was observed in the wild type did not occur in har1-7 , but it was significantly enhanced in tml (Fig. S12d). To further verify the relationship between the AON and LjPLP4 , double mutants of Ljplp4-4-10 and har1-7 or tml were generated and were inoculated with rhizobia. The results showed that the number of infection threads significantly increased in Ljplp4-4-10 har1-7 but not in Ljplp4-4-10 tml , when compared to har1-7 or tml single mutants, respectively ( Fig. 5g, i ). There was no significant increase in the number of nodules when Ljplp4 mutation was introduced in both har1-7 and tml , consistent with the Ljplp4 single mutant ( Figs. 5h, j ). These results suggest that the suppressive function of LjPLP4 is partially HAR1-independent. Rhizobial infection-dependent PI(4,5)P 2 accumulation is mediated by LjPLP4 and LjPIP5Ks The quantitative analysis using LC-MS showed that PI, PIP, and PIP 2 accumulated in wild-type roots upon rhizobial infection ( Fig. 1c-e ). Therefore, we measured the total amounts of PI, PIP, and PIP 2 in Ljplp4 and Ljpip5k6 mutants. The results showed that the amounts of PI, PIP, and PIP 2 did not increase in Ljplp4 and Ljpip5k6 as observed in the wild type (Fig. S13). Analysis of LjPIP5K4 and LjPIP5K6 loss-of-function mutants suggested that PI(4,5)P 2 , among phosphatidylinositol phosphates, might have an important function in rhizobial infection ( Fig. 4e, g ). To examine specific changes in PI(4,5)P 2 during rhizobial infection, we generated a transgenic L. japonicus MG-20 expressing 2xCHERRY-1xTUBBY-C (TUBBY), a fluorescent marker of PI(4,5)P 2 ( Simon et al ., 2014 ). Strong signal was observed in the apex of root hairs (Fig. S14a). When roots were inoculated with M. loti , the signal was observed in the epidermis. In contrast, almost no signal was observed in the cortex (Fig. S14b). These findings indicate that accumulation of PI(4,5)P 2 is induced in the root epidermis, where rhizobia enter host roots. Furthermore, when inoculated with M. loti carrying green fluorescent protein (GFP), PI(4,5)P 2 accumulation was often found to be stronger in the epidermal cells where infection threads formed and in their adjacent cells, than in distal cells ( Fig. 6a ; Movie S1). Moreover, in the epidermal cells with stronger TUBBY signals, infection threads elongated horizontally often without defined direction ( Fig. 6a ), or became entangled and forming clumps ( Fig. 6b ). Infection threads in these cells seemed unable to penetrate the cortex from the epidermis. Interestingly, the TUBBY signal was shown to be much weaker where infection threads had penetrated cortex ( Fig. 6c, d ; Movie S2). The TUBBY signal in nodules was found to be absent where rhizobial infection had occurred and stronger in surrounding cells that were not infected with rhizobia ( Fig. 6e ). Because the number of infection threads increased in Ljplp4 , Ljpip5k4 , and Ljpip5k6 mutants ( Figs. 2b,c , 4e,g ), we further verify the presence of PI(4,5)P 2 in these mutants with TUBBY. The signal was almost absent in the cells where infection thread developed, indicating accumulation of PI(4,5)P 2 was not induced by rhizobial infection ( Fig. 6f-i ). Download figure Open in new tab Figure 6. PI(4,5)P 2 accumulation inhibits rhizobial invasion into root hair cells and elongation into the cortex. a-e. L. japonicus MG-20 wild-type expressing TUBBY, a PI(4,5)P 2 marker, was infected with M. loti expressing GFP. The root epidermal cells (a,b), the epidermal and the cortex cells observed from horizontally (c), nodule primordium observed from above (d), immature nodule (e). a. White arrow indicates a TUBBY signal in an infected cell, yellow arrow indicates a TUBBY signal in the vicinity cells of infected cells, white arrowhead indicates infection thread that failed to penetrate to the cortex. b. White arrow indicates TUBBY signal in an infected cell; white arrowhead indicates infection thread that failed and forming clumps. c. White arrow indicates weakened accumulation of a TUBBY signal around infection threads penetrating to the cortex. Ep; Epidermis, Co; Cortex. d. White arrow indicates a TUBBY signal accumulated in the cells surrounding the infected region; Yellow arrow indicates a TUBBY signal is absent in the infected cell; The white arrowhead indicate infection threads that have branched off and are extending into cortex cell. e. White arrow indicates strong accumulation of a TUBBY signal on the surface of rhizobium-infected nodules. white arrowhead indicates infection threads invading cortical cells and rhizobia infecting cells inside the nodule. f-i . L. japonicus hairy roots expressing TUBBY with Venus as a transformation marker in wild type (f), Ljplp4 (g), Ljpip5k4 (h) or Ljpip5k6 roots (i), were infected with M. loti expressing GFP. Scale bars is 100 µm (a-i). Discussion In this study, RNA-seq analysis revealed rhizobial infection-dependent expression of PI signaling-related genes in L. japonicus roots ( Fig. 1a ). LC-MS analysis showed rhizobial infection-dependent increases in PI, PIP, and PIP 2 ( Fig. 1c–e ). In the LC-MS analysis, quantifying individual phosphorylation variants in PIP and PIP 2 was technically difficult. However, because there was no increase in total PIP 2 in the Ljpip5k6 mutant, PI(4,5)P 2 likely accounts for much of the increase in PIP 2 observed in these LC-MS data (Fig. S13) because PIP5K has been shown to specifically produce PI(4,5)P 2 ( Muftuoglu et al., 2016 ). In addition, observation of PI(4,5)P 2 dynamics in rhizobia infected roots by TUBBY, a PI(4,5)P 2 marker, revealed that ectopic PI(4,5)P 2 accumulation was closely related to rhizobial infection ( Fig. 6 ). Although we cannot rule out the possibility that PI and PIP function in rhizobial infection, the fact that increase of rhizobial infection not only in LjPLP4 but also in LjPIP5K4 and LjPIP5K6 mutants ( Figs. 2b, c and 4e, g ) suggests that the shift from PI(4)P to PI(4,5)P 2 has a suppressive effect on rhizobial infection. Regarding the Sec14 domain present in LjPLP4, two functions have been postulated. First , in vitro lipid transport studies have shown that the Sec14 domain-containing protein transports PI and phosphatidylcholine (PC) across membranes ( Kapranov et al ., 2001 ; Schaaf et al ., 2008 ). Second, in a model predicted based on the crystal structure, Sec14 binds to PI and PIPs on the membrane, thereby forming a structure that exposes PI and PIP to PI4K and PIP5K to be able to function efficiently ( Kf de Campos & Schaaf, 2017 ). The real-time PCR and RNA-seq analyses performed in this study revealed that increased expression of LjPLP4 1 week after rhizobial inoculation ( Fig. 1a , S1b). PIP strip assays revealed that LjPLP4 binds relatively strongly to PI(3)P, PI(4)P, and PI(4,5)P 2 among PI and phosphatidylinositol phosphates present in plant cells ( Fig. 3i, j ). These findings also suggest that LjPLP4 facilitates PI(4)P and PI(4,5)P 2 production by PI4K and PIP5K. This idea is consistent with the fact that the expression of one of PI4K genes (LotjaGi2g1v0442900) is significantly induced by rhizobial infection, along with three LjPIP5Ks ( Fig. 1a ). In addition, LjPLP4 is localized mainly at the PM and the endoplasmic reticulum (ER) and, to a lesser degree, to the Golgi bodies ( Fig. 3a–g ). The function of LjPLP4 in the ER and the Golgi bodies remains unclear, but as LjPLP4 has been shown to be localized at PM in the root epidermal cells, which are the main contact tissue with soil microorganisms, it probably functions to synthesize PI(4)P and PI(4,5)P 2 . Observations of PI (4,5)P 2 by TUBBY showed a hardly detectable signal in the cortex (Fig. S13b). This may be related to the fact that LjPLP4 is not PM-localized in the cortex ( Fig. 3a, b ). The expression pattern analysis using GUS staining revealed that LjPLP4 was expressed in the epidermis and the cortex in inoculated roots, while LjPIP5K4 and LjPIP5K6 were expressed specifically in rhizoial infected cells. The differential expression pattern suggests that LjPLP4 is involved not only with LjPIP5K4 and LjPIP5K6 but also with other PIP5Ks. LjPIP5K4 and LjPIP5K6 were expressed in the nodule primordium. The results suggest that LjPIP5K4 and LjPIP5K6 may be involved in nodule development. However, the increased number of nodules observed in the Ljpip5k4 and Ljpip5k6 mutants appears to result from an increased number of ramifying infection threads in these mutants. These results seem to conflict with the finding that PI(4,5)P 2 was absent in nodule primordium ( Fig. 6e ). It has been reported that diacylglycerol (DAG) accumulates in the nodules in M. truncatula ( Zhang et al., 2020 ). Because PI(4,5)P 2 is hydrolyzed by phospholipase C (PLC) to produce DAG and IP 3 , the PI(4,5)P 2 generated by LjPIP5K and LjPIP5K6 may contribute to nodule formation by being converted into DAG and/or IP 3 . In animal cells, it is known that PI(4, 5)P 2 cleavage by phospholipase C (PLC) releases inositol 1,4,5-trisphosphate (IP 3 ), which functions as a second messenger to trigger Ca 2+ channel opening ( Kania et al ., 2017 ). Furthermore, in M. truncatula , the IP 3 inducer Mastoparan analog (Mas7) treatment induces calcium fluctuations similar to NF-induced Ca 2+ spiking ( Sun et al ., 2007 ). In this regard, we investigated whether the absence of LjPLP4 affected Ca 2+ spiking induced by NFs using a YC 2.60 sensor but found no significant variation in the spiking (Fig. S6). Thus, it is unlikely that Ca 2+ spiking is affected by LjPLP4 -induced changes in PI dynamics. The NIN -dependent suppression mechanism of rhizobial infection has been demonstrated previously ( Soyano et al ., 2014 ; Yoro et al ., 2020 ). The real-time PCR analysis with the nin-9 mutant found that the expression of LjPLP4 , LjPIP5K4 , and LjPIP5K6 required NIN ( Fig. 5a-c ). In addition, in the LjPLP4 , LjPIP5K4 , and LjPIP5K6 mutants overexpressing NIN , the number of infection threads was significantly higher than in wild-type plants overexpressing NIN ( Fig. 5e ). The above results strongly suggest that the suppression of rhizobial infection by LjPLP4 , LjPIP5K4 , and LjPIP5K6 is partly regulated by NIN . Expression analysis in this study showed that the Ljplp4 mutation did not affect the expression of CLE-RS1 (Fig. S12c). NIN regulates expression levels of downstream genes, including CLE-RS1 , in a rhizobial symbiosis-specific manner ( Soyano et al ., 2014 ). The Ljplp4 har1 double mutant showed an increase in the number of infection threads compared with the Ljplp4 single mutant ( Fig. 5i ). In the har1-7 mutant, NIN is expressed even in the absence of rhizobial infection ( Akamatsu et al., 2021 ). Therefore, the gene expression analysis in har1-7 may not have detected an increase in PLP4 expression (Fig. S12d). The results suggest that the suppression of rhizobial infection mediated by LjPLP4 is likely independent of HAR1 . In the Ljplp4 tml double mutant, no increase in the number of infection threads was observed compared to the tml single mutant ( Fig. 5e ). Gene expression analysis found that LjPLP4 was induced in a TML -independent manner (Fig. S12d). The above results suggest that while LjPLP4 is TML -independent in terms of gene expression, LjPLP4 has a functional connection with TML in the suppression of rhizobial infection. PI(4,5)P 2 has been shown to accumulate at the tip of infection threads in the epidermis in M. truncatula ( Lace et al., 2023 ). Previous studies have suggested that PI(4,5)P 2 plays a role in regulating the cytoskeleton and pectin by accumulating at sites of tip growth ( Ischebeck et al ., 2008 ; Hirano et al ., 2018 ). Thus, PI(4,5)P 2 may promote elongation of infection threads in root hairs. On the other hand, in this study, we showed that in L. japonicus , the epidermal cells in the wild type in which infection threads are aborted show the clear accumulation of PI(4,5)P 2 to the PM ( Fig. 6a, b ). In addition, in LjPLP4 , LjPIP5K4 and LjPIP5K6 mutants, the accumulation of PI(4,5)P 2 in the epidermis were weaker than in the wild type ( Fig. 6f-i ). Moreover, analysis of the rhizobial infection phenotype in LjPLP4 , LjPIP5K4 and LjPIP5K6 mutants suggests that PI(4,5)P 2 negatively regulates rhizobial infection ( Figs. 2b, c , 4e, g ). The above results suggest that accumulation of PI(4,5)P 2 to the PM likely facilitates the suppression of rhizobial infection. In M. truncatula , the infection chamber formed at the tip of root hairs during rhizobial infection becomes enlarged as the first step of infection thread formation (Furnier et al ., 2015). The enlarged membrane is then polarized by a protein complex containing RPG, VPY and LIN and proceeds to invade in the direction of the root center. PI(4,5)P 2 accumulates at the tip of the polarized infection thread, which is crucial for the elongation of infection thread with correct direction ( Liu et al., 2019 ; Lace et al., 2023 ). Based on this, it is hypothesized that the strong accumulation of PI(4,5)P 2 to the PM causes the loss of polarity, preventing the penetration of infection threads into the cortex. Furthermore, quantitative analysis of infection events revealed a decrease in the number of MCs in the LjPLP4 , LjPIP5k4 , and LjPIP5K6 mutants, suggesting that the PI(4,5)P 2 accumulation to the PM inhibited the entry of rhizobia into the root hair cells. How PI(4,5)P 2 accumulates to the PM and suppresses excessive rhizobial infection is currently unknown. One possibility is that excess PI, PIP, and PIP 2 are negatively charged and thereby may impair the recruitment of factors necessary for rhizobial infection, inhibiting rhizobial infection. These questions must be investigated in future research studies. In host plant immune responses to pathogenic fungi and oomycetes, the importance of PI(4,5)P 2 or PI(4)P at the infection site is becoming clear ( Shimada et al ., 2019 ; Qin et al ., 2020 ; Zarreen et al ., 2023 ). As the local accumulation likely promotes pathogen infection, it has been thought that pathogens can regulate PI(4,5)P 2 production and/or localization in host cells by contacting their invasive hyphae ( Shimada et al ., 2019 ). Although no upregulation of LjPLP4 was observed during P. palmivora infection, the lesion length of P. palmivora infection, and P. palmivora RNA content were markedly suppressed in the LjPLP4 mutant, suggesting that LjPLP4 plays a positive role in P. palmivora infection ( Fig. 1b , 2g–i ). The gene expression of PI4Ks (LotjaGi3g1g1v0712000_LC, LotjaGi3g0396100) was induced in P. palmivora infection, but not in rhizobial infection ( Fig. 1b ), suggesting that these PI4K might function together with LjPLP4 in defense responses. These results indicate that a similar function to ectopic PI(4,5)P 2 accumulation at the EHM observed during powdery mildew infection reported in Arabidopsis is also present in L. japonicus and that it requires LjPLP4 . In summary ( Fig. 7 ), PI signaling-related genes are upregulated in rhizobial-infected L. japonicus roots. Among them, LjPLP4 causes PI(4,5)P 2 accumulation in the root epidermis. The accumulation of PI(4,5)P 2 in the epidermis by inoculation of rhizobia inhibits the invasion of rhizobia into root hairs. The expression of LjPLP4 , LjPIP5k4 and LjPIP5k6 regulated by NIN suggests that these responses are likely to be rhizobial infection-specific phenomena. Although the direct interaction of LjPLP4 and LjPIP5Ks has not been demonstrated in this study, co-localization and requirement for accumulation of PI(4,5)P 2 suggest that they may function together in the epidermal cells where infection threads formed. In the cells where PI(4,5)P 2 does not accumulate or is degraded, rhizobia invade into root hairs. Furthermore, inhibition due to increased PI(4,5)P 2 also affects elongation of infection threads. Only a few infection threads that avoid the accumulation of PI(4,5)P 2 are able to proceed with infection into the cortex. Download figure Open in new tab Figure 7. Schematic model of the pathway of the suppression system by PI(4,5)P 2 The accumulation of PI(4,5)P 2 in the epidermis by inoculation of rhizobia inhibits the invasion of rhizobia into the root hairs. In the cells where PI(4,5)P 2 does not accumulate or is degraded, rhizobia invade into the root hairs. Furthermore, the accumulation of PI(4,5)P 2 also affects elongation of infection threads with correct direction. Only a few infection threads that avoid accumulation of PI(4,5)P 2 are able to penetrate the cortex. The green color indicates infection thread, and the magenta color indicates PI(4,5)P 2 . CONFLICT OF INTEREST The authors declare that they have no conflicts of interest. Author contributions A.A designed the study. A.A, M.H and N.T wrote the manuscript. T.I contributed to the PIs measurements. H.T and Y.K contributed to data collection. All authors contributed to the interpretation of data and reviewed the manuscript. DATA AVAILABILITY STATEMENT RNA-Seq data used in this publication have been deposited in DDBJ Sequence Read Archive (DRA) at the DNA Data Bank of Japan (DDBJ; http://www.ddbj.nig.ac.jp/ ) under the accession number DRR585568-DRR585579. ACKNOWLEDGEMENTS This work was supported by JSPS KAKENHI Grant Numbers 20K15426 22K06288 (to A.A) 23K17998 (to N. T), an Individual Special Research Fund (Kwansei Gakuin University, Japan, 2023), Hyogo Science and Technology, the Sumitomo Foundation and the Joint Usage/Research Center, the Institute of Plant Science and Resources (Okayama University, Japan). The CRISPR/Cas9 vectors were kindly provided by Dr. Masaki Endo (NARO, Japan). We thank Dr. Masayoshi Kawaguchi (National Institute for Basic Biology, Japan) and Dr. Takuya Suzaki (University of Tsukuba) for kindly providing nin-9 , tml , har1-7 and cyclops-6 mutants. We thank Dr. Ayaka Hieno for the lecture on P. palmivora culture methods. We thank Mr. Hideki Nishimura for his technical support for P. palmivora culture. We acknowledge the NGS core facility of the Genome Information Research Center at the Research Institute for Microbial Diseases of Osaka University for the support in RNA sequencing and data analysis. pCMB-GAr was a gift from Maria Harrison (Addgene plasmid # 61173 ; http://n2t.net/addgene:61173 ; RRID:Addgene_61173) and tdTomato-CTT was a gift from Dr. Minoru Nagano (Ritsumeikan University). Funder Information Declared JSPS KAKENHI , 20K15426 , 22K06288 , 23K17998 REFERENCES ↵ Akamatsu A , Nagae M , Nishimura Y , Romero Montero D , Ninomiya S , Kojima M , Takebayashi Y , Sakakibara H , Kawaguchi M , Takeda N . 2021 . Endogenous gibberellins affect root nodule symbiosis via transcriptional regulation of NODULE INCEPTION in Lotus japonicus . Plant J 105 : 1507 – 1520 . OpenUrl CrossRef PubMed ↵ Akamatsu A , Nagae M , Takeda N . 2022 . The CYCLOPS Response Element in the NIN Promoter Is Important but Not Essential for Infection Thread Formation During Lotus japonicus -Rhizobia Symbiosis . Mol Plant Microbe Interact 35 ( 8 ): 650 – 658 . OpenUrl CrossRef PubMed ↵ Broughton WJaD , M. Y . 1971 . Control of leghemoglobin synthesis in snake bean . Biochem. J . 125 : 1075 – 1080 . OpenUrl Abstract / FREE Full Text ↵ Buttery BR , Park J . 1989 . EFFECTS OF NITROGEN, INOCULATION AND GRAFTING ON EXPRESSION OF SUPERNODULATION IN A MUTANT OF Phuseolus vulgaris L . Can. J. Plant Sci . 70 : 375 – 381 OpenUrl ↵ Cai T , Shu Q , Liu P , Niu L , Guo X , Ding X , Xue P , Xie Z , Wang J , Zhu N , et al. 2016 . Characterization and relative quantification of phospholipids based on methylation and stable isotopic labeling . J Lipid Res 57 : 388 – 397 . OpenUrl Abstract / FREE Full Text ↵ Cerri MR , Wang QH , Stolz P , Folgmann J , Frances L , Katzer K , Li XL , Heckmann AB , Wang TL , Downie JA , et al. 2017 . The ERN1 transcription factor gene is a target of the CCaMK/CYCLOPS complex and controls rhizobial infection in Lotus japonicus . New Phytologist 215 ( 1 ): 323 – 337 . OpenUrl CrossRef PubMed ↵ Clark J , Anderson KE , Juvin V , Smith TS , Karpe F , Wakelam MJO , Stephens LR , Hawkins PT . 2011 . Quantification of PtdInsP(3) molecular species in cells and tissues by mass spectrometry . Nature Methods 8 : 267 – U120 . OpenUrl CrossRef PubMed ↵ Ferguson BJ , Li D , Hastwell AH , Reid DE , Li Y , Jackson SA , Gresshoff PM . 2014 . The soybean ( Glycine max ) nodulation-suppressive CLE peptide, GmRIC1, functions interspecifically in common white bean ( Phaseolus vulgaris ), but not in a supernodulating line mutated in the receptor PvNARK . Plant Biotechnol J 12 ( 8 ): 1085 – 1097 OpenUrl CrossRef PubMed Web of Science ↵ Fournier J , Teillet A , Chabaud M , Ivanov S , Genre A , Limpens E , de Carvalho-Niebel F , Barker DG. 2015 . Remodeling of the infection chamber before infection thread formation reveals a two-step mechanism for rhizobial entry into the host legume root hair . Plant Physiol 167 ( 4 ): 1233 – 1242 . OpenUrl Abstract / FREE Full Text ↵ Fuechtbauer W , Yunusov T , Bozsoki Z , Gavrin A , James EK , Stougaard J , Schornack S , Radutoiu S . 2018 . LYS12 LysM receptor decelerates Phytophthora palmivora disease progression in Lotus japonicus . Plant Journal 93 : 297 – 310 . OpenUrl CrossRef PubMed ↵ Gage DJ . 2004 . Infection and invasion of roots by symbiotic, nitrogen-fixing rhizobia during nodulation of temperate legumes . Microbiol Mol Biol Rev 68 : 280 – 300 . OpenUrl Abstract / FREE Full Text ↵ Gao JP , Liang W , Jiang S , Yan Z , Zhou C , Wang E , Murray JD . 2023 . NODULE INCEPTION activates gibberellin biosynthesis genes during rhizobial infection . New Phytol 239 ( 2 ): 459 – 465 . OpenUrl CrossRef PubMed ↵ Hirano T , Konno H , Takeda S , Dolan L , Kato M , Aoyama T , Higaki T , Takigawa-Imamura H , Sato MH . 2018 . Ptdlns(3,5)P-2 mediates root hair shank hardening in Arabidopsis . Nature Plants 4 : 888 -+. OpenUrl CrossRef PubMed ↵ Ischebeck T , Stenzel I , Heilmann I . 2008 . Type B phosphatidylinositol-4-phosphate 5-kinases mediate Arabidopsis and Nicotiana tabacum pollen tube growth by regulating apical pectin secretion . Plant Cell 20 ( 12 ): 3312 – 3330 . OpenUrl Abstract / FREE Full Text ↵ Ito Y , Nishizawa-Yokoi A , Endo M , Mikami M , Toki S . 2015 . CRISPR/Cas9-mediated mutagenesis of the RIN locus that regulates tomato fruit ripening . Biochemical and Biophysical Research Communications 467 : 76 – 82 . OpenUrl CrossRef PubMed ↵ Ivanov S , Harrison MJ . 2014 . A set of fluorescent protein-based markers expressed from constitutive and arbuscular mycorrhiza-inducible promoters to label organelles, membranes and cytoskeletal elements in Medicago truncatula . Plant J 80 : 1151 – 1163 . OpenUrl CrossRef PubMed ↵ Ivanov S , Harrison MJ . 2019 . Accumulation of phosphoinositides in distinct regions of the periarbuscular membrane . New Phytol 221 ( 4 ): 2213 – 2227 . OpenUrl CrossRef PubMed ↵ Kamal N , Mun T , Reid D , Lin JS , Akyol TY , Sandal N , Asp T , Hirakawa H , Stougaard J , Mayer KFX , et al. 2020 . Insights into the evolution of symbiosis gene copy number and distribution from a chromosome-scale Lotus japonicus Gifu genome sequence . DNA Res 27 ( 3 ). ↵ Kania E , Roest G , Vervliet T , Parys JB , Bultynck G . 2017 . IP(3) Receptor-Mediated Calcium Signaling and Its Role in Autophagy in Cancer . Front Oncol 7 : 140 . OpenUrl CrossRef PubMed ↵ Kapranov P , de Bruijn FJ , Szczyglowski K. 1997 . Novel, highly expressed late nodulin gene (LjNOD16) from Lotus japonicus . Plant Physiol 113 : 1081 – 1090 . OpenUrl Abstract ↵ Kapranov P , Routt SM , Bankaitis VA , de Bruijn FJ , Szczyglowski K. 2001 . Nodule-specific regulation of phosphatidylinositol transfer protein expression in Lotus japonicus . Plant Cell 13 : 1369 – 1382 . OpenUrl Abstract / FREE Full Text ↵ Karimi M , Inze D , Depicker A . 2002 . GATEWAY TM vectors for Agrobacterium-mediated plant transformation . Trends in Plant Science 7 : 193 – 195 . OpenUrl CrossRef PubMed Web of Science ↵ Kf de Campos M , Schaaf G. 2017 . The regulation of cell polarity by lipid transfer proteins of the SEC14 family . Curr Opin Plant Biol 40 : 158 – 168 . OpenUrl CrossRef PubMed ↵ Lace B , Su C , Invernot Perez D , Rodriguez-Franco M , Vernie T , Batzenschlager M , Egli S , Liu CW , Ott T . 2023 . RPG acts as a central determinant for infectosome formation and cellular polarization during intracellular rhizobial infections . Elife 12 . ↵ Lei Y , Lu L , Liu HY , Li S , Xing F , Chen LL . 2014 . CRISPR-P: A Web Tool for Synthetic Single-Guide RNA Design of CRISPR-System in Plants . Molecular Plant 7 : 1494 – 1496 . OpenUrl CrossRef PubMed Web of Science ↵ Li H , Handsaker B , Wysoker A , Fennell T , Ruan J , Homer N , Marth G , Abecasis G , Durbin R , Genome Project Data Processing S . 2009 . The Sequence Alignment/Map format and SAMtools . Bioinformatics 25 ( 16 ): 2078 – 2079 . OpenUrl CrossRef PubMed Web of Science Li Y , Pei Y , Shen Y , Zhang R , Kang M , Ma Y , Li D , Chen Y . 2022 . Progress in the Self-Regulation System in Legume Nodule Development-AON (Autoregulation of Nodulation) . Int J Mol Sci 23 ( 12 ). ↵ Liu C-W , Breakspear A , Stacey N , Findlay K , Nakashima J , Ramakrishnan K , Liu M , Xie F , Endre G , de Carvalho-Niebel F , et al. 2019 . A protein complex required for polar growth of rhizobial infection threads . Nature Communications 10 : 2848 . OpenUrl CrossRef PubMed ↵ Langdon WB . 2015 . Performance of genetic programming optimised Bowtie2 on genome comparison and analytic testing (GCAT) benchmarks . BioData Min 8 ( 1 ): 1 . OpenUrl CrossRef PubMed ↵ Maekawa T , Maekawa-Yoshikawa M , Takeda N , Imaizumi-Anraku H , Murooka Y , Hayashi M . 2009 . Gibberellin controls the nodulation signaling pathway in Lotus japonicus . Plant Journal 58 ( 2 ): 183 – 194 . OpenUrl CrossRef PubMed Web of Science ↵ Madsen EB , Antolin-Llovera M , Grossmann C , Ye JY , Vieweg S , Broghammer A , Krusell L , Radutoiu S , Jensen ON , Stougaard J , et al. 2011 . Autophosphorylation is essential for the in vivo function of the Lotus japonicus Nod factor receptor 1 and receptor-mediated signalling in cooperation with Nod factor receptor 5 . Plant Journal 65 : 404 – 417 . OpenUrl CrossRef PubMed Web of Science ↵ Magori S , Oka-Kira E , Shibata S , Umehara Y , Kouchi H , Hase Y , Tanaka A , Sato S , Tabata S , Kawaguchi M . 2009 . TOO MUCH LOVE, a Root Regulator Associated with the Long-Distance Control of Nodulation in Lotus japonicus . Molecular Plant-Microbe Interactions 22 : 259 – 268 . OpenUrl CrossRef PubMed Web of Science ↵ nisiMotohashi K. 2017 . Seamless Ligation Cloning Extract (SLiCE) Method Using Cell Lysates from Laboratory Escherichia coli Strains and its Application to SLiP Site-Directed Mutagenesis . Methods Mol Biol 1498 : 349 – 357 . OpenUrl CrossRef PubMed ↵ Maekawa T , Kusakabe M , Shimoda Y , Sato S , Tabata S , Murooka Y , Hayashi M . 2008 . Polyubiquitin promoter-based binary vectors for overexpression and gene silencing in Lotus japonicus . Mol Plant Microbe Interact 21 ( 4 ): 375 – 382 . OpenUrl CrossRef PubMed Web of Science ↵ Miri M , Janakirama P , Huebert T , Ross L , McDowell T , Orosz K , Markmann K , Szczyglowski K . 2019 . Inside out: root cortex-localized LHK1 cytokinin receptor limits epidermal infection of Lotus japonicus roots by Mesorhizobium loti . New Phytol 222 ( 3 ): 1523 – 1537 . OpenUrl CrossRef PubMed ↵ Miyata K , Kawaguchi M , Nakagawa T . 2013 . Two Distinct EIN2 Genes Cooperatively Regulate Ethylene Signaling in Lotus japonicus . Plant and Cell Physiology 54 : 1469 – 77 OpenUrl CrossRef PubMed ↵ Muftuoglu Y , Xue Y , Gao X , Wu D , Ha Y . 2016 . Mechanism of substrate specificity of phosphatidylinositol phosphate kinases . Proc Natl Acad Sci U S A 113 ( 31 ): 8711 – 8716 . OpenUrl Abstract / FREE Full Text ↵ Murray JD , Karas BJ , Sato S , Tabata S , Amyot L , Szczyglowski K . 2007 . A cytokinin perception mutant colonized by Rhizobium in the absence of nodule organogenesis . Science 315 ( 5808 ): 101 – 104 . OpenUrl Abstract / FREE Full Text ↵ Nagai T , Yamada S , Tominaga T , Ichikawa M , Miyawaki A . 2004 . Expanded dynamic range of fluorescent indicators for Ca by circularly permuted yellow fluorescent proteins . Proceedings of the National Academy of Sciences , USA 101 : 10554 – 10559 . OpenUrl Abstract / FREE Full Text ↵ Nagano M , Ueda H , Fukao Y , Kawai-Yamada M , Hara-Nishimura I . 2020 . Generation of Arabidopsis lines with a red fluorescent marker for endoplasmic reticulum using a tail-anchored protein cytochrome b5 -B . Plant Signal Behav 15 : 1790196 . OpenUrl CrossRef PubMed ↵ Nishida H , Tanaka S , Handa Y , Ito M , Sakamoto Y , Matsunaga S , Betsuyaku S , Miura K , Soyano T , Kawaguchi M , et al. 2018 . A NIN-LIKE PROTEIN mediates nitrate-induced control of root nodule symbiosis in Lotus japonicus . Nat Commun 9 : 499 . OpenUrl CrossRef PubMed ↵ Noack LC , Jaillais Y . 2020 . Functions of Anionic Lipids in Plants . Annu Rev Plant Biol 71 : 71 – 102 . OpenUrl CrossRef PubMed ↵ Okamoto S , Ohnishi E , Sato S , Takahashi H , Nakazono M , Tabata S , Kawaguchi M . 2009 . Nod Factor/Nitrate-Induced CLE Genes that Drive HAR1-Mediated Systemic Regulation of Nodulation . Plant and Cell Physiology 50 : 67 – 77 . OpenUrl CrossRef PubMed Web of Science ↵ Okamoto S , Shinohara H , Mori T , Matsubayashi Y , Kawaguchi M . 2013 . Root-derived CLE glycopeptides control nodulation by direct binding to HAR1 receptor kinase . Nat Commun 4 : 2191 . OpenUrl CrossRef PubMed ↵ Oldroyd GE , Engstrom EM , Long SR . 2001 . Ethylene inhibits the Nod factor signal transduction pathway of Medicago truncatula . Plant Cell 13 ( 8 ): 1835 – 1849 . OpenUrl Abstract / FREE Full Text ↵ Oldroyd GED . 2013 . Speak, friend, and enter: signalling systems that promote beneficial symbiotic associations in plants . Nat Rev Microbiol 11 : 252 – 263 . OpenUrl CrossRef PubMed ↵ Offringa I.A , Melchers L.S. , Regensburg-Tuink A.J. , Costantino P. , Schilperoort R.A. , Hooykaas P.J . 1986 . Complementation of Agrobacterium tumefaciens tumor-inducing aux mutants by genes from the T(R)-region of the Ri plasmid of Agrobacterium rhizogenes . Proceedings of the National Academy of Sciences 83 : 6935 – 6939 OpenUrl Abstract / FREE Full Text ↵ Peleg-Grossman S , Volpin H , Levine A . 2007 . Root hair curling and Rhizobium infection in Medicago truncatula are mediated by phosphatidylinositide-regulated endocytosis and reactive oxygen species . J Exp Bot 58 ( 7 ): 1637 – 1649 . OpenUrl CrossRef PubMed Web of Science ↵ Penmetsa RV , Cook DR . 1997 . A Legume Ethylene-Insensitive Mutant Hyperinfected by Its Rhizobial Symbiont . Science 275 ( 5299 ): 527 – 530 . OpenUrl Abstract / FREE Full Text ↵ Qin L , Zhou Z , Li Q , Zhai C , Liu L , Quilichini TD , Gao P , Kessler SA , Jaillais Y , Datla R , et al. 2020 . Specific Recruitment of Phosphoinositide Species to the Plant-Pathogen Interfacial Membrane Underlies Arabidopsis Susceptibility to Fungal Infection . Plant Cell 32 : 1665 – 1688 . OpenUrl Abstract / FREE Full Text ↵ Rae AE , Rolland V , White RG , Mathesius U . 2021 . New methods for confocal imaging of infection threads in crop and model legumes . Plant Methods 17 : 24 . OpenUrl CrossRef PubMed ↵ Rahman MZ , Uematsu S , Coffey MD , Uzuhashi S , Suga H , Kageyama K . 2014 . Re-evaluation of Japanese Phytophthora isolates based on molecular phylogenetic analyses . Mycoscience 55 : 314 – 327 . OpenUrl CrossRef ↵ Rey T , Chatterjee A , Buttay M , Toulotte J , Schornack S . 2015 . Medicago truncatula symbiosis mutants affected in the interaction with a biotrophic root pathogen . New Phytol 206 : 497 – 500 . OpenUrl CrossRef PubMed ↵ Robert G , Munoz N , Alvarado-Affantranger X , Saavedra L , Davidenco V , Rodriguez-Kessler M , Estrada-Navarrete G , Sanchez F , Lascano R . 2018 . Phosphatidylinositol 3-kinase function at very early symbiont perception: a local nodulation control under stress conditions? Journal of Experimental Botany 69 : 2037 – 2048 . OpenUrl CrossRef PubMed ↵ Schaaf G , Ortlund EA , Tyeryar KR , Mousley CJ , Ile KE , Garrett TA , Ren J , Woolls MJ , Raetz CR , Redinbo MR , et al. 2008 . Functional anatomy of phospholipid binding and regulation of phosphoinositide homeostasis by proteins of the sec14 superfamily . Mol Cell 29 : 191 – 206 . OpenUrl CrossRef PubMed Web of Science ↵ Schauser L , Roussis A , Stiller J , Stougaard J . 1999 . A plant regulator controlling development of symbiotic root nodules . Nature 402 : 191 – 195 . OpenUrl CrossRef PubMed Web of Science ↵ Shimada TL , Betsuyaku S , Inada N , Ebine K , Fujimoto M , Uemura T , Takano Y , Fukuda H , Nakano A , Ueda T . 2019 . Enrichment of Phosphatidylinositol 4,5-Bisphosphate in the Extra-Invasive Hyphal Membrane Promotes Colletotrichum Infection of Arabidopsis thaliana . Plant and Cell Physiology 60 : 1514 – 1524 . OpenUrl CrossRef PubMed ↵ Simon ML , Platre MP , Assil S , van Wijk R , Chen WY , Chory J , Dreux M , Munnik T , Jaillais Y. 2014 . A multi-colour/multi-affinity marker set to visualize phosphoinositide dynamics in Arabidopsis . Plant J 77 : 322 – 337 . OpenUrl CrossRef PubMed Web of Science ↵ Singh S , Katzer K , Lambert J , Cerri M , Parniske M . 2014 . CYCLOPS, A DNA-Binding Transcriptional Activator, Orchestrates Symbiotic Root Nodule Development . Cell Host & Microbe 15 : 139 – 152 . OpenUrl CrossRef PubMed Web of Science ↵ Soyano T , Akamatsu A , Takeda N , Watahiki MK , Goh T , Okuma N , Suganuma N , Kojima M , Takebayashi Y , Sakakibara H , et al. 2024 . Periodic cytokinin responses in Lotus japonicus rhizobium infection and nodule development . Science 385 : 288 – 294 . OpenUrl CrossRef PubMed ↵ Soyano T , Hirakawa H , Sato S , Hayashi M , Kawaguchi M . 2014 . Nodule Inception creates a long-distance negative feedback loop involved in homeostatic regulation of nodule organ production . Proceedings of the National Academy of Sciences , USA 111 : 14607 – 14612 . OpenUrl Abstract / FREE Full Text ↵ Stiller J , Martirani L , Tuppale S , Chian RJ , Chiurazzi M , Gresshoff PM . 1997 . High frequency transformation and regeneration of transgenic plants in the model legume Lotus japonicus . Journal of Experimental Botany 48 : 1357 – 1365 . OpenUrl CrossRef Web of Science ↵ Sun J , Miwa H , Downie JA , Oldroyd GED . 2007 . Mastoparan activates calcium spiking analogous to nod factor-induced responses in Medicago truncatula root hair cells Plant Physiology 144 : 695 – 702 . OpenUrl Abstract / FREE Full Text ↵ Suzaki T , Yano K , Ito M , Umehara Y , Suganuma N , Kawaguchi M . 2012 . Positive and negative regulation of cortical cell division during root nodule development in Lotus japonicus is accompanied by auxin response . Development 139 : 3997 – 4006 . OpenUrl Abstract / FREE Full Text ↵ Trapnell C , Williams BA , Pertea G , Mortazavi A , Kwan G , van Baren MJ , Salzberg SL , Wold BJ , Pachter L. 2010 . Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation . Nat Biotechnol 28 ( 5 ): 511 – 515 . OpenUrl CrossRef PubMed Web of Science ↵ Tsikou D , Yan Z , Holt DB , Abel NB , Reid DE , Madsen LH , Bhasin H , Sexauer M , Stougaard J , Markmann K . 2018 . Systemic control of legume susceptibility to rhizobial infection by a mobile microRNA . Science 362 ( 6411 ): 233 – 236 . OpenUrl Abstract / FREE Full Text ↵ Wopereis J , Pajuelo E , Dazzo FB , Jiang Q , Gresshoff PM , De Bruijn FJ , Stougaard J , Szczyglowski K. 2000 . Short root mutant of Lotus japonicus with a dramatically altered symbiotic phenotype . Plant J 23 ( 1 ): 97 – 114 . OpenUrl CrossRef PubMed Web of Science ↵ Yoro E , Suzaki T , Kawaguchi M . 2020 . CLE-HAR1 Systemic Signaling and NIN-Mediated Local Signaling Suppress the Increased Rhizobial Infection in the daphne Mutant of Lotus japonicus . Molecular Plant-Microbe Interactions 33 : 320 – 327 . OpenUrl CrossRef PubMed ↵ Yoro E , Suzaki T , Toyokura K , Miyazawa H , Fukaki H , Kawaguchi M . 2014 . A Positive Regulator of Nodule Organogenesis, NODULE INCEPTION, Acts as a Negative Regulator of Rhizobial Infection in Lotus japonicus . Plant Physiology 165 : 747 – 758 . OpenUrl Abstract / FREE Full Text ↵ Zarreen F , Kumar K , Chakraborty S . 2023 . Phosphoinositides in plant-pathogen interaction: trends and perspectives . Stress Biol : 4 . ↵ Zhang G , Ahmad MZ , Chen B , Manan S , Zhang Y , Jin H , Wang X , Zhao J . 2020 . Lipidomic and transcriptomic profiling of developing nodules reveals the essential roles of active glycolysis and fatty acid and membrane lipid biosynthesis in soybean nodulation . Plant J 103 ( 4 ): 1351 – 1371 . OpenUrl CrossRef PubMed View the discussion thread. Back to top Previous Next Posted June 30, 2025. Download PDF Supplementary Material Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. You are going to email the following Rhizobial infection-specific accumulation of phosphatidylinositol 4,5-bisphosphate inhibits the excessive infection of rhizobia in Lotus japonicus Message Subject (Your Name) has forwarded a page to you from bioRxiv Message Body (Your Name) thought you would like to see this page from the bioRxiv website. Your Personal Message CAPTCHA This question is for testing whether or not you are a human visitor and to prevent automated spam submissions. Share Rhizobial infection-specific accumulation of phosphatidylinositol 4,5-bisphosphate inhibits the excessive infection of rhizobia in Lotus japonicus Akira Akamatsu , Toshiki Ishikawa , Hiroto Tanaka , Yoji Kawano , Makoto Hayashi , Naoya Takeda bioRxiv 2025.06.26.661640; doi: https://doi.org/10.1101/2025.06.26.661640 Share This Article: Copy Citation Tools Rhizobial infection-specific accumulation of phosphatidylinositol 4,5-bisphosphate inhibits the excessive infection of rhizobia in Lotus japonicus Akira Akamatsu , Toshiki Ishikawa , Hiroto Tanaka , Yoji Kawano , Makoto Hayashi , Naoya Takeda bioRxiv 2025.06.26.661640; doi: https://doi.org/10.1101/2025.06.26.661640 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Plant Biology Subject Areas All Articles Animal Behavior and Cognition (7618) Biochemistry (17635) Bioengineering (13859) Bioinformatics (41846) Biophysics (21401) Cancer Biology (18534) Cell Biology (25422) Clinical Trials (138) Developmental Biology (13352) Ecology (19860) Epidemiology (2067) Evolutionary Biology (24285) Genetics (15582) Genomics (22463) Immunology (17700) Microbiology (40298) Molecular Biology (17141) Neuroscience (88424) Paleontology (666) Pathology (2825) Pharmacology and Toxicology (4813) Physiology (7633) Plant Biology (15107) Scientific Communication and Education (2042) Synthetic Biology (4284) Systems Biology (9808) Zoology (2267)