OsMYB1 antagonizes OsSPL14 to mediate rice resistance to brown planthopper and Xanthomonas oryzae pv. oryzae

OsMYB1 antagonizes OsSPL14 to mediate rice resistance to brown planthopper and Xanthomonas oryzae pv. oryzae | 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 OsMYB1 antagonizes OsSPL14 to mediate rice resistance to brown planthopper and Xanthomonas oryzae pv. oryzae Bo Sun, Yuan Zhong, Zhihuan Tao, Lin Zhu, Xuexia Miao, Zhenying Shi, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5172835/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 26 Dec, 2024 Read the published version in Plant Cell Reports → Version 1 posted 5 You are reading this latest preprint version Abstract In their natural habitats, plants are concurrently attacked by different biotic factors. Xanthomonas oryzae pv . oryzae ( Xoo ) is a pathogen that severely deteriorates rice yield and quality, and brown planthopper (BPH; Nilaparvata lugens ) is a rice specific insect pest with the damage topping other pathogens. Although genes for respective resistance to BPH and Xoo have been widely reported, few studies pay attention to simultaneous resistance to both. In this study, we identified a MYB transcription factor, OsMYB1, which exhibited diverse transcriptional regulatory capabilities and a negative regulatory role in resistance to both BPH and Xoo . Biochemical and genetic analysis proved OsMYB1 to be a TF that could interact with OsSPL14, a positive regulator of rice resistance to Xoo . OsSPL14 mutants showed increased sensitivity to BPH, suggesting that OsSPL14 is contrary to OsMYB1 in regulating rice resistance to these two biotic stresses. Consistently, OsMYB1 and OsSPL14 displayed opposite functions in regulating defense-related genes. OsMYB1 can form transcription regulation complexes with repressor OsJAZs instead of co-repressor TOPLESS to possibly realize its transcriptional repression function. Taken together, we concluded that two interacting TFs in rice, OsMYB1 and OsSPL14, played antagonistic roles in regulating resistance to BPH and Xoo . MYB SPL Brown planthopper Xanthomonas oryzae pv. oryzae. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Key message OsMYB1 negatively mediates rice resistance to brown planthopper and rice blight. Additionally, OsMYB1 interacts with OsSPL14 and antagonizes its function by oppositely regulating downstream resistance-related genes. Introduction Rice is one of the most important crops worldwide, but it is susceptible to various biotic and abiotic stresses throughout its lifetime, seriously affecting rice quality and yield. Resistance breeding is considered to be a cost-effective and environmentally friendly approach to enhance crop resistance. Therefore, it is crucial to explore and understand the endogenous resistance-related genes in rice, both in terms of their identification and underlying mechanisms. Xanthomonas oryzae pv. oryzae ( Xoo ), a bacterial pathogen, causes rice blight through infection and tissue damage on the rice leaves, while brown planthopper (BPH; Nilaparvata lugens ) is a major insect pest, causes severe crop damage due to sucking of sap and oviposition in stem tissues, both severely impacting rice crop yield and health. Transcription factors (TFs) have been reported to play important roles in plant immunity and serve as a shared resource to help plants respond quickly to a variety of biotic stresses by activating different immune signals (Tsuda and Somssich 2015 ). Therefore, screening TFs involved in the regulation of multiple resistances can not only enhance our understanding of complex plant immune systems but also provide new insights for broad-spectrum pest and disease resistance breeding. The MYB superfamily is one of the largest TF families in plants, playing vital roles in regulating plant development, secondary metabolite biosynthesis, and stress response (Wu, et al. 2022 ). Specially, in tomato, SlMyb33 facilitates the transcription of the resistance gene SlSw5a to enhance resistance against the tomato leaf curl new delhi virus (Sharma, et al. 2021 ). In einkorn wheat, TuMYB46L regulates defense against powdery mildew by influencing the expression of the ethylene biosynthesis gene TuACO3 (Zheng, et al. 2020 ). In rice, OsMYB108 and OsMYB102 negatively regulate lignin biosynthesis, leading to decreased resistance to bacterial disease Xoo (Lin, et al. 2022 ). OsMYBS1 binds directly to the promoter of bsr-d1 and represses its transcription, thereby enhancing rice resistance to fungal diseases caused by M. oryzae (Li, et al. 2017 ). OsMYB30 enhances lignin content in sclerenchyma cells by activating the expression of Os4CL3 and Os4CL5 in the phenylpropanoid metabolic pathway. This, in turn, helps to prevent fungal penetration of rice leaves by M. oryzae (Li, et al. 2020). In addition to regulating plant resistance to diseases, MYB TFs also protect plants from infestation by herbivorous insects. For example, overexpression of CmMYB15 provides chrysanthemum resistance to piercing-sucking mouthpart aphids by regulating lignin biosynthesis (An, et al. 2019 ). Similarly, OsMYB30 can promote the transcription of OsPAL6 and OsPAL8 in the phenylpropanoid metabolic pathway to enhance the levels of SA and lignin in rice and help rice defend BPH (He, et al. 2020 ). In Nicotiana attenuate , NaMYB8 regulates the accumulation of phenylpropanoid-polyamine conjugates, which are essential for local and systemic defense against insect herbivores with chewing mouthparts (Kaur, et al. 2010 ). Notably, an increasing number of studies have revealed that a single MYB TF may regulate plant resistance to multiple biotic stresses. In Arabidopsis , overexpression of the TF AtMYB44 facilitates Botrytis cinerea infection (Shi, et al. 2011 ), but AtMYB44 positively modulates disease resistance to Pseudomonas syringae through the salicylic acid signaling pathway (Zou, et al. 2013 ). Besides, AtMYB44 regulates resistance against the green peach aphid and diamondback moth by activating EIN2-affected defenses (Lü, et al. 2013 ). MYB TF BOS1 regulates Arabidopsis resistance to different types of pathogens including necrotrophic B. cinerea and Alternaria brassicicola , biotrophic pathogens Pseudomonas syringae pv tomato and the oomycete parasite Peronospora parasitica (Mengiste, et al. 2003 ). Plant SQUAMOSA promoter-binding protein-like (SPLs) TFs, which share a highly conserved DNA-binding domain (SBP domain), are a family of plant-specific TFs that regulate flowering time, leaf initiation, and other developmental processes (Preston and Hileman 2013 ). Like MYB TFs, SPLs are vital in regulating plant resistance to various biotic stresses. In Arabidopsis , SPL9 regulates the balance between defense and growth by interacting with the jasmonate signaling (Mao, et al. 2017 ). In pepper, CaSBP12 negatively regulates the defense response against Phytophthora capsica (Zhang, et al. 2019 ). In rice, both OsSPL7 and OsSPL14 have been implicated in regulating resistance against various diseases, such as Xoo (Liu, et al. 2019 ) and rice blast disease (Wang, et al. 2018 ). Furthermore, IPA1 can bind to the promoter of the pathogen defense gene OsWRKY45 and activate its expression, thereby enhancing rice resistance to M. oryzae (Wang, et al. 2018 ). Moreover, the functions of some SPL TFs are highly conserved across multiple species and finely regulated by miR156 (Wang and Wang 2015 ; Wang and Zhang 2017 ; Xu, et al. 2016 ). Previous studies have demonstrated that miR156 negatively regulates rice resistance against Xoo and BPH (Ge, et al. 2018 ; Liu, et al. 2019 ). In plants, different TFs can form dimers by interacting with each other. This phenomenon further expands their functionality in regulating transcription. In rice, OsMYC2 interacts with OsMYB22 to synergistically activate the transcription of OsCEBIP , leading to enhanced resistance to M. oryzae (Qiu, et al. 2022 ). In Arabidopsis , MYB70 interacts with and enhances the stabilization of ABI5, thereby increases the expression of downstream genes related to seed germination (Wan, et al. 2021 ). However, ABI5 hinders MYB49 from binding to its downstream target genes by interacting with MYB49 (Zhang, et al. 2019 ). Moreover, ERF4 and MYB52 regulate downstream gene expression by antagonizing each other's DNA-binding ability. This interaction then allows for the fine-tuning of pectin de-methylesterification in the seed coat (Ding, et al. 2021 ). Furthermore, the key TF BES1 in the BR signaling pathway represses the transcriptional activity of MYB34, MYB51, and MYB122 by physically interacting with them, disrupting the expression of glucosinolate biosynthesis genes, resulting in decreased plant resistance to herbivorous insects (Liao, et al. 2020 ). These results suggest that R2R3 MYB TFs can play a synergistic or antagonistic role by interacting with other TFs. Both MYB and SPL TFs have been reported to regulate plant resistance. However, the extent to which MYB synergizes or antagonizes SPL in plant resistance through their interaction with each other remains relatively unexplored. In this study, we study the functions of OsMYB1 in rice resistance to BPH and Xoo , together with the underlying molecular mechanisms. OsMYB1 negatively regulated resistance to BPH and Xoo . Subcellular localization and transcriptional activation assays proved OsMYB1 to be a TF. Using yeast two-hybrid (Y2H) assay and bimolecular fluorescence complementation (BiFC) assay, we identified the interaction between OsMYB1 and OsSPL7/14 in rice. In contrast to OsMYB1 , OsSPL14 mutants were sensitive to BPH. Furthermore, OsMYB1 affects the expression of resistance-related genes in opposite ways compared to OsSPL14. Taken together, our results showed that OsMYB1 functions antagonistically with OsSPL14 to negatively regulate rice resistance to BPH and Xoo . Materials and methods Plant and insect materials The wild-type (WT) rice used in this study was ZH11 ( Oryza sativa L. subsp. japonica cv Zhonghua no. 11, ZH11). Rice plants were planted either in a glasshouse with a 10h: 14h, light: dark photoperiod or in a paddy field in Shanghai, China, under natural conditions during the summer. The BPH population was initially collected from rice fields in Shanghai and maintained on BPH-susceptible rice cultivar Taichung Native 1 (TN1) plants in a climate-controlled room at a temperature of 26 ± 2°C with a 12 h: 12 h, light: dark cycle and 80% relative humidity. Plasmid construction and plant transformation To produce MYB1-overexpressing (OE) transgenic plants, the CDS of OsMYB1 was amplified by PCR using KOD-plus DNA polymerase (Toyobo, Tokyo, Japan) and then cloned into the p1301-35S-Nos vector through digestion by XbaI and KpnI by using the CloneExpress Ultra One Step Cloning Kit (C115-02; Vazyme, Nanjing, China) to generate the OsMYB1OE plasmid. The sgRNA sequence for OsMYB1 was designed online ( http://skl.scau.edu.cn ) and CRISPR (Clustered regularly interspaced short palindromic repeats)/Cas9 constructs were generated according to a published protocol (Ma, et al. 2015 ). All primers used in this study were listed in Table S1 . The OsMYB1OE, OsMYB1KO and OsSPL14KO plasmids were transferred into A. tumefaciens strain EHA105, then rice transformation was carried out using the Agrobacterium-mediated method in Biorun Biosciences Company (Hiei, et al. 1994 ). OsMYBOE1 transgenic plants were characterized by cloning the Hygromycin B gene from the genomic DNA, and their expression levels were further measured by qRT-PCR. OsMYB1KO and OsSPL14KO plants were characterized by cloning their DNA fragments from their genomic DNA, then the types of mutation were identified by sequencing. BPH resistance detection and analysis For the small population assay, approximately 25 rice plants per line were grown in plastic pots for 3 weeks. After this period, 4–6 second to third instar BPH nymphs were added to each plant. The plant status was examined daily until most of the seedlings in each line had withered, and then the seedling mortality rate was recorded. Pathogen inoculation for field experiments Rice flag leaves were inoculated with Xoo strains at the booting stage using the leaf-clipping method, as described previously (Shen, et al. 2011 ). The Xoo strain used in this study is PXO99A. Disease symptoms were assessed by measuring the relative lesion length 14 days after inoculation. Quantitative real-time polymerase chain reaction (qRT-PCR) Total RNA was extracted using TRIzol (Invitrogen). Then, 2 µg of total RNA was reverse transcribed into cDNA using the ReverTra Ace™qPCR RT Master Mix with gDNA Remover (Toyobo). The qRT-PCR analysis was performed using the SYBR Green Real-time PCR Master Mix Kit (Toyobo), and the actin (LOC_Os03g50885) and UBQ (LOC_Os01g22490) genes were selected as internal references. Collect three biological replicates for each sample, and calculate the mean and standard deviation. Yeast two-hybrid (Y2H) assay For the Y2H assay, the target coding sequence (CDSs) was amplified by PCR using gene-specific primers (Table S1 ) and then inserted into pGADT7/pGBKT7 (Clontech, Mountain View, CA, USA). To detect protein-protein interactions, we conducted assays following the instructions provided by the manufacturer (YC1002; Weidi Biotechnology, Shanghai, China). Clones were grown on deficient medium, lacking either Trp and Leu or Trp, Leu, Ade, and His. Luciferase complementation assay (LCA) and bimolecular fluorescence complementation (BiFC) assay For the LCA, the tested CDSs were amplified by PCR and inserted into pCAMBIA-35S-nLuc and pCAMBIA-35S-cLuc. For the BiFC assay, the tested CDSs were amplified by PCR and inserted into pCAMBIA1300-35S-nYFP and pCAMBIA1300-35S-cYFP. All recombinant plasmids are inserted into the A. tumefaciens strain GV3101 (pSoup-p19). A. tumefaciens overnight cultures were centrifuged at 5000 g for 5 minutes and then resuspended in infiltration buffer (10 mM MgCl2, 150 µM Acetosyringone, and 10 mM MES at pH 5.6). Adjust the optical density to 1.0 at a wavelength of 600 nm. A. tumefaciens suspension was incubated for 2 hours at room temperature and then infiltrated into healthy N. benthamiana leaves using a 1 ml needle-free syringe. The LUC signal was detected 48 hours after infiltration using a CCD camera and a 150 µg ml − 1 fluorescein solution. Confocal microscopy (LSM 880; Zeiss) was used to detect the YFP signal. Subcellular localization PCR amplification of gene CDSs was performed on the pCAMBIA1301-35s-eGFP in N. benthamiana for transient expression. GFP and DAPI signals were observed using confocal microscopy (LSM 880; Zeiss) at 48 hours. Transient transcription assay The sequences encoding GAL4BD, GAL4BD-OsMYB1, and GAL4-VP16 were amplified by PCR and cloned into p1301-35S-Nos to generate the effector plasmids. A sequence consisting of six repeats of the GAL4-binding upstream activating sequence (6×UAS), TATA-Box, and 35S promoter was synthesized and inserted into pGreenII0800-LUC to create the 35S-6×UAS-TATA-LUC reporter plasmid. The full-length OsMYB1 and OsSPL14 CDSs were amplified by PCR and inserted into pCAMBIA1300-35s-3×FLAG or pCAMBIA1300-35s-3×GFP to generate the effector plasmids. The 2 kb OsWRKY45 promoter sequence was cloned into pGreenII0800-LUC to generate the OsWRKY45 pro-LUC reporter plasmid. The effector and reporter plasmids were inserted into A. tumefaciens strain GV3101 (pSoup-p19) and then transiently expressed in N. benthamiana as described above. At 40–48 h post infiltration, the leaves were examined using a CCD camera and a 150 µg ml − 1 luciferin solution. Or according to the manufacturer's instructions (Promega), using a luminometer quantified the luciferase activity. The statistical analysis was performed as previously described (Wang, et al. 2021 ), considering that the state of each N. benthamiana leaf was different, the negative control on each leaf was regarded as a calibrator and set as one, the two-tailed paired Student's t -test was used to compare the difference in mean ± SD and a P-value of ≤ 0.05. Statistical analysis The two-sample Student's t -test was used to compare the difference in mean ± SD and a P-value of ≤ 0.05 was considered significant for qRT-PCR and the small population assays. Accession numbers Sequence data for the genes described herein are available in the China Rice Data Center databases under the following accession nos.: OsMYB1 , LOC_Os01g03720; OsSPL7 , LOC_Os04g46580; OsSPL13 , LOC_Os07g32170; OsSPL14 , LOC_Os08g39890; OsMYB22 , LOC_Os01g65370; OsJAZ3 , LOC_Os08g33160; OsJAZ4 , LOC_Os09g23660; OsPR4 , LOC_Os11g37970; OsWRKY45 , LOC_Os05g25770; Ubiquitin , LOC_Os01g22490; ACTIN , LOC_Os03g50885. Results OsMYB1 responded to BPH infestation and Xoo infection Previously, we demonstrated that OsMYB22 forms a complex with co-repressor TOPLESS proteins to inhibit the expression of F3'H , thereby enhancing rice resistance against BPH (Sun, et al. 2023 ). To search the proteins that could interact with OsMYB22, we used yeast two hybrid (Y2H) and identified OsMYB1 as an interacting partner of OsMYB22, which was further confirmed by BiFC assay (Fig. S1 ). We next detected the expression of OsMYB1 in various rice tissues. The result showed that OsMYB1 has a tissue-wide gene expression pattern, and the highest expression level was observed in the stem. (Fig. 1 A). To study the function of OsMYB1 in rice resistance, we further detected the expression of OsMYB1 upon BPH infestation and Xoo infection. It was revealed that OsMYB1 gene exhibits significantly repressed expression trends at multiple time points by both BPH infestation (Fig. 1 B) and Xoo infection (Fig. 1 C), respectively. These findings suggested that OsMYB1 may play a role in regulating the interaction between rice and these two kinds of biotic factors. OsMYB1 showed variety characteristics of a TF To analyze the characteristics of the OsMYB1 protein, we first studied the subcellular localization of the MYB1-eGFP fusion protein by expressing it in N. benthamiana leaves using Agrobacterium-mediated transformation, it was revealed that the fluorescence signal of MYB1-eGFP overlapped with DAPI (Fig. 2 A), indicating a nuclear localized character. Meanwhile, OsMYB1 proteins showed transcriptional activation in yeast, with the function being situated to the C-terminals (Fig. 2 B). In addition, GAL4BD-MYB1 showed inhibitory effects on reporter gene in N. benthamiana , indicating a potential role of OsMYB1 in transcriptional repression in plants (Fig. 2 C, D). These findings collectively indicated OsMYB1 exhibits v ariety characteristics of a plant TF and can function as a transcriptional repressive regulator in planta. OsMYB1 negatively regulated rice resistance to BPH To explore the function of OsMYB1 , overexpression lines were generated using Agrobacterium-mediated genetic transformation. OsMYB1OE-1 and OsMYB1OE-11 lines showed greatly promoted expression of OsMYB1 compared with the wild type (WT) (Fig. 3 A). In BPH resistance test, both OsMYB1OE-1 and OsMYB1OE-11 plants died earlier than WT after BPH infestation. Moreover, the mortalities were higher for OsMYB1OE-1 and OsMYB1OE-11 plants (Fig. 3 B, C; Fig. S2 ), suggesting a negative regulatory function of OsMYB1 in rice resistance to BPH. Meanwhile, we constructed homozygous knockout lines of OsMYB1 using CRISPR/CAS9 technology, and two homozygous lines, OsMYB1KO-2 and OsMYB1KO-20 were got. In OsMYB1KO-2 line, a T was inserted, while in line OsMYB1KO-20, a C was deleted (Fig. 3 D). However, neither OsMYB1KO-2 nor OsMYB1KO-20 showed significant difference to WT in BPH resistance test (Fig. 3 E, F; Fig. S2 ). Based on data from the Plant Transcription Factor Database ( https://planttfdb.gao-lab.org/ ), the MYB family in rice consists of 130 members. Among them, 15 MYB proteins share the closest evolutionary relationship with OsMYB1, some of them are up-regulated in OsMYB1KO plants (Fig. S3 A). Therefore, the lack of significant difference in BPH resistance between OsMYB1KO-2, OsMYB1KO-20, and WT might be due to possible functional redundancy among these MYBs. OsMYB1 negatively regulated rice resistance to Xoo We further investigated the function of OsMYB1 in resistance to Xoo using the leaf-cutting method. After 14 days post inoculation, the OsMYB1OE-1 and OsMYB1OE-11 lines exhibited a significant increase in lesion length compared to the WT, while the OsMYB1KO-2 and OsMYB1KO-20 showed a significant decrease in lesion length (Fig. 4 A). And statistical analysis of the lesion length further verified the result (Fig. 4 B). Thus, OsMYB1 showed a negative regulatory role in rice resistance to Xoo . OsMYB1 interacted with SPLs In Arabidopsis , AtSPL9 inhibits the formation of the MBW complex PAP1-TT8-TTG1 by interacting with MYB, thereby counteracting downstream genes (Gou, et al. 2011 ). In phylogenetic analysis, it was revealed that OsMYB1 was homologous to AtMYB62 (AT1G68320) and AtMYB116 (AT1G25340) (Fig. S3 B), and the amino acids of AtMYB62, AtMYB116 and OsMYB1 showed high similarity (Fig. S3 C). It is predicted that AtMYB62 can interact with AtSPL4 ( https://thebiogrid.org/ ) (Fig. S4 A). Both OsSPL7 and OsSPL14 were reported to regulate resistance to rice blight (Liu, et al. 2019 ). However, OsSPL14 plays a broader role in stress resistance, including rice blast resistance (Wang et al., 2018 ) and tolerance to abiotic stresses such as drought and chilling (Chen et al., 2023 ; Jia et al., 2022 ). This wider functional spectrum makes OsSPL14 a more compelling candidate for further study. Additionally, phylogenetic analysis shows that OsSPL13 shares high sequence similarity in DNA binding domain with both OsSPL7 and OsSPL14 (Fig. S4 B, C). Based on these observations, we tested the potential interactions between OsMYB1 and these three OsSPL proteins, with a focus on OsSPL14 due to its broader role in stress responses. In Y2H assay, it was revealed that OsMYB1 could interact with OsSPL7 and OsSPL14, but not OsSPL13 in yeast (Fig. 5 A). Both OsSPL7 and OsSPL14 have been previously identified as positive regulators of rice resistance to abiotic stresses such as cold and drought tolerance (Chen, et al. 2023 ; Jia, et al. 2022 ) and biotic stresses such as M. oryzae and Xoo (Liu, et al. 2019 ; Wang, et al. 2018 ). We further used Luciferase complementation assay (LCA) to verify the interaction. Co-infiltration of bacterium harboring both OsMYB1 and OsSPL7, and OsMYB1 and OsSPL14 showed strong inflorescence by LCA and BiFC assays, while those infiltrations of other combinations did not (Fig. 5 B, C). Knockout of OsSPL14 decreased rice resistance to BPH OsSPL14 was reported to negatively regulate rice resistance to Xoo (PXO99A) (Liu et al., 2019 ). We further investigated the role of OsSPL14 in resistance to BPH. First, OsSPL14 transcript was detected in the leaf and sheath (Fig. S5 ). And in response to BPH infestation, OsSPL14 transcript was mainly up-regulated at multiple time points (Fig. 6 A). Next, OsSPL14 homozygous knockout lines, OsSPL14KO-15 and OsSPL14KO-19, were generated using CRISPR/CAS9 technology. In OsSPL14KO-15, a T was inserted, while in OsSPL14KO-19, a G was inserted (Fig. 6 B). Consistent with the previously reported phenotypes in development, the OsSPL14KO plants exhibited an increased tiller number and reduced plant height (Fig. S6 ) (Song, et al. 2017 ). In BPH resistance evaluation, it was revealed that both OsSPL14KO-15 and OsSPL14KO-19 lines died earlier than WT after BPH infestation (Fig. 6 C). In accordance, the morality of OsSPL14KO-15 and OsSPL14KO-19 plant were much higher than that of the WT (Fig. 6 D), indicating a positive regulatory role of OsSPL14 in rice defense against BPH. Overall, OsSPL14 and OsMYB1 have opposing functions in regulating rice resistance to these two kinds of biotic stresses. OsMYB1 and OsSPL14 antagonistically regulated the expression of defense-related genes To investigate the molecular mechanism of the antagonizing role of OsMYB1 and OsSPL14 in rice resistance, we tested the expression of downstream defense-related genes, OsPR4 and OsWRKY45 , in OsSPL14 and OsMYB1 related transgenic plants (Fig. 7 A, B). Compared with WT, the expression of OsPR4 and OsWRKY45 in the OsSPL14KO plants was reduced, but increased in the OsMYB1KO plants. Besides, their expression displayed significantly reduced expression in the OsMYB1OE plants. Notably, OsWRKY45 is a key downstream target gene of OsSPL14 that associated with rice resistance (Wang, et al. 2018 ), we hypothesis that OsMYB1 regulates rice resistance by inhibiting OsWRKY45 . Furthermore, to explore the interplay between OsMYB1 and OsSPL14 in regulating OsWRKY45 , we conducted transient expression assay using the OsWRKY45 promoter-driven reporter gene LUC (Fig. 7 C). It was revealed that OsSPL14 promoted, while OsMYB1 inhibited the expression of LUC driven by OsWRKY45 promoter (Fig. 7 C, D). Furthermore, when co-expressing OsSPL14 and OsMYB1 , the expression of LUC was decreased compared with expressing OsSPL14 alone, suggesting that OsMYB1 can antagonize OsSPL14 by interacting with it. These results suggest an antagonistic relationship between OsMYB1 and OsSPL14 in the regulation of rice immune responses. OsMYB1 interacted with repressor JAZs but not co-repressor TOPLESS Now that OsMYB1 might function as a transcription inhibitor, we further investigate the underlying molecular mechanism. A putative L×L×L type EAR motif was identified near the N-terminus of OsMYB1 (Fig. S3 C), indicative of recruiting co-repressors TOPLESS. Consequently, we tested if there is any interaction between OsMYB1 and the three rice co-repressor TOPLESS proteins, TPL, TPR1, and TPR2. However, Y2H experiments demonstrated that OsMYB1 did not interact with any of the TOPLESS proteins in yeast (Fig. 8 A). Other, JAZs are typical inhibitors in the jasmonic acid (JA) signaling pathway (Wasternack and Song 2017 ). We checked if OsMYB1 interacts with some JAZs. We used Y2H and revealed that the clones harboring both OsMYB1 and OsJAZ3, and OsMYB1 and OsJAZ4 could grow on the SD-Leu-Trp-His-Ade medium, while those controls could not (Fig. 8 B). In LCA, co-infiltration of bacterium harboring both OsMYB1 and OsJAZ3, OsMYB1 and OsJAZ4 showed strong inflorescence, while those infiltrations of other combinations did not (Fig. 8 C). The results indicated that OsMYB1 may form a complex with JAZ3/JAZ4 to function as a transcriptional repressor complex. Discussion As an important component of plant immunity, TFs play key roles in mediating downstream signaling processes, enabling plants to rapidly respond to a wide range of biotic stresses (Tsuda and Somssich 2015 ). Among them, MYB family TFs have been proven to be involved in regulating plant resistance to multiple biotic stresses (He, et al. 2020 ; Li, et al. 2020; Lin, et al. 2022 ; Sharma, et al. 2021 ), and one single MYB TF has been demonstrated to be able to regulate resistance to different kinds of biotic stresses (Lü, et al. 2013 ; Mengiste, et al. 2003 ). As the most notorious insect pest and pathogen respectively, BPH and Xoo can attack rice plants simultaneously in a field environment, identifying genes that can help plants survive better under interlaced adversity factors can not only further our knowledge of molecular mechanism on comprehensive immunity, but also provide a solid theoretical foundation for rice resistance breeding. In this study, we found that the MYB family TF OsMYB1 is involved in regulating rice resistance to both BPH (Fig. 3 B, C) and Xoo (Fig. 4 ), indicating OsMYB1 to be a comprehensive regulator in plant immunity against different kinds of adverse factors. However, the OsMYB1KO plants showed difference in resistance to BPH and Xoo , indicating that the resistance mechanism to these two kinds of biotic stresses might differ (Figs. 3 E, F, 4 A, B). Additionally, rice contains 130 MYB TFs, each with unique expression patterns across tissues and responses to different biotic stresses, indicating they might differ in assignment for resistance to different biotic stresses. MYB family TFs can interact with other TFs, such as the AP2 family, the bHLH family, and the SPL family (Ding, et al. 2021 ; Gou, et al. 2011 ; Qiu, et al. 2022 ), and play a synergistic or antagonistic regulatory role on downstream genes. Here, we found that OsMYB1 interacts with OsSPL7 and OsSPL14 in yeast and N. benthamiana (Fig. 5 ). In rice, SPL TFs play a crucial role in rice defense. As an example, OsSPL7 and OsSPL14 positively regulate rice resistance to Xoo (Liu, et al. 2019 ), and OsSPL14 mediates rice resistance to rice blast (Wang, et al. 2018 ). Besides, chilling-induced phosphorylation of IPA1 by OsSAPK6 activates chilling tolerance responses in rice (Jia, et al. 2022 ), and IPA1 improves drought tolerance by activating SNAC1 (Chen, et al. 2023 ), suggesting that OsSPL14 plays diverse functions in rice resistance to different stresses. In addition, as target of miR156, SPLs are highly conserved across different species (Wang and Wang 2015 ). Thus, we focused on OsSPL14 to reveal the interaction between OsMYB1 and OsSPL14 in regulating rice resistance to BPH and Xoo . Here we proved that OsSPL14 positively regulated BPH resistance (Fig. 6 ), meanwhile, OsSPL14 was reported to positively regulate resistance to Xoo (Liu, et al. 2019 ). Thus, OsSPL14 is a positive regulator in rice immunity against different kinds of adverse factors that function antagonistically with OsMYB1. To the contrary, miR156 plays a negative role in regulating rice resistance to BPH and Xoo (Ge, et al. 2018 ; Liu, et al. 2019 ), further proving the function of OsmiR156/OsSPL14 module in rice immunity. OsMYB1 has some characters of a TF (Fig. 2 ). In N. benthamiana , OsMYB1 exhibits transcriptional repression (Fig. 2 D). Expression of the defense-related marker genes, such as Os PR4 and OsWRKY45 were influenced in the OsMYB1OE and OsMYB1KO plants (Fig. 7 B). Specifically, the transcription of OsWRKY45 , which activated by OsSPL14 (Wang, et al. 2018 ), was inhibited by OsMYB1 (Fig. 7 D). By transient transcription assays, OsSPL14 can activate the expression of LUC driven by the OsWRKY45 promoter, however, when co-expressing OsMYB1 with OsSPL14, the expression of LUC was decreased, which might be the underlying mechanism of the opposing roles of OsMYB1 and OsSPL14 in regulating rice immunity (Fig. 7 D). Altogether, OsMYB1 antagonizes with OsSPL14 in the regulation of rice resistance to BPH and Xoo. There are several main specific forms of antagonism between TFs when they interact with each other. By influencing each other's DNA-binding capacity, ERF4 and MYB52 oppositely regulate downstream gene expression through physical interaction (Ding, et al. 2021 ). BES1 in the BR signaling pathway interacts with MYB34/51/122 and suppresses its transcriptional activity, disrupting the expression of glucosinolate biosynthesis genes, resulting in a reduction of plant resistance against herbivorous insects (Liao, et al. 2020 ). By competing for the same binding site on downstream gene promoters, MYBS1 and MYBS2 competitively regulate the expression of the αAmy gene in opposite ways, thereby controlling the balance of sugar content in rice (Chen, et al. 2019 ). Nevertheless, the underlying molecular mechanism of the antagonistic interaction between OsMYB1 and OsSPL14 remains unclear and requires further investigation. To further explore the molecular mechanism of transcriptional repression of OsMYB1, we found that OsMYB1 contains a putative repressive EAR motif in its N-terminal region, which can interact with co-repressors TOPLESS to form a transcription repressor complex (Plant, et al. 2021 ). In consistency, MYB22 interacts with TOPLESS through its EAR motif (Sun, et al. 2023 ). However, no interaction between OsMYB1 and TOPLESS was detected (Fig. 8 A), implying that OsMYB1 might function as a repressive TF through an alternative mechanism. JA is a crucial phytohormone that is central in regulating plant defense responses against biotic stresses, such as herbivorous insects and pathogenic microorganisms. In rice, key genes in the JA biosynthesis pathway, including OsAOS1 , OsLOX10 , and OsOPR10 , positively influence resistance to Xoo (Hou, et al. 2019 ; Zhou, et al. 2022 ; Xu, et al. 2024 ), while OsAOC enhances resistance to BPH (Guo, et al. 2014 ). In the JA signal transduction pathway, OsCOI1-RNAi plants exhibited reduced resistance to Xoo (Yang, et al. 2009), and both coi1 and coi2 mutants demonstrated increased susceptibility to BPH attacks (Xu, et al. 2021 ; Wang, et al. 2023 ). The Key TF OsMYC2 has been reported to positively mediate rice resistance to both Xoo and BPH (Uji, et al. 2016 ; Xu, et al. 2021 ). As a necessary part of JA signal transduction, JAZ repressors can cooperate with different TFs to repress the transcription of downstream genes. In tartary buckwheat, FtJAZ1 can assist FtMYB11 in inhibiting the expression of rutin biosynthesis pathway-related genes (Zhou, et al. 2017 ). In Arabidopsis , the JAZs protein can interact with the R2R3 DNA binding domain of MYB21/24 through its ZIM domain (Song, et al. 2011 ). In rice, COI1a, COI1b, and OsCOI2 interact with specific JAZ proteins in the presence of JA-Ile, JA-Val, and JA-Leu, with slight variations in interaction patterns among them (Fu, et al. 2022 ; Wang, et al. 2023 ). Notably, OsJAZ3 and OsJAZ4 are consistently recognized by all COIs, highlighting their broad responsiveness to JA signal transduction. Accordingly, we found that OsMYB1 can interact with OsJAZ3 and OsJAZ4 (Fig. 8 B, C), implying that OsMYB1 might form a transcription repressor complex with OsJAZs, which provides a potential possibility for MYB1 to exert a molecular mechanism of transcriptional repression. Furthermore, the interactions between OsMYB1 and its partners (OsMYB22, OsSPL14, and OsJAZs) were supported by yeast two-hybrid (Y2H) and BiFC/LCA assays, which are recognized as reliable, albeit preliminary, methods for demonstrating protein-protein interactions (Yu et al., 2024 ; Zhu et al., 2024 ). Therefore, additional in vivo or in vitro validation techniques, such as co-immunoprecipitation (Co-IP) or pull-down assays, would further strengthen these findings. Statements & Declarations Funding This work was supported by grants from the Shanghai Chongming Agriculture Science and Innovation Project (Grant No. 2023CNKC-01-04), Shanghai Science and Technology Innovation Action Plan (No. 22N41900300). Competing Interests The authors declare no conflict of interest. Author contributions BS and ZY performed the experiments and analyzed the data. XM and HL designed the research. BS and ZS wrote the paper. ZT and LZ revised the paper. All authors read and approved the final manuscript. 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(A) Y2H assay of OsMYB1 and OsMYB22. (B) BiFC assay for the interaction between OsMYB1 and OsMYB22 in N. benthamiana . BF, bright field. Bars represent 20 μm. Fig. S2 The status of the OsMYB1OE, OsMYB1KO, OsSPL14KO, and WT plants before and after BPH infestation. (A) The status of two lines of the OsMYB1OE plants and WT plants before BPH infestation. (B) The status of two lines of the OsMYB1OE plants and WT plants after BPH infestation. (C) The status of two lines of the OsMYB1KO plants and WT plants before BPH infestation. (D) The status of two lines of the OsMYB1KO plants and WT plants after BPH infestation. (E) The status of two lines of the OsSPL14KO plants and WT plants before BPH infestation. (F) The status of two lines of the OsSPL14KO plants and WT plants after BPH infestation. Bars in Figure S2: 6.5 cm. Fig. S3 Phylogenetic tree and amino acid sequence analysis of OsMYB1 proteins. (A) qRT-PCR analysis of the expression of other OsMYB genes in the OsMYB1KO and WT plants (n = 3). The error bars represent ± standard deviation of three biological replicates, ** represents significant differences in comparison with WT as determined by Student’s t-test ( P < 0.01). (B) The phylogenetic tree was generated using the full-length amino acid sequences of OsMYB1 and the Arabidopsis MYB family members according to the maximum-likelihood method in MEGA7.0. The number in each branch means the name of each protein sequence. (C) Alignment of OsMYB1 and two of its homologous proteins from Arabidopsis . Amino acid sequences were aligned by NCBI Multiple Alignment. Fig. S4 Prediction of possible interacting proteins of OsMYB1. (A) The interaction network of AtMYB62 by BioGrid. (B) The phylogenetic tree was generated using DNA binding domain of OsSPLs from the Plant Transcription Factor Database (https://planttfdb.gao-lab.org/) (C) Alignment of OsSPL7/13/14 with their DNA binding domains. Fig. S5 The expression analysis of OsSPL14 in different rice tissues. Fig. S6 Growth phenotype of the OsSPL14KO and WT plants in the field. Cite Share Download PDF Status: Published Journal Publication published 26 Dec, 2024 Read the published version in Plant Cell Reports → Version 1 posted Editorial decision: Accept 18 Dec, 2024 Reviewers agreed at journal 03 Dec, 2024 Reviewers invited by journal 03 Dec, 2024 Editor assigned by journal 03 Dec, 2024 First submitted to journal 02 Dec, 2024 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. 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(B) qRT-PCR analysis of \u003cem\u003eOsMYB1\u003c/em\u003e transcripts after BPH infestation (n = 3). (C) qRT-PCR analysis of \u003cem\u003eOsMYB1\u003c/em\u003e transcripts after infection by \u003cem\u003eXoo\u003c/em\u003e strain PXO99A (n = 3). The error bars represent ± standard deviation of three biological replicates, * and ** in (B) and (C) represent significant differences in comparison with “0 h” as determined by Student’s \u003cem\u003et-\u003c/em\u003etest (P \u0026lt; 0.05 and P \u0026lt; 0.01, respectively).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5172835/v1/6d5ac4900ac89e0ad0f19aa8.png"},{"id":70696586,"identity":"9b8cf584-bc62-4cfe-bbb8-5ac299a31ce7","added_by":"auto","created_at":"2024-12-05 17:38:45","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":514667,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSubcellular localization and the transcriptional activity of OsMYB1 protein\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Localization of the MYB1-eGFP in \u003cem\u003eN. benthamiana\u003c/em\u003e. The nucleus was visualized by DAPI staining. Bars: 10 μm. (B) Transactivation activity assay of OsMYB1 and truncated OsMYB1 (the N-terminal and the C-terminal) in yeast. The truncation of OsMYB1 was indicated. (C) A schematic representation of the constructs used in testing the transactivation activity of OsMYB1 in planta. (D) Quantitative analysis of the relative luciferase activity (as indicated by the ratio of LUC to REN) in the samples presented in (C) (n = 5). The LUC/REN ratio of the negative control (GAL4BD) was used as a calibrator and set as 1. Each value represents the mean ± standard deviation of five biological replicates. * and ** in (D) represent significant differences in comparison with “GAL4BD” as determined by the two-tailed paired Student’s \u003cem\u003et\u003c/em\u003e-test (P \u0026lt; 0.05 and P \u0026lt; 0.01, respectively).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5172835/v1/75a95b4189422eea08295c74.png"},{"id":70696025,"identity":"d578ee7d-7123-45f7-a2f5-38957096143d","added_by":"auto","created_at":"2024-12-05 17:30:45","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":720216,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBPH resistance tests of the OsMYB1OE and OsMYB1KO plants\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) qRT-PCR analysis of the expression of \u003cem\u003eOsMYB1\u003c/em\u003e gene in the OsMYB1OE and WT plants (n = 3). (B) The status of the OsMYB1OE and WT plants after small population test. (C) Mortality of the OsMYB1OE and WT plants presented in (B) (n = 4). The error bars in (A) and (C) represent ± standard deviation, * and ** in (A) and (C) represent significant differences in comparison with “WT” as determined by Student’s \u003cem\u003et\u003c/em\u003e-test (P \u0026lt; 0.05 and P \u0026lt; 0.01, respectively). (D) Schematic representation of the edited sites in the OsMYB1KO plants. (E) Status of the OsMYB1KO plants and WT plants after small population test. (F) Mortality of the OsMYB1KO and WT plants presented in (E) (n = 4). The error bars in (F) represent ± standard deviation. Bars in (B) and (E): 6.5 cm.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5172835/v1/776babb74bc3e29493c1ac04.png"},{"id":70696635,"identity":"6124756f-2070-453f-84dc-8e61408fcf78","added_by":"auto","created_at":"2024-12-05 17:38:46","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":297562,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eXoo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eresistance tests of the OsMYB1OE and OsMYB1KO plants\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) The lesion length on the leaves of two lines of the OsMYB1OE plants, two lines of the OsMYB1KO lines and WT plants after \u003cem\u003eXoo\u003c/em\u003einfection. Bar: 1 cm. (B) Statistical analysis of the lesion length after PXO99A inoculation presented in (A). Whiskers: min to max, * and ** represent significant differences in comparison with “WT” as determined by Student’s \u003cem\u003et\u003c/em\u003e-test (P \u0026lt; 0.05 and P \u0026lt; 0.01, respectively).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-5172835/v1/7a699af38cdb9d90be9155fb.png"},{"id":70696043,"identity":"d24d07be-8006-4eb3-a62e-805a3d062fab","added_by":"auto","created_at":"2024-12-05 17:30:47","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":644027,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOsMYB1 interacts with OsSPL7/14\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Y2H assays to determine the interaction between OsMYB1 and several OsSPLs. (B) LCA assays to determine the interaction between OsMYB1 and OsSPL7, and OsMYB1 and OsSPL14 in \u003cem\u003eN. benthamiana\u003c/em\u003e. (C) BiFC assays to determine the interaction between OsMYB1 and OsSPL7, and OsMYB1 and OsSPL14 in \u003cem\u003eN. benthamiana\u003c/em\u003e. The nucleus was visualized by DAPI staining. Bars: 25 μm.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-5172835/v1/5aa69a7151ea562d739eaebb.png"},{"id":70696680,"identity":"8d42ba6b-c8f7-4707-9f79-8f702ac4b466","added_by":"auto","created_at":"2024-12-05 17:38:50","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":482791,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe BPH resistance tests of the OsSPL14KO plants\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) qRT-PCR analysis of the\u003cem\u003e OsSPL14\u003c/em\u003etranscripts after BPH infestation (n = 3). (B) Schematic representation of the edited sites in the OsSPL14KO plants. (C) The status of two lines of the OsSPL14KO plants and WT plants after small population tests. Bar: 6.5 cm. (D) Mortality of the OsSPL14KO and WT plants presented in (C) (n = 4). The error bars represent ± standard deviation, and **represents significant differences in comparison with “WT” as determined by Student’s \u003cem\u003et\u003c/em\u003e-test (P \u0026lt; 0.01).\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-5172835/v1/5132b4b9a1f5fc838989a5f6.png"},{"id":70696023,"identity":"b52829b4-0b8d-44a9-a954-91cde6976eb7","added_by":"auto","created_at":"2024-12-05 17:30:44","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":138093,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOsMYB1 and OsSPL14 antagonistically regulated the expression of defense-related genes.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) qRT-PCR analysis of the expression of defense-related genes \u003cem\u003eOsPR4\u003c/em\u003e and \u003cem\u003eOsWRKY45 \u003c/em\u003ein the OsSPL14KO and WT plants (n = 3). (B) qRT-PCR analysis of the expression of defense-related \u003cem\u003eOsPR4\u003c/em\u003e and \u003cem\u003eOsWRKY45\u003c/em\u003egenes in the OsMYB1OE, OsMYB1KO and WT plants (n = 3). * and ** represent significant differences in comparison with “WT” as determined by the student’s \u003cem\u003et\u003c/em\u003e-test (P \u0026lt; 0.05 and P \u0026lt; 0.01, respectively). (C) A schematic representation of the constructs used in testing the regulation of OsMYB1 and OsSPL14 on the promoter of \u003cem\u003eOsWRKY45\u003c/em\u003e in planta. (D) Quantitative analysis of the relative luciferase activity (as indicated by the ratio of LUC to REN) (n = 4). The LUC/REN ratio of the negative control (FLAG or GFP) was used as a calibrator and set as 1. * and ** in (A) and (B) represent significant differences in comparison with “WT” as determined by the student’s \u003cem\u003et\u003c/em\u003e-test (P \u0026lt; 0.05 and P \u0026lt; 0.01, respectively). * and ** in (C) and (D) represent significant differences as determined by the two-tailed paired Student’s \u003cem\u003et\u003c/em\u003e-test (P \u0026lt; 0.05 and P \u0026lt; 0.01, respectively).\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-5172835/v1/54df64537b765575d391c6f1.png"},{"id":70696581,"identity":"3299d89a-8702-4ab8-9535-57dc6089557e","added_by":"auto","created_at":"2024-12-05 17:38:44","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":690638,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBiochemical analysis of the interaction between OsMYB1 and repressors/co-repressors.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Y2H assays of OsMYB1 and the three rice TOPLESS proteins. (B) Y2H assays of OsMYB1 and OsJAZ3/4. (C) LCA assays of OsMYB1 and OsJAZ3/4 in \u003cem\u003eN. benthamiana\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-5172835/v1/fdb9ce18ff2ce3c6bfb8a1d9.png"},{"id":72640740,"identity":"6e40ea87-717d-4eb7-99db-ee36f5272169","added_by":"auto","created_at":"2024-12-30 16:09:16","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5040038,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5172835/v1/6492b7f7-312e-4908-86b4-3f3dd6bceb78.pdf"},{"id":70696022,"identity":"1e4e39bf-6473-47f4-83b5-e85550db5837","added_by":"auto","created_at":"2024-12-05 17:30:44","extension":"doc","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":19585238,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupporting Information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTable S1 Primer sequences used in this study\u003c/p\u003e\n\u003cp\u003eFig. S1 OsMYB1 was identified as one of the interactors of OsMYB22.\u003c/p\u003e\n\u003cp\u003e(A) Y2H assay of OsMYB1 and OsMYB22. (B) BiFC assay for the interaction between OsMYB1 and OsMYB22 in \u003cem\u003eN. benthamiana\u003c/em\u003e. BF, bright field. Bars represent 20 μm.\u003c/p\u003e\n\u003cp\u003eFig. S2 The status of the OsMYB1OE, OsMYB1KO, OsSPL14KO, and WT plants before and after BPH infestation.\u003c/p\u003e\n\u003cp\u003e(A) The status of two lines of the OsMYB1OE plants and WT plants before BPH infestation. (B) The status of two lines of the OsMYB1OE plants and WT plants after BPH infestation. (C) The status of two lines of the OsMYB1KO plants and WT plants before BPH infestation. (D) The status of two lines of the OsMYB1KO plants and WT plants after BPH infestation. (E) The status of two lines of the OsSPL14KO plants and WT plants before BPH infestation. (F) The status of two lines of the OsSPL14KO plants and WT plants after BPH infestation. Bars in Figure S2: 6.5 cm.\u003c/p\u003e\n\u003cp\u003eFig. S3 Phylogenetic tree and amino acid sequence analysis of OsMYB1 proteins.\u003c/p\u003e\n\u003cp\u003e(A) qRT-PCR analysis of the expression of other OsMYB genes in the OsMYB1KO and WT plants (n = 3). The error bars represent ± standard deviation of three biological replicates, ** represents significant differences in comparison with WT as determined by Student’s t-test ( P \u0026lt; 0.01). (B) The phylogenetic tree was generated using the full-length amino acid sequences of OsMYB1 and the \u003cem\u003eArabidopsis\u003c/em\u003e MYB family members according to the maximum-likelihood method in MEGA7.0. The number in each branch means the name of each protein sequence. (C) Alignment of OsMYB1 and two of its homologous proteins from \u003cem\u003eArabidopsis\u003c/em\u003e. Amino acid sequences were aligned by NCBI Multiple Alignment.\u003c/p\u003e\n\u003cp\u003eFig. S4 Prediction of possible interacting proteins of OsMYB1.\u003c/p\u003e\n\u003cp\u003e(A) The interaction network of AtMYB62 by BioGrid. (B) The phylogenetic tree was generated using DNA binding domain of OsSPLs from the Plant Transcription Factor Database (https://planttfdb.gao-lab.org/) (C) Alignment of OsSPL7/13/14 with their DNA binding domains.\u003c/p\u003e\n\u003cp\u003eFig. S5 The expression analysis of \u003cem\u003eOsSPL14\u003c/em\u003e in different rice tissues.\u003c/p\u003e\n\u003cp\u003eFig. S6 Growth phenotype of the OsSPL14KO and WT plants in the field.\u003c/p\u003e","description":"","filename":"SupportingInformation1203.doc","url":"https://assets-eu.researchsquare.com/files/rs-5172835/v1/b562a0400365472013f51f89.doc"}],"financialInterests":"","formattedTitle":"OsMYB1 antagonizes OsSPL14 to mediate rice resistance to brown planthopper and Xanthomonas oryzae pv. oryzae","fulltext":[{"header":"Key message","content":"\u003cp\u003eOsMYB1 negatively mediates rice resistance to brown planthopper and rice blight. Additionally, OsMYB1 interacts with OsSPL14 and antagonizes its function by oppositely regulating downstream resistance-related genes.\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003eRice is one of the most important crops worldwide, but it is susceptible to various biotic and abiotic stresses throughout its lifetime, seriously affecting rice quality and yield. Resistance breeding is considered to be a cost-effective and environmentally friendly approach to enhance crop resistance. Therefore, it is crucial to explore and understand the endogenous resistance-related genes in rice, both in terms of their identification and underlying mechanisms. \u003cem\u003eXanthomonas oryzae\u003c/em\u003e pv. \u003cem\u003eoryzae\u003c/em\u003e (\u003cem\u003eXoo\u003c/em\u003e), a bacterial pathogen, causes rice blight through infection and tissue damage on the rice leaves, while brown planthopper (BPH; \u003cem\u003eNilaparvata lugens\u003c/em\u003e) is a major insect pest, causes severe crop damage due to sucking of sap and oviposition in stem tissues, both severely impacting rice crop yield and health. Transcription factors (TFs) have been reported to play important roles in plant immunity and serve as a shared resource to help plants respond quickly to a variety of biotic stresses by activating different immune signals (Tsuda and Somssich \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Therefore, screening TFs involved in the regulation of multiple resistances can not only enhance our understanding of complex plant immune systems but also provide new insights for broad-spectrum pest and disease resistance breeding.\u003c/p\u003e \u003cp\u003eThe MYB superfamily is one of the largest TF families in plants, playing vital roles in regulating plant development, secondary metabolite biosynthesis, and stress response (Wu, et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Specially, in tomato, SlMyb33 facilitates the transcription of the resistance gene \u003cem\u003eSlSw5a\u003c/em\u003e to enhance resistance against the tomato leaf curl new delhi virus (Sharma, et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In einkorn wheat, TuMYB46L regulates defense against powdery mildew by influencing the expression of the ethylene biosynthesis gene \u003cem\u003eTuACO3\u003c/em\u003e (Zheng, et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In rice, OsMYB108 and OsMYB102 negatively regulate lignin biosynthesis, leading to decreased resistance to bacterial disease \u003cem\u003eXoo\u003c/em\u003e (Lin, et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). OsMYBS1 binds directly to the promoter of \u003cem\u003ebsr-d1\u003c/em\u003e and represses its transcription, thereby enhancing rice resistance to fungal diseases caused by \u003cem\u003eM. oryzae\u003c/em\u003e (Li, et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). OsMYB30 enhances lignin content in sclerenchyma cells by activating the expression of \u003cem\u003eOs4CL3\u003c/em\u003e and \u003cem\u003eOs4CL5\u003c/em\u003e in the phenylpropanoid metabolic pathway. This, in turn, helps to prevent fungal penetration of rice leaves by \u003cem\u003eM. oryzae\u003c/em\u003e (Li, et al. 2020). In addition to regulating plant resistance to diseases, MYB TFs also protect plants from infestation by herbivorous insects. For example, overexpression of \u003cem\u003eCmMYB15\u003c/em\u003e provides chrysanthemum resistance to piercing-sucking mouthpart aphids by regulating lignin biosynthesis (An, et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Similarly, OsMYB30 can promote the transcription of \u003cem\u003eOsPAL6\u003c/em\u003e and \u003cem\u003eOsPAL8\u003c/em\u003e in the phenylpropanoid metabolic pathway to enhance the levels of SA and lignin in rice and help rice defend BPH (He, et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In \u003cem\u003eNicotiana attenuate\u003c/em\u003e, NaMYB8 regulates the accumulation of phenylpropanoid-polyamine conjugates, which are essential for local and systemic defense against insect herbivores with chewing mouthparts (Kaur, et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Notably, an increasing number of studies have revealed that a single MYB TF may regulate plant resistance to multiple biotic stresses. In \u003cem\u003eArabidopsis\u003c/em\u003e, overexpression of the TF AtMYB44 facilitates \u003cem\u003eBotrytis cinerea\u003c/em\u003e infection (Shi, et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), but \u003cem\u003eAtMYB44\u003c/em\u003e positively modulates disease resistance to \u003cem\u003ePseudomonas syringae\u003c/em\u003e through the salicylic acid signaling pathway (Zou, et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Besides, \u003cem\u003eAtMYB44\u003c/em\u003e regulates resistance against the green peach aphid and diamondback moth by activating EIN2-affected defenses (L\u0026uuml;, et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). MYB TF BOS1 regulates \u003cem\u003eArabidopsis\u003c/em\u003e resistance to different types of pathogens including necrotrophic \u003cem\u003eB. cinerea\u003c/em\u003e and \u003cem\u003eAlternaria brassicicola\u003c/em\u003e, biotrophic pathogens \u003cem\u003ePseudomonas syringae pv tomato\u003c/em\u003e and the oomycete parasite \u003cem\u003ePeronospora parasitica\u003c/em\u003e (Mengiste, et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2003\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ePlant SQUAMOSA promoter-binding protein-like (SPLs) TFs, which share a highly conserved DNA-binding domain (SBP domain), are a family of plant-specific TFs that regulate flowering time, leaf initiation, and other developmental processes (Preston and Hileman \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Like MYB TFs, SPLs are vital in regulating plant resistance to various biotic stresses. In \u003cem\u003eArabidopsis\u003c/em\u003e, SPL9 regulates the balance between defense and growth by interacting with the jasmonate signaling (Mao, et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). In pepper, CaSBP12 negatively regulates the defense response against \u003cem\u003ePhytophthora capsica\u003c/em\u003e (Zhang, et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In rice, both \u003cem\u003eOsSPL7\u003c/em\u003e and \u003cem\u003eOsSPL14\u003c/em\u003e have been implicated in regulating resistance against various diseases, such as \u003cem\u003eXoo\u003c/em\u003e (Liu, et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) and rice blast disease (Wang, et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Furthermore, IPA1 can bind to the promoter of the pathogen defense gene \u003cem\u003eOsWRKY45\u003c/em\u003e and activate its expression, thereby enhancing rice resistance to \u003cem\u003eM. oryzae\u003c/em\u003e (Wang, et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Moreover, the functions of some SPL TFs are highly conserved across multiple species and finely regulated by miR156 (Wang and Wang \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Wang and Zhang \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Xu, et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Previous studies have demonstrated that miR156 negatively regulates rice resistance against \u003cem\u003eXoo\u003c/em\u003e and BPH (Ge, et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Liu, et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In plants, different TFs can form dimers by interacting with each other. This phenomenon further expands their functionality in regulating transcription. In rice, OsMYC2 interacts with OsMYB22 to synergistically activate the transcription of \u003cem\u003eOsCEBIP\u003c/em\u003e, leading to enhanced resistance to \u003cem\u003eM. oryzae\u003c/em\u003e (Qiu, et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In \u003cem\u003eArabidopsis\u003c/em\u003e, MYB70 interacts with and enhances the stabilization of ABI5, thereby increases the expression of downstream genes related to seed germination (Wan, et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). However, ABI5 hinders MYB49 from binding to its downstream target genes by interacting with MYB49 (Zhang, et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Moreover, ERF4 and MYB52 regulate downstream gene expression by antagonizing each other's DNA-binding ability. This interaction then allows for the fine-tuning of pectin de-methylesterification in the seed coat (Ding, et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Furthermore, the key TF BES1 in the BR signaling pathway represses the transcriptional activity of MYB34, MYB51, and MYB122 by physically interacting with them, disrupting the expression of glucosinolate biosynthesis genes, resulting in decreased plant resistance to herbivorous insects (Liao, et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). These results suggest that R2R3 MYB TFs can play a synergistic or antagonistic role by interacting with other TFs.\u003c/p\u003e \u003cp\u003eBoth MYB and SPL TFs have been reported to regulate plant resistance. However, the extent to which MYB synergizes or antagonizes SPL in plant resistance through their interaction with each other remains relatively unexplored. In this study, we study the functions of \u003cem\u003eOsMYB1\u003c/em\u003e in rice resistance to BPH and \u003cem\u003eXoo\u003c/em\u003e, together with the underlying molecular mechanisms. OsMYB1 negatively regulated resistance to BPH and \u003cem\u003eXoo\u003c/em\u003e. Subcellular localization and transcriptional activation assays proved OsMYB1 to be a TF. Using yeast two-hybrid (Y2H) assay and bimolecular fluorescence complementation (BiFC) assay, we identified the interaction between OsMYB1 and OsSPL7/14 in rice. In contrast to \u003cem\u003eOsMYB1\u003c/em\u003e, \u003cem\u003eOsSPL14\u003c/em\u003e mutants were sensitive to BPH. Furthermore, OsMYB1 affects the expression of resistance-related genes in opposite ways compared to OsSPL14. Taken together, our results showed that \u003cem\u003eOsMYB1\u003c/em\u003e functions antagonistically with \u003cem\u003eOsSPL14\u003c/em\u003e to negatively regulate rice resistance to BPH and \u003cem\u003eXoo\u003c/em\u003e.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePlant and insect materials\u003c/h2\u003e \u003cp\u003eThe wild-type (WT) rice used in this study was ZH11 (\u003cem\u003eOryza sativa\u003c/em\u003e L. subsp. \u003cem\u003ejaponica\u003c/em\u003e cv Zhonghua no. 11, ZH11). Rice plants were planted either in a glasshouse with a 10h: 14h, light: dark photoperiod or in a paddy field in Shanghai, China, under natural conditions during the summer.\u003c/p\u003e \u003cp\u003eThe BPH population was initially collected from rice fields in Shanghai and maintained on BPH-susceptible rice cultivar Taichung Native 1 (TN1) plants in a climate-controlled room at a temperature of 26 ± 2°C with a 12 h: 12 h, light: dark cycle and 80% relative humidity.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePlasmid construction and plant transformation\u003c/h3\u003e\n\u003cp\u003eTo produce MYB1-overexpressing (OE) transgenic plants, the CDS of \u003cem\u003eOsMYB1\u003c/em\u003e was amplified by PCR using KOD-plus DNA polymerase (Toyobo, Tokyo, Japan) and then cloned into the p1301-35S-Nos vector through digestion by XbaI and KpnI by using the CloneExpress Ultra One Step Cloning Kit (C115-02; Vazyme, Nanjing, China) to generate the OsMYB1OE plasmid.\u003c/p\u003e \u003cp\u003eThe sgRNA sequence for \u003cem\u003eOsMYB1\u003c/em\u003e was designed online (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://skl.scau.edu.cn\u003c/span\u003e\u003cspan address=\"http://skl.scau.edu.cn\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e)\u003c/span\u003e and CRISPR (Clustered regularly interspaced short palindromic repeats)/Cas9 constructs were generated according to a published protocol (Ma, et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). All primers used in this study were listed in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eThe OsMYB1OE, OsMYB1KO and OsSPL14KO plasmids were transferred into \u003cem\u003eA. tumefaciens\u003c/em\u003e strain EHA105, then rice transformation was carried out using the Agrobacterium-mediated method in Biorun Biosciences Company (Hiei, et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1994\u003c/span\u003e). OsMYBOE1 transgenic plants were characterized by cloning the Hygromycin B gene from the genomic DNA, and their expression levels were further measured by qRT-PCR. OsMYB1KO and OsSPL14KO plants were characterized by cloning their DNA fragments from their genomic DNA, then the types of mutation were identified by sequencing.\u003c/p\u003e\n\u003ch3\u003eBPH resistance detection and analysis\u003c/h3\u003e\n\u003cp\u003eFor the small population assay, approximately 25 rice plants per line were grown in plastic pots for 3 weeks. After this period, 4–6 second to third instar BPH nymphs were added to each plant. The plant status was examined daily until most of the seedlings in each line had withered, and then the seedling mortality rate was recorded.\u003c/p\u003e\n\u003ch3\u003ePathogen inoculation for field experiments\u003c/h3\u003e\n\u003cp\u003eRice flag leaves were inoculated with \u003cem\u003eXoo\u003c/em\u003e strains at the booting stage using the leaf-clipping method, as described previously (Shen, et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). \u003cem\u003eThe Xoo\u003c/em\u003e strain used in this study is PXO99A. Disease symptoms were assessed by measuring the relative lesion length 14 days after inoculation.\u003c/p\u003e\n\u003ch3\u003eQuantitative real-time polymerase chain reaction (qRT-PCR)\u003c/h3\u003e\n\u003cp\u003eTotal RNA was extracted using TRIzol (Invitrogen). Then, 2 µg of total RNA was reverse transcribed into cDNA using the ReverTra Ace™qPCR RT Master Mix with gDNA Remover (Toyobo). The qRT-PCR analysis was performed using the SYBR Green Real-time PCR Master Mix Kit (Toyobo), and the actin (LOC_Os03g50885) and UBQ (LOC_Os01g22490) genes were selected as internal references. Collect three biological replicates for each sample, and calculate the mean and standard deviation.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eYeast two-hybrid (Y2H) assay\u003c/h2\u003e \u003cp\u003eFor the Y2H assay, the target coding sequence (CDSs) was amplified by PCR using gene-specific primers (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) and then inserted into pGADT7/pGBKT7 (Clontech, Mountain View, CA, USA). To detect protein-protein interactions, we conducted assays following the instructions provided by the manufacturer (YC1002; Weidi Biotechnology, Shanghai, China). Clones were grown on deficient medium, lacking either Trp and Leu or Trp, Leu, Ade, and His.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eLuciferase complementation assay (LCA) and bimolecular fluorescence complementation (BiFC) assay\u003c/h3\u003e\n\u003cp\u003eFor the LCA, the tested CDSs were amplified by PCR and inserted into pCAMBIA-35S-nLuc and pCAMBIA-35S-cLuc. For the BiFC assay, the tested CDSs were amplified by PCR and inserted into pCAMBIA1300-35S-nYFP and pCAMBIA1300-35S-cYFP.\u003c/p\u003e \u003cp\u003eAll recombinant plasmids are inserted into the \u003cem\u003eA. tumefaciens\u003c/em\u003e strain GV3101 (pSoup-p19). A. tumefaciens overnight cultures were centrifuged at 5000 g for 5 minutes and then resuspended in infiltration buffer (10 mM MgCl2, 150 µM Acetosyringone, and 10 mM MES at pH 5.6). Adjust the optical density to 1.0 at a wavelength of 600 nm. \u003cem\u003eA. tumefaciens\u003c/em\u003e suspension was incubated for 2 hours at room temperature and then infiltrated into healthy \u003cem\u003eN. benthamiana\u003c/em\u003e leaves using a 1 ml needle-free syringe. The LUC signal was detected 48 hours after infiltration using a CCD camera and a 150 µg ml\u003csup\u003e− 1\u003c/sup\u003e fluorescein solution. Confocal microscopy (LSM 880; Zeiss) was used to detect the YFP signal.\u003c/p\u003e\n\u003ch3\u003eSubcellular localization\u003c/h3\u003e\n\u003cp\u003ePCR amplification of gene CDSs was performed on the pCAMBIA1301-35s-eGFP in \u003cem\u003eN. benthamiana\u003c/em\u003e for transient expression. GFP and DAPI signals were observed using confocal microscopy (LSM 880; Zeiss) at 48 hours.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eTransient transcription assay\u003c/h2\u003e \u003cp\u003eThe sequences encoding GAL4BD, GAL4BD-OsMYB1, and GAL4-VP16 were amplified by PCR and cloned into p1301-35S-Nos to generate the effector plasmids. A sequence consisting of six repeats of the GAL4-binding upstream activating sequence (6×UAS), TATA-Box, and 35S promoter was synthesized and inserted into pGreenII0800-LUC to create the 35S-6×UAS-TATA-LUC reporter plasmid. The full-length OsMYB1 and OsSPL14 CDSs were amplified by PCR and inserted into pCAMBIA1300-35s-3×FLAG or pCAMBIA1300-35s-3×GFP to generate the effector plasmids. The 2 kb \u003cem\u003eOsWRKY45\u003c/em\u003e promoter sequence was cloned into pGreenII0800-LUC to generate the \u003cem\u003eOsWRKY45\u003c/em\u003epro-LUC reporter plasmid.\u003c/p\u003e \u003cp\u003eThe effector and reporter plasmids were inserted into \u003cem\u003eA. tumefaciens\u003c/em\u003e strain GV3101 (pSoup-p19) and then transiently expressed in \u003cem\u003eN. benthamiana\u003c/em\u003e as described above. At 40–48 h post infiltration, the leaves were examined using a CCD camera and a 150 µg ml\u003csup\u003e− 1\u003c/sup\u003e luciferin solution. Or according to the manufacturer's instructions (Promega), using a luminometer quantified the luciferase activity.\u003c/p\u003e \u003cp\u003eThe statistical analysis was performed as previously described (Wang, et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), considering that the state of each \u003cem\u003eN. benthamiana\u003c/em\u003e leaf was different, the negative control on each leaf was regarded as a calibrator and set as one, the two-tailed paired Student's \u003cem\u003et\u003c/em\u003e-test was used to compare the difference in mean ± SD and a P-value of ≤ 0.05.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe two-sample Student's \u003cem\u003et\u003c/em\u003e-test was used to compare the difference in mean ± SD and a P-value of ≤ 0.05 was considered significant for qRT-PCR and the small population assays.\u003c/p\u003e \u003cp\u003eAccession numbers\u003c/p\u003e \u003cp\u003eSequence data for the genes described herein are available in the China Rice Data Center databases under the following accession nos.: \u003cem\u003eOsMYB1\u003c/em\u003e, LOC_Os01g03720; \u003cem\u003eOsSPL7\u003c/em\u003e, LOC_Os04g46580; \u003cem\u003eOsSPL13\u003c/em\u003e, LOC_Os07g32170; \u003cem\u003eOsSPL14\u003c/em\u003e, LOC_Os08g39890; \u003cem\u003eOsMYB22\u003c/em\u003e, LOC_Os01g65370; \u003cem\u003eOsJAZ3\u003c/em\u003e, LOC_Os08g33160; \u003cem\u003eOsJAZ4\u003c/em\u003e, LOC_Os09g23660; \u003cem\u003eOsPR4\u003c/em\u003e, LOC_Os11g37970; \u003cem\u003eOsWRKY45\u003c/em\u003e, LOC_Os05g25770; \u003cem\u003eUbiquitin\u003c/em\u003e, LOC_Os01g22490; \u003cem\u003eACTIN\u003c/em\u003e, LOC_Os03g50885.\u003c/p\u003e \u003c/div\u003e "},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eOsMYB1\u003c/b\u003e \u003cb\u003eresponded to BPH infestation and\u003c/b\u003e \u003cb\u003eXoo\u003c/b\u003e \u003cb\u003einfection\u003c/b\u003e\u003c/p\u003e\u003cp\u003ePreviously, we demonstrated that OsMYB22 forms a complex with co-repressor TOPLESS proteins to inhibit the expression of \u003cem\u003eF3'H\u003c/em\u003e, thereby enhancing rice resistance against BPH (Sun, et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). To search the proteins that could interact with OsMYB22, we used yeast two hybrid (Y2H) and identified OsMYB1 as an interacting partner of OsMYB22, which was further confirmed by BiFC assay (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eWe next detected the expression of \u003cem\u003eOsMYB1\u003c/em\u003e in various rice tissues. The result showed that OsMYB1 has a tissue-wide gene expression pattern, and the highest expression level was observed in the stem. (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). To study the function of OsMYB1 in rice resistance, we further detected the expression of \u003cem\u003eOsMYB1\u003c/em\u003e upon BPH infestation and \u003cem\u003eXoo\u003c/em\u003e infection. It was revealed that \u003cem\u003eOsMYB1\u003c/em\u003e gene exhibits significantly repressed expression trends at multiple time points by both BPH infestation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB) and \u003cem\u003eXoo\u003c/em\u003e infection (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC), respectively. These findings suggested that \u003cem\u003eOsMYB1\u003c/em\u003e may play a role in regulating the interaction between rice and these two kinds of biotic factors.\u003c/p\u003e\u003ch2\u003eOsMYB1 showed variety characteristics of a TF\u003c/h2\u003e\u003cp\u003eTo analyze the characteristics of the OsMYB1 protein, we first studied the subcellular localization of the MYB1-eGFP fusion protein by expressing it in \u003cem\u003eN. benthamiana\u003c/em\u003e leaves using Agrobacterium-mediated transformation, it was revealed that the fluorescence signal of MYB1-eGFP overlapped with DAPI (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), indicating a nuclear localized character. Meanwhile, OsMYB1 proteins showed transcriptional activation in yeast, with the function being situated to the C-terminals (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). In addition, GAL4BD-MYB1 showed inhibitory effects on reporter gene in \u003cem\u003eN. benthamiana\u003c/em\u003e, indicating a potential role of OsMYB1 in transcriptional repression in plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, D). These findings collectively indicated OsMYB1 exhibits \u003cb\u003ev\u003c/b\u003eariety characteristics of a plant TF and can function as a transcriptional repressive regulator in planta.\u003c/p\u003e\u003cp\u003e \u003cb\u003eOsMYB1\u003c/b\u003e \u003cb\u003enegatively regulated rice resistance to BPH\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo explore the function of \u003cem\u003eOsMYB1\u003c/em\u003e, overexpression lines were generated using Agrobacterium-mediated genetic transformation. OsMYB1OE-1 and OsMYB1OE-11 lines showed greatly promoted expression of \u003cem\u003eOsMYB1\u003c/em\u003e compared with the wild type (WT) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). In BPH resistance test, both OsMYB1OE-1 and OsMYB1OE-11 plants died earlier than WT after BPH infestation. Moreover, the mortalities were higher for OsMYB1OE-1 and OsMYB1OE-11 plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, C; Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e), suggesting a negative regulatory function of \u003cem\u003eOsMYB1\u003c/em\u003e in rice resistance to BPH. Meanwhile, we constructed homozygous knockout lines of \u003cem\u003eOsMYB1\u003c/em\u003e using CRISPR/CAS9 technology, and two homozygous lines, OsMYB1KO-2 and OsMYB1KO-20 were got. In OsMYB1KO-2 line, a T was inserted, while in line OsMYB1KO-20, a C was deleted (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). However, neither OsMYB1KO-2 nor OsMYB1KO-20 showed significant difference to WT in BPH resistance test (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE, F; Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). Based on data from the Plant Transcription Factor Database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://planttfdb.gao-lab.org/\u003c/span\u003e\u003cspan address=\"https://planttfdb.gao-lab.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), the MYB family in rice consists of 130 members. Among them, 15 MYB proteins share the closest evolutionary relationship with OsMYB1, some of them are up-regulated in OsMYB1KO plants (Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eA). Therefore, the lack of significant difference in BPH resistance between OsMYB1KO-2, OsMYB1KO-20, and WT might be due to possible functional redundancy among these MYBs.\u003c/p\u003e\u003cp\u003e \u003cb\u003eOsMYB1\u003c/b\u003e \u003cb\u003enegatively regulated rice resistance to\u003c/b\u003e \u003cb\u003eXoo\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWe further investigated the function of \u003cem\u003eOsMYB1\u003c/em\u003e in resistance to \u003cem\u003eXoo\u003c/em\u003e using the leaf-cutting method. After 14 days post inoculation, the OsMYB1OE-1 and OsMYB1OE-11 lines exhibited a significant increase in lesion length compared to the WT, while the OsMYB1KO-2 and OsMYB1KO-20 showed a significant decrease in lesion length (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). And statistical analysis of the lesion length further verified the result (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Thus, \u003cem\u003eOsMYB1\u003c/em\u003e showed a negative regulatory role in rice resistance to \u003cem\u003eXoo\u003c/em\u003e.\u003c/p\u003e\u003ch2\u003eOsMYB1 interacted with SPLs\u003c/h2\u003e\u003cp\u003eIn \u003cem\u003eArabidopsis\u003c/em\u003e, AtSPL9 inhibits the formation of the MBW complex PAP1-TT8-TTG1 by interacting with MYB, thereby counteracting downstream genes (Gou, et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). In phylogenetic analysis, it was revealed that OsMYB1 was homologous to AtMYB62 (AT1G68320) and AtMYB116 (AT1G25340) (Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eB), and the amino acids of AtMYB62, AtMYB116 and OsMYB1 showed high similarity (Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eC). It is predicted that AtMYB62 can interact with AtSPL4 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://thebiogrid.org/\u003c/span\u003e\u003cspan address=\"https://thebiogrid.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) (Fig. \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003eA). Both OsSPL7 and OsSPL14 were reported to regulate resistance to rice blight (Liu, et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). However, OsSPL14 plays a broader role in stress resistance, including rice blast resistance (Wang et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) and tolerance to abiotic stresses such as drought and chilling (Chen et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Jia et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). This wider functional spectrum makes OsSPL14 a more compelling candidate for further study. Additionally, phylogenetic analysis shows that OsSPL13 shares high sequence similarity in DNA binding domain with both OsSPL7 and OsSPL14 (Fig. \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003eB, C). Based on these observations, we tested the potential interactions between OsMYB1 and these three OsSPL proteins, with a focus on OsSPL14 due to its broader role in stress responses. In Y2H assay, it was revealed that OsMYB1 could interact with OsSPL7 and OsSPL14, but not OsSPL13 in yeast (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Both OsSPL7 and OsSPL14 have been previously identified as positive regulators of rice resistance to abiotic stresses such as cold and drought tolerance (Chen, et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Jia, et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) and biotic stresses such as \u003cem\u003eM. oryzae\u003c/em\u003e and \u003cem\u003eXoo\u003c/em\u003e (Liu, et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Wang, et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). We further used Luciferase complementation assay (LCA) to verify the interaction. Co-infiltration of bacterium harboring both OsMYB1 and OsSPL7, and OsMYB1 and OsSPL14 showed strong inflorescence by LCA and BiFC assays, while those infiltrations of other combinations did not (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, C).\u003c/p\u003e\u003cp\u003e \u003cb\u003eKnockout of\u003c/b\u003e \u003cb\u003eOsSPL14\u003c/b\u003e \u003cb\u003edecreased rice resistance to BPH\u003c/b\u003e\u003c/p\u003e\u003cp\u003eOsSPL14 was reported to negatively regulate rice resistance to \u003cem\u003eXoo\u003c/em\u003e (PXO99A) (Liu et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). We further investigated the role of \u003cem\u003eOsSPL14\u003c/em\u003e in resistance to BPH. First, \u003cem\u003eOsSPL14\u003c/em\u003e transcript was detected in the leaf and sheath (Fig. \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003e). And in response to BPH infestation, \u003cem\u003eOsSPL14\u003c/em\u003e transcript was mainly up-regulated at multiple time points (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Next, \u003cem\u003eOsSPL14\u003c/em\u003e homozygous knockout lines, OsSPL14KO-15 and OsSPL14KO-19, were generated using CRISPR/CAS9 technology. In OsSPL14KO-15, a T was inserted, while in OsSPL14KO-19, a G was inserted (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Consistent with the previously reported phenotypes in development, the OsSPL14KO plants exhibited an increased tiller number and reduced plant height (Fig. \u003cspan refid=\"MOESM6\" class=\"InternalRef\"\u003eS6\u003c/span\u003e) (Song, et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). In BPH resistance evaluation, it was revealed that both OsSPL14KO-15 and OsSPL14KO-19 lines died earlier than WT after BPH infestation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). In accordance, the morality of OsSPL14KO-15 and OsSPL14KO-19 plant were much higher than that of the WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD), indicating a positive regulatory role of \u003cem\u003eOsSPL14\u003c/em\u003e in rice defense against BPH. Overall, OsSPL14 and OsMYB1 have opposing functions in regulating rice resistance to these two kinds of biotic stresses.\u003c/p\u003e\u003ch2\u003eOsMYB1 and OsSPL14 antagonistically regulated the expression of defense-related genes\u003c/h2\u003e\u003cp\u003eTo investigate the molecular mechanism of the antagonizing role of \u003cem\u003eOsMYB1\u003c/em\u003e and \u003cem\u003eOsSPL14\u003c/em\u003e in rice resistance, we tested the expression of downstream defense-related genes, \u003cem\u003eOsPR4\u003c/em\u003e and \u003cem\u003eOsWRKY45\u003c/em\u003e, in \u003cem\u003eOsSPL14\u003c/em\u003e and \u003cem\u003eOsMYB1\u003c/em\u003e related transgenic plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA, B). Compared with WT, the expression of \u003cem\u003eOsPR4\u003c/em\u003e and \u003cem\u003eOsWRKY45\u003c/em\u003e in the OsSPL14KO plants was reduced, but increased in the OsMYB1KO plants. Besides, their expression displayed significantly reduced expression in the OsMYB1OE plants. Notably, \u003cem\u003eOsWRKY45\u003c/em\u003e is a key downstream target gene of OsSPL14 that associated with rice resistance (Wang, et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), we hypothesis that OsMYB1 regulates rice resistance by inhibiting \u003cem\u003eOsWRKY45\u003c/em\u003e. Furthermore, to explore the interplay between OsMYB1 and OsSPL14 in regulating \u003cem\u003eOsWRKY45\u003c/em\u003e, we conducted transient expression assay using the \u003cem\u003eOsWRKY45\u003c/em\u003e promoter-driven reporter gene \u003cem\u003eLUC\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). It was revealed that OsSPL14 promoted, while OsMYB1 inhibited the expression of \u003cem\u003eLUC\u003c/em\u003e driven by \u003cem\u003eOsWRKY45\u003c/em\u003e promoter (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC, D). Furthermore, when co-expressing \u003cem\u003eOsSPL14\u003c/em\u003e and \u003cem\u003eOsMYB1\u003c/em\u003e, the expression of \u003cem\u003eLUC\u003c/em\u003e was decreased compared with expressing \u003cem\u003eOsSPL14\u003c/em\u003e alone, suggesting that OsMYB1 can antagonize OsSPL14 by interacting with it. These results suggest an antagonistic relationship between OsMYB1 and OsSPL14 in the regulation of rice immune responses.\u003c/p\u003e\u003ch2\u003eOsMYB1 interacted with repressor JAZs but not co-repressor TOPLESS\u003c/h2\u003e\u003cp\u003eNow that OsMYB1 might function as a transcription inhibitor, we further investigate the underlying molecular mechanism. A putative L×L×L type EAR motif was identified near the N-terminus of OsMYB1 (Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eC), indicative of recruiting co-repressors TOPLESS. Consequently, we tested if there is any interaction between OsMYB1 and the three rice co-repressor TOPLESS proteins, TPL, TPR1, and TPR2. However, Y2H experiments demonstrated that OsMYB1 did not interact with any of the TOPLESS proteins in yeast (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA). Other, JAZs are typical inhibitors in the jasmonic acid (JA) signaling pathway (Wasternack and Song \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). We checked if OsMYB1 interacts with some JAZs. We used Y2H and revealed that the clones harboring both OsMYB1 and OsJAZ3, and OsMYB1 and OsJAZ4 could grow on the SD-Leu-Trp-His-Ade medium, while those controls could not (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB). In LCA, co-infiltration of bacterium harboring both OsMYB1 and OsJAZ3, OsMYB1 and OsJAZ4 showed strong inflorescence, while those infiltrations of other combinations did not (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC). The results indicated that OsMYB1 may form a complex with JAZ3/JAZ4 to function as a transcriptional repressor complex.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eAs an important component of plant immunity, TFs play key roles in mediating downstream signaling processes, enabling plants to rapidly respond to a wide range of biotic stresses (Tsuda and Somssich \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Among them, MYB family TFs have been proven to be involved in regulating plant resistance to multiple biotic stresses (He, et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Li, et al. 2020; Lin, et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Sharma, et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), and one single MYB TF has been demonstrated to be able to regulate resistance to different kinds of biotic stresses (Lü, et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Mengiste, et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). As the most notorious insect pest and pathogen respectively, BPH and \u003cem\u003eXoo\u003c/em\u003e can attack rice plants simultaneously in a field environment, identifying genes that can help plants survive better under interlaced adversity factors can not only further our knowledge of molecular mechanism on comprehensive immunity, but also provide a solid theoretical foundation for rice resistance breeding. In this study, we found that the MYB family TF OsMYB1 is involved in regulating rice resistance to both BPH (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, C) and \u003cem\u003eXoo\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), indicating OsMYB1 to be a comprehensive regulator in plant immunity against different kinds of adverse factors. However, the OsMYB1KO plants showed difference in resistance to BPH and \u003cem\u003eXoo\u003c/em\u003e, indicating that the resistance mechanism to these two kinds of biotic stresses might differ (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE, F, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, B). Additionally, rice contains 130 MYB TFs, each with unique expression patterns across tissues and responses to different biotic stresses, indicating they might differ in assignment for resistance to different biotic stresses.\u003c/p\u003e\u003cp\u003eMYB family TFs can interact with other TFs, such as the AP2 family, the bHLH family, and the SPL family (Ding, et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Gou, et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Qiu, et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), and play a synergistic or antagonistic regulatory role on downstream genes. Here, we found that OsMYB1 interacts with OsSPL7 and OsSPL14 in yeast and \u003cem\u003eN. benthamiana\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). In rice, SPL TFs play a crucial role in rice defense. As an example, OsSPL7 and OsSPL14 positively regulate rice resistance to \u003cem\u003eXoo\u003c/em\u003e (Liu, et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), and OsSPL14 mediates rice resistance to rice blast (Wang, et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Besides, chilling-induced phosphorylation of IPA1 by OsSAPK6 activates chilling tolerance responses in rice (Jia, et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), and IPA1 improves drought tolerance by activating SNAC1 (Chen, et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), suggesting that OsSPL14 plays diverse functions in rice resistance to different stresses. In addition, as target of miR156, SPLs are highly conserved across different species (Wang and Wang \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Thus, we focused on OsSPL14 to reveal the interaction between OsMYB1 and OsSPL14 in regulating rice resistance to BPH and \u003cem\u003eXoo\u003c/em\u003e. Here we proved that OsSPL14 positively regulated BPH resistance (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e), meanwhile, OsSPL14 was reported to positively regulate resistance to \u003cem\u003eXoo\u003c/em\u003e (Liu, et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Thus, OsSPL14 is a positive regulator in rice immunity against different kinds of adverse factors that function antagonistically with OsMYB1. To the contrary, miR156 plays a negative role in regulating rice resistance to BPH and \u003cem\u003eXoo\u003c/em\u003e (Ge, et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Liu, et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), further proving the function of OsmiR156/OsSPL14 module in rice immunity.\u003c/p\u003e\u003cp\u003eOsMYB1 has some characters of a TF (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). In \u003cem\u003eN. benthamiana\u003c/em\u003e, OsMYB1 exhibits transcriptional repression (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Expression of the defense-related marker genes, such as Os\u003cem\u003ePR4\u003c/em\u003e and \u003cem\u003eOsWRKY45\u003c/em\u003e were influenced in the OsMYB1OE and OsMYB1KO plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). Specifically, the transcription of \u003cem\u003eOsWRKY45\u003c/em\u003e, which activated by OsSPL14 (Wang, et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), was inhibited by OsMYB1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). By transient transcription assays, OsSPL14 can activate the expression of \u003cem\u003eLUC\u003c/em\u003e driven by the \u003cem\u003eOsWRKY45\u003c/em\u003e promoter, however, when co-expressing OsMYB1 with OsSPL14, the expression of \u003cem\u003eLUC\u003c/em\u003e was decreased, which might be the underlying mechanism of the opposing roles of OsMYB1 and OsSPL14 in regulating rice immunity (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD).\u003c/p\u003e\u003cp\u003eAltogether, OsMYB1 antagonizes with OsSPL14 in the regulation of rice resistance to BPH and \u003cem\u003eXoo.\u003c/em\u003e There are several main specific forms of antagonism between TFs when they interact with each other. By influencing each other's DNA-binding capacity, ERF4 and MYB52 oppositely regulate downstream gene expression through physical interaction (Ding, et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). BES1 in the BR signaling pathway interacts with MYB34/51/122 and suppresses its transcriptional activity, disrupting the expression of glucosinolate biosynthesis genes, resulting in a reduction of plant resistance against herbivorous insects (Liao, et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). By competing for the same binding site on downstream gene promoters, MYBS1 and MYBS2 competitively regulate the expression of the \u003cem\u003eαAmy\u003c/em\u003e gene in opposite ways, thereby controlling the balance of sugar content in rice (Chen, et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Nevertheless, the underlying molecular mechanism of the antagonistic interaction between OsMYB1 and OsSPL14 remains unclear and requires further investigation.\u003c/p\u003e\u003cp\u003eTo further explore the molecular mechanism of transcriptional repression of OsMYB1, we found that OsMYB1 contains a putative repressive EAR motif in its N-terminal region, which can interact with co-repressors TOPLESS to form a transcription repressor complex (Plant, et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In consistency, MYB22 interacts with TOPLESS through its EAR motif (Sun, et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). However, no interaction between OsMYB1 and TOPLESS was detected (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA), implying that OsMYB1 might function as a repressive TF through an alternative mechanism. JA is a crucial phytohormone that is central in regulating plant defense responses against biotic stresses, such as herbivorous insects and pathogenic microorganisms. In rice, key genes in the JA biosynthesis pathway, including \u003cem\u003eOsAOS1\u003c/em\u003e, \u003cem\u003eOsLOX10\u003c/em\u003e, and \u003cem\u003eOsOPR10\u003c/em\u003e, positively influence resistance to \u003cem\u003eXoo\u003c/em\u003e (Hou, et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Zhou, et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Xu, et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), while \u003cem\u003eOsAOC\u003c/em\u003e enhances resistance to BPH (Guo, et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). In the JA signal transduction pathway, OsCOI1-RNAi plants exhibited reduced resistance to \u003cem\u003eXoo\u003c/em\u003e (Yang, et al. 2009), and both \u003cem\u003ecoi1\u003c/em\u003e and \u003cem\u003ecoi2\u003c/em\u003e mutants demonstrated increased susceptibility to BPH attacks (Xu, et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Wang, et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The Key TF OsMYC2 has been reported to positively mediate rice resistance to both \u003cem\u003eXoo\u003c/em\u003e and BPH (Uji, et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Xu, et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). As a necessary part of JA signal transduction, JAZ repressors can cooperate with different TFs to repress the transcription of downstream genes. In tartary buckwheat, FtJAZ1 can assist FtMYB11 in inhibiting the expression of rutin biosynthesis pathway-related genes (Zhou, et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). In \u003cem\u003eArabidopsis\u003c/em\u003e, the JAZs protein can interact with the R2R3 DNA binding domain of MYB21/24 through its ZIM domain (Song, et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). In rice, COI1a, COI1b, and OsCOI2 interact with specific JAZ proteins in the presence of JA-Ile, JA-Val, and JA-Leu, with slight variations in interaction patterns among them (Fu, et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Wang, et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Notably, OsJAZ3 and OsJAZ4 are consistently recognized by all COIs, highlighting their broad responsiveness to JA signal transduction. Accordingly, we found that OsMYB1 can interact with OsJAZ3 and OsJAZ4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB, C), implying that OsMYB1 might form a transcription repressor complex with OsJAZs, which provides a potential possibility for MYB1 to exert a molecular mechanism of transcriptional repression. Furthermore, the interactions between OsMYB1 and its partners (OsMYB22, OsSPL14, and OsJAZs) were supported by yeast two-hybrid (Y2H) and BiFC/LCA assays, which are recognized as reliable, albeit preliminary, methods for demonstrating protein-protein interactions (Yu et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Zhu et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Therefore, additional in vivo or in vitro validation techniques, such as co-immunoprecipitation (Co-IP) or pull-down assays, would further strengthen these findings.\u003c/p\u003e"},{"header":"Statements \u0026 Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by grants from the Shanghai Chongming Agriculture Science and Innovation Project (Grant No. 2023CNKC-01-04), Shanghai Science and Technology Innovation Action Plan (No. 22N41900300).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBS and ZY performed the experiments and analyzed the data. XM and HL designed the research. BS and ZS wrote the paper. ZT and LZ revised the paper. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAn C, Sheng LP, Du XP, Wang YJ, Zhang Y, Song AP, Jiang JF, Guan ZY, Fang WM, Chen FD, Chen SM (2019) Overexpression of CmMYB15 provides chrysanthemum resistance to aphids by regulating the biosynthesis of lignin. Hortic Res 6:84\u003c/li\u003e\n \u003cli\u003eChen FH, Zhang HM, Li H, Lian L, Wei YD, Lin YL, Wang LN, He W, Cai QH, Xie HG, Zhang H, Zhang JF (2023) IPA1 improves drought tolerance by activating SNAC1 in rice. 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Funct Plant Biol 40(3):304-313\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":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"plant-cell-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pcre","sideBox":"Learn more about [Plant Cell Reports](https://www.springer.com/journal/299)","snPcode":"299","submissionUrl":"https://submission.nature.com/new-submission/299/3","title":"Plant Cell Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"MYB, SPL, Brown planthopper, Xanthomonas oryzae pv. oryzae.","lastPublishedDoi":"10.21203/rs.3.rs-5172835/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5172835/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIn their natural habitats, plants are concurrently attacked by different biotic factors. \u003cem\u003eXanthomonas oryzae \u003c/em\u003epv\u003cem\u003e. oryzae\u003c/em\u003e (\u003cem\u003eXoo\u003c/em\u003e) is a pathogen that severely deteriorates rice yield and quality, and brown planthopper (BPH; \u003cem\u003eNilaparvata lugens\u003c/em\u003e) is a rice specific insect pest with the damage topping other pathogens. Although genes for respective resistance to BPH and \u003cem\u003eXoo\u003c/em\u003e have been widely reported, few studies pay attention to simultaneous resistance to both. In this study, we identified a MYB transcription factor, OsMYB1, which exhibited\u003cem\u003e \u003c/em\u003ediverse transcriptional regulatory capabilities and a negative regulatory role in resistance to both BPH and \u003cem\u003eXoo\u003c/em\u003e. Biochemical and genetic analysis proved OsMYB1 to be a TF that could interact with OsSPL14, a positive regulator of rice resistance to \u003cem\u003eXoo\u003c/em\u003e. \u003cem\u003eOsSPL14\u003c/em\u003e mutants showed increased sensitivity to BPH, suggesting that \u003cem\u003eOsSPL14\u003c/em\u003e is contrary to\u003cem\u003e OsMYB1\u003c/em\u003ein regulating rice resistance to these two biotic stresses. Consistently, OsMYB1 and OsSPL14 displayed opposite functions in regulating defense-related genes. OsMYB1 can form transcription regulation complexes with repressor OsJAZs instead of co-repressor TOPLESS to possibly realize its transcriptional repression function. Taken together, we concluded that two interacting TFs in rice, OsMYB1 and OsSPL14, played antagonistic roles in regulating resistance to BPH and \u003cem\u003eXoo\u003c/em\u003e.\u003c/p\u003e","manuscriptTitle":"OsMYB1 antagonizes OsSPL14 to mediate rice resistance to brown planthopper and Xanthomonas oryzae pv. oryzae","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-05 17:30:38","doi":"10.21203/rs.3.rs-5172835/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Accept","date":"2024-12-19T04:04:16+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2024-12-04T01:57:37+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-12-04T00:17:46+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-12-03T12:19:09+00:00","index":"","fulltext":""},{"type":"submitted","content":"Plant Cell Reports","date":"2024-12-02T21:26:17+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"plant-cell-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pcre","sideBox":"Learn more about [Plant Cell Reports](https://www.springer.com/journal/299)","snPcode":"299","submissionUrl":"https://submission.nature.com/new-submission/299/3","title":"Plant Cell Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"28abce63-760c-4d0a-b59d-c09e517d71fa","owner":[],"postedDate":"December 5th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-12-30T16:04:07+00:00","versionOfRecord":{"articleIdentity":"rs-5172835","link":"https://doi.org/10.1007/s00299-024-03411-8","journal":{"identity":"plant-cell-reports","isVorOnly":false,"title":"Plant Cell Reports"},"publishedOn":"2024-12-26 15:57:13","publishedOnDateReadable":"December 26th, 2024"},"versionCreatedAt":"2024-12-05 17:30:38","video":"","vorDoi":"10.1007/s00299-024-03411-8","vorDoiUrl":"https://doi.org/10.1007/s00299-024-03411-8","workflowStages":[]},"version":"v1","identity":"rs-5172835","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5172835","identity":"rs-5172835","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}