The gain-of-function mutation of the rice auxin-signaling repressor, OsIAA13, induces rice bacterial blight resistance through activating jasmonic acid-mediating defense system

The gain-of-function mutation of the rice auxin-signaling repressor, OsIAA13, induces rice bacterial blight resistance through activating jasmonic acid-mediating defense system | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (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],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article The gain-of-function mutation of the rice auxin-signaling repressor, OsIAA13, induces rice bacterial blight resistance through activating jasmonic acid-mediating defense system Go Suzuki, Aina Murakami, Yutaro Moriyasu, Manatsu Fukuda, Yuya Uji, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5800788/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 12 Apr, 2025 Read the published version in Journal of General Plant Pathology → Version 1 posted 4 You are reading this latest preprint version Abstract Jasmonic acid (JA) is involved in the regulation of rice defense responses against Xanthomonas oryzae pv. oryzae ( Xoo ). JA also affects other plant hormone signaling to maximize the JA-induced defense responses in rice. In this study, we investigated the JA- and auxin-mediating defense system using a gain-of-function mutant of OsIAA13, a suppressor of auxin signaling, in rice. The expression of some auxin-responsive expansin s was downregulated in the Osiaa13 mutants. The Osiaa13 mutants showed a JA-hypersensitive phenotype. The expression of some JA-responsive defense-related genes such as the lignin biosynthesis gene, OsPrx38 , was upregulated in the Osiaa13 mutants. Lignin content was higher in the Osiaa13 mutants than in the wild type plants. The expression of OsPrx38 was downregulated after IAA treatment. Furthermore, some antibacterial compounds against Xoo accumulated in the Osiaa13 mutants. These results suggest that OsIAA13 has an important role in disease resistance against Xoo by regulating JA- and auxin-mediating defense system in rice. Auxin jasmonic acid lignin volatile Xanthomonas oryzae pv. oryzae Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Rice ( Oryza sativa L.) is one of the most important food crops worldwide and is a model plant for molecular studies of other monocotyledonous species. Rice bacterial blight caused by hemibiotrophic Xanthomonas oryzae pv. oryzae ( Xoo ) is one of the serious diseases in rice-growing countries where the population is increasing. Xoo is a vascular pathogen, where Xoo -infected xylem vessels in rice leaves are filled by a Xoo -producing extracellular polysaccharide, which is an important virulent factor (Kumar et al. 2020). To protect against the infection and spread of Xoo in leaves, rice acquires multiple defense systems regulated by plant hormones. A plant hormone, jasmonic acid (JA), has been reported to play an important role in resistance against Xoo (Zhong et al. 2024). JA induces production of antibacterial compounds that suppress Xoo growth (Kiryu et al. 2018; Kiyama et al. 2021; Tanaka et al. 2014; Yoshitomi et al. 2016). Treatment with Xoo -secreting cell wall-degrading enzymes and melatonin activates JA signaling and induces resistance against Xoo in rice (Chen et al. 2020; Ranjan et al. 2015). Some JA-responsive transcription factors (TFs) such as OsMYC2, OsWRKY45, and OsbHLH034 upregulate expression of many defense-related genes, which are involved in resistance against Xoo (Hui et al. 2019; Onohata and Gomi 2020; Uji et al. 2016; Wang et al. 2021). Transgenic rice plants overexpressing rice NOVEL INTERACTOR OF JAZ1 ( OsNINJA1 ), which acts as a repressor of OsMYC2, are more susceptible to Xoo (Kashihara et al. 2019). A JA-responsive TF, RICE EARLY RESPONSIVE TO JASMONATE1 (RERJ1), positively regulates production of a monoterpene, linalool, which acts as a signaling molecule to induce resistance against Xoo in rice (Taniguchi et al. 2014; Valea et al. 2022). Conversely, JA biosynthesis and its signaling are suppressed in rice after inoculation with virulent Xoo (An et al. 2022; Hou et al. 2019, Tariq et al. 2019). Xoo initiates the activity of OsWRKY72, which directly represses the expression of the JA biosynthesis gene, allene oxide synthase1 ( OsAOS1 ), to increase susceptibility of Xoo in rice (Hou et al. 2019). XopM, an effector secreted into rice cells from Xoo , directly interacts with OsAOS3 to repress JA biosynthesis (Li et al. 2024). JA has been reported to affect other plant hormone signaling involved in the rice defense response against Xoo (Zhong et al. 2024). It has been demonstrated that JA interacts antagonistically with abscisic acid (ABA) in rice (Nahar et al. 2012; Taniguchi et al. 2023), and ABA negatively affects on Xoo resistance in rice (Xu et al. 2013). A NAC-type TF, ONAC066, positively regulates Xoo resistance by suppressing ABA signaling in rice (Liu et al. 2018b). Treatment with ABA suppresses production of JA and downregulates the expression of the JA-responsive genes in rice (Nahar et al. 2012; Xie et al. 2018). Conversely, treatment with JA also suppresses production of ABA and downregulates the expression of the ABA-responsive genes in rice (Taniguchi et al. 2023), suggesting that JA induces Xoo resistance by repressing ABA signaling in rice. Auxin negatively affects Xoo resistance in rice (Zhong et al. 2024). Treatment with indole-3-acetic acid (IAA) increases susceptibility against Xoo in rice (Ding et al. 2008; Suzuki et al. 2022), and Xoo itself secretes IAA to promote the infection process (Fu et al. 2011). Reduction of auxin levels in rice results in increased resistance against Xoo (Li et al. 2015; Li et al. 2016). After inoculation with Xoo , the expression of auxin-responsive expansins , which loosen the cell wall, is upregulated and downregulated in the susceptible and resistant rice plants, respectively (Ding et al. 2008). The overexpression of the auxin-responsive expansins results in increased susceptibility against Xoo in rice (Ding et al. 2008; Uji et al. 2024). Auxin signaling is strictly controlled by AUX/IAA proteins, which act as repressors by interacting with TFs named auxin response factors (ARFs) (Cancé et al. 2022). Degradation of AUX/IAA proteins triggers the activation of ARFs to regulate gene expressions involved in auxin signaling (Cancé et al. 2022). Recently, JA has also been reported to repress auxin signaling in Xoo resistance in rice (Uji et al. 2024). Rice plants overexpressing a JA-responsive MYB-type TF ( JMTF1 ) show similar phenotypes to auxin-insensitive mutants of rice (Uji et al. 2024). Furthermore, JMTF1 negatively affects auxin signaling by upregulating expression of an AUX/IAA gene, OsIAA13 , in Xoo resistance in rice (Uji et al. 2024). The rice gain-of-function mutant, Osiaa13 , shows increased resistance against Xoo (Kitomi et al. 2012; Uji et al. 2024). These results suggest that JA and auxin signaling antagonistically interact through OsIAA13 in Xoo resistance in rice. However, there is no information about JA signaling affected by OsIAA13 in rice resistance against Xoo . In the present study, we investigated OsIAA13-mediating auxin/JA signaling, which is involved in rice resistance against Xoo , using the Osiaa13 mutant. Materials and Methods Plant and bacterial materials The growth conditions for rice plants ( Oryza sativa L.) and Xoo (strain 7174) were as previously described by Kashihara et al. (2022). The Osiaa13 mutant and its background wild type (cv. Taichung 65; T65) were as previously described by Kitomi et al. (2012). Rice plants were grown from seeds under greenhouse conditions (25 ± 1°C, 60–80% relative humidity). The Xoo strain T7174 was cultured on a nutrient agar slant (Becton, Dickinson and Co., Sparks, MD, USA) containing 0.5% sucrose at 25°C for 48 h. Hormone treatments To examine the effects of plant hormones, rice plants were grown to the four-leaf stage in a growth chamber in Kimura-B liquid medium (Sato et al. 1996) at 25°C with 24 h light. The plants were then incubated in the same medium supplemented with 100 µM JA and 10 µM IAA (Sigma-Aldrich, St. Louis, MO, USA) following the method of JA treatment as previously described by Yamada et al. (2012). Reverse transcription-quantitative PCR (RT-qPCR) Total RNA was extracted from the rice leaf blades after each treatment using TRIzol Regent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Four leaf blades were used per replicate. Fourth-leaf blades were used for the RT-qPCR analysis. RT-qPCR was performed using TB Green Premix Ex Taq Mixture (Takara) in a Thermal Cycler Dice TP800 System (Takara) according to the manufacturer’s instructions. The transcript levels of each gene were normalized to those of actin ( Os11g06390 ) and compared with those of the respective mock-treated control or the WT to calculate the fold change in expression levels as previously described by Gomi et al. (2010). Each data point contains four biological replicates. The sequences of the gene-specific primers used in RT-qPCR are shown in Table S1 . Measurement of the chlorophyll content Four leaf blades were used per replicate. Leaf blades (0.1 g fresh weight) treated with 100 µM JA for 4 days were homogenized in 1 mL of 80% acetone and centrifuged at 3,000 g for 10 min. The specific chlorophyll content was determined as previously described by Yamada et al. (2012). Each data point contains four biological replicates. Subcellular localization assay To construct OsPrx38 - DsRed , the ORF of OsPrx38 ( Os03g13210 ) without the stop codon was amplified by PCR and subcloned into the corresponding sites of the pE7133-DsRed vector (Onohata and Gomi 2020); this fused DsRed in-frame to the C-terminus of OsPrx38. The vectors were expressed in onion epidermal cells using a particle bombardment system (PDS-1000/He; BioRad, Hercules, USA) as previously described by Kim et al. (2009). We used a KEYENCE BIOREVO BZ-9000 microscope (KEYENCE, Osaka, Japan) and a BZ filter TRITC (excitation wavelength: 540/25 nm; absorption wavelength: 605/55 nm; dichroic mirror wavelength: 565 nm) to observe DsRed fluorescence. The onion epidermal cells were plasmolyzed by immersion in a 0.5 M mannitol solution for 30 min. Measurement of the lignin content Four leaf blades were used per replicate. Fully opened fourth-leaf blades were used. Lignin content was determined by the method of Suzuki et al. (2009). Measurement of the volatiles Three leaf blades were used per replicate. Fully-opened fourth-leaf blades were ground to a powder in liquid nitrogen and then transferred to a 15 mL glass vial containing 1 mL of 1% NaCl and immediately weighed. The headspace of the vial was collected in a water bath at 50°C using a SPME fiber for GC-MS. GC-MS analysis was performed using a GCMS-QP2010SE (Shimadzu, Kyoto, Japan) fitted with a DB-WAX column (60 m × 0.25 mm, 0.25 µm film thickness; J and W Scientific, Folsom, CA, USA) under the same conditions as previously described by Kiryu et al. (2018). The compounds were identified by comparing their mass spectra to those in a database (Wiley10). Each peak area of volatiles in the leaf blades was calculated based on the total ion current (TIC) chromatograms. Analysis of the antibacterial activity of the volatile compounds Xoo was cultured in YT broth (0.5% Bacto-yeast extract, 1% Bacto-tryptone, pH 6.8; Becton Dickinson and Co.) containing volatile compounds at 100 µM for 6 h. The same amount of DMSO was used as a control; 100 µM ( E )-2-hexenal and methyl salicylate (MeSA) (Wako, Osaka, Japan) were used as known positive and negative controls, respectively (Tanaka et al. 2014). The media were incubated in a shaker at 28°C, 200 rpm, and changes in OD 600 were measured after 6 h. Results Analysis of the auxin-responsive genes in the Osiaa13 mutants We first investigated the expression levels of ARF s in the Osiaa13 mutants. The rice genome contains at least 25 OsARFs (Shen et al. 2010), but OsARF6, OsARF11, OsARF16, OsARF17, OsARF19, OsARF21, and OsARF25 were selected because they are considered to be transcriptional activators of auxin signaling in rice (Shen et al. 2010). Among the selected OsARFs, the expressions of OsARF6 and OsARF16 were significantly downregulated in the Osiaa13 mutants (Fig. 1 a). We next investigated the expression levels of selected auxin-responsive expansin s ( OsEXP s), which are involved in the auxin-mediated suppression of resistance against Xoo in rice (Ding et al. 2008; Uji et al. 2024), in the Osiaa13 mutants. The expressions of OsEXPA4 , OsEXPA8 , and OsEXPB3 were significantly downregulated in the Osiaa13 mutants (Fig. 1 b). Analysis of the JA-related phenotype in the Osiaa13 mutants JA upregulates expression of OsIAA13 to repress auxin signaling in rice (Uji et al. 2024), suggesting that JA sensitivity can be altered in the Osiaa13 mutant. Thus, chlorophyll (Chl) content was measured after JA treatment because JA is known to promote the reduction of Chl content in rice (Yamada et al. 2012). The levels of Chl significantly decreased in the Osiaa13 mutants as early as two days after treatment with JA, unlike in the WT plants (Fig. 2 ). We next investigated the expression levels of thirteen JA-responsive defense-related genes in the Osiaa13 mutants. The expression of five genes [ peroxidase ( OsPrx38 ) ( Os03g13210 ), Bowman–Birk trypsin inhibitor ( Os01g03320 ), beta 1,3-glucanase ( Os01g71340 ), beta glucanase ( Os01g51570 ), and proteinase inhibitor I25 ( Os08g10570 )] were significantly upregulated in the Osiaa13 mutants (Fig. 3 a). OsPrx38 has been reported to be an extracellular protein and is involved in lignin biosynthesis in plants (Kidwai et al. 2019). We also confirmed the extracellular localization of OsPrx38 in plant cells (Fig. 3 b). Furthermore, the content of lignin was found to have significantly increased in the Osiaa13 mutants relative to the WT plants (Fig. 3 c). The expression levels of the five OsIAA13-responsive JA-related genes in response to auxin were investigated. The expression of only OsPrx38 was significantly downregulated after 10 µM IAA treatment for 24 h (Fig. 3 d). Analysis of the volatiles in the Osiaa13 mutants We next measured the volatile compounds in the Osiaa13 mutants because JA positively regulates production of antibacterial volatile compounds against Xoo (Tanaka et al. 2014). As a result, six volatile compounds [( E , E )-2,4-heptadienal, ( E )-2-hexenal, hexanal, ( Z )-3-hexenal, 5,9-undecadien-2-one, and ( E )-4-oxohex-2-enal] were highly accumulated in the Osiaa13 mutants relative to the WT plants (Fig. 4 a). Both ( E )-2-hexenal and ( E , E )-2,4-heptadienal have been shown to exhibit antibacterial activity against Xoo (Gomi et al. 2010; Tanaka et al. 2014). Among other unidentified volatiles, we selected a commercially available compound, hexanal, and analyzed its antibacterial activity against Xoo . ( E )-2-Hexenal and MeSA were used as positive and negative controls, respectively (Tanaka et al. 2014). When hexanal was added to liquid cultures of Xoo , a significant negative effect was not observed on the growth of Xoo (Fig. 4 b). Discussion OsIAA13 has been reported to play a critical role in the development of roots in rice (Kitomi et al. 2012; Yamauchi et al. 2019; Yamauchi et al. 2024). However, there is no information about the role of OsIAA13 in the shoots of rice. In this study, we first revealed that the dominant mutation of OsIAA13 affects both JA and auxin signaling in rice leaf blades. The expression of three auxin-responsive expansin s, OsEXPA4 , OsEXPA8 , and OsEXPB3 , was downregulated in the Osiaa13 mutants. Expansins are divided into four subfamilies [α-expansin (EXPA), β-expansin (EXPB), expansin-like A (EXPLA), and expansin-like B (EXPLB)] based on their phylogenetic relationship (Kende et al. 2004). They are secretory proteins and their main function is to loosen the cell wall for regulating plant development (Marowa et al. 2016). Some expansins have been reported to play an important role in the auxin-mediated suppression of resistance against Xoo in rice (Ding et al. 2008; Uji et al. 2024). The expression of auxin-responsive expansin s after inoculation with Xoo is upregulated and downregulated in susceptible and resistant rice plants, respectively (Ding et al. 2008). Xoo -derived auxin has been suggested to upregulate the expression of expansin s, thereby facilitating pathogen entry and allowing increased nutrient leakage (De Vleesschauwer et al. 2013). The OsEXPA1 -, OsEXPA4 -, OsEXPA5 -, and OsEXPA10 -overexpressing rice transgenic plants exhibit increased susceptibility against Xoo (Ding et al. 2008; Uji et al. 2024). Furthermore, the expression of OsEXPA4 has recently been reported to be downregulated by JA treatment (Uji et al. 2024), suggesting that regulation of OsEXPA4 by JA/auxin has an important role in disease resistance against Xoo in rice. The expressions of OsARF6 and OsARF16 were significantly downregulated in the Osiaa13 mutants, suggesting that these ARFs are regulated by OsIAA13 and involved in regulating the expression of OsEXPA4 , OsEXPA8 , and OsEXPB3 . Further analysis of the direct binding activity of OsARF6/OsARF16 to the promoter of these expansin genes is needed to reveal the role of the OsARF6/OsARF16-regulated defense response against Xoo in rice. The Osiaa13 mutants exhibited a hypersensitive phenotype to JA. Furthermore, some JA-responsive defense-related genes such as proteinase inhibitors , glucanases , and a peroxidase , OsPrx38 , were constitutively upregulated in the Osiaa13 mutants. Transgenic rice plants overexpressing the proteinase inhibitor BBTI4 have been reported to exhibit increased resistance to Xoo (Pang et al. 2013), suggesting that constitutive upregulation of some proteinase inhibitors in Osiaa13 mutants may result in increased resistance to Xoo . Upregulation of glucanase has been reported in Xoo -resistant transgenic rice plants, although the function of the glucanase in the defense response was not clear (Gupta et al. 2022). Further study is needed to reveal the role of glucanase in disease resistance against Xoo in the Osiaa13 mutant. The expression of OsPrx38 was significantly upregulated in the Osiaa13 mutants. OsPrx38 is a secretory-type class III peroxidase involved in the biosynthesis of lignin (Liu et al. 2018a). The overexpression of OsPrx38 in Arabidopsis results in increased lignin content (Kidwai et al. 2019). OsbHLH034 regulates the expression of OsPrx38 and the lignin content significantly increases in the OsbHLH034 -overexpressing rice plants compared with that in the WT (Onohata and Gomi 2020). Consistent with these findings, the lignin content in the Osiaa13 mutants also increased. Lignin is known to have an important role in Xoo resistance (Bart et al. 2010; Hilaire et al. 2001; Kashihara et al. 2020; Song et al. 2016; Suzuki et al. 2022; Uji et al. 2024). Xoo is a vascular pathogen and stays within the xylem vessels throughout the disease interaction (Tabei 1967). There is contact between Xoo and living cells, such as xylem parenchyma cells, through the pit membranes that separate the xylem lumen from the xylem parenchyma cells. Inoculation with an avirulent Xoo strain triggers the thickening of xylem secondary walls and reduces the pit diameter, resulting in the reduction of Xoo access to the xylem parenchyma cells (Hilaire et al. 2001). Lignin is an essential component of the plant response leading to the thickening of the xylem secondary walls. OsPrx38 has been reported to be secreted into the xylem vessels in rice (Aki et al. 2008), suggesting that OsPrx38 plays an important role in lignin biosynthesis for xylem secondary-wall thickening. These results suggest that the enhanced Xoo resistance in the Osiaa13 mutants was caused by an increased physical barrier resulting from OsPrx38-mediated accumulation of lignin. Furthermore, the expression of OsPrx38 was revealed to be downregulated by IAA, suggesting that auxin represses the expression of OsPrx38 . The role of Xoo -derived auxin may be to suppress the production of lignin at the xylem secondary-wall, thereby facilitating pathogen penetration on the pit. Some volatile compounds accumulated in the Osiaa13 mutants. Among them, ( E , E )-2,4-heptadienal is induced by JA (Taniguchi et al. 2014). Both ( E , E )-2,4-heptadienal and ( E )-2-hexenal are known to exhibit antibacterial activity against Xoo (Gomi et al. 2010; Tanaka et al. 2014). ( E )-4-Oxohex-2-enal is known to act as a non-specific toxin for many organisms because it reacts with DNA bases to form adducts, such as deoxyguanosine and deoxycytidine (Kasai et al. 2005; Kawai et al. 2010; Noge et al. 2012). Hexanal, ( Z )-3-hexenal, and ( E )-2-hexenal are C 6 volatile compounds derived from lipoxygenase-hydroperoxide lyase (LOX-HPL) pathway (Noordermeer et al. 2001). Furthermore, JA and the C 7 volatile compound ( E , E )-2,4-heptadienal are derived from the LOX pathway (Bhowmik et al. 2023; Howe and Schilmiller 2002). These results suggest that the LOX pathway involved in the production of these compounds may be constitutively activating in the Osiaa13 mutants, and OsIAA13-dependent auxin signaling may negatively regulate the LOX pathway in rice. In conclusion, we suggest that OsIAA13 has an important role in disease resistance against Xoo by regulating JA- and auxin-mediating defense system in rice. Further study is needed to reveal the mechanism of that interaction between JA and auxin in the disease resistance against Xoo in rice. Declarations Compliance with Ethical Standards This article does not contain any studies with human participants or animals performed by any of the authors. Conflicts of interest The authors declare that they have no conflict of interest. Funding This work was supported in part by a Funding Program for Next Generation World-Leading Researchers from the Japan Society for Promotion of Science (No. GS022) and JSPS KAKENHI (No.15K07313). Author Contributions KG and IY designed the research project. GS, AM, YM, MF, YU, TO, and YF performed the experiments. GS and KG wrote the manuscript. All the authors reviewed and approved the manuscript. 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Plant Cell Environ 37:451–461. https://doi.org/10.1111/pce.12169 Taniguchi S, Takeda A, Kiryu M, Gomi K (2023) Jasmonic acid-induced β-cyclocitral confers resistance to bacterial blight and negatively affects abscisic acid biosynthesis in rice. Int J Mol Sci 24:1704. https://doi.org/10.3390/ijms24021704 Tariq R, Ji Z, Wang C, Tang Y, Zou L, Sun H, Chen G, Zhao K (2019) RNA-Seq analysis of gene expression changes triggered by Xanthomonas oryzae pv. oryzae in a susceptible rice genotype. Rice 12:44. https://doi.org/10.1186/s12284-019-0301-2 Uji Y, Suzuki G, Fujii Y, Kashihara K, Yamada S, Gomi K (2024) Jasmonic acid (JA)-mediating MYB transcription factor1, JMTF1, coordinates the balance between JA and auxin signaling in the rice defense response. Physiologia Plantarum 176: e14257. https://doi.org/10.1111/ppl.14257 Uji Y, Taniguchi S, Tamaoki D, Shishido H, Akimitsu K, Gomi K (2016) Overexpression of OsMYC2 results in the up-regulation of early JA-responsive genes and bacterial blight resistance in rice. 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Front Plant Sci 12:802758. https://doi.org/10.3389/fpls.2021.802758 Xie K, Li L, Zhang H, Wang R, Tan X, He Y, Hong G, Li J, Ming F, Yao X, Yan F, Sun Z, Chen J (2018) Abscisic acid negatively modulates plant defence against rice black-streaked dwarf virus infection by suppressing the jasmonate pathway and regulating reactive oxygen species levels in rice.Plant Cell Environ 41:2504-2514. https://doi.org/10.1111/pce.13372 Xu J, Audenaert K, Höfte M, De Vlesschauwer D (2013) Abscisic Acid Promotes Susceptibility to the Rice Leaf Blight Pathogen Xanthomonas oryzae pv oryzae by Suppressing Salicylic Acid-Mediated Defenses. PLoS One 8:e67413. https://doi.org/10.1371/journal.pone.0067413 Yamada S, Kano A, Tamaoki D, Miyamoto A, Shishido H, Miyoshi S, Taniguchi S, Akimitsu K, Gomi K (2012) Involvement of OsJAZ8 in jasmonate-induced resistance to bacterial blight in rice. Plant Cell Physiol 53:2060–2072. https://doi.org/10.1093/pcp/pcs145 Yamauchi T, Tanaka A, Inahashi H, Nishizawa NK, Tsutsumi N, Inukai Y, Nakazono M. (2019) Fine control of aerenchyma and lateral root development through AUX/IAA- and ARF-dependent auxin signaling. Proc Natl Acad Sci USA 116:20770-20775. https://doi.org/10.1073/pnas.1907181116 Yamauchi T, Tanaka A, Nakazono M, Inukai Y (2024) Age-dependent analysis dissects the stepwise control of auxin-mediated lateral root development in rice. Plant Physiol194:819-831. https://doi.org/10.1093/plphys/kiad548 Yoshitomi K, Taniguchi S, Tanaka K, Uji Y, Akimitsu K, Gomi K (2016) Rice terpene synthase 24 ( OsTPS24 ) encodes a jasmonate-responsive monoterpene synthase that produces an antibacterial γ-terpinene against rice pathogen. J Plant Physiol 191:120–126. https://doi.org/10.1016/j.jplph.2015.12.008 Zhong Q, Xu Y, Rao Y (2024) Mechanism of rice resistance to bacterial leaf blight via phytohormones. Plants 13:2541. https://doi.org/10.3390/plants13182541 Supplementary Files SupplementaryTable1Primersequences.xlsx Cite Share Download PDF Status: Published Journal Publication published 12 Apr, 2025 Read the published version in Journal of General Plant Pathology → Version 1 posted Reviewers agreed at journal 16 Jan, 2025 Reviewers invited by journal 14 Jan, 2025 Editor assigned by journal 10 Jan, 2025 First submitted to journal 09 Jan, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5800788","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":401941443,"identity":"4a5daa5a-b3aa-4171-81f2-e13d1b8f110b","order_by":0,"name":"Go Suzuki","email":"","orcid":"","institution":"Kagawa University Faculty of Agriculture Graduate School of Agriculture: Kagawa Daigaku Nogakubu Daigakuin Nogaku Kenkyuka","correspondingAuthor":false,"prefix":"","firstName":"Go","middleName":"","lastName":"Suzuki","suffix":""},{"id":401941444,"identity":"e29e6193-d36a-48c9-bcc6-91db86f6aa27","order_by":1,"name":"Aina 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05:47:21","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5800788/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5800788/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10327-025-01227-2","type":"published","date":"2025-04-12T16:05:08+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":73929569,"identity":"7da24f13-f833-4d95-b616-84e7f1d3f1fb","added_by":"auto","created_at":"2025-01-16 05:42:05","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":22115,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnalysis of auxin-responsive genes in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eOsiaa13\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e mutant.\u003c/strong\u003e (a) The expression of \u003cem\u003eOsARF \u003c/em\u003egenes in the \u003cem\u003eOsiaa13\u003c/em\u003emutant. Values are means ± SE (n = 3). (b) The expression of \u003cem\u003eexpansin\u003c/em\u003es\u003cem\u003e \u003c/em\u003ein the \u003cem\u003eOsiaa13\u003c/em\u003e mutant. Values are means ± SE (n = 4). (a, b) Data were analyzed using Student’s \u003cem\u003et\u003c/em\u003e-test. Asterisks indicate significant differences from WT plants at \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5800788/v1/28e8e7319275a1348cac2e74.png"},{"id":73930782,"identity":"b9f0d291-ec5c-41e5-81c9-e2a1bccd9aad","added_by":"auto","created_at":"2025-01-16 06:06:05","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":17520,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eOsiaa13\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e mutant exhibits a hypersensitive phenotype to JA. \u003c/strong\u003eTotal chlorophyll content in the leaf blades of WT and \u003cem\u003eOsiaa13 \u003c/em\u003emutants after treatment for 2 days with 100 μM JA. Values are means ± SE (n = 3 for WT; n = 4 for \u003cem\u003eOsiaa13 \u003c/em\u003emutants). Data were analyzed using the Tukey-Kramer test. Different letters indicate significant differences at \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5800788/v1/ce75ef8e89732df6c6f0eb90.png"},{"id":73929571,"identity":"723faec9-5ad2-4740-9ec5-10f0679cc9af","added_by":"auto","created_at":"2025-01-16 05:42:05","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":66817,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eOsiaa13\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e mutant accumulates lignin.\u003c/strong\u003e (a) The expression of JA-responsive defense-related genes\u003cem\u003e \u003c/em\u003ein \u003cem\u003eOsiaa13\u003c/em\u003e mutant. Values are means ± SE (n = 3). (b) Subcellular localization of OsPrx38. OsPrx38-DsRed vector was transferred into onion epidermal cells. DsRed, fluorescence images of DsRed protein; Bright, light-microscopy images. Scale bars = 100 μm. Circles show significant plasmolyzed region after treatment with 0.5 M mannitol for 30 min. (c) Lignin content in the leaf blades of WT plants and \u003cem\u003eOsiaa13\u003c/em\u003e mutants. Values are means ± SE (n = 4). (d) Expression of selected JA-responsive defense-related genes after treatment with 10 μM IAA for 24 h. Values are means ± SE (n = 4). (a, c, and d) Data were analyzed using Student’s \u003cem\u003et\u003c/em\u003e-test. Asterisks indicate significant differences from WT plants at \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5800788/v1/e83a853af60884fc4d1032a2.png"},{"id":73929574,"identity":"18319779-69ce-4dae-8b36-87c7cd5112da","added_by":"auto","created_at":"2025-01-16 05:42:05","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":28476,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eOsiaa13\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e mutant accumulates antibacterial compounds.\u003c/strong\u003e (a) Accumulated volatile compounds in the \u003cem\u003eOsiaa13\u003c/em\u003e mutants. Values are means ± SE (n = 6). Three leaf blades were used for each replicate. n.d., not detected. (b) Effect of 100 µM hexanal on growth of \u003cem\u003eXoo\u003c/em\u003e measured as change in OD\u003csub\u003e600\u003c/sub\u003e after 6 h. Values are means ± SE\u003cstrong\u003e \u003c/strong\u003eof 4 independent experiments. (\u003cem\u003eE\u003c/em\u003e)-2-Hexanal and MeSA were used as positive and negative controls, respectively (Tanaka et al. 2014). (a, b) Data were analyzed using Student’s \u003cem\u003et\u003c/em\u003e-test. Asterisks indicate significant differences from WT or mock-treated plants at \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-5800788/v1/c7b04e579027f31454a27c91.png"},{"id":80559064,"identity":"0ac8d5ea-dc53-43e0-8ea3-cde31f0fe2fc","added_by":"auto","created_at":"2025-04-14 16:17:37","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":978997,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5800788/v1/df9e7737-d26e-4188-b7fc-2242ee83cb15.pdf"},{"id":73930474,"identity":"5664cc69-9a4f-4ac1-a808-63efea467fa6","added_by":"auto","created_at":"2025-01-16 05:58:05","extension":"xlsx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":12978,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable1Primersequences.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5800788/v1/43f139a298417903476a2dbd.xlsx"}],"financialInterests":"","formattedTitle":"The gain-of-function mutation of the rice auxin-signaling repressor, OsIAA13, induces rice bacterial blight resistance through activating jasmonic acid-mediating defense system","fulltext":[{"header":"Introduction","content":"\u003cp\u003eRice (\u003cem\u003eOryza sativa\u003c/em\u003e L.) is one of the most important food crops worldwide and is a model plant for molecular studies of other monocotyledonous species. Rice bacterial blight caused by hemibiotrophic \u003cem\u003eXanthomonas oryzae\u003c/em\u003e pv. \u003cem\u003eoryzae\u003c/em\u003e (\u003cem\u003eXoo\u003c/em\u003e) is one of the serious diseases in rice-growing countries where the population is increasing. \u003cem\u003eXoo\u003c/em\u003e is a vascular pathogen, where \u003cem\u003eXoo\u003c/em\u003e-infected xylem vessels in rice leaves are filled by a \u003cem\u003eXoo\u003c/em\u003e-producing extracellular polysaccharide, which is an important virulent factor (Kumar et al. 2020). To protect against the infection and spread of \u003cem\u003eXoo\u003c/em\u003e in leaves, rice acquires multiple defense systems regulated by plant hormones.\u003c/p\u003e \u003cp\u003eA plant hormone, jasmonic acid (JA), has been reported to play an important role in resistance against \u003cem\u003eXoo\u003c/em\u003e (Zhong et al. 2024). JA induces production of antibacterial compounds that suppress \u003cem\u003eXoo\u003c/em\u003e growth (Kiryu et al. 2018; Kiyama et al. 2021; Tanaka et al. 2014; Yoshitomi et al. 2016). Treatment with \u003cem\u003eXoo\u003c/em\u003e-secreting cell wall-degrading enzymes and melatonin activates JA signaling and induces resistance against \u003cem\u003eXoo\u003c/em\u003e in rice (Chen et al. 2020; Ranjan et al. 2015). Some JA-responsive transcription factors (TFs) such as OsMYC2, OsWRKY45, and OsbHLH034 upregulate expression of many defense-related genes, which are involved in resistance against \u003cem\u003eXoo\u003c/em\u003e (Hui et al. 2019; Onohata and Gomi 2020; Uji et al. 2016; Wang et al. 2021). Transgenic rice plants overexpressing rice \u003cem\u003eNOVEL INTERACTOR OF JAZ1\u003c/em\u003e (\u003cem\u003eOsNINJA1\u003c/em\u003e), which acts as a repressor of OsMYC2, are more susceptible to \u003cem\u003eXoo\u003c/em\u003e (Kashihara et al. 2019). A JA-responsive TF, RICE EARLY RESPONSIVE TO JASMONATE1 (RERJ1), positively regulates production of a monoterpene, linalool, which acts as a signaling molecule to induce resistance against \u003cem\u003eXoo\u003c/em\u003e in rice (Taniguchi et al. 2014; Valea et al. 2022). Conversely, JA biosynthesis and its signaling are suppressed in rice after inoculation with virulent \u003cem\u003eXoo\u003c/em\u003e (An et al. 2022; Hou et al. 2019, Tariq et al. 2019). \u003cem\u003eXoo\u003c/em\u003e initiates the activity of OsWRKY72, which directly represses the expression of the JA biosynthesis gene, \u003cem\u003eallene oxide synthase1\u003c/em\u003e (\u003cem\u003eOsAOS1\u003c/em\u003e), to increase susceptibility of \u003cem\u003eXoo\u003c/em\u003e in rice (Hou et al. 2019). XopM, an effector secreted into rice cells from \u003cem\u003eXoo\u003c/em\u003e, directly interacts with OsAOS3 to repress JA biosynthesis (Li et al. 2024).\u003c/p\u003e \u003cp\u003eJA has been reported to affect other plant hormone signaling involved in the rice defense response against \u003cem\u003eXoo\u003c/em\u003e (Zhong et al. 2024). It has been demonstrated that JA interacts antagonistically with abscisic acid (ABA) in rice (Nahar et al. 2012; Taniguchi et al. 2023), and ABA negatively affects on \u003cem\u003eXoo\u003c/em\u003e resistance in rice (Xu et al. 2013). A NAC-type TF, ONAC066, positively regulates \u003cem\u003eXoo\u003c/em\u003e resistance by suppressing ABA signaling in rice (Liu et al. 2018b). Treatment with ABA suppresses production of JA and downregulates the expression of the JA-responsive genes in rice (Nahar et al. 2012; Xie et al. 2018). Conversely, treatment with JA also suppresses production of ABA and downregulates the expression of the ABA-responsive genes in rice (Taniguchi et al. 2023), suggesting that JA induces \u003cem\u003eXoo\u003c/em\u003e resistance by repressing ABA signaling in rice.\u003c/p\u003e \u003cp\u003eAuxin negatively affects \u003cem\u003eXoo\u003c/em\u003e resistance in rice (Zhong et al. 2024). Treatment with indole-3-acetic acid (IAA) increases susceptibility against \u003cem\u003eXoo\u003c/em\u003e in rice (Ding et al. 2008; Suzuki et al. 2022), and \u003cem\u003eXoo\u003c/em\u003e itself secretes IAA to promote the infection process (Fu et al. 2011). Reduction of auxin levels in rice results in increased resistance against \u003cem\u003eXoo\u003c/em\u003e (Li et al. 2015; Li et al. 2016). After inoculation with \u003cem\u003eXoo\u003c/em\u003e, the expression of auxin-responsive \u003cem\u003eexpansins\u003c/em\u003e, which loosen the cell wall, is upregulated and downregulated in the susceptible and resistant rice plants, respectively (Ding et al. 2008). The overexpression of the auxin-responsive \u003cem\u003eexpansins\u003c/em\u003e results in increased susceptibility against \u003cem\u003eXoo\u003c/em\u003e in rice (Ding et al. 2008; Uji et al. 2024). Auxin signaling is strictly controlled by AUX/IAA proteins, which act as repressors by interacting with TFs named auxin response factors (ARFs) (Canc\u0026eacute; et al. 2022). Degradation of AUX/IAA proteins triggers the activation of ARFs to regulate gene expressions involved in auxin signaling (Canc\u0026eacute; et al. 2022).\u003c/p\u003e \u003cp\u003eRecently, JA has also been reported to repress auxin signaling in \u003cem\u003eXoo\u003c/em\u003e resistance in rice (Uji et al. 2024). Rice plants overexpressing a JA-responsive MYB-type TF (\u003cem\u003eJMTF1\u003c/em\u003e) show similar phenotypes to auxin-insensitive mutants of rice (Uji et al. 2024). Furthermore, JMTF1 negatively affects auxin signaling by upregulating expression of an AUX/IAA gene, \u003cem\u003eOsIAA13\u003c/em\u003e, in \u003cem\u003eXoo\u003c/em\u003e resistance in rice (Uji et al. 2024). The rice gain-of-function mutant, \u003cem\u003eOsiaa13\u003c/em\u003e, shows increased resistance against \u003cem\u003eXoo\u003c/em\u003e (Kitomi et al. 2012; Uji et al. 2024). These results suggest that JA and auxin signaling antagonistically interact through OsIAA13 in \u003cem\u003eXoo\u003c/em\u003e resistance in rice. However, there is no information about JA signaling affected by OsIAA13 in rice resistance against \u003cem\u003eXoo\u003c/em\u003e. In the present study, we investigated OsIAA13-mediating auxin/JA signaling, which is involved in rice resistance against \u003cem\u003eXoo\u003c/em\u003e, using the \u003cem\u003eOsiaa13\u003c/em\u003e mutant.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePlant and bacterial materials\u003c/h2\u003e \u003cp\u003eThe growth conditions for rice plants (\u003cem\u003eOryza sativa\u003c/em\u003e L.) and \u003cem\u003eXoo\u003c/em\u003e (strain 7174) were as previously described by Kashihara et al. (2022). The \u003cem\u003eOsiaa13\u003c/em\u003e mutant and its background wild type (cv. Taichung 65; T65) were as previously described by Kitomi et al. (2012). Rice plants were grown from seeds under greenhouse conditions (25 \u0026plusmn; 1\u0026deg;C, 60\u0026ndash;80% relative humidity). The \u003cem\u003eXoo\u003c/em\u003e strain T7174 was cultured on a nutrient agar slant (Becton, Dickinson and Co., Sparks, MD, USA) containing 0.5% sucrose at 25\u0026deg;C for 48 h.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eHormone treatments\u003c/h3\u003e\n\u003cp\u003eTo examine the effects of plant hormones, rice plants were grown to the four-leaf stage in a growth chamber in Kimura-B liquid medium (Sato et al. 1996) at 25\u0026deg;C with 24 h light. The plants were then incubated in the same medium supplemented with 100 \u0026micro;M JA and 10 \u0026micro;M IAA (Sigma-Aldrich, St. Louis, MO, USA) following the method of JA treatment as previously described by Yamada et al. (2012).\u003c/p\u003e\n\u003ch3\u003eReverse transcription-quantitative PCR (RT-qPCR)\u003c/h3\u003e\n\u003cp\u003eTotal RNA was extracted from the rice leaf blades after each treatment using TRIzol Regent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer\u0026rsquo;s instructions. Four leaf blades were used per replicate. Fourth-leaf blades were used for the RT-qPCR analysis. RT-qPCR was performed using TB Green Premix Ex \u003cem\u003eTaq\u003c/em\u003e Mixture (Takara) in a Thermal Cycler Dice TP800 System (Takara) according to the manufacturer\u0026rsquo;s instructions. The transcript levels of each gene were normalized to those of \u003cem\u003eactin\u003c/em\u003e (\u003cem\u003eOs11g06390\u003c/em\u003e) and compared with those of the respective mock-treated control or the WT to calculate the fold change in expression levels as previously described by Gomi et al. (2010). Each data point contains four biological replicates. The sequences of the gene-specific primers used in RT-qPCR are shown in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e\n\u003ch3\u003eMeasurement of the chlorophyll content\u003c/h3\u003e\n\u003cp\u003eFour leaf blades were used per replicate. Leaf blades (0.1 g fresh weight) treated with 100 \u0026micro;M JA for 4 days were homogenized in 1 mL of 80% acetone and centrifuged at 3,000 \u003cem\u003eg\u003c/em\u003e for 10 min. The specific chlorophyll content was determined as previously described by Yamada et al. (2012). Each data point contains four biological replicates.\u003c/p\u003e\n\u003ch3\u003eSubcellular localization assay\u003c/h3\u003e\n\u003cp\u003eTo construct \u003cem\u003eOsPrx38\u003c/em\u003e-\u003cem\u003eDsRed\u003c/em\u003e, the ORF of \u003cem\u003eOsPrx38\u003c/em\u003e (\u003cem\u003eOs03g13210\u003c/em\u003e) without the stop codon was amplified by PCR and subcloned into the corresponding sites of the pE7133-DsRed vector (Onohata and Gomi 2020); this fused DsRed in-frame to the C-terminus of OsPrx38. The vectors were expressed in onion epidermal cells using a particle bombardment system (PDS-1000/He; BioRad, Hercules, USA) as previously described by Kim et al. (2009). We used a KEYENCE BIOREVO BZ-9000 microscope (KEYENCE, Osaka, Japan) and a BZ filter TRITC (excitation wavelength: 540/25 nm; absorption wavelength: 605/55 nm; dichroic mirror wavelength: 565 nm) to observe DsRed fluorescence. The onion epidermal cells were plasmolyzed by immersion in a 0.5 M mannitol solution for 30 min.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eMeasurement of the lignin content\u003c/h2\u003e \u003cp\u003eFour leaf blades were used per replicate. Fully opened fourth-leaf blades were used. Lignin content was determined by the method of Suzuki et al. (2009).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMeasurement of the volatiles\u003c/h3\u003e\n\u003cp\u003eThree leaf blades were used per replicate. Fully-opened fourth-leaf blades were ground to a powder in liquid nitrogen and then transferred to a 15 mL glass vial containing 1 mL of 1% NaCl and immediately weighed. The headspace of the vial was collected in a water bath at 50\u0026deg;C using a SPME fiber for GC-MS. GC-MS analysis was performed using a GCMS-QP2010SE (Shimadzu, Kyoto, Japan) fitted with a DB-WAX column (60 m \u0026times; 0.25 mm, 0.25 \u0026micro;m film thickness; J and W Scientific, Folsom, CA, USA) under the same conditions as previously described by Kiryu et al. (2018). The compounds were identified by comparing their mass spectra to those in a database (Wiley10). Each peak area of volatiles in the leaf blades was calculated based on the total ion current (TIC) chromatograms.\u003c/p\u003e\n\u003ch3\u003eAnalysis of the antibacterial activity of the volatile compounds\u003c/h3\u003e\n\u003cp\u003e \u003cem\u003eXoo\u003c/em\u003e was cultured in YT broth (0.5% Bacto-yeast extract, 1% Bacto-tryptone, pH 6.8; Becton Dickinson and Co.) containing volatile compounds at 100 \u0026micro;M for 6 h. The same amount of DMSO was used as a control; 100 \u0026micro;M (\u003cem\u003eE\u003c/em\u003e)-2-hexenal and methyl salicylate (MeSA) (Wako, Osaka, Japan) were used as known positive and negative controls, respectively (Tanaka et al. 2014). The media were incubated in a shaker at 28\u0026deg;C, 200 rpm, and changes in OD\u003csub\u003e600\u003c/sub\u003e were measured after 6 h.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eAnalysis of the auxin-responsive genes in the\u003c/b\u003e \u003cb\u003eOsiaa13\u003c/b\u003e \u003cb\u003emutants\u003c/b\u003e\u003c/p\u003e \u003cp\u003eWe first investigated the expression levels of \u003cem\u003eARF\u003c/em\u003es in the \u003cem\u003eOsiaa13\u003c/em\u003e mutants. The rice genome contains at least 25 OsARFs (Shen et al. 2010), but OsARF6, OsARF11, OsARF16, OsARF17, OsARF19, OsARF21, and OsARF25 were selected because they are considered to be transcriptional activators of auxin signaling in rice (Shen et al. 2010). Among the selected OsARFs, the expressions of \u003cem\u003eOsARF6\u003c/em\u003e and \u003cem\u003eOsARF16\u003c/em\u003e were significantly downregulated in the \u003cem\u003eOsiaa13\u003c/em\u003e mutants (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe next investigated the expression levels of selected auxin-responsive \u003cem\u003eexpansin\u003c/em\u003es (\u003cem\u003eOsEXP\u003c/em\u003es), which are involved in the auxin-mediated suppression of resistance against \u003cem\u003eXoo\u003c/em\u003e in rice (Ding et al. 2008; Uji et al. 2024), in the \u003cem\u003eOsiaa13\u003c/em\u003e mutants. The expressions of \u003cem\u003eOsEXPA4\u003c/em\u003e, \u003cem\u003eOsEXPA8\u003c/em\u003e, and \u003cem\u003eOsEXPB3\u003c/em\u003e were significantly downregulated in the \u003cem\u003eOsiaa13\u003c/em\u003e mutants (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e \u003cb\u003eAnalysis of the JA-related phenotype in the\u003c/b\u003e \u003cb\u003eOsiaa13\u003c/b\u003e \u003cb\u003emutants\u003c/b\u003e\u003c/p\u003e \u003cp\u003eJA upregulates expression of \u003cem\u003eOsIAA13\u003c/em\u003e to repress auxin signaling in rice (Uji et al. 2024), suggesting that JA sensitivity can be altered in the \u003cem\u003eOsiaa13\u003c/em\u003e mutant. Thus, chlorophyll (Chl) content was measured after JA treatment because JA is known to promote the reduction of Chl content in rice (Yamada et al. 2012). The levels of Chl significantly decreased in the \u003cem\u003eOsiaa13\u003c/em\u003e mutants as early as two days after treatment with JA, unlike in the WT plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe next investigated the expression levels of thirteen JA-responsive defense-related genes in the \u003cem\u003eOsiaa13\u003c/em\u003e mutants. The expression of five genes [\u003cem\u003eperoxidase\u003c/em\u003e (\u003cem\u003eOsPrx38\u003c/em\u003e) (\u003cem\u003eOs03g13210\u003c/em\u003e), \u003cem\u003eBowman\u0026ndash;Birk trypsin inhibitor\u003c/em\u003e (\u003cem\u003eOs01g03320\u003c/em\u003e), \u003cem\u003ebeta 1,3-glucanase\u003c/em\u003e (\u003cem\u003eOs01g71340\u003c/em\u003e), \u003cem\u003ebeta glucanase\u003c/em\u003e (\u003cem\u003eOs01g51570\u003c/em\u003e), and \u003cem\u003eproteinase inhibitor I25\u003c/em\u003e (\u003cem\u003eOs08g10570\u003c/em\u003e)] were significantly upregulated in the \u003cem\u003eOsiaa13\u003c/em\u003e mutants (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). OsPrx38 has been reported to be an extracellular protein and is involved in lignin biosynthesis in plants (Kidwai et al. 2019). We also confirmed the extracellular localization of OsPrx38 in plant cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). Furthermore, the content of lignin was found to have significantly increased in the \u003cem\u003eOsiaa13\u003c/em\u003e mutants relative to the WT plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). The expression levels of the five OsIAA13-responsive JA-related genes in response to auxin were investigated. The expression of only \u003cem\u003eOsPrx38\u003c/em\u003e was significantly downregulated after 10 \u0026micro;M IAA treatment for 24 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eAnalysis of the volatiles in the\u003c/b\u003e \u003cb\u003eOsiaa13\u003c/b\u003e \u003cb\u003emutants\u003c/b\u003e\u003c/p\u003e \u003cp\u003eWe next measured the volatile compounds in the \u003cem\u003eOsiaa13\u003c/em\u003e mutants because JA positively regulates production of antibacterial volatile compounds against \u003cem\u003eXoo\u003c/em\u003e (Tanaka et al. 2014). As a result, six volatile compounds [(\u003cem\u003eE\u003c/em\u003e,\u003cem\u003eE\u003c/em\u003e)-2,4-heptadienal, (\u003cem\u003eE\u003c/em\u003e)-2-hexenal, hexanal, (\u003cem\u003eZ\u003c/em\u003e)-3-hexenal, 5,9-undecadien-2-one, and (\u003cem\u003eE\u003c/em\u003e)-4-oxohex-2-enal] were highly accumulated in the \u003cem\u003eOsiaa13\u003c/em\u003e mutants relative to the WT plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). Both (\u003cem\u003eE\u003c/em\u003e)-2-hexenal and (\u003cem\u003eE\u003c/em\u003e,\u003cem\u003eE\u003c/em\u003e)-2,4-heptadienal have been shown to exhibit antibacterial activity against \u003cem\u003eXoo\u003c/em\u003e (Gomi et al. 2010; Tanaka et al. 2014). Among other unidentified volatiles, we selected a commercially available compound, hexanal, and analyzed its antibacterial activity against \u003cem\u003eXoo\u003c/em\u003e. (\u003cem\u003eE\u003c/em\u003e)-2-Hexenal and MeSA were used as positive and negative controls, respectively (Tanaka et al. 2014). When hexanal was added to liquid cultures of \u003cem\u003eXoo\u003c/em\u003e, a significant negative effect was not observed on the growth of \u003cem\u003eXoo\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eOsIAA13 has been reported to play a critical role in the development of roots in rice (Kitomi et al. 2012; Yamauchi et al. 2019; Yamauchi et al. 2024). However, there is no information about the role of OsIAA13 in the shoots of rice. In this study, we first revealed that the dominant mutation of OsIAA13 affects both JA and auxin signaling in rice leaf blades. The expression of three auxin-responsive \u003cem\u003eexpansin\u003c/em\u003es, \u003cem\u003eOsEXPA4\u003c/em\u003e, \u003cem\u003eOsEXPA8\u003c/em\u003e, and \u003cem\u003eOsEXPB3\u003c/em\u003e, was downregulated in the \u003cem\u003eOsiaa13\u003c/em\u003e mutants. Expansins are divided into four subfamilies [α-expansin (EXPA), β-expansin (EXPB), expansin-like A (EXPLA), and expansin-like B (EXPLB)] based on their phylogenetic relationship (Kende et al. 2004). They are secretory proteins and their main function is to loosen the cell wall for regulating plant development (Marowa et al. 2016). Some expansins have been reported to play an important role in the auxin-mediated suppression of resistance against \u003cem\u003eXoo\u003c/em\u003e in rice (Ding et al. 2008; Uji et al. 2024). The expression of auxin-responsive \u003cem\u003eexpansin\u003c/em\u003es after inoculation with \u003cem\u003eXoo\u003c/em\u003e is upregulated and downregulated in susceptible and resistant rice plants, respectively (Ding et al. 2008). \u003cem\u003eXoo\u003c/em\u003e-derived auxin has been suggested to upregulate the expression of \u003cem\u003eexpansin\u003c/em\u003es, thereby facilitating pathogen entry and allowing increased nutrient leakage (De Vleesschauwer et al. 2013). The \u003cem\u003eOsEXPA1\u003c/em\u003e-, \u003cem\u003eOsEXPA4\u003c/em\u003e-, \u003cem\u003eOsEXPA5\u003c/em\u003e-, and \u003cem\u003eOsEXPA10\u003c/em\u003e-overexpressing rice transgenic plants exhibit increased susceptibility against \u003cem\u003eXoo\u003c/em\u003e (Ding et al. 2008; Uji et al. 2024). Furthermore, the expression of \u003cem\u003eOsEXPA4\u003c/em\u003e has recently been reported to be downregulated by JA treatment (Uji et al. 2024), suggesting that regulation of \u003cem\u003eOsEXPA4\u003c/em\u003e by JA/auxin has an important role in disease resistance against \u003cem\u003eXoo\u003c/em\u003e in rice.\u003c/p\u003e \u003cp\u003eThe expressions of \u003cem\u003eOsARF6\u003c/em\u003e and \u003cem\u003eOsARF16\u003c/em\u003e were significantly downregulated in the \u003cem\u003eOsiaa13\u003c/em\u003e mutants, suggesting that these ARFs are regulated by OsIAA13 and involved in regulating the expression of \u003cem\u003eOsEXPA4\u003c/em\u003e, \u003cem\u003eOsEXPA8\u003c/em\u003e, and \u003cem\u003eOsEXPB3\u003c/em\u003e. Further analysis of the direct binding activity of OsARF6/OsARF16 to the promoter of these expansin genes is needed to reveal the role of the OsARF6/OsARF16-regulated defense response against \u003cem\u003eXoo\u003c/em\u003e in rice.\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eOsiaa13\u003c/em\u003e mutants exhibited a hypersensitive phenotype to JA. Furthermore, some JA-responsive defense-related genes such as \u003cem\u003eproteinase inhibitors\u003c/em\u003e, \u003cem\u003eglucanases\u003c/em\u003e, and a \u003cem\u003eperoxidase\u003c/em\u003e, \u003cem\u003eOsPrx38\u003c/em\u003e, were constitutively upregulated in the \u003cem\u003eOsiaa13\u003c/em\u003e mutants. Transgenic rice plants overexpressing the proteinase inhibitor \u003cem\u003eBBTI4\u003c/em\u003e have been reported to exhibit increased resistance to \u003cem\u003eXoo\u003c/em\u003e (Pang et al. 2013), suggesting that constitutive upregulation of some proteinase inhibitors in \u003cem\u003eOsiaa13\u003c/em\u003e mutants may result in increased resistance to \u003cem\u003eXoo\u003c/em\u003e. Upregulation of \u003cem\u003eglucanase\u003c/em\u003e has been reported in \u003cem\u003eXoo\u003c/em\u003e-resistant transgenic rice plants, although the function of the glucanase in the defense response was not clear (Gupta et al. 2022). Further study is needed to reveal the role of glucanase in disease resistance against \u003cem\u003eXoo\u003c/em\u003e in the \u003cem\u003eOsiaa13\u003c/em\u003e mutant.\u003c/p\u003e \u003cp\u003eThe expression of \u003cem\u003eOsPrx38\u003c/em\u003e was significantly upregulated in the \u003cem\u003eOsiaa13\u003c/em\u003e mutants. OsPrx38 is a secretory-type class III peroxidase involved in the biosynthesis of lignin (Liu et al. 2018a). The overexpression of \u003cem\u003eOsPrx38\u003c/em\u003e in \u003cem\u003eArabidopsis\u003c/em\u003e results in increased lignin content (Kidwai et al. 2019). OsbHLH034 regulates the expression of \u003cem\u003eOsPrx38\u003c/em\u003e and the lignin content significantly increases in the \u003cem\u003eOsbHLH034\u003c/em\u003e-overexpressing rice plants compared with that in the WT (Onohata and Gomi 2020). Consistent with these findings, the lignin content in the \u003cem\u003eOsiaa13\u003c/em\u003e mutants also increased. Lignin is known to have an important role in \u003cem\u003eXoo\u003c/em\u003e resistance (Bart et al. 2010; Hilaire et al. 2001; Kashihara et al. 2020; Song et al. 2016; Suzuki et al. 2022; Uji et al. 2024). \u003cem\u003eXoo\u003c/em\u003e is a vascular pathogen and stays within the xylem vessels throughout the disease interaction (Tabei 1967). There is contact between \u003cem\u003eXoo\u003c/em\u003e and living cells, such as xylem parenchyma cells, through the pit membranes that separate the xylem lumen from the xylem parenchyma cells. Inoculation with an avirulent \u003cem\u003eXoo\u003c/em\u003e strain triggers the thickening of xylem secondary walls and reduces the pit diameter, resulting in the reduction of \u003cem\u003eXoo\u003c/em\u003e access to the xylem parenchyma cells (Hilaire et al. 2001). Lignin is an essential component of the plant response leading to the thickening of the xylem secondary walls. OsPrx38 has been reported to be secreted into the xylem vessels in rice (Aki et al. 2008), suggesting that OsPrx38 plays an important role in lignin biosynthesis for xylem secondary-wall thickening. These results suggest that the enhanced \u003cem\u003eXoo\u003c/em\u003e resistance in the \u003cem\u003eOsiaa13\u003c/em\u003e mutants was caused by an increased physical barrier resulting from OsPrx38-mediated accumulation of lignin. Furthermore, the expression of \u003cem\u003eOsPrx38\u003c/em\u003e was revealed to be downregulated by IAA, suggesting that auxin represses the expression of \u003cem\u003eOsPrx38\u003c/em\u003e. The role of \u003cem\u003eXoo\u003c/em\u003e-derived auxin may be to suppress the production of lignin at the xylem secondary-wall, thereby facilitating pathogen penetration on the pit.\u003c/p\u003e \u003cp\u003eSome volatile compounds accumulated in the \u003cem\u003eOsiaa13\u003c/em\u003e mutants. Among them, (\u003cem\u003eE\u003c/em\u003e,\u003cem\u003eE\u003c/em\u003e)-2,4-heptadienal is induced by JA (Taniguchi et al. 2014). Both (\u003cem\u003eE\u003c/em\u003e,\u003cem\u003eE\u003c/em\u003e)-2,4-heptadienal and (\u003cem\u003eE\u003c/em\u003e)-2-hexenal are known to exhibit antibacterial activity against \u003cem\u003eXoo\u003c/em\u003e (Gomi et al. 2010; Tanaka et al. 2014). (\u003cem\u003eE\u003c/em\u003e)-4-Oxohex-2-enal is known to act as a non-specific toxin for many organisms because it reacts with DNA bases to form adducts, such as deoxyguanosine and deoxycytidine (Kasai et al. 2005; Kawai et al. 2010; Noge et al. 2012). Hexanal, (\u003cem\u003eZ\u003c/em\u003e)-3-hexenal, and (\u003cem\u003eE\u003c/em\u003e)-2-hexenal are C\u003csub\u003e6\u003c/sub\u003e volatile compounds derived from lipoxygenase-hydroperoxide lyase (LOX-HPL) pathway (Noordermeer et al. 2001). Furthermore, JA and the C\u003csub\u003e7\u003c/sub\u003e volatile compound (\u003cem\u003eE\u003c/em\u003e,\u003cem\u003eE\u003c/em\u003e)-2,4-heptadienal are derived from the LOX pathway (Bhowmik et al. 2023; Howe and Schilmiller 2002). These results suggest that the LOX pathway involved in the production of these compounds may be constitutively activating in the \u003cem\u003eOsiaa13\u003c/em\u003e mutants, and OsIAA13-dependent auxin signaling may negatively regulate the LOX pathway in rice.\u003c/p\u003e \u003cp\u003eIn conclusion, we suggest that OsIAA13 has an important role in disease resistance against \u003cem\u003eXoo\u003c/em\u003e by regulating JA- and auxin-mediating defense system in rice. Further study is needed to reveal the mechanism of that interaction between JA and auxin in the disease resistance against \u003cem\u003eXoo\u003c/em\u003e in rice.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCompliance with Ethical Standards\u003c/h2\u003e \u003cp\u003eThis article does not contain any studies with human participants or animals performed by any of the authors.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConflicts of interest\u003c/strong\u003e \u003cp\u003eThe authors declare that they have no conflict of interest.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was supported in part by a Funding Program for Next Generation World-Leading Researchers from the Japan Society for Promotion of Science (No. GS022) and JSPS KAKENHI (No.15K07313).\u003c/p\u003e\u003ch2\u003eAuthor Contributions\u003c/h2\u003e \u003cp\u003eKG and IY designed the research project. GS, AM, YM, MF, YU, TO, and YF performed the experiments. GS and KG wrote the manuscript. All the authors reviewed and approved the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eWe are grateful to Dr. Kazuya Akimitsu (Kagawa University) for his continuous support and helpful suggestions. We thank Dr. H. Kaku (National Institute of Agrobiological Sciences) for providing the \u003cem\u003eXoo\u003c/em\u003e strain. We also thank Dr. I. Kataoka (Kagawa University), M. Satoh (National Agricultural Research Center for Kyushu Okinawa Region, NARO) and Dr. H. Kanno (NARO) for laying the foundation of part of this study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAki T, Shigyo M, Nakano R, Yoneyama T, Yanagisawa S (2008) Nano scale proteomics revealed the presence of regulatory proteins including three FT-Like proteins in phloem and xylem saps from rice. 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Plants 13:2541. https://doi.org/10.3390/plants13182541\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-general-plant-pathology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jgpp","sideBox":"Learn more about [Journal of General Plant Pathology](http://link.springer.com/journal/10327)","snPcode":"10327","submissionUrl":"https://www.editorialmanager.com/jgpp/default2.aspx","title":"Journal of General Plant Pathology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Auxin, jasmonic acid, lignin, volatile, Xanthomonas oryzae pv. oryzae","lastPublishedDoi":"10.21203/rs.3.rs-5800788/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5800788/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eJasmonic acid (JA) is involved in the regulation of rice defense responses against \u003cem\u003eXanthomonas oryzae\u003c/em\u003e pv. \u003cem\u003eoryzae\u003c/em\u003e (\u003cem\u003eXoo\u003c/em\u003e). JA also affects other plant hormone signaling to maximize the JA-induced defense responses in rice. In this study, we investigated the JA- and auxin-mediating defense system using a gain-of-function mutant of OsIAA13, a suppressor of auxin signaling, in rice. The expression of some auxin-responsive \u003cem\u003eexpansin\u003c/em\u003es was downregulated in the \u003cem\u003eOsiaa13\u003c/em\u003e mutants. The \u003cem\u003eOsiaa13\u003c/em\u003e mutants showed a JA-hypersensitive phenotype. The expression of some JA-responsive defense-related genes such as the lignin biosynthesis gene, \u003cem\u003eOsPrx38\u003c/em\u003e, was upregulated in the \u003cem\u003eOsiaa13\u003c/em\u003e mutants. Lignin content was higher in the \u003cem\u003eOsiaa13\u003c/em\u003e mutants than in the wild type plants. The expression of \u003cem\u003eOsPrx38\u003c/em\u003e was downregulated after IAA treatment. Furthermore, some antibacterial compounds against \u003cem\u003eXoo\u003c/em\u003e accumulated in the \u003cem\u003eOsiaa13\u003c/em\u003e mutants. These results suggest that OsIAA13 has an important role in disease resistance against \u003cem\u003eXoo\u003c/em\u003e by regulating JA- and auxin-mediating defense system in rice.\u003c/p\u003e","manuscriptTitle":"The gain-of-function mutation of the rice auxin-signaling repressor, OsIAA13, induces rice bacterial blight resistance through activating jasmonic acid-mediating defense system","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-01-16 05:42:00","doi":"10.21203/rs.3.rs-5800788/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2025-01-16T05:51:42+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-01-14T10:07:06+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-01-10T07:25:01+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of General Plant Pathology","date":"2025-01-10T00:46:46+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-general-plant-pathology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jgpp","sideBox":"Learn more about [Journal of General Plant Pathology](http://link.springer.com/journal/10327)","snPcode":"10327","submissionUrl":"https://www.editorialmanager.com/jgpp/default2.aspx","title":"Journal of General Plant Pathology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"329eb4a7-06f6-4300-a05f-d547b62d8257","owner":[],"postedDate":"January 16th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-04-14T16:16:26+00:00","versionOfRecord":{"articleIdentity":"rs-5800788","link":"https://doi.org/10.1007/s10327-025-01227-2","journal":{"identity":"journal-of-general-plant-pathology","isVorOnly":false,"title":"Journal of General Plant Pathology"},"publishedOn":"2025-04-12 16:05:08","publishedOnDateReadable":"April 12th, 2025"},"versionCreatedAt":"2025-01-16 05:42:00","video":"","vorDoi":"10.1007/s10327-025-01227-2","vorDoiUrl":"https://doi.org/10.1007/s10327-025-01227-2","workflowStages":[]},"version":"v1","identity":"rs-5800788","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5800788","identity":"rs-5800788","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}